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https://openalex.org/W4302308302	https://zenodo.org/record/5835271/files/1117mechatroj04.pdf	English	null	IMPLEMENTATION OF KALMAN FILTER ON VISUAL TRACKING USING PID CONTROLLER	Zenodo (CERN European Organization for Nuclear Research)	2,017	cc-by	5,904	Mechatronics and Applications: An International Journal (MECHATROJ), Vol. 1, No.1, January 2017 Mechatronics and Applications: An International Journal (MECHATROJ), Vol. 1, No.1, January 2017 KEYWORDS Kalman filter, PID, bilinear transformation, z-transform ABSTRACT This paper explain the design of visual control system which equip Kalman filter as an additional sub- system to predict object movement. It is a method to overcome some weakness, such as low range view of camera and low FPS (Frame per Second). It alsoassist the system to track a fast moving object. The system is implemented to 2 motor servos, which are move on horizontal and vertical axis. Digital PID (Proportional, Integral, and Derivative) controller is used in the system, and bilinear transformation is used to approximate the value of derivative in transforming the analogue to digital controller on z- transform.In conclusion, we can get the value of system responses time from both motor servos. The rise time and settling time of motor servo in horizontal axis are 0.402s and 1.63s, and vertical axis’s responses are 0.38s and 1.34s. IMPLEMENTATION OF KALMAN FILTER ON VISUAL TRACKING USING PID CONTROLLER Abdurrahman,F.1, Gunawan Sugiarta2 and Feriyonika3 Department of Electrical Engineering, Bandung State of Polytechnic, Bandung, Indonesia 1. INTRODUCTION In this digital era, robotic technologies are implementing vision-based control, such as object tracking [1], face tracking [2], and position-based control [3]. It was developed as one of the control method in robotic community. Researchers keep learning and involving the control system to transform itas close as human capability. Recent paper describes the vision-based control and computer vision as numerical theory [4], unfortunately there are lack of paper which describedand design the system in a more practical way. There are many add-on systems in vision-based control to improve accuracy, such as image filter and predictive filter. To implement such systems, which will be used onvarious environment with lots of noises, the method should therefore always be develop to reduce noises. So, there are many researchers in this field to improve the system’s capability. Gian Luca designedimage-based visual servo using geometry [5] in mobile robot and combined it with kinematic design.This paper will explain about a visual tracking system controlled by visual control using PID (Proportional, integral, and derivative) control law to move plants in horizontal and vertical axis. It also combined with Kalman filter to predict the object movement within camera frame. 41 Mechatronics and Applications: An International Journal (MECHATROJ), Vol. 1, No.1, January 201 The purpose of the system is to predict the object movement, so it can make the system work in a better performance to predict the object movement while it is not detected. The system will not search the object while it was outside the range of vision of the system without using kalman filter. The control law (PID), filter, and feature extraction will be designed in one processor. We will use 2-D in feature extraction, there are two axis which are being the feature for control system. The feature extraction method is contour in color detection, however it will not be discussed in this paper. This paper will concern to the design in development of visual control in robotics using the predictive filter. This paper proposedon how to implement and design the system in a more practicalway.According to the passages above, we canconclude that the advantages of the proposed system are: − The digital PID algorithm will be designed using z-transform which make it easier to understand and implement. − Kalman filter will be used to predict object movement, so it will help the system’s responsewhen tracking faster object. 1. INTRODUCTION Kalman filter is useful on system arrangement using low resolution and low FPS camera. It will be useful if the camera frame which used in the system doesn’t have enough resolution and bad fps (frame per second) [6]. This paper is organized in 5 sections. Section 2 and 3 explain about the digital control system design and Kalman filter theory. Section 4 will discussed about the design from the control system perspective and the next section contains results and data analysis comparingdata between system with Kalman filter and nowithout Kalman filter. Finally, the last section is about the conclusion of all the system. 2. DIGITAL CONTROL SYSTEM DESIGN In this section, we will review the digital control system. Z-transform can be used to design the digital control system. To transform the analogue to digital system, the system must following these requirements [7]: − A stable analogue system must transform to a stable digital system. − The frequency response of the digital system must closely resemble the frequency response of the analogue system in the frequency range 0 2 ⁄ where is the sampling frequency. There are many methods to transform the analogue to digital system, consist of: 2.2 Bilinear Transform Equation 2.6 is the relation between Laplace and z-transform [7] using bilinear transformation = (2.6) (2.6) The relation can be found from the equality z = using the first-order approximation on the equation 2.7 where the constant c = 2/T = ln = [ ] (2.7) (2.7) 2.1 Differencing Method The system can be represented by a transfer function or differential equation. Numerical analysis provides standard approximations of the derivative so as to obtain the solution to a differential equation. There are 2 types of the differencing method [7], such as: Forward differencing − Forward differencing g In forward differencing the derivative approach will found through the basic equation 2.1: In forward differencing the derivative approach will found through the basic equation 2 42 Mechatronics and Applications: An International Journal (MECHATROJ), Vol. 1, No.1, January 2017 Mechatronics and Applications: An International Journal (MECHATROJ), Vol. 1, No.1, January 2017 = [ + 1 − ] (2.1) (2.1) (2.1) After that, the equation will be transformed into z-domain, so the equation 2.2 will be produced: After that, the equation will be transformed into z-domain, so the equation 2.2 will be produced: → [ −1] (2.2) (2.2) Based on this method, we could find the relation between Laplace and z-transform. The relation showed on the equation 2.3,with T is a time sampling Based on this method, we could find the relation between Laplace and z-transform. The relation showed on the equation 2.3,with T is a time sampling → (2.3) (2.3) − Backward differencing The other differencing method is backward differencing with the approximation of the derivative is showed on the equation 2.4 = [ − −1 ] (2.4) (2.4) The equation will be transformed into z domain, so we can get the relation between Laplace and z domain in equation 2.5 The equation will be transformed into z domain, so we can get the relation between Laplace and z domain in equation 2.5 The equation will be transformed into z domain, so we can get the relation between Laplace and z domain in equation 2.5 Laplace and z domain in equation 2.5 Laplace and z domain in equation 2.5 → (2.5) (2.5) 2.2 Bilinear Transform 3. DISCRETE KALMAN FILTER The method determine variable R is various, variable can be considered as a constant value or measured using the equation. g q # = & +(& +( + , = + #- −+ & = . − #+ & 3. DISCRETE KALMAN FILTER Kalman filter is essentially a set of mathematical equations that implement a predictor-corrector type estimator that is optimal in the sense that it minimizes the estimated error covariance. There are 2 general processes to implemented, such as time update and measurement update. The time update equations can be thought of as predictor equation, while the measurement update equations can be thought of as corrector equation. Indeed the final estimation algorithm resembles that of a predictor-corrector algorithm for solving numerical problems [8] as shown in Figure 1. 43 Mechatronics and Applications: An International Journal (MECHATROJ), Vol. 1, No.1, January 201 Figure 1. Process of Kalman Filter Figure 1. Process of Kalman Filter Figure 1. Process of Kalman Filter Kalman filter can be use in visual control to track the object. Normally, a tracking algorithm would be able to locate the object anywhere within the image at any point in time. However, only a limited region of the image is searched. Visual tracking will be failed if the object was moving to outside the region of camera frame and movetoo fast. Kalman filter can predict the object movement and solve these problems [6]. To implement discrete Kalman filter [8], we need to write down these equations into algorithm − Time update First task is to determine the prediction value (xk), followed by error covariance (pk). There are other variables, such as A, B, uk, and Q. These variables can be assumed as constant variables. − Time update First task is to determine the prediction value (xk), followed by error covariance (pk). There are other variables, such as A, B, uk, and Q. These variables can be assumed as constant variables. = !"# + $%# & = !& '!( + ) − Measurement update The process continued to determine Kalman gain (kk), error covariance (pk), and prediction value (xk). Xk will be the output of Kalman filter and used as feedback in control system. H can be determined as identity matrix, furthermore R is standard deviation. The method determine variable R is various, variable can be considered as a constant value or measured using the equation. − Measurement update The process continued to determine Kalman gain (kk), error covariance (pk), and prediction value (xk). Xk will be the output of Kalman filter and used as feedback in control system. H can be determined as identity matrix, furthermore R is standard deviation. 4. SYSTEM DESIGN The system was designed to control 2 plants, consist of servo in horizontal axis and servo in vertical axis. These were controlled using PID controller and Kalman filter as feedback to move camera. The output of camera is an image frame which is proceed by computer vision technology before proceed to Kalman filter. 44 4.1 Digital PID Controller 9 >0 −>2. . 9 >0 + <0. : >0 + <1. . : >0 + <2. . : >0 % = A AB %[ −1] −A AB %[ −2] + CB AB [] + C AB [ −1] + C AB [ −2] (4.9) (4.9) The control system is a control loop, so Ziegler-Nichols type 2 was used to find the PID parameter. However, we need to tune it manually to get the best responses time. This method proposed as practical procedure to find the parameter properly. Mechatronics and Applications: An International Journal (MECHATROJ), Vol. 1, No.1, January 201 echatronics and Applications: An International Journal (MECHATROJ), Vol. 1, No.1, January 2017 Figure 2. Block Diagram of Control System PID controller (horizontal axis) Kalman Filter Feature Extraction + - Set Point (x,y) PWM converter (horizontal axis) + - Controller Motor DC Camera Servo (horizontal axis) Image Frame PID controller (vertical axis) PWM converter (vertical axis) + - Controller Motor DC Servo (vertical axis) Vertical position Horizontal position x coordinate y coordinate Coordinate (x,y) x coordinate y coordinate Figure 2. Block Diagram of Control System Figure 2 is the block diagram of the control system that explained on the proceeding section. Each block in the system has their own function. Set point has 2 values, there are x and y coordinate, which are proceed by their own PID block. Then, the output PID will be converted into degree and give instructions to servo to move to certain position. Servo has its own controller to activate the DC motor, so motor servo can determine its position. A Camera will be attached to a servo, therefore the servo’s position also shows the camera frame. The object will be recognized using image processing algorithm in feature extraction and release the x and y coordinate. Figure 3 is illustrating the hardware of the system using two servos (white) and the camera (green) so the system can move the camera in 2 axis, such as horizontal and vertical axis. Figure 3. Horizontal and Vertical of Motor Servos Figure 3. Horizontal and Vertical of Motor Servos Kalman filter will proceed both coordinates in the same time, it means the coordinates will be merged. Kalman filter will be designed using the program. Finally, the output of the filter will be used as feedback in the whole system. Subsection below will explain 2 most important components in control system, such as digital PID (Proportional, Integral, and Derivative) controller and Kalman filter. 45 echatronics and Applications: An International Journal (MECHATROJ), Vol. 1, No.1, January 2017 4.1 Digital PID Controller Z-transform was implemented in the digital control system to design the algorithm. Equation 4.1 is the PID basic equation in Laplace. / = 0 + 12 + 3 (4.1) (4.1) According to the equation, z-transform was applied using bilinear transform, then it will produced the equation 4.2. There is Ts variable in the equation, which is a time sampling. The time sampling has been measured with the value of 0.09second. According to the equation, z-transform was applied using bilinear transform, then it will produced the equation 4.2. There is Ts variable in the equation, which is a time sampling. The time sampling has been measured with the value of 0.09second. 4 5 = 0 + 167 + 3. 7 (4.2) 9 : = 0 −1 + 1 + 12. + 1 + 3. ;. 2. −1 −1 + 1 → <0 + <1 + <2 >0 + >1 + >2 (4.2) : Equation 4.3 is the PID equation in z domain before simplified. We can get six variables, consist of b0, b1, b2, a0, a1, and a2. Each variable will be determined using the equation 4.3 until equation 4.8. <0 = 0 + 12. + 2. 3. ; (4.3) <1 = ?. ; −4. 3. ; (4.4) <2 = −0 + 12. + 2. 3. ; (4.5) >0 = 1 (4.6) >1 = 0 (4.7) >2 = −1 (4.8) (4.8) Therefore, these equations can be substituted to be the final equation, which is equation 4.9 to implementPID (Proportional, Integral, and Derivative) algorithm in digital. Therefore, these equations can be substituted to be the final equation, which is equation 4.9 to implementPID (Proportional, Integral, and Derivative) algorithm in digital. >0. 9 + >1. . 9 + >2. . 9 = <0. : + <1. . : + <2. . : 9 = −>1. . 9 >0 −>2. . 9 >0 + <0. : >0 + <1. . : >0 + <2. . : >0 % = A AB %[ −1] −A AB %[ −2] + CB AB [] + C AB [ −1] + C AB [ −2] (4.9) >0. 9 + >1. . 9 + >2. . 9 = <0. : + <1. . : + <2. . : 9 = −>1. . B = system velocity There are 2 variables that can be changed to adjust the filter’s responses, i.e. Q and R. Best responses will be depend on plant and the capability of the algorithm. Q will be a random value depend on the environment, however R will be found using the basic equation of standard deviation, there is: "̅ = "B + " + " + "E + "F : 5 "̅ = "B + " + " + "E + "F : 5 I = JK L̅ M (4.15) I = JK L̅ M (4.15) (4.15) We take 5 samples to find the standard deviation. 5 samples were the most appropriate among all. If we sample more than 5, a minor change will not affect the system. 4.2 Kalman Filter Kalman filter was designed to estimate the moving object which caught in camera frame. This filter is really beneficial in the system with a small visual angle of camera. Equations below 46 Mechatronics and Applications: An International Journal (MECHATROJ), Vol. 1, No.1, January 2017 explain the Kalman filter process, there are 2 general process in the algorithm, such as prediction which is explained by the equation 4.10 until equation 4.11 and correction. = !"# + $%# (4.10) & = !& '!( + ) (4.11) # = & +(& +( + , (4.12) = + #- −+ (4.13) & = . − #+ & (4.14) = prediction value & = error prediction %# = control input A = transition matrix B = system velocity Kk = Kalman gain H = transformation matrix R = standard deviation of noise measurement Q = proses noise - = input kalman 5. RESULT AND ANALYSIS In this section, we present the simulation and analysed the data. There are 2 types of data which will be analysed, consist of filter data and PID parameters. MATLAB R2013a is used to monitoring the data in graphs. Kalman filter need to analyse before the control system. According to Figure 4, Kalman filter has fast responses, however Kalman filter can search the object whether the object is not detected. Kalman filter will search the object depend on the last velocity of the object. The filter is also used to reduce noises (simulated by shaking the object), nevertheless there are no noises in system, so it will not be used. 47 Mechatronics and Applications: An International Journal (MECHATROJ), Vol. 1, No.1, January 2017 Figure 4. Kalman response (first tuning) Input kalman Output kalman Object is not detected on camera frame Object is shaking Figure 4. Kalman response (first tuning) Input kalman Output kalman Object is not detected on camera frame Object is shaking Figure 4. Kalman response (first tuning) We determine the Q equal to 5.0 and R equal to standard deviation for each 5 sampling data. If we increase Q to 20, we will get a faster response than before as shown inFigure 5.On actual process we implemented the dynamic camera (camera is moving), a slow responses Kalman will only helpful in the static camera. Fast responses are not good enough in dynamic camera, because plant will move the camera outside the moving range harshly if the object is not detected. Input kalman Output kalman Object is not detected on camera frame Object is shaking Figure 4. Kalman response (first tuning) We determine the Q equal to 5.0 and R equal to standard deviation for each 5 sampling data. If we increase Q to 20, we will get a faster response than before as shown inFigure 5.On actual process we implemented the dynamic camera (camera is moving), a slow responses Kalman will only helpful in the static camera. Fast responses are not good enough in dynamic camera, because plant will move the camera outside the moving range harshly if the object is not detected. We determine the Q equal to 5.0 and R equal to standard deviation for each 5 sampling data. 5. RESULT AND ANALYSIS After some experiments, we conclude that PD control law should be used to control the position of motor servos, so Ki is changed to 0. Control system moves 2 motor servo, such as: Table 1. PID (Horizontal Axis) Parameter using Z-N method Table 1. PID (Horizontal Axis) Parameter using Z-N method KP TI TD KI KD 2.188 1.051923 0.075938 2.08 0.318026 Table 2. PID (Vertical Axis) Parameter using Z-N method KP TI TD KI KD 1.902 0.36413 0.100342 5.2234 0.37146 We control motor servo, which has its own controller. After some experiments, we conclude that PD control law should be used to control the position of motor servos, so Ki is changed to 0. Control system moves 2 motor servo, such as: 5. RESULT AND ANALYSIS If we increase Q to 20, we will get a faster response than before as shown inFigure 5.On actual process we implemented the dynamic camera (camera is moving), a slow responses Kalman will only helpful in the static camera. Fast responses are not good enough in dynamic camera, because plant will move the camera outside the moving range harshly if the object is not detected. Figure 5. Kalman response (second tuning) S l d h Q h ld l 5 0 Af h l f Q h b fi d h l Figure 5. Kalman response (second tuning) Figure 5. Kalman response (second tuning) So, we can conclude that Q should equal to 5.0. After the value of Q has been fixed, the control system will be designed. According to the section 3, we will use a digital PID (Proportional, Integral, and Derivative) controller in bilinear transform. First of all, the controller will be designed using Ziegler-Nichols type 2. We need to find the ultimate gain using P controller in this method, we should made the system oscillating constantly, 48 Mechatronics and Applications: An International Journal (MECHATROJ), Vol. 1, No.1, January 2017 and then we got two variables, such as ultimate gain andPeriod of oscillation. After that, we will find the PID parameters using these equations: and then we got two variables, such as ultimate gain andPeriod of oscillation. After that, we will find the PID parameters using these equations: 0 = 0.6 O % (5.1) ;? = P% ∶2 (5.2) ;3 = P% ∶8 (5.3) 0 = 0.6 O % (5.1) ;? = P% ∶2 (5.2) ;3 = P% ∶8 (5.3) (5.3) According to the measurement using the equation 5.1 until equation 5.3, we got the PID parameter shown in Table 1 for horizontal axis and Table 2 for vertical axis. According to the measurement using the equation 5.1 until equation 5.3, we got the PID parameter shown in Table 1 for horizontal axis and Table 2 for vertical axis. Table 1. PID (Horizontal Axis) Parameter using Z-N method KP TI TD KI KD 2.188 1.051923 0.075938 2.08 0.318026 Table 2. PID (Vertical Axis) Parameter using Z-N method KP TI TD KI KD 1.902 0.36413 0.100342 5.2234 0.37146 We control motor servo, which has its own controller. Table 2. PID (Vertical Axis) Parameter using Z-N method PID (Horizontal Axis) Parameter after Manual Tuning without Kalman Filter Kp Ki Kd Settling Time Rise Time Overshoot 1.188 0 0.318 1.38 0.39 5.43% Figure 6 (B) is a PID response without Kalman filter. However, the system without Kalman filter has a faster response which has smaller settling time, rise time, and overshoot. - Motor servo in vertical axis Figure 7(A) is the system’s response of motor servo in vertical axis. According to the Table 5, system has a better result than horizontal axis with a smaller overshoot. There are also noises in the system, which has a same problem with motor servo in horizontal axis. Table 3. PID (Horizontal Axis) Parameter after Manual Tuning Table 4. PID (Horizontal Axis) Parameter after Manual Tuning without Kalman Filter Figure 6 (B) is a PID response without Kalman filter. However, the system without Kalman filter has a faster response which has smaller settling time, rise time, and overshoot. - Motor servo in vertical axis Figure 7(A) is the system’s response of motor servo in vertical axis. According to the Table 5, system has a better result than horizontal axis with a smaller overshoot. There are also noises in the system, which has a same problem with motor servo in horizontal axis. - Motor servo in vertical axis Figure 7(A) is the system’s response of motor servo in vertical axis. According to the Table 5, system has a better result than horizontal axis with a smaller overshoot. There are also noises in the system, which has a same problem with motor servo in horizontal axis axis. (A) (B) Figure 7. (A) PID Response (vertical axis) (B) PID Response without Kalman Filter Table 5. PID (Vertical Axis) Parameter after Manual Tuning Kp Ki Kd Settling Time Rise Time Overshoot 1.402 0 0.274 1.34 0.38 11.9% Table 6. PID (Vertical Axis) Parameter after Manual Tuning Without Kalman Filter Kp Ki Kd Settling Time Rise Time Overshoot 1.402 0 0.274 1.1700 0.47 18.9% Set Point System response Object is moving Noises Set Point System Response Object is Moving Noise (A) Set Point System response Object is moving Noises (A) (B) Figure 7. (A) PID Response (vertical axis) (B) PID Response without Kalman Filter Set Point System response Object is moving Noises Set Point System Response Object is Moving Noise (B) Set Point System Response Object is Moving Noise (B) (A) Figure 7. Table 2. PID (Vertical Axis) Parameter using Z-N method Table 2. PID (Vertical Axis) Parameter using Z-N method Table 2. PID (Vertical Axis) Parameter using Z-N method KP TI TD KI KD 1.902 0.36413 0.100342 5.2234 0.37146 We control motor servo, which has its own controller. After some experiments, we conclude that PD control law should be used to control the position of motor servos so Ki is changed to 0 KP TI TD KI KD 1.902 0.36413 0.100342 5.2234 0.37146 We control motor servo, which has its own controller. After some experiments, we conclude that PD control law should be used to control the position of motor servos, so Ki is changed to 0. Control system moves 2 motor servo, such as: We control motor servo, which has its own controller. After some experiments, we conclude that PD control law should be used to control the position of motor servos, so Ki is changed to 0. Control system moves 2 motor servo, such as: - Motor servo in horizontal axis - Motor servo in horizontal axis Figure 5 (A) shows the system responses using Kalman filter. There are a lot of noises because the environment condition which affect the image processing, furthermore there is a state where the object moving outside the camera frame, then Kalman filter try to find the object. Table 3 is the result of system’s response in horizontal axis using Kalman filter. Figure 5 (A) shows the system responses using Kalman filter. There are a lot of noises because the environment condition which affect the image processing, furthermore there is a state where the object moving outside the camera frame, then Kalman filter try to find the object. Table 3 is the result of system’s response in horizontal axis using Kalman filter. Figure 6. (A) PID Response (horizontal axis) (B) PID Response without Kalman Filter Set Point System response Object is moving Noises Set Point System Response Object is Moving Noise Set Point System Response Object is Moving Noise Figure 6. (A) PID Response (horizontal axis) (B) PID Response without Kalman Filter 49 Mechatronics and Applications: An International Journal (MECHATROJ), Vol. 1, No.1, January 2017 Table 3 PID (Horizontal Axis) Parameter after Manual Tuning Mechatronics and Applications: An International Journal (MECHATROJ), Vol. 1, No.1, January 2017 Table 3. PID (Horizontal Axis) Parameter after Manual Tuning Kp Ki Kd Settling Time Rise Time Overshoot 1.188 0 0.318 1.63 0.402 16.49% Table 4. Table 2. PID (Vertical Axis) Parameter using Z-N method (A) PID Response (vertical axis) (B) PID Response without Kalman Filter Table 5. PID (Vertical Axis) Parameter after Manual Tuning Kp Ki Kd Settling Time Rise Time Overshoot 1.402 0 0.274 1.34 0.38 11.9% Table 6. PID (Vertical Axis) Parameter after Manual Tuning Without Kalman Filter Kp Ki Kd Settling Time Rise Time Overshoot 1.402 0 0.274 1.1700 0.47 18.9% Table 5. PID (Vertical Axis) Parameter after Manual Tuning 50 Mechatronics and Applications: An International Journal (MECHATROJ), Vol. 1, No.1, January 2017 Meanwhile, system response without Kalman filter has been taken and it has a faster response than using Kalman filter. We can see on the Table 6 that a system without Kalman filter has a smaller settling time, but bigger overshoot. Meanwhile, system response without Kalman filter has been taken and it has a faster response than using Kalman filter. We can see on the Table 6 that a system without Kalman filter has a smaller settling time, but bigger overshoot. According to the explanation, the response without Kalman filter is better than the otherwise. The reason is the feedback is not the real-time position, but it is the predictive position. It was predicted based on the object movement, therefore the response will be slower. Figure 8. Error in Kalman Filter Graph -100 -50 0 50 100 150 200 1 20 39 58 77 96 115 134 153 172 191 210 229 248 267 286 305 324 343 362 Pixel Time (s) Error in Kalman FIlter Coordinate Kalman Error Figure 8. Error in Kalman Filter Graph Figure 8 explain about the error between the real coordinateof the object and Kalman’s output in x axis. We can see in some points, there are errors between real coordinate and Kalman’s output. The error happened when the object is moving. Theerror graphs have an overshoot, which is signed by a blue circle,the reason is Kalman filter need to adapt with the environment. It won’t happened if the camera was in static condition. It is the reason why the settling time was slower than without using Kalman filter. Figure 8 explain about the error between the real coordinateof the object and Kalman’s output in x axis. We can see in some points, there are errors between real coordinate and Kalman’s output. The error happened when the object is moving. 6. SUMMARY AND CONCLUSION The paper proposed the designed system which is easier to implement. It was written in a more practical way and less numerical theory. The final response we can get is settling time equal to 1.34s and rise time 0.38 for vertical axis. Beside, the horizontal axis has settling time and rise time in 1.603s and 0.402s. The result shows the difference between a system using Kalman filter and without Kalman filter. We have a faster and better result when it is not using Kalman filter. However, there are some advantages for the system using Kalman filter, such as: - Control system can predict the object movement when the object moves to outside the camera frame. - Kalman filter can estimate the object position when the object moves behind another object. So, the conclusion is the system will be better using kalman filter. In the implementation, the system will be used to aim an object, and the ability to predict the object movement is really needed. It has a slower settling time than without using kalman filter, because the system need to adapt to the environment when the camera is moving. Table 2. PID (Vertical Axis) Parameter using Z-N method Theerror graphs have an overshoot, which is signed by a blue circle,the reason is Kalman filter need to adapt with the environment. It won’t happened if the camera was in static condition. It is the reason why the settling time was slower than without using Kalman filter. (A) (B) (C) Figure 9. Object Movement (A) Initial State (B) Predict the Object (C) Final State (B) (C) (A) (A) (C) (B) Figure 9. Object Movement (A) Initial State (B) Predict the Object (C) Final State The benefit of Kalman filter is able to predict the object movement, which is illustrated in Figure 9. Kalman output is signed by red square, and the object is green square. On the Figure 9(B), the object was blocked by another object and the system can’t detect the object, however the system still predicted the movement. Although it has no better response, kalman filter can be used in this state and the object position can be estimated while the object was blocked. 51 Mechatronics and Applications: An International Journal (MECHATROJ), Vol. 1, No.1, January 201 ACKNOWLEDGEMENTS Thank you for all the parties involved. To our friends in Electronics Engineering of Bandung State of Polytechnic, lecturers, and my beloved parents. This paper won’t finish completely without all of their help and support. I hope, this paper could be a trigger for many students and contribution to the technology development in the international world. [8] Welch, Greg & Bishop, Gary (2001) An Introduction to the Kalman Filter, University of North Carolina, ACM, Inc. REFERENCES [1] Garibotto, Giovanni & Buemi, Francesco (2015) Object Detection and Tracking from fixed and mobile platforms, …, …. [1] Garibotto, Giovanni & Buemi, Francesco (2015) Object Detection and Tracking from fixed and mobile platforms, …, …. p f [2] Parmar, D.N. & Mehta, B. B. (2013) “Face Recognition Methods & Applications”, International Journal Computer Technology and Applications, Vol. 4, No.1, pp84-87. [2] Parmar, D.N. & Mehta, B. B. (2013) “Face Recognition Methods & Applications”, International Journal Computer Technology and Applications, Vol. 4, No.1, pp84-87. [3] Thuilot, Benoit. Martinet, Philippe & Cordesses, Lionel (2002) Position based visual servoing : keeping the object in the field of vision, International Conference on Robotics & Automation, IEEE. [3] Thuilot, Benoit. Martinet, Philippe & Cordesses, Lionel (2002) Position based visual servoing : keeping the object in the field of vision, International Conference on Robotics & Automation, IEEE. [4] Burlacu, Adrian & Lazar, Corneliu (2008) Image Based Controller for Visual Servoing Systems, Buletinul Institutului Politehnic Din Iasi, Universitatea Tehnica ,,Gheorghe Asachi’’ din Iasi. [5] Mariottini, G. Luca. Oriolo, Giuseppe & Prattchizzo, Domenico (2007) “Image-Based Visual Servoing for Nonhonomic Mobile Robots Using Epipolar Geometry”, IEEE Transactions on Robotics, Vol. 23, No.1, pp87-100. pp [6] Funk, Nathan (2003) A Study of the Kalman Filter Applied to Visual Tracking, Project for CMPUT 652, University of Alberta. ] Fadali, M. Sami & Visioli, Antonio (2013) Digital Control Engineering Analysis and Design Second Edition,…, Elesevier Inc. ] Welch, Greg & Bishop, Gary (2001) An Introduction to the Kalman Filter, University of North Carolina, ACM, Inc. 52 Mechatronics and Applications: An International Jour Mechatronics and Applications: An International Journal (MECHATROJ), Vol. 1, No.1, January 201 nal (MECHATROJ), Vol. 1, No.1, January 201 Dr. YB Gunawan Sugiarta Born in 1961 and currently work as a lecture title from Bandung Institute of Technology (ITB) as follow: Electronics Engineering (1999), Field Programmable Gate Array System Design (2003), Image Processing System Design (2009), and currently contributing in rese processing design as a lecture in Bandung State of Polytechnic. Granted title from Bandung Institute of Technology (ITB) as follow: Electronics Engineering (1999), Field Programmable Gate Array System Design (2003), Image Processing System Design (2009), and currently contributing in research program on image Fajar Abdurrahman Born in 1994 and started to study in Bandung State of Polytechnic since 2012. He worked in a business consultant from 2013 quitted from his job and started to focus on his the middle of 2015. He will graduate from his university in 2016 Born in 1994 and started to study in Bandung State of Polytechnic since 2012. He worked in a business consultant from 2013 – 2015 as a part timer job, however he quitted from his job and started to focus on his education and electronic research since the middle of 2015. He will graduate from his university in 2016 of August. Feriyonika ST., M.Sc.Eng Born in 1985 in Sumatra, Indonesia. Studied as Mada University. Then, he granted master degree in control engineering at National Taiwan University of Science and Technology in 2009. Currently worked and contributing in research program on instrument and control system in Bandung State of Polytechnic. Sumatra, Indonesia. Studied as the electrical engineering in Gadjah Then, he granted master degree in control engineering at National Taiwan University of Science and Technology in 2009. Currently worked and contributing in research program on instrument and control system in Bandung State of 53
W2032325380.txt	https://zenodo.org/records/2249626/files/article.pdf	de		Einige mikrochemische Reaktionen desβ-Eucains	Analytical and bioanalytical chemistry/Analytical & bioanalytical chemistry	1,922	public-domain	415	3. Auf Pharmazie beziigliche. 365 wird wiederholt mit Wasser ausgesptilt und das Sptilwasser zu dem Filtrat yon der dem Verbrennungszylinder benachbarten AbsorptionsrShre gegeben. Die Glasperlen werden wiederholt mit Wasser nachgewaschen. Zu den beiden Filtraten mit den Waschwassern werden je 50 ccm ~/5o-Schwefels/i,ure und einige Siedesteinchen gegeben und die mit kleinen Trichtern versehenen Kolben auf einem Asbestdrahtnetz so lunge erhitzt: bis die F1Qssigkeitsmengen etwa 25 ccm betragen. Nach dem Erkalten wird naeh Zugabe'von Phenolphthaleiu mit ~/so-Kalilauge bis zur bleibenden Rotfiirbung titriert. Mit 1 oder 2 Tropfen ~/5~-Schwefels~are wird die Rotfarbung entfernt, hierauf nach ZugalJe yon 5 Tropfen 10% iger KaliumchromatlOsung mit ~/5o-SilbernitratlSsung titriert, tst a die Anzahl der verbrauchten Kubikzentimeter ~/5o-SilbernitratlSsung, s die verbrannte Benzaldehydmenge in Grammen, dann betr~igt der Chlorgehalt des betreffenden Benzaldeh~'ds in P r o z e n t e n - a . 0,071 s S c h i m m e l & Co. batten h~ufig Gelegenheit, nach der mitgeteilten Methode, deren Zuverl~ssigkeit durch Versuche mit nattirlichem blausi~urehaitigem und blausiiurefreiem BittermandelOl, kiinstliChem Benzaldehyd und Benzaldehyd yon bekanntem Chlorgehalt erprobt worden war, Benzaldehyde mit gurantiertem Chlorgehalt, sowie chlorfreie -echte~ BittermandelOle a u f ihren Chlorgehalt nachzupriifen und kounten auf Grund der ermittelten Werte feststellen, dass kaum eines der untersuchten Muster den Angaben der betreffenden Lieferanten eutsprach. Zum Teil waren sogar recht erhebliehe Abweichungen vorhanden, die nach der Meinung der Autoren nut dadureh ihre Erkl~rung finden, dass' nach den fiir die Untersuehung herangezogenen Methoden nur das Chlor der Seitenkette, nieht aber das im Kern enthaltene bestimmt worden war. Mit dem ~Terfahren yon S e h i m m e l & Co. dtirfte wohl einem inzwischen recht ftihlbar gewordenen Mangel abgeholfen sein. Vielleicht wird sich die Methode in der Teehnik ganz allgemein ftir die quantitative ttalogenbestimmung yon halogenhaltigen, organischeu Verbinduagen eignen, w a s einen Vorteil gegeniiber der Methode nach C a r i u s bedeuten wiirde, da d~e Bestimmung bequemer und in kiirzerer Zeit ausftihrbar ist. Bei leieht brennbaren KSrpern dlirfte die u keinerlei Schwierigkeiten bieten, w~thrend schwerbrennbare, bezw. feste KSrper mit Alkohol oder einer ~ihnlich leicht brennbaren Fliissigkeit zu mischen w~iren. F. Stadlmayr. l~,inige mikroehemische Reak~i0nen des ~-Eucains teilt C. H. St e p h e n s o n ~) mit. Nach seiner Angabe ist eine gesitttigte, w~sserige Pikrins~urelSsung ~ das empfindliehste Reagens; L~sungen 1 : 1000 geben wohtausgebildete Rosetten. Eine 5 ~ Natriumsalicylat10sung gibt mit einer Eucainlssuug yon der Konzentration 1 : 1000 keinen 1) Amer, Journ. Pharm. Assoc. 10, 17~: (1921); durch Pharm. Zentralhal]e 62, 531 (1921).
https://openalex.org/W2754927003	https://www.epj-conferences.org/10.1051/epjconf/201714604001/pdf	English	null	General description of fission observables: The GEF code	EPJ web of conferences	2,017	cc-by	4,487	1. Introduction of the different observables, and the stochastic and the self-consistent models are presently restricted to a rather small number of about 4 or 5 degrees of freedom that can explicitly be treated. This contribution is dedicated to the presentation of a general description of ﬁssion observables (the GEF model code) and its application in the domain of nuclear data. The model is characterized by a number of theoretical ideas and hypotheses that form a ﬁrm frame, linking practically all ﬁssion quantities of all ﬁssioning systems among each other. These links include not only the different ﬁssion quantities of one ﬁssioning system, but also their variation with the mass, nuclear charge, excitation energy, and angular momentum of the ﬁssioning system. This theoretical frame allows to trace back the values of practically all ﬁssion quantities of hundreds of ﬁssioning systems over the large energy range from spontaneous ﬁssion up to excitation energies of 100 MeV to a consistent description with only about 100 parameters. These parameters have physical meaning, like energies, temperatures and oscillator frequencies. The values of these parameters have been determined by adjustment to the large body of relevant empirical data. The present article focuses on the dependences of the different ﬁssion quantities and the method of parameter determination in order to understand the ability of the GEF model to provide reliable predictions for ﬁssioning nuclei for which no experimental data exist. Moreover, the ability to establish covariances and its suitability for validation and evaluation are illustrated. A comprehensive documentation of the GEF model code can be found elsewhere [6]. Karl-Heinz Schmidt1,a, and Beatriz Jurado1,b, and Christelle Schmitt2,c 1 CENBG, CNRS/IN2 P3, Chemin du Solarium, BP. 120, 33175 Gradignan, France 2 GANIL, Bd. Henri Becquerel, BP. 55027, 14076 Caen Cedex 05, France Abstract. The GEF model code is described. It has been developed during the last years with the aim to cover practically all ﬁssion quantities of a large number of ﬁssioning systems over a wide range of excitation energy with a semi-empirical description without the need for further adjustment to experimental data of speciﬁc systems. The basic theoretical ideas and the method of the parameter determination are explained, a short overview on selected results is given, and the application for evaluation and validation of nuclear data is demonstrated with a few examples. 2. Theoretical ideas and hypotheses In this section, some of the main theoretical ideas and hypotheses of the GEF model are listed. 2.1. Fission barriers The GEF model combines to some extent the good reproduction of experimental data obtained by purely empirical models (e.g. Ref. [1]) with the predictive power of stochastic models (based on the numerical solution of the Langevin equations or the random-walk approach) (e.g. Refs. [2,3]), or fully microscopic self-consistent models (e.g. Ref. [4]). Of course, the GEF model misses the microscopic basis, but, due to its theoretical frame with adjusted parameter values, it allows a generalization of the empirical information in an extended region not too far from well investigated systems. This is comparable to the liquid-drop model, which is still the basis for the most accurate atomic-mass values provided by the macroscopic-microscopic approach [5]. Additional assets of the GEF model are the consistent description of nearly all ﬁssion quantities with their correlations, while the empirical systematics only provide separate descriptions Fission barriers are calculated by use of the topographic theorem [7] as the sum of the macroscopic barrier and the additional binding energy by the empirical ground-state shell correction. This approach avoids the uncertainties of the theoretical shell-correction energies and allows to discriminate between different macroscopic models [8]. c⃝The 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/). a e-mail: schmidt-erzhausen@t-online.de b e-mail: jurado@cenbg.in2p3.fr c e-mail: schmitt@ganil.fr y y the outer saddle [9]. These shells are assumed to be the same for all ﬁssioning systems. It is the superposition of different shells and the interaction with the macroscopic c⃝The 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/). a e-mail: schmidt-erzhausen@t-online.de b e-mail: jurado@cenbg.in2p3.fr c e-mail: schmitt@ganil.fr EPJ Web of Conferences 146, 04001 (2017) ND2016 EPJ Web of Conferences 146, 04001 (2017) ND2016 EPJ Web of Conferences 146, 04001 (2017) ND2016 DOI: 10.1051/epjconf/201714604001 3. Method of parameter determination In this section, the main aspects of the parameter determination are described. They illustrate the far- reaching inﬂuence of a speciﬁc model parameter on different observables in nearly all ﬁssioning systems. The parameters were determined by minimizing the deviations of the model from measured ﬁssion-fragment A distributions, independent yields, isomeric ratios, total kinetic energies (TKEs), and the multiplicities of prompt and delayed neutrons, and the dependencies between these quantities. We restrict ourselves in the following to the properties of the three most intense ﬁssion channels. More details and the speciﬁc numerical values of the parameters are found in Ref. [6]. Figure 2. Deviation of the mean nuclear charge of isobaric chains from the UCD value for different cases for the system 235U(nth,f). Dashed line: UCD value. Full line: Macroscopic value at scission. Open symbols: Values from GEF before prompt-neutron emission as a function of pre-neutron mass. Full symbols: Values from GEF after prompt-neutron emission as a function of post-neutron mass. The ﬁgure is taken from Ref. [6]. Figure 2. Deviation of the mean nuclear charge of isobaric chains from the UCD value for different cases for the system 235U(nth,f). Dashed line: UCD value. Full line: Macroscopic value at scission. Open symbols: Values from GEF before prompt-neutron emission as a function of pre-neutron mass. Full symbols: Values from GEF after prompt-neutron emission as a function of post-neutron mass. The ﬁgure is taken from Ref. [6]. 2.2. Fission channels The ﬁssion channels are related to the statistical population of quantum oscillators in the mass-asymmetry degree of freedom that form the ﬁssion valleys. The quantum oscillator of each channel is characterized by three parameters (position, depth, and curvature) that are traced back to the macroscopic potential (symmetric ﬁssion channel SL) and to shells in the proton and neutron subsystems of both fragments (ﬁssion channels S1 and S2), which are assumed to be effective already little beyond the outer saddle [9]. These shells are assumed to be the same for all ﬁssioning systems. It is the superposition of different shells and the interaction with the macroscopic EPJ Web of Conferences 146, 04001 (2017) DOI: 10.1051/epjconf/201714604001 EPJ Web of Conferences 146, 04001 (2017) ND2016 Figure 1. Mean positions of the standard S1 and S2 ﬁssion channels in atomic number (upper part) and neutron number (lower part) deduced from measured ﬁssion-fragment A and Z distributions. The shape of the symbol denotes the element as given in the legend of the ﬁgure. Data from Ref. [18] are marked by solid symbols. The values of S1 (S2) for the isotopes of a given element are connected by dashed (full) lines and marked by red (blue) symbols. The ﬁgure is taken from Ref. [18]. potential that create the mass distributions which differ for different systems [10]. These shells also determine the shapes (mainly the quadrupole deformation) of the nascent fragments at scission. According to Strutinsky- type calculations, the fragment shapes are found to be a linearly increasing function of the number of protons, respectively neutrons, in regions between closed spherical shells [11]. Also the charge-polarization (deviation of the N/Z degree of freedom at scission – mean value and ﬂuctuations – from the ‘UCD’ value of the ﬁssioning nucleus) is treated by the corresponding quantum oscillator [12]. 2.3. Energy sorting By the inﬂuence of pairing correlations, the nuclear temperature below the critical pairing energy is assumed to be constant [13]. Therefore, the di-nuclear system between saddle and scission consists of two coupled microscopic thermostates [14]. This leads to a sorting process of the available intrinsic energy and of unpaired nucleons before scission [15–17]. Figure 1. Mean positions of the standard S1 and S2 ﬁssion channels in atomic number (upper part) and neutron number (lower part) deduced from measured ﬁssion-fragment A and Z distributions. The shape of the symbol denotes the element as given in the legend of the ﬁgure. Data from Ref. [18] are marked by solid symbols. The values of S1 (S2) for the isotopes of a given element are connected by dashed (full) lines and marked by red (blue) symbols. The ﬁgure is taken from Ref. [18]. Figure 1. Mean positions of the standard S1 and S2 ﬁssion channels in atomic number (upper part) and neutron number (lower part) deduced from measured ﬁssion-fragment A and Z distributions. The shape of the symbol denotes the element as given in the legend of the ﬁgure. Data from Ref. [18] are marked by solid symbols. The values of S1 (S2) for the isotopes of a given element are connected by dashed (full) lines and marked by red (blue) symbols. The ﬁgure is taken from Ref. [18]. 3.1. Location of ﬁssion channels According to Strutinsky-type calculations [11], the asymmetric ﬁssion channel S1 is caused by the spherical shell closures in Z = 50 and N = 82, and the S2 channel, which has the largest yield in the actinides, is related to a shell near N = 88 at large deformation (β ≈0.5). Therefore, one expects that the S1 ﬁssion channel is located near A = 132, while the S2 channel appears close to N = 88. A detailed analysis of available A and Z distributions, however, revealed that the S1 and the S2 channels appear close to Z = 52 and Z = 55, respectively, and that A and N vary accordingly with the size of the ﬁssioning system, see Fig. 1. This unexpected result is taken as an empirical information without a deeper understanding at this time. 132Sn, while the depth of the S2 ﬁssion valley with respect to the macroscopic potential is the same for all systems. The shapes of the mass distributions are well described by assuming a Gaussian for the S1 channel and a rectangle convoluted with a Gaussian for the S2 channel. This implies that the width of the rectangle appears as an additional parameter. 3.4. Charge polarization There is no direct experimental information available on the charge polarization at scission, but it can be determined indirectly. Figure 2 demonstrates, how the experimentally accessible Zmean −ZUC D values as a function of post-neutron mass are linked by the mass-dependent prompt-neutron multiplicity to the Zmean −ZUC D values (the charge polarization at scission) as a function of 3.2. Yields and shapes of ﬁssion channels The fragment-mass dependence of the prompt-neutron multiplicities is well described by assuming the same linear dependence of the quadrupole deformation with the number of protons in the nascent fragments for all ﬁssioning systems. This is consistent with the constant position of the ﬁssion channels in Z, as mentioned above. It explains also the major part of the increasing prompt-neutron multiplicity, for example from 235U(nth,f) The yield of a speciﬁc ﬁssion channel is expected to be proportional to the exponential of the binding energy at the bottom of the respective ﬁssion channel. Indeed, a good description of the relative yields of the ﬁssion channels is obtained, when the depth of the S1 ﬁssion valley decreases approximately as a linear function with increasing distance of the N/Z of the ﬁssioning system from the N/Z value of 2 EPJ Web of Conferences 146, 04001 (2017) ND2016 DOI: 10.1051/epjconf/201714604001 ND2016 Figure 3. Evaluated [19] and measured [20] mass distributions (black symbols) of ﬁssion fragments in comparison with the result of the GEF model (blue symbols). The green lines show the calculated contributions from the different ﬁssion channels (SL, S1, S2, and the super-asymmetric ﬁssion channel SA). The ﬁgure is taken from Ref. [21] with kind permission of The European Physical Journal (EPJ). For details see Ref. [21]. Figure 3. Evaluated [19] and measured [20] mass distributions (black symbols) of ﬁssion fragments in comparison with the result of the GEF model (blue symbols). The green lines show the calculated contributions from the different ﬁssion channels (SL, S1, S2, and the super-asymmetric ﬁssion channel SA). The ﬁgure is taken from Ref. [21] with kind permission of The European Physical Journal (EPJ). For details see Ref. [21]. to 252Cf(sf), due to the enhanced production of fragments on the right wing of the light fragment component. pre-neutron mass. The trend of the latter quantity follows the macroscopic values in the light and the heavy fragment group, but an additional roughly constant polarization that shifts the light fragments to more neutron-deﬁcient and the heavy fragments to more neutron-rich isotopes is necessary to reproduce the experimental post-neutron values. 4.1. Fission-fragment yields Table 1. Mean properties of prompt and delayed neutrons. (En is the incoming-neutron energy. Mean energy E prompt and multiplicity νprompt refer to prompt neutrons emitted from the fragments. The decay data from JEFF 3.1.1 were used to calculate νdelayed.) In Fig. 3, calculated mass distributions are compared with empirical data for a few selected systems. The Chi-squared deviations between GEF results and the evaluation of Ref. [19] for all mass distributions are shown in Fig. 5. The majority of the Chi-squared values are close to unity, demonstrating the good reproduction of the data by the GEF model. Most of the large Chi-squared values are caused by issues in the evaluation (see Ref. [6]). Some of those will be considered more closely in Sect. 5. System En E prompt νprompt νdelayed [MeV] [MeV] 233U(n,f) thermal 2.02(1) 2.36(1) 0.77(9) 233U(n,f) 5 2.06(1) 3.10(2) 0.79(16) 235U(n,f) thermal 2.00(1) 2.42(2) 1.60(10) 235U(n,f) 5 2.06(1) 3.18(2) 1.48(12) 238U(n,f) 5 2.01(1) 3.05(2) 3.51(14) 237Np(n,f) thermal 2.02(1) 2.38(6) 1.47(7) 237Np(n,f) 5 2.08(1) 3.12(2) 1.05(5) 238Np(n,f) thermal 2.02(1) 2.57(6) 1.82(15) 238Np(n,f) 5 2.09(1) 3.36(3) 1.40(7) 239Pu(n,f) thermal 2.08(1) 2.80(4) 0.68(4) 239Pu(n,f) 5 2.13(1) 3.57(5) 0.61(3) 241Pu(n,f) thermal 2.06(1) 2.88(5) 1.42(5) 241Pu(n,f) 5 2.12(2) 3.70(4) 1.16(5) 252Cf(s,f) —— 2.16(2) 3.76(2) 0.76(12) 4. Selected results In this section, some typical results of the GEF code are presented and, if available, compared with measured or evaluated data. 3 3 EPJ Web of Conferences 146, 04001 (2017) DOI: 10.1051/epjconf/201714604001 Figure 4. Upper panels: experimental prompt-ﬁssion-neutron energy spectra (black lines and error bars) for 235U(nth,f) [22] (left part) and 252Cf(sf) [23] (right part) in comparison with the result of the GEF model (red histograms) in logarithmic scale. In the lower panels, the spectra have been normalized to a Maxwellian with T = 1.32 MeV and T = 1.42 MeV, respectively. The ﬁgure is modifed from Ref. [6]. Figure 4. Upper panels: experimental prompt-ﬁssion-neutron energy spectra (black lines and error bars) for 235U(nth,f) [22] (left part) and 252Cf(sf) [23] (right part) in comparison with the result of the GEF model (red histograms) in logarithmic scale. In the lower panels, the spectra have been normalized to a Maxwellian with T = 1.32 MeV and T = 1.42 MeV, respectively. The ﬁgure is modifed from Ref. [6]. 4.2. Prompt-neutron emission The energy spectra of the prompt neutrons are well reproduced by the GEF model without further adjustments. Figure 4 shows a comparison for the two systems with the best experimental information. Critical ingredients like level densities and transmission coefﬁcients are directly taken from literature (see Ref. [6]). Also the gamma competition, which has an additional inﬂuence on the 4 DOI: 10.1051/epjconf/201714604001 EPJ Web of Conferences 146, 04001 (2017) Figure 5. Chi-squared deviations of 57 mass distributions calculated with GEF from evaluated data [19] in a logarithmic binning. The height of the histogram represents the number of cases per bin. The ﬁgure is taken from Ref. [6]. Table 2. Mean properties of prompt gamma emission. (En is the incoming-neutron energy, Eγ and Nγ are the average gamma energy and multiplicity, and Etot is the total gamma energy in one ﬁssion event.) energy and multiplicity, and Etot is the total gamma energy in one ﬁssion event.) System En Eγ Nγ Etot [MeV] [MeV] 233U(n,f) thermal 1.00(2) 6.8(5) 6.75(40) 233U(n,f) 5 1.00(1) 7.4(4) 7.38(33) 235U(n,f) thermal 0.94(1) 6.9(3) 6.44(20) 235U(n,f) 5 0.94(1) 7.5(4) 7.03(27) 238U(n,f) 5 0.87(2) 7.1(4) 6.21(27) 237Np(n,f) thermal 0.94(2) 6.8(5) 6.42(33) 237Np(n,f) 5 0.94(2) 7.3(6) 6.89(38) 238Np(n,f) thermal 0.92(3) 6.8(6) 6.27(35) 238Np(n,f) 5 0.92(2) 7.4(5) 6.78(31) 239Pu(n,f) thermal 0.94(1) 6.9(3) 6.54(18) 239Pu(n,f) 5 0.94(1) 7.5(4) 7.09(26) 241Pu(n,f) thermal 0.90(2) 7.0(4) 6.23(27) 241Pu(n,f) 5 0.90(2) 7.6(6) 6.81(38) 252Cf(s,f) —— 0.85(2) 7.2(3) 6.14(14) Figure 6. Distribution of the ratio between the experimental and calculated IR for several tens of fragment isomers and ﬁssioning nuclei. System En Eγ Nγ Etot [MeV] [MeV] 233U(n,f) thermal 1.00(2) 6.8(5) 6.75(40) 233U(n,f) 5 1.00(1) 7.4(4) 7.38(33) 235U(n,f) thermal 0.94(1) 6.9(3) 6.44(20) 235U(n,f) 5 0.94(1) 7.5(4) 7.03(27) 238U(n,f) 5 0.87(2) 7.1(4) 6.21(27) 237Np(n,f) thermal 0.94(2) 6.8(5) 6.42(33) 237Np(n,f) 5 0.94(2) 7.3(6) 6.89(38) 238Np(n,f) thermal 0.92(3) 6.8(6) 6.27(35) 238Np(n,f) 5 0.92(2) 7.4(5) 6.78(31) 239Pu(n,f) thermal 0.94(1) 6.9(3) 6.54(18) 239Pu(n,f) 5 0.94(1) 7.5(4) 7.09(26) 241Pu(n,f) thermal 0.90(2) 7.0(4) 6.23(27) 241Pu(n,f) 5 0.90(2) 7.6(6) 6.81(38) 252Cf(s,f) —— 0.85(2) 7.2(3) 6.14(14) Figure 5. Chi-squared deviations of 57 mass distributions calculated with GEF from evaluated data [19] in a logarithmic binning. The height of the histogram represents the number of cases per bin. The ﬁgure is taken from Ref. [6]. shape of the prompt-neutron spectrum, is treated without speciﬁc adjustments [6]. Table 1 lists calculated average quantities of the prompt and the delayed neutrons for a few selected systems. 1 The values of the total gamma energy given in Ref. [6] are not correct and should be replaced by the values given in Table 2. 4.2. Prompt-neutron emission The uncertainties refer only to the uncertainties of the parameters of the GEF model, which are speciﬁc to the ﬁssion process. Additional uncertainties, for example by the nuclear level density or the decay data, are not included. As expected from the good reproduction of the prompt- neutron energy spectra in Fig. 4, the mean prompt-neutron energies for 233,235U(nth,f) and 239Pu(nth,f) agree with the recent evaluation of Ref. [24] within the estimated error bars. The prompt-neutron multiplicities for 235U(nth,f), 239,241Pu(nth,f), 252Cf(sf), 238U(n,f) and 239Pu(n,f) with En = 5 MeV agree with the evaluated data (ENDF/B- VII.1, [25,26]) within the estimated error bars or slightly beyond. The values for 233U(nth,f) and 237,238Np(nth,f) deviate by up to 0.2 units. These discrepancies should be considered in a more comprehensive analysis, may be including a re-examination of the data underlying the evaluations. Figure 6. Distribution of the ratio between the experimental and calculated IR for several tens of fragment isomers and ﬁssioning nuclei. 4.4. Isomeric ratios Many isomers exist among the ﬁssion products and play an important role for the calculation of the decay heat after reactor shutdown. Furthermore, the beta-delayed neutron- emission probability from the isomeric state can be an order of magnitude different from that of the ground state. Thus, proper simulation of the effect of delayed neutrons in reactors requires accurate knowledge of the population of isomeric states in ﬁssion. Measurements of isomeric yield ratios are also important for simulations of the astrophysical r-process. The isomeric ratio (IR) predicted by the GEF model depends on the properties of the ﬁssioning nucleus, namely its excitation energy and spin, as well as on the properties of the ﬁssion fragment, that are its mass, Z, deformation, and difference between its isomeric-state and ground-state spins and binding energies. In the GEF code, it is essentially assumed that the angular momentum of the fragments is created by the statistical population of single-particle and collective states according to the fragment temperature at scission. The energies and spins of the isomeric states as possibly populated in the calculations are taken from empirical data. References Figure 8. Fission-fragment mass distribution of 255Fm(nth,f) from Ref. [19] and ENDF/B-VII (black open symbols) compared with the GEF result (full blue symbols). [1] A.C. Wahl, Report LA-13928 (Los Alamos National Laboratory, 2002) [2] M.D. Usang et al., Phys. Rev. C 94, 044602 (2016) [3] J. Randrup and P. M¨oller, Phys. Rev. C 88, 064606 (2013) [4] D. Regnier, N. Dubray, N. Schunck, and M. Verriere, Phys. Rev. C 93, 054611 (2016) [5] A. Sobiczewski, Y.A. Litvinov, Phys. Rev. C 89, 024311 (2014) [6] K.-H. Schmidt, B. Jurado, Ch. Amouroux, and Ch. Schmitt, Nucl. Data Sheets 131, 107 (2016) Figure 8. Fission-fragment mass distribution of 255Fm(nth,f) from Ref. [19] and ENDF/B-VII (black open symbols) compared with the GEF result (full blue symbols). [7] W.D. Myers and W.J. Swiatecki, Nucl. Phys. A 601, 14 (1996) [8] A. Keli´c and K.-H. Schmidt, Phys. Lett. B 634 (2006) 362 [9] U. Mosel and H.W. Schmitt, Nucl. Phys. A 165, 73 (1971) this observable by the code. A further critical discussion can be found in [6]. [10] K.-H. Schmidt, A. Keli´c, and M.V. Ricciardi, Europh. Lett. 83, 32001 (2008) [11] B.D. Wilkins, E.P. Steinberg, and R.R. Chasman, Phys. Rev. C 14, 1832 (1976) 4.3. Prompt-gamma emission The most important quantity related to prompt-gamma emission in nuclear-reactor technology is the total gamma energy per ﬁssion event. Table 2 lists this quantity together with the mean gamma energy and the gamma multiplicity per ﬁssion as calculated with the GEF code. The uncertainties of the multiplicities and total energies are mostly caused by an assumed uncertainty of 10% (standard deviation) in the fragment angular momenta. The values that can be compared with experimental data agree within the given uncertainties and the scattering of the experimental values (see e.g. tables XIV, XV and XVI of Ref. [6])1. Only the gamma multiplicity (and in consequence the total gamma energy) for 252Cf(sf) is systematically too low by about 10%. This problem that is probably caused by underestimated fragment angular momenta for this system requires further investigation. The achievement of GEF for IRs is summarized in Fig. 6, where the distribution of the ratio between the experimental and calculated IR is shown, including a large sample of representative fragment isomeric states and ﬁssioning systems. It demonstrates the good description of 5 DOI: 10.1051/epjconf/201714604001 EPJ Web of Conferences 146, 04001 (2017) Figure 7. Evidence for a Pu contaminant in a 237Np target. The ﬁssion-fragment mass distribution of the system 237Np(nth,f) from Ref. [19] and ENDF/B-VII (black crosses with error bars) in comparison with the result of the GEF code for a pure 237Np target (upper ﬁgure, blue full points) and for a composite target (40% ﬁssion from 237Np and 60% ﬁssion from 239Pu) (lower ﬁgure, blue full points). In addition, the contribution from the assumed 239Pu contaminant is shown separately in the lower ﬁgure (open red symbols). The ﬁgure is taken from Ref. [6]. 6. Conclusion The GEF code reproduces a large variety of ﬁssion observables with a good precision in a consistent way without further adjustment to speciﬁc ﬁssioning systems. With this global approach, the model is able to predict essentially all the observables associated to the ﬁssion process. In contrast to most existing models, GEF is able to provide accurate predictions for ﬁssioning nuclei for which no experimental data are available. The consistent description of all ﬁssion quantities permits establishing correlations between all of them and makes the model a valuable tool for application in validation and evaluation of nuclear data. Figure 7. Evidence for a Pu contaminant in a 237Np target. The ﬁssion-fragment mass distribution of the system 237Np(nth,f) from Ref. [19] and ENDF/B-VII (black crosses with error bars) in comparison with the result of the GEF code for a pure 237Np target (upper ﬁgure, blue full points) and for a composite target (40% ﬁssion from 237Np and 60% ﬁssion from 239Pu) (lower ﬁgure, blue full points). In addition, the contribution from the assumed 239Pu contaminant is shown separately in the lower ﬁgure (open red symbols). The ﬁgure is taken from Ref. [6]. Figure 7. Evidence for a Pu contaminant in a 237Np target. The ﬁssion-fragment mass distribution of the system 237Np(nth,f) from Ref. [19] and ENDF/B-VII (black crosses with error bars) in comparison with the result of the GEF code for a pure 237Np target (upper ﬁgure, blue full points) and for a composite target (40% ﬁssion from 237Np and 60% ﬁssion from 239Pu) (lower ﬁgure, blue full points). In addition, the contribution from the assumed 239Pu contaminant is shown separately in the lower ﬁgure (open red symbols). The ﬁgure is taken from Ref. [6]. The development of the GEF model code has been supported by the Nuclear-Energy Agency of the OECD as well as by the European Commission within the Sixth Framework Programme through EFNUDAT (project No. 036434), within the Seventh Framework Programme through Fission-2010- ERINDA (project No. 269499), and CHANDA (project No. 605203), and by the GSI/IN2P3-CNRS collaboration agreement 04-48. [22] N.V. Kornilov et al., Nucl. Sci. Eng. 165, 117 (2010) [23] W. Mannhart, INDC(NDS)-220 (1989) 305, IAEA, Vienna [24] R. Capote et al., Nucl. Data Sheets 131, 1 (2016) [25] R.W. Mills, “Fission product yield evaluation”, PhD thesis, University of Birmingham, 1995 [26] V.V. Malinovskij, V.G. Vorob’eva, and B.D. Kuz’minov, Report INDC(CCP)-239, IAEA, Vienna, Austria, 1985 [25] R.W. Mills, “Fission product yield evaluation”, PhD thesis, University of Birmingham, 1995 5. Application for validation and evaluation [12] H. Nifenecker, J. Physique Lett. 41, 47 (1980) [13] K.-H. Schmidt and B. Jurado, Phys. Rev. C 86, 044322 (2012) In this section, the use of GEF for validation and evaluation of ﬁssion data is demonstrated with a few examples. More examples can be found in Ref. [6]. [14] K.-H. Schmidt and B. Jurado, Phys. Rev. Lett. 104, 212501 (2010) [15] K.-H. Schmidt and B. Jurado, Phys. Rev. C 83, 014607 (2011) Figure 7 shows a comparison of the ﬁssion-fragment mass distribution of 237Np(nth,f) from ENDF/B-VII in comparison with the GEF result. The discrepancies in the light-fragment group can be explained by a contamination of the 237Np target with a contribution of 15 ppm of 239Pu, as demonstrated in the lower part. [16] K.-H. Schmidt and B. Jurado, Phys. Rev. C 83, 061601 (2011) [17] B. Jurado and K.-H. Schmidt, J. Phys. G: Nucl. Part. Phys. 42, 055101 (2015) 18] C. B¨ockstiegel et al., Nucl. Phys. A 802, 12 (200 [19] T.R. England and B.F. Rider, Report ENDF-349, LA-UR-94-3106 (Los Alamos National Laboratory, 1994) Another indication for an erroneous evaluation result, probably due to scarce data, is shown in Fig. 8. There is an appreciable shift in the position of the light fragment peak, and the mean value of the mass spectrum of 126.5 lets only room for the emission of 2 prompt neutrons, while a value of 4.91 is expected from GEF. [20] E.K. Hulet et al., Phys. Rev. C 40, 770 (1989) [21] K.-H. Schmidt and B. Jurado, Eur. Phys. J. A 51, 176 (2015) 6 EPJ Web of Conferences 146, 04001 (2017) EPJ Web of Conferences 146, 04001 (2017) ND2016 DOI: 10.1051/epjconf/201714604001 [22] N.V. Kornilov et al., Nucl. Sci. Eng. 165, 117 (2010) [25] R.W. Mills, “Fission product yield evaluation”, PhD thesis, University of Birmingham, 1995 [25] R.W. Mills, “Fission product yield evaluation”, PhD thesis, University of Birmingham, 1995 [26] V.V. Malinovskij, V.G. Vorob’eva, and B.D. Kuz’minov, Report INDC(CCP)-239, IAEA, Vienna, Austria, 1985 7
https://openalex.org/W4362627826	https://figshare.com/articles/journal_contribution/Supplementary_Data_Figure_S1_from_Somatic_HLA_Class_I_Loss_Is_a_Widespread_Mechanism_of_Immune_Evasion_Which_Refines_the_Use_of_Tumor_Mutational_Burden_as_a_Biomarker_of_Checkpoint_Inhibitor_Response/22535728/1/files/39999004.pdf	English	null	Supplementary Data Figure S1 from Somatic HLA Class I Loss Is a Widespread Mechanism of Immune Evasion Which Refines the Use of Tumor Mutational Burden as a Biomarker of Checkpoint Inhibitor Response	null	2,023	cc-by	1,099	Germline HLA-I allele count = 6 (n = 182, mOS = 10.8) Germline HLA-I allele count < 6 (n = 58, mOS = 10.8) Data Figure S1. HLA-I germline zygosity has no impact on patient survival in ICI-treated NSCLC. a) Overall survival of all non-squamous NSCLC patients in the real-world clinico-genomic cohort from start of second-line ICI monotherapy, stratified by number of germline unique HLA-I alleles. The mOS was the same for both cohorts, regardless of germline HLA-I allele count (germline HLA-I allele count = 6 (n=182): mOS 10.8 [7.5-14.0]; germline HLA- I allele count < 6: mOS 10.8 [4.8-18.3]). HR for germline HLA-I allele count = 6: 1.00 [0.73-1.50], P = 0.8. b) Overall survival of non-squamous NSCLC patients in the real-world clinico-genomic cohort with no evidence of somatic HLA-I LOH from start of second-line ICI monotherapy, stratified by number of germline unique HLA-I alleles. The mOS for patients with a germline HLA-I allele count = 6 (n=141) was 11.9 months [8.8-15.9] and the mOS for patients with a germline allele count < 6 (n=39) was 7.1 months [3.7-19.2]. HR for germline HLA-I allele count = 6: 0.91 [0.60-1.40], P = 0.7. For panels a and b, significance is determined by log-rank test and significant (P < 0.05) associations are labeled with an asterisk. b) ( ) b) P = 0.7 Germline HLA-I allele count = 6 (n = 141, mOS = 11.9) Germline HLA-I allele count < 6 (n = 39, mOS = 7.1) Overall Survival (OS) Probability Time (Months) Supplementary Data Figure S1. HLA-I germline zygosity has no impact on patient survival in ICI-treated NSCLC. a) Overall survival of all non-squamous NSCLC patients in the real-world clinico-genomic cohort from start of second-line ICI monotherapy, stratified by number of germline unique HLA-I alleles. The mOS was the same for both cohorts, regardless of germline HLA-I allele count (germline HLA-I allele count = 6 (n=182): mOS 10.8 [7.5-14.0]; germline HLA- I allele count < 6: mOS 10.8 [4.8-18.3]). HR for germline HLA-I allele count = 6: 1.00 [0.73-1.50], P = 0.8. b) Overall survival of non-squamous NSCLC patients in the real-world clinico-genomic cohort with no evidence of somatic HLA-I LOH from start of second-line ICI monotherapy, stratified by number of germline unique HLA-I alleles. The mOS for patients with a germline HLA-I allele count = 6 (n=141) was 11.9 months [8.8-15.9] and the mOS for patients with a germline allele count < 6 (n=39) was 7.1 months [3.7-19.2]. HR for germline HLA-I allele count = 6: 0.91 [0.60-1.40], P = 0.7. Germline HLA-I allele count = 6 (n = 182, mOS = 10.8) Germline HLA-I allele count < 6 (n = 58, mOS = 10.8) Data Figure S1. HLA-I germline zygosity has no impact on patient survival in ICI-treated NSCLC. Germline HLA-I allele count = 6 (n = 182, mOS = 10.8) Germline HLA-I allele count < 6 (n = 58, mOS = 10.8) Data Figure S1. HLA-I germline zygosity has no impact on patient survival in ICI-treated NSCLC. Supplementary Data Figure S1. HLA-I germline zygosity has no impact on patient survival in ICI-treated NSCLC. Supplementary Data Figure S1. HLA-I germline zygosity has no impact on patient survival in ICI-treated NSCLC. a) Germline HLA-I allele count = 6 (n = 182, mOS = 10.8) Germline HLA-I allele count < 6 (n = 58, mOS = 10.8) P = 0.8 Overall Survival (OS) Probability Time (Months) b) P = 0.7 Germline HLA-I allele count = 6 (n = 141, mOS = 11.9) Germline HLA-I allele count < 6 (n = 39, mOS = 7.1) Overall Survival (OS) Probability Time (Months) Supplementary Data Figure S1. HLA-I germline zygosity has no impact on patient su Supplementary Data Figure S1. HLA-I germline zygosity has no impact on patient su a) Overall survival of all non-squamous NSCLC patients in the real-world clinico-genomic ICI monotherapy, stratified by number of germline unique HLA-I alleles. The mOS was th regardless of germline HLA-I allele count (germline HLA-I allele count = 6 (n=182): mOS I allele count < 6: mOS 10.8 [4.8-18.3]). HR for germline HLA-I allele count = 6: 1.00 [0.7 survival of non squamous NSCLC patients in the real world clinico genomic cohort with n a) Germline HLA-I allele count = 6 (n = 182, mOS = 10.8) Germline HLA-I allele count < 6 (n = 58, mOS = 10.8) P = 0.8 Overall Survival (OS) Probability Time (Months) a) a) Germline HLA-I allele count = 6 (n = 182, mOS = 10.8) Germline HLA-I allele count < 6 (n = 58, mOS = 10.8) P = 0.8 Overall Survival (OS) Probability Time (Months) b) Germline HLA-I allele count = 6 (n = 141, mOS = 11.9) Germline HLA-I allele count < 6 (n = 39, mOS = 7.1) ility Time (Months) b) P = 0.7 Germline HLA-I allele count = 6 (n = 141, mOS = 11.9) Germline HLA-I allele count < 6 (n = 39, mOS = 7.1) Overall Survival (OS) Probability Time (Months) Supplementary Data Figure S1. HLA-I germline zygosity has no impact on patient survival in ICI-treated NSCLC. Germline HLA-I allele count = 6 (n = 182, mOS = 10.8) Germline HLA-I allele count < 6 (n = 58, mOS = 10.8) Data Figure S1. HLA-I germline zygosity has no impact on patient survival in ICI-treated NSCLC. For panels a and b, significance is determined by log-rank test and significant (P < 0.05) associations are labeled with an asterisk. Supplementary Data Figure S1. HLA-I germline zygosity has no impact on patient survival in ICI-treated NSCLC. a) Overall survival of all non-squamous NSCLC patients in the real-world clinico-genomic cohort from start of second-line ICI monotherapy, stratified by number of germline unique HLA-I alleles. The mOS was the same for both cohorts, regardless of germline HLA-I allele count (germline HLA-I allele count = 6 (n=182): mOS 10.8 [7.5-14.0]; germline HLA- I allele count < 6: mOS 10.8 [4.8-18.3]). HR for germline HLA-I allele count = 6: 1.00 [0.73-1.50], P = 0.8. b) Overall survival of non-squamous NSCLC patients in the real-world clinico-genomic cohort with no evidence of somatic HLA-I LOH from start of second-line ICI monotherapy, stratified by number of germline unique HLA-I alleles. The mOS for patients with a germline HLA-I allele count = 6 (n=141) was 11.9 months [8.8-15.9] and the mOS for patients with a germline allele count < 6 (n=39) was 7.1 months [3.7-19.2]. HR for germline HLA-I allele count = 6: 0.91 [0.60-1.40], P = 0.7. For panels a and b, significance is determined by log-rank test and significant (P < 0.05) associations are labeled with an asterisk.
https://openalex.org/W4392918722	https://andjournal.sgu.ru/sites/andjournal.sgu.ru/files/text-pdf/2024/02/2004no5p003.pdf	Russian	null	Quantum Arnold diffusion in rippled channel at the presence of alternating electric field	Izvestiâ vysših učebnyh zavedenij. Prikladnaâ nelinejnaâ dinamika	2,005	cc-by	6,283	УДК 517.9 Изв. вузов «ПНД», т.12, № 5, 2004 КВАНТОВАЯ ДИФФУЗИЯ АРНОЛЬДА _ В КАНАЛЕ С ГОФРИРОВАННОЙ ГРАНИЦЕЙ В ПРИСУТСТВИИ ПЕРЕМЕННОГО ЭЛЕКТРИЧЕСКОГО ПОЛЯ В.Я. Демиховский, А.И. Малышев Изучается квантовая диффузия Арнольда в модели частицы, движущейся в двумерном канале с гофрированной границей и в переменном электрическом поле. Построен оператор эволюции системы за произвольное число периодов и рассчитана скорость квантовой диффузии вдоль одного из резонансов связи для различной амплитуды гофрировки и напряженности электрического поля. Обнаружены два квантовых эффекта: остановка диффузии на больших временах наблюдения вследствие динамической локализации и подавление диффузии в условиях, когда число квантовых состояний, попадающих в присепаратрисный стохастический слой, становится порядка единицы. При произвольных значениях параметров задачи CKOpOCTh квантовой диффузии Арнольда оказывается меныше скорости классической диффузии. Изучению классической и квантовой динамики частиц, двиЖжущихся B каналах с гофрированной границей (периодических бильярдах), посвящено большое число работ. Полученные в них результаты весьма полезны как для понимания природы квантового хаоса вообще, так и для интерпретации ряда экспериментов, проведенных в недавнее время с низкоразмерными мезоскопи- ческими структурами. Классическая динамика и транспорт электронов B двумерном канале с синусоидально-модулированной границей изучались в работе [1], где для канала конечной длины был рассчитан коэффициент прозрачности, а также длина траектории при различных значениях глубины модуляции. Квантовые состояния в бесконечном двумерном канале переменной толщины были получены в работе [2], где соответствие между классическим и квантовым подходами устанавливалось путем построения функций Хушими. Кроме этого была исследована статистика межуровневых расстояний зонного спектра, как B регулярном, так и в хаотическом режимах. Следует отметить также работу [3], где, в частности, было установлено, что статистика собственных функций, а также локальная плотность состояний в периодических бильярдах не соответствует предсказаниям теории случайных матриц. В настоящее время периодические бильярды, в которых движение электронов HOCHT квазиклассический характер, могут быть реализованы экспериментально. Так, в работе [4] описана изготовленная методом электронной литографии последовательность пятнадцати квантовых TOUEK, образующих подобный бильярд. В такой системе исследовались также транспортные и магнитотранспортные процессы [5]. Настоящая — работа — посвящена Puc. 1. Двумерный канал и траектория движения B нем материальной TOYKM в присутствии электрического поля, направленного по ocuz. В подобный бильярд. В такой системе исследовались также транспортные и магнитотранспортные процессы [5]. Настоящая — работа — посвящена изучению квантовой диффузии Арнольда для частицы, движущейся в двумерном канале, одна из стенок которого ровная, а другая имеет синусоидальную форму. На частицу действует также внешнее — периоди- ческое поле, ориентированное поперек канала (рис. 1). Учитывается взаимо- отсутствие поля движение происходит IO прямым линиям действие трех резонансов: резонанса связи, который имеет место при рациональном соотношении времени движения поперек канала и времени пролета одного периода гофрировки вдоль канала, а также двух резонансов системы с внешним полем. * Термин «диффузия Арнольда» был позже введен Б.В. Чириковым. КВАНТОВАЯ ДИФФУЗИЯ АРНОЛЬДА _ В КАНАЛЕ С ГОФРИРОВАННОЙ ГРАНИЦЕЙ В ПРИСУТСТВИИ ПЕРЕМЕННОГО ЭЛЕКТРИЧЕСКОГО ПОЛЯ Подобная система может быть реализована экспериментально с использованием мезоскопической полупроводниковой структуры с модули- рованной поверхностью, помещенной в монохроматическое электромагнитное поле. Puc. 1. Двумерный канал и траектория движения B нем материальной TOYKM в присутствии электрического поля, направленного по ocuz. В отсутствие поля движение происходит IO прямым линиям Классическая диффузия Арнольда была теоретически предсказана в 1964 году в работе [6}. Суть этого универсального динамического явления, как известно, заключается в следующем. В 2М-мерном фазовом пространстве резонансы, определяемые соотношением (m-w)=0, где @ - это набор частот, а вектор 1IN имеет целочисленные компоненты, образуют — (2М-1)-мерные поверхности. В то же время КАМ-поверхности являются №М-мерными [7]. Условие, при котором возможно пересечение стохастических слоев, окружающих различные резонансы, можно получить из простых геометрических соображений. Для того чтобы резонансные поверхности He были изолированы друг от друга инвариантными поверхностями, необходимо, чтобы их размерности отличались более чем на единицу, TO есть должно выполняться условие: N<(2N - 1) -1. Отсюда получаем №>2. Takum образом, пересечение стохастических слоев различных резонансов является общим свойством систем с UHCIOM степеней свободы больше двух. Пересекаясь друг с другом, резонансы образуют в фазовом пространстве единую всюду плотную «паутину». Медленно диффундируя вдоль стохастических слоев этой сети, за достаточно долгое время система может уйти от своего начального состояния очень далеко. Как было отмечено [6], такая нестабильность - диффузия Арнольда - является универсальной в том смысле, что не существует критической величины возмущения, необходимой для её возникновения, хотя скорость диффузии стремится K нулю при уменьшении амплитуды возмущения. Классическая диффузия Арнольда изучалась в связи с задачей динамики Классическая диффузия Арнольда изучалась в связи с задачей динамики трех гравитационно взаимодействующих тел [8, 9], динамики галактик [10] и движения элементарных частиц в ускорителе [11], а также в связи с задачей о сильно возбужденном атоме водорода, находящемся в скрещенных электрическом и магнитном полях [12]. Диффузия Арнольда для классической частицы, движущейся в трехмерном канале, одна из границ которого промодулирована B двух взаимно нперпендикулярных направлениях, рассматривалась B монографии Лихтенберга и Либермана [7]. В работе [13] диффузия Арнольда изучалась B квазиклассическом приближении в так называемой модели стохастической накачки. _ @ В наших предыдущих работах [14], выполненных совместно с Ф.М. В наших предыдущих работах [14], выполненных совместно с Ф.М. Израилевым, изучалась квантовая диффузия Арнольда в системе, состоящей U3 двух взаимодействующих нелинейных осцилляторов, на один U3 которых действует переменное электрическое поле (аналогичная классическая система была подробно рассмотрена Чириковым [15]). Было установлено, что диффузионная эволюция квантового состояния имеет место лишь для начальных условий, лежащих в области сепаратрисы. КВАНТОВАЯ ДИФФУЗИЯ АРНОЛЬДА _ В КАНАЛЕ С ГОФРИРОВАННОЙ ГРАНИЦЕЙ В ПРИСУТСТВИИ ПЕРЕМЕННОГО ЭЛЕКТРИЧЕСКОГО ПОЛЯ В соответствии с классической теорией обнаружена экспоненциальная зависимость скорости диффузии от параметра, определяющего интенсивность взаимодействия осцилляторов. При малой константе связи, когда число квантовых состояний, попадающих в область классического стохастического слоя, оказывается порядка единицы, диффузия Арнольда отсутствует. Нами обнаружен также эффект подавления квантовой диффузии на больших временах, что является проявлением динамической локализации, подобной андерсеновской локализации B случайных средах. Настоящая работа построена следующим образом. Первый раздел посвящен Настоящая работа построена следующим образом. Первый раздел посвящен классической диффузии Арнольда в двумерном канале с периодически модулированной границей в присутствии переменного электрического поля. Рассчитан — классический — коэффициент — диффузии. Bo втором — разделе сформулирована квантовая модель и описана процедура расчета стационарных состояний, описывающих систему на резонансе связи. Третий раздел посвящен изучению аппарата, описывающего квантовую динамику в присутствии внешнего двухчастотного электрического поля. Здесь определен оператор эЭволюции за период поля, рассчитаны квазиэнергетические состояния. В четвертом разделе рассчитана скорость квантовой диффузии Арнольда по резонансу связи. 1. Классическая система 3 наглядно иллюстрирует Здесь - хорошо — заметно — множество резонансов, возникающих при рацио- нальном соотношении времени движения поперек KaHama (туда и обратно) Г, и времени пролета одного периода гофрировки вдоль канала Т. Для — их — характеристики — удобно использовать параметр n=I/T, (или n=w, /w ). Заметим, что на рис. 2 показана лишь та часть — фазовой плоскости, — которая — соответствует частицам, движущимся в положитель- HOM направлении OCH X. Рис. 3 наглядно иллюстрирует Рис. 3 наглядно иллюстрирует механизм — диффузии — Арнольда B рассматриваемой системе. Здесь показано расположение некоторых из резонансных поверхностей (B данном случае - прямых линий) в пространстве частот @, @, которые связаны с безразмерной кинетической энергией £ соотношением 2 2 - о2 + (0dn)* = 2тЁ, (2) (2) тде т - масса материальной точки, принимаемая — далее — за — единицу. Заметим, что энергия здесь измеряется в единицах т)/(2тг)?. Каждый из приведенных резонансов имеет свою ширину, однако переход из стохасти- ческого слоя одного резонанса B стохастический слой другого (поперек слоя и вдоль изоэнергетической поверх- ности) HEBO3MOXKeH, TaK KaK они изолированы друг от друга КАМ- поверхностями. 'Такой переход был бы возможен лишь при перекрытии двух резонансов в условиях сильного хаоса. Перемещение траектории вдоль стохас- тического слоя (на рис. 3 это направление показано двумя стрелками) невозможно в силу сохранения энергии. Однако при наличии внешнего, пере- менного во времени поля последнее ограничение снимается и становится возможной диффузия вдоль резонанса Puc. 2. Структура фазового пространства при a=0.01, d=1 (верхняя полуплоскость). Ilo вертикальной OCH OTJIOXKEH — тангенс — угла отражения 1. Классическая система В отсутствие внешнего поля в периоды между соударениями частица движется по прямолинейным траекториям, и €€ динамику удобно изучать с помощью отображений, связывающих между собой последовательные значения углов отражения а, и координат точек отражения от нижней границы Х, (см. рис.1) ау = @, - датс!в((А2л/ D)sin (2л /1)), X, ., =X +2Dtgo,, +Atgao, (cos(2nX /1 n+l ау = @, - датс!в((А2л/ D)sin (2л /1)), o X, ., =X +2Dtgo,, +Atgao, (cos(2nX /1) + cos(2nX, /1)). n+l o Здесь D - средняя ширина канала, A - амплитуда гофрировки. Заметим, что это отображение является точным B отличие от аналогичного отображения, приведенного, например, в книге [7]. Процедура линеаризации (1) вблизи стационарных TOYEK позволяет перейти K стандартному отображению й определить как частоты колебаний на резонансах, так и условия их перекрытия. Для упрощения записи в дальнейшем будем использовать безразмерные Здесь D - средняя ширина канала, A - амплитуда гофрировки. Заметим, что это отображение является точным B отличие от аналогичного отображения, приведенного, например, в книге [7]. Процедура линеаризации (1) вблизи стационарных TOYEK позволяет перейти K стандартному отображению й определить как частоты колебаний на резонансах, так и условия их перекрытия. Для упрощения записи в дальнейшем будем использовать безразмерные Для упрощения записи в дальнейшем будем использовать безразмерные переменные, измеряя все длины в единицах //2л. 'Гаким образом, далее Х=1/Эл-х, где х - безразмерная продольная координата. Поступая аналогично со всеми величинами, имеющими размерность длины, введем d, а и Z - безразмерные ширину канала, амплитуду гофрировки и поперечную координату, соответственно. Для того чтобы работать в дальнейшем с безразмерными единицами, необходимо определить также единицу времени /) и единицу массы т Рис. 2 дает представление о структуре фазового пространства системы (1). Рис. 2 дает представление о структуре фазового пространства системы (1). Рис. 2 дает представление о структуре фазового пространства системы (1). Ж e N= O‘gn S T х Puc. 2. Структура фазового пространства при a=0.01, d=1 (верхняя полуплоскость). Ilo вертикальной OCH OTJIOXKEH — тангенс — угла отражения ‚ Здесь - хорошо — заметно — множество резонансов, возникающих при рацио- нальном соотношении времени движения поперек KaHama (туда и обратно) Г, и времени пролета одного периода гофрировки вдоль канала Т. Для — их — характеристики — удобно использовать параметр n=I/T, (или n=w, /w ). Заметим, что на рис. 2 показана лишь та часть — фазовой плоскости, — которая — соответствует частицам, движущимся в положитель- HOM направлении OCH X. Рис. ‚ CBS3H. С Следует отметить, что в отсутствие внешнего переменного поля движение материальной точки в канале может осуществляться по одной траектории с разными скоростями. В процессе диффузии Арнольда частица остается на резонансе связи, HO при этом компоненты ее скорости v, и У, будут приблизительно пропорциснально варьироваться. =1 — 100 200 300 400 500 Фх› Рис. 3. Взаимное расположение резонансов связи И ИЗзоэнергетической поверхности. Здесь @=л, "7ЪЁ =160000 Внешнее поле U(z,t)=-f;z(cosQ, 1+ +с059,7) порождает свои резонансы B фазовом — пространстве системы — на частотах w =Q, и о,=©,. Их положение =1 также показано на рис. 3 шттриховыми линиями. В простейшем случае можно учитывать взаимодействие лишь трех резонансов: резонанса CBA3M и двух резонансов системы с внешним полем. Заметим, что это лишь часть паутины Арнольда, заполняющей все фазовое пространство. — Выбирая — начальные условия, например, в области стохасти- ческого слоя резонанса сВвязи 7m=1, — можно — ваблюдать 3a TeM, Kak 100 200 300 400 500 Фх› изображающая точка диффундирует под действием поля вдоль него. Для того Рис. 3. Взаимное расположение резонансов связи чтобы обеспечить стохастичность на И ИЗзоэнергетической поверхности. Здесь @=л, сепа-ратрисах отдельных резонансов и в "7ЪЁ =160000 то же время избежать их перекрытия, мы в дальнейшем будем полагать выполненным соотношение / /а=1000. =1 — 100 200 300 400 500 Фх› Рис. 3. Взаимное расположение резонансов связи И ИЗзоэнергетической поверхности. Здесь @=л, "7ЪЁ =160000 Для получения количественной информации о динамике системы был численно рассчитан коэффициент диффузии Арнольда. Для этого использовались отображения, в которых было учтено внешнее переменное электрическое поле, действующее на частицу в канале. Ввиду громоздкости формул мы не приводим здесь явный вид этих отображений. Обратимся сразу K результатам. Поскольку мы владеем информацией о системе лишь в те моменты времени, когда материальная точка ударяется о стенки канала, а они, вообще говоря, слабо коррелируют с периодом внешнего поля, для расчета коэффициента диффузии в безразмерных единицах имеет смысл использовать следующее соотношение: (Ёі+1 - Ё:) 2 W аее \| ©® i i+1 а величин. Y среднение Здесь проводится B два этапа. ©® Поясним смысл входящих сюда величин. Y среднение Здесь проводится B два этапа. Все время наблюдения за системой (например, 10° отображений) разбивается на N отрезков — времени — длительностью Af, соответствующей, например, 10° отображений. Величина Ё, в (4) имеет смысл среднего значения энергии на одном таком интервале, а (Af+Ar,,)/2 - расстояния по времени между соседними интервалами. Верхняя черта в формуле (4) означает усреднение выражения, стоящего в круглых скобках, по интервалам. CBS3H. С Нижний индекс, lgN, при коэффициенте диффузии D указывает на число временных интервалов, на которые разбито все время наблюдения. Столь громоздкая процедура определения коэффициента диффузии Арнольда позволяет снизить эффект случайных колебаний величины энергии системы [15]. Результаты наших расчетов коэффициента классической диффузии Поясним смысл входящих сюда величин. Y среднение Здесь проводится B два этапа. Все время наблюдения за системой (например, 10° отображений) разбивается на N отрезков — времени — длительностью Af, соответствующей, например, 10° Поясним смысл входящих сюда величин. Y среднение Здесь проводится B два этапа. Все время наблюдения за системой (например, 10° отображений) разбивается на N отрезков — времени — длительностью Af, соответствующей, например, 10° отображений. Величина Ё, в (4) имеет смысл среднего значения энергии на одном таком интервале, а (Af+Ar,,)/2 - расстояния по времени между соседними Результаты наших расчетов коэффициента классической диффузии приведены ниже на рис. 7 (см. раздел 4). Отчетливо видно, что в ДОВОЛЬНО широком интервале значений амплитуды гофрировки а (30<1/a?<50), где коэффициенты ) и [, практически равны друг другу, эволюция имеет диффузионный характер, причем скорость диффузии приблизительно линейно убывает с ростом величины 1/gV2 Это согласуется с общей теорией, развитой B — [15]. Появление заметных различий между D, и D, указывает на изменение характера временной эволюции. Так, различия в области 1/4?>70 вызваны тем, что здесь ширина стохастического слоя резонанса связи становится кра_йне малой, отчего эволюция системы становится квазипериодической. Расхождение между D, и D, и их резкий pocT (на порядок) в области Ма!?=6 связаны с перекрытием резонанса связи N=1 с резонансами на частотах ©2, и Q, в условиях сильного хаоса. Здесь в игру вступает механизм перемещения траектории поперек стохастических слоев этих резонансов, на фоне которого слабая диффузия Арнольда вдоль слоя перестает играть заметную роль. 2. Квантовые стационарные состоявия. Резонанс связи Для определения стационарных состояний частицы, движущейся B двумерном канале с гофрированной границей, удобно перейти K HOBBIM координатам, в которых границы являются плоскими, а граничные условия достаточно — простыми. Однако при этом — гамильтониан — приобретает дополнительные слагаемые, зависящие от координат и содержащие операторы дифференцирования [16]. Пусть поверхность канала в безразмерных переменных задана уравнениями z,=0 и z,=d+acosx, причем потенциал внутри канала равен нулю, а на границе обращается в бесконечность. В этом случае преобразование координат, выпрямляющее границы, будет иметь вид х =x, #' = #/(а + acosx). (5) (5) Метрический тензор такого преобразования есть Метрический тензор такого преобразования есть 1 ax'sin x'/(d + acos х') 8 = _ . (6) ax'sin x'/(d + асовх') (1 + ах”? sin®x")/(d + acos х')? разованное уравнение Шредингера будет иметь вид (6) Преобразованное уравнение Шредингера будет иметь вид 1 .М, L2 gy аВ, () е@ дх, дх’, запишется как () а условие нормировки запишется как а условие нормировки запишется как * о125 — Jwpg?ds =, . (8) (8) Здесь и всюду далее мы положили, что безразмерная постоянная Планка равна единице. На новой плоской границе потенциал бесконечен и выполняются нулевые граничные условия: (X' ‚0)=ч›‚.(цх'‚1)=0. Если ограничиться линейными по а/а членами, то при записи гамильтониана можно считать, что X'=x и z'=z/d. Тогда он примет вид H=H (x,2) + Ulx.z), (9) где 2 @ й =- (24 2), дх* д2° (10) ^ а@ 2 ‚ д? д cosx , U= — \| 2cosx — - 2sinx-z - COSX * 2 — - - sin х — 2а 2 дхд2 д: 2 Х H=H (x,2) + Ulx.z), (9) 2 @ й =- (24 2), дх* д2° (10) H=H (x,2) + Ulx.z), (9) где 2 @ й =- (24 2), дх* д2° (10) (9) где где (10) (10) ^ а@ 2 ‚ д? д cosx , U= — \| 2cosx — - 2sinx-z - COSX * 2 — - - sin х — 2а 2 дхд2 д: 2 Х (10) ^ а@ 2 ‚ д? д cosx , U= — \| 2cosx — - 2sinx-z - COSX * 2 — - - sin х — 2а 2 дхд2 д: 2 Х A Чтобы найти стационарные состояния гамильтониана Н, запишем уравнение ^ Шредингера в представлении гамильтониана Н). Спектр и волновые функции невозмущенной задачи имеют вид A Чтобы найти стационарные состояния гамильтониана Н, запишем уравнение ^ Шредингера в представлении гамильтониана Н). Спектр и волновые функции невозмущенной задачи имеют вид E %(x) =y (R4m?Id), ц (n2) = [U(Ld)] e sin (птг), — (11) (11) где L - полная безразмерная длина канала, L>>2. Ввиду периодичности задачи по X, точная во где L - полная безразмерная длина канала, L>>2. Ввиду периодичности задачи по X, точная во Ввиду периодичности задачи по X, точная волновая функция должна иметь блоховский вид, где квазиволновой вектор изменяется в пределах первой зоны Бриллюэна, определяемой неравенствами - 1/, <k< 1/2 При этом будет удобно работать в схеме приведенных 30H, полагая k=k+n, где n=0,£1,12... Представляя волновую функцию в виде двойного ряда Представляя волновую функцию в виде двойного ряда Wi(x,z) =2, ,, Са( (12) (12) запишем стадионарное уравнение Шредингера (7) ¢ учетом (9) и (10) как (13) где где U o=@ ) (}(x z )y dxdz = (& + n)m, п)т К + п’),т Ы (& + n)m (& + п) (& + л') + (14) (бп‚ б 8, ттИ(т? а условие нормировки запишется как Уровни, расположенные над сепаратрисной областью, крайне слабо расщеплены (с высокой степенью точности их можно считать двукратно вырожденными) и в классике соответствуют «вращениям» на резонансе в противоположных направлениях. В соответствии с такой структурой спектра стационарные состояния на резонансе связи удобно характеризовать с помощью двух индексов - номера группы д и номера уровня внутри каждой группы 5. В этом случае энергетический спектр системы может быть записан в следующем виде: тно. Точка сгущения уровней соответствует классической сепаратрисе нелиней- ного резонанса. Уровни, расположенные над сепаратрисной областью, крайне слабо расщеплены (с высокой степенью точности их можно считать двукратно вырожденными) и в классике соответствуют «вращениям» на резонансе в противоположных направлениях. В соответствии с такой структурой спектра стационарные состояния на резонансе связи удобно характеризовать с помощью двух индексов - номера группы д и номера уровня внутри каждой группы 5. В этом случае энергетический спектр системы может быть записан в следующем виде: E =0 g+ EM, (16) -2} (16) E =0 g+ EM, -2} M 7] где Ет есть Матье-подобный спектр группы с номером g. а условие нормировки запишется как -т®)(-1)" +m [(1+2(k+n))s л +1+(1-2(k+n)]€>n, k- mm U o=@ ) (}(x z )y dxdz = (& + n)m, п)т К + п’),т Ы (& + n)m (& + п) (& + л') + (14) = -а/(2сі){л2т2/сі2(бп‚ б 8, ттИ(т? -т®)(-1)" +m [(1+2(k+n))s л +1+(1-2(k+n)]€>n, k- mm Изучаемый далее режим - режим резонанса связи N=1 - соответствует случаю, (14) k- Изучаемый далее режим - режим резонанса связи N=1 - соответствует случаю, когда — выполняется условие @, =@,, T o, =Ё w1 (O-E, (k) &+п)+М) н што—Ето+1 -E, = 2(2т,+1)/(2@). Поскольку мы будем работать в кназиклассическом режиме, полагая п)>>1 и т)>>1, то можно считать, что @ ~l<+n0 и о, =тт/@. Квантовые состояния на резонансе связи удобно характеризовать НОВЫМИ индексами /= л- п) И р=т + (m-m,), а полную энергию отсчитывать от уровня Е° - (k). В этом случае уравнение Шредингера (13) примет вид E(k)c*, = pw, С Yy (PP + @ &) (p-rP)ct 2, U, ck, . L+n0-r,p~r+m0 k+ny-r',p'-r+my = пр Далее для простоты положим d=n и будем рассматривать состояния B центре зоны Бриллюэна, считая k=0. При этом @ —пО‚ . =My, откуда следует лу=т. В этом то случае уравнение Шредингера приобретает более компактный вид рг+гЭс„ +2Z, И, nyor-ring it g Сир (15) раг- ицы, Egs / нной что групп рой. пами уппы что вого овни тан- 0.0 ектра ЭЗдесь = (рто + рЧ2 - рг+гЭс„ +2Z, И, nyor-ring it g Сир (15) Рассчитанный с помощью (15) фраг- мент энергетического спектра частицы, Egs / движущейся в канале с гофрированной границей, показан на рис. 4. Видно, что спектр COCTOMT из отдельных групп уровней €O схожей — структурой. Расстояние между соседними группами составляет ,. Внутри каждой группы спектр напоминает спектр Матье, что довольно THIMYHO — для — квантового нелинейного резонанса. Нижние уровни расположены практически эквидистан- 0.0 Рис. 4. Фрагмент энергетического спектра системы (9) вблизи резонанса связи. ЭЗдесь a=0.01, d=m, k=0 (15) Рассчитанный с помощью (15) фраг- мент энергетического спектра частицы, движущейся в канале с гофрированной границей, показан на рис. 4. Видно, что спектр COCTOMT из отдельных групп уровней €O схожей — структурой. Расстояние между соседними группами составляет ,. Внутри каждой группы спектр напоминает спектр Матье, что довольно THIMYHO — для — квантового нелинейного резонанса. Нижние уровни расположены практически эквидистан- Рис. 4. Фрагмент энергетического спектра системы (9) вблизи резонанса связи. ЭЗдесь a=0.01, d=m, k=0 тно. Точка сгущения уровней соответствует классической сепаратрисе нелиней- ного резонанса. 3. Эволюция квантовых состояний 3.1. Оператор эволюции. Далее обратимся к рассмотрению эволюции состояний на резонансе связи в присутствии внешнего переменного поля. Ero частоты выберем так, чтобы выполнялось соотношение @, —-(9 +2,)2. B численных экспериментах полагалось: w, —400 Q=350 и Q,=450. Таким образом, B силу кратности частот внешнее поле, % следоватеш‚но и гамильтониан имели период Т= 7х2л/©, = 9х2л/0,=0.125664. Известно, что в TOM случае, когда оператор Гамильтона периодически Известно, что в TOM случае, когда оператор Гамильтона периодически зависит от времени, то есть H(t+T)—H(t) решение нестационарного уравнения Шредингера, согласно теореме Флоке, можно представить в виде р (x,z,0) = exp(-ith)uQ(x,z,t), р (x,z,0) = exp(-ith)uQ(x,z,t), где uy(x,z,6)=u,(x,z,t+T) - так называемая квазиэнергетическая функция, e, - квазиэнергия. Ё(вазиэнергетическиАе функции u, (x,z,t) являются собственными функциями оператора эволюции U(T) за период поля Т, причем каждой такой функции соответствует собственное значение ехр(-1,!) где uy(x,z,6)=u,(x,z,t+T) - так называемая квазиэнергетическая функция, e, - квазиэнергия. Ё(вазиэнергетическиАе функции u, (x,z,t) являются собственными функциями оператора эволюции U(T) за период поля Т, причем каждой такой функции соответствует собственное значение ехр(-1,!) U(T)u(x,2,8) = exp(-ie iy (x,7.0). (17) (17) Для исследования эволюции нашей системы необходимо найти явный вид ^ оператора U(T). Для этого представим квазиэнергетическую функцию в виде двойного ряда U (x,Z) = 2 ﬁQqswkq S(X’Z)’ где р „(х 2) - найденные в предыдущем разделе стационарные функции частицы в канале с гофрированной границей (12). В последней — формуле у квазиэнергетической функции опущен аргумент ;, поскольку она определена лишь в моменты времени, кратные периоду поля Т. Последнее соотношение позволяет перейти в уравнении (17) к матричному представлению. В результате такого перехода получим, что коэффициенты AL „з являюТся собственными векторами матрицы (, (T), и могут быть найдены вместе с собственными значениями путем ее диагонализации. Для построения матрицы оператора эволюции можно воспользоваться процедурой, онисанной B наших предыдущих работах\|]4} Ее смысл COCTOUT B следующем. ПОДСИСТВУСМ оператором (М&ТРИЦСИ) 45 s(T) на некоторое начальное состояние С 0‘70=б оЧбЫ (99 = U = 9 qsq s' (Т)С '4' ° q,s;qo,sD(T) Cq,s oo, (18) (18) 10 Коэффициенты С „о могут быть найдены путем численного интегрирования нестационарного уравнения Шредивтера на промежутке времени от () до T, при этом они образуют столбец матриды U, .. ‚ T). Повторяя 3Ty процедуру для различных начальных условии С ‘N— Bq q%”,, где g#q,, $#S, МЫ ЗаПОЛНИМ, TaKUM образом, всю матрицу U, . (7). Приводя полученную матрицу оператора эволюции K диагональному ВИДу, Приводя полученную матрицу оператора эволюции K диагональному ВИДу, находим спектр квазиэнергий ¢, и квазиэнергетические функции А‹Ы_ после чего сам оператор эволюции можно представить B виде Оззе„(Г) = 94® ACT exp(-ieyT). (19) (19) Для того, чтобы вычислить оператор эволюции за N периодов внешнего поля, нужно возвести (19) в степень N и воспользоваться соотношением ортогональности квазиэнергетических функций. В результате будем иметь U, (МТ) = 2,42 А ®' exp (-ie NT). Теперь для TOro, чтобы проследить за эволюцией начального состояния вида С Jo(0)=8 ;55 HA сколь угодно больших временах, достаточно подействовать 90 матрицеи ЦОр (МТ) на функцию (столбец) начального состояния. 3.2. Резонансное приближение. р (x,z,0) = exp(-ith)uQ(x,z,t), Чтобы выполнить намеченную программу, необходимо, прежде всего, решить нестационарное уравнение Шредингера B представлении функций у^ (x,2) ioC, Jot = ((nnoq + ЕМ )С ааЛ уе ав ая (СОЗ @, + COs Q1) C . (20) (20) Для этого, совершая преобразование вида С, (= bqs(t)exp[—i(mnoq + EMW)], (21) (21) перейдем от функций С ‹п(г) к функциям b, (¢), которые удовлетворяют системе уравнений ! idb, Jdt = f 2, 2, . (СОЗО + COsQ,)b , „ехр\|- i(w, (q - ) +(EM, - Ё“, Nl (22) idb, Jdt = f 2, 2, . (СОЗО + COsQ,)b , „ехр\|- i(w, (q - ) +(EM, - Ё“, Nl (22) (22) Теперь B так называемом резонансном приближении в сумме по ¢’ в правой части (22), учитывая, что @, —(Q {+@,)/2, сохраним лишъ медленно осциллирующие слагаемые с 4'=4+1 и B результате придем к следующему уравнению: idb, [dt = - ficos(®QH2)Z {z, . 1 b 1o ехр[-і(ЕМ ~EY )i + q,5; #+1,5° ° g+l,s g+l,s (23) b ,exp[-i(EMq_LS,-EMq,S)t]}, + zq,s; ¢-1,s""q-1,s -0 (23) где 3Q=1Q -0 Очевидный Очевидный интерес представляет строение матриц Z, „а + 1» определяющих переходы между состояниями S и $' соседних групп уровней. сНа рис. 5 схематично показана внутренняя структура матрицы z, , .. Шшкалой интенсивности серого цвета указан порядок величин ),.! В данном случае состояния внутри групп упорядочены так, что низший уровень имеет индекс s=0, а остальные уровни B порядке возрастания энергии имеют индексы $=1, -1, 2, -2, ... и т.д. Исходя из произведенного упорядочивания состояний внутри групп, можно Исходя из произведенного упорядочивания состояний внутри групп, можно сделать ряд важных замечаний. Довольно очевидно, что центр «креста» на рис. 5 соответствует переходам между самыми низкими состояниями в группах н „ — ° (имеющими индекс s=0), соответствую- щими — центру — резонанса — сВязи. Матричные элементы, лежащие на широкой главной диагонали ближе K краям матрицы, определяют переходы между состояниями, лежащими над областью сгущения уровней (сепа- ратрисой). В соответствии с дву- кратным вырождением этих состояний области данной матрицы , $'>10 и „ 5, 5'<-10 симметричны — относительно — ° центра. В обоих перечисленных случаях Р матричные элементы довольно быстро ис. 5. МВТРИЧНЫС элементы Z 39 ‚„»‚ ©опреде- o ляющие переходы между группамиг{—б и 4=1, то убывают с удалением от главной есть вдоль резонанса связи. Параметры Te же, ДИагонали, TO есть C возрастанием что и на рис. 4 разности 15 - 5'!. На концах «креста» видны заметные уширения - эти области соответствуют (имеющими индекс s=0), соответствую- щими — центру — резонанса — сВязи. Матричные элементы, лежащие на широкой главной диагонали ближе K краям матрицы, определяют переходы между состояниями, лежащими над областью сгущения уровней (сепа- ратрисой). В соответствии с дву- кратным вырождением этих состояний области данной матрицы , $'>10 и 5, 5'<-10 симметричны — относительно центра. В обоих перечисленных случаях матричные элементы довольно быстро o убывают с удалением от главной ДИагонали, TO есть C возрастанием разности 15 - 5'!. уширения - эти области соответствуют (имеющими индекс s=0), соответствую- щими — центру — резонанса — сВязи. Матричные элементы, лежащие на широкой главной диагонали ближе K краям матрицы, определяют переходы между состояниями, лежащими над областью сгущения уровней (сепа- ратрисой). В соответствии с дву- кратным вырождением этих состояний области данной матрицы , $'>10 и 5, 5'<-10 симметричны — относительно центра. В обоих перечисленных случаях матричные элементы довольно быстро o убывают с удалением от главной ДИагонали, TO есть C возрастанием разности 15 - 5'!. На концах «креста» видны заметные уширения - эти области соответствуют сепаратрисной области резонанса. где 3Q=1Q -0 Очевидный Величины матричных элементов здесь достаточно велики, поэтому для ё-образного начального условия на сепаратрисе возможны практически равновероятные переходы B широкую по числу уровней область спектра соседних групп. Гаким образом, легко понять, что во внешнем переменном поле переходы между присепаратрисными состояниями разных групп будут происходить с болышей интенсивностью, нежели между другими состояниями. Именно данный факт и определяет скорость диффузии Арнольда в стохастическом сепаратрисном слое вдоль резонанса связи. ’ 4. Квантовая диффузия вдоль резонанса связи В нашей модели квантовая диффузия Арнольда вдоль резонанса связи проявляется как перераспределение начального состояния по группам уровней с разными 4. В этом случае количественной характеристикой скорости диффузии может служить дисперсия распределения состояний по группам. Для различных начальных состояний нами вычислялась зависимость дисперсии — энергии (I:I)zzmnozAq от времени / или числа периодов М. Здесь A =Z (¢-9)°Z\|C, P c_]—E g2 \|C, „Ё. На рис. 6 изображена зависимость A „(М) для трех различных начальных условий: нижняя кривая соответствует начальному — состоянию, выбранному вблизи дентра резонанса связи, средняя кривая - начальному условию на одном из надсепаратрисных состояний, а верхняя кривая - начальному условию, соответствующему одному из сепаратрисных уровней. График демонстрирует явное различие между эволюциями во времени данных начальных состояний. Так, если для под- и надсепаратрисных состояний величина A, квазипериодически осциллирует около некоторого значения, TO для состояний, - отвечающих сепаратрисе резонанса связи величина А, после некоторого времени, соответствующего классическому времени стохастизации, испытывая небольшие осцилляции, в среднем возрастает. Определение среднего наклона графика позволяет найти коэффициент квантовой диффузии Арнольда. Результаты представлены на рис. 7. Подобно тому, что было отмечено в наших предыдущих работах [14], здесь величина коэффициента квантовой диффузии D, также оказывается на 1.5-2.0 порядка ниже классических результатов. ПрИ этом сравнении, правда, нужно иметь B виду и то, что флуктуации классического коэффициента диффузии составляют, как минимум, плюс-минус полпорядка. В нашей модели квантовая диффузия Арнольда вдоль резонанса связи проявляется как перераспределение начального состояния по группам уровней с разными 4. В этом случае количественной характеристикой скорости диффузии может служить дисперсия распределения состояний по группам. Для различных начальных состояний нами вычислялась зависимость дисперсии — энергии (I:I)zzmnozAq от времени / или числа периодов М. Здесь A =Z (¢-9)°Z\|C, P c_]—E g2 \|C, „Ё. На рис. 6 изображена зависимость A „(М) для трех различных 12 gD 10.01 50 Dq оо 80 148 R т т Кц т — д 20 40 6%92 Dy Рис. 7. Зависимость коэффициента д'иффузии — Арнольда в классической (D и D ) и квантовой (D ) системах от амплитуды гофрировки ОДНОЙ ИЗ стенок канала а. Здесь E(t=0)=160000, =350, _.450 fo/a =1000, ширина канала d=mu gD 10.01 50 Dq оо 80 148 R т т Кц т — д 20 40 6%92 Dy Рис. 7. Зависимость коэффициента д'иффузии Арнольда в классической (D и D ) и квантовой (D ) системах от амплитуды гофрировки ОДНОЙ ИЗ стенок канала а. 4. Квантовая диффузия вдоль резонанса связи Здесь E(t=0)=160000, =350, _.450 fo/a =1000, ширина канала d=mu Необходимо отметить также, что зависимости ,, D, и D от величины гофрировки в области 10<1/а\?<20 практически линеины и имеют одинаковый наклон. При 1/а!?<10 расчет коэффициента квантовой диффузии, к сожалению, был крайне затруднен необходимостью учета громадного числа состояний (ввиду большой ширины всех рассматриваемых резонансов и близости границы их перекрытия). При 1/а'?>20 участок линейного роста дисперсии A (N) отсутствовал. Последнее обстоятельство, очевидно, связано с тем, что при слабой гофрировке в область классического стохастического слоя резонанса связи попадает всего 1-2 квантовых состояния. Понятно, что B этом случае говорить O какой-то «квантовой стохастизации» не имеет смысла, отчего и квантовая диффузия Арнольда отсутствует. W3 данных, приведенных на рис. 6, также следует, что через 200-300 W3 данных, приведенных на рис. 6, также следует, что через 200-300 периодов поля после своего начала квантовая диффузия останавливается и величина А, начинает осциллировать около некоторого среднего значения (на рисунке оно показано горизонтальной штриховой линией). Такое поведение, очевидно, можно рассматривать как одно W3 проявлений Такое поведение, очевидно, можно рассматривать как одно W3 проявлений динамической локализации, которая впервые была онисана в модели одномерного ротатора с д-образными толчками [17, 18]. Позднее была установлена связь этого явления с локализацией Андерсона [19]. В нашем случае можно сказать, что динамическая локализация связана C локализацией — квазиэнергетических сепаратрисных функций в д-пространстве. В силу этого в разложении любого начального состояния по квазиэнергетическим функциям эффективно содержится ограниченное число членов, что в конечном итоге приводит K насышщению диффузии и квазипериодическим осцилляциям. Следует подчеркнуть, что в данном случае динамическая локализация имеет место в системе с числом степеней свободы N=2.5, в то время как B случае ротатора с периодическими толчками N=1.5. Необходимо подчеркнуть также, что в нашем случае динамическая локализация наблюдается в условиях слабого хаоса на сепаратрисах, а не глобального Xaocd, как в случае ротатора с 5-толчками. Настоящая работа поддержана грантом РФФИ (npoexm № 03-02-17054), программой «Университеты Poccuu» (№ ур.01.01.022), а также ФНП «Династия». 13 Библиографический список 1. Luna-Acosta G.A., Krokhin A.A., Rodriguez M.A., Hernandez-Tejeda P.H. Classical chaos and ballistic transport in а mesoscopic channel // Phys. Rev. В. 1996. Vol. 54. P. 11410. 2. Luna-Acosta G.A., Na K., Reichl L.E., Krokhin A.A. Band structure аоа quantum Poincare sections of a classically chaotic quantum rippled channel // Phys. Rev. Е. 1996. Vol. 53. P. 3271; Luna-Acosta G.A., Rodriguez M.A., Krokhin A.A., Na K., апа Mendez R.A. Quantum and classical ballistic transport in а chaotic 2D electron channel // Rev. Мех. Fis. 1998. Vol. 44. Р. 7. Mendez R.A. Quantum and classical ballistic transport in а chaotic 2D electron channel Rev. Мех. Fis. 1998. Vol. 44. Р. 7. 3. Luna-Acosta G.A., Mendez-Bermudez J.A., Izrailev F.M. Periodic chaotic billiards: Quantum-classical correspondence in energy space // Phys. Rev. Е. 2001. Vol. 64. P. 036206. 4. Kouwenhoven L.P. et al. Transport through a finite one-dimensional crystal // 4. Kouwenhoven L.P. et al. Transport through a finite one-dimensional crystal // Phys. Rev. Lett. 1990. Vol. 65. P. 361. 5. Lent C.S., Leng M. Magnetic edge states in а corrugated quantum channel // J. 5. Lent C.S., Leng M. Magnetic edge states in а corrugated quantum channel // J. Appl. Phys. 1991. Vol. 70. P. 3157. 6. Арнольд В.И. O неустойчивости динамических систем CO MHOTHMH 6. Арнольд В.И. O неустойчивости динамических систем CO MHOTHMH степенями свободы // ДАН СССР. 1964. Т. 156. С. 9. 7. Лихтенберг А., Либерман М. Регулярная и стохастическая динамика. M.: 7. Лихтенберг А., Либерман М. Регулярная и стохастическая динамика. M.: Мир, 1984. 8. Ferraz-Mello S., Sessin W. Resonances in the motion of planets, satellites and asteroids. Sao Paulo: Brazil, 1985. . 9. Reichl L.E. The Transition to Chaos. New-York: Springer-Verlag, 1992. 9. Reichl L.E. The Transition to Chaos. New-York: Springer-Verlag, 1992. 10. Binney J., Tremaine S. Galactic Dynamics. Princeton: Princeton Univ 9. Reichl L.E. The Transition to Chaos. New-York: Springer-Verlag, 1992. 10. Binney J., Tremaine S. Galactic Dynamics. Princeton: Princeton University Press, 1987. 10. Binney J., Tremaine S. Galactic Dynamics. Princeton: Princeton University Press, 1987. 11. Nonlinear dynamics aspects of particle accelerators // Lecture Notes in Physics. Press, 1987. 11. Nonlinear dynamics aspects of particle accelerators // Lecture Notes in Physics. Berlin: Springer-Verlag, 1986. Vol. 247. Press, 1987. 11. Nonlinear dynamics aspects of particle accelerators // Lecture Notes in Physics. Berlin: Springer-Verlag, 1986. Vol. 247. 11. Nonlinear dynamics aspects of particle accelerators // Lecture Notes in Physics. Библиографический список Berlin: Springer-Verlag, 1986. Vol. 247. 12. Milczewski J. von, Diercksen G.H.F., Uzer T. Computation of the Arnold web 12. Milczewski J. von, Diercksen G.H.F., Uzer T. Computation of the Arnold web for the hydrogen atom in crossed electric and magnetic fields // Phys. Rev. Lett. 1996. Vol. 76. P. 2890. 13. Leitner D.M., Wolynes P.G. Quantization of the stochastic pump model of 13. Leitner D.M., Wolynes P.G. Quantization of the stochastic pump model of nold diffusion // Phys. Rev. Lett. 1997. Vol. 79. P. 55. 14. Demikhovskii V.Ya., Izrailev F.M., апа Malyshev А. Manifestation оЁ 14. Demikhovskii V.Ya., Izrailev F.M., апа Malyshev А. Manifestation оЁ Arnold diffusion in quantum systems // Phys. Rev. Lett. 2002. Vol. 88. P. 154101; Demikhovskii V.Ya., Izrailev F.M., апа Malyshev A.I. Quantum Armold diffusion in а simple nonlinear system // Phys. Rev. E. 2002. Vol. 66. P. 036211. 15. Chirikov B.V. A universal instability оё many dimensional oscillator systems // simple nonlinear system // Phys. Rev. E. 2002. Vol. 66. P. 036211. 15. Chirikov B.V. A universal instability оё many dimensional oscillator systems // Phys. Rep. 1979. Vol. 52. P. 263. 15. Chirikov B.V. A universal instability оё many dimensional oscillator systems // Phys. Rep. 1979. Vol. 52. P. 263. 16. Демиховский В.Я., Потапенко С.Ю., Сатанин А.М.Электронный спектр 16. Демиховский В.Я., Потапенко С.Ю., Сатанин А.М.Электронный спектр в системах с периодически модулированной поверхностью // Физика и техника полупроводников. 1983. Т. 17. С. 213. 17. Casati G., Chirikov B.V., Izrailev F.M., апа Ford J. Stochastic behavior оё а quantum pendulum under а periodic perturbation // Lecture Notes т Physics. Springer- Verlag, Berlin. 1979. Vol. 93. P. 334. _ 18. Chirikov B.V., Izrailev F.M., апа Shepelyansky D.L. Dynamical stochasticity in classical and quantum mechanics // Sov. Sci. Rev. 1981. Vol. 2. P. 209. 19. Fishman 5. et al. Chaos, quantum recurrence and Anderson localization // 19. Fishman 5. et al. Chaos, quantum recurrence and Anderson localization // Phys. Rev. Lett. 1989. Vol. 49. P. 509. Поступила в редакцию 01.11.2004 Нижегородский государственный университет Нижегородский государственный университет 14 QUANTUM ARNOLD DIFFUSION IN RIPPLED CHANNEL AT THE PRESENCE OF ALTERNATING ELECTRIC FIELD V.Ya. Demikhovskii, A.I. Malyshev We study quantum Arnold diffusion for the particle. moving in the rippled channel at the presence of periodic external electric field. The evolution operator for arbitrary number of field periods and diffusion rate was calculated for different ripple amplitude апа electric field intensity. Two new effects which limit quantum Arnold diffusion have been observed - the diffusion suppression due to dynamical localization and diffusion stop in the case when the number of quantum states corresponding to the classical near separatrix chaotic region has the order of unity. For any model parameters the quantum diffusion coefficient prove to be smaller then classical one. Демиховский Валерий Яковлевич - родился в Харькове (1938), окончил Горьковский государственный университет (1961). После окончания ГГУ работает на кафедре теоретической физики физического факультета. Защитил диссертацию на соискание ученой степени доктора физико- математических наук (1979). Основная область интересов - теория конденсированного состояния, физика низкоразмерных квантовых структур, квантовый хаос. Опубликовал более 150 научных работ, в TOM числе монографию «Физика квантовых низкоразмерных структур» (М.: Логос, 2000, в соавторстве с Г.А. Вугальтером). Заслуженный деятель науки Российской Федерации, Соросовский профессор. Мальшиев Александр Игоревич - родился в г. Бор Нижегородской ® области (1978), окончил Нижегородский государственный университет (2001). Работает на кафедре теоретической физики физического факультета ННГУ в должности ассистента и учится на заочном отделении аспирантуры. Основная область научных интересов - квантовый хаос и квантовая диффузия Арнольда. Стипендиат ФНП «Династия». ® Мальшиев Александр Игоревич - родился в г. Бор Нижегородской области (1978), окончил Нижегородский государственный университет (2001). Работает на кафедре теоретической физики физического факультета ННГУ в должности ассистента и учится на заочном отделении аспирантуры. Основная область научных интересов - квантовый хаос и квантовая диффузия Арнольда. Стипендиат ФНП «Династия». 15
https://openalex.org/W4362459711	https://figshare.com/articles/journal_contribution/supplementary_Figure_Legends_1-2_from_Dose-Dense_Chemotherapy_Improves_Mechanisms_of_Antitumor_Immune_Response/22397472/1/files/39843165.pdf	English	null	supplementary Figure Legends 1-2 from Dose-Dense Chemotherapy Improves Mechanisms of Antitumor Immune Response	null	2,023	cc-by	335	Supplemental Figure 1. Single-agent DD chemotherapy is superior to MTD in treating cisplatin-resistant tumor, R HM-1 Supplemental Figure 1. Single-agent DD chemotherapy is superior to MTD in treating cisplatin-resistant tumor, R HM-1 Supplemental Figure 1. Single-agent DD chemotherapy is superior to MTD in treating cisplatin-resistant tumor, R HM-1 To a lesser degree, single-agent DD chemotherapy also exhibited better anti-tumor effect in mice bearing R HM-1 cell tumors. R HM-1 cells (1x106) were injected subcutaneously (s.c.) into the female (C57BL/6, C3H/He) F1 mice (5 in each group, day 0). On day 4, mice started chemotherapy with paclitaxel and cisplatin delivered intraperitoneally (i.p.) in either single-agent paclitaxel (16 mg/kg in DD, 40 mg/kg in MTD) or cisplatin (10 mg/kg in DD, 25 mg/kg in MTD) at the 3-day (DD) and 10-day (MTD) intervals for 7 courses (DD) and 3 courses (MTD). Control group mice were treated with PBS in 3-day interval. Better therapeutic efficacy was shown in mice treated by both DD chemotherapies with cisplatin and paclitaxel (p=0.002 and *p=0.0003 for paclitaxel and cisplatin, respectively, DD versus MTD) motherapy had significantly less tumor loading (#p=0.022, DD versus MTD). DD Supplemental Figure 2. DD chemotherapy is superior to MTD in treating intraperitoneal cisplatin-resistant ovarian tumor Supplemental Figure 2. DD chemotherapy is superior to MTD in treating intraperitoneal cisplatin-resistant ovarian tumor DD chemotherapy exhibited better anti-tumor effect in a mouse intraperitoneal ovarian tumor model. Mouse ovarian tumor ID8 cells (5×105/mice), an aggressive cell line originating from MOSEC cell (19), were injected intraperitoneally (i.p.) into C57BL/6 mice (day 0). On day 7, mice were treated by DD, MTD, or control PBS. ID8 cells were engineered with luciferase and thus tumor growth could be monitored by non-invasive bioluminescent imaging. Photography of tumor image represented by strength of luminescence illustrated that mice receiving DD chemotherapy had significantly less tumor loading (#p=0.022, DD versus MTD). DD chemotherapy exhibited better therapeutic effect against intraperitoneal cisplatin-resistant tumor. There was no statistically significant difference of the tumor loading between mice receiving MTD chemotherapy and control PBS.
https://openalex.org/W2587454028	https://europepmc.org/articles/pmc5316558?pdf=render	English	null	Incidence of invasive salmonella disease in sub-Saharan Africa: a multicentre population-based surveillance study	The Lancet. Global health/The lancet. Global health	2,017	cc-by	15,106	Summary Summary Background Available incidence data for invasive salmonella disease in sub-Saharan Africa are scarce. Standardised, multicountry data are required to better understand the nature and burden of disease in Africa. We aimed to measure the adjusted incidence estimates of typhoid fever and invasive non-typhoidal salmonella (iNTS) disease in sub- Saharan Africa, and the antimicrobial susceptibility proﬁ les of the causative agents. Lancet Glob Health 2017; 5: e310–23 See Comment page e236 International Vaccine Institute, SNU Research Park, Seoul, South Korea (F Marks PhD, V von Kalckreuth MD, L M Cruz Espinoza MD, J F Deerin MPH, J Im MSc, H J Jeon BA, F Konings PhD, B Ley PhD, C Nichols MPH, G D Pak MSc, U Panzner MSc, J K Park PhD, S E Park MIS, H Schütt-Gerowitt MD, H J Seo BA, K Thriemer PhD, M R Warren MSc, Prof J D Clemens MD, T F Wierzba PhD); Bandim Health Project, Bissau, Guinea-Bissau (P Aaby DMSc, M Bjerregaard-Andersen PhD, S V Løfberg MD); Research Center for Vitamins and Vaccines, Copenhagen, Denmark (P Aaby, M Bjerregaard-Andersen, S V Løfberg); Kumasi Centre for Collaborative Research in Tropical Medicine, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana (Prof Y Adu-Sarkodie PhD, N Sarpong MD); Faculty of Medicine, University of Gezira, Wad Medani, Sudan (M A El Tayeb MD, Prof N Gasmelseed PhD); Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA (M Ali PhD); Armauer Hansen Research Institute, Addis Ababa, Ethiopia (A Aseffa PhD, M Teferi MSc, B Yeshitela MSc); Oxford University Clinical Research Unit, Ho Chi Minh City, Vietnam (Prof S Baker PhD, J I Campbell FIBMS, N V M Hoang MSc); Kilimanjaro Christian Medical Centre, Moshi, Tanzania (H M Biggs MD, Prof J A Crump MD, J T Hertz MD); Division of Infectious Diseases and International Health, Duke Methods We established a systematic, standardised surveillance of blood culture-based febrile illness in 13 African sentinel sites with previous reports of typhoid fever: Burkina Faso (two sites), Ethiopia, Ghana, Guinea-Bissau, Kenya, Madagascar (two sites), Senegal, South Africa, Sudan, and Tanzania (two sites). We used census data and health-care records to deﬁ ne study catchment areas and populations. Eligible participants were either inpatients or outpatients who resided within the catchment area and presented with tympanic (≥38·0°C) or axillary temperature (≥37·5°C). Inpatients with a reported history of fever for 72 h or longer were excluded. Funding Bill & Melinda Gates Foundation. Copyright © The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY lice Summary We also implemented a health-care utilisation survey in a sample of households randomly selected from each study area to investigate health-seeking behaviour in cases of self-reported fever lasting less than 3 days. Typhoid fever and iNTS disease incidences were corrected for health-care- seeking behaviour and recruitment. Findings Between March 1, 2010, and Jan 31, 2014, 135 Salmonella enterica serotype Typhi (S Typhi) and 94 iNTS isolates were cultured from the blood of 13 431 febrile patients. Salmonella spp accounted for 33% or more of all bacterial pathogens at nine sites. The adjusted incidence rate (AIR) of S Typhi per 100 000 person-years of observation ranged from 0 (95% CI 0–0) in Sudan to 383 (274–535) at one site in Burkina Faso; the AIR of iNTS ranged from 0 in Sudan, Ethiopia, Madagascar (Isotry site), and South Africa to 237 (178–316) at the second site in Burkina Faso. The AIR of iNTS and typhoid fever in individuals younger than 15 years old was typically higher than in those aged 15 years or older. Multidrug-resistant S Typhi was isolated in Ghana, Kenya, and Tanzania (both sites combined), and multidrug-resistant iNTS was isolated in Burkina Faso (both sites combined), Ghana, Kenya, and Guinea-Bissau. Interpretation Typhoid fever and iNTS disease are major causes of invasive bacterial febrile illness in the sampled locations, most commonly aﬀ ecting children in both low and high population density settings. The development of iNTS vaccines and the introduction of S Typhi conjugate vaccines should be considered for high-incidence settings, such as those identiﬁ ed in this study. Funding Bill & Melinda Gates Foundation. Incidence of invasive salmonella disease in sub-Saharan Africa: a multicentre population-based surveillance study Florian Marks, Vera von Kalckreuth, Peter Aaby, Yaw Adu-Sarkodie, Muna Ahmed El Tayeb, Mohammad Ali, Abraham Aseﬀ a, Stephen Baker, Holly M Biggs, Morten Bjerregaard-Andersen, Robert F Breiman, James I Campbell, Leonard Cosmas, John A Crump, Ligia Maria Cruz Espinoza, Jessica Fung Deerin, Denise Myriam Dekker, Barry S Fields, Nagla Gasmelseed, Julian T Hertz, Nguyen Van Minh Hoang, Justin Im, Anna Jaeger, Hyon Jin Jeon, Leon Parfait Kabore, Karen H Keddy, Frank Konings, Ralf Krumkamp, Benedikt Ley, Sandra Valborg Løfb erg, Jürgen May, Christian G Meyer, Eric D Mintz, Joel M Montgomery, Aissatou Ahmet Niang, Chelsea Nichols, Beatrice Olack, Gi Deok Pak, Ursula Panzner, Jin Kyung Park, Se Eun Park, Henintsoa Rabezanahary, Raphaël Rakotozandrindrainy, Tiana Mirana Raminosoa, Tsiriniaina Jean Luco Razaﬁ ndrabe Emmanuel Sampo, Heidi Schütt-Gerowitt, Amy Gassama Sow, Nimako Sarpong, Hye Jin Seo, Arvinda Sooka, Abdramane Bassiahi Soura, Adama Tall, Mekonnen Teferi, Kamala Thriemer, Michelle R Warren, Biruk Yeshitela, John D Clemens, Thomas F Wierzba Articles Articles Articles Added value of this study y Original data collected in TSAP represent the most comprehensive standardised analysis done in sub-Saharan Africa of the incidence and antimicrobial resistance patterns of invasive salmonella infections. The results describe the incidence estimated, adjusted by health-care-seeking behaviour, and antimicrobial susceptibility of typhoid fever and iNTS diseases from 13 sites in ten sub-Saharan Africa countries. For typhoid fever disease, we estimate that the overall incidence is two to three times higher than a previous estimate (10–100 cases per 100 000 people), and is in some settings similar to data from Asia, where the burden is known to be very high. The data also revealed that children aged 2–14 years bear the greatest burden of the disease. For iNTS disease, the data also reﬂ ect a high incidence, especially in young children, and in speciﬁ c sites (Ghana) the incidence could be more than ﬁ ve times that previously estimated. Salmonella infections are a major cause of global morbidity and mortality; however, substantial knowledge gaps exist with regards to the distribution and incidence of disease caused by Salmonella enterica serotype Typhi and invasive non-typhoidal salmonella (iNTS) disease in sub-Saharan Africa. Before the Typhoid Fever Surveillance in Africa Program (TSAP), estimates of typhoid fever incidence data from Africa were available from four vaccine trials and one population-based study in Kenya. Other estimates of invasive salmonella infections originated from diﬀ erent descriptions of bacteraemia in febrile patients in The Gambia, Malawi, Mozambique, and Kenya. These few, unstandardised, published data are not suﬃ cient for understanding the burden of the disease in sub-Saharan Africa. In 2008, WHO expressed the necessity for more epidemiological information to estimate the incidence and antimicrobial susceptibility of invasive salmonella disease. Consequently, in January, 2009, the International Vaccine Institute (Seoul, South Korea) and the Kenya Medical Research Institute (Kiliﬁ , Kenya) co-hosted a meeting with ﬁ ve other international institutions and 28 investigators from 14 research sites across sub-Saharan Africa. The purpose of the meeting was to review existing data on invasive salmonella infections in sub-Saharan Africa and surveillance infrastructure from sites, and to discuss the way forward to investigate invasive salmonella in the African region. These 28 investigators and the ﬁ ve international institutions presented their data on invasive bacterial disease, focusing on invasive salmonellosis. Articles University Medical Center, Durham, NC, USA (H M Biggs, Prof J A Crump, J T Hertz); Centers for Disease Control and Prevention, Nairobi, Kenya (R F Breiman MD, L Cosmas MPH, B S Fields PhD, J M Montgomery PhD); WHO- Kenya Country Office, Nairobi, Kenya (L Cosmas); Global Health Institute, Emory University, Atlanta, GA, USA (R F Breiman); Duke Global Health Institute, Duke University, Durham, NC, USA (Prof J A Crump); Centre for International Health, University of Otago, Dunedin, New Zealand (Prof J A Crump); Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany (D M Dekker PhD, A Jaeger BA, R Krumkamp DrPH, Prof J May MD); German Center for Infection Research, Hamburg—Borstel—Lü beck, Germany (R Krumkamp, D M Dekker, Prof J May, N Sarpong); Faculty of Science, University of Hafr Al Batin, Hafr Al Batin, Saudi Arabia (Prof N Gasmelseed); Schiphra Hospital, Ouagadougou, Burkina Faso (L P Kabore MSc); National Institute for Communicable Diseases, Johannesburg, South Africa (K H Keddy MMed, A Sooka MSc); Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa (K H Keddy); Global and Tropical Health Division, Menzies School of Health Research, Charles Darwin University, Australia (B Ley, K Thriemer); Institute of Tropical Medicine, Eberhard-Karls University Tübingen, Tübingen, Germany (C G Meyer MD); Duy Tan University, Da Nang, Vietnam (C G Meyer); National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA (E D Mintz MD); Institute Pasteur de Dakar, Dakar, Senegal (A A Niang MSc, Prof A G Sow PhD, A Tall PhD); Center for Clinical Research, Kenya Medical Research Institute, Nairobi, Kenya (B Olack MPHE); Laboratory of Microbiology, University of Antananarivo, Antananarivo, Madagascar (H Rabezanahary MD, R Rakotozandrindrainy MD, Evidence before this study We did a literature search using PubMed with the following search terms: (“typhoid” OR “typhoid fever” OR “Salmonella Typhi” OR “S Typhi” OR “salmonella infection” OR “enteric fever” OR “non-typhoidal salmonella” OR “NTS”) AND (“incidence”OR “rate” OR “frequency” OR “prevalence” OR “morbidity” OR “burden” OR “surveillance” OR “epidemiology”). We restricted publication dates from Dec 31, 1995, to July 30, 2016, and no language restrictions were applied. The date of our last search was July 30, 2016. Research in context The TSAP was created to address the knowledge gaps on the incidence and antimicrobial resistance patterns of invasive salmonella infections at diﬀ erent countries with previous reports of typhoid fever cases in sub-Saharan Africa. TSAP created a network of 13 surveillance sites across ten countries, and implemented cross-sectional studies to investigate the health-care-seeking behaviour of the populations under surveillance. Introduction to cause 21·7 million illnesses and 217 000 fatalities annually, and iNTS disease is estimated to cause 3·4 million illnesses and 681 000 fatalities annually.1,2 Substantial knowledge gaps exist regarding the distribution of typhoid fever and iNTS disease in Africa. The few existing studies,4–8 reported over diﬀ ering time periods and using various protocols, have been extrapolated and contribute to existing typhoid fever estimates, which limits international generalisability. The scarcity of data in sub-Saharan Africa prompted to cause 21·7 million illnesses and 217 000 fatalities annually, and iNTS disease is estimated to cause 3·4 million illnesses and 681 000 fatalities annually.1,2 Salmonella infections contribute substantially to global morbidity and mortality.1,2 The best described invasive salmonella serovars are Salmonella enterica serotype Typhi (S Typhi), causing typhoid fever, and S enterica serotype Paratyphi A, B, and C (S Paratyphi A, B, and C), which cause paratyphoid fever. Other non-typhoidal salmonella (NTS) serovars that typically cause self-limiting diarrhoea can also cause systemic infections, refered to as invasive NTS (iNTS) disease.3 Globally, typhoid fever is estimated Substantial knowledge gaps exist regarding the distribution of typhoid fever and iNTS disease in Africa. The few existing studies,4–8 reported over diﬀ ering time periods and using various protocols, have been extrapolated and contribute to existing typhoid fever estimates, which limits international generalisability. The scarcity of data in sub-Saharan Africa prompted www.thelancet.com/lancetgh Vol 5 March 2017 e310 Articles Added value of this study Implications of all the available evidence The results of this study underscore the need for preventive measures, including vaccines, improved sanitation and hygiene, malaria control, antiretroviral therapy programmes, and improved nutrition. The results also emphasise that the implementation of eﬀ ective antimicrobials might be impaired by the presence and potential increase of drug-resistance salmonella strains in the region. The advent of typhoid conjugate vaccines might provide more powerful tools to control typhoid fever; the ﬁ rst vaccine, which was manufactured in India, has already been submitted to WHO for prequaliﬁ cation. Data from this study will be included in the GAVI Alliance review of potential subsidies for typhoid fever vaccines in 2017; their recommendation will be crucial for the deployment of these vaccines. Hence, an urgent need exists to understand the pragmatic aspects of vaccine targeting and delivery, particularly given the burden of disease in children, the associated risk factors, and the focal nature of the disease. Further assessment of the incidence in infants (0–5 months vs 6–11 months) and data on severe typhoid fever or iNTS, including mortality, is crucial to determine the potential eﬀ ect of future vaccines. Our follow-on study—Severe Typhoid in Africa (SETA)—which investigates severe typhoid burden, is underway. The data indicated the presence of typhoid fever and iNTS disease; however, the studies were not standardised in design, data collection, and laboratory techniques. The meeting concluded that unless standardised methods of data collection and diagnostic procedure were used across countries, and patterns of health-care utilisation were understood and accounted for, the real disease burden of invasive salmonella infections in the region would remain unclear. As a result, a consortium was established and members agreed to form a network of surveillance sites in sub-Saharan Africa in areas with previous reports of cases of typhoid fever. WHO, in 2008, to request more epidemiological information to reliably estimate the incidence of typhoid fever and iNTS disease and the antimicrobial WHO, in 2008, to request more epidemiological information to reliably estimate the incidence of typhoid fever and iNTS disease and the antimicrobial susceptibilities of the corresponding organisms.9 Consequently, between 2010, and 2014, we established 13 surveillance sites across sub-Saharan Africa in e311 www.thelancet.com/lancetgh Vol 5 March 2017 Articles locations where typhoid fever had been previously reported. Added value of this study This network formed the Typhoid Fever Surveillance in Africa Program (TSAP) and served as a platform to implement standardised surveillance of febrile illness and cross-sectional studies to investigate the health-care-seeking behaviour of the surveyed populations.10–12 Here, we present the adjusted incidence estimates of typhoid fever and iNTS disease and the antimicrobial susceptibility proﬁ les of the causative agents at the 13 selected surveillance sites. T J L Razafindrabe PhD); Institut Supérieur des Sciences de la Population, University of Ouagadougou, Ouagadougou, Burkina Faso (A B Soura PhD); Schiphra Hospital, Ouagadougou, Burkina Faso (E Sampo MSc); Institute of Medical Microbiology, University of Cologne, Cologne, Germany (H Schütt-Gerowitt); University Cheikh Anta Diop de Dakar, Dakar, Senegal (Prof A G Sow); International Centre for Diarrheal Disease Research, Bangladesh, Dhaka, Bangladesh (Prof J D Clemens); and University of California Fielding School of Public Health, Los Angeles, CA, USA (Prof J D Clemens) experienced in micro biological laboratory research.10 13 sites in ten countries were selected (ﬁ gure 1), four of which already had established surveillance systems: Pietermaritzburg, South Africa; Asante Akim North, Ghana; Moshi Urban District and Moshi Rural District, Tanzania; and Kibera, Kenya. Four sites were part of the International Network for the Demographic Evaluation of Populations and Their Health (INDEPTH): Polesgo and Nioko II, Burkina Faso; Butajira, Ethiopia; and Bandim, Guinea-Bissau. These sites had functional Health and Demographic Surveillance Systems (HDSS) in place.13 Additional surveillance sites were Isotry and Imerintsiatosika, Madagascar; Pikine, Senegal; and East Wad Medani, Sudan. The surveillance system in Kibera was established before TSAP with an active, population- based surveillance component. Home visits were done once every 2 weeks to screen for febrile patients and encourage visits to the aﬃ liated health-care facility. Active surveillance in Kibera was continued throughout T J L Razafindrabe PhD); Institut Supérieur des Sciences de la Population, University of Ouagadougou, Ouagadougou, Burkina Faso (A B Soura PhD); Schiphra Hospital, Ouagadougou, Burkina Faso (E Sampo MSc); Institute of Medical Microbiology, University of Cologne, Cologne, Germany (H Schütt-Gerowitt); University Cheikh Anta Diop de Dakar, Dakar, Senegal (Prof A G Sow); International Centre for Diarrheal Disease Research, Bangladesh, Dhaka, Bangladesh (Prof J D Clemens); and University of California Fielding School of Public Health, Los Angeles, CA, USA (Prof J D Clemens) Correspondence to: Dr Florian Marks, International Vaccine Institute, SNU Research Park, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, South Korea fmarks@ivi.int www.thelancet.com/lancetgh Vol 5 March 2017 Study design, site selection, and participants Total patients recruited from study area: met inclusion criteria (n=13 358) Total patients analysed: resided in study area, met inclusion criteria, AND had complete blood results (n=13 431) Study database records Health-care facility records* Patients with febrile diseases from study area Surveillance site Site Country 918 756 1251 976 1501 918 763 1251 977 1501 4204† 2080† 1251 1361 1788 Nioko II Polesgo Kibera Imerintsia- tosika Isotry Burkina Faso Kenya Madagascar 2651 1021 644 680 2651 1054 650 684 5822 1533 574 1436‡ Asante Akim North Bandim East Wad Medani Moshi Ghana Guinea-Bissau Sudan Tanzania 847 1058 1128 847 1058 1204 NA‡ NA‡ NA‡ Butajira Pikine Pietermaritz- burg Ethiopia Senegal South Africa Figure 2: Visits to health-care facilities and recruitment of patients during surveillance period at each site NA=not available. Data on health facility visits were collected retrospectively, after completion of surveillance period. Diagnosis of febrile illnesses was used at sites when temperature of patients was not recorded. †Number estimated by the proportion of the population under demographic surveillance at each respective site. ‡In Tanzania, before Nov 11, 2011, every ﬁ fth eligible patient was recruited; from Nov 11, 2011, every second eligible patient was recruited. This recruitment pattern was applied to this number. with the exception of Sudan, where manual culturing with daily subculturing for up to 5 days was instituted. Gram staining and bacterial identiﬁ cation were done with standard microbiological techniques.14 Quality control of preanalytical processes included time and temperature control measures, during which every blood culture bottle was collected, transported, and placed into the incubator. Quality control of analytical processes included sterility and function control of culture media, controls of biochemical reactions, and antimicrobial susceptibility testing. For the quality control of manual culturing in Sudan, additionally, blood culture bottles were inoculated weekly with a suspension containing Escherichia coli or Staphylococcus aureus references. Inoculated blood culture bottles were incubated overnight and veriﬁ ed for growth by subculture. TSAP. All other sites implemented passive surveillance.10 The ethics committees of all collaborating institutions and the International Vaccine Institute (Seoul, South Korea) approved the study protocol. The catchment area for each site was determined through health-care facility records and through accessible administrative and demographic data.11 We determined the population of each catchment area using the latest census or the INDEPTH database. We categorised sites as urban, rural, or other using setting classiﬁ cations at each site. For the study protocol see http://www.ivi.int/?page_id= 12479&uid=922&mod= document Study design, site selection, and participants We used a multicentre, population-based, prospective surveillance study design. Selection of the surveillance sites in sub-Saharan Africa was not random; locations were eligible if they had evidence of previous typhoid fever, a laboratory infrastructure suitable for blood culture, an onsite health-care facility, and staﬀ Correspondence to: Dr Florian Marks, International Vaccine Institute, SNU Research Park, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, South Korea fmarks@ivi.int Figure 1: Sites participating in the Typhoid Fever Surveillance in Africa Program East Wad Madani (Wad Madani) Sudan Population: 46 857 Butajira Ethiopia Population: 62 213 Kibera (Nairobi) Kenya Population: 29 314 Moshi Urban District (Moshi) Tanzania Population: 184 292 Moshi Rural District (Moshi) Tanzania Population: 466 737 Isotry (Antananarivo) Madagascar Population: 70 323 Imerintsiatosika Madagascar Population: 46 381 Pietermaritzburg South Africa Population: 391 830 Asante Akim North Ghana Population: 140 694 Polesgo (Ouagadougou) Burkina Faso Population: 7574 Bandim (Bissau) Guinea-Bissau Population: 87 525 Pikine (Dakar) Senegal Population: 342 178 Nioko II (Ouagadougou) Burkina Faso Population: 17 754 Pikine (Dakar) Senegal Population: 8 Pikine (Dakar) Senegal Figure 1: Sites participating in the Typhoid Fever Surveillance in Africa Program e312 Articles Articles Figure 2: Visits to health-care facilities and recruitment of patients during surveillance period at each site NA=not available. Data on health facility visits were collected retrospectively, after completion of surveillance period. Diagnosis of febrile illnesses was used at sites when temperature of patients was not recorded. †Number estimated by the proportion of the population under demographic surveillance at each respective site. ‡In Tanzania, before Nov 11, 2011, every ﬁ fth eligible patient was recruited; from Nov 11, 2011, every second eligible patient was recruited. This recruitment pattern was applied to this number. Study design, site selection, and participants Surveillance was implemented in each study location for a period of at least 12 months and recruitment occurred at primary, secondary, and tertiary health-care facilities. Recruitment was open to outpatients and inpatients who visited any of the health-care facilities participating in TSAP, who resided within the catchment area and presented with tympanic (≥38·0°C) or axillary temperature (≥37·5°C). Inpatients with a reported history of fever for 72 h or longer were excluded, as were patients with residence outside of the catchment area. Asante Akim North recruited children younger than age 15 years only; other sites recruited patients of all ages. Written informed consent preceded recruitment and clinical appraisal forms were completed for all participants. g g y Contaminants were deﬁ ned as organisms not typically associated with bloodstream infections; these included non-pathogens and those more commonly associated with commensal skin microbiota, including coagulase- negative Staphylococci, Bacillus spp, and Micrococcus spp. Antimicrobial susceptibility testing was done by disc diﬀ usion according to Clinical and Laboratory Standards Institute15 standards for ampicillin, amoxicillin-clavulanic acid, chloramphenicol, co-trimoxazole, ceftriaxone, and ciproﬂ oxacin. Multidrug resistance was deﬁ ned as resistance to ampicillin or amoxicillin-clavulanic acid, chloramphenicol, and co-trimoxazole. Isolates with intermediate susceptibility were classiﬁ ed as resistant. Malaria blood smears were routinely done, except in South Africa. In Ethiopia, rapid diagnostic tests (SD BIOLINE Malaria Ag Pf/Pv, SD Standard Diagnostics, Yongin, South Korea) were used in addition to routine malaria blood smears. Laboratory procedures We standardised laboratory, quality control, and blood sample collection procedures across sites.10 Blood (5–10 mL for adults; 1–3 mL for children) was inoculated into aerobic blood culture bottles and incubated in an automated blood culture system (BD BACTEC, Becton- Dickinson, USA, or BacT/ALERT, BioMérieux, France), e313 www.thelancet.com/lancetgh Vol 5 March 2017 Articles atient demographics atients analysed, N§§ 918 756 1021 1058 2651 644 847 976 1501 1128 406 274 1251 Median age, years (IQR) 4 (1–12) 7 (3–21) 3 (1–7) 22 (14–32) 2 (0–5) 15 (9–32) 11 (5–25) 20 (9–32) 26 (17–40) 3 (1–29) 7 (1–29) 19 (2–39) 7 (4–14) –1 years, n (% of N) 247 (27%) 117 (15%) 369 (36%) 9 (1%) 1114 (42%) 2 (<1%) 74 (9%) 66 (7%) 12 (1%) 427 (38%) 114 (28%) 67 (24%) 99 (8%) –4 years, n (% of N) 235 (26%) 148 (20%) 271 (27%) 23 (2%) 841 (32%) 41 (6%) 124 (15%) 87 (9%) 58 (4%) 209 (19%) 62 (15%) 37 (14%) 312 (25%) –14 years, n (% of N) 228 (25%) 252 (33%) 274 (27%) 255 (24%) 696 (26%) 275 (43%) 303 (36%) 184 (19%) 234 (16%) 95 (8%) 56 (14%) 26 (9%) 539 (43%) 15 years, n (% of N) 208 (23%) 239 (32%) 107 (10%) 771 (73%) NA 326 (51%) 346 (41%) 639 (65%) 1197 (80%) 397 (35%) 174 (43%) 144 (53%) 301 (24%) emale patients, n % of N) 467 (51%) 404 (53%) 487 (48%) 468 (44%) 1204 (45%) 348 (54%) 433 (51%) 570 (58%) 997 (66%) 586 (52%) 211 (52%) 149 (54%) 622 (50%) npatients, n (% of N) 66 (7%) NA¶¶ 224 (22%) 241 (23%) 2651 (100%) NA¶¶ 31 (4%) NA¶¶ NA¶¶ 1128 (100%) 220 (54%) 156 (57%) NA¶¶ (Table 1 continues on next page) Nioko II, Burkina Faso Polesgo, Burkina Faso Bandim, Guinea- Bissau Pikine, Senegal Asante Akim North, Ghana East Wad Medani, Sudan Butajira, Ethiopia Imerintsiato- sika, Madagascar Isotry, Madagascar Pietermaritz- burg, South Africa Moshi Urban District, Tanzania Moshi Rural District, Tanzania Kibera, Kenya* Surveillance sites Type of health-care facility (IPD, OPD) 1 hospital (IPD, OPD) 1 health-care centre (OPD) 1 hospital, 1 health-care centre (IPD, OPD) 1 hospital, 3 health-care centres (IPD, OPD) 1 hospital (IPD) 3 health- care centres (OPD) 1 hospital, 3 health-care centres (IPD, OPD) 1 health-care centre (OPD) 1 health-care centre (OPD) 1 hospital (IPD) 1 hospital (IPD, OPD) 1 hospital (IPD, OPD) 1 health-care centre (OPD) Setting† Semi-urban Semi-urban Urban Urban and urban slum Urban and rural Urban Semi-urban and rural Rural Urban Urban Urban Rural Urban slum Population density, people per km² 2204 5163 17 078 16 695 121 7209 6545 225 29 301 1191 3069 332 77 000 Surveillance period (months)‡ April, 2012, to September, 2013 (18) April, 2012, to September, 2013 (18) December, 2011, to April, 2013 (17) December, 2011, to April, 2013 (17) March, 2010, to May, 2012 (27) July, 2012, to July, 2013 (13) May, 2012, to January, 2014 (21) November, 2011, to June, 2013 (20) February, 2012, to May, 2013 (16) February, 2012, to January, 2014 (24) September, 2011, to May, 2013 (21) September, 2011, to May, 2013 (21) January, 2012, to December, 2013 (24) Source of catchment population HDSS 2011§ HDSS 2011§ HDSS 2011§ Ministry of Health 2012¶ Census 2010\|\| Census 2008** HDSS 2012§ Ministry of Health 2010¶ Ministry of Health 2010¶ Census 2010†† Census 2012‡‡ Census 2012f KEMRI/CDC 2012g Collaborating research institution UoO UoO BHP IPD§ KCCR/BNITM UoG AHRI UoA UoA NICD KCMC/Duke KCMC/ Duke KEMRI/US-CDC Health-care utilisation survey and person-years of observation calculation KEMRI/US-CDC=Kenya Medical Research Institute/US Centers for Disease Control and Prevention, Nairobi. IPD=inpatient department. OPD=outpatient department. HDSS=Health and Demographic Surveillance System. KEMRI=Kenya Medical Research Institute. NA=not available. In Kibera, active population mobilisation was done in addition to passive surveillance. †Setting reﬂ ects the classiﬁ cation commonly used at each site and does not refer to a standard deﬁ nition. ‡Surveillance activities were scheduled for 12 months in Burkina Faso, Guinea-Bissau, Senegal, Sudan, Ethiopia, and Madagascar and for 24 months in Ghana, Kenya, South Africa, and Tanzania. If funds allowed, the scheduled period was extended. §Population data were provided from the HDSS country oﬃ ce. ¶Population data for Senegal and Madagascar were provided by Ministry of Health. Population data correspond to the 2012 population census and 2010 estimated population for the area, respectively. \|\|Population data for Ghana were obtained from the Ghana Statistical Service, 2010 population, and housing census. It includes 53 towns distributed in what is now Asante Akim North and Central. Population data for Sudan were provided by the Statistics Department, Population Center, University of Gezira, Sudan, and correspond to year 2008. ††Population data for South Africa were provided by the Statistics Department in South Africa and corresponds to the 2011 census. ‡‡Population data for Tanzania were provided by the National Bureau of Statistics and correspond to the 2012 population and housing census. §§Patients who met inclusion criteria, consented to take part in the study, and had a blood culture taken and a documented blood culture result. ¶¶Recruitment health-care facility providing outpatient services only. \|\|\|\|Positive for non-contaminant isolates. *Denominator diﬀ ers from all blood cultures analysed because of missing values. Malaria results are based on blood smears, except for the site in Butajira (52% of patients positive for malaria were diagnosed with malaria rapid tests). The health-care-seeking behaviour of the populations under surveillance was investigated with the assumption that access to the TSAP health-care facility was non- uniform throughout the population.16,17 A standardised and pretested health-care utilisation survey was implemented in a representative sample of households randomly selected from each study area.11 We investigated health-care-seeking behaviour in cases of self-reported fever lasting less than 3 days. The ﬁ rst choice of health- care facility in cases of fever was categorised by age- stratiﬁ ed groups and used to calculate the proportion of individuals from the catchment population who visited this TSAP health-care facility. Articles UoO=University of Ouagadougou, Ouagadougou. BHP=Bandim Health Project, Bissau. IPD=Institute Pasteur de Dakar, Dakar. KCCR/BNITM=Kumasi Centre for Collaborative Research in Tropical Medicine, Kumasi/Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany. UoG=University of Gezira, Wad Medani. AHRI=Armauer Hansen Research Institute, Addis Ababa. UoA=University of Antananarivo, Antananarivo. NICD=National Institute for Communicable Diseases, Johannesburg. KCMC/Duke=Kilimanjaro Christian Medical Center, Moshi/Duke University Medical Center, Durham, NC, USA. KEMRI/US-CDC=Kenya Medical Research Institute/US Centers for Disease Control and Prevention, Nairobi. IPD=inpatient department. OPD=outpatient department. HDSS=Health and Demographic Surveillance System. KEMRI=Kenya Medical Research Institute. NA=not available. In Kibera, active population mobilisation was done in addition to passive surveillance. †Setting reﬂ ects the classiﬁ cation commonly used at each site and does not refer to a standard deﬁ nition. ‡Surveillance activities were scheduled for 12 months in Burkina Faso, Guinea-Bissau, Senegal, Sudan, Ethiopia, and Madagascar and for 24 months in Ghana, Kenya, South Africa, and Tanzania. If funds allowed, the scheduled period was extended. §Population data were provided from the HDSS country oﬃ ce. ¶Population data for Senegal and Madagascar were provided by Ministry of Health. Population data correspond to the 2012 population census and 2010 estimated population for the area, respectively. \|\|Population data for Ghana were obtained from the Ghana Statistical Service, 2010 population, and housing census. It includes 53 towns distributed in what is now Asante Akim North and Central. Population data for Sudan were provided by the Statistics Department, Population Center, University of Gezira, Sudan, and correspond to year 2008. ††Population data for South Africa were provided by the Statistics Department in South Africa and corresponds to the 2011 census. ‡‡Population data for Tanzania were provided by the National Bureau of Statistics and correspond to the 2012 population and housing census. §§Patients who met inclusion criteria, consented to take part in the study, and had a blood culture taken and a documented blood culture result. ¶¶Recruitment health-care facility providing outpatient services only. \|\|\|\|Positive for non-contaminant isolates. Denominator diﬀ ers from all blood cultures analysed because of missing values. Malaria results are based on blood smears, except for the site in Butajira (52% of patients positive for malaria were diagnosed with malaria rapid tests). Health-care utilisation survey and person-years of observation calculation This proportion constituted an adjustment factor to correct incidences. The time at risk in person-years of observation (PYO) stratiﬁ ed by age was calculated using the adjusted population. In HDSS sites, each resident contributed to PYO for the time present in the study area during the recruitment period. In non-HDSS sites, we calculated PYO by projecting the catchment population from the start to the end of the study recruitment period, and multiplied the calculated average population by the number of years of surveillance duration. www.thelancet.com/lancetgh Vol 5 March 2017 (Table 1 continues on next page) e314 www.thelancet.com/lancetgh Vol 5 March 2017 Articles Articles Health-care utilisation survey and person-years of observation calculation \|\|Population data for Ghana were obtained from the Ghana Statistical Service, 2010 population, and housing census. It includes 53 towns distributed in what is now Asante Akim North and Central. Population data for Sudan were provided by the Statistics Department, Population Center, University of Gezira, Sudan, and correspond to year 2008. ††Population data for South Africa were provided by the Statistics Department in South Africa and corresponds to the 2011 census. ‡‡Population data for Tanzania were provided by the National Bureau of Statistics and correspond to the 2012 population and housing census. §§Patients who met inclusion criteria, consented to take part in the study, and had a blood culture taken and a documented blood culture result. ¶¶Recruitment health-care facility providing outpatient services only. \|\|\|\|Positive for non-contaminant isolates. *Denominator diﬀ ers from all blood cultures analysed because of missing values. Malaria results are based on blood smears, except for the site in Butajira (52% of patients positive for malaria were diagnosed with malaria rapid tests). Nioko II, Burkina Faso Polesgo, Burkina Faso Bandim, Guinea- Bissau Pikine, Senegal Asante Akim North, Ghana East Wad Medani, Sudan Butajira, Ethiopia Imerintsiato- sika, Madagascar Isotry, Madagascar Pietermaritz- burg, South Africa Moshi Urban District, Tanzania Moshi Rural District, Tanzania Kibera, Kenya (Continued from previous page) Laboratory results Total blood culture, N 918 756 1021 1058 2651 644 847 976 1501 1128 406 274 1251 Total contaminated blood cultures, n (% of N) 220 (24%) 145 (19) 125 (12%) 96 (9%) 182 (7%) 54 (8%) 90 (11%) 6 (1%) 49 (3%) 192 (17%) 8 (2%) 13 (5%) 16 (1%) Total positive blood cultures, n (% of N)\|\|\|\| 29 (3%) 31 (4) 30 (3%) 31 (3%) 175 (7%) 16 (2%) 26 (3%) 11 (1%) 30 (2%) 51 (5%) 17 (4%) 11 (4%) 110 (9%) Positive for malaria, n (% of all patients tested)* 430/908 (47%) 444/744 (60%) 206/525 (39%) 297/1058 (28%) 1139/2651 (43%) 254/632 (40%) 110/822 (13%) 19/955 (2%) 2/274 (1%) 0 4/406 (1%) 2/274 (1%) 226/956 (24%) UoO=University of Ouagadougou, Ouagadougou. BHP=Bandim Health Project, Bissau. IPD=Institute Pasteur de Dakar, Dakar. KCCR/BNITM=Kumasi Centre for Collaborative Research in Tropical Medicine, Kumasi/Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany. UoG=University of Gezira, Wad Medani. AHRI=Armauer Hansen Research Institute, Addis Ababa. UoA=University of Antananarivo, Antananarivo. NICD=National Institute for Communicable Diseases, Johannesburg. KCMC/Duke=Kilimanjaro Christian Medical Center, Moshi/Duke University Medical Center, Durham, NC, USA. Health-care utilisation survey and person-years of observation calculation Laboratory results Total blood culture, N 918 756 1021 1058 2651 644 847 976 1501 1128 406 274 1251 Total contaminated blood cultures, n (% of N) 220 (24%) 145 (19) 125 (12%) 96 (9%) 182 (7%) 54 (8%) 90 (11%) 6 (1%) 49 (3%) 192 (17%) 8 (2%) 13 (5%) 16 (1%) Total positive blood cultures, n (% of N)\|\|\|\| 29 (3%) 31 (4) 30 (3%) 31 (3%) 175 (7%) 16 (2%) 26 (3%) 11 (1%) 30 (2%) 51 (5%) 17 (4%) 11 (4%) 110 (9%) Positive for malaria, n (% of all patients tested)* 430/908 (47%) 444/744 (60%) 206/525 (39%) 297/1058 (28%) 1139/2651 (43%) 254/632 (40%) 110/822 (13%) 19/955 (2%) 2/274 (1%) 0 4/406 (1%) 2/274 (1%) 226/956 (24%) UoO=University of Ouagadougou, Ouagadougou. BHP=Bandim Health Project, Bissau. IPD=Institute Pasteur de Dakar, Dakar. KCCR/BNITM=Kumasi Centre for Collaborative Research in Tropical Medicine, Kumasi/Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany. UoG=University of Gezira, Wad Medani. AHRI=Armauer Hansen Research Institute, Addis Ababa. UoA=University of Antananarivo, Antananarivo. NICD=National Institute for Communicable Diseases, Johannesburg. KCMC/Duke=Kilimanjaro Christian Medical Center, Moshi/Duke University Medical Center, Durham, NC, USA. KEMRI/US-CDC=Kenya Medical Research Institute/US Centers for Disease Control and Prevention, Nairobi. IPD=inpatient department. OPD=outpatient department. HDSS=Health and Demographic Surveillance System. KEMRI=Kenya Medical Research Institute. NA=not available. In Kibera, active population mobilisation was done in addition to passive surveillance. †Setting reﬂ ects the classiﬁ cation commonly used at each site and does not refer to a standard deﬁ nition. ‡Surveillance activities were scheduled for 12 months in Burkina Faso, Guinea-Bissau, Senegal, Sudan, Ethiopia, and Madagascar and for 24 months in Ghana, Kenya, South Africa, and Tanzania. If funds allowed, the scheduled period was extended. §Population data were provided from the HDSS country oﬃ ce. ¶Population data for Senegal and Madagascar were provided by Ministry of Health. Population data correspond to the 2012 population census and 2010 estimated population for the area, respectively. \|\|Population data for Ghana were obtained from the Ghana Statistical Service, 2010 population, and housing census. It includes 53 towns distributed in what is now Asante Akim North and Central. Population data for Sudan were provided by the Statistics Department, Population Center, University of Gezira, Sudan, and correspond to year 2008. ††Population data for South Africa were provided by the Statistics Department in South Africa and corresponds to the 2011 census. Health-care utilisation survey and person-years of observation calculation ‡‡Population data for Tanzania were provided by the National Bureau of Statistics and correspond to the 2012 population and housing census. §§Patients who met inclusion criteria, consented to take part in the study, and had a blood culture taken and a documented blood culture result. ¶¶Recruitment health-care facility providing outpatient services only. \|\|\|\|Positive for non-contaminant isolates. Denominator diﬀ ers from all blood cultures analysed because of missing values. Malaria results are based on blood smears, except for the site in Butajira (52% of patients positive for malaria were diagnosed with malaria rapid tests). , 9 5 5 5 9 5 5 Total contaminated blood cultures, n (% of N) 220 (24%) 145 (19) 125 (12%) 96 (9%) 182 (7%) 54 (8%) 90 (11%) 6 (1%) 49 (3%) 192 (17%) 8 (2%) 13 (5%) 16 (1%) Total positive blood cultures, n (% of N)\|\|\|\| 29 (3%) 31 (4) 30 (3%) 31 (3%) 175 (7%) 16 (2%) 26 (3%) 11 (1%) 30 (2%) 51 (5%) 17 (4%) 11 (4%) 110 (9%) Positive for malaria, n (% of all patients tested)* 430/908 (47%) 444/744 (60%) 206/525 (39%) 297/1058 (28%) 1139/2651 (43%) 254/632 (40%) 110/822 (13%) 19/955 (2%) 2/274 (1%) 0 4/406 (1%) 2/274 (1%) 226/956 (24%) UoO=University of Ouagadougou, Ouagadougou. BHP=Bandim Health Project, Bissau. IPD=Institute Pasteur de Dakar, Dakar. KCCR/BNITM=Kumasi Centre for Collaborative Research in Tropical Medicine, Kumasi/Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany. UoG=University of Gezira, Wad Medani. AHRI=Armauer Hansen Research Institute, Addis Ababa. UoA=University of Antananarivo, Antananarivo. NICD=National Institute for Communicable Diseases, Johannesburg. KCMC/Duke=Kilimanjaro Christian Medical Center, Moshi/Duke University Medical Center, Durham, NC, USA. KEMRI/US-CDC=Kenya Medical Research Institute/US Centers for Disease Control and Prevention, Nairobi. IPD=inpatient department. OPD=outpatient department. HDSS=Health and Demographic Surveillance System. KEMRI=Kenya Medical Research Institute. NA=not available. In Kibera, active population mobilisation was done in addition to passive surveillance. †Setting reﬂ ects the classiﬁ cation commonly used at each site and does not refer to a standard deﬁ nition. ‡Surveillance activities were scheduled for 12 months in Burkina Faso, Guinea-Bissau, Senegal, Sudan, Ethiopia, and Madagascar and for 24 months in Ghana, Kenya, South Africa, and Tanzania. If funds allowed, the scheduled period was extended. §Population data were provided from the HDSS country oﬃ ce. ¶Population data for Senegal and Madagascar were provided by Ministry of Health. Population data correspond to the 2012 population census and 2010 estimated population for the area, respectively. Discussion This study identiﬁ ed Salmonella as a major cause of invasive bacterial febrile illness across sub-Saharan Africa, aﬀ ecting children aged 2–14 years rather than adults, and arising in both high-population and low- population density settings. Other major causes of invasive bacterial febrile illnesses varied by country; E coli and S aureus were the most frequent non- Salmonella pathogens isolated from blood. y y p ) With the exception of East Wad Medani, Salmonella spp were isolated from the blood of febrile patients at all sites (135 S Typhi and 94 iNTS isolates), which accounted for 33% or more of all isolated bacteria in all but four sites (East Wad Medani, Pietermaritzburg, Butajira, and Isotry). Seasonal variation was not observed at any site (data not shown). The most common iNTS serovars were S enterica serotype Typhimurium (38 [40%] of 94), S enterica serotype Enteriditis (11 [12%] of 94), and S enterica serotype Dublin (10 [11%] of 94). The highest AIRs for typhoid fever in the 15 years or younger age group were observed in Polesgo, Kibera, and Asante Akim North (table 2). S Paratyphi A (three isolates) was isolated in Senegal only. p g Results from previous studies18,19 suggest that typhoid fever in some sub-Saharan Africa settings occurs predominately in urban settlements with high- population densities, and that disease incidence could have been overestimated by the use of the Widal test. Our study, done using a standardised protocol in both urban and rural settings, indicated high incidences of typhoid fever and iNTS in areas with high-population and low-population densities. Separate analyses done at the Ghana site conﬁ rmed this observation and revealed a higher disease incidence in children living in rural areas than in those living in urban areas.20 Furthermore, we observed variable incidences of typhoid fever and iNTS among neighbouring populations in Burkina Faso, and in the same populations in Kenya and Ghana in consecutive years, indicating a focal nature and a ﬂ uctuating burden of iNTS disease. Among age groups of children younger than 15 years, the highest AIR for typhoid fever was observed in children aged 2–4 years from Polesgo, Asante Akim North, Moshi Urban District, and Kibera, and in children aged 5–14 years from Kibera and Polesgo (table 2). Statistical analysis y We established a multicountry database using FoxPro software. We excluded patients from the analysis who were recruited during pilot testing, failed to meet inclusion criteria, or had incomplete laboratory results. We estimated incidences per 100 000 PYO. Conﬁ rmed invasive salmonella cases, stratiﬁ ed by age group (0–1 years, 2–4 years, 5–14 years, and ≥15 years), were adjusted by the speciﬁ c age-group recruitment proportion. We calculated this proportion by dividing the number of patients with complete data (numerator) by the total number of patients in the study area who had been diagnosed with a febrile illness at a recruitment facility during the surveillance period (denominator). We used health-care facility records, reviewed at the end of the surveillance activities, to estimate the number of patients diagnosed with a febrile illness. The catchment population in PYO, adjusted by health-care-seeking behaviour, was used as the denominator in crude and adjusted incidence rates (AIR). The 95% CI for AIR was derived on the log-scale and exponentiated. We used the error factor (exp[1·96/√adjusted cases]) to calculate the lower (adjusted rate/error factor) and upper (adjusted rate × error factor) 95% CIs. At the sites in Senegal, Ethiopia, and South Africa, incomplete health-care facility records did not allow for the estimation of the recruitment proportion and calculation of AIRs; for these sites we present crude rates. AIRs for typhoid fever and iNTS were assessed for all other sites. Diﬀ erences in proportions of blood cultures positive for a pathogen between study years were assessed with the χ² test (SAS, version 9.3). Table 1: Demographics and laboratory results of www.thelancet.com/lancetgh Vol 5 March 2017 e315 Articles Role of the funding source Role of the funding source adults (table 2), except for the sites in Pikine, Moshi Rural District, and Kibera. The AIR for iNTS among children aged 2–4 years was highest in Nioko II, Polesgo, and Asante Akim North. The AIR for iNTS in children younger than 15 years was less than 100 per 100 000 PYO in Kibera, Imerintsiatosika, and in both sites in Tanzania. No iNTS was isolated from sites in Sudan, South Africa, Ethiopia, and Isotry. The funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had ﬁ nal responsibility for the decision to submit for publication. Results Between March 1, 2010, and Jan 31, 2014, we recruited 13 558 patients from 13 sites who met the inclusion criteria and resided in the catchment areas (ﬁ gures 1, 2). We excluded data from 127 (1%) patients because of incomplete laboratory results; data from 13 431 patients were analysed, and 8582 patients (64%) were younger than 15 years (table 1). All patients had one blood culture sample analysed at recruitment and 11 421 (85%) were screened for malaria parasites (table 1). The proportion of contaminated blood cultures ranged from less than 1% in Imerintsiatosika to 24% in Nioko II. The proportion of blood cultures that yielded non- contaminant bacteria varied between sites, ranging from 1% in Imerintsiatosika to 9% in Kibera (table 1). In total, 568 non-contaminant bacteria were isolated from blood samples of febrile patients. The most frequent non-contaminant bacteria isolated were S Typhi (135 [24%]), NTS (94 [17%]), S aureus (70 [12%]), E coli (47 [8%]), and Streptococcus pneumoniae (43 [8%]). Of the sites with at least 2 years of surveillance (Asante Akim North, Kibera, and Pietermaritzburg), the proportion of blood cultures that were pathogen positive diﬀ ered signiﬁ cantly between study years in Kibera only (12% at year 1 and 5% at year 2; p<0·0001; χ² test). p y The antimicrobial susceptibility proﬁ les of S Typhi and iNTS isolates diﬀ ered between sites (table 3). Overall, 47% of S Typhi isolates and 48% of iNTS isolates were multidrug resistant. Most multidrug-resistant S Typhi isolates were obtained at the sites in Kenya, Ghana, and Tanzania (both sites combined). Multidrug- resistant iNTS isolates were isolated at the sites in Burkina Faso (both combined), Ghana, Guinea- Bissau, and Kenya (table 3). S Typhi isolates that had reduced ciproﬂ oxacin susceptibility were cultured in Kenya and South Africa, only; one ciproﬂ oxacin- resistant S Paratyphi A organism was isolated in Senegal. Ciproﬂ oxacin-resistant iNTS was similarly uncommon, isolated only in Burkino Faso (once at the Nioko II site) and in Ghana. One iNTS isolate in Kenya was resistant to ceftriaxone (table 3). www.thelancet.com/lancetgh Vol 5 March 2017 Discussion The AIR for typhoid fever in adults (aged ≥15 years) was less than 70 per 100 000 PYO at all sites except Moshi Urban District, Kibera, and Polesgo (table 2). A previous global estimate of the burden of typhoid fever indicated that south-central and east-central Asia had the highest incidences of typhoid fever with more than 100 cases per 100 000 people annually; Africa was estimated to have a medium incidence (10–100 cases per 100 000).1 The AIR for typhoid fever estimated in our ( ) iNTS organisms were more frequently isolated from infants (0–1 years) or children aged 2–4 years than from e316 Articles Articles Articles Proportion of individuals from study population visiting recruitment facility in case of fever (95% CI) PYO estimation Recruitment proportion Salmonella Typhi iNTS Study population Study population adjusted by health-seeking behaviour PYO Crude cases Crude incidence per 100 000 PYO Cases adjusted for recruitment Adjusted incidence per 100 000 PYO (95% CI) Crude cases Crude incidence per 100 000 PYO Cases adjusted for recruitment Adjusted incidence per 100 000 PYO (95% CI) Nioko II, Burkina Faso 0–1 years 81% (74–88) 2208 1788 2097 247/1297 (19%) 0 0 0·0 0 (0–0) 3 143 15·8 753 (460–1233) 2–4 years 81% (75–86) 1823 1477 2097 235/1259 (19%) 1 48 5·3 251 (107–590) 3 143 16·0 753 (460–1233) 5–14 years 81% (78–84) 4295 3479 4889 228/889 (26%) 4 82 15·4 315 (191–519) 3 61 12·0 236 (133–420) <15 years NA 8326 6744 9083 NA 5 55 20·6 227 (148–350) 9 99 43·1 475 (352–640) ≥15 years 81% (79–83) 9428 7637 10 676 208/759 (27%) 0 0 0·0 0 (0–0) 1 9 4·0 35 (13–96) All ·· 17 754 14 381 19 759 NA 5 25 20·6 104 (68–161) 10 51 46·8 237 (178–316) Polesgo Burkina Faso 0–1 years 46% (39–54) 10 852 4992 5198 206/631 (33%) 0 0 0·0 0 (0–0) 5 96 15·2 291 (176–482) 2–4 years 43% (37–48) 7307 3142 3866 175/359 (49%) 1 26 2·0 53 (13–208) 1 26 2·0 53 (13–208) 5–14 years 42% (41–48) 19 905 8360 11 101 187/380 (49%) 1 9 2·0 18 (5–72) 2 18 4·0 53 (14–97) <15 years NA 38 064 16 494 20 165 NA 2 10 4·1 20 (8–53) 8 40 21·3 116 (69–161) ≥15 years 45% (43–47) 62 694 28 212 37 109 105/163 (64%) 1 3 1·6 4 (1–20) 0 0 0·0 0 (0–0) All NA 100 758 44 706 57 274 NA 3 5 5·6 10 (4–22) 8 14 21·3 37 (24–57) (Table 2 continues on next page) (Table 2 continues on next page) ≥15 years 81% (79–83) 9428 7637 10 676 208/759 (27%) 0 0 0·0 0 (0–0) 1 9 4·0 35 (13–96) All ·· 17 754 14 381 19 759 NA 5 25 20·6 104 (68–161) 10 51 46·8 237 (178–316) Polesgo, Burkina Faso 0–1 years 92% (86–99) 896 824 929 117/475 (25% 0 0 0·0 0 (0–0) 1 108 4·0 431 (162–1147) 2–4 years 83% (76–89) 856 710 992 148/466 (32%) 6 605 18·8 1890 (1202–2972) 2 202 6·0 630 (288–1380) 5–14 years 87% (83–91) 1734 1509 2104 252/510 (49%) 5 238 10·2 485 (263–896) 0 0 0·0 0 (0–0) <15 years NA 3486 3043 4025 NA 11 273 29·0 719 (500–1035) 3 75 10·3 255 (138–470) ≥15 years 87% (84–89) 4088 3557 4917 239/629 (38%) 2 41 5·3 107 (46–252) 1 20 3·0 54 (16–179) All NA 7574 6600 8942 NA 13 145 34·2 383 (274–535) 4 45 12·9 144 (83–249) Bandim Guinea Bissau 0–1 years 92% (86–99) 896 824 929 117/475 (25% 0 0 0·0 0 (0–0) 1 108 4·0 431 (162–1147) 2–4 years 83% (76–89) 856 710 992 148/466 (32%) 6 605 18·8 1890 (1202–2972) 2 202 6·0 630 (288–1380) 5–14 years 87% (83–91) 1734 1509 2104 252/510 (49%) 5 238 10·2 485 (263–896) 0 0 0·0 0 (0–0) <15 years NA 3486 3043 4025 NA 11 273 29·0 719 (500–1035) 3 75 10·3 255 (138–470) ≥15 years 87% (84–89) 4088 3557 4917 239/629 (38%) 2 41 5·3 107 (46–252) 1 20 3·0 54 (16–179) All NA 7574 6600 8942 NA 13 145 34·2 383 (274–535) 4 45 12·9 144 (83–249) Polesgo, Burkina Faso 0–1 years 92% (86–99) 896 824 929 117/475 (25% 0 0 0·0 0 (0–0) 1 108 4·0 431 (162–1147) 2–4 years 83% (76–89) 856 710 992 148/466 (32%) 6 605 18·8 1890 (1202–2972) 2 202 6·0 630 (288–1380) 5–14 years 87% (83–91) 1734 1509 2104 252/510 (49%) 5 238 10·2 485 (263–896) 0 0 0·0 0 (0–0) <15 years NA 3486 3043 4025 NA 11 273 29·0 719 (500–1035) 3 75 10·3 255 (138–470) ≥15 years 87% (84–89) 4088 3557 4917 239/629 (38%) 2 41 5·3 107 (46–252) 1 20 3·0 54 (16–179) All NA 7574 6600 8942 NA 13 145 34·2 383 (274–535) 4 45 12·9 144 (83–249) Bandim, Guinea-Bissau 0–1 years 46% (39–54) 10 852 4992 5198 206/631 (33%) 0 0 0·0 0 (0–0) 5 96 15·2 291 (176–482) 2–4 years 43% (37–48) 7307 3142 3866 175/359 (49%) 1 26 2·0 53 (13–208) 1 26 2·0 53 (13–208) 5–14 years 42% (41–48) 19 905 8360 11 101 187/380 (49%) 1 9 2·0 18 (5–72) 2 18 4·0 53 (14–97) <15 years NA 38 064 16 494 20 165 NA 2 10 4·1 20 (8–53) 8 40 21·3 116 (69–161) ≥15 years 45% (43–47) 62 694 28 212 37 109 105/163 (64%) 1 3 1·6 4 (1–20) 0 0 0·0 0 (0–0) All NA 100 758 44 706 57 274 NA 3 5 5·6 10 (4–22) 8 14 21·3 37 (24–57) (Table 2 continues on next page) case of fever (95% CI) Study population Study population adjusted by health-seeking behaviour PYO Crude cases Crude incidence per 100 000 PYO Cases adjusted for recruitment Adjusted incidence per 100 000 PYO (95% CI) Crude cases Crude incidence per 100 000 PYO Cases adjusted for recruitment Adjusted incidence per 100 000 PYO (95% CI) Nioko II, Burkina Faso 0–1 years 81% (74–88) 2208 1788 2097 247/1297 (19%) 0 0 0·0 0 (0–0) 3 143 15·8 753 (460–1233) 2–4 years 81% (75–86) 1823 1477 2097 235/1259 (19%) 1 48 5·3 251 (107–590) 3 143 16·0 753 (460–1233) 5–14 years 81% (78–84) 4295 3479 4889 228/889 (26%) 4 82 15·4 315 (191–519) 3 61 12·0 236 (133–420) <15 years NA 8326 6744 9083 NA 5 55 20·6 227 (148–350) 9 99 43·1 475 (352–640) ≥15 years 81% (79–83) 9428 7637 10 676 208/759 (27%) 0 0 0·0 0 (0–0) 1 9 4·0 35 (13–96) All ·· 17 754 14 381 19 759 NA 5 25 20·6 104 (68–161) 10 51 46·8 237 (178–316) Polesgo, Burkina Faso 0–1 years 92% (86–99) 896 824 929 117/475 (25% 0 0 0·0 0 (0–0) 1 108 4·0 431 (162–1147) 2–4 years 83% (76–89) 856 710 992 148/466 (32%) 6 605 18·8 1890 (1202–2972) 2 202 6·0 630 (288–1380) 5–14 years 87% (83–91) 1734 1509 2104 252/510 (49%) 5 238 10·2 485 (263–896) 0 0 0·0 0 (0–0) <15 years NA 3486 3043 4025 NA 11 273 29·0 719 (500–1035) 3 75 10·3 255 (138–470) ≥15 years 87% (84–89) 4088 3557 4917 239/629 (38%) 2 41 5·3 107 (46–252) 1 20 3·0 54 (16–179) All NA 7574 6600 8942 NA 13 145 34·2 383 (274–535) 4 45 12·9 144 (83–249) Bandim Guinea Bissau e317 www.thelancet.com/lancetgh Vol 5 March 2017 Articles Proportion of individuals from study population visiting recruitment facility in case of fever (95% CI) PYO estimation Recruitment proportion Salmonella Typhi iNTS Study population Study population adjusted by health-seeking behaviour PYO Crude cases Crude incidence per 100 000 PYO Cases adjusted for recruitment Adjusted incidence per 100 000 PYO (95% CI) Crude cases Crude incidence per 100 000 PYO Cases adjusted for recruitment Adjusted incidence per 100 000 PYO (95% CI) (Continued from previous page) Asante Akim North, Ghana 0–1 years 16% (14–18) 11 222 1760 4080 41% 2 49 4·9 120 (49–290) 29 711 70·7 1733 (1373–2188) 2–4 years 16% (13–18) 8086 1268 2940 41%* 13 442 31·7 1079 (762–1528) 23 782 56·1 1908 (1469–2479) 5–14 years 16% (15–17) 34 439 5415 12 554 623/1657 (38%) 15 119 39·5 314 (230–430) 7 56 18·4 147 (93–232) <15 years NA 53 747 8443 19 574 NA 30 153 76·1 389 (310–486) 59 301 145·3 742 (631–873) ≥15 years NA NA† NA NA NA NA† NA NA NA NA† NA NA NA All NA NA† NA NA NA NA† NA NA NA NA† NA NA NA Pikine, Senegal‡§ 0–1 years 39% (32–46) 20 120 7837 11 194 NA 0 0 NA NA 0 0 NA NA 2–4 years 37% (33–41) 30 180 11 097 15 851 NA 0 0 NA NA 0 0 ·· NA 5–14 years 31% (28–34) 96 152 29 807 42 577 NA 3 7 NA NA 1 5 ·· NA <15 years NA 146 452 48 741 69 623 NA 3 4 NA NA 0 0 ·· NA ≥15 years 30% (28–31) 195 726 58 718 83 874 NA 4 5 NA NA 3 6 ·· NA All NA 342 178 107 459 153 496 NA 7 5 NA NA 4 5 ·· NA East Wad Medani, Sudan§ 0–1 years 23% (14–32) 2377 537 589 2/85 (2%) 0 0 0·0 0 (0–0) 0 0 0·0 0 (0–0) 2–4 years 22% (15–29) 3566 781 857 29/108 (27%) 0 0 0·0 0 (0–0) 0 0 0·0 0 (0–0) 5–14 years 25% (21–28) 11 071 2735 2999 160/234 (68%) 0 0 0·0 0 (0–0) 0 0 0·0 0 (0–0) <15 years NA 17 014 4053 4445 NA 0 0 0·0 0 (0–0) 0 0 0·0 0 (0–0) ≥15 years 29% (27–31) 29 843 8684 9525 131/147 (89%) 0 0 0·0 0 (0–0) 0 0 0·0 0 (0–0) All NA 46 857 12 737 13 970 NA 0 0 0·0 0 (0–0) 0 0 0·0 0 (0–0) Butajira, Ethiopia§ 0–1 years 69% (59–78) 2266 1563 2798 NA 0 0 NA NA 0 0 NA NA 2–4 years 62% (55–69) 3398 2107 3771 NA 0 0 NA NA 0 0 NA NA 5–14 years 65% (61–69) 14 015 9110 16 305 NA 1 6 NA NA 0 0 NA NA <15 years NA 19 679 12 780 22 874 NA 1 4 NA NA 0 0 NA NA ≥15 years 65% (62–68) 42 545 28 080 50 257 NA 2 4 NA NA 0 0 NA NA All NA 62 224 40 860 73 131 NA 3 4 NA NA 0 0 NA NA (Table 2 continues on next page) Proportion of individuals from study population visiting recruitment facility in case of fever (95% CI) PYO estimation Recruitment proportion Salmonella Typhi iNTS Study population Study population adjusted by health-seeking behaviour PYO Crude cases Crude incidence per 100 000 PYO Cases adjusted for recruitment Adjusted incidence per 100 000 PYO (95% CI) Crude cases Crude incidence per 100 000 PYO Cases adjusted for recruitment Adjusted incidence per 100 000 PYO (95% CI) (Continued from previous page) Asante Akim North, Ghana 0–1 years 16% (14–18) 11 222 1760 4080 41%* 2 49 4·9 120 (49–290) 29 711 70·7 1733 (1373–2188) 2–4 years 16% (13–18) 8086 1268 2940 41%* 13 442 31·7 1079 (762–1528) 23 782 56·1 1908 (1469–2479) 5–14 years 16% (15–17) 34 439 5415 12 554 623/1657 (38%) 15 119 39·5 314 (230–430) 7 56 18·4 147 (93–232) <15 years NA 53 747 8443 19 574 NA 30 153 76·1 389 (310–486) 59 301 145·3 742 (631–873) ≥15 years NA NA† NA NA NA NA† NA NA NA NA† NA NA NA All NA NA† NA NA NA NA† NA NA NA NA† NA NA NA Pikine, Senegal‡§ 0–1 years 39% (32–46) 20 120 7837 11 194 NA 0 0 NA NA 0 0 NA NA 2–4 years 37% (33–41) 30 180 11 097 15 851 NA 0 0 NA NA 0 0 ·· NA 5–14 years 31% (28–34) 96 152 29 807 42 577 NA 3 7 NA NA 1 5 ·· NA <15 years NA 146 452 48 741 69 623 NA 3 4 NA NA 0 0 ·· NA ≥15 years 30% (28–31) 195 726 58 718 83 874 NA 4 5 NA NA 3 6 ·· NA All NA 342 178 107 459 153 496 NA 7 5 NA NA 4 5 ·· NA East Wad Medani, Sudan§ 0–1 years 23% (14–32) 2377 537 589 2/85 (2%) 0 0 0·0 0 (0–0) 0 0 0·0 0 (0–0) 2–4 years 22% (15–29) 3566 781 857 29/108 (27%) 0 0 0·0 0 (0–0) 0 0 0·0 0 (0–0) Proportion of individuals from study population visiting recruitment facility in case of fever (95% CI) PYO estimation Recruitment proportion Salmonella Typhi iNTS Study population Study population adjusted by health-seeking behaviour PYO Crude cases Crude incidence per 100 000 PYO Cases adjusted for recruitment Adjusted incidence per 100 000 PYO (95% CI) Crude cases Crude incidence per 100 000 PYO Cases adjusted for recruitment Adjusted incidence per 100 000 PYO (95% CI) (Continued from previous page) Asante Akim North, Ghana 0–1 years 16% (14–18) 11 222 1760 4080 41%* 2 49 4·9 120 (49–290) 29 711 70·7 1733 (1373–2188) 2–4 years 16% (13–18) 8086 1268 2940 41%* 13 442 31·7 1079 (762–1528) 23 782 56·1 1908 (1469–2479) 5–14 years 16% (15–17) 34 439 5415 12 554 623/1657 (38%) 15 119 39·5 314 (230–430) 7 56 18·4 147 (93–232) <15 years NA 53 747 8443 19 574 NA 30 153 76·1 389 (310–486) 59 301 145·3 742 (631–873) ≥15 years NA NA† NA NA NA NA† NA NA NA NA† NA NA NA All NA NA† NA NA NA NA† NA NA NA NA† NA NA NA Pikine Senegal‡§ East Wad Medani, Sudan§ 0–1 years 23% (14–32) 2377 537 589 2/85 (2%) 0 0 0·0 0 (0–0) 0 0 0·0 0 (0–0) 2–4 years 22% (15–29) 3566 781 857 29/108 (27%) 0 0 0·0 0 (0–0) 0 0 0·0 0 (0–0) 5–14 years 25% (21–28) 11 071 2735 2999 160/234 (68%) 0 0 0·0 0 (0–0) 0 0 0·0 0 (0–0) <15 years NA 17 014 4053 4445 NA 0 0 0·0 0 (0–0) 0 0 0·0 0 (0–0) ≥15 years 29% (27–31) 29 843 8684 9525 131/147 (89%) 0 0 0·0 0 (0–0) 0 0 0·0 0 (0–0) All NA 46 857 12 737 13 970 NA 0 0 0·0 0 (0–0) 0 0 0·0 0 (0–0) Butajira, Ethiopia§ 0–1 years 69% (59–78) 2266 1563 2798 NA 0 0 NA NA 0 0 NA NA 2–4 years 62% (55–69) 3398 2107 3771 NA 0 0 NA NA 0 0 NA NA 5–14 years 65% (61–69) 14 015 9110 16 305 NA 1 6 NA NA 0 0 NA NA <15 years NA 19 679 12 780 22 874 NA 1 4 NA NA 0 0 NA NA ≥15 years 65% (62–68) 42 545 28 080 50 257 NA 2 4 NA NA 0 0 NA NA All NA 62 224 40 860 73 131 NA 3 4 NA NA 0 0 NA NA (Table 2 continues on next page) (Table 2 continues on next page) iNTS e318 www.thelancet.com/lancetgh Vol 5 March 2017 Articles Articles Articles Proportion of individuals from study population visiting recruitment facility in case of fever (95% CI) PYO estimation Recruitment proportion Salmonella Typhi iNTS Study population Study population adjusted by health-seeking behaviour PYO Crude cases Crude incidence per 100 000 PYO Cases adjusted for recruitment Adjusted incidence per 100 000 PYO (95% CI) Crude cases Crude incidence per 100 000 PYO Cases adjusted for recruitment Adjusted incidence per 100 000 PYO (95% CI) (Continued from previous page) Moshi Rural District, Tanzania 0–1 years 4% (0–11)¶ 24 289 390 693 79%* 0 0 0·0 0 (0–0) 0 0 0·0 0 (0–0) 2–4 years 2% (0–4)\|\| 25 281 406 721 79%* 0 0 0·0 0 (0–0) 0 0 0·0 0 (0–0) 5–14 years 13% (10–16) 118 219 15 487 27 508 79%* 2 (4) 15 5·1 18 (8–44) 0 0 0·0 0 (0–0) <15 years NA 167 789 16 283 28 922 NA 2 (4) 14 5·1 18 (7–42) 0 0 0·0 0 (0–0) ≥15 years 2% (1–2) 298 948 5172 9186 79%* 1 (2) 22 2·5 28 (8–95) 1 (2) 22 2·5 28 (8–95) All NA 466 737 21 454 38 108 NA 3 (6) 16 7·6 20 (10–41) 1 (2) 5 2·5 7 (2–23) Moshi Urban District, Tanzania 0–1 years 7% (0–19)¶ 10 406 335 595 79%* 0 0 0·0 0 (0–0) 1 (2)** 336 2·5 427 (125–1461) 2–4 years 2% (0–6)\|\| 10 831 348 618 79%* 1 (5)** 809 6·4 1028 (472–2237) 0 0 0·0 0 (0–0) 5–14 years 13% (8–19) 37 309 4850 8615 79%* 2 (7) 81 8·9 103 (54–199) 0 0 0·0 0 (0–0) <15 years NA 58 546 5533 9828 NA 3 (12) 122 15·2 155 (94–256) 1 (2)** 20 2·5 26 (8–88) Proportion of individuals from study population visiting recruitment facility in case of fever (95% CI) PYO estimation Recruitment proportion Salmonella Typhi iNTS Study population Study population adjusted by health-seeking behaviour PYO Crude cases Crude incidence per 100 000 PYO Cases adjusted for recruitment Adjusted incidence per 100 000 PYO (95% CI) Crude cases Crude incidence per 100 000 PYO Cases adjusted for recruitment Adjusted incidence per 100 000 PYO (95% CI) (Continued from previous page) Moshi Rural District, Tanzania 0–1 years 4% (0–11)¶ 24 289 390 693 79%* 0 0 0·0 0 (0–0) 0 0 0·0 0 (0–0) 2–4 years 2% (0–4)\|\| 25 281 406 721 79%* 0 0 0·0 0 (0–0) 0 0 0·0 0 (0–0) 5–14 years 13% (10–16) 118 219 15 487 27 508 79%* 2 (4) 15 5·1 18 (8–44) 0 0 0·0 0 (0–0) <15 years NA 167 789 16 283 28 922 NA 2 (4) 14 5·1 18 (7–42) 0 0 0·0 0 (0–0) ≥15 years 2% (1–2) 298 948 5172 9186 79%* 1 (2) 22 2·5 28 (8–95) 1 (2) 22 2·5 28 (8–95) All NA 466 737 21 454 38 108 NA 3 (6) 16 7·6 20 (10–41) 1 (2) 5 2·5 7 (2–23) Moshi Urban District, Tanzania 0–1 years 7% (0–19)¶ 10 406 335 595 79%* 0 0 0·0 0 (0–0) 1 (2)** 336 2·5 427 (125–1461) 2–4 years 2% (0–6)\|\| 10 831 348 618 79%* 1 (5)** 809 6·4 1028 (472–2237) 0 0 0·0 0 (0–0) 5–14 years 13% (8–19) 37 309 4850 8615 79%* 2 (7) 81 8·9 103 (54–199) 0 0 0·0 0 (0–0) <15 years NA 58 546 5533 9828 NA 3 (12) 122 15·2 155 (94–256) 1 (2)** 20 2·5 26 (8–88) ≥15 years 2% (0–3) 125 746 2138 3796 79%* 3 (6) 158 7·6 201 (99–408) 0 0 0·0 0 (0–0) All NA 184 292 7671 13 626 NA 6 (18) 132 22·9 168 (111–253) 1 (2)** 15 2·5 19 (5–64) Kibera, Kenya†† 0–1 years 42% (38–47) 3467 1456 2031 99/99 (100%) 3 148 3·0 148 (48–458) 1 49 1·0 49 (7–350) 2–4 years 39% (36–43) 3070 1197 2039 312/312 (100%) 10 490 10·0 490 (264–912) 1 49 1·0 49 (7–348) 5–14 years 43% (39–47) 7514 3231 5722 539/539 (100%) 28 489 28·0 489 (338–709) 1 17 1·0 17 (2–124) <15 years NA 14 051 5884 9792 NA 41 419 41·0 419 (308–569) 3 31 3·0 31 (10–95) ≥15 years 35% (32–38) 15 263 5342 9228 301/301 (100%) 13 141 13·0 141 (82–243) 3 33 3·0 33 (10–101) All NA 29 314 11 227 19 020 NA 54 284 54·0 284 (217–371) 6 32 6·0 32 (14–70) Imerintsiatosika, Madagascar 0–1 years 28% (20–37) 3424 753 1287 66/85 (78%) 0 0 0·0 0 (0–0) 1 78 1·3 100 (18–562) 2–4 years 19% (14–25) 5136 1130 1932 87/101 (86%) 0 0 0·0 0 (0–0) 0 0 0·0 0 (0–0) 5–14 years 18% (15–20) 13 188 2374 4057 184/256 (72%) 5 123 6·9 171 (81–360) 0 0 0·0 0 (0–0) <15 years NA 21 748 4257 7276 NA 5 69 6·9 95 (45–201) 1 14 1·3 18 (3–99) ≥15 years 17% (15–19) 24 632 4187 7153 639/919 (70%) 1 14 1·4 20 (4–103) 0 0 0·0 0 (0–0) All NA 46 380 8444 14 429 NA 6 42 8·4 58 (29–114) 1 7 1·3 9 (2–50) (Table 2 continues on next page) (Table 2 continues on next page) e319 www.thelancet.com/lancetgh Vol 5 March 2017 Articles Proportion of individuals from study population visiting recruitment facility in case of fever (95% CI) PYO estimation Recruitment proportion Salmonella Typhi iNTS Study population Study population adjusted by health-seeking behaviour PYO Crude cases Crude incidence per 100 000 PYO Cases adjusted for recruitment Adjusted incidence per 100 000 PYO (95% CI) Crude cases Crude incidence per 100 000 PYO Cases adjusted for recruitment Adjusted incidence per 100 000 PYO (95% CI) (Continued from previous page) Isotry, Madagasar 0–1 years 6% (1–12) 3204 192 261 12/14 (86%) 0 0 0·0 0 (0–0) 0 0 0·0 0 (0–0) 2–4 years 10% (5–14) 4805 481 653 58/65 (89%) 0 0 0·0 0 (0–0) 0 0 0·0 0 (0–0) 5–14 years 9% (7–11) 16 386 1475 2005 234/288 (81%) 1 50 1·2 62 (11–359) 0 0 0·0 0 (0–0) <15 years NA 24 395 2147 2919 NA 1 34 1·2 42 (7–247) 0 0 0·0 0 (0–0) ≥15 years 9% (7–11) 45 928 4134 5621 1197/1421 (84%) 2 36 2·4 42 (12–151) 0 0 0·0 0 (0–0) All NA 70 323 6281 8540 NA 3 35 3·6 42 (15–119) 0 0 0·0 0 (0–0) Proportion of individuals from study population visiting recruitment facility in case of fever (95% CI) PYO estimation Recruitment proportion Salmonella Typhi iNTS Study population Study population adjusted by health-seeking behaviour PYO Crude cases Crude incidence per 100 000 PYO Cases adjusted for recruitment Adjusted incidence per 100 000 PYO (95% CI) Crude cases Crude incidence per 100 000 PYO Cases adjusted for recruitment Adjusted incidence per 100 000 PYO (95% CI) (Continued from previous page) Isotry, Madagasar 0–1 years 6% (1–12) 3204 192 261 12/14 (86%) 0 0 0·0 0 (0–0) 0 0 0·0 0 (0–0) 2–4 years 10% (5–14) 4805 481 653 58/65 (89%) 0 0 0·0 0 (0–0) 0 0 0·0 0 (0–0) 5–14 years 9% (7–11) 16 386 1475 2005 234/288 (81%) 1 50 1·2 62 (11–359) 0 0 0·0 0 (0–0) <15 years NA 24 395 2147 2919 NA 1 34 1·2 42 (7–247) 0 0 0·0 0 (0–0) ≥15 years 9% (7–11) 45 928 4134 5621 1197/1421 (84%) 2 36 2·4 42 (12–151) 0 0 0·0 0 (0–0) All NA 70 323 6281 8540 NA 3 35 3·6 42 (15–119) 0 0 0·0 0 (0–0) Pietermaritzburg, South Africa§ 0–1 years 11% (5–17) 13 990 1511 3055 NA 0 0 NA NA 0 0 NA NA 2–4 years 7% (3–12) 20 985 1490 3013 NA 0 0 NA NA 0 0 NA NA 5–14 years 16% (13–19) 62 313 10 157 20 537 NA 0 0 NA NA 0 0 NA NA <15 years NA 97 288 13 158 26 605 NA 0 0 NA NA 0 0 NA NA ≥15 years 15% (13–17) 294 542 43 887 88 739 NA 2 2 NA NA 0 0 NA NA All NA 391 830 57 045 115 344 NA 2 2 NA NA 0 0 NA NA Study population was adjusted for health-seeking behaviour and crude cases were adjusted for recruitment proportion (number of patients analysed divided by number of patients with febrile illness from study area who visited a recruitment health facility, multiplied by 100). Articles Multidrug-resistant iNTS isolates were isolated at several sites and have been isolated in sub-Saharan Africa previously.18,25,26 Furthermore, a single iNTS isolate from Kibera showed resistance to ceftriaxone. Genomic analyses27 have described the spread of S Typhi haplotype H58 into Africa, a multidrug- resistant strain associated with reduced ciproﬂ oxacin susceptibility. The susceptibility patterns observed in our study are concerning, particularly because some anti microbial-resistant S Typhi can have a selective ﬁ tness advantage.28 Concerted measures are needed to monitor the emergence of ﬂ uoroquinolone-resistant Salmonella.29–32 study reveals a higher burden than previously estimated.1 Four sites had an overall AIR for typhoid fever of more than 100 per 100 000 PYO, ﬁ ve sites had an AIR for typhoid fever of more than 100 per 100 000 PYO in children younger than 15 years, and six sites had an AIR for typhoid fever of more than 100 per 100 000 PYO in at least one age group. Similar to the Diseases of the Most Impoverished programme done in Asia,21 our results show that children aged 2–14 years bear the greatest burden of typhoid fever. Notably, our data indicate that the AIR for typhoid fever at TSAP sites was equal to or even greater than incidences reported in ﬁ ve Asian countries in the early 2000s.21,22 study reveals a higher burden than previously estimated.1 Four sites had an overall AIR for typhoid fever of more than 100 per 100 000 PYO, ﬁ ve sites had an AIR for typhoid fever of more than 100 per 100 000 PYO in children younger than 15 years, and six sites had an AIR for typhoid fever of more than 100 per 100 000 PYO in at least one age group. Similar to the Diseases of the Most Impoverished programme done in Asia,21 our results show that children aged 2–14 years bear the greatest burden of typhoid fever. Articles Burkina Faso Guinea-Bissau Senegal* Ghana Ethiopia Madagascar South Africa Tanzania Kenya All Total S Typhi isolates, N 18 3 7 30 3 9 2 9 54 135 Isolate with antimicrobial resistance, n (%)† Ampicillin 0 NR NR 20 (67%) 2 (67%) NR 0 8 (89%) 41 (76%) 71 (53%) Amoxicillin-clavulanic acid 0 NR NR 3 (10%) 0 NR 0 4 (44%) 24 (44%) 31 (23%) Chloramphenicol 2 (11%) NR NR 23 (77%) 0 NR 0 5 (56%) 43 (80%) 73 (54%) Co-trimoxazole 2 (11%) NR NR 24 (80%) 0 NR 0 8 (89%) 43 (80%) 77 (57%) Ceftriaxone 0 NR NR 0 0 NR 0 0 0 0 Ciproﬂ oxacin 0 NR NR 0 0 NR 1 (50%) 0 11 (20%) 12 (9%) Multidrug resistance‡ 0 NR NR 19 (63%) 0 NR 0 5 (56%) 40 (74%) 64 (47%) Total iNTS isolates, N 14 8 4 59 0 1 0 2 6 94 Isolate with antimicrobial resistance, n (%)† Ampicillin 10 (71%) 1 (13%) NR 38 (64%) NR NR NR 0 2 (33%) 51 (54%) Amoxicillin-clavulanic acid 3 (21%) 0 NR 9 (15%) NR NR NR 0 2 (33%) 14 (15%) Chloramphenicol 12 (86%) 1 (13%) NR 34 (58%) NR NR NR 0 1 (17%) 48 (51%) Co-trimoxazole 13 (93%) 1 (13%) NR 34 (58%) NR NR NR 0 2 (33%) 50 (53%) Ceftriaxone 0 0 NR 0 NR NR NR 0 1 (17%) 1 (1%) Ciproﬂ oxacin 1 (7%) 0 NR 2 (3%) NR NR NR 0 0 3 (3%) Multidrug resistance‡ 10 (71%) 1 (13%) NR 33 (56%) NR NR NR 0 1 (17%) 45 (48%) Resistant isolates are reported per country, rather than per site. No Salmonella enterica serotype Typhi (S Typhi) or iNTS isolates were cultured in Sudan. iNTS=invasive non-typhoidal salmonella. NR=no resistant isolates identiﬁ ed. Seven S Typhi, four iNTS, and three S enterica serotype Paratyphi (S Paratyphi) isolates. One of the S Paratyphi isolates was resistant to ciproﬂ oxacin. †Includes isolates fully and intermediately resistant against the respective drug, as deﬁ ned by the Clinical Laboratory and Standards Institute guidelines 2013.15 ‡Deﬁ ned as resistance against ampicillin or amoxicillin AND chloramphenicol AND co-trimoxazole. Table 3: Antimicrobial resistance patterns of Salmonella enterica serotype Typhi and iNTS isolates across sites Pietermaritzburg. www.thelancet.com/lancetgh Vol 5 March 2017 (Table 2 continues on next page) iNTS=invasive non-typhoidal salmonella. NA=not available. PYO=person-years of observation. Recruitment portion was not available for each age strata. Broader values were applied to each stratum. †Target population for surveillance activities in Ghana included patients younger than 15 years of age; patients aged 15 years or older were not recruited. ‡Three Salmonella Paratyphi A were identiﬁ ed at this site, but are not included in this table. §No salmonella was isolated in Sudan. Missing data on recruitment patterns in Senegal, Ethiopia, and South Africa did not allow calculation of adjusted incidences. Crude rates are presented. ¶This proportion applies to age group <1 year, and it was used to adjust the study population by health-seeking behaviour. \|\|This proportion applies to age group 1–4 years, and it was used to adjust the study population by health-seeking behaviour. The adjusted populations in age groups <1 year and 1–4 years were added to estimate the total adjusted population age group 0–4 years. Subsequently, the percentage of children <2 years reported by the 2012 national census was applied to derive age groups 0–1 years and 2–4 years. *Crude cases have been adjusted for recruitment pattern unique to the site in Tanzania: before Nov 11, 2011, every ﬁ fth eligible patient was recruited; from Nov 11, 2011, every second eligible patient was recruited. Adjusted cases (presented inside parentheses) were used to calculate crude rate. ††Active population mobilisation was done, in addition to passive surveillance. e320 www.thelancet.com/lancetgh Vol 5 March 2017 Articles Resistant isolates are reported per country, rather than per site. No Salmonella enterica serotype Typhi (S Typhi) or iNTS isolates were cultured in Sudan. iNTS=invasive non-typhoidal salmonella. NR=no resistant isolates identiﬁ ed. Seven S Typhi, four iNTS, and three S enterica serotype Paratyphi (S Paratyphi) isolates. One of the S Paratyphi isolates was resistant to ciproﬂ oxacin. †Includes isolates fully and intermediately resistant against the respective drug, as deﬁ ned by the Clinical Laboratory and Standards Institute guidelines 2013.15 ‡Deﬁ ned as resistance against ampicillin or amoxicillin AND chloramphenicol AND co-trimoxazole. Articles Fourth, the proportion of the catchment population using the TSAP health-care facilities for febrile illness was low in some sites, and antimicrobial treatment before blood collection and its potential eﬀ ect on blood culture sensitivity were not assessed. Fifth, the classiﬁ cation of the settings as either urban, rural, semi-urban, or urban-slum reﬂ ects the classiﬁ cation commonly used at each site and does not refer to a standard deﬁ nition; instead, the population density of each site is presented to make setting comparisons. Sixth, sites with no previous experience of blood collection for blood culture assessment had a higher incidence of contamination than sites with previous experience of blood collection (South Africa, Ghana, Tanzania, and Kenya); these incidences might have led to errors in clinical interpretation and uncertainty to distinguish between clinically signiﬁ cant bacteraemia and contamination. Available isolates and blood samples collected from participants were PCR tested at the reference lab to minimise misclassiﬁ cation of isolated organisms. Seventh, the site in Ghana recruited only children younger than 15 years and the proportion of recruited inpatients varied greatly across all sites. Finally, data on disease severity, complications, mortality, and HIV status were not assessed because these were not primary study objectives. Despite these limitations, this multisite study, the largest study of typhoid fever and iNTS done across sub-Saharan Africa to date, provides the most current and accurate incidence ﬁ gures for these major infectious diseases across the continent and has substantial implications for their control. disease. Third, given the vast number of patients (and restricted diagnostics capacity), not every patient with a history of fever was enrolled—eg, at sites where inpatients were recruited, patients with a fever for 72 h or longer were excluded to minimise the inclusion of patients pretreated with antimicrobials and to maximise blood culture yield. Fourth, the proportion of the catchment population using the TSAP health-care facilities for febrile illness was low in some sites, and antimicrobial treatment before blood collection and its potential eﬀ ect on blood culture sensitivity were not assessed. Fifth, the classiﬁ cation of the settings as either urban, rural, semi-urban, or urban-slum reﬂ ects the classiﬁ cation commonly used at each site and does not refer to a standard deﬁ nition; instead, the population density of each site is presented to make setting comparisons. Contributors FM and TFW contributed to study conception and design, analysis of data, interpretation of results, and drafting and editing of the paper. FK, JM, UP, VvK, EDM, and JDC contributed to study conception and design, data interpretation, and editing of the paper. MA, GDP, LMCE, VvK, and JKP contributed to data analysis. KT and BL contributed to study conception and design, data acquisition in the ﬁ eld, interpretation of the results, and editing of the paper. VvK, LMCE, SEP, CGM, CN, and JI drafted the manuscript and contributed to interpretation of results and editing of the paper. RFB, MA, FK, JM, UP, TFW, VvK, PA, YA-S, AA, MB-A, JAC, LMCE, JFD, NG, JTH, JI, HJJ, KHK, JMM, RK, RR, AGS, SEP, HJS, AS, MT, MRW, BY, MAET, HMB, LC, AJ, SVL, TMR, NS, and AT contributed to data acquisition in the ﬁ eld, interpretation of results, and editing of the paper. SB, JIC, UP, DMD, BSF, LPK, AAN, NVMH, BO, HR, TJLR, ES, HS-G, and AS contributed to laboratory work, interpretation of results, and editing of the paper. All authors read and approved the ﬁ nal draft. We surmise that the incidence of invasive salmonella infections among children in sub-Saharan Africa is much higher than previously estimated, underscoring the need for preventive measures. Therefore, until access to safe drinking water and improved sanitation is greatly expanded, the prevention of typhoid fever will require immunisation and eﬀ ective treatment options.38 The advent of new typhoid fever conjugate vaccines might provide more powerful tools for disease control; the ﬁ rst typhoid fever conjugate vaccine (Bharat Biotech, Hyderabad) has been submitted to WHO for prequaliﬁ cation. Data from TSAP will be incorporated into the GAVI Alliances’ review of potential subsidies for typhoid fever vaccines in 2017; their recommendation will be crucial for deployment of these vaccines. Hence, the need to understand the pragmatic aspects of vaccine targeting and delivery is pressing, particularly given the burden of disease in children, the associated risk factors, and the focal and unpredictable nature of the disease. Articles Sixth, sites with no previous experience of blood collection for blood culture assessment had a higher incidence of contamination than sites with previous experience of blood collection (South Africa, Ghana, Tanzania, and Kenya); these incidences might have led to errors in clinical interpretation and uncertainty to distinguish between clinically signiﬁ cant bacteraemia and contamination. Available isolates and blood samples collected from participants were PCR tested at the reference lab to minimise misclassiﬁ cation of isolated organisms. Seventh, the site in Ghana recruited only children younger than 15 years and the proportion of recruited inpatients varied greatly across all sites. Finally, data on disease severity, complications, mortality, and HIV status were not assessed because these were not primary study objectives. Despite these limitations, this multisite study, the largest study of typhoid fever and iNTS done across sub-Saharan Africa to date, provides the most current and accurate incidence ﬁ gures for these major infectious diseases across the continent and has substantial implications for their control. Similarly, in the absence of vaccines targeting iNTS disease, prevention will require a major investment in infrastructure for diagnosis and eﬀ ective treatment of iNTS disease. When appropriate diagnosis and treatment are available, the use of eﬀ ective antimicrobials might be impaired by the presence and potential increase of multidrug-resistant salmonella. Further assessment of incidences in infants (0–5 months vs 6–11 months) and data on severe typhoid fever or iNTS, including mortality, is crucial to determine the potential eﬀ ect of future vaccines. We are currently undertaking a follow-on study—Severe Typhoid in Africa (SETA)—which investigates severe typhoid burden. g y We conclude that typhoid fever and iNTS disease are major agents of invasive bloodstream infections in urban and rural locations, aﬀ ecting children more commonly than adults across sub-Saharan Africa. Immunisation of high-risk age groups with existing and new vaccines should be a priority. The next generation of epidemiological studies in sub-Saharan Africa needs to provide better data regarding the severity and mortality of typhoid fever and iNTS to guide the introduction of new typhoid and iNTS vaccines. Lastly, the accelerated development and introduction of iNTS vaccines needs to become a fundamental goal on the global health agenda. www.thelancet.com/lancetgh Vol 5 March 2017 Articles Notably, our data indicate that the AIR for typhoid fever at TSAP sites was equal to or even greater than incidences reported in ﬁ ve Asian countries in the early 2000s.21,22 For iNTS disease, we observed an AIR equal or higher than previously estimated and a bimodal age distribution with very young children and adults being the key age group for symptomatic infection.2 This age distribution diﬀ ered from that observed for typhoid fever, in which children aged 2–14 years were the most aﬀ ected, and emphasises substantial diﬀ erences in the epidemiology of typhoid fever and iNTS disease. Malaria, malnutrition, and HIV infections have been reported to be associated with iNTS disease in Africa.23 At TSAP sites, a higher AIR for iNTS was observed in children with a malaria positivity rate of 30% or more than in those with a lower positivity rate; this observation was conﬁ rmed in a separate analysis.24 We made all eﬀ orts to minimise bias; however, our study has some limitations. First, we did not adjust the disease incidences for blood culture sensitivity, which is approximately 40–60% of bone marrow culture.33–37 This correction factor is inconsistently applied in studies and, if applied here, the incidences presented would double. The restricted sensitivity of blood culture to detect Salmonella pathogens applies to other bacterial pathogens as well—ie, S pneumoniae and Haemophilus inﬂ uenzae type b—however, those are universally recognised as important infections for which vaccines are cost-eﬀ ective, and vaccination programmes have been established. Second, our results represent incidence in sites selected because of their previous reports on typhoid fever. The site selection strategy limits the generalisability of the AIR to other locations and might result in the reduced detection of iNTS Results of our study identiﬁ ed a high prevalence of resistance against ﬁ rst-line antimicrobials in both S Typhi and iNTS infections. Reduced susceptibility to ciproﬂ oxacin was identiﬁ ed in S Typhi from Kibera and e321 Articles disease. Third, given the vast number of patients (and restricted diagnostics capacity), not every patient with a history of fever was enrolled—eg, at sites where inpatients were recruited, patients with a fever for 72 h or longer were excluded to minimise the inclusion of patients pretreated with antimicrobials and to maximise blood culture yield. References 1 Crump JA, Luby SP, Mintz ED. The global burden of typhoid fever. Bull World Health Organ 2004; 82: 346–53. 22 Owais A, Sultana S, Zaman U, Rizvi A, Zaidi AK. Incidence of typhoid bacteremia in infants and young children in southern coastal Pakistan. Pediatr Infect Dis J 2010; 29: 1035–39. 2 Ao TT, Feasey NA, Gordon MA, Keddy KH, Angulo FJ, Crump JA. Global burden of invasive non-typhoidal Salmonella disease. Emerg Infect Dis 2015; 21: e941–49. 23 Feasey NA, Dougan G, Kingsley RA, Heyderman RS, Gordon MA. Invasive non-typhoidal salmonella disease: an emerging and neglected tropical disease in Africa. Lancet 2012; 379: 2489–99. 3 Langridge GC, Nair S, Wain J. Nontyphoidal Salmonella serovars cause diﬀ erent degrees of invasive disease globally. J Infect Dis 2009; 199: 602–03. 24 Park SE, Pak GD, Aaby P, et al. The relationship between invasive non-Typhoidal Salmonella disease, other bloodstream infections, and malaria in sub-Saharan Africa. Clin Infect Dis 2016; 62: S23. 4 Mogasale V, Maskery B, Ochiai RL, et al. Burden of typhoid fever in low-income and middle-income countries: a systematic, literature-based update with risk-factor adjustment. Lancet Global Health 2014; 2: e570–80. 25 Takem EN, Roca A, Cunnington A. The association between malaria and non-typhoid Salmonella bacteraemia in children in sub-Saharan Africa: a literature review. Malar J 2014; 13: 400. 5 Buckle GC, Walker CLF, Black RE. Typhoid fever and paratyphoid fever: systematic review to estimate global morbidity and mortality for 2010. J Global Health 2012; 2: 010401. 26 Mengo DM, Kariuki S, Muigai A, Revathi G. Trends in Salmonella enterica serovar Typhi in Nairobi, Kenya from 2004 to 2006. J Infect Dev Ctries 2010; 4: 393–96. 6 Nielsen, MV, Sarpong, N, Krumkamp, R et al. Incidence and characteristics of bacteremia among children in rural Ghana. PLoS One 2012; 7: e44063. 27 Kariuki S, Revathi G, Kiiru J, et al. Typhoid in Kenya is associated with a dominant multidrug-resistant Salmonella enterica serovar Typhi haplotype that is also widespread in southeast asia. J Clin Microbiol 2010; 48: 2171–76. 7 Breiman RF, Cosmas L, Njuguna H, et al. Population-based incidence of typhoid fever in an urban informal settlement and a rural area in Kenya: implications for typhoid vaccine use in Africa. PLoS One 2012; 7: e29119. 28 Dougan G, Baker S. Salmonella enterica serovar Typhi and the pathogenesis of typhoid fever. Annu Rev Microbiol 2014; 68: 317–36. References 8 Marks F, Adu-Sarkodie Y, Hünger F, et al. Typhoid fever among children, Ghana. Emerg Infect Dis 2010; 16: 1796. 29 Kaur J. Increasing antimicrobial resistance and narrowing therapeutics in Typhoidal Salmonellae. J Clin Diagn Res 2013; 7: 576–79. 9 No authors listed. Typhoid vaccines: WHO position paper. Wkly Epidemiol Rec 2008; 83: 49–59. 30 Vlieghe ER, Phe T, De Smet B, et al. Azithromycin and ciproﬂ oxacin resistance in Salmonella bloodstream infections in cambodian adults. PLoS Negl Trop Dis 2012; 6: e1933. 10 von Kalckreuth V, Konings F, Aaby P, et al. The Typhoid Fever Surveillance in Africa Program (TSAP): clinical, diagnostic, and epidemiological methodologies. Clin Infect Dis 2016; 62: S9. 31 Koirala KD, Thanh DP, Thapa SD, et al. Highly resistant Salmonella enterica serovar typhi with a novel gyra mutation raises questions about the long-term eﬃ cacy of older ﬂ uoroquinolones for treating typhoid fever. Antimicrob Agents Chemother 2012; 56: 2761–62. 11 Panzner U, Pak GD, Aaby P, et al. The utilization of healthcare facilities in the Typhoid Fever Surveillance in Sub-Saharan Africa Program (TSAP). Clin Infect Dis 2016; 62: S56. 12 Clemens JD. Meeting on establishment of consortium to study invasive salmonellosis in sub-Saharan Africa. Emerg Infect Dis 2009; 15: e2. 32 Baker, S. A return to the pre-antimicrobial era? Science 2015; 347: 1064–66. 13 Sankoh O, Byass P. The INDEPTH network: ﬁ lling vital gaps in global epidemiology. Int J Epidemiol 2012; 41: 579–88. 33 Keddy KH, Sooka A, Letsoalo ME, et al. Sensitivity and speciﬁ city of typhoid fever rapid antibody tests for laboratory diagnosis at two sub-Saharan African sites. Bull World Health Organ 2011; 89: 640–47. 14 Murray P, Baro EJ. Chapter 42: Enterobacteriaceae—introduction and identiﬁ cation. Manual of Clinical Microbiology 9th edn. Washington, DC: ASM Press, 2007: 649–69. 34 Akoh JA. Relative sensitivity of blood and bone marrow cultures in typhoid fever. Trop Doct 1991; 21: 174–76. 15 Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; 23rd informational supplement; M100-S23. Wayne, PA: Clinical and Laboratory Standards Institute, 2013. 35 Parry CM, Wijedoru L, Arjyal A, Baker S. The utility of diagnostic tests for enteric fever in endemic locations. Expert Rev Anti Infect Ther 2011; 9: 7111–15. 16 Burton DC, Flannery B, Onyango B, et al. Healthcare-seeking behaviour for common infectious disease-related illnesses in rural Kenya: a community-based house-to-house survey. J Health Popul Nutr 2011; 29: 61–70. Declaration of interests FM, JAC, TFW, and RFB report grants from Bill & Melinda Gates Foundation during the conduct of the study. All other authors declare no competing interests. 21 Ochiai RL, Acosta CJ, Danovaro-Holliday MC, et al. A study of typhoid fever in ﬁ ve asian countries: Disease burden and implications for controls. Bull World Health Organ 2008; 86: 260–68. Acknowledgments This study was supported by the Bill & Melinda Gates Foundation (OPPGH5231). The ﬁ ndings and conclusions contained within are our own and do not necessarily reﬂ ect positions or policies of the Bill & Melinda Gates Foundation or the US Centers for Disease Control and Prevention. International Vaccine Institute acknowledges its donors, including the South Korea and the Swedish International Development Cooperation Agency (Sida). Research infrastructure at the Moshi site was supported by the US National Institutes of Health (R01TW009237; U01 AI062563; R24 TW007988; D43 PA-03–018; U01 AI069484; U01 AI067854; P30 AI064518), and by the UK Biotechnology and Biological Sciences Research Council (BB/J010367). SB is a Sir Henry Dale Fellow, jointly funded by the Wellcome Trust and the Royal Society (100087/Z/12/Z). We are grateful to Sooyoung Kwon for her invaluable administrative support of the project. We also thank all patients who consented to participate and hospital and clinic staﬀ for their support. We especially acknowledge those who personally contributed to the implementation and execution of the study, additional to routine clinical work. Without the eﬀ orts of dedicated ﬁ eld staﬀ this research would not have been possible. additional to routine clinical work. Without the eﬀ orts of dedicated ﬁ eld staﬀ this research would not have been possible. additional to routine clinical work. Without the eﬀ orts of dedicated ﬁ eld staﬀ this research would not have been possible. e322 Articles Articles Articles 20 Cruz Espinoza LM, Nichols C, Adu-Sarkodie Y, et al. Variations of invasive Salmonella infections by population size in Asante Akim North Municipal, Ghana. Clin Infect Dis 2016; 62: S17. Declaration of interests References 36 Wain J, Hosoglu S. The laboratory diagnosis of enteric fever. J Infect Dev Ctries 2008; 2: 421–25. 37 Baker S, Sarwar Y, Aziz H, et al. Detection of Vi-negative Salmonella enterica serovar Typhi in the peripheral blood of patients with typhoid fever in the Faisalabad region of Pakistan. J Clin Microbiol 2005; 43: 4418–25. 17 Bigogo G, Audi A, Aura B, Aol G, Breiman RF, Feikin DR. Health-seeking patterns among participants of population-based morbidity surveillance in rural western Kenya: implications for calculating disease rates. Int J Infect Dis 2010; 14: e967–73. 38 Verma R, Bairwa M, Chawla S, Prinja S, Rajput M. New generation typhoid vaccines: an eﬀ ective preventive strategy to control typhoid fever in developing countries. Hum Vaccin 2011; 7: 883–85. 18 Tabu C, Breiman RF, Ochieng B, et al. Diﬀ ering burden and epidemiology of non-Typhi Salmonella bacteremia in rural and urban Kenya, 2006–2009. PLoS One 2012; 7: e31237. 19 Mweu E, English M. Typhoid fever in children in Africa. Trop Med Int Health 2008; 13: 532–40. e323 www.thelancet.com/lancetgh Vol 5 March 2017
https://openalex.org/W4387623106	https://www.scielo.br/j/pab/a/48hPbjcKzW3XynPSm9DHrTd/?lang=en&format=pdf	English	null	Development of Israeli mango cultivars in the Brazilian semiarid region	Pesquisa Agropecuária Brasileira	2,023	cc-by	7,245	Pomology/ Original Article Pomology/ Original Article Pomology/ Original Article Development of Israeli mango cultivars in the Brazilian semiarid region Abstract – The objective of this work was to evaluate the initial adaptive performance of Israeli mango tree cultivars grown in the submedian region of the São Francisco Valley, Brazil. The experiment was carried out from January 2019 to July 2020 using seedlings of the Omer and Shelly cultivars, at six months after transplanting, at a 3×6 m spacing. The experimental design was randomized complete blocks in a 2×3 factorial arrangement, corresponding to the two Israeli mango tree cultivars and the number of branches after formative pruning (three, four, and five branches), with four replicates. Biometric, biochemical, and photosynthetic variables were analyzed, differing between the evaluation times after pruning. The Omer cultivar is more vigorous than Shelly, and formative pruning with three, four, and five branches is recommended for both mango cultivars under the cultivation conditions of the São Francisco Valley. Maria Jany Kátia Loiola Andrade(1) , Alexandre Santos de Oliveira(2) , Walber Felix dos Santos(2) , Luan dos Santos Silva(3 ) , Jenilton Gomes da Cunha(1) , Antônio Gustavo de Luna Souto(2) and Ítalo Herbert Lucena Cavalcante(2) (1) Universidade Federal do Piauí, Rodovia BR-135, Km 3, Planalto Horizonte, CEP 64900-000 Bom Jesus, PI, Brazil. E-mail: katiaengagronoma@gmail.com, jeniltongomes@hotmail.com (2) Universidade Federal do Vale do São Francisco, Rodovia BR 407, Km 12, Projeto de Irrigação Nilo Coelho, Lote 543, s/no, CEP 56300-000 Petrolina, PE, Brazil. E-mail: alexandre.sanoli@hotmail.com, Walber.felix@hotmail.com, gusluso@hotmail.com, italo.cavalcante@univasf.edu.br (3) Universidade Estadual do Norte Fluminense Darcy Ribeiro, Avenida Alberto Lamego, no 2.000, Parque California, CEP 28013-602 Campos dos Goytacazes, RJ, Brazil. E-mail: luan_agronomia@hotmail.com Corresponding author Received November 03, 2022 Accepted April 05, 2023 How to cite ANDRADE, M.J.K.L.; OLIVEIRA, A.S. de; SANTOS, W.F. dos; SILVA, L. dos S; CUNHA, J.G. da; SOUTO, A.G. de L.; CAVALCANTE, I.H.L. Development of Israeli mango cultivars in the Brazilian semiarid region. Pesquisa Agropecuária Brasileira, v.58, e03173, 2023. DOI: https://doi.org/10.1590/S1678-3921. pab2023.v58.03173. Maria Jany Kátia Loiola Andrade(1) , Alexandre Santos de Oliveira(2) , Walber Felix dos Santos(2) , Luan dos Santos Silva(3 ) , Jenilton Gomes da Cunha(1) , Antônio Gustavo de Luna Souto(2) and Ítalo Herbert Lucena Cavalcante(2) Index terms: Mangifera indica, formative pruning, gas exchange, 'Omer', 'Shelly'. Desenvolvimento de cultivares de mangueiras israelenses no semiárido brasileiro (2) Universidade Federal do Vale do São Francisco, Rodovia BR 407, Km 12, Projeto de Irrigação Nilo Coelho, Lote 543, s/no, CEP 56300-000 Petrolina, PE, Brazil. E-mail: alexandre.sanoli@hotmail.com, Walber.felix@hotmail.com, gusluso@hotmail.com, italo.cavalcante@univasf.edu.br Resumo – O objetivo deste trabalho foi avaliar o desempenho adaptativo inicial de cultivares de mangueiras israelenses cultivadas na região do Submédio do Vale do São Francisco, Brasil. O experimento foi realizado de janeiro de 2019 a julho de 2020, tendo-se utilizado mudas das cultivares Omer e Shelly, aos seis meses após o transplante, em espaçamento de 3×6 m. O delineamento experimental foi em blocos ao acaso, em arranjo fatorial 2×3, correspondente às duas cultivares de mangueiras israelenses e ao número de ramos após a poda de formação (três, quatro e cinco ramos), com quatro repetições. Foram analisadas variáveis biométricas, bioquímicas e fotossintéticas, que diferiram entre as épocas de avaliação após as podas. A cultivar Omer é mais vigorosa que a Shelly, e a poda formativa com três, quatro e cinco ramos é recomendada para ambas as cultivares de manga nas condições de cultivo do Vale do São Francisco. (3) Universidade Estadual do Norte Fluminense Darcy Ribeiro, Avenida Alberto Lamego, no 2.000, Parque California, CEP 28013-602 Campos dos Goytacazes, RJ, Brazil. E-mail: luan_agronomia@hotmail.com Termos para indexação: Mangifera indica, poda de formação, trocas gasosas, 'Omer', 'Shelly'. How to cite ANDRADE, M.J.K.L.; OLIVEIRA, A.S. de; SANTOS, W.F. dos; SILVA, L. dos S; CUNHA, J.G. da; SOUTO, A.G. de L.; CAVALCANTE, I.H.L. Development of Israeli mango cultivars in the Brazilian semiarid region. Pesquisa Agropecuária Brasileira, v.58, e03173, 2023. DOI: https://doi.org/10.1590/S1678-3921. pab2023.v58.03173. Maria Jany Kátia Loiola Andrade(1) , Alexandre Santos de Oliveira(2) , Walber Felix dos Santos(2) , Luan dos Santos Silva(3 ) , Jenilton Gomes da Cunha(1) , Antônio Gustavo de Luna Souto(2) and Ítalo Herbert Lucena Cavalcante(2) (1) Universidade Federal do Piauí, Rodovia BR-135, Km 3, Planalto Horizonte, CEP 64900-000 Bom Jesus, PI, Brazil. E-mail: katiaengagronoma@gmail.com, jeniltongomes@hotmail.com This is an open-access article distributed under the Creative Commons Attribution 4.0 International License How to cite ANDRADE, M.J.K.L.; OLIVEIRA, A.S. de; SANTOS, W.F. dos; SILVA, L. dos S; CUNHA, J.G. da; SOUTO, A.G. de L.; CAVALCANTE, I.H.L. Development of Israeli mango cultivars in the Brazilian semiarid region. Pesquisa Agropecuária Brasileira, v.58, e03173, 2023. DOI: https://doi.org/10.1590/S1678-3921. pab2023.v58.03173. (3) Universidade Estadual do Norte Fluminense Darcy Ribeiro, Avenida Alberto Lamego, no 2.000, Parque California, CEP 28013-602 Campos dos Goytacazes, RJ, Brazil. E-mail: luan_agronomia@hotmail.com Materials and Methods The experiment was conducted during the vegetative stage of seedlings of the Omer and Shelly cultivars, six months after transplanting, from January 2019 to July 2020, in a commercial orchard of Fazenda Le Bourdet, located in the Maniçoba irrigation perimeter, in the municipality of Juazeiro, in the state of Bahia, Brazil (9º20'05.6"S, 40º14'41.1"W). According to Köppen’s classification, the climate is BshW, tropical semiarid, hot, with a rainy season in summer and a high evaporation, mean annual temperature of 26ºC, and mean rainfall of 481.7 mm. The soil is classified as a Ferralsol according to World Reference Base for Soil Resources (IUSS Working Group WRB, 2015). For the adaptation of mango to the environmental conditions of Brazilian semiarid regions, intensive management practices, such as pruning, fertilization, irrigation, and floral induction, are necessary for the expression of the crop’s production potential (Santos et al., 2013; Cavalcante et al., 2018; Oldoni et al., 2018). This is especially important for the São Francisco Valley region, located in the semiarid Brazilian Northeast, which represents 69% of national production and 90% of mango exports (Anuário..., 2022). The Omer and Shelly Israeli mango cultivars were provided by Israel’s Agricultural Research Organization of Volcani Institute. Seedlings of both cultivars were transplanted on 6/22/2018, four months after grafted when reaching a height of 60 cm, spaced at 3×6 m. Each plant was irrigated by a surface emitter (micro-sprinkler) at an individual flow of 2.33 L h-1, following the water requirements of the crop (Lipan et al., 2021). The main mango cultivars produced in the São Francisco Valley are Tommy Atkins, Keitt, Kent, Haden, and Palmer from the United States (Mouco & Lima Neto, 2018; Anuário..., 2022). Although these cultivars are known in the international market and well exported, Brazilian producers are expanding their niche markets to other distribution centers, which require fruits with different characteristics from those traditionally offered (Mouco & Lima Neto, 2018). Sixty days before the seedlings were transplanted, the soil was prepared using two plowing and harrowing operations. The preparation of the pits and the application of fertilizers for the initial stage of the crop were carried out according to the methodologies of Silva (2009) and to the demands of the crop considering the regional cultivation conditions (Cavalcante et al., 2018). Introduction Mango (Mangifera indica L.) is a fruit tree adapted to tropical and subtropical climates that develops well under conditions with high solar radiation incidence and temperatures (Fitchett et al., 2016), low water availability, and high evaporative demand (Khanum et al., 2020). Pesq. agropec. bras., Brasília, v.58, e03173, 2023 DOI: 10.1590/S1678-3921.pab2023.v58.03173 This is an open-access article distributed under the Creative Commons Attribution 4.0 International License M.J.K.L. Andrade et al. 2 2 grown in the submedian region of the São Francisco Valley, Brazil. However, the expected increases in temperature cause several abiotic stresses, reducing photosynthesis due to a rapid stomatal closure when the energy demand (carbohydrates) of the plant increases (Mudo et al., 2020), decreasing photochemical efficiency (Yanhui et al., 2020), and degrading chloroplasts by the excessive production of reactive oxygen species (Qiu et al., 2019; Zhang et al., 2022). The harmful effects of these stresses vary depending on the plant species, adaptation level, exposure time, and phenological stage (Hassan et al., 2022). However, the expected increases in temperature cause several abiotic stresses, reducing photosynthesis due to a rapid stomatal closure when the energy demand (carbohydrates) of the plant increases (Mudo et al., 2020), decreasing photochemical efficiency (Yanhui et al., 2020), and degrading chloroplasts by the excessive production of reactive oxygen species (Qiu et al., 2019; Zhang et al., 2022). The harmful effects of these stresses vary depending on the plant species, adaptation level, exposure time, and phenological stage (Hassan et al., 2022). Pesq. agropec. bras., Brasília, v.58, e03173, 2023 DOI: 10.1590/S1678-3921.pab2023.v58.03173 Materials and Methods Among the new mango cultivars that have shown good acceptance in the consumer market, the Shelly and Omer Israeli cultivars stand out (Lima Neto, 2020), being recently introduced in the São Francisco Valley, but still with no management recommendations. Cultivar Shelly, a result of the cross between Tommy Atkins and Kent (Cohen et al., 2016), has round fruit, with a weight ranging from 350 to 700 g, a juicy and firm pulp without fibers, and an orange color with a red blush (Lavi et al., 1996). Produced from open pollination, cultivar Omer is a hybrid of the Zillate cultivar and is characterized by an oval shape and average weight of 450 g, with a purple and bright-red skin color, little fiber, a light aroma, and a sweet flavor (Cohen et al., 2016). The cultural practices were those recommended by Lopes et al. (2003) for mango cultivation under the conditions of the study region, including pruning, nutritional management via fertigation, harvest point, and control of invasive plants, pests, and diseases. Throughout the experiment, nutrients were supplied to the plants via fertigation. During the plant cycle, the applied amounts were: 1,480 g nitrogen in the form of urea (45% nitrogen) and calcium nitrate (14% nitrogen), 515 g calcium from calcium nitrate (28% calcium), 631 g magnesium from magnesium sulfate (14% magnesium), 674 g potassium in the form of potassium sulfate (51% potassium), and 645 g phosphorus from The objective of this work was to evaluate the initial adaptive performance of Israeli mango tree cultivars Pesq. agropec. bras., Brasília, v.58, e03173, 2023 DOI: 10.1590/S1678-3921.pab2023.v58.03173 Development of Israeli mango cultivars Development of Israeli mango cultivars 3 monoammonium phosphate (48% P2O5). In addition to the mentioned fertilizers, 96 mL fulvic acids (1.0 L ha‑1) were added, containing 10% organic carbon, 11% nitrogen, 1.0% K2O, 6.0% phosphorous acid, and 33% fulvic acid. determined in mature leaves from the penultimate vegetation flow, following the methodology described in Dubois et al. (1956), whereas soluble starch in the branches was determined by the method of Hodge & Hofreiter (1962). Photosynthetic exchanges were measured between 9 and 11 a.m. in the same leaves selected for biochemical determination, using the Li-6400XT infrared gas analyzer (Li-COR Biosciences, Lincoln, NE, USA), coupled to the Li-6400XT portable frequency- modulated light fluorometer (Li-COR Biosciences, Lincoln, NE, USA), at 1,500 μmol photons per square meter per second (artificial light source). Materials and Methods These exchanges were expressed by the variables net photosynthesis (A), stomatal conductance (gs), internal CO2 concentration (Ci), transpiration (E), and water use efficiency (WUE = A/E). Micronutrients were provided by biweekly foliar sprays using a complete fertilizer, containing 10% nitrogen, 8.0% P2O5, 8.0% K2O, 1.0% calcium, 0.5% magnesium, 0.5% boron, 0.2% copper, and 0.5% manganese and zinc. The experimental design was randomized complete blocks in a 2×3 factorial arrangement, corresponding to the two Israeli mango cultivars (Omer and Shelly) and the number of branches left after formative pruning (three, four, and five branches), with four replicates and five plants per plot, in an experimental area of 2,160 m2. The first pruning and topping of the plants were performed below the third node and at a height of 0.7 m above ground level, on 12/28/2018, 189 days after transplanting (DAT). For the first formation pruning of cultivars Omer and Shelly, stems were selected on 1/21/2019, at 213 DAT, and, subsequently, pruned on 3/27/2019 and 4/5/2019, at 247 and 256 DAT, respectively. To determine the effects of formative pruning, biometric, biochemical, and photosynthetic exchange variables were simultaneously evaluated at 31 days after the third pruning on 7/30/2019, at 39 days after the fourth pruning on 11/19/2018, at 22 days after the fifth pruning on 1/10/2020, and at 31 days after the sixth pruning on 7/7/2020. The data were tested for normal distribution and homogeneity of variance using Shapiro-Wilk’s test. Subsequently, the analysis of variance was performed using the F-test, at 5% probability. The means referring to the Israeli cultivars and the number of branches after formation pruning were compared by Tukey’s test, 5% probability. The data were analyzed with the R statistical software (R Core Team, 2022). Pesq. agropec. bras., Brasília, v.58, e03173, 2023 DOI: 10.1590/S1678-3921.pab2023.v58.03173 M.J.K.L. Andrade et al. respectively. In addition, branch diameter, shoot length, and leaf number differed in the last vegetative flow, which could be attributed to plant development stage, prevailing air temperature, water availability, plant vigor, cultivar, and several other external and internal factors (Kavati, 2004). Cultivar Omer was more vigorous than Shelly after all pruning regarding branch diameter, but only up to the third pruning for number of branches. According to Gollan & Aro (2020), a large leaf area is associated with a greater potential for capturing light for photosynthesis under ideal water, light, and nutrient conditions, increasing carbohydrate production for the formation of organs, such as fruits. Regarding the number of branches after each formative pruning, the maintenance of three branches resulted in a higher number of shoots and longer shoot length after the third and fifth formative pruning, Table 1. Analysis of variance and mean test for the biometric variables of the Omer and Shelly Israeli mango (Mangifera indica) cultivars as a function of number of branches after each formative pruning(1). t letters differ by Tukey’s test, at 5% probability. ** and Significant at 1 and 5% probability, respectively. nsNonsignificant Pesq. agropec. bras., Brasília, v.58, e03173, 2023 DOI: 10.1590/S1678-3921.pab2023.v58.03173 ans followed by different letters differ by Tukey’s test, at 5% probability. * and Significant at 1 and 5% probability, respe Results and Discussion The biometric variables were more influenced by the Israeli cultivars, which differed significantly from the third to sixth formative pruning, than by number of branches (Table 1). However, number of branches had a significant effect on number of shoots in the third pruning, shoot length in the fifth pruning, and crown volume in the third to fifth pruning. The following biometric variables were analyzed from the third pruning onwards: number of shoots emitted per branch after each formative pruning; number of newly mature leaves in the last vegetative flow of each branch where the formative pruning was performed; branch diameter, measured using a digital caliper; and crown volume, obtained with the equation [((L/2) × (E/2) × π) × (A)]/3, where π = 3.1416, L is the superior distance between branches, E is the mean thickness of the two branches, and A is crown height (Rossi et al., 2004). Cultivar Omer was superior to Shelly for all biometric variables, except for shoot length between the fourth and sixth formative pruning. The obtained results indicate that the Omer cultivar has a denser and more robust crown due to the presence of more shoots and leaves, whereas Shelly has a more vertical growth characteristic. This is an interesting finding since, although Shelly is not a new cultivar, there are no known studies on its growth habit. This is not the case, however, for cultivar Omer, which showed a greater vigor and leaf area when compared with cultivars Aya, Katuri, and Maya in Egypt, as well as a higher yield in the first year (Haseeb et al., 2020). Regarding the biochemical variables, the indexes of chlorophyll a, b, and total, expressed in the leaf chlorophyll index (Falker chlorophyll index, FCI), were measured between 7 and 9 a.m. using the CFL1030 ClorofiLOG chlorophyll meter (Falker, Porto Alegre, RS, Brazil). Total soluble carbohydrate contents were Pesq. agropec. bras., Brasília, v.58, e03173, 2023 DOI: 10.1590/S1678-3921.pab2023.v58.03173 Pesq. agropec. bras., Brasília, v.58, e03173, 2023 DOI: 10.1590/S1678-3921.pab2023.v58.03173 4 M.J.K.L. Andrade et al. M.J.K.L. Andrade et al. Bars with the same lowercase letters do not differ for cultivars Omer and Shelly within each number of branches after formative pruning. Bars with the same uppercase letters do not differ for number of branches within each Israeli mango cultivar by Tukey’s test, at 5% probability. For number of shoots, a significant difference was only observed after the third pruning, with higher values for cultivar Omer. Moreover, the third and fifth formative pruning showed an interaction between cultivar and number of branches for this variable (Figure 1). After the third formative pruning, cultivar Omer had a higher number of shoots, regardless of the number of branches, whereas Shelly presented a higher number of shoots when it had three branches (Figure 1 A). After the fifth formative pruning, the highest number of shoots was reached when cultivar Omer had four branches. However, the number of branches alone did not affect the number of shoots for both mango cultivars (Figure 1 B). Leaf chlorophyll content was higher for cultivar Shelly after the third formative pruning, whereas the chlorophyll index showed an opposite behavior after the fourth and sixth pruning, with higher values for cultivar Omer (Table 2). This is explained by the chlorophyll homeostasis that occurs as a phenotypic response to the environmental and management conditions to which the plants are exposed (Dhami et al., 2022). The aforementioned result is associated with the biometric variables since cultivar Omer not only has a higher vegetative vigor but also a higher capacity to synthesize chlorophyll pigments after the fourth pruning (Table 1). Therefore, the Omer cultivar is better adapted to the growing environment, as high chlorophyll a contents represent a key physiological adaptation for the high photosynthetic efficiency of plants (Luo et al., 2019). According to Dhami et al. (2022), pigment homeostasis is directly affected by the genetic trait of the plant, which tends to change metabolic pathways to maintain leaf chlorophyll content. Figure 1. Number of shoots in the third (A) and fifth (B) formative pruning of the Omer and Shelly Israeli mango (Mangifera indica) cultivars as a function of number of branches. Bars with the same lowercase letters do not differ for cultivars Omer and Shelly within each number of branches after formative pruning. Bars with the same uppercase letters do not differ for number of branches within each Israeli mango cultivar by Tukey’s test, at 5% probability. M.J.K.L. Andrade et al. Source of variation Diameter (mm) Shoot length (cm) Number of leaves Crown volume (m3) Number of shoots Third prunning Cultivars 2.15ns 71.88 63.53* 36.24* 103.88* Omer 7.43a 19.85a 4.67a 0.002a 4.46a Shelly 7.24a 16.77b 3.14b 0.001b 2.71b Branches 2.54ns 4.25ns 4.39ns 5.38* 8.55* Three 7.54a 19.01a 4.31a 0.001a 4.81a Four 7.30a 18.21a 3.73a 0.002ab 3.38b Five 7.21a 17.72a 3.68a 0.001b 3.28b Cultivars × branches 0.62ns 0.21ns 0.60ns 1.61ns 4.05* Fourth prunning Cultivars 9.58* 11.13* 182.96* 14.33* 0.81ns Omer 14.23a 6.15b 5.66a 0.007a 2.65a Shelly 13.02b 6.55a 3.72b 0.004b 2.55a Branches 2.72ns 5.06ns 0.56ns 34.24* 2.29ns Three 14.18a 6.61a 4.63a 0.009a 2.75a Four 13.62a 6.25a 4.65a 0.001b 2.59a Five 13.06a 6.18a 4.81a 0.007a 2.46a Cultivars × branches 3.26ns 0.03ns 3.14ns 1.87ns 0.18ns Fifth prunning Cultivars 0.49ns 11.13* 0.48ns 23.24* 2.67ns Omer 12.60a 6.15b 7.62a 0.013a 3.24a Shelly 12.39a 6.55a 8.56a 0.009b 3.06a Branches 1.15ns 5.05* 1.1ns 7.35* 0.17ns Three 12.66a 6.61a 7.21a 0.013a 3.15a Four 12.65a 6.25b 7.58a 0.011ab 3.11a Five 12.18a 6.18b 9.51a 0.009b 3.18a Cultivars × branches 1.52ns 0.03ns 0.89ns 0.46ns 4.12* Sixth prunning Cultivars 9.62* 11.21* 2.51ns 24.55* 3.85ns Omer 9.65a 6.15b 5.89a 0.24a 4.05a Shelly 8.61b 6.55a 5.43a 0.16b 4.53a Branches 0.95ns 5.06ns 0.34ns 1.81ns 0.86ns Three 8.80a 6.61a 5.62a 0.222a 4.06a Four 9.35a 6.25b 5.83a 0.202a 4.44a Five 9.23a 6.18b 5.54a 0.185a 4.36a Cultivars × branches 0.06ns 0.02ns 0.42ns 2.21ns 1.4ns (1)Means followed by different letters differ by Tukey’s test, at 5% probability. ** and Significant at 1 and 5% probability, respectively. nsNonsignificant. Table 1. Analysis of variance and mean test for the biometric variables of the Omer and Shelly Israeli mango (Mangifera indica) cultivars as a function of number of branches after each formative pruning(1). Development of Israeli mango cultivars Development of Israeli mango cultivars 5 significantly among the different number of branches for Shelly. Crown volume was influenced by the factor number of branches from the third to fifth formative pruning, being larger for cultivar Omer, which had more shoots and leaves. For this variable, plants with three branches showed higher values than those with five branches after the third and fifth formative pruning and with four branches after the fourth formative pruning. Figure 1. Number of shoots in the third (A) and fifth (B) formative pruning of the Omer and Shelly Israeli mango (Mangifera indica) cultivars as a function of number of branches. Pesq. agropec. bras., Brasília, v.58, e03173, 2023 DOI: 10.1590/S1678-3921.pab2023.v58.03173 M.J.K.L. Andrade et al. 6 Therefore, after the third formative pruning, there was a lack of starch response with the increase in leaf chlorophyll content, which could be attributed to the greater development of shoots, considered drains of high energy demand (Richardson et al., 2021). In this scenario, plants need to maintain high levels of photosynthetic rates after pruning to meet the demand for energy (sugars) necessary for the formation of new reproductive structures (Lopes et al., 2021; Sanches et al., 2023). Despite the varying leaf chlorophyll indices of the Israeli mango cultivars, similar effects were expected for the contents of carbohydrate starch in the branches. Starch and total soluble carbohydrates did not differ between both cultivars in the third pruning (Table 2). In addition, the highest starch contents of 0.208 and 0.129 µg g-1 fresh matter of cultivars Omer and Shelly were found in the fourth formative pruning, coinciding with the moment of the lowest number of shoots, i.e., 2.65 and 2.55, respectively (Table 1). Table 2. Analysis of variance and mean test for the biochemical variables of the Omer and Shelly Israeli mango (Mangifera indica) cultivars as a function of number of branches after each formative pruning(1). Table 2. Analysis of variance and mean test for the biochemical variables of the Omer and Shelly Israeli mango (Mangifera indica) cultivars as a function of number of branches after each formative pruning(1). t letters differ by Tukey’s test, at 5% probability. * and Significant at 1 and 5% probability, respectively. nsNonsignificant M.J.K.L. Andrade et al. The leaf chlorophyll a index was influenced by the interaction cultivar × number of branches after the sixth formative pruning (Figure 2). This finding shows that cultivar Omer had the highest chlorophyll index, regardless of the number of branches. Comparing number of branches, the FCI was the highest for cultivar Omer with four branches, but did not differ Pesq. agropec. bras., Brasília, v.58, e03173, 2023 DOI: 10.1590/S1678-3921.pab2023.v58.03173 6 M.J.K.L. Andrade et al. Pesq. agropec. bras., Brasília, v.58, e03173, 2023 DOI: 10.1590/S1678-3921.pab2023.v58.03173 Development of Israeli mango cultivars 7 Development of Israeli mango cultivars Regarding number of branches, in general, the mango cultivars grown in the São Francisco Valley region (Keitt, Kent, Haden, and Tommy Atkins) are shaped by leaving three branches for crown opening, fruit mass distribution, and phytosanitary management (Kavati, 2004; Anuário..., 2020). However, further researches are necessary on the production of carbohydrates during the development of the mango canopy, an essential subsidy for tissue formation. The interaction between cultivars and number of branches is possibly related to the fact that the plants were in the final process of crown formation in the sixth pruning, when an intense branch reduction was no longer needed (Sanjay et al., 2010). Du Toit et al. (2020) highlighted that there is a tendency to favor the rapid growth of vegetative structures, largely due to the presence of more mature leaves with a higher chlorophyll accumulation. Three branches can also be recommended for the studied Israeli cultivars when considering the starch and carbohydrate contents after the fifth and sixth formative pruning, respectively. With three branches, the plants from both cultivars produced more starch and carbohydrates than those with four, but similar amounts to those with five branches after the fifth and sixth formation pruning, respectively. Therefore, the relationship between number of branches and the production of organic solutes in mango was not completely elucidated. Furthermore, mango is a C3 photosynthetic cycle plant that does not have mechanisms for CO2 contraction, which leads to an energy loss due to the oxygenase activity in Rubisco (photorespiration) under adverse conditions (Beerling & Royer, 2011). Therefore, the higher the chlorophyll content, the better the plant will adapt to tropical conditions since it can more efficiently absorb light energy in a shorter period, which, in theory, explains the greater vigor of cultivar Omer (Table 2). This cultivar showed higher values of A, gs, Ci, and WUE (Table 3), as well as superiority in photosynthesis in the fourth formative pruning and in WUE until the fifth formative pruning. Figure 2. Chlorophyll a index of leaves of the Omer and Shelly Israeli mango (Mangifera indica) cultivars as a function of number of branches after the sixth formative pruning. Bars with the same lowercase letters do not differ for cultivars Omer and Shelly within each number of branches after formative pruning. Pesq. agropec. bras., Brasília, v.58, e03173, 2023 DOI: 10.1590/S1678-3921.pab2023.v58.03173 M.J.K.L. Andrade et al. Source of variation Falker chlorophyll index (FCI) Starch (µg g-1 fresh mass) Carbohydrates (mmol g-1 fresh mass) Chlorophyll a Chlorophyll b Total chlorophyll Third prunning Cultivars 14.87 8.15* 13.67* 0.85ns 2.77ns Omer 37.67b 16.02b 53.71b 0.147a 152.84a Shelly 39.53a 18.37a 57.91a 0.137a 197.95a Branches 1.09ns 1.22ns 1.51ns 0.93ns 0.46ns Three 38.2a 16.67a 54.88a 0.152a 179.34a Four 38.54a 16.81a 55.35a 0.133a 188.96a Five 39.06a 18.11a 57.17a 0.142a 157.89a Cultivars × branches 0.13ns 0.98ns 0.74ns 0.89ns 0.47ns Fourth prunning Cultivars 219.91* 0.46ns 40.14* 5.2* 1.71ns Omer 46.42a 14.16a 60.58ª 0.208a 204.78a Shelly 38.9b 13.54a 52.44b 0.129b 225.27a Branches 0.15ns 0.32ns 0.29ns 0.792ns 1.25ns Three 42.47a 13.61a 56.08ª 0.199a 197.45a Four 42.67a 14.36a 57.19ª 0.152a 223.03a Five 42.83a 13.57a 56.24ª 0.154a 224.61a Cultivars × branches 0.28ns 0.05ns 0.06ns 0.41ns 3.62ns Fifth prunning Cultivars 0.03ns 0.09ns 0.06ns 2.78ns 3.92ns Omer 43.81a 18.91a 62.71a 0.173a 235.01a Shelly 43.83a 18.61a 62.44a 0.147a 204.04a Branches 0.53ns 0.64ns 0.43ns 5.68* 2.14ns Three 43.84a 18.16a 62.00a 0.195a 196.97a Four 43.68a 19.53a 63.22a 0.133b 234.18a Five 43.93a 18.57a 62.51a 0.153ab 227.44a Cultivars × branches 2.00ns 0.21ns 0.39ns 0.12ns 0.07ns Sixth prunning Cultivars 118.31* 0.021ns 33.58* 1.81ns 0.1ns Omer 43.01a 22.53a 65.54a 0.16a 204.96a Shelly 35.35b 22.41a 57.75b 0.15a 202.01a Branches 3.84* 0.001ns 1.0ns 5.01* 5.41* Three 39.13ab 22.43a 61.57a 0.18a 220.65a Four 40.41a 22.48a 62.87a 0.13b 183.41b Five 38.01b 22.48a 60.49a 0.15ab 206.41ab Cultivars × branches 4.75* 0.79ns 2.36ns 0.26ns 0.35ns (1)Means followed by different letters differ by Tukey’s test, at 5% probability. ** and Significant at 1 and 5% probability, respectively. nsNonsignificant. Pesq. agropec. bras., Brasília, v.58, e03173, 2023 DOI: 10.1590/S1678-3921.pab2023.v58.03173 Development of Israeli mango cultivars Bars with the same uppercase letters do not differ for number of branches within each Israeli mango cultivar by Tukey’s test, at 5% probability. For cultivar Tommy Atkins in tropical semiarid conditions, Mudo et al. (2020) concluded that high transpiration rates and internal CO2 concentrations are positively related to good plant development, although the observed WUE was low. For the Palmer cultivar, Souza et al. (2016) found that the reduction in transpiration is associated with an increased stomatal resistance and, therefore, with water limitation. In the present study, there was no significant difference between cultivars regarding transpiration until the fifth pruning, indicating that the higher WUE of Omer is related to its greater photosynthetic activity with a lower water use, a desired condition for crops in semiarid regions (Li et al., 2022). Cultivar Shelly had a lower and slower adaptive response in the semiarid region of the São Francisco Valley, showing higher values of A, gs, E, and WUE only after the sixth formative pruning. Figure 2. Chlorophyll a index of leaves of the Omer and Shelly Israeli mango (Mangifera indica) cultivars as a function of number of branches after the sixth formative pruning. Bars with the same lowercase letters do not differ for cultivars Omer and Shelly within each number of branches after formative pruning. Bars with the same uppercase letters do not differ for number of branches within each Israeli mango cultivar by Tukey’s test, at 5% probability. A significant effect of number of branches was only observed in the fourth formative pruning, with increments in Ci and WUE in plants with five branches (Table 3). These plants showed a higher internal CO2 concentration than those with four branches but did not differ significantly from those with three. In general, the number of branches had little influence on gas exchange, except for Ci in the fourth Pesq. agropec. bras., Brasília, v.58, e03173, 2023 DOI: 10.1590/S1678-3921.pab2023.v58.03173 M.J.K.L. Andrade et al. 8 photosynthesis rate was maintained at high levels, which, associated with water loss control, led to a higher WUE by the plants (Carreiro et al., 2022). Considering that the environmental conditions of the experimental site were similar, the higher WUE presented by cultivar Omer in the third to the fifth formative pruning is another indicative of its greater acclimatization capacity to the São Francisco Valley in comparison with Shelly (Figure 3). However, in pruning and gs without pruning. n; and WUE, water use efficiency. * and Significant at 1 and 5% probability, respectively. nsNonsignificant. nt letters differ by Tukey’s test, at 5% probability. A, net photosynthesis; gs, stomatal conductance; Ci, internal CO followed by different letters differ by Tukey s test, at 5% probability. A, net photosynthesis; gs, stomatal conductan ation; E, transpiration; and WUE, water use efficiency. * and Significant at 1 and 5% probability, respectively. nsNonsig Development of Israeli mango cultivars This effect was considered isolated because these two variables have a strong relationship since stomatal opening and closing (gs) regulate the entry of carbon into plants (Ci) (Carreiro et al., 2022). In the present study, the Omer mango cultivar presented a faster acclimatization process between the third and fifth pruning, mainly because there was no stomatal limitation in the CO2 inflow and the Table 3. Analysis of variance and mean test for gas exchange of the Omer and Shelly Israeli mango (Mangifera indica) cultivars as a function of number of branches after each formative pruning(1). Source of variation A gs Ci E WUE -------------------------------(μmol CO2 m-2 s-1)------------------------------- (mmol H2O m-2 s-1) (μmol CO2 mmol-1 H2O) Third prunning Cultivars 1.076ns 11.77 18.58* 0.12ns 46.26* Omer 17.83a 0.24a 252.01a 3.87a 0.06a Shelly 18.53a 0.19b 216.14b 3.92a 0.05b Branches 0.35ns 0.81ns 1.24ns 0.23ns 2.05ns Three 18.56a 0.21a 226.72a 3.94a 0.05a Four 18.07a 0.23a 232.88a 3.92a 0.06a Five 17.93a 0.21a 242.62a 3.84a 0.05a Cultivars × branches 0.029ns 0.07ns 0.244ns 0.30ns 0.08ns Fourth prunning Cultivars 11.14* 7.17* 0.153ns 1.32ns 41.00* Omer 15.58a 0.16a 213.59a 6.19a 0.03a Shelly 12.45b 0.13b 217.19a 5.81a 0.02b Branches 1.77ns 0.09ns 4.41* 1.09ns 6.59* Three 13.56a 0.15a 220.90ab 6.34a 0.02b Four 15.25a 0.14a 196.57b 5.77a 0.02ab Five 13.24a 0.15a 228.71a 5.88a 0.03a Cultivars × branches 3.96ns 1.08ns 1.23ns 1.12ns 1.24* Fifth prunning Cultivars 0.038ns 2.25ns 12.96* 0.17ns 6.54* Omer 18.39a 0.17a 203.26a 4.68a 0.04a Shelly 18.59a 0.16a 186.52b 4.57a 0.03b Branches 0.03ns 0.63ns 2.63ns 0.41ns 0.11ns Three 18.66a 0.175a 199.84a 4.79a 0.04a Four 18.46a 0.163a 187.51a 4.48a 0.04a Five 18.35a 0.171a 197.33a 4.61a 0.04a Cultivars × branches 2.38ns 1.74ns 3.42ns 1.73ns 1.00ns Sixth prunning Cultivars 30.57* 6.43* 0.05ns 22.30* 2.95ns Omer 13.03b 0.11b 176.66a 2.89b 0.04a Shelly 16.93a 0.14a 179.96a 4.19a 0.03a Branches 1.33ns 0.41ns 0.36ns 0.8ns 0.21ns Three 14.21a 0.12a 180.36a 3.33a 0.04a Four 15.16a 0.12a 169.48a 3.54a 0.03a Five 15.58a 0.13a 185.10a 3.75a 0.04a Cultivars × branches 3.02ns 0.53ns 0.08ns 1.22ns 0.08ns (1)Means followed by different letters differ by Tukey’s test, at 5% probability. A, net photosynthesis; gs, stomatal conductance; Ci, internal CO2 concentration; E, transpiration; and WUE, water use efficiency. ** and Significant at 1 and 5% probability, respectively. nsNonsignificant. Table 3. Analysis of variance and mean test for gas exchange of the Omer and Shelly Israeli mango (Mangifera indica) cultivars as a function of number of branches after each formative pruning(1). Table 3. followed by different letters differ by Tukey’s test, at 5% probability. A, net photosynthesis; gs, stomatal conductan ti E t i ti d WUE t ffi i * d Si ifi t t 1 d 5% b bilit ti l nsN i (1)Means followed by different letters differ by Tukey’s test, at 5% probability. A, net photosynthesis; gs, stomatal conductance; Ci, internal CO2 concentration; E, transpiration; and WUE, water use efficiency. * and *Significant at 1 and 5% probability, respectively. nsNonsignificant. References ANUÁRIO BRASILEIRO DE HORTI & FRUTI 2020. Santa Cruz do Sul: Gazeta Santa Cruz, 2020. 104p. ANUÁRIO BRASILEIRO DE HORTI&FRUIT 2022. Santa Cruz do Sul: Gazeta Santa Cruz, 2022. 96p. BEERLING, D.J.; ROYER, D.L. Convergent cenozoic CO2 history. Nature Geoscience, v.4, p.418-420, 2011. DOI: https://doi.org/10.1038/ngeo1186. Figure 3. Water use efficiency values for the Omer and Shelly Israeli mango (Mangifera indica) cultivars as a function of number of branches after the fourth formative pruning. Bars with the same lowercase letters do not differ for cultivars Omer and Shelly within each number of branches after formative pruning. Bars with the same uppercase letters do not differ for number of branches within each Israeli mango cultivar by Tukey’s test, at 5% probability. CARREIRO, D. de A.; AMARIZ, R.A. e; SANCHES, L.G.; LOBO, J.T.; PAIVA NETO, V.B. de; CAVALCANTE, Í.H.L. Gas exchanges and photosynthetic pigments of 'Tommy Atkins' mango as a function of fenpropimorph. Revista Brasileira de Engenharia Agrícola e Ambiental, v.26, p.239-247, 2022. DOI: https://doi.org/10.1590/1807-1929/agriambi.v26n4p239-247. CAVALCANTE, Í.H.L.; SANTOS, G.N.F. dos; SILVA, M.A. da; MARTINS, R. dos S.; LIMA, A.M.N.; MODESTO, P.I.R.; ALCOBIA, A.M.; SILVA, T.R. de S.; AMARIZ, R.A. e; BECKMANN-CAVALCANTE, M.Z. A new approach to induce mango shoot maturation in Brazilian semi-arid environment. Journal of Applied Botany and Food Quality, v.91, p.281-286, 2018. DOI: https://doi.org/10.5073/JABFQ.2018.091.036. the sixth pruning, both cultivars showed the same efficiency, indicating that Shelly, even if later, can adapt as well as Omer regarding WUE. Moreover, cultivar Omer presented the highest WUE under all number of branches after the fourth formative pruning, which shows that the number of branches had little influence on the photosynthetic efficiency of the plant in relation to water vapor losses. COHEN, Y.; SAADA, D.; DOR, R.; KEINAN, A.; NOY, M. Set of elite new Israeli mango cultivars. AgroIsrael, v.2, p.64-69, 2016. DHAMI, N.; POGSON, B.J.; TISSUE, D.T.; CAZZONELLI, C.I. A foliar pigment-based bioassay for interrogating chloroplast signalling revealed that carotenoid isomerisation regulates chlorophyll abundance. Plant Methods, v.18, art.18, 2022. DOI: https://doi.org/10.1186/s13007-022-00847-5. DU TOIT, E.S.; SITHOLE, J.; VORSTER, J. Pruning intensity influences growth, flower and fruit development of Moringa oleifera Lam. under sub-optimal growing conditions in Gauteng, South Africa. South African Journal of Botany, v.129, p.448- 456, 2020. DOI: https://doi.org/10.1016/j.sajb.2019.11.033. DU TOIT, E.S.; SITHOLE, J.; VORSTER, J. Pruning intensity influences growth, flower and fruit development of Moringa oleifera Lam. under sub-optimal growing conditions in Gauteng, Development of Israeli mango cultivars Analysis of variance and mean test for gas exchange of the Omer and Shelly Israeli mango (Mangifera indica) cultivars as a function of number of branches after each formative pruning(1). Pesq. agropec. bras., Brasília, v.58, e03173, 2023 DOI: 10.1590/S1678-3921.pab2023.v58.03173 Development of Israeli mango cultivars 9 Figure 3. Water use efficiency values for the Omer and Shelly Israeli mango (Mangifera indica) cultivars as a function of number of branches after the fourth formative pruning. Bars with the same lowercase letters do not differ for cultivars Omer and Shelly within each number of branches after formative pruning. Bars with the same uppercase letters do not differ for number of branches within each Israeli mango cultivar by Tukey’s test, at 5% probability. aA aA aA bB bA 0.000 0.005 0.010 0.015 0.020 0.025 0.030 Three Four Five Water use eﬃciency ( mol CO mmol H O) 2 2 -1 Branches Omer Shelly aA aA aA bB bA 0.000 0.005 0.010 0.015 0.020 0.025 0.030 Three Four Five Water use eﬃciency ( mol CO mmol H O) 2 2 -1 Branches Omer Shelly Shelly under the cultivation conditions of the São Francisco Valley. Acknowledgments To Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), for financing, in part, this study (Finace Code 001); and to Fazenda Le Bourdet, for the infrastructure and support in conducting the experiment. Conclusions (Ed.). Methods in carbohydrate chemistry. New York: Academic Press, 1962. p.380-394. MUDO, L.E.D.; LOBO, J.T.; CARREIRO, D. de A.; CAVACINI, J.A.; SILVA, L. dos S.; CAVALCANTE, Í.H.L. Leaf gas exchange and flowering of mango sprayed with biostimulant in semi- arid region. Revista Caatinga, v.33, p.332-340, 2020. DOI: https://doi.org/10.1590/1983-21252020v33n206rc. IUSS WORKING GROUP WRB. World Reference Base for Soil Resources 2014: international soil classification system for naming soils and creating legends for soil maps: update 2015. Rome: FAO, 2015. (FAO. World Soil Resources Reports, 106). OLDONI, F.C.A.; LIMA, A.M.N.; CAVALCANTE, Í.H.L.; SOUSA, K. dos S.M. de; CARNEIRO, M.A.; CARVALHO, I.R.B. de. Boron fertilizing management on fruit production and quality of mango cv. Palmer in semiarid. Revista Brasileira de Fruticultura, v.40, e-622, 2018. DOI: https://doi.org/10.1590/0100-29452018622. KAVATI, R. Manejo da parte aérea da mangueira. In: ROZANE, D.E.; DAREZZO, R.J.; AGUIAR, R.L.; AGUILERA, G.H.A.; ZAMBOLIM, L. Manga: produção integrada, industrialização e comercialização. Viçosa: UFV, 2004. p.303-320. QIU, Z.; ZHU, L.; HE, L.; CHEN, D.; ZENG, D.; CHEN, G.; HU, J.; ZHANG, G.; REN, D.; DONG, G.; GAO, Z.; SHEN, L. ZHANG, Q.; GUO, L.; QIAN, Q. DNA damage and reactive oxygen species cause cell death in the rice local lesions 1 mutant under high light and high temperature. New Phytologist, v.222, p.349-365, 2019. DOI: https://doi.org/10.1111/nph.15597. KHANUM, Z.; TIZNADO-HERNÁNDEZ, M.E.; ALI, A.; MUSHARRAF, S.G.; SHAKEEL, M.; KHAN, I.A. Adaptation mechanism of mango fruit (Mangifera indica L. cv. Chaunsa White) to heat suggest modulation in several metabolic pathways. Royal Society of Chemistry Advances, v.10, p.35531-35544, 2020. DOI: https://doi.org/10.1039/d0ra01223h. R CORE TEAM. R: a language and environment for statistical computing. Vienna: R Foundation for Statistical Computing, 2022. LAVI, U.; KAUFMAN, D.; SHARON, D.; GAZIT, S.; TOMER, E. 'Shelly': a new mango cultivar. HortScience, v.32, p.138-138, 1997. DOI: https://doi.org/10.21273/HORTSCI.32.1.138. RICHARDSON, A.; EYRE, V.; KASHUBA, P.; ELLINGHAM, D.; JENKINS, H.; NARDOZZA, S. Early shoot development affects carbohydrate supply and fruit quality of red-fleshed Actinidia chinensis var. chinensis 'Zes008'. Agronomy, v.11, art.66, 2021. DOI: https://doi.org/10.3390/agronomy11010066. LI, H.; WEI, M.; DONG, L.; HU, W.; XIONG, J.; SUN, Y.; SUN, Y.; YAO, S.; GONG, H.; ZHANG, Y.; HOU, Q.; WANG, X.; XIE, S.; ZHANG, L.; AKRAM, M.A.; RAO, Z.; DEGEN, A. A.; NIKLAS, K.J.; YE, J.-S.; DENG, J. Leaf and ecosystem water use efficiencies differ in their global-scale patterns and drivers. Agricultural and Forest Meteorology, v.319, art.108919, 2022. DOI: https://doi.org/10.1016/j.agrformet.2022.108919. ROSSI, A. de; FACHINELLO, J.C.; RUFATO, L.; PARISOTTO, E.; PICOLOTTO, L.; KRUGER, L.R. Conclusions 1. The Shelly and Omer Israeli mango (Mangifera indica) cultivars show a good development during formation pruning under the growing conditions of the semiarid São Francisco Valley, Brazil. South Africa. South African Journal of Botany, v.129, p.448- 456, 2020. DOI: https://doi.org/10.1016/j.sajb.2019.11.033. DUBOIS, M.; GILLES, K.A.; HAMILTON, J.K.; REBERS, P.A.; SMITH, F. Colorimetric method for determination of sugars and related substances. Analitycal Biochemistry, v.28, p.350-356, 1956. DOI: https://doi.org/10.1021/ac60111a017. 2. Cultivar Omer is more vigorous for the conditions of the São Francisco Valley, but Shelly is able to adapt later regarding water use efficiency. FITCHETT, J.; GRAB, S.W.; THOMPSON, D.I. Temperature and tree age interact to increase mango yields in the Lowveld, South Africa. South African Geographical Journal, v.98, p.105-117, 2016. DOI: https://doi.org/10.1080/03736245.2014.924874. 3. The formative pruning with three, four, and five branches is indicated for mango cultivars Omer and Pesq. agropec. bras., Brasília, v.58, e03173, 2023 DOI: 10.1590/S1678-3921.pab2023.v58.03173 M.J.K.L. Andrade et al. 10 da Produção Integrada de Manga. Petrolina: Embrapa Semi- Árido, 2003. 72p. (Embrapa Semi-Árido. Documentos, 183). GOLLAN, P.J.; ARO, E.-M. Photosynthetic signalling during high light stress and recovery: targets and dynamics. Philosophical Transactions of the Royal Society B, v.375, art.20190406, 2020. DOI: https://doi.org/10.1098/rstb.2019.0406. GOLLAN, P.J.; ARO, E.-M. Photosynthetic signalling during high light stress and recovery: targets and dynamics. Philosophical Transactions of the Royal Society B, v.375, art.20190406, 2020. DOI: https://doi.org/10.1098/rstb.2019.0406. LOPES, R. de C.; PEREIRA, R.N.; SILVA, L. dos S.; LOBO, J.T.; AMARIZ, R.A. e; CAVALCANTE, Í.H.L. Impact of first mechanical fructification pruning on mango orchards. International Journal of Fruit Science, v.21, p.1059-1072, 2021. DOI: https://doi.org/10.1080/15538362.2021.1989358. HASEEB, G.M.; GHOUNIM, I.E.-S.; HMMAM, I.; MUSTAFA, M.R. Evaluation of four newly introduced mango (Mangifera indica L.) cultivars grown under El-Giza conditions. Plant Archives, v.20, p.9405-9410, 2020. LUO, Y.Y.; LI, R.X.; JIANG, Q.S.; BAI, R.; DUAN, D. Changes in the chlorophyll content of grape leaves could provide a physiological index for responses and adaptation to UV‐C radiation. Nordic Journal of Botany, v.37, p.2101-2115, 2019. DOI: https://doi.org/10.1111/njb.02314. HASSAN, M.U.; RASOOL, T.; IQBAL, C.; ARSHAD, A.; ABRAR, M.; ABRAR, M.M.; HABIB-UR-RAHMAN, M.; NOOR, M.A.; SHER, A.; FAHAD, S. Linking plants functioning to adaptive responses under heat stress conditions: a mechanistic review. Journal of Plant Growth Regulation, v.41, p.2596-2613, 2022. DOI: https://doi.org/10.1007/s00344-021-10493-1. MOUCO, M.A. do C.; LIMA NETO, F.P. A mangueira no Vale do São Francisco. Boletim Frutícola, v.1, p.1-11, 2018. HODGE, J.E.; HOFREITER, B.T. Determination of reducing sugars and carbohydrates. In: WHISTLER, R.L.; WOLFROM, M.L. Pesq. agropec. bras., Brasília, v.58, e03173, 2023 DOI: 10.1590/S1678-3921.pab2023.v58.03173 Conclusions Comportamento do pessegueiro 'Granada' sobre diferentes porta-enxertos. Revista Brasileira de Fruticultura, v.26, p.446-449, 2004. DOI: https://doi.org/10.1590/S0100-29452004000300018. LIMA NETO, F.P. Mangueira: melhoramento genético, variedades e mercado. In: SIMPÓSIO ONLINE DE FRUTICULTURA, 2020, Brasília. Simpósio. Brasília: SBF: SBCTA: Embrapa, 2020. SANCHES, L.G.; SANTOS, A.J. da S.; CARREIRO, D. de A.; CUNHA, J.G. da; LOBO, J.T.; CAVALCANTE, Í.H.L.; PAIVA NETO, V.B. de. Biochemical responses in 'Kent' mango grown in Brazilian semi-arid region under different doses of triacontanol. Revista Brasileira de Engenharia Agrícola e Ambiental, v.27, p.309-316, 2023. DOI: https://doi.org/10.1590/1807-1929/agriambi. v27n5p309-316. LIPAN, L.; CARBONELL-PEDRO, A.A.; CÁRCELES RODRÍGUEZ, B.; DURÁN-ZUAZO, V.H.; TARIFA, D.F.; GARCÍA-TEJERO, I.F.; GÁLVEZ RUIZ, B.; CUADROS TAVIRA, S.; MUELAS, R.; SENDRA, E.; CARBONELL- BARRACHINA, Á.A.; HERNÁNDEZ, F. Can sustained deficit irrigation save water and meet the quality characteristics of mango? Agriculture, v.11, art.448, 2021. DOI: https://doi.org/10.3390/agriculture11050448. SANJAY, K.S.; SANJAY, K.S.; RAM, R.S. Effects of pruning intensity on the biochemical status of shoot buds in three mango (Mangifera indica L.) cultivars planted at high density. The Journal of Horticultural Science and Biotechnology, v.85, LOPES, P.R.C.; HAJI, F.N.P.; MOREIRA, A.N.; MATTOS, M.A. de A. (Ed.). Normas técnicas e documentos de acompanhamento LOPES, P.R.C.; HAJI, F.N.P.; MOREIRA, A.N.; MATTOS, M.A. LOPES, P.R.C.; HAJI, F.N.P.; MOREIRA, A.N.; MATTOS, M.A. de A. (Ed.). Normas técnicas e documentos de acompanhamento de A. (Ed.). Normas técnicas e documentos de acompanhamento Pesq. agropec. bras., Brasília, v.58, e03173, 2023 DOI: 10.1590/S1678-3921.pab2023.v58.03173 Development of Israeli mango cultivars 11 v.46, p.442-449, 2016. DOI: https://doi.org/10.1590/1983- 40632016v4642829. p.483-490, 2010. DOI: https://doi.org/10.1080/14620316.2010.115 12702. p.483-490, 2010. DOI: https://doi.org/10.1080/14620316.2010.115 12702. YANHUI, C.; HONGRUI, W.; BEINING, Z.; SHIXING, G.; ZIHAN, W.; YUE, W.; HUIHUI, Z.; GUANGYU, S. Elevated air temperature damage to photosynthetic apparatus alleviated by enhanced cyclic electron flow around photosystem I in tobacco leaves. Ecotoxicology and Environmental Safety, v.204, art.111136, 2020. DOI: https://doi.org/10.1016/j. ecoenv.2020.111136. SANTOS, M.R. dos; MARTINEZ, M.A.; DONATO, S.L.R. Gas exchanges of 'Tommy Atkins' mango trees under different irrigation treatments. Bioscience Journal, v.29, p.1141-1153, 2013. SILVA, F.C. da. (Ed.). Manual de análises químicas de solos, plantas e fertilizantes. 2.ed. rev. e ampl. Brasília: Embrapa Informação Tecnológica, 2009. 627p. SOUZA, M.A. de; MÉSQUITA, A.C.; SIMÕES, W.L.; FERREIRA, K.M.; ARAUJO, E.F.J. Physiological and biochemical characterization of mango tree with paclobutrazol application via irrigation. Pesquisa Agropecuária Tropical, ZHANG, H.; ZHU, J.; GONG, Z.; ZHU, J.-K. Abiotic stress responses in plants. Nature Reviews Genetics, v.23, p.104-119, 2022. DOI: https://doi.org/10.1038/s41576-021-00413-0. Pesq. agropec. bras., Brasília, v.58, e03173, 2023 DOI: 10.1590/S1678-3921.pab2023.v58.03173
https://openalex.org/W2009405377	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0050626&type=printable	English	null	Assessing Risk Factors for Migraine: Differences in Gender Transmission	PloS one	2,012	cc-by	3,819	Introduction sensitivity to environmental stressors or a greater genetic loading for migraine [10,11]. Complex diseases have a high impact on human health and a high population incidence [1]. Migraine, a highly prevalent disease, is one example among many others [2]. Migraine with (MA) and without aura (MO) are the most common forms of this disease. It is a disabling disease leading to a diminished quality of life in both migraineurs and their relatives [3]. Our aim now was to assess which specific factors from our migraine families are contributing to the increased risk for this disorder, taking into account that observations within the same family are not independent. Carolina Lemos1,2, Isabel Alonso1,2, Jose´ Barros3, Jorge Sequeiros1,2, Jose´ Pereira-Monteiro1,2,3, Denisa Mendonc¸a2,4, Alda Sousa1,2 1 UnIGENe, Instituto Biologia Molecular Celular, Universidade do Porto, Porto, Portugal, 2 Instituto Cieˆncias Biome´dicas Abel Salazar, Universidade do Porto, Porto Portugal, 3 Servic¸o de Neurologia, Centro Hospitalar do Porto, Hospital de Santo Anto´nio, Porto, Portugal, 4 Instituto de Sau´de Pu´blica, Universidade do Porto, Porto Portugal Materials and Methods The consistent finding of an increased risk for relatives of migraineurs suggests that genetic factors may be implicated in the most common forms of the disease [2]. We also found a substantial familial risk of migraine for first-degree relatives in a sample of Portuguese migraineurs, which has led us to conclude that migraine could be strongly due to genetic factors [4]. Abstract Aim: Our aim was to assess which specific factors are contributing to an increased risk of migraine in a group of 131 Portuguese families. Methods: We studied 319 first-degree relatives, using a multilevel approach to account for the dependency among members from the same family. We included in the model relative’s gender, the proband’s gender and age-at-onset, to evaluate if any of these variables were associated with relative’s affection status. We also included in the model proband’s migraine subtype. We further assessed female and male transmissions within the proband nuclear family. Results: Relatives’ gender was found to be a risk factor for migraine (Odds Ratio = 2.86; 95% CI = 1.75–4.67), with females at a higher risk. When splitting probands according to their migraine subtype, we found that none of the variables studied contributed to relatives of MA-probands affection-status. Our results also show a significant difference between proband’s transmission and the gender of the parents and offspring. Conclusions: With this study, we showed that gender is truly a risk factor for migraine and that a gender-biased transmission is also observed. This reinforce the importance of identifying genes associated with migraine that are modulated by genes located in the sex chromosomes and the study of mitochondrial DNA or X-chromosome and hormonal-related effects associated with migraine susceptibility. Editor: Gianluigi Forloni, ‘‘Mario Negri’’ Institute for Pharmacological Research, Italy Received August 30, 2012; Accepted October 22, 2012; Published November 21, 2012 Copyright: 2012 Lemos 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 study was supported by grants of Fundac¸a˜o para a Cieˆncia e Tecnologia, FCT (POCTI-034390/99/FCT)and Sociedade Portuguesa de Cefaleias. CL was the recipient of a FCT fellowship (SFRH/BD/17761/2004). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding was received for this study. Competing Interests: The authors have declared that no competing interests exist. E-mail: clclemos@ibmc.up.pt Assessing Risk Factors for Migraine: Differences in Gender Transmission Carolina Lemos1,2*, Isabel Alonso1,2, Jose´ Barros3, Jorge Sequeiros1,2, Jose´ Pereira-Monteiro1,2,3, Denisa Mendonc¸a2,4, Alda Sousa1,2 Data Collection Sample collection was described in a previous study of our group [4]. A diagnostic interview of probands and first-degree relatives (parents, sibs and offspring) was performed using the same structured questionnaire based on the diagnostic criteria of the ‘‘International Headache Society’’ (IHS). To avoid a selection bias towards affected relatives, family members were contacted regard- less of the information provided by the proband about their affection status. The first edition of the IHS criteria (ICHD-I) [12] was used before 2004; when revising the diagnosis using the In a previous study, a lower age at onset in probands was found to be a predictor of migraine familial aggregation [5]. Lifetime prevalence of migraine is increased in females compared to males, with a female:male ratio ranging from 2:1 to 4:1 in several populations [6,7,8,9]. Several hypotheses have been raised for this female predominance such as neurobiological factors, increased November 2012 \| Volume 7 \| Issue 11 \| e50626 1 PLOS ONE \| www.plosone.org Gender as a Migraine’s Risk Factor Table 1. Risk factors of migraine: results from the multilevel model, adjusted for relative’s age at observation (Odds ratio, 95% CI). Table 1. Risk factors of migraine: results from the multilevel model, adjusted for relative’s age at observation (Odds ratio, 95% CI). Table 1. Risk factors of migraine: results from the multilevel model, adjusted for relative’s age at observation (Odds ratio, 95% CI). Variables Migraine Probands MO Probands MA Probands Proband’s gender 0.79 (0.39–1.62) 0.61 (0.22–1.74) 1.16 (0.41–3.31) Proband’s age at onset 0.75 (0.42–1.33) 0.50 (0.23–1.10) 1.21 (0.48–3.09) Relative’s gender 2.86 (1.75–4.67) 4.15 (2.06–8.34) 1.86 (0.90–3.83) Proband’s migraine subtype 0.78 (0.45–1.36) – – Relative’s gender, proband’s gender and age-at-onset were included in the model as possible predictors of relatives’ affection status. Proband’s migraine subtype was also included as a predictor in an additional model. doi:10.1371/journal.pone.0050626.t001 Relatives. Interviews were conducted with 182 first-degree relatives of MO-probands (114 women and 68 men) and 137 first- degree relatives of MA-probands (79 women and 58 men). second edition (ICHD-II) [13] no differences were found in patients’ diagnosis (data not shown). second edition (ICHD-II) [13] no differences were found in patients’ diagnosis (data not shown). In MO-probands group, 116 were affected while 66 were non- migraineurs. In the MA-probands-group, 81 family members were affected while 56 were not. Age of relatives was included in the model to adjust for this variable, since migraine is an age- dependent trait. Data Collection From the total of first-degree relatives, 112 were parents, 139 were siblings whereas 68 were offspring. Risk Factors After adjusting for the remaining variables, relatives’ gender was found to be a risk factor for migraine (OR = 2.86; 95% CI = 1.75– 4.67) (Table 1), with females first-degree relatives at higher risk than males. Variables related with proband’s, gender and age-at- onset were not risk factors for migraine (p.0.05). When introducing in the model proband’s migraine subtype this variable also did not influence relative’s affection status (p.0.05). We also assessed female and male transmissions within the proband nuclear family. We excluded from this analysis bilinear transmissions, i.e. transmissions where both the mother and the father were affected. When splitting probands according to their migraine subtype, we found that none of the variables studied contributed to relatives of MA-probands affection-status. Conversely, gender of relatives of MO-probands was associated with their affection status (OR = 4.15; 95% CI = 2.06–8.34) (Table 1). Maternal and Paternal Transmissions Relatives’ gender was found to be a risk factor for migraine, with females being at a 3-fold higher risk than males. Therefore we hypothesized that gender ratio in migraine families could be due to a biased transmission. In order to explore this hypothesis we analysed female and male transmissions within the proband’s nuclear family. Our result showed a significant difference between proband’s transmission and the gender of the offspring (Table 2) as we found that daughters are more affected than expected (X2 (1) = 6.91, p = 0.009). We included in the model relative’s gender, proband’s gender and age-at-onset as independent variables, to evaluate if any of these variables were associated with relative’s affection status in our families. We also included in the model proband’s migraine subtype and relative’s age-at-observation. Independent variables were adjusted by analyzing their possible effects on the outcome (affection status) altogether in the model. The multilevel general- ised linear model was fit using MLwiN 1.10 software. ( ) We also found that mothers of probands are more affected than expected when compared to proband’s fathers (X2 (1) = 22.41, p,0.001) (Table 2). Categorical data were compared by a chi-square test, using SPSS version 16.0 for Windows. A 5% significance level was used in all analyses. We also compared the ratio of affected fathers, mothers and siblings. We found a higher ratio of affected mothers (78%) and siblings (61%) than affected fathers (30%), with mothers being affected two to three times more than fathers. Ethics Statement Participants gave their written informed consent and the Ethics Committee of CHP,HSA approved the project. Data Analysis Probands were classified according to their migraine subtype. Proband’s age at onset was dichotomized (,16, 16+ years) according to the criterion of Stewart et al [5]. Relatives were divided into three groups according to their age-at-observation (,30, 30–59, 60+ years) in order to obtain a homogeneous distribution of the individuals in each group, and were also stratified by gender, since migraine is an age and gender- dependent trait. Statistical Analysis A multilevel generalised linear analysis using a logit model for a binary variable of outcome (affected/non-affected) was con- ducted to account for the non-independency among members from the same family, with relatives nested within the families. The multilevel modelling has been described as an advantageous tool for modelling data with a hierarchical structure [14]. Demographic Data Although mtDNA may not explain by itself the gender differences found, since migraine is a complex disease with several genetic factors involved, variants in mtDNA or in nuclear genes affecting mitochondrial mechanisms could influence migraine susceptibility. Also it has been suggested that an impairment of mitochondrial metabolism could lower the threshold for migraine attacks [17]. As suggested by Boles et al, we assessed if offspring of probands would be differentially affected and in fact we found that daughters of probands are more affected than expected. Other authors found evidence of a migraine susceptibility locus on chromosome 6 which could explain the daughters’ increased frequency [18,19,20,21]. Another hypothesis is a sex-conditioned genetic model with sex chromosomes influencing the expression of genes in autosomes, which may explain the different prevalence in female and male family members [22]. This was already observed in other human traits as genetic baldness, where both males and females are affected but with different ratios. Furthermore, specific inter-chromosome interactions have been observed in some neurological diseases where the mutation responsible for the disease located in one chromosome may modify the expression of genes located in another chromosome [22]. Additionally, in our sample we found an enrichment of the G allele of rs6951030 in the STX1A gene for female migraineurs only, which reinforces a gender-specific susceptibility in migraine [23]. Our findings showed that, as expected, females had a higher risk of migraine than men. Our next aim was then to assess if there was a gender-biased transmission. We found that mothers of probands were more frequently affected than expected. This biased trans- mission could be explained by a maternally inherited factor such as mitochondrial DNA (mtDNA) [15,16]. We also found that the ratio of affected probands’ fathers is lower than the ratio of affected mothers and siblings which is an evidence in favour of a maternally inherited factor, according to Boles et al [15]. Although mtDNA may not explain by itself the gender differences found, since migraine is a complex disease with several genetic factors involved, variants in mtDNA or in nuclear genes affecting mitochondrial mechanisms could influence migraine susceptibility. Also it has been suggested that an impairment of mitochondrial metabolism could lower the threshold for migraine attacks [17]. Acknowledgments We would like to thank all patients and their families for participating in this study. Migraine presents different gender thresholds, with males having a higher threshold [11,24]. Female steroids play an important role in migraine pathophysiology and can also explain the differential gender ratio found for this disorder since they are involved in mechanisms related in migraine pathophysiology, such as in neuronal excitability, in the synthesis and release of nitric oxide (NO) and neuropeptides such as calcitonin-gene related Demographic Data With this study, we reinforce the importance of identifying genes associated with migraine that are modulated by genes located in the sex chromosomes and the study of mtDNA or X- chromosome and hormonal-related effects. These studies will be crucial to bring some light into migraine’s susceptibility and in particular, gender-specific liability. Author Contributions Conceived and designed the experiments: CL AS. Performed the experiments: CL JB JPM. Analyzed the data: CL IA DM AS. Contributed reagents/materials/analysis tools: JPM JS AS. Wrote the paper: CL AS. Acquired individual and family data: JB JPM. Review the manuscript for intellectual content: IA JB JS JPM DM AS. 5. Stewart WF, Bigal ME, Kolodner K, Dowson A, Liberman JN, et al. (2006) Familial risk of migraine: variation by proband age at onset and headache severity. Neurology 66: 344–348. 6. Cucurachi L, Devetak M, Torelli P, Lambru G, Manzoni GC (2006) Gender ratio of migraine without aura: observations over time. Neurol Sci 27: 47–50. 7. Tellez-Zenteno JF, Garcia-Ramos G, Zermeno-Pohls F, Velazquez A (2005) Demographic, clinical and comorbidity data in a large sample of 1,147 patients with migraine in Mexico City. J Headache Pain 6: 128–134. 8. Radtke A, Neuhauser H (2009) Prevalence and burden of headache and migraine in Germany. Headache 49: 79–89. Demographic Data Our findings regarding risk factors for migraine subtypes lead us also to hypothesize that in our sample, gender-risk factors may not be associated with MA susceptibility, while in MO, hormonal events could influence the risk of having migraine, showing a conjunction of environmental and genetic factors, as suggested previously by Russell et al [28]. Although our sample size can be a limitation of our study, we had some special concerns in the design of the study and in the sample ascertainment. In this study we have taken into account intrafamilial correlations, by using a statistical analysis which corrects for this fact. Male probands are in small number due to a lower frequency of migraine in males and although the number of first-degree relatives of male probands is smaller than female probands, this was not due to an ascertainment bias, since probands were selected regardless of their family history. Furthermore, as described previously [4], some first-degree relatives were contacted by telephone, a method that has been described as a valid instrument [9,29] and that circumvent the possibility of an unbalanced ratio of affected females coming to interviews. Hence, by telephone, both females and males were contacted, avoiding a female preponderance bias. g [ ] As suggested by Boles et al, we assessed if offspring of probands would be differentially affected and in fact we found that daughters of probands are more affected than expected. Other authors found evidence of a migraine susceptibility locus on chromosome 6 which could explain the daughters’ increased frequency [18,19,20,21]. Another hypothesis is a sex-conditioned genetic model with sex chromosomes influencing the expression of genes in autosomes, which may explain the different prevalence in female and male family members [22]. This was already observed in other human traits as genetic baldness, where both males and females are affected but with different ratios. Furthermore, specific inter-chromosome interactions have been observed in some neurological diseases where the mutation responsible for the disease located in one chromosome may modify the expression of genes located in another chromosome [22]. Additionally, in our sample we found an enrichment of the G allele of rs6951030 in the STX1A gene for female migraineurs only, which reinforces a gender-specific susceptibility in migraine [23]. 1. Rannala B (2001) Finding genes influencing susceptibility to complex diseases in the post-genome era. Am J Pharmacogenomics 1: 203–221. 2. Wessman M, Terwindt GM, Kaunisto MA, Palotie A, Ophoff RA (2007) Migraine: a complex genetic disorder. Lancet Neurol 6: 521–532. 3. Terwindt GM, Ferrari MD, Tijhuis M, Groenen SM, Picavet HS, et al. (2000) The impact of migraine on quality of life in the general population: the GEM study. Neurology 55: 624–629. 4. Lemos C, Castro MJ, Barros J, Sequeiros J, Pereira-Monteiro J, et al. (2009) Familial clustering of migraine: further evidence from a Portuguese study. Headache 49: 404–411. 4. Lemos C, Castro MJ, Barros J, Sequeiros J, Pereira-Monteiro J, et al. (2009) Familial clustering of migraine: further evidence from a Portuguese study. Headache 49: 404–411. 3. Terwindt GM, Ferrari MD, Tijhuis M, Groenen SM, Picavet HS, et al. (2000) The impact of migraine on quality of life in the general population: the GEM study. Neurology 55: 624–629. Demographic Data Probands. A total of 131 probands were enrolled in this study (104 women and 27 men, mean age 6 SD, 34.4612.7 years). Regarding age-at-onset, 60 probands had an age-at-onset below 16 years old, while 71 showed migraine after that age. When classifying probands according to their migraine subtype, 85 probands had MO while 46 had MA. Familial aggregation is well established for common migraine, nevertheless, several familial factors may be contributing for these increased risk. Our aim was to search for migraine predictors in a sample of Portuguese migraine families. In a previous study, a lower age at onset in probands was associated with relative’s affection status [5]; in contrast, in our PLOS ONE \| www.plosone.org November 2012 \| Volume 7 \| Issue 11 \| e50626 2 November 2012 \| Volume 7 \| Issue 11 \| e50626 Gender as a Migraine’s Risk Factor Table 2. Distribution of affected and non-affected offspring and parents according gender. Table 2. Distribution of affected and non-affected offspring and parents according gender. Offspring Affected (%) Non-affected (%) Total Parents Affected (%) Non-affected (%) Total Daughters 27 (75) 9 (25) 36 Mothers 60 (78) 17 (22) 77 Sons 14 (44) 18 (56) 32 Fathers 11 (31) 24 (69) 35 doi:10.1371/journal.pone.0050626.t002 peptide (CGRP). Also, the serotonergic, adrenergic and GABA- ergic systems are also modulated by female steroids. Furthermore, some variants in female hormones receptors, such as estrogens and progesterone receptors have been found to be associated with migraine susceptibility [25,26,27]. study, the proportion of affected relatives was independent of proband’s age at onset. After adjusting for the remaining variables, gender was found to be the only risk factor for migraine, while proband’s age at onset and relative’s age at contact were not. p g g Our findings showed that, as expected, females had a higher risk of migraine than men. Our next aim was then to assess if there was a gender-biased transmission. We found that mothers of probands were more frequently affected than expected. This biased trans- mission could be explained by a maternally inherited factor such as mitochondrial DNA (mtDNA) [15,16]. We also found that the ratio of affected probands’ fathers is lower than the ratio of affected mothers and siblings which is an evidence in favour of a maternally inherited factor, according to Boles et al [15]. 2. Wessman M, Terwindt GM, Kaunisto MA, Palotie A, Ophoff RA (2007) Migraine: a complex genetic disorder. Lancet Neurol 6: 521–532. 1. Rannala B (2001) Finding genes influencing susceptibility to complex diseases in the post-genome era. Am J Pharmacogenomics 1: 203–221. 8. Radtke A, Neuhauser H (2009) Prevalence and burden of headache and migraine in Germany. Headache 49: 79–89. 7. Tellez-Zenteno JF, Garcia-Ramos G, Zermeno-Pohls F, Velazquez A (2005) Demographic, clinical and comorbidity data in a large sample of 1,147 patients with migraine in Mexico City. J Headache Pain 6: 128–134. Gender as a Migraine’s Risk Factor Gender as a Migraine’s Risk Factor 9. Russell MB, Rasmussen BK, Thorvaldsen P, Olesen J (1995) Prevalence and sex-ratio of the subtypes of migraine. Int J Epidemiol 24: 612–618. 20. Anttila V, Kallela M, Oswell G, Kaunisto MA, Nyholt DR, et al. (2006) Trait components provide tools to dissect the genetic susceptibility of migraine. Am J Hum Genet 79: 85–99. 10. Aloisi AM (2003) Gonadal hormones and sex differences in pain reactivity. Clin J Pain 19: 168–174. 21. Anttila V, Nyholt DR, Kallela M, Artto V, Vepsalainen S, et al. (2008) Consistently replicating locus linked to migraine on 10q22-q23. Am J Hum Genet 82: 1051–1063. 11. Low NC, Cui L, Merikangas KR (2007) Sex differences in the transmission of migraine. Cephalalgia 27: 935–942. 12. (1988) Headache Classification Committee of the International Headache Society. Classification and diagnostic criteria for headache disorders, cranial neuralgias and facial pain. Cephalalgia 8: 1–96. 22. Wang XP, Liu JM, Zhao YB (2008) Migraine: sex-influenced trait model? Med Hypotheses 71: 14–21. 23. Lemos C, Pereira-Monteiro J, Mendonca D, Ramos EM, Barros J, et al. (2010) Evidence of syntaxin 1A involvement in migraine susceptibility: a Portuguese study. Arch Neurol 67: 422–427. g p p g 13. (2004) Headache Classification Subcommittee of the International Headache Society. The International Classification of Headache Disorders, 2nd edition. Cephalalgia 24 (suppl 1): 1–160. 24. Kidd KK, Spence MA (1976) Genetic analyses of pyloric stenosis suggesting a specific maternal effect. J Med Genet 13: 290–294. p g ( pp ) 14. Greenland S (2000) Principles of multilevel modelling. Int J Epidemiol 29: 158– 167. 25. Oterino A, Toriello M, Cayon A, Castillo J, Colas R, et al. (2008) Multilocus analyses reveal involvement of the ESR1, ESR2, and FSHR genes in migraine. Headache 48: 1438–1450. 15. Boles RG, Gardner A (2008) Sex ratios and mitochondrial genetics in migraine. Cephalalgia 28: 1001–1002; author reply 1002. Cephalalgia 28: 1001–1002; author reply 1002. 16. Montagna P (2000) Molecular genetics of migraine headaches: a review. Cephalalgia 20: 3–14. 26. Colson NJ, Lea RA, Quinlan S, MacMillan J, Griffiths LR (2004) The estrogen receptor 1 G594A polymorphism is associated with migraine susceptibility in two independent case/control groups. Neurogenetics 5: 129–133. 17. Sparaco M, Feleppa M, Lipton RB, Rapoport AM, Bigal ME (2006) Mitochondrial dysfunction and migraine: evidence and hypotheses. Cephalalgia 26: 361–372. independent case/control groups. Neurogenetics 5: 129–133. 27. November 2012 \| Volume 7 \| Issue 11 \| e50626 References 3 PLOS ONE \| www.plosone.org November 2012 \| Volume 7 \| Issue 11 \| e50626 PLOS ONE \| www.plosone.org Gender as a Migraine’s Risk Factor Colson NJ, Lea RA, Quinlan S, MacMillan J, Griffiths LR (2005) Investigation of hormone receptor genes in migraine. Neurogenetics 6: 17–23. 27. Colson NJ, Lea RA, Quinlan S, MacMillan J, Griffiths LR (20 of hormone receptor genes in migraine. Neurogenetics 6: 17– 18. Nyholt DR, Curtain RP, Griffiths LR (2000) Familial typical migraine: significant linkage and localization of a gene to Xq24–28. Hum Genet 107: 18–23. 28. Russell MB, Iselius L, Olesen J (1996) Migraine without aura and migraine with aura are inherited disorders. Cephalalgia 16: 305–309. 29. Launer LJ, Terwindt GM, Ferrari MD (1999) The prevalence and character- istics of migraine in a population-based cohort: the GEM study. Neurology 53: 537–542. 19. Nyholt DR, Dawkins JL, Brimage PJ, Goadsby PJ, Nicholson GA, et al. (1998) Evidence for an X-linked genetic component in familial typical migraine. Hum Mol Genet 7: 459–463. PLOS ONE \| www.plosone.org November 2012 \| Volume 7 \| Issue 11 \| e50626 PLOS ONE \| www.plosone.org 4
https://openalex.org/W2963845396	https://europepmc.org/articles/pmc6639924?pdf=render	English	null	Hallux valgus correction utilising a modified short scarf osteotomy with a magnesium biodegradable or titanium compression screws – a comparative study of clinical outcomes	BMC musculoskeletal disorders	2,019	cc-by	6,207	(2019) 20:334 (2019) 20:334 Atkinson et al. BMC Musculoskeletal Disorders (2019) 20:334 https://doi.org/10.1186/s12891-019-2717-7 Atkinson et al. BMC Musculoskeletal Disorders https://doi.org/10.1186/s12891-019-2717-7 Open Access Hallux valgus correction utilising a modified short scarf osteotomy with a magnesium biodegradable or titanium compression screws – a comparative study of clinical outcomes Henry Dushan Atkinson1,2* , Shahnawaz Khan2, Yasha Lashgari2 and Andreas Ziegler3 Henry Dushan Atkinson1,2* , Shahnawaz Khan2, Yasha Lashgari2 and Andreas Ziegle Abstract Background: Biodegradable implants reduce the likelihood of further surgery for hardware removal and reduce the risks of associated infection and allergy. The purpose of this study is to evaluate the clinical efficacy and determine the comparability of biodegradable magnesium alloy MgYREZr (MAGNEZIX® CS) compression screw fixation compared with standard titanium screw fixation in the surgical treatment of hallux valgus deformity. Methods: Eleven patients undergoing corrective surgery for hallux valgus utilising biodegradable magnesium screws and a control group of 25 patients undergoing corrective hallux valgus surgery with standard titanium screws were reviewed at a median of 19 months (range 12–30 months). PROM scores (Manchester-Oxford Foot Questionnaire (MOXFQ), Foot and Ankle Outcomes Instrument (FAOI) and the EQ-5D-3 L) were recorded preoperatively and at latest follow-up. Results: The results between the two groups were broadly similar, with the Magnesium and Titanium patients showing similar patterns in the various domains in the MOXFQ, the FAOI and the EQ-5D-3 L. Most patients reported a near full shoe comfort score, and EQ-5D-3 L scores were significantly improved in both patient groups (with most patients reporting a full score). Foot pain and foot function improved irrespective of the scoring systems and patients in both groups demonstrated significantly improved scores following the surgery (p < 0.05). Notably, there were no significant differences when comparing the post-operative scores between the groups for any individual scoring parameter. No impairment to quality of life was recorded. There were no intra or post-operative complications. There were no problems encountered through the use of the bioabsorbable screws. Conclusion: Biodegradable magnesium-based compression screws appeared to be safe in this study and are an effective fixation device in the treatment of hallux valgus deformity with clinical outcomes similar to standard titanium screw fixation. Keywords: Hallux valgus, Magnesium, Biodegradable implant, Bioabsorbable implant, Bunion, Scarf osteotomy, Metatarsal displacement osteotomy * Correspondence: dusch1@gmail.com 2North Middlesex University Hospital, Sterling Way, London N18 1QX, UK Full list of author information is available at the end of the article Background demonstrating a hallux valgus deformity. Inclusion cri- teria were patients who were keen to try the biodegrad- able device in place of a routine titanium screw, and the availability of the magnesium screws on the day of sur- gery. Patients underwent thorough informed consent and accepted that metal screws might still have been used if the osteotomy fixation was not deemed adequate. Patients also understood that metal staples would be used for an Akin closing wedge proximal phalangeal osteotomy if this was performed. Exclusion criteria were patients with moderate or severe radiological hallux rigi- dus, those with complex foot deformities requiring sur- gery involving multiple toes, and those who had clear radiographic signs of osteopenia. The results were com- pared with a control group of 25 consecutive patients undergoing corrective surgery using standard titanium screws for isolated hallux valgus deformity at the same centre over the same period. All patients were operated only by the senior author in order to minimise variation in the surgical technique. The Institutional review board approved the study protocol, and this study was carried out in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. g Magnesium plates and screws were first used experi- mentally in the fixation of osteotomies in 1938 [1, 2], though their use remains very limited. MAGNEZIX® CS was the first compression screw to obtain CE marking and was approved for clinical use in 2013. The literature assessing their clinical efficacy is currently limited [3, 4]. The advantages of magnesium-based biodegradable screws have been demonstrated in animal trials [5, 6]. Studies identifying their safe use in humans have also been performed [4, 7–11]. The authors of some human in-vivo studies have suggested that the magnesium potentiates osteoblastic activity and provides a biomechanically stable construct promoting osteo-synthesis [6, 10, 12–16]. Magnesium plates and screws were first used experi- mentally in the fixation of osteotomies in 1938 [1, 2], though their use remains very limited. MAGNEZIX® CS was the first compression screw to obtain CE marking and was approved for clinical use in 2013. The literature assessing their clinical efficacy is currently limited [3, 4]. The advantages of magnesium-based biodegradable screws have been demonstrated in animal trials [5, 6]. Studies identifying their safe use in humans have also been performed [4, 7–11]. Background The authors of some human in-vivo studies have suggested that the magnesium potentiates osteoblastic activity and provides a biomechanically stable construct promoting osteo-synthesis [6, 10, 12–16]. Currently, most standard compression screws are composite alloys of non-absorbable materials, typically titanium and steel [17]. The implants that remain in-situ are not completely biologically inert and have the poten- tial to provoke inflammatory reactions and contact aller- gies well beyond the time of their insertion; nickel and aluminium allergies in particular have been widely re- ported [18–22]. Thus, when using these metal devices, there is a potential of them having to be removed through further surgery [23]. The risk of infection and inflammatory/allergic re- sponses can be potentially reduced through the use of bio- degradable magnesium-based implants [7, 9, 10, 20, 21]. Additionally, magnesium implants reduce artefact on CT scans and have limited noise on magnetic resonance im- aging, and therefore do not preclude the use of these im- aging modalities, if required [24]. © The Author(s). 2019 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. Atkinson et al. BMC Musculoskeletal Disorders (2019) 20:334 Atkinson et al. BMC Musculoskeletal Disorders (2019) 20:334 Page 2 of 8 Atkinson et al. BMC Musculoskeletal Disorders (2019) 20:334 Page 2 of 8 Surgical technique P i Patients were operated in a supine position under general anaesthesia or a local anaesthetic ankle block, with a single dose of intravenous antibiotics and an ankle tourniquet. A medial incision was used, and the capsule was longitudinally incised creating a long dor- sal and plantar capsular flap. A small “pocket” was created on the plantar aspect between the capsule and the plantar soft-tissues to facilitate sesamoid bal- ancing (on capsular closure). The bunion was re- moved using an oscillating saw, and any peripheral osteophytes were removed. An “inside” lateral release was performed though the osteotomy site, releasing the M. adductor hallucis and the lateral sesamoid ligaments. Hardware removal is among the most common of or- thopaedics procedures and represents a significant cost to the health economy at large. The risks of anaesthesia, scarring and infection are also increased with recurrent surgery [23, 25]. Foot and ankle implants are more com- monly associated with local soft tissue irritation due to their subcutaneous location and their use in weight bear- ing areas. Biodegradable implants reduce the likelihood of further surgery for hardware removal and reduce the risks of associated infection and allergy [3, 4, 8, 10]. g A distal 1st metatarsal “short scarf” osteotomy was performed in a routine fashion. The displacement/“shift” was held with a small towel clip and maintained with a 1.1 mm temporary Kirschner wire (K-wire). The correc- tion was verified under mini c-arm fluoroscopic guid- ance. The K-wire was then overdrilled using a 2.5 mm cannulated drill down to the far plantar cortex. The K- wire was then over drilled using the countersink (In2bones). A MAGNEZIX® CS (3.2 mm diameter) com- pression screw of an appropriate length was then inserted to maintain the osteotomy position, while also applying compression (Fig. 1). A “vest-over-pants” cap- sulorrhaphy of the medial capsule was then made. This paper assesses the clinical efficacy of biodegrad- able magnesium screws compared with standard titan- ium screws for hallux valgus corrective surgery using a short scarf displacement metatarsal osteotomy. Study design Eleven consecutive patients undergoing corrective sur- gery with MAGNEZIX® CS biodegradable screws for hal- lux valgus deformity over a 15-month period (February 2016–May 2017) were included in this single centre retrospective study. The indication for hallux valgus sur- gery was bunion-associated pain with radiographs Atkinson et al. BMC Musculoskeletal Disorders (2019) 20:334 Page 3 of 8 Fig. 1 MAGNEZIX® CS 3.2 Compression Screw (Source: Syntellix AG) Fig. 1 MAGNEZIX® CS 3.2 Compression Screw (Source: Syntellix AG) radiographic features of magnesium-based biodegrad- able screws. Bunion corrections in the control patient group were fixed using a single 3.0 mm diameter titanium compression screw, predrilled with a 2 mm drill over a 1 mm K-wire. Scoring systems Ten of the 11 Magnesium screw patients (91%) and 23 of the 25 Titanium screw patients (92%) also had an Akin closing wedge osteotomy of the 1st proximal phal- anx; which the lead author typically performs in the ma- jority of patients for the purposes of toe cosmesis. The Akin osteotomies were fixed using a titanium 8 mm 26° staple. Validated scoring instruments were used to assess pa- tient satisfaction and clinical efficacy of the surgery. The Manchester–Oxford Foot Questionnaire, and the Foot and Ankle Outcome Instrument were used to assess foot function, and the EQ-5D index was used to determine quality of life parameters [26–30]. Scores were recorded in accordance with published surveys and were Post-operative rehabilitation Fig. 2 a and b Pre-operative AP and lateral radiographs Patients were mobilised fully weightbearing in a heel- wedge shoe for 6 weeks, strictly elevating the foot for the first 7 days. Patients were encouraged to gently manipulate their great toes with dorsal and plantar stretches from 7 days. Skin sutures were removed after 2 weeks. After 6 weeks patients were clinically assessed, radiographs were taken, and patients were then mobilised in normal flat footwear. There were no adverse radiographic findings seen in any patient, and all patients were routinely discharged 6 weeks post-operatively. No patient required physiotherapy for toe stiffness. The authors could not justify rou- tinely repeating radiographs after 3, 6, and 12 months given the unnecessary patient exposure to radiation; unless there was a clinical indication to do so. Previ- ous small studies have looked at the radiological (X- ray) and MRI biodegradation of magnesium-based screws in the treatment of hallux valgus and have found that the screws completely degrade by 2–3 years after surgery without any sequelae [8–11]. Patients were contacted at a median of 19 months post-operatively, and at a minimum of 12 months (range 12–30 months) for the purposes of this study. Patients were interviewed by phone and all 36 patients returned their fully completed questionnaires. Fi 2 d b P i AP d l l di h One MAGNEZIX® patient who was re-referred back to our unit for other unrelated reasons had repeat ra- diographs taken of their operated foot at 12 months following the index surgery. This patient’s radiographs are presented in Figs. 2, 3 and 4 and show the typical Fig. 2 a and b Pre-operative AP and lateral radiographs Page 4 of 8 Atkinson et al. BMC Musculoskeletal Disorders (2019) 20:334 (2019) 20:334 Fig. 3 a and b Post-operative AP and lateral radiographs taken at six weeks follow up Fig. 4 a and b Post-operative AP and lateral radiographs taken at 12 months follow up Fig. 4 a and b Post-operative AP and lateral radiographs taken at 12 months follow up Fig. 3 a and b Post-operative AP and lateral radiographs taken at six weeks follow up were operated on a day-case basis. The mean operative time was 35 min. There were no intra-operative or post- operative complications noted. There were no wound- healing issues, no infections and no clinical adverse events to report. Post-operative rehabilitation Of the 25 titanium patients 23 were female (92%). Mean age at the time of surgery was 41 years (range (26–72 years). Right foot/left foot ratio was 16:9. Ten pa- tients had a mild (15–20 degree) and 15 a moderate (21–40 degree) hallux valgus deformity. Mean HVA was 27.3 degrees ±7.0 degrees All patients were operated on a day-case basis. The mean operative time was 34 min. There were no intra-operative or post-operative compli- cations noted. There were no wound-healing issues, no infections and no adverse events to report. No patient from either group required revision surgery or surgery to remove hardware. Fig. 3 a and b Post-operative AP and lateral radiographs taken at six weeks follow up performed by an independent observer. All patients gave their written informed consent to be included in the study. A patient information leaflet was provided. Statistics The Wilcoxon test for paired data was used for analysing non-parametric data, and a significance level of 0.05 was deemed significant. Hodges-Lehmann confidence inter- vals were calculated for preoperative – postoperative values. Statistical calculations were performed using R (r-project.org). Post-operative imaging at 6 weeks did not identify any loss of correction compared with the intra-operative im- aging in any patient. Radiological assessment did not identify any screw loosening or migration. The MAGNE- ZIX® implant is less radiopaque than a titanium screw. Figs. 2, 3 and 4 demonstrate the images from one of the patients; 1a) pre-operative AP and 1b) lateral radiograph, 2a) post-operative AP and 2B0 lateral radiographs at the 6 week review (demonstrating the hydrogen gas bubble), 3a) post-operative AP and 3b) lateral radiograph at 12 month review. Results In terms of foot function, the FAOI scale demon- strated a marked improvement from a mean of 82.82 to 96.89 (p < 0.004) in the MAGNEZIX® patient group (Table 1). Similar patterns were observed in the control group, where all the parameters also significantly improved. In the titanium patients, the MOXFQ score parameters for walking/standing, foot pain and social interaction (20.29.26.6,38.5 respectively) all improved significantly fol- lowing surgery (7.14, 5.2,4.75) with foot pain and social interaction similarly showing the greatest levels of improvement. The FAOI scale also significantly improved from a mean of 84.82 to 95.53 (p < 0.001) (Table 2). all improved significantly following surgery (6.82,10.45, 1.14) in the MAGNEZIX® patient group; with foot pain and social interaction showing the greatest levels of improve- ment. In terms of foot function, the FAOI scale demon- strated a marked improvement from a mean of 82.82 to 96.89 (p < 0.004) in the MAGNEZIX® patient group (Table 1). Similar patterns were observed in the control group, where all the parameters also significantly improved. In the titanium patients, the MOXFQ score parameters for walking/standing, foot pain and social interaction (20.29.26.6,38.5 respectively) all improved significantly fol- lowing surgery (7.14, 5.2,4.75) with foot pain and social interaction similarly showing the greatest levels of When comparing the magnesium and titanium patient groups, the clinical scores were broadly similar. Most patients reported a near full shoe comfort score, and EQ-5D-3 L scores were significantly improved in both patient groups (with most patients reporting a full score). Foot pain and foot function improved irrespective of the scoring systems and patients in both groups demon- strated significantly improved scores following the sur- gery (p < 0.05). Notably, there were no significant differences when comparing the post-operative scores Table 2 Preoperative and postoperative score values for each dimension of the Manchester-Oxford Foot Qu the Foot and Ankle Outcome Index (FAOI) and quality of life (EQ-5D-3 L) in the titanium patient cohort. N = 2 Table 2 Preoperative and postoperative score values for each dimension of the Manchester-Oxford Foot Questionnaire (MOXFQ), the Foot and Ankle Outcome Index (FAOI) and quality of life (EQ-5D-3 L) in the titanium patient cohort. Results Of the 11 MAGNEZIX® patients 9 were female (82%). Mean age at the time of surgery was 38 years (range (25–51 years). Right foot/left foot ratio was 6:5. Four pa- tients had a mild (15–20 degree) and 7 a moderate (21– 40 degree) hallux valgus deformity. Mean hallux valgus angle (HVA) was 28.7 degrees ±5.2 degrees. All patients The MOXFQ score parameters for walking/standing, foot pain and social interaction (33.44,36.36,42.61 respectively) Atkinson et al. BMC Musculoskeletal Disorders (2019) 20:334 Page 5 of 8 Atkinson et al. BMC Musculoskeletal Disorders (2019) 20:334 Page 5 of 8 Page 5 of 8 Table 1 Preoperative and postoperative score values for each dimension of the Manchester-Oxford Foot Questionnaire (MOXFQ), the Foot and Ankle Outcome Index (FAOI) and quality of life (EQ-5D-3 L) in the MAGNEZIX® patient cohort. N = 11 Score Pre-OP Post-OP Median diff pre – post P-value MOXFQ Walking/standing 33.44 ± 11.21 6.82 ± 8.06 26.79 (17.86–33.93) 0.004 Foot pain 36.36 ± 9.51 10.45 ± 9.34 25.00 (20.00–32.50) 0.004 Social interaction 42.61 ± 8.30 1.14 ± 2.53 40.63 (34.38–46.88) 0.004 Index 37.47 ± 8.47 6.14 ± 6.01 31.35 (25.95–36.07) 0.004 FAOI Core scale 82.82 ± 4.64 96.89 ± 2.91 −14.04 (−16.88 – −11.25) 0.004 Core scale mormative 41.62 ± 3.77 53.03 ± 2.36 −11.39 (−13.69 – −9.12) 0.004 Shoe comfort scale 43.64 ± 20.38 82.73 ± 24.12 −38.32 (−55.00 – −22.50) 0.004 Shoe norm 39.75 ± 6.91 53.00 ± 8.18 −13.27 (−18.64 – −7.63) 0.004 EQ-5D-3 L Index 0.83 ± 0.11 1.00 ± 0.00 −0.22 (−0.26 – −0.20) 0.012 VAS 80.00 ± 12.38 88.91 ± 11.84 −9.00 (−15.00 – −4.00) 0.006 Displayed are means ± standard deviations; Pre-op Preoperative, Post-op Postoperative; Median diff pre – post: difference of medians plus 95% Hodges-Lehmann confidence interval Table 1 Preoperative and postoperative score values for each dimension of the Manchester-Oxford Foot Questionnaire (MOXFQ), the Foot and Ankle Outcome Index (FAOI) and quality of life (EQ-5D-3 L) in the MAGNEZIX® patient cohort. N = 11 d postoperative score values for each dimension of the Manchester-Oxford Foot Questionnaire (MOXFQ), ome Index (FAOI) and quality of life (EQ-5D-3 L) in the MAGNEZIX® patient cohort. N = 11 all improved significantly following surgery (6.82,10.45, 1.14) in the MAGNEZIX® patient group; with foot pain and social interaction showing the greatest levels of improve- ment. Displayed are means ± standard deviations; Pre-op Preoperative, Post-op Postoperative; Median diff pre – post: difference of medians plus 95% Hodges-Lehmann confidence interval andard deviations; Pre-op Preoperative, Post-op Postoperative; Median diff pre – post: difference of medians plus 95% Hodges-Lehman Displayed are means ± standard deviations; Pre-op Preoperative, Post-op Postoperative; Median diff pre – post: difference of medians plus 95% Hodges-Lehmann confidence interval Results N = 25 Score Pre-OP Post-OP Median diff pre – post P-value MOXFQ Walking/standing 20.29 ± 13.35 7.14 ± 8.38 14.29 (10.71–17.86) 0.001 Foot pain 26.60 ± 13.05 5.2 ± 6.84 20.00 (17.50–25.00) 0.001 Social interaction 38.50 ± 17.56 4.75 ± 10.09 34.38 (28.12–40.63) 0.001 Index 28.46 ± 13.48 5.70 ± 7.56 22.80 (19.40–26.37) 0.001 FAOI Core scale 84.82 ± 5.25 95.53 ± 2.25 −10.71 (−12.50 – −8.75) 0.001 Core scale normative 43.25 ± 4.26 51.93 ± 1.82 −8.69 (−10.14 – −7.10) 0.001 Shoe comfort scale 41.60 ± 18.41 83.20 ± 22.12 −40.00 (−55.00 – −22.50) 0.001 Shoe normative 39.06 ± 6.24 53.16 ± 7.50 −13.56 (−16.95 – −11.02) 0.001 EQ-5D-3 L Index 0.86 ± 0.24 0.96 ± 0.12 −0.27 (−0.62 – −0.19) 0.014 VAS 81.52 ± 11.82 90.68 ± 9.45 −10.00 (−12.50 – −6.50) 0.001 Displayed are means ± standard deviations; Pre-op Preoperative, Post-op Postoperative; Median diff pre – post: difference of medians plus 95% Hodges-Lehmann confidence interval ostoperative score values for each dimension of the Manchester-Oxford Foot Questionnaire (MOXFQ), e Index (FAOI) and quality of life (EQ-5D-3 L) in the titanium patient cohort. N = 25 Page 6 of 8 Page 6 of 8 Atkinson et al. Discussion This small study has demonstrated that corrective surgery using a modified short scarf osteotomy is clinically effective for isolated minimal and moderate isolated bunion deformities with patients improving significantly in the MOXFQ, FAOI, and EQ-5D-3 L scoring modalities. This study has also demonstrated that the biodegradable MAGNEZIX® CS compression screw is clinically effective, and it was safely used for the fixation of a short scarf displacement metatarsal osteotomy in hallux valgus corrective surgery. The MAGNEZIX® CS compression screw has shown simi- lar results to the standard titanium fixation screws used in the control group, with clinical results also comparable with the literature [4, 8–11, 16]. The pa- tient reported outcomes in this study are also com- parable to existing data relating MAGNEZIX® screws; these other MAGNEZIX® studies also found that pa- tients suffered no long-term pain symptoms, no pro- cedural infections, no loss of fixation position and with high levels of satisfaction [7–10, 16, 31, 32]. This implant offers similar fixation to conventional ti- tanium screws, with the benefit of not requiring hardware removal [7, 16]. Routine implant removal represents a sig- nificant burden on the health economy and carries with it the risks of infection scarring and possible neurovascular injury [23, 25, 33]. Magnesium-based screws confer the advantage of biodegrading via corrosion rather than con- ventional implants that hydrolyse [4, 6, 10, 34, 35]. Corro- sion reduces the inflammatory response associated with screw absorption and is less irritant to surrounding tis- sues, thus minimising osteolysis [3, 4, 10]. Histological analyses performed in animal studies demonstrated the time course over which the implants degrade [2, 6, 36]. In one study at 12 months magnesium screws had com- pletely reabsorbed replaced by bony ingrowth and potas- sium crystals; importantly these potassium crystals are biologically inert and do not affect bone formation sug- gesting that once fully degraded the bone micro architec- ture ostensibly returns to normal [6]. Med diff: difference of medians plus 95% Hodges-Lehmann confidence interval; p p-value from Wilcoxon test for independent samples Med diff: difference of medians plus 95% Hodges-Lehmann confidence interval; p p-value from Wilcoxon test for independent samples between the groups for any individual scoring parameter (Table 3). Results BMC Musculoskeletal Disorders (2019) 20:334 Table 3 A comparison of magnesium and titanium patient groups pre-surgery (Pre), post-surgery (Post), and the changes between pre- and post-surgery (Post-pre) for each dimension and index for the Manchester-Oxford Foot Questionnaire (MOXFQ), the Foot and Ankle Outcome Index (FAOI) and quality of life (EQ-5D-3 L) Score / domain Comparison Med diff pre – post P MOXFQ Walking/standing Pre −14.29 (−25.00 – −3.57) 0.008 Post 0.00 (−3.57–3.57) 0.874 Post-pre 14.28 (7.14–21.43) 0.001 Foot pain Pre −10.00 (−20.00 – −5.00) 0.015 Post -5.00 (−15.00–0.00) 0.088 Post-pre 5.00 (0.00–10.00) 0.114 Social interaction Pre −6.25 (−18.75–6.25) 0.283 Post 0.00 (0.00–0.00) 0.416 Post-pre 6.25 (0.00–18.75) 0.068 Index Pre −11.13 (−19.76 – −0.77) 0.036 Post −0.48 (−6.19–2.38) 0.653 Post-pre 8.63 (3.15–14.17) 0.008 FAOI Core scale Pre 2.42 (−1.75 – −6.17) 0.229 Post −1.75 (−3.25–0.50) 0.099 Post-pre −3.42 (−6.67 – −0.08) 0.036 Core scale normative Pre 1.96 (−1.42–5.00) 0.229 Post −1.42 (−2.64–0.41) 0.099 Post-pre −2.77 (−5.41 – −0.07) 0.038 Shoe comfort scale Pre 0.00 (−15.00–10.00) 0.943 Post 0.00 (−10.00–10.00) 0.939 Post-pre 0.00 (−10.00–25.00) 0.931 Shoe normative Pre 0.00 (−5.08–3.39) 0.943 Post 0.00 (−3.39–3.39) 0.939 Post-pre 0.00 (−3.39–8.47) 0.904 EQ-5D-3 L Index Pre 0.052 (0.00–0.20) 0.233 Post 0.00 (0.00–0.00) 0.252 Post-pre −0.16 (−0.20–0.00) 0.079 VAS Pre 3.00 (−7.00–11.00) 0.667 Post 0.00 (−5.00–10.00) 0.931 Post-pre 0.00 (−5.00–6.00) 0.877 Med diff: difference of medians plus 95% Hodges-Lehmann confidence interval; p p-value from Wilcoxon test for independent samples worse levels of pre-operative symptoms in these specific parameters, while achieving similar post-operative scores. These included the MOXFQ walking/standing post-pre scores, and the MOXFQ post-pre index scores. Pre-operative MOXFQ foot pain was also greater in the magnesium patients. The authors highlight that this lar- ger improvement should not be interpreted as demon- strating any superiority of Magnesium fixation, as the final clinical scores are similar and not significantly dif- ferent between the two patient groups. Funding Funding No funds have been received for this study. Funding No funds have been received for this study. Consent for publication The patients (all adults) all gave their explicit written consent to have all their outcome data, images and demographics included in this review writeup and for publication. Ethics approval and consent to participate This is a retrospective review of a cohort of patients. All patients had undergone routine diagnosis and were treated with routine surgery. The patients were not randomised to the treatment, the treatment was not blinded. Each patient with the bioabsorbable screw willingly elected to the use of the new screw device after receiving printed information and after informed consent. The patients all gave their written consent to have all their outcome data, images and demographics included in this review writeup. This project received Ethical Approval from our Research Institution, The Sports Orthopaedics Research Foundation in 2017. Author details 1 1Sports Orthopaedics Research Foundation, 31 Old Broad Street, London EC2N 1HT, UK. 2North Middlesex University Hospital, Sterling Way, London N18 1QX, UK. 3University of KwaZulu-Natal, Pietermaritzburg, South Africa. Though very encouraging, this is a small cases series and prior to the widespread adoption of this device it re- mains imperative that equivalence is demonstrated in comparison to the current gold standard; and the au- thors would recommend and support a multicentre ran- domised prospective trial assessing its efficacy. Received: 23 May 2019 Accepted: 15 July 2019 Received: 23 May 2019 Accepted: 15 July 2019 Availability of data and materials All the data analyses from this study are included in this published article in the Tables 1, 2, 3. The raw datasets generated are not publicly available due a lack of an appropriate online repository but are available from the corresponding author on reasonable request. There were no complications or intraoperative tech- nical problems encountered, however, the authors would like to stress that the material characteristics of these biodegradable screws are different to those of the con- ventional metal screws. There is a learning curve in their use. The screws are not self-drilling or self-tapping, and the countersink drill must be used in every case; though pre-tapping is not necessary prior to screw insertion. The MAGNEZIX® CS compression screws have a Young’s modulus similar to natural bone, but lower than titanium screws. Hence, the screw heads are more prone to fatigue or fracture on insertion (compared with the conventional metal bi-compression screws), particularly if large torque is applied. These screws should be inserted with care and with reduced torque to prevent sheering of the implant. Once familiar we found that the handling of these screws was not greatly different to the metal screw equivalents, and once inserted the compres- sion and stability of the osteotomy appeared clinically comparable to that which we see with titanium implants. References 1. McBride E. Absorbable metal in bone surgery. J Am Med Assoc. 1938; 111(27):2464–7. 2. Song G, Atrens A. Understanding magnesium corrosion—a framework for improved alloy performance. Adv Eng Mater. 2003;5(12):837–58. 2. Song G, Atrens A. Understanding magnesium corrosion—a framework for improved alloy performance. Adv Eng Mater. 2003;5(12):837–58. Abbreviations CS C i CS: Compression screw; CT: Computed tomography; EQ-5D-3 L: EuroQol Group generic health status; FAOI: Foot and Ankle Outcomes Instrument; MOXFQ: Manchester-Oxford Foot Questionnaire; PROM: Patient Rated Outcome Measure 8. Plaass C, von Falck C, Ettinger S, Sonnow L, Calderone F, Weizbauer A, et al. Bioabsorbable magnesium versus standard titanium compression screws for fixation of distal metatarsal osteotomies - 3 year results of a randomized clinical trial. J Orthop Sci. 2018;23(2):321–7. Acknowledgements g The Authors would like to thank Mr. Albano Gega for his technical support and surgical assistance. The Authors would like to thank Mr. Albano Gega for his technical support and surgical assistance. 9. Acar B, Kose O, Turan A, Unal M, Kati YA, Guler F. Comparison of bioabsorbable magnesium versus titanium screw fixation for modified distal Chevron osteotomy in hallux valgus. Biomed Res Int. 2018:5242806. https:// doi.org/10.1155/2018/5242806. Competing interests AZ is statistical consultant to several medical device companies, including Procon Gmbh, Roche Diabetes Care, Sachtleben GmbH, Syntellix AG. The other authors declare that they have no competing interests. Conclusion improved alloy performance. Adv Eng Mater. 2003;5(12):837–58. 3. Seitz J, Eifler R, Bach F, Maier H. Magnesium degradation products: effects on tissue and human metabolism. J Biomed Mater Res A. 2013;102(10):3744–53. 4. Seitz J, Lucas A, Kirschner M. Magnesium-based compression screws: a novelty in the clinical use of implants. JOM. 2016;68(4):1177–82. The MAGNEZIX® CS compression screw is a biodegrad- able implant made of a magnesium alloy (MgYREZr). Fixation of displacement 1st metatarsal osteotomies in the surgical management of hallux valgus with the MAGNEZIX® CS metal biodegradable compression screw provides adequate fixation with good patient-re- ported outcomes comparable with more conventional metal fixation devices. p y p g ; ( ) 3. Seitz J, Eifler R, Bach F, Maier H. Magnesium degradation products: effects on tissue and human metabolism. J Biomed Mater Res A. 2013;102(10):3744–53. 4. Seitz J, Lucas A, Kirschner M. Magnesium-based compression screws: a 3. Seitz J, Eifler R, Bach F, Maier H. Magnesium degradation products: effects on tissue and human metabolism. J Biomed Mater Res A. 2013;102(10):3744–53. h b d 4. Seitz J, Lucas A, Kirschner M. Magnesium-based compression screws: a novelty in the clinical use of implants. JOM. 2016;68(4):1177–82. 5. Revell P, Damien E, Zhang X, Evans P, Howlett C. The effect of magnesium ions on bone bonding to hydroxyapatite coating on titanium alloy implants. Key Eng Mater. 2003;254-256:447–50. 6. Waizy H, Diekmann J, Weizbauer A, Reifenrath J, Bartsch I, Neubert V, et al. In vivo study of a biodegradable orthopedic screw (MgYREZr-alloy) in a rabbit model for up to 12 months. J Biomater Appl. 2014;28(5):667–75. 7. Windhagen H, Radtke K, Weizbauer A, Diekmann J, Noll Y, Kreimeyer U, et al. Biodegradable magnesium-based screw clinically equivalent to titanium screw in hallux valgus surgery: short term results of the first prospective, randomized, controlled clinical pilot study. Biomed Eng Online. 2013;12(1):62. Discussion The magnesium patients did however have signifi- cantly greater improvement in several of the “post-pre” scoring parameters in the MOXFQ; this was largely due the magnesium patients having suffered significantly During the natural corrosion process of magnesium, hydrogen is created, and this forms a temporary radio- lucency, either seen around the magnesium screw or in Page 7 of 8 Atkinson et al. BMC Musculoskeletal Disorders (2019) 20:334 Page 7 of 8 Atkinson et al. BMC Musculoskeletal Disorders (2019) 20:334 the neighbouring dorsal soft-tissues. This resolves of its own accord without sequelae [10]. Corrosion analysis in- dicates that MAGNEZIX implants hold stability for the initial 6–12 weeks and are completely corroded within 2–3 years [4, 9, 12, 16]. Funding No funds have been received for this study. Funding No funds have been received for this study. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 15. Li R, Kirkland N, Truong J, Wang J, Smith P, Birbilis N, et al. The influence of biodegradable magnesium alloys on the osteogenic differentiation of human mesenchymal stem cells. J Biomed Mater Res A. 2014;102(12):4346–57. 16. Kose O, Turan A, Unal M, Acar B, Guler F. Fixation of medial malleolar fractures with magnesium bio-absorbable headless compression screws: short-term clinical and radiological outcomes in eleven patients. Arch Orthop Trauma Surg. 2018;138(8):1069–75. 17. Staiger M, Pietak A, Huadmai J, Dias G. Magnesium and its alloys as orthopedic biomaterials: a review. Biomaterials. 2005;27(9):1728–34. 17. Staiger M, Pietak A, Huadmai J, Dias G. Magnesium and its alloys as orthopedic biomaterials: a review. Biomaterials. 2005;27(9):1728–34. 18. Lalor P, Revell P, Gray A, Wright S, Railton G, Freeman M. Sensitivity to titanium. A cause of implant failure? J Bone Joint Surg. 1991;73(1):25–8. 18. Lalor P, Revell P, Gray A, Wright S, Railton G, Freeman M. Sensitivity to titanium. A cause of implant failure? J Bone Joint Surg. 1991;73(1):25–8. 19. Wever D, Veldhuizen A, Sanders M, Schakenraad J, van Horn J. Cytotoxic, allergic and genotoxic activity of a nickel-titanium alloy. Biomaterials. 1997; 18(16):1115–20. 20. Hallab N, Merritt K, Jacobs JJ. Metal sensitivity in patients with Orthopaedic implants. J Bone Joint Surg Am. 2001;83(3):428–36. 21. Harloff T, Hönle W, Holzwarth U, Bader R, Thomas P, Schuh A. Titanium allergy or not? Impurity of titanium implant materials. Health. 2010;2(4):306–10. 21. Harloff T, Hönle W, Holzwarth U, Bader R, Thomas P, Schuh A. Titanium allergy or not? Impurity of titanium implant materials. Health. 2010;2(4):306–10. . Basko-Plluska JL, Thyssen JP, Schalock PC. Cutaneous and systemic 22. Basko-Plluska JL, Thyssen JP, Schalock PC. Cutaneous and systemic hypersensitivity reactions to metallic implants. Dermatitis. 2011;22(2):65–79. 23. Schepers T, Van Lieshout E, de Vries M, Van der Elst M. Complications of syndesmotic screw removal. Foot Ankle Int. 2011;32(11):1040–4. 24. Belenko L, Könneker S, Wacker F, von Falck C. Biodegradable magnesium Herbert screw in different modalities—image quality and artefacts; 2015. Poster presentation, C-2339, ECR p 25. Hanson B, van der Werken C, Stengel D. Surgeons' beliefs and perceptions about removal of orthopaedic implants. BMC Musculoskelet Disord. 2008;9(1):73. 26. Dawson J, Coffey J, Doll H, et al. A patient-based questionnaire to assess outcomes of foot surgery: validation in the context of surgery for hallux valgus. Qual Life Res. 2006;15(7):1211–22. 27. Authors’ contributions HA was the operating surgeon and Lead Author. AZ performed the statistical analyses. YL ran the patient questionnaires. SK and HA wrote and prepared the manuscript with input from AZ and YL. All the authors approved the final version of this manuscript. 10. Klauser H. Internal fixation of three-dimensional distal metatarsal I osteotomies in the treatment of hallux valgus deformities using biodegradable magnesium screws in comparison to titanium screws. Foot Ankle Surg. 2018;(18):30030–4. https://doi.org/10.1016/j.fas.2018.02.005. Page 8 of 8 Page 8 of 8 Atkinson et al. BMC Musculoskeletal Disorders (2019) 20:334 Page 8 of 8 Atkinson et al. BMC Musculoskeletal Disorders (2019) 20:334 (2019) 20:334 35. Ezechieli M, Ettinger M, König C, Weizbauer A, Helmecke P, Schavan R, et al. Biomechanical characteristics of bioabsorbable magnesium-based (MgYREZr-alloy) interference screws with different threads. Knee Surg Sports Traumatol Arthrosc. 2014;24(12):3976–81. 11. Choo JT, Lai SHS, Tang CQY, Thevendran G. Magnesium-based bioabsorbable screw fixation for hallux valgus surgery - a suitable alternative to metallic implants. Foot Ankle Surg. 2018;(18):30392–8. https://doi.org/10.1016/j.fas.2018.09.001. 12. Witte F, Kaese V, Haferkamp H, Switzer E, Meyer-Lindenberg A, Wirth C, et al. In vivo corrosion of four magnesium alloys and the associated bone response. Biomaterials. 2005;26(17):3557–63. 36. Erdmann N, Bondarenko A, Hewicker-Trautwein M, Angrisani N, Reifenrath J, Lucas A, et al. Evaluation of the soft tissue biocompatibility of MgCa0.8 and surgical steel 316L in vivo: a comparative study in rabbits. Biomed Eng Online. 2010;9(1):63. 36. Erdmann N, Bondarenko A, Hewicker-Trautwein M, Angrisani N, Reifenrath J, Lucas A, et al. Evaluation of the soft tissue biocompatibility of MgCa0.8 and surgical steel 316L in vivo: a comparative study in rabbits. Biomed Eng Online. 2010;9(1):63. 13. Witte F, Hort N, Vogt C, Cohen S, Kainer K, Willumeit R, et al. Degradable biomaterials based on magnesium corrosion. Curr Opinion Solid State Mater Sci. 2008;12(5–6):63–72. 14. Janning C, Willbold E, Vogt C, Nellesen J, Meyer-Lindenberg A, Windhagen H, et al. Magnesium hydroxide temporarily enhancing osteoblast activity and decreasing the osteoclast number in peri-implant bone remodelling☆. Acta Biomater. 2010;6(5):1861–8. 35. Ezechieli M, Ettinger M, König C, Weizbauer A, Helmecke P, Schavan R, et al. Biomechanical characteristics of bioabsorbable magnesium-based (MgYREZr-alloy) interference screws with different threads. Knee Surg Sports Traumatol Arthrosc. 2014;24(12):3976–81. 36. Erdmann N, Bondarenko A, Hewicker-Trautwein M, Angrisani N, Reifenrath J, Lucas A, et al. Evaluation of the soft tissue biocompatibility of MgCa0.8 and surgical steel 316L in vivo: a comparative study in rabbits. Biomed Eng Online. 2010;9(1):63. Publisher’s Note Dawson J, Doll H, Coffey J, Jenkinson C. Responsiveness and minimally important change for the Manchester-Oxford foot questionnaire (MOXFQ) compared with AOFAS and SF-36 assessments following surgery for hallux valgus. Osteoarthr Cartil. 2007;15(8):918–31. 28. Dawson J, Boller I, Doll H, et al. The MOXFQ patient-reported questionnaire: assessment of data quality, reliability and validity in relation to foot and ankle surgery. Foot (Edinb). 2011;21(2):92–102. 29. Dawson J, Boller I, Doll H, et al. Responsiveness of the Manchester-Oxford foot questionnaire (MOXFQ) compared with AOFAS, SF-36 and EQ5D assessments following foot or ankle surgery. J Bone Joint Surg (Br). 2012; 94(2):215–21. 30. Maher A, Kilmartin T. An analysis of Euroqol EQ-5D and Manchester Oxford foot questionnaire scores six months following podiatric surgery. J Foot Ankle Res. 2012;5(1):17. 31. Matsusue Y, Nakamura T, Suzuki S, Iwasaki R. Biodegradable pin fixation of osteochondral fragments of the knee. Clin Orthop Relat Res. 1996;322:166–73. 32. Modrejewski C, Plaaß C, Ettinger S, Caldarone F, Windhagen H, Stukenborg- Colsman C, von Falck C, Belenko L. Degradation behavior of bioresorbable magnesium implants in distal metatarsal 1 osteotomies in MRIDegradation behavior of magnesium-alloy srews after distal metatarsal osteotomies in MRI, Foot & Ankle 2015;13(3):156-61. 33. Brown OL, Dirschl DR, Obremskey WT. Incidence of hardware-related pain and its effect on functional outcomes after open reduction and internal fixation of ankle fractures. J Orthop Trauma. 2001;15(4):271–4. 34. Amass W, Amass A, Tighe B. A review of biodegradable polymers: uses, current developments in the synthesis and characterization of biodegradable polyesters, blends of biodegradable polymers and recent advances in biodegradation studies. Polym Int. 1998;47(2):89–144. 34. Amass W, Amass A, Tighe B. A review of biodegradable polymers: uses, current developments in the synthesis and characterization of biodegradable polyesters, blends of biodegradable polymers and recent advances in biodegradation studies. Polym Int. 1998;47(2):89–144.
https://openalex.org/W1550152596	https://www.intechopen.com/citation-pdf-url/32049	English	null	A New Scientific Formulation of Tajweed Rules for E-Learning of Quran Phonological Rules	InTech eBooks	2,012	cc-by	6,814	A New Scientific Formulation of Tajweed Rules for E-Learning of Quran Phonological Rules Yahya O. Mohamed Elhadj1, Mohamed Aoun-Allah2, Imad A. Alsughaiyer2 and Abdallah Alansari3 1Information Technology Deanship, 2College of Computer & Information Sciences, 3College of Arabic Language, Al-Imam Muhammad Ibn Saud Islamic University, Riyadh, Kingdom of Saudi Arabia Selection of our books indexed in the Book Citation Index in Web of Science™ Core Collection (BKCI) Interested in publishing with us? Contact book.department@intechopen.com Numbers displayed above are based on latest data collected. For more information visit www.intechopen.com Open access books available Countries delivered to Contributors from top 500 universities International authors and editors Our authors are among the most cited scientists Downloads We are IntechOpen, the world’s leading publisher of Open Access books Built by scientists, for scientists 14% 191,000 210M TOP 1% 154 7,200 12 www.intechopen.com 1. Introduction Recitation of the Holy Quran is governed by a variety of rules called "Tajweed rules" (Correct pronunciation of the Holy Quran). Reciting the Holy Quran in the appropriate way is very important for all Muslims and is indispensable in Islamic worshiping such as prayers. So, teaching how to recite it correctly was transmitted, since its revelation to the prophet (PBUH), orally from teachers to learners throughout generations. Such a method has been considered as the only way to learn it until the twentieth century, where technology produced recording systems and electronic devices that are able to keep both text and sound of the Quran with tajweed rules. Since then, it becomes possible to listen Quranic recitations recorded from authentic reciters. Many computer-programs have been then appeared to assist novice learners by listening while following corresponding text on the screen. However, efforts spent by the computer scientists in general for the sake of the noble Quran are still limited and have been concentrated only on the direct application of the Information Technology techniques, such as storing, listening, searching, etc, without using more elaborated techniques in the domain. This work is a part of a project aiming to build a computerized-environment for learning the Holy Quran and its sciences (Computerized teaching of the Holy Quran "CTHQ") (Elhadj et al., 2010a). Our objective in the CTHQ project was to improve computerization of the Holy Quran by introducing advanced techniques and methodologies. Four main tracks have been designed to carry out this project. In the first track, an environment for teaching how to memorize the Holy Quran in a manner similar to the usual way followed in the Quranic inculcation schools was proposed (Alsughayeir & Elhadj, 2006; Elhadj, 2010). In the second track, automatic speech recognition technologies have been used to teach how to recite the Holy Quran correctly (Alghamdi et al., 2007; Elhadj et al., 2012, 2010b, 2009; Elhadj, 2009a, 2009b). In the third track, techniques for determining the similarity (tashaboh) between verses (ayahs) of the noble Quran were investigated (Alsughayeir & Ohali, 2007). Computer- tools have been developed for analyzing the text of the noble Quran based on complete words and their stems in order to link similar verses (Alsughayeir et al., 2009; Elhadj et al., www.intechopen.com 198 E-Learning – Engineering, On-Job Training and Interactive Teaching 2009c, 2009d). 1. Introduction In the fourth track, our focus was on the automatic processing of tajweed rules by proposing a mathematical formulation of these rules that can be easily processed by a machine (Elhadj & Aoun-Allah, 2011; Elhadj et al., 2009e). In this chapter, we discuss how this kind of formulation was proposed, its advantages and benefits, and how it was programmed in an efficient plug-in engine. The development of an e-learning system for tajweed rules that uses this engine will also be presented. A general integrated environment for self-learning of the holy Quran and its sciences including Tajweed will be also briefly introduced. This chapter proceeds as follows. Sections 2, 3, and 4 deal with Tajweed where the first one presents an introduction. Section 3 surveys previous efforts to computerization of Tajweed whereas Section 4 concentrates on our new formulation. We discuss in Section 5 a prerequisite to our engine of automatic processing of Tajweed rules, which is detailed in Section 6. Our Tajweed Learning System (ETaj) will be presented in the Section 7 and a couple of its important interfaces will be given in Section 8. Finally, before concluding this chapter, we shortly present a global learning environment for Quran and its sciences composed of several subsystems including Tajweed teaching system. 2. Overview of Tajweed rules Reading of the Holy Quran is quite different from the reading of a normal Arabic text due to the pronunciation rules that have to be respected during the recitation. As an example, consider the duration of vowels that may vary from 2 to 6 times that of a single consonant depending on the context. Indeed, there are two kinds of vowels in the Arabic language, short vowels which are keystrokes placed either above or below the preceding consonant and long vowels which denotes a certain repetition of short vowels. There are also other tajweed rules related to: a. types of nasalization (called "ghunnah Δ ,)"غن a. types of nasalization (called "ghunnah Δ ,)"غن b. heaviness and lightness (called respectively "tafkheem "تفΨيمand "tarqeeq ,)"تήقيقwhich means making some sounds emphatic or non-emphatic, c. types of stops which means making a voiceless break at a Quranic word for a brief moment (called "waqf ,)"وقف d. degrees of vibrations or unrest (called "qalqalah ΔϠ )"قϠقwhich means producing the voiced stop consonants with a schwa-like sound at the end, e. etc. d. degrees of vibrations or unrest (called "qalqalah ΔϠ )"قϠقwhich means producing the voiced stop consonants with a schwa-like sound at the end, To preserve the Holy Quran from any alteration in its pronunciation and then to guarantee its perfect reading, early Muslim scholars described Quranic recitation methods very accurately by textual rules as well as sound ones. These descriptions are classified into categories depending on some sound properties or features. These categories are often summarized as follows: a. category "Noon sakinah" & "Tanween" ( )بΎΏ النوϥ السΎكنΔ والتنوين b. category "Meem sakinah" (Δ )بΎΏ الϤيم السΎكن c. category "Qalqalah" (ΔϠ )بΎΏ القϠق d. category "Tafkheem" & "Tarqeeq" ( )بΎΏ التفΨيم والتήقيق e. category “Al Mad” (lengthening ΪϤ ) بΎΏ ال f. etc. a. category "Noon sakinah" & "Tanween" ( )بΎΏ النوϥ السΎكنΔ والتنوين b. category "Meem sakinah" (Δ )بΎΏ الϤيم السΎكن c. category "Qalqalah" (ΔϠ )بΎΏ القϠق d. category "Tafkheem" & "Tarqeeq" ( )بΎΏ التفΨيم والتήقيق e. category “Al Mad” (lengthening ΪϤ ) بΎΏ ال f. etc. a. category "Noon sakinah" & "Tanween" ( )بΎΏ النوϥ السΎكنΔ والتنوين b. category "Meem sakinah" (Δ )بΎΏ الϤيم السΎكن www.intechopen.com 199 A New Scientific Formulation of Tajweed Rules for E-Learning of Quran Phonological Rules Each category has a set of rules governing the pronunciation of underlying sounds. 3. Previous efforts related to computerization of Tajweed Teaching how to apply Tajweed rules during Quranic recitation has been done through teachers who pronounce the Quranic sounds accurately. With the era of computers, it becomes possible to computerize the learning process of Tajweed, but this need an appropriate description of its rules. To the best of our knowledge, there was no attempt to describe them by a scientific algorithm, which could be processed automatically by a machine. This was a conclusion from a large survey we conducted on the Quranic software currently available on the market, which was published in (Alsughayeir & Elhadj, 2006). The methodology employed to conduct this study consisted of collecting a maximum number of Quranic applications, either for desktop, web-based or hand-held applications. Each application was separately studied and evaluated in order to know the characteristics and services it offers. It appeared that softwares developed for the sake of the Holy Quran are still very limited either in their objectives or in the term of availability and relevance of features. Regarding the Tajweed by itself, it was only given as a small part of the surveyed programs. It is offered, at almost all programs, in a classical manner as textual lessons with some graphical explanation of sound production. No dynamic interaction was proposed to allow, neither efficient learning nor any kinds of intelligent processing. 2. Overview of Tajweed rules More details regarding the classification of Tajweed rules can be obtained from the official site of King Fahd Complex for the Printing of Holy Quran at this link: www.qurancomplex.org. b. “C+1” or “C-1” represent respectively the character (or characters separated by a comma) immediately following (rep. preceding) the character treated. 4. New formulation of Tajweed rules In order to find a scientific formulation of tajweed rules, we studied them thoroughly with the assistance of an expert in the domain and finally we came out with the conclusion that almost all Tajweed rules described textually by scholars could be written in a scientific manner and consequently automatically treated by a computer. Our study concluded that to extract the tajweed rule for any letter the maximum number of words concerned by the rule is two; i.e. either the previous or the following word. And in terms of letters, there are at most 6 letters concerned by the rule which are either preceding or following the letter itself. Moreover, many rules are applicable only to a letter at the end of a word which is easily detectable by comparing the next letter to a space character. For instance, in the category of “Noon Sakinah” & “Tanween” if any letter “Noon Sakinah” or diacritic “Tanween” appears at the end of a word followed by any of these characters “ ي ”ϥ و ل ϡ رin the following word then the 2 letters should be assimilated. Moreover, this category is subdivided into two sections where the assimilation could be with “Ghunnah” (a sound effect) or without when the letter is followed respectively by “ ”ϥ و ϡ يor “ .”ل رWe proposed a whole scientific formulation of such textual description of rules into machine readable rules as indicated in the example in Table 1 taking in consideration the following notations: a. “C” means the character treated and “S” its diacritic b. “C+1” or “C-1” represent respectively the character (or characters separated by a comma) immediately following (rep. preceding) the character treated. www.intechopen.com 200 E-Learning – Engineering, On-Job Training and Interactive Teaching c. And in the same way we define “C+2”, “C+3”,… or “C-2”, “C-3”,… according to the extent of the rule. d. “” is used as a wildcard character to replace any letter or diacritic. y e. “Space” represents a space character (between two words). f. “Text” is human comprehensible message to be displayed to the user if needed. f. “Text” is human comprehensible message to be displayed to the user if needed. p g p y g. And finally, the “Ruling” of the rule which denotes its name. g. d a y, t e u g o t e u e c de otes ts a e. 4. New formulation of Tajweed rules C ϥ = ; S = ْ◌; C+1 = Space; S = ; C+2 ϥ,ϡ,ي,و = ;S= ; Text = تΪغم النوϥ السΎكنΔ بغ نΔ في حήوف )ي،و،ϡ،ϥ( Ruling = IdghamGhunnah C = ; S = ˲ ˱ ˳◌; C+1 = Space; S = * ; C+2 = ϥ,ϡ, ;ي,وS = * ; Text = يُΪغم التنوين بغنΔ في حήوف )ي،و،ϡ،ϥ( Ruling = IdghamGhunnah (Table 1.B) (Table 1.A) C-1 = * ; S = ˱◌ ; C = ; ϯ اS = * ; C+1 = Space ; S = * ; C+2 = ϥ,ϡ, ;ي,وS = * ; Text = يُΪغم التنوين بغنΔ في حήوف)ي،و،ϡ،ϥ( Ruling = IdghamGhunnah C = ϡ ; S = ْ◌ ; C+1 = Space ; S = * ; C+2 = ϡ ; S = * ; Text = ΎتُΪغم الϤيم في الϤيم إΩغΎم˱Ύ شفوي بغنΔ Ruling = IdghamGhunnah (Table 1.D) (Table 1.C) Table 1. Rules describing a couple of cases of assimilation with Ghunnah (Table 1.C) Table 1. Rules describing a couple of cases of assimilation with Ghunnah Table 1.A (resp. Table 1.B) describes a rule stating that if any character with a “tanween” diacritic (resp. a “Noon” character with “Sakn” diacritic) at the end of a word followed by a word starting by any of “ ”ϥ و ϡ يletters, then there is assimilation with Ghunnah. Table 1.D describes a special case of the rule in Table 1.B where “tanween” is followed by a “lenghthening” at the end of the word. Finally, Table 1.C deals with a “Meem” character with “Sakin” diacritic as last letter in a word followed by “Meem” character with any diacritic as first letter in the next word and where the ruling is the same as previously. Using this technique of writing “tajweed” rules, we finished describing those of 5 most important categories (or chapters): “Al Mad” (lengthening), “qalqala” (unrest), “Noon Sakinah” & “Tanween”, “Meem Sakinah”, and "Tafkheem" & "Tarqeeq". This results in a total number of almost 200 rules. Moreover, any advanced tajweed rule could be easily added by writing it as described earlier in the rules text file. Notice that we faced many difficulties in writing our rule set as described above. The most important difficulty was the overlap of rules. This situation happens when more than one rule could be triggered for one letter. www.intechopen.com 4. New formulation of Tajweed rules For instance, in the word “ ”السϤΎءwe have a lengthening of obligatory 4 cycles but when it is the last word in a verse it changes to optionally 2, 4 or 6 cycles. To overcome this overlap, we added to each rule a priority in order to avoid the conflict between triggered rules and by choosing only the rule with the highest priority. www.intechopen.com 201 A New Scientific Formulation of Tajweed Rules for E-Learning of Quran Phonological Rules 5. Need of a textual version of the Holy Quran with full diacritics The Holy Quran is generally written in a special font which could not be edited in text editor software. Consequently, a text editable version of the Holy Quran that contains no missing diacritic was necessary to get our automatic processing works correctly. However, this textual editable version as we need it was almost inexistent. Therefore, we looked for the most agreed version to which we manually added full diacritics and then it was given to scholars for revision and validation. 6. Automatic processing of Tajweed rules: Taj Engine (TajE) The Tajweed rule set described in section 4 is interpreted by an inference engine (TajE) able to deal with our rulings formulation and that can be used alone (See Figure 1) or integrated with other components for both identification and verification of Tajweed rules in any Quranic verse (or even any Arabic diacritized text). Fig. 1. Structure of the Taj Engine Taj Engine Tajweed Rules Quranic Verse Tajweed Rulings Taj Engine Tajweed Rulings Quranic Verse Tajweed Rules Fig. 1. Structure of the Taj Engine Fig. 1. Structure of the Taj Engine This inference engine works like an expert system inference engine in forward chaining and one level inference. In other words, for each character of the treated text, our engine tests all rules once to find the rule or those rules that could be triggered. Conflicting rules are treated by the rule priority explained in Section 4. As presented earlier, TajE engine could be used either for identification or for verification of Tajweed rulings. The first option (identification) aims to extract Tajweed rulings from Quranic verses. It is very helpful in different situation, especially to assist a student during his memorization of the Holy Quran. The second option (verification) of the TajE engine is to ensure if a Tajweed ruling really occurred in a specific place in the Quranic verse (See Figure 2). This is also a very important property as it can be used in an interactive manner for teaching and correcting Tajweed exercises. A detailed typical exercise scenario is described in the following section. www.intechopen.com E-Learning – Engineering, On-Job Training and Interactive Teaching 202 Fig. 2. Taj Engine used for verification Taj Engine Tajweed Rules Quranic Verse Errors and Explanations Proposed Tajweed Rulings Missing Tajweed Rulings Errors and Explanations Missing Tajweed Rulings Errors and Explanations Quranic Taj Engine Tajweed Rules Fig. 2. Taj Engine used for verification 7. Tajweed learning system: ETaj Our main objective is to design an easy to use e-learning system for Tajweed using our developed TajE engine. It is intended to be used by students to help them learning, verifying their knowledge, and to train them on Tajweed for the noble intention of correctly reciting the Holy Quran. The proposed system has been designed to include the following features: a. General Rules of Recitation and its Ethics: these are stored guidelines about general rules of recitation like position of stops and their rulings. Reading ethics like “Estiatha ΓΫΎ ”اإستعand “Basmalah ΔϠϤ ,”الΒسetc. are also included. ”اإستand “Basmalah ΔϠϤ ,”الΒسetc. are also included. b. Tajweed Rulings: this option offers to the student rich text lessons about Tajweed rulings along with examples in text and audio formats. The student could interact with by pausing, stopping or replaying the audio files. c. Exercises: they cover all Tajweed categories and are taken by student using TajE engine described in the previous section. A typical scenario is given below. Another option for examination is offered to students to pass examinations in a manner very similar to exercises but in a less helpful way and by assigning a final mark to the learner. Exercises and examination options of ETaj system are implemented as follows Exercises and examination options of ETaj system are implemented as follows: a. A student starts by choosing the Tajweed category he wants to practice or be tested on. b. According to his choice, all rulings under the category will be displayed with a specific color associated to each ruling. c. Besides, a verse or a couple of verses chosen randomly will be displayed too. We notice here that the selection of verses is totally random among all Quran verses and having a fixed minimum number of the selected category rulings. This property offers to our system a great dynamic aspect. The selection process is either done on the fly by searching the Holy Quran using first option of the TajE engine to extract verses with the minimum number of rules, or by selecting them from a database of ruling occurrences already filled using our TajE engine beforehand. Obviously, the first technique is slower than the second but requires less storage space. 7. Tajweed learning system: ETaj However, we chose the second www.intechopen.com 203 A New Scientific Formulation of Tajweed Rules for E-Learning of Quran Phonological Rules technique for the simple reason that is the fastest way since a non-negligible treatmen is done beforehand and also to guarantee a good response time for the system. technique for the simple reason that is the fastest way since a non-negligible treatment is done beforehand and also to guarantee a good response time for the system. g g p y d. At this moment, the student is invited to select letters where Tajweed rulings appear. This process is achieved by the learner by choosing a color (i.e. a category ruling) and then selecting the appropriate letters. The student is free to change or to remove his selections until he decides to validate his choices. d. At this moment, the student is invited to select letters where Tajweed rulings appear. This process is achieved by the learner by choosing a color (i.e. a category ruling) and then selecting the appropriate letters. The student is free to change or to remove his selections until he decides to validate his choices. e. Once validated, choices are treated automatically by TajE engine and a report is displayed to the student containing the incorrect and missing selections along with the necessary explanations (i.e. the field “Text” in the rule, See section 4). e. Once validated, choices are treated automatically by TajE engine and a report is displayed to the student containing the incorrect and missing selections along with the necessary explanations (i.e. the field “Text” in the rule, See section 4). Figures 3 and 4 show respectively the structure of the proposed ETaj system and its architecture. Moreover, we aim to add to the system a new feature allowing the user to specify the “narration” which will influence all the above features. We mention that our current system supports only “Hafs” narration. 8. Most important interfaces of the ETaj system ETaj has been designed to be easy to use and to allow great interaction with the user. It is implemented in the .Net framework using C# and JavaScript as well as other advanced technologies. Our database is implemented using MySql as a database management system as it is free and it offers very excellent features and performance. The most important interfaces of the system are introduced in the following sections. Fig. 3. Structure of the ETaj system ھϤزة الϮصل أحϜΎϡ التجϮيد آΩاΏ التاوة التϤΎرين مΨΎرج الحروف ϝϮ الϤقطϮع والϤϮص القϠقϠة التفΨيم والترقيق الϤد والقصر أحϜΎϡ الϤيم السΎكنة السΎكنةϥϮ النϡΎϜأح التجϮيد Fig. 3. Structure of the ETaj system www.intechopen.com E-Learning – Engineering, On-Job Training and Interactive Teaching 204 Fig. 4. Architecture of the ETaj system If we access the system, the following menu will appear to the user: Fig. 5. Main menu of the ETaj system www.intechopen.com E-Learning – Engineering, On-Job Training and Interactive Teaching 204 Fig. 4. Architecture of the ETaj system If we access the system, the following menu will appear to the user: Fig. 5. Main menu of the ETaj system If we access the system, the following menu will appear to the user: If we access the system, the following menu will appear to the user: Fig. 5. Main menu of the ETaj system www.intechopen.com 205 A New Scientific Formulation of Tajweed Rules for E-Learning of Quran Phonological Rules A New Scientific Formulation of Tajweed Rules for E-Learning of Quran Phonological Rules 205 From this main menu, we can navigate to different options in the system. If we click, for example one of the tajweed rulings, a page like the following is displayed: Fig. 6. Tajweed rulings page Three main parts are enclosed in this page: one for the tajweed category and its rulings that can be navigated one after another (Figure 6 (a)), the content of the current ruling (Figure 6 (b)), and examples of the ruling (Figure 6 (c)): Fig. 6(a). Tajweed category and its rulings From this main menu, we can navigate to different options in the system. If we click, for example one of the tajweed rulings, a page like the following is displayed: From this main menu, we can navigate to different options in the system. If we click, for example one of the tajweed rulings, a page like the following is displayed: Fig. 6. 8. Most important interfaces of the ETaj system Tajweed rulings page Three main parts are enclosed in this page: one for the tajweed category and its rulings that can be navigated one after another (Figure 6 (a)), the content of the current ruling (Figure 6 (b)), and examples of the ruling (Figure 6 (c)): Fig. 6. Tajweed rulings page Three main parts are enclosed in this page: one for the tajweed category and its rulings that can be navigated one after another (Figure 6 (a)), the content of the current ruling (Figure 6 (b)), and examples of the ruling (Figure 6 (c)): Three main parts are enclosed in this page: one for the tajweed category and its rulings that can be navigated one after another (Figure 6 (a)), the content of the current ruling (Figure 6 (b)), and examples of the ruling (Figure 6 (c)): Fig. 6(a). Tajweed category and its rulings Fig. 6(a). Tajweed category and its rulings www.intechopen.com E-Learning – Engineering, On-Job Training and Interactive Teaching 9. General integrated environment for self-learning of the Holy Quran The Tajweed learning system is a part of a fully integrated environment developed for self- learning of the holy Quran and its sciences. This environment comprises four subsystems in addition to the ETaj system: subsystem for learning how to recite and memorise the holy Quran, subsystem for studying the similarity between Quranic terms and verses, subsystem providing the most relevant features that learners of the holy Quran may need such as (Erab"Ώ ,"اإعήاNuzoul “ ,”اسΒΎΏ النزولTafseer “ή ,”التفسيEjaz“ ,”اإعجΎί العϠϤيetc.), and finally a reach library providing authentic sources from which the relevant features are taken. The three main subsystems will be briefly described in the following sections to get picture of this environment. E-Learning – Engineering, On-Job Training and Interactive Teaching 206 g g g g g Fig. 6(b). Content of a ruling Fig. 6(c). Examples of a tajweed ruling Now, if we click on the "exercises" option, we will be redirected to a page where we can choose the category of tajweed we want to practice (Figure 7): Fig. 6(b). Content of a ruling Fig. 6(b). Content of a ruling Fig. 6(c). Examples of a tajweed ruling Fig. 6(c). Examples of a tajweed ruling Fig. 6(c). Examples of a tajweed ruling Now, if we click on the "exercises" option, we will be redirected to a page where we can choose the category of tajweed we want to practice (Figure 7): Now, if we click on the "exercises" option, we will be redirected to a page where we can choose the category of tajweed we want to practice (Figure 7): Fig. 7. Exercise selection page Once the category is selected, a page like that of Figure 8 is displayed and the user is asked to navigate the category rulings and to select their occurrences in the ayah that has been randomly chosen. This is done in the same manner as explained previously in section 7. Fig. 7. Exercise selection page Once the category is selected, a page like that of Figure 8 is displayed and the user is asked to navigate the category rulings and to select their occurrences in the ayah that has been randomly chosen. This is done in the same manner as explained previously in section 7. Once the category is selected, a page like that of Figure 8 is displayed and the user is asked to navigate the category rulings and to select their occurrences in the ayah that has been randomly chosen. This is done in the same manner as explained previously in section 7. www.intechopen.com A New Scientific Formulation of Tajweed Rules for E-Learning of Quran Phonological Rules 207 Fig. 8. Exercise page Fig. 8. Exercise page Fig. 8. Exercise page After finishing determining places of ruling occurrences, a page result (see Figure 9) is displayed After finishing determining places of ruling occurrences, a page result (see Figure 9) is displayed Fig. 9. Page Result Fig. 9. Page Result Fig. 9. Page Result Fig. 9. Page Result 9.1 The memorization subsystem: E-halaga The E-halaga system is designed based-on the Quran memorization halaga “ϥ ”حϠقΎΕ تحفيظ القήآ approach (E-halaga means electronic halaga). It simulates the real one in having tutors, www.intechopen.com 208 E-Learning – Engineering, On-Job Training and Interactive Teaching supervisor, and registered students. E-halaga allows four main types of users with specific roles: administrator, supervisor, tutor, and student. Roles are distributed hierarchically to allow a great flexibility over the system. The creation of the E-halaga is done by its supervisor, which is in turn created by the system administrator. The supervisor is responsible of running the E-halaga in terms of adding or deleting tutors, distributing learners, etc. Since the E-halaga is a simulation of the real halaga, the learner have to specify a daily amount (memorization section) to be memorized starting from somewhere in the Quran (starting ayah/page). As the learner progresses, he needs to have in parallel a revision program for the memorized parts to improve their quality (revision section). So, the registration in the E-halaga system requires specifying the following parameters: starting ayah/page, memorization section and its length, revision section and its length (at least the double of the memorization section), and previously memorized sections before joining the system. Once registered, the learner will obtain a user name and password to access the system. If he logs into the system, he will be redirected to the memorization/revision part where he can find a list of useful options. He can browse his own profile and change it as needed, measure his performance (number of sections perfected, times of failure in every section, duration of memorizing), print out his transcript, etc. The learner can listen to an ideal recitation taken from one of the famous reciters stored in the system as a reference for perfect recitation. After listening to the reference recitation several times, or reciting directly, the learner can test his memory by clicking on an icon to record the section. Then the pages of the section will disappear to test his memory. Clicking on the button of “end recording” will display again the section pages allowing the learner to verify his memorization. The learner can repeat this process (recording and verifying) till the perfection of memorization. Once the learner approves his recording, the audio file is uploaded to the server and the learner will not be able to modify it. 9.1 The memorization subsystem: E-halaga The tutor is then notified with an unmarked recorded section of that learner. After the tutor marks the section, the learner can benefit from audio and/or textual remarks explaining his mistakes. Figure 10 shows the architecture of the E- halaga system and we invite the reader to consult (Elhadj, 2010) for more details. 9.2 The similarity search engine The similarity search engine is a component devoted to determine the similarity between Quranic verses (and Quranic terms). To build this system, we started preparing an authentic fully diacritised textual version of the Holy Quran since such one was not available for the use in the domain of research. Next, the focus was on a manual morphological analysis of this version. Each word in the Holy Quran is split into four parts, prefixes, stem, root and suffixes, and then stored in an indexed database. Words are kept in their original context, which means Quranic verses (Elhadj et al., 2009c, 2009d). A semi-automatic environment for morphological analysis has been developed (see figure 11). Full-text searching techniques were investigated and then computer-programs have been developed for analyzing the text of the noble Quran based on complete words and their stems in order to link similar verses (Alsughayeir & Ohali, 2007; Alsughayeir et al., 2009). Figure 12 shows the engine interface. www.intechopen.com 209 A New Scientific Formulation of Tajweed Rules for E-Learning of Quran Phonological Rules A New Scientific Formulation of Tajweed Rules for E Learning of Quran Phonological Rules 209 Fig. 10. E-halaga architecture Fig. 11. Interface of the Semi-automatic Morphological Analyzer Fig. 10. E-halaga architecture Fig. 10. E-halaga architecture Fig. 11. Interface of the Semi-automatic Morphological Analyzer Fig. 11. Interface of the Semi-automatic Morphological Analyzer www.intechopen.com E-Learning – Engineering, On-Job Training and Interactive Teaching 210 Fig. 12. Interface of the Similarity Search Engine Fig. 12(a). Interface of the Quran Sciences Subsystem 9.3 The Quran sciences subsystem It is a system letting the user navigates the Holy Quran and its main sciences. The main features offered are taken from authentic books, which were linked with the holy Quran through a full indexing of Quranic ayahs using advanced techniques and methodologies. Information are kept in their original sources (books), but can be easily retrieved and quickly processed. The books can be navigated, downloaded, etc., from a library, which represents another subsystem of this environment. Possibility of updates is well considered. Fig. 12. Interface of the Similarity Search Engine Fig. 12. Interface of the Similarity Search Engine Fig. 12(a). Interface of the Quran Sciences Subsystem Fig. 12(a). Interface of the Quran Sciences Subsystem Fig. 12(a). Interface of the Quran Sciences Subsystem 9.3 The Quran sciences subsystem It is a system letting the user navigates the Holy Quran and its main sciences. The main features offered are taken from authentic books, which were linked with the holy Quran through a full indexing of Quranic ayahs using advanced techniques and methodologies. Information are kept in their original sources (books), but can be easily retrieved and quickly processed. The books can be navigated, downloaded, etc., from a library, which represents another subsystem of this environment. Possibility of updates is well considered. www.intechopen.com 211 A New Scientific Formulation of Tajweed Rules for E-Learning of Quran Phonological Rules 11. Future works In near future, we plan to improve ETaj system by organizing tajweed rulings in levels with incremental degree of complexity as it is followed in the real teaching of tajweed. Learners have to go through levels one by one. At the end of each level a self examination or testing will be initiated by the system and the learner will not be allowed to go further until he masters the current level. This means that a kind of authentication needs to be added to the system to follow students in their process of learning. As another future work, we plan to expand our TajE engine to cover other Quranic narrations as we currently covered just "Hafs" narration. 12. Acknowledgements This work was supported by King Abdulaziz City for Sciences and Technology (www.kacst.edu.sa), as part of the CTHQ Project, under the grant number AT-25-113, Saudi Arabia. We thank all the other project team members, which actively participated in the elaboration of the whole project. 10. Conclusion In this chapter, we firstly presented our work related to the textual specification of Tajweed rules in order to come up with a machine-readable formulation of these rules. An appropriate and easily extendable rule set has then been proposed. A fully diacriticised textual version of the Holy Quran has also been prepared to be used with our new-tajweed formulations. Next, we focused on the implementation of these formulations in a fast and efficient plug-in Tajweed Engine (TajE) that can be integrated in different kinds of systems for teaching the Holy Quran and its sciences. TajE is able to handle rulings in two different ways: identification and verification. The identification option is intended to extract Tajweed rulings from Quranic verses. The verification option, may serve to ensure that a specific Tajweed ruling really occurred in a certain place in the Quranic verse, which is very useful. An e-learning system for self learning of tajweed (ETaj) is next built on the top of the tajweed engine (TajE). It provides different options allowing learners to get maximum benefit. The first important option of the system gives general rules and ethics of learning the Holy Quran. The second option offers a well designed component for learning tajweed rulings using a convenient and attractive manner of presenting the content of rulings along with textual examples as well as sound ones. The third option is very important as it provides a full dynamic interaction with the system to practice tajweed rulings. At our best knowledge, both TajE and ETaj systems are the first of their kinds developed for automatic processing of Tajweed in a full interactive manner. They are now ready to be used and can help mastering this important field of Quranic Sciences. 13. References Alghamdi, M., Elhadj, Y.O.M., & Alkanhal, M. (2007). Manual System to Segment and Transcribe Arabic Speech, Proceedings of IEEE/ICSPC'07, Dubai, UAE, November 24-27, 2007. Alghamdi, M., Elhadj, Y.O.M., & Alkanhal, M. (2007). Manual System to Segment and Transcribe Arabic Speech, Proceedings of IEEE/ICSPC'07, Dubai, UAE, November 24-27, 2007. www.intechopen.com 212 E-Learning – Engineering, On-Job Training and Interactive Teaching Alsughayeir, I.A., Khorsi, A.M., Alansari, A.M., & Ohali, Y.M.. (2009). Search Engine for the similarity in Quranic Terms (in Arabic), Proceedings of Int. Conf. on the Glorious Quran and Contemporary Technologies, King Fahd Complex for the Printing of the Holy Quran, Almadinah, Saudi Arabia, October 13-15, 2009. Alsughayeir, I.A. & Ohali, Y.M. (2007). Similarity in Quranic Terms: computer-study (in Arabic), Proceedings of ITRAS'07, Riyadh, Saudi Arabia, March 6-7, 2007. Alsughayeir, I.A. & Elhadj, Y.O.M. (2006). Computerized Quran Products: State-Of-Art (in Arabic), Proceedings of STCEX'06, Riyadh, Saudi Arabia, December 2-6, 2006. Elhadj, Y.O.M., Alghamdi, M., AlKanhal, M. & Alansari, A.M. (2012). Towards an Automatic Corrector of Quranic Recitation Integreated within an Environment for Self-Learning of the Holy Quran (In Arabic). To be appeared in Computer Research Journal published by the Federation of Arab Scientific Research Councils, Vol. 11, No.1. Elhadj, Y.O.M. & Aoun-Allah, M. (2011). A Machine-Readable Formulation of Tajweed Rules for Fast & Efficient Processing, Proceedings of the ICIST'11 International Conference, tebessa, Algeria, April 24-26, 2011 Elhadj, Y.O.M., Alsughayeir, I.A., Alghamdi, M., Alkanhal, M., Ohali, Y.M. & Alansari, A.M. (2010). Computerized teaching of the Holy Quran (in Arabic), Final Technical Report, King Abdulaziz City for Sciences and Technology (KACST), Riyadh, Saudi Arabia, 2010. Elhadj, Y.O.M., Alghamdi, M., AlKanhal, M. & Alansari, A.M. (2010). Automatic Recognition of Quranic Sounds in the Recitation (in Arabic), Proceedings of 6th Int. Conf. on Arabic Computing (ICCA10), Hammat – Tunisia, May 20-21, 2010. f p g y Elhadj, Y.O.M. (2010). E-Halagat: an E-Learning System for Teaching the Holy Quran, TOJET Journal, Vol. 9, No 1, 2010. Elhadj, Y.O.M. (2009). Sound Database with Perfect Reading of the Last Part of the Holy Quran, IJCSNS journal, Vol. 9, No. 7. Elhadj, Y.O.M. (2009). Preparation of speech database with perfect reading of the lat part of the Holy Quran (in Arabic), Proceedings of the 3rd IEEE International Conference on Arabic Language Processing (CITAL'09), Rabat, Morocco, May 4-5, 2009. Elhadj, Y.O.M., Alghamdi, M., Alkanhal, M. & Alansari, A.M. (2009). 13. References Sound Corpus of a part of the noble Quran (in Arabic), Proceedings of Int. Conf. on the Glorious Quran and Contemporary Technologies, King Fahd Complex for the Printing of the Holy Quran, Almadinah, Saudi Arabia, October 13-15, 2009. Elhadj, Y.O.M., Alsughayeir, I.A., Khorsi, A.M. & Alansari, A.M. (2009). Morphology Analysis of the Holy Quran (in Arabic), Journal of Computer Science and Engineering in Arabic, Vol. 3, No 1. Elhadj, Y.O.M., Alsughayeir, I.A., Khorsi, A.M. & Alansari, A.M. (2009). An Indexed Database for Quran Morphology (in Arabic), Proceedings of the 5th International Conference on Computer Science Practice in Arabic, Rabat –Morocco, May 10-11, 2009. Elhadj, Y.O.M., Aoun-Allah, M., Alansari, A.M. & Alsughayeir, I.A. (2009). Interactive learning System for Tajweed (in Arabic), Proceedings of Int. Conf. on the Glorious Quran and Contemporary Technologies, King Fahd Complex for the Printing of the Holy Quran, Almadinah, Saudi Arabia, October 13-15, 2009. www.intechopen.com www.intechopen.com E-Learning - Engineering, On-Job Training and Interactive Teaching Edited by Dr. Sergio Kofuji ISBN 978-953-51-0283-0 Hard cover, 238 pages Publisher InTech Published online 14, March, 2012 Published in print edition March, 2012 Adaptive E-learning was proposed to be suitable for students with unique profiles, particular interests, and from different domains of knowledge, so profiles may consider specific goals of the students, as well as different preferences, knowledge level, learning style, rendering psychological profile, and more. Another approach to be taken into account today is the self-directed learning. Unlike the adaptive E-learning, the Self- directed learning is related to independence or autonomy in learning; it is a logical link for readiness for E- learning, where students pace their classes according to their own needs.This book provides information on the On-Job Training and Interactive Teaching for E-learning and is divided into four sections. The first section covers motivations to be considered for E-learning while the second section presents challenges concerning E- learning in areas like Engineering, Medical education and Biological Studies. New approaches to E-learning are introduced in the third section, and the last section describes the implementation of E-learning Environments. How to reference In order to correctly reference this scholarly work, feel free to copy and paste the following: Yahya O. Mohamed Elhadj, Mohamed Aoun-Allah, Imada A. Alsughayeir and Abdallah Alansari (2012). A New Scientific Formulation of Tajweed Rules for E-Learning of Quran Phonological Rules, E-Learning - Engineering, On-Job Training and Interactive Teaching, Dr. Sergio Kofuji (Ed.), ISBN: 978-953-51-0283-0, InTech, Available from: http://www.intechopen.com/books/e-learning-engineering-on-job-training-and- interactive-teaching/a-new-scientific-formulation-of-tajweed-rules-for-e-learning-of-quran-phonological-rules InTech China Unit 405, Office Block, Hotel Equatorial Shanghai No.65, Yan An Road (West), Shanghai, 200040, China Phone: +86-21-62489820 Fax: +86-21-62489821 InTech China Unit 405, Office Block, Hotel Equatorial Shanghai No.65, Yan An Road (West), Shanghai, 200040, China Phone: +86-21-62489820 Fax: +86-21-62489821 InTech Europe University Campus STeP Ri Slavka Krautzeka 83/A 51000 Rijeka, Croatia Phone: +385 (51) 770 447 Fax: +385 (51) 686 166 www.intechopen.com © 2012 The Author(s). Licensee IntechOpen. This is an open access artic stributed under the terms of the Creative Commons Attribution 3.0 cense, which permits unrestricted use, distribution, and reproduction in ny medium, provided the original work is properly cited. © 2012 The Author(s). Licensee IntechOpen. This is an open access article distributed under the terms of the Creative Commons Attribution 3.0 License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
https://openalex.org/W1991297540	https://periodicos.uem.br/ojs/index.php/ActaSciBiolSci/article/download/6974/6974	English	null	Acute toxicity of pyrazosulfuron-ethyl and permethrin to juvenile Litopenaeus vannamei	Acta Scientiarum. Biological Sciences	2,011	cc-by	5,223	Acute toxicity of pyrazosulfuron-ethyl and permethrin to juvenile Litopenaeus vannamei Lemos de Mello1, Hilton Amaral Júnior1, Silvano Garcia1 and Luis Vinatea2* Giovanni Lemos de Mello1, Hilton Amaral Júnior1, Silvano Garcia1 and Lu 1Empresa de Pesquisa Agropecuária e Extensão Rural de Santa Catarina, Florianópolis, Santa Catarina, Brazil. 2Universidade Federal de Santa Catarina, Campus Reitor João David Ferreira Lima, s/n, 88040-970, Trindade, Florianópolis, Santa Catarina, Brazil.Author for correspondence. E-mail: luis.vinatea@pq.cnpq.br ABSTRACT. The objective of this study was to determine the LC50 (96h) of two pesticides: Sirius® 250 SC herbicide of the pyrazosulfuron-ethyl group, and Talcord® insecticide of the permethrin group, on juvenile Litopenaeus vannamei. Shrimp total hemocyte count (THC) was also determined as an indication of physiological alterations caused by the pesticides. Juvenile shrimp (5.0 ± 0.5 g) were exposed to the following concentrations: 0, 0.1, 1.0, 10, 100 and 1000 μg L-1 Sirius® 250 SC; and 0, 0.001, 0.01, 0.1, 1.0 and 10 μg L-1 Talcord®. The Talcord® LC50 (96h) was of 0.00933 μg L-1 or 9.33 ng L-1. There were no significant changes in the THC between control and test groups. No Sirius® 250 SC concentrations tested killed more than 50% of the shrimp; therefore, the herbicide was considered not toxic to the juveniles. However, the THC showed significant differences between the control and test groups, suggesting sublethal effects to L. vannamei juveniles. According to the results, the insecticide Talcord® is highly lethal for L. vannamei and the herbicide Sirius® 250 SC was not lethal in the concentrations tested but showed sublethal effects as lower THC. The results demonstrate the risks involved in farming L. vannamei shrimp near rice cultures where these pesticides are routinely used. Key words: toxicology, pesticides, shrimp culture, Litopenaeus vannamei. RESUMO. Toxicidade aguda de pirazossulfurom-etílico e permethrin em juvenis de camarão branco Litopenaeus vannamei. O objetivo deste trabalho foi determinar a CL50 (96h) dos agroquímicos Sirius® 250 SC, herbicida à base de pirazossulfurom-etílico, e Talcord®, inseticida à base de permethrin, em juvenis de Litopenaeus vannamei, bem como avaliar possíveis alterações fisiológicas por meio da contagem total de hemócitos (CTH) dos camarões. Juvenis de L. vannamei (5,0 ± 0,5 g) foram expostos às seguintes concentrações dos agroquímicos: Sirius® 250 SC, 0; 0,1; 1; 10; 100 e 1.000 μg L-1, e Talcord®, 0; 0,001; 0,01; 0,1; 1 e 10 μg L-1. A CL50 (96h) do inseticida Talcord® foi de 0,00933 μg L-1 ou 9,33 ng L-1. DOI: 10.4025/actascibiolsci.v33i1.6974 DOI: 10.4025/actascibiolsci.v33i1.6974 Acute toxicity of pyrazosulfuron-ethyl and permethrin to juvenile Litopenaeus vannamei Não houve alterações significativas da CTH entre as médias dos grupos-controle e dos submetidos ao inseticida. Com base nas concentrações testadas do herbicida Sirius® 250 SC, não foi possível determinar a CL50 (96h), assim, este produto não foi considerado tóxico para os juvenis de L. vannamei. Porém, a CTH dos camarões expostos ao herbicida demonstrou diferenças significativas entre as médias do controle e dos tratamentos, o que evidenciou efeito subletal. Os resultados permitem concluir que o inseticida Talcord® é altamente letal para os juvenis de L. vannamei e o herbicida Sirius® 250 SC, apesar de não ter a mesma toxicidade, apresenta efeito subletal relacionado com a diminuição na CTH. Os resultados sugerem a existência de riscos em se cultivar L. vannamei nas proximidades de fazendas de arroz, em que defensivos agrícolas são usados rotineiramente. Palavras-chave: toxicologia, pesticidas, carcinicultura, Litopenaeus vannamei. Acta Scientiarum. Biological Sciences Introduction have reported critical concentrations of restricted or forbidden chlorinated and organophosphate pesticides in the water, sediments and shrimp samples (GALINDO-REYES et al., 1999). In a study along an estuarine environment in Belgium, high concentrations of polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) were found in marine benthic organisms (VOORSPOELS et al., 2004). Wirth et al. (2001) reported that the deterioration of water quality and pesticide Marine shrimp farming has developed in areas traditionally dedicated to agriculture where pesticides are widely used (ROQUE et al., 2005). According to Galindo-Reyes et al. (2000), in coastal ecosystems of Sinaloa, in northwest Mexico, shrimp farms may be in risk as intensive agriculture of about 32 different cultures of fruits, vegetables and cereals is practiced with the use of large amounts of pesticides in crop protection. Studies in coastal ecosystems in Mexico Maringá, v. 33, n. 1, p. 1-6, 2011 2 Mello et al. contamination within Florida Bay, USA, have affected crustacean recruitment. equilibrium is reached, but it depends on the concentration and the physical and chemical characteristics of the compound (RESGALLA et al., 2002). Acute toxicity of high concentrations in a short period of time is generally assessed by the Median Lethal Concentration (LC50) after 96h of exposure, which is defined as the concentration that kills 50% of the organisms exposed for 96h to the test-compound (AMWEG et al., 2005). Shrimps can be affected by a number of products and substances used by man in aquaculture or other agricultural activities, as is the case of herbicides and insecticides. Insecticides are particularly toxic to shrimps as they are very close to insects in evolution (KRIEGER, 2001). In a study by the US Environmental Protection Agency Laboratory (EPA), penaeid shrimps were more sensitive than fishes or mollusks to the toxic effects of most pesticides and it was further suggested that pesticides in the water or in the soil compromises the shrimp immune system and triggers the outbreak of infectious diseases (ROQUE et al., 2005). Little information is available on the toxicity of pesticides to shrimp in farming ponds. Up to date, there is no data on the toxicity of most of the registered pesticides to marine organisms (ROBINSON, 1999). Introduction Studies on the subject have demonstrated that high concentrations of pesticides causes physiological and osmoregulatory alterations, which leads to reduced growth and consequent mortality of the farmed animals (GALINDO- REYES et al., 1996; HUANG et al., 2004; LUND et al., 2000). Pyrazosulfuron-ethyl is the active compound of the herbicide Sirius® 250 SC efficiently used against a broad range of annual and temporary weeds, especially those with large and long leaves, and applied at very low concentrations at pre- or post- germination (NAKAGOME et al., 2006). Permethrin, the active compound of Talcord®, is efficient against a broad range of pest insects, particularly Lepidoptera and Choleoptera in cotton, fruits, tobacco, tomato, vegetables, and grapes by contact and action in the insect’s stomach. It is also efficient against a wide variety of ectoparasites (lice) and flying insects (GARCIA et al., 2001). y g ( ) Studies have described the biochemical and physiological alterations, e.g., reduced growth and survival, caused by pesticides to shrimp embryos, larvae and juveniles (GALINDO-REYES et al., 1996; 2000; 2002; HUANG et al., 2004; LUND et al., 2000). Usually, chronic stress can induce physiological compensations such as change in respiration rate and energy consumption, which can be related to growth, based on the concept that the energy exceeding the amount required for maintenance will be used for growth (GALINDO-REYES et al., 1996). Galindo- Reyes et al. (2000) reported L. vannamei reduced oxygen consumption in water contaminated with sublethal concentrations of the organochlorine pesticides Diazinon, Folidol and Gusathion. In southern Brazil several pesticides are registered and indicated for use in flooded rice culture, but information on the toxicity of such products for non-target organisms is scarce, only toxicity tests of some pesticides to organisms that are not commonly found in flooded rice fields are available (RESGALLA et al., 2002). According to these authors, studies on the toxicity of insecticides and herbicides used in flooded rice culture to freshwater fish species are lacking. Flooded rice culture is socially and economically important to the State of Santa Catarina because it involves more than 8,000 families and farmers in more than 130,000 hectares. However, most of the farmers use herbicides at least once in a cycle to control weeds, one of the main setbacks that have limited the growth of the rice production. Introduction Chemical control of weeds using herbicides has been widely used in rice cultures because it is a practical, efficient and fast method. In most of the rice culture farms in southern Brazil, flooding follows pesticide application or, as in many cases, e.g., in the pre- germinated system, pesticides are used directly in the flooding water (IRGA, 2001). In the present study, the acute toxicity of two pesticides to juvenile L. vannamei was assessed. The pesticides were used during the 2003/2004 rice crop in flooded rice farms located on the estuarine system in south Santa Catarina State, Brazil. Additionally, during the exposure period chronic effects such as alterations in behavior, physiology and immune system were assessed by the total hemocyte count (THC) of the surviving shrimp. Acta Scientiarum. Biological Sciences Results and discussion Figure 1 shows the probability of mortality of juvenile Litopenaeus vannamei shrimp after 96h exposure to the insecticide permethrin, including the lower and upper limits of the confidence interval. The pesticide concentrations tested in this study (Table 1) were based on previous studies. To obtain the test concentrations calculations were based on the concentration of the active ingredients of each pesticide. For each pesticide five concentrations were tested in triplicate plus a control. Four shrimp were randomly stocked in each replicate bucket. Each 300-L tank with four buckets represented one test-concentration with three replicates and a control replicate. Each pesticide was tested only once. 0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 0.01 0.04 0.07 0.1 0.25 0.4 0.55 Probability Permethrin Concentration (µg L-1) Dose Lower limit Upper limit LC50 Figure 1. Relationship between the shrimp mortality probability and the permethrin concentration, including the lower and upper limits of the confidence interval after 96h exposure. 0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 0.01 0.04 0.07 0.1 0.25 0.4 0.55 Probability Permethrin Concentration (µg L-1) Dose Lower limit Upper limit LC50 Table 1. Concentrations of the herbicide Sirius® 250 SC (Pyrazosulfuron-ethyl) and the insecticide Talcord® (Permethrin) used to test the toxicity to juvenile Litopenaeus vannamei shrimp (5.0 ± 0.5 g). Probability Sirius® 250 SC (μg L-1) Talcord® (μg L-1) 0 (control) 0 (control) 0.1 0.001 1 0.01 10 0.1 100 1 1000 10 Sirius® 250 SC (μg L-1) Talcord® (μg L-1) 0 (control) 0 (control) 0.1 0.001 1 0.01 10 0.1 100 1 1000 10 Figure 1. Relationship between the shrimp mortality probability and the permethrin concentration, including the lower and upper limits of the confidence interval after 96h exposure. Based on the statistical analysis, the LC50 (96h) for Talcord® is equivalent to 0.00933 μg L-1 or 9.33 ng L-1. It is important to notice that this concentration refers to the commercial product and not to the active principle permethrin, which, in this case, would be of 0.002333 μg permethrin L-1 or 2.33 ng permethrin L-1. Although a high concentration of the herbicide Sirius® 250 SC was tested, i.e., 1 mg L-1 (1000 μg L-1), no LC50 (96h) was determined. According to the probit analysis, Experiment lasted 96h and mortality was registered every 12h. For standardization purposes, a shrimp was considered dead when the body was still and opaque. Marine shrimp rice’s pesticide toxicity Marine shrimp rice’s pesticide toxicity 3 3 recommended for rice culture but it has been used by a farmer in the city of Jaguaruna (Santa Catarina State, Brazil). feces were not siphoned out from the units, only aeration was adjusted every 6h. Daily water renovation was of 100% in the pyrazosulphuron- ethyl (Sirius® 250 SC) group because shrimp exposed to permethrin (Talcord®) died before the first 24h. Shrimp biomass was estimated at 0.35 g L-1 (dry weight). Total hemocyte count was the immune parameter analyzed at the end of the experiment in the LCM Microscopy Laboratory. Hemolymph samples were collected from shrimp with a plastic syringe (0.1 mL) and placed in a anti- clotting solution (1:4) (MAS: 27 mM sodium citrate, 336 mM sodium chloride, 115 mM glucose, 9 mM EDTA, pH 7.0) and hemocytes immediately counted with the aid of a Neubauer chamber. Juvenile shrimp L. vannamei, mean weight 5.0 ± 0.5 g, were taken from a pre-nursery, Marine Shrimp Laboratory (LCM), Department of Aquaculture, Federal University of Santa Catarina, and tested in the laboratory. Shrimp were acclimated for one week prior the tests in two 300-L tanks at 26°C with constant aeration and fed ad libitum with a commercial compound feed (35% crude protein). Fifty-percent of the water was changed every day. Dead shrimp or shrimp with stress signs (colorless abdomen) or disease (necrosis in carapace or appendices) were discarded. During acclimation, salinity was gradually adjusted to 20% and temperature to 24°C to simulate the shrimp farming conditions in the estuaries in south Santa Catarina State. The statistical analysis to find the LC50 was performed by the inverse accumulated distribution of the normal function or probit analysis (FINNEY, 1971). Total hemocytes count was analyzed by the Kruskal-Wallis non- parametric test. The culture system of the trials was semi-static, twenty 10-L buckets were individually aerated and each stocked with four shrimp. Each group of four buckets was placed in a 300-L tank water bath. The seawater used in the experiment was the same pumped from Moçambique beach, east Florianópolis shore (Santa Catarina Island) and supplied to LCM. Acta Scientiarum. Biological Sciences Material and methods The toxicity of the herbicide Sirius® 250 SC (Pyrazosulfuron-ethyl) (IHARABRAS, Sorocaba, São Paulo State, Brazil) and the insecticide Talcord® (Permethrin) (BASF S.A., Brazil) widely used in the 2003/2004 crop of flooded rice were tested on juvenile shrimp. The use of Talcord® is not One of the risks of using pesticides is to directly or indirectly affect non-target organisms by contamination of their habitat or feeding source. Pesticide absorption in fish is passive until Maringá, v. 33, n. 1, p. 1-6, 2011 Acta Scientiarum. Biological Sciences Results and discussion Shrimp were fed according to feed consumption at an approximate rate of 3% biomass per day. To avoid any disturbance, uneaten feed or Maringá, v. 33, n. 1, p. 1-6, 2011 Acta Scientiarum. Biological Sciences 4 Mello et al. bottom of the tank shivering and died quickly. This behavior can be a sign of the neuromuscular disturbance caused by the insecticide on acetylcholinesterase (AChE), an enzyme involved in the deactivation of acetylcholine at nerve endings, preventing continuous nerve firing, which is vital for normal functioning of sensory neuromuscular systems (COMOGLIO et al., 2005). Studies have demonstrated that many organophosphorus and carbamate insecticides are effective AChE inhibitors (GALGANI et al., 1992; KUMAR; CHAPMAN, 1998; KEY; FULTON, 2006). The inhibition of AChE in estuarine organisms has been established as an indicator of the contamination by insecticides (FULTON; KEY, 2001). Similarly, in the grass shrimp Palaemonetes pugio the inhibition of AchE is a relevant biomarker of the exposure to insecticides (KEY; FULTON, 2002). In addition to organophosphorus and carbamate insecticides, other classes of environmental contaminants, heavy metals and agrochemicals have shown to be potential inhibitors of AChE in organisms exposed to such chemicals (HABIG et al., 1988). the LC50 (96h) for the compound would be 2427 μg L-1 or 2.43 mg L-1, with a confidence interval from 0.53 to 2600 mg L-1, demonstrating that the value is not reliable. For the pesticides studied, the safety indexes are shown in Table 2. Table 2. Values of LC50 (96h) determined in this study for the pesticides Sirius® 250 SC and Talcord® for juvenile L. vannamei, and recommended concentrations, safety index, and toxicology class of the pesticides according to the manufacturers. Parameters Sirius® 250 SC Talcord® Chemical group Pyrazosulfuron-ethyl Permethrin LC50 (96h) > 1000 (mg L-1) 0.00000933 (mg L-1) Recommended concentration 0.08 (mg L-1) 0.000008 (mg L-1) Safety index 12500 1.17 Toxicology class IV III Parameters Sirius® 250 SC Talcord® Chemical group Pyrazosulfuron-ethyl Permethrin LC50 (96h) > 1000 (mg L-1) 0.00000933 (mg L-1) Recommended concentration 0.08 (mg L-1) 0.000008 (mg L-1) Safety index 12500 1.17 Toxicology class IV III Parameters Sirius® 250 SC Talcord® Chemical group Pyrazosulfuron-ethyl Permethrin LC50 (96h) > 1000 (mg L-1) 0.00000933 (mg L-1) Recommended concentration 0.08 (mg L-1) 0.000008 (mg L-1) Safety index 12500 1.17 Toxicology class IV III The mean THCs of shrimp exposed to Talcord® are presented in Table 3. Acta Scientiarum. Biological Sciences Results and discussion Hemocytes were counted in surviving shrimp, i.e., in the control group and at two lower concentrations. Twenty-five percent of the shrimp stocked in each concentration were sampled (five from a total of 20 shrimp and three of 12 shrimp per treatment group). The THC of shrimp exposed to Talcord® was not significantly different between the control group and the two concentrations in which shrimp survived. First, possibly because of the low concentrations tested (0.001 and 0.01 μg L-1) that might not have altered the number of hemocytes per milliliter of hemolymph in a short period of exposure. Second, permethrin causes alterations in the shrimp nervous and muscular systems and does not interfere in the production of defense cells. And third, the high variability in the data as the number of shrimp sampled for THC was low. The THC does not seem to be one of the best hemato- immune parameters to be used as a tool to assess alterations in the immune system caused by a contaminant. Although the high individual variability in the number of cells it is mostly used to determine the health status of crustaceans (LE MOULLAC et al., 1998). Table 3. Values are means (± standard deviation) of Total Hemocyte Count (THC) of juvenile shrimp Litopenaeus vannamei that survived the exposure to Talcord®. Talcord® Concentration (mg L-1) Mean THC (cell mL-1) 0 269,058.35 ± 169,242.20 a 0.001 138,737.00 ± 109,370.30 a 0.01 123,967.25 ± 46,824.96 a Mean values followed by the same superscript letter are not significantly different by the Kruskal-Wallis test. Table 3. Values are means (± standard deviation) of Total Hemocyte Count (THC) of juvenile shrimp Litopenaeus vannamei that survived the exposure to Talcord®. Mean THC of shrimp exposed to the herbicide Sirius® 250 SC are presented in Table 4. Twenty- five percent of the shrimp were sampled. Table 4. Values are means (± standard deviation) of total hemocyte count (THC) of shrimp Litopenaeus vannamei exposed to the herbicide Sirius® 250 SC and survived. Sirius® 250 SC Concentrations (mg L-1) Mean THC (cells mL-1) 0 428,262.40 ± 133,429.35 a 0.1 294,155.92 ± 236,290.71 a 1 275,212.50 ± 87,319.30 a 10 123,579.58 ± 72,871.784 b 100 119,415.42 ± 43,869.10 b 1000 97,463.75 ± 131,065.85 b Mean values followed by the same superscript letter are not significantly different by the Kruskal-Wallis test. References AMWEG, E. L.; WESTON, D. P.; UREDA, N. M. Use and toxicity of pyrethroid pesticides in the Central Valley, California, USA. Environmental Toxicology and Chemistry, v. 24, n. 4, p. 966-972, 2005. Nevertheless, reduction in the THC of shrimp exposed to Sirius® 250 SC (57.5%) as compared to the control group demonstrate that it has a sublethal effect and such reduction in the number of defense cells may leave shrimp more susceptible to infectious diseases. The mortality of only six shrimp of a total of 80 shrimp exposed to the herbicide Sirius® 250 SC could be related to acute effects, or to sublethal effects that interfere in biochemical and physiological processes in the shrimp, despite the low toxicity. COMOGLIO, L.; AMIM, O.; ROQUE, A.; BETANCOURT-LOZANO, M.; ANGUAS, D.; HARO, B. M. Evaluation of sublethal biomarkers in Litopenaeus vannamei on foodborne exposure to methyl parathion. Ecotoxicology and Environmental Safety, v. 62, n. 1, p 66-74, 2005. CRIPE, G. Comparative acute toxicities of several pesticides and metals to Mysidopsis bahia and postlarval Penaeus duorarum. Environmental Toxicology and Chemistry, v. 13, n. 11, p 1867-1872, 1994. CRIPE, G. Comparative acute toxicities of several pesticides and metals to Mysidopsis bahia and postlarval Penaeus duorarum. Environmental Toxicology and Chemistry, v. 13, n. 11, p 1867-1872, 1994. FAO-Food and Agriculture Organization. Pesticide residues in food, toxicological evaluations. Rome: Food and Agriculture Organization of the United Nations and World Health Organization, 1999. FAO-Food and Agriculture Organization. Pesticide residues in food, toxicological evaluations. Rome: Food and Agriculture Organization of the United Nations and World Health Organization, 1999. No substrate was used in the toxicity tests of this study. Toxicity of agrochemicals can be enhanced by the presence of sediment (HOLMES et al., 2008). Hartman and Martin (1984) tested the toxicity of glyphosate to Daphnia pulex and the LC50 (48h) was of 3.2 mg L-1 with sediment and of 7.9 mg L-1 without it. FINNEY, D. J. Probit Analysis. 3rd ed. Cambridge: Cambridge University Press, 1971. FLECK, N. G. Controle de plantas daninhas na cultura do arroz irrigado através da aplicação de herbicidas com ação seletiva. Porto Alegre: Editora do Autor, 2000. Imgrund (2003) reported that products with safety indexes > 20 present lower risks of environmental impact. Marine shrimp rice’s pesticide toxicity 5 should not be exposed to concentrations above 0.93 ng permethrin L-1. of the southern estuarine complex in the State of Santa Catarina, i.e., 20 trillion L of water (20,000 ha) it means that 187 L of the insecticide could kill all indigenous crustaceans in the lagoons of the estuary (this estimation did not take into account the dilution by the daily tide water renewal). of the southern estuarine complex in the State of Santa Catarina, i.e., 20 trillion L of water (20,000 ha) it means that 187 L of the insecticide could kill all indigenous crustaceans in the lagoons of the estuary (this estimation did not take into account the dilution by the daily tide water renewal). Results and discussion Sirius® 250 SC Concentrations (mg L-1) Mean THC (cells mL-1) 0 428,262.40 ± 133,429.35 a 0.1 294,155.92 ± 236,290.71 a 1 275,212.50 ± 87,319.30 a 10 123,579.58 ± 72,871.784 b 100 119,415.42 ± 43,869.10 b 1000 97,463.75 ± 131,065.85 b Mean values followed by the same superscript letter are not significantly different by the Kruskal-Wallis test. The high sensitivity of juvenile L. vannamei to the insecticide Talcord® can be explained by the phylogeny between shrimp and insects, with many similarities, e.g., in the nervous and muscular systems. In general, penaeid shrimp are highly sensitive to permethrin, differently from mollusks that are much more resistant (FAO, 1999; IMGRUND, 2003). Cripe (1994) suggested that crustaceans are more sensitive to agrochemicals during molts, especially the larval forms, as molts occur more frequently in the early life stages.The LC50 (96h) of 0.00933 μg L-1 determined for Talcord® is an extremely low concentration and it is an environmental concern. Considering the total volume Mean values followed by the same superscript letter are not significantly different by the Kruskal-Wallis test. In the shrimp group exposed to Talcord®, LC50 (96h) was of 0.00933 μg L-1 but it can also be considered LC50 (12h) as shrimp mortality was observed a few minutes after the insecticide was added into the water. In the groups exposed to higher concentrations, shrimp were highly agitated and constantly jumping out the water immediately after exposure and a few seconds later they were lying on the Maringá, v. 33, n. 1, p. 1-6, 2011 Acta Scientiarum. Biological Sciences Conclusion The permethrin-based insecticide tested in this study was highly toxic to juvenile Litopenaeus vannamei. Although the herbicide Sirius® 250 SC did not show mortalities above 50% at a concentration of 1,000 μg L-1 it is important to observe that the time of exposure was of only 96h and a longer exposure could result in sublethal or lethal effects affecting shrimp feeding and growth. Studies have demonstrated low toxicity of pyrazosulfuron-ethyl to aquatic animals. Fleck (2000) studied the same compound in rainbow trout Oncorhynchus mykiss and reported LC50 (96h) > 180 mg L-1. On the contrary, Resgalla et al. (2002) reported a LC50 (96h) of 0.32 pyrazosulfuron-ethyl mg L-1 for carp Cyprinus carpio. The herbicide pyrazosulfuron-ethyl did not present acute toxicity but reduced significantly the total hemocyte count in shrimp. The results indicated the potential risk of contamination in farming Litopenaeus vannamei near rice farms that use agrochemicals routinely. Acta Scientiarum. Biological Sciences Mello et al. GALINDO-REYES, J. G.; FOSSATO, V. U.; VILLAGRANA-LIZARRAGA, C.; DOLCI, F. Pesticides in water, sediments, and shrimp from a Coastal Lagoon off the Gulf of California. Marine Pollution Bulletin, v. 38, n. 9, p. 837-841, 1999. LE MOULLAC, G.; SOYEZ, C.; SAULNIER, D.; ANSQUER, J. C.; LEVY, P. Effect of hypoxic stress on the immune response and the resistance to vibriosis of the shrimp Penaeus stylirostris. Fish and Shellfish Immunology, v. 8, n. 8, p. 621-629, 1998. GALINDO-REYES, J. G.; MEDINA, J. A.; VILLAGRANA-LIZARRAGA, C. Physiological and biochemical changes in shrimp larvae (Penaeus vannamei) i i d i h hl i i id i GALINDO-REYES, J. G.; MEDINA, J. A.; VILLAGRANA-LIZARRAGA, C. Physiological and biochemical changes in shrimp larvae (Penaeus vannamei) intoxicated with organochlorine pesticides. Marine Pollution Bulletin, v. 32, n. 12, p. 872-875, 1996. LUND, S. A.; FULTON, M. H.; KEY, P. B. The sensitivity of grass shrimp, Palaemonetes pugio, embryos to organophosphate pesticide induced to g p p p acetylcholinesterase inhibition. Aquatic Toxicology, v. 48, n. 2-3, p. 127-134, 2000. intoxicated with organochlorine pesticides. Marine Pollution Bulletin, v. 32, n. 12, p. 872-875, 1996. GARCIA, E.; GARCIA, A.; BARBAS, B. Validated HPLC method for quantifying permethrin in pharmaceutical NAKAGOME, F.; NOLDIN, J.; RESGALLA, C. Toxicidade aguda e análise de risco de herbicidas e inseticidas utilizados na lavoura do arroz irrigado sobre o cladócero Daphnia magna. Pesticidas: Revista de Ecotoxicologia e Meio Ambiente, v. 16, n. 1, p. 93-100, 2006. formulations. Journal of Pharmaceutical and Biomedical Analysis, v. 24, n. 5, p. 999-1004, 2001. HABIG, C.; DIGIULIO, R. T.; ABOU-DONIA, M. B. Comparative properties of channel catfish Ictalurus punctatus and blue crab Callinectes sapidus acetylcholinesterases. Comparative Biochemistry and Physiology Part C: Comparative Pharmacology, v. 91, n. 2, p. 293-300, 1988. RESGALLA, C.; NOLDIN, J. A.; SANTOS, A. L.; SATO, G.; EBERHARDT, D. S. Toxicidade aguda de herbicidas e inseticida utilizados na cultura do arroz irrigado sobre juvenis de carpa (Cyprinus carpio). Pesticidas: Revista Ecotoxicologia e Meio Ambiente, v. 12, n. 1, p. 59-68. 2002. HARTMAN, W. A.; MARTIN, D. B. Effect of suspended bentonite clay on the acute toxity of gliphosate to Daphnia pulex and Lemna minor. Bulletin of Environmental Contamination and Toxity, v. 33, n. 1, p. 355-361, 1984. ROBINSON, P. W. The toxicity of pesticides and organics to mysid shrimps can be predicted from Daphia spp. toxicity data. Water Research, v. 33, n. 6, p. 1545-1549, 1999. Received on May 4, 2009. Accepted on September 22, 2009. License information: 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 work is properly cited. Mello et al. HOLMES, R. W.; ANDERSON, B. S.; PHILLIPS, B. M.; HUNT, J. W.; CRANE, D. B.; MEKEBRI, A.; CONNOR, V. Statewide investigation of the role of pyrethroid pesticides in sediment toxicity in California’s urban waterways. Environmental Science and Technology, v. 42, n. 18, p. 7003-7009, 2008. HUANG, D. J.; WANG, S. Y.; CHEN, H. C. Effects of the endocrine disrupter chemicals chlordane and lindane on the male green neon shrimp (Neocaridina denticulata). Chemosphere, v. 57, n. 11, p. 1621-1627, 2004. ROQUE, A.; ABAD, S.; BETANCOURT-LOZANO, M.; GARCIA DE LA PARRA, L. M.; BAIRD, D.; GUERRA-FLORES, A. L.; GOMEZ-GIL, B. Evaluation of the susceptibility of the cultured shrimp Litopenaeus vannamei to vibriosis when orally exposed to the insecticide methyl parathion. Chemosphere, v. 60, n. 1, p. 126-134, 2005. SPRAGUE, J. Measurement of pollutant toxicity to fish III. Sublethal effects and safe concentrations. Water Research, v. 5, n. 6, p. 245-266, 1971. IMGRUND, H. Environmental fate of permethrin. Sacramento: California Department of Pesticide Regulation, Environmental Monitoring Branch, 2003. IRGA-Instituto Rio-Grandense de Arroz. Arroz irrigado: recomendações técnicas da pesquisa para o Sul do Brasil. Porto Alegre: Instituto Rio-Grandense do Arroz, 2001. IMGRUND, H. Environmental fate of permethrin. Sacramento: California Department of Pesticide Regulation, Environmental Monitoring Branch, 2003. VOORSPOELS, S.; COVACI, A.; MAERVOET, J.; DE MEESTER, I.; SCHEPENS, P. Levels and profiles of PCBs and OCPs in marine benthic species from the Belgian North Sea and the Western Scheldt Estuary. Marine Pollution Bulletin, v. 49, n. 5-6, p. 393-404, 2004. IRGA-Instituto Rio-Grandense de Arroz. Arroz irrigado: recomendações técnicas da pesquisa para o Sul do Brasil. Porto Alegre: Instituto Rio-Grandense do Arroz, 2001. KEY, P. B.; FULTON, M. H. Characterization of cholinesterase activity in tissues of the grass shrimp Palaemonetes pugio. Pesticide Biochemistry and Physiology, v. 72, n. 3, p. 186-192, 2002. KEY, P. B.; FULTON, M. H. Characterization of cholinesterase activity in tissues of the grass shrimp Palaemonetes pugio. Pesticide Biochemistry and Physiology, v. 72, n. 3, p. 186-192, 2002. KEY, P. B.; FULTON, M. H. Correlation betwen 96-h mortality and 24-h acetylcholinesterase inhibition in three grass shrimp larval life stages. Ecotoxicology and Environmental Safety, v. 63, n. 3, p. 389-392, 2006. WIRTH, E. F.; LUND, S. A.; FULTON, M. H.; SCOTT, G. I. Determination of acute mortality in adults and sublethal embryo responses of Palaemonetes pugio to endosulfan and methoprene exposure. Aquatic Toxicology, v. 53, n. 1, p. 9-18, 2001. KEY, P. Acta Scientiarum. Biological Sciences References For the herbicide Sirius® 250 SC, the safety index was very high, much above the concentrations recommended for use in agriculture, and three times higher than the safety index determined by Resgalla et al. (2002) for juvenile carp. On the other hand, the insecticide Talcord® presented a low safety index, indicating a higher risk to the environment. According to the calculated safety index, the Talcord® concentration used in rice culture (8 ng L-1) is close to the LC50 (96h) of 9.33 ng L-1 determined in this study for juvenile L. vannamei, demonstrating the risk of farming shrimp L. vannamei in areas close to rice farms. The safety level of a compound recommended by Sprague (1971) for aquatic organism is equivalent to 10% of the LC50, therefore, farmed shrimp FULTON, M. H.; KEY, P. B. Acetylcholinesterase inhibition in estuarine fish and invertebrates as an indicator of organophosphorus inseticides exposure and ff i l T i l d Ch i FULTON, M. H.; KEY, P. B. Acetylcholinesterase inhibition in estuarine fish and invertebrates as an indicator of organophosphorus inseticides exposure and effects. Environmental Toxicology and Chemistry, v. 20, n. 1, p. 37-45, 2001. effects. Environmental Toxicology and Chemistry, v. 20, n. 1, p. 37-45, 2001. GALGANI, F.; BOCQUENE, G.; CARDIOU, Y. Evidence of variation in cholinesterase activity in fish along a pollution gradient in the North Sea. Marine Ecology Progress Series, v. 91, n. 1, p. 77-82, 1992. GALINDO-REYES, J. G.; DALLA-VENEZIA, L.; LAZCANO-ALVAREZ, G.; RIVAS-MENDOZA, H. Enzymatic and osmoregulative alterations in white shrimp Litopenaeus vannamei exposed to pesticides. Chemosphere, v. 40, n. 3, p. 233-237, 2000. GALINDO-REYES, J. G.; DALLA-VENEZIA, L.; LAZCANO-ALVAREZ, G. Effect of some organophosphorus pesticides on oxygen consumption of shrimp, Litopenaeus vannamei. Ecotoxicology and Environmental Safety, v. 52, n. 2, p. 134-136, 2002. Maringá, v. 33, n. 1, p. 1-6, 2011 Acta Scientiarum. Biological Sciences Acta Scientiarum. Biological Sciences 6 Mello et al. Mello et al. B.; FULTON, M. H. Correlation betwen 96-h mortality and 24-h acetylcholinesterase inhibition in three grass shrimp larval life stages. Ecotoxicology and Environmental Safety, v. 63, n. 3, p. 389-392, 2006. grass shrimp larval life stages. Ecotoxicology and Environmental Safety, v. 63, n. 3, p. 389-392, 2006. KRIEGER, R. Handbook of pesticide toxicology - agents. 2nd ed. San Diego: Academic Press, 2001. KUMAR, A.; CHAPMAN, J. C. Profenofos toxicity to the rainbow fish Melanotaenia duboulayi. Environmental Toxicology and Chemistry, v. 17, n. 9, p. 1799-1806, 1998. Maringá, v. 33, n. 1, p. 1-6, 2011 Acta Scientiarum. Biological Sciences
https://openalex.org/W4221078745	https://pure.eur.nl/files/81816175/cancers_14_01346_with_cover.pdf	English	null	Reproducibility of Gene Expression Signatures in Diffuse Large B-Cell Lymphoma	Cancers	2,022	cc-by	10,018	Article * Correspondence: m.nijland@umcg.nl; Tel.: +31-50-361-2354 Citation: Plaça, J.R.; Diepstra, A.; Los, T.; Mendeville, M.; Seitz, A.; Lugtenburg, P.J.; Zijlstra, J.; Lam, K.; da Silva, W.A., Jr.; Ylstra, B.; et al. Reproducibility of Gene Expression Signatures in Diffuse Large B-Cell Lymphoma. Cancers 2022, 14, 1346. https://doi.org/10.3390/ cancers14051346 Citation: Plaça, J.R.; Diepstra, A.; Los, T.; Mendeville, M.; Seitz, A.; Lugtenburg, P.J.; Zijlstra, J.; Lam, K.; da Silva, W.A., Jr.; Ylstra, B.; et al. Reproducibility of Gene Expression Signatures in Diffuse Large B-Cell Lymphoma. Cancers 2022, 14, 1346. https://doi.org/10.3390/ cancers14051346 Simple Summary: Multiple gene expression signatures with biological or prognostic subgroups have been published in diffuse large B-cell lymphoma (DLBCL). With exception of the cell of origin (COO) classiﬁer, these were not validated in independent cohorts. The aim of the study was to reproduce four gene expression signatures capturing multiple biological subgroups using the NanoString platform. In addition, we aimed to identify potential associations between the signatures and portray the heterogeneity of DLBCL. We show that, in an independent cohort of clinically well-deﬁned patients, these signatures can co-occur in the same patient and that each classiﬁer captures a different aspect of the biological heterogenous panorama of DLBCL. Beside COO, there is clear evidence of different immune and MYC signatures. A direct comparison in our cohort showed that these signatures reﬂect independent biological features. More comparative studies with gene expression proﬁles need to be conducted to enable a further integration and to help develop new taxonomy systems for clinical utility. Academic Editors: Blanca Scheijen and Alexandar Tzankov Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Abstract: Multiple gene expression proﬁles have been identiﬁed in diffuse large B-cell lymphoma (DL- BCL). Besides the cell of origin (COO) classiﬁer, no signatures have been reproduced in independent studies or evaluated for capturing distinct aspects of DLBCL biology. We reproduced 4 signatures in 175 samples of the HOVON-84 trial on a panel of 117 genes using the NanoString platform. The four gene signatures capture the COO, MYC activity, B-cell receptor signaling, oxidative phosphorylation, and immune response. Performance of our classiﬁcation algorithms were conﬁrmed in the original datasets. We were able to validate three of the four GEP signatures. The COO algorithm resulted in 94 (54%) germinal center B-cell (GCB) type, 58 (33%) activated B-cell (ABC) type, and 23 (13%) unclassiﬁed cases. Article Reproducibility of Gene Expression Signatures in Diffuse Large B-Cell Lymphoma Jessica Rodrigues Plaça 1,2 , Arjan Diepstra 1 , Tjitske Los 3 , Matías Mendeville 3, Annika Seitz 1, Pieternella J. Lugtenburg 4, Josée Zijlstra 5 , King Lam 6, Wilson Araújo da Silva, Jr. 2,7 , Bauke Ylstra 3 , Daphne de Jong 3, Anke van den Berg 1 and Marcel Nijland 8,* 1 Department of Pathology and Medical Biology, University Medical Center Groningen, University of Groningen, 9712 Groningen, The Netherlands; jessicaplaca@usp.br (J.R.P.); a.diepstra@umcg.nl (A.D.); a.seitz@umcg.nl (A.S.); a.van.den.berg01@umcg.nl (A.v.d.B.) p g ( ); g ( ); g g ( ) 2 Center for Cell-Based Therapy, National Institute of Science and Technology in Stem Cel Therapy (INCT/CNPq), Ribeirão Preto 14051-060, Brazil; wilsonjr@usp.br py q j p 3 Department of Pathology, Cancer Center Amsterdam, Amsterdam UMC, 1105 Amsterdam, The Netherlands; g.t.los@amsterdamumc.nl (T.L.); m.mendeville@amsterdamumc.nl (M.M.); b.ylstra@vumc.nl (B.Y.); d.dejong2@amsterdamumc.nl (D.d.J.) 4 Department of Hematology, Erasmus MC Cancer Institute, University Medical Center, 3015 Rotterdam, The Netherlands; p.lugtenburg@erasmusmc.nl 5 Department of Hematology, Amsterdam UMC, 1105 Amsterdam, The Netherlands; j.zijlstra@amsterdamumc.nl 6 Department of Pathology, Erasmus MC, 3015 Rotterdam, The Netherlands; k.lam@erasmusmc.nl 6 Department of Pathology, Erasmus MC, 3015 Rotterdam, The Netherlands; k.lam@erasmusmc.nl 7 Department of Genetics, Ribeirão Preto Medical School, University of São Paulo, p gy, , , ; 7 Department of Genetics, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto 14049-900, Brazil , 8 Department of Hematology, University Medical Center Groningen, University of Groningen, 9712 Groningen, The Netherlands cancers cancers Reproducibility of Gene Expression Signatures in Diffuse Large B-Cell Lymphoma Jessica Rodrigues Plaça, Arjan Diepstra, Tjitske Los, Matías Mendeville, Annika Seitz, Pieternella J. Lugtenburg, Josée Zijlstra, King Lam, Wilson Araújo da Silva, Jr., Bauke Ylstra et al. Jessica Rodrigues Plaça, Arjan Diepstra, Tjitske Los, Matías Mendeville, Annika Seitz, Pieternella J. Lugtenburg, Josée Zijlstra, King Lam, Wilson Araújo da Silva, Jr., Bauke Ylstra et Special Issue Molecular Characterization of Hematological Tumors Edited by Dr. Blanca Scheijen cancers 1. Introduction Diffuse large B-cell lymphoma not otherwise speciﬁed (DLBCL NOS) is a heteroge- neous disease that accounts for 40% of all mature B-cell neoplasms [1]. While the outcome of patients with a low-risk disease as determined by the clinical International Prognostic Index (IPI) score is excellent, the prognosis of patients with high-risk DLBCL remains dismal with 40% of patients failing ﬁrst-line treatment with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP) [2]. p The cell-of-origin (COO) concept was ﬁrst published in 2000, dividing DLBCL based on gene expression proﬁles (GEP) in germinal center B-cell (GCB) type, activated B-cell (ABC) type, and unclassiﬁed cases [3]. The COO subgroups were shown to have distinct features indicating involvement of different oncogenic pathways. Patients with ABC-type DLBCL showed an inferior outcome in a retrospective setting [3]. So far, clinical studies targeted towards speciﬁc oncogenetic characteristics of ABC-type DLBCL patients, e.g., combining the small molecules bortezomib, ibrutinib, and lenalidomide to R-CHOP have not been successful to improve outcome, which underpins that a simple dichotomy to deﬁne DLBCL does not sufﬁciently capture the oncogenetic complexity of this disease [4–6]. Moreover, about 15% of DLBCL cases remain unclassiﬁed and these cases do not have other characteristic aberrations that can advise the treatment of these patients [3]. In 2017, the World Health Organization (WHO) classiﬁcation categorized high-grade B-cell lymphomas with an MYC rearrangement combined with a BCL2 and/or BCL6 rearrangements separately as double hit high-grade B-cell lymphoma (HGBCL DH) and DLBCL cases with high-grade morphology that lack these concurrent hits as HGBCL NOS [1]. The poor outcome in HGBCL patients justiﬁed a dedicated treatment approach within this group, those cases with MYC immunoglobulin heavy or light chain gene (MYC- IG) rearrangements were especially shown to have an inferior prognosis [7]. Despite the use of more intensive chemotherapy and small molecules for patients with HGBCL DH, randomized clinical trials are lacking [8,9]. From 2010 onward, the focus in unraveling the biology has been on deciphering the mu- tational landscape of DLBCL using next generation sequencing (NGS) approaches [10–14]. The subgrouping as based on the observed mutational proﬁles showed enrichment of speciﬁc mutations for either ABC or GCB subsets. Although the mutational patterns are not mutually exclusive, they were at least partially correlated to COO, but much less to the presence of MYC rearrangements [10–14]. In parallel to the mutational landscape studies, multiple GEP studies have identiﬁed distinct biological DLBCL subgroups [15–29]. The MYC-classiﬁer revealed 77 cases with a high MYC-activity score (44%) and this MYC-high signature was observed more frequently in ABC as compared to GCB DLBCL (68% vs. 32%, p < 0.00001). The host response (HR) signature of the consensus clustering was present in 55 (31%) patients, while the B-cell receptor signaling, and oxidative phosphorylation clusters could not be reproduced. The overlap of COO, consensus cluster and MYC activity score differentiated six gene Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/cancers Cancers 2022, 14, 1346. https://doi.org/10.3390/cancers14051346 Cancers 2022, 14, 1346 2 of 15 expression clusters: GCB/MYC-high (12%), GCB/HR (16%), GCB/non-HR (27%), COO-Unclassiﬁed (13%), ABC/MYC-high (25%), and ABC/MYC-low (7%). In conclusion, the three validated signatures identify distinct subgroups based on different aspects of DLBCL biology, emphasizing that each classiﬁer captures distinct molecular proﬁles. expression clusters: GCB/MYC-high (12%), GCB/HR (16%), GCB/non-HR (27%), COO-Unclassiﬁed (13%), ABC/MYC-high (25%), and ABC/MYC-low (7%). In conclusion, the three validated signatures identify distinct subgroups based on different aspects of DLBCL biology, emphasizing that each classiﬁer captures distinct molecular proﬁles. Keywords: diffuse large B-cell lymphoma; gene expression proﬁles; reproducibility 1. Introduction These studies have generated proﬁles related to tumor cell characteristics including MYC activity and the micro-environment composi- tion. However, the biological relevance and clinical impact of these gene signatures have not resulted into incorporation in clinical trials. Around the start of this study, twelve different GEP classiﬁers had been published [15–26]. The aim of this study was to reproduce four biology driven gene expression signa- tures in a large cohort of clinically well annotated DLBCL NOS/HGBCL samples from the HOVON-84 trial using the NanoString platform, which permits robust ampliﬁcation free GEP analysis of RNA from formalin-ﬁxed parafﬁn-embedded tissue with minimal background signal [30]. Selection of the four gene expression signatures was based on the biological features presenting COO, MYC activity, oxidative phosphorylation (Ox- Cancers 2022, 14, 1346 3 of 15 3 of 15 Phos), B-cell receptor (BCR) signaling, and the micro-environment as well as the potential reproducibility of the classiﬁers (FFPE based, number of genes, and availability of algo- rithms) [15–18]. In addition, we studied whether the reproducible gene expression proﬁles are independent of each other and whether their combined use can indicate distinct DLBCL NOS/HGBCL subgroups. Finally, we tested potential associations with clinical features in a well-deﬁned population of patients with DLBCL NOS/HGBCL. 2. Materials and Methods 2.1. Patient Cohort HOVON-84 is a multicentric, randomized phase III trial, with no beneﬁt of the intensi- ﬁcation of rituximab combined with 2-weekly CHOP chemotherapy in patients with newly diagnosed DLBCL. At the time of the study HGBCL DH was not considered a distinct entity and as such was included in the trial [31]. The study was conducted in accordance with the ethical guidelines mandated by the Declaration of Helsinki and approved by all relevant institutional review boards or ethical committees. Written informed consent, including use of biopsy material for research purposes, was obtained from all patients. The HOVON-84 trial included 574 patients and good quality NanoString (Seattle, WA, USA) data could be generated for 175 patients. This cohort forms the core of the present study. In the other 399/574 patients, no representative formalin-ﬁxed parafﬁn embedded (FFPE) biopsy material was available for this study (blocks not available for study, blocks exhausted, or insufﬁcient quality) or NanoString data were of insufﬁcient quality. q y g q y Clinical characteristics of the 175 HOVON-84 patients studied in this report as well as the characteristics of the total cohort and the original GEP signatures cohorts are listed in Table S1. No statistically signiﬁcant differences were observed between the cases included in the present study and the entire HOVON-84 cohort, making the samples used in this study a representation of the entire cohort. (Gender p-value = 0.5; Age p-value = 0.5; Stage p-value = 0.3; LDH levels p-value = 0.1; aaIPI p-value = 0.2; OS p-value = 0.06; COO p-value = 0.9.) 2.2. Immunohistochemistry Immunohistochemistry (IHC) was performed as part of previous studies by the Lunen- burg Lymphoma Biomarker Consortium (North Bethesda, MD, USA) [7,32] and available for 167 DLBCL patients for CD10, MUM1, and BCL6. In addition, BCL2 and MYC IHC was performed for 161 DLBCL patients using routine diagnostic procedures on tissue microar- rays. Scoring of CD10, MUM1, and BCL6 staining and subsequent classiﬁcation as GCB or non-GCB was performed according to the Hans algorithm [33]. MYC IHC was scored as the percentage of positive tumor cells as estimated by an experienced hematopathologist in 10% increments. Lymphomas were deﬁned as double expressors (DE) based on MYC positivity in ≥40% and BCL2 positivity in ≥50% of the tumor cells [34]. For correlation to the MYC gene signature as published by Carey et al. [16], we used a cutoff of ≥50% positive tumor cells for MYC-High and <50% for MYC-Low consistent with the cutoff as deﬁned in this paper. For MHC-II (HLA-II), IHC was performed on tissue microarrays and cores were scored for intensity of staining. No or weak staining was classiﬁed as MHC-II low and all other cases were classiﬁed as MHC-II high [35]. 2.4. Gene Expression Proﬁling For a total of 175 samples, we were able to obtain sufﬁcient good quality RNA with FFPE RNeasy Kit (Qiagen, Hilden, Germany) for analysis on the NanoString Platform. The core set of probes for 117 genes (see Supplementary Table S2 for a complete overview) was hybridized to 100–200 ng of RNA for 16 h at 65 ◦C. Samples were loaded on an nCounter SPRINT Cartridge and processed on the nCounter SPRINT™Proﬁler. The expression data were analyzed using Nanostring’s nSolver analysis software (version 3.0). Registered counts passing the standard QC parameters were used for further analysis. The normalized data were scaled and transformed to log2. 2.6. MYC Activity Score To reproduce the MYC activity score we used the selection and bioinformatics strategy as reported by Carey et al. [16], since the algorithm is not publicly available. In brief, we used their original training cohort as training set of the elastic net classiﬁer. This training set included 14 cases scored as MYC-low based on positive staining in <40% of the tumor cells and 16 cases as MYC-high based on positive staining in >60% of the tumor cells. The classiﬁer was subsequently applied to the HOVON-84 (n = 175) test set. q y pp The training dataset was normalized with the R package NanoStringNorm [37], con- sidering the sum of the expression values to estimate the technical assay variation, the mean to estimate background count levels, and the sum of the six housekeeping genes to normalize for the RNA sample content. In addition, the data were log2 transformed. The alfa and gamma parameters were set at 0.1 and the classiﬁcation accuracy was assessed with the Leave One Out Cross Validation (LOOCV), as in the original publication. A cutoff of 0.5 was used to stratify the tumors with high and low MYC activity score. The importance of each gene was calculated based on combinations of the absolute values of the weights as reported by Gevrey et al. [38]. All the analyses were conducted with the R package caret [39]. The spearman’s correlation was used to evaluate the association between the MYC activity score and MYC IHC values and the predictions were compared with the outcome of the IHC staining. 2.5. COO Classiﬁer For COO classiﬁcation, raw counts obtained by NanoString gene expression analysis for all genes of the algorithm were uploaded at the Lymphoma/Leukemia Molecular Proﬁling Project (LLMPP) website (https://llmpp.nih.gov/LSO/LYMPHCX/lymphcx_ predict.cgi, accessed on 12 September 2017) to run the Lymph2Cx classiﬁer [15]. 2.3. Detection of Chromosomal Translocations in BCL2, BCL6, and MYC Fluorescence in situ hybridization (FISH) for MYC, BCL2, and BCL6 was performed on 152, 148, and 153 cases, respectively, with break apart probes from Vysis LSI, Abbott (Chicago, IL, USA). Scoring was performed as described previously [7,32]. In addition to the FISH, targeted NGS was performed for 140 samples to identify structural variants (SV) in MYC, BCL2, and BCL6 using the protocols as previously de- scribed [36]. The SV information was combined with the FISH results to classify cases as HGBCL DH, regarding all cases with a positive result for either FISH (8) or NGS (7) or both (125) as positive. 4 of 15 Cancers 2022, 14, 1346 2.4. Gene Expression Proﬁling 3.1. Study Design In addition to the widely used COO signature to classify DLBCL cases (Scott et al. 2014 [15]), we prioritized three additional signatures that were NanoString based, since it is a reproducible technology by different laboratories, available at that time, and reﬂected different biological aspects. The three selected signatures included MYC activity score (Carey et al. 2015 [16]), Monti consensus clustering (Monti et al. 2005 [17]), and the immune- ratio signature (Keane et al. 2015 [18]). As the COO classiﬁer and immune ratio classiﬁers were both based on a limited number of genes, we included all genes and applied the published algorithms. For the two much larger classiﬁers, i.e., MYC activity score and the consensus clustering, with algorithms that had to be re-designed, we followed a different approach. We ﬁrst recreated the clustering and/or classiﬁcation algorithms and tested their performance on the originally reported cohorts, with the original set of genes. To make a subsequent clinical application feasible, we reduced the gene list, by prioritizing the genes with the strongest contributions to the algorithms and applied the validated algorithms on the original cohorts to establish the effectivity of our selected gene set. 2.7. Monti Consensus Clustering Brieﬂy, the three consensus clustering approaches applied were Hierarchical Clus- tering (HC) considering the Euclidean distance, Self-Organizing Maps (SOM) with the R packages ConsensusClusterPlus [40] and Kohonen [41], and the Gaussian Finite Mixture Models algorithm (which represents the probabilistic clustering (PC)) using the R package mclust [42]. To deﬁne the best number of clusters, we used 80% of resampling on 200 repli- cates for each clustering algorithm, as in the original paper. Consensus matrices including two to nine clusters were built and evaluated by the relative change in area under CDF curves or Bayesian Information Criterion (BIC) metrics. Confusion matrices were used to determine the number of samples assigned to similar clusters by any 2 algorithms. HOVON-84 samples with the same classiﬁcation by all three algorithms (“meta- consensus”) were deﬁned as samples belonging to the main clusters. For the remaining HOVON-84 samples, we built a naive-Bayes classiﬁer with the R package caret [39]. The naïve-Bayes classiﬁer was ﬁrst trained with the samples from the original meta-consensus clustering study and subsequently used to predict the cluster membership for the remaining HOVON-84 samples, similar to the approach applied in the original publication. 5 of 15 Cancers 2022, 14, 1346 2.9. Statistical Analysis To compare categorical data, we used Fisher’s Exact Test or the X2 test, where ap- plicable. The Kaplan–Meier method was used to estimate the overall survival (OS) and progression free survival (PFS). Univariable and multivariable Cox proportional hazard regression models and Wald p-values were used to evaluate the prognostic impact and statistical signiﬁcance. All the analyses were performed in R 3·6·2 [43]. We did not sep- arately analyze patients per study treatment arm since PFS and OS were similar, and treatment regimens differed on Rituximab-dose only. Patients with signiﬁcant therapy protocol violations were not included. 2.8. Immune Ratio 2.8. Immune Ratio To reproduce the prognostic marker based on the expression ratio between immune effectors and inhibitory (immune checkpoint) genes, we followed the approach as published by Keane et al. [18]. We decided to focus on their main ﬁnding, which was the prognostic signiﬁcance of the CD4 × CD8 to CD163:CD68 × PD-L1 ratio. This immune ratio was additive and independent to the revised-IPI and COO in the original paper. The ratio was calculated using the log2 scaled gene expression values and to assess the prognostic value of this ratio in the HOVON-84 cohort we used the Keane proposed cut-off (−0.278958829) to stratify samples into high and low expression ratio subgroups. 3.2. Performance of the MYC Activity Score Using a Subset of the Genes The MYC activity score algorithm was ﬁrst reproduced in the Carey cohort using the original set of 61 genes (Figure S1A). Next, we tested the validated algorithm on our subset consisting of 34 genes (Figure S1B). Although the impact of the genes in the classiﬁer was different from the original publication for both gene sets [16], MYC had the highest impact consistent with the original paper. We observed a good correlation between the MYC activity score and the percentage of tumor cells staining positive for MYC in the Carey training set cases using both the initial gene set and the subset included in our analysis (Figure S1C,D). Moreover, we observed a perfect match of the MYC activity score with the MYC expression as determined by IHC in the training set (Table S4). 3.3. Performance of the Monti Consensus Clustering Algorithm Using a Subset of the Genes We ﬁrst reproduced the Monti consensus clustering into Oxidative phosphorylation (OxPhos), B-cell Receptor/Proliferation (BCR), and Host response (HR) groups using the dataset of Monti et al. [17]. The three algorithms revealed three subgroups consistent with the original Monti publication using 1112 annotated genes from the 2118 microarray probes. Meta-consensus clustering revealed an initial classiﬁcation of 115 out of 176 samples Cancers 2022, 14, 1346 6 of 15 (Figure S2A). This showed that our algorithm correctly recapitulates the original clustering patterns as reported by Monti et al. [17]. After successful reproduction of the original clustering pattern, we evaluated the performance of the algorithm on our selected subset of genes, which included probes ranking in the top 50 most relevant probes to deﬁne each of the three biologic clusters, as speciﬁed by Monti. In total, this gene set comprised 47 out of 1112 annotated genes (12 out of 342 OxPhos related genes, 14 out of 344 BCR/Proliferation related genes, and 21 out of 427 HR related genes). This revealed for 130 of the 176 samples of the Monti cohort a consistent clustering, indicating that our selection of genes correctly assigned the majority of the samples to the three clusters (Figure S2B). 3.4. COO Classiﬁer in HOVON-84 Application of the COO classiﬁer revealed 94 (54%) GCB, 58 (33%) ABC, and 23 (13%) unclassiﬁed cases (Figure 1). According to Hans classiﬁcation, 91 cases (54%) were classiﬁed as GCB and 76 (46%) as non-GCB. We observed a signiﬁcant association (p < 0.00001) between the COO classiﬁer and the Hans algorithm (Table S3). Sensitivity and speciﬁcity values were 91% and 84% for ABC and non-GCB comparison and 84% and 91% for GCB classes, as previous reported [44]. We also found that ABC cases were enriched in older patients (>60 years) (p < 0.002), as reported by Klapper et al. in 2012 [45]. Figure 1. Heatmap showing relative expression levels of the COO genes used to classify cases using the Lymph2Cx algorithm. A clearly distinct gene expression pattern can be observed for ABC and GCB subtype DLBCL cases. Figure 1. Heatmap showing relative expression levels of the COO genes used to classify cases using the Lymph2Cx algorithm. A clearly distinct gene expression pattern can be observed for ABC and GCB subtype DLBCL cases. 3.5. MYC Activity Score in HOVON-84 3.5. MYC Activity Score in HOVON-84 For the HOVON-84 cohort, we classiﬁed 77 cases (44%) as MYC high and 98 (56%) cases as MYC low (Figure 2A). The sensitivity and speciﬁcity values relative to the MYC IHC score based on staining in at least 50% of the tumor cells were 0.65 and 0.65, respec- tively. The negative and positive predictive values were 0.82 and 0.43, respectively, for the identiﬁcation of MYC IHC expression (Table S4). A signiﬁcant correlation (R 0.493; Fisher exact test p = 0.006) was observed for the MYC activity score and the percentage of tumor cells staining positive for MYC in the HOVON-84 cohort (Figure 2B). 7 of 15 Cancers 2022, 14, 1346 Spearman’s correlation between MYC activity score and MYC Figure 2. Results of the MYC activity classiﬁer in the HOVON-84 cohort: (A) Heatmap for relative expression of the proﬁling panel including the relative contribution of each gene to the classiﬁer (horizontal, shaded bar graph on the left) and the MYC activity score for the HOVON-84 cohort (line graph on top of the ﬁgure). (B) Spearman’s correlation between MYC activity score and MYC IHC expression for the 161 samples of the HOVON-84 cohort. ND, Not Done; NE, Not Evaluable. Spearman’s correlation between MYC activity score and MYC Figure 2. Results of the MYC activity classiﬁer in the HOVON-84 cohort: (A) Heatmap for relative expression of the proﬁling panel including the relative contribution of each gene to the classiﬁer (horizontal, shaded bar graph on the left) and the MYC activity score for the HOVON-84 cohort (line graph on top of the ﬁgure). (B) Spearman’s correlation between MYC activity score and MYC IHC expression for the 161 samples of the HOVON-84 cohort. ND, Not Done; NE, Not Evaluable. The high-activity MYC group was enriched for DE (p < 0.00001) and ABC-type (p < 0.00001) lymphoma. There was no association between the MYC activity score and HGBCL DH. Thus, the MYC activity score could be validated in the HOVON-84 cohort and showed a clear correlation with DE and ABC-type lymphomas. 3.6. Monti Consensus Clustering in HOVON-84 – – For the HOVON-84 cases, application of the validated algorithms revealed two as the most optimal number of clusters (Figure S3A,B). The meta-consensus clustering exhibited a consistent subgroup for all three algorithms for 67 (38%) HOVON-84 cases. These cases were characterized by two proﬁles: a larger cluster (43 samples–24%) with high expression of both BCR/proliferation and Oxphos genes (BCR/Proliferation/Oxphos-high cases) and a cluster (24 samples–14%) characterized by a high expression of HR genes (HR-high cases). Thus, in contrast to the ﬁndings of Monti [17], the non-HR cases were not characterized by a differential expression of BCR/proliferation and Oxphos genes. Next, we followed the same strategy as reported by Monti [18] to deﬁne the most likely cluster for the remaining 108 (62%) HOVON-84 cases. This revealed a consensus BCR/proliferation/Oxphos-high cluster signature for 77 (44%) samples and a consensus HR cluster-signature for 31 (18%) samples. In total 120 (77 + 43) cases were classiﬁed as BCR/proliferation/OxPhos-high and 55 (24 + 31) cases (31%) as HR cluster (Figure 3). 8 of 15 8 of 15 Cancers 2022, 14, 1346 Figure 3. Heatmap showing the relative expression levels of BCR/Proliferation, Host Response (HR) and Oxphos genes used to reproduce the Monti consensus clustering. The HR cluster was validated in 55/175 HOVON-84 cases; the remaining cases showed low expression of HR genes, but no distinct clustering based on BCR/Proliferation and Oxphos genes. Figure 3. Heatmap showing the relative expression levels of BCR/Proliferation, Host Response (HR) and Oxphos genes used to reproduce the Monti consensus clustering. The HR cluster was validated in 55/175 HOVON-84 cases; the remaining cases showed low expression of HR genes, but no distinct clustering based on BCR/Proliferation and Oxphos genes. Figure 3. Heatmap showing the relative expression levels of BCR/Proliferation, Host Response (HR) and Oxphos genes used to reproduce the Monti consensus clustering. The HR cluster was validated in 55/175 HOVON-84 cases; the remaining cases showed low expression of HR genes, but no distinct clustering based on BCR/Proliferation and Oxphos genes. The clusters were distributed across all three COO groups, with an enrichment of BCR/proliferation/Oxphos-high cluster in ABC cases (p = 0.02). In summary, the HR cluster, but not the BCR/Proliferation and Oxphos clusters could be validated in the HOVON-84 cohort. 3.7. Immune-Ratio Classiﬁer The immune ratio [18] revealed a ratio under the cut-off for 74 (42%) of the HOVON-84 samples (Figure S4). 3.8. Comparison of the Reproduced GEPs – – – Next, we compared the four expression signatures to establish a potential overlapping or shared biology. We focused on the overlap among the three larger GEP proﬁles and separately analyzed a potential overlap with the immune-ratio signature. The mutual impact of the COO, MYC, and the HR group of the Monti consensus clustering signatures, is shown in Figure 4. The overall picture indicated that the three proﬁles reﬂect different aspects of lymphoma biology, with no clear overlap. Most ABC cases were characterized by high MYC activity (45/58–77.6%; p < 0.00001), whereas the consensus HR-cluster was uncommon (12/58–20% samples) and showed no clear pattern in relation to the MYC signa- ture (p = 0.44). The GCB samples largely consisted of MYC-low activity cases (73/94–77.7%; p < 0.00001), with in about one third of the cases a consensus HR-cluster (31/94–33%). The smaller GCB/MYC-high group was enriched for DH (p = 0.019), DE (p = 0.41) and MYC im- mune positive cases (p = 0.002). About half of the cases in the COO-Unclassiﬁed cases were high MYC activity (11/23–48%) and consensus HR cases (12/23–52%). Thus, the MYC and consensus clustering proﬁles within the COO-Unclassiﬁed cases showed an intermediate proﬁle and did not indicate a closer association with either ABC or GCB-type DLBCL. Cancers 2022, 14, 1346 9 of 15 9 of 15 GCB/MYC−high GCB/HR GCB/non−HR Unclassified ABC/MYC−high ABC/MYC−low Lymph2Cx MYC activity score Consensus Cluster OS events Lym ph2C x GCB Unclassified ABC M Y C activity score High Low C onsensus C luster Host Response−high BCR/Proliferation/Oxphos−high O S events alive dead Figure 4. Overlap of the gene expression signatures that were validated in the HOVON-84 cohort. The three signatures show no clear overlap and together are likely to capture different aspects of DLBCL biology. OS events were observed in each of the six clusters, with a slight enrichment in the ABC/MYC-high group. Figure 4. Overlap of the gene expression signatures that were validated in the HOVON-84 cohort. The three signatures show no clear overlap and together are likely to capture different aspects of DLBCL biology. OS events were observed in each of the six clusters, with a slight enrichment in the ABC/MYC-high group. 3.8. Comparison of the Reproduced GEPs The high immune-ratio subgroup was associated with the HR consensus cluster (OR = 2.82; p = 0.003) and with the high MYC activity cluster (OR = 0.387; p = 0.003) while no association was found with the COO classiﬁer (Figure S5). We evaluated the correlation of MHC-II IHC with the different gene signatures as proposed by Ennishi et al. [45]. We did not identify an association of MHC-II-IHC high and HLA-II-IHC with COO, MYC activity score, Monti consensus clustering, and immune-ratio signatures. 3.9. Prognostic Impact of Validated Signatures – Consistent with previous publications, poor aaIPI, which does not consider age, advanced age (>60 years), the COO ABC-type, and the high MYC activity score were signiﬁcantly associated with poor ﬁve-years OS in a univariate analysis (Figure 5A–C and Figure 6A). The Hans non-GCB subgroup was associated with poor survival in HOVON-84 samples (p = 0.01) (Figure S6A). The HR cluster of the Monti consensus clustering had no impact on survival consistent with the original report (Figure 5D). In contrast to the original paper, we could not validate the prognostic relevance of the immune-ratio classiﬁer (Hazard ratio 1.6; p = 0.2) (Figure 5E). Other MYC molecular features known to impact patient’s survival based on the literature such as high MYC IHC expression and DE and DH events had no impact on ﬁve-years OS (Figure S6B–D). 5E). Other MYC molecular features known to impact pa Multivariate analysis including the four variables signiﬁcant in the univariate analysis, i.e., aaIPI, age, GCB versus ABC, and MYC activity score showed that only the COO ABC-type remained prognostic (Hazard ratio 3.06; p = 0.023) (Figure 6B). 10 of 15 Cancers 2022, 14, 1346 Cancers 2022, 14, 1346 10 – Figure 5. Kaplan–Meier curves showing overall survival of 175 patients from the HOVON-84 co According to (A) the aaIPI, (B) the COO classiﬁcation deﬁned by the Lymph2Cx algorithm, (C Monti consensus clusters, (D) the MYC activity classiﬁer, (E) the immune-ratio subgroups. – Figure 5. Kaplan–Meier curves showing overall survival of 175 patients from the HOVON-84 cohort: According to (A) the aaIPI, (B) the COO classiﬁcation deﬁned by the Lymph2Cx algorithm, (C) the Monti consensus clusters, (D) the MYC activity classiﬁer, (E) the immune-ratio subgroups. 11 of 15 Cancers 2022, 14, 1346 Figure 6. Five-year OS of HOVON-84 patients: (A) Forest plot with the univariate effect of the clinical variables and GEP signatures. (B) Forest plot with the multivariate effect of clinical variables and GEP signatures. In this cohort, only the COO as deﬁned by the Lymph2Cx remains signiﬁcant. Figure 6. Five-year OS of HOVON-84 patients: (A) Forest plot with the univariate effect of the clinical variables and GEP signatures. (B) Forest plot with the multivariate effect of clinical variables and GEP signatures. In this cohort, only the COO as deﬁned by the Lymph2Cx remains signiﬁcant. Figure 6. Five-year OS of HOVON-84 patients: (A) Forest plot with the univariate effect of the clinical variables and GEP signatures. 3.9. Prognostic Impact of Validated Signatures (B) Forest plot with the multivariate effect of clinical variables and GEP signatures. In this cohort, only the COO as deﬁned by the Lymph2Cx remains signiﬁcant. 4. Discussion Nevertheless, comparison of different algorithms is needed to select the best algorithm for clinical application. pp The HR cluster was the most eminent proﬁle identiﬁed by Monti et al. [17] and highlights the tumor microenvironment as a deﬁning feature. HR cases had increased expression of genes associated with T-cell-mediated immune responses, the classical com- plement pathway, coregulated inﬂammatory mediators, and connective tissue components. A micro-environment-based GE proﬁling described by Lenz et al. [20] (macrophage 1 (M1) and macrophage 2 signatures (M2)) showed a clear distribution among the COO subgroups. Although we did not include the genes to validate the M1 and M2 signatures, the inﬂammatory response described by Monti et al. [17] is characterized by and recapitu- lates this proﬁle. More recently, a novel interest for micro-environment-based GEP using CIBERSORT [50] or single-cell RNA-seq analysis has arisen. The clinical value of the HR signature might become more important with the rise of a whole new range of therapies, including chimeric antigen receptor T cells (CAR T-cells) and bispeciﬁc monoclonal anti- bodies, where the nature of the micro-environment is likely to be important for a durable clinical response [51]. p Recently, three additional signatures associated with MYC and tumor microenviron- ment were reported [27–29]. However, the limited overlap with our gene panel precluded validation of these signatures. Most likely, part of these studies basically looks at similar underlying biology, including two different MYC classiﬁers but using a different set of genes [16,28]. There was no evident overlap among the COO, HR, and MYC activity subgroups, emphasizing that each classiﬁer captures a different aspect of the biological het- erogeneous panorama. In a recent review, a reclassiﬁcation of DLBCL based on molecular genetics and gene expression proﬁling was proposed by Ennishi et al. [48]. We now show experimental evidence supporting their proposed subgrouping, with GCB-type DLBCL samples being split in three subgroups as high MYC activity non-HR cases and low MYC activity score splitting in either HR or non-HR cases. Similarly, ABC DLBCL were mainly characterized by high MYC activity scores. However, we did not ﬁnd any associations with MHC-II expression in the HOVOV-84 cohort. So, our data mostly support the newly proposed classiﬁcation by Ennishi et al. and emphasizes the importance of biology-driven molecular subgroups. Proﬁling of a dedicated subset of genes has become feasible using the Nanostring gene expression system even on FFPE tissue samples containing poor quality RNA. 4. Discussion In this NanoString-based GEP-proﬁling study we used a selected set of genes to validate previously published signatures. With this limited set of genes, we were able to faithfully reproduce the classiﬁcations of the original MYC activity score and Monti consensus clustering algorithms. Besides the COO, we also reproduced the HR cluster of the Monti consensus clustering and the MYC activity signature in the well-deﬁned HOVON- 84 study population. We were not able to reproduce the BCR or Oxphos signatures of the Monti consensus clustering. In contrast to the original study, we did not observe a signiﬁcant difference in survival for the immune-response ratio [18]. g p Although COO is well established, its prognostic value has been disputed [46]. The poor survival as observed for ABC type DLBCL has been used as a starting point for the design of several clinical trials. Combination of lenalidomide and ibrutinib in relapsed DLBCL showed efﬁcacy particularly in patients with Hans-based non-GCB type DLBCL, supporting clinical relevance of the COO concept [47]. Molecular subclassiﬁcation with or without considering the COO might be used for the design of more focused clinical trials to improve the outcome of speciﬁc DLBCL subgroups [12,13,48]. There are several ways to categorize DLBCL cases based on MYC status: i.e., DH (FISH), DE (IHC), and GEP classiﬁers. The more recently published DH GEP signature enables identiﬁcation of cases with cryptic MYC rearrangements [28,49]. The biological rationale underlying the MYC activity GEP signatures is evident, since this enables captur- ing of the indirect activity of MYC; although, implementation of such a proﬁle in clinical practice warrants further development. We were able to reproduce the Carey [16] MYC classiﬁer, but the impact of the genes was different from the original paper. A possible explanation for this difference might be that our cohort includes cases with the entire spectrum of percentage positive cells, whereas the training set from Carey has been selected for cases with more extreme IHC-based MYC scores. The differences in the spectrum of MYC scores possibly explains the weaker correlation and lower positive predictive value for identifying HGBCL-DH in the HOVON-84 samples. The high MYC activity group Cancers 2022, 14, 1346 12 of 15 12 of 15 showed a poor outcome, while DH cases had no impact on OS, probably because we ob- served a limited number of events in the HOVON-84 cohort of patients with a HGBCL-DH. 4. Discussion We show a reliable classiﬁcation of DLBCL cases with multiple gene expression signatures even when using a limited set of genes. Further validation studies are required to link the established signatures to more recently published GEP and mutational signatures and elucidate the complete spectrum of the very heterogenous group of DLBCL. Beyond the biological relevance, COO and MYC activity gene expression signatures had an impact on survival. This highlights the potential of combining different classiﬁers to improve the identiﬁcation of high-risk cases and emphasizes the need to integrate these signatures in future clinical trials. The limited gene set required to generate the signatures in combination with the freely available algorithms enables a strait forward and cost-effective implementation. Moreover, combining multiple GEP may lead to improved stratiﬁcation of patients into speciﬁc molecular subgroups that may be sensitive to speciﬁc targeted therapeutics. 5. Conclusions Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and Good Clinical Practice guidelines. The trial was approved by the medical ethics committee of the Erasmus Medical Center Rotterdam (EudraCT 2006-005174-42, NTR1014). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: Gene expression data are available under request. The clustering algo- rithms are available at: https://github.com/jessicaplaca/HOVON84 (accessed on 27 December 2021). Acknowledgments: The authors would like to thank the Hemato-Oncologie voor Volwassenen Nederland (HOVON) for providing the samples, the molecular diagnostics team of the University Medical Center Groningen (UMCG) for their support with the FISH analysis. In addition, the authors would like to acknowledge the support provided by Staudt’s Laboratory at NCI/NIH Bethesda, Maryland, USA, for online analysis of Lymph2Cx raw data for COO characterization. Conﬂicts of Interest: The authors declare no conﬂict of interest. 5. Conclusions In conclusion, we showed that COO, MYC activity score, and the HR cluster of the Monti consensus clustering were reproduced in the HOVON-84 cohort. These three signatures identify distinct subgroups based on different aspects of DLBCL biology, em- phasizing that each classiﬁer captures distinct molecular proﬁles, offering a framework for clinical trials. Cancers 2022, 14, 1346 13 of 15 Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/cancers14051346/s1, Figure S1: MYC activity classiﬁer for the original Carey training cohort, Figure S2: Consensus clusters in original Monti cohort, Figure S3: Identiﬁcation of consensus clusters in the HOVON-84 cohort using 47 selected genes following the approach as published by Monti, Figure S4: Reproduction of the immune ratio. Distribution of the CD4CD8:(CD163:CD68)PD-L1 immuno-ratio for HOVON-84 cohort, Figure S5: Overlap of Immune ratio, COO classiﬁer, and Consensus Cluster signatures in the HOVON-84 cohort, Figure S6: Kaplan–Meier curves for overall survival of the HOVON-84 cohort for (a) the COO classiﬁcation deﬁned by Hans; Table S1: Overview of characteristics of the HOVON-84 and previously published cohorts, Table S2: List of genes used to classify the four GEP using quantiﬁcation by the Nanostring platform, Table S3: Comparison of cell of origin (COO) allocation between COO classiﬁer and Hans algorithm, Table S4: Performance of MYC activity classiﬁer in the Carey training and HOVON-84 test sets. Author Contributions: J.R.P. performed the GEP analysis and writing. M.N., A.v.d.B., A.D. and W.A.d.S.J. were involved in study design and drafting the manuscript. M.M. and T.L. performed the SV analysis supervised by B.Y., A.S. performed FISH, extracted RNA, and generated the NanoString data. P.J.L. was the principal investigator of the HOVON-84 study, and J.Z. was the study coordinator of the HOVON-84 study, K.L., A.D. and D.d.J. performed centralized histology review. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Dutch Cancer Society (KWF Alpe d’Huzes project: VU2014- 5711), the UMCG. This study was also ﬁnanced in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES)—Finance Code 001 and the São Paulo Research Foundation (FAPESP) (W.A.d.S.J. grant #2013/08135–2). Funding: This research was funded by Dutch Cancer Society (KWF Alpe d’Huzes project: VU2014- 5711), the UMCG. This study was also ﬁnanced in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES)—Finance Code 001 and the São Paulo Research Foundation (FAPESP) (W.A.d.S.J. grant #2013/08135–2). yp g y p , , [ ] 7. Rosenwald, A.; Bens, S.; Advani, R.; Barrans, S.; Copie-Bergman, C.; Elsensohn, M.H.; Natkunam, Y.; Calaminici, M.; Sander, B.; Baia, M.; et al. Prognostic Signiﬁcance of MYC Rearrangement and Translocation Partner in Diffuse Large B-Cell Lymphoma: A Study by the Lunenburg Lymphoma Biomarker Consortium. J. Clin. Oncol. 2019, 37, 3359–3368. [CrossRef] g y p J [ ] 6. Vitolo, U.; Witzig, T.E.; Gascoyne, R.D.; Scott, D.W.; Zhang, Q.; Jurczak, W.; Özcan, M.; Hong, X.; Zhu, J.; Jin, J.; et al. ROBUST: First report of phase III randomized study of lenalidomide/R-CHOP (R2-CHOP) vs placebo/R-CHOP in previously untreated ABC-type diffuse large B-cell lymphoma. Hematol. Oncol. 2019, 37, 36–37. [CrossRef] 1. Swerdlow, S.; Campo, E.; Harris, N. WHO Classiﬁcation of Tumours of Haematopoietic and Lymphoid Tissues; WHO: Rome, Italy, 2017. 2. Sehn, L.H.; Berry, B.; Chhanabhai, M.; Fitzgerald, C.; Gill, K.; Hoskins, P.; Klasa, R.; Savage, K.J.; Shenkier, T.; Sutherland, J.; et al. The revised International Prognostic Index (R-IPI) is a better predictor of outcome than the standard IPI for patients with diffuse large B-cell lymphoma treated with R-CHOP. Blood 2007, 109, 1857–1861. [CrossRef] [PubMed] 3. Alizadeh, A.A.; Eisen, M.B.; Davis, R.E.; Ma, C.; Lossos, I.S.; Rosenwald, A.; Boldrick, J.C.; Sabet, H.; Tran, T.; Yu, X.; et al. Distinct types of diffuse large B-cell lymphoma identiﬁed by gene expression proﬁling. Nature 2000, 403, 503–511. [CrossRef] [PubMed] 4. Leonard, J.P.; Kolibaba, K.S.; Reeves, J.A.; Tulpule, A.; Flinn, I.W.; Kolevska, T.; Robles, R.; Flowers, C.R.; Collins, R.; DiBella, N.J.; et al. Randomized Phase II Study of R-CHOP with or Without Bortezomib in Previously Untreated Patients with Non-Germinal Center B-Cell-Like Diffuse Large B-Cell Lymphoma. J. Clin. Oncol. 2017, 35, 3538–3546. [CrossRef] [PubMed] 5. Younes, A.; Sehn, L.H.; Johnson, P.; Zinzani, P.L.; Hong, X.; Zhu, J.; Patti, C.; Belada, D.; Samoilova, O.; Suh, C.; et al. Randomized Phase III Trial of Ibrutinib and Rituximab Plus Cyclophosphamide, Doxorubicin, Vincristine, and Prednisone in Non-Germinal Center B-Cell Diffuse Large B-Cell Lymphoma. J. Clin. Oncol. 2019, 37, 1285–1295. [CrossRef] g y p 3. Alizadeh, A.A.; Eisen, M.B.; Davis, R.E.; Ma, C.; Lossos, I.S.; Rosenwald, A.; Boldrick, J.C.; Sabet, H.; Tran, T.; Yu, X.; et al. Distinct types of diffuse large B-cell lymphoma identiﬁed by gene expression proﬁling. Nature 2000, 403, 503–511. [CrossRef] [PubMed] 4. Leonard, J.P.; Kolibaba, K.S.; Reeves, J.A.; Tulpule, A.; Flinn, I.W.; Kolevska, T.; Robles, R.; Flowers, C.R.; Collins, R.; 4. Leonard, J.P.; Kolibaba, K.S.; Reeves, J.A.; Tulpule, A.; Flinn, I.W.; Kolevska, T.; Robles, R.; Flowers, C.R.; Collins, R.; DiBella, N.J.; et al. Randomized Phase II Study of R-CHOP with or Without Bortezomib in Previously Untreated Patients with Non-Germinal Center B-Cell-Like Diffuse Large B-Cell Lymphoma. J. Clin. Oncol. 2017, 35, 3538–3546. [CrossRef] [PubMed] 5 Younes A ; Sehn L H ; Johnson P; Zinzani PL ; Hong X ; Zhu J ; Patti C ; Belada D ; Samoilova O ; Suh C ; et al Randomized with Non Germinal Center B Cell Like Diffuse Large B Cell Lymphoma. J. Clin. Oncol. 2017, 35, 3538 3546. [CrossRef] [PubMed] 5. Younes, A.; Sehn, L.H.; Johnson, P.; Zinzani, P.L.; Hong, X.; Zhu, J.; Patti, C.; Belada, D.; Samoilova, O.; Suh, C.; et al. Randomized Phase III Trial of Ibrutinib and Rituximab Plus Cyclophosphamide, Doxorubicin, Vincristine, and Prednisone in Non-Germinal Center B-Cell Diffuse Large B-Cell Lymphoma. J. Clin. Oncol. 2019, 37, 1285–1295. [CrossRef] g y p 3. Alizadeh, A.A.; Eisen, M.B.; Davis, R.E.; Ma, C.; Lossos, I.S.; Rosenwald, A.; Boldrick, J.C.; Sabet, H.; Tran, T.; Yu, X.; et al. Distinct types of diffuse large B-cell lymphoma identiﬁed by gene expression proﬁling. Nature 2000, 403, 503–511. [CrossRef] [PubMed] 4. Leonard, J.P.; Kolibaba, K.S.; Reeves, J.A.; Tulpule, A.; Flinn, I.W.; Kolevska, T.; Robles, R.; Flowers, C.R.; Collins, R.; DiBella, N.J.; et al. Randomized Phase II Study of R-CHOP with or Without Bortezomib in Previously Untreated Patients with Non-Germinal Center B-Cell-Like Diffuse Large B-Cell Lymphoma. J. Clin. Oncol. 2017, 35, 3538–3546. [CrossRef] [PubMed] Y A S h h X h C l d S l O S h C l d d DiBella, N.J.; et al. Randomized Phase II Study of R-CHOP with or Without Bortezomib in Previously Untreated Patients with Non-Germinal Center B-Cell-Like Diffuse Large B-Cell Lymphoma. J. Clin. Oncol. 2017, 35, 3538–3546. [CrossRef] [PubMed] 5. Younes, A.; Sehn, L.H.; Johnson, P.; Zinzani, P.L.; Hong, X.; Zhu, J.; Patti, C.; Belada, D.; Samoilova, O.; Suh, C.; et al. Randomized Phase III Trial of Ibrutinib and Rituximab Plus Cyclophosphamide, Doxorubicin, Vincristine, and Prednisone in Non-Germinal Center B-Cell Diffuse Large B-Cell Lymphoma. J. Clin. Oncol. 2019, 37, 1285–1295. [CrossRef] References Carey, C.D.; Gusenleitner, D.; Chapuy, B.; Kovach, A.E.; Kluk, M.J.; Sun, H.H.; Crossland, R.E.; Bacon, C.M.; Rand, V.; Dal Cin, P.; et al. Molecular classiﬁcation of MYC-driven B-cell lymphomas by targeted gene expression proﬁling of ﬁxed biopsy specimens. J. Mol. Diagn. 2015, 17, 19–30. [CrossRef] p y p J g 17. Monti, S.; Savage, K.J.; Kutok, J.L.; Feuerhake, F.; Kurtin, P.; Mihm, M.; Wu, B.; Pasqualucci, L.; Neuberg, D.; Aguiar, R.C.; et al. Molecular proﬁling of diffuse large B-cell lymphoma identiﬁes robust subtypes including one characterized by host inﬂammatory response. Blood 2005, 105, 1851–1861. [CrossRef] 18. Keane, C.; Vari, F.; Hertzberg, M.; Cao, K.A.; Green, M.R.; Han, E.; Seymour, J.F.; Hicks, R.J.; Gill, D.; Crooks, P.; et al. Ratios of T-cell immune effectors and checkpoint molecules as prognostic biomarkers in diffuse large B-cell lymphoma: A population-based study. Lancet Haematol. 2015, 2, e445–e455. [CrossRef] y 19. Rosenwald, A.; Wright, G.; Chan, W.C.; Connors, J.M.; Campo, E.; Fisher, R.I.; Gascoyne, R.D.; Muller-Hermelink, H.K.; Smeland, E.B.; Giltnane, J.M.; et al. The use of molecular proﬁling to predict survival after chemotherapy for diffuse large-B-cell lymphoma. N. Engl. J. Med. 2002, 346, 1937–1947. [CrossRef] g 20. Lenz, G.; Wright, G.; Dave, S.S.; Xiao, W.; Powell, J.; Zhao, H.; Xu, W.; Tan, B.; Goldschmidt, N.; Iqbal, J.; et al. Stromal gene signatures in large-B-cell lymphomas. N. Engl. J. Med. 2008, 359, 2313–2323. [CrossRef] 21. Shipp, M.A.; Ross, K.N.; Tamayo, P.; Weng, A.P.; Kutok, J.L.; Aguiar, R.C.; Gaasenbeek, M.; Angelo, M.; Reich, M.; Pinkus, G.S.; et al. Diffuse large B-cell lymphoma outcome prediction by gene-expression proﬁling and supervised machine learning. Nat. Med. 2002, 8, 68–74. [CrossRef] g 22. Chan, F.C.; Telenius, A.; Healy, S.; Ben-Neriah, S.; Mottok, A.; Lim, R.; Drake, M.; Hu, S.; Ding, J.; Ha, G.; et al. An RCOR1 loss-associated gene expression signature identiﬁes a prognostically signiﬁcant DLBCL subgroup. Blood 2015, 125, 959–966. [CrossRef] [PubMed] 23. Dybkær, K.; Bøgsted, M.; Falgreen, S.; Bødker, J.S.; Kjeldsen, M.K.; Schmitz, A.; Bilgrau, A.E.; Xu-Monette, Z.Y.; Li, L.; Bergkvist, K.S.; et al. Diffuse large B-cell lymphoma classiﬁcation system that associates normal B-cell subset phenotypes with prognosis. J. Clin. Oncol. 2015, 33, 1379–1388. [CrossRef] [PubMed] 24. Li, C.; Kim, S.W.; Rai, D.; Bolla, A.R.; Adhvaryu, S.; Kinney, M.C.; Robetorye, R.S.; Aguiar, R.C. Copy number abnormalities, MYC activity, and the genetic ﬁngerprint of normal B cells mechanistically deﬁne the microRNA proﬁle of diffuse large B-cell lymphoma. Blood 2009, 113, 6681–6690. References Analysis of the coding genome of diffuse large B-cell lymphoma. Nat. Genet. 2011, 43, 830–837. [CrossRef] y g g g y p 11. Reddy, A.; Zhang, J.; Davis, N.S.; Mofﬁtt, A.B.; Love, C.L.; Waldrop, A.; Leppa, S.; Pasanen, A.; Meriranta, L.; Karjalainen- Lindsberg, M.L.; et al. Genetic and Functional Drivers of Diffuse Large B Cell Lymphoma. Cell 2017, 171, 481–494.e15. [CrossRef] 11. Reddy, A.; Zhang, J.; Davis, N.S.; Mofﬁtt, A.B.; Love, C.L.; Waldrop, A.; Leppa, S.; Pasanen, A.; Meriranta, L.; Karjalainen- Lindsberg, M.L.; et al. Genetic and Functional Drivers of Diffuse Large B Cell Lymphoma. Cell 2017, 171, 481–494.e15. [CrossRef] 12. Schmitz, R.; Wright, G.W.; Huang, D.W.; Johnson, C.A.; Phelan, J.D.; Wang, J.Q.; Roulland, S.; Kasbekar, M.; Young, R.M.; Shaffer, A.L.; et al. Genetics and Pathogenesis of Diffuse Large B-Cell Lymphoma. N. Engl. J. Med. 2018, 378, 1396–1407. [CrossRef] [PubMed] 11. Reddy, A.; Zhang, J.; Davis, N.S.; Mofﬁtt, A.B.; Love, C.L.; Waldrop, A.; Leppa, S.; Pasanen, A.; M Lindsberg, M.L.; et al. Genetic and Functional Drivers of Diffuse Large B Cell Lymphoma. Cell 2017, 171 12. Schmitz, R.; Wright, G.W.; Huang, D.W.; Johnson, C.A.; Phelan, J.D.; Wang, J.Q.; Roulland, S.; Kasbekar, M.; Young, R.M.; Shaffer, A.L.; et al. Genetics and Pathogenesis of Diffuse Large B-Cell Lymphoma. N. Engl. J. Med. 2018, 378, 1396–1407. [CrossRef] [PubMed] g g g g et al. Genetics and Pathogenesis of Diffuse Large B-Cell Lymphoma. N. Engl. J. Med. 2018, 378, 1396–14 Med] 13. Chapuy, B.; Stewart, C.; Dunford, A.J.; Kim, J.; Kamburov, A.; Redd, R.A.; Lawrence, M.S.; Roemer, M.G.M.; Li, A.J.; Ziepert, M.; et al. Molecular subtypes of diffuse large B cell lymphoma are associated with distinct pathogenic mechanisms and outcomes. Nat. Med. 2018, 24, 679–690. [CrossRef] [PubMed] 14. Lacy, S.E.; Barrans, S.L.; Beer, P.A.; Painter, D.; Smith, A.G.; Roman, E.; Cooke, S.L.; Ruiz, C.; Glover, P.; Van Hoppe, S.J.L.; et al. Targeted sequencing in DLBCL, molecular subtypes, and outcomes: A Haematological Malignancy Research Network report. Blood 2020, 135, 1759–1771. [CrossRef] [PubMed] [ ] [ ] 15. Scott, D.W.; Wright, G.W.; Williams, P.M.; Lih, C.J.; Walsh, W.; Jaffe, E.S.; Rosenwald, A.; Campo, E.; Chan, W.C.; Connors, J.M.; et al. Determining cell-of-origin subtypes of diffuse large B-cell lymphoma using gene expression in formalin-ﬁxed parafﬁn-embedded tissue. Blood 2014, 123, 1214–1217. [CrossRef] [PubMed] p , , [ ] [ ] 16. References 1. Swerdlow, S.; Campo, E.; Harris, N. WHO Classiﬁcation of Tumours of Haematopoietic and Lymphoid Tissues; WHO: Rome, Italy, 2017. 2. Sehn, L.H.; Berry, B.; Chhanabhai, M.; Fitzgerald, C.; Gill, K.; Hoskins, P.; Klasa, R.; Savage, K.J.; Shenkier, T.; Sutherland, J.; et al. The revised International Prognostic Index (R-IPI) is a better predictor of outcome than the standard IPI for patients with diffuse large B-cell lymphoma treated with R-CHOP. Blood 2007, 109, 1857–1861. [CrossRef] [PubMed] g y p 3. Alizadeh, A.A.; Eisen, M.B.; Davis, R.E.; Ma, C.; Lossos, I.S.; Rosenwald, A.; Boldrick, J.C.; Sabet, H.; Tran, T.; Yu, X.; et al. Distinct types of diffuse large B-cell lymphoma identiﬁed by gene expression proﬁling. Nature 2000, 403, 503–511. [CrossRef] [PubMed] 4. Leonard, J.P.; Kolibaba, K.S.; Reeves, J.A.; Tulpule, A.; Flinn, I.W.; Kolevska, T.; Robles, R.; Flowers, C.R.; Collins, R.; DiBella, N.J.; et al. Randomized Phase II Study of R-CHOP with or Without Bortezomib in Previously Untreated Patients with Non-Germinal Center B-Cell-Like Diffuse Large B-Cell Lymphoma. J. Clin. Oncol. 2017, 35, 3538–3546. [CrossRef] [PubMed] 5. Younes, A.; Sehn, L.H.; Johnson, P.; Zinzani, P.L.; Hong, X.; Zhu, J.; Patti, C.; Belada, D.; Samoilova, O.; Suh, C.; et al. Randomized Phase III Trial of Ibrutinib and Rituximab Plus Cyclophosphamide, Doxorubicin, Vincristine, and Prednisone in Non-Germinal Center B-Cell Diffuse Large B-Cell Lymphoma. J. Clin. Oncol. 2019, 37, 1285–1295. [CrossRef] g y p 6. Vitolo, U.; Witzig, T.E.; Gascoyne, R.D.; Scott, D.W.; Zhang, Q.; Jurczak, W.; Özcan, M.; Hong, X.; Zhu, J.; Jin, J.; et al. ROBUST: First report of phase III randomized study of lenalidomide/R-CHOP (R2-CHOP) vs placebo/R-CHOP in previously untreated ABC-type diffuse large B-cell lymphoma. Hematol. Oncol. 2019, 37, 36–37. [CrossRef] 14 of 15 14 of 15 Cancers 2022, 14, 1346 8. Casan, J.M.; Barraclough, A.; Shortt, J.; Hawkes, E.A. Dose-adjusted EPOCH-R therapy in MYC-rearranged diffuse large B-cell lymphoma: Not yet the standard of care. Lancet Haematol. 2019, 6, e119. [CrossRef] 9. Chamuleau, M.E.D.; Burggraaff, C.N.; Nijland, M.; Bakunina, K.; Mous, R.; Lugtenburg, P.J.; Dierickx, D.; van Imhoff, G.W.; Vermaat, J.S.P.; Marijt, E.A.F.; et al. Treatment of patients with MYC rearrangement positive large B-cell lymphoma with R-CHOP plus lenalidomide: Results of a multicenter HOVON phase II trial. Haematologica 2019, 105, 2805–2812. [CrossRef] p p g 10. Pasqualucci, L.; Trifonov, V.; Fabbri, G.; Ma, J.; Rossi, D.; Chiarenza, A.; Wells, V.A.; Grunn, A.; Messina, M.; Elliot, O.; et al. References [CrossRef] 25. Sha, C.; Barrans, S.; Care, M.A.; Cunningham, D.; Tooze, R.M.; Jack, A.; Westhead, D.R. Transferring genomics to the clinic: Distinguishing Burkitt and diffuse large B cell lymphomas. Genome Med. 2015, 7, 64. [CrossRef] [PubMed] 26. Masqué-Soler, N.; Szczepanowski, M.; Kohler, C.W.; Spang, R.; Klapper, W. Molecular classiﬁcation of mature aggressive B-cell lymphoma using digital multiplexed gene expression on formalin-ﬁxed parafﬁn-embedded biopsy specimens. Blood 2013, 122, 1985–1986. [CrossRef] [PubMed] 27. Ciavarella, S.; Vegliante, M.C.; Fabbri, M.; De Summa, S.; Melle, F.; Motta, G.; De Iuliis, V.; Opinto, G.; Enjuanes, A.; Rega, S.; et al. Dissection of DLBCL microenvironment provides a gene expression-based predictor of survival applicable to formalin-ﬁxed parafﬁn-embedded tissue. Ann. Oncol. 2018, 29, 2363–2370. [CrossRef] p 28. Ennishi, D.; Jiang, A.; Boyle, M.; Collinge, B.; Grande, B.M.; Ben-Neriah, S.; Rushton, C.; Tang, J.; Thomas, N.; Slack, G.W.; et al. Double-Hit Gene Expression Signature Deﬁnes a Distinct Subgroup of Germinal Center B-Cell-Like Diffuse Large B-Cell Lymphoma. J. Clin. Oncol. 2019, 37, 190–201. [CrossRef] [PubMed] 15 of 15 15 of 15 Cancers 2022, 14, 1346 29. Staiger, A.M.; Altenbuchinger, M.; Ziepert, M.; Kohler, C.; Horn, H.; Huttner, M.; Hüttl, K.S.; Glehr, G.; Klapper, W.; Szczepanowski, M.; et al. A novel lymphoma-associated macrophage interaction signature (LAMIS) provides robust risk prognostication in diffuse large B-cell lymphoma clinical trial cohorts of the DSHNHL. Leukemia 2020, 34, 543–552. [CrossRef] p g g y p , , [ ] 30. Veldman-Jones, M.H.; Brant, R.; Rooney, C.; Geh, C.; Emery, H.; Harbron, C.G.; Wappett, M.; Sharpe, A.; Dymond, M.; Barrett, J.C.; et al. Evaluating Robustness and Sensitivity of the NanoString Technologies nCounter Platform to Enable Multi- plexed Gene Expression Analysis of Clinical Samples. Cancer Res. 2015, 75, 2587–2593. [CrossRef] p p y p [ ] 31. Lugtenburg, P.J.; Brown, P.d.N.; Holt, B.v.d.; D’Amore, F.A.; Koene, H.R.; Jongh, E.d.; Fijnheer, R.; Esser, J.W.v.; Böhmer, L.H.; Pruijt, J.F. Rituximab-CHOP with early rituximab intensiﬁcation for diffuse large B-cell lymphoma: A randomized phase 3 trial of the HOVON and the Nordic Lymphoma Group (HOVON-84). J. Clin. Oncol. 2020, 38, 3377–3387. [CrossRef] 32. Salles, G.; de Jong, D.; Xie, W.; Rosenwald, A.; Chhanabhai, M.; Gaulard, P.; Klapper, W.; Calaminici, M.; Sander, B.; Thorns, C.; et al. Prognostic signiﬁcance of immunohistochemical biomarkers in diffuse large B-cell lymphoma: A study from the Lunenburg Lymphoma Biomarker Consortium. Blood 2011, 117, 7070–7078. [CrossRef] [PubMed] g y p 33. References Bioinformaticsi 2010, 26, 1572–1573. [CrossRef] , , [ ] R.; Kruisselbrink, J. Flexible Self-Organizing Maps in kohonen 3.0. J. Stat. Softw. 2018, 87, 1–18. [CrossRef] 41. Wehrens, R.; Kruisselbrink, J. Flexible Self-Organizing Maps in kohonen 3.0. J. Stat. Softw. 2018, 87, 1–1 42. Scrucca, L.; Fop, M.; Murphy, T.B.; Raftery, A.E. mclust 5: Clustering, Classiﬁcation and Density Estimation Using Gaussian Finite Mixture Models. R J. 2016, 8, 289–317. [CrossRef] [PubMed] 43. Team, R.C. R: A Language and Environment for Statistical Computing; R Foundation for Statistical Computing: Vienna, Austria, 2019. 44. Yoon, N.; Ahn, S.; Yong Yoo, H.; Jin Kim, S.; Seog Kim, W.; Hyeh Ko, Y. Cell-of-origin of diffuse large B-cell lymphomas determined by the Lymph2Cx assay: Better prognostic indicator than Hans algorithm. Oncotarget 2017, 8, 22014–22022. [CrossRef] [PubMed] , g g f p g; p g , , 44. Yoon, N.; Ahn, S.; Yong Yoo, H.; Jin Kim, S.; Seog Kim, W.; Hyeh Ko, Y. Cell-of-origin of diffuse large B-cell lymphomas determined by the Lymph2Cx assay: Better prognostic indicator than Hans algorithm. Oncotarget 2017, 8, 22014–22022. [CrossRef] [PubMed] by the Lymph2Cx assay: Better prognostic indicator than Hans algorithm. Oncotarget 2017, 8, 22014–22022. [CrossRef] [PubMed] 45. Klapper, W.; Kreuz, M.; Kohler, C.W.; Burkhardt, B.; Szczepanowski, M.; Salaverria, I.; Hummel, M.; Loefﬂer, M.; Pellissery, S.; Woessmann, W.; et al. Patient age at diagnosis is associated with the molecular characteristics of diffuse large B-cell lymphoma. Blood 2012, 119, 1882–1887. [CrossRef] [PubMed] 46. Staiger, A.M.; Ziepert, M.; Horn, H.; Scott, D.W.; Barth, T.F.E.; Bernd, H.W.; Feller, A.C.; Klapper, W.; Szczepanowski, M.; Hummel, M.; et al. Clinical Impact of the Cell-of-Origin Classiﬁcation and the MYC/ BCL2 Dual Expresser Status in Diffuse Large B-Cell Lymphoma Treated Within Prospective Clinical Trials of the German High-Grade Non-Hodgkin’s Lymphoma Study Group. J. Clin. Oncol. 2017, 35, 2515–2526. [CrossRef] p 47. Goy, A.; Ramchandren, R.; Ghosh, N.; Munoz, J.; Morgan, D.S.; Dang, N.H.; Knapp, M.; Delioukina, M.; Kingsley, E.; Ping, J.; et al. Ibrutinib plus lenalidomide and rituximab has promising activity in relapsed/refractory non-germinal center B-cell-like DLBCL. Blood 2019, 134, 1024–1036. [CrossRef] 48. Ennishi, D.; Hsi, E.D.; Steidl, C.; Scott, D.W. Toward a New Molecular Taxonomy of Diffuse Large B-cell Lymphoma. Cancer Discov. 2020, 10, 1267–1281. [CrossRef] 49. Hilton, L.K.; Tang, J.; Ben-Neriah, S.; Alcaide, M.; Jiang, A.; Grande, B.M.; Rushton, C.K.; Boyle, M.; Meissner, B.; Scott, D.W.; et al. References Hans, C.P.; Weisenburger, D.D.; Greiner, T.C.; Gascoyne, R.D.; Delabie, J.; Ott, G.; Müller-Hermelink, H.K.; Campo, E.; Braziel, R.M.; Jaffe, E.S.; et al. Conﬁrmation of the molecular classiﬁcation of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray. Blood 2004, 103, 275–282. [CrossRef] g y 34. Hu, S.; Xu-Monette, Z.Y.; Tzankov, A.; Green, T.; Wu, L.; Balasubramanyam, A.; Liu, W.M.; Visco, C.; Li, Y.; Miranda, R.N.; et al. MYC/BCL2 protein coexpression contributes to the inferior survival of activated B-cell subtype of diffuse large B-cell lymphoma and demonstrates high-risk gene expression signatures: A report from The International DLBCL Rituximab-CHOP Consortium Program. Blood 2013, 121, 4021–4031. [CrossRef] [PubMed] g 35. Nijland, M.; Veenstra, R.N.; Visser, L.; Xu, C.; Kushekhar, K.; van Imhoff, G.W.; Kluin, P.M.; van den Berg, A.; Diepstra, A. HLA dependent immune escape mechanisms in B-cell lymphomas: Implications for immune checkpoint inhibitor therapy? Oncoimmunology 2017, 6, e1295202. [CrossRef] [PubMed] gy 36. Mendeville, M.; Roemer, M.G.M.; van den Hout, M.; Los-de Vries, G.T.; Bladergroen, R.; Stathi, P.; Hijmering, N.J.; Rosenwald, A.; Ylstra, B.; de Jong, D. Aggressive genomic features in clinically indolent primary HHV8-negative effusion-based lymphoma. Blood 2019, 133, 377–380. [CrossRef] 37. Waggott, D.; Chu, K.; Yin, S.; Yin, S.; Wouters, B.G.; Wouters, B.; Liu, F.-F.; Liu, F.; Boutros, P.C.; Boutros, P.C. NanoStringNorm: An extensible R package for the pre-processing of NanoString mRNA and miRNA data. Bioinformatics 2012, 28, 1546–1548. [CrossRef] 38. Gevrey, M.; Dimopoulos, I.; Lek, S. Review and comparison of methods to study the contribution of variables in artiﬁcial neural network models. Ecol. Model. 2003, 160, 249–264. [CrossRef] 37. Waggott, D.; Chu, K.; Yin, S.; Yin, S.; Wouters, B.G.; Wouters, B.; Liu, F.-F.; Liu, F.; Boutros, P.C.; Boutros, P.C. NanoStringNorm: An extensible R package for the pre-processing of NanoString mRNA and miRNA data. Bioinformatics 2012, 28, 1546–1548. [CrossRef] 38. Gevrey, M.; Dimopoulos, I.; Lek, S. Review and comparison of methods to study the contribution of variables in artiﬁcial neural 38. Gevrey, M.; Dimopoulos, I.; Lek, S. Review and comparison of methods to study the contribution of va network models. Ecol. Model. 2003, 160, 249–264. [CrossRef] 39. Kuhn, M. Building Predictive Models in R Using the caret Package. J. Stat. Softw. 2008, 28, 1–26. [CrossRef] 39. Kuhn, M. Building Predictive Models in R Using the caret Package. J. Stat. Softw. 2008, 28, 1–26. [CrossRef] 40. Wilkerson, M.D.; Hayes, D.N. ConsensusClusterPlus: A class discovery tool with conﬁdence assessments and item tracking. References The double-hit signature identiﬁes double-hit diffuse large B-cell lymphoma with genetic events cryptic to FISH. Blood 2019, 134, 1528–1532. [CrossRef] 50. Chen, B.; Khodadoust, M.S.; Liu, C.L.; Newman, A.M.; Alizadeh, A.A. Proﬁling Tumor Inﬁltrating Immune Cells with CIBERSORT. Methods Mol. Biol. 2018, 1711, 243–259. [CrossRef] 51. Qin, J.S.; Johnstone, T.G.; Baturevych, A.; Hause, R.J.; Ragan, S.P.; Clouser, C.R.; Jones, J.C.; Ponce, R.; Krejsa, C.M.; Salmon, R.A.; et al. Antitumor Potency of an Anti-CD19 Chimeric Antigen Receptor T-Cell Therapy, Lisocabtagene Maraleucel in Combination with Ibrutinib or Acalabrutinib. J. Immunother. 2020, 43, 107–120. [CrossRef]
https://openalex.org/W3169861924	https://iris.unive.it/bitstream/10278/3740609/1/2021_Bertin_Carrino_Pantalone.pdf	English	null	Do standard classifications still represent European welfare typologies? Novel evidence from studies on health and social care	Social science & medicine	2,021	cc-by	10,237	A R T I C L E I N F O Keywords: Classification Meta-analysis Health policy Social policy Public policy Welfare regime Public economics Due to the profound changes that have characterised welfare systems, the representativeness of standard welfare classifications such as Esping-Andersen’s Three Worlds of Welfare (TWW) have been questioned. In response to concerns that welfare services do not share a common rationale across policy areas, new typologies focused on sub-areas of welfare provision have been introduced. Still, there is little evidence on whether such policy-specific typologies are (i) consistent with the standard TWW classifications; and (ii) consistent across policy areas. i i We reviewed 22 recent studies which identified welfare typologies in 12 European countries focusing on economically relevant areas such as healthcare and social care. We build novel indices of “welfare similarity” to measure the extent to which welfare systems have been grouped together in previous studies. Our findings are twofold: first, healthcare and social care policies are characterised by the coexistence and overlap of multiple regimes, i.e., a hybridisation of the original TWW taxonomy. Second, countries classifications are substantially different between healthcare and social care, which highlights the lack of coherence in welfare systems rationales across policy areas. Our findings suggest that comparative analyses of welfare systems should narrow their focus on policy-specific areas, which may prove more informative than general classifications of welfare states. original EA classification or whether they have led to a reduction in the internal homogeneity and consistency of the EA classification. Do standard classifications still represent European welfare typologies? Novel evidence from studies on health and social care Giovanni Bertin a, Ludovico Carrino a,b, Marta Pantalone a, a Ca’ Foscari, University of Venice, Department of Economics, Fondamenta S. Giobbe - Cannaregio 873, 30121, Venezia, Italy b King’s College London, Department of Global Health & Social Medicine, Aldwych 40, London, WC2R 2LS, UK Social Science & Medicine 281 (2021) 114086 Social Science & Medicine 281 (2021) 114086 Available online 29 May 2021 0277-9536/© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). * Corresponding author. E-mail addresses: giovanni.bertin@unive.it (G. Bertin), ludovico.carrino@kcl.ac.uk (L. Carrino), marta.pantalone@unive.it (M. Pantalone). https://doi.org/10.1016/j.socscimed.2021.114086 Received in revised form 29 April 2021; Accepted 26 May 2021 Available online 29 May 2021 0277-9536/© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1016/j.socscimed.2021.114086 Received in revised form 29 April 2021; Accepted 26 May 2021 * Corresponding author. E-mail addresses: giovanni.bertin@unive.it (G. Bertin), ludovico.carrino@kcl.ac.uk (L. Carrino), marta.pantalone@unive.it (M. Pantalone). * Corresponding author. E-mail addresses: giovan 2. Classifications of evolving welfare systems: background and hypotheses A large stream of recent literature has discussed the complexity and multidimensionality of the transition process within welfare regimes (H¨ausermann, 2012; Jensen, 2011). Following Jensen (2011), the development of welfare states can be explained according to three different perspectives. i The first, the ideological perspective, suggests that welfare reforms are the outcome of an interaction and bargaining process between competing welfare-state ideologies (conservative, democratic or lib eral). The complexity of such interaction makes it often hard to place such policy changes within the classic EA taxonomy. Moreover, the context within which welfare reforms take place is in constant evolution and characterised by unstable equilibria and non-linear developments, reflecting the instability of recent political processes, including the dissolution of massive ideological blocks. This has led to the overlap and merging of different welfare perspectives and rationales, as in the case of liberal neo-welfarism, which results from an overlap between laissez- faire theories and the theoretical grounds at the base of welfare sys tems (Ferrera, 2013). Moreover, several authors have advocated the need to move beyond the debate on stereotypical political ideologies regarding social protection (e.g., the left as the advocate of social pro tection and the right as the driver of social spending cuts; see H¨ausermann, 2012), in favour of a differentiation of policies that aim (or not) to shift public intervention from old to new social risks. Driven by concerns on the limitations of the welfare-as-a-whole classifications, a large body of literature has identified policy-specific welfare typologies through comparative analyses of, for example, healthcare (Bambra, 2005; B¨ohm et al., 2013; Jensen, 2008; Joumard et al., 2010; Reibling, 2010; Wendt, 2009, 2014) and social care policies (Bambra, 2004, 2007; Bettio and Plantenga, 2004; Boje and Ejrnæs, 2012; Chau and Sam, 2013; Cho, 2014; Jensen, 2008; Kautto, 2002; Kraus et al., 2010; Leitner, 2003; Th´evenon, 2011; Verbeek-Oudijk et al., 2014). However, to the best of our knowledge, no study has yet ana lysed: (i) the extent to which the cluster structures resulting from policy-specific studies overlap with the standard EA classification (hybridisation hypothesis); and (ii) whether the typologies emerging from studies focusing on different policy-areas are consistent or not (incoherence hypothesis). i This paper aims to fill this gap by performing a meta-analysis of studies that produced classifications of, respectively, healthcare and social care systems in 12 European countries in the first decade of the 2000s. G. Bertin et al. Social Science & Medicine 281 (2021) 114086 Most meta-analyses of welfare classifications typically include mul tiple areas of social policy (i.e., the “welfare-as-a-whole”). For example, Powell et al. (2020) reviewed studies that focused on the welfare state as-a-whole rather than on specific services. Ferragina and Seeleib-Kaiser (2011) included papers which focused on cash transfers and a mix of indicators related to the concepts of “decommodification”, “social stratification” and “defamilisation”. Similarly, Buhr and Stoy (2015) jointly reviewed studies on social care, healthcare and education pol icies. By considering several policy areas at once, the aforementioned studies implicitly assume that, within the same country, the provision of welfare services across different policy areas share a homogeneous and coherent rationale. Alternatively stated, each welfare regime is assumed to reflect a set of values coherently realised in each policy area. How ever, recent studies have suggested that such assumption is unlikely to hold. l Our findings are twofold: first, we highlight the coexistence and overlap of multiple regimes in both healthcare and social care policies, which results in a hybridisation of the original EA classification. Second, we find that countries classifications are substantially different between healthcare and social care policies, which provides evidence for the lack of coherence of welfare provision rationales across policy areas. Our results are relevant for both the academic and policy debate, as they suggest that classifications of welfare systems should narrow their focus on specific policy areas, which are not necessarily in line with standard classifications. Moreover, comparative analysis based on policy-specific welfare typologies may prove more informative to poli cymakers than welfare-as-a-whole classifications. i The study is structured as follows. Section 2 reviews the existing debates on how transformations in welfare systems can affect welfare classifications. Section 3 outlines the data and methods used to generate the welfare similarity index. Section 4 presents the results of the welfare similarity analysis, while Section 5 concludes by discussing our findings in light of the existing debates. In an influential work, Kasza (2002) highlighted that welfare states exhibit significant inconsistencies across different areas of intervention within and between countries (incoherence hypothesis). As the welfare-as-a-whole taxonomy ignores such variation, Kasza deemed it unable to capture the complex motives that inform each country’s welfare programs and argued in favour of policy-specific typologies. G. Bertin et al. Other scholars have suggested that the welfare-as-a-whole classification is not well suited for studying specific welfare areas such as healthcare, as it lacks focus on social and healthcare services (Bambra, 2005; Wendt, 2009). Similarly, comparative studies have shown incoherence across policy classifications due to different time frames and cultural orienta tions (Saraceno and Keck, 2010). For example, while healthcare policies in United Kingdom and Italy have been sometimes linked to social democratic regimes (as they are universalistic), their social care and pension policies reflect cultural models akin to the liberal (UK) and corporatist (Italy) typologies (Bertin and Carradore, 2015). 1. Introduction The beginning of this century has been characterised by the redefi nition of welfare systems in all European countries, involving a phase of stagnation and review that, at least in some cases, has undermined its original logic (Arts and Gelissen, 2002; Bonoli and Natali, 2012; Ellison and Fenger, 2013; H¨ausermann, 2010; Hemerijck, 2012). Most comparative studies on welfare systems employ, as a benchmark, the Esping-Andersen (EA) Three Worlds of Welfare classification, which was originally conceived in the phase of maximum expansion of welfare policies (Esping-Andersen, 1990, 1999). The relevance of EA’s classifi cation has been questioned due to the profound changes that have characterised, and still characterise, welfare systems. However, there is a limited understanding of how welfare regimes evolve and adapt over time and how particular regimes can be extended from the original core nations to other countries originally belonging to different groups in EA’s typology (Powell and Barrientos, 2015), resulting in a partial hybridisation of the original EA regimes. This paper provides novel evidence on whether the transformations observed in European welfare states might be considered as an evolution within the boundaries of the i The recent literature points to a lack of consensus on how welfare states in OECD countries have been classified during the 2000s. Two recent studies performed a meta-analyses of existing welfare state clas sifications, and came to opposite conclusions: Powell et al. (2020)’s review resulted in a mixed picture of hybrid EA regimes; conversely, Buhr and Stoy (2015) confirmed the validity of EA’s classification. Similarly, Ferragina and Seeleib-Kaiser (2011) classified countries based on a wide set of welfare-provision indicators, providing evidence in favour of EA’s three worlds of welfare capitalism. This conflicting evi dence should be read in light of the ongoing debate on the theoretical and empirical foundations of the analysis of welfare regimes. Some authors have highlighted that the lack of a clear definition for crucial concepts, e.g., “regime” and “commodification”, has led to inconsistent operationalisations across different analyses (Bambra, 2006; Castles and Mitchell, 1993; Powell, 2015; Rice, 2013). Other scholars have high lighted how welfare regimes classifications often lack consistency in the choice of statistical indicators and methods (Barrientos, 2015; Powell and Barrientos, 2015; Y¨orük et al., 2019). G. Bertin et al. 3.1.1. Study design and inclusion criteria 3.1.1. Study design and inclusion criteria We performed a meta-analysis of studies that, since 2000, have proposed a classification of healthcare (HC) and social care (SC) policies. Healthcare policies are one of the largest areas of social welfare and concern the provision of health services to persons in need. Social care policies comprise a set of services aimed at helping families throughout the risks they encounter during their life course, e.g., childbirth, work life balance, the aging of a parent and the need for long-term care. While the analysis by Jensen (2011) was mainly focused on the role of the state, seen as an open system influenced by external environ mental factors, we acknowledge that the inherent complexity of welfare systems extends beyond the dynamics between civil society and the state, requiring further consideration of the role of communities and social relationships, namely, the stakeholders involved in the welfare regimes’ dynamics (H¨ausermann, 2010; Levy, 1999; Vail, 2010). Moreover, third-sector organisations, including volunteering organisa tions, may affect welfare regimes independently from the role of the state. l We followed several selection criteria for the literature review. First, we included original articles, published conference papers and books published in English following a peer-review process; research reports from accredited and internationally recognised organisations were included. Second, to enhance comparability, we selected studies that employed data from the first decade of the 2000s, which can be considered as the end of the expansion phase of welfare systems. We believe this interval allows for an acceptable equilibrium in the trade-off between a narrow time-frame (which enhances comparability) and a large number of studies (which enhances the results’ robustness). Our results are robust to restricting the time-frame to a shorter interval, excluding older studies. Third, we focused on research works that included a classification of healthcare or social care systems. All the aforementioned factors influence the development processes of welfare systems and emphasise their complexity and discontinuity. In this paper, we argue that all the complexity factors highlighted in the literature point towards a non-linear transition process in welfare re gimes, which is highly affected by the dynamics between stakeholders representing different ideologies and social preferences. As the balance of power often shifts over time, the evolution of welfare regimes is often inconsistent. Moreover, although shifts in bargaining power are likely to impact policy makers’ decisions, they cannot completely overturn the existing systems. 3.1.3. Outcomes i According to Esping-Andersen himself, welfare regimes do not exist in pure form. Rather, they are an approximation of the most prevailing characteristics within a cluster of countries (Esping-Andersen, 1990). While the EA classification outlines a taxonomy of ideal-types that never find full realisation in reality, it constitutes a crucial interpretative framework, where the key element is the similarity of the welfare re gimes within clusters. However, the recent debate have highlighted how such similarities may be weakening. We identified 19 research outputs and a total of 22 welfare classifi cation analyses (7 in healthcare and 15 in social care, with 3 papers performing 2 analyses each). Table 1 summarises the data sources and methods implemented in the reviewed analyses. The data cover both European and non-European countries from 2000 to 2010, with two analyses employing data slightly outside this interval. Most of the reviewed studies produce welfare clusters through hierarchical cluster analysis (HCA), though some included non-hierarchical cluster analysis, principal component analysis or other logical methods. We checked that our results are robust to excluding studies employing non-statistical methodologies (logical methods). Our analysis naturally stems from this debate and is aimed to investigate whether the body of research carried out at the end of the evolutionary phase of welfare regimes confirms the similarities between states, or whether it highlights the presence of hybridisation processes which weakened the similarities between welfare systems. Specifically, we aim at testing the following hypotheses: The sets of indicators used to classify welfare states vary widely across papers. In total, 39 indicators were used for the classification of healthcare systems, while 55 indicators were used for social care systems (full details are available in Tables 1 and 2 in the Electronic supple mentary material). H1: hybridisation hypothesis. National welfare systems which consolidated in the first part of the 21st century and originally belonged to the same welfare regime, show low degree of similarities. In partic ular, we will consider the seminal EA regimes, i.e., Liberal (United States, Canada, Australia, United Kingdom), Conservative (Deutschland, Austria, France, the Netherlands), and Social democratic (Sweden, Denmark, Norway), and we will test whether welfare systems originally belonging to the same regime are less similar to each other and more or equally similar to systems belonging to other regimes. 3.1.2. Search strategy We employed electronic database searches (in PubMed, SCOPUS, Sage Journal Online and ScienceDirect) with the following keywords: ‘welfare state regimes/typologies’, ‘social policy’, ‘welfare services’, ‘healthcare/social care systems’, ‘cluster analysis’, ‘classification’. We also included papers/reports referred to by the outcomes of the database search, which complied with our inclusion criteria. 3. Methods consistent with specialised and knowledge-intensive economies. Indeed, although these characteristics have always been particularly present in social democratic countries, human capital formation has become more central in all advanced economies since the 1990s. Moreover, welfare systems must face new risks that have emerged from ongoing societal transformations (Hemerijck, 2012; Pestieau and Lefebvre, 2018; Salt kjel, 2018; Taylor-Gooby, 2004). 3.1.1. Study design and inclusion criteria Therefore, the result of the evolution processes up to the 1990s decade is the implementation of policies that may be groun ded in very different ideologies and social preferences, that is, a hybridisation of welfare regimes (Bertin and Pantalone, 2018; Ciccia, 2017; Yang et al., 2020). Furthermore, specific welfare policies (e.g., health, social care) have often been developed under different time frames and following different cultural and political rationales (hence, regimes), even within the same country (Bertin and Carradore, 2015). 2. Classifications of evolving welfare systems: background and hypotheses Social Science & Medicine 281 (2021) 114086 2. Classifications of evolving welfare systems: background and hypotheses Both policy areas have substantial economic relevance: in OECD countries in 2015–2017, the public expenditure for healthcare and so cial care (defined as family support, especially for children and older people, see Jensen, 2008), averaged 5.7% and 2.3% of GDP, respectively (OECD, 2019). We build an index of “welfare similarity” to capture, separately for the social care and healthcare sectors, the extent to which welfare systems have been grouped together in the reviewed papers. In order to build a robust index of similarity, our analysis focuses on countries which have been extensively included in welfare classification studies. As most of the existing literature overwhelmingly focused on mature welfare regimes in Europe, our main country selection includes Austria, Belgium, Denmark, Finland, France, Germany, Ireland, Italy, the Netherlands, Spain, Sweden and United Kingdom. We improve upon previous meta-analyses (Buhr and Stoy, 2015; Powell et al., 2020) in that we introduce a novel methodological approach that accounts for the variation in the number of times a country is analysed in the reviewed literature. We show that failing to account for this variation may lead to biased results. Second, the neo-institutional perspective links welfare-state changes to the ability of institutional factors (e.g., vested interests and veto powers) to facilitate or delay the expansion of individual policy areas. By underlying the role of institutions in preserving the status quo (Bonoli, 2001), the neo-institutional approach to public policies highlights how institutions often seek their own self-preservation. Such institutions may attempt to influence the development of the system on the basis of their history and well-established features, thus potentially becoming a strong factor of resistance to change. These processes are associated with the dynamics of political consensus and policy development. For example, Weaver (1986) argued that politicians are more focused on blame avoidance (i.e., avoiding criticisms for unpopular choices) than on credit claiming (i.e., being praised for taking popular actions). These dynamics end up consolidating both the resistance to change and the political and cultural matrix of welfare systems. According to the third, neo-functionalist, perspective, the changes in welfare regimes are related to the evolution (and adaptation ability) of economic systems. Jensen, building on the work of Iversen and Stephens (2008), argued that coordinated market economies in social democratic welfare states will increase the demand for childcare and education policies as well as for active labour market policies. Such policies are 2 G. Bertin et al. Table 1 Outcomes of the literature review. Outcomes of the literature review. Policy Paper Data sources, year(s) Area of analysis Method Countries included healthcare Bambra (2005) OECD 1998 WHO 2002 EXP, C.COV, SUP Logical typology EU1 (-ES) AS1 CA CH NO USA Jensen (2008) OECD 2001 EXP HCA EU1 AS1 CA NO USA Wendt (2009) OECD 2007 EXP, ENT, GOV HCA EU1 GR LU PT Reibling (2010) OECD 2009 Legislation review 2003 COST, ENT, GOV, SUP HCA EU1 (-IE) CH CZ GR PL PT Joumard et al. (2010) OECD 2008 EXP, COST, ENT, C.COV, GOV, SUP HCA EU1 AS1 CA CH CZ GR HU IS KR LU NO PL PT SK TR B¨ohm et al. (2013) Legislation review WHO 2008 OECD 2008 GOV Logical typology (logic tree) EU1 AS1 CA CH CZ EE HU IL IS KR LU NO PL PT SI SK Wendt (2014) OECD 2010 EXP, GOV HCA EU1 AS1 CA CH CZ EE GR HU IL IS KR LU NO PL PT SI SK TR USA social care Kautto (2002) (2 classifications) Eurostat 2000 EXP HCA; Logical typology EU1 GR NO PR Boje and Ejrnæs (2012) ESS 2008 LEAVE, EXP, S.COV HCA EU1 BG CZ EE GR HU IS LT LU LV NO PL PT SI SK Leitner (2003) (2 analyses) OECD 1995, 1996, 2000, 2001 LEAVE, EXP, S.COV Logical typology EU1 GR LU PT Bettio and Plantega (2004) ECHP 1996 OECD 2001 LEAVE, EXP, S.COV, SUP Logical typology EU1 GR LU PT Bambra (2004) OECD 1998 UN 1999, 2000 LEAVE, FEM Logical typology EU1 (-ES) AS1 CA CH NO USA Bambra (2007) UN, 2005 LEAVE, FEM HCA EU1 AS1 CA CH GR NO PT Jensen (2008) OECD 2001 EXP HCA EU1 AS1 CA NO USA Kraus et al. (2010) (2 classifications) EC 2009 Legislation review EXP, S.COV, ENT, COST, GOV Logical typology Non-hierarchical cluster analysis EU1 (-IE) BG CZ EE HU LT LV PL PT RO SI SK Thevenon (2011) OECD 2005 LEAVE, EXP, S.COV Principal component analysis EU1 AS1 CA CH CZ GR HU IS KR LU NO PL PT SK USA Chau and Sam (2013) UN 2010 Taiwan National Statistics 2009, 2010 LEAVE, FEM HCA EU1 (-ES) AS1 CA CH HK KR NO SG TW USA Cho (2014) OECD 2012 LEAVE, EXP, FEM HCA EU1 AS1 CH GR NO PT USA Verbeek-Oudijk et al. Legend: OECD = Organisation for Economic Cooperation and Development; WHO = World Health Organisation; ESS = European Social Survey; SHARE = Survey of Health Ageing and Retirement in Europe; EC = European Commission; ECHP = European Community Household Panel; UN = United Nations. COST = cost sharing; C.COV = care coverage; S.COV = service coverage; EXP = expenditure and funding sources; ENT = entitlement; FEM = gendered indicators; GOV = governance; LEAVE = parental leave; SUP = care supply. HCA: hierarchical cluster analysis. EU1: AT BE DE DK FR IT NL SE. AS1: AU JP NZ. 3.1.3. Outcomes i i The 39 healthcare indicators can be categorised in six areas: expenditure and funding sources – EXP (e.g., social spending measures as % of GDP); governance – GOV (e.g., degree of decentralisation to sub- national government bodies); cost sharing – COST (e.g., visits to GPs and specialists); entitlement to receive care - ENT (e.g., complexity of GP access procedures); care coverage - C.COV (e.g., population covered by the healthcare system); and supply – SUP (e.g., number of GPs/physi cians/specialists/nurses per capita). H2: incoherence hypothesis. The classifications of countries vary substantially depending on the specific welfare policy considered. In particular, due to the different time-frames and rationales in which health and social care policies were structured and reformed, we hypothesise that the degree of similarities between countries with respect to their healthcare systems is different than with respect to their social care systems. The 55 social care indicators can be categorised in eight areas: parental leave – LEAVE (e.g., duration of parental leave); expenditure – EXP (e.g., public spending on childcare and elderly care services); gender issues – FEM (e.g., gender–employment gap); service coverage – S.COV (e.g., % of people aged over 65 receiving home-care services); entitlement to receive care – ENT (e.g., implementation of means 3 Social Science & Medicine 281 (2021) 114086 G. Bertin et al. G. Bertin et al. Table 1 (2014) SHARE (2010–2011) EXP, GOV Logical typology EU1 (–FI–IE-UK) CH CZ EE HU PL PT SI Legend: OECD = Organisation for Economic Cooperation and Development; WHO = World Health Organisation; ESS = European Social Survey; SHARE = Survey of Health Ageing and Retirement in Europe; EC = European Commission; ECHP = European Community Household Panel; UN = United Nations. COST = cost sharing; C.COV = care coverage; S.COV = service coverage; EXP = expenditure and funding sources; ENT = entitlement; FEM = gendered indicators; GOV = governance; LEAVE = parental leave; SUP = care supply. HCA: hierarchical cluster analysis. EU1: AT BE DE DK FR IT NL SE. AS1: AU JP NZ. Legend: OECD = Organisation for Economic Cooperation and Development; WHO = World Health Organisation; ESS = European Social Survey; SHARE = Survey of Health Ageing and Retirement in Europe; EC = European Commission; ECHP = European Community Household Panel; UN = United Nations. COST = cost sharing; C.COV = care coverage; S.COV = service coverage; EXP = expenditure and funding sources; ENT = entitlement; FEM = gendered indicators; GOV = governance; LEAVE = parental leave; SUP = care supply. HCA: hierarchical cluster analysis. EU1: AT BE DE DK FR IT NL SE. AS1: AU JP NZ. Table 2 Results for healthcare similarity. at be De Dk es fi fr ie it nl se uk at – 0.71 0.71 0.14 0.17 0.14 0.71 0.33 0.29 0.29 0.29 0.14 be – 0.57 0.14 0.17 0.14 1.00 0.33 0.29 0.43 0.29 0.14 de – 0.14 0.17 0.14 0.57 0.33 0.29 0.43 0.14 0.14 dk – 0.67 0.57 0.14 0.50 0.43 0.43 0.57 0.86 es – 0.83 0.17 0.20 0.17 0.33 0.50 0.50 fi – 0.14 0.17 0.14 0.14 0.57 0.43 fr – 0.33 0.29 0.43 0.29 0.14 ie – 1.00 0.50 0.33 0.67 it – 0.43 0.29 0.57 nl – 0.14 0.43 se – 0.57 uk – 3.2. Relative index of welfare similarity and sample selection We build an index of “welfare similarity” to capture, separately for the social care and healthcare sectors, the extent to which welfare sys tems have been grouped together in the papers we reviewed (i.e., found to share substantial common characteristics), as suggested in recent analyses (Buhr and Stoy, 2015; Powell et al., 2020). However, we depart from previous methodologies in that we factor in the country-specific number of observations (i.e., the number of analyses a specific country appears in). We thereby show that failing to account for this information may lead to biased results. wi,j = Wi.j Ni,j ⃒⃒⃒⃒⃒⃒ with Ni,j ≥0.666 (N), ∀i, jN = 15 for social care studiesN wi,j = Wi.j Ni,j ⃒⃒⃒⃒⃒⃒ with Ni,j ≥0.666 (N), ∀i, jN = 15 for social care studiesN = 7 for healthcare studies = 7 for healthcare studies Let Ni be the number of analyses which include country i. The maximum number of country-specific observations corresponds to Ni; that is, max (Ni) = N, where N = 15 for social care and N* = 7 for healthcare. Furthermore, we define Ni,j as the overall number of times the dyad made of countries i,j is included in a study (e.g., countries i and j jointly appear in the same study), with Ni,j = Nj,i and wi,j = wj,i. Only a small subset of countries appear in all studies, as described in Tables 3 and 4 in the Electronic supplementary material. European countries appear more frequently than non-European countries, which translates to a clear geographical selection in terms of the country-dyad observa tions: European countries are much more likely to be simultaneously present in an analysis than non-European countries. i (1) For example, if countries i and j were simultaneously analysed in 10 studies and linked together in two (i.e., 20% of the times they are ana lysed together), then wi,j = 0.2. However, the interpretation of wi,j may be misleading if Ni,j varies substantially across pairs (i,j). For example, two countries a,b which appear jointly in just one study and are linked together would have Na,b = 1, Wa,b = 1 and wa,b = 1. 3.2. Relative index of welfare similarity and sample selection Similarly, two countries c,d which appear jointly in 15 studies and are always linked together would have Nc,d = 15 and Wc,d = 15 but still have wc,d = 1. Although the relative similarity is the same, the evidence emerging for countries c and d is arguably more robust than for countries a and b, as the former is based on 15 studies, while the latter is based on one study. Following Buhr and Stoy (2015) and Powell et al. (2020), we define a variable Wi,j (= Wj,i) which counts how often countries i and j were grouped (linked) in the same welfare cluster (for any i and j). Such a measure is, however, likely to be affected by the fact that both the number of total appearances Ni and the number of joint appearances Nij largely differ across countries. For example, among social care studies, Canada is included in the same welfare cluster as Austria three times (WCA,AT = 3), while France and Austria are clustered together six times (WFR,AT = 6); however, Canada and Austria appear together in just five studies (NCA,AT = 5), while France and Austria appear in all studies (NFR, AT = 15). Hence, the higher number of links (in absolute terms) between Austria and France may be a direct result of the difference in the number of joint observations. However, when comparing the absolute number of links Wi,j to the number of joint appearances Nij, Austria is linked to Canada 60% of the times that they are studied together (3/5), while Austria is only linked to France 40% of the times (6/15). Therefore, adopting Wi,j as a measure of similarity may bias the results. To avoid this distortion, we therefore introduce a relative measure of similarity wi, j that, for any pair of countries i and j, compares the number of joint classifications Wi,j to the number of joint appearances of i and j, namely, Nij. To allow for a consistent interpretation of the w index, we restrict our sample to country-dyads with a sufficiently high number of appearances. Specifically, we select our sample of countries in such a way that for any pair of countries i,j, they are jointly present in at least two-thirds of the total number of studies. That is, Ni,j ≥0.666(N ), ∀i,j. Table 4 Table 4 Descriptive statistics for similarity index. Similarity index (a) HEALTH (66 links) (b) SOCIAL CARE (66 links) Minimum 0.14 0 25th percentile 0.17 0.13 Median 0.33 0.27 75th percentile 0.5 0.42 Maximum 1 0.87 Descriptive statistics for similarity index. Table 2 explained by the relative broadness of the categories “healthcare” and “social care” (e.g., social care includes both child care and elderly care), as it persists even among studies that focused on similar sub-categories of welfare provision. For example, among studies on elderly care, Kraus et al. (2010) considered public expenditure for long-term care (LTC) services and information on and benefits entitlement and quality assurance, while Verbeek-Oudijk et al. (2014) included public expen diture for non-institutional LTC services, and an indicator on the balance of care responsibilities across public, family and market providers. In the testing); cost sharing indicators – COST; services supply – SUP (e.g., formal and informal supply of care); and governance – GOV (e.g., formal responsibility for long-term care). explained by the relative broadness of the categories “healthcare” and “social care” (e.g., social care includes both child care and elderly care), as it persists even among studies that focused on similar sub-categories of welfare provision. For example, among studies on elderly care, Kraus et al. (2010) considered public expenditure for long-term care (LTC) services and information on and benefits entitlement and quality assurance, while Verbeek-Oudijk et al. (2014) included public expen diture for non-institutional LTC services, and an indicator on the balance of care responsibilities across public, family and market providers. In the The vast majority of indicators are used in only one or two studies, while five indicators were shared by three or more studies. The most recurrent indicators are GP registration and GP remuneration (included in three healthcare studies), maternity leave duration (six social care studies), maternity leave compensation and female labour participation rate (three social care studies). This fragmentation may not be fully 4 Social Science & Medicine 281 (2021) 114086 G. Bertin et al. results section, we discuss how such heterogeneity in the indicators se lection might affect our results. wi,j = Wi.j Ni,j ⃒⃒⃒⃒⃒⃒ with Ni,j ≥0.666 (N), ∀i, jN = 15 for social care studiesN = 7 for healthcare studies (1) Table 4 Descriptive statistics for similarity index. Similarity index (a) HEALTH (66 links) (b) SOCIAL CARE (66 links) Minimum 0.14 0 25th percentile 0.17 0.13 Median 0.33 0.27 75th percentile 0.5 0.42 Maximum 1 0.87 3.2. Relative index of welfare similarity and sample selection For the social care sector, where N = 15, we therefore include any country which has at least 10 joint appearances with any other country in the sample. After applying such restriction, a wi,j score of 1 implies that countries i and j were simultaneously clustered together 100% of the time they were analysed together, corresponding to a minimum of 10 times, and a maximum of 15 times. For the healthcare sector, our threshold corre sponds to five joint appearances (that is, 66.6% of N* = 7). i By applying our inclusion criteria, we are left with a set of 12 Eu ropean countries, identical for both the social care and healthcare sec tors: Austria, Belgium, Denmark, Finland, France, Germany, Ireland, Italy, the Netherlands, Spain, Sweden and United Kingdom. These countries share at least 12 joint appearances for social care (with the exception of Ireland and Spain, which share 10 appearances), and at least 6 joint appearances for healthcare. Although our inclusion criterion is arbitrary, our results are robust to the adoption of a stricter inclusion threshold, limiting the sample to countries that are jointly present in at least 80% of the studies with any other country (available upon request from the authors); or looser j We therefore introduce a relative measure of similarity wi,j between countries i and j, defined as: 4.1. Healthcare Among the Mediterranean countries, a similar pattern emerges: Italy and Spain have a similarity score of 0.69; however, Italy is also linked to Austria, Ireland and the Netherlands in 40% of the studies, while Spain is linked to Ireland in 40% of the studies. The similarity score among continental countries ranges between 0.4 and 0.67, suggesting that they are grouped in the same policy cluster in only around half the analyses; moreover, Austria and Belgium have slightly lower similarity scores with Ireland and United Kingdom (0.43). The results for countries traditionally in the liberal EA welfare group reinforce the aforementioned evidence for the existence of hybrid wel fare typologies. The similarity score for United Kingdom and Ireland (0.58) is only slightly higher than that between United Kingdom and France (0.50), United Kingdom and Austria (0.43) and United Kingdom and Finland (0.43); moreover, it is only slightly higher than the score between Ireland and continental (Austria and Belgium) and The similarity score among continental countries ranges between 0.4 and 0.67, suggesting that they are grouped in the same policy cluster in only around half the analyses; moreover, Austria and Belgium have slightly lower similarity scores with Ireland and United Kingdom (0.43). i Second, focusing on the countries traditionally included in the Scandinavian regime (Sweden, Denmark and Finland), the similarity coefficient does not exceed 0.57. Moreover, some of these countries strongly show links with liberal or continental countries. Spain has a similarity index of 0.5 with Sweden and 0.83 with Finland. Denmark and United Kingdom have a similarity index of 0.86. The results for countries traditionally in the liberal EA welfare group reinforce the aforementioned evidence for the existence of hybrid wel fare typologies. The similarity score for United Kingdom and Ireland (0.58) is only slightly higher than that between United Kingdom and France (0.50), United Kingdom and Austria (0.43) and United Kingdom and Finland (0.43); moreover, it is only slightly higher than the score between Ireland and continental (Austria and Belgium) and Third, liberal countries such as United Kingdom and Ireland share a similarity score of 0.67, yet United Kingdom is, as mentioned, more strongly linked to Denmark (0.86) and similarly linked to Italy (0.57) and Spain (0.5). Table 3 Table 3 Results for social care similarity. at be De Dk es fi fr ie it nl se uk at – 0.53 0.60 0.00 0.23 0.21 0.40 0.42 0.40 0.53 0.00 0.43 be – 0.67 0.13 0.15 0.07 0.67 0.42 0.27 0.47 0.13 0.29 de – 0.13 0.08 0.21 0.67 0.25 0.20 0.40 0.13 0.36 dk – 0.00 0.50 0.27 0.00 0.00 0.20 0.87 0.14 es – 0.17 0.15 0.40 0.69 0.23 0.00 0.25 fi – 0.36 0.08 0.07 0.07 0.50 0.43 fr – 0.33 0.13 0.47 0.27 0.50 ie – 0.42 0.42 0.00 0.58 it – 0.40 0.00 0.21 nl – 0.27 0.36 se – 0.14 uk – Results for social care similarity. 5 G. Bertin et al. Social Science & Medicine 281 (2021) 114086 thresholds, including countries that are present in at least 3 studies with any other country (Supplementary Material 2). the Netherlands. Moreover, the Netherlands is another example of the absence of a clear similarity pattern, as it shows comparable similarity scores with countries traditionally belonging to very different regimes, such as Italy (0.43), Ireland (0.5) and Belgium (0.43). 4.1. Healthcare The results for the similarity structures in social care studies, shown in Fig. 2, point to the coexistence of multiple regimes that are not clearly differentiated, yet in a different way than for the healthcare sector (re sults are summarised in Table 3). Table 6 in the Electronic supplemen tary material reports the absolute number of links between all the countries included in the reviewed papers. We summarise our results for the healthcare similarity analysis by plotting a similarity network in Fig. 1. The strength of the association between any two countries is represented by the thickness of the segment which links them and expressed explicitly as a ratio coefficient (which can be easily converted to percentage terms) in Table 2, following the definition in equation (1). Table 5 in the Electronic sup plementary material reports the absolute number of links between all the countries included in the reviewed papers. We also report some descriptive statistics relating to the distribution of the similarity index in Table 4. As reported in Table 4(b), the median similarity index is 0.27, indi cating that half of the country dyads are classified in the same welfare regime fewer than 30% of the times (which corresponds to roughly four studies). As the maximum similarity index is 0.87, no dyad can be considered a “pure system”. However, unlike for healthcare policies, we find dyads with zero similarity. Overall, our results highlight the lack of a “pure” overlap between the EA typology and health-care typologies. i Scandinavian countries exhibit low, though mostly non-zero, simi larity scores with non-Scandinavian countries. However, while Denmark and Sweden have a high similarity score of 0.87, they have a weaker link with Finland (0.5), thus highlighting the lack of a “pure” cluster within the EA Nordic regime. First, the descriptive statistics in Table 4 column (a) show that, among the 12 countries in the sample, no pair of countries have a zero similarity coefficient, as the minimum similarity score is 0.14 (i.e., all countries have at least one link with any other country). Moreover, the median similarity score is 0.33. Given that any country dyad appears in at least six studies, this means that half of the country dyads are clus tered in the same healthcare regime at least twice (33%). As such, these results suggest that healthcare systems are more hybrid than the EA classification would suggest. 4. Results However, a relatively more stable picture emerges for the conti nental regime countries (Austria, Belgium, France and Germany). These continental countries share high similarity scores, ranging between 0.57 and 1. Conversely, the similarity between the continental countries and the remaining countries always lies below 0.43. We now show the results of the welfare similarity analysis, separately for healthcare and social care policies. We aim to understand (1) the extent to which the typologies identified by the existing literature on healthcare and social care systems overlap with the EA classification, and (2) whether the emerging typologies are consistent between healthcare and social care studies. 4.4.1. Could the choice of indicators affect our findings? 4.4.1. Could the choice of indicators affect our findings? i The hybrid clusters emerging from our results may be, in principle, an artifact of the choice of indicators employed in the specific analyses if, for example, studies employing similar indicators lead to similar country-clusters. Hence, we might wonder whether our findings would be robust to an alternative choice of welfare indicators. However, we argue that this concern is not relevant to our analysis, as the studies we reviewed employ very different indicators (Section 3.1). For example, United Kingdom and Denmark are paired together in six out of seven healthcare studies, which employ a total of 20 indicators. Fourteen in dicators are uniquely used by single studies (not shared by other studies). Five indicators are shared by two studies; one indicator is shared by three studies on of GPs). Within social care policies, Spain and Ireland are clustered together in four studies, which employ a total of 19 indicators, each of them employed by just one study. 4.4.3. Could the selected time-frame affect our findings? Our review includes studies using data from the 2000s, with some study also employing data from 1998 or 2012. This time interval is usually referred as the “post-expansion” era of welfare state evolution. However, scholars have noted that since the end of the 1990s, a process of rationalisation has been put in place, which has resulted in a number of welfare state reforms. We might therefore be concerned that the hybridisation might result from mixing studies from the early and the late 2000s. We therefore replicated our analysis on the studies using data from 2005 onward (the majority of the studies), and obtained re sults entirely in line with our main findings, suggesting that they are not driven by the studies timeframe. Second, a similar change can be identified for Liberal countries. While United Kingdom and Ireland are similarly linked in both health care (0.67) and social care policies (0.58), both are much more strongly linked with the Nordic block and Italy in healthcare than in social care analyses. For United Kingdom, the similarity scores substantially change with respect to Denmark (HC 0.85; SC 0.14), Sweden (HC 0.57; SC 0.14) and Italy (HC 0.57; SC 0.21). Ireland’s similarity index with Denmark (HC 0.5; SC 0) and Italy (HC 1; SC 0.42) exhibits a similar drop. 4.4.4. Could a larger country selection affect our findings? Our main analysis focused on 12 European countries which have been jointly included in at least 66% of the reviewed studies (section 3.2). However, our main findings are robust to broadening the analysis to countries which are less often included in the reviewed studies. Specifically, when enlarging the sample to 21 countries, covering Southern, Eastern and Northern Europe, all of our main findings are confirmed (results and methods are available in the Supplementary Material Section 2). However, as such results are partially based on countries appearing in a small number of studies, we prudently consider them as less robust than our main findings. 4.1. Healthcare Ireland has a much stronger link to Italy (coefficient of 1, meaning that the two countries are always classified together) than to United Kingdom, while having a 0.5 similarity index with Denmark and Fig. 2. Frequency of country pairs grouped into the same cluster with respect to social care policies (Bastian et al., 2009). 6 United Kingdom, while having a 0.5 similarity index with Denmark and Fig. 1. Frequency of country pairs being grouped into the same cluster with respect to healthcare policies (Bastian et al., 2009). Fig. 2. Frequency of country pairs grouped into the same cluster with respect to social care policies (Bastian et al., 2009). Fig. 1. Frequency of country pairs being grouped into the same cluster with respect to healthcare policies (Bastian et al., 2009). Fig. 1. Frequency of country pairs being grouped into the same cluster with respect to healthcare policies (Bastian et al., 2009). Fig. 2. Frequency of country pairs grouped into the same cluster with respect to social care policies (Bastian et al., 2009). 6 Social Science & Medicine 281 (2021) 114086 G. Bertin et al. Mediterranean (Italy) countries, with an index of around 0.4. field journals and books (the vast majority), or in working paper series edited by world renowned organisations with strong quantitative focus. Although the assessment of the quality of a method adopted is ultimately subjective, we believe that the peer-review process that such studies underwent before publication should already be a partial guarantee of their value. Second, we performed a robustness test by arbitrarily excluding from the reviewed classifications those which were not resulting from a statistical algorithm (7 classifications). The resulting similarity indices, available upon request, entirely confirm our main findings, suggesting that they are not an artifact of the variation in methods. Similar to what emerged from the healthcare analysis, the Netherlands is paired with countries belonging to different regimes (continental, Mediterranean and liberal), with an index score of around 0.5 (around half the analyses). 5. Discussion and conclusion While the seminal work by Esping-Andersen (EA) has provided a classification of welfare systems during their developing phase in the second half of the previous century, welfare states have, since then, undergone major transformation processes in most Western countries, which might have weakened the representativeness of Esping-Ander sen’s classification. Our study stems from two major results in the existing literature. On the one hand, previous studies have hypothesised a progressive hybridisation of welfare typologies, which results in wel fare systems borrowing characteristics from more than one regime. On the other hand, public welfare systems have been shown to follow different rationales (e.g., liberal, corporatist and social democratic) in different areas of service provision, even within the same country. On the other hand, the variability in the choice of the indicators might explain the hybrid classification of countries. Although essentially not empirically testable, we argue that this explanation would be in line with our starting hypotheses. In a context of welfare systems hybrid isation, particular sub-sections of the healthcare or social care systems might follow different rationales. Hence, studies employing different indicators to characterise health or social care services could capture such heterogeneity. In other words, should the observed hybrid clusters be due to the variability in the choice of the indicators, this would confirm, rather than contradict, our starting hypotheses. In this paper, we provide novel evidence that the recent literature focusing on healthcare and social care systems in Europe has identified country-clusters which are, to different extents, not fully overlapping with Esping-Andersen’s classification. Our main findings are twofold: first, we provide evidence for a progressive hybridisation of healthcare and social care systems classifications across European countries (H1: hybridisation). With respect to healthcare systems, our results highlight the absence of clear clusters across European countries. This is mainly due to the inconsistencies in the classifications provided by the reviewed studies. For example, both Sweden and the Netherlands are never unanimously classified together with any other country in the sample. Moreover, most studies group together countries which originally belonged to different EA typologies. For example, Mediterranean- welfare countries such as Spain and Italy are often clustered together with Finland, Denmark and Ireland which belong to a social democratic 4.4.2. Could different cluster methodologies affect our findings? 4.3. Comparisons across welfare areas Overall, the comparison between the findings for healthcare policies and social care policies highlights that some traditional EA welfare ty pologies are particularly unstable. First, a substantial change emerges in the similarity network of Mediterranean countries. Within healthcare, Italy and Spain are very rarely linked together (similarity score of 0.17), while both countries share substantial similarities with (different) Nordic and Liberal coun tries. However, within social care policies, such links are almost non- existent. Specifically, stark differences appear in the similarity index between Spain and Denmark (HC 0.67; SC 0), Spain and Finland (HC 0.83; SC 0.17), Spain and Sweden (HC 0.5; SC 0) and Spain and United Kingdom (HC 0.5; SC 0.25); as well as between Italy and Denmark (HC 0.43; SC 0); Italy and Ireland (HC 1; SC 0.42) and Italy and United Kingdom (HC 0.57; SC 0.21). Conversely, Italy and Spain share a higher similarity score in social care policies (0.69), than in healthcare (0.17). References Arts, W., Gelissen, J., 2002. Three worlds of welfare capitalism or more? A state-of-the- art report. J. Eur. Soc. Pol. 12 (2), 137–158, 10.1177%2F0952872002012002114. Bambra, C., 2004. The worlds of welfare: illusory and gender blind? Soc. Pol. Soc. 3 (3), 201–211. https://doi.org/10.1017/S147474640400171X. Bambra, C., 2005. Worlds of welfare and the health care discrepancy. Soc. Pol. Soc. 4 (1), 31–41. https://doi.org/10.1017/S1474746404002143. i Bambra, C., 2005. Worlds of welfare and the health care discre Bambra, C., 2006. Research Note: decommodification and the worlds of welfare revisited. J. Eur. Soc. Pol. 16 (1), 73–80, 10.1177%2F0958928706059835. y y y Our study provides two relevant contributions for both the academic and the policy debate on welfare policies. First, we highlight that, against a background of changing nature of welfare systems, the comparative analysis and classification of welfare policies should devote more focus to the complexity of the unfolding developments of welfare systems and their transformations, which are not necessarily continuous and constantly in line with standard classifications. The relevance of welfare systems classifications is strongly dependent on the ability of such classification to capture the characteristics of the system itself. In our view, our findings underline the importance of narrowing the focus of welfare regime analyses on specific policy areas, to enhance the relevance of the classifications themselves. With respect to health and social care policies in particular, our findings suggest that the existing studies lack consistency in the choice of dimensions and indicators for classification purposes, and do not come to an accepted taxonomy. Hence, further research is needed to strengthen the specificity of health- care and social care systems’ studies, for example, by including more specific indicators which might better capture the transformation pro cesses which are interesting welfare systems in the last decades. i Bambra, C., 2007. Defamilisation and welfare state regimes: a cluster analysis. Int. J. Soc. Welfare 16 (4), 326–338. https://doi.org/10.1111/j.1468-2397.2007.00486.x. Barrientos, A., 2015. ‘A veritable mountain of data and years of endless statistical manipulation’: methods in the three worlds and after. Soc. Pol. Soc. 14 (2), 259–270. https://doi.org/10.1017/S1474746414000578. Bastian, M., Heymann, S., Jacomy, M., 2009. Gephi: an Open Source Software for Exploring and Manipulating Networks. Third international AAAI conference on weblogs and social media. https://doi.org/10.13140/2.1.1341.1520. Bertin, G., Carradore, M., 2015. Differentiation of welfare regimes: the case of Italy. Int. J. Soc. Welfare. https://doi.org/10.1111/ijsw.12183. Bertin, G., Pantalone, M., 2018. 4.4.2. Could different cluster methodologies affect our findings? i A. In principle, the observed hybridisation of welfare clusters could be affected by the heterogeneity in the methods used by the reviewed studies, rather than depicting an actual heterogeneity across welfare systems. We believe this not to be a concern for our findings, for two main reasons. First, our review only selected studies published in top 7 G. Bertin et al. Social Science & Medicine 281 (2021) 114086 research, which often have to rely on general classifications of countries welfare systems while studying specific policy areas, due to data limi tations (e.g., Carrieri et al., 2017; Floridi et al., 2021). Our findings show that broad classifications might underestimate the hybridisation of countries welfare systems in specific policy areas. Hence policy-specific welfare typologies may prove more informative to academics and poli cymakers than general classification of the welfare state as a whole. and liberal welfare tradition, respectively. While the healthcare systems of central-Europe countries (the corporatist regime) exhibit a stronger joint similarity, they are often clustered together with countries belonging to different EA typologies. Similar conclusions can be drawn from social care studies. On the one hand, the reviewed studies are never unanimous in classifying any two countries in the same cluster. On the other hand, several countries exhibit a very low degree of similarity with any other country in the sample. This is particularly evident for continental-regime countries, such as Austria and the Netherlands, as well as for social democratic (Finland) and liberal (Ireland and United Kingdom) countries. It is still possible to identify clusters of countries which are consistent with the original EA classifications. Still, even within these clusters, the degree of similarity is far from unanimous across studies, and typical clusters (such as Spain and Italy; Belgium, Germany and France; Sweden and Denmark) are only identified by roughly two-thirds of the studies. All in all, these results seem to confirm the hybridisation hypothesis. i Finally, we note that, due to the country selection in our study, our findings are relevant with respect to the core set of mature European welfare states. Further comparative research is needed to broaden the perspective beyond the Western European regimes, for example, to Eastern Europe, American and Asian countries. Declaration of competing interest None declared. Acknowledgments Ludovico Carrino is supported by the Economic and Social Research Council (ESRC) (grant number: ES/S01523X/1, IN-CARE project). The views expressed are those of the author(s) and not necessarily those of the ESRC or King’s College London. There are several reasons for the observed lack of consistency across studies in the classification of welfare systems. First, from a methodo logical perspective, we note that only a few studies employed a similar set of indicators (Powell et al., 2020; Y¨orük et al., 2019). While this methodological fragmentation could partially explain the lack of strong links between countries that originally belonged to a well-defined wel fare regime, it can hardly explain our findings of hybrid welfare clusters, where countries from different EA regimes are consistently grouped together. Moreover, we have shown that our results are not sensitive to the methodology employed by the reviewed studies. A second reason lies in the complexity of the unfolding developments of welfare systems (Bonoli, 2001; Jensen, 2011), whose transformations have not been continuous nor constant. Such transformations are the outcome of power dynamics that change overtime, including the resistance to changes in welfare institutions, and the reactions to changes in societal needs and preferences. Hence, the hybridisation of welfare character istics across systems is likely to be an outcome of these dynamics. Authors’ contribution Giovanni Bertin: Conceptualization, Methodology, Writing - Orig inal Draft, Writing - Review & Editing. Ludovico Carrino: Conceptu alization, Methodology, Software, Writing - Original Draft, Writing - Review & Editing. Marta Pantalone: Conceptualization, Methodology, Software, Writing - Original Draft, Writing - Review & Editing. i Second, we show that welfare typologies and similarities can sub stantially differ across policy areas (H2: incoherence). For example, the Spanish healthcare system is clustered together with Finland’s and Denmark’s, suggesting the coexistence of characteristics from both the Mediterranean and social democratic regimes. Another Mediterranean country, Italy, has similarly been linked with Ireland and United Kingdom with respect to its healthcare system. However, within social care policies, Spain and Italy are found to be closely linked. This suggests that the rationales for welfare provision, even within a country, are often inconsistent across policy areas, therefore providing support for the incoherence hypothesis. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.socscimed.2021.114086. References Comparing hybrid welfare systems: the differentiation of health and social care policies at the regional level in Italy. Italian Sociol. Rev. 8 (1) https://doi.org/10.13136/isr.v8i1.146. Bettio, F., Plantenga, J., 2004. Comparing care regimes in Europe. Fem. Econ. 10 (1), 85–113. https://doi.org/10.1080/1354570042000198245. B¨ohm, K., Schmid, A., G¨otze, R., et al., 2013. Five types of OECD healthcare systems: empirical results of a deductive classification. Health Pol. 113 (3), 258–269. https:// doi.org/10.1016/j.healthpol.2013.09.003. g j p Boje, T.P., Ejrnæs, A., 2012. Policy and practice: the relationship between family policy regime and women’s labour market participation in Europe. Int. J. Sociol. Soc. Pol. 32 (9/10), 589–605. https://doi.org/10.1108/01443331211257670. Bonoli, G., 2001. Political Institutions, Veto Points, and the Process of Welfare State Adaptation. The new politics of the welfare state, pp. 238–264. https://doi.org/ 10.1093/0198297564.003.0009. Second, our study suggests that enhancing the specificity of welfare classifications might be relevant for future comparative empirical Bonoli, G., Natali, D., 2012. The Politics of the New Welfare State. Oxford University Press. https://doi.org/10.1093/acprof:oso/9780199645244.001.0001. 8 8 G. Bertin et al. Social Science & Medicine 281 (2021) 114086 Buhr, D., Stoy, V., 2015. More than just welfare transfers? A review of the scope of Esping-Andersen’s welfare regime typology. Soc. Pol. Soc. 14 (2), 271–285. https:// doi.org/10.1017/S1474746414000542. Kautto, M., 2002. Investing in services in West European welfare states. J. Eur. Soc. Pol. 12 (1), 53–65, 10.1177%2F0952872002012001636. Kraus, M., Riedel, M., Mot, E., et al., 2010. A Typology of Long-Term Care Systems in Europe. ENEPRI Working Paper (91). g Carrieri, V., Di Novi, C., Orso, C.E., 2017. Home sweet home? Public financing and inequalities in the use of home care services in Europe. Fisc. Stud. 38 (3), 445–468. https://doi.org/10.1111/j.1475-5890.2017.12138. Leitner, S., 2003. Varieties of familialism: the caring function of the family in comparative perspective. Eur. Soc. 5 (4), 353–375. https://doi.org/10.1080/ 1461669032000127642. Castles, F.G., Mitchell, D., 1993. Worlds of welfare and families of nations. Famil. Nat.: Patt. Publ. Pol. Western Democr. 93 (94,112). Levy, J.D., 1999. Vice into virtue? Progressive politics and welfare reform in continental Europe. Polit. Soc. 27 (2), 239–273, 10.1177%2F0032329299027002004. Chau, R.C., Sam, W., 2013. Defamilisation of twenty-two countries: its implications for the study of East Asian welfare regime. Soc. Pol. Soc. 12 (3), 355–367. https://doi. org/10.1017/S1474746412000577. OECD, 2019. Social Expenditure Update 2019. Public social spending is high in many OECD countries. g Cho, E.Y.-N., 2014. Defamilization typology re-examined: Re-measuring the economic independence of women in welfare states. J. Eur. Soc. Pol. 24 (5), 442–454. References https:// doi.org/10.1017/S1474746412000577. Pestieau, P., Lefebvre, M., 2018. The Welfare State in Europe: Economic and Social Perspectives. Oxford University Press. i Powell, M., 2015. A Re-specification of the welfare state’: conceptual issues in ‘the three worlds of welfare capitalism. Soc. Pol. Soc. 14 (2), 247–258. https://doi.org/ 10.1017/S1474746414000529. i Ciccia, R., 2017. A two-step approach for the analysis of hybrids in comparative social policy analysis: a nuanced typology of childcare between policies and regimes. Qual. Quantity 51 (6), 2761–2780. https://doi.org/10.1007/s11135-016-0423-1. Powell, M., Barrientos, A., 2015. Introduction: twenty five years of the welfare modelling business. Soc. Pol. Soc. 14 (2), 241–245. https://doi.org/10.1017/ S147474641400058X. Ellison, M., Fenger, M., 2013. Introduction:‘New’welfare in practice: trends, challenges and dilemmas. Soc. Pol. Soc. 12 (4), 547–552. https://doi.org/10.1017/ S1474746413000298. Powell, M., Y¨orük, E., Bargu, A., 2020. Thirty years of the three worlds of welfare capitalism: a review of reviews. Soc. Pol. Adm. 54 (1), 60–87. https://doi.org/ 10.1111/spol.12510. Esping-Andersen, G., 1990. The Three Worlds of Welfare Capitalism. Princeton University Press. y Esping-Andersen, G., 1999. Social Foundations of Postindustrial Economies. OUP, Oxford. https://doi.org/10.1093/0198742002.001.0001. Reibling, N., 2010. Healthcare systems in Europe: towards an incorporation of patient access. J. Eur. Soc. Pol. 20 (1), 5–18, 10.1177%2F0958928709352406. Ferragina, E., Seeleib-Kaiser, M., 2011. Thematic Review: welfare regime debate: past, present, futures? Pol. Polit. 39 (4), 583–611. https://doi.org/10.1332/ 030557311X603592. Rice, D., 2013. Beyond welfare regimes: from empirical typology to conceptual ideal types. Soc. Pol. Adm. 47 (1), 93–110. https://doi.org/10.1111/spol.12001. Saltkjel, T., 2018. Welfare resources and social risks in times of social and economic change: a multilevel study of material deprivation in European countries. Eur. J. Soc. Work 21 (5), 639–652. https://doi.org/10.1080/13691457.2017.1320525. Ferrera, M., 2013. Welfare-state transformations: from neo-liberalism to liberal neo- welfarism? In: Thatcher, M., Schmidt, V.A. (Eds.), Resilient Liberalism in Europe’s Political Economy. Cambridge University Press, Cambridge, pp. 77–111. https://doi. org/10.1017/CBO9781139857086.005. Saraceno, C., Keck, W., 2010. Can we identify intergenerational policy regimes in Europe? Eur. Soc. 12 (5), 675–696. https://doi.org/10.1080/ 14616696.2010.483006. Floridi, G., Carrino, L., Glaser, K., 2021. Socioeconomic inequalities in home-care use across regional long-term care systems in Europe. J. Gerontol.: Series B 76 (1), 121–132. Taylor-Gooby, P., 2004. New Risks, New Welfare: the Transformation of the European Welfare State. Oxford University Press. https://doi.org/10.1093/ 019926726X.001.0001. H¨ausermann, S., 2010. The Politics of Welfare State Reform in Continental Europe: Modernization in Hard Times. Cambridge University Press. https://doi.org/ 10.1017/CBO9780511750588. Th´evenon, O., 2011. Family policies in OECD countries: a comparative analysis. Popul. Dev. Rev. 37 (1), 57–87. https://doi.org/10.1111/j.1728-4457.2011.00390.x. References H¨ausermann, S., 2012. The Politics of Old and New Social Policies. The politics of the new welfare state, pp. 111–132. https://doi.org/10.1093/acprof:oso/ 9780199645244.001.0001. Vail, M., 2010. Recasting Welfare Capitalism: Economic Adjustment in Contemporary France and Germany. Temple University Press. Verbeek-Oudijk, D., Woittiez, I., Eggink, E., et al., 2014. Who Cares in Europe? A Comparison of Long-term Care for the over 50s in Sixteen European Countries. Hemerijck, A., 2012. When Changing welfare states and the Eurocrisis meet. Sociologica 6 (1). https://doi.org/10.2383/36887, 0-0. Weaver, R.K., 1986. The politics of blame avoidance. J. Publ. Pol. 6 (4), 371–398. https://doi.org/10.1017/S0143814X00004219. g Iversen, T., Stephens, J.D., 2008. Partisan politics, the welfare state, and three worlds of human capital formation. Comp. Polit. Stud. 41 (4–5), 600–637, 10.1177% 2F0010414007313117. Wendt, C., 2009. Mapping European healthcare systems: a comparative analysis of financing, service provision and access to healthcare. J. Eur. Soc. Pol. 19 (5), 432–445, 10.1177%2F0958928709344247. Jensen, C., 2008. Worlds of welfare services and transfers. J. Eur. Soc. Pol. 18 (2), 151–162, 10.1177%2F0958928707087591. Wendt, C., 2014. Changing healthcare system types. Soc. Pol. Adm. 48 (7), 864–882. https://doi.org/10.1111/spol.12061. Jensen, C., 2011. Determinants of welfare service provision after the Golden Age. Int. J. Soc. Welfare 20 (2), 125–134. https://doi.org/10.1111/j.1468-2397.2009.00667.x. i Yang, Jj, Kim, H.S., Choi, S.E., et al., 2020. What makes hybrid insourcing successful: a new public–private partnership model for social welfare services. Asian Soc. Work Pol. Rev. 14 (1), 11–21. https://doi.org/10.1111/aswp.12188. Joumard, I., Andr´e, C., Nicq, C., 2010. Health Care Systems: Efficiency and Institutions. https://doi.org/10.1787/5kmfp51f5f9t-en. Y¨orük, E., ¨Oker, ˙I., Yıldırım, K., et al., 2019. The variable selection problem in the three worlds of welfare literature. Soc. Indicat. Res. 144 (2), 625–646. https://doi.org/ 10.1007/s11205-019-02070-7. Kasza, G.J., 2002. The illusion of welfare ‘regimes’. J. Soc. Pol. 31 (2), 271–287. https:// doi.org/10.1017/S0047279401006584.
https://openalex.org/W1967947451	https://europepmc.org/articles/pmc2363365?pdf=render	English	null	Vascular endothelial growth factor, platelet-derived endothelial cell growth factor and angiogenesis in non-small-cell lung cancer	British journal of cancer	2,000	cc-by	6,015	Vascular endothelial growth factor, platelet-derived endothelial cell growth factor and angiogenesis in non-small-cell lung cancer 1University Department of Oncology, Leicester Royal Infirmary, Leicester LE1 5WW, UK; 2Department of Radiotherapy & Oncology, Laboratory of Cancer Biology, University Hospital of Iraklion, Iraklion 71110, Crete, Greece; Department of 3Cellular Science and 4Imperial Cancer Research Fund Medical Oncology Unit, Oxford Radcliffe Hospitals Trust, Oxford OX3 7LJ, UK Summary High microvessel density, an indirect measure of angiogenesis, has been shown to correlate with increased tumour size, lymph node involvement and poor prognosis in non-small-cell lung cancer (NSCLC). Tumour cell vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PD-ECGF) expression correlate with angiogenesis and a poor outcome in this disease. In a retrospective study VEGF and PD-ECGF expression and microvessel density were evaluated immunohistochemically in surgically resected specimens (T1–3, N0–2) from 223 patients with operable NSCLC using the VG1, P-GF.44C and JC70 monoclonal antibodies respectively. High VEGF immunoreactivity was seen in 104 (46.6%) and PD-ECGF in 72 (32.3%) cases and both were associated with high vascular grade tumours (P = 0.009 and P = 0.05 respectively). Linear regression analysis revealed a weak positive correlation between VEGF and PD-ECGF expression in cancer cells (r = 0.21; P = 0.002). Co-expression of VEGF and PD-ECGF was not associated with a higher microvessel density than VEGF or PD-ECGF only expressing tumours. Furthermore a proportion of high vascular grade tumours expressed neither growth factor. Univariate analysis revealed tumour size, nodal status, microvessel density and VEGF and PD-ECGF expression as significant prognostic factors. Tumour size (P < 0.02) and microvessel density (P < 0.04) remained significant on multivariate analysis. In conclusion, VEGF and PD-ECGF are important angiogenic growth factors and have prognostic significance in NSCLC. Furthermore the study underlines the prognostic significance of microvessel density in operable NSCLC. © 2000 Cancer Research Campaign Keywords: vascular endothelial growth factor; platelet-derived endothelial cell growth factor; angiogenesis; non-s ndothelial growth factor; platelet-derived endothelial cell growth factor; angiogenesis; non-small cell lung cancer 1996; Giatromanolaki et al, 1998; Oshika et al, 1998; Fontanini et al, 1999). Angiogenesis is essential for tumour growth beyond 1–2 mm in diameter and plays a central role in the metastatic spread of malig- nant disease. Previous retrospective and prospective studies in non-small-cell lung cancer (NSCLC) have clearly demonstrated that angiogenesis, assessed by microvessel counting, is an impor- tant prognostic factor in operable NSCLC, high microvessel counts being associated with disease spread and a poor survival (Giatromanolaki et al, 1996; Fontanini et al, 1997). British Journal of Cancer (2000) 82(8), 1427–1432 © 2000 Cancer Research Campaign British Journal of Cancer (2000) 82(8), 1427–1432 © 2000 Cancer Research Campaign n © 2000 Cancer Research Campaign DOI: 10.1054/ bjoc.1999.1129, available online at http://www.idealibrary.com on DOI: 10.1054/ bjoc.1999.1129, available online at http://www.idealibrary.com on Received 29 March 1999 Revised 11 November 1999 Accepted 11 November 1999 Correspondence to: KJ O’Byrne Vascular endothelial growth factor, platelet-derived endothelial cell growth factor and angiogenesis in non-small-cell lung cancer Platelet-derived endothelial cell growth factor (PD-ECGF) is a non-heparin binding angiogenic factor initially isolated from platelets. Subsequent studies showed that PD-ECGF is a 90 kDa homodimer and is thymidine phosphorylase (TP) (Ishikawa et al, 1989; Moghaddam and Bicknell, 1992). Transfection of the PD-ECGF gene into transformed fibroblasts in nude mice results in neo-angiogenesis (Ishikawa et al, 1989). Stimulation of endothelial cell migration in vitro and enhancement of tumour growth in vivo have also been reported (Moghaddam et al, 1995). The precise mechanism by which PD-ECGF promotes angio- genesis is unclear. PD-ECGF hydrolyses thymidine to thymine and 2′-deoxy-D-ribose-1-phosphate. 2′-deoxy-D-ribose-1 phophate is dephosphorylated to 2′ deoxy-D-ribose which is angiogenic in the chicken-chorioallantoic membrane assay (Moghaddam et al, 1995). In normal lung, PD-ECGF expression is invariably seen in alveolar macrophages. Bronchiolar epithelium occasionally shows positive immunoreactivity. Bronchial basal and differenti- ated columnar cells are weakly positive. Weak immunoreactivity is also seen in stromal fibroblasts (Giatromanolaki et al, 1997). Recent studies indicate that tumour cell, but not stromal cell, PD-ECGF immunoreactivity correlates with angiogenesis and prognosis in NSCLC, high expression being associated with angiogenesis and a poor outcome (Koukourakis et al, 1998). Vascular endothelial growth factor (VEGF) is a potent and specific endothelial cell mitogenic and migratory, and vascular permeability, factor. Although produced from the same gene at least six different isoforms of 121, 145, 148, 165, 189 and 206 amino acids respectively have been identified (Ferrara and Davis- Smyth, 1997; Whittle et al, 1999). In the normal lung, VEGF may be expressed by bronchiolar and differentiated columnar epithelium and alveolar macrophages. Stromal fibroblasts and macrophages are only occasionally positive. In NSCLC tumours, VEGF-expressing blood vessels are identified in > 50% of cases. However, VEGF immunoreactivity is seen in < 10% of infiltrating lymphocytes (Giatromanolaki et al, 1998; Turley et al, 1998). Recent studies indicate that VEGF expression correlates with high microvessel counts and a poor prognosis in NSCLC (Volm et al, Received 29 March 1999 Revised 11 November 1999 Accepted 11 November 1999 Correspondence to: KJ O’Byrne The co-expression of VEGF and PD-ECGF may enhance neovascularization in solid tumours (Toi et al, 1995; Maeda et al, 1997; Ikeda et al, 1999). In this retrospective study we report our 1427 1428 KJ O’Byrne et al was performed using the streptavidin-biotin-peroxidase (Dako, UK) technique. Sections were dewaxed and incubated in 0.5% H2O2 in methanol for 30 min. VEGF immunohistochemistry VEGF immunoreactivity was evaluated employing the VG1 monoclonal antibody and the horseradish peroxidase technique as previously described (Giatromanolaki et al, 1998; Turley et al, 1998). The VG1 monoclonal antibody, which recognizes the 121, 165 and 189 isoforms of VEGF, was raised using recombinant VEGF 189 protein and the specificity of the antibody was confirmed using COS cells transfected with cDNA coding for VEGF 121, 165 and 189 protein and by Western blot studies. Sections were dewaxed and incubated in 0.5% hydrogen peroxide (H2O2) in methanol for 30 min. After microwaving and washing in phosphate-buffered saline (PBS), sections were incubated with the primary antibody for 60 min. After washing in PBS for 5 min, sections were incubated with goat anti-mouse immunoglobulin (1:2000) for 30 min (Dako, UK), washed again with PBS for 5 min and incubated with rabbit anti-goat immunoglobulin (1:100) for 30 min. The peroxidase reaction was developed using diaminobenzidine (Sigma Fast tablets) as chromogen and sections were counterstained with haematoxylin. Normal rabbit immunoglobulin-G was substituted for primary antibody as the negative control (same concentration as the test antibody). Taking into account the extent of positive staining, we divided our cases into two categories: low reactivity (0–70% positive cells) and high immunoreactivity (> 70% positive cells). Intra- and interobserver variability In previous reports we have demonstrated that intraobserver vari- ability was minimal for vascular grade and PD-ECGF with the second assessment correlating with the first for all observers (r = 0.91, P < 0.006 and r = 0.96, P < 0.001 respectively). Similarly the interobserver variability of three investigators was low (r = 0.94, P < 0.001 for vascular grade and r = 0.91, P < 0.008 for PD-ECGF) (Koukourakis et al, 1998). We made a similar observation for interobserver variability for VEGF (r > 0.93, P < 0.0001) (Giatromanolaki et al, 1998). Therefore intra- and interobserver variability were not formally assessed in this study. Vascular endothelial growth factor, platelet-derived endothelial cell growth factor and angiogenesis in non-small-cell lung cancer After washing in Tris-buffered saline (TBS), sections were incubated in normal human serum (1:10) for 20 min. Sections were then washed with TBS for 5 min and incubated with the undiluted primary antibody for 30 min. After washing in TBS for 5 min, sections were incubated with biotinylated goat anti-mouse immunoglobulin (1:200) for 30 min (Dako, UK). After incubation with the streptavidin–biotin complex–horseradish peroxidase (Dako, UK) for 30 min, the peroxidase reaction was developed using diaminobenzidine (Sigma Fast tablets) as chromogen, and sections were counter- stained with haematoxylin. Normal rabbit immunoglobulin-G was substituted for primary antibody as the negative control. Alveolar macrophages were used as a positive internal control (Giatromanolaki et al, 1997). findings on VEGF and PD-ECGF expression in an expanded series of 223 cases of NSCLC including T3 and N2 tumours. In particular the study evaluates the impact of the co-expression of these factors on angiogenesis and overall survival. RESULTS Of the 223 tumours studied 156 were squamous cell carcinomas and 67 adenocarcinomas. Forty-three patients had stage 1a, 77 stage 1b, 22 stage IIa, 52 stage IIb and 29 stage IIIa NSCLC tumours. Survival data was available for 183 patients, patients dying within 60 days of surgery being excluded to avoid bias from peri-operative death. The median follow-up at the time of analysis for patients alive was 3.5 years (range 1.5–7 years). Angiogenesis assessment The JC70 monoclonal antibody (Dako) recognizing CD31 (platelet/endothelial cell adhesion molecule; PECAM-1) was used for microvessel staining on 5-µm paraffin-embedded tissue sections using the alkaline phosphatase/anti-alkaline phosphatase (APAAP) procedure as previously described (Giatromanolaki et al, 1996). Microvessel counting was used for angiogenesis assessment. The areas of the highest vascularization were chosen at low power (×100) and microvessel counting performed on three chosen ×250 fields to establish the highest density within the tumour. The microvessel score was the sum of the vessel counts obtained in these three fields. Microvessels adjacent to normal lung were excluded from the appraisal. Vessels with a clearly defined lumen or well defined linear vessel shape, but no single endothelial cells, were counted. Microvessel scores ≥75 defined high vascular grade disease, and < 75, low vascular grade tumours. This cut-off point was based on a previous study where microvessel scores of 75 or higher defined a group of cases with the highest death rate as compared with other cut-off points (Giatromanolaki et al, 1998). Tumour cell component was assessed for PD-ECGF expression by the intensity and extent of staining (Figure 1D). Two staining patterns of PD-ECGF immunoreactivity were considered: low reactivity (< 50% of cancer cells stained or diffuse weak reac- tivity) and high reactivity (strong staining in > 50% cells). Statistics Statistical analysis and graphic presentation were performed using the Stata 3.1 (Stata Corporation, Texas, USA) and the GraphPad Prism 2.01 package. The unpaired two-tailed t-test or Fisher’s exact test was employed to test for relationships between categor- ical tumour variables as appropriate. Linear regression analysis was used to assess correlation between continuous variables. Survival curves were plotted using the method of Kaplan–Meier, and the log-rank test was used to determine statistical differences between life tables. A Cox proportional hazard model was used to assess the effects of patient and tumour variables on overall survival. A P-value < 0.05 was considered significant. © 2000 Cancer Research Campaign PD-ECGF immunoreactivity high TP/low VEGF (26 patients) C. low TP/high VEGF (55 patients) D. high TP/high VEGF (34 patients) 0 500 1000 1500 2000 2500 100 80 60 40 20 0 Overall survival (%) C Days Figure 1 Kalpan–Meier’s survival curves. (A) VG = vascular grade: high vascular grade vs low vascular grade disease; (B). VEGF = vascular endothelial growth factor: high VEGF vs low VEGF immunoreactive tumours; (C). TP = thymidine phosphorylase which is PD-ECGF: high TP (PD-ECGF) vs low TP (PD-ECGF) immunoreactive tumours; (D) TP(PD-ECGF)–/VEGF– vs TP(PD-ECGF)+/VEGF– vs TP(PD-ECGF) ECGF)–/VEGF+ vs TP(PD-ECGF)+/ VEGF+ immunoreactive tumour subsets Table 1 Correlation of vascular grade with tumour parameters in 223 patients with stage I–IIIA non-small-cell lung cancer Microvessel score Parameters No. of patients Low High P-value Histology Squamous cell 156 94 62 0.44 Adenocarcinoma 67 44 23 T-stage T1 68 46 22 T2 135 86 49 0.007 T3 20 6 14 N-stage N0 127 92 35 N1 77 39 38 0.0005 N2 19 7 12 Grade 1/2 109 70 39 0.44 3 114 68 46 VEGF Negative 119 83 36 0.009 Positive 104 55 49 PD-ECGF Negative 151 100 51 0.05 Positive 72 38 34 Table 1 Correlation of vascular grade with tumour parameters in 223 patients with stage I–IIIA non-small-cell lung cancer VEGF positive (high immunoreactivity) tumours accounted for 104/223 (46.6%) cases examined and were associated with high vascular grade disease (P = 0.009) (Table 1). High PD-ECGF tumour cell immunoreactivity was seen in 72/223 (32.3%) cases and likewise correlated with angiogenesis (P = 0.05) (Table 1). There was a weak positive correlation between VEGF and PD- ECGF expression (r = 0.21, P = 0.002). Eighteen of the 41 (44%) NSCLC tumours with high VEGF and PD-ECGF immunoreactivity had high vascular grade disease. This was equivalent to the proportion of VEGF only and PD-ECGF only positive tumours associated with high microvessel counts (31/63; 49% and 16/31; 48% respectively). As compared to angiogenic growth factor negative disease, positive tumours were strongly associated with high vascular grade (20/88: 22.7% vs 65/135: 48.1%; P = 0.001). Although overall no association was found between VEGF and/or PD-ECGF expression and stage of disease, high PD-ECGF expression was seen more frequently in stage 1b as compared to stage 1a NSCLC tumours (P = 0.02). Survival analyses Univariate analysis of survival showed that T-stage (T2 vs T1; P = 0.01: T3 vs T2 and T1; P = 0.0001), N-stage (N2 vs N1; P = 0.02: N2 vs N0; P = 0.0001: N1 vs N0; P = 0.002) and vascular grade (high vs low; P = 0.0001) were the most significant prognostic variables (Table 2) (Figure 1A). Both high tumour cell VEGF and PD-ECGF immunoreactivity were of prognostic signi- ficance (P = 0.02) (Table 2) (Figure 1B, C). Combined expression of VEGF and PD-ECGF did not identify a worse prognostic PD-ECGF immunoreactivity PD-ECGF expression in NSCLC tissue sections was assessed with the P-GF.44C monoclonal antibody as previously described (Giatromanolaki et al, 1997; Koukourakis et al, 1998). Staining British Journal of Cancer (2000) 82(8), 1427–1432 © 2000 Cancer Research Campaign © 2000 Cancer Research Campaign VEGF, PD-ECGF and angiogenesis in NSCLC 1429 0 500 1000 1500 2000 2500 100 80 60 40 20 0 Overall survival (%) A Months Low VG (118 patients) High VG (65 patients) P = 0.0001 0 500 1000 1500 2000 2500 100 80 60 40 20 0 Overall survival (%) A Months Low VG (118 patients) High VG (65 patients) P = 0.0001 0 500 1000 1500 2000 2500 100 80 60 40 20 0 Overall survival (%) B Days Low VEGF (94 patients) High VEGF (89 patients) P = 0.02 0 500 1000 1500 2000 2500 100 80 60 40 20 0 Overall survival (%) C Days low TP (123 patients) high TP (60 patients) P = 0.02 0 500 1000 1500 2000 2500 100 80 60 40 20 0 Overall survival (%) D Days A vs. B; P = 0.006 A vs. C; P = 0.005 A vs. D; P = 0.01 A vs B,C,D; P = 0.001 A. low TP/low VEGF (68 patients) B. high TP/low VEGF (26 patients) C. low TP/high VEGF (55 patients) D. high TP/high VEGF (34 patients) Figure 1 Kalpan–Meier’s survival curves. (A) VG = vascular grade: high vascular grade vs low vascular grade disease; (B). VEGF = vascular endothelial growth factor: high VEGF vs low VEGF immunoreactive tumours; (C). TP = thymidine phosphorylase which is PD-ECGF: high TP (PD-ECGF) vs low TP (PD-ECGF) immunoreactive tumours; (D) TP(PD-ECGF)–/VEGF– vs TP(PD-ECGF)+/VEGF– vs TP(PD-ECGF) ECGF)–/VEGF+ vs TP(PD-ECGF)+/ VEGF+ immunoreactive tumour subsets 0 500 1000 1500 2000 2500 100 80 60 40 20 0 Overall survival (%) B Days Low VEGF (94 patients) High VEGF (89 patients) P = 0.02 Low VEGF (94 patients) High VEGF (89 patients) Overall survival (%) P = 0.02 0 500 1000 1500 2000 2500 100 80 60 40 20 0 Overall survival (%) D Days 0 500 1000 1500 2000 2500 100 80 60 40 20 0 Overall survival (%) D Days A vs. B; P = 0.006 A vs. C; P = 0.005 A vs. D; P = 0.01 A vs B,C,D; P = 0.001 A. low TP/low VEGF (68 patients) B. Vascular grade, VEGF and PD-ECGF Furthermore the periphery of the tumour, traditionally the area where blood vessels are counted, does not always contain the highest number of vessels (Schor et al, 1998). between angiogenesis and outcome as high microvessel counts are strongly correlated with tumour size and nodal status as shown in the present study and our earlier work (Giatromanolaki et al, 1996). Finally, although there is intense debate on the subject (Fox et al, 1995), a recent study in lung tumours has shown consider- able heterogeneity of vasculature in NSCLC specimens even within blocks from the same region of the tumour. Furthermore the periphery of the tumour, traditionally the area where blood vessels are counted, does not always contain the highest number of vessels (Schor et al, 1998). Although the numbers were relatively small the study included sufficient patients to analyse the impact of VEGF and PD-ECGF, and microvessel density on outcome in stage Ia to IIb disease (stage Ia, 37; stage Ib, 67; stage IIa, 19; and stage IIb, 44 patients). High vascular grade was associated with a poor prognosis in the stage IIb disease subgroup only (P = 0.0005). Over-expression of either or both of the angiogenic growth factors analysed was also associated with a poor outcome in stage IIb disease as compared to VEGF and PD-ECGF negative NSCLC (P = 0.02). A number of studies have demonstrated that VEGF expression is associated with angiogenesis and/or has prognostic significance in solid tumours including breast (Fox et al, 1995), colorectal (Amaya et al, 1997), gastic (Maeda et al, 1997; Takahashi et al, 1998) and pancreatic cancer (Ikeda et al, 1999). The results of the present work confirm previous findings of an important role for tumour cell VEGF expression in the angiogenic process of, and as a prognostic factor for, NSCLC (Volm et al, 1996; Giatromanolaki et al, 1998). VEGF overexpression was not associated with a particular stage of disease or with a negative outcome in any given disease stage subgroup (stage Ia to IIb). However, when all 183 patients were taken into consideration, univariate analysis revealed high VEGF immunoreactivity to be associated with a poor prognosis (P = 0.02). The VG1 monoclonal antibody employed in the current study detects the VEGF 121, 165 and 189 isoforms (Turley et al, 1998). Vascular grade, VEGF and PD-ECGF The non-heparin binding VEGF 121 and the basic, heparin binding VEGF 165 are freely secreted from producing cells whilst the longer isoforms VEGF 189 and 206 are generally cell associated. Employing reverse transcription-poly- merase chain reaction (RT-PCR) and in situ hybridization tech- niques, VEGF 121, VEGF 165 and VEGF 189 have been found to be associated with angiogenesis and/or a poor prognosis in NSCLC (Oshika et al, 1998; Fontanini et al, 1999). No association was found between histology and tumour grade and any of the parameters evaluated. Vascular grade, VEGF and PD-ECGF Eighty-nine of the 223 tumours (39.9%) were of high vascular grade. High microvessel counts were associated with advanced T-stage (P = 0.007) and node-positive (P = 0.0005) disease (Table 1). British Journal of Cancer (2000) 82(8), 1427–1432 © 2000 Cancer Research Campaign © 2000 Cancer Research Campaign 1430 KJ O’Byrne et al 1430 KJ O’Byrne et al Table 2 Univariate analysis of all variables analysed in the study Parameter Hazard ratio P-value Histology Adenocarcinoma vs squamous 1.36 0.21 T-stage T3 vs T2 3.63 0.0001 T3 vs T1 6.29 0.0001 T2 vs T1 1.87 0.01 N-stage N2 vs N1 2.29 0.02 N2 vs N0 4.15 0.0001 N1 vs N0 1.95 0.002 Grade 3 vs 1/2 1.36 0.14 Vascular grade High vs low 2.34 0.0001 VEGF Positive vs negative 1.63 0.02 PD-ECGF Positive vs negative 1.51 0.02 Table 2 Univariate analysis of all variables analysed in the study Table 3a Multivariate analysis of all tumour variables analysed in the study Hazard ratio Variable t ratio P-value Significant? Histology 1.254 0.212 No T stage 2.421 0.017 Yes N status 1.509 0.133 No Grade 0.8698 0.386 No CD31 2.080 0.039 Yes VEGF 1.529 0.128 No PD-ECGF 0.6769 0.499 No Table 3b Multivariate analysis of variables significant at univariate analysis but VEGF/PD-ECGF considered as a single variable Hazard ratio Variable t ratio P-value Significant? T stage 2.574 0.011 Yes N status 1.504 0.134 No CD31 2.056 0.041 Yes VEGF/PD-ECGF 1.941 0.054 No Table 3a Multivariate analysis of all tumour variables analysed in the study subgroup of patients beyond that seen with VEGF or PD-ECGF alone (Figure 1D). T-stage (P < 0.02) and vascular grade (P < 0.04) were independent predictors of outcome on multivariate analysis when all parameters were considered (Table 3a). When analysed as a single variable, the presence of an angiogenic growth factor vs negative tumours approached significance in a multi- variate model (P = 0.054) (Table 3b). between angiogenesis and outcome as high microvessel counts are strongly correlated with tumour size and nodal status as shown in the present study and our earlier work (Giatromanolaki et al, 1996). Finally, although there is intense debate on the subject (Fox et al, 1995), a recent study in lung tumours has shown consider- able heterogeneity of vasculature in NSCLC specimens even within blocks from the same region of the tumour. © 2000 Cancer Research Campaign REFERENCES Amaya H, Tanigawa N, Lu C, Matsumura M, Shimomatsuya T, Horiuchi T and Muraoka R (1997) Association of vascular endothelial growth factor expression with tumor angiogenesis, survival and thymidine phosphorylase/platelet derived endothelial cell growth factor expression in human colorectal cancer. Cancer Lett 119: 227–235 Apolinario RM, van der Valk P, de Jong JS, Deville W, van Ark-Otte J, Dingemans A-MC, van Mourik JC, Postmus PE, Pinedo HM and Giaccone G (1997) Prognostic value of the expression of p53, bcl-2, and bax oncoproteins, and neovascularisation in patients with radically resected non-small cell lung cancer. J Clin Oncol 15: 2456–2466 These findings indicate that VEGF may be a suitable target for novel therapies in the management of NSCLC. Inhibition of VEGF activity with monoclonal antibodies (Kim et al, 1993), soluble VEGF receptors (Lin et al, 1998) and VEGF-receptor 2 (Flk-1/KDR) monoclonal antibodies (Skobe et al, 1997) has shown promise in experimental in vivo models with inhibition of angiogenesis, tumour growth and/or tumour cell invasion being recorded. Arenberg DA, Polverini PJ, Kunkel SL, Shanafelt A, Hesselgesser J, Horuk R and Strieter RM (1997) The role of CXC chemokines in the regulation of angiogenesis in non-small cell lung cancer. J Leukoc Biol 62: 554–562 Budman DR, Meropol NJ, Reigner B, Creaven PJ, Lichtman SM, Bergham E, Behr J, Gordon RJ, Osterwalder B and Griffin T (1998) Preliminary studies of a novel oral fluoropyrimidine carbamate: capecitabine. J Clin Oncol 16: 1795–1802 Chandrachud LM, Pendleton N, Chisholm DM, Horan MA and Schor AM (1997) Relationship between vascularity, age and survival in non-small cell lung cancer. Br J Cancer 76: 1367–1375 As outlined earlier PD-ECGF is thymidine phosphorylase (TP), an important enzyme in the activation of 5-fluorouracil (5-FU) to fluorodeoxyuridine. TP also plays a role in converting 5-FU pro- drugs, including 5′-deoxy-5-fluorouridine and the novel oral fluo- ropyrimidine carbamate, capecitabine, to their active metabolites (Patterson et al, 1995; Budman et al, 1998). A recent report by our group demonstrated that there was a significant improvement in both relapse-free and overall survival in PD-ECGF/TP-positive as compared with PD-ECGF/TP-negative breast cancer patients treated with adjuvant cyclophosphamide, methotrexate and 5-FU (CMF) chemotherapy. This was felt to be due not only to the acti- vation of 5-FU but to the enhancement of the effectiveness of methotrexate by TP (Fox et al, 1997). VEGF, PD-ECGF and angiogenesis in NSCLC 1431 VEGF, PD-ECGF and angiogenesis in NSCLC 1431 this regard. Apart from the more established angiogenic growth factors such as VEGF and PD-ECGF recent evidence has suggested a role for tissue factor (TF) (Koomagi and Volm, 1998), heparin-binding growth-associated molecule (HB-GAM) (Jager et al, 1997) and CXC chemokines (Arenberg et al, 1997) in the induction of angiogenesis in NSCLC. As such other novel anti- angiogenic agents, including angiostatin, endostatin, TNP-470, thalidomide and the matrix metalloproteinase inhibitors (MMPIs) may have a role to play in the treatment of NSCLC (Harris, 1998; Macaulay et al, 1999). disease where PD-ECGF overexpression has been seen in a proportion of all solid tumours studied to date including breast (Toi et al, 1995), colorectal (Amaya et al, 1997), gastric (Maeda et al, 1997; Takahashi et al, 1998), oesophageal (Igarashi et al, 1998) and pancreatic (Ikeda et al, 1999) cancers. In general, PD-ECGF is associated with angiogenesis and/or advanced disease. Furthermore overexpression may predict for poor survival (Takebayashi et al, 1996; Ikeda et al, 1999). However, PD-ECGF immunoreactivity, be it in the malignant, inflammatory and/or stromal cells of the tumour, is not always associated with a poor outcome emphasizing the relatively weak prognostic power of this marker (Toi et al, 1995; Igarashi et al, 1998). In conclusion, VEGF and PD-ECGF are important angiogenic growth factors in NSCLC. The detection of a subset of high vascular grade tumours not dependent on either VEGF or PD- ECGF indicates that other angiogenic growth factors, such as bFGF, HB-GAM and TF, may have an important role in the patho- genesis of NSCLC. We observed a weak positive correlation between tumour VEGF and PD-ECGF expression (r = 0.21; P = 0.002). Similar observa- tions have been made in colorectal cancer (Amaya et al, 1997). Although not seen in our study synergy between VEGF and PD-ECGF in determining the intensity of angiogenesis has been reported for breast, gastric and pancreatic cancer (Toi et al, 1995; Maeda et al, 1997; Ikeda et al, 1999). A correlation between tumour cell VEGF and PD-ECGF-positive CD68 infiltrating inflammatory cells has been observed in gastric cancer, the vessel count being significantly higher in tumours expressing both growth factors as compared to those expressing each growth factor alone (Takayashi et al, 1998). Furthermore, a significant associa- tion between co-expression of both angiogenic factors with the presence of hepatic metastases has been described in this disease (Maeda et al, 1997). ACKNOWLEDGEMENTS This work was supported by the Institute of Cancer Studies, Leicester and the Imperial Cancer Research Fund, UK and the Tumour and Angiogenesis Research Group, Greece. This work was supported by the Institute of Cancer Studies, Leicester and the Imperial Cancer Research Fund, UK and the Tumour and Angiogenesis Research Group, Greece. VEGF, PD-ECGF and angiogenesis in NSCLC 1431 We found that high immunoreactivity of either or both of the angiogenic growth factors analysed conferred a poor prognosis in stage IIb disease (P = 0.02). This may reflect the contribution of these growth factors to the angiogenic process which itself is associated with a particularly poor outcome in the stage IIb patients studied (P = 0.0005). DISCUSSION The results support the findings of previous studies indicating that the intensity of intratumoural angiogenesis, as assessed by tumour microvessel counts, has prognostic significance and plays an important role in the pathogenesis of NSCLC (Giatromanolaki et al, 1996; Fontanini et al, 1997). This is in keeping with observa- tions in other solid tumours including breast (Fox et al, 1995), colorectal (Amaya et al, 1997) and gastric cancer (Maeda et al, 1996; Takahashi et al, 1998). It is important to note, however, that not all studies have found a positive association between microvessel density and prognosis (Apolinario et al, 1997; Chandrachud et al, 1997; Pezzella et al, 1997). Indeed in the largest of these studies tumours with an alve- olar pattern, with no parenchymal destruction and the alveolar septae still present, had a worse outcome than those tumours with an angiogenic pattern. This observation indicates that if an appro- priate blood supply is available, an NSCLC tumour may exploit it and grow within the lung without the need for neovascularization (Pezzella et al, 1997). The study was in stage I NSCLC only. This may, to some extent, account for the lack of an association The present study confirms our earlier work demonstrating that high PD-ECGF overexpression has a weak association with angio- genesis and prognosis, the latter on univariate analysis only. The findings are in keeping with the results of studies in malignant © 2000 Cancer Research Campaign British Journal of Cancer (2000) 82(8), 1427–1432 © 2000 Cancer Research Campaign REFERENCES J Pathol 181: 196–199 Moghaddam A and Bicknell R (1992) Expression of platelet-derived endothelial cell growth factor in Escherichia coli and confirmation of its thymidine phosphorylase activity. Biochemistry 31: 12141–12146 Giatromanolaki A, Koukourakis MI, Kakolyris S, Turley H, O’Byrne KJ, Scott PAE, Pezzella F, Georgoulias V, Harris AL and Gatter KC (1998) Vascular endothelial growth factor, wild-type p53 and angiogenesis in early operable non-small cell lung cancer. Clin Cancer Res 4: 3017–3024 Moghaddam A, Zhang HT, Fan TPD, Hu D-E, Lees V, Turley H, Fox SB, Gatter KC, Harris AL and Bicknell R (1995) Thymidine phosphorylase is angiogenic and promotes tumour growth. Proc Natl Acad Sci USA 92: 998–1002 non-small cell lung cancer. Clin Cancer Res 4: 3017–3024 Harris AL (1998) Are angiostatin and endostatin cures for cancer? Commentary. Lancet 351: 1598–1599 Oshika Y, Nakamura M, Tokunaga T, Ozeki Y, Fukushima Y, Hatanaka H, Abe Y, Yamazaki H, Kijima H, Tamaoki N and Ueyama Y (1998) Expression of cell- associated isoform of vascular endothelial growth factor 189 and its prognostic relevance in non-small cell lung cancer. Int J Oncol 12: 541–544 Ikeda N, Adachi M, Taki T, Huang C, Hashida H, Takabayashi A, Sho M, Nakajima Y, Kanehiro H, Hisanaga M, Nakano H and Miyaka M (1999) Prognostic significance of angiogenesis in human pancreatic cancer. Br J Cancer 79: 1553–1563 relevance in non-small cell lung cancer. Int J Oncol 12: 541–544 Patterson AV, Zhang H, Moghaddam A, Bicknell R, Talbot DC, Stratford IJ and Harris AL (1995) Increased sensitivity to the prodrug 5′-deoxy-5-fluorouridine and modulation of 5-fluoro-2′-deoxyuridine sensitivity in MCF-7 cells transfected with thymidine phosphorylase. Br J Cancer 72: 669–675 Igarashi M, Dhar DK, Kubota H, Yamamoto A, El-Assal O and Nagasue N (1998) The prognostic significance of microvessel density and thymidine phosphorylase expression in squamous cell carcinoma of the esophagus. Cancer 82: 1225–1232 Pezzella F, Pastorino U, Tagliabue E, Andreola S, Sozzi G, Gasparini G, Menard S, Gatter KC, Harris AL, Fox S, Buyse M, Pilotti S, Pierotti M and Rilke F (1997) Non-small-cell lung carcinoma tumor growth without morphological evidence of neo-angiogenesis. Am J Pathol 151: 1417–1423 Ishikawa F, Miyazono K, Hellman U, Dexler H, Wernstedt C, Hagiwara K, Usuki K, Takaku F, Risau W and Heldin CH (1989) Identification of angiogenic activity and the cloning and expression of platelet-derived endothelial cell growth factor. REFERENCES Nature 338: 557–562 Schor AM, Pazouki S, Morris J, Smither RL, Chandrachud LM and Pendleton N (1998) Heterogeneity in microvascular density in lung tumours: comparison with normal bronchus. Br J Cancer 77: 946–951 Jager R, Noll K, Havemann K, Pfluger KH, Knabbe C, Rauvala H and Zugmaier G (1997) Differential expression and biological activity of the heparin-binding growth-associated molecule (HB-GAM) in lung cancer cell lines. Int J Cancer 73: 537–543 Skobe M, Rockwell P, Goldstein N, Vosseler S and Fusenig NE (1997) Halting angiogenesis suppresses carcinoma cell invasion. Nat Med 3: 1222–1227 Takahashi Y, Bucana CD, Akagi Y, Liu W, Cleary KR, Mai M and Ellis LM (1998) Significance of platelet-derived endothelial cell growth factor in the angiogenesis of human gastric cancer. Clin Cancer Res 4: 429–434 Kim KJ, Li B, Winer J, Armanini M, Gillett N, Phillips HS and Ferrara N (1993) Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo. Nature 362: 841–844 Takebayashi Y, Akiyama S, Akiba S, Yamada K, Miyadera K, Sumizawa T, Yamada Y, Murata F and Aikou T (1996) Clinicopathologic and prognostic significance of an angiogenic factor, thymidine phosphorylase, in human colorectal carcinoma. J Natl Cancer Inst 88: 1110–1117 Koomagi R and Volm M (1998) Tissue-factor expression in human non-small-cell lung carcinoma measured by immunohistochemistry: correlation between tissue factor and angiogenesis. Int J Cancer 79: 19–22 Koukourakis MI, Giatromanolaki A, Kakolyris S, O’Byrne KJ, Apostolikas N, Skarlatos J, Gatter KC and Harris AL (1998) Different pattern of stromal and cancer cell thymidine phosphorylase reactivity in non small cell lung cancer. Impact on tumour neoangiogenesis and survival. Br J Cancer 77: 1696–1703 Toi M, Hosina S, Taniguchi T, Yamamoto Y, Ishitsuka H and Tominaga T (1995) Vascular endothelial growth factor and platelet derived endothelial cell growth factor are frequently coexpressed in highly vascularized human breast cancer. Clin Cancer Res 1: 961–964 Turley H, Scott PAE, Watts VM, Bicknall R, Harris AL and Gatter KC (1998) Expression of VEGF in routinely fixed material using a new monoclonal antibody VG1. J Pathol 186: 313–318 Lin P, Sanka S, Shan S, Dewhirst MW, Ploverini PJ, Quinn TQ and Peters KG (1998) Inhibition of tumor growth by targeting tumor endothelium using a soluble vascular endothelial growth factor receptor. Cell Growth Diff 9: 49–58 van Zandwijk N and Giaccone G 1996 Treatment of metastatic non-small cell lung cancer. REFERENCES Given the overexpression of PD-ECGF/TP seen in NSCLC, the combination of fluoropyrim- idines with the more widely used cytotoxics, such as the plat- inums, taxanes, vinca alkaloids and topoisomerase I inhibitors, gemcitabine and ifosphamide, in the treatment of NSCLC should be evaluated further (van Zandwijk and Giaccone, 1996). Ferrara N and Davis-Smyth T (1997) The biology of vascular endothelial growth factor. Endoc Rev 18: 4–25 Fontanini G, Lucchi M, Vignati S, Mussi A, Ciardiello F, De Laurentiss M, De Placido S, Basolo F, Angeletti CA and Bevilacqua G (1997) Angiogenesis as a prognostic indicator of survival in non-small cell lung carcinoma: a prospective study. J Natl Cancer Inst 89: 881–886 Fontanini G, Boldrini L, Chine S, Pisaturo F, Basolo F, Calcinai A, Lucchi M, Mussi A, Angeletti CA and Bevilacqua G (1999) Expression of vascular endothelial growth factor mRNA in non-small cell lung carcinomas. Br J Cancer 79: 363–369 Fox SB, Leek RD, Weekes MP, Whitehouse RM, Gatter KC and Harris AL (1995) Quantification and prognostic value of breast cancer angiogenesis: comparison of microvessel density, Chalkley count and computer image analysis. J Pathol 177: 275–283 Fox SB, Engels K, Comley M, Whitehouse RM, Turley H, Gatter KC and Harris AL (1997) Relationship of elevated tumour thymidine phosphorylase in node- positive breast carcinomas to the effects of adjuvant CMF. Ann Oncol 8: 271–275 Giatromanolaki A, Koukourakis M, O’Byrne K, Fox S, Whitehouse R, Talbot DC, Harris AL and Gatter K (1996) Prognostic value of angiogenesis in operable non-small cell lung cancer. J Pathol 179: 80–88 Giatromanolaki A, Koukourakis M, O’Byrne K, Fox S, Whitehouse R, Talbot DC, Harris AL and Gatter K (1996) Prognostic value of angiogenesis in operable non-small cell lung cancer. J Pathol 179: 80–88 Giatromanolaki A, Koukourakis M, Comley M, Kaklamanis L, Turley H, O’Byrne K, Harris AL and Gatter K (1997) Platelet derived endothelial cell growth In the present study we have clearly demonstrated that a propor- tion of high vascular grade tumours do not express either VEGF or PD-ECGF indicating the importance of other angiogenic factors in non-small cell lung cancer. J Pathol 179: 80–88 Giatromanolaki A, Koukourakis M, Comley M, Kaklamanis L, Turley H, O’Byrne K, Harris AL and Gatter K (1997) Platelet derived endothelial cell growth © 2000 Cancer Research Campaign British Journal of Cancer (2000) 82(8), 1427–1432 1432 KJ O’Byrne et al factor (thymidine phosphorylase) expression in lung cancer. British Journal of Cancer (2000) 82(8), 1427–1432 © 2000 Cancer Research Campaign REFERENCES Curr Opin Oncol 8: 120–125 Volm M, Koomagi R and Mattern J (1996) Interrelationships between microvessel density, expression of VEGF and resistance to doxoresistance of non-small-cell lung carcinoma. Anticancer Res 16: 213–218 Maeda K, Kang SM, Ogawa M, Onoda N, Sawada T, Nakata B, Kato Y, Chung YS and Sowa M (1997) Combined analysis of vascular endothelial growth factor and platelet-derived endothelial cell growth factor expression in gastric carcinoma. Int J Cancer 74: 545–550 Whittle C, Gillespie K, Harrison R, Mathieson PW and Harper SJ (1999) Heterogeneous vascular endothelial growth factor (VEGF) isoform mRNA and receptor mRNA expression in human glomeruli, and the identification of VEGF148 mRNA, a novel truncated splice variant. Clin Sci (Colch) 97: 303–312 Macaulay VM, O’Byrne KJ, Saunders MP, Long L, Gleeson F, Puttick R, Harris AL and Talbot DC (1999) Phase I study of intrapleural batimastat (BB-94), a matrix metalloproteinase inhibitor, in the treatment of malignant pleural effusions. Clin Cancer Res 5: 513–520 British Journal of Cancer (2000) 82(8), 1427–1432 © 2000 Cancer Research Campaign
https://openalex.org/W4393101919	https://jurnalilmiah.ici.ac.id/index.php/JP/article/download/369/138	Indonesian	null	EFEKTIFAS EDUKASI DALAM PENCEGAHAN DIABETES MELITUS DI DESA GENENG POLOKARTO SUKOHARJO	Deleted Journal	2,023	cc-by-sa	2,763	Abstrak Latar Belakang: Pencegahan dini perlu dilakukan untuk deteksi dini terhadap penyakit kronis seperti DM yang mempunyai faktor risiko baik karena pola hidup tidak sehat dan faktor keturunan. Deteksi dini DM dapat dilakukan melalui skrining dengan pemeriksaan kadar gula darah sewaktu. Selain itu, keberhasilan dalam pencegahan timbulnya DM dan pengendalian kadar gula darah pada penderita DM tergantung pada prilaku masyarakat. Perubahan prilaku menuju pola hidup sehat dalam rangka pencegahan dan pengendalian DM yang benar akan dapat diwujudkan apabila masyarakat mempunyai pengetahuan yang cukup tentang DM serta pencegahannya. Tujuan dari kegiatan ini adalah menambah informasi kesehatan kepada masyarakat tentang edukasi pencegahan penyakit Diabetes Melitus. Metode dilakukan dengan ceramah, diskusi dan tanya jawab sedangkan media menggunakan leaflet. Edukasi yang disampaikan meningkatkan pengetahuan masyarakat tentang edukasi pencegahan resiko diabetes, sehingga masyarakat secara mandiri dapat mencegah risiko penyakit diabetes melitus yang merupakan penyakit degeneratif dan diramalkan meningkat setiap tahunnya. Kata Kunci : Diabetes Melitus, Edukasi Kata Kunci : Diabetes Melitus, Edukasi Jurnal Pengabdian Kepada Masyarakat Citra Delima Volume 1, Nomor 1 Tahun 2023 E-ISSN : 3046-7497 Doi: 10.33862/jp.v1i1.369 Jurnal Pengabdian Kepada Masyarakat Citra Delima Volume 1, Nomor 1 Tahun 2023 E-ISSN : 3046-7497 Doi: 10.33862/jp.v1i1.369 Ikrima Rahmasari1, Ady Irawan AM2, Niken Luthfiyanti3, Iswanti Purwaningsih Email Korespondensi: Ikrima_rahmasari@udb.ac.id Ikrima Rahmasari1, Ady Irawan AM2, Niken Luthfiyanti3, Iswanti Purwaningsih4 Email Korespondensi: Ikrima_rahmasari@udb.ac.id 1,2S1 Keperawatan, Universitas Duta Bangsa Surakarta, Indonesia 3S1 Farmasi Keperawatan, Universitas Duta Bangsa Surakarta, Indonesia 4DIII Keperawatan, Politeknik Kesehatan Karya Husada Yogyakarta, Indones 1,2S1 Keperawatan, Universitas Duta Bangsa Surakarta, Indonesia 3S1 Farmasi Keperawatan, Universitas Duta Bangsa Surakarta, Indonesia 4DIII Keperawatan, Politeknik Kesehatan Karya Husada Yogyakarta, Indonesi Abstract Background: Early prevention needs to be done for early detection of chronic diseases such as DM which have good risk factors due to unhealthy lifestyles and heredity. Early detection of DM can be done through screening by checking blood sugar levels at the time. In addition, success in preventing the onset of DM and controlling blood sugar levels in people with DM depends on people's behavior. Changes in behavior towards a healthy lifestyle in the context of correct DM prevention and control will be realized if the community has sufficient knowledge about DM and its prevention. The purpose of this activity is to add health information to the public about education on the prevention of Diabetes Mellitus. The method is carried out by lectures, discussions and questions and answers while the media uses leaflets. The education delivered increases public knowledge about diabetes risk prevention education, so that people can independently prevent the risk of diabetes mellitus which is a degenerative disease and is predicted to increase every year. Keywords: Diabetes Millitus, Education. Keywords: Diabetes Millitus, Education. \| 1 Jurnal Pengabdian Kepada Masyarakat Citra Delima Volume 1, Nomor 1 Tahun 2023 E-ISSN : 3046-7497 Doi: 10.33862/jp.v1i1.369 Jurnal Pengabdian Kepada Masyarakat Citra Delima Volume 1, Nomor 1 Tahun 2023 E-ISSN : 3046-7497 Doi: 10.33862/jp.v1i1.369 dengan komponen genetic dan linkungan yang sama kuat dalam proses timbulnya penyakit tersebut. Pengaruh faktor genetik terhadap penyakit ini dapat terlihat jelas dengan tingginya penderita diabetes yang berasal dari orang tua yang memiliki riwayat diabetes mellitus sebelumnya. Diabetes tipe II adalah sekelompok gangguan heterogen dengan karakteristik derajat resistensi insulin yang bervariasi, gangguan sekresi insulin, dan peningkatan produksi glukosa. Diabetes tipe II diawali dengan suatu periode abnormalitas homeostasis glukosa, yang dikenal sebagai impaired fasting glucose (IFG) atau impaired glucose tolerance (IGT) (Fauci AS., et al, 2018). Pendahuluan Diabetes melitus (DM) merupakan suatu kelompok penyakit metabolik dengan karakteristik hiperglikemia yang terjadi karena kelainan sekresi insulin, kerja insulin, atau kedua-duanya. DM dapat diklasifikasikan menjadi beberapa tipe yakni, DM tipe 1, DM tipe 2, DM tipe 2 merupakan salah satu jenis yang paling banyak ditemukan yaitu lebih dari 90-95% (Petersmann et al., 2019). Data IDF tahun 2017 menunjukkan bahwa jumlah pasien diabetes miitus di Indonesia pada kelompok umur antara 20-79 tahun pada tahun 2019 diperkirakan sebanyak 7 juta yang menempatkan Indonesia padaurutan ke 9, sedangkan pada tahun 2030 diperkirakan jumlahnya meningkat menjadi 12 juta dan menempatkan Indonesia pada urutan ke-6 (Wang et al., 2022). Gejala dan tanda DM ditandai dengan keadaan hiperglikemia yaitu kondisi kadar glukosa dalam darah seseorang melebihi kadar normal yang diperbolehkan. Menurut Soliman et al., (2020) dua hal melatarbelakangi keadaan tersebut yaitu jumlah insulin yang kurang dan keadaan resistensi insulin atau kualitas insulinnya tidak baik. Pada keadaan kedua, meskipun insulin dan reseptor insulin ada, tetapi karena ada kelainan pada sel organ, maka glukosa tidak dapat masuk ke dalam organ untuk dibakar. Akibatnya glukosa tetap berada Menurut konsensus Pengelolaan Diabetes melitus di Indonesia penyuluhan dan perencanaan makan merupakan pilar utama penatalaksanaan DM. Oleh karena itu perencanaan makan dan penyuluhannya kepada pasien DM haruslah mendapat perhatian yang besar (Wicaksana et al., 2020). Mayoritas terjadinya DM yaitu DM tipe 2, DM tipe 2 merupakan penyakit multifaktorial \| 2 Jurnal Pengabdian Kepada Masyarakat Citra Delima Volume 1, Nomor 1 Tahun 2023 E-ISSN : 3046-7497 Doi: 10.33862/jp.v1i1.369 Jurnal Pengabdian Kepada Masyarakat Citra Delima Volume 1, Nomor 1 Tahun 2023 E-ISSN : 3046-7497 Doi: 10.33862/jp.v1i1.369 membutuhkan insulin untuk bertahan hidup. Pada DM tipe 1, fase gangguan kadar glukosa darah membutuhkan insulin untuk bertahan hidup, sedangkan DM tipe 2 dan tipe lainnya, kebutuhan insulin hanya untuk pengontrolan saja, bahkan beberapa tidak membutuhkan insulin(Wang et al., 2022). di pembuluh darah, sehingga kadarnya meningkat dalam darah. Berbagai keluhan dapat ditemukan pada penderita diabetes mellitus. Kecurigaan adanya diabetes perlu dipikirkan apabila terdapat keluhan klasik diabetes berupa: poliuri, polidipsi, polifagi, dan penurunan berat badan tanpa penyebab yang jelas. Keluhan lain yang mungkin ditemukan dapat berupa mudah lelah, gatal pada kulit, pandangan kabur, kesemutan, dan disfungsi ereksi pada laki-laki (Byrne et al., 2017). DM tipe 2 disebabkan oleh kondisi hiperglikemia yang tidak terdeteksi secara spesifik pada pada gejala awal dan berkembang secara bertahap. Pada kondisi ini, pasien mengalami peningkatan risiko terhadap komplikasi makrovaskuler dan mikrovaskuler. Pendahuluan Diperkirakan usia penyakit DM rata-rata mencapai 5-8 tahun saat seseorang terdiagnosa penyakit tersebut. Selain DM tipe 1 dan tipe 2, klasifikasi lainnya menurut adalah DM Gestasional dan DM tipe lainnya yang disebabkan antara lain oleh defek genetik fungsi sel beta, defek genetik kerja insulin, penyakit eksokrin pankreas, endokrinopati, karena obat/zat kimia, infeksi, sebab imunologi yang jarang, dan sindrom genetik lain yang berkaitan dengan DM(Belete et al., 2021). Menurut American Diabetes Association kondisi glukosa dalam darah terbagi dua yaitu Normoglycemia (kadar glukosa dalam darah normal sesuai dengan standar yang berlaku) dan Hyperglycemia (kadar glukosa dalam darah melebihi standar yang berlaku) (Chung et al., 2020). Kondisi hiperglikemia sendiri terbagi atas dua kondisi yaitu Pre-diabetes dan Diabetes Melitus. Prediabetes ditandai dengan kejadian Impaired Glucose Tolerance atau Gannguan Toleransi Glukosa (GTG), atau Impaired Fasting Glucose atau Gangguan Glukosa Puasa. Sedangkan kondisi Diabetes Melitus meliputi tiga kondisi yaitu tidak membutuhkan insulin, membutuhkan insulin untuk pengontrolan, dan Kondisi bukan DM disebut juga Pre-diabetes. Kondisi ini adalah kondisi dimana seseorang mengalami gangguan toleransi glukosa akan tetapi tidak \| 3 \| 3 Jurnal Pengabdian Kepada Masyarakat Citra Delima Volume 1, Nomor 1 Tahun 2023 E-ISSN : 3046-7497 Doi: 10.33862/jp.v1i1.369 jauh hari. Tidak ada salahnya untuk mengadopsi gaya hidup sehat sejak masih berusia muda, tidak perlu menunggu hingga usia lanjut dan adanya penyakit menghampiri. Oleh karena itu, edukasi sangat penting untuk meningkatkan pengetahuan masyarakat. menujukkan gejala-gejala DM. Gangguan Toleransi Glukosa atau Impaired Fasting Glucose adalah kondisi seseorang yang memiliki level glukosa puasa 101-125mg/dL (Soliman et al., 2020). Seseorang yang dinyatakan pre-diabetes memiliki risiko yang relatif tinggi untuk berkembang menjadi DM. Gangguan Toleransi Glukosa berhubungan dengan sindrom metabolik yang meliputi obesitas, dislipidemia, dan hipertensi. Upaya pencegahan DM meliputi pencegahan primer yang bertujuan mencegah timbulnya penyakit DM, pencegahan sekunder bertujuan mencegah timbulnya penyulit, meskipun telah terjadi penyakit DM; dan pencegahan tersier untuk mencegah terjadi kecacatan lebih kanjut, meskipun telah terjadi penyakit DM (Waspadji, 2018). Metode Pelaksanaan Metode yang digunakan dalam meningkatkan pengetahuan tentang pencegahan DM adalah penyuluhan tentang pencegahan DM. Pelaksanaan kegiatan ini memiliki beberapa tahapan, yaitu tahapan persiapan tim, diskusi edukasi dengan peserta dilanjutkan pada tahapan diskusi dan tanya jawab terkait dengan pentingnya pencegahan penyakit diabetes sejak dini. Untuk mempermudah dalam kegiatan pengabdian ini, maka penulis membuat alur kegiatan sebagai berikut : Menurut Centers for Disease Control and Prevention (CDC) Singh et al., (2020), ada beberapa cara yang sebenarnya dapat diterapkan untuk mencegah datangnya diabetes bertamu dalam hidup kita, antara lain mengetahui penyebab diabetes. Rajin melakukan riset mengenai apa saja penyebab diabetes, faktor pemicu dan gejala yang mengarah pada penyakit diabetes adalah solusi awal untuk mengantisipasi dari 1) Koordinasi dengan bidan desa dan kader, 2) Penemuan masalah dan tersusun program, 3) Edukasi tentang pencegahan DM, 4) Monitoring dan Evaluasi. 4) Monitoring dan Evaluasi. Kegiatan ini dilakukan pada bulan Juni 2023 dengan jumlah peserta 28 orang. Pelaksanaan dilakukan selama 60 menit. Dengan waktu 10 menit untuk \| 4 Jurnal Pengabdian Kepada Masyarakat Citra Delima Volume 1, Nomor 1 Tahun 2023 E-ISSN : 3046-7497 Doi: 10.33862/jp.v1i1.369 Delima Gambar 1. Edukasi Pencegahan Diabetes Melitus Di Desa Geneng Polokarto Sukoharjo pengambilan data pengetahuan pre dan post dan 40 menit untuk pemberian materi edukasi pencegahan penyakit DM. Media yang digunakan dalam pendidikan kesehatan ini adalah mengguankan bantuan LCD projektor dan leaflet. Materi yang akan diberikan meliputi pengertian, penyebab, tanda dan gejala DM, pola makan serta cara pencegahan DM. Intrument menggunakan kuesioner pengetahuan untuk melihat pengetahuan peserta terkait DM, kuesioner diisi sebelum dan sesudah pemberian materi edukasi. Gambar 1. Edukasi Pencegahan Diabetes Melitus Di Desa Geneng Polokarto Sukoharjo American Diabetes Association (ADA) menyatakan bahwa seseorang yang bekerja memiliki manfaat yang besar karena kadar glukosa darah dapat terkontrol melalui aktivitas fisik serta mencegah terjadi komplikasi (Powers et al., 2020). Faktor pekerjaan mempengaruhi resiko besar terjadinya diabetes mellitus, pekerjaan dengan aktivitas fisik yang ringan akan menyebabkan kurangnya pembakaran energi oleh tubuh sehingga kelebihan energi dalam tubuh akan disimpan dalam bentuk lemak dalam tubuh yang mengakibatkan obesitas yang merupakan salah satu faktor resiko DM (Suiraoka, 2017). Hasil dan Pembahasan Pengabdian kepada masyarakat dengan judul edukasi pencegahan diabetes mellitus di Desa Geneng Polokarto Sukoharjo. Mereka mengatakan bahwa DM merupakan penyakit gula yang dapat menyebabkan luka khususnya pada kaki sampai diamputasi. Warga juga mengatakan bahwa disekitarnya jarang yang memiliki penyakit DM. Mereka menganggap bahwa kegiatan/ aktivitas yang dilakukan warga sekitar dapat mencegah terjadinya DM. Sebagian besar mereka bekerja di sawah dan sebagai buruh pasar, sehingga dalam sehari berjalan jauh. Hasil kegiatan edukasi menunjukkan bahwa pengetahuan peserta yang kurang baik sebelum diberikan edukasi sebanyak 8 orang atau 28,6%, sedangkan peserta yang berpengetahuan baik sebelum di edukasi sebanyak 20 orang \| 5 Jurnal Pengabdian Kepada Masyarakat Citra Delima Volume 1, Nomor 1 Tahun 2023 E-ISSN : 3046-7497 Doi: 10.33862/jp.v1i1.369 atau 71,4%. Setelah diberikan edukasi tentang pencegahan DM didapatkan hasil terdapat peningkatan pengetahuan tentang pencegahan DM, dibuktikan oleh sebanyak 27 orang berpengatahuan baik dengan presentasi 96,4% dan hanya 1 orang yang memiliki pengetahuan kurang baik yaitu 3,6%. Hal ini dikarenakan pendengaran peserta kurang baik dan kurang kooperatif saat diberikan penjelasan, kemungkinan karena faktor usia. Namun peserta tersebut sangat antusias terhadap kegiatan yang dilakukan. atau 71,4%. Setelah diberikan edukasi tentang pencegahan DM didapatkan hasil terdapat peningkatan pengetahuan tentang pencegahan DM, dibuktikan oleh sebanyak 27 orang berpengatahuan baik dengan presentasi 96,4% dan hanya 1 orang yang memiliki pengetahuan kurang baik yaitu 3,6%. Hal ini dikarenakan pendengaran peserta kurang baik dan kurang kooperatif saat diberikan penjelasan, kemungkinan karena faktor usia. Namun peserta tersebut sangat antusias terhadap kegiatan yang dilakukan. diabetik. Angiopati ini dibagi menjadi dua yaitu makroangiopati (makrovaskuler) dan mikroangiopati (mikrovaskulaer). Penyulit makrovaskuler meliputi ginjal (penyakit ginjal kronik) dan retina mata (terjadi kebutaan). Sedangkan penyulit mikrovaskuler meliputi: pembuluh darah jantung (penyakit jantung koroner), pembuluh darah kaki (luka sukar sembuh), dan pembuluh darah otak (stroke). Keduanya dapat terjadi bersamaan (tidak saling terpisah) dan bukan berrati tidak terjadi sekaligus (Aikins, 2016). Upaya pencegahan DM dapat dilakukan dengan pendekatan kepada penduduk/ populasi/ komunitas. Pendekatan ini berupaya mengubah dan memperbaiki gaya hidup agar menguntungkan, dan mencegah terhadap timbulnya DM atau penyulit kroniknya. Pendekatan ini dilakukan pada pencegahan primer dan sekunder. Upaya pencegahan yang kedua dengan pendekatan perorangan. Pendekatan ini dilakukan pada mereka yang berisiko tinggi mengidap DM dan pada pasien/ penyandang DM, dilakukan pada pencegahan primer, sekunder, dan tersier. Hasil dan Pembahasan Penyulit kronik DM pada dasarnya terjadi pada semua pembuluh darah di tubuh atau disebut angiopati Tindakan yang dilakukan untuk usaha pencegahan primer meliputi penyuluhan mengenai perlunya pengaturan gaya hidup sehat sedini mungkin dengan berpedoman pada mempertahankan pola makan sehari-hari yang sehat dan seimbang, yaitu meningkatkan konsumsi sayuran dan buah, membatasi makanan tinggi lemak dan karbohidrat sederhana, dan mempertahankan berat badan normal/idaman sesuai dengan umur dan tinggi badan. Melakukan kegiatan jasmani yang cukup sesuai dengan umur dan kemampuan dan menghindari obat yang bersifat diabetogenik (Powers et al., 2020). Pada pencegahan sekunder antara lain \| 6 Jurnal Pengabdian Kepada Masyarakat Citra Delima Volume 1, Nomor 1 Tahun 2023 E-ISSN : 3046-7497 Doi: 10.33862/jp.v1i1.369 untuk jangka pendek melakukan deteksi dini penyakit DM dengan kegiatan penyaringan (general check up) glukosa darah terutama pada mereka yang memiliki faktor risiko tinggi. Sedangkan upaya jangka panjang pencegahan sekunder adalah mencegah timbulnya penyulit kronik dalam bentuk mikroangiopati, makroangiopati, dan neuropati. Byrne, H., Caulfield, B., & De Vito, G. (2017). Effects of Self-directed Exercise Programmes on Individuals with Type 2 Diabetes Mellitus: A Systematic Review Evaluating Their Effect on HbA1c and Other Metabolic Outcomes, Physical Characteristics, Cardiorespiratory Fitness and Functional Outcomes. In Sports Medicine (Vol. 47, Issue 4, pp. 717– 733). Springer International Publishing. https://doi.org/10.1007/s40279- 016-0593-y Chung, W. K., Erion, K., Florez, J. C., Hattersley, A. T., Hivert, M. F., Lee, C. G., McCarthy, M. I., Nolan, J. J., Norris, J. M., Pearson, E. R., Philipson, L., McElvaine, A. T., Cefalu, W. T., Rich, S. S., & Franks, P. W. (2020). Precision Medicine in Diabetes: A Consensus Report from the American Diabetes Association (ADA) and the European Association for the Study of Diabetes (EASD). Diabetes Care, 43(7), 1617–1635. https://doi.org/10.2337/dci20-0022 Kesimpulan dan Saran Setelah dilakukan edukasi efektif tentang pencegahan diabetes melitus dapat disimpulkan bahwa terdapat peningkatan pengetahuan tentang DM serta pencegahannya. Fauci, AS., et al., (2018). Harrisons Principles ofInternal Medicine. 17th ed. USA: The McGraw Hill Companies, Inc. 2018; pp. 338. Daftar Pustaka iki Aikins, Ama de-Graft, dan Charles Agyemang. (2016). Introduction: Addressing the Chronic Noncommunicable Disease Burden in Low-and-Middle-income Countries, Ama de-Graft Aikins dan Charles Agyemang, eds. Chronic Non-communicable Disease in Low and Middleincome Countries. London: CAB Publishing. Petersmann, A., Müller-Wieland, D., Müller, U. A., Landgraf, R., Nauck, M., Freckmann, G., Heinemann, L., & Schleicher, E. (2019). Definition, Classification and Diagnosis of Diabetes Mellitus. Experimental and Clinical Endocrinology and Diabetes, 127, S1–S7. https://doi.org/10.1055/a-1018- 9078 Belete, R., Ataro, Z., Abdu, A., & Sheleme, M. (2021). Global prevalence of metabolic syndrome among patients with type I diabetes mellitus: a systematic review and meta-analysis. In Diabetology and Metabolic Syndrome (Vol. 13, Issue 1). BioMed Central Ltd. https://doi.org/10.1186/s13098- 021-00641-8 Powers, M. A., Bardsley, J. K., Cypress, M., Funnell, M. M., Harms, D., Hess- Fischl, A., Hooks, B., Isaacs, D., Mandel, E. D., Maryniuk, M. D., Norton, A., Rinker, J., Siminerio, L. M., & Uelmen, S. (2020). Diabetes Self-management Education and Support in Adults With Type 2 Diabetes: A Consensus Report of \| 7 Jurnal Pengabdian Kepada Masyarakat Citra Delima Volume 1, Nomor 1 Tahun 2023 E-ISSN : 3046-7497 Doi: 10.33862/jp.v1i1.369 the. https://doi.org/10.2337/figshare.12 098571 Si h A G Gh h A & Penyakit Degeneratif Nuha Medika. Wang, H., Li, N., Chivese, T., S H Y L H the. https://doi.org/10.2337/figshare.12 098571 the. https://doi.org/10.2337/figshare.12 098571 Penyakit Degeneratif. Yogyakarta: Nuha Medika. Wang, H., Li, N., Chivese, T., Werfalli, M., Sun, H., Yuen, L., Hoegfeldt, C. A., Elise Powe, C., Immanuel, J., Karuranga, S., Divakar, H., Levitt, Na. A., Li, C., Simmons, D., & Yang, X. (2022). IDF Diabetes Atlas: Estimation of Global and Regional Gestational Diabetes Mellitus Prevalence for 2021 by International Association of Diabetes in Pregnancy Study Group’s Criteria. Diabetes Research and Clinical Practice, 183. https://doi.org/10.1016/j.diabres.20 21.109050 Singh, A. K., Gupta, R., Ghosh, A., & Misra, A. (2020). Diabetes in COVID-19: Prevalence, pathophysiology, prognosis and practical considerations. Diabetes and Metabolic Syndrome: Clinical Research and Reviews, 14(4), 303– 310. https://doi.org/10.1016/j.dsx.2020. 04.004 Soliman, A., Nair, A. P., Al Masalamani, M. S., De Sanctis, V., Abu Khattab, M. A., Alsaud, A. E., Sasi, S., Ali, E. A., Hassan, O. A., Iqbal, F. M., Nashwan, A. J., Fahad, J., El Madhoun, I., & Yassin, M. (2020). Prevalence, clinical manifestations, and biochemical data of type 2 diabetes mellitus versus nondiabetic symptomatic patients with COVID-19: A comparative study. Acta Biomedica, 91(3), 1–9. https://doi.org/10.23750/abm.v91i3 .10214 Wicaksana, A. L., Hertanti, N. S., Ferdiana, A., & Pramono, R. B. (2020). Diabetes management and specific considerations for patients with diabetes during coronavirus diseases pandemic: A scoping review. Diabetes and Metabolic Syndrome: Clinical Research and Reviews, 14(5), 1109–1120. https://doi.org/10.1016/j.dsx.2020. 06.070 Suiraoka, I.P. (2012). Penyakit Degeneratif:Mengenal, Mencegah Dan Mengurangi Faktor Risiko, Suiraoka, I.P. (2012). Penyakit Degeneratif:Mengenal, Mencegah Dan Mengurangi Faktor Risiko, \| 8
https://openalex.org/W4251794381	https://figshare.com/articles/journal_contribution/Self-determination_theory_interventions_versus_usual_care_in_people_with_diabetes_a_protocol_for_a_systematic_review_with_meta-analysis_and_trial_sequential_analysis/20679376/1/files/36893815.pdf	English	null	Self-Determination Theory Interventions Versus Usual Care in People With Diabetes: A Protocol for a Systematic Review With Meta-Analysis and Trial Sequential Analysis	Research Square (Research Square)	2,020	cc-by	11,025	Self-determination theory interventions versus usual care in people with diabetes: a protocol for a systematic review with meta-analysis and trial sequential analysis AUTHOR(S) A S Mathiesen, M J Rothmann, Vibeke Zoffmann, J C Jakobsen, C Gluud, J Lindschou, M Due-Christensen, Bodil Rasmussen, E Marqvorsen, T Thomsen Self-determination theory interventions versus usual care in people with diabetes: a protocol for a systematic review with meta-analysis and trial sequential analysis AUTHOR(S) PROTOCOL Open Access Self-determination theory interventions versus usual care in people with diabetes: a protocol for a systematic review with meta- analysis and trial sequential analysis Anne Sophie Mathiesen1,2, Mette Juel Rothmann2,3,4,5, Vibeke Zoffmann6,7, Janus Christian Jakobsen8,9, Christian Gluud8, Jane Lindschou8, Mette Due-Christensen10,11, Bodil Rasmussen2,5,7, Emilie Marqvorsen6 and Thordis Thomsen12,13 Abstract Background: Existing self-management and behavioural interventions for diabetes vary widely in their content, and their sustained long-term effectiveness is uncertain. Autonomy supporting interventions may be a prerequisite to achieve ‘real life’ patient engagement and more long-term improvement through shared decision-making and collaborative goal setting. Autonomy supportive interventions aim to promote that the person with diabetes’ motivation is autonomous meaning that the person strives for goals they themselves truly believe in and value. This is the goal of self-determination theory and guided self-determination interventions. Self-determination theory has been reviewed but without assessing both benefits and harms and accounting for the risk of random errors using trial sequential analysis. The guided self-determination has not yet been systematically reviewed. The aim of this protocol is to investigate the benefits and harms of self-determination theory-based interventions versus usual care in adults with diabetes. Methods/design: We will conduct the systematic review following The Cochrane Collaboration guidelines. This protocol is reported according to the PRISMA checklist. A comprehensive search will be undertaken in the CENT RAL, MEDLINE, EMBASE, LILACS, PsycINFO, SCI-EXPANDED, CINAHL, SSCI, CPCI-S and CPCI-SSH to identify relevant trials. We will include randomised clinical trials assessing interventions theoretically based on guided self- determination or self-determination theory provided face-to-face or digitally by any healthcare professional in any setting. The primary outcomes will be quality of life, mortality, and serious adverse events. The secondary will be diabetes distress, depressive symptoms and adverse events not considered serious. Exploratory outcomes will be glycated haemoglobin and motivation. Outcomes will be assessed at the end of the intervention and at maximum follow-up. The analyses will be performed using Stata version 16 and trial sequential analysis. Two authors will (Continued on next page) 10536/DRO/DU:30147226 Downloaded from Deakin University’s Figshare repository Deakin University CRICOS Provider Code: 00113B Mathiesen et al. Systematic Reviews (2021) 10:12 https://doi.org/10.1186/s13643-020-01566-5 Correspondence: anne.sophie.mathiesen@regionh.dk Correspondence: anne.sophie.mathiesen@regionh.dk 1Department of Endocrinology, Center for Cancer and Organ Diseases, Copenhagen University Hospital, Rigshospitalet, Blegdamsvej 9, 2100 Copenhagen, Denmark Full list of author information is available at the end of the article Background detection period for unrecognised type 2 diabetes in people with low educational status infers a prolonged time for the complications of diabetes to develop [11]. Diabetes affects 425 million people worldwide, and of these, type 2 diabetes accounts for 90% [1]. The preva- lence and incidence of both type 1 and type 2 diabetes are rapidly increasing [1]. Likewise, the ageing popula- tion contributes to a substantial rise in the number of people with diabetes. Unhealthy lifestyle behaviours and body mass index may explain up to 45% of the social inequality in type 2 diabetes [4]. Within type 1 diabetes, no social gradient exists [9], but still some interventions may potentially broaden the gap between social groups [12–15]. In this systematic review, we plan to investigate a potential dif- ferential impact of included interventions because social inequality is an issue in people with type 2 diabetes. As the interventions under investigation require a level of literacy and language skills they may potentially further increase inequity [14]. Type 1 diabetes is caused by an autoimmune reaction where the body’s immune system attacks the insulin- producing beta cells in the islets of the pancreas gland [1]. Consequently, the body produces little to no insulin [1]. Thus, people with type 1 diabetes depend on mul- tiple daily insulin injections and on managing multiple self-care tasks to maintain a glucose level close to the normal range. The disease can develop at any age and around half of people with type 1 diabetes are diagnosed in adulthood [2]. The human and economic drain from diabetes is ex- cessive; not only because of direct costs but also due to indirect costs like managing complications of diabetes, sick days and early retirement. From a socioeconomic viewpoint, there appears to be convincing incentives to invest in people with diabetes and comorbidities, as the expenses for each individual disease may accumulate, resulting in total costs that exceed the expenses for each individual disease [16]. Due to this effect, the return of investment is often underestimated when intervening in these patients [16]. Type 2 diabetes is caused by a genetic disposition in combination with a sedentary lifestyle and overweight [3]. These risk factors lead to insulin resistance, which initially prompts an increase in insulin production but causes decreased insulin secretion over time. More than 80% are overweight at the time of diagnosis [4]. Background The risk of developing type 2 diabetes increases from 50 to 75% when one or both parents, respectively, have type 2 dia- betes [5]. With age being the single largest risk factor for developing type 2 diabetes, the number of people living with type 2 diabetes and various combinations of comor- bidities is also increasing. Systematic review registration: PROSPERO CRD42020181144 Systematic review registration: PROSPERO CRD42020181144 Keywords: Type 1 diabetes, Type 2 diabetes, Self-determination theory, Guided self-determination method, Quality of life, Diabetes distress, Depressive symptoms, Glycated haemoglobin, Health education tools, Psychosocial support (Continued from previous page) (Continued from previous page) independently screen, extract data from and perform risk of bias assessment of included studies using the Cochrane risk of bias tool. Certainty of the evidence will be assessed by GRADE. independently screen, extract data from and perform risk of bias assessment of included studies using the Cochrane risk of bias tool. Certainty of the evidence will be assessed by GRADE. y y Discussion: Self-determination theory interventions aim to promote a more autonomous patient engagement and are commonly used. It is therefore needed to evaluate the benefit and harms according to existing trials. Systematic review registration: PROSPERO CRD42020181144 Discussion: Self-determination theory interventions aim to promote a more autonomous patient engagement and are commonly used. It is therefore needed to evaluate the benefit and harms according to existing trials. © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 13 Mathiesen et al. Systematic Reviews (2021) 10:12 (2021) 10:12 Mathiesen et al. Systematic Reviews Description of the interventions Diabetes self-management defined as leading a healthy lifestyle, measuring blood glucose, taking medicine and receiving support from healthcare professionals and own social network is considered crucial to reduce develop- ment of complications of diabetes and increase quality of life [17]. Complications to diabetes include macrovascular com- plications such as ischaemic stroke or coronary heart disease [6]. Microvascular complications comprise retin- opathy, neuropathy, and nephropathy [7]. Whilst tight glycaemic control is associated with reduced micro- vascular complications [6, 7], this association is less clear in relation to macrovascular complications [8]. Due to the early onset of type 1 diabetes, complications of type 1 diabetes are more susceptible to develop [9]. In people with type 2 diabetes, macrovascular complications are associated with age, male sex, obesity, dyslipidaemia, and smoking [10]. Up to one-third of people with type 2 dia- betes have developed one or more complications of type 2 diabetes at the time of diagnosis [10]. A longer pre- The diabetes management plan for people with dia- betes should consider the person’s age, cognitive abil- ities, literacy, social and financial situation, cultural factors, diabetes complications and comorbidities, health priorities and preferences of care [18]. Autonomy sup- porting interventions may lead to satisfactory diabetes self-management because lifestyle changes are easier to accomplish and maintain if the person’s motivation is autonomous meaning that they strive for goals they themselves truly believe in and value [19]. Thus, Page 3 of 13 Mathiesen et al. Systematic Reviews (2021) 10:12 Page 3 of 13 Mathiesen et al. Systematic Reviews important to change, thus becoming able to express their thoughts in communication with the healthcare professionals. Guided self-determination intervention is likely to improve clinical outcomes through the follow- ing pathways [32, 37]: increased perceived autonomy support from the healthcare professionals, a higher fre- quency of self-monitored blood glucose, increased per- ceived competence in managing diabetes, decreased diabetes-related distress and ultimately improved gly- caemic control [29–32]. autonomy interventions may be a prerequisite to achieve ‘real life’ engagement and more long-term improvement of the person with diabetes through shared decision- making and collaborative goal setting. Within autonomy interventions, intrinsic motivation is a key concept as it is connected to success to reach and sustain treatment targets [20, 21]. Existing diabetes self-management interventions and interventions focusing on behaviour change vary widely in their content, and their sustained long-term effective- ness is uncertain [22, 23]. Description of the interventions Reviews suggest that interven- tions that are grounded in behavioural change theory are more effective than those that are not [21, 24]. Educa- tional interventions, psychological interventions and health educational tools are based on different theoret- ical grounding, training, clinical skills, and are delivered by different specialists in diverse settings. Educational in- terventions use didactic and enhanced learning methods to improve self-management of diabetes by reducing identifiable gaps in knowledge [25]. Psychological ther- apies use the therapeutic alliance between patient and therapist, in which the patient’s problems are under- stood in terms of emotions, cognitions, and behaviours [26], yet, psychological interventions have not proven ef- fective on glycated haemoglobin (HbA1c) in people with type 1 [27] or type 2 diabetes [22]. Health educational tools that aim at translating person-centred care into practice and finding ways to enhance intrinsic motiv- ation may lead to greater long-term behaviour change than tools solely relying on external motivation [28]. This is the goal of the guided self-determination method [29–32] and self-determination theory [33]. According to the self-determination theory, when the three basic psychological needs: competence, autonomy and relatedness are satisfied; this leads to enhanced au- tonomous motivation and mental health [33, 38, 39]. Self-determination theory proposes a continuum for the internalisation of motivation, whereby people become more autonomous (or self-determined) to engage in be- haviours over time. The pathways of mechanisms are built on a theoretical model [33], which argues, first, that social-contextual events (e.g. feedback, communications, rewards) that conduce towards feelings of competence during action can enhance intrinsic motivation for that action. Accordingly, optimal challenges, tailored feed- back, and lack of demeaning evaluations are hypothe- sised to facilitate intrinsic motivation and thereby promote autonomy [33]. Why is it important to do this systematic review We conducted preliminary literature searches in PubMed and the Cochrane Database of Systematic Re- views using the search terms: diabetes, theory-based in- terventions, self-determination theory, guided self- determination and person-centred in different combina- tions. From these searches, we identified three reviews including studies that provided self-determination theory for behaviour change in the health domain [20, 21, 40]. None of the reviews were systematic reviews. The three reviews investigating self-determination theory [20, 21, 40], included trials investigating the effect of the self- determination theory-based intervention assessing at least one self-determination theory variable. Description of the interventions All three re- views [20, 21, 40] included trials from different popula- tions, primarily with healthy people and multiple experimental designs. Nevertheless, whether an improve- ment can be attributed to the intervention, it can only be established in randomised clinical trials. An overview of the characteristics of the three reviews is shown in Table 1. None of the reviews had a registered or pub- lished protocol, none were based on unrestricted searches and bias risk was only assessed in two reviews, using domains adopted from the Cochrane Handbook for conducting and reporting systematic reviews and meta-analyses [52, 53]. None of the reviews controlled the risks of random errors using trial sequential analysis, We aim to assess the effects of the autonomy- supportive inventions: guided self-determination interven- tion by Zoffmann [29–31] and self-determination theory by Deci and Ryan [33]. Guided self-determination is an empowerment-based method recognised as a life-skills ap- proach clinically applicable in patient-provider relation- ships. The guided self-determination method was empirically developed on the basis of grounded theory [29–32] and formal theories including self-determination theory and life skills theory. The self-determination theory is based on comprehensive empirical research [33]. For transparency, the guided self-determination method and self-determination theory are described according to the model of analyses based on the criteria proposed by Gra- ham et al. [34–36] shown in Supplementary Material Table 1. Methods This protocol has been registered in the PROSPERO database ID nr. CRD42020181144 on 5 July 2020, and is reported according to the Preferred Reporting Items for Systematic reviews and Meta-analysis Protocols (PRIS MA-P) 2015 statement [56] (Checklist as Additional file 1). Regarding guided-self-determination, we found no sys- tematic reviews but we identified four randomised clin- ical trials providing guided self-determination for people with diabetes [32, 50, 54, 55]. Of these, one randomised clinical trial investigating the effect of guided self- determination in young adults with type 1 diabetes iden- tified a larger effect on HbA1c and diabetes distress at follow-up compared to immediately after the interven- tion in women, but not in men [54]. Due to the limita- tions of the existing reviews outlined in Table 1 and the fact that guided self-determination intervention method had not yet been systematically reviewed, we find it justi- fied to conduct a systematic review including trial se- quential analysis and GRADE for assessing the potential of a long-term effect, specifically targeting people with diabetes. Criteria for considering studies for this review Types of studies We will include randomised clinical trials and cluster randomised trials irrespective of publication status, re- ported outcomes, publication date, publication type, and language conducted in any setting for assessment of benefits and harms. We will not include quasi- randomised studies or observational studies [52]. How the interventions might work In the guided self-determination approach, the person with diabetes has a primary role preparing for consulta- tions at home, filling in reflection sheets. This means that the person needs to clarify and prioritise what is Mathiesen et al. Systematic Reviews (2021) 10:12 Mathiesen et al. Systematic Reviews (2021) 10:12 Page 4 of 13 Page 4 of 13 Objective the outcomes reported were limited to specific self- determination theory constructs, and none of the re- views assessed adverse effects. In the review of Ntou- manis et al. [20], the authors concluded that changes in autonomous motivation and perceptions of need support were associated with small positive changes in health behaviours at the end of the intervention, but small to medium changes at follow-up, which may indicate the potential of a sustained behaviour change [20]. the outcomes reported were limited to specific self- determination theory constructs, and none of the re- views assessed adverse effects. In the review of Ntou- manis et al. [20], the authors concluded that changes in autonomous motivation and perceptions of need support were associated with small positive changes in health behaviours at the end of the intervention, but small to medium changes at follow-up, which may indicate the potential of a sustained behaviour change [20]. The objective is to investigate the benefits and harms of guided self-determination and self-determination theory interventions versus usual care in people with diabetes. Assessment time points The primary assessment time points for all outcomes will be closest to the end of intervention. We will sec- ondly assess all outcomes at maximum follow-up. Types of interventions Experimental interventions theoretically based on guided self-determination or self-determination theory provided face-to-face or digitally by any healthcare professional in any setting. The trials must refer to either guided self- determination or self-determination theory as their pri- mary theoretical framework. Additionally, the trials must use the reflection sheets and the communication forms that are basic to the guided self-determination method. Search methods for identification of studies Electronic searches Quality of life (continuous data) measured by either any validated diabetes-specific questionnaire such as the diabetes quality of life [58] or any validated gen- eric outcome measure such as the WHO-5 ques- tionnaire [59]. Quality of life (continuous data) measured by either any validated diabetes-specific questionnaire such as the diabetes quality of life [58] or any validated gen- eric outcome measure such as the WHO-5 ques- tionnaire [59]. We will search Cochrane Central Register of Controlled Trials (CENTRAL), Medical Literature Analysis and Re- trieval System Online (MEDLINE), Excerpta Medical database (EMBASE), Latin American and Caribbean Health Sciences Literature (LILACS), PsycINFO, Science Citation Index Expanded (SCI-EXPANDED), Cumulative Index to Nursing and Allied Health Literature (CINA HL), Social Sciences Citation Index (SSCI), Conference Proceedings Citation Index—Science (CPCI-S), and Conference Proceedings Citation Index—Social Science & Humanities (CPCI-SSH) to identify relevant trials. We will search all databases from their inception to the present. For a detailed example of the search strategy ap- plied in Medline, see Additional file 2. The search strat- egy for the remaining databases will be given at the review stage. All-cause mortality (dichotomous data) y Proportion of participants with one or more serious adverse events (dichotomous data), defined as any untoward medical occurrence that resulted in death, was life-threatening, required hospitalisation or prolonging of existing hospitalisation and resulted in persistent or significant disability or jeopardised the patient [60]. If the trialists do not use the ICH-GCP definition, we will include the data if the trialists use the term “serious adverse event.” If the trialists do not use the ICH-GCP definition nor use the term serious adverse event, then we will also include the data, if the event clearly fulfils the ICH-GCP defin- ition for a serious adverse event. Proportion of participants with one or more serious adverse events (dichotomous data), defined as any untoward medical occurrence that resulted in death, was life-threatening, required hospitalisation or prolonging of existing hospitalisation and resulted in persistent or significant disability or jeopardised the patient [60]. If the trialists do not use the ICH-GCP definition, we will include the data if the trialists use the term “serious adverse event.” If the trialists do not use the ICH-GCP definition nor use the term serious adverse event, then we will also include the data, if the event clearly fulfils the ICH-GCP defin- ition for a serious adverse event. Searching other resources We will contact the authors of included studies asking for unpublished randomised trials. The reference lists of relevant publications and systematic reviews will be checked for any unidentified randomised trials. Further, we will search for ongoing trials on the following: Explorative outcomes HbA1c (continuous data). HbA1c (continuous data). Motivation measured by the 21-items Treatment Self-Regulation Questionnaire (TSRQ) consists of three subscales measuring the patient’s reasons for taking diabetes medication, checking glucoses, fol- lowing diet and exercising regularly: (I) autonomous, originating from the self, (II) controlled, pressured or coerced by intrapsychic or interpersonal forces or (III) a-motivated, without intention to change and often feeling unable to change (continuous data). Types of participants People with a diagnosis of type 1 diabetes or type 2 dia- betes as defined by trialists. The participants should be described as adolescents or adults by trialists. Trials Table 1 Self-determination theory reviews Ng et al. [40] Gillison et al. [21] Ntoumanis et al. [20] Designs included 184 independent datasets, primarily non- experimental design 74 studies including a control group (59 of randomised clinical trials (RCTs) or cluster RCTs) 73 independent datasets, 58 RCTs (20 of these were cluster RCTs) Number of trials including people with diabetes Seven trials [32, 41–46] One trial [47] Six trials [45, 47–51] Registered in PROSPERO No No No Protocol published No No No Restricted searches PsycINFO, PsycARTICLES and PubMed, citation searches (ISI web of knowledge) Web of Science, PsychINFO, PubMed, Cochrane Database, DARE, Biomed Central, Sociological abstracts, ProQuest PsycINFO, PsycARTICLES and PubMed/Medline Assessment of bias risk Not performed A modified version of the Cochrane risk of bias tool (random group allocation, treatment allocation concealment, groups similar at baseline, blinded outcome assessor, intention-to- treat analyses, risk of bias) A modified version of the Cochrane risk of bias tool (random group allocation, group allocation concealment, blinded outcome assessor, handling of missing data, selective reporting, other bias) Assessment of random errors, using trial sequential analysis No No No Outcomes Specific self- determination theory constructs Specific self-determination theory constructs Specific self-determination theory constructs Assessment of adverse effects No No No No Specific self-determination theory constructs No No Page 5 of 13 Mathiesen et al. Systematic Reviews (2021) 10:12 Mathiesen et al. Systematic Reviews Health Questionnaire (PHQ-9) [63] or the hospital anxiety and depression scale [64]. including participants described as children will be excluded. Health Questionnaire (PHQ-9) [63] or the hospital anxiety and depression scale [64]. Proportion of participants with at least one adverse event (dichotomous data) not considered serious [60]. Control group interventions Control interventions may be ‘no intervention’, wait list or standard care as defined by trialists (e.g. stand- ard healthcare provision). We will also accept atten- tion placebo control [57], which is a control intervention that is not related to enhancing auton- omy support but include a similar number of contacts with the interventionists [57]. ClinicalTrials.gov (www.clinicaltrials.gov) Google Scholar (https://scholar.google.dk/) The Turning Research into Practice (TRIP) Database (https://www.tripdatabase.com/) Outcomes For each outcome, we will extract the number of ana- lysed participants, the number of participants lost to follow-up/withdrawals/crossover in the experimental and the control group. Control group intervention All potentially eligible trials identified in the literature searches will be imported into the systematic review management programme, Covidence [68]. Two authors (ASM) and a co-author will independently screen po- tentially eligible studies on title and abstract. All full- text studies will be retrieved and independently assessed by the two reviewers. Reasons for exclusion will be reported. Any disagreements will be solved by discussion or by consulting a third author. Trial se- lection will be displayed in a flow diagram according to the PRISMA-P [56]. We will extract the following data: type of control group intervention, treatment duration of control group, num- ber of sessions (or dose), intensity and treatment format provided to the control group. Any reported beneficial and harmful effects of the control intervention will be derived and described. Co-intervention characteristics We will extract the following data: type of co- intervention, treatment duration of co-intervention, number of sessions (or dose) and treatment format. Intervention group We will extract the following data: type of intervention, treatment duration of intervention group, number of sessions (or dose), intensity and treatment format pro- vided to the intervention group. Data extraction and management Two authors will independently extract data from in- cluded trials. Disagreements will be solved by discussion or by consulting a third author. We will assess duplicate publications and companion papers of a trial together, to evaluate all data simultaneously (to maximise correct Data collection and analysis We will conduct the review following The Cochrane Collaboration guidelines [52]. The analyses will be per- formed by the use of Review manager 5.3 [65]. The ana- lyses will be performed using trial sequential analysis [66] and Stata version 16 [67]. Education and training of the interventionists The intervention could be provided by any intervention- ist. Data on who is providing the intervention will be ex- tracted. The training of the interventionists providing the method will be reported. Assessment of fidelity will be reported. Participants characteristics and diagnosis The World Health Organisation (WHO) International We will extract the following data: number of rando- mised participants in each intervention group, adherence to intervention, age range (mean or median), sex ratio, type of diabetes, diabetes treatment, number of comor- bidities (complications of diabetes/other comorbidities) and socioeconomic status/educational level. Clinical Trials Registry Platform (ICTRP) search portal (http://apps.who.int/trialsearch/) Cochrane Database of Systematic Reviews http://www.evidencebasedpsychotherapies.org/index. php?id=25 Clinical Trials Registry Platform (ICTRP) search portal (http://apps.who.int/trialsearch/) Cochrane Database of Systematic Reviews http://www.evidencebasedpsychotherapies.org/index. php?id=25 Additionally, we will hand search conference abstracts from diabetes conferences for relevant trials and con- sider relevant-for-the-review unpublished and grey lit- erature trials if we identify these. Reference lists of reviews and meta-analyses retrieved from the searches will also be screened. The latest search will be performed in June 2020 supplemented with ongoing alerts from the databases when new studies within the search matrix are published. We will end inclusion in June 2020. Trial characteristics Trial characteristics The following data will be extracted: trial design (paral- lel, factorial, or crossover), number of intervention groups, lengths of follow-up, risk of bias components, and inclusion and exclusion criteria. China Food and Drug Administration (CFDA) (http://eng.cfda.gov.cn/WS03/CL0755/) Medicines and Healthcare Products Regulatory Agency (https://www.gov.uk/government/ organisations/medicines-and-healthcare-products- regulatoryagency) Secondary outcomes Diabetes distress (continuous data) measured with any validated instruments such as the diabetes distress scale or the problem areas in diabetes scale [61, 62]. ClinicalTrials.gov (www.clinicaltrials.gov) Google Scholar (https://scholar.google.dk/) The Turning Research into Practice (TRIP) Database (https://www.tripdatabase.com/) ClinicalTrials.gov (www.clinicaltrials.gov) Google Scholar (https://scholar.google.dk/) The Turning Research into Practice (TRIP) Database (https://www.tripdatabase.com/) Depressive symptoms (continuous data) measured with any validated instruments such as the Patient Page 6 of 13 Mathiesen et al. Systematic Reviews (2021) 10:12 Mathiesen et al. Systematic Reviews European Medicines Agency (EMA) (http://www. ema.europa.eu/ema/) data extraction and bias assessment). We will contact all trial authors to specify any missing data, which may not be reported sufficiently or at all in the publication. US Food and Drug Administration (FDA) (www.fda. gov) Incomplete outcome data p Unclear risk of bias: study authors did not describe the method used to conceal the allocation, so intervention allocations may have been foreseen before, or during, enrolment Low risk of bias: missing data were unlikely to make treatment effects depart from plausible values. The study used adequate methods, such as multiple imputation, to handle missing data or had < 5% missing data. High risk of bias: it is likely that investigators who assigned participants knew the allocation sequence g Unclear risk of bias: information was insufficient to assess whether missing data in combination with the method used to handle missing data were likely to induce bias on the results. Risk of bias assessment Risk of bias in included RCTs will be assessed based on the domains described below [52, 69–79]. This assess- ment will be done separately for each outcome and com- parison and will then be considered in relation to overall reliability of the evidence. This will be done in pairs by two independent review authors (ASM and co-author). Blinding of participants and personnel Low risk of bias: either of the following: no blinding or incomplete blinding, but review authors judged that the outcome was unlikely to have been influenced by lack of blinding or blinding of participants and key study personnel ensured, and it was unlikely that the blinding could have been broken High risk of bias: results were likely to be biassed due to missing data. Allocation concealment Allocation concealment High risk of bias: either of the following: no blinding of outcome assessment, and the outcome measurement was likely to be influenced by lack of blinding; or blinding of outcome assessment, but likely that the blinding could have been broken, and the outcome measurement was likely to be influenced by lack of blinding. Low risk of bias: participant allocations could not have been foreseen in advance of, or during, enrolment. A central and independent randomisation unit controlled the allocation. Investigators were unaware of the allocation sequence (e.g. if the allocation sequence was hidden in sequentially numbered, opaque and sealed envelopes) Notes Funding of the trial and notable conflicts of interest of the trial authors will be reported. Unusual reporting of outcome data will be noted in the ‘Characteristics of Page 7 of 13 Mathiesen et al. Systematic Reviews (2021) 10:12 Mathiesen et al. Systematic Reviews Unclear risk of bias: either of the following: insufficient information to permit judgement of ‘low risk’ or ‘high risk’; or the trial did not address this outcome included studies’ table. Two reviewers (ASM and co- author) will independently extract and transfer data into Review Manager [65]. Disagreements will be solved through discussion or by consulting a third author. High risk of bias: either of the following: no blinding or incomplete blinding, and the outcome was likely to have been influenced by lack of blinding; or blinding of key study participants and personnel attempted, but likely that the blinding could have been broken, and the outcome was likely to have been influenced by lack of blinding Random sequence generation Low risk of bias: either of the following: no blinding of outcome assessment, but review authors judged that the outcome measurement was not likely to be influenced by lack of blinding (we will consider self- reported questionnaires more prone to be affected by lack of blinded outcome assessor and hba1c less likely to be affected by lack of blinded outcome as- sessor) or blinding of outcome assessment ensured, and unlikely that the blinding could have been broken. Low risk of bias: study authors performed sequence generation using computer random number generation or a random numbers table. Drawing lots, tossing a coin, shuffling cards, and throwing dice were adequate if an independent person not otherwise involved in the study performed them. y p Unclear risk of bias: study authors did not specify the method of sequence generation. High risk of bias: sequence generation method was not random or quasi-randomised. Such studies will be excluded for the assessment of benefits. Unclear risk of bias: either of the following: insufficient information to permit judgement of ‘low risk’ or ‘high risk’; or the trial did not address this outcome. Differences between protocol and the review Assessment of reporting bias If ten or more trials are included, we will use a funnel plot to visually assess reporting bias [82]. We are aware of the limitations of a funnel plot (i.e. a funnel plot as- sesses bias due to the small sample size). From this in- formation, we assess possible reporting bias. For dichotomous outcomes, we will test asymmetry with the Harbord test [83] if I2 is less than 0.1 and with the Rücker test if I2 is more than 0.1. For continuous out- comes, we will use the regression asymmetry test [84] and the adjusted rank correlation [85]. Any deviations between the published protocol and the review will be reported in the ‘Differences between the protocol and the review’ section of the systematic review. Continuous outcomes We will calculate the mean differences (MDs) and con- sider calculating the standardised mean difference (SMD) with 95% CI for continuous outcomes. We will also calculate trial sequential analysis-adjusted Cis. We will include randomised clinical trials only. If a trial use a crossover design, only data from the first period will be included [52, 86]. We will include cluster- randomised trials after adjusting the original sample size of the trial to the effective sample size using the intracluster correlation coefficient from the ‘design ef- fect’ [52]. Therefore, there will not be any unit of ana- lyses issues. Selective outcome reporting Low risk of bias: a protocol is published, or a trial has been registered in a trial register (e.g. Mathiesen et al. Systematic Reviews Mathiesen et al. Systematic Reviews (2021) 10:12 Page 8 of 13 clinicaltrials.gov) before or at the time the trial is begun, and the outcome called for in the protocol or trial registration is reported on. and for assessment of risk of bias). Secondly, we will in- vestigate the effects of missing data in sensitivity ana- lyses, specified below. Unclear risk of bias: study authors did not report all pre-defined outcomes fully, or it was unclear whether study authors recorded data on these outcomes. Measures of treatment effect Dichotomous outcomes We will calculate risk ratios (RRs) with 95% confidence interval (CI) for dichotomous outcomes and the trial se- quential analysis-adjusted CIs. Dichotomous outcomes We will not impute missing values for any outcomes in our primary analysis. In our sensitivity analyses, we will impute data. High risk of bias: study authors did not report one or more pre-defined outcomes. High risk of bias: study authors did not report one or more pre-defined outcomes. Other bias Continuous outcomes ll l We will primarily analyse scores assessed at single time points. If only changes from baseline Low risk of bias: the trial appeared free of other factors that could have put it at risk of bias scores are reported, we will analyse the results to- gether with follow-up scores [52]. If standard deviations (SDs) are not reported, we will calculate the SDs using trial data or Review Manager [65]. We will not use intention-to-treat data if the original paper did not con- tain such data. We will impute missing values for the continuous outcomes in the sensitivity analyses. Unclear risk of bias: the trial may or may not have been free of other factors that could have put it at risk of bias High risk of bias: other factors in the trial could have put it at risk of bias We will judge a trial to be at low overall risk of bias if assessed as having low risk of bias in all of the above do- mains. We will judge a trial to be at high overall risk of bias if assessed as having unclear or high risk of bias in one or more of the above domains. Assessment of heterogeneity To assess any sign of heterogeneity, we will investi- gate forest plots visually. Secondly, we will assess the presence of statistical heterogeneity by chi2 test (threshold P < 0.10) and measure the quantities of heterogeneity by the I2 statistic [80, 81]. Further, we will investigate possible heterogeneity through sub- group analyses and may ultimately decide that a meta-analysis is not warranted [52]. We will assess the domains ‘blinding of outcome as- sessment’, ‘incomplete outcome data’, and ‘selective out- come reporting’ for each outcome result. Thus, we can assess the bias risk for each outcome assessed in addition to each trial. Our primary conclusion will be based on the results of our primary outcome results with overall low risk of bias. Data synthesis Meta-analysis difference of the observed SD/2, an alpha of 1.4% for all outcomes, a beta of 10%, and the observed diversity as suggested by the trials in the meta-analysis. y We will undertake the meta-analysis according to the recommendations stated in the Cochrane Handbook for Systematic Reviews of Interventions [52], Keus et al. [87] and the eight-step assessment suggested by Jakobsen et al. [88]. Both random-effects meta-analyses [89] and fixed-effect meta-analyses [90] will be used for assessing a potential intervention effect. We will use the more conservative point estimate of the two [88], which is the estimate closest to zero effect. If the two estimates are similar, we will use the estimate with the widest CI. We assess a total of three primary, three secondary out- comes and two explorative outcomes, and we will there- fore consider a P value of 0.014 or less as the threshold for statistical significance [88]. We will use the eight- step procedure to assess if the thresholds for significance are crossed [88]. As stated, our primary conclusion will be based on results with a low risk of bias [88]. Where multiple trial arms are reported in a trial, we will include the arm(s) relevant for the objective of the review. If two comparisons are combined in the same meta-analysis, we will halve the control group to avoid double- counting [52]. Trials with a factorial design will be in- cluded. If a quantitative synthesis is not appropriate due to a small number of trials eligible for inclusion or con- siderable heterogeneity, the results will be reported narratively. Subgroup analysis and integration of heterogeneity Subgroup analysis The subgroup analyses are moderator analyses that explore effect heterogeneity. Results from subgroup ana- lyses should therefore be interpreted cautiously. The fol- lowing exploratory subgroup analyses will be conducted when analysing the primary outcomes (Quality of life, mortality and serious adverse events): Intervention 6. Trials investigating self-determination theory com- pared to trials investigating guided self- determination method. 7. Trials with an experimental intervention above and below the mean difference in intervention length. 8. Trials investigating individual interventions compared to trials investigating group interventions. For dichotomous outcomes, we will estimate the re- quired information size based on the observed propor- tion of patients with an outcome in the control group (the cumulative proportion of patients with an event in the control groups relative to all patients in the control groups), a relative risk reduction of 20%, an alpha of 1.4% for all our outcomes, a beta of 10%, and the ob- served diversity as suggested by the trials in the meta- analysis. For continuous outcomes, we will use the ob- served SD in the trial sequential analysis, a mean 9. Type of control intervention (no intervention, standard care or placebo attention control) Trial sequential analysis 3. Number of co-morbidities defined as complications of diabetes or other chronic conditions [99]. Traditional meta-analysis runs the risk of random errors due to sparse data and repetitive testing of accumulating data when updating reviews. We wish to control the risks of type I errors and type II errors and thereby the risk of potential false-positive findings of meta-analyses [91]. We will therefore perform trial sequential analysis on the outcomes, in order to calculate the required in- formation size (that is, the number of participants needed in a meta-analysis to detect or reject a certain intervention effect) and the cumulative Z curve’s breach of relevant trial sequential monitoring boundaries [66, 91–98]. More information on trial sequential analysis can be found in the trial sequential analysis manual [98] and at http://www.ctu.dk/tsa/. 4. Effect in men compared to women. 5. Effect in adolescents (13 to 18 years) compared to adults >18 years. Dealing with missing data As specified above, all trial authors will be contacted to obtain any relevant missing data (i.e. for data extraction Page 9 of 13 Mathiesen et al. Systematic Reviews (2021) 10:12 Mathiesen et al. Systematic Reviews Participants 1. Type of diabetes. Trials including participants with type 1 compared to trials including participants with type 2. 2. Socioeconomic status defined according to trialists, e.g. educational level. Low socioeconomic status is an umbrella term including educational level and household income which will be used as a proxy for equity if information on educational level is not reported [52]. An equity-focused review must present both relative and absolute differences be- tween groups [15]. We will investigate trials includ- ing participants with low socioeconomic status compared to trials including participants with high socioeconomic status. y y Dichotomous data This is a protocol for a systematic review that aims at synthesising the evidence for the beneficial and harmful effects of guided self-determination or self- determination theory interventions for people with dia- betes and comorbidity in any healthcare setting assessed in randomised clinical trials. To assess the potential impact of the missing data for di- chotomous outcomes, we will perform the two following sensitivity analyses on both the primary and secondary outcomes. 1. ‘Best-worst-case’ scenario: we will assume that all participants lost to follow-up in the experimental group did not die, had no serious adverse events or non-serious adverse events. We will assume the op- posite for all participants lost to follow-up in the control group The primary outcomes will be quality of life, mortality, serious adverse events, the secondary outcomes diabetes distress, depressive symptoms and non-serious adverse events and the explorative outcomes HbA1c and motivation. This protocol has several strengths. The predefined methodology is based on the Cochrane Handbook for Systematic Reviews of Interventions [52], the eight-step assessment suggested by Jakobsen et al. [88], trial se- quential analysis [66] and GRADE assessment [52, 100]. As such, this protocol considers both risks of random errors and risks of systematic errors. 2. ‘Worst-best-case’ scenario: we will assume that all participants lost to follow-up in the experimental group died, had a serious adverse event or a non- serious adverse event. We will assume the opposite for all participants lost to follow-up in the control group. The limitations of this protocol include the potential for large heterogeneity as a result of including both type 1 and type 2 diabetes and all ways of delivery and inter- ventionists. Therefore, we may ultimately decide that a meta-analysis should be avoided. Moreover, diabetes management always consists of multiple treatment ele- ments [18] and it is likely that different interventions have different effects. Accordingly, if we show a differ- ence between the interventions applying self- determination or self-determination method compared strategies, it will be difficult to conclude what exactly caused the difference in effect. To minimise this limita- tion, ten subgroup analyses are planned, but results of subgroup analyses should always be interpreted with great caution. The large number of comparisons that in- crease the risk of type 1 error must also be noted. This issue will be considered when interpreting the results. y y Dichotomous data A further limitation is our exclusion of quasi-randomised studies and observational studies in the assessments of adverse events. By focusing on randomised clinical trials that are unlikely to identify late and rare ad- verse events, we run the risks of focusing too much on benefits and too little on harms. Therefore, if we identify benefits of the interventions, new systematic reviews focusing on the risks of harms in quasi- randomised studies and observational studies should be conducted to achieve a more balanced evaluation of benefits and harms. We will present the results of both scenarios in our review. Risk of bias 10. Trials at overall high risk of bias compared to trials at overall low risk of bias We will use the formal test for subgroup interactions in Stata [83]. Page 10 of 13 Mathiesen et al. Systematic Reviews (2021) 10:12 Continuous data When analysing the robustness of a continuous ‘benefi- cial’ outcome, e.g. quality of life, we will impute the group mean plus two SDs of the group mean. When dealing with a ‘harmful outcome’, we will impute the group mean minus two SDs of the group mean [88]. We will present the results of both scenarios in our review. Other post hoc sensitivity analyses might be warranted if unexpected clinical or statistical heterogeneity is identi- fied during the analysis of the review results [76]. q y Summary of findings table We will assess the quality of the evidence on the primary outcomes (Quality of life, mortality, serious adverse events), the secondary outcomes (diabetes distress, de- pressive symptoms and non-serious adverse events) and the explorative outcomes (HbA1c and motivation) using the five GRADE considerations: risk of bias, consistency, imprecision, indirectness and publication bias. We will assess imprecision using trial sequential ana- lysis or if that is not an option, two authors, ASM and TT, will independently evaluate the quality of the evi- dence using GRADEpro GDT [100], recommended by the Cochrane Handbook for Systematic Reviews of inter- ventions [52]. Potential disagreements will be solved by an arbiter (co-author). We will report all decisions to downgrade the quality of studies by footnotes to add to the transparency of the decisions. The findings tables will be based on trials with low risk of bias and the re- sults based on all trials. Regarding the health equity subgroup analyses, it has been reported that only 20% report a differential impact of interventions that may increase the social gradient [15]. Thus, we might find that studies do not report on the proxy equity measures for evaluating a differential impact of the interventions described in this protocol [15]. Page 11 of 13 Page 11 of 13 Mathiesen et al. Systematic Reviews (2021) 10:12 Mathiesen et al. Systematic Reviews (2021) 10:12 Further, we expect that no trials will have blinded treatment interventionists and participants. Even though blinding of participants should be relatively easy, blind- ing of treatment providers is theoretically possible but problematic to carry out, especially in psychosocial inter- ventions [57]. Received: 25 June 2020 Accepted: 17 December 2020 Acknowledgements 8. Kelly TN, et al. Systematic review: glucose control and cardiovascular disease in type 2 diabetes. Ann Intern Med. 2009;151(6):394–403. y 9. Chiang JL, et al. Type 1 diabetes through the life span: a position statement of the American Diabetes Association. Diabetes Care. 2014;37(7):2034–54. 9. Chiang JL, et al. Type 1 diabetes through the life span: a position statement of the American Diabetes Association. Diabetes Care. 2014;37(7):2034–54. Supplementary Information 3. Liu SY, et al. Genetic vulnerability to diabetes and obesity: does education offset the risk? Soc Sci Med. 2015;127:150–8. 3. Liu SY, et al. Genetic vulnerability to diabetes and obesity: does education offset the risk? Soc Sci Med. 2015;127:150–8. y The online version contains supplementary material available at https://doi. org/10.1186/s13643-020-01566-5. 4. Stringhini S, et al. Contribution of modifiable risk factors to social inequalities in type 2 diabetes: prospective Whitehall II cohort study. BMJ. 2012;345:e5452. Additional file 1. PRISMA-P 2015 Checklist. Additional file 2. Search strategy for self-determination theory interven- tions versus usual care in adults with diabetes (Anne Sophie Mathiesen). Additional file 3: Table 1. The translational potential of the guided self-determination and the self-determination theory. Additional file 1. PRISMA-P 2015 Checklist. Additional file 2. Search strategy for self-determination theory interven- tions versus usual care in adults with diabetes (Anne Sophie Mathiesen). Additional file 1. PRISMA-P 2015 Checklist. Additional file 2. Search strategy for self-determination theory interven- tions versus usual care in adults with diabetes (Anne Sophie Mathiesen). 5. Lee HY, et al. Different socioeconomic inequalities exist in terms of the prevention, diagnosis and control of diabetes. Eur J Public Health. 2015; 25(6):961–5. 6. Chatterjee S, Khunti K, Davies MJ. Type 2 diabetes. Lancet. 2017;389(10085): 2239–51. 6. Chatterjee S, Khunti K, Davies MJ. Type 2 diabetes. Lancet. 2017;389(10085): 2239–51. Additional file 3: Table 1. The translational potential of the guided self-determination and the self-determination theory. Additional file 3: Table 1. The translational potential of the guided self-determination and the self-determination theory. 7. Gregg EW, Sattar N, Ali MK. The changing face of diabetes complications. Lancet Diabetes Endocrinol. 2016;4(6):537–47. 7. Gregg EW, Sattar N, Ali MK. The changing face of diabetes complications. Lancet Diabetes Endocrinol. 2016;4(6):537–47. 7. Gregg EW, Sattar N, Ali MK. The changing face of diabetes complications. Lancet Diabetes Endocrinol. 2016;4(6):537–47. 8. Kelly TN, et al. Systematic review: glucose control and cardiovascular disease in type 2 diabetes. Ann Intern Med. 2009;151(6):394–403. 9. Chiang JL, et al. Type 1 diabetes through the life span: a position statement of the American Diabetes Association. Diabetes Care. 2014;37(7):2034–54. Lancet Diabetes Endocrinol. 2016;4(6):537–47. 8. Kelly TN, et al. Systematic review: glucose control and cardiovascular disease in type 2 diabetes. Ann Intern Med. 2009;151(6):394–403. 9. Chiang JL, et al. Type 1 diabetes through the life span: a position statement of the American Diabetes Association. Diabetes Care. 2014;37(7):2034–54. References 1. Karuranga S, Fernandes JR, Huang Y, Malanda B. IDF Diabetes Atlas; 2017. 1. Karuranga S, Fernandes JR, Huang Y, Malanda B. IDF Diabetes Atlas; 2017. 2. Thomas NJ, et al. Frequency and phenotype of type 1 diabetes in the first six decades of life: a cross-sectional, genetically stratified survival analysis from UK Biobank. Lancet Diabetes Endocrinol. 2018;6(2):122–9. 1. Karuranga S, Fernandes JR, Huang Y, Malanda B. IDF Diabetes Atlas; 2017. 2. Thomas NJ, et al. Frequency and phenotype of type 1 diabetes in the first six decades of life: a cross-sectional, genetically stratified survival analysis from UK Biobank. Lancet Diabetes Endocrinol. 2018;6(2):122–9. 2. Thomas NJ, et al. Frequency and phenotype of type 1 diabetes in the first six decades of life: a cross-sectional, genetically stratified survival analysis from UK Biobank. Lancet Diabetes Endocrinol. 2018;6(2):122–9. Funding 12. Dennick K, Bridle C, Sturt J. Written emotional disclosure for adults with type 2 diabetes: a primary care feasibility study. Prim Health Care Res Dev. 2015;16(2):179-87. The study is funded by the Novo Nordisk Foundation, Steno Collaborative grant, grant number: NNF10OC0057720. The Novo Nordisk Foundation has not been involved in the design and will not be involved in the collection of data, analyses, interpretation of data or in writing up the manuscript. 13. Bambra C, et al. Tackling the wider social determinants of health and health inequalities: evidence from systematic reviews. J Epidemiol Community Health. 2010;64(4):284–91. Competing interests 18. Davies MJ, et al. Management of hyperglycaemia in type 2 diabetes, 2018. A consensus report by the American Diabetes Association (ADA) and the European Association for the Study of Diabetes (EASD). Diabetologia. 2018;61:2461–98. The authors declare that they have no competing interests. Consent for publication Not applicable 17. ADA. American Diabetes Association (2018) 6. Glycemic targets: standards of medical care in diabetes—2018. 2018 13. August 2019. Authors’ contributions 10. Gedebjerg A, et al. Prevalence of micro- and macrovascular diabetes complications at time of type 2 diabetes diagnosis and associated clinical characteristics: a cross-sectional baseline study of 6958 patients in the Danish DD2 cohort. J Diabetes Complications. 2018;32(1):34–40. 10. Gedebjerg A, et al. Prevalence of micro- and macrovascular diabetes complications at time of type 2 diabetes diagnosis and associated clinical characteristics: a cross-sectional baseline study of 6958 patients in the Danish DD2 cohort. J Diabetes Complications. 2018;32(1):34–40. ASM wrote up the protocol with regular supervision from MJR, VZ, TT, JL, CG and JCJ. JL, CG and JCJ wrote the ‘Methods’ section. MDC contributed with expert knowledge on type 1 diabetes and psychosocial interventions. BR and EM read and commented on the final manuscript before it was submitted for publication. All authors read and approved the final manuscript. 11. Maindal HT, Skriver MV, et al. Comorbidity and lack of education countered participation in a diabetes prevention self-management programme. J Nurs Healthcare Chronic Ill. 2011;3:293–301. Availability of data and materials 14. Lorenc T, et al. What types of interventions generate inequalities? Evidence from systematic reviews. J Epidemiol Community Health. 2013;67(2):190–3. 14. Lorenc T, et al. What types of interventions generate inequalities? Evidence from systematic reviews. J Epidemiol Community Health. 2013;67(2):190–3. We will publish all data including code in the supplementary material of the systematic review. 15. Welch V, et al. PRISMA-Equity 2012 extension: reporting guidelines for systematic reviews with a focus on health equity. Plos Med. 2012;9(10): e1001333. 15. Welch V, et al. PRISMA-Equity 2012 extension: reporting guidelines for systematic reviews with a focus on health equity. Plos Med. 2012;9(10): e1001333. Ethics approval and consent to participate Not applicable Ethics approval and consent to participate 16. Cortaredona S, Ventelou B. The extra cost of comorbidity: multiple illnesses and the economic burden of non-communicable diseases. BMC Med. 2017; 15(1):216. 16. Cortaredona S, Ventelou B. The extra cost of comorbidity: multiple illnesses and the economic burden of non-communicable diseases. BMC Med. 2017; 15(1):216. Author details 1D f Author details 1Department of Endocrinology, Center for Cancer and Organ Diseases, Copenhagen University Hospital, Rigshospitalet, Blegdamsvej 9, 2100 Copenhagen, Denmark. 2Steno Diabetes Center Odense, Odense University Hospital, Odense, Denmark. 3Department of Endocrinology, Odense University Hospital, Odense, Denmark. 4Department of Clinical Research, University of Southern Denmark, Odense, Denmark. 5School of Nursing and Midwifery, Faculty of Health, Deakin University, Melbourne, Australia. 6The Research Unit Women’s and Children’s Health, The Julie Marie Center, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark. 7Sector of Health Services Research, Department of Public Health, University of Copenhagen, Copenhagen, Denmark. 8Copenhagen Trial Unit, Centre for Clinical Intervention Research, Rigshospitalet, Copenhagen, Denmark. 9Department of Regional Health Research, The Faculty of Health Sciences, University of Southern Denmark, Odense, Denmark. 10Faculty of Nursing, Midwifery and Palliative Care, King’s College London, London, UK. 11Steno Diabetes Center Copenhagen, The Capital Region of Denmark, Copenhagen, Denmark. 12Department of Anaesthesiology, Herlev University Hospital, Herlev, Denmark. 13Department of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark. 19. Sheldon KME. AJ, Goal striving, need satisfaction, and longitudinal well- being: the self-concordance model. J Person Soc Psychol. 1999;76(3):486–97. 20. Ntoumanis N, et al. A meta-analysis of self-determination theory-informed intervention studies in the health domain: effects on motivation, health behavior, physical, and psychological health. Health Psychol Rev. 2020:1–31. 21. Gillison FB, et al. A meta-analysis of techniques to promote motivation for health behaviour change from a self-determination theory perspective. Health Psychol Rev. 2019;13(1):110–30. 22. Chew BH, et al. Psychological interventions for diabetes-related distress in adults with type 2 diabetes mellitus. Cochrane Database Syst Rev. 2017;9: CD011469. 23. Dombrowski SU, et al. Long term maintenance of weight loss with non- surgical interventions in obese adults: systematic review and meta-analyses of randomised controlled trials. BMJ. 2014;348:g2646. 24. Prestwich A, et al. Does theory influence the effectiveness of health behavior interventions? Meta-analysis. Health Psychol. 2014;33(5):465–74. 25. Stenov V, et al. An ethnographic investigation of healthcare providers’ approaches to facilitating person-centredness in group-based diabetes education. Scand J Caring Sci. 2017. Page 12 of 13 Page 12 of 13 Mathiesen et al. Systematic Reviews (2021) 10:12 26. Winkley K, et al. Psychological interventions to improve glycaemic control in patients with type 1 diabetes: systematic review and meta-analysis of randomised controlled trials. BMJ. 2006;333(7558):65. control and life skills: a randomized clinical trial in adolescents with type 1 diabetes. Trials. 2014;15:321. control and life skills: a randomized clinical trial in adolescents with type 1 diabetes. Trials. 2014;15:321. 51. 51. Author details 1D f Williams GC, Lynch M, Glasgow RE. Computer-assisted intervention improves patient-centered diabetes care by increasing autonomy support. Health Psychol. 2007;26(6):728–34. 27. Winkley K, et al. Systematic review and meta-analysis of randomized controlled trials of psychological interventions to improve glycaemic control in children and adults with type 1 diabetes. Lancet. 2004;363:1589-97. 52. Cochrane Handbook for Systematic Reviews of Interventions: Cochrane Book Series. The Cochrane Collaboration, ed. J.P.G. Higgins, S. Vol. 5.1.0. 2011 (updated March 2011). Wiley; The Cochrane Collaboration. 28. Phillips, A.S. and C.A. Guarnaccia, Self-determination theory and motivational interviewing interventions for type 2 diabetes prevention and treatment: a systematic review. J Health Psychol, 2017: p. 1359105317737606. 2011 (updated March 2011). Wiley; The Cochrane Collaboration 53. Higgins JP, et al. The Cochrane Collaboration’s tool for assessing risk of bias in randomised trials. BMJ. 2011;343:d5928. 29. Zoffmann V, Harder I, Kirkevold M. A person-centered communication and reflection model: sharing decision-making in chronic care. Qual Health Res. 2008;18(5):670–85. 54. Zoffmann V, Vistisen D, Due-Christensen M. Flexible guided self- determination intervention for younger adults with poorly controlled type 1 diabetes, decreased HbA1c and psychosocial distress in women but not in men: a real-life RCT. Diabet Med. 2015;32(9):1239–46. 30. Zoffmann V, Kirkevold M. Life versus disease in difficult diabetes care: conflicting perspectives disempower patients and professionals in problem solving. Qual Health Res. 2005;15(6):750–65. 55. Mohn J, et al. The effect of guided self-determination on self-management in persons with type 1 diabetes mellitus and HbA1c >/=64 mmol/mol: a group-based randomised controlled trial. BMJ Open. 2017;7(6):e013295. 31. Zoffmann V, Kirkevold M. Relationships and their potential for change developed in difficult type 1 diabetes. Qual Health Res. 2007;17(5):625–38. 56. Moher D, et al. Preferred reporting items for systematic review and meta- analysis protocols (PRISMA-P) 2015 statement. Syst Rev. 2015;4:1. 32. Zoffmann V, Lauritzen T. Guided self-determination improves life skills with type 1 diabetes and A1C in randomized controlled trial. Patient Educ Couns. 2006;64(1-3):78–86. 57. Popp L, Schneider S. Attention placebo control in randomized controlled trials of psychosocial interventions: theory and practice. Trials. 2015;16:150. 33. Ryan RM, Deci EL. Self-determination theory and the facilitation of intrinsic motivation, social development, and well-being. Am Psychol. 2000;55(1):68–78. 58. Shen W, et al. Development and validation of the Diabetes Quality of Life Clinical Trial Questionnaire. Med Care. 1999;0(4 Suppl Lilly):As45–66. 34. Graham ID, et al. Lost in knowledge translation: time for a map? J Contin Educ Health Prof. 2006;26(1):13–24. 59. Bonsignore M, et al. Author details 1D f Validity of the five-item WHO Well-Being Index (WHO- 5) in an elderly population. Eur Arch Psychiatry Clin Neurosci. 2001; 251(Suppl 2):II27–31. 35. Graham ID, Tetroe J, K.T.T.R. Group. Some theoretical underpinnings of knowledge translation. Acad Emerg Med. 2007;14(11):936–41. 60. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, ICH harmonised guideline: integrated addemdum to ICH E6 (R1): guideline for good clinical practice (ICH-GCP). . 2015. 36. Zoffmann V, et al. Translating person-centered care into practice: a comparative analysis of motivational interviewing, illness-integration support, and guided self-determination. Patient Educ Couns. 2016;99(3): 400–7. 61. Polonsky WH, et al. Assessment of diabetes-related distress. Diabetes Care. 1995;18(6):754–60. 37. Zoffmann, V., A. Prip, and A.W. Christiansen, Dramatic change in a young woman’s perception of her diabetes and remarkable reduction in HbA1c after an individual course of Guided Self-Determination. BMJ Case Rep, 2015. 2015. 62. Fisher L, et al. When is diabetes distress clinically meaningful?: establishing cut points for the Diabetes Distress Scale. Diabetes Care. 2012;35(2):259–64. 63. Kroenke K, Spitzer RL, Williams JB. The PHQ-9: validity of a brief depression severity measure. J Gen Intern Med. 2001;16(9):606–13. 38. Ryan RMD, Edward L. Self-determination theory - basic psychological needs in motivation, development and wellness. Taylor & Francis-Asia Pacific: The Guilford Press; 2017. severity measure. J Gen Intern Med. 2001;16(9):606–13. 64. Bjelland I, et al. The validity of the Hospital Anxiety and Depression S An updated literature review. J Psychosom Res. 2002;52(2):69–77. 64. Bjelland I, et al. The validity of the Hospital Anxiety and Depression S An updated literature review. J Psychosom Res. 2002;52(2):69–77. 39. Bryant J, et al. A systematic review and meta-analysis of the effectiveness of behavioural smoking cessation interventions in selected disadvantaged groups. Addiction. 2011;106(9):1568–85. 65. (RevMan)., R.M. 2014., The Nordic Cochrane Centre, Copenhagen: The Cochrane Collaboration. 66. TSA - Trial Sequential Analysis. [Web page] 2020 27-01-2020]; Available from: http://ctu.dk/tsa/. 40. Ng JY, et al. Self-determination theory applied to health contexts: a meta- analysis. Perspect Psychol Sci. 2012;7(4):325–40. 67. software:, S.S.s. 2019, StataCorp LLC; : http://www.stata.com. 41. Gensichen J, et al. Physician support for diabetes patients and clinical outcomes. BMC Public Health. 2009;9:367. 68. Covidence Systematic Review Software. Melbourne: Veritas Health Innovation. https://www.covidence.org/. 42. Julien E, Senecal C, Guay F. Longitudinal relations among perceived autonomy support from health care practitioners, motivation, coping strategies and dietary compliance in a sample of adults with type 2 diabetes. J Health Psychol. 2009;14(3):457–70. 69. Author details 1D f Schulz KF, et al. Empirical evidence of bias. Dimensions of methodological quality associated with estimates of treatment effects in controlled trials. JAMA. 1995;273(5):408–12. 70. Moher D, et al. Does quality of reports of randomised trials affect estimates of intervention efficacy reported in meta-analyses? Lancet. 1998;352(9128): 609–13. 43. Shigaki C, et al. Motivation and diabetes self-management. Chronic Illn. 2010;6(3):202–14. 44. Sweet SN, et al. Understanding physical activity in adults with type 2 diabetes after completing an exercise intervention trial: a mediation model of self-efficacy and autonomous motivation. Psychol Health Med. 2009;14(4): 419–29. 71. Kjaergard LL, Villumsen J, Gluud C. Reported methodologic quality and discrepancies between large and small randomized trials in meta-analyses. Ann Intern Med. 2001;135(11):982–9. 72. Gluud LL, et al. Correction: reported methodologic quality and discrepancies between large and small randomized trials in meta-analyses. Ann Intern Med. 2008;149(3):219. 45. Williams GC, Freedman ZR, Deci EL. Supporting autonomy to motivate patients with diabetes for glucose control. Diabetes Care. 1998;21(10):1644–51. 46. Williams GC, et al. Reducing the health risks of diabetes: how self- determination theory may help improve medication adherence and quality of life. Diabetes Educ. 2009;35(3):484–92. 73. Wood L, et al. Empirical evidence of bias in treatment effect estimates in controlled trials with different interventions and outcomes: meta- epidemiological study. BMJ. 2008;336(7644):601–5. 47. Juul L, et al. Effectiveness of a training course for general practice nurses in motivation support in type 2 diabetes care: a cluster-randomised trial. Plos One. 2014;9(5):e96683. 74. Savovic J, et al. Influence of reported study design characteristics on intervention effect estimates from randomised controlled trials: combined analysis of meta-epidemiological studies. Health Technol Assess. 2012;16(35):1–82. 48. Nansel TR, et al. Improving dietary quality in youth with type 1 diabetes: randomized clinical trial of a family-based behavioral intervention. Int J Behav Nutr Phys Act. 2015;12:58. 48. Nansel TR, et al. Improving dietary quality in youth with type 1 diabetes: randomized clinical trial of a family-based behavioral intervention. Int J Behav Nutr Phys Act. 2015;12:58. 75. Savovic J, et al. Influence of reported study design characteristics on intervention effect estimates from randomized, controlled trials. Ann Intern Med. 2012;157(6):429–38. 49. Vanroy J, et al. Short- and long-term effects of a need-supportive physical activity intervention among patients with type 2 diabetes mellitus: a randomized controlled pilot trial. PLoS One. 2017;12(4):e0174805. 76. Hrobjartsson A, et al. Bias due to lack of patient blinding in clinical trials. Author details 1D f Methods for combining randomized clinical trials: strengths and limitations. Stat Med. 1987;6(3):341–50. 91. Imberger G, et al. False-positive findings in Cochrane meta-analyses with and without application of trial sequential analysis: an empirical review. BMJ Open. 2016;6(8):e011890. 92. Wetterslev J, et al. Trial sequential analysis may establish when firm evidence is reached in cumulative meta-analysis. J Clin Epidemiol. 2008; 61(1):64–75. 93. Brok J, et al. Trial sequential analysis reveals insufficient information size and potentially false positive results in many meta-analyses. J Clin Epidemiol. 2008;61(8):763–9. 94. Brok J, et al. Apparently conclusive meta-analyses may be inconclusive--trial sequential analysis adjustment of random error risk due to repetitive testing of accumulating data in apparently conclusive neonatal meta-analyses. Int J Epidemiol. 2009;38(1):287–98. 94. Brok J, et al. Apparently conclusive meta-analyses may be inconclusive--trial sequential analysis adjustment of random error risk due to repetitive testing of accumulating data in apparently conclusive neonatal meta-analyses. Int J Epidemiol. 2009;38(1):287–98. 95. Thorlund K, et al. Can trial sequential monitoring boundaries reduce spurious inferences from meta-analyses? Int J Epidemiol. 2009;38(1):276–86. 95. Thorlund K, et al. Can trial sequential monitoring boundaries reduce spurious inferences from meta-analyses? Int J Epidemiol. 2009;38(1):276–86. 96. Wetterslev J, et al. Estimating required information size by quantifying diversity in random-effects model meta-analyses. BMC Med Res Methodol. 2009;9:86. 97. Thorlund K, Anema A, Mills E. Interpreting meta-analysis according to the adequacy of sample size. An example using isoniazid chemoprophylaxis for tuberculosis in purified protein derivative negative HIV-infected individuals. Clin Epidemiol. 2010;2:57–66. 98. Thorlund K, e.a. User manual for trial sequential analysis (TSA). 2011. 98. Thorlund K, e.a. User manual for trial sequential analysis (TSA). 2011. 99. Piette JD, Kerr EA. The impact of comorbid chronic conditions on diabetes care. Diabetes Care. 2006;29(3):725–31. 99. Piette JD, Kerr EA. The impact of comorbid chronic conditions on diabetes care. Diabetes Care. 2006;29(3):725–31. 100. GDT, G., GRADEpro Guideline Development Tool [Software]. 2015, McMaster University, 2015 (developed by Evidence Prime, Inc.). https://gradepro.org/. 100. GDT, G., GRADEpro Guideline Development Tool [Software]. 2015, McMaster University, 2015 (developed by Evidence Prime, Inc.). https://gradepro.org/. Author details 1D f A systematic review of trials randomizing patients to blind and nonblind sub- studies. Int J Epidemiol. 2014;43(4):1272–83. 76. Hrobjartsson A, et al. Bias due to lack of patient blinding in clinical trials. A systematic review of trials randomizing patients to blind and nonblind sub- studies. Int J Epidemiol. 2014;43(4):1272–83. 50. Husted GR, et al. Effect of guided self-determination youth intervention integrated into outpatient visits versus treatment as usual on glycemic 50. Husted GR, et al. Effect of guided self-determination youth intervention integrated into outpatient visits versus treatment as usual on glycemic Page 13 of 13 Mathiesen et al. Systematic Reviews (2021) 10:12 Mathiesen et al. Systematic Reviews (2021) 10:12 77. Hrobjartsson A, et al. Observer bias in randomized clinical trials with measurement scale outcomes: a systematic review of trials with both blinded and nonblinded assessors. CMAJ. 2013;185(4):E201–11. 78. Hrobjartsson A, et al. Observer bias in randomized clinical trials with time- to-event outcomes: systematic review of trials with both blinded and non- blinded outcome assessors. Int J Epidemiol. 2014;43(3):937–48. 79. Savovic J, et al. Association between risk-of-bias assessments and results of randomized trials in Cochrane reviews: the ROBES Meta-Epidemiologic Study. Am J Epidemiol. 2018;187(5):1113–22. y 80. Higgins JP, Thompson SG. Quantifying heterogeneity in a meta-analysis. Stat Med. 2002;21(11):1539–58. 80. Higgins JP, Thompson SG. Quantifying heterogeneity in a meta-analysis. Stat Med. 2002;21(11):1539–58. 81. Higgins JP, et al. Measuring inconsistency in meta-analyses. BMJ. 2003; 327(7414):557–60. 81. Higgins JP, et al. Measuring inconsistency in meta-analyses. BMJ. 2003; 327(7414):557–60. 82. Sterne JA, et al. Recommendations for examining and interpreting funnel plot asymmetry in meta-analyses of randomised controlled trials. BMJ. 2011; 343:d4002. 83. Harbord RM, Egger M, Sterne JA. A modified test for small-study effects in meta-analyses of controlled trials with binary endpoints. Stat Med. 2006; 25(20):3443–57. 84. Egger M, et al. Bias in meta-analysis detected by a simple, graphical test. BMJ. 1997;315(7109):629–34. 85. Begg CB, Mazumdar M. Operating characteristics of a rank correlation test for publication bias. Biometrics. 1994;50(4):1088–101. 86. Elbourne DR, et al. Meta-analyses involving cross-over trials: methodological issues. Int J Epidemiol. 2002;31(1):140–9. 87. Keus F, et al. Evidence at a glance: error matrix approach for overviewing available evidence. BMC Med Res Methodol. 2010;10:90. 88. Jakobsen JC, et al. Thresholds for statistical and clinical significance in systematic reviews with meta-analytic methods. BMC Med Res Methodol. 2014;14:120. 89. DerSimonian R, Laird N. Meta-analysis in clinical trials. Control Clin Trials. 1986;7(3):177–88. 90. Demets DL. 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https://openalex.org/W2997596599	https://jfr.sbu.ac.ir/article_97765_8d497a8aae2c4828ce30cc8da1e36a91.pdf	Chinese	null	Relation of character strengths and adolescence attachment to parent and peer with conflict to father and mother	Faṣlnāmah-i khānavādah/pizhūhī.	2,019	cc-by	8,950	M. Heydari, Ph.D. Department of Psychology, Shahid Beheshti University, Tehran, Iran درﻳﺎﻓﺖ ﻣﻘﺎﻟﻪ: 3/9/ 96 درﻳﺎﻓﺖ ﻧﺴﺨﻪ اﺻﻼح ﺷﺪه : 13 /5/ 98 ﭘﺬﻳﺮش ﻣﻘﺎﻟﻪ : 16 /7/ 98 درﻳﺎﻓﺖ ﻣﻘﺎﻟﻪ: 3/9/ 96 درﻳﺎﻓﺖ ﻧﺴﺨﻪ اﺻﻼح ﺷﺪه : 13 /5/ 98 ﭘﺬﻳﺮش ﻣﻘﺎﻟﻪ : 16 /7/ 98 ﭘﺬﻳﺮش ﻣﻘﺎﻟﻪ : 16 /7/ 98 ﭼﻜﻴﺪه ﭘﮋوﻫﺶ ﺣﺎﺿﺮ ﺑﺎ ﻫﺪف ﭘﻴﺶ ﺑﻴﻨﻲ ﺗﻮاﻧﻤﻨﺪي ﻫﺎي ﻣﻨﺶ ﻧﻮﺟﻮاﻧﺎن و دﻟﺒﺴﺘﮕﻲ ﺑﻪ واﻟﺪﻳﻦ و ﻫﻤﺴﺎﻻن ﺑﺮ اﺳﺎس ﺗﻌﺎرض ﺑﺎ ﭘﺪر و ﻣﺎدر ﺑﺮرﺳﻲ ﺷﺪ . در ﻣﻄﺎﻟﻌﻪ ﻫﻤﺒﺴﺘﮕﻲ ﺟﺎﻣﻌﻪ آﻣﺎري ﭘﮋوﻫﺶ ﺷﺎﻣﻞ داﻧﺶ آﻣﻮزان دﺧﺘﺮ ﭘﺎﻳﻪ ﻧﻬﻢ از ﻣﻨﺎﻃﻖ4 ، 3 و10 ﺷﻬﺮ ﺗﻬﺮان اﺳﺖ .195 داﻧﺶ آﻣﻮز ﭘﺎﻳﻪ ﻧﻬﻢ از ﺳﻪ ﻣﻨﻄﻘﻪ ﺗﻬﺮان ﺑﺎ روش ﻧﻤﻮﻧﻪ ﮔﻴﺮي در دﺳﺘﺮس اﻧﺘﺨﺎب ﺷﺪﻧﺪ و ﺑﻪ ﻧﺴﺨﻪ ﻛﻮﺗﺎه اﺑﺰار ﺗﻮاﻧﻤﻨﺪي ﻫﺎي ﻣﻨﺶ )SECS ( ، ﻧﺴﺨﻪ ﻛﻮﺗﺎه ﺗﻌﺎرض ﻧﻮﺟﻮان ﺑﺎ واﻟﺪﻳﻦ) CBQ ( و ﻣﻘﻴﺎس دﻟﺒﺴﺘﮕﻲ ﺑﻪ واﻟﺪﻳﻦ و ﻫﻤﺴﺎﻻن )IPPA (ﭘﺎﺳﺦ دادﻧﺪ . Z. Khosrojerdi, Ph.D. Candidate Department of Psychology, Shahid Beheshti University, Tehran, Iran 347 Journal of Family Research Vol.15(3): 347-360; 2019 / !"# $% / &'%( ./01 : '52 3 '46 ﭘﻴﺶ ﺑﻴﻨﻲ ﺗﻮاﻧﻤﻨﺪي ﻣ ﻨﺶ ﻧﻮﺟﻮاﻧﺎن و دﻟﺒﺴﺘﮕﻲ ﺑﻪ واﻟﺪﻳﻦ و ﻫﻤﺴﺎﻻن ﺑﺮاﺳﺎس ﺗﻌﺎرض ﺑﺎ ﭘﺪر و ﻣﺎدر 347 Journal of Family Research Vol.15(3): 347-360; 2019 / !"# $% / &'%( ./01 : '52 3 '46 ﭘﻴﺶ ﺑﻴﻨﻲ ﺗﻮاﻧﻤﻨﺪي ﻣ ﻨﺶ ﻧﻮﺟﻮاﻧﺎن و دﻟﺒﺴﺘﮕﻲ ﺑﻪ واﻟﺪﻳﻦ و ﻫﻤﺴﺎﻻن ﺑﺮاﺳﺎس ﺗﻌﺎرض ﺑﺎ ﭘﺪر و ﻣﺎدر 347 Journal of Family Research Vol.15(3): 347-360; 2019 347 Journal of Family Research Vol.15(3): 347-360; 2019 Abstract In the present study aims to predict the adolescent character strengths and atta- chment to parents and peers based on con- flicts with parents. The statistical popula- tion of the study consisted of ninth grade female students from the municipal distri- cts 4, 3, and 10 of Tehran. 195 students fr- om the 9th grade were selected from three areas of Tehran using available sampling method and responded to the Short Mea- sure of Character Strengths (SMCS), Con- flict Behavior Questionnaire (CBQ) and the inventory of parent and peer attachm- ent (IPPA). ﻧﻮﻳﺴﻨﺪه ﻣﺴﺌﻮل : ﺗﻬﺮان، داﻧﺸﮕﺎه ﺷﻬﻴﺪ ﺑﻬﺸﺘﻲ، داﻧﺸﻜﺪه روان - ﺷﻨﺎﺳﻲ و ﻋﻠﻮم ﺗﺮﺑﻴﺘﻲ، ﮔﺮوه روان ﺷﻨﺎﺳﻲ ﭘﺴﺖ اﻟﻜﺘﺮوﻧﻴﻜﻲ : com . gmail @ 1400 zahra . khosrojerdi Corresponding author: Department of Psycho- logy, Faculty of Psychology and Education, Univ- ersity of Alzahra, Tehran, Iran. ﻧﺘﺎﻳﺞ ﺗﺤﻠﻴﻞ رﮔﺮﺳﻴﻮن ﭼﻨﺪﮔﺎﻧﻪ ﻧﺸﺎن داد ﻛﻪ ﭘﻴﺶ ﺑﻴﻨﻲ ،ﺗﻌﺎﻣﻞ ﻋﺪاﻟﺖ و ﺗﻌﺎرض ﺑﺎ ﭘﺪر ﺑﺎ دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﭘﺪر، ﻋﺪاﻟﺖ اﻋﺘﺪال و ﺗﻌﺎﻟﻲ، ﻣﺜﺒﺖ و ﻣﻌﻨﺎدار ﺑﻮدﻧﺪ) 001 /0 P< .( ﻫﻢ ﭼﻨﻴﻦ راﺑﻄﻪ ﺗﻌﺎﻣﻞ ﺗﻌﺎﻟﻲ و ﺗﻌﺎرض ﺑﺎ ﻣﺎدر ﺑﺎ دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﻣﺎدر، ﺧﺮد، اﻧﺴﺎﻧﻴﺖ، ﻋﺪاﻟﺖ و ﺗﻌﺎﻟﻲ، ﻣﺜﺒﺖ و ﻣﻌﻨﺎدار ﺑﻮدﻧﺪ) 001 /0 P< .( ﺑﻨﺎﺑﺮاﻳﻦ ﻣﻲ ﺗﻮان ﻧﺘﻴﺠﻪ ﮔﺮﻓﺖ ﺗﻮاﻧﻤﻨﺪي ﻫﺎي ﻣﻨﺶ و دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﭘﺪر و ﻣﺎدر در ﻛﺎﻫﺶ ﺗﻌﺎرض ﻧﻮﺟﻮان ﺑﺎ واﻟﺪﻳﻦ اﺛﺮات ﻣﺜﺒﺖ دارد و ﺗﻌﺎﻣﻞ ﺗﻮاﻧﻤﻨﺪي ﻫﺎي ﻣﻨﺶ ﺑﺎ ﺗﻌﺎرض زﻣﻴﻨﻪ ﺳﺎز ﺣﻞ ﺗﻌﺎرض اﺳﺖ . The results of multiple regression analysis showed that prediction of the interaction of justice and conflict with father with attachment to father, justice, temperance and transcendence were positive and significant (P>0/001). Also relationship interaction of transcendence and conflict with the mother with attachment to the mother, wisdom, love, justice and transce- ndence was positive and significant (P>0/001). Therefore, it can be concluded that character strengths and parenting atta- chment have a positive effect on reducing adolescent conflict with parents. And the interaction of character strengths with co- nflict is the basis of conflict resolution. ﻛﻠﻴﺪواژه ﻫﺎ: ﺗﻌﺎرض، ﺗﻮاﻧﻤﻨﺪي ﻣﻨﺶ، دﻟﺒﺴﺘﮕ ﻲ واﻟﺪﻳﻦ و ﻫﻤﺴﺎﻻن ﻛﻠﻴﺪواژه ﻫﺎ: ﺗﻌﺎرض، ﺗﻮاﻧﻤﻨﺪي ﻣﻨﺶ، دﻟﺒﺴﺘﮕ ﻲ واﻟﺪﻳﻦ و ﻫﻤﺴﺎﻻن Keywords: Conflict, Character Strengths, Attachment to Parent and Peer. ﻣﻘﺪﻣﻪ ﭼﺎرﭼﻮب ﻣﻔﻬﻮﻣﻲ ﻏﺎﻟﺐ ﻣﻄﺎﻟﻌﺎت ﻋﻠﻤﻲ در ﺗﺤﻮل ﻧﻮﺟﻮاﻧﻲ ﻣﺒﺘﻨﻲ ﺑﺮ ﺗﻮﻓﺎن و ﻓﺸﺎر 1 ﺑﻮده اﺳﺖ . ﻧﻘﺺ دﻳﺪﮔﺎه ﻫﺎي ﻣﺮﺗﺒﻂ ﺑﺎ وﻳﮋﮔﻲ ﻫﺎي اﻳﻦ دوره ﺑﺮ ﻣﺪل ﺗﻘﻠﻴﻞ ﺑﻴﻮﻟﻮژﻳﻜﻲ ﺑﻠﻮغ ﻣﺒﺘﻨﻲ ﺑﻮده ﻛﻪ ﻧﻮﺟﻮان را ﺑﺎ ﭼﺎﻟﺶ ﻫﺎ و ﻣﺸﻜﻼت ﺗﻮﺻﻴﻒ ﻣﻲ ﻛﺮده ﺳﺖ ا . در اواﻳﻞ ﺳﺎل 2000 ﺳﻠﻴﮕﻤﻦ، ﭘﻴﺘﺮﺳﻮن و ﭘﺎرك2 ﺑﺮ اﻳﻦ ﺑﺎور ﺑﻮدﻧﺪ ﻛﻪ ﺑﺎﻳﺪ در ﺗﺤﻘﻴﻘﺎت ﻣﺒﺘﻨﻲ ﺑﺮ رﺷﺪ ﻧﻮﺟﻮاﻧﻲ ﺗﻮﺟﻪ ﺑﻴﺸﺘﺮي ﺑﻪ ﻣﻄﺎﻟﻌﻪ ﻋﻠﻤﻲ ﺗﻮاﻧﻤﻨﺪي ﻫﺎي ﻣﻨﺶ 3 و ﻓﻀﺎﺋﻞ ﺷﻮد ﻛﻪ آن ﻫﺎ را ﺑﺮاي ﺷﻜﻮﻓﺎﻳﻲ ﺗﻮاﻧﺎﺗﺮ ﻣﻲ ﻛﻨﺪ ﻧﺎﮔﻲ )4 ، 2015 .( ﺗﻮاﻧﻤﻨﺪي ﻫﺎي ﻣﻨﺶ در ﻣﻨﺶ ﺧﻮب5 اﺛﺮﮔﺬار اﺳ . ﺖ ﻣﻨﺶ6 ﺑﻪ ﺟﻨﺒﻪ ﻫﺎﻳﻲ از ﺻﻔﺎت ﻣﺜﺒﺖ ﺷﺨﺼﻴﺘﻲ و رﻓﺘﺎري اﺷﺎره دارد ﻛﻪ از ﻧﻈﺮ اﺧﻼﻗﻲ ارزﺷﻤﻨﺪﻧﺪ . ﻣﻨﺶ ﺧﻮب ﻫﺴﺘﻪ روان ﺷﻨﺎﺳﻲ ﺗﺤﻮل ﻣﺜﺒﺖ اﺳﺖ ) ﮔﻴﻠﻤﻦ، ﻫﺎﺑﻨﺮ، اﺳﻜﺎت، ﻓﺮﻻﻧﻚ 7 ، 2009 .( روان ﺷﻨﺎﺳﻲ ﻣﺜﺒﺖ و ﺗﺤﻮل ﻣﺜﺒﺖ ﻧﻮﺟﻮاﻧﻲ ﺑﺮ ﻋﻮاﻣﻠﻲ ﻣﺘﻤﺮﻛﺰ اﺳﺖ ﻛﻪ ﻛﻮدك و ﻧﻮﺟﻮان را رﺷﺪ و ﭘﺮورش دﻫ )ﺪ رﻳﭻ، واﺑﺮ، ﭘﺎرك، ﭘﻴﺘﺮﺳﻮن 8 ، 2014 .( ﺗﻮاﻧﻤﻨﺪي ﻫﺎي ﻣﻨﺶ ﺑﻪ ﻋﻨﻮان ﺻﻔﺎت ﻣﺜﺒﺖ در ﺗﻔﻜﺮ، اﺣﺴﺎس و رﻓﺘﺎر ﺗﻌﺮﻳﻒ ﻣﻲ ﺷﻮد ) ﻟﻮن ﺗﺎﭘﻮﻟﻮ، ﺗﺮﻳﻠﻴﻮ ، 2012 .( ﺑﺮﺧﻲ از ﺗﻮاﻧﻤﻨﺪي ﻫﺎي ﻣﻨﺶ ﻣﺎﻧﻨﺪ ﻣﻬﺮﺑﺎﻧﻲ، اﻣﻴﺪ، ﻫﻮش ﻫﻴﺠﺎﻧﻲ و ﺧﻮدﻛﻨﺘﺮﻟﻲ ﻣﻲ ﺗﻮاﻧﺪ اﺛﺮات ﻣﻨﻔﻲ اﺳﺘﺮس و ﺗﺮوﻣﺎ را ﭘﻴﺸﮕﻴﺮي ﻛﺮد ه و ﺑﺎ ﻛﺎﻫﺶ ﻣﺸﻜﻼﺗﻲ ﻣﺎﻧﻨﺪ ﺳﻮء ﻣﺼﺮف اﻟﻜﻞ و ﺳﻴﮕﺎر، ﺧﺸﻮﻧﺖ، اﻓﺴﺮدﮔﻲ و اﻓﻜﺎر ﺧﻮدﻛﺸﻲ ﻫﻤﺮاه اﺳﺖ ) ﮔﻴﻠﻤﻦ ﻫﺎﺑﻨﺮ، اﺳﻜﺎت، ﻓﺮﻻﻧﻚ ، 2009 .( از ﺗﺤﺮﻛﺎت ﻣﺒﺘﻜﺮاﻧﻪ ﺟﻨﺒﺶ روان ﺷﻨﺎﺳﻲ ﻣﺜﺒﺖ ﮔﺮاﻳﻲ، ﮔﺴﺘﺮش ﻃﺒﻘﻪ ﺑﻨﺪي ﺗﻮاﻧﻤﻨﺪي ﻫﺎﺳﺖ ﺑﻪ ﻛﻪ درك ﻫﺮﭼﻪ دﻗﻴﻖ ﺗﺮ ﻓﻀﺎﻳﻞ آدﻣﻲ ﺑﻪ ﻃﻮر ﺧﺎص 349 / ﭘﻴﺶ ﺑﻴﻨﻲ ﺗﻮاﻧﻤﻨﺪي ﻣﻨﺶ ﻧﻮﺟﻮاﻧﺎن و دﻟﺒﺴﺘﮕﻲ ﺑﻪ واﻟﺪﻳﻦ و/... ﻣﻲ ﭘﺮداز .د ﻃﺒﻘﻪ ﺑﻨﺪي ﭘﻴﺸﻨﻬﺎدي ﭘﻴﺘﺮﺳﻮن و ﺳﻠﻴﮕﻤﻦ )2004 ( در ﺑﻴﺴﺖ و ﭼﻬﺎر ﺗﻮاﻧﻤﻨﺪي ﻣﻨﺸﻲ و ﺷﺶ ﻓﻀﻴﻠﺖ ﺧﺮد10 ، ﺷﺠﺎﻋﺖ ، اﻧﺴﺎﻧﻴﺖ ، ﻋﺪاﻟﺖ ، ﻣﻴﺎﻧﻪ روي و ﺗﻌﺎﻟﻲ ﮔﺮوه ﺑﻨﺪي ﺷﺪ . اﻳﻦ ﻓﻀﺎﻳﻞ ﺷﺶ ﮔﺎﻧﻪ در ﺑﺮرﺳﻲ زﻣﻴﻨﻪ ﻫﺎي ﺗﺎرﻳﺨﻲ آﺷﻜﺎر ﺷﺪﻧﺪ . ﺗﻮاﻧﻤﻨﺪي ﻫﺎي ﻣﻨﺶ ﻋﻮاﻣﻞ روان ﺷﻨﺎﺧﺘﻲ، ﻓﺮاﻳﻨ ﺪ و ﺳﺎزﻛﺎرﻫﺎﻳﻲ اﺳﺖ ﻛﻪ ﻓﻀﺎﺋﻞ را ﺗﻌﺮﻳﻒ ﻣﻲ ﻛﻨﻨﺪ . ﻣﻘﺪﻣﻪ آن ﻫﺎ روش ﻫﺎي ﻗﺎﺑﻞ ﺗﺸﺨﻴﺺ ﺑﺮاي ﻧﻤﺎﻳﺶ ﻳﻚ ﻳﺎ ﭼﻨﺪ ﻣﻮرد از ﻓﻀﺎﺋﻞ ﻫﺴﺘﻨﺪ ) ﭘﻴﺘﺮﺳﻮن، ﺳﻴﮕﻠﻤﻦ، 2004 .( ﺑﺎ ﺗﻮﺟﻪ ﺑﻪ اﻫﻤﻴﺖ ﺗﻮاﻧﻤﻨﺪي ﻫﺎي ﻣﻨﺶ در رﺷﺪ دوره ﻧﻮﺟﻮاﻧﻲ، اﻓﺰاﻳﺶ ﺗﻮﺟﻪ در زﻣﻴﻨﻪ آﻣﻮزش و ﭘﮋوﻫﺶ از ﺳﻮي ﺳﻴﺎﺳﺖ ﮔﺬاران، ﺑﺮﻧﺎﻣﻪ ر ﻳﺰان و ﻣﺘﻮﻟﻴﺎن اﻣﻮر ﺗﻌﻠﻴﻤﻲ و ﺗﺮﺑﻴﺘﻲ ﺿﺮوري ﺑﻪ ﻧﻈﺮ ﻣﻲ رﺳﺪ ) ﻧﺎﮔﻲ، 2015 .( در دوران ﻧﻮﺟﻮاﻧﻲ ﻣﺎﻫﻴﺖ رواﺑﻂ ﺧﺎﻧﻮادﮔﻲ ﻛﻴﻔﻴﺖ ﺧﺎﺻﻲ ﭘﻴﺪا ﻣﻲ ﻛﻨﺪ ﻛﻪ ﺑﺎ ﻇﻬﻮر اﻓﺰاﻳﺶ ﺗﻌﺎرض ﺑﻴﻦ ﻧﻮﺟﻮان و واﻟﺪﻳﻦ ﻫﻤﺮاه اﺳﺖ . ﺗﺤﻘﻴﻘﺎت ﻧﺸﺎن ﻣﻲ دﻫﺪ ﺗﻌﺎرض واﻟﺪ ـ ﻧﻮﺟﻮان وﻗﺎﻳﻊ روزﻣﺮه زﻧﺪﮔﻲ ﺧﺎﻧﻮادﮔﻲ را در ﺑﺮ ﻣﻲ ﮔﻴﺮد . اﮔﺮﭼﻪ اﻳﻦ روﻧﺪ اﺟﺘﻨﺎب ﻧﺎﭘﺬﻳﺮ اﺳﺖ، وﻟﻲ ﻣﻲ ﺗﻮاﻧﺪ ﺑﺮاي واﻟﺪﻳﻦ و ﻧﻮﺟﻮاﻧﺎن ﻧﮕﺮاﻧﻲ اﻳﺠﺎد ﻛﻨﺪ . ﺗﺮدﻳﺪي ﻧﻴﺴﺖ ﺑﺎ اﻓﺰاﻳﺶ ﺳﻦ و ﻧﻴﺎز ﻧﻮﺟﻮان ﺑﻪ اﺳﺘﻘﻼل ﺑﻴﺸﺘﺮ، واﻟﺪﻳﻦ ﺑﻪ ﻓﺮزﻧﺪان ﻧﻮﺟﻮان ﺧﻮد آزادي ﺑﻴﺸﺘﺮي اﻋﻄﺎ ﻣﻲ ﻛﻨﻨﺪ . ﺑﺪﻳﻦ ﺗﺮﺗﻴﺐ ﻣﻲ ﺗﻮان ﮔﻔﺖ ﻛﺸﻤﻜﺶ ﻣﻴﺎن وا ﻟﺪﻳﻦ و ﻧﻮﺟﻮاﻧﺎن ﺑﻪ ﻣﻨﺰﻟﻪ ﺧﺪﺷﻪ دار ﺷﺪن راﺑﻄﻪ ﻋﺎﻃﻔﻲ ﻳﺎ ﺟﺪاﻳﻲ ﺑﻴﻦ آﻧﺎن ﻧﻴﺴﺖ، ﺑﻠﻜﻪ ﺗﺎ ﺣﺪودي ﻻزﻣﻪ ﺗﺤﻮل و ﻛﺴﺐ اﺳﺘﻘﻼل اﺳﺖ ) اﺣﺪي16 ، 1380 .( ﺗﻌﺎرض واﻟﺪ ـ ﻧﻮﺟﻮان ﭘﻴﺶ ﺑﻴﻨﻲ ﻛﻨﻨﺪه ﺑﻬﺰﻳﺴﺘﻲ ﻧﻮﺟﻮان و واﻟﺪﻳﻦ اﺳﺖ ) ﮔﺮﻳﺴﺘﻴﺰ، ﺑﺮﻛﻮﻳﻚ، ﺟﺎﻛﻮدﻳﻚ، ﮔﻮردﻧﺎ 17 ، 2011 ( و ﻓﻀﻴﻠﺖ ﻫﺎ ﻣﻲ ﺗﻮا ﻧﻨﺪ راﻫﮕﺸﺎﻳﻲ ﺑﺮاي ﺣﻞ ﺗﻌﺎرض ﺑﺎﺷﻨﺪ . ﻧﻈﺮﻳﻪ ﺣﻞ ﺗﻌﺎرض ﺑﻴﺎن ﻣﻲ ﻛﻨﺪ ﻓﻀﻴﻠﺖ ﻫﺎ ﺑﻪ ﻋﻨﻮان ﭘﺎﻳﻪ ﻫﺎي اﻣﻦ اﺧﻼﻗﻲ ﻣﻲ ﺗﻮاﻧﻨﺪ ﺑﻪ ﺣﻞ ﻣﺸﻜﻼت ﻛﻤﻚ ﻛﻨﻨﺪ ) اﺳﻜﺎت ﻛﺎري18 ، 2007 .( ﻣﺎﻧﻨﺪ ﻋﺪاﻟﺖ ﻛﻪ ﺑﻪ ﻋﻨﻮان ﻓﻀﻴﻠﺘﻲ ﻣﺎ را در رواﺑﻂ ﺑﻴﻦ ﻓﺮدي ﺗﻮاﻧﺎ ﻣﻲ ﺳﺎزد و ﭘﺮورش دﻫﻨﺪه ي ﻣﻬﺎرت ﻫﺎي ﺳﺎزﻧﺪه در ﺣﻞ ﺗﻌﺎرض اﺳﺖ . ﺗﻌﺎﻟﻲ ﻓﻀﻴﻠﺘﻲ ﻛﻪ ﻣﺸﻜﻼت را ﺑﺮاي ﻣﺎ ﻛﻢ رﻧﮓ ﺗﺮ ﺟﻠﻮه ﻣﻲ دﻫﺪ ) ﻟﻴﻜﻮﻧﺎ19 ، 2013 .( در اﻳﻦ راﺳﺘﺎ ﺗﻮاﻧﻤﻨﺪي ﻫﺎ و ﻓﻀﺎﺋﻞ اﺛﺮات ﺳﻮدﻣﻨﺪي ﺑﺮ ﺗﻌﺎرض واﻟﺪ ـ ﻧﻮﺟﻮان دارﻧﺪ . در ﺣﻮزه ﺗﻌﺎرض ﻣﻌﻤﻮﻻً ﺑﺮﺧﻲ از ﺗﻮاﻧﻤﻨﺪي ﻫﺎ ﻣﺎﻧﻨﺪ اﻣﻴﺪ، ﺧﻮش ﺑﻴﻨﻲ ﻣﻮرد ﺑﺮرﺳﻲ ﻗﺮار ﮔﺮﻓﺘﻪ اﺳﺖ، در ﺣﺎﻟﻲ ﻛﻪ اﻳﻦ ﺗﻮاﻧﻤﻨﺪي ﻫﺎ و ﻓﻀﺎﺋﻞ درﺑﺮﮔﻴﺮﻧﺪه ﻇﺮﻓﻴﺖ ﻣﺎ در ﻛﻤﻚ ﺑﻪ ﺧﻮدﻣﺎن و دﻳﮕﺮان اﺳﺖ و زﻣﻴﻨﻪ ﺳﺎز اﺛﺮات ﻣﺜﺒﺖ ﻫﺴﺘﻨﺪ ﺑﻪ وﻳﮋه زﻣﺎﻧﻲ ﻛﻪ از ﭼﻨﻴﻦ ﻇﺮﻓﻴﺘﻲ اﺳﺘﻔﺎده ﻣﻲ ﺷﻮد، ﻣﻲ ﺗﻮان ﺑﺮ ﻧﻘﺶ ﺗﻌﺎﻣﻠﻲ آن ﻫﺎ ﻧﻴﺰ ﺗﺄﻛﻴﺪ ﻛﺮد . ﻣﻘﺪﻣﻪ دﻟﺒﺴﺘﮕﻲ اﻳﻤﻦ ﭘﻴﺶ ﺑﻴﻨﻲ ﻛﻨﻨﺪه ﻣﻬﺎرت ﻫﺎي ﻣﻘﺎﺑﻠﻪ اي ﺳﺎزﻧﺪه اﺳﺖ و ﻧﻮﺟﻮان از ارﺗﺒﺎط ﻣﺜﺒﺖ ﺑﺎ واﻟﺪﻳﻦ ﺑﻴﺸﺘﺮ ﻟﺬت ﻣﻲ ﺑﺮد و ﺗﺠﺮﺑﻪ ﺗﻌﺎرض ﻛﻤﺘﺮي ﺑﺎ ﺧﺎﻧﻮاده و ﻫﻤﺴﺎﻻن ﺧﻮد دارد ) ﻫﻤﺎن ﻣﻨﺒﻊ (. ﺷﻮد و ﭼﻨﻴﻦ ارﺗﺒﺎﻃﻲ ﻧﻴﺰ ﺑﻪ ﻧﻮﺑﻪ ﺧﻮد ﭘﻴﺶ ﺑﻴﻨﻲ ﻛﻨﻨﺪه ﺗﺤﻮل ﺑﻌﺪي ﻓﺮد در ﺳﺎزﮔﺎري ﻳﺎ ﻧﺎﺳﺎزﮔﺎري در زﻧﺪﮔﻲ ﻓﺮدي و اﺟﺘﻤﺎﻋﻲ اوﺳﺖ . ﺑﻪ ﻋﻨﻮان ﻣﺜﺎل دﻟﺒﺴﺘﮕﻲ اﻳﻤﻦ، اﻧﻌﻄﺎف ﭘﺬﻳﺮي ﺿﺮوري را در اﺧﺘﻴﺎر ﻧﻮﺟﻮان ﻗﺮار ﻣﻲ دﻫﺪ ﺗﺎ ﺑﺘﻮاﻧﺪ در ﻣﺪﻳﺮﻳﺖ اﺳﺘﺮس و رواﺑﻂ ﺗﻄﺒﻴﻘﻲ و ﻣﺜﺒﺖ ﺑﺎ دﻳﮕﺮان ﺑﻪ ﺧﻮﺑﻲ ﻋﻤﻞ ﻛﻨﺪ . در ﻣﻘﺎﺑﻞ دﻟﺒﺴﺘﮕﻲ ﻧﺎاﻳﻤﻦ ﻣﺸﻜﻼت اﺟﺘﻤﺎﻋﻲ و ﻫﻴﺠﺎﻧﻲ را اﻓﺰاﻳﺶ ﻣﻲ دﻫﺪ و در ﺧﻮدﺗﻨﻈﻴﻤﻲ و رﻓﺘﺎرﻫﺎي ﺿﺪاﺟﺘﻤﺎﻋﻲ در ﻛﻮدﻛﻲ و ﻧﻮﺟﻮاﻧﻲ ﻣﺸﻜﻼﺗﻲ ﺑﻪ ﻫﻤﺮاه دارد ) ﻧﺎﮔﻲ، 2015 ( . ﻣﻮرﺗﻲ و ﭘﻠﺪ24 ) 2004 ( ﺑﺮاﻳﻦ ﺑﺎور ﻫﺴﺘﻨﺪ ﻛﻪ دﻟﺒﺴﺘﮕﻲ اﻳﻤﻦ در ﻧﻮﺟﻮان ﻣﺎﻧﻨﺪ اواﻳﻞ ﻛﻮدﻛﻲ ﻫﻤﺎن ﺗﺄﺛﻴﺮ ﺑﺮ رﺷﺪ را دارد . دﻟﺒﺴﺘﮕﻲ ﻳﻚ ﭘﺎﻳﻪ اﻣﻦ ﻛﺎوش و رﺷﺪﺷﻨﺎﺧﺘﻲ، اﺟﺘﻤﺎﻋﻲ و ﺷﺎﻳﺴﺘﮕﻲ ﻫﻴﺠﺎﻧﻲ را ﭘﺮورش ﻣﻲ دﻫﺪ . ﻣﻄﺎﻟﻌﺎت ﻧﺸﺎن ﻣﻲ دﻫﺪ ﻧﻮﺟﻮاﻧﺎﻧﻲ ﻛﻪ دﻟﺒﺴﺘﮕﻲ ﻧﺎاﻳﻤﻦ دارﻧﺪ، اﺣﺘﻤﺎﻻً ﺑﻴﺸﺘﺮ درﮔﻴﺮ ﻣﺼﺮف ﻣﻮاد و رﻓﺘﺎرﻫﺎي ﭘﺮﺧﻄﺮ ﺟﻨﺴﻲ ﻣﻲ ﺷﻮﻧﺪ ) ﻣﻮرﺗﻲ، 2004 .( ،دﻟﺒﺴﺘﮕﻲ اﻳﻤﻦ در دﺧﺘﺮان ﺑﺎ ﻣﺸﻜﻼت ﻛﻤﺘﺮي ﻣﺎﻧﻨﺪ اﺿﻄﺮاب، اﻓﺴﺮدﮔﻲ ﺑﻲ ﺗﻮﺟﻬﻲ، ﻣﺸﻜﻼت اﻓﻜﺎر، اﺧﺘﻼل ﺳﻠﻮك، ﺑﺰﻫﻜﺎري و ﭘﺮﺧﺎﺷﮕﺮي ﻫﻤﺮاه اﺳﺖ . دﻟﺒﺴﺘﮕﻲ اﻳﻤﻦ ﭘﻴﺶ ﺑﻴﻨﻲ ﻛﻨﻨﺪه ﻣﻬﺎرت ﻫﺎي ﻣﻘﺎﺑﻠﻪ اي ﺳﺎزﻧﺪه اﺳﺖ و ﻧﻮﺟﻮان از ارﺗﺒﺎط ﻣﺜﺒﺖ ﺑﺎ واﻟﺪﻳﻦ ﺑﻴﺸﺘﺮ ﻟﺬت ﻣﻲ ﺑﺮد و ﺗﺠﺮﺑﻪ ﺗﻌﺎرض ﻛﻤﺘﺮي ﺑﺎ ﺧﺎﻧﻮاده و ﻫﻤﺴﺎﻻن ﺧﻮد دارد ) ﻫﻤﺎن ﻣﻨﺒﻊ (. در اﻳﻦ ﻣﻴﺎن ﻳﻜﻲ از رواﺑﻂ و ﭘﻴﻮﻧﺪﻫﺎي ﻋﺎﻃﻔﻲ ﻛﻪ در ﺳﺎل ﻫﺎي ﻧﻮﺟﻮاﻧﻲ و ﺟﻮاﻧﻲ ﺑﺮاي اﻓﺮاد اﻫﻤﻴﺖ ﺑﻴﺸﺘﺮي ﭘﻴﺪا ﻣﻲ ﻛﻨﺪ، راﺑﻄﻪ و ﭘﻴﻮﻧﺪﻫﺎي ﻋﺎﻃﻔﻲ ﺑﺎ دوﺳﺘﺎ ن و ﻫﻤﺴﺎﻻن اﺳﺖ . در واﻗﻊ اﻓﺮاد ﺑﺎ ورود ﺑﻪ دﻧﻴﺎي ﻧﻮﺟﻮاﻧﻲ، ﺟﻮاﻧﻲ و ﺑﺮﺧﻮرداري از اﺳﺘﻘﻼل، ﺑﻪ ﻫﻤﺴﺎﻻن ﺧﻮد روي ﻣﻲ آورﻧﺪ و ﺑﺮﻗﺮاري رواﺑﻂ ﺣﻤﺎﻳﺘﻲ و ﻋﺎﻃﻔﻲ ﺑﺎ آﻧﺎن داراي اﻫﻤﻴﺖ اﺳﺖ و زﻣﺎن ﺑﻴﺸﺘﺮي را ﺑﺎ دوﺳﺘﺎن و ﻫﻤﺴﺎﻻن ﺧﻮد ﺳﭙﺮي ﻣﻲ ﻛﻨﻨﺪ . ﻣﻘﺪا ر زﻣﺎﻧﻲ ﻛﻪ ﻧﻮﺟﻮان ﺑﺎ ﻫﻤﺴﺎﻻ ن ﺳﭙﺮي ﻣﻲ ﻛﻨﺪ، زﻣﺎن ﺑﻮدن ﺑﺎ واﻟﺪﻳﻦ و اﻋﻀﺎي ﺧﺎﻧﻮاده را ﻣﻲ ﺗﻮاﻧﺪ ﺗﺤﺖ اﻟﺸﻌﺎع ﻗﺮار دﻫﺪ ) ﻓﻴﻮﻟﻴﻜﻨﻲ، اﮔﻠﺰ 25 ، 1993 .( ﻫﻤﭽﻨﻴﻦ ﭼﮕﻮﻧﮕﻲ ﺗﻌﺎﻣﻼت ﻧﻮﺟﻮاﻧﺎن ﺑﺎ ﻫﻤﺴﺎﻻن ﻣﺘﺎﺛﺮ از رواﺑﻂ او ﺑﺎ ﺧﺎﻧﻮاده اﺳﺖ . ﻛﻮدك و ﻧﻮﺟﻮان ﻣﺪﻳﺮﻳﺖ ﺗﻌﺎرض را از ﻃﺮﻳﻖ ﺗﺠﺮﺑﻪ ﺑﺎ ﻣﺮاﻗﺐ اﺻﻠﻲ ﻳﺎد ﻣﻲ ﮔﻴﺮد و در ﺗﻨﻈﻴﻢ ﻫﻴﺠﺎﻧﺎت در ﺳﺎل ﻫﺎي ﺑﻌﺪي زﻧﺪﮔﻲ ﺗﺎﺛﻴﺮﮔﺬار اﺳﺖ . ﻣﻘﺪﻣﻪ ﺗﻮاﻧﻤﻨﺪي ﻫﺎي ﻣﻨﺶ ﺑﺎ ﺟﻨﺒﻪ ﻫﺎي ﻣﺜﺒﺖ زﻧﺪﮔﻲ اﻓﺮاد، ﺷﺎ ﻳﺴﺘﮕﻲ اﺟﺘﻤﺎﻋﻲ و ﺗﻌﺎﻣﻼت ﻟﺬت ﺑﺨﺶ ﺑﻴﻦ ﻓﺮدي در ارﺗﺒﺎط اﺳﺖ ) ﺷﻮﺷﺎﻧﻲ، ﺷﺎورﺗﺮ20 ، 2016 .( ﺑﻨﺎﺑﺮاﻳﻦ در دوران ﻧﻮﺟﻮاﻧﻲ ﻋﻼوه ﺑﺮ وﺟﻮد ﺗﻌﺎرض واﻟﺪ ـ ﻧﻮﺟﻮان ﺑﺎﻳﺪ ﺑﻪ دﻟﺒﺴﺘﮕﻲ21 آﻧﺎن ﺑﺎ واﻟﺪﻳﻦ ﺷﺎن ﻧﻴﺰ ﺗﻮﺟﻪ ﻛﺮد . دﻟﺒﺴﺘﮕﻲ ﻣﻲ ﺗﻮاﻧﺪ زﻣﻴﻨﻪ ﺳﺎز ﺗﺤﻮل ﻧﻮﺟﻮاﻧﻲ ﺑﺎﺷﺪ . ﭼﺮا ﻛﻪ ﺗﻔﻜﺮات اﻣ ﻴﺪﺑﺨﺶ در ﻓﻀﺎي ﺧﺎﻧﻮاده رﺷﺪ ﻣﻲ ﻛﻨﺪ . اواﻳﻞ ﻧﻮﺟﻮاﻧﻲ ﻇﺮﻓﻴﺖ ﺷﻨﺎﺧﺘﻲ ﺑﺮاي دروﻧﻲ ﺳﺎزي ﭘﻴﺎﻣﺪﻫﺎي واﻟﺪﻳﻨﻲ اﺳﺖ ﻛﻪ ﻛﻴﻔﻴﺖ زﻧﺪﮔﻲ آﻧﺎن را ارزﻳﺎﺑﻲ ﻣﻲ ﻛﻨﺪ . ﻛﻴﻔﻴﺖ و ﻧﻮع دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﻋﻨﻮان ﻣﻨﺒﻊ ﻛﻠﻴﺪي ﺑﺮاي ﺗﻔﻜﺮات اﻣﻴﺪﺑﺨﺶ اﺳﺖ ﻛﻪ ﺑﺮ ﺑﻬﺰﻳﺴﺘﻲ ﻧﻮﺟﻮاﻧﺎن ﻣﺆﺛﺮ ﻫﺴﺘﻨﺪ ) ﻫﺎﺑﻨﻴﺮ، ﻫﻴﻠﺰ 22 ، 2013 .(دﻟﺒﺴﺘﮕﻲ در اﻳﺠﺎد رواﺑﻂ ﻗﺎﺑﻞ اﻋﺘﻤﺎد ﺑﺎ واﻟﺪ ﻳﻦ و اﺣﺴﺎس ﻧﺰدﻳﻜﻲ، ارزش ﻫﺎي ﻣﺸﺘﺮك و ﻫﻤﺎﻧﻨﺪﺳﺎزي ﺑﺎ دﻧﻴﺎي اﺟﺘﻤﺎﻋﻲ اﺛﺮﮔﺬار اﺳﺖ . ﺑﺮ اﻳﻦ اﺳﺎس ﺗﺠﺮﺑﻪ دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﺑﺮوز رﻓﺘﺎرﻫﺎي ﻣﻌﻴﻨﻲ در ﻛﻮدك، ﻣﺤﻴﻂ و ارﺗﺒﺎﻃﺎت ﺟﺪﻳﺪ ﻣﻨﺠﺮ ﻣﻲ ﺷﻮد ) ﻣﻠﻚ ﭘﻮر ، 2007 .( ﭘﮋوﻫﺶ ﻫﺎي ﻣﺮﺗﺒﻂ ﺑﺎ دﻟﺒﺴ ﺘﮕﻲ ﻧﺸﺎن داده اﻧﺪ ﭼﮕﻮﻧﮕﻲ ﻛﻴﻔﻴﺖ ﻣﺮاﻗﺒﺖ واﻟﺪﻳﻦ ﻣﻲ ﺗﻮاﻧﺪ ﻣﻨﺠﺮ ﺑﻪ ﻳﻚ ارﺗﺒﺎط اﻣﻦ ﻳﺎ ﻧﺎاﻣﻦ 350 / / !"# $% / &'%( / / ﺷﻮد و ﭼﻨﻴﻦ ارﺗﺒﺎﻃﻲ ﻧﻴﺰ ﺑﻪ ﻧﻮﺑﻪ ﺧﻮد ﭘﻴﺶ ﺑﻴﻨﻲ ﻛﻨﻨﺪه ﺗﺤﻮل ﺑﻌﺪي ﻓﺮد در ﺳﺎزﮔﺎري ﻳﺎ ﻧﺎﺳﺎزﮔﺎري در زﻧﺪﮔﻲ ﻓﺮدي و اﺟﺘﻤﺎﻋﻲ اوﺳﺖ . ﺑﻪ ﻋﻨﻮان ﻣﺜﺎل دﻟﺒﺴﺘﮕﻲ اﻳﻤﻦ، اﻧﻌﻄﺎف ﭘﺬﻳﺮي ﺿﺮوري را در اﺧﺘﻴﺎر ﻧﻮﺟﻮان ﻗﺮار ﻣﻲ دﻫﺪ ﺗﺎ ﺑﺘﻮاﻧﺪ در ﻣﺪﻳﺮﻳﺖ اﺳﺘﺮس و رواﺑﻂ ﺗﻄﺒﻴﻘﻲ و ﻣﺜﺒﺖ ﺑﺎ دﻳﮕﺮان ﺑﻪ ﺧﻮﺑﻲ ﻋﻤﻞ ﻛﻨﺪ . در ﻣﻘﺎﺑﻞ دﻟﺒﺴﺘﮕﻲ ﻧﺎاﻳﻤﻦ ﻣﺸﻜﻼت اﺟﺘﻤﺎﻋﻲ و ﻫﻴﺠﺎﻧﻲ را اﻓﺰاﻳﺶ ﻣﻲ دﻫﺪ و در ﺧﻮدﺗﻨﻈﻴﻤﻲ و رﻓﺘﺎرﻫﺎي ﺿﺪاﺟﺘﻤﺎﻋﻲ در ﻛﻮدﻛﻲ و ﻧﻮﺟﻮاﻧﻲ ﻣﺸﻜﻼﺗﻲ ﺑﻪ ﻫﻤﺮاه دارد ) ﻧﺎﮔﻲ، 2015 ( . ﻣﻮرﺗﻲ و ﭘﻠﺪ24 ) 2004 ( ﺑﺮاﻳﻦ ﺑﺎور ﻫﺴﺘﻨﺪ ﻛﻪ دﻟﺒﺴﺘﮕﻲ اﻳﻤﻦ در ﻧﻮﺟﻮان ﻣﺎﻧﻨﺪ اواﻳﻞ ﻛﻮدﻛﻲ ﻫﻤﺎن ﺗﺄﺛﻴﺮ ﺑﺮ رﺷﺪ را دارد . دﻟﺒﺴﺘﮕﻲ ﻳﻚ ﭘﺎﻳﻪ اﻣﻦ ﻛﺎوش و رﺷﺪﺷﻨﺎﺧﺘﻲ، اﺟﺘﻤﺎﻋﻲ و ﺷﺎﻳﺴﺘﮕﻲ ﻫﻴﺠﺎﻧﻲ را ﭘﺮورش ﻣﻲ دﻫﺪ . ﻣﻄﺎﻟﻌﺎت ﻧﺸﺎن ﻣﻲ دﻫﺪ ﻧﻮﺟﻮاﻧﺎﻧﻲ ﻛﻪ دﻟﺒﺴﺘﮕﻲ ﻧﺎاﻳﻤﻦ دارﻧﺪ، اﺣﺘﻤﺎﻻً ﺑﻴﺸﺘﺮ درﮔﻴﺮ ﻣﺼﺮف ﻣﻮاد و رﻓﺘﺎرﻫﺎي ﭘﺮﺧﻄﺮ ﺟﻨﺴﻲ ﻣﻲ ﺷﻮﻧﺪ ) ﻣﻮرﺗﻲ، 2004 .( ،دﻟﺒﺴﺘﮕﻲ اﻳﻤﻦ در دﺧﺘﺮان ﺑﺎ ﻣﺸﻜﻼت ﻛﻤﺘﺮي ﻣﺎﻧﻨﺪ اﺿﻄﺮاب، اﻓﺴﺮدﮔﻲ ﺑﻲ ﺗﻮﺟﻬﻲ، ﻣﺸﻜﻼت اﻓﻜﺎر، اﺧﺘﻼل ﺳﻠﻮك، ﺑﺰﻫﻜﺎري و ﭘﺮﺧﺎﺷﮕﺮي ﻫﻤﺮاه اﺳﺖ . ﻣﻘﺪﻣﻪ ﺗﺤﻘﻴﻘﺎت ﻧﺸﺎن ﻣﻲ دﻫﺪ دﻟﺒﺴﺘﮕﻲ ﻧﺎاﻳﻤﻦ واﻟﺪ ـ ﻛﻮدك ﺑﺎ رﻓﺘﺎر ﻣﺸﻜﻞ آﻓﺮﻳﻦ26 ﻣﺮﺗﺒﻂ اﺳﺖ . دﻳﻜﻴﻦ و اﺳﭙﻨﺮ 27 ﺑﻴﺎن ﻣﻲ ﻛﻨﻨﺪ ﺑﺴﻴﺎري از رﻓﺘﺎرﻫﺎي ﻣﺸﻜﻞ زا و ﺗﻌﺎرﺿﻲ ﺑﻪ دﻧﺒﺎل راﻫﺒﺮدﻫﺎي دﻟﺒﺴﺘﮕﻲ ﺑﺎ ﻣﺮاﻗﺒﺎن ﺑﻮﺟﻮد ﻣﻲ آﻳﺪ اﮔﺮ . واﻟﺪﻳﻦ ﭘﺎﺳﺨﮕﻮ ﺑﺎﺷﻨﺪ و ﺑﻪ ﮔﺮﻣﻲ و ﻗﺎﺑﻞ اﻃﻤﻴﻨﺎن ﺑﺎ ﻓﺮزﻧﺪ ﺧﻮد رﻓﺘﺎر ﻛﻨﻨﺪ، ﻣﻮﺟﺐ رﻓﺘﺎرﻫﺎي ﻣﻨﺎﺳﺐ در آن ﻫﺎ ﻣﻲ ﺷﻮﻧﺪ . و اﮔﺮ در ﻃﻲ رﺷﺪ و ﺗﺤﻮل ﻓﺮد، ﭘﺎﺳﺦ واﻟﺪﻳﻦ ﻣﻮﺟﺐ دﻟﺒﺴﺘﮕﻲ ﻧﺎاﻳﻤﻦ ﺷﻮد، رﻓﺘﺎرﻫﺎي آن ﻫﺎ دوﺳﻮﮔﺮا، ﻏﻴﺮﻗﺎﺑﻞ اﻃﻤﻴﻨﺎن و ﺗﻌﺎرﺿﻲ اﺳﺖ و اﻓﺰاﻳﺶ ﺗﻌﺎرض را اﻳﺠﺎد ﻣﻲ ﻛﻨ داﺑﺲ )ﺪ 28 ، 2013 .( در ﺗﻌﺎﻣﻼت واﻟﺪﻳﻦ و ﻓﺮزﻧﺪان زﻣﺎﻧﻲ ﻛﻪ ارﺗﺒﺎﻃﺎت ﺑﻪ ﺻﻮرت اﻧﺘﻘﺎد ﻣﺪاوم از ﻳﻜﺪﻳﮕﺮ، رﻓﺘﺎرﻫﺎي ﻧﺎﺧﻮﺷﺎﻳﻨﺪي ﻣﺎﻧﻨﺪ ﻓﺮﻳﺎد و ﻧﻈﺮات ﻣﻨﻔﻲ و ﻣﺸﺎﺟﺮات ﻛﻼﻣﻲ ﺑﺎﺷﺪ، ﺗﻌﺎرض ﺗﺸﺪﻳﺪ ﻣﻲ ﺷﻮد، در ﺣﺎﻟﻲ ﻛﻪ در ﺳﻄﺢ ﻧﺮﻣﺎل ﺗﻌﺎرض واﻟﺪ ـ ﻛﻮدك ﺳﺎزﮔﺎري و اﻓﺰاﻳﺶ ﻣﻬﺎرت ﻫﺎي ﺣﻞ ﻣﺴﺎﻟﻪ را ﭘﺮورش ﻣﻲ دﻫﺪ ) ﻓﻴﻮﻟﻴﻜﻨﻲ، اﮔﻠﺰ، 1993 .( زﻣﺎﻧﻲ ﻛﻪ ﺗﻌﺎرض واﻟﺪﻳﻦ و ﻓﺮزﻧﺪان ﺑﻪ ﺷﻜﻞ ﻧﺎدرﺳﺘﻲ ﻣﺪﻳﺮﻳﺖ ﻣﻲ ﺷﻮد، ﺗﺸﺪﻳﺪ ﺧﺼﻮﻣﺖ ﭘﻴﺎﻣﺪ آن اﺳﺖ و ﻧﺎﺳﺎزﮔﺎري رﻓﺘﺎري را ﻣﻨﺠﺮ ﻣﻲ ﺷﻮد و ﺑﻬﺰﻳﺴﺘﻲ روان ﺷﻨﺎﺧﺘﻲ ﻧﻮﺟﻮان را ﻛﺎﻫﺶ ﻣﻲ دﻫﺪ ) ﭘﺎراﻛﺎردوﻧﺎ، ﻳﻦ، آﻧﺪوﻧﻲ29 ، 2017 .( اﺳﺘﻴﻨﺒﺮگ )2001 ( ﻣﻄﺮح ﻣﻲ ﻛﻨﺪ ﭼﺎﻟﺸﻲ ﻛﻪ ﺑﺮاي واﻟﺪﻳﻦ و ﻧﻮﺟﻮان رخ ﻣﻲ دﻫﺪ، ﺗﻼﺷﻲ ﺑﺮاي ﻣﻘﺎﺑﻠﻪ ﺑﺎ ﻣﺎﻫﻴﺖ در ﺣﺎل ﺗﻐﻴﻴﺮ رواﺑﻂ آﻧﺎن اﺳﺖ . ﺗﻤﺎﻳﻼت و ﺗﺠﺮﺑﻴﺎت ﻣﺘﻔﺎوت ﺑﺎ روﺣﻴﺎت ﻧﻮﺟﻮان و واﻟﺪﻳﻦ ﻣﻮﺟﺐ اﻓﺰاﻳﺶ درﮔﻴﺮي ﻣﻲ ﺷﻮد . 351 / ﭘﻴﺶ ﺑﻴﻨﻲ ﺗﻮاﻧﻤﻨﺪي ﻣﻨﺶ ﻧﻮﺟﻮاﻧﺎن و دﻟﺒﺴﺘﮕﻲ ﺑﻪ واﻟﺪﻳﻦ و/... 351 / ﭘﻴﺶ ﺑﻴﻨﻲ ﺗﻮاﻧﻤﻨﺪي ﻣﻨﺶ ﻧﻮﺟﻮاﻧﺎن و دﻟﺒﺴﺘﮕﻲ ﺑﻪ واﻟﺪﻳﻦ و/... اﻛﺜﺮ ﺗﺤﻘﻴﻘﺎت ﺗﻮاﻧﻤﻨﺪي ﻫﺎي ﻣﻨﺶ، درﺑﺮﮔﻴﺮﻧﺪه ﻧﻤﻮﻧﻪ ﺑﺰرﮔﺴﺎﻻن اﺳ )ﺖ ﭘﻴﺘﺮﺳﻮن، ﺳﻠﻴﮕﻤﻦ، 2007 ( و ﺗﺤﻘﻴﻘﺎت ﻛﻤﺘﺮي ﺑﻪ ﻛﻮدك و ﻧﻮﺟﻮان اﺧﺘﺼﺎص ﻳﺎﻓﺘﻪ اﺳﺖ ) ﺑﺎرﻛﺮ، ﻣﻴﻨﺘﻮن 30 ، 2012 .( ﻣﺤﻘﻘﺎن ﺑﺎ ﻣﺸﺨﺺ ﻛﺮدن ﺗﻮاﻧﻤﻨﺪي ﻫﺎي ﻣﻨﺶ اﻣﻴﺪوارﻧﺪ درك ﺑﻬﺘﺮي از ﺑﻬﺰﻳﺴﺘﻲ، ﺷﺎدي و ﻋﻤﻠﻜﺮدﻫﺎي ﺧﻮش ﺑﻴﻨﺎﻧﻪ در ﻧﻮﺟﻮان اﻳﺠﺎد ﺷﻮد ) ﺗﻮﻧﺮ، ﻫﺎزﻟﻢ، راﺑﻴﻨﺴﻮن، ﺑﺎﻳﺞ 31 ، 2012 .( از آﻧﺠﺎ ﻛﻪ ﺗﺤﻘﻴﻘﺎت ﻛﻤﺘﺮي ﺑﻪ ﺑﺮرﺳﻲ ﺗﻮاﻧﻤﻨﺪي ﻫﺎ و ﻓﻀﺎﺋﻞ و اﺛﺮات ﺗﻌﺎﻣﻠﻲ آن ﺑﺮ ﺗﻌﺎرض ﻧﻮﺟﻮان ﭘﺮداﺧﺘﻪ اﺳﺖ، ﺿﺮوري اﺳﺖ اﻳﻦ اﻣﺮ ﻣﻮرد ﺗﻮﺟﻪ ﻗﺮار ﮔﻴﺮد . اﻳﻦ در ﺣﺎﻟﻲ اﺳﺖ ﻛﻪ ﻧﻮﺟﻮاﻧﻲ اﺣﺘﻤﺎﻻً دوراﻧﻲ ﻫﻤﺮاه ﺑﺎ ﺗﻌﺎرض اﺳﺖ و ﻧﻘﺶ دﻟﺒﺴﺘﮕﻲ ﺑﺪﻟﻴﻞ اﺛﺮﮔﺬاري ﺑﺮ رواﺑﻂ واﻟﺪ - ﻛﻮدك ﻧﻴﺰ ﻣﻮرد ﺗﻮﺟﻪ ﻗﺮار ﮔﺮﻓﺘﻪ اﺳﺖ . ﻣﻘﺪﻣﻪ ﻟﺬا ﺗﻮاﻧﻤﻨﺪي ﻫﺎ و ﻓﻀﺎﺋﻞ ﻣﻲ ﺗﻮاﻧﻨﺪ ﺗﺴﻬﻴﻞ ﻛﻨﻨﺪه رواﺑﻂ ﺑﻬﺘﺮي ﺑﺮاي ﻧﻮﺟﻮان و واﻟﺪﻳﻦ ﺑﺎﺷﻨﺪ ﺗﺎ دﻟﺒﺴﺘﮕﻲ اﻳﻤﻦ ﺗﺮ و ﺗﻌﺎرﺿﺎت ﻛﻤﺘﺮي را در ﭘﻲ داﺷﺘﻪ ﺑﺎﺷﻨﺪ . در اﻳﻦ راﺳﺘﺎ ﺿﺮورت دارد ﻣﺘﻐﻴﺮﻫﺎي اﺳﺎﺳﻲ دوران ﻧﻮﺟﻮاﻧﻲ از ﺟﻤﻠﻪ دﻟﺒﺴﺘﮕﻲ ﺑﻪ واﻟﺪﻳﻦ و ﻫﻤﺴﺎﻻن و ﺗﻌﺎرض ﺑﺎ ﭘﺪر و ﻣﺎدر در ارﺗﺒﺎط ﺑﺎ ﺗﻮاﻧﻤﻨﺪي ﻫﺎي ﻣﻨﺶ ﺑﺮرﺳﻲ ﺷﻮد . ﺑﺎ ﺗﻮﺟﻪ ﺑﻪ اﻧﺪك ﺑﻮدن ﭘﮋوﻫﺶ اﻧﺠﺎم ﺷﺪه ﭘﻴﺮاﻣﻮن ﺗﻮاﻧﻤﻨﺪي ﻫﺎي ﻣﻨﺶ، دﻟﺒﺴﺘﮕﻲ و ﺗﻌﺎرض، ﻫﺪف ﭘﮋوﻫﺶ ﺣﺎﺿﺮ ﺑﺮرﺳﻲ اﻳﻦ ﻣﺘﻐﻴﺮﻫﺎﺳﺖ . ﺑﻪ اﻳﻦ ﻣﻨﻈﻮر ﺗﻮاﻧﻤﻨﺪي ﻫﺎي ﻣﻨﺶ و دﻟﺒﺴﺘﮕﻲ ﺑﻪ واﻟﺪﻳﻦ و ﻫﻤﺴﺎﻻن ﺑﻪ ﻋﻨﻮان ﻣﺘﻐﻴﺮﻫﺎ ي ﭘﻴﺶ ﺑﻴﻦ و ﺗﻌﺎرض ﺑﻪ ﭘﺪر و ﻣﺎدر ﺑﻪ ﻋﻨﻮان ﻣﺘﻐﻴﺮ ﻣﻼك در ﻧﻈﺮ ﮔﺮﻓﺘﻪ ﺷﺪه اﺳﺖ . از ﺑﻴﻦ روش ﻫﺎي رﮔﺮﺳﻴﻮن روش ﭘﺴﺮوﻧﺪه اﺳﺘﻔﺎده ﺷﺪ؛ ﭼﺮاﻛﻪ ﻳﻚ روﻳﻜﺮد ﻣﺪل ﺳﺎزي اﺳﺖ ﻛﻪ ﺑﻪ ﻋﻨﻮان ﻳﻚ ﻓﻦ اﻛﺘﺸﺎﻓﻲ ﻣﻲ ﺗﻮاﻧﺪ ﺑﺮاي ﺣﺬف ﻣﺘﻐﻴﺮﻫﺎي زاﺋﺪ ﺑﻪ ﻣﻨﻈﻮر ﺗﻤﺮﻛﺰ ﺑﺮ ﭘﮋوﻫﺶ ﻫﺎي آﻳﻨﺪه ﻣﻔﻴﺪ ﺑﺎﺷﺪ ) ﺗﺎﺑﺎﻛﻨﻴﻚ، ﻓﻴﺪل 32 ، 1395 .( ﺑﻨﺎﺑﺮاﻳﻦ در اﻳﻦ ﭘﮋوﻫﺶ ﺑﺎ اﺳﺘﻔﺎده از اﻳﻦ روش ﺑﺘﻮاﻧﺪ ﻣﺘﻐﻴﺮﻫﺎي اﺛﺮﮔﺬار ﻓﻀﺎﺋﻞ و دﻟﺒﺴﺘﮕﻲ واﻟﺪﻳﻦ و ﻫﻤﺴﺎﻻن ﺑﺮ ﺗﻌﺎرض واﻟﺪﻳﻦ را ﻣﺸﺨﺺ ﺳﺎزد . ﻟﺬا ﻫﺪف ﭘﮋوﻫﺶ اﻳﻦ اﺳﺖ ﻛﻪ دﻟﺒﺴﺘﮕﻲ ﺑﻪ واﻟﺪﻳﻦ و ﻫﻤﺴﺎﻻن ﭘﻴﺶ ﺑﻴﻨﻲ ﻛﻨﻨﺪه ﺗﻌﺎرض ﺑﺎ ﭘﺪر و ﻣﺎدر اﺳﺖ؟ و ﺗﻮاﻧﻤ ﻨﺪي ﻫﺎي ﻣﻨﺶ ﭘﻴﺶ ﺑﻴﻨﻲ ﻛﻨﻨﺪه ﺗﻌﺎرض ﺑﺎ ﭘﺪر و ﻣﺎدر اﺳﺖ؟ ﺟﺎﻣﻌﻪ آﻣﺎري، ﻧﻤﻮﻧﻪ و روش اﺟﺮاي ﭘﮋوﻫﺶ ﭘﮋوﻫﺶ ﺣﺎﺿﺮ ﺗﻮﺻﻴﻔﻲ و از ﻧﻮع ﻫﻤﺒﺴﺘﮕﻲ اﺳﺖ . ﺟﺎﻣﻌﻪ آﻣﺎري ﭘﮋوﻫﺶ ﺷﺎﻣﻞ داﻧﺶ آﻣﻮزان دﺧﺘﺮ ﭘﺎﻳﻪ ﻧﻬﻢ از ﻣﻨﺎﻃﻖ 4 ، 3 و10 ﺷﻬﺮ ﺗﻬﺮان، در ﻧﻴﻤﺴﺎل دوم ﺗﺤﺼﻴﻠﻲ1397 - 1396 اﺳﺖ . ﻧﻤﻮﻧﻪ آﻣﺎري در اﻳﻦ ﭘﮋوﻫﺶ ﺷﺎﻣﻞ 195 داﻧﺶ آﻣﻮز دﺧﺘﺮ )27 ./ درﺻﺪ از ﻣﻨﻄﻘﻪ4 ، 18 /0 درﺻﺪ از ﻣﻨﻄﻘﻪ3 و55 /0 درﺻﺪ از ﻣﻨﻄﻘﻪ10 ( ﺑﺎ ﻣﻴﺎﻧﮕﻴﻦ ﺳﻨﻲ12 / 15 اﺳﺖ . ﻧﻤﻮﻧﻪ ﮔﻴﺮي ﺑﺎ روش در دﺳﺘﺮس اﻧﺘﺨﺎب ﺷﺪﻧﺪ . ﻣﺎﻳﻠﺰ و ﺷﻮﻟﻴﻦ 33 ) 1395 ( ﺑﺮاي ﺗﻌﻴﻴﻦ ﺣﺠﻢ ﻧﻤﻮﻧﻪ ﻻزم ﺟﻬﺖ آزﻣﻮن R2 در ﻣﺪل رﮔﺮﺳﻴﻮن ﭘﻴﺸﻨﻬﺎد ﻛﺮدﻧﺪ ﻛﻪ ﺣﺠﻢ ﻧﻤﻮﻧﻪ ﺑﺎﻳﺪ ﺑﻴﺸﺘﺮ از K + 104 ، ﺑﺎﺷﺪK ﺑﻴﺎﻧﮕﺮ ﺗﻌﺪاد ﻣﺘﻐﻴﺮﻫﺎي ﻣﺴﺘﻘﻞ اﺳﺖ . در اﻳﻦ ﭘﮋوﻫﺶ ﻣﻼك ﻫﺎي ورود، زﻧﺪﮔﻲ ﺑﺎ ﭘﺪر و ﻣﺎدر و ﺗﺤﺼﻴﻞ در ﭘﺎﻳﻪ ﻧﻬﻢ ﺗﺤﺼﻴﻠﻲ و ﻣﻼك ﺧﺮوج، ﻋﺪم ﭘﺎﺳﺦ دﻫﻲ ﺑﻪ ﺳﺆاﻻت ﭘﺮﺳﺸﻨﺎﻣﻪ ﺑﻮد . اﺑﺰار ﭘﮋوﻫﺶ ﺑﺰ رﭘﮋوﺶ ﻧﺴﺨﻪ ﻛﻮﺗﺎه اﺑﺰار ﺗﻮاﻧﻤﻨﺪي ﻫﺎ ي ﻣﻨﺶ 34 ) SMCS :( اﻳﻦ ﭘﺮﺳﺸﻨﺎﻣﻪ ﺷﺎﻣﻞ24 آﻳﺘﻢ ﺧﻮدارزﻳﺎﺑﻲ در ﻣﻮرد ﺗﻮاﻧﻤﻨﺪي ﻫﺎي ﻣﻨﺶ اﺳﺖ . ﻓﺮﻧﻬﺎم و ﻟﺴﺘﺮ35 ) 2012 ( ﺑﺮ اﺳﺎس ﻧﺴﺨﻪ اﺻﻠﻲ ﻓﻬﺮﺳﺖ ﺗﻮاﻧﻤﻨﺪي ﻫﺎي ﻣﻨﺸﻲ ) ﭘﻴﺘﺮﺳﻮن و ﺳﻠﻴﮕﻤﻦ، 2004 ( ﻧﺴﺨﻪ ﻛﻮﺗﺎه را آن ﺗﻮﺳﻌﻪ دادﻧﺪ . ﺑﻴﺴﺖ وﭼﻬﺎر ﺗﻮاﻧﻤﻨﺪي ﺑﻪ ﺻﻮرت ﺧﻮدارزﻳﺎﺑﻲ و 352 / / !"# $% / &'%( / / ﻣﺎﻧﻨﺪ ﺗﻮزﻳﻊ ﻧﺮﻣﺎل از 55 ﺗﺎ145 ﻧﻤﺮه ﮔﺬاري ﻣﻲ ﺷﻮد . در ﻣﻄﺎﻟﻌﻪ ﻓﺮﻧﻬﺎم و ﻟﺴﺘﺮ )2012 ( ﻧﺘﺎﻳﺞ ﺗﺤﻠﻴﻞ ﻋﺎﻣﻠﻲ اﻛﺘﺸﺎﻓﻲ ﺑﺎ دو ﺷﻴﻮه ﭼﺮﺧﺶ ﻣﺘﻌﺎﻣﺪ و ﻣﺘﻤﺎﻳﻞ ﻧﺸﺎن داد ﻛﻪ 24 ﺗﻮاﻧﻤﻨﺪي ﻫﺎي ﻣﻨﺶ ﻫﻤﺎﻧﻨﺪ ﻧﺴﺨﻪ اﺻﻠﻲ داراي ﺷﺶ ﻓﻀﻴﻠﺖ اﺻﻠﻲ ﻫﺴﺘﻨﺪ . ﺿﺮﻳﺐ ﻫﻤﺴﺎﻧﻲ دروﻧﻲ ﻓﻀﺎﺋﻞ ﺧﺮد، ﺷﺠﺎﻋﺖ، اﻧﺴﺎﻧﻴﺖ، ﻋﺪاﻟﺖ، اﻋﺘﺪال و ﺗﻌﺎﻟﻲ ﺑﻪ ﺗﺮﺗﻴﺐ 66 /0 ، 55 /0 ، 52 /0 ، 50 /0 ، 55 /0 ، 76 /0 ﺑﻪ دﺳﺖ آﻣﺪﻧﺪ ﺗﻮ . اﻧﻤﻨﺪي ﻣﻨﺶ ﻫﻤﺴﻮ ﺑﺎ ﻧﺴﺨﻪ اﺻﻠﻲ ﺑﻪ ﺷﺶ ﻓﻀﻴﻠﺖ ﺧﺮد )6 ﺳﺆال ( ، ﺷﺠﺎﻋﺖ )3 ﺳﺆال ( ، اﻧﺴﺎﻧﻴﺖ )2 ﺳﺆال ( ، ﻋﺪاﻟﺖ )3 ﺳﺆال ( ، اﻋﺘﺪال )3 ﺳﺆال ( ، ﺗﻌﺎﻟﻲ )7 ﺳﺆال ( ﺗﻘﺴﻴﻢ ﻣﻲ ﺷﻮد . در ﻣﻄﺎﻟﻌﻪ ﺷﻜﺮي ﺗﺤﻠﻴﻞ ﻋﺎﻣﻞ ﺗﺎﻳﻴ ﺪي ﻣﺠﺬور ﺧﻲ دو 19 / 542 ، ﺷﺎﺧﺺ ﻣﺠﺬور ﺧﻲ دو ﺑﺮ درﺟﻪ آزادي29 /2 ، ﺷﺎﺧﺺ ﺑﺮازش ﻣﻘﺎﻳﺴﻪ اي89 /0 ، ﺷﺎﺧﺺ ﺑﺮازش ﻧﻴﻜﻮﻳﻲ86 /0 ﺑﻮد . رواﻳﻲ ﭘﺮﺳﺸﻨﺎﻣﻪ در ﻧﻤﻮﻧﻪ داﻧﺸﺠﻮﻳﺎن اﻳﺮاﻧﻲ از ﺳﺎﺧﺘﺎر ﺷﺶ ﻋﺎﻣﻠﻲ ﻧﺴﺨﻪ ﻛﻮﺗﺎه اﺑﺰار ﺗﻮاﻧﻤﻨﺪي ﻫﺎي ﻣﻨﺶ ﺑﻪ ﻃﻮر ﺗﺠﺮﺑﻲ ﺣﻤﺎﻳﺖ ﻛﺮد . ﺿﺮاﻳﺐ ﻫﻤﺴﺎﻧﻲ دروﻧﻲ آن ﻫﺎ ﺑﻪ ﺗﺮﺗﻴﺐ 66 /0 ، 55 /0 ، 52 /0 ، 48 /0 ، 55 /0 ، 76 /0 ﮔﺰارش ﺷﺪه اﺳﺖ . در ﭘﮋوﻫﺶ ﺣﺎﺿﺮ ﺿﺮاﻳﺐ ﻫﻤﺴﺎﻧﻲ دروﻧﻲ ﻓﻀﺎﻳﻞ ﺑﻪ ﺗﺮﺗﻴﺐ 67 /0 ، 61 /0 ، 66 /0 ، 65 /0 ، 54 /0 ، 69 /0 ﺑﻪ دﺳﺖ آﻣﺪ . ﻧﺴﺨﻪ ﻛﻮﺗﺎه ﭘﺮﺳﺸﻨﺎﻣﻪ ﺗﻌﺎرض ﻧﻮﺟﻮان ﺑﺎ واﻟﺪﻳﻦ 36 ) CBQ :( ﭘﺮﺳﺸﻨﺎﻣﻪ رﻓﺘﺎر ﻣﺘﻌﺎرض در ﺳﺎل 1979 ﺗﻮﺳﻂ ﭘﺮﻳﻨﺰ37 و ﻫﻤﻜﺎران ﺳﺎﺧﺘﻪ ﺷﺪ . ﻧﺴﺨﻪ اﺻﻠﻲ آن داراي 75 ﮔﻮﻳﻪ اﺳﺖ . ادارك ﻧﻮﺟﻮان از ﻣﻴﺰان ﺗﻌﺎرض و راﺑﻄﻪ ﻣﻨﻔﻲ ﺑﺎ واﻟﺪﻳﻨﺶ را ﻣﻮرد ارزﻳﺎﺑﻲ ﻗﺮار داد . ﻧﺴﺨﻪ ﻛﻮﺗﺎه اﻳﻦ ﻣﻘﻴﺎس ﻣﻴﺰان ﺗﻌﺎرض ﻧﻮﺟﻮان ﺑﺎ واﻟﺪﻳﻨﺶ را ﻣﻲ ﺳﻨﺠﺪ . اﻳﻦ ﭘﺮﺳﺸﻨﺎﻣﻪ داراي دو ﻓﺮم ﺟﺪاﮔﺎﻧﻪ، 20 ﮔﻮﻳﻪ ﻳﻜﻲ ﺑﻪ ﻣﺎدر و دﻳﮕﺮي ﺑﻪ ﭘﺪر ﻣﺮﺑﻮط اﺳﺖ . ﻳﺎﻓﺘﻪ ﻫﺎ ﺟﺪول1 :ﻣﺎﺗﺮﻳﺲ ﻫﻤﺒﺴﺘﮕﻲ ﺗﻌﺎرض ﺑﺎ واﻟﺪﻳﻦ، دﻟﺒﺴﺘﮕﻲ ﺑﻪ واﻟﺪﻳﻦ و ﻫﻤﺴﺎﻻن و ﺗﻮاﻧﻤﻨﺪي ﻫﺎي ﻣﻨﺶ 11 10 9 8 7 6 5 4 3 2 1 S M ﻣﺘﻐﻴﺮ 1 99 /5 52 / 23 1 - ﺗﻌﺎرض ﭘﺪر 1 13 /0 3/5 06 / 23 2 - ﺗﻌﺎرض ﻣﺎدر 1 60 /0 14 /0 - 33 / 18 44 / 95 3 - دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﻣﺎدر 1 42 /0 22 /0 - 60 /0 - 55 / 24 98 / 88 4 - دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﭘﺪر 1 30 /0 40 /0 19 /0 - ** 22 /0 - 34 / 16 64 / 94 5 - دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﻫﻤﺴﺎﻻن 1 * 15 /0 10 /0 20 /0 - 19 /0 - 11 /0 32 / 71 62 / 684 6 -ﺧﺮد 1 ** 51 /0 1/0 13 /0 11 /0 9/0 - * 17 /0 09 / 45 65 / 344 7 -ﺷﺠﺎﻋﺖ 1 * 15 /0 30 /0 24 /0 23 /0 22 /0 21 /0 - 10 /0 - 49 / 31 65 / 246 8 -اﻧﺴﺎﻧﻴﺖ 1 36 /0 51 /0 54 /0 08 /0 10 /0 * 17 /0 ** 20 /0 - * 23 /0 - 20 / 49 66 / 338 9 -ﻋﺪاﻟﺖ 1 42 /0 23 /0 41 /0 35 /0 01 /0 1/0 23 /0 24 /0 - 12 /0 83 / 46 95 / 330 10 -اﻋﺘﺪال 1 40 /0 47 /0 41 /0 48 /0 51 /0 09 /0 13 /0 19 /0 ** 24 /0 - 13 /0 - 46 / 86 59 / 823 11 -ﺗﻌﺎﻟﻲ P< 05 /0 P< 01 /0 در ﺟﺪول2 ﻧﺘﺎﻳﺞ ﺗﺤﻠﻴﻞ وارﻳﺎﻧﺲ ﺑﺎ روش ﭘﺴﺮوﻧﺪه43 اﺟﺮا ﺷﺪ . ﺗﺤﻠﻴﻞ در ﻫﻔﺖ ﮔﺎم اﻧﺠﺎم ﺷﺪ ﻛﻪ در ﺷﺶ ﮔﺎم اول ﺑﻪ ﺗﺮﺗﻴﺐ ﻣﺘﻐﻴﺮﻫﺎي ﺧﺮد، ﺗﻌﺎر ض ﺑﺎ ﻣﺎدر، ﺷﺠﺎﻋﺖ، دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﻫﻤﺴﺎﻻن، اﻧﺴﺎﻧﻴﺖ از ﻣﻌﺎدل ﺧﺎرج ﺷﺪ، در ﮔﺎم ﻫﻔﺘﻢ ﻣﺘﻐﻴﺮﻫﺎي دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﭘﺪر، ﻋﺪاﻟﺖ، اﻋﺘﺪال، ﺗﻌﺎﻟﻲ و ﺗﻌﺎﻣﻞ ﻋﺪاﻟﺖ و ﺗﻌﺎرض ﺑﺎ ﭘﺪر ﺑﻪ ﻋﻨﻮان ﻣﺘﻐﻴﺮﻫﺎي ﭘﻴﺶ ﺑﻴﻨﻲ ﻛﻨﻨﺪه در ﻣﻌﺎدل ﺑﺎﻗﻲ ﻣﺎﻧﺪﻧﺪ . ﻳﺎﻓﺘﻪ ﻫﺎ ﺟﺪول 1 اﻃﻼﻋﺎت ﺗﻮﺻﻴﻔﻲ ﻣﻴﺎﻧﮕﻴﻦ و اﻧﺤﺮاف اﺳﺘﺎﻧﺪارد ﻣ ﺘﻐﻴﺮﻫﺎي اﺻﻠﻲ ﭘﮋوﻫﺶ ) ،ﺗﻮاﻧﻤﻨﺪي ﻫﺎي ﻣﻨﺶ دﻟﺒﺴﺘﮕﻲ و ﺗﻌﺎرض ( را در داﻧﺶ آﻣﻮزان ﻧﺸﺎن ﻣﻲ دﻫﺪ . ﺑﺎﻻﺗﺮﻳﻦ ﻣﻴﺎﻧﮕﻴﻦ ﺗﻌﺎﻟﻲ و ﻛﻤﺘﺮﻳﻦ آن ﺗﻌﺎرض ﺑﺎ ﻣﺎدر اﺳﺖ . ﺑﺎﻻﺗﺮﻳﻦ ﺳﻄﺢ اﻧﺤﺮاف اﺳﺘﺎﻧﺪارد ﺗﻌﺎﻟﻲ و ﻛﻤﺘﺮﻳﻦ آن ﺗﻌﺎرض ﺑﺎ ﻣﺎدر اﺳﺖ . ﻫﻢ ﭼﻨﻴﻦ ﺿﺮاﻳﺐ ﻫﻤﺒﺴﺘﮕﻲ ﺗﻮاﻧﻤﻨﺪي ﻫﺎي ﻣﻨﺶ ) ﺧﺮد، ﺷﺠﺎﻋ ﺖ، اﻧﺴﺎﻧﻴﺖ، ﻋﺪاﻟﺖ، اﻋﺘﺪال و ﺗﻌﺎﻟﻲ ( ، دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﻣﺎدر، ﭘﺪر و ﻫﻤﺴﺎﻻن و ﺗﻌﺎرض ﺑﺎ ﭘﺪر و ﻣﺎدر را ﻧﺸﺎن ﻣﻲ دﻫﺪ . ﻫﻤﺎن ﻃﻮر ﻛﻪ ﻣﻼﺣﻈﻪ ﻣﻲ ﻛﻨﻴﺪ، ﺑﻴﻦ ﺑﺮﺧﻲ ﻣﺘﻐﻴﺮﻫﺎي ﭘﮋوﻫﺶ راﺑﻄﻪ ﻣﻌﻨﺎداري وﺟﻮد دارد . ﺑﻴﻦ ﻋﺪاﻟﺖ، اﻋﺘﺪال و ﺗﻌﺎﻟﻲ ﺑﺎ دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﭘﺪر و ﻫﻤﺴﺎﻻن و ﺧﺮد ﺑﺎ ﺗﻌﺎرض و دﻟ ﺒﺴﺘﮕﻲ ﺑﻪ ﭘﺪر راﺑﻄﻪ ﻣﻌﻨﺎداري وﺟﻮد ﻧﺪارد . اﺑﺰار ﭘﮋوﻫﺶ 353 / ﭘﻴﺶ ﺑﻴﻨﻲ ﺗﻮاﻧﻤﻨﺪي ﻣﻨﺶ ﻧﻮﺟﻮاﻧﺎن و دﻟﺒﺴﺘﮕﻲ ﺑﻪ واﻟﺪﻳﻦ و/... در ﭘﮋوﻫﺶ واﺣﺪي و ﻓﺘﺤﻲ42 ) 1389 ( ﻣﻴﺰان ﭘﺎﻳﺎﻳﻲ آن ﻣﻮرد ﺑﺮرﺳﻲ ﻣﺠﺪد ﻗﺮار ﮔﺮﻓﺖ . ﺑﻌﺪ از ﺗﺄﻳﻴﺪ و ﻣﻄﻠﻮب ﺑﻮدن رواﻳﻲ ﺻﻮري و ﻣﺤﺘﻮاﻳﻲ، ﭘﺎﻳﺎﻳﻲ دروﻧﻲ ﭘﺮﺳﺸﻨﺎﻣﻪ ﺑﺎ روش آﻟﻔﺎي ﻛﺮوﻧﺒﺎخ ﺑﺮاي ﻣﻘﻴﺎس دﻟ ﺒﺴﺘﮕﻲ ﻣﺎدر، ﭘﺪر و ﻫﻤﺴﺎﻻن ﺑﻪ ﺗﺮﺗﻴﺐ 85 /0 ، 83 /0 و86 /0 ﺑﻮد . در ﭘﮋوﻫﺶ ﺣﺎﺿﺮ ﻧﻴﺰ ﺑﻪ ﻣﻨﻈﻮر ﭘﺎﻳﺎﻳﻲ از روش آﻟﻔﺎي ﻛﺮوﻧﺒﺎخ ﺑﻪ ﺗﺮﺗﻴﺐ دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﻣﺎدر92 /0 ، دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﭘﺪر95 /0 و دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﻫﻤﺴﺎﻻن90 /0 ﺑﻮد . اﺑﺰار ﭘﮋوﻫﺶ ﭘﺮﺳﺸﻨﺎﻣﻪ رﻓﺘﺎر ﻣﺘﻌﺎرض وﻳﮋه ﻧﻮﺟﻮاﻧﺎن اﺳﺖ و آزﻣﻮدﻧﻲ ﺑﺎﻳﺪ ﺑﺎ اﻧﺘﺨﺎب ﻳﻜﻲ از ﭘﺎﺳﺦ ﻫﺎي ﺻﺤﻴﺢ ﻳﺎ ﻏﻠﻂ، ﻣﻮاﻓﻘﺖ ﻳﺎ ﻣﺨﺎﻟﻔﺖ ﺧﻮد را ﺑﺎ ﻫﺮﻳﻚ از ﻋﺒﺎرات ﻣﺸﺨﺺ ﺳﺎزد . ﻧﻤﺮه ﺑﺎﻻﺗﺮ در اﻳﻦ ﭘﺮﺳﺸﻨﺎﻣﻪ، ﻧﺸﺎﻧﮕﺮ ﺗﻌﺎرض ﺑﻴﺸﺘﺮ ﻧﻮﺟﻮان ﺑﺎ ﭘﺪر و ﻣﺎدرش اﺳﺖ . ﭘﺎﻳﺎﻳﻲ ﺣﺎﺻﻞ از روش ﺑﺎزآزﻣﺎﻳﻲ ﺑﺮاي ﻧﺴﺨﻪ ﻧﻮﺟﻮان 84 /0 ﺑﻮده اﺳﺖ . ﻧﺘﻴﺠﻪ ﭘﮋوﻫﺶ روﺑﻴﻦ و ﻓﺎﺳﺘﺮ 38 ) 1989 ( ﻧﺸﺎن داد ﭘﺮﺳﺸﻨﺎﻣﻪ رﻓﺘﺎر ﻣﺘﻌﺎرض ﻣﻲ ﺗﻮاﻧﺪ ﺧﺎﻧﻮاده ﻫﺎي داراي ﻣﺸﻜﻼت ﺑﺎﻟﻴﻨﻲ را از ﺧﺎﻧﻮاده ﻫﺎي ﺳﺎﻟﻢ ﺟﺪا ﺳﺎزد ﻛﻪ ﮔﻮﻳﺎﻳﻲ اﻋﺘﺒﺎر اﻓﺘﺮاﻗﻲ اﻳﻦ اﺑﺰار اﺳﺖ . ﭘﮋوﻫﺶ ﺷﺮﻳﻌﺘﻲ 39 ) 1394 ( ﻧﺸﺎن داد ﺣﻤﺎﻳﺖ و ﮔﺮﻣﻲ ﻣﺎدر و ﭘﺪر ﺑﻪ ﻃﻮر ﻏﻴﺮﻣﺴﺘﻘﻴﻢ و ﺑﺎ واﺳﻄﻪ ﮔﺮي ﺗﻌﺎرض ﻣﺎدر و ﭘﺪر ﺑﺎ ﻧﻮﺟﻮان ﺑﺎ ﺷﻴﻮه ﻫﺎي اﺑﺮاز ﺧﺸﻢ راﺑﻄﻪ دارد ﻛﻪ ﺣﺎﻛﻲ از رواﻳﻲ اﻳﻦ اﺑﺰار اﺳﺖ . ﻫﻢ ﭼﻨﻴﻦ ﺿﺮﻳﺐ آﻟﻔﺎي ﻛﺮوﻧﺒﺎخ ﺑﺮاي ﻛﻞ ﺳﺆاﻻت ﺗﻌﺎرض ﺑﺎ ﻣﺎدر 91 /0 و ﺗﻌﺎرض ﺑﺎ ﭘﺪر89 /0 ﺑﻮد . در ﭘﮋوﻫﺶ ﺣﺎﺿﺮ ﻧﻴﺰ ﻫﻤﺴﺎ ﻧﻲ دروﻧﻲ ﺗﻌﺎرض ﻧﻮﺟﻮان ﺑﺎ ﭘﺪر94 /0 و ﺑﺎ ﻣﺎدر93 /0 اﺳﺖ . ﭘﺮﺳﺸﻨﺎﻣﻪ دﻟﺒﺴﺘﮕﻲ ﺑﻪ واﻟﺪﻳﻦ و ﻫﻤﺴﺎﻻن 40 ) IPPA ( : اﻳﻦ ﭘﺮﺳﺸﻨﺎﻣﻪ ﺗﻮﺳﻂ آرﻣﺴﺪن و ﮔﺮﻳﻦ ﺑﺮگ41 ) 1987 ( ﺑﺮاي ارزﻳﺎﺑﻲ ادراك ﻧﻮﺟﻮان از اﺑﻌﺎد ﺷﻨﺎﺧﺘﻲ و ﻋﺎﻃﻔﻲ ﻣﺜﺒﺖ و ﻣﻨﻔﻲ راﺑﻄﻪ ﺑﺎ واﻟﺪﻳﻦ و دوﺳﺘﺎن ﻧﺰدﻳﻚ ﺳﺎﺧﺘﻪ ﺷﺪ . ﺷﺎﻣﻞ 75 ﮔﻮﻳﻪ ﭘﻨﺞ ﮔﺰﻳﻨﻪ اي از ﻛﺎﻣﻼً ﻣﺨﺎﻟﻔﻢ ﺗﺎ ﻛﺎﻣﻼً ﻣﻮاﻓﻘﻢ اﺳﺖ . دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﻣﺎدر 25 ﮔﻮﻳﻪ اول، دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﭘﺪر25 ﮔﻮﻳﻪ دوم و دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﻫﻤﺴﺎﻻن25 ﮔﻮﻳﻪ ﺳﻮم را ﻣﻲ ﺳﻨﺠﺪ . اﻋﺘﺒﺎر دروﻧﻲ اﻳﻦ آزﻣﻮن ﺗﻮﺳﻂ آرﻣﺴﺪن و ﮔﺮﻳﻦ ﺑﺮگ )1987 ( ﺑﺎ روش ﺑﺎزآزﻣﺎﻳﻲ در ﻓﺎﺻﻠﻪ ﺳﻪ ﻫﻔﺘﻪ روي ﻳﻚ ﻧﻤ ﻮﻧﻪ 27 ﻧﻔﺮي از آزﻣﻮدﻧﻲ ﻫﺎ20 - 18 ﺳﺎﻟﻪ اﻧﺠﺎم ﺷﺪ . ﻣﻴﺰان ﻫﻤﺴﺎﻧﻲ دروﻧﻲ دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﻣﺎدر 87 /0 ، دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﭘﺪر 89 /0 و دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﻫﻤﺴﺎﻻن92 /0 ﺑﻮد ) آرﻣﺴﺪن و ﮔﺮﻳﻨﺒﺮگ، 1987 .( ﻫﻤﺴﺎﻧﻲ دروﻧﻲ اﻳﻦ ﭘﺮﺳﺸﻨﺎﻣﻪ در اﻳﺮان ﺗﻮﺳﻂ ﻧﺼﺮﺗﻲ در ﺳﺎل 1383 اﻧﺠﺎم ﺷﺪ و ﻣﻴﺰان ﺿﺮﻳﺐ آﻟﻔﺎي ﻛﺮوﻧﺒﺎخ د ر دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﻣﺎدر 82 /0 ، دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﭘﺪر83 /0 و دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﻫﻤﺴﺎﻻن92 /0 ﺑﻮد . 353 در ﭘﮋوﻫﺶ واﺣﺪي و ﻓﺘﺤﻲ42 ) 1389 ( ﻣﻴﺰان ﭘﺎﻳﺎﻳﻲ آن ﻣﻮرد ﺑﺮرﺳﻲ ﻣﺠﺪد ﻗﺮار ﮔﺮﻓﺖ . اﺑﺰار ﭘﮋوﻫﺶ ﺑﻌﺪ از ﺗﺄﻳﻴﺪ و ﻣﻄﻠﻮب ﺑﻮدن رواﻳﻲ ﺻﻮري و ﻣﺤﺘﻮاﻳﻲ، ﭘﺎﻳﺎﻳﻲ دروﻧﻲ ﭘﺮﺳﺸﻨﺎﻣﻪ ﺑﺎ روش آﻟﻔﺎي ﻛﺮوﻧﺒﺎخ ﺑﺮاي ﻣﻘﻴﺎس دﻟ ﺒﺴﺘﮕﻲ ﻣﺎدر، ﭘﺪر و ﻫﻤﺴﺎﻻن ﺑﻪ ﺗﺮﺗﻴﺐ 85 /0 ، 83 /0 و86 /0 ﺑﻮد . در ﭘﮋوﻫﺶ ﺣﺎﺿﺮ ﻧﻴﺰ ﺑﻪ ﻣﻨﻈﻮر ﭘﺎﻳﺎﻳﻲ از روش آﻟﻔﺎي ﻛﺮوﻧﺒﺎخ ﺑﻪ ﺗﺮﺗﻴﺐ دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﻣﺎدر92 /0 ، دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﭘﺪر95 /0 و دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﻫﻤﺴﺎﻻن90 /0 ﺑﻮد . ﻳﺎﻓﺘﻪ ﻫﺎ ﺟﺪول 1 اﻃﻼﻋﺎت ﺗﻮﺻﻴﻔﻲ ﻣﻴﺎﻧﮕﻴﻦ و اﻧﺤﺮاف اﺳﺘﺎﻧﺪارد ﻣ ﺘﻐﻴﺮﻫﺎي اﺻﻠﻲ ﭘﮋوﻫﺶ ) ،ﺗﻮاﻧﻤﻨﺪي ﻫﺎي ﻣﻨﺶ دﻟﺒﺴﺘﮕﻲ و ﺗﻌﺎرض ( را در داﻧﺶ آﻣﻮزان ﻧﺸﺎن ﻣﻲ دﻫﺪ . ﺑﺎﻻﺗﺮﻳﻦ ﻣﻴﺎﻧﮕﻴﻦ ﺗﻌﺎﻟﻲ و ﻛﻤﺘﺮﻳﻦ آن ﺗﻌﺎرض ﺑﺎ ﻣﺎدر اﺳﺖ . ﺑﺎﻻﺗﺮﻳﻦ ﺳﻄﺢ اﻧﺤﺮاف اﺳﺘﺎﻧﺪارد ﺗﻌﺎﻟﻲ و ﻛﻤﺘﺮﻳﻦ آن ﺗﻌﺎرض ﺑﺎ ﻣﺎدر اﺳﺖ . ﻫﻢ ﭼﻨﻴﻦ ﺿﺮاﻳﺐ ﻫﻤﺒﺴﺘﮕﻲ ﺗﻮاﻧﻤﻨﺪي ﻫﺎي ﻣﻨﺶ ) ﺧﺮد، ﺷﺠﺎﻋ ﺖ، اﻧﺴﺎﻧﻴﺖ، ﻋﺪاﻟﺖ، اﻋﺘﺪال و ﺗﻌﺎﻟﻲ ( ، دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﻣﺎدر، ﭘﺪر و ﻫﻤﺴﺎﻻن و ﺗﻌﺎرض ﺑﺎ ﭘﺪر و ﻣﺎدر را ﻧﺸﺎن ﻣﻲ دﻫﺪ . ﻫﻤﺎن ﻃﻮر ﻛﻪ ﻣﻼﺣﻈﻪ ﻣﻲ ﻛﻨﻴﺪ، ﺑﻴﻦ ﺑﺮﺧﻲ ﻣﺘﻐﻴﺮﻫﺎي ﭘﮋوﻫﺶ راﺑﻄﻪ ﻣﻌﻨﺎداري وﺟﻮد دارد . ﺑﻴﻦ ﻋﺪاﻟﺖ، اﻋﺘﺪال و ﺗﻌﺎﻟﻲ ﺑﺎ دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﭘﺪر و ﻫﻤﺴﺎﻻن و ﺧﺮد ﺑﺎ ﺗﻌﺎرض و دﻟ ﺒﺴﺘﮕﻲ ﺑﻪ ﭘﺪر راﺑﻄﻪ ﻣﻌﻨﺎداري وﺟﻮد ﻧﺪارد . اﺑﺰار ﭘﮋوﻫﺶ ﺟﺪول1 :ﻣﺎﺗﺮﻳﺲ ﻫﻤﺒﺴﺘﮕﻲ ﺗﻌﺎرض ﺑﺎ واﻟﺪﻳﻦ، دﻟﺒﺴﺘﮕﻲ ﺑﻪ واﻟﺪﻳﻦ و ﻫﻤﺴﺎﻻن و ﺗﻮاﻧﻤﻨﺪي ﻫﺎي ﻣﻨﺶ 11 10 9 8 7 6 5 4 3 2 1 S M ﻣﺘﻐﻴﺮ 1 99 /5 52 / 23 1 - ﺗﻌﺎرض ﭘﺪر 1 13 /0 3/5 06 / 23 2 - ﺗﻌﺎرض ﻣﺎدر 1 * 60 /0 14 /0 - 33 / 18 44 / 95 3 - دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﻣﺎدر 1 42 /0 22 /0 - 60 /0 - 55 / 24 98 / 88 4 - دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﭘﺪر 1 30 /0 40 /0 19 /0 - 22 /0 - 34 / 16 64 / 94 5 - دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﻫﻤﺴﺎﻻن 1 * 15 /0 10 /0 20 /0 - 19 /0 - 11 /0 32 / 71 62 / 684 6 -ﺧﺮد 1 ** 51 /0 1/0 13 /0 11 /0 9/0 - * 17 /0 09 / 45 65 / 344 7 -ﺷﺠﺎﻋﺖ 1 * 15 /0 30 /0 24 /0 23 /0 22 /0 21 /0 - 10 /0 - 49 / 31 65 / 246 8 -اﻧﺴﺎﻧﻴﺖ 1 36 /0 51 /0 54 /0 08 /0 10 /0 * 17 /0 ** 20 /0 - * 23 /0 - 20 / 49 66 / 338 9 -ﻋﺪاﻟﺖ 1 42 /0 23 /0 41 /0 35 /0 01 /0 1/0 23 /0 24 /0 - 12 /0 83 / 46 95 / 330 10 -اﻋﺘﺪال 1 40 /0 47 /0 41 /0 48 /0 51 /0 09 /0 13 /0 19 /0 ** 24 /0 - 13 /0 - 46 / 86 59 / 823 11 -ﺗﻌﺎﻟﻲ P< 05 /0 *P< 01 /0 در ﺟﺪول2 ﻧﺘﺎﻳﺞ ﺗﺤﻠﻴﻞ وارﻳﺎﻧﺲ ﺑﺎ روش ﭘﺴﺮوﻧﺪه43 اﺟﺮا ﺷﺪ . ﺗﺤﻠﻴﻞ در ﻫﻔﺖ ﮔﺎم اﻧﺠﺎم ﺷﺪ ﻛﻪ در ﺷﺶ ﮔﺎم اول ﺑﻪ ﺗﺮﺗﻴﺐ ﻣﺘﻐﻴﺮﻫﺎي ﺧﺮد، ﺗﻌﺎر ض ﺑﺎ ﻣﺎدر، ﺷﺠﺎﻋﺖ، دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﻫﻤﺴﺎﻻن، اﻧﺴﺎﻧﻴﺖ از ﻣﻌﺎدل ﺧﺎرج ﺷﺪ، در ﮔﺎم ﻫﻔﺘﻢ ﻣﺘﻐﻴﺮﻫﺎي دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﭘﺪر، ﻋﺪاﻟﺖ، اﻋﺘﺪال، ﺗﻌﺎﻟﻲ و ﺗﻌﺎﻣﻞ ﻋﺪاﻟﺖ و ﺗﻌﺎرض ﺑﺎ ﭘﺪر ﺑﻪ ﻋﻨﻮان ﻣﺘﻐﻴﺮﻫﺎي ﭘﻴﺶ ﺑﻴﻨﻲ ﻛﻨﻨﺪه در ﻣﻌﺎدل ﺑﺎﻗﻲ ﻣﺎﻧﺪﻧﺪ . 98 /0 از وارﻳﺎﻧﺲ ﺗﻐﻴﻴﺮات ﻣﻼك ﺗﻌﺎرض ﺑﺎ ﭘﺪر ﺗﻮﺳﻂ / ﭘﻴﺶ ﺑﻴﻨﻲ ﺗﻮاﻧﻤﻨﺪي ﻣﻨﺶ ﻧﻮﺟﻮاﻧﺎن و دﻟﺒﺴﺘﮕﻲ ﺑﻪ واﻟﺪﻳﻦ و/... ﻳﺎﻓﺘﻪ ﻫﺎ 98 /0 از وارﻳﺎﻧﺲ ﺗﻐﻴﻴﺮات ﻣﻼك ﺗﻌﺎرض ﺑﺎ ﭘﺪر ﺗﻮﺳﻂ ﺗﻐ ﻴﻴﺮات ﻣﺘﻐﻴﺮﻫﺎي ﭘﻴﺶ ﺑﻴﻦ ﺗﺒﻴﻴﻦ ﻣﻲ ﺷﻮد ﺑﺎ 1869,350 =f در ﺳﻄﺢ )001 /0 < P ( ﻣﻌﻨﺎدار اﺳﺖ . ﻧﺘﺎﻳﺞ ﺗﺤﻠﻴﻞ رﮔﺮﺳﻴﻮن در ﺟﺪول زﻳﺮ آﻣﺪه اﺳﺖ . 354 / / !"# $% / &'%( / / ﺟﺪول2 : ﻧﺘﺎﻳﺞ ﺗﺤﻠﻴﻞ رﮔﺮﺳﻴﻮن ﺑﺮاي ﭘﻴﺶ ﺑﻴﻨﻲ ﺗﻌﺎرض ﺑﺎ ﭘﺪر ﺑﺮ اﺳﺎس ﺗﻌﺎﻣﻞ ﻋﺪاﻟﺖ و ﺗﻌﺎرض ﺑﺎ ﭘﺪر، دﻟﺒﺴﺘﮕﻲ ﺑﻪ واﻟﺪﻳﻦ و ﺗﻮاﻧﻤﻨﺪي ﻫﺎي ﻣﻨﺶ sig t SE b ﻣﺘﻐﻴﺮﻫﺎي ﭘﻴﺶ ﺑﻴﻦ 001 /0 742 /3 - 040 /0 003 /0 01 /0 - دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﭘﺪر 001 /0 219 / 46 - 598 /0 002 /0 073 /0 - ﻋﺪاﻟﺖ 005 /0 823 /2 33 /0 001 /0 004 /0 اﻋﺘﺪال 03 /0 151 /2 - 025 /0 001 /0 002 /0 - ﺗﻌﺎﻟﻲ 001 /0 477 / 94 057 /1 001 /0 003 /0 ﻋﺪاﻟﺖ × ﺗﻌﺎرض ﺑﺎ ﭘﺪر د ر ﺟﺪول 3 ، ﺗﺤﻠﻴﻞ در ﺷﺶ ﮔﺎم اﻧﺠﺎم ﺷﺪ ﻛﻪ در ﭘﻨﺞ ﮔﺎم اول ﺑﻪ ﺗﺮﺗﻴﺐ ﻣﺘﻐﻴﺮﻫﺎي ﺗﻌﺎرض ﺑﻪ ﭘﺪر اﻋﺘﺪال، دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﭘﺪر، دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﻫﻤﺴﺎﻻن و ﺷﺠﺎﻋﺖ از ﻣﻌﺎدﻟﻪ ﺧﺎرج ﺷﺪ، در ﮔﺎم ﺷﺸﻢ ﻣﺘﻐﻴﺮﻫﺎي دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﻣﺎدر، ﺧﺮد، اﻧﺴﺎﻧﻴﺖ، ﻋﺪاﻟﺖ، ﺗﻌﺎﻟﻲ و ﺗﻌﺎﻣﻞ دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﻣﺎدر و ﺗﻌﺎﻟﻲ ﺑﻪ ﻋﻨ ﻮان ﻣﺘﻐﻴﺮﻫﺎي ﭘﻴﺶ ﺑﻴﻨﻲ ﻛﻨﻨﺪه در ﻣﻌﺎدﻟﻪ ﺑﺎﻗﻲ ﻣﺎﻧﺪﻧﺪ . ﺿﺮﻳﺐ ﺗﻌﻴﻴﻦ ﻣﺘﻐﻴﺮﻫﺎي ﺑﺎﻗﻲ ﻣﺎﻧﺪه ﺑﺎ ﻣﺘﻐﻴﺮ ﻣﻼك ﺗﻌﺎرض ﺑﺎ ﻣﺎدر 07 /0 ﺑﺎ1971,14 =f در ﺳﻄﺢ )001 /0 < P (ﻣﻌﻨﺎدار اﺳﺖ . ﻧﺘﺎﻳﺞ ﺗﺤﻠﻴﻞ رﮔﺮﺳﻴﻮن در ﺟﺪول زﻳﺮ آﻣﺪه اﺳﺖ . ﻳﺎﻓﺘﻪ ﻫﺎ ﺟﺪول 3 :ﻧﺘﺎﻳﺞ ﺗﺤﻠﻴﻞ رﮔﺮﺳﻴﻮن ﺑﺮاي ﭘﻴﺶ ﺑﻴﻨﻲ ﺗﻌﺎرض ﺑﺎ ﻣﺎ در ﺑﺮ اﺳﺎس ﺗﻌﺎﻣﻞ ﻋﺪاﻟﺖ و ﺗﻌﺎرض ﺑﺎ ﻣﺎدر، دﻟﺒﺴﺘﮕﻲ ﺑﻪ واﻟﺪﻳﻦ و ﺗﻮاﻧﻤﻨﺪي ﻫﺎي ﻣﻨﺶ Sig t B SE b ﻣﺘﻐﻴﺮﻫﺎي ﭘﻴﺶ ﺑﻴﻦ 02 /0 267 /2 - 026 /0 003 /0 007 /0 دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﻣﺎدر 004 /0 939 /2 034 /0 001 /0 003 /0 ﺧﺮد 009 /0 631 /2 - 027 /0 - 002 /0 005 /0 - اﻧﺴﺎﻧﻴﺖ 009 /0 808 /2 - 0032 /0 - 001 /0 004 /0 - ﻋﺪاﻟﺖ 001 /0 96 / 38 - 47 /0 - 001 /0 030 /0 - ﺗﻌﺎﻟﻲ 001 /0 79 / 85 98 /0 - 001 /0 001 /0 دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﻣﺎدر × ﺗﻌﺎﻟﻲ ﺟﺪول 3 :ﻧﺘﺎﻳﺞ ﺗﺤﻠﻴﻞ رﮔﺮﺳﻴﻮن ﺑﺮاي ﭘﻴﺶ ﺑﻴﻨﻲ ﺗﻌﺎرض ﺑﺎ ﻣﺎ در ﺑﺮ اﺳﺎس ﺗﻌﺎﻣﻞ ﻋﺪاﻟﺖ و ﺗﻌﺎرض ﺑﺎ ﻣﺎدر، دﻟﺒﺴﺘﮕﻲ ﺑﻪ واﻟﺪﻳﻦ و ﺗﻮاﻧﻤﻨﺪي ﻫﺎي ﻣﻨﺶ Sig t B SE b ﻣﺘﻐﻴﺮﻫﺎي ﭘﻴﺶ ﺑﻴﻦ 02 /0 267 /2 - 026 /0 003 /0 007 /0 دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﻣﺎدر 004 /0 939 /2 034 /0 001 /0 003 /0 ﺧﺮد 009 /0 631 /2 - 027 /0 - 002 /0 005 /0 - اﻧﺴﺎﻧﻴﺖ 009 /0 808 /2 - 0032 /0 - 001 /0 004 /0 - ﻋﺪاﻟﺖ 001 /0 96 / 38 - 47 /0 - 001 /0 030 /0 - ﺗﻌﺎﻟﻲ 001 /0 79 / 85 98 /0 - 001 /0 001 /0 دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﻣﺎدر × ﺗﻌﺎﻟﻲ ﺑﺤﺚ و ﻧﺘﻴﺠﻪ ﮔﻴﺮي ﻫﺪف ﭘﮋوﻫﺶ ﺣﺎﺿﺮ ﭘﻴﺶ ﺑﻴﻨﻲ ﺗﻮاﻧﻤﻨﺪي ﻫﺎي ﻣﻨﺶ ﻧﻮﺟﻮاﻧﺎن و دﻟﺒﺴﺘﮕﻲ ﺑﻪ واﻟﺪﻳﻦ و ﻫﻤﺴﺎﻻن ﺑﺮ اﺳﺎس ﺗﻌﺎرض ﺑﺎ ﭘﺪر و ﻣﺎدر اﺳﺖ . ﻧﺘﺎﻳﺞ ﻧﺸﺎن داد ﺑﻴﻦ دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﻣﺎدر و ﺗﻌﺎرض ﺑﻪ ﻣﺎدر راﺑﻄﻪ ﻣﺜﺒﺖ وﺟﻮد دارد از . ﻃﺮف دﻳﮕﺮ دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﭘﺪر راﺑﻄﻪ ﻣﻨﻔﻲ ﺑﺎ ﺗﻌﺎرض ﺑﻪ ﻣﺎدر را ﻧﺸﺎن داد . ﻫﻢ ﭼﻨﻴﻦ دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﻫﻤﺴﺎﻻن ارﺗﺒﺎط ﻣﻨﻔﻲ ﺑﺎ ﺗﻌﺎرض واﻟﺪﻳﻦ دارد ﻛﻪ ﻧﺸﺎن دﻫﻨﺪه اﻳﻦ اﺳﺖ، ﻧﻮﺟﻮان در اﻳﻦ دوران ﻧﻴﺎزﻣﻨﺪ دﻟﺒﺴﺘﮕﻲ ﺑﻪ واﻟﺪ ﻳﻦ و ﻫﻤﺴﺎﻻن اﺳﺖ . ﺧﺮد، اﻧﺴﺎﻧﻴﺖ، ﻋﺪاﻟﺖ، اﻋﺘﺪال و ﺗﻌﺎﻟﻲ ارﺗﺒﺎط ﻣﻨﻔﻲ ﺑﺎ ﺗﻌﺎرض ﺑﻪ ﻣﺎدر دارد . اﻧﺴﺎﻧﻴﺖ، ﻋﺪاﻟﺖ، اﻋﺘﺪال و ﺗﻌﺎﻟﻲ راﺑﻄﻪ ﻣﺜﺒﺘﻲ ﺑﺎ دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﻣﺎدر دارد . ﺧﺮد و اﻧﺴﺎﻧﻴﺖ ﻧﻴﺰ راﺑﻄﻪ ﻣﺜﺒﺘﻲ ﺑﺎ دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﻫﻤﺴﺎﻻن ﻧﺸﺎن داد . ﻳﺎﻓﺘﻪ ﻫﺎ ﺧﺮد ﻧﻮﻋﻲ داﻧﺎﻳﻲ اﺳﺖ ﻛﻪ ﺑﺎ داﺷﺘﻦ آن ﻓﺮ د ﻣﻲ ﺗﻮاﻧﺪ ﺑﺴﻴﺎري از داﺷﺘﻪ ﻫﺎي ﺧﻮد را ﺑﺎ دﻳﮕﺮان ﻗﺴﻤﺖ ﻛﻨﺪ و ﻣﻮﺟﺒﺎت ژرف ﻧﮕﺮي در ﻓﺮد ﻣﻲ ﺷﻮد ) ﭘﻴﺘﺮﺳﻮن، ﺳﻴﻠﮕﻤﻦ، 2004 .( ﻧﻮﺟﻮان ﺑﺎ وﺟﻮد ﭼﻨﻴﻦ وﻳﮋﮔﻲ ﻣﻲ ﺗﻮاﻧﺪ از ﺗﻌﺎرض ﻫﺎي ﺑﻴﻦ ﺧﻮد و واﻟﺪﻳﻦ ﺑﺮداﺷﺖ ﺑﻬﺘﺮي داﺷﺘﻪ ﺑﺎﺷﺪ و ﻲﻮ ﺮي رﺮ ژر وﻮﺒ ﺮن ﻦ ﭘﺮﻮن ﻧﻮﺟﻮان ﺑﺎ وﺟﻮد ﭼﻨﻴﻦ وﻳﮋﮔﻲ ﻣﻲ ﺗﻮاﻧﺪ از ﺗﻌﺎرض ﻫﺎي ﺑﻴﻦ ﺧﻮد و واﻟﺪﻳﻦ ﺑﺮداﺷﺖ ﺑﻬﺘﺮي داﺷﺘﻪ ﺑﺎﺷﺪ و وﺟﻮد دﻳﺪﮔﺎه ﻣﺘﻔﺎوت ﺧﻮد و واﻟﺪﻳﻦ را درك ﻛﻨﺪ . اﻧﺴﺎﻧﻴﺖ ، ﭘﺮورش ﻋﺸﻖ ﺑﻪ ﺧﻮد و دﻳﮕﺮان ﺑﺎ اﻓﺰاﻳﺶ اﺣﺴﺎس 355 / ﭘﻴﺶ ﺑﻴﻨﻲ ﺗﻮاﻧﻤﻨﺪي ﻣﻨﺶ ﻧﻮﺟﻮاﻧﺎن و دﻟﺒﺴﺘﮕﻲ ﺑﻪ واﻟﺪﻳﻦ و/... 355 / ﭘﻴﺶ ﺑﻴﻨﻲ ﺗﻮاﻧﻤﻨﺪي ﻣﻨﺶ ﻧﻮﺟﻮاﻧﺎن و دﻟﺒﺴﺘﮕﻲ ﺑﻪ واﻟﺪﻳﻦ و/... ارﺗﺒﺎط اﺟﺘﻤﺎﻋﻲ و ﻣﺜﺒﺖ ﺑﻮدن ﻧﺴﺒﺖ ﺑﻪ دﻳﮕﺮان و ﻋﻮاﻃﻒ ﻣﺜﺒﺖ ﻣﺸﺨﺺ ﻣﻲ ﺷﻮد ) ﻧﻴﻤﻴﻚ44 ، 2013 .( ﭼﻨﻴﻦ ﻓﻀﻴﻠﺘﻲ در ﻧﻮﺟﻮان ﺟﻬﺖ دﻫﻨﺪه ﭘﺮورش رواﺑﻂ دوﺳﺘﺎﻧﻪ ﺑﺎ واﻟﺪﻳﻦ اﺳﺖ و ﺳﭙﺮي در ﺑﺮاﺑﺮ ﺷﺪت ﺗﻌﺎرض اﺳﺖ و ﺑﻪ اﻳﺠﺎد ﻋﺸﻖ ﻋﻤﻴﻖ ﺗﺮي ﻛﻤﻚ ﻣﻲ ﻛ ﻨﺪ . ﻋﺪاﻟﺖ ﭘﺎﻳﻪ و اﺳﺎس زﻧﺪﮔﻲ اﺟﺘﻤﺎﻋﻲ ﺳﺎﻟﻢ را درﺑﺮ ﻣﻲ ﮔﻴﺮد ) ﭘﻴﺘﺮﺳﻮن، ﺳﻴﻠﮕﻤﻦ، 2004 .( از اﻳﻦ رو ﻧﻮﺟﻮان ﻧﻴﺎز دارد ﻋﻼوه ﺑﺮ ﺑﻮدن ﺑﺎ واﻟﺪﻳﻦ ﺑﺎ ﻫﻤﺴﺎﻻن ﺧﻮد ﻧﻴﺰ زﻣﺎﻧﻲ را ﺻﺮف ﻛﻨﺪ . ﭼﻨﻴﻦ ﺧﺼﻴﺼﻪ اي، ﻧﻮﺟﻮان را در ﺗﻌﺎرض ﻛﻤﺘﺮي ﺑﺎ واﻟﺪﻳﻦ و ﻫﻤﺴﺎﻻن ﻗﺮار ﻣﻲ دﻫﺪ و داﺷﺘﻦ دﻟﺒﺴﺘﮕ ﻲ اﻳﻤﻦ ﺗﻀﻤﻴﻦ ﻛﻨﻨﺪه ﭼﻨﻴﻦ رواﺑﻄﻲ ﺑﺎ واﻟﺪﻳﻦ و ﻫﻤﺴﺎﻻن اﺳﺖ . ﻋﺪاﻟﺖ در ﺗﻌﺪﻳﻞ ﻣﺸﻜﻼت ﻣﺆﺛﺮ اﺳﺖ ﺗﺎ ﻧﻮﺟﻮان از ﺑﺮداﺷﺖ ﺳﻮﮔﻴﺮاﻧﻪ در رواﺑﻂ ﺧﻮد و واﻟﺪﻳﻦ ﻣﺼﻤﻮن ﺑﻤﺎﻧﺪ . اﻋﺘﺪال ﻣﺒﺘﻨﻲ ﺑﺮ ﺧﻮدﻛﻨﺘﺮﻟﻲ رﻓﺘﺎري و ﻫﻴﺠﺎﻧﻲ اﺳﺖ ﻛﻪ در ﺑﺮاﺑﺮ زﻳﺎده روي، رﻓﺘﺎر و ﻫﻴﺠﺎﻧﺎت را ﺗﻨﻈﻴﻢ ﻣﻲ ﻛﻨﺪ اﻳ . ﻦ ﺗﻮاﻧﻤﻨﺪي ارﺗﺒﺎط ﻗﻮي ﺑﺎ راﻫﺒﺮدﻫﺎي ﻣﻘﺎﺑﻠﻪ اي در ﻣﻘﺎﺑﻞ ﺷﺮاﻳﻂ اﺳﺘﺮس آور و ﺗﻌﺎرض دارد ) ﺷﻮﺷﺎﻧﻲ، ﺷﺎورﺗﺰ، 2016 .( ﻟﺬا ﺑﻪ ﻧﻮﺟﻮان ﻛﻤﻚ ﻣﻲ ﻛﻨﺪ در ﺑﺮاﺑﺮ ﺗﻌﺎرﺿﺎﺗﻲ ﻛﻪ در ارﺗﺒﺎط ﺑﺎ واﻟﺪﻳﻦ ﺧﻮد ﭘﻴﺶ ﻣﻲ آﻳﺪ رﻓﺘﺎر و ﻫﻴﺠﺎﻧﺎت ﺧﻮد را ﺑﻬﺘﺮ ﻛﻨﺘﺮل ﻛﻨﺪ . ﺗﻌﺎدل ﺑﻴﻦ ﻓﻜﺮ و ﻫﻴﺠﺎن در ﺑﻬﺒﻮ د رواﺑﻂ واﻟﺪﻳﻦ و ﻧﻮﺟﻮان اﺛﺮات ﺑﺴﺰاﻳﻲ دارد . ﻳﺎﻓﺘﻪ ﻫﺎ ﺗﻌﺎﻟﻲ ﻣﻌﻨﺎ و ﻫﺪﻓﻲ ﺑﺮﺗﺮ ﺑﺮاي زﻧﺪﮔﻲ ﻓﺮد اﻳﺠﺎد ﻣﻲ ﻛﻨﺪ و ﻧﮕﺮاﻧﻲ روزﻣﺮه را ﻛﻢ رﻧﮓ ﻣﻲ ﺳﺎزد ) ﭘﻴﺘﺮﺳﻮن، ﺳﻴﻠﮕﻤﻦ، 2004 .( وﺟﻮد ﻓﻀﻴﻠﺘﻲ ﻫﻢ ﭼﻮن ﺗﻌﺎﻟﻲ ﻛﻪ در ﺑﺮاﺑﺮ ﮔﻴﺮﻧﺪه ﻣﻌﻨﻮﻳﺖ اﺳﺖ، ﻧﮕﺮاﻧﻲ ﻫﺎ و ﺗﻌﺎرﺿﺎت ﭘﻴﺶ آﻣﺪه را ﻛﻤﺮﻧﮓ ﺗﺮ ﻣﻲ ﺳﺎز د و ﺑﻪ او ﻛﻤﻚ ﻣﻲ ﻛﻨﺪ ﺗﺎ ﺧﻮد را ﻣﺘﺼﻞ ﺑﻪ ﻗﺪرت واﻻﺗﺮ و ﺑﺮﺗﺮ ﺑﺪاﻧﺪ و ﻓﺸﺎر رواﻧﻲ ﻧﺎﺷﻲ از ﺗﻌﺎرض ﻛﺎﻫﺶ ﭘﻴﺪا ﻛﻨﺪ . از ﺳﻮي دﻳﮕﺮ ﺧﺮد، اﻧﺴﺎﻧﻴﺖ، ﻋﺪاﻟﺖ، اﻋﺘﺪال و ﺗﻌﺎﻟﻲ، ارﺗﺒﺎط ﻣﺜﺒﺘﻲ ﺑﺎ ﻳﻜﺪﻳﮕﺮ دارﻧﺪ . ﻫﻢ ﭼﻨﻴﻦ ﺷﺠﺎﻋﺖ راﺑﻄﻪ ﻣﺜﺒﺘﻲ ﺑﺎ ﺗﻌﺎرض ﭘﺪر دارد . ﭘﻴﺘﺮﺳﻮن و ﺳﻴﻠﮕﻤﻦ ) 2004 ( ﺷﺠﺎﻋﺖ را اراده ي ﻗﻮي ﻣﻲ داﻧﻨﺪ ﻛﻪ ﺑﺮاي رﺳﻴﺪن ﺑﻪ ﻫﺪف در ﻣﻮاﺟﻬﻪ ﺑﺎ ﻣﺸﻜﻼت دروﻧﻲ و ﺑﻴﺮوﻧﻲ، ﻓﺮد را ﻣﺠﻬﺰ ﻣﻲ ﻛﻨﺪ . از ﺟﻤﻠﻪ وﻳﮋﮔﻲ ﻫﺎي ﻧﻮﺟﻮاﻧﻲ ﺗﻌﺎرض و ﻛﺸﻤﻜﺶ ﺑﺮاي ﺑﻪ دﺳﺖ آوردن اﺳﺘﻘﻼل اﺳﺖ، ﺷﺠﺎﻋﺖ ﺑﻪ ﻧﻮﺟﻮان ﻛﻤﻚ ﻣﻲ ﻛﻨﺪ ﺗﺎ ﺑﺮاي دﺳﺘﻴﺎﺑﻲ ﺑﻪ اﺳﺘﻘﻼل ﺗﻼش ﻛﻨﺪ ﻛﻪ ﮔﺎﻫﻲ ﺗﻮأم ﺑ ﺎ ﺗﻌﺎرض ﺑﺎ واﻟﺪﻳﻦ اﺳﺖ و ﺑﺮاي اﻳﻨﻜﻪ اﺣﺴﺎس اﻣﻨﻴﺖ ﺧﻮد را ﺣﻔﻆ ﻛﻨﺪ، دﻟﺒﺴﺘﮕﻲ ﺑﺎ ﻫﻤﺴﺎﻻن را داراﺳﺖ . ﻧﻈﺮﻳﻪ دﻟﺒﺴﺘﮕﻲ ﺑﻴﺎن ﻣﻲ ﻛﻨﺪ ﺗﺠﺮﺑﻪ ﻫﺎي اﻣﻦ ﺑﺎ واﻟﺪﻳﻦ راﻫﻨﻤﺎي ﺗﺠﺮﺑﻪ ﻫﻴﺠﺎﻧﻲ ﻧﻮﺟﻮان اﺳﺖ ) ﺑﺎﻟﺒﻲ45 ، 1988 .( ﻧﻮﺟﻮان ﻫﺎﻳﻲ ﻛﻪ ﺑﺎ واﻟﺪﻳﻦ درﺑﺎره دﻳﺪﮔﺎه ﻫﺎ و ﻣﺸﻜﻼﺗﺸﺎن ﮔﻔﺖ وﮔﻮ ﻣ ﻲ ﻛﻨﻨﺪ و ﺗﻼش ﻣﻲ ﻛﻨﻨﺪ ﻣﺴﺎﺋﻞ ﺧﻮد را ﺣﻞ ﻛﻨﻨﺪ، ﻛﻤﺘﺮ ﺧﺸﻢ و اﺟﺘﻨﺎب از ﺣﻞ ﻣﺴﺄﻟﻪ را ﺑﻪ ﻛﺎر ﻣﻲ ﺑﺮﻧﺪ . ﺑﻨﺎﺑﺮاﻳﻦ ﺑﻴﺎن ﻫﻤﺪﻟﻲ ﻧﻪ ﺗﻨﻬﺎ در ارﺗﺒﺎط ﻧﻮﺟﻮان ﺑﺎ ﭘﺪر ﮔﺴﺘﺮش ﻣﻲ ﻳﺎﺑﺪ، ﺑﻠﻜﻪ اﺳﺘﻘﻼل ﻣﻨﺎﺳﺒﻲ در ﭼﺎﻟﺶ ﻫﺎي ﺧﻮد ﺑﺎ ﻣﺎدر ﻧﻴﺰ ﻧﺸﺎن ﻣﻲ دﻫﺪ ) ﻫﺮﺷﻨﺒﺮگ، داوﻳﻞ، ﻳﻮﻧﺪا، اﺳﺘﺎر، ﻻﻛﺮ و ﻫﻤﻜﺎران 46 ، 2012 .( ﻟﺬا ﻫﻤﺎن ﮔﻮﻧﻪ ﻛﻪ ﻣﻮرﺗﻲ )2004 ( ،ﻣﻄﺮح ﻛﺮد دﻟﺒﺴﺘﮕﻲ ﻣﻲ ﺗﻮاﻧﺪ ﭘﺎﻳﻪ ﻫﺎي اﻣﻦ رﺷﺪ اﺟﺘﻤﺎﻋﻲ، ﻋﺎﻃﻔﻲ را اﻳﺠﺎد ﻛﻨﺪ . ﻫﺎﺑﻨﻴﺰ )2013 ( ﻧﻴﺰ ﺑﻴﺎن ﻣﻲ ﻛﻨﺪ ﺗﺤﻮل ﻣﺜﺒﺖ ﻣﻲ ﺗﻮاﻧﺪ ﺗﻔﻜﺮات اﻣﻴﺪﺑﺨﺶ را در ﻣﺤﻴﻂ ﺧﺎﻧﻪ ﺑﺮاي ﻧﻮﺟﻮاﻧﺎن ﻣﻴﺴﺮ ﺳﺎزد . ﺑﺮ اﺳﺎس ﻧﻮع رواﺑﻄﻲ ﻛﻪ ﻧﻮﺟﻮان ﺑﺎ ﭘﺪر و ﻣﺎدر ﺧﻮد دارد، ﺗﻌﺎرض ﺑﻴﻦ آن ﻫﺎ ﻣﺘﻔﺎوت اﺳﺖ ﭼﺮا ﻛﻪ ﻫﺮ ﻛﺪام رواﺑﻂ ﻋﺎﻃﻔﻲ، ﻫﻴﺠﺎﻧﻲ، اﺟﺘﻤﺎﻋﻲ ﻣﺘﻔﺎوﺗﻲ را ﺑﺮاي ﻧﻮﺟﻮان اﻳﻔﺎ ﻣﻲ ﻛﻨﻨﺪ . در اﻳﻦ ﭘﮋوﻫﺶ ﻧﺸﺎن داده ﺷﺪ ﻧﻮﺟﻮاﻧﻲ ﻛﻪ از دﻟﺒﺴﺘﮕﻲ اﻣﻦ ﺗﺮ ﺑﺎ ﭘﺪر و ﻣﺎدر ﺧﻮد ﺑﺮﺧﻮردار اﺳﺖ، ﺧﺮد، اﻧﺴﺎﻧﻴﺖ، ﻋﺪاﻟﺖ، اﻋﺘﺪال و ﺗﻌﺎﻟﻲ را ﻫﻢ زﻣﺎن دارا ﻫﺴﺘﻨﺪ و ﺗﻌﺎرض ﻛﻤﺘﺮي را ﻧﺴﺒﺖ ﺑﻪ واﻟﺪﻳﻦ ﺧﻮد ﻧﺸﺎن ﻣﻲ دﻫﻨﺪ . ﻳﺎﻓﺘﻪ ﻫﺎ ﺑﻪ ﻋﺒﺎرﺗﻲ اﺣﺴﺎس اﻣﻨﻴﺖ در ارﺗﺒﺎط ﻧﻮﺟﻮان ﺑﺎ واﻟﺪﻳﻦ ﺑﺎ ﺗﻮاﻧﺎﻳﻲ رﺷﺪ اﺳﺘﻘﻼل و ﺣﻤﺎﻳﺖ واﻟﺪﻳﻦ 356 / / !"# $% / &'%( / / ﻣﺮﺗﺒﻂ اﺳﺖ . ﻫﻤﺎن ﻃﻮر ﻛﻪ ﻫﺮﺷﻨﺒﺮگ )2012 ( ﺑﻴﺎن ﻛﺮد اﻋﺘﻤﺎد ﻣﻮﺟﺐ ﭘﺬﻳﺮش ﻧﻮﺟﻮان ﻣﻲ ﺷﻮد . ﭼﻴﺰي ﻛﻪ در اﻳﻦ ﭘ ﮋوﻫﺶ ﺑﺴﻴﺎر ﻣﺘﻤﺎﻳﺰ ﺑﻪ ﻧﻈﺮ ﻣﻲ رﺳﺪ، داﺷﺘﻦ ﻓﻀﻴﻠﺖ ﻫﺎي ﻫﻤﭽﻮن ﺧﺮد، اﻧﺴﺎﻧﻴﺖ، ﻋﺪاﻟﺖ، اﻋﺘﺪال و ﺗﻌﺎﻟﻲ ﺗﻌﺎرض را ﻛﻢ رﻧﮓ ﻣﻲ ﻛﻨﺪ . ﻓﻀﺎﺋﻞ ﭘﺎﻳﻪ و ﺣﺎﻟﺖ ﻫﺎي اﻧﺴﺎﻧﻲ اﺳﺖ و ﻫﻤﺎﻫﻨﮓ ﺑﻮدن ﺑﺎ ﭼﻨﻴﻦ ﻓﻀﻴﻠﺖ ﻫﺎﻳﻲ ﻓﺮد را ﺑﻪ زﻧﺪﮔﻲ ﺧﻮب روان ﺷﻨﺎﺧﺘﻲ ﻫﺪاﻳﺖ ﻣﻲ ﻛﻨﺪ ) ﭘﻴﺘﺮﺳﻮن، ﺳﻴﻠﮕﻤﻦ، 2004 .( از ﺳﻮ ي دﻳﮕﺮ ﻃﺒﻖ ﻧﻈﺮ ﺑﺎﻟﺒﻲ دﻟﺒﺴﺘﮕﻲ ﭘﻴﻮﻧﺪ ﻋﺎﻃﻔﻲ ﺑﺎ ﻣﺮاﻗﺐ اﺳﺖ ﻛﻪ ﭼﮕﻮﻧﮕﻲ ﻛﻴﻔﻴﺖ آن در ﻣﺮاﺣﻞ ﺑﻌﺪي رﺷﺪ ﺗﺄﺛﻴﺮﮔﺬار اﺳﺖ ) ﻣﻠﻚ ﭘﻮر، 2007 .( دﻟﺒﺴﺘﮕﻲ ﻧﻮﺟﻮان ﺑﻪ واﻟﺪﻳﻦ در اﻳﻦ دوران ﻣﻤﻜﻦ اﺳﺖ ﺑﺎ ﻣﺸﻜﻼﺗﻲ ﻫﻤﺮاه ﺷﻮد، ﭼﺮا ﻛﻪ ﺑﺎﻳﺪ ﻫﻤﺰﻣﺎن ﺑﺎ ﺣﻔﻆ ارﺗﺒﺎط ﺑﺎ واﻟﺪﻳﻦ ﺑﻪ ﺑﺮرﺳﻲ ﻧﻘﺶ ﻫﺎي ﺟﺪﻳﺪ اﺟﺘﻤﺎﻋﻲ ﺑﭙﺮدازد و از ﺳﻮي دﻳﮕﺮ، ﺷﻜﻞ ﮔﻴﺮي دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﻫﻤﺴﺎﻻن در اﻳﻦ دوران ﻧﻴﺰ وﺟﻮد دارد ﻛﻪ ﮔﺬر ﺳﺎﻟﻢ ﺑﻪ اﺳﺘﻘﻼل ﺟﻮاﻧﻲ را ﻣﻤﻜﻦ ﻣﻲ ﺳﺎزد ﺗﺎ در ﻣﻌﺮض ارﺗﺒﺎﻃﺎت ﻫﻴﺠﺎﻧﻲ ﻣﻨﺎﺳﺐ ﻗﺮار ﮔﻴﺮد . ﺗﺤﻘﻴﻘﺎت ﻧﺸﺎن ﻣﻲ دﻫﺪ ﻛﻴﻔﻴﺖ دﻟﺒﺴﺘﮕﻲ ﺑﺎ ﺗﻮﺟﻪ ﺑﻪ ﻧﻘﺶ ﭘﺪر و ﻣﺎدر ﻣﺘﻔﺎوت اﺳﺖ ) ﻣﻮرﺗﻲ، ﭘﻠ ﺪ، 2004 ( ،ﻫﻤﺎن ﮔﻮﻧﻪ ﻛﻪ در اﻳﻦ ﭘﮋوﻫﺶ ﻧﺸﺎن داده ﺷﺪ دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﭘﺪر ﺑﺎ ﻋﺪاﻟﺖ اﻋﺘﺪال و ﺗﻌﺎﻟﻲ ﻣﺸﺨﺺ ﺷﺪه اﺳﺖ، در ﺣﺎﻟﻲ ﻛﻪ دﻟﺒﺴﺘﮕﻲ ﺑﻪ ﻣﺎدر ﺑﺎ ﺧﺮد، اﻧﺴﺎﻧﻴﺖ، ﻋﺪاﻟﺖ و ﺗﻌﺎﻟﻲ ﻣﺸﺨﺺ ﺷﺪه اﺳﺖ . ﻧﻈﺮﻳﻪ دﻟﺒﺴﺘﮕﻲ ﺑﻴﺎن ﻣﻲ ﻛﻨﺪ ﺗﺠﺮﺑﻪ ﺑﺎ ﻧﺰدﻳﻜﺎن ﺑﻪ وﻳﮋه ﻣﺮاﻗﺒﻴﻦ در ﺷﻜﻞ ﮔﻴﺮي ﺑﺎزﻧﻤﺎﻳﻲ ذﻫﻨﻲ اﺛﺮﮔﺬار اﺳﺖ و راﻫﻨﻤﺎﻳﻲ ﺑﺮاي ﺗﻔﺴﻴﺮ و ﺑﺮﻧﺎﻣﻪ رﻳﺰي ﺑﻴﻦ ﻓﺮدي ﺑﺎ دﻳﮕﺮان را ﻫﺪاﻳﺖ ﻣﻲ ﻛﻨﺪ . ﻳﺎﻓﺘﻪ ﻫﺎ ﺑﻨﺎﺑﺮاﻳﻦ ﻣﻬﻢ اﺳﺖ ﻣﺪل ﻫﺎي دﻟﺒﺴﺘﮕﻲ و ﺑﺎزﻧﻤﺎﻳﻲ ذﻫﻨﻲ ﻧﻮﺟﻮان ﻣﻮرد ﺗﺤﻠﻴﻞ ﻗﺮار ﮔﻴﺮد و ﺑﻪ آن ﻫﺎ ﻛﻤﻚ ﺷﻮد ﺗﺎ ﻇﺮﻓﻴﺖ ﺧﻮد را ﺑﺮاي ﺣﻞ ﺗﻌﺎرض ﺗﻐﻴﻴﺮ دﻫﻨﺪ ) ﮔﺎرﺳﻴﺎ ـ روﻳﺰ، رودرﻳﮕﻮ، ﺟﻮﻳﺲ ، ﻫﺮﻧﺪزﮔﺎﺑﺮﻳﻞ، ﻣﮕﻮاز و ﻫﻤﻜﺎران 47 ، 2012 .( ﻟﺬا ﺿﺮورت ﻣﻄﺎﻟﻌﻪ ﺗﻮاﻧﻤﻨﺪي ﻫﺎي ﻣﻨﺶ ﻣﺎﻧﻨﺪ ﻋﺪاﻟﺖ، ﺷﻬﺎﻣﺖ ﺑﺎ ﻓﺮزﻧﺪﭘﺮوري و ﺗﺮﺑﻴﺖ ﻛﻪ ﭘﺮورش دﻫﻨﺪه آن ﻫﺎﺳﺖ اﺣﺴﺎس ﻣﻲ ﺷﻮد ) واﺗﺮز، 2014 .( درك رﺷﺪ ﺗﻮاﻧﻤﻨﺪي ﻫﺎي ﻣﻨﺶ و ﻋﻮاﻣﻞ ﻣﺆﺛﺮ ﺑﺮ رﺷﺪ ﻛﻮدك و ﻧﻮﺟﻮان ﺗﻼﺷﻲ اﺳﺖ ﻛﻪ ﺗﻮﺳﻂ ﻣﻄﺎﻟﻌﺎت ﺳﻴﻠﮕ ﻤﻦ و ﭘﻴﺘﺮﺳﻮن در ﺳﺮاﺳﺮ زﻧﺪﮔﻲ ﺷﺮوع )ﺷﺪ ﻫﺮﺷﻨﺒﺮگ، داوﻳﻞ، ﻳﻮﻧﺪا، اﺳﺘﺎر، ﻻﻛﺮ و ﻫﻤﻜﺎران، 2012 ( ﻛﻪ ﻣﻲ ﺗﻮان آن را ﺑﺎ ﻋﻮاﻣﻞ ﻣﺆﺛﺮ در دروان رﺷﺪ ﻛﻮدك و ﻧﻮﺟﻮان ﻣﻮرد ﺑﺮرﺳﻲ ﻗﺮار داد . ﻫﻤﺎن ﮔﻮﻧﻪ ﻛﻪ اﻳﻦ ﭘﮋوﻫﺶ ﻧﺸﺎن داد ﻫﺮ ﭼﻘﺪر ﻓﺮزﻧﺪان دﻟﺒﺴﺘﮕﻲ اﻳﻤﻦ ﺗﺮي ﺑﺎ واﻟﺪﻳﻦ ﺧﻮد داﺷﺘﻪ ﺑﺎ ﺷﻨﺪ، رﻓﺘﺎرﻫﺎي ﺳﺎزﮔﺎراﻧﻪ آن ﻫﺎ ﺑﻴﺸﺘﺮ و ﺗﻌﺎرض ﻛﻤﺘﺮ ي وﺟﻮد دارد . ز ر ر ر ن ﺮ رض ﺮ و ر وﻮ از ﻣﺤﺪودﻳﺖ ﻫﺎي ﭘﮋوﻫﺶ ﺣﺎﺿﺮ اﻳﻨﻜﻪ ﻧﻮﺟﻮاﻧﺎن ﭘﺴﺮ ﺷﺮﻛﺖ ﻧﺪاﺷﺘﻨﺪ، در ﺣﺎﻟﻲ ﻛﻪ در ﻣﻄﺎﻟﻌﺎت، ﺗﻔﺎوت ﻫﺎﻳﻲ در ﺗﻮاﻧﻤﻨﺪي ﻫﺎي ﻣﻨﺶ دﺧﺘﺮ و ﭘﺴﺮ وﺟﻮد دارد . از اﻳﻦ رو، ﭘﻴﺸﻨﻬﺎد ﻣﻲ ﺷﻮد در ﭘﮋوﻫﺶ ﻫﺎي آﺗﻲ اﻳﻦ ﻣﻘﻮﻟﻪ ﻣﻮرد ﺗ ﻮﺟﻪ ﻗﺮار ﮔﻴﺮد . ﻫﻢ ﭼﻨﻴﻦ ﭘﺮورش ﺗﻮاﻧﻤﻨﺪي ﻫﺎي ﻣﻨﺶ در ﺳﻄﺢ رﺿﺎﻳﺖ و ﺷﺎدﻛﺎﻣﻲ ﻧﻮﺟﻮان و ﻛﺎﻫﺶ ﺗﻌﺎرض ﻧﻘﺶ ﻣﻬﻤﻲ دارد . ﭘﻴﺸﻨﻬﺎد ﻣﻲ ﺷﻮد در ﭘﮋوﻫﺶ ﻫﺎي ﺑﻌﺪي ﺑﻪ ﻣﻘﻮﻟﻪ ﭘﺮورش ﻣﻬﺎرت ﺗﻮاﻧﻤﻨﺪي ﻫﺎي ﻣﻨﺶ ﺗﻮﺟﻪ ﺑﻴﺸﺘﺮي ﺷﻮد . ﻧﻘﺶ ﺗﻌﺎﻣﻠﻲ ﻓﻀﺎﺋﻞ در ﻛﻢ رﻧﮓ ﻛﺮدن ﺗﻌﺎرض ﻫﺎ ﻧﻴﺰ ﺷﺎﻳﺎن ﺗﻮﺟﻪ اﺳﺖ ﻛﻪ ﻣ ﻲ ﺗﻮاﻧﺪ در ﭘﮋوﻫﺶ ﻫﺎي ﺑﻌﺪي ﻣﺪﻧﻈﺮ ﻗﺮار ﮔﻴﺮد . ﻫﻢ ﭼﻨﻴﻦ ﺑﻪ دﻟﻴﻞ اﻳﻨﻜﻪ ﻧﻮﺟﻮان زﻣﺎن زﻳﺎدي را در ﻣﺪرﺳﻪ ﺳﭙﺮي ﻣﻲ ﻛﻨﺪ آﺷﻨﺎﻳﻲ آﻧﺎن ﺑﺎ ﺗﻮاﻧﻤﻨﺪي ﻫﺎي ﻣﻨﺶ، راﻫﺒﺮدﻫﺎي آن، ﭼﮕﻮﻧﮕﻲ ﭘﺮورش و ﺮﻴﺗﺄﺛ آن در زﻧﺪﮔﻲ ﻓﺮدي، ﺧﺎﻧﻮادﮔﻲ، اﺟﺘﻤﺎﻋﻲ ﻣﻲ ﺗﻮاﻧﺪ ﺑﺴﻴﺎر ﻛﻤﻚ ﻛﻨﻨﺪه ﺑﺎﺷﺪ . از ﺳﻮي دﻳﮕﺮ آﮔ ﺎﻫﻲ واﻟﺪﻳﻦ از ﻣﺒﺎﺣﺚ ﺗﻮاﻧﻤﻨﺪي ﻫﺎ و ﻓﻀﺎﺋﻞ در ﻛﻤﻚ ﺑﻪ ﺣﻞ ﺗﻌﺎرض و اﺛﺮات آن را در روﻳﺎروﻳﻲ ﺑﺎ ﻧﻮﺟﻮان دوﭼﻨﺪان ﻛﻨﺪ . ﻫﻢ ﭼﻨﻴﻦ ﺗﻮﺟﻪ ﺑﻪ رﺷﺪ ﺗﻮاﻧﻤﻨﺪي ﻫﺎ و ﻓﻀﺎﺋﻞ در ﻣﺮاﺣﻞ رﺷﺪ ﻛﻮدك و ﻧﻮﺟﻮان ﺑﻪ ﻫﻤﺮاه ﻋﻮاﻣﻞ ﻣﺆﺛﺮ ﺗﺤﻮﻟﻲ و رﺷﺪي ﻧﻮﺟﻮان اﺛﺮات ﻣﺎﻧﺪﮔﺎري روي ﻧﻮﺟﻮان و ﺧﺎﻧﻮاده دارد . ﻫﻢ ﭼﻨﻴﻦ ﻣﻨﺒﻊ ﺗﻌﺎرض از دﻳﺪﮔﺎه واﻟﺪﻳﻦ در ارﺗﺒﺎط ﺑﺎ 357 / ﭘﻴﺶ ﺑﻴﻨﻲ ﺗﻮاﻧﻤﻨﺪي ﻣﻨﺶ ﻧﻮﺟﻮاﻧﺎن و دﻟﺒﺴﺘﮕﻲ ﺑﻪ واﻟﺪﻳﻦ و/... ﻣﻮرد ﺗﻮﺟﻪ ﻗﺮار ﮔﻴﺮد . ﭘﻲ ﻧﻮﺷﺖ ﻫﺎ ﭘﻲ ﻧﻮﺷﺖ ﻫﺎ 1. Storm and Stress 30. Burke, Minton 2. Peterson, Seligman & Park 31. Toner, Haslam, Robinson, Paige 3. Character Strengths 32. Tabachnick, Fidell 4. Ngai 33. Miles & Shevlin 5. Good Character 6. Character 34. Short Measure of Character Strength (SMCS) 7. Gilman, Huebner, Scott. Furlong 35. Furnham, Lester 8. Ruch, Weber, Park, Peterson 36. Conflict Behavior Questionnaire 9. Leontopoulou, Triliva 37. Prinz 10. Wisdom 38. Robin, Foster 11. Courage 39. Shariati 12. Humanity 13. Justice 40. The Inventory of Parent and Peer Attachment 14. Temperance 41. Armsden , Greenberg 15. Transcendence 42. Vahedi, Fathi 16. Ahadi 43. Backward 17. Kerestes, Brkovic, Jagodic, Gordana 44. Niemiec 18. Scott Curry 45. Bowlby 19. Lickona 20. Shoshani, Shwartz 46. Hershenberg, Davila, Yoneda, Starr, Loker, et. al 21. Attachment 22. Huebner, Hills 47. García-Ruiz, Rodrigo, José. Hernández- Cabrera. Máiquez. Et. al 23. Malekpour 48. Ghamari, Ghamrygandooani 24. Moretti, Peled 49. Shariati, Emami-Pour 25. Fuligni, Eccles 50. Noronha, Martins 26. Behavior Problems 27. DeKlyen , Speltz 28. Daubs 29. Parra - Cardona, Yen. Anthony ﭘﻲ ﻧﻮﺷﺖ ﻫﺎ 1. Storm and Stress 30. Burke, Minton 2. Peterson, Seligman & Park 31. Toner, Haslam, Robinson, Paige 3. Character Strengths 32. Tabachnick, Fidell 4. Ngai 33. Miles & Shevlin 5. Good Character 6. Character 34. Short Measure of Character Strength (SMCS) 7. Gilman, Huebner, Scott. Furlong 35. Furnham, Lester 8. Ruch, Weber, Park, Peterson 36. Conflict Behavior Questionnaire 9. Leontopoulou, Triliva 37. Prinz 10. Wisdom 38. Robin, Foster 11. Courage 39. Shariati 12. Humanity 13. Justice 40. The Inventory of Parent and Peer Attachment 14. Temperance 41. Armsden , Greenberg 15. Transcendence 42. Vahedi, Fathi 16. Ahadi 43. Backward 17. Kerestes, Brkovic, Jagodic, Gordana 44. Niemiec 18. Scott Curry 45. Bowlby 19. Lickona 20. Shoshani, Shwartz 46. Hershenberg, Davila, Yoneda, Starr, Loker, et. al 21. Attachment 22. Huebner, Hills 47. García-Ruiz, Rodrigo, José. Hernández- Cabrera. Máiquez. Et. al 23. Malekpour 48. Ghamari, Ghamrygandooani 24. Moretti, Peled 49. Shariati, Emami-Pour 25. Fuligni, Eccles 50. Noronha, Martins 26. Behavior Problems 27. DeKlyen , Speltz 28. Daubs 29. Parra - Cardona, Yen. Anthony 1. Storm and Stress 2. Peterson, Seligman & Park 3. Character Strengths 4. Ngai 5. Good Character 6. Character 7. Gilman, Huebner, Scott. Furlong 8. Ruch, Weber, Park, Peterson 9. Leontopoulou, Triliva 10. Wisdom 11. Courage 12. Humanity 13. Justice 14. Temperance 15. Transcendence 16. Ahadi 17. Kerestes, Brkovic, Jagodic, Gordana 18. ﻳﺎﻓﺘﻪ ﻫﺎ ﺑﺴﻴﺎري از ﭘﮋوﻫﺶ ﻫﺎ در ﻣﻮرد ﺗﻌﺎرض از ﺑﻌﺪ ﻋﻤﻠﻜﺮد ﺧﺎﻧﻮاده ) ﻗﻤﺮي، ﻗﻤﺮي ﮔﻨﺪواﻧﻲ 48 ، 1393 ( و ﺷ ﻴﻮه ﻫﺎي ﻓﺮزﻧﺪﭘﺮوري ) ﺷﺮﻳﻌﺘﻲ، اﻣﺎﻣﻲ ﭘﻮر 49 ، 1394 ( ﺻﻮرت ﮔﺮﻓﺘﻪ اﺳﺖ . ﭘﮋوﻫﺶ ﺣﺎﺿﺮ از ﺣﻮزه روان ﺷﻨﺎﺳﻲ ﻣﺜﺒﺖ وارد ﺷﺪه اﺳﺖ . از ﺳﻮي دﻳﮕﺮ ﺗﻮاﻧﻤﻨﺪي ﻫﺎي ﻣﻨﺶ در ﺣﻮزه ﻫﺎي اﺛﺮﮔﺬار ﻣﺨﺘﻠﻔﻲ ﻣﺎﻧﻨﺪ رﺿﺎﻳﺖ از زﻧﺪﮔﻲ، ﺷﺎدي ) ﭘﻴﺘﺮﺳﻮن، رﻳﭻ، ﺑﺮﻣﺎﻧﺎ، ﭘﺎرك، ﺳﻴﻠﮕﻤﻦ، 2007 ، ﻧﻮرن ﻫﺎ، ﻣﺎرﺗﻴﻨﺰ50 ، 2016 ( و ﺑﻬﺰﻳﺴﺘﻲ ) ﭘﺎرك، ﭘﻴﺘﺮﺳﻮن، ﺳﻴﻠﮕﻤﻦ، 2004 ( ﻛﺎرآﻣﺪي ﺧﻮد را ﺑﻪ اﺛﺒﺎت رﺳﺎﻧﺪه اﺳﺖ، در ﺣﺎﻟﻲ ﻛﻪ ﭘﮋوﻫﺶ ﺣﺎﺿﺮ در راﺳﺘﺎي ﺧﻼء واﻟﺪ ـ ﻧﻮﺟﻮان اراﺋﻪ ﺷﺪه اﺳﺖ . ﺗﻮاﻧﻤﻨﺪي ﻫﺎ در رواﺑﻂ ﻧﺰدﻳﻚ واﻟﺪ، ﻧﻮﺟﻮان، دﻟﺒﺴﺘﮕﻲ و ﺗﻌﺎرض ﻧﻮﺟﻮاﻧﺎن ﺑﺎ واﻟﺪﻳﻦ ﻣﻮرد ﺑﺮرﺳﻲ ﻗﺮار ﮔﺮﻓﺖ ﺗﺎ ﺑﺘﻮاﻧﺪ ﻧﻘﺶ ﺗﻮاﻧﻤﻨﺪي ﻫﺎ و ﻓﻀﺎﻳﻞ را در ﻣﺤﻴﻂ ﺧﺎﻧﻪ ﭘﺮرﻧﮓ ﺗﺮ ﻧﺸﺎن دﻫﺪ ﺗﺎ ﺷﺎدي، ﺑﻬﺰﻳﺴﺘﻲ را ﺑﺮاي اﻋﻀﺎي ﺧﺎﻧﻮاده ﻓﺮاﻫﻢ ﺳﺎزد . ﭘﻲ ﻧﻮﺷﺖ ﻫﺎ Lickona, T. (2013). Raising children of character: ten things parents can do. https://www 2.-cortland.edu/dotAsset/0d9f1b26-48ad-4b67-8e73-1a031fb45d51.pdf ﭘﻲ ﻧﻮﺷﺖ ﻫﺎ Scott Curry 19. Lickona 20. Shoshani, Shwartz 21. Attachment 22. Huebner, Hills 23. Malekpour 24. Moretti, Peled 25. Fuligni, Eccles 26. Behavior Problems 27. DeKlyen , Speltz 28. Daubs 29. Parra - Cardona, Yen. Anthony 46. Hershenberg, Davila, Yoneda, Starr, 47. García-Ruiz, Rodrigo, José. Hernández- Cabrera. Máiquez. Et. al 48. Ghamari, Ghamrygandooani 49. Shariati, Emami-Pour 49. Shariati, Emami-Pour 50. Noronha, Martins 50. Noronha, Martins 358 / / !"# $% / &'%( / ﻣﻨﺎﺑﻊ اﺣﺪي، ح . ، ﺟﻤﻬﺮي، ف . )1380 .(رواﻧﺸﻨﺎﺳﻲ رﺷﺪ ، ﻧﻮﺟﻮاﻧﻲ، ﺑﺰرﮔﺴﺎﻟﻲ ) ﺟﻮاﻧﻲ، ﻣﻴﺎﻧﺴﺎﻟﻲ، ﭘﻴﺮي (، ﺗﻬﺮان : اﻧﺘﺸﺎرات ﭘﺮدﻳﺲ . ﺗﺎﺑﺎﻛﻨﻴﻚ، ب . ، ﺟﻲ، ف . ، ﻟﻴﻨﺪا، اس . )2006 .( ﻛﺎرﺑﺮد آﻣﺎر ﭼﻨﺪﻣﺘﻐﻴﺮي . ﺗﺮﺟﻤﻪ ب . اﻳﺰاﻧﻠﻮ، و . ﻓﺮزاد، ح . ﺣﺴﻦ آﺑﺎدي، و ح . ﺣﺒﻴﺒﻲ )1395 (ﺗﻬ ﺮان : اﻧﺘﺸﺎرات رﺷﺪ . ﺷﺮﻳﻌﺘﻲ، س . ، و اﻣﺎﻣﻲ ﭘﻮر، س . )1394 ( ، ﻧﻘﺶ واﺳﻄﻪ اي ﺗﻌﺎرض واﻟﺪﻳﻦ- ﻧﻮﺟﻮان در راﺑﻄﻪ ﺑﺎ ﺳﺒﻚ ﻫﺎي ﻓﺮزﻧﺪﭘﺮوري ادارك ﺷﺪه و اﺑﻌﺎد اﺑﺮاز ﺧﺸﻢ . ﻓﺼﻠﻨﺎﻣﻪ ﺧﺎﻧﻮاده و ﭘﮋوﻫﺶ . 4) 11 ( ، 22 - 7. ﻗﻤﺮي، م . ، ﻗﻤﺮي ﮔﻨﺪواﻧﻲ، آ . )1393 .( راﺑﻄﻪ ﻋﻤﻠﻜﺮد ﺧﺎﻧﻮاده ﺑﺎ ﺗﻌﺎرض واﻟﺪ- ﻧﻮﺟﻮان در ﺑﻴﻦ داﻧﺶ آﻣﻮزان ﻣﻘﻄﻊ راﻫﻨﻤﺎﻳﻲ . ﻓﺼﻠﻨﺎﻣﻪ ﻓﺮﻫﻨﮕﻲ ـ ﺗﺮﺑﻴﺘﻲ زﻧﺎن و ﺧﺎﻧﻮاده، 26 )8( ، 174 - 157 . ﻣﺎﻳﻠﺰ، ج . ، ﺷﻮﻟﻴﻦ، م . )1999 .( رﮔﺮﺳﻴﻮن و ﻫﻤﺒﺴﺘﮕﻲ ﻛﺎرﺑﺮدي . ﺗﺮﺟﻤﻪ، ع . ﻛﻴﺎﻣﻨﺶ و م . ﻛﺒﻴﺮي )1395 .(ﺗﻬﺮان : اﻧﺘﺸﺎرات ﺟﻬﺎد داﻧﺸﮕﺎﻫﻲ واﺣﺪ ﻋﻼﻣﻪ ﻃﺒﺎﻃﺒﺎﺋﻲ . واﺣﺪي، ش . ، ﻓﺘﺤ ﻲ، آ . )1389 .( راﺑﻄﻪ ﺑﻴﻦ دﻟﺒﺴﺘﮕﻲ ﺑﻪ واﻟﺪﻳﻦ و ﻫﻤﺴﺎﻻن ﺑﺎ ﺟﺪاﻳﻲ روان ﺷﻨﺎﺧﺘﻲ داﻧﺸﺠﻮﻳﺎن دﺧﺘﺮ و ﭘﺴﺮ ﺟﺪﻳﺪاﻟﻮرود . ﻓﺼﻠﻨﺎﻣﻪ ﻋﻠﻤﻲ- ﭘﮋوﻫﺸﻲ داﻧﺸﮕﺎه ﺗﺒﺮﻳﺰ ، 17 )5( ، 183 - 151 . / ﻣﻨﺎﺑﻊ ﺷﺮﻳﻌﺘﻲ، س . ، و اﻣﺎﻣﻲ ﭘﻮر، س . )1394 ( ، ﻧﻘﺶ واﺳﻄﻪ اي ﺗﻌﺎرض واﻟﺪﻳﻦ- ﻧﻮﺟﻮان در راﺑﻄﻪ ﺑﺎ ﺳﺒﻚ ﻫﺎي ﻓﺮزﻧﺪﭘﺮوري ادارك ﺷﺪه و اﺑﻌﺎد اﺑﺮاز ﺧﺸﻢ . ﻓﺼﻠﻨﺎﻣﻪ ﺧﺎﻧﻮاده و ﭘﮋوﻫﺶ . 4) 11 ( ، 22 - 7. ﻗﻤﺮي، م . ، ﻗﻤﺮي ﮔﻨﺪواﻧﻲ، آ . )1393 .( راﺑﻄﻪ ﻋﻤﻠﻜﺮد ﺧﺎﻧﻮاده ﺑﺎ ﺗﻌﺎرض واﻟﺪ- ﻧﻮﺟﻮان در ﺑﻴﻦ داﻧﺶ آﻣﻮزان ﻣﻘﻄﻊ راﻫﻨﻤﺎﻳﻲ . ﻓﺼﻠﻨﺎﻣﻪ ﻓﺮﻫﻨﮕﻲ ـ ﺗﺮﺑﻴﺘﻲ زﻧﺎن و ﺧﺎﻧﻮاده، 26 )8( ، 174 - 157 . ﻣﺎﻳﻠﺰ، ج . ، ﺷﻮﻟﻴﻦ، م . )1999 .( رﮔﺮﺳﻴﻮن و ﻫﻤﺒﺴﺘﮕﻲ ﻛﺎرﺑﺮدي . ﺗﺮﺟﻤﻪ، ع . ﭘﻲ ﻧﻮﺷﺖ ﻫﺎ https://www 2.-cortland.edu/dotAsset/0d9f1b26-48ad-4b67-8e73-1a031fb45d51.pdf 359 / ﭘﻴﺶ ﺑﻴﻨﻲ ﺗﻮاﻧﻤﻨﺪي ﻣﻨﺶ ﻧﻮﺟﻮاﻧﺎن و دﻟﺒﺴﺘﮕﻲ ﺑﻪ واﻟﺪﻳﻦ و/... Leontopoulou, S., Triliva, S. (2012). Explorations of subjective wellbeing and character strengths among a Greek University student sample. International Journal of Well- being, 2(3), 251-270. g Loker, T., Tony, X. T., & Raffaele, M. L., & Dedrick, R. (2012). Adopted Chinese girls’ character strengths from preschool to school age: A longitudinal exploratory study. Michigan Family Review, 16(1), 38-55. g y Kerestes, G., Brkovic, I., & Jagodic, G. K. (2011). Predictors of psychological well- being of adolescents’ parents. Journal of Happiness Studies, 759-773. Malekpour, M. (2007). Effects of attachment on early and later development. The British Journal of Developmental Disabilities, 2(53), 81-95. Miles, J., & Shevlin, M. (1999). Applying Regression & Correlation (A. Kiamanesh, & M. Kabiri Trans.).Tehran: Jahaddaneshgahi. AllamehTabaTabae Publication [in Per- sian]. Moretti, M. Peled, M. (2004). Adolescent-parent attachment: Bonds that support healthy development. Paediatr Child Health, 8(9), 551-555. Niemiec, R. M. (2013). VIA character strengths: Research and practice (The first 10 years). In H. H. Knoop & A. Delle Fave (Eds.), Well-being and cultures: Perspecti- ves on positive psychology (pp. 11-30). New York: Springer. Noronha, A. P., & Martins, D. F. (2016). Associations between character strengths and life satisfaction: A study with college students. Colombian psychology act, 19(2), 28-52. Park, N., Peterson, Ch., & Seligman, M. (2004). Strengths of character and well-being. Journal of Social and Clinical Psychology, 5(23), 603-619. Parra-Cardona, J., Yen, H. H., & Anthony, J. C. (2017). Epidemiological research on parent child conflict in the United States: subgroup variations by place of birth and ethnicity,http://dx.doi.org/10.7717/peerj.2905. Peterson, Ch., & Seligman, M. (2004). Character Strengths and Virtues: A Handbook and Classification. Oxford: Oxford university press. Peterson, Ch. Ruch, W. Beermann, U. Park, & Martin, E. P. (2007). Strengths of chara- cter, or-ientations to happiness, and life satisfaction. The Journal of Positive Psych- ology, 2(3), 149-156. Prinz, R. J., Foster, Sh., Kent, R. N., & O'Leary, K. D. (1979). Multivariate assessment of conflict distressed and nondistressed mother- adolescent dyads. Journal of applied behavior analysis, 12(1), 691-700. Ruch, W., Weber, M., Park, N., & Peterson, Ch. (2014). Character strengths in children and adolescents. European Journal of Psychological Assessment, 1(30), 57-64. Robin, A. L., & Foster, S. L. (1989). Negotiating parent-adolescent conflict: A behav- ioral-family systems approach. New York: Guilford Press. Shariati, S., Emami-Pour, S. (2014). ﭘﻲ ﻧﻮﺷﺖ ﻫﺎ ﻛﻴﺎﻣﻨﺶ و م . ﻛﺒﻴﺮي )1395 .(ﺗﻬﺮان : اﻧﺘﺸﺎرات ﺟﻬﺎد داﻧﺸﮕﺎﻫﻲ واﺣﺪ ﻋﻼﻣﻪ ﻃﺒﺎﻃﺒﺎﺋﻲ . ﻣﺎﻳﻠﺰ، ج . ، ﺷﻮﻟﻴﻦ، م . )1999 .( رﮔﺮﺳﻴﻮن و ﻫﻤﺒﺴﺘﮕﻲ ﻛﺎرﺑﺮدي . ﺗﺮﺟﻤﻪ، ع . ﻛﻴﺎﻣﻨﺶ و م . ﻛﺒﻴﺮي )1395 .(ﺗﻬﺮان : اﻧﺘﺸﺎرات ﺟﻬﺎد داﻧﺸﮕﺎﻫﻲ واﺣﺪ ﻋﻼﻣﻪ ﻃﺒﺎﻃﺒﺎﺋﻲ . واﺣﺪي، ش . ، ﻓﺘﺤ ﻲ، آ . )1389 .( راﺑﻄﻪ ﺑﻴﻦ دﻟﺒﺴﺘﮕﻲ ﺑﻪ واﻟﺪﻳﻦ و ﻫﻤﺴﺎﻻن ﺑﺎ ﺟﺪاﻳﻲ روان ﺷﻨﺎﺧﺘﻲ داﻧﺸﺠﻮﻳﺎن دﺧﺘﺮ و ﭘﺴﺮ ﺟﺪﻳﺪاﻟﻮرود . ﻓﺼﻠﻨﺎﻣﻪ ﻋﻠﻤﻲ- ﭘﮋوﻫﺸﻲ داﻧﺸﮕﺎه ﺗﺒﺮﻳﺰ ، 17 )5( ، 183 - 151 . Ahadi, H. Jomejri, F. (2001). [Developmental Psychology Adolescence, Adulthood (Early, Middle, Late)], Tehran, Pardis Publication [in Persian]. Armsden, B. N & Greenberg, M. T. (1987). The Inventory of parent and peer attachment: Individual differences and their relationship to psychological well-being in Adolescence. Journal of Youth and adolescence, 16(5), 427-454. f ( ) Bowlby, J. (1973). Attachment and Loss, Sadness and Depression. New York: Basic Books. Burke, J., Minton, S. J. (2015). Positive psychology in guidance counselling. National Centre for Guidance in Education (NCGE), 22(1), 5-22. f Daubs, C. (2013). Adolescent behavior problems and interparental conflict: The Moderating role of Parent – child attachment. PhD thesis in Psychology, University of North Texas. Fuligni, A. J., & Eccles, J. S. (1993). Perceived parent-child relationships and early adolescents' orientation toward peers. Developmental Psychology, 4(29), 622-632. Furnham, A., & Lester, D. (2012). The development of a short measure of character strength, European Journal of Psychological Assessment, 28 (2), 95-101. García-Ruiz, M., José Rodrigo, M., & Hernández-Cabrera, J. (2012). Resolution of parent-child conflicts in the adolescence. Eur J Psychol Educ, 28(2), 173-188. Ghamari, M., Ghamrygandooani, A. (1393). [Relationship between family performance and parent-adolescent conflict among middle-level students]. Cultural-Educational Quarterly of Women and Family, 26(8), 157-174. [in Persian]. y f y Gilman, R., Huebner, E. S. & Furlong, M. j. (2009). Handbook of Positive Psychology in Schools. London: Routledge. Hershenberg, R., Davila, J., Yoneda, A., Starr, L. R., & Loker, M. (2012). Adopted Chinese girls’ character strengths from preschool to school age: A longitudinal exploratory study. Michigan Family Review, 16(1), 38-55, p y y g y Huebner, X. U., Jiang, E., Scott, H., & Kimberly, J. (2013). Parent attachment and eaely adolescents’ life satisfaction: The mediating effect of hope. Psychology in the Schools, 50(4), 340-352. ( ) Lickona, T. (2013). Raising children of character: ten things parents can do. ( ) Vahedi, Sh., & Fathi, A. (2010). [Relationship between attachment to parents and peers with psychological separation of female and male students of New Year]. Journal of Research in Tabriz University, 17(5), 151-183 [in Persian]. g y ff ( ) Waters, L. (2014). Strength-based parenting and life satisfaction in teenagers. Social Sci- ences Research Journal, 11(2), 28-39. Toner, E., Haslam, N., Robinson, J., & Paige, Williams. (2012). Character strengths and wellbeing in adolescence: Structure and correlates of the Values in Action Inventory of Strengths for Children. Personality and Individual Differences, 52(5), 642-657. Tabachnick, B., & Fidell, L. S. (2006). Multivariate analysis (B. Isanlou., F.Valiallah, H. Hasanabadi, & M. Habibi Trans.). Tehran: roshd Publication [in Persian]. ﭘﻲ ﻧﻮﺷﺖ ﻫﺎ [The role of parental-adolescent conflict mediators in relation to perceived parenting styles and aspects of anger]. Family Quarterly and Research, 11(4), 7-22 [in Persian]. Steven Sek-yum Ngai (2015). Parental bonding and character strengths among Chinese adol-escents in Hong Kong. International Journal of Adolescence and Youth, 3(20), 317-333. Shoshani, A., & Shwartz, L. (2016). From character strengths to children’s well-being: development and validation of the character strengths inventory for elementary school children. Frontiers in Psychology, 2(9), 74-88. 360 / / !"# $% / &'%( / / 360 / / !"# $% / &'%( / / Toner, E., Haslam, N., Robinson, J., & Paige, Williams. (2012). Character strengths and wellbeing in adolescence: Structure and correlates of the Values in Action Inventory of Strengths for Children. Personality and Individual Differences, 52(5), 642-657. g Waters, L. (2014). Strength-based parenting and life satisfaction in teenagers. Social Sci- ences Research Journal, 11(2), 28-39. Vahedi, Sh., & Fathi, A. (2010). [Relationship between attachment to parents and peers with psychological separation of female and male students of New Year]. Journal of Research in Tabriz University, 17(5), 151-183 [in Persian].
https://openalex.org/W2322335065	https://periodicos.ufba.br/index.php/crh/article/download/20031/12692	Portuguese	null	MOVIMENTOS NACIONAIS DE TRABALHADORES E CONEXÕES TRANSNACIONAIS: a evolução da arquitetura das forças sociais do trabalho no neoliberalismo	Caderno CRH	2,016	cc-by	15,123	C 2 [N.T. Maquila: local de trabalho baseado na combina- ção entre instrumentos de produção estrangeiros e força de trabalho local mal remunerada. O que é produzido nas maquilas geralmente é exportado]. MOVIMENTOS NACIONAIS DE TRABALHADORES E CONEXÕES TRANSNACIONAIS: a evolução da arquite- tura das forças sociais do trabalho no neoliberalismo DOSSIÊ DOSSIÊ Peter Evans* A era neoliberal minou os direitos dos trabalhadores e o poder das forças sociais do trabalho a nível na- cional, mas foi caracterizada, também, como uma era do novo “transnacionalismo do movimento dos tra- balhadores”. Mudanças conjunturais a nível nacional foram fundamentais para aumentar a abertura às alianças transnacionais. Uma análise das campanhas evidencia isso. Avaliar as conexões entre movimentos nacionais de trabalhadores e a nova infraestrutura organizacional que emergiu no neoliberalismo é um ponto de partida necessário para construir teorias mais apuradas sobre as dinâmicas das contestações das forças sociais do trabalho ao capital global. Palavras-chave: Trabalhadores. Sindicatos globais. Transnacionalismo. Sindicatos nacionais. Neoliberalismo. * University of California e Institute for International Studies da Brown University. Department of Sociology. 410 Barrows Hall Berkeley CA 94720. pevans@berkeley.edu 1 Este artigo esteve tempo suficiente em preparação para acumular mais débitos do que posso expressar aqui, mas, seja-me permitido, ao menos, mencionar poucos dos que contribuíram. O artigo beneficiou-se fundamentalmente dos conhecimentos, ideias e sugestões de Mark Anner, Jessica Champagne, Eli Friedman, Kjeld Jakobsen, Carolyn Kazdin, Robert Lawson, and Jamie McCallum, que parti- ciparam do Workshop sobre ‘New Strategies For Building Transnational Labor Solidarity’, ocorrido na Brown Uni- versity’s Watson Institute for International Studies, no ou- tono de 2012. Katy Fox-Hodess e Pablo Gaston brindaram a assistência editorial e acadêmica, ademais de suas pró- prias intuições. Publicado anteriormente em Peter Evans. National Labor Movements and Transnational Connec- tions: Global Labor’s Evolving Architecture Under Neo- liberalism. Global Labor Journal, V. 5, N. 3, setembro de 2014. Tradução de Igor Peres Jerônimo. Revisão técnica de Marco Aurélio Santana e Ruy Braga. * University of California e Institute for International Studies da Brown University. Department of Sociology. 410 Barrows Hall Berkeley CA 94720. pevans@berkeley.edu http://dx.doi.org/10.1590/S0103-49792015000300002 * University of California e Institute for International Studies da Brown University. Department of Sociology. 410 Barrows Hall Berkeley CA 94720. pevans@berkeley.edu MOVIMENTOS NACIONAIS DE TRABALHADORES ... movimentos dos trabalhadores em nível na- cional. Estudos das dinâmicas das campanhas globais (por exemplo, Bronfenbrenner, 2007) esclarecem as conexões transfronteiriças, mas normalmente não apresentam argumentos so- bre como as trajetórias políticas a nível nacio- nal contribuem para seus triunfos ou fracassos. chamado de “novo transnacionalismo do mo- vimento dos trabalhadores”. Esforços para do- cumentar e explicar a emergência de um “novo internacionalismo” começaram a proliferar na virada do milênio, como parte da onda gene- ralizada de otimismo pós-Seattle (e.g. Mazur, 2000; Munck, 2002; Waterman, 2001). Em ge- ral, as explicações enfatizaram as oportunida- des e incentivos criados pela emergência de uma economia política mais globalizada.3 As análises dos efeitos do contexto na- cional costumaram focar mais em como o estar localizado numa economia nacional privile- giada mina o transnacionalismo do movimen- to dos trabalhadores. Os estudos que docu- mentam a capitulação das forças sociais do tra- balho aos moldes do imperialismo Americano são um exemplo (por exemplo, Sciples, 2010). Não faltam, tampouco, análises de como a promessa do privilégio político local draga os movimentos dos trabalhadores do Sul Global na direção de coalizões dominadas pelo capi- tal, fragilizando projetos mais amplos de soli- dariedade de classe (e.g. Chibber, 2007). Que as raízes nacionais possam produzir efeitos negativos sobre o trabalhismo transnacional, especialmente quando estas conferem privilé- gio, é evidente. Mas, uma análise equilibrada deveria examinar, também, as possibilidades de sinergias positivas entre movimentos de trabalhadores situados distintamente As condições gerais criadas pela globali- zação neoliberal são, certamente, decisivas para o destino do trabalhismo. Porém, os movimentos nacionais das forças sociais do trabalho continu- am sendo os componentes mais importantes do movimento dos trabalhadores ao nível global, e a arquitetura geral da solidariedade das forças sociais do trabalho, igualmente global, depende de como as estratégias nacionais se orquestram. A possibilidade desta orquestração depende, por sua vez, das características dinâmicas dos cam- pos políticos com os quais se depara o trabalhis- mo em cada contexto nacional. O movimento nacional dos trabalhadores pode aproveitar as diferenças entre os terrenos globais nos quais opera em vez de deixar a diferença minar a soli- dariedade? Como as mudanças nas conjunturas nacionais afetam a abertura do trabalhismo na- cional às alianças transnacionais? Salvador, v. 28, n. 75, p. 457-478, Set./Dez. 2015 A literatura que aborda estas questões continua pouca desenvolvida. MOVIMENTOS NACIONAIS DE TRABALHADORES ... Com algumas notáveis exceções (por exemplo, Anner, 2011; McCallum, 2013), são poucos os estudos sobre o movimento dos trabalhadores ao nível global no neoliberalismo que esclareçam a evolução das interações entre movimentos dos trabalha- dores em níveis nacionais. Análises históricas comparativas de amplitude (por exemplo, Sil- ver, 2003) se dedicam mais a ressaltar as ma- neiras pelas quais o deslocamento geográfico da produção global afeta a mobilização em diferentes países do que as conexões entre os 4 Como qualquer época, o neoliberalismo combina um conjunto característico de regras econômicas, estratégias e estruturas com mudanças na hierarquia geopolítica e econômica dos Estados-nação. Para uma discussão geral Caderno CRH 3 Para algumas análises mais recentes sobre a evolução do novo transnacionalismo do movimento dos trabalhadores ver Evans (2010), Kay (2010), Munck (2010) and McCallum, J. (2013). Tarrow (2005) e Evans (2008) inserem o novo trans- nacionalismo do movimento dos trabalhadores no contexto da evolução geral dos movimentos sociais transnacionais. APRESENTAÇÃO1 ras continuam perpetuando o mito de que em seu país – e particularmente na indústria da maquila2 – é impossível organizar, e que os sindicatos não serão tolerados em nenhuma circunstância. Por isso a vitória na Russel é tão importante. Prova que é possível organizar nas maquilas” (MSN, 2010). Evangelina Argueta e seus companhei- ros de militância do ramo de vestuário da Cen- tral General de los Trabajadores (CGT) hon- durenha, ao forçar a gigante Russel Athletics, sediada nos Estados Unidos, a negociar um contrato em 2010, borraram a imagem conven- cional de vítimas passivas que caracterizava os trabalhadores de baixo salário. Segundo Argueta, “os líderes dos negócios em Hondu- 1 A luta para organizar a Russel foi forjada por décadas de incansável trabalho em Hon- duras por militantes como Argueta, mas foi, também, uma vitória para a organização trans- nacional, possibilitada por uma rede transre- gional de Organizações Não-Governamentais voltadas ao mundo do trabalho e sindicatos que ligaram Honduras e os Estados Unidos. Casos como estes tornam evidente a possibi- lidade de construir uma atuação coletiva arti- culada das forças sociais do trabalho que ligue distintos territórios nacionais. Embora Honduras pareça ser um lugar improvável para a observação de tendências da militância global, muitos considerariam o caso da Russel um exemplo do que é normalmente 457 http://dx.doi.org/10.1590/S0103-49792015000300002 do neoliberalismo, ver Evans e Sewell (2013). Para aque- les interessados em situar a lógica geopolítica específica da época neoliberal numa visão teórica geral sobre como as lógicas territoriais de poder interagem com a demanda do capital por lucro, o trabalho de Giovanni Arrighi (por exemplo, 1990, 1994, 1996) oferece uma lente poderosa. CONEXÕES NACIONAIS E TRANS- NACIONALISMO DO MOVIMENTO DOS TRABALHADORES Explorar as maneiras pelas quais as dife- renças nacionais podem traduzir-se em siner- gias positivas propícias ao transnacionalismo do movimento dos trabalhadores é o objetivo deste artigo. Ele é, também, uma resposta ao enigma do porquê exemplos deste novo trans- nacionalismo deveriam proliferar sob a égide do neoliberalismo, um regime geopolítico im- placavelmente hostil ao trabalho.4 458 Peter Evans fronteiras, trata-se de mudança potencialmen- te significativa nas possibilidades para o trans- nacionalismo das forças sociais do trabalho. Mesmo sob ataque, o movimento dos trabalha- dores estadunidense ainda comanda recursos mais elevados que a maioria dos movimentos dos trabalhadores no Sul Global. O declínio do poder político das forças sociais do trabalho ao nível nacional, soma- do à sua habilidade para distribuir benefícios econômicos aos seus membros, é uma das mais salientes características da era neolibe- ral. A densidade sindical caiu (especialmente no Norte); a legislação anti-sindical floresceu (particularmente nos Estados Unidos); a pre- cariedade aumentou (em ambos, Norte e Sul) (ver Standing, 2011). Contudo, a despeito dos reveses do movimento dos trabalhadores ao ní- vel nacional, novas conexões entre movimen- tos de trabalhadores nacionais e novas formas de organização global dos trabalhadores que facilitam tais ligações continuaram emergindo. Um segundo deslocamento estrutural, complementar, que foi chamado de “levante do Sul” (ver UNDP, 2013), aumentou a habilidade dos movimentos dos trabalhadores, ao menos em alguns dos maiores países do Sul Global, para expandir sua visão e enxergar para além das fronteiras nacionais. O tipo totalmente globalizado de capital que estes movimentos confrontam em seus próprios terrenos nacio- nais é um estímulo importante para o transna- cionalismo. A capacidade para agir sobre estes incentivos depende tanto da força organizacio- nal interna quanto da posição política do mo- vimento dos trabalhadores ao nível nacional. As alianças transnacionais brasileiras são o melhor exemplo no século XXI do transnacio- nalismo possibilitado por este deslocamento. A explicação das mudanças estruturais e estratégias que facilitaram a habilidade do movimento dos trabalhadores para usar siner- geticamente as diferenças complementa as ex- plicações globais do novo transnacionalismo do movimento dos trabalhadores e equilibra os argumentos que enxergam as diferenças nacio- nais em termos predominantemente negativos. Ajuda, também, a entender por que analistas encontram novas instâncias do novo transna- cionalismo do movimento dos trabalhadores em meio a um clima tão adverso. 6 A “barriga da besta” [belly of the beast] foi uma denomi- nação anti-imperialista muito usada, referente aos Estados Unidos, que remonta a José Martí. Para um uso do termo particularmente interessante ver a referência ao sindica- lista guatemalteco Homero Fuentes usada por Cesar Ro- driguez (2007: 68); ver citação também em Evans, 2010: 366 (nota 30). MOVIMENTOS NACIONAIS DE TRABALHADORES ... O corpo da análise é composto por três seções, sendo o foco das duas primeiras os efeitos dos deslocamentos estruturais nas con- junturas nacionais e o da terceira as mudan- ças globais complementares. Na primeira se- ção, detenho-me na variedade de campanhas internacionais que envolvem os sindicatos estadunidenses. Eles variam das campanhas contra as oficinas precárias5 na indústria do vestuário até esforços atuais do United Auto Workers (UAW) para transnacionalizar seus esforços organizativos na indústria automobi- lística. Essa seção mostra o quanto a abertura para a solidariedade transnacional por parte dos sindicatos estadunidenses pode facilitar as lutas dos sindicatos locais em países pequenos como Honduras e Libéria. Ao mesmo tempo, mostra como países maiores no Sul Global, como o Brasil, por exemplo, podem tornar-se aliados significativamente importantes para os sindicatos do Norte. curicor (G4S), como descrito por McCallum (2013). Construído sobre laços organizacionais entre a Union Network International (UNI), um Global Union (Sindicato Global) com raízes na Europa e o Service Employees Internationl Union (SEIU), um dos mais destacados prati- cantes das campanhas de estilo estaduniden- se, a campanha do G4S combinou um Acordo Marco-Global de estilo europeu com uma cam- panha corporativa agressiva que incluiu mobi- lização de base numa série de países. A análise de McCallum sobre a dinâmica das campanhas na Índia e na África do Sul reforça a importân- cia do contexto político nacional, ilustrando, no caso da África do Sul, como uma campanha global pode contribuir para a revitalização sin- dical a nível nacional. A seção conclusiva retorna à questão de até que ponto podemos generalizar a partir dos casos específicos revisados nas três seções precedentes e quais poderiam ser suas impli- cações para o futuro. Os tipos de conexões si- nergéticas entre movimentos dos trabalhado- res nacionais analisados aqui seriam efêmeros ou tenderiam a persistir e se espalhar? Quão significante e robusto são seus efeitos na ar- quitetura geral do movimento global dos traba- lhadores? Quão vulneráveis são à fragilização ocasionada por futuras mudanças na estrutura da economia política global? p A segunda seção foca na promessa cria- da pelo crescimento dos movimentos dos tra- balhadores no Sul Global, usando o caso do Brasil como exemplo principal. O Brasil ofere- ce um horizonte no que diz respeito à dinami- zação de estratégias e formas organizacionais no movimento dos trabalhadores global. MOVIMENTOS NACIONAIS DE TRABALHADORES ... O envolvimento extensivo do Brasil nos conse- lhos de fábrica de estilo europeu ao redor do mundo, em redes empresariais e nos Acordos Marco-Globais (Global Framework Agreement) complementa sua participação nas campanhas internacionais ao estilo estadunidense, in- cluindo as organizadas pelo UAW e o United Steelworkers (USW). Caderno 5 [N.T. “sweatshop”. Oficinas pequenas e precárias onde se trabalha em péssimas condições e com baixa remunera- ção. Costuma empregar força de trabalho feminina e imi- grante.] CONEXÕES NACIONAIS E TRANS- NACIONALISMO DO MOVIMENTO DOS TRABALHADORES Minha aná- lise se concentrará em dois deslocamentos es- truturais que facilitaram o uso sinergético das diferenças nacionais e nas mudanças das es- tratégias das forças sociais do trabalho a nível global que facilitaram a conexão de movimen- tos para além das fronteiras nacionais. C CRH S l d 28 75 457 478 S t /D 2015 O desenvolvimento da organização e da estratégia a nível global complementou estes dois deslocamentos estruturais nas posições nacionais, tornando mais fácil para o trabalho coordenar ações entre múltiplos terrenos nacio- nais. Esforços mais agressivos e melhor organiza- dos, por conta das Federações Sindicais Globais (GUFs), podem facilitar a integração das estra- tégias de construção institucional, tradicional- mente vinculadas aos sindicatos europeus com agressivas campanhas corporativas transfrontei- riças, associadas aos sindicatos estadunidenses (ver Bronfenbrenner, 2007). Ao mesmo tempo, o florescimento dos Global Framework Agree- ments (GFAs) fornece novos instrumentos com os quais as forças sociais do trabalho poderiam começar a tentar construir sua própria versão da governança global (ver McCallum, 2013). Estes desenvolvimentos, a nível global, de- pendem do engajamento dos movimentos dos trabalhadores nacionais para funcionar, mas ajudam, também, a torná-lo mais provável. Primeiro, argumentarei que ataques vio- lentos ao trabalho, que fazem parte do declínio da economia nacional estadunidense, estimu- laram a emergência de novas perspectivas e es- tratégias transnacionais no movimento dos tra- balhadores estadunidense. Se o deslocamento de uma hegemonia nacional ascendente para uma descendente, nos Estados Unidos, esti- mulou o interesse pelas alianças para além das 459 MOVIMENTOS NACIONAIS DE TRABALHADORES ... 7 Ver Evans (2010, p. 358). Remetendo-se, inicialmente, à técnica corporativista da “chantagem patronal”, que utili- za locais onde o movimento dos trabalhadores é fraco para minar a sua posição onde este é forte, o termo sugere a pos- sibilidade de se fazer o inverso – expandir o poder dos tra- balhadores debilitados em seu próprio local de trabalho, através de sua conexão com trabalhadores de locais onde o trabalhismo é mais forte. [N.T. traduzimos “whipsawing” como “chantagem patronal” quando a palavra é usada sem UM NOVO TRANSNACIONALISMO NA “BARRIGA DA BESTA”?6 As iniciativas transnacionais atuais, le- vadas a cabo pelos sindicatos estadunidenses, devem superar o ressentimento e o ceticismo gerado pela conivência destes últimos com a supressão das organizações militantes de tra- balhadores pelo mundo durante o apogeu da CRH, Salvador, v. 28, n. 75, p A terceira seção mostra como as mesmas inovações institucionais que o movimento dos trabalhadores brasileiro julgou útil facilitaram esforços de organização entre vários países. Esta seção destaca a interação dos sindicatos nacionais e globais na campanha para organi- zar os guardas de segurança do Group 4 Se- 460 Peter Evans arrogância imperial (ver Scipes, 2010). Ainda assim, examinadas em si, as novas iniciativas transnacionais que envolvem os sindicatos estadunidenses parecem refletir um desloca- mento significativo nas atitudes relacionadas à importância de se construir alianças com ou- tros movimentos de trabalhadores nacionais. tadunidense oferece um apoio útil aos traba- lhadores em Honduras e Libéria. Por outro, as alianças com os movimentos de trabalhadores brasileiros, politicamente mais seguros, aju- dam os sindicatos estadunidenses sob ataque. Em campanhas como a da Russell, a emergência de novos atores organizacionais como os United Students Against Sweat- shops (USAS) e do Workers Rights Consor- tium (WRC) foi crucial para colocar na mira as oficinas precárias, sendo parte integrante das campanhas, entretanto, o apoio dos sindicatos tradicionais (Rodriguez, 2007). Do papel histó- rico da Union of Needletrades, Industrial and Textile Employess (UNITE), na emergência dos United Students Against Sweatshops (USAS), ao papel crucial de Jeff Hermanson8 nas nego- ciações da campanha da Russell, houve uma simbiose entre os organizadores que trabalha- vam para os sindicatos e ativistas conectados com novas organizações. E, ao contrário de muitas iniciativas da parte de ONGs transna- cionais (ver Seidman, 2007), as campanhas contra as oficinas precárias como aquela con- tra a Russell estão em “aliança com” ao invés de atuarem “em nome dos” trabalhadores in- surgentes no Sul, os quais definem suas lutas em termos de demandas sindicais – reconhe- cimento, barganha coletiva, salários decentes, trabalho digno e proteção contra as represálias empresariais. Tanto o declínio da economia domés- tica estadunidense quanto o terreno político doméstico cambiante associado com o neoli- beralismo ajudaram a estimular a receptivida- de do movimento de trabalhadores estaduni- dense às estratégias transnacionais. 8 Hermanson foi um militante veterano do International La- dies’ Garment Workers Union (ILGWU) da indústria de ves- tuário e trabalhou também na América Latina com o AFL- CIO Solidarity Center (ver também Anner, 2013, p. 32). complemento. No caso do uso acompanho de “reverse” op- tamos por traduzi-la como “chantagem patronal ao revés”]. Caderno CRH 10 Para uma análise completa da composição complexa das redes envolvidas, ver Rodriguez (2007). Para uma visão interna detalhada da campanha Kukdong, uma campanha anterior exitosa que contribuiu para a “aprendizagem ins- titucional” que tornou possível a vitória da Russell, ver Hermanson (2004). Salvado 9 Fundada no Alabama em 1902, a Russel juntou-se a Fruit of the Loom no império Berkshire Hathaway de Warren Buffet, em 2006. UM NOVO TRANSNACIONALISMO NA “BARRIGA DA BESTA”?6 No início dos anos 1990, quando a administração de Clinton, que as forças sociais do trabalho su- punham ser sua aliada, demonstrou seu firme apoio à versão global do internacionalismo do capital, garantindo a aprovação do North Free Trade Agreement (NAFTA) pelo do congresso, estava claro que as fundações políticas de uma estratégia nacionalista haviam terminado. A subserviência bipartidária às prioridades cor- porativas transformou em quimeras os sonhos de uma proteção nacionalista. Ao mesmo tem- po, o neoliberalismo reforçou a agressividade do capital nos Estados Unidos, deixando claro que, sem novas estratégias, as forças sociais do trabalho americanas definhariam. Minha amostra ilustrativa das novas iniciativas que emergiram no neoliberalismo começa com a campanha na Russell Athletics, o apogeu do ativismo transnacional contra ofi- cinas precárias. Em seguida, discutimos as ini- ciativas internacionais empreendidas por dois sindicatos industriais clássicos dos Estados Unidos – o USW e o UAW. Além de defender o “novo transnacionalismo” no movimento de trabalhadores estadunidense, a seção ilustra dois tipos distintos de chantagem patronal ao revés.7 Por um lado, o internacionalismo es- A vitória de 2010 na Russel, descri- ta por Evangelina Argueta (ver acima), já foi reconhecida de forma incontestável pelos militantes das oficinas precárias do vestuário como a maior vitória já obtida (Greenhouse, 2009) e como “o maior acordo nas manufatu- ras da América Central” (Graham, 2010). Um dos maiores produtores de vestuário estadu- nidense e principal empregador da indústria exportadora mais importante de Honduras, a 461 MOVIMENTOS NACIONAIS DE TRABALHADORES ... MOVIMENTOS NACIONAIS DE TRABALHADORES ... Russell nunca havia assinado um contrato com qualquer sindicato em seus 100 anos de ope- ração nos Estados Unidos.9 O acordo de 2010 incluiu a reintegração de 1.200 trabalhadores do vestuário numa nova empresa sindicalizada (Jerzees Nuevo Dia), uma promessa de neutrali- dade da parte da Russell, e o acesso para os mi- litantes a outras de suas fábricas em Honduras, que empregam cerca de 10.000 trabalhadores. relíquia dos velhos tempos nos quais uma boa lei do trabalho era considerada um sinal da “modernidade”, era bastante progressista. A utilização era risível, mas contar com as leis nos livros ainda era uma vantagem, dado que os códigos de conduta nos Estados Unidos re- queriam conformidade com as leis do trabalho locais. As manufaturas do vestuário hondure- nhas, tanto domésticas quanto de propriedade estrangeira, eram consideradas fortemente ata- das aos mercados estadunidenses e, portanto, vulneráveis às suas ameaças. A vitória dependeu da convergência oportuna entre diversos fatores que tornaram a Russell vulnerável – de sua dependência de produtos oriundos da produção diretamente contratada na América Central à sua depen- dência do nicho de vestimenta universitária nos Estados Unidos.10 Mais importante, ainda, dependeu de uma reserva de habilidade estraté- gica acumulada e distribuída entre a rede trans- nacional de organizações de trabalhadores. Uma militância movimento dos traba- lhadores local forte e a dependência da elite em relação aos mercados estadunidenses cria- ram um potencial para a aliança transnacio- nal. Quando os trabalhadores hondurenhos tomaram a decisão estratégica de construir laços com aliados transnacionais, havia uma estrutura à qual podiam conectar-se (Anner, 2013, p. 31). Eles se conectaram a uma arqui- tetura contra as oficinas precárias que estava longe de ser perfeita. A Fair Labor Association (FLA), cuja missão consistia, teoricamente, em persuadir os manufatureiros estadunidenses a cumprir os códigos de conduta, ignorou, num primeiro momento, as reclamações dos traba- lhadores hondurenhos (Anner, 2013, p. 32). A despeito disto, a USAS foi exitosa em ameaçar o mercado de vestimenta universitária, bastan- te lucrativo para a Russell. r, v. 28, n. 75, p. 457-478, Set./Dez. 2015 g ç À primeira vista, Honduras era um terreno hostil para um triunfo organizativo no século XXI. A dominação empresarial sobre o Estado hondurenho persistiu, mesmo duran- te o governo populista de Mel Zelaya. Peter Evans O caso Russell é excepcional, mas não é único. A luta exitosa para formar um sindi- cato independente na Bridgestone-Firestone, nas plantações de seringueiras da Libéria, é uma variação dos recursos organizacionais sediados nos Estados Unidos para pressionar as operações estrangeiras de uma corporação estadunidense num país pequeno e pobre. As condições nas plantações eram tão execráveis que o International Labor Rights Forum (ILRF) processou a Bridgestone-Firestone em 2005 por impor “condições de trabalhos semelhantes às escravas” (ILRF, 2005). O USW, que organiza os trabalhadores estadunidenses da Bridgestone- Firestone, teve seus próprios problemas com a companhia e viu uma oportunidade de colocar a Firestone na defensiva. trabalhadores militantes em países pequenos e pobres, dependentes economicamente dos Estados Unidos, puderam alavancar conexões transnacionais com os trabalhadores america- nos. Tais alianças transnacionais ajudaram a mudar o território nacional. Os movimentos dos trabalhadores, que foram por eles revigo- rados, adquiriram uma nova possibilidade de se tornarem atores políticos locais. Estes casos ilustram uma abertura às alianças transnacionais por parte dos sindica- tos estadunidenses cuja origem radica da in- dústria de bens de consumo, onde os conflitos a respeito da disposição geográfica dos postos de trabalho foram tradicionalmente conside- rados uma barreira para a construção de uma solidariedade Norte-Sul (Evans, 2010, p. 355). Eles são interessantes, também, porque são construídos sobre alianças entre sindicatos tradicionais e as novas ONGs, vinculadas ao mundo do trabalho, cujo estilo organizacional e ideológico é tido como dissonante em relação ao sindicalismo tradicional, o que sugere que conexões transnacionais e conexões que ligam estilos organizativos devem estar sinergetica- mente relacionadas (Anner and Evans, 2004). Quando os trabalhadores das plantações fizeram uma greve sem o apoio do seu sindi- cato,11 os sindicatos da USW Bridgestone-Fi- restone nos Estados Unidos coletaram fundos de apoio aos trabalhadores grevistas. Depois, quando o recém-formado Firestone Agricul- tural Worker Union of Liberia (FAWUL) dis- putou uma eleição contra o sindicato amare- lo estabelecido da Firestone, o USW, a ICEM (International Federation of Chemical, Energy, Mine and General Workers’ Unions) e o AFL- CIO (American Federation of Labor/Congress of Industrial Organizations) Solidarity Center forneceram apoio e a FAWUL ganhou a eleição internacionalmente monitorada (Kazdin). 11 [N.T. “wildcat strike”. Trata-se de uma greve de trabalha- dores geralmente sindicalizados que, contudo, não conta com o apoio da entidade.] MOVIMENTOS NACIONAIS DE TRABALHADORES ... O gol- pe militar de Estado, que derrubou Zelaya em 2009, reforçou a atmosfera de repressão, na qual a violência contra os ativistas do movi- mento dos trabalhadores era frequente. A elite empresarial estava permeada pela ideologia anticomunista tradicional, na qual sindicatos e comunismo eram tomados como indistintos. Olhando mais de perto, Honduras era um ambiente mais favorável para a organi- zação do que parecia. A militância do movi- mento dos trabalhadores era uma tradição há muito estabelecida. Organizadores inconfor- mados como Argueta atuaram neste ambiente repressivo com uma efetividade surpreenden- te. A legislação trabalhista hondurenha, uma À primeira vista, Honduras era um terreno hostil para um triunfo organizativo no século XXI. A dominação empresarial sobre o Estado hondurenho persistiu, mesmo duran- te o governo populista de Mel Zelaya. O gol- pe militar de Estado, que derrubou Zelaya em 2009, reforçou a atmosfera de repressão, na qual a violência contra os ativistas do movi- mento dos trabalhadores era frequente. A elite empresarial estava permeada pela ideologia anticomunista tradicional, na qual sindicatos e comunismo eram tomados como indistintos. No momento do acordo, aproxima- damente 110 universidades finalizaram seu contrato com a Russell e a USAS começou a buscar seus maiores clientes não universitá- rios (Anner, 2013, p. 34). A vitória final foi um produto transnacional que utilizou fontes institucionais do próprio ambiente da Russell para compensar os empresários recalcitrantes locais e a falta de uma atuação estatal confiá- vel em Honduras. Os representantes da CGT foram aos Estados Unidos para envolver o em- presariado da matriz da Russell nas negocia- ções finais. O acordo final foi viabilizado por conta de uma arbitragem orquestrada nos Es- tados Unidos. v. 28, n. 75, p. 457-478, Set./Dez Olhando mais de perto, Honduras era um ambiente mais favorável para a organi- zação do que parecia. A militância do movi- mento dos trabalhadores era uma tradição há muito estabelecida. Organizadores inconfor- mados como Argueta atuaram neste ambiente repressivo com uma efetividade surpreenden- te. A legislação trabalhista hondurenha, uma 462 Peter Evans MOVIMENTOS NACIONAIS DE TRABALHADORES ... tia anunciaram uma comissão para explorar a formação de um único sindicato Norte Ameri- cano. O sindicato único Norte Americano não se materializou, mas, se este tivesse sido o caso, agregaria, aproximadamente, 200.000 Mineros aos quase 800.000 membros do USW nos Esta- dos Unidos e no Canadá, criando um dos maio- res sindicatos no hemisfério. importado. Porém, seu apoio aos trabalhadores liberianos da plantação de seringueiras foi con- sistente, ao mesmo tempo, com uma história de iniciativas transnacionais. A luta exitosa dos Steelworkers (sindicato dos metalúrgicos) con- tra Marc Rich e o empresariado da Ravenswood Aluminium é uma das campanhas transnacio- nais mais célebres da memória recente (Juravi- ch and Brofenbrenner, 1999).12 Se a aliança USW-Mineros ilustra ainda mais os possíveis benefícios do novo transna- cionalismo estadunidense para os sindicatos do Sul, o apoio que o USW recebeu como con- sequência de sua filiação ao Gerdau Workers World Council (GWWC) é um dos melhores exemplos de como os sindicatos estaduniden- ses podem se tornar beneficiários da chanta- gem patronal ao revés. Mais relevante, ainda, para o ponto de vista desta análise, é a aliança do USW com os sindicatos dos mineiros mexicanos, o SNT- MMSSRM (Sindicato Nacional de Trabaja- dores Mineros, Metalúrgicos, Siderúrgicos y Similares de la República Mexicana), conhe- cido como os Mineros (Davis, 2012). A relação USW-Mineros, não somente ilustra a abertura da USW às alianças transnacionais, como, também, mostra o quanto uma crescente re- ceptividade às alianças transnacionais por par- te de sindicatos, outrora corporativos no Sul, pode ser um complemento crítico às iniciati- vas transnacionais vindas do Norte. O estímulo inicial para o GWWC foi a agressiva expansão nacional e global do maior produtor privado de aço do Brasil, a Gerdau, que, em 2003, tornou-se a quarta maior produ- tora de aço do mundo, com filiais na América do Norte e na Europa (Gray, 2009, p. 89). No Brasil, a Confederação Nacional dos Metalúr- gicos, filiada à Central Única dos Trabalhado- res (CUT) percebeu que não podia defender seus direitos e salários contra a Gerdau com base nas lutas individuais e locais contra os empresários de plantas individuais. Eles construíram, primeiro, uma rede nacional de trabalhadores da Gerdau. Trocas entre os tra- balhadores da USW canadense, fundada pela “SteelWorkers Humanity Fund”, também do Canadá, iniciadas em 1997, principiaram uma iniciativa transnacional. Caderno 12 A USW já havia criado um sindicato transnacional em 2008, através da criação, juntamente com o britânico Uni- te the Union, de 1.5 milhões de membros, de uma nova entidade chamada Workers Uniting. Peter Evans Nes- te caso, o apoio dos aliados estadunidenses foi menos fundamental do que no caso da Russel, em parte devido ao fato de o recém-eleito go- verno de Ellen Johnson Sirleaf haver sido fa- vorável aos trabalhadores da Firestone, mas a formação de um sindicato independente, com 5.000 membros no setor exportador mais im- portante do país, foi um marco semelhante à vitória na Russell. Infelizmente, apenas um número limita- do de países se encaixa no perfil de Honduras e Libéria. As vitórias nos países onde tal é o caso, mesmo se multiplicadas, podem, dificilmente, desequilibrar a balança do poder global na dis- puta entre o movimento dos trabalhadores e o capital. A menos que o conjunto de novas co- nexões nacionais inclua uma gama mais ampla de países, seu impacto na arquitetura das forças sociais do trabalho global será mínimo. Uma mirada mais ampla sobre o USW mostra como um conjunto amplo de alianças transnacionais, que conectam uma série de paí- ses, pode emergir a despeito da relação tradicio- nalmente ambivalente de um país rico com seu sindicato de bens de consumo. A USW definiu sua agenda antiglobalização através do dumping ao aço importado no porto de Seattle, em 1999, e deu as boas vindas às tentativas da adminis- tração Bush de impor quotas ao aço brasileiro Os casos, hondurenho e liberiano, de- monstraram que, sob o neoliberalismo, os 463 MOVIMENTOS NACIONAIS DE TRABALHADORES ... MOVIMENTOS NACIONAIS DE TRABALHADORES ... Em 2003, os trabalha- dores da Gerdau do Brasil, Argentina, Chile, Uruguai, Canadá e Estados Unidos reuniram- se para discutir a possibilidade de construir uma rede transnacional (Gay, 2009, p. 81-92). CRH, Salvador, v. 28, n. 75, p. 457-478, Set./Dez. 2015 Confrontados com a repressão governa- mental à icônica greve de 1989, na mina de co- bre em Cananea, os Mineros do México não es- tavam, ainda, prontos para assumir as alianças com os sindicatos estadunidenses como parte de sua estratégia. Em 2005, uma série de ata- ques virulentos vindos do Estado mexicano mu- dou suas opiniões. Eles assinaram uma aliança estratégica com o USW, filiado tanto à Interna- tional Metalworkers Federation (IMF) quanto à International Federation of Chemical, Energy, Mine and General Workers’ Unions (ICEM), e sustentaram uma greve de solidariedade de um dia em apoio à greve do USW na subsidiária es- tadunidense do Grupo México (Davis, 2012, p. 506). No ano seguinte, as perseguições do go- verno forçaram o presidente dos Mineros, Na- poleon Gomez Urrutia, a exilar-se no Canadá, onde contou com o apoio do USW. Em 2010, o presidente do USW, Leo Gerard, e Gomez Urru- Ao mesmo tempo, a produção de aço nos Estados Unidos estava se transferindo para fábricas pequenas, geridas por firmas hostis ao movimento dos trabalhadores em estados onde vigem leis antisindicais, no sul do país, debi- litando a habilidade do USW para organizar, ou mesmo manter, os contratos existentes. Os 464 Peter Evans problemas do USW e os esforços da rede da Gerdau convergiram em 2005, quando conver- sas sobre contratos numa planta da Gerdau, em Beaumont, Texas, terminaram em uma inter- rupção da produção depois que a Ameristeel, subsidiária da Gerdau, demandou, como parte de sua “melhor oferta derradeira”, demissões de férias, hora extra e direitos de antiguidade. em tirar proveito do ambiente antisindicato do sul estadunidense. Eles mitigaram as motivações econômicas para que os trabalhadores se organi- zassem oferecendo bons salários em comparação com os padrões locais vigentes. A resposta de King a esta missão impos- sível deu-se em várias frentes. Na planta da VW (Volkswagen) em Chattanooga, Tenessee, o UAW desenvolveu uma estratégia baseada na chantagem patronal ao revés, tradicionalmente estruturada, baseada no poder institucionali- zado que a IG Metall havia construído em sua base alemã. 14 Embora seja legítimo ligar a estratégia da UAW à presi- dência de King (e à sua antiga influência como um diretor organizacional), minha afirmação sobre a forma estratégi- ca de pensar é baseada, exclusivamente, em minha inter- pretação das ações e declarações públicas de King e da UAW, não em comunicações pessoais. 13 Deve-se sublinhar que a GWWC é tanto uma aliança Sul-Sul quanto Sul-Norte, preocupada, igualmente, com os efeitos da expansão da Gerdau para o resto da Amé- rica Latina. Uma campanha recente contra as demissões em companhias adquiridas pela Gerdau na Colômbia é um exemplo. 16 O esforço da UAW em 2001 para organizar a planta da Nissan em Smyrma, Tenneesse, terminou em derrota por uma margem de 2 a 1 (Aschoff, 2014). Os críticos de es- querda não estavam impressionados com a margem, atri- buindo a derrota à inabilidade da UAW para conectar-se aos trabalhadores de base (Aschoff, 2014; Early, 2014). S l d 15 88 das 104 plantas da VW ao redor do mundo possuem conselhos de trabalhadores e a lei estadunidense faz da existência de um sindicato um pré-requisito para a exis- tência de um conselho de fábrica. Ver Automotive News, 10/7/2013. 18 Ver a nota conjunta (em discrepância com o editorial do jornal [op. ed]) das lideranças dos metalúrgicos da Força e da CUT em Folha de São Paulo, 19 de novembro, 2012 pg. 3. Como sublinha Anner (2003, 2011), a Força tem sido, tradicionalmente, menos internacionalista que a CUT, sendo esta ação de articulação, portanto, um indicador importante do aprofundamento dos laços institucionais da UAW no Brasil. MOVIMENTOS NACIONAIS DE TRABALHADORES ... ção na Canton cresceria dramaticamente. se focou na planta da Nissan em Canton, Mis- sissippi, na qual trabalhadores comuns cos- tumavam habitar a metade superior da tabela de distribuição de renda e um argumento eco- nômico por parte dos sindicatos não ganharia muita adesão (Greenhouse, 2013). Como alter- nativa, a campanha focou no sindicato como um veículo capaz de dar voz aos trabalhadores em suas vidas no trabalho. O UAW concebeu as reclamações por mais voz no trabalho como o próximo passo para a inconclusa agenda de direitos civis no Mississippi, o que surtiu efei- to para a força de trabalho predominantemen- te afro-americana da planta. Com esta aborda- gem, a campanha ganhou o apoio da National Association for the Advancement of Colored People (NAACP) local (Compa, 2013). Mudar o ambiente político para a Nissan no Brasil requereria mais do que uma campa- nha para angariar apoio para uma greve. De- mandaria um investimento de longo prazo para criar laços com o movimento dos trabalhadores brasileiro e despertar o interesse da mídia e da cultura de massa brasileira a respeito da base antisindical das transplantas no Sul dos Esta- dos Unidos. O UAW investiu em duas organiza- ções no Brasil. Bob King passou uma semana no Brasil, em meados de 2012, para marcar a aber- tura do escritório da UAW no Brasil, discursou no congresso nacional da CUT, e encontrou-se com o presidente da câmara dos deputados, Marco Maia (também um antigo metalúrgico). Quatro meses depois, o UAW bancou um estan- de na exposição de carros em São Paulo para construir relações com a mídia local. Por atraente que pudesse ser a campa- nha a nível local, a UAW percebeu que, en- quanto a batalha fosse levada a cabo somente em Canton, exerceria pouca influência sobre o empresariado global da Nissan. Quando os trabalhadores da Nissan se reuniram no Tuga- loo College, no início de 2013, Vagner Freitas, o presidente da CUT brasileira, estava entre os presentes. Trazer o presidente da CUT à Canton não foi somente um exercício de “diplomacia trabalhista”. Na estratégia global da Nissan para construir um nicho de mercado, o Brasil era um elemento crucial, fazendo deste último um elemento importante também para o UAW. Vencer no Sul dos Estados Unidos dependeu da construção de uma aliança durável entre os países do Sul Global. Caderno CR 17 De 2005 a 2011, o mercado automobilístico brasileiro marcou 12% de crescimento ao ano, comparado com cer- ca de 2% nos Estados Unidos. No ano fiscal de 2011, as vendas da Nissan quase dobraram no Brasil (Nissan 2012 Annual Report, p. 16). MOVIMENTOS NACIONAIS DE TRABALHADORES ... Como um sistema de conselho tole- rante aos sindicatos tornou-se parte integrante do modus operandi da VW ao redor do mundo e, pela lei estadunidense, requer um sindicato, o IG Metall e o UAW puderam persuadir a VW a permanecer neutra nas eleições sindicais.15 O USW percebeu que uma campanha de conscientização teria de estender-se ao país de origem da Gerdau para ser efetiva, e os Me- talúrgicos da CUT estavam preparados para oferecer solidariedade. Sem tentar recapitular a história desta longa e dramática campanha (Gay, 2009, p. 98-122), basta dizer que, pres- sionada tanto em casa quanto por uma cam- panha apoiada na rede da GWWC, o empresa- riado da Gerdau no Brasil decidiu, finalmente, que a abordagem linha dura antitrabalhismo dos empresários da Ameristeel nos Estados Unidos era contra-produtiva. Em 2007, a USW podia negociar contratos nas plantas onde a produção havia sido interrompida.13 O resultado foi uma confrontação do poder econômico transnacional com a cultura política local. A companhia deveria permane- cer neutra, mas os políticos locais no Tenessee foram loquazes em sua condenações à UAW como “estrangeira”, no Sul, e da Volkswagen como “antiamericana” em seu afã por conquis- tar votos. No fim, a chantagem patronal ao re- vés quase funcionou, mas não muito. O UAW perdeu a eleição por uma margem de 86 votos (712 a 626), aproximando-se mais dos esforços anteriores, menos apoiados transnacionalmen- te, para organizar as transplantas do Sul, mas segue sendo uma derrota e uma indicação dos limites da capacidade das estratégias transna- cionais para desequilibrar a balança local das forças políticas nos Estados Unidos.16 Outro sindicato tradicional de bens de consumo, o UAW, busca, atualmente, uma va- riação ainda mais interessante da estratégia da chantagem patronal ao revés. Em 2010, o novo presidente da UAW, Bob King, fez da sindicali- zação das montadoras estrangeiras (conhecidas como “transplantas”), no Sul dos Estados Uni- dos, seu principal objetivo industrial.14 Fazia sentido. A menos que possa sindicalizar estas plantas, o UAW está condenado a assistir ao nú- mero de trabalhadores das indústrias encolher. O problema com a iniciativa de King estava em que soava como um projeto estilo “missão impossí- vel”. Apesar de sindicalizadas em seus países de origem, as firmas estrangeiras estavam contentes A outra estratégia de King para organi- zar as transplantas foi mais inovadora, embora também fosse “uma missão impossível”. Ele 465 Ca q Annual Report, p. 16). MOVIMENTOS NACIONAIS DE TRABALHADORES ... A mensagem de King ao Brasil era a de que os sindicatos estadunidenses admiravam seus colegas brasileiros e ganhariam em apren- der com eles, que o declínio do poder dos sindi- catos nos Estados Unidos estava tornando cada vez mais precária sua classe trabalhadora e que as alianças transnacionais deveriam ser parte da resposta. Os líderes do trabalhismo brasilei- ro estavam impressionados com o fato de que o UAW houvesse começado construindo laços e foram, por isso, mais simpáticos à campanha. A ênfase nos direitos civis na campanha do Mississippi também repercutiu no Brasil ( Donizetti, 2013). Quando a CUT anunciou seu acordo de 2013 com a AFL-CIO, “combater as práticas antisindicato na planta da Nissan no Mississippi” foi a única ação conjunta concreta destacada.18 Em Outubro de 2013, o ex-presi- dente do Brasil, Luís Inácio Lula da Silva, escre- veu uma carta ao presidente da Nissan Gohsn, dizendo que, embora a considerasse “uma com- panhia global impressionante”, que “mantinha Salvador, v. 28, n. 75, p. 457-4 O Brasil era o quarto maior mercado auto- mobilístico no mundo e crescia com mais veloci- dade que os mercados no Norte Global.17 Era um mercado no qual a Nissan viu uma oportunidade para expandir-se. Se o UAW pudesse lograr um acordo plausível para fazer do sucesso da expan- são planejada da Nissan no Brasil uma parte de sua barganha, o custo de impedir a sindicaliza- 466 Peter Evans boas relações com os sindicatos no Brasil e em outros países”, estava profundamente preocu- pado com a campanha antisindicato, que con- duzia a Nissan nos Estados Unidos, e esperava uma “ação reparadora” (Lula, 2013). arenas de contestação sociopolítica, na qual as lutas nacionais do movimento dos trabalhadores são entrelaçadas às estratégias transnacionais. O Brasil é o caso óbvio para explorar o poten- cial para interações positivas entre tais terrenos e a arquitetura das forças sociais do trabalho ao nível global. Há duas questões aqui. Primeiro, que papel devem cumprir os movimentos dos trabalhadores nos maiores países do Sul global para construir uma arquitetura das forças sociais do trabalho ao nível global? Segundo, e recipro- camente, que tipo de papel deve cumprir uma arquitetura mais efetiva das forças sociais do tra- balho para provocar inflexões positivas na evolu- ção destes terrenos nacionais? MOVIMENTOS NACIONAIS DE TRABALHADORES ... Criar um sindicato local com alguns mi- lhares de trabalhadores no Mississippi seria tão consequente para a economia política local quanto foi o estabelecimento de um sindicato independente para os 5.000 trabalhadores da plantação de seringueiras para a economia po- lítica local da Libéria, restando saber se a estra- tégia de King vencerá. Mesmo que a pressão do Brasil sobre a Nissan seja efetiva, a ferocidade local da oposição política será proporcional à magnitude do efeito de sindicalização. Vitória ou derrota, a iniciativa permanece um exem- plo da consciência dos sindicatos tradicionais industriais estadunidenses a respeito do papel essencial que devem cumprir as alianças glo- bais na sobrevivência da estratégia doméstica. O papel do Brasil, no suporte aos es- forços nascentes dos sindicatos estaduniden- ses para construir alianças transnacionais, já foi ressaltado, mas o volume das alianças transnacionais do Brasil envolveu sindicatos europeus, redes empresariais, acordos de padrões internacionais e Federações Sindicais Globais (Global Union Federations - GUFs) es- treitamente ligados aos sindicatos europeus. As primeiras alianças transnacionais do Brasil foram construídas em torno de lutas com o re- gime militar brasileiro, a respeito dos direitos sindicais nos anos 1970 (Anner, 2011, p. 125). Por exemplo, os sindicalistas do IG Metall, que trabalhavam na Volkswagen na Alemanha, apoiaram os esforços dos trabalhadores da planta brasileira da Volkswagen em sua luta para conquistar direitos sindicais básicos. Na medida em que o UAW, assim como o USW, é um sindicato cujas tradições e cultu- ra política são enraizadas na indústria clássica de bens de consumo, estes exemplos possuem implicações que estão para além da simples si- nalização a uma abertura crescente às alianças transnacionais da parte do trabalhismo estadu- nidense. Ambos os exemplos sugerem que os velhos argumentos de que os conflitos sobre a distribuição geográfica dos postos de trabalho em indústrias de bens de consumo minam a possibilidade de iniciativas transnacionais de- veriam ser revisados. Depois, as lutas econômicas ganharam protagonismo. Os líderes dos sindicatos bra- sileiros aprenderam o alemão e se tornaram participantes ativos nos conselhos de traba- lho globais das companhias automobilísti- cas alemães, tanto da Volkswagen quanto da Daimler.19 Este trabalho gerou frutos em 2001 quando a liderança da CUT usou as conexões alemãs para denunciar a intransigência dos empresários de subsidiárias localizadas em C 19 Em 2002, o Brasil possuía mais membros que qualquer outro país nos Volkswagen Worldwide works Council, ex- cluindo-se a Alemanha (Rüb, 2002: 23). MOVIMENTOS NACIONAIS DE TRABALHADORES ... desejam aderir. Apoiou, também, o projeto “CUT- Multi” (Ação de confrontação às multinacionais), que foi concebido para criar redes entre todos os sindicatos (dentro e fora da CUT) que organizam trabalhadores em uma corporação multinacional particular (Jakobsen, 2007:154). Embora se tenha focado intensamente nas filiais das companhias da Dutch (Akzo-Nobel, Phillips etc.), a CUT-Mul- ti estendeu-se para outras multinacionais de ou- tras origens. Uma das redes empresariais mais desenvolvidas é a rede BASF (Badische Anilin und Soda-fabrik, uma Companhia Química Ale- mã) que se estendeu pela América Latina e foi ativada em apoio a U.S. Steel Workers na Améri- ca do Sul. Em 2005, novamente com o apoio da FNV, a metodologia de pesquisa do Observatório foi compartilhada com organizações em seis ou- tros países da América Latina, terminando por criar a “Latin American Network for Research on Multinational Companies” (REDLAT) (Veiga e Jakobsen, 2011: 92-93). São Paulo, efetivar negociações com a sede da VW em Wolfsburg, sediar a produção de um novo modelo no Brasil e mitigar demissões planejadas (Anner, 2011, p. 128). As alianças Alemanha-Brasil também produziram ações solidárias para impedir que a companhia com- pensasse perdas de produção por conta das greves com o aumento de horas extras no Bra- sil (Anner, 2011, p. 130-131). Alianças com outros sindicatos e a parti- cipação em conselhos de trabalho foram com- plementadas pelo uso dos Acordos-Marcos Globais (IFAs/GFAs), concebidos para vincular operações pelo mundo aos padrões aceitos pela companhia matriz em sua sede. Como os con- selhos de trabalho ao redor do mundo, os IFAs são um dispositivo característico da Europa. Concebido em termos bastante genéricos, os IFAs são “capengas” na falta de um poder sindical complementar, mas, no contexto da organização local e das redes transnacionais do movimento dos trabalhadores, podem ser ferramentas valiosas (Fitcher; Helsen, 2011; McCallum, 2013; Stevis; Boswell, 2007, 2008). O Brasil também entendeu que as GUFs podem cumprir um papel doméstico útil. As GUFs devem manter-se ostensivamente neu- tras face à multiplicidade das confederações que surgiram no ambiente relativamente sim- pático ao movimento dos trabalhadores no Bra- sil. Desta forma, as GUFs podem cumprir, com frequência, um papel útil na negociação das campanhas mais amplas. A “Campanha para o trabalho decente na Copa do Mundo da FIFA de 2014” da Building and Wood Worker’s Inter- nationl (BWI) é um bom exemplo. AS ARTICULAÇÕES TRANSNACIO- NAIS DO “SUL INSURGENTE” Assim como o Brasil cumpre um impor- tante papel na visão da Nissan sobre seus lucros globais, os maiores países do Sul Global são cen- trais para a busca geral do capital por expandir lucros globais. Estes países não são somente “grandes economias dinâmicas com uma gran- de influência política” (UNDP, 2013); eles são 467 MOVIMENTOS NACIONAIS DE TRABALHADORES ... MOVIMENTOS NACIONAIS DE TRABALHADORES ... Demandou a construção de uma campanha por parte dos sindicatos da construção, filiados a cinco con- federações brasileiras diferentes, algo que teria sido difícil para a CUT ou qualquer outra con- federação individual brasileira. Da mesma for- ma, a Public Services International (PSI) se vê engajada, frequentemente, nas negociações di- plomáticas entre os sindicatos brasileiros, filia- dos às confederações distintas, que trabalham na saúde ou em outros campos dos serviços. p. 457 478, Set./Dez. 2015 O uso que fizeram os metalúrgicos bra- sileiros do IFA da Daimler, assinado em 2002, exemplifica sua possível utilidade. Como o IFA da Daimler vale tanto para os fornecedores quanto para as próprias filiais, os metalúrgicos da Mercedes no Brasil puderam usá-lo como um instrumento para fortalecer o poder sindi- cal nas plantas fornecedoras menos organiza- das, através da luta contra as violações da IFA nos fornecedores, fazendo os empresários da Mercedes intervirem em prol de sua aplicação (Fichter e Helfen, 2011:99-100). Caderno CRH, Salvador, v. 28, n. 75, p Os sindicalistas brasileiros complemen- taram amplas colaborações com as Federações Europeias com a construção de alianças dentro das empresas individuais. A longa história da colaboração entre a Dutch Federation of trade Unions (FNV) e a CUT é um bom exemplo. Tra- balhando com o Instituto Observatório Social da CUT, a FNV apoiou a pesquisa sobre o cumpri- mento dos padrões fundamentais de trabalho por parte das corporações multinacionais às quais O Brasil demonstra como um terreno favorável pode tanto estimular articulações de redes internacionais de trabalhadores quanto 468 Peter Evans e sua utilização acelerou rapidamente, no neo- liberalismo (Bronfenbrenner, 2007; Munck, 2010), uma mudança bem-vinda com relação à quietude relativa da “era de ouro do capitalis- mo” do pós-segunda guerra. delas se beneficiar. Mostra como um movi- mento dos trabalhadores bem organizado pode engajar-se tanto em campanhas de confronto associadas à postura combativa do movimento dos trabalhadores estadunidense em relação às empresas intransigentes, quanto no foco de construção institucional, atribuído aos sindi- catos europeus. A habilidade do movimento dos trabalhadores no Brasil para lidar com os dois tipos de estratégias é a marca registrada de sua sagacidade para tornar-se um nexo central para as redes transnacionais de trabalhadores. Tradicionalmente, as estruturas sindi- cais internacionais, que deveriam prover a espinha dorsal de tal projeto, foram subfinan- ciadas pelos sindicatos nacionais (Jakobsen, 2001). MOVIMENTOS NACIONAIS DE TRABALHADORES ... Os tempos difíceis do neoliberalismo promoveram iniciativas suficientes para que os movimentos nacionais dos trabalhadores investissem recursos suficientes nas estrutu- ras sindicais globais? Há esperança de que es- tas organizações se tornarão, por sua vez, mais efetivas em articular a interação dos movimen- tos nacionais? Os céticos rechaçarão ambas as possibilidades, mas poderão estar perdendo algumas oportunidades promissoras. O Brasil oferece um modelo proveitoso para o movimento global dos trabalhadores, que pode aproveitar-se do fortalecimento das forças sociais do trabalho nos maiores países do Sul Global, mas, também, ilustra o útil pa- pel da infraestrutura global, tanto como apoio para as campanhas dentro dos territórios na- cionais quanto de suporte à construção de co- nexões entre os movimentos nacionais de tra- balhadores. As conexões baseadas numa con- juntura política nacional ou em alianças bila- terais devem ser complementadas com redes e campanhas entre vários países, que, por sua vez, requerem um aparelho global mais desen- volvido do que aquele com o qual o movimen- to dos trabalhadores pôde contar no passado. As GUFs, a concretização setorial do movimento sindical global, evoluíram desde seus dias como “International Trade Secre- tariats”. No início da “era de ouro do capita- lismo” pós-segunda guerra, o International Trade Secretariats era tímido e setorialmente especializado. A expansão destas organizações setoriais globais se dá em contraste com a re- lativa estagnação na filiação sindical ao nível nacional. Planejam um papel mais ativo, não só em terrenos favoráveis como o Brasil, mas, também, em campanhas entre vários países.20 20 Focar o lado convencional da arquitetura global me força a negligenciar as organizações entre vários países menos convencionais como a SIGTUR (Southern Initiative on Globalization and Trade Union Rights), às quais perten- cem a CUT, a COSATU e a KCTU (Korean Confederation of Trade Unions) e que requerem uma análise separada. Ver Webster, Lambert and Bezuidenhout, 2008; Evans, 2010. 21 Estas comparações de horas extras são um pouco impre- cisas já que se referem somente aos filiados à Confedera- tion of Free Trade Unions (ICFTU) e à International Trade Union Confederation (ITUC). Faltam-nos dados sobre as organizações setoriais filiadas à World Federation of Trade MOVIMENTOS NACIONAIS DE TRABALHADORES ... IFA pode ser integrado a uma campanha corpo- rativa entre vários países é a campanha G4S para organizar os guardas de segurança. A campanha G4S é, geralmente, tomada como o arquétipo da campanha corporativa do início do século XXI. Ela mostra como a combinação entre os IFAS e campanhas globais depende da construção de uma arquitetura internacional correspondente, capaz de combinar esforços mais enraizados de um conjunto diverso de sindicatos nacionais com o escopo de negociação mundial de um Sindicato Global. Evidencia que as estratégias globais são importantes no agora globalizado setor de serviços, bem como na manufatura. E fornece ideias úteis sobre a maneira pela qual territórios nacionais moldam e podem ser mol- dados pelas campanhas globais. riamente, um indicador da perspicácia estra- tégica ou da efetividade mobilizacional, mas disponibiliza capacidades e recursos para as iniciativas estratégicas. As GUFs de hoje são enormes. A IndustriALL, o sindicato global híbrido, possui 50.000.000 de membros e 800 sindicatos nacionais filiados ao redor do mun- do. Os dois maiores setores de serviços das GUFs – PSI e UNI – possuem juntos 40.000.000 membros e 1.500 sindicatos filiados. Junto com o novo tamanho e escopo das GUFs, cresceu o interesse em forçar campa- nhas para assinar os IFAs ou GFAs, como os IFAs discutidos pela Volkswagen e Daimler no Brasil. O primeiro IFA foi assinado em 1988; na virada do milênio, havia, ainda, somente 8; em 2006, havia 55 (Stevis e Boswell, 2007: 112-113). Provou-se quase impossível fazer com que as companhias sediadas nos Estados Unidos assinassem os IFAS (Fitcher e Helfen, 2011; Stevis e Boswell, 2007, 2008), mas o IFA do International Metal Federation de 2012 com a Ford propõe que o novo transnacionalismo dos Estados Unidos deva incluir a pressão para os IFAs nas firmas multinacionais sediadas nos Estados Unidos (IMF, 2012). A análise de Jamie McCallum (2013) da campanha G4S nos fornece uma imagem teórica provocativa de que como funcionou a campanha tanto ao nível global quanto ao nível nacional. Os atores-chave nas análises de McCallum incluíam a Rede Sindical Inter- nacional (Union Network InternationalUnion Network International - UNI), a maior do setor de serviços das GUFs, o SEIU, uma campeã es- tadunidense de campanhas corporativas agres- sivas, a South African Transport and Allied Works Union (SATAWU), a Indian National Trade Union Congress (INTUC), e o Center of Indian Trade Unions (CITU). no CRH, Salvador, v. 28, n. 75, p. CONECTANDO MÚLTIPLOS TERRI- TÓRIOS NACIONAIS Embora a densidade sindical tenha caí- do na maior parte dos países ao redor do mun- do, com a filiação decrescendo em termos ab- solutos em alguns países, a filiação e o número de membros das GUFs continuam a subir até atingir cinco vezes o índice seu tamanho na metade do século.21 O tamanho não é, necessa- Os impérios de corporações multinacio- nais nunca se limitam a conectar unicamente dois mercados nacionais. O movimento global de trabalhadores tampouco pode dar-se por sa- tisfeito com as conexões bilaterais. As conexões nacionais devem ser eventualmente imersas em estruturas mais amplas se querem exercer um poder real na economia global. O movimento dos trabalhadores deve construir sua própria versão da governança global. As campanhas corporativas, para além das fronteiras, redes empresariais, IFAs, e GUFs, são todas formas capazes de ligar múltiplos terrenos nacionais, 469 MOVIMENTOS NACIONAIS DE TRABALHADORES ... MOVIMENTOS NACIONAIS DE TRABALHADORES ... 457-478, Set./Dez. 2015 Na medida em que um IFA possui juris- dições que são tão multinacionais quanto as companhias e GUFs que as assinaram, a verda- deira questão está em se podem difundir gan- hos obtidos em ambientes onde o movimento dos trabalhadores é mais forte para os terrenos nacionais menos favoráveis. Stevis e Boswell (2007, p. 175) argumentam que as “chances de que os IFAs fortaleçam os organizados e orga- nizem os não-organizados dependerão ampla- mente de se se integram às estratégias para além das fronteiras, tais como as campanhas de cons- cientização”. Em suma, eles apontam para uma combinação entre uma construção institucional de estilo europeu e as campanhas corporativas de estilo estadunidense como uma maneira de tornar os IFAs uma ferramenta mais potente para o movimento global de trabalhadores. Como o maior mercado do G4S, a Índia, era um elemento crucial na campanha global, a África do Sul e uma série de outros países no Sul Global cumpriram um importante papel também. Contudo, as origens da campanha do G4S não foram a Índia ou a África do Sul, mas a convicção do SEIU de que somente uma cam- panha global poderia quebrar barreiras que a impediam de organizar o maior empregador de guardas de segurança dos Estados Unidos. Par- tes decisivas da liderança do SEIU decidiram, como relatou Harold Myerseon (2009), “se vo- cês querem organizar os guardas da segurança de Chicago, terão de organizar todo o planeta”. As origens do novo transnacionalismo Cadern O melhor estudo de caso sobre como um Unions (WFTU) e à World Confederation of Unions (WCL). 470 Peter Evans General Secretary em 2010, substituindo o co- fundador do UNI que estava se aposentando, Philip Bowyer, exemplifica a nova geração de lideranças. Transformar uma variada gama de sindicatos a nível nacional num poder coleti- vo que poderia forçar a G4S a assinar um IFA dependeu da aproximação da estrutura orga- nizacional englobante do UNI no intuito de compor uma campanha organizacional entre vários países. Assim, o papel do UNI exempli- fica a possibilidades de transformar as GUFs em veículos para as campanhas organizacio- nais entre diversos países. neste setor de serviço são notavelmente simila- res às dinâmicas já descritas nos casos que en- volvem os automóveis e o aço. MOVIMENTOS NACIONAIS DE TRABALHADORES ... Assim como o USW e o UAW tiveram de encontrar estratégias globais para confrontar o poder dos capitais globais sobre seus membros nos Estados Uni- dos, a divisão do SEIU Property Services viu- se enfrentando empregadores que não eram mais locais, ou mesmo nacionais, mas globais. Quando o G4S, sediado na Inglaterra, adquiriu a Wackenhut, uma das maiores empregadoras de guardas de segurança dos Estados Unidos, o ímpeto para montar uma campanha global para organizar os guardas de segurança nos Es- tados Unidos foi posto em movimento. Uma campanha global foi a condição sine qua non para trazer a G4S para a mesa de negociação, mas a campanha teve que ser luta- da em territórios nacionais distintos. Cada cam- panha nacional refletia um contexto político e uma história sindical local. Ao mesmo tempo, ser parte de uma campanha global gerou um impacto nos sindicatos nacionais envolvidos. A análise de McCallum da África do Sul e da Índia mostra como a política nacional pode di- recionar uma campanha global em dois tipos de campanhas bastante diferentes, em contextos nacionais distintos, bem como a forma como a participação na campanha global pode remode- lar as estratégias dos sindicatos nacionais. A lógica de construir parcerias transna- cionais foi, entretanto, muito distinta no caso do G4S do caso da campanha do UAW e do USW. Nenhum mercado isolado de um país era suficientemente importante a ponto de forçar o G4S a negociar. A campanha teve de ser fei- ta em múltiplos terrenos, não precisando, por isso, de aliados que fossem individualmente poderosos. Trabalhar através do UNI e do SEIU construiu uma estratégia Lilliputianos versus Gulliver, na qual os sindicatos oriundos de contextos múltiplos, nenhum deles particular- mente forte em seus contextos nacionais, con- vergiram para forçar a corporação Gulliver a negociar. Os sindicatos, num conjunto impres- sionante de países, foram envolvidos, variando de países no Norte, como os Estados Unidos e a Inglaterra, aos maiores países do Sul, como Indonésia, Índia e África do Sul, e aos países menores no Sul como Malawi e Ghana. Na África do Sul, a campanha global in- centivou a revitalização do sindicato compa- triota nacional, o SATAWU (McCallum 2011, 2013: cap. 4). Os funcionários da UNI que trabalham na África do Sul enfatizaram a or- ganização que foi fundamental para ajudar o SATAWU a transformar-se de um sindicato de serviços num sindicato militante. MOVIMENTOS NACIONAIS DE TRABALHADORES ... zação na Índia, como a SEWA (Self-Employed Women’s Association) (Agarwala, 2013). Em vez de demandar da companhia diretamente, a campanha terminou trabalhando por “uma legislação nacional que pudesse estabelecer padrões para os guardas de segurança em toda a indústria, estendendo efetivamente aspectos do acordo global à arena política indiana” (Mc- Callum, 2013, p. 138). que superou a recalcitrância dos empresários locais. MacCallum (2013: 118) cita um organi- zador local dizendo, “essa é minha cópia do acordo global. É como uma Bíblia. Quando os empresários me dizem para sair, eu mostro isso a eles [o GFA]. Quando os trabalhadores têm medo de se juntar, mostro isso a eles. Quan- do as pessoas me dizem que não tem direito, aponto para isso”. Embora a África do Sul seja muito ampla para que se reivindique um efeito transforma- dor a nível nacional, comparado com a organi- zação dos trabalhadores da Firestone na Libéria ou da vitória da Russel em Honduras, os efei- tos nacionais desta campanha global ilustram como a mobilização global pode contribuir para a revitalização do sindicato a nível nacional, mesmo em países maiores do Sul Global. A relação recíproca entre a mobilização nacional e global é clara na campanha G4S: os terrenos nacionais moldam o caráter e a efe- tividade das campanhas globais, mas as cam- panhas globais podem moldar, também, os contornos do terreno nacional. A campanha sublinha, uma vez mais, a importância da es- tratégia do Sul Global. Sem as mobilizações multinacionais por uma série de países no Sul Global e a evidência convincente dos salários e condições de trabalho degradadas que estes movimentos trouxeram aos escritórios dos re- presentantes em Londres, é improvável que o empresariado global tivesse cedido. Finalmen- te, a campanha G4S traz à tona, uma vez mais, a mistura que vimos no Brasil: a utilização ao estilo europeu das estruturas sindicais Globais e de acordos de escopo também global, combi- nados com um estilo agressivo, de confronto, da campanha corporativa americana. A Índia provou ser um terreno áspero para a construção de alianças transnacionais. A colcha de retalhos das confederações sindicais politicamente divididas da Índia fez da constru- ção de uma campanha nacional unificada um desafio tão grande quanto aquele de construir uma campanha de escopo continental na Áfri- ca ou na América Latina. MOVIMENTOS NACIONAIS DE TRABALHADORES ... Os militantes do UNI na Índia tentando construir uma aliança entre o Congress of Indian Trade Unions (CITU), com grande participação do Partido Comunista, pre- dominantemente marxista, em Bengal West e o Congress afiliado Indian Trade Union Congress (INTUC) no Sul da Índia. Problemas e conflitos abundaram e a campanha geral teve um êxito apenas limitado a nível nacional. MOVIMENTOS NACIONAIS DE TRABALHADORES ... A participa- ção da SATAWU na campanha ajudou a esti- mular a organização de mais 3.000 guardas de segurança, aumentando em 40% o número de organizados desta categoria. A peça organizacional chave foi o UNI. Fundado em 2000 e tendo assinado seu pri- meiro GFAs em 2001, na primavera de 2013, o UNI havia assinado 48 acordos globais, mais que qualquer outra GUF. A base organizativa agressiva do UNI é uma consequência dire- ta tanto do apoio que esta recebeu do SEIU quanto da infusão de lideranças com expe- riência em trabalhar nas campanhas do SEIU. Christy Hoffman, que veio para o UNI com uma extensa experiência e se tornou Deputy C CRH S l d Os efeitos locais da assinatura do Acor- do Marco-Global pela alta administração da G4S em Londres também corroborou o poten- cial das GFAs. Longe de ser uma peça abstrata do papel global, o GFA revigorou os militan- tes locais, dando-lhes um senso de autoridade 471 MOVIMENTOS NACIONAIS DE TRABALHADORES ... CONEXÕES TRANSNACIONAIS E MOVIMENTOS DE TRABALHADO- RES NA ERA NEOLIBERAL Mas as ramificações deste deslocamen- to vão além das estratégias cambiantes destes dois movimentos nacionais de trabalhadores. Elas envolvem uma ordem caleidoscópica de casos, envolvendo uma série de países tanto do Norte quanto do Sul. Juntos, estes casos corroboram uma vi- são expandida a respeito das possibilidades de chantagens patronais ao revés. Os exemplos tradicionais de chantagem patronal ao revés são: o movimento nacional de trabalhadores no Sul ganha poder construindo laços com o do Norte; tanto os sindicatos brasileiros quanto os sindicatos estadunidenses ganham poder cons- truindo conexões com sindicatos mais solida- mente institucionalizados na Europa. Porém, os casos Brasil-Estados Unidos mostram um tipo de chantagem patronal ao revés no qual a força do Sul pode resultar em benefício para o Norte. No caso dos Estados Unidos, as duradou- ras políticas antitrabalhistas geraram novos in- centivos para que os sindicatos olhassem para as alianças transnacionais. Embora parcial, este deslocamento criou oportunidades para outros movimentos nacionais. As vitórias da Hondu- ran textile workers sobre a Russell athletics e a Liberian Plantatio Workers sobre a Bridgestone- Firestone mostram a utilidade da abertura do movimento dos trabalhadores estadunidense para as iniciativas transnacionais, ao menos no caso de países pequenos e pobres, onde o capi- tal local é dependente da economia dos Estados Unidos. O investimento organizacional da SEIU na campanha G4S ilustra como uma orientação mais transnacional nos Estados Unidos pode contribuir para campanhas mais amplas. A des- peito do declínio de seus recursos ao nível na- cional, o movimento americano de trabalhado- res ainda detém poder e recurso suficiente para ser um útil aliado para outros movimentos. Ao mesmo tempo, estes casos escla- recem a interação das campanhas globais e contextos políticos nacionais historicamente enraizados. Os contextos políticos nacionais continuam o maior determinante da sorte dos movimentos. Eles não são somente “variáveis independentes” que moldam possibilidades de ação transnacional. Eles podem ser, também, remodelados em prol do movimento dos tra- balhadores por campanhas globais. Os esfor- ços transnacionais podem catalisar vitórias e revigorar as forças sociais do trabalho no nível nacional – notadamente em países menores, como mostra Honduras e Libéria, e a um ní- vel setorial mais específico em países maiores, como mostra o efeito revitalizador da campa- nha do G4S sobre o SATAWU. O Brasil mostra como um terreno favorável pode tanto estimu- lar as articulações das redes transnacionais de trabalhadores quanto se beneficiar delas. CONEXÕES TRANSNACIONAIS E MOVIMENTOS DE TRABALHADO- RES NA ERA NEOLIBERAL Os esfor- ços transnacionais podem catalisar vitórias e revigorar as forças sociais do trabalho no nível nacional – notadamente em países menores, como mostra Honduras e Libéria, e a um ní- vel setorial mais específico em países maiores, como mostra o efeito revitalizador da campa- nha do G4S sobre o SATAWU. O Brasil mostra como um terreno favorável pode tanto estimu- lar as articulações das redes transnacionais de trabalhadores quanto se beneficiar delas. O desenvolvimento de novas conexões en- fez do movimento dos trabalhadores brasileiro um aliado valioso. Isto ficou particularmen- te claro no caso do apoio da Gerdau Workers World Council ao USW no caso Ameristeel, e também se aplica aos efeitos potenciais do apoio brasileiro à campanha da UAW Nissan, no Mississippi. Mas o papel do Brasil nas re- des Sul-Sul – incluindo o GWWC e o REDLAT (Latin American Network for research on Mul- tinational Companies) - também é importante. Juntos, estes casos corroboram uma vi- são expandida a respeito das possibilidades de chantagens patronais ao revés. Os exemplos tradicionais de chantagem patronal ao revés são: o movimento nacional de trabalhadores no Sul ganha poder construindo laços com o do Norte; tanto os sindicatos brasileiros quanto os sindicatos estadunidenses ganham poder cons- truindo conexões com sindicatos mais solida- mente institucionalizados na Europa. Porém, os casos Brasil-Estados Unidos mostram um tipo de chantagem patronal ao revés no qual a força do Sul pode resultar em benefício para o Norte. Ao mesmo tempo, estes casos escla- recem a interação das campanhas globais e contextos políticos nacionais historicamente enraizados. Os contextos políticos nacionais continuam o maior determinante da sorte dos l de considerar suas implicações atuais e futu- ras para o movimento global de trabalhadores. fez do movimento dos trabalhadores brasileiro um aliado valioso. Isto ficou particularmen- te claro no caso do apoio da Gerdau Workers World Council ao USW no caso Ameristeel, e também se aplica aos efeitos potenciais do apoio brasileiro à campanha da UAW Nissan, no Mississippi. Mas o papel do Brasil nas re- des Sul-Sul – incluindo o GWWC e o REDLAT (Latin American Network for research on Mul- tinational Companies) - também é importante. Foquei, particularmente, em como os deslocamentos das posições nacionais resul- taram num crescimento da participação nas alianças transnacionais em dois países: o de- clínio dos Estados Unidos e um Brasil emer- gente. CONEXÕES TRANSNACIONAIS E MOVIMENTOS DE TRABALHADO- RES NA ERA NEOLIBERAL Caderno CRH, Salvador, v. 28, n. 75, p. 457-4 A despeito destes problemas, dois as- pectos da campanha na Índia merecem men- ção especial. Primeiro, mesmo que frustrado, em última instância, a Indian Security Workers Organizaing Initiative (ISWOI), organizado pela campanha G4S, foi um dos primeiros es- forços do tipo para ligar o fosso que separava a CITU e a INTUC. Segundo, numa amostra do poder do contexto político local, a campanha na Índia desenvolveu sua própria estratégia específica, consistente com as estratégias de outras campanhas bastante distintas de organi- A ideia de que novos tipos de transna- cionalismo do movimento de trabalhadores emergiram na era neoliberal não é uma quime- ra. O neoliberalismo não impediu a emergên- cia de novas conexões entre as forças sociais do trabalho nacionais. Uma fonte das novas possibilidades foi a mudança de posições dos terrenos nacionais na economia política glo- bal. Outra foi a construção de novas estratégias e estruturas a nível global. Tendo explorado es- tas possibilidades com algum detalhe, é hora 472 Peter Evans fez do movimento dos trabalhadores brasileiro um aliado valioso. Isto ficou particularmen- te claro no caso do apoio da Gerdau Workers World Council ao USW no caso Ameristeel, e também se aplica aos efeitos potenciais do apoio brasileiro à campanha da UAW Nissan, no Mississippi. Mas o papel do Brasil nas re- des Sul-Sul – incluindo o GWWC e o REDLAT (Latin American Network for research on Mul- tinational Companies) - também é importante. Juntos, estes casos corroboram uma vi- são expandida a respeito das possibilidades de chantagens patronais ao revés. Os exemplos tradicionais de chantagem patronal ao revés são: o movimento nacional de trabalhadores no Sul ganha poder construindo laços com o do Norte; tanto os sindicatos brasileiros quanto os sindicatos estadunidenses ganham poder cons- truindo conexões com sindicatos mais solida- mente institucionalizados na Europa. Porém, os casos Brasil-Estados Unidos mostram um tipo de chantagem patronal ao revés no qual a força do Sul pode resultar em benefício para o Norte. Ao mesmo tempo, estes casos escla- recem a interação das campanhas globais e contextos políticos nacionais historicamente enraizados. Os contextos políticos nacionais continuam o maior determinante da sorte dos movimentos. Eles não são somente “variáveis independentes” que moldam possibilidades de ação transnacional. Eles podem ser, também, remodelados em prol do movimento dos tra- balhadores por campanhas globais. MOVIMENTOS NACIONAIS DE TRABALHADORES ... novas estratégias e estruturas a nível global. A es- tratégia de construção de instituições como o IFA/ GFA, conselhos de fábrica pelo mundo e redes empresariais cresceu rapidamente. Campanhas organizadas por vários países, que estão no centro da questão, não podem reivindicar o mesmo cres- cimento acelerado, mas a campanha G4S ilustra o acúmulo de experiência em como organizar mobilizações entre vários países. O movimento global dos trabalhadores continua “aprendendo na prática” a nível global, no neoliberalismo. Os sindicatos brasileiros estavam profundamente en- gajados com os modelos europeus de organizar, vinculados aos GFAs e aos Sindicatos Globais, mas estiveram igualmente abertos ao estilo ame- ricano de fazer campanhas nos casos da Gerdau e da Nissan. A campanha da G4S foi um híbrido que juntou os dois modelos. tornarão características marcantes da contes- tação das forças sociais do trabalho ao capital nas décadas que virão? As circunstâncias que produziram as inúmeras campanhas descritas aqui são pouco idiossincráticas. Decorrem da interação entre terrenos nacionais que são integrantes da es- trutura geral da economia política global. Ao mesmo tempo, estas conexões transnacionais não fluem, simplesmente, das posições nacio- nais na estrutura da economia política global. Elas dependem, especificamente, de histórias nacionais. A sustentação e o reforço das estru- turas e estratégias que emergiram a nível global dependem do apoio e do engajamento dos mo- vimentos de trabalhadores ao nível nacionais e, por isso, são igualmente dependentes de um agregado de trajetórias políticas nacionais. Os efeitos projetados do “Sul insurgen- te” fornecem uma boa ilustração da importân- cia das trajetórias nacionais específicas. Por um lado, há boas razões estruturais para espe- rar que o Sul continue a tornar-se mais impor- tante para o transnacionalismo do movimento dos trabalhadores. O Brasil ilustra como uma conjuntura nacional favorável pode ampliar um efeito geral. Porém, mesmo no caso do Bra- sil, não se pode assumir que o futuro será uma extensão das tendências passadas. Os analistas pessimistas argumentariam que o atual papel internacional do movimento de trabalhadores brasileiro representa um apogeu que dificil- mente se sustentará, seja porque a CUT per- deu sua visão militante, que foi fundamental tanto para a política doméstica quanto para seu internacionalismo (Sluyter-Beltrao, 2010), seja porque o movimento dos trabalhadores brasileiros como um todo está preso a uma ar- madilha corporativista que minará a vontade para investir energia em campanhas combati- vas, sejam nacionais ou globais (Braga, 2012). Caderno CR 22 Um dos desenvolvimentos mais interessantes, cuja dis- cussão tive de deixar de lado aqui, envolve os laços entre trabalhismo e outros movimentos sociais transnacionais (ver, por exemplo, Anner e Evans, 2004; Tarrow, 2005; Evans, 2008; Munck, 2010; Smith e Wiest, 2012). CONEXÕES TRANSNACIONAIS E MOVIMENTOS DE TRABALHADO- RES NA ERA NEOLIBERAL O movimento de trabalhadores brasilei- ro aprendeu cedo o valor das alianças trans- nacionais, durante suas lutas contra o regime militar. A globalização do capital sediado no Brasil somou-se aos incentivos criados pela predominância continuada das corporações transnacionais, tornando o movimento de trabalhadores brasileiro ainda mais aberto às conexões com outros movimentos nacionais. A abertura para as estratégias transnacionais, combinada com capacidades políticas e orga- nizacionais construídas por décadas de lutas, O desenvolvimento de novas conexões en- tre movimentos globais de trabalhadores na era neoliberal foi complementado pela construção de 473 MOVIMENTOS NACIONAIS DE TRABALHADORES ... MOVIMENTOS NACIONAIS DE TRABALHADORES ... Em termos gerais, a erosão do poder do trabalhismo movimento dos trabalhadores no nível nacional em muitos países cobrou seu pre- ço, mas o desenvolvimento de estratégias e co- nexões globais continua.22 A evolução da arqui- tetura global não foi definida pela simples soma das conjunturas de suas partes nacionais. Novas conexões nacionais e aprendizagem prática glo- bal produziram resultados concretos para os tra- balhadores em muitos países. O pulular constan- te de novos exemplos de transnacionalismo das forças sociais do trabalho sob a égide do regime neoliberal hostil pode não acarretar uma “grande transformação” do movimento dos trabalhadores ao nível global (Munck, 2002), mas, tampouco autorizam um “pessimismo teimoso” (Burawoy, 2010). Temperado cuidadosamente, o otimismo cético é sempre uma resposta razoável às ambi- guidades da evidência. Salvador, v. 28, n. E as implicações para o futuro? Os exem- plos de transnacionalismo aqui considerados representam um ponto evanescente criado por conjunturas idiossincráticas no período neoli- beral? Ou, estas iniciativas provavelmente se Considerando a evolução dos movimen- tos dos trabalhadores em outros grandes países do Sul Global, cujas trajetórias políticas pare- ciam igualmente promissoras nos anos 1980, mas que terminaram por não cumprir um pa- 474 Peter Evans estadunidenses para contribuir para os esfor- ços transnacionais durante a fase ascensional da hegemonia americana. Como os exemplos dos esforços de solidariedade alemã na indús- tria automobilística tanto no Brasil quanto nos Estados Unidos ilustram, esta tradição conti- nua. O declínio do movimento dos trabalha- dores europeu durante o século XXI não pa- rece haver estimulado um deslocamento na direção de um aumento do transnacionalismo, comparável com aquele que argumentei ter ocorrido nos Estados Unidos. Em suma, não podemos sustentar que o declínio econômico e os ambientes políticos nacionais mais hostis às forças sociais do trabalho empurrarão o mo- vimento dos trabalhadores no Norte para fora. pel como o do Brasil, destaca-se ainda mais a importância das trajetórias políticas nacionais. A África do Sul é um caso concreto.23 A des- peito dos apelos contínuos a um papel políti- co mais ativo pelas bases dentro da COSATU (Congress of South African Trade Unions) (ver, por exemplo, Sikwebu, 2013), as forças sociais do trabalho, na prática, foram relegadas a ser um parceiro Junior numa aliança política cuja agenda diverge grandemente das prioridades das fileiras e do histórico da COSATU. MOVIMENTOS NACIONAIS DE TRABALHADORES ... Em suma, a habilidade dos grandes mo- vimentos de trabalhadores no Sul para trazer contribuições chave para a arquitetura geral das forças sociais do trabalho global é uma possibi- lidade que depende das trajetórias políticas na- cionais. É possível que os deslocamentos políti- cos futuros em outros países do Sul Global – em algum lugar da África do Sul à Coréia – expan- dam possibilidades de construir articulações transnacionais. Mas, também é possível que o movimento de trabalhadores brasileiros se torne mais dividido e menos engajado transnacional- mente. A evolução futura da economia política provavelmente apresentará aos movimentos de trabalhadores do Sul Global oportunidades de cumprir um papel mais amplo no transnaciona- lismo, mas a possibilidade de captação destas oportunidades dependerá das trajetórias políti- cas nacionais. O movimento dos trabalhadores pode cumprir um papel na modelagem destas trajetórias não em sua determinação. Tampouco é linear a conexão entre declí- nio da hegemonia e crescimento do transnacio- nalismo do movimento dos trabalhadores nos Estados Unidos. Um declínio acelerado poderia minar a capacidade do movimento dos trabalha- dores estadunidense para ser um aliado útil. Isso deveria provocar um giro interno, particular- mente se os esforços transnacionais atuais não geram frutos aos trabalhadores estadunidenses. A maior fonte de incerteza com respeito ao futuro do transnacionalismo do movimento dos trabalhadores não vem, claro, dos Estados Unidos, mas do outro lado da equação da he- gemonia em transformação. Se a hegemonia emergente da China resultar na expansão global da repressão bastante efetiva das organizações trabalhistas independentes por parte do Partido -Estado, as perspectivas para o movimento dos trabalhadores ao nível global seriam cinzentas. Neste cenário distópico, o regime neoliberal hostil poderia aparecer, retrospectivamente, como não sendo o apogeu da política antitraba- lho, mas como sendo a última “janela de opor- tunidade” para as forças sociais do trabalho. Um ponto similar pode ser assinalado com relação ao deslocamento em direção ao transnacionalismo no movimento de trabalha- dores estadunidense. Argumentei que o declí- nio neoliberal dos Estados Unidos teve o efeito de pressionar as forças sociais do trabalho es- tadunidense em direção ao transnacionalismo, mas, insisto, os efeitos do declínio da hegemo- nia não devem ser tomados genericamente. 23 Sobre paralelos entre as fases anteriores da luta dos trabalhadores no Brasil e na África do Sul, ver Seidman (1994). REFERÊNCIAS tinuidade de um conjunto de oportunidades basicamente semelhantes àquelas oferecidas pela era neoliberal é uma projeção razoável. Deste modo, faz sentido focar na lição que po- demos aprender das experiências das décadas recentes sobre as dinâmicas que moldam a ar- quitetura global das forças sociais do trabalho. AGARWALA, R. Informal labor, formal politics, and dignified discontent in India. Cambridge: Cambridge University Press. 2013. ANNER, M. Industrial structure, the state, and ideology: Shaping labor transnationalism in the Brazilian auto industry. Social Science History. v. 27, n. 4, p. 603-634, 2003. _______. Solidarity transformed: labor responses to globalization and crisis in Latin America. Ithaca: Cornell University-ILR Press. 2011. A inabilidade para fornecer predições claras acerca das trajetórias futuras não anula o interesse na reflexão sobre as articulações en- tre os movimentos dos trabalhadores em níveis nacionais nos últimos vinte anos como meio de entender a evolução da arquitetura das forças sociais do trabalho em nível global. A incerteza a respeito das tendências do futuro tampouco deveria distrair das implicações de se focar nas conexões dos movimentos nacionais de traba- lhadores para as análises estratégicas emprega- das nos estudos globais sobre o trabalho. _______. Workers power in global value chains: fighting sweatshop practices at Russell, Nike and Knights Apparel. In: FAIRBROTHER, P.; HENNEBERT, M.A.; LEVESQUE, C. (eds.). Transnational trade unionism building union power. New York: Routledge. 2013. _______ EVANS, P. Building bridges across a double-divide: alliances between U.S. and Latin American labor and NGOs. Development in Practice. v. 14, n. 1-2, p. 34-47, 2004. ARRIGHI, G. The three hegemonies of historical capitalism. Review. XIII, v. 3, p. 365-408. 1990. _______. The long Twentieth Century. Money, power, and the origins of our times. London: Verso. 1994. _______. Workers of the world at century’s end. Review. XIX, v.3, p. 335-351, 1996. _______. Adam Smith in Beijing: Lineages of the Twenty- First Century. London: Verso. 2007. Aschoff, N. (2014) ‘Tennessee Car Sick Blues’, Jacobin. February, [Online]. Available at https://www.jacobinmag.com/2014/02/ tennessee-car-sick-blues/, [Accessed: 9 June 2014]. Automotive News. (2013) ‘VW labor chief says Chattanooga model hinges on works panel,’ (October 7), [Online]. Available at www.autonews.com/article/20131007/ OEM01/ 131009897, [Accessed: 9 June 2014]. Salvador, v. 28, n. 75, p. 457-478, Set./Dez. 2015 As teorias pessimistas sobre o padeci- mento do trabalho nas mãos no neoliberalismo devem ser reconciliadas com as teorias otimis- tas do novo transnacionalismo neste campo. MOVIMENTOS NACIONAIS DE TRABALHADORES ... Os movimentos dos trabalhadores europeus possuem um recorde melhor que os sindicatos Não há garantias de que as conexões na- cionais desenvolvidas no neoliberalismo não sejam desgastadas ou revertidas pela mudança política nas arenas nacionais ou deslocamen- tos na hierarquia do poder nacional que estru- turam a economia política global, mas a con- 475 MOVIMENTOS NACIONAIS DE TRABALHADORES ... Recebido para publicação em 26 de maio de 2015 Aceito em 10 de agosto de 2015 REFERÊNCIAS Integrar a análise de interações entre o movi- mento de trabalhadores nacional com as aná- lises tanto do que está ocorrendo nos terrenos nacionais quanto ao nível global é uma forma de fazê-lo. Uma teoria do movimento dos tra- balhadores global excessivamente ancorada no nível global será sempre parcial e equívoca. A atenção dada à construção de blocos nacionais tampouco deveria ser limitada à descrição das maneiras pelas quais as diferenças nacionais podem subverter a solidariedade transnacional. O potencial para as sinergias positivas, criadas pela diferença nacional, merece igual atenção. Investigar a variedade de maneiras pelas quais os movimentos de trabalhadores nacionais po- dem se conectar entre si, positiva ou negativa- mente, é uma fundação necessária para melho- res teorias da evolução da contestação das for- ças sociais do trabalho ao capital global. BRAGA, R. A política do precariado: do populismo à hegemonia lulista. São Paulo: Boitempo. 2012. BRONFENBRENNER, K. (ed.) Global unions: challenging transnational capital through cross-border campaigns. Ithaca: Cornell University-ILR Press. 2007. BURAWOY, M. From Polanyi to Pollyanna: the false optimism of global labor studies. Global Labour Journal. v. 1, n. 2, p. 301-313, 2010. CHIBBER, V. Into the fold: labor’s incorporation into the Indian Political Economy. In: GOLDFIELD, Michael; BANERJEE, Debdas (eds.). Labour, globalization, and the state. London: Routledge. 278, 2007. COMPA, L. (in collaboration with the NAACP Mississippi State Conference). (2013) ‘Choosing Rights: Nissan in Canton, Mississippi, and Workers’ Freedom of Association under International Human Rights Standards’, [Online]. Available at http://dobetternissan.org/ 2013/10/compa- report, [Accessed: 9 June 2014]. DAVIS, B. The struggle of the national mine, metal and steel workers union of the Mexican Republic. In: Toledo, E.G. (ed.) La situación del trabajo en México: el trabajo en la crisis. Madrid, Spain: Plaza y Valdes. 2012. DONIZETTI, P. Sonhos violados, Rede Brazil. [Online]. Available at www.redebrasilatual.com. br/revistas/81/ trabalho/, [Accessed: 23 July 2013]. 2013. EARLY, S. VW to UAW: so long partner? CounterPunch. (February 19), [Online]. Available at http://www. counterpunch.org/2014/02/19/vw-to-uaw-so-long- partner/, [Accessed: 9 June 2014]. 2014 EVANS, P.B. Is an alternative globalization possible?. Politics & Society. v. 36, n. 2, p. 271-305, 2008. . Is it labor’s turn to globalize? Twenty-First 476 Peter Evans MUNCK, R. Globalization and labour: the new ‘Great Transformation’. London: ZED Books. 2002. Century opportunities and strategic responses. Global Labour Journal. v. 1, n. 3, p. 352-379, 2010. _______. Globalization and the labour movement: challenges and responses. Global Labour Journal. v.1, n. 2, p. REFERÊNCIAS IMF (INTERNATIONAL METALWORKERS FEDERATION) (2012) ‘International framework agreement: Agreed upon social rights and social responsibility principles’, Ford Motor Company and Global IMF I. Ford Global Information Sharing Network, April 25, 2012. SILVER, B.J. Forces of labor: workers’ movements and globalization since 1870. Cambridge: Cambridge University Press. 2003. JAKOBSEN, K. Rethinking the International Confederation of free trade unions and its Inter-American regional organization. Antipode. v. 33, n. 3, p. 363-383, 2001. SLUYTER-BELTRÃO, J. Rise and decline of Brazil’s new unionism: the politics of the Central Unica dos Trabalhadores. Oxford: Oxford University Press. .2010. _______. Estratégia sindical frente às empresas multinacionais. Nueva Sociedad, 211 Sep./Oct., p. 145-159. 2007. SMITH, J.; WIEST, D. Social movements in the world- system: the politics of crisis and transformation. New York: Russell Sage Foundation. 2012. Standing, G. The precariat: the new dangerous class. London: Bloomsbury Academic. 2011. JURAVICH, T.; BRONFENBRENNER, K. Ravenswood: the steelworkers victory and the revival of American labor. Ithaca: Cornell University-ILR Press. 1999. KAY, T. NAFTA and the politics of labor transnationalism. Cambridge: Cambridge University Press. 2010. 8, Set./Dez. 2 STEVIS, D.; BOSWELL, T. International framework agreements: opportunities and challenges for global unionism. In: BRONFENBRENNER, K. (ed.) Global unions: challenging transnational capital through cross-border campaigns. Ithaca: Cornell University-ILR Press. 2007. KAZDIN, C. What lessons from the USW’s Transnational Solidarity Campaigns? Presentation at workshop on ‘New Strategies For Building Transnational Labor Solidarity’ November 8th Watson Institute, Brown University, Providence RI. 2012. . 457-47 _______. Globalization and labor: democratizing global governance. Plymouth: Rowman and Littlefield Publishing. 2008. LULA [LUIZ INÁCIO DA SILVA]. Letter to NISSAN (October, 10), [Online]. 2013. Available at http://dobetternissan. org/2013/10/former-brazil-president-lulas-letter-to-nissan, [Accessed: 9 June 2014] . 75, p TARROW, S. The new transnational activism. New York: Cambridge University Press. 2005. MSN (MAQUILA SOLIDARITY NETWORK) Evangelina Argueta of the Maquila Solidarity Network speaks about her life as a union organizer, [Online]. 2010. Available at http://en.maquilasolidarity. org/node/964, [Accessed: 23 July 2013]. v. 28, n UNDP (UNITED NATIONS DEVELOPMENT PROGRAM). Human development report: the rise of the South. New York: UN. 281. 2013. alvador, VEIGA, J.P.C.; JAKOBSEN, K. A cooperação laranja – verde amarela: A parceiria FNV e CUT. São Paulo, Brazil: CEDOC-CUT. 2011. MAZUR, J. Labour’s New Internationalism. Foreign Affairs. v. 79. n. 1, p. 79-93. 2000. McCallum, J. ‘Trade union renewal and labor transnationalism in South Africa: The case of SATAWU’, WorkingUSA: The Journal of Labor and Society. v. 14, June, p. 161-176, 2011. REFERÊNCIAS 218-232, 2010. _______; SEWELL, W.H. ‘Neoliberalism: Policy regimes, international regimes, and social effects. In: HALL, P; LAMONT, M. (eds.) Social resilience in the neoliberal era. Cambridge: Cambridge University Press. 2013. MYERSON, H. Where are the workers? The American Prospect. v. 20, n. 2, p. 20. 280, 2009. FICHTER, M.; HELFEN, M. Going local with global policies: implementing international framework agreements in Brazil and the United States. In: PAPADAKIS, K. (ed.) Shaping global industrial relations: the impact of international framework agreements. New York: Palgrave Macmillan. 2011. RODRIGUEZ-GARAVITO, C.A. Sewing resistance: transnational organizing, global governance, and labor rights in the U.S.-Caribbean Basin apparel industry (1990- 2005). Unpublished Ph.D. Dissertation, Department of Sociology, University of Wisconsin, Madison, WI. 2007. GRAHAM, A. Massive victory for Honduran workers. Against the Current. v. 24, n. 3, p. 21-22. 2010. Gray, C. Metalúrgicos sem fronteiras: Building a global union at Gerdau. Unpublished MA Thesis, Cornell University. 2009. ROMBALDI, M. Internacionalização do sindicalismo no Brasil: um estudo sobre os setores metalúrgico e de telecomunicações. Tese de Doutorado, Departamento de Sociologia, Universidade de São Paulo, São Paulo, Brazil. 2012. GREENHOUSE, S. Labor fight ends in win for students. The New York Times. November, 18. 2009. RÜB, S. World works councils and other forms of global employee representation in transnational undertakings. Working Paper 55. Dusseldorf: Hans-Böckler-Stiftung. 2002. _______. At a Nissan Plant in Mississippi, a Battle to Shape the U.A.W.’s Future,’ The New York Times. [Online]. 2013. Available at http://www.nytimes.com/2013/10/07/business/ at-a-nissan-plant-in-mississippi-a-battle-to-shape-the- uaws-future.html, [Accessed: 9 June 2014]. Hermanson, J. (2004) ‘Global corporations, global campaigns: The struggle for justice at Kukdong International in Mexico’ Paper presented at Cornell University Conference on Transnational Labor Contention, April 9-11, 2004. 279 SCIPES, K. AFL-CIO’s secret war against developing country workers: solidarity or sabotage? Lanham, MD: Lexington Books. 2010. SEIDMAN, G. Manufacturing militance: workers’ movements in Brazil and South Africa, 1970-1985. Berkeley, CA: University of California Press. 1994. INTERNATIONAL LABOR RIGHTS FORUM (ILRF). Liberia: Firestone Sued Over ‘Slave’ Plantation. International Labor Rights Forum. [Online]. Available at http://www.laborrights.org, [Accessed: 4 December 2005]. _______. Beyond the boycott: labor rights, human rights, and transnational activism. New York: Russell Sage Foundation. 2007. SIKWEBU, D. Notes from a Trade Unionist: Cosatu and its Affiliates as Democratising Agents or Contingent Democrats?’ Rethinking Development and Inequality. v. 2, p. 63-67, 2013). REFERÊNCIAS RH Sa WATERMAN, P. Trade Union Internationalism in the Age of Seattle. Antipode. v. 33, n. 3, p. 312-336, 2001. o C WEBSTER, E., LAMBERT, R.;BEZUIDENHOUT. A. Grounding globalization: labor in the age of insecurity. Malden: Blackwell. 2008. MCCALLUM, J. Global unions, local power: the new spirit of transnational labor organizing. Ithaca: Cornell University Press. 2013. 477 MOVIMENTOS NACIONAIS DE TRABALHADORES ... Key-Words: Workers. Global Unions. Labor transnationalism. Labor national movements. Neoliberalism. Peter Evans The neoliberal era has undermined worker’s rights and labor’s power at the national level, but has also been characterized as an era of “the new labor transnationalism”. Shifting fortunes at the national level have been fundamental to expanding openness to transnational alliances. An analysis of campaigns displays that. Assessing the connections among national labor movements and the new global organizational infrastructure that have emerged under neoliberalism is a necessary foundation for building better theories of labor’s evolving contestation with global capital. L’ère néolibérale a miné les droits des travailleurs et le pouvoir des forces sociales du travail au niveau national mais a aussi été caractérisée comme l’ère d’un nouveau “transnationalisme du mouvement des travailleurs”. Les changements conjoncturels au niveau national ont joué un rôle fondamental pour amplifier l’ouverture à des alliances transnationales. Une analyse des campagnes permet de le mettre en évidence. Faire l’évaluation des liens existants entre les mouvements nationaux des travailleurs et la nouvelle infrastructure organisationnelle qui a surgi au sein du néolibéralisme est un point de départ nécessaire à la construction de théories plus précises concernant les dynamiques de contestation des forces sociales du travail envers le capital mondial. Key-Words: Workers. Global Unions. Labor transnationalism. Labor national movements. Neoliberalism. Mots-clés: Travailleurs. Syndicats mo Transnationalisme. Syndicats na Néolibéralisme. Mots-clés: Travailleurs. Syndicats mondiaux. Transnationalisme. Syndicats nationaux. Néolibéralisme. Caderno CRH, Salvador, v. 28, n. 75, p. 457-478, Set./Dez. 2015 Peter Evans - Professor emérito de Sociologia da University of California, Berkeley e Senior Research Fellow da Watson Institute for International Studies da Brown University. Também trabalha como coordenador do Grupo e Trabalho sobre Movimentos de Trabalhadores da Associação Internacional de Sociologia (ISA). Seu trabalho anterior centrava-se na economia política do desenvolvimento comparado no Sul global. Recentemente vem pesquisando os esforços dos movimentos sociais para mobilizar transnacionalmente uma “globalização contra-hegemônica”. O movimento global dos trabalhadores é um foco central em seu trabalho, como exemplifica o artigo aqui publicado e seu texto de 2010 editado no Global Labour Journal ‘Is it Labor’s Turn to Globalize?’ Caderno CRH, Salvador, v Peter Evans - Professor emérito de Sociologia da University of California, Berkeley e Senior Research Fellow da Watson Institute for International Studies da Brown University. Também trabalha como coordenador do Grupo e Trabalho sobre Movimentos de Trabalhadores da Associação Internacional de Sociologia (ISA). Seu trabalho anterior centrava-se na economia política do desenvolvimento comparado no Sul global. Peter Evans Recentemente vem pesquisando os esforços dos movimentos sociais para mobilizar transnacionalmente uma “globalização contra-hegemônica”. O movimento global dos trabalhadores é um foco central em seu trabalho, como exemplifica o artigo aqui publicado e seu texto de 2010 editado no Global Labour Journal ‘Is it Labor’s Turn to Globalize?’ 478
https://openalex.org/W4321159414	https://zenodo.org/record/8144313/files/PhysRevResearch.5.L012021.pdf	English	null	Noisy intermediate-scale quantum computing algorithm for solving an $n$-vertex MaxCut problem with log($n$) qubits	arXiv (Cornell University)	2,021	cc-by	6,385	Noisy intermediate-scale quantum computing algorithm for solving an n-vertex MaxCut problem with log(n) qubits Marko J. Ranˇci´c * TotalEnergies, Nano-INNOV Bt. 861, 8, Boulevard Thomas Gobert, 91120 Palaiseau, France (Received 2 March 2022; accepted 20 December 2022; published 15 February 2023) Quantum computers are devices, which allow more efﬁcient solutions of problems as compared to their classical counterparts. As the timeline to developing a quantum-error corrected computer is unclear, the quantum computing community has dedicated much attention to developing algorithms for currently available noisy intermediate-scale quantum computers (NISQ). Thus far, within NISQ, optimization problems are one of the most commonly studied and are quite often tackled with the quantum approximate optimization algorithm (QAOA). This algorithm is best known for computing graph partitions with a maximal separation of edges (MaxCut), but can easily calculate other problems related to graphs. Here, I present a novel quantum optimization algorithm, which uses exponentially less qubits as compared to the QAOA while requiring a signiﬁcantly reduced number of quantum operations to solve the MaxCut problem. Such an improved performance allowed me to partition graphs with 32 nodes on publicly available 5 qubit gate-based quantum computers without any preprocessing such as division of the graph into smaller subgraphs. These results represent a 40% increase in graph size as compared to state-of-art experiments on gate-based quantum computers such as Google Sycamore. The obtained lower bound is 54.9% on the solution for actual hardware benchmarks and 77.6% on ideal simulators of quantum computers. Furthermore, large-scale optimization problems represented by graphs of a 128 nodes are tackled with simulators of quantum computers, again without any predivision into smaller subproblems and a lower solution bound of 67.9% is achieved. The study presented here paves way to using powerful genetic optimizer in synergy with quantum computers. DOI: 10.1103/PhysRevResearch.5.L012021 Introduction. A universal quantum computer has been the holy grail of quantum technology [1]. Such a device would allow more efﬁcient searching through databases [2], prime number factorization [3], and more efﬁcient solutions of systems of linear equations [4], just to name a few. How- ever, universal quantum computers require millions of qubits with quantum error correction implemented and with an im- plementation timeline, which is difﬁcult to predict [5]. On the other hand, devices with up to 127 noisy qubits are readily available. This steered the scientiﬁc community to- wards exploring potential computational advantages, which such devices could bring. PHYSICAL REVIEW RESEARCH 5, L012021 (2023) PHYSICAL REVIEW RESEARCH 5, L012021 (2023) Letter Letter Letter *marko.rancic@totalenergies.com Published by the American Physical Society Noisy intermediate-scale quantum computing algorithm for solving an n-vertex MaxCut problem with log(n) qubits In the rapidly expanding ﬁeld of noisy intermediate-scale quantum computing (NISQ) [6,7] two algorithms stand out in prospect: the variational quantum eigensolver (VQE) [8,9] and the aforementioned quantum approximate optimization algorithm (QAOA) [10–12]. The VQE is mainly applied to problems in chemistry and material science while the QAOA is best known for computing graph partitions with a maximal separation of edges (MaxCut), but can easily calculate other properties of graphs, such as MaxIn- dependent set and the partition problem, just to name a few. Given a graph G = (V, E) comprising of \|V \| vertices and \|E\| nodes the QAOA requires n = \|V \| qubits and p(\|E\| + \|V \|) quantum operations to implement the ansatz for the MaxCut problem [11,12]. Here, p is the phenomenological depth pa- rameter. In this paper a variational MaxCut algorithm requiring n = ⌈log2 \|V \|⌉qubits is introduced, where ⌈⌉stands for the ceiling function. For example if x = 2.1, ⌈x⌉= 3, if x = 2.7, ⌈x⌉= 3. In similarity with all other NISQ algorithms the algorithm presented here iteratively improves a trial solution (ansatz) in a hybrid quantum-classical optimization loop. The ansatz is implemented with at most 2n −2n + 5 single qubit gates and at most 2n −2 two qubit CNOT gates (in total up to 2n+1 −2n + 3 gates). Exploiting the fact that large graphs can be treated with the algorithm at a low resource overhead I demonstrate the calculation of a MaxCut of a graph of 32 nodes on a publicly available device of only 5 qubits. This is a 40% increase in graph size compared to state-of-the-art experiments with QAOA on gate-based quantum comput- ers such as the Google Sycamore [13,14]. The algorithm presented here opens perspective for immediate quantum speedup with contemporary quantum processors, given that the quantum hardware community is still some years away from producing processors with hundreds of qubits required for quantum speedup with QAOA [15]. Furthermore, a graph of 128 nodes is partitioned on contemporary simulators of quantum computers. With this methodology, simulators of Published by the American Physical Society under the terms of the Creative Commons Attribution 4.0 International license. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI. L012021-1 Published by the American Physical Society 2643-1564/2023/5(1)/L012021(6) PHYSICAL REVIEW RESEARCH 5, L012021 (2023) MARKO J. RAN ˇCI ´C FIG. Noisy intermediate-scale quantum computing algorithm for solving an n-vertex MaxCut problem with log(n) qubits I can therefore substitute any binary variable θ1 taking values 0 or 1 with θ1 →R f (x, 0, m). The second function R f (x, 1, m) = 0 for 0 ⩽x ⩽π/2 and π ⩽x ⩽3π/2 and R f (x, 1, m) = 1 for π/2 < x < π and 3π/2 < x < 2π. Therefore, I substitute the second binary variable θ2 →R f (x, 1, m) (see green-dashed line in Fig. 1). By substituting all θ1, . . . , θ\|V \|−1 variables with R f (x, 0, m), . . . , R f (x, \|V \| −2, m) I have mapped the \|V \| −1 dimensional binary optimization problem to one-dimensional multimodal continual variable optimization problem. The role of x0(q, m) is to center all R f ’s to 0.5 at x = 0, consequently leading to a more regular optimization landscape (avoiding the where x0(q, m) = arcsin[ln ( −ln(0.5))/2m−q] and the integer m ⩾\|V \| and 0 ⩽q ⩽\|V \| −2. R f is deﬁned in such a way to be n differentiable and to have a minimum (maximum) at 0(1) and rapidly changing multi-oscillatory behavior in between extremas. It should be noted that this deﬁnition is not by any means unique. Assume a graph where \|V \| ≫1, consequently m is a large number. R f (x, 0, m) is such a function, which is mostly 0 in the region of 0 ⩽x ⩽π and rapidly changes to 1 in the region π < x ⩽2π (see red line in Fig. 1). I can therefore substitute any binary variable θ1 taking values 0 or 1 with θ1 →R f (x, 0, m). The second function R f (x, 1, m) = 0 for 0 ⩽x ⩽π/2 and π ⩽x ⩽3π/2 and R f (x, 1, m) = 1 for π/2 < x < π and 3π/2 < x < 2π. Therefore, I substitute the second binary variable θ2 →R f (x, 1, m) (see green-dashed line in Fig. 1). By substituting all θ1, . . . , θ\|V \|−1 variables with R f (x, 0, m), . . . , R f (x, \|V \| −2, m) I have mapped the \|V \| −1 dimensional binary optimization problem to one-dimensional multimodal continual variable optimization problem. Noisy intermediate-scale quantum computing algorithm for solving an n-vertex MaxCut problem with log(n) qubits 1. Equation (1) for q = 0 red, q = 1 dashed green and q = 2 dotted black for m = 4. quantum computers become a powerful tool for graph par- titioning, being able to tackle graphs of hundreds of nodes without dividing the problem into smaller subgraphs, such as the work done in [16] or focusing on correlation between pairs of classical independent variables such as work done in Ref. [17]. The study presented here maps a MaxCut cost function to multimodal multidimensional cost function, which can be evaluated on quantum processing units (QPUs). In the subse- quent step it relies on the powerful class of global optimizers called “genetic optimizers,” which are proven to excel in ﬁnd- ing minima of multidimensional multimodal black-box cost functions [18–20]. FIG. 1. Equation (1) for q = 0 red, q = 1 dashed green and q = 2 dotted black for m = 4. Methodology. a. Variable reduction. In this subsection a variable reduction technique compatible with the MaxCut problem is going to be presented. This is done in order to make the problem amendable to classical optimizers, which cannot easily treat multidimensional data such as for example the sur- rogate model EGO optimizer [21]. Also, as shown in Fig. S2 within the Supplemental Material (SM) [22], variable reduc- tion yields better results for lower graph densities and lower result variance for all graph densities. Due to the fact that the binary optimization problem of ﬁnding a MaxCut is NP-hard, a ﬁrst approach to approximately solve the problem would be to linearly relax the problem. Meaning that instead of assum- ing that binary optimization variables in the MaxCut problem are integers 0 or 1, one assumes that they are continuous variables [0,1]. In the ﬁeld of semideﬁnite programming [23] a different, more efﬁcient approach is taken, binary variables are substituted with vectors. Such classical method of approxi- mately solving the MaxCut problem is state-of-the-art and has a maximum possible performance guarantee of α = 0.87856 as proved by Goemans and Williamson and can be performed in polynomial time [23]. potential splitting of minima around 0.0 and 1.0). It is derived by setting R f (0, q, m) = 0.5 for an arbitrary x0(q, m). f b. The algorithm. Noisy intermediate-scale quantum computing algorithm for solving an n-vertex MaxCut problem with log(n) qubits A Laplacian of a graph L(G), where G = (V, E) is a \|V \|×\|V \| matrix with \|V \| positive terms on the diagonal and 2\|E\| off-diagonal terms. The ith diagonal term of the graph Laplacian corresponds to the number of connections the node i has with remaining nodes in the graph and the i jth off-diagonal term of the matrix is the negative weight between the ith and the jth node. Similarly to a real-valued Hamiltonian in quantum me- chanics, the graph Laplacian is symmetric, furthermore it has a spectrum (eigenvalue range) between 0 and its largest eigenvalue. Now, I introduce a partition vector V of length \|V \| with ith term equaling +1 if ith vertex of G belongs to the ﬁrst subgraph in the graph partition and ith term −1 if the ith node belongs to the second sub-graph in the graph partition. Then, the number of cuts in the graph bipartition is Ncuts = VT LV/4 [24], and this formula is a central piece of the algorithm presented here. By ﬁnding the vector V, which maximizes Ncuts a MaxCut of the graph is found. Vector V has 2\|V \| possible values and there is no known algorithm which can exactly ﬁnd V, which maximizes Ncuts with a computa- tional complexity, which is a polynomial of \|V \|. Here an alternative approach compatible with quantum computing is presented. Let me now introduce a continuous, differentiable function of the following form R f (x, q, m) = exp −exp 2m−q sin (2qx + x0(q, m)) , (1) Now I present the structure of the algorithm for a preselec- tion of r optimization variables x1 . . . xr. where x0(q, m) = arcsin[ln ( −ln(0.5))/2m−q] and the integer m ⩾\|V \| and 0 ⩽q ⩽\|V \| −2. R f is deﬁned in such a way to be n differentiable and to have a minimum (maximum) at 0(1) and rapidly changing multi-oscillatory behavior in between extremas. It should be noted that this deﬁnition is not by any means unique. Assume a graph where \|V \| ≫1, consequently m is a large number. R f (x, 0, m) is such a function, which is mostly 0 in the region of 0 ⩽x ⩽π and rapidly changes to 1 in the region π < x ⩽2π (see red line in Fig. 1). where the number of variables r is between 1 ⩽r ⩽2n and r mod 2 = 0, and mr ⩾2n/r + 2. Pi ∈{II, IX, IY, IZ, XI, XX, XY, XZ,Y I,Y X,YY, Y Z,Y I, ZX, ZY, ZZ}. (5) In Fig. 2, I represent a simple example of a graph with four nodes. For instance the node 1 is connected with two other nodes [Fig. 2(a)]. Therefore, the energy level 1 lies at an energy E = 2 in Fig. 2(b). Node 1 is connected with nodes 2 and 3 so the level 1 is coupled with levels 2 and 3 in the energy scheme. Such logic applies for all nodes of any graph. The algorithm searches for a unitary transformation of the Hamiltonian, which maximizes the number of cuts. (5) It should be noted that all tensor products, which are complex in nature (involving odd numbers of Y Pauli matrices per tensor product) yield a zero trace due to the real nature of the graph Laplacian in our study. Furthermore, in our case the graph Laplacian is a real Hermitian matrix and is thus symmetric, so only the decomposition for lower or upper triangular parts could be performed. Finally, such decom- positions are easily parallelized on HPC architectures as all computations are independent one of another. c. Circuit depth, computational complexity, and quantum advantage. Given a graph with \|V \| nodes n = ⌈log2(\|V \|)⌉ qubits are required to implement the algorithm. The multi- control multitarget qubit gate on n qubits required to realize the diagonal gate U in Eq. (3) can be straightforwardly re- alized with Grey codes [25] or in the context of follow up work [26] at a cost of (23/48)×4n −(3/2)×2n + 4/3 CNOT gates. However, exploiting the fact that U is a diagonal gate and following on the works of [26] and [27] it can be realized with 2n −2 CNOT gates. This means that the ansatz is imple- mented with less than 2\|V \| two qubit CNOT gates, which is in stark contrast with the QAOA ansatz, which requires p\|E\| two qubit gates, where p is the depth parameter. Given that \|E\| ≫\|V \| the algorithm presented here is much more efﬁcient in the number of two qubit gates as compared to even the lowest depth p = 1 QAOA. p p On a quantum computer the computational complexity of evaluating Eq. (3) is O(\|V \|3) [one power of \|V \| coming from the ansatz and up to \|V \|2 summands yielding L(G)]. 5. The number of cuts is calculated as 5. The number of cuts is calculated as H = 1 4 4n<2\|V \|2 i=1 Tr(Pi H)Pi, (4) (4) Ncuts = 2n−2⟨0\|HGU †L(G)UHG\|0⟩. (3) Ncuts = 2n−2⟨0\|HGU †L(G)UHG\|0⟩. (3) 6. Variational parameter x is adjusted with a classical opti- mizer and steps 3-5 are repeated until a maximum is reached. where Pi are all possible combinations of tensor products of the Pauli group composed of {I, X,Y, Z} acting on n qubits. It should be noted that the process of decomposing a Her- mitian matrix into sum of Paulis [see Eq. (4)] is completed in O(\|V \|2): up to \|V \|2 summands, \|V \|3 for the matrix-matrix product of Pi and H and \|V \| for computing the trace. For example, for n = 2 qubits (up to \|V \| = 4 vertex graph) 4n = \|V \|2 = 16 possible products exist where Pi are all possible combinations of tensor products of the Pauli group composed of {I, X,Y, Z} acting on n qubits. It should be noted that the process of decomposing a Her- mitian matrix into sum of Paulis [see Eq. (4)] is completed in O(\|V \|2): up to \|V \|2 summands, \|V \|3 for the matrix-matrix product of Pi and H and \|V \| for computing the trace. For example, for n = 2 qubits (up to \|V \| = 4 vertex graph) 4n = \|V \|2 = 16 possible products exist The algorithm presented here maps the MaxCut problem of a graph G = (V, E) comprising of \|V \| vertices and \|E\| edges to a problem of \|V \| energy levels coupled with \|E\| coupling terms described by a Hamiltonian L(G). The weight between the nodes wi j becomes a coupling strength between energy levels i and j. Energy levels i and j are residing at an energy equal to the connectivity of the node i and j respectively. Noisy intermediate-scale quantum computing algorithm for solving an n-vertex MaxCut problem with log(n) qubits The role of x0(q, m) is to center all R f ’s to 0.5 at x = 0, consequently leading to a more regular optimization landscape (avoiding the 1. Trivial unconnected vertices are added to the graph so it has a dimension, which is a power of two. 2. The graph Laplacian L(G) is represented as a sum of tensor products of unitary matrices, and denoted as L(G) in such form. The decomposition is a prerequisite for measuring the expectation value of the graph Laplacian on a QPU (step 5) and also follows the logic of the implementation in IBMQ’s Qiskit. 3. If a graph has \|V \| nodes a Hadamard gate is applied to ⌈log2 \|V \|⌉qubits. This operation is denoted with HG. g2 q p 4. A variational ansatz in a form of diagonal gate is applied U = diag(exp (iπRf(x1, 0, mr)), .. × exp (iπR f (x1, 2n/r, mr)), . . . exp (iπR f (xr, 0, mr)), .. × exp (iπR f (xr, 2n/r, mr)), 1, . . . 1), (2) where the number of variables r is between 1 ⩽r ⩽2n and r mod 2 = 0, and mr ⩾2n/r + 2. U = diag(exp (iπRf(x1, 0, mr)), .. × exp (iπR f (x1, 2n/r, mr)), . . . exp (iπR f (xr, 0, mr)), .. × exp (iπR f (xr, 2n/r, mr)), 1, . . . 1), (2) U = diag(exp (iπRf(x1, 0, mr)), .. × exp (iπR f (x1, 2n/r, mr)), . . . exp (iπR f (xr, 0, mr)), .. × exp (iπR f (xr, 2n/r, mr)), 1, . . . 1), (2) (2) L012021-2 NOISY INTERMEDIATE-SCALE QUANTUM COMPUTING … PHYSICAL REVIEW RESEARCH 5, L012021 (2023) FIG. 2. (a) A simple graph where wi j are the off-diagonal ele- ments of a graph Laplacian. (b) The mapping of the graph to a set of coupled energy levels. TABLE I. A table summarizing the difference between the ap- proach presented here and QAOA. Complexities are given for one evaluation step and n = ⌈log2 \|V \|⌉. QAOA New alg. New alg. d−regular gr. Ansatz O(p(\|E\| + \|V \|)) O(\|V \|) O(\|V \|) Algorithm O(p(\|E\| + \|V \|)) O(\|V \|3) O(\|V \|d2(d + log∗n)) Qubit nr. \|V \| n n Connectivity graph inspired all-in-all all-in-all FIG. 2. (a) A simple graph where wi j are the off-diagonal ele- ments of a graph Laplacian. Noisy intermediate-scale quantum computing algorithm for solving an n-vertex MaxCut problem with log(n) qubits (b) The mapping of the graph to a set of coupled energy levels. FIG. 2. (a) A simple graph where wi j are the off-diagonal ele- ments of a graph Laplacian. (b) The mapping of the graph to a set of coupled energy levels. was performed. Mathematically, the Pauli basis decomposi- tion of a general Hermitian matrix H is conducted in the following fashion: was performed. Mathematically, the Pauli basis decomposi- tion of a general Hermitian matrix H is conducted in the following fashion: Pi ∈{II, IX, IY, IZ, XI, XX, XY, XZ,Y I,Y X,YY, Y Z,Y I, ZX, ZY, ZZ}. (5) It should also be noted that the way of calculating the number of cuts of a graph on a quantum computer as given by Eq. (3) and can also be applicable to a plethora of algorithms handling different aspects of graph cuts, not only MaxCut with the genetic optimizer as done in this study. FIG. 3. The number of cuts of a 4-node graph obtained with 2 qubits on a simulator of quantum computers (left) and an actual quantum device (right). Results. In Fig. 3, I compare the output of a simulator with the output of publicly available IBMQ Santiago. Although pure dephasing, shot noise and relaxation may distort the optimization landscape, maxima are clearly noticed although equal local maxima become unequal. The Ncuts is estimated for 100 equidistant values of x. required to preform the algorithm presented here requires an all-in-all connectivity between qubits for optimal perfor- mance. On the other hand, QAOA performs best when the qubits are connected in the same way as the nodes of the graph [13]. I further present results obtained by benchmarking ran- domly generated 3-regular graphs of 32 nodes on actual quantum computers and simulators and randomly generated 3-regular graphs of 128 nodes on a simulator of quantum com- puters (Qiskit). Intensive testing showed that the algorithm performs best when the number of variables is kept at 8 or 16 for graphs of the size 32–128 nodes. Intensive numerical testing also showed that a genetic optimizer is best suited for ﬁnding the maximum of the function—not too surpris- ing as genetic optimizer is indeed best used for multimodal cost functions. On top of the genetic algorithm, a number of classical optimizers were tried (COBYLA, Neldear-Mead, As of 2021 the second-most powerful supercomputer in the world is Fugaku [32] with 158 967 nodes each hav- ing 32 Gb of RAM. In total, this supercomputer can store 158 967×32 Gigabytes of data, equaling to it being able to handle 5× 1015 bytes of data. Commonly one requires 8 bytes to store a real number [33,34], indicating that 0.64× 1015 double precision numbers can be stored in the RAM memory in such a device. Given that a graph Lapla- cian is a square matrix, the dimensions of a weighted graph Laplacian, which such a supercomputer could handle is √ 0.64×1015× √ 0.64×1015 = 25.2×106×25.2× 106. Pi ∈{II, IX, IY, IZ, XI, XX, XY, XZ,Y I,Y X,YY, Y Z,Y I, ZX, ZY, ZZ}. (5) This number, although polynomial in \|V \| can still be quite high for large graphs and thus would require an estimation of the expectation value of the large number of summands on a quan- tum computer. However, simulating a d−sparse Hamiltonian (d−regular graph) is done in maximally O(d2(d + log∗n)) queries with the so-called star decomposition of the Hamil- tonian [29,30], and efﬁciently simulating a Hamiltonian is equivalent to calculating an expectation value of a Hamilto- nian [31]. So for d−regular graphs every step in the heuristic algorithm executes in O(\|V \|d2(d + log∗⌈log \|V \|⌉)), which is smaller than the classical O(\|V \|2) for small d. Further reduc- tions of the number of measurements could be conducted by the command group_paulis=True in Pauli expectation class of qiskit. This allows grouping of Pauli strings, which com- mute into same measurements. Also, with larger processors the tasks could be parallelized certain qubits just estimating certain sets of Pauli stings. The algorithm presented here is a heuristic, meaning that its depth is case dependent. However, the quantum imple- mentation of the heuristic can be compared with its classical counterpart for every step of the evaluation. A classical computing variant of Eq. (3) is a vector-matrix-vector multi- plication. For a \|V \|×\|V \| matrix the computational complexity of such an evaluation is O(\|V \|2). In the course of this study the graph Laplacian is de- composed into sum of Pauli matrices with Pennylanne’s qml.Hermitian function [28], afterwards a conversion to Qiskit In Table I, I summarize the main differences between the algorithm presented here and the QAOA. The diagonal gates L012021-3 PHYSICAL REVIEW RESEARCH 5, L012021 (2023) MARKO J. RAN ˇCI ´C FIG. 3. The number of cuts of a 4-node graph obtained with 2 qubits on a simulator of quantum computers (left) and an actual quantum device (right). available at IBM and Google) could handle a 225×225 = 33.5×106×33.5× 106 graph Laplacian. Of course, noise would be a limiting factor in handling such sizes of optimiza- tion problems in the pre-error correction era. However, next year is going to be the year in which large scale error mitiga- tion is going to be implemented on IBMQ systems [5]. This will increase the depth of circuits, which could be executed on IBM QPUs to a larger level, which is difﬁcult to estimate at this point. [4] A. W. Harrow, A. Hassidim, and S. Lloyd, Quantum Algorithm for Linear Systems of Equations, Phys. Rev. Lett. 103, 150502 (2009). [5] IBM quantum roadmap, Research IBM com, https://www.ibm. com/quantum/roadmap. [6] J. R. McClean, J. Romero, R. Babbush, and A. Aspuru-Guzik, The theory of variational hybrid quantum-classical algorithms, New J. Phys. 18, 023023 (2016). [7] J. Preskill, Quantum computing in the NISQ era and beyond, Quantum 2, 79 (2018). Pi ∈{II, IX, IY, IZ, XI, XX, XY, XZ,Y I,Y X,YY, Y Z,Y I, ZX, ZY, ZZ}. (5) On the other hand side, a 25 qubit device (such as those already FIG. 4. Randomly generated 3-regular graphs of 32 nodes. Nodes belonging to different partitions are marked in green and orange respectively and the MaxCut value is written on top of the graph. Graphs are randomly initialized by Python’s networkx package where seed 20 is used for graphs (a)–(c) and seed 30 for graphs (e)–(g). GW stands for Goemans-Williamson. FIG. 4. Randomly generated 3-regular graphs of 32 nodes. Nodes belonging to different partitions are marked in green and orange respectively and the MaxCut value is written on top of the graph. Graphs are randomly initialized by Python’s networkx package where seed 20 is used for graphs (a)–(c) and seed 30 for graphs (e)–(g). GW stands for Goemans-Williamson. FIG. 4. Randomly generated 3-regular graphs of 32 nodes. Nodes belonging to different partitions are marked in green and orange respectively and the MaxCut value is written on top of the graph. Graphs are randomly initialized by Python’s networkx package where seed 20 is used for graphs (a)–(c) and seed 30 for graphs (e)–(g). GW stands for Goemans-Williamson. L012021-4 PHYSICAL REVIEW RESEARCH 5, L012021 (2023) NOISY INTERMEDIATE-SCALE QUANTUM COMPUTING … TABLE II. A table summarizing the results of benchmarking graphs of 128 nodes on a quantum simulator. A random seed in Python is used to generate graph instances. GW stands for the Goemans-Williamson algorithm. for these three graphs I stayed in the domain of quantum sim- ulators. For the ﬁrst graph Table II seed 7: 0.679 ⩽r ⩽0.773, second graph Table II seed 8: 0.743 ⩽r ⩽0.846, third graph Table II seed 9: 0.709 ⩽r ⩽0.807. Values do not converge as nicely as for smaller graphs likely because the genetic algorithm gets trapped in a local minimum with increasing system size. These results are visually represented in Fig. S3 in the SM [22]. seed Maxcut (GW) Maxcut (New alg.) 7 172 133 8 162 137 9 166 134 A d-regular graph with \|V \| nodes has d×\|V \|/2 edges [35]. An average random bipartition of such a graph is d×\|V \|/4 [36], or in the case of 3-regular graphs with 32 nodes the average random bipartition is 24. So both the quantum hardware results and the simulator results stay above the average random bipartition value. An aver- age random bipartition of a 3-regular graph of 128 nodes is 3×128/4 = 96. Pi ∈{II, IX, IY, IZ, XI, XX, XY, XZ,Y I,Y X,YY, Y Z,Y I, ZX, ZY, ZZ}. (5) Basin-Hopping, Particle Swarm, EGO). Further details about the setting of the genetic optimizer can be found in the SM [22]. For the case of 3-regular graphs of 32 nodes variable re- duction is preformed so that the optimization landscape has 8 variables. The MaxCut is calculated with 5 qubits. The Goemans-Williamson algorithm (GW) is a classical approx- imate algorithm for the MaxCut problem has a performance guarantee of 0.87856 [23]. I deﬁne the approximation ratio of the algorithm presented here with respect to the exact solution as Conclusions. In conclusion I have presented a novel al- gorithm for noisy intermediate-scale quantum computers requiring logarithmically less qubits and signiﬁcantly less quantum gates as compared to the contemporary state-the- of-art algorithm—QAOA. I went through to calculate the MaxCut of a randomly generated 3-regular graph of 32 nodes, a 40% increase compared to experiments of state-of-the-art gate-based quantum computers (Google Sycamore). I did so with publicly available IBM hardware, and obtained a lower bound of 54.9% on the solution for actual hardware bench- marks and 77.6% on ideal simulators of quantum computers. Furthermore, I calculated the MaxCut of a 3-regular graph of 128 nodes with quantum simulator obtaining a lower bound of 67.9% on the solution and with no pre-processing of the graph what-so-ever. as 0.87856 MaxCut/MaxCutGW ⩽r ⩽MaxCut/MaxCutGW, (6) W (6) where MaxCut is the value obtained with the algorithm presented here and MaxCutGW is the value obtained with Goemans-Williamson. For the ﬁrst set of graphs in Figs. 4(a)– 4(c) the algorithm performed on a simulator of quantum computers yields 0.776 ⩽r ⩽0.884 and the algorithm exe- cuted on IBMQ Quito 0.552 ⩽r ⩽0.628. For the second set of graphs in Figs. 4(d)–4(f) the algorithm preformed on a sim- ulator of quantum computers yields 0.857 ⩽r ⩽0.975 and the algorithm executed on IBMQ Quito 0.549 ⩽r ⩽0.625. For both realizations there is a clear difference between actual hardware benchmarks and ideal simulation. I assume that the main reason for this is the distortion of the optimization land- scape due to pure dephasing and relaxation. I expect that shot noise contributed less as the expectation value was estimated for the maximally allowed 8192 shots. where MaxCut is the value obtained with the algorithm presented here and MaxCutGW is the value obtained with Goemans-Williamson. For the ﬁrst set of graphs in Figs. [3] P. W. Shor, Algorithms for quantum computation: Discrete log- arithms and factoring, in Proceedings 35th Annual Symposium on Foundations of Computer Science (IEEE Computer Society, NW Washington, DC, 1994), pp. 124–134. [2] L. K. Grover, A fast quantum mechanical algorithm for database search, in Proceedings of the Twenty-Eighth Annual ACM Symposium on Theory of Computing (Association for Computing Machinery, New York, NY, 1996), pp. 212–219. [1] R. P. Feynman, Simulating physics with computers, Int. J. Theor. Phys. 21, 467 (1982). [1] R. P. Feynman, Simulating physics with computers, Int. J. Theor. Phys. 21, 467 (1982). [2] L. K. Grover, A fast quantum mechanical algorithm for database search, in Proceedings of the Twenty-Eighth Annual ACM Symposium on Theory of Computing (Association for Computing Machinery, New York, NY, 1996), pp. 212–219. [3] P. W. Shor, Algorithms for quantum computation: Discrete log- arithms and factoring, in Proceedings 35th Annual Symposium on Foundations of Computer Science (IEEE Computer Society, NW Washington, DC, 1994), pp. 124–134. Pi ∈{II, IX, IY, IZ, XI, XX, XY, XZ,Y I,Y X,YY, Y Z,Y I, ZX, ZY, ZZ}. (5) 4(a)– 4(c) the algorithm performed on a simulator of quantum computers yields 0.776 ⩽r ⩽0.884 and the algorithm exe- cuted on IBMQ Quito 0.552 ⩽r ⩽0.628. For the second set of graphs in Figs. 4(d)–4(f) the algorithm preformed on a sim- ulator of quantum computers yields 0.857 ⩽r ⩽0.975 and the algorithm executed on IBMQ Quito 0.549 ⩽r ⩽0.625. For both realizations there is a clear difference between actual hardware benchmarks and ideal simulation. I assume that the main reason for this is the distortion of the optimization land- scape due to pure dephasing and relaxation. I expect that shot noise contributed less as the expectation value was estimated for the maximally allowed 8192 shots. Output from quantum computers and codes to support the claim are available at zenodo [38]. Acknowledgments. I acknowledge discussions with Adrien Suau, Vedran Dunjko, Thomas Bäck, and Zaid Allybokus, with the latter especially in the domain of use cases to which the algorithm may be applied. I am thankful to the EU for funding within the H2020 project ⟨NE\|AS\|QC⟩. M.J.R. en- visioned, developed, and conducted the whole study himself. The work presented here is a part of a broader provisional patent claim “Method for optimizing a functioning relative to a set of elements and associated computer program product” submission number EP21306155.9 submitted on 26.8.2021. with Marko J. Ranˇci´c and Zaid Allybokus being listed as inventors. y For the case of 3-regular graphs of 128 nodes variable reduction is performed so that the optimization landscape has 16 variables. The MaxCut is calculated with seven simulated qubits under the assumption of no noise processes. Given that devices larger than ﬁve qubits are unavailable to the author, The author declares no further competing interests. [1] R. P. Feynman, Simulating physics with computers, Int. J. Theor. Phys. 21, 467 (1982). [2] L. K. Grover, A fast quantum mechanical algorithm for database search, in Proceedings of the Twenty-Eighth Annual ACM Symposium on Theory of Computing (Association for Computing Machinery, New York, NY, 1996), pp. 212–219. [3] P. W. Shor, Algorithms for quantum computation: Discrete log- arithms and factoring, in Proceedings 35th Annual Symposium on Foundations of Computer Science (IEEE Computer Society, NW Washington, DC, 1994), pp. 124–134. L012021-5 MARKO J. RAN ˇCI ´C PHYSICAL REVIEW RESEARCH 5, L012021 (2023) [8] A. Peruzzo, J. McClean, P. Shadbolt, M.-H. Yung, X.-Q. Zhou, P. J. Love, A. Aspuru-Guzik, and J. L. Pi ∈{II, IX, IY, IZ, XI, XX, XY, XZ,Y I,Y X,YY, Y Z,Y I, ZX, ZY, ZZ}. (5) Obrien, A vari- ational eigenvalue solver on a photonic quantum processor, Nat. Commun. 5, 4213 (2014). [22] See Supplemental Material at http://link.aps.org/supplemental/ 10.1103/PhysRevResearch.5.L012021 for the optimizer set- tings and larger graph results. [23] M. X. Goemans and D. P. Williamson, Improved approximation algorithms for maximum cut and satisﬁability problems using semideﬁnite programming, J. ACM 42, 1115 (1995). [9] J. Tilly, H. Chen, S. Cao, D. Picozzi, K. Setia, Y. Li, E. Grant, L. Wossnig, I. Rungger, G. H. Booth et al., The variational quantum eigensolver: A review of methods and best practices, Phys. Rep. 986, 1 (2022). [24] A. Pothen, H. D. Simon, and K.-P. Liou, Partitioning sparse matrices with eigenvectors of graphs, SIAM J. Matrix Anal. Appl. 11, 430 (1990). [10] E. Farhi, J. Goldstone, and S. Gutmann, A quantum approxi- mate optimization algorithm, arXiv:1411.4028. [25] M. Möttönen, J. J. Vartiainen, V. Bergholm, and M. M. Salomaa, Quantum Circuits for General Multiqubit Gates, Phys. Rev. Lett. 93, 130502 (2004). [11] M. Cerezo, A. Arrasmith, R. Babbush, S. C. Benjamin, S. Endo, K. Fujii, J. R. McClean, K. Mitarai, X. Yuan, L. Cincio et al., Variational quantum algorithms, Nat. Rev. Phys. 3, 625 (2021). [26] V. V. Shende, S. S. Bullock, and I. L. Markov, Synthesis of quantum-logic circuits, IEEE Trans. Compu.-Aided Des. Integr. Circuits Syst. 25, 1000 (2006). [12] L. Zhou, S.-T. Wang, S. Choi, H. Pichler, and M. D. Lukin, Quantum Approximate Optimization Algorithm: Performance, Mechanism, and Implementation on Near-Term Devices, Phys. Rev. X 10, 021067 (2020). [27] E. Malvetti, R. Iten, and R. Colbeck, Quantum circuits for sparse isometries, Quantum 5, 412 (2021). [28] V. Bergholm, J. Izaac, M. Schuld, C. Gogolin, M. S. Alam, S. Ahmed, J. M. Arrazola, C. Blank, A. Delgado, S. Jahangiri et al., Pennylane: Automatic differentiation of hybrid quantum- classical computations, arXiv:1811.04968. [13] M. P. Harrigan, K. J. Sung, M. Neeley, K. J. Satzinger, F. Arute, K. Arya, J. Atalaya, J. C. Bardin, R. Barends, S. Boixo et al., Quantum approximate optimization of non-planar graph problems on a planar superconducting processor, Nat. Phys. 17, 332 (2021). [29] D. W. Berry, G. Ahokas, R. Cleve, and B. C. Sanders, Efﬁ- cient quantum algorithms for simulating sparse Hamiltonians, Commun. Math. Phys. 270, 359 (2007). [14] The reader should be made aware of the state-of-the-art QAOA experiments with 40 trapped ion qubits [37]. Pi ∈{II, IX, IY, IZ, XI, XX, XY, XZ,Y I,Y X,YY, Y Z,Y I, ZX, ZY, ZZ}. (5) However, here the focus was on ﬁnding the ground state of the linear Ising model rather than optimizing a partition of a realistic graph. [30] A. M. Childs and R. Kothari, Simulating sparse Hamiltonians with star decompositions, in Conference on Quantum Computa- tion, Communication, and Cryptography (Springer, New York, 2010), pp. 94–103. [15] G. G. Guerreschi and A. Y. Matsuura, Qaoa for max-cut re- quires hundreds of qubits for quantum speed-up, Sci. Rep. 9, 6903 (2019). [31] E. Knill, G. Ortiz, and R. D. Somma, Optimal quantum mea- surements of expectation values of observables, Phys. Rev. A 75, 012328 (2007). [16] J. Li, M. Alam, and S. Ghosh, Large-scale quantum approximate optimization via divide-and-conquer, arXiv:2102.13288. [32] The top 500 supercomputers list, https://www.top500.org/lists/ top500/2022/11/, 2022. [17] B. Tan, M.-A. Lemonde, S. Thanasilp, J. Tangpanitanon, and D. G. Angelakis, Qubit-efﬁcient encoding schemes for binary optimisation problems, Quantum 5, 454 (2021). [33] D. Goldberg, What every computer scientist should know about ﬂoating-point arithmetic, ACM Comput. Surv. 23, 5 (1991). [34] M. L. Overton, Numerical Computing with IEEE Floating Point Arithmetic (SIAM, Philadelphia, 2001). [18] F. Hoffmeister and T. Bäck, Genetic algorithms and evolution strategies: Similarities and differences, In International Confer- ence on Parallel Problem Solving from Nature (Springer, New York, 1990), pp. 455–469. [35] J. M. Aldous and R. J. Wilson, Graphs and Applications: An Introductory Approach (Springer Science & Business Media, New York, 2003). [36] R. Motwani and P. Raghavan, Randomized Algorithms (Cam- bridge University Press, Cambridge, 1995). [19] N. N. Glibovets and N. M. Gulayeva, A review of niching ge- netic algorithms for multimodal function optimization, Cybern. Syst. Anal. 49, 815 (2013). [37] G. Pagano, A. Bapat, P. Becker, K. S. Collins, A. De, P. W. Hess, H. B. Kaplan, A. Kyprianidis, W. L. Tan, C. Baldwin et al., Quantum approximate optimization of the long-range Ising model with a trapped-ion quantum simulator, Proc. Natl. Acad. Sci. U.S.A. 117, 25396 (2020). [20] N. Casas, Genetic algorithms for multimodal optimization: A review, arXiv:1508.05342. [21] M. A. Bouhlel, J. T. Hwang, N. Bartoli, R. Lafage, J. Morlier, and J. R. R. A. Martins, A python surrogate model- ing framework with derivatives, Adv. Eng. Softw. 135, 102662 (2019). [38] M. J. Rancic, An exponentially more efﬁcient optimization algorithm for noisy quantum computers, zenodo (2021), doi: 10.5281/zenodo.5592834. L012021-6
https://openalex.org/W2924396932	https://eprints.gla.ac.uk/202344/1/202344.pdf	English	null	A phenotypically plastic magic trait promoting reproductive isolation in sticklebacks?	bioRxiv (Cold Spring Harbor Laboratory)	2,019	cc-by	5,283	Evolutionary Ecology (2020) 34:123–131 https://doi.org/10.1007/s10682-019-10015-2 Evolutionary Ecology (2020) 34:123–131 https://doi.org/10.1007/s10682-019-10015-2 ORIGINAL PAPER * Colin E. Adams colin.adams@glasgow.ac.uk Abstract This study identifies one possible mechanism whereby gene flow is interrupted in popula- tions undergoing evolutionary divergence in sympatry; this is an important issue in evo- lutionary biology that remains poorly understood. Variation in trophic morphology was induced in three-spined stickleback by exposing them from an early age either to large benthic or to small pelagic prey. At sexual maturity, females given a choice between two breeding males, showed positive assortative mate choice for males raised on the same diet as themselves. The data indicate that this was mediated through a preference for males with trophic morphology similar to that of fish with which the females were familiar (from their pre-testing holding tanks). In trials where the female did not choose the most familiar male, the evidence suggests that either she had difficulty discriminating between two similar males or was positively choosing males with more extreme morphologies (more benthic- like or pelagic-like). This study has shown for the first time that expression of a plastic trait induced at an early age, not only results in specialisation for local foraging regimes but can also play a significant role in mate choice. This is equivalent to an environmentally induced, plastic version of the “magic traits” that promote ecologically-driven divergence in sympatry, hence the proposed descriptor “plastic magic trait”. Keywords Assortative mating · Epigenetics · Reproductive isolation · Trophic polymorphism Keywords Assortative mating · Epigenetics · Reproductive isolation · Trophic polymorphism A phenotypically plastic magic trait promoting reproductive isolation in sticklebacks? Received: 23 March 2019 / Accepted: 24 October 2019 / Published online: 8 November 2019 © The Author(s) 2019 colin.adams@glasgow.ac.uk 1 Scottish Centre for Ecology and the Natural Environment, IBAHCM, University of Glasgow, Rowardennan, Glasgow G63 0AW, UK 2 Present Address: Facultad de Ciencias, Universidad Autónoma del Estado de México, Instituto Literario no. 100 Colonia Centro, CP 50000 Toluca, Mexico 1 Scottish Centre for Ecology and the Natural Environment, IBAHCM, University of Glasgow, Rowardennan, Glasgow G63 0AW, UK Introduction The process whereby gene flow is interrupted in populations undergoing evolutionary divergence when in sympatry is an important issue in evolutionary biology that remains poorly understood. One potential mechanism is where an ecologically important trait which is under divergent selection also contributes to reproductive isolation and is thus 0123456789) 1 3 56789) 3 Evolutionary Ecology (2020) 34:123–131 124 a so-called “magic trait” (Gavrilets 2004; Servedio et al. 2011). Although evidence of such traits in nature is sparse (Servedio et al. 2011), magic traits are usually envisaged as inherited and linked to mate choice through pleiotropy. However, much research interest has focused on the possible role of phenotypic plasticity in the initiation of evolutionary change through the development of discrete alternative phenotypes (West-Eberhard 1989, 2003; Fitzpatrick 2012; Skulason et al. 2019). If expressed alternative phenotypic traits induced by the environment through plasticity, also form part of the mate choice system of the diverging organism, then assortative mating resulting from mate choice based on such traits have the potential to generate reproductive barriers between individuals expressing different phenotypes (Fitzpatrick 2012). Such traits may thus act as a magic trait without the requirement of pleiotropy. Discrete alternative phenotypes associated with foraging, or trophic polymorphisms [sensu Skúlason and Smith (Skulason and wund 1995)], have been strongly implicated in sympatric speciation events (Dieckmann and Doebeli 1999). Divergent morphological traits are often the result of foraging conditions experienced during development (Day and McPhail 1996; Adams et al. 2003), so can only result in evolutionary change if mecha- nisms exist that result in gene pool segregation (West-Eberhard 1989; Smith and Skulason 1996; Schluter 2003). Here we explore one possible mechanism, namely morph-specific mate choice by breeding females. Using the three-spined stickleback (Gasterosteus acu- leatus) as a model system, we present an example of a developmentally-plastic, trophic specialisation acting as a magic trait generating reproductive isolation and suggest a mech- anism through which this comes about.i Trophic polymorphism is particularly common among freshwater fishes including sticklebacks; it often takes the form of co-existing but discrete phenotypes with morpho- logical and behavioural specialisations for feeding on benthic invertebrates in the littoral zone or zooplankton in the pelagic zone (Skulason and Smith 1996; Adams and Hunting- ford 2002a; Proulx and Magnan 2004). Diet treatments 240 juvenile three-spined sticklebacks fry (5–9 mm length) were collected by dip nets from a small freshwater pond in Scotland (56° 3′ N; 004° 21′ W) and transported to rearing facilities at the Scottish Centre for Ecology and the Natural Environment (SCENE), Glas- gow University, Loch Lomond. Fish were assigned randomly in groups of 40 to 6 rearing aquaria (21L) and raised in the laboratory for 11 months, during which time they were fed twice daily to satiation on one of two diet treatments known to induce differences in trophic morphology (Day and McPhail 1996). Half of the groups were fed on frozen Daph- nia spp in a bag hanging at the water surface, simulating pelagic prey; the rest were fed on frozen chironomid prey placed on the bottom of the tank, simulating benthic prey. Introduction Typically, the benthic form is robust, with a large mouth, small eyes and few short gill rakers, while the pelagic form is lightly built, with a relatively small mouth, large eyes and longer and more numerous gill rakers (Adams et al. 1998; Adams and Huntingford 2002a). Although in some cases the two sympatric forms are fully reproductively isolated, more commonly reproductive isolation is partial, weak or non-existent (Schluter and McPhail 1992; Hendry et al. 2009). Sticklebacks also exhibit scope for the expression of characteristics under phenotypic plasticity (Wund et al. 2008, 2012; Garduño-Paz et al. 2010; Baker et al. 2013) including plasticity in morphological characteristics that define sympatric forms described from the wild (Day and McPhail 1996; Garduño-Paz et al. 2010). The main aim of this study was, having induced variable trophic morphology in three- spined sticklebacks from a single population by manipulating early feeding regimes, to determine whether these plastic, diet-induced differences in trophic morphology were asso- ciated with different patterns of mate choice. A second aim was to seek possible behav- ioural mechanisms that might explain the observed patterns of mating. 1 3 1 Evolutionary Ecology (2020) 34:123–131 125 Analysis of induced morphological differences After 10 months, the sticklebacks were anaesthetised with benzocaine and photographed on their left side with a Canon EOS digital 350D camera (8.0 megapixels). Female fish that were gravid and male fish which were coloured were identified as sexually mature. All fish used in mate choice experiments were re-photographed at 11 months immediately follow- ing the mate choice experiments. Body shape was quantified on the basis of 20 landmarks (Fig. 1), placed using the program “tpsUtil” ad digitised using the program “tpsDig2” (Rohlf 2000). Generalized least squares procrustes superimposition was used to translate, scale and rotate raw landmark coordinates to minimize the summed, squared, inter-land- mark distances among fish; this procedure removes the effect of fish size (Rohlf and Slice 1990). Relative Warp Analysis was conducted on the partial warp scores to reduce the number of informative shape variables (Rohlf 2000). The second relative warp (analogous to a Principal Component), which explained 13% of the total shape variation, separated traits typical of pelagic and benthic feeders (Day and McPhail 1996; Fig. 1). Mate choice trials A female was deemed to have chosen a male if she spent at least 60% of the total time of the trial near that male. Association time has been shown to be a strong predictor of eventual mate choice in this species in a number of other studies (McLellan and McPhail 1990; Rowland et al. 1995; Milinski et al. 2005; Rick and Bakker 2008). Males and females were used maximally in four tri- als on different days; males were re-used in fresh combinations so that the female was never exposed to the same pair of males. Although male pairs were matched in size (by fork length) as nearly as possible, small discrepancies between pairs remained. Retro- spective analysis detected no significant difference in body size (fork length) between chosen and rejected males (mean ± SE size differences between accepted and rejected males = 0.04 cm ± 0.02 paired t test: t = 1.68, p = 0.10).i Body shape, as defined by the second relative warp varied markedly both between and within diets (Fig. 1). Effects of sex (ANOVA: F1,60 = 3.11; p = 0.08) and of sex by diet (ANOVA: F1,60 = 2.86; p = 0.09) were not significant. However, there was a highly significant difference in morphology between the chironomid fed (mean ± SE = 6.753 ±0.23) and Daphnia fed sticklebacks (mean ± SE = 4.82 + 0.34. ANOVA: F1,60 = 22.2; p < 0.0001). The higher scores of fish fed on the benthic diet reflected shorter heads, shorter maxillary bones, smaller eyes and deeper bodies. This score was transformed to create only positive values (by adding 6 and multiplying by 100) and hereafter this dimension of shape variation is referred to as the pelagic–ben- thic (PB) shape score. Lower PB scores indicate shapes tending towards a more typical of a pelagic foraging fish; higher scores tending towards a more benthic foraging fish shape (Fig. 1). To enable testing of the body shape of chosen and rejected males, the difference in PB shape score between the chosen male and the rejected male for each pair was determined as: male PB score difference = PB score chosen male − PB score rejected male. Thus a large positive number in this metric indicates the choice of a male from the pair with a higher PB score; thus tending towards a shape more typical of a benthic foraging fish. Mate choice trials Twenty eight females (21 from the chironomid diet and 7 from the Daphnia. diet) and 36 males (21 from the chironomid diet and 15 from the Daphnia diet) were used in trials of mate choice. Female mate choice was examined using a well-tested method- ology, widely used in previous studies (Milinski and Bakker 1990; Kraak and Bakker Fig. 1 The landmark configurations used in the morphometric analysis of trophic morphology in stickle- backs. The landmarks are connected to aid visualisation of fish shape. Arrows represent vectors describ- ing deformations that change the mean shape of sticklebacks fed on benthic prey compared with the mean shape of those fed on pelagic prey Fig. 1 The landmark configurations used in the morphometric analysis of trophic morphology in stickle- backs. The landmarks are connected to aid visualisation of fish shape. Arrows represent vectors describ- ing deformations that change the mean shape of sticklebacks fed on benthic prey compared with the mean shape of those fed on pelagic prey 3 Evolutionary Ecology (2020) 34:123–131 126 1998; Boulcott et al. 2005; Rick et al. 2006; Rick and Bakker 2008; Heuschele et al. 2009) in which a single gravid female was placed alone in an aquarium (35 × 25 × 20 cm, screened on 3 sides), allowed to settle for 12 h and was then presented simultane- ously with two breeding males in equally-sized sections (25 × 35 × 20 cm) of an adjacent aquarium and thus not subject to olfactory cues. During trials, females could see both males, but the females had no olfactory contact with males and the two males were sep- arated by an opaque partition and so did not have visual contact with each other. In any trial, the female was presented with one Daphnia-fed and one chironomid-fed male. To avoid effects of size and familiarity with specific males, the two males in any given trial were size-matched as far as possible and importantly taken from a different rearing tank from the female. To avoid females potentially making choices based on nest construc- tion, males were not provided with nesting material. Each trial lasted for 5 min, during which, the time the female spent on the side of the tank adjacent to each male was recorded. Three replicates of each pairing trial were conducted, swapping the male position each time. Mate choice trials Conversely a large negative number in the difference between cho- sen and rejected male PB scores indicates a choice of male with a shape tending towards that typical of a more pelagic forging fish. i In nature, female sticklebacks review a number of males before selecting a nest in which to lay her eggs and the females in this study were always tested with different pairs of unfa- miliar males. However female identity was included as a random factor in mixed effects models examining female mate choice. A total of 96 trials of mate choice by females was analysed using “R” (R Development Core Team 2017). 1 3 Evolutionary Ecology (2020) 34:123–131 127 Mate choice in females exposed to benthic and pelagic diets To test for the effect of the previous diet experience of the female on female mate choice, the PB score difference between male pairs in each trial was categorised as either a nega- tive value, indicating the choice of a male with a more pelagic-like body shape, or posi- tive, indicating a choice of the male from the pair with a more benthic-like body shape. The previous diet experience of the female (benthic (chironomid) or pelagic (Daphnia) diet exposure) was used as a fixed factor and female I.D. as random factor, in a mixed effects model with a binomial (positive or negative male PB score difference) probability distribu- tion. Female diet exposure significantly predicted the choice of male body shape in pair- wise tests (p < 0.01) indicating non-random mating on the basis of previous diet exposure. However the total explained variance was low (6.8%) and this effect was mostly driven by females with previous exposure of a pelagic prey diet choosing the male with the more pelagic body shape (a negative male PB score difference) on 66% of occasions (Table 1). In contrast, females exposed to the benthic prey diet chose the male from the pair with the more benthic body shape (a positive male PB score difference) on only 54% of occasions. Behavioural mechanisms of mate choice as random factor, in a mixed effects model with a binomial probability distribution.ii The mean PB score of fish from the tank from which the female originated significantly predicted the female’s choice of male (p < 0.02; r2 = 6.9%). Indicating that females are choosing of males on the basis of body shapes with which they are familiar. Despite this a considerable amount of the variation in the choice of male was not explained by the body shape with which they were familiar. One possible explanation is that in trails where females chose males that were not closer in body shape to those with which she was familiar (from the same rearing tank) and thus she may have had difficulty discriminating between body shapes. If this was the case, then one expectation would be that in such trials the two males are likely to be closer in body shape (similar PB scores) to each other than in trials where the female chose the male closest to that with which she is familiar. This was tested; the PB score difference between males in trials where the female chose the most familiar shape (2.22 ± 1.36; mean ± S.D.) was significantly greater (t test: p = 0.041) than the PB score difference between males from trails where she chose the less familiar male (1.85 ± 0.94). This indicates that in at least some trails females may have had difficulty distinguishing between males of similar body shape. Another possible explanation for the outcome of those trails where the female did not chose the male with a body shape closest to the mean of fish that she was familiar with, is that she may avoid choosing males with extreme body shapes even if they are closer to the body shape with which she is familiar. To test this the Extreme Shape Index was calculated as the deviation in body shape for chosen and rejected male from the average of all fish combined (that is the PB score of each fish—the mean of all fish combined irrespective of sign. As these data deviated significantly from normality, the Extreme Shape Index for chosen and rejected males from trials where the female chose the male with a shape that she was less familiar with, were compared in a paired Wilcoxon test. Behavioural mechanisms of mate choice To explore possible behavioural mechanisms for the observed female preference by diet, mate choice data were analysed in more detail. To test the possibility that females are making a choice of male based on their own body shape, the male PB score difference in each trial, categorised as either a negative value (indicating the choice of a male of a more pelagic-like body shape), or a positive value (indicating a choice of the male with a more benthic-like body shape) was modelled using female PB score as a fixed factor and female I.D. as random factor, in a mixed effects model with a binomial probability distribution. Female body shape did not predict the choice of male (p < 0.45). Thus the assortative mat- ing effect predicted by female previous diet exposure does not appear to be driven by the body shape of the female.l Another possible behavioural mechanism by which rearing diet might influence a female stickleback’s mate choice is through previous experience of the fish with which she was reared, whose shape will, on average, reflect their common rearing diet. To test this pos- sibility, we took advantage of the variability in PB scores between rearing tanks on a given diet. The male PB score difference in each trial, categorised as either a negative value, or a positive value was modelled using the mean PB score of fish in the tank from which the 1 3 Table 1 The frequency with which the male with the higher pelagic–benthic score (more benthic-like) was chosen and rejected by female sticklebacks exposed to alternative diets (a pelagic like diet (Daphnia) and a benthic-like diet (chironomid larvae) Female diet Male with more positive P–B score χ2 DF p Chosen Rejected Benthic 34 29 0.4 1 > 0.53 Pelagic 9 24 6.8 1 < 0.01 le 1 The frequency with which the male with the higher pelagic–benthic score (more benthic-like) was sen and rejected by female sticklebacks exposed to alternative diets (a pelagic like diet (Daphnia) and a thic-like diet (chironomid larvae) Evolutionary Ecology (2020) 34:123–131 128 female was drawn, as a fixed covariate and female I.D. as random factor, in a mixed effects model with a binomial probability distribution.ii female was drawn, as a fixed covariate and female I.D. Behavioural mechanisms of mate choice In these trials the cho- sen male was much more likely to have a more extreme body shape (i.e. a higher Extreme Shape Index) (2.09 ± 1.08; mean ± S.D.) than the rejected male (1.01 ± 0.86). Discussion Our results confirm the findings of previous studies demonstrating a plastic response of morphological traits to rearing diet in three-spined sticklebacks (Day and McPhail 1996). More significantly, they have demonstrated for the first time that exposure to different diets during the juvenile phase can influence the mating preferences shown by breeding females. Thus, females reared on the pelagic diet tended to prefer the male with a more pelagic-like morphology; females reared on a benthic diet however mated randomly with respect to trophic morphology. Thus there is partial assortative mating by diet-induced phenotype. Unlike the case of assortative mating on the basis of diet specialisation in the mustard leaf beetle, which appears to use olfactory cues to identify mates (Geiselhardt et al. 2012) the sticklebacks in this experiment only had visual cues are available to them. However, it is quite possible that olfactory cues might also have affected mate choice had they been avail- able. In addition, the effect reported here did not result from female familiarity with spe- cific individual males, as females were never tested with males from the same rearing tank. 1 3 1 Evolutionary Ecology (2020) 34:123–131 129 Additionally we show that mate choice was not dependent directly of the female’s own trophic morphology. Arguably, this is not surprising, since it is difficult to see how a female stickleback could know what her own morphology is like. Instead the differ- ences in mate choice must be a consequence (direct or indirect) of the experience of being raised on a pelagic or a benthic diet. Making use of the significant variation in morphology between and within rearing tanks exposed to different and the same diets, we show that the expressed morphology of other fish with which the female is familiar (from the same rearing tank) is a good predictor of mate choice. It is highly likely that in the wild also sticklebacks grow up with fish exploiting a similar diet to themselves and thus with similar diet-induced morphology, as individuals exploiting the same for- aging resources are more likely to come into contact with each other, than those that do not share a common diet (Garduño-Paz and Adams 2010). Despite a clear tendency for assortative mating by trophic morphology, females quite often made the opposite choice. Discussion This was most often the case when the difference between the two males was relatively small, but also occurred when if the morphology of the predicted choice male was of an extreme benthic or pelagic-type morphology. One can envisage at least two plausible mechanistic explanations for this, which are not mutually exclusive. It may be that, rather than responding to familiarity per se, females have learned about the foraging efficacy of fish with the range of morphologies that she has experienced during development. If this were the case, then females might actively choose males of a more extreme morphology, which may well be more efficient at forag- ing on the two alternative diets presented, even if this morphology is less common in her previous experience familiar to her. We are not able to test directly this possibility using the data from this study. Although coexisting trophic morphs are thought to be an important step in evolution- ary divergence in sympatry (Skulason et al. 1999), speciation however is unlikely to be completed without some mechanism for morph-specific assortative mating (Skulason et al. 1999). Several routes though which this might occur have been suggested. For example specialist morphs might occupy different habitats. Olafsdóttir et al. (2006) for example, showed that sticklebacks specialising in living in habitats with little vegeta- tion had reduced nest building behaviour and as a result weed-living specialists from the same lake mated assortatively with other weed-living specialists when using nest quality as a mate choice criterion. Disruptive sexual selection is also known to play a significant role in the divergence of recently evolved African cichlid species (Stelkens et al. 2008). Here uniquely we demonstrate assortative mating on the basis of morpho- logical traits that frequently express as discrete forms in the wild, have strong functional significance for resource acquisition (Adams and Huntingford 2002b) are thought to be under strong selection pressure and the expression of which is significantly modulated by plasticity effects. This result indicates that trophic morphology is both a plastic and a magic trait for sticklebacks, thus that pleiotropy may not always be required for traits to operate as magic traits. Acknowledgements We thank Rona Brennan for technical support. M.V.G-P. was supported by a Mexican Council for Science and Technology (CONACYT) scholarship (Grant No. 342451). Acknowledgements We thank Rona Brennan for technical support. M.V.G-P. was supported by a Mexican Council for Science and Technology (CONACYT) scholarship (Grant No. 342451). Compliance with ethical standards Conflict of interest The authors declare no conflicts of interest. 3 3 1 Evolutionary Ecology (2020) 34:123–131 130 Ethical approval This study was conducted in accordance with UK legislation under Home Office Licence Number: PPL 70/8794. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 Interna- tional 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. References Adams C, Huntingford F (2002a) Inherited differences in head allometry in polymorphic charr from Loch Rannoch, Scotland. J Fish Biol 60:515–520. https://doi.org/10.1006/jfbi.2002.1867if Adams C, Huntingford F (2002b) The functional significance of inherited differences in feeding morphol- ogy in a sympatric polymorphic population of Arctic charr. Evol Ecol 16:15–25 Adams CE, Fraser D, Huntingford FA et al (1998) Trophic polymorphism amongst Arctic charr from Loch Rannoch, Scotland. J Fish Biol 52:1259–1271 Adams CE, Woltering C, Alexander G (2003) Epigenetic regulation of trophic morphology through feed- ing behaviour in Arctic charr, Salvelinus alpinus. Biol J Linn Soc 78:43–49. https://doi.org/10.104 6/j.1095-8312.2003.00126.x j Baker JA, Räsänen K, Moore JS, Hendry AP (2013) Genetic and plastic contributions to trait divergence between parapatric habitats: female life-history traits in three-spine stickleback within the Misty Lake system. Evol Ecol Res 15:473–487 y Boulcott PD, Walton K, Braithwaite VA (2005) The role of ultraviolet wavelengths in the mate-choice decisions of female three- spined sticklebacks. J Exp Biol 208:1453–1458. https://doi.org/10.1242/ jeb.01569ffi j Day T, McPhail J (1996) The effect of behavioural and morphological plasticity on foraging efficiency in the threespine stickleback (Gasterosteus sp.). Oecologia 108:380–388 p p g Dieckmann U, Doebeli M (1999) On the origin of species by sympatric speciation. Nature 400:354–357 Dieckmann U, Doebeli M (1999) On the origin of species by sympatric speciation. Nature 400:354–357 Fit t i k B (2012) U d i t d f h t i l ti it f l i l i ti I t patrick B (2012) Underappreciated consequences of phenotypic plasticity for ecological speciation. Int J Ecol 2012:256017 Garduño-Paz MV, Adams CE (2010) Discrete prey availability promotes foraging segregation and early divergence in Arctic charr, Salvelinus alpinus. Hydrobiologia 650:15–26. https://doi.org/10.1007/ s10750-009-0055-8 Garduño-Paz MV, Couderc S, Adams CE (2010) Habitat complexity modulates phenotype expression through developmental plasticity in the threespine stickleback. Biol J Linn Soc 100:407–413. https:// doi.org/10.1111/j.1095-8312.2010.01423.x g j Gavrilets S (2004) Fitness landscapes and the origin of species. Princeton University Press, Princeton g j Gavrilets S (2004) Fitness landscapes and the origin Geiselhardt S, Otte T, Hilker M, Letters E (2012) Looking for a similar partner: host plants shape mating preferences of herbivorous insects by altering their contact pheromones. Ecol Lett 15:971–977. https:// doi.org/10.1111/j.1461-0248.2012.01816.x g j Hendry AP, Huber SK, De León LF et al (2009) Disruptive selection in a bimodal population of Darwin’s finches. Proc Biol Sci 276:753–759. References https://doi.org/10.1098/rspb.2008.1321 i p g p Heuschele J, Mannerla M, Gienapp P, Candolin U (2009) Environment-dependent use of mate choice cues in sticklebacks. Behav Ecol 20:1223–1227. https://doi.org/10.1093/beheco/arp123 p g p Kraak S, Bakker T (1998) Mutual mate choice in sticklebacks: attractive males choose big females, which lay big eggs. Anim Behav 56:859–866i McLellan D, McPhail J (1990) Experimental investigations of the evolutionary significance of sexually dimorphic nuptial colouration in Gasterosteus aculeatus (L.): the relationship between male colour and female behaviour. Can J Zool 68:482–492 Milinski M, Bakker TCM (1990) Female sticklebacks use male coloration in mate choice and hence avoid parasitized males. Nature 344:330–333. https://doi.org/10.1038/344330a0fi Milinski M, Griffiths S, Wegner K et al (2005) Mate choice decisions of stickleback females predictably modified by MHC peptide ligands. Proc Natl Acad Sci 102:4414–4418 i Olafsdóttir GA, Ritchie MG, Snorrason SS (2006) Positive assortative mating between recently described sympatric morphs of Icelandic sticklebacks. Biol Lett 2:250–252. https://doi.org/10.1098/ rsbl.2006.0456 1 3 Evolutionary Ecology (2020) 34:123–131 131 Proulx R, Magnan P (2004) Contribution of phenotypic plasticity and heredity to the trophic polymorphism of lacustrine brook charr (Salvelinus fontinalis). Evol Ecol Res 6:503–522 Proulx R, Magnan P (2004) Contribution of phenotypic plasticity and heredity to the trophic polymorphi of lacustrine brook charr (Salvelinus fontinalis). Evol Ecol Res 6:503–522 R Development Core Team (2017) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna R Development Core Team (2017) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna p g Rick IP, Bakker TCM (2008) Color signaling in conspicuous red sticklebacks: do ultraviolet signals surpass others? BMC Evol Biol 8:1–9. https://doi.org/10.1186/1471-2148-8-189f Rick IP, Bakker TCM (2008) Color signaling in conspicuous red sticklebacks: do ultraviolet signals surpass others? BMC Evol Biol 8:1–9. https://doi.org/10.1186/1471-2148-8-189 p g k IP, Modarressie R, Bakker TCM (2006) UV wavelengths affect female mate choice in three-spined ti kl b k A i B h 71 307 313 htt //d i /10 1016/j b h 2005 03 039 p g Rick IP, Modarressie R, Bakker TCM (2006) UV wavelengths affect female mate choice in three-spined sticklebacks. Anim Behav 71:307–313. https://doi.org/10.1016/j.anbehav.2005.03.039 p g Rick IP, Modarressie R, Bakker TCM (2006) UV wavelengths affect female mate choice in three-spined sticklebacks. Anim Behav 71:307–313. https://doi.org/10.1016/j.anbehav.2005.03.039 Rick IP, Modarressie R, Bakker TCM (2006) UV wavelengths affect female mate choice in three spin sticklebacks. Anim Behav 71:307–313. https://doi.org/10.1016/j.anbehav.2005.03.039 f sticklebacks. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. References https:// doi.org/10.1111/j.1095-8312.2011.01815.x 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. 1 3 References Anim Behav 71:307–313. https://doi.org/10.1016/j.anbehav.2005.03.039 Rohlf F (2000) Statistical power comparisions among alternative morphometric methods. Am J Phys Anthropol 111:463–478 Rohlf F, Slice D (1990) Extensions of the Procrustes method for the optimal superimposition of landmarks. Syst Zool 39:40–59f Rowland W, Bolyard K, Halpern A (1995) The dual effect of stickleback nuptial coloration on rivals: man ulation of a graded signal using video playback. Anim Behav 50:267–272 Schluter D (2003) Frequency dependent natural selection during character displacement in sticklebacks. Evolution 57:1142–1150 Schluter D, McPhail JD (1992) Ecological character displacement and speciation in sticklebacks. Am Nat 140:85–108 Servedio MR, Van Doorn GS, Kopp M et al (2011) Magic traits in speciation: “magic” but not rare? Trends Ecol Evol 26:389–397. https://doi.org/10.1016/j.tree.2011.04.005 Servedio MR, Van Doorn GS, Kopp M et al (2011) Magic traits in speciation: Ecol Evol 26:389–397. https://doi.org/10.1016/j.tree.2011.04.005 Skulason S, Smith TB (1995) Resource polymorphisms in vertebrates. Trends Ecol Evol 10:366–370 Skulason S, Smith TB (1996) The ecology of resource polymorphism in vertebrates—reply. Trends Ecol Evol 11:26. https://doi.org/10.1016/0169-5347(96)81063-9i Skulason S, Smith TB (1996) The ecology of resource polymorphism in Evol 11:26. https://doi.org/10.1016/0169-5347(96)81063-9 Skulason S, Snorrason S, Jonsson B (1999) Sympatric morphs populations and speciation in freshwater fish with emphasis on arctic charr. In: Magurran A, May R (eds) Evolution of biological diversity. Oxford University Press, Oxford, pp 70–92 Skulason S, Parsons KJ, Svanback R et al (2019) A way forward with eco evo devo: an extended theory of resource polymorphism with postglacial fishes as model systems. Biol Rev 94:1786–1808. https://doi. org/10.1111/brv.12534ii g ith TB, Skulason S (1996) Evolutionary significance of resource polymorphisms in fishes, amphibians and birds. Annu Rev Ecol Syst 27:111–133 Stelkens RB, Pierotti MER, Joyce DA et al (2008) Disruptive sexual selection on male nuptial coloration in an experimental hybrid population of cichlid fish. Philos Trans R Soc Lond B 363:2861–2870. https:// doi.org/10.1098/rstb.2008.0049 West-Eberhard MJ (1989) Phenotypic plasticity and the origins of diversity. Annu Rev Ecol Syst 20:249–278 West-Eberhard MJ (2003) Developmental plasticity and evolution. Oxford University Press, Oxfordl Wund M, Baker J, Clancy B et al (2008) A test of the “flexible stem” model of evolution: ancestral plastic- ity, genetic accommodation, and morphological divergence in the threespine stickleback radiation. Am Nat 172:449–462 Wund MA, Valena S, Wood S, Baker JA (2012) Ancestral plasticity and allometry in threespine stickleback reveal phenotypes associated with derived, freshwater ecotypes. Biol J Linn Soc 105:573–583.
https://openalex.org/W4295213522	https://www.mdpi.com/2223-7747/11/18/2350/pdf?version=1662695442	English	null	Quantitative Proteomics and Functional Characterization Reveal That Glutathione Peroxidases Act as Important Antioxidant Regulators in Mulberry Response to Drought Stress	Plants	2,022	cc-by	14,236	Citation: Zhang, M.; Li, W.; Li, S.; Gao, J.; Gan, T.; Li, Q.; Bao, L.; Jiao, F.; Su, C.; Qian, Y. Quantitative Proteomics and Functional Characterization Reveal That Glutathione Peroxidases Act as Important Antioxidant Regulators in Mulberry Response to Drought Stress. Plants 2022, 11, 2350. https:// doi.org/10.3390/plants11182350 Keywords: mulberry (Morus alba L.); drought stress; quantitative proteomics; antioxidant enzymes; glutathione peroxidase (GPX); reactive oxygen species (ROS) Academic Editors: Candida Vannini and Guido Domingo Minjuan Zhang 1 , Wenqiang Li 2, Shuaijun Li 1,2, Junru Gao 2, Tiantian Gan 1 , Qinying Li 2, Lijun Bao 1 , Feng Jiao 1, Chao Su 1,* and Yonghua Qian 1,* Minjuan Zhang 1 , Wenqiang Li 2, Shuaijun Li 1,2, Junru Gao 2, Tiantian Gan 1 , Qinying Li 2, Lijun Bao 1 , Feng Jiao 1, Chao Su 1,* and Yonghua Qian 1,* 1 The Sericultural and Silk Research Institute, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China 2 State Key Laboratory of Crop Stress Biology in Arid Areas, College of Life Sciences, Northwest A&F University, Yangling 712100, China * Correspondence: suchao503@126.com (C.S.); qyh@nwsuaf.edu.cn (Y.Q.) 1 The Sericultural and Silk Research Institute, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China y g g 2 State Key Laboratory of Crop Stress Biology in Arid Areas, College of Life Sciences, Northwest A&F University, Yangling 712100, China y g g * Correspondence: suchao503@126.com (C.S.); qyh@nwsuaf.edu.cn (Y.Q.) y g g * Correspondence: suchao503@126.com (C.S.); qyh@nwsuaf.edu.cn (Y.Q.) Abstract: Mulberry (Morus alba L.) has been an economically important food crop for the domesticated silkworm, Bombyx mori, in China for more than 5000 years. However, little is known about the mechanism underlying mulberry response to environmental stress. In this study, quantitative proteomics was applied to elucidate the molecular mechanism of drought response in mulberry. A total of 604 differentially expressed proteins (DEPs) were identiﬁed via LC-MS/MS. The proteomic proﬁles associated with antioxidant enzymes, especially ﬁve glutathione peroxidase (GPX) isoforms, as a scavenger of reactive oxygen species (ROS), were systematically increased in the drought-stressed mulberry. This was further conﬁrmed by gene expression and enzymatic activity. Furthermore, overexpression of the GPX isoforms led to enhancements in both antioxidant system and ROS- scavenging capacity, and greater tolerance to drought stress in transgenic plants. Taken together, these results indicated that GPX-based antioxidant enzymes play an important role in modulating mulberry response to drought stress, and higher levels of GPX can improve drought tolerance through enhancing the capacity of the antioxidant system for ROS scavenging. plants plants plants plants plants Quantitative Proteomics and Functional Characterization Reveal That Glutathione Peroxidases Act as Important Antioxidant Regulators in Mulberry Response to D ht St Minjuan Zhang 1 , Wenqiang Li 2, Shuaijun Li 1,2, Junru Gao 2, Tiantian Gan 1 , Qinying Li 2, Lijun Bao 1 , Feng Jiao 1, Chao Su 1,* and Yonghua Qian 1,* 1. Introduction The leaves of mulberry (Morus alba L.) have served as the unique feed for sericulture (silkworms) over thousands of years in China. However, mulberry trees are distributed all over the world. To some extent, this is due to strong environmental adaptability of the trees. In recent years, Chinese scientists of sericulture have proposed to expand the application of mulberry in ecological management and in diversiﬁed product development [1–3]. More and more mulberry trees have been planted in arid and semi-arid areas, especially in the northwestern of China, for ecological restoration [4–6]. Therefore, it is necessary to elucidate the mechanism underlying mulberry’s response and tolerance to drought stress. 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. Drought stress generally affects a vast range of morphological and physiological traits in plants, and reduces photosynthesis, leaf water potential, stem sap ﬂow and stomatal conductance [7]. Physiologically, the common effect of drought stress is that the stress disturbs cellular water balance and cellular redox state, resulting in osmotic and oxidative stresses in plants [8,9]. Abscisic acid (ABA)-mediated signal pathways are believed to be the core of plant responses to drought stress, largely via osmotic adjustment [9–11]. Drought stress can lead to excessive generation and accumulation of reactive oxygen species (ROS) causing oxidative damage to cellular components [12,13]. ROS themselves behave Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/plants Plants 2022, 11, 2350. https://doi.org/10.3390/plants11182350 Plants 2022, 11, 2350 2 of 18 as signaling molecules to trigger extensive biochemical and molecular effects that are essential for plant growth and development [14,15]. As ROS can be continuously produced in plant cells, the balance between the production and removal of ROS will be perturbed under stress conditions, evoking oxidative signaling to activate intrinsic mechanisms for ROS scavenging [16,17]. In plants, ROS scavenging medicated by superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione peroxidase (GPX) constitutes the most important mechanism for plant antioxidant defense against abiotic stresses such as drought [18,19]. 1. Introduction Therefore, activation and enhancement of the antioxidant system for the scavenging of excessive ROS are usually associated with increased tolerance of plants to abiotic stresses [16,17,20]. Plant GPXs are non-haeme thiol peroxidases that catalyze the reduction of H2O2 (or organic hydroperoxides) to water or the respective alcohols using reduced glutathione or thioredoxin [21,22]. GPXs were suggested to be a putative link between the glutathione-based and the thioredoxin-based detoxifying systems [23–25]. They possess some functional overlaps with peroxiredoxin (Prx) and glutathione transferases (GSTs), with respect to the maintenance of H2O2 homeostasis by elimination of peroxides, and are involved in the regulation of redox homeostasis by maintaining the thiol–disulﬁde balance [24,26,27]. There is increasing evidence to suggest that GPXs may play crucial roles in plant protection against both biotic and abiotic stresses [23,25,28]. Over the past few decades, remarkable achievements have been made in investigating the genetic and molecular mechanisms of plant abiotic-stress sensing and signaling [29]. However, due to slow reproduction, difﬁculties in genetic manipulation and larger plant size, etc., the genetic and molecular approaches extensively used in model plants are usu- ally inefﬁcient in investigating woody trees. The mechanism remains largely unknown in most tree species, such as mulberry. Transcriptomic and proteomic approaches could effectively identify target genes, proteins or signaling pathways that regulate plant growth, development and environmental stresses. Based on high-throughput proteomic technolo- gies, a large number of proteins have been identiﬁed to be closely associated with stress responses in trees [30–32]. Isobaric tags for relative and absolute quantitation (iTRAQ), which has high sensitivity, good reproducibility, wide range, high-throughput analysis and other advantages, is a powerful proteomic technology to identify and quantify the levels of relevant sets of proteins [33]. In this study, iTRAQ-based quantitative proteomics was applied to identify differ- entially expressed proteins in the mulberry by experimentally withholding water. Our objective was to determine relevant proteins and/or pathways that are positively correlated with drought response in the mulberry. Combined with biochemical measurements and gene functional characterization, our results revealed that thiol-dependent antioxidant path- ways, especially GPX isoforms, are most responsive to drought stress and overexpression of the GPX genes signiﬁcantly increase the drought tolerance of transgenic plants. These ﬁndings will provide a foundation to screen drought-tolerant germplasm in mulberry. 2.1. Physiological and Biochemical Characterization of the Mulberry under Drought Stress The mulberry plants were drought stressed by withholding water for 11 days. Soil water content in the pots growing drought-stressed plants was reduced about 4.6% after 6 days of withholding water and further reduced to below the detection limit of the instrument after 8 days of withholding water, whereas soil water content in the pots of non-stressed plants was not less than 22%. After 11 days of withholding water, the top leaves (1st to 3rd leaf) in each plant exhibited visible dehydration, the middle leaves (5th to 7th leaf) exhibited severe dehydration, and the bottom leaves were extremely dehydrated, yellowed and even fell (Figure 1A). Compared with ~90% relative water content (RWC) in non-stressed plants, the RWC in drought-stressed plants were about 76.1, 62 and 30.2% in top, middle and bottom leaves, respectively (Figure 1B). Compared with that in non- stressed plants, drought stress caused signiﬁcant reductions in the total chlorophyll content Plants 2022, 11, 2350 3 of 18 30.2% non 3 of 18 30.2% non (Figure 1C) and the soluble sugar content (Figure 1D), respectively. The malondialdehyde and free proline contents were signiﬁcantly increased under drought stress (Figure 1E,F). The content of soluble protein was not signiﬁcantly changed under the drought stress (Figure 1G). tent (Figure 1C) and the soluble sugar content (Figure 1D), respectively. The malondial- dehyde and free proline contents were significantly increased under drought stress (Fig- ure 1E,F). The content of soluble protein was not significantly changed under the drought stress (Figure 1G). Figure 1. Phenotypes and physiological analyses of the mulberry under drought stress. (A) The phenotypes in control (non-stress) and drought-stressed plants. Mulberry plants were subjected to drought stress by withholding water for 11 days, while the control plants were irrigated regularly. Bars = 8 cm. (B) Relative water content (RWC) in top leaves (1st to 3rd leaf), middle leaves (5th to 7th leaf) and bottom leaves (9th to 12th leaf) of control and drought-stressed plants. (C−G) Total chlorophyll content (C), soluble sugar content (D), malondialdehyde content (E), proline content (F) and soluble proteins (G) in top leaves of control and drought-stressed plants. Data are means ± SD with five biological replicates. Asterisks represent statistically significant differences between con- trol and drought-treated plants by Student’s t-test ( p < 0.01, * p < 0.001). Figure 1. Phenotypes and physiological analyses of the mulberry under drought stress. 2.1. Physiological and Biochemical Characterization of the Mulberry under Drought Stress Asterisks represent statistically signiﬁcant differences between control and drought-treated plants by Student’s t-test ( p < 0.01, * p < 0.001). 2.1. Physiological and Biochemical Characterization of the Mulberry under Drought Stress (A) Th phenotypes in control (non-stress) and drought-stressed plants. Mulberry plants were subjected t drought stress by withholding water for 11 days, while the control plants were irrigated regularl Bars = 8 cm. (B) Relative water content (RWC) in top leaves (1st to 3rd leaf), middle leaves (5th t 7th leaf) and bottom leaves (9th to 12th leaf) of control and drought-stressed plants. (C–G) Tota chlorophyll content (C), soluble sugar content (D), malondialdehyde content (E), proline content (F and soluble proteins (G) in top leaves of control and drought-stressed plants. Data are means ± SD with ﬁve biological replicates. Asterisks represent statistically signiﬁcant differences between contro and drought-treated plants by Student’s t-test ( p < 0.01, * p < 0.001). Figure 1. Phenotypes and physiological analyses of the mulberry under drought stress. (A) The Figure 1. Phenotypes and physiological analyses of the mulberry under drought stress. (A) The Figure 1. Phenotypes and physiological analyses of the mulberry under drought stress. (A) The phenotypes in control (non-stress) and drought-stressed plants. Mulberry plants were subjected to drought stress by withholding water for 11 days, while the control plants were irrigated regularly. Bars = 8 cm. (B) Relative water content (RWC) in top leaves (1st to 3rd leaf), middle leaves (5th to 7th leaf) and bottom leaves (9th to 12th leaf) of control and drought-stressed plants. (C−G) Total chlorophyll content (C), soluble sugar content (D), malondialdehyde content (E), proline content (F) and soluble proteins (G) in top leaves of control and drought-stressed plants. Data are means ± SD with five biological replicates. Asterisks represent statistically significant differences between con- trol and drought-treated plants by Student’s t-test ( p < 0.01, * p < 0.001). Figure 1. Phenotypes and physiological analyses of the mulberry under drought stress. (A) The phenotypes in control (non-stress) and drought-stressed plants. Mulberry plants were subjected to drought stress by withholding water for 11 days, while the control plants were irrigated regularly. Bars = 8 cm. (B) Relative water content (RWC) in top leaves (1st to 3rd leaf), middle leaves (5th to 7th leaf) and bottom leaves (9th to 12th leaf) of control and drought-stressed plants. (C–G) Total chlorophyll content (C), soluble sugar content (D), malondialdehyde content (E), proline content (F) and soluble proteins (G) in top leaves of control and drought-stressed plants. Data are means ± SD with ﬁve biological replicates. 2.2. Quantitative Proteomic Analysis of the Mulberry under Drought Stress 2.2. Quantitative Proteomic Analysis of the Mulberry under Drought Stress To investigate the changes of mulberry proteome in response to drought stress, iTRAQ-based quantitative proteomics was used. The proteomes were labeled with iTRAQ reagent and then quantified by LC-MS/MS. The length for most of the peptides was dis- tributed between 7 and 21 amino acids (Figure 2A), which agrees with the property of tryptic peptides and means that sample preparation reaches standard. Mass errors, To investigate the changes of mulberry proteome in response to drought stress, iTRAQ- based quantitative proteomics was used. The proteomes were labeled with iTRAQ reagent and then quantiﬁed by LC-MS/MS. The length for most of the peptides was distributed between 7 and 21 amino acids (Figure 2A), which agrees with the property of tryptic peptides and means that sample preparation reaches standard. Mass errors, meaning the mass accuracy of MS data, were checked for all the identiﬁed peptides. Distribution of mass errors in 0~10 also reached standard and were sufﬁcient for the further analyses (Figure 2B). Principal component analysis (PCA) showed that the datasets from both non- stress and drought-stress samples clustered within their own functional group (Figure 2C). A summary of the MS/MS spectrum analysis, including total spectrums, matched spec- trums, peptides and unique peptides, are shown (Figure 2D). Based on the MS/MS data, Plants 2022, 11, 2350 4 of 18 2495 proteins were identiﬁed (Figure 2D), of which 2058 proteins were quantiﬁed in the drought-stress and non-stress samples of mulberry (Table S1). W 5 2495 proteins were identiﬁed (Figure 2D), of which 2058 proteins were quantiﬁed in the drought-stress and non-stress samples of mulberry (Table S1). 5 Figure 2. iTRAQ-based quantitative proteomics of the mulberry under drought stress. (A−C distribution of peptide length (A), the peptide mass tolerance (B) and principal component sc plot analysis (C) for all peptides. (D) The MS/MS spectrum database search analysis summary Figure 2. iTRAQ-based quantitative proteomics of the mulberry under drought stress. (A–C) The distribution of peptide length (A), the peptide mass tolerance (B) and principal component scatter plot analysis (C) for all peptides. (D) The MS/MS spectrum database search analysis summary. gure 2. iTRAQ-based quantitative proteomics of the mulberry under drought stress. (A−C) istribution of peptide length (A), the peptide mass tolerance (B) and principal component sc ot analysis (C) for all peptides. (D) The MS/MS spectrum database search analysis summary Figure 2. iTRAQ-based quantitative proteomics of the mulberry under drought stress. 2.2. Quantitative Proteomic Analysis of the Mulberry under Drought Stress 2.2. Quantitative Proteomic Analysis of the Mulberry under Drought Stress (A–C) The distribution of peptide length (A), the peptide mass tolerance (B) and principal component scatter plot analysis (C) for all peptides. (D) The MS/MS spectrum database search analysis summary. Statistical analysis revealed that 604 proteins changed in abundance signiﬁcantly (Table S2); these were deﬁned as differential expressed proteins (DEPs). In these DEPs, 278 proteins were up-regulated, and 326 proteins were down-regulated (Table S2). Ac- cording to the results of euKaryotic Ortholog Groups (KOG) of protein comparison, the DEPs were annotated to 22 classiﬁcations, including posttranslational modiﬁcation, protein turnover, chaperones, translation, ribosomal structure, and biogenesis, etc. (Figure 3A). Gene ontology (GO) enrichment analysis revealed that molecular functions of the DEPs were signiﬁcantly enriched in terms of carbohydrate binding, hydrolase activity, disul- ﬁde oxidoreductase activity, polysaccharide binding, polygalacturonase inhibitor activity, antioxidant activity, molecular function regulator, and glutathione disulﬁde oxidoreduc- tase activity (Figure 3B). In cellular component category, the DEPs were signiﬁcantly enriched in terms of anchored component of membrane, anchored component of plasma membrane, and intrinsic component of plasma membrane, etc. (Figure 3B). In biological process category, the DEPs were signiﬁcantly enriched in terms of positive regulation of signal transduction, response to hydrogen peroxide, cellular oxidant detoxiﬁcation, etc. (Figure 3B). 5 of 18 6 of 19 Plants 2022, 11, 2350 Plants 2022, 11, x FOR Figure 3. Functional classification and enrichment analysis of the differentially expressed proteins (DEPs) from iTRAQ-based quantitative proteomics. (A−D) EuKaryotic Ortholog Groups classifica- tion (A), Gene ontology enrichment (B), KEGG pathway enrichment (C) and InterPro protein do- main enrichment (D). Figure 3. Functional classiﬁcation and enrichment analysis of the differentially expressed pro- teins (DEPs) from iTRAQ-based quantitative proteomics. (A–D) EuKaryotic Ortholog Groups classiﬁcation (A), Gene ontology enrichment (B), KEGG pathway enrichment (C) and InterPro protein domain enrichment (D). Figure 3. Functional classification and enrichment analysis of the differentially expressed proteins (DEPs) from iTRAQ-based quantitative proteomics. (A−D) EuKaryotic Ortholog Groups classifica- tion (A), Gene ontology enrichment (B), KEGG pathway enrichment (C) and InterPro protein do- main enrichment (D). Figure 3. Functional classiﬁcation and enrichment analysis of the differentially expressed pro- teins (DEPs) from iTRAQ-based quantitative proteomics. (A–D) EuKaryotic Ortholog Groups classiﬁcation (A), Gene ontology enrichment (B), KEGG pathway enrichment (C) and InterPro protein domain enrichment (D). 2.3. 2.2. Quantitative Proteomic Analysis of the Mulberry under Drought Stress 2.2. Quantitative Proteomic Analysis of the Mulberry under Drought Stress Abundance of Antioxidant Enzymes Especially GPX Isoforms Were Systematically Increased in the Mulberry under Drought Stress Proteomic analysis showed that 43 DEPs are associated with cellular redox and anti- oxidant system, accounting for 7% of the total number of DEPs (Table S3). A great number of enzymatic and non-enzymatic antioxidants were changed in protein abundance under drought stress (Table S3). It is showed that the thiol-dependent antioxidants, including four peroxiredoxins (W9QVC2, W9SEV0, W9QVC2, W9SEV0), five GPXs (W9QHE0, W9QT41, W9QH65, W9RT74 and W9SDB3), five thioredoxins (W9SDD7, W9SW93, W9RMD9, W9R4T1 and W9R4T1), five glutaredoxins (W9S7L1, W9QZP8, W9SBA6, W9SCK7, W9SCK7), two glutathione S-transferases (W9RCX8 and W9S168) and a perox- idase (W9SE23) were all up regulated under drought stress (Table 1) A L ascorbate oxi Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that 9 KEGG pathways, including protein processing in endoplasmic reticulum (ER), glutathione metabolism, galactose metabolism, glycerolipid metabolism, MAPK sig- naling pathway, arachidonic acid metabolism, etc., were signiﬁcantly enriched (Figure 3C). As shown in KEGG map (Figure S1), several key steps in glutathione metabolism pathway were affected by drought stress. It implied that glutathione peroxidases (GPXs: EC 1.11.1.9 and EC 1.11.1.12) were signiﬁcantly up-regulated, while L-ascorbate peroxidase (APX: EC 1.11.1.11) was signiﬁcantly down-regulated (Figure S1). The result of InterPro protein domain enrichment indicated that alcohol dehydrogenase GroES-like domain, glycosyl hhdrolases family 17, Glutaredoxin, zinc-binding dehydrogenase and thaumatin family, etc., were signiﬁcantly enriched (Figure 3D). dase (W9RQI7) and a superoxide dismutase SodC (W9SBU2) were significantly down- regulated in protein abundance (Table 1). The results implied that thiol-dependent anti- 2.3. Abundance of Antioxidant Enzymes Especially GPX Isoforms Were Systematically Increased in the Mulberry under Drought Stress dase (W9RQI7) and a superoxide dismutase SodC (W9SBU2) were significantly down- regulated in protein abundance (Table 1). The results implied that thiol-dependent anti- 2.3. Abundance of Antioxidant Enzymes Especially GPX Isoforms Were Systematically Increased in the Mulberry under Drought Stress oxidants, especially the GPX isoforms, were significantly induced in the mulberry under drought stress. Analysis of the mulberry genome has identified 6 typical GPX isoform genes, the MaGPX1~6 [34]. A phylogenetic tree of GPX isoforms from mulberry (MaGPXs), Arabidop- sis (AtGPXs) and rice (OsGPXs) were shown and indicated the similarities of these GPX members (Figure S2). We further investigated the gene expression of MaGPXs in mulberry Neo-Ichinose under drought stress. Compared with the non-stressed mulberry plants, the drought stress induces about 3~8 folds increases in mRNA expression in MaGPX1, Proteomic analysis showed that 43 DEPs are associated with cellular redox and an- tioxidant system, accounting for 7% of the total number of DEPs (Table S3). A great number of enzymatic and non-enzymatic antioxidants were changed in protein abun- dance under drought stress (Table S3). It is showed that the thiol-dependent antiox- idants, including four peroxiredoxins (W9QVC2, W9SEV0, W9QVC2, W9SEV0), ﬁve GPXs (W9QHE0, W9QT41, W9QH65, W9RT74 and W9SDB3), ﬁve thioredoxins (W9SDD7, W9SW93, W9RMD9, W9R4T1 and W9R4T1), ﬁve glutaredoxins (W9S7L1, W9QZP8, W9SBA6, W9SCK7, W9SCK7), two glutathione S-transferases (W9RCX8 and W9S168) and a peroxi- Plants 2022, 11, 2350 6 of 18 dase (W9SE23), were all up-regulated under drought stress (Table 1). A L-ascorbate oxidase (W9RQI7) and a superoxide dismutase SodC (W9SBU2) were signiﬁcantly down-regulated in protein abundance (Table 1). The results implied that thiol-dependent antioxidants, espe- cially the GPX isoforms, were signiﬁcantly induced in the mulberry under drought stress. dase (W9SE23), were all up-regulated under drought stress (Table 1). A L-ascorbate oxidase (W9RQI7) and a superoxide dismutase SodC (W9SBU2) were signiﬁcantly down-regulated in protein abundance (Table 1). The results implied that thiol-dependent antioxidants, espe- cially the GPX isoforms, were signiﬁcantly induced in the mulberry under drought stress. Table 1. The differentially expressed proteins (DEPs) of antioxidant system enzymes in mulberry under drought stress. dase (W9RQI7) and a superoxide dismutase SodC (W9SBU2) were significantly down- regulated in protein abundance (Table 1). The results implied that thiol-dependent anti- 2.3. Abundance of Antioxidant Enzymes Especially GPX Isoforms Were Systematically Increased in the Mulberry under Drought Stress Protein ID Protein Description Fold Change p Value Regulation MW [kDa] Coverage [%] Unique Peptides a PSMs Subcellular Localization W9S7L1 Glutaredoxin domain-containing protein 2.96 0.000474 Up 13.3890 14.4 2 2 Chloroplast W9SE23 Peroxidase 2.85 0.000120 Up 36.4880 13.6 4 27 Chloroplast W9QHE0 Glutathione peroxidase 2.48 0.000983 Up 18.4420 13.4 1 21 Cytoplasm W9QT41 Glutathione peroxidase 2.27 0.003078 Up 18.9620 38.2 8 20 Mitochondria W9QH65 Glutathione peroxidase 2.04 0.000421 Up 26.5190 18.6 5 45 Chloroplast W9RCX8 Glutathione S-transferase 2.02 0.001778 Up 26.7850 48.3 9 74 Cytoplasm W9RT74 Glutathione peroxidase 1.99 0.000374 Up 20.5060 32.1 5 21 Cytoplasm W9SDB3 Glutathione peroxidase 1.97 0.000211 Up 26.0130 29.2 5 37 Chloroplast W9SDD7 Thioredoxin 1.85 0.000336 Up 13.2220 22.7 3 9 Chloroplast W9QZP8 Glutaredoxin-C5 1.77 0.013193 Up 18.9360 26.1 3 9 Chloroplast W9SBA6 Glutaredoxin domain-containing protein 1.69 0.001711 Up 15.0310 30.8 3 34 Chloroplast W9S168 Glutathione S-transferase 1.68 0.000215 Up 39.9490 30.5 11 69 Chloroplast W9SW93 Thioredoxin-like 3-1 1.67 0.000097 Up 21.2490 11.5 2 3 Cytoplasm W9RMD9 Thioredoxin-like fold containing protein 1.66 0.000454 Up 35.4970 13 5 17 Vacuolar membrane W9SCK7 Monothiol glutaredoxin-S16 1.57 0.001539 Up 32.5700 14.4 3 10 Chloroplast W9QVC2 Peroxiredoxin 1.56 0.004432 Up 22.4790 42.1 7 52 Chloroplast, mitochon- dria W9SEV0 Peroxiredoxin 1.54 0.000664 Up 17.2870 36.4 6 44 Cytoplasm W9R4T1 Thioredoxin O1 1.53 0.002092 Up 28.3650 12.3 4 13 Chloroplast W9SCK7 Monothiol glutaredoxin-S16 1.57 0.001539 Up 32.5700 14.4 3 10 Chloroplast W9QVC2 Peroxiredoxin 1.56 0.004432 Up 22.4790 42.1 7 52 Chloroplast, mitochon- dria W9SEM5 Ferredoxin-thioredoxin reductase, catalytic chain 1.56 0.002258 Up 16.2540 40 6 14 Chloroplast W9SEV0 Peroxiredoxin 1.54 0.000664 Up 17.2870 36.4 6 44 Cytoplasm W9R4T1 Thioredoxin O1 1.53 0.002092 Up 28.3650 12.3 4 13 Chloroplast W9SVF9 Monothiol glutaredoxin-S7 0.64 0.006054 Down 20.0390 9.8 1 1 Chloroplast W9RQI7 L-ascorbate oxidase-like protein 0.64 0.007718 Down 60.3300 4.6 2 3 Chloroplast W9SBU2 SodC protein 0.62 0.005244 Down 29.3360 34.2 6 89 Chloroplast W9RYX2 Thioredoxin reductase 0.53 0.004546 Down 56.9680 8.1 3 4 Chloroplast a peptide spectrum matches. Table 1. The differentially expressed proteins (DEPs) of antioxidant system enzymes in mulberry under drought stress. Analysis of the mulberry genome has identiﬁed 6 typical GPX isoform genes, the MaGPX1~6 [34]. A phylogenetic tree of GPX isoforms from mulberry (MaGPXs), Arabidopsis (AtGPXs) and rice (OsGPXs) were shown and indicated the similarities of these GPX members (Figure S2). We further investigated the gene expression of MaGPXs in mulberry Neo-Ichinose under drought stress. dase (W9RQI7) and a superoxide dismutase SodC (W9SBU2) were significantly down- regulated in protein abundance (Table 1). The results implied that thiol-dependent anti- 2.3. Abundance of Antioxidant Enzymes Especially GPX Isoforms Were Systematically Increased in the Mulberry under Drought Stress Compared with the non-stressed mulberry plants, the drought stress induces about 3~8 folds increases in mRNA expression in MaGPX1, MaGPX2, MaGPX3 and MaGPX5 (Figure 4A). This is consistent with the proteomic data that the protein abundance of GPX isoforms was signiﬁcantly increased in mulberry Neo-Ichinose under drought stress. According to the proteomic result, the proteins abundance of MaGPX1 (W9QH65), MaGPX2 (W9RT74), MaGPX3 (W9QHE0), MaGPX4 (W9SDB3) and MaGPX5 Plants 2022, 11, 2350 7 of 18 7 of 18 (W9QT41) were increased, while MaGPX6 (W9QS90) was not changed under drought stress (Figure 4B). These results implied that drought stress increases the abundance of GPX proteins correlated with increased expression of GPX genes. W 8 of 19 Figure 4. The expression levels of MaGPXs genes and proteins in the mulberry under drought stress. (A) Relative mRNA levels of MaGPX1, MaGPX2, MaGPX3, MaGPX4, MaGPX5, and MaGPX6 in mulberry seedlings under drought stress. The data represents the ratio between drought-treated plants and non-treated plants. qRT-PCR was performed with three biological replicates each with three technical replicates. (B) Fold change in MaGPXs protein levels between drought stress and non-stress. The data are selected from iTRAQ-based quantitative proteomics. Figure 4. The expression levels of MaGPXs genes and proteins in the mulberry under drought stress. (A) Relative mRNA levels of MaGPX1, MaGPX2, MaGPX3, MaGPX4, MaGPX5, and MaGPX6 in mulberry seedlings under drought stress. The data represents the ratio between drought-treated plants and non-treated plants. qRT-PCR was performed with three biological replicates each with three technical replicates. (B) Fold change in MaGPXs protein levels between drought stress and non-stress. The data are selected from iTRAQ-based quantitative proteomics. Figure 4. The expression levels of MaGPXs genes and proteins in the mulberry under drought stress. Figure 4. The expression levels of MaGPXs genes and proteins in the mulberry under drought stress. (A) Relative mRNA levels of MaGPX1, MaGPX2, MaGPX3, MaGPX4, MaGPX5, and MaGPX6 in mulberry seedlings under drought stress. The data represents the ratio between drought-treated plants and non-treated plants. qRT-PCR was performed with three biological replicates each with three technical replicates. (B) Fold change in MaGPXs protein levels between drought stress and non-stress. The data are selected from iTRAQ-based quantitative proteomics. (A) Relative mRNA levels of MaGPX1, MaGPX2, MaGPX3, MaGPX4, MaGPX5, and MaGPX6 in mulberry seedlings under drought stress. The data represents the ratio between drought-treated plants and non-treated plants. dase (W9RQI7) and a superoxide dismutase SodC (W9SBU2) were significantly down- regulated in protein abundance (Table 1). The results implied that thiol-dependent anti- 2.3. Abundance of Antioxidant Enzymes Especially GPX Isoforms Were Systematically Increased in the Mulberry under Drought Stress qRT-PCR was performed with three biological replicates each with three technical replicates. (B) Fold change in MaGPXs protein levels between drought stress and non-stress. The data are selected from iTRAQ-based quantitative proteomics. 2.4. Enzymatic Activity of GPX was Increased in the Mulberry under Drought Stress 2.4. Enzymatic Activity of GPX Was Increased in the Mulberry under Drought Stress We determined the enzymatic activities of antioxidants including SOD, APX, CAT, GPX, peroxidase (POD), glutathione reductase (GR) and glutathione S-transferase (GST) in the mulberry. The enzymatic activities of SOD, CAT and peroxidase (POD) were not significantly changed after drought stress (Figure 5A–C). The enzymatic activity of APX was decreased under drought stress (Figure 5D), implying that the ascorbate–glutathione (AsA-GSH) cycle was depressed under drought stress. The enzymatic activity of GPX was significantly increased (Figure 5E), and the content of GSH was also increased (Figure 5F), indicating the activation of the GPX cycle in the mulberry under drought stress. The en- zymatic activity of glutathione reductase (GR) was not changed (Figure 5G), but the ac- tivity of glutathione S-transferase (GST) was significantly decreased under drought stress (Figure 5H). Moreover, the H2O2 content was not significantly changed after drought stress (Figure 5I). These results demonstrated that drought stress may enhance the GPX- based antioxidant system in the mulberry. We determined the enzymatic activities of antioxidants including SOD, APX, CAT, GPX, peroxidase (POD), glutathione reductase (GR) and glutathione S-transferase (GST) in the mulberry. The enzymatic activities of SOD, CAT and peroxidase (POD) were not signiﬁcantly changed after drought stress (Figure 5A–C). The enzymatic activity of APX was decreased under drought stress (Figure 5D), implying that the ascorbate–glutathione (AsA-GSH) cycle was depressed under drought stress. The enzymatic activity of GPX was signiﬁcantly increased (Figure 5E), and the content of GSH was also increased (Figure 5F), indicating the activation of the GPX cycle in the mulberry under drought stress. The enzymatic activity of glutathione reductase (GR) was not changed (Figure 5G), but the activity of glutathione S-transferase (GST) was signiﬁcantly decreased under drought stress (Figure 5H). Moreover, the H2O2 content was not signiﬁcantly changed after drought stress (Figure 5I). These results demonstrated that drought stress may enhance the GPX-based antioxidant system in the mulberry. 2.5. Ectopic Overexpression of Mulberry GPX Isoforms Confered Drought Resistance in Transgenic Arabidopsis 2.5. Ectopic Overexpression of Mulberry GPX Isoforms Confered Drought Resistance in Transgenic Arabidopsis To further investigate the mulberry GPX isoforms, we produced transgenic Arabidopsis by overexpressing the six GPX genes, MaGPX1 to MaGPX6, respectively. A schematic diagram of the MaGPXs expression vectors are shown in Figure 6A. To obtain ectopic over- expression lines, hygromycin-resistant transgenic plants were identiﬁed by GUS staining (Figure S3A). Overexpressions of the MaGPX genes in transgenic plants were conﬁrmed by qRT-PCR (Figure S3B). Before drought stress, no phenotypic difference was observed between the control (containing empty vector) and the overexpression transgenic plants (Figure 6B, left). After 14 days of withholding water, the control, OE-MaGPX1, OE-MaGPX2, OE-MaGPX4 and OE-MaGPX6 transgenic lines were absolutely wilting and dried, but the OE-MaGPX3 and OE-MaGPX5 transgenic lines were still alive (Figure 6B, middle). After re-watering for 5 days, the OE-MaGPX3 and OE-MaGPX5 plants recovered to normal growth, but the other transgenic lines and the control were not recovered from drought stress (Figure 6B, right). The result revealed that the overexpression of mulberry GPX isoforms MaGPX3 and MaGPX5 resulted in an increased tolerance of transgenic plants to drought stress. Plants 2022, 11, 2350 8 of 18 ss may Figure 5. Activities of antioxidant enzymes and the H2O2 content in control (non-stress) and drought- stressed plants. (A–I) Enzymatic activities of SOD (A), CAT (B), POD (C), APX (D), GPX (E), GR (G), GST (H) and GSH content (F), and the H2O2 content (I) in top leaves of control and drought-stressed mulberry plants. Data are means ± SD with ﬁve biological replicates. Asterisks represent statistically signiﬁcant differences between control and drought-stressed plants by Student’s t-test (* p < 0.05, p < 0.01, * p < 0.001). Figure 5. Activities of antioxidant enzymes and the H2O2 content in control (non-stress) and drought p ( ) y ( ), ( ), ( ), ( ), ( ), ( ), GST (H) and GSH content (F), and the H2O2 content (I) in top leaves of control and drought-stressed mulberry plants. Data are means ± SD with ﬁve biological replicates. Asterisks represent statistically signiﬁcant differences between control and drought-stressed plants by Student’s t-test (* p < 0.05, p < 0.01, * p < 0.001). We further evaluated drought tolerance of OE-MaGPX3 and OE-MaGPX5 transgenic lines at the ﬂowering stage (Figure 6C, left). 2.5. Ectopic Overexpression of Mulberry GPX Isoforms Confered Drought Resistance in Transgenic Arabidopsis After 12 days of withholding water, the control displayed a severe wilting and dry phenotype, but the OE-MaGPX3 and OE-MaGPX5 lines showed a relatively mild phenotype (Figure 6C, middle). A week after re-watering, all the OE-MaGPX3 and OE-MaGPX5 transgenic plants were recovered, but all the controls were not able to recover (Figure 6C, right). These data revealed that the ectopic overexpression of MaGPX3 and MaGPX5 leads to the greater resistance of transgenic plants to drought stress. We measured relative water content (RWC) and rate of water loss (RWL) in the control, OE-MaGPX3 and OE-MaGPX5 transgenic lines. It was showed that OE-MaGPX3 and OE-MaGPX5 transgenic plants had signiﬁcantly higher RWC (Figure 6D) and lower RWL (Figure 6E) as compared with that of control under non-stress condition. The drought resistance of the transgenic lines was also conﬁrmed by a mannitol-induced drought-stress experiment (Figure S4). The mannitol treatment caused a signiﬁcant decrease in seedling length in the controls, but no decrease in the OE-MaGPX3 and OE-MaGPX5 transgenic lines (Figure S4). We further evaluated drought tolerance of OE-MaGPX3 and OE-MaGPX5 transgenic lines at the ﬂowering stage (Figure 6C, left). After 12 days of withholding water, the control displayed a severe wilting and dry phenotype, but the OE-MaGPX3 and OE-MaGPX5 lines showed a relatively mild phenotype (Figure 6C, middle). A week after re-watering, all the OE-MaGPX3 and OE-MaGPX5 transgenic plants were recovered, but all the controls were not able to recover (Figure 6C, right). These data revealed that the ectopic overexpression of MaGPX3 and MaGPX5 leads to the greater resistance of transgenic plants to drought stress. We measured relative water content (RWC) and rate of water loss (RWL) in the control, OE-MaGPX3 and OE-MaGPX5 transgenic lines. It was showed that OE-MaGPX3 and OE-MaGPX5 transgenic plants had signiﬁcantly higher RWC (Figure 6D) and lower RWL (Figure 6E) as compared with that of control under non-stress condition. The drought resistance of the transgenic lines was also conﬁrmed by a mannitol-induced drought-stress experiment (Figure S4). The mannitol treatment caused a signiﬁcant decrease in seedling length in the controls, but no decrease in the OE-MaGPX3 and OE-MaGPX5 transgenic lines (Figure S4). We further evaluated drought tolerance of OE-MaGPX3 and OE-MaGPX5 transgenic lines at the ﬂowering stage (Figure 6C, left). After 12 days of withholding water, the control displayed a severe wilting and dry phenotype, but the OE-MaGPX3 and OE-MaGPX5 lines showed a relatively mild phenotype (Figure 6C, middle). 2.5. Ectopic Overexpression of Mulberry GPX Isoforms Confered Drought Resistance in Transgenic Arabidopsis A week after re-watering, all the OE-MaGPX3 and OE-MaGPX5 transgenic plants were recovered, but all the controls were not able to recover (Figure 6C, right). These data revealed that the ectopic overexpression of MaGPX3 and MaGPX5 leads to the greater resistance of transgenic plants to drought stress. We further evaluated drought tolerance of OE-MaGPX3 and OE-MaGPX5 transgenic lines at the ﬂowering stage (Figure 6C, left). After 12 days of withholding water, the control displayed a severe wilting and dry phenotype, but the OE-MaGPX3 and OE-MaGPX5 lines showed a relatively mild phenotype (Figure 6C, middle). A week after re-watering, all the OE-MaGPX3 and OE-MaGPX5 transgenic plants were recovered, but all the controls were not able to recover (Figure 6C, right). These data revealed that the ectopic overexpression of MaGPX3 and MaGPX5 leads to the greater resistance of transgenic plants to drought stress. We measured relative water content (RWC) and rate of water loss (RWL) in the control, OE-MaGPX3 and OE-MaGPX5 transgenic lines. It was showed that OE-MaGPX3 and OE-MaGPX5 transgenic plants had signiﬁcantly higher RWC (Figure 6D) and lower RWL (Figure 6E) as compared with that of control under non-stress condition. The drought resistance of the transgenic lines was also conﬁrmed by a mannitol-induced drought-stress experiment (Figure S4). The mannitol treatment caused a signiﬁcant decrease in seedling length in the controls, but no decrease in the OE-MaGPX3 and OE-MaGPX5 transgenic lines (Figure S4). We measured relative water content (RWC) and rate of water loss (RWL) in the control, OE-MaGPX3 and OE-MaGPX5 transgenic lines. It was showed that OE-MaGPX3 and OE-MaGPX5 transgenic plants had signiﬁcantly higher RWC (Figure 6D) and lower RWL (Figure 6E) as compared with that of control under non-stress condition. The drought resistance of the transgenic lines was also conﬁrmed by a mannitol-induced drought-stress experiment (Figure S4). The mannitol treatment caused a signiﬁcant decrease in seedling length in the controls, but no decrease in the OE-MaGPX3 and OE-MaGPX5 transgenic lines (Figure S4). Plants 2022, 11, 2350 9 of 18 rexpres- olerance Figure 6. Overexpression of mulberry MaGPX3 and MaGPX5 enhances drought tolerance in trans- genic Arabidopsis. (A) Drought tolerance of transgenic Arabidopsis overexpressing six mulberry Figure 6. Overexpression of mulberry MaGPX3 and MaGPX5 enhances drought tolerance in transgenic Arabidopsis. (A) Drought tolerance of transgenic Arabidopsis overexpressing six mul- berry GPX genes, MaGPX1, MaGPX2, MaGPX3, MaGPX4, MaGPX5 and MaGPX6 at the seedling stage. 2.5. Ectopic Overexpression of Mulberry GPX Isoforms Confered Drought Resistance in Transgenic Arabidopsis Overexpression of mulberry MaGPX3 and MaGPX5 enhances drought tolerance in transgenic Arabidopsis. (A) Drought tolerance of transgenic Arabidopsis overexpressing six mul- berry GPX genes, MaGPX1, MaGPX2, MaGPX3, MaGPX4, MaGPX5 and MaGPX6 at the seedling stage. Phenotypes of control and the transgenic lines (OE-MaGPXs) before drought (3-week-old plants), after withholding water for 14 days, and after recovery for 5 days are shown, respectively. Bar = 4 cm. (B) Drought tolerance of the control, OE-MaGPX3 and OE-MaGPX5 transgenic lines at the ﬂowering stage. The phenotypes of control and transgenic lines before drought (40-day-old plants), after withholding water for 12 days, and after recovery for 7 days are shown, respectively. Three independent T3 lines were used in each case. Bar = 4 cm. (C) Relative water content (RWC) of leaves of the control, OE-MaGPX3 and OE-MaGPX5 transgenic plants in non-stress condition. (D) Rate of water loss (RWL) of leaves of the control, OE-MaGPX3 and OE-MaGPX5 transgenic plants in non-stress condition. Data are means ± SD with six biological replicates. Asterisks rep- resent statistically signiﬁcant differences between control and transgenic plants by Student’s t-test (* p < 0.05, ** p < 0.01). 2.5. Ectopic Overexpression of Mulberry GPX Isoforms Confered Drought Resistance in Transgenic Arabidopsis Phenotypes of control and the transgenic lines (OE-MaGPXs) before drought (3-week-old plants), after withholding water for 14 days, and after recovery for 5 days are shown, respectively. Bar = 4 cm. (B) Drought tolerance of the control, OE-MaGPX3 and OE-MaGPX5 transgenic lines at the ﬂowering stage. The phenotypes of control and transgenic lines before drought (40-day-old plants), after withholding water for 12 days, and after recovery for 7 days are shown, respectively. Three independent T3 lines were used in each case. Bar = 4 cm. (C) Relative water content (RWC) of leaves of the control, OE-MaGPX3 and OE-MaGPX5 transgenic plants in non-stress condition. (D) Rate of water loss (RWL) of leaves of the control, OE-MaGPX3 and OE-MaGPX5 transgenic plants in non-stress condition. Data are means ± SD with six biological replicates. Asterisks rep- resent statistically signiﬁcant differences between control and transgenic plants by Student’s t-test (* p < 0.05, ** p < 0.01). Figure 6. Overexpression of mulberry MaGPX3 and MaGPX5 enhances drought tolerance in trans- genic Arabidopsis. (A) Drought tolerance of transgenic Arabidopsis overexpressing six mulberry Figure 6. Overexpression of mulberry MaGPX3 and MaGPX5 enhances drought tolerance in transgenic Arabidopsis. (A) Drought tolerance of transgenic Arabidopsis overexpressing six mul- berry GPX genes, MaGPX1, MaGPX2, MaGPX3, MaGPX4, MaGPX5 and MaGPX6 at the seedling stage. Phenotypes of control and the transgenic lines (OE-MaGPXs) before drought (3-week-old plants), after withholding water for 14 days, and after recovery for 5 days are shown, respectively. Bar = 4 cm. (B) Drought tolerance of the control, OE-MaGPX3 and OE-MaGPX5 transgenic lines at the ﬂowering stage. The phenotypes of control and transgenic lines before drought (40-day-old plants), after withholding water for 12 days, and after recovery for 7 days are shown, respectively. Three independent T3 lines were used in each case. Bar = 4 cm. (C) Relative water content (RWC) of leaves of the control, OE-MaGPX3 and OE-MaGPX5 transgenic plants in non-stress condition. (D) Rate of water loss (RWL) of leaves of the control, OE-MaGPX3 and OE-MaGPX5 transgenic plants in non-stress condition. Data are means ± SD with six biological replicates. Asterisks rep- resent statistically signiﬁcant differences between control and transgenic plants by Student’s t-test (* p < 0.05, ** p < 0.01). Figure 6. Overexpression of mulberry MaGPX3 and MaGPX5 enhances drought tolerance in trans- genic Arabidopsis. (A) Drought tolerance of transgenic Arabidopsis overexpressing six mulberry Figure 6. 2.6. Ectopic Overexpression of Mulberry GPX Isoforms Improve Both Antioxidant Enzymatic Activities and ROS Scavenging in Transgenic Arabidopsis under Drought Stress 2.6. Ectopic Overexpression of Mulberry GPX Isoforms Improve Both Antioxidant Enzymatic Activities and ROS Scavenging in Transgenic Arabidopsis under Drought Stress To investigate why overexpression of MaGPX3 and MaGPX5 increases the tolerance of transgenic plants to drought stress, we analyzed the activities of the antioxidant system and ROS accumulation in transgenic plants. Under both non-stress and drought-stress conditions, GPX enzymatic activities were signiﬁcantly higher in the OE-MaGPX3 and OE-MaGPX5 transgenic lines than in the control (Figure 7A). Compared with that of non- stressed plants, GPX activities showed 311% and 300% increases in OE-MaGPX3 and OE-MaGPX5 transgenic lines, but a 109% increase in the control under drought stress (Figure 7A). These results revealed that overexpression of MaGPX3 and MaGPX5 may Plants 2022, 11, 2350 10 of 18 10 of 18 strongly increase enzymatic activity of GPX in transgenic plants under non-stress and drought-stress conditions. EW 11 of 19 Figure 7. Antioxidant enzyme activities and ROS levels in the control, OE-MaGPX3 and OE- MaGPX5 transgenic Arabidopsis plants. (A–G) Enzymatic activities of GPX (A), APX (B), SOD (C), CAT (D), POD (E), GR (F) and GST (G) in leaves of control, OE-MaGPX3 and OE-MaGPX5 trans- genic lines. (H) Content of hydrogen peroxide (H2O2) in leaves of control, OE-MaGPX3 and OE- MaGPX5 transgenic lines. For sampling, the plants were subjected to drought stress by withholding water for 6 days, while the non-stressed plants were grown in normal conditions. Data are means ± SD with three biological replicates (three independent transgenic lines) and each with three tech- nical replicates. Letters indicate significant differences between means, determined using Duncan’s multiple range test (5% α). (I) Histochemical staining of hydrogen peroxide (H2O2) in drought- stressed and non-stressed plants by 3,3-diaminobenzidine (DAB) assay. (J) Histochemical staining of superoxide anion (O2−) in drought-stressed and non-stressed plants by nitrobluetetrazolium (NBT) assay. Three independent T3 transgenic lines were used in each case. The presence of H2O2 and O2− were assessed by appearance of brown color and dark blue color after staining with DAB and NBT, respectively. Bars = 1 cm (I,J). Figure 7. Antioxidant enzyme activities and ROS levels in the control, OE-MaGPX3 and OE-MaGPX5 transgenic Arabidopsis plants. (A–G) Enzymatic activities of GPX (A), APX (B), SOD (C), CAT (D), POD (E), GR (F) and GST (G) in leaves of control, OE-MaGPX3 and OE-MaGPX5 transgenic lines. (H) Content of hydrogen peroxide (H2O2) in leaves of control, OE-MaGPX3 and OE-MaGPX5 transgenic lines. 2.6. Ectopic Overexpression of Mulberry GPX Isoforms Improve Both Antioxidant Enzymatic Activities and ROS Scavenging in Transgenic Arabidopsis under Drought Stress For sampling, the plants were subjected to drought stress by withholding water for 6 days, while the non-stressed plants were grown in normal conditions. Data are means ± SD with three biological replicates (three independent transgenic lines) and each with three technical replicates. Letters indicate signiﬁcant differences between means, determined using Duncan’s multiple range test (5% α). (I) Histochemical staining of hydrogen peroxide (H2O2) in drought-stressed and non-stressed plants by 3,3-diaminobenzidine (DAB) assay. (J) Histochemical staining of superoxide anion (O2−) in drought-stressed and non-stressed plants by nitrobluetetrazolium (NBT) assay. Three independent T3 transgenic lines were used in each case. The presence of H2O2 and O2−were assessed by appearance of brown color and dark blue color after staining with DAB and NBT, respectively. Bars = 1 cm (I,J). Figure 7. Antioxidant enzyme activities and ROS levels in the control, OE-MaGPX3 and OE- Figure 7. Antioxidant enzyme activities and ROS levels in the control, OE-MaGPX3 and OE-MaGPX5 Figure 7. Antioxidant enzyme activities and ROS levels in the control, OE-MaGPX3 and OE- MaGPX5 transgenic Arabidopsis plants. (A–G) Enzymatic activities of GPX (A), APX (B), SOD (C), CAT (D), POD (E), GR (F) and GST (G) in leaves of control, OE-MaGPX3 and OE-MaGPX5 trans- genic lines. (H) Content of hydrogen peroxide (H2O2) in leaves of control, OE-MaGPX3 and OE- MaGPX5 transgenic lines. For sampling, the plants were subjected to drought stress by withholding water for 6 days, while the non-stressed plants were grown in normal conditions. Data are means ± SD with three biological replicates (three independent transgenic lines) and each with three tech- nical replicates. Letters indicate significant differences between means, determined using Duncan’s multiple range test (5% α). (I) Histochemical staining of hydrogen peroxide (H2O2) in drought- stressed and non-stressed plants by 3,3-diaminobenzidine (DAB) assay. (J) Histochemical staining of superoxide anion (O2−) in drought-stressed and non-stressed plants by nitrobluetetrazolium (NBT) assay. Three independent T3 transgenic lines were used in each case. The presence of H2O2 and O2− were assessed by appearance of brown color and dark blue color after staining with DAB and NBT, respectively. Bars = 1 cm (I,J). Figure 7. Antioxidant enzyme activities and ROS levels in the control, OE-MaGPX3 and OE-MaGPX5 transgenic Arabidopsis plants. (A–G) Enzymatic activities of GPX (A), APX (B), SOD (C), CAT (D), POD (E), GR (F) and GST (G) in leaves of control, OE-MaGPX3 and OE-MaGPX5 transgenic lines. 2.6. Ectopic Overexpression of Mulberry GPX Isoforms Improve Both Antioxidant Enzymatic Activities and ROS Scavenging in Transgenic Arabidopsis under Drought Stress (H) Content of hydrogen peroxide (H2O2) in leaves of control, OE-MaGPX3 and OE-MaGPX5 transgenic lines. For sampling, the plants were subjected to drought stress by withholding water for 6 days, while the non-stressed plants were grown in normal conditions. Data are means ± SD with three biological replicates (three independent transgenic lines) and each with three technical replicates. Letters indicate signiﬁcant differences between means, determined using Duncan’s multiple range test (5% α). (I) Histochemical staining of hydrogen peroxide (H2O2) in drought-stressed and non-stressed plants by 3,3-diaminobenzidine (DAB) assay. (J) Histochemical staining of superoxide anion (O2−) in drought-stressed and non-stressed plants by nitrobluetetrazolium (NBT) assay. Three independent T3 transgenic lines were used in each case. The presence of H2O2 and O2−were assessed by appearance of brown color and dark blue color after staining with DAB and NBT, respectively. Bars = 1 cm (I,J). Enzymatic activity of APX was significantly higher in OE-MaGPX3 and OE-MaGPX5 than in the control under both non-stress and drought-stress conditions (Figure 7B). Un- der non-stress conditions, the enzymatic activities of SOD, CAT, POD, GR and GST were not significantly higher in the OE-MaGPX3 or OE-MaGPX5 transgenic lines than in the control; however, under drought-stress condition, the enzymatic activities of SOD, CAT, POD, GR and GST were significantly higher in the OE-MaGPX3 and OE-MaGPX5 trans- genic lines than in the control (Figure 7C–G). These results revealed that overexpression of MaGPX3 and MaGPX5 not only increases the enzymatic activity of GPX, but also im- proves the activities of other antioxidant system enzymes in transgenic plants exposed to Enzymatic activity of APX was signiﬁcantly higher in OE-MaGPX3 and OE-MaGPX5 than in the control under both non-stress and drought-stress conditions (Figure 7B). Under non-stress conditions, the enzymatic activities of SOD, CAT, POD, GR and GST were not signiﬁcantly higher in the OE-MaGPX3 or OE-MaGPX5 transgenic lines than in the control; however, under drought-stress condition, the enzymatic activities of SOD, CAT, POD, GR and GST were signiﬁcantly higher in the OE-MaGPX3 and OE-MaGPX5 transgenic lines than in the control (Figure 7C–G). These results revealed that overexpression of MaGPX3 and MaGPX5 not only increases the enzymatic activity of GPX, but also improves the activ- ities of other antioxidant system enzymes in transgenic plants exposed to drought stress. Plants 2022, 11, 2350 11 of 18 11 of 18 ROS accumulation was then investigated in the control, OE-MaGPX3 and OE-MaGPX5 transgenic plants. 2.6. Ectopic Overexpression of Mulberry GPX Isoforms Improve Both Antioxidant Enzymatic Activities and ROS Scavenging in Transgenic Arabidopsis under Drought Stress The content of hydrogen peroxide (H2O2) was signiﬁcantly lower in OE- MaGPX3 and OE-MaGPX5 transgenic lines than in the control under both non-stress and drought-stress conditions (Figure 7H). It was showed that drought stress caused a larger increase in hydrogen peroxide content in the control, but a smaller increase in hydrogen peroxide content in the overexpression lines (Figure 7H). Although drought stress caused signiﬁcant increases in hydrogen peroxide content in the control (105% increases), OE- MaGPX3 (69% increase) and OE-MaGPX5 (50% increase), the hydrogen peroxide levels in drought-treated overexpression lines were as low as that in the non-stressed control plants (Figure 7H), indicating enhanced ROS (hydrogen peroxide) scavenging by overexpressing MaGPX3 and MaGPX5. DAB and NBT histochemical staining experiments were further performed to investi- gate ROS (hydrogen peroxide and superoxide) accumulation. Under non-stress conditions, no visible difference in the DAB and NBT staining of leaves was observed between the overexpression transgenic plants and the control plants (Figure 7I,J), indicating that the levels of hydrogen peroxide and superoxide were not different between the overexpression plants and control plants. Under drought-stress conditions, however, the overexpression lines were signiﬁcantly less stained by DAB and NBT as compared with that of the control (Figure 7I,J), indicating that overexpression of MaGPX3 and MaGPX5 led to less accumula- tion of hydrogen peroxide and superoxide in transgenic plants under drought stress. The results revealed that the overexpression of MaGPX3 and MaGPX5 could increase antioxi- dant enzymatic activities and lead to less ROS accumulation in drought-stressed plants. 3. Discussion In this study, drought stress led to a signiﬁcant decrease in APX activity as well as re- duced APX protein abundance in the mulberry. This may imply that APX-mediated ROS scavenging was depressed in the mulberry under drought stress. It also suggests that the APX-mediated ROS scavenging pathway plays a less important role in the mulberry-plant defense against drought stress. g g The drought stress resulted in signiﬁcant increases in GPX gene expression, GPX isoenzymes abundance as well as GPX enzymatic activity in the mulberry under drought stress. This is consistent with some previous studies that GPX was up-regulated in response to environmental stresses [22,43,44]. Given that the mulberry genome encodes 6 GPX mem- bers, the results indicated that mulberry GPX isoenzymes, rather than other antioxidant system enzymes (ie. APX), were extensively induced in mulberry under drought stress. We suggest that GPX could play a prominent role in regulating antioxidant defense against drought stress in the mulberry. Some investigations suggest that the subcellular compartmentalization of antioxidant system enzymes (i.e., GPX isoenzymes) is very important for plant cells to modulate ROS concentration and protect plants from oxidative stress [23,25,40,42]. Our previous study indicated that mulberry GPX members may be present in different subcellular compartments [34]. In this study, ﬁve mulberry GPX isoforms were increased in mRNA expression, protein abundance and enzymatic activity, suggesting that mulberry GPX isoforms were uniformly enhanced in different subcellular compartments to maintain ROS level during drought stress. This consideration is partly supported by the result that drought stress only induced a slight increase in H2O2 level (no signiﬁcance) in the mulberry Neo-Ichinose (Figure 5I). In this respect, it seems that the mulberry variety Neo-Ichinose, as a drought-tolerant variety, possess an efﬁcient antioxidant system that may alleviate drought-induced ROS overproduction in some degree. As a major family of the thiol-based ROS scavenging enzymes, GPX isoforms were initially described as catalyzing the reduction of hydrogen peroxide and lipid peroxides using reduced GSH as electron donor [45,46]. The overexpression of MaGPX3 and MaGPX5 led to less accumulation of ROS in drought-stressed transgenic plants, indicating that the GPX isoforms play a positive role in detoxifying ROS. There is increasing evidence that GPX isoforms are not only regulated by various abiotic stresses, but also involved in modulating plant tolerance to environmental stresses [24,27,47,48]. 3. Discussion For most plants, drought stress can affect a vast range of morphological and physio- logical traits, such as reductions in photosynthesis, leaf water potential, stem sap ﬂow and stomatal conductance [7]. In this study, compared with non-stressed plants, leaf RWC, total chlorophyll content, and soluble sugar content were decreased, but malondialdehyde and proline contents were increased in the stressed mulberry plants, indicating that drought stress reduces water transport, photosynthesis and carbohydrate metabolism, and causes cellular dehydration leading to osmotic and oxidative stress in mulberry. This is consistent with previous studies in other tree species, i.e., Maclura pomifera [35], Prunus sargentii and Larix kaempferi [36]. pf In this study, iTRAQ-based quantitative proteomics was used to investigate the molec- ular mechanism of mulberry’s response to drought stress. We identiﬁed 43 DEPs associated with cellular redox, including many antioxidant system enzymes that are responsible for ROS scavenging or ROS-scavenger recovering (Table S3). The abundance of antioxidant system enzymes such as SOD, GPX, Trx, Grx, GST and APX were extensively changed in the mulberry under drought stress (Table 1). Since most of these antioxidant proteins were upregulated by drought stress, the results suggest that drought stress activates the antioxidant system in the mulberry. y y Under abiotic stress, especially environmental stress (i.e., drought), a plant pro- duces more ROS than it needs [18]; however, the plant also produces more antioxidants, ﬂavonoids, and secondary metabolites which play the role in protecting the plant for detox- ifying ROS and maintaining protein and amino-acid stabilization. It is generally accepted that ROS scavenging or detoxifying medicated by antioxidant system such as SOD, CAT, APX and GPX constitutes the most important mechanism of plant defense against abiotic stresses [17–19]. As important ROS scavengers, SOD converts superoxide into hydrogen peroxide (H2O2); APX, GPX, and CAT may detoxify H2O2 to H2O [37]. APX catalyzes the conversion of H2O2 into H2O by the oxidation of ascorbate to monodehydroascorbate (MDA); however, GPX detoxiﬁes H2O2 into H2O by oxidation of glutathione (GSH) to oxidized GSH [16]. In some cases, it is considered that plant GPX isoforms use thioredoxin (Trx) rather than GSH in the reduction of H2O2 and lipid hydroperoxides [23,26]. In fact, the levels of antioxidant system enzymes, i.e., APX and GPX could be differentially regulated Plants 2022, 11, 2350 12 of 18 12 of 18 in plant responses to environmental stresses and/or during normal growth [20,27,38–42]. 3. Discussion Some previous studies suggest that transgenic plants with additional copies of GPX have higher resistance to stresses; conversely, the plants with the knockout of individual GPX genes are less tolerant to the stress [42]. For example, the overexpression of Synechocystis GPX in Arabidopsis can reinforce the tolerance of transgenic plants to oxidative, chilling, salinity and drought stresses [49]. Overexpression of Rhodiola crenulata GPX5 affects the regulation of multiple biochemical pathways and increases the drought tolerance in Salvia miltiorrhiza [50]. These investigations are consistent with our result that the overexpression of mulberry MaGPX3 and MaGPX5 increase drought tolerance in plants. This suggests that MaGPX3 and MaGPX5 may play a positive role in modulating mulberry’s tolerance to drought stress. Taken together, we considered that a higher abundance of GPX isoforms is associated with greater antioxidant abilities and higher tolerance to drought stress. Levels of GPX gene expression and enzymatic activity may be used as an index to evaluate drought-tolerant germplasm in mulberry. 4.2. Drought Treatments Mulberry plants were treated by withholding water for 11 days, while non-stressed plants were still watered with 1000 mL of water for each pot every two days. To evaluate the drought degree, soil volumetric water content was measured using a soil moisture content analyzer (TZS-IIW, Hangzhou TOP Instrument Co., Ltd., Hangzhou, China) every two days. After withholding of water for 11 days, the bottom leaves of plant were extremely dehydrated and even fell. For proteomic, physiological and antioxidant enzymatic mea- surements, the second and third leaves from top of plant were collected and immediately frozen in liquid nitrogen and stored at −80 ◦C until use. Biochemical measurements were performed with six biological replicates, and one pot was sampled as one replicate. To evaluate drought tolerance of transgenic Arabidopsis plants with overexpression of mul- berry GPX genes, 3-week-old Arabidopsis plants (T3 generation) were subjected to drought stress by withholding water for 14 days, and then followed by re-watering for 5 days. To further conﬁrm drought tolerance, 40-day-old Arabidopsis plants were subjected to drought stress by withholding water for 12 days and then followed by recovery for 7 days. For measurement of antioxidant system enzymes, the seedlings of Arabidopsis plants were subjected to drought stress by withholding water for 6 days when the control plants were lightly wilting. Leaves from non-treated and drought-treated plants were sampled and immediately frozen in liquid nitrogen and stored at −80 ◦C until use. Mannitol-induced drought-stress experiment was applied by germinating of seeds on 1/2 MS agar medium supplemented with 0 or 200 mmol L−1 mannitol. After 10 days of culture, the lengths of seedlings were measured. 4.1. Plant Materials and Growth Conditions 4.1. Plant Materials and Growth Conditions The Neo-Ichinose, a drought- and salt-tolerant mulberry variety (Morus alba L.), origi- nally introduced from Japan, was obtained from the mulberry germplasm resources pool in the Sericultural and Silk Research Institute of Northwest A&F University (Yangling, Shaanxi, China). One-year-old grafted seedlings of mulberry were transplanted into plastic pots (45 cm diameter and 30 cm height) for 2 weeks. One plant was planted in a pot with Plants 2022, 11, 2350 13 of 18 15 kg of soil. All plants were watered with 1000 mL of water every two days prior to the initiation of drought stress. The plants were grown in a greenhouse with a 14-hour-light (34 ◦C)/10-hour-dark (28 ◦C) cycle and 40–50% relative humidity. 15 kg of soil. All plants were watered with 1000 mL of water every two days prior to the initiation of drought stress. The plants were grown in a greenhouse with a 14-hour-light (34 ◦C)/10-hour-dark (28 ◦C) cycle and 40–50% relative humidity. Seeds of overexpression transgenic Arabidopsis thaliana lines and control (Columbia-0 background) were germinated on 1/2 MS agar medium for 5 days. After germination, seedlings with equal size were selected and transferred into square pots with seedling medium (1:1 mixture of peat and vermiculite). The seedlings were incubated with 16 h light/8 h dark cycles at 22 ◦C in a growth chamber. 4.3. Biochemical Measurements W2, after wiping off water following a 4 h water saturation of excised leaves at room temperature; and W0, after subsequently drying leaves for 12 h at 70 ◦C. RWC (%) = (W1 −W0)/(W2 −W0) × 100%. To determine RWL, detached leaves were initially soaked in water for 1 h, after which excess water was removed and, thereafter, leaves were weighed every 30 min. For antioxidant enzymatic activities, the leaves were ground in liquid nitrogen and assayed for activities of antioxidant enzymes including GPX, APX, SOD, CAT, POD, GR, GST and content of GSH by commercially available antioxidant enzyme kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to manufacturer’s instructions. The detailed experimental procedures for antioxidant enzyme activities are available in Methods S1. 4.3. Biochemical Measurements Contents of malondialdehyde, free proline, soluble sugar, soluble proteins and total chlorophyll were determined as described previously [51,52]. Relative water content (RWC) and rate of water loss (RWL) were measured as described by previous protocols [53]. To determine RWC, three leaf weights were taken: W1, immediately after leaf excision; W2, after wiping off water following a 4 h water saturation of excised leaves at room temperature; and W0, after subsequently drying leaves for 12 h at 70 ◦C. RWC (%) = (W1 −W0)/(W2 −W0) × 100%. To determine RWL, detached leaves were initially soaked in water for 1 h, after which excess water was removed and, thereafter, leaves were weighed 30 i F ti id t ti ti iti th l d i li id Contents of malondialdehyde, free proline, soluble sugar, soluble proteins and total chlorophyll were determined as described previously [51,52]. Relative water content (RWC) and rate of water loss (RWL) were measured as described by previous protocols [53]. To determine RWC, three leaf weights were taken: W1, immediately after leaf excision; W2, after wiping off water following a 4 h water saturation of excised leaves at room temperature; and W0, after subsequently drying leaves for 12 h at 70 ◦C. RWC (%) = (W1 Contents of malondialdehyde, free proline, soluble sugar, soluble proteins and total chlorophyll were determined as described previously [51,52]. Relative water content (RWC) and rate of water loss (RWL) were measured as described by previous protocols [53]. To determine RWC, three leaf weights were taken: W1, immediately after leaf excision; W2, after wiping off water following a 4 h water saturation of excised leaves at room temperature; and W0, after subsequently drying leaves for 12 h at 70 ◦C. RWC (%) = (W1 −W0)/(W2 −W0) × 100%. To determine RWL, detached leaves were initially soaked in water for 1 h, after which excess water was removed and, thereafter, leaves were weighed every 30 min. For antioxidant enzymatic activities, the leaves were ground in liquid nitrogen and assayed for activities of antioxidant enzymes including GPX, APX, SOD, CAT, POD, GR, GST and content of GSH by commercially available antioxidant enzyme kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to manufacturer’s instructions. The detailed experimental procedures for antioxidant enzyme activities are available in Methods S1. 4.6. RNA Extraction and Quantitative RT-PCR Total RNAs were isolated from mulberry and Arabidopsis leaves using RNA Extraction Kit (TaKaRa, Dalian, China) according to the manufacturer’s protocol. The extracted RNAs were treated with RNase-free DNase I (TaKaRa, Dalian, China) for eliminating genomic DNA. The ﬁrst strand of cDNA was synthesized using the PrimeScript RT Reagent Kit (TaKaRa, Dalian, China). Quantitative RT-PCR (qRT-PCR) was performed with the SYBR® Premix Ex Taq™II (TaKaRa, Dalian, China) on the Bio-Rad CFX96 Real-Time PCR System. The relative expression levels of MnGPX genes were obtained by normalization to internal reference gene AtACT1 in transgenic Arabidopsis or MaACT1 in mulberry and calculated using the 2–∆∆Ct method. The primers used for qRT-PCR analysis are listed in Table S3. 4.5. Ectopic Overexpression of Mulberry GPX Genes in Arabidopsis The open reading frame (ORF) sequence of MaGPX1 to MaGPX6 were subcloned into a binary plasmid pCAMBIA1301, respectively, under the control of CaMV35S promoter as we described previously [34]. The recombinant constructs and pCAMBIA1301 vector (control) were introduced into Agrobacterium strain GV3101, respectively, and then transformed into Arabidopsis thaliana Col-0 using the ﬂoral dip method. To identify the overexpression lines, T1 transgenic Arabidopsis plants were screened on 1/2 MS agar plates supplemented with hygromycin (30 mg L−1) and then used for GUS staining analysis. To determine expression levels of MaGPX genes, positive T1 plants were further subjected to RNA extraction and quantitative RT-PCR. Hygromycin-resistant screening was continued for the T2 and T3 transgenic lines. 4.4. iTRAQ-Based Quantitative Proteomics and Bioinformatic Analysis Isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative pro- teomics was performed using a customer service by Jingjie PTM-Biolabs Co., Ltd. (Hangzhou, China). In brief, total proteins were extracted from mulberry leaves and then subjected to trypsin digestion. After trypsin digestion, iTRAQ labeling for three drought-treated samples and three control samples were performed according to the manufacturer’s pro- Plants 2022, 11, 2350 14 of 18 14 of 18 tocol with iTRAQ Reagent-8 Plex Multiplex Kit (AB SCIEX, Framingham, MA, USA). iTRAQ-labeled peptides were fractionated by HPLC and then analyzed by LC-MS/MS (Thermo Scientiﬁc Q Exactive Plus, Waltham, MA, USA). MS data were analyzed using MaxQuant software (version 1.5.2.8, https://www.maxquant.org/, (accessed on 2018)), followed by database searching and bioinformatics analysis. Tandem mass spectra were searched against UniProt database concatenated with reverse decoy database. Trypsin/P was speciﬁed as cleavage enzyme allowing up to 2 missing cleavages, 4 modiﬁcations per peptide and 5 charges. Mass error was set to 10 ppm for precursor ions and 0.02 Da for fragment ions. Carbamidomethylation on Cys was speciﬁed as ﬁxed modiﬁcation and oxidation on Met and acetylation on protein N-terminal were speciﬁed as variable modiﬁcations. False discovery rate (FDR) threshold was speciﬁed at 1% and ion score was set >20. Minimum peptide length was set at 6. All the other parameters were set to default values. The relative quantitation of proteins was performed according to the quantitative ratio between drought-treated and control samples. A p value < 0.05 from t-tests and a fold change >1.5 or <0.67 (1/1.5) were set as the thresholds for differentially expressed protein (DEP). Gene ontology (GO; http://www.geneontology.org/, (accessed on 2019)) and Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.genome.jp/kegg/, (accessed on 2019)) databases were used to classify and group the identiﬁed proteins, and Fisher’s exact tests were used to deﬁne signiﬁcantly enriched GO terms and pathways using all the DEPs as target sets. Protein domain annotation was performed using InterProScan software (http://www.ebi.ac.uk/interpro/, (accessed on 2019)). A detailed experimental procedure for iTRAQ-based quantitative proteomics is available in Methods S2. tocol with iTRAQ Reagent-8 Plex Multiplex Kit (AB SCIEX, Framingham, MA, USA). iTRAQ-labeled peptides were fractionated by HPLC and then analyzed by LC-MS/MS (Thermo Scientiﬁc Q Exactive Plus, Waltham, MA, USA). MS data were analyzed using MaxQuant software (version 1.5.2.8, https://www.maxquant.org/, (accessed on 2018)), followed by database searching and bioinformatics analysis. 4.4. iTRAQ-Based Quantitative Proteomics and Bioinformatic Analysis Tandem mass spectra were searched against UniProt database concatenated with reverse decoy database. Trypsin/P was speciﬁed as cleavage enzyme allowing up to 2 missing cleavages, 4 modiﬁcations per peptide and 5 charges. Mass error was set to 10 ppm for precursor ions and 0.02 Da for fragment ions. Carbamidomethylation on Cys was speciﬁed as ﬁxed modiﬁcation and oxidation on Met and acetylation on protein N-terminal were speciﬁed as variable modiﬁcations. False discovery rate (FDR) threshold was speciﬁed at 1% and ion score was set >20. Minimum peptide length was set at 6. All the other parameters were set to default values. The relative quantitation of proteins was performed according to the quantitative ratio between drought-treated and control samples. A p value < 0.05 from t-tests and a fold change >1.5 or <0.67 (1/1.5) were set as the thresholds for differentially expressed protein (DEP). Gene ontology (GO; http://www.geneontology.org/, (accessed on 2019)) and Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.genome.jp/kegg/, (accessed on 2019)) databases were used to classify and group the identiﬁed proteins, and Fisher’s exact tests were used to deﬁne signiﬁcantly enriched GO terms and pathways using all the DEPs as target sets. Protein domain annotation was performed using InterProScan software (http://www.ebi.ac.uk/interpro/, (accessed on 2019)). A detailed experimental procedure for iTRAQ-based quantitative proteomics is available in Methods S2. 5. Conclusions In this study, quantitative proteomics combined with biochemical analyses and gene functional characterization were used to investigate the mechanism of mulberry tree’s response to drought stress. Drought stress reduces water transport, photosynthesis, and carbohydrate metabolism in the mulberry, and causes cellular dehydration, and osmotic and oxidative stress. Quantitative proteomic and biochemical analyses reveal that drought stress causes extensive change in antioxidant-system enzymes, especially a systematic upregulation of ﬁve GPX isoforms and increased antioxidant activity in the mulberry, suggesting a prominent role of GPX in regulating antioxidant defense against drought stress. More importantly, overexpression of the GPX members leads to greater tolerance to drought stress, enhanced capacity of antioxidant system and less accumulation of ROS in transgenic plants. Our results suggest that a higher abundance of GPX isoforms is associated with greater antioxidant abilities and higher tolerance to drought stress in mulberry. These ﬁndings will provide a new approach for identifying drought-tolerant germplasm in mulberry. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/plants11182350/s1, Methods S1. Assays for antioxidant enzyme activities and GSH content. Methods S2. Experimental procedures for iTRAQ-based quantitative proteomics. Table S1. All proteins quantiﬁed by iTRAQ proteomics and annotation summary. Table S2. All differentially expressed proteins (DEPs) identiﬁed by iTRAQ-based quantitative pro- teomics. Table S3. The primers used for qRT-PCR analysis in this study. Figure S1. KEGG pathway for glutathione metabolism (map00480). The enzyme’s EC number displayed with red color for up-regulation and green color for down-regulation in mulberry under drought stress. Phospholipid- hydroperoxide glutathione peroxidase [EC:1.11.1.12]; glutathione peroxidase [EC:1.11.1.9]; L-ascorbate peroxidase [EC:1.11.1.11]; glutathione S-transferase [EC:2.5.1.18]; spermidine synthase [EC:2.5.1.16]. Figure S2. Phylogenetic analysis of glutathione peroxidase (GPX) proteins of mulberry, rice and Arabidopsis. The tree was constructed using the neighbour-joining method of clustalW, with 1000 bootstraps. Accession numbers for rice and Arabidopsis are as follows: AtGPX1 (At2g25080), AtGPX2 (At2g31570), AtGPX3 (At2g43350), AtGPX4 (At2g48150), AtGPX5 (At3g63080), AtGPX6 (At4g11600), AtGPX7 (At4g31870), AtGPX8 (At1g63460). OsGPX1 (Os04g46960), OsGPX2 (Os03g24380), OsGPX3 (Os02g44500), OsGPX4 (Os06g08670) and OsGPX5 (Os11g18170). Figure S3. (A) Identiﬁcation of positive transgenic Arabidopsis lines by GUS staining. L1 to L5 indicate ﬁve independent transgenic lines for each genotype. (B) Gene expression levels of MaGPXs in the Arabidopsis transgenic plants. Data are means ± SD with three replicates. Figure S4. 4.7. ROS Measurement and Histochemical Staining ROS is a collective term that includes both oxygen free radicals (i.e., superoxide) and nonradicals (i.e., hydrogen peroxide). Hydrogen peroxide (H2O2) content was assessed us- ing a commercial hydrogen peroxide assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to manufacturer’s instructions. H2O2 bound to molybdenum acid forms a complex, measured at 405 nm, from which the content of H2O2 was calculated. The visualization of H2O2 and superoxide anion (O2.-) in leaves were detected by 3,3′- Plants 2022, 11, 2350 15 of 18 15 of 18 diaminobenzidine (DAB) and nitroblue tetrazolium chloride (NBT) histochemical staining, respectively [54]. 5. Conclusions (A) Phenotypes of the control, OE-MaGPX3 and OE-MaGPX5 transgenic lines germinated on 1/2 MS agar medium supplemented with 200 mmol.L−1 mannitol or no mannitol supplement and cultured for 10 days. Bar = 15 mm. (B) The seedling length of control, OE-MaGPX3 and OE-MaGPX5 transgenic lines. Data are means ± SD with ﬁve biologi- cal replicates. Letters indicate signiﬁcant differences between means, determined using Duncan’s multiple range test (5% α). Author Contributions: Conceived and designed the experiments, M.Z. and W.L.; performed the experiments, M.Z. and S.L.; contributed to reagents/materials/analytical tools, J.G., T.G., Q.L., L.B. and F.J.; analyzed the data, M.Z., W.L. and S.L.; wrote and revised the paper, M.Z. and W.L.; supervision, Y.Q. and C.S.; project administration, Y.Q. and C.S. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by the Natural Science Basic Research Project in Shaanxi Province (2022JM-121, 2019JM-156), the National Natural Science Foundation of China (32070197 and 31570181), and the China Agriculture Research System (CARS-18). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Plants 2022, 11, 2350 16 of 18 Data Availability Statement: The iTRAQ-based proteomic dataset has been submitted to ProteomeX- change (http://www.proteomexchange.org/, (accessed on 1 December 2019)) via the PRIDE database (Project accession: PXD010227; reviewer account details: username: reviewer20085@ebi.ac.uk; pass- word: 9RNDiGXZ). Acknowledgments: We thank Jingjie PTM-Biolabs (Hangzhou, China) for providing technical sup- port in iTRAQ-based quantitative proteomics. We thank Yingjun Cui (University of Missouri, Columbia, MO, USA) for reading and revising the manuscript. Conﬂicts of Interest: The authors declare that they have no conﬂict of interest. 1. He, X. Progress of comprehensive utilization of mulberry resources and development countermeasure. Acta Sericologica Sin. 2005, 31, 4–7. Impact of drought stress on photosynthetic response, leaf water potent in two cultivars of bi-leader apple trees (Malus × domestica Borkh.). Sci. Hortic. 2019, 246, 535–543. [CrossR in two cultivars of bi-leader apple trees (Malus × domestica Borkh.). Sci. Hortic. 2019, 246, 535–543. [CrossRef] 8. Huang, G.T.; Ma, S.L.; Bai, L.P.; Zhang, L.; Ma, H.; Jia, P.; Liu, J.; Zhong, M.; Guo, Z.F. Signal transduction during cold, salt, and drought stresses in plants Mol Biol Rep 2012 39 969 987 [CrossRef] pp ( ) , , [ ] 8. Huang, G.T.; Ma, S.L.; Bai, L.P.; Zhang, L.; Ma, H.; Jia, P.; Liu, J.; Zhong, M.; Guo, Z.F. Signal transduction during cold, salt, and drought stresses in plants. Mol. Biol. Rep. 2012, 39, 969–987. [CrossRef] g p p 9. Zhu, J.K. Abiotic stress signaling and responses in plants. Cell 2016, 167, 313–324. [CrossRef] 9. Zhu, J.K. Abiotic stress signaling and responses in plants. Cell 2016, 167, 313–324. [CrossRef] Zhu, J.K. Abiotic stress signaling and responses in pla 10. Raghavendra, A.S.; Gonugunta, V.K.; Christmann, A.; Grill, E. ABA perception and signalling. Trends Plant Sci. 2010, 15, 395–401. [CrossRef] 11. Yoshida, T.; Mogami, J.; Yamaguchi-Shinozaki, K. ABA-dependent and ABA-independent signaling in r in plants. Curr. Opin. Plant Biol. 2014, 21, 133–139. [CrossRef] [PubMed] p . Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci. 2002, 7, 405–410. [CrossRef] 12. Mittler, R. Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci. 2002, 7, 405–410. [CrossRef] 13. Zhang, H.; Zhao, Y.; Zhu, J.K. Thriving under stress: How plants balance growth and the stress response. Dev. Cell 2020, 55, 529–543. [CrossRef] [PubMed] 14 S ki N K it k S Mittl R Mill G ROS d d i lli i th f l t t bi ti t Pl t C ll 14. Suzuki, N.; Koussevitzky, S.; Mittler, R.; Miller, G. ROS and redox signalling in the response of plan Environ. 2012, 35, 259–270. [CrossRef] 15. Baxter, A.; Mittler, R.; Suzuki, N. ROS as key players in plant stress signalling. J. Exp. Bot. 2014, 65, 1229–1240. [CrossRef] 16 A l K Hi H R i i M b li id i d i l d i A R Pl Bi l 2004 55 15. Baxter, A.; Mittler, R.; Suzuki, N. ROS as key players in plant stress signalling. J. Exp. Bot. 2014, 65, 15. Baxter, A.; Mittler, R.; Suzuki, N. References 1. He, X. Progress of comprehensive utilization of mulberry resources and development countermeasure. Acta Sericologica Sin. 2005, 31, 4–7. 1. He, X. Progress of comprehensive utilization of mulberry resources and development countermeasure. Acta Sericologica Sin. 2005, 31, 4–7. 1. He, X. Progress of comprehensive utilization of mulberry resources and development countermeasure. Acta Sericologica Sin. 2005, 31, 4–7. 2. Qin, J.; He, N.J.; Wang, Y.; Xiang, Z.H. Ecological issues of mulberry and sustainable development. J. Resour. Ecol. 2012, 3, 330–339. 3. Yin, H.; Wang, L.; Liu, G.; Huang, G.; Wei, L.; Wu, J. Effect of mulberry (Morus alba Linn) on soil and water conservation. Agric. Sci. Technol. 2016, 17, 2308–2311. 2. Qin, J.; He, N.J.; Wang, Y.; Xiang, Z.H. Ecological issues of mulberry and sustainable development. J. Resour. Ecol. 2012, 3, 330–339. 3. Yin, H.; Wang, L.; Liu, G.; Huang, G.; Wei, L.; Wu, J. Effect of mulberry (Morus alba Linn) on soil and water conservation. Agric. Sci. Technol. 2016, 17, 2308–2311. 2. Qin, J.; He, N.J.; Wang, Y.; Xiang, Z.H. Ecological issues of mulberry and sustainable development. J. Resour. Ecol. 2012, 3, 330 339. 3. Yin, H.; Wang, L.; Liu, G.; Huang, G.; Wei, L.; Wu, J. Effect of mulberry (Morus alba Linn) on soil and water conservation. Agric. Sci. Technol. 2016, 17, 2308–2311. 3. Yin, H.; Wang, L.; Liu, G.; Huang, G.; Wei, L.; Wu, J. Effect of mulberry (Morus alba Linn) on soil and water conservation. Agric. Sci. Technol. 2016, 17, 2308–2311. 4. Wang, J.; Ma, S.; Cao, Y.; Li, X.; Song, Y. The advantage of the mulberry using in the forestry’s sustainable development. Chin. Agric. Sci. Bull. 2004, 20, 72–73. g 5. Su, C.; Xue, Z.M.; Jiao, F. Cultivation features and ecological protective functions of adaptive mulberry germplasm resources in Loess Plateau of Northern Shaanxi. Acta Sericologica Sin. 2012, 38, 146–151. J g p p y g p Plateau of Northern Shaanxi. Acta Sericologica Sin. 2012, 38, 146–151. g 6. Lu, H.; Ding, T.Y.; Zuo, S.C.; Wu, L.L.; Ji, D.F.; Qin, J. Mulberry resource utilization and development d ll d” lb d X A S i l i Si 39 1 1 g 6. Lu, H.; Ding, T.Y.; Zuo, S.C.; Wu, L.L.; Ji, D.F.; Qin, J. Mulberry resource utilization and development strategy of “Ecologically and Economically Integrated” mulberry industry in Xinjiang. Acta Sericologica Sin. 2013, 39, 166–170. g y y j g g G.; Yoon, T.-M. Impact of drought stress on photosynthetic response, leaf water potential, and stem sap ﬂow i-leader apple trees (Malus × domestica Borkh.). Sci. Hortic. 2019, 246, 535–543. [CrossRef] Bhusal, N.; Han, S. G.; Yoon, T. M. 1. He, X. Progress of comprehensive utilization of mulberry resources and development countermeasure. Acta Sericologica Sin. 2005, 31, 4–7. Bhusal, N.; Lee, M.; Reum Han, A.; Han, A.; Kim, H.S. Responses to drought stress in Prunus sargentii and Larix kaempferi seedlings using morphological and physiological parameters. For. Ecol. Manag. 2020, 465, 118099. [CrossRef] g g p g p y g p g 37. Sofo, A.; Scopa, A.; Nuzzaci, M.; Vitti, A. Ascorbate peroxidase and catalase activities and their genetic regulation in plants subjected to drought and salinity stresses. Int. J. Mol. Sci. 2015, 16, 13561–13578. [CrossRef] [PubMed] j g y 38. Caverzan, A.; Passaia, G.; Rosa, S.B.; Ribeiro, C.W.; Lazzarotto, F.; Margis-Pinheiro, M. Plant responses to stresses: Role of ascorbate peroxidase in the antioxidant protection. Genet. Mol. Biol. 2012, 35, 1011–1019. [CrossRef] 39. Chen, M.; Li, K.; Li, H.; Song, C.P.; Miao, Y. The glutathione peroxidase gene family in Gossypium hirsutum: Genome-wide identiﬁcation, classiﬁcation, gene expression and functional analysis. Sci. Rep. 2017, 7, 44743. [CrossRef] 40. Attacha, S.; Solbach, D.; Bela, K.; Moseler, A.; Wagner, S.; Schwarzlander, M.; Aller, I.; Muller, S.J.; Meyer, A.J. Glutathione peroxidase-like enzymes cover ﬁve distinct cell compartments and membrane surfaces in Arabidopsis thaliana. Plant Cell Environ. 2017, 40, 1281–1295. [CrossRef] 41. Pandey, S.; Fartyal, D.; Agarwal, A.; Shukla, T.; James, D.; Kaul, T.; Negi, Y.K.; Arora, S.; Reddy, M.K. Abiotic stress tolerance in plants: Myriad roles of ascorbate peroxidase. Front. Plant Sci. 2017, 8, 581. [CrossRef] [PubMed] 42. Bobrovskikh, A.; Zubairova, U.; Kolodkin, A.; Doroshkov, A. Subcellular compartmentalization of the plant antioxidant system: An integrated overview. PeerJ 2020, 8, e9451. [CrossRef] [PubMed] 43. Kim, Y.J.; Jang, M.G.; Noh, H.Y.; Lee, H.J.; Sukweenadhi, J.; Kim, J.H.; Kim, S.Y.; Kwon, W.S.; Yang, D.C. Molecular characterization of two glutathione peroxidase genes of Panax ginseng and their expression analysis against environmental stresses. Gene 2014, 535, 33–41. [CrossRef] [PubMed] 44. Diao, Y.; Xu, H.; Li, G.; Yu, A.; Yu, X.; Hu, W.; Zheng, X.; Li, S.; Wang, Y.; Hu, Z. Cloning a glutathione peroxidase gene from Nelumbo nucifera and enhanced salt tolerance by overexpressing in rice. Mol. Biol. Rep. 2014, 41, 4919–4927. [CrossRef] [PubMed] 44. Diao, Y.; Xu, H.; Li, G.; Yu, A.; Yu, X.; Hu, W.; Zheng, X.; Li, S.; Wang, Y.; Hu, Z. Cloning a glutathione peroxidase gene from Nelumbo nucifera and enhanced salt tolerance by overexpressing in rice. Mol. Biol. Rep. 2014, 41, 4919–4927. [CrossRef] [PubMed] 45. Vieira Dos Santos, C.; Rey, P. Plant thioredoxins are key actors in the oxidative stress response. Trends Plant Sci. 1. He, X. Progress of comprehensive utilization of mulberry resources and development countermeasure. Acta Sericologica Sin. 2005, 31, 4–7. ROS as key players in plant stress signalling. J. Exp. Bot. 2014, 65, 1229–1240. [CrossRef] 16. Apel, K.; Hirt, H. Reactive oxygen species: Metabolism, oxidative stress, and signal transduction. Annu. Rev. Plant Biol. 2004, 55, 15. Baxter, A.; Mittler, R.; Suzuki, N. ROS as key players in plant stress signalling. J. Exp. Bot. 2014, 65, 1229–1240. [CrossRef] 16. Apel, K.; Hirt, H. Reactive oxygen species: Metabolism, oxidative stress, and signal transduction. Annu. Rev. Plant Biol. 2004, 55, 373–399. [CrossRef] y p y p g g p 16. Apel, K.; Hirt, H. Reactive oxygen species: Metabolism, oxidative stress, and signal transduction. Annu. Rev. Plant Biol. 2004, 55, 373–399. [CrossRef] 17. Waszczak, C.; Carmody, M.; Kangasjarvi, J. Reactive oxygen species in plant signaling. Annu. Rev. Pla [CrossRef] 18. Halliwell, B. Reactive species and antioxidants. Redox biology is a fundamental theme of aerobic life. Plant Physiol. 2006, 141, 312–322. [CrossRef] 19. Foyer, C.H.; Shigeoka, S. Understanding oxidative stress and antioxidant functions to enhance photosynthesis. Plant Physiol. 2011, 155, 93–100. [CrossRef] 20. Ozyigit, I.I.; Filiz, E.; Vatansever, R.; Kurtoglu, K.Y.; Koc, I.; Ozturk, M.X.; Anjum, N.A. Identiﬁcation and comparative analysis of H2O2-scavenging enzymes (ascorbate peroxidase and glutathione peroxidase) in selected plants employing bioinformatics approaches. Front. Plant Sci. 2016, 7, 301. [CrossRef] pp 21. Gaber, A.; Ogata, T.; Maruta, T.; Yoshimura, K.; Tamoi, M.; Shigeoka, S. The involvement of Arabidopsis glutathione peroxidase 8 in the suppression of oxidative damage in the nucleus and cytosol. Plant Cell Physiol. 2012, 53, 1596–1606. [CrossRef] [PubMed] 2 22. Islam, T.; Manna, M.; Reddy, M.K. Glutathione peroxidase of Pennisetum glaucum (PgGPx) is a functional Cd2+ dependent peroxiredoxin that enhances tolerance against salinity and drought stress. PLoS ONE 2015, 10, e0143344. [CrossRef] [PubMed] 23. Navrot, N.; Collin, V.; Gualberto, J.; Gelhaye, E.; Hirasawa, M.; Rey, P.; Knaff, D.B.; Issakidis, E.; Jacquot, J.P.; Rouhier, N. Plant glutathione peroxidases are functional peroxiredoxins distributed in several subcellular compartments and regulated during biotic and abiotic stresses. Plant Physiol. 2006, 142, 1364–1379. [CrossRef] [PubMed] y 24. Passaia, G.; Margis-Pinheiro, M. Glutathione peroxidases as redox sensor proteins in plant cells. Plant Sci. 2015, 234, 22–26. [CrossRef] [PubMed] 25. Paiva, A.; Passaia, G.; Lobo, A.; Jardim-Messeder, D.; Silveira, J.; Margis-Pinheiro, M. Mitochondrial glutathione peroxidase (OsGPX3) has a crucial role in rice protection against salt stress. Environ. Exp. Bot. 2019, 158, 12–21. [CrossRef] 17 of 18 Plants 2022, 11, 2350 26. 1. He, X. Progress of comprehensive utilization of mulberry resources and development countermeasure. Acta Sericologica Sin. 2005, 31, 4–7. Iqbal, A.; Yabuta, Y.; Takeda, T.; Nakano, Y.; Shigeoka, S. Hydroperoxide reduction by thioredoxin-speciﬁc glutathione peroxidase isoenzymes of Arabidopsis thaliana. FEBS J. 2006, 273, 5589–5597. [CrossRef] [PubMed] y p 27. Bela, K.; Horvath, E.; Galle, A.; Szabados, L.; Tari, I.; Csiszar, J. Plant glutathione peroxidases: Emerging role of the antioxidant enzymes in plant development and stress responses. J. Plant Physiol. 2015, 176, 192–201. [CrossRef] [PubMed] 28. Zhou, H.; Zhang, F.; Zhai, F.; Su, Y.; Zhou, Y.; Ge, Z.; Tilak, P.; Eirich, J.; Finkemeier, I.; Fu, L.; et al. Rice GLUTATHIONE PEROXIDASE1-mediated oxidation of bZIP68 positively regulates ABA-independent osmotic stress signaling. Mol. Plant 2022, 15, 651–670. [CrossRef] , [ ] 29. Gong, Z.Z.; Xiong, L.M.; Shi, H.Z.; Yang, S.H.; Herrera-Estrella, L.R.; Xu, G.H.; Chao, D.Y.; Li, J.R.; Wang, P.Y.; Qin, F.; et al. Plant abiotic stress response and nutrient use efﬁciency. Sci. China Life Sci. 2020, 63, 635–674. [CrossRef] p y 30. Wang, X.L.; Cai, X.F.; Xu, C.X.; Wang, Q.H.; Dai, S.J. Drought-responsive mechanisms in plant leaves revealed by proteomics. Int. J. Mol. Sci. 2016, 17, 1706. [CrossRef] 31. Krol, A.; Weidner, S. Changes in the proteome of grapevine leaves (Vitis vinifera L.) during long-term drought stress. J. Plant Physiol. 2017, 211, 114–126. [CrossRef] [PubMed] y , , [ ] [ ] 32. Shen, Z.J.; Chen, J.; Ghoto, K.; Hu, W.J.; Gao, G.F.; Luo, M.R.; Li, Z.; Simon, M.; Zhu, X.Y.; Zheng, H.L. Proteomic analysis on mangrove plant Avicennia marina leaves reveals nitric oxide enhances the salt tolerance by up-regulating photosynthetic and energy metabolic protein expression. Tree Physiol. 2018, 38, 1605–1622. [CrossRef] [PubMed] gy p p y 33. Martinez-Esteso, M.J.; Casado-Vela, J.; Selles-Marchart, S.; Pedreno, M.A.; Bru-Martinez, R. Differential plant proteome analysis by isobaric tags for relative and absolute quantitation (iTRAQ). Methods Mol. Biol. 2014, 1072, 155–169. [CrossRef] [PubMed] y g q 34. Li, S.J.; Zhang, M.J.; Meng, C.T.; Su, C.; Dong, X.L.; Chen, K.M.; Li, W.Q. Gene cloning and expression analysis of the MaGPX family in Morus alba L. Acta Bot. Boreali-Occident. Sin. 2019, 39, 991–1000. y 35. Khaleghi, A.; Naderi, R.; Brunetti, C.; Maserti, B.E.; Salami, S.A.; Babalar, M. Morphological, physioc responses of Maclura pomifera to drought stress. Sci. Rep. 2019, 9, 19250. [CrossRef] ri, R.; Brunetti, C.; Maserti, B.E.; Salami, S.A.; Babalar, M. Morphological, physiochemical and antioxidant a pomifera to drought stress. Sci. Rep. 2019, 9, 19250. [CrossRef] 36. 53. Li, W.Q.; Zhang, M.J.; Gan, P.F.; Qiao, L.; Yang, S.Q.; Miao, H.; Wang, G.F.; Zhang, M.M.; Liu, W.T.; Li, H.F.; et al. CLD1/SRL1 modulates leaf rolling by affecting cell wall formation, epidermis integrity and water homeostasis in rice. Plant J. 2017, 92, 904–923. [CrossRef] [PubMed] 54. Ramel, F.; Sulmon, C.; Bogard, M.; Couee, I.; Gouesbet, G. Differential patterns of reactive oxygen species and antioxidative mechanisms during atrazine injury and sucrose-induced tolerance in Arabidopsis thaliana plantlets. BMC Plant Biol. 2009, 9, 28. [CrossRef] [PubMed] 1. He, X. Progress of comprehensive utilization of mulberry resources and development countermeasure. Acta Sericologica Sin. 2005, 31, 4–7. 2006, 11, 329–334. [CrossRef] [PubMed] 45. Vieira Dos Santos, C.; Rey, P. Plant thioredoxins are key actors in the oxidative stress response. Trends P [CrossRef] [PubMed] 46. Geigenberger, P.; Thormahlen, I.; Daloso, D.M.; Fernie, A.R. The unprecedented versatility of the plant thioredoxin system. Trends Plant Sci. 2017, 22, 249–262. [CrossRef] 47. Milla, M.A.R.; Maurer, A.; Huete, A.R.; Gustafson, J.P. Glutathione peroxidase genes in Arabidopsis are ubiquitous and regulated by abiotic stresses through diverse signaling pathways. Plant J. 2003, 36, 602–615. [CrossRef] y g g g p y 48. Miao, Y.; Lv, D.; Wang, P.; Wang, X.C.; Chen, J.; Miao, C.; Song, C.P. An Arabidopsis glutathione peroxidase functions as both a redox transducer and a scavenger in abscisic acid and drought stress responses. Plant Cell 2006, 18, 2749–2766. [CrossRef] y g g g p y Miao, Y.; Lv, D.; Wang, P.; Wang, X.C.; Chen, J.; Miao, C.; Song, C.P. An Arabidopsis glutathione peroxida redox transducer and a scavenger in abscisic acid and drought stress responses Plant Cell 2006 18 2749 2 Y.; Lv, D.; Wang, P.; Wang, X.C.; Chen, J.; Miao, C.; Song, C.P. An Arabidopsis glutathione peroxidase func redox transducer and a scavenger in abscisic acid and drought stress responses. Plant Cell 2006, 18, 2749–2766. [CrossRef] 49. Gaber, A.; Yoshimura, K.; Yamamoto, T.; Yabuta, Y.; Takeda, T.; Miyasaka, H.; Nakano, Y.; Shigeoka, S. Glutathione peroxidase-like protein of Synechocystis PCC 6803 confers tolerance to oxidative and environmental stresses in transgenic Arabidopsis. Physiol. Plant 2006, 128, 251–262. [CrossRef] 50. Zhang, L.; Wu, M.; Teng, Y.; Jia, S.; Yu, D.; Wei, T.; Chen, C.; Song, W. Overexpression of the Glutathione Peroxidase 5 (RcGPX5) gene from Rhodiola crenulata increases drought tolerance in Salvia miltiorrhiza. Front. Plant Sci. 2018, 9, 1950. [CrossRef] 51. Toscano, S.; Farieri, E.; Ferrante, A.; Romano, D. Physiological and biochemical responses in two ornamental shrubs to drought stress. Front. Plant Sci. 2016, 7, 645. [CrossRef] [PubMed] 52. Zhong, T.; Zhang, L.; Sun, S.; Zeng, H.; Han, L. Effect of localized reduction of gibberellins in different tobacco organs on drought stress tolerance and recovery. Plant Biotechnol. Rep. 2014, 8, 399–408. [CrossRef] Plants 2022, 11, 2350 18 of 18 18 of 18
https://openalex.org/W2956840440	https://www.research-collection.ethz.ch/bitstream/20.500.11850/356132/3/document.pdf	English	null	CellSIUS provides sensitive and specific detection of rare cell populations from complex single-cell RNA-seq data	Genome biology	2,019	cc-by	18,728	ETH Library Publication date: 2019-07-17 Permanent link: https://doi.org/10.3929/ethz-b-000356132 Author(s): ( ) Wegmann, Rebekka ; Neri, Marilisa; Schuierer, Sven; Bilican, Bilada; Hartkopf, Huyen; Nigsch, Florian; Mapa, Felipa; Waldt, Annick; Cuttat, Rachel; Salick, Max R.; Raymond, Joe; Kaykas, Ajamete; Roma, Guglielmo; Gubser Keller, Caroline © The Author(s). 2019 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. CellSIUS provides sensitive and specific detection of rare cell populations from complex single-cell RNA-seq data Rebekka Wegmann1,3†, Marilisa Neri1†, Sven Schuierer1, Bilada Bilican2,5, Huyen Hartkopf2, Florian Nigsch1, Felipa Mapa2, Annick Waldt1, Rachel Cuttat1, Max R. Salick2,4, Joe Raymond2, Ajamete Kaykas2,4, Guglielmo Roma1 and Caroline Gubser Keller1* Abstract We develop CellSIUS (Cell Subtype Identification from Upregulated gene Sets) to fill a methodology gap for rare cell population identification for scRNA-seq data. CellSIUS outperforms existing algorithms for specificity and selectivity for rare cell types and their transcriptomic signature identification in synthetic and complex biological data. Characterization of a human pluripotent cell differentiation protocol recapitulating deep-layer corticogenesis using CellSIUS reveals unrecognized complexity in human stem cell-derived cellular populations. CellSIUS enables identification of novel rare cell populations and their signature genes providing the means to study those populations in vitro in light of their role in health and disease. Keywords: Single-cell RNA sequencing, Data analysis, Rare cell types, Clustering, Software, Benchmarking, Human pluripotent stem cells, Cortical development, Choroid plexus, Lineage mapping Background data emerged. These comprise tools for cell-centric ana- lyses, such as unsupervised clustering for cell type identifi- cation [14–16], analysis of developmental trajectories [17, 18], or identification of rare cell populations [8, 9, 19], as well as approaches for gene-centric analyses such as differential expression (DE) analysis [20–22]. Single-cell RNA sequencing (scRNA-seq) enables genome-wide mRNA expression profiling with single-cell granularity. With recent technological advances [1, 2] and the rise of fully commercialized systems [3], throughput and availability of this technology are increasing at a fast pace [4]. Evolving from the first scRNA-seq dataset measuring gene expression from a single mouse blasto- mere in 2009 [5], scRNA-seq datasets now typically include expression profiles of thousands [1–3] to more than one million cells [6, 7]. One of the main applications of scRNA-seq is uncovering and characterizing novel and/or rare cell types from complex tissue in health and disease [8–13]. Whereas a large number of computational methods tailored to scRNA-seq analysis are available, comprehen- sive performance comparisons between those are scarce. This is mainly due to the lack of reference datasets with known cellular composition. Prior knowledge or synthetic data are commonly used to circumvent the problem of a missing ground truth. Here, we generated a benchmark dataset of ~ 12,000 single-cell transcriptomes from eight human cell lines to investigate the performance of scRNA-seq feature selection and clustering approaches. Strikingly, results highlighted a methodology gap for sensitive and specific identification of rare cell types. To fill this gap, we developed a method which we called CellSIUS (Cell Subtype Identification from Upregulated gene Sets). For complex scRNA-seq datasets containing both abundant and rare cell populations, we propose a two-step approach consisting of an initial coarse From an analytical point of view, the high dimensionality and complexity of scRNA-seq data pose significant chal- lenges. Following the platform development, a multitude of computational approaches for the analysis of scRNA-seq * Correspondence: caroline.gubser_keller@novartis.com †Rebekka Wegmann and Marilisa Neri have contributed equally to this work 1Novartis Institutes for Biomedical Research, Basel, Switzerland Full list of author information is available at the end of the article Originally published in: Originally published in: Genome Biology 20(1), https://doi.org/10.1186/s13059-019-1739-7 Genome Biology 20(1), https://doi.org/10.1186/s13059-019-1739-7 This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use. Wegmann et al. Genome Biology (2019) 20:142 https://doi.org/10.1186/s13059-019-1739-7 Page 2 of 21 Page 2 of 21 Wegmann et al. Genome Biology (2019) 20:142 clustering step followed by CellSIUS. Using synthetic and biological datasets containing rare cell populations, we showed that CellSIUS outperforms existing algorithms in both specificity and selectivity for rare cell type and their transcriptomic signature identification. In addition, and in contrast to existing approaches, CellSIUS simultaneously reveals transcriptomic signatures indicative of rare cell type’s function(s). single-cell to bulk expression profiles was used for cell type assignment as described in the “Methods” section (Fig. 1a, b). Cells that did not pass quality control (QC) or could not be unambiguously assigned to a cell line (614 cells, ~ 5%) were discarded, leaving 11,678 cells of known cell type (Fig. 1c and Additional file 1: Figure S1, Table S1). We assembled a modular workflow for the analysis of scRNA-seq data (Fig. 2a). The quality control, normalization, and marker gene identification modules were based on recent publications and described in methods. For a data-driven choice of the most appropriate feature selection method upstream of the clustering module, we compared methods using either a mean-variance trend to find highly variable genes (HVG, [27]) or a depth-adjusted negative binomial model (DANB [28]) for selection of genes with unexpected dropout rates (NBDrop) or dispersions (NBDisp). Using linear modeling as implemented in the plotExplanatoryVariables function from scater [29], we quantified the influence of these feature selection methods on the contribution of four predictors to the total observed variance: cell line, total counts per cell, total detected features per cell, and predicted cell cycle phase (Fig. 2b). Results highlighted that (i) for HVG selected genes, cell line accounted for 10% of the total variance only; (ii) for NBDisp and NBDrop selected genes, the percentage of total variance explained by cell line increased to 37% and 47%, respec- tively, with half of the selected features common to both methods; (iii) genes selected only by NBDisp were gener- ally low expressed (data not shown), highlighting a draw- back of variance-based feature selection [28]; and (iv) NBDrop selected features showed an increased contribu- tion of library size (i.e., total detected features and total counts per cell) to the total variance. Results Investigation of feature selection and clustering approaches for scRNA-seq data reveals a methodology gap for the detection of rare cell populations For our benchmark dataset, the number of total features co-varied with cell type and cell cycle indicating that library size is partially dependent on the cell line (Additional file 1: Figure S1), and thus determined by both technical and biological factors. Therefore, and because in our benchmark dataset, the genes selected by NBDrop explained more cell-line- based variance, we compared some of the most recent or widely used clustering methods after feature selection using NBDrop. To exemplify the use of CellSIUS, we applied the work- flow and our two-step clustering approach to complex biological data. We profiled the gene expression of 4857 human pluripotent stem cell (hPSC)-derived cortical neu- rons generated by a 3D spheroid differentiation protocol. Analysis of this in vitro model of corticogenesis revealed distinct progenitor, neuronal, and glial populations con- sistent with developing human telencephalon. Trajectory analysis identified a lineage bifurcation point between Cajal-Retzius cells and layer V/VI cortical neurons, which was not clearly demonstrated in other in vitro hPSC models of corticogenesis [23–26]. Importantly, CellSIUS revealed known as well as novel rare cell populations that differ by migratory, metabolic, or cell cycle status. These include a rare choroid plexus (CP) lineage, a population that was either not detected, or detected only partly by existing approaches for rare cell type identification. We experimentally validated the presence of CP neuroepi- thelia in our 3D cortical spheroid cultures by confocal microscopy and validated the CP-specific signature gene list output from CellSIUS using primary pre-natal human data. For the CP lineage in particular and other identified rare cell populations in general, the signature gene lists output from CellSIUS provide the means to isolate these populations for in vitro propagation and characterization of their role in neurological disorders. Investigation of feature selection and clustering approaches for scRNA-seq data reveals a methodology gap for the detection of rare cell populations To assess and compare the performance of some of the most recent and widely used feature selection and clus- tering methodologies for scRNA-seq data, we generated a scRNA-seq dataset with known cellular composition generated from mixtures of eight human cell lines. To this end, a total of ~ 12,000 cells from eight human cell lines (A549, H1437, HCT116, HEK293, IMR90, Jurkat, K562, and Ramos) were sequenced using the 10X Genomics Chromium platform [3]. Cells were processed in batches containing mixtures of two or three cell lines each. One of the cell lines was present in two separate batches and indicated that technical batch effects were minor as compared to the biological variability (Fig. 1). To infer cell type identity, we profiled each cell line indi- vidually using bulk RNA sequencing. Correlation of the For the clustering module, we investigated seven un- supervised clustering methods for scRNA-seq data (SC3 [15], Seurat [1], pcaReduce, hclust [30], mclust [31], DBSCAN [32], MCL [33, 34], Additional file 1: Table S2) by in silico subsampling of our dataset of known composition in two subsets with different cell type pro- portions (later referred to as subset 1 and subset 2, Fig. 2c–e, Additional file 1: Table S1). Subset 1 consisted of 4999 cells from eight cell types with abundance varying between 2 and 32%. Subset 2 consisted of 3989 cells with two major cell populations including 90% of all cells of Wegmann et al. Genome Biology (2019) 20:142 Page 3 of 21 A B C Fig. 1 Generation of a scRNA-seq dataset with known cellular composition. a Schematic illustration of the experimental setup. Eight human cell lines were individually profiled by bulk RNA-seq and mixed in four batches containing mixtures of two or three cell lines each for scRNA-seq profiling. Correlation of the single-cell to bulk expression profiles was used for cell type assignment as described in the Methods section. b Visualization of correlations between single-cell and bulk expression profiles for each batch. The top row represents cell type assignment. Single cells were assigned to the cell type correlating most with their expression profile as described in the Methods section. Cells with z- scored correlations below 0.2 were not assigned to any cluster. Cells that correlate strongly with more than one bulk expression profile likely represent doublets and were excluded from future analyses. Investigation of feature selection and clustering approaches for scRNA-seq data reveals a methodology gap for the detection of rare cell populations c Heatmap of gene expression values, clustered by their Pearson’s correlation across rows (genes) and columns (cells). The color bars indicate the cell type and the corresponding batch. Only the top 10% genes selected by NBDrop are shown A A A B C C B C Fig. 1 Generation of a scRNA-seq dataset with known cellular composition. a Schematic illustration of the experimental setup. Eight human cell lines were individually profiled by bulk RNA-seq and mixed in four batches containing mixtures of two or three cell lines each for scRNA-seq profiling. Correlation of the single-cell to bulk expression profiles was used for cell type assignment as described in the Methods section. b Visualization of correlations between single-cell and bulk expression profiles for each batch. The top row represents cell type assignment. Single cells were assigned to the cell type correlating most with their expression profile as described in the Methods section. Cells with z- scored correlations below 0.2 were not assigned to any cluster. Cells that correlate strongly with more than one bulk expression profile likely represent doublets and were excluded from future analyses. c Heatmap of gene expression values, clustered by their Pearson’s correlation across rows (genes) and columns (cells). The color bars indicate the cell type and the corresponding batch. Only the top 10% genes selected by NBDrop are shown Fig. 1 Generation of a scRNA-seq dataset with known cellular composition. a Schematic illustration of the experimental setup. Eight human cell lines were individually profiled by bulk RNA-seq and mixed in four batches containing mixtures of two or three cell lines each for scRNA-seq profiling. Correlation of the single-cell to bulk expression profiles was used for cell type assignment as described in the Methods section. b Visualization of correlations between single-cell and bulk expression profiles for each batch. The top row represents cell type assignment. Single cells were assigned to the cell type correlating most with their expression profile as described in the Methods section. Cells with z- scored correlations below 0.2 were not assigned to any cluster. Cells that correlate strongly with more than one bulk expression profile likely represent doublets and were excluded from future analyses. c Heatmap of gene expression values, clustered by their Pearson’s correlation across rows (genes) and columns (cells). The color bars indicate the cell type and the corresponding batch. Investigation of feature selection and clustering approaches for scRNA-seq data reveals a methodology gap for the detection of rare cell populations Only the top 10% genes selected by NBDrop are shown this subset, four medium to low abundant (between 1% and 5%), and two rarer cell types with abundances below 1%, containing 3 (0.08%) and 6 (0.15%) cells, respectively. We applied each clustering method to the complete data- set as well as to both subsets, using principal component analysis (PCA) [35, 36] to project the original expression values to vectors in a lower dimensional space and calcu- lating all distances based on these projections. For all clustering methods, we adjusted parameters such that they resulted in the expected number of 8 clusters. We then assessed the quality of the classification by calculating the adjusted Rand index (ARI) [37] between assignment and true cell line annotation. methods and mclust failed to identify the different cell types correctly and resulted in average ARI of 0.85 (SC3), 0.78 (pcaReduce), and 0.69 (mclust) (Fig. 1g). On subset 2, all methods failed to correctly identify rarer (6 cells, 0.16% of total cells) cell types (Fig. 1h). DBSCAN achieved the highest ARI (0.99) classifying rare cells as outliers (“border points”). All other methods merged rare cells with clusters of abundant cell types resulting in lower ARI of 0.98 (hclust on Euclidean distance), 0.96 (MCL), 0.96 (hclust on correlation distance), and 0.76 (Seurat). In conclusion, and consistently with a recent review describing the challenges in unsupervised clustering of single-cell RNA-seq data [16], our results showed that most clustering methods performed well in identifying populations defined by more than 2% of total cells. Yet, none of the methods could identify rarer populations, highlighting the need for dedicated tools tailored to detecting rare cell types. On the full dataset, most methods resulted in a perfect assignment (Fig. 2f) with only two of the stochastic methods—pcaReduce and mclust—yielding a lower ave- rage ARI of 0.90 and 0.92. In contrast, on subset 1, where cell type proportions were no longer equal, k-means-based Page 4 of 21 Wegmann et al. Genome Biology (2019) 20:142 A B C D E F G H Fig. 2 Performance assessment of feature selection and clustering methods. a Overview of the computational analysis workflow. b Benchmarking of feature selection methods. Investigation of feature selection and clustering approaches for scRNA-seq data reveals a methodology gap for the detection of rare cell populations In each case, the top 10% of features were selected using either a mean-variance trend to find highly variable genes (HVG, left) or a depth-adjusted negative binomial model (DANB) followed by selecting genes with unexpected dropout rates (NBDrop, middle) or dispersions (NBDisp, right). Plots show the percentage of variance explained by each of the four predictors to the total observed variance: cell line, total counts per cell, total detected features per cell, and predicted cell cycle phase. The blue dashed line indicates the average for the predictor cell line. c–e tSNE projections of the full dataset (c) and two sub-sampled datasets with unequal proportions between different cell lines (d, e). f–h Comparison of clustering assignments by different methods on the full dataset (f), subset 1 (g), and subset 2 (h). Stochastic methods (SC3, mclust, pcaReduce) were run 25 times. Bars and indicated values represent mean adjusted rand index (ARI), and dots correspond to results from individual runs. All other methods are deterministic and were run only once A B A A B B B D C D C E E G F H F H G Fig. 2 Performance assessment of feature selection and clustering methods. a Overview of the computational analysis workflow. b Benchmarking of feature selection methods. In each case, the top 10% of features were selected using either a mean-variance trend to find highly variable genes (HVG, left) or a depth-adjusted negative binomial model (DANB) followed by selecting genes with unexpected dropout rates (NBDrop, middle) or dispersions (NBDisp, right). Plots show the percentage of variance explained by each of the four predictors to the total observed variance: cell line, total counts per cell, total detected features per cell, and predicted cell cycle phase. The blue dashed line indicates the average for the predictor cell line. c–e tSNE projections of the full dataset (c) and two sub-sampled datasets with unequal proportions between different cell lines (d, e). f–h Comparison of clustering assignments by different methods on the full dataset (f), subset 1 (g), and subset 2 (h). Stochastic methods (SC3, mclust, pcaReduce) were run 25 times. Bars and indicated values represent mean adjusted rand index (ARI), and dots correspond to results from individual runs. All other methods are deterministic and were run only once Development of CellSIUS for rare cell population identification and characterization genes gm1, gm2, …, gmj that exhibit a bimodal distribution of expression values with a fold change above a certain threshold (fc_within) across all cells within Cm are identi- fied by one-dimensional k-means clustering (with k = 2). For each candidate gene gmi, the mean expression in the second mode is then compared to this gene’s mean expression level outside Cm (fc_between), considering only cells that have non-zero expression of gmi to avoid biases To overcome the abovementioned limitations, we deve- loped a novel method to identify rare cell populations which we called CellSIUS (Cell Subtype Identification from Upregulated gene Sets). CellSIUS takes as input the expression values of N cells grouped into M clus- ters (Fig. 3a). For each cluster Cm, candidate marker Wegmann et al. Genome Biology (2019) 20:142 Page 5 of 21 A A A B C Fig. 3 Development and benchmarking of CellSIUS. a Schematic overview of CellSIUS. Starting from an initial assignment of N cells in M clusters (i), within each cluster, genes with a bimodal distribution are identified (ii) and only genes with cluster-specific expression are retained (iii). Among the candidate genes, sets with correlated expression patterns are identified by graph-based clustering (iv). Cells are assigned to subgroups based on their average expression of each gene set (v). b, c Performance comparison of CellSIUS to GiniClust2 and RaceID3 in detecting cells from sub-clusters and their signatures. b Recall, precision, and true negative rate (TNR) with regard to the detection of rare cells in synthetic data when varying the number of rare cells from 2 (0.2%) to 100 (10%) c Recall, precision, and true negative rate (TNR) with regard to the detection of outlier genes (gene signature) in synthetic data when varying and the number of signature genes from 2 to 100 C B B C Fig. 3 Development and benchmarking of CellSIUS. a Schematic overview of CellSIUS. Starting from an initial assignment of N cells in M clusters (i), within each cluster, genes with a bimodal distribution are identified (ii) and only genes with cluster-specific expression are retained (iii). Among the candidate genes, sets with correlated expression patterns are identified by graph-based clustering (iv). Cells are assigned to subgroups based on their average expression of each gene set (v). b, c Performance comparison of CellSIUS to GiniClust2 and RaceID3 in detecting cells from sub-clusters and their signatures. CellSIUS outperforms existing algorithms in the identification of rare cell populations We first compared CellSIUS performance to RaceID3 [38] and GiniClust2 [19] using a synthetic dataset. Briefly, we used the expression values of 1000 K562 cells from our dataset to estimate the parameters for the simulation and generated two homogeneous populations of 500 cells (later referred to as clusters 1 and 2). We confirmed the mean-variance and mean-dropout relationships, library sizes, and percentage of zero counts per cells and per gene were similar to the underlying real data (Additional file 1: Figure S2a-f). For this data, both CellSIUS and GiniClust correctly identified the two predefined clusters whereas RaceID3 detected a large number of false positives (Additional file 1: Figure S2 g). To systematically investigate the stability of Cell- SIUS’ output to parameter changes, we repeated the above-described analysis when varying fc_within, fc_ between and corr_cutoff (Additional file 1: Figure S3; Methods). Results that highlighted the stability of both sensitivity and specificity are across a wide range of parameters. In summary, using synthetic data, we showed an in- creased sensitivity and specificity of our algorithm for rare cell type identification and outlier gene identifi- cation compared to GiniClust2 and RaceID3 (Fig. 3b, c) and demonstrated robustness to parameter choices (Additional file 1: Figure S3). We then assessed each algorithm’s ability to detect an increasingly rare cell type by adding between 2 and 100 (0.2–10% of the cluster size) cells of a third type to the two homogenous populations described above. This new synthetic cell type was generated by increasing the log2 expression values of 20 randomly selected genes by an average of 2.5. g We next benchmarked CellSIUS’ specificity and selectiv- ity using our dataset of known cell composition, randomly subsampling 100 HEK293 cells and 125 Ramos cells, and including 2, 5, or 10 Jurkat cells. Only cells assigned to be in cell cycle phase G1 were considered to ensure within- cluster homogeneity. To simulate varying degrees of tran- scriptional difference between the rare cell type (Jurkat) and its closest more abundant cell type (Ramos), we adapted an approach recently presented by Crow et al. [39] (Fig. 4a). Briefly, from the initial dataset, 25 Ramos cells were held out. Subsequently, an increasing fraction of gene expression values in the Jurkat cells were replaced by the respective values in the held out Ramos cells, thus diluting the Jurkat-specific gene expression profile and making the Jurkat cells more and more similar to Ramos. CellSIUS outperforms existing algorithms in the identification of rare cell populations Using this approach, we generated datasets with two equally sized abundant populations (HEK293 and Ramos, 100 cells each) and one rare population (Jurkat, varying between 2, 5, and 10 cells). We pre- defined two initial clusters: cluster 1 contained all HEK293 cells and cluster 2 combined the two lymphomas (Ramos and Jurkat). We compared (i) recall as the fraction of rare cells correctly assigned to new clusters, i.e., the number of correctly identified rare cells divided by the total number of rare cells; (ii) precision as the fraction of true rare cells among all cells not assigned to the two main clusters; and (iii) true negative rate (TNR) as the fraction of abundant cells that were correctly assigned to the two main clusters. To enable a more direct comparison between the methods, benchmarking analyses were carried out with a predefined initial clustering for all approaches. CellSIUS had a recall of 1 for rare cell populations consisting of more than 2 cells. In contrast Gini- Clust2 did not identify any rare cell populations and RaceID3 recalled only ~ 50% of true positives (Fig. 3b, top panel). Additionally, CellSIUS exhibited a TNR of 1.0 and thus a precision of 1.0 (except in the one case where no true positives were recovered). While GiniClust2’s TNR was also 1.0, the precision could not be defined due to the lack of identification of true and false positives. RaceID3 had a low TNR (mean = 0.95, sd = 0.01), resulting in low precision (mean = 0.1, sd = 0.1) (Fig. 3b, middle and bot- tom panel). We then repeated this comparison for the identification of signature genes. To this end, we ge- nerated a second set of populations. Briefly, the number of rare cells was fixed at 20 (~ 2% of total cells), and we increased the log2 expression values of between 2 and 100 genes by 2.5 on average. We compared (i) recall, (ii) We then tested the ability of CellSIUS, RaceID3, and GiniClust2 to identify rare cell types for varying incidence (i.e., total number of rare cells) and subtlety (i.e., fraction of Jurkat genes replaced by Ramos genes). We assessed the recall (Fig. 4b) and precision (Fig. 4c) as above. Results showed a high sensitivity of all three methods for very subtle transcriptional signatures (99.5% of genes replaced, corresponding to 230 unperturbed genes) and low incidence (down to two cells except for GiniClust2). Development of CellSIUS for rare cell population identification and characterization Finally, in contrast to both RaceID3 and GiniClust2, CellSIUS directly returns a gene signature for each of the new cell subpopulations recovered. CellSIUS considers only cells that have non-zero expression for the selected marker genes. Finally, in contrast to both RaceID3 and GiniClust2, CellSIUS directly returns a gene signature for each of the new cell subpopulations recovered. precision, and (iii) TNR as above but with respect to genes. In comparison to CellSIUS, GiniClust2 showed a poor performance (Fig. 3c, top panel), consistent with failing to detect rare cell population. In contrast, RaceID3 performed slightly better than CellSIUS in terms of recall, however, with a precision cost. Whereas both precision and TNR were 1.0 for CellSIUS, RaceID3 had a low TNR (0.5) and consequently a low precision (mean = 0.012, sd = 0.007) (Fig. 3c, top and bottom panels). Development of CellSIUS for rare cell population identification and characterization b Recall, precision, and true negative rate (TNR) with regard to the detection of rare cells in synthetic data when varying the number of rare cells from 2 (0.2%) to 100 (10%) c Recall, precision, and true negative rate (TNR) with regard to the detection of outlier genes (gene signature) in synthetic data when varying and the number of signature genes from 2 to 100 arising from stochastic zeroes. Only genes with sig- nificantly higher expression within the second mode of Cm (by default, at least a twofold difference in mean ex- pression) are retained. For these remaining cluster-specific candidate marker genes, gene sets with correlated expres- sion patterns are identified using the graph-based cluster- ing algorithm MCL. MCL does not require a pre-specified number of clusters and works on the gene correlation network derived from single-cell RNAseq data and detects communities in this network. These (gene) communities are guaranteed to contain genes that are co-expressed, by design. In contrast, in a k-means clustering with a pre- specified k, we cannot be sure that all genes within all clusters are co-expressed to the same degree: genes are assigned the closest centroid, but this is only a relative measure. Thus, by using communities of a gene corre- lation network, with a pre-specified correlation threshold, we can be sure that those communities (if such exist) satisfy the criteria of containing correlated genes. In a last step, cells within each cluster Cm are assigned to sub- groups by one-dimensional k-means clustering of their average expression of each gene set. The overall idea behind CellSIUS is similar to RaceID3 [38] and GiniClust2 [19], two recent methods for the identification of rare cell types in scRNA-seq datasets. All of these algorithms combine a global clustering with a second assignment method tailored to the identifi- cation of rare cell types. However, in contrast to existing methods, CellSIUS requires candidate marker genes to be cluster specific, and therefore, we hypothesized that our method will be more specific and less sensitive to genes that co-vary with confounders such as the total number of detected genes per cell. To overcome biases associated to the high dropout rates in scRNA-seq, Page 6 of 21 Wegmann et al. Genome Biology (2019) 20:142 Wegmann et al. Genome Biology (2019) 20:142 CellSIUS considers only cells that have non-zero expression for the selected marker genes. CellSIUS outperforms existing algorithms in the identification of rare cell populations Next, we adjusted the subtlety of the transcriptional difference between the rare cells and their closest neighbor (cell type 2) by swapping a fraction of gene expression values in the type 2 cells with the corresponding value in the left-out rare cells. We then pre-defined an initial cluster assignment as cluster 1 = type 1, cluster 2 = the union of type 2 and rare cells and assessed whether different algorithms for detecting rare cell types are able to correctly classify the rare cells as such. b, c Comparison of CellSIUS to GiniClust2 and RaceID3 for varying incidence of the rare cell type and varying subtlety of the transcriptional signature here, we used 100 HEK293 cells as type 1, 100 Ramos cells as type 2, and up to 10 Jurkat cells as the rare cell type and we swapped between 0 and 99.5% of gene expression values. For each algorithm, we assessed the recall (b), i.e., the fraction of correctly identified rare cells, and precision (c), i.e., the probability that a cell which is classified as rare is actually a rare cell. d tSNE projection of subset 2 of the cell line dataset, colored by CellSIUS assignment. Cluster numbers correspond to the main clusters identified by MCL, clusters labeled x.sub indicate the CellSIUS subgroups. Symbols correspond to the cell line annotation. e Violin plot showing the main markers identified by CellSIUS, grouped by cluster Fig. 4 CellSIUS benchmarking on cell line data. a Schematic overview of dataset perturbations. Starting from a dataset containing three cell types (abundant cell type 1, abundant cell type 2, and rare cell type), we first generated a defined number of rare cells by subsampling. In addition, we partitioned the type 2 cells in two, leaving out 25 cells from the dataset for later use. Next, we adjusted the subtlety of the transcriptional difference between the rare cells and their closest neighbor (cell type 2) by swapping a fraction of gene expression values in the type 2 cells with the corresponding value in the left-out rare cells. We then pre-defined an initial cluster assignment as cluster 1 = type 1, cluster 2 = the union of type 2 and rare cells and assessed whether different algorithms for detecting rare cell types are able to correctly classify the rare cells as such. CellSIUS outperforms existing algorithms in the identification of rare cell populations However, CellSIUS exhibited high precision Wegmann et al. Genome Biology (2019) 20:142 Page 7 of 21 Fig. 4 CellSIUS benchmarking on cell line data. a Schematic overview of dataset perturbations. Starting from a dataset containing three cell types (abundant cell type 1, abundant cell type 2, and rare cell type), we first generated a defined number of rare cells by subsampling. In addition, we partitioned the type 2 cells in two, leaving out 25 cells from the dataset for later use. Next, we adjusted the subtlety of the transcriptional difference between the rare cells and their closest neighbor (cell type 2) by swapping a fraction of gene expression values in the type 2 cells with the corresponding value in the left-out rare cells. We then pre-defined an initial cluster assignment as cluster 1 = type 1, cluster 2 = the union of type 2 and rare cells and assessed whether different algorithms for detecting rare cell types are able to correctly classify the rare cells as such. b, c Comparison of CellSIUS to GiniClust2 and RaceID3 for varying incidence of the rare cell type and varying subtlety of the transcriptional signature here, we used 100 HEK293 cells as type 1, 100 Ramos cells as type 2, and up to 10 Jurkat cells as the rare cell type and we swapped between 0 and 99.5% of gene expression values. For each algorithm, we assessed the recall (b), i.e., the fraction of correctly identified rare cells, and precision (c), i.e., the probability that a cell which is classified as rare is actually a rare cell. d tSNE projection of subset 2 of the cell line dataset, colored by CellSIUS assignment. Cluster numbers correspond to the main clusters identified by MCL, clusters labeled x.sub indicate the CellSIUS subgroups. Symbols correspond to the cell line annotation. e Violin plot showing the main markers identified by CellSIUS, grouped by cluster Fig. 4 CellSIUS benchmarking on cell line data. a Schematic overview of dataset perturbations. Starting from a dataset containing three cell types (abundant cell type 1, abundant cell type 2, and rare cell type), we first generated a defined number of rare cells by subsampling. In addition, we partitioned the type 2 cells in two, leaving out 25 cells from the dataset for later use. CellSIUS outperforms existing algorithms in the identification of rare cell populations b, c Comparison of CellSIUS to GiniClust2 and RaceID3 for varying incidence of the rare cell type and varying subtlety of the transcriptional signature here, we used 100 HEK293 cells as type 1, 100 Ramos cells as type 2, and up to 10 Jurkat cells as the rare cell type and we swapped between 0 and 99.5% of gene expression values. For each algorithm, we assessed the recall (b), i.e., the fraction of correctly identified rare cells, and precision (c), i.e., the probability that a cell which is classified as rare is actually a rare cell. d tSNE projection of subset 2 of the cell line dataset, colored by CellSIUS assignment. Cluster numbers correspond to the main clusters identified by MCL, clusters labeled x.sub indicate the CellSIUS subgroups. Symbols correspond to the cell line annotation. e Violin plot showing the main markers identified by CellSIUS, grouped by cluster Wegmann et al. Genome Biology (2019) 20:142 Page 8 of 21 (88.4% on average), in comparison to GiniClust2 (51.6% on average) and RaceID3 (15.6% on average). from the dataset for downstream analyses. CR cells expressed DCX, CALB2, STMN2, and MAPT consistently with developing mouse and human cortex (Fig. 5b) [49– 51]. The robust expression of FOXG1 in the general popu- lation (Additional file 1: Figure S5a) and the expression of PAX6, EMX2, and LHX2 in NPs (Fig. 5b) indicated our differentiation protocol mainly generates cells with dorsal telencephalic identity [52]. g g Having shown that CellSIUS is more sensitive and specific for the identification of rare cell types and outlier genes using synthetic and simulated biological data, we tested its ability to reveal transcriptomic signatures indi- cative of rare cell type’s function(s). We applied CellSIUS to subset 2 of our dataset of known composition (Additional file 1: Table S1) with 6 clusters predefined using MCL (Fig. 4d). CellSIUS identified three subgroups (Jurkat, H1437, and a small subgroup of IMR90 cells) within the 6 initial clusters characterized by upregulation of three or more genes (Fig. 4e). CellSIUS outperforms existing algorithms in the identification of rare cell populations Notably, the two stron- gest signatures were obtained for the two subgroups corresponding to Jurkat and H1437 cells with top marker genes consistent with previous knowledge: CD3G and CD3D, both of which are known T cell markers [40] being the top markers for Jurkat (T cell lymphoma), and TFF1 and BPIFA2, both shown to function in the respiratory tract [41, 42] being the top markers for H1437 (lung adenocarcinoma, epithelial/glandular cell type). p y [ ] Applying CellSIUS to this data identified 7 subpopula- tions (Fig. 5c, d). Notably, within the mixed glial cells (G), CellSIUS identified a rare subgroup (1.1% of total population, G.sub_1) characterized by a signature of 10 genes. Nine of those ((TRPM3, PTGDS, TTR, CXCL14, HTR2C, WIF1, IGFBP7, MT1E, DLK1) are known to be enriched in primary pre-natal human choroid plexus (CP) (Fig. 5e) compared to the other tissues from the de- veloping human cortex (harmonizome database [47, 48] using a cutoff of 1.3 for the standardized value, corre- sponding to a Benjamini-Hochberg-corrected p adjusted < 0.05). This G.sub_1 population is therefore consistent with the formation of CP, a secretory neuroepithelial tissue that produces cerebrospinal fluid (CSF) and that has multiple origins along the rostro-caudal axis of the developing nervous system including the dorsal telence- phalic midline [53]. We further validated the presence of CP neuroepithelia in our 3D human cortical cultures by confocal microscopy analysis. Using neurosphere cryo- sections, we demonstrated co-localization of canonical CP marker transthyretin (TTR) with prostaglandin D2 synthase (PTGDS), another CP enriched protein de- scribed in primary mouse and human tissue, in a limited number of cells located almost exclusively on the per- iphery of neurospheres (Fig. 5f). Collectively, these results suggest that the 3D spheroid human cortical differentiation protocol described here can generate developmentally relevant cell types and that CellSIUS can identify rare cell populations within the heteroge- neity and complexity of stem cell-based models. Taken together, these results show that CellSIUS outperforms existing methods in identifying rare cell populations and outlier genes from both synthetic and biological data. In addition, CellSIUS simultaneously reveals transcriptomic signatures indicative of rare cell type’s function. Application to hPSC-derived cortical neurons generated by 3D spheroid directed-differentiation approach As a proof of concept, we applied our two-step approach consisting of an initial coarse clustering step followed by CellSIUS to a high-quality scRNA-seq dataset of 4857 hPSC-derived cortical neurons generated by a 3D cortical spheroid differentiation protocol generated using the 10X Genomics Chromium platform [3] (Additional file 1: Figure S4a and Table S3; see the “Methods” section). During this in vitro differentiation process, hPSCs are expected to commit to definitive neuroepithelia, restrict to dorsal telencephalic identity, and generate neocortical progenitors (NP), Cajal-Retzius (CR) cells, EOMES+ inter- mediate progenitors (IP), layer V/VI cortical excitatory neurons (N), and outer radial-glia (oRG) (Additional file 1: Figure S4b). We confirmed that our 3D spheroid protocol generates cortical neurons with expected transcriptional identity that continue to mature upon platedown with expression of synaptic markers and features of neuronal connectivity at network level [43] (Additional file 1: Figure S4c, d, e, and see the “Methods” section). y p y CellSIUS identified a second subgroup in the mixed glial cells (G) characterized by high expression levels of glycolytic enzymes (G.sub_2, 2.6%) (Fig. 5c, d and Additional file 1: Figure S6a). Analysis between G.sub_2 and the rest of the G cells revealed upregulation of HOPX, PTPRZ1, CLU, BCAN, ID4, and TTYH1 in the main group, a transcriptional signature consistent with developing human outer radial glia (oRG) [54], (Add- itional file 1: Figure S6a Additional file 2: Table S4). oRG cells also upregulated mitochondrial genes (Additional file 2: Table S4) that are crucial for oxidative phosphory- lation, highlighting the metabolic difference between these two groups. We hypothesize the G.sub_2 subgroup to be a progenitor population that is located closer to the hypoxic interior of neurospheres, a common feature of the 3D spheroid differentiation protocols. Initial coarse-grained clustering using MCL identified four major groups of cells that specifically express known markers for NPs [44], mixed glial cells (G), CR cells [45], and neurons (N) [46] (Fig. 5a, b). A small population of contaminating fibroblasts (0.1% of total cells) was removed Wegmann et al. Genome Biology (2019) 20:142 Page 9 of 21 A C E F D B Fig. 5 Characterization of hPSC-derived cortical excitatory neurons by scRNA-seq. a tSNE projection of 4857 single-cell transcriptomes of hPSC- derived neuronal cell types after 86 days of differentiation. Unsupervised clustering using MCL groups cells into four major classes: Neurons (N), neuroepithelial progenitors (NP), mixed glial cells (G), and Cajal-Retzius cells (CR). Application to hPSC-derived cortical neurons generated by 3D spheroid directed-differentiation approach In addition, a small population of fibroblasts (Fib) is identified. b The identified cell populations are characterized by expression of known markers for the expected cell types. Expression values are shown as log2 (normalized UMI counts + 1). c tSNE projection, colored by CellSIUS assignment. Main clusters are denoted .main, subclusters .sub. d Mean expression of each marker gene set identified by CellSIUS, projected onto the same tSNE map as shown in a. The top markers are indicated for each gene sets; numbers in brackets refer to how many additional genes are part of the marker gene set. e Comparison of the gene signature uncovered by CellSIUS to genes found to be enriched (p < 0.05) in choroid plexus of the fourth ventricle according to harmonizome [47, 48]. f Single optical sections of neurosphere cryosections acquired by confocal microscopy showing co-localization of TTR and PTGDS in cells predominantly on the periphery of neurospheres (panel left—composite image of a neurosphere; panels right—split images from a different neurosphere) B A A B C D D C E F Fig. 5 Characterization of hPSC-derived cortical excitatory neurons by scRNA-seq. a tSNE projection of 4857 single-cell transcriptomes of hPSC- derived neuronal cell types after 86 days of differentiation. Unsupervised clustering using MCL groups cells into four major classes: Neurons (N), neuroepithelial progenitors (NP), mixed glial cells (G), and Cajal-Retzius cells (CR). In addition, a small population of fibroblasts (Fib) is identified. b The identified cell populations are characterized by expression of known markers for the expected cell types Expression values are shown as log E F E Fig. 5 Characterization of hPSC-derived cortical excitatory neurons by scRNA-seq. a tSNE projection of 4857 single-cell transcriptomes of hPSC- derived neuronal cell types after 86 days of differentiation. Unsupervised clustering using MCL groups cells into four major classes: Neurons (N), neuroepithelial progenitors (NP), mixed glial cells (G), and Cajal-Retzius cells (CR). In addition, a small population of fibroblasts (Fib) is identified. b The identified cell populations are characterized by expression of known markers for the expected cell types. Expression values are shown as log2 (normalized UMI counts + 1). c tSNE projection, colored by CellSIUS assignment. Main clusters are denoted .main, subclusters .sub. d Mean expression of each marker gene set identified by CellSIUS, projected onto the same tSNE map as shown in a. Application to hPSC-derived cortical neurons generated by 3D spheroid directed-differentiation approach The top markers are indicated for each gene sets; numbers in brackets refer to how many additional genes are part of the marker gene set. e Comparison of the gene signature uncovered by CellSIUS to genes found to be enriched (p < 0.05) in choroid plexus of the fourth ventricle according to harmonizome [47, 48]. f Single optical sections of neurosphere cryosections acquired by confocal microscopy showing co-localization of TTR and PTGDS in cells predominantly on the periphery of neurospheres (panel left—composite image of a neurosphere; panels right—split images from a different neurosphere) Fig. 5 Characterization of hPSC-derived cortical excitatory neurons by scRNA-seq. a tSNE projection of 4857 single-cell transcriptomes of hPSC- derived neuronal cell types after 86 days of differentiation. Unsupervised clustering using MCL groups cells into four major classes: Neurons (N), neuroepithelial progenitors (NP), mixed glial cells (G), and Cajal-Retzius cells (CR). In addition, a small population of fibroblasts (Fib) is identified. b The identified cell populations are characterized by expression of known markers for the expected cell types. Expression values are shown as log2 (normalized UMI counts + 1). c tSNE projection, colored by CellSIUS assignment. Main clusters are denoted .main, subclusters .sub. d Mean expression of each marker gene set identified by CellSIUS, projected onto the same tSNE map as shown in a. The top markers are indicated for each gene sets; numbers in brackets refer to how many additional genes are part of the marker gene set. e Comparison of the gene signature uncovered by CellSIUS to genes found to be enriched (p < 0.05) in choroid plexus of the fourth ventricle according to harmonizome [47, 48]. f Single optical sections of neurosphere cryosections acquired by confocal microscopy showing co-localization of TTR and PTGDS in cells predominantly on the periphery of neurospheres (panel left—composite image of a neurosphere; panels right—split images from a different neurosphere) Page 10 of 21 Page 10 of 21 Wegmann et al. Genome Biology (2019) 20:142 Wegmann et al. Genome Biology (2019) 20:142 In addition, CellSIUS identified a subgroup of NP cells (NP.sub, 10.6%) defined by upregulation of cell-cycle- related genes such as HMGB2, TOP2A, and MKI67 (Fig. 5c, d, Additional file 1: Figure S6a) as well as a sub- group of CR cells (CR.sub, 0.8%) characterized by SEMA3E, BTG1, and PCDH11X (Fig. Application to hPSC-derived cortical neurons generated by 3D spheroid directed-differentiation approach 5b and Additional file 1: Figure S6A) which may represent CR cells at a different stage of migration [55–57]. sub-population (Additional file 1: Figure S5d-e) could suggest patterning towards cortico-spinal motor neurons (CSMNs). However, the presence of NTS, which encodes a 13-amino acid neuropeptide called neurotensin highly expressed in the hypothalamus and amygdala, is not in line with the overall transcriptional identity as discussed above. Analysis of a recently published scRNA-seq dataset from different regions and developmental stages of the human cortex [46] revealed that only a few cells derived from the fetal primary visual cortex (age 13 pcw) express NTS (Additional file 1: Figure S7). The limited number of cells in our dataset limits any firm conclusions. Finally, CellSIUS revealed a split in the neuronal popu- lation (N), identifying 2 groups, N.sub_2 (8.6%) and N.sub_1 (16.7%) (Fig. 5c, d, Additional file 1: Figure S6a) . In addition to NHLH1 and PPP1R17 known to be enriched in immature neurons [54], N.sub_2 expressed EOMES (Additional file 1: Figure S5b), a well-characterized marker of cortical intermediate progenitors [46, 54] that give rise to TBR1+ cortical neurons (Additional file 1: Figure S5c) and is likely a mixed population of interme- diate progenitors and immature neurons. In contrast, markers identified by CellSIUS for the N.sub_1 neuronal population were unexpected. Although co-expression of FEZF2, CRYM, PCDH17, and RUNX1T1 in this cortical neuronal population is consistent with recent scRNA-seq data from the developing human cortex (Additional file 1: Figure S7b, EN-V1–1: Early-born deep-layer/sub-plate excitatory neurons, EN-PFC1: Early-born deep-layer/sub- plate excitatory neurons prefrontal cortex), robust NTS expression in developing cortical neurons has not been reported so far to the best of our knowledge. The expres- sion of FEZF2 (Additional file 1: Figure S5d) in this culture which is consistent with the general dorsal telencephalic identity of these cells and co-expression of FEZF2 and BCL11B (CTIP2) in this particular post-mitotic neuronal y To further characterize the transition from progenitors to the two different neuronal cell types (CR cells and all N populations), we applied Monocle for trajectory analysis to a subset of the cells corresponding to these three identities. This analysis revealed a tree with two branches (Fig. 6a). As expected, cells progress from the tree root which is composed of progenitors via the NHLH1high/PPP1R17high population towards either N (branch 1) or CR cells (branch 2). Comparison of CellSIUS, RaceID3, and Giniclust2 performance for rare cell type identification in hPSC- derived cortical neurons To get an understanding of how CellSIUS, GiniClust2, and RaceID3 differ in the identification of rare cell types from a complex dataset, we compared their output when run on the cortical neuron datasets. Because a classic benchmarking is not possible here due to the lack of a ground truth, we instead focus on comparing the ability of each algorithm to reveal experimentally validated signatures or cell types known from literature. As before, we used the same initial of 4 main clusters identified by MCL (Fig. 5a) for all algorithms. GiniClust2 resulted in a total of 20 clusters. The main differences between GiniClust2 and CellSIUS (Additional file 1: Figure S6b) results can be summarized as follows: (i) GiniClust2 generated clusters that merge major known cell types (for example cluster 14 merges G, G.sub_1 (=CP), G.sub_2, N, N.sub_1 (late neurons) and N.sub_2 (early neurons)), and (ii) GiniClust2 did not detect CP (G.Sub_1), cycling NPs (NP.sub) nor the well-described immature neurons (N.sub_2). In contrast, hclust in combination with dynamicTree- Cut, MCL, and DBSCAN resulted in accurate cluster assignments across all subsampled datasets. Strikingly, none of the methods we tested was able to identify rare cell types (< 1% in this dataset). It is worth noting that although DBSCAN does classify rare cell types as border points, it did however not reliably identify these popula- tions for two reasons: (i) additional cells that did not belong to the rare populations are also classified as border points; (ii) DBSCAN does not perform well if there are points connecting clusters, which is often the case in scRNA-seq datasets. In summary, our com- parison of clustering methods is consistent with a recent review describing the challenges in unsupervised cluster- ing of single-cell RNA-seq data [16], highlighting the methodology gap for detecting rare cell types. RaceID3 with default settings resulted in a total of > 50 clusters, consistent with the high false-positive rate observed with synthetic and cell line data. With a more stringent outlier probability cutoff (10−20), RaceID3 identi- fied 10 clusters with a similar overall assignment to CellSIUS (Additional file 1: Figure S6c). However, if RaceID3 did partly detect CP (G.Sub_1), it also split the CP cluster identified by CellSIUS across several other clusters with the majority of cells assigned to either cluster 3 (19 CP together with 4 other cells) or cluster 5 (mixed with a large number of G, N, and NP cells). Comparison of CellSIUS, RaceID3, and Giniclust2 performance for rare cell type identification in hPSC- derived cortical neurons The CP markers PTGDS and TTR are co-expressed in 49/53 CP cells identified by CellSIUS but only in 19/54 CP cells identified by RaceID3 suggesting that RaceID3 incorrectly assigned most of the CP cells to a merged G/NP/N cluster. In addition, and similarly to GiniClust2, RaceID3 did identify neither cycling NPs (NP.sub) nor the above-described progenitors and immature neurons population (N.sub_2). To overcome these limitations, we developed CellSIUS, a novel algorithm that takes initial coarse clusters as input and identifies rare cell subtypes based on correlated gene sets specific to subpopulations. Based on our comparison of clustering methods above, we used MCL as our default clustering method: MCL showed a high accuracy in the comparison to other methods, requires fewer parameter choices than hclust for defining the number of clusters, and, unlike DBSCAN, assigns all points to clusters. The overall idea behind CellSIUS is similar to RaceID3 [38] and GiniClust2 [19], two recent methods for the identification of rare cell types in scRNA-seq datasets. All of these algorithms combine a global clustering with a second assignment method which is tailored to finding rare cell types. There are however important differences between the approaches which are at the basis of CellSIUS’ superior performance for both rare cell type as well as outlier genes’ identification in terms of specifi- city and selectivity. In summary, these results indicate superior performance with regard to specificity and sensitivity of CellSIUS com- pared to other approaches when applied to the complex and heterogeneous data generated here and demonstrate the algorithm’s ability to identify rare populations within major cell types that differ by their metabolic state, cell cycle phase, or migratory state. Application to hPSC-derived cortical neurons generated by 3D spheroid directed-differentiation approach Along the trajectory, the NP marker VIM decreases gradually whereas NHLH1 increases up to the branch point, then decreases again (Fig. 6b). The CR branch ends with cells expressing high levels of RELN, and the N branch is characterized by gradual increase of FEZF2 expression and ending in the N.sub_1 population (Fig. 6b). Notably, at the very tip of this branch, we also find a very small number of cells expressing LDB2 and DIAPH3 which are markers of CSMNs in the mouse [58]. It is plausible that, given more time, this population may eventually give rise to CSMNs with a more defined transcriptional signature. A B Fig. 6 Monocle analysis of the NP, N, and CR cluster. a Consistent with the subgroup assignment by CellSIUS, monocle orders cells on a trajectory from NP via immature neurons (N_early) to either mature N or CR cells. b Gene expression along pseudotime. Shown is a marker for NPs (VIM), immature neurons (NHLH1), N.sub_2 (FEZF2), and CR cells (RELN) B B A B A Fig. 6 Monocle analysis of the NP, N, and CR cluster. a Consistent with the subgroup assignment by CellSIUS, monocle orders cells on a trajectory from NP via immature neurons (N_early) to either mature N or CR cells. b Gene expression along pseudotime. Shown is a marker for NPs (VIM), immature neurons (NHLH1), N.sub_2 (FEZF2), and CR cells (RELN) Page 11 of 21 Wegmann et al. Genome Biology (2019) 20:142 Wegmann et al. Genome Biology (2019) 20:142 Wegmann et al. Genome Biology (2019) 20:142 Our findings suggest that in our dataset, for unsuper- vised feature selection, the DANB methods implemented in the M3Drop package outperformed HVG. While all clustering methods tested performed equally well on data with balanced and abundant cell populations, k-means and model-based methods performed poorly on subsampled datasets with unequal cell type pro- portions, typically splitting clusters containing many cells while merging those containing few cells. This is likely a consequence of feature selection and PCA-based di- mensionality reduction prior to clustering where these methods select or assign weights to genes based on mean expression and variance across the whole cell population, which are both low if a gene is specifically expressed in a small subset of cells only. Application to hPSC-derived cortical neurons generated by 3D spheroid directed-differentiation approach Comparison of CellSIUS, RaceID3, and Giniclust2 performance for rare cell type identification in hPSC- derived cortical neurons Comparison of CellSIUS, RaceID3, and Giniclust2 performance for rare cell type identification in hPSC- derived cortical neurons Discussion g g To exemplify the added value of CellSIUS over existing approaches in a real-world setting, we applied the work- flow and our two-step clustering approach to a complex biological dataset consisting of hPSC-derived neurons. We identified major neural cell types of early human cortico- genesis such as cycling and quiescent NPs, EOMES+ IPs, CR cells, immature and mature neurons with a transcrip- tional identity indicative of layer V/VI neurons, and oRG. Overall, the transcriptional fingerprint of each major group was in line with a recent scRNA-seq data set from the developing human cortex. CellSIUS analysis also revealed a transcriptional signature in the mature neu- ronal population that deviates from the expected cortical trajectory, typified by the high expression levels of NTS detected in N.sub_1, highlighting the importance of unbiased characterization of hPSC differentiation plat- forms at single-cell level. Single-cell trajectory analysis of NP, CR, and N cells using Monocle revealed a pseudo- temporal order of progenitors gradually differentiating into neurons, with a lineage split between Cajal-Retzius cells and FEZF2+ neurons. GiniClust2 runs two independent clustering steps on the same data. The first clustering aims at capturing global structure of the data by running a k-means clustering on the expression of genes with a high Fano factor. This is motivated by the fact that a high Fano factor is associated with genes that are differentially expressed between abundant cell types. The second clustering is performed by running a density-based clus- tering on genes with a high Gini index which is typically associated with genes being differentially expressed between rare and abundant cells. In a final step, the results of both clustering are merged based on a weighted consensus association. The main differences to CellSIUS are as follows: (i) the selection of the genes for the rare cell type assignment is performed using a global metric (i.e., the Gini coefficient across the whole data- set), whereas CellSIUS takes into account the in- formation on the global clustering (e.g., considers only cluster-specific genes), and (ii) the final assignment is a weighted average of the results from both clustering steps, whereas we use a two-step approach consisting of an initial coarse clustering step followed by CellSIUS for the identification of rare cell types and outlier genes. Discussion We generated a benchmark dataset of ~ 12,000 single- cell transcriptomes from 8 cell lines to compare the per- formance of some of the most recent and widely used scRNA-seq feature selection and clustering approaches. RaceID3’s initial step is a k-medoids clustering, followed by outlier cell identification in each cluster in four steps: (i) calibration of a background model of gene expression by fitting a negative binomial distribution to the mean and Page 12 of 21 Page 12 of 21 Wegmann et al. Genome Biology (2019) 20:142 Page 12 of 21 Wegmann et al. Genome Biology (2019) 20:142 variance of each gene in each cluster; (ii) identification of outlier cells by calculating for each gene and each cell the probability of observing this expression value under the assumption of the background model; (iii) merging of potential outlier cells into new clusters based on the similarity of their gene expression; and (iv) definition of new cluster centers for both the original and the outlier clusters. In a final step, cells are assigned to the cluster they are closest to. In contrast to CellSIUS, RaceID3 does not require the outlier genes to be cluster specific; con- sequently, it may select genes that co-vary with technical confounders such as the total number of detected genes per cell. In addition, whereas CellSIUS only considers subcluster-specific genes to assign cells to final clusters, the final cluster assignment in RaceID3 is done based on the similarity of each cell’s whole transcriptomic signature to each cluster center. In cases where the distance between the outlier cluster and neighboring clusters is small, this leads to a high number of false positives, with many cells initially not identified as outliers being merged into the nearest outlier cluster. to help disentangle technical and biological variability and increase the precision of rare cell type identification, it comes with the limitation of potentially missing rare cell types spread over multiple clusters. This issue could be addressed by iteratively merging the most similar clusters and re-running CellSIUS for each initial cluster definition. A further consideration is CellSIUS’ output sensitivity to initial cluster assignments. In practice, this should only be an issue if there is no clear global structure in the data and cluster assignments are not consistent between different clustering methods and/ or parameter settings. In such cases, one could use a consensus assignment from a combination of different clustering assignments. Discussion Importantly, CellSIUS identified known as well as novel rare cell types within the major groups, such as putative CP (G.sub_1), a population that was either not detected, or detected only partly by existing approaches for rare cell type identification. Single-cell RNA-seq data usually contains a small fraction of doublets, i.e., tran- scriptomes derived from two or more cells, which could form artifactual clusters. Our results do not indicate the presence of doublet-driven clusters—each subcluster has its own unique markers. In addition, most of the sub- population signatures represent biological function that is supported by the literature. Finally, we experimentally validated the presence of CP neuroepithelia in our 3D cortical spheroid cultures by confocal microscopy and validated the CP-specific signature gene list identified by CellSIUS using primary pre-natal human data. For the CP lineage in particular and other identified rare cell populations in general, the signature gene lists output from CellSIUS provide the means to isolate these Enforcing gene signatures to be cluster-specific comes with the promise to overcome some technical biases, e.g., different number of detected genes between cells, differences in the total number of counts per cell or normalization artifacts. For example, normalization may lead to artificially high counts for abundant transcripts in cells that have overall few detected genes. These genes are, however, present across different clusters and would therefore not be considered a valid signature. While restricting to cluster-specific signatures has the potential Page 13 of 21 Page 13 of 21 Wegmann et al. Genome Biology (2019) 20:142 Wegmann et al. Genome Biology (2019) 20:142 Cell 3′ Gel Bead and Library Kit according to CG00052_ SingleCell3’ReagentKitv2UserGuide_RevB. GEM-RT was performed in a Bio-Rad PTC-200 Thermal Cycler with semi-skirted 96-well plate (Eppendorf, P/N 0030 128.605): 53 °C for 45 min and 85 °C for 5 min, held at 4 °C. After RT, GEMs were broken and the single strand cDNA was cleaned up with DynaBeads® MyOne™Silane Beads (Life Technologies P/N, 37002D). cDNA was amplified using a Bio-Rad PTC-200 Thermal cycler with 0.2-ml 8-strip non- Flex PCR tubes, with flat Caps (STARLAB, P/N I1402– 3700): 98 °C for 3 min; cycled 12x: 98 °C for 15 s, 67 °C for 20 s, and 72 °C for 1 min; 72 °C for 1 min; and held at 4 °C. Amplified cDNA product was cleaned up with the SPRIse- lect Reagent Kit (0.6X SPRI). Discussion Indexed sequencing libraries were constructed using the reagents in the Chromium Single Cell 3′ library kit V2 (10x Genomics P/N-120237), following these steps: (1) fragmentation, end-repair and A-tailing; (2) post fragmentation, end-repair, and A-tailing double sided size selection with SPRIselect Reagent Kit (0.6X SPRI and 0.8X SPRI); (3) adaptor ligation; (4) post-ligation cleanups with SPRIselect (0.8X SPRI); (5) sample index PCR using the Chromium Multiplex kit (10x Genomics P/N-120262); (6) post sample index double sided size selection—with SPRIselect Reagent Kit (0.6X SPRI and 0.8X SPRI). The barcode se- quencing libraries were quantified using a Qubit 2.0 with a Qubit™dsDNA HS Assay Kit (Invitrogen P/N Q32854), and the quality of the libraries was performed on a 2100 Bioanalyzer from Agilent using an Agilent High Sen- sitivity DNA kit (Agilent P/N 5067–4626). Sequencing libraries were loaded at 10 pM on an Illumina HiSeq2500 populations for in vitro propagation and characterization of their role in neurological disorders. Conclusions In this study, we present CellSIUS, a novel method to identify and characterize rare cell types from complex scRNA-seq datasets. Benchmarking of CellSIUS on syn- thetic data and a large dataset with known cell compo- sition generated from 8 human cell lines demonstrated the high sensitivity and specificity of CellSIUS over existing approaches. Characterization of a novel human pluripotent cell differentiation protocol recapitulating deep-layer corticogenesis in vitro using scRNA-seq and CellSIUS revealed previously unrecognized complexities in human stem cell-derived cellular populations. Impor- tantly, CellSIUS enabled identification of known and novel rare cell populations and their signature gene list pro- viding the means to study those populations in vitro in light of their role in health and disease. Human cell lines For the benchmarking dataset, 8 different human cell lines from the ATCC biorepository have been used (Table 1). Cell lines were shown to be mycoplasma free using the Mycoalert kit from Lonza. Single-cell RNA-sequencing of cell lines Single-cell RNA-sequencing of cell lines Cellular suspensions were loaded on a 10x Genomics Chro- mium Single Cell instrument to generate GEMs. Single-cell RNA-seq libraries were prepared using GemCode Single Table 1 Cell lines and culture conditions used in this study Table 1 Cell lines and culture conditions used in this study Cell line Gender Cell type Tissue of origin Obtained from Culture conditions A549 M Alveolar basal epithelial (adherent) Lung adenocarcinoma ATCC CCL-185 ATCC F12K (ATCC, P/N 30-2004) +10% FCS (AMIMED, P/N 2-01F36-I). H1437 M Epithelial/glandular (adherent) Lung adenocarcinoma, derived from metastatic site: pleural effusion ATCC CRL-5872 RPMI (Invitrogen, P/N A1049101) +10% FBS (ATCC, P/N SCRR-30-2020) HCT116 M Epithelium-like (adherent) Colon carcinoma ATCC CCL-247 ATCC McCoy's 5A (ATCC, P/N 30-2007) + 10% FCS (AMIMED, P/N 2-01F36-I) HEK293 F Epithelial (adherent) Transformed cell line, derived from embryonic kidney ATCC, P/N CRL-1573 ATCC EMEM (ATCC, P/N 30-2003) +10% FCS (AMIMED, P/N 2-01F36-I) IMR90 F Fibroblast (adherent) Fetal lung ATCC CRL-186 ATCC EMEM (ATCC, P/N 30-2003) 10% FCS (AMIMED, P/N 2-01F36-I) Jurkat M T cell (suspension) Childhood T acute lymphoblastic leukemia ATCC, P/N TIB-152 RPMI (Invitrogen, P/N 61870-044) + 10% FCS (AMIMED, P/N 2-01F36-I) K562 F Undifferentiated, lymphoblast with granulocyte/erythrocyte/ monocyte characteristics (suspension) Chronic myelogenous leukemia, BCR-ABL1 positive ATCC, P/N CRL-1573 RPMI (Invitrogen, P/N 61870-044) + 10% FCS (AMIMED, P/N 2-01F36-I). Ramos M B cell (suspension) Burkitt’s lymphoma ATCC, P/N CRL-1596 Batch 3: RPMI (Invitrogen, P/N A1049101) +10% FBS (ATCC, P/N SCRR-30-2020) Batch 4: RPMI (Invitrogen, P/N 61870-044) + 10% FCS (AMIMED, P/N 2-01F36-I) Page 14 of 21 Wegmann et al. Genome Biology (2019) 20:142 Wegmann et al. Genome Biology (2019) 20:142 (Corning, 354,008), and Matrigel (Corning, 354,230) coated plates. (Corning, 354,008), and Matrigel (Corning, 354,230) coated plates. (Corning, 354,008), and Matrigel (Corning, 354,230) coated plates. (Corning, 354,008), and Matrigel (Corning, 354,230) coated plates. with 2 × 50 paired-end kits using the following read length: 26 cycles Read1, 8 cycles i7 Index, and 98 cycles Read2. The CellRanger suite (2.0.2) was used to generate the aggregated gene expression matrix from the BCL files generated by the sequencer based on the hg38 Cell Ranger human genome annotation files. Immunofluorescence and cryosectioning Immunofluorescence and cryosectioning Cells were fixed with 4% PFA, permeabilized with 0.2% Triton X-100 at room temperature, and then blocked in 3% goat serum, followed by incubation with primary (TBR1 - Abcam, ab31940; CTIP2 – Abcam, ab18465; β-3 tubulin – Biolegend, 801,202; PSD-95 – Synaptic Systems, 124,011; Synaptophysin 1 – Synaptic Systems, 101,002; Transthyretin – Novus Biologicals, NBP2–52575, Pros- taglandin D Synthase (PTGDS) – Abcam, ab182141) and secondary antibodies (Alexa Flours, Invitrogen). The nuclei were counter-stained with 49,6-diamidino-2-pheny- lindole (DAPI, Sigma). Cryosectioning of neurospheres was performed as previously described [61]. Cells were imaged using an Observer D1 (Zeiss) microscope or Olympus SD-OSR spinning-disk confocal microscope (60x oil immersion). The images were processed using Zen 2 (Zeiss), MetaMorph, or Image J (brightness and contrast adjustments, thresholding for composite images) and assembled using Adobe Photoshop CS6. Characterization of cortical neurons generated by the 3D spheroid protocol Generation of layer V/VI neuronal populations was con- firmed by immuno-fluorescence analysis of D86 cultures upon dissociation and plating, showing robust expression of deep-layer cortical neuronal markers TBR1 and CTIP2 (Additional file 1: Figure S4c). Cortical neurons generated by the 3D spheroid protocol co-cultured with rat glia for 4 weeks were positive for pre- and post-synaptic markers Synaptophysin I and PSD-95 (Additional file 1: Figure S4d). Calcium imaging by FDSS 7000EX platform demonstrated spontaneous intracellular calcium oscillations, indi- cating that spontaneous firing was synchronized between the majority of the cortical neurons in the 96-wells (Additional file 1: Figure S4e). Bulk RNA-sequencing of cell lines For each individual cell line, RNA was isolated from 5 × 105 cells using the RNeasy Micro kit (Qiagen, Cat# 74104). The amount of RNA was quantified with the Agilent RNA 6000 Nano Kit (Agilent Technologies, Cat# 5067–1511). RNA sequencing libraries were prepared using the Illumina TruSeq RNA Sample Prep kit v2 and sequenced using the Illumina HiSeq2500 platform. Samples were sequenced to a length of 2 × 76 base-pairs. Read pairs were mapped to the Homo sapiens genome (GRCh38) and the human gene transcripts from Ensembl version 87 [59] by using an in- house gene quantification pipeline [60]. Genome and tran- script alignments were used to calculate gene counts based on Ensembl gene IDs. Calcium imaging 086), 1 μL 10% Tween 20 (Bio-Rad, 1,610,781), and 1 μL Additive A (10X, 220,074). The solution was then incu- bated for 1 min at room temperature and placed back onto the magnetic separator. Thirty-five microliters of eluted sample was transferred to a new tube strip. cDNA amplification reaction mix was prepared from 8 μL water, 50 μL Amplification Master Mix (10X, 220,125), 5 μL cDNA Additive (10X, 220,067), and 2 μL cDNA Primer Mix (10X, 220,106). Sixty-five microliters of amplification master mix was added to the sample, mixed 15 times via pipetting, and briefly centrifuged. The sample then underwent 12 amplification cycles (15 s at 98 °C, 20 s at 67 °C, 1 min at 72 °C). 086), 1 μL 10% Tween 20 (Bio-Rad, 1,610,781), and 1 μL Additive A (10X, 220,074). The solution was then incu- bated for 1 min at room temperature and placed back onto the magnetic separator. Thirty-five microliters of eluted sample was transferred to a new tube strip. cDNA amplification reaction mix was prepared from 8 μL water, 50 μL Amplification Master Mix (10X, 220,125), 5 μL cDNA Additive (10X, 220,067), and 2 μL cDNA Primer Mix (10X, 220,106). Sixty-five microliters of amplification master mix was added to the sample, mixed 15 times via pipetting, and briefly centrifuged. The sample then underwent 12 amplification cycles (15 s at 98 °C, 20 s at 67 °C, 1 min at 72 °C). g g The intracellular Ca2+ oscillations in human cortical neuron and rat glia co-cultures were assessed using the FLIPR Calcium 6 Kit (Molecular Devices LLC, San Jose, California). Briefly, 96-well Greiner μ-clear plates (655097) were seeded with 2500 rat glia (Lonza, R- CXAS-520) per well in Ph IV media and cultured for 7 days. Human cortical neurospheres were dissociated with papain as described above at DIV 56, and 50,000 single cells per well were plated on rat glia in phase IV media. Co-cultures were maintained for 4 weeks with twice-weekly 50% medium exchange. Cells were loaded with calcium 6 dye for an hour which was reconstituted in imaging buffer (NaCl 2.5 mM, KCl 125 mM, KH2PO4 1.25 mM, CaCl2 2 mM, MgCl2 2 mM, HEPES (acid) 25 mM, D-glucose 30 mM, pH 7.4, filter-sterilized). Differentiation of cortical excitatory neurons from human pluripotent stem cells in suspension H9-hESCs (WA09) were obtained from WiCell and maintained in TeSR-E8 medium (Stemcell Tech., 05990) on tissue-culture plates coated with vitronectin (Gibco, A14700). hESCs were passaged using ReLeSR (Stemcell Tech., 05873) to dissociate into cell clumps and were replated in E8 plus thiazovivin (Selleckchem, S1459) at 0.2 μM. H9-hESC line was free of mycoplasma and was tested using the Mycoalert detection kit (Lonza). g y hESCs were changed to mTesR1 (Stemcell Tech., 85, 850) media when they were 70–80% confluent and maintained in mTesR1 for a minimum of 2 days before confluent monolayer of hESCs were neurally converted by changing the media to phase I (Additional file 1: Table S5). Seven days post induction, cells were disso- ciated to single-cell suspension with Accutase (Gibco A1110501), seeded at 1.5E6 cells/mL in spinner flasks with phase II media (Additional file 1: Table S5) supple- mented with 2 μM Thiazovivin and 10 ng/mL FGF2 (Peprotech, 100-18B) (final) and incubated at 37 °C on a micro-stir plate at 40 rpm for 4 days. Media was then changed to phase III (Additional file 1: Table S5), and neurospheres were further cultured for 17 days at 60 rpm, changing media 50% twice a week. On day 28, media were changed to phase IV (Additional file 1: Table S5) and cultures were maintained 21 more days with 50% media change twice a week. From day 49 on- wards, cultures were switched to Ph IV media for main- tenance. Neurospheres were dissociated with Papain kit (Worthington) at day 86 for single-cell RNAseq or neu- ronal platedowns on laminin (Sigma, L2020), fibronectin g p Antibody validation: TBR1: validated on Mouse Hippocampus Tissue Lysate, Rat Hippocampus Tissue Lysate, Human cerebral cortex. CTIP2: validated by IHC on adult mouse hippocampus and adult mouse spinal cord and by ICC on neonatal mouse hippocampal cultured neurons. b3-tubulin: Quality control tested by formalin-fixed paraffin-embedded immunohistochemical staining. PSD-95: Knock-out verified, validated by IF on rat hippocampal neurons. Synaptophysin I: Does not cross-react with other synaptophysins, validated by IF on hippocampal neurons. TTR: Validated by IF analysis of A549 and MCF-7 cells and IHC of human liver tissue. PTGDS: Validated by IF on HEPG2 cells and IHC on human prostate tissue. All information is from supplier product data sheets. Page 15 of 21 Wegmann et al. Genome Biology (2019) 20:142 Wegmann et al. Genome Biology (2019) 20:142 Calcium imaging Kinetics of Ca2+ oscillations were determined as fluorescence intensity at 540 nm following excitation at 480 using the FDSS 7000EX Functional Drug Screening System (Hamamatsu) maintained at a constant 37 °C throughout the assay. A total of 3000 reads per assay were recorded. The exposure time per read was 100 ms with sensitivity set to 1. SPRIselect beads (Beckman Coulter, B23318) were then applied at 0.6X, and solution was mixed 15 times via pipetting. The sample was incubated at room temperature for 5 min, placed onto a magnetic separator, and washed twice with 80% ethanol. Sample was air- dried for 2 min and eluted in 40.5 μL Buffer EB. cDNA yield was measured on a 2100 Bioanalyzer (Agilent, G2943CA) via DNA High Sensitivity Chip (Agilent, 5067–4626). Fragmentation mix was prepared at 4 °C from 10 μL fragmentation enzyme blend (10X, 220,107) and 5 μL fragmentation buffer (10X, 220,108). Thirty-five microli- ters of sample cDNA was then added to the chilled frag- mentation mix. Sample was incubated for 5 min at 32 °C, then 30 min at 65 °C to conduct enzymatic fragmenta- tion, end repair, and A-tailing. Sample was then purified using 0.6X SPRIselect reagent (see above). Adaptor ligation mix was prepared from 17.5 μL water, 20 μL ligation buffer (10X, 220,109), 10 μL DNA ligase (10X, 220,110), and 2.5 μL Adaptor Mix (10X, 220,026). The ligation mix was added to 50 μL of sample and mixed 15 times via pipetting. Sample was then incubated for 15 min at 20 °C to conduct the ligation. The sample was purified using 0.8X SPRIselect reagent (see above). Sample index PCR mix was prepared from 8 μL water, 50 μL Amplification Master Mix (10X, 220,125), and 2 μL SI-PCR Primer (10X, 220,111). 60 μL sample index PCR mix, 30 μL purified sample, and 10 μL of sample index (10X, 220,103) were combined and mixed 15 times via pipetting. Indexing was conducted via 9 cycles of 20 s at 98 °C, 30 s at 54 °C, then 20 s at 72 °C. Sample was purified via double-sided SPRI selection at 0.6X and 0.8X, respectively. Sample was then quantified via DNA High Sensitivity Chip. Single-cell RNA-sequencing of neuronal cells g q g Cells were resuspended to 1 million cells/mL and run through the 10X Chromium, Version 2, single-cell RNA- seq pipeline per vendor’s instructions. Reverse transcrip- tion master mix was prepared from 50 μL RT reagent mix (10X, 220,089), 3.8 μL RT primer (10X, 310,354), 2.4 μL additive A (10X, 220,074), and 10 μL RT enzyme mix (10X, 220,079). 4.3 μL cell solution was mixed with 29.5 μL H2O and 66.2 μL reverse transcription master mix. Ninety-microliter sample was loaded onto the 10X Single Cell 3′ Chip along with 40 μL barcoded gel beads and 270 μL partitioning oil, and the microfluidics system was run to match gel beads with individual cells. The droplet solution was then slowly transferred to an 8-tube strip, which was immediately incubated for 45 min at 53 °C to perform reverse transcription, then 5 min at 85 °C. The sample was treated with 125 μL recovery agent (10X, 220,016), which was then removed along with the partitioning oil. Two hundred microliters of cleanup solution containing 4 μL DynaBeads MyOne Silane Beads (Thermo Fisher, 37002D), 9 μL water, 182 μL Buffer Sample Clean Up 1 (10X, 220,020), and Additive A (10X, 220,074) was added to the sample, and the solution was mixed 5 times by pipetting and allowed to incubate at room temperature for 10 min. Beads were separated via magnetic separator and supernatant was removed. While still on the magnetic separator, the beads were then washed twice with 80% ethanol. The separator was then removed and the beads were resuspended in 35.5 μL elution solution consisting of 98 μL Buffer EB (Qiagen, 19, Additional quantification was conducted via KAPA Li- brary Quantification Kit (Illumina, KK4828–07960166001). Sample was diluted at 10-fold increments from 1:100 to 1: 1,000,000, and mixed 1:9 with KAPA qPCR mix. qPCR was conducted on a Viia7 qPCR machine (Life Technologies). Sample was then sequenced on a HiSeq 4000 (Illumina) using 2 × 50-cycle SBS kits (Illumina, FC-410-1001). Page 16 of 21 Page 16 of 21 Wegmann et al. Genome Biology (2019) 20:142 Wegmann et al. Genome Biology (2019) 20:142 Sample library was diluted to 2 nM in EB buffer with 1% PhiX spike-in. Five microliters nondenatured library was then mixed with 5 μL 0.1 N NaOH, then vortexed and briefly centrifuged. Software requirements and scRNA-seq workflow Software requirements and scRNA-seq workflow is derived from the second-order polynomial appro- ximating the gene-wise variance as a function of mean expression. For genes exhibiting Poissonian behavior (i.e., equal mean and variance), we set λ to a fixed value of 1010. All computational analysis was carried out using R v. 3.4.1 with Bioconductor v. 3.5. We assembled a modular workflow for the analysis of scRNA-seq data that contains five modules: (i) quality control, (ii) data normalization, (iii) feature selection, (iv) clustering, and (v) identification of marker genes (Fig. 2a). Based on re- cent publications, the quality control and normalization modules were based on the popular scater [29] and scran [62] packages. Scran was set as the default normalization based on a recent benchmarking study by Vallejos et al. [63] showing that scran was superior for recovering true size factors compared to other methods. For the marker gene identification module we used the Wilcoxon test [64] by default and provided wrappers to MAST [21] and Limma-trend [65], based on Soneson et al.’s [66] compre- hensive assessment of a large number of DE analysis methods for their performance for controlling type I and type II error rates while being scalable to large datasets. Main cell populations were obtained by permutation of the expression values of 100 randomly chosen genes with mean counts larger than 2. Cell subgroups characterized by high expression of a small set of marker genes were generated by replacing the base mean values μi in a small set of genes with low expression (μi < 0.1) by a value of 2x where x N ð2:5; 1Þ. Thus, the upregulated genes exhibit a log2 fold change of 2.5 on average. Simulating varying degrees of subtlety in transcriptional differences An initial small dataset was subsampled from the bench- marking (8 human cell lines) dataset, comprising 100 HEK293, 125 Ramos, and between 10 Jurkat cells. We used scran to predict cell cycle stage and only included cells in G1 phase. Single-cell RNA-sequencing of neuronal cells Denaturing was conducted at room temperature for exactly 8 min, then stopped via the addition of 5 μL 200 mM Tris-HCl pH 8.0 (Fluka, 93,283). Sample was mixed, briefly centrifuged, and placed on ice. ExAmp reaction mix (Illumina, PE-410- 1001) was prepared, added to the sample, and clus- tering was done on a HiSeq 4000 flow cell via cBot2 (Illumina). The library was then sequenced with paired-end reagents, with 26xRead 1 cycles, 8xi7 index cycles, and 98xRead 2 cycles. μij ¼ θ j μi and dispersion1 and dispersion1 λij ¼ μ2 ij σ2 i −μij A second-order polynomial was fit to the sample variance as a function of the mean in logarithmic space as described in [8]. This polynomial served as an esti- mate of the global mean-variance relationship. Replacing the term σ2 i in the equation above with this estimate, the dispersion can be expressed as a function of μij: The 10X Cell Ranger 1.3.1 pipeline was utilized to con- vert raw BCL files to cell-gene matrices. FASTQ files were aligned to the GRCh37.75 human reference genome, UMI-filtered, and barcodes were matched via the CellRan- ger count script. λij ¼ μ2 ij f μij −μij where f μij ¼ 2^ a log2 μij ^2 þ b log2 μij þ c Generation of synthetic data A synthetic dataset was generated based on estimated parameters for the gene-wise mean μi and variance σ2 i from experimentally determined counts of 1000 K562 cells from our benchmarking dataset. From this initial dataset, 25 Ramos cells were held out. From the remaining dataset (100 HEK293, 100 Ramos, 10 Jurkat), datasets with varying incidence of a rare cell type and subtlety (i.e., degree of difference to closest neighbor) of its transcriptional signature were generated in silico, following an approach recently described by Crow et al. [39]: First, a number of Jurkat cells (i.e., inci- dence of 2, 5, or 10) were sampled from the initial data- set. Then, to simulate varying degrees of transcriptional difference between the rare cell type (Jurkat) and its closest abundant cell type (Ramos), an increasing Because gene expression within each cell is typi- cally not independent but cells that have high/low count number for one gene also tend to have high/ low counts for another, we sampled for each cell j a scaling factor θj such that log2ðθ jÞ N ð0; 0:25Þ, as described in [62]. Simulated counts for gene i and cell j were generated by sampling from a negative binomial with mean Page 17 of 21 Page 17 of 21 Wegmann et al. Genome Biology (2019) 20:142 Wegmann et al. Genome Biology (2019) 20:142 fraction of gene expression values, ranging from 0 to 0.995 in steps of 0.05 (0.045 for the very last step) in the Jurkat cells were replaced by the respective values in the held out Ramos cells. This fraction of replaced expression values is referred to as subtlety. percentage of cells (1–2%), which most likely correspond to cell doublets. Furthermore, for the cell line mixes, IMR90/HCT116 and A549/Ramos additional potential doublets were identified and excluded from the cell line assignment employing a visual inspection of the tSNE plot by looking for (small) clusters of cells having high correlation to both cell lines as well as a high UMI count (Additional file 1: Table S3). fraction of gene expression values, ranging from 0 to 0.995 in steps of 0.05 (0.045 for the very last step) in the Jurkat cells were replaced by the respective values in the held out Ramos cells. This fraction of replaced expression values is referred to as subtlety. Generation of synthetic data This procedure was repeated 5 times for each in- cidence of the rare cell type and each value of the subtlety parameter. The performance of CellSIUS, GiniClust2, and RaceID3 was evaluated in terms of recall, precision and true nega- tive rate (TNR) for each configuration. To this end, a con- fusion matrix between the true cell type and the predicted cell type was generated. “Main clusters” were defined as the two clusters containing the majority of the HEK293 and Ramos cells, respectively. The TPR was then defined as the fraction of Jurkat cells that were not assigned to the main clusters, precision was defined as the fraction of Jurkat cells among all cells not assigned to the two main clusters, and the TNR was defined as the frac- tion of HEK293 and Ramos cells that were assigned to the main clusters. After cell type annotation, the raw count matrices from all four batches were concatenated. Cells that had not passed the initial QC or could not be annotated were discarded. The gene filtering step described above was then repeated for the aggregated dataset, leaving a final cleaned dataset containing a total of 12,718 genes and 11,678 cells. Data pre-processing Initial pre-processing was applied to each batch of cell lines separately prior to annotating cell types. First, cells were filtered based on the total number of detected genes, total UMI counts, and the percentage of total UMI counts attributed to mitochondrial genes. Cutoffs were set individually per batch based on the overall distributions (Additional file 1: Table S5). Cell type annotation where nij denotes the elements that are common between Xi and Yj, and ai, bj are the total number of elements in Xi and Yj, respectively. First, the top 10% overdispersed genes were selected using the NBDrop method described in [28]. Cell types were then annotated based on Pearson’s correlation of the expression profile (log2(normalized counts+ 1)) of the selected features with bulk RNA-seq data obtained for each individual cell line (Fig. 1a, b). For the batches 1–3 that contained only two cell lines each, the Pearson’s correlation coefficients were scaled to z-scores prior to the assignment, and for batch 4, the raw correlation values were used instead. A cell was then assigned to the cell line with the highest value unless this maximum was below 0.2 or if the second highest value was within 5% of the maximum in which case no assignment was given. We found that the latter applied only to a small Benchmarking of clustering approaches Second, genes have to present with at least 3 UMIs in at least one cell. After this initial QC, remaining outlier cells were identified and removed using the plotPCA function from the scater [29] R package with detect_out- liers set to TRUE. The accuracy of each prediction was assessed by the ad- justed rand index (ARI). Given two partitions X = X1, … , Xm and Y = Y1, … , Yk of a set S with n elements, the ARI is defined as: Data were normalized using scran [62], including a first clustering step as implemented in the quickCluster func- tion and with all parameters set to their default values. ARI ¼ P ij nij 2 −P i ai 2 P j bj 2 = n 2 1 2 X i ai 2 þ X j bj 2 − X i ai 2 X j bj 2 = n 2 Dimensionality reduction and calculation of distance matrix The original expression (log2(normalized counts + 1) coordinates were projected into low-dimensional space by PCA, using an implicitly restarted Lanczos method as implemented in the irlba [36] R package. The number of dimensions to retain was determined by visual inspec- tion of a scree plot. It was 10 for all cell line data and 12 for the neuron dataset, and the first k principal com- ponents accounted for 40–50% of the total variance in each case. Cell-cell distances (Euclidean or Pearson, Additional file 1: Table S2) were then calculated on these projections. characterized by a minimum of min_n_genes (default: 3 genes) are considered. characterized by a minimum of min_n_genes (default: 3 genes) are considered. counts) into two groups (“low” and “high”). Candi- date marker genes are selected according to three criteria: (i) the average expression fold change between “low” and “high” is at least 2 on a log2- scale, (ii) less than a user defined percentage (50% by default) of all cells in cluster Cj fall in the “high” category, and (iii) there is a significant difference (t test and Benjamini-Hochberg correction, p value < 0.1) between the “low” and “high” expression values. Identification of rare cell types with RaceID and Giniclust RaceID3 [38] was obtained from GitHub (dgrun/ RaceID3_StemID2, version as of March 26th 2018). Analysis was run with all parameters at their default values, except that we fixed the initial clusters (RaceID@k- part) instead of determining them by k-medoids. On bio- logical data (cell line subset 2 and neuronal population), we in addition changed the probability threshold to 10−20 and set the minimum number of outlier genes (outlg) to 3. This adjustment was made because the default cutoffs in RaceID are not very stringent and resulted in extensive overclustering of the data. 2. 2. Testing cluster specificity: For the list of candidate genes, it is assessed whether the cell subgroup expressing them is specific to cluster Cj. Required for each gene gi are (i) a significant difference in the expression of gi in cells with “high” expression compared to cells not in Cj (t test and FDR correction, p value < 0.1) and (ii) the average expression fold change between all cells with “high” expression and all other cells with non-zero expression of gi to be at least 1 on a log2-scale. GiniClust2 [19] was obtained from GitHub (dtsoucas/ GiniClust2, version as of 4 May 2018). All analysis was run with dataset-specific parameters: MinPts = 3, eps = 0.45, k = 2 for the simulated data, and MinPts = 3, eps = 0.45, k = 8 for the cell line dataset. All other parameters were set to their defaults. p g g 3. Identification of correlated gene sets: For each cluster Cj, the correlation matrix of the expression of all candidate genes g1, . . , n across all cells in cluster Cj is transformed into a graph where genes correspond to nodes and edges are weighted by correlations between them. Trajectory analysis using monocle l l Analysis was run using monocle version 2.4.0. As input, the counts of the top 10% genes selected by NBDrop were used. Prior to monocle analysis, all genes annotated with the GO term cell cycle (GO:0007049) as well as mito- chondrial genes and genes encoding ribosomal proteins were removed from the dataset. All parameters were set to default values. Endnotes 1 1We use this nomenclature in order to be consistent with the definition in R. Note that there is an alternative nomenclature, which defines α = 1/λ as dispersion and is used in edgeR [67] and DESeq2 [68]. Additional file 1: Figure S1. tSNE visualization of potential confounders in cell line dataset. Figure S2. Generation of synthetic scRNA-seq data. Figure S3. Parameter sensitivity analysis of CellSIUS. Figure S4. In vitro differentiation of hPSCs into cortical excitatory neurons. Figure S5. hPSC- derived cortical neurons express characteristic marker genes. Figure S6. Identification of cell subgroups in neuronal populations. Figure S7. Comparison of neuronal population markers to scRNA-seq data from the developing human cortex. Table S1. Composition of full and subsampled cell line datasets. Table S2. Overview of clustering algorithms benchmarked in this study. Table S3. Medium composition for the in vitro differentiation of cortical excitatory neurons from human pluripotent stem cells in suspension. Table S5. Sequencing statistics and QC cutoffs per batch. (PDF 3452 kb) Additional file 2: Table S4. DE analysis between subclusters and main clusters in the neuroscience dataset. The file contains one sheet per comparison. All sheets are listed below: G.sub_1_vs_all_G: compares the G.sub_1 population to all other cells in the G cluster. G.sub_2_vs_all_G: compares the G.sub_2 population to all other cells in the G cluster. G.sub_3_vs_all_G: compares the G.sub_3 population to all other cells in characterized by a minimum of min_n_genes (default: 3 genes) are considered. Edges with weights below a fixed threshold are assigned a weight of 0. By default, this threshold is set to the 95th percentile of all correlations if this value lies between 0.35 and 0.5, and to the lower and upper bound if it is below or above, respectively. The lower bound is set such that it is higher than the maximum of all gene-wise correlations on simulated data from an entirely homogeneous population, which serves as an estimate of the background correlation. Setting an upper bound ensures that gene sets are not falsely split in cases where all candidate genes are highly correlated. Subsequently, MCL [33, 34] is used to identify correlated gene sets, denoted sjk, where j is the index of the main cluster and k the index of the gene set within this cluster. CellSIUS CellSIUS detects cell subpopulations and their gene signatures (Fig. 3a). Starting from an initial partitioning of N cells into m clusters C1, … , Cm, the method identi- fies cell subpopulations and their signatures as follows: 1. Identification of genes with bimodal expression: For each gene gi, within each cluster Cj, a one- dimensional k-means clustering is used to partition the cellular expression levels (log2 normalized UMI Page 18 of 21 Page 18 of 21 Wegmann et al. Genome Biology (2019) 20:142 Not applicable. 10. Tirosh I, Izar B, Prakadan SM, Wadsworth MH, Treacy D, Trombetta JJ, et al. Dissecting the multicellular ecosystem of metastatic melanoma by single- cell RNA-seq. Science. 2016;352:189–96 Available from: https://doi.org/10. 1126/science.aad0501. Review history 5. Tang F, Barbacioru C, Wang Y, Nordman E, Lee C, Xu N, et al. mRNA-Seq whole-transcriptome analysis of a single cell. Nat Methods. 2009;6:377–82 Available from: https://doi.org/10.1038/nmeth.1315. The review history for this manuscript is available as Additional file 3. Acknowledgements 3. Zheng GXY, Terry JM, Belgrader P, Ryvkin P, Bent ZW, Wilson R, et al. Massively parallel digital transcriptional profiling of single cells. Nat Commun. 2017;8:14049 Available from: https://doi.org/10.1038/ ncomms14049. We thank our Novartis colleagues: John Reece-Hoyes, Kushal Joshi, Qiong Wang, and Dojna Shkoza for providing the cell lines; Walter Carbone and Judith Knehr for help with sequencing; Anthony Sonrel and Somesh Sai for discussion about the analytical approach; and Jeremy Jenkins for scientific discussions. 4. Svensson V, Vento-Tormo R, Teichmann SA. Exponential scaling of single- cell RNA-seq in the last decade. Nature Protocols. 2018;13:599–604 Available from: https://doi.org/10.1038/nprot.2017.149. Abbreviations ARI Ad d R 1. Macosko EZ, Basu A, Satija R, Nemesh J, Shekhar K, Goldman M, et al. Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets. Cell. 2015;161:1202–14 Available from: https://doi.org/10.1016/j.cell. 2015.05.002. ARI: Adjusted Rand index; CP: Choroid plexus; CR: Cajal-Retzius; CSF: Cerebrospinal fluid; DANB: Depth-adjusted negative binomial; DE: Differential expression; G: Glia; GC: Glycolytic cell; GMM: Gaussian mixture model; hPSC: Human pluripotent stem cell; HVG: High variance gene; IP: Intermediate progenitor; N: Neuron; NP: Neocortical progenitor; oRG: Outer radial glia; PCA: Principal component analysis; scRNA-seq: Single-cell RNA sequencing 2. Klein AM, Mazutis L, Akartuna I, Tallapragada N, Veres A, Li V, et al. Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells. Cell. 2015;161:1187–201 Available from: https://doi.org/10.1016/j.cell.2015.04. 044. Authors’ contributions 6. Han X, Wang R, Zhou Y, Yuan G-C, Chen M, Correspondence GG, et al. Mapping the mouse cell atlas by Microwell-Seq. Cell. 2018;172:1091–107 Available from: https://doi.org/10.1016/j.cell.2018.02.001. MN, FN, and RW developed CellSIUS and implemented the computational workflow. AW and RC sequenced the human cell lines for the benchmarking study. RW performed the benchmarking analysis. RW, MN, MS, BB, and CGK analyzed and interpreted the neuroscience data. HN, MS, and JR performed the experiments. MF, BB, and AK designed the experiments. MN, GR, SS, AJ, BB, and CGK contributed to the conception of the studies and the interpretation of data. RW, MN, BB, MS, AK, and CGK wrote the manuscript. All authors examined the results and approved the final version of the manuscript. 7. Cao J, Spielmann M, Qiu X, Huang X, Ibrahim DM, Hill AJ, et al. The single- cell transcriptional landscape of mammalian organogenesis. Nature. 2019; 566:496–502 Available from: https://doi.org/10.1038/s41586-019-0969-x. 8. Grün D, Lyubimova A, Kester L, Wiebrands K, Basak O, Sasaki N, et al. Single- cell messenger RNA sequencing reveals rare intestinal cell types. Nature. 2015;525:251–5 Available from: https://doi.org/10.1038/nature14966. 9. Jiang L, Chen H, Pinello L, Yuan G-C. GiniClust: detecting rare cell types from single-cell gene expression data with Gini index. Genome Biol. 2016; 17:144 Available from: https://doi.org/10.1186/s13059-016-1010-4. Competing interests All authors are, or were, employees or affiliates of the Novartis Pharma AG. Additional files Additional file 1: Figure S1. tSNE visualization of potential confounders in cell line dataset. Figure S2. Generation of synthetic scRNA-seq data. Figure S3. Parameter sensitivity analysis of CellSIUS. Figure S4. In vitro differentiation of hPSCs into cortical excitatory neurons. Figure S5. hPSC- derived cortical neurons express characteristic marker genes. Figure S6. Identification of cell subgroups in neuronal populations. Figure S7. Comparison of neuronal population markers to scRNA-seq data from the developing human cortex. Table S1. Composition of full and subsampled cell line datasets. Table S2. Overview of clustering algorithms benchmarked in this study. Table S3. Medium composition for the in vitro differentiation of cortical excitatory neurons from human pluripotent stem cells in suspension. Table S5. Sequencing statistics and QC cutoffs per batch. (PDF 3452 kb) Additional file 2: Table S4. DE analysis between subclusters and main clusters in the neuroscience dataset. The file contains one sheet per comparison. All sheets are listed below: G.sub_1_vs_all_G: compares the G.sub_1 population to all other cells in the G cluster. G.sub_2_vs_all_G: compares the G.sub_2 population to all other cells in the G cluster. G.sub_3_vs_all_G: compares the G.sub_3 population to all other cells in 4. Assigning cells to subgroups: For each cluster Cj and each gene set sjk, a one-dimensional k-means is run on the mean expression of sjk. Cells falling in the “high” mode of this clustering are assigned to a new cluster Cjk. j 5. Final cluster assignment: Cells are assigned to a final cluster which is the combination of all subgroups they belong to. This means if a cell belongs to two subgroups A and B, it will be assigned to a new subgroup AB. The gene signatures for this new subgroup correspond to the union of gene signatures A and B. Only subgroups Page 19 of 21 Page 19 of 21 Wegmann et al. Genome Biology (2019) 20:142 Consent for publication Not applicable. Consent for publication Not applicable. the G cluster. CR.sub_vs_all_CR: compares the CR.sub population to all other cells in the CR cluster. NP.sub_vs_all_NP: compares the NP.sub population to all other cells in the NP cluster. N.sub_1_vs_all_N: compares the N.sub_1 population to all other cells in the N cluster. N.sub_2_vs_all_N: compares the N.sub_2 population to all other cells in the N cluster. Each sheet contains the following columns: Gene_id: Ensembl gene ID. Mean_exprs: Mean expression [log2(normalized counts + 1)] across the whole dataset. Mean_in_subgroup: Mean expression in the respective subgroup. Additional files Pval, adj_pval: p value (Wilcoxon test), adj_pval is adjusted p value (Benjamini-Hochberg). Log2fc: Fold change, calculated as the difference in mean[log2(normalized counts + 1)]. DE_flag: is TRUE if abs(log2fc) > 0.5 and adj_pval < 0.05. Chr, symbol, eg, gene_biotype, description: Additional gene info (chromosome, gene symbol, entrez gene identifier, gene biotype, short description of gene function). (XLSX 8049 kb) the G cluster. CR.sub_vs_all_CR: compares the CR.sub population to all other cells in the CR cluster. NP.sub_vs_all_NP: compares the NP.sub population to all other cells in the NP cluster. N.sub_1_vs_all_N: compares the N.sub_1 population to all other cells in the N cluster. N.sub_2_vs_all_N: compares the N.sub_2 population to all other cells in the N cluster. Each sheet contains the following columns: Gene_id: Ensembl gene ID. Mean_exprs: Mean expression [log2(normalized counts + 1)] across the whole dataset. Mean_in_subgroup: Mean expression in the respective subgroup. Pval, adj_pval: p value (Wilcoxon test), adj_pval is adjusted p value (Benjamini-Hochberg). Log2fc: Fold change, calculated as the difference in mean[log2(normalized counts + 1)]. DE_flag: is TRUE if abs(log2fc) > 0.5 and adj_pval < 0.05. Chr, symbol, eg, gene_biotype, description: Additional gene info (chromosome, gene symbol, entrez gene identifier, gene biotype, short description of gene function). (XLSX 8049 kb) Author details 1 1Novartis Institutes for Biomedical Research, Basel, Switzerland. 2Novartis Institutes for Biomedical Research, Cambridge, USA. 3Present Address: Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland. 4Present Address: Insitro, San Francisco, USA. 5Present Address: Flagship Pioneering, Cambridge, USA. Availability of data and materials ScRNA-seq data of human cell lines have been deposited in the NCBI Short Read Archive (SRA) under accession number SRA: PRJNA484547 [69]. ScRNA-seq data of differentiation of cortical excitatory neurons from human pluripotent stem cells in suspension have been deposited in the NCBI Short Read Archive (SRA) under accession number SRA: PRJNA545246 [70]. The workflow written in the R programming language is deposited in GitHub (https://github.com/Novartis/scRNAseq_workflow_benchmark) and Zenodo (DOI: https://doi.org/10.5281/zenodo.3237742) [71]. The code, vignette, and an example dataset for the computational workflow are included in the repository. 11. Villani A-C, Satija R, Reynolds G, Sarkizova S, Shekhar K, Fletcher J, et al. Single-cell RNA-seq reveals new types of human blood dendritic cells, monocytes, and progenitors. Science. 2017;356:eaah4573 Available from: https://doi.org/10.1126/science.aah4573. 11. Villani A-C, Satija R, Reynolds G, Sarkizova S, Shekhar K, Fletcher J, et al. Single-cell RNA-seq reveals new types of human blood dendritic cells, monocytes, and progenitors. Science. 2017;356:eaah4573 Available from: https://doi.org/10.1126/science.aah4573. 12. Shalek Alex K, Rahul S, Joe S, Trombetta John J, Dave G, Diana L, et al. Single-cell RNA-seq reveals dynamic paracrine control of cellular variation. Nature. 2014;510:363–9 Available from: https://doi.org/10.1038/nature13437. 13. Regev A, Teichmann SA, Lander ES, Amit I, Benoist C, Birney E, et al. The human cell atlas. Elife. 2017;6:e27041 Available from: https://doi.org/10.7554/ eLife.27041. The CellSIUS is deposited in GitHub (https://github.com/Novartis/CellSIUS) [72] and Zenodo (DOI: https://doi.org/10.5281/zenodo.3237749) [73] as a standalone R package. It requires R ≥3.4.1 and uses an external installation of the Markov Clustering Algorithm (MCL) [33, 34]. The R implementation is platform independent; the external MCL runs on any UNIX platform. The codes and processed data to reproduce the analyses presented here are uploaded in Zenodo (https://doi.org/10.5281/zenodo.3238275) [74]. All th l d it i d th “A h Li 2 0” The CellSIUS is deposited in GitHub (https://github.com/Novartis/CellSIUS) [72] and Zenodo (DOI: https://doi.org/10.5281/zenodo.3237749) [73] as a standalone R package. It requires R ≥3.4.1 and uses an external installation of the Markov Clustering Algorithm (MCL) [33, 34]. The R implementation is platform independent; the external MCL runs on any UNIX platform. 14. žurauskiene J, Yau C. pcaReduce: Hierarchical clustering of single cell transcriptional profiles. BMC Bioinformatics. 2016;17:140 Available from: https://doi.org/10.1186/s12859-016-0984-y. The codes and processed data to reproduce the analyses presented here are uploaded in Zenodo (https://doi.org/10.5281/zenodo.3238275) [74]. All the open source released repositories are under the “Apache License 2 0” 15. Received: 7 May 2019 Accepted: 13 June 2019 Additional file 3: Review history (DOCX 58 kb) Ethics approval and consent to participate Not applicable. 13. Regev A, Teichmann SA, Lander ES, Amit I, Benoist C, Birney E, et al. The human cell atlas. Elife. 2017;6:e27041 Available from: https://doi.org/10.7554/ eLife.27041. Availability of data and materials Kiselev VY, Kirschner K, Schaub MT, Andrews T, Yiu A, Chandra T, et al. SC3: consensus clustering of single-cell RNA-seq data. Nat Methods. 2017;14:483– 6 Available from: https://doi.org/10.1038/nmeth.4236. 15. Kiselev VY, Kirschner K, Schaub MT, Andrews T, Yiu A, Chandra T, et al. SC3: consensus clustering of single-cell RNA-seq data. Nat Methods. 2017;14:483– 6 Available from: https://doi.org/10.1038/nmeth.4236. The codes and processed data to reproduce the analyses presented here are uploaded in Zenodo (https://doi.org/10.5281/zenodo.3238275) [74]. All the open source released repositories are under the “Apache License 2.0”. All the open source released repositories are under the “Apache License 2.0”. 16. Kiselev VY, Andrews TS, Hemberg M. Challenges in unsupervised clustering of single-cell RNA-seq data. Nat Rev Genet. 2019;1 Available from: https:// doi.org/10.1038/s41576-018-0088-9. 16. Kiselev VY, Andrews TS, Hemberg M. Challenges in unsupervised clustering of single-cell RNA-seq data. Nat Rev Genet. 2019;1 Available from: https:// doi.org/10.1038/s41576-018-0088-9. Ethics approval and consent to participate Not applicable. Ethics approval and consent to participate Not applicable. Page 20 of 21 Page 20 of 21 Wegmann et al. Genome Biology (2019) 20:142 Wegmann et al. Genome Biology (2019) 20:142 17. Setty M, Tadmor MD, Reich-Zeliger S, Angel O, Salame TM, Kathail P, et al. Wishbone identifies bifurcating developmental trajectories from single-cell data. Nat Biotechnol. 2016;34:637–45 Available from: https://doi.org/10.1038/ nbt.3569. 39. Crow M, Paul A, Ballouz S, Huang ZJ, Gillis J. Characterizing the replicability of cell types defined by single cell RNA-sequencing data using MetaNeighbor. Nat Commun. 2018;9:884 Available from: https://www. nature.com/articles/s41467-018-03282-0. 18. Qiu X, Mao Q, Tang Y, Wang L, Chawla R, Pliner HA, et al. Reversed graph embedding resolves complex single-cell trajectories. Nat Methods. 2017;14: 979–82 Available from: http://www.nature.com/doifinder/10.1038/nmeth.4402. 40. Kuhns MS, Badgandi HB. Piecing together the family portrait of TCR-CD3 complexes. Immunol Rev. 2012;250:120–43 Available from: https://doi.org/ 10.1111/imr.12000. 19. Tsoucas D, Yuan G-C. GiniClust2: a cluster-aware, weighted ensemble clustering method for cell-type detection. Genome Biol. 2018;19:58 Available from: https://doi.org/10.1186/s13059-018-1431-3. 41. Nourani MR, Farajpour Z, Najafi A, Imani Fooladi AA. Trefoil factor family 1 is involved in airway remodeling of mustard lung. Iran J Allergy Asthma Immunol. 2016;15:275–82 Available from: https://www.ncbi.nlm.nih.gov/pubmed/27921407. 20. Kharchenko Peter V, Lev S, Scadden DT. Bayesian approach to single-cell differential expression analysis. Nat Meth. 2014;11:740–2 Available from: https://doi.org/10.1038/nmeth.2967. 42. Prokopovic V, Popovic M, Andjelkovic U, Marsavelski A, Raskovic B, Gavrovic- Jankulovic M, et al. Isolation, biochemical characterization and antibacterial activity of BPIFA2 protein. Arch Oral Biol Pergamon. 2014;59:302–9 Available from: https://doi.org/10.1016/j.archoralbio.2013.12.005. 21. Availability of data and materials Finak G, McDavid A, Yajima M, Deng J, Gersuk V, Shalek AK, et al. MAST: a flexible statistical framework for assessing transcriptional changes and characterizing heterogeneity in single-cell RNA sequencing data. Genome Biol. 2015;16:278 Available from: http://www.ncbi.nlm.nih.gov/pubmed/26653891. 43. Kuijlaars J, Oyelami T, Diels A, Rohrbacher J, Versweyveld S, Meneghello G, et al. Sustained synchronized neuronal network activity in a human astrocyte co-culture system. Sci Rep. 2016;6:36529 Available from: https:// doi.org/10.1038/srep36529. 22. Korthauer KD, Chu LF, Newton MA, Li Y, Thomson J, Stewart R, et al. A statistical approach for identifying differential distributions in single-cell RNAseq experiments. Genome Biol. 2016;17(1):222 Available from: https:// doi/org/10.1186/s13059-016-1077-y 44. Pollen AA, Nowakowski TJ, Shuga J, Wang X, Leyrat AA, Lui JH, et al. Low- coverage single-cell mRNA sequencing reveals cellular heterogeneity and activated signaling pathways in developing cerebral cortex. Nat Biotechnol. 2014;32:1053–8 Available from: https:/doi.org/10.1038/nbt.2967. 23. Johnson MB, Wang PP, Atabay KD, Murphy EA, Doan RN, Hecht JL, et al. Single-cell analysis reveals transcriptional heterogeneity of neural progenitors in human cortex. Nat Neurosci. 2015;18:637–46 Available from; https://doi.org/10.1038/nn.3980. 45. Frotscher M. Cajal-Retzius cells, Reelin, and the formation of layers. Curr Opin Neurobiol. 1998;8(5):570–5 Available from: https://doi.org/10.1016/ S0959-4388(98)80082-2. 46. Nowakowski TJ, Bhaduri A, Pollen AA, Alvarado B, Mostajo-Radji MA, Di Lullo E, et al. Spatiotemporal gene expression trajectories reveal developmental hierarchies of the human cortex. Science. 2017;358:1318–23 Available from: https://doi.org/10.1126/science.aap8809. 24. Camp JG, Badsha F, Florio M, Kanton S, Gerber T, Wilsch-Bräuninger M, et al. Human cerebral organoids recapitulate gene expression programs of fetal neocortex development. Proc Natl Acad Sci. 2015;112(5):15672–7 Available from: https://doi.org/10.1073/pnas.1520760112. 25. Bardy C, Van Den Hurk M, Kakaradov B, Erwin JA, Jaeger BN, Hernandez RV, et al. Predicting the functional states of human iPSC-derived neurons with single-cell RNA-seq and electrophysiology. Mol Psychiatry. 2016;21:1573–88 Available form: https://doi.org/10.1038/mp.2016.158. 47. Rouillard AD, Gundersen GW, Fernandez NF, Wang Z, Monteiro CD, McDermott MG, et al. The harmonizome: a collection of processed datasets gathered to serve and mine knowledge about genes and proteins. Database (Oxford). 2016;2016:baw100 Available from: https://doi.org/10. 1093/database/baw100. 26. Handel AE, Chintawar S, Lalic T, Whiteley E, Vowles J, Giustacchini A, et al. Assessing similarity to primary tissue and cortical layer identity in induced pluripotent stem cell-derived cortical neurons through single-cell transcriptomics. Hum Mol Genet. 2016;25:989–1000 Available from: https:// doi.org/10.1093/hmg/ddv637. 48. Miller JA, Ding SL, Sunkin SM, Smith KA, Ng L, Szafer A, et al. Transcriptional landscape of the prenatal human brain. Nature. Availability of data and materials 2014;508:199–206 Available from: https://doi.org/10.1038/nature13185. 49. Meyer G, Perez-Garcia CG, Gleeson JG. Selective expression of doublecortin and LIS1 in developing human cortex suggests unique modes of neuronal movement. Cereb Cortex. 2002;12:1225–36 Available from: https://doi.org/ 10.1093/cercor/12.12.1225. 27. Brennecke P, Anders S, Kim JK, Kołodziejczyk AA, Zhang X, Proserpio V, et al. Accounting for technical noise in single-cell RNA-seq experiments. Nat Methods. 2013;10:1093–5 Available from: https://doi.org/10.1038/nmeth.2645. 28. Andrews TS, Hemberg M. M3drop: dropout-based feature selection for scRNASeq. Bioinformatics. 2018:1–3 Available from: https://doi.org/10.1093/ bioinformatics/bty1044. 50. Gonzalez-Gomez M, Meyer G. Dynamic expression of calretinin in embryonic and early fetal human cortex. Front Neuroanat. 2014;8:41 Available from: https://doi.org/10.3389/fnana.2014.00041. 51. Martinez-Galan JR, Moncho-Bogani J, Caminos E. Expression of calcium- binding proteins in layer 1 Reelin-Immunoreactive cells during rat and mouse neocortical development. J Histochem Cytochem. 2014;62:60–9 Available from: https://doi.org/10.1369/0022155413509381. 29. McCarthy DJ, Campbell KR, Lun ATL, Wills QF. Scater: pre-processing, quality control, normalization and visualization of single-cell RNA-seq data in R. Bioinformatics. 2017;33:1179–86 Available from: https://doi.org/10.1093/ bioinformatics/btw777. 52. Molyneaux BJ, Arlotta P, JRL M, Macklis JD. Neuronal subtype specification in the cerebral cortex. Nat Rev Neurosci. 2007;8:427–37 Available from: https://doi.org/10.1038/nrn2151. 30. Langfelder P, Zhang B, Horvath S. Defining clusters from a hierarchical cluster tree: the Dynamic Tree Cut package for R. Bioinformatics. 2008: 24(5)719-20 Available from: https://doi.org/10.1093/bioinformatics/btm563 53. Lun MP, Monuki ES, Lehtinen MK. Development and functions of the choroid plexus-cerebrospinal fluid system. Nat Rev Neurosci. 2015;16:445–57 Available from: https://doi.org/10.1038/nrn3921. 31. Fraley C, Raftery AE. Model-based clustering, discriminant analysis and density estimation. J Am Stat Assoc. 2002;97:611–31. 32. Ester M, Kriegel HP, Sander J, Xu X. A density-based algorithm for discovering clusters in large spatial databases with noise. Proc 2nd Int Conf Knowl Discov data min; 1996. p. 226–31. Available from: https://www.aaai. org/Papers/KDD/1996/KDD96-037.pdf 54. Pollen AA, Nowakowski TJ, Chen J, Retallack H, Sandoval-Espinosa C, Nicholas CR, et al. Molecular identity of human outer radial glia during cortical development. Cell. 2015;163:55–67 Available from: https://doi.org/10. 1016/j.cell.2015.09.004. 33. van Dongen S. Graph clustering by flow simulation. PhD thesis, University of Utrecht; 2000 Avalaible from https://dspace.library.uu.nl/handle/1874/848 55. Cooper JA. Molecules and mechanisms that regulate multipolar migration in the intermediate zone. Front Cell Neurosci. 2014;8:386 Available from: https://doi.org/10.3389/fncel.2014.00386. 34. Enright AJ, Van Dongen S, Ouzounis CA. An efficient algorithm for large- scale detection of protein families. Nucleic Acids Res. 2002;30:1575–84 Available from: https://doi.org/10.1093/nar/30.7.1575. 56. Chen G, Sima J, Jin M, Wang KY, Xue XJ, Zheng W, et al. Availability of data and materials Semaphorin-3A guides radial migration of cortical neurons during development. Nat Neurosci. 2008;11:36–44 Available from: https://doi.org/10.1038/nn2018. 35. Mardia K, Kent J, Bibby J. Multivariate analysis. London: Acad Press; 1979. 36. Baglama J, Reichel L. Augmented implicitly restarted Lanczos Bidiagonalization methods. SIAM J Sci Comput. 2005;27:19–42 Available from: http://epubs.siam.org/doi/10.1137/04060593X. 57. Priddle TH, Crow TJ. Protocadherin 11X/Y a human-specific gene pair: an immunohistochemical survey of fetal and adult brains. Cereb Cortex. 2013; 23:1933–41 Available from: https://doi.org/10.1093/cercor/bhs181. 37. Hubert L, Arabie P. Comparing partitions. J Classif. 1985;2:193–218. 58. Lodato S, Molyneaux BJ, Zuccaro E, Goff LA, Chen HH, Yuan W, et al. Gene co-regulation by Fezf2 selects neurotransmitter identity and connectivity of corticospinal neurons. Nat Neurosci. 2014;17:1046–54 Available from: https:// doi.org/10.1038/nn.3757. 38. Grün D, Muraro MJ, Boisset J-C, Wiebrands K, Lyubimova A, Dharmadhikari G, et al. De novo prediction of stem cell identity using single-cell transcriptome data. Cell Stem Cell. 2016;19:266–77 Available from: https:// doi.org/10.1016/j.stem.2016.05.010. Page 21 of 21 Wegmann et al. Genome Biology (2019) 20:142 Wegmann et al. Genome Biology (2019) 20:142 59. Cunningham F, Amode MR, Barrell D, Beal K, Billis K, Brent S, et al. Ensembl 2015. Nucleic Acids Res. 2015;43:D662–9. 60. Schuierer S, Roma G, et al. Nucleic Acids Res. 2016;44(16):e132 Available from: https://doi.org/10.1093/nar/gkw538. 61. Bilican B, Livesey MR, Haghi G, Qiu J, Burr K, Siller R, et al. Physiological normoxia and absence of EGF is required for the long-term propagation of anterior neural precursors from human pluripotent cells. PLoS One. 2014; 9(1):e85932 Available from: https://doi.org/10.1371/journal.pone.0085932. 62. Lun AT, Bach K, Marioni JC. Pooling across cells to normalize single-cell RNA sequencing data with many zero counts. Genome Biol. 2016;17:75 Available from: https://doi.org/10.1186/s13059-016-0947-7. 63. Vallejos CA, Risso D, Scialdone A, Dudoit S, Marioni JC. Normalizing single- cell RNA sequencing data: challenges and opportunities. Nat Methods. 2017; 14:565–71 Available from: https://doi.org/10.1038/nmeth.4292. 64. Wilcoxon F. Individual comparisons by ranking methods. Biom Bull. 1945;1:80. 65. Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, et al. Limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015;43:e47 Available from: https://doi.org/10.1093/nar/gkv007. 66. Soneson C, Robinson MD. Bias, robustness and scalability in single-cell differential expression analysis. Nat Methods. 2018;15:255–61 Available from: http://www.nature.com/doifinder/10.1038/nmeth.4612. 67. Robinson MD, McCarthy DJ, Smyth GK. edgeR: a bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010;26:139–40 Available from: https://doi.org/10.1093/ bioinformatics/btp616. 68. Love MI, Huber W, Anders S. Availability of data and materials Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014; Available from: https://doi.org/10.1186/s13059-014-0550-8. 69. Novartis Institutes for Biomedical Research. Single cell RNA-sequencing of human cell lines performed for the benchmarking of single cell transcriptome analytics. Sequence Read Archive. 2019; Available from: https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA484547. 70. Novartis Institutes for Biomedical Research. Neurons were made from H9 ESCs using a directed differentiation protocol in spinner flasks. After 86 DIV, cells were dissociated and run through the 10X Genomics Chromium single cell RNAseq platform. Sequence Read Archive. 2019; Available from: https:// www.ncbi.nlm.nih.gov/sra/?term=PRJNA545246. 70. Novartis Institutes for Biomedical Research. Neurons were made from H9 ESCs using a directed differentiation protocol in spinner flasks. After 86 DIV, cells were dissociated and run through the 10X Genomics Chromium single cell RNAseq platform. Sequence Read Archive. 2019; Available from: https:// www.ncbi.nlm.nih.gov/sra/?term=PRJNA545246. 71. Novartis Institutes for Biomedical Research. scRNAseq_workflow_benchmark: single cell RNAseq data analysis workflow. Zenodo. 2019; Available from: https://doi.org/10.5281/zenodo.3237742. 72. Novartis Institutes for Biomedical Research. CellSIUS: Cell Subtype Identification from Upregulated gene Sets. Github Repository. 2019; Available from: https://github.com/Novartis/CellSIUS. 73. Novartis Institutes for Biomedical Research. CellSIUS: Cell Subtype Identification from Upregulated gene Sets. Zenodo. 2019; Available from: https://doi.org/10.5281/zenodo.3237749. 74. Novartis Institutes for Biomedical Research. CellSIUS provides sensitive and specific detection of rare cell populations from complex single cell RNA-seq data: Codes and processed data. Zenodo. 2019; Available from: https://doi. org/10.5281/zenodo.3238275. Wegmann et al. Genome Biology (2019) 20:142 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
https://openalex.org/W2069900668	https://zenodo.org/records/1530686/files/article.pdf	English	null	RE-INFECTION IN SYPHILIS.	Lancet	1,909	public-domain	3,182	321 the syllabus represents about one-fourth of the educational value of the Minor qualification. Dr. Levy has done is of great interest as bearing directly upon the method we have advocated. We find ourselves unable to agree with Dr. Donald J. Munro as to there being any disadvantages whatever in practice, dependent upon the fact that ether and chloroform have different rates of vaporisation, provided that the mixture we advocate be administered as we have described. Instead of meeting with irregular effects, as might theoretically be supposed, we have obtained regular and equable results. Dr. J. D. Mortimer criticises our use of the terms open" and semi- open." We think Dr. Levy’s letter will justify the distinction we draw between these two expressions. We are interested to learn that Dr. Desborough Brodie has employed the method we advocate, and with equally good results. Dr. Alexander Brown asks whether it is our opinion that the administration of C.E. mixture by the open method should supersede its administration by means of an inhaler devised by one of us. The fact is that when that inhaler was introduced the possibility of obtaining adequate anaesthesia in all subjects by means of the C.E. mixture administered from a, Skinner’s mask was not fully appreciated, and the passage in the work referred to by Dr. Alexander Brown therefore needs slight modification. At the same time, the inhaler in question, by reason of its wide air-inlet, is greatly to be preferred in point of safety to others of the Rendle type. Now, however, that the practic- ability of obtaining adequate ansesthesia by a simple Skinner’s frame of particular dimension and construction is estab- lished, there is not that necessity for a semi-open inhaler with wide air-inlet that previously existed. Finally, we come to Dr. G. A. H. Barton’s remarks. After directing attention to all absence of novelty in our contribution- which, we are sure, he will see is our misfortune rather than our fault-he suggests one or two improvements in our method. These, however, appear to us to be rather on the side of complexity than on that of simplicity. We do not ourselves attach great importance to rapidity of induction, except in certain special types of subject, believing that the best kind of anaesthesia durigzg operation is to be obtained by gradual rather than by rapid induction methods. At the same time, we tender him and all your other correspondents our best thanks for discussing a subject which we think is of import- tance to all who administer ansesthetics, and particularly to those practitioners to whom simplicity of procedure specially commends itself.-We are Sir yours faithfully t M e Minor qualification. We therefore respectfully suggest that the following condition be embodied in your reply to the Society of Apothecaries ;- y p y y p That whilst the Pharmaceutical Society is willing to register can- didates as pharmaceutical chemists or chemists and druggists who can show that they have passed an examination equivalent to the major and minor examinations respectively, conducted by any responsible examining body such as the Society of Apothecaries,’ ’ The Civil Service Commissioners,’ or any Colonial body, they would call the attention of the Society of Apothecaries to the fact that the present certificate is not ’evidence satisfactory to the Council of the Pharmaceutical Society that they are persons of sufficient skill and knowledge to be so registered,’ and that the mere fact of holding the Assistants’ Certificate for a period of five years as suggested does not satisfy such requirements." y y We submit that the Society of Apothecaries is bound to institute a higher examination before submitting to the Pharmaceutical Society any claim for registration. We feel it would be a gross subversion of the spirit and intention of the law as expressed in the Pharmacy Act, 1908, and a standing injustice to all present holders of the Major and Minor qualifications, as well as a sacrifice of the best interests of pharmacy in the future to grant to any person without corresponding training, individual effort, and financial outlay, registration under the Pharmacy Act. We have every confidence that you will give that careful and sympathetic consideration which our request demands having regard to the fact that the qualification is to many assistant pharmacists the sole means of livelihood. S. CARLTON FARRER, M.P.S. W. J. FINDLAY. VICTOR BOTTOMLEY. EDWIN A. LENTON, M.P.S. JOSEPH M. DOWTY, M.P.S. (S J. WILSON. J. WILSON. LEONARD STEVENSON, M.P.S. LEONARD STEVENSON, M.P.S. LEONARD STEVENSON, M.P.S. EDW. S. FRANCIS, M.P.S. STEVENSON, EDW. S. FRANCIS, M.P.S. JAS. J. B. WALDRON, M.P.S. WALDRON, C. TERRY HOLLOWAY, M.P.S. Birmingham, July 11th, 1909. To the Editor of THE LANCET. SiR,-The annotation in THE LANCET of July 24th, p. 243, under this heading raises the question as to whether the medical man in attendance during the last illness of a person has the power to decide whether he shall give a cer- tificate of death or not. In that annotation you write: ’’ The far more frequent cause of complaint against medical men arises out of the withholding of certificates which cannot conscientiously be granted, but which in the opinion of the coroner or the jury would save the necessity of an inquest." As a matter of fact, the medical man has no such discre- tionary power. It is obligatory upon him to certify the cause of death even if violent. Section 20 of the Births and Deaths Registration Act, 1874 (37 and 38 Vict., c. 88), is most explicit upon this point. By that section it is enacted :- faithfully, FREDERIC HEWITT. J. BLUMFELD. faithfully, FREDERIC HEWITT. J. BLUMFELD. J. BLUMFELD. July 19th, 1909. [COPY.] [COPY.] To the Pharmaceutical Society, London. y, We, the undersigned, officials and executive members of the National Union of Assistant Pharmacists, have carefully considered the com- munication sent to you by the Society of Apothecaries asking for the admission of persons holding their Assistants’ Certificate to the rank and privilege of pharmaceutical chemists. hfully, EDWIN J. TOYE. you quote. am, Sir, yours faithfully, July 26th, 1909. EDWIN J. TOYE. you quote. am, Sir, yours faithfully, July 26th, 1909. EDWIN J. TOYE. you quote. July 26th, 1909. privilege pharmaceutical chemists. On behalf of our members, who are the section in pharmacy most vitally affected by the proposition made therein, and as members of the Pharmaceutical Society and registered chemists and druggists, we hereby emphatically ptotest against their entrance on any such terms ; and we respectfully urge upon you as our representatives to secure to us the utmost possible protection from such unfair competition A CERTIFICATE OF THE CAUSE OF DEATH FOLLOWED BY AN INQUEST. Q To the Editor of THE LANCET. To the Edito’J’ of THE LANCET. To the Edito’J’ of THE LANCET. SIR,-I am instructed to forward you a copy of the protes which has been sent to the Pharmaceutical Society on behal of our union.-I am, Sir, yours faithfully, g It is a great pity that this fact is not fully appreciated and acted upon by medical men. The duty of the medical man would simply be that of signing a certificate in all cases of death, whether due to natural or violent causes. The un- pleasant, responsible, and unremunerated work of deciding whether an appeal to the coroner is necessary would thus fall upon the registrar, where it is meant to fall by the Act. The medical man would then be saved those awkward situations you quote I am Sir, yours faithfully, am, Sir, yours faithfully, S. CARLTON FARRER, M.P.S., Birmingham, July 13th, 1909. Honorary General Secretary. y y, S. CARLTON FARRER, M.P.S., Birmingham, July 13th, 1909. Honorary General Secretary. S. CARLTON FARRER, M.P.S., Birmingham, July 13th, 1909. Honorary General Secretary. Birmingham, July 13th, 1909. Birmingham, July 13th, 1909. NATIONAL UNION OF ASSISTANT PHARMACISTS. 1 tf In case of death of any person who has been attended during his last illness by a registered general practitioner, that practitioner shall sign, and give to some person required by this Act to give information concerning the death, a certificate stating to the best of his knowledge and belief the cause of death. . 321 To the Editor of THE LANCET. Organisation. Each division of the Territorial Army is commanded by a General Officer (G.O.C.), and on his staff is appointed an Administrative Medical Officer (A.M.O.) with the rank of colonel, who is a Territorial medical officer. The G.O.C. is the responsible head of the entire division, and the A.M.O. is responsible to him for the efficiency of all the medical ersonnel and equipment in his division for war, as well as for the physical efficiency and health of the entire division. He is the recognised adviser to the G.O.C. on all questions connected with the health and sanitation of the division, and he is also commanding officer (O.C.) of all the Territorial R.A.M.C. in the division. To assist the A.M. 0. two staff officers (medical) are allowed, one being a retired field officer of the R.A.M.C., and the other a specialist sanitary officer or medical officer of health from the Territorial Medical Force. A permanent office and clerical establishment are also allotted. Each unit (regiment, battery, &c.) in the division has a Territorial medical officer attached, who is responsible for the physical and medical efficiency of his unit and also for the sanitation of the area they occupy. The Senior Medical Officer of each brigade (S.M.O.) ’is responsible in the same way for the brigade as a whole. Orders connected with general medical questions and sanita- tion affecting the entire division are issued by the A.M.O. in the name of the G. 0. C., while purely professional orders are issued in his own name. Likewise orders affecting brigades alone or units alone are issued in the name of their respective commanders by the S.M.O. or M.O. respectively, and purely professional orders in their own names. The A.M.O. of a division, S.M.O. of brigade, and M.O. of regiments make inspections of their respective charges, each having in view sanitation of area, physique of the men, their freedom from disease, ventilation, clothing, cleanliness, food, and purity of drinking water. The sick and wounded are treated in hospitals (field ambulances, general hospitals, &c.), and while in hospital the patients are under the command of the medical officer in charge. Each hospital is a unit in itself and bears the same relationship to the G.O.C. and brigade commander as any other unit under their charge. To the Editor of THE LANCET. To the Editor of THE LANCET. competition. Section 4, Clause B, of the Pharmacy Act, 1908, requires the Council of the Pharmaceutical Society to satisfy itself " that they are persons of sufficient skill and knowledge to be so registered," and we submit that the preeent holders of the Apothecaries Assistants’ Certificate do not fulfil this requirement for the following reasons :- SIR,-If you will excuse this belated letter I should like to make a few comments on Mr. Hutchinson’s interesting and instructive article in THE LANCET of May 29th on Auto- inoculation and Reinfection in Syphilis, which I have only just seen. In the first place, it is only fair to the memory of Ricord to mention that in later life he recanted some of his earlier statements-for instance, the non-contagious- ness of secondary lesions. With regard to the question of e-infection, he made the following remarks in a letter to Mr. W. Acton (published in the British Medical Journal, 1872, Vol. II., p. 228) : "Now that we have authentic requirement following (1) All persons of 18 years are eligible for the certificate, which limits the possibilities of adequate practical training with theoretical knowledge. requirement following (1) All persons of 18 years are eligible for the certificate, which limits the possibilities of adequate practical training with theoretical knowledge. knowledge. (2) The whole period of training need not exceed six months, as con- trasted with the three years minimum required of candidates for the Minor qualification knowledge. (2) The whole period of training need not exceed six months, as con- trasted with the three years minimum required of candidates for the Minor qualification. Minor qualification. (3) No preliminary examination is demanded, thus reducing the educational standard below that which would be required for entrance to any profession. r ’ qualification. (3) No preliminary examination is demanded, thus reducing the educational standard below that which would be required for entrance to any profession. r ’ to any profession. (4) The skill and knowledge required for examination as published inl any profession. s faithfully, W. H. PEARCE, Secretary. am, Sir, yours faithfully, W. H. PEARCE, Secretary. , y Paddington Green Children’s Hospital, London, W., July 24th, 1909. , y Paddington Green Children’s Hospital, London, W., July 24th, 1909. * The authority for the statement was the preliminary report of the Education Committee of the London County Council.-ED. L. * The authority for the statement was the preliminary report of the Education Committee of the London County Council.-ED. L. 10 the Editor of THE LANCET. SIR,-In THE LANCET of July 17th there is an interest- ing annotation (p. 164) on a vegetable source of iron. The observations of two French workers are quoted as showiI1g that the dried root of the dock, Rumex obtetsifoliecs, contains 0 - 447 per cent. of iron in a combination analogous to the ferric derivatives of the nucleones. It is perhaps worth while observing that such a preparation of iron with nucleins as a base has recently been obtained which contains as much as 8 - 0 per cent. of iron. It is called Zer A.woli, and I can speak with confidence as to its value in the treatment of anaemic girls.-I am, Sir, yours faithfully g , , yours faithfully, A. BUTLER HARRIS, M.A., M.B. Oxon. Loughton, Essex, July Hbth, 1SOH. A. BUTLER HARRIS, M.A., M.B. Oxon. Loughton, Essex, July Hbth, 1SOH. To the Editor of THE LANCET. (4) The skill and knowledge required for examination as published inl 2 322 examples of fresh contagions of indurated chancre, with consecutive evolution of the whole series of constitutional symptoms, this proves that patients have been cured, just as the possibility of contracting small-pox afresh or of vaccination again taking proves that the first variolous or vaccine influence has ceased." Mr. Hutchinson attributes the intermittent method of mercurial treatment to Ricord, but I think it was Fournier who introduced the "chronic intermittent treatment" of syphilis. Whether, as Mr. Hutchinson suggests, this method is less efficacious in .curing syphilis than the continuous method, and consequently less likely to be followed by re-infection, is a moot point which would require much evidence to decide. With regard to infection of the subjects of inherited syphilis with the acquired disease, the late Professor Tamowsky published a number of cases of this kind which he called binary syphilis," and considered that this combination has a more lethal effect on the offspring of such subjects than inherited syphilis by itself. As I pointed out in my book on " Syphilology and Venereal Disease," it may be possible for a patient who has had two attacks of syphilis to be suffering from the effects of both attacks at the same time, for parasyphilitic effects, such as tabes or general paralysis due to the first attack, may appear after the date of re-infection. It is true that Krafft- ,Ebing obtained negative results from the inoculation of .general paralytics with syphilis, but I believe authentic cases of syphilitic chancres in the subjects of general paralysis have been recorded.-I am Sir, yours faithfully, ; ; To the Editor of THE LANCET. Three field ambulances are allotted to each division, one to each of its brigades ; the mounted brigade field ambulance is a smaller unit. The remaining medical unit is the general hospital which trains with the division in peace but belongs to the corps troops in war. S1R,-Referring to Dr. J. K. Fowler’s letter in THE LANCET of July 24th (p. 254), I remember well a case of Malta fever .1 attended three and a half years ago at Cannes. I made careful inquiries at the time and I think that a similar sequence of events to that recorded was not very uncommon with regard to the Riviera generally about that time. The evidence is not in my own experience and I give the im- pression resulting from my inquiries for what it is worth. In my own case, which was also an undoubted one, I was quite unable to find the source of infection, though I tried care- fully to do so.-I am, Sir, yours faithfully, . , i , : p , fully , S , yours faithfully, --- R. NEVILLE HART, M.D. Cantab. .Bournemouth, July 24th, 1909. , yours faithfully, --- R. NEVILLE HART, M.D. Cantab. am, yours faithfully, - A. MARIUS WILSON, M.D. Durh. ’ ’ Cape Town, July 6th, 1909. I. ORGANISATION.-DUTIES IN CAMP. THE following notes are intended as an I appreciation " of the medical organisation of the Territorial Army, in so far as routine duties and sanitation in camps of exercise are con- cerned, and the relationship of these to active service in the field. Questions connected with the removal of sick and wounded will not be included. eco ded. am, Sir, yours faithfully, C. F. MARSHALL. am, Sir, yours faithfully, C. F. MARSHALL. am, Sir, yours faithfully, C. F. MARSHALL. ithfully, C. F. MARSHALL. St. John’s Wood Park, N.W., July 25th, 1909. St. John’s Wood Park, N.W., July 25th, 1909. St. John’s Wood Park, N.W., July 25th, 1909. To the Editor of THE LANCET. To the Editor of THE LANCET. SIR,-The attention of my committee has been drawn to the statement in the published report of the meeting of the Education Committee of the London County Council held on July 14th, referred to in THE LANCET of July 17th, that the Paddington Green Children’s Hospital will take 20 ophthalmic cases a week." I am requested to inform you that this statement is incorrect, and that no such offer has been made from this hospital m this hospital. I am, Sir, yours faithfully, ’ ’ Cape Town, July 6th, 1909. TROPICAL ADENITIS AND PLAGUE. To the -Editor of THE LANCET. SiR,-In connexion with the annotation on Tropical Adenitis and Plague which appeared in THE LANCET of June 12th (p. 1701), I may say that, prior to our plague visitation, we had many cases of bubo which were certainly not plague buboes nor the usual ordinary bubo-simple or venereal. I mentioned this fact to Professor Simpson, and he told me that in Calcutta these premonitory buboes had also been noticed just before a plague outbreak. just plague I am, Sir yours faithfully, corps troops The P.M.O. of an army corps, the A.M.O. of a division, the S.M.O. of a brigade, and the M.O. of a unit are the respective advisers of their commanders on all questions j plague I am, Sir yours faithfully, , yours faithfully, - A. MARIUS WILSON, M.D. Durh. ’ ’ Cape Town, July 6th, 1909.
https://openalex.org/W2966259847	https://earth-planets-space.springeropen.com/track/pdf/10.1186/s40623-019-1059-x	English	null	A comprehensive paleomagnetic study from the last Plinian eruptions of Popocatepetl volcano: absolute chronology of lavas and estimation of emplacement temperatures of PDCs	Earth, planets and space	2,019	cc-by	12,877	FULL PAPER Open Access A comprehensive paleomagnetic study from the last Plinian eruptions of Popocatepetl volcano: absolute chronology of lavas and estimation of emplacement temperatures of PDCs Nayeli Pérez‑Rodríguez1, Juan Morales2* , Avto Goguitchaichvili2 and Felipe García‑Tenorio Abstract Three lava flows (Buenavista, Xalitzintla and Nealtican) and pyroclastic density currents (Lorenzo and Pink Pumice) from two Popocatepetl Plinian eruptions were sampled for paleomagnetic dating. A detailed rock-magnetic charac‑ terization of the lavas, scoria clasts and pottery shards intercalated between the volcanic deposits was also carried out. Reliable results, both in direction and in intensity, were obtained for the Nealtican lava flow, which enabled its full-vector paleomagnetic dating using the archaeo_dating tool together with the global paleosecular variation model SHA.DIF.14 k, obtaining an age interval between 1040 AD and 1140 AD (95% probability confidence level), in good agreement with its associated 14C age. The well-grouped paleomagnetic direction of the seven specimens from two different scoria clasts of the Lorenzo Pumice pyroclastic density current suggests that clasts were emplaced hot, at a temperature that seems to have almost completely erased the original remanent magnetization of the clasts. This fact is supported by the reheating of the underlying pottery shards, evidenced as a clear secondary low-tem‑ perature range (room temperature to 350 °C) component at the orthogonal vector plots. Similarly, the three mean clusters directions obtained for site PO-2 (Pink Pumice)—roughly concentrated around the present geomagnetic field—suggest also a high emplacement temperature. Also, the first archeointensity dating of a pottery shard within the pyroclastic density current is reported. Finally, results of the rock-magnetic and paleomagnetic dating of the last Plinian eruptions from the Popocatepetl volcano, applied to different volcanic materials (lava and pyroclastic density currents), show the usefulness of these nonconventional and alternative techniques in the study of the eruptive activ‑ ity of volcanoes. rds: Popocatepetl volcano, Plinian eruptions, Paleomagnetism, Paleointensity, Emplacement tempe Correspondence: jmorales@geofisica.unam.mx 2 Laboratorio Interinstitucional de Magnetismo Natural (LIMNA) y Servicio Arqueomagnético Nacional (SAN), Instituto de Geofísica, UNAM, Unidad Michoacán, Campus Morelia, Antigua Carretera a Pátzcuaro No. 8701 Col. Ex‑Hacienda de San José de la Huerta, 58190 Morelia, Michoacán, Mexico Full list of author information is available at the end of the article © The Author(s) 2019. 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. Correspondence: jmorales@geofisica.unam.mx 2 Laboratorio Interinstitucional de Magnetismo Natural (LIMNA) y Servicio Arqueomagnético Nacional (SAN), Instituto de Geofísica, UNAM, Unidad Michoacán, Campus Morelia, Antigua Carretera a Pátzcuaro No. 8701 Col. Ex‑Hacienda de San José de la Huerta, 58190 Morelia, Michoacán, Mexico Full list of author information is available at the end of the article Pérez‑Rodríguez et al. Earth, Planets and Space (2019) 71:80 https://doi.org/10.1186/s40623-019-1059-x Pérez‑Rodríguez et al. Earth, Planets and Space (2019) 71:80 https://doi.org/10.1186/s40623-019-1059-x Pérez‑Rodríguez et al. Earth, Planets and Space (2019) 71:80 https://doi.org/10.1186/s40623-019-1059-x Introduction although less common, some paleomagnetic studies were carried out to estimate emplacement temperatures of pyroclastic deposits from Santorini, Greece (e.g., McClel- land and Druitt 1989; Bardot 2000; Tema et al. 2015) and from Vesuvius, Italy (e.g., Zanella et al. 2008; Di Vito et al. 2009). Popocatepetl is the riskiest volcano in Mexico, with nearly one million people living within a radius of 40 km (Macías 2005). It is located 65 km southeast from Mexico City—with almost 9 million inhabitants—and forms the southern end of the Sierra Nevada, consisting of Tlaloc, Telapón, Teyotl, Iztaccihuatl and Popocatepetl volcano. Popocatepetl means in Nahuatl “the smoking mountain”; this refers to the fact that, during the pre-Hispanic era, the Aztecs observed it in activity on several occasions. Given the very recent ages of the lava flows, there are the only available techniques that can be used for dating. In this work, rock-magnetic and paleomagnetic analy- ses were performed on lava flows and, for the first time, in pyroclastic material from the last big eruptions (Lorenzo Pumice and Pink Pumice) and ceramics embedded within these deposits of the NE section of the Popocatepetl vol- cano. We most mention, however, that the scarcity of accessible outcrops due to ~ one thousand years of reveg- etation impedes a widespread sampling throughout the eruptive succession, while the expansion of agricultural activities and rural settlements the finding of archeologi- cal vestiges. Nevertheless, the obtained data have yield important preliminary results. Its eruptive history has been very explosive throughout the last 23 ka BP, recording at least five Plinian eruptions. In this period, the volcano experienced three destructive events; the last one occurred at 14 ka BP and gave rise to the construction of the present-day cone (Sosa-Cebal- los et al. 2015). During the Holocene, the Popocatepetl had two eruptions that occurred during pre-Hispanic periods: Lorenzo Pumice (~ 2100 BP) and Pink Pumice (~ 1000 BP), which had implications on the human set- tlements in the basin of Puebla (Plunket and Uruñuela 1998). The eruptions were dated by the 14C method in soils that inter-stratify with pyroclastic materials (Robin 1984; Siebe et al. 1996a; Plunket and Uruñuela 1998; Panfil et al. 1999; Siebe and Macías 2006; Arana-Salinas et al. 2010). However, several inconsistencies regarding the 14C results and the lithological descriptions have not allowed to establish a definitive eruptive history (Siebe et al. 1995). Geological frameworkh The trans-Mexican volcanic belt (TMVB) is a volcanic arc that crosses from east to west the central part of the Mexican territory. It is the result of the subduction of the Rivera and Cocos plates along the trenches of the Acapulco plate (Gómez-Tuena et al. 2005). It is usually divided into three sectors differentiated by their type of volcanism and its chemical composition: The west, cen- tral and east sectors (Ferrari 2000). The Popocatepetl vol- cano is part of the Sierra Nevada, located in the eastern sector of the TMVB; this sector is limited by the Taxco- Queretaro system and is characterized by containing large stratovolcanoes, calderas and complex domes of andesitic to rhyolitic composition. Worth mentioning is that the radiocarbon method does not date the volcanic product directly, but the organic matter associated with an event of interest. On the contrary, the paleomagnetic method allows to obtain the absolute age of the lava flow’s cooling moment based on the comparison of the geomagnetic field logged by the magnetic minerals within the volcanic rocks against a set of paleosecular variation (PSV) master curves established for the same geographical region, or by using geomag- netic field models calculated for the geographical region under study. Popocatepetl is a stratovolcano with a truncated crater made up of an alternation of andesitic and dacitic lava flows and pyroclastic deposits. In the last 5-ka BP, the vol- cano has presented three major Plinian eruptions: Ochre Pumice or Upper Pre-ceramic Plinian eruption (UPCPE, ~ 5000 year BP), Lorenzo Pumice or Lower Ceramic Plin- ian eruption (LCPE, ~ 2150 year BP) and Pink Pumice or Upper Ceramic Plinian eruption (UCPE, ~ 1100 year BP) (Siebe et al. 1996b). The three eruptions started with the emission of small amounts of ash and pyroclastic den- sity currents. Subsequently occurred phreatomagmatic explosions, whose remaining flows spread radially at high speeds on the slopes of the volcano, which culminated in a paroxysmal phase with the emergence of a great Plinian column, pumice fall and the emplacement of pyroclastic density currents after the collapse of the column, end- ing with extensive lahars (Siebe et al. 1996a). Contempo- raneously to the explosive eruptions, or at intermediate Nevertheless, few paleomagnetic studies have been devoted to the study of the Popocatepetl lava flows (e.g., Carrasco-Nuñez et al. 1986; Conte et al. 2004; Kosterov et al. Page 2 of 21 Pérez‑Rodríguez et al. Earth, Planets and Space (2019) 71:80 Pérez‑Rodríguez et al. Earth, Planets and Space Introduction In addition, it should be noted that the lim- ited scope of lava flows (3 km from the top of the crater, on average) and the low impact they have on the evalua- tion of volcanic hazards has caused a low interest in their study, with little information generated on these. Geological frameworkh 2009), while no paleodirection neither paleointen- sity studies have been reported on its pyroclastic deposits up to now, although successfully paleosecular variation studies have been reported on pyroclastic deposits from Mt. St. Helens, USA, Volcán Láscar, Chile, Volcán de Colima, Mexico and Vesuvius, Italy (e.g., Hagstrum and Champion 2002; Paterson et al. 2010). Additionally, Pérez‑Rodríguez et al. Earth, Planets and Space (2019) 71:80 Pérez‑Rodríguez et al. Earth, Planets and Space Page 3 of 21 Fig. 1 Overview Google Earth image of the Popocatepetl volcano deposits/flows. Stars denote the location of the five paleomagnetic sites (PO-1– PO-5). Sites PO-1 and PO-2 correspond to pyroclastic density currents (Lorenzo and Pink Pumice, respectively), while PO-3 (Nealtican lava flow), PO-4 (Xalitzintla lava flow) and PO-5 (Buenavista dacite) correspond to lava flows. See text for details Fig. 1 Overview Google Earth image of the Popocatepetl volcano deposits/flows. Stars denote the location of the five paleomagnetic sites (PO-1– PO-5). Sites PO-1 and PO-2 correspond to pyroclastic density currents (Lorenzo and Pink Pumice, respectively), while PO-3 (Nealtican lava flow), PO-4 (Xalitzintla lava flow) and PO-5 (Buenavista dacite) correspond to lava flows. See text for details Although the Xalitzintla—a light gray coloration lava (Fig. 3a)—and Nealtican—a darker coloration lava with reddish hues (Fig. 3b)—flows are thought to correspond to two eruptive pulses of the same event, since both are between the same volcano-sedimentary deposits but have different colors, we decided to sample both flows since they would log the same paleomagnetic record, and to check which one would be more suitable for paleo- magnetic determinations. stages, the Popocatepetl volcano has also presented effu- sive activity: Lava flows tapped through the central vent at the summit and fissural lava flows of andesitic compo- sition (Schaaf et al. 2005). Methodology Field workh The three major Plinian eruptions were described at out- crops in the northeastern sector of the volcano, located at the towns of Buenavista, San Nicolás de los Ranchos, Santiago Xalitzintla, Tetimpa and Nealtican (Fig. 1), to obtain a composite stratigraphic column of the study area (Fig. 2). Paleomagnetic sampling of three lava flows and scoria clasts of two pyroclastic density currents (PDC) was carried out in field by using a water-cooled portable gasoline-powered rock coring drill. On average, eight standard one-inch paleomagnetic cores (6–12 cm long) were obtained for each lava flow (Buenavista dacite, Xal- itzintla and Nealtican fissural lavas), which were distrib- uted consistently both horizontally and vertically over the outcrops (Fig. 3). Special care was dedicated to sampling only those blocks without evidence of post-emplacement dislodg- ment. We must note, however, that sampled outcrop at site PO-4 was already suspected to be tilted during field work, but since this was the best one that we could find at this flow, we decided, nonetheless, to give it a try. l In case of the two PDCs under the sequence of the Pink Pumice and Lorenzo Pumice, 20 paleomagnetic cores (8–12 cm long) were obtained by sampling five scoria clasts; nine cores from two clasts from the first sequence and 11 cores from three clasts from the second one (Fig. 4). This was possible due to the semi-consolidated Pérez‑Rodríguez et al. Earth, Planets and Space (2019) 71:80 Pérez‑Rodríguez et al. Earth, Planets and Space Page 4 of 21 a b c 5m 1m c b a DEPOSIT TYPE Soil Nealtican - Xalitzintla ﬁsural lava ﬂow Nealtican - Lahar 1 PDC rich scoria Buenavista lava ﬂow CHARACTERISTICS Contains abundant roots Contains ceramic fragments and reworked material with roots Site PO-1 Dacitic composition, located in Buenavista locality. Site PO-5 Andesitic basaltic composi- tion, gray coloration, contains ol + pg phenocrystals and abundant xenoliths. Lava ﬂow in Xalitzintla locality has a darker coloration than the Nealtican locality lava. Perhaps, this corresponds to two different eruptive pulses. Sites PO-3 and PO-4 DEPOSIT TYPE CHARACTERISTICS Nealtican - Lahar 2 Soil PDC rich pumice Pumice fall 1 Pumice fall 2 Pumice fall 3 Pumice fall Surge Gray soil Yellow soil Pumice fall Surge Black soil Reverse gradation, %20 of lithics and 20 % of crystals Reverse gradation, normal stratﬁcation. Stratum of surge at the top Reverse gradation, integrated by pg + px. Methodology Field workh Stratum of surge at the top Composed by pink and gray pumice, ﬁne sand size matrix and scoria fragments. Site PO-2 Contains ceramic fragments Traction carpet structures Yellow, brown, pink and cream colorations pumice Presence of carbonized organic matter Contains ceramic Contains lithic fragments Ocher pumice 9475 ± 150 y BP 4645 ± 60 y BP 2330 ± 195 y BP Lithics Pomez Scoria Organic matter Ceramic fragments Legend (675 ± 60) AD (1095 ± 155) AD Fig. 2 Composite stratigraphic column of the study area. See text for details c a ig. 2 Composite stratigraphic column of the study area. See text for details Fig. 3 Photographs of the different lava flows sampled. a Site PO-4, b site PO-3 and c site PO-5 hotographs of the different lava flows sampled. a Site PO-4, b site PO-3 and c site PO-5 Fig. 3 Photographs of the different lava flows sampled. a Site PO-4, b site PO-3 and c site PO-5 Pérez‑Rodríguez et al. Earth, Planets and Space (2019) 71:80 Page 5 of 21 Fig. 4 a Photograph of the scoria clast (site PO-1). b View of a scoria clast Fig. 4 a Photograph of the scoria clast (site PO-1). b View of a scoria clast state of the PDCs. A total of 44 drilled cores were obtained. Measurement of the azimuth and dip of in- place cores was carried out by means of a precision core orienting fixture, with coupled magnetic compass. Paired sun-compass orientation was carried out when possible. were pressed into salt pellets to treat them as standard paleomagnetic specimens, obtaining 47 archeomagnetic specimens in total, which were dedicated to rock-mag- netic experiments, archeointensity (AI) determinations, and to investigate emplacement temperatures of the pyroclastic flows. See below for details. Also, eight ceramic fragments—1–5 cm long—(Fig. 5a) were collected from a reworked floor of pink tones at the top of the recorded stratigraphic sequence, below the eruptive UCPE. The whole sequence covering the stratum from which the ceramics were obtained has a thickness of approximately 1.5 m (Fig. 5b). Based on the color and type of material of the different ceramic shards, these could belong to the same ceramic style. Nine ceramic fragments of varying sizes—between 2 and 7 cm long—(Fig. Methodology Field workh 5c) were unearthed from a gray colored soil at the base of the LCPE eruptive sequence, approxi- mately 60 cm from the surface, which were covered by a pyroclastic density current and an agricultural soil (Fig. 5d). Differences in the color of the fragments can be observed; thus, they probably correspond to different ceramic artifacts.i yl Bulk magnetic susceptibility (k) measurements were obtained by means of a Bartington MS2B dual-frequency sensor attached to a Bartington MS2 susceptibility meter. Rock-magnetic experiments were performed in air, using a variable field translation balance (AVFTB) from Mag- netic Measurements Ltd, to identify the magnetic min- eralogy (remanence carriers) and their thermal stability, and to investigate the suitability of the material (lavas and scoria) to obtain reliable paleointensity (PI) deter- minations. These experiments included: (1) isothermal remanence magnetization (IRM) acquisition curves, (2) hysteresis loops, (3) backfield curves and (4) thermo- magnetic curves. Saturation remanent magnetizations (Mrs), saturation magnetizations (Ms) and coercivity fields (Bc) were retrieved after correction for paramag- netic contribution of hysteresis cycles with applied fields up to ± 0.7 T. Coercivities of remanence (Bcr) were deter- mined by applying progressively increasing backfields after saturation. Thermomagnetic curves were obtained between room temperature and 600 °C. Detailed tum- bling stepwise alternating field (AF) demagnetization up to 100 mT (using an AGICO LDA 3 equipment) and thermal demagnetization (using an ASC Scientific TD48 furnace) up to 600 °C of samples were performed to iso- late the characteristic remanent magnetization (ChRM) of the samples from the three lava flows and from clast of the scoria flows. Natural remanent magnetization (NRM) and laboratory induced magnetizations (TRMs) were Although in the first case it was not possible to recog- nize what kind of vase the shards come from due to their small and irregular shape, in the second one some of the shards likely come from an ornamental tripod vase. In both cases, however, the shards should come from a sim- ple (rough) manufacturing process. Laboratory procedures Drilled cores were divided into two or more stand- ard cylindrical paleomagnetic samples (1-inch diam- eter × 1-inch height), obtaining a total of 65 specimens. The bigger pottery shards recovered from both pumice sequences were additionally broken into at least 6 speci- mens. These specimens, together with the smaller shards, Pérez‑Rodríguez et al. Earth, Planets and Space (2019) 71:80 Page 6 of 21 Pérez‑Rodríguez et al. Earth, Planets and Space Fig. 5 Different ceramic shards studied. a Ceramic shards buried from the UCPE pyroclastic density current. b View of the UCPE pyroclastic density current. c Ceramic shards buried from the LCPE pyroclastic density current. d View of the LCPE pyroclastic density current Fig. 5 Different ceramic shards studied. a Ceramic shards buried from the UCPE pyroclastic density current. b View of the UCPE pyroclastic density current. c Ceramic shards buried from the LCPE pyroclastic density current. d View of the LCPE pyroclastic density current Fig. 5 Different ceramic shards studied. a Ceramic shards buried from the UCPE pyroclastic density current. b View of the UCPE pyroclastic density current. c Ceramic shards buried from the LCPE pyroclastic density current. d View of the LCPE pyroclastic density current measured in all cases by means of an AGICO dual speed JR6 spinner magnetometer.h shards after each double heating-step were also added to the protocol. Specimens were subjected to the Thellier–Coe (TC) method of paleointensity (PI) determinations (Thellier and Thellier 1959; Coe 1967) at Laboratorio Interin- stitucional de Magnetismo Natural (LIMNA) facilities. The experiments were carried out using a ASC Scientific TD48-SC furnace; all heating/cooling steps were per- formed in air. Ten temperature steps were distributed through the entire temperature range (from room tem- perature to 560 °C) with reproducibility better than 2 °C between two heating steps. The laboratory field strength was set to (45.0 ± 0.5) µT along the cylindrical axis of the paleomagnetic cores. Partial thermoremanent mag- netization (pTRM) checks, carried out every third tem- perature step, as well as pTRM tail checks determinations (Riisager and Riisager 2001) at 350 °C and magnetic sus- ceptibility measurements of lava specimens and ceramic As stated by Chauvin et al. (2000), archeomagnetic objects that have an anisotropic shape, such as tiles and bricks, are often characterized by a strong mag- netic anisotropy. Laboratory procedures Such anisotropic shape deviates the direction of the TRM acquired by these objects during initial cooling from the local direction of the geomag- netic field (e.g., Tema 2009), while their TRM inten- sity depends on the direction in which the local field is applied. In order compensate the AI determinations on the ceramic shard investigated we followed the adjust- ment technique described by Selkin et al. (2000), which requires the measurement of the remanence anisotropy tensor χARM. At the end of the Thellier–Coe experi- ments, the specimens were given an ARM (180 mT AC field; 0.30 mT DC field) along 6 axial directions (± X, ± Y and ± Z)—using an LDA 5 AF demagnetizer coupled to Pérez‑Rodríguez et al. Earth, Planets and Space (2019) 71:80 Pérez‑Rodríguez et al. Earth, Planets and Space Page 7 of 21 Fig. 6 Variation of the NRM intensity and magnetic susceptibility for the three studied lavas and scoria clast, together with lines of constant ratio (Q = NRM/κ·H); the ratio between remanent and induced magnetizations, where κ is the initial magnetic susceptibility and H = 33 Am−1 the magnetic field intensity a PAM 1 anhysteretic/pulse magnetizer from AGICO— following the C-mode protocol described in the ARM— Brief Practical Guide Application note (AGICO Prints 2018). Between each ARM step, an AF demagnetization at 200 mT was performed to be used as a baseline. The remanence anisotropy tensor χARM was determined using the Anisoft5 software (Anisoft5 2018). Finally, the ani- sotropy correction factors (fARM) were estimated—as the ratio between the remanence acquired in a unit field par- allel to the ancient field and the remanence acquired in a unit field in the laboratory field direction—and the AI values corrected. Aimed to quantifying the cooling rate (CR) effect in our samples, the CR dependence of TRM was investigated applying a modified protocol to that described by Chauvin et al. (2000). At the end of the AI experiments, all speci- mens were heated two more times at the highest tem- perate reached during the AI experiment (560 °C) under the same laboratory field used during it. Last measure- ment (in field step) of the AI experiment was designated as TRM1. Then, a second TRM (TRM2) was induced to all the samples, but using this time a longer cooling time (~ 6 h). Laboratory procedures Lastly, a third TRM (TRM3) was created using the same cooling time as that used during the TRM1 creation (~ 45 min). The cooling rate factor fCR was calculated as the ratio between the intensity acquired during a long and a short cooling time: fCR = TRM2/TRM1. Changes in TRM acquisition capacity were estimated by means of the per- centage variation between the intensity acquired during the same cooling time (fAC = TRM3/TRM1). The cooling rate correction was only applied when the corresponding change in TRM acquisition capacity was close to 1 and fCR > 1 (Morales et al. 2009). Fig. 6 Variation of the NRM intensity and magnetic susceptibility for the three studied lavas and scoria clast, together with lines of constant ratio (Q = NRM/κ·H); the ratio between remanent and induced magnetizations, where κ is the initial magnetic susceptibility and H = 33 Am−1 the magnetic field intensity 10 < Q < 100—mainly those of site PO-05—. As will be discussed later, acquisition of strong (secondary) isother- mal remanence magnetizations (IRMs)—evidenced by high Q ratios—could impede to retrieving the character- istic (original) remanent magnetization (ChRM) acquired during cooling of the lava. Results from isothermal remanence magnetization (IRM) acquisition curves show that all volcanic samples, except those from the Xalitzintla lava flow (site PO-4), got saturation at fields between 200 and 250 mT, which suggest the dominance of low coercivity (titano-) magnetite miner- als of pseudo-single-domain (PSD) grain size. Those from site PO-4 did it at fields up to 500 mT, suggesting the pres- ence of hematite (upper part of Fig. 7). Ceramic samples got saturation also at fields above 300 mT, pointing to the dominance of medium coercivity (titanomagnetite) min- erals of single-domain (SD) and/or PSD grain size (lower part of Fig. 7), which is considered as a suitable magnetic mineralogy for paleointensity (PI) determinations. Emplacement temperature estimation of PDCs relies on the identification of two components of magnetiza- tion within the “accidental scoria clasts” (clasts of the existing volcanic structure): the original, higher tempera- ture component, which will be randomly oriented for an assemblage of clasts, and a lower temperature compo- nent that will consistently align with the Earth’s magnetic field at the time of emplacement (Paterson et al. 2010). Laboratory procedures The highest temperature at which the low-temperature component is still present provides an estimate of the emplacement temperature of the clast during a conven- tional stepwise thermal demagnetization process. Estimation of the domain state (grain size) of the mag- netic mineralogy is of fundamental importance since only non-interacting SD grains follow the laws of partial ther- moremanences (pTRMs) of Thellier (Thellier and Thellier 1959), on which the Thellier-type experiments are based (see Dunlop 2011 for a review). On the other hand, the shape—pot-bellied or wasp-waisted—of a hysteresis curve yields also an insight of the composition—single or multiple magnetic phases—of the rock sample. Hyster- esis curves for volcanic materials present both pot-bellied (site PO-1 and site PO-3) and wasp-waisted (site PO-2, Rock‑magnetic experiments NRM intensity versus magnetic susceptibility vari- ation for the three studied lavas and scoria clasts, together with lines of constant Königsberger’s ratio (Q), is shown in Fig. 6. Most samples lie between lines with 1 < Q < 10, while some few others between lines with Pérez‑Rodríguez et al. Earth, Planets and Space (2019) 71:80 Pérez‑Rodríguez et al. Earth, Planets and Space Page 8 of 21 Fig. 7 Representative examples of isothermal remanent magnetization (IRM) acquisition curves for the three lava flows and scoria clast sampled (upper plots) and three ceramic fragments (lower plots) Fig. 7 Representative examples of isothermal remanent magnetization (IRM) acquisition curves for the three lava flows and scoria clast sampled (upper plots) and three ceramic fragments (lower plots) samples from sites PO-1, PO-3 and PO-4 (3.7, 4.0 and 3.3) × 10−5 Am2 kg−1, while that for site PO-5 reaches up to 1.9 × 10−4 Am2 kg−1. Site PO-2, on the contrary, pre- sents the lowest values of Ms (9.0 × 10−5 Am2 kg−1) and Mrs (8.4 × 10−6 Am2 kg−1). Magnetization ratios Mrs/Ms versus coercivity ratios Bcr/Bc are displayed in a Day plot (Day et al. 1977). While lava samples plot mainly in the site PO-4 and site PO-5) shapes, with different values of paramagnetic contribution (Fig. 8). Saturation mag- netization Ms of (3.1, 2.4, 4.1 and 7.1) × 10−4 Am2 kg−1 was obtained for these samples from sites PO-1, PO-3, PO-4 and PO-5, respectively, with coercive force Bc val- ues of (8.7, 13.0, 5.16 and 15.1) mT. Remanent satura- tion magnetization Mrs values are quite similar for the Pérez‑Rodríguez et al. Earth, Planets and Space (2019) 71:80 Page 9 of 21 Fig. 8 Representative examples of hysteresis plots for the two scorias of the two PDCs sampled (upper plots) and lava flows (lower plots) l ) d l fl (l l ) Fig 8 Representative examples of hysteresis p Fig. 8 Representative examples of hysteresis plots for the two scorias of the two PDCs sampled (upper plots) and lava flows (lower plots) PSD region, scoria clasts show a tendency toward multi- domain (MD) grain size (Fig. 10a). However, it is worth mentioning that Day plots are not a very reliable means of determining domain structure. Rock‑magnetic experiments In the case of the ceramic samples, all magnetic param- eters retrieved from the rock-magnetic experiments (AVFTB), and their corresponding ratios, are quite simi- lar for three representative pottery shards (on average, Bc = 9 mT; Bcr = 40 mT; Ms = 1.7 10−4 Am2 kg−1; Mrs = 2.3 Fig. 9 Representative examples of hysteresis plots for the three ceramic fragments Fig 9 Representative examples of hysteresis plot for the three ceramic fragments Fig. 9 Representative examples of hysteresis plots for the three ceramic fragments Fig. 9 PSD region, scoria clasts show a tendency toward multi- domain (MD) grain size (Fig. 10a). However, it is worth mentioning that Day plots are not a very reliable means of determining domain structure. In the case of the ceramic samples, all magnetic param- eters retrieved from the rock-magnetic experiments (AVFTB), and their corresponding ratios, are quite simi- lar for three representative pottery shards (on average, Bc = 9 mT; Bcr = 40 mT; Ms = 1.7 10−4 Am2 kg−1; Mrs = 2.3 g p p y p g In the case of the ceramic samples, all magnetic param- eters retrieved from the rock-magnetic experiments (AVFTB), and their corresponding ratios, are quite simi- lar for three representative pottery shards (on average, Bc = 9 mT; Bcr = 40 mT; Ms = 1.7 10−4 Am2 kg−1; Mrs = 2.3 PSD region, scoria clasts show a tendency toward multi- domain (MD) grain size (Fig. 10a). However, it is worth mentioning that Day plots are not a very reliable means of determining domain structure. Pérez‑Rodríguez et al. Earth, Planets and Space (2019) 71:80 Pérez‑Rodríguez et al. Earth, Planets and Space Page 10 of 21 Fig. 10 Corresponding Day plot (Mrs saturation remanence, Ms saturation magnetization, Bcr remanence of coercivity, Bc coercive force) for the lava flows, scoria clast (a) and ceramic fragments (b) Fig. 10 Corresponding Day plot (Mrs saturation remanence, Ms saturation magnetization, Bcr remanence of coercivity, Bc coercive force) for the lava flows, scoria clast (a) and ceramic fragments (b) M–T curves for the Nealtican and Xalitzintla lavas are highly reversible (Fig. 11c, d), while that corresponding to the Buenavista lava is rather irreversible (Fig. 11e). How- ever, we note that from this experiment it cannot be ascer- tained at which temperature started the alteration. Rock‑magnetic experiments Results of thermomagnetic experiments—analyzed using the Rock- MagAnalyzer 1.0 software (Leonhardt 2006)—indicate the presence of one, two and up to four minerals phases, with quite variable Curie temperatures (Tc), for the Nealtican, Xalitzintla and Buenavista lavas, respectively (Fig. 11c– e). The mineral phase for the Nealtican lava shows a Tc ~ 520 °C. Corresponding mineral phases for the Xalitz- intla lava show Tc’s of ~ 400 and 540 °C. Buenavista lava, on the contrary, show minerals phases with Tc of ~ 160, 270, 360 and 560 °C. These Tc spectra suggest the coexistence of Ti–rich and Ti-poor titanomagnetite minerals, as well as the probably presence of maghemite (Tc ~ 300 °C). 10−5 Am2 kg−1; Mrs/Ms = 0.14; and Bcr/Bc = 4.6) (Figs. 9, 10b). Although ceramic samples show a tendency toward MD grain size, again, day plots are not a very reliable mean of determining domain structure.h Thellier-type experiments are basically an in-labora- tory stepwise recreation of the process that led origin (in nature) to the (thermo-) remanent magnetization acquired by a volcanic rock during cooling. As so, this stepwise recreation requires a multiheating process at increasing temperatures up to the Curie temperature of the magnetic mineralogy of the volcanic material, which could alter it giving raise to the creation of (secondary) chemical magnetizations. A qualitative—but also quanti- tative—way of estimating the degree of alteration expe- rienced by a sample due to heating is provided by the reversibility of the M–T curves. Temperature variation of magnetization (M–T curve) for the PDC PO-1 presents a markedly decrease at a temperature below 100 °C. After this temperature, it gradually increases up to ~ 350 °C (Fig. 11a). Continuous decrease till 600 °C does not allow a clear identification of the Curie temperature (Tc) of the mineral phase responsible for the magnetization. Cooling branch closely follows that of the heating one up to ~ 450 °C; after that, it departs of the heating one. Nonetheless, the very low temperature phase (~ 100 °C) appears again during cooling. On the contrary, corre- sponding M–T curve for the PDC PO-2 presents a mark- edly decrease up to a temperature below 300 °C, after which gradually deceases till 600 °C (Fig. 11b). Three dif- ferent Tc can be identify: 330 °C, 490 °C and 574 °C, the last one corresponding to very low Ti titanomagnetite. Rock‑magnetic experiments In the case of the ceramics, corresponding thermomag- netic curves are highly reversible (Fig. 12b, c), except that coming from Nealtican, which is somewhat irreversible (Fig. 12a). All curves show the presence of two to three magnetic phases: the lower one with Tc ~ 200 °C, the intermediate with Tc between 350 and 470 °C, while the third one with Tc around 500–560 °C (Fig. 12a–c). Paleodirection and paleointensity determinations While paleodirection is normally parallel to that of the magnetizing Earth’s field—and is measured directly using a magnetometer—paleointensity is only proportional to the field strength of the Earth’s field present during cool- ing and its determination is an indirect estimation. How- ever, during their geological past—and also in laboratory Pérez‑Rodríguez et al. Earth, Planets and Space (2019) 71:80 Page 11 of 21 Fig. 11 High-field-induced magnetization versus temperature (M–T) plots for the two scorias of the two PDCs (a, b) and the three lavas (c–e) Fig. 11 High-field-induced magnetization versus temperature (M–T) plots for the two scorias of the two PDCs (a, b) and the three lavas (c–e) duced magnetization versus temperature (M–T) plots for the two scorias of the two PDCs (a, b) and the three lavas (c–e) Fig. 11 High-field-induced magnetization versus temperature (M–T) plots for the two scorias of the two PDCs (a, b) a Fig. 12 High-field-induced magnetization versus temperature (M–T) plots for the three ceramic fragments (a–c) studied from Popocatepetl ( ) di d f P l agnetization versus temperature (M–T) plots for the three ceramic fragments (a–c) studied from Popocatepetl Fig. 12 High-field-induced magnetization versus temperature (M–T) plots for the three ceramic fragments (a–c) studied f Stepwise demagnetization and paleodirectionsf Stepwise demagnetization and paleodirectionsf and during the course of the experiments—volcanic rocks are prone to the acquisition of secondary magneti- zations that can mask the (original) characteristic rema- nent magnetization (ChRM). Therefore, the isolation of the ChRM by means of alternating field (AF) or thermal demagnetization processes is required. Pilot specimens, representative of the different sam- ples under study (lavas, scoria clast and ceramic shards), were selected for both alternating field (AF) and thermal demagnetization experiments. Site-mean directions were investigated using the Remasoft 3.0 software (Chadima and Hrouda 2006). Pilot specimens, representative of the different sam- ples under study (lavas, scoria clast and ceramic shards), were selected for both alternating field (AF) and thermal demagnetization experiments. Site-mean directions were investigated using the Remasoft 3.0 software (Chadima and Hrouda 2006). Pérez‑Rodríguez et al. Earth, Planets and Space (2019) 71:80 Pérez‑Rodríguez et al. Earth, Planets and Space Page 12 of 21 Fig. 13 Representative normalized orthogonal vector plots (Zijderveld diagrams) of stepwise thermally demagnetized samples from a site PO-1, b site PO-2, c site PO-3, d site PO-4 and e site PO-5. Labels along horizontal curves denote the temperature during the demagnetization step. See text for more details Fig. 13 Representative normalized orthogonal vector plots (Zijderveld diagrams) of stepwise thermally demagnetized samples from a site PO-1, b site PO-2, c site PO-3, d site PO-4 and e site PO-5. Labels along horizontal curves denote the temperature during the demagnetization step. See text for more details Fig. 13 Representative normalized orthogonal vector plots (Zijderveld diagrams) of stepwise thermally demagnetized samples from a site PO-1, b site PO-2, c site PO-3, d site PO-4 and e site PO-5. Labels along horizontal curves denote the temperature during the demagnetization step. See text for more details Pyroclastic density currents origin (Fig. 13c), which could be removed at fields/ temperatures below 10 mT/150 °C. Nonetheless, it was possible to obtain a stable end direction in such cases, comparable to those specimens unaffected by such over- prints. Corresponding mean direction (Dec = 338.9°, Inc = 32.2°, α95 = 6.4°) is shown in Fig. 14c. Although specimens from site PO-4 also displayed mainly a sin- gle paleomagnetic component (Fig. 13d), an anomalous mean direction (Dec = 267.7°, Inc = − 5.0°, α95 = 6.8°) was obtained (Fig. 14d). In the case of site PO-5 most of the specimens displayed some strong secondary compo- nents—likely of viscous (VRM) and/or isothermal origin (IRM)—and erratic directions throughout the thermal demagnetization process were obtained (Fig. 13e). They were, nonetheless, able to be removed at fields/tempera- tures below 10 mT/150 °C. As a result of their presence, a quite scattered mean direction was obtained (Fig. 14e). Table 1 displays the mean ChRM directions obtained for the three lava flows and two PDCs studied. Specimens of site PO-1 displayed mainly a single pale- omagnetic component directed through the origin, accompanied by an initial weak overprint of possibly vis- cous origin (Fig. 13a) which could be removed at fields/ temperatures below 10 mT/150 °C. On the contrary, those from site PO-2 presented a composite-trace Zijder- veld diagram, with at least two components which were not fully demagnetized at 580 °C (Fig. 13b). Attempts to retrieving mean direction from the PDCs (sites PO-1 and PO-2) yielded a mean direction (Dec = 337.3°, Inc = 49.2°, α95 = 4.5°) for the first PDC, and a three clusters plot with well-differentiated directions for each cluster for the sec- ond PDC (Fig. 14a, b, respectively). Lava flows Most of the specimens of site PO-3 displayed mainly a single paleomagnetic component, occasionally accom- panied by an initial weak overprint of possibly viscous Pérez‑Rodríguez et al. Earth, Planets and Space (2019) 71:80 Pérez‑Rodríguez et al. Earth, Planets and Space Page 13 of 21 Fig. 14 Stereo plots showing the characteristic remanent magnetization (ChRM) directions for the studied pyroclastic density currents (a and b) and lava flows (c–e). Flow mean directions are shown by the red dots, together with their corresponding 95% confidence angles Fig. 14 Stereo plots showing the characteristic remanent magnetization (ChRM) directions for the studied pyroclastic density currents (a and b) and lava flows (c–e). Flow mean directions are shown by the red dots, together with their corresponding 95% confidence angles Fig. 14 Stereo plots showing the characteristic remanent magnetization (ChRM) directions for the studied pyroclastic den and lava flows (c–e). Flow mean directions are shown by the red dots, together with their corresponding 95% confidence Fig. 15 Representative normalized orthogonal vector plots (Zijderveld diagrams) of stepwise thermally demagnetized samples from a ceramic sherd cL2, b ceramic sherd dL2 and c ceramic sherd dL3. Labels along horizontal curves denote the temperature during the demagnetization step. See text for more details Fig. 15 Representative normalized orthogonal vector plots (Zijderveld diagrams) of stepwise thermally demagnetized samples from a ceramic sherd cL2, b ceramic sherd dL2 and c ceramic sherd dL3. Labels along horizontal curves denote the temperature during the demagnetization step. See text for more details 300–350 °C. Nonetheless, its primary component is directed toward the origin (Fig. 15b). Finally, ceramic sherd labeled dL3 showed a behavior alike that of dL2, although its NRM reduced only to almost 50% at 560 °C (Fig. 15c). Ceramics Ceramic sherd labeled cL2 displayed also a single paleo- magnetic component, infrequently accompanied by an initial weak overprint of possibly viscous origin (Fig. 15a), which could be removed at fields/temperatures below 10 mT/150 °C. Ceramic sherd labeled dL2 presented clearly a stronger secondary component, removed at Pérez‑Rodríguez et al. Earth, Planets and Space (2019) 71:80 Page 14 of 21 Table 1 Site-mean directions for the Popocatepetl paleomagnetic sites, with sampling coordinates Sites with an asterisk denote pyroclastic density current (PDC) N number of recovered drill cores, n number of specimens used for the calculation of site-mean direction, R unit vector sum, k precision parameter, α95 95 per cent confidence level, Dec declination, Inc inclination Site Field code Latitude Longitude N/n R k α95 Dec (°) Inc (°) Xalitzintla PO-1* (I) 19°04′55.15″ 98°31′55.50″ 3/2 2.0 210.6 17.3 343.4 47.9 Xalitzintla PO-1* (II) 19°04′55.15″ 98°31′55.50″ 6/5 4.98 188.2 5.6 335.1 49.4 Xalitzintla PO-2* (I) 19°05′02.28″ 98°31′28.74″ 5/5 4.96 100.0 7.7 357.8 13.6 Xalitzintla PO-2* (II) 19°05′02.28″ 98°31′28.74″ 4/4 3.96 74.80 10.7 341.2 44.7 Xalitzintla PO-2* (III) 19°05′02.28″ 98°31′28.74″ 2/2 2.00 1781.0 5.9 19.5 29.0 Nealtican PO-3 19°02′42.18″ 98°26′46.50″ 7/7 6.93 88.70 6.4 338.9 32.2 Xalitzintla PO-4 19°05′10.38″ 98°31′50.46″ 8/5 4.97 127.17 6.8 267.7 − 5.0 Buenavista PO-5 19°05′21.30″ 98°36′37.50″ 8/4 3.93 41.74 14.4 347.6 36.3 Table 1 Site-mean directions for the Popocatepetl paleomagnetic sites, with sampling coordinates ections for the Popocatepetl paleomagnetic sites, with sampling coordinates Sites with an asterisk denote pyroclastic density current (PDC) py y ( ) N number of recovered drill cores, n number of specimens used for the calculation of site-mean direction, R unit vector sum, k precision parameter, α95 95 per cent confidence level, Dec declination, Inc inclination Table 2 Thellier–Coe paleointensity results for site PO-3 (Neáltican lava flow) δT temperature interval used for the intensity determination, N number of heating steps used for the intensity determination, β = σm/m (m slope of the best fit line, σm standard deviation of m), f fraction of extrapolated NRM used for intensity determination, g gap factor, q quality factor as defined by Coe et al. (1978), δ(CK), δ(TR), MADanc and class as defined by Leonhardt et al. Ceramics (2004), Hraw raw archeointensity value, σH standard deviation of H, f cooling rate correction factor, Hcrc cooling rate- corrected archeointensity Specimen ΔT (°C) N β f g q δ(CK) δ(TR) MADanc Class Hraw (μT) ± σH (μT) f Hcrc (μT) 21A 150–560 11 0.04 0.59 0.86 12.2 5.9 3.3 2.1 B 55.4 2.29 1.11 49.9 22A 200–560 10 0.03 0.90 0.87 30.0 2.7 7.7 0.9 B 59.7 1.55 1.08 55.2 23B 200–560 10 0.02 0.91 0.87 32.4 5.1 7.0 1.1 B 59.5 1.46 1.02 58.3 24C 200–560 10 0.06 0.71 0.77 9.3 6.4 7.0 1.1 B 61.8 3.61 1.03 60.0 25A 250–560 9 0.05 0.76 0.78 12.5 0.3 7.8 1.5 A 63.6 3.04 1.04 61.2 26B 250–560 8 0.04 0.69 0.83 15.7 7.9 4.4 1.5 B 60.8 2.24 1.04 58.4 27A 250–560 9 0.03 0.75 0.85 23.5 6.5 4.1 1.2 B 61.6 1.65 1.05 58.6 Mean = 60.3 57.4 1σ = 2.6 3.8 Table 2 Thellier–Coe paleointensity results for site PO-3 (Neáltican lava flow) δT temperature interval used for the intensity determination, N number of heating steps used for the intensity determination, β = σm/m (m slope of the best fit line, σm standard deviation of m), f fraction of extrapolated NRM used for intensity determination, g gap factor, q quality factor as defined by Coe et al. (1978), δ(CK), δ(TR), MADanc and class as defined by Leonhardt et al. (2004), Hraw raw archeointensity value, σH standard deviation of H, f cooling rate correction factor, Hcrc cooling rate- corrected archeointensity Paleointensities mean value is 57.4 ± 3.8 μT (n = 7). Results for the site PO-3 lava flow are listed in Table 2, together with their corresponding quality parameters, while a representative Arai plot is shown in Fig. 16a. In spite of the non-promising rock-magnetic results of site PO-5 lava flow and Nealtican ceramic sherds for PI experiments, we decided to give them a chance and sub- jected also these materials to paleointensity determina- tions, specially to the Nealtican ceramic sherds since these are unique samples. No positive results from the site PO-4 and site PO-5 specimens could be obtained. In the case of the site PO-5 lava flow, unsuccessful results could be attributed to (1), strong secondary components of some specimens likely caused by lightning strikes. This fact is evidenced at the NRM versus k plot (Fig. 6), where specimens from the Buenavista lava plot between the constant lines Q = 10 and Q = 100. (2) Points at the Arai plot define a typi- cal concave-up behavior, characteristic of multidomain remanence carriers, and (3) the significant contribution to the magnetization carried likely by Ti maghemite, which transforms to Ti magnetite upon heating up to 600 °C. In the case of the site PO-4 lava flow, Arai plots are characterized by a relatively fast NRM lost without corresponding TRM acquisition for the six specimens that failed to produce a PI estimate. Seven, six and six specimens from the site PO-3 (Neal- tican), site PO-4 (Xalitzintla) and site PO-5 (Buenavista) lava flows, respectively, were treated with the TC pro- tocol. In the case of the PDCs, nine specimens coming from two different scoria clasts (site PO-1) and twelve specimens coming from three different scoria clasts (PO- 2) were analyzed using the same protocol. Data were ana- lyzed with the ThellierTool4.0 software (Leonhardt et al. 2004). All analyzed specimens from the site PO-3 lava flow yielded reliable results, with associated mean frac- tion (f), gap (g) and quality (q) parameters of 0.71, 0.84 and 16.0, respectively. Mean raw PI for this lava flow is 60.3 ± 2.6 μT (n = 7), while the cooling rate-corrected Pérez‑Rodríguez et al. Earth, Planets and Space (2019) 71:80 Pérez‑Rodríguez et al. Earth, Planets and Space Page 15 of 21 For the Lorenzo Pumice (site PO 1) 9 specimens pro (site PO 2) nine out of 12 specimens produced reliable Fig. Paleointensities 16 a–c Representative Thellier–Coe NRM lost versus pTRM gained (Arai) plots for the different studied flows. Neáltica lava (a), Lorenzo Pumice flow (b) and summit of Lorenzo Pumice (c). Filled circles correspond to data points obtained at each double heating step used for the best fit regression line. Open circles correspond to data points not considered for the best fit calculation. Inset shows the NRM lost (pTRM gain) versus temperature at each step. Triangles represent the pTRM checks presentative Thellier–Coe NRM lost versus pTRM gained (Arai) plots for the different studied flows. Neáltica lava (a), Lorenzo Pumice Fig. 16 a–c Representative Thellier–Coe NRM lost versus pTRM gained (Arai) plots for the different studied flows. Neáltica lava (a), Lorenzo Pumice flow (b) and summit of Lorenzo Pumice (c). Filled circles correspond to data points obtained at each double heating step used for the best fit regression line. Open circles correspond to data points not considered for the best fit calculation. Inset shows the NRM lost (pTRM gain) versus temperature at each step. Triangles represent the pTRM checks Fig. 16 a–c Representative Thellier–Coe NRM lost versus pTRM gained (Arai) plots for the different studied flows. Neáltica lava (a), Lorenzo Pumice flow (b) and summit of Lorenzo Pumice (c). Filled circles correspond to data points obtained at each double heating step used for the best fit regression line. Open circles correspond to data points not considered for the best fit calculation. Inset shows the NRM lost (pTRM gain) versus temperature at each step. Triangles represent the pTRM checks For the Lorenzo Pumice (site PO-1), 9 specimens pro- duced reliable results. However, 3 specimens yielded a high PI value of 69.2 ± 4.2 μT, while the other 6 speci- mens a lower PI value of 43.8 ± 2.8 μT. A representa- tive Arai plot is shown in Fig. 16b. For the Pink Pumice (site PO-2), nine out of 12 specimens produced reliable results. In this case, PI values of 54.9 ± 3.4 μT (N = 5), 45.4 ± 2.8 μT (N = 3) and 62.9 ± 3.5 μT (N = 2) were obtained for the three different sampled clasts. Results for the sites PO-1 and PO-2 are listed in Table 3. Pérez‑Rodríguez et al. Paleointensities Earth, Planets and Space (2019) 71:80 Page 16 of 21 Table 3 Thellier–Coe paleointensity results for sites PO-1 and PO-2 (pyroclastic density currents) Data in italics not used for PI calculations ΔT temperature interval used for the intensity determination, N number of heating steps used for the intensity determination, β = σm/m, f fraction of extrapolated NRM used for intensity determination, g gap factor, q quality factor as defined by Coe et al. (1978), δ(CK), δ(TR), MADanc and class as defined by Leonhardt et al. Paleointensities (2004), Hraw raw archeointensity value, σH standard deviation of H, f cooling rate correction factor, Hcrc cooling rate-corrected archeointensity Specimen ΔT (°C) N β f g q δ(CK) δ(TR) MADanc Class Hraw (μT) ± σH (μT) f Hcrc (μT) Site PO-1 Clast 1 1B 250–560 9 0.05 0.52 0.60 7.7 1.4 5.9 1.5 C 65.41 2.68 1.00 65.18 2B 250–560 9 0.07 0.42 0.68 4.1 4.4 2.9 1.2 B 69.93 4.83 1.02 66.59 3A 400–560 6 0.04 0.29 0.54 4.0 4.7 5.7 1.4 C 73.74 2.91 1.00 73.37 Mean = 69.7 69.2 1σ = 4.2 4.2 Clast 2 4A 250–560 9 0.01 0.50 0.59 3.0 3.2 4.4 1.1 B 44.29 1.39 1.00 44.29 4B 250–560 9 0.04 0.57 0.55 7.7 3.6 4.8 0.9 A 41.23 1.86 1.00 41.23 5A 250–560 9 0.05 0.69 0.44 5.9 2.6 5.3 1.1 B 44.32 1.67 1.08 41.01 6A 300–560 8 0.03 0.87 0.77 19.0 0.4 8.6 1.1 C 42.15 2.19 1.02 41.50 8A 250–560 9 0.03 0.88 0.82 26.8 9.0 6.8 1.7 C 42.42 1.30 1.00 42.51 9A 250–560 9 0.05 0.91 0.81 15.4 1.6 9.7 0.9 C 45.17 2.40 1.00 45.17 Mean = 44.5 43.8 1σ = 3.7 2.8 Site PO-2 Clast 1 10A 200–540 8 0.06 0.72 0.77 9.1 18.0 5.5 1.3 C 57.7 3.84 1.00 57.7 11A 150–515 7 0.12 0.36 0.66 1.9 0.5 2.8 1.2 B 57.6 7.1 1.01 57.0 12A 200–515 9 0.05 0.37 0.67 4.6 7.6 2.8 1.0 B 58.7 3.14 1.04 56.4 13A 200–515 8 0.10 0.38 0.65 2.5 4.2 4.5 0.9 B 59.2 5.93 1.05 56.4 14A 150–515 8 0.12 0.32 0.64 1.8 3.7 6.5 0.7 B 50.1 5.91 1.01 49.8 Mean = 56.4 54.9 1σ = 4.1 3.4 Clast 2 15A 200–515 9 0.07 0.35 0.78 3.9 6.3 1.5 1.8 B 48.05 3.33 1.04 46.2 16B 300–560 9 0.09 0.35 0.79 3.0 42.9 3.6 1.5 C 50.00 4.67 1.05 47.6 17A 250–560 7 0.08 0.31 0.85 3.1 62.0 4.4 1.1 C 42.74 3.59 1.01 42.3 18A N/R – – – – – – – – – – – Mean = 46.9 45.4 1σ = 3.8 2.8 Clast 3 19B 350–540 7 0.07 0.37 0.82 4.0 9.4 5.7 4.1 B 68.68 4.28 1.05 65.4 20A 200–540 9 0.05 0.49 0.87 8.3 7.6 4.9 2.1 B 64.10 1.46 1.06 60.5 20B N/R – – – – – – – – – Mean = 66.4 62.9 1σ = 3.2 3.5 Table 3 Thellier–Coe paleointensity results for sites PO-1 and PO-2 (pyroclastic density currents) Data in italics not used for PI calculations ΔT temperature interval used for the intensity determination, N number of heating steps used for the intensity determination, β = σm/m, f fraction of extrapolated NRM used for intensity determination, g gap factor, q quality factor as defined by Coe et al. Paleointensities (1978), δ(CK), δ(TR), MADanc and class as defined by Leonhardt et al. (2004), Hraw raw archeointensity value, σH standard deviation of H, f cooling rate correction factor, Hcrc cooling rate-corrected archeointensity ΔT temperature interval used for the intensity determination, N number of heating steps used for the intensity determination, β = σm/m, f fraction of extrapolated NRM used for intensity determination, g gap factor, q quality factor as defined by Coe et al. (1978), δ(CK), δ(TR), MADanc and class as defined by Leonhardt et al. (2004), Hraw raw archeointensity value, σH standard deviation of H, f cooling rate correction factor, Hcrc cooling rate-corrected archeointensity Only three out of the eight analyzed ceramics frag- ments—four unearthed from the Lorenzo Pumice (labeled dL) and four at its summit (labeled cL)—yielded reliable PI results. As in the case of samples from the lava flow site PO-3, symmetrical NRM lost and pTRM gained plots were obtained during the TC paleointensity experiments. Mean quality parameters of f = 0.61, g = 0.82 and q = 13.1, and an associated anisotropy-corrected mean PI = 39.1 ± 1.5 μT (N = 6) were obtained for the fragment cL2 at the summit. For the ceramic fragment dL2, mean quality parameters of f = 0.47, g = 0.80 and q = 8.1, and an associated anisotropy- corrected mean PI = 30.7 ± 2.5 μT (N = 5) were obtained, Pérez‑Rodríguez et al. Earth, Planets and Space (2019) 71:80 Page 17 of 21 Table 4 Thellier–Coe paleointensity results for ceramic fragments Spec: cL2: summit of Lorenzo pumice; dL2 and dL3: below Lorenzo pumice ΔT temperature interval used for the intensity determination, N number of heating steps used for the intensity determination, β = σm/m, f fraction of extrapolated NRM used for intensity determination, g gap factor, q quality factor as defined by Coe et al. (1978), δ(CK), δ(TR), MADanc and class as defined by Leonhardt et al. Paleointensities (2004), Hraw raw archeointensity value, σH standard deviation of Hraw, fani anisotropy correction factor, Hani anisotropy-corrected archeointensity ΔT temperature interval used for the intensity determination, N number of heating steps used for the intensity determination, β = σm/m, f fraction of extrapolated NRM used for intensity determination, g gap factor, q quality factor as defined by Coe et al. (1978), δ(CK), δ(TR), MADanc and class as defined by Leonhardt et al. (2004), Hraw raw archeointensity value, σH standard deviation of Hraw, fani anisotropy correction factor, Hani anisotropy-corrected archeointensity Carrasco et al. 2014) calculated for the geographical posi- tion of the sites. This model is the latest developed using all the available paleomagnetic data for their correspond- ing time intervals and applying the classical modeling approach, i.e., the spherical harmonic analysis in space and the penalized cubic B-splines in time. Full-vector paleomagnetic dating of site PO-3 yields the single time interval of AD 1038–AD 1139 as the most probable age for the lava’s cooling moment (Fig. 17). Paleointensity dating of pottery shard cL2 (Fig. 18) yields a time interval of AD 584–AD 823, locating it in time at the Epiclassic period. Carrasco et al. 2014) calculated for the geographical posi- tion of the sites. This model is the latest developed using all the available paleomagnetic data for their correspond- ing time intervals and applying the classical modeling approach, i.e., the spherical harmonic analysis in space and the penalized cubic B-splines in time. Full-vector paleomagnetic dating of site PO-3 yields the single time interval of AD 1038–AD 1139 as the most probable age for the lava’s cooling moment (Fig. 17). Paleointensity dating of pottery shard cL2 (Fig. 18) yields a time interval of AD 584–AD 823, locating it in time at the Epiclassic period. while for the ceramic fragment dL3 corresponding mean quality parameters of f = 0.36, g = 0.83 and q = 7.1, and an associated anisotropy-corrected mean PI = 40.1 ± 1.3 μT (N = 2) were obtained. PI results for the ceramic fragments are listed in Table 4, together with their corresponding quality parameters, and a representative Arai plot is shown in Fig. 16c. For the other five ceramics fragments erratic behavior of the NRM was evidenced after each zero-field step during the TC experiments. PI estimates were abnor- mally low or high. Paleointensities Results of magnetic susceptibility meas- urements of lava specimens after each double heating-step during the Thellier-Coe PI experiments are shown in Additional file 1. Those corresponding to ceramic shards are shown in Additional file 2. Paleointensities (2004), Hraw raw archeointensity value, σH standard deviation of Hraw, fani anisotropy correction factor, Hani anisotropy-corrected archeointensity Spec ΔT (°C) N β f g q δ(CK) δ(TR) MADanc Class Hraw (μT) ± σH (μT) fani Hani cL2–7 150–515 10 0.06 0.59 0.86 8.4 15.6 1.1 0.8 C 47.1 2.87 0.865304 40.8 cL2–8 250–515 8 0.03 0.55 0.84 14.6 15.3 0.8 1.3 C 43.7 1.47 0.894808 39.1 cL2–9 250–515 8 0.04 0.61 0.84 11.7 7.8 1.3 1.2 B 43.4 1.67 0.875342 38.0 cL2–10 150–515 10 0.05 0.73 0.86 12.8 6.9 2.8 1.7 B 43.5 2.06 0.866909 37.7 cL2–11 350–515 6 0.05 0.58 0.78 8.9 2.0 6.0 1.9 A 47.7 2.30 0.861864 41.1 cL2–12 350–515 6 0.02 0.60 0.77 22.0 5.2 5.1 2.3 B 39.8 0.81 0.954895 38.0 Mean = 44.2 39.1 1σ = 2.9 1.5 dL2–30 350–540 7 0.11 0.51 0.82 3.9 6.4 1.7 7.8 B 31.8 3.5 0.953086 30.3 dL2–31 350–515 6 0.06 0.43 0.78 5.2 2.2 7.5 4.9 B 38.9 2.5 0.976721 38.0 dL2–32 N/R 0 – – – – – – – – – 0.975110 31.8 dL2–33 350–515 6 0.04 0.47 0.77 9.0 3.7 6.8 3.5 B 32.4 1.3 0.981138 28.0 dL2–34 350–540 7 0.10 0.53 0.80 4.3 7.2 16.0 5.8 C 32.6 3.2 0.859148 30.6 dL2–35 350–475 4 0.06 0.30 0.66 3.4 3.9 4.6 3.4 C 34.2 2.0 0.896152 Mean = 34.0 31.7 1σ = 2.9 3.7 dL3–36 300–515 7 0.04 0.27 0.76 4.9 2.7 2.2 1.6 C 54.6 2.2 0.932281 50.9 dL3–38 300–515 7 0.10 0.27 0.70 1.9 2.3 0.3 1.6 C 50.1 5.1 0.906250 45.4 dL3–39 N/R – – – – – – – – C – – 0.964768 54.9 dL3–40 N/R – – – – – – – – – – dL3–41 300–475 5 0.08 0.21 0.72 1.9 2.8 2.0 1.9 56.9 4.5 Mean = 53.9 50.4 1σ = 3.4 4.8 Table 4 Thellier–Coe paleointensity results for ceramic fragments ΔT temperature interval used for the intensity determination, N number of heating steps used for the intensity determination, β = σm/m, f fraction of extrapolated NRM used for intensity determination, g gap factor, q quality factor as defined by Coe et al. (1978), δ(CK), δ(TR), MADanc and class as defined by Leonhardt et al. Paleomagnetic datingh Upper panels: red thick curves show the variation in time of the directional (declination and inclination) and magnitude (intensity) components of the paleomagnetic field as determined from the SHA.DIF.14 k model, while blue thick horizontal lines represent the mean declination, inclination and intensity of the ancient field determined in laboratory. All curves/lines are shown with their corresponding 95% confidence intervals (thin curves/lines above and below the corresponding thick curves/line). Central panels: The corresponding probability density functions are shown as shaded peaks, together with the 95% confidence level highlighted by horizontal green lines. The combined (Dec, Inc and intensity) probability density is shown at the lower most right panel In a previous study, Conte et al. (2004) reported the paleodirectional and paleointensity study of 16 Pop- ocatepetl lava flows, for which a mean paleodirection with D = 345.7°, I = 35.4°, k = 21, α95 = 8.5°, N = 15 was obtained. However, most of these lavas were sampled at the northern sector of the edifice, while ours at the northeastern flank. Mean paleodirection obtained for site PO-3 agrees with that reported by Conte et al. (2004). In regard to the unexpected high inclination value (49.2°) obtained for site PO-1, we note that five out of the 16 lava flows studied by Conte et al. (2004) present even higher inclination values (48.9°–69.5°). On the other hand, only four individual lava flows (fourteen samples) yielded acceptable paleointensity estimates which yielded mean- flow paleointensity values in the range from 30.2 ± 7.3 to 46.9 ± 5.6 μT. while the other two (unsuitable) lava flows at ~ 2700 (site PO-4) and above 3400 m a.s.l. (site PO-5), respectively.h The several strong secondary components shown by samples coming from site PO-5 (Fig. 13e), explained as of isothermal origin (IRMs), could be well explained in terms of lightings—very common at high altitudes—also evidenced by the high values at the Königsberger plot (Fig. 6). Moreover, the rather irreversible M–T plot of the Buenavista lava and the multiple minerals phases with Tc of ~ 160, 270, 360 and 560 °C could account for the unsuccessful results obtained for this lava flow. l On the other hand, the relatively fast NRM lost without corresponding TRM acquisition for the six specimens that failed to produce a PI estimate for site PO-4 could be explained by irreversible motion of domain walls during successive heating and cooling experiments (Levi 1977). Paleomagnetic datingh Volcanic rocks are recognized as good geomagnetic field recorders (e.g., Morales et al. 2010 and references therein), and although at field work the lava flows sam- pled looked fresh and without evidence of alteration, only one out of the three lava flows sampled yielded reliable results, both in direction and in intensity. This successful flow (site PO-3) locates at an altitude of ~ 2300 m a.s.l., The most probable date for the cooling of the analyzed lava flows (paleomagnetic dating) was determined by assessment of the corresponding probability density function (PDF) obtained by the of use the Matlab tool archaeo_dating, developed by Pavón Carrasco et al. (2011), along with the global model SHA.DIF.14 k (Pavón Pérez‑Rodríguez et al. Earth, Planets and Space (2019) 71:80 (2019) 71:80 Pérez‑Rodríguez et al. Earth, Planets and Space Page 18 of 21 Fig. 17 Full-vector paleomagnetic dating of the Nealtican (1 k) lava flow. Upper panels: red thick curves show the variation in time of the directional (declination and inclination) and magnitude (intensity) components of the paleomagnetic field as determined from the SHA.DIF.14 k model, while blue thick horizontal lines represent the mean declination, inclination and intensity of the ancient field determined in laboratory. All curves/lines are shown with their corresponding 95% confidence intervals (thin curves/lines above and below the corresponding thick curves/line). Central panels: The corresponding probability density functions are shown as shaded peaks, together with the 95% confidence level highlighted by horizontal green lines. The combined (Dec, Inc and intensity) probability density is shown at the lower most right panel Fig. 17 Full-vector paleomagnetic dating of the Nealtican (1 k) lava flow. Upper panels: red thick curves show the variation in time of the directional (declination and inclination) and magnitude (intensity) components of the paleomagnetic field as determined from the SHA.DIF.14 k model, while blue thick horizontal lines represent the mean declination, inclination and intensity of the ancient field determined in laboratory. All curves/lines are shown with their corresponding 95% confidence intervals (thin curves/lines above and below the corresponding thick curves/line). Central panels: The corresponding probability density functions are shown as shaded peaks, together with the 95% confidence level highlighted by horizontal green lines. The combined (Dec, Inc and intensity) probability density is shown at the lower most right panel Fig. 17 Full-vector paleomagnetic dating of the Nealtican (1 k) lava flow. Paleomagnetic datingh Alternatively, this behavior may result from irreversible variations of coercive force (Kosterov and Prévot 1998) at low temperatures, and can be interpreted as transforma- tion from a single-domain “metastable” state to a multid- omain state, which results in large NRM lost without any correlated pTRM acquisition. stimation of emplacement temperatures of PDCs In a paleomagnetic and rock-magnetic investigation car- ried out on scoria clasts sampled from three historically active volcanoes—Láscar in the Chilean Andes, Mt. St. Helens, USA and Vesuvius, Italy—Paterson et al. (2010) Pérez‑Rodríguez et al. Earth, Planets and Space (2019) 71:80 Pérez‑Rodríguez et al. Earth, Planets and Space (2019) 71:80 Page 19 of 21 Fig. 18 Paleointensity dating for the pottery shard cL2 based on a mean PI = 38.9 ± 2.6 μT obtained from five specimens. Description of the different panels as in Fig. 17 Fig. 18 Paleointensity dating for the pottery shard cL2 based on a mean PI = 38.9 ± 2.6 μT obtained from five specimens. Description of the different panels as in Fig. 17 highlighted the usefulness of lithic clasts from pyroclastic deposits for paleointensity determination. the emplacement resulted in a slightly scattered mean direction. In this investigation, seven out of nine specimens coming from two different scoria clasts of PDC PO-1 yielded a well-constrained direction (Dec = 337.3°, Inc = 49.2°, α95 = 4.5°), although with a high inclination. Three specimens yielded a high PI value of 69.2 ± 4.2 μT, while the other 6 specimens yielded a lower PI value of 43.8 ± 2.8 μT. Single-magnetization Zijderveld plots and well-grouped paleomagnetic direction of the seven specimens from the two different scoria clasts of PDC PO-1 (Lorenzo Pumice, Fig. 15a) suggest that clasts were emplaced hot, at a temperature that seems to have com- pletely reset the original magnetization of the clasts, and then cooled in situ after deposition. Nonetheless, one or both lithologies seem to have experienced alteration due to reheating, anisotropy of remanence effects affected different to both clasts, or a combination of both cases resulted in different intensities records of the Earth’s magnetic field present during emplacement. Pottery shard cL2 seems to have not experienced a post-deposition heating, as evidenced by the single-mag- netization vectorial plot (Fig. 15a) (see also Additional file 3: THD cL Ceramics). Nonetheless, it should be con- sidered that despite the high temperature of the PDC the pottery fragments were not re-heated because they were covered (protected) by the soil. y Pottery shards dL2 and dL3, unearthed below the— hot-emplaced—Lorenzo Pumice deposit, show results of the reheating as a clear secondary low-temperature range (room temperature to ~ 300–350 °C) component at the orthogonal plot (Fig. 15b) (see also Additional file 4: THD dL Ceramics). Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. stimation of emplacement temperatures of PDCs Likely, this reheating could be acquired during its continues usage as an utilitarian piece. How- ever, as above mentioned, some of the shards likely come from an ornamental tripod vase. Moreover, differences in the color of the fragments can be observed; thus, they probably correspond to different ceramic artifacts. There- fore, most ceramics shards must come from an ornamen- tal (heated just once during its elaboration) rather than from a utilitarian artifact. i In case of site PO-2 (Pink Pumice), the three clusters roughly concentrated around the present geomagnetic field (Fig. 15b). This could suggest that the PDC was emplaced also at high temperature—as high as that esti- mated for site PO-1—and that movement during or after Pérez‑Rodríguez et al. Earth, Planets and Space (2019) 71:80 Pérez‑Rodríguez et al. Earth, Planets and Space (2019) 71:80 Page 20 of 21 Author details 1 1 Posgrado en Ciencias de la Tierra, UNAM, Unidad Michoacán, Campus More‑ lia, Antigua Carretera a Pátzcuaro No. 8701 Col. Ex‑Hacienda de San José de la Huerta, 58190 Morelia, Michoacán, Mexico. 2 Laboratorio Interinstitucional de Magnetismo Natural (LIMNA) y Servicio Arqueomagnético Nacional (SAN), Instituto de Geofísica, UNAM, Unidad Michoacán, Campus Morelia, Antigua Carretera a Pátzcuaro No. 8701 Col. Ex‑Hacienda de San José de la Huerta, 58190 Morelia, Michoacán, Mexico. 3 Instituto de Geofísica, UNAM, Unidad Michoacán, Campus Morelia, Antigua Carretera a Pátzcuaro No. 8701 Col. Ex‑Hacienda de San José de la Huerta, 58190 Morelia, Michoacán, Mexico. Additional file 2. Susceptibility vs Temperature of ceramic shards. Excel file with the results of magnetic susceptibility measurements of the ceramic shards after each double heating-step during the Thellier-Coe paleointensity experiments. At the top of the first sheet are shown the raw data, while at the bottom part the normalized susceptibility values. In the second sheet the normalized Susceptibility vs Temperature plots for each ceramic shard analyzed are shown. Additional file 3. THD cL2 Ceramics: Thermal demagnetization results for representative shards buried by the UCPE pyroclastic density cur‑ rent. Figure includes normalized intensity decay curves (left), Zijderveld diagrams (middle) and corresponding equal-area projections (right). Most specimens displayed mainly a single paleomagnetic component directed through the origin, accompanied by an initial weak overprint of possibly viscous origin which could be removed at fields/temperatures below 10 mT/150 °C. Symbols: Zijderveld diagram: full/open dot, declination/ apparent inclination; equal-area projection: full/open dot, positive/nega‑ tive inclination. Received: 25 February 2019 Accepted: 11 July 2019 Received: 25 February 2019 Accepted: 11 July 2019 Abbreviations TMVB: trans-Mexican volcanic belt; UPCPE: Upper Pre-ceramic Plinian eruption; LCPE: Lower Ceramic Plinian eruption; UCPE: Upper Ceramic Plinian eruption; AI: archeointensity; PI: paleointensity; AVFTB: advance variable field translation balance; AF: alternating field; ARM: anhysteretic remanent magnetization; ChRM: characteristic remanent magnetization; IRM: isothermal remanent magnetization; NRM: natural remanent magnetization; TRM: thermorema‑ nent magnetization; pTRM: partial thermoremanent magnetization; TC: Thellier–Coe; Tc: Curie temperature; CR: cooling rate; SD: single domain; PSD: pseudo-single domain; MD: multidomain; Dec: declination; Inc: inclination; PDF: probability density function. Additionally, first archeointensity dating of pot- tery shards within pyroclastic density currents of Pop- ocatepetl volcano is reported. As claimed in most paleointensity investigations, alter- ation both in nature or during laboratory heating and the influence of MD grains were the main factors of failure to experimentally determine the paleointensity in the other two lava flows. Authors’ contributionsi NP-R participated in the field work, sample preparation and measurement of samples. JM participated in the field work, supervised the experimental pro‑ cedures and participated in the analysis and interpretation of results and the writing of the manuscript. AG participated and supported the field work via his project. FG-T participated in the field work. All authors read and approved the final manuscript. Conclusions Additional file 4. THD dL2 Ceramics: Thermal demagnetization results for representative shards buried by the LCPE pyroclastic density current. Figure includes normalized intensity decay curves (left), Zijderveld dia‑ grams (middle) and corresponding equal-area projections (right). Arrows indicate the temperature step corresponding to the reheating of the ceramic shards. Symbols: Zijderveld diagram: full/open dot, declination/ apparent inclination; equal-area projection: full/open dot, positive/nega‑ tive inclination. Additional file 4. THD dL2 Ceramics: Thermal demagnetization results for representative shards buried by the LCPE pyroclastic density current. Figure includes normalized intensity decay curves (left), Zijderveld dia‑ grams (middle) and corresponding equal-area projections (right). Arrows indicate the temperature step corresponding to the reheating of the ceramic shards. Symbols: Zijderveld diagram: full/open dot, declination/ apparent inclination; equal-area projection: full/open dot, positive/nega‑ tive inclination. New paleomagnetic results from Popocatepetl volcano are reported. Full-vector (direction and intensity) paleo- magnetic dating of the Nealtican lava obtained (for the first time) confirms the 14C date for this fissural lava flow. Reliable new paleointensity results on scoria clasts from PDCs were obtained (also for the first time) for Pop- ocatepetl volcano. Estimated emplacement temperatures for the PDCs of the Lorenzo and Pink Pumice indicate that clasts were emplaced hot, above the Tc of magnetite (580 °C). Acknowledgements h h d The exhaustive and constructive revisions of two anonymous reviewers are greatly acknowledged. l Finally, results of the rock-magnetic and paleomagnetic dating of the last Plinian eruptions from Popocatepetl volcano, applied to different volcanic materials—lava and pyroclastic density currents—show the usefulness of these nonconventional and alternative techniques in the study of the eruptive activity of volcanoes. Both scoria clast and pottery shard within PDCs provide use- ful information about the ancient geomagnetic field, as well as emplacement temperatures of pyroclastic density currents. Competing interests Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Funding AG is grateful for financial support of UNAM-PAPIIT Project 101717. Additional files Ethics approval and consent to participate Ethics approval and consent to participate Not applicable. Not applicable. Additional file 1. Susceptibility vs Temperature of ceramic shards. Excel file with the results of magnetic susceptibility measurements of the lava specimens after each double heating-step during the Thellier-Coe paleointensity experiments. At the top of the first sheet are shown the raw data, while at the bottom part the normalized susceptibility values. In the second sheet the normalized Susceptibility vs Temperature plots for each ceramic shard analyzed are shown. References CO Boletín de la Sociedad Geológica Mexicana LVII(3):227–233 Siebe C, Macías JL, Abrams M, Obenholzner J (1996a) La destrucción de Cacaxtla y Cholula: un suceso en la historia eruptiva del Popocatépetl. Ciencias 41:36–45 Hagstrum JT, Champion DE (2002) A Holocene paleosecular variation record from 14C dated volcanic rocks in western North America. J Geophys Res 107:2025. https://doi.org/10.1029/2001JB000524(B1) Siebe C, Macías JL, Abrams M, Obenholzner J (1996b) Repeated volcanic disaster in Prehispanic time at Popocatépetl, central México: past key to the future? Geology 24(5):399–402 p g Kosterov A, Prévot M (1998) Possible mechanisms causing failure of Thellier palaeointensity experiments in some basalts. Geophys J Int 134:554–572 h h l h Kosterov A, Prévot M (1998) Possible mechanisms causing failure of Thellier palaeointensity experiments in some basalts. Geophys J Int 134:554–572 Sosa-Ceballos G, Macías JL, García-Tenorio F, Layer P, Schaaf P, Solís-Pichardo G, Arce JL (2015) El Ventorrillo, a paleostructure of Popocatépetl volcano: insights from geochronology and geochemistry. Bull Volc 77:2–20f Kosterov A, Conte G, Goguitchaichvili A, Urrutia-Fucugauchi J (2009) Low- temperature magnetic properties of andesitic rocks from Popocatepetl stratovolcano, Mexico. Earth Planets Space 61:133–142. https://doi. org/10.1186/BF03352893 Tema E (2009) Estimate of the magnetic anisotropy effect on the archaeomag‑ netic inclination of ancient bricks. Phys Earth Planet Inter 176:213–223 Tema E (2009) Estimate of the magnetic anisotropy effect on the archaeomag‑ netic inclination of ancient bricks. Phys Earth Planet Inter 176:213–223 Tema E, Zanella E, Pavón-Carrasco FJ, Kondopoulou D, Pavlides S (2015) Palaeo‑ magnetic analysis on pottery as indicator of the pyroclastic flow deposits temperature: new data and statistical interpretation from the Minoan eruption of Santorini, Greece. Geophys J Int 203:33–47 Leonhardt R (2006) Analyzing rock magnetic measurements: the Rock-MagAn‑ alyzer 1.0 software. Comput Geosci 32:1420–1431 Leonhardt R, Heunemann C, Krása D (2004) Analyzing absolute paleointensity determinations: acceptance criteria and the software ThellierTool4.0. Geochem Geophys Geosyst 5(12):Q12016. https://doi.org/10.1029/2004G C000807 y Thellier E, Thellier O (1959) Sur l’intensité du champ magnétique terrestre dans le passé historique et géologique. Ann Géophys 15:285–376 Thellier E, Thellier O (1959) Sur l’intensité du champ magnétique terrestre dans le passé historique et géologique. Ann Géophys 15:285–376 Zanella E, Gurioli L, Lanza R, Sulpizio R, Bontempi M (2008) Deposition temperature of the AD 472 Pollena pyroclastic density current deposits, Somma-Vesuvius, Italy. Bull Volcanol 70:1237–1248 Levi S (1977) The effect of magnetite particle size on paleointensity determina‑ tion of the geomagnetic field. References CO Phys Earth Planet Inter 13:245–259 Macías JL (2005) Geología e historia eruptiva de algunos de los grandes volcanes activos de México. Boletín de la Sociedad Geológica Mexicana, Volumen Conmemorativo del Centenario LVII(3):395–399 References CO J Geophys Res 83:1740–1756 Riisager P, Riisager J (2001) Detecting multidomain magnetic grains in Thellier palaeointensity experiments. Phys Earth Planet Inter 125:111–117 Riisager P, Riisager J (2001) Detecting multidomain magnetic grains in Thellier palaeointensity experiments. Phys Earth Planet Inter 125:111–117 Robin C (1984) Le Volcan Popocatépetl (Mexique): strecture, evolution pétrologique et risques. Bull Volcanol 47:1 y y Robin C (1984) Le Volcan Popocatépetl (Mexique): strecture, evolution pétrologique et risques. Bull Volcanol 47:1 y Conte G, Urrutia-Fucugauchi J, Goguitchaichvili A, Soler-Arechalde AM, Morton-Bermea O (2004) Paleomagnetic Study of Lavas from the Pop‑ ocatepetl Volcanic Region, Central Mexico. Int Geol Rev 46(2004):210–225 Schaaf P, Stimac J, Siebe C, Macías JL (2005) Geochemical evidence for mantle origin and crustal processes from products of Popocatépetl and sur‑ rounding monogenetic volcanoes. J Petrol 46:1243–1282 Day R, Fuller M, Schmidt V (1977) Hysteresis properties of titanomagnetites: grain-size and compositional dependence. Phys Earth Planet Inter 13:260–266 Selkin PA, Gee JS, Tauxe L, Meurer WP, Newell AJ (2000) The effect of rema‑ nence anisotropy on paleointensity estimates: a case study from the Archean Stilleater Complex. Earth Planet Sci Lett 183:403–416 Di Vito MA, Zanella E, Gurioli L, Lanza R, Sulpizio R, Bishop J, Evdokia T, Boenzi G, Laforgia E (2009) The Afragola settlement near Vesuvius, Italy: the destruction and abandonment of a Bronze Age village revealed by archaeology, volcanology and rock-magnetism. Earth Planet Sci Lett 277:408–421 Siebe C, Macías JL (2006) Volcanic hazards in the Mexico City metropolitan area from eruptions at Popocatépetl, Nevado de Toluca, and Jocotitlán stratovolcanoes and monogenetic scoria cones in The Sierra Chichinaut‑ zin Volcanic Field. The Geological Society of America, Boulder, pp 1–33 Dunlop DJ (2011) Physical basis of the Thellier-Thellier and related paleoin‑ tensity methods. Phys Earth Planet Inter 187(3–4):118–138. https://doi. org/10.1016/j.pepi.2011.03.006 Siebe C, Macías JL, Abrams M, Rodríguez S, Castro R, Delgado H (1995) Quater‑ nary explosive volcanism and pyroclastic deposits in east central Mexico: implications for future hazards. In: Chacko J (ed) Guidebook for the 1995 annual meeting of the Geological Society of America. Geological Society of America, New Orleans, Louisiana, pp 1–47 Ferrari L (2000) Avances en el conocimiento de la Faja Volcánica Trans‑ mexicana durante la última década. Boletín de la Sociedad Geológica Mexicana LIII:84–92 Gómez-Tuena A, Orozco-Esquivel MT, Ferrari L (2005) Petrogénesis Ígnea de la Faja Volcánica Transmexicana. References CO References AGICO Prints (2018) Anisotropy of magnetic remanence a brief practical guide application note. https://www.agico.com/. Accessed 6 Sep 2018 Page 21 of 21 Page 21 of 21 Pérez‑Rodríguez et al. Earth, Planets and Space (2019) 71:80 (2019) 71:80 Anisoft5 (2018) Anisotropy data browser. Version 5.1.01. https://www.agico .com/text/software/anisoft/anisoft.php. Accessed 6 Apr 2018 archeointensity determination on Pre-Columbian pottery from Chiapas, Mesoamerica. Earth Planets Space. https://doi.org/10.1186/BF03352887 archeointensity determination on Pre-Columbian pottery from Chiapas, Mesoamerica. Earth Planets Space. https://doi.org/10.1186/BF03352887 Morales J, Zhao X, Gogichaishvili A (2010) Geomagnetic field intensity from Kilauea 1955 and 1960 lava flows: toward a better understanding of paleointensity. Stud Geophys Geod 54:561–574 Arana-Salinas L, Siebe C, Macías JL (2010) Dynamics of the ca. 4965 14C BP “Ochre Pumice” Plinian eruption of Popocatépetl volcano, México. J Volcanol Geoth Res 192:212–231 Volcanol Geoth Res 192:212–231 Bardot L (2000) Emplacement temperature determinations of proximal pyro‑ clastic deposits on Santorini Greece, and their implications. Bull Volcanol 61:450–467 Panfil M, Gardner T, Hirth K (1999) Late Holocene stratigraphy of the Tetimpa archaeological sites, northeast flank of Popocatépetl volcano, central Mexico. GSA Bull 111(2):204–218 Carrasco-Nuñez G, Silva L, Delgado-Granados H, Urrutia-Fucugauchi J (1986) Geologia y paleomagnetismo del Popocatepetl: Mexico, DF. UNAM Publ Ser Inv Inst Geofis, No 33 Paterson GA, Muxworthy AR, Roberts AP, Mac Niocaill C (2010) Assessment of the usefulness of scoria clasts from pyroclastic deposits for paleointensity determination. J Geophys Res 115:B03104. https://doi.org/10.1029/2009J B006475 Chadima M, Hrouda F (2006) Remasoft 3.0—a user-friendly paleomagnetic data browser and analyzer. Travaux Géophysiques XXVII:20–21 Pavón Carrasco FJ, Rodríguez-González J, Osete ML, Torta JM (2011) A Matlab tool for archeomagnetic dating. J Archaeol Sci 38:408–419i Chauvin A, Garcia Y, Lanos P, Laubenheimer F (2000) Paleointensity of the geomagnetic field recovered on archaeomagnetic sites from France. Phys Earth Planet Inter 120:111–136 Pavón Carrasco FJ, Osete ML, Torta JM, De Santis A (2014) A geomagnetic field model for the holocene based on archeomagnetic and lava flow data. Earth Planet Sci Lett 388:98–109 Coe RS (1967) Paleo-intensities of the Earth’s magnetic field determined from tertiary and quaternary rocks. J Geophys Res 72(12):3247–3262 Plunket P, Uruñuela G (1998) Preclassic household patterns preserved under Volcanic Ash at Tetimpa, Puebla, Mexico. Latin Am Antiq 9(4):287–309 Coe RS, Grommé S, Mankinen EA (1978) Geomagnetic paleointensities from radiocarbon dated lava flows on Hawaii and the question of the Pacific nondipole low. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. McClelland E, Druitt TH (1989) Palaeomagnetic estimates of emplacement temperatures of pyroclastic deposits on Santorini Greece. Bull Volcanol 51:16–27 Morales J, Goguitchaichvili A, Acosta G, González-Morán T, Alva-Valdivia L, Robles-Camacho J, Hernández-Bernal MS (2009) Magnetic properties and
https://openalex.org/W2794622105	https://cyberleninka.ru/article/n/increasing-the-efficiency-of-some-antibiotics-on-penetrating-bacteria-cell-membrane/pdf	English	null	Increasing the efficiency of some antibiotics on penetrating bacteria cell membrane	Ukrainian journal of ecology	2,018	cc-by	6,874	Introduction Antibiotic molecules are able for stopping or killing the growth of, microorganisms, including both fungus and bacteria which are called "bacteriostatic" and "bactericidal" respectively (Genc, 2008). Antibiotics are specific chemical compound which is produced through living organisms and can inhibit the life processes of the various organisms and tissues. The first antibiotics were extracted from “micro-tissues” but some are now received from especial plants or animals. Over 2,000 antibiotics have been known and characterized up to now, but only a few of them are used in high quality medicines (Boxall, 2004). Antibiotic molecules are able for stopping or killing the growth of, microorganisms, including both fungus and bacteria which are called "bacteriostatic" and "bactericidal" respectively (Genc, 2008). Antibiotics are specific chemical compound which is produced through living organisms and can inhibit the life processes of the various organisms and tissues. The first antibiotics were extracted from “micro-tissues” but some are now received from especial plants or animals. Over 2,000 antibiotics have been known and characterized up to now, but only a few of them are used in high quality medicines (Boxall, 2004). Although sulfonamides (Lakshmi, 2013), groups were the first antimicrobial (Epand, 2010), to be developed for several of techniques, recently Triclosan, Baxdela, Ticarcillin are extensively applied for antibacterial agents in the modern drug deliveries. Therefor those of them within sulfonamide-based compounds are most important for the second antimicrobial agents and in adition, those are widely used in human and veterinary medicine for preventing of bacterial (Epand, 2010) infectious diseases. An important but often disregarded aspect of antibiotic use is the fate of antibiotic residues entering the environment (Boxall, 2004). In this study increasing the efficiency of those antibiotics on penetrating to bacteria’s cell membrane to control both gram positive and negative treatment has been investigated through halogenated functionalizing. Penicillin which was discovered and characterized by Alexander Fleming in September 1928 has been used in the treatment towards bacteria invasion. Fleming who was working at St. In recent decades; pharmaceutical antibiotics were recognized and sensitized as emerging soil pollutants while the compounds such as sulfonamides and tetracycline reach agricultural land mostly through infected dung from medicated chattels used as muck (Ghosh, 2011). Although sulfonamides (Lakshmi, 2013), groups were the first antimicrobial (Epand, 2010), to be developed for several of techniques, recently Triclosan, Baxdela, Ticarcillin are extensively applied for antibacterial agents in the modern drug deliveries. Increasing the efficiency of some antibiotics on penetrating bacteria cell membrane Sara Shahriari1, Majid Monajjemi2*, Karim Zare1 1Department of Chemistry, Science and Research Branch, Islamic Azad University, Tehran, Iran 2Department of Chemical Engineering, Central Tehran Branch, Islamic Azad University, Tehran, Iran E-mail: m_monajjemi@srbiau.ac.ir Submitted: 18.01.2018. Accepted: 08.03.2018 By this work, it has been concluded that through halogenated functionalizing of Sulfonamide, Triclosan, Baxdela, Ticarcillin, Ampicillin, and Clavulanic acid we are able to control the treatment of those antibiotics against Gram-positive or negative bacteria of various ecologies. The efficiency of Sulfonamide, Triclosan, Baxdela, Ticarcillin, Ampicillin and Clavulanic acid in viewpoint of NMR shielding and S-NICS methods have been studied as a drug delivery approach. We exhibit some halogenated compounds of those antibiotics, specific chemical derived produced, which are primarily against “gram positive” bacteria (due to higher percentage of “peptidoglycan protein” in the cell membranes). Since Gram-positive bacteria which made of peptidoglycan has a very thick cell wall, some of the antibiotics can penetrate gram (+) and some others cannot. In this work, a list of antibiotics and their halogenated deviated have been set-upped due to the Gram-positive bacteria. A list of halogenated compounds had been reported based on mechanism of the S-layer with two different attachments; for gram (+) it is attached to the peptidoglycan and for gram (-) directly to the outer layers of bacteria. Key words: Sulfonamide; Triclosan; Baxdela; Ticarcillin; Ampicillin and Clavulanic acid; NMR and S-NIC ORIGINAL ARTICLE ORIGINAL ARTICLE Ukrainian Journal of Ecology, 2018, 8(1), 671–679 doi: 10.15421/2018_265 Ukrainian Journal of Ecology, 2018, 8(1), 671–679 doi: 10.15421/2018_265 Introduction Therefor those of them within sulfonamide-based compounds are most important for the second antimicrobial agents and in adition, those are widely used in human and veterinary medicine for preventing of bacterial (Epand, 2010) infectious diseases. An important but often disregarded aspect of antibiotic use is the fate of antibiotic residues entering the environment (Boxall, 2004). In this study increasing the efficiency of those antibiotics on penetrating to bacteria’s cell membrane to control both gram positive and negative treatment has been investigated through halogenated functionalizing. Penicillin which was discovered and characterized by Alexander Fleming in September 1928 has been used in the treatment towards bacteria invasion. Fleming who was working at St. In recent decades; pharmaceutical antibiotics were recognized and sensitized as emerging soil pollutants while the compounds such as sulfonamides and tetracycline reach agricultural land mostly through infected dung from medicated chattels used as muck (Ghosh, 2011). As an important antibiotic sulfonamide can be mentioned, this is commonly used drug in primary care practice. Reaction to Sulfonamide (Owa, 1999) Antibiotic is relatively common as compared to other antimicrobials (Chohan, 2008). The hypersensitivities reaction, consisting of fever and non-urticarial rash, usually develop seven up to fourteen days after the medication initiation. The term “sulfa” refers to a derivative of an antimicrobial agent (Epand, 2010), “sulfanilamide”. In this work a list of antibiotics has been reported based on mechanism of the S-layer with two different attachments; for gram (+) it is attached to the peptidoglycan and for gram (-) directly to the outer layers of bacteria. By this work, it has been concluded that through halogenated functionalizing of Sulfonamide (Narasaiah, 2008), Triclosan, Baxdela Ticarcillin, Ampicillin and Clavulanic acid we are able to control the treatment of those antibiotics against Gram-positive or negative bacteria. We exhibit some Ukrainian Journal of Ecology 672 compounds of those antibiotics, specific chemical derived produced, which are primarily against “gram positive” bacteria (due to higher percentage of “peptidoglycan protein” in the cell membranes). Since Gram-positive bacteria which made of peptidoglycan has a very thick cell wall, some of the antibiotics can penetrate gram (+) and some others cannot. In this work, a list of antibiotics and their halogenated deviated have been set-upped due to the Gram-positive bacteria. Fig. 1. Representation of the cell membranes of Gram (+) and Gram (-) for bacteria Gram(+) such as Mycobacterium and Nocardia is included of peptidoglycan, Polysaccharides, Ribitol, Glycerol and Glycolipids and Gram(-) Such as E Coli is included of LPS, Lipoprotein, Porins and peptidoglycan Bacteria with single membrane are known as Gram (+) including a thick peptidoglycan Layers with attached proteins and different glycol-polymers like poly-saccharides and teichoic31. Those layers encase their cytoplasmic of membrane and take up the crystal violet stain used in the Gram staining method, as is known by the names. In gram (-) Bacteria’s cell, the cytoplasmic region is restricted by considerable thin peptidoglycan layers over-layed by an asymmetrical phospholipids bilayer and lipo- poly-saccharides containing some proteins. In common, higher molecules of PE are placed in the cell membranes (Monajjemi, 2015 Cell membrane...) of Gram (-), while the cytoplasmic of Gram (+) is rich in PG. The lipid compound of various Gram (-) and Gram (+) are discussed in result section based on halogenated functionalizing of mentioned antibiotics. Introduction We have exhibited the especial properties of those antibiotics in view point of NMR shielding and S-NICS methods (Derakhshandeh, Monajjemi, 2017) for delivering in cell membrane (Monajjemi, 2015) via QM/MM and ab-initio methods. In eukaryotic cell’s phospholipids, the prokaryotic plasma consists of mainly one kind of phospholipid. For bacterial (Epand, 2010) cells the phospholipids are to a huge extended phosphate-dyl-ethanolamine (PE) (Fig. 1). The residual lipids are negatively charged at physiologically situations, where phosphate-dyl-glycerol (PG), or its derivatives as the same DPG (di-phosphate-idyl-glycerol) or CL, “Cardiolipine” are prevailing. In addition, the lipid structures also based on whether the bacterium belongs to the class of Gram (-) or Gram (+) bacteria (Fig. 1). The bacterial membrane (Goldfine, 1984) shortcoming sterol and have followed other means of strengthening their combination. compounds of those antibiotics, specific chemical derived produced, which are primarily against “gram positive” bacteria (due to higher percentage of “peptidoglycan protein” in the cell membranes). Since Gram-positive bacteria which made of peptidoglycan has a very thick cell wall, some of the antibiotics can penetrate gram (+) and some others cannot. In this work, a list of antibiotics and their halogenated deviated have been set-upped due to the Gram-positive bacteria. We have exhibited the especial properties of those antibiotics in view point of NMR shielding and S-NICS methods (Derakhshandeh, Monajjemi, 2017) for delivering in cell membrane (Monajjemi, 2015) via QM/MM and ab-initio methods. In eukaryotic cell’s phospholipids, the prokaryotic plasma consists of mainly one kind of phospholipid. For bacterial (Epand, 2010) cells the phospholipids are to a huge extended phosphate-dyl-ethanolamine (PE) (Fig. 1). The residual lipids are negatively charged at physiologically situations, where phosphate-dyl-glycerol (PG), or its derivatives as the same DPG (di-phosphate-idyl-glycerol) or CL, “Cardiolipine” are prevailing. In addition, the lipid structures also based on whether the bacterium belongs to the class of Gram (-) or Gram (+) bacteria (Fig. 1). The bacterial membrane (Goldfine, 1984) shortcoming sterol and have followed other means of strengthening their combination. Fig. 1. Representation of the cell membranes of Gram (+) and Gram (-) for bacteria Gram(+) such as Mycobacterium and Nocardia is included of peptidoglycan, Polysaccharides, Ribitol, Glycerol and Glycolipids and Gram(-) Such as E Coli is included of LPS, Lipoprotein, Porins and peptidoglycan (S-NICS method and NMR shielding): The study of net components of a membrane can help understanding of basic biological membrane structures, interaction and mechanisms with proteins insertion and the environment of other components (Boxall, 2004). Although, precise structure of a bilayer that is in the biological pertaining fluid phase is not able for obtaining experimental data, fluctuation of this kind of bilayer indicates correct structure. Molecular modeling is a strong tool for guiding the interpretation of experimental section. The credit of simulation, in other words, might be measured against existing experimental results. There are several techniques such as deuterium NMR quadrupol splitting that can give certain results of electrostatics surfaces per lipid membrane thickness (Haque, 1984). The absence of experimental data is reversed in molecular modeling of lipid membranes (Seelig, 1974), due to force field parameterization. Tight level ab-initio estimation which is needed for definition and parameterization of force fields, presently allows evaluation of those heavy atoms for gaining accurate results. The mentioned method clearly has been discussed in our previous work (Derakhshandeh, 2017; Monajjemi, 2015) Cell membrane) about a statistical approach in NMR shielding and nucleus independent chemical shifts S-NICS which was based on the treatment of asymmetry (η) and skew (κ) parameters. In this work, we have investigated this method via computing the statistical nucleus-independent chemical shifts in view point of probes motions in the sphere of de-shielding and shielding spaces of some hereto rings in sensitive antibiotics- Ecoli complexes. Ukrainian Journal of Ecology, 8(1), 2018 673 Ecological impact of unemployment on rural-urban migration Fig. 2. Optimized (B3LYP-D3/TZP) structures of Triclosan, Sulfonamid, Baxdela and Floriated of Baxdela through two F atoms (number 1 & 5) instead of two (OH) groups, Using “Bq” inside the rings for S-NICS calculations Fig. 2. Optimized (B3LYP-D3/TZP) structures of Triclosan, Sulfonamid, Baxdela and Floriated of Baxdela through two F atoms (number 1 & 5) instead of two (OH) groups, Using “Bq” inside the rings for S-NICS calculations The NMR (Monajjemi, 2014) parameters including isotropic shielding (σiso), anisotropic shielding (σaniso) and chemical shift (δ) were also evaluated after the optimized geometries of all antibiotics structures. The default gauges-including atomic orbital (GIAO) orbitals were used to obtain molecular magnetic susceptibilities, NMR shielding with Gaussian 09 in all calculations (Berlin, 1978). Ukrainian Journal of Ecology, 8(1), 2018 Ukrainian Journal of Ecology, 8(1), 2018 674 Ukrainian Journal of Ecology Fig. 3. (S-NICS method and NMR shielding): NMR Shielding Changing of F2-Baxdela compare to Baxdela and Cl-Triclosan compare to Triclosan Fig. 4. F7-Clavulanic Acid and Cl5-Ampicillin for S-NICS calculations including dummy atoms Fig. 5. NMR Shielding Changing of F7-Clavulanic acid compare to Clavulanic acid and Ampicillin Fig. 3. NMR Shielding Changing of F2-Baxdela compare to Baxdela and Cl-Triclosan compare to Triclosan ig. 3. NMR Shielding Changing of F2-Baxdela compare to Baxdela and Cl-Triclosan compare to Triclosa Fig. 4. F7-Clavulanic Acid and Cl5-Ampicillin for S-NICS calculations including dummy atoms Fig. 4. F7-Clavulanic Acid and Cl5-Ampicillin for S-NICS calculations including dummy atoms Fig. 5. NMR Shielding Changing of F7-Clavulanic acid compare to Clavulanic acid and Ampicillin Fig. 5. NMR Shielding Changing of F7-Clavulanic acid compare to Clavulanic acid and Ampicillin The positive and negative value of this function correspond to the relationships between ∇2𝜌 (density of electrons) and valence shell electron pair repulsion (Monajjemi, 2015) (VSEPR) model, chemical bond type, electron localization and chemical reactivity respectively, which have been built by Bader (Bader, 1990). Becke (Becke and Edgecombe,1990) noted that spherically averaged like-spin conditional pair probability has direct correlation with the Fermi hole and then suggested electron localization function (ELF). Savin et al (1971) have reinterpreted the ELF in view point of kinetic energies (see also Lu, 2012), which makes ELF also explaining for Kohn-Sham DFT wave-function. Ukrainian Journal of Ecology, 8(1), 2018 675 Ecological impact of unemployment on rural-urban migration They show which D(r) reveals the excess kinetic energies densities caused by Pauli repulsion (Monajjemi, 2015), while D0can be considered as Thomas-Fermi kinetic energies density. Localized orbital locator (LOL) is another function for locating high localization regions likewise ELF, defined by Schmider and Becke (see Lu, 2012) Fig. 6. “ElF” profile and LOL curves of Triclosan in water They show which D(r) reveals the excess kinetic energies densities caused by Pauli repulsion (Monajjemi, 2015), while D0can be considered as Thomas-Fermi kinetic energies density. Localized orbital locator (LOL) is another function for locating high localization regions likewise ELF, defined by Schmider and Becke (see Lu, 2012) Fig. 6. “ElF” profile and LOL curves of Triclosan in water Computational details By this work we have modeled a narrow of membrane thickness (Haque, 1984) including di-palmitoyl-phosphatidyl-choline or (DPPC)n through QM/MM calculation using charm force fields. Each segment was designed of 60 lipid molecules surrounding with water molecules. (S-NICS method and NMR shielding): In this investigation, differences in force fields are accomplished by comparing the several energies through using AMBER, OPLS and CHARMM force fields. We have investigated density functional theory with the van der Waals densities functional for modeling the exchange-correlation of DPPC units in membrane. Tight & post-HF ab-initio calculation has applied to model the exchange-correlation energies of the hetero rings of the antibiotics structures. The double ζ-basis set with polarization orbitals (DZP) were used for the hetero rings. The charges and electrostatic potential-derived charges of the halogens were also estimated using Merz-Kollman-Singh, chelp56, or chelpG57. Calculations were performed using packages of Gaussian 09 and GAMESS. The ONIOM methods including 3 levels from high calculation (H), medium (M), and low (L) have been accomplished in this study. The B3LYP-D3/TZP, CAM-B3LYP and M06 methods are used for high layer of the model and the semi empirical methods of “Pm3MM” including pseudo=CEP and “Pm6” are used for the medium and low layers, respectively. In the calculations, we also have mainly focused on getting the optimized results for each item from “advanced DFT” methods including the “m06-L”, “m062x”, “m06-L”, and “m06-HF” which are novel Meta hybrid DFTB. SPSS “Statistical Package for the Social Sciences” has applied for editing and analyzing all sorts of our S-NICS data of the heterocyclic antibiotics in this work. The semi empirical methods have been used to treat the non-bonded interactions between two parts of upper lateral phospholipids side (𝑃+) and downer lateral phospholipids side ( 𝑃−). The interaction (Monajjemi, 2010; 2012) energy for membrane as a capacitor (Monajjemi M 2014) was calculated in all items according to the equation: They show which D(r) reveals the excess kinetic energies densities caused by Pauli repulsion (Monajjemi, 2015), while D0can be considered as Thomas-Fermi kinetic energies density. Localized orbital locator (LOL) is another function for locating high localization regions likewise ELF, defined by Schmider and Becke (see Lu, 2012) They show which D(r) reveals the excess kinetic energies densities caused by Pauli repulsion (Monajjemi, 2015), while D0can be considered as Thomas-Fermi kinetic energies density. Localized orbital locator (LOL) is another function for locating high localization regions likewise ELF, defined by Schmider and Becke (see Lu, 2012) Fig. 6. “ElF” profile and LOL curves of Triclosan in water Computational details The interaction (Monajjemi, 2010; 2012) energy for membrane as a capacitor (Monajjemi, M, 2014) was calculated in all items according to the equation: By this work we have modeled a narrow of membrane thickness (Haque, 1984) including di-palmitoyl-phosphatidyl-choline or (DPPC)n through QM/MM calculation using charm force fields. Each segment was designed of 60 lipid molecules surrounding with water molecules. In this investigation, differences in force fields are accomplished by comparing the several energies through using AMBER, OPLS and CHARMM force fields. We have investigated density functional theory with the van der Waals densities functional for modeling the exchange-correlation of DPPC units in membrane. Tight & post-HF ab-initio calculation has applied to model the exchange-correlation energies of the hetero rings of the antibiotics structures. The double ζ-basis set with polarization orbitals (DZP) were used for the hetero rings. The charges and electrostatic potential-derived charges of the halogens were also estimated using Merz-Kollman-Singh, chelp56, or chelpG57. Calculations were performed using packages of Gaussian 09 and GAMESS. The ONIOM methods including 3 levels from high calculation (H), medium (M), and low (L) have been accomplished in this study. The B3LYP-D3/TZP, CAM-B3LYP and M06 methods are used for high layer of the model and the semi empirical methods of “Pm3MM” including pseudo=CEP and “Pm6” are used for the medium and low layers, respectively. In the calculations, we also have mainly focused on getting the optimized results for each item from “advanced DFT” methods including the “m06-L”, “m062x”, “m06-L”, and “m06-HF” which are novel Meta hybrid DFTB. SPSS “Statistical Package for the Social Sciences” has applied for editing and analyzing all sorts of our S-NICS data of the heterocyclic antibiotics in this work. The semi empirical methods have been used to treat the non-bonded interactions between two parts of upper lateral phospholipids side (𝑃+) and downer lateral phospholipids side ( 𝑃−). The interaction (Monajjemi, 2010; 2012) energy for membrane as a capacitor (Monajjemi, M, 2014) was calculated in all items according to the equation: 𝐸𝑆(𝑒𝑉) = {𝐸𝐶−(∑ (𝐷𝑃𝑃𝐶+)𝑖 60 𝑖=1 + ∑ (𝐷𝑃𝑃𝐶−)𝑖 40 𝑖=1 ) + ∑𝐴𝑛𝑡𝑖𝑏𝑖𝑜𝑡𝑖𝑐𝑠 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑠} +𝐸𝐵𝑆𝑆𝐸 Where the “∆𝐸𝑆” is the stabilities energy of membrane-antibiotics system (Haque, 1984). Where the ∆𝐸𝑆 is the stabilities energy of membrane antibiotics system (Haque, 1984). Fig. 7. Generated CP path and surfaces of Ticarcillin for S-NICS and SPSS Calculations Fig. 7. Computational details p By this work we have modeled a narrow of membrane thickness (Haque, 1984) including di-palmitoyl-phosphatidyl-choline or (DPPC)n through QM/MM calculation using charm force fields. Each segment was designed of 60 lipid molecules surrounding with water molecules. In this investigation, differences in force fields are accomplished by comparing the several energies through using AMBER, OPLS and CHARMM force fields. We have investigated density functional theory with the van der Waals densities functional for modeling the exchange-correlation of DPPC units in membrane. p By this work we have modeled a narrow of membrane thickness (Haque, 1984) including di-palmitoyl-phosphatidyl-choline or (DPPC)n through QM/MM calculation using charm force fields. Each segment was designed of 60 lipid molecules surrounding with water molecules. In this investigation, differences in force fields are accomplished by comparing the several energies through using AMBER, OPLS and CHARMM force fields. We have investigated density functional theory with the van der Waals densities functional for modeling the exchange-correlation of DPPC units in membrane. Tight & post-HF ab-initio calculation has applied to model the exchange-correlation energies of the hetero rings of the antibiotics structures. The double ζ-basis set with polarization orbitals (DZP) were used for the hetero rings. The charges and electrostatic potential-derived charges of the halogens were also estimated using Merz-Kollman-Singh, chelp56, or chelpG57. Calculations were performed using packages of Gaussian 09 and GAMESS. The ONIOM methods including 3 levels from high calculation (H), medium (M), and low (L) have been accomplished in this study. The B3LYP-D3/TZP, CAM-B3LYP and M06 methods are used for high layer of the model and the semi empirical methods of “Pm3MM” including pseudo=CEP and “Pm6” are used for the medium and low layers, respectively. In the calculations, we also have mainly focused on getting the optimized results for each item from “advanced DFT” methods including the “m06-L”, “m062x”, “m06-L”, and “m06-HF” which are novel Meta hybrid DFTB. SPSS “Statistical Package for the Social Sciences” has applied for editing and analyzing all sorts of our S-NICS data of the heterocyclic antibiotics in this work. The semi empirical methods have been used to treat the non-bonded interactions between two parts of upper lateral phospholipids side (𝑃+) and downer lateral phospholipids side ( 𝑃−). Computational details Generated CP path and surfaces of Ticarcillin for S-NICS and SPSS Calculations Ukrainian Journal of Ecology, 8(1), 2018 676 Ukrainian Journal of Ecology Fig. 8. Molecular Dynamic optimization on simulation of Membrane /protein/antibiotics Fig. 8. Molecular Dynamic optimization on simulation of Membrane /protein/antibiotics Result and Discussion 0 -0.4 22 Cl 15.3 -22.9 0.8 150.9 157.3 -0.34 22 Cl 108. 1 -112.1 0.0 162.4 160. 5 -0.25 20 F -5.2 -7.8 0.9 27.7 29.1 0.17 20 F 117. 1 -125.7 0.1 149.9 141. 1 -0.44 21 F -6.2 -9.3 0.3 26.13 29.7 0.181 21 F 113. 8 -120.7 0.1 162.9 168. 3 -0.45 23 F 9.9 -20.1 0.5 26.6 114.2 -0.13 23 F 227. 0 -240.6 0.1 246.93 248. 3 -0.67 10 N 4.2 -5.8 0.8 156.3 157.2 -0.36 10 N 62.5 93.78 0.2 63.7 65.2 7 0.32 15 N 11.4 -16.3 0.8 157.2 156.1 -0.33 15 N 64.6 97.0 0.4 67.0 67.9 0.23 24 O 96.5 144.8 0.7 126.8 66.4 -0.16 24 O 92.8 139.2 0.2 79.2 78.2 0.13 27 O -13.0 -19.6 0.5 145.1 147.0 -0.21 27 O 4.6 -6.0 0.7 29.9 30.1 0.16 1 C -6.1 -9.2 0.5 27.1 30.7 0.14 1 C -4.9 -7.4 0.8 6.8 30.0 0.12 11 C 2.5 -2.7 0.4 30.8 29.6 0.15 11 C Based on the data presented in Tables 1-2, isotropy and asymmetry (η) have been calculated for Cl3-Triclosan, F3-Baxdela, Cl6- Ampicillin, and F7-Clavulanic acid. Gram (+) such as Mycobacterium and Nocardia includes the peptidoglycan, Polysaccharides, Ribitol, Glycerol and Glycolipids and Gram (-) such as E Coli including LPS, Lipoprotein, Porins and peptidoglycan. The Molecular Dynamic optimization of those antibiotics with E-coli [gram (-) classification] membrane /protein / antibiotics, have been ESP), isotropy, span and aromaticity of some atoms of Baxdela in gas phase and solvent media Table 2. S-NICS, Charge (ESP), isotropy, span and aromaticity of some atoms of Cl6-Ampicillin in gas phase and solvent media F3-Baxdela in water F3-Baxdela in gas phase Ω δ ∆ η S-NICS σ- iso charge atom Ω δ ∆ η S-NICS σiso charge atom 113. 7 -120.6 0.1 154.5 167. 0 -0.4 22 Cl 15.3 -22.9 0.8 150.9 157.3 -0.34 22 Cl 108. 1 -112.1 0.0 162.4 160. 5 -0.25 20 F -5.2 -7.8 0.9 27.7 29.1 0.17 20 F 117. 1 -125.7 0.1 149.9 141. 1 -0.44 21 F -6.2 -9.3 0.3 26.13 29.7 0.181 21 F 113. 8 -120.7 0.1 162.9 168. 3 -0.45 23 F 9.9 -20.1 0.5 26.6 114.2 -0.13 23 F 227. 0 -240.6 0.1 246.93 248. Result and Discussion In this work, we have modeled and simulated those mentioned antibiotic’s properties through QM/MM calculations based on specific chemical derived produced, which are primarily against “gram positive” bacteria. Since Gram-positive bacteria which made of peptidoglycan has a very thick cell wall, some of the antibiotics can penetrate gram (+) and some others cannot. The data and results are listed in 7 figures and 5 tables. By this investigation, we have exhibited a statistical method by computing of nucleus-independent chemical shifts in point of probes (BQ) motions around the center of shielding and de-shielding spaces of antibiotic’s hetero-rings. In the previous works, it has been exhibited that S-NICS approach is a suitable method for calculation of the aromaticity (Monajjemi, 2012) in non-benzene rings such as those halogenated antibiotics which are important index for Membrane /Protein / Antibiotics interactions (Monajjemi, 2010; 2013) – see Fig. 8. Fig. 9. Membrane simulation including 120 molecules of DPPC phospholipids. Fig. 9. Membrane simulation including 120 molecules of DPPC phospholipids. Ukrainian Journal of Ecology, 8(1), 2018 Ecological impact of unemployment on rural-urban migration 677 Table1. S-NICS, Charge (ESP), isotropy, span and aromaticity of some atoms of Baxdela in gas phase and solvent media Cl3-Triclosan in water Cl3-Triclosan gas phase Ω δ ∆ η S-NICS σiso charg e atom Ω δ ∆ η S-NICS σiso Charge Atom 24.09 36.13 0.28 110.5 120. 2 0.242 14 Cl -91.7 -137.6 0.8 7 52.5 50.9 0.20 14 Cl 8.87 13.3 0.59 23.6 25.7 0.13 16 Cl -7.93 -11.98 0.9 8 162.0 159. -0.35 16 Cl -13.4 29.7 8.6 144.5 168. 5 -0.45 14 Cl 7.37 -6.2 0.1 3 28.05 26.0 0.168 14 Cl 6.7 10.15 0.53 30.1 29.7 0.13 17 O -7.3 -11.0 0.7 0 9.37 29.3 0.18 17 O -26.9 9.53 5.9 129.7 130. 3 -0.19 13 O 3.04 -4.19 0.8 3 34.56 30.1 0.15 13 O 24.09 36.13 0.28 122.1 120. 2 0.242 1 C 8.24 -7.2 0.1 8 12.3 25.9 0.16 1 C 33.6 50.4 0.58 250.4 245. 2 -0.69 5 C 7.7 -10.5 0.8 0 11.68 28.6 0.17 5 C Table 2. S-NICS, Charge (ESP), isotropy, span and aromaticity of some atoms of Cl6-Ampicillin in gas phase and solvent media F3-Baxdela in water F3-Baxdela in gas phase Ω δ ∆ η S-NICS σ- iso charge atom Ω δ ∆ η S-NICS σiso charge atom 113. 7 -120.6 0.1 154.5 167. Result and Discussion 3 -0.67 10 N 4.2 -5.8 0.8 156.3 157.2 -0.36 10 N 62.5 93.78 0.2 63.7 65.2 7 0.32 15 N 11.4 -16.3 0.8 157.2 156.1 -0.33 15 N 64.6 97.0 0.4 67.0 67.9 0.23 24 O 96.5 144.8 0.7 126.8 66.4 -0.16 24 O 92.8 139.2 0.2 79.2 78.2 0.13 27 O -13.0 -19.6 0.5 145.1 147.0 -0.21 27 O 4.6 -6.0 0.7 29.9 30.1 0.16 1 C -6.1 -9.2 0.5 27.1 30.7 0.14 1 C -4.9 -7.4 0.8 6.8 30.0 0.12 11 C 2.5 -2.7 0.4 30.8 29.6 0.15 11 C Based on the data presented in Tables 1-2, isotropy and asymmetry (η) have been calculated for Cl3-Triclosan, F3-Baxdela, Cl6- Ampicillin, and F7-Clavulanic acid. Gram (+) such as Mycobacterium and Nocardia includes the peptidoglycan, Polysaccharides, Ribitol, Glycerol and Glycolipids and Gram (-) such as E Coli including LPS, Lipoprotein, Porins and peptidoglycan. The Molecular Dynamic optimization of those antibiotics with E-coli [gram (-) classification] membrane /protein / antibiotics, have been calculated through the QM/MM simulation with CHARMM force fields and anisotropy has been yielded from the equation (4). We have modeled a section of membrane (Haque, 1984) systems including di-palmitoyl-phosphatidyl -choline (DPPC)n via those mentioned methods using Monte Carlo (Fig. 2) (Elsagh et al., 2016). Each system was combined of sixty lipids surrounding with water (Seelig, 1974). Thermodynamic averages were calculated from those methods, as the minimum-energy structures which indicate the resistance of membrane /protein/antibiotics. E Coli which includes LPS, Lipoprotein, Porins and peptidoglycan (Figs 8, 9), is some famous bacteria in Gram (-) groups. By this work, through halogenation of antibiotics (Table 3), we exhibited that by changing the aromaticity (total S-NICS value) (Monajjemi, 2012) the bacterial (Epand, 2010) resistance might change due to a relation between S-NICS and resistance. This study is also focused on the electron density of Halogens which is replaced with hydrogen of Ampicillin, Clavulanic acid, Imipeneme, Penicillin, and Ticarcillin in point of view in S-NICS method. The largest Based on the data presented in Tables 1-2, isotropy and asymmetry (η) have been calculated for Cl3-Triclosan, F3-Baxdela, Cl6- Ampicillin, and F7-Clavulanic acid. Gram (+) such as Mycobacterium and Nocardia includes the peptidoglycan, Polysaccharides, Ribitol, Glycerol and Glycolipids and Gram (-) such as E Coli including LPS, Lipoprotein, Porins and peptidoglycan. Result and Discussion The Molecular Dynamic optimization of those antibiotics with E-coli [gram (-) classification] membrane /protein / antibiotics, have been calculated through the QM/MM simulation with CHARMM force fields and anisotropy has been yielded from the equation (4). We have modeled a section of membrane (Haque, 1984) systems including di-palmitoyl-phosphatidyl -choline (DPPC)n via those mentioned methods using Monte Carlo (Fig. 2) (Elsagh et al., 2016). Each system was combined of sixty lipids surrounding with water (Seelig, 1974). Thermodynamic averages were calculated from those methods, as the minimum-energy structures which indicate the resistance of membrane /protein/antibiotics. E Coli which includes LPS, Lipoprotein, Porins and peptidoglycan (Figs 8, 9), is some famous bacteria in Gram (-) groups. By this work, through halogenation of antibiotics (Table 3), we exhibited that by changing the aromaticity (total S-NICS value) (Monajjemi, 2012) the bacterial (Epand, 2010) resistance might change due to a relation between S-NICS and resistance. This study is also focused on the electron density of Halogens which is replaced with hydrogen of Ampicillin, Clavulanic acid, Imipeneme, Penicillin, and Ticarcillin in point of view in S-NICS method. The largest Based on the data presented in Tables 1-2, isotropy and asymmetry (η) have been calculated for Cl3-Triclosan, F3-Baxdela, Cl6- Ampicillin, and F7-Clavulanic acid. Gram (+) such as Mycobacterium and Nocardia includes the peptidoglycan, Polysaccharides, Ribitol, Glycerol and Glycolipids and Gram (-) such as E Coli including LPS, Lipoprotein, Porins and peptidoglycan. The Molecular Dynamic optimization of those antibiotics with E-coli [gram (-) classification] membrane /protein / antibiotics, have been calculated through the QM/MM simulation with CHARMM force fields and anisotropy has been yielded from the equation (4). We have modeled a section of membrane (Haque, 1984) systems including di-palmitoyl-phosphatidyl -choline (DPPC)n via those mentioned methods using Monte Carlo (Fig. 2) (Elsagh et al., 2016). Each system was combined of sixty lipids surrounding with water (Seelig, 1974). Thermodynamic averages were calculated from those methods, as the minimum-energy structures which indicate the resistance of membrane /protein/antibiotics. E Coli which includes LPS, Lipoprotein, Porins and peptidoglycan (Figs 8, 9), is some famous bacteria in Gram (-) groups. By this work, through halogenation of antibiotics (Table 3), we exhibited that by changing the aromaticity (total S-NICS value) (Monajjemi, 2012) the bacterial (Epand, 2010) resistance might change due to a relation between S-NICS and resistance. Conclusions A good result of the theoretical analysis of antibiotics- S-NICS methods is the stable model for drug designing. In this work, a relation between aromaticity and resistance of antibiotics has been exhibited. This resistance is due to membrane potential changing with different compositions of antibiotics-lipids interaction or further affects the interactions with antimicrobial peptides (Epand, 2010). Result and Discussion This study is also focused on the electron density of Halogens which is replaced with hydrogen of Ampicillin, Clavulanic acid, Imipeneme, Penicillin, and Ticarcillin in point of view in S-NICS method. The largest Ukrainian Journal of Ecology, 8(1), 2018 678 Ukrainian Journal of Ecology electron localization is located on halogen atoms which indicate the suitable changing of aromaticity and consequently the resistance. Table 3. The relation between resistance and S-NICS for E-coli Class of antibiotics Antibiotics Halogenated antibiotics S-NICS (+) increasing aromaticity (-) decreasing aromaticity Resistance based ∆𝐺 interaction (-) decreasing (+) increasing Penicillin Ampicillin Amoxicillin Clavulanic acid Cl6-Ampicillin + - F7-Clavulanic acid + + Ticarcillin Carbapenems imipenem F3-imipenem - + Quinolones Triclosan Norfloxacin Ofloxacin Cl3-Triclosan - + sulfonamides Sulfonamide Cl2F2Sulfonamide + - Fluoroquinolone Baxdela F3-Baxdela - + electron localization is located on halogen atoms which indicate the suitable changing of aromaticity and consequently the resistance. Table 3. The relation between resistance and S-NICS for E-coli Acknowledgment g We are thanks of center research in IAU for providing computers equipment and software References Bader, R.F.W. (1990). Atoms in Molecule: A quantum Theory. Oxford Univ. Press, Oxford. , ( ) q y , Becke, A.D., Edgecombe, K.E. (1990). A simple measure of electron localization in atomic and molecular systems. The Journal of Chemical Physics 92, 5397. https://doi.org/10.1063/1.458517 Becke, A.D., Edgecombe, K.E. (1990). A simple measure of electron localization in atomic and molecula Becke, A.D., Edgecombe, K.E. (1990). A simple measure of electron localization in atomic and of Chemical Physics 92, 5397. https://doi.org/10.1063/1.458517 Boxall, A. B. A., Fogg, L. A., Blackwell, P. A., Kay,P., Pemberton,E.J., Croxford, A. (2004) Veterinary medicines in the environment," Reviews of Environmental Contamination and Toxicology, 180, 1-91. https://doi.org/10.1007/0-387-21729-0_1 Chohan,Z.H. (2009), Synthesis of organometallic-based biologically active compounds: In vitro antibacterial, antifungal and cytotoxic properties of some sulfonamide incorporated ferrocences, Journal of Enzyme Inhibition and Medicinal Chemistry, 24(1), 169–175 https://doi.org/10.1080/14756360801948766 Boxall, A. B. A., Fogg, L. A., Blackwell, P. A., Kay,P., Pemberton,E.J., Croxford, A. (2004) Veterinary medicines in the environment," Reviews of Environmental Contamination and Toxicology, 180, 1-91. https://doi.org/10.1007/0-387-21729-0_1 Chohan,Z.H. (2009), Synthesis of organometallic-based biologically active compounds: In vitro antibacterial, antifungal and cytotoxic properties of some sulfonamide incorporated ferrocences, Journal of Enzyme Inhibition and Medicinal Chemistry, 24(1), 169–175 https://doi.org/10.1080/14756360801948766 ( ) p g Derakhshandeh, M., Monajjemi, M (2017), NMR Shielding and S-NICS Investigation for Imipenem,Penicillin G, Ticarcillin, Ampicillin and Clavulanic acid in viewpoint of BioNano technology. Orient J Chem, 33(2), 664-675 https://doi.org/10.13005/ojc/330214 Elsagh, A., Zare, K., Monajjemi, M. (2016). An electrochemical study of POPC phospholipid bilayers in a cell membrane. Oriental Journal of Chemistry, 32(5), 2585-2598. https://doi.org/10.13005/ojc/320530 Epand, R. F., Pollard, J. E., Wright, J. O., Savage, P. B., Epand, R.M. (2010). Depolarization, bacterial membrane composition, and the antimicrobial action of ceragenins, Antimicrobial agents and Chemotherapy, 54(9), 3708–3713. https://doi.org/10.1128/AAC.00380-10 Epand, R. F., Pollard, J. E., Wright, J. O., Savage, P. B., Epand, R.M. (2010). Depolarization, bacterial membrane composition, and the antimicrobial action of ceragenins, Antimicrobial agents and Chemotherapy, 54(9), 3708–3713. https://doi.org/10.1128/AAC.00380-10 p g Farese, R.V., Ishizuka, T., Standaert, M.L., Cooper, D.R. (1991). Sulfonylureas activate glucose transport and protein kinase C in rat adipocytes, Metabolism, 40(2), 196-200. https://doi.org/10.1016/0026-0495(91)90174-U Genc, Y., Ozkanca, R., Bekdemir, Y. (2008). Antimicrobial activity of some sulfonamide derivatives on clinical isolates of Staphylococus aureus, Ann Clin Microbiol Antimicrob, 7, 7-17. https://doi.org/10.1186/1476-0711-7-17 Ghosh, A.K., Anderson. D.D. (2011). Tetrahydrofuran, tetrahydropyran, triazoles and related heterocyclic derivatives as HIV protease inhibitors. Future Med Chem, 3(9), 1181–1197. Citation: Sara Shahriari, Majid Monajjemi, Karim Zare (2018). Increasing the efficiency of some antibiotics on penetrating bacteria cell membrane Ukrainian Journal of Ecology, 8(1), 671–679. This work is licensed under a Creative Commons Attribution 4.0. License References Quantum investigation of non-bonded interaction between the B15N15 ring and BH2NBH2 (radical, cation, anion) systems: a nano molecular motor. Struct Chem, 23,551–580. https://doi.org/10.1007/s11224-011-9895-8 Monajjemi, M. (2012). Quantum investigation of non-bonded interaction between the B15N15 ring and BH2NBH2 (radical, cation, anion) systems: a nano molecular motor. Struct Chem, 23,551–580. https://doi.org/10.1007/s11224-011-9895-8 Monajjemi, M. (2013). Non bonded interaction between BnNn (stator) and BN B(rotor) systems: A quantum rotation in IR region. Chemical Physics, 425, 29-45. https://doi.org/10.1016/j.chemphys.2013.07.014 Monajjemi, M. (2013). Non bonded interaction between BnNn (stator) and BN B(rotor) systems: A quantum rotation in IR region. Chemical Physics, 425, 29-45. https://doi.org/10.1016/j.chemphys.2013.07.014 g y p g j p y Monajjemi, M., Boggs, J.E. (2013). A New Generation of BnNn Rings as a Supplement to Boron Nitride Tubes and Cages. J. Phys. Chem. A, 117, 1670−1684. https://doi.org/10.1021/jp312073q Monajjemi, M., Boggs, J.E. (2013). A New Generation of BnNn Rings as a Supplement to Boron Nitrid emi, M., Boggs, J.E. (2013). A New Generation of BnNn Rings as a Supplement to Boron Nitride Tubes a A 117 1670−1684 https://doi org/10 1021/jp312073q Monajjemi, M., Boggs, J.E. (2013). A New Generation of BnNn Rings as a Supplement to Boron Nitride Tubes and Cages. J. Phys. Chem. A, 117, 1670−1684. https://doi.org/10.1021/jp312073q Monajjemi, M., Lee, V.S., Khaleghian, M., Honarparvar, B., Mollaamin, F. (2010). Theoretical Description of Electromagnetic Nonbonded Interactions of Radical, Cationic, and Anionic NH2BHNBHNH2 Inside of the B18N18 Nanoring. J. Phys. Chem C, 114, 15315. https://doi.org/10.1021/jp104274z Monajjemi, M., Lee, V.S., Khaleghian, M., Honarparvar, B., Mollaamin, F. (2010). Theoretical Description of Electromagnetic Nonbonded Interactions of Radical, Cationic, and Anionic NH2BHNBHNH2 Inside of the B18N18 Nanoring. J. Phys. Chem C, 114, 15315. https://doi.org/10.1021/jp104274z p g jp Monajjemi, M., Mohammadian, N.T. (2015). S-NICS: An Aromaticity Criterion for Nano Molecules. J. Comput. Theor. Nanosci, 12(11), 4895-4914. https://doi.org/10.1166/jctn.2015.4458 p g jp Monajjemi, M., Mohammadian, N.T. (2015). S-NICS: An Aromaticity Criterion for Nano Molecules. J. Comput. Theor. Nanosci, 12(11), 4895-4914. https://doi.org/10.1166/jctn.2015.4458 Monajjemi, M., Robert Wayne, Jr., Boggs, J.E. (2014). NMR contour maps as a new parameter of carboxyl's OH groups in amino acids recognition: A reason of tRNA amino acid conjugation Chemical Physics 433(3) 1 11 p g jp Monajjemi, M., Mohammadian, N.T. (2015). S-NICS: An Aromaticity Criterion for Nano Molecules. J. Comput. Theor. Nanosci, 12(11), 4895-4914. https://doi.org/10.1166/jctn.2015.4458 12(11), 4895 4914. https://doi.org/10.1166/jctn.2015.4458 Monajjemi, M., Robert Wayne, Jr., Boggs, J.E. (2014). References https://doi.org/10.4155/fmc.11.68 Goldfine, H., (1984), Bacterial membranes and lipid packing theory. Journal of lipid research, 25(13), 1501–1507 Haeberlen, U. (1976) In Advances in Magnetic Resonance, Suppl. 1 Academic Press, New York Haque, M.A., Russell, N. J., (2004). Strains of bacillus cereus vary in the phenotypic adaptation of their membrane lipid composition in response to low water activity, reduced temperature and growth in rice starch. Microbiology, 150(5), 1397– 1404. https://doi.org/10.1099/mic.0.26767-0 Herzfeld, J., Berger, A.E. (1980). Sideband intensities in NMR spectra of samples spinning at the magic angle. J. Chem. Phys, 73(12), 6021. https://doi.org/10.1063/1.440136 Farese, R.V., Ishizuka, T., Standaert, M.L., Cooper, D.R. (1991). Sulfonylureas activate glucose transport and protein kinase C in rat adipocytes, Metabolism, 40(2), 196-200. https://doi.org/10.1016/0026-0495(91)90174-U Genc, Y., Ozkanca, R., Bekdemir, Y. (2008). Antimicrobial activity of some sulfonamide derivatives on clinical isolates of Staphylococus aureus, Ann Clin Microbiol Antimicrob, 7, 7-17. https://doi.org/10.1186/1476-0711-7-17 Ghosh, A.K., Anderson. D.D. (2011). Tetrahydrofuran, tetrahydropyran, triazoles and related heterocyclic derivatives as HIV protease inhibitors. Future Med Chem, 3(9), 1181–1197. https://doi.org/10.4155/fmc.11.68 Goldfine, H., (1984), Bacterial membranes and lipid packing theory. Journal of lipid research, 25(13), 1501–1507 Haeberlen, U. (1976) In Advances in Magnetic Resonance, Suppl. 1 Academic Press, New York Haque, M.A., Russell, N. J., (2004). Strains of bacillus cereus vary in the phenotypic adaptation of their membrane lipid composition in response to low water activity, reduced temperature and growth in rice starch. Microbiology, 150(5), 1397– 1404. https://doi.org/10.1099/mic.0.26767-0 Herzfeld, J., Berger, A.E. (1980). Sideband intensities in NMR spectra of samples spinning at the magic angle. J. Chem. Phys, 73(12) 6021 https://doi org/10 1063/1 440136 p g Herzfeld, J., Berger, A.E. (1980). Sideband intensities in NMR spectra of samples spinning at the magic angle. J. Chem. Phys, 73(12), 6021. https://doi.org/10.1063/1.440136 p g Herzfeld, J., Berger, A.E. (1980). Sideband intensities in NMR spectra of samples spinning at the magic angle. J. Chem. Phys, 73(12), 6021. https://doi.org/10.1063/1.440136 Ukrainian Journal of Ecology, 8(1), 2018 679 Ecological impact of unemployment on rural-urban migration Lakshmi, R.S.V., Naresh, K., Raju, C.N. (2013). New sulfonamide and carbamate derivatives of 4-(oxiran-2-ylmethoxy)- 9Hcarbazole: Synthesis, characterization, antimicrobial and antioxidant activities, Der Pharmacia Lettre, 5(1), 221-231 Lu, T., Chen. F. (2012). Multiwfn: A Multifunctional Wavefunction Analyzer. J. Comp. Chem, 33, 580-592. https://doi org/10 1002/jcc 22885 Monajjemi, M. (2014). Metal-doped graphene layers composed with boron nitride–graphene as an insulator: a nano-capacitor Journal of Molecular Modeling, 20, 2507. https://doi.org/10.1007/s00894-014-2507-y Monajjemi, M. (2012). References NMR contour maps as a new parameter of carboxyl's OH groups in amino acids recognition: A reason of tRNA–amino acid conjugation. Chemical Physics, 433(3), 1-11. https://doi.org/10.1016/j.chemphys.2014.01.017 Monajjemi, M., Robert Wayne, Jr., Boggs, J.E. (2014). NMR contour maps as a new parameter of carboxyl's OH groups in amino acids recognition: A reason of tRNA–amino acid conjugation. Chemical Physics, 433(3), 1-11. https://doi org/10 1016/j chemphys 2014 01 017 Monajjemi, M., Robert Wayne, Jr., Boggs, J.E. (2014). NMR contour maps as a new parameter of carboxyl's OH groups in amino acids recognition: A reason of tRNA–amino acid conjugation. Chemical Physics, 433(3), 1-11. p g j p y Monajjemi, M. (2015). Cell membrane causes the lipid bilayers to behave as variable capacitors: A resonance with self- induction of helical proteins. Biophysical Chemistry, 207,114–127. https://doi.org/10.1016/j.bpc.2015.10.003 Monajjemi, M. (2015). Cell membrane causes the lipid bilayers to behave as variable capacitors: A resonance with self- induction of helical proteins. Biophysical Chemistry, 207,114–127. https://doi.org/10.1016/j.bpc.2015.10.003 Monajjemi, M. (2015). Non-covalent attraction of B2N(-,0) and repulsion of B2N(+) in the BnNn ring: a quantum rotatory due to an external field. Theor. Chem. Ass, 134, 77. DOI: 10.1007/s00214-015-1668-9. Monajjemi, M. (2015). Non-covalent attraction of B2N(-,0) and repulsion of B2N(+) in the BnNn ring: a quantum rotatory due to an external field. Theor. Chem. Ass, 134, 77. DOI: 10.1007/s00214-015-1668-9. https://doi.org/10.1007/s00214-015-1668-9 y Owa, T., Yoshino, H., Okauchi, T., Yoshimatsu, K., Ozawa, Y., Hata Sugi, N., Nagasu, T., Koyanagi, N., Kitoh, N. (1999). Discovery of Novel Antitumor Sulfonamides Targeting G1 Phase of the Cell Cycle. J. Med. Chem., 42(19), 3789–3799. https://doi.org/10.1021/jm9902638 Savin, A., Becke, A. D., Flad, J., Nesper, R., Preuss, H. and von Schnering, H. G. (1991), A New Look at Electron Localization. Angew. Chem. Int. Ed. Engl., 30, 409–412. https://doi.org/10.1002/anie.199104091 Savin, A., Becke, A. D., Flad, J., Nesper, R., Preuss, H. and von Schnering, H. G. (1991), A New Look at Electron Localization. Angew. Chem. Int. Ed. Engl., 30, 409–412. https://doi.org/10.1002/anie.199104091 Seelig, J., Niederberger, W. (1974). Two pictures of a lipid bilayer. Comparison between deuterium label and spin-label experiments, Biochemistry, 13(8), 1585–1588. https://doi.org/10.1021/bi00705a005 Seelig, J., Niederberger, W. (1974). Two pictures of a lipid bilayer. Comparison between deuterium label and spin-label experiments, Biochemistry, 13(8), 1585–1588. https://doi.org/10.1021/bi00705a005 Ukrainian Journal of Ecology, 8(1), 2018
https://openalex.org/W4284968979	https://wwwnc.cdc.gov/eid/article/28/8/pdfs/22-0726.pdf	English	null	Imported Monkeypox from International Traveler, Maryland, USA, 2021	Emerging infectious diseases	2,022	cc-by	737	COMMENT LETTERS Address for correspondence: Joshua J. Anzinger, Department of Microbiology, The University of the West Indies, Kingston, Jamaica; email: joshua.anzinger@uwimona.edu.jm and Prevention. Public health investigation for a single case of monkeypox can be intensive and complicated; case-patient contacts outside of the hospital must be identified, monitored, and poten tially given 1 of the 2 orthopoxvirus vaccines of fered for postexposure prophylaxis in the United States (3–5). ( ) Factors that should raise suspicion for monkey pox in a patient with related signs and symptoms include history of travel outside of the United States to a country with confirmed cases or where monkey pox virus is endemic, contact with a person with a similar-appearing rash or who has received a diag nosis of confirmed or probable monkeypox, contact with Africa-endemic wild animal or pet species (liv ing or dead), or use of a product derived from those animals (e.g., game meat, creams, lotions, powders). Monkeypox should also be considered in patients with close or intimate contact with persons in social networks experiencing high monkeypox activity, in cluding men who have sex with men who meet part ners through a website, digital application, or social event. Prompt consultation with public health au thorities is essential for providing clinical guidance, expedititing testing and treatment, and preventing secondary cases (3). DOI: https://doi.org/10.3201/eid2808.220726 DOI: https://doi.org/10.3201/eid2808.220726 To the Editor: Costello et al. described a patient in Maryland, USA, with a diffuse vesicular rash initially diagnosed as disseminated varicella zoster virus (VZV) infection. Only after a biopsy revealed unexpected findings was monkeypox suspected (1). Monkeypox is commonly confused with VZV in countries where both infections are endemic. High fever, lymphadenopathy, and a deep-seated, well- circumscribed, umbilicated rash in the same stage of development (i.e., macule, papule, vesicle, or scab) in distinct anatomic locations are characteristic of monkeypox (2). Although the patient in Maryland experienced lymphadenopathy and rash with um bilicated lesions suggestive of monkeypox, he was afebrile, denied other prodromal signs and symp toms (e.g., headache and chills) that typically pre cede monkeypox rash, and improved while receiv ing intravenous acyclovir, features more consistent with VZV. However, the unusual clinical signs and symptoms experienced by this patient were similar to those observed in other patients in the evolving 2022 multinational monkeypox response. Address for correspondence: Faisal Syed Minhaj, Centers for Disease Control and Prevention, 1600 Clifton Rd NE, Mailstop 24- 12, Atlanta, GA 30327-4027, USA; email: fminhaj@cdc.gov COMMENT LETTERS COMMENT LETTERS Imported Monkeypox from International Traveler, Maryland, USA, 2021 Faisal S. Minhaj, Agam K. Rao, Andrea M. McCollum Author affiliation: Centers for Disease Control and Prevention, Atlanta, Georgia, USA Faisal S. Minhaj, Agam K. Rao, Andrea M. McCollum Author affiliation: Centers for Disease Control and Prevention, Atlanta, Georgia, USA 1. Costello V, Sowash M, Gaur A, Cardis M, Pasieka H, Wortmann G, et al. Imported monkeypox from international traveler, Maryland, USA, 2021. Emerg Infect Dis. 2022;28:1002–5. https://doi.org/10.3201/eid2805.220292 References 1. Costello V, Sowash M, Gaur A, Cardis M, Pasieka H, Wortmann G, et al. Imported monkeypox from international traveler, Maryland, USA, 2021. Emerg Infect Dis. 2022;28:1002–5. https://doi.org/10.3201/eid2805.220292 p g 2. Osadebe L, Hughes CM, Shongo Lushima R, Kabamba J, Nguete B, Malekani J, et al. Enhancing case definitions for surveillance of human monkeypox in the Democratic Republic of Congo. PLoS Negl Trop Dis. 2017;11:e0005857. https://doi.org/10.1371/journal.pntd.0005857 3. Rao AK, Schulte J, Chen T-H, Hughes CM, Davidson W, Neff JM, et al.; July 2021 Monkeypox Response Team. Monkeypox in a traveler returning from Nigeria—Dal las, Texas, July 2021. MMWR Morb Mortal Wkly Rep. 2022;71:509–16. https://doi.org/10.15585/mmwr.mm7114a1 4. Petersen BW, Damon IK, Pertowski CA, Meaney-Delman D, Guarnizo JT, Beigi RH, et al. Clinical guidance for smallpox vaccine use in a postevent vaccination program. MMWR Recomm Rep. 2015;64(RR-02):1–26. p ( ) 5. Rao AK, Petersen BW, Whitehill F, Razeq JH, Isaacs SN, Merch linsky MJ, et al. Use of JYNNEOS (smallpox and monkeypox vaccine, live, nonreplicating) for preexposure vaccination of persons at risk for occupational exposure to orthopoxviruses: recommendations of the Advisory Committee on Immunization Practices—United States, 2022. MMWR Morb Mortal Wkly Rep. 2022;71:734–42. https://doi.org/10.15585/mmwr.mm7122e1 Because differential diagnosis can be challeng ing, public health authorities should be consulted promptly when monkeypox is possible. US Labo ratory Response Network laboratories (https:// emergency.cdc.gov/lrn) can enable rapid testing of specimens (e.g., lesions swab), and pathogen-spe cific antiviral medications can be acquired through consultation with the Centers for Disease Control 1738 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 28, No. 8, August 2022
https://openalex.org/W2496872689	https://bmcinfectdis.biomedcentral.com/counter/pdf/10.1186/s12879-016-1695-8	English	null	Acinetobacter spp. are associated with a higher mortality in intensive care patients with bacteremia: a survival analysis	BMC infectious diseases	2,016	cc-by	6,548	Leão et al. BMC Infectious Diseases (2016) 16:386 DOI 10.1186/s12879-016-1695-8 Leão et al. BMC Infectious Diseases (2016) 16:386 DOI 10.1186/s12879-016-1695-8 Open Access © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: It has been challenging to determine the true clinical impact of Acinetobacter spp., due to the predilection of this pathogen to colonize and infect critically ill patients, who often have a poor prognosis. The aim of this study was to assess whether Acinetobacter spp. bacteremia is associated with lower survival compared with bacteremia caused by other pathogens in critically ill patients. Methods: This study was performed at Hospital das Clínicas, University of São Paulo, Brazil. There are 12 intensive care units (ICUs) in the hospital: five Internal Medicine ICUs (emergency, nephrology, infectious diseases and respiratory critical care), three surgical ICU (for general surgery and liver transplantion), an Emergency Department ICU for trauma patients, an ICU for burned patients, a neurosurgical ICU and a post-operative ICU. A retrospective review of medical records was conducted for all patients admitted to any of the ICUs, who developed bacteremia from January 2010 through December 2011. Patients with Acinetobacter spp. were compared with those with other pathogens (Klebsiella pneumoniae, Staphylococcus aureus, Enterobacter spp., Enterococcus spp., Pseudomonas aeruginosa). We did a 30-day survival analysis. The Kaplan-Meier method and log-rank test were used to determine the overall survival. Potential prognostic factors were identified by bivariate and multivariate Cox regression analysis. Results: One hundred forty-one patients were evaluated. No differences between patients with Acinetobacter spp. and other pathogens were observed with regard to age, sex, APACHE II score, Charlson Comorbidity Score and type of infection. Initial inappropriate antimicrobial treatment was more frequent in Acinetobacter bacteremia (88 % vs 51 %). Bivariate analysis showed that age > 60 years, diabetes mellitus, and Acinetobacter spp. infection were significantly associated with a poor prognosis. Multivariate model showed that Acinetobacter spp. infection (HR = 1.93, 95 % CI: 1.25–2.97) and age > 60 years were independent prognostic factors. Conclusion: Acinetobacter is associated with lower survival compared with other pathogens in critically ill patients with bacteremia, and is not merely a marker of disease severity. Keywords: Acinetobacter, Bacteremia, Intensive care units, Survival analysis, Prognosis Some investigators found high mortality rates in intensive care unit (ICU) patients with Acinetobacter bacteremia: 61.6 % in Israel [2], 65.5 % in Brazil [3] and 43.4 % in the United States [4]. Acinetobacter spp. are associated with a higher mortality in intensive care patients with bacteremia: a survival analysis Aline C. Q. Leão1, Paulo R. Menezes2, Maura S. Oliveira3 and Anna S. Levin1,3,4 Aline C. Q. Leão1, Paulo R. Menezes2, Maura S. Oliveira3 and Anna S. Levin1,3,4 Background It has been challenging to determine the true clinical impact of Acinetobacter spp., due to the predilection of this pathogen to colonize and infect critically ill patients, who often have a poor prognosis irrespective of secondary infective complications [1]. When outcomes from Acinetobacter baumannii were compared directly with those of patients who had bacteremia caused by other organisms, a significantly higher mortality was noted for A. baumannii [2, 5]. However, none of these studies used a formal, standardized method to adjust for severity of illness or comorbidities, such as APACHE or Charlson score. Another study * Correspondence: aleao@usp.br 1Department of Infectious Diseases and LIM 54, University of São Paulo, São Paulo, Brazil Full list of author information is available at the end of the article * Correspondence: aleao@usp.br 1Department of Infectious Diseases and LIM 54, University of São Paulo, São Paulo, Brazil Full list of author information is available at the end of the article Page 2 of 8 Leão et al. BMC Infectious Diseases (2016) 16:386 Page 2 of 8 involving trauma patients showed no difference in mor- tality comparing infections by Acinetobacter and by other pathogens [6]. Tonacio et al. [7] found 30 % of mortality in patients with Acinetobacter spp. infections and trauma was a marker of good prognosis in those patients. invasive devices and antimicrobials after the diagnosis of bacteremia; initial site of infection and treatment; time elapsed from admission in the ICU to diagnosis of bacteremia; Pitt Bacteremia Score [12]; presence of septic shock; and number of organ failures. Acute organ failures (cardiovascular, respiratory, renal, hematologic or central nervous system) were defined using the defini- tions of Zimmerman et al. [13]. The patients’ underlying diseases analyzed were: diabetes mellitus, liver cirrhosis, cancer, transplant recipient, HIV infection, chronic renal disease, obstructive pulmonary disease, trauma, and sys- temic arterial hypertension. We also analyzed the Charlson Comorbidity Score [14]. Some studies observed growing resistance among other gram-negative and gram-positive pathogens that cause healthcare-associated infections. Rice [8] reported these as the “ESKAPE” pathogens: Enterococcus faecium, Staphylo- coccus aureus, Klebsiella pneumoniae, Acinetobacter bau- mannii, Pseudomonas aeruginosa and Enterobacter species [8, 9]. These pathogens cause an increasing number of healthcare-associated infections with significant morbidity and mortality, with are often associated with ICU admission [10]. Bacteremias were classified as primary and secondary bloodstream infections. Microbiology The clinical microbiology laboratory made the identifi- cation and antimicrobial susceptibility test of the se- lected pathogens using VITEK 2® (bioMerieux VITEK, Hazelwood, MO, USA). The breakpoints were those de- fined by the Clinical and Laboratory Standards Institute (CLSI) [15, 16]. The automatic identification method VITEK 2® (bioMerieux VITEK, Hazelwood, MO, USA) showed the results of Acinetobacter as Acinetobacter baumannii- calcoaceticus complex. This complex includes other pathogenic species besides Acinetobacter baumannii, such as A. calcoaceticus, A. tjernbergiae (sp. 3), A. ursingii (sp.13). As the isolates were not available for further identification, we chose to refer to the micro- organism as Acinetobacter spp. Patients with Acinetobacter spp. bacteremia were com- pared with patients with bacteremia caused by other path- ogens (Klebsiella pneumoniae, Staphylococcus aureus, Enterobacter spp., Enterococcus spp., Pseudomonas aerugi- nosa). We selected these agents for comparison, as they were healthcare-associated pathogens of epidemiologic importance, had high antibiotic resistance rates, and were the predominant healthcare-associated pathogens in the hospital. We evaluated prognostic factors associated with mor- tality. The following variables were assessed: sex; age; APACHE II score [12] on admission to ICU; use of Background Primary bloodstream infections were those associated with the use of a central line or those with an unknown or unclear initial site. Secondary bloodstream infections were regarded as those with a clear source of bacteremia other than a central line. Sources of secondary bacteremia were identified by cultures of samples (urine, tracheal secretions, intra-abdominal samples, etc.) obtained from distant sites that yielded the same pathogen with an identical resistance pattern. Distant sites were sites where an infection was diag- nosed other than a central line (pneumonia, surgical site, urinary tract, skin and soft tissue, others). The aim of this study was to evaluate whether bacteremia caused by Acinetobacter spp. was associated with lower sur- vival compared with bacteremia caused by other prevalent pathogens in critically ill patients. Methods This study was performed at Hospital das Clínicas, University of São Paulo, Brazil, a 2200-bed tertiary- care teaching hospital. There are 12 ICUs in the hospital; five Internal Medicine ICUs (emergency, nephrology, infectious diseases and respiratory critical care), three surgical ICU (for general surgery and liver transplantion), an Emergency Department ICU for trauma patients, an ICU for burned patients, a neurosurgical ICU and a post- operative ICU. Antibiotic treatment was deemed initial appropriate antibiotic treatment (IAAT) if the initially prescribed anti- biotic regimen was active against the identified pathogen, based on in vitro susceptibility testing, and administered within two days following the blood culture collection. All other regimens were classified as initial inappropriate anti- biotic treatment (IIAT). A retrospective review of medical records was conducted for all patients admitted to the ICUs who developed bacteremia from January 2010 through December 2011. The inclusion of the patients was based on notifications of nosocomial infections made by the Hospital Infection Control Team according to CDC/NHSN criteria [11]. All hospitalized patients with bacteremia by the selected pathogens were included in the study if the blood cultures were obtained > 48 h after admission to the ICU. In pa- tients with recurrent bacteremia, only the first episode was included. Polymicrobial infections were excluded. Data analysis We initially conducted a descriptive analysis comparing patients with Acinetobacter spp. bacteremia and patients Leão et al. BMC Infectious Diseases (2016) 16:386 Page 3 of 8 Page 3 of 8 Table 1 General characteristics of the entire cohort of patients with bacteremia acquired in intensive care units Acinetobacter spp. Other pathogensa Total Number of patients (%) 59 (42) 82 (58) 141 (100) Age Mean (SD) 52 (18) 56 (16) 54 (17) Median (overall range) 51 (17–92) 57 (18–85) 56 (17–92) Male sex (%) 42 (71) 45 (55) 87 (62) APACHE II score Mean (SD) 20 (7) 20 (9) 20 (8) Median (overall range) 20 (7–40) 19 (0–41) 19 (0–41) CHARLSON score Mean (SD) 3 (3) 3 (3) 3 (3) Median (overall range) 2 (0–10) 3 (0–11) 3 (0–11) Co-morbid condition (%) Diabetes mellitus 14 (24) 17 (21) 31 (22) Liver cirrhosis 18 (31) 10 (12) 28 (20) Cancer 12 (20) 21 (26) 33 (23) Solid organ transplant 17 (29) 13 (16) 30 (21) Liver transplant 14 (24) 10 (12) 24 (17) Kidney transplant 3 (5) 3 (4) 6 (4) Hematopoietic cell transplant 0 (0) 2 (2) 2 (1) HIV infection 3 (5) 6 (7) 9 (6) Chronic renal disease 15 (25) 16 (20) 31 (22) Obstructive pulmonar disease 4 (7) 8 (10) 12 (9) Trauma 8 (14) 7 (9) 15 (11) Systemic arterial hypertension 19 (32) 37 (45) 56 (40) ICU length of stay previous to bacteremia (in days) Mean (SD) 11 (14) 17 (36) 15 (29) Median (overall range) 7 (2–82) 9 (2–314) 8 (2–314) Initial site of infection Bloodstream 43 (73) 58 (71) 101 (72) Pneumonia 3 (5) 11 (13) 14 (10) Surgical site 5 (8) 6 (7) 11 (8) Urinary tract 1 (2) 2 (2) 3 (2) Skin and soft tissue 3 (5) 0 (0) 3 (2) Other 4 (7) 5 (7) 9 (6) SD standard deviation, ICU intensive care unit aIncludes Klebsiella pneumoniae (n: 24), Staphylococcus aureus (n: 21), Enterobacter spp. (n: 15), Enterococcus spp. (n: 12), Pseudomonas aeruginosa (n: 10) Table 1 General characteristics of the entire cohort of patients with bacteremia acquired in intensive care units with bacteremia caused by other pathogens. Baseline characteristics and outcomes were described using sum- mary (mean, standard deviation, median, minimum and maximum) for quantitative variables and absolute and relative frequencies for qualitative variables. We did a 30-day survival analysis. Data analysis For overall survival time, we estimated median survival time according to the characteristics of interest using the Kaplan-Meier function and compared survival rates among the categories using the log-rank test. The bivariate Cox regression was chosen to calculate the hazard ratio (HR) in survival analysis, with a 95 % confidence interval. It was estimated the multiple Cox regression model with the variables with descriptive level in bivariate tests less than 0.10 (p <0.10) and considered with biological plausibility. The tests were done at 5 % significance level. In the case of variables that we considered measured similar characteristics, only one variable was included in the model. Statistical analyses were performed using SPSS (Version 19.0). and carbapenem resistance in Pseudomonas aeruginosa was 30 %; 27 % in Klebsiella pneumoniae and 7 % in Enterobacter spp. isolates. Results The variables: number of organ failures; septic shock; Pitt Bacteremia Score (which evaluates the severity of the bacteremia), mechanical ventilation and use of central venous line were excluded from the bivariate and multivariate analyses because they were considered intrinsically correlated with the event death and not proper prognostic factors. We verified that these factors, excluded from the multivariate analysis, were statistically associated with the outcome, except for use of central venous line (data not shown). Most patients with diabetes mellitus were older than Fig. 2 Cumulative survival after episodes of Acinetobacter spp. bacteremia and bacteremia caused by other gram-negative pathogens. The curve was illustrated with the Kaplan-Meier method (log-rank test, p = 0.033) 60 years, thus the variable diabetes mellitus was also not included in the multivariate analysis. Among the cases of bacteremia by Acinetobacter spp. most re- ceived IIAT (88 %) thus we did not enter this variable into the model. Thus, in the model of Cox regression analysis we evaluated the following variables: age divided into the following strata: ≤60 years or > 60 years; sex; liver cirrhosis; obstructive pulmonary disease; and Acinetobacter spp. bacteremia. The multi- variate model showed that Acinetobacter spp. infec- tion (HR: 1.93, 95 % CI 1.25–2.97) and age > 60 years were statistically associated with mortality (Table 3). Fig. 1 Cumulative survival after episodes of Acinetobacter spp. bacteremia and bacteremia caused by other pathogens. The curve was illustrated with the Kaplan-Meier method (log-rank test, p = 0.005) Results The mean number of organ failures for Acineto- bacter spp. was 2.1 (SD: 1.2) and for other pathogens was 1.67 (SD: 1.3). The cumulative survival curves of the patients according to pathogen are shown in Figs. 1 and 2. The bivariate analysis (Table 2) showed that age >60 years, Acinetobacter spp. infection, and diabetes mellitus were significantly associated with a poor prognosis. The following variables also presented p < 0.10 in the bivariate analysis: sex; liver cirrhosis; ob- structive pulmonary disease, and IIAT. The variables: number of organ failures; septic shock; Pitt Bacteremia Score (which evaluates the severity of the bacteremia), mechanical ventilation and use of central venous line were excluded from the bivariate and multivariate analyses because they were considered intrinsically correlated with the event death and not proper prognostic factors. We verified that these factors, excluded from the multivariate analysis, were statistically associated with the outcome, except for use of central venous line (data not shown). Most patients with diabetes mellitus were older than Fig. 2 Cumulative survival after episodes of Acinetobacter spp. bacteremia and bacteremia caused by other gram-negative pathogens. The curve was illustrated with the Kaplan-Meier method (log-rank test, p = 0.033) Initial inappropriate antibiotic treatment was adminis- tered to 88 % of patients with Acinetobacter spp. and 51 % of patients with other pathogens. More patients with Acinetobacter spp. developed septic shock (81 % vs 52 %); needed mechanical ventilation within 24 h of the diagnosis of bacteremia (88 % vs 66 %); and required a central venous line (97 % vs 85 %). Patients with Acineto- bacter spp. bacteremia had a higher mortality when compared with bacteremia by the other pathogens (73 % vs 50 %). The mean Pitt Bacteremia Score for Acineto- bacter spp. was 7 (SD: 4) and for other pathogens was 4 (SD: 3). The mean number of organ failures for Acineto- bacter spp. was 2.1 (SD: 1.2) and for other pathogens was 1.67 (SD: 1.3). The cumulative survival curves of the patients according to pathogen are shown in Figs. 1 and 2. The bivariate analysis (Table 2) showed that age >60 years, Acinetobacter spp. infection, and diabetes mellitus were significantly associated with a poor prognosis. The following variables also presented p < 0.10 in the bivariate analysis: sex; liver cirrhosis; ob- structive pulmonary disease, and IIAT. Results Three hundred forty-nine patients presented with the selected pathogens bacteremia during the 2-year study period (128 Acinetobacter spp., 55 Klebsiella pneumo- niae, 40 Pseudomonas aeruginosa, 33 Enterobacter spp., 49 Staphylococcus aureus and 44 Enterococcus spp.). 208 were excluded (99 had previous positive blood cultures, 68 had polymicrobial bacteremia, 27 had blood cultures ob- tained ≤48 h after admission in the ICU, nine had incom- plete records and five had unavailable records). Thus 141 patients were evaluated (59 with Acinetobacter spp. bacteremia and 82 with bacteremia caused by other patho- gens). The other pathogens were K. pneumoniae (n: 24), S. aureus (n: 21), Enterobacter spp. (n: 15), Entero- coccus spp. (n: 12) and P. aeruginosa (n: 10). Patient characteristics by pathogen are detailed in Table 1. No differences between Acinetobacter spp. and other pathogens were observed with regard to age, sex, APACHE II score, Charlson Comorbidity Score, dur- ation of hospitalization in the ICU prior to bacteremia and initial site of infection. A detailed analysis of back- ground disease demonstrated no difference between the two groups of patients. Chronic diseases were frequent, including systemic arterial hypertension, cancer, chronic renal disease, diabetes mellitus, solid organ transplants, liver cirrhosis, trauma, obstructive pulmonary disease, HIV infection and hematopoietic stem cell transplantation. Both groups of pathogens presented high rates of resistance to antibiotics. Most Acinetobacter spp. were resistant to carbapenems (92 %) and susceptible to colis- tin (95 %). Among the other pathogens, resistance to methicillin was 71 % among Staphylococcus aureus; among Enterococcus spp. 83 % were vancomycin-resistant (VRE); and carbapenem resistance in Pseudomonas aeruginosa was 30 %; 27 % in Klebsiella pneumoniae and 7 % in Enterobacter spp. isolates. Leão et al. BMC Infectious Diseases (2016) 16:386 Page 4 of 8 Initial inappropriate antibiotic treatment was adminis- tered to 88 % of patients with Acinetobacter spp. and 51 % of patients with other pathogens. More patients with Acinetobacter spp. developed septic shock (81 % vs 52 %); needed mechanical ventilation within 24 h of the diagnosis of bacteremia (88 % vs 66 %); and required a central venous line (97 % vs 85 %). Patients with Acineto- bacter spp. bacteremia had a higher mortality when compared with bacteremia by the other pathogens (73 % vs 50 %). The mean Pitt Bacteremia Score for Acineto- bacter spp. was 7 (SD: 4) and for other pathogens was 4 (SD: 3). Discussion Our study was conducted to evaluate prognostic factors, especially Acinetobacter spp. infection, in patients with bacteremia acquired in ICU. We con- cluded that patients who had Acinetobacter spp. bacteremia presented a significantly worse prognosis, independently of severity of the clinical condition and other potential confounders. Another important as- pect was the short period of time between Acineto- bacter bacteremia and death. The increase in the number of infections caused by multidrug-resistant bacteria, especially gram-negative bacilli, is one of the most important issues in modern healthcare [17]. Among several gram-negative bacilli, non-fermentative organisms such as Pseudomonas aeru- ginosa and Acinetobacter baumannii are the most prob- lematic because of their high frequency and wide Fig. 1 Cumulative survival after episodes of Acinetobacter spp. bacteremia and bacteremia caused by other pathogens. The curve was illustrated with the Kaplan-Meier method (log-rank test, p = 0.005) Leão et al. Discussion BMC Infectious Diseases (2016) 16:386 Page 6 of 8 Table 2 Bivariate analysis of prognostic factors of patients with bacteremia acquired in intensive care units (Continued) ICU length of stay previous to bacteremia > 8 days Yes 20 8.90–31.10 0.78 0.51–1.21 37/65 (57) 0.27 No 9 5.20–12.80 1 47/76 (62) Initial site of infection Primary bloodstream Yes 13 4.71–21.29 1.01 0.63–1.62 60/101 (59) 0.97 No 9 2.98–15.02 1 24/40 (60) Pneumonia Yes 8 0.67–15.33 1.32 0.68–2.56 10/14 (71) 0.41 No 13 4.88–21.13 1 74/127 (58) Surgical site Yes 13 4.22–21.78 1.22 0.58–2.52 76/130 (58) 0.60 No 9 3.53–14.47 1 8/11 (73) Urinary tract Yes a a 0.05 0.00–10.63 0/3 (0) 0.27 No a a 1 84/138 (61) Skin and soft tissue Yes 1 a 1.40 0.34–5.71 2/3 (67) 0.64 No 12 5.27–18.73 1 82/138 (59) Acinetobacter spp. Yes 2 0.00–4,51 1.85 1.21–2.85 43/59 (73) 0.005 No 24 19.42–28,58 1 41/82 (50) IIAT Yes 9 4.46–13.54 1.53 0.98–2.36 50/73 (68) 0.057 No 24 7.06–40.95 1 34/68 (50) CI Confidence interval, HR Hazard Ratio, ICU intensive care unit, IIAT initial inappropriate antimicrobial treatment aNot possible to calculate median time and confidence interval Table 2 Bivariate analysis of prognostic factors of patients with bacteremia acquired in intensive care units (Continued) Table 2 Bivariate analysis of prognostic factors of patients with bacteremia acquired in intensive care units (Continued) CI Confidence interval, HR Hazard Ratio, ICU intensive care unit, IIAT initial inappropriate antimicrobial treatment aNot possible to calculate median time and confidence interval organisms responsible for bacteremias (22 %), and most of Acinetobacter spp. were resistant to carbapenems. spectrum of antimicrobial resistance. This leads to a limited therapeutic armamentarium against them [18, 19]. At our hospital, from January, 2010 through December 2011, 14 % of all episodes of bacteremia were polymicrobial. Of all monomicrobial episodes, most were caused by gram- negative organisms. The rank order of the major patho- gens shows that Acinetobacter spp. were the principal Administering appropriate initial antibiotic therapy is essential in the treatment of septic patients [20] and is associated with lower mortality rate in patients with Acinetobacter spp. bacteremia [21, 22]. Our study found that 92 % of the Acinetobacter spp. isolates were carbapenem-resistant and, in most cases, colistin was the only available antimicrobial agent to treat these serious infections. The time required for identification of Acinetobacter spp. Discussion BMC Infectious Diseases (2016) 16:386 Page 5 of 8 Table 2 Bivariate analysis of prognos Variables Median survival time Age > 60 years Yes 5 No 20 Sex Male 9 Female 25 APACHE II Score > 20 Yes 10 No 15 CHARLSON Score > 3 Yes 15 No 11 Co-morbid condition Diabetes mellitus Yes 7 No 16 Liver cirrhosis Yes 5 No 16 Cancer Yes 18 No 10 Solid organ transplant Yes 5 No 16 Hematopoietic stem cell transplant Yes 7 No 11 HIV infection Yes a No 10 Chronic renal disease Yes 10 No 13 Obstructive pulmonary disease Yes 8 No 12 Trauma Yes a No 10 Systemic arterial hypertension Yes 8 No 15 f patients with bacteremia acquired in intensive care units 95 % CI HR 95 % CI Death/total (%) P 0.00–10.82 1.67 1.08–2.58 37/53 (70) 0.02 11.07–28.93 1 47/88 (53) 4.32–13.69 1.49 0.95–2.35 56/87 (64) 0.08 9.67–40.34 1 28/54 (52) 3.48–16.52 1.11 0.72–1.70 39/60 (65) 0.64 5.10–24.9 1 45/81 (56) 1.78–28.22 1.18 0.76–1.84 31/48 (65) 0.46 5.32–16.68 1 53/93 (57) a 1.67 1.03–2.70 23/31 (74) 0.03 6.86–25.14 1 61/110 (55) 0.01–9.99 1.58 0.96–2.59 21/28 (75) 0.07 4.30–27.70 1 63/113 (56) 0.00–39.72 0.87 0.52–1.46 18/33 (55) 0.59 5.01–14.99 1 66/108 (61) 0.98–9.02 1.31 0.80–2.15 21/30 (70) 0.28 7.22–24.78 1 63/111 (57) a 0.87 0.12–6.26 1/2 (50) 0.89 4.21–17.79 1 83/139 (60) a 0.61 0.22–1.66 4/9 (44) 0.33 4.12–15.88 1 80/132 (61) 0.56–19.45 1.30 0.79–2.14 21/31 (68) 0.29 3.92–22.08 1 63/110 (57) 0.00–21.58 1.73 0.92–3.26 11/12 (92) 0.09 3.85–20.15 1 73/129 (57) a 0.57 0.25–1.30 6/15 (40) 0.18 3.27–16.73 1 78/126 (62) 2.39–13.61 1.16 0.75–1.80 33/56 (59) 0.51 6.39–23.61 1 51/85 (60) Table 2 Bivariate analysis of prognostic factors of patients with bacteremia acquired in intensive care units Variables Median survival time (days) 95 % CI HR 95 % CI Death/total (%) P Age > 60 years Yes 5 0.00–10.82 1.67 1.08–2.58 37/53 (70) 0.02 No 20 11.07–28.93 1 47/88 (53) Sex Male 9 4.32–13.69 1.49 0.95–2.35 56/87 (64) 0.08 Female 25 9.67–40.34 1 28/54 (52) APACHE II Score > 20 Yes 10 3.48–16.52 1.11 0.72–1.70 39/60 (65) 0.64 No 15 5.10–24.9 1 45/81 (56) CHARLSON Score > 3 Yes 15 1.78–28.22 1.18 0.76–1.84 31/48 (65) 0.46 No 11 5.32–16.68 1 53/93 (57) Co-morbid condition Diabetes mellitus Yes 7 a 1.67 1.03–2.70 23/31 (74) 0.03 No 16 6.86–25.14 1 61/110 (55) Liver cirrhosis Yes 5 0.01–9.99 1.58 0.96–2.59 21/28 (75) 0.07 No 16 4.30–27.70 1 63/113 (56) Cancer Yes 18 0.00–39.72 0.87 0.52–1.46 18/33 (55) 0.59 No 10 5.01–14.99 1 66/108 (61) Solid organ transplant Yes 5 0.98–9.02 1.31 0.80–2.15 21/30 (70) 0.28 No 16 7.22–24.78 1 63/111 (57) Hematopoietic stem cell transplant Yes 7 a 0.87 0.12–6.26 1/2 (50) 0.89 No 11 4.21–17.79 1 83/139 (60) HIV infection Yes a a 0.61 0.22–1.66 4/9 (44) 0.33 No 10 4.12–15.88 1 80/132 (61) Chronic renal disease Yes 10 0.56–19.45 1.30 0.79–2.14 21/31 (68) 0.29 No 13 3.92–22.08 1 63/110 (57) Obstructive pulmonary disease Yes 8 0.00–21.58 1.73 0.92–3.26 11/12 (92) 0.09 No 12 3.85–20.15 1 73/129 (57) Trauma Yes a a 0.57 0.25–1.30 6/15 (40) 0.18 No 10 3.27–16.73 1 78/126 (62) Systemic arterial hypertension Yes 8 2.39–13.61 1.16 0.75–1.80 33/56 (59) 0.51 No 15 6.39–23.61 1 51/85 (60) Leão et al. Funding The research and the article were supported by the own funds of authors. Discussion suggested that in vitro and in vivo virulence characteristics differed among indi- vidual strains of the ACB complex [30], which provides further evidence of the impact of genospecies on the out- come of Acinetobacter bacteremia. In our retrospective study, we could not identify these factors, but the evalu- ation of species and virulence factors in future epidemio- logical and clinical studies of Acinetobacter infections may be important. (p = 0.057). In the multivariate model, Acinetobacter was associated with poor prognosis, but IIAT may have a part in explaining why Acinetobacter cases had a worse prognosis. Our data show the high mortality of infections caused by carbapenem-resistant Acinetobacter spp. Based on our findings, we suggest that early initiation of treatment including colistin is important to improve survival in ICUs where infections by these isolates are frequent. Our results also suggest the need for more effective antibiotic stewardship programs to avoid unnecessary treatment with broadly active antibacterial therapy that selects for carbapenem resistance. New infection pre- vention strategies and technologies are needed against these infections. Several limitations of this study are noteworthy. Because it is a single-center study, our findings may be attributable to institution-specific variables and may not reflect the epidemiology of different centers or geographical areas. The study was retrospective and some patients were excluded because of incomplete data. Molecular identifi- cation of the isolates was not performed to identify the genomic species of Acinetobacter. Some studies suggest that Acinetobacter spp. are op- portunistic pathogens that affect patients who are more likely to die because of the severity of their prior disease [23–25]. Blot et al. [26] compared Acinetobacter baumannii bacteremia with matched controls and found that Acinetobacter baumannii was not an independent pre- dictor for mortality. In another single-center experience [6], Acinetobacter baumannii infection, including multidrug- resistant isolates, the impact on mortality in a cohort of trauma patients was not conclusive. However, Acineto- bacter baumannii infection was associated with a longer intensive care unit stay and a higher rate of organ failures. Availability of data and materials Complete data will be provided upon request by the corresponding author (aleao@usp.br). Abbreviations b ACB, Acinetobacter calcoaceticus–baumannii; CLSI, Clinical and Laboratory Standards Institute; IAAT, initial appropriate antibiotic treatment; ICU, intensive care unit; HR, hazard ratio; SD, standard deviation; VRE, vancomycin-resistant Enterococcus Acknowledgements ld l k h We would like to thank the Hospital Infection Control Team for allowing us to use the database of nosocomial infection notification. The median survival of the Acinetobacter group was only two days, thus suggesting the severity of the infec- tion. In our study, the median of Pitt Bacteremia Score was higher in the Acinetobacter spp. group. Rhee et al. suggested that the Pitt bacteremia score is an excellent tool for assessing not only crude mortality, but also mor- tality that is attributed to sepsis in ICU-admitted pa- tients [12]. Authors’ contributions ACQL contributed to the acquisition and synthesis of the data and drafted the original manuscript, which was then amended with suggestions by all authors. ASL and PRM contributed to the conception and design of the work. ASL contributed to the general supervision. ACQL and PRM contributed to the data analysis. ACQL, MSO and ASL contributed to the interpretation of data and discussion of results. All authors read and approved the final manuscript. Some investigators found high mortality rates in ICU patients with Acinetobacter bacteremia (43.4 to 61.6 %) [2–4]. Virulence factors and genotypes of Acinetobacter may have an important role in differences in mortality. Few clinical data are available on the relationship between genospecies and outcome of Acinetobacter bacteremia. Conclusions Our study adds to the existing evidence and the results support that Acinetobacter is associated with lower survival compared with other pathogens in critically ill patients with bacteremia, and is not merely a marker of disease severity. In a review article, Peleg et al. [1] showed that the studies on prognosis of Acinetobacter infections lacked an adequate evaluation of the patients’ severity of under- lying condition. Thus, in our study, we used formal and standardized methods to adjust for severity of illness and comorbidities (APACHE II and Charlson Score). Surprisingly, these variables and the underlying diseases were not significant prognostic factors. These findings support that the high mortality caused by this serious healthcare-associated pathogen cannot be attributed only to underlying conditions and that Acinetobacter infections are not merely markers of the severity of the patients’ clinical condition. Discussion by culture and for identifying car- bapenem resistance was greater than the maximum time (48 h) defined in the present study for beginning the appropriate therapy. Table 3 Multivariate model of prognostic factors of patients with bacteremia acquired in intensive care units Crude HR 95 % CI Adjusted HR 95 % CI P Acinetobacter spp. 0.003 No 1 1 Yes 1.85 1.21–2.85 1.93 1.25–2.97 Age 0.012 ≤60 years 1 1 > 60 years 1.67 1.08–2.58 1.75 1.13–2.70 CI Confidence interval, HR Hazard Ratio Table 3 Multivariate model of prognostic factors of patients with bacteremia acquired in intensive care units Crude HR 95 % CI Adjusted HR 95 % CI P Acinetobacter spp. 0.003 Table 3 Multivariate model of prognostic factors of patients with bacteremia acquired in intensive care units Without microbiological information as a guide, only 12 % of patients with Acinetobacter spp. bacteremia re- ceived effective drugs within 48 h, possibly contributing to the high mortality rate in these patients. In the bivariate analysis of prognostic factors, IIAT appears to be associated with mortality, with a borderline significance CI Confidence interval, HR Hazard Ratio Page 7 of 8 Leão et al. BMC Infectious Diseases (2016) 16:386 Page 7 of 8 Park et al. [27] compared the clinical features, antimicro- bial resistance, and outcome of bacteremia caused by Acinetobacter baumannii versus non-baumannii of the Acinetobacter calcoaceticus–baumannii (ACB) complex. The study found that the species, rather than the anti- biotic resistance, affected mortality, in accordance with other studies [28, 29]. Peleg et al. suggested that in vitro and in vivo virulence characteristics differed among indi- vidual strains of the ACB complex [30], which provides further evidence of the impact of genospecies on the out- come of Acinetobacter bacteremia. In our retrospective study, we could not identify these factors, but the evalu- ation of species and virulence factors in future epidemio- logical and clinical studies of Acinetobacter infections may be important. Park et al. [27] compared the clinical features, antimicro- bial resistance, and outcome of bacteremia caused by Acinetobacter baumannii versus non-baumannii of the Acinetobacter calcoaceticus–baumannii (ACB) complex. The study found that the species, rather than the anti- biotic resistance, affected mortality, in accordance with other studies [28, 29]. Peleg et al. Consent for publication Not applicable. Consent for publication Not applicable. 16. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing: twenty-first informational supplement. Wayne: CLSI; 2011. Document No. M100-S21. 17. Peleg AY, Hooper DC. Hospital-acquired infections due to gram-negative bacteria. N Engl Med. 2010;362:1804–13. Ethics approval and consent to participate 18. Tam VH, Rogers CA, Chang KT, Weston JS, Caeiro JP, Garey JW. Impact of multidrug-resistant Pseudomonas aeruginosa bacteremia on patient outcomes. Antimicrob Agents Chemother. 2010;54:3717–22. The study protocol was reviewed and approved by the Institutional Review Board of Hospital das Clínicas, University of São Paulo, Brazil (Number 0796/11). All data were analyzed on an aggregated basis. The identities of patients and their data remained anonymous. A written informed consent was not required, because the research was retrospective, presents no more than minimal risk of harm to participants and involves no procedure. 19. Karageorgopoulos DE, Falagas ME. Current control and treatment of multidrug-resistant Acinetobacter baumannii infections. Lancet Infect Dis. 2008;8:751–62. 20. Kumar A, Roberts D, Wood KE, Light B, Parrillo JE, Sharma S, et al. Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock. Crit Care Med. 2006; 34:1589–96. Author details 1 f 1Department of Infectious Diseases and LIM 54, University of São Paulo, São Paulo, Brazil. 2Department of Preventive Medicine, University of São Paulo, São Paulo, Brazil. 3Infection Control Department, Hospital das Clínicas, University of São Paulo, São Paulo, Brazil. 4Instituto de Medicina Tropical, University of São Paulo, São Paulo, Brazil. 21. Falagas ME, Kasiakou SK, Rafailidis PI, Zouglakis G, Morfou P. Comparison of mortality of patients with Acinetobacter baumannii bacteremia receiving appropriate and inappropriate empirical therapy. J Antimicro Chemother. 2006;57:1251–4. 22. Kwon KT, Oh WS, Song JH, Chang HH, Jung SI, Kim SW, et al. Impact of imipenem resistance on mortality in patients with Acinetobacter bacteremia. J Antimicrob Chemother. 2007;59:525–30. Received: 26 June 2015 Accepted: 5 July 2016 Received: 26 June 2015 Accepted: 5 July 2016 23. Garnacho J, Sole-Violan J, Sa-Borges M, Diaz E, Rello J. Clinical impact of pneumonia caused by Acinetobacter baumannii in intubated patients: a matched cohort study. Crit Care Med. 2003;31:2478–82. References 1. Peleg AY, Seifert H, Paterson DL. Acinetobacter baumannii: emergence of a successful pathogen. Clin Microbiol Rev. 2008;21:538–82. 1. Peleg AY, Seifert H, Paterson DL. Acinetobacter baumannii: emergence of a successful pathogen. Clin Microbiol Rev. 2008;21:538–82. 24. Kaul R, Burt JA, Cork L, Dedier H, Garcia M, Kennedy C, et al. Investigation of a multiyear multiple critical care unit outbreak due to relatively drug- sensitive Acinetobacter baumannii: risk factors and attributable mortality. J Infect Dis. 1996;174:1279–87. 2. Robenshtok E, Paul M, Leibovici L, Fraser A, Pitlik S, Ostfeld I, et al. The significance of Acinetobacter baumannii bacteremia compared with Klebsiella pneumonia bacteremia: risk factors and outcomes. J Hosp Infect. 2006;64:282–7. 25. Zaragoza R, Artero A, Camarena JJ, Sancho S, Gonzalez R, Nogueira JM. The influence of inadequate empirical antimicrobial treatment on patients with bloodstream infections in an intensive care unit. Clin Microbiol Infect. 2003;9:412–8. 3. Marra AR, Camargo LF, Pignatari AC, Sukiennik T, Behar PR, Medeiros EA, et al. Nosocomial bloodstream infections in Brazilian hospitals: analysis of 2,563 cases from a prospective nationwide surveillance study. J Clin Microbiol. 2011;49:1866–71. 26. Blot S, Vandewoude K, Colardyn F. Nosocomial bacteremia involving Acinetobacter baumannii in critically ill patients: a matched cohort study. Intensive Care Med. 2003;29:471–5. 4. Wisplinghoff H, Bischoff T, Tallent SM, Seifert H, Wenzel RP, Edmond MB. Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study. Clin Infect Dis. 2004;39:309–17. 4. Wisplinghoff H, Bischoff T, Tallent SM, Seifert H, Wenzel RP, Edmond MB. Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study. Clin Infect Dis. 2004;39:309–17. 27. Park KH, Shin JH, Lee SY, Kim SH, Jang MO, Kang SJ, et al. The clinical characteristics, carbapenem resistance, and outcome of Acinetobacter bacteremia according to genospecies. PLoS One. 2013;8:e65026. 5. Jerassy Z, Yinnon AM, Mazouz-Cohen S, Benenson S, Schlesinger Y, Rudensky B, et al. Prospective hospital-wide studies of 505 patients with nosocomial bacteremia in 1997 and 2002. J Hosp Infect. 2006;62:230–6. 5. Jerassy Z, Yinnon AM, Mazouz-Cohen S, Benenson S, Schlesinger Y, Rudensky B, et al. Prospective hospital-wide studies of 505 patients with nosocomial bacteremia in 1997 and 2002. J Hosp Infect. 2006;62:230–6. 28. Chuang YC, Sheng WH, Li SY, Lin YC, Wang JT, Chen YC, et al. Influence of genospecies of Acinetobacter baumannii complex on clinical outcomes of patients with Acinetobacter bacteremia. Clin Infect Dis. 2011;52:352–60. 6. References Eberle BM, Schnüriger B, Putty B, Bamparas G, Kobayashi L, Inaba K, et al. The impact of Acinetobacter baumannii infections on outcome in trauma patients: a matched cohort study. Crit Care Med. 2010;38:2133–8. 6. Eberle BM, Schnüriger B, Putty B, Bamparas G, Kobayashi L, Inaba K, et al. The impact of Acinetobacter baumannii infections on outcome in trauma patients: a matched cohort study. Crit Care Med. 2010;38:2133–8. 29. Wisplinghoff H, Paulus T, Lugenheim M, Stefanik D, Higgins PG, Edmond MB, et al. Nosocomial bloodstream infections due to Acinetobacter baumannii, Acinetobacter pittii and Acinetobacter nosocomialis in the United States. J Infect. 2012;64:282–90. 7. Tonacio AC, Oliveira MS, Malbouisson LM, Levin AS. Trauma is associated with a better prognosis in intensive care patients with Acinetobacter infections. Infection. 2014;42:89–95. 7. Tonacio AC, Oliveira MS, Malbouisson LM, Levin AS. Trauma is associated with a better prognosis in intensive care patients with Acinetobacter infections. Infection. 2014;42:89–95. 30. Peleg AY, de Breiji A, Adams MD, Cerqueira GM, Mocali S, Galardini M, et al. The success of Acinetobacter species: genetic, metabolic and virulence attributes. PLoS One. 2012;7:e46984. 8. Rice LB. Federal funding for the study of antimicrobial resistance in nosocomial pathogens: no ESKAPE. J Infect Dis. 2008;197:1079–81. 8. Rice LB. Federal funding for the study of antimicrobial resistance in nosocomial pathogens: no ESKAPE. J Infect Dis. 2008;197:1079–81. 9. Boucher HW, Talbot GH, Bradley JS, Edwards JE, Gilbert D, Rice LB, et al. Bad Bugs, No Drugs: No ESKAPE! An update from the Infectious Diseases Society of America. Clin Infect Dis. 2009;48:1–12. 10. Jung JY, Park MS, Kim SE, Park BH, Son JY, Kim EY, et al. Risk factors for multi-drug resistant Acinetobacter baumannii bacteremia in patients with colonization in the intensive care unit. BMC Infect Dis. 2010;10:228. 11. Horan TC, Andrus M, Dudeck MA. CDC/NHSN surveillance definition of health care-associated infection and criteria for specific types of infections in the acute care setting. Am J Infect Control. 2008;36:309–32. Competing interests Th h d l h The authors declare that they have no competing interests. The authors declare that they have no competing interests. Page 8 of 8 Page 8 of 8 Page 8 of 8 Leão et al. BMC Infectious Diseases (2016) 16:386 Submit your next manuscript to BioMed Central and we will help you at every step: 12. Rhee JY, Kwon KT, Ki HK, Shin SY, Jung DS, Chung DR, et al. Scoring systems for prediction of mortality in patients with intensive care unit-acquired sepsis: a comparison of the Pitt bacteremia score and the Acute Physiology and Chronic Health Evaluation II scoring systems. Shock. 2009;31:146–50. • 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: 13. Zimmerman JE, Knaus WA, Sun X, Wagner DP. Severity stratification and outcome prediction for multisystem organ failure and dysfunction. World J Surg. 1996;20:401–5. 14. Murray SB, Bates DW, Long Ngo MS, Ufberg JW, Shapiro NI. Charlson Index is associated with one-year mortality in emergency department patients with suspected infection. Acad Emerg Med. 2006;13:530–6. 14. Murray SB, Bates DW, Long Ngo MS, Ufberg JW, Shapiro NI. Charlson Index is associated with one-year mortality in emergency department patients with suspected infection. Acad Emerg Med. 2006;13:530–6. 15. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing: twentieth informational supplement. Wayne: CLSI; 2010. Document No. M100-S20. 15. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing: twentieth informational supplement. Wayne: CLSI; 2010. Document No. M100-S20.
https://openalex.org/W3027383508	https://repositorio.ul.pt/bitstream/10451/43925/1/ICS_EDoBu_Representacoes.pdf	Portuguese	null	Representações e ancoragens sociais do novo coronavírus e do tratamento da COVID-19 por brasileiros	Estudos de Psicologia	2,020	cc-by	10,611	Representações e ancoragens sociais do novo coronavírus e do tratamento da COVID-19 por brasileiros Representations and social anchorages of the new coronavirus and the COVID-19 treatment by Brazilians Emerson Araújo DO BÚ1 0000-0003-3864-3872 Maria Edna Silva de ALEXANDRE2 0000-0003-3610-7208 Viviane Alves dos Santos BEZERRA2 0000-0001-9178-2957 Roseane Christhina da Nova SÁ-SERAFIM3 0000-0001-6751-6421 Maria da Penha de Lima COUTINHO2 0000-0003-3961-2402 Do Bú, E. A., Alexandre, M. E. S., Bezerra, V. A. S., Sá-Serafin, R. C. N., & Coutinho, M. P. L. (2020). Representações e ancoragens sociais do novo coronavírus e do tratamento da COVID-19 por brasileiros. Estudos de Psicologia (Campinas), 37, e200073. http:// dx.doi.org/10.1590/1982-0275202037e200073 Estud. psicol. I Campinas I 37 I e200073 2020 ▼ ▼ ▼ ▼ ▼ 1 Universidade de Lisboa, Instituto de Ciências Sociais. Av. Prof. Aníbal Bettencourt 9, 1600-189, Lisboa, Portugal. Correspondência para/Correspondence to: E.A. DO BÚ. E-mail: <dobuemerson@gmail.com>. 2 Universidade Federal da Paraíba, Centro de Ciências Humanas, Letras e Artes, Programa de Pós-Graduação em Psicologia Social. João Pessoa, PB, Brasil. 3 Universidade Federal de Campina Grande, Centro de Ciências Biológicas e da Saúde, Unidade Acadêmica de Psicologia. Campina Grande, PB, Brasil. Como citar este artigo/How to cite this article Do Bú, E. A., Alexandre, M. E. S., Bezerra, V. A. S., Sá-Serafin, R. C. N., & Coutinho, M. P. L. (2020). Representações e ancoragens sociais do novo coronavírus e do tratamento da COVID-19 por brasileiros. Estudos de Psicologia (Campinas), 37, e200073. http:// dx.doi.org/10.1590/1982-0275202037e200073 ▼ ▼ ▼ ▼ ▼ SEÇÃO TEMÁTICA \| THEMATIC SECTION CONTRIBUIÇÕES DA PSICOLOGIA NO CONTEXTO DA PANDEMIA DA COVID-19 \| CONTRIBUTIONS OF PSYCHOLOGY IN THE CONTEXT OF THE COVID-19 PANDEMIC Representações e ancoragens sociais do novo coronavírus e do tratamento da COVID-19 por brasileiros Representations and social anchorages of the new coronavirus and the COVID-19 treatment by Brazilians Emerson Araújo DO BÚ1 0000-0003-3864-3872 Maria Edna Silva de ALEXANDRE2 0000-0003-3610-7208 Viviane Alves dos Santos BEZERRA2 0000-0001-9178-2957 Roseane Christhina da Nova SÁ-SERAFIM3 0000-0001-6751-6421 Maria da Penha de Lima COUTINHO2 0000-0003-3961-2402 Resumo SEÇÃO TEMÁTICA \| THEMATIC SECTION CONTRIBUIÇÕES DA PSICOLOGIA NO CONTEXTO DA PANDEMIA DA COVID-19 \| CONTRIBUTIONS OF PSYCHOLOGY IN THE CONTEXT OF THE COVID-19 PANDEMIC Representações e ancoragens sociais do novo coronavírus e do tratamento da COVID-19 por brasileiros http://dx.doi.org/10.1590/1982-0275202037e200073 http://dx.doi.org/10.1590/1982-0275202037e200073 Abstract This study aimed to apprehend the genesis of the Social Representations of the new coronavirus, as well as of the treatment of the COVID-19, considering Brazilian people’s different social anchorages. For that purpose, an online questionnaire was answered by 595 participants, predominantly female (69.9%) and from the Northeastern region of Brazil (64.9%). The data collected allowed analyzes of Descending Hierarchical Classifications, indicating that the new coronavirus Social Representations genesis is marked by concerns regarding its dissemination and its psychosocial and affective implications. On the other hand, the representational field of the treatment emphasizes the remission or alleviation of symptoms caused by COVID-19. Given the differences between social groups, the Social Representations variations identified in this research indicate that future interventions should consider each group’s specificities in the dissemination of representations and social practices aiming at containing the pandemic state. Keywords: Coronavirus; Treatment; Social psychology; Health psychology. A Coronavirus Disease 2019 (COVID-19) tem despertado a atenção mundial, ocupando significativo espaço na mídia, na hipermídia e, sobretudo, nas conversações cotidianas de diferentes grupos sociais (Correia, Ramos, & Bathen, 2020). Trata-se de um problema de saúde coletiva, com sérias implicações para a saúde pública, que tem provocado modificações no estilo de vida da população, principalmente no que tange às interações sociais entre pares, dada a recomendação do distanciamento físico para prevenção e contenção do vírus (Brooks et al., 2020; Duan & Zhu, 2020; Fiorillo & Gorwood, 2020). O referido fenômeno, primeiramente, passou a fazer parte da dinâmica social dos chineses em meados de dezembro de 2019. Todavia, em poucos meses, o Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) se espalhou pelos cinco continentes, levando a Organização Mundial da Sáude a declará-lo como uma emergência de saúde pública internacional, dado o seu estado pandêmico (Velavan & Maeyer, 2020; Xu et al., 2020; World Human Organization [WHO], 2020). Em termos operacionais, o SARS-CoV-2 provoca a COVID-19, que consiste em uma doença causada por uma grande família de coronavírus, microrganismo que afeta humanos e atua como agente infeccioso com alto índice de contágio e mortalidade (Velavan & Maeyer, 2020; WHO, 2020). A sua transmissão ocorre de pessoa para pessoa de forma rápida, e seu controle representa um grande desafio (Xu et al., 2020). Prevalentemente, as pessoas com diagnóstico de COVID-19 desenvolvem uma síndrome respiratória aguda, classificada em leve, moderada ou grave. Resumo Objetivou-se neste estudo apreender a gênese das representações sociais do novo coronavírus, bem como do tratamento da COVID-19, considerando-se diferentes ancoragens sociais de brasileiros. Contou-se com 595 participantes, predominantemente do sexo feminino (69,9%) e da região Nordeste do Brasil (64,9%). Os dados, coletados através de um questionário online, permitiram análises de Classificações Hierárquicas Descendentes, indicando que a gênese das representações sociais do novo coronavírus é marcada por preocupações relativas à sua disseminação e implicações psicossociais e afetivas. Já o campo representacional do tratamento enfatiza a remissão ou a amenização dos sintomas causados pela COVID-19. As variações nas representações sociais identificadas nesta pesquisa, em função dos diferentes grupos sociais, indicam que futuras intervenções devem considerar as especificidades de cada um deles na disseminação de representações e práticas sociais direcionadas para conter o estado pandêmico. Palavras-chave: Coronavírus; Psicologia social; Psicologia da saúde; Tratamento. ▼ ▼ ▼ ▼ ▼ 1 Universidade de Lisboa, Instituto de Ciências Sociais. Av. Prof. Aníbal Bettencourt 9, 1600-189, Lisboa, Portugal. Correspondência para/Correspondence to: E.A. DO BÚ. E-mail: <dobuemerson@gmail.com>. eral de Campina Grande, Centro de Ciências Biológicas e da Saúde, Unidade Acadêmica de Psicologia. Campin . CC BY CC CC BY CC BY 2020 Abstract Os fatores de risco mais preponderantes para a agudização dos casos são as doenças cardiovasculares, metabólicas, pulmonares, hepáticas e renais (Villegas-Chiroque, 2020). Dados epidemiológicos indicam que 80% da população infectada apresenta quadros de pneumonia atípica de leve a moderada, 15% evoluem para uma pneumonia grave e 5% dos casos podem desenvolver a Severe Acute Respiratory Syndrome (SARS, Síndrome Respiratória Aguda Grave). Na fase crítica da doença, muitos desenvolvem sepse (infecção generalizada no organismo humano), entram em choque e morrem (Velavan & Maeyer, 2020; Villegas-Chiroque, 2020). Em relação à sintomatologia, as pessoas infectadas apresentam sintomas respiratórios e gastrointestinais após um período de incubação que varia de cinco a catorze dias (Huang, Wuang, Xingwang, Ren, & Zao, 2020). Sua clínica inclui, principalmente, febre ao início do quadro infecioso, tosse seca e dispneia (dificuldade para respirar). Adicionalmente, a pessoa contaminada pode queixar-se de mialgia, fadiga, mal-estar e diarreia (Velavan & Maeyer, 2020). No que tange à suscetibilidade de ocorrência da COVID-19, estudos apontam que homens idosos e imunodeprimidos são os mais suscetíveis (Velavan & Maeyer, 2020; Villegas-Chiroque, 2020). As crianças, por sua vez, são menos vulneráveis à contaminação pelo vírus. Todavia, crianças e jovens, quando infectados, podem permanecer assintomáticas e funcionarem como agentes transmissores do SARS- CoV-2 para outras pessoas (Li et al., 2020). 2 E.A. DO BÚ et al. Estud. psicol. I Campinas I 37 I e200073 2020 Além das questões epidemiológicas, faz-se importante pontuar também os aspectos relacionados às medidas profiláticas para a prevenção e o controle da velocidade de contágio do novo coronavírus. Nessa direção, as principais recomendações são o distanciamento físico, o confinamento domiciliar, a prática de higiene das mãos, o uso de máscaras e a detecção precoce de pessoas infectadas (Adhikari et al., 2020; Duan & Zhu, 2020). Quanto ao tratamento para a COVID-19, o que se tem até o momento são planos terapêuticos de suporte para a sintomatologia que ela provoca. Como ainda não se dispõe de um tratamento farmacológico com eficácia terapêutica e evidências científicas comprovadas em larga escala, o que existe são estudos preliminares que apresentam uma possibilidade interventiva. Ademais, não há estudos conclusivos sobre a imunização (Adhikari et al., 2020; Mahase, 2020). Esse panorama desperta preocupação na população mundial, desencadeia ou potencializa desajustes socioafetivos e transtornos psicológicos preexistentes. Abstract Assim, as pessoas ficam mais suscetíveis ao medo, a sensações de insegurança e impotência, a quadros de ansiedade, depressão e até tentativas de suicídio (Fiorillo & Gorwood, 2020; Duan & Zhu, 2020). Com base nas elucidações anteriormente descritas, nota-se que o núcleo temático sobre o SARS-CoV-2 e a doença que ele provoca (COVID-19) tornou-se alvo de especulação e estudo das mais diversas áreas do conhecimento, dentre elas a Epidemiologia, a Infectologia, a Saúde Pública e a Psicologia. Portanto, estudar o SARS-CoV-2 e a COVID-19 à luz da Psicologia Social e da Saúde, bem como pensar esses enunciados como signos ou correspondentes simbólicos, inerentes às práticas sociais, permite ao pesquisador apreender o modo como as pessoas se organizam socialmente diante da possibilidade de adoecer e tratar-se (Sá-Serafim, 2013). Dessa forma, considerando-se a importância do saber do senso comum na compreensão dos temas em saúde (Oliveira, 2000), este artigo busca responder aos seguintes questionamentos: quais são os elementos que compõem a gênese das representações sociais do novo coronavírus e da COVID-19 para brasileiros? Quais são suas compreensões sobre o tratamento de pessoas diagnosticadas com esse vírus? Para responder a tais questões, toma-se como referência a perspectiva psicossociológica, nomeadamente a Teoria das Representações Sociais proposta por Moscovici (2017). Justifica-se recorrer a esse arcabouço teórico no presente estudo porque se entende que as Representações Sociais são consideradas como princípios que organizam as práticas sociais e as relações simbólicas entre as pessoas frente a objetos sociais que as perpassam (Doise, 2001; Moscovici, 2017). Sabe-se que a gênese de uma representação social se dá por meio de dois processos formadores de natureza social e cognitiva: a ancoragem e a objetivação. Na ancoragem, o indivíduo, em face de um objeto desconhecido, busca em sua memória conteúdos, eventos e pessoas que conhece e os transforma enquanto protótipos, comparando-os com o novo que se interpela. Assim, na ancoragem, assimila-se o novo ao que já existe. Por sua vez, no processo de objetivação, reproduz-se um conceito desconhecido/abstrato da realidade, transferindo-o para um patamar concreto, visível, tangível e “palpável”. Nesses dois processos, então, transforma-se o não familiar em familiar (Moscovici, 2017). 3 REPRESENTAÇÕES SOCIAIS DO NOVO CORONAVÍRUS No cenário de formação das representações sociais, a mídia apresenta fundamental importância, uma vez que transmite códigos normativos de comunicação e conduta (Moscocivi, 2017). Método Trata-se de um estudo misto, quantitativo e qualitativo, descritivo e exploratório, ancorado no aporte teórico da Teoria das Representações Sociais (Doise, 2001; Moscovici, 2017). A amostra da pesquisa foi composta de maneira não probabilística, por conveniência, mediante a participação de 595 brasileiros, na faixa etária de 18 a 78 anos (M = 29,30; DP = 10,10), predominantemente do sexo feminino (69,9%), residentes na região Nordeste do Brasil (64,9%). Em relação ao grau de escolaridade e à renda, 48,9% dos participantes possuíam curso superior e 30,1% auferiam renda de até dois salários mínimos. Quanto aos instrumentos, utilizou-se um Questionário Sociodemográfico com questões relacionadas a idade, sexo, grau de escolaridade, renda e concentração por região do país; e outro questionário que apresentou a Técnica de Associação Livre de Palavras (TALP), contendo os seguintes estímulos indutores: coronavírus e tratamento de pessoas com coronavírus. Faz-se importante pontuar que a TALP consiste em uma técnica projetiva, que se organiza sobre a evocação de respostas dos participantes, a partir de estímulos indutores previamente definidos pelo pesquisador, possibilitando, assim, identificar universos semânticos relacionados a um objeto ou fenômeno social (Coutinho & Do Bú, 2017). Para a coleta de dados, gerou-se um formulário online com os instrumentos supramencionados, divulgado por meio das redes sociais Facebook, WhatsApp e Instagram. Os participantes, após concordarem que eram maiores de 18 anos, brasileiros e que se apresentavam disponíveis para participar da pesquisa voluntariamente, responderam aos dois questionários. Estes ficaram disponíveis para respostas durante cinco dias, entre 14 e 19 de março de 2020, sendo que no dia 14 havia 98 casos da COVID-19 confirmados no Brasil e, no dia 19 de março, 621 casos confirmados e seis mortes no país. Os dados sociodemográficos foram processados por meio do software IBM®SPSS® Statistics (versão 26), que permitiu realizar análises descritivas. Já para os dados da TALP, utilizou-se o software Interface de R pour les Analyses Multidimensionnelles de Textes et de Questionnaires, que viabilizou o desenvolvimento da análise de Classificação Hierárquica Descendente (CHD). Destaca-se que a CHD, a partir da análise da relação (testes de χ2) entre as palavras evocadas pelos participantes do presente estudo, possibilitou a construção de eixos de significados acerca do novo coronavírus, por meio de classes inter-relacionadas de vocábulos, que se configuram como substância bruta para a análise qualitativa da presente pesquisa. Abstract Acerca dessas questões, ressalta-se que, cotidianamente, tem-se verificado, nos mais variados meios de comunicação e interações entre pares, a veiculação e as trocas de informações formais e informais acerca do SARS-CoV-2, bem como da COVID-19. Nesse sentido, sugere-se que tais informações podem ter participação na forma como os brasileiros têm criado e compartilhado representações acerca dos objetos sociais mencionados. Em face do exposto, o presente estudo direciona esforços para compreender como brasileiros se apropriaram do conhecimento sobre o SARS-CoV-2, bem como do tratamento, ainda que especulativo, da COVID-19, assim que ela começou a fazer parte efetiva de sua dinâmica social, isto é, logo nas três primeiras semanas em que foram testados e confirmados casos com tal diagnóstico no Brasil. Desse modo, compreendendo-se que o campo representacional de um dado objeto/fenômeno é construído a partir das Estud. psicol. I Campinas I 37 I e200073 2020 interações das diferentes ancoragens sociais da população com ele (Doise, 2001), objetiva-se, no estudo descrito a seguir, apreender as representações sociais dos objetos supramencionados, a partir de diferentes ancoragens sociais de brasileiros (variáveis sociodemográficas). Acredita-se que tais ancoragens atuarão como ideias de força na construção do pensamento social sobre os fenômenos em questão. Método Além disso, essa análise propiciou a observação da construção de cada classe, por meio das relações entre as variáveis de ancoragem (definidas aqui como as características sociodemográficas dos participantes) e as suas evocações (Camargo & Justo, 2018). Por fim, fez-se uma análise do conteúdo emergido em cada eixo e classes de palavras oriundas das CHD. Buscou-se, nessa análise qualitativa dos dados, apontar aspectos etimológicos das palavras evocadas e relacioná-los com o que a literatura sobre o tema constata, bem como consideraram-se aspectos contextuais do Brasil na atualidade, de modo a evidenciar e compreender sentidos e significados que são criados e compartilhados pelos participantes do presente estudo em face do novo coronavírus e do tratamento, ainda que especulativo, da COVID-19. 4 E.A. DO BÚ et al. 4 E.A. DO BÚ et al. Os procedimentos de coleta de dados seguiram todas as recomendações éticas para esse tipo de pesquisa (CAAE: 30616720.9.0000.0008), conforme preza a Resolução nº 510/2016 do Conselho Nacional de Saúde Brasileiro (Ministério da Saúde, 2016). Estud. psicol. I Campinas I 37 I e200073 2020 Resultados Os resultados referentes aos campos representacionais do novo coronavírus e do tratamento da COVID-19 serão apresentados a partir da formação dos eixos temáticos da CHD e de suas respectivas classes, destacando-se também as variáveis de ancoragem social significativas para a formação destas. Os resultados referentes aos campos representacionais do novo coronavírus e do tratamento da COVID-19 serão apresentados a partir da formação dos eixos temáticos da CHD e de suas respectivas classes, destacando-se também as variáveis de ancoragem social significativas para a formação destas. Estud. psicol. I Campinas I 37 I e200073 2020 g gi p ç Figura 1. Campo representacional e ancoragens sociais do novo coronavírus. Brasil, 2020. Nota: p ≤ 0,05; p ≤ 0,001. UCE: Unidades de Contextos Elementar. Resultados Palavras f X² Palavras f X² Palavras f X² Palavras f X² Álcool 87 132,35 Contágio 80 40,55 China 86 68,77 Cuidado 104 23,73 Máscara 63 54,13 Morte 136 28,21 Epidemia 64 30,00 Prevenção 109 17,06 Aglomeração 20 21,63 Vírus 156 24,71 Morcego 5 26,88 Precaução 19 16,98 Respiratória 6 19,13 Mundo 102 16,10 Crise 43 12,90 Saúde 48 14,66 Infecção 39 17,66 Pandemia 161 15,85 Europa 11 12,68 Empatia 9 14,33 Covid 6 10,74 Idoso 22 14,23 Itália 4 10,61 Medo 145 14,15 Sintomas 10 7,99 Respirador 5 12,82 Brasil 11 7,42 Perigo 57 11,58 Isolamento 207 6,19 Gripe 94 11,80 Vírus 156 6,23 Desinformação 10 11,15 Quarentena 152 4,81 Espirro 14 9,23 Perigoso 12 6,17 Proteção 22 1,01 Respiratório 4 3,15 Pulmão 8 8,76 Doença 247 5,05 Fake News 6 9,49 Propagação 4 3,15 Doença 247 7,23 Globo 7 3,90 Mídia 11 8,66 Coletivo 4 3,15 Doente 5 6,67 Incerteza 7 3,90 Cuidados 42 8,13 Notícia 4 3,15 Respiração 9 6,67 Globalização 15 3,57 Transmissão 27 6,89 Jornal 4 3,15 SARS 4 4,34 Presidente 4 3,54 Vulnerabilidade 4 6,30 Sabão 4 3,15 Difícil 21 4,06 Alarde 4 3,54 Atenção 16 6,15 UTI 33 2,73 Quarentena 152 3,89 Jornal 4 3,54 Preocupação 39 5,39 Alarme 8 2,47 Rápido 14 3,36 Sério 4 3,54 Informação 13 5,12 Mortes 25 2,33 Preocupação 39 3,08 Pavor 6 5,01 Controle 21 2,14 Cura 36 4,47 Fé 8 4,42 Consciência 8 4,42 Leitos 8 4,42 Histeria 12 3,95 Economia 22 3,90 Contaminação 35 3,70 Desespero 35 3,70 Letalidade 5 3,56 Imunidade 31 3,48 Higiene 81 3,43 Classe 2 (15,8% UCE) Origem, vetores de transmissão e focos de disseminação do coronavírus Definição, disseminação e prevenção do coronavírus Implicações psicossociológicas e afetivas do coronavírus Sexo feminino* Região Sul* Entre 3 e 4 salários mínimos* Classe 3 (39,0% UCE) Ancoragens Sociais Ancoragens Sociais Ancoragens Sociais Região Norte* Sexo masculino** Região Centro- Oeste* Classe 4 (16,9% UCE) Estratégias de enfrentamento do coronavírus e canais de informação Classe 1(26,3% UCE) Caracterização, sintomas fisiológicos e abrangência do coronavírus 2 2 2 2 Figura 1. Campo representacional e ancoragens sociais do novo coronavírus. Brasil, 2020. Nota: p ≤ 0,05; p ≤ 0,001. UCE: Unidades de Contextos Elementar. Resultados Palavras f X² Palavras f X² Palavras f X² Palavras f X² Álcool 87 132,35 Contágio 80 40,55 China 86 68,77 Cuidado 104 23,73 Máscara 63 54,13 Morte 136 28,21 Epidemia 64 30,00 Prevenção 109 17,06 Aglomeração 20 21,63 Vírus 156 24,71 Morcego 5 26,88 Precaução 19 16,98 Respiratória 6 19,13 Mundo 102 16,10 Crise 43 12,90 Saúde 48 14,66 Infecção 39 17,66 Pandemia 161 15,85 Europa 11 12,68 Empatia 9 14,33 Covid 6 10,74 Idoso 22 14,23 Itália 4 10,61 Medo 145 14,15 Sintomas 10 7,99 Respirador 5 12,82 Brasil 11 7,42 Perigo 57 11,58 Isolamento 207 6,19 Gripe 94 11,80 Vírus 156 6,23 Desinformação 10 11,15 Quarentena 152 4,81 Espirro 14 9,23 Perigoso 12 6,17 Proteção 22 1,01 Respiratório 4 3,15 Pulmão 8 8,76 Doença 247 5,05 Fake News 6 9,49 Propagação 4 3,15 Doença 247 7,23 Globo 7 3,90 Mídia 11 8,66 Coletivo 4 3,15 Doente 5 6,67 Incerteza 7 3,90 Cuidados 42 8,13 Notícia 4 3,15 Respiração 9 6,67 Globalização 15 3,57 Transmissão 27 6,89 Jornal 4 3,15 SARS 4 4,34 Presidente 4 3,54 Vulnerabilidade 4 6,30 Sabão 4 3,15 Difícil 21 4,06 Alarde 4 3,54 Atenção 16 6,15 UTI 33 2,73 Quarentena 152 3,89 Jornal 4 3,54 Preocupação 39 5,39 Alarme 8 2,47 Rápido 14 3,36 Sério 4 3,54 Informação 13 5,12 Mortes 25 2,33 Preocupação 39 3,08 Pavor 6 5,01 Controle 21 2,14 Cura 36 4,47 Fé 8 4,42 Consciência 8 4,42 Leitos 8 4,42 Histeria 12 3,95 Economia 22 3,90 Contaminação 35 3,70 Desespero 35 3,70 Letalidade 5 3,56 Imunidade 31 3,48 Higiene 81 3,43 Classe 2 (15,8% UCE) Origem, vetores de transmissão e focos de disseminação do coronavírus Definição, disseminação e prevenção do coronavírus Implicações psicossociológicas e afetivas do coronavírus Sexo feminino* Região Sul* Entre 3 e 4 salários mínimos* Classe 3 (39,0% UCE) Ancoragens Sociais Ancoragens Sociais Ancoragens Sociais Região Norte* Sexo masculino** Região Centro- Oeste* Classe 4 (16,9% UCE) Estratégias de enfrentamento do coronavírus e canais de informação Classe 1(26,3% UCE) Caracterização, sintomas fisiológicos e abrangência do coronavírus 2 2 2 2 Palavras f X² Palavras f X² Palavras f X² Palavras f X² Álcool 87 132,35 Contágio 80 40,55 China 86 68,77 Cuidado 104 23,73 Máscara 63 54,13 Morte 136 28,21 Epidemia 64 30,00 Prevenção 109 17,06 Aglomeração 20 21,63 Vírus 156 24,71 Morcego 5 26,88 Precaução 19 16,98 Respiratória 6 19,13 Mundo 102 16,10 Crise 43 12,90 Saúde 48 14,66 Infecção 39 17,66 Pandemia 161 15,85 Europa 11 12,68 Empatia 9 14,33 Covid 6 10,74 Idoso 22 14,23 Itália 4 10,61 Medo 145 14,15 Sintomas 10 7,99 Respirador 5 12,82 Brasil 11 7,42 Perigo 57 11,58 Isolamento 207 6,19 Gripe 94 11,80 Vírus 156 6,23 Desinformação 10 11,15 Quarentena 152 4,81 Espirro 14 9,23 Perigoso 12 6,17 Proteção 22 1,01 Respiratório 4 3,15 Pulmão 8 8,76 Doença 247 5,05 Fake News 6 9,49 Propagação 4 3,15 Doença 247 7,23 Globo 7 3,90 Mídia 11 8,66 Coletivo 4 3,15 Doente 5 6,67 Incerteza 7 3,90 Cuidados 42 8,13 Notícia 4 3,15 Respiração 9 6,67 Globalização 15 3,57 Transmissão 27 6,89 Jornal 4 3,15 SARS 4 4,34 Presidente 4 3,54 Vulnerabilidade 4 6,30 Sabão 4 3,15 Difícil 21 4,06 Alarde 4 3,54 Atenção 16 6,15 UTI 33 2,73 Quarentena 152 3,89 Jornal 4 3,54 Preocupação 39 5,39 Alarme 8 2,47 Rápido 14 3,36 Sério 4 3,54 Informação 13 5,12 Mortes 25 2,33 Preocupação 39 3,08 Pavor 6 5,01 Controle 21 2,14 Cura 36 4,47 Fé 8 4,42 Consciência 8 4,42 Leitos 8 4,42 Histeria 12 3,95 Classe 2 (15,8% UCE) Origem, vetores de transmissão e focos de disseminação do coronavírus Definição, disseminação e prevenção do coronavírus Implicações psicossociológicas e afetivas do coronavírus Classe 3 (39,0% UCE) Ancoragens Sociais Ancoragens Sociais Região Norte* Sexo masculino** Região Centro- Oeste* Classe 4 (16,9% UCE) Estratégias de enfrentamento do coronavírus e canais de informação Classe 1(26,3% UCE) Caracterização, sintomas fisiológicos e abrangência do coronavírus 2 2 2 2 Palavras f X² Palavras f X² Palavras f X² Palavras f X² Álcool 87 132,35 Contágio 80 40,55 China 86 68,77 Cuidado 104 23,73 Máscara 63 54,13 Morte 136 28,21 Epidemia 64 30,00 Prevenção 109 17,06 Aglomeração 20 21,63 Vírus 156 24,71 Morcego 5 26,88 Precaução 19 16,98 Respiratória 6 19,13 Mundo 102 16,10 Crise 43 12,90 Saúde 48 14,66 Infecção 39 17,66 Pandemia 161 15,85 Europa 11 12,68 Empatia 9 14,33 Covid 6 10,74 Idoso 22 14,23 Itália 4 10,61 Medo 145 14,15 Sintomas 10 7,99 Respirador 5 12,82 Brasil 11 7,42 Perigo 57 11,58 Isolamento 207 6,19 Gripe 94 11,80 Vírus 156 6,23 Desinformação 10 11,15 Quarentena 152 4,81 Espirro 14 9,23 Perigoso 12 6,17 Proteção 22 1,01 Respiratório 4 3,15 Pulmão 8 8,76 Doença 247 5,05 Fake News 6 9,49 Propagação 4 3,15 Doença 247 7,23 Globo 7 3,90 Mídia 11 8,66 Coletivo 4 3,15 Doente 5 6,67 Incerteza 7 3,90 Cuidados 42 8,13 Notícia 4 3,15 Respiração 9 6,67 Globalização 15 3,57 Transmissão 27 6,89 Jornal 4 3,15 SARS 4 4,34 Presidente 4 3,54 Vulnerabilidade 4 6,30 Sabão 4 3,15 Difícil 21 4,06 Alarde 4 3,54 Atenção 16 6,15 UTI 33 2,73 Quarentena 152 3,89 Jornal 4 3,54 Preocupação 39 5,39 Alarme 8 2,47 Rápido 14 3,36 Sério 4 3,54 Informação 13 5,12 Mortes 25 2,33 Preocupação 39 3,08 Pavor 6 5,01 Controle 21 2,14 Cura 36 4,47 Fé 8 4,42 Consciência 8 4,42 Leitos 8 4,42 Histeria 12 3,95 Economia 22 3,90 Contaminação 35 3,70 Classe 2 (15,8% UCE) Origem, vetores de transmissão e focos de disseminação do coronavírus Definição, disseminação e prevenção do coronavírus Implicações psicossociológicas e afetivas do coronavírus Classe 3 (39,0% UCE) Ancoragens Sociais Ancoragens Sociais Região Norte* Sexo masculino** Região Centro- Oeste* Classe 4 (16,9% UCE) Estratégias de enfrentamento do coronavírus e canais de informação Classe 1(26,3% UCE) Caracterização, sintomas fisiológicos e abrangência do coronavírus 2 2 2 2 l lavras f X² Palavras f X² hina 86 68,77 Cuidado 104 23,73 demia 64 30,00 Prevenção 109 17,06 rcego 5 26,88 Precaução 19 16,98 Crise 43 12,90 Saúde 48 14,66 uropa 11 12,68 Empatia 9 14,33 tália 4 10,61 Medo 145 14,15 rasil 11 7,42 Perigo 57 11,58 Vírus 156 6,23 Desinformação 10 11,15 rigoso 12 6,17 Proteção 22 1,01 oença 247 5,05 Fake News 6 9,49 Globo 7 3,90 Mídia 11 8,66 erteza 7 3,90 Cuidados 42 8,13 alização 15 3,57 Transmissão 27 6,89 sidente 4 3,54 Vulnerabilidade 4 6,30 larde 4 3,54 Atenção 16 6,15 ornal 4 3,54 Preocupação 39 5,39 Sério 4 3,54 Informação 13 5,12 cupação 39 3,08 Pavor 6 5,01 Cura 36 4,47 Fé 8 4,42 Consciência 8 4,42 Leitos 8 4,42 Histeria 12 3,95 Economia 22 3,90 Contaminação 35 3,70 Desespero 35 3,70 Letalidade 5 3,56 Imunidade 31 3,48 Higiene 81 3,43 Classe 2 (15,8% UCE) m, vetores de transmissão e cos de disseminação do coronavírus Implicações psicossociológicas e afetivas do coronavírus Sexo feminino** Região Sul* Entre 3 e 4 salários mínimos* Classe 3 (39,0% UCE) Ancoragens Sociais Ancoragens Sociais Sexo masculino** Região Centro- Oeste* 2 2 Palavras f X² Cuidado 104 23,73 Prevenção 109 17,06 Precaução 19 16,98 Saúde 48 14,66 Empatia 9 14,33 Medo 145 14,15 Perigo 57 11,58 Desinformação 10 11,15 Proteção 22 1,01 Fake News 6 9,49 Mídia 11 8,66 Cuidados 42 8,13 Transmissão 27 6,89 Vulnerabilidade 4 6,30 Atenção 16 6,15 Preocupação 39 5,39 Informação 13 5,12 Pavor 6 5,01 Cura 36 4,47 Fé 8 4,42 Consciência 8 4,42 Leitos 8 4,42 Histeria 12 3,95 Economia 22 3,90 Contaminação 35 3,70 Desespero 35 3,70 Letalidade 5 3,56 Imunidade 31 3,48 Higiene 81 3,43 Implicações psicossociológicas e afetivas do coronavírus Sexo feminino** Região Sul* Entre 3 e 4 salários mínimos* Classe 3 (39,0% UCE) Ancoragens Sociais 2 Palavras f X² Contágio 80 40,55 Morte 136 28,21 Vírus 156 24,71 Mundo 102 16,10 Pandemia 161 15,85 Idoso 22 14,23 Respirador 5 12,82 Gripe 94 11,80 Espirro 14 9,23 Pulmão 8 8,76 Doença 247 7,23 Doente 5 6,67 Respiração 9 6,67 SARS 4 4,34 Difícil 21 4,06 Quarentena 152 3,89 Rápido 14 3,36 eminação e prevenção do coronavírus o Classe 1(26,3% UCE) Caracterização, sintomas fisiológicos e abrangência do coronavírus 2 Palavras f X² Álcool 87 132,35 Máscara 63 54,13 Aglomeração 20 21,63 Respiratória 6 19,13 Infecção 39 17,66 Covid 6 10,74 Sintomas 10 7,99 Isolamento 207 6,19 Quarentena 152 4,81 Respiratório 4 3,15 Propagação 4 3,15 Coletivo 4 3,15 Notícia 4 3,15 Jornal 4 3,15 Sabão 4 3,15 UTI 33 2,73 Alarme 8 2,47 Mortes 25 2,33 Controle 21 2,14 Definição, dissem Ancoragens Sociais Região Norte* Classe 4 (16,9% UCE) Estratégias de enfrentamento do coronavírus e canais de informação 2 5 REPRESENTAÇÕES SOCIAIS DO NOVO CORONAVÍRUS Figura 1. Campo representacional e ancoragens sociais do novo coronavírus (SARS-CoV-2) No que se refere às evocações dos participantes do presente estudo em face do estímulo “coronavírus”, a CHD reteve 79,6% das Unidades de Contexto Elementar (UCE) do corpus e permitiu identificar quatro classes distintas que compõem o campo representacional do objeto social em questão (Figura 1). Essas classes se apresentam em dois diferentes eixos. O primeiro eixo, intitulado “Definição, disseminação e prevenção do coronavírus”, subdivide-se em dois subconjuntos, em que, à direita, encontram-se as classes 1 e 2 e, em oposição, a classe 4. O segundo eixo é composto apenas pela classe 3 e diz respeito às “Implicações psicossociológicas e afetivas do coronavírus” para brasileiros. A segunda classe, intitulada “Origem, vetores de transmissão e focos de disseminação do coronavírus”, concentra 15,80% das UCE e aborda (majoritariamente a partir de participantes de sexo masculino e da região Centro-Oeste do Brasil as redes de sentido construídas acerca do objeto social em questão. Observa-se nesta classe um intervalo de radicais e vocábulos entre χ² = 68,77 (China) e χ² = 3,08 (preocupação). A primeira classe (Caracterização, sintomas fisiológicos e abrangência do coronavírus), com 28,27% das UCEs do corpus, não associou-se a nenhuma das “variáveis de ancoragem” (características sociodemográficas) a priori estabelecidas. Nesse sentido, apresenta objetivações que são consensuais ao grupo de atores sociais deste estudo acerca da COVID-19. Nesta classe, verificaram-se radicais e palavras no intervalo de χ² = 40,55 (contágio) a χ² = 3,36 (rápido). Em oposição às classes 1 e 2, mas ainda no eixo “Definição, disseminação e prevenção do coronavírus”, encontra-se a classe 4 (Estratégias de enfrentamento do coronavírus e canais de informação), com 16,9% de retenção de UCE do corpus e intervalo de radicais/palavras de χ² = 132,35 (álcool) a χ² = 3,13 (sabão). Essa classe apresenta-se como característica das evocações dos participantes residentes no Norte do Brasil. No eixo 2, evidencia-se a classe 3, que versa acerca das “Implicações sociais, psicológicas e afetivas do coronavírus” para a população brasileira, estando associada, majoritariamente, aos participantes do sexo feminino, com renda entre três e quatro salários mínimos e moradores da região Sul do Brasil. Obteve-se nesta classe o intervalo de radicais e evocações de χ² = 23,73 (cuidado) a χ² = 3,43 (higiene). Campo representacional e ancoragens sociais do novo coronavírus (SARS-CoV-2) Além de apreender como os participantes deste estudo representam o novo fenômeno que os interpela (SARS-CoV-2), buscou-se também identificar suas representações em relação ao tratamento de pessoas com a COVID-19, considerando, ainda, as diferenças em função das variáveis de ancoragem. Destarte, a seguir, apresenta-se a análise relativa ao estímulo “tratamento de pessoas com coronavírus”. Resultados Campo representacional e ancoragens sociais do novo coronavírus. Brasil, 2020. Nota: p ≤ 0,05; p ≤ 0,001. UCE: Unidades de Contextos Elementar. Figura 1. Campo representacional e ancoragens sociais do novo coronavírus. Brasil, 2020. Nota: p ≤ 0,05; *p ≤ 0,001. UCE: Unidades de Contextos Elementar. 5 Estud. psicol. I Campinas I 37 I e200073 Estud. psicol. I Campinas I 37 I e200073 Campo representacional e ancoragens sociais do tratamento de pessoas com coranírus (COVID-19) O material coletado, processado a partir da análise de CHD, deu origem ao dendrograma exposto na Figura 2. Tal análise considerou 87,2% do total das UCE e originou dois eixos. O primeiro eixo, designado “Definições e desafios socioeconômicos face ao tratamento de COVID-19”, subdividiu-se e aglutinando as classes 1 (Estratégias de contenção e automedicação), e 2 (Implicações psicossociais e econômicas para o tratamento), em oposição à classe 3 (Itens de proteção, suporte pessoal e assistência médico-hospitalar); enquanto que o segundo eixo “Busca da cura do COVID-19: Instituições e Agentes responsáveis”, à esquerda, gerou a Classe 4. 6 E.A. DO BÚ et al. 6 E.A. DO BÚ et al. A respeito do eixo 1, especificamente quanto às “Estratégias de contenção e automedicação”, verifica-se o intervalo de radicais e evocações entre χ² = 147,23 (repouso) e χ² = 3,07 (medicação). A característica sociodemográfica dos brasileiros que mais contribuiu para essa classe foi a renda, entre um e dois salários mínimos. No que tange às ‘Implicações psicossociais e econômicas para o tratamento”, evidenciadas pela Estud. psicol. Campo representacional e ancoragens sociais do tratamento de pessoas com coranírus (COVID-19) I Campinas I 37 I e200073 2020 ras f X² Palavras f X² nça 20 45,60 Hospital 104 77,83 a 43 37,72 Máscara 73 35,83 tais 18 31,92 Álcool 99 27,62 uisa 27 31,05 Quarentena 167 24,65 ina 6 20,48 Médico 19 20,52 eiros 6 20,48 Remédio 29 19,93 de 62 17,18 China 76 16,19 rno 18 15,48 Lavar 23 16,11 o 4 13,60 Vacina 98 10,04 mas 10 12,85 Sabão 7 9,63 cia 8 12,52 Casa 23 9,58 mento 8 12,52 Gravidade 9 9,06 S 108 11,87 Leito 4 8,74 e 136 11,10 Sistema 4 8,74 os 48 10,64 Oxigênio 4 8,74 11 10,60 Teste 4 8,74 cia 14 9,59 Isolado 4 8,74 ão 7 9,49 UTI 30 7,00 rro 5 9,34 Exame 10 6,97 dade 5 9,34 Medicamento 10 6,97 ante 10 7,97 Itália 20 5,29 s 10 7,97 Perigoso 20 5,29 cos 10 7,97 Vitamina 16 4,66 mação 6 6,60 Brasil 14 4,36 da 4 6,21 Mídia 12 4,07 ção 32 6,11 Cama 4 3,52 ade 7 4,73 na 98 4,15 a 5 3,95 po 5 3,95 tização 8 3.39 Acima de 8 salários mínimos Ensino Superior* Ancoragens Sociais a da cura da COVID- 19: ões e agentes responsáveis Definições e desa Classe 4 (21,7% UCE) Classe 3 (22,5% UCE) Itens de proteção, suporte pessoal e assistência médico- hospitalar 2 2 Palavras f X² Palavras f X² Palavras f X² Palavras f X² Esperança 20 45,60 Hospital 104 77,83 Repouso 91 147,23 Higiene 95 28,21 Cura 43 37,72 Máscara 73 35,83 Hidratação 21 46,69 Responsabilidad e 25 27,37 Hospitais 18 31,92 Álcool 99 27,62 Água 18 43,94 Cuidados 44 26,15 Pesquisa 27 31,05 Quarentena 167 24,65 Alimentação 27 40,12 Medicamentos 17 18,92 Medicina 6 20,48 Médico 19 20,52 Descanso 18 36,77 Higienização 21 15,98 Enfermeiros 6 20,48 Remédio 29 19,93 Isolamento 233 32,32 Medicação 20 9,66 Saúde 62 17,18 China 76 16,19 Internação 16 30,32 Reclusão 12 9,58 Governo 18 15,48 Lavar 23 16,11 Paracetamol 6 19,57 Atenção 16 7,66 Alívio 4 13,60 Vacina 98 10,04 Dipirona 4 12,99 Proteção 25 7,55 Sintomas 10 12,85 Sabão 7 9,63 Antitérmico 8 11,85 Limpeza 22 7,50 Urgência 8 12,52 Casa 23 9,58 Remédios 21 9,99 Evitar 4 6,67 Investimento 8 12,52 Gravidade 9 9,06 Pulmão 10 7,45 Pobres 4 6,67 SUS 108 11,87 Leito 4 8,74 Imunidade 30 6,82 Confinamento 4 6,67 Morte 136 11,10 Sistema 4 8,74 Paciência 8 6,78 Risco 43 6,49 Idosos 48 10,64 Oxigênio 4 8,74 Nebulização 4 5,88 Cuidado 108 6,11 Fé 11 10,60 Teste 4 8,74 Chá 4 5,88 Contato 18 5,58 Ciência 14 9,59 Isolado 4 8,74 Líquido 4 5,88 Tristeza 10 4,73 Solução 7 9,49 UTI 30 7,00 Contágio 79 5,73 Colapso 5 4,30 Socorro 5 9,34 Exame 10 6,97 Consciência 9 5,15 Respeito 5 4,30 Solidariedade 5 9,34 Medicamento 10 6,97 Tosse 33 4,83 Coletividade 5 4,30 Importante 10 7,97 Itália 20 5,29 Controle 27 4,60 Prevenção 115 4,10 Deus 10 7,97 Perigoso 20 5,29 Gripe 90 4,44 Calma 8 3,77 Médicos 10 7,97 Vitamina 16 4,66 Vitamina 16 3,68 Precaução 21 3,38 Desinformação 6 6,60 Brasil 14 4,36 Desespero 32 3,61 Isolamento 233 3,09 Dúvida 4 6,21 Mídia 12 4,07 Calma 8 3,12 Infecção 32 6,11 Cama 4 3,52 Todos 8 3,12 Dificuldade 7 4,73 Medicação 20 3,07 Vacina 98 4,15 Vida 5 3,95 Grupo 5 3,95 Conscientização 8 3.39 Ancoragens Sociais Acima de 8 salários mínimos* Ensino Superior* Ente 1e 2 salários mínimos* Até um salário mínimo* Ensino médio* Ancoragens Sociais Ancoragens Sociais Busca da cura da COVID- 19: instituições e agentes responsáveis Definições e desafios socieconômicos face ao tratamento da COVID- 19 Classe 4 (21,7% UCE) Classe 3 (22,5% UCE) Itens de proteção, suporte pessoal e assistência médico- hospitalar Classe 1(34,8% UCE) Estratégias de contenção e automedicação Classe 2 (20,8% UCE) Implicações psicossociais e econômicas para o tratamento 2 2 2 2 Responsabilidade Palavras f X² Esperança 20 45,60 Cura 43 37,72 Hospitais 18 31,92 Pesquisa 27 31,05 Medicina 6 20,48 Enfermeiros 6 20,48 Saúde 62 17,18 Governo 18 15,48 Alívio 4 13,60 Sintomas 10 12,85 Urgência 8 12,52 Investimento 8 12,52 SUS 108 11,87 Morte 136 11,10 Idosos 48 10,64 Fé 11 10,60 Ciência 14 9,59 Solução 7 9,49 Socorro 5 9,34 Solidariedade 5 9,34 Importante 10 7,97 Deus 10 7,97 Médicos 10 7,97 Desinformação 6 6,60 Dúvida 4 6,21 Infecção 32 6,11 Dificuldade 7 4,73 Vacina 98 4,15 Vida 5 3,95 Grupo 5 3,95 Conscientização 8 3.39 Doente 8 3,39 A S i i Busca da cura da COVID- 19: instituições e agentes responsáveis Classe 4 (21,7% UCE) 2 X² Palavras f X² Palavras f X² P 45,60 Hospital 104 77,83 Repouso 91 147,23 H 37,72 Máscara 73 35,83 Hidratação 21 46,69 Resp 31,92 Álcool 99 27,62 Água 18 43,94 C 31,05 Quarentena 167 24,65 Alimentação 27 40,12 Med 20,48 Médico 19 20,52 Descanso 18 36,77 Hig 20,48 Remédio 29 19,93 Isolamento 233 32,32 Me 17,18 China 76 16,19 Internação 16 30,32 R 15,48 Lavar 23 16,11 Paracetamol 6 19,57 A 13,60 Vacina 98 10,04 Dipirona 4 12,99 P 12,85 Sabão 7 9,63 Antitérmico 8 11,85 L 12,52 Casa 23 9,58 Remédios 21 9,99 12,52 Gravidade 9 9,06 Pulmão 10 7,45 P 11,87 Leito 4 8,74 Imunidade 30 6,82 Con 11,10 Sistema 4 8,74 Paciência 8 6,78 10,64 Oxigênio 4 8,74 Nebulização 4 5,88 C 10,60 Teste 4 8,74 Chá 4 5,88 C 9,59 Isolado 4 8,74 Líquido 4 5,88 T 9,49 UTI 30 7,00 Contágio 79 5,73 C 9,34 Exame 10 6,97 Consciência 9 5,15 R 9,34 Medicamento 10 6,97 Tosse 33 4,83 Co 7,97 Itália 20 5,29 Controle 27 4,60 Pr 7,97 Perigoso 20 5,29 Gripe 90 4,44 7,97 Vitamina 16 4,66 Vitamina 16 3,68 Pre 6,60 Brasil 14 4,36 Desespero 32 3,61 Iso 6,21 Mídia 12 4,07 Calma 8 3,12 6,11 Cama 4 3,52 Todos 8 3,12 4,73 Medicação 20 3,07 4,15 3,95 3,95 Acima de 8 salários mínimos* Ensino Superior* Ente 1e 2 salários mínimos* Ancoragens Sociais Ancoragens Sociais D- 19: onsáveis Definições e desafios socieconômicos face ao tratamento da CO E) Classe 3 (22,5% UCE) Itens de proteção, suporte pessoal e assistência médico- hospitalar Classe 1(34,8% UCE) Estratégias de contenção e automedicação e 2 2 2 Resp Palavras f X² Pal Repouso 91 147,23 Hig Hidratação 21 46,69 Respon Água 18 43,94 Cuid Alimentação 27 40,12 Medica Descanso 18 36,77 Higien Isolamento 233 32,32 Medi Internação 16 30,32 Rec Paracetamol 6 19,57 Ate Dipirona 4 12,99 Pro Antitérmico 8 11,85 Lim Remédios 21 9,99 Ev Pulmão 10 7,45 Po Imunidade 30 6,82 Confin Paciência 8 6,78 Ri Nebulização 4 5,88 Cui Chá 4 5,88 Co Líquido 4 5,88 Tris Contágio 79 5,73 Col Consciência 9 5,15 Res Tosse 33 4,83 Colet Controle 27 4,60 Prev Gripe 90 4,44 Ca Vitamina 16 3,68 Prec Desespero 32 3,61 Isola Calma 8 3,12 Todos 8 3,12 Medicação 20 3,07 Ente 1e 2 salários mínimos* Ancoragens Sociais ios socieconômicos face ao tratamento da COVID Classe 1(34,8% UCE) Estratégias de contenção e automedicação Im eco 2 Respon Palavras f X² Palavras f X² Repouso 91 147,23 Higiene 95 28,21 Hidratação 21 46,69 Responsabilidad e 25 27,37 Água 18 43,94 Cuidados 44 26,15 Alimentação 27 40,12 Medicamentos 17 18,92 Descanso 18 36,77 Higienização 21 15,98 Isolamento 233 32,32 Medicação 20 9,66 Internação 16 30,32 Reclusão 12 9,58 Paracetamol 6 19,57 Atenção 16 7,66 Dipirona 4 12,99 Proteção 25 7,55 Antitérmico 8 11,85 Limpeza 22 7,50 Remédios 21 9,99 Evitar 4 6,67 Pulmão 10 7,45 Pobres 4 6,67 Imunidade 30 6,82 Confinamento 4 6,67 Paciência 8 6,78 Risco 43 6,49 Nebulização 4 5,88 Cuidado 108 6,11 Chá 4 5,88 Contato 18 5,58 Líquido 4 5,88 Tristeza 10 4,73 Contágio 79 5,73 Colapso 5 4,30 Consciência 9 5,15 Respeito 5 4,30 Tosse 33 4,83 Coletividade 5 4,30 Controle 27 4,60 Prevenção 115 4,10 Gripe 90 4,44 Calma 8 3,77 Vitamina 16 3,68 Precaução 21 3,38 Desespero 32 3,61 Isolamento 233 3,09 Calma 8 3,12 Todos 8 3,12 Medicação 20 3,07 Ancoragens Sociais Ente 1e 2 salários mínimos* Até um salário mínimo* Ensino médio* Ancoragens Sociais fios socieconômicos face ao tratamento da COVID- 19 Classe 1(34,8% UCE) Estratégias de contenção e automedicação Classe 2 (20,8% UCE) Implicações psicossociais e econômicas para o tratamento 2 2 Responsabilidade Palavras f X² Higiene 95 28,21 Responsabilidad e 25 27,37 Cuidados 44 26,15 Medicamentos 17 18,92 Higienização 21 15,98 Medicação 20 9,66 Reclusão 12 9,58 Atenção 16 7,66 Proteção 25 7,55 Limpeza 22 7,50 Evitar 4 6,67 Pobres 4 6,67 Confinamento 4 6,67 Risco 43 6,49 Cuidado 108 6,11 Contato 18 5,58 Tristeza 10 4,73 Colapso 5 4,30 Respeito 5 4,30 Coletividade 5 4,30 Prevenção 115 4,10 Calma 8 3,77 Precaução 21 3,38 Isolamento 233 3,09 Ancoragens Sociais Até um salário mínimo* Ensino médio* o da COVID- 19 Classe 2 (20,8% UCE) Implicações psicossociais e econômicas para o tratamento 2 Responsabilidade 7 REPRESENTAÇÕES SOCIAIS DO NOVO CORONAVÍRUS Estud. Discussão As análises das Classificações Hierárquicas Descendentes para os dois estímulos indutores utilizados neste estudo (coronavírus e tratamento de pessoas com coronavírus) permitiram identificar a gênese constitutiva de seus respectivos campos representacionais. Ademais, também foi verificada a suposição de que as variáveis sociodemográficas se configuravam como ideias de força para a construção de contextos representacionais específicos. Desse modo, a partir dos resultados apresentados, é possível reflexionar sobre os campos representacionais dos referidos objetos sociais e, ainda, observar as diferenciações na forma de representá-los em função das variáveis de ancoragem dos participantes. Especificamente no que se refere aos resultados elucidados por meio da CHD do estímulo indutor coronavírus, observou-se que a classe 1 (Caracterização, sintomas fisiológicos e abrangência do novo coronavírus) apresenta o que foi mais consensual na representação do objeto entre os diferentes grupos sociais desta pesquisa. Nesse sentido, definiu-se o SARS-CoV-2, no momento de coleta dos dados, como um vírus de rápido alastramento pelo mundo (pandemia), com sintomas e forma de contágio característicos de uma gripe (espirro). Tal vírus parece estar associado àquele que provoca uma doença nos pulmões, que pode evoluir, principalmente em idosos, para um quadro de dificuldade respiratória, necessitar do auxílio de respiradores para o seu tratamento e levar à morte. Destarte, demonstra-se uma apropriação do que tem sido veiculado na literatura, principalmente biomédica, acerca do SARS-CoV-2 e do que ele provoca em pessoas contaminadas (Huang et al., 2020; Villegas-Chiroque, 2020). A segunda classe (Origem, vetores de transmissão e focos de disseminação do coronavírus), ainda que contida no mesmo subconjunto que a classe 1, apresenta especificidades dos participantes do sexo masculino e daqueles residentes no Centro-Oeste do Brasil. Para esses grupos, o vírus SARS-CoV-2 surge na China como uma epidemia e, dada a globalização, dissemina-se para a Europa, com especial foco na Itália, chegando, finalmente, ao Brasil. O novo vírus interpela-se para esses grupos como uma incerteza, sendo responsável por uma doença perigosa, que tem sido veiculada na mídia (jornais; Globo – emissora de televisão aberta no Brasil). Ademais, ancora-se a sua transmissão através dos morcegos, o que pode ser justificado dado ao fato histórico, vivenciado em meados de 2003, de que esse mamífero fora indicado como um provável agente de disseminação da SARS (Watanabe et al., 2010). Campo representacional e ancoragens sociais do tratamento de pessoas com coranírus (COVID-19) psicol. I Campinas I 37 I e200073 2020 Figura 2. Campo representacional e ancoragens sociais do tratamento de pessoas com coronavírus (COVID-19). Brasil, 2020. Nota: p ≤ 0,05. UCE: Unidades de Contextos Elementar. Vida 5 3,95 Grupo 5 3,95 Conscientização 8 3.39 Doente 8 3,39 Região Sudeste Acima de 8 salários mínimos* Ensino Superior* Ente 1e 2 salários mínimos* Ancoragens Sociais g classe 2 da CHD, representativa junto a participantes com Ensino Médio, assim como com renda de até um salário mínimo, destacam-se os radicais e extratos de discurso que objetivam o tratamento no intervalo de χ² = 28,21 (higiene) a χ² = 3,09 (isolamento). Em oposição às classes 1 e 2, no eixo 1, encontra-se a classe 3 (Itens de proteção, suporte pessoal e assistência médico-hospitalar) com intervalo de radiais e evocações que variou de χ² = 77,83 (hospital) a Figura 2. Campo representacional e ancoragens sociais do tratamento de pessoas com coronavírus (COVID-19). Brasil, 2020. Nota: p ≤ 0,05. UCE: Unidades de Contextos Elementar. Figura 2. Campo representacional e ancoragens sociais do tratamento de pessoas com coronavírus (COVID-19). Brasil, 2020. Nota: p ≤ 0,05. UCE: Unidades de Contextos Elementar. classe 2 da CHD, representativa junto a participantes com Ensino Médio, assim como com renda de até um salário mínimo, destacam-se os radicais e extratos de discurso que objetivam o tratamento no intervalo de χ² = 28,21 (higiene) a χ² = 3,09 (isolamento). Em oposição às classes 1 e 2, no eixo 1, encontra-se a classe 3 (Itens de proteção, suporte pessoal e assistência médico-hospitalar) com intervalo de radiais e evocações que variou de χ² = 77,83 (hospital) a Estud. psicol. I Campinas I 37 I e200073 2020 χ² = 3,52 (cama). Tal classe desvela evocações dos participantes considerados de maior poder monetário para o contexto brasileiro, com renda per capita superior a oito salários mínimos e Ensino Superior completo. No segundo e último eixo da CHD em análise, encontra-se a classe 4 (Busca da cura da COVID-19: instituições e agentes responsáveis), este eixo fora constituído, majoritariamente, por pessoas que são da região Sudeste do Brasil, com intervalo entre radicais e extratos textuais de χ² = 45,60 (esperança) a χ² = 3,39 (doente). Estud. psicol. I Campinas I 37 I e200073 Discussão Por outro lado, a classe 3 – que aparece sozinha, constituindo um eixo em oposição às demais classes – destaca-se por tangenciar questões relativas às implicações sociais, psicológicas e afetivas, não se restringindo aos aspectos biomédicos. Na referida classe, percebe-se a preocupação coletiva com a prevenção da COVID-19, para além de sua necessidade pessoal, o que foi objetivado pelo termo empatia, que coloca em perspectiva a existência de outras pessoas pelas quais se pode experimentar esse sentimento. Assim, o cuidado profilático é uma ação realizada para si, mas também para a proteção da coletividade. Nesse sentido, salienta-se que Hoffman (2003) considera a empatia como uma variável afetiva preditora de comportamentos pró-sociais. Chama atenção também, nessa classe, a preocupação com as fakes news, fenômeno típico do contexto brasileiro, que gera um conjunto de desinformações sobre objetos sociais, como o novo coronavírus, contribuindo para a construção de representações sociais disfuncionais. Ainda, destacam-se as implicações psicológicas que estão associadas à nova dinâmica social imposta pelo SARS-CoV-2. Consoante a isso, a gravidade e as incertezas relativas a esse fenômeno social provocam emoções e estados psicológicos como o medo, o desespero e até mesmo o pavor. Sublinha-se que implicações psicológicas também já foram observadas em outros contextos sociais diante do novo coronavírus (Duan & Zhu, 2020; Fiorillo & Gorwooad, 2020). As variáveis de ancoragens mais significativas nessa classe (sexo feminino, região Sul e renda per capita entre três e quatro salários mínimos), revelam marcadores importantes na forma de representar as implicações do SARS-CoV-2. O sexo feminino, representativo dessa classe, pode estar relacionado ao fato de serem as mulheres que culturalmente mais se preocupam com a prevenção de doenças e, portanto, que mais buscam os serviços de saúde, em comparação com os homens (Botton, Cúnico, & Strey, 2017). A variável alusiva à região Sul do Brasil, considerada aquela que dispõe de maior poder aquisitivo, parece indicar que, quando as necessidades materiais de sobrevivência estão supostamente garantidas, é possível ampliar o olhar para as diligências de cunho coletivo e subjetivo (Qiu et al., 2020). 9 REPRESENTAÇÕES SOCIAIS DO NOVO CORONAVÍRUS No que se refere ao estímulo “tratamento de pessoas com coronavírus”, nota-se que o campo representacional desse objeto é constituído, majoritariamente, por elementos que remetem seja a indicações que podem ser seguidas para a remissão ou a amenização dos sintomas causados pela COVID-19, seja a estratégias de prevenção que devem ser adotadas frente à doença. Discussão Outro aspecto importante dessa classe relaciona-se ao papel simbólico e pivô da figura dos chefes de estado – presidentes – e seus posicionamentos neste momento de crise e disseminação do SARS-CoV-2. Contextualizando-se no cenário atual brasileiro, a evocação do termo presidente pode ou não se vincular aos posicionamentos do governo de Jair Bolsonaro, contrários às recomendações de quarentena feitas pelas instituições internacionais de saúde para contenção do vírus (WHO, 2020; International Federation of Red Cross and Red Crescent Societies, 2019). Em linhas gerais, o discurso desse governo parece justificar a não-quarentena, por questões econômicas. Em pronunciamentos e entrevistas veiculados na mídia e hipermídia, seus apoiadores sugerem que um número aceitável de vidas podem ser perdidas, desde que a economia não pare. Os idosos, nesse contexto, na condição de grupo de maior risco, parecem também ser desconsiderados. Isso pode estar relacionado ao fato de que, em grande proporção, eles se encontram fora do contexto laboral, sendo representados como incapazes e improdutivos (Araújo, Sá, & Amaral, 2011) para 8 E.A. DO BÚ et al. Estud. psicol. I Campinas I 37 I e200073 2020 a economia. Além disso, tais posicionamentos podem estar ancorados, ainda, na concepção de que apenas os mais fortes sobrevivem (seleção natural), na qual, sendo tal grupo considerado “fraco”, pode morrer. Acerca dessas questões, sugere-se que estudos futuros busquem compreender de forma sistematizada o papel de chefes de estado em contextos pandêmicos, bem como os valores e as ideologias que ancoram posicionamentos da população em face do SARS-CoV-2. Em oposição às classes 1 e 2, destacam-se as “Estratégias de enfrentamento do coronavírus e canais de informação”, como características das evocações dos participantes residentes no Norte do Brasil. Para eles, a utilização de álcool, sabão e máscaras, bem como o isolamento, o evitamento de aglomerações e a quarentena mostram-se como fatores de prevenção e proteção frente à propagação do vírus. Cabe destacar, ainda, o papel da mídia – jornais impressos e televisivos, bem como revistas de circulação nacional – nessa apropriação (Simoneau & Oliveira, 2015), uma vez que, de forma incisiva, tem feito a divulgação de notícias com dados de novos casos e pontos de disseminação, assim como a transmutação de estudos desenvolvidos em todas as partes do mundo para a população. Estud. psicol. I Campinas I 37 I e200073 Discussão Observa-se que, na classe 1 (Estratégias de contenção e automedicação), a ancoragem social que mais contribuiu para a sua composição foi a renda (entre um e dois salários mínimos). Para os participantes com esse perfil socioeconômico, o tratamento de pessoas com coronavírus perpassa aspectos de prevenção e contenção mais relacionados a ações de cunho pessoal/individual. Nessa classe, destacam-se as palavras paracetamol, dipirona, remédios, antitérmico, nebulização e chá. Esses elementos podem indicar que a automedicação é uma prática comum entre as pessoas que apresentam os sintomas da COVID-19. Apesar 2020 2020 de as medicações mencionadas pelos participantes serem apontadas como eficazes no combate aos sintomas leves causados pelo coronavírus, esse é um dado que chama a atenção, pois a automedicação pode acarretar efeitos negativos, sobretudo para aqueles que compõem o grupo de risco, como idosos e pessoas com doenças respiratórias que, em geral, fazem uso de outros medicamentos (fenômeno da polifarmácia), o que pode provocar efeitos colaterais devido à interação entre os fármacos (Secoli, Marquesini, Fabretti, Corona, & Romano-Lieber, 2018). Destaca-se, ainda, nessa classe o isolamento como uma forma de contenção da doença enfatizada pelos participantes. Aqui, faz-se importante compreender qual a definição de isolamento dos respondentes, tendo em vista que essa resposta de saúde pública ao surto da COVID-19 pode ter sérias implicações psicológicas, como o aumento da ansiedade e dos níveis de estresse (Duan & Zhu, 2020; Xiang et al., 2020). Salienta-se que os órgãos e as autoridades de saúde enfatizam que não se trata de um isolamento social, mas sim de um distanciamento físico, estimulando o uso de canais de comunicação seguros para a manutenção do contato entre as pessoas, como forma de diminuir as consequências do distanciamento (Duan & Zhu, 2020; Fiorillo & Gorwooad, 2020). A classe 2, intitulada “Implicações psicossociais e econômicas para o tratamento”, foi representativa para aqueles respondentes com escolaridade até o ensino médio e com renda de até um salário mínimo. Para esses participantes, as estratégias de cuidado e prevenção frente ao novo coronavírus se ancoram em termos que indicam a necessidade de ações mais amplas, com ênfase no bem-estar coletivo. Destacam-se os elementos responsabilidade, cuidados, atenção, proteção, respeito, coletividade e prevenção. Estud. psicol. I Campinas I 37 I e200073 Discussão 10 2020 específicos, como lavar, e a itens de cuidado também típicos, o que demonstra o conhecimento e a clareza desse grupo a respeito das medidas de prevenção e contenção do novo coronavírus, recomendadas pela comunidade científica (Adhikari et al., 2020). específicos, como lavar, e a itens de cuidado também típicos, o que demonstra o conhecimento e a clareza desse grupo a respeito das medidas de prevenção e contenção do novo coronavírus, recomendadas pela comunidade científica (Adhikari et al., 2020). Entretanto, vale salientar que, apesar de ter maior acesso a itens de proteção e ao cuidado médico- -hospitalar, esse grupo não está isento das consequências psicológicas que a COVID-19 pode provocar. Estudos demonstram que pessoas com ensino superior tendem a sentir mais angústia em situações de emergências de saúde pública, provavelmente devido à alta autoconsciência que possuem de sua saúde (Qui et al., 2020). Já a classe 4, constituinte única do eixo denominado a “Busca da cura da COVID-19: instituições e agentes responsáveis”, revela o momento típico vivenciado pelos participantes no período da coleta dos dados deste estudo. Trata-se de um cenário em que o tratamento e a imunização dessa doença ainda são perpassados por incertezas e especulações (Mahase, 2020). Essa classe é mais representativa dos participantes da região Sudeste que, vale ressaltar, foi onde primeiro foram testados e confirmados os casos de pessoas com a COVID-19 no Brasil (Macedo, Ornellas, & Bomfim, 2020). O fato de, naquele momento, a doença fazer parte mais íntima da experiência dos participantes do Sudeste do que daqueles de outras regiões, desperta-lhes a necessidade de controle de tal realidade, o que contribui diretamente para a formação de representações sociais objetivadas a partir dos seguintes aspectos: possíveis fontes (ciência e pesquisa), locais de tratamento (hospitais e SUS) e menção aos responsáveis pelo agenciamento da cura do novo coronavírus (governo, Deus, enfermeiros e médicos). Nesse sentido, as objetivações servem ao propósito de transformar uma realidade abstrata, como é o caso do tratamento da COVID-19, em algo cognoscível (Moscovici, 2017), ao menos do ponto de vista da esperança. Em linhas gerais, indica-se que, a partir da TALP, os participantes que compuseram este estudo puderam compartilhar e nomear cognições e comportamentos forjados em meio social frente ao vírus SARS-CoV-2 e à doença que ele provoca, bem como às questões, ainda que especulativas, de seu tratamento. Discussão Nota-se que as particularidades das condições socioeconômicas desses respondentes encontram-se refletidas nessa classe, quando se observam elementos como pobres, risco, colapso e tristeza, enfatizando a situação de vulnerabilidade em que se encontram as pessoas de tal estatuto socioeconômico, tendo em vista que, em sua grande maioria, não têm acesso a itens de proteção e não podem parar suas atividades laborais, o que faz aumentar seus riscos de contaminação. A esse respeito, um estudo realizado por Qiu et al. (2020), que buscou investigar o sofrimento psíquico na população geral da China durante a epidemia de COVID-19, demonstrou que os trabalhadores que precisavam se deslocar diariamente para o serviço experimentaram o mais alto nível de sofrimento psíquico, quando comparados àqueles que foram dispensados de suas atividades ou que estavam trabalhando em home office. Os altos níveis de estresse desses trabalhadores estavam relacionados à preocupação com a exposição ao vírus no transporte público para o trabalho, com a diminuição do tempo de trabalho e com a consequente diminuição de renda. Esses dados corroboram as observações do presente estudo, demonstrando que, a depender do estatuto socioeconômico que ocupam, as pessoas estarão preocupadas não apenas com a prevenção e a contenção de uma determinada doença, nesse caso específico a COVID-19. Portanto, cabe às autoridades competentes levar em consideração a existência de uma realidade em que, ao lado da doença, as pessoas experimentam e incorporam várias incertezas cotidianas, associadas às suas condições precárias de vida, que afetam sua sobrevivência. Tratando-se da classe 3 (Itens de proteção, suporte pessoal e assistência médico-hospitalar), que foi construída, essencialmente, pelas evocações dos participantes de alto poder aquisitivo para o contexto brasileiro (renda per capita superior a oito salários mínimos) e com ensino superior completo. Verifica-se que esses participantes enfatizam itens específicos de proteção, tais como máscara, álcool e vitamina, e, além disso, evocam elementos que apontam para a possibilidade de realização do cuidado em casa, como cama, que se diferencia de leito, que também aparece na classe, mas está relacionado ao cuidado médico-hospitalar. Tal como nas classes anteriores, as ancoragens sociais permitem observar que determinadas ações de cuidado estão restritas ao imaginário social daqueles que têm acesso a condições para realizar tratamentos específicos e obter determinados produtos. Nota-se que os elementos que compõem essa classe referem-se a termos 10 E.A. DO BÚ et al. Discussão Nesse direcionamento, destaca-se o potencial dessa ferramenta de pesquisa para responder aos questionamentos propostos, permitindo capturar os elementos embrionários das RS do novo coronavírus e do tratamento da COVID-19. Visando-se ampliar o espectro de compreensão das crenças, percepções, opiniões, ideias e práticas específicas das diferentes regiões do país acerca dos objetos sociais em estudo, sugere-se que pesquisas futuras visem ampliar, equalizar e homogeneizar as amostras de participantes, seja em relação ao sexo (uma vez que o presente estudo dispôs majoritariamente de mulheres), seja em relação à região do país (pois participaram, aqui, mais pessoas da região Nordeste). 11 REPRESENTAÇÕES SOCIAIS DO NOVO CORONAVÍRUS Além dessas questões de organização metodológica, novos estudos devem buscar compreender o papel da simpatia ideológica e da empatia no contexto da COVID-19. Um possível modelo a ser testado seria a relação entre a simpatia ideológica (conservadores versus progressistas), a empatia e os comportamentos pró-sociais frente às pessoas contaminadas pelo SARS-CoV-2. Sugerem-se também estudos que busquem compreender a associação entre o poder aquisitivo e a percepção de vulnerabilidade ao novo coronavírus. Salienta-se, ainda, que as análises deste estudo podem ser úteis para fundamentar estratégias interventivas por órgãos governamentais e não governamentais, bem como pela mídia, frente à COVID-19. Para fins de intervenção por tais agentes, aponta-se a necessidade de considerar as especificidades dos diferentes grupos sociais na forma como se apropriam de um saber reificado, típico do universo científico, como é o caso do novo coronavírus, e o transformam em um saber do senso comum. Isso porque o guia de leitura de uma mesma mensagem, por grupos distintos, é baseado nos elementos disponíveis em sua realidade e em sua experiência social. Logo, considerar tais aspectos pode garantir, ao menos, uma comunicação cognoscível, capaz de instrumentalizar a construção e a disseminação de representações e práticas sociais que confluam para a prevenção e a contenção do novo coronavírus no cenário brasileiro. Estud. psicol. I Campinas I 37 I e200073 2020 Contribuição Todos os autores participaram da concepção e delineamento do trabalho e participação na discussão dos resultados; redação do manuscrito e revisão crítica do seu conteúdo e aprovação da versão final do manuscrito. Referências Adhikari, S. P, Meng, S., Wu, Y. -J. Mao, Y. P., Ye, R. -X., Wang, Q. Z., ... Zhou, H. (2020). Epidemiology, causes, clinical manifestation and diagnosis, prevention and control of coronavirus disease (COVID-19) during thee earle outbreal period: a scoping review. Infectious Diseases of Poverty, 9(1), 1-12. http://dx.doi.org/10.1186/s40249-020-00646-x Adhikari, S. P, Meng, S., Wu, Y. -J. Mao, Y. P., Ye, R. -X., Wang, Q. Z., ... Zhou, H. (2020). Epidemiology, causes, clinical manifestation and diagnosis, prevention and control of coronavirus disease (COVID-19) during thee earle outbreal period: a scoping review. Infectious Diseases of Poverty, 9(1), 1-12. http://dx.doi.org/10.1186/s40249-020-00646-x Araújo, L., Sá, E. C. N., & Amaral, E. B. (2011). Corpo e velhice: um estudo das representações sociais entre homens idosos. Psicologia: Ciência e Profissão, 31(3), 468-481. http://dx.doi.org/10.1590/S1414-98932011000300004 Araújo, L., Sá, E. C. N., & Amaral, E. B. (2011). Corpo e velhice: um estudo das representações sociais entre homens idosos. Psicologia: Ciência e Profissão, 31(3), 468-481. http://dx.doi.org/10.1590/S1414-98932011000300004 Botton, A., Cúnico, S. D., & Strey, M. N. (2017). Diferenças de gênero no acesso aos serviços de saúde: problematizações necessárias. Revista Mudanças Psicologia da Saúde, 25(1), 67-72. http://dx.doi.org/10.15603/2176-1019/mud. v25n1p67-72 Botton, A., Cúnico, S. D., & Strey, M. N. (2017). Diferenças de gênero no acesso aos serviços de saúde: problematizações necessárias. Revista Mudanças Psicologia da Saúde, 25(1), 67-72. http://dx.doi.org/10.15603/2176-1019/mud. v25n1p67-72 Brooks, S. K., Webster R. K., Smith L. E., Woodland L., Wessely, S., Greenberg, N., & Rubin, G. J. (2020). The psychological impact of quarantine and how to reduce it: rapid review of the evidence. The Lancet, 395(10227), 912-920. http:// dx.doi.org/10.1016/S0140-6736(20)30460-8 Camargo, B. V., & Justo A. M. (2018). Tutorial para uso do software de análise textual IRAMUTEQ. Florianópolis: Universidade Federal de Santa Catarina. Recuperado de http://iramuteq.org/documentation/fichiers/tutoriel-portugais-22-11-2018 Correia, M. I. D. T., Ramos, R. F., & Bahten, L. C. V. (2020). Os cirurgiões e a pandemia do COVID-19. Revista Colégio Brasileiro de Cirurgiões, 47(1), 1-6. http://dx.doi.org/10.1590/0100-6991e-20202536 Coutinho, M. P. L., & Do Bú, E. (2017). A técnica de associação livre de palavras sobre o prisma do Software tri-deux- mots (version 5.2). Revista Campo do Saber, 3(1), 219-242. Recuperado de http://periodicos.iesp.edu.br/index.php/ campodosaber/article/view/72/58 Do Bú, E. A., Alexandre, M. E. S., Bezerra, V. A. S., Sá-Serafin, R. C. N., & Coutinho, M. P. L. (2020). Representações e ancoragens sociais do novo coronavírus e do tratamento da COVID-19 por brasileiros. Scielo Preprints. Versão 1. http://doi.org/10.1590/SciELOPreprints.120 Doise, W. (2001). Direitos do homem e força das ideias. Referências Lisboa: Livros Horizontes. W. (2001). Direitos do homem e força das ideias. Lisboa: Livros Horizontes. Duan, L., & Zhu, G. (2020). Psychological interventions for people affected by the COVID-19 epidemic. The Lancet, 7(4), 300-302. http://dx.doi.org/10.1016/S2215-0366(20)30073-0 Fiorillo, A., & Gorwood, P. (2020). The consequences of the COVID-19 pandemic on mental health and implications for clinical practice. European Psychiatry, 63(1), 1-4. http://dx.doi.org/10.1192/j.eurpsy.2020.35 International Federation of Red Cross and Red Crescent Societies. (2019). Psychological first aid. Recuperado de https:// media.ifrc.org/ifrc/emergency/global-covid-19/ Hoffman, M. L. (2003). Empathy and moral development: implications for caring and justice. New York: University Press. Huang, C., Wuang, Y., Xingwang, L., Ren, L., & Zao, J. (2020). Clinical feature of patients infected with 2019 novel coronavirus in Wuhan, China. The Lancet, 395(10223), 497-506. http://dx.doi.org/10.1016/S0140- 6736(20)30183-5 Li, Q., Guan, X., Wu, P., Wang, X., Zhou, L., Tong, Y., … Feng, Z. (2020). Early transmission dynamics in Wuhan, China, of novel coronavirus–infected pneumonia. New England Journal of Medicine, 382, 1199-1207. http://dx.doi.org/10.1056/ NEJMoa2001316 Macedo, Y. M., Ornellas, J. L., & Bomfim, H. F. (2020). COVID – 19 no Brasil: o que se espera para população subalternizada? Revista Encantar: Educação, Cultura e Sociedade, 2, 1-10. http://dx.doi.org/10.5935/encantar.v2.0001 Mahase, E. (2020). COVID-19: what treatments are being investigated? BMJ, 368(1252), 1-2. http://dx.doi.org/10.1136/ bmj.m1252 Ministério da Saúde. (Brasil). (2016). Resolução nº 510, de 7 de abril de 2016. Estabelece as diretrizes e normas regulamentadoras de pesquisas envolvendo seres humanos. Diário Oficial da União. Brasília: Autor. Recuperado de http://www.in.gov.br/materia/-/asset_publisher/Kujrw0TZC2Mb/content/id/22917581 covici, S. (2017). A Psicanálise, sua imagem e seu público. Petrópolis: Vozes. Moscovici, S. (2017). A Psicanálise, sua imagem e seu público. Petrópolis: Vozes. Oliveira, D. C. (2000). Social representations and public health care: subjectivity as a participant in the daily routine in health care. Revista de Ciências Humanas, 2, 47-65. http://dx.doi.org/10.5007/%25x 12 Estud. psicol. I Campinas I 37 I e200073 2020 Qui, J., Shen, B., Zhao, M., Wang, Z., Xie, B., & Xu, Y. (2020). A nationwide survey of psychological distress among Chinese people in the COVID-19 epidemic: implications and policy recommendations. General Psychiatry, 33, 1-3 http://dx.doi.org/10.1136/gpsych-2020-100213 Sá-Serafim, R. C. N. (2013). Corpo mastectomizado e representações sociais: rede de significações que conduzem a ação (Tese de doutorado não-publicada). Universidade Federal da Paraíba. Recuperado de https://repositorio.ufpb. br/jspui/handle/tede/6961 Secoli, S. R., Marquesini, E. A., Fabretti, S, C., Corona, L. P., & Romano-Lieber, N. S. (2018). Tendência da prática de automedicação entre idosos brasileiros entre 2006 e 2010: estudo SABE. Referências Revista Brasileira de Epidemiologia, 21(2), 1-14. http://dx.doi.org/10.1590/1980-549720180007.supl.2 Simoneau, A., & de Oliveira, D. (2015). Representações sociais e meios de comunicação: produção do conhecimento científico em periódicos brasileiros. Psicologia e Saber Social, 3(2), 281-300. http://dx.doi.org/10.12957/psi.saber. soc.2014.14478 Velavan, T, P., & Mayer, C. G. (2020). The COVID-19 epidemic. Tropical Medicine and International Health, 25(3), 278- 280 http://dx.doi.org/10.1111/tmi.13383 Villegas-Chiroque, M. (2020). Pandemia de COVID-19: pelea o huye. Revista Experiencia en Medicina del Hospital Regional Lambayeque, 6(1). http://dx.doi.org/10.37065/rem.v6i1.424 Watanabe, S., Masangkay, J. S., Nagata, N., Morikawa, S. Mizutani, T., Fukushi, S., ... Akashi, H. (2010). Bat Coronaviruses and Experimental Infection of Bats, the Philippines. Emerging Infectious Diseases,16(8), 1217-1223. http://dx.doi. org/10.3201/eid1608.100208 World Human Organization. (2020). Coronavirus disease (COVID-19): situation dashboard. Geneve: Author. Retrieved from dehttps://experience.arcgis.com/experience/685d0ace521648f8a5beeeee1b9125cd Xiang, Y., Yang, Y., Li, W., Zhang, L., Zhang, Q., & Cheung, T. (2020). Timely mental health care for the 2019 novel coronavirus outbreak is urgently needed. The Lancet, 7(3), 227-229. http://dx.doi.org/10.1016/S2215-0366(20)30046-8 Xu, H., Zhong, L., Deng, J., Peng, J., Dan, H., Zeng, X., … Chen, Q. (2020). High expression of ACE2 receptor of 2019-nCoV on the epithelial cells of oral mucosa. International Journal of Oral Science, 12(8). http://dx.doi.org/10.1038/s41368-0 20-0074-x Recebido em: abril 13, 2020 Aprovado: abril 23, 2020 13 REPRESENTAÇÕES SOCIAIS DO NOVO CORONAVÍRUS Estud. psicol. I Campinas I 37 I e200073 2020
https://openalex.org/W4242341391	https://acp.copernicus.org/articles/21/16609/2021/acp-21-16609-2021.pdf	English	null	Comment on acp-2021-420	null	2,021	cc-by	18,232	A climatology of trade-wind cumulus cold pools and their link to mesoscale cloud organization Correspondence: Raphaela Vogel (raphaela.vogel@lmd.ipsl.fr) Received: 18 May 2021 – Discussion started: 1 June 2021 Revised: 15 September 2021 – Accepted: 4 October 2021 – Published: 12 November 2021 Overall, we ﬁnd cold-pool periods to be ∼90 % cloudier rel- ative to the average winter trades. Also, the wake of cold pools is characterized by above-average cloudiness, suggest- ing that mesoscale arcs enclosing broad clear-sky areas are an exception. A better understanding of how cold pools in- teract with and shape their environment could therefore be valuable to understand cloud cover variability in the trades. Abstract. We present a climatology of trade cumulus cold pools and their associated changes in surface weather, verti- cal velocity and cloudiness based on more than 10 years of in situ and remote sensing data from the Barbados Cloud Ob- servatory. Cold pools are identiﬁed by abrupt drops in surface temperature, and the mesoscale organization pattern is clas- siﬁed by a neural network algorithm based on Geostation- ary Operational Environmental Satellite 16 (GOES-16) Ad- vanced Baseline Imager (ABI) infrared images. We ﬁnd cold pools to be ubiquitous in the winter trades – they are present about 7.8 % of the time and occur on 73 % of days. Cold pools with stronger temperature drops (1T ) are associated with deeper clouds, stronger precipitation, downdrafts and humidity drops, stronger wind gusts and updrafts at the on- set of their front, and larger cloud cover compared to weaker cold pools, which agrees well with the conceptual picture of cold pools. The rain duration in the front is the best predictor of 1T and explains 36 % of its variability. 1 Introduction Satellite images in the trades usually show very beautiful and diverse cloud structures over the dark blue ocean. Recurrent features in these images are mesoscale arcs of cumuli that encircle either clear-sky areas or extensive stratiform cloud decks. The mesoscale arcs result from spreading cold pools that have favourable conditions at their gust front for trigger- ing new convection. Convective cold pools are generated by the evaporation of precipitation into unsaturated downdrafts, spreading out at the surface as a density current. Cold pools are not only important for the triggering of new and often deeper convection (Schlemmer and Hohenegger, 2014; Feng et al., 2015; Rowe and Houze, 2015), but might also play a role in regulating cloud cover in the trades – a regime re- sponsible for much of the uncertainty in climate sensitivity (Bony and Dufresne, 2005; Vial et al., 2013). Here we use ground-based in situ and remote sensing data from the Bar- bados Cloud Observatory (BCO) to study the climatology of trade-wind cumulus cold pools and to investigate its link to the pattern of mesoscale cloud organization. The mesoscale organization pattern has a strong inﬂu- ence on the occurrence frequency of cold pools. Fish has the largest cold-pool fraction (12.8 % of the time), followed by Flowers and Gravel (9.9 % and 7.2 %) and lastly Sugar (1.6 %). Fish cold pools are also signiﬁcantly stronger and longer-lasting compared to the other patterns, while Gravel cold pools are associated with signiﬁcantly stronger updrafts and deeper cloud-top height maxima. The diel cycle of the occurrence frequency of Gravel, Flowers, and Fish can ex- plain a large fraction of the diel cycle in the cold-pool occur- rence as well as the pronounced extension of the diel cycle of shallow convection into the early afternoon by cold pools. Atmos. Chem. Phys., 21, 16609–16630, 2021 https://doi.org/10.5194/acp-21-16609-2021 © Author(s) 2021. This work is distributed under the Creative Commons Attribution 4.0 License. R. Vogel et al.: Trade cumulus cold-pool climatology Many studies addressing oceanic cold pools have focused on deep convection (Zuidema et al., 2017). In the trades, de- tailed case studies for 2 weeks of the Rain in Cumulus over the Ocean (RICO) campaign have advanced our understand- ing of cold pools from shallow convection (Zuidema et al., 2012). They showed that the deepest clouds and strongest radar signals occurred in the moistest tercile of water vapour paths and that precipitation-driven downdrafts can introduce additional gradients in the thermodynamic structure. More recently, analyses of data from the Elucidating the Role of Clouds-Circulation Coupling in Climate (EUREC4A) ﬁeld campaign (Bony et al., 2017; Stevens et al., 2021), which took place in January and February 2020 upstream Barba- dos, revealed that cold pools are frequent in the winter trades and can be well detected from soundings due to their very shallow mixed layers (Touzè-Peiffer et al., 2021). What is missing is a long-term climatology of trade cumulus cold pools along with a description of the changes in cloud proper- ties and sub-cloud layer dynamics associated with the cold- pool passages. Such a climatology is particularly pertinent given the need for a reference dataset for comparison against increasingly available high-resolution simulations (Stevens et al., 2019; Rochetin et al., 2021). This paper presents the ﬁrst long-term climatology of trade-wind cumulus cold pools and addresses the following research questions. 1. How frequent are cold pools in the trade cumulus regime, and with what changes in the surface meteo- rology, cloudiness, and vertical velocity are they associ- ated? 2. How do cold-pool characteristics covary with the pat- tern of mesoscale organization? We use more than 10 years of surface meteorology and ground-based remote sensing data from 2011 to 2021 col- lected at the BCO (Stevens et al., 2016). Clouds, their pre- cipitation, and therefore likely also cold pools at the BCO were shown to be representative across the trades (Medeiros and Nuijens, 2016). Cold pools are identiﬁed by abrupt drops in surface temperature, and the pattern of mesoscale organi- zation is classiﬁed by a neural network algorithm based on infrared satellite images (Schulz et al., 2021). To focus on trade cumulus cold pools, we limit most of our analysis to the winter regime from December to April, as in summer the intertropical convergence zone is often close to Barbados and convection is much deeper (Brueck et al., 2015). Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. 16610 R. Vogel et al.: Trade cumulus cold-pool climatology Renewed interest in trade cumulus cold pools is also motivated by recent advances in characterizing patterns of mesoscale cloud organization. Stevens et al. (2020) classi- ﬁed 900 satellite images in the North Atlantic trades and identiﬁed four prominent patterns of mesoscale cloud orga- nization – Sugar, Gravel, Flowers, and Fish. The horizon- tal structure of the latter three patterns is intrinsically linked to the occurrence of mesoscale arcs and hence cold pools. The four patterns differ not only in their horizontal struc- ture, but also in cloud cover, cloud depth, and precipitation (Bony et al., 2020; Schulz et al., 2021; Vial et al., 2021). These differences likely also manifest in different cold-pool characteristics. Furthermore, cold pools might play differ- ent roles in creating and maintaining these patterns. For the Fish pattern with its very large-scale ﬁsh-bone structures that are tightly linked to extratropical dry intrusions (Aemiseg- ger et al., 2021; Schulz et al., 2021), cold pools are likely to give the cloudy part its skeletal structure, while the over- all system is forced by the large-scale dynamics into its lin- ear alignment. Observations of drizzling stratocumulus of- ten show cold pools being dragged along with a larger sys- tem without initiating its mesoscale organization (Wilbanks et al., 2015). Contrastingly, for the Gravel pattern, the large- scale inﬂuence may be less important and also more homo- geneous. Thus, cold pools likely play an important role in creating and maintaining this pattern, similar to the strong inﬂuence of rain (and indirectly also cold pools) on the tran- sition from closed- to open-cell stratocumulus (Xue et al., 2008; Wang and Feingold, 2009; Glassmeier and Feingold, 2017). Before we can understand the different roles that cold pools play in these patterns, we need to understand whether and how cold-pool characteristics differ among them. Section 2.1 presents the data sources and explains the cold-pool detection algorithm and the selection criteria. In Sect. 3, we present the cold-pool climatology and analyse the temporal structure of cold-pool passages and the associated changes in meteorology and cloudiness. Section 4 discusses differences between the cold-pool properties of the different mesoscale organization patterns. Conclusions are presented in Sect. 5. 2.1.3 Cloud radar Vertical proﬁles of hydrometeors (including both cloud and rain droplets) at 10 s temporal and 30 m vertical resolution are derived from two 35.5 GHz (Ka-band) Doppler cloud radars. Radar returns with an equivalent radar reﬂectivity lower than −50 dBZ are removed to eliminate signal from sea salt aerosol (Klingebiel et al., 2019). To identify con- nected 2D cloud objects, a cloud segmentation algorithm is applied (Konow, 2020). Radar reﬂectivity is converted to a binary mask and morphological closing is applied to re- move noise from measurement interruptions. The resulting mask is used to identify cloud objects using connected com- ponent analysis with 8-connectivity. A minimum cloud size of 4 pixels is applied, and everything smaller than 4 pixels is discarded as clutter. To focus on clouds connected to the trade-wind layer, only cloud objects with a lowest cloud-base height (CBHID) smaller than 4 km are considered in the anal- ysis. We derive 1 min time series of both the average vertical velocity in the sub-cloud layer (SCL) as the mean over 15 range gates from 75 to 495 m (wSCL) and the vertical veloc- ity near the sub-cloud layer top at 450 m as the mean over the four range gates from 405 to 495 m (w450). Doppler li- dar vertical velocities are commonly considered reliable also in rainy periods (see e.g. Zhu et al., 2021). We did not en- counter problems with the Doppler lidar retrievals in rainy periods, and Figs. 2 and 3h will show that the negative verti- cal velocities associated with downdrafts are generally well captured. 2.1.4 Doppler lidar The MRR is a vertically pointing frequency-modulated continuous-wave radar operating at 24 GHz (K band). The MRR data have a temporal resolution of 1 min and a range gate of 30 m up to a height of 3 km. Rain rates lower than 0.03 mm h−1 are below the noise level and set to zero. We derive the mean rain rate (RR) and the rain intensity (Rint, i.e. the instantaneous rain rate during periods of rain) for a speciﬁed period from data at 325 m above ground (the low- est level with reliable data). The MRR is also used to com- pute the rain frequency (Rfreq), which is set to 1 when a RR > 0.05 mm h−1 is measured in at least ﬁve range gates in the lowest 3 km (following Nuijens et al., 2014). A few in- stances with unrealistically large RR exceeding 200 mm h−1 are set to NA (not applicable). The vertical velocity in the sub-cloud layer is measured by two Halo Photonics Streamline Pro Doppler wind lidar sys- tems (Päschke et al., 2015) at 30 m vertical resolution. The Doppler lidars measure vertical velocities of up to ±20 m s−1 with a 1500 nm laser at altitudes from about 50 m to 1 km, de- pending on the atmospheric conditions and the aerosol load- ing. The precision is < 20 cm s−1 for a signal-to-noise ratio (SNR) of −17 dB. Measurements with a SNR smaller than −18.3 dB are discarded. Data from the ﬁrst system that was operated in vertically pointing mode with a temporal reso- lution of 1.3 s are used from March 2016 to October 2019. A second system has been operated in horizontally scanning mode since February 2019 and has a temporal resolution of 3 s, with two out of seven proﬁles measured in vertically pointing mode. Vertical data from this second lidar are used from November 2019 to March 2021. 2.1.1 Surface meteorology A Vaisala WXT520 sensor mounted on a 5 m mast measures temperature, relative humidity, barometric pressure, wind speed, and wind direction. We discard temperature measure- ments exceeding 35 ◦C and pressure measurements lower than 980 hPa, as they are outside the expected range of vari- ability at the BCO. R. Vogel et al.: Trade cumulus cold-pool climatology (CCprcp, the same threshold as in Klingebiel et al., 2021). A given 1 min HF proﬁle can only count to one of the three cat- egories, such that e.g. a 2 km-deep cloud with CBH < 300 m will only be counted in the CCprcp category. Note that the above classiﬁcation into the different CBH categories does not consider the cloud objects, and subsequent HF proﬁles are classiﬁed independently. A similar analysis accounting for the cloud objects by classifying CC contributions of dif- ferent cloud objects by their CBHID is shown in Appendix A. (CCprcp, the same threshold as in Klingebiel et al., 2021). A given 1 min HF proﬁle can only count to one of the three cat- egories, such that e.g. a 2 km-deep cloud with CBH < 300 m will only be counted in the CCprcp category. Note that the above classiﬁcation into the different CBH categories does not consider the cloud objects, and subsequent HF proﬁles are classiﬁed independently. A similar analysis accounting for the cloud objects by classifying CC contributions of dif- ferent cloud objects by their CBHID is shown in Appendix A. 2.1 BCO data We use in situ and ground-based remote sensing data from the BCO (Stevens et al., 2016), which has been operated by the Max Planck Institute for Meteorology together with the Caribbean Institute for Meteorology and Hydrology since April 2010. The BCO is located atop a 17 m cliff on an eastward promontory of Barbados called Deebles Point (13.16◦N, 59.43◦W) and samples nearly undisturbed At- lantic trade-wind conditions. We have used surface meteo- rology and micro-rain radar (MRR) data since January 2011, cloud radar data since January 2012, and Doppler lidar data from March 2016 until March 2021. All data are aggregated into 1 min averages. The instruments used and meteorolog- ical variables derived are explained in the following. More details about the BCO and its instrumentation can be found in Nuijens et al. (2014) and Stevens et al. (2016). https://doi.org/10.5194/acp-21-16609-2021 Atmos. Chem. Phys., 21, 16609–16630, 2021 16611 2.2 Machine learning classiﬁcation of mesoscale cloud organization patterns The BCO data at 1 min resolution are considered con- temporaneous with the nearest 30 min neural network classi- ﬁcation. If a given pattern is present for more than 75 % of the duration of a cold pool, the cold pool is categorized by this pattern. Figure 1. Illustration of the cold-pool detection algorithm. (a) 11 min ﬁltered Tﬁl (thick line) and 1 min raw surface temper- ature (thin line), and (bottom) ﬁltered temperature difference δT along with the threshold of −0.05 K used (dashed). The detected cold-pool fronts and wakes are indicated in dark grey (tmax to tmin) and light grey (tmin to tend), with the corresponding 1T indicated at the top. The dark red lines in (a) show the analysis periods used for computing the diagnostics (see Sect. 2.5). At any given time, multiple rectangles of different sizes of the same and different patterns can occur. Multiple rectan- gles of the same pattern are combined and counted only once, while multiple rectangles of different patterns are counted separately. This leads to locations in the images, and hence data in the meteorological time series, being classiﬁed e.g. as both Gravel and Flowers. Excluding situations with mul- tiple patterns only marginally inﬂuences the results but re- duces the sample size considerably (as previously noted in Vial et al., 2021). Ambiguities in the classiﬁcation can be physical – for example due to regime transitions or similar- ities between patterns – or related to ambiguities introduced to the neural network by disagreement in the human classiﬁ- cations. The occurrence of multiple patterns can be reduced if a stricter threshold is used for the agreement score repre- senting the conﬁdence of the neural network prediction (here set to 0.4 as in Schulz et al., 2021; Vial et al., 2021), but this again reduces the sample size. ring within 20 min of the previous minimum are com- bined if the temperature does not rise by more than 0.5 K above the previous minimum in between. 3. tend: the end of a cold pool is deﬁned either as the minimum of (a) the time when the ﬁltered tempera- ture ﬁrst exceeds its minimum by 1T/e, where 1T = Tmax −Tmin is the total ﬁltered temperature drop in the cold pool and e is Euler’s number, or (b) the onset of the next cold pool. 2.2 Machine learning classiﬁcation of mesoscale cloud organization patterns The pattern of mesoscale cloud organization at the BCO for the period January 2018 to March 2021 is classiﬁed by a neural network algorithm applied to infrared satellite images from the Geostationary Operational Environmental Satellite 16 (GOES-16). We use brightness temperature retrievals ev- ery 30 min from the 10.35 µm channel at a spatial resolution of 2 km from the Advanced Baseline Imager (ABI) Level- 1b data product (GOES-R Calibration Working Group and GOES-R Series Program, 2017), over a large domain includ- ing Barbados (45–66◦W, 9.3–23.3◦N). From the remaining clouds, we derive 1 min averaged time series of the hydrometeor fraction (HF), cloud-base height (CBH), cloud-top height (CTH), and projected cloud cover (CC). Following Nuijens et al. (2014), CC is fur- ther split up into contributions from cloud segments with different CBH, which represent cloudiness near the lifting- condensation level (CClcl; 300 m < CBH ≤1 km) and cloudi- ness aloft such as stratiform layers or edges of deeper cumuli (CCaloft; 1 km < CBH ≤4 km). We also introduce a third category of precipitating cloud segments if CBH ≤300 m The neural network based on the RetinaNet algorithm (Lin et al., 2017) was initially trained on and applied to visible https://doi.org/10.5194/acp-21-16609-2021 Atmos. Chem. Phys., 21, 16609–16630, 2021 R. Vogel et al.: Trade cumulus cold-pool climatology Figure 1. Illustration of the cold-pool detection algorithm. (a) 11 min ﬁltered Tﬁl (thick line) and 1 min raw surface temper- ature (thin line), and (bottom) ﬁltered temperature difference δT along with the threshold of −0.05 K used (dashed). The detected cold-pool fronts and wakes are indicated in dark grey (tmax to tmin) and light grey (tmin to tend), with the corresponding 1T indicated at the top. The dark red lines in (a) show the analysis periods used for computing the diagnostics (see Sect. 2.5). R. Vogel et al.: Trade cumulus cold-pool climatology R. Vogel et al.: Trade cumulus cold-pool climatology 16612 images in Rasp et al. (2020) and later retrained and applied to infrared images by Schulz et al. (2021). The use of in- frared images also allows study of the diurnal cycle of the mesoscale organization (Vial et al., 2021). The classiﬁcations of the neural network are rectangles of various sizes that be- long to either the Sugar, Gravel, Flowers, or Fish pattern. We select every classiﬁed rectangle that overlaps with the BCO location. Periods without a classiﬁcation are labelled as “No”. 2.2 Machine learning classiﬁcation of mesoscale cloud organization patterns If using condition (a) or (b) leads to any temperature between tmin and tend being smaller than Tmin −0.15 K, then tend is deﬁned as (c) the time when the ﬁltered temperature ﬁrst decreases again af- ter increasing for some time following tmin. Cold pools with tend deﬁned by (a) are referred to as recovered. 2.4 Example cases Time series of example cold-pool days along with corre- sponding satellite images are shown for every pattern in Fig. 2 and shed some light on the differences in the cold-pool characteristics of the four patterns. The two Sugar cold pools stem from isolated precipitating deeper cumuli. The satellite image captures the deeper cloud over the BCO at the time of the ﬁrst cold pool and also indicates some organization of the cumuli in lines upstream the BCO, while the canonical Sugar ﬁelds of shallow cumuli pass further north. The textbook- like Gravel example day is characterized by many short and often weak cold pools quickly following each other, inter- spersed by stronger cold pools. The stronger cold pools are associated with the presence of strongly precipitating deeper clouds (note that the radar did not work prior to 12:00 LT). The many cold pools present on this day clearly imprint their signature on the satellite image in the form of mesoscale arcs. As mentioned in Sect. 2.2, the organization pattern def- inition is somewhat ambiguous. Also among the example days shown in Fig. 2, multiple cloud patterns pertain to some cold pools. For the Flowers case, the 2 h at the beginning and end of the period shown are also classiﬁed, respectively, as Gravel and Fish. In the Sugar case, only the period be- tween 09:00 and 16:00 LT is exclusively classiﬁed as Sugar, while the periods before and after are also partly classiﬁed as Gravel. Most surprisingly, the textbook Gravel day is also en- tirely classiﬁed as Flowers, and also setting a stricter agree- ment score of 0.5 leaves half of the day co-classiﬁed as Flow- ers. This indicates that distinguishing Gravel from Flowers can be particularly challenging (as also shown in Vial et al., 2021). The Fish day is very conﬁdently classiﬁed and no other pattern is detected for the entire day. The cold pools on the Flowers day are associated with the large cloud system whose stratiform layer reaches the BCO at 10:00 LT. Three cold pools are directly associated with the large system, with the ﬁrst one starting at 11:00 LT, show- ing a very strong 1T of −3.85 K. The large system has rain rates up to 3.6 mm h−1 and is preceded by a weaker cold pool at 09:30 LT associated with the very thin mesoscale arc visible in the satellite image. R. Vogel et al.: Trade cumulus cold-pool climatology difﬁculty in deﬁning the end of the cold-pool wake is illus- trated in the Fish case: the cold pool starting shortly before 16:00 LT lasts until well after 18:00 LT, but the temperature drop near 17:00 LT causes a premature end of the cold pool. A temperature drop of this magnitude could also be caused by the diel cycle in temperature. The cold-pool end deﬁni- tion could be improved by an additional rain or downdraft requirement to more robustly distinguish between cold-pool activity and other processes. Because most analyses and di- agnostics computed in this study focus entirely on the cold- pool front (see next section), not fully representing the wake of rare long-lasting cold pools is a minor issue that could only inﬂuence the overall cold-pool fraction and the duration statistics. 2.4 Example cases This ﬁrst weak cold pool goes along with a strong increase in humidity of 1.3 g kg−1. The Fish day features a 6 h-long cold pool associated with steady and intense rain (maximum RR of 11.6 mm h−1), continued strong downdrafts, and very high humidity throughout its en- tire duration. The temperature fully recovers within about 20 min of the cold-pool end, and 3 h later two subsequent pronounced cold pools follow that are again characterized by continued precipitation and downdrafts. The satellite im- age shows the ﬁsh-bone-like cloud band typically associated with the Fish pattern, which is strongly connected to trailing cold fronts of extratropical origins (Aemisegger et al., 2021; Schulz et al., 2021). The more front-like character of the Fish cold pools with steady showers and downdrafts is clearly ev- ident. While most of the cooling is expected to stem from the evaporating precipitation, we cannot rule out a small cooling contribution related to a larger-scale temperature contrast be- tween the south and north of the Fish cloud band (see also Schulz et al., 2021). 2.3 Cold-pool detection algorithm We detect cold pools by identifying abrupt drops in the BCO surface temperature time series following Vogel (2017). We ﬁrst ﬁlter the 1 min averaged temperature time series with an 11 min running average. We then classify all ﬁltered 1 min temperature drops δT = Tﬁl(t) −Tﬁl(t −1) < −0.05 K (per minute) as a cold-pool candidate (see Fig. 1 for an illustra- tion). For every candidate cold pool, we detect the time of the cold-pool front onset (tmax), the time of the minimum tem- perature (tmin), and the end of the cold pool (tend) as follows. The period between tmax and tmin is referred to as the cold- pool front and the period between tmin and tend as the cold- pool wake. p Our cold-pool detection algorithm is similar to the one used by de Szoeke et al. (2017) but with the important mod- iﬁcation that we only identify cold pools for situations with abrupt temperature drops exceeding our threshold of δT < −0.05 K. With our algorithm we thus ﬁlter out both turbu- lent ﬂuctuations and advective or diurnal patterns of temper- ature variability. The threshold of δT < −0.05 K is subjec- tively chosen based on visual impression and represents dis- tinct variations in temperature. For an 11 min averaging win- dow, a δT of −0.05 K corresponds to about 2 % of the data. Figure 2 shows example cold pools for all patterns and illus- trates the algorithm. In the next subsection we brieﬂy discuss the strengths and weaknesses of the algorithm based on these examples. 1. tmax: the onset of the cold-pool front tmax is deﬁned as the last instance of δT > 0 K within 20 min before the initial abrupt temperature drop with δT < −0.05 K. If the temperature is falling continuously in this period, tmax is chosen as the time of the maximum temperature (that is, 20 min before the abrupt temperature drop). We refer to the smoothed temperature at tmax as Tmax. 2. tmin: the time of the minimum ﬁltered temperature Tmin marks the end of the cold-pool front and is identiﬁed as the minimum of contiguous temperature minima. Sub- sequent candidate cold pools with δT < −0.05 K occur- https://doi.org/10.5194/acp-21-16609-2021 Atmos. Chem. Phys., 21, 16609–16630, 2021 16613 2.5 Selection criteria and diagnostics For the subsequent analyses, we apply a number of selection criteria to make the comparison of cold pools more robust. That is, we only consider cold pools with 1T < −0.4 K and less than two missing values in the ﬁltered temperature time series during the entire cold-pool duration (set all with 9234 cold pools). For the analyses of the cold-pool properties we further apply a criterion of no non-recovered cold pool in the hour prior to the cold-pool onset (set noprev with 8772 cold pools), which selects cold pools moving into an initially undisturbed atmosphere that is not modiﬁed by previous con- vection. Except for Appendix B, we also focus on the dry winter regime from December to April (set noprevWI with 3889 cold pools), which is characterized by steady easter- lies, subsiding large-scale motion in the free troposphere and the predominance of shallow trade-wind convection (Brueck et al., 2015). The example cases highlight how well the detection algo- rithm works in these diverse situations. Abrupt strong tem- perature drops are reliably detected, successive fronts sensi- bly combined into one single cold pool, and even the 6 h-long cold pool with frontal character on the Fish day is correctly identiﬁed. All these selection criteria reduce the cold-pool sample size considerably. They represent a trade-off between ensur- ing a robust and unbiased sample to address our research questions while not being unnecessarily strict and remov- ing too many cold pools. The selection criteria are thus somewhat subjective and differ among studies. For example, Chandra et al. (2018) used the criterion of no rain in the hour The examples also indicate some challenges of the cold- pool identiﬁcation. Although they look like cold pools, some temperature drops on the Gravel and Sugar days are not iden- tiﬁed as cold pools because they are either not abrupt enough (δT ≥−0.05 K) or not strong enough (1T ≥−0.4 K). The https://doi.org/10.5194/acp-21-16609-2021 Atmos. Chem. Phys., 21, 16609–16630, 2021 R. Vogel et al.: Trade cumulus cold-pool climatology 16614 2. BCO time series and satellite images for 18 h of 4 cold-pool days representative of the four patterns. Shown are time series o emperature and speciﬁc humidity, MRR rain rate, and time–height plots of Doppler lidar vertical velocity and radar reﬂect vel day the radar did not work prior to 12:00 LT, and the ﬁrst ceilometer cloud base (CBH1) is shown instead. R. Vogel et al.: Trade cumulus cold-pool climatology 16615 prior to the cold-pool onset to select cold pools unmodiﬁed by previous convection, whereas we achieve the same goal with the criterion of no non-recovered cold pool in the prior hour, which excludes less cold pools in our case (i.e. about 2500 additional cold pools would be discarded with the cri- terion of Chandra et al., 2018). Instead of focusing on the winter regime, we could have also set a criterion based on the cloud-top height to focus on trade cumulus cold pools. However, as this would restrict the analysis to periods when the radar is running and – as we are relying on single-site measurements – the parent convection might not move over the BCO in its entirety, we would likely exclude too many cold pools with a CTH criterion without even being sure that periods of deep convection are really excluded. Despite the rather strict criteria applied here, the long time series leads to a much larger number of cold pools analysed than in previous studies. Table 1. Table showing median ± IQR of various cold-pool proper- ties for the noprevWI set of cold pools as well as the 25 % strongest (1T < −1.39 K) and weakest (1T > −0.61 K) cold pools of this set. The computation of the diagnostics is explained in Sect. 2.5. 3 Cold-pool climatology In this section we present the climatology of trade cumulus cold pools detected at the BCO for the winter seasons of the years 2011–2021. The ﬁrst subsection presents general statistics, followed by a discussion of the composite tempo- ral structure of the cold pools in Sect. 3.2. The diel cycle of cold-pool statistics is shown in Sect. 3.3. While our focus lies on the winter regime, Appendix B also brieﬂy discusses the seasonal cycle of the cold-pool statistics. p g p y If not mentioned differently, diagnostics for each cold pool are computed either as the minimum difference (1Xmin) or maximum difference (1Xmax) of a variable X across the cold-pool duration, e.g. 1Xmax = max(X((tmax+1) : tend)− X(tmax)). If the cold-pool wake lasts longer than 20 min, the diagnostics are computed only until 20 min after tmin (instead of until tend) to prevent problems in case of a poorly de- ﬁned cold-pool end. Similarly, Xmean or Xmax are the mean or maximum of variable X over the same analysis period (in- dicated in dark red in Fig. 1). For the Doppler lidar vertical velocities, we diagnose wmaxSCL (wmax450) as the maximum wSCL (w450) in the ﬁrst half of the front (including the last 10 min before tmax) and wminSCL as the minimum wSCL in the second half of the front (including the ﬁrst 10 min after tmin). Unless otherwise stated, the surface meteorology diag- nostics are computed from the 11 min ﬁltered time series. R. Vogel et al.: Trade cumulus cold-pool climatology noprevWI Strong Weak # 3889 972 972 1T (K) −0.89 ± 0.78 −1.82 ± 0.67 −0.5 ± 0.1 1Tunﬁl (K) −1.2 ± 0.8 −2.16 ± 0.66 −0.79 ± 0.17 1qmin (g kg−1) −0.43 ± 0.65 −0.55 ± 0.81 −0.36 ± 0.54 1qmax (g kg−1) 0.2 ± 0.41 0.29 ± 0.51 0.12 ± 0.3 1θe,min (K) −2.05 ± 2.08 −3.3 ± 2.25 −1.35 ± 1.35 1θv,min (K) −0.96 ± 0.81 −1.92 ± 0.7 −0.55 ± 0.14 1pmax (hPa) 0.09 ± 0.29 0.2 ± 0.44 0.04 ± 0.19 1Umax (m s−1) 1.14 ± 1.55 2 ± 1.97 0.7 ± 0.99 1Umax.unﬁl (m s−1) 2.81 ± 2.36 4 ± 2.52 2.02 ± 1.69 1wdirmean (◦) 0.48 ± 12.57 3.33 ± 18.34 −0.32 ± 8.59 Rint (mm h−1) 0.9 ± 1.76 1.45 ± 2.42 0.41 ± 0.95 RRmean (mm h−1) 0.05 ± 0.38 0.39 ± 1.06 0 ± 0.04 CTHmax (km) 3.04 ± 1.11 3.56 ± 1.2 2.66 ± 0.96 CTHmean (km) 2.32 ± 0.88 2.74 ± 0.81 2.03 ± 0.89 wminSCL (m s−1) −0.55 ± 1.56 −1.89 ± 2.42 −0.27 ± 0.51 wmaxSCL (m s−1) 0.91 ± 0.62 1.1 ± 0.7 0.78 ± 0.54 wmax450 (m s−1) 0.98 ± 0.81 1.27 ± 0.99 0.79 ± 0.66 Length (km) 13.3 ± 9.5 18.6 ± 10.9 10 ± 6 1tnextcp (min) 117 ± 426 85 ± 245 158 ± 725 Dur (min) 33 ± 22 47 ± 29 25 ± 12 Front dur (min) 19 ± 12 29 ± 19 15 ± 4 Another potential sampling issue regarding the single-site measurements is that it is not clear at which stage of its life cycle we sample the cold pool and where we sample it with respect to its centre. Assuming azimuthally symmetric wind variations around the cold-pool centre, which in case of lit- tle wind shear is a good approximation (Touzè-Peiffer et al., 2021), the change in wind direction from the mean direction prior to the cold-pool onset could give a hint as to the location relative to the cold-pool centre. Due to our large sample size, a potential random bias is likely to be small. The inﬂuence of wind shear on the propagation direction and characteristics of cold pools is an interesting topic for a future study. 2.5 Selection criteria and diagnostics The x ax me and the detected cold-pool fronts and wakes are indicated in grey and light grey, with 1T indicated at the bottom. Visible rom 10–15◦N, 60–55◦W are from MODIS Aqua (Sugar day) and GOES-16 ABI (other days), with the respective record d by the orange lines in the temperature panels. The BCO is located near the easternmost tip of Barbados (outlined in yello Figure 2. BCO time series and satellite images for 18 h of 4 cold-pool days representative of the four patterns. Shown are time series of ﬁltered surface temperature and speciﬁc humidity, MRR rain rate, and time–height plots of Doppler lidar vertical velocity and radar reﬂectivity. On the Gravel day the radar did not work prior to 12:00 LT, and the ﬁrst ceilometer cloud base (CBH1) is shown instead. The x axis shows local time and the detected cold-pool fronts and wakes are indicated in grey and light grey, with 1T indicated at the bottom. Visible satellite images from 10–15◦N, 60–55◦W are from MODIS Aqua (Sugar day) and GOES-16 ABI (other days), with the respective recording times indicated by the orange lines in the temperature panels. The BCO is located near the easternmost tip of Barbados (outlined in yellow). Atmos. Chem. Phys., 21, 16609–16630, 2021 https://doi.org/10.5194/acp-21-16609-2021 3.2 Composite temporal structure The average cold-pool duration is 33 min, of which a bit more than half of the time pertains to the front. Multiply- ing the duration by the surface wind speed yields a median cold-pool length larger than 13.3 km. This median cold-pool duration and length may seem small compared to satellite imagery, in which mesoscale cold-pool arcs can easily span 100 km. Also, the largest 2 % of cold pools are hardly larger than 40 km. The smaller cold-pool sizes found here are likely due to the algorithm sampling mostly the edge of the cold pools and due to the challenges of deﬁning the cold-pool end purely based on the surface temperature time series (see dis- cussion in Sect. 2.4). Figure 3 shows the composite mean temporal structure of the perturbations associated with the cold-pool passages. To fa- cilitate the comparison of different cold pools, we use a nor- malized time coordinate within the cold-pool front with val- ues after tmax and before tmin mapped onto 20 points (the me- dian front duration), similar to previous studies (Young et al., 1995; de Szoeke et al., 2017; Zuidema et al., 2017). The temperature of the composite-mean cold pool, after increasing slightly before tmax, decreases rapidly in the front to −1.15 K and recovers by 1T/e within 16 min after tmin (not shown). The temperature remains about 0.5 K below Tmax in the hour after the frontal passage. The temperature drop in the front of the 25 % strongest cold pools is by deﬁ- nition stronger but with a mean tendency of −0.070 K min−1 also more than twice as abrupt compared to the weakest cold pools. The strongest cold pools also take longer to recover than the weakest ones. The IQR shows that all these medians are associated with substantial variability, especially for the humidity and rain variables. However, focusing on the winter regime generally reduces the IQR of the diagnostics compared to all seasons (not shown), suggesting that this criterion indeed results in a more homogeneous cold-pool sample representative of the trade cumulus regime. The temporal structure of the speciﬁc humidity response is intriguing. The composite-mean humidity starts to increase already 8 min before tmax and increases by about 0.2 g kg−1 until tmax. R. Vogel et al.: Trade cumulus cold-pool climatology 16616 Table 1 presents statistics of the most important cold-pool properties for the set of winter cold pools with no non- recovered cold pool in the prior hour (noprevWI). It shows that 50 % of the cold pools have a temperature drop ex- ceeding 0.9 K across the front (the unﬁltered temperature drop is 0.3 K stronger), a 1qmax exceeding 0.2 g kg−1 and a 1qmin below −0.43 g kg−1, decreases in θe and θv exceed- ing −2.1 K and −0.96 K, respectively, a 1pmax exceeding 9 Pa, and a 1Umax larger than 1.14 m s−1 (with the unﬁl- tered anomaly being more than twice as large). The median rain intensity measured by the MRR is 0.9 mm h−1. Further- more, 50 % of the cold pools are associated with a maximum cloud-top height exceeding 3 km and wmaxSCL and wminSCL of 0.9 m s−1 and −0.55 m s−1 near the onset and end of the front, respectively. (R2 = 0.357). That the accumulated rain amount in the front explains less variability in 1T (R2 = 0.21) than the rain du- ration indicates that the rain intensity is of secondary im- portance. Another important predictor of 1T is the down- draft strength wminSCL (R2 = 0.23), which together with the front duration explains 50 % of the variability in 1T for the noprevWI set. The CTH usually scales with the precipita- tion amount for trade cumuli (Byers and Hall, 1955; Kubar et al., 2009; Nuijens et al., 2009), and CTHmax also explains some variability in 1T (R2 = 0.10). That the rain duration, the downdraft strength, and the maximum CTH also distin- guish the cold-pool properties well indicates that the parent convection triggering the cold pool is sampled well by the single-point measurements. 3.1 General statistics In total we detect 3889 cold pools that meet the criteria of 1T < −0.4 K and less than two missing values in Tﬁl in the winter seasons considered. We ﬁnd that cold pools are very frequent at the BCO, and on 73 % of days at least one cold pool is detected. The BCO is on average affected by cold pools during 7.8 % of the day (i.e. 112 min) and by a cold- pool front during 4.4 % of the day. The median daily cold- pool fractions are about one-third smaller than the means mentioned, indicating that there are some days with a very large cold-pool fraction. The mean daily cold-pool fraction of 8.6 % for January and February 2011–2021 is also very close to the 7 % found by Touzè-Peiffer et al. (2021) dur- ing the EUREC4A campaign in January and February 2020, despite their very different method deﬁning cold pools in at- mospheric soundings based on a mixed-layer depth criterion. Along with most diagnostics and composites, we show the standard error (SE), which measures how well the median or mean of a given sample can be estimated. The SE of the median is computed as IQR/√n, where IQR represents the inter-quartile range and n the sample size, and the SE of the mean as σ/√n, where σ is the standard deviation. As not all instruments were running all the time, some diagnostics are only available for a subset of the cold pools, and the sample size is adjusted accordingly when computing the SE. https://doi.org/10.5194/acp-21-16609-2021 Atmos. Chem. Phys., 21, 16609–16630, 2021 https://doi.org/10.5194/acp-21-16609-2021 3.2 Composite temporal structure Vogel et al.: Trade cumulus cold-pool climatology 16617 Figure 3. Composite mean temporal structure of anomalies with respect to the cold-pool onset (tmax) for the surface properties (a) tempera- ture, (b) speciﬁc humidity, (c) equivalent potential temperature, (d) relative humidity, and (e) wind speed as well as absolute values of (f) the MRR rain frequency and (g) rain rate and (h) the vertical velocity at 450 m height. The black line shows the mean structure of all cold pools matching the noprevWI criterion, and the red and blue lines show the mean for the 25 % strongest and weakest cold pools, respectively. The dotted lines show the mean ± 1 SE. Vertical and horizontal reference lines are added to indicate tmax, tmin, and 0. R. Vogel et al.: Trade cumulus cold-pool climatology R. Vogel et al.: Trade cumulus cold-pool climatology R. Vogel et al.: Trade cumulus cold-pool climatology 16617 Figure 3. Composite mean temporal structure of anomalies with respect to the cold-pool onset (tmax) for the surface properties (a) tempera- ture, (b) speciﬁc humidity, (c) equivalent potential temperature, (d) relative humidity, and (e) wind speed as well as absolute values of (f) the MRR rain frequency and (g) rain rate and (h) the vertical velocity at 450 m height. The black line shows the mean structure of all cold pools matching the noprevWI criterion, and the red and blue lines show the mean for the 25 % strongest and weakest cold pools, respectively. The dotted lines show the mean ± 1 SE. Vertical and horizontal reference lines are added to indicate tmax, tmin, and 0. pre-front humidity increase could be moisture convergence ahead of the front (Schlemmer and Hohenegger, 2016). cold pools (Touzè-Peiffer et al., 2021). Another reason in cases of more strongly precipitating cold pools might be con- tinued evaporation of precipitation, which would cool and moisten the air in the cold-pool wake and thus speed up the humidity recovery but slow down the temperature recovery. The temporal structure of the equivalent potential temper- ature (Fig. 3c) is similar to the humidity structure but with a stronger drop across the front and a stronger difference be- tween the weaker and stronger cold pools governed by the temperature drops. The relative humidity signal in the front is mostly governed by the temperature decrease, with RH be- ing 8 % larger at tmin for the strongest cold pools. 3.2 Composite temporal structure In the ﬁrst quarter of the front, the humidity in- creases by another 0.2 g kg−1 before it drops to its minimum of −0.25 g kg−1 at tmin, which is hardly lower than the pre- front value. The speciﬁc humidity response of the strongest cold pools only differs signiﬁcantly from the weakest cold pools at tmin, with the humidity drop at tmin being about −0.4 g kg−1 and thus about twice as strong as the drop for the weakest cold pools. If the entire set of cold pools includ- ing the summer season with deeper convection is used, the strongest cold pools have a signiﬁcantly weaker positive hu- midity anomaly at the beginning of the front and a signif- icantly faster and stronger humidity reduction at tmin com- pared to the weakest cold pools (see Fig. B1c–d). Table 1 also compares the median ± IQR of the 25 % strongest and weakest cold pools in terms of 1T . The strongest cold pools last longer, follow each other more quickly (lower 1tnextcp), and are associated with deeper clouds, more rain, stronger downdrafts, humidity drops and wind gusts, and larger positive vertical velocities at the be- ginning of the front compared to weaker cold pools. This agrees well with the conceptual picture of deeper clouds pro- ducing more rain and having a larger potential for rain evap- oration, which drives stronger downdrafts that bring down more dry air from further aloft and which induces a stronger cooling and a stronger gust front that is associated with stronger rising motion at its leading edge. Similar but slightly smaller differences between stronger and weaker cold pools are found when comparing cold pools associated with the 25 % strongest versus weakest downdrafts or the 25 % deep- est versus shallowest CTHmax (not shown). The humidity recovers much more quickly than the tem- perature and remains slightly elevated compared to its pre- front value in the hour after. The fast humidity recovery might be due to the trapping of surface moisture ﬂuxes in the anomalously shallow mixed layer typically associated with The rain duration in the front is the diagnostic that ex- plains most variability in 1T (R2 = 0.364). Rain duration is well correlated (R = 0.47) with the front duration, which itself explains a comparable amount of variability in 1T https://doi.org/10.5194/acp-21-16609-2021 Atmos. Chem. Phys., 21, 16609–16630, 2021 R. R. Vogel et al.: Trade cumulus cold-pool climatology For the vertical velocity averaged over the entire sub-cloud layer (wSCL), the picture is similar, but the peak wmaxSCL is slightly smaller for the strongest cold pools and more similar compared to the weaker cold pools (Table 1). The cloud radars at the BCO also allow study of how the cloud properties change across the cold-pool passage (Fig. 4). The mean cloud-top height (CTH) increases rapidly by ∼500 m after the cold-pool onset and peaks at the end of the front. CTH remains elevated by about 300 m com- pared to the pre-front value in the following hour. The 25 % strongest cold pools are associated with signiﬁcantly deeper clouds throughout the entire period shown, especially so at the end of the front, when the CTH is on average higher than 3300 m. The cloud-base height (CBH) starts to decrease al- ready slightly before tmax and reaches its minimum near the end of the front at ∼500 m. This decrease is due to the more frequent precipitation with very low echo-base heights and is most pronounced for the strongest cold pools. p g p The total hydrometeor cover (CC) increases rapidly at the beginning of the cold-pool front, remains about 25 % larger compared to the pre-front value inside the front, and decreases slowly in the wake. The mean CC of the 25 % strongest cold pools reaches nearly 100 % at the end of the front and is signiﬁcantly larger than the CC of the weakest cold pools during the entire period shown, especially so in the wake. In Fig. 4d–f the cloud cover is split into contri- butions from cloud segments with different CBH by consid- ering all 1 min hydrometeor fraction proﬁles independently. This shows that the enhanced CC of the strongest cold pools in the prior hour is entirely due to cloud segments with CBH above 1 km (CCaloft), whereas the enhanced CC in the front and wake of the strongest cold pools is mostly due to pre- cipitating cloud segments with CBH below 300 m. The rapid increase in CClcl up to its peak at tmax strongly contributes to the CC increase at the edge of the front. This peak is also larger for the strongest cold pools, consistent with their larger w450 at tmax. R. Vogel et al.: Trade cumulus cold-pool climatology 16618 Figure 3f–g show the composite mean Rfreq and RR mea- sured by the MRR. Both rain variables increase rapidly after the onset of the cold pool, peak towards the middle or end of the front, and start to decrease shortly before tmin. The strongest cold pools have much larger rain rates and rain fre- quencies during the entire front compared to the weakest cold pools, and the rain frequency of the strongest cold pools also remains strongly elevated until more than an hour after tmin. the value at tmax in the wake. We hypothesize that this dif- ference can be explained by the stronger cold pools during DYNAMO travelling further away from their parent convec- tion, such that they continue to push forward into the mean wind. What strengthens cold pools in the trades despite the shallower parent convection is the drier cloud layer and free troposphere compared to the deep convective regions, which facilitates evaporation of precipitation and can strengthen downdrafts (Chandra et al., 2018). g y min The last panel of Fig. 3h shows the Doppler lidar verti- cal velocity averaged over four 30 m range gates with mean height of 450 m (w450). The mean w450 peaks at the edge of the front with about 0.25 m s−1 and decreases rapidly to −0.3 m s−1 near the end of the front, reﬂecting updrafts trig- gered at the cold-pool gust front and downdrafts driven by the evaporating precipitation inside the front, respectively. The median wmax450 at the gust front edge (see Table 1) is at 1 m s−1 much larger than the averaged hourly in-cloud vertical velocities near cloud base measured by the BCO Doppler radar, which has a peak density at 0.2 m s−1 and maxima of 0.6 m s−1 (Klingebiel et al., 2021). 1 m s−1 also marks the upper tail of cloud-base averaged updraft vertical velocities at the BCO (see Fig. 4b of Sakradzija and Klinge- biel, 2020). This suggests that the gust-front vertical velocity maxima are very relevant for triggering new convection in the trade cumulus regime. The strongest cold pools have sig- niﬁcantly stronger downdrafts and also updrafts compared to the weakest cold pools (see also Table 1), the latter highlight- ing the potentially enhanced triggering of new convection by stronger cold pools. 3.2 Composite temporal structure The initial increase in humidity at the edge of the front at the BCO might be explained by enhanced surface ﬂuxes due to the strengthening winds (Langhans and Romps, 2015; Torri and Kuang, 2016), by moisture advection (Schlemmer and Hohenegger, 2016), or by an accumulation of moisture from evaporation of precipitation of the parent convection, which was pushed to the edge of the front (Tompkins, 2001). Analyses of the various isotope measurements made during the EUREC4A ﬁeld campaign (Stevens et al., 2021) might help elucidate the origin of these moisture rings, as suggested by idealized large-eddy simulations (Torri, 2021). This could also help understand why cloud-resolving models seem to have difﬁculties in representing the humidity structure in the cold-pool front correctly (Chandra et al., 2018). As discussed by de Szoeke et al. (2017), the humidity increase just before tmax might be mostly due to the increasing saturation-speciﬁc humidity associated with the increasing temperature before tmax (as seen by the relative humidity anomaly in panel d be- ing slightly below zero) and as such likely also related to the way we identify Tmax. A potential dynamical reason for the The in-front wind speed increase has a maximum in the middle of the front, conﬁrming that the interval between tmax and tmin catches the front characterized by a vortical over- turning internal circulation. After the frontal passage, the wind speed decreases slightly below the pre-front level. As the cold pools spread into a strong background easterly ﬂow (mean wind direction at tmax is 86◦, not shown), the wind speed anomalies show that the cold pools on average push forward into the wind until tmax, and backward after tmin. For some cold pools, tmin might thus mark the center of the di- vergent ﬂow and indicate that the parent convection passed over the BCO. The strengthening winds in the front and the slackening winds in the wake are again signiﬁcantly more pronounced for the strongest cold pools, with a maximum of 1.5 m s−1 and a minimum smaller than −0.5 m s−1 in the front and wake compared to the value at tmax. https://doi.org/10.5194/acp-21-16609-2021 Atmos. Chem. Phys., 21, 16609–16630, 2021 R. Vogel et al.: Trade cumulus cold-pool climatology R. Vogel et al.: Trade cumulus cold-pool climatology R. Vogel et al.: Trade cumulus cold-pool climatology 16619 Figure 4. (a–f) Same as Fig. 3 but for (a) cloud-top height, (b) cloud-base height, (c) total cloud cover, and the contribution to total cloud cover from (d) CCprcp, (e) CClcl, and (f) CCaloft. Also indicated is the climatological mean value for the winter periods of 2012–2020. (g–i) Composite mean temporal structure of vertical hydrometeor fraction (HF) proﬁles for all noprevWI cold pools as well as the 25 % strongest and weakest. The thin dashed line at 600 m height marks the typical base of the cumulus layer. 16619 16619 Figure 4. (a–f) Same as Fig. 3 but for (a) cloud-top height, (b) cloud-base height, (c) total cloud cover, and the contribution to total cloud cover from (d) CCprcp, (e) CClcl, and (f) CCaloft. Also indicated is the climatological mean value for the winter periods of 2012–2020. (g–i) Composite mean temporal structure of vertical hydrometeor fraction (HF) proﬁles for all noprevWI cold pools as well as the 25 % strongest and weakest. The thin dashed line at 600 m height marks the typical base of the cumulus layer. They show that cold-pool periods are much cloudier than the average winter conditions at the BCO, with the average in- front CC being twice as large as the 10-year climatological mean. Cold-pool periods also have much deeper clouds than the climatological mean of about 2 km, which is expected as it needs deeper precipitating clouds to form cold pools. The enhanced CC in the wake of cold pools compared to the long-term mean is nevertheless surprising, as convection might be expected to be suppressed in the cold-pool wake. Mesoscale arcs encircling vast decks of deeper cumuli with stratiform layers therefore seem more representative for pe- riods of cold-pool activity than the more classical picture of trade cumulus cold pools as mesoscale arcs enclosing broad clear-sky areas. pool characteristics cannot be disentangled. Some informa- tion about different cloud types populating the cold-pool front or wake can be derived by analysing the CC contri- butions grouped by the overall CBH of the segmented cloud objects (CBHID; see Appendix A and Sect. 2.1). We ﬁnd that the peak in CClcl at tmax is mainly due to edges of precipitat- ing clouds that have CBH > 300 m. R. Vogel et al.: Trade cumulus cold-pool climatology CClcl and CCaloft are lower at the end of the front for the strongest cold pools, as the lowest CBH is mostly below 300 m and the cloud segments thus count to the CCprcp category. (Note that a given time can only count to one of the three categories.) As already shown in Table 1, Fig. 3 shows that the strongest cold pools are also the driest and the rainiest, and have the strongest wind and vertical velocity anomalies in the front. The relationships and timings discussed are mostly the same when considering all cold pools meeting the no- prev criterion (i.e. also including summer periods), just with larger anomalies and the differences mentioned above for the humidity structure. The mean temporal structure for all vari- ables – except for the speciﬁc humidity and partly for the wind speed – is also similar to previous observations of trop- ical deep convective cold pools during the DYNAMO ﬁeld campaign (de Szoeke et al., 2017; Chandra et al., 2018), just with slightly larger mean across-front temperature and humidity decreases (−1.3 K and −0.6 g kg−1 during DY- NAMO compared to −1.15 K and −0.25 g kg−1 at the BCO) and larger mid-front wind speed increases (about 1.5 m s−1 compared to 1 m s−1) during DYNAMO due to the deeper convection. Furthermore, during DYNAMO the increases in speciﬁc humidity at the beginning of the front are hardly present, and the wind speed remains elevated by 0.4 m s−1 in the wake of the DYNAMO cold pools (de Szoeke et al., 2017), whereas at the BCO the wind speed decreases below Overall, Fig. 4 indicates that clouds and rain are nearer tmin in the strongest cold pools and nearer tmax for the weak- est cold pools. A potential explanation for this observation is that stronger cold pools are running further ahead of their stronger parent convection, while the weaker parent convec- tion of the weaker cold pools might have already dissipated. However, drawing such conclusions from single-point ob- servations is tricky, as the inﬂuence of the life-cycle stage and the overall cold-pool strength on the observed cold- https://doi.org/10.5194/acp-21-16609-2021 Atmos. Chem. Phys., 21, 16609–16630, 2021 R. Vogel et al.: Trade cumulus cold-pool climatology Assuming that this cloud population represents the clouds evident as mesoscale arcs in satellite imagery, this suggests that the cloudiness at the gust front is mostly characterized by well-developed precip- itating clouds. The cloud-type analysis also shows that more than half of the CCaloft in the cold-pool wake is part of large precipitating clouds and is not from detached stratiform lay- ers. This is also suggested by the time–height plots of the composite-mean hydrometeor fraction shown in Fig. 4g–i. These panels nicely summarize what was discussed in the previous paragraphs and again highlight the differences be- tween the 25 % strongest and weakest cold pools in terms of the cloud response. They show that cold-pool periods are much cloudier than the average winter conditions at the BCO, with the average in- front CC being twice as large as the 10-year climatological mean. Cold-pool periods also have much deeper clouds than the climatological mean of about 2 km, which is expected as it needs deeper precipitating clouds to form cold pools. The enhanced CC in the wake of cold pools compared to the long-term mean is nevertheless surprising, as convection might be expected to be suppressed in the cold-pool wake. Mesoscale arcs encircling vast decks of deeper cumuli with stratiform layers therefore seem more representative for pe- riods of cold-pool activity than the more classical picture of trade cumulus cold pools as mesoscale arcs enclosing broad clear-sky areas. pool characteristics cannot be disentangled. Some informa- tion about different cloud types populating the cold-pool front or wake can be derived by analysing the CC contri- butions grouped by the overall CBH of the segmented cloud objects (CBHID; see Appendix A and Sect. 2.1). We ﬁnd that the peak in CClcl at tmax is mainly due to edges of precipitat- ing clouds that have CBH > 300 m. Assuming that this cloud population represents the clouds evident as mesoscale arcs in satellite imagery, this suggests that the cloudiness at the gust front is mostly characterized by well-developed precip- itating clouds. The cloud-type analysis also shows that more than half of the CCaloft in the cold-pool wake is part of large precipitating clouds and is not from detached stratiform lay- ers. This is also suggested by the time–height plots of the composite-mean hydrometeor fraction shown in Fig. 4g–i. R. Vogel et al.: Trade cumulus cold-pool climatology These panels nicely summarize what was discussed in the previous paragraphs and again highlight the differences be- tween the 25 % strongest and weakest cold pools in terms of the cloud response. Despite the various signiﬁcant differences between the strongest and weakest cold pools highlighted in the previ- ous paragraphs, there is a lot of variability among individ- ual cold pools. The variability is illustrated in Fig. 5, which shows the temporal structure of the most important vari- ables for individual cold pools ranked according to their 1T . Especially the individual differences in humidity and wind Figure 4a–f also indicate the respective mean CTH, CBH, and CC for all the winter months of the period 2012–2021. https://doi.org/10.5194/acp-21-16609-2021 Atmos. Chem. Phys., 21, 16609–16630, 2021 R. Vogel et al.: Trade cumulus cold-pool climatology 16620 5. Temporal structure of individual cold pools ranked according to their 1T . Shown are all cold pools of noprevWI that have uments running. The panels show anomalies relative to the cold-pool onset (tmax) for (a) temperature, (b) speciﬁc humidity, and speed as well as absolute values of (d) the MRR rain frequency, (e) the cloud-top height, and (f) the vertical velocity averaged over cloud layer. Figure 5. Temporal structure of individual cold pools ranked according to their 1T . Shown are all cold pools of noprevWI that have all instruments running. The panels show anomalies relative to the cold-pool onset (tmax) for (a) temperature, (b) speciﬁc humidity, and (c) wind speed as well as absolute values of (d) the MRR rain frequency, (e) the cloud-top height, and (f) the vertical velocity averaged over the sub-cloud layer. R. Vogel et al.: Trade cumulus cold-pool climatology 16621 R. Vogel et al.: Trade cumulus cold-pool climatology 16621 Figure 6. Daily cycles of important cold-pool diagnostics. (a) Mean ± 1 SE of hourly cold-pool frequency as well as the number of cold pools per hour, (b) 1T , (c) 1qmin, (d) 1Umax, (e) CTHmax, (f) MRR RRmean, (g) wminSCL, (h) wmax450, and (i) CCtot, with cold pools associated with a speciﬁc hour according to their tmax. In panels (b)–(i) the lines represent the 25 %, 50 %, and 75 % quartiles of the respective variables and the shading represents the median ± 1 SE. Also indicated in green is the median climatological background diel cycle of 30 min values of (e) maximum CTH, (g) minimum wSCL, (h) maximum w450, and (i) mean CC, shifted by the mean difference of the climatological median compared to the cold-pool median to ease reading. Due to the infrequent rain, the median climatological RRmean is always 0 and omitted in panel (f). Figure 6. Daily cycles of important cold-pool diagnostics. (a) Mean ± 1 SE of hourly cold-pool frequency as well as the number of cold pools per hour, (b) 1T , (c) 1qmin, (d) 1Umax, (e) CTHmax, (f) MRR RRmean, (g) wminSCL, (h) wmax450, and (i) CCtot, with cold pools associated with a speciﬁc hour according to their tmax. In panels (b)–(i) the lines represent the 25 %, 50 %, and 75 % quartiles of the respective variables and the shading represents the median ± 1 SE. Also indicated in green is the median climatological background diel cycle of 30 min values of (e) maximum CTH, (g) minimum wSCL, (h) maximum w450, and (i) mean CC, shifted by the mean difference of the climatological median compared to the cold-pool median to ease reading. Due to the infrequent rain, the median climatological RRmean is always 0 and omitted in panel (f). 2019). The cold-pool occurrence remains strongly elevated between 02:00 and 15:00 LT, with a peak at 14:00 LT. mum temperatures near 05:00 and 12:00 LT, respectively, not shown), but this should not affect the cloudy cold-pool peri- ods much and would contribute to lower 1T in the morning. Other diagnostics like 1qmax and Rint do not show a pro- nounced diel variability (not shown). Also, most cold-pool diagnostics show a pronounced diel variability. 3.3 Diel cycle in the front and beginning of the wake can by far exceed the mean differences among the strongest and weakest cold pools shown in Fig. 3. Notable is again the occurrence of pronounced negative values of the wind speed anomaly af- ter tmin, which suggests that some cold pools push backward into the mean wind. Tendencies of more frequent (and in- tense) rain, deeper clouds, and stronger downdrafts near tmin of the stronger cold pools are nevertheless clearly evident. Especially the downdraft strength seems to be systematically increasing for stronger temperature drops. Besides showing the CTH, Fig. 5e also gives an indication of the CC, again illustrating how cloudy the cold-pool periods are. The long time series also allows study of the variability of the cold-pool frequency and characteristics at the daily timescale. Figure 6 shows the diel variability of cold-pool properties for the noprevWI set. There are clearly fewer cold pools and a lower hourly cold-pool frequency between 16:00 and 22:00 LT compared to the rest of the day. Just before midnight, the cold-pool frequency strongly increases in re- sponse to the nighttime increase in cloud cover, cloud depth, and rain rate (see green lines in panels e–i and Vial et al., Atmos. Chem. Phys., 21, 16609–16630, 2021 https://doi.org/10.5194/acp-21-16609-2021 R. Vogel et al.: Trade cumulus cold-pool climatology 16621 R. Vogel et al.: Trade cumulus cold-pool climatology Atmos. Chem. Phys., 21, 16609–16630, 2021 R. Vogel et al.: Trade cumulus cold-pool climatology 16622 tern labels are available from January 2018 to March 2021, and using the noprevWI criterion leaves 1332 cold pools to be analysed. much larger values (as indicated in the respective legends). For the vertical velocity diagnostics, the amplitude of the diel cycle is also much larger compared to the background clima- tology. The discussion of the 4 example cold-pool days in Fig. 2 already shed some light on the differences in the cold-pool characteristics of the four patterns. For a statistical compari- son, Fig. 7 shows distributions of several cold-pool properties for the different patterns, including the “No” category and the union of the ﬁve categories (“tot”). The most pronounced dif- ference among the patterns lies in the occurrence frequency of cold pools. Most cold pools detected at the BCO pertain to the Gravel pattern, followed by Fish and Flowers (Fig. 7a). As expected, only a few cold pools are detected during Sugar periods. Many cold pools are also associated with the No cat- egory. The period of enhanced cold-pool occurrence between 23:00 and 15:00 LT and its peak at 14:00 LT extends much further into the day compared to the period of enhanced mean background rain rate between about 03:00 and 06:00 LT typ- ical for the trades (Nuijens et al., 2009; Vial et al., 2019), suggesting that cold pools help extend the diel cycle of shal- low convection into the early afternoon. Also, the diel cycle of cloud cover seems to be slightly extended in cold-pool pe- riods, with CCtot decreasing below the diel mean about 4 h later compared to the climatological CC. The likely reason for the extension of convection into the afternoon is that the cold pools are successful in triggering new convection, which can again induce new cold pools that trigger further con- vection. Thereby, cold pools reinforce each other, which is supported by the shorter median interval between subsequent cold pools of 121 min between 07:00 and 14:00 LT compared to 182 min between 22:00 and 04:00 LT. When we look at the fraction of time a cold pool is ob- served in a given pattern (Fig. 7b), the picture changes and the Fish pattern is associated with the largest cold-pool frac- tion (12.8 % of the time), followed by Flowers and Gravel (9.9 % and 7.2 %, respectively). Again, Sugar has clearly the lowest cold-pool fraction (1.6 %). R. Vogel et al.: Trade cumulus cold-pool climatology The cold-pool fraction of the No category in winter is at 6.4 % also substantial. Fig- ure 7b also shows the cold-pool fractions using different se- lection criteria, namely that cold pools attributed to multiple patterns are excluded (“.only”) and that all noprev cold pools from all seasons are used (“all”). The four patterns remain distinct in their cold-pool fractions independent of the cri- teria considered. Only for Sugar (and to a small extent also Flowers) do these criteria change the cold-pool fraction. The No category is particularly sensitive to the inclusion of all seasons, as in summer with more frequent deep convection most cold pools pertain to the No category (not shown). The importance of cold pools in triggering new convection is well established in the literature and can occur through me- chanic lifting or moisture accumulations, both of which are favoured when multiple cold pools collide (Rotunno et al., 1988; Tompkins, 2001; Feng et al., 2015; Torri and Kuang, 2019; Meyer and Haerter, 2020). Rochetin et al. (2021) also found a diel cycle of cold-pool occurrence near Barbados in realistic simulations with the ICON model at 2.5 km grid spacing, however, without the pronounced extension into the afternoon observed at the BCO (see their Fig. 16d). This might be due to gust front vertical velocities being poorly resolved at kilometre-scale resolutions, which leads to too little convective triggering and deﬁcits in rain and convective organization, as simulations over Germany showed (Hirt and Craig, 2021). That Gravel has the largest number of cold pools but only the third largest cold-pool fraction is partly because Gravel is the most frequent pattern at the BCO (a total of 178 d out of the 18 winter months considered, compared to 113 Fish, 78 Flowers, and 72 Sugar days) and partly because Gravel cold pools on average last 6 min shorter than Fish cold pools (Fig. 7c). Fish has the signiﬁcantly longest-lasting cold pools of all patterns, which is tightly linked to frequent long-lasting rain events. Cold pools in the Fish pattern also follow each other most rapidly, with a median of 124 min separating in- dividual cold pool fronts (Fig. 7d). Also for Flowers and Gravel, cold pools follow each other quickly, whereas much more time passes between cold pools for Sugar and No. The same picture emerges when considering the cold-pool length (i.e. R. Vogel et al.: Trade cumulus cold-pool climatology the duration multiplied by the surface wind speed): Fish cold pools are with a median size of 13.8 km slightly larger than Gravel and Flowers cold pools (both about 12.6 km, not shown). Vial et al. (2021) also ﬁnd the diel cycle of trade cumuli to be strongly linked to the diel cycle in the occurrence fre- quency of the mesoscale organization patterns. Whether the cold-pool characteristics and their diel cycles are related to the pattern of mesoscale organization will be discussed in the next section. R. Vogel et al.: Trade cumulus cold-pool climatology During nighttime between about midnight and 04:00 LT, cold pools are associated with signiﬁcantly deeper clouds, stronger mean rain rates, stronger downdrafts and up- drafts, larger CC, and slightly stronger humidity drops and weaker wind gusts compared to daytime cold pools between about 08:00 and 16:00 LT. There is also a hint of slightly stronger 1T during nighttime compared to daytime, but nei- ther in the median nor in the 25 % quartiles is this diel cy- cle signiﬁcant. It is somewhat surprising that we ﬁnd no pronounced diel cycle in 1T , despite the diel cycle of e.g. wminSCL and CTHmax. There is a climatological background diel cycle in temperature of about 1.2 K due to the daytime solar heating of the ground at the BCO (minimum and maxi- The pronounced diel variability in the cold-pool frequency and most diagnostics is not surprising given the distinct diel cycle in trade cumulus cloudiness discussed in detail in Vial et al. (2019) based on realistic high-resolution simulations and both BCO and satellite observations. The diel cycle of trade cumuli is characterized by larger CC and deeper clouds at the end of the night and smaller CC and shallower clouds in the afternoon. This is evident in the background clima- tological diel cycles indicated in Fig. 6e–i. The diel cycles of most cold-pool diagnostics have a similar phase and also amplitude to their background diel cycles but are shifted to https://doi.org/10.5194/acp-21-16609-2021 Atmos. Chem. Phys., 21, 16609–16630, 2021 R. Vogel et al.: Trade cumulus cold-pool climatology R. Vogel et al.: Trade cumulus cold-pool climatology 16623 Figure 7. Distributions of various cold-pool diagnostics conditioned on the organization patterns. (a) Number of cold pools, (b) cold-pool fraction, (c) cold-pool duration, (d) time since tmin of the last cold pool, (e) 1T , (f) 1qmin, (g) 1qmax, (h) 1Umax, (i) MRR Rint, (j) CTHmax, (k) wmax450, and (l) wminSCL. The different symbols in panels (c)–(l) represent the 25 %, 50 %, and 75 % quartiles of the respective variables, the solid lines represent the median ± 1 SE, and the dotted horizontal reference lines show the median of the entire set (“tot”). Besides the cold pools matching the noprevWI criterion, panel (b) also shows the fraction of cold pools for all seasons (“all”) and excluding periods with multiple organization patterns (“WI.only” and “all.only”). Figure 7. Distributions of various cold-pool diagnostics conditioned on the organization patterns. (a) Number of cold pools, (b) cold-pool fraction, (c) cold-pool duration, (d) time since tmin of the last cold pool, (e) 1T , (f) 1qmin, (g) 1qmax, (h) 1Umax, (i) MRR Rint, (j) CTHmax, (k) wmax450, and (l) wminSCL. The different symbols in panels (c)–(l) represent the 25 %, 50 %, and 75 % quartiles of the respective variables, the solid lines represent the median ± 1 SE, and the dotted horizontal reference lines show the median of the entire set (“tot”). Besides the cold pools matching the noprevWI criterion, panel (b) also shows the fraction of cold pools for all seasons (“all”) and excluding periods with multiple organization patterns (“WI.only” and “all.only”). Gravel cold pools are associated with signiﬁcantly larger CTHmax and stronger updrafts compared to the other patterns (Fig. 7j, k). For the humidity and rain diagnostics (Fig. 7f, g, i), the differences between Gravel, Flowers, and Fish cold pools are minor. Sugar cold pools generally have the weakest cold-pool signatures. Contrastingly, the cold pools of the No category show no signiﬁcant differences compared to Gravel, Fish, and Flowers for most statistics. These results are largely insensitive to both an increase in the neural network agree- ment score (i.e. using 0.5 compared to 0.4; see Sect. 2.2) and to the exclusion of multiple patterns (except for Sugar and to some degree also Flowers, whose sample sizes become very small, not shown). whereas the onset of the cold-pool front is much more clearly evident for the Gravel and even more for the Sugar CC (see also the time–height composites in Fig. 4 Relationship of cold-pool characteristics with mesoscale organization patterns In this section we investigate whether the cold-pool frequency and characteristics depend on the pattern of mesoscale cloud organization. For this we condition the cold pools on the organization pattern present at the BCO. As ex- plained in Sect. 2.2, a cold pool is attributed to a pattern if it is present during > 75 % of the cold-pool duration. As mul- tiple patterns can be present at the same time, two (or rarely even three) patterns can be attributed to one cold pool. Pat- Figure 7e–i show the differences in the surface meteorol- ogy, rain, and cloud response associated with cold pools for the different patterns. Fish has the strongest median 1T and the strongest downdrafts of all patterns (Fig. 7e, l) and also a stronger 1Umax compared to Gravel and Flowers (Fig. 7h). Atmos. Chem. Phys., 21, 16609–16630, 2021 https://doi.org/10.5194/acp-21-16609-2021 R. Vogel et al.: Trade cumulus cold-pool climatology Shown are (a) total CC, the contribution to total CC from (b) CClcl and (c) CCaloft, and (d) the CTH. The dotted lines show the mean ± 1 SE. Also indicated on the far left of panels (a)–(d) are the climatological mean values per pattern for the corresponding winter periods of 2018–2021. Panels (e)–(h) show the mean temporal structure of the vertical hydrometeor fraction proﬁles for the four patterns. change from Gravel, followed closely by Flowers and Fish. The two patterns with the largest cold-pool fraction, Fish and Flowers, also have the largest mean cloud object sizes (Bony et al., 2020), suggesting that cloud size and cold-pool occur- rence are positively correlated (Schlemmer and Hohenegger, 2014). a diel cycle of the cold-pool fraction with a peak in the after- noon (Fig. 9c), which is broadly in phase with the occurrence frequency. The diel cycle in the cold-pool fraction might be due to cold pools lasting a while once they are formed, which is supported by the much weaker diel cycles of the cold-pool front fraction (dashed lines in Fig. 9c). Once present, cold pools often trigger new cold pools, as suggested by the 33 % shorter interval between subsequent fronts during daytime compared to nighttime (see discussion in Sect. 3.3). From the present analyses, it is difﬁcult to disentangle causal rela- tionships between the pattern occurrence, cold pools, and the diel cycle. It is also difﬁcult to pin down the evolution from one pattern to another and the role of cold pools therein. As the number of cold pools per pattern and hour is quite low (especially in the case of Flowers), more data are needed to draw robust conclusions on this. As mentioned before, Vial et al. (2021) ﬁnd the diel cycle of trade cumuli to be strongly linked to the diel cycle in the occurrence frequency of the mesoscale organization patterns. Figure 9a shows strong diel variations of the number of cold pools associated with the different patterns. These variations are strongly connected to the diel cycles in the occurrence frequency of the patterns (Fig. 9b and Vial et al., 2021). The maximum number of Gravel cold pools occurs just after mid- night, followed by Flowers around 07:00 LT and Fish cold pools at 10:00 LT. The number of Sugar cold pools is very low throughout the day. R. Vogel et al.: Trade cumulus cold-pool climatology 8e–h). The CC in the wake of Sugar cold pools also decreases most rapidly back to its pre-front value. Fish tends to have the deepest mean CTH associated with the cold-pool periods, closely followed by Gravel and Flowers. Again, the mean CTH of Gravel and Sugar cold pools increase most rapidly in the front compared to the other patterns. For all patterns, the cold-pool periods are characterized by signiﬁcantly deeper clouds and larger CC compared to the pattern average (indicated by the dashed lines on the far left of Fig. 8a–d). Nevertheless, the climatological dif- ferences in CC, CCaloft, and CTH among the different pat- terns (Schulz et al., 2021; Vial et al., 2021; Bony et al., 2020) also remain during cold-pool periods, indicating the robust- ness of the pattern-speciﬁc cloud macrophysical properties. The climatological differences in CTH of the patterns cor- respond very well to their differences in the cold-pool frac- tion (Fig. 7b), with Sugar having clearly the shallowest mean CTH and the lowest cold-pool fraction, separated by a step Figure 8 shows the differences in the temporal structures of cloud properties associated with the cold-pool passages for the four patterns. Fish and Flowers have the largest CC (Fig. 8a) due to larger contributions of CCaloft (Fig. 8c), which are associated with frequent stratiform layers near 1.5–2 km (Fig. 8f, h). CClcl (Fig. 8b) and CCprcp (not shown) instead are fairly similar among the patterns. The CC of Fish cold pools hardly changes across the cold-pool passage, https://doi.org/10.5194/acp-21-16609-2021 Atmos. Chem. Phys., 21, 16609–16630, 2021 16624 R. Vogel et al.: Trade cumulus cold-pool climatology Figure 8. Composite mean temporal structure of the four organization patterns and the No category. Shown are (a) total CC, the contribution to total CC from (b) CClcl and (c) CCaloft, and (d) the CTH. The dotted lines show the mean ± 1 SE. Also indicated on the far left of panels (a)–(d) are the climatological mean values per pattern for the corresponding winter periods of 2018–2021. Panels (e)–(h) show the mean temporal structure of the vertical hydrometeor fraction proﬁles for the four patterns. R. Vogel et al.: Trade cumulus cold-pool climatology R. Vogel et al.: Trade cumulus cold-pool climatology 16624 Figure 8. Composite mean temporal structure of the four organization patterns and the No category. 5 Conclusions This paper presents a long-term climatology of trade cumulus cold pools based on more than 10 years of in situ and ground- based remote sensing data from the Barbados Cloud Obser- vatory (BCO; Stevens et al., 2016). Cold pools are detected by abrupt drops in low-pass-ﬁltered temperature time series, and their associated changes in surface meteorology, cloudi- ness, and sub-cloud layer dynamics are extracted. The cold- pool climatology is combined with a neural network classi- ﬁcation of the four mesoscale organization patterns Sugar, Gravel, Flowers, and Fish (Stevens et al., 2020) based on GOES-16 ABI infrared images (Schulz et al., 2021). To fo- cus on trade cumulus cold pools, most analyses are restricted to the set of 3889 cold pools detected in the dry winter regime from December to April that have no non-recovered cold pool in the hour prior to their onset. The strength of the cold-pool signature depends strongly on the intensity of the temperature drops (1T ). The rain du- ration in the front is the best predictor of 1T and explains 36 % of its variability. We ﬁnd that the minimum vertical ve- locity averaged over the sub-cloud layer and the maximum cloud-top height also distinguish stronger and weaker cold pools very well. Cold pools with stronger 1T are associ- ated with deeper clouds, stronger precipitation, downdrafts, and humidity drops, stronger wind gusts and updrafts at the edge of the front, and larger cloud cover compared to cold pools with weaker 1T . Stronger cold pools also last sig- niﬁcantly longer and follow each other more quickly than weaker cold pools, indicating that they are likely more suc- cessful in triggering new convection than weaker cold pools. We ﬁnd clouds and rain to be nearer to the temperature min- imum in the strongest cold pools, whereas they tend to be nearer the onset of the front in weaker cold pools. This sug- gests that stronger cold pools are running further ahead of their stronger parent convection, while the clouds forming the weaker cold pools might have already dissipated. We ﬁnd cold pools to be ubiquitous in the winter trades – they are present about 7.8 % of the time, and on more than 73 % of days at least one cold pool is detected. R. Vogel et al.: Trade cumulus cold-pool climatology 16625 Figure 9. Daily cycles of (a) number of cold pools, (b) hours of data for the different organization patterns, and (c) hourly fraction in cold pool (solid) and in cold-pool front (dashed). A 5-hourly running mean is applied to smooth the data. The diel means are indicated on the left-hand side of each panel. Figure 9. Daily cycles of (a) number of cold pools, (b) hours of data for the different organization patterns, and (c) hourly fraction in cold pool (solid) and in cold-pool front (dashed). A 5-hourly running mean is applied to smooth the data. The diel means are indicated on the left-hand side of each panel. (Fig. 7e), which might compensate for the opposite expecta- tion due to the diel phasing of CTHmax and wminSCL. cloud-top height increases, cloud-base height decreases (due to the very frequent precipitation), and cloud cover increases with the cold-pool onset. Cloudiness at the gust front is mostly due to cloud segments near the lifting-condensation level that pertain to larger precipitating cloud objects. Simi- larly, cloud segments with bases above 1 km in the cold-pool wake are mostly part of large precipitating clouds and are not from detached stratiform layers. R. Vogel et al.: Trade cumulus cold-pool climatology The pattern-associated diel phasing of the cold-pool num- ber might give a clue about why 1T varies little on the daily timescale (Fig. 6c), although the diel cycle of most cold- pool properties would suggest that 1T should be stronger at night compared to day. The daytime Fish pattern has signiﬁ- cantly stronger 1T compared to the nighttime Gravel pattern Figure 9a suggests that the extension of the diel cycle of convection into the early afternoon due to cold pools may largely be explained by the Fish pattern, together with a substantial contribution of the No category to the peak at 14:00 LT. Despite the strong connection between the diel phasings of Fig. 9a–b, especially the Fish pattern also shows Atmos. Chem. Phys., 21, 16609–16630, 2021 https://doi.org/10.5194/acp-21-16609-2021 R. Vogel et al.: Trade cumulus cold-pool climatology Atmos. Chem. Phys., 21, 16609–16630, 2021 R. Vogel et al.: Trade cumulus cold-pool climatology 16626 This study paves the way for more in-depth analyses of the cold-pool properties and their relation to the environment in the trades. Especially the complex humidity signals deserve a more detailed investigation, also using data from the recent EUREC4A ﬁeld campaign (Stevens et al., 2021) and from re- alistic large-eddy simulations. Together with the vertical ve- locity statistics, the humidity anomalies can help shed light on the triggering of new convection at the cold-pool front. Additional measurements of the mixed-layer depth from ra- diosondes and the Raman or Doppler lidar could help re- ﬁne the cold-pool end deﬁnition, which is only poorly con- strained by the surface temperature data. Such additional data could also provide interesting insight into the cold-pool re- covery process. A systematic matching with satellite imagery would also help collocate the clouds sampled at the BCO with the broader view of the entire cold pool seen from space. cloud cover, slightly stronger humidity drops, and weaker wind gusts associated with nighttime compared to daytime cold pools. The phase of these diel signatures is consistent with their background climatological diel cycle but shifted to much larger values. The diel amplitude of the vertical ve- locity maxima and minima is also greater during cold-pool periods. p In the wet summer regime, cold pools are about 30 % more frequent relative to the average winter regime. Summer cold pools are also associated with signiﬁcantly stronger temper- ature and humidity drops, deeper clouds, and stronger down- drafts – consistent with the frequent deep convection and stronger precipitation of this season (Brueck et al., 2015). On the other hand, the summer cold pools have weaker updrafts and humidity maxima at the beginning of the front, suggest- ing that they might be less effective in triggering new con- vection. While the temporal structures of cold-pool passages for most meteorological variables in both seasons resemble those of previous observations of tropical deep convective cold pools (de Szoeke et al., 2017; Chandra et al., 2018; Zuidema et al., 2017), especially the humidity structure and also the generally larger anomalies render the summer cold pools more similar to the deep convective cold pools from previous studies. Overall, we ﬁnd that the cold-pool periods are about 90 % cloudier relative to the average winter trades. The larger cloudiness is mostly due to larger cloud cover from precipi- tating and stratiform cloud segments. R. Vogel et al.: Trade cumulus cold-pool climatology Also, the wake of cold pools is characterized by above-average cloudiness, indicat- ing that the classical image of trade cumulus cold pools as mesoscale arcs enclosing broad clear-sky areas is rather the exception than the rule. Our study suggests that a better un- derstanding of how trade cumulus cold pools interact with and shape their environment is important for understanding the variability in cloud cover and cloud organization in the trade-wind regime. We also analysed whether the cold-pool frequency and characteristics depend on the pattern of mesoscale cloud or- ganization. The most pronounced difference among the pat- terns lies in the occurrence frequency of cold pools, with Fish having the largest cold-pool fraction (12.8 % of the time), followed by Flowers and Gravel (9.9 % and 7.2 %, respec- tively). As expected, the cold-pool fraction of Sugar is neg- ligible (1.6 %). Fish cold pools last signiﬁcantly longer than cold pools from all the other patterns, and they are also asso- ciated with the strongest temperature drops and downdrafts. Gravel cold pools are associated with the strongest updrafts at the cold-pool onset and the deepest cloud-top height max- ima. 5 Conclusions The aver- age cold-pool passage is characterized by a 0.9 K tempera- ture drop, a 0.2 g kg−1 humidity increase just before and an- other 0.2 g kg−1 humidity increase right after the front onset, followed by a −0.4 g kg−1 humidity decrease at the end of the front, wind speed and pressure increases of 1.15 m s−1 and 9 Pa, and rain intensities of 0.9 mm h−1. The vertical velocity at the sub-cloud layer top shows pronounced max- ima of 1 m s−1 near the cold-pool onset, which lies in the upper tail of cloud-base averaged updraft velocities at the BCO (Sakradzija and Klingebiel, 2020) and thus is very relevant for triggering new convection. The second half of the front is marked by sub-cloud layer averaged downdrafts of −0.55 m s−1. Strong signals of cold-pool passages are also found for all cloud macrophysical properties analysed: The cold-pool frequency and characteristics show pro- nounced diel variability. There are signiﬁcantly fewer cold pools and a lower cold-pool frequency between 16:00 and 22:00 LT compared to the rest of the day. We ﬁnd that cold pools extend the diel cycle of convection into the early after- noon, with a peak in both the cold-pool number and fraction at 14:00 LT. Also, most cold-pool diagnostics show a pro- nounced diel cycle, with signiﬁcantly deeper clouds, stronger mean rain rates, stronger downdrafts and updrafts, larger https://doi.org/10.5194/acp-21-16609-2021 Atmos. Chem. Phys., 21, 16609–16630, 2021 https://doi.org/10.5194/acp-21-16609-2021 R. Vogel et al.: Trade cumulus cold-pool climatology 16627 Figure A1. Same as Fig. 3 but for (a) CCID.tot, (b) CCID.prcp, (c) CCID.lcl, and (d) CCID.aloft for all cold pools of noprevWI and the 25 % strongest and weakest cold pools. Figure A1. Same as Fig. 3 but for (a) CCID.tot, (b) CCID.prcp, (c) CCID.lcl, and (d) CCID.aloft for all cold pools of noprevWI and the 25 % strongest and weakest cold pools. Figure A1. Same as Fig. 3 but for (a) CCID.tot, (b) CCID.prcp, (c) CCID.lcl, and (d) CCID.aloft for all cold pools of noprevWI and the 25 % strongest and weakest cold pools. discussed in Sect. 3. However, wmax450 is signiﬁcantly lower by 0.2 m s−1 and 1qmax by 0.1 g kg−1 in summer compared to winter, indicating that cold pools in summer might be less successful in triggering new convection. Furthermore, CCtot of summer cold pools is also signiﬁcantly smaller com- pared to winter cold pools by about 10 %. The differences in the cold-pool characteristics between the summer and win- ter regimes are not surprising, as the summer regime is re- ferred to as the wet season in Barbados and characterized by frequent deep convection and much larger precipitation (Brueck et al., 2015). When excluding periods of deep con- vection (deﬁned by the presence of a radar signal between 4.5 and 8 km), the number of cold pools detected in summer strongly decreases compared to winter, and the median sum- mer cold pool also becomes weaker compared to the median winter cold pool (not shown). pool sets shown. For the 25 % strongest cold pools, the end of the front is entirely covered by precipitating clouds. CCID.lcl in Fig. A1c for all sets is relatively stable at about 17.5 % before the cold-pool onset, decreases abruptly after tmax to a minimum near tmin, and then slowly recovers back to the pre-front value. CCID.lcl shows the strongest impact when the cloud objects are considered through the CBHID and thus the strongest difference to the structure of CClcl (Fig. 4e). The absence of a peak in CCID.lcl near tmax indicates that the CClcl peak there is almost entirely due to edges of precipitat- ing clouds with a CBH > 300 m and not due to (not-yet or) non-precipitating trade cumuli. The temporal structure of CCID.aloft resembles the struc- ture of CCaloft (Fig. R. Vogel et al.: Trade cumulus cold-pool climatology 4f) yet with substantially lower coverage, as most cloud segments with CBH > 1 km are connected to a precipitating core. This shows that nearly half of the CCaloft in the cold-pool wake is part of large precipitating clouds and not from detached stratiform layers. Code and data availability. The BCO data used in the analysis and other Supplement information that may be useful for reproduc- ing the present study are available from the ﬁrst author on re- quest. The GOES-16 ABI data are publicly available online at https://doi.org/10.7289/V5BV7DSR (GOES-R Calibration Work- ing Group and GOES-R Series Program, 2017). The satellite im- ages in Fig. 2 are retrieved from the imagery of the Earth Ob- serving System Data and Information System (EOSDIS) World- view application (https://wvs.earthdata.nasa.gov, NASA, 2021) and from the NASA ATOMIC-EUREC4A GOES-16 ABI im- agery (https://satcorps.larc.nasa.gov/cgi-bin/site/showdoc?docid= 22&lkdomain=Y&domain=FEXP-ATOMIC-SATIMG, NASA Sat- CORPS group, 2021). Appendix A: Cloud cover contributions from different types of cloud objects The contributions to total cloud cover from clouds at dif- ferent height levels can either be computed by classify- ing every radar proﬁle independently based on its CBH (see Fig. 4d–f) or – if a cloud segmentation mask is avail- able – by classifying the entire cloud objects according to their CBHID (i.e. their overall lowest CBH). As both ap- proaches can provide valuable insights, Fig. A1 also shows the temporal structure of the cold-pool signatures for the latter classiﬁcation method. For this, the cloud cover is again split up into contributions from precipitating clouds with CBHID ≤300 m (CCID.prcp), LCL clouds (CCID.lcl; 300 m < CBHID ≤1 km), and stratiform clouds (CCID.aloft; 1 km < CBHID ≤4 km). The difference between CCID.prcp and CCprcp is that edges or slanted sides of precipitating clouds that have a CBH > 300 m are counted in their entirety to the CCID.prcp category, while they would be counted in the CClcl or CCaloft category if the cloud ID was not considered. Due to the potential presence of cloud objects at different heights, the sum of the three height categories (CCID.tot) can be larger than 1. Given the distinct diel cycle in the occurrence frequency of the four patterns found in Vial et al. (2021), it is not sur- prising that we ﬁnd strong diel variations of the number of cold pools associated with the different patterns. The maxi- mum number of Gravel cold pools occurs around midnight, followed by Flowers around 07:00 LT and Fish cold pools around 10:00 LT, in line with the diel cycles in the occurrence frequency of the patterns. The Gravel, Flowers, and Fish cold pools can thus explain a large fraction of the diel cycle in the cold-pool occurrence as well as their extension into the early afternoon. Note also that the unclassiﬁed cold pools have a non-negligible contribution to the peak at 14:00 LT. Interest- ingly, the climatological differences in the cloud cover and cloud-top height among the different patterns are also present during cold-pool periods – the overall cloud cover and cloud- top height for all patterns are just enhanced compared to their respective climatological values. CCID.prcp already starts to increase before tmax and contin- ues to increase until the middle of the front for all the cold- Atmos. Chem. Phys., 21, 16609–16630, 2021 https://doi.org/10.5194/acp-21-16609-2021 R. Vogel et al.: Trade cumulus cold-pool climatology R. Appendix A: Cloud cover contributions from different types of cloud objects Vogel et al.: Trade cumulus cold-pool climatology R. Vogel et al.: Trade cumulus cold-pool climatology 16628 Figure B1. Monthly and seasonal distribution of important cold-pool diagnostics. (a) Daily cold-pool frequency, (b) 1T , (c) 1qmin, (d) 1qmax, (e) CTHmax, (f) MRR Rint, (g) wminSCL and (h) wmax450, and (i) CCtot. The lines represent the 25 %, 50 %, and 75 % quar- tiles of the respective variables, the shading represents the median ± 1 SE, and the points show the average distribution for the 5 winter (w; December–April) and summer months (s; July–November). Figure B1. Monthly and seasonal distribution of important cold-pool diagnostics. (a) Daily cold-pool frequency, (b) 1T , (c) 1qmin, (d) 1qmax, (e) CTHmax, (f) MRR Rint, (g) wminSCL and (h) wmax450, and (i) CCtot. The lines represent the 25 %, 50 %, and 75 % quar- tiles of the respective variables, the shading represents the median ± 1 SE, and the points show the average distribution for the 5 winter (w; December–April) and summer months (s; July–November). Competing interests. The contact author has declared that neither they nor their co-authors have any competing interests. Financial support. This research has been supported by the Euro- pean Research Council (ERC) under the European Union’s Hori- zon 2020 research and innovation programme (grant no. 694768) for Raphaela Vogel, by the German Research Foundation DFG HALO Priority Program (grant no. HALO SPP 1294) for Heike Konow, and by the NOAA Climate Program Ofﬁce award (grant no. NA19OAR4310379) for Paquita Zuidema. Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Review statement. This paper was edited by Farahnaz Khosrawi and reviewed by three anonymous referees. Acknowledgements. Many thanks go to the Tropical Cloud Obser- vation group at MPI for maintaining the BCO infrastructure. We thank Nicolas Rochetin, Ludovic Touzé-Peiffer, Cathy Hohenegger, Bjorn Stevens, and the EUREC4A science team at LMD and MPI for interesting discussions. Raphaela Vogel thanks Louise Nuijens for motivation to look at cold pools at the end of the PhD thesis. We thank the editor Farahnaz Khosrawi and three anonymous referees for their helpful comments. Appendix B: Seasonal cycle of cold-pool characteristics While this study focuses on the cold-pool climatology of the winter regime, it is also interesting to look at the seasonal cycle of the cold-pool characteristics at the BCO. Using all cold pools of the noprev category, we ﬁnd the largest median daily cold-pool frequency in the summer months from July to November and another peak in January (Fig. B1a). Only 13 % of days have no cold pool at all in summer, compared to 27 % in winter. The same monthly variability is found for the cold-pool front frequency but with 45 % lower values due to the shorter duration of the front compared to the entire cold pool (not shown). Author contributions. RV initiated the project in exchange with PZ. HK prepared the radar cloud mask and HS the neural network clas- siﬁcations of the organization patterns. RV developed the cold- pool detection algorithm, performed the analyses, and wrote the manuscript. All the co-authors contributed to the interpretation of the results and commented on the manuscript. Author contributions. RV initiated the project in exchange with PZ. HK prepared the radar cloud mask and HS the neural network clas- siﬁcations of the organization patterns. RV developed the cold- pool detection algorithm, performed the analyses, and wrote the manuscript. All the co-authors contributed to the interpretation of the results and commented on the manuscript. Figure B1b–i show the monthly distributions of various cold-pool properties as well as averages over the 5 win- ter and summer months, respectively. They show that the summer cold pools are on average characterized by signiﬁ- cantly stronger 1qmin, CTHmax, and Rint as well as slightly stronger 1T and wminSCL, consistent with the relationships https://doi.org/10.5194/acp-21-16609-2021 Atmos. Chem. Phys., 21, 16609–16630, 2021 R. Vogel et al.: Trade cumulus cold-pool climatology References Aemisegger, F., Vogel, R., Graf, P., Dahinden, F., Villiger, L., Jansen, F., Bony, S., Stevens, B., and Wernli, H.: How Rossby wave breaking modulates the water cycle in the North At- lantic trade wind region, Weather Clim. Dynam., 2, 281–309, https://doi.org/10.5194/wcd-2-281-2021, 2021. Atmos. Chem. Phys., 21, 16609–16630, 2021 Atmos. Chem. Phys., 21, 16609–16630, 2021 Atmos. Chem. Phys., 21, 16609–16630, 2021 R. Vogel et al.: Trade cumulus cold-pool climatology 16629 Konow, H.: BCO Cloudmask Code, Zenodo [code], https://doi.org/10.5281/zenodo.4312818, 2020. Bony, S. and Dufresne, J.-L.: Marine boundary layer clouds at the heart of tropical cloud feedback uncertainties in climate models, Geophys. Res. Lett., 32, L20806, https://doi.org/10.1029/2005GL023851, 2005. Kubar, T. L., Hartmann, D. L., and Wood, R.: Understanding the Importance of Microphysics and Macrophysics for Warm Rain in Marine Low Clouds. Part I: Satellite Observations, J. Atmos. Sci., 66, 2953–2972, https://doi.org/10.1175/2009JAS3071.1, 2009. p g Bony, S., Stevens, B., Ament, F., Bigorre, S., Chazette, P., Crewell, S., Delanoë, J., Emanuel, K., Farrell, D., Flamant, C., Gross, S., Hirsch, L., Karstensen, J., Mayer, B., Nuijens, L., Ruppert, J. H., Sandu, I., Siebesma, P., Speich, S., Szczap, F., Totems, J., Vogel, R., Wendisch, M., and Wirth, M.: EUREC4A: A Field Campaign to Elucidate the Couplings Between Clouds, Convection and Circulation, Surv. Geophys., 38, 1529–1568, https://doi.org/10.1007/s10712-017-9428-0, 2017. S., Hirsch, L., Karstensen, J., Mayer, B., Nuijens, L., Ruppert, Langhans, W. and Romps, D. M.: The origin of water vapor rings in tropical oceanic cold pools, Geophys. Res. Lett., 42, 7825–7834, 2015. J. H., Sandu, I., Siebesma, P., Speich, S., Szczap, F., Totems, Lin, T., Goyal, P., Girshick, R. B., He, K., and Dollár, P.: Fo- cal Loss for Dense Object Detection, CoRR, arXiv [preprint], arXiv:1708.02002 2017. Bony, S., Schulz, H., Vial, J., and Stevens, B.: Sugar, Gravel, Fish, and Flowers: Dependence of Mesoscale Patterns of Trade-Wind Clouds on Environmental Conditions, Geophys. Res. Lett., 47, e2019GL085988, https://doi.org/10.1029/2019GL085988, 2020. Medeiros, B. and Nuijens, L.: Clouds at Barbados are representa- tive of clouds across the trade wind regions in observations and climate models, P. Natl. Acad. Sci. USA, 113, E3062–E3070, https://doi.org/10.1073/pnas.1521494113, 2016. Brueck, M., Nuijens, L., and Stevens, B.: On the seasonal and syn- optic time-scale variability of the north atlantic trade wind re- gion and its low-level clouds, J. Atmos. Sci., 72, 1428–1446, https://doi.org/10.1175/JAS-D-14-0054.1, 2015. Meyer, B. and Haerter, J. O.: Mechanical Forcing of Con- vection by Cold Pools: Collisions and Energy Scal- ing, J. Adv. Model. Earth Sy., 12, e2020MS002281, https://doi.org/10.1029/2020MS002281, 2020. Byers, H. R. and Hall, R. K.: A census of cumulus-cloud height versus precipitation in the vicinity of Puerto Rico during the winter and spring of 1953–1954, J. Atmos. Sci., 12, 176–178, https://doi.org/10.1175/1520- 0469(1955)012<0176:ACOCCH>2.0.CO;2, 1955. NASA: Worldview Application, available at: https://worldview. earthdata.nasa.gov, last access: 21 March 2021. R. Vogel et al.: Trade cumulus cold-pool climatology NASA SatCORPS group, 2021: NASA Langley Sat- CORP support for ATOMIC-EUREC4A, available at: https://satcorps.larc.nasa.gov/cgi-bin/site/showdoc?docid= 22&lkdomain=Y&domain=FEXP-ATOMIC-SATIMG, last access: 21 March 2021. Chandra, A. S., Zuidema, P., Krueger, S., Kochanski, A., de Szoeke, S. P., and Zhang, J.: Moisture Distributions in Tropical Cold Pools From Equatorial Indian Ocean Observations and Cloud- Resolving Simulations, J. Geophys. Res.-Atmos., 123, 11445– 11465, https://doi.org/10.1029/2018JD028634, 2018. Nuijens, L., Stevens, B., and Siebesma, A. P.: The environment of precipitating shallow cumulus convection, J. Atmos. Sci., 66, 1962–1979, 2009. de Szoeke, S. P., Skyllingstad, E. D., Zuidema, P., and Chan- dra, A. S.: Cold Pools and Their Inﬂuence on the Tropi- cal Marine Boundary Layer, J. Atmos. Sci., 74, 1149–1168, https://doi.org/10.1175/JAS-D-16-0264.1, 2017. Nuijens, L., Serikov, I., Hirsch, L., Lonitz, K., and Stevens, B.: The distribution and variability of low-level cloud in the North Atlantic trades, Q. J. Roy. Meteor. Soc., 140, 2364–2374, https://doi.org/10.1002/qj.2307, 2014. Feng, Z., Hagos, S., Rowe, A. K., Burleyson, C. D., Martini, M. N., and de Szoeke, S. P.: Mechanisms of convective cloud orga- nization by cold pools over tropical warm ocean during the AMIE/DYNAMO ﬁeld campaign, J. Adv. Model. Earth Sy., 7, 357–381, https://doi.org/10.1002/2014MS000384, 2015. Päschke, E., Leinweber, R., and Lehmann, V.: An assessment of the performance of a 1.5 µm Doppler lidar for operational vertical wind proﬁling based on a 1-year trial, Atmos. Meas. Tech., 8, 2251–2266, https://doi.org/10.5194/amt-8-2251-2015, 2015. Glassmeier, F. and Feingold, G.: Network approach to patterns in stratocumulus clouds, P. Natl. Acad. Sci. USA, 114, 10578– 10583, https://doi.org/10.1073/pnas.1706495114, 2017. Rasp, S., Schulz, H., Bony, S., and Stevens, B.: Combining Crowd- sourcing and Deep Learning to Explore the Mesoscale Organiza- tion of Shallow Convection, B. Am. Meteorol. Soc., 101, E1980– E1995, https://doi.org/10.1175/BAMS-D-19-0324.1, 2020. GOES-R Calibration Working Group and GOES-R Series Program: NOAA GOES-R Series Advanced Baseline Imager (ABI) Level 1b Radiances, NOAA National Centers for Environmental Infor- mation [code], https://doi.org/10.7289/V5BV7DSR, 2017. Rochetin, N., Hohenegger, C., Touzè-Peiffer, L., and Ville- franque, N.: A Physically Based Deﬁnition of Convectively Generated Density Currents: Detection and Characterization in Convection-Permitting Simulations, J. Adv. Model. Earth Sy., 13, e2020MS002402, https://doi.org/10.1029/2020MS002402, 2021. Hirt, M. and Craig, G. C.: A cold pool perturbation scheme to improve convective initiation in convection- permitting models, Q. J. Roy. Meteor.Soc., 147, 2429–2447, https://doi.org/10.1002/qj.4032, 2021. Rotunno, R., Klemp, J. B., and Weisman, M. L.: A Theory for Strong, Long-Lived Squall Lines, J. At- mos. Sci., 45, 463–485, https://doi.org/10.1175/1520- 0469(1988)045<0463:ATFSLL>2.0.CO;2, 1988. Klingebiel, M., Ghate, V. https://doi.org/10.5194/acp-21-16609-2021 Atmos. Chem. Phys., 21, 16609–16630, 2021 https://doi.org/10.5194/acp-21-16609-2021 https://doi.org/10.5194/acp-21-16609-2021 R. Vogel et al.: Trade cumulus cold-pool climatology 16630 muli by isolating the main controlling factors of the mass ﬂux distribution, Q. J. Roy. Meteor. Soc., 146, 254–266, https://doi.org/10.1002/qj.3671, 2020. Touzè-Peiffer, L., Vogel, R., and Rochetin, N.: Detecting cold pools from soundings during EUREC4A, arXiv [preprint], arXiv:2104.09146, 2021. Vial, J., Dufresne, J. L., and Bony, S.: On the interpretation of inter- model spread in CMIP5 climate sensitivity estimates, Clim. Dy- nam., 41, 3339–3362, https://doi.org/10.1007/s00382-013-1725- 9, 2013. Schlemmer, L. and Hohenegger, C.: The Formation of Wider and Deeper Clouds as a Result of Cold-Pool Dynamics, J. At- mos. Sci., 71, 2842–2858, https://doi.org/10.1175/JAS-D-13- 0170.1, 2014. Schlemmer, L. and Hohenegger, C.: Modiﬁcations of the atmo- spheric moisture ﬁeld as a result of cold-pool dynamics, Q. J. Roy. Meteor. Soc., 142, 30–42, https://doi.org/10.1002/qj.2625, 2016. Vial, J., Vogel, R., Bony, S., Stevens, B., Winker, D. M., Cai, X., Ho- henegger, C., Naumann, A. K., and Brogniez, H.: A New Look at the Daily Cycle of Trade Wind Cumuli, J. Adv. Model. Earth Sy., 11, 3148–3166, https://doi.org/10.1029/2019MS001746, 2019. Schulz, H., Eastman, R., and Stevens, B.: Characterization and Evolution of Organized Shallow Convection in the Down- stream North Atlantic Trades, J. Geophys. Res.-Atmos., 126, e2021JD034575, https://doi.org/10.1029/2021JD034575, 2021. Vial, J., Vogel, R., and Schulz, H.: On the daily cycle of mesoscale cloud organization in the winter trades, Q. J. Roy. Meteor. Soc., 147, 2850–2873, https://doi.org/10.1002/qj.4103, 2021. Vogel, R.: The inﬂuence of precipitation and convective orga- nization on the structure of the trades (Doctoral disserta- tion, Universität Hamburg), Berichte zur Erdsystemforschung, https://doi.org/10.17617/2.2503092, 2017. Stevens, B., Farrell, D., Hirsch, L., Jansen, F., Nuijens, L., Serikov, I., Brügmann, B., Forde, M., Linne, H., Lonitz, K., and Prospero, J. M.: The Barbados Cloud Observatory: Anchoring Investiga- tions of Clouds and Circulation on the Edge of the ITCZ, B. Am. Meteorol. Soc., 97, 787–801, https://doi.org/10.1175/BAMS-D- 14-00247.1, 2016. Wang, H. and Feingold, G.: Modeling Mesoscale Cel- lular Structures and Drizzle in Marine Stratocumulus. Part I: Impact of Drizzle on the Formation and Evo- lution of Open Cells, J. Atmos. Sci., 66, 3237–3256, https://doi.org/10.1175/2009JAS3022.1, 2009. Stevens, B., Satoh, M., Auger, L., Biercamp, J., Bretherton, C. S., Chen, X., Düben, P., Judt, F., Khairoutdinov, M., Klocke, D., Kodama, C., Kornblueh, L., Lin, S.-J., Neumann, P., Putman, W. M., Röber, N., Shibuya, R., Vanniere, B., Vidale, P. R. Vogel et al.: Trade cumulus cold-pool climatology P., Naumann, A. K., Ditas, F., Pöhlker, M. L., Pöhlker, C., Kandler, K., Konow, H., and Stevens, B.: Re- mote Sensing of Sea Salt Aerosol below Trade Wind Clouds, J. Atmos. Sci., 76, 1189–1202, https://doi.org/10.1175/JAS-D-18- 0139.1, 2019. Rowe, A. K. and Houze Jr., R. A.: Cloud organization and growth during the transition from suppressed to active MJO conditions, J. Geophys. Res.-Atmos., 120, 10324–10350, https://doi.org/10.1002/2014JD022948, 2015. Klingebiel, M., Konow, H., and Stevens, B.: Measuring shallow convective mass ﬂux proﬁles in the trade wind region, J. At- mos. Sci., 78, 3205–3214, https://doi.org/10.1175/JAS-D-20- 0347.1, 2021. Sakradzija, M. and Klingebiel, M.: Comparing ground-based observations and a large-eddy simulation of shallow cu- https://doi.org/10.5194/acp-21-16609-2021 Atmos. Chem. Phys., 21, 16609–16630, 2021 R. Vogel et al.: Trade cumulus cold-pool climatology L., Wedi, N., and Zhou, L.: DYAMOND: the DYnamics of the Atmospheric general circulation Modeled On Non-hydrostatic Domains, Progress in Earth and Planetary Science, 6, 61 pp., https://doi.org/10.1186/s40645-019-0304-z, 2019. Wilbanks, M. C., Yuter, S. E., De Szoeke, S. P., Brewer, W. A., Miller, M. A., Hall, A. M., and Burleyson, C. D.: Near-Surface Density Currents Observed in the Southeast Paciﬁc Stratocumulus-Topped Marine Boundary Layer, Mon. Weather Rev., 143, 3532–3555, 2015. Xue, H., Feingold, G., and Stevens, B.: Aerosol effects on clouds, precipitation, and the organization of shal- low cumulus convection, J. Atmos. Sci., 65, 392–406, https://doi.org/10.1175/2007JAS2428.1, 2008. Stevens, B., Bony, S., Brogniez, H., Hentgen, L., Hohenegger, C., Kiemle, C., L’Ecuyer, T. S., Naumann, A. K., Schulz, H., Siebesma, P. A., Vial, J., Winker, D. M., and Zuidema, P.: Sugar, gravel, ﬁsh and ﬂowers: Mesoscale cloud patterns in the trade winds, Q. J. Roy. Meteor. Soc., 146, 141–152, https://doi.org/10.1002/qj.3662, 2020. Young, G. S., Perugini, S. M., and Fairall, C. W.: Convec- tive Wakes in the Equatorial Western Paciﬁc during TOGA, Mon. Weather Rev., 123, 110–123, https://doi.org/10.1175/1520- 0493(1995)123<0110:CWITEW>2.0.CO;2, 1995. Stevens, B., Bony, S., Farrell, D., et al.: EUREC4A, Earth Syst. Sci. Data, 13, 4067–4119, https://doi.org/10.5194/essd-13-4067- 2021, 2021. Zhu, Z., Kollias, P., Yang, F., and Luke, E.: On the Es- timation of In-Cloud Vertical Air Motion Using Radar Doppler Spectra, Geophys. Res. Lett., 48, e2020GL090682, https://doi.org/10.1029/2020GL090682, 2021. Tompkins, A. M.: Organization of Tropical Convection in Low Vertical Wind Shears: The Role of Cold Pools, J. Atmos. Sci., 58, 1650–1672, https://doi.org/10.1175/1520- 0469(2001)058<0529:OOTCIL>2.0.CO;2, 2001. Zuidema, P., Li, Z., Hill, R. J., Bariteau, L., Rilling, B., Fairall, C., Brewer, W. A., Albrecht, B., and Hare, J.: On Trade Wind Cumulus Cold Pools, J. Atmos. Sci., 69, 258–280, https://doi.org/10.1175/JAS-D-11-0143.1, 2012. Torri, G.: On the Isotopic Composition of Cold Pools in Radiative-Convective Equilibrium, J. Geophys. Res.-Atmos., 126, e2020JD033139, https://doi.org/10.1029/2020JD033139, 2021. Zuidema, P., Torri, G., Muller, C., and Chandra, A.: A Survey of Precipitation-Induced Atmospheric Cold Pools over Oceans and Their Interactions with the Larger-Scale Environment, Surv. Geophys., 38, 1283–1305, https://doi.org/10.1007/s10712-017- 9447-x, 2017. Torri, G. and Kuang, Z.: Rain evaporation and moist patches in trop- ical boundary layers, Geophys. Res. Lett., 43, 9895–9902, 2016. Torri, G. and Kuang, Z.: On Cold Pool Collisions in Tropical Boundary Layers, Geophys. Res. Lett., 46, 399–407, 2019. Atmos. Chem. Phys., 21, 16609–16630, 2021 https://doi.org/10.5194/acp-21-16609-2021
https://openalex.org/W2980539484	https://europepmc.org/articles/pmc6834201?pdf=render	English	null	Disinfection and Sterilization Using Plasma Technology: Fundamentals and Future Perspectives for Biological Applications	International journal of molecular sciences	2,019	cc-by	13,461	Received: 8 October 2019; Accepted: 21 October 2019; Published: 21 October 2019 Abstract: Recent studies have shown that plasma can eﬃciently inactivate microbial pathogens such as bacteria, fungi, and viruses in addition to degrading toxins. Moreover, this technology is eﬀective at inactivating pathogens on the surface of medical and dental devices, as well as agricultural products. The current practical applications of plasma technology range from sterilizing therapeutic medical devices to improving crop yields, as well as the area of food preservation. This review introduces recent advances and future perspectives in plasma technology, especially in applications related to disinfection and sterilization. We also introduce the latest studies, mainly focusing on the potential applications of plasma technology for the inactivation of microorganisms and the degradation of toxins. Keywords: discharge; disinfection; inactivation; sterilization; toxins 1. Introduction Irving Langmuir ﬁrst coined the term ‘plasma’ in 1927 to describe an ionized gas [1]. Early applications of plasma technology mainly focused in the ﬁeld of engineering, such as nuclear fusion and plasma etching [2–4]. However, over the past 20 years, there has been a plethora of patents and scientiﬁc papers describing the microbicidal properties of plasma [5]. Recently accumulated knowledge has led to improvements in the eﬃciency of the disinfection and sterilization using plasma technology and a growing awareness of its potential utility [5–7]. In this review, we summarize the fundamentals of the methods for plasma generation and their application, as well as the eﬃcacy of these disinfection/sterilization methods against various microorganisms. Furthermore, we also discuss the possible future application of this technology in the area of medicine and dentistry as well as agriculture. Akikazu Sakudo 1,, Yoshihito Yagyu 2 and Takashi Onodera 3 Akikazu Sakudo 1,, Yoshihito Yagyu 2 and Takashi Onodera 3 1 Faculty of Veterinary Medicine, Okayama University of Science, Imabari, Ehime 794-8555, Japan 2 Department of Electrical and Electric Engineering, National Institute of Technology Sasebo College, Nagasaki 857-1193, Japan; yyagyu@sasebo.ac.jp 3 Research Center for Food Safety, Graduate School of Agricultural and Life Sciences, the University of Tokyo Bunkyo-ku, Tokyo 113-8657, Japan; takashi.onodera@riken.jp * Correspondence: akikazusakudo@gmail.com 1 Faculty of Veterinary Medicine, Okayama University of Science, Imabari, Ehime 794-8555, Japan 2 Department of Electrical and Electric Engineering, National Institute of Technology Sasebo College, Nagasaki 857-1193, Japan; yyagyu@sasebo.ac.jp 3 Research Center for Food Safety, Graduate School of Agricultural and Life Sciences, the University of Tokyo Bunkyo-ku, Tokyo 113-8657, Japan; takashi.onodera@riken.jp * Correspondence: akikazusakudo@gmail.com International Journal of Molecular Sciences International Journal of Molecular Sciences 2. Fundamentals of Plasma and Methods for Its Generation There are three commonly encountered states of matter: solid, liquid, and gas. When a solid is heated, it transforms into a liquid and then from a liquid into a gas. If enough energy is applied to gas, it becomes an ionized gas known as plasma, which represents the fourth fundamental state of matter [8]. The plasma contains reactive chemical species such as electrons, ions, neutral molecules, and atoms, as well as charged species [9]. In addition, the emission of radiation occurs in the ultraviolet (UV) as well as visible and near-infrared regions during plasma generation. Int. J. Mol. Sci. 2019, 20, 5216; doi:10.3390/ijms20205216 www.mdpi.com/journal/ijms www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, 5216 2 of 17 A state of plasma could be typically classiﬁed according to temperature [10–17] (Table 1). In a high-temperature plasma, which is a strong or fully ionized plasma, the temperature of the electrons Te and ions Tion are the same, so they are in thermal equilibrium with each other by collision due to thermal motion. The gas temperature Tgas of high-temperature plasma and thermal plasma is too extreme for treating living organisms. Alternatively, in non-thermal plasma, comprising partially ionized plasmas, the temperature of the electrons Te is much higher than that of the ions Tion and neutrons Tn. The energy transfer of the kinematics of a collision between electrons (light particles) and ions or neutrals (heavy particles) tends to be very slow by elastic collision, but electron-electron collisions readily achieve thermodynamic equilibrium. Therefore, the ionized gas temperature keeps the normally ambient temperature in non-thermal plasma. As a result, the gas temperature of non-thermal plasma remains low, making it suitable for biological applications. Table 1. Typical classiﬁcation of plasma [10,17]. Classiﬁcation Temperature [K] Electron Density [m−3] Discharge Type Examples High-temperature plasma (Equilibrium plasma) Te ≈Tion ≈Tgas = 106–108 ne ≥1020 Laser fusion Tokamak Fusion plasma for energy Thermal plasma (Quasi-equilibrium plasma) Te ≈Tion ≈Tn ≈ Tgas ≤2 × 104 ne ≥1020 Arc plasma, Plasma torch, Radio-frequency (RF) Plasma, Microwave plasma etc. Radiation, welding and cutting, Waste treatment, Material processing, etc. Non-thermal plasma (Non-equilibrium plasma) Te ≥Tion ≥Tn ≈ Tgas = 300–1000 ne ≈1010 Glow discharge, Corona discharge, atmospheric pressure plasma jet (APPJ), dielectric barrier discharge (DBD), micro-hollow cathode discharge (MHCD), Plasma needle, Low-pressure plasma etc. 2. Fundamentals of Plasma and Methods for Its Generation Ozonizer, Plasma medicine, Volatile organic compound (VOC) treatment, Plasma agriculture, Surface modiﬁcations (coating, etching, activation, cleaning, nitration, etc.), Illumination (plasma screen, ﬂuorescent lamps, etc.) Te = electron temperature, Tion = ion temperature, Tgas = gas temperature, ne = electron density. Table 1. Typical classiﬁcation of plasma [10,17]. Table 1. Typical classiﬁcation of plasma [10,17]. Electrical discharge methods commonly utilized for non-thermal plasma generation in biological applications are generally categorized into one of the following: glow discharge, corona discharge, atmospheric pressure plasma jet (APPJ), dielectric barrier discharge (DBD), micro-hollow cathode discharge (MHCD), DC discharge, pulse discharge, or high/low-frequency discharge (Table 2). The type of discharge depends on the frequency of the power source, such as direct current (DC) and alternating current (AC) discharge, as well as ambient gas pressure, such as low-pressure and atmospheric pressure plasma, and the precise shape and conﬁguration of the electrodes [9]. In addition, the waveform may also aﬀect the type of discharge. Diﬀerent types of plasma can be used in various biological ﬁelds, including disinfection/sterilization. Int. J. Mol. Sci. 2019, 20, 5216 3 of 17 Table 2. Various types of electrical discharge methods for non-thermal plasma generation. Table 2. Various types of electrical discharge methods for non-thermal plasma generation. Discharge Type * Representative Conditions (V, A, Freq, Gas) Pressure Gas Temperature Application References Direct current (DC) corona discharge 5–30 kV direct current (DC) (positive and negative); 10–250 µA; dry or wet; O2, N2, Ar, He at 10 L/min 1 atm Room temperature Biomedical applications [18] Atmospheric pressure plasma jet (APPJ) microwave P = 2.5 W; 2.45 GHz; He/O2/N2 at 2.0/1.2/1.5 L/min 1 atm Max. 50.8 ◦C on a dentin surface; 20 ◦C on an agar surface Biomedical applications [19] Dielectric barrier discharge (DBD) (Flexible sheet-type) ±2.5 kV; 5 kHz; air, humidity 64.4% 1 atm Approximately 50 ◦C Biomedical applications [20] Micro-hollow cathode discharge (MHCD) jet 1.5–2.5 kV DC; 20 mA; air (0.1–8 L/min) >1 atm Room temperature (220 mL/min); >55 ◦C (5 mm from nozzle, 220 mL/min) Medical applications [21] Pin-to-hole spark discharge (PHD) plasma 4 kV DC; average ~1.8 J/pulse 1 atm 9030 ± 320 K (by Boltzmann calculation) Medical applications (wound healing) [22] * These types of electrical discharge are for atmospheric pressure plasmas. Non-thermal plasma is easy to obtain under low-pressure conditions because the collisions between electrons, ions, and neutral molecules occur infrequently. 3. Inactivation of Microorganisms by Plasma 3. Inactivation of Microorganisms by Plasma Some microorganisms such as bacteria, viruses, and fungi act as pathogens and cause diseases. There is a resistance hierarchy of microorganisms against disinfection/sterilization that can be divided into the following five categories: most resistant, highly resistant, intermediately resistant, less resistant, and very susceptible [43]. The most resistant infectious agents are prions (proteinaceous infectious particles), which are the causative agents of prion diseases, such as the Creutzfeldt–Jakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, and chronic wasting disease (CWD) in cervids (deer family). Bacterial spores, protozoan oocysts, and helminth eggs are categorized as highly resistant microorganisms. Intermediately resistant microorganisms include mycobacteria, protozoan cysts, small non-enveloped viruses, and fungal spores. Vegetative bacteria, protozoa, helminths, fungi, and algae, as well as large non-enveloped viruses are less resistant. Enveloped viruses such as human immunodeficiency virus (HIV) are generally highly susceptible to various disinfectants. However, in reality, the situation is more complex. For example, although the enveloped virus Some microorganisms such as bacteria, viruses, and fungi act as pathogens and cause diseases. There is a resistance hierarchy of microorganisms against disinfection/sterilization that can be divided into the following ﬁve categories: most resistant, highly resistant, intermediately resistant, less resistant, and very susceptible [43]. The most resistant infectious agents are prions (proteinaceous infectious particles), which are the causative agents of prion diseases, such as the Creutzfeldt–Jakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, and chronic wasting disease (CWD) in cervids (deer family). Bacterial spores, protozoan oocysts, and helminth eggs are categorized as highly resistant microorganisms. Intermediately resistant microorganisms include mycobacteria, protozoan cysts, small non-enveloped viruses, and fungal spores. Vegetative bacteria, protozoa, helminths, fungi, and algae, as well as large non-enveloped viruses are less resistant. Enveloped viruses such as human immunodeﬁciency virus (HIV) are generally highly susceptible to various disinfectants. However, in reality, the situation is more complex. For example, although the enveloped virus HIV is very sensitive to disinfection and is rapidly inactivated even at room temperature without any treatment, the influenza virus (another enveloped virus) can remain infectious for up to 48 h. Orthopoxviruses (e.g., smallpox, vaccinia) and filoviruses (e.g., Ebola virus, Marburg virus) are all enveloped viruses that remain infectious for up to several weeks. Furthermore, susceptibility against However, in reality, the situation is more complex. 2. Fundamentals of Plasma and Methods for Its Generation in the post discharging area due to their short half-life at ambient temperature. In the case of plasma- treated solutions, freezing can extend the storage time and minimize the loss of reactive chemical species [37]. Figure 1. Types of plasma treatment can be classified as follows. (a) Discharging area is in contact with the sample, or (b) the sample is in contact with plasma transferred to the target site from the discharging area. Alternatively, (c) solutions previously subjected to plasma treatment could be used as reagents to apply to samples. Photographic images of the different modes of treatment are shown in the upper section with corresponding illustrations in the lower section. Figure 1. Types of plasma treatment can be classiﬁed as follows. (a) Discharging area is in contact with the sample, or (b) the sample is in contact with plasma transferred to the target site from the discharging area. Alternatively, (c) solutions previously subjected to plasma treatment could be used as reagents to apply to samples. Photographic images of the diﬀerent modes of treatment are shown in the upper section with corresponding illustrations in the lower section. Figure 1. Types of plasma treatment can be classified as follows. (a) Discharging area is in contact with the sample, or (b) the sample is in contact with plasma transferred to the target site from the discharging area. Alternatively, (c) solutions previously subjected to plasma treatment could be used as reagents to apply to samples. Photographic images of the different modes of treatment are shown in the upper section with corresponding illustrations in the lower section. Figure 1. Types of plasma treatment can be classiﬁed as follows. (a) Discharging area is in contact with the sample, or (b) the sample is in contact with plasma transferred to the target site from the discharging area. Alternatively, (c) solutions previously subjected to plasma treatment could be used as reagents to apply to samples. Photographic images of the diﬀerent modes of treatment are shown in the upper section with corresponding illustrations in the lower section. 2. Fundamentals of Plasma and Methods for Its Generation Low-pressure plasma can be generated by a low breakdown voltage in a vacuum chamber evacuated with a vacuum pump. Low-pressure plasma systems are important for the manufacture of semiconductor components. Furthermore, research into low-pressure plasma systems has also focused on the decontamination and sterilization of medical devices [23,24]. Although low pressure plasma can generate high concentrations of active species with a uniform glow plasma, it involves high maintenance costs because of the requirement for a vacuum system. Atmospheric pressure plasma requires a high voltage and relatively high temperature due to frequent collisions between electrons and ions accompanying the high particle density [9]. However, it is possible to generate plasma under non-thermal conditions by using a pulse discharge and APPJ [25], DBD [26], and ﬂoating electrode barrier discharge (FE-DBD) [27,28], or MHCD [21,29]. These non-thermal conditions allow applications involving exposure of the plasma with tissues such as skin [30]. Similarly, non-thermal plasma can be used to disinfect agricultural products and medical devices with relatively little impact on their structural integrity [31,32]. Alternatively, the plasma could be transferred to a target site where the object for treatment is located using a plasma afterglow (Figure 1). Indirect treatment using solutions treated with plasma, known as “plasma-activated water (PAW)” [33], “plasma-activated medium (PAM)” [34], “plasma-stimulated medium (PSM)” [35], “plasma-treated water (PTW)” [36,37], “plasma-treated phosphate-buﬀered saline (pPBS)” [38], or “non-thermal plasma-conditioned media (NTP media)” [39], is also possible. The constituents in these solutions react with samples and act as disinfectants [40] or anti-cancer agents [41]. In cases where samples are in contact with plasma bulk in the discharging area, plasma components such as UV radiation and reactive chemical species directly interact with the samples. Thus, short-life reactive chemical species, such as reactive oxygen species (ROS) and reactive nitrogen species (RNS), eﬃciently interact with the sample components [42]. By contrast, in cases where the sample is in contact with plasma bulk away from the discharging area, the contribution of UV radiation is signiﬁcantly lower. In addition, there is a greatly reduced concentration of reactive chemical species in the post discharging Int. J. Mol. Sci. 2019, 20, 5216 efficiently interact w contact with plasma 4 of 17 in is area due to their short half-life at ambient temperature. In the case of plasma-treated solutions, freezing can extend the storage time and minimize the loss of reactive chemical species [37]. 3. Inactivation of Microorganisms by Plasma 3. Inactivation of Microorganisms by Plasma For example, although the enveloped virus HIV is very sensitive to disinfection and is rapidly inactivated even at room temperature without any treatment, the inﬂuenza virus (another enveloped virus) can remain infectious for up to 48 h. Orthopoxviruses (e.g., smallpox, vaccinia) and ﬁloviruses (e.g., Ebola virus, Marburg virus) are all enveloped viruses that remain infectious for up to several weeks. Furthermore, susceptibility against biocides depends on the environment where the microorganisms are present (e.g., blood, serum, spinal ﬂuid, saliva). Certain soils can prevent drying and stabilize the viral structure, extending the survival time of viruses. For example, viruses normally interact with external materialss including proteins, lipids, salts, and cell debris. In other cases, viruses cause the aggregation of host cells [44] or make aggregates themselves [45], which could reduce the virucidal eﬀect of disinfectants. Therefore, the susceptibility of microorganisms to disinfection/sterilization should be examined under a range of diﬀerent conditions. Int. J. Mol. Sci. 2019, 20, 5216 spinal fluid, saliva). C survival time of virus 5 of 17 he g The most impressive results in terms of microbicidal activity have come from studies using bacteria, including bacterial spores [4–49]. Although some bacteria can form bioﬁlms in certain environments, this does not prevent their successful inactivation by plasma treatment [50,51]. Furthermore, the recent emergence of drug-resistant pathogens is now a serious public health concern that has been acknowledged by both the World Health Organization (WHO) [52] and the US Centers for Disease Control (CDC) [53]. The extensive and indiscriminate use of antibiotics may have altered the environmental microbiome, contributing to the emergence of drug-resistant bacteria [54]. Consequently, there is an urgent requirement to devise novel methods to eliminate these multidrug-resistant bacteria from the food production process. Plasma treatment is an especially promising method because the mechanism of bactericidal action is unlikely to diﬀer between multidrug-resistant and normal bacteria. The main mechanisms of bactericidal action in plasma are thought to involve exposure to reactive chemical species for which multidrug-resistant bacteria are unlikely to be resistant [55]. Furthermore, plasma pre-treatment enhances the sensitivity of methicillin-resistant Staphylococcus aureus to antibiotics [56]. or make aggregates themselves [45], which could reduce the virucidal effect of disinfectants. Therefore, the susceptibility of microorganisms to disinfection/sterilization should be examined under a range of different conditions. The most impressive results in terms of microbicidal activity have come from studies using bacteria, including bacterial spores [4–49]. 3. Inactivation of Microorganisms by Plasma 3. Inactivation of Microorganisms by Plasma Although some bacteria can form biofilms in certain environments, this does not prevent their successful inactivation by plasma treatment [50,51]. Furthermore, the recent emergence of drug-resistant pathogens is now a serious public health concern that has been acknowledged by both the World Health Organization (WHO) [52] and the US Centers for Disease Control (CDC) [53]. The extensive and indiscriminate use of antibiotics may have altered the environmental microbiome, contributing to the emergence of drug-resistant bacteria [54]. Consequently, there is an urgent requirement to devise novel methods to eliminate these multidrug- resistant bacteria from the food production process. Plasma treatment is an especially promising method because the mechanism of bactericidal action is unlikely to differ between multidrug- resistant and normal bacteria. The main mechanisms of bactericidal action in plasma are thought to involve exposure to reactive chemical species for which multidrug-resistant bacteria are unlikely to be resistant [55]. Furthermore, plasma pre-treatment enhances the sensitivity of methicillin-resistant Several reports suggest that plasma can be eﬀective in inactivating fungi [57–59]. However, our own investigations have shown that the viable cell number of Aspergillus brasiliensis was not signiﬁcantly impacted after 5 min plasma treatment using a nitrogen gas plasma device, BLP-TES (bi-polar and low-pressure plasma-triple eﬀects sterilization) [57], whereas Salmonella enterica serovar Abony was completely inactivated after employing the same treatment regime [55] (Figure 2). Indeed, a 15 min treatment with nitrogen gas plasma was required to reduce the viability of A. brasiliensis. Thus, by comparison to bacteria, extended treatment time with nitrogen gas plasma must be used to inactivate fungi. p p y Staphylococcus aureus to antibiotics [56]. Several reports suggest that plasma can be effective in inactivating fungi [57–59]. However, our own investigations have shown that the viable cell number of Aspergillus brasiliensis was not significantly impacted after 5 min plasma treatment using a nitrogen gas plasma device, BLP-TES (bi- polar and low-pressure plasma-triple effects sterilization) [57], whereas Salmonella enterica serovar Abony was completely inactivated after employing the same treatment regime [55] (Figure 2). Indeed, a 15 min treatment with nitrogen gas plasma was required to reduce the viability of A. brasiliensis. Thus, by comparison to bacteria, extended treatment time with nitrogen gas plasma must be used to inactivate fungi. Figure 2. Direct plasma treatment inactivates bacteria and fungi. 3. Inactivation of Microorganisms by Plasma 3. Inactivation of Microorganisms by Plasma (a) A sample of Salmonella was treated with plasma using the BLP-TES (bi-polar and low-pressure plasma-triple effects sterilization) device, which generates nitrogen gas plasma using a fast high-voltage pulse by a static induction (SI) thyristor power supply, for the indicated time. Colony-forming units (CFU) per ml of culture reduced with the plasma treatment in a time-dependent manner. (b) Viable cell numbers of Aspergillus reduced after plasma treatment using the BLP-TES device. Differences where * p < 0.05 and ** p < 0.01 versus control (0 min) were considered significant. Modified from Maeda et al. 2015 [55] and Sakudo et al. 2017 [57] with permission from Elsevier. The resistance of fungi to plasma treatment has also been studied Soušková et al reported the Figure 2. Direct plasma treatment inactivates bacteria and fungi. (a) A sample of Salmonella was treated with plasma using the BLP-TES (bi-polar and low-pressure plasma-triple eﬀects sterilization) device, which generates nitrogen gas plasma using a fast high-voltage pulse by a static induction (SI) thyristor power supply, for the indicated time. Colony-forming units (CFU) per ml of culture reduced with the plasma treatment in a time-dependent manner. (b) Viable cell numbers of Aspergillus reduced after plasma treatment using the BLP-TES device. Diﬀerences where * p < 0.05 and ** p < 0.01 versus control (0 min) were considered signiﬁcant. Modiﬁed from Maeda et al. 2015 [55] and Sakudo et al. 2017 [57] with permission from Elsevier. Figure 2. Direct plasma treatment inactivates bacteria and fungi. (a) A sample of Salmonella was treated with plasma using the BLP-TES (bi-polar and low-pressure plasma-triple effects sterilization) device, which generates nitrogen gas plasma using a fast high-voltage pulse by a static induction (SI) thyristor power supply, for the indicated time. Colony-forming units (CFU) per ml of culture reduced with the plasma treatment in a time-dependent manner. (b) Viable cell numbers of Aspergillus reduced after plasma treatment using the BLP-TES device. Differences where * p < 0.05 and ** p < 0.01 versus control (0 min) were considered significant. Modified from Maeda et al. 2015 [55] and Sakudo et al. 2017 [57] with permission from Elsevier. Th f f l h l b d d S šk á l d h Figure 2. Direct plasma treatment inactivates bacteria and fungi. 3. Inactivation of Microorganisms by Plasma 3. Inactivation of Microorganisms by Plasma (a) A sample of Salmonella was treated with plasma using the BLP-TES (bi-polar and low-pressure plasma-triple eﬀects sterilization) device, which generates nitrogen gas plasma using a fast high-voltage pulse by a static induction (SI) thyristor power supply, for the indicated time. Colony-forming units (CFU) per ml of culture reduced with the plasma treatment in a time-dependent manner. (b) Viable cell numbers of Aspergillus reduced after plasma treatment using the BLP-TES device. Diﬀerences where * p < 0.05 and ** p < 0.01 versus control (0 min) were considered signiﬁcant. Modiﬁed from Maeda et al. 2015 [55] and Sakudo et al. 2017 [57] with permission from Elsevier. Figure 2. Direct plasma treatment inactivates bacteria and fungi. (a) A sample of Salmonella was t t d ith l i th BLP TES (bi l d l l t i l ff t t ili ti ) Figure 2. Direct plasma treatment inactivates bacteria and fungi. (a) A sample of Salmonella was treated thyristor power supply, for the indicated time. Colony-forming units (CFU) per ml of culture reduced with the plasma treatment in a time-dependent manner. (b) Viable cell numbers of Aspergillus reduced after plasma treatment using the BLP-TES device. Differences where * p < 0.05 and ** p < 0.01 versus control (0 min) were considered significant. Modified from Maeda et al. 2015 [55] and Sakudo et al. 2017 [57] with permission from Elsevier. g g g p g g g p y ( ) y power supply, for the indicated time. Colony-forming units (CFU) per ml of culture reduced with the plasma treatment in a time-dependent manner. (b) Viable cell numbers of Aspergillus reduced after plasma treatment using the BLP-TES device. Diﬀerences where * p < 0.05 and ** p < 0.01 versus control (0 min) were considered signiﬁcant. Modiﬁed from Maeda et al. 2015 [55] and Sakudo et al. 2017 [57] with permission from Elsevier. The resistance of fungi to plasma treatment has also been studied. Soušková et al. reported the susceptibility of fungi is different between species, including Aspergillus oryzae, Cladosporium The resistance of fungi to plasma treatment has also been studied. Soušková et al. reported the susceptibility of fungi is diﬀerent between species, including Aspergillus oryzae, Cladosporium sphaerospermum, and Penicillium crustosum despite no signiﬁcant diﬀerences in susceptibility against plasma generated by corona discharge among bacteria, including Escherichia coli and Staphylococcus epidermidis [58–60]. 3. Inactivation of Microorganisms by Plasma 3. Inactivation of Microorganisms by Plasma For e a le la a i a ti atio of the yea t like al ae P otothe a o fii [69] a d ate bo e hel i th Furthermore, a DBD plasma torch inactivated the non-enveloped virus, feline calicivirus [66]. Inactivation of viruses was achieved by a relatively short exposure to plasma. According to the U.S. Environmental Protection Agency (USEPA) “Guide Standard and Protocol for Testing Microbiological Water Puriﬁers,” the minimum performance standards of the inactivation eﬃciency are a six-log reduction/inactivation of bacteria, or a four-log reduction/inactivation of viruses [67]. Treatment using nitrogen gas plasma generated by BLP-TES showed an approximate two-log reduction in inﬂuenza virus titer after 1 min and four-log reduction of virus titer of adenovirus within 4 min [61,62]. A 1-min treatment with the DBD plasma torch resulted in a greater than two-log reduction of virus titer for feline calicivirus [66]. Lengthening the treatment to 2 min reduced the viral titer to an undetectable level (3.81 × 104 ± 1.58 × 103 median tissue culture infectious dose (TCID50)/mL at 0 min; below the detection limit at 2 min). These results suggest that the 2 min treatment meets the performance standards set by USEPA as outlined earlier. However, as far as the authors are aware, there are a limited number of studies on the inactivation of plant viruses using plasma [68]. Furthermore, there are no published studies on the plasma inactivation of viroid’s, which are infectious RNAs that cause plant diseases. Indeed, to develop a plasma disinfection system for the agricultural sector, it would be necessary to determine the eﬀectiveness of this technology on a range of plant pathogens. example, plasma inactivation of the yeast-like algae Prototheca zopfii [69] and water-borne helminth Schistosoma japonicum [70], Acanthamoeba species (spp.), and other ocular pathogens, as well as water- borne protozoan enteroparasite Cryptosporidium parvum when combined with pulsed UV [71] was Plasma is also eﬀective against other microorganisms besides the ones mentioned above. For example, plasma inactivation of the yeast-like algae Prototheca zopﬁi [69] and water-borne helminth Schistosoma japonicum [70], Acanthamoeba species (spp.), and other ocular pathogens, as well as water-borne protozoan enteroparasite Cryptosporidium parvum when combined with pulsed UV [71] was conﬁrmed. These results suggest that plasma has the potential to inactivate cysts and protozoal oocysts as well as trophozoites of protozoon parasites. 3. Inactivation of Microorganisms by Plasma 3. Inactivation of Microorganisms by Plasma (b) Nitrogen gas plasma treatment with the BLP-TES device resulted in a decrease in viral titer [plaque forming units (PFU) per ml] of adenovirus. Differences where * p < 0.05 versus control (0 min) were considered significant. Cited from Sakudo et al. 2013 [61] and Sakudo, Toyokawa, and Imanishi 2016 [62] under the terms of the Creative Commons Attribution license. values reduced after treatment with low-pressure nitrogen gas plasma using the BLP-TES device. (b) Nitrogen gas plasma treatment with the BLP-TES device resulted in a decrease in viral titer [plaque forming units (PFU) per ml] of adenovirus. Diﬀerences where * p < 0.05 versus control (0 min) were considered signiﬁcant. Cited from Sakudo et al. 2013 [61] and Sakudo, Toyokawa, and Imanishi 2016 [62] under the terms of the Creative Commons Attribution license. Furthermore, a DBD plasma torch inactivated the non-enveloped virus, feline calicivirus [66]. Inactivation of viruses was achieved by a relatively short exposure to plasma. According to the U.S. Environmental Protection Agency (USEPA) “Guide Standard and Protocol for Testing Microbiological Water Purifiers,” the minimum performance standards of the inactivation efficiency are a six-log reduction/inactivation of bacteria, or a four-log reduction/inactivation of viruses [67]. Treatment using nitrogen gas plasma generated by BLP-TES showed an approximate two-log reduction in influenza virus titer after 1 min and four-log reduction of virus titer of adenovirus within 4 min [61,62]. A 1-min treatment with the DBD plasma torch resulted in a greater than two-log reduction of virus titer for feline calicivirus [66]. Lengthening the treatment to 2 min reduced the viral titer to an undetectable level (3.81 × 104 ± 1.58 × 103 median tissue culture infectious dose (TCID50) /mL at 0 min; below the detection limit at 2 min). These results suggest that the 2 min treatment meets the performance standards set by USEPA as outlined earlier. However, as far as the authors are aware, there are a limited number of studies on the inactivation of plant viruses using plasma [68]. Furthermore, there are no published studies on the plasma inactivation of viroid’s, which are infectious RNAs that cause plant diseases. Indeed, to develop a plasma disinfection system for the agricultural sector, it would be necessary to determine the effectiveness of this technology on a range of plant pathogens. Plasma is also effective against other microorganisms besides the ones mentioned above. 3. Inactivation of Microorganisms by Plasma 3. Inactivation of Microorganisms by Plasma Among these fungi, Aspergillus displayed the greatest resistance to plasma inactivation, possibly due to the presence of spores. Therefore, the resistance of fungi to plasma treatment appears to be related to spore generation. The resistance of fungi to plasma treatment has also been studied. Soušková et al. reported the susceptibility of fungi is different between species, including Aspergillus oryzae, Cladosporium The resistance of fungi to plasma treatment has also been studied. Soušková et al. reported the susceptibility of fungi is diﬀerent between species, including Aspergillus oryzae, Cladosporium sphaerospermum, and Penicillium crustosum despite no signiﬁcant diﬀerences in susceptibility against plasma generated by corona discharge among bacteria, including Escherichia coli and Staphylococcus epidermidis [58–60]. Among these fungi, Aspergillus displayed the greatest resistance to plasma inactivation, possibly due to the presence of spores. Therefore, the resistance of fungi to plasma treatment appears to be related to spore generation. Several studies have investigated the eﬀect of plasma on the inactivation of both enveloped and non-enveloped viruses. Representative studies showed that nitrogen plasma generated by BLP-TES 6 of 17 ma Int. J. Mol. Sci. 2019, 20, 5216 inactivation, possibly treatment appears to inactivated enveloped viruses, such as the inﬂuenza virus [61] and respiratory syncytial virus (RSV) [42], as well as non-enveloped viruses, such as the adenovirus [62] (Figure 3). In addition, there are several studies using bacteriophages as model objects of viral inactivation by plasma [63–65]. non-enveloped viruses. Representative studies showed that nitrogen plasma generated by BLP-TES inactivated enveloped viruses, such as the influenza virus [61] and respiratory syncytial virus (RSV) [42], as well as non-enveloped viruses, such as the adenovirus [62] (Figure 3). In addition, there are several studies using bacteriophages as model objects of viral inactivation by plasma [63–65]. dies using bacteriophages as model objects of viral inactivation by plasma [63–65]. [ ] p [ ] ( g ) several studies using bacteriophages as model objects of viral inactivation by plasma [63–65]. Figure 3. Direct plasma treatment inactivates viruses. (a) Viral titers of median tissue culture infectious dose (TCID50) per ml were calculated in duplicate (No 1 and No 2) for the influenza virus. The TCID50 values reduced after treatment with low-pressure nitrogen gas plasma using the BLP-TES device. (b) Nitrogen gas plasma treatment with the BLP-TES device resulted in a decrease in viral titer [plaque forming units (PFU) per ml] of adenovirus. 3. Inactivation of Microorganisms by Plasma 3. Inactivation of Microorganisms by Plasma Differences where * p < 0.05 versus control (0 min) were considered significant. Cited from Sakudo et al. 2013 [61] and Sakudo, Toyokawa, and Imanishi 2016 [62] under the terms of the Creative Commons Attribution license. F h DBD l h i i d h l d i f li li i i [66] Figure 3. Direct plasma treatment inactivates viruses. (a) Viral titers of median tissue culture infectious dose (TCID50) per ml were calculated in duplicate (No 1 and No 2) for the inﬂuenza virus. The TCID50 values reduced after treatment with low-pressure nitrogen gas plasma using the BLP-TES device. (b) Nitrogen gas plasma treatment with the BLP-TES device resulted in a decrease in viral titer [plaque forming units (PFU) per ml] of adenovirus. Diﬀerences where * p < 0.05 versus control (0 min) were considered signiﬁcant. Cited from Sakudo et al. 2013 [61] and Sakudo, Toyokawa, and Imanishi 2016 [62] under the terms of the Creative Commons Attribution license. Figure 3. Direct plasma treatment inactivates viruses. (a) Viral titers of median tissue culture infectious dose (TCID50) per ml were calculated in duplicate (No 1 and No 2) for the influenza virus. The TCID50 values reduced after treatment with low-pressure nitrogen gas plasma using the BLP-TES device. (b) Nitrogen gas plasma treatment with the BLP-TES device resulted in a decrease in viral titer [plaque forming units (PFU) per ml] of adenovirus. Differences where * p < 0.05 versus control (0 min) were considered significant. Cited from Sakudo et al. 2013 [61] and Sakudo, Toyokawa, and Imanishi 2016 [62] under the terms of the Creative Commons Attribution license. Figure 3. Direct plasma treatment inactivates viruses. (a) Viral titers of median tissue culture infectious dose (TCID50) per ml were calculated in duplicate (No 1 and No 2) for the inﬂuenza virus. The TCID50 values reduced after treatment with low-pressure nitrogen gas plasma using the BLP-TES device. (b) Nitrogen gas plasma treatment with the BLP-TES device resulted in a decrease in viral titer [plaque forming units (PFU) per ml] of adenovirus. Diﬀerences where * p < 0.05 versus control (0 min) were considered signiﬁcant. Cited from Sakudo et al. 2013 [61] and Sakudo, Toyokawa, and Imanishi 2016 [62] under the terms of the Creative Commons Attribution license. The TCID50 values reduced after treatment with low pressure nitrogen gas plasma using the BLP TES device. 3. Inactivation of Microorganisms by Plasma 3. Inactivation of Microorganisms by Plasma Differences where p < 0.01 versus control (0 min) were considered significant. (a) and (b) are cited from Sakudo et al. [57] with permission from Elsevier, while (c) and (d) are cited from Sakudo et al. [74] under the terms of the Creative Commons Attribution 4.0 International license. Figure 4. Direct plasma treatment inactivates toxins. Quantitative measurement of aﬂatoxin B1 (AFB1) (a,b), Shiga toxin 1 (Stx1) (c), Shiga toxin 2 (Stx2) (D) after low-pressure nitrogen gas plasma treatment with BLP-TES at 1.5 kpps for the indicated times (a,c,d) and at 0–1.5 kpps for 15 min (b) was performed by an enzyme-linked immunosorbent assay (ELISA) using an MytoJudge Total Aﬂatoxin kit (NH Foods Ltd.) and a RIDASCREEN® Verotoxin kit (R-Biopharm AG, Darmstadt). Diﬀerences where p < 0.01 versus control (0 min) were considered signiﬁcant. (a) and (b) are cited from Sakudo et al. [57] with permission from Elsevier, while (c) and (d) are cited from Sakudo et al. [74] under the terms of the Creative Commons Attribution 4.0 International license. Thus, plasma has applications not only for disinfection/sterilization but also for the degradation of toxins [75]. This includes not only exotoxins but also endotoxins, lipopolysaccharides (LPS) by inactivating their lipid A [76]. Consequently, plasma technology is a novel advanced disinfection/sterilization system that can simultaneously inactivate pathogens and their associated toxins. The inactivation mechanisms of action of plasma remain to be determined [5,6,31,77]. The Thus, plasma has applications not only for disinfection/sterilization but also for the degradation of toxins [75]. This includes not only exotoxins but also endotoxins, lipopolysaccharides (LPS) by inactivating their lipid A [76]. Consequently, plasma technology is a novel advanced disinfection/sterilization system that can simultaneously inactivate pathogens and their associated toxins. The inactivation mechanisms of action of plasma remain to be determined [5,6,31,77]. The mechanisms depend on the types of gases used to generate the plasma. In the case of nitrogen gas plasma, at least three major mechanisms (reactive chemical species, UV radiation exposure, and electric fields) are thought to be involved [31]. In addition, etching effects may also contribute, especially in the case of oxygen plasmas [78]. Specifically, shrinking of bacterial spores were observed in oxygen gas plasma-treated spores but not in nitrogen gas plasma-treated spores [79–81]. 3. Inactivation of Microorganisms by Plasma 3. Inactivation of Microorganisms by Plasma Although prions are known to be the most resistant pathogens, they are nonetheless inactivated by radio-frequency (RF) plasma treatment using an Ar/O2 gas mixture [72] and by plasma from a microwave discharge [73]. In addition, plasma treatment can eﬃciently degrade toxins produced by both bacteria and fungi (Figure 4). 7 of 17 g ma Int. J. Mol. Sci. 2019, 20, 5216 an Ar/O2 gas mixture t t t ffi i y g p y g ( g ) Figure 4. Direct plasma treatment inactivates toxins. Quantitative measurement of aflatoxin B1 (AFB1) (a,b), Shiga toxin 1 (Stx1) (c), Shiga toxin 2 (Stx2) (D) after low-pressure nitrogen gas plasma treatment with BLP-TES at 1.5 kpps for the indicated times (a,c,d) and at 0–1.5 kpps for 15 min (b) was performed by an enzyme-linked immunosorbent assay (ELISA) using an MytoJudge Total Aflatoxin kit (NH Foods Ltd.) and a RIDASCREEN® Verotoxin kit (R-Biopharm AG, Darmstadt). Differences where p < 0.01 versus control (0 min) were considered significant. (a) and (b) are cited from Sakudo et al. [57] with permission from Elsevier, while (c) and (d) are cited from Sakudo et al. [74] under the terms of the Creative Commons Attribution 4.0 International license. Figure 4. Direct plasma treatment inactivates toxins. Quantitative measurement of aﬂatoxin B1 (AFB1) (a,b), Shiga toxin 1 (Stx1) (c), Shiga toxin 2 (Stx2) (D) after low-pressure nitrogen gas plasma treatment with BLP-TES at 1.5 kpps for the indicated times (a,c,d) and at 0–1.5 kpps for 15 min (b) was performed by an enzyme-linked immunosorbent assay (ELISA) using an MytoJudge Total Aﬂatoxin kit (NH Foods Ltd.) and a RIDASCREEN® Verotoxin kit (R-Biopharm AG, Darmstadt). Diﬀerences where p < 0.01 versus control (0 min) were considered signiﬁcant. (a) and (b) are cited from Sakudo et al. [57] with permission from Elsevier, while (c) and (d) are cited from Sakudo et al. [74] under the terms of the Creative Commons Attribution 4.0 International license. Figure 4. Direct plasma treatment inactivates toxins. Quantitative measurement of aflatoxin B1 (AFB1) (a,b), Shiga toxin 1 (Stx1) (c), Shiga toxin 2 (Stx2) (D) after low-pressure nitrogen gas plasma treatment with BLP-TES at 1.5 kpps for the indicated times (a,c,d) and at 0–1.5 kpps for 15 min (b) was performed by an enzyme-linked immunosorbent assay (ELISA) using an MytoJudge Total Aflatoxin kit (NH Foods Ltd.) and a RIDASCREEN® Verotoxin kit (R-Biopharm AG, Darmstadt). 4. Future Perspectives in Agriculture and the Food Industry There are extensive applications of plasma technology in the ﬁeld of agriculture and in the food sector. For example, plasma technology could be applied to the disinfection of foods, packaging materials and equipment as well as agricultural sources such as seeds, fertilizer, water, and soil. Agricultural products, such as fruits and vegetables, are prone to contamination from agricultural sources, including seeds, fertilizers, water, and soil. Moreover, the agricultural products come into contact with dust, insects, animal feces, ﬁeld workers, and equipment during pre-harvest/harvest, transport, packaging and food processing stages of the supply chain. These individual risk factors may contribute to a microbial hazard. The application of innovative disinfection techniques, including plasma technology, may help reduce the potential risk from these factors. A recent review has shown that food products subjected to plasma disinfection are becoming widespread, which include fresh fruits, vegetables, dry fruits, nuts, seeds, spices, eggshells, as well as protein products such as meat and cold cuts [82]. However, further advances in plasma disinfection technology are required before this method can be applied to the food industry (e.g., food processing and distribution system as well as agricultural products). g p We anticipate the wide-ranging application of plasma technology in the ﬁeld of agriculture once the equipment has been fully developed. The current priority is the development of an eﬃcient open-air system suitable for disinfecting both large objects and high numbers of samples. Moreover, such a device will be readily scalable. Based on this background, we recently developed a novel roller conveyer plasma device (Figure 5). Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 9 of 17 Figure 5. (a) Schematic representation of a roller conveyer plasma device for the continuous disinfection of fruits and vegetables using atmospheric pressure plasma. As an example, oranges are shown. (b) Oranges on rollers during operation of the device. (c) Enlarged image of (b) showing the plasma (Arrow) generated between the orange and roller during operation of the device. The image is modified from Toyokawa et al. 2017 [83] with permission from Elsevier. I dditi iti l f t t id i th f li ti f thi l t h l Th Figure 5. (a) Schematic representation of a roller conveyer plasma device for the continuous disinfection of fruits and vegetables using atmospheric pressure plasma. As an example, oranges are shown. (b) Oranges on rollers during operation of the device. 4. Future Perspectives in Agriculture and the Food Industry (c) Enlarged image of (b) showing the plasma (Arrow) generated between the orange and roller during operation of the device. The image is modiﬁed from Toyokawa et al. 2017 [83] with permission from Elsevier. Figure 5. (a) Schematic representation of a roller conveyer plasma device for the continuous disinfection of fruits and vegetables using atmospheric pressure plasma. As an example, oranges are shown. (b) Oranges on rollers during operation of the device. (c) Enlarged image of (b) showing the plasma (Arrow) generated between the orange and roller during operation of the device. The image is modified from Toyokawa et al. 2017 [83] with permission from Elsevier. Figure 5. (a) Schematic representation of a roller conveyer plasma device for the continuous disinfection of fruits and vegetables using atmospheric pressure plasma. As an example, oranges are shown. (b) Oranges on rollers during operation of the device. (c) Enlarged image of (b) showing the plasma (Arrow) generated between the orange and roller during operation of the device. The image is modiﬁed from Toyokawa et al. 2017 [83] with permission from Elsevier. In addition, a critical factor to consider is the safe application of this novel technology. The European Commission stated that there are no restrictions in the regulations regarding the use of plasma as an electronic preservative practice for organic foods [84]. However, plasma treatment of aqueous solutions can potentially generate hydrogen peroxide, nitrates, and nitrites [85,86]. These compounds might react to form other toxic compounds such as peroxynitrous acid. Therefore, a comprehensive evaluation of the effect of plasma on foods and human health is necessary before this new disinfection technology can be fully utilized. Indeed, a range of applications of plasma technology in the field of agriculture is currently being assessed. Recently, the use of plasma technology has been reported to enhance seed germination and the This plasma device is well suited to the disinfection of fruits and vegetables during sorting on rollers [83]. The apparatus is an original design that generates atmospheric plasma by the mechanism of DBD. This unique plasma apparatus is composed of rolling electrodes comprising a plastic rod (diameter = 30 mm) covered with a thin aluminum and silicon sheet positioned at an interval of 50 mm between the high -voltage electrode and earth electrode. The high-voltage electrode is then connected to an alternating power supply. 3. Inactivation of Microorganisms by Plasma 3. Inactivation of Microorganisms by Plasma Overall, reactive chemical species, UV radiation, and electric fields contribute to the a ti i obial effe t of la a de e di o the ty e of a e a ell a the ethod e loyed to The inactivation mechanisms of action of plasma remain to be determined [5,6,31,77]. The mechanisms depend on the types of gases used to generate the plasma. In the case of nitrogen gas plasma, at least three major mechanisms (reactive chemical species, UV radiation exposure, and electric ﬁelds) are thought to be involved [31]. In addition, etching eﬀects may also contribute, especially in the case of oxygen plasmas [78]. Speciﬁcally, shrinking of bacterial spores were observed in oxygen gas plasma-treated spores but not in nitrogen gas plasma-treated spores [79–81]. antimicrobial effects of plasma, depending on the type of gases as well as the methods employed to generate the plasma. Reactive chemical species seem to be the principal inactivation factor in most Overall, reactive chemical species, UV radiation, and electric ﬁelds contribute to the antimicrobial eﬀects of plasma, depending on the type of gases as well as the methods employed to generate the plasma. Reactive chemical species seem to be the principal inactivation factor in most cases, although this may vary depending on the method of plasma generation and whether the sample is exposed to direct or indirect plasma treatment. In addition, inactivation mechanisms may vary depending on the target microorganism. However, it should be noted that most studies on the inactivation mechanisms of plasma have been conducted using bacterial spores. Therefore, further studies are required on the inactivation of various microorganisms using plasma to understand the underlying mechanisms involved fully. Int. J. Mol. Sci. 2019, 20, 5216 8 of 17 4. Future Perspectives in Agriculture and the Food Industry Plasma is generated in the silicon sheet when electrically conductive samples, such as fruits and vegetables as well as metals, make contact with both the high-voltage Int. J. Mol. Sci. 2019, 20, 5216 9 of 17 electrode and earth electrode. Our ﬁndings suggest the device could have practical applications for the disinfection of agricultural products during the sorting process on rollers. Disinfection of Xanthomonas campestris p.v. campestris-contaminated cabbage leaves using the roller conveyer plasma device has been achieved. In addition, our preliminary study has shown that the surface of Penicillium-contaminated oranges could be disinfected using the device (Sakudo and Yagyu, unpublished results). Nonetheless, to achieve broad applicability with a variety of agricultural food products, the device needs to be scaled-up and its performance fully evaluated. In addition, a critical factor to consider is the safe application of this novel technology. The European Commission stated that there are no restrictions in the regulations regarding the use of plasma as an electronic preservative practice for organic foods [84]. However, plasma treatment of aqueous solutions can potentially generate hydrogen peroxide, nitrates, and nitrites [85,86]. These compounds might react to form other toxic compounds such as peroxynitrous acid. Therefore, a comprehensive evaluation of the eﬀect of plasma on foods and human health is necessary before this new disinfection technology can be fully utilized. Indeed, a range of applications of plasma technology in the ﬁeld of agriculture is currently being assessed. Recently, the use of plasma technology has been reported to enhance seed germination and the growth of plants [87]. In addition, the removal of volatile organic compounds, such as ethylene gas, by plasma treatment may be useful during the transportation of agricultural products in containers [88]. Therefore, the application of plasma technology could also contribute to higher crop yields as well as the preservation of foods. 6. Conclusions In conclusion, plasma disinfection covers almost all of the resistance hierarchy of microorganisms. The susceptibility to the plasma of microorganisms categorized as being most resistant, highly resistant, intermediate resistant, less resistant, and very susceptible, have already been studied. Therefore, the applicability of plasma technology in disinfection/sterilization is potentially wide-ranging (Figure 6). However, to date, no studies investigating plasma treatment of viroids have been reported. Investigation of the eﬀect of plasma on various microorganisms would potentially contribute to further expanding the applicability of this technology. In addition to the ﬁeld of agriculture and medicine, plasma technology has also been utilized in a range of environmental applications, including water puriﬁcation and remediation, as well as the treatment of exhaust gases [108,109]. We anticipate that the utilization of this technology will continue to expand. Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 11 of 17 Figure 6. Recent and potential applications of plasma disinfection technology in the field of agriculture, medicine, dentistry, and environment. In the agricultural field, plasma technology could be applied to the disinfection of foods and packaging materials as well as agricultural sources such as seeds, fertilizers, waters and soils. In the medical field, plasma is useful for disinfection/sterilization of medical devices, as well as the degradation of toxins and other pathological contaminants. Potential applications of this technology also includes skin antisepsis as well as the treatment of pathogen- based skin diseases. In dentistry, plasma treatment has been used for microbicidal decontamination, including root canal disinfection and tooth disinfection. Plasma technology may also be utilized in the environmental field, including cleaning of wastewater as well as the treatment of exhaust gases. Figure 6. Recent and potential applications of plasma disinfection technology in the ﬁeld of agriculture, medicine, dentistry, and environment. In the agricultural ﬁeld, plasma technology could be applied to the disinfection of foods and packaging materials as well as agricultural sources such as seeds, fertilizers, waters and soils. In the medical ﬁeld, plasma is useful for disinfection/sterilization of medical devices, as well as the degradation of toxins and other pathological contaminants. Potential applications of this technology also includes skin antisepsis as well as the treatment of pathogen-based skin diseases. In dentistry, plasma treatment has been used for microbicidal decontamination, including root canal disinfection and tooth disinfection. 5. Future Perspectives in Medicine and Dentistry 2019, 20, 5216 10 of 17 10 of 17 and tumor tissues that resulted in cell killing and tumor-shrinking eﬀects [41,101–103]. In addition, PAM is reported to inhibit the MAP (Mitogen-activated protein) kinase (MAPK) pathway, which is an important signaling pathway for cell proliferation [104]. Cell death is induced by the suppression of these signaling cascades [105]. Moreover, ROS and RNS in plasma are key factors for the induction of cancer cell death, although the mechanisms of action have not been fully elucidated [106,107]. 5. Future Perspectives in Medicine and Dentistry Sterilization of medical instruments contaminated with pathogens is crucial in preventing secondary infections. Currently, medical instruments are sterilized by autoclaving, gamma-ray treatment, UV exposure, and the use of gases such as ethylene oxide, hydrogen peroxide, formaldehyde, peracetic acid [43]. Each sterilization method has both advantages and disadvantages. Autoclaving is relatively quick, highly penetrative, and generates no toxic residues, but temperatures of 121 ◦C could damage the material being sterilized. Treatment with gamma-rays is highly penetrative, and involves low temperatures with no associated residues, but it could induce changes in the properties of the materials and is a relatively slow process. UV treatment is fast, low cost with no toxic residues, and involves low temperatures, but the eﬀectiveness of the sterilization is poor and may result in damage to the material. Chemical treatments are low temperature and eﬀective, but these procedures generally involve the use of toxic gases that may be carcinogenic and ﬂammable. Furthermore, these gases sometimes induce unwanted biochemical changes. Although novel techniques have been developed, such as chemical treatment with supercritical carbon dioxide as well as freeze-drying and other methods, these procedures are often ineﬀective and may damage the material being sterilized. Thus, it is necessary to evaluate each sterilization method for a particular purpose carefully. Plasma technology is a promising new method that enables rapid processing at low temperatures without any associated chemical residues [6]. Plasma is eﬀective against a broad spectrum of pathogens, including bacterial spores and prions, both of which display a high level of resistance to chemical and physical treatments [47,89]. The potential of plasma technology in medical and dental applications is extremely broad. As well as disinfection/sterilization of medical and dental devices, the technology could be used to treat beds, desks, and ﬂoors [90]. Plasma technology may also have therapeutic potential [91–93]. Therapeutic uses include the treatment of skin diseases [94], blood coagulation [95] as well as dental treatment [96] and applications in dermatology such as chronic wound healing [97]. Recently, the potential application of plasma technology as a novel anticancer therapy has been assessed [98]. Induction of cancer cell death by both direct and indirect exposure of plasma has been reported [99,100]. However, as actual therapy, plasma is often diﬃcult to apply to cancer cells. As an alternative approach, a plasma-irradiated medium (PAW, PAM, PSM, PTW, pPBS, NTP media) has been used for the treatment of cancer cells Int. J. Mol. Sci. 6. Conclusions Plasma technology may also be utilized in the environmental ﬁeld, including cleaning of wastewater as well as the treatment of exhaust gases. Figure 6. Recent and potential applications of plasma disinfection technology in the field of agriculture, medicine, dentistry, and environment. In the agricultural field, plasma technology could be applied to the disinfection of foods and packaging materials as well as agricultural sources such as seeds, fertilizers, waters and soils. In the medical field, plasma is useful for disinfection/sterilization of medical devices, as well as the degradation of toxins and other pathological contaminants. Potential applications of this technology also includes skin antisepsis as well as the treatment of pathogen- based skin diseases. In dentistry, plasma treatment has been used for microbicidal decontamination, including root canal disinfection and tooth disinfection. Plasma technology may also be utilized in the environmental field, including cleaning of wastewater as well as the treatment of exhaust gases. Figure 6. Recent and potential applications of plasma disinfection technology in the ﬁeld of agriculture, medicine, dentistry, and environment. In the agricultural ﬁeld, plasma technology could be applied to the disinfection of foods and packaging materials as well as agricultural sources such as seeds, fertilizers, waters and soils. In the medical ﬁeld, plasma is useful for disinfection/sterilization of medical devices, as well as the degradation of toxins and other pathological contaminants. Potential applications of this technology also includes skin antisepsis as well as the treatment of pathogen-based skin diseases. In dentistry, plasma treatment has been used for microbicidal decontamination, including root canal disinfection and tooth disinfection. Plasma technology may also be utilized in the environmental ﬁeld, including cleaning of wastewater as well as the treatment of exhaust gases. Figure 6. Recent and potential applications of plasma disinfection technology in the field of agriculture, medicine, dentistry, and environment. In the agricultural field, plasma technology could be applied to the disinfection of foods and packaging materials as well as agricultural sources such as seeds, fertilizers, waters and soils. In the medical field, plasma is useful for disinfection/sterilization of medical devices, as well as the degradation of toxins and other pathological contaminants. Potential applications of this technology also includes skin antisepsis as well as the treatment of pathogen- based skin diseases. In dentistry, plasma treatment has been used for microbicidal decontamination, including root canal disinfection and tooth disinfection. 6. Conclusions Plasma technology may also be utilized in the environmental field, including cleaning of wastewater as well as the treatment of exhaust gases. Figure 6. Recent and potential applications of plasma disinfection technology in the ﬁeld of agriculture, medicine, dentistry, and environment. In the agricultural ﬁeld, plasma technology could be applied to the disinfection of foods and packaging materials as well as agricultural sources such as seeds, fertilizers, waters and soils. In the medical ﬁeld, plasma is useful for disinfection/sterilization of medical devices, as well as the degradation of toxins and other pathological contaminants. Potential applications of this technology also includes skin antisepsis as well as the treatment of pathogen-based skin diseases. In dentistry, plasma treatment has been used for microbicidal decontamination, including root canal disinfection and tooth disinfection. Plasma technology may also be utilized in the environmental ﬁeld, including cleaning of wastewater as well as the treatment of exhaust gases. Int. J. Mol. Sci. 2019, 20, 5216 11 of 17 Finally, it should be mentioned that an increase in the disinfection eﬃciency and improved cost performance is required before the true potential of plasma technology can be fully realized. This may be achieved by optimization of the plasma generating conditions, including the use of diﬀerent gas mixtures and careful control of the relative humidity as well as plasma generation methods. Author Contributions: For A.S., Y.Y., and T.O. contributed to the conceptualization and wrote the manuscri Funding: This work was supported by JSPS (Japan Society for the Promotion of Science) KAKENHI Grant number JP16K04997 as well as Grant-in-aid from Takahashi Industrial and Economic Research Foundation. Conﬂicts of Interest: The authors declare no conﬂict of interest. Abbreviations AC Alternating current APPJ Atmospheric plasma jet BLP-TES Bi-polar and low- pressure plasma-triple eﬀects sterilization BSE Bovine spongiform encephalopathy CDC Centers for Disease Control CJD Creutzfeldt-Jakob disease CWD Chronic wasting disease DBD Dielectric barrier discharge DC Direct current ELISA Enzyme-linked immunosorbent assay FE-DBD Floating electrode barrier discharge HIV Human immunodeﬁciency virus LPS Lipopolysaccharides MAP Mitogen-activated protein MAPK MAP kinase MHCD Micro hollow cathode discharge ne Electron density NTP Non-thermal plasma PAM Plasma-activated medium PAW Plasma-activated water PHD Pin-to-hole spark discharge pPBS Plasma-treated phosphate-buﬀered saline PSM Plasma-stimulated medium PTW Plasma-treated water RF Radio-frequency RNS Reactive nitrogen species ROS Reactive oxygen species RSV Respiratory syncytial virus Te Electron temperature Tgas Gas temperature Tion Ion temperature Tn Neutron temperature USEPA U.S. Environmental Protection Agency UV Ultraviolet VOC Volatile organic compound WHO World Health Organization References 1. Langmuir, I. Oscillations in ionized gases. Proc. Natl. Acad. Sci. USA 1928, 14, 627–637. [CrossRe 2. Ichimaru, S. Nuclear fusion in dense plasmas. Rev. Mod. Phys. 1993, 65, 255–259. [CrossRef] 3. Team, J. Fusion energy production from a deuterium-tritium plasma in the JET tokamak. Nu AC Alternating current APPJ Atmospheric plasma jet BLP-TES Bi-polar and low- pressure plasma-triple eﬀe BSE Bovine spongiform encephalopathy CDC Centers for Disease Control CJD Creutzfeldt-Jakob disease CWD Chronic wasting disease DBD Dielectric barrier discharge DC Direct current ELISA Enzyme-linked immunosorbent assay FE-DBD Floating electrode barrier discharge HIV Human immunodeﬁciency virus LPS Lipopolysaccharides MAP Mitogen-activated protein MAPK MAP kinase MHCD Micro hollow cathode discharge ne Electron density NTP Non-thermal plasma PAM Plasma-activated medium PAW Plasma-activated water PHD Pin-to-hole spark discharge pPBS Plasma-treated phosphate-buﬀered saline PSM Plasma-stimulated medium PTW Plasma-treated water RF Radio-frequency RNS Reactive nitrogen species ROS Reactive oxygen species RSV Respiratory syncytial virus Te Electron temperature Tgas Gas temperature Tion Ion temperature Tn Neutron temperature USEPA U.S. Environmental Protection Agency UV Ultraviolet VOC Volatile organic compound WHO World Health Organization 1. Langmuir, I. Oscillations in ionized gases. Proc. Natl. Acad. Sci. USA 1928, 14, 627 637. [CrossRef] [PubMed] 2. Ichimaru, S. Nuclear fusion in dense plasmas. Rev. Mod. Phys. 1993, 65, 255–259. [CrossRef] 3. Team, J. Fusion energy production from a deuterium-tritium plasma in the JET tokamak. Nuc. Fus. 1992, 32, 187–203. [CrossRef] References 2002, 74, 327–335. [CrossRef] 14. Bonnizzoni, G.; Vassallo, E. Plasma physics and technology; industrial applications. Vacuum 2002, 64, 327–336. [CrossRef] 3. Heberlein, J. New approaches in thermal plasma technology. Pure Appl. Chem. 2002, 74, 327–335. [Cross 14. Bonnizzoni, G.; Vassallo, E. Plasma physics and technology; industrial applications. Vacuum 2002, 64, 327–336. [CrossRef] 15. Hippler, R.; Kersten, H.; Schmidt, M.; Schoenbach, K.H. Low Temperature Plasmas. Fundamentals, Technologies, and Techniques, 2nd ed.; Wiley-VCH: Weinheim, Germany, 2008. 16. Meichsner, J.; Schmidt, M.; Schneider, R.; Wagner, H.E. Nonthermal Plasma Chemistry and Physics; CRC Press, Taylor & Francis Group: London, UK, 2013. 17. Taylor, P.R.; Pirzada, S.A. Thermal plasma processing of materials: A review. Adv. Perform. Mater. 1994, 1, 35–50. [CrossRef] 18. Dobrynin, D.; Friedman, G.; Fridman, A.; Starikovskiy, A. Inactivation of bacteria using dc corona discharge: Role of ions and humidity. New J. Phys. 2011, 13, 103033. [CrossRef] [PubMed] 19. Rupf, S.; Lehmann, A.; Hannig, M.; Schafer, B.; Schubert, A.; Feldmann, U.; Schindler, A. Killing of adherent oral microbes by a non-thermal atmospheric plasma jet. J. Med. Microbiol. 2010, 59, 206–212. [CrossRef] [PubMed] 20. Eto, H.; Ono, Y.; Ogino, A.; Nagatsua, M. Low-temperature sterilization of wrapped materials using ﬂexible sheet-type dielectric barrier discharge. Appl. Phys. Lett. 2008, 93, 221502. [CrossRef] 21. Kolb, J.F.; Mohamed, A.A.H.; Price, R.O.; Swanson, R.J.; Bowman, A.; Chiavarini, R.L.; Stacey, M.; Schoenbach, K.H. Cold atmospheric pressure air plasma jet for medical applications. Appl. Phys. Lett. 2008, 92, 241501. [CrossRef] 22. Dobrynin, D.; Arjunan, K.; Fridman, A.; Friedman, G.; Morss Clyne, A. Direct and controllable nitric oxide delivery into biological media and living cells by a pin-to hole spark discharge (PHD) plasma. J. Phys. D Appl. Phys. 2011, 44, 075201. [CrossRef] 23. Rossi, F.; Kylian, O.; Rauscher, H.; Gilliland, D.; Sirghi, L. Use of a low-pressure plasma discharge for the decontamination and sterilization of medical devices. Pure Appl. Chem. 2008, 80, 1939–1951. [CrossRef] 23. Rossi, F.; Kylian, O.; Rauscher, H.; Gilliland, D.; Sirghi, L. Use of a low-pressure plasma discharge for the decontamination and sterilization of medical devices. Pure Appl. Chem. 2008, 80, 1939–1951. [CrossRef] 24. Stapelmann, K.; Fiebrandt, M.; Raguse, M.; Awakowicz, P.; Reitz, G.; Moeller, R. Utilization of low-pressure decontamination and sterilization of medical devices. Pure Appl. Chem. 2008, 80, 1939–1951. [CrossRef] 24. Stapelmann, K.; Fiebrandt, M.; Raguse, M.; Awakowicz, P.; Reitz, G.; Moeller, R. Utilization of low-pressure plasma to inactivate bacterial spores on stainless steel screws. References 1. Langmuir, I. Oscillations in ionized gases. Proc. Natl. Acad. Sci. USA 1928, 14, 627–637. [CrossRef] [PubMed] 2. Ichimaru, S. Nuclear fusion in dense plasmas. Rev. Mod. Phys. 1993, 65, 255–259. [CrossRef] 3. Team, J. Fusion energy production from a deuterium-tritium plasma in the JET tokamak. Nuc. Fus. 1992, 32, 187–203. [CrossRef] 1. Langmuir, I. Oscillations in ionized gases. Proc. Natl. Acad. Sci. USA 1928, 14, 627–637. [CrossRef] [PubMed] 2. Ichimaru, S. Nuclear fusion in dense plasmas. Rev. Mod. Phys. 1993, 65, 255 259. [CrossRef] 3. Team, J. Fusion energy production from a deuterium-tritium plasma in the JET tokamak. Nuc. Fus. 1992, 32, 187–203. [CrossRef] 3. Team, J. Fusion energy production from a deuterium-tritium plasma in the JET tokamak. Nuc. Fus. 1992, 32, 187–203. [CrossRef] Int. J. Mol. Sci. 2019, 20, 5216 12 of 17 12 of 17 4. Coburn, J.W.; Winters, H.F. Plasma etching-A discussion of mechanisms. J. Vac. Sci. Technol. 1979, 16, 391–403. [CrossRef] 5. Sakudo, A.; Shintani, H. Sterilization and Disinfection by Plasma: Sterilization Mechanisms, Biological and Medical Applications (Medical Devices and Equipment); Nova Science Publishers: New York, NY, USA, 2010. 6. Shintani, H.; Sakudo, A. Gas Plasma Sterilization in Microbiology: Theory, Applications, Pitfalls and New Perspectives; Caister Academic Press: London, UK, 2016. 7. Sakudo, A.; Shintani, H.; Yagyu, Y. Plasma sterilization. In Block’s Disinfection, Sterilization, and Preservation; McDonnell, G.E., Ed.; Lippincott Williams & Wilkins: Philadelphia, PA, USA, 2019; in press. 7. Sakudo, A.; Shintani, H.; Yagyu, Y. Plasma sterilization. In Block s Disinfection, Sterilization, and Preservation; McDonnell, G.E., Ed.; Lippincott Williams & Wilkins: Philadelphia, PA, USA, 2019; in press. McDonnell, G.E., Ed.; Lippincott Williams & Wilkins: Philadelphia, PA, USA, 2019; in press. 8. Nandkumar, N. Plasma-The fourth state of matter. Int. J. Sci. Technol. Res. 2014, 3, 49–52. 9. Fridman, A. Plasma Chemistry; Cambridge University Press: Cambridge, UK, 2012. Nehra, V.; Kumar, A.; Dwivedi, H. Atmospheric non-thermal plasma sources. Int J. Eng. 2008, 2, 53–68. 11. Boulos, M.I.; Fauchais, P.; Pfender, E. Thermal Plasma: Fundamental and Applications, Vol. 1; Plenum Press: New York, NY, USA, 1994. 12. Boulos, M.I. New frontiers in thermal plasma processing. Pure Appl. Chem. 1996, 68, 1007–1010. [CrossRef] 12. Boulos, M.I. New frontiers in thermal plasma processing. Pure Appl. Chem. 1996, 68, 1007–1010. [CrossRef] 13. Heberlein, J. New approaches in thermal plasma technology. Pure Appl. Chem. 2002, 74, 327–335. [CrossRef] 13. Heberlein, J. New approaches in thermal plasma technology. Pure Appl. Chem. References Astrobiology 2013, 13, 597–606. [CrossRef] 25. Teschke, M.; Kedzierski, J.; Finantu-Dinu, E.G.; Korzec, D.; Engemann, J. High-speed photographs of a dielectric barrier atmospheric pressure plasma jet IEEE Trans Plasma Sci 2005 33 310–311 [CrossRef] 24. Stapelmann, K.; Fiebrandt, M.; Raguse, M.; Awakowicz, P.; Reitz, G.; Moeller, R. Utilization of low-pressure plasma to inactivate bacterial spores on stainless steel screws. Astrobiology 2013, 13, 597–606. [CrossRef] 24. Stapelmann, K.; Fiebrandt, M.; Raguse, M.; Awakowicz, P.; Reitz, G.; Moeller, R. Utilization of low-pressure plasma to inactivate bacterial spores on stainless steel screws. Astrobiology 2013, 13, 597–606. [CrossRef] 25. Teschke, M.; Kedzierski, J.; Finantu-Dinu, E.G.; Korzec, D.; Engemann, J. High-speed photographs of a 5. Teschke, M.; Kedzierski, J.; Finantu-Dinu, E.G.; Korzec, D.; Engemann, J. High-speed photographs dielectric barrier atmospheric pressure plasma jet. IEEE Trans. Plasma Sci. 2005, 33, 310–311. [CrossRef 26. Kalghatgi, S.U.; Fridman, G.; Cooper, M.; Nagaraj, G.; Peddinghaus, M.; Balasubramanian, M.; Vasilets, V.N.; Gutsol, A.F.; Fridman, A.; Friedman, G. Mechanism of blood coagulation by nonthermal atmospheric pressure dielectric barrier discharge plasma. IEEE Trans. Plasma Sci. 2007, 35, 1559. [CrossRef] 27. Fridman, G.; Peddinghaus, M.; Balasubramanian, M.; Ayan, H.; Fridman, A.; Gutsol, A.; Brooks, A. Blood coagulation and living tissue sterilization by ﬂoating-electrode dielectric barrier discharge in air. Plasma Chem. Plasma Process. 2006, 26, 425. [CrossRef] 28. Cooper, M.; Fridman, G.; Fridman, A.; Joshi, S. Biological responses of Bacillus stratosphericus to ﬂoating electrode-dielectric barrier discharge plasma treatment. J. Appl. Microbiol. 2010, 109, 2039. [CrossRef] [PubMed] Int. J. Mol. Sci. 2019, 20, 5216 13 of 17 29. Schoenbach, K.; Verhappen, R.; Tessnow, T.; Peterkin, F.; Byszewski, W. Microhollow cathode discharges. Appl. Phys. Lett. 1996, 68, 13. [CrossRef] 30. Fridman, G.; Fridman, G.; Gutsol, A.; Shekhter, A.B.; Vasilets, V.N.; Fridman, A. Applied plasma medicine. Plasma Process. Polym. 2008, 5, 503–533. [CrossRef] 31. Shintani, H.; Sakudo, A.; Burke, P.; McDonnell, G. Gas plasma sterilization of microorganisms and mechanisms of action. Exp. Ther. Med. 2010, 1, 731–738. [CrossRef] [PubMed] 32. Pankaj, S.K.; Wan, Z.; Keener, K.M. Eﬀects of cold plasma on food quality: A review. Foods 2018, 7, 4. [CrossRef] [PubMed] 33. Kamgang-Youbi, G.; Herry, J.M.; Meylheuc, T.; Brisset, J.L.; Bellon-Fontaine, M.N.; Doubla, A.; Naitali, M. Microbial inactivation using plasma-activated water obtained by gliding electric discharges. Lett. Appl. Microbiol. 2009, 48, 13–18. [CrossRef] [PubMed] 34. Duan, J.; Lu, X.; He, G. References The selective eﬀect of plasma activated medium in an in vitro co-culture of liver cancer and normal cells. J. Appl. Phys. 2017, 121, 013302. [CrossRef] 35. Yan, D.; Talbot, A.; Nourmohammadi, N.; Cheng, X.; Canady, J.; Sherman, J.; Keidar, M. Principles of using cold atmospheric plasma stimulated media for cancer treatment. Sci. Rep. 2015, 5, 18339. [CrossRef] 36. Park, J.Y.; Park, S.; Choe, W.; Yong, H.I.; Jo, C.; Kim, K. Plasma-functionalized solution: A potent antimicrobial agent for biomedical applications from antibacterial therapeutics to biomaterial surface engineering. ACS Appl. Mater. Interfaces 2017, 9, 43470–43477. [CrossRef] f 37. Ikawa, S.; Tani, A.; Nakashima, Y.; Kitano, K. Physicochemical properties of bactericidal plasma-treated water. J. Phys. D Appl. Phys. 2016, 49, 425401. [CrossRef] 38. Van Boxem, W.; Van der Paal, J.; Gorbanev, Y.; Vanuytsel, S.; Smits, E.; Dewilde, S.; Bogaerts, A. Anti-cancer capacity of plasma-treated PBS: Eﬀect of chemical composition on cancer cell cytotoxicity. Sci Rep. 2017, 7, 16478. [CrossRef] [PubMed] 39. Liedtke, K.R.; Bekeschus, S.; Kaeding, A.; Hackbarth, C.; Kuehn, J.P.; Heidecke, C.D.; von Bernstorﬀ, W.; von Woedtke, T.; Partecke, L.I. Non-thermal plasma-treated solution demonstrates antitumor activity against pancreatic cancer cells in vitro and in vivo. Sci. Rep. 2017, 7, 8319. [CrossRef] [PubMed] 40. Balan, G.G.; Rosca, I.; Ursu, E.L.; Doroftei, F.; Bostanaru, A.C.; Hnatiuc, E.; Nastasa, V.; Sandru, V.; Stefanescu, G.; Trifan, A.; et al. Plasma-activated water: A new and eﬀective alternative for duodenoscope reprocessing. Infect. Drug Resist. 2018, 11, 727–733. [CrossRef] [PubMed] 41. Tanaka, H.; Mizuno, M.; Ishikawa, K.; Nakamura, K.; Utsumi, F.; Kajiyama, H.; Kano, H.; Maruyama, S.; Kikkawa, F.; Hori, M. Cell survival and proliferation signaling pathways are downregulated by plasma-activated medium in glioblastoma brain tumor cells. Plasma Med. 2012, 2, 207–220. [CrossRef] 42. Sakudo, A.; Toyokawa, Y.; Imanishi, Y.; Murakami, T. Crucial roles of reactive chemical species in modiﬁcation of respiratory syncytial virus by nitrogen gas plasma. Mater. Sci. Eng. C Mater. Biol. Appl. 2017, 74, 131–136. [CrossRef] 43. McDonnell, G.E. Antisepsis, Disinfection, and Sterilization; ASM Press: Washington, DC, USA, 2007. 44. Knipe, D.M.; Howley, P.M.; Griﬃn, D.E.; Lamb, R.A.; Martin, M.A. Fields Virology, 5th ed.; Lippincott Williams & Wilkins: Philadelphia, PA, USA, 2006. 45. Yokoyama, T.; Murai, K.; Murozuka, T.; Wakisaka, A.; Tanifuji, M.; Fujii, N.; Tomono, T. Removal of small non-enveloped viruses by nanoﬁltration. Vox Sang. 2004, 86, 225–229. [CrossRef] 6. Niemira, B.A. References Cold plasma reduction of Salmonella and Escherichia coli O157:H7 on almonds using amb pressure gases. J. Food Sci. 2012, 77, M171–M175. [CrossRef] 47. Klampﬂ, T.G.; Isbary, G.; Shimizu, T.; Li, Y.F.; Zimmermann, J.L.; Stolz, W.; Schlegel, J.; Morﬁll, G.E.; Schmidt, H.U. Cold atmospheric air plasma sterilization against spores and other microorganisms of clinical interest. Appl. Environ. Microbiol. 2012, 78, 5077–5082. [CrossRef] 48. Sung, S.J.; Huh, J.B.; Yun, M.J.; Chang, B.M.; Jeong, C.M.; Jeon, Y.C. Sterilization eﬀect of atmospheric pressure non-thermal air plasma on dental instruments. J. Adv. Prosthodont 2013, 5, 2–8. [CrossRef] 49. Tian, Y.; Sun, P.; Wu, H.; Bai, N.; Wang, R.; Zhu, W.; Zhang, J.; Liu, F. Inactivation of Staphylococcus aureus and Enterococcus faecalis by a direct-current, cold atmospheric-pressure air plasma microjet. J. Biomed. Res. 2010, 24, 264–269. [CrossRef] 50. Vandervoort, K.G.; Brelles-Marino, G. Plasma-mediated inactivation of Pseudomonas aeruginosa bioﬁlms grown on borosilicate surfaces under continuous culture system. PLoS ONE 2014, 9, e108512. [CrossRef] [PubMed] Int. J. Mol. Sci. 2019, 20, 5216 14 of 17 51. Niemira, B.A.; Boyd, G.; Sites, J. Cold plasma rapid decontamination of food contact surfaces contaminated with Salmonella bioﬁlms. J. Food Sci. 2014, 79, M917–M922. [CrossRef] [PubMed] 52. WHO (World Health Organization). Antimicrobial Resistance. Available online: http://www.who.int/ mediacentre/factsheets/fs194/en/ (accessed on 24 June 2019). 53. CDC (The US Centers for Disease Control and Prevention). National Antimicrobial Resistance Monitoring System for Enteric Bacteria (NARMS). Available online: https://www.cdc.gov/narms/index.html (accessed on 24 June 2019). 54. Krauland, M.G.; Marsh, J.W.; Paterson, D.L.; Harrison, L.H. Integron-mediated multidrug resistance in a global collection of nontyphoidal Salmonella enterica isolates. Emerg Infect. Dis. 2009, 15, 388–396. [CrossRef] [PubMed] 55. Maeda, K.; Toyokawa, Y.; Shimizu, N.; Imanishi, Y.; Sakudo, A. Inactivation of Salmonella by nitrogen gas plasma generated by a static induction thyristor as a pulsed power supply. Food Control. 2015, 52, 54–59. [CrossRef] 56. Guo, L.; Xu, R.; Zhao, Y.; Liu, D.; Liu, Z.; Wang, X.; Chen, H.; Kong, M.G. Gas Plasma pre-treatment increases antibiotic sensitivity and persister eradication in methicillin-resistant Staphylococcus aureus. Front. Microbiol. 2018, 9, 537. [CrossRef] 57. Sakudo, A.; Toyokawa, Y.; Misawa, T.; Imanishi, Y. Degradation and detoxiﬁcation of aﬂatoxin B1 using nitrogen gas plasma generated by a static induction thyristor as a pulsed power supply. Food Control. 2017, 73B, 619–626. [CrossRef] 58. Scholtz, V.; Pazlarova, J.; Souskova, H.; Khun, J.; Julak, J. Nonthermal plasma-A tool for decontamination and disinfection. Biotechnol. Adv. 2015, 33, 1108–1119. [CrossRef] 59. References Short communication: Cold atmospheric plasma inactivation of Prototheca zopﬁi isolated from bovine milk. J. Dairy Sci. 2018, 101, 118–122. [CrossRef] [PubMed] 0. Wang, X.Q.; Wang, F.P.; Chen, W.; Huang, J.; Bazaka, K.; Ostrikov, K.K. Non-equilibrium plasma preven of Schistosoma japonicum transmission. Sci. Rep. 2016, 6, 35353. [CrossRef] 71. Hayes, J.; Kirf, D.; Garvey, M.; Rowan, N. Disinfection and toxicological assessments of pulsed UV and pulsed-plasma gas-discharge treated-water containing the waterborne protozoan enteroparasite Cryptosporidium parvum. J. Microbiol. Methods 2013, 94, 325–337. [CrossRef] 72. Baxter, H.C.; Campbell, G.A.; Whittaker, A.G.; Jones, A.C.; Aitken, A.; Simpson, A.H.; Casey, M.; Bountiﬀ, L.; Gibbard, L.; Baxter, R.L. Elimination of transmissible spongiform encephalopathy infectivity and decontamination of surgical instruments by using radio-frequency gas-plasma treatment. J. Gen. Virol. 2005, 86, 2393–2399. [CrossRef] 73. von Keudell, A.; Awakowicz, P.; Benedikt, J.; Raballand, V.; Yanguas-Gil, A.; Opretzka, J.; Flötgen, C.; Reuter, R.; Byelykh, L.; Halfmann, H.; et al. Inactivation of bacteria and biomolecules by low pressure plasma discharges. Plasma Process. Polym. 2010, 7, 327–352. [CrossRef] 74. Sakudo, A.; Imanishi, Y. Degradation and inactivation of Shiga toxins by nitrogen gas plasma. AMB Express 2017, 7, 77. [CrossRef] [PubMed] 75. McCombs, G.B.; Darby, M.L. New discoveries and directions for medical, dental and dental hygiene research: Low temperature atmospheric pressure plasma. Int. J. Dent. Hyg. 2010, 8, 10–15. [CrossRef] [PubMed] 76. Shintani, H.; Shimizu, N.; Imanishi, Y.; Sekiya, T.; Tamazawa, K.; Taniguchi, A.; Kido, N. Inactivation of microorganisms and endotoxins by low temperature nitrogen gas plasma exposure. Biocontrol. Sci. 2007, 12, 131–143. [CrossRef] [PubMed] 77. Liao, X.; Liu, D.; Xiang, Q.; Ahn, J.; Chen, S.; Ye, X.; Ding, T. Inactivation mechanisms of non-thermal plasma on microbes: A review. Food Control. 2017, 75, 83–91. [CrossRef] 78. Traba, C.; Chen, L.; Liang, J.F. Low power gas discharge plasma mediated inactivation and removal of bioﬁlms formed on biomaterials. Curr. Appl. Phys. 2013, 13, S12–S18. [CrossRef] 79. Kylian, O.; Sasaki, T.; Rossi, F. Plasma sterilization of Geobacillus stearothermophilus by O2:N2 RF inductively coupled plasma. Eur Phys. J. Appl. Phys. 2006, 34, 139–142. [CrossRef] 80. Rossi, F.; Kylian, O.; Hasiwa, M. Decontamination of surfaces by low pressure plasma discharges. Plasma Process. Polym. 2006, 3, 431–442. [CrossRef] 81. Rossi, F.; Kylian, O.; Hasiwa, M. Mechanisms of sterilization and decontamination of surfaces by low-pressure plasma. References Soušková, H.; Scholtz, V.; Julák, J.; Savická, D. The fungal spores survival under the low-temperature plasma. In Plasma for Bio-Decontamination, Medicine and Food Security (NATO Science for Peace and Security Series A: Chemistry and Biology); Machala, Z., Hensel, K., Akishev, Y., Eds.; Springer: New York, NY, USA, 2012; pp. 57–66. 60. Souskova, H.; Scholtz, V.; Julak, J.; Kommova, L.; Savicka, D.; Pazlarova, J. The survival of micromycetes and yeasts under the low-temperature plasma generated in electrical discharge. Folia Microbiol. (Praha) 2011, 56, 77–79. [CrossRef] 61. Sakudo, A.; Shimizu, N.; Imanishi, Y.; Ikuta, K. N2 gas plasma inactivates inﬂuenza virus by inducing changes in viral surface morphology, protein, and genomic RNA. Biomed. Res. Int. 2013, 2013, 694269. [CrossRef] 62. Sakudo, A.; Toyokawa, Y.; Imanishi, Y. Nitrogen gas plasma generated by a static induction thyristor as a pulsed power supply inactivates adenovirus. PLoS ONE 2016, 11, e0157922. [CrossRef] 63. Alshraiedeh, N.H.; Alkawareek, M.Y.; Gorman, S.P.; Graham, W.G.; Gilmore, B.F. Atmospheric pressure, nonthermal plasma inactivation of MS2 bacteriophage: Eﬀect of oxygen concentration on virucidal activity. J. Appl. Microbiol. 2013, 115, 1420–1426. [CrossRef] [PubMed] 64. Guo, L.; Xu, R.; Gou, L.; Liu, Z.; Zhao, Y.; Liu, D.; Zhang, L.; Chen, H.; Kong, M.G. Mechanism of virus inactivation by cold atmospheric-pressure plasma and plasma-activated water. Appl. Environ. Microbiol. 2018, 84. [CrossRef] [PubMed] 65. Wu, Y.; Liang, Y.; Wei, K.; Li, W.; Yao, M.; Zhang, J.; Grinshpun, S.A. MS2 virus inactivation by atmospheric-pressure cold plasma using diﬀerent gas carriers and power levels. Appl. Environ. Microbiol. 2015, 81, 996–1002. [CrossRef] [PubMed] 66. Yamashiro, R.; Misawa, T.; Sakudo, A. Key role of singlet oxygen and peroxynitrite in viral RNA damage during virucidal eﬀect of plasma torch on feline calicivirus. Sci. Rep. 2018, 8, 17947. [CrossRef] [PubMed] 67. USEPA (US Environmental Protection Agency). Registration Division Oﬃce of Pesticide Program, Criteria and Standards Division Oﬃce of Drinking Water, Guide Standard and Protocol for Testing Microbiological Water Puriﬁers. Available online: https://nepis.epa.gov/Exe/ZyPDF.cgi/9102362E.PDF?Dockey=9102362E.pdf (accessed on 24 June 2019). 68. Filipic, A.; Primc, G.; Zaplotnik, R.; Mehle, N.; Gutierrez-Aguirre, I.; Ravnikar, M.; Mozetic, M.; Zel, J.; Dobnik, D. Cold atmospheric plasma as a novel method for inactivation of potato virus Y in water samples. Food Environ. Virol. 2019, 11, 220–228. [CrossRef] [PubMed] Int. J. Mol. Sci. 2019, 20, 5216 15 of 17 69. Tyczkowska-Sieron, E.; Markiewicz, J.; Grzesiak, B.; Krukowski, H.; Glowacka, A.; Tyczkowski, J. References 2. Fridman, A.; Friedman, G. Plasma Medicine; Wiley y 93. Laroussi, M.; Kong, M.G.; Morﬁll, G.; Stolz, W. Plasma Medicine: Applications of Low-Temperature Gas. Plasmas in Medicine and Biology; Cambridge University Press: Cambridge, UK, 2012. 94. Wu, A.S.; Kalghatgi, S.; Dobrynin, D.; Sensenig, R.; Cerchar, E.; Podolsky, E.; Dulaimi, E.; Paﬀ, M.; Wasko, K.; Arjunan, K.P.; et al. Porcine intact and wounded skin responses to atmospheric nonthermal plasma. J. Surg. Res. 2013, 179, e1–e12. [CrossRef] 95. Shahbazi Rad, Z.; Abbasi Davani, F. Experimental investigation on electrical characteristics and dose measurement of dielectric barrier discharge plasma device used for therapeutic application. Rev. Sci. Instrum. 2017, 88, 043504. [CrossRef] 96. Ballout, H.; Hertel, M.; Doehring, J.; Kostka, E.; Hartwig, S.; Paris, S.; Preissner, S. Eﬀects of plasma jet, dielectric barrier discharge, photodynamic therapy and sodium hypochlorite on infected curved root canals. J. Biophotonics 2018, 11, e201700186. [CrossRef] 97. Gan, L.; Zhang, S.; Poorun, D.; Liu, D.; Lu, X.; He, M.; Duan, X.; Chen, H. Medical applications of nonthermal atmospheric pressure plasma in dermatology. J. Dtsch. Dermatol. Ges. 2018, 16, 7–13. [CrossRef] 98. Choi, J.S.; Kim, J.; Hong, Y.J.; Bae, W.Y.; Choi, E.H.; Jeong, J.W.; Park, H.K. Evaluation of non-thermal plasma-induced anticancer eﬀects on human colon cancer cells. Biomed. Opt. Express 2017, 8, 2649–2659. [CrossRef] [PubMed] 99. Lin, A.; Gorbanev, Y.; De Backer, J.; Van Loenhout, J.; Van Boxem, W.; Lemiere, F.; Cos, P.; Dewilde, S.; Smits, E.; Bogaerts, A. Non-thermal plasma as a unique delivery system of short-lived reactive oxygen and nitrogen species for immunogenic cell death in melanoma cells. Adv. Sci. (Weinh) 2019, 6, 1802062. [CrossRef] [PubMed] 100. Azzariti, A.; Iacobazzi, R.M.; Di Fonte, R.; Porcelli, L.; Gristina, R.; Favia, P.; Fracassi, F.; Trizio, I.; Silvestris, N.; Guida, G.; et al. Plasma-activated medium triggers cell death and the presentation of immune activating danger signals in melanoma and pancreatic cancer cells. Sci. Rep. 2019, 9, 4099. [CrossRef] [PubMed] 101. Tanaka, H.; Mizuno, M.; Toyokuni, S.; Maruyama, S. Cancer therapy using non-thermal atmospheric pressure plasma with ultra-high electron density. Phys. Plasmas 2015, 22, 122004. [CrossRef] 102. Hattori, N.; Yamada, S.; Torii, K.; Takeda, S.; Nakamura, K.; Tanaka, H.; Kajiyama, H.; Kanda, M.; Fujii, T.; Nakayama, G.; et al. Eﬀectiveness of plasma treatment on pancreatic cancer cells. Int. J. Oncol. 2015, 47, 1655–1662. [CrossRef] 103. References In Advanced Plasma Technology; D’Agostino, R., Favia, P., Kawai, Y., Ikegami, H., Sato, N., Areﬁ-Khonsari, F., Eds.; Wiley-VCH Verlag GmbH &Co.: Weinheim, Germany, 2008; pp. 319–340. 82. Pignata, C.; D’Angelo, D.; Fea, E.; Gilli, G. A review on microbiological decontamination of fresh produce with nonthermal plasma. J. Appl. Microbiol. 2017, 122, 1438–1455. [CrossRef] 83. Toyokawa, Y.; Yagyu, Y.; Misawa, T.; Sakudo, A. A new roller conveyer system of non-thermal gas plasma as a potential control measure of plant pathogenic bacteria in primary food production. Food Control. 2017, 72A, 62–72. [CrossRef] 84. European Commission. Plasma gas technique as electronic preservation practice of organic food and feed, EGTOP/2014, Directorate-General for Agriculture and Rural Development, Directorate, B. Multilateral relations, quality policy, B.4. Organics, Expert Group for Technical Advice on Organic Production EGTOP, Final Report on Food (III). Available online: https://ec.europa.eu/agriculture/organic/sites/orgfarming/ﬁles/ docs/body/egtop-ﬁnal-report-food-iii_en.pdf (accessed on 26 April 2018). 85. Naitali, M.; Kamgang-Youbi, G.; Herry, J.M.; Bellon-Fontaine, M.N.; Brisset, J.L. Combined eﬀects of long-living chemical species during microbial inactivation using atmospheric plasma-treated water. Appl. Environ. Microbiol. 2010, 76, 7662–7664. [CrossRef] 86. Traylor, M.J.; Pavlovich, M.J.; Karim, S.; Hait, P.; Sakiyama, Y.; Clark, D.S.; Graves, D.B. Long-term antibacterial eﬃcacy of air plasma-activated water. J. Phys. D Appl. Phys. 2011, 44, 472001. [CrossRef] 87. Ito, M.; Oh, J.S.; Ohta, T.; Shiratani, M.; Hori, M. Current status and future prospects of agricultural applications using atmospheric-pressure plasma technologies. Plasma Process. Polym. 2017, 15, e1700073. [CrossRef] Int. J. Mol. Sci. 2019, 20, 5216 16 of 17 88. Nishimura, J.; Takahashi, K.; Takaki, K.; Koide, S.; Suga, M.; Orikasa, T.; Teramoto, Y.; Uchino, T. Removal of ethylene and by-products using dielectric barrier discharge with Ag nanoparticle-loaded Zeolite for keeping freshness of fruits and vegetables. Trans. Mater. Res. Soc. Jpn. 2016, 41, 41–45. [CrossRef] 9. Itarashiki, T.; Ohshiro, S.; Sakudo, A.; Hayashi, N. Current trend of medical sterilization and disinfec methods using plasmas. J. Plasma Fus. Res. 2015, 91, 505–513. 90. Shiely, F.; Fallon, D.; Casey, C.; Kerins, D.M.; Eustace, J.A. Trial of a novel plasma gas disinfection system (Radica) to reduce mattress residual bacterial contamination in the acute hospital setting: A preliminary study. Ir J. Med. Sci. 2017, 186, 17–21. [CrossRef] 91. Šimonˇcicová, J.; Kryštofová, S.; Medvecká, V.; ˇDurišová, K.; Kaliˇnáková, B. Technical applications of plasma treatments: Current state and perspectives. Appl. Microbiol. Biotechnol. 2019, 103, 5117–5129. [CrossRef] p p pp 92. Fridman, A.; Friedman, G. Plasma Medicine; Wiley-Blackwell: Oxford, UK, 2013. References Utsumi, F.; Kajiyama, H.; Nakamura, K.; Tanaka, H.; Mizuno, M.; Ishikawa, K.; Kondo, H.; Kano, H.; Hori, M.; Kikkawa, F. Eﬀect of indirect nonequilibrium atmospheric pressure plasma on anti-proliferative activity against chronic chemo-resistant ovarian cancer cells in vitro and in vivo. PLoS ONE 2013, 8, e81576. [CrossRef] 104. Nakamura, K.; Peng, Y.; Utsumi, F.; Tanaka, H.; Mizuno, M.; Toyokuni, S.; Hori, M.; Kikkawa, F.; Kajiyama, H. Novel Intraperitoneal Treatment With Non-thermal plasma-activated medium inhibits metastatic potential of ovarian cancer cells. Sci. Rep. 2017, 7, 6085. [CrossRef] 105. Sato, T.; Yokoyama, M.; Johkura, K. A key inactivation factor of HeLa cell viability by a plasma ﬂow. J. Phys. D Appl. Phys. 2011, 44, 372001. [CrossRef] 106. Machala, Z.; Tarabova, B.; Hensel, K.; Spetlikova, E.; Sikurova, L.; Lukes, P. Formation of ROS and RNS in water electro-sprayed through transient spark discharge in air and their bactericidal eﬀects. Plasma Process. Polym. 2013, 2013, 649–659. [CrossRef] Int. J. Mol. Sci. 2019, 20, 5216 17 of 17 17 of 17 107. Kurake, N.; Tanaka, H.; Ishikawa, K.; Kondo, T.; Sekine, M.; Nakamura, K.; Kajiyama, H.; Kikkawa, F.; Mizuno, M.; Hori, M. Cell survival of glioblastoma grown in medium containing hydrogen peroxide and/or nitrite, or in plasma-activated medium. Arch. Biochem. Biophys. 2016, 605, 102–108. [CrossRef] 08. Hashim, S.A.; Samsudin, F.N.; Wong, C.S.; Abu Bakar, K.; Yap, S.L.; Mohd Zin, M.F. Non-thermal plasma air and water remediation. Arch. Biochem. Biophys. 2016, 605, 34–40. [CrossRef] [PubMed] 109. Magureanu, M.; Piroi, D.; Mandache, N.B.; David, V.; Medvedovici, A.; Bradu, C.; Parvulescu, V.I. Degradation of antibiotics in water by non-thermal plasma treatment. Water Res. 2011, 45, 3407–3416. [CrossRef] [PubMed] © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W4280628515	https://napier-surface.worktribe.com/file/2873145/1/Approaching%20the%20domesticated%20plant%20holobiont%20from%20a%20community%20evolution%20perspective	English	null	Approaching the domesticated plant holobiont from a community evolution perspective	Microbiology	2,022	cc-by	4,590	INTRODUCTION In recent years, we have come to appreciate that as single individuals can have heritable phenotypes, the effect of these phenotypes on a community or at ecosystem level can also lead to heritable community and ecosystem phenotypes [1]. Because micro-organisms are ubiquitous in virtually all environments on Earth, plant and animal species do not evolve in isolation but are colonized by a rich and diverse microbial community [2]. Therefore, the effect of a heritable host phenotype can lead to a heritable component of the host microbiome. Potentially, the host and its microbiome, a holobiont, can be subject to community selection, which may lead to community evolution [3]. Community evolution is the ‘outcome of selection operating at multiple levels that results in the differential survival and proliferation of communities’ [4]. Through community evolution, specific microbial community phenotypes could arise, which may provide positive microbe-to-host effects. One advantage of approaching plant–microbe interactions from a community selection perspective is that community selection does not make any assumptions about the level at which selection is occurring or whether selection acts on the holobiont as a unit [4]. In this approach, we simply define the holobiont as a community of organisms and micro-organisms in which community selection can occur [4]. Central to theories of community evolution is the concept of heritability. This aspect is often confused with inheritance, but they refer to two different processes. Inheritance is the vertical transmission of members of the microbiome from one generation to the other. Heritability measures how much of the variation in the microbiome can be attributed to host genetic variation [5] and it can be calculated for single microbial members or at a community level. As we will see later, we argue that heritability, when expanded to a community level, can provide useful insight into the evolution of the holobiont in agricultural settings. INSIGHT REVIEW Soldan et al., Microbiology 2022;168:001188 DOI 10.1099/mic.0.001188 Approaching the domesticated plant holobiont from a community evolution perspective Abstract Plants establish a pivotal relationship with their microbiome and are often conceptualized as holobionts. Nonetheless, holobiont theo- ries have attracted much criticism, especially concerning the fact that the holobiont is rarely a unit of selection. In previous work, we discussed how the plant microbiome can be considered to be an ‘ecosystem on a leash’, which is subject to the influence of natural selection acting on plant traits. We proposed that in domesticated plants the assembly of the plant microbiome can usefully be concep- tualized as being subject to a ‘double leash’, which encompasses both the effect of artificial selection imposed by the domesticator on plant traits and the leash from the plant to the microbiome. Here we approach the domesticated plant holobiont, simply defined as a community of organisms, from a community evolution point of view, and show how community heritability (a measure of community selection) complements the ‘double-leash’ framework in providing a community-level view of plant domestication and its impact on plant–microbe interactions. We also propose simple experiments that could be performed to investigate whether plant domestication has altered the potential for community selection at the holobiont level. Approaching the domesticated plant holobiont from a community evolution perspective Riccardo Soldan1,, Marco Fusi2 and Gail M. Preston1, Riccardo Soldan1,, Marco Fusi2 and Gail M. Preston1, Riccardo Soldan1,, Marco Fusi2 and Gail M. Preston1, g Correspondence: Riccardo Soldan, riccardosoldan@hotmail.it; Gail M. Preston, gail.preston@plants.ox.ac.uk Abbreviations: H2C, broad-sense community heritability; IIGEs, interspecific indirect genetic effects; NMDS, non-metric multidimensional scaling. 001188 © 2022 The Authors This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution. Received 24 January 2022; Accepted 24 April 2022; Published 17 May 2022 Author affiliations: 1Department of Plant Sciences, University of Oxford, Oxford, UK; 2Edinburgh Napier University, School of Applied Sciences, Edinburgh, UK. g Correspondence: Riccardo Soldan, riccardosoldan@hotmail.it; Gail M. Preston, gail.preston@plants.ox.ac.uk Abbreviations: H2C broad-sense community heritability; IIGEs interspecific indirect genetic effects; NMDS non-metric multidimensional scaling y p p y Author affiliations: 1Department of Plant Sciences, University of Oxford, Oxford, UK; 2Edinburgh Napier Univer Edinburgh, UK. 1 Received 24 January 2022; Accepted 24 April 2022; Published 17 May 2022 Author affiliations: 1Department of Plant Sciences, University of Oxford, Oxford, UK; 2Edinburgh Napier University, School of Applied Sciences, Edinburgh, UK. *Correspondence: Riccardo Soldan, riccardosoldan@hotmail.it; Gail M. Preston, gail.preston@plants.ox.ac.uk Abbreviations: H2C, broad-sense community heritability; IIGEs, interspecific indirect genetic effects; NMDS, non-metric multidimensional scaling. 001188 © 2022 The Authors This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution. Received 24 January 2022; Accepted 24 April 2022; Published 17 May 2022fi BROAD-SENSE COMMUNITY HERITABILITY Broad-sense community heritability (H2 C) expands the concept of broad-sense heritability calculated for single microbial members to a community level [6]. In practice, it measures how much of the variation at a community level is explained by the host genotype. This can be achieved by condensing information on microbial composition into univariate scores using an ordination method, such as non-metric 1 Soldan et al., Microbiology 2022;168:001188 multidimensional scaling (NMDS), and estimating how much of the variation in the NMDS score can be attributed to the genetic variance of the host through an ANOVA. multidimensional scaling (NMDS), and estimating how much of the variation in the NMDS score can be attributed to the genetic variance of the host through an ANOVA. Broad-sense community heritability is important because finding a statistically significant effect of the host genotype in influencing microbial community composition provides evidence of community selection [7]. In other words, it provides evidence of genetic covariance between host and microbiome, documenting a heritable effect on the host-associated microbiome due to plant phenotypes. When community selection occurs, generating holobionts with differential survival, community evolution could lead to specific host– microbiome interactions spanning from mutualism to parasitism. Practically, broad-sense community heritability can be calculated through common garden experiments [7], in which for several plant genotypes and replicates of plant genotypes (clones) community data is measured (e.g. abundance of lichens, insects, micro-organisms) [8–10] and the ANOVA on the NMDS score is calculated, with the host genotype being the explanatory variable. In this approach, the value of community heritability is the R2 of the ANOVA, and the P-value given by the factor plant genotype provides the likeliness of the null hypothesis being true. Conceptually, broad-sense community heritability is proportional to the product of the broad-sense heritability of the host phenotypic traits (‍H2 θ‍) (for example, root length, root architecture, and so forth) and the intensity of the host phenotypic effect at the microbial community level (also referred to as interspecific indirect genetic effects, IIGEs) (‍γ‍), relative to the total selection (‍γ + En‍), that is the sum of the community-level selection intensity (the effect of IIGEs) and other sources of variation in the microbiome community (‍En‍) (for example, soil chemical and physical properties) [7]. H2 C ∝H2 θ γ γ+En‍ IIGEs could be seen as equivalent to the host-to-microbe effects used in the framework of Foster et al. FFECT OF PLANT DOMESTICATION ON BROAD-SENSE COMMUNITY HERITAf In our recent work [12] we conceptualized how the domesticated plant microbiome could differ from that of wild progenitors due to an effect of domesticated plant phenotypes by expanding the ‘ecosystem on a leash’ framework of Foster et al. [13]. We proposed that in domesticated plants the plant microbiome can be conceptualized as being subject to a ‘double leash’ that includes the effect of artificial selection imposed by the domesticator on plant traits and the effect of plant traits on the microbiome. We predicted a reduction in positive-microbe-to-host effects when domesticated plant phenotypes arose due to artificial selection. However, we did not discuss how our double-leash framework could be investigated at a community level. In this sense, broad- sense community heritability could help us to understand how community evolution may have been affected in domesticated vs wild plants. This is important because community evolution could lead to new community phenotypes (holobiont phenotypes) that could have important agricultural repercussions. For example, as discussed below, loss of plant heritable phenotypic variation, often associated with plant domestication, could lead to a reduction in community evolution potential, which in turn could result in plant holobionts that are less resilient to environmental stresses than their wild progenitors. We propose that plant domestication could lower broad-sense community heritability of the host-microbiome by (1) reducing community-level selection on the microbiome (‍γ‍), and (2) increasing other potential sources of variation in the host–microbiome community (‍En‍). BROAD-SENSE COMMUNITY HERITABILITY [9], however, IIGEs can be more generally applied to any community interactions. When host phenotypic effects on microbial communities (IIGEs) are weak (low γ), and the proportion of total selection due to other sources of variation is relatively high (high ‍En‍), broad-sense community heritability approaches zero [7]. For example, low microbial broad-sense community heritability could be the result of transplanting plants into different soils, with the consequence that the soil microbiome is the main determinant of the rhizos- phere microbiome [11] (the rhizosphere is the region in the vicinity of plant roots that is inhabited by a distinctive population of microorganisms which is influenced by plant roots). Under these conditions, any IIGEs resulting from certain root phenotypic traits could be overcome by stronger drivers of microbiome assembly, in this case, the different soils (high En), and, the rhizosphere microbiome would have low broad-sense community heritability. Effect of plant domestication on community-level selection (‍γ‍) One of the main consequences of plant domestication has been a reduction in plant genetic diversity (domestication bottleneck) [14]. Phenotypic traits in wild ancestors with potentially high IIGEs on the microbial community could have been lost due to the domestication bottleneck [15], particularly if these traits did not contribute positively to the plant phenotype from the domesticators’ point of view [12]. The effect of domesticated plant phenotypic traits on microbiome assembly, such as root length and plant height have been investigated for wheat [16] and common bean [17], showing that plant traits can have strong IIGEs. The probability of losing plant phenotypic traits with high IIGEs would increase with the strength of the domestication bottleneck (Fig. 1a), as the more genetic diversity is lost, the more likely it is that plants will lose heritable plant traits, which exert IIGEs. Additionally, artificial selection could have directly selected against certain plant phenotypes if those traits were unsuitable for an agricultural ecosystem. For example, the reduction of plant secondary metabolites to make plants more palatable or less toxic 2 Soldan et al., Microbiology 2022;168:001188 Fig. 1. Effect of plant domestication on broad-sense community heritability depends on the combined effect of the domestication bottleneck and the IIGEs of the host phenotypes. (a) A reduction in plant genetic diversity due to domestication can lead to loss of host phenotypes with strong IIGEs (e.g. concentration of secondary metabolites), which results in a reduction in community heritability of the microbiome. This could be exacerbated when traits subject to artificial selection in domesticated plant phenotypes exert low IIGEs on the microbial communities (e.g. traits selected for aesthetic purposes). (b) When domesticated plant phenotypes exert IIGEs, loss of plant phenotypes due to the domestication bottleneck could be compensated, assuming there are sufficient heritable phenotypic variations to observe the effect of these plant phenotypes. Under these conditions, a reduction in broad-sense community heritability may not occur. Axes for loss of genetic diversity and IIGEs refer to relative loss/gain (range 0–1). The x-axis for broad-sense community heritability is also relative (range 0–1) and represents the total microbial community variation condensed into NMDS scores that can be attributed to host genotypes (see main text for details). Fig. 1. Effect of plant domestication on broad-sense community heritability depends on the combined effect of the domestication bottleneck and the IIGEs of the host phenotypes. Effect of plant domestication on community-level selection (‍γ‍) (a) A reduction in plant genetic diversity due to domestication can lead to loss of host phenotypes with strong IIGEs (e.g. concentration of secondary metabolites), which results in a reduction in community heritability of the microbiome. This could be exacerbated when traits subject to artificial selection in domesticated plant phenotypes exert low IIGEs on the microbial communities (e.g. traits selected for aesthetic purposes). (b) When domesticated plant phenotypes exert IIGEs, loss of plant phenotypes due to the domestication bottleneck could be compensated, assuming there are sufficient heritable phenotypic variations to observe the effect of these plant phenotypes. Under these conditions, a reduction in broad-sense community heritability may not occur. Axes for loss of genetic diversity and IIGEs refer to relative loss/gain (range 0–1). The x-axis for broad-sense community heritability is also relative (range 0–1) and represents the total microbial community variation condensed into NMDS scores that can be attributed to host genotypes (see main text for details). is a common feature of domesticated crops [18]. Secondary metabolites are known to play an important role in host–microbe interactions [19, 20], possibly exerting high IIGEs. Thus, we might generally predict to observe a reduction in both community-level selection and in broad-sense community herit- ability as a consequence of domestication, which is consistent with the double-leash hypothesis. However, an essential condition for community selection to occur is the existence of heritable plant phenotypic variations exerting IIGEs. If domesticated plant phenotypes exert strong host-to-microbe effects, but there is little or no heritable plant phenotypic variation in IIGEs across individuals, community selection at holobiont level cannot occur. However, providing there is heritable phenotypic variation, broad-sense community heritability of the domesticated plant microbiome would not necessarily decrease with the strength of the domestication bottleneck as the loss of plant phenotypes could be balanced out by strong IIGEs induced by domesticated plant phenotypes (Fig. 1b). t of plant domestication on other sources of variation in the microbiome community (‍E The effects of domestication on plant phenotypes and the plant microbiome also need to be interpreted in the context of agricul- tural environments, in which agricultural inputs such as fertilization or soil management practices can have a larger effect on the host-associated microbiome than IIGEs of host traits, thus increasing ‍En‍ [21]. For example, inorganic nitrogen applications have been shown to affect both taxonomical and functional profiles of rhizosphere microbial communities of wheat [22]. At the same time, intensive agriculture reduces soil microbiome alpha-diversity, creating biotic homogenisation [23]. This could ultimately limit the number of community members affected by IIGEs. MICROBIOME BROAD-SENSE COMMUNITY HERITABILITY OF DIFFERENT PLANT MICROBIOME BROAD-SENSE COMMUNITY HERITABILITY OF DIFFERENT PLANT COMPARTMENTS PRACTICAL IMPLICATIONS Approaching the holobiont from a community evolution perspective could enable us to better understand the evolution of the holobiont and identify plant traits leading to the assembly of specific microbial communities. This view is different but comple- mentary to what we have described in our double-leash framework, which focuses on the host individual level. The double-leash framework posits that when domesticated plant phenotypes are selected through artificial selection by the domesticator and they have no consequences for plant fitness, these traits are unlikely to have evolved as an attempt of the host to control the microbiota and receive positive microbe-to-host effects. The community evolution perspective also supports the conceptual development of scenarios in which domestication could lead to reduced community selection through a reduction in IIGEs, irrespective of whether interactions have positive implications for the host, and requires us to consider the possibility that a loss of genetic diversity could lead to scenarios where heritable variation in plant phenotypic traits exerting IIGEs is insufficient for community selection to occur. One criticism could be that it is unlikely that important IIGEs were lost during domestication as the domesticator would have selected against ‘compromised’ holobionts. However, domesticators cannot predict future conditions that would benefit from lost IIGEs that at the time selection was exerted were not important. For example, root trait variation is reduced in domesticated plants [26]. Selection of domesticated plants with reduced root phenotypic variation compared to wild plants may not have major consequences at present. Nonetheless, environmental stresses such as drought are becoming more frequent as a result of climate change [27]. The introduction of higher variation in root phenotypes through crossing with wild progenitors could enable the evolution of holobionts more tolerant to environmental stress as a result of community (holobiont) selection, where enough root trait variation exerting IIGEs exists. Importantly, viewing plant domestication through the lens of community evolution facilitates the design of experiments to detect and quantify IIGEs, which will not only help us to understand whether domestication has led to a loss in plant phenotypic varia- tion that was exerting strong IIGEs, but could also enable us to identify plant traits responsible for certain aspects of microbiome assembly. MICROBIOME BROAD-SENSE COMMUNITY HERITABILITY OF DIFFERENT PLANT COMPARTMENTS While in some contexts it is useful to consider broad-sense community heritability at the level of the whole plant, it is also important to consider that different host compartments can be colonized by different, but interconnected microbial communities [11, 24]. Therefore, a host phenotypic trait (e.g. leaf tannin concentration) could have different IIGEs on microbial communities 3 3 Soldan et al., Microbiology 2022;168:001188 inhabiting different host compartments [e.g. the phyllosphere (above ground plant surfaces that can be colonized by micro- organisms) microbiome compared to the rhizosphere microbiome]. In this example, while the phyllosphere microbiome could be affected directly by tannin concentrations in leaves, the rhizosphere microbiome could be also affected as leaves fall on the ground, where they contribute to and are decomposed by the soil microbiota. In turn, the seed microbiome could be affected by changes in the assembly of the phyllosphere and rhizosphere microbiome, which both contribute micro-organisms to the seed microbiome. At the same time, even within the same plant compartment, microbial communities change dynamically based on the plant developmental stage [25]. For this reason, microbial communities inhabiting different plant compartments at different devel- opmental stages will have different broad-sense community heritability. The domestication process will, therefore, differen- tially affect microbial broad sense community heritability depending on the host compartment, developmental stage and the interaction of host compartments. The effect of IIGEs is also likely to be stronger where community diversity is lower (e.g. endophytes vs non-endophytes) [13] (endophytes are micro-organisms living inside a plant compartment). For this reason, we would expect broad-sense community heritability to be higher for endophytic microbial communities, which can then lead to stronger community-level selection. PRACTICAL IMPLICATIONS A simple initial experiment could be to grow wild and domesticated plants in a small geographical area in their correspondent ecosystems, that is natural ecosystems for wild plants (or as close as possible to natural) and agricultural for domesticated plants. Plant genotypes should be selected to encompass the population genetic diversity of the species being studied (Fig. 2a). Each genotype would be replicated paying attention that replicates of the same genotype are identical (clones or highly homozygous individuals). The microbiome of different plant compartments would then be sampled and analysed for both wild and domesti- cated plant populations. This simple experiment could help us understand (i) how wild and domesticated plants differ in terms of community selection by plant compartment, (ii) whether domesticated plants have lower broad-sense community heritability compared to wild progenitors. This would help demonstrate whether domestication has led to (i) a reduction in IIGEs, (ii) insufficient heritable plant phenotypic variation to detect IIGEs, or (iii) a combination of both. Moreover, these results could lead to the identification of plant compartments for which a reduction of broad-sense community heritability has occurred in domesticated plants compared to wild types. In the hypothetical example shown in Fig. 2a, broad- sense community heritability of the root compartment in wild-type is higher than in domesticated plants. In this scenario, subsequent experiments involving progeny from crosses and backcrosses between wild and domesticated plants and genome- wide association studies would help to identify which plant genotypes and phenotypes are exerting IIGEs (Fig. 2b). An alternative approach would be to examine correlations between plant traits and microbiome assembly, as reported in common bean for root length [17]. 4 4 Soldan et al., Microbiology 2022;168:001188 References 1. Bailey JK, Schweitzer JA, Ubeda F, Koricheva J, LeRoy CJ, et al. From genes to ecosystems: a synthesis of the effects of plant genetic factors across levels of organization. Philos Trans R Soc Lond B Biol Sci 2009;364:1607–1616. 13. Foster KR, Schluter J, Coyte KZ, Rakoff-Nahoum S. The evolu- tion of the host microbiome as an ecosystem on a leash. Nature 2017;548:43–51. 2. McFall-Ngai M, Hadfield MG, Bosch TCG, Carey HV, Domazet-Lošo T, et al. Animals in a bacterial world, a new imperative for the life sciences. Proc Natl Acad Sci U S A 2013;110:3229–3236. 14. Diamond J. Evolution, consequences and future of plant and animal domestication. Nature 2002;418:700–707. 3. Simon J-C, Marchesi JR, Mougel C, Selosse M-A. Host-microbiota interactions: from holobiont theory to analysis. Microbiome 2019;7:5. 15. Brown TA. Is the domestication bottleneck a myth? Nat Plants 2019;5:337–338. 16. Hassani MA, Özkurt E, Franzenburg S, Stukenbrock EH. Ecolog- ical assembly processes of the bacterial and fungal micro- biota of wild and domesticated wheat species. Phytobiomes J 2020;4:217–224. 4. Whitham TG, Allan GJ, Cooper HF, Shuster SM. Intraspecific genetic variation and species interactions contribute to community evolution. Annu Rev Ecol Evol Syst 2020;51:587–612. 5. Henry LP, Bruijning M, Forsberg SKG, Ayroles JF. The microbiome extends host evolutionary potential. Nat Commun 2021;12:5141. 17. Pérez-Jaramillo JE, Carrión VJ, Bosse M, Ferrão LFV, de Hollander M, et al. Linking rhizosphere microbiome composition of wild and domes- ticated Phaseolus vulgaris to genotypic and root phenotypic traits. ISME J 2017;11:2244–2257. 6. Whitham TG, Bailey JK, Schweitzer JA, Shuster SM, Bangert RK, et al. A framework for community and ecosystem genetics: from genes to ecosystems. Nat Rev Genet 2006;7:510–523. 18. Moreira X, Abdala-Roberts L, Gols R, Francisco M. Plant domesti- cation decreases both constitutive and induced chemical defences by direct selection against defensive traits. Sci Rep 2018;8:1–11. 7. Shuster SM, Lonsdorf EV, Wimp GM, Bailey JK, Whitham TG. Community heritability measures the evolutionary consequences of indirect genetic effects on community structure. Evolution 2006;60:991–1003. 19. Korenblum E, Dong Y, Szymanski J, Panda S, Jozwiak A, et al. Rhizosphere microbiome mediates systemic root metabo- lite exudation by root-to-root signaling. Proc Natl Acad Sci U S A 2020;117:3874–3883. 8. Lamit LJ, Lau MK, Naesborg RR, Wojtowicz T, Whitham TG, et al. Genotype variation in bark texture drives lichen commu- nity assembly across multiple environments. Ecology 2015;96:960–971. 20. Kudjordjie EN, Sapkota R, Steffensen SK, Fomsgaard IS, Nicolaisen M. Fig. 2. An experimental framework to enhance community evolution potential in domesticated crops. (a) Analysis of whether broad-sense community heritability is reduced in domesticated plants. Wild and domesticated plants are grown in their correspondent ecosystems [natural ecosystem for wild plants (or as close as possible to natural) and agricultural for domesticated plants]. Plant genotypes (Gn) should be selected to encompass the population genetic diversity of the species being studied. Replicates of the same genotype should be clones or highly homozygotic individuals. Broad-sense community heritability of different plant compartments is then assessed. In the example shown, both the phyllosphere and rhizosphere compartments of domesticated plants have lower broad-sense community heritability than wild progenitors, but the difference in broad-sense community heritability between wild and domesticated plants is more pronounced for the rhizosphere (>>). (b) Backcrossing, GWAS, and multivariate statistics aimed at linking plant traits to microbiome assembly can help to identify plant genotypes and phenotypes exerting IIGEs. (c) Traits responsible for IIGEs are reintroduced into domesticated plants, reestablishing heritable phenotypic variation leading to community selection. (d) Once genetic variation associated with IIGEs is reintroduced into domesticated crops, community evolution can lead to the emergence of new holobionts with new environmental potentials. For example, selection schemes could include the application of stresses, such as drought, and select the holobionts with higher potential in terms of yield, drought resistance, or any other trait of interest. 5 Soldan et al., Microbiology 2022;168:001188 Having found evidence of loss of IIGEs, introgression of wild germplasm into domesticated populations could reintroduce vari- ation in IIGEs and repristinate the potential for community evolution in domesticated plants to occur (Fig. 2c). Subsequently, experiments aimed at selecting the best performing holobionts in certain environmental conditions, such as drought, could be performed (Fig. 2d). The feature for selection could be crop yield or any other trait of interest. The proposed approach would facilitate selection at the holobiont level by first addressing whether domestication has reduced community selection potential. Conflicts of interest l The authors declare that they have no conflicts of interest. l The authors declare that they have no conflicts of interest. 12. Soldan R, Fusi M, Cardinale M, Daffonchio D, Preston GM. The effect of plant domestication on host control of the microbiota. Commun Biol 2021;4:936. CONCLUDING REMARKS Approaching the holobiont, defined as host and host-associated microbiome, from a community level perspective avoids many of the debated aspects of holobiont theories, such as co-evolution and inheritance. In the context of plant domestication, approaching the holobiont from a community-level prospect could give us a more holistic view of plant–microbe interactions in the agricultural ecosystem, pave the way to the identification of plant traits exerting strong IIGEs and cast light on the assembly of domesticated plant microbiomes. Funding information Funding information R.S. is supported by Biotechnology and Biological Sciences Research Council (BBSRC) grant BB/M011224/1, the Oxford Interdisciplinary Bioscience Doctoral Training Partnership (Doctoral Training Centre, University of Oxford) and the Ermenegildo Zegna’s founder scholarship. This project was also supported by BBSRC grant BB/R009236/1 awarded to G.M.P. Author contributions R.S. conceived the idea; R.S. and G.M.P. wrote the manuscript with inputs from MF. All authors critically revised the manuscript.l 25. Moroenyane I, Mendes L, Tremblay J, Tripathi B, Yergeau É. Plant Compartments and developmental stages modulate the balance between niche-based and neutral processes in soybean micro- biome. Microb Ecol 2021;82:416–428. 24. Costello EK, Lauber CL, Hamady M, Fierer N, Gordon JI, et al. Bacte- rial community variation in human body habitats across space and time. Science 2009;326:1694–1697. 26. Isaac ME, Nimmo V, Gaudin ACM, Leptin A, Schmidt JE, et al. Crop domestication, root trait syndromes, and soil nutrient acquisition in organic agroecosystems: a systematic review. Front Sustain Food Syst 2021;5:5. 27. Spinoni J, Vogt JV, Naumann G, Barbosa P, Dosio A. Will drought events become more frequent and severe in Europe? Int J Climatol 2018;38:1718–1736. References Maize synthesized benzoxazinoids affect the host associated micro- biome. Microbiome 2019;7:59. 9. Compson ZG, Hungate BA, Whitham TG, Meneses N, Busby PE, et al. Plant genotype influences aquatic‐terrestrial ecosystem linkages through timing and composition of insect emergence. Ecosphere 2016;7:e01331. 21. Hartman K, van der Heijden MGA, Wittwer RA, Banerjee S, Walser J-C, et al. Cropping practices manipulate abundance patterns of root and soil microbiome members paving the way to smart farming. Microbiome 2018;6:14. 10. Ferrier SM, Bangert RK, Hersch-Green EI, Bailey JK, Allan GJ, et al. Unique arthropod communities on different host-plant genotypes results in greater arthropod diversity. Arthropod Plant Interact 2012;6:187–195. 22. Kavamura VN, Hayat R, Clark IM, Rossmann M, Mendes R, et al. Inorganic nitrogen application affects both taxonomical and predicted functional structure of wheat rhizosphere bacterial communities. Front Microbiol 2018;9:9. 11. Bulgarelli D, Rott M, Schlaeppi K, Ver Loren van Themaat E, Ahmadinejad N, et al. Revealing structure and assembly cues for Arabidopsis root-inhabiting bacterial microbiota. Nature 2012;488:91–95. 23. Olden JD, Leroy Poff N, Douglas MR, Douglas ME, Fausch KD. Ecological and evolutionary consequences of biotic homogeniza- tion. Trends Ecol Evol 2004;19:18–24. 6 Soldan et al., Microbiology 2022;168:001188 7
https://openalex.org/W2590205493	https://jcmr-online.biomedcentral.com/counter/pdf/10.1186/s12968-017-0340-z	English	null	Comparison of CT and CMR for detection and quantification of carotid artery calcification: the Rotterdam Study	Journal of cardiovascular magnetic resonance	2,016	cc-by	6,426	© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Comparison of CT and CMR for detection and quantification of carotid artery calcification: the Rotterdam Study Blerim Mujaj1, Andrés M. Arias Lorza2, Arna van Engelen2, Marleen de Bruijne2,4, Oscar H. Franco1, Aad van der Lugt3, Meike W. Vernooij1,3 and Daniel Bos1,3,5* Mujaj et al. Journal of Cardiovascular Magnetic Resonance (2017) 19:28 DOI 10.1186/s12968-017-0340-z Mujaj et al. Journal of Cardiovascular Magnetic Resonance (2017) 19:28 DOI 10.1186/s12968-017-0340-z Mujaj et al. Journal of Cardiovascular Magnetic Resonance (2017) 19:28 DOI 10.1186/s12968-017-0340-z RESEARCH Open Access Comparison of CT and CMR for detection and quantification of carotid artery calcification: the Rotterdam Study Blerim Mujaj1, Andrés M. Arias Lorza2, Arna van Engelen2, Marleen de Bruijne2,4, Oscar H. Franco1, Aad van der Lugt3, Meike W. Vernooij1,3 and Daniel Bos1,3,5* Abstract Background: Carotid artery atherosclerosis is an important risk factor for stroke. As such, quantitative imaging of carotid artery calcification, as a proxy of atherosclerosis, has become a cornerstone of current stroke research. Yet, population-based data comparing the computed tomography (CT) and cardiovascular magnetic resonance (CMR) for the detection and quantification of calcification remain scarce. Methods: A total of 684 participants from the population-based Rotterdam Study underwent both a CT and CMR of the carotid artery bifurcation to quantify the amount of carotid artery calcification (mean interscan interval: 4.9 ± 1.2 years). We investigated the correlation between the amount of calcification measured on CT and CMR using Spearman’s correlation coefficient, Bland-Altman plots, and linear regression. In addition, using logistic regression modeling, we assessed the association of CT and CMR based calcification volumes with a history of stroke. Results: We found a strong correlation between CT and CMR based calcification volumes (Spearman’s correlation coefficient:0.86, p-value ≤0.01). Bland-Altman analyses showed a good agreement, though CT based calcification volumes were systematically larger. Finally, calcification volume assessed with either imaging modality was associated with a history of stroke with similar effect estimates (odds ratio (OR) per 1-SD increase in calcification volume: 1.52 (95% CI:1.00;2.30) for CT, and 1.47 (95% CI:1.01;2.14) for CMR. Conclusion: CT based and CMR based volumes of carotid artery calcification are highly correlated, but CMR based calcification is systematically smaller than those obtained with CT. Despite this difference, both provide comparable information with regard to a history of stroke. Keywords: CT, CMR, Carotid artery, Atherosclerosis, Calcification, Stroke * Correspondence: d.bos@erasmusmc.nl 1Department of Epidemiology, Erasmus MC, University Medical Center, Office Na 2824k, PO Box 2040, 3000 CA Rotterdam, The Netherlands 3Department of Radiology and Nuclear Medicine, Erasmus MC University Medical Center, Rotterdam, The Netherlands Full list of author information is available at the end of the article Background of atherosclerosis [6]. An important advantage of CT and CMR is that both modalities offer possibilities for detailed characterization and quantification of the ath- erosclerotic plaque [7]. The mostly studied characteristic of the atherosclerotic plaque is calcification, given that it is one of the most prominent plaque characteristics and represents a reliable marker of the underlying plaque burden [8]. For the visualization of calcification, non- contrast CT is acknowledged to be superior to any other imaging modality [9]. Yet, thanks to rapid technological advances, non-contrast CMR now also allows for the detection and quantification of calcification in the ath- erosclerotic plaque [10] and has the major advantage over CT that it does not involve radiation exposure. Atherosclerosis located at the bifurcation of the carotid artery is an important risk factor for stroke [1–5]. As such, quantification of the severity of carotid athero- sclerosis has become an increasingly important topic in stroke research. Multiple non-invasive imaging tech- niques, including ultrasound, computed tomography (CT), and cardiovascular magnetic resonance (CMR), are currently available to obtain measures of the extent * Correspondence: d.bos@erasmusmc.nl 1Department of Epidemiology, Erasmus MC, University Medical Center, Office Na 2824k, PO Box 2040, 3000 CA Rotterdam, The Netherlands 3Department of Radiology and Nuclear Medicine, Erasmus MC University Medical Center, Rotterdam, The Netherlands Full list of author information is available at the end of the article Page 2 of 7 Mujaj et al. Journal of Cardiovascular Magnetic Resonance (2017) 19:28 Mujaj et al. Journal of Cardiovascular Magnetic Resonance (2017) 19:28 Page 2 of 7 Mujaj et al. Journal of Cardiovascular Magnetic Resonance (2017) 19:28 Moreover, with CMR it is possible to visualize additional plaque characteristics such as intraplaque hemorrhage or lipid-rich necrotic core which provide unique add- itional information on the disease. Despite these poten- tial advantages of CMR, it remains unclear whether calcification volumes obtained with CMR are compar- able to those measured with CT. Against this back- ground, we set out to quantify and compare CT-based and CMR-based carotid artery calcification in terms of absolute volumes and with respect to the history of stroke as a relevant clinical outcome, in participants from the population-based Rotterdam Study. Assessment of CT-based calcification We performed a non-enhanced CT-examination (16-or 64-slice MDCT Somatom Sensation, Siemens, Forchheim, Germany) that reached from the aortic arch to the intra- cranial vasculature, to visualize calcification in the extra- cranial carotid arteries. Background The detailed information regarding the scan protocol is described elsewhere [12]. In short, the following scan parameters were used: 16 x 0.75 mm colli- mation, 120 kVp, 100 effective mAs, and 0.5 s rotation time, with a normalized pitch of 1. Images were recon- structed with an effective slice width of 1 mm, a recon- struction interval of 0.5 mm, and a medium sharp convolution kernel [12]. Calcification in the extra-cranial carotid artery was measured bilaterally within three cen- timeters proximal and distal of the bifurcation and was automatically quantified with dedicated commercially available software (syngo calcium scoring, Siemens, Germany) [12]. Calcification volumes in both carotid arter- ies were expressed in cubic millimeters (mm3) [13] (Fig. 1). Assessment of CT-based calcification Assessment of CT-based calcification Assessment of CMR-based calcification Assessment of CMR-based calcification CMR of the carotid arteries was performed on a single 1.5-T scanner (GE Healthcare, Milwaukee, WI, USA) with a dedicated bilateral phased-array surface coil (Machnet, Eelde, The Netherlands). The high-resolution images were obtained using a standardized protocol [14]. First, both carotids were identified by means of two-dimensional (2D) time-of-flight MR angiography. Second, high-resolution CMR sequences were planned to image the carotid bifurcations on both sides. These sequences consisted of four 2D sequences in the axial plane, namely a proton density weighted (PDw)-fast spin echo (FSE)-black blood (BB) sequence (in-plane reso- lution 130/160 x 130/128 = 0.8 x 1 cm); a PDw-FSE-BB with an increased in-plane resolution (in-plane reso- lution 130/224 x 130/160 = 0.5 x 0.8 cm); a PDw-echo planar image (EPI) sequence (in-plane resolution 130/ 160 x 70/160 = 0.8 x 0.4 cm); and a T2w-EPI sequence (in-plane resolution 130/160 x 70/160 = 0.8 x 0.4 cm). Additionally, we performed two 3D sequences, namely a 3D-T1w-gradient echo (GRE) sequence (in-plane reso- lution 180/192 x 180/180 = 0.9 x 1 cm), and a 3D phased-contrast MR angiography (in-plane resolution From October 2007 onwards, carotid CMR was in- corporated in the Rotterdam Study. Between 2007 and 2012, we invited 2,666 participants to undergo CMR of the carotid arteries to study atherosclerotic disease. These participants were selected on the basis of the presence of atherosclerosis in at least one ca- rotid artery on ultrasound examination (defined as intima-media thickness >2.0 mm in one or both ca- rotid arteries), which is regularly performed in all Rotterdam Study participants. In total 1,982 partici- pants underwent carotid CMR. From these 1,982, 808 participants had also undergone a CT-examination. Due to image artifacts or low image quality (n = 31, or errors in the CMR registration process needed for analysis (n = 93) 124 participants were excluded, leav- ing 684 participants with usable CT and CMR data for the current study. The mean time interval be- tween CT scan and CMR scan was 4.9 years (standard deviation 1.2 years). Fig. 1 Example of calcification in the left carotid artery bifurcation (indicated by the red star) on CT (left image) and on CMR (middle image; PDw-FSE-BB sequence, and right image; magnitude image of the 3D-phase contrast sequence) Fig. Methods Setting This study was carried out within the framework of the Rotterdam Study, a prospective population-based study among middle-aged and elderly persons [11]. Between 2003 and 2006, all participants that visited the research center were invited to undergo multi- detector computed tomography (MDCT) to quantify vascular calcification in multiple vessels, including the carotid artery bifurcation [12]. In total 2,524 partici- pants were scanned. Assessment of history of stroke At study entry, all participants were interviewed and a history of stroke was assessed. Moreover, after enroll- ment, all participants are continuously followed for the occurrence of stroke [16]. All potential stroke events were reviewed by research physicians and verified by an experienced stroke neurologist [17]. At the time of CT scan, 38 participants had suffered a prior stroke [16]. Results Table 1 shows the baseline characteristics of the study population. The mean age of participants at the time of CT examination was 68.1 years (SD: 6.1 years). There were 41.5% female participants. We found no calcification in 60 participants (8.8%). There were no instances in which calcification was found on either CT or CMR and not on the other modality. The mean Ln-transformed cal- cification volume for CT was 3.98 mm3 (SD: 1.86 mm3), and 2.70 mm3 (SD: 1.36 mm3) for CMR. Table 1 Baseline characteristics of study participants Sample size 684 Woman 41.5% Age, years at CT scan 68.8 ± 6.1 Age, years at CMR scan 74.2 ± 6.1 CT calcification volumes, mm3a 3.98 ± 1.87a CMR calcification volumes, mm3a 2.70 ± 1.37a Smoking (current) 40.2% Systolic blood pressure (mm/Hg) 146.81 ± 19.46 Diastolic blood pressure (mm/Hg) 79.84 ± 10.85 Diabetes Mellitus 13.3% Serum total cholesterol (mmol/L) 5.6 ± 0.9 HDL cholesterol (mmol/L) 1.4 ± 0.3 Antihypertensive medication use 37.7% Statin medication use 31.1% Stroke events 5.6% Values are means with standard deviations for continuous variables and percentages for dichotomous or categorical variables Abbreviation: CT computed tomography, HDL high-density lipoprotein, CMR cardiovascular magnetic resonance aLn-transformed volumes (Ln(calcification volume + 1 mm3)) Assessment of CMR-based calcification 1 Example of calcification in the left carotid artery bifurcation (indicated by the red star) on CT (left image) and on CMR (middle image; PDw-FSE-BB sequence, and right image; magnitude image of the 3D-phase contrast sequence) Mujaj et al. Journal of Cardiovascular Magnetic Resonance (2017) 19:28 Page 3 of 7 180/256 x 180/128 = 0.7 x 1.4 cm) (Additional file 1: Table S3). The total scanning time was approximately 30 min [14]. Calcification was evaluated bilaterally within three centimeters proximal and distal of the bi- furcation [12]. All calcification measurements on CMR were performed by one trained physician under the supervision of an experienced neuroradiologist. We performed an intra- and inter-observer reproducibility analysis on a random set of 30 CMR examinations. The intra- and inter-agreement was very good [Cohens’ Kappa : 0.91 (95% CI 0.82–0.99) and 0.94 (95% CI 0.86– 0.99)], respectively. We defined calcification as a hypoin- tense region in the plaque on all sequences. We manu- ally annotated and segmented calcification in all plaques using a standardized approach. First, we pre-processed all images using a method that has been described ex- tensively before [15]. This starts with a bias correction to reduce the intensity inhomogeneity characteristic in CMR [15]. Subsequently, the carotid artery in all images was rigidly registered to the black-blood image space using the Elastix tool [15]. For the registration of the sequences, a Region Of Interest (ROI) around the artery in black-blood was used. This ROI was obtained semi- automatically by uniformly growing an extracted carotid artery centerline, which requires three marked seed points at the common, internal and external parts of the artery [15]. Then calcification was manually delineated in every consecutive slice using an annotation tool developed in Mevislab (MeVisLab, MeVis Medical Solutions AG). Fourth, the total volume of calcification was calculated by counting the number of voxels within the annotated areas and multiplying this by the voxel volume (Fig. 1). This provided volumes of calcification in cubic millimeters. 180/256 x 180/128 = 0.7 x 1.4 cm) (Additional file 1: Table S3). The total scanning time was approximately 30 min [14]. Calcification was evaluated bilaterally within three centimeters proximal and distal of the bi- furcation [12]. All calcification measurements on CMR were performed by one trained physician under the supervision of an experienced neuroradiologist. We performed an intra- and inter-observer reproducibility analysis on a random set of 30 CMR examinations. Assessment of CMR-based calcification The intra- and inter-agreement was very good [Cohens’ Kappa : 0.91 (95% CI 0.82–0.99) and 0.94 (95% CI 0.86– 0.99)], respectively. We defined calcification as a hypoin- tense region in the plaque on all sequences. We manu- ally annotated and segmented calcification in all plaques using a standardized approach. First, we pre-processed all images using a method that has been described ex- tensively before [15]. This starts with a bias correction to reduce the intensity inhomogeneity characteristic in CMR [15]. Subsequently, the carotid artery in all images was rigidly registered to the black-blood image space using the Elastix tool [15]. For the registration of the sequences, a Region Of Interest (ROI) around the artery in black-blood was used. This ROI was obtained semi- automatically by uniformly growing an extracted carotid artery centerline, which requires three marked seed points at the common, internal and external parts of the artery [15]. Then calcification was manually delineated in every consecutive slice using an annotation tool developed in Mevislab (MeVisLab, MeVis Medical Solutions AG). Fourth, the total volume of calcification was calculated by counting the number of voxels within the annotated areas and multiplying this by the voxel volume (Fig. 1). This provided volumes of calcification in cubic millimeters. CT-based and CMR-based calcification volumes while adjusting for the time interval between the scans. Given the substantial time interval between the CT and CMR examinations, we furthermore performed a sensitivity analysis in which we analyzed the correlation between CT-based and CMR-based calcification volumes only for those persons with an interval equal or less than 3 years (n = 128). We performed post-hoc sensitivity analysis while adjusting for CT-scanner type also. Third, we assessed the agreement between CT-based and CMR-based calcification volumes using a Bland- Altman analysis. Fourth, as a proof-of-principle, we in- vestigated the association of CT-based and CMR-based calcification volumes (per 1-SD increase) related with a history of stroke using logistic regression while adjust- ing for age, sex and the time interval between CT and CMR, and studied whether the results were comparable for both modalities All analyses were carried out using IBM SPSS Statistics version 21 (International Business Machines Corporation, Armonk, New York). Statistical analysis Due to skewed distributions of the calcification data, we used natural log (Ln) transformed values after we added 1.0 mm3 to the non-transformed data in order to deal with calcification scores of zero (Ln (calcification volume +1.0 mm3)) [16]. Our analysis strategy consisted of four steps. First, we investigated the correlation of CT-based calcification volumes with CMR-based calcification vol- umes using Spearman’s correlation coefficient. Second, we used linear regression to assess the relation between Page 4 of 7 Mujaj et al. Journal of Cardiovascular Magnetic Resonance (2017) 19:28 We found a strong correlation between CT and CMR calcification volumes (Spearman’s correlation coeffi- cient:0.86) (Fig. 2, Additional file 1: Table S1, and Additional file 1: Table S2). This correlation was simi- lar when we investigated the left and right side separ- ately (Additional file 1: Table S1). After performing linear regression with adjustment for the time interval between the CT and CMR scan, the prominent relation between CT-based and CMR-based calcification vol- umes remained present [beta per 1-SD increase in CT- based calcification volume: 0.65 (95% confidence interval (CI): 0.63;0.68)]. After performing the analyses in those persons with a time interval between the scans of less or equal to 3 years, the association between CT-based and CMR-based calcification volumes was similar [beta per 1-SD increase in CT-based calcification volume: 0.65 (95% CI: 0.58;0.72)]. Adjustment for CT-scanner type did not influence the results (data not shown). CMR-based volumes of carotid artery calcification are highly correlated, but CMR-based calcification is system- atically smaller than those obtained with CT. Despite this difference, both provide comparable information with regard to a history of stroke. g y We found that CT-based and CMR-based calcification volumes were highly correlated. Yet, we also found that the volumes measured with CMR were systematically smaller than those measured on CT. This was especially interesting in light of the fact that the CMR was per- formed on average 4 years later than the CT. Given that our scanning protocol on CT was specifically designed for the visualization of vascular calcification combined with that CT is currently the gold standard for the assessment calcification, it is likely that with CMR the amount of calcification is systematically underestimated [6]. The reason for this could the differences between CT-based and CMR-based calcification volume may be explained by differences in image analysis to a certain extent. Statistical analysis Additionally, differences in spatial resolution be- tween CT and CMR might be a potential explanation for this difference. In this light, it is important to note that CT images were analyzed automatically using dedicated commercially available software, whilst CMR images were analyzed manually for the presence and amount of calcification. To our knowledge, there are no studies that have compared CT and CMR on the detection and quantification of carotid artery using a non-invasive population-based approach. Previous research performed on the comparison between CT and CMR in 50 patients with recent TIA or minor stroke, demonstrated a correl- ation between CT-based and CMR-based calcification volumes of the only p: 0.55 [18]. We demonstrate that with the use of dedicated CMR-multi-sequences for the Figure 3 shows the Bland-Altman plot for the relation between the absolute differences in Ln-transformed calci- fication volumes and the mean of the two measurements of 1.27 mm3 (standard deviation: 0.92). We found that the CT-based calcification volumes were consistently larger than those obtained from CMR. When investigating the relationship between calcifica- tion and a history of stroke, we found that both CT-based and CMR-based calcification volumes were associated with a history of stroke [CT - odds ratio per 1-SD increase: 1.52 (95% CI: 1.00; 2.30), CMR – odds ratio per 1-SD increase: 1.47 (95% CI: 1.01; 2.14)] (Table 2). Discussion In this large population-based sample of persons with subclinical atherosclerosis, we found that CT-based and Fig. 2 Scatter plot of Ln-transformed CT-based and CMR-based calcification volumes, indicating a positive correlation between both detected and quantified calcification volumes Fig. 2 Scatter plot of Ln-transformed CT-based and CMR-based calcification volumes, indicating a positive correlation between both detected and quantified calcification volumes Fig. 2 Scatter plot of Ln-transformed CT-based and CMR-based calcification volumes, indicating a positive correlation between both detected and quantified calcification volumes Mujaj et al. Journal of Cardiovascular Magnetic Resonance (2017) 19:28 Page 5 of 7 Fig. 3 Bland-Altman plot of the difference of CT-based and CMR-based Ln-transformed total calcification volumes, with a mean absolute difference (bold continues line) and 95% confidence interval of mean differences (dashed lines) Fig. 3 Bland-Altman plot of the difference of CT-based and CMR-based Ln-transformed total calcification volumes, with a mean absolute difference (bold continues line) and 95% confidence interval of mean differences (dashed lines) detection of calcification the correlation between CT- based and CMR-based calcification volume is substan- tially improved. Finally, another important topic to con- sider with regard to the difference between CT and CMR is the blooming effect of calcifications which is known to occur on CT [19]. Especially for calcifications with very high Hounsfield units, a gradient over multiple adjacent pixels is necessary to reach a low Hounsfield unit. This effect may lead to slight overestimation of the calcification area. On the other hand, CMR is known to underestimate the amount of calcification, because a certain amount of calcification is required before the MR-signal disappears. In this context, it is important to acknowledge that possible micro-calcifications in the atherosclerotic plaque may be missed [20]. Importantly, despite the fact that CMR systematically underestimates the amount of calcification compared to CT, we found comparable risk estimates for CT-based and CMR-based calcification volumes with respect to a history of stroke. This suggests that when assessing clinical outcomes, the value of CMR-based calcification is similar to that of CT. Our findings have implications that should be consid- ered in the choice for CMR or CT for the assessment of vascular calcification. First, while assessing atheroscler- osis with CMR it is directly possible to visualize other plaque characteristics in addition to calcification, in- cluding intra-plaque hemorrhage and lipid-rich nec- rotic core which provide unique additional information on the disease. Discussion Yet, we would like to emphasize that in all instances the CT-scan was made before the CMR- scan and that calcification is a plaque component that generally remains present and shows only very slow pro- gression over time [23, 24]. Therefore, it seems unlikely that the amount of calcification at the time of CMR would differ substantially from that at the time of the CT. This is further supported by the fact that adjustment for the time interval did not change the results; and secondly by our finding that CMR volumes were consistently estimated somewhat smaller than CT volumes, whereas a large influence of the time interval would induce an opposite difference. Another potential limitation is that we used two types of MDCT scanners (16-slice and 64-slice) to assess calcification. Yet, adjustment for scanner-type did not change the association. 4.9 years. We acknowledge that the interscan interval rep- resents a potential limitation of the current study and that during this interval there may have been slight changes in plaque composition. Yet, we would like to emphasize that in all instances the CT-scan was made before the CMR- scan and that calcification is a plaque component that generally remains present and shows only very slow pro- gression over time [23, 24]. Therefore, it seems unlikely that the amount of calcification at the time of CMR would differ substantially from that at the time of the CT. This is further supported by the fact that adjustment for the time interval did not change the results; and secondly by our finding that CMR volumes were consistently estimated somewhat smaller than CT volumes, whereas a large influence of the time interval would induce an opposite difference. Another potential limitation is that we used two types of MDCT scanners (16-slice and 64-slice) to assess calcification. Yet, adjustment for scanner-type did not change the association. Author details 1 1Department of Epidemiology, Erasmus MC, University Medical Center, Office Na 2824k, PO Box 2040, 3000 CA Rotterdam, The Netherlands. 2Biomedical Imaging Group Rotterdam, Departments of Medical Informatics, Radiology, and Nuclear Medicine, Erasmus MC University Medical Center, Rotterdam, The Netherlands. 3Department of Radiology and Nuclear Medicine, Erasmus MC University Medical Center, Rotterdam, The Netherlands. 4Department of Computer Science, University of Copenhagen, Copenhagen, Denmark. 5 Conclusion In summary, CT-based and CMR-based volumes of carotid artery calcification are highly correlated, but CMR-based calcification is systematically smaller than those obtained with CT. Despite this difference, both provide comparable information with regard to a his- tory of stroke. Authors' information Not applicable. Authors' information Not applicable. Authors' information Not applicable. Availability of data and materials Data can be obtained upon request. Requests should be directed towards the management team of the Rotterdam Study (secretariat.epi@erasmusmc.nl), which has a protocol for approving data requests. Because of restrictions based on privacy regulations and informed consent of the participants, data cannot be made freely available in a public repository. Ethics approval and consent to participate The Rotterdam Study has been approved by the Medical Ethics Committee of the Erasmus MC and by the Dutch Ministry of Health, Welfare and Sports, implementing the “Wet Bevolkings Onderzoek: ERGO (Population Screening Act: Rotterdam Study)”. All participants provided written informed consent to participate in the study and to obtain information from their treating physicians. References CI: Confidence interval; CMR: Cardiovascular magnetic resonance; CT: Computed tomography; ERGO: Erasmus Rotterdam Gezondheid Onderzoek; MDCT: Multi detector computed tomography; OR: Odds ratio; SD: Standard deviation; TIA: Transient ischemic attack 1. Mozaffarian D, Benjamin EJ, Go AS, Arnett DK, Blaha MJ, Cushman M, Das SR, de Ferranti S, Despres JP, Fullerton HJ, et al. Executive Summary: Heart Disease and Stroke Statistics-2016 Update: A Report From the American Heart Association. Circulation. 2016;133:447–54. 2. Kwee RM. Systematic review on the association between calcification in carotid plaques and clinical ischemic symptoms. J Vasc Surg. 2010;51:1015–25. 3. Donnan GA, Fisher M, Macleod M, Davis SM. Stroke. Lancet. 2008;371:1612–23. 4. Lusis AJ. Atherosclerosis. Nature. 2000;407:233–41. 5. Libby P, Ridker PM, Hansson GK. Progress and challenges in translating the biology of atherosclerosis. Nature. 2011;473:317–25. 6. Owen DR, Lindsay AC, Choudhury RP, Fayad ZA. Imaging of atherosclerosis. Annu Rev Med. 2011;62:25–40. 7. Golledge J, Siew DA. Identifying the carotid 'high risk' plaque: is it still a riddle wrapped up in an enigma? Eur J Vasc Endovasc Surg. 2008;35:2–8. 8. Nandalur KR, Baskurt E, Hagspiel KD, Finch M, Phillips CD, Bollampally SR, Kramer CM. Carotid artery calcification on CT may independently predict stroke risk. AJR Am J Roentgenol. 2006;186:547–52. 9. Chalela JA. Evaluating the carotid plaque: going beyond stenosis. Cerebrovasc Dis. 2009;27 Suppl 1:19–24. 10. Truijman MT, Kooi ME, van Dijk AC, de Rotte AA, van der Kolk AG, Liem MI, Schreuder FH, Boersma E, Mess WH, van Oostenbrugge RJ, et al. Plaque At RISK (PARISK): prospective multicenter study to improve diagnosis of high-risk carotid plaques. Int J Stroke. 2014;9:747–54. 1. Mozaffarian D, Benjamin EJ, Go AS, Arnett DK, Blaha MJ, Cushman M, Das SR, de Ferranti S, Despres JP, Fullerton HJ, et al. Executive Summary: Heart Disease and Stroke Statistics-2016 Update: A Report From the American Heart Association. Circulation. 2016;133:447–54. 1. Mozaffarian D, Benjamin EJ, Go AS, Arnett DK, Blaha MJ, Cushman M, Das SR, de Ferranti S, Despres JP, Fullerton HJ, et al. Executive Summary: Heart Disease and Stroke Statistics-2016 Update: A Report From the American Heart Association. Circulation. 2016;133:447–54. 2. Kwee RM. Systematic review on the association between calcification in carotid plaques and clinical ischemic symptoms. J Vasc Surg. 2010;51:1015–25. 3 Donnan GA Fisher M Macleod M Davis SM Stroke Lancet 2008;371:1612–23 Competing interests Dr. Franco works in ErasmusAGE, a center for aging research across the life course funded by Nestlé Nutrition (Nestec Ltd.); Metagenics Inc.; and AXA. The other authors report no potential conflicts of interest. Received: 21 January 2017 Accepted: 10 February 2017 Received: 21 January 2017 Accepted: 10 February 2017 Acknowledgments h d d The dedication, commitment, and contribution of the inhabitants, general practitioners, and pharmacists of the Ommoord district to the Rotterdam Study are gratefully acknowledged. 5. Libby P, Ridker PM, Hansson GK. Progress and challenges in translating the biology of atherosclerosis. Nature. 2011;473:317–25. 5. Libby P, Ridker PM, Hansson GK. Progress and challenges in translating the biology of atherosclerosis. Nature. 2011;473:317–25. Discussion Second, CMR has the major advantage over CT that it does not involve harmful radiation exposure. Third, the systematic underestimation of cal- cification on CMR may pose a problem, specifically in situations where one is particularly interested in the exact amount of calcification. Fourth, drawbacks of CMR, in general, are its absolute contraindications (i.e. metal objects in the body), and the fact that CMR is more time-consuming, more expensive and less widely available than CT. Taken together, the pros and cons of both imaging modalities should be carefully considered for all research and clinical applications involving the assessment of vascular calcification. As a proof of principle, we investigated the association of CT-based and CMR-based calcification with a history of stroke and found that both related to this outcome with comparable effect estimates. We chose history of stroke because the relationship between carotid artery cal- cification and stroke has been well-established [16, 21, 22]. Table 2 Association of calcification volumes with stroke Odds ratio (95% CI) p-value Model 1 CT calcification volumes 1.63 (1.09–2.46) 0.01 CMR calcification volumes 1.55 (1.07–2.24) 0.01 Model 2 CT calcification volumes 1.52 (1.00–2.30) 0.04 CMR calcification volumes 1.47 (1.01–2.14) 0.04 Model 1 - scan time difference Model 2 –adjusted for age, sex and scan time difference. Values represent odd ratios with 95% CI per 1 standard deviation increase in calcification volumes Abbreviation: CT computed tomography, CMR cardiovascular magnetic resonance Table 2 Association of calcification volumes with stroke Odds ratio (95% CI) p-value Table 2 Association of calcification volumes with stroke The strengths of our study include the relatively large sample size of community-dwelling individuals, all with varying degrees of carotid atherosclerosis, and the stan- dardized assessment of calcification volumes on both modalities. Yet, some limitations should also be taken into account of which the first is the time interval between the CT scan and the CMR scan, with a mean interval of Mujaj et al. Journal of Cardiovascular Magnetic Resonance (2017) 19:28 Page 6 of 7 Page 6 of 7 Page 6 of 7 4.9 years. We acknowledge that the interscan interval rep- resents a potential limitation of the current study and that during this interval there may have been slight changes in plaque composition. Additional file Additional file 1: Table S1–S3. (Relation between calcification volume on CT and CMR), (Relation between calcification volume on CT and CMR, between the subjects with <3 years and >3 years difference on CT and CMR scans). (Parameters of the CMR Protocol). (DOCX 18 kb) Additional file 1: Table S1–S3. (Relation between calcification volume on CT and CMR), (Relation between calcification volume on CT and CMR, between the subjects with <3 years and >3 years difference on CT and CMR scans). (Parameters of the CMR Protocol). (DOCX 18 kb) 5Department of Clinical Epidemiology, Harvard TH Chan School of Public Health, Boston, USA. Authors' contributions Conceptualization: BM, DB, AVL, OHF. Data curation: BM, DB. Formal analysis: BM. Investigation: BM. Methodology:BM, AAL, AVE, MDB, DB. Supervision: DB, OHF. Visualization: BM. Writing – original draft: BM. Writing – review & editing: BM, AAL, AVE, MDB, OHF, AVL, MWV, DB. All authors read and approved the final manuscript. Conceptualization: BM, DB, AVL, OHF. Data curation: BM, DB. Formal analysis: BM. Investigation: BM. Methodology:BM, AAL, AVE, MDB, DB. Supervision: DB, OHF. Visualization: BM. Writing – original draft: BM. Writing – review & editing: BM, AAL, AVE, MDB, OHF, AVL, MWV, DB. All authors read and approved the final manuscript. Conceptualization: BM, DB, AVL, OHF. Data curation: BM, DB. Formal analysis: BM. Investigation: BM. Methodology:BM, AAL, AVE, MDB, DB. Supervision: DB, OHF. Visualization: BM. Writing – original draft: BM. Writing – review & editing: BM, AAL, AVE, MDB, OHF, AVL, MWV, DB. All authors read and approved the final manuscript. References 1. Mozaffarian D, Benjamin EJ, Go AS, Arnett DK, Blaha MJ, Cushman M, Das SR, de Ferranti S, Despres JP, Fullerton HJ, et al. Executive Summary: Heart Disease and Stroke Statistics-2016 Update: A Report From the American Heart Association. Circulation. 2016;133:447–54. 2. Kwee RM. Systematic review on the association between calcification in carotid plaques and clinical ischemic symptoms. J Vasc Surg. 2010;51:1015–25. 3. Donnan GA, Fisher M, Macleod M, Davis SM. Stroke. Lancet. 2008;371:1612–23. 4. Lusis AJ. Atherosclerosis. Nature. 2000;407:233–41. 5. Libby P, Ridker PM, Hansson GK. Progress and challenges in translating the biology of atherosclerosis. Nature. 2011;473:317–25. 6. Owen DR, Lindsay AC, Choudhury RP, Fayad ZA. Imaging of atherosclerosis. Annu Rev Med. 2011;62:25–40. 7. Golledge J, Siew DA. Identifying the carotid 'high risk' plaque: is it still a riddle wrapped up in an enigma? Eur J Vasc Endovasc Surg. 2008;35:2–8. 8. Nandalur KR, Baskurt E, Hagspiel KD, Finch M, Phillips CD, Bollampally SR, Kramer CM. Carotid artery calcification on CT may independently predict stroke risk. AJR Am J Roentgenol. 2006;186:547–52. 9. Chalela JA. Evaluating the carotid plaque: going beyond stenosis. Cerebrovasc Dis. 2009;27 Suppl 1:19–24. 10. Truijman MT, Kooi ME, van Dijk AC, de Rotte AA, van der Kolk AG, Liem MI, Schreuder FH, Boersma E, Mess WH, van Oostenbrugge RJ, et al. Plaque At RISK (PARISK): prospective multicenter study to improve diagnosis of high-risk carotid plaques. Int J Stroke. 2014;9:747–54. Funding h 6. Owen DR, Lindsay AC, Choudhury RP, Fayad ZA. Imaging of atherosclerosis. Annu Rev Med. 2011;62:25–40. The Rotterdam Study is supported by the Erasmus MC and Erasmus University Rotterdam; the Netherlands Organization for Scientific Research (NWO); the Netherlands Organization for Health Research and Development (ZonMw); the Research Institute for Diseases in the Elderly (RIDE); the Netherlands Genomics Initiative (NGI); the Ministry of Education, Culture and Science, the Ministry of Health, Welfare and Sports; the European Commission (DG XII); and the Municipality of Rotterdam. 7. Golledge J, Siew DA. Identifying the carotid 'high risk' plaque: is it still a riddle wrapped up in an enigma? Eur J Vasc Endovasc Surg. 2008;35:2–8. 8. Nandalur KR, Baskurt E, Hagspiel KD, Finch M, Phillips CD, Bollampally SR, Kramer CM. Carotid artery calcification on CT may independently predict stroke risk. AJR Am J Roentgenol. 2006;186:547–52. 8. Nandalur KR, Baskurt E, Hagspiel KD, Finch M, Phillips CD, Bollampally SR, Kramer CM. Carotid artery calcification on CT may independently predict stroke risk. AJR Am J Roentgenol. 2006;186:547–52. 9. Chalela JA. Evaluating the carotid plaque: going beyond stenosis. Cerebrovasc Dis. 2009;27 Suppl 1:19–24. Mr. Mujaj is supported by Erasmus Mundus Western Balkans (ERAWEB), a project funded by the European Commission. Mr. Mujaj is supported by Erasmus Mundus Western Balkans (ERAWEB), a project funded by the European Commission. 10. Truijman MT, Kooi ME, van Dijk AC, de Rotte AA, van der Kolk AG, Liem MI, Schreuder FH, Boersma E, Mess WH, van Oostenbrugge RJ, et al. Plaque At RISK (PARISK): prospective multicenter study to improve diagnosis of high-risk carotid plaques. Int J Stroke. 2014;9:747–54. 10. Truijman MT, Kooi ME, van Dijk AC, de Rotte AA, van der Kolk AG, Liem MI, Schreuder FH, Boersma E, Mess WH, van Oostenbrugge RJ, et al. Plaque At RISK (PARISK): prospective multicenter study to improve diagnosis of high-risk carotid plaques. Int J Stroke. 2014;9:747–54. None of the funders had any role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; and preparation, review, or approval of the manuscript. None of the funders had any role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; and preparation, review, or approval of the manuscript. Page 7 of 7 Mujaj et al. Journal of Cardiovascular Magnetic Resonance (2017) 19:28 11. Funding h Hofman A, Brusselle GG, Darwish Murad S, van Duijn CM, Franco OH, Goedegebure A, Ikram MA, Klaver CC, Nijsten TE, Peeters RP, et al. The Rotterdam Study: 2016 objectives and design update. Eur J Epidemiol. 2015;30:661–708. 12. Odink AE, van der Lugt A, Hofman A, Hunink MG, Breteler MM, Krestin GP, Witteman JC. Association between calcification in the coronary arteries, aortic arch and carotid arteries: the Rotterdam study. Atherosclerosis. 2007;193:408–13. 13. van den Bouwhuijsen QJ, Bos D, Ikram MA, Hofman A, Krestin GP, Franco OH, van der Lugt A, Vernooij MW. Coexistence of Calcification, Intraplaque Hemorrhage and Lipid Core within the Asymptomatic Atherosclerotic Carotid Plaque: The Rotterdam Study. Cerebrovasc Dis. 2015;39:319–24. 14. van den Bouwhuijsen QJ, Vernooij MW, Hofman A, Krestin GP, van der Lugt A, Witteman JC. Determinants of magnetic resonance imaging detected carotid plaque components: the Rotterdam Study. Eur Heart J. 2012;33:221–9. 15. Arias-Lorza AM, Petersen J, van Engelen A, Selwaness M, van der Lugt A, Niessen WJ, de Bruijne M. Carotid Artery Wall Segmentation in Multispectral MRI by Coupled Optimal Surface Graph Cuts. IEEE Trans Med Imaging. 2016;35:901–11. 16. Bos D, Portegies ML, van der Lugt A, Bos MJ, Koudstaal PJ, Hofman A, Krestin GP, Franco OH, Vernooij MW, Ikram MA. Intracranial carotid artery atherosclerosis and the risk of stroke in whites: the Rotterdam Study. JAMA Neurol. 2014;71:405–11. 17. Wieberdink RG, Poels MM, Vernooij MW, Koudstaal PJ, Hofman A, van der Lugt A, Breteler MM, Ikram MA. Serum lipid levels and the risk of intracerebral hemorrhage: the Rotterdam Study. Arterioscler Thromb Vasc Biol. 2011;31:2982–9. 18. Kwee RM, Teule GJ, van Oostenbrugge RJ, Mess WH, Prins MH, van der Geest RJ, Ter Berg JW, Franke CL, Korten AG, Meems BJ, et al. Multimodality imaging of carotid artery plaques: 18 F-fluoro-2-deoxyglucose positron emission tomography, computed tomography, and magnetic resonance imaging. Stroke. 2009;40:3718–24. 19. de Weert TT, Ouhlous M, Meijering E, Zondervan PE, Hendriks JM, van Sambeek MR, Dippel DW, van der Lugt A. In vivo characterization and quantification of atherosclerotic carotid plaque components with multidetector computed tomography and histopathological correlation. Arterioscler Thromb Vasc Biol. 2006;26:2366–72. 20. Baheza RA, Welch EB, Gochberg DF, Sanders M, Harvey S, Gore JC, Yankeelov TE. Detection of microcalcifications by characteristic magnetic susceptibility effects using MR phase image cross-correlation analysis. Med Phys. 2015;42:1436–52. 21. Bos D, Ikram MA, Elias-Smale SE, Krestin GP, Hofman A, Witteman JC, van der Lugt A, Vernooij MW. Mujaj et al. Journal of Cardiovascular Magnetic Resonance (2017) 19:28 Funding h Calcification in major vessel beds relates to vascular brain disease. Arterioscler Thromb Vasc Biol. 2011;31:2331–7. 22. Rennenberg RJ, Kessels AG, Schurgers LJ, van Engelshoven JM, de Leeuw PW, Kroon AA. Vascular calcifications as a marker of increased cardiovascular risk: a meta-analysis. Vasc Health Risk Manag. 2009;5:185–97. 23. van Gils MJ, Bodde MC, Cremers LG, Dippel DW, van der Lugt A. Determinants of calcification growth in atherosclerotic carotid arteries; a serial multi-detector CT angiography study. Atherosclerosis. 2013;227:95–9. 24. van Gils MJ, Vukadinovic D, van Dijk AC, Dippel DW, Niessen WJ, van der Lugt A. Carotid atherosclerotic plaque progression and change in plaque composition over time: a 5-year follow-up study using serial CT angiography. AJNR Am J Neuroradiol. 2012;33:1267–73. 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 Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries
https://openalex.org/W3042543808	https://durham-repository.worktribe.com/preview/1264563/31581.pdf	English	null	The crystal engineering of radiation-sensitive diacetylene cocrystals and salts	Chemical science	2,020	cc-by	12,452	aDurham University, Department of Chemistry, Lower Mountjoy, Stockton Road, Durham, DH1 3LE, UK. E-mail: jon.steed@durham.ac.uk bAshland LLC, 1005 Route 202/206, Bridgewater, NJ 08807, USA † Electronic supplementary information (ESI) available: XRPD, IR, Raman, DSC, SS NMR and unit cell data. CCDC 2000830–2000837. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/d0sc02540b Cite this: Chem. Sci., 2020, 11, 8025 Amy V. Hall, a Dmitry S. Yuﬁt,a David C. Apperley,a Larry Senak,b Osama M. Musa,b David K. Hoodb and Jonathan W. Steed a All publication charges for this article have been paid for by the Royal Society of Chemistry In this work we develop photoreactive cocrystals/salts of a commercially-important diacetylene, 10,12- pentacosadiynoic acid (PCDA, 1) and report the ﬁrst X-ray crystal structures of PCDA based systems. The topochemical reactivity of the system is modiﬁed depending on the coformer used and correlates with the structural parameters. Crystallisation of 1 with 4,40-azopyridine (2), 4,40-bipyridyl (3), and trans-1,2- bis(4-pyridyl)ethylene (4) results in unreactive 2 : 1 cocrystals or a salt in the case of 4,40-bipiperidine (5). However, salt formation with morpholine (6), diethylamine (7), and n-butylamine (8), results in highly photoreactive salts 12$7 and 1$8 whose reactivity can be explained using topochemical criteria. The salt 1$6 is also highly photoreactive and is compared to a model morpholinium butanoate salt. Resonance Raman spectroscopy reveals structural details of the photopolymer including its conformational disorder in comparison to less photoactive alkali metal salts and the extent of solid state conversion can be monitored by CP-MAS NMR spectroscopy. We also report an unusual catalysis in which amine evaporation from photopolymerised PCDA ammonium salts eﬀectively acts as a catalyst for polymerisation of PCDA itself. The new photoreactive salts exhibit more reactivity but decreased conjugation compared to the commercial lithium salt and are of considerable practical potential in terms of tunable colours and greater range in UV, X-ray, and g-ray dosimetry applications. Received 5th May 2020 Accepted 18th July 2020 DOI: 10.1039/d0sc02540b rsc.li/chemical-science highlight the importance of molecular organisation in the topochemical reaction.7,8 Chemical Science View Article Online View Journal \| View Issue This journal is © The Royal Society of Chemistry 2020 The crystal engineering of radiation-sensitive diacetylene cocrystals and salts† Cite this: Chem. Sci., 2020, 11, 8025 Introduction The solid state 1,4-addition polymerisation of diacetylenes can be initiated by radiation and heat, and results in a conjugated ene–yne polymeric chain. The reaction is thought to only occur if the topochemical parameters of the diacetylene packing are optimal. The rst report of topochemical reactivity in the solid state was in an alkene system described by Schmidt in 1964 who suggested that carbon double bonds must be separated by a maximum distance of 4.2 ˚A for successful polymerisation.1 In 1969, Wegner reported the rst example of diacetylene poly- merisation in the solid state,2,3 while 15 years later, Enkelmann proposed strict criteria for diacetylene reactivity, whereby adjacent diacetylene monomers will react when the reactive groups are separated by a C1–C40 contact distance (d) of #3.8 ˚A, a translational period repeat spacing (r) of #4.9 ˚A, along with an orientation angle (q) to the crystal axis at an optimum value of 45 (Scheme 1).4–6 The diacetylene polymerisation parameters The monomer-to-polymer transition is clearly observed by a colour change from colourless to blue, due to the rearrange- ment of the diacetylene monomers to give an ene–yne chro- mophore. The blue colour is due to p–p transitions in the ordered, conjugated chain with the reorganisation of the chains controlling the degree of diacetylene polymerisation.9 Addi- tional external stimuli on the polymerised diacetylene (poly- diacetylene) such as extended heating,10–12 pH change,10,13–16 treatment with organic solvents,10,17–21 mechanical stress,22,23 and ligand–receptor interactions,24,25 can cause the poly- diacetylenes to exhibit a range of colours from blue, to red, to yellow.15 These chromic changes can be explained by a confor- mational rearrangement within the polydiacetylene assembly Scheme 1 The topochemical parameters for diacetylene 1,4-addition polymerisation requires the tilt angle (q) of the monomers to be 45, the C1–C40 distance (r) to be #3.8 ˚A, and the translational repeat distance (d) to be #4.9 ˚A to yield a polydiacetylene. Scheme 1 The topochemical parameters for diacetylene 1,4-addition polymerisation requires the tilt angle (q) of the monomers to be 45, the C1–C40 distance (r) to be #3.8 ˚A, and the translational repeat distance (d) to be #4.9 ˚A to yield a polydiacetylene. This journal is © The Royal Society of Chemistry 2020 Chem. Introduction Sci., 2020, 11, 8025–8035 \| 8025 Edge Article View Article Online Edge Article View Article Online Chemical Science which disrupts the conjugated backbone, causing reduced overlap of the p orbitals, resulting in a widening of the HOMO– LUMO energy gap and hence the polydiacetylene absorbing light at a higher energy.12,26,27 The commercially important diacetylene, 10,12-pentacosadiynoic acid (PCDA, 1), is used to provide a colourimetric change in practical chemosensors,28–33 biosensors,24,34–36 and dosimeters.37–40 Although PCDA is some- what photoreactive, further tuning of its photoresponse is of considerable interest, especially for radiation dosimetry appli- cations. Covalent modication oﬀers a viable strategy to PCDA analogues with a tuned photoresponse.41–43 However, since the solid state reactivity of dialkynes depends on their crystal packing arrangement, a simpler strategy is to address the dia- lkyne reactivity through modication of non-covalent interac- tions by cocrystal or salt formation.44–47 Whether a cocrystal or salt will form depends on the diﬀerence in pKa of the two components. For a cocrystal, the DpKa must be <2–3 log units, while salt formation is expected for a greater diﬀerence.48,49 Cocrystals of carboxylic acids can be prepared using the robust hydrogen-bonded COOH/Npyridine heterosynthon, while salts can be based on ammonium complexes of more basic amines.50–54 In this work we explore the relationship between structure and photochemistry for PCDA (1) with three diﬀerent pyridine-containing coformers 4,40-azopyridine (2), 4,40-bipyr- idyl (3), and trans-1,2-bis(4-pyridyl)ethylene (4) and compare the photoreactivity of the resulting cocrystals with aliphatic amine salts of 4,40-bipiperidine (5), morpholine (6) diethylamine (7), and n-butylamine (8) (Scheme 2). To date no X-ray structure information has been reported for PCDA or related photoactive surfactant-like molecules despite its commercial importance and hence no structure–reactivity relationship has been elucidated. generally regarded as diﬃcult. However, preliminary powder X- ray diﬀraction (PXRD) analysis indicated that compound 1 is crystalline with a lamellar structure (ESI Fig. S1†). Slow evapo- ration of an acetone solution of PCDA gave high a quality sample suitable for analysis at the I19 beamline at the Diamond Light Source, Oxfordshire. Some decay of photosensitive 1 in the high-intensity synchrotron X-ray beam was noted, however, a structure determination was successfully carried out. The X- ray structure of 1 revealed a centrosymmetric structure (P1 space group) based on the well-known OH/O R2 2(8) ring hydrogen-bonded carboxylic acid dimer synthon55,56 (Fig. 1). Results and discussion The X-ray crystal structure of PCDA is unknown and the prep- aration of single crystals of such surfactant-like compounds is Introduction The structure is based on a head-to-head bilayer arrangement of molecules and the aliphatic substituents at either end of the dialkyne unit adopt an anti-conformation. The crystallographic c-axis of 46.647(3) ˚A is long, which is also apparent in the PXRD pattern, which shows a lamellar progression of low-angle peaks from 1.9–17.1 2q, all correlating to reections in the (00l) plane (Fig. S1†).57 The structure conforms to the topochemical postulate for photoreactivity with a translational repeat distance of #4.9 ˚A, at 4.574(1) ˚A, along with a tilt angle of 44.7 that very close to the desired value of 45. The inter-alkyne C1–C40 distance between adjacent molecules of 1 is 3.712(1) ˚A, which is close to the upper limit of the topochemical postulate for alkyne reactivity (a maximum distance of 3.8 ˚A).3 These values are consistent with the limited observed solid state photoreactivity of 1 which, while it gradually turns visually blue upon exposure to UV and X-ray, the actual photoconversion as monitored by CP-MAS 13C NMR spectroscopy displays no alkene peaks, implying less than 1% even upon prolonged exposure (Fig. S2– S5†). Therefore, the question is raised as to whether the top- ochemistry of 1 can be engineered to provide an altered and tunable radiation response of use in radiation dosimetry by incorporating 1 into a cocrystal or a salt. The lithium salt of PCDA has been reported in this context in the patent litera- ture,58 however, no X-ray crystal structure has been obtained to understand the photoreactivity of the salt. As a result, organic coformers potentially oﬀer a structural insight to photo- reactivity, along with improved versatility and more facile pro- cessing because of enhanced solubility. Edge Article I19 beamline at the Diamond Light Source at 100 K, while crystals of 12$4 were analysed on a Bruker D8 Venture diﬀrac- tometer at 120 K. The two materials are isostructural and crys- tallise in the monoclinic space group P21/c. The X-ray structures of cocrystals 12$3 and 12$4 consist of hydrogen bonds between the carboxylic acid hydrogen atom of 1 and the pyridyl nitrogen atom of the coformer at an O/N distance of 2.652(4) ˚A in 12$3 (Fig. 3a) and 2.6579(17) ˚A in 12$4 (Fig. 3b). Interestingly the dialkyne moieties in both structures adopt a syn-conformation, in contrast to the anti-conformation in 1 and 12$2 indicating that subtle modication of conformer can have a signicant eﬀect on crystal packing mode. The X-ray structures of 12$3 and 12$4 and their packing diagrams are shown in Fig. 3c and d. The ethylene bond of 12$4 is disordered over two positions. The C1– C40 inter-alkyne distances between adjacent PCDA molecules in both cocrystals are both within the topochemical postulate at distances of 3.730(1) ˚A and 3.726(2) ˚A, respectively, although they are longer than those found in 1 and 12$2, because of the syn conformation of the dialkyne fragments. However, the tilt angle of PCDA in the cocrystals and the translational repeat distance for 12$3 is 47.4 and 5.442(1) ˚A, respectively, with similar values for 12$4 with a tilt angle of 1 in the cocrystal at 47.3 and a repeat distance of 5.449(2) ˚A. Therefore, neither of the isostructural cocrystals are expected to be photoreactive, as only one out of three of the parameters are within the top- ochemical postulate. The syn conformation of the dialkyne substituents allows an interdigitated, bilayer packing arrange- ment which translates to the much longer crystallographic c axes which encompass four folded molecules in the cocrystals of 3 and 4 as opposed to two extended molecules in 12$2. cocrystallisation of 1 and 2 in acetone. No other stoichiometries were attempted. Aer the evaporation of solvent at room temperature for one week, plate-shaped crystals of 12$2 formed and were analysed by single crystal X-ray diﬀraction (SC-XRD). The structure of 12$2 reveals a 2 : 1 stoichiometry with the diacetylene substituents in anti-conformation, analogous to the structure of 1, with OH/N hydrogen bonds from the carboxylic acid protons of 1 to the pyridyl nitrogen atoms of 2 (Fig. 2a). PCDA cocrystals Grinding PCDA (1) and 4,40-azopyridine (2) in a Retsch MM 200 mixer mill for 1 hour in a 2 : 1 ratio, respectively (which reects the single hydrogen bond donor group of 1 and the two hydrogen bond acceptor groups of 2), gave a powder of cocrystal 12$2, which was characterised by PXRD and used for seeding the Fig. 1 The X-ray structure of 1 showing the carboxylic acid dimer (O/ O distance of 2.6581(9) ˚A) and the anti-conformation of the diac- etylene substituents. The C1–C40 distance and intermolecular repeat distance are denoted by d and r, respectively. Scheme 2 Structures of PCDA (1), with coformers 4,40-azopyridine (2), 4,40-bipyridyl (3), trans-1,2-bis(4-pyridyl)ethylene (4), and salt formers 4,40-bipiperidine (5), morpholine (6), diethylamine (7) and n- butylamine (8). Fig. 1 The X-ray structure of 1 showing the carboxylic acid dimer (O/ O distance of 2.6581(9) ˚A) and the anti-conformation of the diac- etylene substituents. The C1–C40 distance and intermolecular repeat distance are denoted by d and r, respectively. 8026 \| Chem. Sci., 2020, 11, 8025–8035 This journal is © The Royal Society of Chemistry 2020 Chemical Science View Article Online Chemical Science View Article Online Chemical Science View Article Online Edge Article The O/N distance of 2.677(4) ˚A is consistent with a strong, carboxylic acid OH/pyridyl hydrogen bond. The carboxylic acid proton was located experimentally in the X-ray structure and is situated on the oxygen atom of the carboxylic acid, ruling out the possibility of salt formation. The unit cell of 12$2 has a shorter crystallographic c-axis of 39.920(2) ˚A compared to 1 itself (by a considerable 6.87 ˚A) implying a more slanted orientation of the lamellar structure (Fig. 2b). Compared to 1, the cocrystal 12$2 also has a signicantly shorter inter-alkyne C1–C40 distance of 3.633(1) ˚A, however, the tilt angle of 1 in the cocrystal is greater than the optimum value at 48.4, along with a translational repeat distance outside of the desired range for topochemical reactivity at 5.354(1) ˚A. This journal is © The Royal Society of Chemistry 2020 Open Access Article. Published on 20 July 2020. Downloaded on 8/25/2020 10:43:31 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Along with SC-XRD, 12$2 was characterised by Diﬀerential Scanning Calorimetry (DSC) which displayed a melting onset temperature of 57 C (Fig. S6†), which is lower than the melting temperatures of the individual components (62 C for 1 (Fig. S7†) and 96 C for 2 (ref. 59)) implying relatively weak interactions. The Fourier-transform infrared (FTIR) spectrum displays a hydrogen-bonded carbonyl stretching band at 1695 cm1, compared to 1692 cm1 in pure 1 implying slightly weaker hydrogen bonding (Fig. S8†). The cocrystal displays signicant anisotropic thermal expansion along the c-axis which increases from 39.33 ˚A to 40.99 ˚A between 120 and 273 K. The diﬀerences in the unit cell made the calculated and experimental PXRD data diﬃcult to compare, although it is clear that the single crystal studied is representative of the bulk material (Fig. S9†). Fig. 3 (a) The X-ray structure of 12$3 with components joined by an OH/N hydrogen bond (O/N distance of 2.6448(9) ˚A) showing the syn-conformation of the dialkyne substituents. (b) The X-ray structure of 12$4 with OH/N hydrogen bonds (O/N distance of 2.6579(17) ˚A) and the diacetylene substituents in a syn-conformation (disorder omitted). Packing diagram of 12$3 in the (c) (010) and (d) (001) crys- tallographic planes. Two further cocrystals 12$3 and 12$4 were synthesised from 4,40-bipyridyl and trans-1,2-bis(4-pyridyl)ethylene, respectively, by grinding the coformers with PCDA in a 2 : 1 ratio for 45 minutes in a mixer mill, to yield the cocrystal in powder form. Samples were characterised by PXRD and then used in seeded crystallisations in acetone. These experiments gave plates of 12$3 and 12$4 aer the evaporation of solvent at room temper- ature for one week. A single crystal of 12$3 was analysed at the Fig. 2 (a) The X-ray structure of 12$2 showing the OH/N hydrogen bond (O/N distance of 2.677(4)) with the diacetylene substituents in an anti-conformation. (b) The packing diagram of 12$2 in the (100) crystallographic plane. Fig. 3 (a) The X-ray structure of 12$3 with components joined by an OH/N hydrogen bond (O/N distance of 2.6448(9) ˚A) showing the syn-conformation of the dialkyne substituents. (b) The X-ray structure of 12$4 with OH/N hydrogen bonds (O/N distance of 2.6579(17) ˚A) and the diacetylene substituents in a syn-conformation (disorder omitted). Packing diagram of 12$3 in the (c) (010) and (d) (001) crys- tallographic planes. Fig. PCDA salts The morpholinium salt 1$6 was crystallised by the slow evaporation of acetone at room temperature, however, due to poor crystal quality aer repeated crystallisation attempts, no SC-XRD analysis of 1$6 could be undertaken. To model the interactions between the two components, the synthesis of the butanoic acid (BuA) salt of 6 was attempted. Large single crys- tals of BuA$6 formed from equimolar amounts of reagents in a sealed ask allowed to stand overnight. The X-ray structure reveals a salt with a butanoate anion and protonated morpho- linium cation (Fig. 5). The structure involves two unique NH/O hydrogen bonding interactions with N/O distances of 2.673(1) ˚A and 2.732(1) ˚A. Based on the similar pKa of 1 and butanoic acid it is possible that 1$6 is also a salt with similar head-group structure, although the relevance of this model system to the PCDA analogue is otherwise limited. Cocrystals of PCDA with bifunctional coformers 2–4 appear to give structures that are unlikely to be photoreactive based on their topochemical metrics. As a result, we examined both mono- and bifunctional coformers with higher basicity inten- ded to deprotonate the PCDA acid functionality and hence alter the hydrogen bonding pattern and change the consequent stacking of the PCDA units. Salt formation was undertaken with a bifunctional diamine (5), a cyclic amine (6), a linear secondary amine (7), and a linear terminal amine (8). PCDA and compounds 5–8 were mechanochemically ground in a mixer mill to give a range of new salt materials as indicated by FTIR analysis. The carboxylate asymmetric carbonyl stretching modes proved to be at lower wavenumbers than in the free acid (1, 1692 cm1) with a carbonyl stretch at 1653 cm1 in 12$5 (Fig. S16†) and 1$6 (Fig. S17†), 1627 cm1 in 12$7 (Fig. S18†), and 1649 cm1 in 1$8 (Fig. S19†), suggesting stronger hydrogen bonding in the salts than the cocrystals and a delocalised carboxylate anion structure. The X-ray structure of 12$5 reveals a salt with two anions of 1 and a dication of double protonated 5 in a 2 : 1 stoichiometry, respectively, consisting of NH/O hydrogen bonds from the amine hydrogen atom of 5 and the oxygen atom of 1, at an N/O distance of 2.717(1) ˚A (Fig. 4). The salt 12$5 crystallises with the same symmetry as 1 and 12$2 in the space group P1, with the crystallographic c-axis at the shortest observed so far at 23.0041(15) ˚A. Open Access Article. Published on 20 July 2020. Downloaded on 8/25/2020 10:43:31 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 3 (a) The X-ray structure of 12$3 with components joined by an OH/N hydrogen bond (O/N distance of 2.6448(9) ˚A) showing the syn-conformation of the dialkyne substituents. (b) The X-ray structure of 12$4 with OH/N hydrogen bonds (O/N distance of 2.6579(17) ˚A) and the diacetylene substituents in a syn-conformation (disorder omitted). Packing diagram of 12$3 in the (c) (010) and (d) (001) crys- tallographic planes. Fig. 2 (a) The X-ray structure of 12$2 showing the OH/N hydrogen bond (O/N distance of 2.677(4)) with the diacetylene substituents in an anti-conformation. (b) The packing diagram of 12$2 in the (100) crystallographic plane. Chem. Sci., 2020, 11, 8025–8035 \| 8027 This journal is © The Royal Society of Chemistry 2020 Open Access Article. Published on 20 July 2020. Downloaded on 8/25/2020 10:43:31 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 5 The X-ray structure of BuA$6 showing two diﬀerent hydrogen bonding interactions. cocrystal is below the desired value (45) at 24.1, and the translational repeat distance of 5.577(2) ˚A is outside the maximum distance for this parameter (#4.9 ˚A) again suggesting limited photoreactivity. Chemical Science Fig. 5 The X-ray structure of BuA$6 showing two diﬀerent hydrogen bonding interactions. Edge Article The DSC thermogram of 12$3 displays a melt onset endo- therm of 73 C (Fig. S10†) (compared to the coformer melt temperatures for 1 and 3 of 62 C and 114 C,60 respectively), while 12$4 exhibits a melting onset temperature of 72 C (Fig. S11†), compared to 150 C for 4.61 The similar melting temperatures for the two cocrystals are expected due to the isostructural nature of these materials. The FTIR spectra for these cocrystals display a hydrogen-bonded carbonyl stretch at 1683 cm1 and 1688 cm1 respectively, compared to 1692 cm1 in pure 1, implying slightly stronger hydrogen bonding (Fig. S12 and S13†). In a similar way to 12$2, cocrystals 12$3 and 12$4 show considerable anisotropic thermal expansion on warming (see Table S1†). This makes the calculated PXRD patterns appear somewhat diﬀerent to the room temperature experi- mental patterns (Fig. S14 and S15†). PCDA salts The C1–C40 inter- alkyne distance between adjacent molecules of 1 is 3.760(2) ˚A, which is within the topochemical postulate for the reactivity of diacetylenes (#3.8 ˚A), however, the tilt angle of 1 in the salt Salts of PCDA with diethylamine (7) and n-butylamine (8) crystallised by slow evaporation of acetone solutions at room temperature. Surprisingly the crystals are highly coloured purple and blue, respectively, consistent with facile photo- polymerisation (Fig. 6). However, the X-ray structure determi- nations reveal salts of unpolymerised PCDA and hence the colouration is likely to be a surface eﬀect. Indeed, cutting a single crystal in half revealed a colourless inner core. The structure of the diethylammonium salt proved to be a salt Fig. 6 Photographs of (a) the purple crystal of 12$7 and (b) the blue crystals of 1$8, taken before X-ray irradiation. Fig. 4 The X-ray structure of the salt cocrystal 12$5 in the (a) (100) and (b) (001) crystallographic planes. Fig. 4 The X-ray structure of the salt cocrystal 12$5 in the (a) (100) and (b) (001) crystallographic planes. Fig. 6 Photographs of (a) the purple crystal of 12$7 and (b) the blue crystals of 1$8, taken before X-ray irradiation. 8028 \| Chem. Sci., 2020, 11, 8025–8035 This journal is © The Royal Society of Chemistry 2020 Chemical Science View Article Online Chemical Science View Article Online Edge Article Chemical Science Fig. 7 The X-ray structure of 12$7 in the (a) (100) and (b) (010) crys- tallographic planes. show signicant photoreactivity, consistent with the sponta- neous surface colouration of the crystals. y DSC analyses of the PCDA salts of 5–8 reveal melt onset endotherms of 107 C for 12$5 (Fig. S20†) (compared to 62 C and 170 C (ref. 62) for the parent components 1 and 5, respectively). This relatively high value likely reects the fact that proton transfer has occurred as well as the higher melting point of the bipiperidine coformer. The morpholinium salt 1$6 has a low melting onset of 44 C (Fig. S21†) consistent with the fact that morpholine is a liquid at room temperature (it boils at 128 C).63 In the same way as 1$6, the DSC thermogram of 12$7 exhibits a melt onset endotherm of 46 C (Fig. S22†), in comparison to the boiling temperature of 55 C for 7,64 while 1$8 displays a melt onset endotherm at 57 C (Fig. Open Access Article. Published on 20 July 2020. Downloaded on 8/25/2020 10:43:31 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The 12$7 structure also has a large c-axis of 57.520(4) ˚A, which is the longest c-axis of all the structures studied reecting the linear, parallel arrangement of the PCDA components. Salts 12$7 and 1$8 have similar C1–C40 inter- alkyne distances of 3.776(2) ˚A and 3.779(1) ˚A, respectively, with tilt angles of 41.9 and 43.7, and translational repeat distances of 4.644(3) ˚A and 4.593(1) ˚A. For these two salts, all three values are well within the optimum values of the top- ochemical postulate, and they are therefore are expected to Open Access Article. Published on 20 July 2020. Downloaded on 8/25/2020 10:43:31 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 7 The X-ray structure of 12$7 in the (a) (100) and (b) (010) crys- tallographic planes. cocrystal that also includes a neutral molecule of 1 (Fig. 7), with formula 12$7. The butylammonium compound is a 1 : 1 salt of formula 1$8. The structure adopts a stacked bilayer arrange- ment (Fig. 8). In 12$7, hydrogen bonding occurs from the ammonium NH hydrogen atoms to the carbonyl oxygen of 1, with an N/O distance of 2.737(1) ˚A. The carboxylic acid group of the neutral PCDA hydrogen bonds to the carboxylate func- tionality on the PCDA anion with a very short O/O distance of 2.444(1) ˚A (the additional hydrogen atom present between PCDA and the PCDA anion is disordered). In the 1 : 1 salt 1$8, there are three diﬀerent hydrogen bond interactions form from the NH3 + cation to the carboxylate oxygen atoms of the PCDA anion, with NH/O distances of 2.671(1) ˚A, 2.725(1) ˚A, and 2.784(1) ˚A. The 12$7 structure also has a large c-axis of 57.520(4) ˚A, which is the longest c-axis of all the structures studied reecting the linear, parallel arrangement of the PCDA components. Salts 12$7 and 1$8 have similar C1–C40 inter- alkyne distances of 3.776(2) ˚A and 3.779(1) ˚A, respectively, with tilt angles of 41.9 and 43.7, and translational repeat distances of 4.644(3) ˚A and 4.593(1) ˚A. For these two salts, all three values are well within the optimum values of the top- ochemical postulate, and they are therefore are expected to cocrystal that also includes a neutral molecule of 1 (Fig. 7), with formula 12$7. The butylammonium compound is a 1 : 1 salt of formula 1$8. The structure adopts a stacked bilayer arrange- ment (Fig. 8). In 12$7, hydrogen bonding occurs from the ammonium NH hydrogen atoms to the carbonyl oxygen of 1, with an N/O distance of 2.737(1) ˚A. The carboxylic acid group of the neutral PCDA hydrogen bonds to the carboxylate func- tionality on the PCDA anion with a very short O/O distance of 2.444(1) ˚A (the additional hydrogen atom present between PCDA and the PCDA anion is disordered). In the 1 : 1 salt 1$8, there are three diﬀerent hydrogen bond interactions form from the NH3 + cation to the carboxylate oxygen atoms of the PCDA anion, with NH/O distances of 2.671(1) ˚A, 2.725(1) ˚A, and 2.784(1) ˚A. Cocrystal and salt response to UV and X-rays The powder of each cocrystal and salt was placed on lter paper in a dark box and exposed to a 6-Watt handheld UV light at 254 nm for varying durations (Fig. 9). It is known that the azo- benzene coformer 2 itself undergoes photoisomerisation to the cis form when irradiated at 365 nm (ref. 66) and so 12$2 was also irradiated at this wavelength in order to probe photoresponse of the coformer component within the cocrystal. While PCDA powder itself gradually darkens from a white to deep blue upon irradiation, all of the cocrystals with coformers 2–4 do not change colour despite the close proximity of the dialkyne functionalities, which are within the distance specied by the topochemical postulate. However, the tilt angles of 1 in the cocrystals, and the translational repeat distances of the coc- rystals are outside of the desired values. The irradiated cocrys- tals were analysed by PXRD, solid-state CP-MAS 13C NMR spectroscopy, and FTIR spectroscopy. This data conrmed that the cocrystal samples do not undergo photopolymerisation aer irradiation (Fig. S24–S32†). However, aer one hour and one day, 12$2 and 12$4 aer one day, display additional peaks in the Fig. 9 The coformer 1 and cocrystals 12$2, 12$3 and 12$4, and salts 12$5, 1$6, 12$7, 1$8 and the lithium salt (Li salt) before and after UV irradiation at 254 nm for diﬀerent durations. The 12$2a sample was irradiated with UV light at 365 nm while 254 nm radiation was used for the 12$2b sample. The initial colour changes to UV radiation show that only 1 and the salt samples visibly change colour with prolonged irradiation, with 12$7 being the most radiation sensitive to the naked eye and experimentally. Fig. 8 (a) The X-ray structure of 1$8 (b) in the (100) crystallographic plane. Fig. 9 The coformer 1 and cocrystals 12$2, 12$3 and 12$4, and salts 12$5, 1$6, 12$7, 1$8 and the lithium salt (Li salt) before and after UV irradiation at 254 nm for diﬀerent durations. The 12$2a sample was irradiated with UV light at 365 nm while 254 nm radiation was used for the 12$2b sample. The initial colour changes to UV radiation show that only 1 and the salt samples visibly change colour with prolonged irradiation, with 12$7 being the most radiation sensitive to the naked eye and experimentally. Fig. This journal is © The Royal Society of Chemistry 2020 PCDA salts S23†), with the salt former 8 boiling at 77 C.65 Open Access Article. Published on 20 July 2020. Downloaded on 8/25/2020 10:43:31 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 20 July 2020. Downloaded on 8/25/2020 10:43:31 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. consistent with the crystal packing revealed by their structures, which both show parameters within the range specied by the topochemical postulate. While structural data is not available for the morpholinium salt, it seems likely that this too is within the topochemical postulate range. The topochemical parame- ters for each compound are summarised in Table 1. p FTIR analysis of the salt cocrystals aer irradiation shows that salts 1$6, 12$7, and 1$8 begin to lose their volatile coformers aer prolonged UV exposure and revert to free carboxylic acids. This is evidenced by the decrease in intensity of the carboxylate asymmetric stretch band nasymm(CO2) of the salt (1653 cm1 in 1$6, 1627 cm1 in 12$7, and 1649 cm1 in 1$8) and the emergence of a free acid peak at 1692 cm1 close to the value of PCDA as the sample is irradiated (Fig. 11). The eﬀect is highly pronounced for the morpholinium salt 1$6 which reverts to free acid aer just one hour while 7 and 8 begin to separate from their respective salts aer one day of irradiation. The resulting carboxylic acid is a mixture of free PCDA and photo- polymer. These ndings are also supported by PXRD analysis of the irradiated salts (Fig. S38–S40†). Interestingly, given the very limited photoreactivity of PCDA itself, salt formation followed by removal of the amine in this way gives an interesting route to the free acid photopolymer and hence transient amine complexation eﬀectively catalyses the photopolymerisation reaction of PCDA itself. The salts were also exposed to ambient conditions for seven days to investigate whether the amine is lost from the salts without irradiation. The FTIR of salts 1$6 and p y y p p y reaction of PCDA itself. The salts were also exposed to ambient conditions for seven days to investigate whether the amine is lost from the salts without irradiation. The FTIR of salts 1$6 and Fig. 10 CP-MAS 13C NMR spectra of 12$5, 1$6, 12$7, and 1$8 after 7 days of UV irradiation at 254 nm, highlighting the alkyne carbons (60– 85 ppm), alkene carbons of the photoreactive salts (100–135 ppm), and carboxylate environment of each salt (175–185 ppm). Fig. Chemical Science Chemical Science PXRD patterns and solid-state NMR spectra that are attributable to 1. This suggests that irradiation decomposes the cocrystals to liberate free 1, particularly in the case of the azobenzene complex 12$2. This behaviour is attributed to the photo- isomerisation of coformer 2 resulting in degradation of the cocrystal. The PXRD pattern and solid-state NMR spectra also imply a less-crystalline material at one hour in 12$2 and show the presence of crystalline 1 aer one day of irradiation. In addition, the solid-state NMR spectroscopic data revealed that even the deep blue colour of the irradiated sample of PCDA is a surface eﬀect and the material undergoes <1% photo- polymerization, implying that the radiation is not penetrating the bulk of the sample. The bipiperidinium salt 12$5 is also not photoresponsive (Fig. S33 and S34†), consistent with the unfavourable translational repeat distance observed in the X-ray structure. From these results, bifunctional salt/coformers in general seem to give rise to a slightly oﬀset packing mode that consistently results in an unfavourable repeat distance and tilt angle and hence essentially no photosensitivity. In contrast, the salts of monofunctional ammonium cations are all highly photoactive. Signicant visual colour change occurs aer just ve minutes of irradiation for the salts 1$6, 12$7 and 1$8 (Fig. 9). Signals assigned to photopolymerised material are clearly visible by CP-MAS 13C NMR spectroscopy (Fig. 10 and S35– S37†). Salt 12$7 shows the greatest sensitivity towards UV radi- ation by CP-MAS 13C NMR spectroscopy with the most signi- cant change occurring in the alkene region (100–140 ppm) of the spectrum corresponding to the ene–yne photopolymer functionality (Fig. 10). However, even in these systems the conversion is slow, and the sharpness of the NMR resonances imply a relatively low degree of oligomerisation. This kind of slow reactivity reects the solid-state nature of the process resulting in poor radiation penetration into the bulk of the sample. However, this gradual response is desirable in dosim- etry applications making these materials of considerable interest. The signicant photoreactivity of 12$7 and 1$8 is Table 1 Topochemical parameters for PCDA cocrystals and salts structurally characterised in this work. The tilt angle (q) is calculated from the orientation of 1 in the cocrystal and salt cocrystal samples PXRD patterns and solid-state NMR spectra that are attributable to 1. Chemical Science This suggests that irradiation decomposes the cocrystals to liberate free 1, particularly in the case of the azobenzene complex 12$2. This behaviour is attributed to the photo- isomerisation of coformer 2 resulting in degradation of the cocrystal. The PXRD pattern and solid-state NMR spectra also imply a less-crystalline material at one hour in 12$2 and show the presence of crystalline 1 aer one day of irradiation. In addition, the solid-state NMR spectroscopic data revealed that even the deep blue colour of the irradiated sample of PCDA is a surface eﬀect and the material undergoes <1% photo- polymerization, implying that the radiation is not penetrating the bulk of the sample. The bipiperidinium salt 12$5 is also not photoresponsive (Fig. S33 and S34†), consistent with the unfavourable translational repeat distance observed in the X-ray structure. From these results, bifunctional salt/coformers in general seem to give rise to a slightly oﬀset packing mode that consistently results in an unfavourable repeat distance and tilt angle and hence essentially no photosensitivity. In contrast, the salts of monofunctional ammonium cations are all highly photoactive. Signicant visual colour change occurs aer just ve minutes of irradiation for the salts 1$6, 12$7 and 1$8 (Fig. 9). Signals assigned to photopolymerised material are clearly visible by CP-MAS 13C NMR spectroscopy (Fig. 10 and S35– S37†). Salt 12$7 shows the greatest sensitivity towards UV radi- ation by CP-MAS 13C NMR spectroscopy with the most signi- cant change occurring in the alkene region (100–140 ppm) of the spectrum corresponding to the ene–yne photopolymer functionality (Fig. 10). However, even in these systems the conversion is slow, and the sharpness of the NMR resonances imply a relatively low degree of oligomerisation. This kind of slow reactivity reects the solid-state nature of the process resulting in poor radiation penetration into the bulk of the sample. However, this gradual response is desirable in dosim- etry applications making these materials of considerable interest. The signicant photoreactivity of 12$7 and 1$8 is Compound d/˚A r/˚A q/ 1 3.712(1) 4.574(1) 44.7 12$2 3.633(1) 5.354(1) 48.4 12$3 3.730(1) 5.442(1) 47.4 12$4 3.726(2) 5.449(2) 47.3 12$5 3.760(2) 5.577(2) 24.1 12$7 3.776(2) 4.644(3) 41.9 1$8 3.779(1) 4.593(1) 43.7 Compound d/˚A r/˚A q/ Cocrystal and salt response to UV and X-rays 9 The coformer 1 and cocrystals 12$2, 12$3 and 12$4, and salts 12$5, 1$6, 12$7, 1$8 and the lithium salt (Li salt) before and after UV irradiation at 254 nm for diﬀerent durations. The 12$2a sample was irradiated with UV light at 365 nm while 254 nm radiation was used for the 12$2b sample. The initial colour changes to UV radiation show that only 1 and the salt samples visibly change colour with prolonged irradiation, with 12$7 being the most radiation sensitive to the naked eye and experimentally. Fig. 8 (a) The X-ray structure of 1$8 (b) in the (100) crystallographic plane. This journal is © The Royal Society of Chemistry 2020 Chem. Sci., 2020, 11, 8025–8035 \| 8029 Edge Article View Article Online Edge Article Edge Article 1$8 display changes consistent with UV irradiation for one day, while salt 12$7 displays total loss of amine from the salt under ambient conditions aer seven days (Fig. 11). the p-bonds of the chromophore when irradiated.68 Salt 1$6 also shows signicantly more visual colour change upon irradiation compared to 1 alone. It is likely that the increased hydrogen bonding in the salt brings the monomers of 1 in a closer spatial arrangement and hence makes it more photosensitive. Aer 100 Gy of X-ray irradiation, salt 12$7 also displays a prominent photopolymer alkyne band at 2097.7 cm1 with minimal residual dialkyne signal (Fig. S4†). The pre-resonance Raman eﬀect is very evident in 12$7 and can be seen in the exaggeration of the ene–yne band in Fig. 12 relative to the dialkyne band in the region of 2250 cm1. Solid-state NMR results indicate about 53% polymerisation (Fig. S47†), however the Raman signal for the colourless monomer is almost invisible. Interestingly in the Raman spectrum of 12$7, the breadth of the ene–yne band, and the presence of an additional alkene peak at 1500 cm1 at slightly higher wavenumber than the typical major 1445 cm1 alkene band indicates multiple conformations of the poly- merised material, and implies some structural diﬀerences in the resulting chromophore suggesting that multiple confor- mations of the polymerised salt exist. For 1$8, Raman analysis of the 100 Gy X-ray irradiated sample shows that the salt grad- ually photopolymerises and has a similar radiation sensitivity as 1 alone, with an ene–yne band at 2098.1 cm1 (Fig. S48†). Additionally, for the photosensitive salts, the C–H wagging progressions arising between 1300 cm1 and 1150 cm1 from the polymer side chains of 1 in the salts change with irradiation to suggest a changed conformational structure when compared to the lithium salt (Fig. 13). The change of the side chain conformation is due to diﬀerence in phase angles of coupled oscillations between methylene groups. These diﬀerences in C–H wagging progressions can be used as an additional conformational tool for detecting the presence of a PCDA polymer. Close examination of the diﬀerences in frequency within the wagging mode progressions may also indicate stresses on the side chains due to their close approach to each other as the polymer is formed. Open Access Article. Published on 20 July 2020. Downloaded on 8/25/2020 10:43:31 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 11 The FTIR spectra of salts 1$6, 12$7, and 1$8 in the range of 1475–1825 cm1 showing the carboxylate environment of the salts with volatile amines as the samples are irradiated with UV light (254 nm), and exposure to ambient conditions (AC) for seven days. Fig. 10 CP-MAS 13C NMR spectra of 12$5, 1$6, 12$7, and 1$8 after 7 days of UV irradiation at 254 nm, highlighting the alkyne carbons (60– 85 ppm), alkene carbons of the photoreactive salts (100–135 ppm), and carboxylate environment of each salt (175–185 ppm). Fig. 11 The FTIR spectra of salts 1$6, 12$7, and 1$8 in the range of 1475–1825 cm1 showing the carboxylate environment of the salts with volatile amines as the samples are irradiated with UV light (254 nm), and exposure to ambient conditions (AC) for seven days. Fig. 11 The FTIR spectra of salts 1$6, 12$7, and 1$8 in the range of 1475–1825 cm1 showing the carboxylate environment of the salts with volatile amines as the samples are irradiated with UV light (254 nm), and exposure to ambient conditions (AC) for seven days. Fig. 10 CP-MAS 13C NMR spectra of 12$5, 1$6, 12$7, and 1$8 after 7 days of UV irradiation at 254 nm, highlighting the alkyne carbons (60– 85 ppm), alkene carbons of the photoreactive salts (100–135 ppm), and carboxylate environment of each salt (175–185 ppm). This journal is © The Royal Society of Chemistry 2020 8030 \| Chem. Sci., 2020, 11, 8025–8035 Chemical Science View Article Online Chemical Science View Article Online Chemical Science View Article Online Edge Article Interestingly, the positions of the ene–yne alkyne bands in the irradiated diethylammonium In addition to UV irradiation, the eﬀect of X-ray on PCDA and its derivatives was also analysed. Free PCDA (1) was irradiated with 100 Gy of X-ray radiation and analysed by Raman spec- troscopy (Fig. 12). This revealed a clear ene–yne polymer alkyne band at 2098.8 cm1 with a small residual dialkyne band at 2253.3 cm1 (Fig. S41†). The enhanced appearance of the 2098.8 cm1 band despite the very limited photoreactivity of free 1 is a reection of a pre-resonance Raman eﬀect since the excitation wavelength of the laser 785 nm overlaps with the absorption band of the photopolymer, resulting in signicant enhancement of the chromophore Raman bands. This is consistent with the visual observation of some blue colouration despite the 13C CP MAS-NMR data that indicate a very low degree of bulk conversion (Fig. S4†). In contrast, when all three cocrystals with coformers 2–4 were irradiated with 10 Gy of X-ray radiation they showed very little photoreactivity, as evidenced by the low intensity peaks in the conjugated ene–yne region (approx. 2100 cm1)67 that exists both before and aer irradia- tion (Fig. S42–S44†). Cocrystal 12$2 has a small ene–yne band present at 2100.4 cm1 compared to the other two cocrystals likely arising from small amounts of 1 photopolymer present as a contaminant in the starting PCDA. Similarly to the cocrystals, salt 12$5 displays a band at 2258.4 cm1 assigned to unreacted dialkyne even aer 100 Gy of X-ray irradiation which further reinforces that the salt is photostable (Fig. S45†). The small ene– yne photopolymer band at 2100.3 cm1 is likely to arise from small amounts of photopolymerised PCDA impurities. On the other hand, salt 1$6 shows impressive sensitivity to X-ray radi- ation as indicated by the presence of the signicant ene–yne band at 2088.1 cm1 (Fig. S46†). This band is signicantly red- shied compared to photopolymerised PCDA, indicating a more planar, conjugated conformation of the chromophore. This is in contrast to 1 alone which exhibits torsional strain on Open Access Article. Published on 20 July 2020. Downloaded on 8/25/2020 10:43:31 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 12 Raman spectra of 1, and cocrystal and salts after X-ray irra- diation. The peaks at approx. 2260 cm1 correlate to the alkyne band of monomeric 1, while the internal alkyne of the photopolymer is displayed at approx. 2100 cm1. Fig. 12 Raman spectra of 1, and cocrystal and salts after X-ray irra- diation. The peaks at approx. 2260 cm1 correlate to the alkyne band of monomeric 1, while the internal alkyne of the photopolymer is displayed at approx. 2100 cm1. Fig. 13 The methylene wagging vibrations of 1 and each cocrystal and salt after X-ray irradiation, analysed by Raman spectroscopy. This journal is © The Royal Society of Chemistry 2020 Chem. Sci., 2020, 11, 8025–8035 \| 8031 Fig. 13 The methylene wagging vibrations of 1 and each cocrystal and salt after X-ray irradiation, analysed by Raman spectroscopy. Fig. 12 Raman spectra of 1, and cocrystal and salts after X-ray irra- diation. The peaks at approx. 2260 cm1 correlate to the alkyne band of monomeric 1, while the internal alkyne of the photopolymer is displayed at approx. 2100 cm1. Fig. 13 The methylene wagging vibrations of 1 and each cocrystal and salt after X-ray irradiation, analysed by Raman spectroscopy. Chem. Sci., 2020, 11, 8025–8035 \| 8031 This journal is © The Royal Society of Chemistry 2020 Edge Article View Article Online Edge Article View Article Online Chemical Science and butylammonium salts of around 2100 cm1 contrast sharply with the value of 2066.3 cm1 obtained for commercial lithium PCDA. This signicantly red-shied value implies a much more planar ‘ordered’ chromophore in the lithium salt and hence while the commercial material exists in an ordered ‘blue state’ the use of the organic salt-formers give a less ordered ‘red state’ photopolymer. The value of 2088 cm1 for the morpholinium salt is somewhere in between and implies that the polymer ordering and hence, potentially, colour may be tunable. radiochromic lms and due to their diﬀerent sensitivities to radiation, can be applied to diﬀerent radiation dose ranges, and therapeutic areas. Preliminary indications suggest that they outperform the commercial lithium salt in terms of photosen- sitivity, while Raman spectroscopy shows that the photopoly- mers are relatively disordered with ene–yne bands in the range 2088–2100 cm1, potentially allowing access to a range of colours. Conclusion Three PCDA cocrystals 12$2, 12$3, and 12$4 have been prepared which were engineered to exhibit the common carboxylic acid/ pyridine supramolecular synthon. The lamellar structures of these surfactant-like compounds exhibit two very diﬀerent packing modes with either a syn or anti conformation of the dialkyne substituents and display considerable thermal expan- sion in the c-axis direction upon warming. The cocrystal with the shortest dialkyne distance of 3.631(6) ˚A is 12$2, which is shorter than in PCDA itself and all cocrystals have inter-alkyne distances within the topochemical postulate. However, they all exhibit very little response to UV and X-ray radiation, because the translational repeat distances are greater than the maximum value. Hence, cocrystallisation appears to signi- cantly stabilise PCDA in the solid state. This observation is somewhat surprising in the light of the photoreactivity of related pyridyl-containing cocrystals of di- and trialkynes.45,69 In contrast, salts of monofunctional amines gave highly photo- reactive materials. For example, the morpholinium salt 1$6 changes from pink-to-blue-to-black in under 10 minutes of UV irradiation. Similarly the salt cocrystal with diethylamine 12$7 and the butylammonium salt 1$8 are both highly radiation sensitive and undergo an impressive lilac-to-black colour change with under 5 minutes of UV irradiation for 12$7 and a pink-to-navy colour change for 1$8, in powder form. Their X- ray crystal structures indicate that these salts adhere to the topochemical postulate. These radiation-sensitive salts are of considerable commercial interest for the development of Mass spectra by the ASAP technique were recorded using a LCT Premier XE mass spectrometer (Waters Ltd) heating approximately 1 mg of powder isothermally at 350 C. Single crystal X-ray data for the cocrystals and salts 12$2, 12$4, 12$5, BuA$6, and 12$7 were collected at 120.0(2) K on a Bruker D8 Venture diﬀractometer (Photon 100 CMOS detector, IuS- microsource focusing mirrors) equipped with Cryostream (Oxford Cryostreams) open-ow nitrogen cryostat and using Mo Ka and Cu Ka radiation with wavelengths of 0.71073 ˚A and 1.54178 ˚A, respectively. The single crystal data for 1$8 was collected at 120.0 K on an XCalibur Agilent, Sapphire3 diﬀrac- tometer equipped with Cryostream 700 nitrogen cryostat and using Mo Ka radiation with a wavelength of 0.71073 ˚A. Single crystal data for 1 and 12$3 were collected at 100.0(2) K at I19 beamline (Dectris Pilatus 2M pixel-array photon-counting detector, undulator, graphite monochromator, l ¼ 0.6889 ˚A) at the Diamond Light Source, Oxfordshire. General 10,12-Pentacosadynoic acid (1) was supplied by Ashland LLC, with all other reagents and solvents purchased from standard commercial sources and used without further purication. IR spectra were measured with a PerkinElmer 100 FT-IR spec- trometer with an uATR attachment. Raman spectra were collected on a PerkinElmer Ramanstation 400F with 5–10 accumulations of 10–60 second scans, using an excitation laser with a wavelength of 785 nm. Solid-state NMR spectra were recorded at 100.63 MHz using a Bruker Avance III HD spec- trometer and a 4 mm magic-angle spinning probe. Spectra were obtained using cross-polarisation with a 20 s recycle delay with 7 ms contact time at ambient probe temperature (approx. 25 C) at a sample spin rate of 10 kHz with 400 repetitions. Spectral referencing was with respect to an external sample of neat tet- ramethylsilane. Diﬀerential scanning calorimetry thermograms were recorded using a PerkinElmer 8500 calorimeter, calibrated using an indium standard, with samples accurately weighed (0.01 mg) into standard aluminium pans. Open Access Article. Published on 20 July 2020. Downloaded on 8/25/2020 10:43:31 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. In an attempt to assess the degree of polymerisation, the irradiated samples were analysed by two diﬀerent mass spec- trometry techniques, matrix-assisted laser desorption/ ionisation (MALDI) and atmospheric solid analysis probe (ASAP). However, neither method was able to fully quantify the amount of polymer present in the samples nor the molecular weight distribution. MALDI did not generate any signals assignable to photopolymerised material. In contrast, the ASAP technique did show peaks assigned to PCDA monomer, dimer, and trimer, however, the distribution of these signals was the same before and aer UV irradiation for one day for PCDA itself and even for 12$7 UV irradiated for 14 days. It is likely that higher molecular weight oligomers are present given the broadness of the CP-MAS NMR spectra, therefore, these are not volatile enough to be detected by mass spectrometry in this way. Similarly, the poor solubility of the polymerised materials made them unsuitable for gel permeation chromatography. Open Access Article. Published on 20 July 2020. Downloaded on 8/25/2020 10:43:31 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The novel feature of amine evaporation over time means that transient ammonium salt formation with a volatile amine eﬀectively catalyses the solid-state photopolymerisation of the relatively unreactive PCDA. Open Access Article. Published on 20 July 2020. Downloaded on 8/25/2020 10:43 This article is licensed under a Creative Commons Attribution 3.0 Un 2), a ¼ 5.3544(3) ˚A, b ¼ 6.8239(4) ˚A, c ¼ 39.920(2) ˚A, a ¼ 87.742(4), b ¼ 88.869(4), g ¼ 75.291(4), V ¼ 1409.64(14) ˚A3, Z ¼ 2, Dc ¼ 1.100 g cm3, F000 ¼ 512.0, Cu Ka radiation, l ¼ 1.54178 ˚A, T ¼ 120 K, 2qmax ¼ 137.98, 33 397 reections collected, 5160 unique (Rint ¼ 0.1445). Final GooF ¼ 1.090, R1 ¼ 0.0994, wR2 ¼ 0.1798, R indices based on 5160 reections with I $ 2s(I) (renement on F2), 312 parameters, 0 restraints, m ¼ 0.522 mm1. PCDA 4,40-bipiperidine salt (12$5). Salt 12$5 was prepared by grinding 1 (0.2 g, 0.53 mmol) and 5 (0.045 g, 0.27 mmol) in a 2 : 1 ratio in a Retsch MM 200 mixer mill for 90 minutes at a frequency of 20 s1 (yield ¼ 0.22 g, 0.40 mmol, 89%). The resulting powder (0.030 g) was combined with ethanol (2 mL) and briey sonicated and leto crystallise by slow evaporation at room temperature. Colourless plate crystals formed aer 2 weeks. Analysis calc. of C60H104N2O4: C 78.55, H 11.43, N 3.05%, found: C 77.94, H 11.36, N 2.83%; FTIR (n/cm1): 2917, 2849, 1692, 1644, 1527, 1467, 1418, 1305, 1266, 1231, 1099, 1011, 921, 868, 807, 719, 639. Crystal data: M ¼ 917.45 g mol1, 0.767 0.314 0.1 mm3, triclinic, space group P1 (no. 2), a ¼ 5.5770(4) ˚A, b ¼ 11.8339(8) ˚A, c ¼ 23.0041(15) ˚A, a ¼ 100.670(2), b ¼ 96.096(2), g ¼ 103.007(2), V ¼ 1436.25(17) ˚A3, Z ¼ 1, Dc- ¼ 1.061 g cm3, F000 ¼ 510.0, Mo Ka radiation, l ¼ 0.71073 ˚A, T ¼ 120 K, 2qmax ¼ 65, 36 532 reections collected, 10 400 unique (Rint ¼ 0.0455). Final GooF ¼ 1.019, R1 ¼ 0.0497, wR2 ¼ 0.1263, R indices based on 10 400 reections with I $ 2s(I) (renement on F2), 312 parameters, 0 restraints, m ¼ 0.064 mm1. PCDA 4,40-bipyridyl cocrystal (12$3). Cocrystal 12$3 was prepared by grinding 1 (0.15 g, 0.40 mmol) and 3 (0.032 g, 0.20 mmol) in a Retsch MM 200 mixer mill for 45 minutes at a frequency of 20 s1 (yield ¼ 0.091 g, 0.20 mmol, 50%). The resulting powder was used as a seed (ca. 0.005 g, 0.011 mmol) for the cocrystallisation of 1 (0.025 g, 0.067 mmol) and 3 (0.053 g, 0.034 mmol) in acetone (2 mL). Open Access Article. Published on 20 July 2020. Downloaded on 8/25/2020 10:43 This article is licensed under a Creative Commons Attribution 3.0 Un PCDA trans-1,2-bis(4-pyridine)ethylene cocrystal (12$4). Cocrystal 12$4 was prepared by grinding 1 (0.15 g, 0.40 mmol) and 4 (0.039 g, 0.21 mmol) in a Retsch MM 200 mixer mill for 45 minutes at a frequency of 20 s1 (yield ¼ 0.12 g, 0.26 mmol, 65%). The resulting powder was used as a seed (ca. 0.005 g, 0.011 mmol) for the cocrystallisation of 1 (0.025 g, 0.067 mmol) and 4 (0.0061 g, 0.033 mmol) in acetone (2 mL). Aer brief sonication, the solution was leto crystallise by slow evapora- tion at room temperature and yielded small colourless block crystals. Analysis calc. of C31H47NO2: C 79.95, H 10.17, N 3.01%, found: C 79.84, H 10.14, N 2.86%; FTIR (n/cm1): 2919, 2850, 1688, 1604, 1471, 1414, 1325, 1252, 1235, 1213, 1183, 1100, 974, 826, 718, 553. Crystal data: M ¼ 465.69 g mol1, 0.36 0.31 0.23 mm3, monoclinic, space group P21/c (no. 14), a ¼ 5.4494(3) ˚A, b ¼ 8.9235(5) ˚A, c ¼ 57.441(3) ˚A, b ¼ 92.643(2), V ¼ 2790.2(3) ˚A3, Z ¼ 4, Dc ¼ 1.109 g cm3, F000 ¼ 1024.0, Mo Ka radiation, l ¼ 0.71073 ˚A, T ¼ 120 K, 2qmax ¼ 56.98, 34 945 reections collected, 6537 unique (Rint ¼ 0.0635). Final GooF ¼ 1.034, R1 ¼ 0.0560, wR2 ¼ 0.1073, R indices based on 6537 reections with I $ 2s(I) (renement on F2), 321 parameters, 1 restraint, m ¼ 0.067 mm1. 10,12-Pentacosadiynoic acid (1). Analysis calc. of C25H42O2: C 80.16, H 11.30%, found: C 80.05, H 11.18%; FTIR (n/cm1): 2956, 2918, 2848, 1692, 1467, 1460, 1417, 1291, 1264, 932, 722. Colourless needle crystals of high quality were grown from the slow evaporation of acetone at room temperature for one week from a failed cocrystallisation experiment with pyrazine. Crystal data: M ¼ 374.58 g mol1, 0.12 0.04 0.01 mm3, triclinic, space group P1 (no. 2), a ¼ 4.5738(3) ˚A, b ¼ 5.3909(3) ˚A, c ¼ 46.647(3) ˚A, a ¼ 88.6499(15), b ¼ 88.5073(14), g ¼ 81.4017(14), V ¼ 1136.64(12) ˚A3, Z ¼ 2, Dc ¼ 1.094 g cm3, F000 ¼ 416.0, synchrotron radiation, l ¼ 0.6889 ˚A, T ¼ 100 K, 2qmax ¼ 58.994, 21 023 reections collected, 6893 unique (Rint ¼ 0.0574). Final GooF ¼ 1.035, R1 ¼ 0.0640, wR2 ¼ 0.1772, R indices based on 6893 reections with I $ 2s(I) (renement on F2), 249 parameters, 0 restraints, m ¼ 0.063 mm1. Conclusion All structures were solved using direct methods and rened by full-matrix least squares on F2 for all data using SHELXL50 and OLEX2 soware.51 All non-hydrogen atoms were rened with anisotropic displacement parameters. CH hydrogen atoms were placed in calculated positions, assigned an isotropic displacement factor that is a multiple of the parent carbon atom and allowed to ride. H atoms attached to oxygen atoms were located on the 8032 \| Chem. Sci., 2020, 11, 8025–8035 This journal is © The Royal Society of Chemistry 2020 Chemical Science View Article Online Chemical Science View Article Online Chemical Science View Article Online Open Access Article. Published on 20 July 2020. Downloaded on 8/25/2020 10:43 This article is licensed under a Creative Commons Attribution 3.0 Un PCDA 4,40-azopyridine cocrystal (12$2). Cocrystal 12$2 was prepared by grinding 1 (0.15 g, 0.40 mmol) and 2 (0.037 g, 0.20 mmol) in a 2 : 1 ratio, respectively, in a Retsch MM 200 mixer mill for 1 hour at a frequency of 20 s1 (yield 0.10 g, 0.22 mmol, 55%). The resulting powder was used as a seed (ca. 0.005 g, 0.011 mmol) for the cocrystallisation of 1 (0.025 g, 0.067 mmol) and 2 (0.006 g, 0.033 mmol) in acetone (2 mL). Aer brief sonication, the solution was leto crystallise by slow evapora- tion at room temperature, which yielded large colourless plate crystals. Analysis for C30H46N2O2 calc.: C 77.19, H 9.94, N 6.01%, found: C 77.19, H 9.93, N 5.32%; FTIR (n/cm1): 2936, 2919, 2850, 1695, 1598, 1470, 1410, 1253, 1214, 1184, 1011, 848, 719, 573. Crystal data: M ¼ 466.69 g mol1, 0.32 0.09 0.02 mm3, triclinic, space group P1 (no. 2), a ¼ 5.3544(3) ˚A, b ¼ 6.8239(4) ˚A, c ¼ 39.920(2) ˚A, a ¼ 87.742(4), b ¼ 88.869(4), g ¼ 75.291(4), V ¼ 1409.64(14) ˚A3, Z ¼ 2, Dc ¼ 1.100 g cm3, F000 ¼ 512.0, Cu Ka radiation, l ¼ 1.54178 ˚A, T ¼ 120 K, 2qmax ¼ 137.98, 33 397 reections collected, 5160 unique (Rint ¼ 0.1445). Final GooF ¼ 1.090, R1 ¼ 0.0994, wR2 ¼ 0.1798, R indices based on 5160 reections with I $ 2s(I) (renement on F2), 312 parameters, 0 restraints, m ¼ 0.522 mm1. PCDA 4,40-azopyridine cocrystal (12$2). Cocrystal 12$2 was prepared by grinding 1 (0.15 g, 0.40 mmol) and 2 (0.037 g, 0.20 mmol) in a 2 : 1 ratio, respectively, in a Retsch MM 200 mixer mill for 1 hour at a frequency of 20 s1 (yield 0.10 g, 0.22 mmol, 55%). The resulting powder was used as a seed (ca. 0.005 g, 0.011 mmol) for the cocrystallisation of 1 (0.025 g, 0.067 mmol) and 2 (0.006 g, 0.033 mmol) in acetone (2 mL). Aer brief sonication, the solution was leto crystallise by slow evapora- tion at room temperature, which yielded large colourless plate crystals. Analysis for C30H46N2O2 calc.: C 77.19, H 9.94, N 6.01%, found: C 77.19, H 9.93, N 5.32%; FTIR (n/cm1): 2936, 2919, 2850, 1695, 1598, 1470, 1410, 1253, 1214, 1184, 1011, 848, 719, 573. Crystal data: M ¼ 466.69 g mol1, 0.32 0.09 0.02 mm3, triclinic, space group P1 (no. This journal is © The Royal Society of Chemistry 2020 Edge Article temperature, which yielded small colourless plate crystals. Analysis calc. of C30H46N2O2: C 79.60, H 10.24, N 3.09%, found: C 79.24, H 10.20, N 3.12%; FTIR (n/cm1): 2919, 2850, 1683, 1600, 1471, 1408, 1365, 1325, 1287, 1253, 1212, 1187, 1071, 1000, 821, 718, 625. Crystal data: M ¼ 452.68 g mol1, 0.12 0.04 0.03 mm3, monoclinic, space group P21/c (no. 14), a ¼ 5.4415(2) ˚A, b ¼ 8.9535(4) ˚A, c ¼ 55.673(3) ˚A, b ¼ 90.8823(10), V ¼ 2712.1(2) ˚A3, Z ¼ 4, Dc ¼ 1.109 g cm3, F000 ¼ 996.0, synchrotron radiation, l ¼ 0.6889 ˚A, T ¼ 100 K, 2qmax ¼ 57.994, 47 696 reections collected, 7923 unique (Rint ¼ 0.0663). Final GooF ¼ 1.069, R1 ¼ 0.0529, wR2 ¼ 0.1510, R indices based on 7923 reections with I $ 2s(I) (renement on F2), 303 parameters, 0 restraints, m ¼ 0.064 mm1. temperature, which yielded small colourless plate crystals. Analysis calc. of C30H46N2O2: C 79.60, H 10.24, N 3.09%, found: C 79.24, H 10.20, N 3.12%; FTIR (n/cm1): 2919, 2850, 1683, 1600, 1471, 1408, 1365, 1325, 1287, 1253, 1212, 1187, 1071, 1000, 821, 718, 625. Crystal data: M ¼ 452.68 g mol1, 0.12 0.04 0.03 mm3, monoclinic, space group P21/c (no. 14), a ¼ 5.4415(2) ˚A, b ¼ 8.9535(4) ˚A, c ¼ 55.673(3) ˚A, b ¼ 90.8823(10), V ¼ 2712.1(2) ˚A3, Z ¼ 4, Dc ¼ 1.109 g cm3, F000 ¼ 996.0, synchrotron radiation, l ¼ 0.6889 ˚A, T ¼ 100 K, 2qmax ¼ 57.994, 47 696 reections collected, 7923 unique (Rint ¼ 0.0663). Final GooF ¼ 1.069, R1 ¼ 0.0529, wR2 ¼ 0.1510, R indices based on 7923 reections with I $ 2s(I) (renement on F2), 303 parameters, 0 restraints, m ¼ 0.064 mm1. diﬀerence map when possible or placed in calculated positions. X-ray powder diﬀraction patterns were performed on glass slides, using a Bruker AXS D8 Advance diﬀractometer, with a Lynxeye Soller PSD Detector, using Cu Ka radiation at a wavelength of 1.5406 ˚A. CCDC deposition numbers 2000830– 2000837.† The powdered cocrystal and salt samples were placed on lter paper in a dark box and exposed to a 6-Watt handheld UV light at 254 nm. Compound 12$2 was also irradiated at 365 nm. The powdered samples were mixed at regular intervals to ensure an even exposure of the bulk to irradiation. Notes and references PCDA diethylamine salt cocrystal (12$7). Salt cocrystal 12$7 was prepared by grinding 1 (0.2 g, 0.53 mmol) and 7 (0.055 mL, 0.53 mmol) in a Retsch MM 200 mixer mill for 45 minutes at a frequency of 20 s1 (yield ¼ 0.22 g, 0.5 mmol, 94%). The resulting powder of 12$7 (0.030 g) was combined with acetone (3 mL) and briey sonicated and leto crystallise by slow evapo- ration at room temperature. Purple plate crystals formed aer 2 weeks to reveal a 2 : 1 (PCDA : diethylamine) stoichiometry with the formula (C25H42O2)$(C25H41O2)$(C4H12N+). Analysis calc. for 12$7: C 78.87, H 11.64, N 1.70%, found: C 77.47, H 11.61, N 1.70%; FTIR (n/cm1): 2923, 2846, 1627, 1461, 1423, 1384, 1068, 1010, 955, 854, 811, 724, 592. Crystal data: M ¼ 822.3 g mol1, 0.5 0.12 0.1 mm3, monoclinic, space group P2/n (no. 13), a ¼ 9.5968(6) ˚A, b ¼ 4.6441(3) ˚A, c ¼ 57.520(4) ˚A, b ¼ 92.590(2), V ¼ 2561.0(3) ˚A3, Z ¼ 2, Dc ¼ 1.066 g cm3, F000 ¼ 916.0, Mo Ka radiation, l ¼ 0.71073 ˚A, T ¼ 120 K, 2qmax ¼ 55.996, 30 938 reections collected, 6051 unique (Rint ¼ 0.0502). Final GooF ¼ 1.049, R1 ¼ 0.0561, wR2 ¼ 0.1217, R indices based on 6051 reections with I $ 2s(I) (renement on F2), 274 parameters, 0 restraints, m ¼ 0.065 mm1. 1 F. L. Hirshfeld and G. M. J. Schmidt, J. Polym. Sci., Part A: Gen. Pap., 1964, 2, 2181–2190. 2 G. Wegner, Z. Naturforsch., B: J. Chem. Sci., 1969, 24, 824–832. 3 G. M. J. Schmidt, Pure Appl. Chem., 1971, 27, 647–678. 4 V. Enkelmann, Adv. Polym. Sci., 1984, 63, 91–136. 5 J. W. Lauher, F. W. Fowler and N. S. Goroﬀ, Acc. Chem. Res., 2008, 41, 1215–1229. 6 S. C. Wang, Y. Li, H. Liu, J. P. Li, T. S. Li, Y. J. Wu, S. Okada and H. Nakanishi, Org. Biomol. Chem., 2015, 13, 5467–5474. 7 R. H. Baughman, J. Appl. Phys., 1972, 43, 4362–4370. 8 R. H. Baughman and K. C. Yee, J. Polym. Sci., Part D: Macromol. Rev., 1978, 13, 219–239. 9 A. Mueller and D. F. O'Brien, Chem. Rev., 2002, 102, 727–757. 10 N. Charoenthai, T. Pattanatornchai, S. Wacharasindhu, M. Sukwattanasinitt and R. Traiphol, J. Colloid Interface Sci., 2011, 360, 565–573. 11 D. J. Ahn, E. H. Chae, G. S. Lee, H. Y. Shim, T. E. Chang, K. Acknowledgements We thank Ashland LLC and the Engineering and Physical Sciences Research Council for studentship funding. We also thank the Diamond Light Source for an award of instrument time on the Station I19 (MT 16117 and CY222240) and the instrument scientists for their support and patience. Addition- ally, we thank Mr W. Douglas Carswell for his assistance with DSC measurements, Dr David Parker for his help and expertise with mass spectrometry experiments, and Dr Andrei Batsanov for collection data of compound 12$5. Open Access Article. Published on 20 July 2020. Downloaded on 8/25/2020 10:43:31 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Morpholinium butanoate salt (BuA$6). Salt 1$6 was prepared by combining butanoic acid (0.05 mL, 0.55 mmol) and 6 (0.047 mL, 0.55 mmol) to give a yellow oil and was leto precipitate slowly overnight in a sealed round-bottom ask to yield large colourless plate crystals (yield ¼ 0.088 g, 0.5 mmol, 91%). Analysis calc. of C8H17NO3: C 54.84, H 9.78, N 7.99%, found: C 54.38, H 10.06, N 7.70%; FTIR (n/cm1): 2961, 2871, 1711, 1545, 1456, 1394, 1379, 1303, 1243, 1195, 1107, 1049, 997, 878, 829, 766, 615. Crystal data: M ¼ 175.22 g mol1, 0.44 0.25 0.21 mm3, monoclinic, space group C2/c (no. 15), a ¼ 20.0926(14) ˚A, b ¼ 8.0678(6) ˚A, c ¼ 11.6061(8) ˚A, b ¼ 97.064(3), V ¼ 1867.1(2) ˚A3, Z ¼ 8, Dc ¼ 1.247 g cm3, F000 ¼ 768.0, Mo Ka radiation, l ¼ 0.71073 ˚A, T ¼ 120 K, 2qmax ¼ 58.994, 13 471 reections collected, 2587 unique (Rint ¼ 0.0313). Final GooF ¼ 1.023, R1 ¼ 0.0356, wR2 ¼ 0.0931, R indices based on 2587 reections with I $ 2s(I) (renement on F2), 177 parameters, 0 restraints, m ¼ 0.094 mm1. Open Access Article. Published on 20 July 2020. Downloaded on 8/25/2020 10:43 This article is licensed under a Creative Commons Attribution 3.0 Un Aer brief sonication, the solution was leto crystallise by slow evaporation at room PCDA morpholine salt (1$6). Salt 1$6 was prepared by grinding 1 (0.2 g, 0.53 mmol) and 6 (0.046 mL, 0.53 mmol) in a Retsch MM 200 mixer mill for 45 minutes at a frequency of 20 s1 (yield ¼ 0.24 g, 0.52 mmol, 98%). The resulting powder of PCDA morpholine salt (1$6). Salt 1$6 was prepared by grinding 1 (0.2 g, 0.53 mmol) and 6 (0.046 mL, 0.53 mmol) in a Retsch MM 200 mixer mill for 45 minutes at a frequency of 20 s1 (yield ¼ 0.24 g, 0.52 mmol, 98%). The resulting powder of Chem. Sci., 2020, 11, 8025–8035 \| 8033 This journal is © The Royal Society of Chemistry 2020 Edge Article View Article Online Chemical Science Chemical Science 1$6 (0.030 g) was combined with acetone (3 mL) and briey sonicated and leto crystallise by slow evaporation at room temperature. Colourless plate crystals formed aer 2 weeks, however, crystallisations with and without seeding yielded poor- quality crystals that did not diﬀract suﬃciently to allow single crystal structure determination. Analysis calc. of C29H51NO3: C 75.45, H 11.14, N 3.03%, found C 75.18, H 11.08, N 2.80%; FTIR (n/cm1): 2917, 2850, 1652, 1515, 1474, 1406, 1297, 1108, 879, 728, 616. 1115, 1095, 1056, 1028, 932, 921, 750, 722, 650. Crystal data: M ¼ 447.72 g mol1, 0.36 0.08 0.05 mm3, monoclinic, space group P21 (no. 4), a ¼ 4.5934(6) ˚A, b ¼ 56.597(9) ˚A, c ¼ 5.5096(6) ˚A, b ¼ 99.130(10), V ¼ 1414.2(3) ˚A3, Z ¼ 2, Dc ¼ 1.051 g cm3, F000 ¼ 500.0, Mo Ka radiation, l ¼ 0.71073 ˚A, T ¼ 120 K, 2qmax ¼ 53.97, 14 179 reections collected, 5663 unique (Rint ¼ 0.1264). Final GooF ¼ 1.026, R1 ¼ 0.1156, wR2 ¼ 0.2163, R indices based on 5663 reections with I $ 2s(I) (renement on F2), 293 parameters, 22 restraints, m ¼ 0.064 mm1. 1$6 (0.030 g) was combined with acetone (3 mL) and briey sonicated and leto crystallise by slow evaporation at room temperature. Colourless plate crystals formed aer 2 weeks, however, crystallisations with and without seeding yielded poor- quality crystals that did not diﬀract suﬃciently to allow single crystal structure determination. Analysis calc. of C29H51NO3: C 75.45, H 11.14, N 3.03%, found C 75.18, H 11.08, N 2.80%; FTIR (n/cm1): 2917, 2850, 1652, 1515, 1474, 1406, 1297, 1108, 879, 728, 616. Open Access Article. Published on 20 July 2020. Downloaded on 8/25/2020 10:43:31 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 58 J. Anyumba, D. F. Lewis, H.-Y. Shih, X. Yu, Europe Pat., EP1614002A4, 2004. 30 Y. J. Gwon, C. Kim and T. S. Lee, Sens. Actuators, B, 2019, 281, 343–349. 31 D. E. Wang, X. H. Gao, G. B. Li, T. Xue, H. Yang and H. Y. Xu, Sens. Actuators, B, 2019, 289, 85–92. 59 C. B. Aakeroy, S. Panikkattu, B. DeHaven and J. Desper, CrystEngComm, 2013, 15, 463–470. 32 S. Ali, F. Ahmed and A. Khatri, Mehran Univ. Res. J. Eng. Technol., 2016, 35, 287–292. 60 A. O. Surov, A. A. Simagina, N. G. Manin, L. G. Kuzmina, A. V. Churakov and G. L. Perlovich, Cryst. Growth Des., 2015, 15, 228–238. 33 M. Kim, Y. J. Shin, S. W. Hwang, M. J. Shin and J. S. Shin, J. Appl. Polym. Sci., 2018, 135, 5. 61 K. Tsaggeos, N. Masiera, A. Niwicka, V. Dokorou, M. G. Siskos, S. Skoulika and A. Michaelides, Cryst. Growth Des., 2012, 12, 2187–2194. 34 J. P. Jeong, E. Cho, D. Yun, T. Kim, I. S. Lee and S. Jung, Polymers, 2017, 9, 9. 62 Sigma-Aldrich, 4,40-Bipiperidine, https:// www.sigmaaldrich.com/catalog/product/aldrich/705845? lang¼en&region¼GB, accessed 27/01/2020, 2020. 35 J. Luo, K. Y. Fu, H. Y. Dong and D. Y. Chen, Chin. J. Chem. Phys., 2016, 29, 749–753. 36 A. Kamphan, C. J. Gong, K. Maiti, S. Sur, R. Traiphol and D. P. Arya, RSC Adv., 2017, 7, 41435–41443. 63 D. Vedal, O. H. Ellestad, P. Klaboe and G. Hagen, Spectrochim. Acta, Part A, 1976, 32, 877–890. 37 P. Sun, Y. C. Fu, J. Hu, N. Hao, W. Huang and B. Jiang, Radiat. Meas., 2016, 85, 116–125. 64 A. Aucejo, S. Loras, R. Munoz, J. Wisniak and H. Segura, J. Chem. Eng. Data, 1997, 42, 1201–1207. 38 S. Devic, N. Tomic and D. Lewis, Phys. Med., 2016, 32, 541– 556. 65 M. Dominguez, S. Martin, H. Artigas, M. C. Lopez and F. M. Royo, J. Chem. Eng. Data, 2002, 47, 405–410. 39 A. Rink, D. F. Lewis, S. Varma, I. A. Vitkin and D. A. Jaﬀray, J. Med. Phys., 2008, 35, 4545–4555. 66 M. Zhu and L. Yu, J. Therm. Anal. Calorim., 2018, 132, 463– 469. 40 Y. S. Soliman, A. A. Abdel-Fattah, A. A. Hamed and A. M. M. Bayomi, Radiat. Phys. Chem., 2018, 144, 56–62. 67 Y. Y. Xu, S. Y. Fu, F. Y. Liu, H. Y. Yu and J. G. Gao, SoMatter, 2018, 14, 8044–8050. 41 K. Open Access Article. Published on 20 July 2020. Downloaded on 8/25/2020 10:43:31 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 47 L. Luo, C. Wilhelm, A. W. Sun, C. P. Grey, J. W. Lauher and N. S. Goroﬀ, J. Am. Chem. Soc., 2008, 130, 7702–7709. N. S. Goroﬀ, J. Am. Chem. Soc., 2008, 130, 7702–7709. 21 M. J. Shin and J. S. Shin, J. Appl. Polym. Sci., 2019, 136, 1–7. 48 W.-Q. Tong and G. Whitesell, Pharm. Dev. Technol., 1998, 3, 215–223. 22 R. W. Carpick, D. Y. Sasaki, M. S. Marcus, M. A. Eriksson and 22 R. W. Carpick, D. Y. Sasaki, M. S. Marcus, M. A. Eriksson and A. R. Burns, J. Phys.: Condens. Matter, 2004, 16, 679–697. 49 D. J. Berry and J. W. Steed, Adv. Drug Delivery Rev., 2017, 117, 3–24. A. R. Burns, J. Phys.: Condens. Matter, 2004, 16, 679–697. 23 H. S. Peng, X. M. Sun, F. J. Cai, X. L. Chen, Y. C. Zhu, G. P. Liao, D. Y. Chen, Q. W. Li, Y. F. Lu, Y. T. Zhu and Q. X. Jia, Nat. Nanotechnol., 2009, 4, 738–741. 50 M. Khan, V. Enkelmann and G. Brunklaus, Cryst. Growth Des., 2009, 9, 2354–2362. 51 G. R. Desiraju, Angew. Chem., Int. Ed. Engl., 1995, 34, 2311– 2327. 24 J. L. Deng, Z. H. Sheng, K. Zhou, M. X. Duan, C. Y. Yu and L. Jiang, Bioconjugate Chem., 2009, 20, 533–537. 52 G. R. Desiraju, J. Am. Chem. Soc., 2013, 135, 9952–9967. 25 D. J. Ahn and J. M. Kim, Acc. Chem. Res., 2008, 41, 805–816. 53 T. R. Shattock, K. K. Arora, P. Vishweshwar and M. J. Zaworotko, Cryst. Growth Des., 2008, 8, 4533–4545. 26 J. Pang, L. Yang, B. F. McCaughey, H. Peng, H. S. Ashbaugh, C. J. Brinker and Y. Lu, J. Phys. Chem. B, 2006, 110, 7221– 7225. 54 T. K. Adalder, R. Sankolli and P. Dastidar, Cryst. Growth Des., 2012, 12, 2533–2542. 27 C. Khanantong, N. Charoenthai, S. Wacharasindhu, M. Sukwattanasinitt, N. Traiphol and R. Traiphol, J. Ind. Eng. Chem., 2018, 58, 258–265. 55 M. C. Etter, Acc. Chem. Res., 1990, 23, 120–126. 56 F. H. Allen, W. D. S. Motherwell, P. R. Raithby, G. P. Shields and R. Taylor, New J. Chem., 1999, 23, 25–34. 28 R. Kassis, M. Bassil and A. Al Choueiry, Mater. Res. Express, 2019, 6, 1–7. 57 M. Okaniwa, Y. Oaki, S. Kaneko, K. Ishida, H. Maki and H. Imai, Chem. Mater., 2015, 27, 2627–2632. 29 Y. L. Su, React. Funct. Polym., 2006, 66, 967–973. Notes and references D. Ahn and J. M. Kim, J. Am. Chem. Soc., 2003, 125, 8976–8977. PCDA n-butylamine salt (1$8). Salt 1$8 was prepared by grinding 1 (0.2 g, 0.53 mmol) and 8 (0.053 mL, 0.53 mmol) in a Retsch MM 200 mixer mill for 45 minutes at a frequency of 20 s1 (yield ¼ 0.23 g, 0.5 mmol, 96%). The resulting powder of 1$8 (0.030 g) was combined with acetone (2 mL) and briey soni- cated. A powder seed (ca. 0.004 g, 0.0089 mmol) was added to the sample and leto crystallise by slow evaporation at room temperature. Blue block crystals formed aer one month. Anal- ysis calc. of C29H53NO2: C 77.79, H 11.93, N 3.13%, found: C 77.50, H 11.84, N 3.08%; FTIR (n/cm1): 2919, 2848, 2675, 2594, 2183, 1650, 1567, 1508, 1461, 1411, 1334, 1309, 1272, 1239, 1201, 12 C. Khanantong, N. Charoenthai, F. Kielar, N. Traiphol and R. Traiphol, Colloids Surf., A, 2019, 561, 226–235. 13 H. Jeon, J. Lee, M. H. Kim and J. Yoon, Macromol. Rapid Commun., 2012, 33, 972–976. 14 G. S. Lee, S. J. Hyun and D. J. Ahn, Macromol. Res., 2018, 26, 566–570. 15 K. Yoo, S. Kim, N. Han, G. E. Kim, M. J. Shin, J. S. Shin and M. Kim, Dyes Pigm., 2018, 149, 242–245. 16 J. P. Yapor, A. Alharby, C. Gentry-Weeks, M. M. Reynolds, A. Alam and Y. V. Li, ACS Omega, 2017, 2, 7334–7342. This journal is © The Royal Society of Chemistry 2020 8034 \| Chem. Sci., 2020, 11, 8025–8035 View Article Online View Article Online Edge Article Chemical Science Chemical Science 17 T. Pattanatornchai, N. Charoenthai, S. Wacharasindhu, M. Sukwattanasinitt and R. Traiphol, J. Colloid Interface Sci., 2013, 391, 45–53. 44 S. M. Curtis, N. Le, F. W. Fowler and J. W. Lauher, Cryst. Growth Des., 2005, 5, 2313–2321. 45 J. J. Kane, R. F. Liao, J. W. Lauher and F. W. Fowler, J. Am. Chem. Soc., 1995, 117, 12003–12004. 18 B. Yoon, S. Lee and J. M. Kim, Chem. Soc. Rev., 2009, 38, 1958–1968. 46 N. S. Goroﬀ, S. M. Curtis, J. A. Webb, F. W. Fowler and J. W. Lauher, Org. Lett., 2005, 7, 1891–1893. , , J , J. W. Lauher, Org. Lett., 2005, 7, 1891–1893. 19 M. J. Shin and J. D. Kim, Langmuir, 2016, 32, 882–888. 47 L. Luo, C. Wilhelm, A. W. Sun, C. P. Grey, J. W. Lauher and 47 L. Luo, C. Wilhelm, A. W. Sun, C. P. Grey, J. W. Lauher and N S GoroﬀJ Am Chem Soc 2008 130 7702 7709 20 R. Kassis, M. Bassil and A. Al Choueiry, Mater. Res. Express, 2019, 6, 1–7. Open Access Article. Published on 20 July 2020. Downloaded on 8/25/2020 10:43:31 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fahsi, J. Deschamps, K. Chougrani, L. Viau, B. Boury, A. Vioux, A. van der Lee and S. G. Dutremez, CrystEngComm, 2013, 15, 4261–4279. 68 M. Wenzel and G. H. Atkinson, J. Am. Chem. Soc., 1989, 111, 6123–6127. 42 H. Matsuzawa, S. Okada, A. Sarkar, H. Matsuda and H. Nakanishi, Polym. J., 2001, 33, 182–189. 69 J. Xiao, M. Yang, J. W. Lauher and F. W. Fowler, Angew. Chem., Int. Ed., 2000, 39, 2216–2219. 43 B. C. Roy and S. Mallik, Org. Lett., 2001, 3, 1877–1879. Chem. Sci., 2020, 11, 8025–8035 \| 8035 This journal is © The Royal Society of Chemistry 2020
https://openalex.org/W4210728383	https://vbn.aau.dk/ws/files/294335982/1_s2.0_S014067361830134X_main.pdf	English	null	Risk thresholds for alcohol consumption: combined analysis of individual-participant data for 599 912 current drinkers in 83 prospective studies	Lancet	2,018	cc-by	12,485	Aalborg Universitet Aalborg Universitet Lancet 2018; 391: 1513–23 This online publication has been corrected. The corrected version first appeared at thelancet.com on May 31, 2018 See Comment page 1460 Other investigators of the Emerging Risk Factors Collaboration are listed in the appendix p 48 Department of Public Health and Primary Care, University of Cambridge, Cambridge, UK (A M Wood PhD, S Kaptoge PhD, A S Butterworth PhD, P Willeit MD, S Warnakula PhD, T Bolton MMath, D S Paul PhD, M Sweeting PhD, S Burgess PhD, S Bell PhD, W Astle PhD, D Stevens MSc, Prof S G Thompson FMedSci, E Di Angelantonio MD, Prof J Danesh FMedSci); Medical University Innsbruck, Innsbruck, Austria (P Willeit, Prof S Kiechl MD); National Centre for Epidemiology and Population Health, Australian National University, Canberra, Australia (E Paige PhD, Prof E Banks PhD); MRC Biostatistics Unit, Cambridge Institute of Public Health, University of Cambridge, Cambridge, UK (S Burgess); NIHR BRC Nutritional Biomarker Laboratory, University of Cambridge, Cambridge, UK (A Koulman PhD); Norwegian Methods We did a combined analysis of individual-participant data from three large-scale data sources in 19 high- income countries (the Emerging Risk Factors Collaboration, EPIC-CVD, and the UK Biobank). We characterised dose–response associations and calculated hazard ratios (HRs) per 100 g per week of alcohol (12·5 units per week) across 83 prospective studies, adjusting at least for study or centre, age, sex, smoking, and diabetes. To be eligible for the analysis, participants had to have information recorded about their alcohol consumption amount and status (ie, non-drinker vs current drinker), plus age, sex, history of diabetes and smoking status, at least 1 year of follow-up after baseline, and no baseline history of cardiovascular disease. The main analyses focused on current drinkers, whose baseline alcohol consumption was categorised into eight predefined groups according to the amount in grams consumed per week. We assessed alcohol consumption in relation to all-cause mortality, total cardiovascular disease, and several cardiovascular disease subtypes. We corrected HRs for estimated long-term variability in alcohol consumption using 152 640 serial alcohol assessments obtained some years apart (median interval 5·6 years [5th–95th percentile 1·04–13·5]) from 71 011 participants from 37 studies. Findings In the 599 912 current drinkers included in the analysis, we recorded 40 310 deaths and 39 018 incident cardiovascular disease events during 5·4 million person-years of follow-up. Risk thresholds for alcohol consumption combined analysis of individual-participant data for 599 912 current drinkers in 83 prospective studies Wood, Angela M; Kaptoge, Stephen; Butterworth, Adam S; Willeit, Peter; Warnakula, Samantha; Bolton, Thomas; Paige, Ellie; Paul, Dirk S; Sweeting, Michael; Burgess, Stephen; Bell, Steven; Astle, William; Stevens, David; Koulman, Albert; Selmer, Randi M; Verschuren, W M Monique; Sato, Shinichi; Njølstad, Inger; Woodward, Mark; Salomaa, Veikko; Nordestgaard, Børge G; Yeap, Bu B; Fletcher, Astrid; Melander, Olle; Kuller, Lewis H; Balkau Beverley; Marmot, Michael; Koenig, Wolfgang; Casiglia, Edoardo; Cooper, Cyrus; Arndt, Volker; Franco, Oscar H; Wennberg, Patrik; Gallacher, John; de la Cámara, Agustín Gómez; Völzke, Henry; Dahm, Christina C; Dale, Caroline E; Bergmann, Manuela M; Crespo, Carlos J; van der Schouw, Yvonne T; Kaaks, Rudolf; Simons, Leon A; Lagiou, Pagona; Schoufour, Josje D; Boer, Jolanda M A; Key, Timothy J; Rodriguez, Beatriz; Moreno-Iribas, Conchi; Davidson, Karina W ; Taylor, James O ; Sacerdote, Carlotta; Wallace, Robert B ; Quirós, J Ramón; Tumino, Rosario; Blazer, II, Dan G ; Linneberg, Allan; Daimon, Makoto; Panico, Salvatore; Howard, Barbara ; Skeie, Guri; Strandberg, Timo ; Weiderpass, Elisabete; Nietert, Paul J ; Psaty, Bruce M; Kromhout, Daan ; Salamanca-Fernandez, Elena; Kiechl, Stefan ; Krumholz, Harlan M ; Grioni, Sara; Palli, Domenico; Huerta, José M; Price, Jackie ; Sundström, Johan ; Arriola, Larraitz; Arima, Hisatomi ; Travis, Ruth C; Panagiotakos, Demosthenes B ; Karakatsani, Anna; Trichopoulou, Antonia; Kühn, Tilman; Grobbee, Diederick E; Barrett-Connor, Elizabeth ; van Schoor, Natasja ; Boeing, Heiner; Overvad, Kim; Kauhanen, Jussi ; Wareham, Nick; Langenberg, Claudia; Forouhi, Nita ; Wennberg, Maria; Després, Jean-Pierre ; Cushman, Mary; Cooper, Jackie A ; Rodriguez, Carlos J ; Sakurai, Masaru ; Shaw, Jonathan E; Knuiman, Matthew ; Voortman, Trudy ; Meisinger, Christa; Tjønneland, Anne; Brenner, Hermann; Palmieri, Luigi; Dallongeville, Jean; Brunner, Eric J ; Assmann, Gerd ; Trevisan, Maurizio ; Gillum, Richard F.; Ford, Ian; Sattar, Naveed; Lazo, Mariana ; Thompson, Simon G; Ferrari, Pietro; Leon, David A ; Davey Smith, George; Peto, Richard; Jackson, Rod ; Banks, Emily ; Di Angelantonio, Emanuele; Danesh, John; Emerging Risk Factors Collaboration/EPIC-CVD/UK Biobank Alcohol Study Group Published in: Lancet DOI (link to publication from Publisher): 10.1016/S0140-6736(18)30134-X Link to publication from Aalborg University Articles Summary y Background Low-risk limits recommended for alcohol consumption vary substantially across different national guidelines. To define thresholds associated with lowest risk for all-cause mortality and cardiovascular disease, we studied individual-participant data from 599 912 current drinkers without previous cardiovascular disease. Risk thresholds for alcohol consumption: combined analysis of individual-participant data for 599 912 current drinkers in 83 prospective studies Angela M Wood, Stephen Kaptoge, Adam S Butterworth, Peter Willeit, Samantha Warnakula, Thomas Bolton, Ellie Paige, Dirk S Paul, Michael Sweeting, Stephen Burgess, Steven Bell, William Astle, David Stevens, Albert Koulman, Randi M Selmer, W M Monique Verschuren, Shinichi Sato, Inger Njølstad, Mark Woodward, Veikko Salomaa, Børge G Nordestgaard, Bu B Yeap, Astrid Fletcher, Olle Melander, Lewis H Kuller, Beverley Balkau, Michael Marmot, Wolfgang Koenig, Edoardo Casiglia, Cyrus Cooper, Volker Arndt, Oscar H Franco, Patrik Wennberg, John Gallacher, Agustín Gómez de la Cámara, Henry Völzke, Christina C Dahm, Caroline E Dale, Manuela M Bergmann, Carlos J Crespo, Yvonne T van der Schouw, Rudolf Kaaks, Leon A Simons, Pagona Lagiou, Josje D Schoufour, Jolanda M A Boer, Timothy J Key, Beatriz Rodriguez, Conchi Moreno-Iribas, Karina W Davidson, James O Taylor, Carlotta Sacerdote, Robert B Wallace, J Ramon Quiros, Rosario Tumino, Dan G Blazer II, Allan Linneberg, Makoto Daimon, Salvatore Panico, Barbara Howard, Guri Skeie, Timo Strandberg, Elisabete Weiderpass, Paul J Nietert, Bruce M Psaty, Daan Kromhout, Elena Salamanca-Fernandez, Stefan Kiechl, Harlan M Krumholz, Sara Grioni, Domenico Palli, José M Huerta, Jackie Price, Johan Sundström, Larraitz Arriola, Hisatomi Arima, Ruth C Travis, Demosthenes B Panagiotakos, Anna Karakatsani, Antonia Trichopoulou, Tilman Kühn, Diederick E Grobbee, Elizabeth Barrett-Connor, Natasja van Schoor, Heiner Boeing, Kim Overvad, Jussi Kauhanen, Nick Wareham, Claudia Langenberg, Nita Forouhi, Maria Wennberg, Jean-Pierre Després, Mary Cushman, Jackie A Cooper, Carlos J Rodriguez, Masaru Sakurai, Jonathan E Shaw, Matthew Knuiman, Trudy Voortman, Christa Meisinger, Anne Tjønneland, Hermann Brenner, Luigi Palmieri, Jean Dallongeville, Eric J Brunner, Gerd Assmann, Maurizio Trevisan, Richard F Gillum, Ian Ford, Naveed Sattar, Mariana Lazo, Simon G Thompson, Pietro Ferrari, David A Leon, George Davey Smith, Richard Peto, Rod Jackson, Emily Banks, Emanuele Di Angelantonio, John Danesh, for the Emerging Risk Factors Collaboration/EPIC-CVD/UK Biobank Alcohol Study Group This online publication has been corrected. The corrected version first appeared at thelancet.com on May 31, 2018 See Comment page 1460 Other investigators of the Emerging Risk Factors Collaboration are listed in the appendix p 48 This online publication has been corrected. The corrected version first appeared at thelancet.com on May 31, 2018 See Comment page 1460 Other investigators of the Emerging Risk Factors Collaboration are listed in the appendix p 48 Department of Public Health and Primary Care, University of Cambridge, Cambridge, UK (A M Wood PhD, S Kaptoge PhD, A S Butterworth PhD, P Willeit MD, S Warnakula PhD, T Bolton MMath, D S Paul PhD, M Sweeting PhD, S Burgess PhD, S Bell PhD, W Astle PhD, D Stevens MSc, Prof S G Thompson FMedSci, E Di Angelantonio MD, Prof J Danesh FMedSci); Medical University Innsbruck, Innsbruck, Austria (P Willeit, Prof S Kiechl MD); National Centre for Epidemiology and Population Health, Australian National University, Canberra, Australia (E Paige PhD, Prof E Banks PhD); MRC Biostatistics Unit, Cambridge Institute of Public Health, University of Cambridge, Cambridge, UK (S Burgess); NIHR BRC Nutritional Biomarker Laboratory, University of Cambridge, Cambridge, UK (A Koulman PhD); Norwegian Introduction Alcohol consumption guidelines vary substantially across the globe.1,2 In the USA, for example, an upper limit of 196 g per week (about 11 standard UK glasses of wine or pints of beer per week) is recommended for men, and an upper limit of 98 g per week is recommended for women.1 Similar recommendations apply in Canada and Sweden.2 By contrast, guidelines in Italy, Portugal, and Spain recommend low-risk limits almost 50% higher than these.1,2 At the other extreme, UK guidelines recommend low-risk limits for men almost half that recommended by US guidelines.1,2 Study design, data sources, and participants We focused our study on current alcohol drinkers for three main reasons. First, alcohol guidelines provide recommendations about low-risk limits only for drinkers (we are unaware of any guidelines that encourage non-drinkers to consume alcohol). Second, a focus on current drinkers should limit potential biases that are difficult to control in observational studies (eg, reverse causality, residual confounding, and unmeasured effect modification) because ex-drinkers include people who might have abstained from alcohol owing to poor health itself,18–20 as well as those who have changed their habits to achieve a healthier lifestyle. Third, never-drinkers might differ systematically from drinkers in ways that are difficult to measure, but which might be relevant to disease causation.21 Such variation in policy might reflect ambiguity about drinking risk thresholds associated with the lowest risk of mortality,3–15 as well as uncertainty about the specific consequences of alcohol consumption, including those related to cardiovascular disease subtypes. For example, recent studies have challenged the concept that moderate alcohol consumption is universally associated with lower cardiovascular disease risk,16,17 but the dose–response associations of alcohol consumption with cardiovascular disease subtypes remain poorly understood. Therefore, to help in the formulation of evidence-based alcohol policy, we analysed individual-participant data from 83 long- term prospective studies in 19 high-income countries. Our aim was to characterise risk thresholds for all-cause mortality and cardiovascular disease subtypes in current drinkers of alcohol. We did a combined analysis of individual-participant data from three large-scale data sources available to our consortium, each constituting purpose-designed pro spective cohort studies with quantitative information about alcohol consumption (appendix p 21). Implications of all the available evidence The chief implication of this study for public policy is to support reductions of alcohol consumption limits in existing guidelines, suggesting that the threshold for lowest risk for all-cause mortality is about 100 g per week (about 5–6 standard UK glasses of wine or pints of beer per week). The chief implication for scientific understanding is the strengthening of evidence that the association between alcohol consumption and total cardiovascular disease risk is actually comprised of several distinct and opposite dose–response curves rather than a single J-shaped association. Introduction First, the Emerging Risk Factors Collaboration (ERFC) is a collaboration of prospective cohort studies with infor mation about a variety of risk factors, cardiovascular disease outcomes, and mortality.22 Of the 102 studies in the ERFC with information about alcohol status, 81 contained information about the quantity of consumption. Second, Funding UK Medical Research Council, British Heart Foundation, National Institute for Health Research, European Union Framework 7, and European Research Council. Funding UK Medical Research Council, British Heart Foundation, National Institute for Health Research, European Union Framework 7, and European Research Council. Funding UK Medical Research Council, British Heart Foundation, National Institute for Health Research, European Union Framework 7, and European Research Council. Institute of Public Health, Oslo, Norway (R M Selmer PhD); National Institute for Public Health and the Environment, Bilthoven, Netherlands (Prof W M M Verschuren PhD, J M A Boer PhD); Julius Centre for Health Sciences and Primary Care, University Medical Center Utrecht, Utrecht, Netherlands (Prof W M M Verschuren, Prof Y T van der Schouw PhD, Prof D E Grobbee MD); Chiba Prefectural Institute of Public Health, Chiba, Japan (Prof S Sato MD); Department of Community Medicine, University of Tromsø, Tromsø, Norway (Prof I Njølstad MD); Nuffield Department of Population Health, Medical Sciences Division, University of Oxford, Oxford, UK (Prof M Woodward PhD, Prof R Peto FRS, Prof T J Key DPhil, R C Travis DPhil); The George Institute for Global Health, University of Sydney, Sydney, NSW, Australia (Prof M Woodward); Bloomberg School of Public Health (Prof M Woodward) and School of Medicine (M Lazo MD), Johns Hopkins University, Baltimore, MD, USA; THL-National Institute for Health and Welfare, Helsinki, Finland (Prof V Salomaa MD); Copenhagen University Hospital, Copenhagen, Denmark (Prof B G Nordestgaard MD); Department of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark (Prof B G Nordestgaard, Prof A Linneberg MD); School of Medicine, University of Western Australia, Perth, WA, Australia (Prof B B Yeap MBBS); Fiona Stanley Hospital, Perth, WA, Australia (Prof B B Yeap); Harry Perkins Institute of Medical Research, Perth, WA, Australia (Prof B B Yeap); London School of Hygiene & Tropical Medicine, London, UK (Prof A Fletcher PhD, Prof D A Leon PhD); Department of Clinical Sciences, Malmö, Lund University, Malmö, Sweden (Prof O Melander MD); Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA, USA (Prof L H Kuller MD); CESP INSERM UMRS 1018, Villejuif Cedex, France (B Balkau PhD); Department of Epidemiology and Public Health, University College London, London, UK (Prof M Marmot FMedSci, Prof E J Brunner PhD, Copyright © The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. pyright © The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 lic Evidence before this study First, it reduced the potentially distorting effects of reverse causality by focusing on current drinkers without previous cardiovascular disease who survived at least 12 months of follow-up. Second, it enhanced generalisability by including individual-participant data from 83 prospective studies in 19 different high-income countries. Third, it used a variety of established and emerging risk factors, enabling investigation of potential confounders and mediators. We searched for prospective epidemiological studies of alcohol consumption investigating disease risk thresholds published in any language up until March 1, 2017 (with no specified earliest date), in PubMed, Scientiﬁc Citation Index Expanded, and Embase using relevant terms (“alcohol”, “mortality”, “survival”, “cardiovascular disease”, “cohort”, and “prospective”). We found many primary reports and literature-based reviews. However, no study had combined the following key features required to achieve reliable estimates of dose–response associations: availability of individual-participant data; quantitative assessment of alcohol consumption levels using validated instruments; periodic re-surveys of alcohol consumption levels; recording of large numbers of deaths (eg, >20 000 deaths); and sufficient detail and power to disaggregate incident cardiovascular disease outcomes into subtypes (eg, >20 000 incident total cardiovascular disease outcomes). Lancet 2018; 391: 1513–23 For all-cause mortality, we recorded a positive and curvilinear association with the level of alcohol consumption, with the minimum mortality risk around or below 100 g per week. Alcohol consumption was roughly linearly associated with a higher risk of stroke (HR per 100 g per week higher consumption 1·14, 95% CI, 1·10–1·17), coronary disease excluding myocardial infarction (1·06, 1·00–1·11), heart failure (1·09, 1·03–1·15), fatal hypertensive disease (1·24, 1·15–1·33); and fatal aortic aneurysm (1·15, 1·03–1·28). By contrast, increased alcohol consumption was log- linearly associated with a lower risk of myocardial infarction (HR 0·94, 0·91–0·97). In comparison to those who reported drinking >0–≤100 g per week, those who reported drinking >100–≤200 g per week, >200–≤350 g per week, or >350 g per week had lower life expectancy at age 40 years of approximately 6 months, 1–2 years, or 4–5 years, respectively. Interpretation In current drinkers of alcohol in high-income countries, the threshold for lowest risk of all-cause mortality was about 100 g/week. For cardiovascular disease subtypes other than myocardial infarction, there were no clear risk thresholds below which lower alcohol consumption stopped being associated with lower disease risk. These data support limits for alcohol consumption that are lower than those recommended in most current guidelines. www.thelancet.com Vol 391 April 14, 2018 1513 Articles Added value of this study The current study combined all the key study design features mentioned above, and afforded several additional advantages. www.thelancet.com Vol 391 April 14, 2018 1514 Articles Articles EPIC-CVD, a ten-country case-cohort study nested in the European Prospective Investigation into Cancer and Nutrition (EPIC) prospective cohort study, had quantitative alcohol information from 22 of its 23 contributing centres.23 Third, UK Biobank—a single large prospective study—had cohort-wide data about quantitative alcohol consumption.24 Therefore, our combined analysis included information from a total of 83 prospective studies that each used broadly similar methods to quantify alcohol consumption, record risk factors, and ascertain cause- specific death and cardiovascular disease events. We harmonised records of alcohol consumption across the contributing studies using a conversion of 1 unit=8 g of pure alcohol to a standard scale of grams per week (appendix pp 1–2), enabling a common analytical approach despite variation in the methods used (eg, self-administered vs interview-led questionnaires; food frequency question naires vs dietary recall surveys), and in consumption scales over different periods of ascertainment. Details of contributing studies are in appendix pp 3–4, 10–11. EPIC-CVD, a ten-country case-cohort study nested in the European Prospective Investigation into Cancer and Nutrition (EPIC) prospective cohort study, had quantitative alcohol information from 22 of its 23 contributing centres.23 Third, UK Biobank—a single large prospective study—had cohort-wide data about quantitative alcohol consumption.24 Therefore, our combined analysis included information from a total of 83 prospective studies that each used broadly similar methods to quantify alcohol consumption, record risk factors, and ascertain cause- specific death and cardiovascular disease events. We harmonised records of alcohol consumption across the contributing studies using a conversion of 1 unit=8 g of pure alcohol to a standard scale of grams per week (appendix pp 1–2), enabling a common analytical approach despite variation in the methods used (eg, self-administered vs interview-led questionnaires; food frequency question naires vs dietary recall surveys), and in consumption scales over different periods of ascertainment. Details of contributing studies are in appendix pp 3–4, 10–11. Added value of this study EPIC-CVD’s case-cohort design (which was used because lipids and other cardiovascular disease biomarkers were measured only in the case-cohort subset and not the full EPIC cohort), the Cox models for cardiovascular disease events were adapted using Prentice weights and stratified by centre.26 For the four case-control studies nested within prospective cohorts of the ERFC, odds ratios were calculated using, as appropriate, conditional or uncon ditional logistic regression models, taking into account relevant matching factors. Study-specific estimates were then pooled across studies by random-effects meta- analysis.27 We tested for violation of the proportional hazards assumption by including time interactions with alcohol consumption. To avoid model overfitting, studies with fewer than five incident cases of a particular outcome were excluded from analyses of that particular outcome. Added value of this study J A Cooper MSc); 92 Deutsches Herzzentrum München, Technische Universität München, Munich, Germany, DZHK (German Centre for Cardiovascular Research), partner site Munich Heart Alliance, Munich, Germany (Prof W Koenig MD); University of Ulm Medical Center, Ulm, Germany (Prof W Koenig); Department of Medicine, University of Padua, Padua, Italy (Prof E Casiglia MD); MRC Lifecourse Epidemiology Unit, University of Southampton, Southampton, UK (Prof C Cooper FMedSci); German Cancer Research Center (DKFZ), Heidelberg, Germany (V Arndt MD, T Kühn PhD, Prof H Brenner MD, Prof R Kaaks MD); Erasmus University Medical Center Rotterdam, Rotterdam, Netherlands (Prof O H Franco MD, J D Schoufour PhD, T Voortman PhD); Department of Public Health and Clinical Medicine, Umeå University, Umeå, Sweden (P Wennberg MD, M Wennberg PhD); Department of Primary Care and Public Health, Cardiff University, Cardiff, UK (Prof J Gallacher PhD); 12 de Octubre Research Institute, CIBERESP, Madrid, Spain (A Gómez de la Cámara MD); Institute for Community Medicine, University Medicine Greifswald, Greifswald, Germany (Prof H Völzke MD); Department of Public Health, Aarhus University, Aarhus, Denmark (C C Dahm PhD, Prof K Overvad MD); Farr Institute of Health Informatics Research, UCL Institute of Health Informatics, University College London, London, UK (C E Dale PhD); German Institute of Human Nutrition, Potsdam–Rehbrüke, Germany (M M Bergmann PhD, Prof H Boeing PhD); School of Community Health, Portland State University, Portland, OR, USA (C J Crespo PhD); St Vincent’s Clinical School, University of New South Wales, Sydney, NSW, Australia (L A Simons MD); Hellenic Health Foundation, Athens, Greece (P Lagiou MD, A Karakatsani MD, Prof A Trichopoulou MD); National and Kapodistrian y p To correct for measurement error and within-person variability in alcohol consumption over time, we estimated long-term average (henceforth, “usual”) alcohol consumption using multi-level regression calibration and information from 152 640 serial assessments in 71 011 individuals from 37 studies. This calculation was achieved either by regressing re-survey measurements (for the repeat alcohol assessments available in the ERFC studies and UK Biobank) or lifetime alcohol consumption measurements (for calculated lifetime alcohol con sumption measurements available in EPIC-CVD) on baseline alcohol consumption, adjusted for duration of follow-up and baseline age, sex, smoking status, history of diabetes, other relevant covariate(s), and with random effects for study and re-survey.28,29 The regression dilution ratio (ie, the calibration slope), which measures the extent of within-person variability,28 was extracted from the calibration model. Added value of this study HRs in this paper relate to usual alcohol consumption levels unless specified otherwise. To be eligible for the analysis, participants had to have information recorded about their alcohol consumption amount and status (ie, non-drinker vs current drinker), plus age, sex, history of diabetes and smoking status, at least 1 year of follow-up after baseline, and no known baseline history of cardiovascular disease (defined as coronary heart disease, other heart disease, stroke, transient ischaemic attack, peripheral arterial disease, or cardiovascular surgery); appendix p 21. The main analyses focused on current drinkers, whose baseline alcohol consumption was categorised into eight predefined groups according to the amount in grams consumed per week: >0–≤25, >25–≤50, >50–≤75, >75–≤100, >100–≤150, >150–≤250, >250–≤350, and >350 g per week. We assessed alcohol consumption in relation to all-cause mortality, total cardiovascular disease, and the following car diovascular disease subtypes (defined in appendix p 5): fatal and non-fatal myocardial infarction; fatal and non- fatal coronary disease excluding myocardial infarction; fatal and non-fatal stroke (including ischaemic, haemorrhagic, subarachnoid, and unclassified subtypes of stroke); fatal and non-fatal heart failure; and mortality from other cardiovascular causes, including cardiac dysrhythmia, hypertensive disease, sudden death, and aortic aneurysm.7,17,25 In analyses of cardiovascular disease subtypes, participants contributed follow-up time until the first outcome recorded (ie, cardiovascular deaths preceded by non-fatal outcomes were not included). Event times were censored at the end of follow-up or death from non-cardiovascular causes. We assessed the shapes of associations for all-cause mortality and cardiovascular disease outcomes by calculating study-specific HRs within the predefined groups of baseline alcohol consumption, pooled them by multivariate random-effects meta-analysis, and plotted them against mean usual (and baseline) alcohol consumption within each group. We estimated 95% CIs for each group (including the reference group) that corresponded to the amount of information underlying each group.30,31 For each major outcome, we determined the best fitting first or second order fractional polynomial32 to describe the association with baseline alcohol consumption (using a 1% significance level as evidence for a second order fractional polynomial over a first order fractional polynomial) using Cox regression models stratified by sex, study, and centre. Further analyses assumed a linear association with alcohol consumption, expressing results per 100 g per week (12·5 units/week) in usual alcohol consumption. To assess the effect of excluding known current drinkers with missing alcohol consumption data, we did a sensitivity analysis using multiple imputation within studies, before combining www.thelancet.com Vol 391 April 14, 2018 Articles ERFC EPIC-CVD UK Biobank Participants with resurveys of alcohol consumption Study level characteristics Location 81 studies in 19 countries 22 centres in 10 European countries England, Scotland, and Wales 37 studies in 15 countries Years of recruitment 1964–2008 1990–2002 2006–10 1964–2010 Year of most recent endpoint follow-up 2013 2009 2016 2016 Participant level characteristics Total participants 356 819 30 702 358 833 89 499 Known current drinkers at baseline 247 504 26 036 326 372 71 011 Weekly baseline alcohol consumption in current drinkers >0–≤25 g per week 53 418 (22%) 7906 (30%) 39 641 (12%) 12 301 (17% [11 g/week vs 36 g/week]‡) >25–≤50 g per week 33 953 (14%) 3704 (14%) 39 334 (12%) 8365 (12% [38 g/week vs 56 g/week]‡) >50–≤75 g per week 26 656 (11%) 2748 (11%) 42 907 (13%) 7322 (10% [63 g/week vs 80 g/week]‡) >75–≤100 g per week 16 557 (7%) 2446 (9%) 36 780 (11%) 6394 (9% [87 g/week vs 98 g/week]‡) >100–≤150 g per week 36 236 (15%) 2602 (10%) 55 815 (17%) 10 051 (14% [126 g/week vs 126 g/week]‡) >150–≤250 g per week 31 645 (13%) 3090 (12%) 60 025 (18%) 12 255 (17% [193 g/week vs 173 g/week]‡) >250–≤350 g per week 23 607 (10%) 1744 (7%) 26 669 (8%) 6927 (10% [303 g/week vs 248 g/week]‡) ≥350 g per week 25 432 (10%) 1796 (7%) 25 201 (8%) 7396 (10% [515 g/week vs 354 g/week]‡) Baseline characteristics restricted to all current drinkers Alcohol consumption (g/week), median (5th–95th percentiles) 87·7 (2·2–522·4) 61·9 (2·6–404·0) 103·9 (11·8–420·8) 105·2 (6·0–482·8) Age (years) at baseline 57·1 (8·7) 55·0 (9·2) 56·5 (8·0) 55·3 (8·2) Sex Male 162 685 (66%) 13 508 (52%) 157 809 (48%) 44 360 (62%) Female 84 819 (34%) 12 528 (48%) 168 563 (52%) 26 651 (38%) Smoking status Not current 161 037 (65%) 17 608 (68%) 293 182 (90%) 50 930 (72%) Current 86 467 (35%) 8428 (32%) 33 190 (10%) 20 081 (28%) History of diabetes No 237 685 (96%) 24 875 (96%) 315 090 (97%) 68 159 (96%) Yes 9819 (4%) 1161 (4%) 11 282 (3%) 2852 (4%) BMI, kg/m² 26·1 (3·8) 26·4 (4·1) 27·0 (4·4) 26·1 (3·8) HDL-C, mmol/L 1·40 (0·41) 1·40 (0·42) Not available* 1·41 (0·41) Total cholesterol, mmol/L 5·80 (1·17) 6·11 (1·16) Not available* 5·78 (1·08) Systolic blood pressure, mm Hg 136·5 (19·0) 138·4 (21·3) 137·9 (18·5) 134·6 (18·4) Major outcomes restricted to current drinkers All-cause mortality events 32 813 784† 6720 6912 All cardiovascular disease 18 791 12 758 7469 11 597 Data are n, n (%), or mean (SD), unless otherwise indicated. Articles ERFC=Emerging Risk Factors Collaboration. EPIC-CVD=European Prospective Investigation into Cancer and Nutrition—Cardiovascular Disease. BMI=body-mass index. HDL-C=high-density-lipoprotein cholesterol. At the time of analysis, measurements of HDL-C and total cholesterol were not available in the UK Biobank. †All-cause mortality events from EPIC derive only from the 13 670 participants in the random sub-cohort of EPIC-CVD, rather than from the entire EPIC prospective study. ‡Mean consumption (g/week) at baseline vs resurvey. Table 1: Study-level and participant-level characteristics of the contributing data sources Data are n, n (%), or mean (SD), unless otherwise indicated. ERFC=Emerging Risk Factors Collaboration. EPIC-CVD=European Prospective Investigation into Cancer and Nutrition—Cardiovascular Disease. BMI=body-mass index. HDL-C=high-density-lipoprotein cholesterol. At the time of analysis, measurements of HDL-C and total cholesterol were not available in the UK Biobank. †All-cause mortality events from EPIC derive only from the 13 670 participants in the random sub-cohort of EPIC-CVD, rather than from the entire EPIC prospective study. ‡Mean consumption (g/week) at baseline vs resurvey. Table 1: Study-level and participant-level characteristics of the contributing data sources adjusted for usual levels of available potential con founders or mediators, including body-mass index (BMI), systolic blood pressure, high-density-lipoprotein chol esterol (HDL-C), low-density-lipoprotein cholesterol (LDL-C), total cholesterol, fibrinogen, and baseline measures for smoking amount (in pack-years), level of education reached (no schooling or primary education only vs secondary education vs university), occupation (not working vs manual vs office vs other), self-reported physical activity level (inactive vs moderately inactive vs moderately active vs active), self-reported general health (scaled 0–1 where low scores indicate poorer health), the data in a meta-analysis. We investigated associations with alcohol type (wine, beer, and spirits), consumption frequency (dichotomised as drinkers who consumed alcohol on ≤2 days per week or those who consumed alcohol on >2 days per week) and episodic heavy drink ing (dichotomised as binge drinkers who consumed ≥100 g per drinking occasion or non-binge drinkers who consumed <100 g per drinking occasion). Table 1: Study-level and participant-level characteristics of the contributing data sources www.thelancet.com Vol 391 April 14, 2018 Statistical analysis Hazard ratios (HRs) for alcohol consumption were calculated separately within each study using Cox regression models, stratified by sex and with adjustment for known confounders: age, smoking status (current vs non-current) and history of diabetes. To account for www.thelancet.com Vol 391 April 14, 2018 1515 Articles Articles Articles Heterogeneity was investigated by grouping studies according to recorded characteristics and through meta-regression, assessed by the I² statistic.33 Evidence of small study effects was assessed visually with funnel plots and by Begg and Mazumdar’s test34 and Egger’s test.35 0 100 200 300 All-cause mortality (40 310 deaths) Cardiovascular disease (39 018 events) 400 0 100 200 300 400 0·9 1·0 1·1 1·2 1·3 1·4 1·5 1·6 Hazard ratio (95% Cl) Usual alcohol consumption (g per week) Usual alcohol consumption (g per week) Methods we used to estimate reductions in life expectancy (years of life lost) are described in the appendix (pp 6–7). Briefly, estimates of cumulative survival from 40 years of age onwards in different categories of baseline alcohol consumption were calculated by applying estimated HRs (specific to age-at-risk) for cause-specific mortality to the detailed mortality component of the US Centers for Disease Control and Prevention’s WONDER database,36 which recorded 10 million deaths (from all causes) in more than 305 million individuals in the USA during 2007–10.37,38 Results were modelled from age 40 years and enabled estimation of years of life lost between light drinkers (defined as those consuming >0–≤100 g/week of alcohol) and pre-defined groups of >100–≤200, >200–≤350, and >350 g per week. This method does not make use of the survival estimates from the modelled data; instead, it makes inferences by estimating age-at-risk specific HRs, which are then combined with external population age-specific mortality rates.39 Figure 1: Associations of usual alcohol consumption with all-cause mortality and the aggregate of cardiovascular disease in current drinkersi Cardiovascular disease was defined as an aggregate of myocardial infarction, coronary heart disease, and stroke. Hazard ratios are adjusted for age, smoking, and history of diabetes, and stratified by sex and EPIC centre. The reference category is the lowest baseline alcohol consumption category (between 0 and 25 g/week). HRs are plotted against the mean usual alcohol consumption in each category. Sizes of the boxes are proportional to the inverse of the variance of the log-transformed hazard ratios. Vertical lines represent 95% CIs. and 39 018 first incident cardiovascular disease out comes, including 12 090 stroke events, 14 539 myocardial infarction events, 7990 coronary disease events exclud ing myocardial infarction, 2711 heart failure events, and 1121 deaths from other cardiovascular diseases (appendix p 13). Articles University of Athens, Athens, Greece (P Lagiou, A Karakatsani, Prof A Trichopoulou); Harvard TH Chan School of Public Health, Boston, MA, USA (P Lagiou); Office of Public Health Studies, University of Hawaii, Honolulu, HI, USA (Prof B Rodriguez MD); Instituto de Salud Pública de Navarra, IdiSNA - Navarra Institute for Health Research, Pamplona, Spain (C Moreno-Iribas PhD); Red de Investigación en University of Athens, Athens, Greece (P Lagiou, A Karakatsani, Prof A Trichopoulou); Harvard TH Chan School of Public Health, Boston, MA, USA (P Lagiou); Office of Public Health Studies, University of Hawaii, Honolulu, HI, USA (Prof B Rodriguez MD); Instituto de Salud Pública de Navarra, IdiSNA - Navarra Institute for Health Research, Pamplona, Spain (C Moreno-Iribas PhD); Red de Investigación en We used regression calibration methods similar to those described above to estimate and adjust for long- term levels of potential confounding factors or mediators in individuals with available information. HRs were 1516 www.thelancet.com Vol 391 April 14, 2018 Articles 0 100 200 300 All-cause mortality (40 310 deaths) Cardiovascular disease (39 018 events) 400 0 100 200 300 400 0·9 1·0 1·1 1·2 1·3 1·4 1·5 1·6 Hazard ratio (95% Cl) Usual alcohol consumption (g per week) Usual alcohol consumption (g per week) Figure 1: Associations of usual alcohol consumption with all-cause mortality and the aggregate of cardiovascular disease in current drinkers Cardiovascular disease was defined as an aggregate of myocardial infarction, coronary heart disease, and stroke. Hazard ratios are adjusted for age, smoking, and history of diabetes, and stratified by sex and EPIC centre. The reference category is the lowest baseline alcohol consumption category (between 0 and 25 g/week). HRs are plotted against the mean usual alcohol consumption in each category. Sizes of the boxes are proportional to the inverse of the variance of the log-transformed hazard ratios. Vertical lines represent 95% CIs. self-reported red meat consumption, and self-reported use of anti-hypertensive drugs. We investigated effect modification with formal tests for interaction, using a 0·1% significance threshold to make some allowance for multiple testing. Articles Baseline alcohol consumption varied substantially across studies, was generally lower in more recent calendar periods of recruitment, and was positively skewed (median 96 g/week [5th–95th percentiles 6–448]; appendix p 22). It was weakly and positively correlated with male sex, smoking status and amount, systolic blood pressure, HDL-C level, fibrinogen, and lower socioeconomic status (appendix pp 23–24). 152 640 serial assessments of alcohol consumption were available for 71 011 participants from 37 studies (median interval between baseline and serial measurements 5·6 years [5th–95th percentiles 1·04–13·5]). Participants with serial measurements were younger, had slightly higher baseline alcohol consumption, and were more likely to be men than those without serial measurements (table 1, appendix p 14). The regression dilution ratio for alcohol consumption was 0·50 (95% CI 0·47–0·52), similar to that for systolic blood pressure (0·52, 0·50–0·55) but lower than that for HDL-C concentration (0·74, 0·72–0·76) in a common set of participants. gi y Analyses used Stata (version 14.2 and 15.1). All p values presented are for 2-sided tests. Role of the funding source The funders of the study did not have any role in the study design, data analysis, or reporting of this manuscript. AMW and SK had full access to the combined dataset, and, together with EDA and JD, had responsibility for the decision to submit the manuscript for publication. www.thelancet.com Vol 391 April 14, 2018 Articles 0·8 1·0 1·2 1·4 1·6 1·8 2·0 2·2 2·4 Hazard ratio (95% Cl) Hazard ratio (95% Cl) All stroke (12 090 events) Myocardial infarction (14 539 events) 0 100 200 300 400 0·8 1·0 1·2 1·4 1·6 1·8 2·0 2·2 2·4 Usual alcohol consumption (g per week) Heart failure (2711 events) 0 100 200 300 400 Usual alcohol consumption (g per week) Deaths from other types of cardiovascular disease (1121 deaths) 0 100 200 300 400 Usual alcohol consumption (g per week) Coronary disease excluding myocardial infarction (7990 events) Figure 2: Associations of usual alcohol consumption with cardiovascular subtypes in alcohol drinkers Hazard ratios are adjusted for age, smoking, and history of diabetes, and stratified by sex and EPIC centre. The reference category is the lowest baseline alcohol consumption category (between 0 and 25g/week). Hazard ratios are plotted against the mean usual alcohol consumption in each category. Studies with fewer than five events of any outcome were excluded from the analysis of that outcome. Sizes of the boxes are proportional to the inverse of the variance of the log-transformed hazard ratios. Vertical lines represent 95% CIs. Deaths from other cardiovascular disease include the following outcomes: cardiac dysrhythmia, hypertensive disease, sudden death, and aortic aneurysm. 0·8 1·0 1·2 1·4 1·6 1·8 2·0 2·2 2·4 Hazard ratio (95% Cl) All stroke (12 090 events) Heart failure (2711 events) Myocardial infarction (14 539 events) Deaths from othertypes of cardiovascular 0 100 200 300 400 Usual alcohol consumption (g per week) Coronary disease excluding myocardial infarction (7990 events) Deaths from other types of cardiovascular disease (1121 deaths) Usual alcohol consumption (g per week) 0 100 200 300 400 Usual alcohol consumption (g per week) Deaths from other types of cardiovascular disease (1121 deaths) alcohol consumption with cardiovascular subtypes in alcohol drinkersi Figure 2: Associations of usual alcohol consumption with cardiovascular subtypes in alcohol drinkers Hazard ratios are adjusted for age, smoking, and history of diabetes, and stratified by sex and EPIC centre. The reference category is the lowest baseline alcohol consumption category (between 0 and 25g/week). Hazard ratios are plotted against the mean usual alcohol consumption in each category. Studies with fewer than five events of any outcome were excluded from the analysis of that outcome. Sizes of the boxes are proportional to the inverse of the variance of the log-transformed hazard ratios. Vertical lines represent 95% CIs. Articles Deaths from other cardiovascular disease include the following outcomes: cardiac dysrhythmia, hypertensive disease, sudden death, and aortic aneurysm. HRs (table 2, appendix pp 15, 30). First, adjustment for HDL-C level weakened the inverse association between alcohol consumption and myocardial infarction, but strengthened the positive association between alcohol consumption and both coronary disease and heart failure. Second, adjustment for systolic blood pressure strengthened the inverse association between alcohol consumption and myocardial infarction, but weakened the positive associations between alcohol consumption and all other cardiovascular disease outcomes. Our analysis confirmed the established association of alcohol consumption with cancers of the digestive system, which did not change after additional adjustment for the factors listed above (appendix p 16). Furthermore, additional adjustment for smoking amount abolished the apparent association of alcohol consumption with lung cancer (appendix pp 16), in line with the accepted view that alcohol consumption does not cause lung cancer.40 After adjustment for age, sex, smoking, and history of diabetes, the amount of alcohol consumed had positive and roughly linear associations with stroke (HR per 100 g/week higher consumption 1·14, 1·10–1·17), coronary disease excluding myocardial infarction (1·06, 1·00–1·11), heart failure (1·09, 1·03–1·15), fatal hypertensive disease (1·24, 1·15–1·33), and fatal aortic aneurysm (1·15, 1·03–1·28; figures 2, 3). By contrast, there was an inverse and approximately log-linear association with myocardial infarction (0·94, 0·91–0·97; figures 2, 3). Stroke associations were similar for fatal and non-fatal outcomes (appendix p 28) and across subtypes (appendix p 29). However, for coronary disease excluding myocardial infarction, associations were stronger for fatal than non-fatal outcomes (appendix p 28). For myocardial infarction, inverse associations were possibly more pronounced with non-fatal than fatal outcomes (figure 3, appendix p 28). After adjustment for age, sex, smoking, and history of diabetes, the amount of alcohol consumed had positive and roughly linear associations with stroke (HR per 100 g/week higher consumption 1·14, 1·10–1·17), coronary disease excluding myocardial infarction (1·06, 1·00–1·11), heart failure (1·09, 1·03–1·15), fatal hypertensive disease (1·24, 1·15–1·33), and fatal aortic aneurysm (1·15, 1·03–1·28; figures 2, 3). By contrast, there was an inverse and approximately log-linear association with myocardial infarction (0·94, 0·91–0·97; figures 2, 3). Stroke associations were similar for fatal and non-fatal outcomes (appendix p 28) and across subtypes (appendix p 29). However, for coronary disease excluding myocardial infarction, associations were stronger for fatal than non-fatal outcomes (appendix p 28). www.thelancet.com Vol 391 April 14, 2018 Results Of the 786 787 participants with sufficient information for inclusion in this consortium, 186 875 (19%) reported not drinking at baseline, leaving 599 912 current drinkers without a history of cardiovascular disease at base line who were eligible for the prespecified principal analysis. The current drinkers were derived from ERFC (247 504 participants), EPIC-CVD (26 036), and the UK Biobank (326 372; table 1). Baseline year of recruitment ranged from 1964 to 2010. The mean age of the participants was 57 years (SD 9). 265 910 (44%) of 599 912 participants were women, and 128 085 (21%) were current smokers (appendix p 12). About 50% reported drinking more than 100 g of alcohol per week, and 8·4% drank more than 350 g per week (table 1). During 5·4 million person-years (median 7·5 years of follow-up [5th–95th percentiles 5·0–18·4]), there were 40 310 deaths from all causes, (including 11 762 vascular and 15 150 neoplastic deaths), For all-cause mortality, there was a positive and curvilinear association with alcohol consumption, with the lowest risk for those consuming below 100 g per week (figure 1, appendix p 25). Associations were similar for men and women (appendix p 26), but weaker at older ages (appendix p 27). There was a J-shaped association for the aggregate of cardiovascular disease outcomes (figure 1, appendix p 25). However, disaggregation showed two opposing sets of associations (figure 2). www.thelancet.com Vol 391 April 14, 2018 1517 Articles Articles Studies with fewer than five events of any outcome were Events/participants All stroke Non-fatal stroke Fatal stroke Ischaemic stroke Haemorrhagic stroke Subarachnoid haemorrhage Unclassiﬁed stroke All myocardial infarction Non-fatal myocardial infarction Fatal myocardial infarction Coronary disease excluding myocardial infarction Non-fatal coronary disease excluding myocardial infarction Fatal coronary disease excluding myocardial infarction Heart failure (fatal and non-fatal) Death from other types of cardiovascular disease Cardiac dysrhythmia Hypertensive disease Sudden cardiac death Aortic aneurysm 12 090/585588 9910/491050 2142/532204 6256/491204 1482/505948 663/412732 3215/527729 14 539/594561 11 706/515377 2748/538117 7990/523548 6000/389976 1889/510147 2711/447436 1121/488122 261/71682 178/383269 283/68002 289/423145 1·14 (1·10–1·17) 1·14 (1·10–1·18) 1·13 (1·07–1·19) 1·13 (1·09–1·18) 1·17 (1·12–1·23) 1·09 (1·00–1·19) 1·13 (1·06–1·20) 0·94 (0·91–0·97) 0·93 (0·90–0·97) 0·99 (0·93–1·05) 1·06 (1·00–1·11) 1·00 (0·97–1·03) 1·11 (1·04–1·18) 1·09 (1·03–1·15) 1·18 (1·07–1·30) 1·17 (0·86–1·60) 1·24 (1·15–1·33) 1·12 (0·90–1·41) 1·15 (1·03–1·28) 12 (0–35) 14 (0–40) 0 (0–35) 8 (0–37) 0 (0–37) 0 (0–58) 14 (0–40) 12 (0–35) 24 (0–45) 8 (0–35) 26 (0–49) 0 (0–52) 12 (0–40) 4 (0–31) 33 (2–53) 63 (35–79) 0 (0–55) 29 (0–63) 0 (0–49) Heterogeneity l2 (95% Cl) Hazard ratio (95% Cl) Lower risk of disease with higher alcohol consumption Higher risk of disease with higher alcohol consumption 1·0 0·8 1·2 1·6 1·4 Figure 3: Hazard ratios for subtypes of cardiovascular outcomes in current drinkers, per 100 g per week higher usual alcohol consumption Heterogeneity l2 (95% Cl) Hazard ratio (95% Cl) Figure 3: Hazard ratios for subtypes of cardiovascular outcomes in current drinkers, per 100 g per week higher usual alcohol consumption Hazard ratios are adjusted for age, smoking, and history of diabetes, and stratified by sex and centre. Studies with fewer than five events of any out excluded from the analysis of that outcome. Figure 3: Hazard ratios for subtypes of cardiovascular outcomes in current drinkers, per 100 g per week higher usual alcohol consumption Hazard ratios are adjusted for age, smoking, and history of diabetes, and stratified by sex and centre. Studies with fewer than five events of any outcome were excluded from the analysis of that outcome. Figure 3: Hazard ratios for subtypes of cardiovascular outcomes in current drinkers, per 100 g per week higher usual alcohol consumption Hazard ratios are adjusted for age, smoking, and history of diabetes, and stratified by sex and centre. Studies with fewer than five events of any outcome were excluded from the analysis of that outcome. Articles For myocardial infarction, inverse associations were possibly more pronounced with non-fatal than fatal outcomes (figure 3, appendix p 28). p g When including never-drinkers and ex-drinkers, we reproduced previously reported U-shaped associations of (i g ) With the following notable exceptions, further adjust ment for additional covariates did not substantially change www.thelancet.com Vol 391 April 14, 2018 1518 Articles Events/participants All stroke Non-fatal stroke Fatal stroke Ischaemic stroke Haemorrhagic stroke Subarachnoid haemorrhage Unclassiﬁed stroke All myocardial infarction Non-fatal myocardial infarction Fatal myocardial infarction Coronary disease excluding myocardial infarction Non-fatal coronary disease excluding myocardial infarction Fatal coronary disease excluding myocardial infarction Heart failure (fatal and non-fatal) Death from other types of cardiovascular disease Cardiac dysrhythmia Hypertensive disease Sudden cardiac death Aortic aneurysm 12 090/585588 9910/491050 2142/532204 6256/491204 1482/505948 663/412732 3215/527729 14 539/594561 11 706/515377 2748/538117 7990/523548 6000/389976 1889/510147 2711/447436 1121/488122 261/71682 178/383269 283/68002 289/423145 1·14 (1·10–1·17) 1·14 (1·10–1·18) 1·13 (1·07–1·19) 1·13 (1·09–1·18) 1·17 (1·12–1·23) 1·09 (1·00–1·19) 1·13 (1·06–1·20) 0·94 (0·91–0·97) 0·93 (0·90–0·97) 0·99 (0·93–1·05) 1·06 (1·00–1·11) 1·00 (0·97–1·03) 1·11 (1·04–1·18) 1·09 (1·03–1·15) 1·18 (1·07–1·30) 1·17 (0·86–1·60) 1·24 (1·15–1·33) 1·12 (0·90–1·41) 1·15 (1·03–1·28) 12 (0–35) 14 (0–40) 0 (0–35) 8 (0–37) 0 (0–37) 0 (0–58) 14 (0–40) 12 (0–35) 24 (0–45) 8 (0–35) 26 (0–49) 0 (0–52) 12 (0–40) 4 (0–31) 33 (2–53) 63 (35–79) 0 (0–55) 29 (0–63) 0 (0–49) Heterogeneity l2 (95% Cl) Hazard ratio (95% Cl) Lower risk of disease with higher alcohol consumption Higher risk of disease with higher alcohol consumption 1·0 0·8 1·2 1·6 1·4 Figure 3: Hazard ratios for subtypes of cardiovascular outcomes in current drinkers, per 100 g per week higher usual alcohol consumption Hazard ratios are adjusted for age, smoking, and history of diabetes, and stratified by sex and centre. Articles 40 50 70 60 80 90 0 1 2 3 4 5 6 Years of life lost (95% Cl) Age (years) 40 50 70 60 80 90 Age (years) Men Women >100–≤200 g/week >200–≤350 g/week >350 g/week Figure 4: Estimated future years of life lost by extent of reported baseline alcohol consumption compared with those who reported consuming >0–≤100 g per week The estimates of cumulative survival from 40 years of age onwards in the alcohol-drinking groups were calculated by applying hazard ratios (specific to age at risk) for all-cause mortality associated with categorised baseline alcohol consumption to US death rates at the age of 40 years or older. Mean usual levels of alcohol consumption within each baseline alcohol consumption category were 56, 123, 208 and 367 g per week, respectively, for the groups >0–≤100 g per week, >100–≤200 g per week, >200–≤350 g per week, and >350 g per week. pp 43–45). There was no evidence of small study effects (appendix p 46). Our data showed no evidence of violation of the proportional hazards assumption. 40 50 70 60 80 90 Age (years) Women 40 50 70 60 80 90 0 1 2 3 4 5 6 Years of life lost (95% Cl) Age (years) Men >100–≤200 g/week >200–≤350 g/week >350 g/week In comparison to those who reported drinking >0–≤100 g (mean usual 56 g) alcohol per week, those who reported drinking >100–≤200 g (mean usual 123 g) per week, >200–≤350 g (mean usual 208 g) per week or >350 g (mean usual 367 g) per week had shorter life expectancy at age 40 years of approximately 6 months, 1–2 years, or 4–5 years respectively (figure 4). Similarly, men who reported consuming above the UK upper limit of 112 g per week had a shorter life expectancy at age 40 years of 1·6 years (95% CI 1·3–1·8), and men who reported drinking above the US upper limit of 196 g per week had a shorter life expectancy at age 40 years of 2·7 years (2·4–3·1) compared with men who reported drinking below these respective upper limits. Thus, men who reported drinking less than 100 g alcohol per week had about a 1–2 years longer life expectancy at age 40 years than those who reported drinking 196 g per week (appendix p 47). Articles Rome, Italy (L Palmieri PhD); Institut Pasteur de Lille, Lille, France (J-P Dallongeville MD); Assmann-Stiftung für Prävention, Münster, Germany (Prof G Assmann MD); The City College of New York, New York, NY, USA (M Trevisan MD); Howard University Hospital, Washington DC, USA (R F Gillum MD); Institute of Cardiovascular & Medical Sciences, University of Glasgow, Glasgow, UK (Prof I Ford PhD, Prof N Sattar FMedSci); International Agency for Research on Cancer, Lyon, France (P Ferrari PhD); MRC Integrative Epidemiology Unit (IEU), University of Bristol, Bristol, UK (Prof G Davey Smith MD); and School of Population Health, The University of Auckland, Auckland, New Zealand (Prof R Jackson PhD) Correspondence to: Dr Angela Wood, Emerging Risk Factors Collaboration and EPIC-CVD Coordinating Centres, Department of Public Health and Primary Care, Strangeways Research Laboratory, University of Cambridge, Cambridge, CB1 8RN, UK amw79@medschl cam ac uk Rome, Italy (L Palmieri PhD); Institut Pasteur de Lille, Lille, France (J-P Dallongeville MD); Assmann-Stiftung für Prävention, Münster, Germany (Prof G Assmann MD); The City College of New York, New York, NY, USA (M Trevisan MD); Howard University Hospital, Washington DC, USA (R F Gillum MD); Institute of Cardiovascular & Medical Sciences, University of Glasgow, Glasgow, UK (Prof I Ford PhD, Prof N Sattar FMedSci); International Agency for Research on Cancer, Lyon, France (P Ferrari PhD); MRC Integrative Epidemiology Unit (IEU), University of Bristol, Bristol, UK (Prof G Davey Smith MD); and School of Population Health, The University of Auckland, Auckland, New Zealand (Prof R Jackson PhD) Correspondence to: Dr Angela Wood, Emerging Risk Factors Collaboration and EPIC-CVD Coordinating Centres, Department of Public Health and Primary Care, Strangeways Research Laboratory, University of Cambridge, Cambridge, CB1 8RN UK Rome, Italy (L Palmieri PhD); Institut Pasteur de Lille, Lille, France (J-P Dallongeville MD); if f Professor John Danesh, Emerging Risk Factors Collaboration and EPIC-CVD Coordinating Centres, Department of Public Health and Primary Care, Strangeways Articles Women who reported drinking above either the UK threshold (112 g per week) or US threshold (98 g per week) had about 1·3 (1·1–1·5) years shorter life expectancy at age 40 years compared with women who reported drinking below these thresholds (appendix p 47). About 20% of the alcohol-related survival difference for men (and slightly less for women) was attributed to excess death from cardiovascular disease (appendix p 47). Similar findings to those for the US population were observed when modelling was based on EU mortality rates (data not shown). Figure 4: Estimated future years of life lost by extent of reported baseline alcohol consumption compared with those who reported consuming >0–≤100 g per week The estimates of cumulative survival from 40 years of age onwards in the alcohol-drinking groups were calculated by applying hazard ratios (specific to age at risk) for all-cause mortality associated with categorised baseline alcohol consumption to US death rates at the age of 40 years or older. Mean usual levels of alcohol consumption within each baseline alcohol consumption category were 56, 123, 208 and 367 g per week, respectively, for the groups >0–≤100 g per week, >100–≤200 g per week, >200–≤350 g per week, and >350 g per week. recorded fewer than five events for a particular outcome (appendix p 36); provided separate analyses of men and women (appendix p 17, appendix p 26); omitted outcomes recorded in the initial 5 years of follow-up (appendix p 18); excluded participants with diabetes or other known chronic diseases at baseline (appendix p 18); and restricted the analyses to studies that recorded both non-fatal and fatal endpoints (appendix p 37). Associations of baseline alcohol consumption with all-cause mortality were stronger in drinkers of beer or spirits than of wine, and in those drinking less frequently (when consuming the same weekly amount), including binge drinkers (appendix p 38). However, people showing these behaviours had higher baseline levels of smoking and other indicators of lower socioeconomic status, suggesting the potential for confounding effects (appendix pp 19–20). For cardio vascular disease subtypes, HRs tended to be higher in beer and spirit drinkers than in wine drinkers, but not significantly so in direct comparisons involving a common set of participants (appendix p 39). www.thelancet.com Vol 391 April 14, 2018 amw79@medschl.cam.ac.uk Articles All stroke Myocardial infarction Coronary disease excluding myocardial infarction Heart failure Deaths from other types of cardio vascular disease Subset of participants with measurement of systolic blood pressure Cohorts/events 70/11 297 73/13 519 46/7789 39/2668 44/1019 Basic adjustment* 1·16 (1·11–1·22) 0·95 (0·91–0·99) 1·06 (1·00–1·12) 1·11 (1·04–1·18) 1·16 (1·06–1·27) Plus adjustment for systolic blood pressure 1·10 (1·06–1·14) 0·91 (0·87–0·94) 1·03 (0·97–1·10) 1·08 (1·02–1·15) 1·14 (1·03–1·25) Subset of participants with measurement of high-density-lipoprotein cholesterol Cohorts/events 56/7982 61/9911 36/3608 29/1886 34/690 Basic adjustment* 1·16 (1·10–1·23) 0·93 (0·88–0·97) 1·07 (0·98–1·17) 1·09 (1·00–1·19) 1·22 (1·06–1·40) Plus adjustment for high-density- lipoprotein cholesterol 1·17 (1·11–1·22) 1·00 (0·96–1·04) 1·13 (1·05–1·22) 1·14 (1·01–1·27) 1·22 (1·08–1·38) Subset of participants with measurement of body-mass index Cohorts/events 68/11 733 71/14 217 43/7761 36/2566 42/1035 Basic adjustment* 1·15 (1·10–1·19) 0·95 (0·91–0·98) 1·06 (1·02–1·12) 1·12 (1·04–1·20) 1·16 (1·06–1·27) Plus adjustment for body-mass index 1·14 (1·10–1·18) 0·94 (0·91–0·97) 1·06 (1·01–1·12) 1·10 (1·03–1·16) 1·16 (1·06–1·27) Data are hazard ratio (95% CI) per 100 g per week higher usual alcohol consumption, unless otherwise indicated. Analyses were restricted to individuals with basic adjustment variables plus the additional variable. Studies with fewer than five events were excluded from the analysis of each outcome. *Basic adjustment includes age, smoking, and history of diabetes, and stratification by sex and centre. Table 2: Hazard ratios for major cardiovascular outcomes in current drinkers, without and with adjustment for usual levels of systolic blood pressure, high-density-lipoprotein cholesterol, or body-mass index alcohol consumption with total cardiovascular disease and all-cause mortality (appendix p 31). However, we observed notable differences in baseline characteristics between never drinkers and current drinkers (eg, in relation to sex, ethnicity, smoking, and diabetes status; appendix p 12), supporting the validity of focusing on current drinkers in our main analysis. We recorded similar findings to those reported above in sensitivity analyses that involved the following approaches: used multiple imputation rather than complete-case analysis (appendix p 32); used fractional polynomials (appendix p 34); used a fixed-effect meta-analysis (appendix p 35); included studies that www.thelancet.com Vol 391 April 14, 2018 1519 Articles Discussioni The main finding of this analysis was that the threshold for lowest risk for all-cause mortality was about 100 g per week. For men, we estimated that long-term reduction of alcohol consumption from 196 g per week (the upper limit recommended in US guidelines) to 100 g per week or below was associated with about 1–2 years of longer life expectancy at age 40 years. Exploratory analyses suggested that drinkers of beer or spirits, as well as binge drinkers, had the highest risk for all-cause mortality. g y Our study has highlighted the complex and diverse potential mechanisms by which alcohol consumption may exert cardiovascular effects.41,42 It has shown that the association between alcohol consumption and total cardiovascular disease risk comprises several distinct and opposite dose–response curves, rather than a single J-shaped association. In particular, whereas higher alcohol consumption was roughly linearly associated with a higher risk of all stroke subtypes, coronary dis ease excluding myocardial infarction, heart failure, and several less common cardiovascular disease subtypes, it was approximately log-linearly associated with a lower risk of myocardial infarction. Our results are concordant with recent observational data and Mendelian ran domisation studies.16,43–46 We noted little heterogeneity in the studies contrib uting results for stroke (I²=12%), myocardial infarc tion (I²=12%), coronary disease excluding myocardial infarction (I²=26%), heart failure (I²=4%) or deaths from other types of cardiovascular disease (I²=33%; figure 3). HRs for the cardiovascular disease outcomes we studied were broadly similar for different geographical regions, decade of study enrolment, by data source (ie, ERFC, EPIC-CVD, and UK Biobank), and alcohol assessment method (appendix pp 40–42). HRs for the cardiovascular disease outcomes were generally higher at younger ages, but did not vary substantially by sex, history of diabetes, proatherogenic lipids, BMI, smoking status, or other individual-level characteristics (appendix Declaration of interests ASB reports grants from European Commission Framework 7 (HEALTH-F2-2012-279233), the European Research Council (268834), the British Heart Foundation (SP/09/002 and RG/08/014 and RG13/13/30194), the UK Medical Research Council (G0800270 and MR/L003120/1), from National Institute for Health Research (through the NIHR Cambridge Biomedical Research Centre), during the conduct of the study; and grants from Merck, Biogen, Bioverativ, Novartis, and Pfizer, outside the submitted work. BMP reports that he serves on the DSMB of a clinical trial funded by Zoll LifeCor and on the Steering Committee of the Yale Open Data Access Project funded by Johnson & Johnson. MD reports grants from Japan Society for the Promotion of Science, during the conduct of the study. EDA reports grants from European Commission Framework 7, the European Research Council, the British Heart Foundation, the UK Medical Research Council, and the National Institute for Health Research, during the conduct of the study; and grants from NHS Blood and Transplant, outside the submitted work. EB reports grants from the National Health and Medical Research Council of Australia, during the conduct of the study. HMK reports a research agreement (through Yale) from Johnson & Johnson (Janssen) and Medtronic to develop methods of clinical trial data sharing; personal fees from UnitedHealth, IBM Watson, Element Science, and Aetna; a personal health information platform from Hugo; grants from the FDA and Medtronic; and contracts from Centers for Medicare & Medicaid Services to develop and maintain measures that are publicly reported, outside the submitted work. JD reports grants from the UK Medical Research Council, the British Heart Foundation, the UK National Institute of Health Research, and the European Commision, during the conduct of the study; personal fees and non-financial support from Merck Sharp and Dohme UK Atherosclerosis, personal fees and non-financial support from Novartis Cardiovascular and Metabolic Advisory Board, grants from the British Heart Foundation, European Research Council, Merck, the National Institute of Health Research, NHS Blood and Transplant, Novartis, Pfizer, the UK Medical Research Council, the Wellcome Trust, and AstraZeneca, and personal fees and non-financial support from Pfizer Nevertheless, our study has some potential limitations. Self-reported alcohol consumption data are prone to bias and are challenging to harmonise across studies conducted over different time periods that used varying instruments and methods to record such data.20,57 We did not, however, identify major differences in results across studies that used differing alcohol measurement instruments. Research Laboratory, University of Cambridge, Cambridge CB1 8RN, UK jd292@medschl.cam.ac.uk See Online for appendix amw79@medschl.cam.ac.uk or Professor John Danesh, Emerging Risk Factors Collaboration and EPIC-CVD Coordinating Centres, Department of Public Health and Primary Care, Strangeways www.thelancet.com Vol 391 April 14, 2018 1520 Articles Our results contribute toward understanding of the basis for these directionally divergent cardiovascular disease associations. For example, our data have suggested that elevated systolic blood pressure could mediate alcohol consumption’s positive association with stroke and coronary disease excluding myocardial infarction.44,47,48 By contrast, pathways related to HDL-C (but not necessarily HDL-C itself49–52) could mediate alcohol consumption’s inverse association with myo cardial infarction. Both blood pressure and HDL-C are known to increase in response to alcohol consumption.50 They have contrasting associations with cardiovascular disease outcomes: the inverse association of HDL-C with cardiovascular disease is substantially stronger for coronary disease than stroke,53,54 whereas the positive association of systolic blood with cardiovascular disease is considerably stronger for stroke than coronary disease.55 However, we did not find convincing evidence that other known risk factors were important mediators or confounders. Research Laboratory, University of Cambridge, Cambridge CB1 8RN, UK jd292@medschl.cam.ac.uk See Online for appendix effects of reverse causation (especially since some contributing studies did not record baseline chronic disease other than cardiovascular disease). Therefore, alternative study designs including randomised trials58 are needed, to control more completely for residual biases (including those related to studying ex-drinkers and never-drinkers). In conclusion, our study shows that among current drinkers, the threshold for lowest risk of all-cause mortality was about 100 g per week. For cardiovascular disease subtypes other than myocardial infarction, there were no clear thresholds below which lower alcohol consumption stopped being associated with a lower disease risk. These data support adoption of lower limits of alcohol consumption than are recommended in most current guidelines. Coordinating centre Coordinating centre Thomas Bolton, Stephen Burgess, Adam S Butterworth, Emanuele Di Angelantonio, Stephen Kaptoge, Lisa Pennells, Catherine Perry, David Stevens, Sarah Spackman, Simon G Thompson, Matthew Walker, Angela M Wood, and John Danesh (principal investigator). Catherine Perry, David Stevens, Sarah Spackman, Simon G Thompson, Matthew Walker, Angela M Wood, and John Danesh (principal investigator). www.thelancet.com Vol 391 April 14, 2018 Contributors ll h h All the authors contributed to data collection, and to the design, analysis, interpretation, and re-drafting of this report. AMW and SK had full access to the combined data and did the statistical analysis. AMW, EDA, and JD drafted the manuscript and had responsibility for submission of the manuscript for publication. Our study’s access to individual-participant data avoided limitations of previous literature-based reviews.56 To limit reverse causality, our study focused on current drinkers without baseline cardiovascular disease and omitted the initial period of follow-up. To limit confounding, our study adjusted for a variety of risk factors. To correct for misclassification in alcohol consumption and covariates, our study also used extensive information on serial assessments. Our results were robust to a variety of sensitivity analyses. Generalisability of the findings was enhanced by inclusion of data from 83 prospective studies based in many different high-income countries recruited between 1964 and 2010. Although alcohol consumption levels declined during this period, HRs were similar over calendar time. Data management team Data management team Thomas Bolton, Catherine Perry, Sarah Spackman, and Matthew Walker. g Thomas Bolton, Catherine Perry, Sarah Spackman, and Matthew Walker. Thomas Bolton, Catherine Perry, Sarah Spackman, and Matthew Wa Acknowledgments The study’s coordinating centre (Emerging Risk Factors Collaboration and EPIC-CVD Coordinating Centres, Department of Public Health and Primary Care, University of Cambridge, Strangeways Research Laboratory, Cambridge, UK) has been underpinned by grants from the UK Medical Research Council (G0800270 and MR/L003120/1), British Heart Foundation (SP/09/002, RG/08/014 and RG13/13/30194), National Institute for Health Research (through the National Institute for Health Research Cambridge Biomedical Research Centre), European Commission Framework 7 (through the EPIC-CVD award; HEALTH-F2-2012-279233), and the European Research Council (through an Advanced Investigator Award to JD; 268834). JD holds a BHF Professorship and NIHR Senior Investigator Award. A study website Funding for the EPIC-InterAct project was provided by the EU FP6 programme (grant number LSHM_CT_2006_037197). A study website includes a list that investigators have provided of funding agencies that have supported individual EPIC centres. A study website includes a list that investigators have provided of funding agencies that have supported individual cohorts of the ERFC contributing to the present consortium. This research has been conducted using the UK Biobank resource (application 21886). We thank Nicola Kerrison and Stephen Sharp (both from the University of Cambridge MRC Epidemiology Unit, Cambridge, UK) for the former’s data management in the EPIC-InterAct subcohort and the latter’s statistical input into d l t f th EPIC CVD’ l ti l id li 15 Xi B, Veeranki SP, Zhao M, Ma C, Yan Y, Mi J. Relationship of alcohol consumption to all-cause, cardiovascular, and cancer-related mortality in U.S. adults. J Am Coll Cardiol 2017; 70: 913–22. 16 Smyth A, Teo KK, Rangarajan S, et al. Alcohol consumption and cardiovascular disease, cancer, injury, admission to hospital, and mortality: a prospective cohort study. Lancet 2015; 386: 1945–54. 17 Bell S, Daskalopoulou M, Rapsomaniki E, et al. Association between clinically recorded alcohol consumption and initial presentation of 12 cardiovascular diseases: population based cohort study using linked health records. BMJ 2017; 356: j909. 18 Jackson R, Broad J, Connor J, Wells S. Alcohol and ischaemic heart disease: probably no free lunch. Lancet 2005; 366: 1911–12. 19 Knott CS, Coombs N, Stamatakis E, Biddulph JP. All cause mortality and the case for age specific alcohol consumption guidelines: pooled analyses of up to 10 population based cohorts. BMJ 2015; 350: h384. 20 Emberson JR, Bennett DA. Effect of alcohol on risk of coronary heart disease and stroke: causality, bias, or a bit of both? Vasc Health Risk Manag 2006; 2: 239–49. Articles Population Research Advisory Panel, outside the submitted work. ML reports grants from National Institutes of Health, during the conduct of the study; grants from National Kidney Foundation, outside the submitted work; and Funding from the National Institutes of Health, Grant 5U10AA025286, to Johns Hopkins University. MS reports grants from the UK Medical Research Council, the British Heart Foundation, the National Institute for Health Research, European Commission Framework 7, and the European Research Council, during the conduct of the study. NvS reports grants from the Netherlands Ministry of Health Welfare and Sports, Directorate of Long-Term Care, during the conduct of the study. OHF reports grants from Nestle and Metagenics, outside the submitted work. PJN reports grants from the NIH, during the conduct of the study. SGT reports grants from the UK Medical Research Council and the British Heart Foundation, during the conduct of the study. SKi reports grants from FFG COMET program: “Research Center of Excellence in Vascular Ageing—Tyrol, VASCage” (K-Project No. 843536) funded by the BMVIT, BMWFW, Wirtschaftsagentur Wien and Standortagentur Tirol, outside the submitted work. SKa reports grants from the UK Medical Research Council and the British Heart Foundation, during the conduct of the study. WK reports personal fees from AstraZeneca, Novartis, Pfizer, The Medicines Company, GSK, DalCor, Sanofi, Berlin-Chemie, Kowa, and Amgen; grants and non-financial support from Roche Diagnostics, Beckmann, Singulex, and Abbott, outside the submitted work. The other authors declare no competing interests. 7 Corrao G, Bagnardi V, Zambon A, La Vecchia C. A meta-analysis of alcohol consumption and the risk of 15 diseases. Prev Med 2004; 38: 613–19. 8 Hvidtfeldt UA, Tolstrup JS, Jakobsen MU, et al. Alcohol intake and risk of coronary heart disease in younger, middle-aged, and older adults. Circulation 2010; 121: 1589–97. 8 9 Patra J, Taylor B, Irving H, et al. Alcohol consumption and the risk of morbidity and mortality for different stroke types—a systematic review and meta-analysis. BMC Public Health 2010; 10: 258. 10 10 Ronksley PE, Brien SE, Turner BJ, Mukamal KJ, Ghali WA. Association of alcohol consumption with selected cardiovascular disease outcomes: a systematic review and meta-analysis. BMJ 2011; 342: d671. 11 Roerecke M, Rehm J. The cardioprotective association of average alcohol consumption and ischaemic heart disease: a systematic review and meta-analysis. Addiction 2012; 107: 1246–60. 12 Bergmann MM, Rehm J, Klipstein-Grobusch K, et al. For the study website of the funding agencies that have supported individual EPIC centres see http://www.phpc. cam.ac.uk/ceu/epic_cvd/ funding-sources/ For the study website of funding agencies that have supported individual cohorts of the ERFC see http://www. phpc.cam.ac.uk/ceu/erfc/list-of- studies/ Articles The association of pattern of lifetime alcohol use and cause of death in the European Prospective Investigation into Cancer and Nutrition (EPIC) study. Int J Epidemiol 2013; 42: 1772–90. 13 Leong DP, Smyth A, Teo KK, et al. Patterns of alcohol consumption and myocardial infarction risk: observations from 52 countries in the INTERHEART case-control study. Circulation 2014; 130: 390–98. 14 Ferrari P, Licaj I, Muller DC, et al. Lifetime alcohol use and overall and cause-specific mortality in the European Prospective Investigation into Cancer and nutrition (EPIC) study. BMJ Open 2014; 4: e005245. Acknowledgments 21 Ng Fat L, Cable N, Marmot MG, Shelton N. Persistent long-standing illness and non-drinking over time, implications for the use of lifetime abstainers as a control group. J Epidemiol Community Health 2014; 68: 71–77. Epidemiology Unit, Cambridge, UK) for the former’s data management in the EPIC-InterAct subcohort and the latter’s statistical input into development of the EPIC-CVD’s analytical guidelines. Epidemiology Unit, Cambridge, UK) for the former’s data management in the EPIC-InterAct subcohort and the latter’s statistical input into development of the EPIC-CVD’s analytical guidelines. 22 Danesh J, Erqou S, Walker M, et al. The Emerging Risk Factors Collaboration: Analysis of individual data on lipid, inflammatory and other markers in over 1.1 million participants in 104 prospective studies of cardiovascular diseases. Eur J Epidemiol 2007; 22: 839–69. References 1 Department of Health. Alcohol Guidelines Review: Report from the guidelines development group to the UK Chief Medical Officers. 2016. https://www.gov.uk/government/uploads/system/uploads/ attachment_data/file/545739/GDG_report-Jan2016.pdf (accessed Feb 5, 2018). 23 Danesh J, Saracci R, Berglund G, et al. EPIC-Heart: The cardiovascular component of a prospective study of nutritional, lifestyle and biological factors in 520,000 middle-aged participants from 10 European countries. Eur J Epidemiol 2007; 22: 129–41. 2 Kalinowski A, Humphreys K. Governmental standard drink definitions and low-risk alcohol consumption guidelines in 37 countries. Addiction 2016; 111: 1293–98. 2 Kalinowski A, Humphreys K. Governmental standard drink definitions and low-risk alcohol consumption guidelines in 37 countries. Addiction 2016; 111: 1293–98. 24 Sudlow C, Gallacher J, Allen N, et al. UK Biobank: an open access resource for identifying the causes of a wide range of complex diseases of middle and old age. PLoS Med 2015; 12: e1001779. 3 Stampfer MJ, Colditz GA, Willett WC, Speizer FE, Hennekens CH. A prospective study of moderate alcohol consumption and the risk of coronary disease and stroke in women. N Engl J Med 1988; 319: 267–73. 3 Stampfer MJ, Colditz GA, Willett WC, Speizer FE, Hennekens CH. A prospective study of moderate alcohol consumption and the risk of coronary disease and stroke in women. N Engl J Med 1988; 319: 267–73. 25 Conen D. Alcohol consumption and incident cardiovascular disease: not just one unifying hypothesis. Eur Heart J 2015; 36: 897–98. 26 Prentice RL. A case-cohort design for epidemiologic cohort studies and disease prevention trials. Biometrika 1986; 73: 1–11. 4 Boffetta P, Garfinkel L. Alcohol drinking and mortality among men enrolled in an American Cancer Society prospective study. Epidemiology 1990; 1: 342–48. 27 Thompson S, Kaptoge S, White I, et al. Statistical methods for the time-to-event analysis of individual participant data from multiple epidemiological studies. Int J Epidemiol 2010; 39: 1345–59. 5 Thun MJ, Peto R, Lopez AD, et al. Alcohol consumption and mortality among middle-aged and elderly U.S. adults. N Engl J Med 1997; 337: 1705–14. 5 Thun MJ, Peto R, Lopez AD, et al. Alcohol consumption and mortality among middle-aged and elderly U.S. adults. N Engl J Med 1997; 337: 1705–14. 28 Fibrinogen Studies Collaboration, Wood AM, White I, Thompson SG, Lewington S, Danesh J. Regression dilution methods for meta-analysis: assessing long-term variability in plasma fibrinogen among 27,247 adults in 15 prospective studies. Int J Epidemiol 2006; 35: 1570–8. 6 Mukamal K, Conigrave K, Mittleman M, et al. For the study website of funding agencies that have supported individual cohorts of the ERFC see http://www. phpc.cam.ac.uk/ceu/erfc/list-of- studies/ Declaration of interests Despite our study’s access to extensive serial alcohol re-surveys from mid-life, our study could not investigate alcohol consumption during the entire life course. Misclassification in outcomes would have diluted dose-response associations, suggesting that true underlying associations of alcohol consumption with cardiovascular disease subtypes are stronger and more divergent than we observed. Because we did not generally have access to additional alcohol-related adverse out comes (eg, non-fatal liver disease, injuries, or psychiatric comorbidities), we probably under-estimated potential benefits associated with lowering alcohol consumption. Because some individuals who reduced, but did not cease, alcohol consumption due to health complications were probably included in our analysis, we cannot exclude the 1521 Articles Articles For the study website of the funding agencies that have supported individual EPIC centres see http://www.phpc. cam.ac.uk/ceu/epic_cvd/ funding-sources/ For the study website of the funding agencies that have supported individual EPIC centres see http://www.phpc. cam.ac.uk/ceu/epic_cvd/ funding-sources/ References Roles of drinking pattern and type of alcohol consumed in coronary heart disease in men. N Engl J Med 2003; 348: 109–18. 6 Mukamal K, Conigrave K, Mittleman M, et al. Roles of drinking pattern and type of alcohol consumed in coronary heart disease in men. N Engl J Med 2003; 348: 109–18. 1522 www.thelancet.com Vol 391 April 14, 2018 Articles 29 Wood AM, Thompson SG, Kostis JB, et al. Correcting for multivariate measurement error by regression calibration in meta-analyses of epidemiological studies. Stat Med 2009; 28: 1067–92. 45 Au Yeung SL, Jiang C, Cheng KK, et al. Moderate alcohol use and cardiovascular disease from Mendelian randomization. PLoS One 2013; 8: e68054. 46 Yun KE, Chang Y, Yun S-C, et al. Alcohol and coronary artery calcification: an investigation using alcohol flushing as an instrumental variable. Int J Epidemiol 2017; 46: 950–62. 30 Easton DF, Peto J, Babiker AG. Floating absolute risk: an alternative to relative risk in survival and case-control analysis avoiding an arbitrary reference group. Stat Med 1991; 10: 1025–35. 47 Taylor AE, Lu F, Carslake D, et al. Exploring causal associations of alcohol with cardiovascular and metabolic risk factors in a Chinese population using Mendelian randomization analysis. Sci Rep 2015; 5: 14005. 31 Plummer M. Improved estimates of floating absolute risk. Stat Med 2004; 23: 93–104. 32 Royston P, Ambler G, Sauerbrei W. The use of fractional polynomials to model continuous risk variables in epidemiology. Int J Epidemiol 1999; 28: 964–74. 48 Chen L, Smith GD, Harbord RM, Lewis SJ. Alcohol intake and blood pressure: a systematic review implementing a Mendelian randomization approach. PLoS Med 2008; 5: e52. 33 Higgins JPT, Thompson SG. Quantifying heterogeneity in a meta-analysis. Stat Med 2002; 21: 1539–58. 49 Zanoni P, Khetarpal SA, Larach DB, et al. Rare variant in scavenger receptor BI raises HDL cholesterol and increases risk of coronary heart disease. Science 2016; 351: 1166–71. 34 Begg CB, Mazumdar M. Operating characteristics of a rank correlation test for publication bias. Biometrics 1994; 50: 1088. 50 Brien SE, Ronksley PE, Turner BJ, Mukamal KJ, Ghali WA. Effect of alcohol consumption on biological markers associated with risk of coronary heart disease: systematic review and meta-analysis of interventional studies. BMJ 2011; 342: d636. 35 Egger M, Davey Smith G, Schneider M, et al. Bias in meta-analysis detected by a simple, graphical test. BMJ 1997; 315: 629–34. References 36 Centers for Disease Control and Prevention, National Center for Health Statistics. Underlying cause of death 1999–2015 on CDC WONDER Online Database (released Dec, 2016). https://wonder. cdc.gov/ucd-icd10.html (accessed Feb 5, 2018). 51 Voight BF, Peloso GM, Orho-Melander M, et al. Plasma HDL cholesterol and risk of myocardial infarction: a mendelian randomisation study. Lancet 2012; 380: 572–80. 37 WHO. WHO Statistical Information System (WHOSIS). Geneva, Switzerland: World Health Organization, 2007. 52 HPS3/TIMI55-REVEAL Collaborative Group. Effects of anacetrapib in patients with atherosclerotic vascular disease. N Engl J Med 2017; 377: 1217–27. 38 UN Population Division. World Population Prospects. New York, NY: United Nations, 2005. 53 The Emerging Risk Factors Collaboration, Di Angelantonio E, Sarwar N, et al. Major lipids, apolipoproteins, and risk of vascular disease. JAMA 2009; 302: 1993–2000. 39 Emerging Risk Factors Collaboration, Di Angelantonio E, Kaptoge S, et al. Association of cardiometabolic multimorbidity with mortality. JAMA 2015; 314: 52–60. 54 Woodward M, Barzi F, Feigin V, et al. Associations between high-density lipoprotein cholesterol and both stroke and coronary heart disease in the Asia Pacific region. Eur Heart J 2007; 28: 2653–60. 40 IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Personal habits and indoor combustions. Volume 100 E. A review of human carcinogens. IARC Monogr Eval Carcinog Risks Hum 2012; 100 (Pt E): 1–538. 55 MacMahon S, Peto R, Collins R, et al. Blood pressure, stroke, and coronary heart disease. Part 1, prolonged differences in blood pressure: prospective observational studies corrected for the regression dilution bias. Lancet 1990; 335: 765–74. 41 Lazo M, Chen Y, McEvoy JW, et al. Alcohol consumption and cardiac biomarkers: The atherosclerosis risk in communities (ARIC) study. Clin Chem 2016; 62: 1202–10. 56 Ahmed I, Sutton AJ, Riley RD. Assessment of publication bias, selection bias, and unavailable data in meta-analyses using individual participant data: a database survey. BMJ 2012; 344: d7762. 42 Kiechl S, Willeit J. Complex association between alcohol consumption and myocardial infarction: always good for a new paradox. Circulation 2014; 130: 383–86. 57 Del Boca FK, Darkes J. The validity of self-reports of alcohol consumption: state of the science and challenges for research. Addiction 2003; 98: 1–12. 43 Holmes MV, Dale CE, Zuccolo L, et al. Association between alcohol and cardiovascular disease: Mendelian randomisation analysis based on individual participant data. BMJ 2014; 349: g4164. 58 Mukamal KJ, Clowry CM, Murray MM, et al. www.thelancet.com Vol 391 April 14, 2018 References Moderate alcohol consumption and chronic disease: the case for a long-term trial. Alcohol Clin Exp Res 2016; 40: 2283–91. 44 Cho Y, Shin S-Y, Won S, Relton CL, Davey Smith G, Shin M-J. Alcohol intake and cardiovascular risk factors: a Mendelian randomisation study. Sci Rep 2015; 5: 18422. 1523 www.thelancet.com Vol 391 April 14, 2018 www.thelancet.com Vol 391 April 14, 2018
https://openalex.org/W1602635397	https://openaccess.wgtn.ac.nz/articles/thesis/The_glacial_history_of_Tongariro_and_Ruapehu_volcanoes_New_Zealand/17012306/2/files/31467083.pdf	English	null	The glacial history of Tongariro and Ruapehu volcanoes, New Zealand	null	2,021	cc-by-sa	104,385	The glacial history of Tongariro and Ruapehu volcanoes, New Zealand. Shaun Robert Eaves Abstract Understanding the drivers and mechanisms of past, natural changes in Earth’s climate is a fundamental goal of palaeoclimate science. Recent advances in cosmogenic surface exposure dating and numerical glacier modelling have greatly improved the utility of geological glacial records for palaeoclimatic reconstruction. Here, I apply these tech- niques to investigate the timing and magnitude of late Quaternary mountain glacier ﬂuctuations on Tongariro massif and Mt. Ruapehu volcanoes in central North Island, New Zealand (39◦S). First, I constrain the local cosmogenic 3He production rate, in order to compare my subsequent 3He moraine chronologies with other well-dated palaeoclimate records. I present a new radiocarbon age for a large debris avalanche event on the northwest slopes of Mt. Ruapehu that occurred at 10.4-10.6 cal. ka BP. Cosmogenic 3He concentra- tions in surﬁcial boulders deposited during this event are consistent with that predicted by a global compilation of similar production rate calibrations. Thus, I conclude that this globally compiled production rate is suitable for cosmogenic 3He exposure age calculations in New Zealand. Exposure ages from moraine boulders on both volcanoes constrain the timing of two periods of glaciation during the last glacial cycle, when the termini of valley glaciers reached c. 1200 m asl. The most recent of these events occurred between c. 31-17 ka, which corresponds with the global Last Glacial Maximum. During this period, the local equilibrium line altitude was depressed by c. 800-1100 m. Numerical model simulations of the glaciers, using a coupled energy balance/ice ﬂow model, suggest that local atmospheric temperature was 4-7 ◦C colder than present. This palaeotem- perature estimate is not greatly impacted by post-glacial topographic change on these active volcanoes. Surface exposure ages from a degraded lateral moraine on Tongariro massif indicate that an earlier period of glaciation, of similar extent to that at the LGM, culminated during Marine Isotope Stage 4. During the last glacial-interglacial transition (c. 18-11 ka), glacial retreat on Mt. Ru- apehu was interrupted by a re-advance during the late-glacial (c. 15-11 ka). Exposure ages for this event exhibit some scatter, likely due to surface processes. Accounting for these processes with a topographic diffusion model yields a best-estimate age of 14-13 ka, corresponding to the Lateglacial reversal in New Zealand. Glacier model experiments indicate this re-advance resulted from a temperature lowering of 2.5-3.4 ◦C relative to present. Comparison with other proxy records suggests that this cooling was most pronounced during summer. Abstract Due to its lower elevation, it is unlikely that glaciers were present on Tongariro massif at this time. The results of this research provide the ﬁrst direct age constraint and quantitative palaeoclimate reconstructions for late Quaternary glacier ﬂuctuations in central North Island, New Zealand. The timing and magnitude of these changes are in good agree- ment with glacial records from the Southern Alps and South America. This suggests that glaciers in the southern mid-latitudes were responding to common climatic forcings at orbital- and millennial-timescales, during the last glacial cycle. Acknowledgments Many people have helped make this research feasible. Firstly, I would like to thank my supervisor, Andrew Mackintosh, for supporting me in pursuit of my research interests. The work presented here would not have been possible without his expert guidance and unwavering enthusiasm for this project. I am also indebted to Andrew for my development as a scientist over the last 3.5 years. He has challenged me to think about the methods of scientiﬁc enquiry, included me in the development of research proposals and peer reviews, and shared with me his wide network of collaborators. For all of this, I am extremely grateful. I thank Brian Anderson for introducing me to glacier modelling and providing advice in the manipulation and application of his model code. Dougal Townsend and Graham Leonard have provided excellent maps, new insights into the geological evolution of the central North Island volcanoes, and continual logistical support for ﬁeldwork. All supervisors have provided valuable, constructive feedback on thesis chapters and manuscripts. I thank all of the other brilliant scientists with whom I have had the opportunity to collaborate. Gisela Winckler and Joerg Schaefer generously hosted me at Lamont- Doherty Earth Observatory in 2013 and granted me the freedom to run samples until the early hours! Sascha Serno, Linda Baker and Roseanne Schwartz also provided expert guidance on sample preparation and noble gas mass spectrometry, as well as a friendly working atmosphere. Brent Alloway has provided invaluable insight to volcanic sedimentology and tephrostratigraphy, as well as elegant stratigraphic logs. Marcus Vandergoes introduced me to the intricacies of radiocarbon sample se- lection and provided radiocarbon measurements. Diane Seward kindly taught me techniques in mineral separation. Kevin Norton inspired me to take a quantitative approach to geomorphological problems and provided great company and coffee on various ﬁeld trips. Regine Hock kindly hosted me in Fairbanks and constructed the superb International Glaciology Summer School, in which I was fortunate to participate. I thank all of the other brilliant scientists with whom I have had the opportunity to collaborate. Gisela Winckler and Joerg Schaefer generously hosted me at Lamont- Doherty Earth Observatory in 2013 and granted me the freedom to run samples until the early hours! Sascha Serno, Linda Baker and Roseanne Schwartz also provided expert guidance on sample preparation and noble gas mass spectrometry, as well as a friendly working atmosphere. Acknowledgments Brent Alloway has provided invaluable insight to volcanic sedimentology and tephrostratigraphy, as well as elegant stratigraphic logs. Marcus Vandergoes introduced me to the intricacies of radiocarbon sample se- lection and provided radiocarbon measurements. Diane Seward kindly taught me techniques in mineral separation. Kevin Norton inspired me to take a quantitative approach to geomorphological problems and provided great company and coffee on various ﬁeld trips. Regine Hock kindly hosted me in Fairbanks and constructed the superb International Glaciology Summer School, in which I was fortunate to participate. The staff of the Antarctic Research Centre and the School of Geography, Environment iii iv and Earth Sciences have created a friendly and productive academic environment. Michelle Dow, Robyn Mcfarlane and Kate King have ensured smooth negotiation of various grant systems, building works and other administrative tasks. It has been a privilege to share the doctoral research experience with other postgraduate students in the ARC and SGEES. I thank Chris Conway for being a great ﬁeld compan- ion and for helpful discussions about lava-ice interactions. Alice Doughty provided invaluable support for exposure dating sample selection and glacier modelling, as well as shared a general fascination with mountain glaciers and Quaternary palaeoclimate. I thank fellow ’Beer Pom’, Richard Jones for useful scientiﬁc discussions and ﬁeld assistance. Bella Duncan has been an ever-present ofﬁcemate, part-time ﬂatmate and full-time friend. Thanks for all the cups of tea and delicious roast dinners! Thanks to Matt Ryan and Ignacio Jara for discussions about Southern Hemisphere palaeoclimate. Emily, Loretta, Juliet, Molly, Prisco, Ari, Julene and many others have also helped make ofﬁce, lab and general life enjoyable. I thank Max Watt for teaching me how to ﬁsh the local waters without ever having to worry about catching anything! I thank my Mum and Dad for their unconditional love and support from the other side of the world. Jenni, thank you for always knowing what is best. And for supplying beautiful artwork (opposite). I can’t wait for our next adventure. I am grateful for support from Victoria University of Wellington Doctoral and Submis- sion Scholarships. I also acknowledge generous ﬁnancial support from the Antarctic Research Centre Endowed Development Fund, VUW Strategic Research Grants, the ST Lee Travel Award, and the Project Tongariro Memorial Award. Contents 1 Introduction 1 1.1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.2 Organisation of this thesis . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1.3 Statement on the contributions made to this thesis by the author, super- visors and collaborators . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2 Background 7 2.1 Glaciers as a palaeoclimate proxy . . . . . . . . . . . . . . . . . . . . . . . 8 2.2 Quaternary palaeoclimate: a brief history of the orbital revolution . . . . 9 2.2.1 Outstanding questions in orbital theory - southern connections . 13 2.3 Millennial-scale oscillations . . . . . . . . . . . . . . . . . . . . . . . . . . 17 2.4 The late Quaternary glacial history of New Zealand (c. 125 - 10 ka) . . . 19 2.4.1 Pre-Last Glacial Maximum (c. 125 - 35 ka) . . . . . . . . . . . . . . 20 2.4.2 Last Glacial Maximum (c. 35 - 18ka) . . . . . . . . . . . . . . . . . 21 2.4.3 Late-Glacial (15 - 11.5 ka) . . . . . . . . . . . . . . . . . . . . . . . 24 2.5 Study site and previous work . . . . . . . . . . . . . . . . . . . . . . . . . 29 2.5.1 Geological setting . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 2.5.2 Present day climatic situation . . . . . . . . . . . . . . . . . . . . . Acknowledgments Finally, thank you to Rewi Newnham (VUW), David Barrell (GNS Science) and Bethan Davies (RHUL, UK) for each taking the time to provide detailed feedback on the content and structure of this thesis, and for providing a stimulating discussion at the oral exam. v v vi vi Contents 31 2.5.3 Contemporary glacierisation . . . . . . . . . . . . . . . . . . . . . 35 2.5.4 Previous work: palaeo-glaciation in Tongariro National Park . . . 43 2.6 Research questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 3 Methodology 47 3.1 Geomorphological mapping . . . . . . . . . . . . . . . . . . . . . . . . . . 47 3.2 Cosmogenic surface exposure dating . . . . . . . . . . . . . . . . . . . . . 50 3.2.1 Cosmic radiation and nuclide production . . . . . . . . . . . . . . 51 3.2.2 Application to palaeoglaciology . . . . . . . . . . . . . . . . . . . 53 3.2.3 Approach used in this thesis . . . . . . . . . . . . . . . . . . . . . 55 3.3 Palaeoclimate reconstruction using glaciers . . . . . . . . . . . . . . . . . 61 3.3.1 Equilibrium line altitude (ELA) reconstruction . . . . . . . . . . . 61 3.3.2 Numerical glacier modelling . . . . . . . . . . . . . . . . . . . . . 62 vii 3 Methodology vii CONTENTS viii CONTENTS pp 4 A test of the cosmogenic 3He production rate in the southwest Paciﬁc (39◦S) 79 4.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 4.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 4.3 Facies architecture and stratigraphy of the Murimotu Formation debris avalanche deposit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 4.4 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 4.4.1 Radiocarbon dating . . . . . . . . . . . . . . . . . . . . . . . . . . 87 4.4.2 3Hecos sample collection, preparation and measurement . . . . . 87 4.4.3 Production rate calculations . . . . . . . . . . . . . . . . . . . . . . 88 4.5 Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 4.5.1 Radiocarbon dating of the Murimotu debris avalanche . . . . . . 90 4.5.2 3Hecos measurements and production rate calibration . . . . . . . 91 4.6 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 5 A cosmogenic 3He chronology of mountain glacier ﬂuctuations in North Is- land, New Zealand (39◦S) during the last glacial cycle. 99 5.1 Abstract . . . . . . . . . . . . . . . . . . . . . 3 Methodology . . . . . . . . . . . . . . . . 99 5.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 5.3 Setting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 5.3.1 Regional climatic situation . . . . . . . . . . . . . . . . . . . . . . 101 5.3.2 Study site and previous work . . . . . . . . . . . . . . . . . . . . . 102 5.4 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 5.4.1 Cosmogenic surface exposure dating . . . . . . . . . . . . . . . . 108 5.4.2 Tephrochronology . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 5.4.3 Equilibrium line altitude reconstruction . . . . . . . . . . . . . . . 112 5.5 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 5.5.1 Cosmogenic 3He results and moraine age interpretation . . . . . 114 5.5.2 Coverbed stratigraphy and tephra major element geochemistry . 121 5.5.3 Equilibrium line altitude reconstruction . . . . . . . . . . . . . . . 126 5.6 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 5.6.1 The Last Glacial Cold Period in central North Island . 3 Methodology . . . . . . 127 5.6.2 Pre-LGCP glaciation . . . . . . . . . . . . . . . . . . . . . . . . . . 129 5.7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 6 Uniform summertime cooling across New Zealand drove glacial readvance during the late-glacial (15-11 ka) 133 6.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 6.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134 CONTENTS ix 6.3 Study site and previous work . . . . . . . . . . . . . . . . . . . . . . . . . 135 6.4 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138 6.4.1 Geomorphological mapping . . . . . . . . . . . . . . . . . . . . . 138 6.4.2 Cosmogenic 3He surface exposure dating . . . . . . . . . . . . . . 138 6.4.3 Glacier modelling . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 6.5 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 6.5.1 Glacial geomorphology and chronology . . . . . . . . . . . . . . . 145 6.5.2 Glacier modelling . . . . . . . . . . . . . . 3 Methodology . . . . . . . . . . . . . . 151 6.6 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155 6.6.1 Glacial chronology . . . . . . . . . . . . . . . . . . . . . . . . . . . 155 6.6.2 Palaeoclimatic reconstruction . . . . . . . . . . . . . . . . . . . . . 162 6.7 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170 7 Temperature change during the Last Glacial Cold Period in central North Island, New Zealand (39 ◦S), inferred from 2D glacier modelling 171 7.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 7.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172 7.2.1 Setting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174 7.3 Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176 7.3.1 Cosmogenic 3He surface exposure dating . . . . . . . . . . . . . . 176 7.3.2 Model input data . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178 7.3.3 Model description . . . . . . . . . . . . . . . . . . . 3 Methodology . . . . . . . . 179 7.3.4 Sensitivity tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181 7.3.5 Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182 7.4 Geomorphological characterisation . . . . . . . . . . . . . . . . . . . . . . 190 7.4.1 Whakapapaiti valley (’WHA’) . . . . . . . . . . . . . . . . . . . . . 190 7.4.2 Mangaturuturu valley (’MTU’) . . . . . . . . . . . . . . . . . . . . 192 7.4.3 Wahianoa valley (’WAH’) . . . . . . . . . . . . . . . . . . . . . . . 192 7.4.4 Mangatoetoenui valley (’MTO’) . . . . . . . . . . . . . . . . . . . . 194 7.4.5 Mangatepopo Valley (’MPO’) . . . . . . . . . . . . . . . . . . . . . 198 7.4.6 Eastern Tongariro massif . . . . . . . . . . . . . . . . . . . . . . . . 198 7.5 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201 7.5.1 Cosmogenic surface exposure dating . . . . . . . . . . . . . . . . 201 7.5.2 Glacier modelling . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202 7.6 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205 7.6.1 Topographic uncertainty . 3 Methodology . . . . . . . . . . . . . . . . . . . . . . . 205 7.6.2 Glacier model uncertainty . . . . . . . . . . . . . . . . . . . . . . . 207 7.6.3 LGCP climate in New Zealand . . . . . . . . . . . . . . . . . . . . 209 7.7 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211 CONTENTS x 8 Synthesis 213 8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 8.2 Original scientiﬁc contributions made by this research . . . . . . . . . . . 213 8.3 Research Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215 8.3.1 Can cosmogenic isotopes be used to constrain past glacial activity on the North Island volcanoes? . . . . . . . . . . . . . . . . . . . . 215 8.3.2 When did glaciers in central North Island reach their maximum extent during the last glacial cycle? . . . . . . . . . . . . . . . . . . 218 8.3.3 How cold was was the Last Glacial Cold Period in central North Island? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220 8.3.4 Did glaciers in central North Island respond to the late-glacial climate reversal in New Zealand? . . . . . . . . . . . . . . . . . . 223 8.4 Further research . . . . . . . . . . . . . . . . . . . . . . . . List of Figures 2.1 Milankovitch cycles: eccentricity, obliquity and axial precession . . . . . 11 2.2 Covariance of austral (77◦S) summer duration and boreal (65◦N) summer insolation intensity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 2.3 Millennial-scale climate variability in ice cores (10-90 ka) . . . . . . . . . 18 2.4 Recalculation of cosmogenic exposure ages from Cobb valley . . . . . . 25 2.5 Geological setting of Tongariro and Ruapehu volcanoes . . . . . . . . . . 32 2.6 Existing radiometric ages of lava ﬂows on Tongariro massif and Mt. Ruapehu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 2.7 New Zealand climatic situation . . . . . . . . . . . . . . . . . . . . . . . . 34 2.8 Climate at Whakapapa Village (1097 m asl), Mt. Ruapehu . . . . . . . . . 36 2.9 Contemporary glaciers on Mt. Ruapehu . . . . . . . . . . . . . . . . . . . 37 2.10 Photograph of Mangaehuehu Glacier, Mt. Ruapehu . . . . . . . . . . . . 39 2.11 Photograph of Summit Plateau, Mt. Ruapehu . . . . . . . . . . . . . . . . 40 2.12 Photograph of Mangatoetoenui Glacier, Mt. Ruapehu . . . . . . . . . . . 41 2.13 Photograph of Crater Lake, Mt. Ruapehu . . . . . . . . . . . . . . . . . . 42 2.14 Photograph of lacustrine sediments in Mangaetoetoenui valley, Mt. Ru- apehu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 3.1 Attenuation of cosmogenic nuclide production due to shielding . . . . . 3 Methodology . . . . . . . . . 225 8.4.1 Reﬁned estimates of local cosmogenic 3He production . . . . . . 225 8.4.2 Development of other cosmogenic nuclides for use in the south west Paciﬁc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226 8.4.3 What caused the Antarctic Cold Reversal? . . . . . . . . . . . . . 227 8.4.4 Holocene glacier ﬂuctuations in the Southern Hemisphere . . . . 228 List of Figures 58 3.2 Ice melt from geothermal heating on active volcanoes . . . . . . . . . . . 71 3.3 Modelled present day ice distribution on Mt. Ruapehu . . . . . . . . . . 75 4.1 Map of existing cosmogenic 3He geological calibration sites . . . . . . . 81 4.2 Map of Murimotu Formation debris avalanche deposit and sample sites 84 4.3 Stratigraphic logs of Murimotu Formation debris avalanche . . . . . . . 85 4.4 Field and laboratory photographs of radiocarbon samples at Murimotu Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 4.5 Cosmogenic 3He samples from Murimotu Formation . . . . . . . . . . . 89 4.6 Radiocarbon results from Murimotu Formation . . . . . . . . . . . . . . . 92 4.7 Calibrated cosmogenic 3He production rates from Murimotu Formation 94 xi LIST OF FIGURES xii 5.1 Maps showing climatic and topographic setting of Mangatepopo valley 102 5.2 The glacial geology of Mangatepopo valley . . . . . . . . . . . . . . . . . 105 5.3 Selected photos of glacial landforms on western Tongariro massif . . . . 106 5.4 Photographs of boulders sampled for cosmogenic 3He surface exposure dating in Mangatepopo valley . . . . . . . . . . . . . . . . . . . . . . . . . 110 5.5 The impact of hypothetical soil/ash burial scenarios on exposure ages in Mangatepopo valley . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 5.6 Stratigraphic log and photos of moraine coverbed in Mangatepopo valley122 5.7 Major element tephra chemistry from rhyolite horizons in Mangatepopo valley . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 5.8 ELA and palaeotemperature reconstruction of the former Mangatepopo glacier . List of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 6.1 Overview of study region on southern Mt. Ruapehu . . . . . . . . . . . . 136 6.2 Examples of boulders from late-glacial moraines sampled for cosmogenic 3He exposure dating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141 6.3 The glacial geology of Mangaehuehu catchment, Mt. Ruapehu . . . . . . 147 6.4 Photographs of sediment underlying moraine LG1 . . . . . . . . . . . . . 148 6.5 The glacial geology of Te Unuunuakapuateariki catchment, Mt. Ruapehu 149 6.6 The glacial geology of Wahianoa catchment, Mt. Ruapehu . . . . . . . . 150 6.7 Late-glacial palaeotemperature-precipitation results from numerical glacier modelling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154 6.8 Seasonal sensitivity analyses of glacier length on Mt. Ruapehu . . . . . . 155 6.9 The impact of hypothetical soil/ash burial scenarios on the LG1 moraine exposure ages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158 6.10 Cosmogenic surface exposure age distribution from Mangaehuehu catch- ment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165 6.11 Histograms of good-ﬁt moraine degradation model results, applied to LG1 moraine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166 7.1 Digital elevation model and Last Glacial Cold Period ice limits of Mt. Ruapehu . . . . . . . List of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 7.2 Topographic reconstruction of Tongariro Volcanic Centre (>15 ka) . . . . 185 7.3 Modelled ice thickness on both volcanoes from step-cooling experiments 186 7.4 Palaeotemperature estimates from glacier modelling - present day topog- raphy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187 7.5 Modelled ice thickness and distribution from select Experiment 2 experi- ments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188 7.6 Palaeotemperature estimates from glacier modelling - pre-15 ka topogra- LIST OF FIGURES IST OF FIGURES xiii 7.7 The glacial geology of Whakapapaiti valley . . . . . . . . . . . . . . . . . 191 7.8 The glacial geology of Mangaturuturu valley . . . . . . . . . . . . . . . . 193 7.9 The glacial geology of Wahianoa valley . . . . . . . . . . . . . . . . . . . 195 7.10 Ice-scoured, striated bedrock in Wahianoa valley . . . . . . . . . . . . . . 196 7.11 The glacial geology of Mangatoetoenui valley . . . . . . . . . . . . . . . . 197 7.12 The glacial geology of eastern Tongariro massif. . . . . . . . . . . . . . . 199 8.1 Photograph of ice needles lifting sediment . . . . . . . . . . . . . . . . . . 218 List of Tables 3.1 Geomorphological mapping workﬂow for this study . . . . . . . . . . . 49 3.2 Empirically derived, seasonal air temperature lapse rates for upland (> 300 m asl) New Zealand . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 3.3 Optimal energy balance and ice ﬂow model parameter settings. . . . . . 72 4.1 Radiocarbon data from organic material entrained within/buried by the Murimotu Formation debris avalanche. . . . . . . . . . . . . . . . . . . . 90 4.2 Elemental composition of pyroxene samples . . . . . . . . . . . . . . . . 95 4.3 Murimotu Formation cosmogenic 3He sample locations, geometrical corrections and noble gas mass spectrometry data . . . . . . . . . . . . . 95 4.4 Calibrated SLHL 3He production rates from the Murimotu Formation . 96 4.5 Cosmogenic 3He/10Be and 3He/14C production ratios for New Zealand 96 5.1 Location and geometry of cosmogenic 3He samples from Mangatepopo valley . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 5.2 Helium isotope data of all samples from Mangatepopo valley . . . . . . 120 5.3 Cosmogenic 3He surface exposure ages for all samples from Mangatepopo valley . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 5.4 Glass shard major element compositions of rhyolitic tephras from the Taurewa and Mangatepopo sections, compared with potential correla- tives from the Okataina Volcanic Centre (OVC). . . . . . . . . . . . . . . . 124 6.1 Moraine boulder cosmogenic 3He sample details (Mangaehuehu and Te Unuunuakapuateariki catchments). . . . . . . . . . . . . . . . . . . . . . . 139 6.2 Helium mass spectrometry data (Mangaehuehu and Te Unuunuakapu- ateariki catchments) . . . LIST OF FIGURES xiv xiv List of Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152 6.3 Exposure ages (ka) for all samples in the Mangaehuehu and Te Un- uunuakapuateariki catchments . . . . . . . . . . . . . . . . . . . . . . . . 153 6.4 Moraine degradation model parameters space and best ﬁt result . . . . . 161 6.5 Best-ﬁt moraine degradation model parameter combinations when indi- vidual variables are prescribed using literature-based values. . . . . . . . 161 xv LIST OF TABLES xvi 6.6 A compilation of chronologies and quantitative palaeoclimate recon- structions for the late-glacial climate reversal in New Zealand. . . . . . . 167 7.1 Cosmogenic 3He surface exposure dating sample details, helium content and exposure ages for Wahianoa valley . . . . . . . . . . . . . . . . . . . 201 7.2 Model-derived LGCP equilibrium line altitudes for glacial catchments in Tongariro National Park . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 1Following Aubry et al. (2009), I use ’annus’ (’a’) to denote absolute time and ’year’ (’yr’) for durations. 1.1 Overview The last c. 1 million years of Earth’s history have been characterised by natural, cyclical changes between glacial and interglacial climate, which occurred over periods of c. 80-120 kyr1. These cycles are paced by astronomically induced changes in the seasonal distribution of insolation (Croll, 1864; Milankovitch, 1941; Hays et al., 1976). However, the response of global climate to orbital forcing exhibits several non-linearities that have thus far deﬁed explanation. At ﬁrst order, a single glacial cycle comprises a long (c. 80-90 kyr) gradual cooling, during which, global mean temperatures decline by 3-4 ◦C (Schneider von Deimling et al., 2006), ice sheets advance over northern Europe and North America (Clark et al., 2009), global mean sea level falls by c. 120-140 m (Fleming et al., 1998; Lambeck et al., 2014) and atmospheric CO2 is reduced by c. 100 ppmv (Monnin et al., 2001). Then, over the course of c. 10 kyr all of this is reversed and an interglacial climate emerges (Denton et al., 2010), before the cycle is repeated. Within these cycles, many high-amplitude oscillations occur over sub-orbital, millennial timescales and appear to be antiphased between the hemispheres (Blunier and Brook, 2001; EPICA Community Members, 2006). Resolving the drivers and mechanisms of these changes remains an outstanding goal of palaeoclimate science. Few opportunities exist to examine the imprint of past ice age climate in the Southern Hemisphere. Where available, climate proxy records from austral mid- to high-latitudes offer insight to past changes of components of the atmospheric and oceanic systems, which are considered to play important roles in driving global climatic change over orbital- to millennial timescales (Anderson et al., 2009; Barker et al., 2009; Denton et al., 2010; Putnam et al., 2010a; Whittaker et al., 2011; De Deckker et al., 2012). The New Zealand landmass is situated at a climatic boundary between sub-tropical and 1 CHAPTER 1. INTRODUCTION 2 2 sub-polar air and water masses. High relief topography, spanning >10◦of latitude (34-47◦S), interacts with prevailing westerly circulation creating steep environmental gradients that are susceptible to small changes in atmospheric and oceanic conditions (Alloway et al., 2007). Thus, palaeoclimate reconstructions from New Zealand can provide unique insight to past changes in the global climate system. The distinctive landscapes created through the erosion, entrainment and deposition of rocks by glaciers provided the ﬁrst clues for Quaternary glacial cycles (Agassiz, 1840). 1.1. OVERVIEW 3 3 Zealand remains relatively restricted. Much work has focused on the central Southern Alps, which afford well-preserved suites of glacial landforms (Barrell, 2011, 2014), as well as pristine, sedimentary archives that contain fossil pollen assemblages and other palaeo-environmental proxies (Vandergoes et al., 2005, 2008). Expanding the spatial range of palaeoclimate records allows consideration of past climatic gradients, which may be indicative of past climate drivers (Carter et al., 2008; Lorrey et al., 2012b; Newn- ham et al., 2012). In North Island, late Quaternary palaeoclimate records predominantly consist of low- altitude sedimentary archives that preserve fossil pollen and diatom assemblages (Newnham and Lowe, 2000; Newnham et al., 2007, 2012; Stephens et al., 2012a,b; Sikes et al., 2013). In conjunction with a well-constrained local tephrostratigraphy (Lowe et al., 2008, 2013), these archives have provided important insight to the timing of palaeoenvironmental change in North Island (Newnham and Lowe, 2000; Hajdas et al., 2006). However, quantitative palaeoclimate reconstruction using such proxies is subject to relatively high uncertainties (Wilmshurst et al., 2007; Newnham et al., 2013). Addi- tion of alternative quantitative palaeoclimate reconstructions from North Island will allow robust assessment of past climatic gradients across the latitudinal length of New Zealand. The volcanic peaks of Tongariro massif and Mt. Ruapehu, in central North Island, formed over the last 250 ka (Gamble et al., 2003), thus were present through at least one full glacial cycle. Mt. Ruapehu is the highest peak in North Island and the only one to intercept the current permanent snowline, with several small cirque glaciers present on its upper slopes (Keys, 1988; Brook et al., 2011). Thus, there is potential to investigate past climate change in this region using glaciers. Indeed, geomorphological evidence for past, more extensive glaciation of the central North Island volcanoes has long been recognised (Taylor, 1927; Mathews, 1967; Topping, 1974; McArthur and Shepherd, 1990; Barrell, 2011). However, the absence of geochronological tools has thus far limited the utility of these glacial records for palaeoclimatic research. Taking advantage of recent developments in surface exposure dating and numeri- cal glacier modelling, my primary aim in this thesis is to constrain the timing and magnitude of glacier ﬂuctuations in central North Island over the late Quaternary, in order to improve understanding of orbital- and millennial-scale climatic drivers. To achieve this aim, I complete the following objectives: 1. 1.1 Overview Mountain glaciers are highly sensitive to changes in climate, in particular temperature and precipitation (Oerlemans, 2001). Changes in mean annual temperature alter the mass balance of glaciers by increasing/decreasing surface melt and changing the ratio of solid to liquid precipitation. Changes in total precipitation alter the amount of precipitation that can fall as snow. New Zealand glaciers are most sensitive to tem- perature changes, with c. 30-80 % increases in precipitation required to balance a 1 ◦C increase in temperature (Oerlemans, 1997; Anderson and Mackintosh, 2006; Anderson et al., 2010; Anderson and Mackintosh, 2012). Positive glacier mass balance increases glacier volume and, if sustained (over timescales of years-to decades), this mass gain typically results in advance of the terminus. Meanwhile glacier retreat occurs following sustained negative mass balance. Thus, geological records of past glacier ﬂuctuations afford insight into past, natural climate variations. Two recent developments have improved the utility of glacial geology for palaeo- climate reconstruction. First, improved understanding of the production (e.g. Goehring et al., 2010; Putnam et al., 2010b; Blard et al., 2013b), extraction and measurement (Schae- fer et al., 2009) of terrestrial cosmogenic nuclides has provided a means to directly date glacial landforms (Balco, 2011). Second, increased understanding of ice-climate relationships (e.g. Oerlemans, 2001), coupled with advances in computing power and the increasing availability of digital datasets, means that numerical glacier mod- els of varying complexity can now be used to investigate the climatic signiﬁcance of past glacier ﬂuctuations (Plummer and Phillips, 2003; Oerlemans, 2005; Kessler et al., 2006). Application of these methods in recent years has resulted in a step-increase in understanding of the timing and climatic signiﬁcance of palaeo- glacier ﬂuctuations worldwide, including in the Southern Alps of New Zealand (e.g. Ivy Ochs et al., 1999; Shulmeister et al., 2005; Anderson and Mackintosh, 2006; Schaefer et al., 2006; Barrows et al., 2007b; Schaefer et al., 2009; McCarthy et al., 2008; Kaplan et al., 2010, 2013; Putnam et al., 2010a, 2012, 2013a,b; Golledge et al., 2012; Barrows et al., 2013; Doughty et al., 2013; Rowan et al., 2013; Rother et al., 2014). Despite this progress, well dated, quantitative palaeoclimatic reconstructions in New 1.1. OVERVIEW Delineate the spatial extent of past mountain glacier ﬂuctuations on the cen- tral North Island volcanoes, through remote- and ﬁeld-based geomorphological CHAPTER 1. INTRODUCTION CHAPTER 1. INTRODUCTION 4 4 mapping of glacial landforms. mapping of glacial landforms. • Field investigations serve to ’ground-truth’ initial, desk-based interpretations, as well as identify sites and collect samples for objective 2 (below). • Field investigations serve to ’ground-truth’ initial, desk-based interpretations, as well as identify sites and collect samples for objective 2 (below). 2. Develop and apply tools to constrain the timing of past glacial activity in central North Island, New Zealand. • Comparison of glacial chronologies with other, well-dated palaeoclimate proxy records provides insight to the relative timing of past changes in cli- mate, at various spatial scales (i.e. regional, hemispheric, inter-hemispheric). Identiﬁcation of leads-lags helps to inform the possible drivers of climatic change. 3. Provide quantitative estimates of past temperature change in central North Island, using a physically-based, numerical model to simulate former glacier extents. • Quantifying palaeoclimatic change using proxy evidence helps to under- stand the drivers and mechanisms causing the observed changes, in two main ways: (i) constraining spatial gradients of change with that predicted by speciﬁc hypotheses; and (ii) comparison with outputs from global climate model experiments. In completing these objectives, I make the following original contributions: • geological constraint of cosmogenic 3He production in the south west Paciﬁc since c. 11 ka • maps of the glacial landform/sediment distribution in central North Island • direct age constraint of glacier ﬂuctuations on both volcanoes over the last glacial cycle • quantitative palaeotemperature estimates for the Last Glacial Maximum and the late-glacial climate reversal • a hypothesis concerning the inter-proxy comparisons and seasonal climate change during the late-glacial • an investigation into the effects of changing topographic boundary conditions for palaeoclimate estimates using glacier models in dynamic landscapes 1.2. ORGANISATION OF THIS THESIS 5 5 1.2 Organisation of this thesis In Chapters 4 - 7, I present original research, designed and carried out by myself (see below for supervisor/collaborator contributions). In each of these chapters, I address a primary research question identiﬁed in Section 2.6. These chapters are formatted in the traditional style of a full scientiﬁc journal article. Some have already been submitted for publication in international journals. At the front end of the thesis, I present a literature review and introduction to the study region, before identifying several research questions to be addressed (Chapter 2). In Chapter 3, I provide descriptions and justiﬁcations for use of the methodologies adopted in this thesis. After the primary data are presented (Chapters 4-7), I readdress the original research questions in light of the results (Chapter 8). 1.3 Statement on the contributions made to this thesis by the author, supervisors and collaborators Research design, data collection, interpretation and presentation, and write up repre- sent my own efforts. This project was supervised by A. Mackintosh (ARC/SGEES), B. Anderson (ARC) and D. Townsend (GNS Science). Supervisor contributions to this thesis primarily include: (i) discussion and contribution of scientiﬁc ideas and con- cepts; (ii) editorial advice on write up of results; (iii) digital datasets; and (iv) research funds for ﬁeldwork and sample costs, as well as overseas travel. Primary supervisor A.Mackintosh is co-author on all manuscripts. The ice-ﬂow and energy-balance models were originally coded by secondary supervisor B. Anderson (Victoria University of Wellington). Anderson is co-author on manuscripts where I have utilised his glacier model code. The research in this thesis also contributes towards a new geological map of Tongariro National Park, with accompanying written descriptions (GNS Science - contract DM-593774). This project is led by external supervisor D. Townsend, who has provided digital datasets and initial geomorphological interpretations. Townsend is co-author on all manuscripts. Below, I have detailed the research manuscripts that comprise this thesis, the co-authors and any publication information. Brief statements describing collaborator contributions are also included. Chapter 4: Eaves, S.R., Winckler, G., Schaefer, J.M., Vandergoes, M., Alloway, B.V., Mackintosh, A., Townsend, D.B., Ryan, M.T., Li, X. A test of the cosmogenic 3He produc- CHAPTER 1. INTRODUCTION 6 tion rate in the south west Paciﬁc (39◦S). Accepted for publication in Journal of Quaternary Science. tion rate in the south west Paciﬁc (39◦S). Accepted for publication in Journal of Quaternary Science. Winckler, Schaefer and I provided cosmogenic 3He measurements. Alloway, Mackin- tosh, Ryan, Vandergoes and I undertook stratigraphic ﬁeld investigations and collected radiocarbon samples. Vandergoes and Li provided radiocarbon measurements. Chapter 5: Eaves, S.R., Mackintosh, A., Winckler, G., Schaefer, J.M., Alloway, B.V., Townsend, D.B. Orbital scale glacier ﬂuctuations in North Island, New Zealand (39◦S) during the last glacial cycle. in prep. Mackintosh and I identiﬁed the sites and undertook palaeoglaciological reconstructions. Winckler, Schaefer and I provided cosmogenic 3He measurements. Alloway and I provided stratigraphic ﬁeld investigations and electron microprobe analysis of tephra. Townsend and I provided geomorphological maps. Chapter 6: Eaves, S.R., Mackintosh, A., Winckler, G., Schaefer, J.M., Anderson, B.M., Doughty, A.M., Townsend, D.B. Uniform summer cooling across New Zealand drove glacier readvance during the late-glacial (15-11 ka). in prep. Mackintosh, Doughty and I carried out ﬁeldwork. Winckler, Schaefer and I provided out cosmogenic 3He measurements. Anderson and Doughty provided guidance on model initialisation. Townsend and I provided geomorphological maps. Chapter 7: Eaves, S.R., Anderson, B.M., Mackintosh, A., Winckler, G., Schaefer, J.M., Townsend, D.B., Leonard, G., Conway, C. Temperature change during the Last Glacial Cold Period in central North Island, New Zealand (39◦S), inferred from 2D glacier modelling. in prep. Winckler, Schaefer and I carried out cosmogenic 3He measurements. Townsend and I provided geomorphological maps. Townsend, Conway and Leonard provided geo- logical reconstructions of LGM topographies based on ﬁeld mapping and radiometric dating. 2.1 Glaciers as a palaeoclimate proxy Glaciers are perennial masses of frozen water that ﬂow under their own weight and form in speciﬁc topoclimatic situations where solid precipitation remains on the ground throughout the year. Thus, they are intimately connected to climate through the strong dependence of glacier mass balance on climatic variables - in particular temperature and precipitation (Oerlemans and Fortuin, 1992; Oerlemans, 2001, 2005). Mass accumu- lation on the glacier surface is predominantly sourced from solid precipitation, which occurs when near surface air temperature is beneath a critical threshold (typically < 2 ◦C). Site-speciﬁc topoclimatic factors can also contribute to snow accumulation, such as avalanching from steep slopes and wind-drifting. Ablation of mountain glacier snow and ice occurs as surface melt, sublimation, erosion by wind, avalanching and lake-calving. Surﬁcial melt of snow and ice is the dominant process causing ablation on land-terminating glaciers in the temperate mid-latitudes (Cuffey and Paterson, 2010). Melt occurs when there is a net ﬂux of energy to the glacier surface and the temperature of the surface is at 0 ◦C (273.15 K). Most of the energy that contributes to melt comes from solar radiation and atmospheric heat content (Cuffey and Paterson, 2010; also see Chapter 3 for a detailed description of surface energy balance components). Energy exchanges at glacier surfaces can be represented by relatively simple, physical mod- els (Hock, 1999). In New Zealand, development and application of energy and mass balance models has shown that net radiation and turbulent heat exchanges provide roughly equal contributions to glacier melt, and that local glacier mass balance is most sensitive to temperature change (Anderson et al., 2010; Anderson and Mackintosh, 2012). Coupling mass balance models to ice-ﬂow models provides a means to quantify the palaeoclimatic information represented in historic and geological glacier length change records (e.g. Oerlemans, 2005; Anderson and Mackintosh, 2006; Doughty et al., 2013). The ﬂow of ice under gravity results in erosion and entrainment of sediment, which is transported down-valley and to the ice margins via a combination of supraglacial, englacial and/or subglacial pathways (Kirkbride, 1995). Erosion, modiﬁcation and deposition of sediment by glaciers creates distinctive sediment facies, landforms and landform assemblages, which can be preserved long after glacial retreat (Benn and Evans, 2010). Empirical observations of geomorphological process-form relationships in glacierised environments have classiﬁed common assemblages of glacial sediments and landforms at a variety of different glaciological settings (e.g. Evans, 2003). Chapter 2 ’What’s past is prologue.’ William Shakespeare (1611) Palaeoclimate (palaeo- from Greek palaios, meaning ’ancient’) research has two funda- mental aims: (i) to reconstruct, both qualitatively and quantitatively, the magnitude and timing of pre-historic changes in climatic variables; and (2) to understand the drivers and mechanisms of these changes. The former is achieved using preserved components of natural environmental systems that are strongly dependent on climate - known as climate proxies (e.g. see Alloway et al., 2007 for a review of key late-Quaternary climate proxy records in New Zealand). The latter increasingly uses numerical models that represent the systems of interest in sets of equations bound by physical and material laws. Combination of climate proxy reconstructions with physically-based models provides a powerful method to understand the workings, interactions and feedbacks of the Earth system and provide context for the rates and magnitudes of contempo- rary climate change (e.g. Masson Delmotte et al., 2013). Furthermore, quantitative palaeoclimate reconstructions from climatic states much different from today provide critical benchmarks for global climate models that are used to predict future change (e.g. Braconnot et al., 2012). Thus, whilst the present may be key to the past, the past can help illuminate the future. In this chapter, I ﬁrst present a short argument for the use of mountain glaciers as a palaeoclimate proxy. Following this, I review the present understanding of late Qua- ternary glacier ﬂuctuations in New Zealand. I then provide an overview of the climatic, glaciological and geological setting of Ruapehu and Tongariro volcanoes, including a review of existing knowledge of the local glacial history. From this review, I identify 7 CHAPTER 2. BACKGROUND CHAPTER 2. BACKGROUND 8 several research questions that are addressed in this thesis. several research questions that are addressed in this thesis. used to reconstruct past styles and extents of glaciation. used to reconstruct past styles and extents of glaciation. Almost all reconstructions of palaeoglacier geometries utilise moraines. ’Moraine’ is a general term that is used in glaciological and palaeoclimatic literature to describe both landforms and sediment facies transported and deposited by glaciers. In this thesis, I use moraine to describe discrete landforms composed of glacially transported sediment deposited at a former ice margin. Transport of sediment from glacier ice to outside of the glacier margins, to form moraines, occurs downstream of the equilibrium line (Benn and Evans, 2010). Where the geometry and mass balance of a particular glacier is in equilibrium with local climate, the continuation of moraine-building pro- cesses can build large (several 10-100s of metres high), linear moraines that delimit the ice margin. Sediment deposited by retreating glaciers forms less distinct and less readily preserved landforms due to the dynamic ice margin. Meanwhile advancing glaciers may override and destroy moraines. Thus, unlike continuous sedimentary archives (e.g. ice cores, tree rings, lacustrine/marine sediments), moraines preserved in the landscape represent a snapshot in time of a former glacial extent. Moraines depicting former ice margins are found on all continents of Earth (e.g. Ehlers et al., 2011), often at high altitude, in areas with few other opportunities for palaeoclimate reconstruction. Substantial progress in the ﬁeld of cosmogenic nuclides over the last two decades has resulted in a range of tools that are now widely accessible and widely applied to con- strain the age of glacial landforms (Balco, 2011). Thus, well-dated moraine sequences provide ideal targets for quantitative palaeoclimatic reconstruction. 2.1 Glaciers as a palaeoclimate proxy Identiﬁcation of preserved glacial landform assemblages, following glacier retreat, can therefore be . ORBITAL REVOLUTIONS 9 9 2.2. used to reconstruct past styles and extents of glaciation. 2.2 Quaternary palaeoclimate: a brief history of the or- bital revolution) The Quaternary is the most recent geological period (2.58 Ma - present; Cohen et al., 2013) and is characterised by cyclical shifts in the Earth’s climate between cold glacial- and warm interglacial-conditions. Glacial geomorphology provided some of the ﬁrst evidence for major climatic cooling in the recent geological past. In the early-mid 19th century, a group of European naturalists (Evans (1887) and Imbrie and Imbrie (1986) review the relative individual contributions) formulated the theory that expanded glaciation was responsible for the distribution of erratic boulders and polished, striated bedrock surfaces observed several kilometres down-valley of contemporary glaciers in the European Alps. This ’Ice Age Theory’ was formalised and widely promoted by Agassiz (1840), who discovered further evidence for former existence and greater extent of ice across parts of the Northern Hemisphere. Whilst this idea had been postu- CHAPTER 2. BACKGROUND 10 lated in various forms by geologists and naturalists for several decades, it remained controversial for two main reasons: (i) it challenged the prevailing ’ﬂood hypothesis’ of the time; and (ii) it lacked any accompanying theory or evidence that could explain the drivers and mechanisms of glacier advance (Imbrie and Imbrie, 1986). Orbital theory of the ice ages attributes past waxing and waning of continental ice sheets to cyclical variations in solar insolation caused by changing orbital geometries. Three main mechanisms are responsible for orbital forcing of the spatial and temporal patterns of insolation. Eccentricity (e; Equation 2.1) describes the deviation of Earth’s solar orbit from a perfect circle (deﬁned as e = 0). Currently, the Earth-Sun distance at aphelion and perihelion is similar and Earth’s orbit is close to circular (e = 0.0167). Variations in the gravitational forces imparted by other planets (mainly Jupiter and Saturn) cause Earth’s eccentricity to vary between a maximum of 0.058 and a minimum of 0.0034 (Figure 2.1A). This has periods of 100 kyr and 400 kyr and has the effect of lengthening summer (winter) in one hemisphere at the expense of summer (winter) in the opposite hemisphere. This oscillation also alters the total annual insolation, although only on the order of <0.2%. More importantly, Earth’s eccentricity cycle modulates the amplitude of the precessional cycle (see below). e = a −p a + p (2.1) (2.1) where a is the Earth-Sun distance at aphelion (currently c. 152 x 106 km) and p is the Earth-Sun distance at perihelion (currently c. 147 x 106 km). 2.2 Quaternary palaeoclimate: a brief history of the or- bital revolution) where a is the Earth-Sun distance at aphelion (currently c. 152 x 106 km) and p is the Earth-Sun distance at perihelion (currently c. 147 x 106 km). Obliquity describes Earth’s axial tilt relative to its orbital plane (currently 23.4◦), which varies by up to 2.4◦over a 41 kyr cycle (Figure 2.1B). This tilt causes the seasons and opposing insolation intensity between the hemispheres. Changes in tilt angle inﬂuence the spatial variation of insolation. Higher tilt results in higher solar radiation receipts at high latitudes. Precession is the change in orientation, or ’wobble’, of Earth’s rotational axis result- ing from the gravitational forces exerted on Earth by the Sun and moon. This cycle has dominant periodicities of 23 kyr and 19 kyr and has the effect of changing the occurrence of the equinoxes and solstices, relative to Earth’s position in its solar orbit. Thus, precession alters the timing of the seasons, relative to perihelion/aphelion. The amplitude of precessional forcing is dictated by the eccentricity cycle (above; Figure 2.2. ORBITAL REVOLUTIONS 11 Figure 2.1: (A) Axial eccentricity; (B) obliquity; (C) precessional index (esinϖ); and (D) mid-summer (Dec. 21) insolation intensity at 39 ◦S (lower panel). Values calculated using the code of Huybers and Eisenman (2006). Figure 2.1: (A) Axial eccentricity; (B) obliquity; (C) precessional index (esinϖ); and (D) mid-summer (Dec. 21) insolation intensity at 39 ◦S (lower panel). Values calculated using the code of Huybers and Eisenman (2006). 2.1A). This effect, together with changing obliquity, dictates changes in the frequency and amplitude of insolation, in time (e.g. see Figure 2.1C, lower panel) and space. Adhemar (1842) was the ﬁrst to call on orbital variations as the driver of past ice ages. He suggested that Agassiz’s glaciation was induced by lengthened boreal win- ters, caused by the precessionally-forced coincidence of Northern Hemisphere winter solstice with aphelion. This theory predicts anti-phased glacial cycles between the hemispheres and Adhemar used the present-day Antarctic ice sheets as evidence to support his idea. Adhemar’s theory was dismissed by Von Humboldt (1852) who stated that the reduced radiation receipts of winter are made up for by increases during summer as a result of the alignment with perihelion. Scottish geologist James Croll took up the orbital theory, building on the discovery that the eccentricity of Earth’s orbit changes with time (Leverrier, 1843). Extending the CHAPTER 2. BACKGROUND 12 eccentricity reconstruction to 3 Ma, Croll (1864) found evidence for a 400 kyr periodic- ity, which modulates the amplitude of the 100 kyr cycle. Combining his eccentricity calculations with precession, Croll (1864) hypothesised that glacial-interglacial cycles followed the 400 kyr cycle, which dictated when aphelion was far enough away to sufﬁciently reduce winter radiation receipts. During these ’glacial periods’, Croll be- lieved precession then prescribed which hemisphere experienced glaciation, through its control on the position of the solstices relative to the orbital plane. Unlike Adhemar (1842), this theory was based on the premise that the reduction in winter insolation intensity, rather than winter duration, is key for glacial inception. Croll (1864) further advanced this hypothesis by considering the feedbacks of continental glaciation on the Earth system. He suggested that increased snowfall would amplify the orbital impacts by increasing the surface albedo and that growing ice masses would disrupt atmospheric and oceanic circulation. Despite some mismatches between his theory and observations (see Sugden, 2014), the majority of Croll’s thinking was almost a century ahead of its time. However, the geochronological methods required to test his ideas about orbital forcing of glacial climates were not developed until the mid-20th century. Thus, despite support from eminent scientists of the time, including Charles Lyell and William Buckland, Croll’s theory could not be tested. In the early-mid 20th century, Serbian mathematician, Milutan Milankovitch, cor- rected and further reﬁned the calculations of his predecessors, adding in the effects of orbital obliquity (see above) and computing the energy balance to estimate the mag- nitude of past radiation change (Milankovitch, 1920, 1941). In contrast to Adhemar and Croll, Milankovitch considered changes in summer radiation receipts at high lati- tude as the main limiting factor for glacial development, due to the strong correlation with summer insolation and the snowline (Milankovitch, 1920; K¨oppen and Wegener, 1924). Milankovitch calculated that summer insolation at 65 ◦N can vary by 20 % when maximum obliquity and eccentricity coincide (e.g. see Figure 2.1, c. 200 ka). Like his predecessors, Milankovitch suffered from a lack of geochronological methods to test his hypothesis. Furthermore, geologists remained sceptical as orbital theory predicted many more ice ages than the four that were chieﬂy preserved in terrestrial glacial and ﬂuvial sequences (e.g. Penck and Br¨uckner, 1909). The development of radiometric geochronological methods (chieﬂy radiocarbon, and U-Th dating) and the extraction of deep-sea sediment cores provided chronologically- constrained, continuous records of palaeoenvironmental change of sufﬁcient length to overcome the preservation issues of terrestrial glacial and ﬂuvial records. Stable oxygen isotope ratios of marine foraminifera preserved in these cores predominantly reﬂect changes in global ice volume (Shackleton, 1967). Emiliani (1955) presented the ﬁrst such ORBITAL REVOLUTIONS 13 2.2. record from a core covering approximately the last 280 kyr, although he interpreted the δ18O signal to reﬂect changes in surface ocean temperatures, rather than ice volume. Nevertheless, this work agreed well with Milankovitch’s prediction of the last glacial maximum occurring c. 20 ka and the last interglacial at c. 100 ka. Further addition of marine cores that largely replicated the results of Emiliani (1955), as well as U-Th dating of coral reefs (Broecker, 1966; Broecker et al., 1968), and terrestrial proxy records such as loess accumulations (e.g. Kukla, 1970), added to the growing support for orbital theory as proposed by Milankovitch. The seminal work of Hays et al. (1976), using spectral analysis, showed the prominence of the precessional, obliquital and eccentrical periodicities in time series of foraminiferal δ18O spanning c. 0-300 ka. This provided conclusive evidence that orbitally-driven changes in insolation played a key role in late Pleistocene global ice volume changes. Extraction of ice cores from Greenland corroborated the broad pattern of cooling, at orbital timescales over the last glacial cycle (Dansgaard et al., 1969, 1982). Compari- son with early ice core records from Camp Vostok in Antarctica showed that the polar ice sheets in each hemisphere exhibit broad covariance in cooling and warming patterns over orbital timescales, which indicated glacial-interglacial cycles were global in extent (Epstein et al., 1970; Lorius et al., 1985; Jouzel et al., 1987). Later, more detailed work would show important differences between the hemispheres at shorter timescales (see Section 2.3). The location of Antarctic ice cores, situated distal to terrestrial CaCO3 dust ﬂuxes, also permitted measurement of atmospheric composition. These measurements showed covariance of Antarctic temperature and atmospheric CO2 (Barnola et al., 1987), which indicated that insolation effects may have been ampliﬁed by radiative forcing from atmospheric changes. This lead to the suggestion that changes in atmospheric greenhouse gases may explain the globally synchronous climatic changes between the hemispheres, despite out of phase insolation (Jouzel et al., 1987; see Section 2.2.1 below). 2.2.1 Outstanding questions in orbital theory - southern connections Discovery of orbital frequencies in palaeoenvironmental proxies by Hays et al. (1976) raised several important questions, and the precise role of insolation changes in driving global climate change over the Pleistocene remains uncertain (see Pierrehumbert, 2010). For example, why does the 100 kyr cycle dominate oxygen isotope spectra during the late Pleistocene, especially given the negligible impact of the 100 kyr eccentricity cycle on insolation (Imbrie et al., 1993)? Why are glacial cycles ’saw-tooth’ in shape (Broecker and Donk, 1970), with long (c. 70-90 kyr) periods of gradual cooling and abrupt returns to interglacial periods, dubbed ‘terminations’, over c. 10 kyr? What caused the shift CHAPTER 2. BACKGROUND 14 from 40 kyr to 100 kyr glacial cycles that occurred at c. 1 Ma? Why has ice volume in both hemispheres varied in broad synchrony, despite anti-phased changes in summer insolation (Broecker, 1978; Mercer, 1984; Lorius et al., 1985)? The latter question is per- tinent to the research presented in this thesis and I review the topic in more detail below. Over orbital timescales, changes in seasonal insolation intensity are anti-phased be- tween the hemispheres, due to the dominance of the precessional component. As a consequence, both Adhemar and Croll explicitly predicted asynchronous glacial advance-retreat cycles between the hemispheres (see above). However, it has been shown convincingly, using a multitude of climatic proxy reconstructions, that Southern Hemisphere climate varied in-phase with the north, over orbital timescales, during the late Pleistocene (Hays et al., 1976; Mercer, 1984; Denton et al., 1999; Petit et al., 1999; Vandergoes et al., 2005; Schaefer et al., 2006; Barrows et al., 2007a; Kawamura et al., 2007; Clark et al., 2009; Putnam et al., 2013b). What drives Southern Hemisphere glacial cycles, if not local summer insolation intensity? This conundrum has been termed a ’ﬂy in the insolation ointment’ (Broecker (1978), cited in Mercer, 1984). Resolving the mechanisms that synchronised the hemispheres over orbital timescales remains a major outstanding question in palaeoclimatic research, and a number of theories have been put forward. Several researchers have assumed that the close resemblance of southern climate proxy records to boreal insolation change implies a northern driver-southern response re- lationship. As such, an interhemispheric pathway, to synchronise the hemispheres, has been sought in the global atmospheric-oceanic system. For example, Imbrie et al. 2.2.1 Outstanding questions in orbital theory - southern connections (1992) and Gildor and Tziperman (2001) suggest cooling of North Atlantic Deepwater (NADW), due to growth of the northern ice sheets, is translated to the Southern Ocean via the thermohaline circulation, leading to increased stratiﬁcation and sea ice develop- ment. A major issue with these theories is that changes in southern proxy records are in phase with, or even lead, northern changes (e.g. Hays et al., 1976; Wolff et al., 2009), whilst translation of northern forcing through the oceans is thought to be subject to a signiﬁcant time lag. A separate agent, with strong potential for closely coupling the hemispheres, is at- mospheric carbon dioxide. Carbon dioxide is well mixed in the global atmosphere (Pierrehumbert, 2010), thus changes are felt instantaneously around the globe. Vari- ations in the atmospheric partial pressure of carbon dioxide (pCO2), as recorded in Antarctic ice cores, closely tracked Antarctic temperature change over the last 800,000 years (Petit et al., 1999; Siegenthaler et al., 2005; L¨uthi et al., 2008), reaching lows of c. 180 ppmv during peak glacial times and highs of c. 280 ppmv during interglacials. 15 2.2. ORBITAL REVOLUTIONS 15 Figure 2.2: Austral spring insolation intensity (blue), austral summer duration (deﬁned as days with average insolation > 250 W m−2) at 77◦S (blue) and boreal summer solstice intensity at 65◦N (black) for the last 1 Ma, calculated using the code of Huybers (2006). Figure 2.2: Austral spring insolation intensity (blue), austral summer duration (deﬁned as days with average insolation > 250 W m−2) at 77◦S (blue) and boreal summer solstice intensity at 65◦N (black) for the last 1 Ma, calculated using the code of Huybers (2006). An estimated 30-70% of temperature decline in the Southern Hemisphere at the LGM can be attributed to the radiative effects of atmospheric pCO2 reduction (Broccoli and Manabe, 1987; Lorius et al., 1990; Schneider von Deimling et al., 2006). It is likely that the ocean acted as the main sink for atmospheric pCO2 during glacial times, due to its large storage capacity (Sigman and Boyle, 2000). The prevailing view is that this oceanic drawdown occurred through increased utilisation of nutrients, phosphate and nitrate, by plants in the surface of the Southern Ocean (i.e. a more efﬁcient biological pump; e.g. Broecker, 1982). However, the driver of this change remains uncertain. 2.2.1 Outstanding questions in orbital theory - southern connections One possibility is that increased fertilisation of the surface ocean by iron input from raised dust ﬂux during peak glacial times facilitated this drawdown (Martin, 1990). However, this scenario is predicated on cooling having already occurred, which would facilitate greater meridional temperature gradients and increased windiness required to increase the land-ocean dust ﬂux (Broecker, 2013). More recently, Broecker (2013) proposed that drawdown of atmospheric pCO2 was the cause of glacial, rather than a consequence. Broecker outlines how density changes in high latitude water masses may result from orbital forcing, which would lead to increased ocean stratiﬁcation and utilisation of phosphate. Precisely resolving the leads-lags between pCO2 and temperature change (e.g. Shakun et al., 2012) for periods before the last termination will provide critical tests for determining the role of pCO2 in glacial cycles. CHAPTER 2. BACKGROUND 16 Alternative theories invoke components of local insolation as drivers of austral glacial cycles. Huybers (2006) argued that insolation integrated over a given intensity thresh- old (related to the sign of surface temperature) is a more relevant index for glacier mass balance, due to the strong relationship between positive degree days and glacier melt (e.g. Ohmura, 2001). Using radiative equilibrium theory, Huybers and Denton (2008) show that radiative equilibrium temperature is more sensitive at lower atmospheric temperatures. Thus, higher temperatures result from weaker, but longer duration summers, as occurs when summer solstice aligns with aphelion. They argue that this is the dominant driver of temperature change in Antarctica at orbital timescales, as feedbacks (e.g. albedo, surface elevation) associated with ice sheet growth and decay are less relevant at the polar-centric Antarctic ice sheets. In contrast, summer insolation intensity is key in the north where ice sheets reached lower latitudes and radiation contributes directly to surface melt and the feedbacks associated with ice sheet growth/decay strongly dictate local temperature. Austral summer duration covaries with northern summer intensity (e.g. Figure 2.2), therefore providing a means of synchronising climate between the hemispheres at orbital timescales (Huybers and Denton, 2008). At the culmination of the LGM, surface temperatures in West Antarctica began rising several millennia prior to those in East Antarctica (Blunier and Brook, 2001; Fudge et al., 2013). This occurrence is interpreted to represent a response to the early break up of sea ice (e.g. 2.3 Millennial-scale oscillations Superimposed on orbital scale glacial cycles, are high amplitude, millennial-scale oscil- lations. These changes occur over timescales too short to have been driven by changes in orbital geometry, therefore they most likely represent internal feedbacks in the global atmospheric-oceanic system. First recognised in Greenland ice cores (Dansgaard et al., 1982, 1984), such millennial-scale oscillations are present in continuous palaeoclimatic proxy records globally (e.g. Arz et al., 1998; McManus et al., 1999; Blunier and Brook, 2001; Wang et al., 2001, 2004), although the shape and timing of these changes differs markedly between the hemispheres (Blunier and Brook, 2001). In Greenland ice cores, 18O/16O (δ18O) ratios indicate abrupt warming events, (> 10◦C over 3-4 decades), which occurred quasi-periodically throughout the last glacial cycle (Dansgaard et al., 1993). Each warming event was followed by slow return to pre-warming conditions lasting c. 1-3 kyr and is known as a ’Dansgaard-Oeschger’ (D-O) cycle (Figure 2.3). Synchronising Antarctic and Greenland ice core records, using methane concentrations in air bubbles, Blunier and Brook (2001) showed that temperatures in Antarctica tend to rise steadily during cold intervals in Greenland and decrease steadily during boreal warm intervals (Figure 2.3). This is best illustrated during the late-glacial chron (15-11 ka). At the onset of the Antarctic Cold Reversal (c. 14.6 ka), deglacial temperature rise in Antarctica reversed, which coincided with rapid warming in Greenland (D-O event 1; Figure 2.3). Warming then resumed in Antarctica when Younger Dryas cooling prevailed in Greenland some 1800 yr later (Figure 2.3). It is widely believed that these millennial scale oscillations, of opposite sign between the hemispheres, represent the spatial redistribution of heat brought about by changes in ocean circulation, although the ultimate driver(s) remain unclear. The bi-polar seesaw hypothesis invokes variability in NADW formation as the mechanism that generates opposing changes in the south (Crowley, 1992; Broecker, 1998). Reduced Atlantic meridional overturning circulation (AMOC) decreases the northward ﬂux of warm surface waters from low-latitudes causing regional cooling in boreal high-latitudes. At the same time, reduced heat export from the South Atlantic leads to local warming (Crowley, 1992). Barker et al. (2009) showed that millennial-scale variability in the South Atlantic exhibits similar timing and rates of opposing change to that of the North Atlantic, thus supporting the theory that the Atlantic basin served as the gateway for interhemispheric transmission of abrupt climate change during the last termination. 2.2.1 Outstanding questions in orbital theory - southern connections Allen et al., 2011; Collins et al., 2012) due to increasing summer duration, which facilitated advection of warmer air masses into the lower elevation ice surfaces in West Antarctica (Fudge et al., 2013). The onset of warming at in West Antarctica also coincides with increasing austral (63◦S) spring insolation. Timmermann et al. (2009) argue that this is an important pacemaker for southern glacial cycles through two mech- anisms: (i) controlling the availability of heat for advection to the Antarctic continent; and more importantly, (ii) controlling ventilation of CO2 from the Southern Ocean. Thus, although there are plausible local drivers of southern climate over glacial cycles, it is difﬁcult to discriminate between different theories when they share essentially the same pattern of change (Huybers, 2009; Figure 2.2). Palaeoclimatic research can inform this debate in two main ways: (i) providing in- creased number of spatially distributed, well-dated quantitative palaeoclimate recon- structions, particularly prior to the LGM, in order to assess the timing and magnitude of change; and (ii) using this information to evaluate global climate model experiments that test speciﬁc hypotheses about possible drivers and mechanisms of past climate change (e.g. Braconnot et al., 2007; He et al., 2013). 2.3. MILLENNIAL-SCALE OSCILLATIONS 17 2.3 Millennial-scale oscillations The Southern Ocean is thought to be slower (on the order of centuries to millennia) to respond to such changes due to thermal inertia and the absence of landmasses to support wave propagation of density anomalies (Stocker and Johnsen, 2003). Stocker 18 CHAPTER 2. BACKGROUND Figure 2.3: (a) GISP2 oxygen isotope record, showing D-O events (numbered); (b) Atmospheric pCO2 from Antarctica (Monnin et al., 2001; Ahn and Brook, 2008); (c) EPICA Dome C deuterium record (EPICA Community Members, 2004), with the Antarctic warming events originally identiﬁed by Blunier and Brook (2001). Vertical grey bars indicate Heinrich events (Heinrich, 1988) using the chronology of Hemming (2004). Figure 2.3: (a) GISP2 oxygen isotope record, showing D-O events (numbered); (b) Atmospheric pCO2 from Antarctica (Monnin et al., 2001; Ahn and Brook, 2008); (c) EPICA Dome C deuterium record (EPICA Community Members, 2004), with the Antarctic warming events originally identiﬁed by Blunier and Brook (2001). Vertical grey bars indicate Heinrich events (Heinrich, 1988) using the chronology of Hemming (2004). and Johnsen (2003) suggest that this muted response of the Southern Ocean explains the different temporal characteristics of millennial-scale temperature changes in Antarctica, relative to Greenland (e.g. Figure 2.3). Several studies have also highlighted the potential role of atmospheric changes for interhemispheric transmittance of abrupt climate changes (Wang et al., 2001; Ander- son et al., 2009; Denton et al., 2010). Changes in equator-pole temperature gradients associated with the changes in winter sea ice extent (e.g. Denton et al., 2005; Ander- son et al., 2009) result in latitudinal displacement of the thermal equator (Wang et al., 2001) and mid-latitude westerlies (Moreno et al., 1999; Toggweiler, 2009). Denton et al. (2010) suggest that southward movement of the southern westerlies during Greenland stadials would help facilitate austral warming via the break-up of sea ice, increased upwelling of warm deep water, and increased southward eddy-driven transport of heat. 2.4. LATE QUATERNARY GLACIATION IN NZ 19 The Bølling-Allerød (Antarctic Cold Reversal) warming (cooling) event in the Northern (Southern) Hemisphere at c. 14.6 ka is a well-studied example of one such millennial scale climate event. Cessation of freshwater forcing in the North Atlantic at the end of Heinrich stadial 1 represents one hypothesised trigger for the resumption of oceanic overturning this time (Liu et al., 2009). Far-ﬁeld sea-level proxy records indicate a rise of c. 2.3 Millennial-scale oscillations 14-18 m in < 500 yrs (14.65-14.31 ka; Meltwater Pulse 1A - MWP1A), which indicates a signiﬁcant freshwater ﬂux to the global ocean coincided with resumed Atlantic overturning (Deschamps et al., 2012). Geological evidence and glaciological modelling suggests northern ice sheets contributed a substantial proportion of MWP1A (Carlson and Clark, 2012; Tarasov et al., 2012). However, a freshwater pulse to the North Atlantic is not consistent with concomitant abrupt intensiﬁcation of AMOC, under the bipolar seesaw model. Weaver et al. (2003) show that freshwater forcing of equivalent magnitude to MWP1A applied to the Southern Ocean provides a viable trigger to restart Atlantic deep water formation and replicate the spatial pattern of northern warming (southern cooling) observed in proxy records at that time. Using an Earth system model, Menviel et al. (2011) show that a combination of southern freshwater forcing and North Atlantic overturning may be jointly responsible for this abrupt climatic transition. However, the proportion of MWP1A derived from Antarctica remains uncertain. Recent geological evidence shows evidence for increased Antarctic discharge at that time (Weber et al., 2014), although glaciological model simulations suggest this may have been a response to resumed AMOC (Golledge et al., 2014). Fur- ther addition of well-dated quantitative palaeoclimatic proxy reconstructions will help to constrain climate model simulations that seek to resolve the drivers and mechanisms of past abrupt climate change over the last glacial cycle. 2.4.1 Pre-Last Glacial Maximum (c. 125 - 35 ka) Evidence for glaciation early in the last glacial cycle is limited and often restricted to exposures of glacioﬂuvial sedimentary sequences, usually found downstream of former ice limits. Using a variety of luminescence techniques to date glacioﬂuvial aggradation episodes in northern Westland, Preusser et al. (2005) constrain pre-LGM glacial advances to c. 111 ka, c. 85 ka and c. 64 ka. East of the Main Divide in Rakaia valley, Shulmeister et al. (2010b) document evidence for glacier ﬂuctuations within a thick sedimentary sequence of glacial - glacioﬂuvial deposits. Using infrared stimulated luminescence dating they constrain the timing of glacier advances to 100 - 90 ka, c. 80 ka, c. 48 ka and c. 40 ka. Luminescence ages from these two studies have uncertainties of 10-20 %. Sutherland et al. (2007) present a cosmogenic 10Be chronology for a series of moraines that document glacier extension of similar magnitude at 4 separate occasions in Cascade valley, southwest New Zealand. In the original paper Sutherland et al. (2007) suggest these moraines formed at 19-22 ka, 58 ± 3 ka, 79 ± 4 ka and 117 ± 4 ka, which appears to correlate with minima in local mid-summer insolation intensity. Sutherland et al. (2007) interpret 4 outliers that cluster at c. 30 ka as representing the onset of deteriorating climatic conditions leading into the LGM. Recalculating these ages with the revised, local cosmogenic 10Be production rate of Putnam et al. (2010b) increases the exposure dates by an average of 17-18 %, which produces moraine ages of 22-25 ka (CA4/CA5), 68 ± 4 ka (CA3 younger), 93 ± 2 ka (CA3 older; wtd. mean, n=5), and 138 ± 6 ka (CA2 younger). The outliers now cluster at c. 35 ka, instead of 30 ka. There are few things to be noted about the revised Cascade valley moraine ages and their relationship to changing insolation patterns. Firstly, the work of Putnam et al. (2010b) represents a calibration of cosmogenic 10Be production rate integrated over the last c. 10 ka, and they also demonstrate good agreement of this revised production rate with independent radiocarbon ages over the last 18 ka. Thus, whilst application of this new production rate to exposure ages from the LGM and younger is robust, application to older features becomes increasingly uncertain. Secondly, all of the recalculated ages of Sutherland et al. 2.4 The late Quaternary glacial history of New Zealand (c. 125 - 10 ka) The record of glaciation in New Zealand dates back to at least the early Pleistocene (Suggate, 1990). However, the active tectonic setting and high erosion rates mean that very little evidence of glacial ﬂuctuations prior to the late Pleistocene is preserved in the modern landscape. Several reviews exist that draw together the fragmentary records of glacial geology and place these within local- to regional- stratigraphic and palaeoclimatic frameworks (Gage and Suggate, 1958; Gage, 1985; Suggate, 1990; Barrell, 2011). In this section, I review evidence for past glacier ﬂuctuations in New Zealand over the last glacial cycle (c. 125 ka to present) with a focus on geochronology and the debates surrounding the associated climatic drivers. CHAPTER 2. BACKGROUND CHAPTER 2. BACKGROUND 20 2.4.1 Pre-Last Glacial Maximum (c. 125 - 35 ka) (2007) are presented assuming no erosion of the sampled surface, as was the protocol in the original paper. The study site experiences high total annual precipitation (c. 5-6 m yr−1; Tait et al., 2006) and it is unlikely that the older samples in this dataset have remained pristine through successive stadial-interstadial cycles. Inclusion of conservative erosion rates impacts signiﬁcantly on the exposure ages of the 21 2.4. LATE QUATERNARY GLACIATION IN NZ 21 older moraines in this dataset and the possible correspondence with potential orbital drivers. For example, assuming erosion of 3 mm kyr−1 (sensu Barrows et al., 2001) would place the CA3 older moraine in the peak glacial conditions of marine oxygen isotope stage (MIS) 6 (c. 130 ka). There is increasing evidence for a major glacial maximum in New Zealand during late MIS 4. In the central Southern Alps, Schaefer et al. (2015) present 50 cosmogenic 10Be surface exposure ages that show the former Pukaki glacier reached its most extensive position of the last glacial cycle at 65 ± 3 ka. This coincides with the evidence presented by Preusser et al. (2005) (above), although recent cosmogenic surface exposure dating indicates that glacier advances in northern Westland were most extensive during MIS 2 (Barrows et al., 2013). In the Tasman mountains, northern South Island, McCarthy et al. (2008) report a basal OSL age of 65 ± 10 ka from glaciolacustrine sediment, which indicates glaciation in late MIS 4 that was slightly greater in extent than at the LGM. Glaciation in this region at this time is also supported when a single cosmogenic 10Be exposure age of Thackray et al. (2009) is recalculated according to Putnam et al. (2010b). Thackray et al. (2009) report cosmogenic 10Be exposure ages of 53 ± 6 ka and 32 ± 4 ka from separate roche moutonee on Mt. Arthur tableland. The ages are increased by 11-13 % by the recalculation, which suggests glacier retreat from these locations at 60 ± 2 ka and 36 ± 2 ka, respectively. There is very little evidence to constrain glacial extent early in MIS 3 (c. 45-60 ka), which suggests that glaciers were less extensive than MIS 2 and MIS 4 advances. In southern Westland, Almond et al. (2001) constrain the timing of glacial advance to between 45-50 ka. This agrees with the 48 ± 3 ka luminescence age obtained by Shul- meister et al. 2.4.1 Pre-Last Glacial Maximum (c. 125 - 35 ka) (2010b) for deposition of an ice-contact debris fan in Rakaia valley, which they interpret as relating to an advancing ice mass. However, ice geometries for these periods remain unconstrained. Recently, Kelley et al. (2014) have shown that the former Pukaki glacier extended into the Pukaki valley at c. 42 ± 1 ka. Few records exist from other catchments to indicate major glaciation at this time. 3. When did deglaciation begin and how rapid was it? 3. When did deglaciation begin and how rapid was it? The onset of cooling in New Zealand leading into the Last Glacial Cold Period (c. 28-18 ka; Alloway et al., 2007; Barrell et al., 2013; LGCP) remains relatively poorly constrained and glacial chronologies for this period are only available from a few sites. Putnam et al. (2013b) have shown that the former Ohau glacier, in central Southern Alps, reached a similar extent to that of the LGCP by 33 ± 1 ka. This early onset of glaciation is further supported by the ﬁndings of Rowan et al. (2012), who present OSL and radiocarbon ages that constrain aggradation of glacioﬂuvial gravels on the Canterbury Plains to two periods, c. 37 - 31 ka and c. 24 - 19 ka. Detailed stratigraphic investigation of moraine coverbeds in northern Westland, combined with radiocarbon dating, led Suggate and Almond (2005) to suggest the earliest advance (their la1/M51) began at c. 34 ka. How- ever, recent surface exposure dating by Barrows et al. (2013) found no evidence for glacier advance prior to 30 ka, or moraine formation prior to c. 27 ka. The majority of glacial chronologies indicate that mountain glaciers in New Zealand attained their maximum extent of the last glacial cycle at c. 28 ka. For example, Kelley et al. (2014) show that the moraines immediately inboard of the 42 ka feature at Lake Pukaki date to 28 ± 1 ka. In the Rangitata and Ashburton catchments, Rother et al. (2014) show that large outlet glaciers attained their maximum extent of the last glacial cycle at c. 28 ka, and subsequently ﬂuctuated inboard of this dated limit. Shulmeister et al. (2010a) (recalculated after Putnam et al., 2010b, by Putnam et al., 2013a) ﬁnd a similar age for outer LGM moraines in the Rakaia valley, which were deposited at c. 28-26 ka. In summary, there is evidence to suggest that glacier advance began prior to c. 33 ka, however moraines of this age are rare, which indicates that subsequent advances were most likely of equal or greater extent. Subsequent to the 33 ka moraine formation at Lake Ohau, Putnam et al. (2013b) show that discrete moraines were emplaced immediately inboard, at 27 ± 1 ka, 23 ± 1 ka and 18 ± 1 ka. Similarly, Kelley et al. (2014) and Schaefer et al. CHAPTER 2. BACKGROUND CHAPTER 2. BACKGROUND 22 2.4.2 Last Glacial Maximum (c. 35 - 18ka) There are three main outstanding questions concerning glacier advances in New Zealand during the Last Glacial Maximum: 1. When did glaciers ﬁrst reach their maximum extent during this stadial? 1. When did glaciers ﬁrst reach their maximum extent during this stadial? 2. What is the timing and magnitude of glacier ﬂuctuations between 30-18 ka? 2. What is the timing and magnitude of glacier ﬂuctuations between 30-18 ka? CHAPTER 2. BACKGROUND 3. When did deglaciation begin and how rapid was it? (2015) document moraine formation at 28 ka, 21 ka and 18 ka at nearby Lake Pukaki. The youngest recalcu- lated ages of Sutherland et al. (2007) indicate that former Cascade alley glacier was extended between c. 29 - 22 ka. In northern Westland, Barrows et al. (2013) show that glaciers deposited moraines on the coastal lowlands at c. 25 ± 1 ka, 21-20 ka and 17 ± 1 ka. In the Rangitata and Ashburton catchments, east of the Main Divide, Rother et al. (2014) show that, between 19-16 ka, the former outlet glaciers were immediately inboard of the 28 ka limits. In summary, it is clear that glaciers across the Southern Alps ﬂuctuated about their extended positions between c. 28 and 18 ka. The recent cos- mogenic surface exposure dating campaigns summarised above have served to afﬁrm 2.4. LATE QUATERNARY GLACIATION IN NZ 23 the general stratigraphic model of previous investigations (e.g. Suggate and Almond, 2005) that suggests glaciers advanced between 30-28 ka, and subsequently reached a similar extent between 23-18 ka. Whilst well-preserved moraine belts record the culmi- nation of some advances during this period, it is unclear how much retreat occurred between moraine forming events. Furthermore, the chronological imprecision of sur- face exposure dating of moraines (mostly due to processes causing geological scatter), means that it is difﬁcult to correlate minor ﬂuctuations across catchments with certainty. Modelling of the entire LGM Southern Alps ice ﬁeld indicates that equilibrium line altitude (ELA) depressions were normally distributed (c. 500 to 1200 m) about a mean of c. 840 m below present (Golledge et al., 2012). ELA depression was greatest in high- precipitation catchments, west of the Main Divide in central Southern Alps, and lower in drier, eastern regions. Manual ELA reconstructions from individual catchments generally fall within this range (e.g. Porter, 1975; McCarthy et al., 2008), although some reconstructions from small cirque glaciers indicate much greater lowering (e.g. Brook et al., 2008; Brook, 2009). Golledge et al. (2012) found a best ﬁt to identiﬁed LGM limits when modern climate was 6-6.5 ◦C cooler and c. 25 % drier than present. This is in good agreement with similar applications at individual catchment scale that typically indicate a cooling of 5-7 ◦C in central Southern Alps (McKinnon et al., 2012; Putnam et al., 2013b; Rowan et al., 2013). 3. When did deglaciation begin and how rapid was it? Currently there are two main schools of thought concerning the onset and rate of deglaciation in New Zealand at the end of the LGCP. Moraine chronologies from large outlet glaciers in central and western Southern Alps indicate rapid withdrawal of ice beginning at c. 17-18 ka. For example, on the west coast, Barrows et al. (2013) show that the former Taramakau glacier withdrew rapidly from the Moana Formation moraines at 17.3 ± 0.5 ka. The former Pukaki glacier retreated from the present-day, southern lake margin at c. 18 ka (Putnam et al., 2010b; Kelley et al., 2014; Schaefer et al., 2015) and subsequently thinned by c. 400 m prior to 16.2 ± 0.9 cal. ka (Barrell and Read, 2014). Similarly, Putnam et al. (2013b) show that the terminus of the neighbouring Ohau glacier withdrew from the southern shoreline of present day lake shortly after 18 ka. Cosmogenic 10Be surface exposure ages from a glacially abraded bedrock knob situated c. 24 km upstream of the southern lake margin, indicate that the Ohau glacier thinned vertically by c. 200 m within c. 300 yr of the onset of retreat. Further to the east, abundant moraines and glacially abraded bedrock outcrops in Rakaia valley afford a more detailed investigation of glacial retreat. Putnam et al. (2013a) show that the glacier terminus withdrew from the innermost LGM moraines at c. 17.8 ± 0.2 ka and retreated c. 58 km up valley over the following c. 2 kyr. This directly contrasts with the earlier results of Shulmeister et al. (2010a), who had suggested the Rakaia glacier retained as CHAPTER 2. BACKGROUND 24 much as 90% of its LGM length until c. 15 ka. Adjusting for different production rates resolves some of the age discrepancies between these two studies, but Putnam et al. (2013a) draw on a far larger and more robust dataset in their rejection of delayed glacier retreat in the Rakaia catchment. In contrast to the aforementioned ﬁndings, Rother et al. (2014) ﬁnd that the former Rangitata glacier, situated c. 25 km south of Rakaia valley, retained up to 80% of its LGM length until c. 15.8 ± 0.4 ka. Rother et al. (2014) argue that the retreat of former outlet glaciers at Pukaki, Ohau and Rakaia was signiﬁcantly inﬂuenced by the formation of proglacial lakes, which may have decoupled glacier mass balance from atmospheric forcing and caused rapid retreat through calving. 3. When did deglaciation begin and how rapid was it? However, this theory does not explain why the Taramakau glacier retreated rapidly from the coastal lowland to the Main Divide at 17.3 ka (Barrows et al., 2013). Nor does it provide an explanation for a deglacial chronology of former valley glacier in northwest South Island (Shulmeister et al., 2005) after recalculation using the updated local cosmogenic 10Be chronology of Putnam et al. (2010b). Shulmeister et al. (2005) present a 10Be chronology from moraine boulders and glacially-abraded bedrock exposures from Cobb valley. Twenty-one ages from 9 sites show good consistency (the authors identify 5 outliers, which fall outside of the arithmetic mean), indicating glacier retreat over a distance of c. 21 km was rapid at this location. Recalculating the 10Be ages according to Putnam et al. (2010b) raises the arithmetic mean of the dataset from 16.4 ± 2.4 ka, to 19.5 ± 3.1 ka (or 19.7 ± 1.5 ka, with the 5 outliers removed). In the original paper, Shulmeister et al. (2005) conclude that: (1) glacier retreat began no earlier than 20 ka; (2) thinning and retreat was well underway by 18 ka; and (3) that the glacier ceased to exist sometime between 14-15 ka. Following recalculation and outlier removal, the ﬁrst two conclusions may still hold true, however there is little evidence to support the claim that the Cobb valley glacier existed post c. 18 ka. Recalculated exposure ages from moraine boulders and bedrock situated 4-15 km downstream from the valley head are indistinguishable suggesting rapid disappearance of this glacier between 18-19 ka. Thus, the glacial chronology from Cobb valley best ﬁts the deglaciation scenario favoured by Putnam et al. (2013a) and Putnam et al. (2013b). The reservoir present in Cobb valley today is man made and Shulmeister et al. (2005) report no evidence for past, natural lake formation over the c. 20 km of valley ﬂoor mapped. Thus it would appear that the Rangitata record is somewhat anomalous amongst deglacial chronologies in New Zealand. 2.4.3 Late-Glacial (15 - 11.5 ka) The occurrence, precise timing and climatic signiﬁcance of glacial advance in New Zealand during the late-glacial chron (c. 15 - 11.5 ka) has proved to be a controver- 25 25 2.4. LATE QUATERNARY GLACIATION IN NZ Figure 2.4: Probability density plots of cosmogenic 10Be exposure ages of Shulmeister et al. (2005) (red) recalculated according to Putnam et al. (2010b) (blue). Outliers identiﬁed by Shulmeister et al. (2005) shown as dashed lines. Figure 2.4: Probability density plots of cosmogenic 10Be exposure ages of Shulmeister et al. (2005) (red) recalculated according to Putnam et al. (2010b) (blue). Outliers identiﬁed by Shulmeister et al. (2005) shown as dashed lines. sial topic. Fitzsimons (1997) reviewed the chronological understanding of late-glacial glacier advance in New Zealand and concluded that several instances of moraines situated between LGM and neoglacial limits had been identiﬁed, but precise chrono- logical control was lacking. For example, glacial tills have been assigned late-glacial ages in Rakaia valley (11.9 - 10 14C ka BP - Burrows and Russell, 1975), Cropp River (10.3 14C ka BP - Basher and McSaveney, 1989) and the Wairau valley (11.3 - 9.1 14C ka BP McCalpin, 1992) based on bracketing radiocarbon dates, in combinations with clast weathering rind thicknesses. On the west coast of South Island, the Waiho Loop moraine rises > 100 m above the Franz Josef Glacier foreland, c. 11 km from the modern glacier snout, and has long been identiﬁed as evidence for late-glacial glacier advance in New Zealand. How- ever, despite many attempts at chronological constraint, the precise timing of moraine formation remains enigmatic. Early attempts at radiocarbon dating wood fragments within glacial till at Canavans Knob, a bedrock outcrop c. 2 km upstream of the Waiho Loop moraine, yielded a range of dates between 11.3 - 12.6 14C ka BP, which were interpreted to represent pre-Younger Dryas glacier advance (Wardle, 1978; Mercer, 1988). Comprehensive re-assessment by Denton and Hendy (1994) placed the timing of glacier advance at 11.1 14C ka BP, leading to a conclusion of coeval glacier advance CHAPTER 2. BACKGROUND 26 between New Zealand and Europe during the Younger Dryas. Turney et al. (2007) note the relatively wide range of dates (11.5-10.7 14C ka BP) produced in the aforementioned studies from Canavans Knob, and use a χ2 test to demonstrate potential contamination of the sample population. 2.4.3 Late-Glacial (15 - 11.5 ka) New dates from a highly weathered Weinmannia racemosa fragment yield an error weighted mean of 11641 ± 16 14C a BP, while a well preserved Metrosideros cf. umbellata dates to 11062 ± 30 14C a BP (Turney et al., 2007). It was concluded that the older sample represents reworking of older wood fragments into the Canavan Knob sequence, likely causing the wide spread of ages seen in previous studies. Furthermore, the well-preserved Metrosideros sample was interpreted to rep- resent tree-growth contemporaneous with late glacial advance of Franz Josef Glacier, therefore providing a maximum age. Calibration of this age (c. 13.1 cal. ka BP) places the time of Franz Josef Glacier advance before the onset of the Younger Dryas, therefore Turney et al. (2007) favour a scenario in which the till at Canavans Knob represents an advance of Franz Josef Glacier at the culmination of the ACR. The glacial till at Canavans Knob cannot be stratigraphically connected to the Waiho Loop moraine, thus the two may not be contemporaneous. Barrows et al. (2007b) present a combination of 4 36Cl and 9 10Be ages from boulders on the moraine surface, which yield an error weighted mean of 10.5 ± 0.2 ka suggesting early Holocene ice retreat from this location. They conclude that the Franz Josef Glacier: (i) was not responding to the same climatic forcing driving advance of European glaciers during the Younger Dryas; and (ii) may have remained extended down-valley for over two millennia after advancing over Canavans Knob, which potentially explains the large volume of the moraine. However, Applegate et al. (2008) challenge this age interpreta- tion using a numerical model of moraine diffusion and cosmogenic nuclide production. They suggest that the samples analysed by Barrows et al. (2007a) do not have consistent exposure histories, with some boulders having been shielded from cosmogenic rays since deposition, therefore a weighted mean age provides a date that is younger than the true age of glacial retreat. Further analysis of this concept by Applegate et al. (2012), using the recalculated ages of Barrows et al. (2007a) after Putnam et al. (2010b), yields a best-ﬁt age of 13.0 ka for the Waiho Loop. Recent work by Barrows et al. (2013) has implied that previous cosmogenic 36Cl production rates were too high. This would support an older age for the Waiho Loop than that presented in Barrows et al. (2007a). 2.4.3 Late-Glacial (15 - 11.5 ka) Some studies have questioned whether the advance of Franz Josef Glacier to Waiho Loop was driven by climatic change, as suggested in a numerical modelling study by Anderson and Mackintosh (2006). Tovar et al. (2008) suggest the lithological compo- sition of the moraine is restricted to angular low-grade metamorphosed sandstone, which only outcrops in a small portion of the catchment. They suggest a rock avalanche 2.4. LATE QUATERNARY GLACIATION IN NZ 27 emplaced on the glacier surface may have decoupled glacier mass balance from local climate and induced glacier advance. However, subsequent glacier modelling analyses have questioned whether this scenario could produce a discrete moraine ridge such as the Waiho Loop (Vacco et al., 2010), or whether a rock avalanche is capable of producing terminus advance of the required magnitude (Alexander et al., 2014). Most recently (Alexander et al., 2014) suggest, based on seismic surveys and ﬂowline modelling, that the Waiho Loop is the product of ice stagnation due to an overdeepened trough. A non-climatic driver for late-glacial advance of Franz Josef Glacier is further weakened by the increasing evidence for synchronous, widespread glacier advance across the Southern Alps at this time. For example, Putnam et al. (2010a) present 24 cosmogenic 10Be exposure ages from the well-preserved Birch Hill moraines in the Pukaki valley, in central South Island. They ﬁnd that the onset of rapid late-glacial glacier retreat was initiated at 13.0 ± 0.3 ka, with > 150 m of glacier thinning in the following few centuries. Putnam et al. (2010a) also present 3 10Be ages from the Macaulay catchment c. 50 km to the NE, indicating glacial retreat from 13.3 ± 0.3 ka, which is synchronous, within error, with the Birch Hill data. Kaplan et al. (2010) report a similar situation of glacier retreat during the Younger Dryas in the Irishman Stream cirque, located 20 km SE of the Birch Hill moraine complex. Cosmogenic 10Be exposure ages from a series of recessional moraine ridges record the retreat of a small cirque glacier beginning at 13.0 ± 0.5 ka, with minor stillstands at 12 ka and 11.5 ka. Recalibrating and combining the Canavans Knob till ages of Denton and Hendy (1994) and Turney et al. (2007), Putnam et al. (2010a) ﬁnd good agreement with cosmogenic moraine chronologies and conclude that glaciers in four separate catchments advanced synchronously, in an event that culminated at c. 13 ka. 2.4.3 Late-Glacial (15 - 11.5 ka) More recently, Kaplan et al. (2013) ﬁnd that a glacier in Whale Stream, in the Ben Ohau range, which is proximal to both the Birch Hill and Irishman Stream sites, was also extended during the late-glacial. Cosmogenic 10Be exposure ages of moraines in this catchment show that the former glacier attained its greatest extent early (15-14 ka) in the Antarctic Cold Reversal, and subsequently underwent gradual retreat through the Younger Dryas and Holocene. Further to the north, a series of stratigraphically-correlated late-glacial moraine ridges have been described lining the N-S trending head of the Bealey/Otira valley at the Main Divide of the Southern Alps, close to Arthurs Pass (Chinn, 1981). In the only study to address the chronology in this valley using absolute dating techniques thus far, Ivy Ochs et al. (1999) derive cosmogenic 10Be exposure ages from latero-frontal moraines situated inboard of Lake Misery. This complex is believed to have been deposited by an eastward ﬂowing glacier sourced from the adjacent Otira valley. Four moraine boulder ages yield a weighted mean age of 11.7 ± 0.3 ka, which led Ivy Ochs et al. (1999) to CHAPTER 2. BACKGROUND 28 support Denton and Hendy’s (1994) conclusion of Younger Dryas glacier response in New Zealand. More recently, Barrows et al. (2007b) recalculated these ages using the CRONUS cosmogenic 10Be production rate (Balco et al., 2008), which placed the retreat of late-glacial ice from Lake Misery at 13.4 ± 0.6 ka, towards the latter stage of the Antarctic Cold Reversal and prior to the Younger Dryas. Barrows et al. (2013) further revise these ages based on Putnam et al. (2010b) and report a mean exposure age of 16.1 ± 0.8 ka (using Lifton et al., 2005 scaling, and NZ3 outlier removed), which places the formation of these moraines prior to the Antarctic Cold Reversal. Using the parameters reported by Ivy Ochs et al. (1999), the ’S555’ beryllium standard (S.Ivy Ochs, pers. comm., 2014) and the ’Li’ model, I can not replicate this recalculated age. Instead, I derive a mean age of 14.8 ± 1.6 ka (n = 5), and 15.4 ± 0.8 ka when sample NZ3 is removed. Using the ’Lm’ scheme, which has been shown to outperform the ’Li’ model (Lifton et al., 2014), yields arithmetic mean ages of 14.6 ± 1.6 ka (n = 5) and 15.2 ± 1.6 ka, respectively. 2.4.3 Late-Glacial (15 - 11.5 ka) It is therefore possible the Misery moraines correlate to the oldest ACR moraine found in Whale Stream by Kaplan et al. (2013), however the low number of samples and the relatively low precision of these early 10Be measurements makes it difﬁcult to discriminate between the latter scenario and the alternative suggested by Barrows et al. (2013). Revisiting this site with the aim of producing a greater number of exposure ages at the precision now afforded by accelerator mass spectrometry could reconcile this existing uncertainty. In summary, recent developments in the ﬁeld of cosmogenic surface exposure dat- ing have been applied across several sites in the central Southern Alps, which have shown that glaciers advanced early in the late-glacial chron, coinciding with the Antarc- tic Cold Reversal. Numerical glacier modelling experiments at some of these sites have shown that this advance occurred when air temperatures were 2-4 ◦C lower than present (Anderson and Mackintosh, 2006; Doughty et al., 2013; Kaplan et al., 2013). Slow retreat, punctuated by minor still stands, occurred through the Younger Dryas. Local improvements in other geochronological techniques, such as tephrochronology has shown that this temporal pattern of relative temperature change is also widely acknowledged in continuous climate proxy archives across New Zealand (Lowe et al., 2013), although outstanding questions remain over the magnitude and spatial pattern of cooling at this time (Newnham et al., 2012). 2.5. STUDY SITE AND PREVIOUS WORK 29 2.5.1 Geological setting Tongariro Volcanic Centre (TgVC) represents an area of predominantly andesitic vol- canism, situated in an active graben at the south-western end of the Taupo Volcanic Centre (TVC), central North Island, New Zealand (Figure 2.5). TgVC consists of four Quaternary andesitic massifs, Kakaramea-Tihia, Pihanga, Tongariro, and Ruapehu, the latter two of which are the focus of this research and are described in more detail below. Mount Ruapehu (39◦17’S, 175◦34’E) is the highest peak (2797 m asl) in North Island, New Zealand. In its current conﬁguration, Ruapehu consists of a c. 110 km3 volcanic cone, currently active through a single vent, Crater Lake (Hackett and Houghton, 1989). Previous geological mapping and petrological studies have identiﬁed four broad periods of geochemically-distinct cone-building activity at Mt. Ruapehu, which were assigned formation status (Hackett and Houghton, 1989; Price et al., 2012). In chronological order, these formations are termed, Te Herenga (250-180 ka); Wahianoa (160-115 ka); Mangawhero (55-15 ka); and Whakapapa (15-0 ka) (Gamble et al., 2003). A compilation of existing radiometric (40K/40Ar and 40Ar/39Ar) ages from lava ﬂows on both volcanoes is shown in Figure 2.6. This ﬁgure shows that the oldest lavas on Mt. Ruapehu (Te Herenga Fm) outcrop on the north western side of the volcano, whilst lavas pertaining to the Wahianoa Fm cone-building episode are concentrated on the south eastern side of the volcano. Several syn-glacial (c. 45-15 ka) lava ﬂows have also been dated from several valleys on Mt. Ruapehu (Figure 2.6). These ﬂows are typically positioned adjacent to the major glacial troughs and display a wide range of cooling fractures consistent with having chilled rapidly against the margins of former, more extensive mountain glaciers (Sp¨orli and Rowland, 2006; Conway et al., 2015). The present-day upper cone of Mt Ruapehu is broad (c. 2 km2) and elongate with a NNE-SSW orientation. The northern portion of this broad summit consists of an ice-ﬁlled depression (Otway et al., 1985), with thick surface debris cover, that forms a c. 1 km2 plateau, surrounded by a protruding rim, with several peaks exceeding 2600 m asl. The southern portion of the summit is dominated by the 0.5 km2 Crater Lake that ﬁlls the active, modern-day, volcanic vent. The acidic, warm lake water currently drains over a bedrock lip at its southern margin, into the deeply incised, eastward-draining Whangaehu river catchment. 2.5.1 Geological setting Lying c.15 km NNE of Ruapheu, the Tongariro massif (39◦08’S, 175◦39’E) represents a coalescence of up to 17 effusive centres, totalling c. 60 km3 in volume (Hobden et al., 30 CHAPTER 2. BACKGROUND 1999). K-Ar dating of lava ﬂows place the initiation of ediﬁce growth at 275-250 ka, while further intensive periods of activity occurred 210-200 ka, 130-75 ka and 25-0 ka (Hobden et al., 1996). Figure 2.6 shows that all of radiometric ages collected from Tongariro massif predate the peak of the last glacial cycle (i.e. > 25 ka). Post-glacial eruptions on Tongariro massif have been sourced from several vents, but primarily from the Holocene-aged cone of Mt. Ngauruhoe (Hobden et al., 2002; Moebis et al., 2011), which today forms the highest peak of the massif. The oldest ages on the massif (c. >260 ka) come from the southern ﬂank, close to the saddle with Mt. Ruapehu. Proximal to this location, a cluster of ages show that cone-building occurred at c. 200 ka in the vicinity of the present-day Mt. Nguaruhoe. Lavas underlying previously identiﬁed moraines (e.g. Mathews, 1967; Topping, 1974) in several valleys such as Man- gatepopo and Waihohonu, are in excess of the c. 90 ka. These ages provide maximum constraining ages for the overlying glacial landforms, which suggest they likely pertain to the last glacial cycle. An additional record of volcanic activity for both volcanoes is preserved on their sur- rounding (and coalescent), 6 - 15 km wide ringplains (Donoghue et al., 1999). Thick ac- cumulations of volcaniclastic material make up the ringplain, predominantly consisting of debris ﬂow, debris avalanche, hyperconcentrated stream ﬂow deposits, interbedded with a mixture of local andesitic and distal rhyolitic tephras (Donoghue and Neall, 2001). The latter are generally well-dated (e.g. Lowe et al., 2008, 2013; Vandergoes et al., 2013), therefore provide chronological constraint for the local tephrostratigraphic record. In particular, the Kawakawa/Oruanui Tephra (KOT; c. 25 ka), Waiohao Tephra (c. 14 ka) and the Taupo Ignimbrite (c. 1.8 ka) are the most widely distributed across TgVC Donoghue et al. (1995). Previous work has developed a detailed local tephros- tratigraphic framework (Topping, 1974; Donoghue et al., 1995, 1997; Donoghue and Neall, 1996; Cronin and Neall, 1997), which allows correlation of volcaniclastic units and providing useful chronostratigraphic marker horizons, which help to constrain the timing of major geomorphic events such as moraine building. 2.5.1 Geological setting In a comprehensive review of the local TgVC tephrostratigraphy, Donoghue et al. (1995) constrain the spatio-temporal patterns of local tephra dispersal and preservation. Isopach maps of locally-sourced tephras consistently show that volcanic products are preferentially transported and preserved to the east of TgVC, which represents the inﬂuence of the prevailing westerly wind. For large eruptive events, thicknesses of individual members within tephra formations range from <0.1 m to c. 1 m within c. 5 km downwind of the source vent. Thus, the most complete sedimentological sequence of local volcanic activity preserved on the ringplain is found to the east of Mt Ruapehu, whilst preservation of TgVC-sourced tephra elsewhere on the ringplain is 2.5. STUDY SITE AND PREVIOUS WORK 31 comparatively low, particularly to the north, south and west of the main vents where palaeosol development is common between tephra horizons and the basal ages of cover bed sequences rarely exceeds c. 10 - 15 ka (Topping, 1973; Donoghue et al., 1995). comparatively low, particularly to the north, south and west of the main vents where palaeosol development is common between tephra horizons and the basal ages of cover bed sequences rarely exceeds c. 10 - 15 ka (Topping, 1973; Donoghue et al., 1995). 2.5.2 Present day climatic situation Situated between 34-47◦S in the south-west sector of the Paciﬁc Ocean, New Zealand spans subtropical and subpolar climes (Figure 2.7), therefore is highly sensitive to regional climatic ﬂuctuations (Newnham et al., 1999). Westerly atmospheric circulation dominates between 30-60◦S and is responsible for the eastward migrating troughs and anticyclones that deﬁne weather variability in New Zealand (Sturman and Tap- per, 1996). Southern Annular Mode (SAM) is an index of the atmospheric surface pressure gradient between high- and mid-latitudes in the Southern Hemisphere and represents an important control on weather variability in New Zealand at inter- to sub-annual timescales (Renwick and Thompson, 2006). During the positive phase of SAM the core of the westerlies move southwards away from New Zealand, resulting in reduced cyclonic activity and drier conditions. During the negative phase, the core of the westerlies is situated further north and wetter conditions prevail in New Zealand, particularly in western regions. At interannual timescales, El Ni˜no Southern Oscillation (ENSO) is the dominant mode of climatic variability in the Paciﬁc region (Salinger et al., 2001). El Ni˜no conditions are associated with enhanced zonal circulation over New Zealand, which ampliﬁes the west-east precipitation gradient and lowers average air temperatures (Salinger and Mullan, 1999). La Ni˜na years are associated with enhanced north-easterly atmospheric ﬂow, which generates a negative north-south precipitation gradient anomaly and generally higher air temperatures. At decadal timescales, ENSO activity is modulated by the Interdecadal Paciﬁc Oscillation (IPO), which switched abruptly from a negative phase (favouring La Ni˜na) to positive phase (favouring El Ni˜no) in 1976/77 (Salinger et al., 2001). Precipitation changes in New Zealand, associated with changes interannual to in- terdecadal regional circulation changes, are spatially variable, due to the interaction of airﬂow with local topography (Salinger, 1980a). Meanwhile, interannual-decadal air temperature anomalies in New Zealand are strongly inﬂuenced by upwind sea surface temperatures (Sutton et al., 2005) and are generally consistent across the country (Salinger, 1980b). Contemporary oceanic inﬂuences on North Island, New Zealand (34 - 41 ◦S) are largely sub-tropical in nature, predominantly originating from an eastward ﬂowing branch of the equatorial-sourced East Australian Current, known as the Tas- man Front, which descends the northeast coast before continuing eastwards along the 32 CHAPTER 2. 2.5.2 Present day climatic situation BACKGROUND gure 2.5: The geological and tectonic setting of Tongariro and Ruapehu volcanoes showi e distribution of lavas and their relative ages (Q1 = 0-12 ka; Q2 = 24-12 ka; Q3 = 59-24 4 = 71-59ka; Q5 = 128-71 ka; Q6 = 128-71 ka; Q7 = 186-128 ka; Q8 = 303-245 ka), morain nd major fault systems of the region. Geological data from the GNS Science QMAP series n and 8 (Townsend et al., 2008; Lee et al., 2011) and tectonic data from the GNS Science Acti ults Database. Inset map shows the location of Tongariro Volcanic Centre at the southern e Taupo Volcanic Zone (TVZ) in central North Island, New Zealand. Figure 2.5: The geological and tectonic setting of Tongariro and Ruapehu volcanoes showing the distribution of lavas and their relative ages (Q1 = 0-12 ka; Q2 = 24-12 ka; Q3 = 59-24 ka; Q4 = 71-59ka; Q5 = 128-71 ka; Q6 = 128-71 ka; Q7 = 186-128 ka; Q8 = 303-245 ka), moraines and major fault systems of the region. Geological data from the GNS Science QMAP series nos. 7 and 8 (Townsend et al., 2008; Lee et al., 2011) and tectonic data from the GNS Science Active Faults Database. Inset map shows the location of Tongariro Volcanic Centre at the southern end of Taupo Volcanic Zone (TVZ) in central North Island, New Zealand. 2.5. STUDY SITE AND PREVIOUS WORK 33 igure 2.6: Existing radiometric ages of lava ﬂows from Tongariro massif and Mt Ruapehu erived using 40K/40Ar (Hobden et al., 1996) and 40Ar/39Ar (italics; Gamble et al., 2003 onway et al., 2015) dating. Other data sources as for previous ﬁgure Figure 2.6: Existing radiometric ages of lava ﬂows from Tongariro massif and Mt Ruapehu derived using 40K/40Ar (Hobden et al., 1996) and 40Ar/39Ar (italics; Gamble et al., 2003; Conway et al., 2015) dating. Other data sources as for previous ﬁgure CHAPTER 2. BACKGROUND 34 northern margin of the Chatham Rise (Figure 2.7). In contrast, South Island (40-47 ◦S) intersects the sub-tropical front (STF), where sub-tropical gyres and sub-antarctic water masses converge, representing a temperature, salinity and nutrient boundary, which deﬁnes the northern margin of the Southern Ocean (Sikes et al., 2009). Consequently, steep, zonal and meridional sea surface temperature (SST) gradients exist across New Zealand. For example, in the far north annual SSTs average c. 20 ◦C compared to c. 2.5.2 Present day climatic situation 10 ◦C in the south (Uddstrom and Oien, 1999). This spatial variability in SSTs strongly inﬂuences terrestrial air temperature anomalies resulting from regional circulation changes. Figure 2.7: Contemporary atmospheric and oceanic circulation in the southwest Paciﬁc (Back- ground imagery from Google Maps - see ﬁgure for primary sources of imagery). Figure 2.7: Contemporary atmospheric and oceanic circulation in the southwest Paciﬁc (Back- ground imagery from Google Maps - see ﬁgure for primary sources of imagery). Mean annual temperature at Whakapapa Village, located at c. 1100 m asl on the NW ﬂank of Ruapehu, is 7.5 ◦C (Figure 2.8a). Mean monthly temperatures range from 3.1 ◦C 2.5. STUDY SITE AND PREVIOUS WORK 35 2.5. in July to 12.6 ◦C in January. Precipitation at this location averages c. 2800 mm annually and is relatively evenly distributed throughout the year, with summer (DJF) totals slightly lower than those in winter (JJA; Figure 2.8a). At Ohakune township (607 m asl), situated to the south of Mt. Ruapehu, the monthly temperature and precipitation patterns broadly mirror those at Whakapapa village (Figure 2.8b), with differences in absolute values likely representing an different orographic inﬂuence on temperature and precipitation between the two sites. Wind measurements at Whakapapa Village (Figure 2.8c) show that westerly wind conditions prevail, with typical ground wind speeds of 1-10 m s−1 and rarely exceeding 20 m s−1. This indicates that local airﬂow in the region is strongly inﬂuenced by regional atmospheric circulation. A signiﬁcant ESE component is also present, with winds from this direction reaching speeds of > 15 m s−1. Figure 2.8c shows that while the westerly winds at Whakapapa are present year round, the ESE component is predominantly a winter (JJA) phenomenon. This suggests that katabatic winds, driven by lower winter air temperatures and seasonal snow cover, play an important role in boundary layer air movement on the mountain. A transition from mountain beech and silver pine tree stands into sub-alpine shrub heath occurs at c. 1500 m asl on the western slopes, and c. 1100 m asl in the east (Wardle, 1991), perhaps reﬂecting spatial differences in moisture availability. This pattern may reﬂect an east-west precipitation gradient across Mt. Ruapehu. Prevailing westerly air masses are intercepted by the volcanic ediﬁce, which produces orographic precipitation on the western slopes and possibly creating a rain-shadow effect on the eastern side. 2.5.2 Present day climatic situation However, the precise magnitude of the climatic gradient is difﬁcult to quantitatively constrain due to the paucity of proximal, empirical climate station data across this transect. 2.5.3 Contemporary glacierisation At 2797 m asl, Mount Ruapehu represents the only peak in the North Island to cur- rently intercept the permanent snowline. The most recent glacier survey on Ruapehu was conducted in 1988 in which, a total of 9 small glacial bodies were identiﬁed as containing <0.2 km3 ice and covering an estimated 4 km2 (Keys, 1988). Much less attention has been paid to contemporary North Island glaciers and their relationship with climate, in comparison to glaciers in the Southern Alps. Interpreting the climatic signiﬁcance of past glacier ﬂuctuations from geological records requires CHAPTER 2. BACKGROUND 36 Figure 2.8: (a,b) 30 yr (1981-2010) mean monthly temperature and precipitation for Whakapapa Village and Ohakune climate stations, respectively (NIWA, 2014); (c) Wind roses derived using 10 yr (2001-2010) of hourly wind speed and direction measurements at Whakapapa Village (NIWA, 2014). Figure 2.8: (a,b) 30 yr (1981-2010) mean monthly temperature and precipitation for Whakapapa Village and Ohakune climate stations, respectively (NIWA, 2014); (c) Wind roses derived using 10 yr (2001-2010) of hourly wind speed and direction measurements at Whakapapa Village (NIWA, 2014). knowledge of present day glacier-climate relationship (e.g. mass balance controls, equilibrium line altitudes). In the following sections, I review the existing glaciological measurements and observations on Mt. Ruapehu, in order to assess the utility of these ice masses as a datum from which to infer past climate change using moraine records. knowledge of present day glacier-climate relationship (e.g. mass balance controls, equilibrium line altitudes). In the following sections, I review the existing glaciological measurements and observations on Mt. Ruapehu, in order to assess the utility of these ice masses as a datum from which to infer past climate change using moraine records. 2.5.3.1 Whakapapa glacier The former Whakapapa Glacier was the best studied ice mass on Mt Ruapehu, primarily due to its easy access via the Bruce Road and Whakapapa skiﬁeld. The ﬁrst written account of this glacier estimated that the glacier terminus lay at approximately 7500 ft (c. 2300 m) above sea level (Taylor, 1927). Few direct glaciological observations/accounts were made in the subsequent three decades, although Krenek (1959) reported that the terminus remained ’more or less stationary’ during the period 1930-1950. The ﬁrst comprehensive morphometric assessment of Whakapapa Glacier was carried out in 1953 by Odell (1955), who reported that the glacier length from the terminus 37 2.5. STUDY SITE AND PREVIOUS WORK Figure 2.9: Ice and snow-patch distribution on Mt Ruapehu, as shown on LINZ Topo50 map and deﬁned by Keys (1988). Selected peaks labeled in black, glaciers labeled in blue. Present-day (2014) ice coverage is reduced from that shown here. Figure 2.9: Ice and snow-patch distribution on Mt Ruapehu, as shown on LINZ Topo50 map and deﬁned by Keys (1988). Selected peaks labeled in black, glaciers labeled in blue. Present-day (2014) ice coverage is reduced from that shown here. position at 2130 m asl to Paretetaitonga col at 2621 m asl was slightly in excess of 1.5 km. In 1954, Krenek (1959) reported a minor (c. 2 m) advance of the glacier terminus and reﬁned the estimate of glacier length to 1.7 km. Detailed observations and repeat ground photography of the Whakapapa Glacier document the great changes that oc- curred in the mid-1950s. Krenek (1959) reported that the 1954 winter accumulation was entirely removed, midway through the following the ablation season (January 1955). By February 1955 the glacier terminus had retreated 68 m, and by the end of the ablation season (April 1955) a total of 94 m retreat had occurred. In addition, surface lowering was measured as 6-8 m at the glacier margin, and 10-15 m on the glacier interior, caus- ing the emergence of a distinct ash layer on the upper glacier, thought to have been deposited by the 1945 eruptions, as well as large areas of bedrock through the lower glacier. The emergence of bedrock enhanced the local melt potential as Krenek (1959) reported that large randklufts (gaps between glacier margin and bedrock) developed at the glacier margins. 2.5.3.1 Whakapapa glacier These effects were replicated during the 1956 ablation season causing a further 65 m of terminus retreat and the additional down-wasting resulted in the Whakapapanui lobe being completely isolated from the main Whakapapa Glacier CHAPTER 2. BACKGROUND 38 by Restful Rocks ridge (Figure 2.9). After this severance, Whakapapa Glacier, became informally known as ’Whakapapaiti glacier’ Keys (1988). by Restful Rocks ridge (Figure 2.9). After this severance, Whakapapa Glacier, became informally known as ’Whakapapaiti glacier’ Keys (1988). Recession continued in 1957, despite being reported as a positive balance year, although overall retreat was vastly reduced in comparison to the preceding two years, with just 6 m retreat measured (Krenek, 1959). Krenek (1959) also reported the existence of an ’inverted’ end of summer snowline in 1957, with bare ice exposed on the upper glacier and net snow accumulation on the lower tongue. It was suggested that wind-driven re-distribution of winter snow likely played a signiﬁcant role in this occurrence. The glacier terminus was recorded as stationary in the 1957-58 balance year (Krenek, 1959). The ﬁrst direct mass balance measurements were carried out on the Whakapapa Glacier in spring 1958. Winter accumulation was measured in snow pits as 86 cm (1971 m asl); 125 cm (2344 m asl); and 145 cm (2454 m asl). Successive negative balance years from 1960-1962 resulted in further downwasting of both the Whakapapa and Whakapapanui glaciers, as recorded in repeat photo surveys (Heine, 1962, 1963). At the end of the 1961-62 balance year the Whakapapa Glacier length was measured as 0.6 km, with the terminus at 2377 m asl; the Whakapapanui glacier was 0.3 km long and terminated at 2256 m asl (Heine, 1962). These measurements are consistent with the New Zealand Map Survey sheet for Ruapehu published in 1963. Field surveying and mass balance in 1968 (Thompson and Kells, 1973) estimated that the Whakapapanui glacier covered an area of 37000 m2, and terminated at 2273 m asl, indicating little change from the previous measurements in 1962. A comprehensive mass balance survey on the Whaka- papanui glacier in the 1968-69 balance year measured a positive balance of c. 2 m snow water equivalent (swe). Thompson and Kells (1973) report that winter accumulation of 1968 was 45% above that of the 1957-68 average, whilst temperatures were c. 1.5 ◦C lower for the budget year. 2.5.3.1 Whakapapa glacier This positive balance was short-lived however, as the following ablation season removed the winter accumulation and 5.3 m of glacier ice, resulting in c. 150 m of terminus recession (Thompson and Kells, 1973). In 1988, the Whakapapa(iti) glacier terminus was recorded at 2400 m, suggesting only minor (c. 100 m) retreat from the measured position in 1962, in contrast to the Whakapapanui which receded c. 460 m and reduced in area by c. 0.017 km2 over the same time period (Keys, 1988). Today, no glacial ice remains in the vicinity of the Paratetaitonga and the Whakapapanui lobe persists as a snow patch. 2.5.3.2 Mangaehuehu Glacier This southward draining cirque glacier occupies a topographic depression beneath Tahurangi, between Skyline Ridge to the north west, and Girdlestone Peak to the east 39 2.5. STUDY SITE AND PREVIOUS WORK 39 Figure 2.10: Mangaehuehu Glacier on the the southern slopes of Mt. Ruapehu. Note the sharp-crested lateral moraines immediately down valley of the current glacier terminus. Highest peak is Tahurangi (2797 m asl). (23 February 2013). Figure 2.10: Mangaehuehu Glacier on the the southern slopes of Mt. Ruapehu. Note the sharp-crested lateral moraines immediately down valley of the current glacier terminus. Highest peak is Tahurangi (2797 m asl). (23 February 2013). (Figure 2.9; and Figure 2.10). Heine (1963) reported the glacier terminus at 6800 ft (c. 2050 m asl). Keys (1988) recorded c. 250 m terminus retreat, and c. 30 m surface lowering in the intervening period, with the glacier now terminating immediately above a steep rock cliff. Brook et al. (2011) analysed interannual changes in the surface area of Mangaehuehu Glacier between the years 1988 and 2007, using oblique aerial photographs. Correlation analysis between the observed glacier area changes and a se- ries of meteorological data and climatic indices found that ablation season temperature is a key control on the interannual ﬂuctuations of glacier size on Ruapehu, which in turn are connected to changes in atmospheric circulation, such as Southern Oscillation. This mirrors ﬁndings from the Southern Alps (Hooker and Fitzharris, 1999; Chinn et al., 2005), suggesting that modern glaciers across the latitudinal range of New Zealand respond to similar climatic forcing over interannual to interdecadal timescales. 2.5.3.3 Summit Plateau Otway et al. (1985) conducted a radio-echo sounding survey on Summit Plateau (Figure 2.9), where it was estimated that 60-130 m of ice ﬁlled two crater-like depressions that cover c. 1 km2 of the northern portion of the mountain top. Keys (1988) estimated Summit Plateau ice volume at 0.05 km2, accounting for just under half of the total ice volume on the mountain. Otway et al. (1985) compare their results to an aerial survey conducted in the mid-1950s and derive mean ablation rates of 0.3 - 1.1 m yr−1 (1955-1985), with higher values towards the southern end of the ice mass. Keys (1988) CHAPTER 2. BACKGROUND 40 Figure 2.11: Panoramic photograph showing Summit Plateau after total melt of the winter snowpack. Photo taken from approx. Glacier Knob (2642 m asl). Tukino Peak at left and Paretetaitonga peak at far right. (25 February 2012). Figure 2.11: Panoramic photograph showing Summit Plateau after total melt of the winter snowpack. Photo taken from approx. Glacier Knob (2642 m asl). Tukino Peak at left and Paretetaitonga peak at far right. (25 February 2012). reported that the downwasting of this ice over this period had lead to the emergence of the rock rim around the plateau, severing the connection and ice ﬂow input to the Man- gatoetoenui and former Whakapapa glaciers and reducing the input to the Whangaehu Glacier (see below). Despite this, several authors have noted that the plateau plays an important role in nourishing the surrounding outlet and cirque glaciers through wind-driven re-distribution of fresh snow (Krenek, 1959; Paulin, 2008). In negative balance years, complete removal of the winter snowpack reveals a thick debris cover overlying the Summit Plateau ice mass, consisting largely of volcanic ash, lapilli and large boulders (Figure 2.11). Comparison of ice ablation beneath this debris cover against the measured ablation of clean ice showed that melt rates are highest where debris cover is c. 70 mm thick, whilst rates are reduced to below that of clean ice where debris cover exceeds 120 mm (Richardson and Brook, 2010). These ﬁndings are broadly consistent with other studies of the affect of debris cover on ice ablation, whereby small debris thicknesses (e.g. 2.5.3.3 Summit Plateau < 100 mm) enhance ablation through reduced albedo and increased absorption of incoming shortwave radiation, whilst thicker debris cover (> 100 mm) has an insulating effect on the underlying ice, reducing the available radiative energy for melting (Brock et al., 2007). 2.5.3.5 Mangatoetoenui Glacier Mangatoetoenui Glacier is situated on the north-east ﬂank Mt Ruapehu and bound by Te Heuheu peak to the north and Summit Plateau crater rim to the west (Figure 2.9). Heine (1963) reported that this south-eastward ﬂowing glacier was partially fed by Summit Plateau ice and terminated at c.7000 ft (c. 2150 m asl). In 1988 this glacier had an area of 0.55 km2, an estimated volume of 0.013 km3 and an average velocity of 24 ± 5 m yr−1 (Keys, 1988). Keys (1988) also estimated that the surface lowered 20 m between 1961 and 1988, which resulted in severance from Summit Plateau ice ﬁeld. 2.5.3.6 Wahianoa Glacier Wahianoa Glacier is positioned on the south-eastern ﬂank of the volcano and is bound by the Girdlestone-Tahurangi ridge to the west and Ringatoto Peak to the east (Figure 2.9). Heine (1963) reported that the head of this glacier was located at 8500 ft (c. 2600 m asl) and terminated at 7120 ft (c. 2150 m asl). Keys (1988) reported that this glacier retreated by c. 160 m between 1962-1988, and calculated a glacier area of 0.47 km2 and a volume of 0.0074 km3. 2.5.3.4 Whangaehu Glacier The Whangaehu Glacier is the largest glacier on the mountain, measured by Keys (1988) to cover 0.76 km2. This glacier is sourced from the southern end of Summit Plateau, close to Dome Ridge, and the debris-covered terminus is located at c. 2100 m asl (Figure 2.9). Keys (1988) reported that the Whangaehu Glacier was the only one to still be fed by ice ﬂow from Summit Plateau, although it was observed that the continuing thinning of the plateau was reducing this input. Ice velocity measurements on the glacier by 2.5. STUDY SITE AND PREVIOUS WORK 41 Figure 2.12: Panoramic photograph showing Mangatoetoenui Glacier beneath Te Heuheu peak (26 February 2012). Figure 2.12: Panoramic photograph showing Mangatoetoenui Glacier beneath Te Heuheu peak (26 February 2012). Paulin (2008) showed that the glacier was ﬂowing at an average of 30 m yr−1 and that ice contribution from Summit Plateau to the Whangaehu Glacier was negligible. Mass balance measurements and end-of-summer snowline (EOSS) observations on the Whangaehu Glacier showed complex and highly variable interannual patterns, likely driven by aeolian redistribution of snow and variations in ablation season temperatures (Paulin, 2008). 2.5.3.7 Mangaturuturu Glacier Mangaturuturu Glacier is westward-draining and is located on the western slopes of Mt Ruapehu (Figure 2.9). Heine (1963) reported that Mangaturuturu Glacier existed between 8450 ft (c. 2600 m asl) and 7200 ft (c. 2200 m asl). Keys (1988) reported that 42 CHAPTER 2. BACKGROUND Figure 2.13: Photograph showing Crater Lake and Crater basin glacier beneath Tahurangi (19 January 2014). Figure 2.13: Photograph showing Crater Lake and Crater basin glacier beneath Tahurangi (19 January 2014). this glacier retreated approximately 240 m since Heine’s (1963) survey and measured the area of this glacier in 1988 to cover 0.42 km2 and contain c. 0.01 km3 of ice. this glacier retreated approximately 240 m since Heine’s (1963) survey and measured the area of this glacier in 1988 to cover 0.42 km2 and contain c. 0.01 km3 of ice. 2.5.3.8 Crater basin and ’unnamed’ glaciers Keys (1988) deﬁned the Crater basin glacier as that draining northwards from un- derneath Tahurangi peak, towards Crater Lake (Figure 2.9; Figure 2.13). This was differentiated from the connected, southward draining ’unnamed glacier’ (see below) located beneath Paretetaitonga peak, on the grounds of signiﬁcant differences in their response to the Crater Lake outburst of 1953. Following a rapid drop in the lake level by > 8 m in 1953, Keys (1988) reports that Crater basin glacier has thinned by up to 90 m. Between 1961-1988, unnamed glacier was in a state of positive mass balance, resulting in a c. 30 m thickening of the glacier tongue, such that it now terminates in a 30 m ice cliff at the northern edge of Crater Lake. Keys (1988) attributes the positive mass balance to the topographic setting, situated on the leeward side of Paretetaitonga, where wind-blown snow accumulates and direct incoming shortwave radiation is reduced. 2.5.3.9 Summary of present day glacier-climate relationship in North Island From the review presented above it is clear that most glaciers on Mt. Ruapehu have experienced net thinning and terminus retreat during the last century, probably in response to increasing ablation season temperatures. The ﬁnding that glacier surface area exhibits signiﬁcant negative correlations with mean ablation season (Nov-Mar) air temperature (Brook et al., 2011), despite some evidence for topoclimatic controls on 2.5. STUDY SITE AND PREVIOUS WORK 43 2.5. glacier mass balance (Krenek, 1959), provides conﬁdence in the use of contemporary equilibrium line altitude observations (e.g. Keys, 1988) to relate geologically-inferred ELA changes to atmospheric temperature anomalies. Furthermore, signiﬁcant correlations with Southern Oscillation Index and end-of- summer-snowline height in the Southern Alps suggests that glaciers in North Island and the Southern Alps respond similarly to changes in atmospheric circulation over interannual to interdecadal timescales (Hooker and Fitzharris, 1999; Brook et al., 2011). This inference is further supported by the detailed observations of length changes at Whakapapa Glacier during the late 1950s. The down-wasting and terminus retreat at Whakapapa Glacier during the 1955/1956 ablation season coincided with one of the strongest La Ni˜na events of the 20th century (Lorrey et al., 2012a). On the west coast of South Island, Franz Josef and Fox glaciers, which exhibit reaction times of 0-5 years in response to a climatic perturbation, reduced in length by 10-15% between 1955- 1965 (Purdie et al., 2014). La Ni˜na events are characterised by positive mean annual temperature anomalies across New Zealand, but higher (lower) precipitation totals in North (South) Island (Sturman and Tapper, 1996). Similar changes of glaciers across both islands during this period, despite potential differences in precipitation anomalies, indicates a common sensitivity to atmospheric temperatures, although process-oriented studies using mass balance models and measurements are required to develop a more comprehensive understanding of these relationships. Comparison of the magnitudes of glacier ﬂuctuations between these two regions over geological timescales may provide useful insight into past changes in regional temperature gradients. 2.5.4 Previous work: palaeo-glaciation in Tongariro National Park In North Island, only Ruapehu is of sufﬁcient altitude to support small modern glaciers; however both Mt. Ruapehu and Tongariro massif display characteristic glaciated val- leys that radiate from the central peaks. Further evidence for former, more extensive glaciation in the TgVC includes: large lateral moraines; striae; cirques; and ice-contact lava ﬂows (Taylor, 1927; Grange and Williamson, 1930; Hackett and Houghton, 1989; Sp¨orli and Rowland, 2006; and references therein). However, such is the lack of un- derstanding of the timing and former extent of these glaciations, that the two major reviews of New Zealand palaeo-glaciation (Suggate, 1990; Barrell, 2011) afford no more than one or two paragraphs to the matter. In the only signiﬁcant publication thus far, McArthur and Shepherd (1990) reconstructed a c. 140 km2 ice mass on Ruapehu, based largely on geomorphological ﬁeld mapping. Moraine ridges were identiﬁed ﬂanking all of the major valleys draining Ruapehu, extending down to c. 1200 m asl. CHAPTER 2. BACKGROUND 44 Figure 2.14: Photograph showing the horizontally-bedded lacustrine sediments in Mangatoe- toenui valley, identiﬁed by Topping (1974) and described by McArthur and Shepherd (1990). (27 February 2012). Figure 2.14: Photograph showing the horizontally-bedded lacustrine sediments in Mangatoe- toenui valley, identiﬁed by Topping (1974) and described by McArthur and Shepherd (1990). (27 February 2012). Additionally, deformed laminated silts and clays situated between inner and outer moraines in Mangatoetoenui valley (Figure 2.14) are thought to represent deposition and subsequent overriding of a proglacial lake, suggesting multiple glacial advances in this region. The major contributions to the understanding of glaciation on Tongariro massif come from Mathews (1967) and Topping (1974), although the primary focus of these studies is volcanic. Mathews (1967) describes the conspicuous moraine ridges of the Man- gatepopo and Waihohonu valleys and tentatively identiﬁes morainic landforms in the Oturere valley. He also describes sediment sections of diamicton containing striated clasts, interpreted as glacial till, from Mangatepopo and Makahikatoa valleys on the western side of the massif. Topping (1974) builds on this work by identifying further diamictons inferred to be glacial till, as well as describing the relationships between moraine ridges and underlying lava ﬂows. Despite this, no detailed ice mass recon- struction has yet been conducted for Tongariro massif. The precise timing of glacial advance(s) on both volcanoes is unknown. Existing research utilises the approximate ages of volcanic products to provide chronostrati- 2.6. RESEARCH QUESTIONS 45 graphic, minimum-maximum bracketing ages. 2.5.4 Previous work: palaeo-glaciation in Tongariro National Park Correlations with the better constrained timings of ice advance on the South Island have also been attempted. Using titano- magnetite analyses, Topping (1974) tentatively identiﬁed the Kawakawa/Oruanui Tephra (25.4 cal. ka BP) in a sequence of deformed lacustrine sediments interbedded with till (Figure 2.14). McArthur and Shepherd (1990) suggest these sediments were deposited by retreating ice correlated with the penultimate LGM advance of the South Island (equivalent to Kumara-2-2 in Suggate and Moar, 1970), and the deformation structures were therefore caused by ice advance correlated with the ﬁnal (Kumara-3) event. McArthur and Shepherd (1990) highlight an alternative scenario in which the silts and tephra are deposited in front of the advancing Kumara-2-2 ice mass and are subsequently overrun and deformed during the same event. This therefore invokes earlier glacial advance on Ruapehu than that proposed by Topping (1974), where the outer moraines that impounded the proglacial lake could be much older (McArthur and Shepherd, 1990). The thick (up to 5 m) ash sequences overlying the lower sides of moraines on Tongariro massif provide useful chronostratigraphic markers (Mathews, 1967; Topping and Kohn, 1973; Topping, 1974). Assuming a constant Holocene sedimentation rate for overly- ing ash, Mathews (1967) calculated that the moraines ridges were formed before at least 4 ka, although he stated that they are more likely to correlate to late Pleistocene glaciations of the South Island. Using titano-magnetite assemblages, Topping and Kohn (1973) identiﬁed the Rerewhakaaitu ash mantling the large moraine ridge on the true right of Mangatepopo valley, which indicates a minimum moraine age of 17.5 ± 0.5 cal. ka BP (Lowe et al., 2013). The maximum ages of these moraines are constrained by underlying lava, which has been dated to > 100 ka (Stipp, 1968; Hobden et al., 1996; Figure 2.6). Younger tephras are found mantling other moraines radiating from Tongariro, although these too provide a minimum age constraint due to the likelihood of older tephras having been stripped off during the harsher stadial climate (Topping, 1974). 2.6 Research questions The idea that Earth has transitioned from glacial to interglacial climates was triggered by recognition of glacial landforms far outside of present day ice limits. Whilst many palaeoclimatic proxies now exist (often of high temporal resolution), records of past glacier ﬂuctuations offer the opportunity to constrain past climate in alpine regions, us- ing a relatively simple physical system. Recent advances in geochronological techniques such as cosmogenic surface exposure dating, and the development of numerical mod- CHAPTER 2. BACKGROUND 46 els of glacial processes now afford the opportunity to obtain temporally-constrained, quantitative estimates of past cooling events. New Zealand offers one of the few places to obtain terrestrial palaeoclimate records in the mid-latitudes of the Southern Hemisphere. Situated at the northern margin of the southern westerly circulation and straddling the modern oceanic subtropical front, local climate proxy records are ideally placed to record changes in these major climatic boundaries thought to be integral to major climatic shifts in recent Earth history. Widening the spatial scope of local glacial-climate reconstructions, which have thus far mainly focused on the Southern Alps, will allow quantitative constraint of past climatic gradients and spatiotemporal variability of past climatic change. With this in mind, I will answer the following research questions (each of which is addressed in one or more speciﬁc thesis chapters): 1. Can cosmogenic isotopes be used to constrain the timing of glaciation on the North Island volcanoes? (Chapter 4) 1. Can cosmogenic isotopes be used to constrain the timing of glaciation on the North Island volcanoes? (Chapter 4) 2. When did glaciers in central North Island advance during the last glacial cycle? (Chapters 5,7) 2. When did glaciers in central North Island advance during the last glacial cycle? (Chapters 5,7) 3. Did glaciers in central North Island respond to the late-glacial climate reversal? (Chapter 6) 4. What does the geological record of glaciation in central North Island tell us about the magnitude of past temperature changes? (Chapters 5,6,7) Chapter 3 The concept of uniformitarianism dictates that past geological activity can all be explained by contemporary, measurable processes with invariant rates, which is embodied in the phrase: the present is key to the past. More recently, the importance of infrequent cataclysmic events punctuating the predominant gradual nature of geologic activity has been identiﬁed (e.g. Bretz, 1969), leading to a redeﬁnition of uniformitarianism, also known as actualism. This PhD research is set within an actualistic framework, adopting largely uniformitarian principles by deﬁning and interpreting the palaeoglaciology of TgVC in terms of empirically observed and analogous glacial processes, whilst accept- ing and questioning the likelihood of variance in process rates. An increasing body of empirically-derived, sedimentological and geomorphological process-response research in glacierised catchments is now available (e.g. Benn and Evans, 2010) for actualistic palaeoglaciological reconstructions using the geomorphological record. Furthermore, improved understanding of ice-ﬂow and glacier-climate interactions (Oerlemans, 2001; Cuffey and Paterson, 2010) has facilitated development of numerical glacier models. The combination of well-dated, geomorphically-constrained glacier reconstructions and numerical glacier modelling provides a powerful tool in deriving quantiﬁed estimates of palaeoclimate (e.g. Anderson and Mackintosh, 2006; Putnam et al., 2013b; Doughty et al., 2013; Kaplan et al., 2013). Using this framework, the following section details the methods that will be employed for palaeoglaciological reconstruction in TgVC. 3.1 Geomorphological mapping Geomorphological mapping is the process by which landforms are identiﬁed, their spatial location recorded and their genesis interpreted, based on the analysis of the land surface morphology and its constitutional geologic materials/structures (Hubbard and Glasser, 2005). The interpretative link to process-form geomorphic systems separates 47 CHAPTER 3. METHODOLOGY 48 this activity from ’morphological mapping’, which simply seeks to describe the form of a land surface (Knight et al., 2011). Table 3.1 outlines the methodological framework for the geomorphological mapping component of this research, based on the suggestions of Knight et al. (2011). The aim of the ﬁeld mapping component is to identify and record the nature and location of glacial geomorphology not detectable at the resolution of the remotely sensed datasets and locate landforms suitable for cosmogenic surface exposure dating (see Section 3.2, below). This is achieved by: • traversing the study locations by foot, recording observations on breaks of slope, sedimentary composition, landform conﬁguration (such as cross-cutting relation- ships, degree of preservation) and morphostratigraphy; • compiling detailed sedimentary logs/descriptions of exposed sections in key landforms to aid genetic interpretation; • using a handheld global positioning system (GPS) unit to locate all observations and ﬁeld photographs. The principle of equiﬁnality is one potential complication to geomorphological inter- pretation. In this instance, equiﬁnality refers to the formation of landforms with the same morphological and/or sedimentological characteristics by two or more different processes (Chorley, 1962). There are several recent examples of such debates (e.g. He- witt, 1999; McColl and Davies, 2011), perhaps stemming from ’...an over reliance on the role of glaciation to interpret constructional ridges and mounds...’ in upland environments (Knight et al., 2011). (McArthur and Shepherd, 1990) acknowledge that moraine-like landforms on active vol- canoes may be created by volcanic processes, although they do not provide any further detail into these processes. From a morphological perspective, volcanic landforms such as lava ﬂow levees and debris avalanche (from sector collapse) mounds can resemble moraine ridges/hummocks. In such cases, the lithological and sedimentological archi- tecture (e.g. Palmer and Neall, 1989) are sufﬁciently different from moraines that ﬁeld investigations can resolve the formative processes. However, other volcanic processes can construct moraine-like landforms, which are less readily differentiated from glacial processes using only morphological and sedimentological enquiry. Volcanic mass movement events (debris ﬂows, hyperconcentrated streamﬂows) can produce levees comprised of poorly sorted, weakly stratiﬁed/unstratiﬁed, angular to sub-rounded sediment, possbly exhibiting striae (Atkins, 2003). 3.1 Geomorphological mapping These are all common characteristics of glacial till. Potential incorporation of glacial sediments within such volcanic mass 3.1. GEOMORPHOLOGICAL MAPPING Table 3.1: Geomorphological mapping workﬂow for this study (based on Kn et al., 2011). Pre-mapping 1. Identify and obtain relevant remote sensing data (topographic maps, aerial photography, digital elevation models) 2. Design and create a GIS database using digital and digitised remote sensing imagery 3. Map large landforms using remotely sensed data, where data resolution permits 4. Identify sites of interest for ﬁeld investigation 5. Create 1:10000-scale paper base maps 6. Obtain permission for access to mapping regions 7. Conduct hazard/risk assessment for the planned mapping activities Syn-mapping 9. Conduct mapping using the stated protocol 10. Consider landform genesis within a multiple working hypothesis frame- work 11. Use hand-held GPS to record important tracks/waypoints 12. Write notes and take photos positioned using the GPS 13. Adhere to health and safety issues / risk assessment Post-mapping 13. Download and integrate GPS data with the existing GIS database 14. Compare ﬁeld and remote sensing mapping data in order to validate remotely sensed observations 15. Write up notes, integrate notes and photos with GPS points in GIS database 16. Digitise ﬁeld mapping data 17. Draw ﬁnal geomorphological map, using analogue or digital carto- 3.1. GEOMORPHOLOGICAL MAPPING 49 1. Identify and obtain relevant remote sensing data (topographic maps, aerial photography, digital elevation models) y g ( p g p p , aerial photography, digital elevation models) 2. Design and create a GIS database using digital and digitised remote sensing imagery 3. Map large landforms using remotely sensed data, where data resolution permits 4. Identify sites of interest for ﬁeld investigation 5. Create 1:10000-scale paper base maps 6. Obtain permission for access to mapping regions 7. Conduct hazard/risk assessment for the planned mapping activities Syn-mapping 9. Conduct mapping using the stated protocol 10. Consider landform genesis within a multiple working hypothesis frame- work 11. Use hand-held GPS to record important tracks/waypoints 12. Write notes and take photos positioned using the GPS 13. Adhere to health and safety issues / risk assessment Post-mapping 13. Download and integrate GPS data with the existing GIS database 14. Compare ﬁeld and remote sensing mapping data in order to validate remotely sensed observations 15. Write up notes, integrate notes and photos with GPS points in GIS database 16. Digitise ﬁeld mapping data 17. 3.1 Geomorphological mapping Draw ﬁnal geomorphological map, using analogue or digital carto- graphic symbols 18. Write/present explanatory notes accompanying the map 19. Apply geomorphological map output to the interpretation of past glacial activity 2. Design and create a GIS database using digital and digitised remote sensing imagery 3. Map large landforms using remotely sensed data, where data resolution permits 9. Conduct mapping using the stated protocol 10. Consider landform genesis within a multiple working hypothesis frame- work 11. Use hand-held GPS to record important tracks/waypoints 12. Write notes and take photos positioned using the GPS 13. Adhere to health and safety issues / risk assessment 19. Apply geomorphological map output to the interpretation of past glacial activity CHAPTER 3. METHODOLOGY 50 movement deposits further complicates glacial vs. volcanic interpretations. movement deposits further complicates glacial vs. volcanic interpretations. For these reasons, it is necessary to consider the individual landform genesis within the context of the wider landform assemblage, i.e. a landsystems approach. A number of glacial landsystems have been described, covering a range of different glaciological settings (Evans, 2003). I consider the temperate ’glaciated valley’ and ’mountain ice ﬁeld’ landsystems (e.g.Benn et al., 2003; Golledge, 2007; Benn and Evans, 2010) most appropriate for this research. These landsystems are characterised by assemblages of erosional (due to basal sliding) and depositional landforms, where geomorphic directional ice ﬂow indicators are generally concordant with contemporary surface slopes (Benn et al., 2003; Golledge, 2007). I consider these landsystem models are appropriate for this research based on: (i) the results of previous geomorphological research in TgVC (Mathews, 1967; Topping, 1974; McArthur and Shepherd, 1990), (ii) the more extensive body of contemporary and palaeoglaciological investigations of glacial sediment-landform assemblages (e.g. Kirkbride and Matthews, 1997; Hambrey and Ehrmann, 2004; Mager and Fitzsimons, 2007; Barrell et al., 2011; Barrell, 2014; Evans et al., 2013) in the Southern Alps, and (iii) existing constraints on the magnitude of past climate changes over the late Quaternary (e.g. Vandergoes et al., 2008; Golledge et al., 2012; Newnham et al., 2012, 2013; Doughty et al., 2013). In summary, I consider the formation of linear, constructional landforms in TgVC within a multiple working hypothesis framework (i.e. glacial vs volcanic). 3.1 Geomorphological mapping I initially classify such landforms as moraine ridges using the diagnostic (but not deterministic) criteria of McArthur and Shepherd (1990): (1) landform is composed of diamicts char- acterised predominantly by sub-angular to sub-rounded, faceted and/or striated clasts of mixed lithology; (2) landform may display overﬂow channels on ridges, possibly caused by glacio-ﬂuvial incision; (3) landform exhibits steep valley-proximal slopes possibly caused by presence of former ice mass; (4) glaciolacustrine deposits may be in- terbedded with diamicton. This initial interpretation is then evaluated in the context of the catchment-scale landform assemblage, using existing process-form and landsystem models from similar glacierised or glaciated landscapes. 3.2 Surface exposure dating using terrestrial cosmogenic nuclides Exposure age dating of geological surfaces using in situ terrestrial cosmogenic nuclides, ﬁrst proposed by Davis and Schaeffer (1955), is now a commonly applied geochrono- COSMOGENIC SURFACE EXPOSURE DATING 51 3.2. logical method used to date geomorphic events that have created distinct terrestrial landforms composed of previously unexposed geological material (Gosse and Phillips, 2001). In situ terrestrial cosmogenic nuclides (henceforth cosmogenic nuclides) are rare isotopes (e.g. 10Be, 36Cl, 3He, 26Al) of elements common on Earth, which are produced in rock-forming minerals at the Earth’s surface via nuclear reactions with a secondary cascade of high-energy particles (neutrons and muons), originating from primary galac- tic cosmic radiation (Gosse and Phillips, 2001). Measurements of the concentration of a stable cosmogenic nuclide (e.g. 3He, 21Ne) in a mineral sample (N) therefore represent a proxy for surface residence time, which can be converted into an exposure age (Texp): Texp = N P (3.1) (3.1) where P is the production rate of the measured nuclide scaled to the sample site (see below). This equation assumes no post-depositional erosion (i.e. loss of nuclides) and no inheritance of nuclides from prior exposure. For radionuclides (i.e. 10Be, 26Al, 36Cl) age calculations must account for radioactive decay using the relevant decay constant (Gosse and Phillips, 2001). 3.2.1 Cosmic radiation and nuclide production Primary galactic cosmic radiation at the top of the atmosphere consists primarily of protons (c. 83 %) and alpha-particles (c. 13 %) and predominantly originates within the Milky Way (Gosse and Phillips, 2001). The primary ray ﬂux is modulated by Earth’s magnetic ﬁeld and, to a lesser extent, solar activity. Primary ray particles are deﬂected by the geomagnetic ﬁeld and only penetrate when momentum exceeds the cutoff rigidity, which is a product of the incident angle and position relative to the geomagnetic ﬁeld (Gosse and Phillips, 2001). Thus, primary ray production is spatially heterogeneous. For example, incoming particles require greater energies to overcome the geomagnetic ﬁeld at lower latitudes compared to high latitudes, as geomagnetic ﬁeld lines near the equator are orientated approximately perpendicular to the average incident angles of incoming primary rays. Particles that overcome this barrier collide with atomic nuclei in the atmosphere to initiate a cascade of secondary radiation. Successive re- actions in this cascade reduce the number and energies of secondary rays that reach Earth’s surface. The neutron ﬂux at the Earth’s surface decreases exponentially with increasing rock/soil depth, such that at depths of c. 2.5 - 3 m the ﬂux is reduced to <1% of that at the surface (Dunai, 2010). Attenuation of the neutron ﬂux with depth is dependent on the density of the overlying material and the energy of the incoming neutron. Production of cosmogenic nuclei in a host rock predominantly (c. 98%) occurs via spallation, where fast and high energy neutrons and muons strike and disintegrate CHAPTER 3. METHODOLOGY 52 target nuclei (Dunai, 2010). A separate mechanism for cosmogenic nuclide production is thermal neutron capture. This occurs when incoming neutrons are slowed down to thermal energies (c. < 0.5eV) and become absorbed by target nuclei (Gosse and Phillips, 2001). Cosmogenic nuclide production via this mechanism varies between nuclides, depending on the abundance and cross sections of target nuclei (Dunai, 2010). Accurate and precise determination of an exposure age from a given cosmogenic nu- clide concentration requires accurate and precise knowledge of the rate of cosmogenic nuclide production (P: Eq. 3.1) at the speciﬁc sample site. As outlined above, P at earth’s surface varies in time and space predominantly due to variances of the geo- magnetic ﬁeld and surface elevation. can also be included in the exposure age calculation. can also be included in the exposure age calculation. 3.2.1 Cosmic radiation and nuclide production Several, commonly applied, physically-based numerical descriptions of these processes exist to predict P at a given atmospheric depth and position relative to the geomagnetic ﬁeld (Lal, 1991; Stone, 2000; Dunai, 2001; Lifton et al., 2005; Desilets et al., 2006; Balco et al., 2008). Minor differences in the treatment of magnetic ﬁeld variability, atmospheric depth and solar variability between these models, results in different estimates of P for the same sample position (Balco et al., 2008). Typically cosmogenic surface exposure ages are reported using all of these scaling schemes, however a single scaling scheme may be preferred where production rates have been derived using an independent geochronometer (e.g. ra- diocarbon), thereby allowing comparison between scaling methods. For example, in the Southern Alps, 10Be exposure ages calculated using the modiﬁed, time-dependent Lal (1991)/Stone (2000) scheme (commonly referred to as ’Lm’ after Balco et al., 2008) produced the best match to associated, independent radiocarbon chronologies (Putnam et al., 2010b). The established scaling schemes require a reference value for P, which refers to pro- duction of the given nuclide at sea-level and high latitude (SLHL). These reference values are derived from empirical calibration studies, where the concentration of the cosmogenic nuclide of interest is measured in a surface of known age (e.g. Putnam et al., 2010b; Goehring et al., 2010). Local production rates derived using such an approach are normalised to SLHL (PSLHL) using each of the scaling schemes, for subsequent applications. Where available, locally calibrated production rates minimise the uncer- tainties that arise from production rate scaling. To aid this process and to help ensure internal consistency between applications, publicly-available cosmogenic exposure age calculators have been produced for individual nuclides (e.g. Balco et al., 2008 for 10Be and 26Al; Goehring et al., 2010 for 3He). These tools allow users to generate exposure age datasets for measurements of the given nuclides, according to the scaling schemes listed above. Furthermore, site-speciﬁc inﬂuences on P derived from ﬁeld observations, such as erosion rates, local shielding by surrounding topography and sample thickness, 53 . COSMOGENIC SURFACE EXPOSURE DATING 3.2. 3.2.2 Application to palaeoglaciology CHAPTER 3. METHODOLOGY 54 The fact that these assumptions do not always hold true was illustrated in the ﬁrst application of this technique to date moraines. Phillips et al. (1990) found that scatter in nuclide concentrations, outside of the measurement uncertainty, increased with the stratigraphic age of the parent moraine landforms. Furthermore, Phillips et al. (1990) found an age reversal, whereby boulders from the stratigraphically oldest moraine returned exposure ages younger than those from a stratigraphically younger, cross- cutting landform. Hallet and Putkonen (1994) developed a topographic diffusion model that simulates moraine cross proﬁle evolution over time, based on the rate of sediment transference from the moraine crest to the moraine slopes. This model also included a boulder weathering component that simulated the shrinkage and eventual loss of moraine boulders due to sub-aerial and sub-surface weathering. Applying this model to the study site of Phillips et al. (1990), Hallet and Putkonen (1994) found that a combi- nation of moraine diffusion and boulder surface erosion could explain the observed distribution of cosmogenic surface exposure ages. Subsequent analyses of large datesets of cosmogenic surface exposure ages from moraine boulders and geomorphic process model experiments have shown that post- depositional disturbance (i.e. moraine and boulder erosion) is the most common source of scatter in cosmogenic moraine chronologies (Putkonen and Swanson, 2003; Putkonen and O’Neal, 2006; Applegate et al., 2008; Heyman et al., 2011; Applegate et al., 2012). This is because moraines are unlithiﬁed and steep-sided at the time of ice withdrawal, and frequently form in environments where gravitational and climatic processes maintain an active land surface. In contrast, moraine boulders displaying evidence for inheritance of cosmogenic nuclides due to prior exposure are relatively rare in temperate glacial environments and often readily identiﬁable as outliers given a sufﬁcient sample size (Putkonen and Swanson, 2003; Heyman et al., 2011; Balco, 2011). It is important to note that moraine exposure age datasets exhibit scatter to varying degrees. The degree of scatter is a product of sample choices and the integrated effects of the aforementioned geological processes, with respect to time. Important parameters in moraine degradation models include boulder erosion rates and the proportionality constant, topographic diffusivity. The latter represents the sediment transfer rate nor- malised by the surface slope. Thus the value of this parameter reﬂects propensity of the substrate to erode and propensity of local climate to cause erosion (e.g. precipitation, freeze-thaw cycles). 3.2.2 Application to palaeoglaciology Cosmogenic surface exposure dating is now ﬁrmly established as the primary geochrono- logical tool for constraining pre-historic glacier ﬂuctuations (see review by Balco, 2011) as it is one of the few techniques that dates the deposition of clasts directly by ice. Glacial erosion and deposition, particularly within temperate glaciers, creates two main situations that can be exploited using cosmogenic nuclides to constrain the timing of past ice ﬂuctuations. First, glaciers quarry and entrain material, which is transported and deposited at ice margins forming moraines. Once deposited, moraine boulders are exposed to the cosmic ray ﬂux. Thus, cosmogenic nuclide concentrations of boulders on the crest of moraines that are now unoccupied by glaciers are assumed to represent the length of time since ice withdrew from that location. Second, temperate glaciers erode their bed through abrasion and quarrying, removing cosmogenic nuclide in- ventories that may have accumulated during prior periods of exposure to the cosmic ray ﬂux. When the shielding ice mass retreats, abraded bedrock begins to accumulate cosmogenic nuclides. Thus, samples from such situations can also be used to infer the time since ice retreat. Since the 1980s, improvements in mass spectrometry, chemical preparation methods and understanding of cosmogenic nuclide production rates and scaling have revolutionised the ability to constrain the timing of past glacier and ice sheet variations (Balco, 2011). For example, the timing of glacier readvance in New Zealand during the late-glacial (c. 15-11 ka BP) represents a prominent case in point of how methodological improvements in cosmogenic surface exposure dating have helped to address key questions in palaeoclimatology (Denton and Hendy, 1994; Ivy Ochs et al., 1999; Kaplan et al., 2010; Putnam et al., 2010b,a, see Chapter 2). Application of cosmogenic surface exposure dating to moraine boulders, with the goal of dating past glacier ﬂuctuations, involves two main assumptions. First, that the sampled surface has remained exposed to the cosmic ray ﬂux in its current position and has not undergone surface erosion since deposition (i.e. glacier retreat). If this assumption is violated, the measured concentration of cosmogenic nuclides will re- turn a surface exposure age for the boulder that is younger than the age of the parent moraine. Second, it is assumed that the sampled moraine boulder surface contained no measurable quantity of cosmogenic nuclides immediately prior to deposition. If this assumption is violated, the measured cosmogenic nuclide concentration will overesti- mate the time since glacier retreat. 3.2.2 Application to palaeoglaciology The role of precipitation in eroding boulder surfaces and moraines is perhaps illustrated by the the excellent internal consistency of recent datasets derived from low precipitation (c. <1 m yr−1) sites in the Southern Alps (e.g. Schaefer et al., 2006; Putnam et al., 2013b; Kelley et al., 2014; Schaefer et al., 2015). Understanding the range of potetial error sources in cosmogenic moraine chronology datasets is of vital COSMOGENIC SURFACE EXPOSURE DATING 55 3.2. importance when designing a sampling campaign and interpreting results that display scatter outside of the analytical uncertainty. importance when designing a sampling campaign and interpreting results that display scatter outside of the analytical uncertainty. 3.2.3 Approach used in this thesis In this thesis I use the cosmogenic nuclide 3He to constrain the timing of glacier ﬂuctu- taions on Tongariro and Ruapehu volcanoes. The primary reason for using this nuclide is the lithology of the moraine boulders at these sites. Cosmogenic 3He is produced and quantitively retained in pyroxene, which is a relatively abundant phenocryst in the local andesitic lavas (Price et al., 2012). Quartz occurs infrequently as isolated xenoliths, but not in sufﬁcient quantities to use 10Be. Cosmogenic 36Cl represents an alternative nuclide that could be used in central North Island. However, the relatively complex production pathways and discrepancies surrounding element-speciﬁc production rates (Stone et al., 1996; Swanson and Caffee, 2001) limits the precision of 36Cl exposure ages - although recent advances are beginning to resolve these issues (Schimmelpfennig et al., 2009). Below I provide a brief introduction to cosmogenic 3He, before outlining the sam- pling protocol and the laboratory procedures used. Below I provide a brief introduction to cosmogenic 3He, before outlining the sam- pling protocol and the laboratory procedures used. 3.2.3.1 Cosmogenic 3-Helium (3He) 3He is a stable helium isotope that is extremely rare on Earth. Cosmogenic production of 3He was ﬁrst suggested by Bauer (1947), who recognised an inverse relationship between meteorite size and 3He content. Bauer postulated that the lower velocities of smaller meteorites resulted in less removal of surface material (and therefore of cosmogenic 3He) relative to larger meteorites with higher velocities. In measuring the 3He/4He ratios of meteorites, Paneth et al. (1952) recognised the chronological potential of this nuclide production. They stated, ”If the absolute amount of helium 3 produced by cosmic rays were known exactly we could calculate the time the meteorite -or at least a particular part of it - had been exposed to their inﬂuence; this would give us a second way for calculating its minimum age.” Despite this early recognition of cosmogenic 3He production in extraterrestrial bodies, the ﬁrst studies to identify excessive 3He/4He ratios in terrestrial material advocated a primordial origin (Tolstikhin et al., 1974). Craig et al. (1979) ﬁrst postulated a cosmic source as an explanation for elevated terrestrial 3He content, however this was not supported until the mid-1980s, following the devel- opment of gas mass spectrometry. Repeated measurements of a surﬁcial sample from a Hawaiian lava ﬂow yielded 3He/4He ratios of up to 418 times meteoric values, whereas drill core samples from c. 160 m depth yielded atmospheric ratios ( Craig and Poreda, CHAPTER 3. METHODOLOGY 56 1986; Kurz, 1986a,b). These depth proﬁles were interpreted to represent cosmogenic 3He production at the surface, which rapidly attenuates with depth. Cosmogenic 3He nuclei can be produced in spallation reactions from all elements except hydrogen (Niedermann, 2002). At the Earth’s surface, cosmogenic 3He production is almost entirely from spallation reactions, with little to no muonic contribution (Lal, 1987; Farley et al., 2006; Dunai, 2010). Another potential production pathway for cos- mogenic 3He is via thermal neutron capture on 6Li, although this is only signiﬁcant in rocks with elevated lithium concentrations (Niedermann, 2002). Thermal neutrons are also produced non-cosmogenically, as the product of alpha-neutron reactions initiated by U and Th decay in the host rock (Niedermann, 2002), therefore nucleogenic 3He production via thermal neutron capture can be signiﬁcant in rocks with old crystallisa- tion ages (Gosse and Phillips, 2001). Helium can also be incorporated in melt and ﬂuid inclusions during mineral crystallisation. 3.2.3.2 Sampling protocol Moraine boulder samples were collected using a portable, 16V rock saw ﬁtted with a segmented, diamond-tipped blade (methodology adapted from Suganuma et al., 2012). In the ﬁeld, each sample was measured and described (e.g. geometry, depositional context, degree of weathering) and photographed from multiple angles. Sample loca- tions and elevations were recorded using a Trimble GeoXH global positioning system, relative to the WGS84 datum. These data were differentially corrected using continuous measurements from GeoNet ’Chateau Observatory’ (’VGOB’) base station (39◦11’ 59” S, 175◦32’ 32”E; 1161 m asl), located within 15 km of all sample locations. Horizontal and vertical post-processed uncertainties for individual sample locations are < 1m. Where possible, samples were collected from several boulders per moraine, in order to assess the presence of geological scatter. I developed a set of criteria for selecting samples for cosmogenic surface exposure dating, with the aim of minimising the potential for scatter due to geological processes (as outlined in Section 3.2.2). Boulder surface integrity Physical and chemical weathering of moraine boulders, which have been exposed for 102 - 105 years, facilitates erosion of boulder surfaces. This process can reduce nuclide concentrations, resulting in exposure age calculations that underestimate the time since boulder exposure began. In this thesis, the integrity of boulder surfaces was assessed using the criteria outlined by Putnam et al. (2013b), who rejected potential samples that displayed one of more of the following: (i) a structure that exhibits signiﬁcant internal jointing/fracturing; (ii) a signiﬁcantly pitted surface, which is indicative of chemical weathering via pooled water; and (iii) boulders that display evidence of signiﬁcant spallation/disintegration. The degree to which the latter has occurred is commonly quantiﬁed through measurement of the protrusion height of resistant mineral (typically quartz) veins on the boulder surface. The premise here being that mineral veins are commonly more resistant than the surrounding groundmass, therefore the difference in height represents the minimum amount of post-depositional boulder surface lowering that has occurred via weathering processes. This data can then be integrated into age calculations to correct for nuclide loss. This technique was not possible in this study, due to the absence of mineral veins in the local igneous rocks. To reduce the potential for errors occurring in this manner, preference was given to boulders with a glassy matrix, which are more resistant to weathering processes. 3.2. COSMOGENIC SURFACE EXPOSURE DATING 3.2. COSMOGENIC SURFACE EXPOSURE DATING 57 3.2.3.1 Cosmogenic 3-Helium (3He) The contribution of 3He from this magmatic source can be determined and corrected for by crushing an aliquot of the sample in vacuo and measuring the 3He/4He ratio of the gas released from the mineral inclusions (Kurz, 1986a). Although produced in all minerals, cosmogenic 3He readily diffuses from many minerals at environmental temperatures and therefore is only useful as a geochronometer in phases with structures suited to its retention (Dunai, 2010). Olivine and pyroxene are the most commonly utilised minerals, although biotite and horn- blende (Amidon and Farley, 2012), and apatite and zircon (Amidon and Farley, 2011) also quantitatively retain helium. Cosmogenic 3He has a number of distinct advantages over other nuclides for surface exposure dating applications. First, it is radiometrically stable, therefore can be applied to date very old surfaces (e.g. Sch¨afer et al., 1999). Second, it has relatively high, and well-studied, production rate (e.g. c. 120 atoms g−1 yr−1 at SLHL (Goehring et al., 2010 compared to c. 4 atoms g−1 yr−1 for 10Be (Putnam et al., 2010b) and low detection limits, therefore can also be applied to very young surfaces (e.g. Fenton and Niedermann, 2014). Third, it is readily retained in common rock-forming minerals such as olivine and pyroxene (Goehring et al., 2010), therefore it can be applied in quartz-deﬁcient litholo- gies that preclude application of the most frequently used cosmogenic 10Be. Fourth, the sample preparation procedures (see below) for 3He measurement are inexpensive in comparison other nuclides (e.g. 36Cl, 10Be, 26Al). 3.2.3.2 Sampling protocol < 2km) to active volcanic COSMOGENIC SURFACE EXPOSURE DATING 59 3.2. vents of the sample sites in this thesis raises the possibility that cosmogenic nuclide production in the geologic past may have been reduced by snowfall and volcanic ashfall, respectively. For example, it is notable that the only previous age constraints for past glacial activity in TgVC are provided by volcanic ash beds that stratigraphically overlie moraines (e.g. (Topping and Kohn, 1973; Topping, 1974)). In this thesis, a number of methods were thus employed to minimise the potential for sampling boulders that had experienced burial and subsequent exhumation. First, boulders surfaces that stood > 50 cm (> 1 m where sampling at elevations < 1400 m asl, where tephra/soil cover is greater) above the moraine surface were preferentially selected. Salient boulder surfaces have greater exposure to wind, which reduces the potential for settling of snow or ﬁne-grained sediment. Furthermore, taller boulders are likely to have buried last/exhumed ﬁrst if burial/exhumation has occurred, therefore exposure ages from taller boulders are more likely to approach the true depositional age. Second, all samples were removed from the highest point of the boulder above the moraine surface, for similar reasons to the previous point. Third, boulders in hollows or surrounded by erosional scarps were avoided, as these characteristics could be indicative of exhumation. Fourth, low-angle (generally < 10◦dip) boulder surfaces were preferred, in order to minimise self-shielding effects. Azimuthal elevations were measured in the ﬁeld using a standard geological compass and clinometer to account for any topographic shielding by the surrounding land surface. These geometric shield- ing corrections were computed using the CRONUS-EARTH calculator (available at: http://hess.ess.washington.edu/). All shielding corrections were < 1%. Estimation of snow cover on sampled boulders is difﬁcult due to the paucity of in- formation concerning snow depths in the pre-instrumental period. Contemporary annual snow cover varies greatly between the elevation range of sampled boulders. The peak winter snowline in the study region typically occurs at c. 1600 m asl al- though snowfall can occur down to 600 m asl, therefore most of the samples in this study are subject to periodic winter snowfall. However, observations of sampled sites in winter, as well as inspection of historical satellite imagery (e.g. USGS Landsat: http://landsatlook.usgs.gov) shows that sample locations < 1800 m asl do not experi- ence signiﬁcant winter snow cover for signiﬁcant periods (e.g. weeks-months). 3.2.3.2 Sampling protocol Shielding (burial / topographic) Boulders that have experienced post-depositional shielding from the cosmic ray ﬂux will have cosmogenic nuclide concentrations that underestimate the age of the parent landform, as penetration of incoming secondary cosmic ray neutrons into the earth surface is exponentially attenuated with depth. 58 CHAPTER 3. METHODOLOGY Figure 3.1: Attenuation of cosmogenic nuclide production due to shielding beneath year round soil cover (ρcover = 1.2 - 2.0 g cm−3) and half a year of snow (ρcover = 0.1 - 0.3 g cm−3) cover of thicknesses 0 cm - 400 cm. Calculated using equations from Gosse and Phillips (2001) - their section 3.7.3 Figure 3.1: Attenuation of cosmogenic nuclide production due to shielding beneath year round soil cover (ρcover = 1.2 - 2.0 g cm−3) and half a year of snow (ρcover = 0.1 - 0.3 g cm−3) cover of thicknesses 0 cm - 400 cm. Calculated using equations from Gosse and Phillips (2001) - their section 3.7.3 The deviation of boulder exposure age from moraine age resulting from shielding is proportional to the density of the overlying material, burial depth and burial time. For example, if a moraine boulder was covered by 1 m of snow (typical density of c. 0.1 - 0.3 g cm−3) for 6 months of the year, this would decrease the cosmogenic nuclide production at the boulder surface by 3 - 8 % (Figure 3.1). By comparison, year-round burial beneath 1 m of soil with a density of c. 1.6 g cm−3 reduces production by c. 65 % (Figure 3.1). Shielding of boulder surfaces can occur beneath a variety of different media with varying thicknesses and densities. For example, a boulder at the surface today, may have been exhumed from the moraine subsurface via denudation of the surrounding moraine matrix (e.g. Hallet and Putkonen, 1994). Alternatively, a boulder at the moraine surface may have experienced post-depositional burial, for example beneath accumulating loess, volcanic ash, soil or snow. The deviation of boulder exposure age from moraine age resulting from shielding is proportional to the density of the overlying material, burial depth and burial time. For example, if a moraine boulder was covered by 1 m of snow (typical density of c. 0.1 The altitude (e.g. > 1000 m asl) and close proximity (e.g. < 2km) to active volcanic The altitude (e.g. > 1000 m asl) and close proximity (e.g. 3.2.3.2 Sampling protocol This is likely because wind transportation of snow favours erosion from topographic high points such as moraine ridge crests (prominent boulders in particular), and deposition in lee side depressions (which were avoided during sampling). In any case, Figure 3.1 shows that extreme scenarios for this location, such as 50 cm of snow for 6 months of the year, would impact the production rate by < 5 %, which is less than the present uncer- tainty in the cosmogenic 3He production rate used to derive exposure ages (Goehring CHAPTER 3. METHODOLOGY 60 et al., 2010). Thus, the role of shielding by snow cover is not considered to be a major source of uncertainty in this study. Boulder rotation The unstable, mobile nature of poorly consolidated moraine sedi- ments can result in rotation of individual boulders, thereby altering the skyward-facing surface of the boulder and reducing the concentration of cosmogenic nuclides, relative to the moraine age. In this thesis, a number of methods were employed to avoid sampling boulders that may have moved since deposition: (i) preference was given to boulders that were visibly embedded in the surrounded moraine matrix, which therefore reduces their potential mobility; (ii) if this criterion could not be met, then preference was given to boulders with a vertical a-axis, as this orientation was deemed to be the least likely result of gravitational settling; (iii) all boulders sampled were on the the crest of the parent moraine, to minimise the potential for post-depositional transport. Inheritance The erosional effects of glacial transport, particularly in temperate, moun- tain glaciers, are commonly assumed to remove nuclides from prior exposure. However, clasts transported short distances, or supraglacially, may not experience sufﬁcient sur- face erosion to remove any existing nuclide inventory. To minimise the likelihood of sampling a boulder with inherited nuclides, boulders exhibiting evidence for wet-based glacial transport and erosion (i.e. faceted sides, striae) were preferred, although such features are not always preserved. 3.2.3.3 Laboratory procedures for mineral separation and cosmogenic 3He mea- surement Thin section analysis showed that the modal pyroxene grain size in samples was 250- 500 µm. Samples were jaw-crushed, rinsed in de-ionised water and dry-sieved to isolate this size fraction. Density (> 3.1 g cm3) and magnetic separation techniques were used to isolate 150-600 mg of pyroxene grains per sample. Following Bromley et al. (2014), separated pyroxenes were ﬁrst leached in 5% hydroﬂuoric (HF) / 2% nitric (HNO3) acid solution for 24 hours, followed by a separate 10% hydrochloric (HCl) acid solution for 24 hours, to remove adhering groundmass particles. Leached pyroxene crystals were visually inspected for purity and wrapped in aluminium foil. Each sample was completely degassed by heating in a furnace to >1300◦C for 15 minutes, during which, released gases were exposed to a liquid-nitrogen chilled, charcoal trap. Extracted gases were exposed to an SAES getter before being collected on a cryogenic cold trap at <15 Kelvin. Helium was then isolated from other noble gases by heating the cold trap to 45 K. Mass spectrometry was conducted using a MAP 215-50 noble gas mass spectrometer at 3.3. PALAEOCLIMATE RECONSTRUCTION USING GLACIERS 61 Lamont-Doherty Earth Observatory, New York relative to the Yellowstone ’Murdering Mudpot’ (MM) helium standard (3He/4He ratio of 16.45Ra, where Ra = 3He/4Heair = 1.384 x 10−6), using the protocol of Winckler et al. (2005). Concentrations of Lithium (Li), Uranium (U) and Thorium (TH) were measured in multiple samples to check for pos- sible errors resulting from helium production via nucleogenic and radiogenic pathways. 3.3 Palaeoclimate reconstruction using glaciers Quantitative palaeoclimatic estimates from geomorphologically constrained glacier reconstructions are calculated using two main methods: (i) ELA reconstruction; and (ii) numerical modelling. 3.3.1.1 Accumulation area ratio (AAR) One of the most commonly used techniques is the Accumulation-Area Ratio (AAR), which utilises the observation that the accumulation area of a glacier represents a ﬁxed proportion of the total glacier area. Accumulation areas of modern glaciers glob- ally, typically occupy 50-80% of the total glacier surface area (Meier and Post, 1962), whilst empirical studies show that New Zealand glaciers most commonly have an accumulation-ablation area ratio of 2:1 (AAR= 0.67; Chinn et al., 2012). To calculate the former steady-state ELA from a reconstructed glacier outline, surface contours are interpolated across the reconstructed glacier surface, including a generalised account of the spatial variation in contour shape induced by extending and compressing glacial ﬂow in the upper and lower glacier respectively. Planimetric areas between surface contours are calculated, with which the AAR-ELA is derived, using reference AAR values (e.g. Porter, 1975). CHAPTER 3. METHODOLOGY 62 mental air temperature lapse rates (e.g. (McCarthy et al., 2008; Putnam et al., 2012)). Although these methods involve several assumptions (Plummer and Phillips, 2003), recent glacier-climate modelling in the Southern Alps (Doughty et al., 2013) has shown results that are comparable with AAR estimations for glaciers with relatively simple geometries (e.g. cirque glaciers; Kaplan et al., 2010, 2013), which provides conﬁdence in using such techniques for palaeoclimatic reconstruction. In this thesis, I use geomorphological observations and cosmogenic surface exposure dating chronologies (see above) to reconstruct former glacier geometries in several catchments on Mt. Ruapehu and Tongariro massif, for several different time periods during the late Quaternary. Sites were chosen based on the preservation of moraine ridge crests, which depict the terminus and lower margins of the former glaciers, whilst erosional landforms such as glacial cirques and glacially-trimmed cliffs provide guidance on former, upper glacier limits. 3.3.1 Equilibrium line altitude (ELA) reconstruction The equilibrium line altitude (ELA) of a glacier is the spatially-averaged altitude at the glacier surface where the climatic mass balance is zero at a given moment, although most commonly at the end of the mass balance year (Cogley et al., 2011). Thus, the ELA represents the transition from net accumulation to net ablation and is strongly related to climate, in particular temperature and solid precipitation (e.g. Ohmura et al., 1992). Increases in air temperature and/or decreases in solid precipitation tend towards more negative glacier mass balance and increases in the ELA, and vice versa. Given this relationship, the ELA is a frequently used metric for tracking contemporary and palaeoclimatic changes in glacierised and glaciated regions, respectively. ELA reconstruction from palaeoglaciers relies on accurate, manual delineations of former ice geometries (assumed to represent steady-state), which are constrained by interpretation of ice-marginal geomorphology (Porter, 1975; Benn et al., 2005). Whilst this is relatively straightforward at former glacier fronts, where lateral and terminal moraines delineate former ice margins, some uncertainty can arise in former accu- mulation areas where the geomorphological imprint of ice boundaries is less clear (e.g. Kaplan et al., 2010). A number of techniques, derived through empirical obser- vation of modern glacier-climate relationships (e.g. Porter, 1975), can be applied to geomorphologically reconstructed glacier outlines, in order to estimate the former steady-state ELA. Typically, several of these methods are applied to any one glacier reconstruction and the results evaluated in light of the catchment-speciﬁc geomorpho- logical and palaeo-glaciological characteristics (Benn and Lehmkuhl, 2000; Benn et al., 2005). Once a palaeo-ELA has been calculated, this can be related to climate using the ELA depression relative to that of local, contemporary glaciers and applying environ- CHAPTER 3. METHODOLOGY 3.3.2 Numerical glacier modelling ELA reconstruction provides a relatively quick and simple method of palaeoclimatic reconstruction from isolated cirque/valley glaciers, however uncertainties increase when dealing with ice caps / ice ﬁelds, where former glacier hypsometries in the accumulation zone are unconstrained by geological data (Plummer and Phillips, 2003). It is possible that small ice caps/ﬁelds, drained by valley outlet glaciers, existed on Tongariro massif and Mt. Ruapehu during the coldest periods of the last glacial cycle, when terrestrial air temperature in New Zealand was depressed by c. 5-8◦C (Golledge 3.3. PALAEOCLIMATE RECONSTRUCTION USING GLACIERS 63 et al., 2012; Putnam et al., 2013b; Newnham et al., 2013). Advances in the understanding of glacier-climate relationships (e.g. Oerlemans, 2001), as well as greater computing power and availability of digital datasets describing topoclimatic boundary conditions in glacierised and glaciated regions have resulted in widespread application of numeri- cal glacier models to investigate past, present and future glacier-climate relationships (e.g. (Oerlemans, 1992; Oerlemans, 2005; Plummer and Phillips, 2003; Anderson et al., 2006; Kessler et al., 2006; Doughty et al., 2013). Given the variety of glacier model com- plexity now available, a model approach can be tailored to suit the research question. For example, glacier mass balance models based on surface energy balance concepts, provide a powerful method for investigating the relative contributions of individual energy ﬂuxes to glacier mass balance, through sensitivity experiments (Plummer and Phillips, 2003; Anderson et al., 2010; Doughty et al., 2013). In this thesis I employ a distributed mass-energy balance model to simulate the effects of changing climate on past glacier mass balance, which is coupled to a two-dimensional ice ﬂow model that updates the glacial geometry based on the mass balance forcing. A full description is provided below, whilst abridged versions are given in the chap- ters that include model applications (Chapters 6,7). For dated moraines (see surface exposure dating section, above), climatic variables temperature and precipitation are experimentally varied, in order to ﬁnd combinations of forcing that result in a modelled ice geometry that matches the geological record (e.g. Plummer and Phillips, 2003; Kessler et al., 2006; Doughty et al., 2013). This approach has a number of advantages. First, the physically-based energy balance model explicitly describes the relationship between climatic variables and the surface energy ﬂuxes that contribute to glacier melt. 3.3.2.1 Energy balance model To simulate ablation, the energy balance equation is solved (Equation 3.2) using a distributed energy balance model (EBM) as developed (Oerlemans, 1992; Anderson et al., 2010) and previously applied in contemporary (Anderson and Mackintosh, 2012) and palaeo-glaciological (Doughty et al., 2013) studies in New Zealand. (3.2) QM = I(1 −α) + L ↓+L ↑+QH + QE + QR + QS + QG (3.2) where QM is the energy available for melt, I is incoming shortwave radiation, α is the albedo of the glacier surface, L ↓is incoming longwave radiation, L ↑is outgoing longwave radiation, QH and QE are sensible and latent heat ﬂuxes respectively, QR is heat input from rain, QS is the sub-surface heat ﬂux and QG is the geothermal heat ﬂux. All terms are represented in W m−2, where positive (negative) values indicate a gain (loss) of energy available for melt. The individual components of the energy balance equation are described below. Input data Terrain elevation data comes from the New Zealand School of Surveying Digital Elevation Model (NZSoSDEM) (Columbus et al., 2011) and is resampled to 100 m resolution. An ice mask depicting the distribution of contemporary glaciers on Mt. Ruapehu is created using the ’snow/ice’ data from the Land Information New Zealand NZMS260 map series. Ice thickness, based on the survey of Keys (1988), is subtracted from the DEM to create an ice-free surface. Climate data for the energy balance and snow accumulation models is from several different sources. Solar radiation and rela- tive humidity are from the Virtual Climate Station Network (VCSN) gridded datasets, sourced from NIWA CliFlo Database (NIWA, 2014). These data were resampled to the model domain resolution using bilinear interpolation. Due to temporal artefacts in the VCSN grids (Anderson and Mackintosh, 2012) that result from discontinuous measurements, present-day wind speed data comes from the National Centers for Environmental Prediction (NCEP) 850 hPa level, reanalysis data (1981-2010; Kalnay et al., 1996). This dataset is scaled against observational data and applied uniformly over the model domain. Following Anderson and Mackintosh (2012) and Doughty et al. (2013), raw temperature and precipitation data is taken from individual climate stations distributed around and within the model domain in order to improve the temperature representation at higher elevations. Temperature grids are created using the method described in Anderson and Mackintosh (2012) and Doughty et al. (2013). CHAPTER 3. METHODOLOGY CHAPTER 3. METHODOLOGY 64 3.3.2 Numerical glacier modelling Other melt models, such as temperature-index models, relate air temperature to melt rates using a proportionality factor, which implicitly includes the individual surface energy ﬂuxes. Despite generally good performance (Hock, 2003), such models are less suitable for palaeotemperature constraint due to the over-dependence of melt on temperature. Furthermore, an energy balance model allows assessment of the uncer- tainty in the temperature estimates that could result from changes in other climatic variables (e.g. precipitation, wind, relative humidity), which are often not available for the past. Second, coupling the mass balance model to the ice-ﬂow model accounts for topoclimatic (e.g. shading, temperature change with altitude) feedbacks that may result from a growing ice mass. Third, the two-dimensional model produces outputs of ice geometry that can be readily compared with moraine maps. Fourth, this style of modelling has previously been used in New Zealand (e.g. Doughty et al., 2013; Kaplan et al., 2013; Rowan et al., 2013), therefore results for the same time periods can be directly compared in order to reconstruct past climatic gradients and test hypotheses that seek to explain the mechanisms of past climate events (e.g. see Chapter 6). 3.3.2.1 Energy balance model First a sea-level reference surface (Tr) is created in a horizontal plane (sensu Tait and Zheng, 2007) by normalising point-based station data (Tst) by station elevation (zst), 3.3. PALAEOCLIMATE RECONSTRUCTION USING GLACIERS 65 using a temperature lapse rate (dT dz ): using a temperature lapse rate (dT dz ): using a temperature lapse rate (dT dz ): Tr = Tst −dT dz zst (3.3) (3.3) We use the empirically-derived, seasonal, upland (> 300 m asl) temperature lapse rate of Norton (1985) (Table 3.2). To obtain air temperature at the surface (Ta) Tr at each grid cell (z) is lapsed back to the elevation determined by the DEM, using the same temperature lapse rate: We use the empirically-derived, seasonal, upland (> 300 m asl) temperature lapse rate of Norton (1985) (Table 3.2). To obtain air temperature at the surface (Ta) Tr at each grid cell (z) is lapsed back to the elevation determined by the DEM, using the same temperature lapse rate: Ta = Tr + dT dz z (3.4) (3.4) Monthly precipitation surfaces are created using individual station data (NIWA, 2014) and a mean annual precipitation surface (Tait et al., 2006), following Anderson and Mackintosh (2012). At each station the monthly proportion of total annual precipita- tion was interpolated across the model grid and then multiplied by the mean annual precipitation surface. Table 3.2: Empirically derived, seasonal air temperature lapse rates for upland ( 300 m asl) New Zealand (Norton, 1985). Season (months) Lapse rate (◦C m−1) Spring (SON) -0.0053 Summer (DJF) -0.0057 Autumn (MAM) -0.0049 Winter (JJA) -0.0048 Shortwave radiation (I) Incoming shortwave radiation (I) is energy derived from the sun at wavelengths 0.2 - 4 µm. At a given point and time on the Earth’s surface, I comprises diffuse (Idif) and direct (Idir) components. The former represents received radiation that has been subject to atmospheric scattering by aerosols (e.g. clouds, pollu- tants, volcanic ash) or topographic reﬂection, whilst the former describes unscattered incoming solar radiation. I reaching a given point on the surface of the Earth is further limited by aspect, slope, altitude and latitude. Under clear skies, I is primarily limited by the zenith angle, whilst atmospheric transmissivity dominates attenuation in over- cast conditions (Cuffey and Paterson, 2010). In maritime, mid-latitude environments, such as New Zealand, the importance of shortwave radiation for glacier melt is reduced by the high frequency of cloud cover (Conway et al., 2014). 3.3.2.1 Energy balance model CHAPTER 3. METHODOLOGY 66 Both direct and diffuse radiation are dependent on the insolation receipt at the top of the atmosphere S, which varies temporally, according to changing orbital geometry. This is taken from the calculations of Huybers and Eisenman (2006) for the speciﬁc time window being studied (e.g. 13 ka for late-glacial simulations - Chapter 6; and 22 ka for LGM simulations - Chapter 7). Direct radiation (Idir) is calculated following Oerlemans (1992): Idir = [0.2 + 0.65(1 −n)]Scosθ (3.5) (3.5) cosθ = cos(β)cosZ + sin(β)sinZcos(ϕsun −ϕslope) (3.6) (3.6) where n is cloudiness calculated according to Anderson et al. (2010), θ is the angle of incidence between the topographic slope angle (β), Z is the solar zenith angle, ϕsun is the the solar azimuth angle and ϕslope is the slope zenith angle (aspect). where n is cloudiness calculated according to Anderson et al. (2010), θ is the angle of incidence between the topographic slope angle (β), Z is the solar zenith angle, ϕsun is the the solar azimuth angle and ϕslope is the slope zenith angle (aspect). For diffuse radiation (Idif), contributions from topographic reﬂectance and aerosols other than water vapour (cloudiness) are neglected, following Oerlemans (1992): Idif = [0.8 −0.65(1 −n)]Ssin π 2 −Z ! (3.7) (3.7) The proportional contributions from Idir and Idif are then adjusted: ortional contributions from Idir and Idif are then adjusted: I = tatc(Idif + Idir) (3.8) (3.8) where ta (Equation 3.9) and tc (Equation 3.10) are transmissivity of air and clouds respectively (Oerlemans, 1992): where ta (Equation 3.9) and tc (Equation 3.10) are transmissivity of air and clouds respectively (Oerlemans, 1992): ta = (0.79 + 0.000024z)            1 −0.08 π 2 −γ π 2            (3.9) (3.9) tc = 1 −(0.41 −0.000065z)n −0.37n2 (3.10) (3.10) where z is surface elevation, and γ is solar elevation. where z is surface elevation, and γ is solar elevation. The contribution of I to the total surface energy, is determined by the albedo (α), or reﬂectivity, of the ground surface. This can vary signiﬁcantly in space and time, accord- ing to the land surface type (e.g. water, snow, vegetation, bare rock). For snow and 3.3. 3.3.2.1 Energy balance model PALAEOCLIMATE RECONSTRUCTION USING GLACIERS 67 ice, albedo depends on factors such as crystal structure, impurity content and water content, all of which change with time (Oerlemans, 1992). Across a glacier α can be as high as 0.98 (i.e. 98% reﬂectance) for fresh snow, or as low as 0.06 for debris-laden ice (Cuffey and Paterson, 2010). Many parameterisation schemes exist for modelling spatial and temporal changes in α (Brock et al., 2000). I parameterise α using the ’ELA-dependent’ method of Oerlemans (1992). First, a ’background albedo’ (αb) is determined: αb = 0.43 + 0.18 π arctan z −zELA + 300 200 ! (3.11) (3.11) where zELA is the equilibrium line altitude. α is then calculated: α = αsnow −(αsnow −αb)e−5d (3.12) (3.12) where αsnow represents the albedo of snow (0.72) and d is equal to snow depth. For areas of glacier cover without snow, the albedo is set to 0.34, a typical value for ice (Cuffey and Paterson, 2010). Longwave radiation (L) Longwave radiation (L) is emitted by any physical object with a temperature above absolute zero (Cuffey and Paterson, 2010). L is supplied to glacier surfaces (L ↓) through radiative emission at infrared wavelengths (c. 4 - 120 µm) from the atmosphere and surrounding terrain, and is lost through emission from the glacier itself (L ↑). Snow absorbs all radiation at infrared wavelengths (Cuffey and Paterson, 2010), thus longwave radiation ﬂux can be described by the Stefan-Boltzmann law for blackbodies: L = ϵσT 4 (3.13) (3.13) where ϵ is the emissivity of the emitting object (i.e. 1 for blackbodies), σ is the Stefan- Boltzmann constant (5.67 x 10−8 W m−2 K−4) and T is the absolute temperature of the emitting object. L ↑is calculated assuming the glacier surface is at melting point and that snow is a perfect radiator (ϵ = 1). Thus, L ↑is assumed constant, at 317 W m−2. Atmospheric water vapour absorbs and re-radiates energy as longwave radiation, and this process can supply a signiﬁcant amount of energy for glacier melt, particularly in humid conditions. In alpine settings, radiation from exposed rocks and sediment CHAPTER 3. METHODOLOGY 68 at glacier margins can also provide a signiﬁcant source of energy (Benn and Evans, 2010). 3.3.2.1 Energy balance model L ↓thus comprises atmospheric and terrain components, which are not perfect radiators therefore they are related to Equation 3.13 by their respective emissivities (ϵ): at glacier margins can also provide a signiﬁcant source of energy (Benn and Evans, 2010). L ↓thus comprises atmospheric and terrain components, which are not perfect radiators therefore they are related to Equation 3.13 by their respective emissivities (ϵ): L ↓= ϵeffσT 4 a v + ϵtσT 4 t (1 −v) (3.14) (3.14) where Ta and Tt are the temperature of air and terrain, respectively. Where covered by snow, Tt is assumed to be 273 K and where not covered Tt is assumed equal to Ta. v is the topographic viewﬁeld, calculated from the DEM, using the algorithm of Corripio (2003). ϵt is the emissivity of terrain, which is set to 0.4 (Plummer and Phillips, 2003), and ϵeff is the effective atmospheric emissivity, calculated according to Konzelmann et al. (1994): ϵeff = ϵc(1 −np) + ϵocnp (3.15) (3.15) I follow Anderson et al. (2010) in using p = 1, based on measurements at Brewster Glacier, New Zealand. Emissivity for overcast conditions (ϵoc) is set to 0.924. ϵc is clear sky emissivity, calculated as (Konzelmann et al., 1994): ϵc = 0.23 + 0.484 ea Ta 1/8 (3.16) (3.16) where ea is vapour pressure. Turbulent ﬂuxes (QH, QE) Sensible (QH) and latent (QE) heat describe energy trans- fers between the atmospheric boundary layer and the glacier surface. Eddies in the atmospheric boundary layer, enhanced by greater wind speed and glacier surface roughness, promote greater turbulent ﬂux exchange (Cuffey and Paterson, 2010; Equa- tions 3.17 and 3.20). QH is the direct conduction of energy between the glacier surface and the overlying air, and the direction of heat transfer is dependent on the sign of the temperature gradient between these media (Equation 3.17). QH = ρaircpkHU(Ta −Tsurf) (3.17) (3.17) where ρair is the density of ambient air, cp is the speciﬁc heat capacity of ambient air, and U is wind speed at height z = 2 m. Tsurf is assumed to be 0◦C. kH is the exchange 3.3. PALAEOCLIMATE RECONSTRUCTION USING GLACIERS 69 3.3. 3.3.2.1 Energy balance model In continental settings average geothermal heat ﬂuxes are c. 50 - 80 mW/m2, which will melt approximately 6 mm water equivalent of temperate ice annually (Figure 3.2). However, geothermal heat ﬂuxes show large spatiotemporal variation, according to age and lithology of the bed, volcanism, crustal thickness and radioactive decay (Van der Veen et al., 2007). In active volcanic regions, convection and advection of heat to the surface via mantle upwelling and redistribution can raise the heat ﬂux by several orders of magnitude (Figure 3.2). In glacierised, active volcanic regions, geothermal heat ﬂuxes on the order of 100 - 105 W m−2 have been inferred from glacier calorimetry (Clarke et al., 1989; Cuffey and Paterson, 2010). If sustained, such heat ﬂuxes can have a non-trivial impact on glacier mass balance, however these extreme cases are typically only sustained over the order of days to weeks (e.g. Gudmundsson et al., 2004). Melt = QG ρLf (3.22) (3.22) Given the spatial and temporal transience of geothermal heating events and the absence of data constraining past volcanic activity in the central North Island, it is not possible to include such effects into the palaeo-glacier simulations. Whilst it is possible that past volcanic activity may have impacted past glacier mass balance and ice dynam- ics, empirical observations from other glacierised, volcanically active regions have demonstrated that these effects are often highly localised and temporally restricted (Gudmundsson et al., 1997; Gudmundsson et al., 2004). It is therefore reasonable to assume that climate is the dominant driver of glacier behaviour over the timescales considered in this thesis. I employ a nominal geothermal ﬂux of 1 W m−2 ( = 10 cm w.e. annual melt; Figure 3.2) and discuss the possible implications of past volcanism for the palaeoclimatic interpretations derived from glacier model experiments. 3.3.2.1 Energy balance model PALAEOCLIMATE RECONSTRUCTION USING GLACIERS 69 coefﬁcient for sensible heat: coefﬁcient for sensible heat: kH = k0 2 log z z0 log z z0H (1 −5.2Rb)2 (3.18) (3.18) where K0 = 0.4 is von K´arm´an’s constant, Z0 and Z0H are the effective roughness length for wind and sensible heat, respectively (Anderson et al., 2010). Rb is the Richardson stability criterion, which accounts for boundary layer stability resulting from temperature inversions and reduced turbulent heat exchange: Rb = g Ta (Ta −Tsurf)(z −z0) U 2 (3.19) (3.19) where g = 9.8 m s−2, is acceleration due to gravity. Rb is applied in Equation 3.18 when R > 0 and U > 1. Latent heat transfer results from phase changes during mass exchange between the atmosphere and the glacier surface. For example, melting snow or ice uses energy at a rate of 334 J g−1 (Lf - the speciﬁc latent heat of fusion of ice), whilst condensation on the surface supplies energy for melt at a rate of 2834 J g−1 (Lv - the speciﬁc latent heat of vapourisation). Phases changes at the surface are dependent on the vapour pressure gradient between the glacier surface and overlying air: QE = 0.622ρicekEULv (q −qs) p (3.20) (3.20) where kE is the exchange coefﬁcient for latent heat (Equation 3.18), q is the vapour pressure of ambient air, qs is the vapour pressure of air at the glacier surface, and p is air pressure. Heat from rainfall (QR) Sensible heat transfer from rainfall comprises a minor pro- portion of glacial energy budgets (Cuffey and Paterson, 2010): QR = cwPTa (3.21) (3.21) where cw is the speciﬁc heat capacity of water (Table 3.3), P is the rain rate. Rainfall temperature is assumed to equal Ta. Sub-surface heat ﬂux (QS) The sub-surface heat ﬂux, also commonly referred to as ground heat ﬂux, describes energy exchanges between the glacier surface and the glacier interior. As I assume that the ice is temperate and that the glacier surface is constantly at the melting point (Oerlemans, 1992), there is no temperature gradient, therefore QS = 0. 70 CHAPTER 3. METHODOLOGY Geothermal heat ﬂux (QG) Geothermal heat describes the energy retained within the Earth, which can contribute to basal melt of glaciers. The contribution of geothermal heat to glacier mass balance is generally considered negligible in comparison to the sur- face ﬂuxes (Cuffey and Paterson, 2010). 3.3.2.2 Accumulation Model All precipitation when Ta < 0.5◦C is assumed to fall as snow (Table 3.3). Potential con- tributions from avalanching are considered to be negligible, due to the low probability of non-glaciated terrain situated above the former valley glaciers on the volcanoes. This is consistent with the present-day, where few peaks exist at elevations greater than the contemporary glaciers on Mt. Ruapehu. The effects of wind redistribution on snow accumulation are also not accounted for in the accumulation model. This represents a 3.3. PALAEOCLIMATE RECONSTRUCTION USING GLACIERS 71 Figure 3.2: Annual melt total of temperate ice estimated for empirically derived geothermal heat ﬂuxes from different geological settings (values taken from summary (p.418) of Cuffey and Paterson, 2010). Melt rates calculated using ice density of 917 kg m−3 and latent heat of fusion = 334 kJ kg−1 (Equation 3.22). Figure 3.2: Annual melt total of temperate ice estimated for empirically derived geothermal heat ﬂuxes from different geological settings (values taken from summary (p.418) of Cuffey and Paterson, 2010). Melt rates calculated using ice density of 917 kg m−3 and latent heat of fusion = 334 kJ kg−1 (Equation 3.22). potential source of uncertainty, as previous mass balance investigations have noted the role of wind as a key control on the spatial variability in mass balance on Mt. Ruapehu (e.g. Krenek, 1959). Modelling of wind-driven snow accumulation is non-trivial (Dadic et al., 2010) and I discuss the potential role of this uncertainty for the palaeoclimatic interpretations made in Chapters 6 and 7. potential source of uncertainty, as previous mass balance investigations have noted the role of wind as a key control on the spatial variability in mass balance on Mt. Ruapehu (e.g. Krenek, 1959). Modelling of wind-driven snow accumulation is non-trivial (Dadic et al., 2010) and I discuss the potential role of this uncertainty for the palaeoclimatic interpretations made in Chapters 6 and 7. 3.3.2.3 Ice ﬂow model Glaciers transfer mass via a combination of internal deformation, basal sliding and bed deformation. These mechanisms form an important component of glacier response to climate change, therefore it is necessary to couple a numerical model describing ice ﬂow to the mass balance model. The material law describing the ﬂow of glacier ice is that of an incompressible, viscous The material law describing the ﬂow of glacier ice is that of an incompressible, viscous CHAPTER 3. METHODOLOGY 72 Table 3.3: Optimal energy balance and ice ﬂow model parameter settings. Parameter Value Source Energy balance model: Temperature lapse rate (δT δz ) Seasonal (Table 3.2) (Norton, 1985) Snow-rain threshold temperature (Ts) 0.5 ◦C (Hendrikx and Hreinsson, 2012) Snow albedo (αsnow) 0.72 (Oerlemans, 1992) Ice albedo (αice) 0.34 (Oerlemans and Knap, 1998) Ice roughness (zice) 0.004 m (Anderson and Mackin- tosh, 2012) Snow roughness (zsnow) 0.001 m (Brock et al., 2006) Modern snowline (zELA) 2483 m asl (Keys, 1988) Ice ﬂow model: Typical sliding velocity (Uc) 50 m yr−1 This study Glen’s ﬂow law coefﬁcient (A) 2.14 x 10−16 Pa−3 a−1 (Paterson, 1994) Glen’s ﬂow law exponent (n) 3 Characteristic driving stress (τc) 100 kPa able 3.3: Optimal energy balance and ice ﬂow model parameter settings. meter Value Source ﬂuid, and can be represented using the general Stokes ﬂow equation for creeping motion: µ∇2u −∇p + f = 0 (3.23) (3.23) where µ is the dynamic viscosity of the ﬂuid, u is ﬂuid velocity, p is pressure and f is an external force. Inertial terms that form part of the full Navier-Stokes equations are neglected due to the slow movement of glacial ice. where µ is the dynamic viscosity of the ﬂuid, u is ﬂuid velocity, p is pressure and f is an external force. Inertial terms that form part of the full Navier-Stokes equations are neglected due to the slow movement of glacial ice. For ice, Equation 3.23 takes the form: For ice, Equation 3.23 takes the form: ∇· τ ′ ij −∇p + ρiceg = 0 (3.24) (3.24) where the viscous force (µ∇2u in Equation 3.23) is represented by the deviatoric stress tensor (τ ′ ij) and the external force (f in Equation 3.23) is the weight of the ice. 3.3.2.3 Ice ﬂow model Solving the full Stokes equation is computationally expensive (Leysinger Vieli and Gudmundsson, 2004), therefore many studies instead employ a simpliﬁed set of equa- tions known as the ’Shallow Ice Approximation’ (SIA; Hutter, 1983; Morland, 1984). The SIA equations were developed for efﬁcient simulation of ice sheets and exploit the low aspect ratio (H/W) of these ice masses, which mean that the driving stress can be 3.3. PALAEOCLIMATE RECONSTRUCTION USING GLACIERS 73 assumed to be entirely balanced by vertical shearing at the bed (Pattyn, 2006). Thus, the effects of lateral shear and longitudinal extension/compression are neglected within the SIA and ice ﬂow is simply related to changes in the surface slope and thickness of ice (Egholm et al., 2011). Whilst this assumption holds for large parts of the polar ice sheets, the SIA may not accurately represent the full stress ﬁelds of mountain glaciers, where bed topography can be complex and ice geometry aspect ratios can be high in comparison to ice sheets. However, comparison of full Stokes and SIA-based models shows that, in mountainous settings, the SIA produces comparable results to methods solving the full stress ﬁeld (Leysinger Vieli and Gudmundsson, 2004; Le Meur et al., 2004). This method is therefore frequently applied in steady-state model simulations of past alpine glaciation (e.g. Plummer and Phillips, 2003; Doughty et al., 2013; Rowan et al., 2013), where uncertainty in the mass balance terms far outweighs that due to ice ﬂow (Greuell, 1992; Leysinger Vieli and Gudmundsson, 2004). In this thesis, I employ a vertically-integrated 2D ice ﬂow model using the SIA (Kessler et al., 2006; Doughty et al., 2013). Ice ﬂow velocity due to internal deformation (Ud) is given as: ⃗Ud = 2 5AH⃗τ n b (3.25) (3.25) where ⃗Ud is vertically-averaged ice velocity from internal deformation, A is Glen’s ﬂow law coefﬁcient, set to A = 2.14 x 10−16 Pa−3 yr−1, ⃗τb is the gravitational driving stress (⃗τb = ρgH∇z), and n is Glen’s ﬂow law exponent, set to n = 3 (Cuffey and Paterson, 2010). Following Kessler et al. (2006), a sliding term is also included: Following Kessler et al. (2006), a sliding term is also included: Us = Uce 1−τc ⃗τb (3.26) (3.26) where Uc is the characteristic sliding velocity, set to Uc = 50 m yr−1, and τc is the gravita- tional driving stress that causes Us, set to τc = 100 kPa (Cuffey and Paterson, 2010). 3.3.2.3 Ice ﬂow model where Uc is the characteristic sliding velocity, set to Uc = 50 m yr−1, and τc is the gravita- tional driving stress that causes Us, set to τc = 100 kPa (Cuffey and Paterson, 2010). dH dt = M −∇· ⃗q (3.27) dH dt = M −∇· ⃗q (3.27) (3.27) where H is ice thickness, t is time, ⃗q is ice ﬂux (⃗Ud + ⃗Us) and M is mass balance. Equation 3.27 evolves glacier geometry through time. Ice velocities are calculated on a grid offset from ice thickness and the ﬂux gradients are used to update ice thickness using a forward explicit time-step (Hindmarsh and Le Meur, 2001). To account for CHAPTER 3. METHODOLOGY 74 boundary effects that may violate mass conservation in the ﬁnite difference formulation (e.g. Plummer and Phillips, 2003), the bed topography is smoothed using a 3x3 moving window to reduce high bed slopes in the upper catchment, and an ice ﬂux correction is applied. For each cell, total ice divergence cannot exceed the total mass contained in the source cell. At each time step a check is carried out to make sure mass is conserved and if not, the excess ice is removed (sensu Plummer and Phillips, 2003). 3.3.2.4 Model assessment As a test of model performance, Figure 3.3 compares modelled, steady state ice extent on Ruapehu, calculated using the energy balance model in combination with the ice ﬂow model (model parameters in Table 3.3), against mapped ice extent and observed glacier ELAs (taken from Keys, 1988). Modelled ice is more extensive than observa- tions in most catchments, particularly for Whangaehu and Whakapapa glaciers. In the Whangaehu catchment, the most likely reason for this mismatch is elevated geothermal heat ﬂux around Crater Lake, which is not accounted for in the model. As Figure 3.3 shows, ice accumulates in the region currently occupied by Crater Lake (which ﬂuctuates in temperature between 20-60 ◦C), which is not the case in reality. The excess ice predicted by the model in this region feeds into several glacier catchments (largely the Whangaehu), causing the observed over prediction. It is notable that modelled ice extent in catchments that do not receive any/as much ice from Crater Lake (i.e. Mangaehuehu, Wahianoa) are better aligned with modern observations. The size of the modelled Whakapapa Glacier is also greater than observed, which is probably due to three reasons. First is the potential contribution from the excess ice in the Crater Lake region as discussed above. Second, the model resolution (100 m) smooths out the narrow rock lip that separates the Whakapapa catchment from Summit Plateau. Field observations show that this topographic constraint currently prevents ice leaving Summit Plateau, but this effect is not captured at the current model resolution. Third, this east facing glacier is exposed to prevailing westerly winds, which may may act to remove snow accumulation from the glacier. This process is not accounted for in the model. Despite the slight over-prediction of modelled ice extent, modelled ELAs of individual glaciers match closely with observed end of summer snowlines (Keys, 1988). The modelled ELAs of the Whangaehu, Mangatoetoenui, Mangaturuturu and Mangaehuehu glaciers all fall within or close to the observed ranges. In summary, there is good agreement between the modelled and observed ice ge- ometries on Mt. Ruapehu. Some important controls on present-day glacier distribution are not represented in the model, which cause some deviation between modelled and 3.3. PALAEOCLIMATE RECONSTRUCTION USING GLACIERS 75 observed ice geometries. However, Crater Lake is believed to have formed in the Late Holocene (c. 3.3.3 Application of these methods in this thesis My goal is to constrain the timing and magnitude of past climate change in central North Island, using glaciers as a climate proxy. The four research chapters apply vari- ous combinations of the 3 methods described above to answer the research questions outlined in Chapter 2. I conducted ﬁeld-based investigations of the glacial geology to increase the accuracy of existing mapping efforts, which have predominantly utilised remote-sensing products for landform interpretation (McArthur and Shepherd, 1990; Barrell, 2011). Coupled with cosmogenic surface exposure dating, this detailed map- ping has shed new light on frequency and magnitude of past glacier ﬂuctuations, as preserved in the local geomorphological record. For example, in Chapter 6 I target a moraine-set previously correlated to the LGCP by McArthur and Shepherd (1990). Field investigations showed that older moraines were present in vegetated regions down val- ley, which may instead pertain to the LGCP. Cosmogenic 3He surface exposure dating supports this interpretation and places the formation of the inboard moraines in the late-glacial (c. 15-11 ka), which represents the northern most expression of glacial activ- ity in New Zealand at this time. In Chapter 5, I use cosmogenic surface exposure dating to directly constrain the formation of moraines in Mangatepopo Valley, which occurred at c. 31-21 ka and c. 60 ka. Geomorphological and geomorphometric investigation of these landforms permitted extrapolation of these ages to glacial landforms across both volcanoes, which provided reﬁned constraint of ice limits in the region during the LGCP. Well deﬁned and well dated former glacial geometries provide useful targets from which to extract quantitative palaeoclimatic information using glacier models. In Chapters 6 and 7, I constrain the range of climatic forcings associated with mapped and dated (or morphostratigraphically correlated) glacial extents, pertaining to the late-glacial (c. 13 ka) and the LGCP in New Zealand (c. 30-18 ka; Barrell et al., 2013). In these glacier simulations, present day temperatures are systematically reduced in order to ﬁnd the degree of cooling, relative to present, that produces a steady-state glacier with a geometry that matches the geological constraints. Modern climate represents a useful datum from which to begin these simulations as it can be well constrained by observations. Each former glacier geometry can be reproduced by a range of combined temperature-precipitation changes. Due to uncertainty in past precipitation change, I present the range of palaeotemperature estimates that accompany precipitation changes of up to ± 50%. 3.3.2.4 Model assessment 3 ka; Donoghue et al., 1997), therefore post-dates the time periods of interest for glacier modelling applications in this thesis. Prior to 3 ka, the active vent location on Mt. Ruapehu is largely unknown and likely to have been transient, based on post-glacial lava distribution (e.g. Hackett and Houghton, 1989). Thus, although this currently represents an important energy source controlling snow accumulation patterns, it is not possible to constrain this for the past. Similarly, whilst small-scale topography not resolved by the model is also currently an important control on ice distribution, this is a smaller issue for previous periods when mountain glaciers were thicker and ice ﬂow was less constrained by topographic undulations at short length scales (i.e. < 102 m). Thus, I conclude that the present day situation is well-represented by the input data and model formulation, which provides a good datum from which to simulate past ice geometries. Figure 3.3: Steady state ice extent and mass balance on Mt. Ruapehu, compared to mapped ice extent and end of summer snowline ranges reported by (Keys, 1988). Figure 3.3: Steady state ice extent and mass balance on Mt. Ruapehu, compared to mapped ice extent and end of summer snowline ranges reported by (Keys, 1988). CHAPTER 3. METHODOLOGY 76 3.3.3 Application of these methods in this thesis In each of these studies, I draw on the existing palaeoclimatic literature (e.g. proxy reconstructions, climate model experiments) to inform whether precipita- tion was likely enhanced or reduced, relative to present. The addition of quantitative palaeoclimate reconstructions in central North Island increases the spatial resolution of palaeotemperature estimates, which are useful for tracing the drivers of past climatic 3.3. PALAEOCLIMATE RECONSTRUCTION USING GLACIERS 77 change. For example, in Chapter 5 I compare my results to similar work from the central Southern Alps (Doughty et al., 2013; Kaplan et al., 2013) to test the theory that the magnitude of atmospheric cooling in North Island during the late-glacial climate reversal (c.13 ka; Barrell et al., 2013) was lower than that in South Island (Newnham et al., 2012). A glacier modelling approach also allows exploration of unconstrained parameters, which improves understanding of both the uncertainty of the palaeoclimate estimates, and glacier-climate-topography relationships. For example, in all model applications I conduct sensitivity tests of energy balance and ice-ﬂow parameters. In these tests, each parameter is varied in turn, in order to quantify the sensitivity of the palaeotemper- ature estimates to the parameter in question. Simpler glacier-climate reconstruction techniques, such as manual ELA reconstruction, have many inherent uncertainties that are often not quantiﬁable, therefore potential errors in the resultant palaeoclimate estimates are poorly constrained and often underestimated (Plummer and Phillips, 2003). In Chapter 6, I design model experiments that quantify the length sensitivity of glaciers on Mt. Ruapehu to climate changes that are unevenly distributed across the mass balance year (i.e. changing seasonality). This results in improved understanding of glacier-climate relationships, which is useful for interpreting the climatic signiﬁcance of moraine records, particularly during abrupt climate events characterised by shifts in seasonality (Vandergoes et al., 2008; Sikes et al., 2013). In Chapter 7, I compare glacier model derived temperature estimates of the LGCP in central North Island using current topography and a geologically-based reconstruction of the land surface during the LGCP. These experiments permit assessment of whether post-glacial volcanism has altered the landscape to a degree that modern topography does not provide suitable boundary conditions for palaeoglacier modelling. CHAPTER 3. METHODOLOGY 78 CHAPTER 3. METHODOLOGY 4.1 Abstract Calculation of surface exposure ages, using in situ cosmogenic nuclides, requires knowl- edge of local production rates. Here, I report an attempt to calibrate cosmogenic 3He production in the south west Paciﬁc region. I present a new radiocarbon chronology that precisely constrains emplacement of the Murimotu Formation, a large debris avalanche deposit in central North Island, New Zealand (c. 830m asl; 39◦S), which occurred c. 10.4-10.6 cal. ka before present. Measurements of cosmogenic 3He in large andesitic blocks deposited at the surface during this event yield a reference sea- level, high-latitude production rate of 120 ± 12 atoms gram−1 yr−1, using the ’Lm’ scaling scheme. This is consistent with a recent global compilation of production rates (Goehring et al., 2010), comprised predominantly of calibration sites located in the Northern Hemisphere. Thus, I conclude that the globally compiled cosmogenic 3He production rate is appropriate for use in the south-west Paciﬁc region. Using indepen- dent, proximal calibrations of cosmogenic isotopes 10Be and 14C from quartz in New Zealand, I derive cosmogenic 3He/10Be and 3He/14C production ratios of 32.2 ± 3.2 and 10.6 ± 1.6, respectively. 4.2 Introduction Cosmogenic surface exposure dating is an important geochronological tool for con- straining the timing of past climatic and geological events. Many cosmogenic nuclides exist (e.g. 10Be, /26Al, /36Cl, /3He,/21Ne), each with a unique set of properties that is 79 CHAPTER 4. 3HE PRODUCTION RATE IN NZ 80 useful in different applications and settings. Cosmogenic 3-Helium (3Hecos) is a noble gas with a number of useful properties for Quaternary research. First, 3Hecos is stable, therefore can be applied to date very old surfaces (e.g. Sch¨afer et al., 1999). Second, it has a relatively high production rate (Goehring et al., 2010; Blard et al., 2013b) coupled with low detection limits, therefore has the potential to be applied to very young sur- faces (e.g. Kurz and Brook, 1994; Fenton and Niedermann, 2014). Third, it is readily retained in common rock-forming minerals such as olivine (Kurz, 1986a) and pyroxene (Bruno et al., 1997; Sch¨afer et al., 1999), therefore it can be applied in quartz-deﬁcient lithologies that preclude application of cosmogenic 10Be. Fourth, sample preparation procedures for 3Hecos (e.g. (Bromley et al., 2014) are relatively inexpensive, both tempo- rally and ﬁnancially, in comparison other nuclides, whilst measurements are made on sector-ﬁeld noble gas mass spectrometers, which are commercially available. Accurate and precise knowledge of the rate at which cosmogenic nuclides are produced at the Earth’s surface is a fundamental requirement for cosmogenic surface exposure dating applications. Geological calibrations of production rates provide increased accu- racy for proximal applications as they integrate past changes in atmospheric pressure, geomagnetic and/or solar modulation effects, thus reducing the uncertainty inherent in existing scaling protocols (Balco et al., 2008). A relatively large number of production rate calibration studies exist for 3Hecos (e.g. Kurz et al., 1990; Cerling and Craig, 1994; Licciardi et al., 1999, 2006; Dunai, 2001; Ackert et al., 2003; Blard et al., 2006, 2013b; Goehring et al., 2010; Amidon and Farley, 2011; Foeken et al., 2012; Fenton et al., 2013). However, these studies are largely restricted to the Northern Hemisphere (Figure 6.1). Recent compilations of these datasets (Goehring et al., 2010; Blard et al., 2013b) show 3Hecos production is consistent over large spatial and temporal scales and suggest 119 ± 9.9 atoms g yr−1 (in pyroxene - Goehring et al., 2010, scaled with the ’Lm’ protocol) as a widely applicable sea-level high-latitude (SLHL) production rate. However, this number and its consistency is challenged by Foeken et al. 4.2 Introduction (2012), who report 3Hecos production rate estimates from a low latitude site in the Cape Verde islands (15◦N; Figure 6.1), which are up to 20% lower than Goehring et al. (2010). At the upper end, Ackert et al. (2003), consider past atmospheric pressure anomalies to explain relatively high (c. 130-139 atoms g yr−1) 3Hecos production rate estimates from Patagonia (47◦S). Given the general paucity of Southern Hemisphere 3Hecos production rate sites (Figure 6.1) and the potential for regional anomalies in cosmogenic nuclide production (e.g. Foeken et al., 2012), I aim to test the 3Hecos production rate of Goehring et al. (2010) in the south west Paciﬁc region. To do this, I target a debris avalanche deposit on the lower northwestern ﬂanks of Ruapehu (Topping, 1974; Palmer and Neall, 1989), a composite andesitic stratovolcano situated in the central North Island of New Zealand (39◦09’S, 4.3. MURIMOTU FORMATION 81 Figure 4.1: Distribution of individual calibration sites (red stars) that make up the existing global 3Hecos production rate (from Goehring et al., 2010). Note the paucity of sites in the eastern and southern hemispheres. The location of this study is shown on the inset map. Figure 4.1: Distribution of individual calibration sites (red stars) that make up the existing global 3Hecos production rate (from Goehring et al., 2010). Note the paucity of sites in the eastern and southern hemispheres. The location of this study is shown on the inset map. 175◦29’E). This deposit includes buried organic horizons and prominently exposed an- desitic boulders that protrude the present-day surface, thus providing an ideal situation to calibrate local 3Hecos production. In this study, I ﬁrst produce a reﬁned, independent radiocarbon chronology for this event, before calibrating 3Hecos production using 3Hecos concentrations measured in pyroxene separated from the associated andesitic boulders. 4.3 Facies architecture and stratigraphy of the Murimotu Formation debris avalanche deposit The Murimotu Formation (MF) is a discrete debris avalanche deposit on the northwest slopes of Mount Ruapehu (Figure 4.2). The hummocky surface topography and oc- currence of large volcanic clasts in this deposit has intrigued geologists for over 120 years (Hill, 1891; Park, 1926; Bossard, 1928; Topping, 1974; Hackett and Houghton, 1989; Townsend et al., 2008). Following observations of the May 18th 1980 syn-eruptive sector collapse of Mount St. Helens (Voight et al., 1981; Glicken, 1991), the MF was recognised as the product of a similar event sourced from the upper ﬂanks of Mount Ruapehu (Hackett and Houghton, 1989; Palmer and Neall, 1989). Identiﬁcation of similar deposits adjacent to Taranaki Volcano in western North Island (Neall, 1979; CHAPTER 4. 3HE PRODUCTION RATE IN NZ 82 Ui et al., 1986; Neall and Alloway, 1986), led to the development of a sedimentary facies scheme applicable for topographically unconstrained debris avalanche deposits (Palmer et al., 1991). The internal structure of debris-avalanche deposits can typically be subdivided into two major components: (1) fragmental rock clasts (FRCs), and (2) matrix. An FRC is deﬁned as a fragmented or deformed piece of lava or layered volcaniclastic material commonly preserving stratiﬁcation and/or intrusive contacts formed within the original volcanic ediﬁce. The most commonly recognised FRC is andesitic lava that is commonly brec- ciated forming a diamicton of homogeneous composition. The scheme of Sundell and Fisher (1985) is used deﬁne the FRC size classes: boulder (0.256 - 10 m), megaboulder (10 - 100 m), block (100 - 1000 m) and megablock (1-10 km). A gravel-sized class of FRCs (0.002 - 0.256 m) is also used. The matrix is referred to as inter-clast matrix and is deﬁned as all the material within the deposit surrounding the FRCs and <0.002 m in diameter. It should not be confused with the matrix of an FRC, which is here termed intra-clast matrix. Inter-clast matrix includes all blended, unsorted, and unstratiﬁed parts of the deposit and consists of material ranging in size from clay to very coarse sand. Incorporated with the interclast matrix are rip-up clasts of plastically distorted soil, peat and tephra layers, clasts with variable rounding, and wood fragments derived from the terrain beneath. Inter-clast matrix is more abundant in inter-mound areas and is predominant in the distal and lateral margins of the deposit. 4.3 Facies architecture and stratigraphy of the Murimotu Formation debris avalanche deposit Three sediment facies are recognised within the MF (Palmer and Neall, 1989), sub- sequently referred to as axial a, axial b, and marginal facies (Palmer et al., 1991). Axial a facies is deﬁned as a mappable area where fragmental rock clasts (FRCs) dominate (2 - 36 m length), with <30% inter-clast matrix, and where the surface topography is dominated by a concentrated area of steep sloping hills and mounds up to 30 m high with basal diameters as much as 300 m. Axial b facies is deﬁned as an area where the proportion of inter-clast matrix is subdominant to dominant (30-90%) relative to FRCs and where the surface topography is dominated by sparsely distributed mounds and hills <10 m high with basal diameters <25 m. This facies corresponds with the mixed block and matrix facies of (Glicken, 1986). Marginal facies is deﬁned as an area where the proportion of inter-clast matrix is dominant (>90%) relative to FRCs and where the surface is without mounds or hills. FRCs protruding at the surface in the areas mapped as the axial facies are the target of our cosmogenic surface exposure measurements. 4.3. MURIMOTU FORMATION 83 Existing age constraint for the MF debris avalanche event is scant. At some outcrops the MF is seen to underlie the time-transgressive Papakai tephra formation (c. 11.7 - 3.4 cal. ka BP; Topping, 1974; Donoghue et al., 1995), and overlie the Taurewa tephra formation (c. 13.8 - 11.2 cal. ka BP; Donoghue et al., 1999). Topping (1974) reports the only existing radiocarbon date from wood entrained within the MF, which places the event at 9.5 ± 0.1 14C ka BP (c. 10.5-11.1 cal. ka BP OxCal v.4.2.3/SHCal13; Bronk Ramsey, 2009; Hogg et al., 2013). Existing age constraint for the MF debris avalanche event is scant. At some outcrops the MF is seen to underlie the time-transgressive Papakai tephra formation (c. 11.7 - 3.4 cal. ka BP; Topping, 1974; Donoghue et al., 1995), and overlie the Taurewa tephra formation (c. 13.8 - 11.2 cal. ka BP; Donoghue et al., 1999). Topping (1974) reports the only existing radiocarbon date from wood entrained within the MF, which places the event at 9.5 ± 0.1 14C ka BP (c. 10.5-11.1 cal. ka BP OxCal v.4.2.3/SHCal13; Bronk Ramsey, 2009; Hogg et al., 2013). 4.3 Facies architecture and stratigraphy of the Murimotu Formation debris avalanche deposit To improve the age constraint for this event, I revisited several key sites from pre- vious studies (Topping, 1974; Palmer and Neall, 1989). Stratigraphic descriptions were conducted to identify the context of radiocarbon samples within the MF facies frame- work (Figure 5.6). At both sampled sites, marginal facies predominates and can be subdivided into an upper oxidised portion (exhibiting strong post-depositional pedo- genic weathering) and a lower anaerobic portion (exhibiting weak post-depositional pedogenic weathering). Fossil tree molds are commonly observed in the weathered upper portion, whilst wood and peaty material is preserved in the lower portion of the MF deposit. A road cutting to the north of Whakapapanui stream (39◦08’S; 175◦30’E; Figure 4.2) presents east- and west-facing exposures on the northeastern margin of the deposit, which were previously sampled for radiocarbon dating by Topping (1974). At this same locality, I collected 5 further samples (Uoa-1, Uoa-2, Uo1, U6, U3a; Table 4.1) of bark and outer tree trunk wood entrained within the lower 1 m of the MF, and a further 3 twig samples (U10, U12, U13; Table 4.1) were collected from a palaeosol imme- diately underlying the MF (Figure 5.6). Another wood sample (S2) was retrieved from one of the several ﬂow-oriented tree trunks incorporated within marginal facies on the south-western margin of the MF (39◦10’S; 175◦28’E; Figure 4.2). Wood samples were targeted to represent short-lived (<50 years) growth material, including twigs, outer bark and growth rings from short-lived species identiﬁed primarily as Dracophyllum sp. (Table 4.1). CHAPTER 4. 3HE PRODUCTION RATE IN NZ 84 Figure 4.2: (A) The lower Murimotu Formation debris avalanche deposit with radiocarbon and 3Hecos sample locations. Sediment facies distribution taken from Palmer and Neall (1989); (B) Inset hillshade digital elevation model showing the location of (A) (dashed red box) in the context of the local volcanic topography. Figure 4.2: (A) The lower Murimotu Formation debris avalanche deposit with radiocarbon and 3Hecos sample locations. Sediment facies distribution taken from Palmer and Neall (1989); (B) Inset hillshade digital elevation model showing the location of (A) (dashed red box) in the context of the local volcanic topography. 85 4.3. MURIMOTU FORMATION Figure 4.3: Volcaniclastic stratigraphy exposed at the Whakapapanui and Whakapapaiti sites showing the position of Murimotu Formation with respect to enveloping ﬂuvio-laharic deposits, local-sourced basaltic-andesitic tephra and distal rhyolitic tephra from the Taupo and Okataina Volcanic Centres. 4.3 Facies architecture and stratigraphy of the Murimotu Formation debris avalanche deposit The occurrence of the rhyolitic Taupo (232 AD), Waiohau (13.6 ka) and Kawakawa/Oruanui (25.4 ka) tephra beds within these sections (as indicated) provides sup- porting chronological control (Lowe et al., 2013; Vandergoes et al., 2013). The positions of radiocarbon samples associated with Murimotu Formation are also indicated. Figure 4.3: Volcaniclastic stratigraphy exposed at the Whakapapanui and Whakapapaiti sites showing the position of Murimotu Formation with respect to enveloping ﬂuvio-laharic deposits, local-sourced basaltic-andesitic tephra and distal rhyolitic tephra from the Taupo and Okataina Volcanic Centres. The occurrence of the rhyolitic Taupo (232 AD), Waiohau (13.6 ka) and Kawakawa/Oruanui (25.4 ka) tephra beds within these sections (as indicated) provides sup- porting chronological control (Lowe et al., 2013; Vandergoes et al., 2013). The positions of radiocarbon samples associated with Murimotu Formation are also indicated. e at Whakapapaiti-south where marginal facies outcrops. Note the ﬂow oriented tree t xposure and yielded an age of 9535 ± 31 14C a BP. (b) Abundant small twigs (arrow - sam immediately beneath the Murimotu Formation at Whakapapanui-north. (c) Example of apanui-north. also note the organic horizon preserved beneath the marginal debris avala Whakapapanui-north, radiocarbon dated to 9341 ± 77 14C a BP. (e) Small twig (U12) 31 14C a BP. (f) Various short-lived twig fragments found orented with debris avalanche Murimotu Formation. One twig (U3a) yielded a radiocarbon age of 9287 ± 33 14C a BP. ere marginal facies outcrops. Note the ﬂow oriented tree t 9535 ± 31 14C a BP. (b) Abundant small twigs (arrow - sam imotu Formation at Whakapapanui-north. (c) Example of rganic horizon preserved beneath the marginal debris avala rbon dated to 9341 ± 77 14C a BP. (e) Small twig (U12) -lived twig fragments found orented with debris avalanche ig (U3a) yielded a radiocarbon age of 9287 ± 33 14C a BP. here marginal facies outcrops. Note the ﬂow oriented tree 9535 ± 31 14C a BP. (b) Abundant small twigs (arrow - sa rimotu Formation at Whakapapanui-north. (c) Example of organic horizon preserved beneath the marginal debris aval arbon dated to 9341 ± 77 14C a BP. (e) Small twig (U12) t-lived twig fragments found orented with debris avalanch wig (U3a) yielded a radiocarbon age of 9287 ± 33 14C a BP. t Whakapapaiti-south w sure and yielded an age o mediately beneath the M anui-north. also note the akapapanui-north, radio 14C a BP. (f) Various sho imotu Formation. 4.4.1 Radiocarbon dating Radiocarbon samples were submitted to the Rafter Radiocarbon Laboratory of GNS Science, Lower Hutt, New Zealand (NZA samples) for analysis by accelerator mass spectrometry (AMS). Sample weights varied between 9-388 mg. Where observed, root hairs were manually removed from sample specimens using tweezers. Chemical pre- treatment involved cellulose extraction and acid-alkali-acid exposure. Sample weights after pre-treatment were 2.1 - 33.7 mg. Carbon dioxide was generated using sealed tube combustion and yields varied between 0.7 - 1.9 mgC. Sample carbon dioxide was converted to graphite by reduction with hydrogen over an iron catalyst. Conventional 14C ages are reported as deﬁned by Stuiver and Polach (1977). 4.4. METHODS 4.4 Methods 4.4. METHODS 4.4. METHODS 87 4.3 Facies architecture and stratigraphy of the Murimotu Formation debris avalanche deposit One t 4.4.2 3Hecos sample collection, preparation and measurement I sampled the surfaces of un-shattered, andesitic boulders embedded in the surface of the axial a facies of the MF (Figure 4.5). Post-depositional erosion and/or shielding of boulder surfaces would result in 3Hecos concentrations that under-represent the period of exposure since emplacement. I restricted sampling to clasts that are well-embedded in the MF and surfaces that remain free from soil or vegetation cover (Figure 4.5). Un- fortunately, few such blocks exist, thus I was restricted to four boulder samples for the 3Hecos measurements. Samples were collected using a portable rock saw and by ham- mer and chisel. All samples were collected from the highest point of the parent boulder and boulders were up to 8 m tall, to minimise the potential for past burial, for example by volcanic ash. Boulder surfaces exhibited <5◦dip relative to horizontal, thereby minimising self-shielding effects. Azimuthal elevations were measured in the ﬁeld using a standard geological compass and clinometer. Sample locations and elevations were recorded using a Trimble GeoXH global positioning system, relative to the WGS84 datum. These data were differentially corrected using continuous measurements from GeoNet ’Chateau Observatory’ (’VGOB’) base station (39◦11’59” S, 175◦32’ 32”E; 1161 m asl), located c. 12 km to the south east. Post-processed horizontal and vertical uncertainties for individual sample locations are <0.4 m and <0.8 m, respectively. Thin section analysis showed pyroxene typically represents c. 10-15 % of the total sample mass, with a modal grain size of c. 250-500 µm. Whole rock samples were jaw-crushed, rinsed in de-ionised water and dry-sieved to isolate the desired size frac- tion. Density and magnetic separation techniques were used to separate c. 550-750 mg of pyroxene of (clino)enstatite pigeonite composition (Table 4.2). Following Bromley CHAPTER 4. 3HE PRODUCTION RATE IN NZ 88 et al. (2014), mineral separates were twice leached in 5 % hydroﬂuoric (HF) / 2 % nitric (HNO3) acid solution for 24 hours to remove adhering groundmass particles. This was followed by a separate 10 % hydrochloric (HCl) acid solution for 24 hours, to remove ﬂuorite precipitates that resulted from exposure to HF. Leached mineral crystals were then visually inspected for purity and wrapped in aluminium foil. Each sample was completely degassed by heating in a furnace to >1300 ◦C for 15 minutes, during which, gases were exposed to a liquid-nitrogen chilled, charcoal trap. 4.4.2 3Hecos sample collection, preparation and measurement Extracted gases were exposed to an SAES getter before being collected on a cryogenic cold trap at <15 Kelvin. Helium was then isolated from other noble gases by heating the cold trap to 45 K. Mass spectrometry was conducted using a MAP 215-50 noble gas mass spectrometer at Lamont-Doherty Earth Observatory, New York relative to the Yellowstone ’Murdering Mudpot’ (MM) helium standard 3He/4He ratio of 16.45Ra, (where Ra = 3He/4Heair = 1.384 x 10−6) using the protocol of Winckler et al. (2005). 4.4.3 Production rate calculations (2006); Li = Lifton et al. (2005); Lm = time-dependent adaptation of Lal (1991). 4.5.1 Radiocarbon dating of the Murimotu debris avalanche Eight out of the nine radiocarbon ages from wood fragments situated within and im- mediately beneath the MF, yield internally consistent ages that range from 9287 ± 33 to 9535 ± 31 14C a BP (Figure 4.2; Table 4.1). These results also agree with the single previous determination of 9540 ± 100 14C a BP (Topping, 1974). Sample ’U6’ yielded an age of 8788 ± 29 14C a BP, which is an outlier according to the Grubbs test (p < 0.05; Grubbs, 1969). This anomalous result may arise from contamination by young carbon, such as inﬁltration of younger rootlets or humic acids, and I exclude this sample from further discussion of this dataset. Table 4.1: Radiocarbon data from organic material entrained withina/buried byb the Murimotu Formation debris avalanche. Sample U6, shown in italics, is an outlier (p<0.05) according to the Grubbs test (Grubbs, 1969). Individual and combined age calibrations were calculated using OxCal v.4.2.3 (Bronk Ramsey, 2009) with the SHCal13 calibration curve (Hogg et al., 2013). Sample ID Lab ID Material δ13C (‰) 14C age ± 2 σ (14C a BP) Whakapapanui stream (north side; east-facing): Uoa1a 40401/1 Bark -26.09 9517 ± 31 Uoa2a 40401/2 Bark -26.17 9498 ± 31 Uo1a 40401/3 Outer growth wood -23.19 9346 ± 31 U6a 40401/6 Bark -22.97 8788 ± 29 U3yra 40401/7 Outer growth wood -28.67 9287 ± 33 U13b 40401/9 Twig -23.81 9410 ± 31 U12b 40401/10 Twig -28.48 9398 ± 31 U10b 40401/11 Twig -37.31 9341 ± 77 Whakapapaiti stream (north side; east-facing): S2a 40401/8 Wood -22.04 9535 ± 31 Table 4.1: Radiocarbon data from organic material entrained withina/buried byb the Murimotu Formation debris avalanche. Sample U6, shown in italics, is an outlier (p<0.05) according to the Grubbs test (Grubbs, 1969). Individual and combined age calibrations were calculated using OxCal v.4.2.3 (Bronk Ramsey, 2009) with the SHCal13 calibration curve (Hogg et al., 2013). 4.4.3 Production rate calculations All calculations are computed using the CRONUS-Earth exposure age calculator MAT- LAB code, as described by Balco et al. (2008), and modiﬁed for 3Hecos by Goehring et al. (2010) (version 1.1). Local surface pressure is calculated according to the standard atmosphere equation using mean sea level pressure and 1000 mb air temperature val- ues taken from NCEP-NCAR reanalysis (www.cdc.noaa.gov/ncep_reanalysis/). 3Hecos concentrations are corrected for sample thickness using a uniform rock density of 2.7 g cm−3 and an effective neutron attenuation length of 160 g cm−2 (Dunne et al., 1999). Field measurements of azimuthal elevations are used to calculate topographic shielding for each sample site, which are negligible (<1 %; Table 4.3). Sample thickness and topographic shielding corrections were computed using CRONUS-EARTH tools (available at: http://hess.ess.washington.edu/). Field investigations showed no obvious signs of erosion and observations from mid-winter showed that any snow accumulation at the sample elevation (c. 800-850 m asl) is short-lived and does not cover boulder surfaces. Similarly any volcanic ashfall from the proximal, active vents, is unlikely to persist on salient boulder tops. Thus, I do not consider surface erosion or past shielding to be a signiﬁcant source of error at the sample sites and make no corrections for these effects. Regional uplift in the central North Island has been calcu- lated as c. 0.6 mm yr-1 for the past 500 ka (Pulford, 2002), which has a negligible impact on helium concentration over the exposure time of our samples. Therefore, I do not include a correction for regional tectonic uplift. I present spallogenic 3Hecos production rates scaled to sea-level and high-latitude (SLHL) according to the ﬁve accepted scaling models and adopt the terminology implemented by Balco et al. (2008): St = Stone (2000) 4.4. METHODS 89 igure 4.5: (a,c,e,g) Parent boulders for samples MM-12-01, MM-12-02, MM-12-03 MM-12-04, respectively; (b,d,f,h) Sampled surfaces for each boulder, respectively. Figure 4.5: (a,c,e,g) Parent boulders for samples MM-12-01, MM-12-02, MM-12-03 and MM-12-04, respectively; (b,d,f,h) Sampled surfaces for each boulder, respectively. 90 CHAPTER 4. 3HE PRODUCTION RATE IN NZ ollowingLal (1991); Du = Dunai (2001); De = Desilets et al. (2006); Li = Lifton et al. 2005); Lm = time-dependent adaptation of Lal (1991). followingLal (1991); Du = Dunai (2001); De = Desilets et al. (2006); Li = Lifton et al. (2005); Lm = time-dependent adaptation of Lal (1991). followingLal (1991); Du = Dunai (2001); De = Desilets et al. 4.5.1 Radiocarbon dating of the Murimotu debris avalanche Radiocarbon dates from these samples therefore are likely to overlap, within measurement uncertainty, with the true age of MF emplacement. Using chi-squared (χ2) to test for a single population, Group 1 samples fail at the 95% conﬁdence limit (χ2(df.=4, n=5) = 49.4, p < 0.05), which indicates that the deviation between individual ages is caused by processes other than measurement uncertainty. The internal variation of Group 2 samples is not signiﬁcant at the 95% conﬁdence interval (χ2(df.=2, n=3) = 0.7, p > 0.05), which suggests that they form a single age popu- lation. The probability density functions of individual calibrated ages (Figure 4.6) show that two sub-populations exist within Group 1. Samples S2, Uoa1 and Uoa2 (Subgroup 1a) are offset in age by c. 400 calendar years from the remaining samples, Uo1 and U3a (Subgroup 1b; Figure 4.6). I also note that Subgroup 1a is offset from the Group 2 samples (Figure 4.6). This offset can be explained by Subgroup 1a samples having died prior to entrainment in the debris avalanche and I discount them from further consideration. Subgroup 1b and Group 2 samples are statistically indistinguishable (χ2(df.=4, n=5) = 9.2, p > 0.05), which supports our interpretation that Group 2 samples closely constrain the age of the MF debris avalanche. Given these stratigraphic and statistical analyses, I consider that Subgroup 1b and Group 2 samples directly date MF emplacement. I combine these samples using the R-combine function implemented within the OxCal program, version 4.2.4 (Bronk Ramsey, 2009). This yields a calendar age of 10644 - 10425 cal. a BP for the debris avalanche event (Figure 4.6). For the purpose of 3Hecos production rate calibration, I use the median of this range (10535 ± 110 cal. a BP), plus 62 years, to account for the offset between the calibrated radiocarbon age in calendar years before AD1950 and 3Hecos measurements, which represent cosmogenic nuclide accumulation until samples were collected in AD2012 (sensu Schaefer et al., 2009). This yields an age of 10597 ± 110 a (before AD2012), for the MF debris avalanche event. 4.5.1 Radiocarbon dating of the Murimotu debris avalanche 13 14 ( gg , ) Sample ID Lab ID Material δ13C (‰) 14C age ± 2 σ (14C a BP) Whakapapanui stream (north side; east-facing): Uoa1a 40401/1 Bark -26.09 9517 ± 31 Uoa2a 40401/2 Bark -26.17 9498 ± 31 Uo1a 40401/3 Outer growth wood -23.19 9346 ± 31 U6a 40401/6 Bark -22.97 8788 ± 29 U3yra 40401/7 Outer growth wood -28.67 9287 ± 33 U13b 40401/9 Twig -23.81 9410 ± 31 U12b 40401/10 Twig -28.48 9398 ± 31 U10b 40401/11 Twig -37.31 9341 ± 77 Whakapapaiti stream (north side; east-facing): S2a 40401/8 Wood -22.04 9535 ± 31 The remaining 8 samples comprise two groups with different stratigraphic relationships with the MF. Group 1 samples (Uoa1, Uoa2, Uo1, U3a and S2) represent organic material (bark and outer growth wood) incorporated within the MF (Figure 3, Figure 4.4), which suggests that the sampled material was entrained by the debris avalanche event. These samples may have been alive or dead at the time of entrainment, therefore the group may contain a mixture of individual radiocarbon decay signatures that provide direct or maximum ages for the debris avalanche event. Group 2 samples (U10, U12 and 5. RESULTS AND DISCUSSION 91 4.5. U13) are from twigs suspended within a c. 3 cm thick, organic-rich horizon immedi- ately underlying the MF at the northern Whakapapanui site (Figure 3). I interpret this horizon to represent a leaf-litter environment that was present at the ground surface when the MF marginal facies was emplaced. Whilst Group 2 ages stratigraphically represent a maximum age for the MF, the dates are from small (1 - 2 mm diameter) twigs, which typically have a short residence time in the forest ﬂoor environment (100 - 101 a; Prescott et al., 1989). Radiocarbon dates from these samples therefore are likely to overlap, within measurement uncertainty, with the true age of MF emplacement. U13) are from twigs suspended within a c. 3 cm thick, organic-rich horizon immedi- ately underlying the MF at the northern Whakapapanui site (Figure 3). I interpret this horizon to represent a leaf-litter environment that was present at the ground surface when the MF marginal facies was emplaced. Whilst Group 2 ages stratigraphically represent a maximum age for the MF, the dates are from small (1 - 2 mm diameter) twigs, which typically have a short residence time in the forest ﬂoor environment (100 - 101 a; Prescott et al., 1989). 4.5.2 3Hecos measurements and production rate calibration Individual 3He concentrations of samples MM-12-01 to MM-12-04 range from 2.63 to 1.94 x 106 at. g−1, with measurement uncertainties of 3-4 % (Table 4.3). Our samples ex- CHAPTER 4. 3HE PRODUCTION RATE IN NZ 92 Figure 4.6: Probability density functions for individually calibrated radiocarbon ages, arranged by stratigraphic relationship with the Murimotu Formation. Brackets 1A and 1B denote sample subgroups (see text for discussion). Vertical grey bar and inset plot depict the calibrated age for emplacement of the Murimotu Formation (see text for details). Plots generated using functions implemented in the OxCal program (Bronk Ramsey, 2009), version 4.2.4 with SHCal13 (Hogg et al., 2013). Figure 4.6: Probability density functions for individually calibrated radiocarbon ages, arranged by stratigraphic relationship with the Murimotu Formation. Brackets 1A and 1B denote sample subgroups (see text for discussion). Vertical grey bar and inset plot depict the calibrated age for emplacement of the Murimotu Formation (see text for details). Plots generated using functions implemented in the OxCal program (Bronk Ramsey, 2009), version 4.2.4 with SHCal13 (Hogg et al., 2013). hibit high 3He/4He ratios (c.80-200Ra; Table 4.3), together with low 4He concentrations (Table 4.3). These characteristics reﬂect: (i) the youth of the parent material (<300 ka; Gamble et al., 2003), which restricts the time for radiogenic accumulation of 4He (and 3He); (ii) sample preparation procedures (e.g. HF-leaching), which result in a higher purity pyroxene separates and absence or suppression of radiogenic 4He (Bromley et al., 2014) and (iii) a negligible magmatic input. Following Dunai et al. (2007), I measured lithium concentrations in our samples to test for nucleogenic 3He (3Hen) produced via thermal neutron reactions (6Li (n,α) 3H 3He). Concentrations are higher than expected and vary between 9-44 ppm (Table 4.2). However, uranium and thorium concentrations are very low (c. 3-80 ppb; Table 4.2), which reduces the potential for thermal neutron production. Furthermore, our samples have young closure ages, individual crystals are large (c. 250-500 µm) and they have undergone HF pre-treatment to remove the outer rims. The arguments above, combined with the fact that there is no correlation between the wide scatter in Li concentrations between samples and the internally consistent 3He concentrations in the samples, lead me to conclude that thermal neutron produced 3He is negligible in this study. Thus, I can robustly assume all 3He to be of cosmogenic origin. 4.5. RESULTS AND DISCUSSION 93 93 CHAPTER 4. 4.5.2 3Hecos measurements and production rate calibration g−1) R/Ra 9.48 ± 0.33 2.55 ± 0.09 181 8.50 ± 0.31 2.29 ± 0.08 77 7.22 ± 0.30 1.94 ± 0.08 79 9.81 ± 0.29 2.63 ± 0.08 199 ion e h-Li b Wo 3 3 5 4 metry He ± −1) 55 ± 0 29 ± 0 94 ± 0 63 ± 0 ion e h-Li b Wo 3 3 5 4 metry He ± −1) 55 ± 0 29 ± 0 94 ± 0 63 ± 0 nd pyroxene classiﬁca n using ICP-OES; U-T i (ppm) Th (ppb) En Fs 4 80 64 33 5 <13a 69 27 <13a 63 32 8 60 63 33 noble gas mass spectro cc- 3He ± 1σ (10−14 cc- STP g−1) 9.48 ± 0.33 8.50 ± 0.31 7.22 ± 0.30 9.81 ± 0.29 e samples from this st t 29 ± 5 20 ± 5 08 ± 5 36 ± 4 23 ± 12 28 ± 8 y) to the global com nam et al., 2010b) an .1 3.2 1.3 .1 2.3 1.1 his s com ) an com b) an 140 ± 4 138 ± 4 152 ± 5 133 ± 4 126 ± 13 124 ± 12 136 ± 14 120 ± 12 M-12-03 removed) 132 ± 9 130 ± 9 143 ± 10 125 ± 8 production rate in pyroxene (3Hecos (NZ); this stu he New Zealand in situ cosmogenic 10Be (10BeNZ; P ) production rates. De Du Li Lm St GLOBAL 1.0 ± 0.1 1.0 ± 0.1 1.0 ± 0.1 1.0 ± 0.1 1.0 ± Z) 32.6 ± 3.3 32.5 ± 3.3 33.0 ± 3.4 32.2 ± 3.2 32.1 10.7 ± 1.4 10.5 ± 1.3 10.8 ± 1.4 10.6 ± 1.3 10.5 n=3) GLOBAL 1.0 ± 0.1 1.0 ± 0.1 1.0 ± 0.1 1.0 ± 0.1 1.0 ± Z) 34.0 ± 2.4 33.8 ± 2.4 34.4 ± 2.5 33.5 ± 2.2 33.4 11.1 ± 1.1 11.0 ± 1.1 11.2 ± 1.2 11.0 ± 1.1 11.0 tion rates for individual and com De Du Li 132 ± 5 130 ± 5 143 122 ± 5 121 ± 5 133 111 ± 5 109 ± 5 120 140 ± 4 138 ± 4 152 126 ± 13 124 ± 12 136 removed) 132 ± 9 130 ± 9 143 ction rate in pyroxene (3Hecos w Zealand in situ cosmogenic 10 uction rates. 4.5.2 3Hecos measurements and production rate calibration 3HE PRODUCTION RATE IN NZ 94 Figure 4.7: : (a) Sea-level, high-latitude (SLHL) production rates (1 sigma uncertainty) for individual 3Hecos measurements, derived using the Lm scaling protocol. (b) Probability density function for the calibrated, SLHL (Lm scaling) 3Hecos production rate, derived using all samples (n=4). Vertical black bar denotes the arithmetic mean, with vertical dashed blue bars indicating the 2 sigma uncertainty range. (c) As for plot (b), but derived with sample MM-12-03 removed. Grey shading denotes globally compiled 3Hecos production rate in pyroxene, derived by Goehring et al. (2010). Figure 4.7: : (a) Sea-level, high-latitude (SLHL) production rates (1 sigma uncertainty) for individual 3Hecos measurements, derived using the Lm scaling protocol. (b) Probability density function for the calibrated, SLHL (Lm scaling) 3Hecos production rate, derived using all samples (n=4). Vertical black bar denotes the arithmetic mean, with vertical dashed blue bars indicating the 2 sigma uncertainty range. (c) As for plot (b), but derived with sample MM-12-03 removed. Grey shading denotes globally compiled 3Hecos production rate in pyroxene, derived by Goehring et al. (2010). xpressed as enstatite y ICP-MS. data 1σ (106 at. R/Ra 09 181 08 77 08 79 08 199 and pyroxene classiﬁcation expressed as enstatite en using ICP-OES; U-Th-Li by ICP-MS. Li (ppm) Th (ppb) En Fs Wo 44 80 64 33 3 15 <13a 69 27 3 9 <13a 63 32 5 38 60 63 33 4 noble gas mass spectrometry data cc- 3He ± 1σ (10−14 cc- STP g−1) 3He ± 1σ (106 at. 4.5.2 3Hecos measurements and production rate calibration De Du Li 1.0 ± 0.1 1.0 ± 0.1 1.0 ± 0.1 32.6 ± 3.3 32.5 ± 3.3 33.0 ± 3.4 10.7 ± 1.4 10.5 ± 1.3 10.8 ± 1.4 1.0 ± 0.1 1.0 ± 0.1 1.0 ± 0.1 34.0 ± 2.4 33.8 ± 2.4 34.4 ± 2.5 11.1 ± 1.1 11.0 ± 1.1 11.2 ± 1.2 RESULTS AND DISCUSSION 97 4.5. When corrected for sample thickness and local shielding, I derive local 3Hecos produc- tion rates of 199 - 253 at. g−1 yr−1, for the given sample locations and elevations (Table 4.3). When scaled to sea-level and high-latitude (SLHL) using the 5 standard scaling protocols, individual production rate estimates for each of the 3Hecos concentrations show good agreement with the compiled pyroxene production rates of Goehring et al. (2010) (e.g. Figure 4.7a). Taking the arithmetic mean 3Hecos concentration of our total sample population, scaled to SLHL (1.28 ± 0.13 x 106 at. g−1), I derive a production rate of 120 ± 12 at. g−1 yr−1 (Table 4.4). This rate is indistinguishable, within uncertainty, from that presented by Goehring et al. (2010) (119 ± 9.9 at. g−1 yr−1 for pyroxene; Figure 4.7a,b; Table 4.5). However, applying the reduced-χ2 (χ2 red) test for all samples gives χ2 red = 8.7, which indicates a low probability that the dataset represents a single population. Figure 4.7b shows that sample MM-12-03 forms a separate peak in the probability distribution for the total population, therefore may represent an outlier. This sample has a 3Hecos concentration that is lower than the range of the other three samples, which could result from post-depositional erosion or previous shielding from 3Hecos production. To test the inﬂuence of this possible outlier on our production rate, I re-calculate the production rates with sample MM-12-03 removed (Table 4.4). This yields a SLHL (Lm scaling) production rate of 125 ± 8 at. g−1 yr−1 (Table 4.4), which remains within the range of Goehring et al. (2010) (Figure 4.7c; Table 4.5). Due to the low number of samples afforded by this study site, it is not possible to conclude with any certainty whether the 3Hecos production rate in New Zealand falls in the centre (e.g. Figure 4.7b) or towards the upper end (e.g. Figure 4.7c) of that presented by Goehring et al. (2010). CHAPTER 4. 3HE PRODUCTION RATE IN NZ 98 To compare these results with New Zealand, I take the arithmetic mean (n=4) of our SLHL 3Hecos production rates in pyroxene and the weighted mean SLHL 10Be production rates in quartz of Putnam et al. (2010b). This yields a 3Hepx / 10Beqz(NZ) production ratio of 32.2 ± 3.2 (Lm scaling; Table 4.5), which is indistinguishable from previously published ratios. Using the proximal in situ cosmogenic 14C production rate of Schimmelpfennig et al. (2012), I derive a cosmogenic 3Hepx / 14Cqz(NZ) ratio of 10.6 ± 1.6 (Lm scaling; Table 4.5). These ratios provide a prediction for future tests of the production rates for each of these nuclides in New Zealand, in situations where quartz and pyroxene co-exist and have simple exposure histories. Such tests do not require independent age constraint. To compare these results with New Zealand, I take the arithmetic mean (n=4) of our SLHL 3Hecos production rates in pyroxene and the weighted mean SLHL 10Be production rates in quartz of Putnam et al. (2010b). This yields a 3Hepx / 10Beqz(NZ) production ratio of 32.2 ± 3.2 (Lm scaling; Table 4.5), which is indistinguishable from previously published ratios. Using the proximal in situ cosmogenic 14C production rate of Schimmelpfennig et al. (2012), I derive a cosmogenic 3Hepx / 14Cqz(NZ) ratio of 10.6 ± 1.6 (Lm scaling; Table 4.5). These ratios provide a prediction for future tests of the production rates for each of these nuclides in New Zealand, in situations where quartz and pyroxene co-exist and have simple exposure histories. Such tests do not require independent age constraint. 4.5.2 3Hecos measurements and production rate calibration However, the close agreement between these rates does indicate that the globally-compiled 3Hecos production rate is appropriate in the south west Paciﬁc region, therefore it can be applied to exposure age and erosion rate applications with increased conﬁdence. Addition of a 3Hecos calibration means that New Zealand now has independent cali- brations of cosmogenic 3He (this study), 10Be (Putnam et al., 2010b) and 14C (Schim- melpfennig et al., 2012) production rates. Ratios of production rates from co-existing minerals, or closely-spaced calibration sites provide insight to cosmogenic nuclide production (Schimmelpfennig et al., 2011). For example, co-existing measurements of cosmogenic 3He and 10Be have shown that, in some regions, the ratio of these nuclides changes with elevation (Gayer et al., 2004; Amidon et al., 2008), which suggests different elevation-dependence of production rates between nuclides. Previous 3Hepx / 10Beqz ratios have been reported from paired measurements in the Himalaya (32.3 ± 0.9 - Amidon et al., 2009, calculated by Blard et al., 2013a) and the high tropical Andes (33.3 ± 0.9 at 4820 m asl; Blard et al., 2013a). Blard et al. (2013a) also derive a 3Hepx/ol / 10Beqz ratio of 31.7 ± 4.1 using globally-compiled, SLHL production rates for each nuclide. 4.6 Conclusion I produced a precise radiocarbon chronology for the Murimotu Formation debris avalanche event on Mt. Ruapehu, which occurred 10.4-10.6 cal. ka BP. Using this independent age constraint, I present a 3Hecos calibration dataset for the south west Paciﬁc region, which is consistent with the existing compilation production rate of Goehring et al. (2010). This rate can therefore be applied to exposure age and erosion rate applications in the south west Paciﬁc region with increased conﬁdence. Future work should aim to reduce the uncertainties of the existing 3Hecos production rate, through the addition of calibration sites with more robust sample sizes. This study, together with previous cosmogenic nuclide production rate calibrations from South Island (10Be in quartz Putnam et al., 2010b; 14C in quartz Schimmelpfennig et al. (2012)) means New Zealand now has independent constraints on each of the following production ratios: 3Hepx / 10Beqz(NZ) = 32.3 ± 3.2 and 3Hepx / 14Cqz(NZ) = 10.6 ± 1.6. 5.2 Introduction Explaining the drivers of Quaternary climate cycles in the Southern Hemisphere re- mains an outstanding goal of palaeoclimate research. New Zealand is one of the few locations in the Southern Hemisphere where terrestrial palaeoclimate can be recon- structed. Furthermore, it is ideally situated to record ﬂuctuations of the southern westerly winds and the oceanic subtropical and subpolar fronts (Figure 6.1), which are considered to have played an important role in past climate dynamics (Denton et al., 2010). Regional tectonic uplift and localised effusive volcanism have resulted in high elevation and high relief topography in both the North and South Island of New Zealand, which support contemporary glaciers spanning latitudes from 39 - 46◦S. Empirical and model-based analyses show contemporary glacier mass balance in New Zealand is most sensitive to changes in atmospheric temperature (Oerlemans, 1997; Anderson and Mackintosh, 2006; Anderson et al., 2010; Brook et al., 2011). Steep mass balance gradients and relatively high ice velocities mean that mass balance changes are translated to the termini of mountain glaciers over timescales of 101 - 102 yrs (Oerle- mans, 1997; Purdie et al., 2014). Geological records of past glacier ﬂuctuations in New Zealand therefore represent an important proxy for past climatic change in the southern mid-latitudes (e.g. Anderson and Mackintosh, 2006; Schaefer et al., 2006, 2009; Kaplan et al., 2010, 2013; Putnam et al., 2010a, 2012, 2013a,b; Golledge et al., 2012; Barrows et al., 2013; Doughty et al., 2013; Rother et al., 2014). Existing reconstructions of late Quaternary glacier ﬂuctuations in New Zealand have predominantly concentrated on large outlet glaciers that drained the central Southern Alps (e.g. Porter, 1975; Suggate and Almond, 2005; Schaefer et al., 2006; Barrows et al., 2013; Putnam et al., 2013b; Rother et al., 2014). Radiocarbon and luminescence dating of peat and loess units, interbedded with glacial till and glacioﬂuvial outwash, have provided the chronostratigraphic framework for this region (Suggate, 1990; Suggate and Almond, 2005). More recently, cosmogenic surface exposure dating of moraines has provided the means to directly constrain the timing and magnitude of past glacier ﬂuctuations (Schaefer et al., 2006; Barrows et al., 2013; Putnam et al., 2013a,b; Rother et al., 2014). During the last glacial cycle, glacier length in the Southern Alps peaked relatively early (c. 32-26 ka; (Suggate and Almond, 2005; Putnam et al., 2013b; Barrows et al., 2013; Rother et al., 2014)), in comparison to global ice volume (Clark et al., 2009). 5.1 Abstract Establishing the timing and magnitude of southern mountain glacier ﬂuctuations prior to, and through the Last Glacial Maximum, helps to identify the drivers and mecha- nisms of past climate change. In this study, I use cosmogenic 3He surface exposure dating and tephrochronology to constrain the timing of past glaciation of Tongariro massif in central North Island, New Zealand (39◦S). Exposure ages from moraine boul- ders show that valley glaciation persisted between c. 31 - 21 ka. This followed an earlier period of glaciation, of similar magnitude to the LGM, that likely culminated late in Marine Isotope Stage 4. The equilibrium line altitude of the reconstructed LGM glacier is c. 1400-1550 m asl, which is c. 930-1080 m lower than at present. This equates to a temperature depression of 5.6 ± 1.1 ◦C, when uncertainties in the ELA reconstruction and temperature lapse rates are considered. Reinterpretation of moraine tephrostratig- raphy, using major element geochemistry analysis, shows that ice retreat and climatic amelioration at the last glacial termination was well underway prior to 14 ka. Good agreement between the timing and magnitude of glacier ﬂuctuations in central North Island and the Southern Alps indicate a response to a common climatic forcing during the last glacial cycle. 99 CHAPTER 5. THE LAST GLACIAL CYCLE CHAPTER 5. THE LAST GLACIAL CYCLE 100 5.2 Introduction At this time, local equilibrium line altitudes were depressed by c. 850 m and tempera- tures were c. 6-6.5◦C colder, relative to present (Golledge et al., 2012). Subsequently, glaciers ﬂuctuated about these positions until at least c. 18 ka before rising tempera- tures resulted in widespread deglaciation (Schaefer et al., 2006; Putnam et al., 2013a,b; Rother et al., 2014). Recent work by Kelley et al. (2014) and Schaefer et al. (2015) have 5.3. SETTING 101 shown that the former Pukaki glacier in central Southern Alps exceeded its LGM extent multiple times during the last glacial cycle, in advances that culminated at c. 42 ± 1 ka and 65 ± 3 ka. Previous work had suggested the occurrence of signiﬁcant pre-LGM glacial advances, but the exact timing of these events was poorly resolved due to low chronological precision or small sample populations (Preusser et al., 2005; McCarthy et al., 2008; Sutherland et al., 2007). Resolving the timing and magnitude of late Quaternary glacier ﬂuctuations in lo- cations outside of the central Southern Alps will provide insight to the synchroneity and climatic gradients of past climate change in this region. In this study, I use cos- mogenic 3He exposure dating, tephrochronology and equilibrium line altitude (ELA) reconstruction techniques, to constrain the timing and magnitude of glacier ﬂuctuations on Tongariro massif, central North Island (39◦S). 5.3.1 Regional climatic situation Situated between 34-47◦S in the south-west sector of the Paciﬁc Ocean, New Zealand spans subtropical and subpolar climes (Figure 6.1), therefore is highly sensitive to regional climatic ﬂuctuations (Newnham et al., 1999). Westerly atmospheric circulation dominates between 30-60◦S and is responsible for the eastward migrating troughs and anticyclones that deﬁne weather variability in New Zealand year-round (Sturman and Tapper, 1996). Meanwhile, interannual-decadal air temperature anomalies in New Zealand are strongly inﬂuenced by upwind sea surface temperatures (Sutton et al., 2005). The latitudinal range and high topographic relief of the New Zealand landmass result in meridional and zonal gradients in air temperature and precipitation respec- tively. Contemporary oceanic inﬂuences on North Island, New Zealand (34 - 41 ◦S) are largely sub-tropical in nature, predominantly originating from an eastward ﬂowing branch of the equatorial-sourced East Australian Current, known as the Tasman Front, which descends the northeast coast before continuing eastwards along the northern margin of the Chatham Rise (Figure 6.1). In contrast, South Island (40-47 ◦S) intersects the sub-tropical front (STF), where sub-tropical gyres and sub-antarctic water masses converge, representing a temperature, salinity and nutrient boundary, which deﬁnes the northern margin of the Southern Ocean (Sikes et al., 2009). Consequently, steep, zonal and meridional sea surface temperature (SST) gradients exist across New Zealand. For example, in the far north annual SSTs average c. 20 ◦C compared to c. 10 ◦C in the south (Uddstrom and Oien, 1999). CHAPTER 5. THE LAST GLACIAL CYCLE 102 Figure 5.1: (A) Contemporary general atmospheric and oceanic circulation in New Zealand and locations of sites referred to in text (background image sourced from National Institute for Water and Atmospheric research (NIWA)). Sites: TgVC = Tongariro Volcanic Centre; TaM = Tararua Mts.; TsM = Tasman Mts.; CSA = Central Southern Alps; CV = Cascade valley. Ocean currents: EAUC = East Australian Current; ECC = East Cape Current; STF = Sub-tropical front; SAF = Sub-Antarctic Front; ACC = Antarctic Circumpolar Current; (B) Hill-shaded digital elevation model (Columbus et al., 2011) of the Tongariro massif and Mt. Ruapehu, with the main study region deﬁned (black outline). The location of proximal tephra reference site at Taurewa and Mt. Ruapehu glacier outlines according to Keys (1988) are also shown. Figure 5.1: (A) Contemporary general atmospheric and oceanic circulation in New Zealand and locations of sites referred to in text (background image sourced from National Institute for Water and Atmospheric research (NIWA)). 5.3.1 Regional climatic situation Sites: TgVC = Tongariro Volcanic Centre; TaM = Tararua Mts.; TsM = Tasman Mts.; CSA = Central Southern Alps; CV = Cascade valley. Ocean currents: EAUC = East Australian Current; ECC = East Cape Current; STF = Sub-tropical front; SAF = Sub-Antarctic Front; ACC = Antarctic Circumpolar Current; (B) Hill-shaded digital elevation model (Columbus et al., 2011) of the Tongariro massif and Mt. Ruapehu, with the main study region deﬁned (black outline). The location of proximal tephra reference site at Taurewa and Mt. Ruapehu glacier outlines according to Keys (1988) are also shown. 5.3.2 Study site and previous work For the glacial reconstruction, I target glacial landform assemblages on the western ﬂanks of the Tongariro massif in central North Island (39◦08’S, 175◦39’E). This volcano forms part of Tongariro Volcanic Centre (TgVC), an area of andesitic volcanism at the southwestern end of Taupo Volcanic Zone. Tongariro massif comprises 17 coalescing volcanic vents, with ages ranging from c. 270 ka to present (Hobden et al., 1996). The local climate, as recorded at Whakapapa village (1097 m asl) located 15 km SSW of Tongariro, is characterised by low seasonal precipitation variability, with winter precipitation averaging 762 mm, compared to 624 mm in summer. Monthly mean 103 5.3. SETTING temperatures range from c. 13 ◦C in February to c. 3 ◦C in July, with an annual average of 7.5 ◦C (1981-2010; NIWA, 2014). Geomorphological evidence for past glaciation on Tongariro massif has long been recognised (Mathews, 1967; Topping, 1974), however lack of chronological constraint has so far precluded any palaeoclimatic interpretation. Mathews (1967) describes con- spicuous, lateral moraines in several valleys radiating from the volcano and suggests that they correlate with late Pleistocene moraines of the Southern Alps, based on their size and degree of preservation. Topping and Kohn (1973) identify two rhyolitic hori- zons in a soil section that overlies a large lateral moraine in the Mangatepopo valley. Using analyses of titano-magnetite assemblages, they correlate the lower horizon to the Rerewhakaaitu Tephra, which erupted from Okataina Volcanic Centre (OVC) c. 17.5 ± 0.5 ka BP (Lowe et al., 2013). This ﬁnding was then used to stratigraphically correlate the upper horizon to the Waiohau Tephra (cf. Donoghue et al., 1995), also sourced from the OVC, at 14.0 ± 0.2 ka (Lowe et al., 2013). On the eastern ﬂanks of Tongariro, Cronin and Neall (1997) identiﬁed both the Waiohau and Rerewhakaaitu Tephras at the base of soils overlying lateral moraines. On nearby Mt. Ruapehu (2797 m asl), situated 15 km to the south west, McArthur and Shepherd (1990) identiﬁed geomorphological features representative of a former ice cap drained by several outlet glaciers that reached c. 1200 m asl. They suggest the former ice mass existed during the LGM, based on the presence of the Kawakawa/Oruanui Tephra (25.4 ± 0.2 ka - Vandergoes et al., 2013) in moraine-bound glaciolacustrine deposits. 5.3.2.1 Mangatepopo Valley The Mangatepopo valley is c. 6 km long and 1 - 2 km wide, with small, westward- ﬂowing under-ﬁt streams draining both the northern and southern valley margins, which converge at the valley mouth to form the Mangatepopo Stream (Figure 5.2). The Mangatepopo Stream ﬂows northwards, before forming a tributary of the Whanganui River, which is a major drainage channel for the eastern-central North Island. At the head of the Mangatepopo valley, South Crater is a large bedrock amphithe- atre, which is 1 km across at its widest point and 250 m deep (Figure 5.2; Figure 5.3a). At 1967 m asl, Mt. Tongariro forms the highest point on the south-facing back wall of South Crater, whilst the Holocene-aged volcanic cone of Mt. Ngauruhoe (2287 m asl) is situated c. 3 km to the south. Following Mathews (1967), I interpret South Crater as a glacial cirque that served as the main accumulation centre for the former Mangatepopo glacier. The ﬂat-bottomed, cirque ﬂoor is c. 1700 m asl, although this contemporary At the head of the Mangatepopo valley, South Crater is a large bedrock amphithe- atre, which is 1 km across at its widest point and 250 m deep (Figure 5.2; Figure 5.3a). At 1967 m asl, Mt. Tongariro forms the highest point on the south-facing back wall of South Crater, whilst the Holocene-aged volcanic cone of Mt. Ngauruhoe (2287 m asl) is situated c. 3 km to the south. Following Mathews (1967), I interpret South Crater as a glacial cirque that served as the main accumulation centre for the former Mangatepopo glacier. The ﬂat-bottomed, cirque ﬂoor is c. 1700 m asl, although this contemporary CHAPTER 5. THE LAST GLACIAL CYCLE 104 elevation represents inﬁlling by an unknown thickness of post-glacial volcanic products. Bedrock spurs forming the mouth of the cirque have been partially overprinted by Mt. Ngauruhoe lavas, however these spurs clearly curve towards the southwest, which indicates the direction of former ice ﬂow from this accumulation zone. To the southwest of Mt. Tongariro peak, the outer, western-facing ﬂank of South Crater exhibits a stacked sequence of truncated lava ﬂows in a steep, but shallow, concave amphitheatre, which is suggestive of headward erosion by a westward-ﬂowing glacier. Field investigations show that bedrock exposures in this region exhibit smoothed surfaces, indicative of glacial abrasion by a temperate ice-mass (Hobden et al., 1996; Figure 5.2). 5.3.2.1 Mangatepopo Valley Several radiometric (40K/40Ar) ages from lavas show that cone-building in the South Crater region occurred c. 70 - 100 ka (Figure 2.6). These dates provide a maximum bracketing age for the creation of the erosional glacial landforms. The surface of the valley ﬂoor is convex, representing post-glacial inﬁlling by lava ﬂows and pyroclastics from the nearby volcanic vents. Both valley sides exhibit truncated lava cliffs, aligned parallel to the west-east trending valley axis, and are interpreted as having been glacially cut. On the northern edge of Pukekaikiore (Figure 5.2), two such vertical cliffs are stacked on top of one another, separated by a clear bench, perhaps indicative of multiple erosive periods, by ice masses of differing thickness. K/Ar dates from Pukekaikiore lavas range between c. 200-100 ka, suggesting emplacement prior to the peak of the last glacial cycle (Hobden et al., 1996). Multiple lateral moraines exist on both valley sides and can be differentiated chronolog- ically, based on morphostratigraphic criteria. This distinction is clearest on the lower portion of the northern valley side where two large lateral moraines have signiﬁcantly different crest heights and widths (Figure 5.3b). Topographic proﬁles, aligned perpen- dicular to the moraine crest orientation illustrate the differing morphologies of these landforms (Figure 5.2B). The outer moraine (M3) is c. 20 m tall, with a highly rounded crest, whilst the inner moraine (M2) is sharp-crested and signiﬁcantly taller. I interpret these geomorphic differences as representing a considerable time gap between the two moraine forming glacial episodes, with the outer, older moraine having undergone a greater amount of degradation. Immediately north of the M3 moraine, a separate linear ridge, c. 500 m in length, is orientated NW-SE and appears to be overlain by M3 at its upstream end. I tenta- tively identify this landform as a moraine ridge, based on the similar appearance to M3, although the unbroken coverbeds on the surface make it difﬁcult to establish the geological composition. If this landform is a moraine, then based on its orientation, it is unclear what the former ice geometery or source area would have been. Due to this 5.3. SETTING 105 igure 5.2: (A) Glacial geology and chronology of western Tongariro massif. Yellow stars repr ent the location of moraine boulder samples for cosmogenic 3He exposure dating. Associate abels are the calculated exposure ages, using Lm scaling - see text for detailed description of ag alculation methods. 5.3.2.1 Mangatepopo Valley (B) Topographic proﬁles across moraines M2 and M3, with 3He exposur ges and sample numbers. Note the greater roundness of the older, M3 crest. ∗Exposure age or M3 are considered minimum ages for moraine formation - see text. rregular relationship to the present day drainage and topography of Tongariro massi Figure 5.2: (A) Glacial geology and chronology of western Tongariro massif. Yellow stars repre- sent the location of moraine boulder samples for cosmogenic 3He exposure dating. Associated labels are the calculated exposure ages, using Lm scaling - see text for detailed description of age calculation methods. (B) Topographic proﬁles across moraines M2 and M3, with 3He exposure ages and sample numbers. Note the greater roundness of the older, M3 crest. ∗Exposure ages for M3 are considered minimum ages for moraine formation - see text. irregular relationship to the present day drainage and topography of Tongariro massif, I tentatively correlate this landform to the penultimate glacial cycle. As much of the irregular relationship to the present day drainage and topography of Tongariro massif, I tentatively correlate this landform to the penultimate glacial cycle. As much of the CHAPTER 5. THE LAST GLACIAL CYCLE 106 Figure 5.3: (a) South Crater cirque, viewed from the upper slopes of Mt. Ngauruhoe; (b) moraines M2 (middle ground, left) and M3 (middle ground, centre), note the difference in morphometry between the two landforms; (c) a view down valley from the upstream end of moraine M1 - note the glacially truncated side of Pukekaikiore in shadow on the far valley side; (d) the inner lateral moraines of Mangatepopo valley clearly depict the former glacier terminus (photo taken from the upper western slopes of Pukekaikiore); (e) coverbeds on the ice-proximal ﬂank of M2 moraine thin towards the crest; (e) the sharp-crested right lateral moraine of the Makahikatoa valley runs diagonally from left to right across this image (photo taken from the upper northern slopes of Pukekaikiore). Figure 5.3: (a) South Crater cirque, viewed from the upper slopes of Mt. 5.3.2.1 Mangatepopo Valley Ngauruhoe; (b) moraines M2 (middle ground, left) and M3 (middle ground, centre), note the difference in morphometry between the two landforms; (c) a view down valley from the upstream end of moraine M1 - note the glacially truncated side of Pukekaikiore in shadow on the far valley side; (d) the inner lateral moraines of Mangatepopo valley clearly depict the former glacier terminus (photo taken from the upper western slopes of Pukekaikiore); (e) coverbeds on the ice-proximal ﬂank of M2 moraine thin towards the crest; (e) the sharp-crested right lateral moraine of the Makahikatoa valley runs diagonally from left to right across this image (photo taken from the upper northern slopes of Pukekaikiore). underlying lava that forms the present Mangatepopo valley was emplaced 70-100 ka, it is possible that the geometry of any ice that existed prior to the last glacial cycle could have been signiﬁcantly different from the most recent period of glaciation. underlying lava that forms the present Mangatepopo valley was emplaced 70-100 ka, it is possible that the geometry of any ice that existed prior to the last glacial cycle could have been signiﬁcantly different from the most recent period of glaciation. The lower portions (<1200 m asl) of both M2 and M3 moraines have continuous 5.3. SETTING 107 coverage by tephra, palaeosols and volcanic loess accumulation (Figure 5.2; Figure 5.3d,e). Between 1200 - 1250 m asl, these coverbeds become discontinuous, with the transitions from bare moraine to the coverbeds marked by erosional scarps, which indicate former, more extensive coverage of the moraine surface at this elevation. The upstream end of moraine M2 (>1250 m asl) is characterised by continuous thin (< 1 m) coverbeds on the lower moraine slope, which thins towards the bare, rocky moraine crest (Figure 5.3e). No erosional scarps exist on the upper portion of this moraine. Directly upstream from moraine M2, moraine M1 is a single, continuous ridge (Figure 5.3c), separated from M2 by a cliff, which represents the downstream limit of an underly- ing lava ﬂow dated by Hobden et al. (1996) to 110 ± 6 ka (Figure 2.6). There is no tephra cover on M1 present today, nor are there any remnants to suggest that coverbeds have been more extensive in the past. It is likely that the greater elevation of M1 prevents the accumulation of ﬁne-grained sediment due to wind exposure and lack of vegetation. 5.3.2.1 Mangatepopo Valley On the southern valley side, a single, sharp-crested lateral moraine extends west- wards for c. 1 km, from Pukekaikiore (Figure 5.2). The crest of this moraine is c. 40 m above the present day valley ﬂoor. Immediately down valley, two left latero-frontal moraines further constrain the former glacier margin. Soil and tephra cover on these moraines precludes sampling for surface exposure dating, although both landforms exhibit relatively sharp crests, which suggests that they correspond to the most recent period of glacier occupation (i.e. M2). The innermost moraines on both valley sides converge downstream at c. 1150 m asl, forming the valley mouth, thereby deﬁning the maximum limit of the most recent period of valley glaciation (Figure 5.3d). CHAPTER 5. THE LAST GLACIAL CYCLE 108 ridge that can be traced down valley to c. 1200 m asl and represents a signiﬁcantly older glacial limit. Based on the morphostratigraphic relationship of these moraines, I correlate them to the outer (M3) and inner (M1 / M2) moraine ridges situated on the northern ﬂank of Mangatepopo Valley (Figure 5.2). 5.3.2.2 Makahikatoa Stream To the south of Mangatepopo valley, the northern headwaters of the Makahikatoa stream drain southwestwards from a northeast-southwest orientated bedrock amphithe- atre, interpreted as a glacially eroded cliffs cut into the south-east ﬂank of Pukekaikiore (Figure 5.2). The eastern limits of this former glacial catchment are ill-deﬁned owing to the Holocene lava ﬂows of Mt. Nguaruhoe, which is situated immediately to the east. A sharp-crested lateral moraine extends from the southern ﬂank of Pukekaikiore, descending in a southwest direction for c. 500 m, terminating at c. 1275 m asl (Figure 5.2; Figure 5.3e). This moraine is paired by a shorter moraine ridge, issuing from a steep bedrock spur that deﬁnes the southern margin of this former glacial catchment. The long, right lateral moraine cross-cuts a signiﬁcantly wider, more rounded moraine CHAPTER 5. THE LAST GLACIAL CYCLE 5.4.1 Cosmogenic surface exposure dating Moraine boulder samples were collected using a portable rock saw ﬁtted with a seg- mented, diamond-tipped blade. Samples were only taken from boulders on the moraine crest (Figure 5.4), and those close to known faults and beneath lava cliffs were avoided. Where possible, I sampled boulders that were partially embedded in the moraine matrix to minimise the likelihood that the boulder has undergone post-depositional rotation. All samples were collected from the highest point of the parent boulder, and all boulders were over 0.6 m tall, thus minimising the potential for past burial by snow and/or volcanic ash. Maximising boulder height was a key priority for sample selection on the lower moraines, which have abundant tephra coverbeds on their lower ﬂanks. Thus, the majority of these samples come from boulders that stand >1.5 m above the moraine surface (Table 5.1). Sampled boulder surfaces exhibited <12◦dip relative to horizontal, thereby minimising potential self-shielding effects. Azimuthal inclinations were measured in the ﬁeld using a compass and clinometer and geometric shielding corrections were computed using the CRONUS-EARTH calculator (available at: http://hess.ess.washington.edu). All shielding corrections were less than 1%. Sample locations and elevations were recorded using a Trimble GeoXH global positioning system, relative to to the WGS84 datum. These data were differentially corrected using continuous measurements from GeoNet ’Chateau Observatory’ (’VGOB’) base station (39◦11’59” S, 175◦32’ 32”E; 1161 m asl), located 8 km south west of Mangatepopo valley. Horizontal and vertical post-processed uncertainties for individual sample locations are < 1m. Samples were jaw-crushed, rinsed in de-ionised water and dry-sieved to isolate the 250- 500 µm size fraction. Density (> 3.1 g cm−3) and magnetic separation techniques were used to separate 150 - 600 mg of pyroxene (clinoenstatite; En27−37, Fo17−31, Wo41−46). Crushing and mineral separation were undertaken using facilities at Victoria University of Wellington. Pyroxene separates were then prepared according to Bromley et al. (2014) at the Cosmogenic Nuclide Laboratory of Lamont-Doherty Earth Observatory (LDEO). 5.4. METHODS 109 Table 5.1: Location and geometry of cosmogenic 3He samples Sample Lat. Long. 5.4.1 Cosmogenic surface exposure dating Altitude Thickness Surface Shielding Boulder height ID (m asl) (cm) strike/dip (cm) MP1201 -39.1362 175.6194 1519 2.0 0 0.998 90 MP1202 -39.1387 175.6124 1429 2.0 210/10 0.998 80 MP1203 -39.1389 175.6117 1429 2.0 004/4 0.998 65 MP1204 -39.1371 175.6177 1481 1.5 0 0.997 70 MP1205 -39.1383 175.6139 1437 2.0 312/8 0.997 60 MP1206 -39.1413 175.5982 1302 2.5 098/10 0.997 165 MP1207 -39.1411 175.5997 1308 2.5 0 0.998 155 MP1208 -39.1392 175.5983 1286 2.5 0 0.998 60 MP1209 -39.1391 175.5980 1280 3.0 258/6 0.998 165 MP1210 -39.1390 175.5979 1275 2.5 296/2 0.998 150 MP1211 -39.1391 175.5969 1266 3.0 124/2 0.998 120 Table 5.1: Location and geometry of cosmogenic 3He samples Mineral separates were ﬁrst leached in 5% hydroﬂuoric (HF) / 2% nitric (HNO3) acid solution for 24 hours, followed by a separate 10% hydrochloric (HCl) acid solution for 24 hours, to remove adhering ground mass particles. Leached pyroxene crystals were visually inspected for purity and wrapped in aluminium foil. Each sample was completely degassed by heating in a furnace to >1300◦C for 15 minutes, during which, released gases were exposed to a liquid-nitrogen chilled, charcoal trap. Extracted gases were exposed to an SAES getter before being collected on a cryogenic cold trap at <15 K. Exclusively helium was then released by heating the cold trap to 45 K. Mass spectrometry was conducted at the LDEO Noble Gas Mass Spectrometry Laboratory using a MAP 215-50 noble gas mass spectrometer. Measurements were made relative to the Yellowstone ’Murdering Mudpot’ (MM) helium standard (3He / 4He ratio of 16.45Ra, where Ra = 3He / 4Heair = 1.384 x 10−6), using the protocol of Winckler et al. (2005). The relatively young crystallisation ages of the samples (< 273 ka; Hobden et al., 1996) explain the low 4He content of the pyroxene crystals (1-3 x 10−9 cm3 (STP) g−1) and the resultant high 3He / 4He ratios, which range between 47 - 311 times Ra (Table 5.2). Following Dunai et al. (2007), I measured lithium, uranium and thorium concentrations in two samples to test for nucleogenic 3He (3Hen) produced via thermal neutron reac- tions (6Li (n,α) 3H 3He). Concentrations for all three elements are low (< 1 ppm), which indicates that 3Hen content is negligible in the samples. Thus, I assume all 3He to be of cosmogenic origin. 5.4.1 Cosmogenic surface exposure dating Attenuation of cosmogenic neutron ﬂux with depth from the surface was corrected for using measured sample thickness, a standard rock density of 2.7 g cm−3 and an atten- uation length of 160 g cm−2 (Dunne et al., 1999). Field observations from mid-winter (July) show that the sampled boulders are not subject to burial by seasonal snow. Snow cover is not quantiﬁable for the geological past, however the topographic prominence CHAPTER 5. THE LAST GLACIAL CYCLE 110 Figure 5.4: Examples of boulders sampled for cosmogenic 3He surface exposure dating. Visib ample bags denote sample position on boulder surface. (A) Sample MP1201 at the eastern e f moraine M1. Photo facing westwards. (B) Sample MP1203 on moraine M1 in the foregroun with Mt. Tongariro (1963 m asl) in the background. Photo facing northeast. (C) MP1206 moraine M2. Note prominent lateral moraines on opposite valley side. Photo facing southwe D) MP1207 on moraine M2. Photo facing east. (E) Sample MP1208 on moraine M3. No he highly diffuse moraine crest, compared to moraine M1. Also, note the Holocene cone Mt. Ngauruhoe (background right). Photo facing east. (F) Samples MP1209 and MP1210 Figure 5.4: Examples of boulders sampled for cosmogenic 3He surface exposure dating. Visible sample bags denote sample position on boulder surface. (A) Sample MP1201 at the eastern end of moraine M1. Photo facing westwards. (B) Sample MP1203 on moraine M1 in the foreground, with Mt. Tongariro (1963 m asl) in the background. Photo facing northeast. (C) MP1206 on moraine M2. Note prominent lateral moraines on opposite valley side. Photo facing southwest. (D) MP1207 on moraine M2. Photo facing east. (E) Sample MP1208 on moraine M3. Note the highly diffuse moraine crest, compared to moraine M1. Also, note the Holocene cone of Mt. Ngauruhoe (background right). Photo facing east. (F) Samples MP1209 and MP1210 on moraine M3. Photo facing east. Figure 5.4: Examples of boulders sampled for cosmogenic 3He surface exposure dating. Visible sample bags denote sample position on boulder surface. (A) Sample MP1201 at the eastern end of moraine M1. Photo facing westwards. (B) Sample MP1203 on moraine M1 in the foreground, with Mt. Tongariro (1963 m asl) in the background. Photo facing northeast. (C) MP1206 on moraine M2. Note prominent lateral moraines on opposite valley side. Photo facing southwest. (D) MP1207 on moraine M2. Photo facing east. (E) Sample MP1208 on moraine M3. 5.4.1 Cosmogenic surface exposure dating Note the highly diffuse moraine crest, compared to moraine M1. Also, note the Holocene cone of Mt. Ngauruhoe (background right). Photo facing east. (F) Samples MP1209 and MP1210 on moraine M3. Photo facing east. 5.4. METHODS 111 of the boulders and moraine crests is not conducive to snow accumulation due to preferential erosion of snow by the wind. Thus, I do not apply any correction to the age calculation for burial by snow. The absence of resistant mineral veins in the local andesites precluded quantitative assessment of post-depositional erosion of boulder surfaces. Although sampled boulders did not exhibit glacial striae, some boulders retain faceted sides and care was also taken to avoid boulders that displayed clear evidence of erosion, such as discolouration, weathering scarps, onion-skin weathering, pitting/water pooling. I therefore choose to present the exposures ages without an erosion correction. Regional uplift in the central North Island has been estimated at c. 0.6 - 1.0 mm yr−1 for the past 500 ka (Pulford, 2002). Integrating this elevation change into the age calculations for boulders exposed during the LGM (c. 20-30 ka) does not alter the results outside the range of the 3He measurement uncertainty. For the oldest samples of this study, integrating this uplift rate has the effect of increasing the exposure ages by 1-2 kyr. I present the age dataset without corrections for this effect, but discuss the implications for the glacial chronology in the text. Exposure ages were computed using the cosmogenic 3He exposure age calculator and globally-compiled, sea-level, high-latitude (SLHL) cosmogenic 3He production rate of Goehring et al. (2010), which is applicable in New Zealand (Chapter 4). All calculations are computed using the CRONUS-Earth exposure age calculator MATLAB code, as described by Balco et al. (2008), and modiﬁed for cosmogenic 3He by Goehring et al. (2010). Elevation and latitude scaling of cosmogenic 3He production to the sample locations was calculated using ﬁve standard scaling models described in Balco et al. (2008) (Table 5.3). Recently, Lifton et al. (2014) have shown that neutron monitor based scaling schemes (Li, Du, De) over-estimate scaling factors, and introduce an updated scaling scheme very similar to the Lm-scaling. Thus, I base the age-calculations and discussion on the Lm-scaling scheme. 5.4.2 Tephrochronology The use of discrete, isochronous, pyroclastic (primarily ash and lapilli) marker horizons as chronological tie-points in sedimentary archives of paleoenvironmental change is well established in New Zealand (e.g. Lowe et al., 2008, 2013). In glaciated landscapes, well-dated tephra layers stratigraphically overlying depositional landforms such as moraines, provide minimum ages for moraine formation, whereas tephra preserved beneath deposits of glacial outwash or till, constrains the maximum age of deposition (e.g. Kirkbride and Dugmore, 2001). Since the work of Topping and Kohn (1973), signif- icant progress has been made to constrain the precise age and geochemical signature of rhyolitic tephra marker horizons in New Zealand (e.g. Lowe et al., 2008, 2013). To test CHAPTER 5. THE LAST GLACIAL CYCLE 112 the interpretation of Topping and Kohn (1973), I revisited their moraine soil section in Mangatepopo valley to sample the lower two rhyolite horizons for electron microprobe (EMP) analysis of glass shard major element compositions. To aid identiﬁcation, I com- pare the geochemistry of the Mangatepopo tephras to that of a discrete rhyolite horizon identiﬁed at Taurewa road cutting (39◦07′19.2′′ S, 175◦31′29.1′′ E, 825 m asl; Figure 6.1), situated c. 5 km west from the study site. This tephra stratigraphically overlies the Kawakawa/Oruanui Tephra (KOT), which was recently dated at this section (c. 25.4 cal. ka; Vandergoes et al., 2013). All samples were wet-sieved to isolate the >63 µm fraction, before individual glass shards were handpicked (n= >18 per sample; Table 5.4) and loaded in an epoxy mount. All major element determinations were made on a JEOL Superprobe (JXA-8230) at Victoria University of Wellington, using the ZAF correction method. Analyses were performed using an accelerating voltage of 15 kV under a static electron beam operating at 8 nA. The electron beam was focused to 10 µm. 5.4. METHODS 113 A number of methods exist to estimate the pELA for former glaciers. The Accumulation Area Ratio (AAR) method is applied most commonly, which is based on the assumption that the accumulation zone of a steady-state glacier represents a ﬁxed proportion of the total glacier area. Accumulation areas of modern glaciers globally, typically occupy 50-80% of the total glacier surface area (Meier and Post, 1962), whilst empirical studies show that New Zealand glaciers most commonly have an accumulation-ablation area ratio of 2:1 (AAR= 0.67; Chinn et al., 2012). A shortcoming of the AAR method is the failure to account for glacier hypsometry (Fur- bish and Andrews, 1984). For former valley and cirque glaciers, reconstructed glacier hypsometry is usually well-constrained by topography, therefore AAR-based pELA estimates are subject to less uncertainty. However, the hypsometry of former valley glaciers sourced from ice caps or plateau ice ﬁelds is less certain due to the absence of geomorphic indicators that constrain ice thickness in the upper catchment. Underesti- mating the planimetric surface area at the glacier head leads to underestimation of the local ELA, as a larger ablation area is required to balance the expanded accumulation zone. Other techniques have been developed to overcome this shortcoming in the AAR method (Furbish and Andrews, 1984; Osmaston, 2005), however these are also reliant on accurate knowledge of the former ice geometry. The glacial geomorphology of the wider Tongariro massif suggests that the former Mangatepopo glacier may have been sourced from a central ice ﬁeld, as several other moraine-lined glacial valleys radiate from the centre of the ediﬁce (Mathews, 1967), thus implying a central source. If this were the case, the AAR method will underestimate the pELA, therefore I consider the AAR-based reconstructions as a maximum estimate of ELA depression from present. Reconstructed pELAs provide a useful metric for comparing the magnitude of past climate change (Porter, 1975). This requires accurate knowledge of the present-day ELA. No glaciers currently exist on Tongariro massif, however small cirque glaciers persist on Mt. Ruapehu (2797 m asl), situated 15 km to the south. In the most recent survey of these glaciers, Keys (1988) reports contemporary ELAs of 2340 - 2650 m asl, based on end of summer snowline surveys. 5.4.3 Equilibrium line altitude reconstruction Palaeoclimatic calculations from reconstructed glaciers rely on accurate delineations of former ice masses, which are constrained by interpretation of ice-marginal geo- morphology (Porter, 1975; Benn et al., 2005). Moraine ridges and glacial till extent clearly depict the terminus and lower margins of the former Mangatepopo glacier, whilst erosional landforms such as glacial cirque and glacially-trimmed cliffs provide guidance on the upper glacier limits. I reconstruct the palaeo-equilibrium line altitude (pELA) of the Mangatepopo glacier for the innermost glacier geometry (c. 21 ka - see below) delineated by the preserved glacial landforms only, as the outboard ice limits are less well preserved. I consider the c. 21 ka glacier margin to be located slightly below M1 moraine on the northern valley side, based on the cosmogenic 3He exposure ages and the topographic relationship of moraines M1 and M2 (Section 5.3.2.1). In the vicinity of Mt. Ngauruhoe, post-glacial volcanic activity has obscured evidence of the former glacial limits, therefore I estimate former ice limits in this region by interpolating between the cirque headwall and the glacially-truncated lava ﬂows of Pukekaikiore (Figure 5.2), using analogous contemporary valley glacier geometries. To estimate the hypsometry of the former glacier, elevation contours are drawn from the intersection of the reconstructed ice margin and the modern topographic contours. Ice surface topog- raphy is reconstructed to represent ﬂow vectors in contemporary glaciers, which are increasingly convergent with distance upstream from the ELA, and increasingly diver- gent downstream (Paterson, 1994). I deﬁne the upper margins of the LGM glacier using the head of South Crater cirque (sensu Kaplan et al., 2010), although, it is possible that additional ice was sourced from an ice ﬁeld centred on Tongariro massif (Section 5.3.2.2). 5.4. METHODS 5.4. METHODS Topoclimatic factors such as wind-driven snow accumulation and topographic shading impart a strong inﬂuence on the mass balance of cirque glaciers (Kuhn, 1995), which can reduce the utility of the ELA as an index for atmospheric temperature change. However, Brook et al. (2011) analysed surface area changes of one glacier on Mt. Ruapehu between AD1988-2008 and found that variations closely follow ablation season temperature changes. This correlation supports the use of end of summer snowline observations on Mt. Ruapehu to relate local pELA reconstructions to past atmospheric temperature change. I take the arith- CHAPTER 5. THE LAST GLACIAL CYCLE 114 metic mean of the midpoints of Keys (1988) observations (2483 ± 55 m asl) to use as a modern (AD1988) ELA datum. metic mean of the midpoints of Keys (1988) observations (2483 ± 55 m asl) to use as a modern (AD1988) ELA datum. 5.5.1.1 Upper moraine (M1) Exposure ages (with internal uncertainties) of the ﬁve boulders sampled from moraine M1 are 30.6 ± 0.7 ka, 26.0 ± 0.6 ka, 23.4 ± 0.5 ka, 22.8 ± 0.9 ka and 16.3 ± 0.5 ka (Table 5.3. I adjudge the youngest sample (MP1203) to be an outlier, based on the preservation of moraine M2 down valley, which dates to 21 ka (see section 5.5.1.2 below). It is unlikely that M2 would be preserved today, if ice of sufﬁcient thickness to be depositing boulder MP1203 at the elevation of M1 was present in Mangatepopo valley at c. 16 ka. Re-examination of ﬁeld descriptions and photographs (e.g. Figure 5.4B) suggests that this boulder may have shed a portion of its surface since deposition, as evidence of fracturing is present, probably caused by preferential weathering along internal cooling joint planes. Post-depositional removal of the boulder surface through this process provides a possible explanation for the anomalous young age. Following omission of this outlier, three possible depositional scenarios can explain the age distribution of the remaining four samples. First, the oldest age (c. 31 ka - MP1201) may represent inheritance of cosmogenic 3He from exposure prior to deposition at its current position on the moraine crest. In this scenario, the true age of M1 would be closer to the remaining three samples, between 26-23 ka. Second, the oldest age (c. 31 ka - MP1201) may represent the true age of M1, with the remaining samples having been subject to post-depositional weathering or moraine degradation processes (e.g. Applegate et al., 2008), causing the surface exposure ages to post-date moraine formation. Third, M1 could represent a composite landform (e.g. Roethlisberger and Schneebeli, 1979) comprising deposits from several glacier ﬂuctuations of similar mag- nitude between 31-23 ka. Based on the existing dataset, it is not possible to unequivocally attribute either one of these scenarios to the M1 moraine, however some points can be made. Analyses of large datasets of moraine boulder surface exposure ages show that only a very small percentage of boulders deposited by non-polar, temperate glaciers exhibit evidence 115 5.5. RESULTS for prior exposure (inheritance) (Putkonen and Swanson, 2003; Heyman et al., 2011). 5.5.1.1 Upper moraine (M1) This ﬁnding is supported locally, by recent dating campaigns in the Southern Alps that have generated several hundred, individual high-precision in situ cosmogenic 10Be exposure ages for moraine boulders (Schaefer et al., 2009; Kaplan et al., 2010; Putnam et al., 2010a, 2013a,b; Kelley et al., 2014), which indicate that any inherited component is typically less than the measurement uncertainty (see Schaefer et al. (2009) for a detailed consideration). The maritime, mid-latitude location of the study site, together with geomorphic evidence for scouring of the glacier bed (Section 5.3.2.1) suggest that former glaciation at this site was warm-based. Thus, whilst it is feasible that the 3He content of MP1201 could represent inheritance from prior exposure, I consider this scenario the least likely explanation for the age distribution. It is more difﬁcult to decipher between the latter two scenarios, however both imply moraine aggradation at c. 31 ka, with the younger ages either representing post-depositional disturbance (scenario 2), or further aggradation from glacial reoccupation at 26 - 23 ka (scenario 3). Whilst scenario 2 is most commonly assumed in such situations, diachronous moraines have also previously been inferred from surface exposure age datasets displaying multiple populations (e.g. Licciardi et al., 2004; Briner, 2009). This is more likely in situations where a glacier is topographically constrained (Roethlisberger and Schneebeli, 1979), such as the Mangatepopo valley. Exposure ages from down-valley (Section 5.5.1.2) suggest valley glaciation endured until c. 21 ka, therefore I suggest the age distribution from M1 most likely represents time-transgressive aggradation through the period c. 31 - 23 ka. Moraine construction would have occurred when the ice surface elevation was sufﬁcient to overcome the prominent lava ﬂow that bounds the northern valley side. 5.5.1.2 Lower moraine - inner (M2) Two boulders were deemed suitable for exposure dating (MP1206 and MP1207; Figure 5.4C & D) and returned ages of 20.0 ± 0.6 ka and 21.4 ± 0.6 ka, respectively. These ages are in close agreement and are stratigraphically coherent with the age of M1 and the tephrostratigraphy (Section 5.5.2). I consider that the arithmetic mean of these ages, 21 ± 1.0 ka to represent the moraine formation age and onset of glacial retreat from this position. This age is several thousand years younger than the age of M1 (above), which is situated directly up-valley, such that in plan view it appears that both M1 and M2 moraines may relate to a single period of moraine building. If M1 and M2 do in fact represent a single event, then the offset in surface exposure ages between the moraines CHAPTER 5. THE LAST GLACIAL CYCLE 116 must be caused by either, (i) inheritance in the M1 samples, or (ii) post-depositional erosion or shielding of the M2 samples. I consider the former unlikely, for the reasons outlined above in Section . Concerning the latter, there is no ﬁeld evidence that either sample from M2 has undergone signiﬁcant erosion of the boulder surface sufﬁcient to raise the true exposure age by several kyr (c. 10 - 20mm kyr−1), therefore I consider this an unlikely source of this systematic offset. Concerning the latter, there is evidence to suggest that soil/tephra cover on M2 moraine has been more extensive in the past, as indicated by the present-day soil distribution and erosional scarps (Figure 5.3d,e). Thus, to assess the possibility that shielding from volcanic ash/soil development has reduced cosmogenic 3He production in the geological past, I use the attenuation rate of cosmogenic nuclide production due to burial (Figure 3.1) to construct hypothetical burial scenarios beneath given thicknesses and burial durations of soil cover (ρ = 1.6 g cm3). The nuclide concentration that would result from the hypothetical burial scenario, according to the attenutation rate of nuclide production depth (Figure 3.1), is subtracted from the measured nuclide concentration to leave the concentration aqcuired through simple exposure. The ’true’ age is then calculated by adding to the simple exposure concentration, the nuclide concentration that would result from simple exposure for a duration equal to the hypothetical burial time. These calculations do not include possible changes in nuclide production with time due to changes in the geomagnetic ﬁeld intensity. 5.5.1.2 Lower moraine - inner (M2) Soil thicknesses represent that overlying the sampled boulder surfaces, which typically sit >1 - 1.5 m above the moraine surface (see Table 5.1). Figure 5.5: (a) The hypothetical ’true’ ages of moraine M2 samples due to shielding of Figure 5.5: (a) The hypothetical true ages of moraine M2 samples due to shielding of cosmo- genic nuclide production beneath sediment (ρ = 1.6 g cm−3) cover of given burial durations and thicknesses; (b) As (a), but using 55 ka as the measured exposure age, thus representing an example from the M3 moraine samples. Soil thicknesses represent that overlying the sampled boulder surfaces, which typically sit >1 - 1.5 m above the moraine surface (see Table 5.1). smaller volume than M2 and is situated on top of a prominent lava ﬂow that outcrops on the valley wall. Given the lack of an obvious M1 correlative moraine outboard of M2 it is likely that, during M1 time, the former Mangatepopo glacier terminated in a similar location to that depicted by the M2 moraine. This scenario could explain the large difference in volume between the two moraines, if M2 construction began in the period 31 - 23 ka, whilst M1 was only intermittently constructed during periods when the glacier thicknesses exceeded the topographic threshold presented by the prominent lava ﬂow. This scenario requires an explanation for why the M1 moraine was not also buried during construction of M2 moraine at c. 21 ka, if the glacier geometry was largely the same. One possibility is that erosion of the glacier bed during between 31 - 23 ka could have resulted in a lower glacier surface by the time the M2 samples were emplaced, leaving the the M1 moraine stranded above the glacier margin. Another possibility is a change in the mass balance regime of the glacier between M1 and M2 construction. For example, higher precipitation during M1 time could have produced a thicker, steeper glacier (e.g. Golledge et al., 2012), whereas reduced precipitation during M2 construction may have produced a thinner glacier with a lower gradient longitudinal proﬁle. This pattern of climatic forcing is observed in speleothem records, whereby the onset of the LGCP was characterised by higher precipitation and milder temperatures, whilst the culmination of the stadial was drier and colder (Whittaker et al., 2011). 5.5.1.2 Lower moraine - inner (M2) Figure 5.5 shows that if the nuclide concentration in the M2 samples actually rep- resents c. 31 kyr with intermittent burial, then the sample surfaces must have been buried by at least 250 cm of soil/tephra for a duration of c. 10 kyr in order to produce the measured cosmogenic 3He concentration. A true M2 moraine age of c. 24 ka re- quires c. 8 kyr beneath 50 cm of soil, or 2 - 3 kyr beneath > 100 cm. These thicknesses represent soil depth on the surface of the boulders, which stand >150 cm above the moraine surface. Thus, even small burial thickness scenarios require at least c. 150 cm of sediment on the moraine surface before nuclide production in the boulder surfaces is reduced. This is inconsistent with the observed coverbed thinning from the base of the moraine slope to the crest (Figure 5.3e), which indicates that soil/tephra thickness on the moraine crest has not exceeded c. 1 m. Furthermore, the time required for tephra and soil accumulation/development is substantial (5 m per 14 kyr on the sheltered lower moraine slope - Figure 5.6), which further restricts the amount of time available to attenuate nuclide production in the samples. In summary, it appears that the difference in age between the M2 and M1 exposure ages is real, as implausible erosion/shielding scenarios are required to raise the M2 ages by several thousand years to align with those for M1. The M1 moraine has a 5.5. RESULTS 117 Figure 5.5: (a) The hypothetical ’true’ ages of moraine M2 samples due to shielding of cosmo- genic nuclide production beneath sediment (ρ = 1.6 g cm−3) cover of given burial durations and thicknesses; (b) As (a), but using 55 ka as the measured exposure age, thus representing an example from the M3 moraine samples. Soil thicknesses represent that overlying the sampled boulder surfaces, which typically sit >1 - 1.5 m above the moraine surface (see Table 5.1). Figure 5.5: (a) The hypothetical ’true’ ages of moraine M2 samples due to shielding of cosmo- genic nuclide production beneath sediment (ρ = 1.6 g cm−3) cover of given burial durations and thicknesses; (b) As (a), but using 55 ka as the measured exposure age, thus representing an example from the M3 moraine samples. 5.5.1.3 Lower moraine - outer (M3) 5.5.1.3 Lower moraine - outer (M3) Samples were collected from four boulders, which returned ages ranging from 58.0 ± 1.0 ka to 46.6 ± 1.0 ka. I consider these ages to be minimum-limiting ages for this landform for the following reasons. First, assuming that this landform was originally sharp-crested like the other, younger moraines in the valley, the rounded cross-section present today (Figure 5.2B) indi- cates substantial post-depositional landform degradation. Such degradation may have caused exhumation of boulders previously shielded from cosmic rays (Hallet and Putko- nen, 1994; Applegate et al., 2012). This degradation probably occurred in vegetation-free, periglacial conditions during MIS 2, when renewed glacial conditions caused glacial re-advance and the deposition of moraines M1 and M2 in the Mangatepopo valley (see above). Previous workers have noted that many of the high-altitude coverbeds in the northern and western regions of TgVC represent post-glacial deposition (Topping, 1973; Donoghue et al., 1995), therefore it is likely that during glacial times the land surface was free of vegetation and soil, which facilitates hillslope diffusion. Second, continual exposure of these andesitic boulders to the relatively high total annual precipitation at this location and elevation (c. 3 m yr−1; NIWA, 2014), over several tens of millennia, is likely to have caused granular disintegration of the boulder surfaces. Such erosion would remove cosmogenic 3He atoms, thus resulting in nuclide concentrations that underestimate the true exposure age of the boulder. For example, boulder surface erosion rates of 1.5 - 5.5 mm kyr−1 would raise the exposure ages of the M3 samples to c. 63 ka, which corresponds with the culmination of a major glacier advance in the Southern Alps (Schaefer et al., 2015). Such rates of rock erosion agree well with the compilation of Portenga and Bierman (2011) which indicates typical long term erosion rates of c. 1 - 10 mm kyr−1 for igneous lithologies in temperate climatic environments. Second, continual exposure of these andesitic boulders to the relatively high total annual precipitation at this location and elevation (c. 3 m yr−1; NIWA, 2014), over several tens of millennia, is likely to have caused granular disintegration of the boulder surfaces. Such erosion would remove cosmogenic 3He atoms, thus resulting in nuclide concentrations that underestimate the true exposure age of the boulder. For example, boulder surface erosion rates of 1.5 - 5.5 mm kyr−1 would raise the exposure ages of the M3 samples to c. 5.5.1.2 Lower moraine - inner (M2) Regardless of the precise cause, the age data presented here indicates that the M2 samples were deposited after construction of the M1 moraine, thus they represent the last time that M2 was occupied by a glacier in the Mangatepopo valley. CHAPTER 5. THE LAST GLACIAL CYCLE 118 5.5.1.3 Lower moraine - outer (M3) 63 ka, which corresponds with the culmination of a major glacier advance in the Southern Alps (Schaefer et al., 2015). Such rates of rock erosion agree well with the compilation of Portenga and Bierman (2011) which indicates typical long term erosion rates of c. 1 - 10 mm kyr−1 for igneous lithologies in temperate climatic environments. Third, the age calculations are uncorrected for potential tectonic uplift, which has been estimated at c. 0.6 - 1.0 mm yr−1 (Pulford, 2002). Including c. 40 - 60 m of uplift into the exposure age calculation serves to lower the time-integrated local production rate, relative to that estimated for the present day sample elevation. Depending on the temporal pattern of uplift over the exposure period, this effect could cause underesti- mation of boulder ages by up to 2 kyr. As with the M2 samples (above), the distribution of tephra and soil coverbeds and erosional scarps on M3 moraine, suggest that this cover was more extensive on the 5.5. RESULTS 119 moraine surface in the geological past. Potential attenuation of cosmogenic nuclide production in the M3 samples can be assessed in two ways. First, Figure 5.5 shows the effect of different burial duration and thickness scenarios on the true age of a boulder that yields a hypothetical exposure age of 55 kyr. Given the height of the boulders (Table 5.1), realistic burial depths are likely <100 cm, thus Figure 5.5 shows that several tens of thousands of years of burial beneath such a soil thickness are required to push the true age of the M3 samples back beyond MIS 4. Second, all of the M3 samples are taken from a similar location on the M3 moraine, thus it is likely that all would have had a similar burial/exhumation history. Thus, the measured nuclide concentrations should correlate with boulder height, as taller boulders would likely be buried last by accumulating soil/ash, and uncovered ﬁrst during subsequent exhumation. This is not the case, as the lowest boulder (MP1208; Table 5.1) on the M3 moraine returned the oldest exposure age, whilst taller boulders produced lower nuclide concentrations. In summary, it is unlikely that the samples on M3 have undergone any shielding due to more extensive soil/tephra cover, thus the most likely scenario is that this moraine was deposited during MIS 4 (c. 60-65 ka). samples from Mangatepopo valley. 5.5.1.3 Lower moraine - outer (M3) He/4He ± R/Ra 155x10−4 7.766x10−6 156 906x10−4 5.171x10−6 138 009x10−4 4.187x10−6 73 376x10−4 1.878x10−5 172 473x10−4 8.758x10−6 178 471x10−5 1.825x10−6 47 111x10−4 3.293x10−6 80 177x10−4 9.399x10−6 301 599x10−4 5.844x10−6 188 702x10−4 7.023x10−6 195 312x10−4 1.482x10−5 311 121 5.5. RESULTS Table 5.3: Exposure ages (ka) with internal (external) uncertainties. Calculated using the ﬁve scaling models of Stone (2000) (’St’), Desilets et al. (2006) (’De’), Dunai (2001) (’Du’), Lifton et al. (2005) (’Li’) and Lal (1991) (’Lm’). Lm ages are presented in bold and are those discussed in text. aSamples from Lower moraine - outer (M3) have been subject to 40-60 m uplift since deposition, therefore ages presented are a minimum estimate. p Sample ID St ± De ± Du ± Li ± Lm ± Upper moraine (M1): MP1201 31.5 0.7 (2.6) 29.1 0.7 (2.9) 29.1 0.7 (2.9) 29.2 0.7 (3.1) 30.6 0.7 (3.5) MP1202 23.7 0.5 (1.9) 22.3 0.5 (2.2) 22.3 0.5 (2.2) 22.5 0.5 (2.4) 23.4 0.5 (2.7) MP1203 16.1 0.5 (1.4) 15.3 0.5 (1.5) 15.3 0.5 (1.6) 15.5 0.5 (1.7) 16.3 0.5 (1.9) MP1204 23.0 0.9 (2.0) 21.6 0.9 (2.2) 21.6 0.9 (2.3) 22.0 0.9 (2.4) 22.8 0.9 (2.7) MP1205 26.3 0.6 (2.1) 24.6 0.5 (2.4) 24.6 0.5 (2.4) 24.8 0.5 (2.6) 26.0 0.6 (3.0) Lower moraine - inner (M2): MP1206 20.0 0.6 (1.7) 19.1 0.5 (1.9) 19.1 0.5 (1.9) 19.3 0.5 (2.1) 20.0 0.6 (2.3) MP1207 21.5 0.6 (1.8) 20.4 0.6 (2.0) 20.4 0.6 (2.1) 20.6 0.6 (2.2) 21.4 0.6 (2.4) Lower moraine -outer (M3)a: MP1208 60.5 1.0 (4.9) 55.4 1.0 (5.4) 55.8 1.0 (5.5) 55.5 0.9 (5.9) 58.0 1.0 (6.4) MP1209 56.0 1.0 (4.5) 50.9 0.9 (4.9) 51.4 0.9 (5.1) 50.9 0.9 (5.4) 53.5 0.9 (6.0) MP1210 49.2 1.0 (4.0) 44.6 0.9 (4.4) 45.0 1.0 (4.5) 44.5 0.9 (4.7) 46.6 1.0 (5.3) MP1211 55.0 1.0 (4.4) 50.0 0.9 (4.9) 50.4 1.0 (5.0) 50.0 0.9 (5.3) 52.3 1.0 (5.9) 5.5.2 Coverbed stratigraphy and tephra major element geochemistry Stratigraphic logging of soil sections overlying the ice-proximal ﬂank of the M2 moraine (Figure 5.6A) permits correlation with the detailed descriptions of Topping and Kohn (1973) and Topping (1974). At c. 450 cm depth I identify a matrix-supported diamicton, predominantly consisting of boulders and cobbles in a silty matrix, which represents the surface of the underlying lateral moraine. A section containing approximately 90 cm of interbedded cm- to dm-thick andesitic ash and lapilli beds, with two yellow- white rhyolitic tephra beds and weakly formed palaeosols, immediately overlies the moraine surface. The lowermost rhyolitic tephra (sample: ’MP833a’) is 0.2-1 cm thick and situated c. 10-20 cm above the moraine surface, immediately overlying a 3 cm thick, coarse (med. sand), dark-grey andesitic tephra (Figure 5.6C). The lower rhyolitic tephra exhibits ductile fold structures (Figure 5.6B), which suggests post-depositional deformation, perhaps via frost-heave. Approximately 40 cm above MP833a, a discontin- uous rhyolitic tephra (sample: ’MP833d’) varies in thickness from 0-3 cm (Figure 5.6C). I identify a distinct cobble-to-ﬁne gravel unit at 350 cm depth, which I correlate to the pebble unit identiﬁed by Topping and Kohn (1973) (described fully in Topping, 1974), therefore I am conﬁdent that these tephra samples correspond with those analysed by Topping and Kohn (1973). A sharp erosional contact separates this coarse unit from a massive, grey/brown silt-clay bed (300-50 cm depth), with abundant rhizomorphs and two, discrete, interbedded pumiceous horizons. The lower pumice bed (at c. 275 cm depth) is c. 10 cm thick and consists of yellow and dark brown coloured clasts, up to 2 cm in diameter. The upper pumice is c. 40-50 cm thick and immediately underlies the modern soil horizon. This pumice is correlated to the Taupo ignimbrite (AD232 ± CHAPTER 5. THE LAST GLACIAL CYCLE 122 Figure 5.6: (A) Stratigraphic log of the Taurewa reference section and the Mangatepopo moraine section originally described by Topping and Kohn (1973), with the samples labelled; (B) The lowermost rhyolite horizon (sample MP833a) displaying evidence for post-depositional deformation; (C) Soil pit showing both rhyolite horizons at the Mangatepopo section. Figure 5.6: (A) Stratigraphic log of the Taurewa reference section and the Mangatepopo moraine section originally described by Topping and Kohn (1973), with the samples labelled; (B) The lowermost rhyolite horizon (sample MP833a) displaying evidence for post-depositional deformation; (C) Soil pit showing both rhyolite horizons at the Mangatepopo section. 5.5.2 Coverbed stratigraphy and tephra major element geochemistry 10; Lowe et al., 2013), based on stratigraphic position, large pumice clasts (up to 10 cm diameter), and abundant charred twigs. Shane (2000) summarised the major element chemistry of post-25 ka rhyolitic tephras in North Island and found that OVC tephras are characterised by higher SiO2 (c. 76-79 wt. %) and lower FeO (c. 1 wt. %), compared to those from the Taupo Volcanic Centre (SiO2 = c. 71-77 wt. %; FeO = 1.5-3.5 wt. %). Using this information, I can assign samples MP833a and MP833d (Table 5.4 ; Figure 5.7) to the OVC with high conﬁdence. This supports the original interpretation of Topping and Kohn (1973), who assigned both to horizons in the Mangatepopo section to OVC-sourced eruptions. However, the major element compositions of MP833a and MP833d are also indistinguishable from one another (Table 5.4; Figure 5.7), which suggests that the upper sample (MP833d) 123 5.5. RESULTS could represent a reworked remnant of MP833a below. This is further supported by the ﬁeld observations of deformation structures within the lower horizon (MP833a), but contradicts the initial interpretation of Topping and Kohn (1973). The geochemical composition of the Taurewa rhyolitic tephra (Sample: 13-16-27; Figure 5.6) is indistin- guishable from the Mangatepopo data, which suggests it represents the same volcanic event (Figure 5.7). To further constrain deglaciation in the Mangatepopo valley, I seek to determine which OVC-sourced event is represented by the Mangatepopo tephra. To do this, I compare the glass shard major element measurements to proximal and distal OVC reference data from Honeycomb Trench (B.V. Alloway, unpub. data) and Waipaoa river basin (Bilderback, 2012; Marden et al., 2014), respectively. Using binary plots of K2O - FeO, the Rerewhakaaitu Tephra can be readily discriminated from the Waiohau Tephra in both the OVC-proximal Honeycomb dataset and distal Waipaoa dataset, based on the presence of high and low K2O populations (Figure 5.7B; Shane et al., 2008). The Mangatepopo and Taurewa data are sufﬁciently different from the Rotorua Tephra for us to rule this out as a possible correlative. Biotite has been noted as a diagnostic component of the ferromagnesian mineral assemblage of the Rerewhakaaitu Tephra (Froggatt and Lowe, 1990) and I do not identify any biotite ﬂakes within the mineral assemblages of the samples. 5.5.2 Coverbed stratigraphy and tephra major element geochemistry Isopach maps of tephra dispersal over North Island (Lowe et al., 2013) indicate a similar thickness (0.5 - 1 cm) for both the Rerewhakaaitu and Waiohau Tephras in the vicinity of the study site, therefore ﬁeld observations of thick- ness are not useful in discriminating between the two. In summary, ﬁeld descriptions and EMP measurements of glass shard major elements suggest that the samples from two rhyolitic tephra horizons likely represent the same event, as opposed to the previous interpretation of two separate events (Topping and Kohn, 1973). Furthermore, the major element composition of this tephra can be corre- lated to the OVC with high conﬁdence. The single K2O population and the absence of biotite is consistent with the known chemical and mineral composition of the Waiohau Tephra, rather than the Rerewhakaaitu Tephra. However, this interpretation is not consistent with the Fe-Ti analyses of Topping and Kohn (1973). Regardless of which speciﬁc event this tephra represents, the ﬁndings are stratigraphically consistent with surface exposure ages from the moraine crest (c. 21 ka; Section 5.5.1.2) and accord with moraine-tephrostratigraphy elsewhere on Tongariro massif (Cronin and Neall, 1997) that suggests moraine abandonment and soil aggradation in response to climatic ame- lioration prior to 14.0 ka. This represents a minimum age for ice-retreat, which likely occurred several thousands of years earlier, as indicated by the cosmogenic surface exposure ages on the moraine crest. CHAPTER 5. THE LAST GLACIAL CYCLE 5.5.3 Equilibrium line altitude reconstruction I derive a pELA (AAR = 0.67; Chinn et al., 2012) for the LGM Mangatepopo glacier of 1410 m asl (Figure 5.8a). This is c. 120 m lower than the maximum elevation of lateral moraine (MELM; Andrews, 1975) M1, which can be used to approximate the pELA associated with the older (c. 31-23 ka), slightly thicker Mangatepopo glacier. The moraine stratigraphy suggests that the Mangatepopo glacier was broadly of similar extent during the period 31-21 ka, therefore the MELM method provides a useful independent test of the AAR-based ELA estimate, particularly as it is not subject to uncertainties in past glacier hypsometry or topographic change. The good agreement between the AAR and MELM methods provides conﬁdence in this ELA reconstruction. The lower value predicted by the AAR supports the interpretation that the glacier sourced ice from a wider accumulation zone than currently appreciated, such as an ice ﬁeld (Rea et al., 1999). It is therefore unlikely that the 21 ka ELA was lower than 1400 m asl. I consider 1400 - 1550 m asl a most-likely estimate of the ELA in Mangatepopo valley during the period 31-21 ka. Using the modern ELA datum of Keys (1988) (2483 ± 55 m asl), this represents an ELA lowering of c. 930-1080 m, relative to present. The ELA on a given glacier is primarily controlled by summer air temperature and winter precipitation, although a range of other energy-balance (insolation, local wind speed, cloudiness, humidity) and topoclimatic (avalanching, snow drifting, topographic shading) factors also contribute (Oerlemans and Fortuin, 1992). Assuming no change in precipitation (see Discussion), it is possible to derive a ﬁrst-order estimate of atmo- spheric temperature change associated with a pELA reconstruction, using a temperature lapse rate. Temperature lapse rates can vary signiﬁcantly in space and time (Minder et al., 2010; Doughty et al., 2013), and ELA-based palaeotemperature reconstructions are sensitive to this value. For example, I calculate an LGM temperature lowering of c. 5.4 ◦C relative to present, when using the mean annual temperature lapse rate for upland (> 300 m) New Zealand (-5.1 ◦C km−1; Norton, 1985). However, using the standard en- vironmental lapse rate (-6.5 ◦C km−1) increases the temperature depression estimate to 7.0 ◦C. CHAPTER 5. THE LAST GLACIAL CYCLE 124 Table 5.4: Glass shard major element compositions of rhyolitic tephras from the Taurewa and Man- gatepopo sections, compared with potential correlatives from the Okataina Volcanic Centre (OVC). Oxide values are recalculated to 100% on a volatile-free basis. Total Fe expressed as FeOt. Mean and 1 standard deviation (italics), based on n analyses. All samples normalised either against glass stan- dard VG-568 or ATHO-G. EMP Analyst: B.V. Alloway, for all samples except ’MP833d’ (S.R. Eaves). Table 5.4: Glass shard major element compositions of rhyolitic tephras from the Taurewa and Man- gatepopo sections, compared with potential correlatives from the Okataina Volcanic Centre (OVC). Oxide values are recalculated to 100% on a volatile-free basis. Total Fe expressed as FeOt. Mean and 1 standard deviation (italics), based on n analyses. All samples normalised either against glass stan- dard VG-568 or ATHO-G. EMP Analyst: B.V. Alloway, for all samples except ’MP833d’ (S.R. Eaves). dard VG-568 or ATHO-G. EMP Analyst: B.V. Alloway, for all samples except ’MP833d’ (S.R. Eaves). SiO2 Al2O3 TiO2 FeO MgO MnO CaO Na2O K2O Cl Total n Mangatepopo moraine: MP833(i) 78.13 12.39 0.15 0.98 0.13 0.05 0.89 3.96 3.23 0.09 98.21 20 0.28 0.12 0.03 0.08 0.01 0.03 0.04 0.14 0.10 0.01 1.28 MP833a(ii) 78.21 12.38 0.14 1 0.12 0.05 0.88 3.84 3.28 0.09 98.2 20 0.29 0.1 0.03 0.14 0.03 0.03 0.04 0.14 0.31 0.02 1.58 MP833d 78.86 12.39 0.14 0.91 0.13 0.05 0.88 3.5 2.99 0.15 99.3 18 0.24 0.10 0.02 0.09 0.03 0.03 0.06 0.14 0.12 0.02 1.00 Standard - VG-568: Sept. 14 2012 75.6 12.2 0.23 3.27 0.1 0.1 1.72 3.75 2.61 nd 99.57 31 0.55 0.12 0.02 0.09 0.01 0.02 0.03 0.1 0.03 0.65 Standard - ATHO-G: Oct. 1 2013 75.57 12.2 0.26 3.27 0.09 0.1 1.7 3.73 2.64 0.02 99.58 48 0.60 0.09 0.03 0.10 0.01 0.03 0.03 0.13 0.05 0.01 0.75 Dec. 2013 75.62 12.2 0.24 3.27 0.09 0.11 1.7 3.73 2.64 0.08 99.68 20 0.36 0.07 0.02 0.09 0.01 0.04 0.02 0.11 0.06 0.06 0.41 plots for Mangatepopo samples and OVC-proximal reference data from Honeycomb Trench mples and OVC-proximal reference data from Honeycomb Tren sam ots for ots fo ots fo CHAPTER 5. THE LAST GLACIAL CYCLE 126 5.5.3 Equilibrium line altitude reconstruction Figure 5.8b shows the cumulative probability distribution of palaeotemperature estimates in Mangatepopo valley derived using the empirically constrained palaeo- (1400-1550 m asl; AAR and MELM) and contemporary-ELAs (2483 ± 55 m asl; Keys, 1988) and a range of possible atmospheric temperature lapse rates, equally weighted between 4 - 7 ◦C km−1. This yields a normally distributed range of palaeotemperature estimates, centred on 5.6 ± 1.1 ◦C (Figure 5.8a), which I consider to be a best-estimate estimate of atmospheric cooling in central North Island between 31-21 ka. 5.6. DISCUSSION 127 Figure 5.8: (a) Area-altitude curve for the reconstructed 21 ka Mangatepopo glacier. Solid line depicts the associated ELA derived using an AAR of 0.67. Grey shading indicates range using the AAR values of 0.5 - 0.8 (Meier and Post, 1962). (b) Cumulative probability distribution function for 21 ka palaeotemperature estimate in the Mangatepopo Valley derived using palaeo- (1400-1550 m asl; AAR and MELM) and contemporary-ELAs (2483 ±55 m asl; Keys, 1988) and a range of possible atmospheric temperature lapse rates, equally weighted between 4 - 7 ◦C km−1. Shaded grey zone indicates 1σ uncertainty interval. 5.8: (a) Area-altitude curve for the reconstructed 21 ka Mangatepopo glacier. Solid line Figure 5.8: (a) Area-altitude curve for the reconstructed 21 ka Mangatepopo glacier. Solid line depicts the associated ELA derived using an AAR of 0.67. Grey shading indicates range using the AAR values of 0.5 - 0.8 (Meier and Post, 1962). (b) Cumulative probability distribution function for 21 ka palaeotemperature estimate in the Mangatepopo Valley derived using palaeo- (1400-1550 m asl; AAR and MELM) and contemporary-ELAs (2483 ±55 m asl; Keys, 1988) and a range of possible atmospheric temperature lapse rates, equally weighted between 4 - 7 ◦C km−1. Shaded grey zone indicates 1σ uncertainty interval. Figure 5.8: (a) Area altitude curve for the reconstructed 21 ka Mangatepopo glacier. Solid line depicts the associated ELA derived using an AAR of 0.67. Grey shading indicates range using the AAR values of 0.5 - 0.8 (Meier and Post, 1962). (b) Cumulative probability distribution function for 21 ka palaeotemperature estimate in the Mangatepopo Valley derived using palaeo- (1400-1550 m asl; AAR and MELM) and contemporary-ELAs (2483 ±55 m asl; Keys, 1988) and a range of possible atmospheric temperature lapse rates, equally weighted between 4 - 7 ◦C km−1. Shaded grey zone indicates 1σ uncertainty interval. CHAPTER 5. THE LAST GLACIAL CYCLE 128 The glacial chronology presented here is in good agreement with the only other glacio- logical reconstruction in North Island, New Zealand. Brook et al. (2008) report cos- mogenic 10Be moraine and bedrock exposure ages of c. 18 ka in the Tararua ranges of southern North Island (c. 40◦S). Recalculating these ages using the local 10Be production rate of Putnam et al. (2010b) yields revised ages of c. 21 ± 2 ka, which is indistinguish- able from the last pulse of glaciation recorded in the Mangatepopo valley. Brook (2009) also identiﬁes the KOT within this moraine, which suggests it represents a composite feature ﬁrst occupied prior to 25.4 ka (Brook and Crow, 2008; Brook, 2009). Recent, high-precision cosmogenic 10Be moraine chronologies from the central Southern Alps show a similar temporal pattern of glacier ﬂuctuations. Local constraint of the 10Be production rate (Putnam et al., 2010b), coupled with favourable topographic situations for moraine preservation and extensive exposure age datasets, have afforded detailed insight to late Quaternary glacier ﬂuctuations in this region. For example, Putnam et al. (2013b) show that the former Ohau glacier deposited terminal moraines at c. 32 ka, c. 27 ka c. 23 ka and c. 18 ka. On the west coast of South Island, Suggate and Almond (2005) suggest glacier advances culminated at c. 28 ka, 22 ka and 19 ka, however recent cosmogenic 10Be surface exposure dating of these moraine sequences has reﬁned the age of these deposits to c. 25 ka, c. 21 ka and c. 17 ka (Barrows et al., 2013). On the eastern side of the Southern Alps, Rother et al. (2014) show that the former Rangitata glacier reached its maximum extent before c. 28 ka, followed by successive ﬂuctuations of slightly lesser extent between 26-19 ka. Thus, the ﬁndings from central North Island add to a growing body of evidence that show glaciers across New Zealand attained their maximum extent in late MIS 3 and ﬂuctuated about this position through the global LGM (26-19 ka; Clark et al., 2009). At the last glacial termination, glacier retreat in central North Island may have be- gun as early as 21.0 ± 2 ka, as indicated by the cosmogenic 3He moraine chronology. Additional constraint from moraine tephrostratigraphy in the Mangatepopo valley, suggests signiﬁcant climatic amelioration prior to deposition of the Waiohau Tephra (c. 14.0 ± 0.2 ka; Lowe et al., 2013). 5.6.1 The Last Glacial Cold Period in central North Island Using in situ cosmogenic 3He surface exposure dating of moraine boulders, I provide the ﬁrst direct chronological constraint for extensive valley glaciation on Tongariro massif during MIS 3 - 2, which began as early as 31 ± 3 ka and persisted until at least c. 21 ± 2 ka. This is well-aligned, within dating errors, with the Last Glacial Cold Period (LGCP; c. 29-18 ka) as identiﬁed in the New Zealand Climate Event Stratigraphy (Barrell et al., 2013). The local ELA depression associated with LGCP glaciation in Man- gatepopo valley was c. 1400-1550 m asl. The timing and magnitude of these changes are in good agreement with previous palaeoenvironmental reconstructions from central North Island. On Mt. Ruapehu, situated c. 15 km to the south of Mangatepopo valley, McArthur and Shepherd (1990) identify geomorphological evidence for a former ice mass with an equilibrium line of 1500-1600 m asl, which agrees well with my pELA reconstruction. McArthur and Shepherd (1990) suggest deformed pro-glacial lake sediments interbedded with moraines on northeast Ruapehu record several glacier ad- vances during the last glacial cycle. Topping (1974) identiﬁed the Kawakawa-Oruanui Tephra within these lake sediments, which places glacier advances either side of 25.4 ka. The latter advance likely correlates to the ﬁnal stand of the Mangatepopo glacier at 21 ± 2 ka, however the lack of direct dating on Mt. Ruapehu means the timing of preceding glacier ﬂuctuations is unconstrained. CHAPTER 5. THE LAST GLACIAL CYCLE Several catchments in the Southern Alps exhibit evidence for moraine formation at c. 20-22 ka (Schaefer et al., 2006; Putnam et al., 2013b; Kelley et al., 2014), however these high-precision chronologies also indicate that glaciers remained at, or close to, the 21 ka limits until at least 18 ka. Given the current precision of the cosmogenic 3He production rate (Chapter 4; Goehring et al., 2010) it is not possible to resolve any time lags in the onset of deglaciation that may exist between North Island and South Island. It is also possible that geomorphological evidence for a ﬁnal, short-lived glacier advance may not be preserved or sampled in the study site. I 129 5.6. DISCUSSION note that multiple, sharp-crested moraines exist on the southern side of Mangatepopo Valley (Figure 5.2), which indicates there may be more structure to the LGM glacial signature in this valley than currently indicated by the chronology. However, thick over- lying soil sequences at these lower altitudes preclude exposure dating of these moraines. The palaeoglacier reconstruction for Mangatepopo valley indicates local atmospheric temperature was reduced by 5.6 ± 1.1 ◦C relative to present (Figure 5.8b) during the LGCP. This estimate assumes that precipitation remained similar to present. Currently there is a paucity of quantitative LGCP precipitation estimates in New Zealand, how- ever proxy-, glacier model-, and climate model-based reconstructions are generally consistent in suggesting that precipitation was similar or slightly reduced, relative to present (Drost et al., 2007; Rojas et al., 2009; Whittaker et al., 2011; Golledge et al., 2012; Lorrey et al., 2012b; Stephens et al., 2012b). Reduced precipitation in central North Island during the LGCP would increase the magnitude of atmospheric cooling required to explain the reconstructed ELA. However, empirical and glacier model-based ev- idence suggests that past and present-day glacier mass balance in New Zealand is relatively insensitive to precipitation change, with precipitation increases of c. 30-80% required to balance 1 ◦C of warming (Oerlemans, 1997; Anderson and Mackintosh, 2006, 2012; Anderson et al., 2010). Thus, the error in the palaeotemperature estimate arising from past precipitation change is relatively insigniﬁcant (< 1 ◦C) in comparison to the uncertainty in the ELA reconstruction and temperature lapse rate. The LGCP temperature estimate for central North Island exhibits good agreement with catchment- and regional-scale glacier model simulations of LGM glaciers in the Southern Alps, which indicate temperature depression of c. CHAPTER 5. THE LAST GLACIAL CYCLE 6-7 ◦C relative to present (Golledge et al., 2012; McKinnon et al., 2012; Putnam et al., 2013b; Rowan et al., 2013). The estimate also agrees with a recent pollen-based assessment, which indicates that average mean annual air temperature in New Zealand was reduced by 6.0 ± 1.9 ◦C dur- ing the LGM (Newnham et al., 2013). Offshore, local sea surface temperature estimates are spatially variable, but converge on a 4-7◦C lowering between c. 30-18 ka (Pahnke and Sachs, 2006; Barrows et al., 2007a; Bostock et al., 2013). 5.6.2 Pre-LGCP glaciation Cosmogenic 3He surface exposure ages from moraine M3 in Mangatepopo valley indi- cate that the former Mangatepopo glacier attained its maximum extent of the last glacial cycle prior to the local LGCP (c. 28-18 ka; Barrell et al., 2013) and global LGM (26-19 CHAPTER 5. THE LAST GLACIAL CYCLE 130 ka; Clark et al., 2009). Surface erosion (e.g. c. 1 - 6 mm kyr−1) and tectonic uplift of these samples, which have been exposed since at least c. 58 ka, means that the exposure ages represent minimum ages for moraine formation and the true moraine age is likely several millennia older (e.g. c. 65-60 ka). Below, I consider this interpretation in the context of other geological records of glacial ﬂuctuations during this time period. Evidence for glacial activity prior to the LGCP is relatively scarce and, where present, the timing is often poorly constrained. However, a recent high-precision cosmogenic 10Be exposure age dataset from the Balmoral moraines in central Southern Alps shows that an advance of the former Pukaki and Tekapo glaciers culminated at 65 ± 3 ka (n = 39, plus three outliers; Schaefer et al., 2015). The Balmoral moraines associated with this advance (Barrell, 2014) are present outside of the well-dated, local LGCP limits (Schaefer et al., 2006; Kelley et al., 2014; Schaefer et al., 2015), thus indicating that the glaciers attained their maximum extent of the last glacial cycle prior to the LGCP. Sutherland et al. (2007) present cosmogenic 10Be surface exposure ages from a suite of moraines preserved on Cascade plateau (44◦S), on the west coast of South Island. The moraine belt immediately outboard of the LGCP limits at this location yielded boulder exposure ages of c. 60 - 68 ka (n = 2, plus one outlier) when recalculated according to Putnam et al. (2010b). These revised ages provide further evidence for a signiﬁcant glacial advance during Marine Isotope Stage (MIS) 4 (Williams et al., 2015). Other glacial records from South Island also indicate glacier expansion around this time, however chronologies are less-precisely constrained, largely relying on bracketing lumi- nescence ages from ice-marginal deposits. For example, McCarthy et al. (2008) present evidence from the Tasman Mountains in northern South Island (41◦S) that suggests two periods of glaciation, of similar magnitude, occurred during MIS 4 and the LGCP. 5.6.2 Pre-LGCP glaciation The MIS 4 glaciation at this location is constrained with optically-stimulated luminescence ages of glacio-lacustrine deposits, which are present outboard of the LGCP limits, and date to 64 ± 10 ka. On the central west coast of South Island, luminescence ages of sand/silt beds interbedded with glacial outwash gravels suggest glacier expansion at c. 85 ka and c. 64 ka (Preusser et al., 2005). However, moraines correlated with these outwash deposits have recently been shown to date to c. 25 ka (Barrows et al., 2013). No moraines of MIS 4 age were recognised by Barrows et al. (2013), therefore they suggest that any such deposits were overrun during the LGCP when glaciers in this catchment attained their maximum extent of the last glacial cycle. There is little evidence for glacier advance in New Zealand between 60 - 45 ka (Williams et al., 2015), during which fall most of the exposure ages from M3 moraine, if taken at face value. Furthermore, continuous climate proxy data indicate that this time period 5.7. CONCLUSIONS 131 5.7. was characterised by relatively mild, interstadial conditions (Shulmeister et al., 2001; Shane and Sandiford, 2003; Whittaker et al., 2011; Williams et al., 2015), which were probably unfavourable for signiﬁcant glacier advance. Offshore, Barrows et al. (2007a) identify intervals of high clastic sediment input between 70 - 60 ka in multiple cores, which are intepreted to represent higher terrestrial erosion rates due to expansion of nearby mountain glaciers. This is followed by a sharp reduction after 60 ka, which is consistent with the terrestrial evidence for climatic amelioration and glacial retreat. Thus, the majority of evidence from glacial records and other climate proxies support the interpretation of glacier advance in central North Island during late MIS 4 (c. 65 - 60 ka), which was of similar extent to the LGCP. 5.7 Conclusions 1. Tongariro massif was last glaciated between 31-21 ka when a central ice ﬁeld fed valley glaciers that extended down to c. 1200 m asl. The onset of glacial retreat occurred at c. 21 ± 2 ka, which is in agreement with the only other moraine ages in North Island (Brook et al., 2008). 1. Tongariro massif was last glaciated between 31-21 ka when a central ice ﬁeld fed valley glaciers that extended down to c. 1200 m asl. The onset of glacial retreat occurred at c. 21 ± 2 ka, which is in agreement with the only other moraine ages in North Island (Brook et al., 2008). 2. During the Last Glacial Cold Period (LGCP), the local equilibrium line altitude was c. 1400-1550 m asl, which is 930-1080 m lower than present. This equates to a best-estimate temperature depression of 5.6 ± 1.1 ◦C, when uncertainties in the ELA reconstruction and temperature lapse rate are considered. The timing and magnitude of glaciation in central North Island is consistent with a growing body of evidence that shows mountain glaciers in Southern Alps attained their maximum LGCP position by 32-28 ka and persisted until the termination (Schaefer et al., 2006; Putnam et al., 2013b; Kelley et al., 2014; Rother et al., 2014). 3. Reinvestigation of the 21 ka moraine coverbed stratigraphy, using ﬁeld observa- tions and major element analysis, indicates that the rhyolitic tephra close to the moraine surface is the Waiohau Tephra. This horizon (c. 14 cal. ka BP; Lowe et al., 2013) provides a minimum limiting age for the onset of climatic amelioration and soil formation at this site. Previous work had suggested that this horizon corresponds to the Rerewhakaaitu Tephra Topping and Kohn (1973). 4. Cosmogenic 3He surface exposure ages from boulders on the crest of a degraded lateral moraine indicate that glaciers on Tongariro massif attained their greatest extent of the last glacial cycle prior to the Last Glacial Maximum. Geological processes such as boulder erosion, exhumation and tectonic uplift mean that these CHAPTER 5. THE LAST GLACIAL CYCLE CHAPTER 5. THE LAST GLACIAL CYCLE 132 ages provide minimum limiting constraint of the moraine age and the true age is likely older by several millennia. This evidence, together with comparison to continuous climate proxy records from North Island and glacier records from Southern Alps, indicates that this event occurred late in Marine Isotope Stage 4. 6.2 Introduction The last glacial termination (c. 18-11 ka BP; Denton et al., 2010) was characterised by millennial-scale climate oscillations that were antiphased between the poles (Blunier and Brook, 2001). In Antarctica, steady deglacial warming from c. 18 -14.7 ka was interrupted by a c. 2 kyr hiatus, which persisted until c. 12.6 ka, known as the ’Antarctic Cold Reversal’ (ACR). In Greenland, this period is marked by an abrupt warming to near-interglacial temperature, followed by steady temperature decline. Resumption of warming in Antarctica at the end of the ACR, coincides with the onset of a c. 1000 yr cold period in Greenland, known as the Younger Dryas (12.7-11.6 ka), during which time local temperature returned to near-glacial levels. A long standing goal of palaeo- climatology has been to document the precise timing and magnitude of these events outside of the polar regions (e.g. Thompson et al., 1995; Putnam et al., 2010a), in order to establish the key drivers and mechanisms of abrupt climate change. In New Zealand, the timing, magnitude and spatial variability of climate events be- tween 15-11 ka has proven controversial. Early work indicated that a millennial-scale cooling event occurred synchronously with the Younger Dryas (Denton and Hendy, 1994; Ivy Ochs et al., 1999), which implied the ACR was restricted to southern high- latitudes. However, improved dating precision (e.g. Schaefer et al., 2009; Putnam et al., 2010b; Lowe et al., 2013) from an increasing number of palaeoclimatic archives now places the ’late-glacial climate reversal’ (LGR) in New Zealand at 13.5-11.6 ka (Alloway et al., 2007; Lowe et al., 2013), which largely overlaps the ACR. Despite improved chronological constraint, the absolute magnitude of cooling and in particular the spatial pattern, remains uncertain. For example, in a latitudinal tran- sect (36-44◦S) of terrestrial sediment cores, pollen-temperature reconstructions indicate northward attenuation of LGR cooling (Newnham et al., 2012), which suggests a steeper than present meridional temperature gradient existed across New Zealand at this time. However, other, multi-proxy, terrestrial records from southern New Zealand show differences in the magnitude of change recorded between proxies, which could repre- sent differences in their seasonal sensitivity (Vandergoes et al., 2008; Sikes et al., 2013). Evidence for latitudinal control on terrestrial cooling across New Zealand is based solely on pollen records, therefore a critical test can be conducted by the addition of quantitative palaeoclimate reconstructions from other proxy archives. 6.1 Abstract The mechanisms of atmospheric cooling in New Zealand during the last glacial - interglacial transition remain uncertain. Improved understanding of the timing and magnitude of climatic variability during this period will help to identify the drivers of abrupt climate change. In this study, I report results from geological mapping and cosmogenic 3He exposure dating, which show evidence for readvance of mountain glaciers on Mt Ruapehu in central North Island, New Zealand (39◦S) during the late glacial chron (15-11 ka). Using a distributed energy balance model, coupled with a 2D ice ﬂow model, I perform a range of experiments and sensitivity analyses to constrain estimates of past temperature associated with the mapped and dated former ice limits. Results show that glaciers in North Island readvanced early in the late glacial in response to a likely temperature cooling of 2.5 - 3.4 ◦C relative to present day, assuming precipitation remained within ± 20% of present. This magnitude of cooling is greater than that recorded in nearby pollen archives, which may reﬂect a seasonal bias between climate proxies. Strong agreement of the results presented here with other summer temperature proxy records (mountain glaciers, chironomids) from the Southern Alps, suggest New Zealand experienced uniform summertime cooling during the late-glacial climate reversal. 133 CHAPTER 6. LATE-GLACIAL READVANCE 134 6.2 Introduction This is important, because constraining the spatial patterns of LGR cooling can inform the driving mecha- nisms. For example, as outlined by Carter et al. (2008), the existence of an enhanced temperature gradient points to a critical role for the sub-tropical front in modulating rapid, millennial scale climate events. 6.3. STUDY SITE AND PREVIOUS WORK 135 In this study, I test this theory by reconstructing the timing and magnitude of glacier ﬂuctuations in central North Island, New Zealand (39◦S). I identify and date a moraine sequence in the central North Island that shows mountain glaciers readvanced in re- sponse to cooling during the LGR. Using numerical glacier modelling to constrain quantitative estimates of the cooling required to generate this advance, I test the follow- ing hypothesis: Hnull: there is no signiﬁcant difference in the magnitude of atmospheric cooling across New Zealand during the LGR, as indicated by numerical glacier modelling. To reject Hnull, evidence must exist for a signiﬁcant difference between the cooling observed in central South Island records (e.g. Anderson and Mackintosh, 2006; Doughty et al., 2013; Kaplan et al., 2013) and the North Island site. If this is the case, then the alternative hypothesis (Halt) states: Halt: there is a signiﬁcant latitudinal difference in the magnitude of cooling during the late- glacial climate reversal, as indicated by numerical glacier modelling. 6.3 Study site and previous work Situated in the southwest sector of the Paciﬁc Ocean, New Zealand represents a rare op- portunity to reconstruct terrestrial palaeoclimate in the ocean-dominated mid-latitudes of the Southern Hemisphere. The longitudinal extent (34-47◦S) and high topographic relief intercepts key components of global atmospheric and oceanic circulation, making this location highly sensitive to past climatic change (Alloway et al., 2007). The zonal, cyclone-bearing southern westerly winds are intercepted by the NNE-SSW trending axial ranges, which generate steep, W-E precipitation gradients across central and southern portions of New Zealand. Meanwhile the sub-tropical front (STF), which deﬁnes the boundary between sub-Antarctic and sub-tropical water masses, augments the north-south temperature gradient that exists across the latitudinal length of New Zealand. For example, northernmost North Island lies within an oceanic domain charac- terised by subtropical water masses delivered from the central equatorial Paciﬁc Ocean via the Tasman Front (Figure 2.7). Meanwhile, central and southernmost portions of New Zealand intercept the STF. South of this boundary, cooler sub-polar water masses bathe the western and southern coasts of South Island. Consequently, a sea surface temperature (SST) gradient exists across the latitudinal length of New Zealand, with average annual SSTs of c. 20◦C in the far north, and c. 10 ◦C in the south (Uddstrom CHAPTER 6. LATE-GLACIAL READVANCE 136 Figure 6.1: Overview map of the study region on southern Ruapehu for this chapter showing regional moraine distribution. Inset is the location of the main part of the ﬁgure (solid rectangle) and the glacier modelling domain (dashed rectangle). Figure 6.1: Overview map of the study region on southern Ruapehu for this chapter showing regional moraine distribution. Inset is the location of the main part of the ﬁgure (solid rectangle) and the glacier modelling domain (dashed rectangle). and Oien, 1999). Mount Ruapehu (2797 m asl; 39◦2’S, 175◦3’E) is a composite, andesite-dacite stratovol- cano situated in the central North Island, at the southern end of the Taupo Volcanic Zone (TVZ). The TVZ is a c. 350 km long, NNE-SSW trending chain of volcanoes caused by subduction of the Paciﬁc Plate beneath the Australian Plate (Cole, 1978). Radiometric (K/Ar) dating of andesitic lava ﬂows constrain the onset of cone-building volcanism at Mt. Ruapehu to at least c. 250 ka (Gamble et al., 2003). Modern local climate, as recorded on the lower NW ﬂank at Whakapapa village (1097 m asl), is characterised by relatively high total annual precipitation and low seasonal precipitation variability (Chapter 2). Monthly mean temperatures range from c. 13 ◦C in February, to c. 3 ◦C in July, with an annual average of 7.5 ◦C (1981-2010; NIWA, 2014). Small glaciers and snow patches persist on the upper reaches of the mountain, generally above c. 2300-2400 m asl, in south-facing topographic hollows. Interannual ﬂuctuations in the mass balance of these ice bodies, as recorded in annual surface area changes, correlates strongly with ablation season temperature and mean end of summer snowlines in the Southern Alps (Brook et al., 2011). 6.3. STUDY SITE AND PREVIOUS WORK 137 Previous studies of past glaciation in central North Island have identiﬁed abundant geomorphological evidence for multiple periods of more extensive glaciation during the late Quaternary (McArthur and Shepherd, 1990; Chapter 5, Chapter 7). On Mt. Ruapehu, McArthur and Shepherd (1990) record large lateral moraines in several catch- ments, likely constructed by a former ice mass with outlet glaciers that reached c. 1200 m asl. McArthur and Shepherd (1990) suggest these deposits pertain to multiple ﬂuctu- ations during the last glacial cycle, based on the presence of the Kawakawa-Oruanui Tephra (25.4 ± 0.2 ka - Vandergoes et al., 2013) interbedded in deformed, moraine- bound glacio-lacustrine deposits. On nearby Tongariro massif, situated 15 km to the north-east, cosmogenic 3He exposure dating of moraines supports this conclusion, indicating signiﬁcant periods of glaciation during late MIS 4 (c. 58 ± 6 ka) and the Last Glacial Cold Period (LGCP; c. 30-21 ka) when snowlines were depressed by c. 1000 m relative to present (Chapter 5). On Tongariro massif, no evidence exists to suggest renewed glacial activity since the LGCP. 6.4.1 Geomorphological mapping Initial geomorphological mapping was conducted using aerial photographs, from which prominent landforms were delineated, such as large moraines, till cover, cirques, tephra coverbeds, lava cliffs and alluvial fans. Field mapping was then undertaken to ground-truth initial interpretations. Field data were recorded using a handheld global positioning system receiver and enlarged 1:50000 scale topographic base maps, then digitised using a Geographic Information System. and Oien, 1999). This is most likely because the topography, prior to Holocene cone growth, was of insufﬁcient relief to intercept the estimated snowlines during the LGR (c. 300-500 m relative to present - Porter, 1975; Kaplan et al., 2010, 2013; see also Chapter 7). However, at c. 2800 m asl and currently glacierised, Mt. Ruapehu is better positioned to record past cooling events of this magnitude. In this study, I target three stream catchments on the southern ﬂanks of Mt. Ruapehu: (i) the glacierised Mangaehuehu catchment, which drains in a south-west direction, originating between Tahurangi (2797 m asl) and Girdlestone (2658 m asl) peaks; (ii) Te Unuunuakapuateariki Stream, which rises at c. 2200 m asl below Girdlestone Peak and drains in a southward direction; and (iii) the deeply incised Wahianoa valley, which drains southeastwards from Girdlestone Peak (Figure 6.1). Recent geological mapping in these catchments has identiﬁed multiple former ice limits, depicted by discrete moraine ridges (Townsend et al., in prep.). Previous studies of the glacial geo- morphology in the Mangaehuehu catchment have resulted in differing interpretations of former ice limits. For example, McArthur and Shepherd (1990) tentatively suggest a distinct moraine pair, which indicates a former ice mass that terminated at c. 1600 m asl, as the late Otiran (45 - 18 ka; Barrell et al., 2011) ice limits in this catchment and they do not recognise any glacial deposits down valley from this location. Conversely, Barrell (2011) suggests late Otiran ice extended further down valley and terminated at c. 1200 m asl. This latter interpretation therefore implies that the moraines identiﬁed by McArthur and Shepherd (1990) represent a glacier readvance/stillstand <18 ka. In the Te Unuunuakapuateariki and Wahianoa catchments, moraines also exist at c. 1800 - 1500 m asl, which may have been deposited synchronously with those in the Mangaehuehu catchment. In this study, I reﬁne the understanding of the glacial stratigraphy of this region using geological mapping, cosmogenic surface exposure dating and numerical CHAPTER 6. LATE-GLACIAL READVANCE 138 glacier modelling. glacier modelling. 6.4.2 Cosmogenic 3He surface exposure dating Moraine boulder samples were collected using a portable, 16V rock saw ﬁtted with a segmented, diamond-tipped blade. Samples were taken only from boulders on the moraine crest (Figure 6.2). Where possible, boulders were partially embedded in the moraine matrix, to minimise the likelihood of post-depositional boulder rotation. All samples were collected from the highest point of the parent boulder, which were > 0.6 m tall, thus minimising the potential for burial by snow and/or volcanic ash (Table 6.1). Azimuthal horizon elevations were measured in the ﬁeld using a standard geological compass and clinometer, and geometric shielding corrections were computed using the CRONUS-EARTH calculator (available at: http://hess.ess.washington.edu/; Balco et al., 2008). All shielding corrections were < 1%. Sample locations and elevations were recorded using a Trimble GeoXH global positioning system, relative to the WGS84 datum. These data were differentially corrected using continuous measurements from GeoNet ’Chateau Observatory’ (’VGOB’) base station (39◦11’59” S, 175◦32’ 32”E; 1161 m asl), located 13 km north of the study site. Horizontal and vertical post-processed uncertainties for individual sample locations are < 1 m. Samples were jaw-crushed, rinsed in de-ionised water and dry-sieved to isolate the 250-500 µm size fraction. Density (> 3.1 g cm−3) and magnetic separation techniques were used to isolate 150-600 mg of pyroxene grains. Following Bromley et al. (2014), separated pyroxenes were ﬁrst leached in 5% hydroﬂuoric (HF) / 2% nitric (HNO3) 6.4. METHODS 139 Table 6.1: Moraine boulder cosmogenic 3He sample details. 6.4.2 Cosmogenic 3He surface exposure dating Sample Latitude Longitude Altitude Thickness Geometry Surface Shielding ID (m a.s.l) (cm) (H x L x W (cm)) strike/dip Upper moraine (LG2): BH1210 -39.311 175.5433 1779 1.5 75 x 220 x 140 294/2 0.998 BH1209 -39.312 175.5424 1769 1.5 110 x 180 x 170 102/8 0.998 BH1211 -39.312 175.5432 1780 2.0 60 x 190 x 100 356/10 0.998 BH1208 -39.313 175.5424 1765 1.5 90 x 140 x 130 048/8 0.998 Lower moraine (LG1): BH1204 -39.318 175.537 1605 2.0 75 x 150 x 100 072/9 0.998 BH1206 -39.318 175.537 1605 2.5 80 x 18 x 120 298/15 0.996 BH1207 -39.318 175.537 1606 1.5 90 x 150 x 140 062/13 0.997 BH1205 -39.318 175.537 1602 2.5 75 x 130 x 100 140/9 0.998 BH1201 -39.316 175.539 1621 1.5 85 x 220 x 200 200/12 0.996 BH1213 -39.315 175.539 1632 2.0 80 x 130 x 110 156/2 0.998 BH1203 -39.316 175.538 1630 2.5 140 x 150 x 100 276/16 0.995 Outboard moraine (LG3): BH1212 -39.312 175.539 1657 2.0 60 x 100 x 100 154/13 0.991 Outboard moraine (LG4): BH1214 -39.315 175.537 1612 1.5 150 x 210 x 140 332/6 0.995 BH1215 -39.315 175.537 1605 1.5 100 x 150 x 130 268/6 0.995 TU1 moraine - left lateral: ME1201 -39.322 175.568 1673 1.5 125 x 340 x 160 072/8 0.996 ME1202 -39.322 175.568 1684 1.5 80 x 260 x 130 0 0.996 ME1203 -39.322 175.568 1689 1.0 130 x 220 x 180 0 0.996 TU2 moraine - right lateral: ME1204 -39.320 175.565 1691 2.0 75 x 150 x 120 214/12 0.994 Table 6.1: Moraine boulder cosmogenic 3He sample details. acid solution for 24 hours, followed by a separate 10% hydrochloric (HCl) acid solution for 24 hours, to remove adhering groundmass particles. Leached pyroxene crystals were visually inspected for purity and wrapped in aluminium foil. Each sample was completely degassed by heating in a furnace to >1300◦C for 15 minutes, during which released gases were exposed to a liquid nitrogen chilled charcoal trap. Extracted gases were exposed to an SAES getter before being collected on a cryogenic cold trap at <15 K. Helium was then isolated from other noble gases by heating the cold trap to 45 K. 6.4.2 Cosmogenic 3He surface exposure dating Mass spectrometry was conducted using a MAP 215-50 noble gas mass spectrometer at Lamont-Doherty Earth Observatory, USA, relative to the Yellowstone ’Murdering Mudpot’ (MM) helium standard (3He/4He ratio of 16.45Ra, where Ra = 3He/4Heair = 1.384 x 10−6), using the protocol of Winckler et al. (2005). The relative youth of the parent lavas from which the samples are derived (< 300 ka) means that the samples have extremely low 4He content and therefore high 3He / 4He ratios, up to 547 times that for air (Table 6.2), meaning any corrections for magmatic 3He would be < 1%. I measured lithium (Li), uranium (U) and thorium (Th) concentrations in three samples (BH1206/07 and ME1204) to check for possible errors resulting from helium production via nucleogenic and radiogenic pathways. Concentrations of these three elements are negligible (< 1 ppm), therefore I assume all measured 3He to be of cosmogenic origin. acid solution for 24 hours, followed by a separate 10% hydrochloric (HCl) acid solution for 24 hours, to remove adhering groundmass particles. Leached pyroxene crystals were visually inspected for purity and wrapped in aluminium foil. Each sample was completely degassed by heating in a furnace to >1300◦C for 15 minutes, during which released gases were exposed to a liquid nitrogen chilled charcoal trap. Extracted gases were exposed to an SAES getter before being collected on a cryogenic cold trap at <15 K. Helium was then isolated from other noble gases by heating the cold trap to 45 K. Mass spectrometry was conducted using a MAP 215-50 noble gas mass spectrometer at Lamont-Doherty Earth Observatory, USA, relative to the Yellowstone ’Murdering Mudpot’ (MM) helium standard (3He/4He ratio of 16.45Ra, where Ra = 3He/4Heair = 1.384 x 10−6), using the protocol of Winckler et al. (2005). The relative youth of the parent lavas from which the samples are derived (< 300 ka) means that the samples have extremely low 4He content and therefore high 3He / 4He ratios, up to 547 times that for air (Table 6.2), meaning any corrections for magmatic 3He would be < 1%. I measured lithium (Li), uranium (U) and thorium (Th) concentrations in three samples (BH1206/07 and ME1204) to check for possible errors resulting from helium production via nucleogenic and radiogenic pathways. Concentrations of these three elements are negligible (< 1 ppm), therefore I assume all measured 3He to be of cosmogenic origin. CHAPTER 6. 6.4.2 Cosmogenic 3He surface exposure dating LATE-GLACIAL READVANCE 140 Exposure ages were calculated using the cosmogenic 3He exposure age calculator and global, sea-level, high-latitude (SLHL) cosmogenic 3He production rate of Goehring et al. (2010), which I have shown to be applicable in New Zealand (Chapter 4). Attenu- ation of cosmogenic neutron ﬂux with depth from the surface was corrected for using measured sample thickness, a standard rock density of 2.7 g cm−3 and an attenuation length of 160 g cm−2 (Dunne et al., 1999). Field observations from mid-winter showed that the salient, ridge-top boulders are not subject to prolonged burial by seasonal snow, therefore no correction was applied to the age calculation. Although sampled boulders did not exhibit glacial striae, some boulders retain faceted sides. Care was taken to avoid boulders that displayed clear evidence of erosion, such as discolouration, weathering scarps, onion-skin weathering, or pitting/water pooling. Thus, I present the exposures ages without an erosion correction. Regional uplift in the central North Island has been estimated for the past 500 ka at < 1 mm yr−1 (Pulford, 2002). This does not alter exposure ages outside of the measurement uncertainty, therefore I do not include a correction for regional tectonic uplift. Elevation and latitudinal scaling of cosmogenic 3He production is undertaken using the ’Lm’ (Lal, 1991/Stone, 2000) model, which outperforms neutron-monitor based schemes (Lifton et al., 2014). 6.4.3.1 Research design In order to test Hnull, I use a distributed energy balance model, coupled with a 2D ice ﬂow model to constrain quantitative estimates of past temperature in central North Island. This model has previously been used to constrain late-glacial temperatures in South Island (Doughty et al., 2013; Kaplan et al., 2013), therefore the results are directly comparable. The model domain is 24 x 21 km, centred on the peak of Mt. Ruapehu (-39.28◦; 175.57◦; Figure 6.1), and has a grid cell resolution of 100 m. I include the whole upper mountain in the simulations in order to capture any potential changes in glacier catchment boundaries that result from a growing ice mass. Modern climate (tem- perature, precipitation, solar radiation, wind speed and relative humidity) is derived from a combination of climate station and reanalysis data, which is summarised into monthly grids (see Section 7.3.2 and Chapter 3). Temperature (∆T) and precipitation (∆P) perturbations are imposed iteratively to ﬁnd the necessary combination required to simulate the Mangaehuehu Glacier so that it reaches steady state and terminates within one grid cell (± 100m) of the downstream end of moraine LG1 (Figure 6.3). ∆P is applied as a percentage change, relative to modern, and is varied between -50% to +50%. All simulations begin from an ice-free domain and are run for 250-300 model years, which is sufﬁcient for glaciers to reach equilibrium following an imposed cli- 141 6.4. METHODS 6.4. METHODS 6.4. METHODS Figure 6.2: (a) Sample BH1209 (photo facing W); (b) Sample BH1211 (photo facing SW); (c) Sample BH1202 (photo facing N); (d) The sampled surface of BH1202; (e) Sample BH1207 (photo facing S); (e) Sample BH1204 (photo facing N). Figure 6.2: (a) Sample BH1209 (photo facing W); (b) Sample BH1211 (photo facing SW); (c) Sample BH1202 (photo facing N); (d) The sampled surface of BH1202; (e) Sample BH1207 (photo facing S); (e) Sample BH1204 (photo facing N). CHAPTER 6. LATE-GLACIAL READVANCE 142 matic perturbation. To assess the impact of key parameter choices on the results, I perform sensitivity tests in which key parameters are systematically varied. Climate perturbations and parameter sensitivity changes are imposed uniformly across the model domain. 6.4.3.2 Input data Terrain elevation data comes from the New Zealand School of Surveying Digital Eleva- tion Model (NZSoSDEM) (Columbus et al., 2011) and is resampled to 100 m resolution. An ice mask is created using the ’snow/ice’ data from the Land Information New Zealand NZMS260 map series. Ice thickness estimates, based on the survey of Keys (1988), are used to create an ice-free elevation model. Climate data for the energy bal- ance and snow accumulation models is from several different sources. Solar radiation and relative humidity are from the Virtual Climate Station Network gridded datasets, sourced from NIWA CliFlo Database (NIWA, 2014). These datasets are resampled to the model domain resolution using bilinear interpolation. Present day wind speed data comes from the National Centers for Environmental Prediction (NCEP) 850 hPa level, reanalysis data (1981-2010; Kalnay et al., 1996). This dataset is scaled against observa- tional data and applied uniformly over the model domain. Following Anderson and Mackintosh (2012) and Doughty et al. (2013), I use raw temperature and precipitation data from individual climate stations distributed around and within the model domain to generate climate grids using the methods detailed in Chapter 3. 6.4.3.3 Mass-/energy-balance model Precipitation is partitioned in to rain and snow, using a temperature threshold (Ts = 0.5◦C). To simulate ablation, I use the energy balance equation (Equation 6.1) within a distributed energy balance model (EBM) as developed (Anderson et al., 2010) and previously applied in contemporary- (Anderson and Mackintosh, 2012) and palaeo- glaciological (Doughty et al., 2013) studies in New Zealand (see Chapter 3 for full description). (6.1) QM = I(1 −α) + L ↓+L ↑+QH + QE + QR + QG (6.1) where QM is the energy available for melt, I is incoming shortwave radiation, L ↓is incoming longwave radiation, L ↑is outgoing longwave radiation, QH and QE are sen- sible and latent heat ﬂuxes respectively, QG is geothermal heat ﬂux and QR is heat input from rain. Sub-surface heat ﬂuxes (QS; Chapter 3) are negated due to the assumption 143 6.4. METHODS that ice is temperate and constantly at the melting point. that ice is temperate and constantly at the melting point. Incoming shortwave radiation (I) comprises both direct and diffuse components (Oer- lemans, 1992). The effect of changing orbital geometry is accounted for by using the insolation (13 ka) calculations of Huybers and Eisenman (2006). Albedo (α) is parame- terised using the ELA-dependent scheme of Oerlemans (1992), whereby α increases with elevation and snow thickness, relative to the equilibrium line altitude. I use αsnow=0.72 (Oerlemans, 1992; Doughty et al., 2013; Table 3.3) and test the inﬂuence of these parameterisation choices in sensitivity tests. Longwave ﬂuxes (L↓, L↑) include the effects of surrounding topography, cloudiness and air temperature (Plummer and Phillips, 2003). Turbulent heat ﬂuxes (QH, QE) are calculated using the bulk method and include the roughness of snow and ice and the Richardson stability criterion (Oer- lemans, 1992; Klok and Oerlemans, 2002; Anderson et al., 2010). Geothermal heat ﬂux (QG) has the potential to contribute signiﬁcantly to the energy balance of snow and ice situated on volcanically active terrain (see Chapter 3 for dis- cussion). However, as QG can vary greatly in space and time, and is unknown for the geological past, it is difﬁcult to accurately parameterise in the model. I use a nominal value of QG = 1 W m −2 applied uniformly over the model domain. I do not include debris cover in the simulations because it is unknown for the time of interest. Historically, debris cover on the glaciers situated on Mt. 6.4.3.3 Mass-/energy-balance model Ruapehu has varied greatly in space and time. During the most recent signiﬁcant volcanic eruptions (AD 1995-96), all glaciers became buried by volcanic products. However, presently only ice bodies with a low surface slope and those situated close to the current volcanc vent, such as the summit plateau and the upper Whangaehu glacier, remain debris covered. Elsewhere on the mountain, steeper glaciers such as Mangatoetoenui and Mangaehuehu now have very little surface debris cover. Thus, evidence suggests debris cover emplaced via volcanic eruptions is transient in the maritime environment (e.g. Kirkbride and Dugmore, 2003) and the effects on energy balance are negligible over geological timescales. I acknowledge that this is a source of uncertainty in the simulations. 6.4.3.4 Ice ﬂow model Ice ﬂow is described using a vertically-integrated, two-dimensional (2D) model based on the shallow ice approximation (SIA) (Plummer and Phillips, 2003; Kessler et al., CHAPTER 6. LATE-GLACIAL READVANCE 144 2006). This formulation assumes ice ﬂow is driven by vertical shear stresses, therefore compressional and tensional (longitudinal) stresses are neglected (Hutter, 1983). I consider that the role of longitudinal stresses on past glacial ﬂow in the Mangaehuehu valley would be low, owing to the low bed slope and absence of steep, bounding valley sides in the lower catchment. Furthermore, several comparison studies between SIA and higher order ice ﬂow models show little difference in ice geometry outputs (e.g. Le Meur et al., 2004; Leysinger Vieli and Gudmundsson, 2004), thus the SIA is commonly applied in mountain glacier environments (e.g. Plummer and Phillips, 2003; Kessler et al., 2006; Doughty et al., 2013). Ice ﬂow velocity due to internal deformation (Ud) is given as: Ice ﬂow velocity due to internal deformation (Ud) is given as: ⃗Ud = 2 5AH⃗τ n b (6.2) (6.2) where A is Glen’s ﬂow law coefﬁcient, set to 2.14 x 10−16 Pyr−3 yr−1 (Paterson, 1994), ⃗τb is the gravitational driving stress (⃗τb = ρgH∇z), and n is Glen’s ﬂow law exponent, set to n = 3. Following Plummer and Phillips (2003) and Kessler et al. (2006), a sliding term is also included: Us = Uce 1−τc ⃗τb (6.3) (6.3) where Uc and and τc are constants that represent average sliding velocity and driving stresses, which are set to Uc = 50 m yr−1 and τc = 100 kPa. where Uc and and τc are constants that represent average sliding velocity and driving stresses, which are set to Uc = 50 m yr−1 and τc = 100 kPa. dH dt = M −∇· ⃗q (6.4) (6.4) where H is ice thickness, t is time, ⃗q is ice ﬂux (⃗Ud + ⃗Us) and M is mass balance. where H is ice thickness, t is time, ⃗q is ice ﬂux (⃗Ud + ⃗Us) and M is mass balance. Equation 6.4 evolves glacier geometry through time. Ice velocities are calculated on a grid offset from ice thickness and the ﬂux gradients are used to update ice thickness using a forward explicit time-step (Hindmarsh and Le Meur, 2001). To account for boundary effects that may violate mass conservation in the ﬁnite difference formulation (e.g. 6.4.3.4 Ice ﬂow model Plummer and Phillips, 2003), the bed topography is smoothed using a 300 x 300 m moving window to reduce high bed slopes in the upper catchment, and an ice ﬂux correction is applied. For each cell, total ice divergence cannot exceed the total mass contained in the source cell. At each timestep it was ensured that this criterion was met and if not, the excess ice was removed (Plummer and Phillips, 2003). 145 6.5. RESULTS 6.5.1.1 Mangaehuehu catchment Multiple moraine ridges, often paired, are present between 0-5 km from the present Mangaehuehu Glacier terminus (c. 2300 m asl; Figure 6.3a). A pair of sharp-crested lateral moraines, which are c. 1 km long and are situated immediately down valley from present-day Mangaehuehu Glacier, represent the most recent episode of moraine- building in this catchment. Between c. 1750 - 1500 m asl, a complex arrangement of moraine ridges indicates the position of a former ice margin. A thick (c. 20-30 m) lava ﬂow dominates the centre of the Mangaehuehu valley at this location, which has recently been dated using 40Ar/39Ar to 20 ± 2 ka (C. Conway / G. Leonard, pers. comm. 2014). A single moraine ridge (LG2 - Figure 6.3a) overlies this lava, and extends down valley to c. 1720 m asl. Four cosmogenic 3He surface exposure ages from boulders on this moraine are stratigraphically consistent with (younger than) the age of the underlying lava and three out of the four samples yield ages between 15-12 ka, with one anomalously young outlier at 7.1 ka (Table 6.5.1.3). Immediately down-valley, an arcuate moraine ridge (LG1) builds from the downstream limit of the prominent lava ﬂow front and terminates at c. 1550 m asl. Four of seven surface exposure ages from boulders on the crest of this moraine date to 14 - 11 ka, with the remaining three spread between 3-7 ka (Table 6.5.1.3). Immediately to the west, two smaller moraine ridges (LG3-4 - Figure 6.3a) represent deposition at a separate lobe of ice that ﬂowed to the west of the prominent lava. Three exposure ages from boulders on these moraines range from 11.1 - 4.5 ka. On the eastern side of the valley a single, wide-crested composite moraine extends continuously from c. 1800 m asl to c. 1600 m asl and represents the eastern margin of the former valley glacier. Further down valley, broad moraine ridges on both valley sides overlie thick (c. 10-20 m) lava bluffs that outcrop in deeply incised gorges. The maximum downstream extent of these moraine deposits is unclear due to soil and forest cover, however interpretation of aerial photographs combined with the ﬁeld investigations suggests ice reached at least c. 1200 m asl, which is consistent with the geomorphological interpretations of Barrell (2011). Moraine LG1 is underlain by c. 5 m of ﬁnely laminated, well-sorted silts and sands CHAPTER 6. 6.5.1.1 Mangaehuehu catchment LATE-GLACIAL READVANCE 146 (herein referred to as rhythmites) with occasional pebbles (<20 cm a-axis) and gravel lenses, as shown in a ﬂuvially-eroded exposure at the base of the landform (Figure 6.3c; Figure 6.4a). Beds within the red-dark grey coloured rhythmites are of variable thickness (< 1 mm to c. 2 cm) and exhibit a range of ductile and brittle deforma- tion structures. Evidence for ductile deformation is abundant, with acute folds that represent 2-3 m horizontal shortening (Figure 6.3c). Reverse faults with centimetre- to decimetre-scale offsets are also prevalent. Sub-vertical clastic dykes crosscut the horizontally-bedded rhythmites and are c. 0.5 - 2 cm wide, several metres long and consist of highly-compacted, well-sorted clay-silt particles (Figure 6.4b,c). Internally, the particles ﬁlling the dykes commonly form sub-mm scale laminae, orientated parallel to the direction of dyke propagation (Figure 6.4b). The direction of dyke emplacement is uncertain due to incomplete exposure. I interpret these sediments to represent deposition in a low-energy, relatively deep water (> 20 m) lacustrine environment, where ﬁne sediment was allowed to settle out of sus- pension and form centimetre to millimetre-scale laminae. The assemblage of polyphase deformation structures is consistent with that expected from post-depositional overrid- ing by an advancing ice mass (Benn and Evans, 2010). The presence of isolated clasts and gravel lenses within the rhythmites, possibly represents deposition of ice-rafted debris, which may indicate that this lake was proglacial. Similar deformed sediments have been described interbedded with till deposits elsewhere on Mt. Ruapehu, which were also interpreted to represent deformation beneath an advancing glacier (McArthur and Shepherd, 1990). Clastic dykes are a common product of glaciotectonism (van der Meer et al., 2009), which, together with the overlying diamicton and moraine ridge, is also consistent with glacial readvance at this site. 6.5.1.2 Te Unuunuakapuateariki catchment The Te Unuunuakapuateariki (TU) stream rises at c. 2100 m asl on the southern ﬂank of Girdlestone peak (2658 m asl) and ﬂows southwards (Figure 6.5a-d). No glacial ice exists at the head of this catchment, however small perennial snow patches are present in hollows at 2300-2400 m asl. Between 1900-1700 m asl, the TU stream drains a relatively narrow (c. 300 m wide) catchment. A prominent outcrop of polished bedrock rises above the modern stream channel at c. 1800 m, indicating past glacial erosion beneath a temperate ice mass. At 1700-1600 m asl, the TU catchment opens into a c. 1 km wide basin. This transition is marked by a complex assemblage of lateral and latero-frontal moraine ridges that demarcate the terminus of a former glacier. Multiple individual moraine crests form a compound mass of glacial till on the eastern side of TU stream, which suggests the margin of the former ice mass oscillated around this 147 6.5. RESULTS Figure 6.3: (a) Map of the glacial geology of the Mangaehuehu catchment on sou (b) photograph showing the boulder-rich surface of moraine LG1 with person the prominent lava ﬂow (see text) in the middle ground and the modern Mang glacier in the background; (c) Photograph of ductile deformation structures w deposits beneath moraine LG1 - location depicted by yellow star in panel (a). Figure 6.3: (a) Map of the glacial geology of the Mangaehuehu catchment on southern Ruapehu; (b) photograph showing the boulder-rich surface of moraine LG1 with person for scale. Note the prominent lava ﬂow (see text) in the middle ground and the modern Mangaehuehu cirque glacier in the background; (c) Photograph of ductile deformation structures within rhythmite deposits beneath moraine LG1 - location depicted by yellow star in panel (a). CHAPTER 6. LATE-GLACIAL READVANCE 148 Figure 6.4: (a) Overview photo showing ﬁne grained-sediment exposures beneath the unsorted moraine LG1 (photo facing NE); (b) Close up photograph of ﬁnely laminated, ﬁne-grained clastic dyke within the ﬁne-grained sediments; (c) photographic example of a sub-vertical clastic dyke within ﬁne-grained sediments beneath moraine LG1. Figure 6.4: (a) Overview photo showing ﬁne grained-sediment exposures beneath the unsorted moraine LG1 (photo facing NE); (b) Close up photograph of ﬁnely laminated, ﬁne-grained clastic dyke within the ﬁne-grained sediments; (c) photographic example of a sub-vertical clastic dyke within ﬁne-grained sediments beneath moraine LG1. position. Samples ME1201 to ME1203 (e.g. 6.5.1.2 Te Unuunuakapuateariki catchment Figure 6.5b,c) were taken from boulders on a rounded moraine ridge crest (TU1 - Figure 6.5a) at the outer margin of this moraine complex. Two of these ages are indistinguishable within measurement uncertainty at c. 14.5 ka, with the other dating to 16.5 ka. On the western side of the stream, a prominent lateral moraine (TU2) has been undercut by the modern stream, which has produced a steep eastern ﬂank of the moraine (Figure 6.5c). 6.5.1.3 Wahianoa catchment Wahianoa valley is a deeply-incised valley with a parabolic cross-section, which drains towards the southeast from Tahurangi peak (2797 m asl; Figure 6.6). Boulder-rich lateral moraine crests line both ﬂanks of the lower 3 km of the valley to c. 1200 m asl, recording glacier ﬂuctuations during the most extensive period of glaciation preserved in this catchment (see Chapter 7). A pair of small (c. 5-10 m high) moraine ridges lies on the valley ﬂoor, approximately 2.5 km up-stream from the valley mouth, and are bisected by the modern Wahianoa River. The left lateral of this pair is a discrete, rounded ridge that has been partly incised by the Wahianoa River, exposing till sections close to the active river channel. Large boulders (> 1m high) are rare on the crest of this ridge and there is a thin mantle of pale-yellow pyroclastic material on the surface. 6.5. RESULTS 149 Figure 6.5: (a) Map of the glacial geology of the Te Unuunuakapuateariki catchment; (b) Samp ME-12-01 on moraine TU1 in the Te Unuunuakapuateariki catchment; (c) Sample ME-12-02 o moraine TU1 in the Te Unuunuakapuateariki catchment. Note the prominent crest of morain TU2 in the background and the prominence of the boulder surfaces above the winter snow cov - photo facing north west; (d) Photograph of the Te Ununukapuateariki moraines complex, pho facing north. Figure 6.5: (a) Map of the glacial geology of the Te Unuunuakapuateariki catchment; (b) Sample ME-12-01 on moraine TU1 in the Te Unuunuakapuateariki catchment; (c) Sample ME-12-02 on moraine TU1 in the Te Unuunuakapuateariki catchment. Note the prominent crest of moraine TU2 in the background and the prominence of the boulder surfaces above the winter snow cover - photo facing north west; (d) Photograph of the Te Ununukapuateariki moraines complex, photo facing north. CHAPTER 6. LATE-GLACIAL READVANCE 150 Figure 6.6: (a) Map of the glacial geology of the upper Wahianoa catchment.; (b) Oblique a photograph (source: D. Townsend) of the region shown in (a). Note the prominent mo assemblage on the valley ﬂoor (dashed box). Red dot and arrow mark the location and orient of photo in (c); (c) Photograph of the prominent true right moraines in the middle Wahi valley - photo facing north Figure 6.6: (a) Map of the glacial geology of the upper Wahianoa catchment.; (b) Oblique aerial photograph (source: D. Townsend) of the region shown in (a). 6.5.1.3 Wahianoa catchment Note the prominent moraine assemblage on the valley ﬂoor (dashed box). Red dot and arrow mark the location and orientation of photo in (c); (c) Photograph of the prominent true right moraines in the middle Wahianoa valley - photo facing north 151 6.5. RESULTS The right lateral of this pair can be distinguished as a linear landform, with a higher abundance of large (> 1 m high) boulders, although does not have a discrete ridge crest. This morainic material continues for c. 1.5 km up valley, ending at a major fork in the modern Wahianoa River. Here, the moraine is underlain by a prominent lava ﬂow, recently dated to 17 ± 1 ka (Conway et al., in prep), which provides a maximum bracketing age for the overlying moraine. The preservation and morphostratigraphic situation of this moraine assemblage indicate they were formed after the deposition of the more extensive moraines down-valley. This suggests a readvance or temporary cessation of glacier retreat of sufﬁcient duration to deposit this volume of material. The head of the Wahianoa valley, above 1700 m asl, is steep, remote terrain and, as such, mapping was restricted to visual inspection from high vantage points in the ﬁeld and aerial photography. Small patches of glacial ice, which are relatively free of supraglacial debris, are present on the uppermost slopes. Immediate down-valley from the present-day Wahianoa Glacier, numerous linear, boulder-topped landforms are identiﬁed (Figure 6.6), which are interpreted as moraine ridges. These appear to track the recession of the Wahianoa Glacier terminus from c. 1800 m asl to its present elevation at c. 2400 m asl. 6.5.2 Glacier modelling Figures 6.7a & b show the range of combined temperature (∆T) and precipitation (∆P) perturbations that produce a steady state ice mass that ﬁts the downstream limit of past glaciation in the Mangaehuehu catchment, as marked by moraine LG1. Using the optimal parameter set (Table 3.3), a cooling of -2.9◦C relative to present is necessary when ∆P = 0. In this scenario, the Mangaehuehu Glacier has a maximum thickness of c. 130 m (Figure 6.7c) and an equilibrium line altitude of c. 1950 m (Figure 6.7d). This is approximately 500 m lower than present, as observed in the last glacier survey (Keys, 1988). Under the optimal model parameter settings the temperature forcing required to simu- late the former Mangaehuehu Glacier depicted by moraine LG1 ranges between ∆T = 2.5 - 3.4◦C, when precipitation is varied by ± 20% from present day (Figure 6.7b). Systematic variation of model parameters causes maximum deviations in ∆T of ± 0.5◦C from the optimal runs. Snow albedo is the most sensitive parameter, with variations in ∆T of ±0.4◦C, when varying albedo by ± 0.05. Increases in the snow temperature threshold used to parameterise accumulation have relatively little impact on ∆T in low CHAPTER 6. LATE-GLACIAL READVANCE 152 Table 6.2: Helium mass spectrometry results. 6.5.2 Glacier modelling (DL = detection limit) SampleID Weight 3He ± 4He ± 3He/4He ± R/Ra (g) (atoms/g) (atoms/g) Mangaehuehu catchment: Upper moraine (LG2): BH1210 0.5385 6.80E+06 1.48E+05 8.98E+09 7.24E+08 7.57E-04 6.32E-05 547 BH1209 0.3358 6.03E+06 1.80E+05 2.85E+10 1.23E+09 2.12E-04 1.11E-05 153 BH1211 0.5494 5.37E+06 1.26E+05 1.42E+10 7.20E+08 3.79E-04 2.12E-05 274 BH1208 0.1471 3.20E+06 2.15E+05 3.20E+10 3.18E+09 9.98E-05 1.20E-05 72 Upper moraine (LG1): BH1204 0.1523 5.54E+06 4.01E+05 3.11E+10 3.11E+09 1.78E-04 2.20E-05 129 BH1206 0.1487 5.31E+06 2.59E+05 0.00E+00 3.32E+09 DL 2.92E-02 - BH1207 0.1504 4.87E+06 2.52E+05 0.00E+00 3.39E+09 DL 5.99E-04 - BH1205 0.3423 4.18E+06 1.40E+05 5.60E+09 1.04E+09 7.46E-04 1.40E-04 539 BH1201 0.3155 2.76E+06 1.19E+05 1.24E+10 1.19E+09 2.23E-04 2.34E-05 161 BH1213 0.2692 2.29E+06 1.21E+05 1.13E+10 1.44E+09 2.04E-04 2.82E-05 147 BH1202 0.3040 1.16E+06 5.60E+04 3.24E+10 1.10E+09 3.57E-04 1.24E-04 258 Lower moraine (ii): BH1212 0.3463 4.56E+06 1.49E+05 1.61E+10 9.86E+08 2.83E-04 1.96E-05 204 Outboard moraine (i): BH1214 0.3197 2.94E+06 1.31E+05 1.34E+10 1.19E+09 2.19E-04 2.17E-05 158 BH1215 0.3268 1.75E+06 9.49E+04 3.24E+10 1.37E+09 5.38E-05 3.71E-06 39 TU catchment: TU1 moraine - left lateral: ME1201 0.5021 6.16E+06 1.81E+05 1.16E+10 5.70E+08 5.33E-04 3.05E-05 385 ME1202 0.4040 6.07E+06 1.68E+05 2.21E+10 1.05E+09 2.75E-04 1.51E-05 199 ME1203 0.5126 7.05E+06 1.58E+05 6.29E+09 7.61E+08 1.12E-03 1.38E-04 810 TU2 moraine - right lateral: ME1204 0.4206 2.81E+06 1.02E+05 2.41E+10 1.09E+09 1.17E-04 6.79E-06 84 precipitation experiments, but results in deviations of up to 0.5◦C when precipitation is increased by >10%, relative to present. Table 6.2: Helium mass spectrometry results. (DL = detection limit) Table 6.2: Helium mass spectrometry results. (DL = detecti precipitation experiments, but results in deviations of up to 0.5◦C when precipitation is increased by >10%, relative to present. Steady-state simulations of the former Mangaehuehu Glacier show excellent agreement between the modelled ice geometries and moraine records in the Te Unuunuakapu- ateariki and Wahianoa catchments (e.g. Figures 6.6 & 6.5). The model experiments predict that climate forcings necessary to match the LG moraines in the Mangaehuehu catchment also produce glaciers that terminate close to the former glacier limits identi- ﬁed in these separate adjacent catchments (e.g. Figures 6.7c & d). To evaluate the sensitivity of Mangaehuehu Glacier length to seasonal climatic change, I ran steady state experiments where either temperature (∆T = -3◦C) or precipitation (∆P = +20%) perturbations were applied to single months, and then 3-month windows, in turn (Figure 6.8). These experiments show that glacier length is most sensitive to temperature decreases during summer months (DJF), in particular December and January. 6.5.2 Glacier modelling The precipitation increases result in lower magnitude glacier advances, com- pared to temperature decreases, and are most important when applied to the months May-October. 153 6.5. RESULTS Table 6.3: Exposure ages (ka) for all samples using the ’St’ (Stone, 2000) and ’Lm’ scal- ing models, with internal (’Int.’) and external (’Ext.’) uncertainties after to Goehring et al. (2010). Sample St Int. Ext. Lm Int. Ext. Mangaehuehu catchment: Upper moraine (LG2): BH1210 14.9 0.3 1.2 15.0 0.3 1.7 BH1209 13.4 0.4 1.1 13.5 0.4 1.6 BH1211 11.9 0.3 1.0 12.0 0.3 1.4 BH1208 7.1 0.5 0.7 7.1 0.5 0.9 Lower moraine (LG1): BH1204 13.9 1.0 1.5 14.0 1.0 1.8 BH1206 13.4 0.7 1.2 13.5 0.7 1.6 BH1207 12.1 0.6 1.1 12.2 0.6 1.5 BH1205 10.5 0.4 0.9 10.6 0.4 1.2 BH1201 6.9 0.3 0.6 6.8 0.3 0.8 BH1213 5.7 0.3 0.5 5.7 0.3 0.7 BH1202 2.8 0.2 0.4 3.0 0.3 0.5 Outer moraine (LG3): BH1212 11.1 0.4 0.9 11.1 0.4 1.3 Outer moraine (LG4): BH1214 7.3 0.3 0.7 7.3 0.3 0.9 BH1215 4.4 0.4 0.4 4.5 0.2 0.6 Te Unuunuakapuateariki catchment: Left lateral (TU1): ME1201 14.6 0.4 1.2 14.7 0.4 1.7 ME1202 14.3 0.4 1.2 14.4 0.4 1.7 ME1203 16.4 0.4 1.3 16.5 0.4 1.9 Right lateral (TU2): ME1204 6.6 0.2 0.6 6.7 0.2 0.8 (∆T) and precipitation (∆P) perturbations that result in steady state glacier in the Mangaehuehu catchment ing a suite of parameter sensitivity tests. Flow model parameters (A = 1 x 10−15 - 1 x 10−18 Pyr−3 yr−1; Uc = of <0.1 ◦C and are not shown; (b) The range of ∆T forcings required to balance ∆P of ± 20 %; (c) Modelled ss balance for ∆T = -2.9 ◦C; ∆P = 0 across the Mangaehuehu, Te Unuunuakapuateariki and Wahianoa 1 and TU2 described in Section 6.5.1 are labelled. state glacier in the Mangaehuehu catchment (A = 1 x 10−15 - 1 x 10−18 Pyr−3 yr−1; Uc = uired to balance ∆P of ± 20 %; (c) Modelled u, Te Unuunuakapuateariki and Wahianoa itation (∆P) perturbations that result in steady ameter sensitivity tests. Flow model parameters re not shown; (b) The range of ∆T forcings requ T = -2.9 ◦C; ∆P = 0 across the Mangaehuehu bed in Section 6.5.1 are labelled. tion (∆P) perturbations that result in steady meter sensitivity tests. 6.5.2 Glacier modelling Flow model parameters not shown; (b) The range of ∆T forcings requ = -2.9 ◦C; ∆P = 0 across the Mangaehueh d in Section 6.5.1 are labelled. ∆T) and precipit ng a suite of para f <0.1 ◦C and are s balance for ∆T and TU2 describ 155 6.6. DISCUSSION Figure 6.8: Top panel: Length increase of the Managehuehu glacier (%) from present, resulting from temperature (∆T = -3 ◦C) and precipitation (∆P = +20%) applied separately to individual months. Bottom panel: the same forcing experiments but applied to 3-month moving windows across the mass balance year. Figure 6.8: Top panel: Length increase of the Managehuehu glacier (%) from present, resulting from temperature (∆T = -3 ◦C) and precipitation (∆P = +20%) applied separately to individual months. Bottom panel: the same forcing experiments but applied to 3-month moving windows across the mass balance year. 6.6.1.1 Cosmogenic 3He chronology Eighteen cosmogenic 3He surface exposure ages of moraine boulders in the Mangae- huehu (Figure 6.10) and Te Unuunuakapuateariki catchments exhibit a high degree of internal scatter (Table 6.5.1.3). Field observations and previous studies (e.g. Putkonen CHAPTER 6. LATE-GLACIAL READVANCE 156 and Swanson, 2003; Heyman et al., 2011; Applegate et al., 2012) lead me to conclude that processes causing incomplete exposure of moraine boulders are the most likely cause of the scatter observed in the dataset. For example, I ﬁnd that the distributions of the Mangaehuehu dataset as a whole (n=14) and for moraine LG1 alone (n=7) are char- acterised by negative skewness (Figures 6.10a,b), which is a useful ﬁrst-order indicator of incomplete exposure (Applegate et al., 2010). Also, multiple studies (Putkonen and Swanson, 2003; Heyman et al., 2011), utilising large empirical datasets (100s-1000s of ages) from a wide range of glaciological and climatic settings, have demonstrated that incomplete exposure is the most common cause of scatter in populations of moraine boulder cosmogenic exposure ages, especially in temperate glacial environments such as the study site. In these studies, the authors suggest that exposure ages should be interpreted as minimum limiting ages and the true moraine age lies closest to the oldest peak in the distribution (e.g. 15-11 ka; Figures 6.10a,b). Extension of these conclusions to the study site is further supported by the ﬁeld observations of striated boulders and glacially-polished bedrock outcrops within the glacial catchment, which indicate that temperate conditions prevailed in the geological past, which decreases the likelihood of cosmogenic nuclide inheritance within moraine boulders. Also, steep valley sides that characterise most alpine glacier catchments and potentially contribute sediment with inherited cosmogenic nuclide components to the glacier surface, are absent from the ﬁeld site. This further reduces the potential for inheritance of cosmogenic 3He, therefore the older ages are more likely to approximate the true age of the moraine. In the Te Unuunuakapuateariki catchment, fewer ages are available to make a robust assessment of possible source of scatter. Extending the previous reasoning suggests the best-estimate age for the outermost moraine (TU1) is 16.4 ± 1.3 ka (Table 6.5.1.3), which is older than those in the Mangaehuehu catchment. However, the remaining two samples from TU1 are consistent with one another, within the measurement uncertainty, at c. 14.6 ± 1.7 ka. 6.6.1.1 Cosmogenic 3He chronology If this is the true age of this outer moraine, then it closely agrees with the oldest ages from LG1 and LG2 moraines in the Mangaeheuehu catchment. 6.6.1.2 Extreme exposure age scatter in Mangaehuehu: Possible causes Post-depositional processes that result in exposure ages to be younger than the true depositional age include, burial beneath sediment cover and erosion of the sampled surface. If samples are buried, then nuclide production is reduced by a factor related to the thickness and density of the overlying material (Figure 3.1). If samples have un- dergone erosion and this is not recognised and accounted for, then the loss of nuclides from the sample is the cause of exposure age underestimation. 6.6. DISCUSSION 157 Using the known attenuation of cosmogenic nuclide production at depth, it is possible to investigate the possible ’true’ age of the moraine for given burial scenarios (see also, Section 5.5.1.2). Figure 6.9 shows that samples that yield simple exposure ages of c. 13 ka, require between c. 5 kyr and 12 kyr of burial beneath soil/tephra of thicknesses 300 cm to 25 cm respectively, in order to explain the measured concentrations if the samples were actually deposited during the preceding cold period in New Zealand (’Otira A’; c. 18 ka; Barrell et al., 2013). Meanwhile, c. 10 kyr of burial beneath soil/tephra at least 100 cm thick is required to explain the nuclide concentrations of LG1 samples that yield simple exposure ages of c. 6 ka, if they were deposited during the LGR and have solely been affected by shielding. Given the extreme paucity of evidence for substantial soil cover on the LG1 moraine in the geological past (e.g. remnant patches), it is unlikely that such shielding scenarios occurred to affect the samples measured in this study. If, as suggested above (Section 6.6.1.1), moraine LG1 was deposited during the LGR at c. 13 ka, then boulder surface erosion rates of c. 10 mm kyr−1 to >100 mm kyr−1 are required to explain the exposure ages from moraine LG1 that, simply interpreted, yield post LGR ages (Table 6.5.1.3). These rates exceed, by up to an order of magnitude, the compiled empirically-derived long-term erosion rates for igneous lithologies in temperate climatic environments (<10 mm kyr−1; Portenga and Bierman, 2011). Thus, it is unlikely that surface erosion is the dominant cause of the extreme scatter shown in the Mangaehuehu dataset. 6.6.1.2 Extreme exposure age scatter in Mangaehuehu: Possible causes Recently, numerical models that describe the impact of moraine degradation and inher- itance processes on cosmogenic nuclide production have been developed and applied to aid moraine age interpretation (Hallet and Putkonen, 1994; Putkonen and Swanson, 2003; Applegate et al., 2008; Applegate et al., 2010). These models simulate the distribu- tion of cosmogenic 10Be exposure ages found on a moraine surface under a prescribed geomorphic scenario and can be used to (i) test whether the observed exposure age distribution can be produced by hillslope diffusion; and (ii) better constrain moraine ages by ﬁtting the modeled distributions to empirical datasets (e.g. Applegate et al., 2008, 2012). I adapt the moraine degradation-cosmogenic nuclide production model developed by Applegate et al. (2010) to simulate the effects of moraine erosion on cosmogenic 3He production (Equation 6.5). For example, 3He is a stable isotope, therefore I remove the decay constant from the nuclide concentration calculation (Applegate et al., 2010, their equation 6/7). Furthermore, I neglect muonic contribution in the calculations (Applegate et al., 2010, their equation 5) and assume all production occurs via spallation CHAPTER 6. LATE-GLACIAL READVANCE 158 Figure 6.9: (a) Modelled ’true’ ages of moraine LG1 samples due to hypothetical shielding of cosmogenic nuclide production beneath sediment (ρ = 1.6 g cm−3) cover of given burial durations and thicknesses. Soil thicknesses represent that overlying the sampled boulder surfaces, which typically sit c. 1 m above the moraine surface (see Table 6.1). Calculated for samples that yield simple exposure ages of c. 13 ka. (b) As for (a), but calculated for samples that yield simple exposure ages of c. 6 ka. Figure 6.9: (a) Modelled ’true’ ages of moraine LG1 samples due to hypothetical shielding of cosmogenic nuclide production beneath sediment (ρ = 1.6 g cm−3) cover of given burial durations and thicknesses. Soil thicknesses represent that overlying the sampled boulder surfaces, which typically sit c. 1 m above the moraine surface (see Table 6.1). Calculated for samples that yield simple exposure ages of c. 13 ka. (b) As for (a), but calculated for samples that yield simple exposure ages of c. 6 ka. (Equation 6.6), as production of cosmogenic 3He via muons is not well constrained and not accounted for in cosmogenic 3He exposure age calculations (Goehring et al., 2010). 6.6.1.2 Extreme exposure age scatter in Mangaehuehu: Possible causes Omitting muon production may cause underestimation of modelled 3He concentra- tions within exhumed boulders, as production via high-energy muons becomes more important with increasing depth, due to higher effective attenuation lengths (Heisinger et al., 2002). However, Applegate et al. (2010) demonstrate that even when potential muonic contribution is considered, the majority of cosmogenic nuclide accumulation for deeply buried boulders is derived from spallation reactions after the boulder reaches the moraine surface. Furthermore, theoretical calculations indicate muons account for 2-3 % of total cosmogenic 3He surface production (Lal, 1987), therefore this is not a signiﬁcant source of uncertainty in the calculations. All other model parameters match those for exposure age calculation (discussed above). The moraine diffusion model is unchanged from Applegate et al. (2010), who give a full description. Cf = Z tf 0 P(d(t))dt. (6.5) (6.5) where Cf is the ﬁnal 3He concentration of surface boulders after simulation of moraine degradation over time t. Cosmogenic nuclide production at depth, P(d), is described as: where Cf is the ﬁnal 3He concentration of surface boulders after simulation of moraine degradation over time t. Cosmogenic nuclide production at depth, P(d), is described as: P(d) = P0exp−d Λ (6.6) (6.6) 159 6.6. DISCUSSION 6.6. DISCUSSION where P0 is the surface production rate scaled (using the ’Lm’ model) to the moraine surface using the calculator of Goehring et al. (2010), −d is depth below the moraine surface, and Λ is the effective attenuation length of cosmic rays (160 g cm −2; Dunne et al., 1999) divided by material density. This model assumes that the distribution of nuclide concentrations of boulders at a moraine surface is a product of boulder exhumation and surface erosion, and that boulders are uniformly distributed throughout the parent till of the moraine (Applegate et al., 2010). These assumptions are therefore violated by compound, wide-crested moraine ridges where topographic diffusion is limited following glacier retreat, or for moraines that exhibit low boulder concentrations (Applegate et al., 2010). I apply the moraine degradation model to moraine LG1, which satisﬁes both criteria. It is relatively sharp-crested (Figure 6.10a) with relatively high (c. 20-25◦) slope angles on both the ice-proximal and ice-distal ﬂanks, therefore there is high potential for down-slope sediment transfer. Furthermore, ﬁeld observations show that the crest and parent till of the moraine is boulder-rich (Figure 6.3). empirical exposure age distributions. empirical exposure age distributions. I ﬁnd modelled exposure age distributions best ﬁt the empirical dataset when moraine age is prescribed as 13.6 ka (Table 6.4; Figure 6.10c). This parameterisation also pro- duces a ﬁnal moraine cross-proﬁle of similar geometry to the empirical measurements (Figure 6.10d). Figure 6.11 presents histograms of parameter values for the upper 5th percentile of KS results (KS<0.2897; n = 3386). This ﬁgure shows moraine ages of 12.4-16.8 ka can produce exposure age distributions that ﬁt well with the empirical age dataset. All model runs where the prescribed moraine age is outside of this range yield KS results that fall outside of the 5th percentile, indicating low probability that the true moraine age is <12.4 ka, or >16.8 ka. The age dataset presented in Figure 6.11 exhibits a unimodal distribution about a mode of 13.8 ka. Topographic diffusivity of c. 0.03 m−2 yr−1 produces the greatest number of modelled age distributions that ﬁt well with the empirical dataset and this rate agrees well with observed diffusivities in alpine landscapes (Table 6.5). Moraine slopes and boulder erosion rates are uniformly distributed (Figure 6.11), which indicates that good-ﬁt model results are insensitive to these parameters. Thus, although the best ﬁt permutation includes a steep initial moraine slope (61◦) and no boulder surface erosion, similar KS statistics result from when lower initial slope angles and higher boulder surface erosion rates are applied (Table 6.5). As a further analysis, I examine the best-ﬁt moraine ages produced when individual parameters are held constant at empirically observed values (Table 6.5). Again these parameterisations yield best-ﬁt ages of 13.6-14.0 ka, with the slightly older best-ﬁt age resulting from the inclusion of boulder surface erosion (c. 3 mm kyr−1). In summary, these results indicate that the cosmogenic 3He surface exposure age distribution of samples from moraine LG1 can be closely reproduced by simulating the combined effects of boulder exhumation and surface erosion on cosmogenic nuclide concentrations. Simulations that prescribe a moraine age of 13.6-14.0 ka yield the closest ﬁt to the empirical exposure age dataset and this result is robust across a range of observed parameter values. This is consistent with the age interpretation presented above, and further supports a late-glacial age for this moraine. 6.6.1.2 Extreme exposure age scatter in Mangaehuehu: Possible causes Apart from moraine age, important parameters in the degradation model are ini- tial moraine height, initial moraine slope and topographic diffusivity, with the latter two imparting most inﬂuence on the derived exposure age distributions (Applegate et al., 2010). Each of these parameters are unknown for pre-historic moraines, there- fore previous applications of this model have varied each parameter values between characteristic values derived from empirical datasets, to search for the parameter com- bination that best ﬁts the empirical exposure age distribution (e.g. Applegate et al., 2012). Barrows et al. (2008) stress the importance of empirical observations to constrain the range of model parameter values, in order to best represent the study site. I use 14 topographic cross-proﬁles (Figure 6.10a), evenly-spaced along the length of moraine LG1, in order to measure the modern height and slope angles. Initial moraine height exerts little inﬂuence on modelled exposure age distributions (Applegate et al., 2010), therefore I keep this constant in the simulations at 60 m. Initial moraine slope angles (i.e. immediately following glacier withdrawal) are uncertain for the past. Present-day slope angles for moraine LG1 are 21◦(Figure 6.10c), which I use as the lower bound in the simulations. The maximum upper bound for initial moraine slope is 61◦, based on empirical measurements of recently vacated (< 100 yr old) moraines in the Swiss Alps (Curry et al., 2009). I iteratively run the model using all permutations (n = 6.8 x 103) of the parameter value ranges listed in Table 6.4. As implemented by Applegate et al. (2012), the Kolmonov-Smimov statistic (KS) is generated for each iteration, which provides a measure between 0 (good) and 1 (poor) of the ﬁt between the modelled and CHAPTER 6. LATE-GLACIAL READVANCE 160 empirical exposure age distributions. 6.6.1.3 Other chronologies of late glacial cooling in New Zealand The inference from cosmogenic 3He dating, that the Mangaehuehu ’LG’ moraines rep- resent a glacial readvance in response to cooling at c. 14-12 ka is supported by several, well-dated terrestrial and marine climate proxy records from across New Zealand 6.6. DISCUSSION 161 Table 6.4: Parameter space and best ﬁt values for simulation of moraine degradation on cosmogenic exposure age distribution for moraine LG1. Parameter Upper bound Lower bound Step size Best ﬁt Moraine age (ka) 15 3 0.1 13.6 Topographic diffusivity (log10(m−2 yr−1)) 1 -3 0.5 1.5 Initial moraine slope (◦) 61 21 5 61 Boulder surface erosion (mm kyr−1) 10 0 1 0 Table 6.5: Best-ﬁt moraine degradation model parameter combinations when indi- vidual variables are prescribed using literature-based values. Parameter Value Source Best- ﬁt age (ka) Best-ﬁt slope (◦) Best-ﬁt diffusivity (m−2 yr−1) Best-ﬁt erosion rate (mm kyr−1) KS Slope 31-41◦ Hallet and Putkonen (1994); Putkonen and Swan- son (2003) 13.6 - 0.1 0 0.2183-0.2293 Topographic diffusvity 0.1 m−2 yr−1 Matsuoka (1998) 13.6 31 - 0 0.2183 Erosion rate 3 mm kyr−1 Barrows et al. (2001) 14.0 61 0.01 - 0.2217 .5: Best-ﬁt moraine degradation model parameter combinations when indi- variables are prescribed using literature-based values. region. In North Island, the Waiohau Tephra (14.0 ± 0.2 ka; Lowe et al., 2013) is a critical chronological marker horizon for the late glacial stratigraphies. Previous latitudinal comparisons of continuous climate proxy records through the last glacial-interglacial transition have suggested there is a time-lag in cooling between the North and South Islands (Newnham et al., 2012), with northern cooling lagging the south. However, recent reﬁnement of the Waiohau Tephra age, which forms the key chronological tie- point in northern records, suggests late glacial cooling was likely synchronous across the latitudinal range of New Zealand (e.g. Table 6.6) and broadly concomitant with the Antarctic Cold Reversal (Lowe et al., 2013) evident in Antarctic ice core records (e.g. Blunier and Brook, 2001; EPICA Community Members, 2006). The best constrained, continuous late-glacial climate proxy record comes from Kaipo Bog (Newnham and Lowe, 2000; Hajdas et al., 2006; Lowe et al., 2013), situated c. 150 km northeast of Mt. Ruapehu. This record forms the chronological type-locality for cold conditions during the lateglacial in the New Zealand Climate Event Stratigraphy (Barrell et al., 2013). At this location, a c. 6.6.1.3 Other chronologies of late glacial cooling in New Zealand 1200 yr climatic reversal is evident, which be- gan soon after deposition of the Waiohau Tephra, and is constrained to 13.8 - 12.6 ka by Bayesian modelling of 20 14C dates (Lowe et al., 2013). Similarly, in pollen stratgraphy CHAPTER 6. LATE-GLACIAL READVANCE 162 at Otamangakau Bog, c. 35 km north of Mt. Ruapehu, Turney et al. (2003) identify a moderate climatic reversal of c. 1000 yr duration, immediately following deposition of the Waiohau Tephra. Further north, in Auckland (37◦S), multi-proxy evidence from several maar records broadly agree that deglacial warming was interrupted by a cli- matic reversal that began at, or slightly before, the time of Waiohau Tephra deposition and lasted c. 700-1000 yrs (Augustinus et al., 2012; Stephens et al., 2012a,b; Sikes et al., 2013). The distribution of the Waiohau Tephra does not extend to South Island, however several climate proxy records, dated using a variety of absolute techniques, constrain a cooling episode of similar timing and duration to those in North Island. In north- ern South Island (41◦S), pronounced negative δ18O excursions in multiple speleothem records imply cooling, which are constrained by uranium-series dating to 13.8-11.7 ka (Hellstrom et al., 1998) and 14.4-13.0 ka (Whittaker et al., 2011). In the central Southern Alps (43-44◦S) multiple, well-dated climate proxy records constrain the timing of a late- glacial reversal. A continuous record of chironomid and pollen change from Boundary Stream tarn, in the Ben Ohau Range, shows cooling 14.2 - 13.2 ka, with the most intense cooling c. 13.7-13.2 ka (Vandergoes et al., 2008). Nearby, an increasing number of high-precision cosmogenic 10Be (cf. Schaefer et al., 2009; Putnam et al., 2010b) exposure chronologies indicate a period of moraine building by mountain glaciers centred on c. 14-13 ka (Kaplan et al., 2010; Putnam et al., 2010a; Kaplan et al., 2013). On the central west coast, Franz Josef Glacier advanced over a prominent bedrock outcrop, Canavans Knob, depositing glacial till with wood fragments that date to c. 13.1 ka (Denton and Hendy, 1994; Turney et al., 2007). Thus, multiple glacial records from the central Southern Alps suggest glaciers advanced at this time in response to cooling, or a hiatus in deglacial warming. In summary, there is increasing evidence for broadly synchronous, and widespread atmospheric cooling in New Zealand between c. 14-12.5 ka in palaeoclimate proxy records. This is consistent with the geomorphological and geochronological evidence presented here from southern Mt. 6.6.1.3 Other chronologies of late glacial cooling in New Zealand Ruapehu, which shows glacier readvance in multiple catchments at this time. 6.6.2.1 Late-glacial palaeoclimatic estimates derived from glacier modelling Glacier model-derived palaeotemperature estimates indicate that a cooling of 2.5 - 3.4 ◦C relative to present is necessary to simulate a steady state glacier that matches the 6.6. DISCUSSION 163 former glacial limits represented by the LG moraines on southern Mt. Ruapehu (Figure 6.7). This temperature range reﬂects uncertainty in past precipitation change of ± 20%. Few precipitation proxy records exist to help further constrain the temperature estimate. Speleothem isotope records from western regions in New Zealand generally point towards increased moisture availability during the late-glacial reversal, which suggests increased precipitation (Hellstrom et al., 1998; Williams et al., 2005; Whittaker et al., 2011). At multiple sites on the west coast (43◦S), Vandergoes and Fitzsimons (2003) conclude that changing pollen taxa assemblages during the period 14.4 - 11.4 ka are also consistent with cooler, wetter conditions. However, multiple palaeoenvironmental proxy records from maar lakes in Auckland (36◦S) yield mixed signals concerning late-glacial precipitation change (Augustinus et al., 2012; Stephens et al., 2012b; Sikes et al., 2013). Pollen, cladocera and diatom assemblages lead Augustinus et al. (2012) and Stephens et al. (2012b) to conclude that the late-glacial climate reversal was a time of relatively dry conditions in northern New Zealand. Meanwhile, carbon isotopes suggest reduced aridity at this time, at least during spring and summer (Sikes et al., 2013). Thus, although most records suggest increased precipitation during the LGR, I consider the relative paucity of proxy records and the spatial and possible seasonal variability between existing records justiﬁes the uncertainty range reported here. I test the impact of the energy balance and ice ﬂow parameter choices on the derived paleotemperatures, in a suite of systematic model sensitivity analyses (Figure 6.7). In model runs with increased precipitation relative to present, increases in the snow-rain temperature threshold (Ts > 0.5◦C) impart a signiﬁcant impact on palaeotemperature estimates (6.7a). This is because higher values for Ts increase the ratio of snow to rain. When combined with greater annual precipitation, modelled accumulation increases signiﬁcantly, thus requiring signiﬁcantly less cooling to achieve the target palaeoglacier geometry (Figure 6.7). Altering snow albedo (αsnow± 0.05) consistently imparts the greatest deviation (± 0.4◦C) from palaeotemperatures derived using the optimal pa- rameter settings. Increasing asnow requires less cooling to match the moraine limits, due to increased reﬂectance of incoming shortwave radiation, and vice versa (Figure 6.7a). 6.6.2.1 Late-glacial palaeoclimatic estimates derived from glacier modelling In reality, αsnow is primarily determined by surface impurity concentrations, as well as snow crystal size (Cuffey and Paterson, 2010), however it is not possible to constrain pre-historic changes in these variables. The proximity of the Mangaehuehu Glacier to an active volcanic vent increases the likelihood of periodic delivery of sediment to the glacier surface, as was evident during historic eruptions. Depending on the thickness of the imposed debris cover, such events could lead to enhanced or retarded surface melt rates (Richardson and Brook, 2010). However, whilst such impacts may have a signiﬁcant impact on the local energy balance in the short term (c. 1-10 yrs), long-term impacts in relatively steep, fast, temperate glaciers are generally low, due to mass loss CHAPTER 6. LATE-GLACIAL READVANCE 164 at the terminus and erosion by surface meltwater (Kirkbride and Dugmore, 2003; Nield et al., 2013). In prescribing modern topographic boundary conditions in the model application presented here, I assume that no signiﬁcant change has occurred in the Mangaehuehu catchment in the last c. 15 ka. This assumption can be assessed in two ways: (i) by reviewing the known geologic events that have occurred over this time period, which may have impacted on the local geomorphology; and (ii) by comparing modelled glacier geometries in other catchments on Mt. Ruapehu to the local moraine distribu- tion. Concerning the ﬁrst point, the preservation of the LG moraines suggests little post-depositional geomorphic disturbance in the lower portion of the former glacial catchment. On the upper mountain, the major geomorphic events in the last 15 ka include, the Murimotu debris avalanche ﬂank collapse (10.4-10.6 cal. ka: Chapter 4; Topping, 1974; Palmer and Neall, 1989) and emplacement of the Whakapapa Formation lava ﬂows (Hackett and Houghton, 1989; Gamble et al., 2003). The major geomorphic imprints of these events are concentrated on the northwest ﬂank of Mt. Ruapehu, well away from the Mangaehuehu catchment. No known lavas of Holocene age exist in the Mangaehuehu catchment (C. Conway/G. Leonard/J. Gamble, pers. comm., 2014). Concerning the second point, ’intermediate’ moraines (i.e. between inferred LGM and modern day ice) exist in several other catchments on southern Ruapehu (e.g. Figure 6.7c). Although some dating uncertainty exists, the excellent agreement between the model simulations and these geologically-inferred glacial limits supports the assumption of topographic stability over the last 15 ka. 6.6. DISCUSSION 165 6.6. CHAPTER 6. LATE-GLACIAL READVANCE 6.6.2.1 Late-glacial palaeoclimatic estimates derived from glacier modelling DISCUSSION 165 Figure 6.10: (a) Histogram and statistics describing the distribution of all moraine exposure ages from the Mangaehuehu catchment (LG moraines); (b) Histogram and statistics describing the distribution of moraine boulder exposure ages from moraine LG1 in the Mangaehuehu catchment; (c) Empirical (red) and modelled (blue) exposure age distribution for moraine LG1. Vertical dashed line depicts the best-ﬁt moraine exposure age (grey shading represents ± 10% uncertainty), as indicated by moraine degradation modelling, according to Applegate et al. (2010); (d) Empirical and modelled topographic cross proﬁles of moraine LG1. Figure 6.10: (a) Histogram and statistics describing the distribution of all moraine exposure ages from the Mangaehuehu catchment (LG moraines); (b) Histogram and statistics describing the distribution of moraine boulder exposure ages from moraine LG1 in the Mangaehuehu catchment; (c) Empirical (red) and modelled (blue) exposure age distribution for moraine LG1. Vertical dashed line depicts the best-ﬁt moraine exposure age (grey shading represents ± 10% uncertainty), as indicated by moraine degradation modelling, according to Applegate et al. (2010); (d) Empirical and modelled topographic cross proﬁles of moraine LG1. CHAPTER 6. LATE-GLACIAL READVANCE 166 Figure 6.11: Histograms of moraine degradation model parameter values (moraine age, initial slope, topographic diffusivity and boulder erosion rate) for the upper 5th percentile of KS results (KS<0.2897; n = 3386) that result from comparison to the LG1 cosmogenic 3He exposure age dataset. Figure 6.11: Histograms of moraine degradation model parameter values (moraine age, initial slope, topographic diffusivity and boulder erosion rate) for the upper 5th percentile of KS results (KS<0.2897; n = 3386) that result from comparison to the LG1 cosmogenic 3He exposure age dataset. and quantitative palaeoclimate reconstructions for the late-glacial climate reversal in Latitude (◦) Onset (ka) End (ka) ∆T (◦C) Dating method ∆P Proxy -37 13.8a 12.8a - tephra/14C ↑ Sediment in ﬂux/diatoms -37 14.5a 13a # tephra/14C ↓ Pollen -37 12.8a - # 14C Pollen -37 13.8a 12.4a - tephra/14C ↑ Pollen/sediment inﬂux -38 13.6 a 12.6a -0.5 - -1±1.5 tephra/14C Pollen -38 13.8 12.6 - tephra/14C/bayesian model - - th Island -39 13.4 11.2 - U/Th ↑ Speleothem -39 14.7a 13.7a - tephra/14C Pollen g -39 14.7a 13.0a - tephra/14C Pollen -39 - c. 6.6.2.2 Testing Hnull: Was there an enhanced latitudinal air temperature gradient across NZ during the late-glacial climate reversal? To test Hnull, I compare the estimated late-glacial cooling of 2.5 - 3.4 ◦C to other well- dated, quantitative palaeoclimate reconstructions (Table 6.6). Few quantitative recon- structions of LGR air temperatures exist for North Island. Those that do exist (e.g. Newnham et al., 2012; Sikes et al., 2013) are derived using a pollen-temperature transfer function that uses a database of pre-deforestation pollen taxa (Wilmshurst et al., 2007) in order to minimise the anthropogenic impacts that distort the present-day vegetation- climate relationship (Norton et al., 1986). Thus, the different reference times from which palaeotemperature estimates are made between proxies (e.g. glaciers - from present day, pollen - from c. AD1300) makes it difﬁcult to compare absolute palaeotemperature estimates. However, this limitation does not prevent inter-proxy comparison of the spa- tial patterns of palaeotemperature reconstructions, which is the focus of the hypothesis test in this study. The glacier model results for Mt. Ruapehu presented here are consistent with ex- isting, quantitative glacier-palaeoclimate reconstructions from South Island (Table 6.6), most of which come from the central Southern Alps (c. 43-44 ◦S). For example, at Franz Josef glacier, Anderson and Mackintosh (2006) use an empirically calibrated degree-day approach, with a 1D glacier ﬂowline model to simulate the climatic forcing required for ice to override the well dated glacial till at Canavan’s Knob (Denton and Hendy, 1994; Turney et al., 2007), c. 12 km down-valley from the present glacier terminus. They ﬁnd that a cooling of c. 2.7 - 3.6 ◦C is necessary to reach this site, when precipitation is varied by ± 20%. Two key uncertainties surround this palaeotemperature estimate. First, the maximum down-valley position of the glacier terminus related to this advance remains uncertain (e.g. Barrows et al., 2007a; Applegate et al., 2008, 2012); further cooling is required to produce a glacier that terminates further down valley, e.g. at the Waiho Loop moraine. Conversely, the simulations conducted by Anderson and Mackintosh (2006) do not include potential ice contributions from the Callery tributary, which, if included, may lower the climatic forcing necessary to force Franz Josef Glacier out past Canavans Knob. Despite these uncertainties, the estimates of Anderson and Mackintosh (2006) are broadly consistent with the estimates of Doughty et al. (2013) and Kaplan et al. (2013), to derive palaeotemperature estimates of 2.3 - 3.2 ◦C and 1.8 - 2.6 ◦C for the well-dated (c. 6.6.2.1 Late-glacial palaeoclimatic estimates derived from glacier modelling 14- 12 -2.5 - -3.4 3He Glacier model -41 13.5 11.1 - U/Th ↑ Speleothem -41 14.4 13 - U/Th ↑ Speleothem -41 13.8 11.7 - U/Th ↑ Speleothem -42 15.0 a 12.9a - tephra/14C Pollen -43 15.0a 12.9a - tephra/14C Pollen -43 14 12.7 - 14C ↑ Pollen -43 - 13.0 - 10Be - Glacier length -43 - 13.0 - 10Be Glacier length -43 15 13.4 # 14C - Pollen r -43 - - - 3 - -4.7 - - Glacier model -44 14.2 13.2 -3 - -4 ±1.4 14C - Chironomids (WA PLS) -44 14.2 13.2 -2 - -3 ±1.4 14C - Chironomids (PLS) -44 - 13 - 10Be - Glacier ELA -44 - - -2.3 - -3.2 10Beb - Glacier model -44 14 -1.8 - -2.6 10Be - Glacier model -52 14.5 12.5 -2 - -2.5 ±1 14C Pollen -52 - 12.5 -2 - -2.5 ±1 14C - Pollen ersal in i ms ment el th th el ds (W s (PLS) el el (W PLS) 168 CHAPTER 6. LATE-GLACIAL READVANCE a Age model uses age for Waiohau Tephra that pre-dates the revision by Lowe et al. (2013) b Dating by Kaplan et al. (2010) (c. 13 ka) a Age model uses age for Waiohau Tephra that pre-dates the revision by Lowe et al. (2013) b Dating by Kaplan et al. (2010) (c. 13 ka) laeotemperature estimates are made relative to a pre-deforestation reference time - see tex 6.6.2.2 Testing Hnull: Was there an enhanced latitudinal air temperature gradient across NZ during the late-glacial climate reversal? 14-12 ka) moraine records in Irishman (Kaplan et al., 2010) 6.6. DISCUSSION 169 and Whale streams, respectively (Table 6.6). The strength of the latter two studies for the present hypothesis test is that the same glacier model approach is used for the South Island (Irishman & Whale Streams) and North Island (this study). This consistent approach minimises the potential for methodologically-based differences in results and indicates uniform temperature change across New Zealand during the LGR. The glacier model results presented here do not support the hypothesis of an enhanced latitudinal temperature gradient across New Zealand during the LGR, therefore I fail to reject Hnull. This poses the question, what causes the discrepancy between the spatial pattern of cooling as indicated by pollen (e.g. Newnham et al., 2012) and glacier records during the LGR in New Zealand? I argue that such conﬂicts represent a difference between the seasonal sensitivity of the two proxies. Glacier mass balance in New Zealand is most sensitive to temperature (Figure 6.8; Anderson and Mackintosh, 2006; Anderson et al., 2010; Anderson and Mackintosh, 2012), due to its strong contribution to glacier melt and the close-proximity of winter temperatures to the snow-rain threshold. Seasonal sensitivity experiments conducted in this study show that glacier length sensi- tivity is most sensitive to temperature changes during the ablation season, as opposed to other periods of the mass balance year (Figure 6.8). This accords with similar work by Oerlemans and Reichert (2000), who demonstrate that mass balance sensitivity to temperature is greatest in months where average air temperatures exceed, or approach 0 ◦C. In temperate, maritime locations such as New Zealand, mass balance sensitivity to temperature is therefore greatest in summer, but changes in the shoulder seasons (autumn/spring) can also be important (Oerlemans and Reichert, 2000). In contrast, the distribution of vegetation taxa in New Zealand is considered to be predominantly controlled by the duration and/or intensity of the cold season, or mean annual temper- ature, due to low resistence to freezing (Wilmshurst et al., 2007; Vandergoes et al., 2008). Thus, it seems glaciers and vegetation in New Zealand may be sensitive to different thresholds in the annual temperature cycle. Used in combination, these proxies may inform past changes in seasonality. Using a similar approach, Vandergoes et al. (2008) compare synchronous assemblage changes of chironomids and pollen through the LGR. CHAPTER 6. LATE-GLACIAL READVANCE 170 6.6.2.2 Testing Hnull: Was there an enhanced latitudinal air temperature gradient across NZ during the late-glacial climate reversal? They ﬁnd a muted pollen signal during the LGR, in comparison to that from chirono- mids (c. ∆T = -2 - -4 ◦C; Table 6.6), which are most sensitive to changes in summer temperatures (Dieffenbacher Krall et al., 2007). This mirrors the relationship observed between glaciers and pollen in the central North Island, and supports the hypothesis that the LGR in New Zealand was a time of reduced seasonality, with greater cooling during summer than winter. Furthermore, it appears that summertime temperatures exhibited a similar latitudinal gradient to present-day across New Zealand during the LGR. The pollen-based LGR palaeotemperature gradient observed by Newnham et al. (2012) may therefore represent a steeper winter temperature gradient during the LGR CHAPTER 6. LATE-GLACIAL READVANCE 6.7 Conclusion Mountain glaciers on Mt. Ruapehu in central North Island, New Zealand, readvanced during the late-glacial climate reversal (Barrell et al., 2013), which is largely coeval with the Antarctic Cold Reversal. Numerical glacier model experiments suggest local glaciers responded to a climate that was 2.5-3.4 ◦C cooler and assuming that precipita- tion remained in the range ± 20%, from present-day. This magnitude of cooling differs from nearby pollen-based reconstructions, therefore I fail to reject a null hypothesis of uniform temperature change across New Zealand during the late-glacial climate reversal. I suggest that differences between the seasonal sensitivity of glacier mass balance and vegetation are the cause of these conﬂicting quantitative palaeoclimatic estimates. These ﬁndings suggest that uniform summer cooling occurred across New Zealand during the LGR, and that perhaps an enhanced north-south winter tempera- ture gradient prevailed at this time. I therefore support the existing hypothesis that the late-glacial climate reversal was a time of reduced seasonality (e.g. Vandergoes et al., 2008). 7.1 Abstract Quantitative climate reconstructions provide important data for tracing the drivers and mechanisms of past, natural climate variability and for constraining possible fu- ture responses of the climate system. Geological evidence of former mountain glacier geometries affords the opportunity to reconstruct palaeoclimate, due to the strong relationship between ice extent and local climate. In this study, I present results from geological mapping, cosmogenic 3He surface exposure dating and numerical glacier modelling, which constrains glacial extent and palaeoclimate in nine catchments on the central North Island volcanoes of New Zealand, during the Last Glacial Cold Period (c. 30-18 ka). Moraine distribution indicates that valley glaciers extended down to c. 1200 m asl, from individual ice ﬁelds centred over Tongariro massif and Mt. Ruapehu volcanoes. Simulations using a 2D coupled energy-balance/ice-ﬂow model show that the mapped ice limits can be reproduced when present day temperatures are reduced by 4 - 7 ◦C. Glacier model experiments using topographic reconstructions that account for the effects of effusive post-glacial volcanism, generally increased the magnitude of cooling required to simulate the former ice limits by up to 0.5 ◦C. The palaeotempera- ture estimates presented here are consistent with independent, proximal temperature reconstructions from fossil pollen assemblages, as well as similar glacier modelling reconstructions from central Southern Alps. This agreement, together with the rela- tively small impact of topographic change, provide increased conﬁdence in the use of 171 CHAPTER 7. LGM GLACIER MODELLING 172 moraine records from volcanically-active domains for palaeoclimatic reconstruction over millennial to orbital timescales. moraine records from volcanically-active domains for palaeoclimatic reconstruction over millennial to orbital timescales. 7.2 Introduction Comparison of palaeoclimatic estimates from such studies permits robust assessment of former climatic gradients, over a range of spatial scales, which can help to identify drivers of past climate change. The Last Glacial Maximum (’LGM’) describes the global sea level low-stand achieved between 26-19 ka, when Northern Hemisphere ice sheets reached their maximum extent of the last glacial cycle (Clark et al., 2009). During this interval, global mountain glacier extent also peaked (Schaefer et al., 2006; Clark et al., 2009), in response to ELA lowering of c. 900-1000 m, relative to present (Broecker and Denton, 1990). In New Zealand, local nomenclature such as ’extended LGM’ (Newnham et al., 2007) or ’Last Glacial Cold Period’ (’LGCP’; Alloway et al., 2007; Barrell et al., 2013) has been introduced to describe the prevailing glacial climatic conditions between c. 30 - 18 ka (e.g. Chapter 5; Vandergoes et al., 2005; Putnam et al., 2013b). Continuous and well-dated climate proxy records have greatly improved understanding of the timing and relative magnitudes of climatic changes in New Zealand through this period (e.g. Barrell et al., 2013). However, quantitative estimates of terrestrial palaeoclimatic variables (namely air temperature and precipitation) are rare. Where available, independent estimates of LGCP climate have shown good agreement across relatively short spatial scales (e.g. central Southern Alps - Golledge et al., 2012; Putnam et al., 2013a,b; Rowan et al., 2013). However, quantitative palaeoclimate reconstructions from elsewhere in New Zealand can differ greatly. For example, McKinnon et al. (2012; their Table 3) summarise all previously published, terrestrial, LGCP temperature estimates for New Zealand, which range from +0.5◦C to -9◦C, relative to present. Such differences may arise from methodologi- cal/chronological uncertainties, or could represent meaningful spatial relationships that represent key climatic processes (e.g. Lorrey et al., 2012b) with the potential to inform the drivers of observed changes. Increasing the number and spatial coverage of quantitative palaeoclimate reconstructions will help to resolve these issues. In Chapter 5, I present geological and geochronological evidence for valley glacia- tion on Tongariro massif, in central North Island, during the LGCP. Reconstruction of the palaeo-ELA associated with this former glacier indicates a lowering of c. 1000 m relative to present, which equates to a temperature depression of c. 5-7 ◦C from modern. However, this estimate remains uncertain for two main reasons. First, former valley glaciers on the central North Island volcanoes were likely fed by central ice ﬁelds. 7.2 Introduction Constraining the magnitude of warming across glacial-interglacial transitions provides fundamental information about the global climate system (e.g. CLIMAP, 1976; Wael- broeck et al., 2009; Bartlein et al., 2011). Such data permit: (i) critical tests of hypotheses that seek to explain the drivers and mechanisms of past natural climate change (Shakun et al., 2012); (ii) constraint of climate sensitivity to radiative forcing (Schmittner et al., 2011; Hargreaves et al., 2012); and (iii) critical assessment of general circulation mod- els that are used to predict future climatic change (Braconnot et al., 2012; Schmidt et al., 2013). Many environmental proxies now exist for palaeoclimatic reconstructions (e.g. Alloway et al., 2007). Mountain glacier length ﬂuctuations primarily reﬂect mass changes resulting from the perturbation of snow accumulation and snow/ice ablation (Oerlemans, 2001). These perturbations are due primarily to changes in air temperature and solid precipitation, and records of past glacial extent provide useful insight to past climatic change. Moraines are landforms comprising unconsolidated sediment that has been transported via glacial ﬂow and deposited at the ice margins (Benn and Evans, 2010). Preservation of such features in the modern landscape delineates past ice geometries which, when coupled with geochronological techniques such as cosmogenic surface exposure dating, provide the opportunity to constrain both the magnitude and timing of palaeoclimatic change (e.g. Chapter 5; Schaefer et al., 2006, 2009; Kaplan et al., 2010; Putnam et al., 2010a). Traditionally, past glacial geometries have been related to palaeoclimate through man- ual reconstruction of former equilibrium line altitude (ELA; e.g. Porter, 1975; Meierding, 1982); however, these methods are subject to several unquantiﬁable sources of error (Plummer and Phillips, 2003). Increased understanding of glacier climate relation- ships (Oerlemans, 2001; Cuffey and Paterson, 2010), coupled with increasing comput- ing power and availability of digital climate datasets now facilitates application of physically-based numerical models to simulate past former glaciers (Plummer and Phillips, 2003; Doughty et al., 2013). Models that describe glacier mass balance and ice-ﬂow provide a glacier-climate transfer function, which allows quantitative palaeo- climatic information to be extracted from geological records of glacier length change (Oerlemans, 2005). This approach affords the opportunity to constrain palaeoclimatic estimates in an objective and consistent manner, at multiple locations where evidence for past glaciation exists, whilst uncertainty bounds can be quantiﬁed through model 7.2. INTRODUCTION 173 sensitivity analyses (e.g. Rowan et al., 2014). 7.2 Introduction The absence of geomorphic evidence to constrain past ice thickness in the accumulation zones means that the hypsometry of former glacier surfaces is uncertain, which can lead to errors in manual ELA reconstruction (Rea et al., 1999). Second, part of the catchment studied in Chapter 5 has undergone considerable post-glacial topographic change, due to effusive volcanism during the Holocene (Moebis et al., 2011). Growth of CHAPTER 7. LGM GLACIER MODELLING 174 volcanic cones and emplacement of lava ﬂows has potentially altered drainage patterns and adds further uncertainty to the reconstruction of former ﬂow divides and glacier surfaces. In this chapter I aim to: (i) provide reﬁned constraint of LGCP glacial limits in central North Island, through direct dating and morphostratigraphic correlation of the chronological results from Chapter 5 to glacial landform assemblages across a wider study domain that encompasses catchments on both Tongariro massif and Mt. Ruapehu volcanoes; (ii) constrain the magnitude of cooling associated with the identiﬁed glacial limits, using a 2D numerical glacier energy-balance/ice-ﬂow model; and (iii) investigate the sensitivity of glacier model derived palaeoclimate estimates to post-glacial topographic change, using an expert-deﬁned reconstruction of the topo- graphic boundary conditions during the LGCP. 7.2.1 Setting Tongariro Volcanic Centre (TgVC) represents the southernmost expression of the Taupo Volcanic Zone, which is a c. 300 km long, northeast trending belt of subduction zone volcanism at the Australian-Paciﬁc plate margin (Cole, 1978). Located in central North Island, New Zealand (39 ◦S 174 ◦E; Figure 7.1), TgVC is dominated by the andesite- dacite stratovolcanic centres of Tongariro massif (including Mt. Tongariro - 1963 m asl and the Holocene cone of Mt. Ngauruhoe - 2291 m asl) and Mt. Ruapehu (2797 m asl). Cone-building volcanism in the region began before c. 275 ka (Stipp, 1968) and both have exhibited effusive activity in historical times (Houghton et al., 1987). The local climate, as recorded at Whakapapa village (1097 m a.s.l.) on the northwest ﬂank of Mt. Ruapehu, is characterised by low seasonal precipitation variability, with winter (JJA) precipitation averaging 762 mm, compared to 624 mm in summer. Monthly mean temperatures range from c. 13 ◦C in February to c. 3 ◦C in July, with an annual average of 7. 5 ◦C (2000-2010; NIWA, 2014). At 2797 m asl, Mt. Ruapehu is the highest peak in North Island and the only to intercept the modern snowline and several small (< 1 km2) cirque glaciers currently exist on the upper mountain slopes. The average equilibrium line altitude for the ﬁve main cirque glaciers on Mt. Ruapehu is c. 2500 m (Keys, 1988). Brook et al. (2011) show that interannual ﬂuctuations in glacial extent on Mt. Ruapehu are inﬂuenced by El Ni˜no Southern Oscillation (ENSO) and the Interdecadal Paciﬁc Oscillation (IPO). This mirrors the pattern of glacier length changes observed in the Southern Alps, where enhanced southwesterly airﬂow during El Ni˜no years results in lower air temperatures and enhanced precipitation, and thus positive glacier mass balance (Hooker and Fitzharris, 1999). 175 7.2. INTRODUCTION igure 7.1: Hillshade digital elevation model depicting the study domain of this research. White dashed lines indicate the LGCP ice mass delineated by McArthur and Shepherd (1990), ashed black line indicate the inferred LGCP ice limits of Barrell (2011) and the solid black lines elineate the mapped ice limits constrained by the ﬁeld investigations and morphostratigraphi- lly correlated to the LGCP in this study. 7.2.1 Setting 1200 m asl. They suggest the former ice mass existed during the LGCP, based on the presence of the Kawakawa-Oruanui tephra (25.4 ± 0.2 ka; Vandergoes et al., 2013) in moraine- bound glacio-lacustrine deposits on the northeast ﬂank of the volcano. In this chapter, I document the glacial geomorphology of the major catchments on Mt. Ruapehu and Tongariro massif, through detailed ﬁeld and desk-based investigations. 7.2.1 Setting Three-letter catchment labels refer to catchments escribed in text: ’MPO’ = Mangatepopo valley; ’MHO’ = Mangahouhounui valley; ’MTA’ Mangatawai valley; ’UNK’ = unnamed valley; ’WAI’ = Waihohonu valley; ’MTO’ = Man- atoetoenui valley; ’WAH’ = Wahianoa valley; ’MTU’ = Mangaturuturu valley’; ’WHA’ = Whakapapanui valley. Inset map shows location of study domain in central North Island, New ealand. Figure 7.1: Hillshade digital elevation model depicting the study domain of this research. White dashed lines indicate the LGCP ice mass delineated by McArthur and Shepherd (1990), dashed black line indicate the inferred LGCP ice limits of Barrell (2011) and the solid black lines delineate the mapped ice limits constrained by the ﬁeld investigations and morphostratigraphi- cally correlated to the LGCP in this study. Three-letter catchment labels refer to catchments described in text: ’MPO’ = Mangatepopo valley; ’MHO’ = Mangahouhounui valley; ’MTA’ = Mangatawai valley; ’UNK’ = unnamed valley; ’WAI’ = Waihohonu valley; ’MTO’ = Man- gatoetoenui valley; ’WAH’ = Wahianoa valley; ’MTU’ = Mangaturuturu valley’; ’WHA’ = Whakapapanui valley. Inset map shows location of study domain in central North Island, New Zealand. CHAPTER 7. LGM GLACIER MODELLING 176 Geomorphic evidence for past glaciation on the central North Island volcanoes has long been recognised (Taylor, 1927; Mathews, 1967; Topping, 1974; McArthur and Shepherd, 1990). On Tongariro massif, Mathews (1967) identiﬁed large (> 30 m high), lateral moraines in several valleys radiating out from a central former ice source and suggested a correlation with late Pleistocene moraines of the Southern Alps, based on moraine size and the degree of preservation. In Chapter 5, I use cosmogenic 3He exposure dating of moraine boulders in Mangatepopo valley on western Tongariro massif to constrain two main periods of glaciation at > 60 ka and c. 31-21 ka, when the local equilibrium line altitude was depressed by c. 1000 m relative to present. The latter of these periods directly coincides with the Last Glacial Cold Period in New Zealand (Barrell et al., 2013). Identiﬁcation of well-dated volcanic ash layers stratigraphically overlying moraines around the Tongariro massif indicates widespread glacial retreat and soil accumulation occurred in response to warming between c. 17.5 - 14 ka (Top- ping and Kohn, 1973; Cronin and Neall, 1997; Chapter 5). On Mt. Ruapehu, situated 15 km to the south west, McArthur and Shepherd (1990) identify geomorphological evidence for a former ice cap drained by several outlet glaciers that reached c. 7.3.1 Cosmogenic 3He surface exposure dating Moraine boulders in the WAH catchment were sampled using a portable rock saw ﬁtted with a segmented, diamond-tipped blade. All samples were collected from the highest point of the parent boulder, and all boulders were over 1 m tall, to minimise the potential for past burial by snow and/or volcanic ash. Azimuthal inclinations were measured in the ﬁeld using a compass and clinometer and geometric shield- ing corrections were computed using the CRONUS-EARTH calculator (available at: http://hess.ess.washington.edu). All shielding corrections were less than 1 % (Table 7.1). Sample locations and elevations were recorded using a Trimble GeoXH global positioning system, relative to the WGS84 datum. These data were differentially cor- rected using continuous measurements from GeoNet ’Chateau Observatory’ (’VGOB’) base station (39◦11’59” S, 175◦32’ 32”E; 1161 m asl), located 10 km to the north of the sample site. Horizontal and vertical post-processed uncertainties for individual sample 7.3. METHODOLOGY 177 locations are < 1 m. Pyroxene crystals (pigeonite; En60−62) were separated at Victoria University of Wellington according to the protocols outlined in Chapter 3 and prepared for mass spectrometry according to Bromley et al. (2014). Helium measurements were conducted at the LDEO Noble Gas Mass Spectrometry Laboratory using a MAP 215-50 noble gas mass spectrometer. Measurements were made relative to the Yellowstone ’Murdering Mudpot’ (MM) helium standard (3He / 4He ratio of 16.45Ra, where Ra = 3He / 4Heair = 1.384 x 10−6), using the protocol of Winckler et al. (2005). The relatively young crystallisation ages (< 300 ka; Gamble et al., 2003) explains the high 3He / 4He ratios (R) of the samples, which range between 76 - 131 times that of Ra. I therefore assume all 3He to be of cosmogenic origin (sensu Bromley et al., 2011). Attenuation of cosmogenic neutron ﬂux with depth from the surface was corrected for using measured sample thickness, a standard rock density of 2.7 g cm−3 and an attenu- ation length of 160 g cm−2 (Dunne et al., 1999). Field observations from peak winter (July/August) show that the sampled boulders are not subject to burial by seasonal snow (e.g. Figure 7.9b). Although this is not quantiﬁable for the geological past, the topographic prominence of the boulders and moraine crests is not conducive to snow accumulation due to wind exposure in the alpine environment, therefore no correction was applied to the age calculation. CHAPTER 7. LGM GLACIER MODELLING 178 7.3.1 Cosmogenic 3He surface exposure dating The absence of resistant mineral veins in the local andesites precluded quantitative assessment of post-depositional erosion of boulder surfaces. Care was taken to avoid boulders that displayed clear evidence of erosion, such as discolouration, weathering scarps, onion-skin weathering, pitting/water pool- ing, therefore I present exposures ages without an erosion correction. Regional uplift in the central North Island has been estimated at c. 0.6 mm yr−1 for the past 500 ka (Pulford, 2002). Integrating this elevation change into the age calculations does not alter the results outside the range of the 3He measurement uncertainty, therefore I present the age dataset without corrections for this effect. Exposure ages were computed using the cosmogenic 3He exposure age calculator and globally-compiled, sea-level, high-latitude (SLHL) cosmogenic 3He production rate of Goehring et al. (2010), which I conﬁrm for New Zealand in Chapter 4. All calculations are computed using the CRONUS- Earth exposure age calculator MATLAB code, as described by Balco et al. (2008), and modiﬁed for cosmogenic 3He by Goehring et al. (2010). Elevation and latitude scaling of cosmogenic 3He production to the sample locations was calculated using the time dependent model of Lal (1991)/Stone (2000) (Lm), which has been shown to outperform the neutron-monitor based scaling schemes (Lifton et al., 2014). CHAPTER 7. LGM GLACIER MODELLING CHAPTER 7. LGM GLACIER MODELLING 7.3. METHODOLOGY 7.3. METHODOLOGY 179 7.3.3.1 Mass-/energy-balance model Precipitation is partitioned into rain and snow, using a temperature threshold (Ts). Snow accumulation occurs in grid cells when temperature falls below this threshold, set to Ts = 0.5◦C. To simulate ablation, the energy balance equation (Equation 7.1) is solved within a distributed energy balance model (EBM) as developed (Anderson et al., 2010) and previously applied in contemporary- (Anderson and Mackintosh, 2012) and palaeo-glaciological (Doughty et al., 2013) studies in New Zealand. QM = I(1 −α) + L ↓+L ↑+QH + QE + QG + QR (7.1) (7.1) where QM is the energy available for melt, I is incoming shortwave radiation,L ↓is incoming longwave radiation, L ↑is outgoing longwave radiation, QH and QE are sensible and latent heat ﬂuxes respectively, QG is geothermal heat ﬂux and QR is heat input from rain. Subsurface heat exchanges are negated (see Chapter 3). Incoming shortwave radiation (I) comprises both direct and diffuse components (Oer- lemans, 1992). The effect of changing orbital geometry is accounted for using the insolation calculations of Huybers and Eisenman (2006). Albedo (α) is parameterised according to the ELA-dependent scheme of Oerlemans (1992), whereby α increases with elevation and snow thickness, relative to the equilibrium line altitude (zELA = 2483 ± 50 m asl; Keys, 1988). Following Doughty et al. (2013), I use αsnow=0.72 and explore the impact of this parameterisation in sensitivity tests. Longwave ﬂuxes (L↓ , L↑) include the effects of surrounding topography, cloudiness and air temperature (Plummer and Phillips, 2003). Turbulent heat ﬂuxes (QH, QE) are calculated using the bulk method and include the roughness of snow and ice and the Richardson stability criterion (Table 3.3; Oerlemans, 1992; Klok and Oerlemans, 2002; Anderson et al., 2010). Incoming shortwave radiation (I) comprises both direct and diffuse components (Oer- lemans, 1992). The effect of changing orbital geometry is accounted for using the insolation calculations of Huybers and Eisenman (2006). Albedo (α) is parameterised according to the ELA-dependent scheme of Oerlemans (1992), whereby α increases with elevation and snow thickness, relative to the equilibrium line altitude (zELA = 2483 ± 50 m asl; Keys, 1988). Following Doughty et al. (2013), I use αsnow=0.72 and explore the impact of this parameterisation in sensitivity tests. Longwave ﬂuxes (L↓ , L↑) include the effects of surrounding topography, cloudiness and air temperature (Plummer and Phillips, 2003). 7.3.2 Model input data Terrain elevation data comes from the New Zealand School of Surveying Digital Eleva- tion Model (NZSoSDEM) (Columbus et al., 2011) and is resampled to 100 m resolution. An ice mask is created using the ’snow/ice’ data from the Land Information New Zealand NZMS260 map series. This mask is assigned ice thickness values based on the survey of Keys (1988) and is used to subtract contemporary ice masses from the DEM. Climate data for the energy balance and snow accumulation models comes from several different sources. Solar radiation and relative humidity are from the Virtual Cli- mate Station Network gridded datasets, sourced from NIWA CliFlo Database (NIWA, 2014). These datasets are resampled to 100 m resolution using bilinear interpolation. Due to temporal bias and spatial artefacts in the VCSN wind dataset (Anderson and Mackintosh, 2012), present day wind speed data comes from the National Centers for Environmental Prediction (NCEP) 850 hPa level, reanalysis data (1981-2010; Kalnay et al., 1996). This dataset is scaled against observational data and applied uniformly over the model domain. Following Anderson and Mackintosh (2012) and Doughty et al. (2013), I use temperature and precipitation data from individual climate stations distributed around and within the model domain (NIWA, 2014). Monthly temperature grids are created according to Anderson and Mackintosh (2012) and Doughty et al. (2013) using the method described in Chapter 3. A sea-level reference surface (Tr) is created in a horizontal plane (sensu Tait and Zheng, 2007) by normalising point-based station data (Tst) by station elevation (zst), using the empirically-derived, seasonal, upland (> 300 m asl) temperature lapse rate ( dT dz ) of Norton (1985) (Table 3.2). To obtain temperature (T) at the elevation of each grid cell at each grid cell (z), Tr is lapsed back to the elevation determined by the DEM, using the same temperature lapse rate (see Chapter 3). Daily variability is introduced to the monthly temperature data via random selec- tion of a normally distributed perturbation value with mean zero and a standard deviation of 2.5 ◦C (sensu Golledge et al., 2012). Monthly precipitation surfaces are created using individual station data (NIWA, 2014) and a mean annual precipitation surface (Tait et al., 2006), following Anderson and Mackintosh (2012). At each station the monthly proportion of total annual precipitation was interpolated across the model grid and then multiplied by the mean annual precipitation surface. 7.3.3.1 Mass-/energy-balance model Turbulent heat ﬂuxes (QH, QE) are calculated using the bulk method and include the roughness of snow and ice and the Richardson stability criterion (Table 3.3; Oerlemans, 1992; Klok and Oerlemans, 2002; Anderson et al., 2010). Raised geothermal heat ﬂuxes (QG) due to active volcanism, have the potential to contribute signiﬁcantly to the ablation of snow and ice in the study site, both today and during the LGCP. However, as such ﬂuxes can vary greatly over relatively short temporal and spatial scales, and are unknown for the geological past, it is difﬁcult to accurately parameterise in the model. I use a nominal value of QG = 1 W m −2 applied uniformly over the model domain (see Chapter 3 for further discussion of geothermal heat ﬂuxes). I do not include surface debris cover in the simulations, also because it is unknown for the LGCP. Historically, debris cover on the glaciers situated on Mt. Ruapehu has varied in space and time. During the most recent volcanic eruptions (AD CHAPTER 7. LGM GLACIER MODELLING 180 1995-96), all glaciers became buried by volcanic products. However, this is quickly incor- porated and presently only ice bodies with a low surface slope and those situated close to the current volcanic vent, such as the Summit Plateau and the upper Whangaehu Glacier, remain partially debris-covered. Elsewhere on the mountain, steeper glaciers such as Mangatoetoenui and Mangaehuehu now have greatly reduced surface debris cover, relative to the immediate aftermath of the eruptions. This evidence accords with studies in Iceland, which suggest that ﬁne-grained debris cover emplaced via volcanic eruptions in maritime regions is highly transient (Kirkbride and Dugmore, 2001). I acknowledge the potential for past geothermal heat cover and debris cover as sources of uncertainty in the simulations and provide further consideration of the speciﬁc impacts that these phenomena may have on the palaeoclimatic reconstructions in the Discussion section (below). 7.3.3.2 Ice ﬂow model Ice ﬂow is described using a vertically-integrated, two-dimensional (2D) model based on the shallow ice approximation (SIA) (Plummer and Phillips, 2003; Kessler et al., 2006). This formulation assumes ice ﬂow is driven by vertical shear stresses, therefore compressional and tensional (longitudinal) stresses are neglected (Hutter, 1983). I consider that the role of longitudinal stresses on past glacial ﬂow in the glacial troughs studied here would be low, owing to the low-angle bed slopes and absence of steep, bounding valley sides that characterise typical alpine glacier environments. Further- more, several comparison studies between SIA and higher order ice ﬂow models show little difference in steady-state ice geometries (e.g. Le Meur et al., 2004; Leysinger Vieli and Gudmundsson, 2004). Thus, the SIA is commonly applied in mountain glacier environments for palaeoclimatic reconstructions, where mass balance imparts greatest uncertainty (e.g. Plummer and Phillips, 2003; Kessler et al., 2006; Doughty et al., 2013). Ice ﬂow velocity due to internal deformation (Ud) is given as: Ice ﬂow velocity due to internal deformation (Ud) is given as: Ice ﬂow velocity due to internal deformation (Ud) is given as: ⃗Ud = 2 5AH⃗τ n b (7.2) (7.2) where ⃗Ud is vertically-averaged ice velocity from internal deformation, A is Glen’s ﬂow law coefﬁcient, set to A = 2.14 x 10−16 Pa−3 yr−1, ⃗τb is the gravitational driving stress (⃗τb = ρgH∇z), and n is Glen’s ﬂow law exponent, set to n = 3 (Cuffey and Paterson, 2010). Following Plummer and Phillips (2003) and Kessler et al. (2006), a sliding term is Following Plummer and Phillips (2003) and Kessler et al. (2006), a sliding term is 7.3. METHODOLOGY 181 METHODOLOGY also included: also included: Us = Uce 1−τc ⃗τb (7.3) (7.3) where Uc is the characteristic sliding velocity, set to Uc = 50 m yr−1, and τc is the gravita- tional driving stress, set to τc = 100 kPa. where Uc is the characteristic sliding velocity, set to Uc = 50 m yr−1, and τc is the gravita- tional driving stress, set to τc = 100 kPa. dH dt = M −∇· ⃗q (7.4) dH dt = M −∇· ⃗q (7.4) (7.4) where H is ice thickness, t is time, ⃗q is ice ﬂux (⃗Ud + ⃗Us) and M is mass balance. where H is ice thickness, t is time, ⃗q is ice ﬂux (⃗Ud + ⃗Us) and M is mass balance. Equation 7.4 evolves glacier geometry through time. 7.3.3.2 Ice ﬂow model Ice velocities are calculated on a grid offset from ice thickness and the ﬂux gradients are used to update ice thickness using a forward explicit time-step (Hindmarsh and Le Meur, 2001). To account for boundary effects that may violate mass conservation in the ﬁnite difference formulation (e.g. Plummer and Phillips, 2003), the bed topography is smoothed using a 300 x 300 m moving window to reduce high bed slopes in minor portions of the upper catchment, and an ice ﬂux correction is applied. For each cell, total ice divergence cannot exceed the total mass contained in the source cell. At each timestep it was ensured that this criterion was met and if not the excess ice was removed (Plummer and Phillips, 2003). 7.3.4 Sensitivity tests To test the sensitivity of the palaeotemperature estimates to model parameter choices, I quantify the deviation of ∆T from the optimal set up, in response to systematic varia- tions applied to several key parameters. Model-based assessments of contemporary glacier mass balance in New Zealand have shown that turbulent ﬂuxes contribute a signiﬁcant proportion of the total energy available for melt (Anderson et al., 2010). In the optimal parameterisation, ice roughness length (zice) is derived from tuning experiments over a domain in the central Southern Alps (Anderson and Mackintosh, 2012), thus I include this parameter in the sensitivity tests. Furthermore, glacier mass balance in this maritime setting is highly sensitive to temperature, due to its control on precipitation phase and strong relationship with several components of the sur- face energy balance (e.g. longwave and turbulent ﬂuxes). Thus, I test the impact of temperature-related parameters such as the lapse rate ( dT dz ) and snow/rain threshold (Ts = 0 - 1.5 ◦C). I also constrain palaeotemperature sensitivity to albedo (αsnow = 0.67-0.77), which limits shortwave radiation. In the ﬂow model, I assess the sensitivity of the characteristic sliding velocity (Uc = 20 - 80 m yr−1) and Glen’s ﬂow law coefﬁcient (A = CHAPTER 7. LGM GLACIER MODELLING 182 1 x 10−15 - 1 x 10−18 Pa−3 yr−1), which principally control ice thickness and can affect the ﬁt to the geologically inferred former ice limits. 1 x 10−15 - 1 x 10−18 Pa−3 yr−1), which principally control ice thickness and can affect the ﬁt to the geologically inferred former ice limits. 7.3.5 Approach The aim of this work is provide a quantitative estimate of temperature change, relative to present day, for the LGCP in central North Island. To do this, I use the glacier model described above to constrain the climatic forcing required to simulate the geologically- constrained (Section 7.2.1) former glacial geometries in the catchments on Mt. Ruapehu (WHA, WAH, MTO and MTU) and Tongariro massif (MPO, MHO, MTA and WAI). This approach overcomes some of the uncertainties in manual ELA reconstruction (e.g. Chapter 5), as ice thickness and distribution is simulated based on the physical principles and interrelation of glacier mass balance and ice ﬂow and does not require assumptions of past accumulation area ratios. Thus, this approach removes the uncer- tainty associated with manual interpolation of ice surfaces in area with few geomorphic constraints. Applying this objective methodology across several catchments permits critical assessment of the possible drivers (e.g. topographic, climatic) of variability in the palaeoclimatic reconstructions. Furthermore, uncertainty related to parameter choice can be quantiﬁed in sensitivity tests. Chronological constraint comes from the cosmogenic surface exposure dating of moraines in the MPO (Chapter 5) and WAH catchments, which have been extrapolated to the current study domain using a morphostratigraphic approach. For example, in the MPO catchment the moraines of LGCP age stand higher above the valley ﬂoor and exhibit steeper slopes and more pronounced ridge crests, in comparison to older (c. 60 ka) moraines. Furthermore, in Chapter 5, I present chronological evidence for post-LGCP moraine formation on Mt. Ruapehu, of restricted extent in comparison to the LGCP limits. Recognition of this late-glacial glacier still-stand has also guided the relative correlation of glacial landforms. The good agreement between multiple, independent mapping of LGCP ice limits in this study domain (e.g. McArthur and Shepherd, 1990; Barrell, 2011, this study) also suggests that the interpretations made here are robust. However, it is possible the mapped former glacier limits are not contemporaneous. For example, multiple continuous climate proxy reconstructions show the prevailing stadial conditions were interrupted by prominent interstadials at 25.4 - 24.5 ka and 22.6 - 21.7 ka (Barrell et al., 2013). It is probable that the glaciers on the central North Island volcanoes would have ﬂuctuated in response to these stadial-interstadial transi- tions therefore the targeted moraines could pertain to separate stadial events during the LGCP. For example, several catchments (e.g. WHA, WAH, MTU, MPO) exhibit 7.3. 7.3.5 Approach METHODOLOGY 183 multiple moraines that depict glacier ﬂuctuations of very similar extent, which I have correlated to the LGCP. In these instances, I simulate the glacier geometry depicted by the outermost LGCP moraine, thereby targeting the peak magnitude of cooling during the LGCP (sensu Golledge et al., 2012). In the following section, I outline the 3 glacier modelling experiments undertaken in this study. 7.3.5.2 Experiment 2 Second, I run steady-state simulations to constrain the combinations of ∆T and ∆P, that produce ice extents that best ﬁt the geological evidence in each individual catchment. Temperature forcing results are presented for three precipitation scenarios, whereby modern precipitation is varied by +25 %, -25 % and 0 % and I discuss the implications for these scenarios below. For efﬁciency, these simulations are run over a smaller domain than for Experiment 1, with a resolution of 100 m. The domains cover the entirety of each individual volcano, in order to capture the potential effects of changing ice-divides that may result from a growing ice mass. Resultant steady-state ice extents were manually compared to the geomorphic constraints (e.g. Figure 7.4 and Figure 7.5). A satisfactory result was considered to have been obtained when the modelled glacier terminus reached within 1 grid cell (100 m) of the downstream limit of the LGCP ice mass, as inferred from geomorphology mapping (above). In some simulations ice spills over the lateral moraines before the glacier terminus reaches the inferred former limit. I discuss the implications and possible reasons for this, below (Section 7.6.2). 7.3.5.1 Experiment 1 First, I carry out an assessment of the patterns of ice growth that result from incremental reductions in temperature over a 30 x 30 km domain (100 m resolution) that includes both Mt. Ruapehu and Tongariro massif. Step-coolings from present (∆T) of -2 to -7 ◦C, at intervals of 1 ◦C, are applied uniformly across the domain and the resultant ice masses are allowed to evolve to steady-state. Equilibrium is achieved when ice volume change (dV/dt) becomes zero, which takes 200-350 model years depending on the magnitude of ∆T. These experiments permit an initial assessment of the patterns of ice growth across both volcanoes and the results will guide the catchment speciﬁc simulations carried out in Experiments 2 and 3 (described below). 7.3.5.3 Experiment 3 Third, I repeat Experiment 2, but use modiﬁed topographic boundary conditions, in order to assess the sensitivity of ∆T to post-glacial topographic change. These simula- tions are run over altered digital elevation models considered representative of LGCP CHAPTER 7. LGM GLACIER MODELLING 184 topography on both volcanoes (Figure 7.2). These surfaces have been reconstructed using the results of a 5-year project to constrain the spatial and temporal geomorphic evolution of this region. For example, compilation of existing mapping (e.g. Gamble et al., 2003; Townsend et al., 2008; Price et al., 2012), together with extensive primary ﬁeld mapping has been used to produce 1:60000 scale maps of the volcanic geology, which delineate individual lava ﬂows. Furthermore, a suite of new, high-precision Ar-Ar ages (Conway et al. in prep), together with existing chronological data (e.g. Gam- ble et al., 2003) and geochemical correlation using extensive documentation of major elements, has broadly constrained the timing of lava emplacement across the domain. To generate palaeo-topographies for these simulations, modern DEMs are manually altered to subtract the convex relief created by post-glacial (< 15 ka), effusive volcanic activity (Figure 7.2). Manual alteration of contour lines was undertaken by experts working at GNS Science who have led the mapping project in this region (D.Townsend, pers. comm.). topography on both volcanoes (Figure 7.2). These surfaces have been reconstructed using the results of a 5-year project to constrain the spatial and temporal geomorphic evolution of this region. For example, compilation of existing mapping (e.g. Gamble et al., 2003; Townsend et al., 2008; Price et al., 2012), together with extensive primary ﬁeld mapping has been used to produce 1:60000 scale maps of the volcanic geology, which delineate individual lava ﬂows. Furthermore, a suite of new, high-precision Ar-Ar ages (Conway et al. in prep), together with existing chronological data (e.g. Gam- ble et al., 2003) and geochemical correlation using extensive documentation of major elements, has broadly constrained the timing of lava emplacement across the domain. To generate palaeo-topographies for these simulations, modern DEMs are manually altered to subtract the convex relief created by post-glacial (< 15 ka), effusive volcanic activity (Figure 7.2). Manual alteration of contour lines was undertaken by experts working at GNS Science who have led the mapping project in this region (D.Townsend, pers. comm.). 7.3.5.3 Experiment 3 5 ka topography, displayed as a difference (in metres) from modern topographic bo u (1 = Whakapapa ﬂows (Hackett and Houghton, 1989); 2 = Holocene ﬂows in ﬂow(s); 5-6 = LGCP-Holocene age ﬂows in and adjacent to Mangatoetoenui), = Tama Lakes explosion craters; 4 = Oturere ﬂows). Blues = surface lowering (n played as a difference (in metres) from modern topographic b ﬂows (Hackett and Houghton, 1989); 2 = Holocene ﬂows i CP-Holocene age ﬂows in and adjacent to Mangatoetoenui) osion craters; 4 = Oturere ﬂows). Blues = surface lowering ( ﬂows ( P-Hol sion c ka topography, di (1 = Whakapapa ﬂow(s); 5-6 = LG = Tama Lakes exp kness (in metres) and extent on Mt. Ruapehu and Tongariro massif resulting from unchanged. Solid and dashed lines represent geologically constrained LGCP ice lim Barrell, 2011). See text for catchment labels. d Tongariro massif resulting fro ogically constrained LGCP ice l res) and extent on Mt. Ruapehu Solid and dashed lines represent ). See text for catchment labels. kness (in me unchanged. Barrell, 201 m present, necessary to simulate inferred LGCP ice geometries in catchments on Mt. Ruap tion change (∆P) scenarios: 0 % (black); +25 % (blue); and -25 % (red) change from pres ere modelled ice thickness spills over ice-marginal landforms, before reaching the geologic ght grey shading depict the pollen-based southern North Island LGCP temperature lower rived using the partial least squares method. Dashed black lines and dark grey shading dep estimate of Golledge et al. (2012) (-6.0 to -6.5 ◦C, when precipitation is reduced by 25 %). r the following parameters: albedo of snow (αsnow), snow temperature threshold (Tsnow), ss (Zice). Dashed lines indicate the mean impact of each sensitivity test. Flow parameters n ∆T. ferred LGCP ice geometries in catchments on Mt. Ruap black); +25 % (blue); and -25 % (red) change from pres ver ice-marginal landforms, before reaching the geologic -based southern North Island LGCP temperature lower es method. Dashed black lines and dark grey shading de -6.0 to -6.5 ◦C, when precipitation is reduced by 25 %) o of snow (αsnow), snow temperature threshold (Tsnow) e mean impact of each sensitivity test. Flow parameter present, necessary to simulate i n change (∆P) scenarios: 0 % e modelled ice thickness spills o grey shading depict the pollen ed using the partial least squar timate of Golledge et al. (2012) he following parameters: albed (Zice). 7.3.5.3 Experiment 3 Dashed lines indicate t ∆T. CHAPTER 7. LGM GLACIER MODELLING 188 Figure 7.5: (a) Modelled, steady-state ice geometry on Mt. Ruapehu when ∆T = -5.2 ◦C and ∆P = 0 (Experiment 2). This represents the best-ﬁt simulation for the WHA catchment; (b) Steady state ice geometry on Tongariro massif when ∆T = -6.7 ◦C and ∆P = 0 (Experiment 2). This is the best ﬁt to the inferred LGCP terminus in the WAI catchment, however note the ice overspill at the lateral margins; (c) Steady state ice geometry on Mt. Ruapehu when ∆T = -5.4 ◦C and ∆P = 0 (Experiment 2). Note the ice overspill in the MTO catchment, despite agreement between the inferred and modelled glacier terminus positions. (d) Steady state ice geometry on Mt. Ruapehu when ∆T = -5.2 ◦C and ∆P = 0 (Experiment 3). Note the improved ﬁt between the inferred and modelled glacier margins on the northern side of the MTO catchment, relative to panel (c). Figure 7.5: (a) Modelled, steady-state ice geometry on Mt. Ruapehu when ∆T = -5.2 ◦C and ∆P = 0 (Experiment 2). This represents the best-ﬁt simulation for the WHA catchment; (b) Steady state ice geometry on Tongariro massif when ∆T = -6.7 ◦C and ∆P = 0 (Experiment 2). This is the best ﬁt to the inferred LGCP terminus in the WAI catchment, however note the ice overspill at the lateral margins; (c) Steady state ice geometry on Mt. Ruapehu when ∆T = -5.4 ◦C and ∆P = 0 (Experiment 2). Note the ice overspill in the MTO catchment, despite agreement between the inferred and modelled glacier terminus positions. (d) Steady state ice geometry on Mt. Ruapehu when ∆T = -5.2 ◦C and ∆P = 0 (Experiment 3). Note the improved ﬁt between the inferred and modelled glacier margins on the northern side of the MTO catchment, relative to panel (c). m present (∆Texperiment3), necessary to simulate inferred LGM ice geometries in catchm iﬁed, pre-15 ka topography. Results shown for three precipitation change (∆P) scena present. Underlined labels on the y-axis represent catchment where modelled ice thick geologically inferred terminus (see text). Vertical black lines and light grey shading d e estimate of Newnham et al. (2013) (6.5 ± 2.0 ◦C) - derived using the partial least squ ict the glacier model derived Southern Alps LGCP temperature estimate of Golledge 5%). 7.3.5.3 Experiment 3 (b) The difference in ∆T between model simulations in Experiment 3 (∆Texperim sary to simulate inferred LGM ice geometries in catchm sults shown for three precipitation change (∆P) scen he y-axis represent catchment where modelled ice thic see text). Vertical black lines and light grey shading d 13) (6.5 ± 2.0 ◦C) - derived using the partial least sq uthern Alps LGCP temperature estimate of Golledge etween model simulations in Experiment 3 (∆Texperim present (∆Texperiment3), ne ﬁed, pre-15 ka topography. resent. Underlined labels on eologically inferred terminu stimate of Newnham et al. t the glacier model derived %). (b) The difference in ∆T CHAPTER 7. LGM GLACIER MODELLING 190 7.4.1 Whakapapaiti valley (’WHA’) Whakapapaiti valley drains towards the northwest from Paretetaitonga peak (2751 m asl) on Mt. Ruapehu (Figure 7.7). The lower valley is 2-3 km wide and is bound by large (c. 50 m high) lateral moraines on both ﬂanks. On the northeastern valley side, a lateral moraine extends for 1.5 km from a prominent lava outcrop at c. 1550 m asl. Further down valley, at c. 1400 m asl, this moraine displays two clear crests, which indicates that this large feature possibly represents a compound landform formed during multiple periods of glacial occupation. Both crests are relatively sharp, which suggests they are of similar age and most likely formed during the last period of major glaciation on Mt. Ruapehu. This interpretation is further supported by the coverbed stratigraphy of the inboard lateral moraine on the true right of the valley (Figure 7.7c). At this location, the Waiohau Tephra (14.0 ± 0.2 ka; Lowe et al., 2013) is prominent c. 80 cm above the moraine surface. The soil proﬁle shows strong similarity to that in the Mangaetepopo valley (Chapter 5), with interbedded, coarse andesitic tephras (4.5 - 3.0 m depth) overlain by a c. 1 m palaeosol and the Taupo pumice (Unit Y; AD 232 ± 10 a; Lowe et al., 2013) near the top of the sequence. Based on their similar morphology and coverbed stratigraphy, I suggest these moraines correlate to the M1 and M2 moraines in the Mangatepopo valley (Chapter 5), which formed between 31-21 ka. On the southwestern side of Whakapapaiti valley, a prominent, but discontinuous lateral moraine marks the outermost limit of glaciation in this catchment. Based on the geometric and topographic similarity with the continuous moraine on the northeastern valley side, I suggest this landform delineates the outermost limit of glaciation during the LGCP. Low-relief, moraine crests on the southeastern valley side, mark minor stillstands during deglaciation (Figure 7.7). of the Whakapapaiti valley; (b) Oblique aerial photo of the Whakapapaiti valley (source: D. Townsend). atigraphically deﬁned LGCP ice limit in the Whakapapaiti valley. Red dashed lines indicate small lateral lstands during deglaciation; (c) Stratigraphic logs of soil proﬁles from moraines in Mangatepopo and ed using rhyolitic tephras. the Whakapapaiti valley; (b) Oblique aerial photo of the Whakapapaiti valley (source: D. Townsend). tigraphically deﬁned LGCP ice limit in the Whakapapaiti valley. 7.4.2 Mangaturuturu valley (’MTU’) The head of this southwestward-draining catchment is occupied by the small (< 0.5 km2) Mangaturuturu cirque glacier, which lies between Paretetaitonga and Tahurangi peaks on the western side of the summit of Mt. Ruapehu (Figure 7.8). At its head, the Mangaturuturu valley is c. 2 km wide and narrows as it descends in a southwest direction for c. 8 km to the inferred LGCP glacial limits. Due to the dense forest cover in the lower portion of this valley, the LGCP limits have been delineated predominantly using aerial imagery and digital elevation models. Glacial till cover is characterised by hummocky topography, often arranged in discontinuous linear ridges, orientated parallel, to sub-parallel with the valley axis. Relatively sharp-crested, but discontinuous lateral moraines are easily depicted in the remotely sensed datasets and provide good constraint on the former ice surface and lateral extent of the LGCP Mangaturuturu outlet glacier (Figure 7.8). Post-glacial ﬂuvial erosion has removed any evidence of the former terminus location, however, projecting the lateral moraines downstream sug- gests that the most extensive former glaciation in this catchment did not descend below c. 1050 m asl. Downstream of c. 1100 m asl, the land surface texture becomes more smooth and grades linearly downslope to the west. This topography is characteristic of ﬂuvial topography and likely comprises interbedded alluvium, volcaniclastic and glacial outwash. The upper catchment is dominated by exposed bedrock outcrops, which commonly display glacially-polished surfaces, particularly above c. 1700 m asl. Post-glacial ﬂu- vial incision has created a deep (c. 50 m) bedrock gorge on the northern valley side. Between 1300-1500 m asl, glacial till overlies thick (c. 10 m) lava ﬂows that outcrop to the north. These deposits, which are situated approximately mid-way between the inferred LGCP limits and and the present ice margin, likely represent a post-LGCP ice extent and have been correlated to the late glacial deposits of the nearby Mangaehuehu catchment (Chapter 6). However, the lack of discernable ridge crests within these deposits precludes precise delineation of the former ice mass at this location. 7.4.1 Whakapapaiti valley (’WHA’) Red dashed lines indicate small lateral stands during deglaciation; (c) Stratigraphic logs of soil proﬁles from moraines in Mangatepopo and d using rhyolitic tephras. Whakapapaiti valley; (b) Oblique aerial photo of the Whakapapaiti valley (source: D. Townsend). of the Whakapapaiti valley; (b) Oblique aerial photo of the Whakapapaiti valley (source: D. Townsend). atigraphically deﬁned LGCP ice limit in the Whakapapaiti valley. Red dashed lines indicate small lateral lstands during deglaciation; (c) Stratigraphic logs of soil proﬁles from moraines in Mangatepopo and al photo of the Whakapapaiti valley (source: D. Townsend). aiti valley; (b) Oblique aerial photo of the Whakapapaiti valley (source: D. Townsend). lly deﬁned LGCP ice limit in the Whakapapaiti valley. Red dashed lines indicate small lateral ring deglaciation; (c) Stratigraphic logs of soil proﬁles from moraines in Mangatepopo and hyolitic tephras. ateral o and eﬁned LGCP ice limit in the Whakapapaiti valley. Red dashed lines indicate small latera deglaciation; (c) Stratigraphic logs of soil proﬁles from moraines in Mangatepopo and tic tephras. f the Whakapa tigraphically d stands during ed using rhyol f the Whakapa atigraphically d lstands during ed using rhyol CHAPTER 7. LGM GLACIER MODELLING 192 7.4.3 Wahianoa valley (’WAH’) Wahianoa valley is a deeply incised, c. 7 km long, glacial valley that drains towards the southeast from the highest peak of Mt. Ruapehu (Tahurangi peak, 2797 m asl). Today, small patches of glacial ice, which are relatively free of supraglacial debris, persist on the uppermost slopes at the valley head. Multiple, boulder-rich lateral moraine crests line both ﬂanks of the lower 3 km of the valley to c. 1200 m asl, recording glacier ﬂuctuations during the most extensive period of glaciation preserved in this catchment. 7.4. GEOMORPHOLOGICAL CHARACTERISATION 193 Figure 7.8: (a) A glacial geological map of the Mangaturuturu valley; (b) Oblique aerial photo of the Mangaturuturu Valley (source: D. Townsend). White dashed line indicates the morphostratigraphically deﬁned LGCP ice limit in the Mangaturuturu valley. Figure 7.8: (a) A glacial geological map of the Mangaturuturu valley; (b) Oblique aerial photo of the Mangaturuturu Valley (source: D. Townsend). White dashed line indicates the morphostratigraphically deﬁned LGCP ice limit in the Mangaturuturu valley. CHAPTER 7. LGM GLACIER MODELLING 194 On the true-left valley side, the morphostratigraphic relationship of these individual ridges indicates at least two moraine-building events of similar extent. Figure 7.9 shows the outermost moraine on the true-left is bisected by a deep gully that has incised c. 20 m into the lateral moraine, which I interpret to represent glacioﬂuvial erosion from supraglacial meltwater runoff whilst the Wahianoa Glacier remained close to its maximum extent. Subsequent to this gully forming, a separate lateral moraine ridge was deposited, across the head of the outbreak gully. This inboard moraine was deposited by a slightly smaller glacier, which appears to have terminated c. 1 km up-valley from the valley mouth. This interpretation is based on the longitudinal angle of the moraine crest, which appears to dip more steeply than the outer moraines formed by a more extensive glacier. All of these moraines display sharp crests and steep lateral slopes, thus I interpret them to be of similar age, which record relatively minor glacier ﬂuctuations during the LGCP. On the lower true-right (south-western) valley side, a single, continuous lateral moraine extends from the valley mouth to approximately 3 km up-valley. The crest of this moraine stands 100-150 m above the valley ﬂoor and is punctuated by several southward- draining gullies that have incised since moraine emplacement. 7.4.3 Wahianoa valley (’WAH’) In contrast to the true left ﬂank of Wahianoa valley (described above), on the true right there is little discern- able structure or stratigraphy to suggest multiple moraine building events. Given the clear evidence for ﬂuctuating ice margins on the true left, the true right is interpreted to represent a compound morainic feature formed by multiple periods of occupation during, and possibly before, the LGCP. Ice-scoured bedrock exposures with parallel striae are preserved on the upper true right hand side of the valley (Figure 7.9; Figure 7.10), which indicates the former presence of a temperature glacier. CHAPTER 7. LGM GLACIER MODELLING 7.4.4 Mangatoetoenui valley (’MTO’) The Mangatoetoenui valley descends towards the northeast from between Te Heuheu (2732 m asl) and Tukino (2720 m asl) peaks on Mt. Ruapehu (Figure 7.11). At the head of this catchment, the Mangatoetoenui Glacier persists in a south-facing hollow and terminates at c. 2350 m asl. A series of sharp-crested, nested lateral moraines extend from the current elevation of the glacier terminus to c. 1900 m asl, which likely represent late Holocene - historical glacier positions. Further down valley, several prominent lava ﬂows are present on the valley ﬂoor between 1300 - 1900 m asl, in this catchment. It is likely that these were emplaced 7.4. GEOMORPHOLOGICAL CHARACTERISATION 195 Figure 7.9: (a) A glacial geological map and cosmogenic 3He surface exposure ages of the Wahianoa valley; (b) Oblique aerial photo of the Wahianoa Valley taken in August 2010 (Source: D. Townsend). White dashed line indicates the morphostratigraphically Figure 7.9: (a) A glacial geological map and cosmogenic 3He surface exposure ages of the Wahianoa valley; (b) Oblique aerial photo of the Wahianoa Valley taken in August 2010 (Source: D. Townsend). White dashed line indicates the morphostratigraphically deﬁned LGCP ice limit in the Wahianoa valley; red dashed line marks inboard LGCP moraine described in text; orange dashed lines mark the inferred late-glacial moraines (Chapter 6). CHAPTER 7. LGM GLACIER MODELLING 196 Figure 7.10: One of several exposures of ice-scoured, striated bedrock on the upper true-right hand side of Wahianoa valley. Compass is orientated parallel to striae, which point down-valley (Wahianoa valley in photo background, with prominent left lateral moraines) Figure 7.10: One of several exposures of ice-scoured, striated bedrock on the upper true-right hand side of Wahianoa valley. Compass is orientated parallel to striae, which point down-valley (Wahianoa valley in photo background, with prominent left lateral moraines) 7.4. GEOMORPHOLOGICAL CHARACTERISATION 197 Figure 7.11: (a) The glacial geology of Mangatoetoenui valley (MTO) on northeastern side of Mt. Ruapehu; (b) An oblique aerial photograph of lower Mangatoetoenui valley (Source:D.Townsend). White dashed line indicates the morphostratigraphically deﬁned LGCP ice limit. White star indicates the location of glaciolacustrine sediments decribed by McArthur and Shepherd (1990). when ice ﬁlled the valley which caused this overthickening (Conway et al in prep) Figure 7.11: (a) The glacial geology of Mangatoetoenui valley (MTO) on northeastern side of Mt. Ruapehu; (b) An oblique aerial photograph of lower Mangatoetoenui valley (Source:D.Townsend). White dashed line indicates the morphostratigraphically deﬁned LGCP ice limit. 7.4.4 Mangatoetoenui valley (’MTO’) White star indicates the location of glaciolacustrine sediments decribed by McArthur and Shepherd (1990). when ice ﬁlled the valley, which caused this overthickening (Conway et al., in prep). This interpretation is supported by radiometric dating (Gamble et al., 2003, C.Conway, 198 CHAPTER 7. LGM GLACIER MODELLING pers. com.) and the presence of ice-contact textures, which have been described in lava outcrops in the upper Mangatoetoenui catchment (Sp¨orli and Rowland, 2006). These thick ﬂows are overlain by glacial till of uncertain age. The maximum extent of former glaciation in the Mangatoetoenui catchment is well- approximated by till distribution and valley shape. Discontinuous lateral moraines and till cover trace both valley sides down-valley to c. 1230 m asl. At this location the glacial limit is clearly deﬁned by a well-preserved terminal moraine, which loops south- wards from the northern valley side. Upstream, the lateral moraines are diffuse and provide approximate constraint of the former lateral margins of the Mangatoetoenui Glacier. For example, large, complex till accumulations, between 1400-1600 m asl on the southern valley side, possibly attest to former interaction between outlet glaciers of the Mangatoetoenui and Whangaehu catchments. 7.4.5 Mangatepopo Valley (’MPO’) Mangatepopo valley is a westward draining catchment on Tongariro massif, which is sourced from South Crater, a glacial cirque in the centre of the volcanic massif. A full description of the glacial geomorphology and chronology of this site is given in Chapter 5, therefore I only provide a summary here. On the northern ﬂank of the upper valley, a continuous moraine ridge was deposited early in the LGCP (c. 31 - 23 ka). Prominent, paired lateral moraines converge at the valley mouth (c. 1200 m asl), which record the ﬁnal LGCP stand of glaciation in this valley at 21 ± 2 ka. The former equilibrium line altitude of this glacier was likely between 1400-1550 m asl, which represents a lowering of c. 900-1100 m from present. Post-glacial volcanic activity, such as the development of present-day Mt. Ngauruhoe cone during the Holocene (Hobden et al., 2002; Moebis et al., 2011) may have signiﬁcantly altered the drainage in the upper Mangatepopo catchment, as well as raising the elevation of valley ﬂoor through aggradation of lava and pyroclastic deposits. 7.4.6 Eastern Tongariro massif glacial geology of eastern Tongariro massif. glacial geology of eastern Tongariro massif. lacia CHAPTER 7. LGM GLACIER MODELLING 200 Several east- to southeastward draining valleys on the eastern ﬂanks on the Tongariro massif exhibit clear evidence for past glacial activity (Figure 7.12). To the north, the Mangahouhounui (’MHO’) valley rises below Rotopaunga peak (1856 m asl) in an east- facing glacial cirque. This c. 2 km wide, former accumulation area narrows down valley and lateral moraines on both valley sides converge at c. 1100 m asl. Possible till cover continues below this, however vegetation cover and the absence of clear topographic structure means that this remains unclear. The diffuse nature of these lowermost de- posits means that they may correlate with the MIS 4 moraine in the Mangatepopo valley (Chapter 5). Immediately to the south of Mangahouhounui valley, is the southeastward draining Mangatawai Stream (’MTA’; Figure 7.12). Clearly deﬁned, continuous lateral moraines line the sides of this underﬁt stream and converge at c. 1100 m asl. The absence of an obvious accumulation area for this former glacier suggests that it was either sourced from an ice ﬁeld/cap over central Tongariro massif, or was an overﬂow outlet of a glacier in the adjacent Oturere valley (’OTU’; Figure 7.12) to the south. It is likely that the Oturere valley was occupied by a valley/outlet glacier during the LGCP, however, post-depositional inﬁlling by lava has obscured the glacial geomorphology in this catchment. The lateral distribution of this valley-ﬁlling lava ﬂow provides a clear indication of the planimetric geometry of the former glacial trough. The downstream limit of the ﬂow coincides with the 1100 m contour. Downstream of this point the Oturere Stream is deeply incised. At the valley margins, glacial till is discontinuous and remnant lateral moraines are diffuse and ill-deﬁned, thus the age of these land- forms remains highly uncertain. Well-preserved lateral moraines are present in small unnamed (’UNK’; Figure 7.12) trough immediately to the south of Oturere. Similar to the Mangatawai Stream catchment, these sharp-crested moraines likely represent the former extent of a small lobe of ice that spilled over from the Oturere catchment. The moraines at this location clearly delineate the former glacier that existed here and terminated at c. 1350 m asl. The Waihohonu valley (’WAI’; Figure 7.12) drains towards the southeast from the Holocene-age volcanic cone of Mt. Ngauruhoe. Large lateral moraines line both valley sides. 7.4.6 Eastern Tongariro massif These are cross-cut by several NE-SW trending faults, which have created vertical offsets in the moraines of up to several metres. A highly diffuse outboard moraine, present on the southwest side of the valley, may correlate to the c. 60 ka moraine of MPO catchment, thus I correlate the better preserved, prominant inboard moraine to the LGCP. This 1.5 - 2 km wide glacial trough has been partially inﬁlled at the head by post-glacial lava and block-and-ash ﬂows from Mt. Ngauruhoe, which may have removed evidence for less extensive ice stands. Similarly, creation of the modern cone of Mt. Ngauruhoe has obscured evidence that delineates the accumulation zone of the former Waihohonu Glacier. Although, the orientation and size of this glacial valley 201 7.5. RESULTS suggests that considerable relief or an ice ﬁeld/cap must have persisted in that general region, in order to have supplied the former valley glacier. suggests that considerable relief or an ice ﬁeld/cap must have persisted in that general region, in order to have supplied the former valley glacier. a External uncertainty shown in parentheses. External uncertainty shown in parentheses. 7.5.2.1 Experiment 1 Figure 7.3 shows steady-state ice thickness results of Experiment 1, conducted over a domain covering both Mt. Ruapehu and Tongariro massif. Also shown are the inferred LGCP ice limits of greater and lesser conﬁdence (Barrell, 2011, modiﬁed by this study). Figure 7.3 shows that modest coolings of 2 - 3 ◦C from present are sufﬁcient to produce a small ice mass on Mt. Ruapehu (see also, Chapter 6), but this temperature change is insufﬁcient to promote signiﬁcant ice accumulation on Tongariro massif. A cooling of 4 ◦C is sufﬁcient to meet the well-deﬁned LGCP limits in the WAH catchment on southeast Ruapehu, however, the termini of other valley glaciers on this volcano remain well upstream of their mapped limits. Meanwhile in this scenario, ice accumulation on Tongariro is restricted to elevations > c. 1900 m asl, therefore remains well short of the mapped LGCP limits. Modelled ice extent approaches the LGCP limits in the remaining three catchments (MTO, WHA, MTU) on Mt. Ruapehu when ∆T = -5 ◦C and these limits are exceeded in all catchments at ∆T = -6 ◦C. On Tongariro massif, modelled ice extent is reaching the geological limits in several catchments (MPO, MHO, UNK) in response to a cooling of 6 ◦C. At ∆T = -7 ◦C, the individual ice masses have merged and extent of this single ice mass exceeds the LGCP limits in all catchments in the domain. In summary, this experiment shows that all mapped LGCP limits can be attained in model simulations with temperature forcings that range between c. -4 ◦C and -7 ◦C, when precipitation remains unchanged from modern. However, the variability between individual catchments that suggest other factors (e.g. topographic change, climatic gradients, dating uncertainty) may be inﬂuencing this result. 7.5.1 Cosmogenic surface exposure dating Cosmogenic 3He surface exposure ages from 3 boulders on the outermost moraine in WAH catchment range from 16.5 - 18.4 ka (Table 7.1). This result places moraine formation late in the LGCP and supports the morphostratigraphic correlation of proxi- mal moraines to this time period (Section 7.4.3). Formation of this outermost moraine late in the LGCP poses the question of whether the WAH glacier attained a similar or more extensive limit early in the LGCP, as seen in the MPO catchment (Chapter 5) and several Southern Alps moraine records (Putnam et al., 2013b; Rother et al., 2014). There is no geomorphic evidence outboard to suggest the WAH glacier was previously further extended. This outermost moraine may represent a composite feature formed over the LGCP, or the WAH glacier was inboard of this position earlier in the LGCP and any moraines pertaining to this time were subsequently overrun and eroded. I favour the former, due to the large size of this moraine and the widespread evidence for early-LGCP cooling of equal/greater magnitude than the late LGCP (e.g. Chapter 5; Barrell et al., 2013; Putnam et al., 2013b; Rother et al., 2014). Table 7.1: Cosmogenic 3He surface exposure dating sample details, helium content and exposure ages for Wahianoa valley. ID Lat. (◦S) Long. (◦E) Alt. (m asl) Thick. (cm) Shield. 3He (10 6 at. g−1 a−1 Exposure age (ka; Lm scaling)a WH1212 39.32 175.62 1374 3 0.994 5.463 ± 0.126 16.5 ± 0.4 (1.9) WH1213 39.32 175.62 1437 1.5 0.995 6.093 ± 0.167 17.2 ± 0.5 (2.0) WH1214 39.32 175.62 1438 2 0.996 6.505 ± 0.183 18.4 ± 0.5 (2.1) able 7.1: Cosmogenic 3He surface exposure dating sample details, helium con nd exposure ages for Wahianoa valley. CHAPTER 7. LGM GLACIER MODELLING 202 7.5.2.2 Experiment 2 The precise ∆T required to simulate the LGCP ice geometries delineated by geological evidence in each catchment described above, range from -4.0 to -6.8 ◦C when precip- itation remains unchanged from present (Figure 7.4a). Steady state equilibrium line altitudes for these simulations range from c. 1380 - 1660 m asl, which represent depres- sions of c. 820 - 1100 m from present (Table 7.2). Imposing a 25 % increase in modern precipitation reduces ∆T by c. 0.6 ◦C for all catchments (Figure 7.4), meanwhile, de- creasing modern precipitation by 25 % requires increases in ∆T of c. 0.8 ◦C (Figure 7.4a). Sensitivity tests of key energy balance parameters impact the palaeotemperature recon- Sensitivity tests of key energy balance parameters impact the palaeotemperature recon- 203 7.5. RESULTS structions by up to ±0.5 ◦C for the chosen ranges (Figure 7.4b). Altering the albedo of snow (αsnow = 0.67 - 0.77) and snow-temperature threshold (Tsnow = 0 - 1.5 ◦C) have the greatest effects (c. ± 0.1 - 0.5 ◦C). Using a temperature lapse rate (dT/dz) of -0.006 ◦C m−1, uniformly applied across all months, decreases ∆T by 0.1 - 0.2 ◦C. Changing the characteristic ice roughness length (Zice = 0.0008 - 0.01 m) also causes deviations in ∆T of ± 0.1 - 0.2 ◦C, relative to the optimal setting. Flow parameters Uc and A have negligible (<0.1 ◦C) impact on ∆T. Findings from Experiment 1 highlighted the variability in the LGCP palaeoclimate re- constructions that exists, both between volcanoes, and between individual catchments. Figure 7.4a shows that the Wahianoa (WAH) catchment on Mt. Ruapehu requires a conspicuously lower amount of cooling to match the identiﬁed LGCP ice limits (∆T = -4.0 ◦C, when ∆P = 0), compared to all other catchments studied (c. -5.2 to -6.8 ◦C, when ∆P = 0). Also, there is an offset in the temperature forcings necessary to simulate the mapped LGCP ice limits between the two volcanoes. Catchments on Tongariro massif range require a cooling of c. -6.0 to -6.8 ◦C (mean = -6.3 ◦C), when precipitation is unchanged, whilst catchments on Mt. Ruapehu require -4.0 to -5.8 ◦C (mean = 5.0 ◦C, or 5.4 ◦C when WAH is removed). Finally, the results presented in Figure 7.4 represent the climatic forcing, from present, required to meet the inferred downstream limits of LGCP glaciation. CHAPTER 7. LGM GLACIER MODELLING 204 7.5.2.3 Experiment 3 Figure 7.6a shows the change in temperature from present required to simulate the mapped LGCP geometries in the nine catchments, using topographic boundary con- ditions that approximate that of the LGCP (Figure 7.2). When precipitation remains unchanged from present, ∆T ranges from c. -4.1 to -7.1 ◦C, which represent differences of +0.1 to -0.5 ◦C from Experiment 2 (Figure 7.6b). In all catchments except one (MTO), more cooling was required, relative to Experiment 2 (Figure 7.6b). This is likely because the majority of the imposed topographic changes involved subtraction of depositional units (Figure 7.2), which increased local surface temperature. The greatest changes in ∆T between Experiment 2 and Experiment 3 occurs in the UNK catchment, where c. 0.5 ◦C of extra cooling is required to simulate the inferred glacial geometries (Figure 7.6b). This change is the result of reduced ﬂux from the vicinity of Mt. Ngauruhoe, caused by the elevation reduction in the accumulation area, following removal of this Holocene cone (Figure 7.2). However, this topographic alteration has less of an impact in the WAI catchment, where ∆T is reduced by 0.2 ◦C, relative to Experiment 2. Whilst removal of Mt. Ngauruhoe may act to channel ice ﬂow into the WAI catchment, the overall reduction in elevation reduces snow accumulation, therefore reducing the impact on ∆T. The imposed topographic changes did not improve the poor ﬁt between modelled ice geometry and the geological constraints, as ice still spills over lateral moraines in the WAI and MTA catchments before reaching the inferred LGCP termini. On Mt. Ruapehu, the major changes to the topography were made in the MTO catch- ment, which is the only catchment where ∆T decreased by c. 0.1 - 0.2 ◦C, relative to Experiment 2 (Figure 7.6b). In this instance, the removal of syn- and post-LGCP lava ﬂows in the upper and middle parts of this catchments has resulted in increased ice ﬂux to the lower valley, despite the overall reduction in elevation. It is also notable that the steady-state ice margins in the MTO catchment exhibit improved ﬁt to the geologically inferred ice limits in Experiment 3 (Figure 7.5d). This is caused by a reduction in the ice ﬂux leaving the catchment through overspill, which helps offset the effect of increased temperature caused by the reduction in bed elevation. In the other catchments on Mt. 7.5.2.2 Experiment 2 In several catchments, ice spills over ice-marginal indicators, such as lateral moraines, before the geologically-constrained termini are reached (Figure 7.5b,c). The possible reasons for the discrepancies, and the potential implications for palaeoclimate estimates are discussed below (Section 7.6.2). Table 7.2: Palaeo-equilibrium line altitudes (pELA) of the simulated LGCP glaciers and the difference from present using (dELAmean (m) = arithmetic mean of Keys (1988) = 2483 m asl; dELA = change from the mid-point of present day ELAs (in parentheses) of individual glaciers given by Keys (1988), where available. Glacier pELA (m asl) dELAmean (m) dELA Modelled ∆T (◦C) MTA 1380 -1103 -6.8 WAI 1390 -1093 -6.7 MHO 1460 -1023 -6.4 MPO 1510 -973 -6.0 UNK 1550 -933 -5.8 MTU 1530 -953 -995 (2450 - 2600 m asl) -5.7 MTO 1580 -903 -870 (2400 - 2500 m asl) -5.3 WHA 1550 -933 -1050 (2550 - 2650 masl) -5.2 WAH 1660 -823 -4.0 Table 7.2: Palaeo-equilibrium line altitudes (pELA) of the simulated LGCP glaciers and the difference from present using (dELAmean (m) = arithmetic mean of Keys (1988) = 2483 m asl; dELA = change from the mid-point of present day ELAs (in parentheses) of individual glaciers given by Keys (1988) where available Table 7.2: Palaeo-equilibrium line altitudes (pELA) of the simulated LGCP glaciers and the difference from present using (dELAmean (m) = arithmetic mean of Keys (1988) = 2483 m asl; dELA = change from the mid-point of present day ELAs (in parentheses) of individual glaciers given by Keys (1988), where available. CHAPTER 7. LGM GLACIER MODELLING CHAPTER 7. LGM GLACIER MODELLING CHAPTER 7. LGM GLACIER MODELLING Ruapehu, ∆T was reduced by 0.1 - 0.2 ◦C relative to Experiment 2. Thus accounting for post-glacial changes in bed topography cannot resolve the anomalous ∆T result in the WAH catchment. Finally, there remains a poor ﬁt between the geologically-inferred lateral ice margins in the MTU catchment and those simulated in Experiment 3. 7.6. DISCUSSION 7.6. DISCUSSION 205 7.6 Discussion Using a numerical glacier model, I investigated the magnitude of temperature depres- sion from present in central North Island during the LGCP and the sensitivity of these results to model parameterisation and changing topographic boundary conditions. The main ﬁndings are as follows: (1) the temperature depression required to simulate the LGCP glacial geometries of individual catchments varies between -4.0 and -6.8 ◦C; (2) there is a systematic offset of >1 ◦C in the model-derived palaeotemperatures associated with LGCP moraines between the two volcanoes; (3) using geologically-constrained reconstructions of LGCP topography has relatively little impact (+0.2 to -0.5 ◦C) on the palaeotemperature reconstruction. Below, I ﬁrst consider the possible sources of uncertainty, before placing the results in context of other, local terrestrial and marine palaeotemperature proxy reconstructions. 7.6.1 Topographic uncertainty Improved constraint of the timing and extent of late Quaternary volcanism in central North Island has allowed a test of the impact that changing topographic boundary conditions have on palaeoclimate estimates for glacier modelling. Using expert-deﬁned topographic reconstructions, informed by recent ﬁeld mapping and radiometric dating, the temperature forcing required to simulate the inferred LGCP glaciers is altered by +0.2 to -0.5 ◦C. The majority of the imposed topographic changes serve to remove post- glacial lava ﬂows that have built volcanic cones (e.g. Mt. Ngauruhoe) or in ﬁlled glacial troughs (e.g. MTO, MPO; Figure 7.2). Subtraction of these features has lowered the glacier bed elevation, which raises the local surface air temperature and explains why most catchments require increased cooling to achieve the LGCP limits in Experiment 3, relative to Experiment 2. This mirrors the ﬁndings of McKinnon et al. (2012), who found that subtraction of post-glacial sedimentary ﬁll from the Pukaki basin, in central Southern Alps, resulted in lower glacier model derived palaeotemperature estimates, relative to studies that used present day bed topography. Conversely, removal of the post-glacial lava ﬂows in the vicinity of the MTO catchment reduced the temperature forcing required to simulate the LGCP ice geometry in that catchment by c. 0.2◦C, relative to Experiment 2. Reduced overspill, as shown by the improved ﬁt between the model output and the lateral moraines, indicates that the retention of ice within the MTO catchment was im- proved by the imposed topographic changes and this effect was sufﬁcient to offset the decreased (increased) accumulation (ablation) induced by land surface lowering. How- CHAPTER 7. LGM GLACIER MODELLING 206 ever, the imposed topographic changes did not improve the ﬁt in all catchments where overspill occurs (e.g. MTA, WAI, MTU), thus it is probable that the LGCP palaeotem- perature estimates presented in Figures 7.4 and 7.6 for these catchments overestimate the true magnitude of temperature lowering associated with the LGCP moraines in these catchments. This interpretation is supported by the fact that these catchments require the greatest magnitude of cooling, relative to present, of all catchments on the respective volcanoes. Discounting the reconstructions from these catchments leaves LGCP palaeotemperature estimate ranges of -4.1 to -5.4 ◦C for Mt. Ruapehu and -6.1 to -6.3 ◦C for Tongariro massif (∆P = 0; Experiment 3). 7.6.2 Glacier model uncertainty Numerical glacier models represent simpliﬁed descriptions of glacier mass balance and ice ﬂow, which often require assumptions about parameters due to lack of empirical con- straint. The sensitivity tests presented in Figure 7.4b, provide a ﬁrst-order assessment of the uncertainty imparted by parameters in the energy balance model. Varying key parameters within acceptable bounds causes deviations in reconstructed temperatures of up to ±0.5 ◦C, which indicates that some of the variability in palaeotemperatures (e.g. WAH catchment) could be explained by spatial heterogeneities in model parameters, which are currently assumed uniform across the model domain. Palaeotemperature estimates are most sensitive to albedo, which cannot be constrained for the past. Heterogeneous spatial distribution of surface albedo during the LGCP, for example differential surface debris thickness, provides a possible explanation for the offset in palaeotemperature reconstructions between the volcanoes, and/or the anomalous ﬁnding in the WAH catchment on Mt. Ruapehu. Debris cover can act to enhance or reduce surface melt on glaciers, depending on the debris thickness, which in turn is dictated by sediment availability. The potential for past inputs of volcanic ash to the glacier surface represents the most likely source of surface debris cover at this study site. Ice-contact structures in lava ﬂows on Mt. Ruapehu (Sp¨orli and Rowland, 2006) show evidence for such interaction, whilst 40Ar/39Ar dating shows that Mt. Ruapehu was active in the 30 - 18 ka time window (Gamble et al., 2003). Richardson and Brook (2010) measured ice ablation rates on Mt. Ruapehu beneath tephra emplaced during the 2007 eruption and found that tephra cover up to 7 cm thick enhanced ablation rates of the underlying ice, relative to that of clean snow. This ’effective thickness’ (at which maximum ablation occurs, relative to clean snow) is relatively high in comparison to other studies, which typically ﬁnd that debris thicknesses of 0-1 cm are sufﬁcient to retard ablation (Brock et al., 2007). Variability in the effective and critical (that at which ablation is reduced relative to clean snow/ice) thicknesses of surﬁcial tephra cover is introduced by differences in tephra properties such as grain size, angularity and density (Nield et al., 2013), which can change with time due to different eruption styles. Kirkbride and Dugmore (2003) assessed the effects of historic volcanic fallout events on outlet glacier behaviour in Iceland. 7.6.1 Topographic uncertainty Thus, accounting for ice overspill and known topographic changes is insufﬁcient to resolve the apparent offset between LGCP palaeotempertaure estimates between these two volcanoes. Identiﬁcation and geometrical constraint of well-preserved post-glacial lava ﬂows can be achieved with relative ease through detailed ﬁeld investigations, however the recognition of post-glacial erosional events (e.g. sector collapse, ﬂuvial incision) and subsequent topographic reconstruction is less straightforward. Some erosional events are identiﬁable in the modern landscape on Mt. Ruapehu (e.g. Murimotu Formation sector collapse at 10.4 - 10.6 cal. ka BP - Chapter 4), however the precise source locations and pre-event topographies remain highly uncertain (e.g. Hackett and Houghton, 1989; Palmer and Neall, 1989; McClelland and Erwin, 2003). Such erosional events act to decrease surface slopes, alter drainage pathways and alter the bed hypsometry, with potential implications for modelled ice distributions and palaeoclimatic recon- struction. For example, the offset in LGCP temperature reconstructions between the glacial catchments of Mt. Ruapehu and Tongariro massif, theoretically could be caused by post-glacial change in the relative altitudes of the two volcanoes. A post-glacial decrease in the summit altitude of Tongariro massif, relative to Mt. Ruapehu, could explain the need for greater cooling on Tongariro massif in the simulations presented here. However, there is little geological evidence to support the notion that Tongariro massif has experienced major post-glacial degradation, nor that Mt. Ruapehu has signiﬁcantly increased in elevation since the LGCP. This absence of evidence, combined with the fact that known changes in topographic boundary conditions had relatively little impact on palaeotemperature reconstructions (Experiment 3), makes it unlikely that post-glacial topographic change is the primary source of this systematic offset. 7.6. DISCUSSION 7.6. DISCUSSION 207 7.6.2 Glacier model uncertainty They found a long-term effect of enhanced surface ablation after a volcanic eruption, caused by re-distribution of tephra in the maritime setting. Enhanced ablation on Mt. Ruapehu by thin tephra cover, or retarded ablation on Tongariro massif by thicker tephra cover, provide possible scenarios that could explain some of the difference in glacier model derived LGCP palaeotemperature reconstructions on the two volcanoes. CHAPTER 7. LGM GLACIER MODELLING 208 Volcanic activity has the potential to affect glacier mass balance and dynamics in several other ways, such as increased ablation by raised geothermal heat ﬂuxes, catas- trophic removal of ice through explosive eruptions and changing sub-glacial hydrology. Thermal energy released by volcanic eruptions also has the potential to ablate con- siderable volumes of snow and ice (Major and Newell, 1989), however such events are relatively short-lived, often persisting on timescales of days to months (e.g. Gud- mundsson et al., 1997). Raised geothermal heat ﬂuxes therefore can complicate the climatic interpretation of glacier ﬂuctuations over annual- to decadal timescales (Rivera et al., 2012; Rivera and Bown, 2013), however climate is the most-likely driver of glacier ﬂuctuations on active volcanoes over centennial to millennial timescales (e.g. Kirkbride and Dugmore, 2001; Mackintosh et al., 2002; Blard et al., 2007; Licciardi et al., 2007; Osborn et al., 2012). This interpretation is supported by the broad internal consistency of palaeotemperature reconstructions found in this study and the agreement of these estimates with independent reconstructions of LGCP climate in New Zealand (Golledge et al., 2012; Newnham et al., 2013). In Chapter 3, I demonstrate the good agreement between contemporary ice distri- bution on Mt. Ruapehu and ice geometries simulated using the 30-year (AD 1981-2010) average climate datasets. This indicates that these datasets provide a useful starting point from which to assess the local LGCP climate anomaly in catchments on Mt. Ru- apehu. No glaciers exist on Tongariro massif today, therefore it is more difﬁcult to assess how representative the modern climate grids are for this volcano. The paucity of high-altitude precipitation data for the present day imparts considerable uncertainty in glacier model applications. Rowan et al. (2014) ﬁnd that uncertainty in present day precipitation distribution imparts uncertainty of up to 25 % in modelled LGCP glacier length in the central Southern Alps, which equates to about 0.5 ◦C in the palaeotemper- ature estimate. 7.6. DISCUSSION 209 (Lorrey et al., 2012b) conclude that winters were characterised by a greater frequency of blocking highs over South Island. This would result in more frequent north-easterlies in central/northern North Island, during the accumulation season, which may have increased precipitation on Tongariro massif at the expense of Mt. Ruapehu. Synoptic type frequency changes are not accounted for in the glacier model simulations and such an effect, at least in part, may explain the differences in palaeotemperature estimates between the volcanoes. The precipitation-temperature relationships presented in Figure 7.4a indicate that precipitation changes of ± 25 % are balanced by temperature changes of ± c. 0.6 - 0.8 ◦C, which is consistent with similar estimates for glaciers South Island (Oerlemans, 1997; Anderson and Mackintosh, 2012). Thus, precipitation on Tongariro would need to be increased by >25 %, relative to Mt. Ruapehu, in order to account for the c. 1.0 - 1.3 ◦C temperature difference associated with the inferred LGCP glacial limits between the two volcanoes. 7.6.2 Glacier model uncertainty The absence of continuous, high mountain ranges and the increased distance from the prevailing westerly storm track means that spatial precipitation gradi- ents in central North Island are much lower than the central Southern Alps. However, it is likely that uncertainty in the present-day spatial precipitation distribution, as well as total precipitation amount, could explain part of the offset in LGCP palaeotemperature estimates between the two volcanoes. Changes in the spatial gradients of precipitation across the model domain, between the LGCP and today, could impart similar uncertainty to the palaeotemperature estimates (Kessler et al., 2006). At present, precipitation-bearing cyclones in central North Island are predominantly advected from the west and orographic interaction of these weather systems with Mt. Ruapehu creates a west-east rain shadow effect on the volcano (Chap- ter 3). In a proxy-data driven assessment of palaeocirculation in New Zealand at 21 ka, 7.6. DISCUSSION Table 7.2). Lowering of the regional ELA to c. 1500 m at the LGCP is insufﬁcient to promote widespread glaciation in the mountain ranges elsewhere in North Island, as few other peaks exceed this elevation. The only existing evidence for LGCP glaciation outside of the central North Island volcanoes comes from the Tararua Ranges in southern North Island, where the local pELA was c. 1100 m asl (Brook et al., 2005; Brook et al., 2008). This is considerably lower than the ELA depression in the present study and elsewhere in New Zealand (Porter, 1975; McCarthy et al., 2008; Golledge et al., 2012), which may represent topo-climatic controls on mass balance of this former cirque glacier, such as wind-driven snow accumulation. The absence of contemporary glaciers in the Tararuas precludes robust spatial comparison of ELA depressions to the results presented here. Several quantitative palaeotemperature estimates from North Island have been made using fossil pollen assemblages, which also indicate LGCP temperature depressions of 4-7 ◦C below present (McGlone and Topping, 1977; Newnham et al., 1989; Newnham et al., 2013; Sandiford et al., 2003; Wilmshurst et al., 2007), which is consistent with the glacier model results. Several glacier-based assessments of LGCP temperature have previously been made for South Island, New Zealand, using a variety of different glacier models. Simulations of the entire Southern Alps iceﬁeld, using the Parallel Ice Sheet Model, indicate that the LGCP was characterised by temperatures 6 - 6.5 ◦C colder than present, coupled with a reduction in precipitation of c. 25 % (Golledge et al., 2012). It is notable that the best-estimate palaeotemperature scenarios did not achieve a good ﬁt between modelled ice extent and the geological evidence in all catchments (Golledge et al., 2012, their Figure 10B). Using a different glacier model with higher grid resolution and a different representation of modern climate, Rowan et al. (2013) and Putnam et al. (2013a) achieve a good model ﬁt in the regions where Golledge et al. (2012) did not (e.g. Rakaia), despite using a similar temperature forcing (∆T = -6.25 to -6.5 ◦C). Using the University of Main Ice Sheet Model, Putnam et al. (2013b) ﬁnd that a cooling of 6.25 ± 0.5 ◦C (with no precipitation change) is required to generate an ice extent that matches well-dated moraines in the Lake Ohau catchment. When precipitation is reduced by 30 % the required cooling increases to 6.9 ◦C. 7.6.3 LGCP climate in New Zealand The glacier modelling experiments presented here suggest that stadial conditions in central North Island were characterised by temperatures 4 to 7 ◦C lower than present (Figure 7.4). Local precipitation change during the LGCP remains poorly constrained, although evidence from climate modelling (Drost et al., 2007; Rojas et al., 2009), previ- ous glacier modelling (Golledge et al., 2012), carbon isotopes in speleothems (Whittaker et al., 2011) and diatoms in maar deposits (Stephens et al., 2012b) indicate that drier than present conditions prevailed across New Zealand at this time. Precipitation reduc- tions of up to 25 % from present require additional cooling of up to 0.8 ◦C to achieve the LGCP glacial geometries in central North Island (Figure 7.4). Such a change in precipitation is likely a maximum estimate given that climate model simulations predict changes in total annual precipitation of < 10 % (e.g. Drost et al., 2007). Steady-state equilibrium line altitudes of the simulated LGCP glaciers fall between c. 1400 - 1650 m asl, which represent depressions of c. 800 - 1100 m, relative to present. This estimate slightly exceeds that of McArthur and Shepherd (1990), who manually reconstructed the LGCP ELAs on Mt. Ruapehu to between 700-900 m below present. This difference likely reﬂects the addition of catchments on Tongariro massif, as well as the methodological differences between the two studies. In Chapter 5, I estimated the ELA of the LGCP glacier in the MPO catchment as c. 1400 - 1550 m asl, using the accumulation area ratio (AAR) and maximum elevation of lateral moraine (MELM) methods, which agrees well with the model simulation presented here (c. 1510 m asl; CHAPTER 7. LGM GLACIER MODELLING CHAPTER 7. LGM GLACIER MODELLING 210 Table 7.2). Table 7.2). Thus, these studies have shown that, despite differences in boundary conditions and formulations for glacier ﬂow, glacier model experiments consistently suggest peak stadial air temperatures during the LGCP were 6-7 ◦C cooler than present across New Zealand. This magnitude of air temperature depression is also in broad agreement with sea surface temperature reconstructions from the New Zealand sector of the Southern Ocean (Barrows et al., 2007a; Bostock et al., 2013). 7.7. CONCLUSION 211 7.7 Conclusion In this chapter I have presented geomorphic mapping and 2D glacier modelling results, which constrain ice distribution and the climatic forcing associated with the Last Glacial Cold Period in central North Island, New Zealand. I have shown that: 1. During the Last Glacial Cold Period in New Zealand, valley glaciers extended to c. 1200 m asl from central ice ﬁelds that covered both Tongariro massif and Mt. Ruapehu in central North Island. 1. During the Last Glacial Cold Period in New Zealand, valley glaciers extended to c. 1200 m asl from central ice ﬁelds that covered both Tongariro massif and Mt. Ruapehu in central North Island. 2. Temperatures 4 - 7 ◦C lower than present are required to simulate geologically constrained glacier extents associated with these ice limits, when precipitation remains unchanged. A decrease in precipitation (as suggested by proxy evidence and climate models) of up to 25 % from present, increases the required tempera- ture change by up to c. 0.8 ◦C. 3. Steady-state ELAs were c. 800-1100 m lower than present in central North Island during the Last Glacial Cold Period, which agrees well with the manual recon- struction of the Mangatepopo valley presented in Chapter 5 (c. 1000 m), as well as the previous determination (700-900 m) of McArthur and Shepherd (1990). 3. Steady-state ELAs were c. 800-1100 m lower than present in central North Island during the Last Glacial Cold Period, which agrees well with the manual recon- struction of the Mangatepopo valley presented in Chapter 5 (c. 1000 m), as well as the previous determination (700-900 m) of McArthur and Shepherd (1990). 4. Accounting for volcanically-induced, post-glacial topographic change generally decreases the elevation of the glacier bed elevation, which increases the magnitude of cooling required to simulate the former ice limits. The imposed topographic changes do not signiﬁcantly change past glacial drainage patterns, although the difﬁculty in reconstructing pre-erosional topographies makes it hard to fully assess this possibility. The impact of topographic change on the temperature reconstructions is variable between catchments, with changes on the order of 0.1 - 0.5 ◦C, relative to the simulations with present day land surface. 5. CHAPTER 7. LGM GLACIER MODELLING 7.7 Conclusion The palaeoclimatic reconstructions here agree well with proximal, pollen-based estimates (Newnham et al., 2013), as well as several similar assessments from glacial records in central Southern Alps (Golledge et al., 2012; Putnam et al., 2013b; Rowan et al., 2013), which all indicate that temperatures were depressed by c. 6 5. The palaeoclimatic reconstructions here agree well with proximal, pollen-based estimates (Newnham et al., 2013), as well as several similar assessments from glacial records in central Southern Alps (Golledge et al., 2012; Putnam et al., 2013b; Rowan et al., 2013), which all indicate that temperatures were depressed by c. 6 212 CHAPTER 7. LGM GLACIER MODELLING ◦C during the Last Glacial Cold Period. CHAPTER 7. LGM GLACIER MODELLING 212 ◦C during the Last Glacial Cold Period. 8.1 Introduction The aim of this thesis is to constrain the magnitude, timing and associated climatic forcing of past mountain glacier ﬂuctuations on New Zealand’s central North Island volcanoes: Mt. Ruapehu and Tongariro massif. In this chapter, I will ﬁrst outline the original scientiﬁc contributions made by the research presented here, before revisiting the research questions identiﬁed from the literature review in Chapter 2. Lastly, I discuss outstanding questions that I consider important for future research, in light of the progress made by this thesis. 8.2 Original scientiﬁc contributions made by this research Well-constrained chronological and palaeoclimatic reconstructions of late Quaternary glacier ﬂuctuations in New Zealand have largely been focused on the central Southern Alps (e.g. Suggate and Almond, 2005; Anderson and Mackintosh, 2006; Schaefer et al., 2006, 2009; Doughty et al., 2013; Putnam et al., 2013a,b), with very few other studies existing outside of this narrow latitudinal band (e.g. Shulmeister et al., 2005; McCarthy et al., 2008; Brook et al., 2008). Using a combination of geomorphological and geological mapping, cosmogenic surface exposure dating and numerical glacier modelling, I have provided new insights into the timing and magnitude of climatic change in central North Island (39◦S) during the Last Glacial Maximum and the late glacial. In Chapter 4, I use a debris avalanche deposit on Mt. Ruapehu to constrain locally the production rate of cosmogenic 3He. In collaboration with VUW and GNS Sci- ence geologists, we constrained the timing of this event, using radiocarbon dating, to 10.4-10.6 cal. ka BP. I made measurements of cosmogenic 3He concentrations in 213 214 CHAPTER 8. SYNTHESIS boulders deposited at the surface during this event, which show that a previous ’glob- ally’ (based primarily on Northern Hemisphere calibration sites) compiled estimate of cosmogenic 3He production (Goehring et al., 2010) is applicable in New Zealand. This ﬁnding means that surface exposure dates and erosion rates, using cosmogenic 3He, can now be calculated with reduced uncertainties, in this region of the world. Although not a speciﬁc aim of this thesis, a separate, important result of this study is the improved age constraint of this major debris avalanche event. The new, robust radiocarbon age reﬁnes previous estimates based on a single radiocarbon date (Top- ping, 1974) and provides useful constraint of post-glacial topographic change on this volcano, which has important implications for palaeoglacier modelling (e.g. Chapter 7). This result is also of use for local geohazard assessment in this active volcanic setting. Application of cosmogenic surface exposure dating to constrain past glacier ﬂuctuations is presented in Chapters 5, 6 and 7. In Chapter 5, I present evidence for two periods of extensive glaciation, when the local ELA was depressed by 930-1080 m relative to present, which occurred during MIS 4 (> 58 ka) and from late MIS 3 (c. 31 ka) through the LGCP. In Chapter 6, I constrain the timing of a glacial re-advance on Mt. Ruapehu during the late-glacial. 8.2 Original scientiﬁc contributions made by this research I adapt a coupled model of cosmogenic nuclide production and moraine diffusion, previously applied to a moraine in South Island (Applegate et al., 2008), to investigate the scatter in this 3He dataset. The results of this work, together with recent advances in local tephrochronology (Lowe et al., 2013), indicate that the North Island cooled in synchrony with South Island and Antarctica during the Antarctic Cold Reversal (c. 13 ka). In Chapter 7, I show that a valley glacier on the southeast side of Mt. Ruapehu was present until at least c. 17-18 ka. This ﬁnding provides constraint for glacier modelling experiments that seek to constrain temperature change in central North Island. Improved age constraint of glacier ﬂuctuations on the central North Island, provides useful targets for quantitative palaeoclimate estimates using glacier model experiments (e.g. Golledge et al., 2012; Doughty et al., 2013). In Chapters 6 and 7, I present the results of glacier model experiments with the aim of constraining the magnitude of air temperature depression, relative to present, associated with the glacial advances in central North Island during the late-glacial (Chapter 6) and LGCP (Chapter 7). Using a coupled, distributed energy balance and 2D ice ﬂow model, I show that a cooling of c. 2.9 ◦C from present is required to simulate the mapped and dated glacial geometry of a late-glacial re-advance on southern Mt. Ruapehu, when precipitation remains unchanged from modern (Chapter 6). Experiments with imposed precipitation changes of ±20% from present, yield corresponding temperature estimates that range between 2.5 and 3.4 ◦C. These ﬁndings provide evidence to suggest that North Island cooling 8.3. RESEARCH QUESTIONS 215 during the Antarctic Cold Reversal was of similar magnitude to that of South Island (e.g. Doughty et al., 2013) and the sub-Antarctic Islands (e.g. McGlone et al., 2010). I conduct seasonal sensitivity tests that show glacier length changes on Mt. Ruapehu are most sensitive to summer temperature change and that reduced seasonality may explain dif- ferences in the magnitude of cooling indicated by different proxies (e.g. glaciers, pollen). In Chapter 7, I use the cosmogenic 3He ages and extensive ﬁeld-based geomorpho- logical investigations to assign ages to undated moraines across the two volcanoes. These moraines are then used as targets in glacier modelling experiments, to constrain quantitative temperature estimates in central North Island for the LGCP. 8.2 Original scientiﬁc contributions made by this research Of the 9 catch- ments studied, 8 yield LGCP air temperature estimates between 5.2 - 6.8 ◦C lower than present, when precipitation remains unchanged. This range of cooling increases to 6.0 - 7.5 ◦C, when precipitation is reduced by 25% from present. Experiments with > 15 ka topographic reconstructions show that these estimates are insensitive to the known topographic changes (i.e. lava ﬂow emplacement) that have occurred since deglaciation. 8.3.1.1 The suitability of cosmogenic 3He Yes. The work presented in this thesis has shown that, not only can 3He be used for surface exposure dating on these volcanoes, but that it is very well suited to the local geology. A key determinant for use of 3He in this study is the local lithology, as the andesitic moraine boulders contain abundant pyroxene phenocrysts (e.g. Price et al., 2012). Whilst cosmogenic 3He is produced in all minerals, it diffuses out of most min- eral phases at environmental temperatures (Niedermann, 2002). However, it has been demonstrated that cosmogenic 3He is quantitatively retained in pyroxene crystals (e.g. Kurz, 1986a) and is routinely measured in this phase (e.g. Bruno et al., 1997; Sch¨afer et al., 1999; Bromley et al., 2009, 2011). The paucity of quartz in the andesite-dacite moraine boulders of the central North Island volcanoes precludes application of 10Be, until methods for extraction of these nuclides from other minerals (e.g. pyroxene) become established. 36Cl is measured in whole rock samples of basaltic rocks, however the complex production pathways restricts the precision at which surface exposure ages can be calculated (Dunai, 2010). CHAPTER 8. SYNTHESIS 216 3He can be cosmogenic, magmatic and nucleogenic in origin, therefore for surface exposure dating applications it is important to resolve the relative contributions from these potential sources. However samples measured in the course of this thesis display extremely high 3He/4He (up to several 100x the atmospheric ratio), together with very low 4He concentrations. These characteristics reﬂect: (i) the youth of the parent material (i.e. < 300 ka; Gamble et al., 2003), which restricts the time for radiogenic accumulation of 3He and 4He; (ii) sample preparation procedures (e.g. HF-leaching), which result in a higher purity pyroxene separates and absence or supression of nucleogenic 4He (Bromley et al., 2014); and (iii) a negligible magmatic input. Furthermore, contributions from nucleogenic 3He, which is produced via thermal neutron reactions on lithium-6 (6Li (n,α) 3H 3He; Dunai et al., 2007), are also negligible, due to the generally low lithium, uranium and thorium concentrations. Samples with elevated Li concentrations exhibit no clear correlation 3He (Chapter 4), probably due to the acid leaching treatment (Bromley et al., 2014). Future applications should continue to assess the potential for non-cosmogenic contributions prior to exposure age / erosion rate calculations. 8.3.1.1 The suitability of cosmogenic 3He The results presented in Chapter 4 show that the time-integrated cosmogenic 3He production rate in New Zealand since 11 ka is well approximated by a globally com- piled production rate sourced mainly from Northern Hemisphere calibration sites (Goehring et al., 2010). This constraint means that cosmogenic 3He concentrations measured in New Zealand can be accurately converted to surface exposure ages. 8.3.1.2 Processes causing scatter in surface exposure ages An interesting ﬁnding from the application of cosmogenic surface exposure dating to moraine boulders on Mt. Ruapehu and Tongariro massif is the common occurrence of scatter, outside of the measurement uncertainty, within sample populations derived from single moraine landforms. Whilst this is not an uncommon occurrence (e.g. Hey- man et al., 2011), the degree of scatter in the datasets generated here is considerably greater than many other datasets generated in New Zealand (e.g. Schaefer et al., 2009; Kaplan et al., 2010, 2013; Putnam et al., 2010a, 2012, 2013a,b; Kelley et al., 2014), thus is worthy of discussion here. Due to the number of samples available, the dataset presented in Chapter 6, from the Mangaehuehu catchment on Mt. Ruapehu, affords the best opportunity to analyse this problem. These samples exhibit a negatively skewed distribution, with a cluster of ages around 14-11 ka and a tail of younger ages. This type of age distribution is commonly observed and is attributed to ’geological processes’ that act to suppress, or 3. RESEARCH QUESTIONS 217 8.3. reduce cosmogenic nuclide concentrations, for example via shielding or erosion (Apple- gate et al., 2012). In Chapter 6, I use a coupled topographic diffusion / cosmogenic 3He production model to show that the scatter observed in the Mangaehuehu dataset can be explained by the process of moraine diffusion, whereby exhumation of previously shielded boulders from within the moraine subsurface generates a population of surface boulders with a negatively skewed distribution of surface exposure ages. Topographic cross-proﬁles across moraines of MIS 4 and MIS 2 age in the Mangatepopo valley (Chapter 5) provide an opportunity to examine this possibility over longer timescales. The older landforms exhibit lower-angle slopes and wider ridge crests, compared to younger moraines, which is indicative of topographic diffusion. Additional support for the moraine diffusion hypothesis comes from the 3He mea- surements of debris avalanche material presented in Chapter 4. These four relatively consistent measurements come from boulders well-embedded within a sediment body that has low surface slopes, therefore is not undergoing diffusion. The coherence of this small dataset, which have a similar exposure duration to the samples from Chapter 6, suggests that moraine diffusion may be the critical process leading to scatter. However, it is important to note that the elevation difference between these two sites is c. 8.3.1.2 Processes causing scatter in surface exposure ages 800-900 m, thus the higher elevation moraine samples are subject to greater annual precipitation totals and lower minimum temperatures, which may promote surface erosion of boul- ders, as well as moraine diffusion through physical weathering (e.g. freeze-thaw cycles). I believe local climate is a major factor causing the observed scatter of moraine boulder surface exposure ages presented here. Support for this notion is provided by com- paring the datasets presented in this thesis to cosmogenic moraine chronologies from the Southern Alps. Annual precipitation totals in the study sites of this thesis are c. 2000-3000 mm (Tait et al., 2006), which may contribute to chemical weathering of boulder surfaces and physical erosion and transport of moraine sediment. The altitude of sample sites (c. 1000-1800 m asl) means that freeze-thaw cycles contribute to physical erosion of moraines (Figure 8.1). Previous study sites in the Mackenzie Basin are lower in elevation and are situated within a rain shadow created by the central Southern Alps, which reduces local potential for boulder and moraine degradation. This interpretation is similar to that suggested by Applegate et al. (2008), who ascribe the scatter observed in the Waiho Loop exposure age dataset of Barrows et al. (2007b) to moraine diffusion processes in the hypermaritime setting. Although I have provided evidence that moraine diffusion contributes towards the scatter in the exposure age datasets, it is difﬁcult to resolve the relative contributions from all possible processes. Surface erosion of sampled boulders is also likely to have 218 CHAPTER 8. SYNTHESIS Figure 8.1: Ice needles (c. 2-4 cm) lifting silt - ﬁne gravel particles on the LG1 moraine in the Mangaehuehu catchment. Field observations from several locations on both volcanoes show this process is capable of lifting large cobbles and small boulders. [7 May 2012] Figure 8.1: Ice needles (c. 2-4 cm) lifting silt - ﬁne gravel particles on the LG1 moraine in the Mangaehuehu catchment. Field observations from several locations on both volcanoes show this process is capable of lifting large cobbles and small boulders. [7 May 2012] occurred over the duration of boulder exposure, however the absence of resistant mineral veins in the local igneous rocks precludes quantitative constraint of this effect. Future work should integrate surface erosion effects into moraine diffusion model experiments. Volcanic processes (e.g. 8.3.1.2 Processes causing scatter in surface exposure ages tephra fall) may also contribute some uncertainty to the observed exposure age distributions, although I have shown in Chapters 5 and 6 that shielding beneath ash fallout is is unlikely to change the main conclusions. 8.3.2.1 Timing of glacier ﬂuctuations Cosmogenic 3He surface exposure ages from Mangatepopo valley show that glacier extent on Tongariro massif peaked prior to 58 ka, probably during MIS 4 (c. 65-60 ka). Renewed valley glaciation occurred by 31 ± 3 ka and remained extended until c. 21 ± 2 ka. The downstream limit of MIS 4 glaciation in Mangatepopo valley is relatively poorly resolved due to the paucity of geomorphic evidence. However, moraine M3 is <100 m outboard of the 21 ka moraine (M2), which suggests similar or slightly greater ice extent prior to the global LGM. 8.3. RESEARCH QUESTIONS 219 Re-investigation of the moraine (M2) tephrostratigraphy in Mangatepopo valley, in- cluding addition of major element analyses of rhyolite horizons, has provided new insight into the timing of deglaciation. The ﬁndings presented in Chapter 5 suggest that the Waiohau Tephra is situated c. 20 cm above the moraine surface, whilst reworked pockets of this tephra are also present c. 30-40 cm higher in the soil sequence. In combi- nation with the cosmogenic surface exposure ages from the moraine crest, this shows that the Mangatepopo glacier retreated from the moraine crest at c. 21 ka, and had undergone signiﬁcant retreat prior to 14 ka. Deformation structures, possibly caused by frost creep processes, indicate probable cold conditions at some time after 14 ka. The timing of glacier ﬂuctuations on Mt. Ruapehu during the last glacial cycle is less well constrained by direct dating. However the close proximity of glaciated catch- ments to Tongariro massif and similar morphology of moraines permits correlation on morphostratigraphic grounds. This is supported by similar soil-tephra stratigraphy found overlying moraines in Whakapapaiti valley, which is used to correlate these features to the LGCP. Cosmogenic surface exposure ages from Wahianoa catchment on southeast Ruapehu indicate valley glaciation persisted until c. 18 ka. 8.3.2.2 Implications for possible climate drivers The chronological results from Tongariro massif indicate that glaciers in central North Island ﬂuctuated in phase with those in the Southern Alps during the last glacial cycle. This adds to a growing body of research that suggests an ’early’ onset, or ’extended’ period of peak glacial conditions in New Zealand c. 30-18 ka (Vandergoes et al., 2005; Alloway et al., 2007; Newnham et al., 2007; Barrell et al., 2013; Putnam et al., 2013b; Rother et al., 2014). Greatest ice extent and cooling appears to have occurred early in this window (c. 30-26 ka), during rising local summer insolation intensity and decreasing summer (and increasing winter) duration (Huybers and Denton, 2008). Furthermore, cold conditions prevailed until at least 18 ka, which coincides with a peak in local summer insolation intensity and a minimum in austral summer duration. Vandergoes et al. (2005) argue that early southern cooling at the onset of the LGCP was driven by the minimum in austral summer insolation intensity at c.32 ka. This explanation accords with the timing of glaciation in central North Island (e.g. moraine M1 - Mangatepopo), however it does not satisfy geological evidence for the contin- uation of glacial conditions through the global LGM (c. 19-26 ka; Clark et al., 2009), when austral summer intensity was increasing (Mercer, 1984). It also does not explain CHAPTER 8. SYNTHESIS 220 recent evidence for extensive glaciation in the Southern Alps at c. 42 ka (Kelley et al., 2014), which also coincides with a local insolation intensity maximum. Newnham et al. (2012) postulate that the extended LGM in New Zealand results from an early manifestation of the bipolar see-saw, whereby warming in Greenland during Greenland Interstadial 3 and 4 (c. 29 - 28 ka), was counterbalanced by increased export of heat from the Southern Ocean (Crowley, 1992; Broecker, 1998). These events fall within the production rate errors of the cosmogenic 3He chronology from Mangatepopo valley, but can not explain the Lake Ohau glacial maximum at 32.5 ± 1 ka (Putnam et al., 2013b). Putnam et al. (2013b) present a hybrid hypothesis, whereby New Zealand glaciers respond to air temperature anomalies driven by stratiﬁcation of the Southern Ocean in response to orbital forcing of winter duration (cf. Huybers and Denton, 2008), coupled with millennial-scale ﬂuctuations of the sub-tropical front (e.g. De Deckker et al., 2012). 8.3.2.2 Implications for possible climate drivers Broecker (2013) hypothesises that glacier ﬂuctuations are fundamentally driven by the radiative effects of changing atmospheric CO2. Further constraint of the timing and magnitude of past glacier ﬂuctuations, particularly prior to MIS 2 (e.g. Kelley et al., 2014; Schaefer et al., 2015), will help to resolve the relative roles of these hypothesised climate drivers. 8.3.3.1 Equilibrium line altitude reconstructions Manual reconstruction of the LGCP valley glacier in Mangatepopo valley indicates the local ELA was between 1400-1550 m asl (MELM; AAR = 0.67). This is c. 930-1080 m lower than present (2483 ± 55 m asl), according to the glacier survey of Keys (1988). Iteratively applying air temperature lapse rates of -4 to -7 ◦C km−1, I calculate a proba- bility distribution of palaeotemperature estimates centred on 5.6 ± 1.1 ◦C (1 σ). 8.3.3.2 Numerical glacier modelling Physically-based numerical glacier modelling of past ice geometries provides a more objective, robust means of constraining past climate variation (Plummer and Phillips, 2003; Doughty et al., 2013). This is especially the case where a paucity of geomorphic evidence can preclude accurate constraint of former ice margins in accumulation areas (Rea et al., 1999). 221 .3. RESEARCH QUESTIONS Matching glacier model outputs to geologically constrained LGCP ice margins in 9 catchments across both volcanoes, I derive palaeotemperature estimates between -4 and -7◦C, relative to present. Palaeo-equilibrium line altitudes (pELAs) for the sim- ulated glaciers range between c. 1400 - 1650 m asl, which represent depressions of 800-1100 m below present. In the Mangatepopo valley, the model-simulated glacier has a pELA of 1510 m asl (970 m below present) and best ﬁts the LGCP moraines when temperature is reduced by 6 ◦C relative to present, and precipitation is held at present day levels. These results agree well with the manual pELA reconstructions presented in Chapter 5 (particularly the MELM estimate), despite the complex ice geometry. Two results stand out from this work. First, the Wahianoa (WAH - Chapter 7) catchment on southeast Ruapehu requires an anomalously low temperature change (c. -4 ◦C) to simulate the LGCP glacier, in comparison with the rest of the catchments studied. Second, estimates of palaeotemperaure change from catchments on Mt. Ruapehu are systematically lower than those from Tongariro massif, by c. 1 ◦C. Accounting for known topographic changes since the LGCP (see below) does not resolve these differ- ences. The result from the Wahianoa catchment is difﬁcult to explain. Cosmogenic surface exposure ages provide reasonable age constraint and there is little geomorphic evidence for greater ice extent outboard of the moraines targeted in Chapter 7. It is possible that topographic change in the upper catchment may have changed the ﬂow divides, but this is difﬁcult to reconstruct and geomorphic evidence is absent. The difference in palaeotemperature estimates between the volcanoes is also some- what enigmatic and reﬂects uncertainty of several aspects of the experiment. A likely candidate for causing this difference is the uncertainty in present-day precipitation distribution and annual totals, which arises due to the paucity of high-altitude cli- mate station measurements. I use empirical precipitation data from > 1000 m asl on Mt. Ruapehu to create the modern climate grids, however no such measurements are available from Tongariro massif. 8.3.3.2 Numerical glacier modelling Thus, the palaeotemperature estimates may reﬂect an underestimation of precipitation on Tongariro. Similarly, changing precipitation pat- terns in the past may also contribute to this difference. Synoptic type frequency change at the LGCP (Lorrey et al., 2012b) may have changed the north-south precipitation gradient, meaning snow accumulation on Tongariro massif may be underestimated in the climate datasets. Finally, poor ﬁt between the modeled ice mass and the moraines in two catchments on Tongariro massif causes overestimation of the temperature forcing. This poor ﬁt may arise from model resolution, post-glacial inﬁlling of valley ﬂoors by volcanic products, or uncertainty in ﬂow parameters. 222 CHAPTER 8. SYNTHESIS 8.3.3.3 Topographic change The glacier simulations described above used models of the present day elevation as input for the glacier bed topography. This assumes that topography during the LGCP was similar to today. However, radiometric dating of lava ﬂows (e.g. Hobden et al., 1996; Gamble et al., 2003) and stratigraphic investigations of tephra and debris avalanche deposits (e.g. Palmer and Neall, 1989; Moebis et al., 2011) suggest that this is not the case. To assess the inﬂuence of possible changes in bed topography since the LGCP, I re-ran model simulations over a reconstructed >15 ka topography created by GNS Science geologists currently researching on the volcanic history of this region. This result generally increased the magnitude of cooling necessary to simulate the LGCP glaciers, by up to 0.5 ◦C. This reﬂects the the general lowering of the bed, caused by removal of post-glacial lava ﬂows. The topographic reconstruction is probably biased towards such changes, which are more readily identiﬁable in the landscape. Furthermore, pre-historic topographies are difﬁcult to constrain, particularly following destructional events. In these simulations, one catchment required lower magnitude cooling, which represents changes to the upper catchment boundaries that have reduced the ice ﬂux into this valley, relative to the simulations using modern topography. Accounting for known topographic changes did not improve the mis-matches in palaeotemperature estimates discussed above. This suggests that, either, (i) some topographic changes are unaccounted for; (ii) the mis-matches represent chronological uncertainties arising from the morphostratigraphic correlation; (iii) climatic gradients were signiﬁcantly different between the LGCP and today; or (iv) some combination of the aforementioned possibilities. However, the relatively minor effects of topographic change on palaeotemperature estimates, as demonstrated here using glacier modelling, does provide increased conﬁdence in the atmospheric cooling estimate of 4-7 ◦C in central North Island, during the LGCP. 8.3. RESEARCH QUESTIONS 223 8.3.4.1 Geomorphological and geochronological evidence In Chapter 6, I present geomorphological evidence from 3 adjacent valleys that contain moraines situated intermediate to the present day glacier termini and the ice limits of the LGCP. In the Mangaehuehu catchment, these moraines (termed ’LG’ in Chapter 6) were previously correlated with the LGCP by McArthur and Shepherd (1990). My own ﬁeld investigations support the interpretation of Barrell (2011) that the former Mangae- huehu glacier extended further down valley during the LGCP, which implies a younger age for the LG moraines. This interpretation is further supported by the glacier model simulations presented in Chapter 7, which predict that the glacier terminus in this catchment extended down valley of the LG moraines, when temperatures are reduced by >4 ◦C. The LG moraines overlie ﬁnely laminated glaciolacustrine sediments that display evidence for ductile and brittle deformation which suggests the Mangaehuehu glacier re-advanced to the location of the LG moraines, following an unknown amount of retreat. Cosmogenic 3He surface exposure ages of the LG moraines yield an arithmetic mean age of 13.4 ± 1.3 ka, after removal of outliers (see above for discussion of scatter). This accords with the best-estimate derived from simulations using a topographic diffusion model (e.g. Applegate et al., 2010). A less robust exposure age dataset from the adjacent Te Unuunuakapuateariki catchment, affords slightly older ages, but these landforms may represent composite features that were occupied multiple times. The glaciers that deposited the dated and inferred late-glacial moraines identiﬁed in this study can be simulated by a numerical glacier model, using a common climatic forcing. This supports the interpretation of coeval deposition. 8.3.4.2 Climatic signiﬁcance Glacier model experiments presented in Chapter 6 show that a change in temperature of -2.9 ◦C (when precipitation remains unchanged), relative to present, is necessary to simulate a steady-state glacial geometry on southern Mt. Ruapehu that accords with the geomorphological and geochronological constraints. As precipitation for this time period is relatively poorly constrained, I show that the likely cooling for this event lies between 2.5 - 3.4 ◦C, which represents the range of temperature change necessary to balance precipitation changes of ± 20% from present. This ﬁnding is indistinguishable CHAPTER 8. SYNTHESIS 224 from that of Doughty et al. (2013) for the ACR glacier in Irishman Stream (44◦S) and similar to that for nearby Whale Stream (Kaplan et al., 2013). The modelling experi- ments in the aforementioned study were conducted using the same model formulations for mass balance and ice ﬂow and forced by climatic data from the same source, which facilitates robust comparison of palaeotemperature estimates. Thus, these ﬁndings indicate that glaciers between 39 - 44◦S register a similar magnitude of cooling (c. 2-3 ◦C) during the late-glacial climate reversal in New Zealand. Recent pollen-based temperature reconstructions indicate a lower amplitude LGR temperature change in North Island, compared to South Island (Newnham et al., 2012). Previous research has suggested the late-glacial climate reversal was characterised by changing seasonality, as indicated by stronger response of summer-sensitive proxies such as chironomids, compared to winter-sensitive proxies such as vegetation (Vander- goes et al., 2008). Numerical model experiments suggest glacier lengths ﬂuctuations on Mt. Ruapehu are most sensitive to summer temperature. This accords with previous, similar investigation of seasonal sensitivity of Franz Josef Glacier (Oerlemans and Reichert, 2000) and suggests moraine records may be biased towards summer tempera- ture changes. Thus, changing seasonality during the late-glacial climate reversal, with greater summer cooling, represents a possible explanation for the discrepancy between glacier- and pollen-based palaeotemperature estimates. Uniform terrestrial cooling (at least during summer) across New Zealand during the ACR does not accord with changes in the oceans at this time. Carter et al. (2008) ﬁnd little to no response of sea surface temperature north of the sub-tropical front through this period. They implicate greater southward ﬂux of sub-tropical water to the vicinity of New Zealand, which is consistent with increasing tropical foram abundances at the site. 8.3.4.2 Climatic signiﬁcance Air temperature anomalies in New Zealand are intimately connected to the oceans, however this connection is bridged by regional atmospheric circulation (Salinger, 1980b). Thus, I hypothesise that the differential oceanic and atmospheric temperature response observed for North Island during the ACR may be reconciled by a synoptic type frequency change, whereby enhanced southerly- and suppressed northerly airﬂow, particularly during summer, lowers air temperatures across New Zealand. Analogous conditions occur today during El Ni˜no events (Jiang et al., 2013). Given the increasing number of well-resolved palaeoclimate reconstructions for this period, future research should aim to test this hypothesis using a regional climate regime classiﬁcation approach (e.g. Lorrey et al., 2012b, 2014; see below). 8.4. FURTHER RESEARCH 225 8.4.2 Development of other cosmogenic nuclides for use in the south west Paciﬁc Cosmogenic chlorine-36 (36Cl) was the ﬁrst in situ cosmogenic nuclide detected in terrestrial material (Davis and Schaeffer, 1955) and remains an important tool for earth science research, particularly in carbonate and basalt domains. In the south west Paciﬁc, 36Cl has been used in surface exposure dating applications to constrain the timing of mountain glacier ﬂuctuations (Barrows et al., 2002, 2007a, 2013; Mackintosh et al., 2006), but has also been widely applied elsewhere in the world to constrain rates of palaeoseismicity (e.g. Schlagenhauf et al., 2010). The half-life of 36Cl (c. 300 ka) also provides an opportunity to investigate complex exposure histories, when paired with other nuclides (e.g. 10Be, 3He). The accuracy of 36Cl surface exposure dates is currently hampered by uncertainty in the production rates. Production of 36Cl occurs via several pathways: spallation on potassium (K) and calcium (Ca), and to a lesser extent, iron (Fe) and titanium (Ti); negative muon capture by K and Ca; and thermal neutron capture by 35Cl (Dunai, 2010). The high abundance of 35Cl in nature and the complex controls on thermal neutron ﬂux are likely to be the main reasons for the existing uncertainty in 36Cl production rates (Schimmelpfennig et al., 2009). Schimmelpfennig et al. (2009) show that 36Cl surface measurements in bulk rock basalt samples are systematically higher than those derived from separated, pre-treated K-feldspar phases in the same sample. They conclude that preferential hosting of 35Cl in the groundmass has been unaccounted for in previous spallation production rate calibration studies and that mineral separates with low Cl content should be used instead of bulk rock samples. These ﬁndings, combined with developments presented in Chapter 4, present an opportunity to further develop cosmogenic 36Cl extraction and constrain production rates for the New Zealand region. Samples from the andesitic megaclasts deposited during the Murimotu Formation debris avalanche contain abundant pyroxene and K-feldspar, which can be easily separated from bulk rock samples using magnetic and density techniques (Chapter 4). Furthermore, the depositional setting and new, robust, independent radiocarbon age constraint of this event, provide an ideal setting for cosmogenic nuclide production rate calibration (Chapter 4). I propose that this site should be utilised, with the following aims: (i) to establish a Cl-decontamination scheme for measurement of 36Cl in pyroxene; and (ii) calibration of local K and Ca spallation production rates of in situ cosmogenic 36Cl. 8.4.1 Reﬁned estimates of local cosmogenic 3He production SYNTHESIS 226 8.4.1 Reﬁned estimates of local cosmogenic 3He production In Chapter 4, I show that the time-integrated (11- 0 ka) production rate of cosmogenic 3He in New Zealand is well approximated by an existing compilation of calibration sites (Goehring et al., 2010). However, the paucity of suitable andesitic samples in the Murimotu Formation debris avalanche precludes reﬁnement of the c. 10% uncertainty associated with the compiled production rate. It is desirable to reduce these uncertain- ties, through further addition of local calibration sites with robust sample sizes, in order to reduce the external uncertainties associated with surface exposure age calculation (e.g. Putnam et al., 2010b). Throughout the developmental stages of my doctoral research, I considered many potential sites for local calibration of cosmogenic 3He in New Zealand. The Muri- motu Formation provided the best potential for development of a robust, independent chronology using radiocarbon, although other possible sites of late-glacial-Holocene age are worthy of future reconnaissance work. For example, Saddle Cone lava ﬂow on the lower, northern ﬂanks of Mt Ruapehu (-39.22◦, 175.61◦), is a widespread deposit that ﬂowed below the altitude of the modern day treeline (c. 1300 m asl), therefore may have buried organic material suitable for radiocarbon dating. On Tongariro massif, the post-glacial, valley-ﬁlling lava ﬂow in the Oturere Valley (-39.17◦, 175.68◦) provides a similar depositional setting. Fluvial incision at the margins of both of these lava bodies affords the opportunity for natural exposure of buried organic horizons. Furthermore, both ﬂows display well-preserved ﬂow structures at the surface, such as pressure ridges, which indicate minimal surface erosion - ideal for cosmogenic surface exposure sampling of the in situ lava. I believe there is little scope for cosmogenic 3He production rate calibration on the cen- tral North Island volcanoes over timescales > c. 15 ka. Lava ﬂows and debris avalanche deposits in this age range have either been subject to previous burial by glacial advance, or exist at sufﬁciently low elevations to have been buried by soil/tephra. On Mt. Taranaki in eastern North Island, debris avalanche deposits of similar ap- pearance to the Murimotu Formation provide another potential site for production rate calibration. Of the the three main debris avalanche formations mapped by Neall (1979), the Pungaraehu and Opua formations probably afford the best opportunity for such work, due to their relative youth (< 23 ka), which means they remain well preserved at the land surface and are potentially dateable within the radiocarbon time frame. CHAPTER 8. 8.4.2 Development of other cosmogenic nuclides for use in the south west Paciﬁc All of the capabilities required to achieve this work (mineral separation, major element analysis, dissolution experi- ments), except for accelerator mass spectrometry, are available at Victoria University of 8.4. FURTHER RESEARCH 227 Wellington. The results would increase the utility of 36Cl for geoscientiﬁc applications in the south west Paciﬁc, particularly in older (several Myr) igneous domains such as Banks Peninsula and the sub-Antarctic Islands, where high uncertainties in cosmogenic 3He may be incurred due to potential magmatic/nucleogenic sources. 8.4.4 Holocene glacier ﬂuctuations in the Southern Hemisphere Constraining the timing and magnitude of glacier ﬂuctuations during the Holocene provides important context for current glacial retreat, as well as important insight to the controls on glacier extent under interglacial climatic conditions. Recent 10Be chronolo- gies from the Southern Alps have noted important differences from the well-established Northern Hemisphere based model of Holocene climate change. For example, whilst northern glaciers were similar to or smaller in extent than present during the early Holocene (e.g. Goehring et al., 2011), moraine chronologies from New Zealand show that glacier termini slowly retreated between their late-glacial and present positions from c. 13 ka until at least c. 6-7 ka (Schaefer et al., 2009; Putnam et al., 2012; Ka- plan et al., 2013). Mueller Glacier achieved its pre-industrial extent by c. 3.5 ka, and ﬂuctuated about this position until the mid-1900s (Schaefer et al., 2009). Meanwhile, glaciers in the Arrowsmith and Ben Ohau ranges also achieved their pre-industrial positions prior to the ’Little Ice Age’ (LIA; c. 1450 - 1850 AD) (Putnam et al., 2012; Kaplan et al., 2013). This contrasts with a large body of evidence from glaciers in the Northern Hemisphere, which were generally more extensive during this historical cold spell than at any other time in the Holocene. Recent compilation of palaeoclimate proxy records for the New Zealand region suggest that the mean climate state for the LIA was characterised by lower temperatures (summer temperatures were depressed by 0.6± 0.3 ◦C), increased precipitation and lower than present sea surface temperatures in the Tasman Sea (Lorrey et al., 2014). This study attributes these conditions to increased frequency of weak El Ni˜no-Modoki and negative Southern Annular Mode. The geomorphological records of glacier ﬂuctuations on Mt. Ruapehu provide the op- portunity to contribute to our understanding of LIA, and potentially pre-LIA, Holocene climate variability in New Zealand. Two catchments in particular contain suites of moraines that constrain Holocene ice geometries - based on historical photographic evidence, morphostratigraphic correlation and preliminary cosmogenic 3He surface exposure dates. In the Mangaehuehu catchment, prominent lateral moraines extend c. 500 m down valley from the current glacier terminus. Historical photographic evidence shows that the surface of the Mangaehuehu glacier was close to these limits in the early 20th century. Older, more fragmentary moraine landforms also exist outboard of these former glacier limits, but upstream of the late-glacial moraines described in Chapter 6. 8.4.3 What caused the Antarctic Cold Reversal? The results presented in Chapter 6 and the recent ﬁndings of several other climate re- construction studies indicate that air temperatures from the sub-Antarctics to northern New Zealand declined by 2 - 3◦C during the Antarctic Cold Reversal (c.14.6 - 12.7 ka). The ultimate cause of this cooling remains unknown. It is commonly accepted that this southern cooling event represents a bipolar seesaw effect, whereby resumption of overturning in the North Atlantic (AMOC) leads to net heat export from the Southern Ocean (Crowley, 1992). But what triggered this? One possibility is that AMOC recovery occurred in response to cessation of freshwater input to the North Atlantic at the end of Heinrich Stadial 1 (Liu et al., 2009). Another possibility is that AMOC was restarted by a meltwater pulse delivered to the Southern Ocean from Antarctica (Weaver et al., 2003). Comparing the effects of each of these hypotheses predicted by coupled atmo- spheric/oceanic circulation models (e.g. Weaver et al., 2003; Liu et al., 2009; He et al., 2013), to proxy data could provide an effective evaluation of these proposed triggers. Quantitative palaeoclimate reconstructions from New Zealand are well situated to help resolve these outstanding questions and there is a growing number of well-dated, point-based proxy records. Glacier model applications such as that presented in Chap- ter 6 and Doughty et al. (2013), conducted at the growing number of sites with dated moraines that depict glacial geometries during the Antarctic Cold Reversal, would permit detailed analysis of the spatial pattern of cooling during this event. Integrating such results with other, well-dated quantitative and qualitative palaeoclimate recon- structions (e.g. Newnham et al., 2012; Stephens et al., 2012a), using an approach such as that of Lorrey et al. (2014), would provide a holistic assessment of temperature, precipitation and synoptic type frequency anomalies that characterised this cooling event. Comparison of these spatially distributed, proxy-driven reconstructions to cli- mate model outputs (e.g. Liu et al., 2009; He et al., 2013) would help to test the existing hypotheses that seek to explain the climatic driver(s) at this time. 228 CHAPTER 8. SYNTHESIS 8.4.4 Holocene glacier ﬂuctuations in the Southern Hemisphere I have a preliminary set of cosmogenic 3He surface exposure dates that suggest the glacier was extended at c. 5-6 ka (outboard moraines) and 0.8-0.2 ka (inboard moraines). On the northwest of Mt. Ruapehu, an even greater number of Holocene moraines are preserved immediately down valley of the present-day Mangatoetoenui glacier. No sampling of boulders on these landforms has yet been undertaken for cosmogenic 8.4. FURTHER RESEARCH 229 surface exposure dating. Constraining the age and ELA depressions/model-based temperature depressions associated with these former glacial geometries will increase the spatial and temporal coverage of quantitative Holocene temperature estimates in New Zealand. Results from such work would provide increased constraint on the amplitude of natural climate ﬂuctuations during global interglacial conditions and help to test hypothesised drivers of Holocene climate change (e.g. Putnam et al., 2012). 230 CHAPTER 8. SYNTHESIS CHAPTER 8. SYNTHESIS Bibliography Ackert, R. P., Singer, B. S., Guillou, H., Kaplan, M. R., and Kurz, M. D. (2003). Long-term cosmogenic 3He production rates from 40Ar/39Ar and K-Ar dated Patagonian lava ﬂows at 47◦S. Earth and Planetary Science Letters, 210(1-2):119–136. Adhemar, J. (1842). Revolutions de la Mer: D´eluges P´eriodiques. Carilian-Goeury et V. Dalmont, Paris, France. Agassiz, L. (1840). ´Etudes sur les glaciers. Jent and Gassmann, Neuchˆatel, France Ahn, J. and Brook, E. J. (2008). Atmospheric CO2 and climate on millennial time scales during the last glacial period. Science, 322(5898):83–85. Alexander, D., Davies, T., and Shulmeister, J. (2014). Formation of the waiho loop terminal moraine, new zealand. Journal of Quaternary Science, 29(4):361–369. Allen, C. S., Pike, J., and Pudsey, C. J. (2011). Last glacial-interglacial sea-ice cover in the SW Atlantic and its potential role in global deglaciation. Quaternary Science Reviews, 30(19-20):2446–2458. Alloway, B. V., Lowe, D. J., Barrell, D. J. A., Newnham, R. M., Almond, P. C., Augustinus, P. C., Bertler, N. A. N., Carter, L., Litchﬁeld, N. J., McGlone, M. S., Shulmeister, J., Vandergoes, M. J., Williams, P. W., et al. (2007). Towards a climate event stratigraphy for New Zealand over the past 30 000 years (NZ-INTIMATE project). Journal of Quaternary Science, 22(1):9–35. Almond, P., Moar, N., and Lian, O. (2001). Reinterpretation of the glacial chronology of South Westland, New Zealand. New Zealand Journal of Geology and Geophysics, 44(1):1–15. Amidon, W. H. and Farley, K. A. (2011). Cosmogenic 3He production rates in apatite, zircon and pyroxene inferred from Bonneville ﬂood erosional surfaces. Quaternary Geochronology, 6(1):10–21. Amidon, W. H. and Farley, K. A. (2012). Cosmogenic 3He and 21Ne dating of biotite and hornblende. Earth and Planetary Science Letters, 313-314(1):86–94. 231 232 BIBLIOGRAPHY Amidon, W. H., Farley, K. A., Burbank, D. W., and Pratt Sitaula, B. (2008). Anomalous cosmogenic 3He production and elevation scaling in the high Himalaya. Earth and Planetary Science Letters, 265(1-2):287–301. Amidon, W. H., Farley, K. A., Burbank, D. W., and Pratt Sitaula, B. (2008). Anomalous cosmogenic 3He production and elevation scaling in the high Himalaya. Earth and Planetary Science Letters, 265(1-2):287–301. Amidon, W. H., Rood, D. H., and Farley, K. A. (2009). Cosmogenic 3He and 21Ne production rates calibrated against 10Be in minerals from the Coso volcanic ﬁeld. Earth and Planetary Science Letters, 280(1-4):194–204. Anderson, B., Lawson, W., Owens, I., and Goodsell, B. (2006). Past and future mass balance of ’Ka Roimata o Hine Hukatere’ Franz Josef Glacier, New Zealand. Bibliography Journal of Glaciology, 52(179):597–607. Anderson, B. and Mackintosh, A. (2006). Temperature change is the major driver of late-glacial and Holocene glacier ﬂuctuations in New Zealand. Geology, 34(2):121–124. Anderson, B. and Mackintosh, A. (2012). Controls on mass balance sensitivity of maritime glaciers in the Southern Alps, New Zealand: The role of debris cover. Journal of Geophysical Research: Earth Surface, 117(F1):F01003. Anderson, B., Mackintosh, A., Stumm, D., George, L., Kerr, T., Winter Billington, A., and Fitzsimons, S. (2010). Climate sensitivity of a high-precipitation glacier in New Zealand. Journal of Glaciology, 56(195):114–128. Anderson, R. F., Ali, S., Bradtmiller, L. I., Nielsen, S. H. H., Fleisher, M. Q., Anderson, B. E., and Burckle, L. H. (2009). Wind-driven upwelling in the southern ocean and the deglacial rise in atmospheric CO2. Science, 323(5920):1443–1448. Andrews, J. (1975). Glacial Systems. An Approach to Glaciers and Their Environments. Duxbury Press, North Scituate, USA. Applegate, P. J., Lowell, T. V., and Alley, R. B. (2008). Comment on ”absence of cooling in New Zealand and the adjacent ocean during the Younger Dryas chronozone”. Science, 320(5877). Applegate, P. J., Urban, N. M., Keller, K., Lowell, T. V., Laabs, B. J. C., Kelly, M. A., and Alley, R. B. (2012). Improved moraine age interpretations through explicit matching of geomorphic process models to cosmogenic nuclide measurements from single landforms. Quaternary Research, 77(2):293–304. Applegate, P. J., Urban, N. M., Laabs, B. J. C., Keller, K., and Alley, R. B. (2010). Modeling the statistical distributions of cosmogenic exposure dates from moraines. Geoscientiﬁc Model Development, 3:293–307. 233 BIBLIOGRAPHY Arz, H. W., P¨atzold, J., and Wefer, G. (1998). Correlated millennial-scale changes in surface hydrography and terrigenous sediment yield inferred from last-glacial marine deposits off northeastern Brazil. Quaternary Research, 50(2):157–166. Arz, H. W., P¨atzold, J., and Wefer, G. (1998). Correlated millennial-scale changes in surface hydrography and terrigenous sediment yield inferred from last-glacial marine deposits off northeastern Brazil. Quaternary Research, 50(2):157–166. Atkins, C. B. (2003). Characteristics of striae and clast shape in glacial and non-glacial environments. Phd thesis, Victoria University of Wellington. Aubry, M.-P., Van Couvering, J. A., Christie-Blick, N., Landing, E., Pratt, B. R., Owen, D. E., and Ferrusquia-Villafranca, I. (2009). Terminology of geological time: Estab- lishment of a community standard. Stratigraphy, 6(2):100–105. Augustinus, P., Cochran, U., Kattel, G., D’Costa, D., and Shane, P. (2012). Late Quater- nary paleolimnology of Onepoto maar, Auckland, New Zealand: implications for the drivers of regional paleoclimate. Quaternary International, 26:389–401. Balco, G. (2011). Bibliography Contributions and unrealized potential contributions of cosmogenic- nuclide exposure dating to glacier chronology, 1990–2010. Quaternary Science Reviews, 30(1):3–27. Balco, G., Stone, J. O., Lifton, N. A., and Dunai, T. J. (2008). A complete and easily accessible means of calculating surface exposure ages or erosion rates from 10Be and 26Al measurements. Quaternary Geochronology, 4:93–107. Barker, S., Diz, P., Vautravers, M. J., Pike, J., Knorr, G., Hall, I. R., and Broecker, W. S. (2009). Interhemispheric Atlantic seesaw response during the last deglaciation. Nature, 457(7233):1097–1102. Barnola, J., Raynaud, D., Lorius, C., and Korotkevich, Y. (1987). Vostok ice core provides 160,000-year record of atmospheric CO2. Nature, 329:408–414. Barrell, D. J. A. (2011). Quaternary glaciers of New Zealand. In Ehlers, J., Gibbard, P., and Hughes, P., editors, Developments in Quaternary Science, volume 15, pages 1047–1064. Elsevier, Amsterdam, The Netherlands. Barrell, D. J. A. (2014). The Balmoral moraines near Lake Pukaki, Southern Alps: a new reference area for the early Otira Glaciation in New Zealand. New Zealand Journal of Geology and Geophysics, 57:442–452. Barrell, D. J. A., Almond, P. C., Vandergoes, M. J., Lowe, D. J., and Newnham, R. M. (2013). A composite pollen-based stratotype for inter-regional evaluation of cli- matic events in New Zealand over the past 30,000 years (NZ-INTIMATE project). Quaternary Science Reviews, 74:4–20. 234 BIBLIOGRAPHY Barrell, D. J. A., Andersen, B. G., and Denton, G. H. (2011). Glacial geomorphology of the central South Island, New Zealand. GNS Science monograph 27. 81 p. + map (5 sheets) + legend (1 sheet). Barrell, D. J. A. and Read, S. A. L. (2014). The deglaciation of Lake Pukaki, South Island, New Zealand–a review. New Zealand Journal of Geology and Geophysics, 57(1):86–101. Barrows, T. T., Almond, P., Rose, R., Keith Fiﬁeld, L., Mills, S. C., and Tims, S. G. (2013). Late Pleistocene glacial stratigraphy of the Kumara-Moana region, West Coast of South Island, New Zealand. Quaternary Science Reviews, 74:139–159. Barrows, T. T., Juggins, S., De Deckker, P., Calvo, E., and Pelejero, C. (2007a). Long-term sea surface temperature and climate change in the Australian-New Zealand region. Paleoceanography, 22(2):PA2215. Barrows, T. T., Lehman, S. J., Fiﬁeld, L. K., and De Deckker, P. (2007b). Absence of cool- ing in New Zealand and the adjacent ocean during the Younger Dryas chronozone. Science, 318(5847):86–89. Barrows, T. T., Lehman, S. J., Fiﬁeld, L. K., and De Deckker, P. (2008). Bibliography Response to comment on ”Absence of cooling in New Zealand and the adjacent ocean during the Younger Dryas chronozone”. Science, 320(5877):746. Barrows, T. T., Stone, J. O., Fiﬁeld, L. K., and Cresswell, R. G. (2001). Late Pleistocene glaciation of the Kosciouszko Massif, Snowy Mountains, Australia. Quaternary Research, 55(2):179–189. Barrows, T. T., Stone, J. O., Fiﬁeld, L. K., and Cresswell, R. G. (2002). The timing of the Last Glacial Maximum in Australia. Quaternary Science Reviews, 21(1-3):159–173. Bartlein, P., Harrison, S., Brewer, S., Connor, S., Davis, B., Gajewski, K., Guiot, J., Harrison Prentice, T., Henderson, A., Peyron, O., et al. (2011). Pollen-based conti- nental climate reconstructions at 6 and 21 ka: a global synthesis. Climate Dynamics, 37(3-4):775–802. Basher, L. and McSaveney, M. (1989). An early Aranuian glacial advance at Cropp River, central Westland, New Zealand. Journal of the Royal Society of New Zealand, 19(3):263–268. Bauer, C. A. (1947). Production of helium in meteorites by cosmic radiation. Physical review, 72(4):354. Benn, D. I. and Evans, D. J. A. (2010). Glaciers and Glaciation. Hodder Education, London, UK. 235 BIBLIOGRAPHY Benn, D. I., Kirkbride, M. P., Owen, L. A., and Brazier, V. (2003). Glaciated valley landsystems. In Evans, D. J. A., editor, Glacial Landsystems, pages 372–406. Hodder Arnold, London, UK. Benn, D. I., Kirkbride, M. P., Owen, L. A., and Brazier, V. (2003). Glaciated valley landsystems. In Evans, D. J. A., editor, Glacial Landsystems, pages 372–406. Hodder Arnold, London, UK. Benn, D. I. and Lehmkuhl, F. (2000). Mass balance and equilibrium-line altitudes of glaciers in high-mountain environments. Quaternary International, 65:15–29. Benn, D. I., Owen, L. A., Osmaston, H. A., Seltzer, G. O., Porter, S. C., and Mark, B. (2005). Reconstruction of equilibrium-line altitudes for tropical and sub-tropical glaciers. Quaternary International, 138-139:8–21. Bilderback, E. L. (2012). Hillslope response to climate-modulated river incision and the role of deep-seated landslides in post-glacial sediment ﬂux: Waipaoa Sedimentary System, New Zealand. Ph.D. Thesis, University of Canterbury, New Zealand. Blard, P.-H., Braucher, R., Lav, J., and Bourl´es, D. (2013a). Cosmogenic 10Be production rate calibrated against 3He in the high Tropical Andes (3800-4900 m, 20-22◦S). Earth and Planetary Science Letters, 382:140–149. Blard, P.-H., Lav´e, J., Pik, R., Wagnon, P., and Bourl`es, D. (2007). Persistence of full glacial conditions in the central Paciﬁc until 15,000 years ago. Nature, 449(7162):591– 594. Blard, P.-H., Lav´e, J., Sylvestre, F., Placzek, C. J., Claude, C., Galy, V., Condom, T., and Tibari, B. (2013b). Bibliography Cosmogenic 3He production rate in the high tropical Andes (3800 m, 20◦S): Implications for the local last glacial maximum. Earth and Planetary Science Letters, 377-378:260–275. Blard, P.-H., Pik, R., Lav, J., Bourls, D., Burnard, P. G., Yokochi, R., Marty, B., and Trusdell, F. (2006). Cosmogenic 3He production rates revisited from evidences of grain size dependent release of matrix-sited helium. Earth and Planetary Science Letters, 247(3-4):222–234. Blunier, T. and Brook, E. J. (2001). Timing of millennial-scale climate change in Antarc- tica and Greenland during the last glacial period. Science, 291(5501):109–112. Bossard, L. (1928). Origin of the conical hills in the neighbourhood of Mount Egmont. N.Z. Journal of Science and Technology, 10:119–124. Bostock, H., Barrows, T., Carter, L., Chase, Z., Cortese, G., Dunbar, G., Ellwood, M., Hayward, B., Howard, W., Neil, H., Noble, T., Mackintosh, A., Moss, P., Moy, A., White, D., Williams, M., and Armand, L. (2013). A review of the AustralianNew Zealand sector of the Southern Ocean over the last 30 ka (Aus-INTIMATE project). Quaternary Science Reviews, 74:35 – 57. 236 BIBLIOGRAPHY BIBLIOGRAPHY Braconnot, P., Harrison, S. P., Kageyama, M., Bartlein, P. J., Masson Delmotte, V., Abe Ouchi, A., Otto Bliesner, B., and Zhao, Y. (2012). Evaluation of climate models using palaeoclimatic data. Nature Climate Change, 2(6):417–424. Braconnot, P., Otto Bliesner, B., Harrison, S., Joussaume, S., Peterchmitt, J.-Y., Abe Ouchi, A., Cruciﬁx, M., Driesschaert, E., Fichefet, T., Hewitt, C., et al. (2007). Results of PMIP2 coupled simulations of the Mid-Holocene and Last Glacial Maximum–Part 1: experiments and large-scale features. Climate of the Past, 3(2):261–277. Bretz, J. H. (1969). The Lake Missoula ﬂoods and the channeled scabland. The Journal of Geology, 77:505–543. Briner, J. P. (2009). Moraine pebbles and boulders yield indistinguishable 10Be ages: A case study from Colorado, USA. Quaternary Geochronology, 4(4):299 – 305. Broccoli, A. and Manabe, S. (1987). The inﬂuence of continental ice, atmospheric CO2, and land albedo on the climate of the last glacial maximum. Climate Dynamics, 1(2):87–99. Brock, B., Rivera, A., Casassa, G., Bown, F., and Acu˜nn, C. (2007). The surface energy balance of an active ice-covered volcano: Villarrica Volcano, southern Chile. Annals of Glaciology, 45(1):104–114. Brock, B. W., Willis, I. C., and Sharp, M. J. (2000). Measurement and parameterization of albedo variations at Haut Glacier d’Arolla, Switzerland. Journal of Glaciology, 46(155):675–688. Brock, B. W., Willis, I. C., and Sharp, M. J. (2006). Measurement and parameterization of aerodynamic roughness length variations at Haut Glacier d’Arolla, Switzerland. Journal of Glaciology, 52(177):281–297. Broecker, W. S. (1966). Absolute dating and the astronomical theory of glaciation. Science, 151(3708):299–304. Broecker, W. S. (1978). The cause of glacial to interglacial climatic change. In Evolution of Planetary Atmospheres and Climatology of the Earth, pages 165–190. Centre National d’Etudes Spatiales, Toulouse, France. Broecker, W. S. (1982). Ocean chemistry during glacial time. Geochimica et Cosmochimica Acta, 46(10):1689–1705. Broecker, W. S. (1998). Paleocean circulation during the last deglaciation: A bipolar seesaw? Paleoceanography, 13(2):119–121. Broecker, W. S. (2013). What Drives Glacial Cycles? Eldigio Press, New York, USA. BIBLIOGRAPHY 237 Broecker, W. S. and Denton, G. H. (1990). The role of ocean-atmosphere reorganizations in glacial cycles. Quaternary Science Reviews, 9(4):305–341. Broecker, W. S. and Denton, G. H. (1990). The role of ocean-atmosphere reorganizations in glacial cycles. Quaternary Science Reviews, 9(4):305–341. Broecker, W. S. and Donk, J. (1970). Insolation changes, ice volumes, and the O18 record in deep-sea cores. Reviews of Geophysics, 8(1):169–198. Broecker, W. S., Thurber, D. L., Goddard, J., Ku, T.-L., Matthews, R., and Mesolella, K. J. (1968). Milankovitch hypothesis supported by precise dating of coral reefs and deep-sea sediments. Science, 159(3812):297–300. Bromley, G. R., Schaefer, J. M., Winckler, G., Hall, B. L., Todd, C. E., and Rademaker, K. M. (2009). Relative timing of last glacial maximum and late-glacial events in the central tropical Andes. Quaternary Science Reviews, 28(23):2514–2526. Bromley, G. R., Winckler, G., Schaefer, J. M., Kaplan, M. R., Licht, K. J., and Hall, B. L. (2014). Pyroxene separation by HF leaching and its impact on helium surface- exposure dating. Quaternary Geochronology, 23:1–8. Bromley, G. R. M., Hall, B. L., Schaefer, J. M., Winckler, G., Todd, C. E., and Rade- maker, K. M. (2011). Glacier ﬂuctuations in the southern Peruvian Andes during the late-glacial period, constrained with cosmogenic 3He. Journal of Quaternary Science, 26(1):37–43. Bronk Ramsey, C. (2009). Bayesian analysis of radiocarbon dates. Radiocarbon, 51(1). Brook, M. S. (2009). Lateral moraine age in Park Valley, Tararua Range, New Zealand. Journal of the Royal Society of New Zealand, 39(2-3):63–69. Brook, M. S. and Crow, T. V. H. (2008). A debris ridge in Park Valley, Tararua Range, New Zealand as evidence for Pleistocene glaciation. New Zealand Journal of Geology and Geophysics, 51(1):23–28. Brook, M. S., Dean, J. F., and Keys, H. J. R. (2011). Response of a mid-latitude cirque glacier to climate over the last two decades: Mangaehuehu Glacier, Mt Ruapehu. Earth Surface Processes and Landforms, 36(14):1973–1980. Brook, M. S., Purdie, H. L., and Crow, T. V. (2005). Valley cross-proﬁle morphology and glaciation in Park Valley, Tararua Range, New Zealand. Journal of the Royal Society of New Zealand, 35(4):399–407. Brook, M. S., Shulmeister, J., Crow, T. V. H., and Zondervan, A. (2008). First cosmogenic 10Be constraints on LGM glaciation on New Zealand’s North Islands: Park Valley, Tararua Range. Journal of Quaternary Science, 23(8):707–712. 238 BIBLIOGRAPHY Bruno, L. A., Baur, H., Graf, T., Signer, P., Wieler, R., et al. (1997). BIBLIOGRAPHY Dating of Sirius Group tillites in the Antarctic dry valleys with cosmogenic 3He and 21 Ne. Earth and Planetary Science Letters, 147(1):37–54. Bruno, L. A., Baur, H., Graf, T., Signer, P., Wieler, R., et al. (1997). Dating of Sirius Group tillites in the Antarctic dry valleys with cosmogenic 3He and 21 Ne. Earth and Planetary Science Letters, 147(1):37–54. Burrows, C. J. and Russell, J. B. (1975). Moraines of the upper Rakaia Valley. Journal of the Royal Society of New Zealand, 5(4):463–477. Carlson, A. E. and Clark, P. U. (2012). Ice sheet sources of sea level rise and freshwater discharge during the last deglaciation. Reviews of Geophysics, 50(4). Carter, L., Manighetti, B., Ganssen, G., and Northcote, L. (2008). Southwest Paciﬁc modulation of abrupt climate change during the Antarctic Cold Reversal–Younger Dryas. Palaeogeography, Palaeoclimatology, Palaeoecology, 260(1):284–298. Cerling, T. E. and Craig, H. (1994). Cosmogenic 3He production rates from 39◦N to 46◦N latitude, western USA and France. Geochimica et Cosmochimica Acta, 58(1):249–255. Chinn, T., Fitzharris, B. B., Willsman, A., and Salinger, M. J. (2012). Annual ice volume changes 1976-2008 for the New Zealand Southern Alps. Global and Planetary Change, 92-93:105–118. Chinn, T., Winkler, S., Salinger, M. J., and Haakensen, N. (2005). Recent glacier advances in Norway and New Zealand: a comparison of their glaciological and meteorological causes. Geograﬁska Annaler: Series A, Physical Geography, 87(1):141–157. Chinn, T. J. H. (1981). Use of rock weathering-rind thickness for Holocene absolute age-dating in New Zealand. Arctic and Alpine Research, 13:33–45. Chorley, R. (1962). Geomorphology and general systems theory. US Government Printing Ofﬁce, Washington DC, USA. Clark, P. U., Dyke, A. S., Shakun, J. D., Carlson, A. E., Clark, J., Wohlfarth, B., Mitrovica, J. X., Hostetler, S. W., and McCabe, A. M. (2009). The Last Glacial Maximum. Science, 325(5941):710–714. Clarke, G. K., Cross, G. M., and Benson, C. S. (1989). Radar imaging of glaciovolcanic stratigraphy, Mount Wrangell caldera, Alaska: interpretation model and results. Journal of Geophysical Research: Solid Earth, 94(B6):7237–7249. CLIMAP, P. M. (1976). The surface of the ice-age earth. Science, 191(4232):1131. Cogley, J., Hock, R., Rasmussen, L., Arendt, A., Bauder, A., Braithwaite, R., Jansson, P., Kaser, G., Moller, M., Nicholson, L., and Zemp, M. (2011). Glossary of Glacier Mass Balance and Related Terms. IHP-VII Technical Documents in Hydrology No. 86, IACS Contribution No. 2, UNESCO-IHP, Paris. BIBLIOGRAPHY 239 Cohen, K., Finney, S., Gibbard PL, and Fan, J.-X. (2013). The ICS International Chronos- tratigraphic Chart. Episodes, 36:199–204. BIBLIOGRAPHY Cohen, K., Finney, S., Gibbard PL, and Fan, J.-X. (2013). The ICS International Chronos- tratigraphic Chart. Episodes, 36:199–204. Cole, J. W. (1978). Andesites of the Tongariro Volcanic Centre, North Island, New Zealand. Journal of Volcanology and Geothermal Research, 3:121–153. Collins, L. G., Pike, J., Allen, C. S., and Hodgson, D. A. (2012). High-resolution reconstruction of southwest Atlantic sea-ice and its role in the carbon cycle during marine isotope stages 3 and 2. Paleoceanography, 27(3):PA3217. Columbus, J., Sirguey, P., and Tenzer, R. (2011). A free, fully accessible 15-m DEM for New Zealand. Survey Quarterly, 66:16–19. Conway, C., Townsend, D., Leonard, G., Wilson, C., Calvert, A., and Gamble, J. (2015). Lava-ice interaction on a large composite volcano: a case study from Ruapehu, New Zealand. Bulletin of Volcanology, 77(21). Conway, J., Cullen, N., Spronken-Smith, R., and Fitzsimons, S. (2014). All-sky radiation over a glacier surface in the Southern Alps of New Zealand: characterizing cloud effects on incoming shortwave, longwave and net radiation. International Journal of Climatology. Corripio, J. G. (2003). Vectorial algebra algorithms for calculating terrain parameters from DEMs and solar radiation modelling in mountainous terrain. International Journal of Geographical Information Science, 17(1):1–23. Craig, H., Poreda, R., Lupton, J. E., Marti, K., and Regnier, S. (1979). Rare gases and hydrogen in josephinite. EOS Trans. Am. Geophys. Union, 60(46):970. Craig, H. and Poreda, R. J. (1986). Cosmogenic 3He in terrestrial rocks: The summit lavas of Maui. Proceedings of the National Academy of Sciences, 83(7):1970–1974. Croll, J. (1864). XIII. On the physical cause of the change of climate during geological epochs. The London, Edinburgh, and Dublin Philosophical Magazine and Journal of Science, 28(187):121–137. Cronin, S. J. and Neall, V. E. (1997). A late quaternary stratigraphic framework for the northeastern Ruapehu and eastern Tongariro ring plains, New Zealand. New Zealand Journal of Geology and Geophysics, 40(2):185–197. Crowley, T. J. (1992). North Atlantic deep water cools the Southern Hemisphere. Paleoceanography, 7(4):489–497. Cuffey, K. and Paterson, W. (2010). The physics of glaciers. Butterworth- Heinemann/Elsevier, Burlington, USA. 240 BIBLIOGRAPHY BIBLIOGRAPHY Curry, A. M., Sands, T. B., and Porter, P. R. (2009). Geotechnical controls on a steep lateral moraine undergoing paraglacial slope adjustment. 320:181–197. Curry, A. M., Sands, T. B., and Porter, P. R. (2009). Geotechnical controls on a steep lateral moraine undergoing paraglacial slope adjustment. 320:181–197. Dadic, R., Mott, R., Lehning, M., and Burlando, P. (2010). Parameterization for wind– induced preferential deposition of snow. Hydrological processes, 24(14):1994–2006. Dansgaard, W., Clausen, H., Gundestrup, N., Hammer, C., Johnsen, S., Kristinsdottir, P., and Reeh, N. (1982). A new Greenland deep ice core. Science, 218(4579):1273–1277. Dansgaard, W., Johnsen, S., Clausen, H., Dahl Jensen, D., Gundestrup, N., Hammer, C., Hvidberg, C., Steffensen, J., Sveinbj¨ornsdottir, A., Jouzel, J., et al. (1993). Evi- dence for general instability of past climate from a 250-kyr ice-core record. Nature, 364(6434):218–220. Dansgaard, W., Johnsen, S., Clausen, H., Dahl Jensen, D., Gundestrup, N., Hammer, C., and Oeschger, H. (1984). North Atlantic climatic oscillations revealed by deep Greenland ice cores. In Hansen, J. E. and Takahashi, T., editors, Climate processes and climate sensitivity, pages 288–298. American Geophysical Union, Washington DC, USA. Dansgaard, W., Johnsen, S. J., Møller, J., and Langway, C. C. (1969). One thousand centuries of climatic record from Camp Century on the Greenland ice sheet. Science, 166(3903):377–380. Davis, R. and Schaeffer, O. A. (1955). Chlorine-36 in nature. Annals of the New York Academy of Sciences, 62(5):107–121. De Deckker, P., Moros, M., Perner, K., and Jansen, E. (2012). Inﬂuence of the tropics and southern westerlies on glacial interhemispheric asymmetry. Nature Geoscience, 5(4):266–269. Denton, G. and Hendy, C. (1994). Younger Dryas age advance of Franz Josef glacier in the southern Alps of New Zealand. Science, 264(5164):1434–1437. Denton, G. H., Alley, R. B., Comer, G. C., and Broecker, W. S. (2005). The role of seasonality in abrupt climate change. Quaternary Science Reviews, 24(10):1159–1182. Denton, G. H., Anderson, R. F., Toggweiler, J. R., Edwards, R. L., Schaefer, J. M., and Putnam, A. E. (2010). The last glacial termination. Science, 328(5986):1652–1656. Denton, G. H., Lowell, T. V., Heusser, C. J., Schl¨uchter, C., Andersen, B. G., Heusser, L. E., Moreno, P. I., and Marchant, D. R. (1999). Geomorphology, stratigraphy, and radiocarbon chronology of Llanquihue drift in the area of the southern Lake District, Seno Reloncavi, and Isla Grande de Chiloe, Chile. Geograﬁska Annaler, Series A: Physical Geography, 81(2):167–229. 241 BIBLIOGRAPHY Deschamps, P., Durand, N., Bard, E., Hamelin, B., Camoin, G., Thomas, A. L., Hender- son, G. BIBLIOGRAPHY M., Okuno, J., and Yokoyama, Y. (2012). Ice-sheet collapse and sea-level rise at the Bolling warming 14,600 [thinsp] years ago. Nature, 483(7391):559–564. Deschamps, P., Durand, N., Bard, E., Hamelin, B., Camoin, G., Thomas, A. L., Hender- son, G. M., Okuno, J., and Yokoyama, Y. (2012). Ice-sheet collapse and sea-level rise at the Bolling warming 14,600 [thinsp] years ago. Nature, 483(7391):559–564. Desilets, D., Zreda, M., and Prabu, T. (2006). Extended scaling factors for in situ cosmogenic nuclides: New measurements at low latitude. Earth and Planetary Science Letters, 246(3-4):265–276. Dieffenbacher Krall, A., Vandergoes, M., and Denton, G. (2007). An inference model for mean summer air temperatures in the Southern Alps, New Zealand, using subfossil chironomids. Quaternary Science Reviews, 26(19-21):2487–2504. Donoghue, S. and Neall, V. (1996). Tephrostratigraphic studies at Tongariro volcanic centre, New Zealand: an overview. Quaternary International, 34:13–20. Donoghue, S., Neall, V., Palmer, A., and Stewart, R. (1997). The volcanic history of Ruapehu during the past 2 millennia based on the record of Tufa Trig tephras. Bulletin of Volcanology, 59(2):136–146. Donoghue, S. L. and Neall, V. E. (2001). Late Quaternary constructional history of the southeastern Ruapehu ring plain, New Zealand. New Zealand Journal of Geology and Geophysics, 44(3):439–466. Donoghue, S. L., Neall, V. E., and Palmer, A. S. (1995). Stratigraphy and chronology of late Quaternary andesitic tephra deposits, Tongariro volcanic Centre, New Zealand. Journal - Royal Society of New Zealand, 25(2):115–206. Donoghue, S. L., Palmer, A. S., McClelland, E., Hobson, K., Stewart, R. B., and Neall, V. E. (1999). The Taurewa Eruptive Epsiode: evidence for climactic eruptions at Ruapehu volcano, New Zealand. Bulletin of Volcanology, 60:223–240. Doughty, A. M., Anderson, B. M., Mackintosh, A. N., Kaplan, M. R., Vandergoes, M. J., Barrell, D. J. A., Denton, G. H., Schaefer, J. M., Chinn, T. J. H., and Putnam, A. E. (2013). Evaluation of Lateglacial temperatures in the Southern Alps of New Zealand based on glacier modelling at Irishman Stream, Ben Ohau Range. Quaternary Science Reviews, 74:160–169. Drost, F., Renwick, J., Bhaskaran, B., Oliver, H., and McGregor, J. (2007). A simulation of New Zealand’s climate during the Last Glacial Maximum. Quaternary Science Reviews, 26(19-21):2505–2525. Dunai, T. J. (2001). Inﬂuence of secular variation of the geomagnetic ﬁeld on production rates of in situ produced cosmogenic nuclides. Earth and Planetary Science Letters, 193(1-2):197–212. 242 BIBLIOGRAPHY Dunai, T. J. (2010). Cosmogenic Nuclides: Principles, concepts and applications in the Earth surface sciences. Cambridge University Press, New York, USA. BIBLIOGRAPHY Dunai, T. J., Stuart, F. M., Pik, R., Burnard, P., and Gayer, E. (2007). Production of 3 He in crustal rocks by cosmogenic thermal neutrons. Earth and Planetary Science Letters, 258(1):228–236. Dunne, J., Elmore, D., and Muzikar, P. (1999). Scaling factors for the rates of production of cosmogenic nuclides for geometric shielding and attenuation at depth on sloped surfaces. Geomorphology, 27(1):3–11. Egholm, D. L., Knudsen, M. F., Clark, C. D., and Lesemann, J. E. (2011). Modeling the ﬂow of glaciers in steep terrains: The integrated second-order shallow ice ap- proximation (iSOSIA). Journal of Geophysical Research: Earth Surface (2003–2012), 116(F2):F02012. Ehlers, J., Ehlers, J., Gibbard, P. L., and Hughes, P. D. (2011). Quaternary glaciations-extent and chronology: a closer look. Elsevier, Amsterdam, The Netherlands. Emiliani, C. (1955). Pleistocene temperatures. The Journal of Geology, 63:538–578. EPICA-Community-Members., Augustin, L., Barbante, C., Barnes, P. R., Barnola, J. M., Bigler, M., Castellano, E., Cattani, O., Chappellaz, J., Dahl-Jensen, D., Delmonte, B., et al. (2004). Eight glacial cycles from an Antarctic ice core. Nature, 429(6992):623–628. EPICA-Community-Members., Barbante, C., Barnola, J., Becagli, S., Beer, J., Bigler, M., Boutron, C., Blunier, T., Castellano, E., Cattani, O., Chappellaz, J., et al. (2006). One- to-one coupling of glacial climate variability in Greenland and Antarctica. Nature, 444(7116):195–198. Epstein, S., Sharp, R. P., and Gow, A. (1970). Antarctic ice sheet: stable isotope analyses of Byrd station cores and interhemispheric climatic implications. Science, 168(3939):1570–1572. Evans, D. (2003). Glacial Landsystems. Hodder Arnold, London, UK. Evans, D. (2003). Glacial Landsystems. Hodder Arnold, London, UK. Evans, D. J., Rother, H., Hyatt, O. M., and Shulmeister, J. (2013). The glacial sedimentol- ogy and geomorphological evolution of an outwash head/moraine-dammed lake, South Island, New Zealand. Sedimentary Geology, 284285:45 – 75. Evans, E. (1887). The authorship of the glacial theory. The North American review, 145:94–97. 243 BIBLIOGRAPHY Farley, K. A., Libarkin, J., Mukhopadhyay, S., and Amidon, W. (2006). Cosmogenic and nucleogenic 3He in apatite, titanite, and zircon. Earth and Planetary Science Letters, 248(1-2):436–446. Farley, K. A., Libarkin, J., Mukhopadhyay, S., and Amidon, W. (2006). Cosmogenic and nucleogenic 3He in apatite, titanite, and zircon. Earth and Planetary Science Letters, 248(1-2):436–446. Farley, K. A., Libarkin, J., Mukhopadhyay, S., and Amidon, W. (2006). Cosmogenic and nucleogenic 3He in apatite, titanite, and zircon. Earth and Planetary Science Letters, 248(1-2):436–446. Fenton, C. R., Mark, D. F., Barfod, D. N., Niedermann, S., Goethals, M. M., and Stuart, F. M. (2013). 40Ar/39Ar dating of the SP and Bar Ten lava ﬂows AZ, USA: Laying the foundation for the SPICE cosmogenic nuclide production-rate calibration project. Quaternary Geochronology, 18:158–172. Fenton, C. R. and Niedermann, S. (2014). Surface exposure dating of young basalts (1–200 ka) in the San Francisco volcanic ﬁeld (Arizona, USA) using cosmogenic 3 He and 21 Ne. Quaternary Geochronology, 19:87–105. Fitzsimons, S. J. (1997). Late-glacial and early Holocene glacier activity in the Southern Alps, New Zealand. Quaternary International, 38:69–76. Fleming, K., Johnston, P., Zwartz, D., Yokoyama, Y., Lambeck, K., and Chappell, J. (1998). Reﬁning the eustatic sea-level curve since the Last Glacial Maximum using far-and intermediate-ﬁeld sites. Earth and Planetary Science Letters, 163(1):327–342. Foeken, J. P. T., Stuart, F. M., and Mark, D. F. (2012). Long-term low latitude cosmogenic 3He production rate determined from a 126ka basalt from Fogo, Cape Verdes. Earth and Planetary Science Letters, 359-360:14–25. Froggatt, P. C. and Lowe, D. J. (1990). A review of late Quaternary silicic and some other tephra formations from New Zealand: their stratigraphy, nomenclature, distribution, volume, and age. New Zealand Journal of Geology and Geophysics, 33(1):89–109. Fudge, T., Steig, E. J., Markle, B. R., Schoenemann, S. W., Ding, Q., Taylor, K. C., McConnell, J. R., Brook, E. J., Sowers, T., White, J. W., et al. (2013). Onset of deglacial warming in West Antarctica driven by local orbital forcing. Nature, 500:440–444. Furbish, D. J. and Andrews, J. T. (1984). Evans, D. (2003). Glacial Landsystems. Hodder Arnold, London, UK. The use of hypsometry to indicate long- term stability and response of valley glaciers to changes in mass transfer. Journal of Glaciology, 30(105):199–211. Gage, M. (1985). Glaciation in New Zealandthe ﬁrst century of research. Quaternary Science Reviews, 4(3):189–214. Gage, M. and Suggate, R. P. (1958). Glacial chronology of the New Zealand Pleistocene. Geological Society of America Bulletin, 69(5):589–598. Gamble, J. A., Price, R. C., Smith, I. E., McIntosh, W. C., and Dunbar, N. W. (2003). 40Ar/39Ar geochronology of magmatic activity, magma ﬂux and hazards at Ruapehu 244 BIBLIOGRAPHY volcano, Taupo Volcanic Zone, New Zealand. Journal of Volcanology and Geothermal Research, 120(34):271 – 287. Gayer, E., Pik, R., Lav´e, J., France-Lanord, C., Bourles, D., and Marty, B. (2004). Cos- mogenic 3he in Himalayan garnets indicating an altitude dependence of the 3he 10be production ratio. Earth and Planetary Science Letters, 229(1):91–104. Gildor, H. and Tziperman, E. (2001). Physical mechanisms behind biogeochemical glacial-interglacial CO2 variations. Geophysical Research Letters, 28(12):2421–2424. Glicken, H. (1986). Mt. St. Helens volcano. Ph.D. Thesis, University of California, Santa Barbara, California. Glicken, H. (1991). Sedimentary architecture of large volcanic-debris avalanches. In Fisher, R. V. and Smith, G. A., editors, Sedimentation in Volcanic Settings, volume 45, pages 99–106. Goehring, B. M., Kurz, M. D., Balco, G., Schaefer, J. M., Licciardi, J., and Lifton, N. (2010). A reevaluation of in situ cosmogenic 3He production rates. Quaternary Geochronology, 5(4):410–418. Goehring, B. M., Schaefer, J. M., Schl¨uchter, C., Lifton, N. A., Finkel, R. C., Jull, A. T., Akc¸ar, N., and Alley, R. B. (2011). The Rhone Glacier was smaller than today for most of the Holocene. Geology, 39(7):679–682. Golledge, N., Menviel, L., Carter, L., Fogwill, C., England, M., Cortese, G., and Levy, R. (2014). Antarctic contribution to meltwater pulse 1A from reduced Southern Ocean overturning. Nature communications, 5. Golledge, N. R. (2007). An ice cap landsystem for palaeoglaciological reconstructions: characterizing the Younger Dryas in western Scotland. Quaternary Science Reviews, 26(1):213–229. Golledge, N. R., Mackintosh, A. N., Anderson, B. M., Buckley, K. M., Doughty, A. M., Barrell, D. J. A., Denton, G. H., Vandergoes, M. J., Andersen, B. G., and Schaefer, J. M. (2012). Last Glacial Maximum climate in New Zealand inferred from a modelled Southern Alps iceﬁeld. Quaternary Science Reviews, 46:30–45. Gosse, J. C. and Phillips, F. M. (2001). Terrestrial in situ cosmogenic nuclides: theory and application. Quaternary Science Reviews, 20(14):1475–1560. Grange, L. and Williamson, J. (1930). Tongariro Subdivision. Evans, D. (2003). Glacial Landsystems. Hodder Arnold, London, UK. NZ Geological Survey 24th Annual Report, pages 10–13. 245 BIBLIOGRAPHY BIBLIOGRAPHY Greuell, W. (1992). Hintereisferner, Austria: mass-balance reconstruction and numerical modelling of the historical length variations. Journal of Glaciology, 38(129):233–244. Grubbs, F. (1969). Procedures for detecting outlying observations in samples. Techno- metrics, 11:1–21. Gudmundsson, M. T., Sigmundsson, F., and Bj¨ornsson, H. (1997). Ice–volcano interac- tion of the 1996 Gj´alp subglacial eruption, Vatnaj¨okull, Iceland. Nature, 389(6654):954– 957. Gudmundsson, M. T., Sigmundsson, F., Bj¨ornsson, H., and H¨ognad´ottir, T. (2004). The 1996 eruption at Gj´alp, Vatnaj¨okull ice cap, Iceland: efﬁciency of heat transfer, ice deformation and subglacial water pressure. Bulletin of Volcanology, 66(1):46–65. Hackett, W. R. and Houghton, B. F. (1989). A facies model for a quaternary andesitic composite volcano: Ruapehu, New Zealand. Bulletin of Volcanology, 51(1):51–68. Hajdas, I., Lowe, D. J., Newnham, R. M., and Bonani, G. (2006). Timing of the late-glacial climate reversal in the Southern Hemisphere using high-resolution radiocarbon chronology for Kaipo bog, New Zealand. Quaternary Research, 65(2):340–345. Hallet, B. and Putkonen, J. (1994). Surface dating of dynamic landforms: young boulders on aging moraines. Science, 265(5174):937–940. Hambrey, M. J. and Ehrmann, W. (2004). Modiﬁcation of sediment characteristics during glacial transport in high-alpine catchments: Mount Cook area, New Zealand. Boreas, 33(4):300–318. Hargreaves, J. C., Annan, J. D., Yoshimori, M., and Abe Ouchi, A. (2012). Can the Last Glacial Maximum constrain climate sensitivity? Geophysical Research Letters, 39(24):L24702. Hays, J. D., Imbrie, J., and Shackleton, N. J. (1976). Variations in the earth’s orbit: Pacemaker of the ice ages. Science, 194(4270):1121–1132. He, F., Shakun, J. D., Clark, P. U., Carlson, A. E., Liu, Z., Otto Bliesner, B. L., and Kutzbach, J. E. (2013). Northern Hemisphere forcing of Southern Hemisphere climate during the last deglaciation. Nature, 494(7435):81–85. Heine, A. (1962). Glacier changes on Mount Ruapehu, New Zealand:1957-1961. In Variations in the regime of existing glaciers. Proceedings of the Symposium of Obergurgl, pages 10–18. Heine, A. J. (1963). Mount Ruapehu ice and snow photo survey. New Zealand Journal of Geology and Geophysics, 6(2):261–267. 246 BIBLIOGRAPHY Heinrich, H. (1988). Origin and consequences of cyclic ice rafting in the northeast Atlantic Ocean during the past 130,000 years. Quaternary research, 29(2):142–152. Heisinger, B., Lal, D., Jull, A. J. T., Kubik, P., Ivy Ochs, S., Neumaier, S., Knie, K., Lazarev, V., and Nolte, E. (2002). Production of selected cosmogenic radionuclides by muons 1. Fast muons. Earth and Planetary Science Letters, 200(3-4):345–355. Hellstrom, J., McCulloch, M., and Stone, J. (1998). BIBLIOGRAPHY A detailed 31,000-year record of climate and vegetation change, from the isotope geochemistry of two New Zealand speleothems. Quaternary Research, 50:167–178. Hemming, S. R. (2004). Heinrich events: Massive late Pleistocene detritus layers of the North Atlantic and their global climate imprint. Reviews of Geophysics, 42(1):RG1005 1–43. Hendrikx, J. and Hreinsson, E. (2012). The potential impact of climate change on seasonal snow in New Zealand: part iiindustry vulnerability and future snowmaking potential. Theoretical and Applied Climatology, 110(4):619–630. Hewitt, K. (1999). Quaternary moraines vs catastrophic rock avalanches in the Karako- ram Himalaya, northern Pakistan. Quaternary Research, 51(3):220–237. Heyman, J., Stroeven, A. P., Harbor, J. M., and Caffee, M. W. (2011). Too young or too old: Evaluating cosmogenic exposure dating based on an analysis of compiled boulder exposure ages. Earth and Planetary Science Letters, 302(1-2):71–80. Hill, H. (1891). Tongariro, Ngauruhoe and Ruapehu as volcanic cones. Report of the third meeting of the Australasian Association for the Advancement of Science. p.162-172. Hindmarsh, R. C. and Le Meur, E. (2001). Dynamical processes involved in the retreat of marine ice sheets. Journal of Glaciology, 47(157):271–282. Hobden, B. J., Houghton, B. F., Davidson, J. P., and Weaver, S. D. (1999). Small and short-lived magma batches at composite volcanoes: Time windows at Tongariro volcano, New Zealand. Journal of the Geological Society, 156(5):865–868. Hobden, B. J., Houghton, B. F., Lanphere, M. A., and Nairn, I. A. (1996). Growth of the Tongariro volcanic complex: New evidence from K-Ar age determinations. New Zealand Journal of Geology and Geophysics, 39(1):151–154. Hobden, B. J., Houghton, B. F., and Nairn, I. A. (2002). Growth of a young, frequently active composite cone: Ngauruhoe volcano, New Zealand. Bulletin of Volcanology, 64(6):392–409. 247 BIBLIOGRAPHY BIBLIOGRAPHY Hock, R. (1999). A distributed temperature-index ice-and snowmelt model including potential direct solar radiation. Journal of Glaciology, 45(149):101–111. Hock, R. (1999). A distributed temperature-index ice-and snowmelt model including potential direct solar radiation. Journal of Glaciology, 45(149):101–111. Hock, R. (2003). Temperature index melt modelling in mountain areas. Journal of Hydrology, 282(1):104–115. Hogg, A., Hua, Q., Blackwell, P., Niu, M., Buck, C., Guilderson, T., Heaton, T., Palmer, J., Reimer, P., Reimer, R., Turney, C., and Zimmerman, S. (2013). SHCal13 Southern Hemisphere calibration, 0-50,000 years cal. BP. Radiocarbon, 55(4). Hooker, B. and Fitzharris, B. (1999). The correlation between climatic parameters and the retreat and advance of Franz Josef Glacier, New Zealand. Global and Planetary Change, 22(1):39–48. Houghton, B., Latter, J., and Hackett, W. (1987). Volcanic hazard assessment for Ru- apehu composite volcano, Taupo volcanic zone, New Zealand. Bulletin of volcanology, 49(6):737–751. Hubbard, B. and Glasser, N. (2005). Field techniques in glaciology and glacial geomorphology. John Wiley & Sons, Chichester, UK. Hutter, K. (1983). Theoretical glaciology: material science of ice and the mechanics of glaciers and ice sheets. Reidel. Huybers, P. (2006). Early pleistocene glacial cycles and the integrated summer insolation forcing. Science, 313(5786):508–511. Huybers, P. (2009). Antarctica’s orbital beat. Science, 325(5944):1085–1086. Huybers, P. and Denton, G. (2008). Antarctic temperature at orbital timescales controlled by local summer duration. Nature Geoscience, 1(11):787–792. Huybers, P. and Eisenman, A. (2006). Integrated summer insolation calculations. NOAA/NCDC Paleoclimatology Program, Data Contribution Series no.2006-079. Imbrie, J., Berger, A., Boyle, E., Clemens, S., Duffy, A., Howard, W., Kukla, G., Kutzbach, J., MartinsonN, D., McIntyre, A., et al. (1993). On the structure and origin of major glaciation cycles. II: The 100,000-year cycle. Paleoceanography, 8(6):699–735. Imbrie, J., Boyle, E., Clemens, S., Duffy, A., Howard, W., Kukla, G., Kutzbach, J., Martinson, D., McIntyre, A., Mix, A., et al. (1992). On the structure and origin of major glaciation cycles 1. Linear responses to Milankovitch forcing. Paleoceanography, 7(6):701–738. 248 BIBLIOGRAPHY Imbrie, J. and Imbrie, K. P. (1986). Ice ages: solving the mystery. Harvard University Press, Massachusetts, USA. Imbrie, J. and Imbrie, K. P. (1986). Ice ages: solving the mystery. Harvard University Press, Massachusetts, USA. Ivy Ochs, S., Schl¨uchter, C., Kubik, P. W., and Denton, G. H. (1999). Moraine exposure dates imply synchronous Younger Dryas glacier advances in the European Alps and in the Southern Alps of New Zealand. Geograﬁska Annaler: Series A, Physical Geography, 81(2):313–323. Jiang, N., Grifﬁths, G., and Lorrey, A. (2013). BIBLIOGRAPHY Inﬂuence of large-scale climate modes on daily synoptic weather types over New Zealand. International Journal of Climatology, 33(2):499–519. Jouzel, J., Genthon, C., Lorius, C., Petit, J., and Barkov, N. (1987). Vostok ice core-A continuous isotope temperature record over the last climatic cycle (160,000 years). Nature, 329:403–408. Kalnay, E., Kanamitsu, M., Kistler, R., Collins, W., Deaven, D., Gandin, L., Iredell, M., Saha, S., White, G., Woollen, J., et al. (1996). The NCEP/NCAR 40-year reanalysis project. Bulletin of the American meteorological Society, 77(3):437–471. Kaplan, M. R., Schaefer, J. M., Denton, G. H., Barrell, D. J. A., Chinn, T. J. H., Putnam, A. E., Andersen, B. G., Finkel, R. C., Schwartz, R., and Doughty, A. M. (2010). Glacier retreat in New Zealand during the Younger Dryas stadial. Nature, 467(7312):194–197. Kaplan, M. R., Schaefer, J. M., Denton, G. H., Doughty, A. M., Barrell, D. J. A., Chinn, T. J. H., Putnam, A. E., Andersen, B. G., Mackintosh, A., Finkel, R. C., et al. (2013). The anatomy of long-term warming since 15 ka in New Zealand based on net glacier snowline rise. Geology, 41(8):887–890. Kawamura, K., Parrenin, F., Lisiecki, L., Uemura, R., Vimeux, F., Severinghaus, J. P., Hutterli, M. A., Nakazawa, T., Aoki, S., Jouzel, J., et al. (2007). Northern Hemi- sphere forcing of climatic cycles in Antarctica over the past 360,000 years. Nature, 448(7156):912–916. Kelley, S. E., Kaplan, M. R., Schaefer, J. M., Andersen, B. G., Barrell, D. J., Putnam, A. E., Denton, G. H., Schwartz, R., Finkel, R. C., and Doughty, A. M. (2014). High-precision 10Be chronology of moraines in the Southern Alps indicates synchronous cooling in Antarctica and New Zealand 42,000 years ago. Earth and Planetary Science Letters, 405:194–206. Kessler, M. A., Anderson, R. S., and Stock, G. M. (2006). Modeling topographic and climatic control of east-west asymmetry in Sierra Nevada glacier length during the Last Glacial Maximum. Journal of Geophysical Research: Earth Surface, 111(F2002). 249 BIBLIOGRAPHY BIBLIOGRAPHY Keys, H. (1988). 1988 Survey of the glaciers on Mt. Ruapehu, Tongariro National Park - A baseline for detecting effects of climate change. Technical Report Science and Research Internal Report No.24, Department of Conservation, Wellington, NZ. Keys, H. (1988). 1988 Survey of the glaciers on Mt. Ruapehu, Tongariro National Park - A baseline for detecting effects of climate change. Technical Report Science and Research Internal Report No.24, Department of Conservation, Wellington, NZ. Kirkbride, M. (1995). Processes of glacial transportation. In Menzies, J., editor, Modern and past glacial environments, pages 147–169. Butterworth-Heinemann, Oxford, UK. Kirkbride, M. and Matthews, D. (1997). The role of ﬂuvial and glacial erosion in landscape evolution: the Ben Ohau Range, New Zealand. Earth Surface Processes and Landforms, 22(3):317–327. Kirkbride, M. P. and Dugmore, A. J. (2001). Timing and signiﬁcance of mid-Holocene glacier advances in Northern and Central Iceland. Journal of Quaternary Science, 16(2):145–153. Kirkbride, M. P. and Dugmore, A. J. (2003). Glaciological response to distal tephra fallout from the 1947 eruption of Hekla, south Iceland. Journal of Glaciology, 49(166):420–428. Klok, E. and Oerlemans, J. (2002). Model study of the spatial distribution of the energy and mass balance of Morteratschgletscher, Switzerland. Journal of Glaciology, 48(163):505–518. Knight, J., Mitchell, W. A., and Rose, J. (2011). Geomorphological Field Mapping. In Smith, M. J., Paron, P., and Grifﬁth, J. S., editors, Geomorphological mapping: methods and applications, pages 151–188. Elsevier, Oxford, UK. Konzelmann, T., van de Wal, R. S., Greuell, W., Bintanja, R., Henneken, E. A., and Abe Ouchi, A. (1994). Parameterization of global and longwave incoming radiation for the Greenland Ice Sheet. Global and Planetary change, 9(1):143–164. K¨oppen, W. P. and Wegener, A. (1924). Die klimate der geologischen vorzeit, volume 1. Gebr¨uder Borntraeger, Stuttgart, Germany. Krenek, L. (1959). Changes in the glaciers of Mt Ruapehu in 1955. New Zealand Journal of Geology and Geophysics, 2(4):643–653. Kuhn, M. (1995). The mass balance of very small glaciers. Zeitschrift f¨ur Gletscherkunde und Glazialgeologie, 31(1):171–179. Kukla, J. (1970). Correlations between loesses and deep-sea sediments. Geologiska F¨oreningen i Stockholm F¨orhandlingar, 92(2):148–180. Kurz, M. and Brook, E. (1994). Surface exposure dating with cosmogenic nuclides. In Beck, C., editor, Dating in exposed and surface contexts, pages 139–159. Univ. NM Press, Albuquerque, NM. 250 BIBLIOGRAPHY Kurz, M. D. (1986a). Cosmogenic helium in a terrestrial igneous rock. Nature, 320(6061):435–439. Kurz, M. D. (1986b). In situ production of terrestrial cosmogenic helium and some applications to geochronology. BIBLIOGRAPHY Geochimica et Cosmochimica Acta, 50(12):2855–2862. Kurz, M. D., Colodner, D., Trull, T. W., Moore, R. B., and O’Brien, K. (1990). Cosmic ray exposure dating with in situ produced cosmogenic 3He: results from young Hawaiian lava ﬂows. Earth and Planetary Science Letters, 97(1-2):177–189. Lal, D. (1987). Production of 3He in terrestrial rocks. Chemical Geology: Isotope Geoscience section, 66(1):89–98. Lal, D. (1991). Cosmic ray labeling of erosion surfaces: in situ nuclide production rates and erosion models. Earth and Planetary Science Letters, 104(2-4):424–439. Lambeck, K., Rouby, H., Purcell, A., Sun, Y., and Sambridge, M. (2014). Sea level and global ice volumes from the Last Glacial Maximum to the Holocene. Proceedings of the National Academy of Sciences, 111(43):15296–15303. Le Meur, E., Gagliardini, O., Zwinger, T., and Ruokolainen, J. (2004). Glacier ﬂow modelling: a comparison of the Shallow Ice Approximation and the full-Stokes solution. Comptes Rendus Physique, 5(7):709–722. Lee, J., Townsend, D., Bland, K., and Kamp, P. (2011). Geology map of the Hawkes Bay area area: scale 1:250,000. Three maps and notes (36 p.). Institute of Geological and Nuclear Sciences 1:250,000 geological map 8. 86p. + 1 folded map. Leverrier, U.-J. (1843). Recherches sur l’orbite de Mercure et sur ses perturbations. D´etermination de la masse de V´enus et du diam`etre du Soleil. Journal de Math´ematiques Pures et Appliqu´ees, pages 273–359. Leysinger Vieli, G. and Gudmundsson, G. (2004). On estimating length ﬂuctuations of glaciers caused by changes in climatic forcing. Journal of Geophysical Research: Earth Surface, 109(F1):F01007. Licciardi, J. M., Clark, P. U., Brook, E. J., Elmore, D., and Sharma, P. (2004). Variable responses of western U.S. glaciers during the last deglaciation. Geology, 32(1):81–84. Licciardi, J. M., Kurz, M. D., Clark, P. U., and Brook, E. J. (1999). Calibration of cosmogenic 3He production rates from Holocene lava ﬂows in Oregon, USA, and effects of the Earth’s magnetic ﬁeld. Earth and Planetary Science Letters, 172(3-4):261– 271. BIBLIOGRAPHY 251 Licciardi, J. M., Kurz, M. D., and Curtice, J. M. (2006). Cosmogenic 3He production rates from Holocene lava ﬂows in Iceland. Earth and Planetary Science Letters, 246(3- 4):251–264. Licciardi, J. M., Kurz, M. D., and Curtice, J. M. (2006). Cosmogenic 3He production rates from Holocene lava ﬂows in Iceland. Earth and Planetary Science Letters, 246(3- 4):251–264. Licciardi, J. M., Kurz, M. D., and Curtice, J. M. (2006). Cosmogenic 3He production rates from Holocene lava ﬂows in Iceland. Earth and Planetary Science Letters, 246(3- 4):251–264. Licciardi, J. M., Kurz, M. BIBLIOGRAPHY D., and Curtice, J. M. (2006). Cosmogenic 3He production rates from Holocene lava ﬂows in Iceland. Earth and Planetary Science Letters, 246(3- 4):251–264. Licciardi, J. M., Kurz, M. D., and Curtice, J. M. (2007). Glacial and volcanic history of Icelandic table mountains from cosmogenic 3He exposure ages. Quaternary Science Reviews, 26(11-12):1529–1546. Lifton, N., Sato, T., and Dunai, T. J. (2014). Scaling in situ cosmogenic nuclide production rates using analytical approximations to atmospheric cosmic-ray ﬂuxes. Earth and Planetary Science Letters, 386:149–160. Lifton, N. A., Bieber, J. W., Clem, J. M., Duldig, M. L., Evenson, P., Humble, J. E., and Pyle, R. (2005). Addressing solar modulation and long-term uncertainties in scaling secondary cosmic rays for in situ cosmogenic nuclide applications. Earth and Planetary Science Letters, 239(1-2):140–161. Liu, Z., Otto Bliesner, B., He, F., Brady, E., Tomas, R., Clark, P., Carlson, A., Lynch Stieglitz, J., Curry, W., Brook, E., et al. (2009). Transient simulation of last deglaciation with a new mechanism for Bølling-Allerød warming. Science, 325(5938):310–314. Lorius, C., Jouzel, J., Raynaud, D., Hansen, J., and Le Treut, H. (1990). The ice-core record: climate sensitivity and future greenhouse warming. Nature, 347(6289):139– 145. Lorius, C., Ritz, C., Jouzel, J., Merlivat, L., and Barkov, N. (1985). A 150,000-year climatic record from Antarctic ice. Nature, 316:591–596. Lorrey, A., Dalu, G., Renwick, J., Diamond, H., and Gaetani, M. (2012a). Reconstructing the South Paciﬁc convergence zone position during the presatellite era: a La Ni˜na case study. Monthly Weather Review, 140(11):3653–3668. Lorrey, A., Fauchereau, N., Stanton, C., Chappell, P., Phipps, S., Mackintosh, A., Ren- wick, J., Goodwin, I., and Fowler, A. (2014). The Little Ice Age climate of New Zealand reconstructed from Southern Alps cirque glaciers: a synoptic type approach. Climate Dynamics, 42(11-12):3039–3060. Lorrey, A. M., Vandergoes, M., Almond, P., Renwick, J., Stephens, T., Bostock, H., Mackintosh, A., Newnham, R. M., Williams, P. W., Ackerley, D., Neil, H., and Fowler, A. M. (2012b). Palaeocirculation across New Zealand during the last glacial maximum at 21 ka. Quaternary Science Reviews, 36:189–213. 252 BIBLIOGRAPHY Lowe, D. J., Blaauw, M., Hogg, A. G., and Newnham, R. M. (2013). Ages of 24 widespread tephras erupted since 30,000 years ago in New Zealand, with re- evaluation of the timing and palaeoclimatic implications of the Lateglacial cool episode recorded at Kaipo bog. Quaternary Science Reviews, 74:170–194. Lowe, D. J., Shane, P. A. R., Alloway, B. V., and Newnham, R. M. (2008). BIBLIOGRAPHY Fingerprints and age models for widespread New Zealand tephra marker beds erupted since 30,000 years ago: a framework for NZ-INTIMATE. Quaternary Science Reviews, 27(1- 2):95–126. L¨uthi, D., Le Floch, M., Bereiter, B., Blunier, T., Barnola, J.-M., Siegenthaler, U., Raynaud, D., Jouzel, J., Fischer, H., Kawamura, K., et al. (2008). High-resolution carbon dioxide concentration record 650,000–800,000 years before present. Nature, 453(7193):379–382. Mackintosh, A., Barrows, T., Colhoun, E., and Fiﬁeld, L. (2006). Exposure dating and glacial reconstruction at Mt. Field, Tasmania, Australia, identiﬁes MIS 3 and MIS 2 glacial advances and climatic variability. Journal of Quaternary Science, 21(4):363–376. Mackintosh, A. N., Dugmore, A. J., and Hubbard, A. L. (2002). Holocene cli- matic changes in Iceland: evidence from modelling glacier length ﬂuctuations at S´olheimaj¨okull. Quaternary International, 91(1):39–52. Mager, S. and Fitzsimons, S. (2007). Formation of glaciolacustrine late pleistocene end moraines in the Tasman Valley, New Zealand. Quaternary Science Reviews, 26(5):743– 758. Major, J. and Newell, C. (1989). Snow and ice perturbation during historical volcanic eruptions and the formation of lahars and ﬂoods. Bulletin of Volcanology, 52:1–27. Marden, M., Betts, H., Palmer, A., Taylor, R., Bilderback, E., and Litchﬁeld, N. (2014). Post-last glacial maximum ﬂuvial incision and sediment generation in the unglaciated Waipaoa catchment, North Island, New Zealand. Geomorphology, 214:283–306. Martin, J. H. (1990). Glacial-interglacial CO2 change: The iron hypothesis. Paleoceanog- raphy, 5(1):1–13. Masson Delmotte, V., Schulz, M., Abe Ouchi, A., Beer, J., Ganopolski, A., Gonzlez Rouco, J., Jansen, E., Lambeck, K., Luterbacher, J., Naish, T., Osborn, T., Otto Bliesner, B., Quinn, T., Ramesh, R., Rojas, M., Shao, X., and Timmermann, A. (2013). Historical Overview of Climate Change. In Stocker, T., Qin, D., Plattner, G.-K., Tignor, M., Allen, S., Boschung, J., Nauels, A., Xia, Y., Bex, V., and Midgley, P., editors, Climate Change 2013: The Physical Science Basis. Contribution of Working Group I to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change. Cambridge University Press, Cambridge, United Kingdom and New York, NY, USA. 253 Meier, M. F. and Post, A. S. (1962). Recent variations in mass net budgets of glaciers in western North America. In International Association of Scientiﬁc Hydrology Publication 58, pages 63–77. BIBLIOGRAPHY Mathews, W. H. (1967). A contribution to the geology of the Mount Tongariro massif, North Island, New Zealand. New Zealand Journal of Geology and Geophysics, 10(4):1027– 1038. Matsuoka, N. (1998). Modelling frost creep rates in an alpine environment. Permafrost and Periglacial Processes, 9(4):397–409. McArthur, J. L. and Shepherd, M. J. (1990). Late Quaternary glaciation of Mt Ruapehu, North Island, New Zealand. Journal - Royal Society of New Zealand, 20(3):287–296. McCalpin, J. P. (1992). Glacial geology of the upper Wairau valley, Marlborough, New Zealand. New Zealand Journal of Geology and Geophysics, 35(2):211–222. McCarthy, A., Mackintosh, A., Rieser, U., and Fink, D. (2008). Mountain glacier chronol- ogy from Boulder Lake, New Zealand, indicates MIS 4 and MIS 2 ice advances of similar extent. Arctic, Antarctic, and Alpine Research, 40(4):695–708. McClelland, E. and Erwin, P. S. (2003). Was a dacite dome implicated in the 9,500 BP collapse of Mt Ruapehu? A palaeomagnetic investigation. Bulletin of Volcanology, 65(4):294–305. McColl, S. and Davies, T. (2011). Evidence for a rock-avalanche origin for ’The Hillocks’ moraine, Otago, New Zealand. Geomorphology, 127(3):216–224. McGlone, M. and Topping, W. . (1977). Aranuian (post-glacial) pollen diagrams from the Tongariro region, North Island, New Zealand. New Zealand Journal of Botany, 15(4):749–760. McGlone, M. S., Turney, C. S. M., Wilmshurst, J. M., Renwick, J., and Pahnke, K. (2010). Divergent trends in land and ocean temperature in the Southern Ocean over the past 18,000 years. Nature Geoscience, 3:622–626. McKinnon, K. A., Mackintosh, A. N., Anderson, B. M., and Barrell, D. J. A. (2012). The inﬂuence of sub-glacial bed evolution on ice extent: A model-based evaluation of the Last Glacial Maximum Pukaki glacier, New Zealand. Quaternary Science Reviews, 57:46–57. McManus, J. F., Oppo, D. W., and Cullen, J. L. (1999). A 0.5-million-year record of millennial-scale climate variability in the North Atlantic. Science, 283(5404):971–975. Meier, M. F. and Post, A. S. (1962). Recent variations in mass net budgets of glaciers in western North America. In International Association of Scientiﬁc Hydrology Publication 58, pages 63–77. 254 BIBLIOGRAPHY Meierding, T. C. (1982). Late Pleistocene glacial equilibrium-line altitudes in the Col- orado Front Range: a comparison of methods. Quaternary Research, 18(3):289–310. Menviel, L., Timmermann, A., Timm, O. E., and Mouchet, A. (2011). Deconstructing the Last Glacial termination: the role of millennial and orbital-scale forcings. Quaternary Science Reviews, 30(9):1155–1172. Mercer, J. (1988). The age of the Waiho Loop terminal moraine, Franz Josef Glacier, Westland. BIBLIOGRAPHY New Zealand Journal of Geology and Geophysics, 31(1):95–99. Mercer, J. H. (1984). Simultaneous climatic change in both hemispheres and similar bipolar interglacial warming: Evidence and implications. Climate Processes and Climate Sensitivity, 29:307–313. Milankovitch, M. (1920). Theorie mathematique des phenomenes thermiques produits par la radiation solaire. Gauthier-Villars, Paris, France. Milankovitch, M. (1941). Kanon der Erdbestrahlung und seine Anwendung auf das Eiszeiten- problem. Royal Serbian Academy, Belgrade. Minder, J. R., Mote, P. W., and Lundquist, J. D. (2010). Surface temperature lapse rates over complex terrain: Lessons from the Cascade Mountains. Journal of Geophysical Research D: Atmospheres, 115(14):D14122. Moebis, A., Cronin, S. J., Neall, V. E., and Smith, I. E. (2011). Unravelling a complex volcanic history from ﬁne-grained, intricate holocene ash sequences at the Tongariro Volcanic Centre, New Zealand. Quaternary International, 246(1):352–363. Monnin, E., Indermhle, A., Dllenbach, A., Flckiger, J., Stauffer, B., Stocker, T. F., Raynaud, D., and Barnola, J. . (2001). Atmospheric CO2 concentrations over the last glacial termination. Science, 291(5501):112–114. Moreno, P. I., Lowell, T. V., Jacobson Jr., G. L., and Denton, G. H. (1999). Abrupt vegeta- tion and climate changes during the Last Glacial Maximum and last termination in the Chilean Lake District: A case study from Canal de la Puntilla (41◦S). Geograﬁska Annaler, Series A: Physical Geography, 81(2):285–311. Morland, L. (1984). Thermomechanical balances of ice sheet ﬂows. Geophysical & Astrophysical Fluid Dynamics, 29(1-4):237–266. Neall, V. (1979). Sheets P19, P20 and P21 - New Plymouth, Egmont and Manaia. 1st ed. Geological map of New Zealand 1:50 000. Three maps and notes (36 p.), New Zealand Department of Scientiﬁc and Industrial Research, Wellington. BIBLIOGRAPHY BIBLIOGRAPHY 255 Neall, V. E. and Alloway, B. V. (1986). International Volcanological Congress, New Zealand. Tour Guide C3: Quaternary volcaniclastics and volcanic hazards of Taranaki. N. Z. Geological Survey record 12. Neall, V. E. and Alloway, B. V. (1986). International Volcanological Congress, New Zealand. Tour Guide C3: Quaternary volcaniclastics and volcanic hazards of Taranaki. N. Z. Geological Survey record 12. Newnham, R. M. and Lowe, D. J. (2000). Fine-resolution pollen record of late-glacial climate reversal from New Zealand. Geology, 28(8):759–762. Newnham, R. M., Lowe, D. J., Giles, T., and Alloway, B. V. (2007). Vegetation and climate of Auckland, New Zealand, since ca. 32 000 cal. yr ago: Support for an extended LGM. Journal of Quaternary Science, 22(5):517–534. Newnham, R. M., Lowe, D. J., and Green, J. D. (1989). Palynology, vegetation and climate of the Waikato lowlands, North Island, New Zealand, since c. 18,000 years ago. Journal of the Royal Society of New Zealand, 19(2):127–150. Newnham, R. M., Lowe, D. J., and Williams, P. W. (1999). Quaternary environmental change in New Zealand: A review. Progress in Physical Geography, 23(4):567–610. Newnham, R. M., McGlone, M., Moar, N., Wilmshurst, J., and Vandergoes, M. (2013). The vegetation cover of New Zealand at the Last Glacial Maximum. Quaternary Science Reviews, 74:202–214. Newnham, R. M., Vandergoes, M. J., Sikes, E., Carter, L., Wilmshurst, J. M., Lowe, D. J., McGlone, M. S., and Sandiford, A. (2012). Does the bipolar seesaw extend to the terrestrial southern mid-latitudes? Quaternary Science Reviews, 36:214–222. Niedermann, S. (2002). Cosmic-ray-produced noble gases in terrestrial rocks: dating tools for surface processes. Reviews in Mineralogy and Geochemistry, 47(1):731–784. Nield, J. M., Chiverrell, R. C., Darby, S. E., Leyland, J., Vircavs, L. H., and Jacobs, B. (2013). Complex spatial feedbacks of tephra redistribution, ice melt and surface roughness modulate ablation on tephra covered glaciers. Earth Surface Processes and Landforms, 38(1):95–102. NIWA (2014). CliFlo: NIWA’s National Climate Database on the Web. Website: http://cliﬂo.niwa.co.nz/. Retrieved 2011-2014. Norton, D., McGlone, M., and Wigley, T. (1986). Quantitative analyses of modern pollen-climate relationships in New Zealand indigenous forests. New Zealand Journal of Botany, 24(2):331–342. Norton, D. A. (1985). A multivariate technique for estimating New Zealand temperature normals. Weather and Climate, 5:64–74. 256 BIBLIOGRAPHY Odell, N. (1955). Mount Ruapehu, New Zealand: Observations on its Crater Lake and Glaciers. Journal of Glaciology, 2(18):601–607. Oerlemans, J. (1992). Climate sensitivity of glaciers in southern Norway: application of an energy-balance model to Nigardsbreen, Hellstugubreen and Alfotbreen. BIBLIOGRAPHY Journal of Glaciology, 38(129):223–232. Oerlemans, J. (1997). Climate sensitivity of Franz Josef Glacier, New Zealand, as revealed by numerical modeling. Arctic and Alpine Research, 29(2):233–239. Oerlemans, J. (2001). Glaciers and climate change. Balkema, Rotterdam, The Netherlands. Oerlemans, J. (2005). Extracting a climate signal from 169 glacier records. Science, 308(5722):675–677. Oerlemans, J. and Fortuin, J. P. F. (1992). Sensitivity of glaciers and small ice caps to greenhouse warming. Science, 258(5079):115–117. Oerlemans, J. and Knap, W. (1998). A 1 year record of global radiation and albedo in the ablation zone of Morteratschgletscher, Switzerland. Journal of Glaciology, 44(147):231– 238. Oerlemans, J. and Reichert, B. K. (2000). Relating glacier mass balance to meteorological data by using a seasonal sensitivity characteristic. Journal of Glaciology, 46(152):1–6. Ohmura, A. (2001). Physical basis for the temperature-based melt-index method. Journal of Applied Meteorology, 40(4):753–761. Ohmura, A., Kasser, P., and Funk, M. (1992). Climate at the equilibrium line of glaciers. Journal of Glaciology, 38(130):397–411. Osborn, G., Menounos, B., Ryane, C., Riedel, J., Clague, J. J., Koch, J., Clark, D., Scott, K., and Davis, P. T. (2012). Latest pleistocene and holocene glacier ﬂuctuations on Mount Baker, Washington. Quaternary Science Reviews, 49:33–51. Osmaston, H. (2005). Estimates of glacier equilibrium line altitudes by the Area Alti- tude, the Area Altitude Balance Ratio and the Area Altitude Balance Index methods and their validation. Quaternary International, 138-139:22–31. Otway, P., Holdsworth, R., Bell, D., and Robertson S. H (1985). Radio echo sounding survey of sub-glacial topography, Mt Ruapehu, 28 March 1985. Technical Report Immediate report, New Zealand Geological Survey, Wairakei, NZ. Pahnke, K. and Sachs, J. P. (2006). Sea surface temperatures of southern midlatitudes 0–160 kyr BP. Paleoceanography, 21(2):PA2003. 257 BIBLIOGRAPHY BIBLIOGRAPHY Palmer, B. and Neall, V. (1989). The Murimotu Formation - 9500 year old deposits of a debris avalanche and associated lahars, Mount Ruapehu, North Island, New Zealand. New Zealand Journal of Geology & Geophysics, 32(4):477–486. Palmer, B. and Neall, V. (1989). The Murimotu Formation - 9500 year old deposits of a debris avalanche and associated lahars, Mount Ruapehu, North Island, New Zealand. New Zealand Journal of Geology & Geophysics, 32(4):477–486. Palmer, B. A., Alloway, B. V., and Neall, V. E. (1991). Volcanic-debris-avalanche deposits in New Zealand - lithofacies organization in unconﬁned, wet-avalanche ﬂows. In Fisher, R. V. and Smith, G. A., editors, Sedimentation in Volcanic Settings, volume 45, pages 89–98. Paneth, F., Reasbeck, P., and Mayne, K. (1952). Helium 3 content and age of meteorites. Geochimica et Cosmochimica Acta, 2(5):300–303. Park, J. (1926). Morainic mounds on the Waimarino Plain, near Ruapehu. Transactions of the N.Z. Institute, 56:382–383. Paterson, W. S. B. (1994). The Physics of Glaciers. Elsevier, Oxford, New York and Tokyo. Pattyn, F. (2006). GRANTISM: An ExcelTM model for Greenland and Antarctic ice-sheet response to climate changes. Computers & geosciences, 32(3):316–325. Paulin, T. (2008). Glaciers and Climate Change, Mount Ruapehu, New Zealand. Mas- ter’s thesis, Victoria University of Wellington, NZ. Penck, A. and Br¨uckner, E. (1909). Die alpen im Eiszeitalter, volume 3. Tauchnitz, Leipzig, Germany. Petit, J. R., Jouzel, J., Raynaud, D., Barkov, N. I., Barnola, J. ., Basile, I., Bender, M., Chappellaz, J., Davis, M., Delaygue, G., Delmotte, M., Kotiyakov, V. M., Legrand, M., Lipenkov, V. Y., Lorius, C., Ppin, L., Ritz, C., Saltzman, E., and Stievenard, M. (1999). Climate and atmospheric history of the past 420,000 years from the Vostok ice core, Antarctica. Nature, 399(6735):429–436. Phillips, F. M., Zreda, M. G., Smith, S. S., Elmore, D., Kubik, P. W., and Sharma, P. (1990). Cosmogenic chlorine-36 chronology for glacial deposits at Bloody Canyon, eastern Sierra Nevada. Science, 248(4962):1529–1532. Pierrehumbert, R. T. (2010). Principles of planetary climate. Cambridge University Press, Massachusetts, USA. Plummer, M. A. and Phillips, F. M. (2003). A 2-D numerical model of snow/ice energy balance and ice ﬂow for paleoclimatic interpretation of glacial geomorphic features. Quaternary Science Reviews, 22(14):1389–1406. Portenga, E. W. and Bierman, P. R. (2011). Understanding Earth’s eroding surface with 10Be. GSA Today, 21(8):4–10. 258 BIBLIOGRAPHY Porter, S. C. (1975). Equilibrium-line altitudes of late Quaternary glaciers in the Southern Alps, New Zealand. Quaternary Research, 5(1):27–47. Prescott, C., Corbin, J., and Parkinson, D. (1989). BIBLIOGRAPHY Input, accumulation and residence times of carbon, nitrogen and phosphorous in four Rocky Mountain coniferous forests. Canadian Journal of Forest Research, 19:489–498. Preusser, F., Andersen, B. G., Denton, G. H., and Schl¨uchter, C. (2005). Luminescence chronology of Late Pleistocene glacial deposits in North Westland, New Zealand. Quaternary Science Reviews, 24(20-21):2207–2227. Price, R. C., Gamble, J. A., Smith, I. E., Maas, R., Waight, T., Stewart, R. B., and Woodhead, J. (2012). The anatomy of an Andesite volcano: a time–stratigraphic study of andesite petrogenesis and crustal evolution at Ruapehu Volcano, New Zealand. Journal of Petrology, 53:2139–2189. Pulford, A. K. (2002). Crustal structure and lithospheric doming: Aspects of deformation along an obliquely convergent plate margin, New Zealand. Ph.D. Thesis, Victoria University of Wellington. Purdie, H., Anderson, B., Chinn, T., Owens, I., Mackintosh, A., and Lawson, W. (2014). Franz Josef and Fox Glaciers, New Zealand: Historic length records. Global and Planetary Change, 121:41–52. Putkonen, J. and O’Neal, M. (2006). Degradation of unconsolidated Quaternary land- forms in the western North America. Geomorphology, 75(3):408–419. Putkonen, J. and Swanson, T. (2003). Accuracy of cosmogenic ages for moraines. Quaternary Research, 59(2):255–261. Putnam, A. E., Denton, G. H., Schaefer, J. M., Barrell, D. J. A., Andersen, B. G., Finkel, R. C., Schwartz, R., Doughty, A. M., Kaplan, M. R., and Schl¨uchter, C. (2010a). Glacier advance in southern middle-latitudes during the Antarctic Cold Reversal. Nature Geoscience, 3(10):700–704. Putnam, A. E., Schaefer, J. M., Barrell, D. J. A., Vandergoes, M., Denton, G. H., Kaplan, M. R., Finkel, R. C., Schwartz, R., Goehring, B. M., and Kelley, S. E. (2010b). In situ cosmogenic 10Be production-rate calibration from the Southern Alps, New Zealand. Quaternary Geochronology, 5(4):392–409. Putnam, A. E., Schaefer, J. M., Denton, G. H., Barrell, D. J. A., Andersen, B. G., Koffman, T. N. B., Rowan, A. V., Finkel, R. C., Rood, D. H., Schwartz, R., Vandergoes, M. J., Plummer, M. A., Brocklehurst, S. H., Kelley, S. E., and Ladig, K. L. (2013a). Warming 259 BIBLIOGRAPHY and glacier recession in the Rakaia valley, Southern Alps of New Zealand, during Heinrich Stadial 1. Earth and Planetary Science Letters, 382:98–110. and glacier recession in the Rakaia valley, Southern Alps of New Zealand, during Heinrich Stadial 1. Earth and Planetary Science Letters, 382:98–110. Putnam, A. E., Schaefer, J. M., Denton, G. H., Barrell, D. J. A., Birkel, S. D., Andersen, B. G., Kaplan, M. R., Finkel, R. C., Schwartz, R., and Doughty, A. M. (2013b). BIBLIOGRAPHY The Last Glacial Maximum at 44◦S documented by a 10Be moraine chronology at Lake Ohau, Southern Alps of New Zealand. Quaternary Science Reviews, 62:114–141. Putnam, A. E., Schaefer, J. M., Denton, G. H., Barrell, D. J. A., Finkel, R. C., Andersen, B. G., Schwartz, R., Chinn, T. J., and Doughty, A. M. (2012). Regional climate control of glaciers in New Zealand and Europe during the pre-industrial Holocene. Nature Geoscience, 5(9):627–630. Rea, B. R., Whalley, W. B., Dixon, T. S., and Gordon, J. E. (1999). Plateau iceﬁelds as contributing areas to valley glaciers and the potential impact on reconstructed ELAs: a case study from the Lyngen Alps, North Norway. Annals of Glaciology, 28(1):97–102. Renwick, J. and Thompson, D. (2006). The southern annular mode and New Zealand climate. Water & Atmosphere, 14(2):24–25. Richardson, J. M. and Brook, M. S. (2010). Ablation of debris-covered ice: some effects of the 25 September 2007 Mt Ruapehu eruption. Journal of the Royal Society of New Zealand, 40(2):45–55. Rivera, A. and Bown, F. (2013). Recent glacier variations on active ice capped volcanoes in the Southern Volcanic Zone (37◦–46◦S), Chilean Andes. Journal of South American Earth Sciences, 45:345–356. Rivera, A., Bown, F., Carri´on, D., and Zenteno, P. (2012). Glacier responses to recent volcanic activity in Southern Chile. Environmental Research Letters, 7(1):014036. Roethlisberger, F. and Schneebeli, W. (1979). Genesis of lateral moraine complexes, demonstrated by fossil soils and trunks; indicators of postglacial climatic ﬂuctuations. In Schl¨uchter, C., editor, Moraines and varves: origin, genesis, classiﬁcation, pages 387– 420. Balkema, Rotterdam, The Netherlands. Rojas, M., Moreno, P., Kageyama, M., Cruciﬁx, M., Hewitt, C., Abe Ouchi, A., Ohgaito, R., Brady, E. C., and Hope, P. (2009). The Southern Westerlies during the last glacial maximum in PMIP2 simulations. Climate Dynamics, 32(4):525–548. Rother, H., Fink, D., Shulmeister, J., Mifsud, C., Evans, M., and Pugh, J. (2014). The early rise and late demise of New Zealands last glacial maximum. Proceedings of the National Academy of Sciences, 111(32):11630–11635. 260 BIBLIOGRAPHY Rowan, A. V., Brocklehurst, S. H., Schultz, D. M., Plummer, M. A., Anderson, L. S., and Glasser, N. F. (2014). Late Quaternary glacier sensitivity to temperature and precipitation distribution in the Southern Alps of New Zealand. Journal of Geophysical Research: Earth Surface, 24:PA2201. Rowan, A. V., Plummer, M. A., Brocklehurst, S. H., Jones, M. A., and Schultz, D. M. (2013). Drainage capture and discharge variations driven by glaciation in the Southern Alps, New Zealand. Geology, 41(2):199–202. Cited By (since 1996):1. Rowan, A. V., Roberts, H. M., Jones, M. A., Duller, G. A., Covey Crump, S. J., and Brocklehurst, S. H. (2012). Optically stimulated luminescence dating of glacioﬂu- vial sediments on the Canterbury Plains, South Island, New Zealand. Quaternary Geochronology, 8:10–22. Salinger, M. and Mullan, A. (1999). New Zealand climate: temperature and precipitation variations and their links with atmospheric circulation 1930–1994. International Journal of Climatology, 19(10):1049–1071. Salinger, M., Renwick, J., and Mullan, A. (2001). Interdecadal paciﬁc oscillation and south paciﬁc climate. International Journal of Climatology, 21(14):1705–1721. Salinger, M. J. (1980a). New Zealand climate: I. Precipitation patterns. Monthly Weather Review, 108(11):1892–1904. Salinger, M. J. (1980b). New Zealand climate: II. Temperature patterns. Monthly Weather Review, 108(11):1905–1912. Sandiford, A., Newnham, R. M., Alloway, B. V., and Ogden, J. (2003). A 28 000-7600 cal yr BP pollen record of vegetation and climate change from Pukaki Crater, northern New Zealand. Palaeogeography, Palaeoclimatology, Palaeoecology, 201(3-4):235–247. Schaefer, J. M., Denton, G. H., Barrell, D. J. A., Ivy Ochs, S., Kubik, P. W., Andersen, B. G., Phillips, F. M., Lowell, T. V., and Schl¨uchter, C. (2006). Near-synchronous interhemispheric termination of the Last Glacial Maximum in mid-latitudes. Science, 312(5779):1510–1513. Schaefer, J. M., Denton, G. H., Kaplan, M., Putnam, A., Finkel, R. C., Barrell, D. J. A., Andersen, B. G., Schwartz, R., Mackintosh, A., Chinn, T., and Schl¨uchter, C. (2009). High-frequency Holocene glacier ﬂuctuations in New Zealand differ from the north- ern signature. Science, 324(5927):622–625. Schaefer, J. M., Putnam, A. E., Denton, G. H., Kaplan, M. R., Birkel, S., Doughty, A. M., Kelley, S., Barrell, D. J. A., Finkel, R. C., Winckler, G., Anderson, R. F., Ninneman, U. S., Barker, S., Schwartz, R., and Schl¨uchter, C. (2015). The Southern Glacial BIBLIOGRAPHY 261 Maximum 65000 years ago and its Unﬁnished Termination. Manuscript submitted for publication. Maximum 65000 years ago and its Unﬁnished Termination. Manuscript submitted for publication. Sch¨afer, J. M., Ivy Ochs, S., Wieler, R., Leya, I., Baur, H., Denton, G. BIBLIOGRAPHY H., and Schl¨uchter, C. (1999). Cosmogenic noble studies in the oldest landscape on earth: Surface exposure ages of the Dry Valleys, Antarctica. Earth and Planetary Science Letters, 167(3-4):215– 226. Schimmelpfennig, I., Benedetti, L., Finkel, R., Pik, R., Blard, P.-H., Bourl`es, D., Burnard, P., and Williams, A. (2009). Sources of in-situ 36Cl in basaltic rocks. Implications for calibration of production rates. Quaternary Geochronology, 4(6):441–461. Schimmelpfennig, I., Schaefer, J. M., Goehring, B. M., Lifton, N., Putnam, A. E., and Barrell, D. J. A. (2012). Calibration of the in situ cosmogenic 14C production rate in New Zealand’s Southern Alps. Journal of Quaternary Science, 27(7):671–674. Schimmelpfennig, I., Williams, A., Pik, R., Burnard, P., Niedermann, S., Finkel, R., Schneider, B., and Benedetti, L. (2011). Inter-comparison of cosmogenic in-situ 3He, 21Ne and 36Cl at low latitude along an altitude transect on the SE slope of Kilimanjaro volcano (3◦S, Tanzania). Quaternary Geochronology, 6(5):425–436. Schlagenhauf, A., Gaudemer, Y., Benedetti, L., Manighetti, I., Palumbo, L., Schim- melpfennig, I., Finkel, R., and Pou, K. (2010). Using in situ Chlorine-36 cosmonuclide to recover past earthquake histories on limestone normal fault scarps: a reappraisal of methodology and interpretations. Geophysical Journal International, 182(1):36–72. Schmidt, G., Annan, J., Bartlein, P., Cook, B., Guilyardi, E., Hargreaves, J., Harrison, S., Kageyama, M., LeGrande, A., Konecky, B., et al. (2013). Using paleo-climate comparisons to constrain future projections in CMIP5. Climate of the Past Discussions, 9:775–835. Schmittner, A., Urban, N. M., Shakun, J. D., Mahowald, N. M., Clark, P. U., Bartlein, P. J., Mix, A. C., and Rosell Mel´e, A. (2011). Climate sensitivity estimated from temperature reconstructions of the Last Glacial Maximum. Science, 334(6061):1385–1388. Schneider von Deimling, T., Ganopolski, A., Held, H., and Rahmstorf, S. (2006). How cold was the last glacial maximum? Geophysical Research Letters, 33(14):L14709. Shackleton, N. J. (1967). Oxygen isotope analyses and Pleistocene temperatures re- assessed. Nature, 215(5096):15–17. Shakun, J. D., Clark, P. U., He, F., Marcott, S. A., Mix, A. C., Liu, Z., Otto Bliesner, B., Schmittner, A., and Bard, E. (2012). Global warming preceded by increasing carbon dioxide concentrations during the last deglaciation. Nature, 484(7392):49–54. 262 BIBLIOGRAPHY Shane, P. (2000). Tephrochronology: A New Zealand case study. Earth Science Reviews, 49(1-4):223–259. Shane, P. (2000). Tephrochronology: A New Zealand case study. Earth Science Reviews, 49(1-4):223–259. Shane, P., Nairn, I. A., Martin, S. B., and Smith, V. C. (2008). Compositional hetero- geneity in tephra deposits resulting from the eruption of multiple magma bodies: Implications for tephrochronology. BIBLIOGRAPHY Quaternary International, 178(1):44 – 53. Shane, P. and Sandiford, A. (2003). Paleovegetation of marine isotope stages 4 and 3 in Northern New Zealand and the age of the widespread Rotoehu tephra. Quaternary Research, 59(3):420–429. Shulmeister, J., Fink, D., and Augustinus, P. C. (2005). A cosmogenic nuclide chronology of the last glacial transition in North-West Nelson, New Zealandnew insights in Southern Hemisphere climate forcing during the last deglaciation. Earth and Planetary Science Letters, 233(3):455–466. Shulmeister, J., Fink, D., Hyatt, O. M., Thackray, G. D., and Rother, H. (2010a). Cosmo- genic 10Be and 26Al exposure ages of moraines in the Rakaia Valley, New Zealand and the nature of the last termination in New Zealand glacial systems. Earth and Planetary Science Letters, 297(3):558–566. Shulmeister, J., Shane, P., Lian, O. B., Okuda, M., Carter, J. A., Harper, M., Dickinson, W., Augustinus, P., and Heijnis, H. (2001). A long late-Quaternary record from Lake Poukawa, Hawkes Bay, New Zealand. Palaeogeography, Palaeoclimatology, Palaeoecology, 176(1):81–107. Shulmeister, J., Thackray, G., Rieser, U., Hyatt, O., Rother, H., Smart, C., and Evans, D. (2010b). The stratigraphy, timing and climatic implications of glaciolacustrine deposits in the middle Rakaia Valley, South Island, New Zealand. Quaternary Science Reviews, 29(17):2362–2381. Siegenthaler, U., Stocker, T. F., Monnin, E., L¨uthi, D., Schwander, J., Stauffer, B., Raynaud, D., Barnola, J.-M., Fischer, H., Masson Delmotte, V., et al. (2005). Stable carbon cycle– climate relationship during the late Pleistocene. Science, 310(5752):1313–1317. Sigman, D. M. and Boyle, E. A. (2000). Glacial/interglacial variations in atmospheric carbon dioxide. Nature, 407(6806):859–869. Sikes, E. L., Howard, W. R., Samson, C. R., Mahan, T. S., Robertson, L. G., and Volkman, J. K. (2009). Southern ocean seasonal temperature and subtropical front movement on the South Tasman Rise in the Late Quaternary. Paleoceanography, 24(2):PA2201. BIBLIOGRAPHY 263 Sikes, E. L., Medeiros, P. M., Augustinus, P., Wilmshurst, J. M., and Freeman, K. R. (2013). Seasonal variations in aridity and temperature characterize changing climate during the last deglaciation in New Zealand. Quaternary Science Reviews, 74:245–256. Sikes, E. L., Medeiros, P. M., Augustinus, P., Wilmshurst, J. M., and Freeman, K. R. (2013). Seasonal variations in aridity and temperature characterize changing climate during the last deglaciation in New Zealand. Quaternary Science Reviews, 74:245–256. Sp¨orli, K. and Rowland, J. (2006). ’Column on column’ structures as indicators of lava/ice interaction, Ruapehu andesite volcano, New Zealand. Journal of Volcanology and Geothermal Research, 157(4):294–310. BIBLIOGRAPHY Stephens, T., Atkin, D., Augustinus, P., Shane, P., Lorrey, A., Street Perrott, A., Nilsson, A., and Snowball, I. (2012a). A late glacial Antarctic climate teleconnection and variable Holocene seasonality at Lake Pupuke, Auckland, New Zealand. Journal of Paleolimnology, 48(4):785–800. Stephens, T., Atkin, D., Cochran, U., Augustinus, P., Reid, M., Lorrey, A., Shane, P., and Street Perrott, A. (2012b). A diatom-inferred record of reduced effective precipitation during the Last Glacial Coldest Phase (28.8-18.0 cal kyr BP) and increasing Holocene seasonality at Lake Pupuke, Auckland, New Zealand. Journal of Paleolimnology, 48(4):801–817. Stipp, J. J. (1968). The geochronology and petrogenesis of the Cenovoic volcanics of the North Island, New Zealand. Ph.D. Thesis, The Australian National University, Canberra. Stocker, T. F. and Johnsen, S. J. (2003). A minimum thermodynamic model for the bipolar seesaw. Paleoceanography, 18(4):PA000920. Stone, J., Evans, J., Fiﬁeld, K., Cresswell, R., and Allan, G. (1996). Cosmogenic chlorine- 36 production rates from calcium and potassium. Radiocarbon, 38(1):170–171. Stone, J. O. (2000). Air pressure and cosmogenic isotope production. Journal of Geophysi- cal Research B: Solid Earth, 105(B10):23753–23759. Stuiver, M. and Polach, H. A. (1977). Discussion; reporting of c-14 data. Radiocarbon, 19(3):355–363. Sturman, A. P. and Tapper, N. J. (1996). The weather and climate of Australia and New Zealand. Oxford University Press, Melbourne, Australia. Suganuma, Y., Miura, H., and Okuno, J. (2012). A new sampling technique for surface exposure dating using a portable electric rock cutter. Antarctic Record, 56(2):85–90. Sugden, D. (2014). James Croll (1821-1890): ice, ice ages and the Antarctic connection. Antarctic Science, 26:604–613. Suggate, R. P. (1990). Late Pliocene and quaternary glaciations of New Zealand. Quater- nary science reviews, 9(2):175–197. 264 BIBLIOGRAPHY Suggate, R. P. and Almond, P. C. (2005). The Last Glacial Maximum (LGM) in western South Island, New Zealand: Implications for the global LGM and MIS 2. Quaternary Science Reviews, 24(16-17):1923–1940. Suggate, R. P. and Almond, P. C. (2005). The Last Glacial Maximum (LGM) in western South Island, New Zealand: Implications for the global LGM and MIS 2. Quaternary Science Reviews, 24(16-17):1923–1940. Suggate, R. P. and Moar, N. T. (1970). Revision of the chronology of the late Otira glacial. New Zealand Journal of Geology and Geophysics, 13(3):742–746. Sundell, K. A. and Fisher, R. V. (1985). Very coarse grained fragmental rocks: A proposed size classiﬁcation. Geology, 13(10):692–695. Sutherland, R., Kim, K., Zondervan, A., and McSaveney, M. (2007). BIBLIOGRAPHY Orbital forcing of mid-latitude Southern Hemisphere glaciation since 100 ka inferred from cosmogenic nuclide ages of moraine boulders from the Cascade Plateau, southwest New Zealand. Bulletin of the Geological Society of America, 119(3-4):443–451. Sutton, P. J. H., Bowen, M., and Roemmich, D. (2005). Decadal temperature changes in the Tasman Sea. New Zealand Journal of Marine and Freshwater Research, 39(6):1321– 1329. Swanson, T. W. and Caffee, M. L. (2001). Determination of 36Cl production rates derived from the well-dated deglaciation surfaces of Whidbey and Fidalgo islands, washington. Quaternary Research, 56(3):366–382. Tait, A., Henderson, R., Turner, R., and Zheng, X. (2006). Thin plate smoothing spline interpolation of daily rainfall for New Zealand using a climatological rainfall surface. International Journal of Climatology, 26(14):2097–2115. Tait, A. and Zheng, X. (2007). Analysis of the spatial interpolation error associated with maps of median annual climate variables. National Institute of Water and Atmospheric Research, Auckland, New Zealand. Tarasov, L., Dyke, A. S., Neal, R. M., and Peltier, W. (2012). A data-calibrated distribution of deglacial chronologies for the North American ice complex from glaciological modeling. Earth and Planetary Science Letters, 315:30–40. Taylor, G. (1927). Notes on the glaciation of Ruapehu. Transactions of the N.Z. Institute, 57:235–236. Thackray, G. D., Shulmeister, J., and Fink, D. (2009). Evidence for expanded middle and late pleistocene glacier extent in northwest Nelson, New Zealand. Geograﬁska Annaler, Series A: Physical Geography, 91(4):291–311. Thompson, L. G., Mosley Thompson, E., Davis, M. E., Lin, P.-N., Henderson, K. A., Cole Dai, J., Bolzan, J. F., and Liu, K.-B. (1995). Late glacial stage and Holocene tropical ice core records from Huascaran, Peru. Science, 269(5220):46–50. BIBLIOGRAPHY 265 Thompson, R. D. and Kells, B. R. (1973). Mass balance studies on the Whakapapanui glacier, New Zealand. The role of snow and ice in hydrology, pages 383–393. Timmermann, A., Timm, O., Stott, L., and Menviel, L. (2009). The Roles of CO2 and Orbital Forcing in Driving Southern Hemispheric Temperature Variations during the Last 21 000 Yr. Journal of Climate, 22(7):1626–1640. Toggweiler, J. R. (2009). Shifting westerlies. Science, 323(5920):1434–1435. Tolstikhin, I., Mamyrin, B., Khabarin, L., and Erlikh, E. (1974). Isotope composition of helium in ultrabasic xenoliths from volcanic rocks of Kamchatka. Earth and Planetary Science Letters, 22(1):75–84. Topping, W. (1973). Tephrostratigraphy and chronology of late Quaternary eruptives from the Tongariro Volcanic Centre, New Zealand. New Zealand Journal of Geology and Geophysics, 16(3):397–423. Topping, W. W. (1974). Some aspects of Quaternary history of Tongariro Volcanic Centre. Ph.D. Thesis, Victoria University of Wellington. Topping, W. W. and Kohn, B. P. (1973). Rhyolitic tephra marker beds in the Tongariro area, North Island, New Zealand. New Zealand Journal of Geology and Geophysics, 16(3):375–395. Tovar, D. S., Shulmeister, J., and Davies, T. (2008). Evidence for a landslide origin of New Zealand’s Waiho Loop moraine. Nature Geoscience, 1(8):524–526. Townsend, D., Vonk, A., and Kamp, P. (2008). Geology map of the Taranaki area: scale 1:250,000. Three maps and notes (36 p.). Institute of Geological and Nuclear Sciences 1:250,000 geological map 7. 77p. + 1 folded map. Turney, C., Roberts, R., De Jonge, N., Prior, C., Wilmshurst, J., McGlone, M., and Cooper, J. (2007). Redating the advance of the New Zealand Franz Josef Glacier during the Last Termination: evidence for asynchronous climate change. Quaternary Science Reviews, 26(25):3037–3042. Turney, C. S. M., McGlone, M. S., and Wilmshurst, J. M. (2003). Asynchronous climate change between New Zealand and the North Atlantic during the last deglaciation. Geology, 31(3):223–226. Uddstrom, M. J. and Oien, N. A. (1999). On the use of high-resolution satellite data to describe the spatial and temporal variability of sea surface temperatures in the New Zealand region. Journal of Geophysical Research C: Oceans, 104(C9):20729–20751. 266 BIBLIOGRAPHY Ui, T., Kawachi, S., and Neall, V. E. (1986). Fragmentation of debris-avalanche mate- rial during ﬂowageevidence from the Pungarehu Formation, Mount Egmont, New Zealand. Journal of Volcanology and Geothermal Research, 27:255264. Vacco, D. A., Alley, R. B., and Pollard, D. (2010). Glacial advance and stagnation caused by rock avalanches. Earth and Planetary Science Letters, 294(1):123–130. van der Meer, J. J. M., Kjr, K. BIBLIOGRAPHY H., Krger, J., Rabassa, J., and Kilfeather, A. A. (2009). Under pressure: clastic dykes in glacial settings. Quaternary Science Reviews, 28(7-8):708–720. Van der Veen, C., Leftwich, T., von Frese, R., Csatho, B., and Li, J. (2007). Subglacial topography and geothermal heat ﬂux: Potential interactions with drainage of the Greenland ice sheet. Geophysical research letters, 34(12):L12501. Vandergoes, M. J., Dieffenbacher Krall, A. C., Newnham, R. M., Denton, G. H., and Blaauw, M. (2008). Cooling and changing seasonality in the southern Alps, New Zealand during the Antarctic cold reversal. Quaternary Science Reviews, 27(5):589–601. Vandergoes, M. J. and Fitzsimons, S. J. (2003). The last glacial-interglacial transition (LGIT) in south Westland, New Zealand: Paleoecological insight into mid-latitude Southern Hemisphere climate change. Quaternary Science Reviews, 22:1461–1476. Vandergoes, M. J., Hogg, A. G., Lowe, D. J., Newnham, R. M., Denton, G. H., Southon, J., Barrell, D. J. A., Wilson, C. J. N., McGlone, M. S., Allan, A. S. R., Almond, P. C., Petchey, F., Dabell, K., Dieffenbacher Krall, A. C., and Blaauw, M. (2013). A revised age for the Kawakawa/Oruanui tephra, a key marker for the Last Glacial Maximum in New Zealand. Quaternary Science Reviews, 74:195–201. Vandergoes, M. J., Newnham, R. M., Preusser, F., Hendy, C. H., Lowell, T. V., Fitzsimons, S. J., Hogg, A. G., Kasper, H. U., and Schl¨uchter, C. (2005). Regional insolation forcing of Late Quaternary climate change in the Southern Hemisphere. Nature, 436(7048):242–245. Voight, B., Glicken, H., Janda, R. J., and Douglass, M. (1981). Catastrophic rockslide avalanche of May 18 (Mount St. Helens). U.S.Geological Survey Professional Paper, 1250:347–377. Von Humboldt, A. (1852). Cosmos: vol. 4 (Translated by Paul, BH, and others). George Bell & Sons, London, UK. Waelbroeck, C., Paul, A., Kucera, M., Rosell Mel´e, A., Weinelt, M., Schneider, R., Mix, A., Abelmann, A., Armand, L., Bard, E., et al. (2009). Constraints on the magnitude and patterns of ocean cooling at the Last Glacial Maximum. Nature Geoscience, 2(2):127–132. 267 BIBLIOGRAPHY Wang, X., Auler, A. S., Edwards, R. L., Cheng, H., Cristalli, P. S., Smart, P. L., Richards, D. A., and Shen, C. . (2004). Wet periods in northeastern Brazil over the past 210 kyr linked to distant climate anomalies. Nature, 432(7018):740–743. Wang, Y.-J., Cheng, H., Edwards, R. L., An, Z., Wu, J., Shen, C.-C., and Dorale, J. A. (2001). A high-resolution absolute-dated late Pleistocene monsoon record from Hulu Cave, China. Science, 294(5550):2345–2348. Wardle, P. (1978). BIBLIOGRAPHY Further radiocarbon dates from Westland National Park and the Omoeroa river mouth, New Zealand. New Zealand Journal of Botany, 16(1):147–152. Wardle, P. (1991). Vegetation of New Zealand. Cambridge University Press, Cambridge, UK. Weaver, A. J., Saenko, O. A., Clark, P. U., and Mitrovica, J. X. (2003). Meltwater pulse 1A from Antarctica as a trigger of the Bølling-Allerød warm interval. Science, 299(5613):1709–1713. Weber, M., Clark, P., Kuhn, G., Timmermann, A., Sprenk, D., Gladstone, R., Zhang, X., Lohmann, G., Menviel, L., Chikamoto, M., et al. (2014). Millennial-scale variability in Antarctic ice-sheet discharge during the last deglaciation. Nature, 510:134–138. Whittaker, T. E., Hendy, C. H., and Hellstrom, J. C. (2011). Abrupt millennial-scale changes in intensity of Southern Hemisphere westerly winds during marine isotope stages 2-4. Geology, 39(5):455–458. Williams, A. J., Stuart, F. M., Day, S. J., and Phillips, W. M. (2005). Using pyroxene microphenocrysts to determine cosmogenic 3He concentrations in old volcanic rocks: An example of landscape development in central Gran Canaria. Quaternary Science Reviews, 24(1-2):211–222. Williams, P. W., McGlone, M., Neil, H., and Zhao, J.-X. (2015). A review of New Zealand palaeoclimate from the Last Interglacial to the global Last Glacial Maximum. Quaternary Science Reviews, 110:92–106. Williams, P. W., Neil, H. L., and Zhao, J. X. (2010). Age frequency distribution and revised stable isotope curves for New Zealand speleothems: Palaeoclimatic implica- tions. International Journal of Speleology, 39(2):99–112. Wilmshurst, J. M., McGlone, M. S., Leathwick, J. R., and Newnham, R. M. (2007). A pre-deforestation pollen-climate calibration model for New Zealand and quantitative temperature reconstructions for the past 18 000 years BP. Journal of Quaternary Science, 22(5):535–547. 268 Wolff, E., Fischer, H., and R¨othlisberger, R. (2009). Glacial terminations as southern warmings without northern control. Nature Geoscience, 2(3):206–209. BIBLIOGRAPHY Winckler, G., Anderson, R. F., and Schlosser, P. (2005). Equatorial Paciﬁc productiv- ity and dust ﬂux during the mid-Pleistocene climate transition. Paleoceanography, 20(4):PA4025. Winckler, G., Anderson, R. F., and Schlosser, P. (2005). Equatorial Paciﬁc productiv- ity and dust ﬂux during the mid-Pleistocene climate transition. Paleoceanography, 20(4):PA4025. Wolff, E., Fischer, H., and R¨othlisberger, R. (2009). Glacial terminations as southern warmings without northern control. Nature Geoscience, 2(3):206–209. Wolff, E., Fischer, H., and R¨othlisberger, R. (2009). Glacial terminations as southern warmings without northern control. Nature Geoscience, 2(3):206–209.
https://openalex.org/W2548098208	https://europepmc.org/articles/pmc5133367?pdf=render	English	null	Non‐small cell lung cancer is characterised by a distinct inflammatory signature in serum compared with chronic obstructive pulmonary disease	Clinical & translational immunology	2,016	cc-by	6,867	Non-small cell lung cancer is characterised by a distinct inﬂammatory signature in serum compared with chronic obstructive pulmonary disease Hanne Astrid Eide1, Ann Rita Halvorsen1, Vandana Sandhu1, Anne Fåne2, Janna Berg1,3, Vilde Drageset Haakensen1, Elin H Kure1,4, Odd Terje Brustugun1,5, Cecilie Essholt Kiserud6, Jon Amund Kyte2,5 and Åslaug Helland1,5 Development of lung cancer is closely related to smoking in a majority of patients. Most smokers, however, do not develop lung cancer in spite of a high mutational load accumulating in the lung tissue. Here we investigate whether a cancer-speciﬁc footprint can be revealed by investigating circulating inﬂammatory markers in patients with non-small cell lung cancer (NSCLC) compared with patients with chronic obstructive pulmonary disease (COPD), both cohorts characterised by similar smoking history. Serum concentrations of 57 cytokines and matrix metalloproteinases (MMPs) from 43 patients with advanced NSCLC were evaluated by multiplex immunoassays and compared with serum samples from 35 patients with COPD. Unsupervised hierarchical clustering and non-parametric analyses were performed. False discovery rate was used to adjust for multiple testing. Clustering of cytokine and MMP concentrations in the serum revealed a distinct separation of the NSCLC patients from the COPD group. Individual concentrations of thymus and activation-regulated cytokine (C-C motif chemokine ligand 17), Gro-b (C-X-C motif chemokine ligand 2 (CXCL2)), CXCL13, interleukin (IL)-1ra, IL-6, IL-8 (CXCL8), IL-16, IL-17A, macrophage migration inhibitory factor (MIF), granulocyte colony-stimulating factor, platelet-derived growth factor subunit B, MMP-2, MMP-8 and MMP-12 were signiﬁcantly different in serum from NSCLC and COPD patients. Moreover, the interferon-γ/IL-10 ratio was lower in cancer patients compared with COPD patients, consistent with a cytokine milieu favouring tumour tolerance. Our results suggest that NSCLC is characterised by a distinct inﬂammatory signature in serum. The different cytokine proﬁles in NSCLC and COPD patients may represent tumour-promoting and tumour-suppressing immune responses developing in response to mucosal inﬂammation and mutations induced by smoking. y g Clinical & Translational Immunology (2016) 5, e109; doi:10.1038/cti.2016.65; published online 2 November 2016 epidemiological and clinical studies.5,6 There is, however, limited knowledge if or how the host immune response inﬂuence whether an individual with COPD develops lung cancer. The severity of lung cancer is well known and lung cancer remains the leading cause of cancer-related death worldwide.1 Treatment and prognosis rely heavily on disease stage at diagnosis. A majority of patients are diagnosed in advanced stages, not eligible for treatment with curative intent. 1Department of Cancer Genetics, Institute for Cancer Research, Oslo University Hospital-The Norwegian Radium Hospital, Oslo, Norway; 2Department for Cell Therapy, Oslo University Hospital-The Norwegian Radium Hospital, Oslo, Norway; 3Department of Medicine, Vestfold Hospital Trust, Tønsberg, Norway; 4Department for Environmental Health and Science, Telemark University College, Bø in Telemark, Norway; 5Department of Oncology, Oslo University Hospital-The Norwegian Radium Hospital, Oslo, Norway and 6Department of Oncology, National Advisory Unit on Late Effects After Cancer Treatment, Oslo University Hospital-The Norwegian Radium Hospital, Oslo, Norway Correspondence: Associate Professor Å Helland, Department of Cancer Genetics, Institute for Cancer Research, Oslo University Hospital-The Norwegian Radium Hospital, Postboks 4953 Nydalen, Oslo 0424, Norway. E-mail: ahelland@medisin.uio.no Received 11 July 2016; revised 26 August 2016; accepted 23 September 2016 OPEN OPEN OPEN Clinical & Translational Immunology (2016) 5, e109; doi:10.1038/cti.2016.65 Ofﬁcial journal of the Australasian Society for Immunology Inc. www.nature.com/cti ORIGINAL ARTICLE Non-small cell lung cancer is characterised by a distinct inﬂammatory signature in serum compared with chronic obstructive pulmonary disease Although scientiﬁc endeavours are comprehen- sive, there is a deﬁnite need for further insight into lung cancer biology aiming at improved diagnostics, more efﬁcient cancer treatment and ultimately an increase in survival. We know that heavy smokers accumulate a high mutational load in the lung tissue, but still only a minority develop cancer. Carcinogenesis is not merely a malignant transformation of cells; it is dependent on multiple interactions with different cell types making up the tumour microenvironment. Cells of the immune system, endothelial cells and ﬁbroblasts are among others found adjacent to malignant cells in a tumour and are of importance in cancer development and progression.7 Genetic events in cells are followed by an interaction with the immune system.2 The immune response can either suppress the development of a tumour by eliminating cancer cells or promote tumour growth by selecting cancer cells that escape the immune Inﬂammation is linked to multiple tumour-promoting effects and is in general recognised to have an important part in cancer evolution.2 The lungs are vulnerable for air-borne environmental factors, and tobacco smoke in particular is implicated in lung inﬂammation.3,4 An association between the inﬂammatory disease chronic obstructive pulmonary disease (COPD) and lung cancer is evident from Distinct inﬂammatory markers in NSCLC HA Eide et al Distinct inﬂammatory markers in NSCLC HA Eide et al 2 control.8 It is evident that immune cells in the tumour microenviron- ment are relevant for tumour characteristics and patient outcome. Tumour-inﬁltrating lymphocytes are of clinical impact in several cancers, including lung cancer where high levels are correlated to better prognosis.9 To target the non-malignant cells in the tumour environment is an interesting approach to the treatment of cancer. Certainly, recent clinical trials with immune-associated mediators have proven efﬁcient in non-small cell lung cancer (NSCLC) treatment.10,11 Proteins and enzymes make up complex networks responsible for cell-to-cell communication in the tumour microenvironment. Cytokines are essential modulators. A shift from an immunological pattern with a T helper type 1 (TH1) orientation to a TH2 pattern mediated by cytokines is reported as a biological event in the carcinogenesis.12 Indeed, increased serum levels of speciﬁc cytokines are associated with a risk of developing lung cancer and is linked to survival both in early- and advanced-stage lung cancer.13–15 Matrix metalloproteinases (MMPs) are proteolytic enzymes originally known to degrade extracellular matrix. Patient and disease characteristics The median age of the NSCLC patients at inclusion was 70 years (range 47–88) and 72% of the patients were male. All NSCLC patients had a history of smoking either as current (26%) or former (74%) smokers. Twenty six of the patients (61%) had not received previous chemotherapy. Fourteen NSCLC patients (33%) were treated with systemic steroids at the time of inclusion. The median age of the COPD patients were 72 years (range 50–87) and 40% were male. All but three patients (91%) had a smoking history. Only 4 patients (11%) in the COPD group received systemic steroids at the time of serum sampling. A majority had grade III COPD disease (46%). The NSCLC and COPD cohorts were fairly well balanced at baseline with no signiﬁcant differences in age and smoking history. A higher percentage of patients in the NSCLC cohort (33%) used systemic steroids compared with the COPD cohort (11%), but this difference was not signiﬁcant. There was, however, a signiﬁcant difference in sex (P=0.006) with a larger percentage of males relative to females in the NSCLC group compared with the COPD group. Baseline characteristics of the NSCLC and COPD patients are shown in Table 1. Non-small cell lung cancer is characterised by a distinct inﬂammatory signature in serum compared with chronic obstructive pulmonary disease Moreover, they also regulate the activity of growth factors, cytokines, cell receptors and other proteases, thus inﬂuencing a variety of inﬂammatory processes and biological activities in cancer.16 Table 1 Characteristics of NSCLC and COPD patients included in the cytokine/MMP analysis Variable NSCLC cohort COPD cohort P-value N = 43 % N = 35 % Age at inclusion (years) Mean/median/range 69/70/47–88 71/72/50–87 0.333a Sex Male 31 72.1 14 40.0 0.006b Female 12 27.9 21 60.0 Smoking history Current 11 25.6 13 37.1 0.057c Former 32 74.4 19 54.3 Never 0 0 3 8.6 Pack years Mean/median/range 34/30/4–144 32/35/12–60 0.698a Use of systemic steroids Yes 14 32.6 4 11.4 0.153b No 29 67.4 31 88.6 COPD grade I 1 2.9 II 10 28.6 III 16 45.7 IV 8 22.8 ECOG performance status 0 8 18.6 1 22 51.2 2 13 30.2 Stage III 11 25.6 IV 32 74.4 Histology Adenocarcinoma 26 60.5 Squamous cell carcinoma 13 30.2 NOS 4 9.3 Previous chemotherapy Yes 17 39.5 No 26 60.5 Abbreviations: COPD, chronic obstructive pulmonary disease; ECOG, Eastern Cooperative Oncology Group; MMP, matrix metalloproteinase; NOS, not otherwise speciﬁed; NSCLC, non-small cell lung cancer. aTwo sided t-test for continuous variables. bFisher’s exact test for categorical variables. cChi sq a e test fo catego ical a iables Table 1 Characteristics of NSCLC and COPD patients included in the cytokine/MMP analysis Here we analyse a comprehensive panel of circulating cytokines and MMPs in patients with advanced-stage NSCLC using COPD patients as a control group. Both cohorts represent current or former smokers. Our aim is to investigate the cancer-speciﬁc footprint in the serum mirroring lung cancer carcinogenesis. Clinical & Translational Immunology Distinct signatures of serum proteins separating NSCLC from COPD patients Box plots of signiﬁcantly different Distinct inﬂammatory markers in NSCLC HA Eide et al MIP−1sigma/CCL15 SCYB16 MPIF−1 CXCL13 MIP−3a/CCL20 MMP−12 MMP−7 MMP−13 CXCL6/GCP−2 MCP−4 I−TAC IP−10 MIG CCL27 SDF−1a+b MDC MCP−1 MCP−2 MIP−1A IL−6 IL−8 CCL11/Eotaxin CX3CL1/Fractalkine IL−10 MIP−3b TARC GM−CSF IL−4 TNF−a CCL26/Eotaxin−3 MCP−3 TECK IFN−g IL−2 CCL1/I−309 IL−1b CXCL5 CXCL1/Gro−a CXCL2/Gro−b PDGF−BB MMP−1 IL−12p70 VEGF CCL24/Eotaxin−2 MMP−3 MMP−10 MMP−8 MMP−9 RANTES TRAIL MMP−2 IL−1ra IL−17A G−CSF IL−16 MIF −5 5 Row Z−Score Color Key Sex (p = 0.035) Smoking (p = 0.271) COPD NSCLC Cluster 1 Cluster 2 3 MIP−1sigma/CCL15 SCYB16 MPIF−1 CXCL13 MIP−3a/CCL20 MMP−12 MMP−7 MMP−13 CXCL6/GCP−2 MCP−4 I−TAC IP−10 MIG CCL27 SDF−1a+b MDC MCP−1 MCP−2 MIP−1A IL−6 IL−8 CCL11/Eotaxin CX3CL1/Fractalkine IL−10 MIP−3b TARC GM−CSF IL−4 TNF−a CCL26/Eotaxin−3 MCP−3 TECK IFN−g IL−2 CCL1/I−309 IL−1b CXCL5 CXCL1/Gro−a CXCL2/Gro−b PDGF−BB MMP−1 IL−12p70 VEGF CCL24/Eotaxin−2 MMP−3 MMP−10 MMP−8 MMP−9 RANTES TRAIL MMP−2 IL−1ra IL−17A G−CSF IL−16 MIF −5 5 Row Z−Score Color Key Sex (p = 0.035) Smoking (p = 0.271) Steroids (p = 0.104) Histology Stage COPD grade COPD NSCLC Cluster 1 Cluster 2 Sex Male Smoking Current Systemic steroids No Histology No Stage III COPD grade 1 Female Former Yes Yes IV 2 Never NOS 3 4 Missing UN Figure 1 Hierarchical clustering of 57 proteins (cytokines and MMPs) in serum samples from 43 patients with NSCLC and 35 patients with COPD. Clinica parameters with tests for signiﬁcant differences between the clusters are visualised below the heat map. AD, adenocarcinoma; NOS, none otherwise speciﬁed; SCC, squamous cell carcinoma; UN, unknown COPD grade. Sex (p = 0.035) Smoking (p = 0.271) Steroids (p = 0.104) Histology Stage COPD grade Male Female Missing Missing Figure 1 Hierarchical clustering of 57 proteins (cytokines and MMPs) in serum samples from 43 patients with NSCLC and 35 patients with COPD. Clinical parameters with tests for signiﬁcant differences between the clusters are visualised below the heat map. AD, adenocarcinoma; NOS, none otherwise speciﬁed; SCC, squamous cell carcinoma; UN, unknown COPD grade. NSCLC patients but did not retain signiﬁcance after correction for multiple testing. IFNγ/IL-10 ratio, however, was signiﬁcantly different between NSCLC and COPD patients (Po0.001), with a median ratio of 0.84 in the NSCLC group versus 1.11 in the COPD cohort (Figure 2). levels of cytokines/MMPs, and a complete table of all proteins investigated in the two cohorts are included in Supplementary Information (Supplementary Figure S1 and Supplementary Table S1). Distinct signatures of serum proteins separating NSCLC from COPD patients p Hierarchical clustering of cytokines and MMPs separated the serum samples into two clusters (Figure 1) illustrating a clear separation of cytokine levels in NSCLC and COPD patients. There was a signiﬁcant difference between the two clusters regarding sex (P=0.035), concurrent with the difference in baseline characteristics with fewer men among the COPD patients. No signiﬁcant differences in smoking history or in the use of systemic steroids were seen between the clusters. Several individual cytokines and MMPs had different concentrations in serum between the two cohorts (Table 2, Figure 2). After correction for multiple testing, NSCLC patients had a signiﬁcant higher median level of thymus and activation regulated cytokine (TARC; C-C motif chemokine ligand 17 (CCL17)), Gro-b (C-X-C motif chemokine ligand 2 (CXCL2)), CXCL13, interleukin (IL)-6, IL-8 (CXCL8), platelet-derived growth factor subunit B (PDGF-BB), MMP-8 and MMP-12 in the serum compared with the COPD controls. For IL-1ra, IL-16, IL-17A, macrophage migration inhibitory factor (MIF), gran- ulocyte colony-stimulating factor (G-CSF) and MMP-2, the median serum concentration value in NSCLC patients was signiﬁcantly lower compared with COPD patients. Box plots of signiﬁcantly different Hierarchical clustering of cytokines and MMPs separated the serum samples into two clusters (Figure 1) illustrating a clear separation of cytokine levels in NSCLC and COPD patients. There was a signiﬁcant difference between the two clusters regarding sex (P=0.035), concurrent with the difference in baseline characteristics with fewer men among the COPD patients. No signiﬁcant differences in smoking history or in the use of systemic steroids were seen between the clusters. Several individual cytokines and MMPs had different concentrations in serum between the two cohorts (Table 2, Figure 2). After correction for multiple testing, NSCLC patients had a signiﬁcant higher median level of thymus and activation regulated cytokine (TARC; C-C motif chemokine ligand 17 (CCL17)), Gro-b (C-X-C motif chemokine ligand 2 (CXCL2)), CXCL13, interleukin (IL)-6, IL-8 (CXCL8), platelet-derived growth factor subunit B (PDGF-BB), MMP-8 and MMP-12 in the serum compared with the COPD controls. For IL-1ra, IL-16, IL-17A, macrophage migration inhibitory factor (MIF), gran- ulocyte colony-stimulating factor (G-CSF) and MMP-2, the median serum concentration value in NSCLC patients was signiﬁcantly lower compared with COPD patients. Associations of cytokine and MMP levels in different clinical subsets of NSCLC patients When omitting all NSCLC and COPD patients using corticosteroids, we observed a distinct serum signature separating NSCLC and COPD patients, similar to the one observed in the entire cohort (Supplementary Table S2). In addition, the levels of IL-10 and MMP-3 were observed to be signiﬁcantly different, with a higher and lower median level in NSCLC patients compared with COPD patients, respectively. The signiﬁcant association of TARC between the patient groups was lost. To the best of our knowledge, this is the ﬁrst study where COPD patients are chosen as controls in a serum cytokine/MMP analysis in NSCLC patients. Several cytokine/MMP studies have previously been conducted with healthy subjects as controls.15,17 All of the NSCLC patients included in our study were either former or current smokers, and there were no signiﬁcant differences in pack years between the NSCLC and COPD cohorts. COPD is an inﬂammatory disease strongly associated with smoking and is therefore likely to have an effect on the cytokine proﬁle in patients. Higher levels of inﬂammatory markers have been reported among former and current smokers as well as among those with a history of chronic bronchitis or emphysema, compared with healthy subjects, supporting that notion.13 Respiratory function tests were not a requirement at inclusion in the Thoracal Radiotherapy and Tarceva (ThoRaT) study. Sixteen of the NSCLC patients reported to have COPD (degree unknown) in their medical history at baseline. Owing to the similar smoking histories in both cohorts, it is likely that even more of the NSCLC patients would be suffering from COPD. COPD patients, on the other hand, were followed for a minimum of 2 years after serum sampling, and none Among NSCLC patients, two MMPs had a signiﬁcant different concentration after correction for multiple testing, when comparing patients using systemic steroids or not (Table 3). MMP-3 (Po0.001) and MMP-9 (P = 0.001) had a higher median concentration in the systemic steroid-user group. Cytokine/MMP levels in other clinical subsets of NSCLC are presented in Supplementary Table S3. Of note, current smokers had a signiﬁcantly higher level of cytokines CCL11 (P = 0.001), Gro-a (CXCL1) (P = 0.001), stromal-derived factor 1 alpha+beta (SDF-1a+b; CXCL12) (P = 0.003) and IL-4 (Po0.001) compared with former smokers. Increased C-reactive protein (CRP) levels correlated with higher levels of multiple pro-inﬂammatory cytokines. The association between CRP, Gro-b (CXCL2) and IL-6 remained statistically signiﬁcant after correction for multiple testing (P = 0.001 and Po0.001, respectively). DISCUSSION In the present study, we have investigated a panel of 57 circulating inﬂammatory markers consisting of cytokines and MMPs in the serum from NSCLC patients with advanced disease. The cytokines and MMPs were chosen based on previously published reports on potential biomarkers in lung cancer, and they represent a broad spectrum of inﬂammatory mediators of special interest in the tumour microenvironment. Obtaining peripheral blood samples is easy compared with samples of lung tumours. The concept of revealing essential markers of disease in blood tests have a particular allure in this cohort of advanced-stage NSCLC patients, where life expectancy is short and good quality of life is important. No difference was seen in the level of IL-1b between the groups, but the IL-1ra concentration in the serum samples was signiﬁcantly lower in the NSCLC group. Accordingly, the IL-1b/IL-1ra ratio in the NSCLC group was signiﬁcantly higher compared with the COPD patients (Po0.001; Figure 3). In the present study, we have investigated a panel of 57 circulating inﬂammatory markers consisting of cytokines and MMPs in the serum from NSCLC patients with advanced disease. The cytokines and MMPs were chosen based on previously published reports on potential biomarkers in lung cancer, and they represent a broad spectrum of inﬂammatory mediators of special interest in the tumour microenvironment. Obtaining peripheral blood samples is easy compared with samples of lung tumours. The concept of revealing essential markers of disease in blood tests have a particular allure in this cohort of advanced-stage NSCLC patients, where life expectancy is short and good quality of life is important. Distinct signatures of serum proteins separating NSCLC from COPD patients Interferon gamma (IFNγ) and IL-10 are considered hallmark cytokines for antitumour TH1 responses and immunological tumour tolerance, respectively. The serum concentration of IFNγ did not vary between NSCLC and COPD patients. The IL-10 levels were higher in Another interesting connection at the biological level in this panel of cytokines was the association between the pro-inﬂammatory protein IL-1b and its naturally occurring antagonist IL-1ra. Clinical & Translational Immunology Distinct inﬂammatory markers in NSCLC HA Eide et al 4 Table 2 Levels of circulating cytokines and MMPs with a signiﬁcant difference in NSCLC and COPD patients Cytokine/MMP NSCLC median COPD median P-value CCL1/I-309 77 89 0.041 MIP-3a (CCL20) 24 17 0.036 MIP-3b (CCL19) 919 606 0.029 TARC (CCL17) 400 253 0.011a Eotaxin (CCL11) 63 72 0.037 Gro-b (CXCL2) 878 514 o0.001a CXCL13 54 36 0.000a Fractalkine (CX3CL1) 254 212 0.025 IL-1ra 434 1079 o0.001a IL-6 27 16 0.002a IL-8 (CXCL8) 31 21 0.000a IL-10 76 56 0.016 IL-16 370 532 o0.001a IL-17A 73 273 o0.001a MIF 808 4895 o0.001a G-CSF 33 127 o0.001a PDGF-BB 3288 2357 0.003a MMP-2 37 242 89 243 o0.001a MMP-8 9436 5072 o0.001a MMP-12 871 347 o0.001a Abbreviations: CCL, C-C motif chemokine ligand; COPD, chronic obstructive pulmonary disease; CXCL, C-X-C motif chemokine ligand; G-CSF, granulocyte colony-stimulating factor; IL, interleukin; MIF, macrophage migration inhibitory factor; MIP, macrophage inﬂammatory protein; MMP, matrix metalloproteinase; NSCLC, non small cell lung cancer; PDGF-BB, platelet- derived growth factor subunit B; TARC, thymus and activation regulated cytokine. Concentrations measured in pg ml−1. aStatistically signiﬁcant P-values retained after correction for multiple. Table 2 Levels of circulating cytokines and MMPs with a signiﬁcant difference in NSCLC and COPD patients −5000 0 5000 0 5 10 15 Median difference between NSCLC and COPD patients −log10(P.value) Gro−b(CXCL2) CXCL13 IL−8(CXCL8) MMP−8 MMP−12 IL−1ra IL−16 IL−17A MIF G−CSF −5000 0 5000 0 5 10 15 Median difference between NSCLC and COPD patients −log10(P.value) Gro−b(CXCL2) CXCL13 IL−8(CXCL8) MMP−8 MMP−12 IL−1ra IL−16 IL−17A MIF G−CSF Median difference between NSCLC and COPD patients Figure 2 Volcano plot illustrating the magnitude and signiﬁcance of the differences in cytokine/MMP serum concentration levels in patients with NSCLC and COPD. Dots marked in red are cytokines/MMPs with a signiﬁcantly different median serum concentration after correction for multiple testing. correction for false discovery rate in NSCLC patients with a previous history of chemotherapy use compared with patients with no prior chemotherapy treatment. Clinical & Translational Immunology Associations of cytokine and MMP levels in different clinical subsets of NSCLC patients No signiﬁcant differences were seen after Clinical & Translational Immunology Distinct inﬂammatory markers in NSCLC HA Eide et al 5 Figure 3 Differences in IFNγ/IL-10 (a) and IL-1b/IL-1ra (b) ratios comparing patients with NSCLC and COPD. Table 3 Cytokine and MMP serum concentration values in the NSCLC patients (N = 43) with a signiﬁcant different distribution in non-steroid users compared with patients using systemic steroids Cytokine/MMP NSCLC cohort P-value No systemic steroids, N = 29, median Systemic steroids, N = 14, median MDC (CCL22) 977 453 0.013 IL-17a 88 56 0.011 MMP-3 5681 15 443 o0.001a MMP-7 3759 2676 0.036 MMP-9 42 768 77 164 0.001a MMP-12 1050 389 0.003 MMP-13 148 75 0.011 Abbreviations: CCL, C-C motif chemokine ligand; IL, interleukin; MDC, macrophage-derived chemokine; MMP, matrix metalloproteinase; NSCLC, non-small cell lung cancer. Concentrations measured in pg ml−1. aStatistically signiﬁcant P-values adjusted for multiple testing. Table 3 Cytokine and MMP serum concentration values in the NSCLC patients (N = 43) with a signiﬁcant different distribution in non-steroid users compared with patients using systemic steroids and no differences in TNFα, VEGF and GM-CSF in comparison with COPD patients. In another study, Barrera et al.15 found higher levels of IL-6, IL-8, IL-12p70, IL-17A and IFNγ comparing an advanced NSCLC cohort with 50% non-smokers, with healthy controls. Again, our results conﬁrm the high levels of IL-6 and IL-8 in NSCLC patients; however, no differences were seen between our cohorts in IL-12p70 or IFNγ concentrations, and IL-17A was found to be lower in our NSCLC group. COPD-associated inﬂammation can lead to elevated serum levels of multiple cytokines and the different observations with regard to some cytokines between our study and those of Karminska et al.17 and Barrera et al.15 may be explained by our use of COPD controls, rather than healthy individuals. The concentration of IL-17A in serum was found approximately fourfold lower in NSCLC versus COPD patients in our study. We cannot, from our data set, decode if this ﬁnding reﬂects elevated levels of IL-17A in the COPD cohort or low levels among the NSCLC patients. Associations of cytokine and MMP levels in different clinical subsets of NSCLC patients Based on current knowledge, the ﬁrst explanation appears more likely, as IL-17 has been found elevated in patients with a range of chronic inﬂammatory disorders.18 One former study revealed elevated levels of IL-17A in serum collected from COPD patients compared with control groups of both healthy smokers and non- smokers; IL-17A was also seen increased in accordance with advancing COPD stages.19 Studies in NSCLC patients have, contradictory to our results, found elevated IL-17A levels in circulation but then again compared with healthy controls.15,20 It is known that naive CD4+ TH precursor cells can differentiate into a variety of different TH subsets, including TH1, TH2, TH17 and regulatory T cells.21 TH17 cells are considered the predominant producer of IL-17. Both tumour- suppressing and tumour-promoting functions have been attributed to IL-17A and TH17 cells, and the role of TH17 cells in tumour immunity remains ambiguous.22 Figure 3 Differences in IFNγ/IL-10 (a) and IL-1b/IL-1ra (b) ratios comparing patients with NSCLC and COPD. of the patients included in theseanalyses developed cancer later. In our study, we aimed at discovering a cytokine and MMP proﬁle in the serum reﬂecting the malignant disease and not a confounding inﬂammatory pattern owing to smoking. The different cohorts have a similar background, the use of a highly carcinogenic substance, but only one group of patients developed cancer. The study investigates serum cytokine proﬁles in a relatively small cohort of patients with advanced NSCLC. Previous chemotherapy could be a possible confounder in the evaluation of the differences in cytokine distribution between NSCLC and certainly untreated COPD patients. Sixty percent of the NSCLC patients included in the study, however, had not received treatment with chemotherapy prior to inclusion. Moreover, when comparing the cytokine concentration level in NSCLC with and without previous chemotherapy, no signiﬁcant differences were found. The immune system has a pivotal role against cancer. The development of a successful immune response depends on the balance between the TH1 (antitumour) and TH2 responses and on the activity of immune cells that inhibit antitumour responses. Clinical & Translational Immunology Associations of cytokine and MMP levels in different clinical subsets of NSCLC patients T cells producing IL-10, in particular type 1 regulatory (Tr1) T cells and TH2 cells, are considered to be key suppressors of antitumour response and it is previously proposed that TH2-type inﬂammation facilitates tumour growth.23 It is further known that TH1 cells produce one particular set of cytokines such as IL-2, IFNγ and TNFα while TH2 cells produce others, for example, IL-4, IL-5, IL-6, IL-10 and IL-13.24 In our study, Many former studies have been conducted with only a few cytokines in parallel in the same individuals, but some have reported on larger panels. Kaminska et al.17 found elevated serum levels of tumour necrosis factor alpha (TNFα), IL-6, IL-8, IL-10, IL-1ra, vascular endothelial growth factor (VEGF), granulocyte-macrophage colony-stimulating factor (GM-CSF) and G-CSF in a study including NSCLC patients of all stages, compared with healthy controls. Elevated levels of IL-6, IL-8 and IL-10 were consistent with our ﬁndings, but contrary to their study, we found lower levels of G-CSF and IL-1ra, Clinical & Translational Immunology Distinct inﬂammatory markers in NSCLC HA Eide et al Distinct inﬂammatory markers in NSCLC HA Eide et al 6 IL-6 and IL-10 concentration in the serum was found to be higher in NSCLC patients, although the differences in IL-10 did not retain signiﬁcance after correction for multiple testing. IFNγ/IL-10 ratio was nonetheless found reduced in NSCLC patients compared with the COPD group, supporting the notion that the TH1 response in NSCLC patients was turned down, whereas the TH2 response was more prominent. These contrasting immune responses developing in different individuals may carry particular importance for the devel- opment of lung cancer. Intriguingly, most heavy smokers do not develop lung cancer in spite of a high mutation load accumulating in the lung tissue. There is increasing evidence suggesting that this resilience is related to the host immune response, known to be particularly potent for controlling tumours with a high mutation load and thus a high neoantigen frequency.25–27 We hypothesise that the different cytokine proﬁles identiﬁed among NSCLC patients and COPD subjects in our study represent a footprint of tumour- promoting versus tumour-suppressing immune responses developing in the host, in response to mucosal inﬂammation and mutations induced by smoking. It would be of interest to assess how this cytokine proﬁle is inﬂuenced by immunological checkpoint inhibitors or other forms of therapy. Study population and data collection The cytokine analyses were performed using serum samples obtained from 43 patients with advanced-stage NSCLC included in an ongoing clinical study (ThoRaT) from December 2011 until June 2015. The patients were referred for palliative radiotherapy. Clinical characteristics of the NSCLC patients were collected from the hospital medical records. Tumours were staged according to the Union for International Cancer Control, Tumour, Node, Metastasis 7. Histopathological evaluations were retrieved from pathology reports. The pro-inﬂammatory cytokine IL-1 has been shown elevated in several cancer types such as breast, colon, melanoma, head and neck as well as lung cancer and is associated with an increase in metastases and poor prognosis.28 We did not observe any difference between serum levels of IL-1b between the NSCLC and COPD cohorts investigated. The NSCLC patients in our study did, however, have a median concentration level of IL-1ra approximately half the value seen in the COPD serum samples. Moreover, the median IL-1ra concentration values were lower in stage IV NSCLC patients compared with patients in stage III, although this did not retain signiﬁcance after correction for multiple testing. IL-1ra is a naturally occurring IL1 antagonist. In IL-1ra-deﬁcient mice, skin tumours developed more rapidly compared with wild-type mice in a model of IL-1-induced carcinogenesis.29 In lung cancer, elevated levels of IL-1ra in serum have previously been found associated with decreased risk of lung cancer.13 Here we discovered that the IL-1b/IL-1ra ratio was elevated in NSCLC patients compared with COPD patients, indicating that the pro-malignant IL-1b expression level was higher in NSCLC patients relative to the antagonist; the ‘cancer-protective’ IL-1ra. Interestingly, therapeutic agents reducing IL-1 activity, such as recombinant IL-1ra, are commercially available for treatment of inﬂammatory diseases and could be an approach in cancer treatment.30 Blood sample processing The blood samples from the NSCLC patients were collected at inclusion in the ThoRaT study, prior to radiotherapy. Blood was collected in serum tubes, kept in room temperature appending coagulation and then processed at 2450 g for 15 min within 1 h after sampling. Finally, the samples were transferred in 250 μl aliquots into cryovials and stored at80 °C until usage. COPD cohort Serum samples from 35 patients with COPD were obtained at the Department of Medicine, Vestfold Hospital Trust, Tønsberg, Norway. Clinical information was acquired from the hospital records (Table 1). All COPD patients included were in a regular follow-up and had no sign of lung cancer prior to blood sampling. The patients were also followed for a minimum of 2 years after blood sampling with no sign of cancer. The serum samples in the COPD cohort were preprocessed under strictly deﬁned and equal conditions as the samples obtained from the NSCLC patients, stored at the same site as the NSCLC samples and processed further at the same centre by the same personnel. Associations of cytokine and MMP levels in different clinical subsets of NSCLC patients Further, it is possible that a pro-cancer cytokine proﬁle precedes the clinical manifestation of NSCLC and can be used for early detection of lung cancer. recognised as important participants in the communication between cancer cells and the non-malignant stroma and can modulate most stages of tumour progression.16 Our study suggests that NSCLC is mirrored by a distinct inﬂammatory signature in the serum. The different cytokine proﬁles in NSCLC and COPD patients may represent tumour-promoting and tumour-suppressing immune responses. Lack of independent validation remains a limitation in the interpretation of the data as well as the relatively small number of patients. However, the results are encouraging for a follow-up in a larger cohort owing to its possible implications for disease etiology, diagnostics and treatment. There is an increasing interest in the use of radiological screening for early detection of lung cancer. In this setting, serum biomarkers may serve as a cost-effective, high-throughput tool for identifying subjects at risk who may be referred to computed tomographic scans. Clinical & Translational Immunology Distinct inﬂammatory markers in NSCLC HA Eide et al Statistical analyses 13 Shiels MS, Pfeiffer RM, Hildesheim A, Engels Ea, Kemp TJ, Park JH et al. Circulating inﬂammation markers and prospective risk for lung cancer. J Natl Cancer Inst 2013; 105: 1871–1880. Data are reported using descriptive statistics with percentages, means, medians and ranges. Unsupervised hierarchical clustering was performed using Spear- man correlation and average linkage with scaled cytokine/MMP values owing to differences in interprotein concentration values. Differences in the clinical groups and between clusters were calculated with two-sided t-tests (assuming equal variance) and chi-squared/Fisher’s exact tests for continuous and categorical data, respectively. Non-parametric tests, Mann–Whitney U-test and Kruskal–Wallis test, were used to explore the differences in individual cytokine levels between NSCLC and COPD patients as well as in different clinical subsets. The median difference between serum levels in the two cohorts was utilised to make the volcano plot, also owing to lack of normally distributed data. The Benjamini–Hochberg false discovery rate was used in order to reduce the possibility for signiﬁcant independent test results by chance alone owing to multiple testing. 14 Bodelon C, Polley MY, Kemp TJ, Pesatori aC, McShane LM, Caporaso NE et al. Circulating levels of immune and inﬂammatory markers and long versus short survival in early-stage lung cancer. Ann Oncol 2013; 24: 2073–2079. 15 Barrera L, Montes-Servin E, Barrera A, Ramirez-Tirado La, Salinas-Parra F, Banales-Mendez JL et al. Cytokine proﬁle determined by data-mining analysis set into clusters of non-small-cell lung cancer patients according to prognosis. Ann Oncol 2014; 26: 428–435. 16 Kessenbrock K, Plaks V, Werb Z. Matrix metalloproteinases: regulators of the tumor microenvironment. Cell 2010; 141: 52–67. 17 Kaminska J, Kowalska M, Kotowicz B, Fuksiewicz M, Glogowski M, Wojcik E et al. Pretreatment serum levels of cytokines and cytokine receptors in patients with non- small cell lung cancer, and correlations with clinicopathological features and prognosis: M-CSF - an independent prognostic factor. Oncology 2006; 70: 115–125. 18 Maddur MS, Miossec P, Kaveri SV, Bayry J. Th17 cells: biology, pathogenesis of autoimmune and inﬂammatory diseases, and therapeutic strategies. Am J Pathol 2012; 181: 8–18. 19 Zhang L, Cheng Z, Liu W, Wu K. Expression of interleukin (IL)-10, IL-17A and IL-22 in serum and sputum of stable chronic obstructive pulmonary disease patients. COPD 2013; 10: 459–465. Data were analysed using the SPSS software package version 21 (SPSS, Chicago, IL, USA). Hierarchical clustering performed in R version 3.2.2 (R Project for Statistical Computing, Vienna, Austria). ACKNOWLEDGEMENTS We thank the Department of Cell Therapy, Oslo University Hospital — The Norwegian Radium Hospital, Oslo, Norway for allowing us to use their facility in order to perform the multiplex bioassays. 28 Lewis AM, Varghese S, Xu H, Alexander HR. Interleukin-1 and cancer progression: the emerging role of interleukin-1 receptor antagonist as a novel therapeutic agent in cancer treatment. J Transl Med 2006; 4: 48. 29 Krelin Y, Voronov E, Dotan S, Elkabets M, Reich E, Fogel M et al. Interleukin-1β-driven inﬂammation promotes the development and invasiveness of chemical carcinogen- induced tumors. Cancer Res 2007; 67: 1062–1071. 30 Dinarello CA. Why not treat human cancer with interleukin-1 blockade? Cancer Metastasis Rev 2010; 29: 317–329. 1 Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer 2014; 136: E359–E386. 31 Qian Q, Wang Q, Zhan P, Peng L, Wei S-Z, Shi Y et al. The role of matrix metalloproteinase 2 on the survival of patients with non-small cell lung cancer: a systematic review with meta-analysis. Cancer Invest 2010; 28: 661–669. 2 Grivennikov SI, Greten FR, Karin M. Immunity, inﬂammation, and cancer. Cell 2010; 140: 883–899. 32 Peng W-J, Zhang J-Q, Wang B-X, Pan H-F, Lu M-M, Wang J. Prognostic value of matrix metalloproteinase 9 expression in patients with non-small cell lung cancer. Clin Chim Acta 2012; 413: 1121–1126. 3 Walser T, Cui X, Yanagawa J, Lee JM, Heinrich E, Lee G et al. Smoking and lung cancer: the role of inﬂammation. Proc Am Thorac Soc 2008; 5: 811–815. 33 Liang Y, Guo S, Zhou Q. Prognostic value of matrix metalloproteinase-7 expression in patients with non-small cell lung cancer. Tumor Biol 2014; 35: 3717–3724. 4 Takahashi H, Ogata H, Nishigaki R, Broide DH, Karin M. Tobacco smoke promotes lung tumorigenesis by triggering IKKbeta- and JNK1-dependent inﬂammation. Cancer Cell 2010; 17: 89–97. 34 Hadler-Olsen E, Winberg JO, Uhlin-Hansen L. Matrix metalloproteinases in cancer: their value as diagnostic and prognostic markers and therapeutic targets. Tumor Biol 2013; 34: 2041–2051. 5 Adcock IM, Caramori G, Barnes PJ. Chronic obstructive pulmonary disease and lung cancer: new molecular insights. Respiration 2011; 81: 265–284. value as diagnostic and prognostic markers and therapeutic targets. Tumor Biol 2013; 34: 2041–2051. 35 Ribbens C, Martin y Porras M, Franchimont N, Kaiser M-J, Jaspar J-M, Damas P et al. Cytokine analyses Serum concentration levels of the cytokines CCL1/I-309, monocyte chemotactic chemokine-1 (MCP-1) (CCL2), MCP-2 (CCL8), MCP-3 (CCL7), MCP-4 (CCL13), RANTES (CCL5), macrophage inﬂammatory protein (MIP) 1alpha (CCL3), MIP-1sigma (CCL15), MIP-3alpha (CCL20), MIP-3beta (CCL19), TARC (CCL17), eotaxin (CCL11), eotaxin-2 (CCL24), eotaxin-3 (CCL26), CCL-21, macrophage-derived chemokine (CCL22), myeloid progenitor inhibitory factor 1 (CCL23), thymus expressed cytokine (CCL25), CCL27, growth-regulated protein alpha (Gro-a) (CXCL1), growth-regulated protein beta (Gro-a) (CXCL2), CXCL5, granulocyte chemotactic protein-2 (CXCL6), monokine induced by gamma interferon (CXCL9), interferon gamma induced protein-10 (CXCL10), interferon inducible T-cell alpha chemoattractant (CXCL11), SDF-1a+b (CXCL12), CXCL13, SCYB16 (CXCL16), fractalkine (CX3CL1), IL-1b, IL-1ra, IL-2, IL-4, IL-6, IL-8, IL-10, IL12p70, IL-16, IL17a, IFNγ, TNFα, TNF-related apoptosis-induced ligand, MIF, G-CSF, GM-CSF, PDGF-BB, VEGF and MMP-1, -2, -3, -7, -8, -9, -12 and -13 were quantiﬁed using a multiplex bioassay (BioRad, Hercules, CA, USA) according to the manufacturer`s instructions. Concentrations were calculated using an eight- parameter standard curve. Each serum sample was run in duplicates and averaged to calculate the concentrations. Several meta-analyses suggest MMPs as prognostic markers in lung cancer, although the results are conﬂicting.31–33 Many previous studies of MMPs comparing lung cancer patients with healthy individuals have also shown contradictory results.34 In our study, a higher level of circulating MMP-8 and MMP-12 was revealed, whereas MMP-2 was reduced in NSCLC compared with COPD patients. When excluding patients using steroids from both cohorts, MMP-3 was also found lower in NSCLC patients. In the NSCLC cohort, MMP-3 and MMP-9 levels were elevated in patients using systemic steroids. It is previously shown that MMP-3 can be induced by steroids.35 MMP-9 may also be released from an increasing amount of neutrophils accompanying treatment with steroids. In COPD, tissue destruction and remodeling are important processes where MMPs are likely to have a central part.36 The most likely explanation for MMPs in the pathogenesis of cancer is also through degradation of extracellular matrix enabling tumour cell invasion and metastasis. However, MMPs are also Clinical & Translational Immunology Distinct inﬂammatory markers in NSCLC HA Eide et al Distinct inﬂammatory markers in NSCLC HA Eide et al 7 The authors declare no conﬂict of interest. 26 Schumacher TN, Schreiber RD. Neoantigens in cancer immunotherapy. Science 2015; 348: 69–74. 27 McGranahan N, Furness AJS, Rosenthal R, Ramskov S, Lyngaa R, Saini SK et al. Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade. Science 2016; 351: 1463–1469. Ethics approval 23 Mantovani A, Allavena P, Sica A, Balkwill F. Cancer-related inﬂammation. Nature 2008; 454: 436–444. This study was approved by the regional ethics committee in Norway (reference number 2012/320) and the institutional review board. A written consent was obtained from all the participants in this study. 24 Abbas AK, Murphy KM SA. Functional diversity of helper T lymphocytes. Nature 1996; 383: 787–793. 25 Rizvi NA, Hellmann MD, Snyder A, Kvistborg P, Makarov V, Havel JJ et al. Mutational landscape determines sensitivity to PD-1 blockade in non-small cell lung cancer. Science 2015; 348: 124–128. Statistical analyses Two sided P-valueso0.05 were considered statistical signiﬁcant. Stricter levels of signiﬁcance due to correction for multiple testing are noted in the text and tables where applicable. 20 Duan M-C, Han W, Jin P-W, Wei Y-P, Wei Q, Zhang L-M et al. Disturbed Th17/Treg balance in patients with non-small cell lung cancer. Inﬂammation 2015; 38: 2156–2165. 21 Zhu J, Yamane H, Paul WE. Differentiation of effector CD4 T cell populations. Annu Rev Immunol 2010; 28: 445–489. 22 Bailey SR, Nelson MH, Himes RA, Li Z, Mehrotra S, Paulos CM. Th17 cells in cancer: the ultimate identity crisis. Front Immunol 2014; 5: 1–13. The Supplementary Information that accompanies this paper is available on the Clinical and Translational Immunology website (http://www.nature. com/cti) CONFLICT OF INTEREST The authors declare no conﬂict of interest. ACKNOWLEDGEMENTS Increased matrix metalloproteinase-3 serum levels in rheumatic diseases: relationship with synovitis and steroid treatment. Ann Rheum Dis 2002; 61: 161–166. 6 Sekine Y, Hata A, Koh E, Hiroshima K. Lung carcinogenesis from chronic obstructive pulmonary disease: characteristics of lung cancer from COPD and contribution of signal transducers and lung stem cells in the inﬂammatory microenvironment. Gen Thorac Cardiovasc Surg 2014; 62: 415–421. 36 Elkington PTG, Friedland JS. Matrix metalloproteinases in destructive pulmonary pathology. Thorax 2006; 61: 259–266. 36 Elkington PTG, Friedland JS. Matrix metalloproteinases in destructive pulmonary pathology. Thorax 2006; 61: 259–266. 7 Balkwill FR, Capasso M, Hagemann T. The tumor microenvironment at a glance. J Cell Sci 2012; 125: 5591–5596. 8 Mittal D, Gubin MM, Schreiber RD, Smyth MJ. New insights into cancer immunoediting and its three component phases-elimination, equilibrium and escape. Curr Opin Immunol 2014; 27: 16–25. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license,userswillneedto obtainpermissionfromthelicense holderto reproduce the material. To view a copy of this license, visit http:// creativecommons.org/licenses/by/4.0/ 9 Bremnes RM, Busund L-T, Kilvær TL, Andersen S, Richardsen E, Paulsen EE et al. The role of tumor inﬁltrating lymphocytes in development, progression and prognosis of non- small cell lung cancer. J Thorac Oncol 2016; 11: 789–800. g 10 Brahmer J, Reckamp KL, Baas P, Crino L, Eberhardt WE, Poddubskaya E et al. Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer. N Engl J Med 2015; 373: 1627–1639. 11 Borghaei H, Paz-Ares L, Horn L, Spigel DR, Steins M, Ready NE et al. Nivolumab versus docetaxel in advanced nonsquamous non-small-cell lung cancer. N Engl J Med 2015; 373: 1627–1639. 12 Ruffell B, DeNardo DG, Affara NI, Coussens LM. Lymphocytes in cancer development: polarization towards pro-tumor immunity. Cytokine Growth Factor Rev 2010; 21: 3–10. r The Author(s) 2016 The Supplementary Information that accompanies this paper is available on the Clinical and Translational Immunology website (http://www.nature. com/cti) The Supplementary Information that accompanies this paper is available on the Clinical and Translational Immunology website (http://www.nature. com/cti) The Supplementary Information that accompanies this paper is available on the Clinical and Translational Immunology website (http://www.nature. com/cti) n that accompanies this paper is available on the Clinical and Translational Immunology website (http://www.nature Clinical & Translational Immunology
https://openalex.org/W4220815081	https://run.unl.pt/bitstream/10362/143498/1/Multidrug_Resistant_Methicillin_Resistant_Coagulase_Negative.pdf	English	null	Multidrug-Resistant Methicillin-Resistant Coagulase-Negative Staphylococci in Healthy Poultry Slaughtered for Human Consumption	Antibiotics	2,022	cc-by	9,137	Citation: Silva, V.; Caniça, M.; Ferreira, E.; Vieira-Pinto, M.; Saraiva, C.; Pereira, J.E.; Capelo, J.L.; Igrejas, G.; Poeta, P. Multidrug-Resistant Methicillin-Resistant Coagulase-Negative Staphylococci in Healthy Poultry Slaughtered for Human Consumption. Antibiotics 2022, 11, 365. https://doi.org/ 10.3390/antibiotics11030365 Academic Editors: Clair L. Firth and Marc Maresca Received: 18 January 2022 Accepted: 3 March 2022 Published: 9 March 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). p y p g 7 CECAV—Veterinary and Animal Research Centre, University of Trás-os-Montes and Alto Douro (UTAD), 5000-801 Vila Real, Portugal; mmvpinto@utad.pt (M.V.-P.); candido.ls95@gmail.com (C.S.) 8 Associate Laboratory for Animal and Veterinary Sciences (AL4AnimalS), 5000-801 Vila Real, Portugal 9 BIOSCOPE Group, LAQV-REQUIMTE, Chemistry Department, Faculty of Science and Technology, NOVA University of Lisbon, 2825-466 Almada, Portugal; jlcm@fct.unl.pt 10 Proteomass Scientiﬁc Society, 2825-466 Costa de Caparica, Portugal * Correspondence: ppoeta@utad.pt 7 CECAV—Veterinary and Animal Research Centre, University of Trás-os-Montes and Alto Douro (UTAD), 5000-801 Vila Real, Portugal; mmvpinto@utad.pt (M.V.-P.); candido.ls95@gmail.com (C.S.) Citation: Silva, V.; Caniça, M.; Ferreira, E.; Vieira-Pinto, M.; Saraiva, C.; Pereira, J.E.; Capelo, J.L.; Igrejas, G.; Poeta, P. Multidrug-Resistant Methicillin-Resistant Coagulase-Negative Staphylococci in Healthy Poultry Slaughtered for Human Consumption. Antibiotics 2022, 11, 365. https://doi.org/ 10.3390/antibiotics11030365 Coagulase-Negative Staphylococci in Healthy Poultry Slaughtered for Human Consumption. Antibiotics 2022, 11, 365. https://doi.org/ 10.3390/antibiotics11030365 Abstract: Coagulase-negative staphylococci are commensals that are known to be prevalent in most environments, and they are also an important reservoir of antimicrobial-resistant genes. Staphylococ- cal infections in animal husbandry are a high economic burden. Thus, we aimed to determine the prevalence and species diversity of methicillin-resistant coagulase-negative staphylococci (MRCoNS) in poultry slaughtered for human consumption and to study the antimicrobial resistance of the isolates. Swab samples were recovered from 220 commercial chickens, homebred chickens and quails. Species identiﬁcation was performed using MALDI-TOF. Antimicrobial susceptibility testing was performed by the disc diffusion method against 14 antimicrobials. The presence of antimicrobial- resistant genes was investigated by polymerase chain reaction. Totals of 11 (19.6%), 13 (20.3%), and 51 (51%) MRCoNS were isolated from commercial chickens, homebred chickens and quails, respectively. S. lentus was isolated from all homebred chickens, whereas 11 S. lentus and 2 S. urealyticus were isolated from commercial chickens. Article Multidrug-Resistant Methicillin-Resistant Coagulase-Negative Staphylococci in Healthy Poultry Slaughtered for Human Consumption Vanessa Silva 1,2,3,4 , Manuela Caniça 5,6 , Eugénia Ferreira 5,6, Madalena Vieira-Pinto 7 , Cândido Saraiva 7 , José Eduardo Pereira 1,7,8, José Luis Capelo 9,10 , Gilberto Igrejas 2,3,4 and Patrícia Poeta 1,4,7,8,* 1 Microbiology and Antibiotic Resistance Team (MicroART), Department of Veterinary Sciences, University of Trás-os-Montes and Alto Douro (UTAD), 5000-801 Vila Real, Portugal; vanessasilva@utad.pt (V.S.); jeduardo@utad.pt (J.E.P.) 1 Microbiology and Antibiotic Resistance Team (MicroART), Department of Veterinary Sciences, University of Trás-os-Montes and Alto Douro (UTAD), 5000-801 Vila Real, Portugal; vanessasilva@utad.pt (V.S.); jeduardo@utad.pt (J.E.P.) 1 Microbiology and Antibiotic Resistance Team (MicroART), Department of Veterinary Sciences, University of Trás-os-Montes and Alto Douro (UTAD), 5000-801 Vila Real, Portugal; vanessasilva@utad.pt (V.S.); jeduardo@utad.pt (J.E.P.) 2 Department of Genetics and Biotechnology, University of Trás-os-Montes and Alto Douro, 5000-801 Vila Real, Portugal; gigrejas@utad.pt 3 Functional Genomics and Proteomics Unit, University of Trás-os-Montes and Alto Douro (UTAD), 5000-801 Vila Real, Portugal 4 LAQV-REQUIMTE, Department of Chemistry, NOVA School of Science and Technology, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal 5 National Reference Laboratory of Antibiotic Resistances and Healthcare Associated Infections (NRL-AMR/HAI), Department of Infectious Diseases, National Institute of Health Dr Ricardo Jorge, Av. Padre Cruz, 1649-016 Lisbon, Portugal; manuela.canica@insa.min-saude.pt (M.C.); eugenia.ferreira@insa.min-saude.pt (E.F.) 6 Centre for the Studies of Animal Science, Institute of Agrarian and Agri-Food Sciences and Technologies, Oporto University, 4051-401 Oporto, Portugal antibiotics antibiotics antibiotics antibiotics antibiotics Keywords: coagulase-negative Staphylococcus; CoNS; antimicrobial resistance; poultry; quails; broilers 1. Introduction Staphylococci colonize the skin and mucous membranes of humans and are consid- ered commensals or opportunistic pathogens [1]. By 2018, 45 species and 24 subspecies of Staphylococcus had been described [2]. Staphylococci are divided into two groups, coagulase- positive (CoPS) and coagulase-negative staphylococci (CoNS), according to their ability to coagulate plasma. CoPS are pathogenic species which have the coagulase enzyme that converts plasma ﬁbrinogen into ﬁbrin [3]. CoNS lack this enzyme and were considered, until recently, to be minor pathogens or apathogenic [4]. CoNS possess fewer virulence factors that participate in the pathogenesis of infection when compared to CoPS, such as S. aureus, but, in the last few decades, CoNS have emerged as common causes of nosocomial infections [4]. Within the CoNS species, S. epidermidis, S. haemolyticus and S. saprophyticus are examples of the most signiﬁcant types of CoNS in human infections [5]. As oppor- tunistic pathogens, CoNS generally cause infection in colonized immunocompromised individuals, patients with catheters and prosthetic implants, dialysis and oncologic pa- tients and neonates [6]. CoNS are responsible for a broad spectrum of infections, such as invasive endocarditis, bacteremia and bone infections [6,7]. In addition, increasing rates of antibiotic resistance have been detected in CoNS, in some cases even greater than for S. aureus, which limits the therapeutic options available [5]. Methicillin resistance in CoNS is usually due to the expression of the mecA gene, which encodes an alternative binding protein 2a (PBP2a) that has a low afﬁnity for β-lactam antibiotics, although some studies have reported the presence the mecC gene, a homologue of mecA [8–10]. The mec genes are located on a mobile genetic element called the Staphylococcal Cassette Chromosome mec (SCCmec). SCCmec elements are more diverse in methicillin-resistant CoNS when compared to S. aureus, and many SCCmec elements could not be typed using multiplex PCR [10]. Tetracycline resistance is also frequently detected in different CoNS species [11]. y q y p CoNS also colonize and infect other mammals besides humans, with S. chromogenes, S. simulans and S. xylosus being the principal cause of infection [11]. CoNS are frequently responsible for arthritis, cow mastitis and, less often, systemic infections in animals [12]. The presence of CoNS has been reported in pets, livestock and wild animals [13–15]. As for quails, the most prevalent MRCoNS were S. urealyticus. Almost all isolates had a multidrug-resistant proﬁle and carried the mecA gene. Most isolates showed resistance to erythromycin, clindamycin, penicillin, tetracycline, ciproﬂoxacin and fusidic acid and harbored the ermA, ermB, ermC, mphC tetK, tetL, tetM and tetO genes. This study showed a frequent occurrence of multidrug resistance in MRCoNS isolated from healthy poultry in Portugal. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Keywords: coagulase-negative Staphylococcus; CoNS; antimicrobial resistance; poultry; quails; broilers Antibiotics 2022, 11, 365. https://doi.org/10.3390/antibiotics11030365 https://www.mdpi.com/journal/antibiotics 2 of 11 Antibiotics 2022, 11, 365 1. Introduction It has been shown that food of animal origin can carry CoNS and other foodborne pathogens and, besides being able to cause infection, CoNS can also cause food poisoning [16]. Both CoPS and CoNS have been associated with avian pathologies such as arthritis, osteomyelitis, pododermatitis, septicemia and blepharitis [17,18]. Nevertheless, the presence of CoPS and CoNS has also been observed in healthy poultry and poultry meat, which may act as reservoirs and vehicles of zoonotic pathogens and antimicrobial resistance [16,19]. The spread of antimicrobial resistance among commensal CoNS in healthy poultry may rep- resent a hazard for human and animal health [11]. Studies reporting the monitorization of antimicrobial-resistant pathogens in poultry and poultry meat have been published, but most studies focus only on S. aureus species [20–24]. The prevalence of antimicrobial- resistant pathogens in poultry, particularly staphylococci, may be due to their high con- sumption of antimicrobials. According to the ESVAC report, in Portugal the population- weighted mean consumption (expressed in milligrams per kilogram of estimated biomass) of antimicrobials was 175.8 mg/Kg in food-producing animals in 2020 [25]. In Portugal, the biomass-corrected consumption of third- and fourth-generation cephalosporins, quinolones, penicillin, macrolides and tetracyclines in food-producing animals was around 0.4, 7.3, 38.9, 20 and 60.4 mg/Kg [25]. Furthermore, all these antimicrobial classes were used in poultry production. Therefore, we aimed to investigate the presence of methicillin-resistant CoNS (MRCoNS) in healthy poultry for human consumption as well as the antimicrobial-resistant phenotypes and genotypes of the isolates. 2. Results The mecA gene was detected in all isolates, including those that were susceptible to cefoxitin. Totals of 11 S. lentus, 21 S. urealyticus, 14 S. sciuri and 3 S. haemolyticus were phenotypically resistant to penicillin, but the mechanism of penicillin resistance could not be identiﬁed. Resistance to aminoglycosides was detected in 40% of the isolates and was mediated by the aph(3′)-IIIa, ant(4′)-Ia and str genes in different combinations. All S. lentus and S. urealyticus were resistant to macrolides and lincosamides, while 14 S. sciuri and 2 S. haemolyticus showed resistance to this antimicrobial class. Macrolide-lincosamide resistant isolates harbored the ermA, ermB, ermC and mphC genes alone or in different combinations: ermB (n = 5); ermC (n = 11); mphC (n = 3); ermC and mphC (n = 27); ermA, ermC and mphC (n = 6); ermB, ermC and mphC (n = 10); ermB and mphC (n = 8); ermA and ermC (n = 1); ermA, ermB, ermC and mphC (n = 1); and ermA, ermB and mphC (n = 1). Tetracycline resistance, which was detected in all S. urealyticus, S. sciuri and S. haemolyticus, and in 25 (69.4%) S. lentus, was mediated by the tetK, tetL, tetM and/or tetO genes. The tetL gene was the most frequent, followed by the tetK. The catp194 encoding resistance to chloramphenicol was detected in one S. lentus isolate. Resistance to trimethoprim-sulfamethoxazole was detected in 10 isolates. Some S. lentus isolates harbored a combination of dfrK and dfrD genes, while S. sciuri and S. haemolyticus carried only the dfrK. One S. sciuri exhibited resistance to linezolid, mediated by the cfr gene. None of the isolates showed resistance to vancomycin. carriage of MRCoNS species was identiﬁed only among quail samples, and the pattern of co-carriage was as follows: Staphylococcus sciuri/S. urealyticus (n = 2), Staphylococcus lentus/S. urealyticus and Staphylococcus lentus/Staphylococcus haemolyticus. A total of 75 MRCoNS were recovered and identiﬁed as S. lentus (n = 26), S. urealyticus (n = 21), S. sciuri (n = 15) and S. haemolyticus (n = 3). S. haemolyticus was exclusively isolated from quails. Chickens, both commercial and homebred, were mainly colonized by S. lentus, while S. urealyticus was the most frequently detected species in quails, followed by S. lentus. Quails were colonized signiﬁcantly more frequently by MRCoNS than homebred chickens. Furthermore, the prevalence of S. lentus and S. urealyticus was signiﬁcantly higher than that of S. haemolyticus. 2. Results Results of the prevalence of each staphylococcal species are shown in Supplementary Figure S1. Table 1. Number of animals sampled, frequency and diversity of CoNS species detected among healthy poultry. Animal Number of Animals Sampled Number of CoNS Carriers (%) Isolates Recovered S. lentus S. urealyticus S. sciuri S. haemolyticus Quails 100 47 (47) 51 15 19 14 3 Commercial chickens 50 13 (26) 13 11 2 - - Homebred chickens 70 11 (15.7) 11 10 - 1 - Total 220 71 (32.3) 75 36 21 15 3 Table 1. Number of animals sampled, frequency and diversity of CoNS species detected among healthy poultry. Table 2 shows the antimicrobial-resistant phenotypes and genotypes of MRCoNS, while the detailed characterization of each isolate is summarized in Supplementary Table S1. The percentage of resistance to each antibiotic is shown in Figure 1. All isolates showed phenotypic and genotypic resistance to antibiotics, with 73 (97.3%) isolates displaying a multidrug-resistant proﬁle since they showed resistance to at least three different classes of antimicrobials. The multidrug-resistance pattern was as follows: 15 (20%) isolates were resistant to 3 classes, 27 (26%) to 4 classes, 17 (22.7%) to 5 classes, 12 (16%) to 6 classes and 2 (2.7%) to 7 classes of antimicrobials. The non-multiresistant isolates were both S. lentus and were isolated from chickens. Both isolates showing resistance to seven antimicrobial classes were isolated from quails. The mecA gene was detected in all isolates, including those that were susceptible to cefoxitin. Totals of 11 S. lentus, 21 S. urealyticus, 14 S. sciuri and 3 S. haemolyticus were phenotypically resistant to penicillin, but the mechanism of penicillin resistance could not be identiﬁed. Resistance to aminoglycosides was detected in 40% of the isolates and was mediated by the aph(3′)-IIIa, ant(4′)-Ia and str genes in different combinations. All S. lentus and S. urealyticus were resistant to macrolides and lincosamides, while 14 S. sciuri and 2 S. haemolyticus showed resistance to this antimicrobial class. Macrolide-lincosamide resistant isolates harbored the ermA, ermB, ermC and mphC genes alone or in different combinations: ermB (n = 5); ermC (n = 11); mphC (n = 3); ermC and mphC (n = 27); ermA, ermC and mphC (n = 6); ermB, ermC and mphC (n = 10); ermB and mphC (n = 8); ermA and ermC (n = 1); ermA, ermB, ermC and mphC (n = 1); and ermA, ermB and mphC (n = 1). 2. Results In this study, the presence of methicillin-resistant CoNS (MRCoNS) was detected in 71 (32.3%) of the 220 birds tested (Table 1). The co-carriage of two different species was identiﬁed in four animals, and 67 birds carried only one staphylococcal species. Co- Antibiotics 2022, 11, 365 3 of 11 Antibiotics 2022, 11, 365 3 of 11 carriage of MRCoNS species was identiﬁed only among quail samples, and the pattern of co-carriage was as follows: Staphylococcus sciuri/S. urealyticus (n = 2), Staphylococcus lentus/S. urealyticus and Staphylococcus lentus/Staphylococcus haemolyticus. A total of 75 MRCoNS were recovered and identiﬁed as S. lentus (n = 26), S. urealyticus (n = 21), S. sciuri (n = 15) and S. haemolyticus (n = 3). S. haemolyticus was exclusively isolated from quails. Chickens, both commercial and homebred, were mainly colonized by S. lentus, while S. urealyticus was the most frequently detected species in quails, followed by S. lentus. Quails were colonized signiﬁcantly more frequently by MRCoNS than homebred chickens. Furthermore, the prevalence of S. lentus and S. urealyticus was signiﬁcantly higher than that of S. haemolyticus. Results of the prevalence of each staphylococcal species are shown in Supplementary Figure S1. Table 1. Number of animals sampled, frequency and diversity of CoNS species detected among healthy poultry. Animal Number of Animals Sampled Number of CoNS Carriers (%) Isolates Recovered S. lentus S. urealyticus S. sciuri S. haemolyticus Quails 100 47 (47) 51 15 19 14 3 Commercial chickens 50 13 (26) 13 11 2 - - Homebred chickens 70 11 (15.7) 11 10 - 1 - Total 220 71 (32.3) 75 36 21 15 3 Table 2 shows the antimicrobial-resistant phenotypes and genotypes of MRCoNS, while the detailed characterization of each isolate is summarized in Supplementary Table S1. The percentage of resistance to each antibiotic is shown in Figure 1. All isolates showed phenotypic and genotypic resistance to antibiotics, with 73 (97.3%) isolates displaying a multidrug-resistant proﬁle since they showed resistance to at least three different classes of antimicrobials. The multidrug-resistance pattern was as follows: 15 (20%) isolates were resistant to 3 classes, 27 (26%) to 4 classes, 17 (22.7%) to 5 classes, 12 (16%) to 6 classes and 2 (2.7%) to 7 classes of antimicrobials. The non-multiresistant isolates were both S. lentus and were isolated from chickens. Both isolates showing resistance to seven antimicrobial classes were isolated from quails. 2. Results sciuri exhibited resistance to linezolid, mediated by the cfr gene. None of the isolates showed resistance to vancomycin. Figure 1. Percentage of resistance to each antibiotic by MRCoNS isolated from poultry. Figure 1. Percentage of resistance to each antibiotic by MRCoNS isolated from poultry. Figure 1. Percentage of resistance to each antibiotic by MRCoNS isolated from poultry. Figure 1. Percentage of resistance to each antibiotic by MRCoNS isolated from poultry. 2. Results Tetracycline resistance, which was detected in all S. urealyticus, S. sciuri and S. haemolyticus, and in 25 (69.4%) S. lentus, was mediated by the tetK, tetL, tetM and/or tetO genes. The tetL gene was the most frequent, followed by the tetK. The catp194 encoding resistance to chloramphenicol was detected in one S. lentus isolate. Resistance to trimethoprim-sulfamethoxazole was detected in 10 isolates. Some S. lentus isolates harbored a combination of dfrK and dfrD genes, while S. sciuri and S. haemolyticus carried only the dfrK. One S. sciuri exhibited resistance to linezolid, mediated by the cfr gene. None of the isolates showed resistance to vancomycin. 4 of 11 Antibiotics 2022, 11, 365 Table 2. Antimicrobial-resistant genes identiﬁed among the CoNS isolated from poultry. Species Number of Isolates Antimicrobial Resistance Phenotype Genotype S. lentus 36 PEN11, FOX4, CIP11, CN2, TOB14, KAN9, ERY35, CD36, TET25, C4, FD12, SXT6 mecA36, ermA8, ermB8, ermC28, mphC29, aph(3′)-IIIa9, ant(4′)-Ia12, str2, tetL19, tetK14, tetO1, tetM2, catp1941, dfrK6, dfrD2 S. urealyticus 21 PEN21, FOX18, CIP3, CN4, TOB6, KAN5, ERY21, CD21, TET21, C3, FD17 mecA21, ermA1, ermB7, ermC19, mphC16, aph(3′)-IIIa5, ant(4′)-Ia2, str2, tetL17, tetK18, tetO13, tetM4 S. sciuri 15 PEN14, FOX6, LNZ1, CIP3, TOB8, KAN4, ERY14, CD14, TET15, C2, FD10, SXT2 mecA15, cfr1, ermB9, ermC7, mphC9, aph(3′)-IIIa3, ant(4′)-Ia7, str1, tetL11, tetK12, tetO2, tetM3, dfrK1 S. haemolyticus 3 PEN3, FOX1, CIP2, TOB2, KAN1, ERY2, CD2, TET3, FD2, SXT2 mecA3, ermB1, ermC2, mphC2, aph(3′)-IIIa2, ant(4′)-Ia1, str1, tetL3, tetK1, dfrK1 Abbreviations. C: chloramphenicol; CD: clindamycin; CIP: ciproﬂoxacin; ERY: erythromycin; FD, fusidic acid; FOX: cefoxitin; PEN: penicillin; SXT: trimethoprim-sulfamethoxazole; TET: tetracycline; CN: gentamicin; KAN: kanamycin; TOB: tobramycin; LNZ: linezolid. Note: the superscript number after each antibiotic and gene indicates the number of strains showing resistance to that antibiotic and harboring that gene, respectively. Antibiotics 2022, 11, x FOR PEER REVIEW 4 of 11 dfrK. One S. sciuri exhibited resistance to linezolid, mediated by the cfr gene. None of the isolates showed resistance to vancomycin. Table 2. Antimicrobial-resistant genes identiﬁed among the CoNS isolated from poultry. Table 2. Antimicrobial-resistant genes identiﬁed among the CoNS isolated from poultry. Abbreviations. C: chloramphenicol; CD: clindamycin; CIP: ciproﬂoxacin; ERY: erythromycin; FD, fusidic acid; FOX: cefoxitin; PEN: penicillin; SXT: trimethoprim-sulfamethoxazole; TET: tetracycline; CN: gentamicin; KAN: kanamycin; TOB: tobramycin; LNZ: linezolid. Note: the superscript number after each antibiotic and gene indicates the number of strains showing resistance to that antibiotic and harboring that gene, respectively. dfrK. One S. Table 2. Antimic 3. Discussion cohnii being the most frequent followed by S. saprophyticus and S. epidermidis [29]. In accordance with our results, Saha et al. found a higher occurrence of S. lentus in poultry samples [30]. Boamah et al. reported a frequency of 42.97% S. sciuri, 35.94% S. lentus, 4.30% S. xylosus, 3.91%, S. haemolyticus 3.91%, 1.95% S. saprophyticus and 0.39% S. cohnii [31]. A study conducted in Brazil found that most CoNS from chickens were S. gallinarum followed by S. simulans [18]. In a report by El-Nagar et al., the majority of CoNS were S. xylosus [32]. Marek et al. found a higher occurrence of S. epidermidis in poultry in Poland [26]. Finally, S. hominis followed by S. xylosus and S. lentus were the most frequently detected species in quail eggs [33]. Yet, most studies have reported the presence of S. sciuri, S. lentus and S. cohnii. It has been shown that some species of CoNS, such as S. sciuri, S. xylosus or S. cohnii, are considered important poultry pathogens, particularly when associated with antimicrobial resistance [29]. Furthermore, most of these CoNS species are considered an issue of meat safety rather than the classical poultry pathogens [29]. p y p g The most common species found among poultry in this study was S. lentus. This species is considered an animal pathogen and has been detected among livestock, pets, wild animals and retail meats [13,16,34,35]. Nevertheless, S. lentus has also been responsible for a wide range of human infections and its clinical relevance seems to be increasing [36]. S. urealyticus was the second most common CoNS species found in poultry and it was mostly detected in quail samples. This CoNS species has been regarded as a commen- sal organism and is not usually involved in severe infections [37]. S. urealyticus strains of animal origin were shown to have multiple phenotypic resistances and carry several antimicrobial resistance genes [38]. All CoNS isolated in this study harbored the mecA gene, and the methicillin resistance of the isolates was conﬁrmed. However, most S. lentus and S. sciuri isolates were phenotypically susceptible to cefoxitin. It has been shown that the staphylococcal species belonging to the S. sciuri group, which include S. sciuri, S. ﬂeurettii, S. lentus, S. stepanovicii and S. vitulinus, carry a close homologue to the mecA gene, which does not confer resistance to β-lactam antibiotics [39]. Accordantly, almost all S. urealyticus had phenotypic resistance to cefoxitin. Table 2. Antimic 3. Discussion g g p y Species Number of Isolates Antimicrobial Resistance Phenotype Genotype S. lentus 36 PEN11, FOX4, CIP11, CN2, TOB14, KAN9, ERY35, CD36, TET25, C4, FD12, SXT6 mecA36, ermA8, ermB8, ermC28, mphC29, aph(3′)-IIIa9, ant(4′)-Ia12, str2, tetL19, tetK14, tetO1, tetM2, catp1941, dfrK6, dfrD2 S. urealyticus 21 PEN21, FOX18, CIP3, CN4, TOB6, KAN5, ERY21, CD21, TET21, C3, FD17 mecA21, ermA1, ermB7, ermC19, mphC16, aph(3′)-IIIa5, ant(4′)-Ia2, str2, tetL17, tetK18, tetO13, tetM4 S. sciuri 15 PEN14, FOX6, LNZ1, CIP3, TOB8, KAN4, ERY14 CD14 TET15 C2 mecA15, cfr1, ermB9, ermC7, mphC9, aph(3′)-IIIa3, ant(4′)-Ia7, str1 tetL11 tetK12 tetO2 tetM3 MRCoNS in livestock was ﬁrst reported in healthy chickens in Japan in 1996. Despite the increasing interest in CoNS in recent years, there is very limited information on their prevalence and resistance proﬁles in poultry production, and information is even more limited regarding MRCoNS. In our study, we investigated the presence of MRCoNS in healthy quails and commercial and homebred chickens. Among the 220 birds tested, 71 (32.3%) carried at least one CoNS, which is in accordance with the results obtained by Marek et al. [26]. CoNS colonized 47% and 20% of the quails and chickens, respectively. This carriage frequency was higher than the one obtained by Younis et al., who found a prevalence of CoNS in quails and chickens of 8.75% and 7.14%, respectively [27]. A study conducted with turkey samples found a frequency of CoNS of 15.6%, which is also lower than the one obtained in this study [28]. Other studies found a higher frequency of CoNS in poultry [18,29]. Nevertheless, it is important to point out that in our study all samples were only screened for the presence of MRCoNS, which may have contributed to a higher frequency of CoNS. Furthermore, some studies focused only on diseased animals Antibiotics 2022, 11, 365 5 of 11 that would most likely have been discarded in the slaughterhouse and would not have reached the ﬁnal consumer. In our study, only four different species of CoNS were detected: S. lentus (n = 26), S. urealyticus (n = 21), S. sciuri (n = 15) and S. haemolyticus (n = 3). The predominant CoNS species found in our study included those commonly found in skin microbiota in chickens [29,30]. The occurrence of the staphylococci species among poultry samples appears to vary widely. Pyzik et al. detected a high number of CoNS species in diseased broiler chickens and turkeys, with S. Table 2. Antimic 3. Discussion Multidrug resistance was exhibited in almost all isolates, which is in accordance with other studies conducted with poultry samples [27–29]. Although the European Union banned the use of antibiotics for growth promotion in livestock in 2006, and several other measures have been taken since then, it is estimated that over 60% of all antimicrobials produced are used in livestock comprising poultry [40]. Higher resistance levels were detected among quails, including two isolates resistant to seven antimicrobial classes, which may be explained by the fact that in Portugal the legislation for antibiotics administration in quails is not as well-regulated as that for other poultry, such as chickens; thus, antibiotics may be administrated indiscriminately to quails, leading to an increase in antimicrobial resistance [20]. Only one isolate, S. sciuri, was resistant to linezolid and carried the cfr gene. This gene was ﬁrst detected in a bovine S. sciuri [41]. Although uncommon, resistance to linezolid mediated by the cfr gene is wor- risome, since this gene confers cross-resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins and streptogramin A antibiotics [42,43]. Studies reporting the cfr gene in poultry identiﬁed it in S. lentus, S. urealyticus, S. arlettae. sciuri and S. simulans [39,44,45]. Furthermore, a low frequency of this gene has been reported in CoNS from poultry [39]. Resistance to macrolides and lincosamides was detected in all isolates, except for one S. sciuri and one S. haemolyticus, and it was mediated by the ermA, ermB, ermC and mphC genes. Both ermC and mphC genes were carried by 56 isolates. Phosphotransferases are encoded by the mphC gene which confers resistance to erythromycin and other macrolides but not Antibiotics 2022, 11, 365 6 of 11 to lincosamides [46]. Nevertheless, the erm genes confer cross-resistance to macrolides, lincosamides and streptogramins B [46]. Although the ermA and ermC genes are the most frequent erm genes in staphylococci, the ermA gene was only detected in the S. lentus and S. urealyticus isolates, while ermB was identiﬁed in all MRCoNS species in this study. Other studies reported similar results for the frequency of erm genes in poultry [28,39]. A study by Syed et al. investigated the resistance of staphylococci in poultry intestines and reported a lower frequency of resistance to macrolides and lincosamides, but the ermC gene was also the most prevalent [47]. Table 2. Antimic 3. Discussion In the same study, resistance to tetracycline was detected in more than half of the isolates encoded by the tetK and tetM genes [47]. In our study, resistance to tetracycline was detected in 85.3% of the isolates, including all S. sciutri, S. urealyticus and S. haemolyticus, and in 25 out of 36 S. lentus, which was similar to the ﬁndings of other studies [28,31,48]. The high frequency of tetracycline resistance in poultry samples may be due to the fact that, according to the ECDC/EFSA/EMA report, tetracycline and penicillin were the most prescribed antibiotics for food-producing animals in 2017 [49]. Among the genes that confer resistance to tetracycline, tetL (n = 50) was the most prevalent, followed by tetK (n = 45), tetO (n = 16) and tetM (n = 9). Similar results were obtained by Lee et al. in a study that investigated the tet genes in poultry meat [16]. In contrast, in a study by Nemeghaire et al. tetM was the most common gene among S. sciuri from healthy chickens [39]. However, due to the lack of studies investigating the prevalence of resistant genes in CoNS from poultry, it is difﬁcult to make a direct comparison. Fusidic acid was detected in 54.6% of the isolates but none of the resistance genes tested were found, which suggests the presence of other resistant genes. Indeed, in a study by Chen et al. none of the fusidic acid-resistant S. urealyticus possessed fusB, fusC or fusD genes; instead, S. urealyticus isolates carried the novel fusF gene, which seems to be an intrinsic factor in S. urealyticus and may not be conserved in another subspecies [50]. Resistance to vancomycin was not detected in this study, which was unsurprising since vancomycin-resistant staphylococci are rare and, as far as we know, in Portugal there is only one study reporting a vancomycin intermediate-resistant S. aureus isolated from a human infection [51]. In general, penicillin and tetracycline are extensively used for the treatment of staphy- lococcal infections in poultry [52]. In our study, we also found higher levels of resistance to those antimicrobial agents. The ingestion of poultry meat contaminated with staphylococci may lead to food poisoning. Furthermore, the handling or ingesting of staphylococci contaminated meat is a potential risk factor for colonization by methicillin-resistant staphy- lococci [53]. Our ﬁndings show that the frequency of multidrug-resistant staphylococci in poultry is alarming and may represent a public health problem. 4.2. Phenotypic Antibiotic Resistance Testing 4.2. Phenotypic Antibiotic Resistance Testing Antibiotic susceptibility proﬁles were determined for all of isolates by the Kirby–Bauer disc diffusion method on Mueller Hinton agar. The tested antibiotics included: cefoxitin (30 µg), chloramphenicol 132 (30 µg), ciproﬂoxacin (5 µg), clindamycin (2 µg), erythromycin (15 µg), fusidic acid (10 133 µg), gentamicin (10 µg), kanamycin (30 µg), linezolid (10 µg), mupirocin (200 µg), penicillin (1 U), tetracycline (30 µg), tobramycin (10 µg), and trimetho- prim/sulfamethoxazole 135 (1.25/23.75 µg). The diameter of the inhibition zones was measured for each antibiotic disk and recorded in millimeters. The interpretation of re- sults followed the recommendations given in the European Committee on Antimicrobial Susceptibility Testing (EUCAST) 2019 guidelines with the exception of kanamycin that followed the Clinical and Laboratory Standards Institute (CLSI) 2017 recommendations. The minimal inhibitory concentrations (MICs) of vancomycin were determined by a stan- dard broth microdilution method in sterile 96-well microplates according to the EUCAST guidelines. Brieﬂy, bacterial suspension was adjusted to 0.5 McFarland standards and then diluted 1:20. Then, 50 µL of Mueller–Hinton broth, 50 µL of the antibiotic dilutions, and 5 µL of the inoculum were mixed and incubated at 37 ◦C for 24 h. Isolates show- ing a vancomycin MIC ≤4 µg/mL were considered susceptible and those showing an MIC > 4 µg/mL were classiﬁed as resistant. The reference strain S. aureus ATCC 25923 was used for quality control. 4.3. DNA Extraction DNA extraction was performed as previously described. Brieﬂy, 2 staphylococci colonies were suspended in 45 µL of Milli-Q water and 5 µL of lysostaphin (1 mg/mL) was added. The samples were incubated at 37 ◦C for 10 min, after which 45 µL of Milli-Q water, 150 µL of Tris-HCl (0.1 M) and 5 µL of proteinase K (2 mg/mL) were added. After 10 min of incubation at 67 ◦C, the samples were boiled at 100 ◦C for 5 min. The DNA was stored at −20 ◦C until use. The spectrophotometric quantiﬁcation of DNA was carried out through the NanoDrop 1000 (Thermo Fisher Scientiﬁc, Waltham, MA, USA) [55]. 4.4. Antimicrobial-Resistant Genes The presence of antimicrobial-resistant genes was investigated in each isolate accord- ing to the phenotypic resistance. The detection of the following antimicrobial-resistant genes was performed in a ProFlexTM PCR system (Applied Biosystems, Waltham, MA, USA): beta-lactams (blaZ, mecA and mecC), linezolid (cfr), aminoglycosides (aac(6′)-aph(2′′), aph(3′)-IIIa, ant(4′)-Ia and str), macrolides and lincosamide (ermA, ermB, ermC, ermT, msr(A/B), mphC, lnuA, lnuB, vgaA and vgaB), tetracycline (tetK, tetM, tetL and tetO), chloramphenicol (fexA, fexB, catpC194, catpC221 and catpC223), fusidic acid (fusB, fusC and fusD) and trimetho- prim/sulfamethoxazole (dfrA, dfrG, dfrK and dfrD). The protocol used for DNA ampliﬁca- tion was as follows: a ﬁnal volume of 50 µL contained 39.7 µL of ultra-pure water, 5 µL 10× complete buffer (Bioron, Römerberg, Germany), 1 µL 25 mM MgCl2, 1 µL deoxynucleotides triphosphate, 1 µL of each primer, 0.3 µL DFS Taq DNA polymerase (Bioron) and 1 µL DNA sample at 10 pg/µL. Primer sequences and PCR programs for the same are given in Table S2. The concentration and purity of the extracted DNA was measured using a spectrophotometer and Nano-DropTM software (Thermo ScientiﬁcTM, Waltham, MA, USA). Positive and negative controls used in all the experiments belonged to the strain collection of the University of Trás-os-Montes and Alto Douro. 4.1. Sample Collection and Bacterial Isolates During the month of February 2020, a total of 220 samples were collected from poultry in a Portuguese slaughterhouse. Swab samples were collected from the cloaca and tra- chea of 100 quails, 50 commercial chickens and 70 homebred chickens. Batches of quails, homebred and commercial chickens arrived at the slaughterhouse 3 days a week and around 36,000 quails, 3500 homebred and 8000 commercial chickens were slaughtered each day. Four samples were recovered from each batch. The swabs were inserted into tubes containing brain heart infusion (BHI) broth with 6.5% of NaCl and incubated at 37 ◦C under aerobic conditions for 24 h. The inoculum was then seeded onto ORSAB agar plates supplemented with 2 mg/mL of oxacillin, incubated at 37 ◦C and examined after 24 h to 48 h. Up to three colonies per plate with different colors and morphology were recovered and further investigated. The staphylococci species identiﬁcation was performed by matrix-assisted laser desorption/ionization time-of-ﬂight coupled to time-of-ﬂight mass spectrometry (MALDI-TOF MS) (Bruker Daltonics, Bremen, Germany) as described by Dubois et al. [54]. 7 of 11 7 of 11 Antibiotics 2022, 11, 365 5. Conclusions MRCoNS are common bacteria found in healthy poultry in Portugal. S. urealyticus seems to be more prevalent in quails, while broiler chickens are more often colonized by S. lentus, indicating a separate epidemiology. The high frequency of MRCoNS isolates in this study may be due to the fact that these bacteria are colonizers of the normal skin ﬂora of animals. However, the multidrug resistance found in almost all isolates indicates that MRCoNS in poultry may be an important reservoir of antimicrobial-resistant genes. This is of great concern for public health, since most antimicrobial resistances detected were antimicrobials commonly used in human medicine. Some measures to overcome antimicrobial resistance in poultry in Portugal should be taken into consideration, such as the education of poultry producers, limiting the availability of antibiotics and the application of strict legislation concerning antimicrobial prescription. Supplementary Materials: The following supporting information can be downloaded at https://www. mdpi.com/article/10.3390/antibiotics11030365/s1: Table S1: Antimicrobial-resistant phenotype and genotype and SCCmec typing of CoNS isolated from poultry. Table S2: Primer pairs used for molecular typing and detection of antimicrobial resistance genes in MRSA strains. Figure S1: Prevalence of each staphylococci specie in poultry samples. References [56–71] are cited in the Supplementary Materials. Author Contributions: Conceptualization, V.S., M.V.-P. and P.P.; methodology, V.S.; validation, V.S., M.C. and P.P.; investigation, V.S.; resources, M.V.-P. and C.S.; data curation, V.S. and E.F.; writing— original draft preparation, V.S.; writing—review and editing, V.S., M.C. and P.P.; visualization, J.E.P.; supervision, J.L.C., G.I. and P.P.; funding acquisition, P.P. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by the R&D Project CAREBIO2: Comparative assessment of antimicrobial resistance in environmental bioﬁlms through proteomics—towards innovative thera- nostic biomarkers, with reference NORTE-01-0145-FEDER-030101 and PTDC/SAU-INF/30101/2017, ﬁnanced by the European Regional Development Fund (ERDF) through the Northern Regional Operational Program (NORTE 2020) and the Foundation for Science and Technology (FCT). This work was supported by the Associate Laboratory for Green Chemistry-LAQV, which is ﬁnanced by national funds from FCT/MCTES (UIDB/50006/2020 and UIDP/50006/2020) and by the projects UIDB/CVT/00772/2020 and LA/P/0059/2020 funded by the Portuguese Foundation for Science and Technology (FCT). Vanessa Silva is grateful to FCT (Fundacão para a Ciência e a Tecnologia) for ﬁnancial support through the PhD grant SFRH/BD/137947/2018. 4.5. Statistical Analysis Pearson’s chi-square test was used compare the carriage of S. sciuri, S. lentus, S. urealyticus and S. haemolyticus between the quails, the homebred chickens and the commercial chickens. The analyses were carried out using IBM SPSS Statistics, Version 26.0 (IBM Corp., Armonk, NY, USA) and signiﬁcance was set at p ≤0.05. 8 of 11 Antibiotics 2022, 11, 365 5. Conclusions Institutional Review Board Statement: The study was conducted according to the Helsinki Declara- tion (ICH-GCP principles), compliance with Schedule Y/ICMR Guidelines, the Oviedo Convention, and approved by the Ethics Committee of the University of Trás-os-Montes e Alto Douro (EC-UTAD, 8 November 2019). Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest. 5. Becker, K.; Heilmann, C.; Peters, G. Coagulase-negative staphylococci. Clin. Microbiol. Rev. 2014, 27, 870–926. [CrossRef] 6. Michalik, M.; Samet, A.; Podbielska-Kubera, A.; Savini, V.; Mi˛edzobrodzki, J.; Kosecka-Strojek, M. Coagulase-negative staphylo- cocci (CoNS) as a signiﬁcant etiological factor of laryngological infections: A review. Ann. Clin. Microbiol. Antimicrob. 2020, 19, 1–10. [CrossRef] References 1. Parlet, C.P.; Brown, M.M.; Horswill, A.R. Commensal staphylococci inﬂuence Staphylococcus aureus skin colonization and disease. Trends Microbiol. 2019, 27, 497–507. [CrossRef] [PubMed] 2. Gherardi, G.; Di Bonaventura, G.; Savini, V. Chapter 1—Staphylococcal Taxonomy. In Pet-to-Man Travelling Staphylococci; Academic Press: Cambridge, MA, USA, 2018; pp. 1–10, ISBN 978-0-12-813547-1. g pp 3. Smith, J.T.; Andam, C.P. Extensive horizontal gene transfer within and between species of coagulase-negative Staphylococcus. Genome Biol. Evol. 2021, 13, evab206. [CrossRef] [PubMed] 5. Becker, K.; Heilmann, C.; Peters, G. Coagulase-negative staphylococci. Clin. Microbiol. Rev. 2014, 27, 870–926. [CrossRef] 6. Michalik, M.; Samet, A.; Podbielska-Kubera, A.; Savini, V.; Mi˛edzobrodzki, J.; Kosecka-Strojek, M. Coagulase-negative staphylo- cocci (CoNS) as a signiﬁcant etiological factor of laryngological infections: A review. Ann. Clin. Microbiol. Antimicrob. 2020, 19, 1–10. [CrossRef] 6. Michalik, M.; Samet, A.; Podbielska-Kubera, A.; Savini, V.; Mi˛edzobrodzki, J.; Kosecka-Strojek, M. Coagulase-negative staphylo- cocci (CoNS) as a signiﬁcant etiological factor of laryngological infections: A review. Ann. Clin. Microbiol. Antimicrob. 2020, 19, 1–10. [CrossRef] 9 of 11 Antibiotics 2022, 11, 365 7. Noshak, M.A.; Rezaee, M.A.; Hasani, A.; Mirzaii, M. The role of the coagulase-negative staphylococci (CoNS) in infective endocarditis; a narrative review from 2000 to 2020. Curr. Pharm. Biotechnol. 2020, 21, 1140–1153. [CrossRef] 8. MacFadyen, A.C.; Harrison, E.M.; Drigo, I.; Parkhill, J.; Holmes, M.A.; Paterson, G.K. A mecC allotype, mecC3, in the CoNS Staphylococcus caeli, encoded within a variant SCCmecC. J. Antimicrob. Chemother. 2019, 74, 547–552. [CrossRef] 9. Loncaric, I.; Kübber-Heiss, A.; Posautz, A.; Ruppitsch, W.; Lepuschitz, S.; Schauer, B.; Feßler, A.T.; Krametter-Frötscher, R.; Harrison, E.M.; Holmes, M.A.; et al. Characterization of mecC gene-carrying coagulase-negative Staphylococcus spp. isolated from various animals. Vet. Microbiol. 2019, 230, 138–144. [CrossRef] 10. Zong, Z.; Peng, C.; Lü, X. Diversity of SCCmec Elements in Methicillin-Resistant Coagulase-Negative Staphylococci Clinical Isolates. PLoS ONE 2011, 6, e20191. [CrossRef] Isolates. PLoS ONE 2011, 6, e20191. [CrossRef] 11. Chaj˛ecka-Wierzchowska, W.; Zadernowska, A.; Nalepa, B.; Sierpi´nska, M.; Łaniewska-Trokenheim, Ł. Coagulase-negative staphylococci (CoNS) isolated from ready-to-eat food of animal origin—Phenotypic and genotypic antibiotic resistance. Food Microbiol. 2015, 46, 222–226. [CrossRef] 12. Argemi, X.; Hansmann, Y.; Prola, K.; Prévost, G. Coagulase-negative staphylococci pathogenomics. Int. J. Mol. Sci. 2019, 20, 1215. [CrossRef] [PubMed] 13. Silva, V.; Pereira, J.E.; Maltez, L.; Ferreira, E.; Manageiro, V.; Caniça, M.; Capelo, J.L.; Igrejas, G.; Poeta, P. Diversity of methicillin- resistant staphylococci among wild Lepus granatensis: First detection of mecA-MRSA in hares. FEMS Microbiol. Ecol. 2019, 96, ﬁz204. References [CrossRef] [PubMed] [ ] [ ] 14. Roberts, M.C.; Garland-Lewis, G.; Trufan, S.; Meschke, S.J.; Fowler, H.; Shean, R.C.; Greninger, A.L.; Rabinowitz, P.M. Distribution of Staphylococcus species in dairy cows, workers and shared farm environments. FEMS Microbiol. Lett. 2018, 365, fny146. [CrossRef] [PubMed] 15. Suepaul, S.; Georges, K.; Unakal, C.; Boyen, F.; Sookhoo, J.; Ashraph, K.; Yusuf, A.; Butaye, P. Determination of the frequency, species distribution and antimicrobial resistance of staphylococci isolated from dogs and their owners in Trinidad. PLoS ONE 2021, 16, e0254048. [CrossRef] [PubMed] 16. Lee, S.I.; Kim, S.D.; Park, J.H.; Yang, S.-J. Species Distribution, Antimicrobial Resistance, and Enterotoxigenicity of Non-aureus Staphylococci in Retail Chicken Meat. Antibiotics 2020, 9, 809. [CrossRef] [PubMed] 17. Huynh, M.; Carnaccini, S.; Driggers, T.; Shivaprasad, H.L. Ulcerative Dermatitis and Valvular Endocarditis Associated with Staphylococcus aureus in a Hyacinth Macaw (Anadorhynchus hyacinthinus). Avian Dis. 2014, 58, 223–227. [CrossRef] [PubMed] 18. Pimenta, R.L.; de Melo, D.A.; Bronzato, G.F.; Souza, V.R.d.S.; Holmström, T.C.N.; de Mattos de Oliveira Coelho, S.; de Silva Coelho I ; de Souza M M S Characterization of Staphylococcus spp isolates and β lactam resistance in broiler chicken production 17. Huynh, M.; Carnaccini, S.; Driggers, T.; Shivaprasad, H.L. Ulcerative Dermatitis and Valvular E Staphylococcus aureus in a Hyacinth Macaw (Anadorhynchus hyacinthinus). Avian Dis. 2014, 58, 223–2 17. Huynh, M.; Carnaccini, S.; Driggers, T.; Shivaprasad, H.L. Ulcerative Dermatitis and Valvular Endocarditis Associated with Staphylococcus aureus in a Hyacinth Macaw (Anadorhynchus hyacinthinus). Avian Dis. 2014, 58, 223–227. [CrossRef] [PubMed] 18 Pi t R L d M l D A B t G F S VR d S H l t ö T C N d M tt d Oli i C lh S d Sil 18. Pimenta, R.L.; de Melo, D.A.; Bronzato, G.F.; Souza, V.R.d.S.; Holmström, T.C.N.; de Mattos de Oliveira Coelho, S.; de Silva Coelho, I.; de Souza, M.M.S. Characterization of Staphylococcus spp. isolates and β-lactam resistance in broiler chicken production. Braz. J. Vet. Med. 2021, 43, e00720. [CrossRef] 19. Bhargava, K.; Zhang, Y. Characterization of methicillin-resistant coagulase-negative staphylococci (MRCoNS) in retail meat. Food Microbiol. 2014, 42, 56–60. [CrossRef] 20. Silva, V.; Vieira-Pinto, M.; Saraiva, C.; Manageiro, V.; Reis, L.; Ferreira, E.; Caniça, M.; Capelo, J.L.; Igrejas, G.; Poeta, P. Prevalence and Characteristics of Multidrug-Resistant Livestock-Associated Methicillin-Resistant Staphylococcus aureus (LA-MRSA) CC398 Isolated from Quails (Coturnix Coturnix Japonica) Slaughtered for Human Consumption. Animals 2021, 11, 2038. [CrossRef] p g p 21. 29. Pyzik, E.; Marek, A.; St˛epie´n-Py´sniak, D.; Urban-Chmiel, R.; Jarosz, Ł.S.; Jagiełło-Pod˛ebska, I. Detection of antibiotic resistance and classical enterotoxin genes in coagulase-negative staphylococci isolated from poultry in Poland. J. Vet. Res. 2019, 63, 183. [CrossRef] 28. Moawad, A.A.; Hotzel, H.; Awad, O.; Roesler, U.; Hafez, H.M.; Tomaso, H.; Neubauer, H.; El-Adawy, H. Evolution of Antibiotic Resistance of Coagulase-Negative Staphylococci Isolated from Healthy Turkeys in Egypt: First Report of Linezolid Resistance. Microorganisms 2019, 7, 476. [CrossRef] References [CrossRef] g p y pp y g 31. Boamah, V.E.; Agyare, C.; Odoi, H.; Adu, F.; Gbedema, S.Y.; Dalsgaard, A. Prevalence and antibiotic resistance of coagulase- negative Staphylococci isolated from poultry farms in three regions of Ghana. Infect. Drug Resist. 2017, 10, 175–183. [CrossRef] 31. Boamah, V.E.; Agyare, C.; Odoi, H.; Adu, F.; Gbedema, S.Y.; Dalsgaard, A. Prevalence and antibiotic resistance of coagulase- negative Staphylococci isolated from poultry farms in three regions of Ghana. Infect. Drug Resist. 2017, 10, 175–183. [CrossRef] 32. El-Nagar, S.; Abd El-Azeem, M.W.; Nasef, S.A.; Sultan, S. Prevalence of toxigenic and methicillin resistant staphylococci in poultry chain production. J. Adv. Vet. Res. 2017, 7, 33–38. g p y p y g f g 32. El-Nagar, S.; Abd El-Azeem, M.W.; Nasef, S.A.; Sultan, S. Prevalence of toxigenic and methicillin resistant staphylococci in poultry chain production. J. Adv. Vet. Res. 2017, 7, 33–38. p 33. Pyzik, E.; Marek, A. Characterization of bacteria of the genus Staphylococcus isolated from the eggs of Japanese quail (Coturnix japonica). Pol. J. Vet. Sci. 2012, 15, 791–792. [CrossRef] [PubMed] 34. Ruzauskas, M.; Couto, N.; Kerziene, S.; Siugzdiniene, R.; Klimiene, I.; Virgailis, M.; Pomba, C. Prevalence, species distribution and antimicrobial resistance patterns of methicillin-resistant staphylococci in Lithuanian pet animals. Acta Vet. Scand. 2015, 57, 27. [CrossRef] [PubMed] 35. Dhaouadi, S.; Souﬁ, L.; Campanile, F.; Dhaouadi, F.; Sociale, M.; Lazzaro, L.; Cherif, A.; Stefani, S.; Elandoulsi, R.B. Prevalence of meticillin-resistant and -susceptible coagulase-negative staphylococci with the ﬁrst detection of the mecC gene among cows, humans and manure in Tunisia. Int. J. Antimicrob. Agents 2020, 55, 105826. [CrossRef] [PubMed] 36. Wu, C.; Zhang, X.; Liang, J.; Li, Q.; Lin, H.; Lin, C.; Liu, H.; Zhou, D.; Lu, W.; Sun, Z.; et al. Characterization of ﬂorfenicol resistance genes in the coagulase-negative Staphylococcus (CoNS) isolates and genomic features of a multidrug-resistant Staphylococcus lentus strain H29. Antimicrob. Resist. Infect. Control 2021, 10, 9. [CrossRef] [PubMed] f 37. Seni, J.; Mshana, S.E.; Msigwa, F.; Iddi, S.; Mazigo, H.; Parkhill, J.; Holmes, M.A.; Paterson, G.K. Draft genome sequence of a multidrug-resistant caprine isolate of Staphylococcus cohnii subsp. urealyticus from Tanzania encoding ermB, tet(K), dfrG, fusF and fosD. J. Glob. Antimicrob. Resist. 2019, 18, 163–165. [CrossRef] 38. Lienen, T.; Schnitt, A.; Hammerl, J.A.; Marino, S.F.; Maurischat, S.; Tenhagen, B.-A. Multidrug-resistant Staphylococcus cohnii and Staphylococcus urealyticus isolates from German dairy farms exhibit resistance to beta-lactam antibiotics and divergent penicillin-binding proteins. Sci. Rep. References 2021, 11, 6075. [CrossRef] p g p p 39. Nemeghaire, S.; Argudín, M.A.; Haesebrouck, F.; Butaye, P. Molecular epidemiology of methicillin-resistant Staphylococcus sciuri in healthy chickens. Vet. Microbiol. 2014, 171, 357–363. [CrossRef] 40. Agyare, C.; Boamah, V.E.; Zumbi, C.N.; Osei, F.B. Antibiotic use in poultry production and its Antimicrobial Resistance—A Global Threat; IntechOpen: London, UK, 2018; pp. 33–50. gy , ; , ; , ; , p y p Antimicrobial Resistance—A Global Threat; IntechOpen: London, UK, 2018; pp. 33–50. 41. Schwarz, S.; Werckenthin, C.; Kehrenberg, C. Identiﬁcation of a plasmid-borne chloramphenicol-ﬂorfenicol resistance gene in h l i i i i b h h [C f] 41. Schwarz, S.; Werckenthin, C.; Kehrenberg, C. Identiﬁcation of a plasmid-borne chloramphenicol-ﬂorf Staphylococcus sciuri. Antimicrob. Agents Chemother. 2000, 44, 2530–2533. [CrossRef] 42. Wendlandt, S.; Shen, J.; Kadlec, K.; Wang, Y.; Li, B.; Zhang, W.-J.; Feßler, A.T.; Wu, C.; Schwarz, S. Multidrug resistance genes in staphylococci from animals that confer resistance to critically and highly important antimicrobial agents in human medicine. Trends Microbiol. 2015, 23, 44–54. [CrossRef] 43. Schoenfelder, S.M.K.; Dong, Y.; Feßler, A.T.; Schwarz, S.; Schoen, C.; Köck, R.; Ziebuhr, W. Antibio coagulase-negative staphylococci in livestock environments. Vet. Microbiol. 2017, 200, 79–87. [CrossRef 44. Wang, Y.; He, T.; Schwarz, S.; Zhao, Q.; Shen, Z.; Wu, C.; Shen, J. Multidrug resistance gene cfr in methicillin-resistant coagulase- negative staphylococci from chickens, ducks, and pigs in China. Int. J. Med. Microbiol. 2013, 303, 84–87. [CrossRef] [PubMed] 44. Wang, Y.; He, T.; Schwarz, S.; Zhao, Q.; Shen, Z.; Wu, C.; Shen, J. Multidrug resistance gene cfr in methicillin-resistant coagulase- 45. He, T.; Wang, Y.; Schwarz, S.; Zhao, Q.; Shen, J.; Wu, C. Genetic environment of the multi-resistance gene cfr in methicillin-resistant coagulase-negative staphylococci from chickens, ducks, and pigs in China. Int. J. Med. Microbiol. 2014, 304, 257–261. [CrossRef] [PubMed] 46. Petinaki, E. Resistance of Staphylococci to Macrolides-Lincosamides-Streptogramins B (MLSB): Epidemiology and Mechanisms of Resistance. In Staphylococcus Aureus; Hemeg, H., Ozbak, H., Afrin, F., Eds.; IntechOpen: Rijeka, Croatia, 2019. g j 47. Syed, M.A.; Ullah, H.; Tabassum, S.; Fatima, B.; Woodley, T.A.; Ramadan, H.; Jackson, C.R. Staphylococci in poultry intestines: A comparison between farmed and household chickens1. Poult. Sci. 2020, 99, 4549–4557. [CrossRef] [PubMed] 48. Hamed, E.A.; Abdelaty, M.F.; Sorour, H.K.; Roshdy, H.; AbdelRahman, M.A.A.; Magdy, O.; Ibrahim, W.A.; Sayed, A.; Mohamed, H.; Youssef, M.I.; et al. References Bernier-Lachance, J.; Arsenault, J.; Usongo, V.; Parent, É.; Labrie, J.; Jacques, M.; Malouin, F.; Archambault, M. Prevalence and characteristics of Livestock-Associated Methicillin-Resistant Staphylococcus aureus (LA-MRSA) isolated from chicken meat in the province of Quebec, Canada. PLoS ONE 2020, 15, e0227183. [CrossRef] p 22. Tang, Y.; Larsen, J.; Kjeldgaard, J.; Andersen, P.S.; Skov, R.; Ingmer, H. Methicillin-resistant and -susceptible Staphylococcus aureus from retail meat in Denmark. Int. J. Food Microbiol. 2017, 249, 72–76. [CrossRef] [PubMed] 23. Okorie-Kanu, O.J.; Anyanwu, M.U.; Ezenduka, E.V.; Mgbeahuruike, A.C.; Thapaliya, D.; Gerbig, G.; Ugwuijem, E.E.; Okorie-Kanu, C.O.; Agbowo, P.; Olorunleke, S.; et al. Molecular epidemiology, genetic diversity and antimicrobial resistance of Staphylococcus aureus isolated from chicken and pig carcasses, and carcass handlers. PLoS ONE 2020, 15, e0232913. [CrossRef] [PubMed] p g [ ] [ ] 24. Bala, H.K.; Igwe, J.C.; Olayinka, B.O.; Olonitola, O.S.; Onaolapo, J.A.; Okafo, C.N. Antibiotic susceptibility proﬁle of Staphylococ- cus aureus isolated from healthy chickens in poultry farms in Kano state, Nigeria. Sky J. Microbiol. Res. 2016, 4, 42–46. 25. European Medicines Agency European Surveillance of Veterinary Antimicrobial Consumption. ‘Sales of Veterinary Antimicrobial Agents in 31 European Countries in 2019 and 2020′ EMA/58183/2021; Publications Ofﬁce of the European Union: Luxembourg, 2021. 26. Marek, A.; Pyzik, E.; St˛epie´n-Py´sniak, D.; Dec, M.; Jarosz, Ł.S.; Nowaczek, A.; Sulikowska, M. Bioﬁlm-Formation Ability and the Presence of Adhesion Genes in Coagulase-Negative Staphylococci Isolates from Chicken Broilers. Animals 2021, 11, 728. [CrossRef] [PubMed] 27. Younis, W.; Sabra, M.; Sayed, H.H. Occurrence and characterization of coagulase positive and negative Staphylococci isolated from Japanese quails and broiler chickens at Qena Governorate, Egypt. SVU-Int. J. Vet. Sci. 2021, 4, 1–15. [CrossRef] p q gyp 28. Moawad, A.A.; Hotzel, H.; Awad, O.; Roesler, U.; Hafez, H.M.; Tomaso, H.; Neubauer, H.; El-Adawy, H. Evolution of Antibiotic Resistance of Coagulase-Negative Staphylococci Isolated from Healthy Turkeys in Egypt: First Report of Linezolid Resistance. Microorganisms 2019, 7, 476. [CrossRef] 29. Pyzik, E.; Marek, A.; St˛epie´n-Py´sniak, D.; Urban-Chmiel, R.; Jarosz, Ł.S.; Jagiełło-Pod˛ebska, I. Detection of antibiotic resistance and classical enterotoxin genes in coagulase-negative staphylococci isolated from poultry in Poland. J. Vet. Res. 2019, 63, 183. [CrossRef] 10 of 11 10 of 11 Antibiotics 2022, 11, 365 30. Saha, O.; Rakhi, N.N.; Istiaq, A.; Islam, I.; Sultana, M.; Hossain, M.A.; Rahaman, M.M. Evaluation of Commercial Disinfectants against Staphylococcus lentus and Micrococcus spp. of Poultry Origin. Vet. Med. Int. 2020, 2020, 8811540. y p y 53. Bortolaia, V.; Espinosa-Gongora, C.; Guardabassi, L. Human health risks associated with antimicrobial Staphylococcus aureus on poultry meat. Clin. Microbiol. Infect. 2016, 22, 130–140. [CrossRef] References Detection of erythromycin-resistant determinants by PCR. Antimicrob. Agents Chemother. 1996, 40, 2562–2566. [CrossRef] 59. Shopsin, B.; Mathema, B.; Alcabes, P.; Said-Salim, B.; Lina, G.; Matsuka, A.; Martinez, J.; Kreiswirth, B.N. Prevalence of agr speciﬁcity groups among Staphylococcus aureus strains colonizing children and their guardians. J. Clin. Microbiol. 2003, 41, 456–459. [CrossRef] [ ] 60. Gomez-Sanz, E.; Torres, C.; Lozano, C.; Fernandez-Perez, R.; Aspiroz, C.; Ruiz-Larrea, F.; Zarazaga, M. Detection, molecular characterization, and clonal diversity of methicillin-resistant Staphylococcus aureus CC398 and CC97 in Spanish slaughter pigs of different age groups. Foodborne Pathog. Dis. 2010, 7, 1269–1277. [CrossRef] g g p g 61. Wondrack, L.; Massa, M.; Yang, B.V.; Sutcliffe, J. Clinical strain of Staphylococcus aureus inactivates an Antimicrob. Agents Chemother. 1996, 40, 992–998. [CrossRef] g a, M.; Yang, B.V.; Sutcliffe, J. Clinical strain of Staphylococcus aureus inactivates and causes efﬂux of macrolides hemother. 1996, 40, 992–998. [CrossRef] 62. Lina, G.; Quaglia, A.; Reverdy, M.E.; Leclercq, R.; Vandenesch, F.; Etienne, J. Distribution of genes encoding resistance to macrolides, lincosamides, and streptogramins among staphylococci. Antimicrob. Agents Chemother. 1999, 43, 1062–1066. [CrossRef] p g g p y g 63. Bozdogan, B.; Berrezouga, L.; Kou, M.S.; Yurek, D.A.; Farley, K.A.; Stockman, B.J.; Leclercq, R. A new resistance gene, linB, conferring resistance to lincosamides by nucleotidylation in Enterococcus faecium HM1025. Antimicrob. Agents Chemother. 1999, 43, 925–929. [CrossRef] 64. Lozano, C.; Aspiroz, C.; Rezusta, A.; Gómez-Sanz, E.; Simon, C.; Gómez, P.; Ortega, C.; Revillo, M.J.; Zarazaga, M.; Torres, C. Identiﬁcation of novel vga(A)-carrying plasmids and a Tn5406-like transposon in meticillin-resistant Staphylococcus aureus and Staphylococcus epidermidis of human and animal origin. Int. J. Antimicrob. Agents 2012, 40, 306–312. [CrossRef] 65. Hammerum, A.M.; Jensen, L.B.; Aarestrup, F.M. Detection of the satA gene and transferability of virginiamycin resistance in Enterococcus faecium from food- animals. FEMS Microbiol. Lett. 1998, 168, 145–151. [CrossRef] [PubMed] 65. Hammerum, A.M.; Jensen, L.B.; Aarestrup, F.M. Detection of the satA gene and transferability of virginiamycin resistance in Enterococcus faecium from food- animals. FEMS Microbiol. Lett. 1998, 168, 145–151. [CrossRef] [PubMed] 66 A t F M A L Y Ah P JŁ J L M d M J L B A ti i bi l tibilit d f 66. Aarestrup, F.M.; Agers, L.Y.; Ahrens, P.; JŁrgensen, J.L.; Madsen, M.; Jensen, L.B. Antimicrobial su resistance genes in staphylococci from poultry. Vet. Microbiol. 2020, 74, 353–364. [CrossRef] 67. Van de Klundert, J.A.M.; Vliegenthart, J.S. References Monitoring of Antimicrobial Susceptibility of Bacteria Isolated from Poultry Farms from 2014 to 2018. Vet. Med. Int. 2021, 2021, 6739220. [CrossRef] 49. European Centre for Disease Prevention and Control (ECDC); European Food Safety Authority (EFSA); European Medicines Agency (EMA). Third joint inter-agency report on integrated analysis of consumption of antimicrobial agents and occurrence of antimicro. EFSA J. 2021, 19, e06712. 50. Chen, H.-J.; Hung, W.-C.; Lin, Y.-T.; Tsai, J.-C.; Chiu, H.-C.; Hsueh, P.-R.; Teng, L.-J. A novel fusidic aci fusF, in Staphylococcus cohnii. J. Antimicrob. Chemother. 2015, 70, 416–419. [CrossRef] 51. Gardete, S.; Aires-De-Sousa, M.; Faustino, A.; Ludovice, A.M.; de Lencastre, H. Identiﬁcation of the First Vancomycin Intermediate- Resistant Staphylococcus aureus (VISA) Isolate from a Hospital in Portugal. Microb. Drug Resist. 2008, 14, 1–6. [CrossRef] 52. Ali, Y.; Islam, M.A.; Muzahid, N.H.; Sikder, M.O.F.; Hossain, M.A.; Marzan, L.W. Characterization, prevalence and antibiogram study of Staphylococcus aureus in poultry. Asian Pac. J. Trop. Biomed. 2017, 7, 253–256. [CrossRef] 53. Bortolaia, V.; Espinosa-Gongora, C.; Guardabassi, L. Human health risks associated with antimicrobial-resistant enterococci and Staphylococcus aureus on poultry meat. Clin. Microbiol. Infect. 2016, 22, 130–140. [CrossRef] 11 of 11 11 of 11 Antibiotics 2022, 11, 365 54. Damien, D.; David, L.; Paul, C.J.; Markus, K.; Olivier, S.P.; Régine, T.; Richard, B.; Julien, D. Identiﬁcation of a Variety of Staphylococcus Species by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry. J. Clin. Microbiol. 2010, 48, 941–945. [CrossRef] , [ ] 55. Silva, V.; Almeida, F.; Carvalho, J.A.; Castro, A.P.; Ferreira, E.; Manageiro, V.; Tejedor-Junco, M.T.; Caniça, M.; Igrejas, G.; Poeta, P. Emergence of community-acquired methicillin-resistant Staphylococcus aureus EMRSA-15 clone as the predominant cause of diabetic foot ulcer infections in Portugal. Eur. J. Clin. Microbiol. Infect. Dis. 2020, 39, 179–186. [CrossRef] [PubMed] g f 56. Zhang, K.; Sparling, J.; Chow, B.L.; Elsayed, S.; Hussain, Z.; Church, D.L.; Gregson, D.B.; Louie, T.; Conly, J.M. New quadriplex PCR assay for detection of methicillin and mupirocin resistance and simultaneous discrimination of Staphylococcus aureus from coagulase-negative staphylococci. J. Clin. Microbiol. 2004, 42, 4947–4955. [CrossRef] [PubMed] 57. Schnellmann, C.; Gerber, V.; Rossano, A.; Jaquier, V.; Panchaud, Y.; Doherr, M.G.; Thomann, A.; Straub, R.; Perreten, V. Presence of new mecA and mph(C) variants conferring antibiotic resistance in Staphylococcus spp. isolated from the skin of horses before and after clinic admission. J. Clin. Microbiol. 2006, 44, 4444–4454. [CrossRef] [PubMed] J 58. Sutcliffe, J.; Grebe, T.; Tait-Kamradt, A.; Wondrack, L. 71. Chen, H.J.; Hung, W.C.; Tseng, S.P.; Tsai, J.C.; Hsueh, P.R.; Teng, L.J. Fusidic acid resistance determinan clinical isolates. Antimicrob. Agents Chemother. 2010, 54, 4985–4991. [CrossRef] References PCR detection of genes coding for aminoglycoside-modifying enzymes. In Diagnostic Molecular Microbiology: Principles and Applications; Persing, D.H., Smith, T.F., Tenover, F.C., White, T.J., Eds.; American Society for Microbiology: Washington, DC, USA, 1993; pp. 547–552. gy g pp 68. Kehrenberg, C.; Schwarz, S. Distribution of Florfenicol Resistance Genes fexA and cfr among Ch Staphylococcus Isolates. Antimicrob. Agents Chemother. 2006, 50, 1156–1163. [CrossRef] [PubMed] 69. Liu, H.; Wang, Y.; Wu, C.; Schwarz, S.; Shen, Z.; Jeon, B.; Ding, S.; Zhang, Q.; Shen, J. A novel phenicol exporter gene, fexB, found in enterococci of animal origin. J. Antimicrob. Chemother. 2012, 67, 322–325. [CrossRef] 69. Liu, H.; Wang, Y.; Wu, C.; Schwarz, S.; Shen, Z.; Jeon, B.; Ding, S.; Zhang, Q.; Shen, J. A novel phenicol exporter gene, fexB, found in enterococci of animal origin. J. Antimicrob. Chemother. 2012, 67, 322–325. [CrossRef] g 70. Mclaws, F.; Chopra, I.; O’Neill, A.J. High prevalence of resistance to fusidic acid in clinical isolates of Staphylococcus epidermidis. J. Antimicrob. Chemother. 2008, 61, 1040–1043. [CrossRef] g 70. Mclaws, F.; Chopra, I.; O’Neill, A.J. High prevalence of resistance to fusidic acid in clinical isolates of Staphylococcus epidermidis. J. Antimicrob. Chemother. 2008, 61, 1040–1043. [CrossRef] 71. Chen, H.J.; Hung, W.C.; Tseng, S.P.; Tsai, J.C.; Hsueh, P.R.; Teng, L.J. Fusidic acid resistance determinants in Staphylococcus aureus clinical isolates. Antimicrob. Agents Chemother. 2010, 54, 4985–4991. [CrossRef] 71. Chen, H.J.; Hung, W.C.; Tseng, S.P.; Tsai, J.C.; Hsueh, P.R.; Teng, L.J. Fusidic acid resistance determinants in Staphylococcus aureus clinical isolates. Antimicrob. Agents Chemother. 2010, 54, 4985–4991. [CrossRef] 71. Chen, H.J.; Hung, W.C.; Tseng, S.P.; Tsai, J.C.; Hsueh, P.R.; Teng, L.J. Fusidic acid clinical isolates. Antimicrob. Agents Chemother. 2010, 54, 4985–4991. [CrossRef]
https://openalex.org/W4294669866	https://www.researchsquare.com/article/rs-936620/latest.pdf	English	null	Somewhere to go: assessing the impact of public restroom interventions on reports of open defecation in San Francisco, California from 2014 to 2020	BMC public health	2,022	cc-by	6,889	Somewhere to go: Assessing the impact of public restroom interventions on reports of open defecation in San Francisco, California from 2014 to 2020 Heather Kathleen Amato (  heather_amato@berkeley.edu ) University of California Berkeley School of Public Health https://orcid.org/0000-0002-0501-075 Methods We evaluated the impact of various public restroom interventions implemented from January 1, 2014 to January 1, 2020 on reports of exposed feces, captured through a 311 municipal service. Publicly available 311 reports of exposed feces were spatially and temporally matched to 31 Pit Stop restroom interventions in ten San Francisco neighborhoods. We conducted an interrupted time-series analysis to compare pre- versus post-intervention rates of feces reports near the restrooms. Conclusions Increased access to public toilets and the addition of restroom attendants reduced fecal contamination in San Francisco, especially in neighborhoods with people experiencing homelessness. Programs that improve access to public restrooms should be evaluated at the neighborhood level in order to tailor sanitation interventions to neighborhood-specific needs. Results Feces reports declined by 12.47 reports per week after the installation of 13 Pit Stop restrooms (p-value = 0.0002). The rate of reports per week declined from the six-month pre-intervention period to the post-intervention period (slope change=-0.024 [95% CI=-0.033, -0.014]). Reports also declined after new restroom installations in the Mission and Golden Gate Park, and after the provision of attendants in the Mission, Castro/Upper Market, and Financial District/South Beach. Background Open defecation due to a lack of access to sanitation facilities remains a public health issue in the United States. People experiencing homelessness face barriers to accessing sanitation facilities, and are often forced to practice open defecation on streets and sidewalks. Exposed feces may contain harmful pathogens posing a significant threat to public health, especially among unhoused persons living near open defecation sites. The City of San Francisco’s Department of Public Works implemented the Pit Stop Program to provide the unhoused and the general public with improved access to sanitation with the goal of reducing fecal contamination on streets and sidewalks. The objective of this study was to assess the impact of these public restroom interventions on reports of exposed feces in San Francisco, California. Research DOI: https://doi.org/10.21203/rs.3.rs-936620/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/12 Introduction Open defecation in San Francisco, California has been highlighted as a problem in recent years.1 However, no rigorous have been conducted to understand how access to public restrooms can potentially mitigate open defecation in this setting. A recent study by Capone et al. suggested that at least 930,000 individuals in the United States lack access to basic sanitation, in striking contrast to previous estimates by the World Health Organization Joint Monitoring Program (JMP) which placed the number at 28,000.2 Critically, Capone’s estimate was the first to include people experiencing homelessness (PEH), who collectively accounted for approximately half of the population lacking access to basic sanitation (460,000). Notably, the JMP has reported that open defecation (disposal of human feces in fields, forests, bushes, open bodies of water, beaches, and other open spaces) and limited sanitation (use of improved facilities shared between two or more households) are nonexistent in the United States.3 Capone et al. argues that all unsheltered PEH should be classified as engaging in open defecation and that most sheltered PEH should be classified as having limited sanitation.2 In 2019, there were an estimated 8,000 PEH in San Francisco, 64% of which were unsheltered.4 Unsheltered PEH rely on public restrooms, homeless service agencies, and privately owned business restrooms for their sanitation needs. Access to these facilities can be restricted by barriers such as limited hours of operation, transportation difficulties when traveling to distant facilities, customer-only policies at businesses, discrimination against PEH by staff members, and insufficient levels of cleanliness, maintenance, and monitoring.1,5 PEH may be forced to practice open defecation, which may have detrimental effects on their physical, mental, and social well-being. Open defecation also constitutes a public health hazard: exposure to fecal contamination in the environment can spread pathogenic infections causing diarrheal and other illnesses.6,7 Limited research has shown that homelessness may be a risk factor for certain infectious diseases in San Francisco.8,9 Unsheltered PEH may be most at risk of exposure to fecal pathogens if they occupy public sidewalks or other spaces where open defecation occurs and do not have reliable access to water or sanitation for hygiene practices. Page 2/12 Page 2/12 Seeking to address these issues and reduce open defecation, the San Francisco Department of Public Works (DPW) began the Pit Stop Program in 2014. This program provides free, public restrooms throughout the city, many of which are staffed with two paid attendants. Reports of Exposed Feces San Francisco 311 reports since 2008 are publicly available at https://datasf.org/opendata/. The 311 reporting system includes a variety of municipal services and several report classification systems to route reports to appropriate agencies. Each report includes the report type (e.g. Human/Animal Waste), responsible agency, date, location (street address and longitude/latitude), status notes, and a photo of the incident (if provided by the individual making the report). Only reports of type Human/Animal Waste were included in this analysis, as these correspond to incidents of exposed feces. To remove duplicate or misclassified reports, we excluded reports with “dup” or “transfer” in the status notes and only included reports from agencies that respond to feces reports. We consulted with DPW staff to help develop and validate these data processing methods. Spatial Analysis We mapped Pit Stop locations from GPS coordinates (provided by the San Francisco DPW) using ArcGIS Online hosted by Esri. For neighborhood boundaries, we used Analysis Neighborhoods polygons created by the San Francisco Department of Public Health, available here: https://data.sfgov.org/Geographic-Locations-and-Boundaries/Analysis-Neighborhoods/p5b7-5n3h. In ArcGIS Online, we created 500-meter walking distance buffers (polygon derived from all 500m routes following pedestrian paths and roads) around each Pit Stop location to capture the number of 311 feces reports within the surrounding area of each intervention. Feces reports were then spatially and temporally matched to each Pit Stop intervention in R version 4.0.210 using the data.table and sf packages.11,12 We identified all 311 feces reports that occurred within a 500m walking distance buffer of each intervention location and within six months before and six months after the intervention start date. Statistical Methods Pit Stop Restroom Interventions We evaluated the impact of San Francisco Pit Stop interventions implemented between January 1, 2014 and January 1, 2020. Information on Pit Stop locations and intervention start dates was provided by the San Francisco Department of Public Works (DPW) upon request. Within the Pit Stop Program, we identified three categories of sanitation interventions: 1) installation of new restroom (the provision of portable staffed Pit Stop facilities in locations where no public restrooms previously existed); 2) provision of attendants (the conversion of previously existing unstaffed public restrooms into staffed Pit Stops); and 3) expansion of service hours (the extension of hours of operation from daytime online to 24 hours per day, a 2019 pilot program). Existing restrooms that were converted to staffed Pit Stops included self-cleaning JC Decaux facilities and Recreation and Park Department facilities. Introduction The attendants ensure the Pit Stop is clean, safe, and adequately stocked with supplies. All Pit Stops are also equipped with waste bins, dog waste bags, and needle disposal boxes. The DPW utilizes a citywide 311 municipal reporting system for individuals to report exposed feces found on public property. Using these reports as a proxy for instances of open defecation, we retrospectively investigated the impacts of different Pit Stop public restroom interventions on reports of exposed feces in San Francisco by analyzing the pre- versus post-intervention change in weekly feces reports near each restroom. Statistical Methods Our main outcome of interest was the number of exposed feces reports per week within 500m of each Pit Stop location. We calculated the means and standard deviations (SD) of reports in a six-month period before the intervention and during the six months after the intervention by intervention type and neighborhood. We obtained p-values from permutation tests (N = 10,000 permutations) using an alpha of 0.05 to determine statistical significance of the difference in sample means.13 We also calculated means and SDs of reports by year and season. We used an interrupted time series approach to further analyze the longitudinal impacts of Pit Stop interventions on reports of exposed feces.14 We assessed longitudinal trends in 311 feces reports per week during the six-month (26-week) period before versus the six-month period after each intervention. We specified the following negative binomial model, appropriate for modeling overdispersion in weekly count data:15 Page 3/12 We estimated the change in the rate of feces reports per week (i.e. the post-intervention slope change) by intervention type, as well as by neighborhood and intervention type. We included neighborhood type in the models stratified by intervention type to adjust for confounding due to spatial dependence of Pit Stops within the same neighborhood. Sandwich estimators were used to calculate robust standard errors and 95% confidence intervals. To establish a transition period between the pre- and post-intervention samples, we removed the 27th week (which included the intervention start date) for each Pit Stop intervention for all analyses. Statistical analyses were conducted in R version 4.0.210 using the following packages: dplyr, perm, Mass, lmtest, and sandwich.16–19 Plots were created using ggplot2 and ggpubr packages.20(p2),21 Results There were 31 Pit Stop interventions implemented across 27 locations between January 1, 2014 and January 1, 2020, including the installation of 13 new restrooms (Table 1; Fig. 1). Existing restrooms were staffed with attendants at 15 locations throughout the study period, and three restrooms expanded their service hours beginning in 2019. The earliest interventions included in the analysis were three new restrooms installed in the Tenderloin on July 15, 2014, and the most recent interventions were the expansion of service hours at three existing restrooms in different neighborhoods on August 16, 2019 (Supplemental Materials, Table S1). The number of exposed feces reports within a 500m walking distance of each Pit Stop intervention ranged from 0-201 reports per week. During the six-year study period, the highest mean number of feces reports per week occurred in the spring (mean = 36.8, SD = 40.2), followed by summer (mean = 35.3, SD = 35.8), winter (mean = 29.6, SD = 29.4), and fall (mean = 28.4, SD = 21.6) (Figure S1). Page 4/12 Page 4/12 Table 1 Pre- versus post-intervention mean feces reports per week by intervention type and neighborhood. No. Pit Stop Interventions No. Weeks Observed a Mean reports per week pre-intervention (SD) Mean reports per week post-intervention (SD) Change in Mean (Δ) p-value b Intervention Type Installation of New Restroom 13 338 49.18 (48.45) 36.71 (27.17) -12.47 0.0002 Provision of Attendants 15 390 22.75 (26.56) 20.87 (16.95) -1.88 0.2296 Expansion of Service Hours 3 78 34.45 (16.92) 46.45 (29.45) 12 0.0016 Neighborhood of Intervention Tenderloin c 11 286 68.01 (45.18) 50.40 (21.89) -17.60 0.0002 Mission c 5 130 26.98 (12.71) 28.24 (11.57) 1.25 0.4068 South of Market (SoMa) c 4 104 37.42 (13.92) 39.38 (21.55) 1.95 0.4406 Castro/Upper Market 4 104 8.99 (5.30) 12.08 (6.19) 3.09 0.0004 Golden Gate Park c 2 52 0.69 (1.04) 0.85 (1.23) 0.15 0.5561 Haight Ashbury c 1 26 1.08 (1.38) 1.85 (1.91) 0.77 0.1242 Bayview Hunters Point c 1 26 2.50 (1.48) 2.35 (1.72) -0.15 0.7953 Sunset Parkside 1 26 0.27 (0.53) 0.65 (0.75) 0.38 0.0606 North Beach 1 26 6.65 (3.67) 15.19 (4.89) 8.54 0.0002 Financial District/South Beach 1 26 3.35 (2.42) 6.50 (4.31) 3.15 0.0030 a Number (No.) of weeks observed is per six-month period (e.g. Results in the Golden Gate Park neighborhood, 26 weeks pre-intervention were compared to 26 weeks post-intervention across 2 Pit Stop interventions, resulting in the comparison of 52 weeks pre-intervention versus 52 weeks post-intervention). b P-values are estimated from nonparametric permutation tests (n = 10,000 permutations) comparing the difference in the sample means post- versus pre-intervention. c Neighborhoods with at least one new restroom installed. SD = standard deviation. Table 1 a Number (No.) of weeks observed is per six-month period (e.g. in the Golden Gate Park neighborhood, 26 weeks pre-intervention were compared to 26 weeks post-intervention across 2 Pit Stop interventions, resulting in the comparison of 52 weeks pre-intervention versus 52 weeks post-intervention). b P-values are estimated from nonparametric permutation tests (n = 10,000 permutations) comparing the difference in the sample means post- versus pre-intervention. c Neighborhoods with at least one new restroom installed. SD = standard deviation. Results from permutation tests are presented in terms of the change in mean feces reports, denoted Δ. The mean number of feces reports near all newly installed Pit Stop restrooms dropped significantly after their installation (Δ =-12.47; p = 0.0002) (Table 1). There was no significant reduction in feces reports near Pit Stop locations where attendants were hired to service the restrooms (Δ=-1.88; p = 0.2296). Though there were only three Pit Stop locations that expanded service hours to 24 hours per day, there was a significant increase in the mean feces reports per week after the expansion of service hours (Δ = 12.00; p = 0.0016). Results from permutation tests are presented in terms of the change in mean feces reports, denoted Δ. The mean number of feces reports near all newly installed Pit Stop restrooms dropped significantly after their installation (Δ =-12.47; p = 0.0002) (Table 1). There was no significant reduction in feces reports near Pit Stop locations where attendants were hired to service the restrooms (Δ=-1.88; p = 0.2296). Though there were only three Pit Stop locations that expanded service hours to 24 hours per day, there was a significant increase in the mean feces reports per week after the expansion of service hours (Δ = 12.00; p = 0.0016). Results Page 5/12 Regression results estimating the post-intervention slope change, denoted Δm, showed there was a significant reduction in the rate of feces reports from the six-month post-intervention period to the pre-intervention period (Δm=-0.024 [95% CI=-0.033, -0.014]) across all locations with new restrooms installed (Table 2, Fig. 2). There was no significant change in the rate of feces reports after the provision of attendants across all locations (Δm=-0.001 [-0.011, 0.008]), while there was an increase in the rate of feces reports following the expansion of service hours (Δm = 0.033 [0.021, 0.044]). Table 2 Pre- versus post-intervention rate of feces reports by intervention type and neighborhood. No. Pit Stop Interventions Total No. Weeks Observed a Pre-Intervention Slope (m) (95% CI) b Post-Intervention Slope Change (Δm) (95% CI) b Intervention Type c Installation of New Restroom 13 676 0.013 (0.006, 0.020) -0.024 (-0.033, -0.014) Provision of Attendants 15 780 0.002 (-0.006, 0.010) -0.001 (-0.011, 0.008) Expansion of Service Hours 3 156 -0.002 (-0.009, 0.006) 0.033 (0.021, 0.044) Neighborhood of Intervention (Installation of New Restroom, only) Tenderloin 5 260 0.020 (0.008, 0.029) -0.035 (-0.049, -0.021) Mission 3 156 0.010 (-0.001, 0.022) -0.015 (-0.029, -0.0005) South of Market (SoMa) 2 104 0.007 (-0.002, 0.015) -0.015 (-0.031, 0.001) Golden Gate Park 1 52 0.027 (-0.079, 0.133) -0.182 (-0.316, -0.047) Haight Ashbury 1 52 -0.054 (-0.117, 0.010) 0.055 (-0.017, 0.128) Bayview Hunters Point 1 52 -0.015 (-0.042, 0.012) 0.0004 (-0.043, 0.044) (Provision of Attendants, only) Tenderloin 5 260 -0.015 (-0.026, -0.005) 0.017 (0.004, 0.030) Mission 2 104 0.016 (-0.003, 0.034) -0.031 (-0.055, -0.008) South of Market (SoMa) 1 52 -0.022 (-0.040, -0.005) 0.046 (0.025, 0.066) Castro/Upper Market 3 156 0.022 (0.007, 0.037) -0.022 (-0.043, -0.001) Golden Gate Park 1 52 0.040 (0.003, 0.078) -0.040 (-0.010, 0.020) Sunset/Parkside 1 52 0.054 (-0.041, 0.148) -0.009 (-0.117, 0.099) North Beach 1 52 0.030 (0.003, 0.058) -0.023 (-0.055, 0.009) Financial District/South Beach 1 52 0.065 (0.032, 0.098) -0.071 (-0.122, -0.021) a No. (number) of weeks observed indicates total number of weeks across both the pre- and post-intervention periods (52 weeks total per Pit Stop intervention). b Estimates are from negative binomial regression models with 95% confidence intervals (CI) calculated from robust standard errors. c Models stratified by intervention include neighborhood as a main effect to adjust for confounding. a No. Discussion This study found that the installation of public restrooms as part of the San Francisco Pit Stop program was associated with a long-term reduction in the rate of reports of exposed feces. The decline in feces reports in the six-month period after the installation of new restrooms was driven by reductions in the Tenderloin, the Mission, Golden Gate Park and, to a lesser extent, SoMa. The provision of attendants at existing restrooms also led to significant reductions in the rate of feces reports in the Mission, the Castro/Upper Market and the Financial District/South Beach. These results support the hypothesis that Pit Stop public restroom interventions help reduce the incidence of open defecation due to improved restroom access for PEH. Additionally, the provision of restroom attendants may improve the cleanliness, maintenance and safety of the facilities, increasing the use of previously unsafe or unsanitary restrooms. This aligns with previous research showing that PEH will often avoid restrooms that are broken down, unclean, not stocked with supplies, or unmonitored (due to safety concerns).1,5 The addition of new restrooms and the provision of attendants may improve access to and the quality of sanitation facilities, thus reducing open defecation for vulnerable populations without access to other sanitation solutions. In 2019, the San Francisco point-in-time count of PEH estimated that District 6, which contains the Tenderloin and SoMa neighborhoods, had 3,656 homeless residents, double the amount in the next-highest district.4 Of these, 1,990 (54%) were unsheltered. Unsheltered individuals lack access to the limited shared sanitation facilities offered by homeless shelters and other housing programs and are more likely to have to resort to open defecation.1,5 Results from this study suggest that improvements in restroom quality and accessibility have a more appreciable impact in areas where the need for them is higher. The Tenderloin and SoMa had the highest number of feces reports compared to other neighborhoods. This suggests that these neighborhoods may have the highest incidence of open defecation, which aligns with the high prevalence of unsheltered PEH in these areas. We found that Pit Stop locations in the Tenderloin had the largest average reduction in reports of exposed feces following the interventions. Despite the high number of feces reports and the high prevalence of homelessness in SoMa, there was only a near-significant decline in the post-intervention rate of feces reports near SoMa Pit Stops. Results (number) of weeks observed indicates total number of weeks across both the pre- and post-intervention periods (52 weeks total per Pit Stop intervention). b Estimates are from negative binomial regression models with 95% confidence intervals (CI) calculated from robust standard errors. c Models stratified by intervention include neighborhood as a main effect to adjust for confounding. Page 6/12 Pit Stop interventions in the Tenderloin neighborhood had the most feces reports, with a mean of 68.01 reports per week (SD = 45.18) pre- intervention (Table 1). Only Pit Stop interventions located in the Tenderloin resulted in a significant reduction in the mean number of nearby feces reports per week (Δ=-17.60, p = 0.0002). There were significant increases in the mean number of feces reports after Pit Stop interventions were implemented in the Castro/Upper Market (Δ = 3.09, p = 0.0004), North Beach (Δ = 8.54, p = 0.0002), and the Financial District/South Beach (Δ = 3.15, p = 0.0030) (Table 1). Pit Stop interventions in the Tenderloin neighborhood had the most feces reports, with a mean of 68.01 reports per week (SD = 45.18) pre- intervention (Table 1). Only Pit Stop interventions located in the Tenderloin resulted in a significant reduction in the mean number of nearby feces reports per week (Δ=-17.60, p = 0.0002). There were significant increases in the mean number of feces reports after Pit Stop interventions were implemented in the Castro/Upper Market (Δ = 3.09, p = 0.0004), North Beach (Δ = 8.54, p = 0.0002), and the Financial District/South Beach (Δ = 3.15, p = 0.0030) (Table 1). Regression results from the interrupted time series analysis identified different changes in long-term trends of feces reports per week when stratified by neighborhood and intervention type. Among Pit Stop locations where new restrooms were installed, the rate of feces reports was significantly lower in the six-months post-intervention compared to the pre-intervention period in the Tenderloin (Δm=-0.035 [-0.049, -0.021]), the Mission (Δm=-0.015 [-0.029, -0.0005]), and Golden Gate Park (Δm=-0.182 [-0.316,-0.047]) (Table 2). The rate of feces reports also declined after new restrooms were installed in SoMa, though the slope change was not statistically significant (Δm=-0.015 [-0.029, 0.001]). Results Among existing restroom locations where attendants were provided, there were significant reductions in the rate of feces reports near Pit Stops in the Mission (Δm=-0.031 [-0.055, -0.008]), the Castro/Upper Market (Δm=-0.022 [-0.043, -0.001]) and the Financial District/South Beach (Δm=-0.071 [-0.122, -0.021]) (Table 2). The rate of feces reports significantly increased after the provision of attendants at existing Pit Stop locations in the Tenderloin (Δm = 0.017 [0.004, 0.030]) and SoMa (Δm = 0.046 [0.025, 0.066]) (Table 2, Fig. 2). Discussion A 2018 study in Atlanta, Georgia detected harmful pathogens in 23% of human fecal samples collected from various open defecation sites.25 Poor sanitation is a known contributing factor to the spread of infectious diseases in communities worldwide.26 Additionally, homelessness has been identified as a potential risk factor for antimicrobial resistant infections in San Francisco, CA and elsewhere.8,27,28 PEH are frequent visitors of emergency rooms, often due to mental health needs or substance abuse, increasing their risk of exposure to drug-resistant pathogens that are difficult to treat.29,30 Individuals who acquire drug-resistant infections in the hospital may spread drug resistance to others in their community, especially where sanitation and hygiene conditions are inadequate. Future studies should identify pathogens in exposed feces in the urban environment of San Francisco and characterize pathogen carriage among PEH to determine the extent to which exposure to human feces drives infections in these vulnerable communities. Mission are most at risk of exposure to feces based on the high number of 311 exposed feces reports. Feces of humans, as well as dogs, may contain harmful pathogens that pose public health risks to the homeless communities in these neighborhoods. A 2018 study in Atlanta, Georgia detected harmful pathogens in 23% of human fecal samples collected from various open defecation sites.25 Poor sanitation is a known contributing factor to the spread of infectious diseases in communities worldwide.26 Additionally, homelessness has been identified as a potential risk factor for antimicrobial resistant infections in San Francisco, CA and elsewhere.8,27,28 PEH are frequent visitors of emergency rooms, often due to mental health needs or substance abuse, increasing their risk of exposure to drug-resistant pathogens that are difficult to treat.29,30 Individuals who acquire drug-resistant infections in the hospital may spread drug resistance to others in their community, especially where sanitation and hygiene conditions are inadequate. Future studies should identify pathogens in exposed feces in the urban environment of San Francisco and characterize pathogen carriage among PEH to determine the extent to which exposure to human feces drives infections in these vulnerable communities. Our analysis has some limitations, the first being the use of 311 Human/Animal Waste reports as a proxy for incidence of open defecation, which may be prone to user error and misclassification. Discussion Reports that are correctly classified as Human/Animal Waste may not correspond to a human open defecation event, but may instead be animal feces (especially dog feces), though we were unable to distinguish between reports for human versus animal feces. Animal feces are an important source of exposure to fecal pathogens that can cause diarrheal diseases and other adverse health effects in humans.31 Pit Stops are equipped with dog waste bags so it is possible that they reduce both human and animal fecal contamination. Further research is warranted to determine the impacts of Pit Stop interventions on reducing animal versus human fecal contamination in San Francisco. Second, season may have played a role in both the incidence of open defecation and the incidence of reporting exposed feces. In this analysis covering six years of data, reports of feces near Pit Stop interventions were highest in the spring and summer months. Existing literature suggests that seasonal and climatic factors of environmental fecal contamination are variable and context-specific. For example, levels of fecal indicator bacteria in surface waters are often higher during hotter, drier months in tropical climates, while levels of fecal indicator bacteria in temperate urban climates may be higher during months with more rainfall.32 This is somewhat consistent with our findings, since spring months in San Francisco are temperate with some rainfall, and the summer months are dry with relatively higher temperatures. Season may also influence the frequency of reporting, as pedestrian traffic may decrease during the colder, rainy months, thereby reducing the chance that someone will encounter and report exposed feces. Third, other time-specific factors, such as changes in public awareness of fecal contamination or the 311 reporting system, may be confounders. Between August and October of 2018, there were at least three events that led to increased media coverage in the San Francisco chronicle and elsewhere: 1) in August, San Francisco DPW announced its plan to create a ‘Poop Patrol’; 2) in September, an online report about 311 feces reports in San Francisco and other major cities called “Doo-Doo, the New Urban Crisis” was published; and 3) in October, the creation of a free phone app called SnapCrap, designed to make 311 feces reporting in San Francisco more user-friendly, was announced (Supplemental Materials, Appendix A). Media events like these may account for some fluctuations in feces reports throughout the study period. Discussion It is possible that there was unmeasured confounding due to changes in public awareness, pedestrian traffic, or misclassification of animal feces as open defecation. While 311 feces reports can be a useful tool to plan and evaluate sanitation interventions, additional research is needed to validate these reports as an accurate and reliable indicator of open defecation over time. This study has several strengths. First, our interrupted time series analysis utilized a multiple baseline design (i.e. interventions beginning on various dates), which inherently controls for time-specific confounding factors. Second, this approach allows for each intervention to serve as its own control during the pre-intervention period, controlling for location-specific factors at each intervention site. Third, we assessed long-term changes in the rate of feces reports per week over a 12-month period, preventing short-term time-specific confounding from biasing our results. The UN General Assembly passed Resolution 64/292 in 2010 (and reaffirmed in 2018) declaring that adequate access to safe water and sanitation are essential human rights.33 California became the first state to legally recognize the human right to water for drinking, cooking and sanitary purposes with the passage of Assembly Bill 685 in 2012.34 However, this bill failed to recognize the human right to access to sanitation, and basic sanitation needs remain unmet in the most vulnerable populations of California. According to the 2019 point-in-time count, there are at least 108,432 unsheltered PEH in the state of California and at least 5,180 unsheltered PEH in the city of San Francisco.4,35 The Pit Stop Program improved access to sanitation facilities in San Francisco neighborhoods with the highest number of unsheltered people. This study provides evidence that a public sanitation program can reduce reports of exposed feces in public spaces, especially in neighborhoods with the greatest need for sanitation facilities. Though the Pit Stop Program attempts to fill the gap in sanitation access in San Francisco despite the lack of state legislation to do so, explicitly recognizing basic sanitation as a human right would drive other cities across California to improve sanitation access for all. Discussion We also observed a significant decline in the post-intervention rate of feces reports in the Mission, which had the third highest mean number of reports of exposed feces near Pit Stop locations. According to the 2019 point-in- time count, there were 643 total PEH including 257 (40%) unsheltered PEH in the Mission District (District 9).4 Though the reported population of PEH in the Mission is much lower than in the Tenderloin and SoMa, the estimated total number of PEH in the Mission District is increasing; District 9’s point-in-time count was 410 in 2015 and 552 in 2017.22,23 The Mission also shares its southern and eastern borders with District 10, which had the second highest point-in-time count in 2019 (1,820 PEH) and a single Pit Stop restroom located in Bayview Hunters Point.4 Notably, there were eleven Pit Stop interventions implemented in the Tenderloin. In contrast, there were only four Pit Stop interventions implemented at three locations in SoMa and five Pit Stop interventions implemented at five locations in the Mission which were spread across a large area. Other city-based studies have documented how sanitation coverage can reduce fecal contamination. In a study of low-income urban neighborhoods of Accra, Ghana, increased spatial clustering of sanitation coverage was associated with reduced environmental fecal bacteria contamination.24 The Tenderloin Pit Stops may have had a greater impact on reducing fecal contamination because more Pit Stop restrooms were clustered together within a smaller area, providing more sanitation facilities within a short walking distance of many PEH. Given the high number of fecal reports observed in SoMa and the Mission, increasing the density of Pit Stop restrooms near known areas with unsheltered people would result in more comprehensive access to sanitation facilities, potentially yielding greater reductions in open defecation. Unsheltered individuals in neighborhoods with poor access to sanitation may be at greater risk of exposure to fecal contamination from open defecation in their surrounding environment. Our results suggest that in San Francisco, unsheltered individuals in the Tenderloin, SoMa, and the Page 7/12 Page 7/12 Mission are most at risk of exposure to feces based on the high number of 311 exposed feces reports. Feces of humans, as well as dogs, may contain harmful pathogens that pose public health risks to the homeless communities in these neighborhoods. Authors’ contributions JG and DM developed the original study concept. DM acquired data and conducted initial data processing and contributed to manuscript writing. HA conducted spatial and statistical data analyses, developed tables and figures, and led manuscript writing. CH advised statistical analysis methods. All authors read and approved the final manuscript. Acknowledgements The authors thank Avery Richards and the San Francisco Department of Public Works for assistance with data acquisition and initial data processing. The authors thank Avery Richards and the San Francisco Department of Public Works for assistance with data acquisition and initial data processing. Page 8/12 Page 8/12 Based on the findings of this analysis, we recommend that the San Francisco Pit Stop Program be expanded to increase sanitation coverage in SoMa and other areas with high numbers of PEH. Unsheltered PEH are often forced to practice open defecation due to inadequate restroom access, potentially increasing their risk of being exposed to dangerous fecal pathogens in their surrounding environment. Allowing such conditions to persist constitutes a violation of basic human rights and human dignity, and poses a significant public health risk. It is imperative that state and local governments in California and elsewhere prioritize effective interventions that improve sanitation access, such as San Francisco’s Pit Stop Program, while simultaneously pursuing measures that improve housing affordability and reduce homelessness. Ethics approval and consent to participate Ethics approval and consent to participate Not applicable. Not applicable. Consent for publication Not applicable. Availability of data and materials Availability of data and materials Availability of data and materials Data will be published in DRYAD when this manuscript is accepted for publication in a scientific journal. Data and code used to conduct this analysis are currently available online at: https://github.com/HeatherKAmato/pit-stop The authors declare that they have no competing interests. The authors declare that they have no competing interests. Funding Not applicable. Authors’ contributions Authors’ contributions References Pebesma E, Bivand R, Racine E, et al. Sf: Simple Features for R.; 2021. Accessed June 23, 2021. https://CRAN.R-project.org/package=sf. 13. Chung E, Romano JP. Exact and asymptotically robust permutation tests. The Annals of Statistics. 2013;41(2):484–507. doi:10.1214/13- AOS1090. 13. Chung E, Romano JP. Exact and asymptotically robust permutation tests. The Annals of Statistics. 2013;41(2):484–507. doi:10.1214/13- AOS1090. 14. Bernal JL, Cummins S, Gasparrini A. Interrupted time series regression for the evaluation of public health interventions: a tutorial. Int J Epidemiol. 2017;46(1):348–55. doi:10.1093/ije/dyw098. 15. Gardner W, Mulvey EP, Shaw EC. Regression analyses of counts and rates: Poisson, overdispersed Poisson, and negative binomial models. Psychol Bull. 1995;118(3):392–404. doi:10.1037/0033-2909.118.3.392. 6. Fay M. Perm: Exact or Asymptotic Permutation Tests.; 2010. Accessed June 23, 2021. https://CRAN. 16. Fay M. Perm: Exact or Asymptotic Permutation Tests.; 2010. Accessed June 23, 2021. https://CRAN.R-project.org/package=perm. 16. Fay M. Perm: Exact or Asymptotic Permutation Tests.; 2010. Accessed June 23, 2021. https://CRAN.R-project.org/package=perm. 17 Ripley B Venables B Bates DM ca 1998) KH (partial port ca 1998) AG (partial port Firth D MASS: Support Functions and Datasets for 17. Ripley B, Venables B, Bates DM ca. 1998) KH (partial port, ca 1998) AG (partial port, Firth D. MASS: Support Functions and Datasets for Venables and Ripley’s MASS.; 2021. Accessed June 23, 2021. https://CRAN.R-project.org/package=MASS. 18. Hothorn T, Zeileis A, Farebrother (pan.f) RW, Cummins (pan.f) C, Millo G, Mitchell D. Lmtest: Testing Linear Regression Models.; 2020. Accessed June 23, 2021. https://CRAN.R-project.org/package=lmtest. 19. Zeileis A, Lumley T, Graham N, Koell S. Sandwich: Robust Covariance Matrix Estimators.; 2021. Accessed June 23, 2021. https://CRAN.R- project.org/package=sandwich. 20. Wickham H, Chang W, Henry L, et al. Ggplot2: Create Elegant Data Visualisations Using the Grammar of Graphics.; 2021. Accessed June 23, 2021. https://CRAN.R-project.org/package=ggplot2. 21. Kassambara A. Ggpubr: “ggplot2” Based Publication Ready Plots.; 2020. Accessed June 23, 2021. https://CRAN.R- project.org/package=ggpubr. 21. Kassambara A. Ggpubr: “ggplot2” Based Publication Ready Plots.; 2020. Accessed June 23, 2021. https://CRAN.R- project org/package=ggpubr 22. 2015 San Francisco Point-in-Time Count and Survey Comprehensive Report. Applied Survey Research;:1–86. Accessed June 24, 2021. 22. 2015 San Francisco Point-in-Time Count and Survey Comprehensive Report. Applied Survey Research;:1–86. Accessed June 24, 2021. https://hsh.sfgov.org/wp-content/uploads/2016/06/2015-San-Francisco-Homeless-Count-Report_0-1.pdf. 23. 2017 San Francisco Homeless Count and Survey Comprehensive Report. Applied Survey Research;:1–80. Accessed June 24, 2021. h l h h f h l d l df 23. 2017 San Francisco Homeless Count and Survey Comprehensive Report. Applied Survey Research;:1–80. Accessed June 24, 2021. https://live-hsh-sf.pantheonsite.io/wp-content/uploads/2017/06/2017-SF-Point-in-Time-Count-General-FINAL-6.21.17-1.pdf. 24. References 1. Frye EA, Capone D, Evans DP. Open Defecation in the United States: Perspectives from the Streets. Environmental Justice. 2019;12(5):226– 30. doi:10.1089/env.2018.0030. 1. Frye EA, Capone D, Evans DP. Open Defecation in the United States: Perspectives from the Streets. Environmental Justice. 2019;12(5):226– 30. doi:10.1089/env.2018.0030. 2. Capone D, Cumming O, Nichols D, Brown J. Water and Sanitation in Urban America, 2017–2019. Am J Public Health. 2020;110(10):1567– 72. doi:10.2105/AJPH.2020.305833. 3. WHO/UNICEF JMP Household. WASH Data. United Nations Children’s Fund and World Health Organization.; 2019. https://washdata.org/data/household#!/table?geo0=country&geo1=USA. 4. 2019 San Francisco Homeless Count and Survey Comprehensive Report. Applied Survey Research:1–77. Accessed June 24, 2021. https://hsh.sfgov.org/wp-content/uploads/2020/01/2019HIRDReport_SanFrancisco_FinalDraft-1.pdf. 5. Lupien S, Liu H, Lobato A, Myerson D, Schwartz B, Polsky C. Basic and Urgent: Realizing the Human Right to Sanitation for Californians Experiencing Homelessness. Environmental Law Clinic, University of California, Berkeley Law; 2018. https://www.law.berkeley.edu/wp- content/uploads/2018/08/FINAL_EJCW.ELC_.Basic_.UrgentReportonAccesstoWaterandSanitationbyHomelessCalifornians.8.8.18.docx.pdf. 6. Bartram J, Cairncross S. Hygiene, Sanitation, and Water: Forgotten Foundations of Health. PLOS Medicine. 2010;7(11):e1000367. d i 10 1371/j l d 1000367 5. Lupien S, Liu H, Lobato A, Myerson D, Schwartz B, Polsky C. Basic and Urgent: Realizing the Human Right to Sanitation for Californians Experiencing Homelessness. Environmental Law Clinic, University of California, Berkeley Law; 2018. https://www.law.berkeley.edu/wp- content/uploads/2018/08/FINAL_EJCW.ELC_.Basic_.UrgentReportonAccesstoWaterandSanitationbyHomelessCalifornians.8.8.18.docx.pdf. 6. Bartram J, Cairncross S. Hygiene, Sanitation, and Water: Forgotten Foundations of Health. PLOS Medicine. 2010;7(11):e1000367. doi:10.1371/journal.pmed.1000367. 7. Mara D, Lane J, Scott B, Trouba D. Sanitation and Health. PLoS Med. 2010;7(11). doi:10.1371/journal.pmed.1000363. 7. Mara D, Lane J, Scott B, Trouba D. Sanitation and Health. PLoS Med. 2010;7(11). doi:10.1371/journal.pmed.1000363. Page 9/12 Page 9/12 8. Charlebois ED, Bangsberg DR, Moss NJ, et al. Population-based community prevalence of methicillin-resistant Staphylococcus aureus in the urban poor of San Francisco. Clin Infect Dis. 2002;34(4):425–33. doi:10.1086/338069. 9. Hennessey KA, Bangsberg DR, Weinbaum C, Hahn JA. Hepatitis A seroprevalence and risk factors among homeless adults in San Francisco: should homelessness be included in the risk-based strategy for vaccination? Public Health Rep. 2009;124(6):813–7. doi:10.1177/003335490912400608. 10. R Core Team. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing; 2020. https://www.R- project.org. 11. Dowle M, Srinivasan A, Gorecki J, et al. Data.Table: Extension of “Data.Frame.”; 2021. Accessed June 23, 2021. https://CRAN.R- project.org/package=data.table. 12. Pebesma E, Bivand R, Racine E, et al. Sf: Simple Features for R.; 2021. Accessed June 23, 2021. h 12. Pebesma E, Bivand R, Racine E, et al. Sf: Simple Features for R.; 2021. Accessed June 23, 2021. https://CRAN.R-project.org/package=sf. 12. References https://digitallibrary.un.org/record/1649518?ln=en. 34. Bill Text - AB-685 State water policy. Accessed October 4. 2020. https://leginfo.legislature.ca.gov/faces/billTextClient.xhtml? bill_id=201120120AB685. 35. 2019 Continuum of Care Homeless Populations and Subpopulations Report - California. US Department of Housing and Urban Development; 2019. https://www.hudexchange.info/programs/coc/coc-homeless-populations-and-subpopulations-reports/. 35. 2019 Continuum of Care Homeless Populations and Subpopulations Report - California. US Department of Housing and Urban Development; 2019. https://www.hudexchange.info/programs/coc/coc-homeless-populations-and-subpopulations-reports/. References Berendes DM, Kirby AE, Clennon JA, et al. Urban sanitation coverage and environmental fecal contamination: Links between the household and public environments of Accra, Ghana. PLoS One. 2018;13(7). doi:10.1371/journal.pone.0199304. 25. Capone D, Ferguson A, Gribble MO, Brown J. Open Defecation Sites, Unmet Sanitation Needs, and Potential Sanitary Risks in Atlanta, Georgia, 2017–2018. Am J Public Health. 2018;108(9):1238–40. doi:10.2105/AJPH.2018.304531. 26. Freeman MC, Garn JV, Sclar GD, et al. The impact of sanitation on infectious disease and nutritional status: A systematic review and meta- analysis. Int J Hyg Environ Health. 2017;220(6):928–49. doi:10.1016/j.ijheh.2017.05.007. 26. Freeman MC, Garn JV, Sclar GD, et al. The impact of sanitation on infectious disease and nutritional status: A systematic review and meta- analysis. Int J Hyg Environ Health. 2017;220(6):928–49. doi:10.1016/j.ijheh.2017.05.007. 27. Young DM. An Epidemic of Methicillin-Resistant Staphylococcus aureus Soft Tissue Infections Among Medically Underserved Patients. Arch Surg. 2004;139(9):947. doi:10.1001/archsurg.139.9.947. 27. Young DM. An Epidemic of Methicillin-Resistant Staphylococcus aureus Soft Tissue Infections Among Medically Underserved Patients. Arch Surg. 2004;139(9):947. doi:10.1001/archsurg.139.9.947. 28. Leibler JH, León C, Cardoso LJP, et al. Prevalence and risk factors for MRSA nasal colonization among persons experiencing homelessness in Boston, MA. J Med Microbiol. 2017;66(8):1183–8. doi:10.1099/jmm.0.000552. 28. Leibler JH, León C, Cardoso LJP, et al. Prevalence and risk factors for MRSA nasal colonization among persons experiencing homelessness in Boston, MA. J Med Microbiol. 2017;66(8):1183–8. doi:10.1099/jmm.0.000552. 29. Mulvey MR, Simor AE. Antimicrobial resistance in hospitals: How concerned should we be? CMAJ. 2009;180(4):408–15. doi:10.1503/cmaj.080239. 29. Mulvey MR, Simor AE. Antimicrobial resistance in hospitals: How concerned should we be? CMAJ. 2009;180(4):408–15. doi:10.1503/cmaj.080239. 30. Kushel MB, Perry S, Bangsberg D, Clark R, Moss AR. Emergency Department Use Among the Homeless and Marginally Housed: Results From a Community-Based Study. Am J Public Health. 2002;92(5):778–84. 31. Penakalapati G, Swarthout J, Delahoy MJ, et al. Exposure to Animal Feces and Human Health: A Systematic Review and Proposed Research Priorities. Environmental Science Technology. 2017;51(20):11537–52. doi:10.1021/acs.est.7b02811. 32. Rochelle-Newall E, Nguyen TMH, Le TPQ, Sengtaheuanghoung O, Ribolzi O. A short review of fecal indicator bacteria in tropical aquatic ecosystems: knowledge gaps and future directions. Front Microbiol. 2015;6. doi:10.3389/fmicb.2015.00308. Page 10/12 33. The Human Rights to Safe Drinking Water and Sanitation: Resolution / Adopted by the Human Rights Council on 27 September 2018. UN Human Rights Council; 2018. https://digitallibrary.un.org/record/1649518?ln=en. 33. The Human Rights to Safe Drinking Water and Sanitation: Resolution / Adopted by the Human Rights Council on 27 September 2018. UN Human Rights Council; 2018. Figure 2 Feces reports by intervention type (A) and by neighborhood for new restroom installations (B) and the provision of attendants (C). Dashed vertical lines indicate the intervention start date. Individual points represent the number of feces reports per week within a 500 meter walking distance buffer of each Pit Stop intervention. Solid horizontal lines represent the slope of weekly feces reports before and after intervention start dates. Only neighborhoods with >10 feces reports per week on average are included in panels B and C. Figure 1 Restroom interventions implemented in San Francisco neighborhoods, 2014-2020. All 27 Pit Stop public restroom locations included in the analysis are shown on this map. Shaded and labeled areas represent neighborhoods. Orange and purple dots indicate multiple interventions occurred at a single Pit Stop restroom location. Restroom interventions implemented in San Francisco neighborhoods, 2014-2020. All 27 Pit Stop public restroom locations included in the analysis are shown on this map. Shaded and labeled areas represent neighborhoods. Orange and purple dots indicate multiple interventions occurred at a single Pit Stop restroom location. Page 11/12 Page 11/12 Figure 2 Feces reports by intervention type (A) and by neighborhood for new restroom installations (B) and the provision of attendants (C). Dashed vertical lines indicate the intervention start date. Individual points represent the number of feces reports per week within a 500 meter walking distance buffer of each Pit Stop intervention. Solid horizontal lines represent the slope of weekly feces reports before and after intervention start dates. Only neighborhoods with >10 feces reports per week on average are included in panels B and C. ighborhood for new restroom installations (B) and the provision of attendants (C). Dashed Individual points represent the number of feces reports per week within a 500 meter walking olid horizontal lines represent the slope of weekly feces reports before and after intervention start ts per week on average are included in panels B and C. SupplementalMaterialsPitStopsand311Reports.docx Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SupplementalMaterialsPitStopsand311Reports.docx Page 12/12
https://openalex.org/W2558541757	http://journal.walisongo.ac.id/index.php/ahkam/article/download/899/892	Latin	null	EVOLUSI IJTIHAD IMAM SYAFI’I : Dari Qawl Qadīm ke Qawl Jadīd	Al-Ahkam/Al-Ahkam	2,016	cc-by-sa	14,552	Ainol Yaqin: Evolusi Ijtihad Imam Syafi’i …. (h. 143-178) Ainol Yaqin: Evolusi Ijtihad Imam Syafi’i …. (h. 143-178) Ainol Yaqin Sekolah Tinggi Agama Islam Negeri (STAIN) Pamekasan e-mail: ainulfairus@ymail.com Abstract This article describes Imam al-Shāfi'i thought in building istinbath Islamic law method and its decition's which evolved from qawl qadīm to qawl jadīd. He is known as the founder of uṣūl al-fiqh science which is arranged systematically-logically and critically. He tried to combine the two schools of thought, those are: Maliki, known as ahl al-ḥadīth that thrives in the Hijaz, and the Hanafi, known as ahl al-ra'y that is entrenched in Iraq. He managed to combine the two schools by taking method good ahl al-ḥadīth and leaving the less ones successfully. Reciprocally, he took the good ahl al-ra’y method and left the poor. It was done for the reason that he had studied with Imam Malik and Muḥammad ibn Ḥasan al-Shaibani, the adherents of the Hanafi schools. The fatwa which he formulated at his living in Iraq is known as qawl qadīm. After he reviewed the fatwa built in Iraq and found the fragility of the arguments as a fundamental, finally, he triggered a new law, namely qawl jadīd which was based on the strong arguments. 1Mannā’ al-Qaṭṭān, Tārīkh al-Tashrī’ al-Islāmī (Beirut: Dār al-Fikr, 1995), h. 262. 2Muhammad Alī al- Sāyis, Tārīkh al-Fiqh al-Islāmī (Beirut: Dār al-Fikr, 1995), h. 73. 3Rif’at Faurī ‘Abdu al-Maṭlab, Muqaddimah al-Taḥqīq al-Umm, Juz I (Beirut: Dār al-Wafa’, 2001), h. 8; Ibn Hajar al-‘Asqalānī, Tawālī al-Ta`sīs (Beirut: Dār al-Kutub al-‘Ilmiah, 1986), h. 55-56; Sālim al- ‘Imrānī, al-Bayān fī Madhhab al-Imām al-Shāfi’i, Jilid I (Beirut: Dār al-Minhāj, t.th.), h. 5; Abū Zakariyā Muḥyiddīn ibn Sharaf al-Nawawī, al-Majmū’ Sharḥ al-Muhadhdhab, Jilid I (Beirut: Dār al-Fikr, t.th.), h. 25; Sulaimān ibn Muḥammad al-Bujairamī, al-Bujairamī ‘alā al-Khaṭīb, Juz I (Beirut: Dār al-Kutub al- ‘Ilmiah, 1996), h. 76; Muḥammad ibn al-Khaṭīb al-Sharbīnī, Mughnī al-Muḥtāj ilā Ma’rifati Ma’ānī `Alfāẓ al-Minhāj, Juz I (Beirut: Dār al-Ma’rifah, 1998), h. 37. [] Artikel ini menjelaskan tentang gagasan Imam Syafi’i dalam membangun metode istinbath hukum Islam dan fatwa-fatwanya yang mengalami evolusi dari qawl qadīm kepada qawl jadīd. Ia dikenal sebagai peletak dasar ilmu ushul fikih yang disusun secara sistematis-logis dan kritis. Ia berupaya memadukan antara dua aliran mazhab yakni Maliki yang dikenal sebagai ahl al-ḥadīth yang tumbuh subur di Hijaz, dan mazhab Hanafi yang dikenal sebagai ahl al-ra’y yang membudaya di Irak. Ia berhasil memadukan kedua aliran tersebut dengan mengambil metode ahl al-ḥadīth yang dinilai baik dan menanggalkan yang kurang baik. Begitu pula, ia mengambil metode ahl al-ra’y yang dipandang baik dan meninggalkan yang kurang baik. Hal ini dilakukan karena ia pernah berguru pada imam Mālik dan Muḥammad ibn Ḥasan al-Shaibanī, penganut mazhab Hanafi. Fatwa-fatwa yang ia rumuskan pada waktu berdomisili di Irak dikenal dengan qawl qadīm. Setelah ia meninjau kembali fatwa-fatwa yang dibangun di Irak dan menemui kerapuhan dalil-dalil yang dibuat pijakan, akhirnya, ia mencetuskan hukum baru, yakni qawl jadīd yang dibangun di atas dalil-dalil yang kuat. Keywords: qawl qadīm, qawl jadīd, perubahan hukum Islam AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 Volume 26, Nomor 2, Oktober 2016 ║143 143 Volume 26, Nomor 2, Oktober 2016 ║ Ainol Yaqin 4Karakterikstik mazhab ahl al-ra’y adalah: pertama, penggunaan ra’yu/akal dalam menetapkan status hukum kasuistik tidak hanya terbatas pada fenomena yang mengemuka pada masa itu. Bahkan mereka juga memprediksikan hukum suatu masalah yang belum terjadi. Karena itu, metodo- logi mereka dikenal sebagai fiqh iftiraḍi atau fiqih pengandaian. Kedua, sangat selektif dan ketat dalam penerimaan suatu hadis dengan membuat persyaratan yang ketat. Dalam penetapan suatu pe- riwayatan hadis, mereka tidak memperbanyak periwayatan hadis dari Nabi, dikhawatirkan ter- jerumus ke dalam hadis-hadis palsu. Hal tersebut menjadikan mereka mengesampingkan pe- riwayatan hadis dan sebaliknya, mereka lebih mengedepankan nalar akal/ra’yu. Mannā’ al-Qaṭṭān, Tārīkh al-Tashrī’ al-Islāmī , h. 271. Pendahuluan Perbedaan pendapat di antara imam mazhab dalam merumuskan hukum Islam merupakan sebuah keniscayaan. Sebab perangkat metodologi ijtihad masing-masing mereka memiliki karakteristik tersendiri sehingga produk hukum yang dicetuskannya pun mesti tidak sama. Disamping itu, sosio- kultural dimana seorang mujtahid memfatwakan hukum berperan besar dalam mewarnai hasil ijtihadnya. Imam Abū Ḥanīfah (80-150 H) hidup di Irak dengan sistem interaksi sosial, muamalah dan budaya masyarakat yang sudah berperadaban. Ruang lingkup ijtihad di Irak lebih luas dan diskursus proble- matika yang mengemuka lebih bervariasi. Sehingga ia lebih cenderung untuk menggunakan akal logika/ra’yu ketika merumuskan hukum suatu masalah.1 Sedangkan Imam Mālik (93-179 H) berdomisili di Madinah dengan budaya masyarakat yang masih kental mempraktekkan ajaran sunnah-sunnah Nabi yang mentradisi secara turun temurun. Sehingga ia dalam berijtihad lebih me- rujuk pada naṣ al-Qur’an dan sunnah-sunnah Nabi secara literal.2 Sementara itu, Imam Syafi’i (150-204 H) sempat berguru pada Imam Mālik di Madinah dan Muḥammad ibn Ḥasan al-Shaibani (w. 189 H) serta fuqahā’ lainnya di Irak sehingga beliau mengenali kehidupan masyarakat Madinah dan masyarakat Irak.3 Riḥlah hidup dan safari intelektual Imam al-Syafī’ī cukup mewarnai kreasi ijtihad yang digagasnya, sehingga pola ijtihad beliau bercirikan moderat. Secara garis besar, pola ijtihad para mujtahid terbagi pada dua macam, yaitu mazhab ahl al-ra’y dan mazhab ahl al-ḥadīth. Mazhab ahl al-ra’y tumbuh ber- kembang di Irak, yang menjadi kiblat pemerintahan Islam dan peradaban pada masa itu, yaitu Baghdad. Mazhab ini dipelopori oleh Imam Abū Ḥanīfah. Pola AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 144 Volume 26, Nomor 2, Oktober 2016 Evolusi Ijtihad Imam Syafi’i …. ijtihad Imam Abū Ḥanīfah bercirikan rasionalistik, dimana ra’yu /akal berperan besar dalam menghasilkan suatu hukum Islam. Hal ini, tidak dapat lepas dari seting sosio-kultural dimana beliau hidup bermasyarakat.4 Dan pada generasi belakangan dikenal dengan mazhab Hanafi. Di belahan daerah lain bersamaan dengan itu, terdapat ulama yang konsisten melestarikan dan memegang erat sunnah Nabi yang membudaya di daerah Madinah. Kelompok ini dikomandani Imam Mālik ibn Anas. Kemudian pada generasi berikutnya dikenal dengan mazhab Māliki. Beliau pernah nyantri pada seorang ulama Madinah yang ber- haluan ahl al-ra’y, bernama Rabī'ah ibn Farrūkh. Tetapi, beliau lebih tertarik menimba ilmu hadis dari Rabī'ah, bukan pemikiran al-ra'yu-nya. “Aḥmad ibn Hanbal berkata: Dahulu kita menjelek-jelekkan ahl al-ra’y, begitu pula sebaliknya. Sampai datanglah Imam al-Syafi’i, beliau menggabungkan keduanya. “ “Aḥmad ibn Hanbal berkata: Dahulu kita menjelek-jelekkan ahl al-ra’y, begitu pula sebaliknya. Sampai datanglah Imam al-Syafi’i, beliau menggabungkan keduanya. “ Lebih lanjut ia menyatakan: Pendahuluan Mazhab Māliki menitikberatkan pada literal bunyi teks dan mengutamakan sunnah daripada al- ra’yu, hingga akhirnya aliran ini dikenal dengan mazhab ahl al-ḥadīth.5 Bertolak dari dua arus mazhab yang tampak berseberangan itu, Imam Syafi’i berupaya memadukan keduanya, dengan mengambil metodologi maz- hab ahl al-ra’y yang dipandangnya baik dan menanggalkan yang kurang baik. Begitu pula, beliau mengambil metodologi mazhab ahl al-ḥadīth yang dianggap baik dan meninggalkan yang kurang baik.6 Hal itu terjadi karena beliau pernah 5Karakteristik mazhab ahl al-ḥadīth: pertama, pengistinbathan hukum suatu masalah hanya merujuk kepada al-Qur’an dan hadis Nabi. Mereka cenderung tidak menyukai penggunaan nalar ra’yu dan juga sangat berhati-hati ketika mengeluarkan fatwa suatu permasalahan. Mereka menegaskan bahwa hukum itu hanya bersandarkan pada fenomena yang terjadi saat ini, seolah-olah menyindir ahl al-ra’y dengan fiqh iftiraḍi-nya. Kedua, naṣ-naṣ hukum Islam, baik al-Qur’an maupun hadis dipahami secara literal-tekstual, serta menganggap hukum sebagai ketentuan ilahi yang tidak dapat dirasionalisasi, sehingga mereka menafikan ‘illat dan hubungan suatu hukum. Al-Sha’bi mengomentari mazhab ini sekaligus menolak gagasan rasionalisme Ibrāhīm al-Nakha’ī, ia menyatakan “Sesuatu yang diriwayatkan dari para sahabat, ambil dan jagalah. Sedangkan sesuatu yang keluar dari hasil nalar akal mereka, buanglah ”. Ibid., h. 271. 6Disebutkan: ﻗ ﺪ ﺑﻦ ﺣﻨﺒﻞ ﻣﺎ زﺎ ﻧﻠﻌﻦ أﻫﻞ ا ﺮأي وﻌﻠﻨﻮﻧﻨﺎ ﺣ ﺟﺎء ا ﺸﺎﻓ ﻓﻤﺰج ﺑﻨﻨﺎ$ﺎل أ . AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 Volume 26, Nomor 2, Oktober 2016 ║145 Volume 26, Nomor 2, Oktober 2016 Ainol Yaqin berguru pada Imam Mālik dan Muḥammad ibn Ḥasan al-Shaibanī serta fuqahā’ Irak lainnya, penganut dan penyebar mazhab Hanafi.7 Dilatarbelakangi safari intelektualnya, Imam Syafi’i berusaha mengkombinasikan manhaj ahl al-ra’y dan manhaj ahl al-ḥadīth untuk membangun mazhab yang beliau gagas sendiri. Ia tidak terlalu ekstrem pada tuntutan naṣ dan tidak pula berlebihan bertumpu pada ra’yu. Namun, ia berupaya mempertemukan seruan naṣ dengan realitas sosial dengan memaksimalkan nalar kritis ijtihadnya. Maka pertentangan antara mazhab ahl al-ra’y dan mazhab ahl al-ḥadīth sebenarnya berakhir saat Imam Syafi’i menggabungkan dua metodologi dalam meng- istinbatkan hukum Islam. Sebagaimana telah diketahui bahwa Imam Syafi’i memiliki dua qawl, yaitu qawl qadīm dan qawl jadīd. Pemetaan istilah tersebut dengan melihat dimana tempat beliau memutuskan hukum. Pendapat Imam Syafi’i yang difatwakan dan ditulis di Irak dikenal dengan qawl qadīm. Pembukuan pe- mikiran tersebut diperoleh dari perdebatan beliau dengan ahli fikih rasionalis ﺪﻳﺚ أن ﺻﺤﻴﺢ ا ﺮأي ﻓﺮع اﻷﺻﻞ، وﻋﻠﻢ أﺻﺤﺎب ا ﺮأي أﻧﻪ ﻻ ﻓﺮع إﻻ ﺑﻌﺪ7ﻓﻌﻠﻢ أﺻﺤﺎب ا اﻷﺻﻞ، وأﻧﻪ ﻻ ﻏ? 8Muhammad Abū al-Zahrah, al-Shāfi’ī, h. 158-160; ‘Abdullāh ibn Yūsuf al-Juwainī, Nihāyah al- Maṭlab fī Dirāyah al-Madhhab, Juz I, h. 162; Rif’at Faurī ‘Abdu al-Maṭlab, Muqaddimah al-Taḥqīq al- Umm, Juz I, h. 10-11; ‘Abdulwāhid ibn Ismā’īl al-Rūyānī, Bahru al-Madhhab fī Furū’ al-Imām al-Shāfi’ī, Juz I (Beirut: Ihyā` al-Turāth al-‘Arabī, 2002), h. 25; Sālim al-‘Imrānī, al-Bayān fī Madhhab al-Imām al- Shāfi’ī, Jilid I, h. 38. Pendahuluan ﻋﻦ ﺗﻘﺪﻳﻢ ا ﺴ; وﺻﺤﻴﺢ اﻵﺛﺎر أوﻻ .ً “Para ahli hadis akhirnya mengetahui bahwa ra’yu yang benar itu cabang dari asal (al-Qur’an dan hadis), dan ahl al-ra’y mengetahui bahwa tidak ada cabang jika tidak ada asal. Maka tidak ada alasan untuk tidak mendahulukan sunnah dan athār yang ṣaḥīḥ.” Qādlī Iyāḍ ibn Mūsa, Tartīb al-Madārij wa Taqrīb al-Masālik (Beirut: Dār al-Kutub al-‘Ilmiyyah, 2001), h. 91. 7al-Syafi’i kecil sebelum berguru kepada Imam Mālik, ia terlebih dahulu meminjam kitab al- Muwaṭṭhā` pada seseorang di Makkah, kemudian ia membaca dan menghafalnya. Baru setelah itu, ia belajar di bawah asuhan sang Imam sampai gurunya itu meninggal. Sementara ilmu fikih dari mazhab ahlu al-ra’y ia peroleh dari Muhammad ibn Hasan al-Syaibanī. Ia menimba ilmu padanya dengan cara menelaah kitab-kitab karyanya, kemudian berdiskusi bersama teman sejawat dan sang guru. Sehingga pada diri beliau terkumpul dua aliran mazhab sekaligus. Muhammad Abū al-Zahrah, al- Shāfi’i (Beirut: Dār al-Fikr al-‘Arabī, 1978), h. 19-25; Ibn Ḥajar al-‘Asqalānī, Tawālī al-Ta`sīs, h. 54; ‘Abdullāh ibn Yūsuf al-Juwainī, Nihāyah al-Maṭlab fī Dirāyah al-Madhhab, Juz I (Jeddah: Dār al-Minhāj, 2007), h. 101; Rif’at Faurī ‘Abdu al-Maṭlab, Muqaddimah al-Taḥqīq al-Umm, Juz I, h. 9; Abdurraḥmān ibn Abī Hātim al-Rāzī, Ādāb al-Shāfi’i wa Manāqibuhu (Beirut: Dār al-Kutub al-‘Ilmiah, 2003), h. 22; Sālim al-‘Imrānī, al-Bayān fī Madhhab al-Imām al-Shāfi’i, jilid I, h. 53; Sulaimān ibn Muḥammad al- Bujairamī, al-Bujairamī ‘alā ‘l-Khaṭīb, Juz I, h. 77. AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 146 Volume 26, Nomor 2, Oktober 2016 Evolusi Ijtihad Imam Syafi’i …. Irak. Di tengah-tengah kesibukannya ia menyempatkan diri mengabadikan pendapatnya dalam lembaran-lembaran kitab yang disebut dengan al-ḥujjah yang secara komprehensif memuat problematika masyarakat Irak yang kom- pleks dengan budaya dan peradabannya. Sedangkan, pendapat Imam Syafi’i dan didiktekan pada muridnya di Mesir dan kemudian dibukukan dikenal dengan al-Umm.8 9Sālim al-‘Imrānī, al-Bayān fī Madhhab al-Imām al-Shāfi’ī, Jilid I, h. 5; Abdul’azhīm Mahmūd al-Dīb, Muqaddimāt Nihāyah al-Maṭlab fī Nihāyah al-Madhhab, h. 115-118; Sulaimān ibn Muhammad al- Bujairamī, al-Bujairamī ‘alā ‘l-Khaṭīb, Juz I, h. 77; Aḥmad ‘Izzi dan ‘Ināyah al-Dimasyqī, Muqaddimah al-Taḥqīq Baḥru al-Madhhab, Juz I, (Beirut: Ihyā` al-Turāth al-‘Arabī, 2002), h. 25-27; Muhammad ibn al-Khaṭīb al-Sharbīnī, Mughnī al-Muḥtāj ilā Ma’rifati Ma’ānī Alfāẓ al-Minhāj, Juz I, h. 38; Abdulhamīd al- Sharwanī, Hawāsyī Tuhfah al-Minhāj bi Sharḥ al-Minhāj, Juz I (Kairo: al-Maktabah al-Tijāriyyah al- Kubra, 2007), h. 54; Aḥmad ibn Salamah al-Qalyūbī, Hāsyiyyah Qalyūbī, Juz I (Kairo: Musthafā al-Bābī al-Halabī, 2007), h. 13; Muḥammad ibn Abī al-‘Abbās al-Ramlī, Nihāyah al-Muntāj ilā Sharḥi al-Minhāj, Juz I (Beirut: Dār al-Fikr, t.th.), h. 19-20. Pengertian Qawl Qadīm dan Qawl Jadīd Istilah qawl qadīm dan qawl jadīd hanya dijumpai dalam khazanah ijtihad Imam Syafi’i. Karena beliau mencetuskan dua produk hukum yang berbeda dalam satu kasus. Pendapat Imam Syafi’i yang digagas dan difatwakan pada waktu ia masih berada di Irak (195-199 H), disebut dengan qawl qadīm, sedangkan hasil ijtihad Imam Syafi’i yang digali dan difatwakan selama ia ber- mukim di Mesir (199-204 H), dikenal dengan qawl jadīd. Kebanyakan pendapat Imam Syafi’i sewaktu menetap di Irak banyak dituliskan dalam al-Risālah al- Qadīmah dan al-Ḥujjah, yang populer dengan sebutan al-Kitāb al-Qadīm. Qawl qadīm yang tertuang dalam kedua kitab tersebut dan fatwa-fatwa Imam Syafi’i yang dimunculkan di Irak diriwayatkan oleh sejumlah murid dan sahabatnya yang berada di Irak, antara lain: Ḥasan ibn Ibrāhīm ibn Muḥammad al-Shabbāh al-Za'farānī (170-260 H), Husaīn ibn Alī al-Karābīsī (w. 240 H), Imam Aḥmad ibn Hanbal (164-241 H), Sulaimān ibn Daud al-Hāshimī (w. 220 H), dan Abū Thūr Ibrāhīm ibn Khālid Yamanī al-Kalabī (170-240 H). Sedangkan qawl jadīd yang dirumuskan Imam Syafi’i setelah beliau berdomisili di Mesir diabadikan dalam beberapa kitab, yaitu: al-Risālah al-Jadīdah, al-Umm, al-Amālī, al-Imlā' dan lain- lain. Qawl jadīd diriwayatkan oleh sejumlah murid dan sahabatnya yang bermukim di Mesir dan sekitarnya, diantaranya: Harmalah ibn Yaḥyā ‘Abdullāh al-Tujībī (166-243 H), al-Rabī' ibn Sulaimān al-Murādī (w. 270 H), 'Abdullāh ibn Zubaīr al-Ḥamīdī (w.219 H), Yūsuf ibn Yaḥyā al-Buwaiṭī (w. 231 H), Abī Ibrāhīm AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 Volume 26, Nomor 2, Oktober 2016 AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 147 Volume 26, Nomor 2, Oktober 2016 Ainol Yaqin Ismā’īl ibn Yaḥyā al-Muzannī (175-264 H), Muḥammad ibn ‘Abdullāh ibn al- Ḥakīm (182-237 H), al-Rabī' ibn Sulaimān al-Jīzī (w. 257 H), Yūnus ibn ‘Abdi al- `A’lā (170-264 H) dan Abū Bakar al-Humaidī (w. 219 H).9 12Imam al-Māwardī, al-Hāwi al-Kabīr, Juz IV, h. 226-227; Imam al-Ṭurmudhī meriwayatkan hadis tersebut melalui rentetan sanad Shadaqah ibn ‘Abdullāh, dari Mūsa ibn Yasar, dari Nāfi' dari Ibn 'Umar dengan sanad marfū'. Imam al-Ṭurmudhī, Sunan al-Ṭurmudhī, Jilid II (Beirut: Dār al-Fikr, 1994), h. 128. Imam Syafi'i menyatakan bahwa Sa'ad ibn Abī Dubāb memberitakan suatu riwayat yang menunjukkan bahwa Nabi tidak memerintah untuk mengambil zakat madu. Muḥammad ibn Idrīs al-Shāfi’ī, al-Umm, Jilid I, h. 39. Zakat Madu Imam Syafi’i memiliki dua pendapat tentang zakat madu. Beliau dalam qawl qadīm-nya berpendapat bahwa madu wajib dikeluarkan zakatnya, karena berdasarkan athār sahabat dan hadis ḍa’īf.10 Sedangkan dalam qawl jadīd ia menyatakan madu tidak wajib dikeluarkan zakatnya karena madu bukan termasuk kategori makanan pokok (qūt) dan didukung hadis ṣaḥīḥ.11 Adapun dalil-dalil ijtihad qawl qadīm adalah: a) Kaum Banī Salamah men- datangi Nabi dengan membawa sepersepuluh kurma milik mereka, kemudian Nabi menerimanya dan memberi perlindungan pada mereka; b) Diriwayatkan dari ‘Abdullāh ibn Ẓayyāb, ia berkata: Saya mendatangi Rasulullah kemudian saya memeluk Islam. Saya berkata pada Rasulullah: Ya, Rasulullah, saya me- 10Qawl qadīm masih terpilah dua pendapat, satu pendapat secara qaṭ'ī tidak mewajibkan zakat madu dan pendapat kedua mewajibkan zakat madu. Pendapat pertama ditegaskan oleh Abū Hāmid, al-Bandanijī, dan ulama' Syāfi'iyyah yang lain. Dikalangan aṣḥāb mengemukakan bahwa pendapat yang ṣaḥīḥ adalah qawl jadīd karena hadis yang dijadikan dalil qawl qadīm adalah lemah (dha'īf). Sementara itu, hadis ḍā'īf tidak dapat dijadikan sandaran pengistinbathan hukum Islam. Abū Zakariyā Muḥyiddīn ibn Sharaf al-Nawawī, al-Majmū' Sharḥ al-Muhadhdhab, Jilid V, h. 415; Muḥam- mad ibn al-Khaṭīb al-Sharbīnī, Mughnī al-Muḥtāj ilā Ma’rifati Ma’ānī `Alfāẓ al-Minhāj, Juz I, h. 566; Abdulhamīd al-Sharwanī, Hawāsyī Tuhfah al-Muḥtāj bi Sharḥi al-Minhāj, Juz III, h. 344. 11Abū Zakariyā Muḥyiddīn ibn Sharaf al-Nawawī, al-Majmū' Sharḥ al-Muhadhdhab, Jilid V, h. 452; Imam al-Māwardī, al-Hāwi al-Kabīr, Juz IV, (Beirut: Dār al-Fikr, 1994), h. 228-229; Muḥammad ibn Muḥammad al-Ghazālī, al-Wasīṭ fī al-Madhhab, Jilid II (Kairo: Dār al-Salām, 1997), h. 458; Abdulkarīm al-Rāfi’ī al-Qazwīnī, al-‘Azīz Sharḥ al-Wajīz, Juz III (Beirut: Dār al-Kutub al-‘Ilmiah, 2007), h. 53. AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 148 Volume 26, Nomor 2, Oktober 2016 Evolusi Ijtihad Imam Syafi’i …. nyarankan pada kaumku untuk menyerahkan harta-harta mereka. Setelah itu, aku menyatakan pada kaumku tentang madu, aku katakan pada mereka keluarkan zakatnya karena buah yang tidak dizakati kurang berkah. Mereka bertanya: Berapa yang wajib dikeluarkan? Aku menjawab: sepersepuluh, ke- mudian aku memungutnya dan aku berikan pada ‘Umar, lalu ‘Umar menerima dan menjadikannya sebagai zakat orang Muslim.12 Dalil-dalil yang dijadikan sandaran qawl jadīd adalah sebagai berikut: Pertama, hadis yang diriwayatkan oleh Abū Mūsa dan Mu'ād. 13Abu Bakar Aḥmad ibn al-Ḥusain al-Bayhaqī, Kitāb al-Sunan al-Ṣaghīr, Jilid I (Beirut: Dār al-Fikr, t.th.), h. 72; 'Alī ibn Umar al-Dāruquṭnī, Sunan al-Dāruquṭnī, Jilid I (Beirut: Dār al-Fikr, t.th.), h. 315; Hasan Sulaimān al-Nūri dan 'Alawī 'Abbās al-Mālikī, Ibānah al-Aḥkām Sharḥ Bulūgh al-Marām, Jld. II (Beirut: Dār al-Fikr, 2000), h. 236; Aḥmad ‘Abdurraḥmān al-Bannā, Bulūgh al-Amālī min Asrār al-Fath al- Rabbānī, Juz IX (Beirut: Dār Ihyā` al-Turāth al-‘Arabī, t.th.), h. 8; Aḥmad ibn ‘Alī ibn Hajar al-‘Asqalānī, al- Mathālib al-‘Āliyyah bi Zawā`idi al-Masānid al-Thamāniyyah, Jilid. V (Riyad: Dār al-‘Āshimah, 1998), h. 489. 15Abū Daud, Sunan Abī Daud, Jilid. II (Beirut: Dār al-Fikr, t.th.), h. 109; Imam al-Shāfi’ī dalam al- Umm secara khusus membahas bab ketidakwajiban zakat madu, Muḥammad ibn Idrīs al-Shāfi’ī, al- Umm, Juz III, h. 98-99. 16Muḥammad ibn Idrīs al-Shāfi’ī, al-Umm, Juz III, h. 98-99. 17Imam al-Māwardī, al-Hāwī al-Kabīr, Juz IV, h. 229. 18Aḥmad ibn Salamah al-Qalyūbī, Hāsyiyyah Qalyūbī, Juz IV, h. 61; Abdulhamīd al-Sharwanī, Hawāsyī Tuhfah al-Minhāj bi Sharḥ al-Minhāj, Juz I, h. 54; Muḥammad ibn al-Khaṭīb al-Sharbīnī, Zakat Madu ﻌﺔC إﻻ ﻣﻦ أرDﻻ ﺗﺄﺧﺬ اﻟﻌ َ ْ َ ٍ ْ َ ِ M ِ َ ْ ُ ْ َ ِ ُ ْ َ : ، واﺨﻞ ، واﻟﻌﻨﺐRﻨﻄﺔ ، وا ﺸﻌ7 ا ِ َ ِ ِ َ ْ َ ِ ْM ِ َ ِ َ ْ ِ ْ . 13 "Janganlah kamu mengambil sepersepuluh (sebagai zakat) melainkan pada empat buah; gandum hinthah, gandum sya'ir, kurma dan anggur." Secara ṣarīh hadis di atas menjelaskan jenis-jenis harta yang wajib di- keluarkan zakatnya sebanyak sepersepuluh, yaitu gandum sya’ir, gandum hinthah, kurma dan anggur.14 Dalam hadis tersebut Nabi tidak menyebutkan madu di antara jenis harta yang wajib dikeluarkan zakatnya. Karena itu, madu bukanlah termasuk jenis barang yang wajib dizakati. 13Abu Bakar Aḥmad ibn al-Ḥusain al-Bayhaqī, Kitāb al-Sunan al-Ṣaghīr, Jilid I (Beirut: Dār al-Fikr, t.th.), h. 72; 'Alī ibn Umar al-Dāruquṭnī, Sunan al-Dāruquṭnī, Jilid I (Beirut: Dār al-Fikr, t.th.), h. 315; Hasan Sulaimān al-Nūri dan 'Alawī 'Abbās al-Mālikī, Ibānah al-Aḥkām Sharḥ Bulūgh al-Marām, Jld. II (Beirut: Dār al-Fikr, 2000), h. 236; Aḥmad ‘Abdurraḥmān al-Bannā, Bulūgh al-Amālī min Asrār al-Fath al- Rabbānī, Juz IX (Beirut: Dār Ihyā` al-Turāth al-‘Arabī, t.th.), h. 8; Aḥmad ibn ‘Alī ibn Hajar al-‘Asqalānī, al- Mathālib al-‘Āliyyah bi Zawā`idi al-Masānid al-Thamāniyyah, Jilid. V (Riyad: Dār al-‘Āshimah, 1998), h. 489. 14al-Ḥasan, Ibn Abī Lailā, al-Thaurī dan ulama’ lainnya menegaskan bahwa kewajiban zakat hanya tertuju pada empat jenis buah, yaitu gandum sya’īr, gandum hinthah, kurma dan anggur. Ketetapan ini merupakan ijma’ ulama, sekalipun pada rinciannya ada perselisihan. Mūsa ibn ‘Iyāḍ, Ikmāl al-Mu’lim bi Fawāidi Muslim, Juz III (Beirut: Dār al-Wafā’, 1998), h. 468; Muḥammad ibn Khalfah al-Wasytānī, Ikmāl Akmāl al-Mu’lim, Juz III (Beirut: Dār al-Kutub al-‘Ilmiah, t.th.), h. 112; Aḥmad ‘Abdurraḥmān al-Bannā, Bulūgh al-Āmalī min Asrār al-Fath al-Rabbānī, Juz IX, h. 9; Aḥmad ibn ‘Umar al-Qurṭubī, al-Mufhim limā Asykala min Talkhīsh Kitāb Muslim, Juz III (Beirut: Dār Ibn Kathīr, 1996), h. 13. AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 Volume 26, Nomor 2, Oktober 2016 149 Volume 26, Nomor 2, Oktober 2016 Ainol Yaqin Kedua, hadis yang diriwayatkan ‘Umar ibn Shu’ib dari ayahnya dari kakek- nya: Kedua, hadis yang diriwayatkan ‘Umar ibn Shu’ib dari ayahnya dari kakek- nya: “Bahwa suatu kaum mendatangi Nabi dengan membawa sepersepuluh kurma, mereka meminta perlindungan pada Nabi. Kemudian, Nabi menyetujui per- mintaan mereka. Pada periode ‘Umar menjabat sebagai khalifah, Sufyan ibn Wahab melayangkan surat yang berisi tentang kasus tersebut. Zakat Madu ‘Umar mem- balasnya dengan ungkapan: jika mereka melakukan padamu sama dengan apa yang mereka perbuat pada Nabi, lindungilah mereka. Akan tetapi, jika mereka tidak melakukannya, tetap lindungi mereka sebab hal itu bukan suatu ke- wajiban.”15 “Bahwa suatu kaum mendatangi Nabi dengan membawa sepersepuluh kurma, mereka meminta perlindungan pada Nabi. Kemudian, Nabi menyetujui per- mintaan mereka. Pada periode ‘Umar menjabat sebagai khalifah, Sufyan ibn Wahab melayangkan surat yang berisi tentang kasus tersebut. ‘Umar mem- balasnya dengan ungkapan: jika mereka melakukan padamu sama dengan apa yang mereka perbuat pada Nabi, lindungilah mereka. Akan tetapi, jika mereka tidak melakukannya, tetap lindungi mereka sebab hal itu bukan suatu ke- wajiban.”15 Ketiga, dari ‘Abdullāh ibn Abū Bakar ia berkata: ‘Umar ibn ‘Abd al-‘Azīz menulis surat pada bapaknya yang sedang berada di Mina. Ia mengingatkan untuk tidak memungut zakat kuda dan madu.16 Al-Māwardī menegaskan jika mengeluarkan zakat madu dipandang wajib oleh syara', mesti sahabat ‘Umar selaku khalifah memerintahkan pada Sufyān ibn Wahab untuk memungut zakat madu dari mereka, sekalipun tidak mem- balas dengan memberi perlindungan pada mereka. Disamping itu, ungkapan ‘Umar "itu hanyalah hujan rizki yang dapat dimakan siapapun yang menyukai- nya" menunjukkan bahwa madu tidak terkena kewajiban beban zakat.17 Jika ditelaah dalil-dalil yang dijadikan dasar ijtihad qawl qadīm dan qawl jadīd, maka kita dapat memahami bahwa berpindahnya Imam Syafi’i dari qawl qadīm pada qawl jadīd lantaran ia menemukan sandaran dalil yang lebih kuat untuk dijadikan pijakan hukum. Dalam qawl qadīm beliau merumuskan hukum berdasarkan athār sahabat, sedangkan dalam qawl jadīd ia mem- bangun nalar ijtihadnya pada hadis Nabi. Oleh karena itu, Imam Syafi’i ber- ijtihad kembali dan membongkar hasil ijtihad sebelumnya yang dipandang berlandaskan pada dalil yang lemah. Ia berkata "Hadis ṣaḥīḥ adalah mazhabku dan tinggalkanlah pendapatku jika berlawanan dengan hadis ṣaḥīḥ".18 Sejalan AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 150 Volume 26, Nomor 2, Oktober 2016 Evolusi Ijtihad Imam Syafi’i …. dengan prinsip ijtihadnya, Imam Syafi’i menetapkan hukum terlebih dahulu bertumpu pada al-Qur’an dan hadis yang telah disepakati. Menurutnya, hukum juga dapat ditetapkan berdasarkan hadis yang diriwayatkan secara aḥad (dari seorang perawi kepada seorang perawi berikutnya). 19Muḥammad ibn Idrīs al-Shāfi’ī, al-Risālah, (Beirut: Dār al-Fikr, 1309 H), h. 599-600. 20Abū Zakariyā Muḥyiddīn ibn Sharaf al-Nawawī, al-Majmū' Sharḥ al-Muhadhdhab, Jilid V, h. 413. 21Imam al-Ṭurmudhī, Sunan al-Ṭurmudhī, Jilid II, h. 128. 22Abū Zakariyā Muḥyiddīn ibn Sharaf al-Nawawī, al-Majmū' Sharḥ al-Muhadhdhab, Jilid V, h. 455. 23HR. Muslim. Muḥammad ibn Idrīs al-Shāfi’ī, al-Umm, Jilid I, h. 39. Zakat Madu Jika al-Qur’an dan hadis tidak terdapat keterangan tentang kasus tersebut, maka dapat berdalil dengan ijma', kemudian alternatif terakhir berdalil pada qiyās.19 Hadis yang memberitakan tentang Bani Salamah yang memuat zakat madu diriwayatkan Abū Daud, Bayhaqī dan lainnya dari ‘Amr ibn Shu’ib, dari bapaknya dari kakeknya dengan jalur sanad yang ḍa'īf.20 Imam al-Ṭurmudhī mengomentari bahwa hadis tersebut tidak layak disandarkan pada Nabi. Hal ini, sejalan dengan pendirian mayoritas ulama.21 Demikian pula, Imam al- Bukhārī mengemukakan tidak terdapat dalil ṣaḥīḥ yang mewajibkan zakat madu. Dengan demikian, hadis dan athār di atas merupakan hadis yang lemah yang tidak bisa dijadikan sebagai pijakan dalil pengistinbathan hukum Islam. Menurut al-Nawawī dan mayoritas ulama bahwa pendapat yang ṣaḥīḥ adalah qawl jadīd, sebab sandaran dalil qawl qadīm tergolong lemah dan tidak terdapat dalil yang berkonotasi wajibnya zakat madu.22 Sementara itu, Imam Syafi’i menegaskan dalam karyanya, al-Umm bahwa madu dan kuda tidak wajib dikeluarkan zakatnya.23 Mughnī al-Muḥtāj ilā Ma’rifati Alfāẓ al-Minhāj, Juz I, h. 38; Sulaimān ibn Muḥammad al-Bujairamī, Bujairamī ‘alā al-Khaṭīb, Juz I, h. 77; Muḥammad ibn Abī ‘Abbās al-Ramlī, Nihāyah al-Muḥtāj Ilā Sharḥ al-Minhāj, Juz I, (Beirut: Dar al-Fikr, t.th), h. 20, Dengan statemen berbeda, tapi subtansi yang sama beliau menyatakan jika kamu menemukan dalam kitabku menyalahi sunnah Rasulullah, ber- peganggah pada sunnah Rasulullah dan tinggalkanlah pendapatku. Abū Zakariyā Muḥyiddīn ibn Sharaf al-Nawawī, al-Majmū' Sharḥ al-Muhadhdhab, Jilid I, h. 104; Muḥammad ibn Muḥammad al- Ghazālī, al-Wasīth fī al-Madhhab, Jilid I, h. 88. Hukum Mengqada' Puasa Orang yang tidak berpuasa di bulan Ramadan lantaran uzur syar'i dan ia tidak sempat mengqada'nya hingga ajal merenggut kehidupannya, maka gugurlah ║ AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 151 Volume 26, Nomor 2, Oktober 2016 p-ISSN: 0854-4603; e-ISSN: 2502-3209 Ainol Yaqin kewajiban puasa baginya. Akan tetapi, seseorang yang mempunyai tanggungan puasa karena ada uzur ataupun tidak dan ada kesempatan untuk mengqada'nya, namun ia melalaikan kesempatan itu hingga ajal merenggut sebelum sempat mengqada' puasa, maka wajib baginya mengqada' puasa. Tentang kewajiban mengqada' puasa dalam persoalaan ini terdapat dua qawl, menurut jumhūr dan qawl qadīm wajib diganti satu mud dalam per-hari yang diambil dari harta tirkah- nya, dan tidak sah bagi wali untuk mengganti puasanya. Sedangkan menurut qawl jadīd, merupakan pendapat yang mukhtār (dipilih) bahwa wali boleh berpuasa untuk mengqada' puasa yang ditinggalkan mayit atau memberi makan sebanyak satu mud pada orang miskin.24 Adapun dalil-dalil yang dijadikan rujukan qawl qadīm adalah sebagai berikut: pertama, hadis yang diriwayatkan 'Āisyah: ﻪVﻨﻪ وW ﻣﻦ ﻣﺎت وﻋﻠﻴﻪ ﺻﻮم ﺻﺎم ُ ُZ ِ َ َ ْ َ َ َ َ َ ِ ْ َ َ َ َْ 25 . "Barang siapa yang mati dan ia meninggalkan tanggungan puasa maka walinya boleh menggantinya". Kedua, hadis yang diriwayatkan Sa'ad ibn Abī Waqqash: ﺻ` اﷲ ﻋﻠﻴﻪ وﺳﻠﻢ ﻓﻘﺎل ﻳﺎرﺳﻮل اﷲ ان ا] ﻣﺎﺗﺖ وﻋﻠﻴﻬﺎ ﺻﻮمa اbﺟﺎء رﺟﻞ إ 24Abū Zakariyā Muḥyiddīn ibn Sharaf al-Nawawī, al-Majmū' Sharḥ al-Muhadhdhab, Jilid VI, h. 369; Sālim al-‘Imrānī, al-Bayān fī Madhhab al-Imām al-Shāfi’ī, Jilid III, h. 546; ‘Abdullāh ibn Yūsuf al-Juwainī, Nihāyah al-Maṭlab fī al-Dirāyah al-Madhhab, Juz IV, h. 61-62; Muḥammad ibn Idrīs al-Shāfi’ī, al-Umm, Jilid III, h. 262; Aḥmad ibn Salamah al-Qalyūbi, Khāsyiyyah Qalyūbi, Juz II, h. 66-67; Muḥyiddīn ibn Sharf al-Nawawī, al-Tanqīh fī Sharḥ al-Wasīth, Jilid II (Kairo: Dār al-Salam, 1997), h. 551-553; Abdulhamīd al- Sharwanī, Tuhfah al-Minhāj bi Sharḥ al-Minhāj, Juz II, h. 435-436; Yaḥyā ibn Sharaf al-Nawawī, Tashhīh al-Tanbīh, Juz I (Beirut: Muassasah al-Risālah, 1996), h. 241; Muḥammad ibn Muḥammad al-Ghazālī, al- Wajīz fī Fiqh al-Imām al-Shāfi’ī, Juz I (Beirut: Dār al-Arqam, 1997), h. 226; Yūsuf al-Fairūz Abādī al- Sharbīnī, al-Muhadhdhab fī Fiqh al-Imām al-Shāfi’ī, Juz I (Beirut: Dār al-Kutub al-‘Ilmiah, 1995), h. 343- 345; Sulaimān al-Bujairamī, al-Bujairamī ‘alā al-Khaṭīb, Juz III, h. 138-139; Muḥammad ibn al-Khaṭīb al- Syarbinī, Mughnī al-Muḥtāj ilā Ma’rifati Ma’ānī Alfāẓ al-Minhāj, Juz I, h. 642. 25Abū al-Ḥusain Muslim al-Naisabūrī, Ṣaḥīḥ Muslim, Jilid I (Beirut: Dār al-Fikr, 1988), h. ﺻ` اﷲ ﻋﻠﻴﻪ وﺳﻠﻢ ﻓﻘﺎل ﻳﺎرﺳﻮل اﷲ ان ا] ﻣﺎﺗﺖ وﻋﻠﻴﻬﺎ ﺻﻮمa اbﺟﺎء رﺟﻞ إ Hukum Mengqada' Puasa 510; Jalāluddīn ‘Abdurraḥmān al-Suyūṭī, al-Tausyīh Sharḥ al-Jāmi’ al-Ṣaḥīḥ, Juz IV (Riyad: Maktabah al- Rusyd, 1998), h. 1450, 'Alī ibn Umar al-Dāruquṭnī, Sunan al-Dāruquṭnī, Jilid I, h. 156; Muḥammad ibn Khalfah al-Wasytanī, Ikmāl Akmāl al-Ma`lam, Juz III, h. 262; Abū Daud Sulaimān al-Sajastanī, Sunan Abi daud, Jild. I, 315; Aḥmad ibn ‘Alī ibn Hajar al-‘Asqalānī, Taghlīq al-Ta’līq ‘Alā Ṣaḥīḥ al-Bukhāri, Jilid III (Beirut: Dār ‘Imār, 1985), h. 189-190, Aḥmad ‘Abdurraḥmān al-Bannā, Bulugh al-Amālī min Asrār al-Fath al-Rabbānī, Juz X, h. 135; Aḥmad ibn ‘Umar al-Qurṭubī, al-Mufhim limā Asykala min Talkhīsh Kitāb Muslim, Juz III, h. 208. AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 152 Volume 26, Nomor 2, Oktober 2016 Evolusi Ijtihad Imam Syafi’i …. Evolusi Ijtihad Imam Syafi’i …. ﻨﺖ ﻗﺎﺿﻴﻪ ﻋﻨﻬﺎ ﻗﺎل ﻧﻌﻢ ﻗﺎل ﻓﺪﻳﻦdﻚ دﻳﻦ أg اh نi ﺷﻬﺮ أﻓﺄﻗﻀﻴﻪ ﻋﻨﻬﺎ ﻓﻘﺎل ﻮ lاﷲ اﺣﻖ أن ﻳﻘ . 26 ﻨﺖ ﻗﺎﺿﻴﻪ ﻋﻨﻬﺎ ﻗﺎل ﻧﻌﻢ ﻗﺎل ﻓﺪﻳﻦdﻚ دﻳﻦ أg اh نi ﺷﻬﺮ أﻓﺄﻗﻀﻴﻪ ﻋﻨﻬﺎ ﻓﻘﺎل ﻮ lاﷲ اﺣﻖ أن ﻳﻘ . 26 "Seseorang mendatangi Nabi kemudian ia bertanya, wahai Rasulullah se- sungguhnya ibuku telah meninggal dan mempunyai tanggungan puasa sebulan, apakah saya boleh mengqada'nya. Nabi menjawab seandainya ibumu mempunyai hutang apakah kamu akan membayarnya, ia menjawab, ya. Kemudian Rasulullah bersabda hutang pada Allah lebih berhak untuk dibayar." Ketiga, hadis yang diriwayatkan Ibnu 'Abbās: Ketiga, hadis yang diriwayatkan Ibnu 'Abbās: رﺳﻮل اﷲbﺮأة إgﺟﺎءت ا ﺻ` اﷲ ﻋﻠﻴﻪ وﺳﻠﻢ ﻓﻘﺎﻟﺖ ﻳﺎرﺳﻮل اﷲ ان أ] ﻣﺎﺗﺖ ن ﻳﺆدى iﻴﻪ اqﻚ دﻳﻦ ﻓﻘﻀﻴg اr نi وﻋﻠﻴﻬﺎ ﺻﻮم ﻧﺬر أﻓﺄﺻﻮم ﻋﻨﻬﺎ ﻗﺎل اﻓﺮأﻳﺖ ﻮ ﻚ gذ ﻚ ﻋﻨﻬﺎ ﻗﺎﻟﺖ ﻧﻌﻢ ﻗﺎل ﻓﺼﻮ] ﻋﻦ ا . 27 "Seorang perempuan mendatangi Rasulullah kemudian bertanya wahai Rasulullah sesungguhnya ibuku telah mati dan ia meninggalkan tanggungan puasa nadzar, apakah saya boleh menggantinya, Rasulullah menjawab bagai- mana pendapatmu seandainya ibumu mempunyai hutang apakah kamu akan melunasinya, ia menjawab ia. Kemudian Rasullullah bersabda berpuasalah untuk mengganti puasa ibumu." Keempat, hadis yang diriwayatkan Barīdah: Keempat, hadis yang diriwayatkan Barīdah: aﺑﻨﺎ اﻧﺎ ﺟﺎ ﺲ ﻋﻨﺪ ا ﺻ` vﺮأة ﻓﻘﺎﻟﺖ ﻳﺎرﺳﻮل اﷲ إg اﷲ ﻋﻠﻴﻪ وﺳﻠﻢ إذ أﺗﺘﻪ ا ﺎرﺔ واﻧﻬﺎ ﻣﺎﺗﺖx ] أh ﺗﺼﺪﻗﺖ . اث ﻗﺎﻟﺖ Rzﻓﻘﺎل وﺟﺐ أﺟﺮك وردﻫﺎ ﻋﻠﻴﻚ ا ن ﻋﻠﻴﻬﺎ ﺻﻮم ﺷﻬﺮ أﻓﺄﺻﻮم ﻋﻨﻬﺎ ﻗﺎل ﺻﻮ] ﻋﻨﻬﺎi ﻳﺎرﺳﻮل اﷲ اﻧﻪ . ﻗﺎﻟﺖ اﻧﻬﺎ ﻢ }ﺞ 26HR. al-Bukhārī dan Muslim. Abu al-Ḥusain Muslim al-Naisaburī, Ṣaḥīḥ Muslim, Jilid I, h. 510; Jalālud- dīn ‘Abdurraḥmān al-Suyūṭī, al-Tausyīh Sharḥ al-Jāmi’ al-Ṣaḥīḥ, Juz IV, h. Hukum Mengqada' Puasa 1450-1451; Abū Daud Sulaimān al-Sajastanī, Sunan Abi daud, Jilid II, h. 237; Muḥammad ibn Khalfah al-Washtanī, Ikmāl Akmāl al-Ma`lam, Juz III, h. 262-273; Aḥmad ‘Abdurraḥmān al-Bannā, Bulūgh al-Amālī min Asrār al-Fath al-Rabbānī, Juz X, h. 136; Aḥmad ibn ‘Umar al-Qurṭubī, al-Mufhim limā Asykala min Talkhīsh Kitāb Muslim, Juz III, h. 210; Aḥmad ibn ‘Alī ibn Hajar al-‘Asqalānī, Taghlīq al-Ta’līq ‘alā Ṣaḥīḥ al-Bukhārī, Jilid III, h. 93. 27HR. al-Bukhārī dan Muslim. Jalāluddīn al-Suyūṭī, Sunan al-Nasā`ī, Jilid IV (Beirut: Dār al-Fikr, 1930), h. 20-21; Jalāluddīn ‘Abdurraḥmān al-Suyūṭī, al-Tausyīh Sharḥ al-Jāmi’ al-Ṣaḥīḥ, Juz IV, h. 1451; Aḥmad ibn ‘Umar al-Qurṭubī, al-Mufhim limā Asykala min Talkhīsh Kitāb Muslim, Juz III, h. 210; Aḥmad ibn ‘Alī ibn Hajar al-‘Asqalānī, Taghlīq al-Ta’līq ‘alā Ṣaḥīḥ al-Bukhārī, Jilid. III, h. 194. AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 153 Volume 26, Nomor 2, Oktober 2016 Ainol Yaqin ~ﻗﻂ أﻓﺎﺣﺞ ﻋﻨﻬﺎ ﻗﺎل ﺣ .ﻋﻨﻬﺎ28 "Ketika saya duduk di samping Rasulullah tiba-tiba seorang perempuan meng- hampiri Rasulullah kemudian berkata wahai Rasulullah sesungguhnya aku ber- sedekah atas nama ibuku pada seorang budak perempuan. Namun ibuku telah wafat, Nabi menjawab, kamu harus membayarnya dan menyerahkan warisan yang menjadi hakmu. Ia bertanya, wahai Rasulullah sesungguhnya ibuku mem- punyai tanggungan puasa satu bulan, apakah saya boleh menggantinya, Rasulullah menjawab, berpuasalah, ia bertanya: ibuku belum pernah berhaji, apakah saya boleh berhaji atas nama ibuku, Rasulullah menjawab: berhajilah ." Kelima, hadis yang diriwayatkan Ibnu Abbās: Kelima, hadis yang diriwayatkan Ibnu Abbās: aﺒﻞ أن ﺗﺼﻮم ﻓﺄﺗﺖ أﺧﺘﻬﺎ ا€ ﺮأة ا‚ﺤﺮ •ﻨﺬرت أن ﺗﺼﻮم ﺷﻬﺮا •ﻤﺎﺗﺖgﺒﺖ اƒرM ِ M َ ُ ْ ْ ْ ُ ْ ْ ْ َ ْ َ َ َ ْ َ ٌ ْ َ َ َ َ َ َ َ َ َ َ ُ ُ َ َ َ َ ً َ َ َ ْ َ َ ْ َ ْ ِ ﻨﻬﺎW ﺮﻫﺎ أن ﺗﺼﻮمgﺻ` ا‡ ﻋﻠﻴﻪ وﺳﻠﻢ وذﻛﺮت ذ ﻚ … ﻓﺄ َ َ َ َ ْ َ َ َ َ ْ َ ُ َ َ َ َ َ َ َ ُ َ َ َ ِ َ ْ َ َ َM ِ ْ ُ M M . 29 "Seorang perempuan mengarungi lautan kemudian ia bernadzar untuk ber- puasa selama satu bulan. Allah mengabulkan nadzarnya, ia tidak sempat berpuasa hingga ajal merenggut. Lalu saudarinya mendatangi Rasulullah dan beliau memerintahkan agar berpuasa untuk menggantinya.". Keenam, ibadah puasa apabila batal dapat diganti dengan membayar kaffarat. 28HR. Muslim. Muḥammad ibn Khalfah al-Wasytanī, Ikmāl Akmāl al-Ma`lam, Juz III, h. 263; Aḥmad ‘Abdurraḥmān al-Bannā, Bulūgh al-Amālī min Asrār al-Fath al-Rabbānī, Juz X, h. 137; Aḥmad ibn ‘Umar al-Qurṭubī, al-Mufhim limā Asykala min Talkhīsh Kitāb Muslim, Juz III, h. 210-211. 28HR. Muslim. Muḥammad ibn Khalfah al-Wasytanī, Ikmāl Akmāl al-Ma`lam, Juz III, h. 263; Aḥmad ‘Abdurraḥmān al-Bannā, Bulūgh al-Amālī min Asrār al-Fath al-Rabbānī, Juz X, h. 137; Aḥmad ibn ‘Umar al-Qurṭubī, al-Mufhim limā Asykala min Talkhīsh Kitāb Muslim, Juz III, h. 210-211. 29Abū Daud Sulaimān al-Sajastānī, Sunan Abī Daud, Jilid II, h. 237. 30HR. Abū Daud dan al-Ṭurmudhī. Muḥammad ibn Yazīd al-Qazwīnī, Sunan Ibn Mājah, Jilid I, (Beirut: Dār al-Fikr, t.th.), h. 558; Aḥmad ‘Abdurraḥmān al-Bannā, Bulūgh al-Amālī min Asrār al-Fath al-Rabbānī, Juz X, h. 137; Abu Bakar Aḥmad ibn al-Ḥusain al-Bayhaqī, Kitāb al-Sunan al-Ṣaghīr, Jilid I, h. 357, Muḥammad ibn `Isa al-Ṭurmudhī, Sunan al-Ṭurmudhī, Juz II, h. 110; ‘Iyāḍ ibn Mūsā ibn ‘Iyāḍ al- Bahshī, Ikmāl al-Mu’lim bi Fawāidi Muslim, Juz IV, h. 104. j 30HR. Abū Daud dan al-Ṭurmudhī. Muḥammad ibn Yazīd al-Qazwīnī, Sunan Ibn Mājah, Jilid I, (Beirut: Dār al-Fikr, t.th.), h. 558; Aḥmad ‘Abdurraḥmān al-Bannā, Bulūgh al-Amālī min Asrār al-Fath al-Rabbānī, Juz X, h. 137; Abu Bakar Aḥmad ibn al-Ḥusain al-Bayhaqī, Kitāb al-Sunan al-Ṣaghīr, Jilid I, h. 357, Muḥammad ibn `Isa al-Ṭurmudhī, Sunan al-Ṭurmudhī, Juz II, h. 110; ‘Iyāḍ ibn Mūsā ibn ‘Iyāḍ al- Bahshī, Ikmāl al-Mu’lim bi Fawāidi Muslim, Juz IV, h. 104. Hukum Mengqada' Puasa Karena itu, ibadah puasa dapat menerima perwakilan karena uzur syara’, sama halnya dengan ibadah haji. Adapun qawl jadīd diperkuat oleh dalil-dalil sebagai berikut: Pertama, hadis dari Ibnu ‘Umar bahwa Rasulullah bersabda: ﺴﻜﻴﻨﺎg ﻦ ‹ ﻳﻮمW ﻀﺎن ﻓﻠﻴﻄﻌﻢgﻣﻦ ﻣﺎت وﻋﻠﻴﻪ ﺻﻮم ر ً َ ْ ِ ِ ْ ْ َ ْ ٍ ِ ْ ْ ْ ْŒ ُ َ َ ِ ُ ُ ْ َ َ َ َ َ َ َ َ َ َ . 30 "Barang siapa yang mati dengan meninggalkan tanggungan puasa Ramadan, hendaklah memberi makan pada orang miskin dalam setiap hari (puasa yang ditinggalkan)". 28HR. Muslim. Muḥammad ibn Khalfah al-Wasytanī, Ikmāl Akmāl al-Ma`lam, Juz III, h. 263; Aḥmad ‘Abdurraḥmān al-Bannā, Bulūgh al-Amālī min Asrār al-Fath al-Rabbānī, Juz X, h. 137; Aḥmad ibn ‘Umar al-Qurṭubī, al-Mufhim limā Asykala min Talkhīsh Kitāb Muslim, Juz III, h. 210-211. 30HR. Abū Daud dan al-Ṭurmudhī. Muḥammad ibn Yazīd al-Qazwīnī, Sunan Ibn Mājah, Jilid I, (Beirut: Dār al-Fikr, t.th.), h. 558; Aḥmad ‘Abdurraḥmān al-Bannā, Bulūgh al-Amālī min Asrār al-Fath al-Rabbānī, Juz X, h. 137; Abu Bakar Aḥmad ibn al-Ḥusain al-Bayhaqī, Kitāb al-Sunan al-Ṣaghīr, Jilid I, h. 357, Muḥammad ibn `Isa al-Ṭurmudhī, Sunan al-Ṭurmudhī, Juz II, h. 110; ‘Iyāḍ ibn Mūsā ibn ‘Iyāḍ al- Bahshī, Ikmāl al-Mu’lim bi Fawāidi Muslim, Juz IV, h. 104. AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 154 Volume 26, Nomor 2, Oktober 2016 Evolusi Ijtihad Imam Syafi’i …. Hadis di atas secara tegas memerintahkan membayar kaffarat dan meng- gugurkan kewajiban qada'. Menurut al-Turmudhī hadis yang diriwayatkan Ibnu ‘Umar tersebut tergolong hadis marfū' dan hanya dalam persoalan ini Ibnu ‘Umar meriwayatkan hadis secara marfū'. Menurut pendapat yang ṣaḥīḥ hadis tersebut termasuk hadis mawqūf.31 Kedua, Nāfi' meriwayatkan dari Ibnu ‘Umar, Kedua, Nāfi' meriwayatkan dari Ibnu ‘Umar, ﻘŽ ﻀﺎن ﺑﻤﺮض ، و ﻢgﻣﻦ أ•ﻄﺮ • ر ْ َ ْ ْ َ َ َ َ َ َ َ ِ َ ٍ َ َ َ ِ َ ْ ﻦW ﺾ ﺣ ﻣﺎت ، أﻃﻌﻢ َ ْ َ َ ْ َ َ َ َM ِ ‹ ﻳﻮم ﻣﺪﻳﻦ ِ ْM ُ ٍ ْ َ Œ ُ . 31Muḥammad ibn `Īsa al-Ṭurmudhī, Sunan al-Ṭurmudhī, Juz II, h. 110. 32Imam al-Māwardī, al-Hāwī al-Kabīr, Juz III, h. 313-315. 32Imam al-Māwardī, al-Hāwī al-Kabīr, Juz III, h. 313-315. 33Ibid. 31Muḥammad ibn `Īsa al-Ṭurmudhī, Sunan al-Ṭurmudhī, Juz II, h. 110. 32Imam al-Māwardī, al-Hāwī al-Kabīr, Juz III, h. 313-315. 33Ibid. Hukum Mengqada' Puasa 32 "Barang siapa tidak berpuasa di bulan Ramadan lantaran sakit dan tidak sempat mengqada' hingga ajal merenggutnya, maka harus memberi makan dua mud dalam per-harinya." "Barang siapa tidak berpuasa di bulan Ramadan lantaran sakit dan tidak sempat mengqada' hingga ajal merenggutnya, maka harus memberi makan dua mud dalam per-harinya." Sanksi satu mud untuk mengganti puasa yang ditinggalkan dan satu mud untuk menebus kelalaiannya. Hal ini berdasarkan ijma' ulama: Pertama, diriwayatkan dari Ibnu Abbās, ‘Umar, ‘Āisyah, mereka berkata: “Barang siapa yang mati dan meninggalkan tanggungan puasa maka harus di- keluarkan kaffarat dari harta tirkahnya, dan tidak boleh pada orang lain untuk menggantinya." Ketetapan ini tidak diperselisihkan oleh seorang pun di kalangan sahabat, maka dapat dikatakan ijma'. Kedua, karena puasa termasuk ibadah yang tidak boleh diwakilkan pada orang lain di waktu hidupnya seseorang dengan alasan apapun. Sebab itu, dengan adanya kematian puasa tidak dapat diwakilkan pada orang lain.33 Imam al-Māwardī mengkritisi dalil-dalil yang dibuat landasan qawl qadīm, yaitu: pertama, petunjuk hadis-hadis yang dijadikan dasar qawl qadīm terarah pada mengganti puasa dengan membayar kaffarat, yaitu memberi makan fakir miskin. Karena hadis-hadis tersebut bertentangan dengan petunjuk hukum hadis lain. Kedua, menganalogikan puasa pada haji tidaklah benar karena hal ini berbeda antara haji dengan puasa, dimana dalam hal ihwal haji dapat digantikan AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 ║1 AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 ║155 ║155 Volume 26, Nomor 2, Oktober 2016 Ainol Yaqin pada orang lain karena lemah. Orang yang tidak mampu berpuasa beban ke- wajiban beralih pada membayar kaffarat, yaitu memberi makan fakir miskin.34 Tampaknya, penilaian ulama Syafi’iyyah berbeda pandangan dalam mem- pertimbangkan kehujjahan hadis-hadis yang dibuat rujukan mazhab Syafi’i. Ada yang menilai dalil-dalil yang dijadikan sandaran qawl qadīm lebih kuat, dan ada pula yang memandang dalil-dalil yang dijadikan pijakan qawl jadīd yang lebih ṣaḥīḥ dan lebih rājih. Imam al-Bayhaqī sebagai ahli hadis memandang dalil-dalil yang dibuat dasar qawl qadīm lebih kuat karena diriwayatkan dari berbagai sanad dengan perawi yang berbeda. Karena itu, ia mempertegas pendapatnya tentang kebolehan wali mengganti puasa yang ditinggalkan mayit, baik puasa ramadhan, nadzar maupun yang lainnya, karena didukung oleh beberapa hadis ṣaḥīḥ dan tidak ditemukan dalil yang bertolak belakang dengan hadis-hadis tersebut. Jadi pendapat ini layak disandarkan pada mazhab Syafi’i, sebab ia sendiri mengungkapkan "Apabila hadis itu ṣaḥīḥ itulah mazhabku maka tinggal- kanlah pendapatku yang bertentangan dengannya."35 Lebih lanjut, al-Bayhaqī mengkritik bahwa hadis yang diriwayatkan Ibnu ‘Umar termasuk hadis mawqūf bukan hadis marfū'. 34Ibid., Juz III, h. 314-315. 35Abū Zakariyā Muḥyiddīn ibn Sharaf al-Nawawī, al-Majmū' Sharḥ al-Muhadhdhab, Jilid I, h. 104; Muḥammad ibn Muḥammad al-Ghazālī, al-Wasīth fī al-Madhhab, Jilid I, h. 88. 36Abū Zakariyā Muḥyiddīn ibn Sharaf al-Nawawī, al-Majmū' Sharḥ al-Muhaddab, Jilid VI, h. 371. 38Muḥammad 'Ajjāj al-Khaṭīb, Uṣūl al-Ḥadīth (Beirut: Dār al-Fikr, 1989), h. 313; disebutkan: Hukum Mengqada' Puasa Hadis tersebut merupakan ungkapan Ibnu ‘Umar yang dimarfu'kan oleh Muḥammad ibn Abdurrahman dari Nāfi' dari Ibnu ‘Umar bahwa Rasulullah memutuskan tentang perkara orang yang meninggal dan mempunyai tanggungan puasa bahwa "ia harus memberi makan separuh sha' gandum dalam perhari". Terdapat dua kesalahan –menurut al-Bayhaqī– pada hadis di atas, yaitu pertama, hadis tersebut adalah hadis mawqūf tapi di- marfu'kan, dan kedua, Ibnu ‘Umar menyatakan satu mud gandum bukan separuh mud.36 Imam al-Bayhaqī menyangkal anggapan sebagian ulama Syafi’iyyah yang mengklaim bahwa hadis Ibnu Abbās dan 'Āisyah dinilai hadis ḍa'īf. Sebab para pakar hadis tetap menṣaḥīḥkan sebuah hadis yang diriwayatkan bertentangan dengan amaliah perawi, dengan cacatan perawi tersebut tergolong orang alim. Sementara itu, Imam al-Māwardī menilai bahwa pendapat qawl jadīd lebih kuat AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 156 Volume 26, Nomor 2, Oktober 2016 Evolusi Ijtihad Imam Syafi’i …. adalah karena ditopang beberapa dalil yang kuat. Tampaknya, pembelaan al- Māwardī atas qawl jadīd karena ia memandang kualitas dalil yang dijadikan rujukan ijtihad. Menurutnya, dalil-dalil yang digunakan Imam Syafi’i untuk mem- bangun qawl qadīm tergolong hadis-hadis ḍa’īf. Karena Imam al-Māwardī belum melihat faktor-faktor yang mendukung keshahīhan dalil-dalil qawl qadīm maka ia tetap menganggap bahwa qawl jadīd lebih kuat dan layak untuk diamalkan.37 Alasan peralihan Imam Syafi’i dari qawl qadīm pada qawl jadīd adalah karena didorong penemuan hadis-hadis yang lebih kuat dan lebih ṣaḥīḥ dari- pada dalil yang dijadikan rujukan qawl qadīm. Perbedaan temuan dalil dan ke- shahihan cukup mewarnai putusan hukum. Hadis-hadis yang dijadikan dalil pendukung qawl qadīm diriwayatkan oleh Imam al-Bukhārī, Imam Muslim dan Imam Abū Daud. Kalangan ulama hadis sepakat bahwa hadis yang di- kodifikasikan dalam ṣaḥīḥ Bukhārī-Muslim tergolong hadis ṣaḥīḥ. Berdasarkan penelitian para ulama sebuah hadis dianggap ṣaḥīḥ oleh Imam al-Bukhārī bilamana rentetan sanadnya benar-benar bersambung yang dapat diketahui dengan pertemuan langsung antara guru dan murid atau tolok ukur minimal diketahui guru dan murid hidup semasa.38 Secara umum, hadis-hadis yang terangkum dalam ṣaḥīḥ Muslim berkualitas ṣaḥīḥ, atau dinilai ṣaḥīḥ oleh mayoritas ulama. Bukan dalam pengertian semua hadis yang terdapat dalam kitab ini berkualitas ṣaḥīḥ, dan bukan berarti hadis-hadis di luar kitab ini tidak ṣaḥīḥ. Imam Muslim menyatakan bahwa ia tidak memasukkan hadis yang hanya dianggap ṣaḥīḥ menurut penilainnya dalam kitab ini, melainkan hadis- hadis yang telah disepakati ulama hadis.39 38Muḥammad 'Ajjāj al-Khaṭīb, Uṣūl al-Ḥadīth (Beirut: Dār al-Fikr, 1989), h. 37Imam al-Māwardī, al-Hāwī al-Kabīr, Juz III, h. 313. 37Imam al-Māwardī, al-Hāwī al-Kabīr, Juz III, h. 313. 40Muḥammad 'Ajjāj al-Khaṭīb, Uṣūl al-Ḥadīth, h. 326. 41Ibid., h. 326-327. 42Muḥammad ibn Idrīs al-Shāfi’ī, al-Risālah, h. 127-128. Hukum Mengqada' Puasa 313; disebutkan: i r ﻬﻢ اﷲ$اﺗﻔﻖ اﻟﻌﻠﻤﺎء ر ان أﺻﺢ ا ﻜﺘﺐ ﺑﻌﺪ اﻟﻘﺮآن ا ﺴﻠﻢ g ﻟﻌﺰﺰ ا ﺼﺤﻴﺤﺎن ا‚ﺨﺎري و و ﺗﻠﻘﺘﻬﻤﺎ اﻻﻣﺔ ﺑﺎﻟﻘﺒﻮل و ﺘﺎب ا‚ﺨﺎري أﺻﺤﻬﻤﺎ و اﻛ™ﻫﻤﺎ ﻓﻮاﺋﺪ و ﻣﻌﺎرف ﻇﺎﻫﺮƒﻀﺔgš ة و . “Menurut kesepakatan ulama' bahwa kitab-kitab hadis paling ṣaḥīḥ setelah al-Qur'an al-'Azīz adalah Bukhārī dan Muslim, dan sepatutnya umat menerimanya. Kitab ṣaḥīḥ Bukhārī adalah paling ṣaḥīḥ dan paling banyak mengandung faidah, pengetahuan baik yang mudah maupun yang rumit. “ Abū Zakariyā Muḥyiddīn ibn Sharah al-Nawawī, Ṣaḥīḥ Muslim bi Sharḥ al-Imām al- Nawawī, Jilid. I (Beirut: Dār al-Fikr, t.th.), h. 14. 39Muḥammad ibn 'Alawī al-Mālikī al-Ḥasanī, al-Manhal al-Laṭīf fī Uṣūl al-Hadīth al-Sharīf (Beirut: Dār al-Fikr, 1978), h. 292. ║ AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 157 Volume 26, Nomor 2, Oktober 2016 157 p-ISSN: 0854-4603; e-ISSN: 2502-3209 Ainol Yaqin Ainol Yaqin Sementara hadis-hadis yang dijadikan rujukan qawl jadīd diriwayatkan oleh Imam al-Ṭurmudhī, Ibnu Mājah dan al-Bayhaqī. Kualitas hadis yang terdapat dalam Sunan al-Ṭurmudhī kebanyakan hadis ḥasan. Karena itu, kitab tersebut populer juga dengan sebutan kitab hadis ḥasan. Menurutnya, hadis ṣaḥīḥ ḥasan nilainya lebih unggul daripada hadis ḥasan, tapi tetap lebih rendah tingkatannya daripada hadis ṣaḥīḥ. Mengenai Sunan Ibnu Mājah, ia tampaknya kurang selektif dalam memasukkan hadis dalam Sunan-nya. Hal itu, terbukti dalam kitabnya terdapat hadis-hadis yang bernilai ḥasan, ḍa’īf, munkar dan kategori hadis ḍa’īf lainnya.40Karena syarat yang dijadikan standar penyeleksian hadis sangat long- gar. Sebelum abad keenam, kitab Sunan Ibnu Mājah tidak dimasukkan dalam jumlah Kutub al-Sittah. Keberadaan Sunan Ibnu Mājah masuk deretan Kutub al- Sittah setelah dilirik Muḥammad Ṭāhir al-Maqdisī di tingkatan terakhir.41 Pengkajian pada dalil-dalil yang dibuat rujukan qawl qadīm dan qawl jadīd berdasarkan analisis penulis bahwa pendapat yang kuat adalah qawl qadīm, sebab hadis yang diriwayatkan Ibnu ‘Umar terperangkap pada hadis mawqūf, tidak sampai pada tingkatan hadis marfū'. Sementara hadis-hadis yang di- ungkapkan qawl qadīm termasuk kategori hadis ṣaḥīḥ. Dalam al-Risālahnya, al- Syafi’i mengemukakan bahwa kontradiksi dalam hadis dapat saja terjadi karena perawi menerima hadis dengan matan yang tidak sempurna atau isi hadis hanya berdasarkan imajinasi perawi. Untuk itu, jika terjadi kontradiksi antara hadis maka dibutuhkan tarjīh. Dengan demikian, dipilihlah hadis yang kualitas materi dan mata rantai sanadnya lebih kuat, sebab hadis yang kontra- diktif, rentang tingkatan kualitasnya tidak sama. Hanya hadis yang dapat di- percaya dan otentik yang dapat diterima berdasarkan bukti lain dari kitab Allah, Sunnah Nabi atau bukti-bukti lain. Jadi, sejatinya tidak akan dijumpai dua hadis yang kontradiksi melainkan mesti ada jalan keluarnya, atau salah satu terdapat bukti kesesuaian dengan kitab Allah, sunnah Nabi, atau petunjuk (dalālah) yang lain.42 Kaitannya dengan pendapat sahabat yang dijadikan hujjah qawl jadīd dapat ditampik oleh perkataan al-Syafi’i, "Bagaimana aku AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 158 Volume 26, Nomor 2, Oktober 2016 Evolusi Ijtihad Imam Syafi’i …. Evolusi Ijtihad Imam Syafi’i …. akan meninggalkan hadis disebabkan pendapat seseorang, yang seandainya aku hidup semasa dengannya mesti aku tantang berdebat".43 Status Mahar yang Rusak Mahar yang diberikan seorang suami pada istrinya merupakan bentuk penghargaan pada istri dan sepenuhnya menjadi hak milik istri. Ulama` ber- selisih pendapat mengenai apa yang menyebabkan mahar beralih status men- jadi hak milik istri? Imam Syafi’i dan Imam Abū Ḥanīfah berpendapat bahwa mahar menjadi milik istri dengan adanya akad nikah, sedangkan Imam Mālik mengemukakan bahwa separuh mahar dimiliki istri dengan adanya akad dan separuhnya lagi dimiliki setelah ia disenggamai. Imam Syafi’i membangun pendapatnya atas logika pemahaman terhadap surat al-Nisā' ayat 4: ﺴﺎء ﺻﺪﻗﺎﺗﻬﻦ ›ﻠﺔœوآﺗﻮا اﻟ ِ ْ َ ًM ِ ِ َ ُ َ َ َ َ ِّ ُ ... ﴿ Ÿ ﴾ "Dan berikanlah mas kawin (mahar) kepada perempuan (yang kamu nikahi) sebagai pemberian yang penuh kerelaan”. 44 Berpijak pada ayat ini ada dua ketetapan yang dapat diperoleh: Pertama, peng'idhafahan seluruh mahar (ṣaduqah) pada istri-istri. Hal ini menunjukkan mahar dapat dimiliki seutuhnya oleh istri setelah akad nikah dilaksanakan. Kedua, amar (perintah) mengarah untuk menyerahkan seluruh mahar pada istri. Sebab suami dapat memiliki dan menikmati tubuh istri se- utuhnya. Karenanya, istri pun berhak memiliki mahar seutuhnya setelah akad nikah dilaksanakan.45 43Tājuddīn Abdul al-Wahhāb al-Subkī, Jam'u al-Jawāmi', Jilid. II (Beirut: Dār al-Fikr, 1982), h. 146. 44QS. al-Nisā: 4. 45Imam al-Māwardī, al-Hāwī al-Kabīr, Juz XII, h. 35-36; Ibn Abbās, Qatadah, Ibn Zaid dan Ibn Juraih menyatakan bahwa khiṭab ini tertuju pada suami-suami. Allah memerintahkan kepada para suami untuk memberikan mahar kepada istri sebagai penghibur. Muḥammad ibn Aḥmad al-Anṣārī al- Qurṭubī, al-Jāmi’ li Aḥkām al-Qur’ān, Juz V (Beirut: Dār al-Fikr, t.th.), h. 23; Muḥammad ibn ‘Umar ibn Ḥusain al-Rāzī, al-Tafsīr al-Kabīr, Jilid IX (Beirut: Dār al-Kutub al-‘Ilmiyyah, 2004), h. 146; Abū Ja’far mengutarakan bahwa suami diperintahkan untuk memberikan mahar kepada istrinya sebagai pemberian wajib. Muḥammad ibn Jārir al-Ṭabarī, Tafsīr al-Ṭabarī, Jilid III (Beirut: Dār al-Kutub al- ‘Ilmiyyah, 2009), h. 583. 43Tājuddīn Abdul al-Wahhāb al-Subkī, Jam'u al-Jawāmi', Jilid. II (Beirut: Dār al-Fikr, 1982), h. 146. 44QS. al-Nisā: 4. 45Imam al-Māwardī, al-Hāwī al-Kabīr, Juz XII, h. 35-36; Ibn Abbās, Qatadah, Ibn Zaid dan Ibn Juraih menyatakan bahwa khiṭab ini tertuju pada suami-suami. Allah memerintahkan kepada para suami untuk memberikan mahar kepada istri sebagai penghibur. Muḥammad ibn Aḥmad al-Anṣārī al- Qurṭubī, al-Jāmi’ li Aḥkām al-Qur’ān, Juz V (Beirut: Dār al-Fikr, t.th.), h. 23; Muḥammad ibn ‘Umar ibn Ḥusain al-Rāzī, al-Tafsīr al-Kabīr, Jilid IX (Beirut: Dār al-Kutub al-‘Ilmiyyah, 2004), h. 146; Abū Ja’far mengutarakan bahwa suami diperintahkan untuk memberikan mahar kepada istrinya sebagai pemberian wajib. , ,J , 47Abū Zakariyā Muḥyiddīn ibn Sharaf al-Nawawī, al-Majmū' Sharḥ al-Muhaddab, Jilid XVI, 342; Muḥyiddīn ibn Sharḥ al-Nawawī, al-Tanqīh fī Sharḥ al-Wasīth, Jilid V, h. 217-218; Muḥammad ibn Idrīs al-Shāfi’ī, al-Umm, Juz VI, h. 157-158; Abū Zakariyā ibn Yaḥyā ibn Sharaf al-Nawawī, Rauḍah al-Thālibīn, 46Imam al-Māwardī, al-Hāwī al-Kabīr, Juz XII, h. 36-37. Status Mahar yang Rusak Muḥammad ibn Jārir al-Ṭabarī, Tafsīr al-Ṭabarī, Jilid III (Beirut: Dār al-Kutub al- ‘Ilmiyyah, 2009), h. 583. Volume 26, Nomor 2, Oktober 2016 AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 159 Volume 26, Nomor 2, Oktober 2016 Ainol Yaqin Sedangkan Imam Mālik mendasarkan pendapatnya pada ra’yu, nalar logika yang dihadapkan pada kasus talak. Dalam masalah talak, istri yang ditalak sebelum disetubuhi berhak mendapat separuh mahar yang telah di- terima. Hal itu menunjukkan bahwa sebelum digauli ia hanya berhak separuh mahar. Selain itu, menyandingkan mahar dengan budu' (vagina) kurang tepat, sebab pemanfataan vagina tidak bisa ditangguhkan, sedangkan pembayaran mahar dapat dilakukan secara cash atau kredit.46 Berkaitan dengan permasalahan ini, apakah mahar yang sudah ditentukan kualitas dan kuantitasnya dalam akad, jika kemudian hari rusak sebelum diberikan pada istri, dapatkah diganti dengan benda yang sejenis atau harus menyamai mahar mithl? Menurut qawl qadīm Imam Syafi’i menyatakan suami wajib mengganti barang yang sama dengan mahar yang pertama sebab pemberian mahar wajib bagi suami. Jika ada benda yang sepadan dengan mahar yang telah diberikan, ia harus membayar dengan barang yang sama persis. Jika mahar yang telah di- serahkan tidak ada padanannya, ia harus mengganti sejumlah nilai mahar yang rusak dengan nomimal yang lebih berharga. Pendapat ini sama dengan pendapat Imam Abū Ḥanīfah, Imam Aḥmad, dan pendapat yang dipilih oleh Abū Hāmid, dan Ibn Shabbāgh. Namun, dalam qawl jadīd Imam Syafi’i ber- pendapat, ia harus membayar mahar mithl. Argumen yang ia angkat karena mahar merupakan 'iwadl (ganti) yang tertentu, tidak bisa ditukar dengan barang lainnya. Sebab syara' melarang pada istri untuk tidak melayani suami dengan alasan mahar yang diterima rusak. Kasus tersebut juga diperkokoh dengan dianalogikan pada masalah jual-beli. Sebagai perbandingan, jika sese- orang membeli baju dengan imbalan sepeda dan ternyata sepeda yang dijadi- kan thaman itu rusak maka ia harus mengganti barang yang seharga dengan sepeda. Pendapat ini yang dipedomani Imam Muzannī, Abū Ishāq al-Marūzī, Qāḍi Abū Ṭayyib.47 AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 160 Volume 26, Nomor 2, Oktober 2016 Evolusi Ijtihad Imam Syafi’i …. Evolusi Ijtihad Imam Syafi’i …. Evolusi Ijtihad Imam Syafi’i …. Juz V (Riyaḍ: Dār ‘Ālam al-Kutub, 2003), h. 576; Abī al-Khair Sālim al-‘Imranī, al-Bayān fī Madhhab al- Imām al-Shāfi’ī, Jilid IX, h. 397; Aḥmad ibn Salamah al-Qalyūbī, Hāsyiyyah Qalyūbī, Juz III, 276, Yūsuf al- Fairūz Abādī al-Shairazī, al-Muhadhdhab fī Fiqh al-Imām al-Shāfi’ī (Beirut: Dār al-Kutub al-‘Ilmiah, 1995), Juz II, h. 465-466; Muḥammad ibn Abdulkarīm al-Rāfi’ī al-Qazwinī, al-‘Azīz Sharḥ al-Wajīz, Juz VIII, h. 235; Muḥammad ibn al-Khaṭīb al-Sharbīnī, Mughnī al-Muḥtāj ilā Ma’rifati Alfāẓ al-Minhāj, Juz III, h. 239. 48Yaḥyā ibn Sharaf al-Nawawī, Rawḍah al-Ṭālibīn, Juz VI, h. 373; Yūsuf al-Fairūz Abādī al-Shairazī, al-Muhadhdhab fī Fiqh al-Imām al-Syāfi’i, Juz III, h. 133; Muḥammad ibn Muḥammad al-Ghazālī, al- Wasīth fī al-Madhhab, Jilid VI, h. 143. 48Yaḥyā ibn Sharaf al-Nawawī, Rawḍah al-Ṭālibīn, Juz VI, h. 373; Yūsuf al-Fairūz Abādī al-Shairazī, al-Muhadhdhab fī Fiqh al-Imām al-Syāfi’i, Juz III, h. 133; Muḥammad ibn Muḥammad al-Ghazālī, al- Wasīth fī al-Madhhab, Jilid VI, h. 143. Hukum Nikah pada Masa 'Iddah Istri yang telah ditalak wajib menjalani masa 'iddah dengan tujuan untuk mengetahui bersihnya rahim (barā’ah al-raḥim) dari sperma (nuṭfah) suami dan 'iddah juga bisa sebagai bentuk rasa duka cita yang mendalam bagi istri yang menjalaninya sebab ditinggal mati oleh suami. Dalam realitanya, ter- kadang dijumpai kasus istri yang sedang berkewajiban menempuh masa ‘iddah, menikah lagi dengan laki-laki lain. Kemudian problem tersebut me- nyisakan sesuatu persolaan hukum menyangkut status nikah dan sanksi atas pelanggarannya. Dalam qawl qadīm Imam Syafi’i berpendapat bahwa istri haram selamanya untuk menikah dengan suami kedua. Sedangkan dalam qawl jadīd beliau mengemukakan istri tidak haram selamanya untuk menikah dengan suami kedua. Jika istri telah habis masa 'iddah dari suami pertama ia boleh merajut kembali ikatan nikah dengan suami kedua. Karena waṭi’ yang dilakukan suami merupakan waṭi' syubhat yang tidak berdampak keharaman nikah.48 Imam Syafi’i membangun qawl qadīm berdasar kepada athār ‘Umar yang diriwayatkan Sa’id ibn Musayyab dan Sulaimān ibn Basyar bahwa Thulaihah bersuami Sayid al-Thaqāfī kemudian ia ditalak bain. Thulaihah menikah lagi pada masa ‘iddah dan ketika ‘Umar mendengar kejadian ini ia memukul keduanya dengan cemeti dan mem-firaq (memisah) keduanya. Kemudian ‘Umar berkata: perempuan mana pun yang menikah pada masa ‘iddah, jika suami yang menikahi belum menjima'nya maka harus dipisah keduanya kemudian ia meneruskan ‘iddah suami yang pertama dan suami kedua dianggap berstatus pelamar. Tetapi, jika suami kedua telah menjima' mesti dipisah keduanya kemudian ia melanjutkan 'iddah suami pertama dan AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 Volume 26, Nomor 2, Oktober 2016 161 Volume 26, Nomor 2, Oktober 2016 Ainol Yaqin ditambah 'iddah suami kedua, dan diharamkan menikahinya untuk selama- nya.49 Imam Syafi’i berkata bahwa Sayid menyerahkan mahar sebagai bentuk 'penghalalan' pada istri. Diriwayatkan juga, dari Yaḥyā ibn Ḥassan dari Jarīr dari 'Aṭā’ ibn Saib dari Zadan Abī ‘Umar dan ‘Alī bahwa ia memutuskan tentang perempuan yang nikah pada masa 'iddah. Isi putusannya harus dipisahkan dan istri berhak mendapatkan mahar serta wajib menyempurnakan 'iddah pertama kemudian ditambah 'iddah kedua.50 Sementara dalam qawl jadīd Imam Syafi’i menjadikan athār Ali yang diriwayatkan Abdul Majīd dari ibnu Juraij berkata Aṭā' menceritakan bahwa seseorang mentalak istrinya kemudian ia menjalani 'iddah. Lalu ada seseorang yang tidak tahu tentang keadaan si istri, ia menikahinya di akhir masa 'iddah dan keduanya tetap hidup seatap. 49Muḥammad ibn Idrīs al-Shāfi’ī, al-Umm, Juz VI, h. 590-591; Ismā’īl ibn ‘Umar ibn Kathīr, Tafsīr al- Qur’ān al-‘Aẓīm, Jilid I (Beirut: Dār Thayyibah, t.th.), h. 640; Abū Zakariyā Muḥyiddīn ibn Sharaf al- Nawawī, al-Majmū' Sharḥ al-Muhadhdhab, Jilid XVIII, h. 191. 50Muḥammad ibn Idrīs al-Syāfi’i, al-Umm, Juz VI, 590; Abū Zakariyā Muḥyiddīn ibn Sharaf al- Nawawī, al-Majmū' Sharḥ al-Muhadhdhab, Jilid XVIII, h. 191. 51Muḥammad ibn Idrīs al-Shāfi’ī, al-Umm, Juz VI, h. 590-591; Abū Zakariyā Muḥyiddīn ibn Sharaf al-Nawawī, al-Majmū' Sharḥ al-Muhadhdhab, Jilid. XVIII, h. 191-193 . Hukum Nikah pada Masa 'Iddah Berita ini sampai pada ‘Alī, lalu ia memisah- kan keduanya dan memerintah pada si istri untuk meneruskan 'iddah yang pertama kemudian memperpanjang masa 'iddah-nya dengan menjalani 'iddah yang kedua. Setelah habis 'iddah yang pertama ia diberi opsi (pilihan) antara menikah dengan suami kedua atau memutus hubungan dengannya.51 Kedua qawl Imam Syafi’i baik qawl qadīm maupun qawl jadīd sama-sama diperkuat athār sahabat. Qawl qadīm dilandaskan pada athār ‘Umar, sedang- kan qawl jadīd ditopang athār ‘Alī. Seperti kesimpulan sebelumnya, berubah- nya alur ijtihad al-Syafi’i dari qawl qadīm pada qawl jadīd karena dilatari perbedaan dalil yang dijadikan hujjah. Ketika beliau di Mesir memeriksa kembali pendapatnya yang difatwakan di Irak dengan temuan dalil baru dan berijtihad kembali. Beliau meninggalkan qawl qadīm karena terdapat sisi kelemahan pada dalil dan berpindah pada qawl jadīd yang dipandang dalilnya lebih kuat. AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 162 Volume 26, Nomor 2, Oktober 2016 Evolusi Ijtihad Imam Syafi’i …. Kedua athār di atas murni hasil ijtihad shabahat terhadap kasus yang dihadapi di tengah-tengah kehidupan masyarakat. Karena itu, memperhatikan budaya, perilaku masyarakat, orang yang menjadi objek fatwa di saat fatwa dikemukakan menjadi faktor krusial guna mengetahui kasus yang sebenarnya. Sejauh ini, penulis menganggap qawl jadīd yang dibangun atas athār ‘Alī lebih kuat karena di lain kesempatan ‘Umar menarik kembali pendapat yang telah difatwakan. Di tengah-tengah khalayak ‘Umar mengatakan: Tinggalkanlah kebodohan-kebodohan dan berpeganglah pada al-Sunnah. Kemudian ‘Umar berpegangan pada pendapat ‘Alī.52 Status Nikah Istri yang Ditinggal oleh Suaminya Bagaimana status ikatan nikah istri yang suaminya hilang, tidak diketahui rimbanya, dan kabar pun tidak kunjung datang? Menyikapi kasus seperti ini, Imam Syafi’i berpendapat dalam qawl qadīm bahwa ia harus menunggu selama empat tahun kemudian menjalani ‘iddah wafat, baru setelah itu boleh menikah lagi dengan laki-laki lain yang telah menjadi pilihannya. Sedangkan dalam qawl jadīd Imam Syafi’i menyatakan bahwa ia tidak boleh menikah dengan laki-laki lain hingga kabar suami secara nyata diketahui sudah mati atau telah mentalaknya.53 Adapun dalil-dalil yang memperkuat qawl qadīm Imam Syafi’i adalah sebagai berikut: Pertama, riwayat ‘Umar ibn Dīnar dari Yaḥyā ibn Ja'dah: أن رﺟﻼ اﺳﺘﻬﻮﺗﻪ ا¡ﻦ ﻓ ﺮأgﻐﺎب ﻋﻦ أ ﺮﻫﺎ g اﷲ ﻋﻨﻪ ﻓﺄ¤ﻄﺎب ر¥ﺗﻪ، ﻓﺄﺗﺖ ﻋﻤﺮ ﺑﻦ ا 52Abū Zakariyā Muḥyiddīn ibn Sharaf al-Nawawī, al-Majmū' Sharḥ al-Muhadhdhab, Jilid XVIII, h.190-193. 52Abū Zakariyā Muḥyiddīn ibn Sharaf al-Nawawī, al-Majmū' Sharḥ al-Muhadhdhab, Jilid XVIII, h.190-193. 53Muḥyiddīn ibn Sharaf al-Nawawī, al-Tanqīh fī Sharḥ al-Wasīth, Jilid VI, h. 148; Yaḥyā ibn Sharaf al-Nawawī, Rawḍah al-Thālibīn, Juz VI, h. 377; Muḥammad ibn Abdulkarīm al-Rāfi’ī al-Qozwinī, al- ‘Azīz Sharḥ al-Wajīz, Juz IX, 484-485; Abdulhamīd al-Sharwanī, Hawāsyī Tuhfah al-Muḥtāj bi Sharḥi al- Minhāj, Juz VIII, h. 253; Muḥammad ibn al-Khaṭīb al-Syarbinī, Mughnī al-Muḥtāj ilā Ma’rifat Ma’ānī Alfādz al-Minhāj, Juz III, h. 520-521; Aḥmad ibn Salamah al-Qalyūbī, Hāsyiyyah al-Qalyūbī, Juz IV, h. 51, Abū Zakariyā Muḥyiddīn ibn Sharaf al-Nawawī, al-Majmū’ Sharḥ al-Muhadhdhab, Jilid XIX, h. 442; Yaḥyā ibn Abilkhair al-‘Imronī, al-Bayān fī Madhhab al-Shāfi’ī, Jilid XI, 44-45; ‘Abdullāh ibn Yūsuf al- Juwainī, Nihāyah al-Maṭlab fī Nihāyah al-Madhhab, Jilid XV, h. 287. ║163 AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 Volume 26, Nomor 2, Oktober 2016 163 Volume 26, Nomor 2, Oktober 2016 Ainol Yaqin ﺮﻫﺎ أن ﺗﻌﺘﺪ ﺛﻢ ﺗ¦وجg§، ﺛﻢ أœﻊ ﺳCأن ﺗﻤﻜﺚ أر .54 "Bahwa seorang laki-laki dikelabui jin kemudian ia lenyap dari penglihatan istrinya, lalu si istri mendatangi ‘Umar ibn Khattāb dan ‘Umar pun memutus- kan ia harus menunggu selama empat tahun, setelah itu ia diperintah untuk ber'iddah. 54Abū Zakariyā Muḥyiddīn ibn Sharaf al-Nawawī, al-Majmū' Sharḥ al-Muhadhdhab, Jilid XV, 155; Imam al-Māwardī, al-Hāwī al-Kabīr, Juz XIV, h. 366. J 55Imam al-Māwardī, al-Hāwī al-Kabīr, Juz XIV, h. 366 ;‘Abdulhamīd al-Sharwanī, Hawāsyī Tuḥfah al-Muḥtāj bi Sharḥi al-Minhāj, Juz VIII, h. 254; Yaḥyā ibn Abilkhair al-‘Imranī, al-Bayān fī Madhhab al- Shāfi’ī, Jilid XI, h. 46. 56Abū Zakariyā Muḥyiddīn ibn Sharaf al-Nawawī, al-Majmū' Sharḥ al-Muhadhdhab, Jilid XV, h. 155-157. y ḥy , j ḥ , J , ; Imam al-Māwardī, al-Hāwī al-Kabīr, Juz XIV, h. 366. 55Imam al-Māwardī, al-Hāwī al-Kabīr, Juz XIV, h. 366 ;‘Abdulhamīd al-Sharwanī, Hawāsyī Tuḥfah al-Muḥtāj bi Sharḥi al-Minhāj, Juz VIII, h. 254; Yaḥyā ibn Abilkhair al-‘Imranī, al-Bayān fī Madhhab al- Shāfi’ī, Jilid XI, h. 46. 56Abū Zakariyā Muḥyiddīn ibn Sharaf al-Nawawī, al-Majmū' Sharḥal-Muhadhdhab, Jilid XV, h. 54Abū Zakariyā Muḥyiddīn ibn Sharaf al-Nawawī, al-Majmū' Sharḥ al-Muhadhdhab, Jilid XV, 155; Imam al-Māwardī, al-Hāwī al-Kabīr, Juz XIV, h. 366. 55Imam al-Māwardī, al-Hāwī al-Kabīr, Juz XIV, h. 366 ;‘Abdulhamīd al-Sharwanī, Hawāsyī Tuḥfah al-Muḥtāj bi Sharḥi al-Minhāj, Juz VIII, h. 254; Yaḥyā ibn Abilkhair al-‘Imranī, al-Bayān fī Madhhab al- Shāfi’ī, Jilid XI, h. 46. 56Abū Zakariyā Muḥyiddīn ibn Sharaf al-Nawawī, al-Majmū' Sharḥ al-Muhadhdhab, Jilid XV, h. 155-157. 57Ibid., Jilid XV, h. 155; Yaḥyā ibn Abilkhair al-‘Imranī, al-Bayān fī Madhhab al-Shāfi’ī, Jilid XI, h. 44- 45. 58Yaḥyā ibn Abilkhair al-‘Imranī, al-Bayān fī Madhhab al-Shāfi’ī, Jilid XI, h. 46. 59Abū Zakariyā Muḥyiddīn ibn Sharaf al-Nawawī, al-Majmū' Sharḥ al-Muhadhdhab, Jilid XV, h. 155. 60Muḥammad ibn Idrīs al-Shāfi’ī, al-Risālah, h. 596-598. Status Nikah Istri yang Ditinggal oleh Suaminya Kemudian diperkenankan untuk menikah lagi dengan laki-laki lain." Kedua, fasakh dengan alasan suami impoten dan tidak mampu menafkahi dibolehkan syara', apalagi persoalaan ini (berkumpul dua mafsadat, yaitu istri tidak memperoleh nafkah lahir dan nafkah batin).55 Dalam riwayat Yaḥyā diceritakan oleh Ibnu Abi Dunyā ia berkata di- riwayatkan Abū Muslim Abdurrahmān Ibnu Yūsuf dari Sufyān ibn 'Uyainah dari ‘Umar ibn Dīnār dari Yaḥyā ibn Ja'dah berkata: “Pada masa ‘Umar ada se- seorang yang dikelabui oleh jin, tentu tidak diketahui apakah ia masih hidup atau sudah mati. Hari-hari berikutnya sang istri mendatangi ‘Umar mengadu keluh resahnya, kemudian beliau menyuruhnya menunggu selama empat tahun. Kemudian ‘Umar menyuruh wali si suami untuk mentalak. Setelah itu, ‘Umar memerintah si istri ber-'iddah dan kawin. Jika dikemudian hari sang suami datang ia diberi pilihan untuk tetap mempertahankan istrinya dan membayar mahar”. Menurut al-Nawawī validitas berita ini tidak dapat di- pertanggungjawabkan karena Yaḥyā tidak semasa dengan ‘Umar. Oleh kerena itu, berita ini terjebak pada khabar munqaṭi'.56 Sedangkan dalil-dalil yang memperkokoh qawl jadīd Imam Syafi’i adalah: Sedangkan dalil-dalil yang memperkokoh qawl jadīd Imam Syafi’i adalah: Pertama, riwayat ‘Alī : Pertama, riwayat ‘Alī : ﻮﺗﻪg ﺗﺼ« ﺣ ﻳﻌﻠﻢ . ﻮﺗﻪg ﺗﺼ« ﺣ ﻳﻌﻠﻢ . "Bersabarlah hingga dia (suami) benar-benar diketahui telah meninggal dunia." 55Imam al-Māwardī, al-Hāwī al-Kabīr, Juz XIV, h. 366 ;‘Abdulhamīd al-Sharwanī, Hawāsyī Tuḥfah al-Muḥtāj bi Sharḥi al-Minhāj, Juz VIII, h. 254; Yaḥyā ibn Abilkhair al-‘Imranī, al-Bayān fī Madhhab al- Shāfi’ī, Jilid XI, h. 46. 56Abū Zakariyā Muḥyiddīn ibn Sharaf al-Nawawī, al-Majmū' Sharḥ al-Muhadhdhab, Jilid XV, h. 155-157. AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 Volume 26, Nomor 2, Oktober 2016 AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 164 Volume 26, Nomor 2, Oktober 2016 Evolusi Ijtihad Imam Syafi’i …. Evolusi Ijtihad Imam Syafi’i …. Kedua, kasus ini tidak bisa disamakan dengan kasus firāq (berpisah) dengan alasan impoten dan sukar menafkahi. Sebab kematiaan bersifat pra- duga, sedangkan impoten dan sukar menafkahi jelas dan nyata.57 Di kalangan aṣḥāb cenderung berpegangan pada qawl jadīd yang di- pandang lebih rājih. Abū Ishāq mengemukakan bahwa istri yang ditinggal suami memulai masa 'iddah sejak putusan hakim dijatuhkan sebab dalam kasus ini perpisahan (firāq) terjadi berdasarkan ijtihad hakim. Sedangkan sebagian aṣḥāb berpendapat ia dapat memulai masa 'iddah ketika suami telah tiada. Status Nikah Istri yang Ditinggal oleh Suaminya Tampaknya, pendapat pertama yang mengungguli karena putusan firāq bergantung pada ijtihad hakim.58 Uraian al-Nawawī atas dalil-dalil qawl qadīm dan qawl jadīd memperjelas bahwa beralihnya Imam Syafi’i dari qawl qadīm pada qawl jadīd karena perbedaan materi dalil dan keshahihan sebuah khabar. Sebab dalil yang di- jadikan rujukan qawl qadīm terperangkap pada khabar munqati' yang mata rantai sanadnya tidak bersambung. Disamping itu, Ju'dah sebagai perawi athār tersebut disangsikan status kesahabatannya. Apakah ia masih mengikuti Nabi atau tidak? Begitu juga, Yaḥyā sebagai perawi athār patut dicurigai kevalidan beritanya karena ia hidup tidak semasa dengan ‘Umar.59 Sejalan dengan pendirian Imam Syafi’i dalam menelaah qawl sahabat bahwa pendapat sahabat yang masih tidak mendapat kata sepakat di kalangan sahabat harus dilakukan tarjīh dengan mencari dalil-dalil pendukung. Pen- dapat sahabat yang harus dipedomani adalah pendapat yang sesuai dengan al- kitab, sunnah, ijma' atau diperkuat qiyas yang lebih ṣaḥīḥ. Lebih lanjut, Imam Syafi’i menegaskan bahwa ia akan berpedoman pada pendapat seorang sahabat, bila masalah yang dihadapi tidak dijumpai keterangannya dalam kitab, sunnah, ijma', atau merujuk pada qiyas.60 ║ AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 Volume 26, Nomor 2, Oktober 2016 165 Ainol Yaqin 61QS. al-Ṭalāq: 1. 62Yaḥyā ibn Abilkhair al-‘Imranī, al-Bayān fī Madhhab al-Shāfi’ī, Jld XI, 74-75; Muḥammad ibn ‘Abdulkarīm al-Rāfi’ī, al-‘Azīz Sharḥ al-Wajīz, Juz IX, h. 261-262; ‘Abdulhamīd al-Sharwanī, Hawāsyī Tuhfah al-Muḥtāj bi Sharḥi al-Minhāj, Juz VIII, h. 511; Aḥmad ibn Salamah al-Qalyūbī, Hāsyiyyah Qalyūbī, Juz IV, h. 55; Yaḥyā ibn Sharaf al-Nawawī, Rawḍah al-Ṭālibīn, Juz VI, h. 393; Shamsuddīn Muḥammad ibn al-Khaṭīb, Mughnī al-Muḥtāj ilā Ma’rifat Sharḥ al-Minhāj, Juz III, h. 529. 63HR. Muslim, Abū Daud, Ibn Mājah, dan al-Nasā'ī. Jalāluddīn al-Suyūṭī, Sunan al-Nasā`ī, Jilid III. h. 437; Muḥammad ibn Yazīd al-Qazwinī, Sunan Ibn Mājah, Jilid II, h. 513; Abū Zakariyā Muḥyiddīn ibn Sharaf al-Nawawī, al-Majmū' Sharḥ al-Muhadhdhab, Jilid XVIII, h. 174-177. 63HR. Muslim, Abū Daud, Ibn Mājah, dan al-Nasā'ī. Jalāluddīn al-Suyūṭī, Sunan al-Nasā`ī, Jilid III. h. 437; Muḥammad ibn Yazīd al-Qazwinī, Sunan Ibn Mājah, Jilid II, h. 513; Abū Zakariyā Muḥyiddīn ibn Sharaf al-Nawawī, al-Majmū' Sharḥ al-Muhadhdhab, Jilid XVIII, h. 174-177. 61QS. al-Ṭalāq: 1. 62Yaḥyā ibn Abilkhair al-‘Imranī, al-Bayān fī Madhhab al-Shāfi’ī, Jld XI, 74-75; Muḥammad ibn ‘Abdulkarīm al-Rāfi’ī, al-‘Azīz Sharḥ al-Wajīz, Juz IX, h. 261-262; ‘Abdulhamīd al-Sharwanī, Hawāsyī Tuhfah al-Muḥtāj bi Sharḥi al-Minhāj, Juz VIII, h. 511; Aḥmad ibn Salamah al-Qalyūbī, Hāsyiyyah Qalyūbī, Juz IV, h. 55; Yaḥyā ibn Sharaf al-Nawawī, Rawḍah al-Ṭālibīn, Juz VI, h. 393; Shamsuddīn Muḥammad ibn al-Khaṭīb, Mughnī al-Muḥtāj ilā Ma’rifat Sharḥ al-Minhāj, Juz III, h. 529. Keluar Rumah di saat Menjalani 'Iddah Evolusi Ijtihad Imam Syafi’i …. keluarlah, datangi kebun kurmamu. Barangkali kamu ingin bershadaqah atau hendak melakukan kebaikan." ‘Umar ibn Khattāb memberi dispensasi pada perempuan yang menjalani 'iddah wafat untuk keluar rumah di hari-hari sucinya. Zaib ibn Hārith secara mutlak membolehkan pada perempuan yang menjalani 'iddah wafat untuk keluar rumah. Sementara ‘Alī lebih longgar lagi, ia membolehkan untuk keluar rumah pada perempuan yang aktivitas kesehariannya bepergian seperti; pebisnis, wanita karir dan lainnya.64 Perbedaan qawl qadīm dan qawl jadīd muncul karena perbedaan wajh istidlāl dan dalil. Dalil al-Qur’an sebagai rujukan qawl jadīd berbentuk nahi' yang mengarah pada pemahaman umum. Keumuman sīghat nahi pada ayat di atas hanya ditakhsis dengan adat istisnā'. Karenanya, tidak menutup peluang ditakhsis dengan dalil lain yang khitabnya lebih spesifik. Berhubung dalil hadis yang dibuat sumber rujukan qawl jadīd terdapat keterkaitan hukum maka dapat dijadikan dalil yang mentakhsis al-Qur’an. Dengan demikian, qawl jadīd berdalil dengan rujukan yang lebih kuat sebab dalil yang mentakhsis ke- umuman dalil lain lebih kuat petunjuk hukumnya (dalālah al-ḥukmi). AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 Volume 26, Nomor 2, Oktober 2016 ║167 Keluar Rumah di saat Menjalani 'Iddah Istri yang ditalak ba`in dan istri yang ber-'iddah wafat tidak boleh keluar rumah selama menjalani masa 'iddah, kecuali ada 'uzur. Hal ini berdasarkan firman Allah: ...ﺮﺟﻦ إﻻ أن ﻳﺄ¬§ ﺑﻔﺎﺣﺸﺔ ﻣﺒﻨﺔ- ﻻ ¯ﺮﺟﻮﻫﻦ ﻣﻦ ®ﻴﻮﺗﻬﻦ وﻻ ٍ ٍ ّ َZِ ِ َ َ َ ِ ِ ِ َ ْ ْ ْ َ َ M ِ َ ْ ُ َ َ َ َ M M ِ ُ ُ ِ ُ ُ ِ ُ .... ﴿ ° ﴾ ”Janganlah kamu keluarkan mereka dari rumahnya dan janganlah (diizinkan) keluar kecuali jika mereka mengerjakan perbuatan keji dan jelas”. 61 ”Janganlah kamu keluarkan mereka dari rumahnya dan janganlah (diizinkan) keluar kecuali jika mereka mengerjakan perbuatan keji dan jelas”. 61 ”Janganlah kamu keluarkan mereka dari rumahnya dan janganlah (diizinkan) keluar kecuali jika mereka mengerjakan perbuatan keji dan jelas”. 61 Jika istri yang beri'ddah mempunyai hajat (kebutuhan) untuk keluar rumah, misalnya berkeinginan membeli barang yang dibutuhkan atau menjual sesuatu, ia tidak boleh keluar di malam hari karena dikhawatirkan membawa hal yang negatif. Apabila istri yang ditalak ba`in bermaksud bepergian di siang hari, dalam hal ini terdapat dua pendapat. Menurut qawl qadīm Imam Syafi’i mengemuka- kan ia tidak boleh keluar rumah di siang hari. Pendapat ini bertumpu pada ayat di atas. Sedangkan dalam qawl jadīd Imam Syafi’i, ia boleh keluar rumah di siang hari.62 Pendapat ini berpijak pada hadis yang diriwayatkan Jābir. ّﻃﻠ ﺎ ﻓﻠﻘﻴﻬﺎ رﺟﻞ ﻓﻨﻬﺎﻫﺎ ﻓﺄ± ﻼ² ﺪ³ ﻘﺖ ﺧﺎﻟ´ ﺛﻼﺛﺎ ﻓﺨﺮﺟﺖ ّ ﺗ ﺻ` اﷲ aﺖ ا ّ ّ ِ ّ ﻣﻨﻪ أو µﻠﻚ ﻟﻌﻠﻚ أن ﺗﺼﺪ² ﺎ اﺧﺮ· ﻓﺠﺪي± ﻋﻠﻴﻪ وﺳﻠﻢ ﻓﺬﻛﺮت ذ ﻚ … ﻓﻘﺎل ّ ّ ِ ِ ّ ّ ْ اRﺗﻔﻌ¸ ﺧ . 63 "Bibiku ditalak tiga, ia keluar rumah untuk mendatangi pohon kurma kemudian ia berpapasan dengan orang dan melarang untuk pergi. Lalu bibiku mendatangi Nabi dan mejelaskan problem yang dialaminya. Nabi bersabda: "Bibiku ditalak tiga, ia keluar rumah untuk mendatangi pohon kurma kemudian ia berpapasan dengan orang dan melarang untuk pergi. Lalu bibiku mendatangi Nabi dan mejelaskan problem yang dialaminya. Nabi bersabda: 63HR. Muslim, Abū Daud, Ibn Mājah, dan al-Nasā'ī. Jalāluddīn al-Suyūṭī, Sunan al-Nasā`ī, Jilid III. h. 437; Muḥammad ibn Yazīd al-Qazwinī, Sunan Ibn Mājah, Jilid II, h. 513; Abū Zakariyā Muḥyiddīn ibn Sharaf al-Nawawī, al-Majmū' Sharḥ al-Muhadhdhab, Jilid XVIII, h. 174-177. AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 166 Volume 26, Nomor 2, Oktober 2016 Evolusi Ijtihad Imam Syafi’i …. AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 Siapa yang Dimaksud “... biyadihi 'uqdat ‘n-nikāḥ...”? Jika suami mentalak istri yang tidak pernah disetubuhinya, ia wajib mem- bayar separuh mahar. Akan tetapi, orang yang mempunyai kuasa atas ikatan nikah ('uqdat al-nikāḥ) dapat memberi dispensasi yang berakibat gugurnya kewajiban menunaikan pembayaran mahar. Hal ini berdasarkan firman Allah dalam surat al-Baqarah ayat 237: ﺒﻞ أن ¬ﻤﺴﻮﻫﻦ وﻗﺪ ﻓﺮﺿﺘﻢ ﻬﻦ ﻓﺮﻀﺔ ﻓﻨﺼﻒ ﻣﺎ ﻓﺮﺿﺘﻢ€ و»ن ﻃﻠﻘﺘﻤﻮﻫﻦ ﻣﻦ ْ ْZ ُ ُ ُ ُ ْ ْ ْ َ َ َ َ َ َ َ َ َ َ َ ْ َ ُ ْ ِ ً َ ْ ِ ِ M M M َ ُ ُ َ َ ِ ِ ُ M َ إﻻM ِ ِﻘﺪة ا½حW ﻌﻔﻮ ا¿ي ®ﻴﺪهŽ ﻌﻔﻮن أوŽ أن ِّ َ ِ ُ ْ َ ُ ِ َ ِ ِ M َ ُ ُ ْ ْ َ َ ْ َ َ َ ۚ وأن ¬ﻌﻔﻮا أﻗﺮب ﻠﺘﻘﻮى ْ َ ٰM ِ ُ َ َ ْ َ َ ُ ْ َ ۚ ﺴﻮاœوﻻ ﺗ َ َ ُ َ َ ﻢÁاﻟﻔﻀﻞ ﺑﻨ ْ َ ُ ْ َ ْ َ ْ َ ۚ ﴿Rإن ا‡ ﺑﻤﺎ ¬ﻌﻤﻠﻮن ﺑﺼ َ ُ َ َ ِ ٌ َ َ ْM ِ َ M ِ ÂÃÄ ﴾ 64Abū Zakariyā Muḥyiddīn ibn Sharaf al-Nawawī, al-Majmū' Sharḥ al-Muhadhdhab, Jilid. XVIII, h. 177. AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 Volume 26, Nomor 2, Oktober 2016 ║167 AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 Ainol Yaqin "Dan jika kamu menceraikan mereka sebelum kamu sentuh (campuri), padahal sesungguhnya kamu sudah menentukan maharnya, maka (bayarlah) separuh dari yang telah kamu tentukan, kecuali jika mereka itu memaafkan (membebaskan) atau dimaafkan oleh orang yang memegang ikatan nikah". 65 Berkenaan dengan pemberiaan dispensasi atas pembayaran mahar, siapa- kah yang dimasud “biyadihi 'uqdat ’n-nikāḥ” pada ayat tersebut? Menurut Qawl qadīm Imam Syafi’i berpendapat bahwa yang dimaksud “biyadihi ‘uqdat ’n- nikāḥ” adalah wali. Pendapat ini dilandaskan pada pemahaman gramatika arab terhadap ayat di atas. Dalam kajian gramatika Arab tidak tepat biyadihi ‘uqdat ’n-nikāḥ ditujukan pada "suami", sebab fi'il muḍāri' di sini mengandung ḍāmir ghā’ib, sedangkan azwāj (suami-suami) telah disebutkan sebelumnya. Jika yang dituju oleh Shāri’ “biyadihi 'uqdat ’n-nikāḥ” adalah suami maka bunyi firman-Nya seperti ini ( أن إ ن أو ا ")kecuali mereka memaafkan atau kalian (suami-suami) memaafkan. ". Wali yang dimaksud di sini adalah bapak atau kakek dari istri yang masih perawan. 65QS. al-Baqarah: 237. 66Ibid., Jilid XVI, 364-365, Yaḥyā ibn Abilkhair al-‘Imranī, al-Bayān fī Madhhab al-Shāfi’ī, Jilid. IX, h. 439-343; Muḥammad ibn ‘Abdulkarīm al-Rāfi’ī, al-‘Azīz Sharḥ al-Wajīz, Juz VIII, h. 320; Muḥammad ibn Muḥammad al-Ghazālī, al-Wasīṭ fī al-Madhhab, Jilid V, h. 260-261; Yaḥyā ibn Sharaf al-Nawawī, Rawḍah al-Ṭālibīn, Juz V, h. 631-632; Aḥmad ibn Salamah al-Qalyūbī, Hāsyiyyah Qalyūbī, Juz III, h. 290; ‘Abdulhamīd al-Sharwanī, Hawāsyī Tuhfah al-Minhāj bi Sharḥ al-Minhāj, Juz VII, h. 408-409; Shamsuddīn Muḥammad ibn al-Khaṭīb, Mughnī al-Muḥtāj ilā Ma’rifat Sharḥ al-Minhāj, Juz III, h. 317; Wahbāh al-Zuhailī, al-Tafsīr al-Munīr, Jld. II (Beirut: Dār al-Fikr, 1998), h. 383; Muḥammad ibn Jarīr al- Siapa yang Dimaksud “... biyadihi 'uqdat ‘n-nikāḥ...”? Syara' menganjurkan pada istri yang sudah dewasa untuk tidak me- nuntut separuh maharnya dibayarkan oleh suami, dengan tujuan supaya sang suami mencintai istri lagi. Ini adalah Pendapat Ibnu Abbās dari kalangan sahabat, al-Ḥasan, Mujāhid, 'Ikrimah, Ṭāwūs dari kalangan tābi'īn, dan Rabī'ah, Mālik, dan Hanbali dari kalangan fuqahā’. Sedangkan dalam qawl jadīd Imam Syafi’i menyatakan bahwa yang dimaksud “biyadihi 'uqdat ’n-nikāḥ” adalah suami. Sebab, wali sedikit pun tidak mempunyai hak memaafkan/memberi dispensasi terhadap sesuatu yang sudah menjadi hak milik istri. Selain itu, pada ayat di atas dapat dibenarkan yang di-khiṭāb adalah suami-suami, sebab dalam ayat lain Allah berfirman dengan khiṭāb hāḍir kemudian Ia mengkhitab dengan khiṭāb ghā’ib.66 Sebagaimana firman-Nya: AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 168 Volume 26, Nomor 2, Oktober 2016 Evolusi Ijtihad Imam Syafi’i …. Evolusi Ijtihad Imam Syafi’i …. ... ﺣ إذا ﻛﻨﺘﻢ • اﻟﻔﻠﻚ وﺟﺮﻦ ﺑﻬﻢ َ ْ َ ِ ِ َ ِ َ َ ْ ُ ْ َ ُ ُ ْ ِ ٰ ِM .... ﴿ ÂÂ ﴾ "... sehingga ketika kamu di dalam kapal, dan meluncurlah (kapal) itu mem- bawa mereka ...."67 "... sehingga ketika kamu di dalam kapal, dan meluncurlah (kapal) itu mem- bawa mereka ...."67 Firman Allah "kecuali mereka (istri) memaafkan", khitab ini tertuju pada istri, menganjurkan mereka untuk 'memaafkan' (mengembalikan) separuh mahar yang telah menjadi haknya supaya suami luluh hatinya sehingga menaruh iba yang mendalam untuk merajut kembali mahligai rumah tangga yang pernah dibina bersama. Ini adalah Pendapat ‘Alī, Jubair dari kalangan sahabat. Syuraih, Sa'īd ibn Jabīr, Sa'īd ibn Musayyab, dan Syi'bī dari kalangan tābi'īn, Sufyān al- Thaurī, Ibnu Abī lailā, dan Abū Ḥanīfah dari kalangan fuqaha'.68 Adapun dalil-dalil yang menopang qawl qadīm adalah sebagai berikut: Pertama, setelah talak terjadi hanyalah wali yang mempunyai hak absolut atas putrinya, karena ia yang mempunyai hak untuk mengawinkan putrinya. Oleh sebab itu, perintah ini diarahkan pada wali semata, dan tidak dapat ditujukan pada suami sebab setelah suami mentalak, ia tidak mempunyai wewenang apa pun. Kedua, wali adalah orang yang mempunyai hak untuk mengakad putrinya, sedangkan suami mempunyai hak untuk istimta' dengan sang istri. Karena itu, makna “biyadihi 'uqdat ’n-nikāḥ” tertuju pada wali lebih relevan dilihat dari redaksi ayatnya. Ketiga, suami berpiutang untuk separuh mahar yang telah menjadi hak istri, yang menjadi hak milik istri yang telah dewasa atau wali sang istri yang masih kecil/perawan. Ṭabarī, Tafsīr al-Ṭabarī, Jilid II, h. 557-565; Muḥammad ibn ‘Umar ibn Ḥusain al-Rāzī, al-Tafsīr al- Kabīr, Jilid III, h. 121-123. 67QS. Yūnus : 22. 68 Imam al-Māwardī, al-Hāwī al-Kabīr, Juz XII, 144-145. Muḥammad ibn Aḥmad al-Anṣārī al- Qurṭubī, al-Jāmi’ li Aḥkām al-Qur’ān, Jilid II, h. 207. 69 Imam al-Māwardī, al-Hāwī al-Kabīr, Juz XII, h. 143-145. Ṭabarī, Tafsīr al-Ṭabarī, Jilid II, h. 557-565; Muḥammad ibn ‘Umar ibn Ḥusain al-Rāzī, al-Tafsīr al- Kabīr, Jilid III, h. 121-123. 67QS. Yūnus : 22. Siapa yang Dimaksud “... biyadihi 'uqdat ‘n-nikāḥ...”? Karena itu, yang lebih berhak memberi 'maaf' (dispensasi) adalah orang yang mempunyai hak bukan orang yang memikul beban kewajiban.69 ║ AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 Volume 26, Nomor 2, Oktober 2016 ║169 AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 ║169 Ainol Yaqin Ainol Yaqin Sedangkan dalil-dalil yang mendukung qawl jadīd adalah sebagai berikut: Sedangkan dalil-dalil yang mendukung qawl jadīd adalah sebagai berikut: Pertama, firman Allah َِِِ َُِْةُ اَح yang dimaksud al-'uqdah adalah suatu ungkapan tentang perkara yang tersimpul. Dari ayat ini dapat ditarik pengertian bahwa istri setelah akad nikah dilangsungkan berada di bawah kekuasaan suami. Kedua, shāri' memerintah pada orang yang memiliki hak untuk memaafkan (memberi dispensasi) pada yang terkena beban kewajiban. Imam Syafi’i berkata: ﻄﺎب ﺑﺎﻟﻌﻔﻮ¥ ان ﻳﺘﻮﺟﻪ اÅ ﻓﺎﻗﺘÆﺎ ﻚ دون ا ﻮzﻠﻚ و ا ﺰوج ﻫﻮ اg و إﻧﻤﺎ ﻳﻌﻔﻮ ﻣﻦ Æ ا ﻮÇﻪ ﻻ إVإ . Æ ا ﻮÇﻪ ﻻ إVإ . ”Bahwasanya yang dapat memaafkan orang yang memiliki dan suamilah yang memiliki istri. Karena itu, khiṭāb ayat di atas lebih pantas dimaksudkan pada suami, bukanlah pada wali.” ”Bahwasanya yang dapat memaafkan orang yang memiliki dan suamilah yang memiliki istri. Karena itu, khiṭāb ayat di atas lebih pantas dimaksudkan pada suami, bukanlah pada wali.” Ketiga, pada hakikatnya hak 'memaafkan' adalah meninggalkan, itu adalah hak suami, karena ia memiliki separuh mahar setelah terjadi talak. Jika ia membiarkan untuk tidak memiliki maka tidak berhak untuk memiliki. Sedang- kan wali mempunyai hak maaf terkait dengan perkara lain, yaitu berupa pemberian jika berwujud benda dan ibra’ jika berada dalam tanggungan. Jadi, pemberiaan maaf berada di tangan suami. Keempat, hak 'memaafkan' jika tertuju pada suami maka bisa mencakup pada semua suami yang mentalak. Sebaliknya, jika hak 'memaafkan' diarahkan pada wali maka hanya mengenai pada sebagian wali dan sebagian istri-istri saja, yakni bapak dan kakek dan hanya tertentu pada istri yang masih kecil lagi perawan. ﺼﻮص¥ ﻣﺎ ﻳﻮﺟﺐ اr ﻠﻪ$ ﻣﻦÉ ﻣﺎ ﻳﻮﺟﺐ اﻟﻌﻤﻮم أوr ﻄﺎب¥ﻞ ا$ . ”Membawa khiṭāb pada sesuatu yang menunjukkan general lebih utama daripada mengarahkan khiṭāb pada sesuatu yang menunjukkan spesifik.” Kelima, firman Allah "! َْى#ِ َُب%ْ&ََأَنْ َ ْ ُا أ khiṭāb ini tidak dialamatkan pada wali karena kualitas ketaqwaan wali terletak pada melindungi harta yang berada dalam kekuasaannya, bukan memaafkan dan membebaskan. 70Muḥammad ibn Aḥmad al-Anṣārī al-Qurṭubī, al-Jāmi’ li Aḥkām al-Qur’ān, Jilid II, h. 206. 71Muḥammad ibn Idrīs al-Shāfi’ī, al-Umm, Juz VI, h.190; Imam al-Māwardī, al-Hāwī al-Kabīr, Juz XII, h. 143-145; Jalāluddīn al-Suyūṭī, al-Durru al-Manthūr fī al-Tafsīr bil al-Ma`thūr, Juz III (Kairo: Markaz Hajr li ‘l-Buḥūth wa al-Dirāsāt al-‘Arabīyah wa al-Islāmiyyah, 2003), h. 29; Ismā’īl ibn ‘Umar ibn Kathīr, Tafsīr al-Qur’ān al-‘Aẓīm, Jilid I, h. 643-645; Ismā’īl ibn Yaḥyā ibn Ismā’īl al-Muzannī, Mukhtaṣar al-Muzannī (Beirut: Dār al-Kutub al-‘Ilmiah, 1998), h. 244. Siapa yang Dimaksud “... biyadihi 'uqdat ‘n-nikāḥ...”? Keenam, ditopang hadis yang diriwayatkan Ibnu Luhai'ah dari ‘Umar ibn Shu’ib dari ayahnya dari kakeknya berkata bahwa Rasulullah bersabda: AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 0║ Volume 26, Nomor 2, Oktober 2016 170║ p-ISSN: 0854-4603; e-ISSN: 2502-3209 Evolusi Ijtihad Imam Syafi’i …. Evolusi Ijtihad Imam Syafi’i …. ﻋﻘﺪ ا½ح ا ﺰوجÉو . 70 ﻋﻘﺪ ا½ح ا ﺰوجÉو . 70 ﻋﻘﺪ ا½ح ا ﺰوجÉو . 70 ”Wali (yang menguasai) ikatan nikah adalah suami..” Ketujuh, ijma' sahabat, diriwayatkan Syuraih dari ‘Alī ibn Abī Ṭālib berkata: Ketujuh, ijma' sahabat, diriwayatkan Syuraih dari ‘Alī ibn Abī Ṭālib berkata: ﻘ ةا½ ا أنا¿ي 71 أن ا¿ي ﺑﻴﺪه ﻋﻘﺪة ا½ح ا ﺰوج . 71 "Sesungguhnya orang yang mempunyai kuasa ikatan nikah adalah suami." Imam Syafi’i merombak kembali hasil ijtihadnya pada waktu berdomisili di Irak karena beliau memiliki nalar ijtihad yang berbeda dengan sebelumnya. Kematangan pola pikir Imam Syafi’i setelah beliau berada di Mesir cukup me- warnai kesimpulan hukum yang beliau cetuskan. Terbukti, meskipun per- soalaan di atas berakar dari ayat yang sama namun memunculkan produk hukum yang berbeda. Hal itu terjadi karena perbedaan pemahaman ijtihad Imam Syafi’i terhadap naṣ al-Qur’an. Mencermati ulasan dalil-dalil di atas dapat ditegaskan bahwa qawl jadīd memiliki sumber rujukan yang lebih jelas dan kuat. Selain berlandaskan analisa gramatika atas al-Qur’an juga dikukuhkan oleh hadis dan ijma' para sahabat. Jadi, peralihan ijtihad Imam Syafi’i dari qawl qadīm pada qawl jadīd karena ditemukan hadis yang sebelumnya tidak diketahui, dan penemuan terhadap qawl sahabat, serta karena perubahan logika pemahaman terhadap sumber hukum dan dalil yang digunakan sebelumnya. 70Muḥammad ibn Aḥmad al-Anṣārī al-Qurṭubī, al-Jāmi’ li Aḥkām al-Qur’ān, Jilid II, h. 206. 72QS. al-Mā`idah : 3. 73Muḥyiddīn ibn Sharaf al-Nawawī, al-Tanqīh fī Sharḥ al-Wasīṭ, Jilid I, h. 233; ‘Abdullāh ibn Yūsuf al-Juwainī, Nihāyah al-Maṭlab fī Nihāyah al-Madhhab, Jilid. I, 29; Yaḥyā ibn Sharḥ al-Nawawī, Rawḍah al-Ṭālibīn, Juz I, h.152; Muḥammad ibn Muḥammad al-Ghazālī, al-Wajīz fī fiqh al-Imām al-Shāfi’ī, Juz I, h. 119; Yūsuf al-Fairuz Abādī al-Shairazī, al-Muhadhdhab fī Fiqh al-Imām al-Shāfi’ī, Juz I, h. 27-28. 74Imam al-Māwardī, al-Hāwī al-Kabīr, Juz I, h. 69; Abū Zakariyā Muḥyiddīn ibn Sharaf al-Nawawī, al-Majmū' Sharḥ al-Muhadhdhab, Jilid I, h. 228-229. 74Imam al-Māwardī, al-Hāwī al-Kabīr, Juz I, h. 69; Abū Zakariyā Muḥyiddīn ibn Sharaf al-Nawawī, al-Majmū' Sharḥ al-Muhadhdhab, Jilid I, h. 228-229. 72QS. al-Mā`idah : 3. Jual Beli Kulit Bangkai yang telah Disamak Dalam qawl qadīm Imam Syafi'i berpendapat bahwa menjual kulit bangkai yang telah disamak hukumnya tidak boleh. Karena dispensasi kebolehan hanya dalam hal memanfaatkan saja, sedangkan hukum mentasarufkan (men- AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 Volume 26, Nomor 2, Oktober 2016 171 Ainol Yaqin distribusikan) tetap haram. Pendapat ini berdasarkan firman Allah dalam surat al-Mā`idah ayat 3: مËﻢ ا ﻤﻴﺘﺔ واÁﺣﺮﻣﺖ ﻋﻠﻴ ُM َ ُ َ َ َ ْ ْ ْ َ ْ ُ ُ َ ِّ ُ .... ﴿ Ã ﴾ مËﻢ ا ﻤﻴﺘﺔ واÁﺣﺮﻣﺖ ﻋﻠﻴ ُM َ ُ َ َ َ ْ ْ ْ َ ْ ُ ُ َ ِّ ُ .... ﴿ Ã ﴾ 73Muḥyiddīn ibn Sharaf al-Nawawī, al-Tanqīh fī Sharḥ al-Wasīṭ, Jilid I, h. 233; ‘Abdullāh ibn Yūsuf al-Juwainī, Nihāyah al-Maṭlab fī Nihāyah al-Madhhab, Jilid. I, 29; Yaḥyā ibn Sharḥ al-Nawawī, Rawḍah al-Ṭālibīn, Juz I, h.152; Muḥammad ibn Muḥammad al-Ghazālī, al-Wajīz fī fiqh al-Imām al-Shāfi’ī, Juz I, h. 119; Yūsuf al-Fairuz Abādī al-Shairazī, al-Muhadhdhab fī Fiqh al-Imām al-Shāfi’ī, Juz I, h. 27-28. "Diharamkan bagimu (memakan) bangkai, darah….. ". 72 "Diharamkan bagimu (memakan) bangkai, darah….. ". 72 "Diharamkan bagimu (memakan) bangkai, darah….. ". 72 Sedangkan dalam qawl jadīd ia berpendapat bahwa menjual kulit yang telah disamak hukumnya boleh.73 Argumentasinya, 'illat najis yang menyebab- kan menjual kulit bangkai tidak dibolehkan itu bisa hilang setelah disamak. Karena 'illat yang melarang telah hilang, sehingga kulit yang disamak menjadi suci dan dapat diperjual-belikan. Sebagaimana khamr bila telah berubah men- jadi cuka hukum menjualnya menjadi boleh dan sah. Imam al-Nawawī menegaskan bahwa yang tidak dibolehkan memanfaat- kan 'ain bangkai, sedangkan memperjual-belikan kulit bangkai sah-sah saja. Aṣḥāb Imam Syafi’i menjawab hujjah qawl qadīm dengan dianalogikan pada budak ummu walad, waqaf dan makanan yang terdapat di daerah peperangan (dār harb). Dianalogikan pada ketidakbolehan menjual ummu walad karena terdapat hak hurriah pada ummu walad. Diqiyaskan pada waqaf karena harta waqaf tidak dapat dimiliki siapa pun sebab telah menjadi milik Allah. Disama- kan dengan makanan di daerah peperangan dibolehkan untuk memenuhi kebutuhan fisik sekedar mencukupi perut. Sedangkan persoalan kulit bangkai berkaitan dengan kenajisannya. Jika sifat najisnya telah tiada maka menjadi suci dan hukum jual-belinya pun menjadi sah. Menurut aṣḥāb bahwa pendapat yang ṣaḥīḥ adalah qawl jadīd. Pendapat ini seirama dengan pendapat Abū Ḥanīfah dan jumhūr ulamā'.74 Tampaknya, faktor yang mempengaruhi adanya perubahan ijtihad Imam Syafi’i dari qawl qadīm pada qawl jadīd karena ia berhujjah dengan qiyas ter- Q 73Muḥyiddīn ibn Sharaf al-Nawawī, al-Tanqīh fī Sharḥ al-Wasīṭ, Jilid I, h. 233; ‘Abdullāh ibn Yūsuf al-Juwainī, Nihāyah al-Maṭlab fī Nihāyah al-Madhhab, Jilid. I, 29; Yaḥyā ibn Sharḥ al-Nawawī, Rawḍah al-Ṭālibīn, Juz I, h.152; Muḥammad ibn Muḥammad al-Ghazālī, al-Wajīz fī fiqh al-Imām al-Shāfi’ī, Juz I, h. 119; Yūsuf al-Fairuz Abādī al-Shairazī, al-Muhadhdhab fī Fiqh al-Imām al-Shāfi’ī, Juz I, h. 27-28. 74Imam al-Māwardī, al-Hāwī al-Kabīr, Juz I, h. 69; Abū Zakariyā Muḥyiddīn ibn Sharaf al-Nawawī, al-Majmū' Sharḥ al-Muhadhdhab, Jilid I, h. 228-229. AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 172 Volume 26, Nomor 2, Oktober 2016 Evolusi Ijtihad Imam Syafi’i …. hadap kasus yang bersumber pada naṣ. Beliau menilai dalil yang menopang qawl qadīm kurang cocok sebab ayat di atas menyebutkan secara umum, yang membuka peluang ditemukannya dalil-dalil lain yang mentakhsisnya. 75 ‘Ali ibn ‘Umar al-Daruquṭnī, Sunan al-Daruquṭnī, Jilid I, h. 39. 76Muḥyiddīn ibn Sharaf al-Nawawī, al-Tanqīh fī Sharḥ al-Wasīṭ, Jilid I, h. 235; Muḥammad ibn Muḥammad al-Ghazālī, al-Wajīz fī fiqh al-Imām al-Shāfi’ī, Juz I, h.119; Shaikh Sulaimān al-Jamal, al- Jamal ‘ala Sharḥi al-Minhāj, Jilid I (Beirut: Dār Ihyā’ al-Turāth al-‘Arabī, t.th.), h. 181; Yūsuf al-Fairūz Abādī al-Shairazī, al-Muhadhdhab fī Fiqh al-Imām al-Shāfi’ī, Juz I, h. 28. 77Abū Zakariyā Muḥyiddīn ibn Sharaf al-Nawawī, al-Majmū' Sharḥ al-Muhadhdhab, Jilid I, h. 229. 76Muḥyiddīn ibn Sharaf al-Nawawī, al-Tanqīh fī Sharḥ al-Wasīṭ, Jilid I, h. 235; Muḥammad ibn Muḥammad al-Ghazālī, al-Wajīz fī fiqh al-Imām al-Shāfi’ī, Juz I, h.119; Shaikh Sulaimān al-Jamal, al- Jamal ‘ala Sharḥi al-Minhāj, Jilid I (Beirut: Dār Ihyā’ al-Turāth al-‘Arabī, t.th.), h. 181; Yūsuf al-Fairūz Abādī al-Shairazī, al-Muhadhdhab fī Fiqh al-Imām al-Shāfi’ī, Juz I, h. 28. 75 ‘Ali ibn ‘Umar al-Daruquṭnī, Sunan al-Daruquṭnī, Jilid I, h. 39. 77Abū Zakariyā Muḥyiddīn ibn Sharaf al-Nawawī, al-Majmū' Sharḥ al-Muhadhdhab, Jilid I, h. 229. Memakan Kulit Bangkai yang Sudah Disamak Kulit bangkai yang sudah disamak dari hewan yang dapat dimakan daging- nya, apakah boleh dimakan atau tetap haram? Menurut qawl qadīm Imam Syafi’i menyatakan bahwa kulit bangkai yang disamak hukumnya haram untuk dimakan. Pendapat ini berdasarkan pada hadis yang diriwayatkan Ibnu Abbās bahwa Nabi bersabda: ّﻫﻼ ﻠﻬﺎd أﺧﺬﺗﻢ إﻫﺎﺑﻬﺎ ﻓﺪﺑﻐﺘﻤﻮه ﻓﺎﻧﺘﻔﻌﺘﻢ ﺑﻪ ﻗﺎ ﻮا اﻧﻬﺎ ﻣﻴﺘﺔ ﻗﺎل إﻧﻤﺎ ﺣﺮم أ . 75 "Tidakkah kalian mengambil dagingnya (bangkai), lalu samak dan kalian dapat memanfaatkan. Para sahabat bertanya: hewan itu sudah mati, Nabi me- responsnya "bangkai yang diharamkan hanya memakannya." "Tidakkah kalian mengambil dagingnya (bangkai), lalu samak dan kalian dapat memanfaatkan. Para sahabat bertanya: hewan itu sudah mati, Nabi me- responsnya "bangkai yang diharamkan hanya memakannya." Sedangkan dalam qawl jadīd ia berpendapat kulit bangkai binatang yang halal dimakan dagingnya, hukum memakan kulit yang telah disamak juga halal. Karena kulit bangkai yang telah suci dengan cara disamak statusnya sama dengan kulit binatang yang disembelih sesuai syara'. Tetapi, kulit bang- kai binatang yang haram dimakan dagingnya meskipun telah disamak kulitnya tetap dihukumi haram memakannya.76 Menurut jumhūr, pendapat yang ṣaḥīḥ adalah pendapat qawl qadīm kerena secara ṣarīh Rasulullah menegaskan bahwa haram memakan daging bangkai. Tetapi, sebagian ulama menṣaḥīḥkan qawl jadīd, diantaranya: al- Qaffāl, al-Fairanī, al-Rauyanī, al-Jurjānī.77 Dengan demikian, perubahan ijtihad Imam Syafi’i dari qawl qadīm pada qawl jadīd jelas dipengaruhi perubahan logika pemahaman atas dalil hukum AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 Volume 26, Nomor 2, Oktober 2016 173 Volume 26, Nomor 2, Oktober 2016 Ainol Yaqin yang digunakannya. Mengalisis hadis di atas melahirkan kesimpulan bahwa qawl jadīd dapat diunggulkan daripada qawl qadīm. Sebab jawaban Nabi "bangkai yang diharamkan hanya memakannya" itu muncul dari pertanyaan sahabat "binatang itu sudah jadi bangkai". Jadi, jelas yang dilarang oleh Nabi memakan bangkai, bukan memakan daging bangkai yang sudah disamak karena dalam hadis Nabi tidak menyinggung hukum itu.78 78‘Abdullāh ibn Yūsuf al-Juwainī, Nihāyah al-Maṭlab fī Nihāyah al-Madhhab, Jilid I, h. 29-30. Kesimpulan Berdasarkan paparan dalil-dalil yang melandasi qawl qadīm dan qawl jadīd, maka dapat dikatakan bahwa perubahan hasil ijtihad Imam Syafi’i dari qawl qadīm pada qawl jadīd disebabkan beberapa faktor, yaitu pertama, dalam bidang ibadah, faktor-faktor yang mempengaruhi perubahan hasil ijtihad Imam Syafi’i disebabkan temuan hadis ṣaḥīḥ (masalah zakat madu), hadis, qawl dan ijma’ shahābah serta nalar al-ra’yu/logika (masalah mengqada’ puasa). Kedua, dalam bidang munākahat, faktor-faktor yang menyebabkan perubahan hasil ijtihad Imam Syafi’i dikarenakan wajh istidlāl/nalar berpikir dan analogi (masalah status mahar yang rusak), athār dan qawl ṣaḥabah yang lebih rājih (masalah hukum nikah pada masa ‘iddah), hadis dan athār ṣaḥabah yang ṣaḥīḥ (masalah nikah istri yang ditinggalkan suami), hadis ṣaḥīḥ yang bersifat khāsh (masalah keluar rumah di saat ‘iddah), nalar al-ra’yu, analisa gramatika bahasa, hadis dan ijma’ ṣaḥabah (masalah biyadihi ‘uqdah al-nikah). Ketiga, dalam bidang mu’āmalah, faktor-faktor yang mempengaruhi per- ubahan hasil ijtihad Imam Syafi’i didasarkan pada qiyas/analogi (masalah jual beli kulit bangkai yang sudah disamak), dan nalar al-ra’yu yang lebih matang (masalah memakan kulit bangkai yang sudah disamak). Selain hal itu, kita pula dapat memetik sikap mulia dan tingginya keilmuan Imam Syafi’i sebagai imam mazhab dan ilmuwan sejati. Sikap-sikap mulia tersebut adalah pertama, rasa ingin tahu dan haus ilmu pengetahuan. Hal ini, tercermin di sepanjang perjalanan hidupnya dihabiskan untuk mencari ilmu, baik dengan cara membaca, menghafal, menulis, berdiskusi, berdebat untuk mencari kebenaran maupun berguru pada syaikh-syaikh ternama. AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 ║ 174 Volume 26, Nomor 2, Oktober 2016 Evolusi Ijtihad Imam Syafi’i …. Kedua, pencari kebenaran bukan pembenaran. Ia tidak merasa malu untuk merombak hasil ijtihadnya karena terjerumus pada dalil-dalil yang lemah. Kemudian membangun kembali di atas dalil-dalil yang lebih kuat dan kokoh. Ketiga, keberaniannya dalam mengakui dan menyadari kesalahan. Fatwa- fatwa yang beliau rumuskan jika terbukti terdapat kesalahan, maka tidak segan untuk ditarik kembali, kemudian dikoreksi dan diubah dengan men- cetuskan fatwa baru.[a] DAFTAR PUSTAKA al-‘Asqalānī, Aḥmad ibn ‘Alī ibnu Hajar, Taghlīq al-Ta’līq ‘alā Ṣaḥīḥ al-Bukhāri, Beirut: Dār ‘Imār, 1985. __________, Tawālī al-Ta`sīs, Beirut: Dār al-Kutub al-‘Ilmiah, 1986. __________, al-Maṭālib al-‘Āliyyah bi Zawā`idi al-Masānid al-Thamāniah, Riyad: Dār al-‘Āshimah, 1998. al-Bayhaqī, Abū Bakar Aḥmad ibn al-Ḥusain, Kitāb al-Sunan al-Ṣaghīr, Beirut: Dār al-Fikr, t.th. al-Bannā, Aḥmad ‘Abdurrahmān, al-Bulūgh al-Amālī min Asrār al-Fatḥ al- Rabbānī, Beirut: Dār Ihyā` al-Turāth al-‘Arabī, t.th. al-Bujairamī, Sulaimān ibn Muḥammad, al-Bujairamī ‘ala ’l-Khaṭīb, Beirut: Dār al-Kutub al-‘Ilmiah, 1996. al- Dāruquṭnī, Alī ibn ‘Umar, Sunan al-Dāruquṭnī, Beirut: Dār al-Fikr, t.th. Daud, Abū, Sunan Abī Daud, Beirut: Dār al-Fikr, t.th. Daud, Abū, Sunan Abī Daud, Beirut: Dār al-Fikr, t.th. al-Ghazālī, Muḥammad ibn Muḥammad, al-Wajīz fī Fiqh al-Imām al-Syafi’i, Beirut: Dār al-Arqam, 1997. al-Ghazālī, Muḥammad ibn Muḥammad, al-Wasīṭ fi ’l-Madhhab, Kairo: Dār al- Salām, 1997. al-Ḥasanī, Muḥammad ibn 'Alawī al-Mālikī, al-Manhal al-Laṭīf fī Uṣūl al-Ḥadīth al-Syarīf, Beirut: Dār al-Fikr, 1978. al-Jamāl, Shaikh Sulaimān, al-Jamāl ‘alā Sharḥ al-Minhāj, Beirut: Dār Ihyā’ al- Turāth al-‘Arabī, t.th. al-‘Imrānī, Sālim, al-Bayān fī Madhhab al-Imām al-Shāfi’i, Beirut; Dār al-Minhāj, t.th. AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 Volume 26, Nomor 2, Oktober 2016 ║175 AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 KAM 854-4603; e-ISSN: 2502-3209 Volume 26, Nomor 2, Oktober 2016 ║175 Ainol Yaqin ‘Iyāḍ, Mūsa ibn, Ikmāl al-Mu’lim bi Fawāidi Muslim, Beirut: Dār al-Wafā’, 1998. al-Juwainī, ‘Abdullāh ibn Yūsuf, Nihāyah al-Maṭlab fī Dirāyah al-Madhhab, Jeddah: Dār al-Minhāj, 2007. Kathīr, Ismā’īl ibn ‘Umar ibn Ibnu, Tafsīr al-Qur’ān al-‘Aẓīm, Beirut: Dār Ṭayyibah, t.th. al-Khaṭīb, Muḥammad 'Ajjāj, Uṣūl al-Ḥadīth, Beirut: Dār al-Fikr, 1989. al-Mālikī, Ḥasan Sulaimān al-Nūri dan 'Alawī 'Abbās, Ibānah al-Aḥkām Sharḥ Bulūgh al-Marām, Beirut: Dār al-Fikr, 2000. al-Maṭlab, Rif’at Faurī ‘Abdu, Muqaddimah al-Taḥqīq al-Umm, Beirut: Dār al- Wafa’, 2001. al-Māwardī, Imām, al-Hāwi al-Kabīr, Beirut: Dār al-Fikr, 1994. Mūsa, Qādlī Iyādl ibn, Tartīb al-Madārij wa Taqrīb al-Masālik, Beirut: Dār al- Kutub al-‘Ilmiah, 2001. al-Muzannī, Ismā’īl ibn Yaḥyā ibn Ismā’īl, Mukhtaṣar al-Muzannī, Beirut: Dār al- Kutub al-‘Ilmiah, 1998. al-Naisabūrī, Abū al-Ḥusain Muslim, Ṣaḥīḥ Muslim, Beirut: Dār al-Fikr, 1988. al-Nawawī, Abū Zakariyā ibn Yaḥyā ibn Sharaf, Rawḍah al-Ṭālibīn, Riyaḍ: Dār ‘Ālam al-Kutub, 2003. al-Nawawī, Abū Zakariyā Muhyiddīn ibn Sharaf, al-Majmū’ Sharḥ al- Muhadhdhab, Beirut: Dār al-Fikr, t.th. al-Nawawī, Abū Zakariyā Muhyiddīn ibn Syarah, Ṣaḥīḥ Muslim bi Sharḥ al- Imām al-Nawawī, Beirut: Dār al-Fikr, t.th. al-Nawawī, Muhyiddīn ibn Syarf, al-Tanqīh fī Sharḥ al-Wasīṭ, Kairo: Dār al- Salam, 1997. al-Nawawī, Yaḥyā ibn Sharaf, Tashhīh al-Tanbīh, Beirut: Muassasah al-Risālah, 1996. al-Ṭabarī, Muḥammad ibn Jārir, Tafsīr al-Ṭabarī, Beirut: Dār al-Kutub al-‘Ilmiah, 2009. AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 DAFTAR PUSTAKA al-Qalyūbī, Aḥmad ibn Salamah, Hāsyiyyah Qalyūbī, Kairo: Musthafā al-Bābī al- Halabī, 2007. al-Qaṭṭān, Mannā’, Tārīkh al-Tashrī’ al-Islāmī, Beirut: Dār al-Fikr, 1995. al-Qazwīnī, Abdulkarīm al-Rāfi’ī, al-‘Azīz Sharḥ al-Wajīz, Beirut: Dār al-Kutub al-‘Ilmiah, 2007. al-Qazwīnī, Muḥammad ibn Yazīd, Sunan Ibn Mājah, Beirut: Dār al-Fikr, t.th. AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 176║ Volume 26, Nomor 2, Oktober 2016 176║ Evolusi Ijtihad Imam Syafi’i …. al-Qazwinī, Muḥammad ibn Yazīd, Sunan Ibn Majah, Beirut: Dār al-Kutub al- ‘Ilmiah, 2009. al-Qurṭubī, Aḥmad ibn ‘Umar, al-Mufhim limā Asykala min Talkhīsh Kitāb Muslim, Beirut: Dār Ibnu Kathīr, 1996. al-Qurṭubī, Muḥammad ibn Aḥmad al-Anṣārī, al-Jāmi’ li Aḥkām al-Qur’ān, Beirut: Dār al-Fikr, t.th. al-Ramlī, Muḥammad ibn Abī ‘Abbās, Nihāyah al-Muḥtāj ilā Sharḥ al-Minhāj, Beirut: Dar al-Fikr, t.th. al-Rāzī, Abdurrahmān ibn Abī Hātim, Ādāb al-Syafi’i wa Manāqibuhu, Beirut: Dār al-Kutub al-‘Ilmiah, 2003. al-Rāzī, Muḥammad ibn ‘Umar ibn Ḥusain, al-Tafsīr al-Kabīr, Beirut: Dār al- Kutub al-‘Ilmiah, 2004. al-Rūyānī, ‘Abdulwāhid ibn Ismā’īl, Bahru al-Mazhab fī Furū’ al-imām al-Syafi’i, Beirut: Ihyā` al-Turāth al-‘Arabī, 2002. al-Sajastanī, Abū Daud Sulaimān, Sunan Abi Daud, Beirut: Dār al-Fikr, t.th. al-Sāyis, Muḥammad Alī, Tārīkh al-Fiqh al-Islāmī, Beirut: Dār al-Fikr, 1995. al-Subkī, Tājuddīn Abdul al-Wahhāb, Jam'u al-Jawāmi', Beirut: Dār al-Fikr, 1982. al-Suyūṭī, Jalāluddīn ‘Abdurrahmān, al-Tausyīh Sharḥ al-Jāmi’ al-ṣaḥīḥ, Riyad: Maktabah al-Rusyd, 1998. al-Suyūṭī, Jalāluddīn, al-Durru al-Manthūr fī al-Tafsīr bil ‘l-Ma`thūr, Kairo: Markaz Ḥajr li ‘l-Buḥūth wa al-Dirāsāt al-‘Arabīyah wa al-Islāmiyyah, 2003. al-Suyūṭī, Jalāluddīn, Sunan al-Nasā`ī, Beirut: Dār al-Fikr, 1930. al-Syafi’i, Muḥammad ibn Idrīs, al-Risālah, Beirut: Dār al-Fikr, 1309 H. al-Syafi’i, Muḥammad ibn Idrīs, al-Umm, Beirut: Dār al-Wafa’, 2001. al-Syairazī, Yūsuf al-Fairūz Abādī, al-Muhadhdhab fī Fiqh al-Imām asy-Syafi’i, Beirut: Dār al-Kutub al-‘Ilmiah, 1995. al-Sharbīnī, Muḥammad ibn al-Khaṭīb, Mughnī al-Muḥtāj ilā Ma’rifati Ma’ānī `Alfāẓ al-Minhāj, Beirut: Dār al-Ma’rifah, 1998 al-Sharbīnī, Yūsuf al-Fairūz Abādī, al-Muhadhdhab fī Fiqh al-Imām al-Syafi’i, Beirut: Dār al-Kutub al-‘Ilmiah, 1995. al-Sharwanī, Abdulhamīd, Hawāsyī Tuhfah al-Minhāj bi Sharḥ al-Minhāj, Kairo: al-Maktabah al-Tijāriyyah al-Kubra, 2007. AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 Volume 26, Nomor 2, Oktober 2016 ║1 177 Volume 26, Nomor 2, Oktober 2016 Ainol Yaqin al-Ṭabarī, Muḥammad ibn Jārir, Tafsīr al-Ṭabarī, Beirut: Dār al-Kutub al-‘Ilmiah, 2009. al-Ṭurmudhī, Imam, Sunan al-Ṭurmudhī, Beirut: Dār al-Fikr, 1994. al-Wasytānī, Muḥammad ibn Khalfah, Ikmāl Akmāl al-Mu’lim, Beirut: Dār al- Kutub al-‘Ilmiah, t.th. Zahrah, Muḥammad Abū, al-Syafi’i, Beirut: Dār al-Fikr al-‘Arabī, 1978. al-Zuḥailī, Wahbāh, al-Tafsīr al-Munīr, Beirut: Dār al-Fikr, 1998. AL-AHKAM p-ISSN: 0854-4603; e-ISSN: 2502-3209 ║ Volume 26, Nomor 2, Oktober 2016 178║ 178
https://openalex.org/W2892093735	https://scielo.conicyt.cl/pdf/maderas/v20n4/0718-221X-maderas-04101.pdf	English	null	Using artificial neural networks in estimating wood resistance	Maderas. Ciencia y tecnología	2,018	cc-by	5,940	531 1Universidade de Brasília (UnB), Departamento de Engenharia Florestal (EFL), Brasília, DF, Brazil. 2Universidade Federal Rural do Semi-Árido (UFERSA), Centro de Ciências Agrárias (CCA), Departamento de Ciências Agronômicas e Florestais (DCAF), Mossoró, RN, Brasil. 3Universidade Federal de Mato Grosso (UFMT), Programa de Pós-Graduação em Ciências Florestais e Ambientais (PPGCFA), Sinop, MT, Brazil. ♠Corresponding author: miguelederpereira@gmail.com Received: 10.04.2017 Accepted: 10.04.2018 DOI: 10.4067/S0718 221X2018005004101 ISSN online 0718 221X USING ARTIFICIAL NEURAL NETWORKS IN ESTIMATING WOOD RESISTANCE Eder Pereira Miguel1,♠, Rafael Rodolfo de Melo2, Laércio Serenini Junior3, Cláudio Henrique Soares Del Menezzi1 ABSTRACT The purpose of this research was to evaluate the potential of Artificial Neural Networks in estimating the properties of wood resistance. In order to do so, a hybrid of eucalyptus (Eucalyptus urograndis) planted in the Northern Region of the State of Mato Grosso was selected and ten trees were collected. Then, four samples of each tree were removed, totaling 40 samples, which were later subjected to non-destructive testing of apparent density, ultrasonic wave propagation velocity, dynamic modulus of elasticity obtained by ultrasound, and Janka hardness. These properties were used as estimators of resistance and compressive strength parallel to fibers, and hardness. Multilayer Perceptron networks were also employed, training 100 of them for each of the evaluated parameters. The obtained results indicated that the use of Artificial Neural Networks is an efficient tool for predicting wood resistance. Keywords: Artificial intelligence, Eucalyptus urograndis, hardness, mechanical properties, non- destructive testing. Maderas. Ciencia y tecnología 20(4): 531 - 542, 2018 DOI: 10.4067/S0718-221X2018005004101 ISSN impresa 0717-3644 ISSN online 0718-221X USING ARTIFICIAL NEURAL NETWORKS IN ESTIMATING WOOD RESISTANCE Maderas. Ciencia y tecnología 20(4): 531 - 542, 2018 DOI: 10.4067/S0718-221X2018005004101 0717-3644 0718-221X ISSN impresa ISSN online INTRODUCTION The genus Eucalyptus is among the most successful commercially-planted tree species in the tropics, mainly due to its rapid growth, species diversity and a wide range of industrial uses. Brazil is one of the countries where clonal forestry of the Eucalyptus genus has developed the most. The Eucalyptus urograndis is a hybrid developed in Brazil by crossing the species E. grandis x E. urophylla. The first plantation of E. urograndis took place in the state of Espírito Santo in 1979, but it was in the 1990s that this species boosted the rate of forest growth, as well as a more homogeneous quality of planted forests. This hybrid is currently one of the most planted forest essences in the country, constituting the basis of Brazilian clonal forestry. One of the most planted clones in the world is H13, which is widely used by several companies to produce wood for cellulose and coal because of its fast growth and 531 Maderas. Ciencia y tecnología 20(4): 531 - 542, 2018 Universidad del B í o - B í o considerably favorable energetic characteristics. Some elementary features of wood are directly related to their properties. Among these are the wood density and its response to the propagation of acoustic waves by non-destructive methods. These characteristics are obtained easily and quickly and can be used to estimate the remaining properties of wood. However, these characteristics are still unknown and need to be studied, demanding suitable techniques that could expedite fast and efficient prediction of such characteristics. Considering this, Artificial Neural Networks (ANNs) could be a promising tool. considerably favorable energetic characteristics. Some elementary features of wood are directly related to their properties. Among these are the wood density and its response to the propagation of acoustic waves by non-destructive methods. These characteristics are obtained easily and quickly and can be used to estimate the remaining properties of wood. However, these characteristics are still unknown and need to be studied, demanding suitable techniques that could expedite fast and efficient prediction of such characteristics. Considering this, Artificial Neural Networks (ANNs) could be a promising tool. Artificial Neural Networks (ANNs) are mathematical models that use artificial intelligence in order to solve certain complex problems. These are formed by simple processing elements which are artificial neurons, and are activated by a function called activation function. INTRODUCTION Neurons are linked together by connections usually associated with coefficients or weights that are adjusted by training algorithms. Neurons are responsible for removing the peculiarities of the database and storing the knowledge of networks. As basic characteristics, networks have adaptive learning, self-organizing capacity and a robust structure with parallel distribution (layers). They are efficient in learning and generalization, and in addition to being tolerant to outliers, they are also able to model different variables and their non-linear relationships, as well as enabling quantitative and qualitative variable modeling (Kuvendziev et al. 2014, Haykin 2001). These premises make ANNs stand out, as their adjustment methodology is based on machine learning regardless of the model (Alves et al. 2017), with wide application in agricultural sciences, engineering and medicine. In practice, they are data modeling tools that are widely used in predictions, mappings and pattern recognition, being able to go beyond human capacity to search large databases and relate them to a specific desirable characteristic at the same time (Goyal 2013). The applicability of artificial neural networks in wood science has already been evaluated by several authors (Tiryaki and Hamzacebi 2014, Tiryaki and Aydin 2014, Okan et al. 2015, Melo and Miguel 2016, Tiryaki et al. 2016, Bardak et al. 2016), which denotes its potentiality. The purpose of this research was to evaluate the potential of ANNs in estimating wood resistance in young individuals of Eucalyptus urograndis, one of the main species used by Brazilian silviculture. Description of the study area and the choice of individuals The wood samples were taken from a Eucalyptus urograndis plantation in the city Sinop, Mato Grosso. They were extracted from a clone selected from a 5-year-old Eucalyptus clonal testing plantation. The test was done in a completely arbitrary design with 4 replicates, using a spacing of 3 x 2 m. To distinguish the clone, ten trees were felled and sampling was performed following the recommendations of the Pan American Standards Commission (COPANT 458, 1972). Prior to the tests, the samples were evaluated through non-destructive testing methods of apparent density, while the dynamic modulus of elasticity was obtained by ultrasound. The hardness, resistance and compressive strength were determined parallel to the fibers. The tests were implemented following the recommendations of the NBR 7190 (Brazilian National Standards Organization – ABNT, 2011). Determining the Dynamic Modulus of Elasticity (Ed) The test was conducted in the Pundit Lab of Proceq, which uses a 24 kHz frequency flat transducer and receiver placed in the longitudinal direction of the samples, and when using a coupling (which in this case was solid Vaseline), ultrasonic waves passed through the wood. The calibration of the instrument was done with a calibration block (25,4 μs) and automatic precision adjustment. The time and wave propagation velocity were documented for calculating the dynamic modulus of elasticity (Ed) 532 Maderas. Ciencia y tecnología 20(4): 531 - 542, 2018 Using artificial neural networks in..: Miguel et al. of the material. The dynamic modulus of elasticity was calculated by Equation 1. 2 5 9,804 10 ap V Ed x ρ − = (1) 2 5 9,804 10 ap V Ed x ρ − = (1) In which: Ed = dynamic modulus of elasticity (MPa); V = wave propagation speed (m/s); ρap = apparent density (kg/m³) Network Selection and Validation The best ANN was selected based on the correlation between observed variables and those estimated by the networks, the stability of the training levels of the networks provided by the software during the training, selection and evaluation phases, which means they must have little variation between them (Miguel et al. 2016), the root-mean-square error in percentage form and a graphical analysis of the residues. The RMSE evaluates the mean squared error between the observed and the estimated values. The lower the RMSE, the better the average precision of the estimates, with the optimal situation being when it is equal to zero (Mehtätalo et al. 2006) (Equation 5). (5) 2 1 1( ) 100 (%) n xi x RMSE n x = − = ∑ In which: is the average of the variables of interest (hardness, modulus of elasticity and modulus of rupture); are the variables predicted by ANN; xi are the individual values of each variable, and “n” are the total number of observations related to each variable. x An independent sample corresponding to 25% of the database samples (10 of them) was randomly selected in order to validate the results. The chosen criteria to validate the adjustments were the Chi- squared test (χ²), an aggregated difference in percentage (AD%) and the graph of the behavior between observed and estimated values. Maderas. Ciencia y tecnología 20(4): 531 - 542, 2018 Universidad del B í o - B í o Maderas. Ciencia y tecnología 20(4): 531 - 542, 2018 In which: = sigmoid activation function; β = estimation of the parameter that determines curve of the sigmoid function; u = potential of the activation function. Resilient propagation was used as the algorithm for this type of activation function and network category, and the training parameters were a learning rate (μ) of 0,2 and a momentum term (η) of 0,9. Network training First, the weights of all the networks were randomly generated (Heaton 2011). Next, the individual update value evolved during the learning process based on the error function. A supervised method was adopted for the network training, in which the input and output variables were indicated for the networks. This is a feedforward method and it uses the algorithm for unidirectional data flow without cycles (Haykin 2001). Network training continued until the error rate was reduced to an acceptable range between the predicted values and the actual values supplied to the network, known as the delta rule, or until the maximum number of times or cycles was reached (Shiblee et al. 2010). The criterion for stopping the training algorithm was reaching the root-mean-square error (RMSE) of less than 1%, or when the root-mean-square error increased again (as suggested by Chen et al. 2014), so the training was finalized when one of the criteria was reached. Adjustments of ANNs For the adjustment of the ANNs, 75% of the information that composed the database of the (30) samples was randomly selected. The input layer was composed of three neurons, one for each independent variable: wood density (ρb), velocity (V0) and dynamic modulus of elasticity (Ed). The output layer was also composed of three neurons, which are (in practice) the response/predictor variables: hardness (fH), modulus of elasticity (Ec0) and modulus of rupture (fc0), with 1000 networks having been trained. The ANN adjustment was performed with the help of the Intelligent Problem Solver (IPS) tool of Statistica 7 software (Statsoft 2007), which standardizes the data between 0-1 and tests several architectures, asserting those that best fit the problem. Only MLP (Multilayer Perceptron) networks were selected for the training. According to Silva et al. (2010), this category of networks allows for the neural layer of output to be composed of different neurons, where each corresponds to one of the process outputs to be mapped. The networks were also made up of a single hidden layer. According to Esquerre (2002), most of the time networks require a single hidden layer to non-linearly solve separable problems. The number of neurons in the hidden layer was also optimized with the Intelligent Problem Solver. An artificial neuron is the information processing unit of an ANN, formed by “n” inputs x1, x2,.. xn (dendrites) and an output “y” (axon). The inputs are associated with weights w1, w2,..., wn which represent the synapses. These can be negative or positive. Currently, a basic model of an artificial neuron can be represented mathematically as Equation 2: ( ) k k Y V ϕ = (2) ( ) k k Y V ϕ = (2) In which: Yk = output of the artificial neuron; = activation function; Vk = linear combiner result, which is Equation 3: m k n n o V x w =∑ (3) m k n n o V x w =∑ (3) The sigmoid function was preferred in activating the ANN training, as it is the most usual in the elaboration of artificial neural networks, and which can approximate any continuous function with precision in well-designed architectures. Mathematically, it is given by Equation 4: 1 ( ) 1 u v expβ ϕ = + (4) 533 Parameters employed evaluating the networks The mean values of density, wave propagation velocity, dynamic modulus of elasticity, hardness, modulus of elasticity and rupture of parallel compression are shown in Table 1. The values observed in 534 Using artificial neural networks in..: Miguel et al. Maderas. Ciencia y tecnología 20(4): 531 - 542, 2018 density were similar to those observed by Bassa et al. (2007) in studying hybrids from the same species at age 7, and found density values of 521 and 543 kg/m³, respectively. density were similar to those observed by Bassa et al. (2007) in studying hybrids from the same species at age 7, and found density values of 521 and 543 kg/m³, respectively. The values of elasticity (Ec0) and resistance (fc0) obtained by the compression test parallel to the fibers can also be observed in Table 1. The mean value of elasticity found in Eucalyptus urograndis was 14721 MPa. Studying Eucalyptus grandis wood at the age of 15 years, Stangerlin et al. (2008) found mean values of elasticity for the compression test parallel to the fibers of 13119 MPa near the marrow, and 16944 MPa near the trunk bark. Table 1. Descriptive statistics of the analyzed variables. Density (kg/m3) Velocity (m/s) Ed (MPa) fH (N) Ec0 (MPa) fc0 (MPa) Minimum 500 4831 12516 1851 8526 35,5 Mean 540 5148 14721 2563 10549 42,9 Maximum 640 5420 18030 3822 13373 52,9 Standard Deviation 2 172,43 1353,34 453,38 897 3,17 CV (%) 5,04 3,35 9,19 17,69 8,51 7,41 Table 1. Descriptive statistics of the analyzed variables. Network training The statistics for precision adjustment of the selected networks in predicting hardness, modulus of elasticity and modulus of tree rupture of Eucalyptus sp. were efficient. The selected ANN presented low variation between the levels of training, selection and evaluation, and ideal results in exhibiting training stability (Binoti et al. 2013). The correlation between observed and estimated hardness, modulus of elasticity and modulus of rupture was of 0,95; 0,96 and 0,97 with the root-mean-square error in percentage form (RMSE%) of 7,52%; 3,43% and 2,56% respectively (Table 2). Thus, the use of neural networks effectively sums the estimates of wood properties, and at the same time dispenses the basic assumptions of conventional mathematical modeling such as normality and linearity between predicted and predictor variables (Egrioglu et al. 2014). These attributes often have to undergo different mathematical transformations to be modeled in a traditional way, and can lead to losses in quality and selection of models (Miguel et al. 2016). In addition to these characteristics, ANNs present certain advantages under conventional techniques, highlighting its generalization capacity, parallelism and the possibility of learning, which result in accurate values according to the outcome of the research. Table 2. Characteristics and performance statistics of artificial neural networks selected to estimate the hardness, modulus of elasticity and modulus of rupture in Eucalyptus sp. wood in Brazil. Network Predictor variables Predicted variables Neurons by layer Adjustment Input Hidden Output LT LS LA  yxx RMSE% ANN Density Velocity Ed fH Ec0 fc0 3 8 3 0,10 0,12 0,11 0,95 0,96 0,97 7,52 3,45 2,56 Ed = dynamic modulus of elasticity, fH = hardness, Ec0 = modulus of elasticity, fc0 = modulus of rupture, LT = levels of training (network acquisition), LS = levels of stop selection (training stop), LA = levels of assessment (trained network quality),  yxx = correlation between observed and estimated variables, RMSE% = root-mean-square error in percentage. 2. Characteristics and performance statistics of artificial neural networks selected to estimate hardness, modulus of elasticity and modulus of rupture in Eucalyptus sp. wood in Brazil. Ed = dynamic modulus of elasticity, fH = hardness, Ec0 = modulus of elasticity, fc0 = modulus of rupture, LT = levels of training (network acquisition), LS = levels of stop selection (training stop), LA = levels of assessment (trained network quality),  yxx = correlation between observed and estimated variables, RMSE% = root-mean-square error in percentage. Network elaboration Consequently, the best ANN was selected from the 30 samples used to train the networks,. It was composed of an input layer that was constituted by three (3) neurons: density (ρ), wave propagation velocity (Vo) and dynamic modulus of elasticity (Ed). The hidden layer was composed of a single layer, as previously defined (Esquerre 2002, Melo and Miguel 2016). According to Cybenko (1989), this single layer is supported by the universal approximation theorem, which is sufficient for an MLP-like network to approach any continuous function. Also, its number of neurons as determined by the Intelligent Problem Solver (IPS) tool was seven (7) neurons. Finally, an output layer constituted by three (3) neurons: hardness (fH), modulus of elasticity (Ec0) and modulus of rupture (fc0), as shown in Figure 1. Figure 1. Graphic scheme of the selected network. 535 Figure 1. Graphic scheme of the selected network. Figure 1. Graphic scheme of the selected network. Maderas. Ciencia y tecnología 20(4): 531 - 542, 2018 Universidad del B í o - B í o Network training Even if the statistical goodness-of-fit criteria presented are suitable indicators for the selection of a neural network, the graphic analysis of residues is fundamental to corroborate them (Draper and Smith 1981), since this type of analysis detects possible underestimation trends in predicting the variables of interest, which are not measured by the statistics that evaluate precision. The graphs of the correlation between observed and estimated variables by the ANN of the properties fH, Ec0 and fc0 with the actual values (A1, B1 and C1), with residue distribution as a percentage (A2, B2 and C2) and error frequency histogram of the trained network (A3, B3 and C3) are shown in Figure 2. It is possible to observe that the selected ANN presents adequate estimated behavior, with flexibility to adhere to the data. The residual graph shows an absence of network trends such as maximum errors of ± 30% for all predicted properties. The evaluation of residues in the form of histograms is an indicated analysis to complement the dispersion graphs and thus avoid misinterpretations, since several points overlap the residual graph. Regarding this, the trained network presented a frequency of adequate errors, with the great majority in the classes varying between -10% and 10% error. The statistics presented in Table 2 indicate accuracy, as they are in accordance with the residual distribution and histograms of errors (Figure 2). Like this, the ANN was able to accurately predict the characteristics fH, Ec0 and fc0 from Eucalyptus sp. wood from the interface of wood density attributes, wave propagation velocity and dynamic modulus of elasticity. 536 Maderas. Ciencia y tecnología 20(4): 531 - 542, 2018 Using artificial neural networks in..: Miguel et al. Figure 2. Adjustment correlation of selected ANN in the prediction of hardness, Ec0 and fc0 (A1, B1 and C1), residual distribution (A2, B2 and C2) and frequency of error histogram (A3, B3 and C3) fo Eucalyptus sp. agglomerated panels, Brazil. Network training 30 35 40 45 50 30 35 40 45 50 Observed MOR Estimated MOR C1 -30 -10 10 30 34 39 45 50 55 Residual (%) Estimated MOR C2 1 14 14 1 0 4 7 11 14 18 -10 to -5 -5 to 0 0 to 5 5 to 10 Frequency C3 -30 -10 10 30 8850 10567 12283 Residual (%) Estimated MOE B2 2 14 13 1 0 4 8 12 16 20 -10 to -5 -5 to 0 0 to 5 5 to 10 Frequency B3 7000 9000 11000 13000 15000 17000 7000 10333 13667 17000 Observed MOE Estimated MOE B1 1750 2500 3250 4000 4750 1750 2500 3250 4000 4750 Observed hardness Estimated hardness A1 -30 -18 -5 8 20 33 1850 2453 3057 3660 Residual (%) Estimated hardness A2 3 11 9 7 0 3 6 9 12 15 -20 to - 10 -10 to 0 0 to 10 10 to 20 Frequency A3 3 11 9 7 0 3 6 9 12 15 -20 to - 10 -10 to 0 0 to 10 10 to 20 Frequency A3 -30 -18 -5 8 20 33 1850 2453 3057 3660 Residual (%) Estimated hardness A2 1750 2500 3250 4000 4750 1750 2500 3250 4000 4750 Observed hardness Estimated hardness A1 2 14 13 1 0 4 8 12 16 20 -10 to -5 -5 to 0 0 to 5 5 to 10 Frequency B3 7000 9000 11000 13000 15000 17000 7000 10333 13667 17000 Observed MOE Estimated MOE B1 -30 -10 10 30 8850 10567 12283 Residual (%) Estimated MOE B2 -30 -10 10 30 34 39 45 50 55 Residual (%) Estimated MOR C2 1 14 14 1 0 4 7 11 14 18 -10 to -5 -5 to 0 0 to 5 5 to 10 Frequency C3 30 35 40 45 50 30 35 40 45 50 Observed MOR Estimated MOR C1 Figure 2. Adjustment correlation of selected ANN in the prediction of hardness, Ec0 and fc0 (A1, B1 and C1), residual distribution (A2, B2 and C2) and frequency of error histogram (A3, B3 and C3) for Eucalyptus sp. agglomerated panels, Brazil. Maderas. Ciencia y tecnología 20(4): 531 - 542, 2018 Maderas. Ciencia y tecnología 20(4): 531 - 542, 2018 Universidad del B í o - B í o In evaluating the estimates of the properties fH, Ec0 and fc0, the values predicted by the network were close to the real values from the mechanical analysis; a concurrence later verified by the Chi-squared test (χ²). Thus, the use of artificial neural networks proved to be a reliable technique in estimating these mechanical properties using density, wave propagation velocity and dynamic modulus of elasticity as predictive attributes (Table 3). The aggregated difference, or the sum of the observed and estimated values, serves as the indicator criterion of under or overestimations. The trained network presented the following positive values: 1,92%; 0,45% and 0,26%, indicating a slight underestimation in predicting the properties fH, Ec0 and fc0 of the agglomerated panels (Table 3). These low values along with the non-significant result of the Chi-squared analysis demonstrate the effectiveness of the neural networks in predicting the panels’ mechanical properties. Table 3. Mean, minimum and maximum values observed and estimated by ANN in the prediction of hardness (fH), modulus of elasticity (Ec0) and modulus of rupture (fc0) of the Eucalyptus sp., Brazil. Variables/properties Minimum Mean Maximum AD (%) (χ²) D.cal (χ²) D.ref Result observed fH 2147 2265 3823 1,92 14,55 16,92 ns estimated fH 2217 2620 3697 observed Ec0 9852 10596 12063 0,45 12,21 ns estimated Ec0 9765 10567 11899 observed fc0 35,49 42,73 47,99 0,26 3,68 ns estimated fc0 36,50 42,41 47,05 (χ²) D.cal = Chi-squared calculated value, (χ²) D.ref = Chi-squared reference value (α = 0,05; 9), the (%) = aggregated difference, ns = not significant at 5%. Neural networks currently represent a promising area for multidisciplinary research (Silva et al. 2010), and the use of ANN in Brazil has become important in estimating measurements of forest stands, as it is considered an efficient and promising technique by several researchers (Leite et al. 2011, Castro et al. 2013, Binoti et al. 2015, Miguel et al. 2015). Nevertheless, studies are scarce when describing the physical and mechanical properties of wood and its engineered products to predict the quality of agglomerated panels (Melo and Miguel 2016). Neural network validation The validation of the trained network statistically confirmed the viability of the use of artificial intelligence (AI) tools in the prediction of Eucalyptus sp. wood properties (Table 3). During the validation process, 10 samples (25%) of the experimental data (not used for the network training) were used, thus proving that samples should be independent of the adjustment base (Zucchini 2000). Next, the data predicted by the ANN was compared with the observed values and later subjected to the Chi- squared test to be validated and to the aggregated difference test in percentage and the mean prediction error (Table 3). 537 Maderas. Ciencia y tecnología 20(4): 531 - 542, 2018 For the validation process, predictive learning ability, compact and homogeneous residuals, and distribution of errors in class intervals close to zero are desirable regardless of the modeling technique, since they demonstrate the ability of the models to estimate the variables of interest with precision. Similar to the training, ANN was effective in predicting fH, Ec0 and fc0 properties of wood, presenting residue adhesion, compactness, and homogeneity. It is also remarkable how the histogram of errors presented the highest frequency close to zero (Figure 3). Finally, the network maintained errors of overestimation and underestimation at less than 30%, once again following the same behavior presented in the training phase. The results found in this research concur with the statements of Egrioglu et al. (2014), when mentioning that ANNs have advantages over conventional techniques due to their generalization capacity, parallelism and possibility of learning. As such, they can extract standards from a certain database (learning) and adequately reapply them in others. Hence, their use is highly recommended. The Chi-squared test evidenced adherence and the aggregated difference (AD) revealed a high accuracy of the selected ANN for simultaneous outputs (three neurons in the output layer) in the prediction of mechanical properties of fH, Ec0 and fc0. Thus, this research is of great value because it positively impacts the prediction of these products’ quality through the exclusive use of non-destructive chemical and mechanical variables (density, velocity and dynamic modulus of elasticity) as predictor 538 Maderas. Ciencia y tecnología 20(4): 531 - 542, 2018 Using artificial neural networks in..: Miguel et al. variables. Figure 3. ANN validation in the correlation information of the adjustment in the prediction of fH, Ec0 and fc0 (A1, B1 and C1), residual distribution (A2, B2 and C2) and frequency error histogram (A3, B3 and C3) for Eucalyptus sp. agglomerated panels, Brazil. Maderas. Ciencia y tecnología 20(4): 531 - 542, 2018 Considering this research it is worth emphasizing that the results obtained and presented are 35 40 45 50 35 40 45 50 Observed MOR Estimated MOR C1 1 3 5 1 0 2 3 5 6 -10 to -5 -5 to 0 0 to 5 5 to 10 Frequency B3 -30 -10 10 30 9000 10500 12000 Residual (%) Estimated MOE B2 7000 9000 11000 13000 15000 7000 9000 110001300015000 Observed MOE Estimated MOE B1 1 4 4 1 0 2 3 5 6 -10 to -5 -5 to 0 0 to 5 5 to 10 Frequency C3 -30 -10 10 30 35 40 45 50 55 Residual (%) Estimated MOR C2 1 4 4 1 0 2 3 5 6 -20 to -10-10 to 0 0 to 10 10 to 20 Frequency A3 -30 -15 0 15 30 1800 2500 3200 3900 Residual (%) Estimated hardness A2 1750 2500 3250 4000 4750 1750 2500 3250 4000 4750 Observed hardness Estimated hardness A1 variables. 1 4 4 1 0 2 3 5 6 -20 to -10-10 to 0 0 to 10 10 to 20 Frequency A3 -30 -15 0 15 30 1800 2500 3200 3900 Residual (%) Estimated hardness A2 1750 2500 3250 4000 4750 1750 2500 3250 4000 4750 Observed hardness Estimated hardness A1 -30 -10 10 30 9000 10500 12000 Residual (%) Estimated MOE B2 7000 9000 11000 13000 15000 7000 9000 110001300015000 Observed MOE Estimated MOE B1 1 3 5 1 0 2 3 5 6 -10 to -5 -5 to 0 0 to 5 5 to 10 Frequency B3 B1 -30 -10 10 30 35 40 45 50 55 Residual (%) Estimated MOR C2 1 4 4 1 0 2 3 5 6 -10 to -5 -5 to 0 0 to 5 5 to 10 Frequency C3 35 40 45 50 35 40 45 50 Observed MOR Estimated MOR C1 Figure 3. ANN validation in the correlation information of the adjustment in the prediction of fH, Ec0 and fc0 (A1, B1 and C1), residual distribution (A2, B2 and C2) and frequency error histogram (A3, B3 and C3) for Eucalyptus sp. agglomerated panels, Brazil. Considering this research, it is worth emphasizing that the results obtained and presented are specific for the evaluated species (Eucalyptus urograndis). CONCLUSIONS Simultaneously-adjusted multilayered perceptron artificial neural networks (three neurons in the output layer) which use information of density, wave propagation velocity and dynamic modulus of elasticity as predictor variables are accurate in estimating mechanical properties of hardness, modulus of elasticity and modulus of rupture of the Eucalyptus urograndis wood, and do not statistically differ from the values obtained through destructive mechanical test. Therefore, Multilayer Perceptron ANNs are a practical and efficient tool in mechanical-technological wood testing. The results indicate that this tool can be used in a practical way by industries of the timber sector to effectively and non-destructively predict the performance of wood parts. Maderas. Ciencia y tecnología 20(4): 531 - 542, 2018 Further studies should be carried out to study other species as well as different ages, and consequently different configurations and architectures of neural networks should be trained. As a suggestion, the insertion of “species” and “age” as categorical variables in the input layer is an interesting alternative, as it may result in a single ANN capable of accurately predicting mechanical characteristics of wood for a range of species at different ages. 539 Universidad del B í o - B í o Maderas. Ciencia y tecnología 20(4): 531 - 542, 2018 Sciences 109(8): 1094-1100. Esquerre, K.P.O.; Mori, M.; Bruns, R.E. 2002. Simulation of an industrial wastewater treatment plant using artificial neural networks and principal components analysis. Brazilian Journal of Chemical Engineering 19(4): 365-370. Goyal, S. 2013. Artificial neural networks in vegetables: a comprehensive review. Scientific Journal of Crop Science 2(7): 75-94. Haykin, S. 2001. Redes neurais: princípios e prática. Porto Alegre: Bookman, 900p. Heaton, J. 2011. Programming Neural Networks with Encog3 in Java. 2ed. St. Louis: Heaton Research Incorporated, 240p. Leite, H. G.; Silva, M. L. M.; Binoti, D. H. B.; Fardin, L.; Takizawa, F. H. 2011. Estimation of inside-bark diameter and heartwood diameter for Tectona grandis Linn. trees using artificial neural networks. European Journal of Forest Research 130(2): 263-269. Kuvendziev A. S.; Lisichkova K.; Zekovic, Z.; Marinkovski, M. 2014. Artificial neural network modelling of supercritical fluid CO2 extraction of polyunsaturated fatty acids from common carp (Cyprinus carpio L.) viscera. J Supercrit Fluids 92(6):242-248. Mehtätalo, L.; Maltamo, M.; Kangas, A. 2006. The use of quantile trees in the prediction of the diameter distribution of a stand. Silva Fennica 40(3):501-516. Miguel, E. P.; Rezende, A. V.; Leal, F. A.; Matricardi, E. A.; Vale, A. T.; Pereira, R. S. 2015. Redes neurais artificiais para a modelagem do volume de madeira e biomassa do cerradão com dados de satélite. Pesquisa Agropecuária Brasileira 50(9): 829-839. Miguel, E.P.; Mota, F. C. M.; Teo, S. J.; Nascimento, R. G. M.; Leal, F.A.; Pereira, R. S.; Rezende, A.V. 2016. Artificial intelligence tools in predicting the volume of trees within a forest stand. African Journal of Agricultural Research 11(21): 1914-1923. Melo, R.R.; Miguel, E.P. 2016. Use of artificial neural networks in predicting particleboard quality parameters. Revista Árvore 40(5): 949-958. Okan, O.T.; Deniz, I.; Tiryaki, S. 2015. Application of artificial neural networks for predicting tensile index and brightness in bleaching pulp. Maderas-Cienc Tecnol 17(3): 571-584. Shiblee, M.D.; Chandra, B.; Kalra, P.K. 2010. Learning of geometric mean neuron model using resilient propagation algorithm. Expert Systems with Applications 37(12): 7449-7455. Silva, I.N.; Spatti, D.H.; Flauzino, R.A. 2010. Redes Neurais Artificiais para engenharia e ciências aplicadas. São Paulo: Artliber. 397p. Stangerlin, D. M.; Calegari, L.; Santini, E. J.; Domingues, J. M. X.; Gatto, D. A.; Melo, R. R. 2008. Determinação do módulo de elasticidade em madeiras por meio de métodos destrutivo e não destrutivo. Revista Brasileira de Ciências Agrárias 3(2): 145-150. Tiryaki, S.; Aydın, A. 2014. REFERENCES Alves, D. P.; Tomaz, R. S.; Laurindo, B. S.; Laurindo, R. D. F.; Silva, F. F.; Cruz, C. D.; Nick, C.; Silva, D. J.H. 2017. Artificial neural network for prediction of the area under the disease progress curve of tomato late blight. Scientia Agricola 74(01): 51-59. Alves, D. P.; Tomaz, R. S.; Laurindo, B. S.; Laurindo, R. D. F.; Silva, F. F.; Cruz, C. D.; Nick, C.; Silva, D. J.H. 2017. Artificial neural network for prediction of the area under the disease progress curve of tomato late blight. Scientia Agricola 74(01): 51-59. Associação Brasileira de Normas Técnicas. ABNT. 2001. Projetos de estruturas de madeira. NBR 7190. Rio de Janeiro. Associação Brasileira de Normas Técnicas. ABNT. 2001. Projetos de estruturas de madeira. NBR 7190. Rio de Janeiro. Bassa, A. G. M. C.; Silva Júnior, F. G.; Sacon, V. M. 2007. Misturas de madeira de Eucalyptus gradis x Eucalyptus urophylla e Pinus taeda para produção de celulose kraft através do Processo Lo- Solids. Scientia Forestalis 75(4): 19-30. Bardak S.; Tiryaki S.; Nemli, G.; Aydın A. 2016. Investigation and neural network prediction of wood bonding quality based on pressing conditions. International Journal of Adhesion and Adhesives 68(5): 115–123. Binoti M. L. M. S.; Binoti, D. H. B.; Leite, H. G. 2013. Aplicação de redes neurais artificiais para estimação da altura de povoamentos equiâneos de eucalipto. Revista Árvore 37(4): 639-645. Binoti, M. L. M. S.; Leite, H. G.; Binoti, D. H. B.; Gleriani, J. M. 2015. Prognose em nível de povoamento de clones de eucalipto empregando redes neurais artificiais. Cerne 21(1): 97-105. Castro, R. V. O.; Soares, C. P. B.; Leite, H. G.; Souza, A. L.; Nogueira, G. S.; Martins, F. B. 2013. Indiv’idual growth model for Eucalyptus Stands in Brazil using artificial neural network. ISRN Forestry: Article ID 196832. Chen, W. C.; Tseng, L.Y.; Wu, C. S. 2014. A unified evolutionary training scheme for single and ensemble of feedforward neural network. Neurocomputing 143(C): 347-361. Comisión Panamericana de Normas Técnicas. COPANT. 1972. Seleção das amostras. COPANT 458. Cybenko, G.V. 1989. Approximation by superpositions of a sigmoidal function. Mathematics of Control, Signals and System 2(4): 303-314. Draper, N. R.; Smith, H. 1981. Applied regression analysis. New York: Jonh Willey & Sons 407 p. Egrioglu, E.; Yolcu, U.; Aladag, C.H.; Bas, E. 2014. Recurrent multiplicative neuron model artificial neural network for non-linear time series forecasting. Procedia - Social and Behavioral 540 Maderas. REFERENCES Ciencia y tecnología 20(4): 531 - 542, 2018 Using artificial neural networks in..: Miguel et al. Universidad del B í o - B í o Sciences 109(8): 1094-1100. An artificial neural network model for predicting compression strength of heat treated woods and comparison with a multiple linear regression model. Construction and Building Materials 62(13): 102-108. Tiryaki, S.; Hamzacebi, C. 2014. Predicting modulus of rupture (MOR) and modulus of elasticity (MOE) of heat treated woods by artificial neural networks. Measurement 49(3): 266–274. Tiryaki, S.; Bardak, S.; Aydın, A.; Nemli, G. 2016. Analysıs of volumetrıc swellıng and shrınkage of heat treated woods: experımental and artıfıcıal neural network modelıng approach. Maderas-Cienc Tecnol 18(3): 477–492. 541 Universidad del B í o - B í o Maderas. Ciencia y tecnología 20(4): 531 - 542, 2018 Universidad del B í o - B í o Maderas. Ciencia y tecnología 20(4): 531 - 542, 2018 Zucchini, W. 2000. An introduction to model selection. Journal of Mathematical Psychology Maderas. Ciencia y tecnología 20(4): 531 - 542, 2018 Maderas. Ciencia y tecnología 20(4): 531 - 542, 2018 Maderas. Ciencia y tecnología 20(4): 531 - 542, 2018 Universidad del B í o - B í o Zucchini, W. 2000. An introduction to model selection. Journal of Mathematical Psychology 44(1): 41-61. 542
https://openalex.org/W4254127830	https://revistas.ucm.es/index.php/ESIM/article/download/37670/36454	Spanish; Castilian	null	Escribir soberano	Escritura e imagen	2,012	cc-by	10,287	Resumen Se aborda la deconstrucción de la soberanía desde el inclinado ángulo del loco soberano; desde una lectura de El Rey Lear de Shakespeare, obra en la que se divi- de dos veces la soberanía, al principio y al final de la pieza, y que abre la posibili- dad de una lectura contemporánea de la misma en torno a dos ejes fundamentales: la locura y la muerte. Dos locuras y tres muertes soberanas. Palabras claves: hostipitalidad, división, deconstrucción, locura, muerte, quizá. Escribir soberano Delmiro Rocha UNED escrituraeimagen@filos.ucm.es Abstract We approach the deconstruction of sovereignty from the slant point of view of the sovereign madman. We base this approach on the reading of King Lear by Shakespeare, a tragedy in which sovereignty is divided twice, at the beginning and the end of the play, and which opens the possibility of a contemporary reading of it around two fundamental axes: madness and death. Two sovereign madnesses and three sovereign deaths. Key words: hospitality, division, deconstruction, madness, death, perhaps. El 4 de Marzo de 1963 en el Collège Philosophique Derrida lee lo siguiente: El 4 de Marzo de 1963 en el Collège Philosophique Derrida lee lo siguiente: El 4 de Marzo de 1963 en el Collège Philosophique Derrida lee lo siguiente: creo que la historicidad en general sería imposible sin una historia de la filosofía y creo que ésta sería imposible a su vez si no hubiese más que hipérbole, por una parte, o si no ISSN: 1885-5687 http://dx.doi.org/10.5209/rev_ESIM.2011.37670 Escritura e imagen Nún. ext. (2011): 23-40 23 Delmiro Rocha Escribir soberano Escribir soberano hubiese, por otra parte, más que estructuras históricas determinadas, Weltanschauungen finitas. La historicidad propia de la filosofía tiene su lugar y se constituye en ese pasa- je, en ese diálogo entre la hipérbole y la estructura finita, entre el exceso sobre la tota- lidad y la totalidad cerrada, en la diferencia entre la historia y la historicidad; es decir, en el lugar, o más bien el momento en que el cogito y todo lo que éste simboliza aquí (locura, desmesura, hipérbole, etc.) se dicen, se reafirman y decaen, se olvidan, de forma necesaria, hasta su reactivación, su despertar en otra ocasión de decir el exceso, que más tarde será también otra decadencia y otra crisis. […] La crisis o el olvido no es quizás el lado accidental sino el destino de la filosofía hablante, que no puede vivir más que encerrando a la locura, pero que moriría como pensamiento, y bajo una violencia aún peor, si a cada instante una nueva palabra, aun encerrando en ella misma, en su pre- sente, al loco del momento, no liberase a la antigua locura. Es sólo gracias a esta opre- sión de la locura como puede reinar un pensamiento-finito, es decir, una historia. Sin atenerse a un momento histórico determinado, sino extendiendo esta verdad a la histo- ricidad en general, se podría decir que el reino de un pensamiento-finito sólo puede esta- blecerse sobre la base del encierro y la humillación y el encadenamiento y la irrisión más o menos disimulada del loco que hay en nosotros, de un loco que sólo puede ser el loco de un logos, como padre, como señor, como rey.1 Extraigo de esta larga cita, a la vez que cerceno su encentadura, el final del final, la cola de su última frase, como si de una disección anatómica se tratase. 1 Derrida, J.: La escritura y la diferencia, Barcelona, anthropos, 1989, pp. 86-87. Traducción de Patricio Peñalver. El 4 de Marzo de 1963 en el Collège Philosophique Derrida lee lo siguiente: Derrida dice: “del loco que hay en nosotros, de un loco que sólo puede ser el loco de un logos, como padre, como señor, como rey”. ¿Qué significa, cómo debemos leer o, en todo caso, cómo podemos interpretar ese “nosotros”? aparentemente es la pri- mera persona del plural con la cual el pronombre personal universaliza la locura como algo intrínseco. Pero justo acompañando a un léxico como “padre”, “señor” o “rey”, ¿no es más bien ese “nosotros” un plural mayestático? Un plural que creó escuela entre escritores de toda índole y que hoy en día se confunde con el plural de modestia (pluralis modestiae) y el plural de autoría (pluralis auctoris). Pero el adje- tivo mayestático deriva del término latín maiestas –atis, que significa “majestuoso”, es decir, con la solemnidad o magnificencia propias de la majestad. Es el plural que la figura del soberano se arroga, incluyendo la figura del Papa, y que engloba bajo su persona la pluralidad de las cosas, cosas que pasan directamente a ser sus cosas. Es decir, la soberanía se constituye a través de un proceso de apropiación que, del todo, hace unidad. La pluralidad mayestática es, entonces, la indivisibilidad del Uno. Términos como “nosotros”, “Nos” o “Vuestra” son otra forma de escribir “soberano”. Pero, prolongando esta digresión inicial, ¿no hay, acaso, en todo escri- bir, una actitud intrínseca e irrechazable de control total, una predisposición, tanto más inconsciente, a dominar la escritura? ¿No es todo acto de escritura un escribir 24 Escritura e imagen Vol. ext. (2011): 23-40 Escritura e imagen Vol. ext. (2011): 23-40 Delmiro Rocha Escribir soberano soberano? Siguiendo a Derrida, como aquí confieso hacer, si la locura es aquello que el pensamiento encarcela para constituirse como tal, a la vez que debe liberar- lo para no perecer “bajo una violencia aún peor”, entonces, si alguien se adelanta y, acusándome de cierta falta de cordura, (me) dice: “éste está loco”, ¿no está acaso, con un gesto doble, coronándome? ciertamente, según el pensamiento de Derrida, colocar las palabras “escribir” y “soberano” la una detrás de la otra y situarlas a la cabeza de un texto, es decir, en el lugar que preside y, todavía sin contexto, gobierna cierto querer decir, parece, cuando menos, arriesgado. Parece haber cierta apariencia de exclusión. Lo sobera- no semeja limitarse hoy a lo que Derrida llamó, hace ya mucho tiempo, la clausura del libro. 2 Shakespeare, W.: El Rey Lear, RBa Ediciones, Barcelona, 2003, p. 81. El 4 de Marzo de 1963 en el Collège Philosophique Derrida lee lo siguiente: El exceso aparecerá más antes que después, en la obra de Shakespeare, bajo la figura de la locura. “En tanto que Nos [dice el rey todavía en plural y con mayúscula, letra capital pues él es todavía la cabeza], nos encaminaremos hacia la muerte”. hay aquí una referencia al desdoble de la personalidad, no un desdoble de personalidad psicoló- gico sino jurídico, es decir, a las dos personas del rey que reconocía el derecho ecle- siástico y que Derrida nos recuerda en el primer volumen del Seminario La bestia y el soberano cuando cita y analiza Los dos cuerpos del rey de Kantorowicz. El rey Lear cede la persona idealis, el rey inmortal, a sus herederos a la vez que encami- na, en un mismo gesto, la persona personalis, el rey mortal, hacia la muerte. Sólo así el rey puede morir, según lo que al menos desde Bodin se entiende como sobe- ranía, esto es, una e indivisible. Ya aquí, desde el inicio de esta pieza de teatro majestuosa, el tema de la muer- te sube a escena para quedarse ahí y no descender jamás. Lo mismo ocurre en los dos volúmenes del Seminario La bestia y el soberano, ese otro gran teatro magis- tral que compone uno de los bestiarios más ricos de nuestro tiempo. La muerte, pues, está en escena o, a lo sumo, acechando a paso de lobo, viendo sin ser vista, entre bastidores. Pues no otra cosa es la soberanía, nos recuerda hobbes, que el derecho sobre la vida y la muerte. hay un detalle estilístico excepcional con el cual el rey Lear asume su muerte mucho antes de que se consuma. consiste en renunciar al plural mayestático a par- tir del momento en que finaliza la cesión del poder soberano. Desde ese instante hasta la última escena del último acto, en la cual el rey Lear muere besando a cordelia, su hija menor, el rey usará siempre la primera persona del singular. La muerte, pues, como posibilidad imposible para el rey. La división del rey en dos cuerpos como condición de imposibilidad para la soberanía. Pero, en El Rey Lear de Shakespeare, el rey no sólo tiene dos cuerpos sino que sufre, además, varias muertes. La primera de ellas, la acabamos de ver, es la renuncia al plural mayestá- tico. Las siguientes serán, sucesivamente, la locura y la muerte de su persona per- sonalis. El 4 de Marzo de 1963 en el Collège Philosophique Derrida lee lo siguiente: El escribir, siguiendo la analogía, sería la apertura del texto. La escritura derridiana, que según su poder diseminante no vuelve al padre (léase logos, señor, rey, etc), haría salir el “loco que hay en nosotros”. Según un gesto habitual en Derrida que consiste en leer con un libro en cada mano, siempre en los márgenes de la literatura y la filosofía, me gustaría abordar la deconstrucción de la soberanía desde el inclinado ángulo del loco soberano. Desde cierta escritura soberana que, tratando quizá de restituir una monarquía metafísica, abre en su escritura aquello mismo que pretende cerrar. Me detengo entonces en una lectura no lineal y necesariamente incompleta del que probablemente es el loco soberano más importante de la literatura occidental: El rey Lear de W. Shakespeare. Derrida jamás ha publicado un texto sobre El rey Lear de Shakespeare. conociendo el interés y la fuerza con que Derrida leía habitualmente a Shakespeare, yo comienzo pidiendo disculpas por esta lectura sucedánea, no especializada y pre- tendidamente deconstructiva que me dispongo a realizar. Lo dicho, pues. q p g En la primera escena del primer acto el rey Lear dice: Sabed que hemos dividido el reino en tres partes. Es nuestro intento formal librar nues- tra vejez del peso de los negocios y pasar ese fardo a hombros más jóvenes, en tanto que Nos, exentos de todo cuidado, nos encaminaremos hacia la muerte…2 Resulta asombrosa la cantidad de elementos que se ponen en juego desde el pri- mer acto. Todo está sobre la mesa, incluso el destino fatal al que ciertamente se encamina el rey. Primero, gráfica y formalmente, el rey habla en plural mayestático, con autoría pero sin modestia, y con el poder absoluto que constituye su palabra arconte. El decir performativo del rey hace ley, es la ley. Pero aquí el performativo se pliega sobre sí mismo al anunciar, desde su poder soberano, que ya no es más soberano, que renuncia a sí mismo. “hemos [es decir, yo, el rey, la totalidad que en mi perso- na se agrupa] decidido dividir el reino en tres partes [es decir, hemos decidido dejar 25 Escritura e imagen Vol. ext. (2011): 23-40 Escritura e imagen Vol. ext. (2011): 23-40 Delmiro Rocha Escribir soberano Escribir soberano de ser el rey]”. autoinmunidad soberana que limita —sujeta a una totalidad cerra- da— al tiempo que excede la soberanía misma. 3 Shakespeare, W.: El Rey Lear, RBa Ediciones, Barcelona, 2003, p. 86. El 4 de Marzo de 1963 en el Collège Philosophique Derrida lee lo siguiente: Pero en la primera de sus muertes el rey Lear, desde el ejercicio de la sobe- ranía que permite el exceso de renunciar a su palabra arconte, se resiste, no obstan- te, a perder una palabra: el nombre de rey. Sólo queremos conservar el nombre de rey y las muestras exteriores de nuestra digni- dad; en cuanto al poder, las rentas y el ejercicio de la monarquía, os lo abandonamos todo, queridos hijos. Y para confirmar la donación que os hago, compartid entre ambos esta corona… (Se quita la corona real y la da a sus yernos)3 26 Escritura e imagen Vol. ext. (2011): 23-40 Escritura e imagen Vol. ext. (2011): 23-40 Delmiro Rocha Escribir soberano Escribir soberano he aquí otro pequeño texto cargado hasta el infinito de reenvíos significantes. El rey Lear quiere conservar el nombre de rey. Primera pregunta: ¿es la palabra “rey” un nombre? Sin duda es un nombre común sustantivo masculino, pero ¿es “rey” un nombre propio? Sin duda se puede escribir, y de hecho se escribe, con mayúscula para darle la altura y la alteza propias de la majestad. Sería, en este caso, el nombre más propio puesto que todo lo engloba en sí mismo. Pero el nombre “rey” funciona como suplemento en cuanto suplanta y subsume al nombre propio de la persona personalis; no obstante sólo lo puede hacer cuando la persona se duplica y pasa a portar además la persona idealis, es decir, no hay rey sin dos cuer- pos. Sin embargo, en el caso del rey Lear, él quiere conservar el nombre de rey a pesar de haber renunciado al poder que erige y constituye dicho nombre. otra vez, como vemos y seguiremos viendo, el exceso de la soberanía, el exceso como sobe- ranía. Pero, para Derrida, tampoco el nombre propio resiste a la lógica de la diffé- rance, incluso diríamos que es el más afectado por ella puesto que su constitución misma se identifica con la lógica apropiante del logos. ¿De qué quiere seguir sien- do propietario el rey Lear: del nombre o de lo que el nombre porta? ¿Se puede por- tar un nombre que no porta nada? ¿Quién porta, en realidad, a quién? En Derrida, no existe el nombre propio fuera de la cadena de reenvíos y remitencias diseminan- tes del lenguaje compuesto por huellas de huellas. Un nombre propio nunca se podría definir por sí mismo. 4 Derrida: Ecografías de la Televisión. Entrevistas filmadas, Buenos aires, Eudeba, 1998, p.137. Los corchetes no son de Derrida sino del autor de este artículo. 5 J. Derrida: “El Bien soberano o estar malo de ganas de soberanía”, Revista Archipiélago 75, 2007, pp. 94-95. El 4 de Marzo de 1963 en el Collège Philosophique Derrida lee lo siguiente: En segun- do lugar, cuando Lear intenta recuperar el poder bajo el nombre de “rey” yendo en contra de la ley que él mismo instauró con su palabra soberana. Una ley que viola la ley. Una soberanía que se divide a sí misma. Pues no hay que olvidar que en 1608, año en que se reconoce la primera edición de El rey Lear y año en que Francia amplia —a la vez que divide— su soberanía con la fundación del Quebec, y tan solo 28 años después de que el filósofo francés J. Bodin publique Los seis libros de la república (1576), en 1608, pues, la pieza de teatro El rey Lear de Shakespeare comienza por ordenar una división de la soberanía. “Sabed que hemos dividido el reino en tres par- tes”, dice el rey Lear en la tercera página de la edición que yo manejo. Y apenas unas líneas más arriba, en la primera intervención del rey Lear en la obra de teatro, éste dice: “Id a buscar al rey de Francia y al duque de Borgoña”. ambos eran los preten- dientes de cordelia, la hija menor del rey, y pretendientes a su vez de la herencia soberana de Lear. La obra comienza, entonces, cuando el rey Lear manda llamar al rey de Francia para proceder al reparto de su soberanía o, lo que es lo mismo, al reparto de sus tres hijas. Parece como si Shakespeare mandase llamar a Bodin (muer- to apenas siete años antes de la primera representación de la obra) para decirle que, frente a su idea de soberanía Una e indivisible defendida desde su puesto en el tribu- nal superior de justicia en el Parlamento de París, él, Shakespeare, va a comenzar por una división de la soberanía. Un exceso doble constituye la exapropiación. Un exceso sobre la propiedad de lo propio. El rey Lear incurre en este exceso bajo una hipérbole doble. En primer lugar, una hipérbole parricida o regicida, esto es, cuando el rey intenta acabar con su cuerpo soberano, persona idealis, volviendo la soberanía contra sí misma. En segun- do lugar, cuando Lear intenta recuperar el poder bajo el nombre de “rey” yendo en contra de la ley que él mismo instauró con su palabra soberana. Una ley que viola la ley. Una soberanía que se divide a sí misma. El 4 de Marzo de 1963 en el Collège Philosophique Derrida lee lo siguiente: Pues no hay que olvidar que en 1608, año en que se reconoce la primera edición de El rey Lear y año en que Francia amplia q p y y q p —a la vez que divide— su soberanía con la fundación del Quebec, y tan solo 28 años después de que el filósofo francés J. Bodin publique Los seis libros de la república (1576), en 1608, pues, la pieza de teatro El rey Lear de Shakespeare comienza por ordenar una división de la soberanía. “Sabed que hemos dividido el reino en tres par- tes”, dice el rey Lear en la tercera página de la edición que yo manejo. Y apenas unas líneas más arriba, en la primera intervención del rey Lear en la obra de teatro, éste dice: “Id a buscar al rey de Francia y al duque de Borgoña”. ambos eran los preten- dientes de cordelia, la hija menor del rey, y pretendientes a su vez de la herencia soberana de Lear. La obra comienza, entonces, cuando el rey Lear manda llamar al rey de Francia para proceder al reparto de su soberanía o, lo que es lo mismo, al reparto de sus tres hijas. Parece como si Shakespeare mandase llamar a Bodin (muer- to apenas siete años antes de la primera representación de la obra) para decirle que, frente a su idea de soberanía Una e indivisible defendida desde su puesto en el tribu- nal superior de justicia en el Parlamento de París, él, Shakespeare, va a comenzar por una división de la soberanía. y y —a la vez que divide— su soberanía con la fundación del Quebec, y tan solo 28 años después de que el filósofo francés J. Bodin publique Los seis libros de la república (1576), en 1608, pues, la pieza de teatro El rey Lear de Shakespeare comienza por ordenar una división de la soberanía. “Sabed que hemos dividido el reino en tres par- tes”, dice el rey Lear en la tercera página de la edición que yo manejo. Y apenas unas líneas más arriba, en la primera intervención del rey Lear en la obra de teatro, éste dice: “Id a buscar al rey de Francia y al duque de Borgoña”. ambos eran los preten- dientes de cordelia, la hija menor del rey, y pretendientes a su vez de la herencia soberana de Lear. Escritura e imagen Vol. ext. (2011): 23-40 El 4 de Marzo de 1963 en el Collège Philosophique Derrida lee lo siguiente: Es decir, el nombre propio desgarra la propiedad del nombre en general, desapropia, desquicia, divide y no produce más conexión esen- cial entre concepto y mundo que la que produce el desvío del significante hacia otros significantes a través de la lógica del signo. En el caso del rey Lear, el nom- bre propio que pretende conservar es el que más desapropia y desbarata el concep- to “propio”, puesto que acaba de perder, en la cesión de su cargo, todas sus propie- dades. El nombre propio porta la muerte y la inscribe allí donde pretende designar una vida exclusiva, es decir, propia. El rey Lear, en un intento desesperado y con- tradictorio, aspira a apropiarse del nombre de rey y de la dignidad de la realeza al mismo tiempo que se expropia a sí mismo del poder soberano. Este doble movi- miento contradictorio de apropiación y expropiación que pretende apropiarse del sentido a la vez que lo deja en su alteridad es lo que Derrida denomina “exapropia- ción”. aquí tenemos, mediante un gesto inverso al que hasta aquí me trajo, a Derrida desde Shakespeare, Ecografías de la Televisión desde la boca del rey Lear: Lo que llamo ‘exapropiación’ [es decir, ese intento contradictorio del rey Lear de inten- tar conservar su nombre] es ese doble movimiento en que me dirijo hacia el sentido con la intención de apropiarme de él [del nombre rey], pero a la vez sé y deseo, lo reconoz- ca o no, que siga siendo extraño para mí, trascendente, otro, que permanezca allí donde hay alteridad [en sus yernos a quienes les cede su corona]. Si pudiera reapropiarme totalmente del sentido, exhaustivamente y sin dejar nada, no habría sentido. Si no quie- ro apropiarme de él en absoluto, tampoco lo hay. así, pues, hace falta (el ‘faltar’ de ese 27 Escritura e imagen Vol. ext. (2011): 23-40 Escritura e imagen Vol. ext. (2011): 23-40 Escritura e imagen Vol. ext. (2011): 23-40 Delmiro Rocha Escribir soberano ‘hace falta’ es la existencia misma en general), un movimiento de apropiación termina- do, una exapropiación“4. Un exceso doble constituye la exapropiación. Un exceso sobre la propiedad de lo propio. El rey Lear incurre en este exceso bajo una hipérbole doble. En primer lugar, una hipérbole parricida o regicida, esto es, cuando el rey intenta acabar con su cuerpo soberano, persona idealis, volviendo la soberanía contra sí misma. El 4 de Marzo de 1963 en el Collège Philosophique Derrida lee lo siguiente: La obra comienza, entonces, cuando el rey Lear manda llamar al rey de Francia para proceder al reparto de su soberanía o, lo que es lo mismo, al reparto de sus tres hijas. Parece como si Shakespeare mandase llamar a Bodin (muer- to apenas siete años antes de la primera representación de la obra) para decirle que, frente a su idea de soberanía Una e indivisible defendida desde su puesto en el tribu- nal superior de justicia en el Parlamento de París, él, Shakespeare, va a comenzar por una división de la soberanía. Una vez más el rey Lear comienza por ejercer el exceso, la soberanía como exceso, y pretende dividir lo indivisible y compartir lo incompartible a través de una donación sin precedentes. Más allá de la fantasía histórica o del mito celta que hace historia, no podemos obviar las palabras de Derrida cuando, sin querer ejercer de teórico político, postula lo siguiente: Y si tuviese que proponer aquí una tesis política, ésta no sería la de la oposición de la soberanía y la no-soberanía como oposición del bien al mal o del bien que es un mal al mal que desea el bien sino otra política de la partición de la soberanía, a saber, de la par- tición de lo incompartible y de la división de lo indivisible.5 El rey Lear propone dos modalidades distintas de división y reparto. Primero 28 Escritura e imagen Vol. ext. (2011): 23-40 Escritura e imagen Vol. ext. (2011): 23-40 Delmiro Rocha Escribir soberano Escribir soberano divide y cede su reino en dos partes para ser compartido por sus dos hijas mayores: Gonerila y Regana. Segundo, dona la monarquía pero quiere seguir conservando la majestad, es decir, el nombre de rey. Podemos ver aquí una deconstrucción de la soberanía que pasa por la división y la partición de lo incompartible. Y aunque de hecho esta división será el desencadenante de la trama y del drama que describe el teatro que nos ocupa, y en el cual por otro lado todavía estamos, esa misma división imposible seguirá constituyendo la condición de imposibilidad para la deconstruc- ción de la soberanía si entendemos, con Derrida, la deconstrucción como una expe- riencia de lo imposible que apuesta por la divisibilidad infinita. 6 Shakespeare 2003, op. cit. (nota 2), pp. 86-87. El 4 de Marzo de 1963 en el Collège Philosophique Derrida lee lo siguiente: El rey Lear, después de dividir su hacienda entre sus dos hijas mayores y des- pués de renunciar a su tercera hija, cordelia, su preferida, rompiendo incluso desde su palabra soberana todo lazo natural y sanguíneo para con ella, situando así la soberanía por encima de toda determinación natural y forzando su exilio a Francia, lugar donde todavía reinaba la monarquía indivisible, el rey comienza a adentrarse en lo que supone la segunda de sus muertes: la locura. Esta catarata de asuntos y cuestiones políticas se ponen en juego en las pocas páginas de la primera escena del primer acto. Es todavía ahí donde el conde de Kent acusa al rey de falta de cordura, para defender la monarquía e intentar evitar el exi- lio de cordelia. aquí comienza la segunda muerte del rey Lear. El Rey Lear (a Kent)- El arco está tendido y la cuerda tirante. Guarda que la flecha te dé. Kent- al contrario, que me dé, aunque la punta penetre hasta el corazón. Kent puede ser descortés porque Lear está demente.6 El conde de Kent, fiel vasallo y defensor acérrimo del rey a lo largo de toda la obra, es el primero en denunciar la enajenación del rey. así, la aparente contradic- ción entre la acusación y la defensa que Kent formula paralelamente se disuelve si entendemos que el conde no ha dejado jamás de ser un siervo leal puesto que a quien se opone no es al rey Lear sino a la locura que se ha apoderado de él y que lo suplanta al tiempo que lo destituye. El conde de Kent sigue encadenado, cual repre- sentante de toda la filosofía occidental, a la articulación metafísica que une la Razón al sentido y al poder. El logos, para ir deprisa, sería lo que ha perdido el rey, su pala- bra soberana. Pero es solamente mediante el discurso supuestamente enajenado del rey como el conde de Kent niega el logos a Lear, dicho de otra forma, lo acusa de mantener un discurso sin Discurso, un logos sin Logos. Y, para eso, es necesario que el discurso de Kent sí sea un discurso, un discurso con Discurso. Kent parece sos- tener un discurso “foucaultiano” al objetivar la locura, pues no puede aceptar una división de la soberanía tan demente como la que el rey Lear pretende. 29 Escritura e imagen Vol. ext. El 4 de Marzo de 1963 en el Collège Philosophique Derrida lee lo siguiente: (2011): 23-40 Escritura e imagen Vol. ext. (2011): 23-40 Delmiro Rocha Escribir soberano Escribir soberano La expresión «decir la locura misma» es contradictoria en sí misma. Decir la locura sin expulsarla en la objetividad es dejarla que se diga ella misma. Pero la locura es, por esencia, lo que no se dice: es, dice profundamente Foucault, «la ausencia de obra».7 El rey muere por segunda vez y hace duelo de sí mismo, de su yo muerto, duelo consigo y por sí, duelo por su persona idealis que ha perecido en la locura. Lamenta la pérdida de su “yo rey“, la muerte de sí mismo. otra vez la soberanía como posi- bilidad para el exceso: Duelo imposible, duelo por sí. Todo trabajo, dice Derrida en Glas, es quizás un trabajo de duelo. a partir de este momento el rey es expulsado de palacio y deambula por un reino doblemente dividido, vaga por el solipsismo de su locura como un robinsón en su isla desierta y en compañía de su bufón. El bufón es la locura oficial, casi un ser humano, como Viernes, impropiamente humano. El bufón es la locura objetivada y aceptada, una locura que se dice a sí misma y que dice algo de sí, la locura que habita en palacio y que produce risas más que temores porque, bajo el sombrero colorido y con campañillas que la identifica, permanece en una prisión conceptual. El rey, en este destierro de sí, solamente se lleva de palacio a su bufón. Dos locuras caminando, dos cuerpos sin cabeza. Dos locuras distintas, teóricamente irreconciliables, que piensan la locura y se dan la mano. como si Derrida y Foucault paseasen por los chiflados callejones de cierta soberanía. cabe resaltar que el rey Lear es acusado de locura fundamentalmente por dos actos excesivos o hiperbólicos, imposibles, pero que muestran la condición de sobe- rano no como una comparación en la cual el rey siempre es más que lo otro (el más que...) sino como un superlativo absoluto (el más...). Es según el exceso como Derrida define lo propio de la soberanía: Lo esencial y propio de la soberanía no es pues la grandura o la altura geométricamen- te medibles, sensibles o inteligibles, sino el exceso, la hipérbole, un exceso insaciable de desbordar cualquier límite determinable: más alto que la altura, más grande que la grandura, etc. 7 Derrida 1989, op. cit (nota 1), p. 63. 8 Derrida, J: Séminaire La bête et le souverain. Volume I (2001-2002), Paris, Galilée, 2008, p. 345- 346. 8 Derrida, J: Séminaire La bête et le souverain. Volume I (2001-2002), Paris, Galilée, 2008, 346. 7 Derrida 1989, op. cit (nota 1), p. 63. 9 Shakespeare 2003, op. cit. (nota 2), pp. 112-114. El 4 de Marzo de 1963 en el Collège Philosophique Derrida lee lo siguiente: Es lo más, lo más que lo que cuenta, lo absolutamente más, el suplemen- to absoluto que excede cualquier comparativo hacia un superlativo absoluto.8 Los dos excesos del rey Lear son: primero, la división y donación de la sobera- nía. Segundo, la renuncia de cordelia negándole su condición de hija suya y situan- do el poder absoluto por encima incluso de la physis. En semejante hipérbole, el rey convierte de un golpe a dos de sus tres hijas here- deras en soberanas a la vez que en madres suyas. al instante el rey es acusado de locura y cae inmediatamente en ella. La locura como muerte viva producirá una ten- 30 Escritura e imagen Vol. ext. (2011): 23-40 Escritura e imagen Vol. ext. (2011): 23-40 Delmiro Rocha Escribir soberano dencia de la vida a la conservación de sí, un nuevo exceso que consiste en volver a la vida, esto es, en recuperar la cordura y el poder que se le asocia. Lear quiere vol- ver a ser el Rey Lear. Será el bufón, figura de la única locura aceptada y circunscri- ta, quien realizará los mayores esfuerzos para expulsar a Lear de cierta locura que no le es propia, que le pertenece en propiedad al bufón. La locura y el exceso jue- gan un papel opuesto y superpuesto, pues son a la vez sinónimos y contrarios. El bufón será el más cuerdo entre los cuerdos, “el loco malicioso”; y el rey, el más loco entre los que quieren ser cuerdos, “el loco inocentón”. El Rey Lear- Eres un loco malicioso. El Bufón- ¿Sabes tú, niño viejo, la diferencia que va de un loco malicioso a un loco ino- centón? l hij l El Rey Lear- Eres un loco malicioso. El Bufón- ¿Sabes tú, niño viejo, la diferencia que va de un loco malicioso a un loco ino- centón? El Rey Lear- Eres un loco malicioso. El Bufón- ¿Sabes tú, niño viejo, la diferenc centón? El Rey Lear- No, hijo mío, enséñamelo. El Bufón- oye. Ya que te han aconsejado que abandones tu corona, olvida, tío, tu trono y abrázate a mi persona. Dos locos verán los ojos de tu cortesana grey: uno en traje de bufón, otro con traje de rey. El Rey Lear- ¿Me llamas loco, hijo mío? El 4 de Marzo de 1963 en el Collège Philosophique Derrida lee lo siguiente: El bufón- has abdicado todos los títulos q Kent- No es tan bobo como parece, milord El Bufón- No, a fe; y eso que hago un ofi los señores y magnates. Me disputan el mo parte. hasta las damas me disputan esta p tareas. Tío, dame un huevo y te daré dos c El rey Lear- ¿cuáles son las dos coronas q El Bufón- Tomaré el huevo, lo partiré por daré las dos cáscaras. cuando partiste en hiciste lo mismo que si en un camino lleno tas. había muy poco seso bajo la corona d corona de oro. Si lo que voy a decir ahora den los azotes al primero que lo niegue. (c Para nosotros los locos el año ha sido muy malo; ya los cuerdos nos envidian y pretenden imitarnos.9 El Rey Lear- Eres un loco malicioso. El Bufón- ¿Sabes tú, niño viejo, la diferencia que va de un loco malicioso a un loco ino- centón? El R L N hij í éñ l uno en traje de bufón, otro con traje de rey. El Rey Lear- ¿Me llamas loco, hijo mío? El bufón- has abdicado todos los títulos que tenías por nacimiento Kent- No es tan bobo como parece, milord. El Bufón- No, a fe; y eso que hago un oficio del cual no quieren dejarme el privilegio los señores y magnates. Me disputan el monopolio de la locura, quieren ellos tener una parte. hasta las damas me disputan esta profesión y cometen usurpaciones sobre mis tareas. Tío, dame un huevo y te daré dos coronas. El rey Lear- ¿cuáles son las dos coronas que me darás? El Bufón- Tomaré el huevo, lo partiré por el medio, me comeré la yema y la clara y te daré las dos cáscaras. cuando partiste en dos tu corona y diste la una y la otra parte, hiciste lo mismo que si en un camino lleno de barro te hubieses cargado el burro a cues- tas. había muy poco seso bajo la corona de tu calvicie el día que hiciste donación de tu corona de oro. Si lo que voy a decir ahora no es de un cuerdo más que de un loco, que den los azotes al primero que lo niegue. (canta) 31 Escritura e imagen Vol. ext. (2011): 23-40 Delmiro Rocha Escribir soberano Escribir soberano El bufón no bromea sino que critica y denuncia. El 4 de Marzo de 1963 en el Collège Philosophique Derrida lee lo siguiente: No se mofa, ni divierte, ni se burla sino que analiza, asevera y postula. Defiende una concepción racionalista de la soberanía clásica sosteniendo que una división de la soberanía ni siquiera un loco se atrevería a hacerla. cierra las puertas a la novedad, al porvenir, y echa el cerrojo de la ortodoxia. La locura del rey Lear, incluso para el bufón chocarrero y cuentis- ta, es excesiva. En efecto, el rey Lear excede y se excede. Pero el exceso, propio de la soberanía, que pone en marcha es un exceso que excede el exceso. Usa el exce- so propiamente soberano para exceder la soberanía misma, para desde su “propia- mente”, desde su “en cuanto tal”, desde su “en sí misma”, salir fuera, al afuera de una totalidad, para expropiarse a sí y plegar la hipérbole. Mucho más allá de una economía soberana el rey Lear trabaja un pensamiento del don. Renuncia incondi- cionalmente a la soberanía para donarla, mediante una ofrenda sin reciprocidad, pues el que todo lo tiene nada puede esperar a cambio, y precisamente eso, “nada”, es lo que puede esperar, espera sin espera. La mayor ofrenda posible, el don del rey Lear al otro, rompiendo ya la unidad, da a la alteridad la alteridad, a ese radicalmen- te otro que es habitualmente lo que está más cerca, justo al lado, en el abismo de un “muy” cerca, o incluso “demasiado” cerca. Es un pensamiento de la vida/la muerte el que atraviesa la cabeza sin cabeza, sin corona y sin cap, del rey Lear. Una auto- decapitación parricida que no mata sino que da paso a la muerte abriendo un porve- nir incalculable. La demasía o el colmo del exceso como soberanía que el rey Lear acomete consiste en hacer o en pretender hacer lo imposible. En dar aquello que es propiamente sí mismo, único e indivisible, y dividirlo en su darse al otro. Este es el poder auto-inmunitario que sobrepasa y viola la incondicionalidad del poder sobe- rano al hacer lo imposible. 10 Derrida, J. : Séminaire La bête et le souverain. Volume II (2002-2003), Paris, Galilée, 2010. p. 328. 11 Shakespeare 2003, op. cit. (nota 2), p. 159. El 4 de Marzo de 1963 en el Collège Philosophique Derrida lee lo siguiente: Derrida pregunta respondiendo en el capítulo nueve del segundo volumen de La bestia y el soberano: “¿Qué es el poder si no puede más que el im-poder, si no puede más que lo que no puede, a saber, lo imposible?”10 Lear, en el intento desesperado de recuperar su poder, propone introducirse en la división y el reparto que él mismo ha llevado a cabo y compartir el poder con sus hijas. Regana, al rechazar esta opción, abre, sin embargo, la puerta a lo imposible: “¿cómo queréis vos que en una misma casa obedezca tanta gente a distintos amos y viva en buena armonía? Es difícil, es casi imposible”11. Lo “casi imposible” per- tenece ciertamente al dominio de lo posible. Regana niega esta posibilidad a Lear, bajo pretexto de imposible, a pesar de que ella misma la entiende como posible, como “casi imposible”. Por otro lado, lo que ella niega por “casi imposible” es pre- cisamente el poder dividido que ella ostenta con su hermana Gonerila. a lo largo de toda la obra este desplazamiento entre imposible y casi imposible es una constante que muestra la división sobre ambos terrenos, siempre (casi) imposible. Este “casi” imposible, este “por poco” o “cerca de” lo imposible, no es simplemente la aproxi- 32 Escritura e imagen Vol. ext. (2011): 23-40 Escritura e imagen Vol. ext. (2011): 23-40 Delmiro Rocha Escribir soberano Escribir soberano mación de lo posible a lo imposible sino también la posibilidad imposible como la adyacencia o la vecindad de lo imposible con lo posible. El arrimo de la imposibi- lidad a lo posible es el arribo del porvenir o del quizá como acontecimiento inaudi- to, el anuncio del porvenir que quizá venga o la llegada del porvenir que viene quizá como quizá, bajo la forma de un peligroso quizá. Pero a Regana lo “casi imposible”, el extremo de lo posible, le basta para confirmar la enajenación de Lear e insistir en que la locura debe estar fuera de palacio, lejos de la residencia del poder, del senti- do y de la razón. Sin embargo, la locura es siempre el huésped de la razón, el pen- sionista o convidado de la cordura. No es un forastero que viene de fuera e invade la pureza del hogar propio sino que habita como anfitrión (segundo significado de huésped) injertando la alteridad y dividiendo toda vivienda. Divivienda; divivir. 12 a lo largo de toda la obra la palabra “cabeza” se repite constantemente. El texto habla de la cabeza, del cap, del cabeza de familia, pero también de la decapitación simbólica, el destronamiento del rey. 13 Shakespeare 2003, op. cit. (nota 2), p. 175. El 4 de Marzo de 1963 en el Collège Philosophique Derrida lee lo siguiente: División de la vivienda y vivir en división. Dos neologis- mos que me permito o me arriesgo a inscribir aquí para acercarme lentamente a la deconstrucción de lo propio de la soberanía que está en marcha, pues la locura en la corte del Rey Lear no se limita a las figuras del bufón y del rey. hasta cinco per- sonajes de palacio serán visitados por la demencia a lo largo de la obra. En el tercer acto, Edgardo, que había abandonado el castillo real engañado so pena de traición, reaparece en escena con “aspecto de loco” en una choza donde el rey Lear, el bufón y el conde de Kent buscan cobijo de la tormenta que se avecina sobre sus cabezas12. “(aparece Edgardo con el aspecto de un loco.)”13 así dice, entre paréntesis, la didascalia o acotación escénica inserta en el medio del texto. No explica necesaria- mente si Edgardo finge o no finge estar loco. Simplemente “aparece Edgardo con el aspecto de un loco”. El aspecto de un loco no es un atributo constitutivo ni de un loco ni de un cuerdo. Edgardo aparece, fenomenalmente, “con el aspecto de un loco”. La apostilla o acotación introduce al tercer demente de la historia. Ningún elemento es ajeno aquí a la realeza o a palacio. Toda la obra de teatro, incluidos sus márgenes escénicos, están acotados al mundo soberano, imperial y principesco. Incluidas las acotaciones escénicas, las apostillas, las didascalias o las cotas, térmi- no antiguo pero que resulta el más justo o ajustado en este caso. [Paréntesis etimológico: La palabra “cota”, que deriva del latín “quota”, es, en una de sus acepciones, un sinónimo de “acotación”. Pero la etimología de la pala- bra “cota” tiene una doble vertiente o fuente significativa. así, la palabra “cota”, que deriva del francés antiguo, significa, en la segunda acepción del diccionario María Moliner, “Vestidura de los reyes de armas, sobre la que están bordados los escudos reales.”] 33 33 Escritura e imagen Vol. ext. (2011): 23-40 Escritura e imagen Vol. ext. (2011): 23-40 Escritura e imagen Vol. ext. (2011): 23-40 Delmiro Rocha Escribir soberano Escribir soberano Double bind de la cota. Las escasas cotas, en la obra de Shakespeare como en todo el teatro clásico, son, en la obra de Derrida y según la deriva etimológica fran- cesa, abundantísimas. El 4 de Marzo de 1963 en el Collège Philosophique Derrida lee lo siguiente: Los símbolos, materiales e inmateriales, de la soberanía mar- can la importancia de la visibilidad para el regio poder. No obstante, el verdadero poder del soberano radica en su (in)visibilidad. Su ser visible/invisible, su ser sin estar. cualidad fantasmática del que es y no es. El “efecto visera” que Derrida desa- rrolla en Espectros de Marx a propósito del padre de hamlet supone la autoridad constituida a través de un ver sin ser visto, del poder de verlo todo porque quien todo lo ve es, asimismo, invisible. asimismo invisible, a-sí mismo invisible, mas no invisible a sí mismo. Invisibilidad para el otro como invisibilidad del otro. El otro teme y siente el poder de la ley porque no la ve, porque sin verla la ve, porque se hace visible en su invisibilidad. Sólo en cuanto invisible la ley es absolutamente visible, imposible de esquivar. División soberana de la cota: por un lado, anotación al margen que no se ve en la representación pero, sin embargo, se ve sin ser vista sobre la escena. Por otro lado, simbología visible del poder regio. así aparece en escena Edgardo, el hijo de Gloucester y aspirante un día al trono. “aparece Edgardo con el aspecto de un loco”. Escondido bajo harapos y refugiado en una choza. Vestimenta y alojamiento contrarios a los símbolos de la realeza. anunciado por una cota, sostiene un discurso enajenado que oculta definitivamen- te su sangre azul, pero será precisamente este detalle el que llame la atención y la admiración de Lear. Dos cuerpos de la realeza que se atraen por la locura que los excluye de la realeza que los atrae. Dos páginas después de que el bufón anuncie “esta noche glaciar nos volverá locos a todos”14, entra en la choza, antorcha en mano, el conde de Gloucester. allí encuentra al rey Lear enajenado, a su hijo Edgardo con aspecto de loco, al bufón de la corte y al conde de Kent al que no reconoce. Gloucester confiesa “poco falta para que yo también me vuelva loco”15. Este grupo de locos serán, no obstante, los que defiendan y representen la cordura en la pieza. En cualquier caso, la locura parece apoderarse de toda la obra y de todos los personajes hasta el desenlace. Gloucester será acusado de loco por el rey Lear16, y el duque de albany lo será a su vez por boca de Gonerila17. 14 Shakespeare 2003, op. cit. (nota 2), p. 178. 15 Ibídem p. 183. 16 Ibídem p. 228. 17 Ibídem p. 211. 18 Ibídem pp. 183-184. 17 Ibídem p. 211. 18 Ibídem pp. 183-184. El 4 de Marzo de 1963 en el Collège Philosophique Derrida lee lo siguiente: al mismo tiempo, el rey Lear tilda a Edgardo, con aspecto de loco y discurso enajenado, de “filósofo”, “sabio tebano”, “noble filósofo”, “mi filó- sofo” y “mi querido ateniense”18. Tanto la locura como “el buen sentido” y la filosofía se encuentran del lado de los personajes caracterizados y/o acusados de demencia. Paralelamente, los actos 17 Ibídem p. 211. 34 Escritura e imagen Vol. ext. (2011): 23-40 Escritura e imagen Vol. ext. (2011): 23-40 Delmiro Rocha Escribir soberano Escribir soberano más ajenos al “buen sentido” o cordura clásica se sitúan en los personajes que jue- gan el papel de juiciosos y cabales. Esta inversión clásica de la trama revela sin embargo aspectos que hacen posible una lectura contemporánea de la obra. Más allá de la interpretación clásica de esta pieza de teatro majestuosa, que normalmente se asume como una escritura que defiende el amor filial, la philia y el respeto al padre (señor y soberano), aquí se ponen en juego temáticas contemporáneas como la divi- sión de la soberanía y la indivisibilidad entre razón y locura. En El Rey Lear de Shakespeare la locura habita en palacio como huésped: a la vez invitado y anfitrión. La hospitalidad y la hostilidad, la acogida y el rechazo del otro, no son simplemente vecinos semánticos sino también coinquilinos. Si bien es cierto que la hospitalidad no es simplemente la ausencia de hostilidad, así como la hostilidad tampoco es simplemente la ausencia de hospitalidad, debería ser igual de cierto, al menos en principio, que allí donde hay, si la hay, hospitalidad habría tam- bién ausencia de hostilidad, y viceversa. Pero esta ausencia es siempre fenoménica o especulativa pues ambos términos se co-implican. El término castellano “hoste” deriva del itálico “oste” y significa “posadero”, y de ahí tenemos hostalero, hostalaje, hospicio, hostal o el inglés hostel; “hostal”, sinónimo de hospedería o posada, establecimiento que ofrece alojamiento y comi- da al otro, deriva a su vez del latín “hospitalis”, exactamente la misma raíz que tene- mos para el término “hospital”, establecimiento que se ocupa de dar cobijo sanita- rio al otro, y que antiguamente significaba “hospitalario”. El 4 de Marzo de 1963 en el Collège Philosophique Derrida lee lo siguiente: asimismo, el término “hoste” (oste) nos conduce al francés hôtel y al castellano “hotel”, que además de ser un hospedaje de mayor categoría y refinamiento que el hostal, era también en los inicios del siglo XV en Francia el nombre otorgado a las lujosas residencias con jardín de los señores y terratenientes, funcionando como sinónimo de “palacio”, “chalet”, “villa” o “quinta”, y reenviando la etimología del lado del poder y de la autoridad. Por este lado, y forzando quizá demasiado la etimología, también encon- tramos en castellano el término “hospodar” que corresponde a una forma rumana o ucraniana y deriva del ruso “gospodar”, que significa “señor”: era el nombre que se daba a los antiguos príncipes soberanos de Moldavia y de Valaquia. Pero el término “hoste”, en castellano, tiene una segunda entrada que deriva directamente del latín “hostis” y significa “enemigo”, “ejército” o “parte de un ejér- cito”, es decir “hueste”, que también deriva del latín “hostis” y significa “tropa” o incluso “partidario”. Si se continúa por esta segunda acepción encontraremos la palabra “hostaje”, del provenzal “ostatge”, que significa “rehén”, así como “hos- tia”, “hostilizar”, “hostil” y “hostilidad”. El término “hoste”, con su doble significación o significación bífida, se sitúa de lleno sobre el terreno gubernativo y abre en un mismo gesto dos vertientes políti- cas: la hospitalidad o la hostilidad como ética fronteriza que marca la relación del Estado con el otro. así como para carl Smith la hostilidad, es decir, el concepto de 35 Escritura e imagen Vol. ext. (2011): 23-40 Escritura e imagen Vol. ext. (2011): 23-40 Escritura e imagen Vol. ext. (2011): 23-40 Delmiro Rocha Escribir soberano “enemigo” (hoste), marca el principio de la política, para Derrida la hospitalidad incondicional no será una opción ética frente a otras sino la ética misma a partir de la cual se toman o se abren las opciones: La hospitalidad es la cultura misma y no es una ética entre otras. El 4 de Marzo de 1963 en el Collège Philosophique Derrida lee lo siguiente: En la medida en que atañe tanto al ethos, a saber, a la morada, al chez-soi (a la casa, al hogar), al lugar de la estancia familiar como a la manera de estar ahí, a la manera de referirse a uno mismo y a los otros, a los otros como los suyos propios o como a unos extranjeros, la ética es hospitalidad, es de arriba a abajo coextensi va con la experiencia de la hospitalidad, cualquiera que sea la forma en que se la abra o se la limite. Pero por esa misma razón, y porque el ser-uno-mismo en casa (la ipseidad misma) supone una acogida o una inclu- sión del otro que uno intenta apropiarse, controlar, dominar, según diferentes modalida- des de la violencia, hay una historia de la hospitalidad, una perversión siempre posible de La ley de la hospita lidad (que puede parecer incondicional) y de las leyes que vie- nen a limitarla, a condicionarla inscribiéndola en un derecho.19 Desde El Sofista y El Político de Platón hasta la introducción a Ser y Tiempo de heidegger, la potestad o inclusión (control y violencia) del extranjero se asienta sobre la autoridad del paterfamilias. La soberanía del poder, potestas, presupone el hogar, la casa, el lugar de lo propio. El dominio o la familia del ipse, que se resume por economía con el sustantivo abstracto “ipseidad”, sigue marcando la primera y la última frontera de lo político. a pesar de la profunda transformación que produ- ce hoy en día el mundo de las telecomunicaciones y todo lo que su defensa o su agravio conlleva, el límite entre lo público y lo privado continúa presuponiendo el chez soi como lo propio, el hogar o la morada donde sólo manda uno, donde manda el Uno. La hospitalidad o la hostilidad, para la tradición que hoy heredamos, segui- ría empezando a partir de ese presupuesto ontológico que otorga a la propiedad pri- vada el poder y control absolutos sobre sí misma. Este presupuesto, además de afianzar una idea capitalística y extender sus raíces hasta el zócalo originario de la “fundación” europea, consolida el triunfo de una historia del sujeto, de la historia del sujeto soberano y dueño de sí. 19 Derrida, J.: Cosmopolitas de todos los países, ¡un esfuerzo más!, Valladolid, cuatro Ediciones, 1996, pp. 41-42. 20 Derrida, J.: La Hospitalidad, Buenos aires, Ediciones La Flor, 3ª ed. 2008, p. 59. El 4 de Marzo de 1963 en el Collège Philosophique Derrida lee lo siguiente: No existe hospitalidad, en sentido clásico, sin soberanía del sí mismo sobre el propio- hogar, pero como tampoco hay hospitalidad sin finitud, la soberanía sólo puede ejercer- se filtrando, escogiendo, por lo tanto excluyendo y ejerciendo violencia. La injusticia, cierta injusticia, incluso cierto perjurio, comienza inmediatamente, desde el umbral del derecho a la hospitalidad.20 36 Escritura e imagen Vol. ext. (2011): 23-40 Escritura e imagen Vol. ext. (2011): 23-40 Delmiro Rocha Escribir soberano Entonces, la violencia es intrínseca a la propia soberanía aunque ésta se decan- te, consciente y premeditadamente, por una ética de la hospitalidad. Esta contradic- ción conduce a la auto-inmunidad de la soberanía: para salvaguardar el lugar (de lo) propio, ya sea la casa familiar o el Estado, y para poder aplicar el derecho de la hos- pitalidad, es preciso no ser hospitalario, es preciso negar o cerrar, de entrada, la entrada al extranjero. Esta es la aporía: no hay lugar propio sin hostilidad de prin- cipio, no hay hospitalidad sin lugar propio. aquello que da lugar y permite el dere- cho a la hospitalidad es la hostilidad que, a su vez, la niega. Derrida enuncia esta aporía con el sintagma francés “pas d´hospitalité”. Imposible traducción, pues en francés “pas” significa a la vez: la partícula negati- va que permite traducir el sintagma como “no existe hospitalidad” o “nada de hos- pitalidad”; y el sustantivo “paso” que anuncia el movimiento de la hospitalidad o la marcha hacia la hospitalidad, “paso de hospitalidad”. Por un lado, no hay hospitalidad sin hostilidad pero, por el otro, la hospitalidad no se abre más allá del sujeto soberano de sí mismo sino que pertenece al propio chez soi, marca la instancia desgarrada de lo propio y divide infinitamente al suje- to; deconstrucción del sujeto. La hostipitalidad comienza en el otro, y el otro divi- de y habita, divive en la divivienda del chez soi. Este proceso hostipitalario es el que (di)vive el rey Lear. El soberano absoluto, el dueño y señor, el paterfamilias de todas las familias sufre este proceso, por lo menos, doblemente doble: Primera doblez: según un gesto de hospitalidad el rey da a sus yernos, [pues no hay que olvidar la lectura machista y misógina que atraviesa la obra: el rey necesi- ta casar a sus hijas para poder dividir el poder], en una donación absoluta, la totali- dad de su reino. 21 Shakespeare 2003, op. cit. (nota 2), p. 266. 22 Ibídem. El 4 de Marzo de 1963 en el Collège Philosophique Derrida lee lo siguiente: En este gesto incluye al otro [sus hijas y especialmente sus yernos y las familias de estos] en su casa. casa que deja de ser su casa (chez soi) en el pro- pio dar. Esta hospitalidad desencadena la hostilidad que constituye la trama hasta su fatal desenlace. al mismo tiempo, la hospitalidad del dar o del incluir al otro en su casa es una hospitalidad hostil, pues se hace a través de la palabra soberana que hace y es la ley. Es decir, la hospitalidad del rey Lear se otorga gracias a la violencia de su ley, no se invita al otro a entrar sino que se le ordena que entre. El rey Lear es dueño de sí mismo, de su casa, de su lugar propio y, por consiguiente, decide sobe- ranamente quién y cuándo debe entrar. Esta hostilidad ontológica permite la hospi- talidad, a su vez hostil, del soberano. Segunda doblez: el cuerpo soberano del rey se divide en razón y locura. huésped y anfitrión, huésped como anfitrión. La hostilidad habita en la casa de la razón, en la cabeza de la familia, y vulnera un supuesto límite externo desde den- tro, desde el adentro que se proyecta hacia afuera. El expulsar, expeler, prohibir, desterrar, deportar, etc. al otro, a la alteridad de lo otro que está dentro como un agente impuro y que viene a contaminar la pacífica convivencia consigo del dueño 37 Escritura e imagen Vol. ext. (2011): 23-40 Escritura e imagen Vol. ext. (2011): 23-40 Escritura e imagen Vol. ext. (2011): 23-40 Delmiro Rocha Escribir soberano que es uno, siempre ha constituido, aun con fines hospitalarios, la violencia de la frontera. Pero la hostipitalidad muestra que esta frontera se divide desde el interior proyectando hacia afuera una ilusión de límite unitario y sólido. Si hay frontera, si creamos frontera, es porque lo que se trata de impedir está ya de alguna forma a ambos lados del límite. No se impide la llegada de lo que no se sabe que llega, de lo que no ha llegado ya. Quizá, tercera doblez: lo que llega sin anuncio y sin visibilidad, sin aviso ni pre- visibilidad posible, es el quizá mismo, el porvenir como llegada imposible del quizá. Este acontecimiento absoluto, este arribante sin ser, llega en El Rey Lear de Shakespeare para deconstruir finalmente la unidad, para dividir sin premeditación ni conciencia lo que inevitablemente se esperaba unitario. El 4 de Marzo de 1963 en el Collège Philosophique Derrida lee lo siguiente: (2011): 23-40 Delmiro Rocha Escribir soberano Escribir soberano albany- Llevad esos cadáveres…Un luto general: he aquí ahora nuestra tarea… (a Kent y a Edgardo.) amigos míos, los más queridos, gobernad ambos este reino y sed sus sos- tenes.23 albany- Llevad esos cadáveres…Un luto general: he aquí ahora nuestra tarea… (a Kent y a Edgardo.) amigos míos, los más queridos, gobernad ambos este reino y sed sus sos- tenes.23 “Gobernad ambos este reino”, dice el Duque de albany, siendo él la autoridad en este particular “estado de excepción”. El Duque, al instituir un nuevo poder gubernativo y evitar así la orfandad del reino, divide una vez más la soberanía. Esta nueva división de la soberanía, lejana a la voluntad del Rey Lear, llega como lo imprevisible mismo, como la venida de lo inaudito. El carácter imposible de la divi- sión soberana se pone en entredicho por segunda vez. La obra empieza y acaba con dicha división. Quizá la imposibilidad que aqueja y limita la división de la soberanía sea sim- plemente el fruto todavía verde que proviene del árbol ontológico de occidente y hunde sus raíces en una tele-teo-onto-logía político-metafísica. Rastrear sus frutos, dar credibilidad a ciertas semillas allí donde pudieran haber florecido, quizá aporte la convicción de que la división de la soberanía no es simplemente imposible sino que arriba o llega como lo imposible, como la metáfora literaria de una narratividad que florece en el agreste terreno de una posibilidad imposible… Detengo aquí esta lectura de El Rey Lear de Shakespeare. Y aquí que estamos, de una manera u otra, heredando a Derrida, siéndole infiel por fidelidad, haciendo un trabajo de duelo, yo, como el rey Lear que hizo múltiples duelos: por su hija cordelia, por su soberanía perdida e incluso por sí mismo; yo quisiera no desapro- vechar la oportunidad de este teatro, de estos minutos que amablemente me han sido concedidos, para manifestar también otro duelo y otra herencia, para traer a otro fantasma que a muchos de los aquí presentes nunca nos ha dejado de asediar, de per- seguir. Este texto, el que ahora leo casi sin improvisación, lo he escrito en lo que era y seguirá siendo la mesa, la silla, el ordenador de Paco Vidarte. como ustedes saben, su tesis doctoral, publicada en francés bajo el título “Derritages. El 4 de Marzo de 1963 en el Collège Philosophique Derrida lee lo siguiente: al final de la obra, el rey Lear recupera por un instante el poder soberano: albany- […] resignaremos en manos de nuestro anciano monarca la autoridad absoluta para que la goce el resto de sus días. (a Edgardo y a Kent.) Vosotros seréis reintegrados en todos vuestros derechos y se os conferirán nuevos honores que habéis merecido.21 El rey consigue recuperar a través de la palabra de albany el poder que perdió a través de su palabra. Pero, justo en ese instante, la soberanía absoluta vuelve a no ser suficiente y el rey Lear muere ante el cuerpo muerto de su hija cordelia: El Rey Lear- ¡Y a mi pobre hija la han estrangulado!... ¡No, no tiene vida! ¿Por qué un perro, un caballo, un ratón, viven, y tú en cambio no respiras?... ¿Ya no te veré más, no! ¡Jamás, jamás, jamás!…Desabrochadme este botón, os lo ruego… ¡Gracias!... ¿Veis, veis? ¡Miradla, miradla!... Sus labios… ¡oh! ¡Mirad, mirad!... (Pega sus labios a los de cordelia y muere.)22 La tercera muerte del Rey Lear, la muerte de su persona personalis, se produce un instante después de que la soberanía se reagrupase de nuevo en un solo cuerpo, produciendo a su vez y en el mismo gesto de la reunión la división interna idealis/personalis. El rey fracasa en su intento de división de la soberanía, sus tres hijas mueren, él recupera el poder y muere acto seguido. Pero este final intensamente dramático guarda todavía una vuelta imprevisible. Quizá el único final inesperado y sorpren- dente. Shakespeare parece estar todavía pensando en la indivisibilidad de la sobera- nía de Bodin cuando escribe las últimas frases de la pieza de teatro. con el rey muerto y el reino, por lo tanto, sin cabeza, huérfano, albany toma por segunda vez en la obra la palabra soberana y dicta la ley que instaurará la futura autoridad: 38 Escritura e imagen Vol. ext. (2011): 23-40 Escritura e imagen Vol. ext. 23 Shakespeare 2003, op. cit. (nota 2), p. 267. 24 Vidarte, P.: Derritages. Une Thèse en déconstruction, L´harmattan, Paris, 2001, pp. 120-121. Escritura e imagen Vol. ext. (2011): 23-40 El 4 de Marzo de 1963 en el Collège Philosophique Derrida lee lo siguiente: Une thèse en déconstruction” es todavía hoy uno de los mejores escritos dedicados a la heren- cia de y en Derrida. Me gustaría cederle a él, a ese amigo que hacía sacar “el loco que hay en nosotros”, los últimos minutos de mi intervención: “Des-cendre(s) de Derrida decíamos hace no mucho en un deseo contradictorio de pyrificación. Inevitable double bind de la herencia que se le impone a todo comentador. Voluntad de purificar, de extractar lo más importante, lo casi indispen- sable: antología, monumento. Impotencia ante la pirificación que ello conlleva, la incineración de los restos del padre que hace imposible la memoria, el recuerdo, la arqueología: cenizas, ruina. Frases que no son más que ceniza, condenadas a la dis- persión, a la diseminación sin retorno al padre, a ningún origen previo, a ningún sentido preexistente del que fueran resto. Restos de restos amenazados de la disipa- 39 Escritura e imagen Vol. ext. (2011): 23-40 Escritura e imagen Vol. ext. (2011): 23-40 Delmiro Rocha Escribir soberano ción más absoluta, que se retiran ante todo intento de apropiación, de contrasigna- tura, obligando a nuestra escritura a prolongarse en un duelo sin sentido, melancó- lico, porque no sabe lo que ha perdido, que quizá sea pura pérdida sin objeto, de un objeto fantasmático llorado con algo de retraso. Pyrificación que no puede sino ser responsable ante la indecidibilidad de preferir éstas a otras cenizas, forzándonos a decidir allí donde la decisión se hace imposible y no puede más que ser sobreveni- da. Verdadera experiencia la de rebuscar afanosamente entre unos restos de no se sabe qué Fénix, mientras proseguimos en nuestra lectura incinerante el inevitable holocausto de la herencia. Ya es demasiado tarde para no pensar a partir de Derrida”24. Y también de Paco. 40 Escritura e imagen Vol. ext. (2011): 23-40 Escritura e imagen Vol. ext. (2011): 23-40
https://openalex.org/W2894382422	https://link.springer.com/content/pdf/10.1007%2F978-981-10-8950-3_31.pdf	English	null	FLOW 800 for Vascular Surgery	Springer eBooks	2,018	cc-by	3,699	31 31 FLOW 800 for Vascular Surgery Yoko Kato, Ittichai Sakarunchai, and Mohsen Nouri 31.2.1 Color-Coded Images These images convert the black and white images of conventional ICG-VA into a gradient where red and blue colors represent arteries and veins, respectively (Fig. 31.1). This can be helpful espe- cially in AVM surgeries to identify feeding arter- ies. Also, if the color of a distal artery changes after clipping of an aneurysm, this might imply blood flow impairment. However, one should be cautious about interpretation of these images. As mentioned earlier, the basis of this software to color a vessel in red or blue is their chronological ICG fluorescence not the real direction of blood flow. This may result in misdiagnosis in some occasions such as when one type of vessel (artery or vein) is not in the field of view (e.g., a deep feeding artery). A good knowledge of anatomy and reviewing details of the vascular pathology before the surgery are mandatory for the surgeon to compensate for these shortcomings. 31.1 Introduction introduced to picture blood vessels during surgi- cal exposure. Most commonly used fluorescent dye is indocyanine green (ICG) which was first used for assessment of hepatic function in severe chronic liver diseases for which it was approved by FDA in 1959 and later for retinal and choroi- dal circulation, liver and renal blood flow, and cardiac output. Microscopic-integrated near- infrared ICG videoangiography (VA) has been used in several cerebrovascular surgeries since 2003. Intraoperative ICG-VA is done by using the commercially available microscopes (e.g., OPMI Pentero, Carl Zeiss, Oberkochen, Germany). This allows the surgeon to obtain an intraoperative angiography in less than 5 min (in contrast with conventional DSA which usually takes about 20 min) without the need to intro- duce any new device (i.e., angiography unit) into the surgical field. The main shortcoming of ICG-VA is that its images are limited to the sur- gical field and identifying arteries from veins is not readily possible. Also, it provides the sur- geon with only anatomical data without any information about the physiology and dynamics of the blood flow. To overcome these limitations, a new image analysis software package was released later to distinguish physical properties of the flow in the vessels and demonstrate semi- quantitative data. Neurovascular surgeries are sophisticated pro- cedures, and a thorough knowledge of the ves- sels before and during the operation is a necessity to prevent inadvertent damage and catastrophic results. Comprehensive monitoring especially real-time evaluation of cerebral blood flow is very helpful for surgery of cerebral aneu- rysms, cerebral arteriovenous malformations (AVMs), and extracranial-intracranial (EC-IC) bypass. It has been shown that addition of intraopera- tive angiography improves the outcome of aneu- rysm and AVM surgeries by approving complete obliteration of the pathology and preventing inadvertent occlusion of the adjacent vessels. Until a decade ago, there was only intraoperative conventional angiography for this purpose, but recently near-infrared fluorescence module that integrated to microscope for neurosurgery was Y. Kato (*) Department of Neurosurgery, Fujita Health University, Toyoake, Aichi, Japan e-mail: kyoko@fujita-hu.ac.jp I. Sakarunchai Division of Neurosurgery, Department of Surgery, Faculty of Medicine, Prince of Songkhla University, Songkhla, Thailand M. Nouri Gundishapour Academy of Neuroscience, Ahvaz, Iran e-mail: nouri@gan-ac.ir 269 © The Author(s) 2019 J. July, E. J. Wahjoepramono (eds.), Neurovascular Surgery, https://doi.org/10.1007/978-981-10-8950-3_31 Y. Kato et al. 270 31.2 FLOW 800 Software area of interest (ROI), and then the curve and some primary parameters are calculated and depicted by the software. Parameters calculated directly by the software include average intensity (shown in arbitrary intensity [AI] units), delay time (i.e., the time interval from 0 to 50% of max- imum fluorescence intensities [MFI]), and the slope of the curve. On the other hand, there are some other indices presented in the literature known to be correlated with perfusion character- istics of tissues which simply can be calculated with an image analysis software such as Image J (version 1.46, National Institute of Health, USA) after delivering the data from the microscope sta- tion (Fig. 31.2). Transit time is the time differ- ence between MFI in artery and brain tissue, and rise time is defined as time during which fluores- cence intensity rises from 10 to 90% of its peak. Other variables that can be measured manually include MFI, time to peak (i.e., from the appear- ance of fluorescence to MFI), and cerebral blood flow index (CBFI) which is defined as ratio of MFI to rise time. These parameters can be calcu- lated for each vessel, and their changes should be tracked throughout the procedure [2]. New image analysis software, namely, FLOW 800 (Carl Zeiss, Oberkochen, Germany) was pre- sented in 2010 that produces intensity diagrams and color mapping. This software works on the assumption that the earlier-arriving ICG in the field belongs to the arteries and those disappear- ing at last belong to the veins and so depicts the vascular field in a color-coded mode [1]. The operator (e.g., the surgeon or assistants) can deter- mine some region of interest (ROI) for the soft- ware which can be further analyzed to draw the intensity diagram. This diagram shows the inten- sity of fluorescence over time in the ROI. More information can be deduced from this diagram after manual analysis by an image analysis soft- ware. The combination of these two processed images in accumulation to the ICG-VA helps the surgeon to judge the anatomy and physiology of the vessels in different situations (see below). 31.3.1 Cerebral AVMs Surgeries During AVM surgeries, ICG-VA can detect ves- sels as small as 0.5 mm in diameter and is very practical for in superficial AVMs. However, the field of angiography is limited to the field of microscopic view. As the surgeon cannot see the deep vessels and the whole structure of the AVM, sometimes it gets difficult to identify arteries and veins. FLOW 800 software can help the surgeon with identification of AVM vessels by producing a color-coded map where red represents feeding arteries and blue stands for drainers (Fig. 31.1). Also, after selecting some ROIs for the software, the intensity analysis curves help to understand the hemodynamics of the vessels and the sur- rounding brain parenchyma. For example, both image types can be used before and after tempo- rary clipping of the arterial feeders to evaluate its effect on the AVM nidal blood flow and as a guide 31.2.2 Intensity Diagram To draw an intensity diagram, we usually use cir- cular or rectangular marks to define the vascular 271 31 FLOW 800 for Vascular Surgery a b c d e f Fig. 31.1 Left occipital arteriovenous malformation (AVM) in a 45-year-old man. (a) Reconstructed com- puted tomography angiogram shows the location, feed- ing artery, and draining vein of the AVM. (b) Before opening the dura, color-coded imaging by FLOW 800 delineated vessels and helped us tailor durotomy. (c) The superficial presentation of the AVM after opening the dura. (d) Color-coded angiography immediately after opening the dura. (e) Color-coded imaging after partial occlusion of the feeders. Note that color changes in the vessels may indicate hemodynamic changes of the AVM. (f) Color-coded angiography after confirming total resection of the malformation b a b a a c d c e Fig. 31.1 Left occipital arteriovenous malformation (AVM) in a 45-year-old man. (a) Reconstructed com- puted tomography angiogram shows the location, feed- ing artery, and draining vein of the AVM. (b) Before opening the dura, color-coded imaging by FLOW 800 delineated vessels and helped us tailor durotomy. (c) The superficial presentation of the AVM after opening the e e f Fig. 31.1 Left occipital arteriovenous malformation (AVM) in a 45-year-old man. (a) Reconstructed com- puted tomography angiogram shows the location, feed- ing artery, and draining vein of the AVM. (b) Before opening the dura, color-coded imaging by FLOW 800 delineated vessels and helped us tailor durotomy. (c) The superficial presentation of the AVM after opening the dura. (d) Color-coded angiography immediately after opening the dura. (e) Color-coded imaging after partial occlusion of the feeders. Note that color changes in the vessels may indicate hemodynamic changes of the AVM. (f) Color-coded angiography after confirming total resection of the malformation to externally validate these data to better define their normal range and predictive capacity. for the next step of the operation. Also, changes in the cerebral parenchymal blood flow can be calculated to evaluate the effect of AVM removal on cerebral perfusion and predict postoperative autoregulation disturbances (e.g., breakthrough phenomenon). However, more data are required to externally validate these data to better define their normal range and predictive capacity. The main limitations of ICG-VA in AVM sur- geries also extend to the FLOW 800 as deeply located vessels are not visualized and comple- mentary DSA is required in such cases [3]. 31.2.2 Intensity Diagram Also, The main limitations of ICG-VA in AVM sur- geries also extend to the FLOW 800 as deeply located vessels are not visualized and comple- mentary DSA is required in such cases [3]. Also, 272 Y. Kato et al. 0 0 200 400 600 800 1000 1200 5 10% MFI 90% MFI MFI 892 AI Time to peak 14.37 s Rise time 6.02 s Delay 7.95 s D CBFI = MFI/rise time 892.00/6.02 = 148.17 AI/s 10 Time (s) Average Intensity AJ 15 Fig. 31.2 Intensity diagram of a vessel after manual image analysis with Image J software. Maximum fluorescence intensity (MFI) is the highest intensity of fluorescence for a defined area, and time to peak is the time interval from the appearance of fluorescence until its peak. Rise time is the time interval when fluorescence intensity rises from 10 to 90% of MFI. Cerebral blood flow index (CBFI) is the ratio of MFI to rise time 0 0 200 400 600 800 1000 1200 5 10% MFI 90% MFI MFI 892 AI Time to peak 14.37 s Rise time 6.02 s Delay 7.95 s D CBFI = MFI/rise time 892.00/6.02 = 148.17 AI/s 10 Time (s) Average Intensity AJ 15 0 0 200 400 600 800 1000 1200 5 10% MFI 90% MFI MFI 892 AI Time to peak 14.37 s Rise time 6.02 s Delay 7.95 s D CBFI = MFI/rise time 892.00/6.02 = 148.17 AI/s 10 Time (s) Average Intensity AJ 15 Fig. 31.2 Intensity diagram of a vessel after manual image analysis with Image J software. Maximum fluorescence intensity (MFI) is the highest intensity of fluorescence for a defined area, and time to peak is the time interval from the appearance of fluorescence until its peak. Rise time is the time interval when fluorescence intensity rises from 10 to 90% of MFI. Cerebral blood flow index (CBFI) is the ratio of MFI to rise time In a recent study, 2 of 12 cases needed adjust- ment of the clip after finding an occlusion of per- forating vessels [4]. Also, after ICG-VA they found 4 cases of incomplete clipping out of 45 (8.9%) who required appropriate readjustment to complete the obliteration. in cases in which the main vessels are covered with blood clot, brain tissue, cottonoid patties, etc., the software may not be accurate in differen- tiating arteries from veins. 31.3.2 Cerebral Aneurysm Surgeries In some cases, atherosclerotic plaque in the aneurysm may make interpretation of ICG-VA very difficult if not impossible. In these cases when there is any doubt in the result of an ICG-VA, we should use other monitoring tech- niques such as micro-doppler or endoscope that can be used to check for residual blood flow in aneurysm sac or incomplete obliteration of the aneurysm neck. In aneurysm surgeries, ICG-VA can demonstrate perforating arteries before and after clipping, check for patency of the distal vessels after clip- ping, and confirm complete aneurysm obliteration. However, there are certain circumstances where checking the anatomy with ICG-VA is not all enough for a safe surgery. Sometimes, despite hav- ing the distal blood flow depicted on angiograms, subtle decrease in flow presents which may com- promise perfusion of the cerebral parenchyma leading to disturbed function of the brain. FLOW 800 may assist the surgeon to calculate the CBFI before and after clipping and by showing him or her the color-coded images where any change in color after clipping regarded as an alarming sign. One of the advantages of ICG-VA over DSA in aneurysm surgery is its real-time nature where the surgeon can manipulate the vessels and adjust the clip just during the angiography, if required. Also, FLOW 800 allows physiological monitor- ing of the clipping procedure both in the vessels and in the surrounding brain tissue. ICG-VA has largely replaced conventional DSA during aneu- rysm surgery in most centers, and attempts to fur- ther clarify the role of FLOW 800 in these procedures are underway. When ICG-VA is repeated at surgery after clipping, the dome of the aneurysm may show some residual ICG illumination from previous injections. This results in confusion as the sur- geon cannot be assured of the total obliteration of the neck. In this occasion, FLOW 800 can dif- ferentiate a residual ICG from incomplete clip- ping: in case of clip shortage, the aneurysm dome appears red in color-coded images, and if the dome is selected as a ROI, a curve that follows an arterial diagram is produced (Fig. 31.3). 31.3.3 Bypass Surgeries Different stages of EC-IC bypass surgery such as to identify the recipient artery, to evaluate patency of the graft, and to detect any possible stenosis are facilitated by ICG-VA. Januszewski 31 FLOW 800 for Vascular Surgery 273 et al. used ICG-VA to analyze the blood flow type after anastomosis which can predict early postoperative graft occlusion [5]. Also, Esposto et al. used ICG-VA to identify arterial territory in temporary or permanent occlusion of the vessels [6]. Although the application of the ICG-VA in bypass evaluation is well estab- lished and widely accepted, the role of FLOW 800 is still unclear. In spite of some available limited data [7] whether pre- to post-bypass 31.4 Expert Opinion Additional hemodynamic analysis with the help of FLOW 800 to the conventional angiographies (e.g., ICG-VA) allows a real-time physiological and anatomical assessment of blood flow during cerebrovascular procedures such as aneurysms, AVMs, and bypass surgeries. Intraoperative imaging of the vessels in vascular surgeries a c d b 0 0 100 200 300 400 500 0.1 s 3.1 s 6.1 s 9.1 s 12.1 s 15.0 s 1 0.00s 14.24s 10.54s 2 3 10 20 30 Time [s] Average Intensity AI 40 Delay = 14.24 s Slope = 13.55 AI/s 50 Fig. 31.3 (a) Surgical view under infrared camera after aneurysm clipping and before indocyanine green (ICG) injection. Note that the aneurysm glows due to previous ICG accumulated in the aneurysm sac. (b) The same view after ICG injection. It is difficult if not impossible to dif- ferentiate previously accumulated ICG in the aneurysm from newly injected in the vessels. This view is consistent with incomplete aneurysm obliteration. (c) Color-coded image by FLOW 800 software demonstrate the aneurysm in red. This is the shortcoming of the software that cannot differentiate a previously injected dye from early arrival of blood flow. (d) The red curve shows the intensity changes inside the aneurysm sac. Constant curve confirms absence of blood flow in the aneurysm. Some sudden changes in intensity are observed which are due to manip- ulation of the aneurysm with suction tip to see behind the aneurysm. 31.3.3 Bypass Surgeries Please note that delay and slope of the curve are calculated by the software b a b a b a c 0.1 s 3.1 s 6.1 s 9.1 s 12.1 s 15.0 s d d 0 0 100 200 300 400 500 1 0.00s 14.24s 10.54s 2 3 10 20 30 Time [s] Average Intensity AI 40 Delay = 14.24 s Slope = 13.55 AI/s 50 in red. This is the shortcoming of the software that cannot differentiate a previously injected dye from early arrival of blood flow. (d) The red curve shows the intensity changes inside the aneurysm sac. Constant curve confirms absence of blood flow in the aneurysm. Some sudden changes in intensity are observed which are due to manip- ulation of the aneurysm with suction tip to see behind the aneurysm. Please note that delay and slope of the curve are calculated by the software Fig. 31.3 (a) Surgical view under infrared camera after aneurysm clipping and before indocyanine green (ICG) injection. Note that the aneurysm glows due to previous ICG accumulated in the aneurysm sac. (b) The same view after ICG injection. It is difficult if not impossible to dif- ferentiate previously accumulated ICG in the aneurysm from newly injected in the vessels. This view is consistent with incomplete aneurysm obliteration. (c) Color-coded image by FLOW 800 software demonstrate the aneurysm et al. used ICG-VA to analyze the blood flow type after anastomosis which can predict early postoperative graft occlusion [5]. Also, Esposto et al. used ICG-VA to identify arterial territory in temporary or permanent occlusion of the vessels [6]. Although the application of the ICG-VA in bypass evaluation is well estab- lished and widely accepted, the role of FLOW 800 is still unclear. In spite of some available limited data [7], whether pre- to post-bypass, ratio of measures such as CBFI or MFI corre- lates with the outcome and requires further comparative studies. 31.4 Expert Opinion Additional hemodynamic analysis with the help of FLOW 800 to the conventional angiographies (e.g., ICG-VA) allows a real-time physiological and anatomical assessment of blood flow during cerebrovascular procedures such as aneurysms, AVMs, and bypass surgeries. Intraoperative imaging of the vessels in vascular surgeries decreases postoperative morbidities and is an inseparable adjunct in almost all centers dealing with such pathologies. This along with other 274 Y. Kato et al. sity in cerebral aneurysm surgery. J Clin Neurosci. 2011;18:1097–100. intraoperative monitoring modalities such as neuroendoscopy and motor-evoked potentials results in superb outcome of the patients in the modern era of cerebrovascular surgeries [8]. Intraoperative DSA is still considered the gold standard procedure especially for AVMs where the deep vessels pose certain concerns during the surgery. However, in most aneurysm and selected AVM surgeries, ICG-VA can replace conven- tional DSA as it is a cheaper technique, requires less time for the procedure, and eliminates expo- sure to radiation. Also, physiological evaluations by FLOW 800 are very useful for objective docu- mentation of the blood flow in aneurysm sac, AVM vessels, and bypass graft. Yet, as these data are semiquantitative, external validation of these measures against standard physiological assess- ments is required to better clarify the value of the numbers calculated by the software and how they can be applied in practice. This will be the topic of future studies. intraoperative monitoring modalities such as neuroendoscopy and motor-evoked potentials results in superb outcome of the patients in the modern era of cerebrovascular surgeries [8]. Intraoperative DSA is still considered the gold standard procedure especially for AVMs where the deep vessels pose certain concerns during the surgery. However, in most aneurysm and selected AVM surgeries, ICG-VA can replace conven- tional DSA as it is a cheaper technique, requires less time for the procedure, and eliminates expo- sure to radiation. Also, physiological evaluations by FLOW 800 are very useful for objective docu- mentation of the blood flow in aneurysm sac, AVM vessels, and bypass graft. Yet, as these data are semiquantitative, external validation of these measures against standard physiological assess- ments is required to better clarify the value of the numbers calculated by the software and how they can be applied in practice. This will be the topic of future studies. 2. Son YJ, Kim JE, Park SB, Lee SH, Chung YS, Yang HJ. Quantitative analysis of intraoperative indocya- nine green video angiography in aneurysm surgery. 31.4 Expert Opinion J Cerebrovasc Endovasc Neurosurg. 2013;15(2):76–84. 3. Ye X, Liu XJ, Ma L, Liu LT, Wang WL, Wang S, Cao Y, Zhang D, Wang R, Zhao JZ, Zhao YL. Clinical value of intraoperative indocyanine green fluorescence video angiography with Flow 800 software in cerebro- vascular surgery. Chin Med J. 2013;126(22):4232–7. 4. Chen SF, Kato Y, Oda J, Kumar A, Watabe T, Imizu S, Oguri D, Sano H, Hirose Y. The application of intraoperative near-infrared indocyanine green video- angiography and analysis of fluorescence intensity in cerebrovascular surgery. Surg Neurol Int. 2011;2:42. 5. Januszewski J, Beecher JS, Chalif DJ, Dehdashti AR. Flow-based evaluation of cerebral revasculariza- tion using near-infrared indocyanine green videoan- giography. Neurosurg Focus. 2014;36(2):1–11. 6. Esposito G, Durand A, Doormaal TV, Regli L. Selective-targeted extra-intracranial bypass sur- gery in complex middle cerebral artery aneurysms: correctly identifying the recipientartery using indo- cyanine green videoangiography. Neurosurgery. 2012;71(ONS Suppl 2):274–85. 7. Uchino H, Nakamura T, Houkin K, Murata J, Saito H, Kuroda S. Semiquantitative analysis of indo- cyanine green videoangiography for cortical perfu- sion assessment in superficial temporal artery to middle cerebral artery anastomosis. Acta Neurochir. 2013;155(4):599–605. Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), 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. References 1. Oda J, Kato Y, Chen SF, Sodhiya P, Watabe T, Imizu S, Oguri D, Sano H, Hirose Y. Intraoperative near-infrared indocyanine green–videoangiography (ICG–VA) and graphic analysis of fluorescence inten- 8. Yamada Y, Kato Y, Nouri M. No overtaking! Take a safe trip to aneurysm. Austin J Cerebrovasc Dis Stroke. 2014;1(2):1. Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), 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 chapter are included in the chapter’s Creative Commons license, unless indicated otherwise in a credit line to the material. 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https://openalex.org/W2900554853	https://digitalscholarship.unlv.edu/cgi/viewcontent.cgi?article=1001&context=wlc_fac_articles	English	null	Cinematic Amnesia as Remembering: Coming Home (2014) and Red Amnesia (2014)	Arts	2,018	cc-by	5,490	Cinematic Amnesia as Remembering: Coming Home (2014) and Cinematic Amnesia as Remembering: Coming Home (2014) and Red Amnesia (2014) Red Amnesia (2014) Ying Bao University of Nevada, Las Vegas, ying.bao@unlv.edu Follow this and additional works at: https://digitalscholarship.unlv.edu/wlc_fac_articles Part of the Arts and Humanities Commons Follow this and additional works at: https://digitalscholarship.unlv.edu/wlc_fac_articles P t f th A t d H iti C World Languages and Cultures Faculty Publications World Languages and Cultures Faculty Publications World Languages and Cultures World Languages and Cultures 1 According to (Baxendale 2004, p. 1480), no fewer than 10 silent movies (before 1926) feature amnesic characters. Garden of Lies is one of the earliest and a trendsetter of nuptial amnesia. Furthermore, The Right of Way is “one the ﬁrst ﬁlms to depict amnesia as the result of an assault and the trigger for starting life afresh.” . . . time is no healer: the patient is no longer here. . . . time is no healer: the patient is no longer here. —T.S. Eliot Repository Citation Repository Citation Bao, Y. (2018). Cinematic Amnesia as Remembering: Coming Home (2014) and Red Amnesia (2014). Arts 7(4) 1-8 Available at: Available at: http://dx.doi.org/10.3390/arts7040083 Available at: Available at: http://dx.doi.org/10.3390/arts7040083 This Article is protected by copyright and/or related rights. It has been brought to you by Digital Scholarship@UNLV with permission from the rights-holder(s). You are free to use this Article in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/ or on the work itself. This Article has been accepted for inclusion in World Languages and Cultures Faculty Publications by an authorized administrator of Digital Scholarship@UNLV. For more information, please contact digitalscholarship@unlv.edu. Cinematic Amnesia as Remembering: Coming Home 2014) and Red Amnesia (2014) Ying Bao Ying Bao Department of World Languages and Cultures, University of Nevada, Las Vegas, NV 89154-5047, USA; ying.bao@unlv.edu Received: 29 September 2018; Accepted: 16 November 2018; Published: 21 November 2018 Abstract: This article examines the trope of amnesia—the crisis of memory—in two recent Chinese-language ﬁlms dealing with traumatic memories of the Cultural Revolution and its aftermath: Zhang Yimou’s Coming Home (Guilai, 2014) and Wang Xiaoshuai’s Red Amnesia (Chuangru zhe, 2014). Cinematic representation of real and symbolic amnesia, I argue, can be an affective way to overcome historical amnesia, both institutionalized by the Party-state and privatized by individuals. By exploring the dynamics between forgetting and remembering at both collective and individual levels, we can reach a deeper understanding of the profound impact of the Cultural Revolution and its present-day repercussions. Keywords: amnesia; China; the Cultural Revolution; memory; trauma Who controls the past . . . controls the future: who controls the present controls the past. —George Orwell, 1984 Article Cinematic Amnesia as Remembering: Coming Home (2014) and Red Amnesia (2014) Article 2. Ruins of Private Memory As China renegotiates its position in the world order with feverish modernization projects and tight political control, memories of the Cultural Revolution, a tumultuous decade of massive violence and repression that displaced and killed millions, have been fading out of Chinese political and cultural life. Still a highly classiﬁed “state secret,” the total number of the upheaval’s victims remains unknown, and non-quantiﬁable devastations have never been fully appraised. The Party-state has been consistently discounting its catastrophic signiﬁcance and denying the people reﬂections on this dark period of Chinese history through heavy-handed censorship and ideological control. Authoritarian politics compounded with consumer economy has created a culture of collective amnesia that keeps younger generations oblivious to the nation’s recent history. Since the early 1990s, Chinese art cinema has been exploring the narrative and allegorical power of amnesia to underscore the fundamental contradictions of historical narrative in modern China. Jiang Wen’s much-discussed directorial debut In the Heat of the Sun (Yangguang canlan de rizi, 1994), for instance, uses an unreliable narrator who interrupts, intervenes, denies, and rewrites the seemingly linear narrative about coming of age during the Cultural Revolution. The dynamic use of an amnesiac voice-over in the ﬁlm creates a double temporality that frames the distant memory of the Cultural Revolution against the recent memory of the 1989 crackdown and marks a double remembering of suppressed memories. Jiang’s later ﬁlm The Sun Also Rises (Taiyang zhaochang shengqi, 2007) furthers his experimentation of disrupted time and memory. It features an amnesiac character, “Crazy Mother,” whose fragmented memory of the past blurs the lines between reality and madness. The ﬁlm’s nonlinear storytelling, dreamlike visual style, eccentric characters, and symbolic objects all accentuate the futile struggles of reconstructing memory disjointed by personal trauma. Emblematized by the absent father, the past can only be reconstructed as a disembodied affect. In the labyrinth of amnesia, broken pieces of the past are delicately pasted together but fall into pieces again in a matter of a sneeze. Different from Jiang’s ﬁlms, which focus on the Cultural Revolution period per se, Zhang Yimou’s Coming Home and Wang Xiaoshuai’s Red Amnesia are more engaged with the aftermath of the Cultural Revolution. 1. Introduction Amnesia has been a common plot device in world cinema since the silent era. From the 1915 ﬁlm The Garden of Lies1 to Chinese time-travel fantasy romance Once Upon a Time (San sheng san shi shi li taohua, 2017), many commercial ﬁlms have used various ploys of amnesia for its dramatic potential to create suspense, provoke emotional responses, and explain away preposterous situations. Because amnesia is closely associated with physical and/or psychological traumas, cinematic representation of amnesia can also be used to tackle issues of historical narrative and serves as an affective vehicle for dealing with remembering and loss. In this article, I examine the trope of amnesia in two recent Chinese art ﬁlms, Coming Home (Guilai, 2014) and Red Amnesia (Chuangru zhe, 2014), with respect to memory and trauma in the aftermath of the Cultural Revolution (1966–1976). The ﬁlms, directed by the leading directors of the Fifth Generation and Sixth Generation Zhang Yimou and Wang Xiaoshuai, respectively, imbue cinematic amnesia with an ethical urgency of historical reﬂection. Amnesia has been forged as a powerful symbol of historical trauma suppressed by political manipulation and personal guilt. By confronting the audience with the devastating consequences of amnesia, the ﬁlms call attention to post-Mao memory crisis and re-engage the concealed and neglected history affectively, thus opening up the possibility of overcoming historical amnesia. Arts 2018, 7, 83; doi:10.3390/arts7040083 www.mdpi.com/journal/arts www.mdpi.com/journal/arts 2 of 8 Arts 2018, 7, 83 2. Ruins of Private Memory Loosely based on the ﬁnal chapters of Yan Geling’s novel The Criminal Lu Yanshi (Lu fan Yanshi, 2011), Coming Home subtly touches upon the untold history of labor camps in northwestern China, where many intellectuals were sent to be “re-educated” through forced labor in the Gobi Desert since the Anti-Rightists Campaign in 1957, and quietly shows how a family is ripped apart and permanently devastated long after the Cultural Revolution has ended and the surviving prisoners returned. Wrapped in a touching love story between a rehabilitated political prisoner and his amnesiac wife, played by veteran actors Chen Daoming and Gong Li, the ﬁlm set a record for box ofﬁce receipts for art ﬁlms in China, grossing 295 million RMB in its ﬁrst two weeks (Chou 2015). However, reading the ﬁlm as a “twilight romance” is reductive and misses the point. A renewed collaboration between Zhang Yimou and Gong Li, Coming Home immediately invokes the intertextual connection to their early work To Live (Huozhe, 1994), also starring Gong Li and dealing with individual lives deeply affected by political turmoil. To Live won the Jury’s Grand Prix at the 1994 Cannes Film Festival but was banned—a form of institutionalized amnesia—in China, and Zhang Yimou was banned from making ﬁlms for two years. In contrast to To Live, which confronts people’s sufferings and survivals against China’s pivotal historical moments directly, Zhang chooses a restrained and elliptical narrative style that exposes only the tip of the historical iceberg in Coming Home. In a 2014 interview, Zhang Yimou said it was his intention to use liubai as a key narrative strategy (Zhang 2014). Liubai, or leaving empty space, is a compositional device in traditional Chinese ink painting that intentionally leaves empty space to prompt affective contemplation. Coming Home’s avoidance of melodramatic treatment of historical traumas is both a political strategy to pass censorship and an aesthetic choice to expand cinematic space affectively. The ﬁlm begins three years before the end of the Cultural Revolution: Lu Yanshi, a former professor who has been imprisoned for over a decade, escapes from the labor camp and tries to 3 of 8 Arts 2018, 7, 83 covertly rendezvous with his wife Feng Wanyu. Their daughter Dandan, a gifted ballerina striving to claim the leading role in revolutionary ballet The Red Detachment of Women, turns him in when she discovers her parents’ plan to meet at the train station. 2. Ruins of Private Memory Three years after the Cultural Revolution has ended, Lu is rehabilitated and allowed to return home. He ﬁnds out that Dandan has become a textile factory worker, ousted from home by Feng for her betrayal, now living in a dormitory, and his wife Feng no longer recognizes him. Diagnosed with an affective amnesia possibly induced by physical injury, psychological trauma, or malnutrition, Feng persistently denies Lu as her husband. On several occasions, she panically mistakes him for an “Ofﬁcer Fang,” a former member of the local Revolutionary Committee who Lu later ﬁnds out had sexually assaulted her during the Cultural Revolution. Aided by Dandan, Lu makes every effort to take care of Feng and restore her elusive memory, including recollecting old photos, playing her favorite tunes, spending time together reading unmailed letters Lu had secretly written in the labor camp, and faithfully accompanying her to the station to “pick up” himself on the ﬁfth day of every month. Gradually, Feng allows Dandan to move back home and accepts Lu as someone she can trust, but still refuses to recognize him as her husband. The ﬁlm ends on a wintry morning many years later. An old and weary Lu accompanies a now-wheelchair-bound Feng in heavy snow at the train station. They are waiting behind a closed gate for the impossible return of Lu himself. Guided by the liubai aesthetics, Coming Home uses minimal dialogues and relies on the scene to convey the emotion. With the exception of Lu’s re-capture at the train station early in the ﬁlm, explicit violence is remarkably absent on-screen in the ﬁlm. Nonetheless, the ﬁlm confronts the audience with a more subtle, resounding, and slow-burning kind of trauma epitomized by Feng’s crisis of memory. “There is certainly a feeling that we are struggling to remember our past,” said Zhang Yimou in another interview (Koepke 2015), after Coming Home was released at Toronto International Film Festival. “I believe that movies are the most potent, powerful form of art; I have this sense of responsibility to use my movies to inﬂuence other people’s [view of the Cultural Revolution], especially young people in today’s China, to let them know more about history.” The adaptation decision of refocusing the ﬁlm’s narrative to Feng’s amnesia reﬂects both the ﬁlmmakers’ aesthetic preference for the liubai technique and their anxiety about collective amnesia. 2. Ruins of Private Memory In a sense, Lu’s attempts of recovering Feng’s memory parallel ﬁlmmakers’ effort of recovering historical memory through artistic endeavors. Feng’s amnesia thus carries symbolic signiﬁcance. The physical and psychological traumas brought by the state violence mercilessly rupture the family life. Within the ﬁlm’s narrative, Feng’s amnesia erases the traumatic episodes and suppresses the painful memory of her husband’s re-capture and Fang’s assault on her. Her failure to recognize the newly released Lu, who has apparently been transformed by his suffering in the labor camp, as her husband, is a symptom of her physical/psychological trauma. However, at the symbolic level, it can also be read as an outright rejection of the post-Cultural Revolution state rhetoric that calls the people to let bygones be bygones and move on. In a particularly poignant scene, when the same Director Li from the district communal committee, who intrudes Feng’s apartment earlier with two cadres from Lu’s labor camp during the Cultural Revolution, tries to convince Feng that Lu is indeed her husband, she says: “Don’t you trust the Party? Am I not a Party representative? Shouldn’t you trust me? In the name of the Party, I assure you that this man right here in front of you truly is your husband Lu Yanshi.” Her crude insertion of state authority into private matters highlights the continuity of China’s authoritarian political system and invites a sense of déjà vu. Feng’s panicky response that the man before her is not her husband, but rather Ofﬁcer Fang, represents a desire to reject the narrative the Party assigned to her. Lost memory parallels lost trust between individuals and between the individual and the state. In this sense, her persistent amnesia embodies both her family’s psycho-physical traumas as well as their crisis of trust with regard to authority and its authenticity. The violent erasure of personal memories by political forces is visually exempliﬁed in Lu’s family album. At the doctor’s suggestion, Lu Yanshi tries to help Feng recover memory by reconstructing a shared past. His ﬁrst effort is to recollect family photos in the hope that a photographic referent of him 4 of 8 Arts 2018, 7, 83 may rekindle a spark of remembering. After all, in pre-digital age photography, as Roland Barthes has observed in Camera Lucida (Barthes 1981, pp. 2. Ruins of Private Memory 76–77), we “can never deny that the thing has been there.” However, Lu discovers every single image of him has been completely cut out from their family album by their daughter Dandan. The memory holes created by the visual removal of an unwelcome father is a choice both personal and institutional. Dandan, who was only three when her father was arrested, had no personal memory of Lu in everyday life. Guided by party propaganda and her own desires for career advancement, Dandan conceived of Lu strictly as the abstract representation of a class enemy. y p y Like empty space left in a traditional Chinese painting, the traumatic events that directly cause Feng’s amnesia mark their absent presence through other details. Feng’s amnesia creates two senses of time. On the one hand, she is nostalgic for the uncontaminated time, the good old days before the trauma; on the other hand, she is hopeful for the promised return of her husband on the ﬁfth day of an unspeciﬁed month. When Lu ﬁnally recovers a photo from their youth picturing two couples in western dress, the other man in the photo has committed suicide during the Cultural Revolution. Feng is unable to recognize the friends, but she immediately identiﬁes Lu. Her affectionate attachment to a pre-Cultural Revolution memory and her unacceptance of post-Cultural Revolution Lu as her husband explain the emotional intensity of her response to the piano music Lu plays to her; the tune Lu softly plays is an adaption of Song of the Fishermen, the theme song of the 1934 eponymous ﬁlm directed by leftist ﬁlmmaker Cai Chusheng, who was tortured and died in 1968. In a surge of emotions, she reaches out her hand to touch Lu’s shoulder. Both in tears, they share an embrace. However, the physical contact instantly triggers the traumatic memory of her being assaulted. Feng slaps Lu and breaks away. Haunted by the unhealed trauma, a substantiated reconstruction of the past is impossible. The intertextual connection not only expands the ﬁlm’s narrative and affective power, but also deepens the tragic loss of the bonding between Lu and Feng. After Lu has gradually won Feng’s trust as a friendly neighbor, Feng entrusts him with the task of reading her husband’s letters from the labor camp. 2 The term “afterwardsness” is coined by Jean Laplanche in his Essays on Otherness (Laplanche 1999), as a translation and extension of Sigmund Freud’s German term Nachträglichkeit. The concept suggests that an earlier event in one’s life can later acquire a meaning. The notion suggests a dual temporal directionality at play in the hermeneutics of trauma: a retrogressive and a progressive direction. 2. Ruins of Private Memory The letters, written in tiny characters on whatever scraps of paper a prisoner could ﬁnd, provide a fragmentary glimpse into the life in the labor camp. Writing is an act of resistance against amnesia. The reading of the letters not only represents the slow process of rebuilding personal connections between Feng and Lu, but also sutures another gap of memory, that of the untold horror stories of the forced labor camp. 3. The Silent Specter of History With a similar sensibility to afterwardsness2 found in Zhang Yimou’s Coming Home, Wang Xiaoshuai’s Red Amnesia, the last installment of his Third Front trilogy, presents a psychological and allegorical take on the problematic legacy of the so-called Third Front Construction (sanxian jianshe). From 1964 to 1971, China secretly carried out a massive urban-to-rural migration program intended to build self-sufﬁcient industrial bases inland for national defense concerns. Thousands of existing factories and their workers were uprooted from the coast and relocated to the remote southwestern and western region, where they endured harsh and isolated conditions for many years. As government funding for such facilities gradually dwindled during the mid-1970s, workers started seeking opportunities to return to their native cities. Ever since the reform of state-owned enterprises started in the 1980s, some factories have moved out of the mountains, some have been dismantled, some have tried to reinvent themselves and remain productive, and others have simply been abandoned (Naughton 1988). The negative impact of the program on China’s economic development, as Naughton (1988, p. 351) points out, is “certainly more far-reaching than the disruption of the Cultural 5 of 8 Arts 2018, 7, 83 Revolution.” Wang’s ﬁlm demonstrates how the specter of this forgotten past returns as postmemory trauma and continues to haunt the present. Red Amnesia follows the everyday life of a retired widow Deng in present day Beijing. When her life of daily chores is disrupted by a series of mysterious silent phone calls and the appearance of a migrant boy in a red cap, her seemingly forgotten past begins to resurface and haunt her like an unburied ghost. A former Third Front factory worker, Deng managed to win the only opportunity of returning to Beijing by informing on her rival Zhao forty years ago. When she revisits the factory in remote Guizhou for the ﬁrst time ever since, she is confronted with not only her past but also the brutal impossibility of atonement. Discontinuity editing in Red Amnesia creates a strong sense of anxiety, confusion, and disorientation that visualizes the affective disruption of past trauma and guilt lurking in the present. The inaudible caller remains silent but persistently demands a hearing. The police and her daughter-in-law dismiss it as the imaginings of a lonely old lady. Deng’s older son Jun suspects a retaliation from his unpaid contract worker. 3. The Silent Specter of History Furthermore, Deng, visibly affected by the silence emotionally, secretly believes it is from the specter of the recently deceased Zhao. For Deng in Red Amnesia, the willful forgetting of her past transgressions gnaws on her conscience and makes its comeback with full force. Her memory of the Third Front factory was directly triggered by the news of Zhao’s death. It has been forty years since Deng managed to leave the remote factory and move back to Beijing. Her younger son Bing, born in Beijing, is totally oblivious of the family history. Since 1992, China has been accelerating its pace toward marketization and urbanization to be assimilated into the global economy. Motivated by ﬁnancial proﬁts, massive real estate projects obliterate sites of memory and recreate its past. In tandem with the privatization of state industries, the state-sponsored workplace-based social beneﬁt system that had supported workers in their old age, ill-health, and during times of economic hardship has gradually vanished, leaving a vast vacuum to ﬁll. This process of rapid social changes has created a huge gap between major cities and hinterlands. Workers who remain in the Third Front towns not only could not beneﬁt from China’s economic reform, but also feel their contribution and sacriﬁce have been unfairly neglected or forgotten. Before his recent death, Zhao had been bedridden for about four decades as a result of a stroke shortly after he learned Deng’s informing on him has dashed his hope of moving back to Beijing. The family still lives in the dilapidated residential compound where Deng and their co-workers had lived in the distant past. Most families have moved out, but the Zhaos, poverty-stricken and left behind, are conﬁned to their past and see no way out. Their children have become migrant workers. Their only grandson, the boy in the red cap, becomes a homicide fugitive driven by a transgenerational hatred toward Deng and the urban life she represents. The boy’s tragedy derives from a transgenerational transmission of trauma, or what Marianne Hirsch calls “postmemory.” Denied the possibility of returning to their native city, those who remain in the Third Front region commonly suffer from loss of identity and dignity. When Deng revisits her former factory in Guizhou, she has a gathering with her old co-workers. Their accents indicate their various immigration backgrounds from Beijing, Shanghai, Sichuan, etc. 3. The Silent Specter of History One old man remarks, “When I am dead, I don’t want to be buried in Shanghai or here. Just sprinkle my ashes into the sky.” Growing up in the declining factory town, the children bear witness to their older generations’ unaddressed wounds of victimization, and they are further marginalized as a result of their inherited disadvantages. Unchecked, the inherited traumatic memory, compounded by their own personal experiences of victimization and disorientation, may manifest in violent ways. The Chinese title of Red Amnesia, Chuangru zhe, means “the Intruder(s).” As Wang (2017) observes, both Deng and the boy are seen intruding into other people’s lives and leaving unwanted marks. However, ultimately, as the ﬁlm reveals in the end, the state itself is the most violent and ubiquitous intruder of private lives. The unpredictable state policies have created a general sense of rootlessness and disorientation. In her native city Beijing, Deng ﬁnds she has turned into an outsider who does not belong to any group. Once she returns to her former factory, she instantly experiences a déjà vu: “I’ve seen this in my dreams: right here by this tree, we are talking about the same things.” Walking Arts 2018, 7, 83 6 of 8 down memory lane, the ﬁlm uses extra-diegetic music to auralize Deng’s affective link to the past: the sound of bugle call, the Chinese rendition of “Ural Rowan Tree” (a popular Soviet song about factory romance), and laughter of children. However, the pan shots reveal only an empty swing and dilapidated buildings. The factory and the apartments have almost completed moving to a new district. The site of her memory is in the imminence of disappearing. Both the socialist regime and global capitalism join hands in pursuit of strategic oblivion. Forgetting the past allows the country to sprint unhindered toward becoming a global economic powerhouse. The youth embrace this loss of history so as to loosen the chains that weigh down older generations. In Red Amnesia, Deng is seen regularly visiting and caring for her elderly mother in a senior home. While Deng seems to have found an attentive audience in her mother, with whom she can share her private thoughts about her grown-up children and her own aging, the mother is always silent. Like the relic of some bygone society, she continues to exist but has no voice of her own. 4. Conclusions: Amnesia and (Im)possibility of Redemption Over forty years have passed since the end of the Cultural Revolution; however, the government has yet to admit responsibility or engage in any meaningful dialogue. Artistic interest in examining the suppressed history has never ceased. Early works tend to simplify historical trauma in a binary discourse of victim versus victimizer. As Wang (2017, p. 55) points out, the representation of the memory of the Cultural Revolution in Chinese cinema has largely accepted a logic of collective victimization. Films like Legend of the Tianyun Mountain (Tianyunshan chuanqi, 1980), The Herdsman (Muma ren, 1982), and Hibiscus Town (Furong zhen, 1988) adopt a melodramatic mode that provides “easy and comforting answers to difﬁcult and complex questions. It offers moral clarity at a time when nothing seems clear” (Pickowicz 2009, p. 321). Coming Home and Red Amnesia are notably more nuanced efforts of confronting the past, its long-lasting impact on the everyday life in the present, state and individual responsibility, and the problematics of forgetting. The history of China’s long 1970s will constantly resurface and haunt the present generations who have seemingly forgotten the past. As scholars have observed from modern Chinese history, a political power such as the Communist Party can manipulate the memory machine, but so too can commercial culture (Liu 2007, p. 26). In Red Amnesia, stark generational differences and regional gaps manifest in everyday details. In the capital city, the retirees like Deng stay in crowded but still decent low-rise apartments presumably assigned to them by their work units during the planned economy period. They commute via packed public transportation, unplug electronics to conserve energy and prevent power leakage, eat preserved food, engage in collective activities like neighborhood watch and choirs, and are eager to provide care to their surviving parents and their children, regardless of whether it is welcomed or not. Eager to forget the past but still embodying it, Deng’s generation is the transmitter and terminator of the Cultural Revolution’s memory. The ambivalent attitude of Deng toward the Cultural Revolution is evident in her reaction on hearing the communal chorus of old revolutionary songs. She approaches the gate of the rehearsal hall, attentively peeks inside while keeping a cautious distance, and soon hurries away. By contrast, the son’s generation is, perhaps willingly, oblivious to their national and familial past. They embrace change and are eager to distance themselves from the memories of the old world. 3. The Silent Specter of History She has stopped passing on memories to younger generations, and so is forgotten by them. Once, Deng brings her grandson with her on a visit. While she is feeding her mother food, the child stands by at a distance with a puzzled look. When they are picked up later, her daughter-in-law grumbles that she should not bring the child to a place like this. To a younger generation caught in the whirlwind of economic growth, the past—both the history and the older generations who embody it—is an inconvenient burden that they are taught to forget with relief. 4. Conclusions: Amnesia and (Im)possibility of Redemption With new lifestyles afforded by the growing material wealth and social tolerance of the capital city, the new generation lives in sterile high-rises, commutes by private car, and enjoys greater ﬁnancial and sexual freedom. Like the daughter-in-law who does not want her son being brought to visit his 7 of 8 Arts 2018, 7, 83 great-grandmother in her senior home, or the younger son who does not understand his mother’s sense of entitlement to intrude in his private life as a gay man, the younger generation in general both passively and actively forget the historical forces that shaped them. However, like Zhao’s alleged spirit, the dead do not rest easy. The shiny facade of modernized life only superﬁcially covers a troubled past which does not hesitate to break out of its prison. The ﬁlm opens with a slow-moving dolly shot capturing a dilapidated brick building with broken windows and the thumping sounds of machinery heard offscreen. It then cuts to the interior of a modern bathroom where a boy takes a shower. The shot of the building reappears a few scenes later when Deng answers a phone call. Serving as a subconscious ﬂashback intrigued by the mysterious phone call, the scene of the dilapidated building reminds the audience of the site of hidden memory. When the ﬁlm ends, the audience realize that the entire ﬁlm is parenthesized by the factory in Guizhou, the haunting site of Deng’s memory. To restore the soul, one must retrieve the memory. If there is any redemption at all, it will come through memory. However, both ﬁlms show the impossibility of redemption. For Feng and Lu in Coming Home, there is nowhere to seek revenge when the government never openly recognizes its past crimes, and the villain Fang, who never appears in the ﬁlm, apparently is in jail for other reasons. In Red Amnesia, when Deng ﬁnally decides to revisit the site of her suppressed memory in Guizhou and apologizes to Zhao’s widow, not only she does not receive forgiveness, she also indirectly causes the unintended death of Zhao’s grandson. Hearing the thumping sound of the boy’s fatal fall, the camera dwells on Deng’s shellshocked face, as she collapses to the ground, overwhelmed by old and new guilts. 4. Conclusions: Amnesia and (Im)possibility of Redemption The location sounds of dog barks and pounding machinery bridges the close-up of Deng’s face to a ﬂashback of the boy gazing back at the camera against the family photo wall in Deng’s apartment that we have seen earlier in the ﬁlm. In the ﬁnal shot of the ﬁlm, the static camera gazes through the remains of the window frame, now beyond repair, into the abandoned factory buildings that have buried the memory of more than one generation of urban youth who were forced to uproot. After a moment of silence, the sorrowful extra-diegetic music swells and brings the ﬁlm to its emotional climax. The location sounds are still audible but withdraw to the background. Toward the end of the credit sequence, the string music, composed by a rock band Ziyue Qiuye (credit as Umeit in the ﬁlm), subtly quotes the motif of a well-known Cultural Revolution song “The Sun is the Reddest, and Chairman Mao is the Dearest.” This commemorative song, composed shortly after Mao’s death, is quoted so ﬂeetingly as almost to escape the notice of the audience. It ends with a slight variation at the end of the second line replacing a rising note with a falling note. This anachronistic leitmotif drives home the past’s tenacity in resurfacing even among mediums or genres attempting to rebel against it; yet at the same time, it also asserts that even the past cannot endure unchanged. Funding: This research received no external funding. Acknowledgments: The author would like to thank the Arts editors and reviewers for their helpful suggestions and the University of Nevada, Las Vegas for the sabbatical assistance that made this project possible. Conﬂicts of Interest: The author declares no conﬂict of interest. Baxendale, Sallie. 2004. Memories Aren’t Made of This: Amnesia at the Movies. BMJ 329: 1480–83. [CrossRef] [PubMed] ﬂ g p y g Chou, Eva Shan. 2015. In Zhang Yimou’s Coming Home History Is Muted but Not Silent. China File, October 2. Available online: http://www.chinaﬁle.com/reporting-opinion/culture/zhang-yimous-coming-home- history-muted-not-silent (accessed on 1 April 2018). References Baxendale, Sallie. 2004. Memories Aren’t Made of This: Amnesia at the Movies. BMJ 329: 1480–83. [CrossRef] [PubMed] Barthes, Roland. 1981. Camera Lucida: Reﬂections on Photography. New York: Hill and Wang. Chou, Eva Shan. 2015. In Zhang Yimou’s Coming Home History Is Muted but Not Silent. China File, October 2. Available online: http://www.chinaﬁle.com/reporting-opinion/culture/zhang-yimous-coming-home- history-muted-not-silent (accessed on 1 April 2018). References Arts 2018, 7, 83 8 of 8 Koepke, Melora. 2015. Coming Home Director Zhang Yimou Jogs China’s Memory. The Georgia Straight, September 30. Available online: http://www.straight.com/movies/546076/coming-home-director-zhang- yimou-jogs-chinas-memory (accessed on 1 April 2018). Laplanche, Jean. 1999. Essays on Otherness. Edited by John Fletcher. Abingdon: Routledge. Liu, Jianmei. 2007. To Join the Commune or Withdraw from it? A Reading of Yan Lianke’s Shouhuo. Modern Chinese Literature and Culture 19: 1–33. Naughton, Barry. 1988. The Third Front: Defence Industrialization in the Chinese Interior. The China Quarterly 115: 351–86. [CrossRef] Pickowicz, Paul. 2009. Melodramatic Representation and the ‘May Fourth’ Tradition of Chinese Cinema. In From May Fourth to June Fourth: Fiction and Film in Twentieth-Century China. Edited by Ellen Widmer and David Der-wei Wang. Cambridge: Harvard University Press, pp. 295–326. Wang, Yanjie. 2017. Ghostly Haunting and Moral Interrogation in Wang Xiaoshuai’s Red Amnesia. Modern Chinese Literature and Culture 29: 34–65. Zhang, Xiaoye. 2014. Duihua Guilai daoyan Zhang Yimou: “Wo buxiang zai chongfu yibian Huozhe” (Conversation with Coming Home’s Director Zhang Yimou: I Don’t Want to Repeat What I have Done in To Live). April 24. Available online: http://news.mtime.com/2014/04/24/1526919.html (accessed on 16 April 2018). © 2018 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W2807977284	https://europepmc.org/articles/pmc6079052?pdf=render	English	null	A survey of Greek women’s satisfaction of postnatal care	AIMS public health	2,018	cc-by	6,800	1 Department of Nursing, Laboratory of Integrated Health Care, University of Peloponnese Sparta, Greece 2 Faculty of Nursing, National & Kapodistrian University of Athens, Athens, Greece 2 Faculty of Nursing, National & Kapodistrian University of Athens, Athens, Greece * Correspondence: Email: vpraxitelisp@gmail.com. AIMS Public Health, 5(2): 158–172. DOI: 10.3934/publichealth.2018.2.158 Received: 28 February 2018 Accepted: 05 June 2018 Published: 12 June 2018 http://www.aimspress.com/journal/aimsph A survey of Greek women’s satisfaction of postnatal care 1 Department of Nursing, Laboratory of Integrated Health Care, University of Peloponn Sparta, Greece eywords: midwifery care; postnatal period; maternal satisfaction; Greece; postpartum ca 1. Introduction The postnatal period is important for the health of the mother, the newborn and the establishment of new family relationships [1–3]. Healthcare professionals have a key role in providing postnatal care for the mother and newborn [4,5]. There is limited research conducted in Greece on the postnatal period, what is available mainly covers aspects of breastfeeding [6,7] and postnatal depression [8,9]. Until now there is no research conducted in Greece to assess the effectiveness of the provided postnatal care and the parents’ satisfaction from the care they receive postnatally. This study aims to fill this research gap. The United Nation’s Global Strategy for Women’s, Children’s and Adolescents’ Health 2016–2030 10 points out the importance of providing quality care in all settings for new mothers and new babies. In light of this strategy postnatal maternity health services in Greece should be assessed and changes should be implemented for the benefit of the new mother, the new baby and the new family. Sadly, postnatal care is frequently not given as much attention as pregnancy and labour [11–14]. During the postnatal period some women express dissatisfaction from the health care services provided [15–18]. Brown et al. 15, in Australia, identified that after labour the new mothers felt that the midwives did not show enough sensitivity to their needs as they seemed to be always in a hurry and did not spend enough time with them. When the women got discharged home from hospital they felt that they did not have enough information and knowledge for how to best look after their baby. Rudman and Waldenström 16 investigated women’s negative views of the postnatal care they received in a hospital in Sweden. The patients reported difficulty in receiving personalised care and help with breastfeeding. Razurel et al. 17 found that the women in Switzerland felt that the education they received during pregnancy did not help them when problems and concerns arouse in the postnatal period. The women expressed their need to have further help and support during the postnatal period from health care professionals. Another study from Vancouver, Canada 18, found that the time the women spent in the postnatal ward did not prepare them adequately for the first weeks with their baby at home. A study in Turkey concluded that new mothers were not sufficiently prepared for the postpartum period 19. * Correspondence: Email: vpraxitelisp@gmail.com. Abstract: Background: The research described in this paper is a cross-sectional study which surveys women who delivered their babies in a regional hospital in Greece to investigate their satisfaction with their postnatal care. This is the first published study which measures satisfaction of postnatal services in Greece. The aim of this study is to determine which factors most influence postnatal satisfaction, which areas are lacking and therefore identify specific areas which should be targeted to improve the performance of health services. Methods: A cross sectional, quantitative study of 300 women who gave birth in a regional Greek hospital between January 2015 and July 2017 were surveyed 40 days after birth using a self-administered questionnaire. The questionnaire contained sociodemographic and clinical characteristic questions and a selection of questions from the WOMen’s views of Birth Postnatal Satisfaction Questionnaire (WOMBPNSQ). Results: This study found that the dimensions with the higher satisfaction scores were ―Professional support‖ and ―Continuity‖. The lower satisfaction scores were for the dimensions ―Woman’s health‖, ―Contraceptive advice‖ and ―Social support‖ indicating that these are areas for improvement. The three dimensions most correlated with general satisfaction were ―Time with woman‖, ―Feeding baby‖ and ―Professional support‖. Conclusions: This study highlights the important role of health professionals showing that they can enhance postnatal satisfaction by spending time with the women, giving guidance on the care of the newborn and baby feeding. Focusing on improving these areas is expected to enhance the quality of postnatal care. 159 2.1. Study setting, participants & selection A total of 300 women who gave birth in a regional Greek public hospital between January 2015 and July 2017 were surveyed 40 days after the birth of their baby to assess their satisfaction with the postnatal care they received. Inclusion criteria were women aged over 18, who delivered a healthy term infant. Mothers were informed that no personally identifying information would be recorded at any point of the survey and verbal informed consent was given by the women before they voluntarily completed the self-administered questionnaire. 375 women were approached during the two and a half year research period and asked to take part to the research, 25 women declined (7%) to participate. Another 50 women did not meet the inclusion criteria (13%) so were also excluded from the study leaving a total of 300 women surveyed; the survey was stopped after 300 completed responses had been received. Excluded women were those under 18 (30 women, 6%), women who had not delivered a healthy term infant (7 women, 2%), or were illiterate and could not complete the questionnaire (13 women, 4%). During the period that study was conducted a total of 850 deliveries were performed in the Greek regional hospital the research took place. The final sample of 300 women was obtained by randomly selecting the post partum women and was considered to be a representative sample of the population of postnatal women of the hospital where the study took place. 1. Introduction Whilst there have been studies of women’s experiences of pregnancy, labour and childbirth in Greece [20–22] there are no studies covering women’s experiences and satisfaction of their postnatal care. Postnatal care in Greece is provided mainly in the hospital. The new mother stays in the hospital in average for four days after the birth of her baby and then she is discharged home. The new mother returns to the hospital at around 40 days after her discharge for the doctor to check her recovery from childbirth. The research described in this paper investigates the satisfaction of the women who delivered in a regional hospital in Greece with their postnatal care to help fill the gap in this area of research and identify areas for improvement in clinical practice. Patient satisfaction is considered to be an important indicator for health care quality and at the same time is a significant quality improvement tool for health care providers [23,24]. AIMS Public Health Volume 5, Issue 2, 158–172. 160 2.2. Questionnaire Ethical approval for this research was granted from the scientific committee of the regional Greek public general hospital (18th Dec 2014, approval number Φ/Γ/2/14962). New mothers completed the questionnaires in the postnatal ward before attending their 40 day postnatal check after birth. The questionnaires included questions, translated to Greek, from the WOMen’s views of Birth Postnatal Satisfaction Questionnaire (WOMBPNSQ), which is a psychometric multidimensional postnatal satisfaction questionnaire 25. The questions used in this study from the WOMBPNSQ questionnaire were translated from English to Greek and back translated from Greek to English by two language experts. Questions not used in this study from the WOMBPNSQ questionnaire 25 where those relating to the dimensions ―Postnatal visiting‖, ―Health visitor care‖ and ―GP care‖ as these services are not usually provided and these professionals are not generally involved in the provision of postnatal care in the Greek National Health Service. Additional questions were added so that correlations with the demographics and the obstetric history of the women could be identified; a list of these questions is given in Tables 1 and 2. Volume 5, Issue 2, 158–172. AIMS Public Health AIMS Public Health 161 Table 1. Demographics of the women surveyed. Number of people surveyed 300 Maternal age, mean (SD) 31.3 (5.4) Marital status No. (%) Married 272 (90.7%) Not Married 28 (9.3%) Educational level No. (%) Primary 28 (9.3%) High School 40 (13.3%) Senior High 98 (32.7%) College Certificate 43 (14.3%) Technological University Degree 41 (13.7%) University Degree 38 (12.7%) Postgraduate Degree 12 (4.0%) Employment Working, No. (%) 109 (36.5%) Not Working, No. (%) 190 (63.5%) If working how many weeks leave do you have? mean (SD) 7.0 (4.4) How many children do you have including the newborn? No. (%) 1 117 (39.0%) 2 84 (28.0%) 3 37 (12.3%) > 3 9 (3.0%) 2.3. Data analysis 2.3. Data analysis Volume 5, Issue 2, 158–172. 2.3. Data analysis Α pilot study, with a sample of 80 new mothers conducted prior to the survey, checked that the questions were understood by the women and verified that the internal reliability of the satisfaction questions was good or acceptable (Cronbach’s Alpha typically above 0.6 and maximum 0.82) 26. A seven point Likert scale was used for the satisfaction questions. Μean values and Standard Deviations (SD) were used for the description of the quantitative variables. Absolute (N) and relative (%) frequencies were used to describe the dichotomous variables. Student’s t-test was used to compare two groups of quantitative variables. The Pearson correlation coefficient (r) was used to measure the correlation between two quantitative variables. The significance of this correlation was checked by calculating the probability ―p‖ that this correlation occurred by chance (the null hypothesis), a p value less than 0.05 was considered to be statistically significant. The internal reliability of the questionnaire was tested using Cronbach’s Alpha. The cross-correlations between the dimensions were investigated by calculating the correlation coefficient between each dimension. The p-value probability that this correlation occurred by random variability was also calculated. Volume 5, Issue 2, 158–172. AIMS Public Health 162 3. Results The 300 women surveyed had a mean age of 31 and a wide range of educational levels, Table 1. Thirty six percent of the women were employed and most (91%) were married. It is common practice to induce women in Greece which explains the relatively high proportion of induced labours (32%). The rate of epidural or spinal anaesthesia was 27%. Of childbirths 53% were natural and the remainder either by forceps (7%), planned (25%) or unplanned caesarean (15%), Table 2. A summary of the main results of the questionnaire is given in Table 3. The results of the questions were grouped into the same satisfaction dimensions used in the original WOMBPNSQ questionnaire 25. The internal reliability of the dimensions was generally good, having internal reliability with Cronbach’s Alpha over 0.6, with maximum 0.858, Table 4. The ―Feeding baby‖ and ―Social support‖ dimensions had less than ideal reliability with Alpha of 0.63 but were retained as these were used in the original WOMBPNSQ study. The dimensions with the higher internal reliability were ―Professional support‖, ―Partner support‖ and ―Time with the woman‖, which all had coefficients above 0.8. The dimensions with the higher mean scores were ―Professional support‖ and ―Continuity‖ which indicate a higher satisfaction in these areas. The dimensions ―Woman’s health‖, ―Contraceptive advice‖ and ―Social support‖ had the lower satisfaction scores indicating that these areas could be improved. It was found that many of the dimensions are cross-correlated with each other and the general satisfaction scale; these cross-correlations are statistically significant as they are unlikely to have occurred by chance (p < 0.001), Table 5. The three dimensions most correlated with general satisfaction were ―Time with woman‖, ―Feeding baby‖ and ―Professional support‖. AIMS Public Health Volume 5, Issue 2, 158–172. Table 3. Results of the questionnaire. Table 2. Prenatal statistics of the women surveyed. Table 2. Prenatal statistics of the women surveyed. In general, would you say your health is: No. (%) Very good 178 (61.0%) Good 108 (37.0%) Moderate 5 (1.7%) Poor 0 (0.0%) Very poor 1 (0.3%) Childbirth Type No. (%) (52.3%) Natural 157 Forceps 22 (7.3%) Emergency Caesarean 44 (14.7%) (25.3%) Planned Caesarean 76 Induced Labour? No. (%) Yes 94 (32.3%) No 197 (67.7%) Epidural anesthesia? No. (%) Yes 78 (27.4%) No 207 (72.6%) Do you breastfeed? No. (%) Yes 269 (90.6%) No 28 (9.4%) If yes, do you breastfeeding exclusively? No. (%) Yes 103 (39.9%) No 155 (60.1%) If not, duration in days of breastfeeding? Mean (SD) 15 (19.1) How many babies born in this birth? No. (%) 1 291 (97.0%) 2 7 (2.3%) Did the baby need to be hospitalized in the neonatal unit? No. (%) Yes 13 (4.3%) No 286 (95.7%) How many days did you stay in the hospital after childbirth? Mean 3.7 (SD) (0.7) Mean (SD) Gestational age, in weeks, at delivery 38.3 (2.4) Mean (SD) Infant birth weight (g) 3207 (465.5) Were there complications in pregnancy? No. (%) Yes 11 (3.7%) No 289 (96.3%) Were there complications in childbirth? No. (%) Yes 14 (4.7%) No 286 (95.3%) AIMS Public Health 163 3. Results Question Mean score Standard deviation My partner/husband could not have supported me any better in any possible way 79% 23% My carers explored adequately with me my contraceptive needs 65% 21% For my postnatal care I always saw the same carer(s) 67% 24% My carers often appeared rushed 27% 18% I needed to be at home much sooner after the birth 42% 25% There are things about the postnatal care system where I received my care that need to be improved 53% 23% Sometimes carers made me feel a little foolish 20% 17% The amount of time that I spent in hospital after my baby was born was about right 74% 17% I would have liked more advice on feeding my baby 38% 24% My partner/husband was the best possible help to me after the baby was born 68% 26% Carers never acted too businesslike and impersonally towards me 80% 17% Carers usually spent plenty of time with me 72% 18% I was given little advice on contraception following the birth of my baby 42% 22% Continued on next page Table 3. Results of the questionnaire. Table 3. Results of the questionnaire. Question AIMS Public Health 164 Question Question Mean score Standard deviation I was in a fair bit of pain in the first few days/weeks after the birth 53% 28% My postnatal care went nearly exactly as I had hoped it would 76% 16% Many different carers provided me with postnatal check ups 59% 24% I was given an excellent explanation of why I experienced after-pains and how I could cope with them 69% 17% My postnatal care just seemed to involve a series of routine procedures 48% 19% I made new friends during the days/weeks after the birth of my baby 56% 21% It would have been so much better if I had had a longer hospital stay after the birth 27% 18% I didn’t need a lot of pain relief after the birth 55% 28% My carers acted professionally at all times 78% 17% The postnatal care that I received was just about perfect 77% 17% Meeting in the postnatal days/weeks other women who had recently given birth was of no use to me 39% 18% Those who provided my postnatal care sometimes hurried too much when they treated me 29% 19% I could have had just a very little more help from my birth partner/husband 31% 24% A little more time being spent on my health would have been welcome 52% 23% I needed more time in hospital to get used to caring for my new baby 26% 18% My carers rarely left me feeling that I didn’t know what was best for my baby 73% 18% It was reassuring to meet other women like me after my baby was born 66% 17% My carers were never insensitive nor lacked understanding 77% 19% I would have liked more chance to talk to my carers for medical advice about care of myself 46% 23% I was given lots of help on how to feed my baby 75% 19% My carers discussed the full range of contraception options with me following the birth of my baby 59% 22% The carers who treated me should sometimes have given me just a little more respect 24% 17% I needed more time with my carers so that they could help me more 35% 23% There are some things about the postnatal care that I received that could have been better 47% 24% After the birth I would have liked more chance to talk to doctors for medical advice 40% 23% All my carers always treated me in the most friendly and courteous manner possible 80% 16% My partner met all my needs after the birth 77% 20% After the birth, carers always had lots of time to discuss problems with me 69% 19% I could have done with more time for my body to adjust after the birth before going home 31% 19% Sometimes carers did what was easier for them and not what seemed best for me 24% 19% Volume 5, Issue 2, 158–172. Volume 5, Issue 2, 158–172. Question AIMS Public Health 165 Table 4. Satisfaction dimensions and comparison with UK survey. Dimension Survey in Greece (these results) Survey in UK (from Ref 25) t-test p value (that Mean of Greece-UK samples different by chance) Mean SD Cronbach’s Alpha Mean SD Cronbach’s Alpha General satisfaction 68.6 16.0 0.764 41.8 21.8 0.848 < 0.001 Inpatient stay 72.2 15.4 0.781 31.0 21.6 0.861 < 0.001 Woman’s health 53.9 18.9 0.757 37.3 19.8 0.825 < 0.001 Contraceptive advice 60.6 17.5 0.746 40.5 23.8 0.855 < 0.001 Feeding baby 71.6 17.9 0.629 41.7 13.6 0.778 < 0.001 Partner support 71.3 20.0 0.821 24.5 21.6 0.839 < 0.001 Social support 61.0 14.1 0.637 49.9 16.6 0.744 < 0.001 Professional support 78.1 13.8 0.858 27.5 18.7 0.744 < 0.001 Pain after birth 73.1 24.7 0.731 53.9 27.8 0.779 < 0.001 Time with woman* 70.1 15.6 0.817 N/A* N/A* N/A* N/A* Continuity 74.2 20.9 0.766 59.4 23.4 0.735 < 0.001 *Dimension not included in WOMBPNQ4 so UK results not published in Ref 25, this survey uses the full set of questions and dimensions from WOMBPNQ3. Table 4. Satisfaction dimensions and comparison with UK survey. Volume 5, Issue 2, 158–172. AIMS Public Health AIMS Public Health 166 Table 5. Cross-correlations of the Satisfaction Dimensions with p-value probabilities given below in (brackets). Question Dimension General satisfaction Inpatient stay Woman’s health Contraceptive advice Feeding baby Partner support Social support Professional support Pain after birth Time with woman Continuity General satisfaction 1.000 0.284 0.588 0.438 0.692 0.344 0.395 0.698 0.191 0.733 0.077 N/A (< 0.001) (< 0.001) (< 0.001) (< 0.001) (< 0.001) (< 0.001) (< 0.001) (< 0.001) (< 0.001) (0.456) Inpatient stay 0.284 1.000 0.422 0.102 0.504 0.150 0.170 0.333 0.297 0.305 0.302 (< 0.001) N/A (< 0.001) (0.077) (< 0.001) (0.009) (0.003) (< 0.001) (< 0.001) (< 0.001) (0.003) Woman’s health 0.588 0.422 1.000 0.444 0.714 0.260 0.141 0.495 0.185 0.583 0.068 (< 0.001) (< 0.001) N/A (< 0.001) (< 0.001) (< 0.001) (0.015) (< 0.001) (0.001) (< 0.001) (0.513) Contraceptive advice 0.438 0.102 0.444 1.000 0.360 0.158 0.266 0.333 0.037 0.460 0.055 (< 0.001) (0.077) (< 0.001) N/A (< 0.001) (0.006) (< 0.001) (< 0.001) (0.523) (< 0.001) (0.595) Feeding baby 0.692 0.504 0.714 0.360 1.000 0.044 0.248 0.699 0.190 0.365 0.255 (< 0.001) (< 0.001) (< 0.001) (< 0.001) N/A (0.662) (0.012) (< 0.001) (0.057) (< 0.001) (0.012) Partner support 0.344 0.150 0.260 0.158 0.044 1.000 0.251 0.348 0.039 0.365 0.102 (< 0.001) (0.009) (< 0.001) (0.006) (0.662) N/A (< 0.001) (< 0.001) (0.503) (< 0.001) (0.321) Social support 0.395 0.170 0.141 0.266 0.248 0.251 1.000 0.409 0.123 0.426 0.055 (< 0.001) (0.003) (0.015) (< 0.001) (0.012) (< 0.001) N/A (< 0.001) (0.033) (< 0.001) (0.594) Professional support 0.698 0.333 0.495 0.333 0.699 0.348 0.409 1.000 0.257 0.746 0.270 (< 0.001) (< 0.001) (< 0.001) (< 0.001) (< 0.001) (< 0.001) (< 0.001) N/A (< 0.001) (< 0.001) (0.008) Pain after birth 0.191 0.297 0.185 0.037 0.190 0.039 0.123 0.257 1.000 0.237 0.153 (< 0.001) (< 0.001) (0.001) (0.523) (0.057) (0.503) (0.033) (< 0.001) N/A (< 0.001) (0.136) Time with woman 0.733 0.305 0.583 0.460 0.691 0.365 0.426 0.746 0.237 1.000 0.087 (< 0.001) (< 0.001) (< 0.001) (< 0.001) (< 0.001) (< 0.001) (< 0.001) (< 0.001) (< 0.001) N/A (0.401) Continuity 0.077 0.302 0.068 0.055 0.255 0.102 0.055 0.270 0.153 0.087 1.000 (0.456) (0.003) (0.513) (0.595) (0.012) (0.321) (0.594) (0.008) (0.136) (0.401) N/A isfaction Dimensions with p-value probabilities given below in (brackets). Table 5. Cross-correlations of the Satisfaction Dimensions with p-value prob Volume 5, Issue 2, 158–172. Question AIMS Public Health 167 No significant correlation was found between any of the satisfaction dimensions and Maternal age, Number of children or Infant birth weight. Education level was found to be statistically significantly correlated (p-value less than 0.0001) with the Partner support dimension with a correlation coefficient of 0.31; this shows that more highly educated women are, generally, more satisfied with the support given to them by their partners than less well educated women. The mean satisfaction value of women who work vs. the women who don’t work was only statistically significantly different (t-test p-value greater than 0.05) for the ―Partner support‖ dimension (mean value 75.6% for working women vs. 69.1% for non-working women, t-test p-value = 0.006). Working women were also found to be older (mean age of working women 32.8, vs. 30.4 not working, t-test p = 0.0001) and more highly educated e.g. more likely to have a university degree or higher (t-test p < 0.0001). Age was correlated with education level with a correlation coefficient of 0.12 (t-test p = 0.047). The findings of this study are that younger women are more likely to breastfeed (mean age of women who breastfed 31.5 vs. 33.5 mean age of women who did not, t-test p that these mean values are different by chance = 0.021). Women who breastfed were also positively correlated with those who gave a higher score on the ―Feeding baby‖ satisfaction dimension, mean score 73.2 for those who breastfed vs. 45.8 for those who didn’t (t-test p = 0.002). Breast feeding women also had a higher general satisfaction compared to those who didn’t (mean 69.4% vs. 60.9%, t-test p = 0.007), higher satisfaction with professional support (mean 79.1% vs. 70.1%, t-test p = 0.001) and higher satisfaction on the ―Time with woman‖ dimension (71.1% vs. 60.4%, t-test p = 0.0006). The fact that women who have not previously given birth are more likely to be induced in Greece is also seen in the statistical analysis. Women who have been induced have a statistically significant (t-test p-value less than 0.01) lower mean number of children than those that have not been induced (1.2 vs. 1.6 respectively). 4. Discussion The dimensions with the higher mean scores were ―Professional support‖ and ―Continuity‖ which indicate a higher satisfaction for new mothers in these areas. ―Professional support‖ and ―Time with woman‖ were found to be the dimensions most strongly correlated with the general satisfaction dimension. These results are in agreement with the results of other studies which found that improved professional support, including continuity of midwifery care, results in higher satisfaction rates of postpartum women [27,28]. This is particularly relevant as multiple studies [29–32] have found that there is frequently a lack of continuity or absence of adequate care in the postpartum period. The dimensions with the lower mean satisfaction scores were ―Woman’s health‖, ―Contraceptive advice‖ and ―Social support‖ which indicate lower satisfaction of new mothers in these areas. These results show that postnatal women need more time from health care professionals to get professional advice about their health and wellbeing. Health care providers should also empower women to mobilise social support that will help them in their parenting role and psychological health. Recent studies [33–35] have shown the important role of health professionals in maternal health promotion and wellness. The findings of this study show that, if health care professionals improve these areas on clinical practice, a better quality postnatal care will be provided to postnatal women to improve new mothers satisfaction on these areas of their postnatal care. Using the same dimensions as the original study enables direct comparison between the women surveyed in this research and those surveyed by Smith 25 in the UK. What is clear from the results is AIMS Public Health Volume 5, Issue 2, 158–172. AIMS Public Health 168 that the women in this study have responded differently to those from the original study, Table 1. This difference is tested statistically and it is found that t-test p values are all less than 0.001 indicating that it is unlikely that the different mean values of satisfaction between the two studies appear because of random variability in the results. However, the values of the internal reliability, as measured by Cronbach’s Alpha, remain within acceptable limits so whilst the level of the women’s satisfaction in each of the dimensions is different from those in the UK, the dimensions themselves remain a valid measurement tool. 4. Discussion In general, the women surveyed in this research report higher levels of satisfaction than those in the UK resulting in higher mean values in all of the dimensions. The replies from the different women in this study also showed lower variability than those in the UK, resulting in a lower standard deviation. There are many possible reasons for the differences in the results of the two studies, like differences in the hospital protocols and policies, differences in the method of collecting the survey results, differences in the level of women’s expectations and differences in the settings of the studies. p g The policy and protocols of the hospital in this study accommodate the family support by allowing the new mother to have one or two people with her 24 hours a day during her hospital stay. It is common for the mother or mother-in-law to stay with the woman during her postnatal stay to assist with the care of the baby. The visiting times as well as the number of visitors are also flexible, so extended family and friends are allowed to visit as and when they like with the agreement of the new mother. On the contrary, this is not the case in the UK. The new mother is not usually allowed to have someone with her 24 hours a day. Even her partner is allowed to visit during the visiting hours. There are typically restrictions on the number of visitors and the new mother is allowed to have only two visitors by her bedside at a time. The flexible visiting times of the new mother may contribute to the increased satisfaction levels in the local Greek hospital in this research compared with UK hospitals. Whilst the questions asked in the survey were the same with the study by Smith 25, translated from English to Greek and back translated from Greek to English, there are differences in the method used to collect the survey replies from the women. Smith 25 sent the questionnaires by post to women who completed the forms and then also returned them by post. In this survey women completed the questionnaires at the hospital. It is not known at this stage how much, if any, this changed the results; a future study could be completed using the postal method for women in Greece. AIMS Public Health 4. Discussion However, it is noted that the Greek postal system is less customer focused than the UK postal system thus women in Greece would be less likely to return the survey. Women in the UK simply need to put the reply questionnaire in the prepaid envelope and post it in one of the many post boxes, whilst women in Greece may have a lengthy wait in the post office to post the envelope with the questionnaire. The hospitals in Greece have significant shortages of staff, equipment and some materials/medication due to the financial crisis [36,37]. With the lack of staff and equipment it might have been expected that women’s satisfaction of their care would be lower in Greece than the UK but this was not found in this study. Perhaps, the fact that the women are aware of the difficult circumstances, and they are also affected by the financial crisis, lowers their expectations which are then exceeded by the hard work of the staff [38,39] resulting in higher levels of satisfaction. The present study in Greece took place at a regional general hospital. The maternity clinic in this hospital includes the delivery suite and the antenatal and postnatal wards on the same floor. The staff working in the clinic is involved with the care of the women from the time they are admitted to the ward in labour through the delivery and their postnatal stay. This gives the women continuity of care as the same health professionals are involved at all stages of their care. However, the UK study AIMS Public Health AIMS Public Health Volume 5, Issue 2, 158–172. 169 covered a variety of maternity services including different care models in different settings. This could have a direct effect on the results produced for the UK study. covered a variety of maternity services including different care models in different settings. This could have a direct effect on the results produced for the UK study. The single location used in this research is a limitation of this study and is likely to be at least part of the reason for the lower standard deviation of the results compared to the UK study. We also note that the setting of this study is different from Urban Greek maternity hospitals. For example, in Athens the maternity hospitals have separate labour wards, antenatal wards and postnatal wards. 5. Conclusions This study surveyed 300 women in a regional hospital in Greece to determine their satisfaction of the postnatal care they received. The findings of this research show that health professionals can play a key role in enhancing satisfaction of new mothers with their postnatal care by addressing their needs and expectations. It is recommended that health care professionals spend quality time with the postnatal women giving them patient centred care and advice on mother’s health, baby care and feeding. There is certainly place for improvement in clinical practice, as the postnatal care provided is not ideal and yet there is a clear need for a more holistic and personalised care. Health care providers should offer holistic care and try not only to deal with problems, but also to promote maternal health and well being to educate new mothers for the parental skills they need to care for their baby and empower women to mobilise social support. These recommendations for changes in clinical practice are expected to improve the satisfaction of the women and at the same time would improve the health and well being of the new parents and the neonate. Acknowledgements Dr Lindsey Smith from the East Somerset Research Consortium kindly provided the questions from the WOMBPNSQ satisfaction scale which was translated into Greek and used for the satisfaction survey. 4. Discussion The new mothers see different healthcare professionals during different stages of their care. A recommendation for future research is to measure maternal satisfaction in the large maternity hospitals in metropolitan areas of Greece. This would enable a comparison of the results of this study with the satisfaction of new mothers in larger population areas of Greece. The findings of this study in a regional Greek Hospital show that the satisfaction levels of new mothers from the postnatal care they receive are good. However, it is also clear that postnatal women need more from their carers, in respect of their general health and wellbeing. The traditional health care model in Greece deals with targeting health problems. Postnatal women need advice on health promotion, contraception and transition to motherhood. The findings of this research have implications for clinical practice as changes should be implemented. Health care professionals should assume their role in health promotion empowering women to acquire the right skills for the postnatal period. References 1. Wilkins C (2006) A qualitative study exploring the support needs of first-time mothers on their journey towards intuitive parenting. Midwifery 22: 169–180. 1. Wilkins C (2006) A qualitative study exploring the support needs of first-time mothers on their journey towards intuitive parenting. Midwifery 22: 169–180. 2. Emmanuel EN, Creedy DK, St JW (2011) Maternal role development: The impact of maternal distress and social support following childbirth. Midwifery 27: 265–272. 2. Emmanuel EN, Creedy DK, St JW (2011) Maternal role development: The impact of maternal distress and social support following childbirth. Midwifery 27: 265–272. 3. Leahy-Warren P, Mccarthy G (2011) Maternal parental self-efficacy in the postpartum period. Midwifery 27: 802–810. 3. Leahy-Warren P, Mccarthy G (2011) Maternal parental self-efficacy in the postpartum period. Midwifery 27: 802–810. 4. Hynes L (1999) The puererium, In: Bennett RV, Brown LK, Myles textbook for midwives, 13th Ed., Edinburgh: Churchill Livingston Ltd, 589–628. 5. Abbott H, Bick D, Mcarthur C (1997) Health after birth, In: Henderson C, Jones K, Essential midwifery, London: Mosby, 285–318. 6. Tavoulari EF, Benetou V, Vlastarakos PV, et al. (2016) Factors affecting breastfeeding duration in Greece: What is important? World J Clin Pediatr 5: 349–357. 7. Bouras G, Mexi-Bourna P, Bournas N, et al. (2013) Mothers’ expectations and other factors affecting breastfeeding at six months in Greece. J Child Health Care 17: 387–396. 8. Koutra K, et al. (2018) Pregnancy, perinatal and postpartum complications as determinants of postpartum depression: The Rhea mother-child cohort in Crete, Greece. Epidemiol Psychiatr Sci 27: 244–255. 9. Gonidakis F, Rabavilas AD, Varsou E, et al. (2008) A 6-month study of postpartum depression and related factors in Athens Greece. Compr Psychiatry 49: 275–282. 10. United Nations (2015) Every Woman Every Child. Saving lives, protecting futures: Progress report on the Global Strategy for Women’s and Children’s Health. United Nations, New York. 11. Bick DE, Rose V, Weavers A, et al. (2011) Improving inpatient postnatal services: Midwives views and perspectives of engagement in a quality improvement initiative. BMC Health Serv Res 11: 293. 12. Bick D, Murrells T, Weavers A, et al. (2012) Revising acute care systems and processes to improve breastfeeding and maternal postnatal health: A pre and post intervention study in one English maternity unit. BMC Pregnancy Childbirth 12: 41. 13. Schmied V, Cooke M, Gutwein R, et al. Conflicts of interest Following the International Committee of Medical Journal Editors Form for Disclosure of Potential Conflicts of Interest 40, the authors have nothing to disclose. AIMS Public Health Volume 5, Issue 2, 158–172. 170 19. Kirca N, Ozcan S (2018) Problems experienced by puerperants in the postpartum period and views of the puerperants about solution recommedations for these problems: A qualitative research. Int J Car Sci 11: 360–370. References (2009) An evaluation of strategies to improve the quality and content of hospital-based postnatal care in a metropolitan Australian hospital. J Clin Nurs 18: 1850–1861. 14. Beake S, Rose V, Bick D, et al. (2010) A qualitative study of the experiences and expectations of women receiving in-patient postnatal care in one English maternity unit. BMC Pregnancy Childbirth 10: 70. 15. Brown SJ, Davey MA, Bruinsma FJ (2005) Women’s views and experiences of postnatal hospital care in the Victorian Survey of Recent Mothers 2000. Midwifery 21: 109–126. 16. Rudman A, Waldenström U (2007) Critical views on postpartum care expressed by new mothers. BMC Health Serv Res 7: 178. 17. Razurel C, Kaiser B, Dupuis M, et al. (2013) Validation of the postnatal perceived stress inventory in a French speaking population of primiparous women. J Obstet Gynecol Neonat Nurs Jognn 42: 685–696. 18. Smith MP (1989) Postnatal concerns of mothers: An update. Midwifery 5: 182–188. Volume 5, Issue 2, 158–172. AIMS Public Health 171 19. Kirca N, Ozcan S (2018) Problems experienced by puerperants in the postpartum period and views of the puerperants about solution recommedations for these problems: A qualitative research. Int J Car Sci 11: 360–370. 19. Kirca N, Ozcan S (2018) Problems experienced by puerperants in the postpartum period and views of the puerperants about solution recommedations for these problems: A qualitative research. Int J Car Sci 11: 360–370. 20. Sapountzi-Krepia D, Tsaloglidou A, Psychogiou M, et al. (2011) Mothers’ experiences of pregnancy, labour and childbirth: A qualitative study in Northern Greece. Int J Nurs Pract 17: 583–590. 21. Chaniotakis IE, Lymperopoulos C (2009) Service quality effect on satisfaction and word of mouth in the health care industry. J Serv Theory Pract 19: 229–242. 22. Tsetsila E, Lavdaniti M, Psychogiou M, et al. (2010) New mothers’ perceptions regarding maternity care services provided in a prefecture of Northern Greece. Int J Car Sci 3: 129–135. 23. Al-Abri R, Al-Balushi A (2014) Patient satisfaction survey as a tool towards quality improvement. Oman Med J 29: 3–7. 23. Al-Abri R, Al-Balushi A (2014) Patient satisfaction survey as a tool towards quality improvement. Oman Med J 29: 3–7. 24. Faley H, Enguidanos ER, Coletti CM, et al. (2014) Patient satisfaction surveys and quality of care: An information paper. Ann Emerg Med 64: 351–357. 25. Smith LF (2011) Postnatal care: Development of a psychometric multidimensional satisfaction questionnaire (the WOMBPNSQ) to assess women’s views. 20. Sapountzi-Krepia D, Tsaloglidou A, Psychogiou M, et al. (2011) Mothers’ experiences of pregnancy, labour and childbirth: A qualitative study in Northern Greece. Int J Nurs Pract 17: 583–590. 39. Kerasidou A, Kingori P, Legido-Quigley H (2016) ―You have to keep fighting‖: Maintaining healthcare services and professionalism on the frontline of austerity in Greece. Int J Equity Health 15: 118. 40. ICMJE (2017) International Committee of Medical Journal Editors Form for Disclosure of Potential Conflicts of Interest. Available from: http://www.icmje.org/conflicts-of-interest/. © 2018 the Author(s), licensee AIMS Press. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0) Volume 5, Issue 2, 158–172. References Br J Gen Pract 61: e628–e637. 26. Panagopoulou V, Hancock J, Tziaferi S (2016) Women’s Satisfaction of Postnatal Care- A Pilot Study in a District Hospital in Greece. 9th Panhellenic, 8th Pan-European Scientific & Professional Nursing Congress, Kalamata, Greece (in Greek). 27. Forster DA, Mclachlan HL, Davey MA, et al. (2016) Continuity of care by a primary midwife (caseload midwifery) increases women’s satisfaction with antenatal, intrapartum and postpartum care: Results from the COSMOS randomised controlled trial. BMC Pregnancy Childbirth 16: 28. 28. Barimani M, Vikström A (2015) Successful early postpartum support linked to management, informational, and relational continuity. Midwifery 31: 811–817. 29. Martin A, Horowitz C, Balbierz A, et al. (2014) Views of women and clinicians on postpartum preparation and recovery. Matern Child Health J 18: 707–713. p p y 30. Bailey S (2010) Postnatal care: Exploring the views of first-time mothers. Comm Prac 31. Rudman A, Waldenström U (2007) Critical views on postpartum care expressed by new mothers. BMC Health Serv Res 7: 178. 32. Cronin C (2003) First-time mothers—identifying their needs, perceptions and experiences. J Clinl Nurs 12: 260–267. 33. Hajimiri K, Shakibazadeh E, Mehrizi AAH, et al. (2018) The impact of general health and social support on health promoting lifestyle in the first year postpartum: The structural equation modelling. Electron Physician 10: 6231–6239. 34. Cornell A, Mccoy C, Stampfel C, et al. (2016) Creating New Strategies to Enhance Postpartum Health and Wellness. Matern Child Health J 20: 39–42. 35. Fahey JO, Shenassa E (2013) Understanding and meeting the needs of women in the postpartum period: The perinatal maternal health promotion model. J Midwifery Womens Health 58: 613–621. 36. Mckee M, Stukler D (2016) Health effects of the financial crisis: Lessons from Greece. Lancet Public Health 1: e40–e41. 37. Filipidis F, Gerovasili V, Millett C, et al. (2017) Medium-term impact of the economic crisis on mortality, health-related behaviours and access to healthcare in Greece. Sci Rep 7: 464223. 38. Nunes S, Rego G, Nunes R (2015) The impact of economic recession on health-care and the contribution by nurses to promote individuals’ dignity. Nurs Inquiry 22: 285–295. Volume 5, Issue 2, 158–172. AIMS Public Health 172 39. Kerasidou A, Kingori P, Legido-Quigley H (2016) ―You have to keep fighting‖: Maintaining healthcare services and professionalism on the frontline of austerity in Greece. Int J Equity Health 15: 118. 39. References Kerasidou A, Kingori P, Legido-Quigley H (2016) ―You have to keep fighting‖: Maintaining healthcare services and professionalism on the frontline of austerity in Greece. Int J Equity Health 15: 118. 40. ICMJE (2017) International Committee of Medical Journal Editors Form for Disclosure of Potential Conflicts of Interest. Available from: http://www.icmje.org/conflicts-of-interest/. 40. ICMJE (2017) International Committee of Medical Journal Editors Form for Disclosure of Potential Conflicts of Interest. Available from: http://www.icmje.org/conflicts-of-interest/. Volume 5, Issue 2, 158–172. AIMS Public Health AIMS Public Health
W2171246493.txt	https://journals.plos.org/ploscompbiol/article/file?id=10.1371/journal.pcbi.1001107&type=printable	en		Human Leg Model Predicts Ankle Muscle-Tendon Morphology, State, Roles and Energetics in Walking	PLOS computational biology/PLoS computational biology	2,011	cc-by	11,787	Human Leg Model Predicts Ankle Muscle-Tendon Morphology, State, Roles and Energetics in Walking Pavitra Krishnaswamy1, Emery N. Brown1,2,3, Hugh M. Herr1,4* 1 Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America, 2 Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America, 3 Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, United States of America, 4 The Media Laboratory, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America Abstract A common feature in biological neuromuscular systems is the redundancy in joint actuation. Understanding how these redundancies are resolved in typical joint movements has been a long-standing problem in biomechanics, neuroscience and prosthetics. Many empirical studies have uncovered neural, mechanical and energetic aspects of how humans resolve these degrees of freedom to actuate leg joints for common tasks like walking. However, a unifying theoretical framework that explains the many independent empirical observations and predicts individual muscle and tendon contributions to joint actuation is yet to be established. Here we develop a computational framework to address how the ankle joint actuation problem is resolved by the neuromuscular system in walking. Our framework is founded upon the proposal that a consideration of both neural control and leg muscle-tendon morphology is critical to obtain predictive, mechanistic insight into individual muscle and tendon contributions to joint actuation. We examine kinetic, kinematic and electromyographic data from healthy walking subjects to find that human leg muscle-tendon morphology and neural activations enable a metabolically optimal realization of biological ankle mechanics in walking. This optimal realization (a) corresponds to independent empirical observations of operation and performance of the soleus and gastrocnemius muscles, (b) gives rise to an efficient load-sharing amongst ankle muscle-tendon units and (c) causes soleus and gastrocnemius muscle fibers to take on distinct mechanical roles of force generation and power production at the end of stance phase in walking. The framework outlined here suggests that the dynamical interplay between leg structure and neural control may be key to the high walking economy of humans, and has implications as a means to obtain insight into empirically inaccessible features of individual muscle and tendons in biomechanical tasks. Citation: Krishnaswamy P, Brown EN, Herr HM (2011) Human Leg Model Predicts Ankle Muscle-Tendon Morphology, State, Roles and Energetics in Walking. PLoS Comput Biol 7(3): e1001107. doi:10.1371/journal.pcbi.1001107 Editor: Karl J. Friston, University College London, United Kingdom Received August 16, 2010; Accepted February 10, 2011; Published March 17, 2011 Copyright: ß 2011 Krishnaswamy 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 supported by the NIH Pioneer Award DP1 OD003646 (to ENB) and the MIT Media Lab (Consortia Accounts 2736448 and 6895867). 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: hherr@mit.edu of common tasks. Electromyography (EMG) has long quantified neurally stimulated electrical activity (activation) in individual muscles, and indicated which MTUs contribute to joint dynamics during the course of a movement [4]. Recently, ultrasonography has resolved ankle plantar flexor and knee extensor MTU strain into muscle strain and tendon strain during walking, running, and jumping [5–7]. Novel approaches using powered exoskeletons to replace leg muscle work have helped estimate the metabolic efficiency of ankle joint actuation in walking [8,9]. Together, the above studies have uncovered critical neural, mechanical and energetic aspects of individual muscle and tendon contributions to joint actuation. However, the abundance of research on the (a) driving objectives underlying and (b) empirical observations on redundancy resolution is not accompanied by a unifying theoretical framework that relates the two. There is a need to explain the breakdown of joint actuation, possibly driven by one or more of the above driving objectives, into observed individual element contributions. Previous studies [10,11] have proposed that the optimality of neural control for prescribed objectives can resolve individual Introduction A common feature in biological neuromuscular systems is the redundancy in joint actuation. Redundancies in actuating a joint with a prescribed force and motion can be classified at three levels. Joints can be actuated by multiple muscle-tendon units (MTUs) working simultaneously. At any instant, energy for MTU work could come from the series elastic tendon or from the active muscle. Each muscle has many sensors and can be controlled by multiple neural pathways acting together. Understanding how these redundancies are resolved in typical joint movements has been a long-standing problem in biomechanics, neuroscience and prosthetics [1,2]. There is a large literature (reviewed in [3]) on objectives that might drive the way humans resolve neuromechanical redundancies. Several objectives ranging from metabolic cost, efficiency, and mechanical economy to fatigue and active muscle volume have been proposed as driving factors. Direct measurements in humans have revealed some details pertaining to the ‘inner workings’ of individual muscles and tendons resulting from the resolution of neuromechanical redundancies in natural execution PLoS Computational Biology \| www.ploscompbiol.org 1 March 2011 \| Volume 7 \| Issue 3 \| e1001107 Leg Model Predicts Muscle Function in Walking muscles. This promises to circumvent the above-mentioned difficulties in obtaining optimal muscle activations. Further, having muscle activation profiles could also enable a more systematic study of the effects of MTU structure (design) on the breakdown of joint actuation amongst individual elements. In other words, estimating muscle activations from the data allows a consideration of both neural control and muscle-tendon design, in tandem, on the operation of individual muscles and tendons. Motivated by the above ideas, we have developed a theoretical framework to (a) address how the load of actuating a joint is shared amongst the many MTUs, (b) elucidate features of leg design and neuromuscular control enabling the breakdown and (c) clarify functional advantages arising from the load sharing. As a case study, we examine ankle joint actuation in human walking. We model the three primary leg MTUs contributing to ankle action in walking (Figure 1). Each MTU is characterized by (a) Hill-type muscle dynamics [15], (b) a common non-linear tendon model [16] and (c) a bilinear excitation-activation relation [3] - all of which are assumed to be internally consistent. These relations are parameterized with a minimal set of twelve muscle-tendon morphological features (representing leg MTU design). We conduct a computational exploration of the muscle-tendon design space for correspondence to well-known biological objectives. Specifically, for each set of system parameters, we actuate the model with joint state and muscle activations from healthy human gait data (Methods) to characterize the resulting joint torque and metabolic consumption. An overview of the modeling scheme is presented in Figure 1. Our results are organized into five sections. First we present our estimates of muscle activations from EMGs recorded during human walking. In the second section, we characterize the leg parameter space by ability to produce human-like ankle torques and economy. We show that there is a unique parameter vector that is able to accomplish both, and that this unique vector corresponds to the maximum metabolic economy. Third, we present the optimal leg parameters, compare them with biological values and discuss their influence on metabolic economy. Fourth, we present model plantar flexor muscle and tendon strain predictions, compare them with two sets of independent empirical recordings and use them to evaluate mechanical power breakdown between muscle and tendon within each MTU. In the fifth section, we present metrics regarding the breakdown of ankle actuation amongst the two different plantar flexors. Author Summary Biological neuromuscular systems are generally able to perform a specified movement task in several ways – as they have significantly more degrees of freedom than mechanical constraints. Understanding how humans resolve these redundancies to drive individual muscles and tendons in typical joint movements is of interest in the fields of biomechanics, neuroscience and prosthetics. Many experimental studies have uncovered neural, mechanical and energetic features of individual muscle and tendon function in common tasks like walking and running. However, a unifying theoretical framework that explains the many independent empirical observations is yet to be established. In this work, we show that leg muscle-tendon morphology and neural co-ordination, together, enable efficient ankle movements in walking. This finding provides quantitative insight into the operation and performance of posterior-leg muscles and tendons in walking, and motivates the idea that different muscle-tendon units take on different mechanical roles to best actuate the ankle in gait. Results reported have implications both for better understanding neuromuscular co-ordination in gait, and for the design of lower limb prosthetic and orthotic technologies. muscle-tendon contributions to joint actuation in walking. These studies model leg MTUs with morphological parameters based on literature estimates, assert a control objective such as tracking biological joint mechanics or minimizing metabolic cost of transport, and obtain optimal muscle activation profiles for the specified objective. While the importance of neural control in determining the operation of individual muscles and tendons is undisputed, such approaches neglect the fact that many sets of activation patterns can correspond to similar values for the driving objective - making it difficult to uniquely resolve individual muscle activation profiles from an overall mechanical or energetic prescription. Further, several control objectives may be operating in tandem to generate neural activations given the highly nonlinear, multi-input multi-output nature of the system - making it difficult to obtain optimal neural activations using a top-down approach. These observations reduce the utility of such approaches for explaining empirical results and making testable predictions on the workings of individual muscles and tendons within the system. An alternative proposal for resolving individual muscle-tendon contributions to joint actuation in walking is found in optimal design. A starting point for such an approach lies in a recent study by Lichtwark & Wilson [12]. They propose that optimal muscletendon design for efficient actuation of an isolated MTU can explain empirically observed muscle and tendon strain profiles within the MTU. The empirically realistic nature of this proposal may directly stem from the well-documented fact that compliant tendons enable muscles to produce force economically [13,14]. However, this proposal does not scale to explain the breakdown of joint actuation amongst individual elements, as the forces produced by individual MTUs are not known a priori for a given joint actuation. Thus, existing optimal control and optimal design approaches are limited, albeit in different ways, by the very joint actuation redundancies they seek to address. Extra sources of information are needed to address this problem. EMG data contains information about muscle activity, and could potentially be used as a source of biologically realistic neural control commands to PLoS Computational Biology \| www.ploscompbiol.org Results Estimating Muscle Activation Muscle activation is an indicator of a muscle’s force-generation capability, indicated by the proportion of troponin bound to calcium [17–19]. It is driven by neurally stimulated electrical activity in the muscle. Since EMG data is a qualitative indicator of muscle electrical activity [4], it contains valuable information about individual muscle activity and can be useful in understanding the breakdown of joint actuation. However, quantitative uses of EMG data have been limited by variability in the signal and measurement artifacts. Here we show that considering dominant biophysical characteristics of the muscle activation build-up along with the randomness inherent in the EMG measurement yields repeatable and reasonable activation estimates. Classic EMG analysis involves rectification and low-pass filtering [20,21]. But low-pass filters smear out the filtered signal, leading to loss of both phase and amplitude information, particularly turn-on and turn-off of muscle activity [22]. Recently Sanger proposed a probabilistic method to resolve the signal variability and noise floor related problems in analyzing EMG 2 March 2011 \| Volume 7 \| Issue 3 \| e1001107 Leg Model Predicts Muscle Function in Walking Figure 1. Schematic of model and experiment. The dynamical model of muscle-tendon units contributing to ankle action in walking is shown. Anatomical correlates of the model are indicated. The soleus and gastrocnemius are collectively referred to as plantar flexor muscles. The red triangles denote muscles, green springs denote tendons, the dashed pink lines denote moment arms and gray rectangles denote body segments. The term ‘leg’ is used as per its anatomical definition throughout this paper. All muscle-tendon units are defined with Hill-type dynamics, ~ trial (tendon slack length, tendon material properties and muscle maximum isometric parameterized with 12 muscle-tendon morphological features m ~ trial , the model was actuated with kinematic and EMG data from healthy subjects. Details on data force). For randomly generated parameter vectors m collection, model dynamics, computation of model torque and metabolic cost are in Methods. The resulting model ankle torque and metabolic consumption were characterized to understand biophysical features underlying the gait data. doi:10.1371/journal.pcbi.1001107.g001 signals [22]. In this paper, the muscle electrical activity x(t) driving the EMG signal was modeled as a jump-diffusion process: dx~E(dW )z(U{x)dNb a jump. The measured EMG signal was modeled as a random process with an exponential density and rate given by 1=x(t): ð1Þ P(emgjx)~ where dW is a diffusion process with rate E, dN is a jump process with frequency b and U represents a uniform distribution indicating that x(t) is a uniform random variable when there is PLoS Computational Biology \| www.ploscompbiol.org exp ({emg=x) x ð2Þ Propagating the probability densities in a classic recursive Bayesian manner, to estimate the x(t) that best describes the 3 March 2011 \| Volume 7 \| Issue 3 \| e1001107 Leg Model Predicts Muscle Function in Walking observed EMG signal, Sanger reported excellent temporal resolution of EMG turn-on/turn-off during forced maximal contraction tasks. However, the biophysical relevance to analyzing EMG from dynamic tasks is limited by (a) the sharp, nearinstantaneous turn-on and turn-off in the Sanger estimates, and (b) the lack of amplitude-buildup when the muscle is on (Figure 2). We attribute this to differences between the modeled jumpdiffusion process and the true buildup of muscle active state in normal tasks (Supplementary Text S2). The constant frequency and uniform amplitude of the jump process [22] compromises the history dependence of active-state buildup, causing sudden jumps in the estimates when the EMG signal turns on/off. Further, the Sanger model has the same jump rate for source and sink or for activation and deactivation. This neglects the differences in activation and deactivation time constants that are critical to muscle activation build-up [19]. Thus the Bayesian approach Figure 2. Estimating muscle activations from EMG data. The rectified EMG signal is shown in blue, the Bayesian estimate of muscle electrical activity x(t) based on [22] is shown in green and activation estimates a(t) obtained by feeding in the Bayesian estimate through bilinear activation dynamics are in red. The step-like feature of the Bayesian estimate is apparent. The muscle activation estimate builds up after the EMG bursts on, and lasts well after the EMG turns off. Step to step variations in the EMG signals are seen, as are their effects on the activation estimates. The position of the leg corresponding to the time axis is shown for interpretation. The leg is shaded during stance (between heel strike and toe off) and transparent during swing. doi:10.1371/journal.pcbi.1001107.g002 PLoS Computational Biology \| www.ploscompbiol.org 4 March 2011 \| Volume 7 \| Issue 3 \| e1001107 Leg Model Predicts Muscle Function in Walking proposed in [22] appears to estimate the times when muscle electrical activity turns on/off, and not the muscle active state because activation dynamics (relating electrical activity to cross bridge formation) are not explicitly included. One way to account for the activation dynamics would be to incorporate them directly into the jump-diffusion model and numerically evaluate a solution. We chose a simpler approximation, and applied the activation dynamics on the muscle electrical activity x(t) from Sanger’s model to estimate muscle active state a(t). Activation dynamics was specified by the classic bilinear form [3]: 2 3 7 da(t) 6 1 1 7 z6 4tact (cz(1{c)x(t))5a(t)~ tact x(t), dt \|ﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ{zﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ} rateconstant where 0vc~ Notable features of the plot include (a) the overall L shape, (b) a vertical boundary evidently representing the minimum energy that model muscles have to expend given the inputs, regardless of torque match and (c) an evidently systematic horizontal boundary below the population representing the best match between model and data. Each point along this horizontal boundary corresponds to a different metabolic consumption for the same level of error between model and human dynamics. A published empirical estimate of the range of metabolic consumption for ankle actuation in walking [8] is indicated, and is seen to be well-approximated by points exhibiting near-minimal economies, close to the the vertical boundary. Remarkably, this overall parameter-space characterization reveals that the empirically observed realization is among the most economical of the many ways to produce human-like torque. Thus the human leg and the nervous system controlling it resolve the load-sharing redundancies in actuating the ankle most economically. Points that best approximate human-like dynamics and optimal human-like metabolics lie near the bottom horizontal and left vertical boundary respectively. Thus points representing a logical intersection of the model’s ability to best produce both human-like dynamics and metabolics lie in a small region at the lower-left corner (indicated by box in Figure 3). Points in this region not only have similar values of the torque and metabolic cost but also have similar values for the morphological parameters defining them. The coefficients of variance amongst parameter values in the corner region, listed in the caption of Figure 3, are low for most of the parameters (details in Supplementary Text S3). Further, all points outside the corner region compromise on either torque match, or economy, or both. Thus, parameter vectors defining the corner region points can be identified computationally by encoding the simultaneous realization of two objectives (torque match and optimal economy) into a multi-objective problem. Solutions for such problems are generally sets of points that simultaneously realize both objectives as best as possible. These solutions, known as Pareto solutions, typically form a frontier along which the two objectives can be traded off against each other to varying degrees. In the special case that both objectives logically intersect at a mathematically sharp corner, there is a single strong Pareto optimal solution that best fulfils both objectives without any tradeoffs. As demonstrated above, our problem resembles this special case - within systematic limits of experimental precision, data variability and functional relevance. Thus it is possible to interpret our problem within the strong Pareto optimal framework, and simplify standard multiobjective optimization methods (such as Aggregate Objective Functions, Pareto ranking, evolutionary algorithms, or costconstraint techniques [23]) to solve for the biologically realistic parameter vectors. Our simplified approach relies on the observation that biologically realistic muscle-tendon morphological parameters ~ b ) should (a) produce the normal (henceforth referred to as m human walking mechanics, and (b) minimize metabolic cost. To ~ b we take a two-step path: (a) restrict the search to solve for m parameter vectors that enable the model to produce human-like torque (horizontal boundary), and (b) look, within the restricted space, for parameter vectors that optimize economy. Thus, the ~ b is akin to a constrained optimization, problem of finding m performed by generating candidate parameter vector populations and iteratively focussing the search on the biologically realistic left corner (Methods). For each of the five subjects, we used the training gait data to obtain activation and joint state estimates, automated the above ð3Þ tact v1 tdeact This differential equation models the history dependence in build-up of muscle activation, and captures differences between activation (tact ) and deactivation (tdeact ) time constants with the ratio c. Notes on the biophysical relevance of our estimation procedure are available in Supplementary Text S2. The muscle activation profiles estimated using our two-step procedure are shown in Figure 2. The intermediate Bayesian estimate x(t) has a step-like shape as it primarily captures the turn on and turn off of the muscle electrical activity measured by EMG. The estimated activations a(t) have profiles that are qualitatively expected from known temporal features of ankle muscle force buildup [4]. Further, the synergistic soleus and gastrocnemius muscles have similar profiles. Random step to step variations in EMG signals do not drastically change the estimated activation profiles. A repeatable ensemble average was obtained in as few as eight trials in cases of minimal motion artifact. The ensemble average estimates (Supplementary Text S2) show little variability in turn-on/turn-off times, and show greater variability in amplitude features (particularly when activation is high). The method and resulting estimates were found to be quite robust to normal, day-to-day variations in electrode placement for a given subject. We used our estimates of neurally stimulated muscle activations observed in walking to conduct the computational exploration (illustrated in Figure 1) of muscle-tendon morphologies. Mechanical and Metabolic Effects of Muscle Activations and MTU Morphologies Using the muscle activations a(t) and joint kinematics hjoint (t) estimated from normal walking data, we actuated the leg muscle~. tendon model M parameterized by a set of morphological features m ~ comprises the tendon reference strain lref , The parameter vector m the tendon shape factor Ksh , the muscle maximum isometric force Fmax and the tendon slack length lsl for each of the three ankle MTUs. We randomly generated sets of leg muscle-tendon parameter ~ (from a uniform distribution with bounds stated in vectors, m Supplementary Text S1), and computed both the model ankle torque profile, tmod (t) and metabolic energy consumed, C, for each set: M(~ m,a(t),hjoint (t))~½tmod (t),C ð4Þ The resulting errors between model and human ankle torques are plotted against the model metabolic consumption (Figure 3). PLoS Computational Biology \| www.ploscompbiol.org 5 March 2011 \| Volume 7 \| Issue 3 \| e1001107 Leg Model Predicts Muscle Function in Walking Figure 3. Relation between model dynamics and metabolics across the leg parameter space for normal gait data. Each point encodes ~ trial , and therefore a different leg morphology. The torque axis is defined relative to human ankle torques in normal a different parameter vector m walking. The metabolic consumption values are calculated as outlined in Methods. The corner region, marked by the box, is the region corresponding to biologically realistic ankle torques and metabolic energies. The dimensions of the box correspond to the normal range (within error) of the ankle torques (from kinetic data obtained in this study) and metabolic energies (from independent studies [8,9]). Points in the corner region have similar values for the morphological parameters defining them. Coefficients of variance indicating spread amongst parameter vectors in this corner region are within 4% for most of the 12 parameters: ½2:31%,7:56%,2:52% for the SOL, GAS and TA reference strains, ½10:9%,49:7%,13:1% for the SOL, GAS and TA shape factors, ½1:44%,1:57%,2:04% for the SOL, GAS and TA slack lengths and ½2:86%,1:79%,3:84% for the SOL, GAS and TA muscle isometric forces respectively (more details in Supplementary Text S3). doi:10.1371/journal.pcbi.1001107.g003 of biologically realistic morphological parameters is dominated by stance-phase activity of the powerful soleus and gastrocnemius muscles, we focus on predictions for these two plantar flexor muscles. Table 1 highlights the MTU structure trends. Notably, the model soleus and gastrocnemius tendon stiffness values (kSOL and kGAS) are quite compliant and lie within literature ranges [12,24]. While the stiffness trends encapsulate effects of parameters Ksh , lref and Fmax , the effect of slack length lsl is captured in a geometric effect described in the last two rows of Table 1. The ratio of muscle rest length lopt to the computed tendon slack length lsl is conserved for both plantar flexor muscles across subjects. This trend is consistent with published human cadaver studies as well [25]. exploration to find corner region parameters (listed in Supplementary Text S3) and defined the model with the optimal vector to be the ‘trained model’. We cross-validated the trained model against variations in input data (Supplementary Text S3) and proceeded to characterize the biological relevance of the trained model morphology. Biologically Realistic Morphologies and Relevance to Metabolic Economy ~ b for each subject The optimal leg morphological features m were seen to fall within physiological ranges [18]. To gain insight into non-apparent features underlying the solution, we extracted both functional and geometrical features that significantly influence muscle-tendon action and the associated metabolics: (a) tendon stiffnesses and (b) muscle-tendon rest length ratios. To compute these metrics, the trained model dynamics were solved numerically (Methods) to obtain muscle lengths lCE , muscle velocities vCE , tendon lengths lSE , muscle-tendon unit force profiles Fm and model ankle torques tmod . Tendon stiffness was approximated as the best fit slope of the tendon force-length relation defined by the computed morphologies Ksh , lref and Fmax (Methods). Only regions of the tendon F –l curve where force was over 10% of the peak force were considered to prevent the nonlinear toe regions from artificially reducing the stiffness estimates. Geometric metrics were computed using the optimized morphological features. Since the ankle metabolic cost guiding identification PLoS Computational Biology \| www.ploscompbiol.org Table 1. Trends in optimal muscle-tendon morphological parameters. Subject 1 2 3 4 5 kSOL [N/mm] 228 291 214 245 337 kGAS [N/mm] SOL l opt l sl GAS l opt l sl 111 103 96 103 150 0:127 0:130 0:127 0:129 0:130 0:119 0:121 0:120 0:117 0:128 doi:10.1371/journal.pcbi.1001107.t001 6 March 2011 \| Volume 7 \| Issue 3 \| e1001107 Leg Model Predicts Muscle Function in Walking tendon length estimates from the model are shown in Figure 4. Across subjects, both soleus and gastrocnemius muscle strains were noticeably less than tendon strains. Plantar flexor tendons are stretched slowly over most of stance, and released quickly before toe-off just as the muscles shorten rapidly. In accordance with observations in the previous section, we see that the optimal morphologies enable the timely storage and release of tendon elastic energy (stretching and shortening of tendons), giving rise to efficient (near-isometric) muscle operation. The model’s plantar flexor muscle strain predictions are qualitatively consistent with trends reported in independent ultrasonography-based in vivo measures [5,6]. Further, the model captures the diversity represented in the in vivo data from different studies. For the gastrocnemius muscle, model profiles (Figure 4, Panel B) are consistent with ultrasound recordings reported in [5] for some subjects, and with the measures from [6] for other subjects (Figure 4, Panel C). Specifically, there are differences in early stance action that appear to arise largely from differences in early stance ankle angle, and orientation of the foot at the moment of ground impact. There are also differences in the degree of peak shortening towards toe-off. Thus, our results suggest that qualitative trend variations among different in vivo measures [5,6] may arise from subject-to-subject gait variations and not necessarily due to differences in the ultrasonography techniques. Beyond these qualitative observations, model soleus muscle peak strains (Figure 4, Panel A) are quantitatively consistent with those published in [5]. But quantitative differences exist between model predictions for the gastrocnemius muscle (Figure 4, Panels B and C) and the two sets of in vivo measurements. Model gastrocnemius peak shortening strains range from 30{35%, while [5] and [6] report peak shortening strains of 9% and 20{25% respectively. To understand the reason for these differences in muscle strains, we studied the tendon and MTU strain profiles. Interestingly, model tendon lengths (Figure 4) and MTU lengths (not displayed) agree quantitatively with both sets of in vivo measures. However, this does not translate to quantitative agreement between model and the in vivo muscle strains. Since muscle length is a geometric function of the tendon and MTU lengths, the quantitative differences can be attributed to inconsistencies between the model’s geometry and the complex in vivo geometry. Sources for discrepancy include (a) dimensions of the subjects studied, and (b) differences between our lumped element model geometry and the true anatomical geometries, arising possibly from the two-dimensional nature of our analyses (no volume or shape considerations) and from other model simplifications like constant pennation angles. Nevertheless, the overall trends in model muscle and tendon strain predictions are robust to these errors, and empirically realistic. The value of our modeling effort extends well beyond enabling comparisons between our theory and published empirical measurements. Difficulties in directly measuring individual muscle and tendon forces within a muscle-tendon unit have precluded resolution of how the total MTU power output breaks down between the muscle and the tendon. Our analysis provides estimates of individual muscle and tendon forces, and therefore enables calculation of muscle power and tendon power within each MTU - as displayed in Figure 5. The most striking feature of these plots is that much of the MTU power arises from the tendons not the muscles. In particular, during the late stance positive power generation period, tendons provide over 80% of the MTU power across subjects. This is consistent with the above observations of tendon strains being much larger than muscle strains for both plantar flexors. Overall, the soleus MTU has higher peak MTU powers than the gastrocnemius MTU. This Next, we sought to understand the significance of the optimal morphologies (specifically as embodied in the above in tendon compliance and the conserved lopt /lsl ratio trends) to metabolic economy. For this, we compared a metabolic efficiency metric accounting for the effects of tendon elasticity against the efficiency of muscle positive work alone. Muscle positive work efficiency was computed based on the metabolic cost of muscle mechanical work during active shortening (Equation 12). For comparison, a net joint level mechanical efficiency based on the total metabolic cost of performing mechanical work at the joint (inclusive of muscle work during active shortening, active lengthening and passive tendon contributions) was calculated (Equation 13). In the latter case, the metabolic and mechanical calculations are not restricted to muscle positive work phases, as the MTU dynamics can allow tendons to perform positive mechanical work at the joint even when the muscle cannot. Table 2 details the resulting muscle positive work efficiency and the overall joint mechanical work efficiency. The average stance phase efficiency of muscles doing positive work (without regard to storage and release of elastic energy) is 0:25+0:06. This is consistent with empirically measured performance of isolated skeletal muscle doing positive work [8,16]. Though the plantar flexor muscles themselves perform at ordinary efficiencies, accounting for tendon elastic energy contributions boosts their efficiency in performing joint mechanical work to a high net ankle mechanical efficiency of 0:68+0:13 during stance phase (Table 2). To ensure this is not an over-estimate due to neglect of tendon viscosity, we recalculated with a nominal viscous loss of 10% of the tendon elastic energy [18] - and obtained a 0:61+0:12 joint work efficiency, still 2:5 times higher than positive muscle work efficiency. The observation that accounting for elastic energy affords a dramatic increase in efficiency of joint work is qualitatively consistent with another recent report [8]. Thus the biologically realistic morphologies correspond to compliant tendons that store and release elastic energy to enhance joint work with little extra metabolic cost to muscles. As the elastic storage and release is timed to allow muscles to work efficiently, there is an optimal tendon slack length lsl that is tuned to muscle optimal length lopt and the input activation profiles. In summary, our exploration of the muscle-tendon morphological space predicts that the optimal muscle-tendon morphologies enable the nervous system to drive ankle muscles in high performance regimes. Muscle and Tendon Operation within Plantar Flexor MTUs We queried the model for further details regarding individual muscle and tendon operation regimes. Plantar flexor muscle and Table 2. Contribution of tendon elasticity to overall metabolic efficiency of joint work. Subject 1 2 3 4 5 Metabolic Cost for Positive Muscle Work (J) 16:0 11:9 5:7 2:6 5:0 3:4 1:2 0:55 1:7 Efficiency of Positive Muscle Work Positive Muscle Mechanical Work (J) 3:2 0:20 0:29 0:20 0:21 0:33 Total Metabolic Cost (J) 20:7 24:4 17:1 13:3 9:9 Net Mechanical Work at Joint (J) 13:9 11:6 11:5 10:5 7:8 Mechanical Efficiency at Joint 0:67 0:48 0:67 0:79 0:79 Efficiencies calculated with and without accounting for mechanical work contributions from the elastic tendon are compared doi:10.1371/journal.pcbi.1001107.t002 PLoS Computational Biology \| www.ploscompbiol.org 7 March 2011 \| Volume 7 \| Issue 3 \| e1001107 Leg Model Predicts Muscle Function in Walking Figure 4. Model predictions of soleus and (medial) gastrocnemius muscle fascicle and tendon lengths. Panel A displays soleus muscle and tendon length predictions, while Panels B and C display gastrocnemius muscle and tendon length predictions for two different subjects. Dashed lines in panels A, B and C represents standard deviations, propagated from standard deviations of data-based activation and muscle-tendon unit length estimates. Notable features across the panels include (a) the relatively small changes in muscle lengths for much of stance, (b) the slow stretch of tendons in midstance, and (c) the rapid recoil of tendons in late stance. These observations are qualitatively consistent with previously published ultrasonography-based measures on walking humans [5,6]. Model muscle-tendon unit lengths (not shown) are directly related to the model muscle lengths and tendon lengths. Soleus and gastrocnemius actions have distinct features. Soleus fascicle strain profile has an eccentric phase between 35{55% GC, while gastrocnemius fascicle length is largely isometric between 30{55% GC. The gastrocnemius fascicle strain profile is variable across subjects during the early stance and toe-off phases of the gait cycle. doi:10.1371/journal.pcbi.1001107.g004 granularity of information motivates a more detailed study of similarities and differences in the operation of the different muscles and tendons. Roles of Plantar Flexors and Load-Sharing The synergistic soleus and gastrocnemius muscles are similar in that they shorten significantly right before toe-off, and move with low velocities, as is expected from their compliant tendons. But there are two easily apparent differences in the movement of these two muscles - during mid and late stance respectively. First, the length estimates of the two plantar flexors are very different in mid-stance (Figure 4). In particular, the soleus lengthens (eccentric operation) during mid-stance while the gastrocnemius appears characteristically isometric (&30%{55% GC). Thus the soleus absorbs mechanical work in mid-stance, while the gastrocnemius holds the tendon in place at the muscle end and does little mechanical work. This observation is consistent with ultrasound literature reports [5,7]. Moreover, there are differences in late stance operation of the two muscles, which are apparent from an analysis of muscle velocities (Figure 6, Panel A). During pre-toe-off shortening, the soleus operates at a peak velocity of 0:15vmax {0:20vmax , while the gastrocnemius operates at a larger peak velocity of 0:25vmax {0:35vmax (pv0:025). These peak toe-off velocities fall in well-recognized ranges. Muscle efficiency is known to peak around vCE &0:17vmax for a wide range of muscle lengths, while muscle mechanical power peaks around v~0:3vmax [13,26]. Within precision of these empirical numbers, our results suggest that stance-end muscle operation may be driven by peak efficiency for the soleus and peak mechanical power for the gastrocnemius. Motivated by this idea, we compared each muscle’s positive mechanical work and metabolic consumption during the positive work phase of late stance. Table 3 reports ratios of positive mechanical work, metabolic energy cost and the resulting efficiencies of the two muscles in late stance. While the relation between soleus and gastrocnemius mechanical work and metabolic cost had varying trends across subjects, soleus is consistently more efficient than the gastrocnemius. In other words, soleus achieves a much bigger bang (mechanical work-wise) for its buck than the gastrocnemius. Further, the fact that the mechanical work ratios are low (0:8{2) for most subjects - despite the fact that soleus is nearly 3 times as large (in cross-section area) as gastrocnemius suggests that the gastrocnemius may be more powerful than soleus on a per fiber basis (due to the velocity difference noted above). The above results argue that soleus may be an economical force PLoS Computational Biology \| www.ploscompbiol.org 8 March 2011 \| Volume 7 \| Issue 3 \| e1001107 Leg Model Predicts Muscle Function in Walking Figure 5. Breakdown of power generation within the soleus and gastrocnemius MTUs. Panel A shows model predictions of soleus muscle, tendon and MTU power, while Panel B shows similar predictions for the gastrocnemius group. Powers are computed along the tendon axis, and considered positive during shortening phases for each element (muscle, tendon and MTU). 100% gait cycle is equivalent to a stridetime of 1.1 seconds for the gait cycle displayed. For both the MTUs, tendons contribute much more to the MTU power output than do muscles - especially during late stance. Soleus has higher peak MTU power than the gastrocnemius MTU primarily because the soleus has a larger cross-section area. doi:10.1371/journal.pcbi.1001107.g005 PLoS Computational Biology \| www.ploscompbiol.org 9 March 2011 \| Volume 7 \| Issue 3 \| e1001107 Leg Model Predicts Muscle Function in Walking Figure 6. Roles and breakdown amongst different muscle-tendon units spanning the ankle. Panel A shows model predictions of soleus and gastrocnemius muscle (fascicle) velocity. Plantar flexor muscle velocities are close to zero through most of stance. Standard deviations (thin dashed lines) are obtained by propagating the standard deviations of the input activations and muscle-tendon lengths through the model. Soleus and gastrocnemius velocities are significantly different right around toe-off. The asterisks (*) indicate a paired difference t-test significance with pv0:025. Panel B shows the breakdown of ankle torque amongst the different muscle-tendon units. Peak gastrocnemius torque is nearly half of the PLoS Computational Biology \| www.ploscompbiol.org 10 March 2011 \| Volume 7 \| Issue 3 \| e1001107 Leg Model Predicts Muscle Function in Walking peak soleus torque. Total model torque is within normal variations of the biological ankle torques for this subject. Since it is only meaningful to evaluate muscle action when muscles are ‘on’ and working, figures are presented for stance phase only. doi:10.1371/journal.pcbi.1001107.g006 The tendon slack lengths balance the capacity for a timely buildup of force in response to the activations against the need to cycle tendon elastic energy for efficient force generation. Finally the optimal tendon material properties make for just the right stiffness values to produce the required joint torque but with just enough compliance to reduce muscle metabolic cost. These features indicate that leg muscles and tendons are designed to enable a metabolically optimal realization of human-like ankle mechanics under neural controls observed in normal walking. Interestingly, the optimal parameters for any one MTU do not arise independently of those for the other MTUs, as both efficiency and torque match are net objectives for all the MTUs operating together. Rather the optimal structural parameters are a solution for the system as a whole to achieve the two objectives. Therefore, unlike the Lichtwark & Wilson study [12] that predicted the most efficient force generating design for an isolated MTU, our results predict the leg structure that most efficiently breaks down ankle torque amongst the different MTUs, and then the muscles and tendons within each MTU - all in an empirically consistent manner. Further, the load-division implied by the optimal leg morphology also reveals a role division amongst the different muscles. The gastrocnemius muscle has a very compliant tendon - allowing it to work isometrically like a clutch in mid-stance, store energy in the tendon slowly and release it rapidly in late stance to produce high mechanical power on a per fiber basis - akin to a catapult (as proposed in [5]). The soleus muscle, on the other hand, has a stiffer tendon and larger maximum isometric force Fmax - making it very inefficient to generate high power by rapid shortening in late stance (as the metabolic cost increases commensurately with shortening velocities and scales with Fmax ). Thus soleus operates at lower muscle velocities in late stance, and can be thought of as an efficient force generator. To our knowledge, this is the first observation of differences in late stance operation of the two plantar flexor muscles in human walking, and remains to be tested in future experiments. Interestingly, previous studies have found differences in energy management by adjacent leg extensor muscles in insect locomotion [27,28]. Thus differences in morphology of adjacent synergistically controlled MTUs may diversify MTU function across species. For smaller muscles like the tibialis anterior, which contribute little torque or mechanical power, the efficiency objective appears flat across the MTU design space. Nevertheless, these muscles may have important roles to play in fine control or sensing - that could be explored in a future study. Much of the biomechanics literature has focused on single objective problems. It has been acknowledged that multiple objectives could be acting in tandem [3]. Our results motivate the novel idea that one overall objective (of economically producing human-like torque) can give rise to different objectives (power, efficiency, control) for each individual element in the system. It is possible that the neural controller may be ‘managing’ the different muscles and tendons spanning the ankle by ‘assigning’ different roles to each - based on their morphology - to efficiently accomplish ankle actuation in walking. In other words, the dynamical interplay between neural control (modeled here with estimated activations from human EMG data) and leg structure (modeled here with MTU morphologies) may in itself be optimal for the overall objective of efficiently generating ankle torque. This idea stands in contrast to previous proposals of the optimality of neural control alone [10,11,29] or of MTU structure alone [12] producing muscle, while gastrocnemius fibers may be more powerful and metabolically demanding than soleus fibers. Details of the metabolic and mechanical powers of the two muscles are available in Supplementary Figure S1. To further elucidate roles of the two plantar flexors, we studied the metabolically optimal breakdown of ankle torque between the two (Figure 6, Panel B). The ratio of peak soleus and gastrocnemius torque contributions to ankle actuation is an average of 2:23+0:23 across subjects. This ratio does not directly follow either the ratios of the optimal Fmax values or the metabolic costs of the two muscles. It is likely due to a combination of the Fmax , metabolic costs and the muscle activations. Interestingly, the most efficient partitioning of ankle torque amongst the synergistic plantar flexor muscles appears commensurate with the ratios of soleus and gastrocnemius stiffness reported in Table 1. This suggests that the soleus and gastrocnemius tendon extensions may be similar, which is just what we see in Figure 4. Finally, the muscle operation and load-sharing results arise ~ along the uniquely from the optimal parameter vectors. A point m horizontal boundary of Figure 3 - that has a greater metabolic consumption than the biologically realistic corner points - also corresponds to (a) different muscle velocities than the optimal corner points (one-to-one relation between metabolic cost and muscle velocities), (b) different forces generated for the same activations, and (c) different (non-optimal) load-sharing solutions (see Supplementary Text S3). Discussion Our results describe how humans resolve redundancies within and between MTUs involved in ankle joint actuation. We have demonstrated that there is a unique leg morphology which (a) most economically relates activations and angles from gait data with torques therein, (b) produces the above data via plantar flexor muscle motions, tendon motions, and metabolic performance that are consistent with experimental observations and (c) resolves empirically inaccessible features ranging from individual muscle forces and metabolic demand to mechanical power and working efficiencies. This morphology (defined by maximum isometric forces, tendon material properties and slack lengths for the ankle MTUs) is anatomically realistic, and Pareto optimal for the two objectives of torque match and efficiency. To understand features of the morphology that enable this multi-objective optimality, we make a few observations about the solution. The optimal muscle isometric forces result in the most efficient breakdown of joint torque amongst the different MTUs. Table 3. Efficiency of positive muscle mechanical work: Soleus vs. gastrocnemius. Subject 1 2 3 4 5 Ratio of Positive Muscle Work (SOL/GAS) 0:95 52:5 1:27 1:56 2:03 Ratio of Metabolic Energy Consumed for Positive Muscle Work (SOL/GAS) 0:85 4:55 0:92 0:84 1:01 Ratio of Positive Muscle Work Efficiencies (SOL/GAS) 1:13 11:5 1:39 1:87 2:01 doi:10.1371/journal.pcbi.1001107.t003 PLoS Computational Biology \| www.ploscompbiol.org 11 March 2011 \| Volume 7 \| Issue 3 \| e1001107 Leg Model Predicts Muscle Function in Walking Experimental Subjects (protocol number 0903003157). All subjects provided written informed consent for the collection of data, subsequent analysis and publication of results. for a prescribed control or performance objective. The interaction between neural control and muscle-tendon unit mechanics may be facilitated by any subset of many neural pathways - particularly reflex pathways. However, there are many possible reflexes (muscle force [30], fascicle length and velocity [31,32]) that can modulate impedance of ankle muscles at any given point in the gait cycle. This neural pathway redundancy has posed a challenge to decipher when and by how much each reflex pathway may contribute to activation of any given muscle. A systematic approach to quantify the role of specific reflexes and resolve this redundancy is desirable. Our framework has implications as a starting point for such an endeavor. Since every reflex pathway is sensitive to specific changes in muscle state (force, length and velocity), inspecting the dominant trends in our muscle state and activation estimates provides insight on possible pathways contributing to the observed state changes. For instance, a period of muscle stretch and low activation followed by a period of isometric behavior and a coincident rise in activation is likely to correspond to a stretch reflex (gastrocnemius in mid-stance period of walking). A period of similarly shaped force and activation profiles may involve positive force feedback (soleus in late stance of walking). Such observations generate hypotheses on how impedance is modulated within the neuromuscular system. Forward dynamical simulations with perturbation analyses could used to test such hypotheses and quantify contributions of different reflexes to legged dynamics. Insights gained from such efforts are of interest for applications in the control of assistive devices [15]. Also, understanding the reflex responses that (along with tendon and MTU dynamics) modulate leg extensor muscle impedance after heel-strike may add perspective to studies on neuro-motor control during mechanical contact [33]. Finally, the framework described in this study also has implications as an analytical tool to probe empirically inaccessible metrics to understand regulation, roles, operation and performance of individual elements in gait. The first steps would be to extend the theory across muscles and joints for walking. Difficulties in obtaining inputs from gait and EMG data for deeper and more proximal muscles could be overcome via a forward dynamical simulation approach wherein both the timings of muscle activity, along with the muscle-tendon morphological parameters, are evaluated for our two objectives. If feedback control loops linking muscle state to activation are also included, perhaps other objectives of dynamical stability could be considered in tandem in a similar framework, to quantitatively characterize the interplay between neural control and leg morphology. Accounting for feedforward contributions to this interplay constitutes an important challenge that needs to be overcome. Another natural extension would be to characterize different tasks with our framework. A question of fundamental interest is to understand whether the same leg morphology is energetically optimal for the neural controls and joint mechanics across tasks. An affirmative answer would suggest that, for any specified task, humans select the joint mechanics that minimizes metabolic cost for the legs they have. A negative outcome would imply that human leg morphology and neuromuscular co-ordination are specifically energetically optimal for self-selected-speed walking. Data Data collection. Kinematic, kinetic and electromyographic (EMG) data were collected at an instrumented motion analysis facility in the MIT Computer Science and Artificial Intelligence Lab. Five healthy adult males participated in the study. After obtaining informed consent, participants were asked to walk barefoot at a self-selected speed (typically around 1:25{1:35m=s). Walking trials within 5% of self-selected speed were accepted. For each participant, a total of 25–30 trials were collected. For two subjects, data were collected on multiple days (with consistent calibrations) to test robustness of the modeling and estimation techniques to day-to-day differences. Standard procedures were used to collect the three types of data synchronously. Kinematic data was obtained using an infrared camera system (16 cameras, VICON motion analysis system, Oxford Metrics, Oxford, UK) to measure three-dimensional locations (precision &1mm) of reflective markers at 120Hz. The markers, 13mm in diameter, were placed at 46 (bilateral) locations on the participant’s body (Helen Hayes model) to track movements during trials. Kinetic data was collected using two back-to-back embedded platforms (Advanced Mechanical Technology, Inc., Watertown, MA) to measure ground reaction force and center of pressure locations (precisions &0:1N and 2mm respectively) at 1080Hz. To ensure natural gait, subjects were not informed about the force-plate locations. Finally, surface EMGs were obtained using a 1080Hz 16 channel EMG system and MA411 20X gain preamplifiers (Motion Lab Systems, Inc., Baton Rouge, LA); and disposable pre-gelled surface bipolar electrodes having 20mm center-to-center spacing (Electrode Store Model BS-24SAF, part # DDN-20). Electrodes were placed on the soleus, medial gastrocnemius, lateral gastrocnemius and tibialis anterior muscles of one randomly chosen leg in the presence of a physician. Obtaining joint motion, muscle-tendon geometries and joint dynamics. Raw marker and force-plate data were analyzed in SIMM (Software for Interactive Musculoskeletal Modeling, MusculoGraphics Inc., Evanston, IL) to obtain joint motion and dynamics. Using biomechanical properties in the SIMM Full Body Dynamic Model, inverse kinematics was performed to calculate joint angles, muscle-tendon lengths and moment arms [25]. Using the SIMM Dynamics Pipeline, inverse dynamics analyses were performed to determine joint torque profiles (arising from muscle-tendon contributions only, no external forces) and full body center-of-mass trajectories. All steady state walking data were split into gait-cycles and timenormalized to percent gait cycle (%GC) coordinates. Walking speed, stride length and timing of key gait cycle events were also calculated using the motion capture data. Estimating muscle activation. EMG data was analyzed in MATLAB(R) (Mathworks, Natick, MA) to estimate the phase and amplitude of the underlying muscle active state. Raw EMG data for each muscle was pre-processed by removing DC offsets, clipping the signal amplitude to within five standard deviations, full-wave rectifying the clipped signal and then normalizing with respect to the peak value of the rectified signal [34]. The preprocessed EMG data was analyzed using the Bayesian algorithm [22], to estimate the neural excitation x(t) for each muscle. The bilinear excitation-activation dynamics (Equation 3) was solved in MATLAB Simulink to estimate muscle activation, a(t). The activation and deactivation time constants governing Equation 3 Methods Ethics Statement This study was conducted in strict accordance with the principles expressed in the Declaration of Helsinki. The study was approved by the MIT Committee on the Use of Humans as PLoS Computational Biology \| www.ploscompbiol.org 12 March 2011 \| Volume 7 \| Issue 3 \| e1001107 Leg Model Predicts Muscle Function in Walking were set to average values specified in [18]. The offset in the minimum estimated amplitude was removed to eliminate the noise-floor (when muscle was not on). Plantar flexor muscle EMGs were occasionally corrupted by motion artifacts. The artifacts were prominent in the neural excitation estimates x(t) around the foot-flat period (8{30% GC for soleus, and 15{30% GC for gastrocnemius). The artifacts were removed using a causal, 100 ms moving average filter applied on the neural excitation estimates right around foot-flat, while preserving the shape of the neural excitation profiles. All gait data for a given subject were split into mutually exclusive training and testing sets. Within each set, ensemble averages and standard deviations of temporal profiles of each variable (joint angle, joint torque, muscle-tendon length, moment arms, and muscle active state) were used for analysis. with each other. The MTU dynamics follows from the interaction between muscle and tendon, described by the first-order implicit nonlinear differential equation below: : FMTU (t)~FSE (lSE ,t)~Fm (a,lCE ,l CE ,t)coshpenn ð6Þ lMTU (t)~lSE (t)zlCE (t)coshpenn ð7Þ where lMTU , denoting MTU length, is related to joint angle hjoint according to the leg geometry (as specified in the SIMM Dynamic Model). Each MTU has seven morphological parameters, three for the muscle dynamics, three for the tendon dynamics, and the pennation angle. Joint dynamics. The overall ankle torque resulting from the three model muscle-tendon units was specified as: Ankle Musculoskeletal Model To investigate the leg dynamics underlying the data, we modeled the major muscle-tendon units contributing to ankle function in normal walking. Anatomically, this corresponds to the big MTUs responsible for ankle joint rotation in the sagittal plane - the soleus and gastrocnemius plantar flexors with the Achilles tendon split amongst them, and the tibialis anterior dorsiflexor (Figure 1). Both the medial and lateral heads of the gastrocnemius muscle were represented as one effective muscle, since they act synergistically in gait. Other muscle-tendon units spanning the ankle joint were not included as their contribution to ankle torques and energetics in normal, level-ground walking is minuscule [4]. The muscle-tendon dynamics actuating the ankle joint are outlined below. Muscle dynamics. Each muscle was modeled as a unidirectional actuator with classic Hill-type dynamics, as in [30]. The muscle model consists of a contractile element (CE) representing active fascicles and a parallel elastic component (PE) representing connective tissue within the muscle. The contractile force FCE develops as a function of muscle active state a, muscle : fiber length lCE and contractile velocity vCE ~l CE . The parallel elastic element was modeled as a unidirectional non-linear spring, with force depending on lCE . Muscle force resulting from both the contractile and parallel elastic elements is denoted by Fm . Musclespecific parameters defining the dynamics include (a) the maximum isometric force Fmax ; (b) the optimum fiber length lopt at which muscle provides the maximum isometric force, a:Fmax for activity level a; and (c) the maximum contractile velocity of the muscle vmax (mainly a function of the muscle’s fiber composition). Tendon dynamics. Each tendon is a non-linear elastic element in series with the corresponding muscle. Of the several approximations to tendon force-length relations in the literature, we chose a general non-linear form [16]: exp FSE (lSE )~Fmax Ksh lref l {1 exp(Ksh ){1 for l~ tmod (t)~ FMTU,i (t)ri (t) i refers to muscle index ð8Þ i where ri (t) represents the time-varying moment arm for muscle i spanning the ankle. The moment arms were obtained from joint angles in the data using the musculoskeletal geometries in SIMM, as detailed in the Data section. The full model dynamics was implemented in MATLAB Simulink, and is defined by 21 muscle-tendon morphological parameters, 7 for each of the 3 MTUs. Model Parameters, Inputs and Outputs For each MTU, we minimized the number of free model parameters by (a) using literature values where they are known to be reliable (vmax [30]), (b) fixing values in documented general ranges when dynamics are insensitive to precise values (hpenn [35]), and (c) taking advantage of inter-relations between parameters (example, muscle optimal length lopt and tendon slack length lsl are inter-related by subject dimensions, so lopt was set to a scaled nominal value from [35]). Set values for the above three parameters for each of the three MTUs are provided (with sensitivity notes) in Supplementary Text S1. The other twelve parameters each correspond to a key morphological feature (slack length, reference strain and force, shape factor) of the three modeled muscle-tendon units. They are known to be difficult to measure in vivo [36], cadaver measurements are rather unreliable [25] and there is no fool-proof procedure for scaling nominal values from literature to subject dimensions [37]. Thus the model is characterized by twelve free ~. parameters, denoted as m ~, The leg muscle-tendon unit dynamics, characterized by m relates neurally commanded muscle activation (a(t)) and joint ~ , active angle hjoint to joint torque tmod . Considering a specified m state profiles for the three muscles and joint angles from the data (or equivalently the muscle-tendon unit lengths lMTU (t)) constrain Equations 5 and 6 for each unit. The relations can be simultaneously solved for lSE and lCE (implicitly vCE ), which in turn can be used to evaluate FCE and FSE for each unit, and thus to calculate the model ankle torque tmod . lSE {lsl w0 ð5Þ lsl where FSE , lSE and l represent tendon force, tendon length and tendon strain with respect to slack length lsl respectively. All parameters defining the tendon model lsl ,Ksh ,lref ,Fmax are morphological. lsl and Fmax capture the dimensions, crosssectional areas and space organization in the muscle-tendon unit. lref and Ksh depend on the material properties and influence tendon stiffness. Parameter lref represents the reference strain at which FSE ~Fmax . Ksh determines the shape and non-linearity of the length-tension curve. Muscle-tendon unit dynamics. Each MTU comprises a muscle and a tendon connected in series, at pennation angle hpenn PLoS Computational Biology \| www.ploscompbiol.org X M(~ m,lMTU (t),a(t),r(t))~½lCE (t),tmod (t) ð9Þ ~ specifies a certain (a) model ankle Each possible vector m torque, (b) distribution of that ankle torque amongst the MTUs, (c) division of mechanical work and MTU strain amongst the ~ associated muscle and tendon. Since each parameter vector m 13 March 2011 \| Volume 7 \| Issue 3 \| e1001107 Leg Model Predicts Muscle Function in Walking exacts different mechanical work from the model muscles, it also causes them to expend different amounts of metabolic energy to actuate the ankle. strained optimization procedure, motivated in the parameter exploration section of Results. Mathematically, the leg muscletendon morphology was specified as that which minimizes metabolic energy required to produce human-like dynamics: Muscle Metabolic Consumption " Muscle metabolic consumption is known to result from the heats of activation, maintenance, shortening, resting, and other molecular processes involved in muscle force generation [38]. While many schemes have been proposed to comprehensively account for these different components [10,12,39], they depend on setting several parameters correctly. To avoid accuracy and sensitivity issues that accompany multi-parameter metabolic calculations, we used empirically-based heat measures from classically accepted and well-reproduced muscle metabolic studies [13,26,38]. The data, reproduced in Supplementary Text S1 (along with sensitivity bands), relates the normalized metabolic power required for isolated muscle activity with the normalized contractile velocity. At any time t, if muscle i is activated to level ai (t), and is contracting at velocity vCE,i (t), then the instantaneous (denormalized) metabolic power consumed by that muscle is: P(t)(~ mi ,t)~p(vCE (~ mi ,t)):½ai (t):Fmax,i :vmax,i ~ b ~arg min C~ m R when a(t)w0:01 enotendon dlCE v0 ð10Þ ð11Þ Fm dlCE P(~ mi ,t) dt when a(t)w0:01 ð12Þ t such that dlCE (t)v0 8 C, if constraint satisfied > > vﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ < ! u Pi~n 2 u cost~ i~1 (tmod (i){tbio (i)) t > otherwise 1000 > : n 2. Muscle-tendon efficiency - accounting for both muscle and tendon contributions to the net ankle joint mechanics: Ð tmod (t) dhjoint (t) Wmech ~ etot ~ Wmet total metabolic cost C ð13Þ ð15Þ ð16Þ where n is the number of points during the stance phase. The penalty drove the optimization down the torque gradient into regions satisfying constraints. In the rare event that the penalty was steep enough to entrap the optimization in a particular feasible region, we used a population segregation approach [41] to diversify the search. Accuracy and robustness of the computational Identifying Muscle-Tendon Parameters Mathematical problem statement. Biologically realistic ~ b were identified via the conmuscle-tendon parameters m PLoS Computational Biology \| www.ploscompbiol.org ð14Þ tbio is the ensemble mean biological ankle torque for a given subject, tbio,sd is the ensemble standard deviation in tbio for the same subject, and atbio,sd represents the narrowest band around the mean human ankle torque curve within which the model torque profile for that subject can lie. In other words a indicates the least RMS error between the model and data torques. Cost is calculated for stance phase only, as swing phase metabolic consumption for ankle function is small and rather flat in the parameter space. Constraint was kept consistent with cost, and imposed point-wise during stance. As numerical errors in starting up the model and splitting up gait cycles make it difficult to satisfy the constraint of matching the steady state biological torque profile during 0–5% GC, the simulation was started at a point in the 0-5%GC range when the model-biological torque matching constraint was fulfilled and ran to completion of one gait cycle from there on out. Constraint violations between 0{5% GC were discounted. Identifying the most economical parameter vector for the best torque match gives the most energetically conservative estimates ~b in the slightly rounded corner region of Figure 3. All for m analyses are reported for this most conservative point, even though the points in the corner region are similar in value and function. Computational algorithm. The parameter space along the constrained landscape (horizontal boundary of Figure 3) was found to be rugged. Hence, a stochastic evolutionary method that could prevent entrapment in local minima was deemed appropriate. We chose the genetic algorithm (MATLAB Direct Search Toolbox) as it was easily integrated with the modeling and data analysis routines. The algorithm was implemented with settings that enabled speedy exploration of a diverse parameter space (Supplementary Text S1). The non-linear constraint was enforced using a simple penalty method [40]. When the constraint was satisfied, the cost was just the metabolic energy. When the constraint was violated, a penalty proportional to the deviations between model and human torque was imposed: 1. Muscle efficiency - accounting for positive mechanical work from the active muscle (during shortening) only: Ð Ci i~1 m,t){tbio (t)jvatbio,sd (t), where 0vav1 jtmod (~ To avoid numerical errors in computing cost, metabolic power was only accounted for when ai (t)w0:01, which is a small enough approximation that it does not affect accuracy. Total metabolic P cost of actuating the ankle muscles in 1 gait cycle is C~ 3i~1 Ci for the three muscles i~1 to 3. In summary, given the data-driven inputs and model dynamics, each set of morphological parameters defining the dynamics specifies a model torque profile, and a model metabolic consumption - as indicated in equation 4. For analysis, two efficiency metrics were calculated for stance phase of the gait cycle: ~ð # where search is restricted to regions R - in the space of all possible ~ - satisfying the non-linear torque-match constraint: m ~ due to the where vCE (t) is implicitly a function of the parameters m specified muscle-tendon dynamics, p(vCE (~ mi ,t)) is the function in Supplementary Text S1, and the maximum isometric force when muscle i is activated to level ai (t) during a natural task is ai (t):Fmax,i . Overall metabolic energy cost for the muscle is the time integral of its instantaneous metabolic power: ð mi ,t) dt C(~ mi )~ P(~ 3 X 14 March 2011 \| Volume 7 \| Issue 3 \| e1001107 Leg Model Predicts Muscle Function in Walking Text S1 Model and Solver Settings. Model parameters motivat- solutions to our problem were cross-checked by (a) inspecting the optimal parameter vectors for correspondence to features of Figure 3, (b) performing repeat runs with different starting points and follow-on gradient descent searches and (c) employing crossvalidation checks against variations in inputs (Supplementary Text S3) as well as model assumptions (Supplementary Text S1) and (d) checking for biological features known from independent experiments (Results). ed from literature, metabolic cost calculation details, bounds and algorithm settings used to identify optimal parameter vectors. Found at: doi:10.1371/journal.pcbi.1001107.s002 (0.45 MB PDF) Text S2 Estimating Muscle Activation. Biophysical interpretation of the estimation procedure, along with estimated activation profiles for the 3 ankle muscles across 5 subjects. Found at: doi:10.1371/journal.pcbi.1001107.s003 (0.71 MB PDF) Supporting Information Text S3 MTU Parameter Space Exploration. Supplementary notes on the biologically realistic points in the corner region, comparisons between corner region points and other points along the boundaries, tabulation of the optimal parameter values for 5 subjects. Found at: doi:10.1371/journal.pcbi.1001107.s004 (0.54 MB PDF) Figure S1 Plantar Flexor Muscle Metabolic Powers and Corresponding MTU Mechanical Powers. Panel A shows model predictions of soleus muscle mechanical power in relation to its tendon and MTU powers. Panel B shows model predictions of gastrocnemius muscle mechanical power in relation to its tendon and MTU powers. Mechanical powers are computed along the tendon axis, and considered positive during shortening phases for each element (muscle, tendon and MTU). Panel C shows metabolic power of soleus and gastrocnemius muscles. 100% gait cycle is equivalent to a stridetime of 1.16 seconds for the gait cycle displayed. Both muscles consume significant metabolic energy even when their MTU is doing negative mechanical work (,15-50%GC). Comparisons between the mechanical and metabolic power trends for the two muscles in late stance are analyzed in Table 3. Found at: doi:10.1371/journal.pcbi.1001107.s001 (1.88 MB EPS) Acknowledgments The authors wish to thank Bruce Deffenbaugh for helpful comments and suggestions. We acknowledge the assistance of Eric Swart M.D., Peter Loan, and Jing Wang in data collection and processing. Author Contributions Conceived and designed the experiments: PK. Performed the experiments: PK. Analyzed the data: PK. Wrote the paper: PK ENB HMH. Guided conception of the study, advised and oversaw the work: HMH ENB. References 1. Yamaguchi G, Morana D, Si J (1995) A computationally efficient method for solving the redundant problem in biomechanics. J Biomech 28: 999–1005. 2. Todorov E (2004) Optimality principles in sensorimotor control. Nat Neurosci 7: 907–915. 3. Zajac FE (1989) Muscle and tendon: properties, models, scaling, and application to biomechanics and motor control. Crit Rev Biomed Eng 17: 359–411. 4. Perry J (1992) Gait Analysis: Normal and Pathological Function. 3rd edition. New York: Academic Press. 5. Ishikawa M, Komi PV, Grey MJ, Lepola V, Bruggemann G (2005) Muscletendon interaction and elastic energy usage in human walking. J Appl Physiol 99: 603–608. 6. Lichtwark G, Bougoulias K, Wilson A (2007) Muscle fascicle and series elastic element length changes along the length of the human gastrocnemius during walking and running. J Biomech 40: 157–164. 7. Fukunaga T, Kubo K, Kawakami Y, Fukashiro S, Kanehisa H, et al. (2001) In vivo behaviour of human muscle tendon during walking. Proc Biol Sci 268: 229–233. 8. Sawicki GS, Ferris DP (2008) Mechanics and energetics of level walking with powered ankle exoskeletons. J Exp Biol 211: 1402–1413. 9. Sawicki GS, Ferris DP (2009) Powered ankle exoskeletons reveal the metabolic cost of plantar flexor mechanical work during walking with longer steps at constant step frequency. J Exp Biol 212: 21–31. 10. Anderson FC, Pandy MG (2001) Dynamic optimization of human walking. J Biomech Eng 123: 381–390. 11. Neptune RR, Sasaki K, Kautz SA (2008) The effect of walking speed on muscle function and mechanical energetics. Gait Posture 28: 135–143. 12. Lichtwark GA, Wilson AM (2008) Optimal muscle fascicle length and tendon stiffness for maximising gastrocnemius efficiency during human walking and running. J Theor Biol 252: 662–673. 13. Hill AV (1938) The heat of shortening and the dynamic constants of muscle. Proc R Soc Lond B Biol Sci 126: 136–195. 14. Roberts TJ (2002) The integrated function of muscles and tendons during locomotion. Comp Biochem Physiol A Mol Integr Physiol 133: 1087–1099. 15. Michael Eilenberg HG, Herr H (2010) Control of a powered ankle-foot prosthesis based on a neuromuscular model. IEEE Trans Neural Syst Rehabil Eng 18: 164–173. 16. McMahon TA (1984) Muscles, Reflexes and Locomotion. Princeton, NJ: Princeton University Press. 17. Hill AV (1970) First and Last Experiments in Muscle Mechanics. Cambridge, UK: Cambridge University Press. 18. Winters J, Woo SE (1990) Multiple Muscle Systems: Biomechanics and Movement Organization. New York: Springer-Verlag. 19. Hatze H (1977) A myocybernetic control model of skeletal muscle. Biol Cybern 25: 103–119. 20. Hogan N, Mann RW (1980) Myoelectric signal processing: Optimal estimation applied to electromyography. IEEE Trans Biomed Eng BME-27: 382–410. PLoS Computational Biology \| www.ploscompbiol.org 21. Hogan N (1976) A review of the methods of processing EMG for use as a proportional control signal. Biomed Eng 11: 81–86. 22. Sanger TD (2007) Bayesian filtering of myoelectric signals. J Neurophysiol 97: 1839–1845. 23. Deb K (2001) Multi-objective optimization using evolutionary algorithms. New York: John Wiley and Sons. 24. Arampatzis A, Monte GD, Karamanidis K, Morey-Klapsing G, Stafilidis S, et al. (2006) Influence of the muscle-tendon unit’s mechanical and morphological properties on running economy. J Exp Biol 209: 3345–3357. 25. Delp SL (1990) Surgery simulation: A computer graphics system to analyze and design musculoskeletal reconstructions of the lower limb [PhD dissertation]. Stanford (California): Stanford University. 26. Hill AV (1964) The efficiency of mechanical power development during muscular shortening and its relation to load. Proc R Soc Lond B Biol Sci 159(975): 319–324. 27. Ahn A, Full R (1999) Muscles stimulated by the same motor neuron function differently in running roaches. Am Zool 39: 117A–118A. 28. Ahn A, Full R (2002) A motor and a brake: two leg extensor muscles acting at the same joint manage energy differently in a running insect. J Exp Biol 205: 379–389. 29. Zajac FE, Neptune RR, Kautz SA (2002) Biomechanics and muscle coordination of human walking part i: Introduction to concepts, power transfer, dynamics and simulation. Gait Posture 16: 215–232. 30. Geyer H, Herr H (2010) A muscle-reflex model that encodes principles of legged mechanics produces human walking dynamics and muscle activities. IEEE Trans Neural Syst Rehabil Eng 18: 263–273. 31. Winter DA (1983) Biomechanical motor patterns in normal walking. J Mot Behav 15: 302–330. 32. Grey MJ, Ladouceur M, Andersen JB, Nielsen JB, Sinkjaer T (2001) Group II muscle afferents probably contribute to the medium latency soleus stretch reflex during walking in humans. J Physiol 534: 925–933. 33. Ivanenko YP, Grasso R, Macellari V, Lacquaniti F (2002) Control of foot trajectory in human locomotion: Role of ground contact forces in simulated reduced gravity. J Neurophysiol 87: 3070–3089. 34. Clancy EA, Morin EL, Merletti R (2002) Sampling, noise-reduction and amplitude estimation issues in surface electromyography. J Electromyogr Kinesiol 12: 1–16. 35. Delp SL, Loan JP, Hoy MG, Zajac FE, Topp EL, et al. (1990) An interactive graphics-based model of the lower extremity to study orthopaedic surgical procedures. IEEE Trans Biomed Eng 37: 757–767. 36. Hof A, den Berg JV (1981) EMG to force processing III: estimation of model parameters for the human triceps surae muscle and assessment of the accuracy by means of a torque plate. J Biomech 14: 779–785. 37. Winby C, Lloyd D, Kirk T (2008) Evaluation of different analytical methods for subject-specific scaling of musculotendon parameters. J Biomech 41: 1682–1688. 15 March 2011 \| Volume 7 \| Issue 3 \| e1001107 Leg Model Predicts Muscle Function in Walking 40. Michalewicz Z (1995) A survey of constraint handling techniques in evolutionary computation methods. In: Proceedings of the 4th Annual Conference on Evolutionary Programming. Cambridge: MIT Press. pp 135–155. 41. Deb K (1998) An efficient constraint handling method for genetic algorithms. Comput Methods Appl Mech Eng 186: 311–338. 38. Woledge RC, Curtin NA, Homsher E (1985) Energetic Aspects of Muscle Contraction. London: Academic Press. 39. Ma SP, Zahalak GI (1991) A distribution-moment model of energetics in skeletal muscle. J Biomech 24: 21–35. PLoS Computational Biology \| www.ploscompbiol.org 16 March 2011 \| Volume 7 \| Issue 3 \| e1001107
https://openalex.org/W3041228885	https://www.epj-conferences.org/articles/epjconf/pdf/2020/13/epjconf_ilrc292020_06004.pdf	English	null	Detection of Complex Terrain-Induced Wind Shear by Doppler Lidar at Beijing Capital International Airport	EPJ web of conferences	2,020	cc-by	2,375	1. INTRODUCTION Wind shear has a great threat to aviation safety according to statistical reports [1]. Wind speed or wind direction changes rapidly during wind shear, which alters the lift of aircraft and causes deviations from the predetermined flight path. On account of the limitations of conventional wind shear detection instruments (such as anemometer networks, weather radars, and wind profilers), lidar has gradually become the popular technology to observe and alert wind shear, with its high spatial-temporal resolutions and flexible scanning modes [2]. Hong Kong International Airport (HKIA), which is often plagued by wind shear due to its special geographical location, introduced lidar to the airport for the first time for routine observations throughout the day and developed an alerting system for extensive researches [2-3]. ABSTRACT wind shear scanning strategies [4-7] and the accuracy of alerting methods [8-10]. And the distribution law of wind shear has been further explored through wind shear statistical characteristics and turbulence feature analysis along the glide path [7-8, 11-12]. Wind shear can occur in a wide range of weather conditions, like terrain effects under stably stratified boundary layer, sea breeze with sunshine, and gust or microburst in severe convection weather. The terrain-induced wind shear becomes difficult for warning due to its high spatial and temporal variability wind field, which plays a dominant role in many factors with accounting for 70% in HKIA [2]. In November 2018, the lidar-based wind shear synchronous experiment was performed at Beijing Capital International Airport (BCIA). In this experiment, aiming at the measurement of the terrain-induced wind shear and the wind field around the runway, the glide path scanning mode, and the RHI strategy were conducted alternately. Radial velocity retrieved from the glide path scanning can obviously present the wakes caused by complex terrain (e.g., hills, tall trees, residential and terminal buildings). The Pulse Coherent Doppler Lidar (PCDL) warned the terrain-induced wind shear, which was verified by the pilot report. The wind field structure around the runway under the wake effect and the building shielding effect is also analyzed. This paper explores the complex terrain-induced wind shear by using Pulse Coherent Doppler Lidar (PCDL) and the wind field structure around the runway under the wake effect and the building shielding effect at Beijing Capital International Airport (BCIA). EPJ Web Conferences 237, 06004 (2020) ILRC 29 EPJ Web Conferences 237, 06004 (2020) ILRC 29 EPJ Web Conferences 237, 06004 (2020) ILRC 29 https://doi.org/10.1051/epjconf/202023706004 DETECTION OF COMPLEX TERRAIN-INDUCED WIND SHEAR BY DOPPLER LIDAR AT BEIJING CAPITAL INTERNATIONAL AIRPORT Xiaoying Liu1, Songhua Wu1, 2, Hongwei Zhang1, Jianjun Zhang3, Zhiqiang He3, Xi Zhang3 1Ocean Remote Sensing Institute, College of Information Science and Engineering, Ocean University of China, Qingdao 266100, China. 2 Laboratory for Regional Oceanography and Numerical Modeling, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao 266237, China. 3 North China Regional Air Traffic Management Bureau of CAAC, Beijing 100621, China. Email: lxy6302@stu.ouc.edu.cn © 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/). DP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 ses/by/4 0/) 2. LIDAR EXPERIMENT AND METHODOLOGY As the largest passenger throughput airport in China, BCIA is located in the center of Beijing. Although there are no high mountains around it, the presence of hills, trees, and buildings makes the wind field more complicated. The weather conditions in Beijing are controlled by a typical monsoon climate. Therefore, the northerly wind and northwesterly wind become prevailing in the winter and early springtime at BCIA, accompanied by more frequent windy weather. BCIA contains three runways, namely 36L/18R, 36R/18L and 01/19, as shown in Fig.1. Statistics data reported by the pilots (from 2015, 2016 and 2018) showed that the wind shear mainly occurred Recently, wind shear studying has made great progress based on lidars in the design of effective EPJ Web Conferences 237, 06004 (2020) ILRC 29 https://doi.org/10.1051/epjconf/202023706004 Fig.3. Wind direction statistics during wind shear at 36L and 01 runway corridor at BCIA (from 2018). Fig.1. The geographical environment of BCIA. Fig.3. Wind direction statistics during wind shear at 36L and 01 runway corridor at BCIA (from 2018). research. With the arrival of the winter monsoon, in November 2018, Lidar2 moved to 01 runway (as shown in Fig.1) to observe the terrain-induced wind shear under prevailing wind. During the experiment, Lidar1 and Lidar2 both adopted the glide path scanning strategies [2, 4] with the coordinate change of azimuth and pitch angles to carry out the synchronous observation. Table 1 lists the detailed specifications of the PCDL. In the experiment, the distance between the two lidar locations and the centerline of 36L and 01 runway are all 70m, so that the velocity along the laser beam can be as nearly equal to the real velocity along the glide path. And then, the radial velocities of lidars were used to construct the headwind profiles experienced by the aircraft along the glide path. Fig.1. The geographical environment of BCIA. in the 36L and 01 runway at BCIA, accounting for 43% and 31% respectively, as shown in Fig.2. Subsequently, the wind direction when the pilots reported the occurrence of wind shear in 36L and 01 runway corridor (from 2018) was analyzed in Fig.3, and the results reveal that the northwester, norther, and wester prevailed. In consideration of the geographical environment around BCIA, complex terrains under the above-mentioned prevailing wind should be responsible for this. 3. RESULTS Lidar has an advantage in exploring the complex terrain-induced wind shear with a rapid change of wind field in the clear sky. Since January 2018, BCIA has introduced two lidars located at 36L (Lidar1) and 18R (Lidar 2) runway for scientific 2. LIDAR EXPERIMENT AND METHODOLOGY The residential buildings, tall trees and small mounds on the west of 36L/18R runway and the terminal buildings which are adjacent west side to 01/19 will cause disturbance to the wind field along the glide path in 36L and 01. Table 1. Specifications of the PCDL system at BCIA. Table 1. Specifications of the PCDL system at BCIA. Parameters Specification Radial Spatial Resolution 30m Measurement Range* 60 to 4000 m Wavelength 1.5 μm Pulse Energy 150 μJ Pulse Repetition Frequency 10 kHz Scanner Positioning Accuracy 0.1° Resolution of Velocity 0.1 m/s * Weather condition related Fig.2. Wind Shear statistics reported by the pilots in different runways at BCIA (from 2015, 2016 and 2018). Fig.2. Wind Shear statistics reported by the pilots in different runways at BCIA (from 2015, 2016 and 2018). 3.1 Complex terrain wakes and wind shear In December each year, northerly and northwest winds are prevalent at BCIA. The runway corridor 01 and 36L are located downwind of complex terrain (as shown in Fig.1), which is susceptible to 2 EPJ Web Conferences 237, 06004 (2020) ILRC 29 https://doi.org/10.1051/epjconf/202023706004 the wakes of the hills (or tall trees) and buildings. Fig.4 shows the obvious influence of wakes formed by the terminal under prevailing wind in 01 runway. The wind strips will disturb the velocity along the glide path and increase the bumpiness of the aircraft with potential hazards. The pilot reported the presence of wind shear in 01 runway at 19:03 (local time) on December 27, 2018, at this time, the background wind field is shown in the right half of Fig.4. Meanwhile, the PCDL also issued a wind shear alert, as illustrates in Fig.5. The velocity dropped from 17.7m/s to 8.9m/s during landing from far afield to the touchdown points, with changing about 8.8m/s (17kt) which is much more than 7.7m/s (15kt) adopted by the International Civil Aviation Organization (ICAO). which depicted the situation is more serious than 01 runway at the same time (If the time difference is less than 1.5min, it is considered to be synchronous between 36L and 01 runways in this paper.) as shown in Fig.5 and Fig.7. In addition to the wake effect, the smaller velocity at the lower altitude (the blue area near the lidar, as shown in Fig.6) should contribute to this serious situation. Fig.7 presents that the higher-altitude velocity was maintained above 15m/s, thus forming a sharp velocity change along the glide path. The pilot reported the presence of wind shear in 01 runway at 19:03 (local time) on December 27, 2018, at this time, the background wind field is shown in the right half of Fig.4. Meanwhile, the PCDL also issued a wind shear alert, as illustrates in Fig.5. The velocity dropped from 17.7m/s to 8.9m/s during landing from far afield to the touchdown points, with changing about 8.8m/s (17kt) which is much more than 7.7m/s (15kt) adopted by the International Civil Aviation Organization (ICAO). Fig.6. Glide path radial velocity of 36L runway on December 27, 2018. Fig.6. Glide path radial velocity of 36L runway on December 27, 2018. Fig.7. Headwind profile of 36L runway on December 27, 2018. Fig.4. Glide path radial velocity of 01 runway on December 27, 2018. Fig.6. 3.1 Complex terrain wakes and wind shear Glide path radial velocity of 36L runway on December 27, 2018. Fig.7. Headwind profile of 36L runway on December 27, 2018. Fig.7. Headwind profile of 36L runway on December 27, 2018. Fig.4. Glide path radial velocity of 01 runway on December 27, 2018. Fig.5. Headwind profile of 01 runway on December 27, 2018. Fig.5. Headwind profile of 01 runway on December 27, 2018. Fig.7. Headwind profile of 36L runway on December 27, 2018. ACKNOWLEDGEMENTS This work was supported by the National Key Research and Development Program of China, grant number 2016YFC1400904 and North China Regional Air Traffic Management Bureau of CAAC. We thank our colleagues including Xiaoye Wang and Xiaomin Chen from Ocean University of China for preparing and conducting the experiment. REFERENCES [1] Hallowell, R.G., et al. Lincoln Laboratory Journal 18 (2) (2010) [1] Hallowell, R.G., et al. Lincoln Laboratory Journal 18 (2) (2010) [2] Shun, C. M., et al. Journal of Atmospheric and Oceanic Technology 25(5): 637-655 (2008) [ ] , , p Oceanic Technology 25(5): 637-655 (2008) [3] Shun, C M, et al. 10th Conference on Aviation, Range, and Aerospace Meteorology (2002) [4] Zhang, H, et al. 19th Coherent Laser Radar Conference (2018) Fig.8. Radial velocity from RHI scanning around the 01/19 runway. [3] Shun, C M, et al. 10th Conference on Aviation, Range, and Aerospace Meteorology (2002) [4] Zhang, H, et al. 19th Coherent Laser Radar Conference (2018) ( ) [5] CHAN, P. W., et al. 12th Conference on Aviation, Range, and Aerospace Meteorology (2006) [6] Chan, P. W, et al. Journal of Atmospheric and Oceanic Technology 29(2): 207-220 (2012) [5] CHAN, P. W., et al. 12th Conference on Aviation, Range, and Aerospace Meteorology (2006) [5] CHAN, P. W., et al. 12th Conference on Aviation, Range, and Aerospace Meteorology (2006) [6] Chan, P. W, et al. Journal of Atmospheric and Oceanic Technology 29(2): 207-220 (2012) [7] Zhang, H, et al. Infrared Physics & Technology 96:113-122 (2019) [6] Chan, P. W, et al. Journal of Atmospheric and Oceanic Technology 29(2): 207-220 (2012) gy ( ) ( ) [7] Zhang, H, et al. Infrared Physics & Technology 96:113-122 (2019) [8] Chan, P. W, et al. Meteorologische Zeitschrift, 20(6): 661-670 (2011) Fig.8. Radial velocity from RHI scanning around the 01/19 runway. [9] Chan, P. W. Meteorologische Zeitschrift 21(2): 193-204 (2012) [10] Lee, Y. F, et al. Meteorological Applications 21(1): 86-93 (2014) 3.2 Complex topography and wind field along the runway 3.2 Complex topography and wind field along the runway In this experiment, the RHI scanning strategy and the glide path scanning mode were designed alternately. For the RHI scanning, the azimuth of PCDL was consistent with the 01/19 runway’s azimuth, and the pitch angle varied between 0 and 45 degrees. Wind shear alerts were issued by the PCDL at 03:40 and 03:47 (local time) on January 04, 2019, respectively. The glide path radial velocity figures showed evident stripes that are Fig.5. Headwind profile of 01 runway on December 27, 2018. During this period, under the influence of strong northwest wind, the velocity in the 36L runway was affected by the wakes of tall trees, hills, and residential buildings, as the stripes formed in Fig.6. Lidar also issued a wind shear signal that the difference of velocity was up to 10.6m/s (20kt) 3 EPJ Web Conferences 237, 06004 (2020) ILRC 29 https://doi.org/10.1051/epjconf/202023706004 the terminal building wakes, which was not shown in this paper. Fig.8 shows the radial velocity retrieved by RHI scanning for the above time period. In this graph, with zero representing the location of PCDL, the positive distances represent the positions at the south of lidar along the runway and vice versa for the northern positions. In the upper graph of the Fig.8, there are two lower-velocity zones in the wind field below the height of 100m at 600m and 1700m account of wake effect. And the flight glide path just passed through the two zones, which were accompanied by the wind shear or turbulence. In the lower part of Fig.8, a wind area of lower-velocity was distributed at the distance of 60m to 1600m below the altitude of 200m, mainly because this area is located on the east side of the terminal building (referring to Fig.1) with strong shielding effect. turbulence. In addition, under the shielding effect of the terminal building, a wide range of lower- velocity was formed at low altitudes. 4. CONCLUSIONS [11] Chan, P. W. 12th Conference on Aviation, Range, and Aerospace Meteorology (2006) Glide path mode designed to detect the wind shear and RHI scanning strategy used to explore the wind field around the runway observed alternately in this synchronous experiment between 36L runway and 01 runway. The stripes appearing in the glide path scanning radial velocity indicate the wake effects produced by complex terrain. The PCDL warned the terrain-induced wind shear, and which was verified by the pilot report. The results show that the difference between higher-velocity and lower-velocity at different altitudes in 36L runway exacerbates the variation of the headwind profile and brings a more severe wind shear situation. The structure around the runway can be obtained by RHI scanning. The complex terrain wake will form lower-velocity areas along the glide path, which will bring the wind shear and [12] Chan, P W. Meteorologische Zeitschrift 19(6): 549-563 (2010)
https://openalex.org/W4248617572	https://www.qeios.com/read/WHSDUJ/pdf	English	null	Stage IV Renal Cell Cancer AJCC v6	Definitions	2,020	cc-by	113	Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Open Peer Review on Qeios Stage IV Renal Cell Cancer AJCC v6 National Cancer Institute National Cancer Institute Qeios ID: WHSDUJ · https://doi.org/10.32388/WHSDUJ Source Source National Cancer Institute. Stage IV Renal Cell Cancer AJCC v6. NCI Thesaurus. Code C4003. Stage IV includes: (T4, N0, M0); (T4, N1, M0); (Any T, N2, M0); (Any T, Any N, M1). T4: Tumor invades beyond Gerota's fascia. N0: No regional lymph node metastases. N1: Metastasis in a single regional lymph node. N2: Metastasis in more than one regional lymph node. M0: No distant metastasis. M1: Distant metastasis. (AJCC 6th ed.) Qeios ID: WHSDUJ · https://doi.org/10.32388/WHSDUJ 1/1
https://openalex.org/W2593525977	https://inria.hal.science/hal-01699733/document	English	null	Multi-party Interactive Visioneering Workshop for Smart Connected Products in Global Manufacturing Industry Considering PLM	IFIP advances in information and communication technology	2,016	cc-by	4,640	To cite this version: Satoshi Goto, Elio Trolio, Osamu Yoshie, Kin’ya Tamaki. Multi-party Interactive Visioneering Work- shop for Smart Connected Products in Global Manufacturing Industry Considering PLM. 13th IFIP International Conference on Product Lifecycle Management (PLM), Jul 2016, Columbia, SC, United States. pp.501-511, 10.1007/978-3-319-54660-5_45. hal-01699733 Multi-party Interactive Visioneering Workshop for Smart Connected Products in Global Manufacturing Industry Considering PLM Satoshi Goto, Elio Trolio, Osamu Yoshie, Kin’ya Tamaki Satoshi Goto, Elio Trolio, Osamu Yoshie, Kin’ya Tamaki Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-01699733 https://inria.hal.science/hal-01699733v1 Submitted on 2 Feb 2018 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 Multi-party Interactive Visioneering Workshop for Smart Connected Products in Global Manufacturing Industry Considering PLM Satoshi Goto1,3,4, Elio Trolio2, Osamu Yoshie3, and Kin’ya Tamaki4 1Business Transformation Management, PTC Japan Co. Ltd., Japan 2IoT Strategy & Business Consulting, ThingWorx Inc., U.S.A. 3Graduate School of Information, Production and Systems, Waseda University, Japan 4Human Innovation Research Center, Aoyama Gakuin University, Japan sgoto@ptc.com, elio.trolio@thingworx.com, yoshie@waseda.jp, kinya.tamaki@gmail.com Abstract. Currently, Internet of Things (IoT) is a dominant technology and a core mechanism for the third Information Technology (IT) revolution. Many benefits are expected to be enabled by implementing the IoT technologies through the product lifecycle management (PLM) process, such as remote monitoring of field service and predictive quality reliability engineering design in R&D. Smart connected products (SCPs) are forecast to produce tremendous business value. However, significant business challenges are associated with SCPs. Manufacturers have difficulty in rapidly launching IoT products in the market. This paper proposes a pragmatic visioneering workshop framework informed by real-world industry practices. The group facilitation for visioneering focuses on identifying the relation between the 26 practical IoT use cases through the PLM process. Moreover, the proposed workshop format will also enable the participants to engage in a discussion and interact with the framework through use case analysis. Keywords: Internet of Things (IoT), PLM Process, Smart Connected Products (SCPs), Multi-Party Interaction, Visioneering Workshop Facilitation 1 Introduction Currently, Internet of Things (IoT) is a dominant technology and is called the third information technology (IT) revolution [7]. The IoT technology enables multiple opportunities and business values through the entire product life cycle management (PLM) process [12]. Remote monitoring of field service and predictive quality reliability engineering design in R&D. Smart factories are alone valuated as a $3.7 trillion dollar industry and are forecast to produce tremendous business value [5]. It is estimated that 30 billion connected “things” will exist by 2020 [4]. Global discrete manufacturing companies such as automotive and high-tech electronics and industrial equipment manufacturers are currently facing significant IoT related business challenges. It is very difficult for these companies to rapidly launch IoT products in the market because of the new complexity derived from the addition of software applications and connectivity components. According to the results of IDC research, it was found that 66% of the discrete manufacturers pursue IoT initiatives and 40% of them are still at the pilot trial stage [4]. In addition, top- level executives are faced with new strategic challenges such as identifying new corporate models to accelerate the investment in R&D. Moreover, they are still struggling to get started. The PLM experts who are assigned the task of IoT promotion in such companies have various individual opinions and pursue different directions. This causes difficulties in choosing a single direction and achieving consensus regarding the development of smart connected products (SCPs). Therefore, companies spend more time in the planning stage of SCPs as compared to general products. In this new era of SCP development, the first critical step is to coordinate the early stages of the PLM process. Thus, a multi-party interactive consensus- building approach is very important; such an approach must be rapid. This paper proposes a pragmatic visioneering workshop framework informed by real-world industry practices. The group facilitation of visioneering focuses on identifying the relation between the key issues and challenges in some of the 26 practical IoT use cases. It identifies how a company can plan an SCP solution and craft a high-level IoT value roadmap chart understanding each phase of the PLM process. This paper also proposes to incorporate a workshop format that will enable participants to engage in a discussion and interact with the framework through customer value chain analysis (CVCA) [3] referring to the IoT use cases as a guide during the group discussion session. 1 Introduction The paper is organized as follows: Section 2 briefly presents the 26 IoT use cases that are categorized through the entire product lifecycle stages. Section 3 proposes a framework of IoT visioneering workshop agenda. A case study of a workshop conducted by a leading global discrete manufacturer is discussed in Section 4. We discuss whether the visioneering framework was valuable to the participants in group facilitation in the SCP concept planning phase. Finally, in Section 5, it is concluded that the workshop provided a benefit of achieving consensus in a shorter time period than that expected by the participants. Moreover, an outlook on this study is also mentioned in the conclusion of this paper. Table 1. IoT Use Cases aligned with PLM processes [11] Category (a.k.a. PLM process) IoT Use Case 2 IoT Use Cases throughout Product Lifecycle Stages To overcome the stuck business situation described in Section 1, templates of the 26 IoT use cases have been developed [9, 10, 11]. These templates are used as a guide to help the stakeholders who seek to understand how to create a business value of SCP solutions in the early stage of product strategy planning. Each use case is defined as a typical IoT practice example that is experienced by hundreds of manufactures through the PLM processes. The 26 use cases are also categorized by six key product lifecycle stages (Table 1) so that the use cases aligned with the PLM process can be recognized. Category (a.k.a. PLM process) IoT Use Case Category (a.k.a. PLM process) A) Marketing and Sales 1. Customer Insights and Opportunities 2. Flexible Billing and Pricing Models 3. New Value Added Services B) Product Development 4. Connected Product Usage Analysis 5. Connected Product Quality Analysis 6. Connected Software Management C) Operations and Manufacturing 7. Asset and Material Tracking 8. Connected Operations Intelligence 9. Unified Key Performance Indicators 10. Real-time Asset Health Monitoring 11. Operations Management Improvements D) Service and Support 12. Monitoring and Diagnostics 13. Remote Service 14. Automated Service Execution 15. Condition-based Predictive Maintenance 16. Connected Service Parts Planning E) Information and Operational Technology 18. Flexible Product and Asset Connectivity 19. Identity and Security Management 20. Scalable IoT Operations Management 21. Seamless IoT Data Integration 22. Automated Analytics and Actions 23. Rapid IoT Application Development F) Customers 24. Usage and Performance Dashboard 25. Customer Self-service 26. Product Personalization The contents of the above 26 use cases are mainly utilized at the proposed visioneering session during the group activity; the participants can clearly determine what they need to focus on for their IoT initiatives. One of the benefits is that it helps the group to quickly understand and easily choose key IoT initiatives in shorter discussion time, for example, in 15–20 min. A more specific description of this is provided in Section 3. 3.2 Proposed Workshop Agenda & Timetable Part 1: Ask for Business Strategies (Value Drivers) Part 1 of the questionnaire asks the questionee about Business Strategies and comprises 6 options (Fig. 1) called “Value Drivers” [9, 10, 11]. These 6 options are organized into two categories. Options 1–3 are based on “Operational Effectiveness” and are aimed at helping to improve the optimization of the operational performance. Options 4-6 are for strategic differentiation. The idea of Part 1 is based on the competitive strategy framework developed by Professor Michael Porter [6, 7]. Part 2: Ask for Current States (Challenges) f g Part 2 is focused on typical common business challenges (Fig. 1). Twenty options are given that comprehensively describe the end users’ problems through the entire product lifecycle process with examples such as the slow pace of product innovation and expensive internal development process for SCP projects. 3.4 Preliminary Questionnaire 3.4 Preliminary Questionnaire y Q A preliminary questionnaire is an efficient approach for obtaining the participant’s individual thoughts and insights in advance and is employed to facilitate the smooth running of the workshop. The following is the proposed format for the questionnaire comprising two parts. 3.1 Background and Aim This workshop is designed for product managers and lead engineers who are working at manufacturing companies. As a background, C-level executives assigned them to be as corporate led IoT product promotion members. However, the workshop members are not always available to work full-time on the assigned mission. Thus, an efficient and more productive approach is required that will enable consensus building over a shorter time. The members need to rapidly provide a single common SCP solution idea that contributes to the executives’ strategic goals. This paper aims to provide a procedure for thinking through facilitated group visioneering approaches in such business situations. 3.2 Proposed Workshop Agenda & Timetable 3.2 Proposed Workshop Agenda & Timetable Table 2 shows the proposed agenda for the visioneering workshop for multi-party participants invited from the various product and service development organizations in the company. The timetable is very compact, and an intensive configuration for such busy participants is a necessary and sufficient condition. The workshop is designed to be completed in a total of 5 hours and is configured in 7 step-by-step sessions. Table 2 shows the proposed agenda for the visioneering workshop for multi-party participants invited from the various product and service development organizations in the company. The timetable is very compact, and an intensive configuration for such busy participants is a necessary and sufficient condition. The workshop is designed to be completed in a total of 5 hours and is configured in 7 step-by-step sessions. Table 2. Proposed Agenda Template for the Visioneering Workshop Round # Session Agenda Interval (min.) Clock Time (as sample) 1 Introduction/Agenda Review 15 13:00–13:15 2 IoT Introduction & Strategy Overview 30 13:15–13:45 3 Global Industry IoT Case Studies 45 13:45–14:30 Break Time 15 14:30–14:45 Group Work for Visioneering Step 1. Identify Stakeholders 4 Step 2. Select major IoT Use Cases 120 14:45–16:45 Step 3. Narrow-down Use Cases Step 4. Craft IoT Value Roadmap Step 5. Set Metrics for IoT Business 5 Group Presentation 15 16:45–17:00 Break Time 15 17:00–17:15 6 IoT Enablement 30 17:15–17:45 7 Wrap-up/Next Steps Discussion 15 17:45–18:00 Total 300 13:00–18:00 Table 2. Proposed Agenda Template for the Visioneering Workshop Web-based Assessment Tool Web-based Assessment Tool The proposed preliminary questionnaire is also available as a Web-based system for the workshop participants so that they can respond to the questionnaire on the Web (https://jp.surveymonkey.com/r/XKQ9ZFV). The questionnaire must be submitted a couple of days prior to the date of the workshop. The option selection for Parts 1 and Part 2 is very easy for the questionee, and it normally takes 15 minutes to complete each Part. The Web system is a freeware that everyone can use on the Web [13]. An Excel sheet is also available for the participants who cannot access the internet environment (Fig. 1). Fig. 1. Preliminary Questionnaire (Part 1 and Part 2) https://jp.surveymonkey.com/r/XKQ9ZFV ig. 1. Preliminary Questionnaire (Part 1 and Part https://jp.surveymonkey.com/r/XKQ9ZFV Fig. 1. Preliminary Questionnaire (Part 1 and Part 2 https://jp.surveymonkey.com/r/XKQ9ZFV 3.5 Design of Group Facilitation for Visioneering Session pp p Step 2: Select Top 6 IoT Use Cases—aligning with corporate Value Drivers Step 2: Select Top 6 IoT Use Cases—aligning with corporate Value Drivers In the second step, the group members will review the 26 use case examples and select 6 use cases. This is to support their conclusion whether their selected product or asset will become worthwhile as a future SCP solution. In addition, they need to understand which of the selected use cases provide business impact for the specific Value Drivers (Business Strategies) based on the preliminary results of Part 1 of the questionnaire. For this step, 15 minutes is an appropriate amount of time. S 3 N d l d U C l i 3 f 6 f b tep 3: Narrow-down selected Use Cases—selecting 3 out of 6 for to be more pecific In the third step, the participants review and prioritize the above selected 6 use cases and select the top 3 use cases. Then, the members will discuss why these use cases were selected. Finally, they will unanimously agree on the most important use case for the first action on the future roadmap. For this step, 10 minutes is an appropriate amount of time. Step 4: Craft IoT Value Roadmap—positioning the Use Cases on the value maturity The members will use the selected top 3 use cases to consider the steps and value maturity. Considering the As-Is situation and examining the result of the preliminary questionnaire, they will create an IoT Value Roadmap to add a To-Be objective and goal for each step [Fig. 2]. For this step, 45 minutes is an appropriate amount of time. Step 5: Set Metrics (KPIs)—qualifying Business Goals p ( ) q fy g During step 5, the group members will identify action items to move forward utilizing the use cases. In parallel, they discuss key metrics (KPIs) for each use case. KPI examples should be provided by the facilitator to the group members. The selected metrics would be significant indicators of whether the planned business transformation is correctly promoted with the SCP solutions that they would develop. Finally, they will draw one single page as a high-level IoT value roadmap putting all of the insights that they discovered through the steps 1–4. For example, how to better qualify selected use cases, from which use case should we begin, and what are the “quick wins” or “strategic values.” For this step, 30 minutes is an appropriate amount of time. 3.5 Design of Group Facilitation for Visioneering Session 3.5 Design of Group Facilitation for Visioneering Session This workshop emphasizes intensive group work (round #4 in Table 2), wherein 4 or 5 people per group and 2 or 3 groups per workshop are reasonable. Moreover, 2 facilitators support all the group activities. During the group session, many debating situations are possible. In one case, each person has his own opinion and may try to push his own idea to others. In another roundtable, the group discussion would be very quiet and low-key; nobody tries to speak up and the participants are just watching each other until someone makes a comment. Either one of these two cases is not always ideal for building a consensus for a single direction. Furthermore, a difficulty in reaching the final goal of the group discussion outcome will be faced in both cases. Therefore, the following five pragmatic steps are proposed as an engineering facilitation methodology aiming to smoothly achieve a consensus for a single direction (Table 3). Table 3. Five Steps for Group Facilitation at a Visioneering Session Table 3. Five Steps for Group Facilitation at a Visioneering Session Step # Group Discussion Topic 1 Identify Stakeholder—utilizing Customer Value Chain Analysis (CVCA) 2 Select Top 6 IoT Use Cases—aligning with corporate Value Drivers 3 Narrow-down the Use Cases—selecting 3 out of 6 for to be more specific 4 Craft IoT Value Roadmap—positioning the Use Cases on the value maturity 5 Set Metrics (KPIs)—qualifying Business Goals Step 1: Identify Stakeholders—utilizing Customer Value Chain Analysis (CVCA) Step 1: Identify Stakeholders—utilizing Customer Value Chain Analysis (CVCA) Using CVCA methodology [3], the group members are encouraged to discuss all the people and processes that impact or depend on the product or asset. First, this requires the group members to select a product or asset to focus the discussion on; the members will select and identify as many stakeholders as possible, such as internal/external and direct/indirect. The roles of the stakeholders should be specific. The discussed stakeholders should then be connected with a line. As a result, a CVCA diagram is drawn surrounding the selected product or asset. This task is aimed to help the members realize that there are many influencers and to expand the value of the product or asset connecting various stakeholders. For this step, 20 minutes is an appropriate amount of time. 3.5 Design of Group Facilitation for Visioneering Session pp p Step 2: Select Top 6 IoT Use Cases—aligning with corporate Value Drivers Tailored IoT Value Roadmap with Maturity Curve p y At the end of the group discussion, a value roadmap is crafted as a one-page summary. Figure 1 is an example that is configured with Value Driver, Value Area, Sensing Information, Challenge, Metric, and IoT Solutions. Fig. 2. Tailored IoT Value Roadmap with Maturity Curve (a Sample Template) Fig. 2. Tailored IoT Value Roadmap with Maturity Curve (a Sample Template) pp p Step 2: Select Top 6 IoT Use Cases—aligning with corporate Value Drivers 3.6 Key Achievements of Visioneering Workshop Through the visioneering session, the following are achieved as group work outcomes recognizing the group members’ efforts. All-hands Intensive Group Presentation Regarding the visioneering session, it is most important to recognize its group efforts. The participants intensively work together during the limited session time such as for 120 minutes. At the end of the group session, the group presentation time is required by the facilitator. The aim of this step is that all of the participants at the workshop are able to get a mutual understanding and compare with other group members’ outcomes. The presentation time for each group is only 5 minutes. It should include the group CVCA diagram and the high-level SCP value roadmap discussed during the group work (Fig. 2). After the presentation time, the audience (other groups) must ask constructive questions to the presenter group (at least 2 questions). Thus, presentation time provides critical insights regarding the value propositions. Regarding the visioneering session, it is most important to recognize its group efforts. The participants intensively work together during the limited session time such as for 120 minutes. At the end of the group session, the group presentation time is required by the facilitator. The aim of this step is that all of the participants at the workshop are able to get a mutual understanding and compare with other group members’ outcomes. The presentation time for each group is only 5 minutes. It should include the group CVCA diagram and the high-level SCP value roadmap discussed during the group work (Fig. 2). After the presentation time, the audience (other groups) must ask constructive questions to the presenter group (at least 2 questions). Thus, presentation time provides critical insights regarding the value propositions. Tailored IoT Value Roadmap with Maturity Curve 4.1 Background and Opportunity Company-X (as anonym) is a leading global manufacturer of specific precision instruments. Company-X’s product development process is globally distributed, e.g., among countries A, B, C, and D. For example, the hardware design team is located in country A, whereas the software application development team is located in country B. These teams have been developing high-quality hardware centric products over a period of time, and the company has built a dominant position in the specific global market. The market is quite oligopolistic and has a high entry barrier because of the severe industry-specific regulations. Because the IoT technology is recently recognized as a disruptive innovation that can transform the existing product functionalities, the boundaries of the competition shift and expand from the exiting industry to a broader system of products. Moreover, there was a threat of a severe battle for Company-X. This was a new competitive era with not only the existing competitors but also with the newly entering cost-competitive emerging companies. p y g p g g p In such a new business transformation, the senior executive officer in charge of a global business unit in Company-X decided to start a “vision definition” for their future IoT-enabled SCP solutions. This required collaboration with the corporate product management team and the local development members who are distributed among the various countries. A critical challenge was how the differences of cultures and opinions among the members can be efficiently controlled to enable the formulation of a single and common future vision in a short time frame such as a half- day internal big meeting. This was an opportunity for our study team to propose our developed visioneering workshop framework to the officer, supporting Company-X’s vision-making initiative as an independent third party. It was a significant empirical study opportunity for us to examine whether the workshop framework can validate our study concept and its assumption. 4.2 Characteristic of Participants The following distributed members were gathered at a single location in country A (Table 4). They came from four different regions around the world and their nationalities and mother tongues were different. To support mutual communication, a dedicated interpreter staff was assigned for translation between English and the local language of country A. Table 4. Attendees List of the Visioneering Workshop at Company-X Table 4. Attendees List of the Visioneering Workshop at Company-X Group Name Participant (individual #) Business title Region (Work location) Mother tongue Group-A (w/ global managers) 1 VP Americas language-a 2 Director EMEA language-b 3 Americas language-a 4 language-a 5 General Mgr. Asia-Oceania language-c 6 EMEA language-b 7 Manager Asia-Oceania language-c Group-B (w/ local managers) 8 General Mgr. Asia-Oceania language-c 9 10 11 Group-C (w/ local engineers) 12 Manager Asia-Oceania language-c 13 Sr. Engineer 14 15 Engineer 4.3 Discussions In this paper, we focus our discussion on the “Group Work for Visioneering” session for Round #4 in Table 2 based on the result for the actual case of Company-X. The developed group facilitation approach has comprehensively provided significant insights to the workshop group members. This allowed them to identify IoT values that they have never previously realized. The following three items were particularly significant discussion points. Well-balanced PLM process as IoT use cases. p The predefined IoT use case templates allowed the group members to provide well- balanced strategy planning workflow in IoT topics and discussions. Although most of the participants were basically from the “engineering department,” they realized the value of selecting some of the IoT initiatives of the product manufacturing and field service processes that were not within their specialties. The initiatives they selected were also well-aligned with the corporate strategy. These potential values would not have been discovered without the use of such templates. In addition, the participants from the “hardware” design team recognized the importance of the value of “software” rather than hardware innovation. Another remarkable contribution by the facilitator was that the 26 use cases were prepared as “26 cards.” This means that the group members enjoyed the group discussion time as if they were playing cards, which had a positive effect by relaxing the participants and enabling them to think about brand new ideas. Doubling productivity vs. negative busyness? g p y g y During the group work session, the facilitator was rigidly measuring the session time with a stopwatch. This brought about a remarkable increase in productivity. Moreover, the predefined timetable was a quite a useful guide for the facilitator. In fact, there was a very positive endorsement from a lead participant in the workshop, “Without such time management and use case templates, we could not complete on time. We would spend twice as much time as we actually did.” On the other hand, the rigid timing also identified some of the participants’ mental stress due to the busyness forced by the facilitator. This should be a topic for improvement in a future study. Multi-linguistic party and challenges on remote facilitation g p y g f Although each group (A, B, and C) comprised people with different backgrounds and cultures from overseas countries, no operationally fatal problems were identified during the group discussion time. All three groups achieved the final conclusions. However, we have to admit the contribution of the professional interpreter’s savvy. Such multinational and multi-linguistic group activities are currently estimated to be increasing. We are still dependent on such a talent of the interpreter for better human communication for the solution of the problems involved in the discussion in such a diverse environment. Furthermore, in this case, another facilitator joined remotely through the Web from his base country. Currently, Web meeting applications such as WebEx on a smartphone are very convenient and cheaper than ever before. Thus, we actually applied a remote facilitation style during round #3 in Table 2. This had a negative influence because it was quite difficult for the remote facilitator to recognize the audiences’ personal perceptions. Generally, it is very important to understand how a remote facilitator can be acceptable in such an unknown situation [2]. This should be improved in the workshop agenda design based on the previous literature and cross-disciplinary studies and research. 5 Conclusions and Future Work We proposed a visioneering workshop approach utilizing the 26 IoT use cases through the PLM process. We have identified some significant values during the proposed group facilitation approach at a global manufacturing company focused on specifically planning an SCP concept as a part of IoT product solution suite. We also recognized that the proposed approach was acceptable for the workshop participants because they were able to achieve a common vision and consensus on a single SCP concept in a shorter time than they initially estimated. For the workshop participants, the largest contribution was made by the ability to use the comprehensive formatted 26 IoT use case examples. The participants clearly imagined future candidates of IoT solutions because the use cases were pragmatic business templates and were demonstrated in the actual industry environment. On the other hand, we need to consider the remote facilitator’s role at the requirement gathering phase described in the literature [2] as a possibility of virtual meeting space with ICT remote environment. As the next step, we are building on the research of previous studies in directions such as visual planning for virtual multi-site teams [1, 8]. Furthermore, we would like to investigate the effects of adopting innovative user experiences such as augmented reality. This would provide a supportive effect for the globally distributed participants as if they worked together in-person in the same workshop room. References 1. Bertilsson, Josefin, Wentzel, G.: Visual Planning. Coordination and Collaboration of Multi- site Teams in Product Development Organisations (2015). 2. Damian, Daniela, E., et al.: An Exploratory Study of Facilitation in Distributed Requirements Engineering. Requirements Engineering8.1, 24-26 (2003) Engineering. Requirements Engineering8.1, 24-26 (2003 3. Donaldson, Krista, M., Ishii, K., Sheri, D., Sheppard: Customer Value Chain Analysis. Research in Engineering Design16.4, 174-183 (2006) 4. IDC InfoBrief sponsored by PTC: Connected Products and Operation. Reshaping the Manufacturing and Operations Landscape (2015) 5. James, M.:The Internet of Things. Mapping the Valu 6. Porter, M.E., Heppelmann, J.E.: How Smart, Connected Products are Transforming Companies. Harvard Business Review93.10, 96-114 (2015) 7. Porter, M.E., Heppelmann, J.E.: How Smart, Connected Products are Transforming Competition. Harvard Business Review92.11, 64-88 (2014) 8. Project Visit, http://www.projectvisit.org/ (visited on 10/1/2015) 9. PTC Inc.: IoT Value Roadmap, http://www.ptc.com/File%20Library/IoT/IoT-Use-Case- eBook.pdf (Visited on 6/10/16) 10. PTC Community, https://www.ptcusercommunity.com/docs/DOC-8646 (visited on 3/19/16) 11. PTC Inc.: To Create Real Business Value You Need to Identify and Prioritize the Specific IoT Use Cases, http://www.ptc.com/internet-of-things/use-cases (visited on 3/1/2016) p p g ( ) 12. Stark, J.: PLM and the IoT (#4): The Opportunities of the IoT. PLM Consultant and Owner, John Stark Associates, https://www.linkedin.com/pulse/plm-iot-4-opportunities-john- stark?trk=mp-reader-card (visited on 1/11/2016) p ( ) 13. Survey Monkey, https://www.surveymonkey.com/mp/aboutus/ (visited on 3/9/2016)
https://openalex.org/W3183935352	https://link.springer.com/content/pdf/10.1007/s00442-021-04992-x.pdf	English	null	Soil carbon and plant richness relationships differ among grassland types, disturbance history and plant functional groups	Oecologia	2,021	cc-by	9,911	Oecologia (2021) 196:1153–1166 https://doi.org/10.1007/s00442-021-04992-x Oecologia (2021) 196:1153–1166 https://doi.org/10.1007/s00442-021-04992-x COMMUNITY ECOLOGY – ORIGINAL RESEARCH Abstract Understanding the relationship of soil carbon storage and species diversity in grasslands can provide insights into manag- ing these ecosystems. We studied relationships among soil C and plant species richness within ~ 9700 ha of grasslands in Colorado, US. Using 141 grassland transects, we tested how soil C was related to plant species richness, grassland type, soil texture, and prairie dog presence. Soil C was significantly, positively related to plant species richness, while native perennial graminoid species richness exhibited an even stronger positive relationship. However, the relationship of soil C and plant richness was not found in all three grassland types studied, but instead was unique to the most common grassland type, mixed grass prairie, and absent from both xeric tallgrass and mesic tallgrass prairie. The presence of a single indicator species, Andropogon gerardii, showed a significant, positive relationship with soil carbon. Our best possible model explained 45% of the variance in soil C using species richness, grassland type, and their interaction. Surprisingly, soil C was negatively related to soil clay, suggesting that surface clays amplify evaporation and water runoff rather than protecting soil organic matter from decomposition. Soil C was negatively related to prairie dog presence, suggesting that prairie dogs do not enhance soil carbon sequestration; in fact, prairie dog occupied sites had significantly lower soil C, likely related to loss of topsoil from prairie dog colonies. Our results suggest that management for species richness provides the co-benefit of soil C storage, and high clay and prairie dog disturbance compromises both. Keywords Soil carbon · Plant species richness · Grasslands · Functional group · Prairie dogs Soil carbon and plant richness relationships differ among grassland types, disturbance history and plant functional groups B. L. Anacker1 · T. R. Seastedt2 · T. M. Halward2 · A. L. Lezberg1 Received: 18 December 2020 / Accepted: 16 July 2021 / Published online: 25 July 2021 © The Author(s) 2021 2 Institute of Arctic and Alpine Research, University of Colorado, Boulder, CO, USA 1 City of Boulder Open Space and Mountain Parks, Boulder, CO, USA * B. L. Anacker AnackerB@bouldercolorado.gov Introduction In our region, preliminary observations of prairie dogs suggest that their activities deplete native vegetation and lead to soil loss when colonies of these animals are constrained within a matrix of extensive agricultural and built environments (Seastedt 2013), while in other regions prairie dogs have been reported to increase soil C (Martinez-Estévez et al. 2013). While research demonstrates that high species richness can enhance ecosystem functioning (Isbell et al. 2011; Til- man et al. 2012; Yang et al. 2019), managing for functional groups or single indicator species may be easier and more effective than managing for total species richness. For exam- ple, a key functional plant trait is the C4 photosynthetic pathway, most often associated with grasses. C4 grasses are expected to contribute more soil C than C3 grasses due to their relatively greater above-ground biomass and below-ground productivity, more recalcitrant tissues and slower decomposition, and higher nitrogen-use efficiency (Yang et al. 2019). However, results are conflicting, with some studies showing the presence of C4 grasses is associ- ated with higher accumulated soil C (Fornara and Tilman 2008; O’Brien et al. 2010), while others show negative or no effect of C4 plant abundance on soil C pools (Mahaney et al. 2008; Hernández et al. 2013; Ampleman et al. 2014). In experimental manipulations, Fornara and Tilman (2008) demonstrated that higher species diversity plots were asso- ciated with greater soil carbon than lower diversity plots, even when C4 grasses were present in the lower diversity plots, suggesting the species richness and/or complemen- tarity was a more important driver of carbon accumula- tion than individual functional groups. Another possibility is that an individual species may have a suite of traits that consistently favor soil C accumulation. If such species can also persist over long-time scales, its presence could hedge against losses of diversity that might reduce soil C. If the soil C-richness relationship differs among plant functional groups or when individual species are present, then manag- ing for species richness within functional groups or man- aging for individual species will better serve the purpose Recent studies underline the importance of account- ing for environmental heterogeneity for landscape-scale, observational studies, where control of environmental and disturbance factors is not feasible (Manning et al. 2019). For example, a recent meta-analysis of small-plot manipu- lations of species richness (i.e., plots ≤ 400 m2) shows that soil carbon storage generally increases with species richness (Weisser et al. Introduction Higher soil C may also be related to the higher temporal stability often associated with richer 2 Institute of Arctic and Alpine Research, University of Colorado, Boulder, CO, USA (0121 3456789) 3 1154 Oecologia (2021) 196:1153–1166 plant communities: their many redundant species (i.e., the insurance effect), high likelihood that some species will do well when others do not (i.e., compensatory dynamics), and increased chance of selecting species that increase soil C from the “species pool” (i.e., the sampling effect) (Tilman 1999; Ives and Carpenter 2007) should help ensure that species-rich plant communities store carbon in all condi- tions and years. Underlying this expectation is the assump- tion that a diverse species mix will enhance the diversity and abundance of litter substrates for decomposition, and root carbon inputs that increase soil microbial activity and biomass (Lange et al. 2015). Moreover, positive feedbacks can occur between ecosystem functioning and species rich- ness, where each new species may add to soil C and therein water holding capacity, in turn favoring the establishment of even more species (Chen et al. 2018; Hoffland et al. 2020; Werner et al. 2020). Importantly, a mechanistic link between soil C and species richness implies that increases or decreases in ecosystem services follow species gains or losses. Of course, variation in soil C across a landscape is not predicted by species richness alone; a more complete assessment should account for landscape-scale heterogeneity in factors like resource availability and disturbance history (Schimel et al. 1994). soil available water that frequently controls plant produc- tivity (e.g., Baldock and Skjemstad 2000; Hook and Burk 2000). Mixed grass prairie may have the lowest water avail- ability due to surface clays and low soil organic matter (as described in our results), followed by xeric tallgrass (which, despite its name, is relatively wet deeper in the soil profile or where gravel mulch reduces evaporation), and with mesic tallgrass having the greatest water availability. The range of conditions of the different grassland types are further described in Table 1.f The presence of prairie dogs also varies spatially, affect- ing soil C storage directly through effects on soil structure and processes (Martinez-Estévez et al. 2013) and/or indi- rectly through changes in plant biomass, diversity, and composition (Beals et al. 2014). Introduction decomposition of these substrates (Jackson et al. 2017). Further, recent findings indicate that C storage is enhanced by increased grassland plant species richness in several situations: in systems recovering from disturbance (Yang et al. 2019), in experimental grasslands (Fornara and Til- man 2008, Steinbeis et al. 2008, Cong et al. 2014, Zou et al. 2019), and in natural ecosystems (Chen et al. 2018). These studies also find that plant life form and variation in species productivity influence C storage. Thus, management deci- sions to preserve grassland diversity have the potential to provide ecosystem services beyond the more obvious plant and wildlife conservation values. The maintenance and enhancement of soil carbon are man- agement imperatives for global sustainability (Minasny et al. 2017; Vermeulen et al. 2019). Grasslands are overachievers at storing soil carbon (Conant et al. 2017). For example, soil C storage is higher in temperate grasslands than temperate forests (Lal 2004) due to relatively high grassland plant bio- mass allocation to roots and relatively slow below-ground Communicated by Brian J. Wilsey. B.L. Anacker and T.R. Seastedt have contributed equally to this work. * B. L. Anacker AnackerB@bouldercolorado.gov 1 City of Boulder Open Space and Mountain Parks, Boulder, CO, USA 2 Institute of Arctic and Alpine Research, University of Colorado, Boulder, CO, USA Communicated by Brian J. Wilsey. B.L. Anacker and T.R. Seastedt have contributed equally to this work. * B. L. Anacker AnackerB@bouldercolorado.gov 1 City of Boulder Open Space and Mountain Parks, Boulder, CO, USA 2 Institute of Arctic and Alpine Research, University of Colorado, Boulder, CO, USA Communicated by Brian J. Wilsey. A relationship between soil C and plant species rich- ness is expected from niche theory. The co-occurrence of plant species with different niches (e.g., grasses vs forbs, native vs exotic, annual vs perennial) should lead to a more complete use of the available soil resources and overall higher carbon sequestration (i.e., niche complementarity; Turnbull et al. 2016). Introduction 2017), while a meta-analysis of 35 observa- tional studies on larger areas reveals about equal numbers of increased, decreased, and neutral relationships between soil C and species richness (van der Plas 2019). Here, we pre- sent a landscape-scale study of the soil C-species richness relationship and evaluate how the soil C-richness relates to landscape heterogeneity in grassland type, soil texture, and prairie dogs (Cynomys ludovicianus). The three grassland types studied here (mixed grass prai- ries, xeric tallgrass prairies, and mesic tallgrass prairies) were adopted for management purposes long before our study began based on dominant plant species composition; so, an interesting question is whether these same designa- tions correspond with differences in resource availability. These grassland types have the potential to vary substan- tially in topography, soil moisture, the composition of parent materials, and soil texture, which all affect soil C storage (Branson et al. 1965; Schimel et al. 1985; Hopkins-Arnold 1998). Soil texture, for example, influences carbon storage directly through variation in the degree of chemical and physical protection of soil organic matter, most often asso- ciated with clay content and indirectly through effects on 1 3 3 1155 Oecologia (2021) 196:1153–1166 Table 1 Attributes of three grassland communities found on City of Boulder lands Grassland type Area (ha) Most frequent native graminoid species Land Use Landscape context and composition Moisture availability Mixed grass prairie 4664 Western wheat (Pascopyrum smithii [Rydb.] Barkworth and D.R. Dewey) Blue grama (Bouteloua gracilis [Kunth] Lag. ex Griffiths) Sideoats grama (Bouteloua curtipendula [Michx.] Torr.) Buffalo grass (Buchloe dactyloides [Nutt.] Engelm.) Livestock grazing Historic tilling Prairie dog occupied Mosaic of diverse plant associations domi- nated by short- and mid-height species like western wheatgrass or needle and thread grass Relatively dry, in part due to high clay con- tent and low organic matter Xeric tallgrass prairie 2310 Big Bluestem Sun sedge (Carex pensylvanica Lam.) Sideoats grama Blue grama Livestock grazing A tallgrass community that occurs in uplands on rocky soils, which can often overlay clay-rich subsoils. Introduction Characterized by tall grass species like big bluestem, little bluestem, and prairie dropseed, often intermixed with species charac- teristic of the Rocky Mountain montane life zone Water stored at depth, available to deeply rooted natives Mesic tallgrass prairie 140 Switchgrass (Panicum virgatum L.) Arctic Rush (Juncus arcticus Willd.) Big Bluestem Yellow Indian grass (Sorghastrum nutans [L.] Nash) Irrigation Haying A tallgrass community that occurs in floodplains and higher terraces where high ground-water tables or flood irriga- tion support big bluestem, switchgrass, and arctic rush Relatively wet, with water available even at shallow depths 1 3 1156 Oecologia (2021) 196:1153–1166 of promoting carbon sequestration and managing for total species richness (c.f., Isbell et al. 2011). upland grassland types of conservation interest not specifi- cally targeted for agricultural use (Table 1). Some of these grassland types contain prairie dog colonies of varying den- sities (Johnson and Collinge 2004), which remain valued by some stakeholders for their role of keystone species (c.f. Kotlier et al. 1999). Our study utilizes an extensive vegetation inventory across three grassland types comprising ~ 9700 ha of pub- licly managed lands to test two hypotheses: Hypothesis 1 Soil C is positively related to plant species richness, even when accounting for landscape heterogeneity in grassland type, soil texture, and prairie dogs. Study area Grasslands managed by the City of Boulder, CO (USA) occupy the plains and foothill regions of Colorado’s Front Range centered at about 40 N latitude and 105 W longitude. The area experiences an average of 513 mm precipitation, with the spring-early summer interval being the wettest (NOAA 2020). Grassland inventory and monitoring efforts have documented about 800 vascular plant species (OSMP 2010). Unlike many other well-studied grasslands, the rain- fall gradient generated by the adjacent Rocky Mountains along with substantial topographical differences and varia- tion in parent material produce grassland communities with different dominant species across relatively short distances. These grasslands, therefore, host a unique and unusually diverse list of species (635 vascular species recorded in 9 years of monitoring), allowing for communities dominated by tallgrass species common to the Eastern US to lie adja- cent to communities dominated by species of the shortgrass steppe or higher elevation montane plant communities (Ves- tal 1914; Livingston 1952; Branson et al. 1965; Moir 1969; Bock and Bock 1998). If these vegetation differences con- tribute to large local differences in carbon storage, Boulder grasslands would be a logical area to test the relationship of soil C and species richness. The magnitude of this effort (16,000 recordings per full data set) meant that not all transects could be sampled on an annual basis. Here, we use vegetation monitoring data from 2016, the last year in which nearly full vegetation sampling was available (158 of 160 total transects) prior to the initia- tion of soil sampling in 2018–2019. A complete vegetation data set collected in the prior year (2015) showed similar patterns when related to our soils data, even though 2015 was much wetter than 2016 (data not shown) and we, there- fore, chose the most recent data set for our analyses. Vegetation monitoring Vegetation transects were located using a Generalized Random-Tessellation Stratified Design (Stevens and Olsen 2004; R software) to achieve a random and spatially bal- anced design of 160 transects stratified across City of Boul- der’s three grassland types. Between July and August of every sampling year, each transect was monitored using a point-intercept technique that recorded the top plant spe- cies or substrate intersected at 0.5 m on either side of every meter mark along a 50-m tape, giving a total of 100 possi- ble intercepts. Intercepts were observed through an optical point projection device (Cover-Point, ESCO Associates) that magnified the point under the cross hairs of a lens, reducing bias in sampling (Buckner 1985). Cover for each species was estimated as the number of respective intercepts across the transect. Transect-level species richness was based on augmenting the species list created during point-intercept sampling with any additional species found while searching the entire 2 m × 50 m belt transect.f Hypothesis 2 Soil C shows even stronger relationships to richness or presence of plant species with adaptations expected to promote accumulation of soil C (e.g., native perennial grasses; species with C4 photosynthetic pathway). 3 Linear models For simplicity, we used soil C as our response variable whenever possible, although we acknowledge that the mech- anistic relationship between soil C and plants is a continuous feedback and therefore edaphic or botanic variables could be used as the response variable. We prefer to use soil C as the response variable because we want to isolate con- ditions/predictors that managers can change to protect or influence carbon sequestration. Further, we note that soil N is correlated with soil C (r2 = 0.87), and the ratio of soil C to N would be of interest to some readers, but we leave this unexplored. Finally, we acknowledge that soil C is involved in complex multi-way feedbacks with environmental traits (e.g., climate, topography, land use) and ecosystem functions (e.g., productivity, herbivory, biodiversity) (e.g., Weisser et al. 2017; van der Plas 2019) beyond the set of predictors we have chosen to measure here. Soils were air dried, root fragments and rocks removed by hand and passed through a 2-mm mesh sieve and stored until analysis. Soil texture (% sand, % silt, % clay) was determined using the Bouyoucos hydrometer method for analyzing the particle size of soils (Texas A and M 2005). A subsample of each soil was pulverized using a Cianflone model 2601 soil pulverizer (Scientific Instruments Corp.). These processed soils were then sent to the Soil, Water & Plant Testing Lab at Colorado State University where they were analyzed for inorganic C content using a pressure transducer (Sherrod et al. 2002) and analyzed for total C and N using the dry combustion method in a Leco furnace CHN Analyzer (Model LECO-CHN-1000). Organic C was calculated by subtracting inorganic C from total C in each sample; hereafter, when we refer to “soil C”, we are refer- ring to % organic soil C. For Hypothesis 1, we derived four predictors (species richness, grassland type, clay, and the presence/absence of prairie dogs) for each transect. To test H1, we fit 6 models (Table 2), including a “full model” with all four predic- tors, 4 constituent models treating each single variable as a predictor, and the best multivariate model. To find the best multivariate model, we ran a candidate set of 33 models and selected the one with the lowest AIC score (see Electronic Supplementary Material 1 for list of models and AIC val- ues). Linear models There appeared to be a non-linear relationship between soil C and species richness, and so we fit a model with a polynomial term for species richness (Fig. 1). Finally, we fit 6 additional models to describe relationships among our four predictors themselves. Outliers The final data used for analyses included 141 transects sampled and analyzed for both soils (2018–2019) and vegetation (2016) that remained after excluding outliers that appeared contaminated with unusual amounts of C, N or both materials, although we cannot be sure. The 141 samples reported were those found within three standard deviations of the corresponding mean (i.e., for C, N, or C:N ratio) calculated without inclusion of obvious outli- ers from the pool of samples. We used SAS (Statistical Analysis System, SAS 9.4, 2017) and R (3.6.0) as our primary analysis tools. Samples were obtained and analyzed from 90 transects in 2018 and 59 additional transects in 2019 (149 total). Eleven of the 160 transects were not sampled and/or ana- lyzed due to restrictions related to conservation, cultural resource protection, or major site disturbance. While sam- pling the top 15 cm tends to emphasize carbon deposi- tion from grasses as opposed to shrubs or forbs (O’Keefe et al. 2019), our assumption here is that this sample rep- resents an index of C found at these sites, an assumption that appears reasonable for grasslands in our area (e.g., Schimel et al. 1985). That study also showed that bulk density across prairie landscapes was relatively constant, implying that soil C values can represent an index of total C in the top 15 cm of soil. Statistical procedures across a 15 cm depth to produce a similar soil volume per sample. While we excluded rocks from our sampling, we acknowledge that plots with many rocks will simply store less Carbon per unit area. Records were kept of the number of probes attempted in obtaining each core (max = 15, at which time the rock hammer was used), and these data were subsequently recorded to be used as an index of site rockiness. Soil sampling Beginning in May of 2018, established vegetation tran- sects were sampled for soils using a procedure that required accommodation for variations in rock cover and rock content. At each transect, a single, composited sample was obtained by sampling eight sites, four each at 10-m intervals on either side of each transect. Sampling loca- tions were located approximately 2 m outside of the center vegetation transect line at each interval. We obtained a 2 cm diameter by 15 cm deep core at each site, producing a composited volume of about 375 cc of soil per tran- sect. In the event that rocks precluded the use of a cor- ing tool, a rock hammer was used to excavate a small pit, and approximately 50–60 cc of soil were scraped evenly The City of Boulder’s Open Space and Mountain Parks (OSMP) department’s Grassland Ecosystem Management Plan (OSMP 2010) describes many goals related to the accommodation of conservation, recreation, and historical agricultural uses. Plan implementation included mapping of vegetation to delineate plant alliances (USNVC Data- base Ver 2.02), aggregating alliances into grassland types (referred to as “conservation targets” in OSMP 2010), and the establishment of an ambitious monitoring program with emphasis on monitoring vegetation composition on the three 1 3 3 1157 Oecologia (2021) 196:1153–1166 Prairie dog activity Prairie dog activity around each transect was determined by ArcGIS spatial analyses as the spatial intersection of vegetation monitoring transects against the prairie dog col- onies mapped in the field cumulatively between 1996 and the fall prior to soil sampling. To map prairie dog colonies, an annual field visit is conducted to mark the perimeter of the colony with a GPS and to confirm the presence of prairie dogs. Prairie dogs were considered present if any part of the transect fell within the boundaries of an active or historic prairie dog colony. Clay was negatively correlated with species richness (r = − 0.33). Thus, we used variance partitioning to appor- tion the variation in soil C among these two predictors (spe- cies richness and clay) as well as the joint/shared effects of the two predictors (Peres-Neto et al. 2006). We used the 1 3 Oecologia (2021) 196:1153–1166 1158 Table 2 Statistical models explaining variance in soil C measured among 141 grassland transects See Figs. 1 and 2 for graphical depictions of relationships and more statistical information. Prairie dog activity See ESM2 for the data file for use in re-running these models to investigate the coefficients and effect sizes The best model is in bold a* P < 0.001; P < 0.01, P < 0.05, ns not significant Hypothesis Model # Model description Model formula with P valuesa R2 AIC df model, df error 1 1 Full model Soil C–species richness + grassland type* + clayns + prairie dog presence/absencens 0.41 337.3 5, 135 1 2 Constituent model 1 Soil C–species richness* 0.21 374.3 1, 139 1 3 Constituent model 2 Soil C–grassland type* 0.33 351.7 2, 138 1 4 Constituent model 3 Soil C–clay* 0.03 403.8 1, 139 1 5 Constituent model 4 Soil C–prairie dog presence/absence* 0.14 386.9 1, 139 1 6 Best model Soil C–species richness* + grassland type* + spe- cies richness X grassland type 0.44 330.0 5, 135 2 7 Models for species subsets Soil C–native species richness* 0.17 381.2 1, 139 2 8 “ Soil C–exotic species richness 0.04 402.1 “ 2 9 “ Soil C–native perennial graminoid species richness* 0.28 361.5 “ 2 10 “ Soil C–exotic perennial graminoid species richness* 0.09 394.9 “ 2 11 “ Soil C–native perennial forb species richness* 0.11 391.4 “ 2 12 “ Soil C–exotic perennial forb species richness 0.06 398.9 “ 2 13 “ Soil C–native annual forb species richnessns 0.0 408.7 “ 2 14 “ Soil C–exotic annual forb species richnessns 0.0 409.2 “ 2 15 “ Soil C–Andropogon gerardii 1/0*** 0.27 364.6 “ “varpart” function of the “vegan” package in R to fit three linear models (soil C–species richness; soil C–clay; soil C–species richness + clay) for this purpose. The procedure then separates the fractions using addition and subtraction as applied to the model adjusted R2 values. In summary, to test Hypothesis 2, we fit 9 models (Table 2): two models predicting soil C from species rich- ness of native or exotic species, 6 models predicting soil C from richness of the 6 functional groups, and one model using the presence/absence of A. Prairie dog activity gerardii.i For Hypothesis 2, we derived 9 predictor variables: native species richness, exotic species richness, species richness for 6 functional groups (native perennial grami- noid [grasses, sedges, rushes], exotic perennial graminoid, native annual forb, exotic annual forb, native perennial forb, exotic perennial forb), and the presence/absence of big bluestem (Andropogon gerardii Vitman) Note: native and exotic annual graminoids were excluded due to their rarity in the dataset; likewise, species classified as “other” (cacti and woody species) were excluded. We chose to focus on A. gerardii (versus other indicator species) for several reasons: (1) it has likely effects on soil C, based on literature show- ing that C4 grasses contribute disproportionately to soil C (O’Brien et al 2010; Fornara and Tilman 2009), (2) its cover is considered an indicator of the condition of our grasslands in our grassland management plan (OSMP 2010), and (3) it is frequent and abundant in our grasslands. Cursory analysis using linear regression (not shown) showed that A. gerar- dii had the strongest relationship to soil C of all species in our dataset (almost 2 × stronger that the 2nd best predictor species). The models used to test our main hypotheses were fit using linear models and least sum of squares (“lm” func- tion in R). We tested if each model met the assumptions of normality of residuals and homogeneity of variance. The assumptions were met for just 6 of the 15 models, but model assumptions could be met via variable transformations or non-parametric tests for all the remaining 9 models, and these adjustments made no difference to the assessment of variable significance (not shown). Therefore, for conveni- ence, we share the results from using conventional linear models and untransformed variables. The derived dataset is available in csv format (ESM2). Results Hypothesis 1 Soil C is positively related to plant species richness, even when accounting for landscape heterogeneity in grassland type, soil texture, and prairie dogs. 1 3 3 1159 Oecologia (2021) 196:1153–1166 Fig. 1 Relationship of soil C and plant species richness, colored by each of three predictors: a grassland type, b soil texture, and c prairie dog presence. In panel a, the solid lines represent the slope parameters from linear regression; the dashed line represents the polynomial fit Fig. 1 Relationship of soil C and plant species richness, colored by each of three predictors: a grassland type, b soil texture, and c prairie dog presence. In panel a, the solid lines represent the slope parameters from linear regression; the dashed line represents the polynomial fit accounted for 4% more of the variance than did the linear response (R2 = 0.25; Fig. 1a) and had a lower AIC value (368.4 vs 374.3). Soil C–grassland type • Species richness was significantly related to grassland type (R2 = 0.31, P < 0.001) Soil C was significantly related to grassland type (R2 = 0.33; Table 2), where mesic tallgrass prairies had the highest soil C (Table 3). Adding grassland type to the model of spe- cies richness more than doubled the regression R2 values (Fig. 1), and a model that included an interaction of species richness and grassland type (ESM1) explained 43% of the variation in soil C. Three candidate models were tied (i.e., within ± 2 AIC units of each other) for “best model”, but based on parsimony, we selected the model with the fewest parameters as our best model: soil C ~ species richness * grassland type. The two other competing models had the additional predictors of prairie dog presence/absence and clay (ESM1) suggesting that prairie dogs and soil clay have a role to play even after accounting for species richness and grassland type. • Species richness was negatively related to clay (b = − 0.40; R2 = 0.10; P < 0.001) • Species richness was negatively related to prairie dog presence (b = − 13.5; R2 0.13; P < 0.001) p • Grassland type was significantly related to clay (R2 = 0.24, P < 0.001)i • Grassland type was significantly related to prairie dogs (they were found almost exclusively in mixed grass prai- ries; chi-squared test: X2 = 29.5; P < 0.001). • Clay was positively related to prairie dog presence (b = 7.2, R2 = 0.05; P < 0.01) • Clay was positively related to prairie dog presence (b = 7.2, R2 = 0.05; P < 0.01) Hypothesis 2 Soil C shows even stronger relationships to plant species richness or presence of species with adapta- tions expected to promote accumulation of soil C. Of note is that when the relationship between soil C and plant richness is analyzed separately by grassland type, only the mixed grass prairie exhibited a significant positive rela- tionship (R2 = 0.26, P < 0.001). The other two grasslands exhibited no pattern between soil C and richness (Xeric: P = 0.41; Mesic P = 0.69). It is possible that the lower sam- ple size and limited range in species richness across xeric and mesic tallgrass prairies prevented the detection of a soil C–richness relationship. Soil C–prairie dogs We found a positive relationship between soil C and plant species richness, despite confounding effects of landscape heterogeneity. The high soil C storage in our species-rich communities may be due to a high degree of niche com- plementarity of functional groups (Fornara and Tilman 2008; Turnbull et al. 2016; Yang et al. 2019), relatively long temporal stability of plant communities (Hector et al. 2010), positive feedbacks of species richness on carbon accumulation as mediated by increased productivity, high above-ground plant and root biomass (Yang et al. 2019), increased diversity of soil organic compounds (El Moujahid et al. 2017) enhanced microbial activity and diversity (Lange et al. 2015), or some other explanation. Regardless of the The past or current presence of prairie dogs produced a significant decline in soil C from 2.7% to 1.8% (R2 = 0.14; P < 0.001; Fig. 1). Prairie dogs were not included in the best model, likely due to the non-orthogonal nature (i.e., correla- tion) between the predictors, as described below. Soil C–species richness We found that soil C was related to species richness (R2 = 0.21, P < 0.0001; Fig. 1). A curvilinear polynomial fit, implying that soil C was highest at intermediate richness, 1 3 Table 3 Average soil and plant characteristics of three grassland communities Grassland Type Soil clay (%) Rockiness (# of core attempts) Soil C (%) Species richness Mixed grass prairie 24.8 3.0 2.0 33.8 Xeric tallgrass prairie 9.6 10.9 3.0 56.0 Mesic tallgrass prairie 12.8 6.4 3.5 43.6 1160 Oecologia (2021) 196:1153–1166 Soil C–grassland type The relationship between soil C and species richness for various combinations of functional groups often resulted in positive relationships with soil C (Fig. 2). The strongest contribution to a soil C relationship was created by native perennial graminoids (R2 = 0.28), but the surprising finding was that the presence or absence of a single C4 species, A. gerardii, was an equally strong predictor (R2 = 0.27; note, species richness was also correlated with the presence/ absence of A. gerardii; R2 = 0.41). All other groupings either contributed less variance to the relationship or were non- significant (Fig. 3). Soil C–soil texture Soil texture, as measured by % clay, was significantly but weakly negatively related to soil C (R2 = 0.03; Table 2). Vari- ance partitioning showed that clay had a much smaller effect on soil C than did species richness: unique effect of clay on soil C, R2 = 0.0; unique effect of species richness on soil C, R2 = 0.18; shared effect of clay and species richness on soil C, R2 = 0.04. These results suggest that texture was not driv- ing patterns of soil C. i Both exotic and native species richness show the overall significant positive relationship with soil C, however the strength of the relationship is much weaker with exotic spe- cies (R2 = 0.04) versus natives (R2 = 0.17). Of interest, there was no correlation between native and introduced species richness (P = 0.64). Correlation among species richness, grassland type, soil texture, and prairie dogs Six additional statistical models describe the relationship among our four predictors: 1 3 Oecologia (2021) 196:1153–1166 1161 1161 Oecologia (2021) 196:1153–1166 exact mechanistic cause of the relationship, the link that we discovered between soil C and species richness implies that addition or loss of ~ 35 species per 100 m2 is associated with the addition or loss of 1% soil C. The relationship between soil C and plant richness was much better described by accounting for grassland type, soil texture, and prairie dogs. Within grassland types, only one community type (mixed grass prairie) showed a strong rela- tionship between soil C and species richness, but it has two- fold higher acreage than the next most widespread grassland type (Table 1). Transects in mixed grass prairie spanned a relatively broad range of soil C and species richness, includ- ing the lowest observed values of C and richness associated with land use legacy effects such as tilling and overgrazing, which may have leveraged the overall soil C–richness rela- tionship. If one interprets C storage as the integrated out- come of inputs and outputs, our results argue that the mixed grass prairie provides fewer inputs due to a combination of factors including reduced species richness, a larger cover by introduced species (which lowers native diversity and con- tributes relatively small amounts of organic matter), reduc- tion in productivity due to land use legacy effects and direct and indirect effects of prairie dogs. These potential factors are consistent in both explaining the mixed grass prairie’s Fig. 2 Relationship between soil carbon and plant species richness in three different grassland types for natives (left column) and exotics (right col- umn) by group (rows). Data are presented for: a, b all species, c, d perennial graminoid, e, f perennial forb and g, h annuals. See Table 2 for statistics. Note that the x axis scales differ for each plot Fig. 3 Soil carbon (mean ± SE) by the presence of Andropogon gerar- dii (big bluestem) (mean ± standard error). A. gerardii was present in 73 of the 141 transects between t species erent natives (left (right col- s). Data are ll species, noid, e, f , h annuals. stics. Note differ for Fig. 3 Soil carbon (mean ± SE) by the presence of Andropogon gerar- dii (big bluestem) (mean ± standard error). A. Fig. 2 Relationship between soil carbon and plant species richness in three different grassland types for natives (left column) and exotics (right col- umn) by group (rows). Data are presented for: a, b all species, c, d perennial graminoid, e, f perennial forb and g, h annuals. See Table 2 for statistics. Note that the x axis scales differ for each plot Correlation among species richness, grassland type, soil texture, and prairie dogs The non-linear pattern also indicates that total richness is not the most relevant predictor of soil C in tallgrass prairies. Prairie dogs are a keystone species (Kotliar et al. 1999), and an important component of high functioning native- dominated grasslands in our region. Some colonies support intact native plant communities and prairie dog presence provides prey and landscape structure necessary for the pres- ence of associated species (OSMP 2010). However, in our study, prairie dog occupied sites had significantly lower soil C, although we note the prairie dog effect on soil C is dif- ficult to disentangle from the effects of species richness and soil clay. Some prairie dog colonies in our area are charac- terized by a high density of burrows and diminished native vegetation, likely related to low predator pressure and the restriction that urbanization places on prairie dog movement (OSMP 2010). These conditions have led to localized loss of topsoil from prairie dog colonies (Seastedt et al. 2013), leaving behind the C-depleted soils that we measured here. A different study on prairie dogs and soil C reported an increase soil C related to the burying of plant material, but further examination of the experimental design and sample analysis indicate that the elevated levels of soil C at depth was the result of a layer of calcium carbonate common in arid and semi-arid environments (Martinez-Estévez et al. 2013). The inverse relationship between soil C and soil clays on these sites was a surprise, but it is worth restating that the importance of clay to soil C in our study was very small. At a regional scale, soil carbon storage in mesic regions is often positively related to soil clay content (e.g., Burke et al. 1989; Schimel et al. 1994; Jobbágy and Jackson 2000). This phe- nomenon assumes that soil C becomes physically protected from further decomposition by sorption to mineral surfaces and aggregate formation. However, there is a limit to the capacity for clays to protect organic C in some environments and not all clay particles are equal in their ability to stabilize soil C because of their diverse mineral properties (Hassink 1997; Percival et al. 2000; Rasmussen et al. 2018). Thus, while we expect that clay content plays a role to enhance carbon storage in our study, the effect is obscured by other factors. Correlation among species richness, grassland type, soil texture, and prairie dogs Less soil C can also reduce water storage (Werner et al. 2020) thereby further reducing plant richness. In any event, the sandier soils will move water deeper into the soils where C4 plants in particular might be able to access this resource, resulting in higher plant productivity and richness and subsequently greater C content of soils. Again, we note that the relation- ship between soil C and clay was only a weak negative cor- relation in our study, but it is at least fair to say that carbon storage was not positively related to soil clay content. reduced soil C content relative to the other two communities, and the pattern observed between soil C and richness within the community itself. According to results from biodiver- sity experiments, these low diversity communities may be expected to have relatively larger losses in productivity (and corresponding declines in soil C) when species are lost as compared to more species-rich communities (Cardinale et al. 2011). In contrast, we suspect that the tallgrass communi- ties are potentially more resource rich, have higher plant available water (Branson et al. 1965), and have been less disturbed over time by historical cattle grazing, tilling and long-term prairie dog occupation.i Some parallels exist between our findings and those of Konza Prairie, a tallgrass prairie site where a landscape gradient generates higher productivity and higher amounts of carbon storage in relatively species-poor mesic lowlands when compared to species-rich xeric tallgrass uplands (Gib- son and Hulbert 1987; Briggs and Knapp 1995; Collins and Calabrese 2012). At that tallgrass site, plant competition for light in productive areas likely restricts a subset of species, and this interpretation may explain the differences observed between the xeric and mesic tallgrass prairies in our study, where plant productivity levels in mesic tallgrass can match or exceed those at the Kansas lowland site (Hopkins-Arnold 1998). Xeric tallgrass sites have the highest richness, but mesic tallgrass sites have the highest soil C. Both the tall- grass prairies have higher richness and soil C than the mixed grass prairies, regardless of the presence or absence of prairie dog communities. The result that species richness peaks at intermediate levels of soil C is reminiscent of the “humped-back” model used to describe the richness-produc- tion relationship observed elsewhere (c.f., Adler et al. 2011; Fraser et al. 2015). Correlation among species richness, grassland type, soil texture, and prairie dogs gerardii was present in 73 of the 141 transects addition or loss of ~ 35 species per 100 m2 is associated with the addition or loss of 1% soil C. The relationship between soil C and plant richness was much better described by accounting for grassland type, soil texture, and prairie dogs. Within grassland types, only one community type (mixed grass prairie) showed a strong rela- tionship between soil C and species richness, but it has two- fold higher acreage than the next most widespread grassland type (Table 1). Transects in mixed grass prairie spanned a relatively broad range of soil C and species richness, includ- ing the lowest observed values of C and richness associated with land use legacy effects such as tilling and overgrazing, which may have leveraged the overall soil C–richness rela- tionship. If one interprets C storage as the integrated out- come of inputs and outputs, our results argue that the mixed grass prairie provides fewer inputs due to a combination of factors including reduced species richness, a larger cover by introduced species (which lowers native diversity and con- tributes relatively small amounts of organic matter), reduc- tion in productivity due to land use legacy effects and direct and indirect effects of prairie dogs. These potential factors are consistent in both explaining the mixed grass prairie’s Fig. 3 Soil carbon (mean ± SE) by the presence of Andropogon gerar- dii (big bluestem) (mean ± standard error). A. gerardii was present in 73 of the 141 transects exact mechanistic cause of the relationship, the link that we discovered between soil C and species richness implies that 1 3 Oecologia (2021) 196:1153–1166 1162 higher percentage of soil water is lost to surface evapora- tion (Sala et al. 1988). However, spring and summer grow- ing season precipitation almost always exceeds 34 cm at our sites (https://psl.noaa.gov/boulder/Boulder.mm.precip. html). Given an average rainfall input of ~ 50 cm in this area, increased clay should support increased nutrient storage and availability that would result in greater plant species rich- ness, leading to greater C deposition in the soil. This is not the case, and we speculate that the increased surface clays, along with the increased bare surfaces found in the mixed grass sites, result in greater surface evaporation and water runoff characteristic of shale-derived soils (Branson et al. 1965), and therefore reduced soil water storage. Correlation among species richness, grassland type, soil texture, and prairie dogs At reduced precipitation levels (i.e., below 34 cm of annual precipitation), high clay content in soils can have a negative effect on plant production because a relatively Our best model explained 43% of the variation in soil C, leaving much of the landscape variation of soil C unex- plained. Land uses, such as fire, grazing and tilling history may account for variation in soil C in our grasslands, as can heterogeneity in soil chemical properties, parent mate- rial, landscape position, presence of other functional groups (e.g., legumes), and plant productivity (Conant et al. 2017; Jackson et al. 2017; Rasmussen et al. 2018). 3 3 1163 Oecologia (2021) 196:1153–1166 Positive soil C–richness relationships were observed within various functional groups, though these were often weaker than relationships between soil C and total species richness, with the one exception of the C4 dominant, A. gerardii. The strong soil C–richness relationship for native perennial graminoids reflects the diverse niches of mem- ber species. When richness is high, the mix of grasses, rushes, and Cyperaceae species in this group can likely exhibit complementarity in both time (e.g., early-season and late-season species; varying rates of litter decompo- sition among C3 and C4 species) and space (e.g., micro- habitat variation in soil moisture availability; separation by rooting depth), thus enhancing carbon accumulation, and reflecting the major importance of graminoids to soil C pools (February et al. 2020). native species richness benefits introduced species richness (Lonsdale 1999). In our case, we expected that exotic spe- cies richness would be favored in ruderal areas with distur- bance, high surface clays, and poor soils, and competitively excluded from resource-rich, intact tallgrass prairies. Our finding means that exotic species make only minor contribu- tions to soil C in these grasslands. Conclusion Supplementary Information The online version contains supplemen- tary material available at https://doi.org/10.1007/s00442-021-04992-x. Acknowledgements Lynn Riedel and Megan Bowes collected the veg- etation data and Marianne Giollito created the monitoring design and assisted with monitoring. We thank Sasha Abcassis, Bryan Sechler, and Claire Gentry for helping obtain and process soil samples. Dr. Jim Ippolito, CSU Soil, Water, and Plant testing Lab, provided access to equipment to measure C. We also thank Julie Larson for reviewing an earlier draft of this paper. Author contribution statement TS and TH developed the research in consultation with ALL and BA. TS and TH collected soil samples and ALL assembled previously collected vegetation data. TH conducted lab analyses, and BA and TS conducted statistical analyses. TS wrote a draft of the manuscript that was then edited by all authors. Funding This research was supported by the City of Boulder and the University of Colorado, Boulder. Data availability The data are available as ESM2. Code availability The code used during the current study is available from the corresponding author on reasonable request. Conclusion Surveys such as ours argue that careful analysis of landscape variables can deepen our understanding of the relationship between ecosystem services and changing plant species richness. The similarly strong relationship between soil C and A. gerardii suggests that big bluestem, like other C4 grasses, contributes disproportionately to soil C (O’Brien et al 2010; Fornara and Tilman 2009). For A. gerardii in our study area, late-season physiological activity during hot, dry conditions that trigger dormancy in other plants, may extend the period of soil C accrual, while plastic- ity in rooting characteristics may facilitate root exploita- tion of microhabitats (Weaver and Darland 1949). High water use efficiency of this species can ensure high rates of carbon gain (Turner et al. 1995), even when these tall- grass communities experience seasonal water stress and periodic drought. These traits, along with tall stature and relatively high above-ground productivity, relatively slower turnover and high C:N ratios of shoot, fine root and coarse below-ground structures (Wedin and Tilman 1990; Craine et al. 2003) and significantly higher fine root biomass when compared to other tallgrass species (Craine et al. 2003) suggest that A. gerardii contributes high C:N carbon sources through both above- and below-ground parts and has a suite of functional traits favoring high C storage. This relationship was not merely an artifact of the strong correlation between A. gerardii presence and species diversity or its utilization of the C4 photosynthetic system. The positive effect of A. gerardii on soil C may best be observed in grassland sites with a long history of A. gerardii occupation such as ours, as compared to younger, restored agricultural sites where the contribution of C4 species to soil C lags behind C3 species more abun- dant early in succession (Mahaney et al. 2008; Hernández et al. 2013). These relationships provide general support that some plant groups and some species appear more important than others in the C storage process. Maintaining soil organic matter in a semi-arid environ- ment by way of plant conservation management practices appears to increase C, nutrient storage and release and increase water holding capacity, but high surface clay con- tent and disturbance by prairie dogs can provide a major challenge to maintaining both soil C and species richness. However, exotic species richness was lower and less vari- able than native species richness; as a result, we may have underestimated the contributions of exotic species to soil C. References Adler PB, Seabloom EW, Borer ET, Hillebrand H, Hautier Y, Hector A et al (2011) Productivity is a poor predictor of plant species richness. Science 333:1750–1753. https://doi.org/10.1126/scien ce.1204498f Fraser LH, Pither J, Jentsch A, Sternberg M, Zobel M, Askariza- deh, et al (2015) Worldwide evidence of a unimodal relation- ship between productivity and plant species richness. Science 349:302–305. https://doi.org/10.1126/science.aab3916fi Ampleman MD, Crawford KM, Fike DA (2014) Differential soil organic carbon storage at forb- and grass-dominated plant com- munities, 33 years after tallgrass prairie restoration. Plant Soil 374:899–913. https://doi.org/10.1007/s11104-013-1916-5 Gibson DJ, Hulbert LC (1987) Effects of fire, topography and year-to- year climatic variation on species composition in tallgrass prairie. Vegetatio 72:175–185. https://doi.org/10.1007/BF00039839 Hassink J (1997) The capacity of soils to preserve organic C and N by their associations with clay and silt particles. Plant Soil 191:77– 87. https://doi.org/10.1023/A:1004213929699 Baldock JA, Skjemstad JO (2000) Role of the soil matrix and min- erals in protecting natural organic materials against biologi- cal attack. Org Geochem 31:697–710. https://doi.org/10.1016/ S0146-6380(00)00049-8f Hector A, Hautier Y, Saner P, Wacker R, Bagchi R, Joshi M et al (2010) General stabilizing effects of plant diversity on grassland produc- tivity through population asynchrony and overyielding. Ecology 91:2213–2220. https://doi.org/10.1890/09-1162.1 Beals SC, Hartley LM, Prevéy JS, Seastedt TR (2014) The effects of black-tailed prairie dogs on plant communities within a com- plex urban landscape: an ecological surprise? Ecology 95:1349– 1359. https://doi.org/10.1890/13-0984.1 Hernández DL, Esch EH, Alster CJ, McKone MJ, Camill P (2013) Rapid accumulation of soil carbon and nitrogen in a prairie resto- ration chronosequence. Soil Sci Soc Am J 77:2029–2038. https:// doi.org/10.2136/sssaj2012.0403fl Bock JH, Bock CE (1998) Tallgrass prairie: remnants and relicts. Great Plains Res 8:213–230 Branson FA, Miller RF, McQueen IS (1965) Plant communities and soil moisture relationships near Denver, Colorado. Ecology 46:311–319. https://doi.org/10.2307/1936334 Hoffland E, Kuyper TW, Comans RMJ, Creamer RE (2020) Eco-func- tionality of organic matter in soils. Plant Soil 455:1–22. https:// doi.org/10.1007/s11104-020-04651-9 Briggs JM, Knapp AK (1995) Interannual variability in primary pro- duction in tallgrass prairie: climate, soil moisture, topographic position, and fire as determinants of aboveground biomass. Amer J Bot 82:1024–1030. https://doi.org/10.2307/2446232 Hook PB, Burke I (2000) Biogeochemistry in a shortgrass landscape: control by topography, soil texture, and microclimate. Ecol- ogy 81:2686–2703. https://doi.org/10.1890/0012-9658(2000) 081[2686:BIASLC]2.0.CO;2 Buckner DL (1985) Point-intercept sampling in revegetation studies: maximizing objectivity and repeatability. Consent for publication Not required for this study. Conant RT, Cerri CEP, Osborne BB, Paustian K (2017) Grassland management impacts on soil carbon stocks: a new synthesis. Ecol Appl 27:662–668. https://doi.org/10.1002/eap.1473 Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, 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/. Cong W-F, van Ruijven J, Mommer L, De Deyn GB, Berendse F, Hof- fland E (2014) Plant species richness promotes soil carbon and nitrogen stocks in grasslands without legumes. J Ecol 102:1163– 1170. https://doi.org/10.1111/1365-2745.12280 g g g 1170. https://doi.org/10.1111/1365-2745.12280 Craine JM, Wedin DA, Chapin FS, Reich PB (2003) Relationship between the structure of root systems and resource use for 11 North American grassland plants. Plant Ecol 165:85–100. https:// doi.org/10.1023/A:1021414615001 El Moujahid L, Le Roux X, Michalet S, Bellvert F, Weigelt A, Poly F (2017) Effect of plant diversity on the diversity of soil organic compounds. PLoS One. https://doi.org/10.1371/journal.pone. 0170494 February E, Pausch J, Higgins SI (2020) Major contribution of grass roots to soil carbon pools and CO2 fluxes in a mesic savanna. Plant Soil 454:207–215. https://doi.org/10.1007/s11104-020-04649-3l Fornara DA, Tilman D (2008) Plant functional composition influences rates of soil carbon and nitrogen accumulation. J Ecol 96:314– 322. https://doi.org/10.1111/j.1365-2745.2007.01345.x Declarations Conflict of interest The authors declare that they have no conflict of interest. Soil C was much more strongly related to native species richness than to exotic species richness, and, in turn, native species richness was not related to exotic species richness. These relationships are potentially at odds with the litera- ture (e.g., Stohlgren et al. 2003). Normally, what benefits Ethical approval Not required for this study. Ethical approval Not required for this study. Ethical approval Not required for this study. Consent to participate Not required for this study. Consent to participate Not required for this study. 3 Oecologia (2021) 196:1153–1166 1164 References In: Proceedings 2nd Annual Meeting American Society Mining and Reclamation 110–113 Hopkins-Arnold AA (1998) Response of Colorado tallgrass prairie to fire, winter grazing, and nitrogen manipulations PhD Dissertation. University of Colorado Boulder Isbell F, Calcagno V, Hector A, Connolly J, Harpole WS, Reich PB et al (2011) High plant diversity is needed to maintain ecosystem ser- vices. Nature 477:199–202. https://doi.org/10.1038/nature10282 Burke ID, Yonker CM, Parton WJ, Cole CV, Flach K, Schimell DS (1989) Texture, climate, and cultivation effects on soil organic-matter content in US grassland soils. Soil Sci Soc Am J 53:800–805. https://doi.org/10.2136/sssaj1989.0361599500 5300030029x Ives AR, Carpenter SR (2007) Stability and diversity of ecosystems. Science 317:58–62. https://doi.org/10.1126/science.1133258 Jackson RB, Lajtha K, Crow SE, Hugelius G, Kramer MG, Pineiro G (2017) The Ecology of soil carbon: pools, vulnerabilities, and biotic and abiotic controls. Annu Rev Ecol Evol Syst 48:419–445. https://doi.org/10.1146/annurev-ecolsys-112414-054234 Cardinale BJ, Matulich KL, Hooper DU, Byrnes JE, Duffy E, Gamfeldt L et al (2011) The functional role of producer diversity in ecosys- tems. Am J Bot 98:572–592. https://doi.org/10.3732/ajb.1000364 s://doi.org/10.1146/annurev-ecolsys-112414-054234 Chen S, Wang W, Xu W, Wang Y, Wan H, Chen D et al (2018) Plant diversity enhances productivity and soil carbon storage. Proc Natl Acad Sci USA 115:4027–4032. https://doi.org/10.1073/pnas. 1700298114fi Jobbágy EG, Jackson RB (2000) The vertical distribution of soil organic carbon and its relation to climate and vegetation. Ecol Appl 10:423–436. https://doi.org/10.1890/1051-0761(2000) 010[0423:TVDOSO]2.0.CO;2f Johnson WC, Collinge SK (2004) Landscape effects on black-tailed prairie dog colonies. Biol Cons 115:487–497. https://doi.org/10. 1016/S0006-3207(03)00165-4 Collins SL, Calabrese LB (2012) Effects of fire, grazing and topo- graphic variation on vegetation structure in tallgrass prairie. J Veg Sci 23:563–575. https://doi.org/10.1111/j.1654-1103.2011. 01369.x 1 3 Oecologia (2021) 196:1153–1166 1165 Kotliar NB, Baker BW, Whicker AD, Plumb G (1999) A critical review of assumptions about the prairie dog as a keystone species. Envi- ron Manag 24:177–192. https://doi.org/10.1007/s002679900225 Schimel DS, Braswell BH, Holland EA, McKeown R, Ojima DS, Painter TH et al (1994) Climatic, edaphic and biotic controls over storage and turnover of carbon in soils. Global Biogeochem Cycles 8:279–293. https://doi.org/10.1029/94GB00993 Lal R (2004) Soil carbon sequestration to mitigate climate change. Geoderma 123:1–22. https://doi.org/10.1016/j.geoderma.2004. 01.032fi Seastedt TR, Hartley L, Nippert J (2013) Ecosystem transformations along the Colorado front range: prairie dog interactions with mul- tiple components of global environmental change. In: Hobbs R, Higgs E, Hall C (eds) Novel ecosystems: intervening in the new ecological world order. Wiley, Chichester, pp 142–149 Lange M, Eisenhauer N, Sierra CA, Bessler H, Engels C, Griffiths R et al (2015) Plant diversity increases soil microbial activity and soil carbon storage. Nat Commun 6:6707. https://doi.org/10.1038/ ncomms7707 Sherrod LA, Dunn G, Peterson GA, Kolberg RL (2002) Inorganic car- bon analysis by modified pressure calcimeter method. J Soil Sci Soc Am J 66:299–305. https://doi.org/10.2136/sssaj2002.2990 Livingston RB (1952) Relict true prairie communities in central Colo- rado. Ecology 33:72–86. https://doi.org/10.2307/1931253 Steinbeiss S, Beßler H, Engels C, Temperton VM, Buchmann N, Roscher C et al (2008) Plant diversity positively affects short-term soil carbon storage in experimental grasslands. Glob Change Biol 14:2937–2949. https://doi.org/10.1111/j.1365-2486.2008.01697.x Lonsdale WM (1999) Global patterns of plant invasions and the con- cept of invasibility. Ecology 80:522–1536. https://doi.org/10. 2307/176544 Mahaney WM, Smemo KA, Gross KL (2008) Impacts of C4 grass introductions on soil carbon and nitrogen cycling in C3-dominated successional systems. Oecologia 157:295–305. https://doi.org/10. 1007/s00442-008-1063-5 Stevens DL, Olson AR (2004) Spatially balanced sampling of natural resources. J Am Stat Assoc 99:262–278. s://doi.org/10.1146/annurev-ecolsys-112414-054234 https://doi.org/10.1198/ 016214504000000250 Manning P, Loos J, Barnes AD, Batary P, Bianchi F, Buchmann N et al (2019) Transferring biodiversity-ecosystem function research to the management of ‘real-world’ ecosystems. In: Eisenhauer N (ed) Advances in ecological research. Academic Press, NY, pp 323–356 Stohlgren TJ, Barnett DT, Kartesz JT (2003) The rich get richer: pat- terns of plant invasions in the United States. Front Ecol Environ 1:11–14. https://doi.org/10.1890/1540-9295(2003)001[0011: TRGRPO]2.0.CO;2 Texas A and M University (2005) On-site wastewater treatment sys- tems: soil particle analysis. Procedure Technical Publication B-6175. https://oaktrust.library.tamu.edu/handle/19691/87299. Accessed 10 Aug 2020 Martinez-Estévez L, Balvanera P, Pacheco J, Ceballos G (2013) Prairie dog decline reduces the supply of ecosystem services and leads to desertification of semiarid grasslands. PLoS One 8:e75229. https://doi.org/10.1371/journal.pone.0075229 Tilman D (1999) The ecological consequences of changes in biodi- versity: a search for general principles. Ecology 80:1455–1474. https://doi.org/10.1890/0012-9658(1999)080[1455:TECOCI]2.0. CO;2 Minasny B, Malone BP, McBratney AB, Angers DA, Arrouays D, Chambers A et al (2017) Soil carbon 4 per mille. Geoderma 292:59–86. https://doi.org/10.1016/j.geoderma.2017.01.002 Moir WH (1969) Steppe communities in the foothills of the Colorado Front Range and their relative productivities. Amer Midl Nat 81:331–340. https://doi.org/10.2307/2423974 Tilman D, Reich PB, Isbell F (2012) Biodiversity impacts ecosystem productivity as much as resources disturbance or herbivory. Proc Nat Acad Sci 109:10394–10397. https://doi.org/10.1073/pnas. 1208240109 p g NOAA (2020) Boulder monthly precipitation. https://psl.noaa.gov/ boulder/Boulder.mm.precip.html. Accessed 12 Aug 2020 Turnbull LA, Isbell F, Purves DW, Loreau M, Hector A (2016) Under- standing the value of plant diversity for ecosystem functioning through niche theory. Proc R Soc B 283:20160536. https://doi. org/10.1098/rspb.2016.0536 O’Brien SL, Jastrow JD, Grimley DA, Gonzalez-Meller MA (2010) Moisture and vegetation controls on decadal-scale accrual of soil organic carbon and total nitrogen in restored grasslands. Glob Change Biol 16:2573–2588. https://doi.org/10.1111/j.1365-2486. 2009.02114.x Turner CL, Kneisler JR, Knapp AK (1995) Comparative gas exchange and nitrogen responses of the dominant C4 grass Andropogon gerardii and five C3 forbs to fire and topographic position in tallgrass prairie during a wet year. Int J Plant Sci 156:216–226. https://doi.org/10.1086/297243 O’Keefe K, Nippert JB, McCulloh KA (2019) Plant water uptake along a diversity gradient provides evidence for complementarity in hydrological niches. Oikos 128:1748–1760. https://doi.org/10. 1111/oik.06529 van der Plas F (2019) Biodiversity and ecosystem functioning in natu- rally assembled communities. Biol Rev 94:1220–1245. https:// doi.org/10.1111/brv.12499 OSMP (2010) Grassland ecosystem management plan. https://bould ercolorado.gov/osmp/grassland-ecosystem-management-plan. Yang Y, Tilman D, Furey G, Lehman C (2019) Soil carbon seques- tration accelerated by restoration of grassland biodiversity. Nat Commun 10:718. https://doi.org/10.1038/s41467-019-08636-w Zhou X, Wu W, Niu K, Du G (2019) Realistic loss of plant species diversity decreases soil quality in a Tibetan alpine meadow. Agri Ecosys Environ 279:25–32. https://doi.org/10.1016/j.agee.2019. 03.019 buffering. J Geophys Res Biogeosci. https://doi.org/10.1029/2019J G005158 s://doi.org/10.1146/annurev-ecolsys-112414-054234 Accessed 12 Aug 2020i Vermeulen S, Bossio D, Lehmann J, Luu P, Paustian K, Webb C et al (2019) A global agenda for collective action on soil carbon. Nature Sustainability 2:2–4. https://doi.org/10.1038/s41893-018-0212-z Percival HJ, Parfitt RL, Scott NA (2000) Factors controlling soil carbon levels in New Zealand grasslands—is clay content important? Soil Sci Soc Am J 64:1623–1630. https://doi.org/10.2136/sssaj2000. 6451623x Vestal AG (1914) Prairie vegetation of a mountain-front area in Colo- rado. Bot Gaz 58:377–400 Peres-Neto PR, Legendre P, Dray S, Borcard D (2006) Variation par- titioning of species data matrices: estimation and comparison of fractions. Ecology 87:2614–2625. https://doi.org/10.1890/0012- 9658(2006)87[2614:VPOSDM]2.0.CO;2 Weaver JE, Darland RW (1949) Soil-root relationships of certain native grasses in various soil types. Ecol Monogr 19:303–338. https:// doi.org/10.2307/1943273f Wedin DA, Tilman D (1990) Species effects on nitrogen cycling: a test with perennial grasses. Oecologia 84:433–441. https://doi.org/10. 1007/BF00328157 Rasmussen C, Heckman K, Wieder WR, Keiluweith M, Lawrence CR, Berhe AA et al (2018) Beyond clay: towards an improved set of variables for predicting soil organic matter content. Biogeochem- istry 137:297–306. https://doi.org/10.1007/s10533-018-0424-3 Weisser WW, Roscher C, Meyer ST, Ebeling A, Luo G, Allan E et al (2017) Biodiversity effects on ecosystem functioning in a 15-year grassland experiment: patterns, mechanisms, and open questions. Basic Appl Ecol 23:1–73. https://doi.org/10.1016/j.baae.2017.06. 002 Sala OE, Parton WJ, Joyce LA, Lauenroth WK (1988) Primary produc- tion of the central grassland region of the United States. Ecology 69:40–45. https://doi.org/10.2307/1943158 Werner WJ, Sanderman J, Melillo JM (2020) Decreased soil organic matter in a long-term soil warming experiment lowers soil water holding capacity and affects soil thermal and hydrological Schimel DS, Stillwell MA, Woodmansee RG (1985) Biogeochemistry of C N and P in a soil catena of the shortgrass steppe. Ecology 66:276–282. https://doi.org/10.2307/1941328 1 3 1166 Oecologia (2021) 196:1153–1166 1 3 3
https://openalex.org/W2103758678	https://ehoonline.biomedcentral.com/counter/pdf/10.1186/2162-3619-2-9	English	null	Bone marrow non-mesenchymal mononuclear cells induce functional differentiation of neuroblastoma cells	Experimental hematology & oncology	2,013	cc-by	2,576	© 2013 Phruksaniyom 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. Experimental Hematology & Oncology Experimental Hematology & Oncology Phruksaniyom et al. Experimental Hematology & Oncology 2013, 2:9 http://www.ehoonline.org/content/2/1/9 Open Access Abstract Less is known about the non-mesenchymal mononuclear cell fraction of human bone marrow on functional adaptation of neuroblastoma cells. Using immunocytochemistry, we showed that bone-marrow mononuclear cell (BMMC)-conditioned medium can induce tyrosine hydroxylase expression in neuroblastoma cells, which is similar to the effect of retinoic acid. Using quantitative RT-PCR, we showed that NGF, CNTF, and BDNF mRNAs were detected in unfractionated BMMC populations from all human donors at different expression levels. Our results suggest that cells of the non-mesenchymal mononuclear cell fraction can induce functional adaptation of neuroblastoma cells, probably via their secreted trophic factors. Keywords: Bone-marrow mononuclear cells, Neuroblastoma cells, Tyrosine hydroxylase, Trophic factors, Neuronal differentiation Keywords: Bone-marrow mononuclear cells, Neuroblastoma cells, Tyrosine hydroxylase, Trophic factors, Neuronal differentiation -marrow mononuclear cells, Neuroblastoma cells, Tyrosine hydroxylase, Trophic factors, Neuronal Bone marrow non-mesenchymal mononuclear cells induce functional differentiation of neuroblastoma cells Chareerut Phruksaniyom1, Permphan Dharmasaroja1* and Surapol Issaragrisil2 Chareerut Phruksaniyom1, Permphan Dharmasaroja1* and Surapol Issaragrisil2 * Correspondence: permphan.dha@mahidol.ac.th 1Department of Anatomy, Faculty of Science, Mahidol University, Rama VI Road, Ratchathewi, Bangkok 10400, Thailand Full list of author information is available at the end of the article © 2013 Phruksaniyom 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. To the editor of tyrosine hydroxylase (TH) in neuroblastoma cells [7]. Using a co-culture method, human MSCs promoted the survival and neuritogenesis of neuroblastoma cells, similar to that of ATRA [8]. Less is known about the mononuclear cell fraction of human bone marrow on functional adaptation of neuroblastoma cells. Many studies investigating the possible therapeutic role of bone marrow-derived stem cells (BMDCs) used a specific subpopulation: the bone marrow mesenchymal stromal cells (MSCs) obtained after several weeks in cul- tures [1], or the mononuclear fraction (BMMC; bone marrow mononuclear cells) obtained immediately after aspiration. Cells of the hematopoietic stem cell fraction, when transplanted into lesions of a developing spinal cord in a chicken embryo, can differentiate into neurons [2]. The capacity of BMMCs to generate neural cells is poorly characterized. Several studies indicate an over- lap in the molecular programs for hematopoiesis and neuropoiesis in mice [3,4]. Primary CD34+ human hematopoietic stem cells (HSCs) have been shown to express mRNA for a number of proteins that are used by neurons [5]. We hypothesize that cells of the BMMC fraction can induce functional adaptation of neuroblastoma cells, probably via their secreted trophic factors. First, we eval- uated the effect of cells of the human BMMC fraction on the expression of TH protein in neuroblastoma cells by culturing SH-SY5Y cells in BMMC-conditioned medium. Human bone marrow samples were aspirated from healthy donors after obtaining informed consent and ethical approval by the Siriraj Ethics Committee of Siriraj Hospital. After isolation, the mononuclear cells were plated at a concentration of 1.5 × 105 cells/ml onto flasks containing low glucose-DMEM supplemented with 10% fetal bovine serum (FBS). After incubation for 72 h, non-adherent cells were collected for culture in MEM/F12 medium supplemented with 10% FBS. After an additional 24 h, non-adherent cells were collected again and cultured in the medium as described above. After 24 h, non-adherent cell-conditioned medium was collected for further experiment. SH-SY5Y cells were Evidence has indicated that human SH-SY5Y neuro- blastoma cells changed into neuron-like phenotypes with reduced proliferation by all-trans retinoic acid (ATRA) [6], and treatment with RA increased protein expression Page 2 of 3 Phruksaniyom et al. Experimental Hematology & Oncology 2013, 2:9 http://www.ehoonline.org/content/2/1/9 Phruksaniyom et al. Experimental Hematology & Oncology 2013, 2:9 http://www.ehoonline.org/content/2/1/9 Figure 1 Immunostaining micrographs using a confocal microscope demonstrate localization of the tyrosine hydroxylase (TH). To the editor Cells were stained with polyclonal antibody against TH and immunostained with Alexa 488-conjugated secondary antibody (green) and nuclei were stained with DAPI (blue). CM, BMMC-conditioned medium; RA, retinoic acid. Figure 1 Immunostaining micrographs using a confocal microscope demonstrate localization of the tyrosine hydroxylase (TH). Cells were stained with polyclonal antibody against TH and immunostained with Alexa 488-conjugated secondary antibody (green) and nuclei were stained with DAPI (blue). CM, BMMC-conditioned medium; RA, retinoic acid. TCATGGATGG; BDNF: ACTCTGGAGAGCGTGAA TGG and ATCCAACAGCTCTTCTATCACG; β-actin: CATGTACGTTGCTATCCAGGC and CTCCTTAATG TCACGCACGAT. The results showed that NGF, CNTF, and BDNF mRNAs were detected in unfractionated BMMC populations from all donors at different expres- sion levels (Figure 2). Their expression levels were rather low, suggesting that not all but only some populations of the cells expressed these trophic factors. TCATGGATGG; BDNF: ACTCTGGAGAGCGTGAA TGG and ATCCAACAGCTCTTCTATCACG; β-actin: CATGTACGTTGCTATCCAGGC and CTCCTTAATG TCACGCACGAT. The results showed that NGF, CNTF, and BDNF mRNAs were detected in unfractionated BMMC populations from all donors at different expres- sion levels (Figure 2). Their expression levels were rather low, suggesting that not all but only some populations of the cells expressed these trophic factors. TCATGGATGG; BDNF: ACTCTGGAGAGCGTGAA TGG and ATCCAACAGCTCTTCTATCACG; β-actin: CATGTACGTTGCTATCCAGGC and CTCCTTAATG TCACGCACGAT. The results showed that NGF, CNTF, and BDNF mRNAs were detected in unfractionated BMMC populations from all donors at different expres- sion levels (Figure 2). Their expression levels were rather low, suggesting that not all but only some populations of the cells expressed these trophic factors. then seeded onto plates containing BMMC-conditioned medium at an initial density of 1 × 104 cells/ml, and cul- tures were maintained for 7 days prior to analysis of TH expression using immunocytochemistry. As a positive control, cells were treated with 10 μM ATRA for 5 days to induce neuronal differentiation. With estimated con- centration of 35 ± 2 × 104 cells/ml for staining, the re- sults showed that BMMC-conditioned medium can induce TH protein expression in neuroblastoma cells (Figure 1C), which is similar to the effect of ATRA (Figure 1B). Untreated cells did not express TH or expressed at very low levels (Figure 1A). Figure 2 Quantitative expression of NGF, CNTF, and BDNF mRNAs in cells of the BMMC fraction obtained from three healthy donors. 20 ng cDNA was used as PCR template. Real-time PCR mixture was prepared with KAPA SYBRW FAST qPCR master mix. β-actin was used as the reference gene. Acknowledgments This work was supported by a grant from Faculty of Science, Mahidol University to PD. Authors’ contributions PD was involved in the design and execution of the experiments, performed data and statistical analyzes, wrote the manuscript and contributed to overall experiment design. CP conducted immunocytochemistry and quantitative RT-PCR. SI provided bone marrow samples and submitted the project for ethical approval. All authors have read and approved the final manuscript. doi:10.1186/2162-3619-2-9 Cite this article as: Phruksaniyom et al.: Bone marrow non-mesenchymal mononuclear cells induce functional differentiation of neuroblastoma cells. Experimental Hematology & Oncology 2013 2:9. Author details 1 1Department of Anatomy, Faculty of Science, Mahidol University, Rama VI Road, Ratchathewi, Bangkok 10400, Thailand. 2Division of Hematology, Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. Received: 14 January 2013 Accepted: 16 March 2013 Published: 3 April 2013 Phruksaniyom et al. Experimental Hematology & Oncology 2013, 2:9 http://www.ehoonline.org/content/2/1/9 Expression of TH, the enzyme involved in the first step of the biosynthesis pathway of dopamine and nor- adrenaline, in SH-SY5Y cells by BMMC-conditioned media suggests the functional differentiation of the cells. ATRA can also induce responsiveness to BDNF in SH- SY5Y cells [12]. Our results imply that expression and secretion of BDNF from cells in the mononuclear frac- tion may explain the similar effects of RA and BMMC- conditioned media. BMDCs constitutively synthesize and secrete NGF, BDNF, and CNTF. Multiple cell types, however, are present in the BMMC fraction. Primary CD34+ human HSCs express mRNA for a number of proteins, including receptors for trophic factors and other mediators involved in the development of neurons [5]. Most of the CD34+ cells are progenitors for myeloid and lymphoid lineages, which express some trophic factors, such as CNTF [11], that were also observed in our quantitative RT-PCR results. Further studies are required to quantify the contribution of trophic factors to BMMC-induced effects on functional adaptation of neuroblastoma cells, which could have clinical relevance in treatment of neuroblastoma. 6. Miloso M, Villa D, Crimi M, Galbiati S, Donzelli E, Nicolini G, et al: Retinoic acid-induced neuritogenesis of human neuroblastoma SH-SY5Y cells is ERK independent and PKC dependent. J Neurosci Res 2004, 75:241–252. 7. Kume T, Kawato Y, Osakada F, Izumi Y, Katsuki H, Nakagawa T, et al: Dibutyryl cyclic AMP induces differentiation of human neuroblastoma SH-SY5Y cells into a noradrenergic phenotype. Neurosci Lett 2008, 443:199–203. 8. Crigler L, Robey RC, Asawachaicharn A, Gaupp D, Phinney DG: Human mesenchymal stem cell subpopulations express a variety of neuro- regulatory molecules and promote neuronal cell survival and neuritogenesis. Exp Neurol 2006, 198:54–64. 9. Caroleo MC, Costa N, Tirassa P, Aloe L: Nerve growth factor produced by activated human monocytes/macrophages is severely affected by ethanol. Alcohol 2004, 34:107–114. 10. Kerschensteiner M, Gallmeier E, Behrens L, Leal VV, Misgeld T, Klinkert WE, et al: Activated human T cells, B cells, and monocytes produce brain- derived neurotrophic factor in vitro and in inflammatory brain lesions: a neuroprotective role of inflammation? J Exp Med 1999, 189:865–870. 10. Kerschensteiner M, Gallmeier E, Behrens L, Leal VV, Misgeld T, Klinkert WE, et al: Activated human T cells, B cells, and monocytes produce brain- derived neurotrophic factor in vitro and in inflammatory brain lesions: a neuroprotective role of inflammation? J Exp Med 1999, 189:865–870. 10. Phruksaniyom et al. Experimental Hematology & Oncology 2013, 2:9 http://www.ehoonline.org/content/2/1/9 Kerschensteiner M, Gallmeier E, Behrens L, Leal VV, Misgeld T, Klinkert WE, et al: Activated human T cells, B cells, and monocytes produce brain- derived neurotrophic factor in vitro and in inflammatory brain lesions: a neuroprotective role of inflammation? J Exp Med 1999, 189:865–870. 11. Su AI, Wiltshire T, Batalov S, Lapp H, Ching KA, Block D, et al: A gene atlas of the mouse and human protein-encoding transcriptomes. Proc Natl Acad Sci USA 2004, 101:6062–6067. 11. Su AI, Wiltshire T, Batalov S, Lapp H, Ching KA, Block D, et al: A gene atlas of the mouse and human protein-encoding transcriptomes. Proc Natl Acad Sci USA 2004, 101:6062–6067. 12. Edsjo A, Lavenius E, Nilsson H, Hoehner JC, Simonsson P, Culp LA, et al: Expression of trkB in human neuroblastoma in relation to MYCN expression and retinoic acid treatment. Lab Invest 2003, 83:813–823. 12. Edsjo A, Lavenius E, Nilsson H, Hoehner JC, Simonsson P, Culp LA, et al: Expression of trkB in human neuroblastoma in relation to MYCN expression and retinoic acid treatment. Lab Invest 2003, 83:813–823. 12. Edsjo A, Lavenius E, Nilsson H, Hoehner JC, Simonsson P, Culp LA, et al: Expression of trkB in human neuroblastoma in relation to MYCN expression and retinoic acid treatment. Lab Invest 2003, 83:813–823. doi:10.1186/2162-3619-2-9 Cite this article as: Phruksaniyom et al.: Bone marrow non-mesenchymal mononuclear cells induce functional differentiation of neuroblastoma cells. Experimental Hematology & Oncology 2013 2:9. Competing interests The authors declare that they have no competing interests. To the editor Each bar represents mean ± SD from triplicate of each sample. The expression levels were scaled relative to the lowest unscaled expression level for the same gene as the sample of interest. i f d Further, we evaluated whether cells of the BMMC frac- tion expressed any trophic factors that could contribute to biochemical adaptation of neuroblastoma cells. Monocytes in the human bone marrow have been shown to produce nerve growth factor (NGF), which plays an important role in neuronal plasticity, maturation, and survival [9]. Human monocytes, T cells, and B cells can secrete brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family that regulates the differentiation and survival of various neuronal popula- tions [10]. Ciliary neurotrophic factor (CNTF), another factor involved in neurogenesis, is also expressed in monocytes, myeloid cells, lymphoblasts, T cells and B cells [11]. Here, we evaluated the mRNA expression of NGF, BDNF, and CNTF in BMMCs using quantitative RT-PCR. The sequences of the sense and antisense primers are as follows: NGF: TAAAAAGCGGCGACT CCGTT and ATTCGCCCCTGTGGAAGATG; CNTF: ACCAGCAGGTGCATTTTACC and GAAACGAAGG Figure 2 Quantitative expression of NGF, CNTF, and BDNF mRNAs in cells of the BMMC fraction obtained from three healthy donors. 20 ng cDNA was used as PCR template. Real-time PCR mixture was prepared with KAPA SYBRW FAST qPCR master mix. β-actin was used as the reference gene. Each bar represents mean ± SD from triplicate of each sample. The expression levels were scaled relative to the lowest unscaled expression level for the same gene as the sample of interest. Figure 2 Quantitative expression of NGF, CNTF, and BDNF mRNAs in cells of the BMMC fraction obtained from three healthy donors. 20 ng cDNA was used as PCR template. Real-time PCR mixture was prepared with KAPA SYBRW FAST qPCR master mix. β-actin was used as the reference gene. Each bar represents mean ± SD from triplicate of each sample. The expression levels were scaled relative to the lowest unscaled expression level for the same gene as the sample of interest. Page 3 of 3 Page 3 of 3 Phruksaniyom et al. Experimental Hematology & Oncology 2013, 2:9 http://www.ehoonline.org/content/2/1/9 References 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 1. Wang S, Qu X, Zhao RC: Clinical applications of mesenchymal stem cells. J Hematol Oncol 2012, 5:19. 1. Wang S, Qu X, Zhao RC: Clinical applications of mesenchymal stem cells. J Hematol Oncol 2012, 5:19. 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: 2. Sigurjonsson OE, Perreault MC, Egeland T, Glover JC: Adult human hematopoietic stem cells produce neurons efficiently in the regenerating chicken embryo spinal cord. Proc Natl Acad Sci USA 2005, 102:5227–5232. • Convenient online submission 3. Terskikh AV, Easterday MC, Li L, Hood L, Kornblum HI, Geschwind DH, et al: From hematopoiesis to neuropoiesis: evidence of overlapping genetic programs. Proc Natl Acad Sci USA 2001, 98:7934–7939. g 4. Goolsby J, Marty MC, Heletz D, Chiappelli J, Tashko G, Yarnell D, et al: Hematopoietic progenitors express neural genes. Proc Natl Acad Sci USA 2003, 100:14926–14931. 5. Steidl U, Bork S, Schaub S, Selbach O, Seres J, Aivado M, et al: Primary human CD34+ hematopoietic stem and progenitor cells express functionally active receptors of neuromediators. Blood 2004, 104:81–88.
https://openalex.org/W2140740550	https://stemcellres.biomedcentral.com/counter/pdf/10.1186/scrt147	English	null	Spatiotemporal evolution of early innate immune responses triggered by neural stem cell grafting	Stem cell research & therapy	2,012	cc-by	9,211	© 2012 Reekmans 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. RESEARCH Open Access Abstract Introduction: Transplantation of neural stem cells (NSCs) is increasingly suggested to become part of future therapeutic approaches to improve functional outcome of various central nervous system disorders. However, recently it has become clear that only a small fraction of grafted NSCs display long-term survival in the (injured) adult mouse brain. Given the clinical invasiveness of NSC grafting into brain tissue, profound characterisation and understanding of early post-transplantation events is imperative to claim safety and efficacy of cell-based interventions. Methods: Here, we applied in vivo bioluminescence imaging (BLI) and post-mortem quantitative histological analysis to determine the localisation and survival of grafted NSCs at early time points post-transplantation. Results: An initial dramatic cell loss (up to 80% of grafted cells) due to apoptosis could be observed within the first 24 hours post-implantation, coinciding with a highly hypoxic NSC graft environment. Subsequently, strong spatiotemporal microglial and astroglial cell responses were initiated, which stabilised by day 5 post-implantation and remained present during the whole observation period. Moreover, the increase in astrocyte density was associated with a high degree of astroglial scarring within and surrounding the graft site. During the two-week follow up in this study, the NSC graft site underwent extensive remodelling with NSC graft survival further declining to around 1% of the initial number of grafted cells. Conclusions: The present study quantitatively describes the early post-transplantation events following NSC grafting in the adult mouse brain and warrants that such intervention is directly associated with a high degree of cell loss, subsequently followed by strong glial cell responses. it is hoped that NSCs, or more committed (progenitor) cells derived thereof, will become part of novel treat- ment options for a variety of CNS disorders. To date, several studies have reported the clinical benefit of NSCs following intraperitoneal, intravenous, intraventri- cular, intrathecal and intra-tissue grafting in various models of neuroinflammation, neurodegeneration or injury [1-4]. However, despite many suggestive literature reports, it still remains elusive whether the actual clini- cal benefit of grafted NSCs is due to cell integration, trophic support, immunomodulation or other yet to be defined mechanisms [5-7]. In this context, preceding work by others [8] and us [9] determined the actual sur- vival of grafted NSCs after at least two weeks to be less then 2% upon direct grafting into healthy or inflamed brain tissue. In addition, our preceding histological ana- lyses indicated the presence of both microglia and Reekmans et al. Stem Cell Research & Therapy 2012, 3:56 http://stemcellres.com/content/3/6/56 Spatiotemporal evolution of early innate immune responses triggered by neural stem cell grafting Kristien Reekmans1,2, Nathalie De Vocht1,2,3, Jelle Praet1,2,3, Erik Fransen4, Debbie Le Blon1,2, Chloé Hoornaert1,2, Jasmijn Daans1,2, Herman Goossens2, Annemie Van der Linden3, Zwi Berneman1,2 and Peter Ponsaerts1,2* * Correspondence: Peter.Ponsaerts@ua.ac.be 1Laboratory of Experimental Hematology, Faculty of Medicine and Health Sciences, University of Antwerp, Universiteitsplein 1, Antwerp-Wilrijk, 2610, Belgium Full list of author information is available at the end of the article Introduction Neural stem cells (NSCs) are defined as a population of self-renewing multipotent progenitor cells present in the developing and adult central nervous system (CNS) [1]. Despite their specific spatiotemporal occurrence in vivo, ex vivo culture expansion of NSCs derived from various sources (for example, embryonic or postnatal brain and embryonic stem cells) was shown to be relatively straightforward. Moreover, given the observation that ex vivo cultured neurosphere-derived NSCs and mono- layer-cultured NSC populations can be triggered to dif- ferentiate into neurons, astrocytes and oligodendrocytes, * Correspondence: Peter.Ponsaerts@ua.ac.be 1Laboratory of Experimental Hematology, Faculty of Medicine and Health Sciences, University of Antwerp, Universiteitsplein 1, Antwerp-Wilrijk, 2610, Belgium Full list of author information is available at the end of the article Reekmans et al. Stem Cell Research & Therapy 2012, 3:56 http://stemcellres.com/content/3/6/56 Page 2 of 10 examination was performed according to previously optimised procedures [9]. Serial 10-μm thick cryosec- tions were obtained from the entire implant region using a Microm HM5000 cryostat (Prosan, Merelbeke, Belgium), consecutively marked and missing slides were noted. For further immunofluorescence analysis of tissue sections, antibody staining was performed as previously described [9,10,12], using the following antibodies: (i) a rabbit anti-mouse ionized calcium binding adaptor molecule 1 (Iba1) antibody (1/200) (Wako chemicals, Osaka, Japan; 019-19714) and (ii) a rabbit anti-pimoni- dazole (Hypoxyprobe-1) antibody (1/200) (HPI Inc., Bur- lington, MA, USA; Pab2627), all in combination with a secondary donkey anti-rabbit AlexaFluor 555 antibody (1/500) (Life Technologies, Carlsbad, CA, USA; A31572), (iv) a rabbit anti-mouse S100B antibody (1/ 200) (Abcam, Cambridge, UK; 52642) in combination with a secondary donkey anti-rabbit AlexaFluor 555 antibody (1/1000) (Life Technologies, Carlsbad, CA, USA; A31572), (v) a mouse anti-mouse GFAP antibody (1/400) (Millipore, Billerica, MA, USA; MAB360) in combination with a secondary goat anti-mouse Alexa- Fluor 555 (1/1000) (Life Technologies, Carlsbad, CA, USA; A21127), and (vi) a chicken anti-mouse MBP anti- body (1/200) (Millipore, Billerica, MA, USA; AB9348) in combination with a secondary donkey anti-chicken DyLight 549 (Jackson Immunoresearch, Suffolk, UK; 703-506-155). The presence of terminal deoxynucleoti- dyl transferase dUTP nick end labelling (TUNEL)+ apoptotic cells was investigated using the In Situ Cell Death Detection Kit TMR Red (Roche, Penzberg, Ger- many; 12156792910), according to manufacturer’s instructions. Nuclear staining was performed using a TOPRO3 deep red stain (1/200) (Life Technologies, Carlsbad, CA, USA). astrocytes within and surrounding the NSC graft site at two weeks post-implantation [9]. Cell implantation experiments NSC genetically engineered with the Luciferase and eGFP reporter proteins (NSC-Luc/eGFP, FVB-back- ground) were cultured and characterised as previously described [10]. For cell implantation experiments, adult female Friend leukemia virus B (FVB)/NCrl mice (n = 33) were obtained from Charles River Laboratories (Wil- mington, MA, USA - strain code 207). Cell implantation of NSC-Luc/eGFP (2.5 × 105 cells in 2 μl PBS) was reproducibly targeted to the right hemisphere at the fol- lowing coordinates relative to bregma: 2 mm posterior, 2 mm lateral, and 2.25 mm ventral. All surgical inter- ventions were performed under sterile conditions, as previously described [9-11]. For all experiments, mice were kept in a normal day-night cycle (12/12) with free access to food and water. All experimental procedures were approved by the Ethics Committee for Animal Experiments of the University of Antwerp (UA) (approval no. 2011/13). Introduction Following these obser- vations, our next research aim was to determine whether the observed glial cell responses: (i) were directly executed against the grafted NSC (or their reporter proteins), or (ii) resulted as a consequence of immediate cell graft mortality. In order to investigate both hypotheses, the present study determined the fol- lowing parameters at multiple early time points (that is, day 0, 1, 3, 5, 7 and 14) post-implantation: (i) the actual survival of grafted NSC, (ii) the occurrence of cellular hypoxia, and (iii) the occurrence and/or maintenance of cell graft-induced glial cell responses. Histological analysis - quantitative analysis In vivo bioluminescence imaging (BLI) was performed at different time points post-implantation (day 1, 3, 5, 7, 10 and 14) using a real-time photon-imager system (Bio- space, Centennial, CO, USA), according to previously optimised procedures [10-12]. Using the M3 Vision soft- ware (Biospace, Centennial, CO, USA), light emission was measured from a fixed region of interest on the mouse head, and values of signal intensity are presented as the average number of photons/s/sr/cm2 over a 3- minute time period. An additional region of interest was drawn on the mouse shoulder and considered as back- ground signal. Histological analysis - quantitative analysis Quantitative analysis of cell graft survival, glial cell responses and cellular hypoxia were performed using NIH ImageJ analysis software (ImageJ) and TissueQuest immunofluorescence analysis software (TissueGnostics GmbH, Vienna, Austria), allowing determination of the following parameters: (i) total graft site volume in mm3, (ii) density of eGFPpos NSC-luc/eGFP within the graft site provided in number of cells/mm3 (six data counts per cell graft analysed), (iii) cell graft survival provided in absolute numbers and as % calculated to the initial number of grafted cells, (iv) density of Iba1pos microglia within the graft site provided in number of cells/mm3 (three data counts per cell graft analysed), (v) density of Iba1pos microglia within the implant border (that is, region extending 100 μm from the implant site) pro- vided in number of cells/mm3 (three data counts per cell graft analysed), (vi) density of S100Bpos astrocytes Histological analysis - immunofluorescence staining Before sacrifice (1.5 hours), mice used in this study were injected intraperitoneally with Hypoxyprobe-1 (HPI Inc, Burlington, MA, USA), according to the manufacturer’s instructions. Preparation of brain tissue for histological Page 3 of 10 Reekmans et al. Statistical analysis All statistical analyses were performed using the statisti- cal package R, version 2.13.1, with linear mixed models fitted using the lme function in the nlme package. Dif- ferences in graft site volume between day 0 and day 14 post-grafting were tested using the Mann-Withney U- test. The evolution over time of NSC graft survival was modelled using piecewise linear regression, taking the knot at day 1 post-implantation. Using this model, sepa- rate regression slopes up to day 1 and beyond day 1 (that is, from on day 3) were estimated and tested for significance. The evolution over time of the obtained in vivo bioluminescence signal intensities, microglia densi- ties, astrocyte densities and the degree of astrogliosis was modelled using piecewise linear mixed model analy- sis. A random intercept for individual was added to the model to account for the dependence between observa- tions from the same individual. Values for biolumines- cence signal intensity, microglia densities and astrocyte densities were log-transformed to obtain a more nor- mally distributed outcome variable. The position of the knot was determined based upon visual inspection of the data. Separate regression slopes before and after the knot were estimated and tested for significance. For all analyses, a P-value < 0.05 was considered statistically significant. Quantitative analysis of neural stem cell graft survival Quantitative analysis of neural stem cell graft survival Histological analyses of brain tissue from cell-grafted mice were performed at day 0 (4 hours post-implanta- tion, n = 5), day 1 (n = 5), day 3 (n = 4), day 5 (n = 4), day 7 (n = 4) and day 14 (n = 4) post-implantation. For this, cryosections were prepared from the whole graft site area and screened for the presence of eGFP-expres- sing NSC-Luc/eGFP implants. Representative histologi- cal images of NSC-Luc/eGFP implants, provided in Figure 2 (first row), already indicate extensive cell death at day 1 post-implantation. The latter is clearly visua- lised by the loss of eGFP expression within NSC-Luc/ eGFP grafts at day 1 as compared to the uniform eGFP expression within NSC-Luc/eGFP grafts at day 0. From day 7 post-implantation, the necrotic core of NSC-Luc/ eGFP implants has disappeared, while remaining NSC- Luc/eGFP have dispersed along the white matter tracts of the capsula externa and corpus callosum. Larger images of those presented in Figure 2 are provided in Figure S1 in Additional file 1. Further quantitative ana- lysis estimated the actual number of grafted NSC-Luc/ eGFP at day 0 post-implantation and the number of sur- viving NSC-Luc/eGFP at day 1, day 3, day 5, day 7 and day 14 post-implantation (Figure 3a). Presented data indicate a dramatic initial cell loss (up to 80%) within the first 24 hours post-implantation (P < 0.0001), which slowly continues between day 1 and 14 post-implanta- tion (P = 0.004). Based on the number of grafted cells at day 0, graft survival was estimated to be 22%, 6%, 5%, 4% and 1% respectively, at day 1, day 3, day 5, day 7 and day 14 post-implantation (Figure 3a inset). Histological analysis - quantitative analysis Stem Cell Research & Therapy 2012, 3:56 http://stemcellres.com/content/3/6/56 within the graft site in number of cells/mm3 (three data counts per cell graft analysed), (vii) density of S100Bpos astrocytes within the implant border provided in num- ber of cells/mm3 (three data counts per cell graft ana- lysed), (viii) the degree of glial fibrillary acidic protein (GFAP)pos astrogliosis within the graft site provided as % astrogliosis (that is, image-covering of GFAP staining) (one data count per cell graft analysed), (ix) the degree of GFAPpos astrogliosis within the implant border pro- vided as % astrogliosis (one data count per cell graft analysed), (x) the percentage of Hypoxyprobe-1pos eGFP- pos NSC versus total eGFPpos NSC within the implant zone (one data count per cell graft analysed). regions on top of the mouse head (Figure 1B) and the mean background BLI signals from fixed control regions on the mouse shoulder (Figure 1B) were plotted versus time post-implantation (Figure 1C). Regression analysis of the data indicates a significant decrease in BLI signal beyond day 3 post-implantation (P < 0.0001), indicative of a progressive decrease of cell viability within NSC- Luc/eGFP grafts. Longitudinal in vivo bioluminescence imaging of neural stem cell grafts Regions of interest are drawn on the mouse head, where NSC-Luc/eGFP were injected, and on the mouse shoulder, considered as background signal. A representative time course image was chosen out of five mice imaged for the whole time course. (C) In vivo BLI- image analysis. Quantitative analysis of in vivo BLI analysis at day 1, 3, 5, 7, 10 and 14 post-implantation. BLI signals (in photons/s/sr/cm2) are provided as the mean (± SEM) for all mice analysed, both from the specific region of interest on the mouse head (red bars) and from the control region of interest on the mouse shoulder (blue bars). Significant differences are described in the results section. Figure 1 Longitudinal in vivo bioluminescence imaging of neural stem cell-Luciferase/enhanced green fluorescent protein (NSC-Luc/ eGFP) grafts. (A) In vitro characterisation of NSC-Luc/eGFP. Left, representative fluorescence microscopy image of Luciferase/eGFP-expressing NSC (NSC-Luc/eGFP) used in this study. Green colour, direct eGFP fluorescence. Middle histogram overlay, representative flowcytometric analysis of parental NSC and NSC-Luc/eGFP. Open black histogram, control background fluorescence in FL-1 green channel from parental NSC. Filled green histogram, direct eGFP fluorescence in FL-1 green channel from NSC-Luc/eGFP. Right, in vitro bioluminescence analysis of 1 × 105 parental NSC and NSC-Luc/eGFP. Data are expressed as photons/s/sr/cm2 from a 5-minute time period (± standard error of the mean (SEM), n = 4). (B) In vivo bioluminescence imaging (BLI) - image aquisition. Representative time course image showing in vivo BLI of mice grafted with 1.5 × 105 NSC-Luc/eGFP in the central nervous system (CNS). Images were acquired at day 1 (n = 25), day 3 (n = 15), day 5 (n = 15), day 7 (n = 10), day 10 (n = 5) and day 14 (n = 5) post-implantation. Regions of interest are drawn on the mouse head, where NSC-Luc/eGFP were injected, and on the mouse shoulder, considered as background signal. A representative time course image was chosen out of five mice imaged for the whole time course. (C) In vivo BLI- image analysis. Quantitative analysis of in vivo BLI analysis at day 1, 3, 5, 7, 10 and 14 post-implantation. BLI signals (in photons/s/sr/cm2) are provided as the mean (± SEM) for all mice analysed, both from the specific region of interest on the mouse head (red bars) and from the control region of interest on the mouse shoulder (blue bars). Significant differences are described in the results section. Longitudinal in vivo bioluminescence imaging of neural stem cell grafts As shown by the representative images in Figure 2 (sec- ond row), it is clear at day 0 and day 1 post-grafting that the majority of grafted NSC-Luc/eGFP are under hypoxic condition as demonstrated by immunostaining of tissue sections for Hypoxyprobe-1 (an in vivo probe for labelling of hypoxic cells). Further quantitative image analysis indeed confirmed a high percentage of hypoxic cells among (still) viable eGFP expressing NSC-Luc/ eGFP at day 0 (mean 74.3% ± standard deviation (SD) 8.3%) and day 1 (54.9% ± 5.5%) post-grafting, which was not detected at later time points post-grafting in the few For cell grafting experiments, we used a previously engi- neered NSC line expressing both the Luciferase and eGFP reporter proteins (Figure 1A), further named as NSC-Luc/eGFP [10]. Following grafting of 1.5 × 105 NSC-Luc/eGFP in the CNS of immune-competent FVB mice (n = 33), longitudinal in vivo BLI was performed at day 1 (n = 25), day 3 (n = 15), day 5 (n = 15), day 7 (n = 10), day 10 (n = 5) and day 14 (n = 5) post-implanta- tion. For quantitative analysis of the observed BLI sig- nals, the mean cell graft-specific BLI signals from fixed Page 4 of 10 Reekmans et al. Stem Cell Research & Therapy 2012, 3:56 http://stemcellres.com/content/3/6/56 Figure 1 Longitudinal in vivo bioluminescence imaging of neural stem cell-Luciferase/enhanced green fluorescent protein (NSC-Luc/ eGFP) grafts. (A) In vitro characterisation of NSC-Luc/eGFP. Left, representative fluorescence microscopy image of Luciferase/eGFP-expressing NSC (NSC-Luc/eGFP) used in this study. Green colour, direct eGFP fluorescence. Middle histogram overlay, representative flowcytometric analysis of parental NSC and NSC-Luc/eGFP. Open black histogram, control background fluorescence in FL-1 green channel from parental NSC. Filled green histogram, direct eGFP fluorescence in FL-1 green channel from NSC-Luc/eGFP. Right, in vitro bioluminescence analysis of 1 × 105 parental NSC and NSC-Luc/eGFP. Data are expressed as photons/s/sr/cm2 from a 5-minute time period (± standard error of the mean (SEM), n = 4). (B) In vivo bioluminescence imaging (BLI) - image aquisition. Representative time course image showing in vivo BLI of mice grafted with 1.5 × 105 NSC-Luc/eGFP in the central nervous system (CNS). Images were acquired at day 1 (n = 25), day 3 (n = 15), day 5 (n = 15), day 7 (n = 10), day 10 (n = 5) and day 14 (n = 5) post-implantation. Longitudinal in vivo bioluminescence imaging of neural stem cell grafts Direct eGFP fluorescence (green) combined with TOPRO3 staining (false colour representation in blue) at day 0, 1, 3, 5, 7 and 14 post-implantation. Representative images were chosen from multiple stained slides (n = 6 to 9 for eGFP/TOPRO3 combination) per mouse analysed at each time point. The provided scale bars indicate 200 μm. Second row, cellular hypoxia. Direct eGFP fluorescence (green) combined with Hypoxyprobe-1staining (red) at day 0, 1, 3, 5, 7 and 14 post-implantation. Representative images were chosen from two to five mice analysed at each time point. The provided scale bars indicate 50 μm. Third, fourth and fifth row, endogenous glial cell behaviour. Direct eGFP fluorescence (green) combined with TOPRO3 staining (false colour representation in blue) and combined with immunofluorescence staining for ionized calcium binding adaptor molecule 1 (Iba1) (red, fourth row), S100 calcium binding protein B (S100B) (red, fifth row) or glial fibrillary acidic protein (GFAP) (red, sixth row) at day 0, 1, 3, 5, 7 and 14 post-implantation. Representative images were chosen from multiple stained slides (n = 3 for eGFP/TOPRO3/Iba1 combination, n = 3 for eGFP/TOPRO3/S100B combination and n = 1 for eGFP/TOPRO3/GFAP) per mouse analysed at each time point (n = 4/5). The provided scale bars indicate 50 μm for Iba1 and S100B images and 200 μm for GFAP images. Sixth row, graft site remodelling. Direct eGFP fluorescence (green) combined with myelin base protein (MBP) staining (red) at day 0, 1, 3, 5, 7 and 14 post-implantation. Representative images were chosen from multiple mice analysed at each time point (n = 2). The provided scale bars indicate 200 μm. Longitudinal in vivo bioluminescence imaging of neural stem cell grafts surviving NSC-Luc/eGFP. Moreover, as shown in Figure 4, already at 4 hours post-grafting (day 0) TUNEL reac- tivity can be observed within the population of grafted NSC-Luc/eGFP. In contrast, at day 1 post-grafting, TUNEL reactivity is highly apparent within the eGFP- negative necrotic core of the NSC-Luc/eGFP graft, but not in the few surviving NSC-Luc/eGFP at the border of the graft. Larger images of those presented in Figure 4 are provided in Figure S2 in Additional file 1. Based on these data, we suggest that excessive cell loss observed within the first 24 hours post-grafting might be (par- tially) initiated by cellular hypoxia (and presumably also lack of nutrients) within the core of NSC-Luc/eGFP grafts, resulting in massive apoptotic cell death. Page 5 of 10 Reekmans et al. Stem Cell Research & Therapy 2012, 3:56 http://stemcellres.com/content/3/6/56 Figure 2 Histological analysis of neural stem cell (NSC) graft survival and endogenous glial cell responses. First row, NSC-Luciferase/ enhanced fluorescent green protein (Luc/eGFP) graft survival. Direct eGFP fluorescence (green) combined with TOPRO3 staining (false colour representation in blue) at day 0, 1, 3, 5, 7 and 14 post-implantation. Representative images were chosen from multiple stained slides (n = 6 to 9 for eGFP/TOPRO3 combination) per mouse analysed at each time point. The provided scale bars indicate 200 μm. Second row, cellular hypoxia. Direct eGFP fluorescence (green) combined with Hypoxyprobe-1staining (red) at day 0, 1, 3, 5, 7 and 14 post-implantation. Representative images were chosen from two to five mice analysed at each time point. The provided scale bars indicate 50 μm. Third, fourth and fifth row, endogenous glial cell behaviour. Direct eGFP fluorescence (green) combined with TOPRO3 staining (false colour representation in blue) and combined with immunofluorescence staining for ionized calcium binding adaptor molecule 1 (Iba1) (red, fourth row), S100 calcium binding protein B (S100B) (red, fifth row) or glial fibrillary acidic protein (GFAP) (red, sixth row) at day 0, 1, 3, 5, 7 and 14 post-implantation. Representative images were chosen from multiple stained slides (n = 3 for eGFP/TOPRO3/Iba1 combination, n = 3 for eGFP/TOPRO3/S100B combination and n = 1 for eGFP/TOPRO3/GFAP) per mouse analysed at each time point (n = 4/5). The provided scale bars indicate 50 μm for Iba1 and S100B images and 200 μm for GFAP images. Sixth row, graft site remodelling. Longitudinal in vivo bioluminescence imaging of neural stem cell grafts Direct eGFP fluorescence (green) combined with myelin base protein (MBP) staining (red) at day 0, 1, 3, 5, 7 and 14 post-implantation. Representative images were chosen from multiple mice analysed at each time point (n = 2). The provided scale bars indicate 200 μm. re 2 Histological analysis of neural stem cell (NSC) graft survival and endogenous glial cell responses. First row d fl (L / GFP) f l D GFP fl ( ) b d h TOPRO3 Figure 2 Histological analysis of neural stem cell (NSC) graft survival and endogenous glial cell responses. First row, NSC-Luciferase/ enhanced fluorescent green protein (Luc/eGFP) graft survival. Direct eGFP fluorescence (green) combined with TOPRO3 staining (false colour representation in blue) at day 0, 1, 3, 5, 7 and 14 post-implantation. Representative images were chosen from multiple stained slides (n = 6 to 9 for eGFP/TOPRO3 combination) per mouse analysed at each time point. The provided scale bars indicate 200 μm. Second row, cellular hypoxia. Direct eGFP fluorescence (green) combined with Hypoxyprobe-1staining (red) at day 0, 1, 3, 5, 7 and 14 post-implantation. Representative images were chosen from two to five mice analysed at each time point. The provided scale bars indicate 50 μm. Third, fourth and fifth row, endogenous glial cell behaviour. Direct eGFP fluorescence (green) combined with TOPRO3 staining (false colour representation in blue) and combined with immunofluorescence staining for ionized calcium binding adaptor molecule 1 (Iba1) (red, fourth row), S100 calcium binding protein B (S100B) (red, fifth row) or glial fibrillary acidic protein (GFAP) (red, sixth row) at day 0, 1, 3, 5, 7 and 14 post-implantation. Representative images were chosen from multiple stained slides (n = 3 for eGFP/TOPRO3/Iba1 combination, n = 3 for eGFP/TOPRO3/S100B combination and n = 1 for eGFP/TOPRO3/GFAP) per mouse analysed at each time point (n = 4/5). The provided scale bars indicate 50 μm for Iba1 and S100B images and 200 μm for GFAP images. Sixth row, graft site remodelling. Direct eGFP fluorescence (green) combined with myelin base protein (MBP) staining (red) at day 0, 1, 3, 5, 7 and 14 post-implantation. Representative images were chosen from multiple mice analysed at each time point (n = 2). The provided scale bars indicate 200 μm. Figure 2 Histological analysis of neural stem cell (NSC) graft survival and endogenous glial cell responses. First row, NSC-Luciferase/ enhanced fluorescent green protein (Luc/eGFP) graft survival. Quantitative analysis of glial cell responses following neural stem cell grafting we investigated the temporal behaviour of microglia and astrocytes following NSC-Luc/eGFP grafting. From the representative data provided in Figure 2 (third row) no direct microglial response can be observed at day 0 post-implantation, as indicated by the absence of Since around 95% of cell graft mortality can be observed within the first 48 hours following NSC-Luc/eGFP graft- ing, glial cell responses are inevitable evoked. Therefore, Reekmans et al. Stem Cell Research & Therapy 2012, 3:56 http://stemcellres.com/content/3/6/56 Page 6 of 10 Figure 3 In vivo cell graft and glial cell behaviour - quantitative analysis. (a) Survival of grafted neural stem cell-Luciferase/enhanced fluorescent green protein (NSC-Luc/eGFP). Presented data are the estimated total number of eGFP-expressing NSC-Luc/eGFP detected within the implant site for each mouse analysed (n = 5 for day 0, n = 5 for day 1, n = 4 for day 3, n = 4 for day 5, n = 4 for day 7 and n = 4 for day 14). The red line indicates the average total number of eGFP-expressing NSC-Luc/eGFP at each time point analysed. The inset graph represents the mean % (± standard error of the mean (SEM)) of NSC-Luc/eGFP graft survival at each time point analysed from day 1 post-implantation. Significant differences are described in the results section. (b-e) Cellular density of ionized calcium binding adaptor molecule 1 (Iba1)pos microglia and S100 calcium binding protein B (S100B)pos astrocytes within the implant site and within the implant border. Presented data are the ln(x)- transformed values of estimated total density of microglia and astrocytes within the implant border for each mouse analysed (n = 4 for day 0, n = 5 for day 1, n = 4 for day 3, n = 5 for day 5, n = 4 for day 7 and n = 4 for day 14). The red line indicates the average total cellular density at each time point analysed. Significant differences are described in the results section. (f) Astrogliosis within the implant site and implant border (both areas are combined here). Presented data indicate the average degree of glial fibrillary acidic protein (GFAP)pos astrogliosis (based on image coverage after GFAP staining) for each mouse analyses (n = 4 for day 0, n = 5 for day 1, n = 4 for day 3, n = 5 for day 5, n = 4 for day 7 and n = 4 for day 14). Quantitative analysis of glial cell responses following neural stem cell grafting The red line indicates the average degree of astrogliosis at each time point analysed. Significant differences are described in the results section. Figure 3 In vivo cell graft and glial cell behaviour - quantitative analysis. (a) Survival of grafted neural stem cell-Luciferase/enhanced fluorescent green protein (NSC-Luc/eGFP). Presented data are the estimated total number of eGFP-expressing NSC-Luc/eGFP detected within the implant site for each mouse analysed (n = 5 for day 0, n = 5 for day 1, n = 4 for day 3, n = 4 for day 5, n = 4 for day 7 and n = 4 for day 14). The red line indicates the average total number of eGFP-expressing NSC-Luc/eGFP at each time point analysed. The inset graph represents the mean % (± standard error of the mean (SEM)) of NSC-Luc/eGFP graft survival at each time point analysed from day 1 post-implantation. Significant differences are described in the results section. (b-e) Cellular density of ionized calcium binding adaptor molecule 1 (Iba1)pos microglia and S100 calcium binding protein B (S100B)pos astrocytes within the implant site and within the implant border. Presented data are the ln(x)- transformed values of estimated total density of microglia and astrocytes within the implant border for each mouse analysed (n = 4 for day 0, n = 5 for day 1, n = 4 for day 3, n = 5 for day 5, n = 4 for day 7 and n = 4 for day 14). The red line indicates the average total cellular density at each time point analysed. Significant differences are described in the results section. (f) Astrogliosis within the implant site and implant border (both areas are combined here). Presented data indicate the average degree of glial fibrillary acidic protein (GFAP)pos astrogliosis (based on image coverage after GFAP staining) for each mouse analyses (n = 4 for day 0, n = 5 for day 1, n = 4 for day 3, n = 5 for day 5, n = 4 for day 7 and n = 4 for day 14). The red line indicates the average degree of astrogliosis at each time point analysed. Significant differences are described in the results section. grafted NSC/Luc-eGFP focused on: (a) the implant site, delineated based on eGFP expression, and (b) the implant border, delineated as a 100-μm border around the implant site. Discussion Over the past years, neural stem cell transplantation has been recognized as a promising novel therapeutic tool to treat CNS disorders for which today, no effective therapies are available. However, successful functional integration of grafted NSCs (or in vitro/in vivo NSC- derived cell types) is one of the major challenges in Quantitative analysis of glial cell responses following neural stem cell grafting (Figure 3d and 3e), S100Bpos astrocyte density signifi- cantly increased within the graft site until day 5 post- grafting (P < 0.0001), with no increase beyond day 5 (P = 0.27). Within the implant border, astrocyte density slightly increased between day 0 and day 14 post-graft- ing (P = 0.0021). All absolute microglia and astrocyte cell densities within and surrounding the graft site at day 0, 1, 3, 5, 7 and 14 are provided in Table 1. Although the number of astrocytes recruited within and surrounding the graft site is less impressive compared to microglia recruitment, staining for GFAP was performed in order to determine GFAPpos astroglial scarring within and surrounding the graft site. Presented data (Figure 3f) clearly indicate that the degree of GFAPpos astroglio- sis within and surrounding the graft site (both areas combined) significantly increases throughout the whole observation period, with the highest increase occurring prior to day 3 post-implantation (P < 0.0001), and a reduced, although significant (P = 0.006), increase beyond day 3 post-implantation. subsequent injection of a cell suspension. As shown by the representative images in Figure 2 (sixth row), it is clear that both the CNS architecture, as visualised by staining for myelin basic protein (MBP), and the NSC- Luc/eGFP graft site, as visualised by the presence of eGFP-expressing cells, undergo significant changes at early stages post-grafting. While initially grafted NSC- Luc/eGFP are clustered together and myelin conforma- tion of the capsula externa is temporally spread out, this gradually remodels as the limited number of surviving NSC-Luc/eGFP spread along the capsula externa/corpus callosum, and the disturbance in myelin conformation partially restores itself. The latter is accompanied with a reduction of graft site volume (that is, the area in which eGFP-expressing cells can be found) from 0.35 ± (SD) 0.09 mm3 at day 0 post-grafting to 0.07 ± 0.07 mm3 at day 14 post-grafting (P = 0.015). Quantitative analysis of glial cell responses following neural stem cell grafting Within the implant border, astrocyte de slightly increased between day 0 and day 14 post- ing (P = 0.0021). All absolute microglia and astr cell densities within and surrounding the graft s day 0, 1, 3, 5, 7 and 14 are provided in Tab Although the number of astrocytes recruited withi surrounding the graft site is less impressive compar microglia recruitment, staining for GFAP was perfo in order to determine GFAPpos astroglial scarring w and surrounding the graft site. Presented data (F 3f) clearly indicate that the degree of GFAPpos astr sis within and surrounding the graft site (both combined) significantly increases throughout the w observation period, with the highest increase occu prior to day 3 post-implantation (P < 0.0001), a reduced, although significant (P = 0.006), inc beyond day 3 post-implantation. Disruption of CNS architecture following neural stem transplantation Inevitably, cell grafting in the CNS will induce da to the CNS architecture due to needle insertion an Table 1 Astroglial and microglial densities following Time post-implantation Day 0 Day 1 Implant site Microglia density 1,293 ± 768 10,017 ± 10,678 1 Astrocyte density 5,382 ± 5,483 7,376 ± 5,653 1 Border zone Microglia density 1,537 ± 1,071 5,367 ± 5,951 2 Astroglial density 22,010 ± 6,202 17,757 ± 3,149 Absolute microglia and astrocyte cell densities (in number of cells/mm and day 14 post-grafting. (Figure 3d and 3e), S100Bpos astrocyte density signifi- cantly increased within the graft site until day 5 post- grafting (P < 0.0001), with no increase beyond day 5 (P = 0.27). Within the implant border, astrocyte density slightly increased between day 0 and day 14 post-graft- ing (P = 0.0021). All absolute microglia and astrocyte cell densities within and surrounding the graft site at day 0, 1, 3, 5, 7 and 14 are provided in Table 1. Although the number of astrocytes recruited within and surrounding the graft site is less impressive compared to microglia recruitment, staining for GFAP was performed in order to determine GFAPpos astroglial scarring within and surrounding the graft site. Presented data (Figure 3f) clearly indicate that the degree of GFAPpos astroglio- sis within and surrounding the graft site (both areas combined) significantly increases throughout the whole observation period, with the highest increase occurring prior to day 3 post-implantation (P < 0.0001), and a reduced, although significant (P = 0.006), increase beyond day 3 post-implantation. Quantitative analysis of glial cell responses following neural stem cell grafting Regarding microglial cell recruitment (Figure 3b and 3c), Iba1pos microglia density significantly increases until day 3 post-implantation within and sur- rounding the graft site (for both P < 0.0001). However, no further increase (or decrease) in microglia density was observed within or surrounding the graft site beyond day 3 post-implantation (respectively P = 0.76 and P = 0.96). Regarding astroglial cell recruitment staining for Iba1pos microglia. However, on day 1 post- implantation, and coinciding with the presence of a large number of necrotic cells, an accumulation of Iba1- pos microglia within and surrounding the graft site can be observed, which remains detectable from day 3 post- implantation. Next, although the number of S100Bpos astrocytes does not appear to change largely over time (Figure 2, fourth row), a high degree of astrogliosis, based on staining for GFAP (Figure 2, fifth row), is apparent at from day 3 post-implantation. Further quan- titative image analysis of glial cell responses evoked by Page 7 of 10 Reekmans et al. Stem Cell Research & Therapy 2012, 3:56 http://stemcellres.com/content/3/6/56 Reekmans et al. Stem Cell Research & Therapy 2012, 3:56 http://stemcellres.com/content/3/6/56 Figure 4 Histological analysis of in vivo neural stem cell (NSC) graft apoptosis. Direct enhanced fluorescent green protein (eGFP) fluorescence (green) combined with TOPRO3 staining (false colour representation in blue) and combined with the control immunofluorescence staining for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) (red, CONTROL STAINING) and the specific immunofluorescence staining for TUNEL+ apoptotic cells (red, TUNEL STAINING) at day 0 and 1 post-implantation. Representative images were chosen from multiple mice analysed at each time point (n = 2). The provided scale bars indicate 100 μm. Figure 4 Histological analysis of in vivo neural stem cell (NSC) graft apoptosis. Direct enhanced fluorescent green protein (eGFP) fluorescence (green) combined with TOPRO3 staining (false colour representation in blue) and combined with the control immunofluorescence staining for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) (red, CONTROL STAINING) and the specific immunofluorescence staining for TUNEL+ apoptotic cells (red, TUNEL STAINING) at day 0 and 1 post-implantation. Representative images were chosen from multiple mice analysed at each time point (n = 2). The provided scale bars indicate 100 μm. (Figure 3d and 3e), S100Bpos astrocyte density si cantly increased within the graft site until day 5 grafting (P < 0.0001), with no increase beyond day = 0.27). Disruption of CNS architecture following neural stem cell transplantation Inevitably, cell grafting in the CNS will induce damage to the CNS architecture due to needle insertion and the Table 1 Astroglial and microglial densities following intracerebral implantation of NSC-Luc/eGFP. Time post-implantation Day 0 Day 1 Day 3 Day 5 Day 7 Day 14 Implant site Microglia density 1,293 ± 768 10,017 ± 10,678 104,712 ± 75555 146,551 ± 188,791 94,803 ± 126,268 24,017 ± 250,036 Astrocyte density 5,382 ± 5,483 7,376 ± 5,653 18,059 ± 12,436 25,515 ± 5,986 28,900 ± 9,807 32,647 ± 15,108 Border zone Microglia density 1,537 ± 1,071 5,367 ± 5,951 24,551 ± 17,042 34,467 ± 33,349 25,062 ± 22,264 41,693 ± 44,628 Astroglial density 22,010 ± 6,202 17,757 ± 3,149 22,798 ± 4,216 25,308 ± 4,943 27,262 ± 5,647 34,603 ± 11,392 Absolute microglia and astrocyte cell densities (in number of cells/mm3 ± SD) within the implant site and the implant border at day 0, day 1, day 3, day 5, day 7 and day 14 post-grafting. Table 1 Astroglial and microglial densities following intracerebral implantation of NSC-Luc/eGFP. Reekmans et al. Stem Cell Research & Therapy 2012, 3:56 http://stemcellres.com/content/3/6/56 Page 8 of 10 whereby autologous reporter-gene modified cellular grafts were immune-tolerated and allogeneic cellular grafts were immune-rejected. However, our early studies did not include full quantitative analysis of in vivo BLI and histology data. Based on our new data showing that NSC grafts already display a high degree of mortality before the initiation of glial cell responses (Figure 3b-f), the observed glial cell responses are more likely to be associated with phagocytocis of cellular debris rather than active recognition of grafted autologous NSC. Nevertheless, based on our preceding data regarding full rejection of allogeneic (but not autologous) cellular grafts, active distinguishment of autologous and allo- geneic cellular grafts by microglia (or astrocytes) will thus most likely be occurring during or after the initial clearance of cellular debris. Following initial activation of glial cell responses, survival of grafted autologous NSC further declines to around 1% by week 2 post- implantation. Moreover, although endogenous microglia and astrocytes are able to produce either pro-inflamma- tory/neurotoxic or anti-inflammatory/neuroprotective factors depending on the type of activation [19,20], it is highly likely that the evoked immune responses follow- ing NSC implantation create an inhibitory pro-inflam- matory environment, thereby further limiting the long- term survival of transplanted NSC [21]. Disruption of CNS architecture following neural stem cell transplantation Of note, addi- tional immunophenotypical stainings were performed to determine the in vivo differentiation potential of the few surviving NSC-Luc/eGFP (data not shown). Results indi- cated that grafted NSC-Luc/eGFP did not change phe- notypic properties between day 0 and day 14 post- grafting, that is, They remained SOX2pos (as NSC mar- ker), S100Bneg (as astrocyte marker), NeuNneg (as neuro- nal marker) and CC1neg (as oligodendrocytes marker). Likewise, proper in vivo differentiation and functional integration of the few surviving NSC is most likely inhibited by an endogenous glial-cell-induced pro- inflammatory immune environment [22-24]. current neuroscience research, and if successful, will have an enormous impact on the future development of cell replacement therapies for CNS disorders. In order to contribute to this understanding, we have investigated the spatiotemporal evolution of NSC graft behaviour and endogenous glial cell responses at early time points post-implantation. With regard to direct grafting of NSCs in CNS tissue, our results demonstrate that this procedure immediately results in: (i) significant disruption of the CNS architec- ture (Figure 2, sixth row) and (ii) substantial cell graft mortality (Figure 3a). First, the observed disruption of CNS architecture (although partially restored over time) will remain inevitable as long as cells (or cell-seeded scaffolds) are directly implanted into CNS tissue. Although alternative administration routes (that is, intraventricular, intrathecal or intravenous) have been suggested to facilitate cell delivery into injured CNS tis- sue, the cell number arriving at the target site is gener- ally nihil to a few representative cells detected by histological analysis [13-15]. Second, apoptotic cell death within the first 24 hours post-grafting (Figure 4) can most likely be explained by the lack of oxygen (Fig- ure 2, second row) and/or nutrients delivered to the NSC graft, which, in fact, is introduced without any structural and/or functional support. More recently, novel biomimetic strategies are emerging aiming to cre- ate a permissive micro environmental niche, thereby enhancing survival, differentiation and integration of grafted NSCs [16-18]. The latter will be inevitable, as a substantial amount of grafted cells need to survive fol- lowing transplantation in order to functionally partici- pate in neuronal circuitries. Disruption of CNS architecture following neural stem cell transplantation Of note, while we used both in vivo BLI (Figure 1c) and post-mortem histological analysis (Figure 3a) to determine cell graft survival, it should be noted that the observed in vivo BLI signals at day 1 and day 3 post-grafting are resulting from respec- tively 20% and 5% of the original number of grafted cells. Although we and others have associated this early in vivo BLI signal with complete cell graft survival, our results now clearly indicate that precaution has to be taken in the interpretation of early in vivo BLI data [10,11]. Nevertheless, in vivo BLI remains an interesting tool for longitudinal assessment of cell graft survival, as progressive cell loss of implanted NSC could clearly be demonstrated from on day 3 post-implantation (Figure 1c). Wi h d h b d li l ll h Abbreviations BLI: bioluminescence imaging; CC1: anti-APC (Adenomatous polyposis coli protein) antibody; CNS: central nervous system; eGFP: enhanced green fluorescent protein; GFAP: glial fibrillary acidic protein; Iba-1: ionized calcium binding adaptor molecule 1; Luc: Luciferase; MBP: myelin basic protein; NeuN: neuronal nuclei; NSC: neural stem cell; PBS: phosphate-buffered saline; S100B: S100 calcium binding protein B; SOX2: sex-determining region Y-box 2; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling. 11. Bergwerf I, De Vocht N, Tambuyzer B, Verschueren J, Reekmans K, Daans J, Ibrahimi A, Van Tendeloo V, Chatterjee S, Goossens H, Jorens PG, Baekelandt V, Ysebaert D, Van Marck E, Berneman ZN, Linden AV, Ponsaerts P: Reporter gene-expressing bone marrow-derived stromal cells are immune-tolerated following implantation in the central nervous system of syngeneic immunocompetent mice. BMC Biotechnol 2009, 9:1. Conclusions Dunnett SB, Rosser AE: Clinical translation of cell transplantation in the brain. Curr Opin Organ Transplant 2011, 16:632-639. 7. Martino G, Pluchino S, Bonfanti L, Schwartz M: Brain regeneration in physiology and pathology: the immune signature driving therapeutic plasticity of neural stem cells. Physiol Rev 2011, 91:1281-1304. 8. Sher F, van Dam G, Boddeke E, Copray S: Bioluminescence imaging of Olig2-neural stem cells reveals improved engraftment in a demyelination mouse model. Stem Cells 2009, 27:1582-1591. 9. Praet J, Reekmans K, Lin D, De Vocht N, Bergwerf I, Tambuyzer B, Daans J, Hens N, Goossens H, Pauwels P, Berneman Z, Van der Linden A, Ponsaerts P: Cell type-associated differences in migration, survival and immunogenicity following grafting in CNS tissue. Cell Transplant 2012. Reekmans K, Praet J, De Vocht N, Daans J, Van der Linden A, Berneman Z, Ponsaerts P: Stem cell therapy for multiple sclerosis: preclinical evidence beyond all doubt? Regen Med 2012, 7:245-259. Acknowledgements This work was supported by research grants G.0136.11 and G.0130.11 (granted to ZB, AvDL and PP) of the Fund for Scientific Research-Flanders (FWO-Vlaanderen, Belgium), in part by a Methusalem research grant from the Flemish government (granted to ZB and HG) and in part by funding received from the European Union’s Seventh Framework Programme (FP7/ 2007-2013) under grant agreement number 278850 (INMiND) (granted to AvDL). Nathalie De Vocht holds a PhD-studentship from the FWO- Vlaanderen. Debbie Le Blon holds a PhD-studentship from the Flemish Institute for Science and Technology (IWT-Vlaanderen). Peter Ponsaerts is a post-doctoral fellow of the FWO-Vlaanderen. This work was supported by research grants G.0136.11 and G.0130.11 (granted to ZB, AvDL and PP) of the Fund for Scientific Research-Flanders (FWO-Vlaanderen, Belgium), in part by a Methusalem research grant from the Flemish government (granted to ZB and HG) and in part by funding received from the European Union’s Seventh Framework Programme (FP7/ 2007-2013) under grant agreement number 278850 (INMiND) (granted to AvDL). Nathalie De Vocht holds a PhD-studentship from the FWO- 12. De Vocht N, Bergwerf I, Vanhoutte G, Daans J, De Visscher G, Chatterjee S, Pauwels P, Berneman Z, Ponsaerts P, Van der Linden A: Labeling of Luciferase/eGFP-expressing bone marrow-derived stromal cells with fluorescent micron-sized iron oxide particles improves quantitative and qualitative multimodal imaging of cellular grafts in vivo. Mol Imaging Biol 2011, 13:1133-1145. Vlaanderen. Debbie Le Blon holds a PhD-studentship from the Flemish Institute for Science and Technology (IWT-Vlaanderen). Peter Ponsaerts is a post-doctoral fellow of the FWO-Vlaanderen. 13. Franchi S, Valsecchi AE, Borsani E, Procacci P, Ferrari D, Zalfa C, Sartori P, Rodella LF, Vescovi A, Maione S, Rossi F, Sacerdote P, Colleoni M, Panerai AE: Intravenous neural stem cells abolish nociceptive hypersensitivity and trigger nerve regeneration in experimental neuropathy. Pain 2012, 153:850-861. Additional material Additional file 1: Histological analysis of neural stem cell (NSC) graft survival, endogenous glial cell responses and in vivo NSC graft apoptosis. This file contains larger images of those presented in Figure 2 (Figure S1) and Figure 4 (Figure S2). Additional file 1: Histological analysis of neural stem cell (NSC) graft survival, endogenous glial cell responses and in vivo NSC graft apoptosis. This file contains larger images of those presented in Figure 2 (Figure S1) and Figure 4 (Figure S2). 10. 10. Reekmans KP, Praet J, De Vocht N, Tambuyzer BR, Bergwerf I, Daans J, Baekelandt V, Vanhoutte G, Goossens H, Jorens PG, Ysebaert DK, Chatterjee S, Pauwels P, Van Marck E, Berneman ZN, Van der Linden A, Ponsaerts P: Clinical potential of intravenous neural stem cell delivery for treatment of neuroinflammatory disease in mice? Cell Transplant 2011, 20:851-869. 10. Reekmans KP, Praet J, De Vocht N, Tambuyzer BR, Bergwerf I, Daans J, Baekelandt V, Vanhoutte G, Goossens H, Jorens PG, Ysebaert DK, Chatterjee S, Pauwels P, Van Marck E, Berneman ZN, Van der Linden A, Ponsaerts P: Clinical potential of intravenous neural stem cell delivery for treatment of neuroinflammatory disease in mice? Cell Transplant 2011, 20:851-869. Author details 1 1Laboratory of Experimental Hematology, Faculty of Medicine and Health Sciences, University of Antwerp, Universiteitsplein 1, Antwerp-Wilrijk, 2610, Belgium. 2Vaccine and Infectious Disease Institute (Vaxinfectio), Faculty of Medicine and Health Sciences, University of Antwerp, Universiteitsplein 1, Antwerp-Wilrijk, 2610, Belgium. 3BioImaging Laboratory, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, Department of Biomedical Sciences, Universiteitsplein 1, Antwerp-Wilrijk, 2610, Belgium. 4StatUa Centre for Statistics, City Campus, University of Antwerp, Prinsstraat 13, Antwerp, 2000, Belgium. 14. Mothe AJ, Bozkurt G, Catapano J, Zabojova J, Wang X, Keating A, Tator CH: Intrathecal transplantation of stem cells by lumbar puncture for thoracic spinal cord injury in the rat. Spinal Cord 2011, 49:967-973. 15. Kim H, Walczak P, Muja N, Campanelli JT, Bulte JW: ICV-transplanted human glial precursor cells are short-lived yet exert immunomodulatory effects in mice with EAE. Glia 2012, 60:1117-1129. 16. Uemura M, Refaat MM, Shinoyama M, Hayashi H, Hashimoto N, Takahashi J: Matrigel supports survival and neuronal differentiation of grafted embryonic stem cell-derived neural precursor cells. J Neurosci Res 2010, 88:542-551. 4StatUa Centre for Statistics, City Campus, University of Antwerp, Prinsstraat 13, Antwerp, 2000, Belgium. Authors’ contributions 17. Zhong J, Chan A, Morad L, Kornblum HI, Fan G, Carmichael ST: Hydrogel matrix to support stem cell survival after brain transplantation in stroke. Neurorehabil Neural Repair 2010, 24:636-644. KR, NDV, JP, HG, AvDL, ZB and PP designed the research study; KR, NDV, JP, DLB, CH and JD performed the research; EF performed the statistical analyses; KR and PP wrote the manuscript. All authors approved the final version of the manuscript. 18. Kim H, Cooke MJ, Shoichet MS: Creating permissive microenvironments for stem cell transplantation into the central nervous system. Trends Biotechnol 2012, 30:55-63. Competing interests 19. Czeh M, Gressens P, Kaindl AM: The yin and yang of microglia. Dev Neurosci 2011, 33:199-209. The authors declare that they have no competing interests. 20. Almad AA, Maragakis NJ: Glia: an emerging target for neurological disease therapy. Stem Cell Res Ther 2012, 3:37. Received: 17 September 2012 Revised: 29 November 2012 Received: 17 September 2012 Revised: 29 November 2012 Accepted: 12 December 2012 Published: 14 December 2012 Accepted: 12 December 2012 Published: 14 December 2012 21. De Vocht N, Lin D, Praet J, Hoornaert C, Reekmans K, Le Blon D, Daans J, Pauwels P, Goossens H, Hens N, Berneman Z, Van der Linden A, Ponsaerts P: Quantitative and phenotypic analysis of mesenchymal stromal cell graft survival and recognition by microglia and astrocytes in mouse brain. Immunobiology 2012. Conclusions The present study quantitatively describes the early post-transplantation events following NSC grafting in the adult mouse brain and warrants that such interven- tion is associated with an immediate high degree of cell loss. The latter is subsequently followed by strong glial cell responses, presumably creating a non-permissive environment, which limit proper migration, differentia- tion and integration of grafted NSC into the host tissue. Unfortunately, these observations are generally not con- sidered when evaluating the potential of pre-clinical cell therapy studies, as most reports lack detailed cell graft survival and/or glial reactivity analysis at early and late time-points post-grafting. Moreover, it is important to note that early NSC graft mortality and subsequent glial With regard to the observed glial cell responses, these are not surprising as the majority (up to 95%) of grafted NSCs already die within the first 24 to 48 hours post- grafting (Figure 3a). However, in our preceding studies regarding autologous and allogeneic stem cell grafting in the CNS of immune competent mice, we suggested that the occurrence of glial cell responses was most likely due to the recognition of the cellular implant itself, Page 9 of 10 Reekmans et al. Stem Cell Research & Therapy 2012, 3:56 http://stemcellres.com/content/3/6/56 neural stem cell biology and transplantation research. Stem Cell Rev 2012, 8:262-278. cell responses themselves might be responsible for many of the observed beneficial effects following cell trans- plantation in CNS disorders [2]. We therefore under- score the current need for a profound characterisation of all cellular and/or molecular interactions following cell grafting in the CNS, as the full potential of NSC transplantation therapy can only be determined follow- ing better understanding (and manipulation) of NSC graft survival and inhibitory immune responses. neural stem cell biology and transplantation research. Stem Cell Rev 2012, 8:262-278. 2. Reekmans K, Praet J, De Vocht N, Daans J, Van der Linden A, Berneman Z, Ponsaerts P: Stem cell therapy for multiple sclerosis: preclinical evidence beyond all doubt? Regen Med 2012, 7:245-259. 3. Willerth SM: Neural tissue engineering using embryonic and induced pluripotent stem cells. Stem Cell Res Ther 2011, 2:17. 4. Feng Z, Gao F: Stem cell challenges in the treatment of neurodegenerative disease. CNS Neurosci Ther 2012, 18:142-148. 5. Ronsyn MW, Berneman ZN, Van Tendeloo VF, Jorens PG, Ponsaerts P: Can cell therapy heal a spinal cord injury? Spinal Cord 2008, 46:532-539. 6. neural stem cell biology and transplantation research. Stem Cell Rev 2012, 8:262-278. 2. Reekmans K, Praet J, De Vocht N, Daans J, Van der Linden A, Berneman Z, Ponsaerts P: Stem cell therapy for multiple sclerosis: preclinical evidence beyond all doubt? Regen Med 2012, 7:245-259. 3. Willerth SM: Neural tissue engineering using embryonic and induced pluripotent stem cells. Stem Cell Res Ther 2011, 2:17. 4. Feng Z, Gao F: Stem cell challenges in the treatment of neurodegenerative disease. CNS Neurosci Ther 2012, 18:142-148. 5. Ronsyn MW, Berneman ZN, Van Tendeloo VF, Jorens PG, Ponsaerts P: Can cell therapy heal a spinal cord injury? Spinal Cord 2008, 46:532-539. 6. Dunnett SB, Rosser AE: Clinical translation of cell transplantation in the brain. Curr Opin Organ Transplant 2011, 16:632-639. 7. Martino G, Pluchino S, Bonfanti L, Schwartz M: Brain regeneration in physiology and pathology: the immune signature driving therapeutic plasticity of neural stem cells. Physiol Rev 2011, 91:1281-1304. 8. Sher F, van Dam G, Boddeke E, Copray S: Bioluminescence imaging of Olig2-neural stem cells reveals improved engraftment in a demyelination mouse model. Stem Cells 2009, 27:1582-1591. 9. Praet J, Reekmans K, Lin D, De Vocht N, Bergwerf I, Tambuyzer B, Daans J, Hens N, Goossens H, Pauwels P, Berneman Z, Van der Linden A, Ponsaerts P: Cell type-associated differences in migration, survival and immunogenicity following grafting in CNS tissue. Cell Transplant 2012. Reekmans et al. Stem Cell Research & Therapy 2012, 3:56 http://stemcellres.com/content/3/6/56 22. Nakanishi M, Niidome T, Matsuda S, Akaike A, Kihara T, Sugimoto H: Microglia-derived interleukin-6 and leukaemia inhibitory factor promote astrocytic differentiation of neural stem/progenitor cells. Eur J Neurosci 2007, 25:649-658. 23. Faijerson J, Tinsley RB, Aprico K, Thorsell A, Nodin C, Nilsson M, Blomstrand F, Eriksson PS: Reactive astrogliosis induces astrocytic differentiation of adult neural stem/progenitor cells in vitro. J Neurosci Res 2006, 84:1415-1424. 24. Worlitzer MM, Bunk EC, Hemmer K, Schwamborn JC: Anti-inflammatory treatment induced regenerative oligodendrogenesis in parkinsonian mice. Stem Cell Res Ther 2012, 3:33. doi:10.1186/scrt147 Cite this article as: Reekmans et al.: Spatiotemporal evolution of early innate immune responses triggered by neural stem cell grafting. Stem Cell Research & Therapy 2012 3:56. 22. Nakanishi M, Niidome T, Matsuda S, Akaike A, Kihara T, Sugimoto H: Microglia-derived interleukin-6 and leukaemia inhibitory factor promote astrocytic differentiation of neural stem/progenitor cells. Eur J Neurosci 2007, 25:649-658. 23. Faijerson J, Tinsley RB, Aprico K, Thorsell A, Nodin C, Nilsson M, Blomstrand F, Eriksson PS: Reactive astrogliosis induces astrocytic differentiation of adult neural stem/progenitor cells in vitro. J Neurosci Res 2006, 84:1415-1424. 24. Worlitzer MM, Bunk EC, Hemmer K, Schwamborn JC: Anti-inflammatory treatment induced regenerative oligodendrogenesis in parkinsonian mice. Stem Cell Res Ther 2012, 3:33. doi:10.1186/scrt147 Cite this article as: Reekmans et al.: Spatiotemporal evolution of early innate immune responses triggered by neural stem cell grafting. Stem Cell Research & Therapy 2012 3:56. 22. Nakanishi M, Niidome T, Matsuda S, Akaike A, Kihara T, Sugimoto H: Microglia-derived interleukin-6 and leukaemia inhibitory factor promote astrocytic differentiation of neural stem/progenitor cells. Eur J Neurosci 2007, 25:649-658. 23. Faijerson J, Tinsley RB, Aprico K, Thorsell A, Nodin C, Nilsson M, Blomstrand F, Eriksson PS: Reactive astrogliosis induces astrocytic differentiation of adult neural stem/progenitor cells in vitro. J Neurosci Res 2006, 84:1415-1424. 24. Worlitzer MM, Bunk EC, Hemmer K, Schwamborn JC: Anti-inflammatory treatment induced regenerative oligodendrogenesis in parkinsonian mice. Stem Cell Res Ther 2012, 3:33. doi:10.1186/scrt147 Cite this article as: Reekmans et al.: Spatiotemporal evolution of early innate immune responses triggered by neural stem cell grafting. Stem Cell Research & Therapy 2012 3:56. References 1. Reekmans K, Praet J, Daans J, Reumers V, Pauwels P, Van der Linden A, Berneman ZN, Ponsaerts P: Current challenges for the advancement of Page 10 of 10 Page 10 of 10 doi:10.1186/scrt147 Cite this article as: Reekmans et al.: Spatiotemporal evolution of early innate immune responses triggered by neural stem cell grafting. Stem Cell Research & Therapy 2012 3:56. Reekmans et al. 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W4390876033.txt	https://vca.univd.edu.ua/index.php/vca/article/download/78/70	uk		КРИМІНОЛОГІЧНА ХАРАКТЕРИСТИКА ОСІБ, ЗАСУДЖЕНИХ ЗА ЗЛОЧИНИ ПРОТИ МОРАЛЬНОСТІ	Vìsnik Krimìnologìčnoï asocìacìï Ukraïni	2,023	cc-by	8,068	ISSN 2304-4756. ВІСНИК КРИМІНОЛОГІЧНОЇ АСОЦІАЦІЇ УКРАЇНИ. 2023. № 3 (30) СУЧАСНІ ТЕОРЕТИЧНІ ТА ПРИКЛАДНІ ПРОБЛЕМИ КРИМІНОЛОГІЇ УДК 343.8 DOI: https://doi.org/10.32631/vca.2023.3.07 Дмитро Євгенійович ЗАЇКА аспірант (Харківський національний університет внутрішніх справ, м. Харків, Україна) КРИМІНОЛОГІЧНА ХАРАКТЕРИСТИКА ОСІБ, ЗАСУДЖЕНИХ ЗА ЗЛОЧИНИ ПРОТИ МОРАЛЬНОСТІ Стаття присвячена комплексному аналізу кримінологічних особливостей осіб, що притягувалися до відповідальності за вчинення злочинів проти моральності. Для виконання поставленої мети автором було проведено дослідження нормативно-правового регулювання, матеріалів 488 вироків суду за ст. 297-304 Кримінального кодексу України та останніх наукових публікацій. На підставі зібраних даних у статті детально проаналізовано кількісно-якісні характеристики осіб, засуджених за злочини проти моральності, їх соціальний статус, освіта, родинні зв'язки та обставин вчинення злочинів. Застосування кримінологічного підходу дозволяє виокремити фактори, які впливають на вчинення злочинів проти моральності, а також визначити можливі шляхи профілактики подібних правопорушень. Автор робить акцент на важливості розуміння механізмів виникнення та динаміки злочинів проти моральності для подальшого удосконалення кримінально-правового регулювання та соціальнопрофілактичних заходів. Результати дослідження можуть слугувати основою для наукових пошуків у сфері кримінології, кримінального та кримінально-виконавчого права, спрямованих на вдосконалення системи протидії злочинам проти моральності та підвищення ефективності соціальних та психологічних програм реабілітації осіб, що вчинили відповідні правопорушення.  86  ISSN 2304-4756. ВІСНИК КРИМІНОЛОГІЧНОЇ АСОЦІАЦІЇ УКРАЇНИ. 2023. № 3 (30) Ключові слова: злочини проти моральності, моральність, кримінологічна характеристика, особа засудженого, злочинність. Постановка проблеми. Злочини проти моральності є загрозою стабільності та безпеки в Україні оскільки характеризуються деструктивним впливом на суспільство, сприяють посиленню конфліктів між різними соціальними групами і підривають довіру до правоохоронних органів і держави в цілому. При цьому, загальний рівень моральності суспільства є одним із головних факторів, що визначає правила та норми поведінки, допомагає уникнути хаосу та внутрішнього розбрату. Виокремлення ключових аспектів кримінологічної характеристики осіб, засуджених за злочини проти моральності, є необхідним етапом для розробки науково обґрунтованих та системних заходів, спрямованих на зменшення рецидиву злочинів проти моральності та соціальну реабілітацію правопорушників. За останні роки дослідженню кримінальних правопорушень проти моральності присвятили свої праці Л. С. Кучанська щодо поняття та системи злочинів проти моральності (2007 р.); О. І. Бандурка щодо кримінальноправової характеристики злочинів проти моральності у сфері статевих стосунків (2010 р.); В. В. Кузнєцов щодо кримінально-правової охорони громадського порядку та моральності (2013 р.); С. Ф. Денисов, В. О. Макаров щодо кримінально-правових санкцій та їх застосування за злочини проти моральності (2017 р.); С. Ф. Денисов та С. Г. Кулик щодо окремих аспектів характеристики та запобігання злочинам проти моральності (2018 р.); А. Р. Топузян щодо кримінально-правової охорони моральності неповнолітніх в Україні (2018 р.); О. М. Ємець щодо захисту суспільної моралі від злочинних посягань (2019 р.); М. В. В’юник, М. В. Карчевський, О. Д. Арланова щодо статистичних показників злочинності в Україні (2020 р.); В. П. Бойченко щодо кримінально-правової охорони суспільної моралі в Україні (2021 р.) та ін. Проте, з урахуванням соціально-політичних перетворень останніх років, динаміки злочинності та змін кримінального законодавства, поточна кримінологічна характеристика осіб, засуджених за злочини проти моральності залишається малодослідженою, що у зумовлює актуальність вибору теми. Метою цієї публікації є дослідження кримінологічної характеристики осіб, засуджених за злочини проти моральності в Україні. Для реалізації вказаної мети було поставлено такі завдання: актуалізувати кримінальноправову характеристику злочинів проти моральності; проаналізувати вироки суду за ст. 297-304 Кримінального кодексу України; дослідити кількісно-якісні показники злочинів проти моральності; вивчити матеріали вже існуючих наукових досліджень; провести порівняльно-правовий аналіз; надати науково обґрунтовані висновки щодо загальної характеристики засуджених за злочини проти моральності. Виклад основного матеріалу. Система злочинів проти моральності, передбачена кримінальним законодавством України, є однією з найбільш  87  ISSN 2304-4756. ВІСНИК КРИМІНОЛОГІЧНОЇ АСОЦІАЦІЇ УКРАЇНИ. 2023. № 3 (30) повних та розвинутих. Цьому, безсумнівно, сприяло відокремлення моральності у якості самостійного об’єкту кримінально-правової охорони1. При проведенні аналізу кількості виявлених правопорушень та вироків суду у справах за ст. 297-304 Кримінального кодексу України було встановлено, що кількість злочинів проти моральності як і злочинів проти громадського порядку характеризується загальною тенденцією до зменшення. Негативна динаміка збільшення прослідковується щодо жорстокого поводження з тваринами, але такий результат пояснюється здебільшого відсутністю належної уваги з боку правоохоронних органів у минулих роках. Сама проблематика набуває дедалі більшого суспільного інтересу, а отже і кількість виявлених випадків і призначених вироків зростає. Новими і майже недослідженими є норми статей 301-1 та 301-2, що з’явились у Кримінальному кодексі лише за законом № 1256-IX від 18.02.2021 та пов’язані із розвитком міжнародних та загальнодержавних програм захисту дітей. Найбільшим у структурі злочинів проти моральності за кількістю виявлених випадків є наруга над могилою, іншим місцем поховання або тілом загиблого. При дослідженні посягання на основні моральні принципи і цінності у сфері духовного і культурного життя суспільства (статті 297, 298, 298-1, 299, 300 та 301-1) було визначено, що спільним суб’єктом злочину за всіма зазначеними статтями є фізична осудна особа, яка досягла віку у 16 років. Щодо ст. 298 КК України, то останні дослідження матеріалів судової практики показали, що суб'єктом вчинення діянь, передбачених у частинах 2, 3 та 4, може бути як стороння особа, так і особа, яка має певний, передбачений нормативно-правовими актами, зв'язок з об'єктом культурної спадщини або пам'яткою національного значення 2. Проте, щодо ч. 5 ст. 298 та ч. 3 ст. 298-1 — будь-яка службова особа, яка вчинила злочин з використанням свого службового становища3. Суб’єктивна сторона злочинів дещо відрізняється. Для засудженого за ст. 298 та 298-1 характерною є наявність як прямого так і непрямого умислу, хоча на практиці більшість правопорушень у сфері використання пам’яток культури здійснюється саме через необережність4. У випадку ст. 297, 299 та 300 необхідною умовою є наявність лише прямого умислу5 6. При цьому щодо ввезення, виготовлення або розповсюдження творів, що пропагують культ насильства і жорстокості, 1 Кучанська Л. С. Поняття та система злочинів проти моральності у кримінальному праві України : автореф. дис. ... канд. юрид. наук : 12.00.08. Київ, 2007. С. 14. 2 Герелюк Т. Б. Кримінально-правова характеристика умисного незаконного знищення, руйнування або пошкодження об'єктів культурної спадщини за законодавством України та країн Східної Європи : дис. ... канд. юрид. наук : 12.00.08. Одеса, 2021. С. 104. 3 Кримінальний кодекс України: закон, кодекс від 05.04.2001 р. № 2341-III. Дата оновлення 05.10.2023. https://zakon.rada.gov.ua/laws/show/2341-14 (дата звернення: 01.11.2023). 4 Каткова Т. Г. Правова охорона культурної спадщини в Україні: монографія. Харків: Право, 2008. С. 109. 5 Кузнецов В. В. Кримінально-правова охорона громадського порядку та моральності : дис. ... док. юрид. наук : 12.00.08. Київ, 2013. С.331, 337 - 339. 6 Сердюк П. П. Кримінологічні та кримінально-правові проблеми ввезення, виготовлення або розповсюдження творів, що пропагують культ насильства і жорстокості : автореф. дис. ... канд. юрид. наук : 12.00.08. Одеса, 2005. С. 13.  88  ISSN 2304-4756. ВІСНИК КРИМІНОЛОГІЧНОЇ АСОЦІАЦІЇ УКРАЇНИ. 2023. № 3 (30) расову, національну чи релігійну нетерпимість та дискримінацію особа має усвідомлювати зміст твору, суспільно небезпечний характер своїх дій і бажати їх вчинення. Крім того, необхідна мета розповсюдження чи збуту зазначених у цій статті предметів1. Дослідження вказують, що у 2019–2023 рр. мало місце суттєве скорочення виявлених фактів наруги над могилою, іншим місцем поховання або над тілом померлого. Якщо у 2013–2018 рр. вона коливалася на рівні 1500–2000 випадків на рік, то в останні роки скоротилася майже у 2 рази та у 2019 р. становила 1085 (–46,6%), а у 2020 р. – 1081 (–0,4%) 2. Схожу динаміку показує і судова статистика. Так, згідно форми 7 річної звітності Судової адміністрації України у 2020 році в Україні за досліджуваною статтею було засуджено 164 особи, а у 2022 лише 87 осіб. Попри прояв явної неповаги до норм моралі та очевидне порушення загальнолюдських правил поведінки, що характерне для будь-якого діяння в межах даного злочину, на відміну від хуліганства, наруга над могилою, іншим місцем поховання або над тілом померлого не має у структурі суб’єктивної сторони складу злочину такого мотиву, як неповага до суспільства. При дослідженні матеріалів кримінальних справ щодо обставин вчинення злочину, передбаченого ст. 297 Кримінального кодексу України, було встановлено, що більшість правопорушень вчинено з корисливих мотивів. У процесі дослідження кримінологічного портрету злочинця, засудженого за ст. 297 Кримінального кодексу України було проаналізовано вибірку із 109 вироків суду та встановлено, що у 82% випадків наруга над могилою була вчинена з метою заволодіння та подальшої реалізації металевих предметів. Злочини вчиняються здебільшого особами чоловічої статі, адже лише 8% з них були вчинені жінками. Варто відзначити, що 44% осіб вже мали судимість та вчинили злочин повторно, що є одним за найбільших показників рецидиву серед кримінальних правопорушень проти громадського порядку та моральності. При цьому 97% засуджених не мали вищої освіти та лише 7% офіційно працевлаштовані. Особа засудженого має здебільшого низький рівень життя та матеріального забезпечення. У більшості випадків вони не працюють, часто мають залежність від алкоголю чи наркотичних засобів, а тому такі посягання мають винятковий характер добування засобів для задоволення своїх життєвих потреб та залежностей. Варто відзначити, що дані дослідження з незначною похибкою відтворюють показники, отримані С. Ф. Денисовим та В. О. Макаровим у 2017 році 3. Щодо ст. 298 та 298-1, то за умов нестабільності сучасного світу, загрози тероризму, природних та техногенних катастроф, міжнаціональних 1 Кримінальне право України: Особлива частина: підручник / за заг. ред. В. В. Сташиса, В. Я. Тація. 4те вид. Харків: Право, 2010. С. 369-376. 2 Вербенський М. Г., Кулик О. Г., Наумова І. В. Кримінальна ситуація в Україні: основні тенденції: 2020 рік : монографія за заг. ред. М. Г. Вербенського. Вінниця: ТВОРИ, 2021. С. 95. 3 Денисов С. Ф., Макаров В. О. Кримінально-правові санкції та їх застосування за злочини проти моральності : монографія. Чернігів : Десна Поліграф, 2017. С. 97-98.  89  ISSN 2304-4756. ВІСНИК КРИМІНОЛОГІЧНОЇ АСОЦІАЦІЇ УКРАЇНИ. 2023. № 3 (30) конфліктів, питання збереження культурної спадщини набувають особливого значення1. Правопорушення безсумнівно є суспільно небезпечними, проте не відноситься до розповсюджених внаслідок особливостей об’єктивної сторони складу злочину2. Так, за останні 10 років судами було призначено лише 11 вироків за ст. 298 та лише 1 за ст. 298-1 (за пошуком доступні 2, але 1 вирок внесено помилково). Близько 16% відкритих кримінальних проваджень доходять до суду та закінчуються обвинувальними вироками. Така ситуація пояснюється тим, що іноді не вдається встановити винних осіб або виявляється, що в діях правопорушників відсутній склад злочину, оскільки та чи інша пам’ятка не занесена до Державного реєстру3. Приблизно 60% посягань на об’єкти культурної спадщини є груповими, з яких групи із 3–6 осіб становлять 40%, групи із 10 і більше осіб – 15–20%. Їх вчиняють особи: від 14 до 18 років – 12%; від 18 до 25 років – 39%; від 25 до 30 років – 21%; від 30 до 40 років – 16%; від 40 до 45 років – 6%; від 45 до 50 років – 5%; старше 50 років – 1%4. Переважна більшість злочинів була вчинена за непрямого умислу, суб’єктом за ст. 298 у 100% випадків виступає чоловік, здебільшого з середньою або професійно-технічною освітою, а за ст. 298-1 жінка, офіційно працевлаштована у підрозділах Національного фонду України, одружена, має вищу освіту. Знищення, руйнування або пошкодження об’єктів культурної спадщини у багатьох випадках вчиняється одноособово раніше не судимими суб’єктами, у яких відсутня стійка антисуспільна спрямованість на вчинення злочинів 5. Щодо кримінологічної характеристики засудженого за жорстоке поводження з тваринами, то основний масив облікованих кримінальних правопорушень – це діяння, передбачене ч. 1 ст. 299 КК України (90%), яке вчинене з хуліганських мотивів (57,0%)6. Злочинцем у 97% випадків є чоловік, характерною є наявність середньої або професійно-технічної освіти, 13% злочинів вчинено пенсіонерами, 29% у стані алкогольного сп’яніння, що є достатньо високим показником у загальній структурі злочинності. При цьому С. Ф. Денисов, М. О. Макаров, О. О. Шуміло та ін. вказують на високий рівень латентності злочину та значний відсоток відмов у порушенні кримінальної справи за фактом жорстокого поводження з тваринами. На думку більшості експертів у галузі зоозахисту, офіційно 1 Каткова Т. Г. Правова охорона культурної спадщини в Україні: монографія. Харків: Право, 2008. С. 12. 2 В’юник М. В., Карчевський М. В., Арланова О. Д. Кримінально-правове регулювання в Україні: реалії та перспективи (аналітичні матеріали) / упоряд. Ю. В. Баулін. Харків : Право, 2020. С. 133. 3 Базелюк В. В. Кримінологічні фактори встановлення кримінальної відповідальності за незаконне проведення пошукових робіт на об’єкті археологічної спадщини, знищення, руйнування або пошкодження об’єктів культурної спадщини. Проблеми законності, 2015. Вип. 128. С. 63-64. 4 Асейкін Р. В. Посягання на об'єкти культурної спадщини: кримінологічна характеристика та запобігання : автореф. дис. ... канд. юрид. наук : 12.00.08. Запоріжжя, 2012. С. 9, 11. 5 Міщенко М. О. Особа злочинця, який вчиняє посягання на культурні цінності. Правові реформи в Україні: реалії сьогодення : Збірники наукових праць, матеріали конференцій (семінарів, круглих столів). 2015. С. 76. 6 Шуміло О. О. Кримінологічна характеристика та запобігання жорстокому поводженню з тваринами : автореф. дис. ... канд. юрид. наук : 12.00.08. Запоріжжя, 2016. С. 3.  90  ISSN 2304-4756. ВІСНИК КРИМІНОЛОГІЧНОЇ АСОЦІАЦІЇ УКРАЇНИ. 2023. № 3 (30) реєструється лише один із десяти тисяч випадків жорстокого поводження з тваринами1 2. Для злочинця, засудженого за ст. 300 КК України характерним є гендерний поділ: усі 100% з досліджених правопорушень були вчинені чоловіками при цьому освіта не має значення, проте характерним є те, що суб’єктом у 38% вчинених правопорушеннях була раніше засуджена особа. Зазвичай злочинні дії полягають у поодинокому продажі програвачів, які за результатами експертиз визнавалися такими, що пропагують культ насильства й жорстокості3. При дослідженні посягання на основні принципи моральності у сфері статевих відносин (статті 301, 302 і 303) було визначено, що загальним суб’єктом злочину за всіма зазначеними статтями є фізична осудна особа, яка досягла 16-ти річного віку4. Суб’єктивна сторона цих злочинів завжди характеризується прямим умислом. Проте, у випадку ст. 301 - особа має усвідомлювати порнографічний зміст твору, зображення чи предмета, суспільно небезпечний характер цього діяння і бажати його вчинення. Обов’язковою ознакою, у тому числі і при зберіганні, є мета збуту чи розповсюдження цих предметів незалежно від мотиву. При цьому в Україні, як і закордоном найбільш поширена мета – розповсюдження, далі збут, не є характерними демонстрація, сприяння оптовим поставкам, використання, оренда, імпорт, експорт, отримання прибутку, транспортування5. Щодо ст. 302 мотиви і мета вчинення злочину можуть бути різними, однак за наявності мети наживи дії винного підлягають кваліфікації за ч. 2 статті. Характерним для ст. 303 є не лише наявність прямого умислу, але і його поєднання з метою втягнення особи у заняття проституцією, а при сутенерстві — наявністю корисливої мети6. Немало дискусій свого часу точилось довкола питання існування кримінальної відповідальності за систиматичне зайняття проституцією, яке у 2006 р. було декриміналізовано. Дослідження показують, що кількість установлених фактів ввезення, виготовлення, збуту і розповсюдження порнографічних предметів (ст. 301) у середньому залишається без змін: у 2014 р. дорівнювала 1152 (+12,8%), у 2016 р. – 925 (–19,7% до рівня 2014 р.), у 2018 р. – 1665 (+80,0% до 2016 р.), у 2019 р. – 1014 (–39,1%) та у 2020 р. – 1153 (+13,7%)7. Суб‘єктом злочину з кримінологічної точки зору у 85% випадків є чоловік, переважно з вищою 1 Денисов С. Ф., Макаров В. О. Кримінально-правові санкції та їх застосування за злочини проти моральності : монографія. Чернігів : Десна Поліграф, 2017. С. 105. 2 Шуміло О. О. Кримінологічна характеристика та запобігання жорстокому поводженню з тваринами : автореф. дис. ... канд. юрид. наук : 12.00.08. Запоріжжя, 2016. С. 3,8. 3 ральності : монографія. Чернігів : Десна Поліграф, 2017. С. 107. 4 Бандурка І. О. Кримінально-правова характеристика злочинів проти моральності у сфері статевих стосунків : автореф. дис. ... канд. юрид. наук : 12.00.08, Запоріжжя, 2010. С. 13. 5 Кузнецов В. В. Кримінально-правова охорона громадського порядку та моральності : дис. ... док. юрид. наук : 12.00.08. Київ, 2013. С.343. 6 Кримінальне право України: Особлива частина: підручник / за заг. ред. В. В. Сташиса, В. Я. Тація. 4те вид. Харків: Право, 2010. С. 377-381. 7 Вербенський М. Г., Кулик О. Г., Наумова І. В. Кримінальна ситуація в Украї ні: основні тенденції: 2020 рік : монографія за заг. ред. М. Г. Вербенського. Вінниця: ТВОРИ, 2021. С. 95-96.  91  ISSN 2304-4756. ВІСНИК КРИМІНОЛОГІЧНОЇ АСОЦІАЦІЇ УКРАЇНИ. 2023. № 3 (30) освітою, що не є характерним для будь-якого іншого злочину вказаної групи. Більшість працюючих вчинили цей злочин задля розваги або, так би мовити, ведучи дорослий спосіб життя, зокрема, знайомлячись у соціальних мережах. Причому 83% із засуджених неодружені1. За результатами проведених опитувань, 71 % користувачів Інтернету заявили про відвідування порно-сайтів щомісячно або частіше. З цього числа опитаних 67 % осіб були у віці від 12 до 30 років 2 3. Злочинне діяння реалізується переважно з використанням мережі інтернет, проте, як зазначає В. П. Бойченко проблема полягає у тому, що повнолітня особа, яка знаходиться у скрутному матеріальному становищі, розміщуючи свої зображення еротичного характеру на веб-сайті чи платформі для повнолітніх за платню має шанси бути виправданою, враховуючи право на недоторканність приватного життя, право на свободу вираження, та право власності на продукти авторського права4. При цьому 96% досліджених злочинів було вчинено особами, що не мають попередньої судимості. Не характерним для злочину є перебування у стані алкогольного чи наркотичного сп‘яніння. Щодо створення або утримання місць розпусти і звідництва, то динаміка загальної кількості зареєстрованих злочинів після зростання у 2014 р. до 509 випадків (+21,2%) наступними роками майже постійно скорочувалася (виключення – 2019 р.: +15,1%) та у 2020 р. знизилася до 163 (–37,1%), тобто за весь цей час зменшилася у 3,1 раза 5. Суб‘єктом злочину у 80% досліджених вироків є жінка, яка має середню або професійно-технічну освіту (87%). У 27% випадків суб‘єкт злочину офіційно працевлаштований на посаді адміністратора банних комплексів чи готелів. З-поміж усіх, лише 7% злочинів було вчинено попередньо засудженими особами. Оскільки приблизно в половині випадків це було звідництво з метою наживи, то можна припускати, що такий рівень рецидиву зумовлений інтересом отримання прибутку, як і сама проституція6. Не характерним є і наявність стану алкогольного сп’яніння. За кількістю зареєстрованих кримінальних правопорушень ситуація з сутенерством схожа на ту, що стосується ввезення, виготовлення, збуту і розповсюдження порнографічних предметів адже зростання мало місце у 2014 р. (303; +17,0%), 2017–2018 рр. (331; +47,8% та 412; +24,5%) та 2020 р. (341; +1,5%) 7. Науковці вказують на тенденції до все більшої латентизації 1 Денисов С. Ф., Макаров В. О. Кримінально-правові санкції та їх застосування за злочини проти моральності : монографія. Чернігів : Десна Поліграф, 2017. С. 109. 2 Шендрик В. В. Боротьба з порнографією: монографія. Харків : Оберіг, 2010. С. 58-59. 3 Шевчук Т. А. Проституція і злочинність : основні взаємозв’язки. Фонові для злочинності явища: запобігання та протидія: зб. тез доп. Всеукр. наук.практ. конф. (м. Харків, 27 квіт. 2018 р.) Харків: ХНУВС, 2018. С.117. 4 Бойченко В. П. Кримінально-правова охорона суспільної моралі в Україні: антропологічний вимір : автореф. дис. ... канд. юрид. наук : 12.00.08. Одеса, 2021. С. 11. 5 Вербенський М. Г., Кулик О. Г., Наумова І. В. Кримінальна ситуація в Україні: основні тенденції: 2020 рік : монографія за заг. ред. М. Г. Вербенського. Вінниця: ТВОРИ, 2021. С. 96. 6 Денисов С. Ф., Макаров В. О. Кримінально-правові санкції та їх застосування за злочини проти моральності : монографія. Чернігів : Десна Поліграф, 2017. С. 112. 7 Вербенський М. Г., Кулик О. Г., Наумова І. В. Кримінальна ситуація в Україні: основні тенденції: 2020 рік : монографія за заг. ред. М. Г. Вербенського. Вінниця: ТВОРИ, 2021. С. 96.  92  ISSN 2304-4756. ВІСНИК КРИМІНОЛОГІЧНОЇ АСОЦІАЦІЇ УКРАЇНИ. 2023. № 3 (30) розглядуваного злочину, що пояснюється, перш за все, тим, що вказаний злочин є так званим «злочином без потерпілих»1. Серед засуджених за сутенерство або втягнення особи в проституцію 78% не мають легального прибутку2. Суб‘єктом злочину у 69% є жінки, для них характерною є наявність середньої або професійно-технічної освіти та відсутність офіційного місця роботи. Усі 100% досліджених злочинів були вчинені вперше, 28% з них групою осіб за попередньою змовою. Не характерним є наявність стану алкогольного чи наркотичного сп‘яніння. Останньою недослідженою групою кримінальних правопорушень є посягання на основні принципи моральності у сфері морального і фізичного розвитку неповнолітніх (ч. 2 ст. 299, частини 2 і 3 ст. 300, частини 2 і 3 ст. 301, ст. 301-1, ст. 301-2, ч. 3 ст. 302, частини 3 і 4 ст. 303, ст. 304). Відповідно до положень постанови пленуму Верховного суду України №2 від 27.02.2004 необхідно враховувати, що відповідальність дорослих осіб за втягнення неповнолітніх у злочинну чи іншу антигромадську діяльність встановлена не тільки ст. 304 Кримінального кодексу України, а й іншими статтями цього Кодексу (ч. 3 ст. 300, ч. 3 ст. 301, ч. 3 ст. 302, ч. 3 ст. 303, ч. 3 ст. 307, ч. 3 ст. 309, ч. 2 ст. 315, ч. 2 ст. 317, статтями 323 і 324), які є спеціальними нормами щодо неї. У разі вчинення злочину, передбаченого спеціальною нормою, кваліфікувати дії винної особи ще й за ст. 304 КК не потрібно. В усіх зазначених вище випадках суб‘єктом злочину є фізична осудна особа, яка на момент вчинення злочину досягла 18-ти річного віку3. Зважаючи на те, що такі злочини можуть бути вчинені тільки з прямим умислом, суди повинні з'ясовувати, чи усвідомлювала доросла особа, що своїми діями втягує неповнолітнього у вчинення злочину або в іншу антигромадську діяльність4. Інтерпретація підвищеного віку суб’єкту злочину ґрунтується, як правило, на твердженнях про особливий характер та специфіку самого діяння, наявність певного життєвого досвіду повнолітніх, певного обсягу соціально значущих знань та навичок5. При цьому щодо ч. 2 ст. 304 слід виділити спеціального суб’єкта - особу, на яку покладено обов’язки щодо виховання потерпілого чи піклування про нього6. Наукових досліджень кримінологічної характеристики незаконного обігу дитячої порнографії за ст. 301-1 Кримінального кодексу України майже не проводилось, оскільки Кримінальний кодекс було доповнено цією 1 Шевчук Т. А. Сутенерство та втягнення особи в заняття проституцією: кримінологічна характеристика та запобігання : автореф. дис. … канд. юрид. наук : 12.00.08. Харків, 2011. 11 с. 2 Денисов С. Ф., Макаров В. О. Кримінально-правові санкції та їх застосування за злочини проти моральності : монографія. Чернігів : Десна Поліграф, 2017. С. 113. 3 Бандурка І. О. Кримінально-правова характеристика злочинів проти моральності у сфері статевих стосунків : автореф. дис. ... канд. юрид. наук : 12.00.08, Запоріжжя, 2010. С. 17. 4 Про застосування судами законодавства про відповідальність за втягнення неповнолітніх у злочинну чи іншу антигромадську діяльність: постанова Верховного Суду України від 27.02.2004 № 2. URL: https://zakon.rada.gov.ua/laws/show/v0002700-04 (дата звернення: 16.10.2021). 5 Топузян А. Р. Кримінально-правова охорона моральності неповнолітніх в Україні : дис. ... канд. юрид. наук : 12.00.08. Харків, 2018. С. 172-173. 6 Кузнецов В. В. Кримінально-правова охорона громадського порядку та моральності : дис. ... док. юрид. наук : 12.00.08. Київ, 2013. С.356.  93  ISSN 2304-4756. ВІСНИК КРИМІНОЛОГІЧНОЇ АСОЦІАЦІЇ УКРАЇНИ. 2023. № 3 (30) статтею лише у 2021 році. Задля проведення кримінологічного аналізу було систематизовано дані за 2021-2022 роки форми статистичної звітності № 6 Державної судової адміністрації України, матеріали 94 вироків суду за 20212023 роки та порівняно з актуальними результатами вітчизняних та зарубіжних наукових досліджень. Субʼєктом злочину є фізична осудна особа, яка досягла 16-ти річного віку. З кримінологічної точки зору у 98,9% випадків злочин був вчинений особами чоловічої статі, 71,2% належать до вікової категорії від 30 до 50 років, 13,3% належать до вікової категорії від 50 до 65 років. Серед засуджених 36,9% з вищою, 63,1% з середньою або професійно-технічною освітою. Аналіз вироків вказує, що 60,8% засуджених були працездатного віку, проте офіційно безробітні, 72,5% - неодружені. Характерно, що більшість злочнів було вчинено особами вперше. Перебування у стані алкогольного чи наркотичного спʼяніння не є нетиповим для вказаного кримінального правопорушення. У абсолютної більшості з-поміж досліджених випадків злочин був вчинений з використанням інформаційно-телекомунікаційних систем чи технологій, що суттєво ускладнює процес виявлення кримінального правопорушення, фіксації та проведення слідчо-розшукових дій. Під час вчинення злочину найчастіше використовуються комп’ютерні пристрої, цифрові фотоапарати, відеокамери та мобільні телефони. За оцінками Міністерства юстиції США у будь-який момент часу існує більше, ніж один мільйон порнографічних зображень дітей в Інтернеті, а кожен день з’являється 200 нових зображень. При цьому, більша частина торгівлі дитячою порнографією відбувається на прихованому рівні в Інтернеті1. До предмету злочину належать: порнографічні тексти – 2,80 %; фотовироби – 33,20 %; зображення – 8,50 %; вироби, знаряддя, сувеніри та побутові предмети – 0,80 %; кіно- або відеопродукція – 35,80 %; звукозаписи – 0,60 %; не фіксована online порнографія – 18,30 %2. Так, вчені зазначають, що переоцінка контактних злочинів, рецидивів та наявності педофілії помірно корелює з підтримкою реєстрації засуджених за злочини, пов’язані з дитячою порнографією і мають високу громадянську підтримку - від 68% до 84%. Поза тим, підтримка лікування замість ув’язнення під час опитування була низькою – 32% і мала слабку негативну кореляцію з передбачуваним ризиком, пов’язаним із рецидивами та поширеністю педофілії3. Для дослідження особи злочинця засудженого за проведення видовищного заходу сексуального характеру за участю неповнолітньої 1 Денисов С. Ф., Макаров В. О. Кримінально-правові санкції та їх застосування за злочини проти моральності : монографія. Чернігів : Десна Поліграф, 2017. С. 47. 2 Телійчук В., Жукоцький В. Отримання інформації з відкритих джерел під час виявлення та документування злочинів, пов’язаних із розповсюдженням порнографічних предметів у мережі “Інтернет”. Науковий вісник Дніпропетровського державного університету внутрішніх справ, 2022. № 1. С. 291. 3 Csepregi K., Kovacks I. Online sexual exploitation of children, in particular the crime of child pornography. Journal of Criminology and Criminal Law, 2022. Vol. 60. P. 1180.  94  ISSN 2304-4756. ВІСНИК КРИМІНОЛОГІЧНОЇ АСОЦІАЦІЇ УКРАЇНИ. 2023. № 3 (30) особи у відкритому доступі ми маємо лише один вирок для ознайомлення (загалом два, проте, один за одним із них інформація заборонена для оприлюднення згідно з пунктом чотири частини першої статті 7 Закону України "Про доступ до судових рішень”), а отже інформація у майбутньому потребуватиме коригування з урахуванням нових матеріалів справ. Поза тим можна стверджувати, що характерною ознакою є використання мережі інтернет та пристроїв відеозв’язку. Суб’єктом даного злочину виступає жінка із середньою освітою, що не працює та раніше не судима. Щодо ст. 304, то динаміка зареєстрованих випадків втягнення неповнолітніх у протиправну діяльність має відносно стабільні показники. Упродовж періоду аналізу їх чисельність постійно зменшувалася (особливо суттєво у 2014 р: –22,1%; 2016 р.: –38,8% та 2019 р.: –34,2%) і загалом скоротилася з 791 у 2013 р. до 154 у 2019 р., тобто у 5,1 раза. У 2020 р. вона трохи зросла до 160 (+3,9%), але все ще залишається низькою1. Кримінологічний портрет злочинця за ст. 304 було досліджено на підставі даних 85 вироків ухвалених суддями України у 2020 - 2023 рр. Лише 3,5% з-поміж загальної кількості складають правопорушення пов’язані з втягненням неповнолітніх у пияцтво, 17,7% втягненням неповнолітніх у жебрацтво та 78,8% з вчиненням злочинів проти власності. З-поміж останніх крадіжки, поєднані з проникненням у житло, інше приміщення чи сховище складають 85 %. Здебільшого неповнолітні (малолітні) використовуються: а) як виконавець чи знаряддя вчинення злочину через менші фізичні розміри і пов’язані з цим додаткові можливості проникнення до житла, приміщень (75 %); б) як засіб убезпечення себе від викриття у злочинній діяльності (13 %); в) для забезпечення функцій пособництва (12 %)2. При втягненні неповнолітніх у злочинну діяльність дорослі особи перебували у таких стосунках з ними: а) знайомі – 20%; б) близькі – 15%; в) у вироках не вказано – 65%3. Серед засуджених високий відсоток розлучених та неодружених (80%), утім, неповні сім’ї все одно стали осередком для дітей втягнення батьками в злочинну чи іншу шкідливу поведінку (28%). Решта ж неповнолітніх із неповних сімей були втягнені в таку діяльність чужими особами4. Характерною особливістю є гендерна залежність складу кримінального правопорушення: лише в 1 з 15 випадків чоловік став суб’єктом втягнення неповнолітнього у жебракування, натомість лише у 7 із 70 випадків жінка стала суб’єктом втягнення неповнолітнього у злочинну діяльність. У 64,6 % випадків винний має одну судимість, у 18,4 % – дві, у 17 % – три і більше. У структурі їх попереднього 1 Вербенський М. Г., Кулик О. Г., Наумова І. В. Кримінальна ситуація в Україні: основні тенденції: 2020 рік : монографія за заг. ред. М. Г. Вербенського. Вінниця: ТВОРИ, 2021. С. 96. 2 Ситнік О. М. Кримінологічна характеристика та запобігання втягненню неповнолітніх у злочинну діяльність в Україні : автореф. дис. ... канд. юрид. наук : 12.00.08. Харків, 2018. С. 7. 3 Однолько І. В. Запобігання втягненню неповнолітніх у злочинну діяльність. Науковий часопис Національної академії прокуратури України, 2016. № 4. С. 152. 4 Денисов С. Ф., Макаров В. О. Кримінально-правові санкції та їх застосування за злочини проти моральності : монографія. Чернігів : Десна Поліграф, 2017. С. 115.  95  ISSN 2304-4756. ВІСНИК КРИМІНОЛОГІЧНОЇ АСОЦІАЦІЇ УКРАЇНИ. 2023. № 3 (30) кримінального досвіду злочини проти власності складають 77 %1. Висновки. Отже, за результатами проведеного дослідження можна констатувати, що за кількісними показниками злочинність у сфері моральності уступає багатьом іншим групам злочинів, проте однозначно становить високу суспільну небезпечність та не може перебувати поза увагою держави. Отримані дані слугують підставою для формулювання рекомендацій щодо вдосконалення чинного законодавства та соціальних програм, спрямованих на профілактику та соціальну реабілітацію засуджених. Важливим є розуміння того, що ефективна боротьба із злочинністю вимагає комплексного підходу, який охоплює як реагування на порушення, так і попередження їх виникнення. У світлі отриманих результатів, з урахуванням обсягу відомостей щодо особи засудженого, пропонується розширити обсяг досліджень, спрямованих на запобігання злочинам проти моральності. СПИСОК ВИКОРИСТАНИХ ДЖЕРЕЛ 1. Csepregi K., Kovacks I. Online sexual exploitation of children, in particular the crime of child pornography. Journal of Criminology and Criminal Law, 2022. Vol. 60. P. 7-12. 2. Асейкін Р. В. Посягання на об'єкти культурної спадщини: кримінологічна характеристика та запобігання : автореф. дис. ... канд. юрид. наук : 12.00.08. Запоріжжя, 2012. 23 с. 3. Базелюк В. В. Кримінологічні фактори встановлення кримінальної відповідальності за незаконне проведення пошукових робіт на об’єкті археологічної спадщини, знищення, руйнування або пошкодження об’єктів культурної спадщини. Проблеми законності, 2015. Вип. 128. С. 59-67. 4. Бандурка І. О. Кримінально-правова характеристика злочинів проти моральності у сфері статевих стосунків : автореф. дис. ... канд. юрид. наук : 12.00.08, Запоріжжя, 2010. 22 с. 5. Бойченко В. П. Кримінально-правова охорона суспільної моралі в Україні: антропологічний вимір : автореф. дис. ... канд. юрид. наук : 12.00.08. Одеса, 2021. 23 с. 6. В’юник М. В., Карчевський М. В., Арланова О. Д. Кримінальноправове регулювання в Україні: реалії та перспективи (аналітичні матеріали) / упоряд. Ю. В. Баулін. Харків : Право, 2020. 212 с. 7. Вербенський М. Г., Кулик О. Г., Наумова І. В. Кримінальна ситуація в Україні: основні тенденції: 2020 рік : монографія за заг. ред. М. Г. Вербенського. Вінниця: ТВОРИ, 2021. 144 с. 8. Герелюк Т. Б. Кримінально-правова характеристика умисного незаконного знищення, руйнування або пошкодження об'єктів культурної 1 Ситнік О. М. Кримінологічна характеристика та запобігання втягненню неповнолітніх у злочинну діяльність в Україні : автореф. дис. ... канд. юрид. наук : 12.00.08. Харків, 2018. С. 8.  96  ISSN 2304-4756. ВІСНИК КРИМІНОЛОГІЧНОЇ АСОЦІАЦІЇ УКРАЇНИ. 2023. № 3 (30) спадщини за законодавством України та країн Східної Європи : дис. ... канд. юрид. наук : 12.00.08. Одеса, 2021. 223 с. 9. Денисов С. Ф., Макаров В. О. Кримінально-правові санкції та їх застосування за злочини проти моральності : монографія. Чернігів : Десна Поліграф, 2017. 328 с. 10. Каткова Т. Г. Правова охорона культурної спадщини в Україні: монографія. Харків: Право, 2008. 216 с. 11. Кримінальне право України: Особлива частина: підручник / за заг. ред. В. В. Сташиса, В. Я. Тація. 4-те вид. Харків: Право, 2010. 608 с. 12. Кримінальний кодекс України: закон, кодекс від 05.04.2001 р. № 2341-III. Дата оновлення 05.10.2023. https://zakon.rada.gov.ua/laws/show/2341-14 (дата звернення: 01.11.2023). 13. Кузнецов В. В. Кримінально-правова охорона громадського порядку та моральності : дис. ... док. юрид. наук : 12.00.08. Київ, 2013. 676 с. 14. Кучанська Л. С. Поняття та система злочинів проти моральності у кримінальному праві України : автореф. дис. ... канд. юрид. наук : 12.00.08. Київ, 2007. 19 с. 15. Міщенко М. О. Особа злочинця, який вчиняє посягання на культурні цінності. Правові реформи в Україні: реалії сьогодення : Збірники наукових праць, матеріали конференцій (семінарів, круглих столів), 2015. С. 75-77. 16. Однолько І. В. Запобігання втягненню неповнолітніх у злочинну діяльність. Науковий часопис Національної академії прокуратури України, 2016. № 4. С. 146–154. 17. Про застосування судами законодавства про відповідальність за втягнення неповнолітніх у злочинну чи іншу антигромадську діяльність: постанова Верховного Суду України від 27.02.2004 № 2. URL: https://zakon.rada.gov.ua/laws/show/v0002700-04 (дата звернення: 16.10.2021). 18. Сердюк П. П. Кримінологічні та кримінально-правові проблеми ввезення, виготовлення або розповсюдження творів, що пропагують культ насильства і жорстокості : автореф. дис. ... канд. юрид. наук : 12.00.08. Одеса, 2005. 21 с. 19. Ситнік О. М. Кримінологічна характеристика та запобігання втягненню неповнолітніх у злочинну діяльність в Україні : автореф. дис. ... канд. юрид. наук : 12.00.08. Харків, 2018. 21 с. 20. Телійчук В., Жукоцький В. Отримання інформації з відкритих джерел під час виявлення та документування злочинів, пов’язаних із розповсюдженням порнографічних предметів у мережі “Інтернет”. Науковий вісник Дніпропетровського державного університету внутрішніх справ, 2022. № 1. С. 290-298. 21. Топузян А. Р. Кримінально-правова охорона моральності неповнолітніх в Україні : дис. ... канд. юрид. наук : 12.00.08. Харків, 2018. 249 с.  97  ISSN 2304-4756. ВІСНИК КРИМІНОЛОГІЧНОЇ АСОЦІАЦІЇ УКРАЇНИ. 2023. № 3 (30) 22. Шевчук Т. А. Проституція і злочинність : основні взаємозв’язки. Фонові для злочинності явища: запобігання та протидія: зб. тез доп. Всеукр. наук.практ. конф. (м. Харків, 27 квіт. 2018 р.) Харків: ХНУВС, 2018. С.117-118. 23. Шевчук Т. А. Сутенерство та втягнення особи в заняття проституцією: кримінологічна характеристика та запобігання : автореф. дис. … канд. юрид. наук : 12.00.08. Харків, 2011. 17 с. 24. Шендрик В. В. Боротьба з порнографією: монографія. Харків : Оберіг, 2010. 215 с. 25. Шуміло О. О. Кримінологічна характеристика та запобігання жорстокому поводженню з тваринами : автореф. дис. ... канд. юрид. наук : 12.00.08. Запоріжжя, 2016. 20 с. Стаття надійшла до редакції 25.10.2023 Dmytro Ye. ZAYIKA (Kharkiv National University of Internal Affairs, Kharkiv, Ukraine) CRIMINOLOGY CHARACTERISTICS OF PERSONS CONVICTED OF CRIMES AGAINST MORALITY The article is devoted to a comprehensive analysis of the criminological characteristics of persons who were prosecuted for committing crimes against morality. To fulfill the set goal, the author conducted a study of legal regulations, materials of 488 court verdicts under Art. 297-304 of the Criminal Code of Ukraine and the latest scientific publications. Based on the collected data, the article analyzes in detail the qualitative characteristics of persons convicted of crimes against morality, their social status, education, family ties, and the circumstances of the crime. The use of the criminological approach allows to single out the factors that influence the commission of crimes against morality, as well as to determine possible ways of prevention of such offenses. The author emphasizes the importance of understanding the mechanisms of occurrence and dynamics of crimes against morality for further improvement of criminal law regulation and social preventive measures. The results of the study can serve as a basis for scientific research in the field of criminology, criminal and criminal law, aimed at improving the system of combating crimes against morality and increasing the effectiveness of social and psychological rehabilitation programs for persons who have committed relevant offenses. Key words: crimes against morality, morality, criminological characteristics, the convicted person, criminality.  98  ISSN 2304-4756. ВІСНИК КРИМІНОЛОГІЧНОЇ АСОЦІАЦІЇ УКРАЇНИ. 2023. № 3 (30) УДК 343.9 DOI: https://doi.org/10.32631/vca.2023.3.08 Сергій Вікторович ЛУК’ЯНЕНКО кандидат юридичних наук (Сумська філія Харківського національного університету внутрішніх справ, м. Суми, Україна) СИСТЕМА ЧИННИКІВ ФОРМУВАННЯ ОСОБИСТОСТІ ЗЛОЧИНЦЯ З’ясовано, що особистість злочинця – це комплекс характеристик людини, що визначають мотивації, цінності, вміння, здібності та інші особистісні якості, що стимулюють злочинну поведінку. Узагальнено, що до чинників, які впливають на формування особистості злочинця, найбільш доцільно віднести наступні: а) соціальні фактори: народження в неблагополучній родині; низький рівень освіти; відсутність роботи, належність до злочинних груп; етнічні та культурні особливості тощо; б) психологічні фактори: знижений рівень емпатії, агресивність, неврівноваженість; низький рівень самоконтролю; депресія; похмілля; тощо; б) емоційні фактори: страх, образа, ненависть, ревнощі, бажання помсти тощо; в) біологічні фактори: генетичні аномалії; психічні розлади; хвороби мозку та інші хвороби; г) ситуаційні фактори: надмірне навантаження; стресові ситуації; важка матеріальна ситуація; наявність зброї тощо. Наголошено на принциповій необхідності осмислення особистості злочинця у новітніх для вітчизняної кримінології злочинах, а також тих, що з огляду на цифровізацію, науково-технічний прогрес та особливі виклики в умовах воєнного стану зазнали суттєвої трансформації. Ключові слова: особа злочинця, злочин, злочинність, злочинна група, чинники, фактори. Постановка проблеми. Особистість злочинця є однією з найголовніших тем кримінологічних досліджень. Власне саме кримінологічне знання зароджувалося навколо осмислення питання внутрішніх основ мислення та діяльності особистості, що стоять за злочинною поведінкою. Розуміння цих основ, розширення масштабу усвідомлення психологічного, соціального, економічного в особистості злочинця породжує розвиток кримінологічного знання. Тому з огляду на специфічні умови воєнного стану, які не лише руйнують українську інфраструктуру та промисловість, а й трансформують мислення людини, позначаючись на найтонших основах свідомості, питання особистості злочинця набуває особливого значення у сучасній кримінологічній доктрині. Проблема розуміння особистості злочинця з огляду на її всеосяжне та засадниче значення у царині кримінології неодноразово ставала предметом дослідження багатьох науковців. Зокрема, їй приділяли увагу О.М. Бандурка, Б.М. Грек, І.М. Даньшин А. П. Закалюк, Л. М. Кононенко, О.М. Костенко,  99  ISSN 2304-4756. ВІСНИК КРИМІНОЛОГІЧНОЇ АСОЦІАЦІЇ УКРАЇНИ. 2023. № 3 (30) О. М. Литвинов, Ю. В. Нікітін, Л.С. Сміян, П. Г. Хоменко та інші вітчизняні та зарубіжні дослідники. Разом із тим, враховуючи принципову цінність того теоретичного матеріалу, що міститься у наукових доробках вказаних дослідників, ми вважаємо, що в умовах цифровізації, науково-технічного прогресу, а останнім часом ще й воєнного стану питання особистості злочинця потребує новітнього осмислення, особливо у ході аналізу воєнних злочинів та цілої низки інших злочинних дій, які набули нових рис після військової агресії. Мета статті полягає у тому, щоб на основі авторського осмислення новітніх аспектів життєдіяльності людини в умовах воєнного стану визначити сутність та структуру особистості злочинця. Задля досягнення вказаної мети необхідно вирішити наступні завдання: розкрити сутність понять злочинець та особистість злочинця; узагальнити наукові думки з приводу структури особистості злочинця. Виклад основного матеріалу. На початку нашого дослідження вважаємо доречним окреслити широту поглядів науковців на проблему особи/особистості злочинця. Авторський колектив у складі М. І. Бажанова, Ю. В. Бауліна, В. І. Борисова та ін. зазначають, що «злочинець – це особа з надзвичайно високим рівнем занепокоєності та невпевненості у собі, імпульсивності та агресивності, відчуженості від суспільних цінностей і корисного спілкування. Це поєднується з високою вразливістю в міжособистісних відносинах, тому такі особи часто застосовують насилля у різних конфліктах. Вони нехтують вимогами правових та моральних норм, відчужені від суспільства та його цінностей, від малих соціальних груп (сім’ї, трудових колективів та ін.), у них погана соціальна пристосовуваність, а відтак виникають чималі труднощі при спробах адаптуватися у тих же малих групах»1. Ми вважаємо погляд науковців досить комплексним, проте ми також стоїмо на тому, що є доволі суперечливою позиція щодо такої деталізації загального визначення даної категорії. Адже, наприклад, елітарній чи білокомірцевій злочинності буде не так властива агресивність, а більше високий рівень освіти та інтелекту в цілому, при цьому, безумовно, із поєднанням корисливості. Тому в ході визначення поняття «злочинець» вважаємо більш доречним апелювати базовими, загальними категоріями, що знаходять своє втілення у різних видах злочинності. С. Ф. Денисов наголошує, що «особа злочинця як різновид особи у загальному розумінні (людини), звісно, повинна мати всі загальні ознаки останньої. Однак кримінологічне вивчення особи злочинця має певну специфіку. Тому треба вказати, що врахування тих чи інших властивостей та якостей особистості повинно залежати не лише від загальних характеристик, а й ураховувати особливості вчиненого злочину або інші 1 Кримінальне право України. Загальна частина: Підручник для юридичних вузів і фак. / М.І.Бажанов, Ю.В.Баулін, В.І.Борисов та ін. За ред. професорів М.І.Бажанова, В.В.Сташиса, В. Я. Тація. Харків: Право, 1997. C. 79-80  100  ISSN 2304-4756. ВІСНИК КРИМІНОЛОГІЧНОЇ АСОЦІАЦІЇ УКРАЇНИ. 2023. № 3 (30) притаманні окремій групі особливостей, наприклад у разі дослідження особи-рецидивіста»1. Поділяючи дану тезу, ми хочемо зауважити на принциповій необхідності осмислення особистості злочинця у новітніх для вітчизняної кримінології злочинах, а також тих, що з огляду на цифровізацію, науковотехнічний прогрес та особливі виклики в умовах воєнного стану зазнали суттєвої трансформації, зокрема шахрайств, пов’язаних із фішингом та вішингом, із проведенням волонтерських зборів для придбання автівок, інших предметів матеріально-технічного забезпечення для Збройних Сил України. Окремого глибинного наукового осмислення потребує також особистість воєнного злочинця, колаборанта, що має принципове значення у ході розслідування воєнних злочинів. А. П. Закалюк визначає особу злочинця як «сукупність соціально типових ознак, які сформувалися у процесі неблагополучного соціального розвитку особи, відрізняються крайньою формою суспільної небезпечності, обумовленою криміногенною мотивацією та кримінальною активністю особи, безпосередньо спричиняють вчинення злочину» 2. Акцентуючи увагу на оригінальності підходу дослідника, ми водночас зазначимо, що категорія «неблагополучний соціальний розвиток» є досить неоднозначною та суперечливою. Адже дослідник не виділяє критерію благополучного та неблагополучного. Варто бути свідомими також, що з часів Ломброзо питання біологічного також має місце в аналізі особистості злочинця поряд із соціальним. Окремо також стоять питання несвідомого, надсвідомого, холотропних станів, архетипних образів, масової свідомості, які мають свій вияв не лише в соціальній площині і мають своєю основою зовсім не соціальні процеси. Цього теж треба бути свідомими у ході широкого осмислення особистості злочинця, яке не зводиться лише до вчиненого особою діяння, особливостей готування та замаху на певний злочин. Окремі із вищеперерахованих категорій можуть мати свої витоки ще навіть до народження людини, накладаючи свій відбиток на все подальше життя. І. М. Даньшин пише, що «особа злочинця це сукупність істотних і стійких соціальних властивостей і ознак, соціально значущих біопсихологічних особливостей індивіда, які, об’єктивно реалізуючись у конкретному вчиненому злочині, надають вчиненому діянню характер суспільної небезпечності, а винній у ньому особі – властивість суспільної небезпечності, в зв’язку з чим вона і притягується до відповідальності, передбаченої кримінальним законом»3. Погоджуючись із більшістю положень даного визначення, ми 1 Денисов С. Ф. Особа злочинця у кримінологічній теорії України. Вісник Кримінологічної асоціації України. 2020. № 1 (22). С. 152-159. 2 Закалюк А.П. Курс сучасної української кримінології: теорія і практика: у 3 кн. Кн. 1. Київ : Видавничий Дім «Ін Юре», 2007. C. 239. 3 Даньшин І.М. Кримінологія: Загальна та Особлива частини: підручник для студентів юрид. спец. вищ. навч. закладів. Харків : Право, 2003. C. 63.  101  ISSN 2304-4756. ВІСНИК КРИМІНОЛОГІЧНОЇ АСОЦІАЦІЇ УКРАЇНИ. 2023. № 3 (30) зауважимо, що дослідник зводить особу злочинця до сукупності властивостей і ознак, що є соціально значущими. Проте, на наше глибоке переконання, дані ознаки і властивості можуть і не бути соціально значущими, тобто тими, що знаходять своє широке поширення у суспільстві, турбують значну кількість людей. Ми стоїмо на тому, що особистість кожного злочинця є унікальною, адже процес виховання у дошкільній установі, навчання у школі, подальшої освіти, соціальне оточення, захоплення, уподобання, морально-етичні якості, внутрішні цінності, ідеологічні установки є у своїй сукупності принципово унікальними на рівні певної особистості. В. Є. Загородній вказує, що до ознак особи злочинця належать: це – сукупність суттєвих та усталених соціальних властивостей та ознак і соціально значущих біопсихічних особливостей індивіду, які, об’єктивно реалізуючись у конкретному злочині, надають вчиненому діянню характер суспільної небезпеки, а винному – властивість суспільної небезпечності, у зв’язку з чим особа притягається до кримінальної відповідальності, передбаченої кримінальним законом. Узагальнене поняття особи злочинця є певною абстракцією, поки воно не наповнюється конкретним змістом, зокрема соціально-демографічною, психологічною та моральною 1 характеристиками . Таким чином, узагальнює автор, особистість злочинця це комплекс особистісних характеристик, що визначають мотивації, цінності, вміння, здібності та інші особистісні якості, що стимулюють злочинну поведінку2. Погляд дослідника органічно втілює в собі ключові підходи до визначення особистості злочинця, проте ми вважаємо, що є досить суперечливим вважати, що цей комплекс зводиться виключно до особистісних якостей. Велику роль в окремих випадках можуть відігравати колективні фактори, які у тому числі враховувалися у ході розробки технологій управління масовою свідомістю. Значну роль у формуванні особистості відіграє родина як первинний соціальний інститут, трудовий колектив, освітнє середовище. Тому ми вважаємо більш точним розглядати особистість злочинця крізь призму як особистісних, так і тих, що мають місце у ході соціальної взаємодії. Ми цілком погоджуємося із позицією М.І. Фіалки, який зазначає, що термін «особа» слід використовувати для позначення конкретної людини в межах тієї чи іншої життєвої ситуації; термін «особистість» необхідно використовувати в межах кримінологічного дослідження як зовнішнього, так і внутрішнього змісту людини, яка або схильна до вчинення кримінального правопорушення, або вчинила кримінальне правопорушення3. 1 Загородній В. Є. Криміналістичне значення відомостей про особу злочинця // Європейський вибір України, розвиток науки та національна безпека в реаліях масштабної військової агресії та глобальних викликів ХХІ століття» : матеріали Міжнар.наук.-практ. конф. (м. Одеса, 17 червня 2022 р.) / за загальною редакцією С. В. Ківалова. Одеса : Видавничий дім «Гельветика», 2022. Т. 2. С. 539-541. 2 Так само. 3 Фіалка М. І. Кримінальний правопорушник: особа чи особистість (до проблеми термінологічного  102  ISSN 2304-4756. ВІСНИК КРИМІНОЛОГІЧНОЇ АСОЦІАЦІЇ УКРАЇНИ. 2023. № 3 (30) Так і ми стоїмо на принциповій важливості виокремлення категорій «особа, яка вчинила злочин» та «особистість злочинця». Вони не є тотожними та співвідносяться як частина та ціле. При чому доречно наголосити, що покликанням кримінології є глибинне дослідження саме особистості злочинця, виведення із разових якісних чи кількісних ознак, характерних для осіб, що вчинили певні злочини, знань із інших соціальних, економічних та правових дисциплін вищого конструкту особистості. І. О. Бандурка вказує, що «поняття особистості злочинця включає в себе цілий комплекс соціально-демографічних, соціально-функціональних (рольових), соціально-психологічних і кримінально-правових ознак, які в тій чи іншій мірі пов’язані з злочинною дією (бездіяльністю), характеризують його суспільну небезпеку, пояснюють причини вчинення злочину»1. На основі проаналізованих поглядів дослідників та авторської позиції ми під особистістю злочинця розуміємо соціальні, біологічні, психологічні, економічні, зовнішні та внутрішні, свідомі, несвідомі, надсвідомі та ті, що знаходять свій вияв у масовій свідомості, характеристики певної людини яка вчинили або потенційно може вчинити кримінальне правопорушення. Загалом, структура особистості злочинця повинна мати всі ознаки, властивості, якості та інші показники, які у своїй сукупності характеризують особу, яка нехтує законом, порушує заборони, притягується до кримінальної відповідальності та заслуговує покарання. При цьому структура особи злочинця повинна відображати не лише різноманітність ознак, що її утворюють, і різновид характеру, але і різну їх роль в етіології злочинної поведінки2. О. М. Литвинов наголошує на тому, що варто бути свідомим динамічної функціональної структури особистості. У ній виділяють чотири типи основних підструктур особистості, які знаходяться одна над одною. Нижньою підструктурою особистості є біологічно обумовлені якості: темперамент, рефлекси, патології психіки або обдарованість людини. Над нею знаходиться підструктура досвіду, тобто отримані упродовж життя знання, вміння, навички, вчинки. Верхньою підструктурою особистості є спрямованість особистості. Це система установок (диспозицій), ціннісні орієнтації, установки на певні види діяльності та вчинків. Всі чотири підструктури особистості приймають участь у регулюванні поведінки людини3. Висновки. Таким чином, підбиваючи підсумок представленого наукового дослідження, слід узагальнити, що до чинників, які впливають на особистість злочинця, найбільш доцільно віднести наступні: закріплення). Вісник Кримінологічної асоціації України. 2021. № 2 (25). С. 117. 1 Бандурка І. О. Кримінологічна характеристика особистості неповнолітнього злочинця. Наше право. 2014. № 6. С. 92-100. 2 Денисов С. Ф. Особа злочинця у кримінологічній теорії України. Вісник Кримінологічної асоціації України. 2020. № 1 (22). С. 152-159. 3 Литвинов О. М. Кримінологія: питання та відповіді : навчальний посібник. Харків : Золота миля, 2015. 324 с.  103  ISSN 2304-4756. ВІСНИК КРИМІНОЛОГІЧНОЇ АСОЦІАЦІЇ УКРАЇНИ. 2023. № 3 (30) - соціальні фактори: народження в неблагополучній родині; низький рівень освіти; відсутність роботи, належність до злочинних груп; етнічні та культурні особливості тощо. - психологічні фактори: знижений рівень емпатії, агресивність, неврівноваженість; низький рівень самоконтролю; депресія; похмілля; тощо. - емоційні фактори: страх, образа, ненависть, ревнощі, бажання помсти тощо; - біологічні фактори: генетичні аномалії; психічні розлади; хвороби мозку та інші хвороби. - ситуаційні фактори: надмірне навантаження; стресові ситуації; важка матеріальна ситуація; наявність зброї тощо. Так, дане питання потребує якнайширшого осмислення у контексті останніх викликів правового режиму воєнного стану. Наголошуємо на принциповій необхідності осмислення особистості злочинця у новітніх для вітчизняної кримінології злочинах, а також тих, що з огляду на цифровізацію, науково-технічний прогрес та особливі виклики в умовах воєнного стану зазнали суттєвої трансформації. Дані питання мають ставати предметами широкого обговорення наукової спільноти, громадськості, творчої інтелігенції, а також представників освітнього середовища з метою спільного опрацювання механізмів виховного впливу на особистість злочинця в контексті викликів сучасності. СПИСОК ВИКОРИСТАНИХ ДЖЕРЕЛ 1. Кримінальне право України. Загальна частина: Підручник для юридичних вузів і фак. / М. І. Бажанов, Ю. В. Баулін, В. І. Борисов та ін. За ред. професорів М. І. Бажанова, В. В. Сташиса, В. Я. Тація. Харків: Право, 1997. 368 с. 2. Денисов С. Ф. Особа злочинця у кримінологічній теорії України. Вісник Кримінологічної асоціації України. 2020. № 1(22). С. 152-159. 3. Закалюк А.П. Курс сучасної української кримінології: теорія і практика: у 3 кн. Кн. 1. Київ : Видавничий Дім «Ін Юре», 2007. 424 с. 4. Даньшин І.М. Кримінологія: Загальна та Особлива частини: підручник для студентів юрид. спец. вищ. навч. закладів. Харків : Право, 2003. 351 с. 5. Загородній В. Є. Криміналістичне значення відомостей про особу злочинця. Європейський вибір України, розвиток науки та національна безпека в реаліях масштабної військової агресії та глобальних викликів ХХІ століття : матеріали Міжнар.наук.-практ. конф. (м. Одеса, 17 червня 2022 р.) / за загальною редакцією С. В. Ківалова. Одеса : Видавничий дім «Гельветика», 2022. Т. 2. С. 539-541. 6. Фіалка М. І. Кримінальний правопорушник: особа чи особистість (до проблеми термінологічного закріплення). Вісник Кримінологічної асоціації України. 2021. № 2(25). С. 108-118.  104  ISSN 2304-4756. ВІСНИК КРИМІНОЛОГІЧНОЇ АСОЦІАЦІЇ УКРАЇНИ. 2023. № 3 (30) 7. Бандурка І. О. Кримінологічна характеристика особистості неповнолітнього злочинця. Наше право. 2014. № 6. С. 92-100. 8. Литвинов О. М. Кримінологія: питання та відповіді : навчальний посібник. Харків : Золота миля, 2015. 324 с. Стаття надійшла до редакції 18.11.2023 Serhii V. LUKYANENKO PhD in Law (Sumy branch of Kharkiv National University of Internal Affairs, Sumy, Ukraine) SYSTEM OF FACTORS FOR FORMING THE PERSONALITY OF A CRIMINAL It was found that the personality of a criminal is a complex of personal characteristics that determine motivations, values, skills, abilities and other personal qualities that stimulate criminal behavior. In general, it is most appropriate to include the following as factors that influence the formation of a criminal's personality: a) social factors: birth in a dysfunctional family; low level of education; lack of work, belonging to criminal groups; ethnic and cultural characteristics, etc.; b) psychological factors: reduced level of empathy, aggressiveness, imbalance; low level of self-control; depression; hangover; etc; b) emotional factors: fear, resentment, hatred, jealousy, desire for revenge, etc.; c) biological factors: genetic abnormalities; mental disorders; brain diseases and other diseases; d) situational factors: excessive workload; stressful situations; difficult financial situation; availability of weapons, etc. It is emphasized the fundamental need to understand the identity of the criminal in the newest crimes for domestic criminology, as well as those that have undergone a significant transformation due to digitalization, scientific and technological progress and special challenges in the conditions of martial law. Key words: the person of the criminal, crime, criminality, criminal group, factors, factors.  105 
https://openalex.org/W4388831470	https://peerj.com/articles/16146v0.3/submission	English	null	Peer Review #5 of "Determining Minnesota bee species’ distributions and phenologies with the help of participatory science (v0.2)"	null	2,023	cc-by	34,957	Manuscript to be reviewed 1 Determining Minnesota bee species’ distributions and 2 phenologies with the help of participatory science 3 4 Colleen D. Satyshur1, Elaine C. Evans2,3, Britt M. Forsberg3, Thea A. Evans1, Robert B. Blair4 1 Determining Minnesota bee species’ distributions and 2 phenologies with the help of participatory science 3 4 Colleen D. Satyshur1, Elaine C. Evans2,3, Britt M. Forsberg3, Thea A. Evans1, Robert B. Blair4 5 6 1 Department of Ecology, Evolution, and Behavior, University of Minnesota, St. Paul, MN, USA 7 2 Department of Entomology, University of Minnesota, St. Paul, MN, USA 8 3 University of Minnesota Extension, University of Minnesota, St. Paul, MN, USA 9 4 Department of Fisheries, Wildlife, and Conservation Biology, University of Minnesota, St. 10 Paul, MN, USA 11 12 Corresponding Author: 13 Colleen Satyshur1 14 100 Ecology Building, 1987 Upper Buford Circle, St. Paul, MN, 55108, USA 15 Email address: csatyshu@umn.edu 16 17 Abstract 18 The Minnesota Bee Atlas project contributed new information about bee distributions, 19 phenologies, and community structure by mobilizing participatory science volunteers to 20 document bees statewide. Volunteers submitted iNaturalist (© California Academy of Sciences 21 2016) photograph observations, monitored nest-traps for tunnel-nesting bees, and conducted 22 roadside observational bumble bee surveys. By pairing research scientists and participatory 23 science volunteers, we overcame geographic and temporal challenges to document the presence, 24 phenologies, and abundances of species. Minnesota Bee Atlas project observations included new 25 state records for Megachile inimica, Megachile frugalis, Megachile sculpturalis, Osmia 26 georgica, Stelis permaculata, and Bombus nevadensis, nesting phenology for 17 species, 27 documentation of bivoltinism for Megachile relativa in Minnesota, and over 500 observations of 28 the endangered species Bombus affinis. We also expanded known ranges for 16 bee species 29 compared with specimens available from the University of Minnesota (UMN) Insect Collection. 30 Surveys with standardized effort across the state found ecological province associations for six 31 tunnel-nesting species and lower bumble bee abundance in the Prairie Parkland ecological 32 i h h L i Mi d F E B dl f F l i l i 17 Abstract 18 The Minnesota Bee Atlas project contributed new information about bee distributions, 19 phenologies, and community structure by mobilizing participatory science volunteers to 20 document bees statewide. Volunteers submitted iNaturalist (© California Academy of Sciences 21 2016) photograph observations, monitored nest-traps for tunnel-nesting bees, and conducted 22 roadside observational bumble bee surveys. By pairing research scientists and participatory 23 science volunteers, we overcame geographic and temporal challenges to document the presence, 24 phenologies, and abundances of species. Minnesota Bee Atlas project observations included new 25 state records for Megachile inimica, Megachile frugalis, Megachile sculpturalis, Osmia 26 georgica, Stelis permaculata, and Bombus nevadensis, nesting phenology for 17 species, 27 documentation of bivoltinism for Megachile relativa in Minnesota, and over 500 observations of 28 the endangered species Bombus affinis. We also expanded known ranges for 16 bee species 29 compared with specimens available from the University of Minnesota (UMN) Insect Collection. 30 Surveys with standardized effort across the state found ecological province associations for six 31 tunnel-nesting species and lower bumble bee abundance in the Prairie Parkland ecological 32 province than the Laurentian Mixed Forest or Eastern Broadleaf Forest ecological provinces, 33 indicating potential benefit of a focus on bumble bee habitat management in the Prairie Parkland. 34 Landcover analysis found associations for four tunnel-nesting species, as well as a possible 35 association of B. affinis with developed areas. These data can inform management decisions 36 affecting pollinator conservation and recovery of endangered species. By engaging over 2,500 37 members of the public, we also promoted conservation action for pollinators through our 38 educational programs and interactions. Determining Minnesota bee species’ distributions and phenologies with the help of participatory science Colleen D. Satyshur Corresp., 1 , Elaine C. Evans 2, 3 , Britt M. Forsberg 2 , Thea A. Evans 1 , Robert B. Blair 4 1 Department of Ecology, Evolution and Behavior, University of Minnesota, St. Paul, Minnesota, United States 2 University of Minnesota Extension, University of Minnesota, St. Paul, Minnesota, United States 3 Department of Entomology, University of Minnesota, St. Paul, Minnesota, United States 4 Department of Fisheries, Wildlife and Conservation Biology, University of Minnesota, St. Paul, Minnesota, United States Corresponding Author: Colleen D. Satyshur Email address: csatyshu@umn.edu The Minnesota Bee Atlas project contributed new information about bee distributions, phenologies, and community structure by mobilizing participatory science volunteers to document bees statewide. Volunteers submitted iNaturalist (© California Academy of Sciences 2016) photograph observations, monitored nest-traps for tunnel-nesting bees, and conducted roadside observational bumble bee surveys. By pairing research scientists and participatory science volunteers, we overcame geographic and temporal challenges to document the presence, phenologies, and abundances of species. Minnesota Bee Atlas project observations included new state records for Megachile inimica, Megachile frugalis, Megachile sculpturalis, Osmia georgica, Stelis permaculata, and Bombus nevadensis, nesting phenology for 17 species, a new documentation of bivoltinism for Megachile relativa in Minnesota, and over 500 observations of the endangered species Bombus aﬃnis. We also expanded known ranges for 16 bee species compared with specimens available from the University of Minnesota (UMN) Insect Collection. Surveys with standardized eﬀort across the state found ecological province associations for six tunnel- nesting species and lower bumble bee abundance in the Prairie Parkland ecological province than the Laurentian Mixed Forest or Eastern Broadleaf Forest ecological provinces, indicating potential beneﬁt of a focus on bumble bee habitat management in the Prairie Parkland. Landcover analysis found associations for four tunnel-nesting species, as well as a possible association of B. aﬃnis with developed areas. These data can inform management decisions aﬀecting pollinator conservation and recovery of endangered species. By engaging over 2500 volunteers, we also promoted conservation action for pollinators through our educational programs and interactions. PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed 1 Determining Minnesota bee species’ distributions and 2 phenologies with the help of participatory science 3 4 Colleen D. Satyshur1, Elaine C. Evans2,3, Britt M. Forsberg3, Thea A. Evans1, Robert B. Blair4 5 6 1 Department of Ecology, Evolution, and Behavior, University of Minnesota, St. Paul, MN, US 7 2 Department of Entomology, University of Minnesota, St. Paul, MN, USA 8 3 University of Minnesota Extension, University of Minnesota, St. Determining Minnesota bee species’ distributions and phenologies with the help of participatory science Colleen D. Satyshur Corresp., 1 , Elaine C. Evans 2, 3 , Britt M. Forsberg 2 , Thea A. Evans 1 , Robert B. Blair 4 Paul, MN, USA 9 4 Department of Fisheries, Wildlife, and Conservation Biology, University of Minnesota, St. 10 Paul, MN, USA 11 12 Corresponding Author: 13 Colleen Satyshur1 14 100 Ecology Building, 1987 Upper Buford Circle, St. Paul, MN, 55108, USA 15 Email address: csatyshu@umn.edu 16 17 Abstract 18 The Minnesota Bee Atlas project contributed new information about bee distributions, 19 phenologies, and community structure by mobilizing participatory science volunteers to 20 document bees statewide. Volunteers submitted iNaturalist (© California Academy of Sciences 21 2016) photograph observations, monitored nest-traps for tunnel-nesting bees, and conducted 22 roadside observational bumble bee surveys. By pairing research scientists and participatory 23 science volunteers, we overcame geographic and temporal challenges to document the presence 24 phenologies, and abundances of species. Minnesota Bee Atlas project observations included new 25 state records for Megachile inimica, Megachile frugalis, Megachile sculpturalis, Osmia 26 georgica, Stelis permaculata, and Bombus nevadensis, nesting phenology for 17 species, 27 documentation of bivoltinism for Megachile relativa in Minnesota, and over 500 observations o 28 the endangered species Bombus affinis. We also expanded known ranges for 16 bee species 29 compared with specimens available from the University of Minnesota (UMN) Insect Collection 30 Surveys with standardized effort across the state found ecological province associations for six 31 tunnel-nesting species and lower bumble bee abundance in the Prairie Parkland ecological 32 province than the Laurentian Mixed Forest or Eastern Broadleaf Forest ecological provinces, 33 indicating potential benefit of a focus on bumble bee habitat management in the Prairie Parklan 34 Landcover analysis found associations for four tunnel-nesting species, as well as a possible 35 association of B. affinis with developed areas. These data can inform management decisions 36 affecting pollinator conservation and recovery of endangered species. By engaging over 2,500 37 members of the public, we also promoted conservation action for pollinators through our 38 educational programs and interactions. 39 40 Introduction 41 While bees are widely recognized for their important role in food security and the maintenance 42 of ecological integrity (Klein et al., 2007; Ollerton, Winfree & Tarrant, 2011), the monitoring 1 Determining Minnes 2 phenologies with th 3 4 Colleen D. Satyshur1, Elaine C. Determining Minnesota bee species’ distributions and phenologies with the help of participatory science Colleen D. Satyshur Corresp., 1 , Elaine C. Evans 2, 3 , Britt M. Forsberg 2 , Thea A. Evans 1 , Robert B. Blair 4 Ev 5 6 1 Department of Ecology, Evolution 7 2 Department of Entomology, Univ 8 3 University of Minnesota Extensio 9 4 Department of Fisheries, Wildlife 10 Paul, MN, USA 11 12 Corresponding Author: 13 Colleen Satyshur1 14 100 Ecology Building, 1987 Upper 15 Email address: csatyshu@umn.edu 16 17 Abstract 18 The Minnesota Bee Atlas project c 19 phenologies, and community struct 20 document bees statewide. Voluntee 21 2016) photograph observations, mo 22 roadside observational bumble bee 23 science volunteers, we overcame g 24 phenologies, and abundances of sp 25 state records for Megachile inimica 26 georgica, Stelis permaculata, and B 27 documentation of bivoltinism for M 28 the endangered species Bombus aff 29 compared with specimens available 30 Surveys with standardized effort ac 31 tunnel-nesting species and lower bu 32 province than the Laurentian Mixed 33 indicating potential benefit of a foc 34 Landcover analysis found associati 35 association of B. affinis with develo 36 affecting pollinator conservation an 37 members of the public, we also pro 38 educational programs and interactio 39 40 Introduction 41 While bees are widely recognized f 42 of ecological integrity (Klein et al 1 Determining Minnesota bee speci 2 phenologies with the help of parti 3 4 Colleen D. Satyshur1, Elaine C. Evans2,3, Britt M. Forsberg 5 6 1 Department of Ecology, Evolution, and Behavior, Univer 7 2 Department of Entomology, University of Minnesota, St. 8 3 University of Minnesota Extension, University of Minnes 9 4 Department of Fisheries, Wildlife, and Conservation Biol 10 Paul, MN, USA 11 12 Corresponding Author: 13 Colleen Satyshur1 14 100 Ecology Building, 1987 Upper Buford Circle, St. Paul 15 Email address: csatyshu@umn.edu 16 17 Abstract 18 The Minnesota Bee Atlas project contributed new informat 19 phenologies, and community structure by mobilizing partic 20 document bees statewide. Volunteers submitted iNaturalist 21 2016) photograph observations, monitored nest-traps for tu 22 roadside observational bumble bee surveys. By pairing rese 23 science volunteers, we overcame geographic and temporal 24 phenologies, and abundances of species. Minnesota Bee At 25 state records for Megachile inimica, Megachile frugalis, M 26 georgica, Stelis permaculata, and Bombus nevadensis, nest 27 documentation of bivoltinism for Megachile relativa in Mi 28 the endangered species Bombus affinis. Determining Minnesota bee species’ distributions and phenologies with the help of participatory science Colleen D. Satyshur Corresp., 1 , Elaine C. Evans 2, 3 , Britt M. Forsberg 2 , Thea A. Evans 1 , Robert B. Blair 4 We also expanded 29 compared with specimens available from the University of 30 Surveys with standardized effort across the state found eco 31 tunnel-nesting species and lower bumble bee abundance in 32 province than the Laurentian Mixed Forest or Eastern Broa 33 indicating potential benefit of a focus on bumble bee habita 34 Landcover analysis found associations for four tunnel-nesti 35 association of B. affinis with developed areas. These data c 36 affecting pollinator conservation and recovery of endanger 37 members of the public, we also promoted conservation acti 38 educational programs and interactions. 39 40 Introduction 41 While bees are widely recognized for their important role i 42 of ecological integrity (Klein et al., 2007; Ollerton, Winfre Manuscript to be reviewed Participatory science contributions can provide complementary and 71 widespread records across locations and time, contributing observations earlier in the season and 72 of a significantly broader distribution than professional datasets alone (van der Wal et al., 2015; 73 Soroye, Ahmed & Kerr, 2018; Dubaić et al., 2022). Structured participatory science projects in 74 North America and Europe have also produced data of sufficient quality to be used in 75 monitoring, conservation, and management (Kremen, Ullman & Thorp, 2011; Appenfeller, 76 Lloyd & Szendrei, 2020; Koffler et al., 2021), documented natural history traits such as nesting 77 and seasonality (Lye et al., 2012; Maher, Manco & Ings, 2019; Olsen et al., 2020) and increased 78 conservation action (Ganzevoort & van den Born, 2021; Griffin et al., 2021). 79 80 In this study, we leveraged the power of participatory science to investigate bee distribution, 81 nesting phenology, and community structure across the state of Minnesota in the U.S. We 82 engaged volunteers in three tiers of sampling rigor: 1) casual observations of all bee species 83 using the mobile app and website iNaturalist.org (© California Academy of Sciences 2016), 2) 84 nest-trap surveys of tunnel-nesting bees, and 3) observational bumble bee surveys. The three 85 tiers of sampling rigor represent increasing levels of volunteer training and commitment and 86 yielded different data types. The iNaturalist observations required minimal training and flexible 43 and baseline information necessary for regional bee conservation is often missing (Cardoso et al., 44 2011; Lebuhn et al., 2013). Without such data on species distributions, habitat associations, and 45 phenology, it is difficult to understand if or how bee communities are changing or how to enact 46 conservation practices. Knowing species distributions and estimates of abundance can help 47 prioritize management and conservation efforts (Cardoso et al., 2011). For example, species with 48 small geographic distributions are at higher risk of extinction (Gaston & Fuller, 2009). Habitat 49 associations are also important because bees are often closely tied to plant communities (Potts et 50 al., 2003; Sheffield & Heron, 2019) and habitat needs such as nest sites (Potts et al., 2003; 51 Harmon-Threatt, 2020). In addition, establishing phenology baselines is important to 52 understanding the ecological role of bee species and how climate change impacts ecosystems 53 now and in the future (Burkle, Marlin & Knight, 2013; Ogilvie & Forrest, 2017). Manuscript to be reviewed 54 55 The importance of baseline information has led to calls for developing national survey and 56 monitoring programs to support state-based pollinator conservation plans (Woodard et al., 2020). 57 While recent efforts list over 500 bee species in Minnesota (Portman et al., 2023), the 58 distribution, population, and life history traits such as nesting phenology, often remain unknown. 59 There are four distinct ecological provinces in the state: Prairie Parklands (PP), Tallgrass Aspen 60 Parklands (TAP), Eastern Broadleaf Forest (EBF), and Laurentian Mixed Forest (LMF) 61 (Minnesota Department of Natural Resources, 2023). The effort and funds required to survey 62 these ecologically different areas of the state for insect pollinators are a challenge. Additionally, 63 commonly used methods for studying insects require extensive specimen collection and 64 taxonomic expertise for species-level identification for most groups, which can also be expensive 65 (Woodard et al., 2020). 66 67 Inviting the public to participate in scientific research can help overcome geographic and 68 temporal challenges of bee monitoring. Here we use the term participatory science (sometimes 69 called citizen science or community science) to indicate volunteer participants who are not 70 monetarily compensated. Participatory science contributions can provide complementary and 71 widespread records across locations and time, contributing observations earlier in the season and 72 of a significantly broader distribution than professional datasets alone (van der Wal et al., 2015; 73 Soroye, Ahmed & Kerr, 2018; Dubaić et al., 2022). Structured participatory science projects in 74 North America and Europe have also produced data of sufficient quality to be used in 75 monitoring, conservation, and management (Kremen, Ullman & Thorp, 2011; Appenfeller, 76 Lloyd & Szendrei, 2020; Koffler et al., 2021), documented natural history traits such as nesting 77 and seasonality (Lye et al., 2012; Maher, Manco & Ings, 2019; Olsen et al., 2020) and increased 78 conservation action (Ganzevoort & van den Born, 2021; Griffin et al., 2021). 79 80 In this study, we leveraged the power of participatory science to investigate bee distribution, 81 nesting phenology, and community structure across the state of Minnesota in the U.S. We 82 engaged volunteers in three tiers of sampling rigor: 1) casual observations of all bee species 83 using the mobile app and website iNaturalist.org (© California Academy of Sciences 2016), 2) 84 nest-trap surveys of tunnel-nesting bees, and 3) observational bumble bee surveys. Manuscript to be reviewed 43 and baseline information necessary for regional bee conservation is often missing (Cardoso et al., 44 2011; Lebuhn et al., 2013). Without such data on species distributions, habitat associations, and 45 phenology, it is difficult to understand if or how bee communities are changing or how to enact 46 conservation practices. Knowing species distributions and estimates of abundance can help 47 prioritize management and conservation efforts (Cardoso et al., 2011). For example, species with 48 small geographic distributions are at higher risk of extinction (Gaston & Fuller, 2009). Habitat 49 associations are also important because bees are often closely tied to plant communities (Potts et 50 al., 2003; Sheffield & Heron, 2019) and habitat needs such as nest sites (Potts et al., 2003; 51 Harmon-Threatt, 2020). In addition, establishing phenology baselines is important to 52 understanding the ecological role of bee species and how climate change impacts ecosystems 53 now and in the future (Burkle, Marlin & Knight, 2013; Ogilvie & Forrest, 2017). 54 55 The importance of baseline information has led to calls for developing national survey and 56 monitoring programs to support state-based pollinator conservation plans (Woodard et al., 2020). 57 While recent efforts list over 500 bee species in Minnesota (Portman et al., 2023), the 58 distribution, population, and life history traits such as nesting phenology, often remain unknown. 59 There are four distinct ecological provinces in the state: Prairie Parklands (PP), Tallgrass Aspen 60 Parklands (TAP), Eastern Broadleaf Forest (EBF), and Laurentian Mixed Forest (LMF) 61 (Minnesota Department of Natural Resources, 2023). The effort and funds required to survey 62 these ecologically different areas of the state for insect pollinators are a challenge. Additionally, 63 commonly used methods for studying insects require extensive specimen collection and 64 taxonomic expertise for species-level identification for most groups, which can also be expensive 65 (Woodard et al., 2020). 66 67 Inviting the public to participate in scientific research can help overcome geographic and 68 temporal challenges of bee monitoring. Here we use the term participatory science (sometimes 69 called citizen science or community science) to indicate volunteer participants who are not 70 monetarily compensated. 40 Introduction 41 While bees are widely recognized for their important role in food security and the maintenance 42 of ecological integrity (Klein et al 2007; Ollerton Winfree & Tarrant 2011) the monitoring 41 While bees are widely recognized for their important role in food security and the maintenance 42 of ecological integrity (Klein et al., 2007; Ollerton, Winfree & Tarrant, 2011), the monitoring PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed As of March 2021, 2,300 users submitted 110 observations of bees in Minnesota to iNaturalist, some of whom specifically contributed to the 111 MN Bee Atlas project and many of whom submitted bee observations that the portal 112 automatically added to the project. Over 1,000 users contributed identifications to MN Bee Atlas 113 iNaturalist records. 114 115 iNaturalist 116 The broadest and simplest level of participation relied on the mobile app and website iNaturalist. 117 This global public biodiversity portal enables individuals to upload locations and evidence of 118 living things, including photos or recordings, which are then identified by the observer, other 119 users, or an algorithmic suggestion based on existing research-grade observations. Each 120 identification is qualified based on a data validation system and considered research-grade if an 121 observation is not of a captive or cultivated species, has a date, photo and location, and two- 122 thirds of users agree on genus and species-level identification. This is not foolproof, as there are 123 no required credentials to add identification. However, there are many knowledgeable iNaturalist 124 users, both professionals and experienced enthusiasts, who spend time identifying iNaturalist 125 observations from others and are integral to the creation of research-quality data. The quality of 126 identification typically grows over time as additional users join the platform and as additional 127 identification experts participate. We examined a subset of research-grade observations from 128 genera that are difficult to identify to species (i.e., Andrena, Lasioglossum, Nomada). These 129 records were verified by expert bee taxonomists, including John Ascher, Jason Gibbs, and Zach 130 Portman. Once identifications reach research-grade, records feed into databases such as GBIF 131 (www.gbif.org). We trained 338 participants who attended workshops to add bee observations to 132 iNaturalist and to identify bees to groups, usually family. Most workshop participants added 133 observations to iNaturalist, with a small percentage becoming regular contributors or identifiers. 134 101 The Minnesota Bee Atlas participatory science project operated between 2016-2020. We 102 recruited volunteers statewide (Fig. 1) by advertising to local volunteer groups and conservation 103 organizations, on social media, and through University of Minnesota web pages. Manuscript to be reviewed As of March 2021, 2,300 users submitted 110 observations of bees in Minnesota to iNaturalist, some of whom specifically contributed to the 111 MN Bee Atlas project and many of whom submitted bee observations that the portal 112 automatically added to the project. Over 1,000 users contributed identifications to MN Bee Atlas 113 iNaturalist records. 114 115 iNaturalist 116 The broadest and simplest level of participation relied on the mobile app and website iNaturalist. 117 This global public biodiversity portal enables individuals to upload locations and evidence of 118 living things, including photos or recordings, which are then identified by the observer, other 119 users, or an algorithmic suggestion based on existing research-grade observations. Each 120 identification is qualified based on a data validation system and considered research-grade if an 121 observation is not of a captive or cultivated species, has a date, photo and location, and two- 122 thirds of users agree on genus and species-level identification. This is not foolproof, as there are 123 no required credentials to add identification. However, there are many knowledgeable iNaturalist 124 users, both professionals and experienced enthusiasts, who spend time identifying iNaturalist 125 observations from others and are integral to the creation of research-quality data. The quality of 126 identification typically grows over time as additional users join the platform and as additional 127 identification experts participate. We examined a subset of research-grade observations from 97 conservation plans and contribute to baseline assessments for evaluating the status of pollinators 98 in Minnesota in the future. 99 100 Materials & Methods 101 The Minnesota Bee Atlas participatory science project operated between 2016-2020. We 102 recruited volunteers statewide (Fig. 1) by advertising to local volunteer groups and conservation 103 organizations, on social media, and through University of Minnesota web pages. Volunteers had 104 various affiliations including the Minnesota Master Naturalist program, Minnesota Department 105 of Natural Resources Scientific and Natural Area stewards, Environmental Learning Centers, 106 nature centers, county natural resource departments, Soil and Water Conservation Districts, 107 native plant nurseries, and federal agencies including the U.S. Forest Service and the U.S. Fish 108 and Wildlife Service. Approximately 150 volunteers engaged with project staff and participated 109 in one of the three protocol areas each field season. Manuscript to be reviewed Volunteers had 104 various affiliations including the Minnesota Master Naturalist program, Minnesota Department 105 of Natural Resources Scientific and Natural Area stewards, Environmental Learning Centers, 106 nature centers, county natural resource departments, Soil and Water Conservation Districts, 107 native plant nurseries, and federal agencies including the U.S. Forest Service and the U.S. Fish 108 and Wildlife Service. Approximately 150 volunteers engaged with project staff and participated 109 in one of the three protocol areas each field season. As of March 2021, 2,300 users submitted 110 observations of bees in Minnesota to iNaturalist, some of whom specifically contributed to the 111 MN Bee Atlas project and many of whom submitted bee observations that the portal 112 automatically added to the project. Over 1,000 users contributed identifications to MN Bee Atlas 113 iNaturalist records. Manuscript to be reviewed The three 85 tiers of sampling rigor represent increasing levels of volunteer training and commitment and 86 yielded different data types. The iNaturalist observations required minimal training and flexible 87 volunteer time commitment. While not appropriate for all bee species, the use of crowd-sourced 88 identifications provided presence data for bee species amenable to identification from 43 and baseline information necessary for regional bee conservation is often missing (Cardoso et al., 44 2011; Lebuhn et al., 2013). Without such data on species distributions, habitat associations, and 45 phenology, it is difficult to understand if or how bee communities are changing or how to enact 46 conservation practices. Knowing species distributions and estimates of abundance can help 47 prioritize management and conservation efforts (Cardoso et al., 2011). For example, species with 48 small geographic distributions are at higher risk of extinction (Gaston & Fuller, 2009). Habitat 49 associations are also important because bees are often closely tied to plant communities (Potts et 50 al., 2003; Sheffield & Heron, 2019) and habitat needs such as nest sites (Potts et al., 2003; 51 Harmon-Threatt, 2020). In addition, establishing phenology baselines is important to 52 understanding the ecological role of bee species and how climate change impacts ecosystems 53 now and in the future (Burkle, Marlin & Knight, 2013; Ogilvie & Forrest, 2017). 43 and baseline information necessary for regional bee conservation is often missing (Cardoso et al., 44 2011; Lebuhn et al., 2013). Without such data on species distributions, habitat associations, and 45 phenology, it is difficult to understand if or how bee communities are changing or how to enact 46 conservation practices. Knowing species distributions and estimates of abundance can help 47 prioritize management and conservation efforts (Cardoso et al., 2011). For example, species with 48 small geographic distributions are at higher risk of extinction (Gaston & Fuller, 2009). Habitat 49 associations are also important because bees are often closely tied to plant communities (Potts et 50 al., 2003; Sheffield & Heron, 2019) and habitat needs such as nest sites (Potts et al., 2003; 51 Harmon-Threatt, 2020). In addition, establishing phenology baselines is important to 52 understanding the ecological role of bee species and how climate change impacts ecosystems 53 now and in the future (Burkle, Marlin & Knight, 2013; Ogilvie & Forrest, 2017). 54 PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed 89 photographs, particularly bumble bees. The nest-trap surveys required more training and a 90 season-long commitment from volunteers. They provided distribution, ecological association, 91 nesting phenology, and nesting biology data for a subset of bees that are often not well 92 represented in other survey methods (Westphal et al., 2008; Staab et al., 2018). Volunteers who 93 worked on bumble bee surveys had in-depth training on bumble bee identification and sampling 94 methods and committed to a more time-intensive sampling protocol. Bumble bee surveys used 95 equal sampling effort across observations to provide abundance and distribution data, as well as 96 indication of habitat associations. Together, these data will inform statewide pollinator 97 conservation plans and contribute to baseline assessments for evaluating the status of pollinators 98 in Minnesota in the future. 89 photographs, particularly bumble bees. The nest-trap surveys required more training and a 90 season-long commitment from volunteers. They provided distribution, ecological association, 91 nesting phenology, and nesting biology data for a subset of bees that are often not well 92 represented in other survey methods (Westphal et al., 2008; Staab et al., 2018). Volunteers who 93 worked on bumble bee surveys had in-depth training on bumble bee identification and sampling 94 methods and committed to a more time-intensive sampling protocol. Bumble bee surveys used 95 equal sampling effort across observations to provide abundance and distribution data, as well as 96 indication of habitat associations. Together, these data will inform statewide pollinator 97 conservation plans and contribute to baseline assessments for evaluating the status of pollinators 98 in Minnesota in the future. 99 100 Materials & Methods 101 The Minnesota Bee Atlas participatory science project operated between 2016-2020. We 102 recruited volunteers statewide (Fig. 1) by advertising to local volunteer groups and conservation 103 organizations, on social media, and through University of Minnesota web pages. Volunteers had 104 various affiliations including the Minnesota Master Naturalist program, Minnesota Department 105 of Natural Resources Scientific and Natural Area stewards, Environmental Learning Centers, 106 nature centers, county natural resource departments, Soil and Water Conservation Districts, 107 native plant nurseries, and federal agencies including the U.S. Forest Service and the U.S. Fish 108 and Wildlife Service. Approximately 150 volunteers engaged with project staff and participated 109 in one of the three protocol areas each field season. 135 Tunnel-nesting bees Nest-traps were distributed across 60 of the 87 Minnesota counties and 160 all four ecological provinces, including 69 in the LMF, 224 in the EBF, 87 in the PP, and two in 161 the TAP ecological provinces (Fig. 1). The Minnesota Department of Natural Resources 162 approved research permit numbers 2016-29, 2016- 4R, 201705, 2017-9R, 201822, and 2018-15R 163 for nest-traps placed in State Parks, State Forests, Scientific and Natural Areas and Wildlife 164 Management Areas. 165 166 We received one homemade nest-trap bundle made from Phragmites stems from one volunteer 167 in Brown County each year between 2016 and 2018. In 2019, the final year, we sent 11 168 additional nest bundles made with hollow or pithy plant stems to selected volunteers to observe 169 nesting with different natural substrates. We made each bundle from stems of one of six native 170 plant species; Asclepias incarnata, Silphium perfoliatum, Arnoglossum atriplicifolium, 171 Helianthus giganteus, Vernonia fasciculata, or Liatris ligulistylis, and placed bundles inside a 172 plastic sleeve with an overhanging roof made from a 64 oz (1.89 liter) beverage bottle. We 173 sealed the backs of the stems with cotton balls and latex. The number of stems per bundle varied 174 due to the size differences between stems. Monitoring protocols were like those used for wood 175 nest-traps. 176 177 In the late fall, volunteers returned nest-traps and stem bundles to the University of Minnesota 178 for overwintering and rearing in a temperature-controlled growth chamber as described in 179 Satyshur et al. (2021). After a four-month period at 5°C, we stimulated emergence by increasing 180 the temperature in steps to a high of 30°C. We covered each nest-trap tunnel entrance with test 136 Tunnel-nesting bees nest in above-ground tunnels in wood or plant stems. Each female builds her 137 own nest by constructing a series of compartments in each of which she stores pollen and nectar 138 and lays a single egg. When the nest is complete, she plugs the tunnel entrance, leaving the 139 young to develop on their own. Different species use different materials for nest plugs. Many 140 species will also nest in artificial nest-traps which can be used to survey species. In this study, 141 participants hung and monitored wood nest-traps in semi-natural habitats on private or public 142 lands from April to October. 115 iNaturalist 116 The broadest and simplest level of participation relied on the mobile app and website iNaturalist. 117 This global public biodiversity portal enables individuals to upload locations and evidence of 118 living things, including photos or recordings, which are then identified by the observer, other 119 users, or an algorithmic suggestion based on existing research-grade observations. Each 120 identification is qualified based on a data validation system and considered research-grade if an 121 observation is not of a captive or cultivated species, has a date, photo and location, and two- 122 thirds of users agree on genus and species-level identification. This is not foolproof, as there are 123 no required credentials to add identification. However, there are many knowledgeable iNaturalist 124 users, both professionals and experienced enthusiasts, who spend time identifying iNaturalist 125 observations from others and are integral to the creation of research-quality data. The quality of 126 identification typically grows over time as additional users join the platform and as additional 127 identification experts participate. We examined a subset of research-grade observations from 128 genera that are difficult to identify to species (i.e., Andrena, Lasioglossum, Nomada). These 129 records were verified by expert bee taxonomists, including John Ascher, Jason Gibbs, and Zach 130 Portman. Once identifications reach research-grade, records feed into databases such as GBIF 131 (www.gbif.org). We trained 338 participants who attended workshops to add bee observations to 132 iNaturalist and to identify bees to groups, usually family. Most workshop participants added 133 observations to iNaturalist, with a small percentage becoming regular contributors or identifiers. 134 PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) 135 Tunnel-nesting bees 135 Tunnel nesting bees 136 Tunnel-nesting bees nest in above-ground tunnels in wood or plant stems. Each female builds her 137 own nest by constructing a series of compartments in each of which she stores pollen and nectar 138 and lays a single egg. When the nest is complete, she plugs the tunnel entrance, leaving the 139 young to develop on their own. Different species use different materials for nest plugs. Many 140 species will also nest in artificial nest-traps which can be used to survey species. In this study, 141 participants hung and monitored wood nest-traps in semi-natural habitats on private or public 142 lands from April to October. Nest-trap design and nest plug descriptions were adapted from The 143 Bees’ Needs (Rose, Scott & Bowers, 2015, V. Scott, personal communication, Feb. 2016). We 144 drilled five tunnels of six different diameters (3.18 mm, 4.76 mm, 6.35 mm, 7.94 mm, 9.53 mm, 145 and 11.11 mm) into blocks of untreated pine or Douglas fir with a cedar shingle roof (Appendix 146 1). We use the term “nest” to mean a tunnel that produced a particular bee species. Different 147 species sometimes build sequential nests in the same tunnel. Occasionally, different individuals 148 from the same species may nest within the same tunnel, but for this study we assumed 149 individuals of the same species within a tunnel were from the same mother. 150 151 With the goal of surveying the whole state, we actively recruited volunteers to hang nest-traps in 152 rural areas and in areas with less existing data. Volunteers attended in-person or online training 153 and received a written instruction manual with photographs of different plug materials. They 154 placed nest-traps in a semi-sunny location facing east or south at a height of 1 to 2 m, with the 155 flexibility to find a mounting site that fit their habitat. Volunteers were instructed to report 156 plugged tunnels or other nest evidence every 2-3 weeks via the project web page. Bee Atlas staff 157 provided feedback on observations via email and newsletters. In 2016, 2017 and 2018, we sent 158 out 120, 129, and 141 nest-traps respectively and 116, 127, and 140 were returned, respectively, 159 for a return rate of 98%. Manuscript to be reviewed 135 Tunnel-nesting bees Manuscript to be reviewed 181 tubes and removed emerging insects daily. Bundles were reared in bags. Some bees appeared to 182 have already emerged by fall 2016, so in 2017 and 2018, we swapped out a few nest-traps with 183 similar plugs in mid-summer and reared them in the lab at ambient temperature. We (CS, TE) 184 identified bees to species using keys (Sandhouse, 1939; Mitchell, 1962; Sheffield et al., 2011; 185 Arduser, 2018; Andrus, Droege & Griswold, 2020a,b,c; Griswold et al., 2020; Nelson & Droege, 186 2020a,b; Orr et al., 2020) and comparisons with previously identified specimens. Jason Gibbs, 187 Michael Orr, Ryan Oram, and Sam Droege confirmed identification of more difficult specimens. 188 We identified wasps using keys (Gibson, Huber & Woolley, 1997; Triplehorn, 2005; Heraty, 189 2008). John Lumen identified all Ichneumonidae and provided consultation on Chalcidoidea. 190 Kocourekia cf. debilis was identified to species using Cao et al. (2017) and verified by Jorge 191 González and Mike Gates. We deposited voucher specimens in the UMN Insect Collection. We 192 included locations of specimens in the UMN Insect Collection database when mapping species 193 distributions. Many UMN Insect Collection specimens did not have latitude or longitude 194 associated with their records. In such cases, we used the location description to estimate the most 195 accurate position possible. We chose the approximate center of geographic areas such as cities 196 and state parks. If only county location was available, we placed the specimen in the 197 approximate center of the county and identified the records as such. 198 199 We examined nesting phenology using volunteer-submitted nest plug observations. For each nest 200 tunnel that produced bee offspring, project staff evaluated observations and assigned a quality 201 value based on clarity and frequency of observations. Higher values were assigned if the full 202 plug observation was clear and consistently observed following formation and if observations 203 were four weeks apart or less. Nest tunnels with high or medium quality values were used in 204 phenological estimations, with 65.1% of observations meeting those criteria. Because volunteers 205 checked approximately every two to three weeks, we could determine that nest completion 206 occurred in the interval between the last date that the volunteer recorded an empty tunnel and the 207 first date with a complete nest plug. Manuscript to be reviewed (2017) and verified by Jorge 191 González and Mike Gates. We deposited voucher specimens in the UMN Insect Collection. We 192 included locations of specimens in the UMN Insect Collection database when mapping species 193 distributions. Many UMN Insect Collection specimens did not have latitude or longitude 194 associated with their records. In such cases, we used the location description to estimate the most 195 accurate position possible. We chose the approximate center of geographic areas such as cities 196 and state parks. If only county location was available, we placed the specimen in the 197 approximate center of the county and identified the records as such. 198 199 We examined nesting phenology using volunteer-submitted nest plug observations. For each nest 200 tunnel that produced bee offspring, project staff evaluated observations and assigned a quality 201 value based on clarity and frequency of observations. Higher values were assigned if the full 202 plug observation was clear and consistently observed following formation and if observations 203 were four weeks apart or less. Nest tunnels with high or medium quality values were used in 204 phenological estimations, with 65.1% of observations meeting those criteria. Because volunteers 205 checked approximately every two to three weeks, we could determine that nest completion 206 occurred in the interval between the last date that the volunteer recorded an empty tunnel and the 207 first date with a complete nest plug. We assumed nests were equally likely to be completed on 208 any particular day in an interval and assigned each day an equal probability. We summed these 209 probabilities over all nests with sufficient quality observations and determined the median date. 210 We also calculated the 0.25 and 0.75 quartile values, which bound a central period when nests 211 were most likely completed. 212 181 tubes and removed emerging insects daily. Bundles were reared in bags. Some bees appeared to 182 have already emerged by fall 2016, so in 2017 and 2018, we swapped out a few nest-traps with 183 similar plugs in mid-summer and reared them in the lab at ambient temperature. We (CS, TE) 184 identified bees to species using keys (Sandhouse, 1939; Mitchell, 1962; Sheffield et al., 2011; 185 Arduser, 2018; Andrus, Droege & Griswold, 2020a,b,c; Griswold et al., 2020; Nelson & Droege, 186 2020a,b; Orr et al., 2020) and comparisons with previously identified specimens. Manuscript to be reviewed Jason Gibbs, 187 Michael Orr, Ryan Oram, and Sam Droege confirmed identification of more difficult specimens. 188 We identified wasps using keys (Gibson, Huber & Woolley, 1997; Triplehorn, 2005; Heraty, 189 2008). John Lumen identified all Ichneumonidae and provided consultation on Chalcidoidea. 190 Kocourekia cf. debilis was identified to species using Cao et al. (2017) and verified by Jorge 191 González and Mike Gates. We deposited voucher specimens in the UMN Insect Collection. We 192 included locations of specimens in the UMN Insect Collection database when mapping species 193 distributions. Many UMN Insect Collection specimens did not have latitude or longitude 194 associated with their records. In such cases, we used the location description to estimate the most 195 accurate position possible. We chose the approximate center of geographic areas such as cities 196 and state parks. If only county location was available, we placed the specimen in the 197 approximate center of the county and identified the records as such. 198 181 tubes and removed emerging insects daily. Bundles were reared in bags. Some bees appeared to 182 have already emerged by fall 2016, so in 2017 and 2018, we swapped out a few nest-traps with 183 similar plugs in mid-summer and reared them in the lab at ambient temperature. We (CS, TE) 184 identified bees to species using keys (Sandhouse, 1939; Mitchell, 1962; Sheffield et al., 2011; 185 Arduser, 2018; Andrus, Droege & Griswold, 2020a,b,c; Griswold et al., 2020; Nelson & Droege, 186 2020a,b; Orr et al., 2020) and comparisons with previously identified specimens. Jason Gibbs, 187 Michael Orr, Ryan Oram, and Sam Droege confirmed identification of more difficult specimens. 188 We identified wasps using keys (Gibson, Huber & Woolley, 1997; Triplehorn, 2005; Heraty, 189 2008). John Lumen identified all Ichneumonidae and provided consultation on Chalcidoidea. 190 Kocourekia cf. debilis was identified to species using Cao et al. (2017) and verified by Jorge 191 González and Mike Gates. We deposited voucher specimens in the UMN Insect Collection. We 192 included locations of specimens in the UMN Insect Collection database when mapping species 193 distributions. Many UMN Insect Collection specimens did not have latitude or longitude 194 associated with their records. In such cases, we used the location description to estimate the most 195 accurate position possible. We chose the approximate center of geographic areas such as cities 196 and state parks. 135 Tunnel-nesting bees Nest-trap design and nest plug descriptions were adapted from The 143 Bees’ Needs (Rose, Scott & Bowers, 2015, V. Scott, personal communication, Feb. 2016). We 144 drilled five tunnels of six different diameters (3.18 mm, 4.76 mm, 6.35 mm, 7.94 mm, 9.53 mm, 145 and 11.11 mm) into blocks of untreated pine or Douglas fir with a cedar shingle roof (Appendix 146 1). We use the term “nest” to mean a tunnel that produced a particular bee species. Different 147 species sometimes build sequential nests in the same tunnel. Occasionally, different individuals 148 from the same species may nest within the same tunnel, but for this study we assumed 149 individuals of the same species within a tunnel were from the same mother. 150 PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed We assumed nests were equally likely to be completed on 208 any particular day in an interval and assigned each day an equal probability. We summed these 209 probabilities over all nests with sufficient quality observations and determined the median date. 210 We also calculated the 0.25 and 0.75 quartile values, which bound a central period when nests 211 were most likely completed. 212 213 Bumble bees 214 We trained volunteers in survey methods and skills to distinguish bumble bees from other 215 insects, determine sex, identify readily distinguishable bumble bee species, and photograph 216 bumble bees to enable identification. Based on regional collections, we estimated that 90% of 217 observations would be readily distinguishable species (Bombus impatiens Cresson, 1863, 218 Bombus bimaculatus Cresson, 1863, Bombus griseocollis (De Geer, 1773), or Bombus ternarius 219 Say, 1837). We adapted survey methods from previous state-wide bumble bee surveys that used 220 lethal collection methods (Golick & Ellis, 2006; McFarland, Richardson & Zahendra, 2015; 221 Richardson et al., 2019). Due to volunteer preferences and the presence of federally protected 222 Bombus affinis Cresson, 1863, we used observational data instead of specimen collections. 223 Forty-four volunteers observed bees at five stops along 39.5-kilometer routes between 10 a.m. 224 and 6 p.m. on days with little or no precipitation, temperatures greater than 15.6 C, and wind 225 speeds less than 32.2 kph. We requested volunteers survey along their route three times each 226 year, between late June and mid-August with at least two weeks between visits. Volunteers 181 tubes and removed emerging insects daily. Bundles were reared in bags. Some bees appeared to 182 have already emerged by fall 2016, so in 2017 and 2018, we swapped out a few nest-traps with 183 similar plugs in mid-summer and reared them in the lab at ambient temperature. We (CS, TE) 184 identified bees to species using keys (Sandhouse, 1939; Mitchell, 1962; Sheffield et al., 2011; 185 Arduser, 2018; Andrus, Droege & Griswold, 2020a,b,c; Griswold et al., 2020; Nelson & Droege, 186 2020a,b; Orr et al., 2020) and comparisons with previously identified specimens. Jason Gibbs, 187 Michael Orr, Ryan Oram, and Sam Droege confirmed identification of more difficult specimens. 188 We identified wasps using keys (Gibson, Huber & Woolley, 1997; Triplehorn, 2005; Heraty, 189 2008). John Lumen identified all Ichneumonidae and provided consultation on Chalcidoidea. 190 Kocourekia cf. debilis was identified to species using Cao et al. Manuscript to be reviewed If only county location was available, we placed the specimen in the 197 approximate center of the county and identified the records as such. 198 199 We examined nesting phenology using volunteer-submitted nest plug observations. For each nest 200 tunnel that produced bee offspring, project staff evaluated observations and assigned a quality 201 value based on clarity and frequency of observations. Higher values were assigned if the full 202 plug observation was clear and consistently observed following formation and if observations 203 were four weeks apart or less. Nest tunnels with high or medium quality values were used in 204 phenological estimations, with 65.1% of observations meeting those criteria. Because volunteers 205 checked approximately every two to three weeks, we could determine that nest completion 206 occurred in the interval between the last date that the volunteer recorded an empty tunnel and the 207 first date with a complete nest plug. We assumed nests were equally likely to be completed on 208 any particular day in an interval and assigned each day an equal probability. We summed these 209 probabilities over all nests with sufficient quality observations and determined the median date. 210 We also calculated the 0.25 and 0.75 quartile values, which bound a central period when nests 211 were most likely completed. Manuscript to be reviewed affinis, Bombus terricola Cresson, 1863, 242 Bombus pensylvanicus (De Geer, 1773)) as listed by the International Union for the Conservation 243 of Nature (Hatfield et al., 2015). Volunteers submitted data through the Bee Atlas website. We 244 (EE) verified identifications for all photo-specimens. Most specimens (89%) were identified by 245 volunteers, with 10% of specimens verified with photographs, and 1% unverifiable due to poor 246 photo quality. Two species, Bombus vagans Smith, 1854 and Bombus sandersoni Franklin, 1913, 247 were grouped because most observations did not include identifying features that enabled 248 separation of these two closely related species from each other. 249 251 We used R (R Core Team, 2022) and Rstudio (Rstudio Team, 2022) for all statistical analyses. 252 We examined differences among ecological provinces for tunnel-nesting bees and bumble bees 253 using generalized linear mixed-effect models in the glmmTMB R package (Brooks et al., 2017) 254 with post-hoc comparisons of estimated marginal means using the R package emmeans (Lenth et 255 al., 2023). We checked all model residuals for overdispersion and heteroscedasticity. We 256 compared overall frequency of tunnel use by nesting bees across the LMF, EBF, and PP with a 257 negative-binomial model to account for the high numbers of zeros in the data. We did not 258 include the TAP since there were only two nest-traps in that province. We also used negative 259 binomial distribution to model annual nest counts per nest-trap per species by ecological 260 province, with year and location as random effects. The location variable grouped nest-traps that 261 were within one kilometer of one another. We selected the following nest-building species for 262 this analysis based on presence in 30 or more nest-traps (10% or more of all nest-traps): 263 Heriades carinata Cresson, 1864, Megachile campanulae (Robertson, 1903), Megachile pugnata 264 Say, 1837, Megachile relativa Cresson, 1878, Megachile rotundata (Fabricius, 1787), Osmia 265 lignaria Say, 1837, Osmia pumila Cresson, 1864, and Osmia tersula Cockerell, 1912. We did not 266 include parasitic species in this analysis due to their correlation with their host species. 267 Megachile campanulae and O. pumila were not recorded by nest-traps in the LMF and were 268 analyzed for PP and EBF only. Manuscript to be reviewed We selected the following nest-building species for 262 this analysis based on presence in 30 or more nest-traps (10% or more of all nest-traps): 263 Heriades carinata Cresson, 1864, Megachile campanulae (Robertson, 1903), Megachile pugnata 264 Say, 1837, Megachile relativa Cresson, 1878, Megachile rotundata (Fabricius, 1787), Osmia 265 lignaria Say, 1837, Osmia pumila Cresson, 1864, and Osmia tersula Cockerell, 1912. We did not 266 include parasitic species in this analysis due to their correlation with their host species. 267 Megachile campanulae and O. pumila were not recorded by nest-traps in the LMF and were 268 analyzed for PP and EBF only. We created models for bumble bees with log-transformed 269 abundance of bumble bees per route per year as the response variable and ecological province as 270 the predictor with year and route as random effects. After preliminary analysis, we changed year 271 from a random to a fixed effect due to singularity. We limited data to include only routes with 272 three completed route runs (a set of five 10-minute observations) within a year, which equaled 227 surveyed 45 of 90 available routes between 2016 and 2020, with 37 routes with three completed 228 route runs per year, and 17 routes surveyed for three or more years (Fig. 1, Table 1). Routes were 229 based on established North American Breeding Bird Survey routes (USGS Patuxent Wildlife 230 Research Center, 2017) because of their accessibility and systematic spread across different 231 ecological areas. For analysis, we combined the single route from the TAP ecological province 232 with routes from the PP ecological province due to the low sample size in this province and 233 ecological similarity. Volunteers chose five stops along a route by finding flower patches with 234 bee activity located at least 1.61km (1 mile) from each other. On average, survey stops were 5.23 235 kilometers apart from each other. Volunteers examined flower patches within 150 meters of the 236 survey stop, collecting bumble bees from flowers into jars for ten minutes of collecting time, and 237 noting the flower's identity. Volunteers placed bees in coolers with ice to avoid risk of bees 238 overheating and to ease photography. 213 Bumble bees 214 We trained volunteers in survey methods and skills to distinguish bumble bees from other 215 insects, determine sex, identify readily distinguishable bumble bee species, and photograph 216 bumble bees to enable identification. Based on regional collections, we estimated that 90% of 217 observations would be readily distinguishable species (Bombus impatiens Cresson, 1863, 218 Bombus bimaculatus Cresson, 1863, Bombus griseocollis (De Geer, 1773), or Bombus ternarius 219 Say, 1837). We adapted survey methods from previous state-wide bumble bee surveys that used 220 lethal collection methods (Golick & Ellis, 2006; McFarland, Richardson & Zahendra, 2015; 221 Richardson et al., 2019). Due to volunteer preferences and the presence of federally protected 222 Bombus affinis Cresson, 1863, we used observational data instead of specimen collections. 223 Forty-four volunteers observed bees at five stops along 39.5-kilometer routes between 10 a.m. 224 and 6 p.m. on days with little or no precipitation, temperatures greater than 15.6 C, and wind 225 speeds less than 32.2 kph. We requested volunteers survey along their route three times each 226 year, between late June and mid-August with at least two weeks between visits. Volunteers PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed We checked all model residuals for overdispersion and heteroscedasticity. We 256 compared overall frequency of tunnel use by nesting bees across the LMF, EBF, and PP with a 257 negative-binomial model to account for the high numbers of zeros in the data. We did not 258 include the TAP since there were only two nest-traps in that province. We also used negative 259 binomial distribution to model annual nest counts per nest-trap per species by ecological 260 province, with year and location as random effects. The location variable grouped nest-traps that 261 were within one kilometer of one another. We selected the following nest-building species for 262 this analysis based on presence in 30 or more nest-traps (10% or more of all nest-traps): 263 Heriades carinata Cresson, 1864, Megachile campanulae (Robertson, 1903), Megachile pugnata 264 Say, 1837, Megachile relativa Cresson, 1878, Megachile rotundata (Fabricius, 1787), Osmia 265 lignaria Say, 1837, Osmia pumila Cresson, 1864, and Osmia tersula Cockerell, 1912. We did not 266 include parasitic species in this analysis due to their correlation with their host species. 267 Megachile campanulae and O. pumila were not recorded by nest-traps in the LMF and were 268 analyzed for PP and EBF only. We created models for bumble bees with log-transformed 269 abundance of bumble bees per route per year as the response variable and ecological province as 270 the predictor with year and route as random effects. After preliminary analysis, we changed year 271 from a random to a fixed effect due to singularity. We limited data to include only routes with 272 three completed route runs (a set of five 10-minute observations) within a year, which equaled 227 surveyed 45 of 90 available routes between 2016 and 2020, with 37 routes with three completed 228 route runs per year, and 17 routes surveyed for three or more years (Fig. 1, Table 1). Routes were 229 based on established North American Breeding Bird Survey routes (USGS Patuxent Wildlife 230 Research Center, 2017) because of their accessibility and systematic spread across different 231 ecological areas. For analysis, we combined the single route from the TAP ecological province 232 with routes from the PP ecological province due to the low sample size in this province and 233 ecological similarity. Volunteers chose five stops along a route by finding flower patches with 234 bee activity located at least 1.61km (1 mile) from each other. Manuscript to be reviewed On average, survey stops were 5.23 235 kilometers apart from each other. Volunteers examined flower patches within 150 meters of the 236 survey stop, collecting bumble bees from flowers into jars for ten minutes of collecting time, and 237 noting the flower's identity. Volunteers placed bees in coolers with ice to avoid risk of bees 238 overheating and to ease photography. Volunteers counted and released readily identifiable 239 individuals and photographed a subset of bees including all bees that were not readily 240 identifiable, all bees belonging to the subgenus Psithyrus other than Bombus citrinus (Smith, 241 1854), and all individuals of conservation concern (B. affinis, Bombus terricola Cresson, 1863, 242 Bombus pensylvanicus (De Geer, 1773)) as listed by the International Union for the Conservation 243 of Nature (Hatfield et al., 2015). Volunteers submitted data through the Bee Atlas website. We 244 (EE) verified identifications for all photo-specimens. Most specimens (89%) were identified by 245 volunteers, with 10% of specimens verified with photographs, and 1% unverifiable due to poor 246 photo quality. Two species, Bombus vagans Smith, 1854 and Bombus sandersoni Franklin, 1913, 247 were grouped because most observations did not include identifying features that enabled 248 separation of these two closely related species from each other. 249 250 Statistical analysis 251 We used R (R Core Team, 2022) and Rstudio (Rstudio Team, 2022) for all statistical analyses. 252 We examined differences among ecological provinces for tunnel-nesting bees and bumble bees 253 using generalized linear mixed-effect models in the glmmTMB R package (Brooks et al., 2017) 254 with post-hoc comparisons of estimated marginal means using the R package emmeans (Lenth et 255 al., 2023). We checked all model residuals for overdispersion and heteroscedasticity. We 256 compared overall frequency of tunnel use by nesting bees across the LMF, EBF, and PP with a 257 negative-binomial model to account for the high numbers of zeros in the data. We did not 258 include the TAP since there were only two nest-traps in that province. We also used negative 259 binomial distribution to model annual nest counts per nest-trap per species by ecological 260 province, with year and location as random effects. The location variable grouped nest-traps that 261 were within one kilometer of one another. Manuscript to be reviewed 227 surveyed 45 of 90 available routes between 2016 and 2020, with 37 routes with three completed 228 route runs per year, and 17 routes surveyed for three or more years (Fig. 1, Table 1). Routes were 229 based on established North American Breeding Bird Survey routes (USGS Patuxent Wildlife 230 Research Center, 2017) because of their accessibility and systematic spread across different 231 ecological areas. For analysis, we combined the single route from the TAP ecological province 232 with routes from the PP ecological province due to the low sample size in this province and 233 ecological similarity. Volunteers chose five stops along a route by finding flower patches with 234 bee activity located at least 1.61km (1 mile) from each other. On average, survey stops were 5.23 235 kilometers apart from each other. Volunteers examined flower patches within 150 meters of the 236 survey stop, collecting bumble bees from flowers into jars for ten minutes of collecting time, and 237 noting the flower's identity. Volunteers placed bees in coolers with ice to avoid risk of bees 238 overheating and to ease photography. Volunteers counted and released readily identifiable 239 individuals and photographed a subset of bees including all bees that were not readily 240 identifiable, all bees belonging to the subgenus Psithyrus other than Bombus citrinus (Smith, 241 1854), and all individuals of conservation concern (B. affinis, Bombus terricola Cresson, 1863, 242 Bombus pensylvanicus (De Geer, 1773)) as listed by the International Union for the Conservation 243 of Nature (Hatfield et al., 2015). Volunteers submitted data through the Bee Atlas website. We 244 (EE) verified identifications for all photo-specimens. Most specimens (89%) were identified by 245 volunteers, with 10% of specimens verified with photographs, and 1% unverifiable due to poor 246 photo quality. Two species, Bombus vagans Smith, 1854 and Bombus sandersoni Franklin, 1913, 247 were grouped because most observations did not include identifying features that enabled 248 separation of these two closely related species from each other. 249 250 Statistical analysis 251 We used R (R Core Team, 2022) and Rstudio (Rstudio Team, 2022) for all statistical analyses. 252 We examined differences among ecological provinces for tunnel-nesting bees and bumble bees 253 using generalized linear mixed-effect models in the glmmTMB R package (Brooks et al., 2017) 254 with post-hoc comparisons of estimated marginal means using the R package emmeans (Lenth et 255 al., 2023). Manuscript to be reviewed Volunteers counted and released readily identifiable 239 individuals and photographed a subset of bees including all bees that were not readily 240 identifiable, all bees belonging to the subgenus Psithyrus other than Bombus citrinus (Smith, 241 1854), and all individuals of conservation concern (B. affinis, Bombus terricola Cresson, 1863, 242 Bombus pensylvanicus (De Geer, 1773)) as listed by the International Union for the Conservation 243 of Nature (Hatfield et al., 2015). Volunteers submitted data through the Bee Atlas website. We 244 (EE) verified identifications for all photo-specimens. Most specimens (89%) were identified by 245 volunteers, with 10% of specimens verified with photographs, and 1% unverifiable due to poor 246 photo quality. Two species, Bombus vagans Smith, 1854 and Bombus sandersoni Franklin, 1913, 247 were grouped because most observations did not include identifying features that enabled 248 separation of these two closely related species from each other. 227 surveyed 45 of 90 available routes between 2016 and 2020, with 37 routes with three completed 228 route runs per year, and 17 routes surveyed for three or more years (Fig. 1, Table 1). Routes were 229 based on established North American Breeding Bird Survey routes (USGS Patuxent Wildlife 230 Research Center, 2017) because of their accessibility and systematic spread across different 231 ecological areas. For analysis, we combined the single route from the TAP ecological province 232 with routes from the PP ecological province due to the low sample size in this province and 233 ecological similarity. Volunteers chose five stops along a route by finding flower patches with 234 bee activity located at least 1.61km (1 mile) from each other. On average, survey stops were 5.23 235 kilometers apart from each other. Volunteers examined flower patches within 150 meters of the 236 survey stop, collecting bumble bees from flowers into jars for ten minutes of collecting time, and 237 noting the flower's identity. Volunteers placed bees in coolers with ice to avoid risk of bees 238 overheating and to ease photography. Volunteers counted and released readily identifiable 239 individuals and photographed a subset of bees including all bees that were not readily 240 identifiable, all bees belonging to the subgenus Psithyrus other than Bombus citrinus (Smith, 241 1854), and all individuals of conservation concern (B. Manuscript to be reviewed Manuscript to be reviewed 273 150 minutes of survey time, to ensure equal sampling across routes. We included all observations 274 of bumble bees. 275 276 We summarized land cover in areas surrounding nest-traps and bumble bee routes using the 2016 277 National Land Cover Database (NLCD) (Dewitz, 2019). We verified land-cover categories by 278 randomly spot checking against aerial photographs across approximately 25% of surveyed areas 279 and checking all areas characterized as barren, as that NLCD category can have a higher error 280 rate (Hollister et al., 2004). Land use surrounding one nest-trap that was near the border with 281 Canada was supplemented with visual assessment from aerial photos because NLCD data was 282 only available for half of the buffer area surrounding the nest-trap site. For tunnel-nesting bees, 283 we examined land cover within a radius of 250 m of nest-traps (Gathmann & Tscharntke, 2002; 284 Steffan-Dewenter et al., 2002). For bumble bees, we examined land cover within a 2 km radius 285 of the center of all bumble bee survey stops and summed them for each route (Hagen, Wikelski 286 & Kissling, 2011; Rao & Strange, 2012). We simplified NLCD land-cover classes to groupings 287 that we consider to be biologically relevant to bee distribution (Holzschuh, Steffan-Dewenter & 288 Tscharntke, 2010; Westerfelt, Weslien & Widenfalk, 2018; Lanterman et al., 2019). We 289 combined deciduous, mixed, and evergreen forest into the forested category, all developed 290 categories into one developed category, grasslands/herbaceous and pasture/hay into the 291 grasslands category, and woody wetlands and emergent herbaceous wetlands into the wetlands 292 category. Crops, open water, and barren were not combined with any other categories. Land use 293 surrounding nest-traps consisted of 28% forested, 20% grasslands, 18% developed, 12% crops, 294 14% wetlands, 6% open water, and 0.3% barren. Land use surrounding bumble bee route stops 295 consisted of 26% crops, 26% forested, 24% wetlands, 11% grassland, 8% developed, 5% open 296 water and <1% barren. 273 150 minutes of survey time, to ensure equal sampling across routes. We included all observations 274 of bumble bees. 275 273 150 minutes of survey time, to ensure equal sampling across routes. We included all observations 274 of bumble bees. 275 276 We summarized land cover in areas surrounding nest-traps and bumble bee routes using the 2016 277 National Land Cover Database (NLCD) (Dewitz, 2019). Manuscript to be reviewed We verified land-cover categories by 278 randomly spot checking against aerial photographs across approximately 25% of surveyed areas 279 and checking all areas characterized as barren, as that NLCD category can have a higher error 280 rate (Hollister et al., 2004). Land use surrounding one nest-trap that was near the border with 281 Canada was supplemented with visual assessment from aerial photos because NLCD data was 282 only available for half of the buffer area surrounding the nest-trap site. For tunnel-nesting bees, 283 we examined land cover within a radius of 250 m of nest-traps (Gathmann & Tscharntke, 2002; 284 Steffan-Dewenter et al., 2002). For bumble bees, we examined land cover within a 2 km radius 285 of the center of all bumble bee survey stops and summed them for each route (Hagen, Wikelski 286 & Kissling, 2011; Rao & Strange, 2012). We simplified NLCD land-cover classes to groupings 287 that we consider to be biologically relevant to bee distribution (Holzschuh, Steffan-Dewenter & 288 Tscharntke, 2010; Westerfelt, Weslien & Widenfalk, 2018; Lanterman et al., 2019). We 289 combined deciduous, mixed, and evergreen forest into the forested category, all developed 290 categories into one developed category, grasslands/herbaceous and pasture/hay into the 291 grasslands category, and woody wetlands and emergent herbaceous wetlands into the wetlands 292 category. Crops, open water, and barren were not combined with any other categories. Land use 293 surrounding nest-traps consisted of 28% forested, 20% grasslands, 18% developed, 12% crops, 294 14% wetlands, 6% open water, and 0.3% barren. Land use surrounding bumble bee route stops 295 consisted of 26% crops, 26% forested, 24% wetlands, 11% grassland, 8% developed, 5% open 296 water and <1% barren. 297 298 We examined the relationship of bees to land cover categories using redundancy analysis (RDA) 299 with presence-absence for tunnel-nesting bees and constrained correspondence analysis (CCA) 300 with abundance for bumble bees using the vegan R package (Oksanen et al., 2020). For the 301 RDA, we used forward selection using permutation tests with 1,000 permutations to select the 302 final model. We removed the land uses crops, wetlands, open water, and barren from the final 303 model due to lack of significance. Manuscript to be reviewed We created models for bumble bees with log-transformed 269 abundance of bumble bees per route per year as the response variable and ecological province as 270 the predictor with year and route as random effects. After preliminary analysis, we changed year 271 from a random to a fixed effect due to singularity. We limited data to include only routes with 272 three completed route runs (a set of five 10-minute observations) within a year, which equaled p p y p 267 Megachile campanulae and O. pumila were not recorded by nest-traps in the LMF and were 268 analyzed for PP and EBF only. We created models for bumble bees with log-transformed 269 abundance of bumble bees per route per year as the response variable and ecological province as 270 the predictor with year and route as random effects. After preliminary analysis, we changed year 271 from a random to a fixed effect due to singularity. We limited data to include only routes with 272 three completed route runs (a set of five 10-minute observations) within a year, which equaled PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed The bundles of Phragmites stems sent by the volunteer in Brown County 352 contained nests of Heriades carinata, Megachile campanulae, Megachile brevis Say, 1837, 353 Megachile rotundata, Megachile mendica Cresson, 1878 and Stelis coarctatus Crawford, 1916. 354 Of the bundles sent out in 2019, Hylaeus mesillae (Cockerell, 1896) emerged from a bundle of 355 Liatris ligulistylis stems in Hennepin County. A bundle of Asclepias incarnata stems in St. Louis 356 County produced Heriades carinata, Hoplitis albifrons (Kirby, 1837), Hylaeus verticalis 357 (Cresson, 1869), Megachile pugnata, Megachile relativa, and O. tersula. Two nest-building bee 358 species were only found in bundles: Megachile brevis and Hoplitis albifrons. 359 360 We displayed species distributions by mapping nest frequency across ecological provinces (Fig. 361 2, Fig. 3, Table 2). Comparison of nest frequency by province showed that total nest-building 319 combined made up about 85% of the research-grade records. Other commonly recorded species 320 included: Agapostemon virescens (Fabricius, 1775) (192 records), Melissodes bimaculatus 321 (Lepeletier, 1825) (165), Halictus ligatus Say, 1837 (123), and Megachile latimanus Say, 1823 322 (118). Some bee species were notably absent in iNaturalist, particularly those in the family 323 Halictidae (19 species were represented in iNaturalist of the 134 species known to be in 324 Minnesota) (Portman et al., 2023). 325 326 The iNaturalist data include research grade records from 79 of the 87 counties in Minnesota (Fig. 327 1). Bombus affinis, the federally endangered rusty patched bumble bee, was frequently identified 328 in iNaturalist data (over 500 observations). Public participants also documented declining 329 bumble bee species (B. terricola and B. pensylvanicus), an introduced species (Megachile 330 sculpturalis Smith, 1853), a newly documented in Minnesota species (Bombus nevadensis 331 Cresson, 1874) (Portman & Dolan, 2022), and a rarely recorded species (Bombus frigidus Smith, 332 1854). 333 334 Tunnel-nesting bees 335 From the 383 nest-traps in this study, we reared a total of 13,062 specimens, which emerged 336 from 1,821 nest tunnels. Specimens included 3,488 solitary nest-building wasps, 1,387 parasitic 337 wasps, and 7,123 bees from 32 species (Table 2, Appendix 3). Five bee species were 338 cleptoparasitic, species that lay eggs in a host bee's nest. Less than one percent of bee-occupied 339 nest tunnels were of introduced species. The bee species that occupied the greatest number of 340 nest tunnels were O. lignaria (484), Heriades carinata (375), O. Manuscript to be reviewed pumila (173), Megachile 341 pugnata (151), Megachile relativa (132), and Megachile campanulae (128). The Minnesota Bee 342 Atlas project also documented rarely collected species, including Megachile lapponica Thomson, 343 1872 and Hylaeus nelumbonis (Robertson, 1890), and four species, Megachile inimica Cresson, 344 1872, Megachile frugalis Cresson, 1872, Osmia georgica Cresson, 1878 and Stelis permaculata 345 Cockerell, 1898, that were new records for the state (Satyshur et al., 2021, 2022). The Minnesota 346 Bee Atlas specimens added six additional species to the UMN Insect Collection, Minnesota's 347 statewide repository. 348 349 The 14 stem bundles produced a total of 382 specimens, including 31 solitary nest-building 350 wasps, 10 parasitic wasps, and 336 bees. There were 13 species of bees, including one 351 cleptoparasitic species. The bundles of Phragmites stems sent by the volunteer in Brown County 352 contained nests of Heriades carinata, Megachile campanulae, Megachile brevis Say, 1837, 353 Megachile rotundata, Megachile mendica Cresson, 1878 and Stelis coarctatus Crawford, 1916. 354 Of the bundles sent out in 2019, Hylaeus mesillae (Cockerell, 1896) emerged from a bundle of 355 Liatris ligulistylis stems in Hennepin County. A bundle of Asclepias incarnata stems in St. Louis 356 County produced Heriades carinata, Hoplitis albifrons (Kirby, 1837), Hylaeus verticalis 319 combined made up about 85% of the research-grade records. Other commonly recorded species 320 included: Agapostemon virescens (Fabricius, 1775) (192 records), Melissodes bimaculatus 321 (Lepeletier, 1825) (165), Halictus ligatus Say, 1837 (123), and Megachile latimanus Say, 1823 322 (118). Some bee species were notably absent in iNaturalist, particularly those in the family 323 Halictidae (19 species were represented in iNaturalist of the 134 species known to be in 324 Minnesota) (Portman et al., 2023). 360 We displayed species distributions by mapping nest frequency across ecological provinces (Fig. 361 2, Fig. 3, Table 2). Comparison of nest frequency by province showed that total nest-building 362 bee tunnel use per trap was similar across the LMF, EBF, and PP (X2 = 2.27, df = 2, p = 0.3216) 363 with a mean ± SE of 4.9 ± 1.5 in the LMF, 4.2 ± 1.2 in the EBF, 3.6 ± 1.4 in the PP (Table 3). 364 O i t l d M hil l ti t i ifi tl f t i th LMF th i 360 We displayed species distributions by mapping nest frequency across ecological provinces (Fig. 361 2, Fig. 3, Table 2). Manuscript to be reviewed For the CCA, we removed the variable crops due to 304 multicollinearity (variance inflation factor >20), the variables open water and barren due to poor 305 correlation (intra-set correlations with axes 1,2, or 3 <0.4), and species accounting for less than 306 5% of the inertia for CCA 1 and 2 (B. citrinus, Bombus insularis (Smith, 1861), and Bombus 307 rufocinctus Cresson, 1863). Significance of the overall CCA and ordination axes was determined 308 with a Monte Carlo permutation test with 999 randomizations. 309 310 Results 311 iNaturalist 312 People will continue contributing observations to iNaturalist indefinitely, but as of 9 March 313 2021, the Minnesota Bee Atlas project included 18,956 records of bees from 2,300 observers. Of 314 these observations, 65.3% (12,384) were research-grade, slightly higher than the 60.8% rate of 315 research-grade observations for bees worldwide in the same period (Appendix 2). Research- 316 grade observations contained 33 genera (7 taken to subgenera) and 128 species. Of the top ten 317 most common species identified to research-grade, nine were bumble bees (Bombus), and the 318 tenth was the western honey bee (Apis mellifera Linnaeus 1758). Bumble bees and honey bees PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed 319 combined made up about 85% of the research-grade records. Other commonly recorded species 320 included: Agapostemon virescens (Fabricius, 1775) (192 records), Melissodes bimaculatus 321 (Lepeletier, 1825) (165), Halictus ligatus Say, 1837 (123), and Megachile latimanus Say, 1823 322 (118). Some bee species were notably absent in iNaturalist, particularly those in the family 323 Halictidae (19 species were represented in iNaturalist of the 134 species known to be in 324 Minnesota) (Portman et al., 2023). 325 326 The iNaturalist data include research grade records from 79 of the 87 counties in Minnesota (Fig. 327 1). Bombus affinis, the federally endangered rusty patched bumble bee, was frequently identified 328 in iNaturalist data (over 500 observations). Public participants also documented declining 329 bumble bee species (B. terricola and B. pensylvanicus), an introduced species (Megachile 330 sculpturalis Smith, 1853), a newly documented in Minnesota species (Bombus nevadensis 331 Cresson, 1874) (Portman & Dolan, 2022), and a rarely recorded species (Bombus frigidus Smith, 332 1854). 333 334 Tunnel-nesting bees 335 From the 383 nest-traps in this study, we reared a total of 13,062 specimens, which emerged 336 from 1,821 nest tunnels. Specimens included 3,488 solitary nest-building wasps, 1,387 parasitic 337 wasps, and 7,123 bees from 32 species (Table 2, Appendix 3). Five bee species were 338 cleptoparasitic, species that lay eggs in a host bee's nest. Less than one percent of bee-occupied 339 nest tunnels were of introduced species. The bee species that occupied the greatest number of 340 nest tunnels were O. lignaria (484), Heriades carinata (375), O. pumila (173), Megachile 341 pugnata (151), Megachile relativa (132), and Megachile campanulae (128). The Minnesota Bee 342 Atlas project also documented rarely collected species, including Megachile lapponica Thomson, 343 1872 and Hylaeus nelumbonis (Robertson, 1890), and four species, Megachile inimica Cresson, 344 1872, Megachile frugalis Cresson, 1872, Osmia georgica Cresson, 1878 and Stelis permaculata 345 Cockerell, 1898, that were new records for the state (Satyshur et al., 2021, 2022). The Minnesota 346 Bee Atlas specimens added six additional species to the UMN Insect Collection, Minnesota's 347 statewide repository. 348 349 The 14 stem bundles produced a total of 382 specimens, including 31 solitary nest-building 350 wasps, 10 parasitic wasps, and 336 bees. There were 13 species of bees, including one 351 cleptoparasitic species. Manuscript to be reviewed 365 the EBF or PP (Table 3). Osmia lignaria nested significantly more frequently in the LMF and 366 EBF than in the PP. Osmia pumila nested significantly more frequently in the EBF than the PP 367 and was absent from the LMF. Heriades carinata, Megachile campanulae, and Megachile 368 pugnata nested significantly more frequently in the PP and EBF than the LMF. Nests of 369 Megachile inermis Provancher, 1888, Hylaeus annulatus (Linnaeus, 1758) and Hylaeus 370 verticalis were infrequent (present in less than 10% of nest-traps) but primarily occurred in the 371 LMF. Megachile rotundata, Megachile mendica, Hylaeus leptocephalus (Morawitz, 1871), and 372 Hylaeus mesillae nests were infrequent, but were primarily found in the southern half of the state 373 across both the PP and EBF. Megachile centuncularis (Linnaeus, 1758) and Heriades variolosa 374 (Cresson, 1872) were also infrequent but found mostly in the PP. The TAP had very few nest- 375 traps, with only one or two nests for the species that were found there (O. lignaria, O. tersula, 376 Megachile relativa, Megachile pugnata, and Heriades carinata). The distributions of the 377 cleptoparasitic bees Coelioxys moesta Cresson, 1864, Coelioxys alternata Say, 1837, Coelioxys 378 modesta Smith, 1854 and S. coarctatus tracked, to a smaller extent, those of their hosts, 379 Megachile relativa, Megachile pugnata, Megachile campanulae, and Heriades carinata, 380 respectively. 381 382 Tunnel-nesting bee abundance and land use were significantly correlated for the first two RDA 383 axes according to the permutation test. Axes RDA1 (eigenvalue=0.05, F=14.69, p<0.001) and 384 RDA2 (eigenvalue=0.02, F=4.99, p<0.001) of the redundancy analysis explained a cumulative 385 97% of the variation (Fig. 4). RDA1 primarily distinguished between grasslands and forest 386 covers and RDA2 primarily distinguished between developed and grasslands (Table 4). Heriades 387 carinata and Megachile pugnata were associated with grassland land cover (Fig. 4). Megachile 388 campanulae was associated with developed land cover. Osmia lignaria was associated with 389 forested land cover. 390 391 Nest phenology data from 1,041 bee nest tunnels representing 17 species was of sufficient 392 quality to include in a summary (Fig. 5). Osmia completed nests earliest, with O. lignaria in 393 May, followed by O. pumila and then O. tersula near the end of June. Osmia georgica had only 394 one nest, which was completed between the middle of May and the end of June. Manuscript to be reviewed Megachile nests 395 were primarily completed between 15 June and 15 August, with most Megachile campanulae, 396 Megachile pugnata, and Megachile relativa completing nests near mid-July, most Megachile 397 inermis and Megachile rotundata completing nests in late July, and most Megachile mendica 398 completing nests near mid-August. We reared Megachile relativa from nest-traps that were 399 brought into the lab during mid-summer, showing this species can have two generations per year 400 in Minnesota and may have two nesting phenology peaks. Megachile centuncularis and 401 Megachile frugalis are represented by only one nest each in late July to August. For Megachile 402 inimica and Megachile lapponica, we have a last empty date but no full plug date, which only 403 indicates nests were completed after about July 7 and 18 respectively. Heriades species primarily 404 completed nests between 23 June and 15 August, with Heriades carinata slightly earlier than 405 Heriades variolosa and Heriades leavitti Crawford, 1913. 406 407 Bumble bees 408 Volunteers recorded 9,186 individuals belonging to 17 bumble bee species during 1,330 10- 409 minute observations at survey stops. Volunteers observed zero bumble bees at 220 out of 1,330 410 survey stops. Volunteers observed no bees across all five survey stops along a route for 10 route 365 the EBF or PP (Table 3). Osmia lignaria nested significantly more frequently in the LMF and 366 EBF than in the PP. Osmia pumila nested significantly more frequently in the EBF than the PP 367 and was absent from the LMF. Heriades carinata, Megachile campanulae, and Megachile 368 pugnata nested significantly more frequently in the PP and EBF than the LMF. Nests of 369 Megachile inermis Provancher, 1888, Hylaeus annulatus (Linnaeus, 1758) and Hylaeus 370 verticalis were infrequent (present in less than 10% of nest-traps) but primarily occurred in the 371 LMF. Megachile rotundata, Megachile mendica, Hylaeus leptocephalus (Morawitz, 1871), and 372 Hylaeus mesillae nests were infrequent, but were primarily found in the southern half of the state 373 across both the PP and EBF. Megachile centuncularis (Linnaeus, 1758) and Heriades variolosa 374 (Cresson, 1872) were also infrequent but found mostly in the PP. The TAP had very few nest- 375 traps, with only one or two nests for the species that were found there (O. lignaria, O. tersula, 376 Megachile relativa, Megachile pugnata, and Heriades carinata). Manuscript to be reviewed Comparison of nest frequency by province showed that total nest-building 362 bee tunnel use per trap was similar across the LMF, EBF, and PP (X2 = 2.27, df = 2, p = 0.3216) 363 with a mean ± SE of 4.9 ± 1.5 in the LMF, 4.2 ± 1.2 in the EBF, 3.6 ± 1.4 in the PP (Table 3). PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed The distributions of the 377 cleptoparasitic bees Coelioxys moesta Cresson, 1864, Coelioxys alternata Say, 1837, Coelioxys 378 modesta Smith, 1854 and S. coarctatus tracked, to a smaller extent, those of their hosts, 379 Megachile relativa, Megachile pugnata, Megachile campanulae, and Heriades carinata, 380 respectively. Manuscript to be reviewed An alternative CCA using presence-absence data instead of abundance 440 data for bumble bees is available in Appendix 4 to address possible aggregation effects from nest 441 proximity. 442 443 Discussion 444 The Minnesota Bee Atlas project was made possible by the contributions of over 2,500 project 445 volunteers and other iNaturalist users across three sampling protocols who recorded 30%, or 151, 446 of the approximately 500 bee species known in Minnesota (Portman et al., 2023). Each sampling 447 protocol contributed different and complementary data, indicating that multiple sampling levels 448 would be useful in future bee monitoring projects. Through iNaturalist, volunteers reported new 449 locations for B. affinis, as well as recording several other rare bumble bees and the first state 450 record of an adventive species. Nest-traps in this project produced baseline range data for 31 451 species, including four new state records, and expanded the known range for 16 of those species. 411 runs, representing seven different routes. Several species of conservation concern were 412 documented, including 17 B. affinis along four routes, 103 B. terricola along 14 routes, and 22 B. 413 pensylvanicus along 11 routes (Table 5). Patterns of abundance from survey routes added 414 information on regional prevalence of bumble bee species in comparison to historic and 415 biodiversity portal records that did not include survey effort (Fig. 6, Fig. 7). For example, while 416 B. rufocinctus was present in records from all four ecoregions, surveys showed that B. 411 runs, representing seven different routes. Several species of conservation concern were 412 documented, including 17 B. affinis along four routes, 103 B. terricola along 14 routes, and 22 B. 413 pensylvanicus along 11 routes (Table 5). Patterns of abundance from survey routes added 414 information on regional prevalence of bumble bee species in comparison to historic and 415 biodiversity portal records that did not include survey effort (Fig. 6, Fig. 7). For example, while 416 B. rufocinctus was present in records from all four ecoregions, surveys showed that B. 417 rufocinctus was most abundant in the EBF. The composition and total bumble bee abundance 418 varied among ecological provinces (Table 5). The most common bumble bees in the EBF were 419 B. impatiens (1,781), B. bimaculatus (1,109), B. vagans group (756), and B. griseocollis (733). 420 The most common bumble bees in the PP were B. griseocollis (102), B. bimaculatus (77), and B. Manuscript to be reviewed 411 runs, representing seven different routes. Several species of conservation concern were 412 documented, including 17 B. affinis along four routes, 103 B. terricola along 14 routes, and 22 B. 413 pensylvanicus along 11 routes (Table 5). Patterns of abundance from survey routes added 414 information on regional prevalence of bumble bee species in comparison to historic and 415 biodiversity portal records that did not include survey effort (Fig. 6, Fig. 7). For example, while 416 B. rufocinctus was present in records from all four ecoregions, surveys showed that B. 417 rufocinctus was most abundant in the EBF. The composition and total bumble bee abundance 418 varied among ecological provinces (Table 5). The most common bumble bees in the EBF were 419 B. impatiens (1,781), B. bimaculatus (1,109), B. vagans group (756), and B. griseocollis (733). 420 The most common bumble bees in the PP were B. griseocollis (102), B. bimaculatus (77), and B. 421 impatiens (55). Bombus ternarius (1,466) and B. vagans group (1,116) were the most common 422 bumble bees in the LMF. Total bumble bee abundance within a route in a year differed among 423 ecological provinces (X2=12.03, df=2,78, p< 0.01) with bee abundance per route lower in the PP 424 than the EBF or the LMF (Fig. 8, Table 6). 425 426 Bumble bee species abundance and land use were significantly correlated for the first three 427 canonical axes according to the Monte Carlo permutation test. Bumble bee species Axes CCA1 428 (eigenvalue=0.60, F=66.32, p<0.001) and CCA2 (eigenvalue=0.09, F=9.62, p<0.001) of the 429 correspondence analysis explained a cumulative 42% of the variation (Fig. 4). CCA1 primarily 430 distinguished between grasslands and wetlands covers and CCA2 primarily distinguished 431 between developed and grassland covers (Table 4). Habitat associations for species with lower 432 abundances may be due to chance (Legendre & Legendre, 2012), leading to caution interpreting 433 habitat associations for these species due to their low abundances: B. affinis (17), and Bombus 434 flavidus Eversmann, 1852 (36). Bombus fervidus, B. griseocollis, and B. bimaculatus were 435 associated with grassland land cover (Fig. 4). Bombus vagans group, B. borealis, and B. terricola 436 were associated with forested land cover. Bombus ternarius, Bombus perplexus Cresson, 1863, 437 and possibly B. flavidus were associated with forested and wetlands land covers. Bombus 438 impatiens, Bombus auricomus (Robertson, 1903), and possibly B. affinis were associated with 439 developed land cover. 407 Bumble bees 408 Volunteers recorded 9,186 individuals belonging to 17 bumble bee species during 1,330 10- 409 minute observations at survey stops. Volunteers observed zero bumble bees at 220 out of 1,330 410 survey stops. Volunteers observed no bees across all five survey stops along a route for 10 route PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed 421 impatiens (55). Bombus ternarius (1,466) and B. vagans group (1,116) were the most common 422 bumble bees in the LMF. Total bumble bee abundance within a route in a year differed among 423 ecological provinces (X2=12.03, df=2,78, p< 0.01) with bee abundance per route lower in the PP 424 than the EBF or the LMF (Fig. 8, Table 6). 416 B. rufocinctus was present in records from all four ecoregions, surveys showed that B. 417 rufocinctus was most abundant in the EBF. The composition and total bumble bee abundance 418 varied among ecological provinces (Table 5). The most common bumble bees in the EBF were 419 B. impatiens (1,781), B. bimaculatus (1,109), B. vagans group (756), and B. griseocollis (733). 420 The most common bumble bees in the PP were B. griseocollis (102), B. bimaculatus (77), and B. 421 impatiens (55). Bombus ternarius (1,466) and B. vagans group (1,116) were the most common 422 bumble bees in the LMF. Total bumble bee abundance within a route in a year differed among 423 ecological provinces (X2=12.03, df=2,78, p< 0.01) with bee abundance per route lower in the PP 424 than the EBF or the LMF (Fig. 8, Table 6). 425 426 Bumble bee species abundance and land use were significantly correlated for the first three 427 canonical axes according to the Monte Carlo permutation test. Bumble bee species Axes CCA1 428 (eigenvalue=0.60, F=66.32, p<0.001) and CCA2 (eigenvalue=0.09, F=9.62, p<0.001) of the 429 correspondence analysis explained a cumulative 42% of the variation (Fig. 4). CCA1 primarily 430 distinguished between grasslands and wetlands covers and CCA2 primarily distinguished 431 between developed and grassland covers (Table 4). Habitat associations for species with lower 432 abundances may be due to chance (Legendre & Legendre, 2012), leading to caution interpreting 433 habitat associations for these species due to their low abundances: B. affinis (17), and Bombus 434 flavidus Eversmann, 1852 (36). Bombus fervidus, B. griseocollis, and B. bimaculatus were 435 associated with grassland land cover (Fig. 4). Bombus vagans group, B. borealis, and B. terricola 436 were associated with forested land cover. Bombus ternarius, Bombus perplexus Cresson, 1863, 437 and possibly B. flavidus were associated with forested and wetlands land covers. Bombus 438 impatiens, Bombus auricomus (Robertson, 1903), and possibly B. affinis were associated with 439 developed land cover. Manuscript to be reviewed An alternative CCA using presence-absence data instead of abundance 440 data for bumble bees is available in Appendix 4 to address possible aggregation effects from nest 441 proximity. 442 443 Discussion 444 The Minnesota Bee Atlas project was made possible by the contributions of over 2,500 project 445 volunteers and other iNaturalist users across three sampling protocols who recorded 30%, or 151, 446 of the approximately 500 bee species known in Minnesota (Portman et al., 2023). Each sampling 447 protocol contributed different and complementary data, indicating that multiple sampling levels 448 would be useful in future bee monitoring projects. Through iNaturalist, volunteers reported new 449 locations for B. affinis, as well as recording several other rare bumble bees and the first state 450 record of an adventive species. Nest-traps in this project produced baseline range data for 31 451 species, including four new state records, and expanded the known range for 16 of those species. 452 We also found ecological province associations for six tunnel-nesting species and landcover 453 associations for four species. Volunteer-collected data provided relative nesting seasonality of 454 bee species and indicated some species with multiple generations per year. Bumble bee surveys 455 examined abundances across ecological provinces, indicating potential benefit of a regional 456 focus on bumble bee habitat management, as well as possible habitat associations for species of 425 426 Bumble bee species abundance and land use were significantly correlated for the first three 427 canonical axes according to the Monte Carlo permutation test. Bumble bee species Axes CCA1 428 (eigenvalue=0.60, F=66.32, p<0.001) and CCA2 (eigenvalue=0.09, F=9.62, p<0.001) of the 429 correspondence analysis explained a cumulative 42% of the variation (Fig. 4). CCA1 primarily 430 distinguished between grasslands and wetlands covers and CCA2 primarily distinguished 431 between developed and grassland covers (Table 4). Habitat associations for species with lower 432 abundances may be due to chance (Legendre & Legendre, 2012), leading to caution interpreting 433 habitat associations for these species due to their low abundances: B. affinis (17), and Bombus 434 flavidus Eversmann, 1852 (36). Bombus fervidus, B. griseocollis, and B. bimaculatus were 435 associated with grassland land cover (Fig. 4). Bombus vagans group, B. borealis, and B. terricola 436 were associated with forested land cover. Bombus ternarius, Bombus perplexus Cresson, 1863, 437 and possibly B. flavidus were associated with forested and wetlands land covers. Bombus 438 impatiens, Bombus auricomus (Robertson, 1903), and possibly B. Manuscript to be reviewed affinis were associated with 439 developed land cover. An alternative CCA using presence-absence data instead of abundance 440 data for bumble bees is available in Appendix 4 to address possible aggregation effects from nest 441 proximity. 442 443 Discussion 444 The Minnesota Bee Atlas project was made possible by the contributions of over 2,500 project 445 volunteers and other iNaturalist users across three sampling protocols who recorded 30%, or 151, 446 of the approximately 500 bee species known in Minnesota (Portman et al., 2023). Each sampling 447 protocol contributed different and complementary data, indicating that multiple sampling levels 448 would be useful in future bee monitoring projects. Through iNaturalist, volunteers reported new 449 locations for B. affinis, as well as recording several other rare bumble bees and the first state 450 record of an adventive species. Nest-traps in this project produced baseline range data for 31 451 species, including four new state records, and expanded the known range for 16 of those species. 452 We also found ecological province associations for six tunnel-nesting species and landcover 453 associations for four species. Volunteer-collected data provided relative nesting seasonality of 454 bee species and indicated some species with multiple generations per year. Bumble bee surveys 455 examined abundances across ecological provinces, indicating potential benefit of a regional 456 focus on bumble bee habitat management, as well as possible habitat associations for species of 426 Bumble bee species abundance and land use were significantly correlated for the first three 427 canonical axes according to the Monte Carlo permutation test. Bumble bee species Axes CCA1 428 (eigenvalue=0.60, F=66.32, p<0.001) and CCA2 (eigenvalue=0.09, F=9.62, p<0.001) of the 429 correspondence analysis explained a cumulative 42% of the variation (Fig. 4). CCA1 primarily 430 distinguished between grasslands and wetlands covers and CCA2 primarily distinguished 431 between developed and grassland covers (Table 4). Habitat associations for species with lower 432 abundances may be due to chance (Legendre & Legendre, 2012), leading to caution interpreting 433 habitat associations for these species due to their low abundances: B. affinis (17), and Bombus 434 flavidus Eversmann, 1852 (36). Bombus fervidus, B. griseocollis, and B. bimaculatus were 435 associated with grassland land cover (Fig. 4). Bombus vagans group, B. borealis, and B. terricola 436 were associated with forested land cover. Bombus ternarius, Bombus perplexus Cresson, 1863, 437 and possibly B. flavidus were associated with forested and wetlands land covers. Manuscript to be reviewed nevadensis, which were not found in the more structured surveys, 471 also illustrate the utility of the large number of observers and widespread observations on the 472 platform. 473 474 A second strength of iNaturalist is that observations are rapidly available, making the platform 475 useful for monitoring adventive species that can be quickly identified to research grade. 476 Previously documented in neighboring states (Parys, Tripodi & Sampson, 2015), Megachile 477 sculpturalis, an introduced species with an expanding range, was recorded for the first time in 478 Minnesota in the first year of the Bee Atlas project. Although it was only recorded once in the 479 Minnesota Bee Atlas iNaturalist project, it is a large and easily recognized bee, and opportunistic 480 participatory science platforms have been important to monitoring its spread in Europe (Le Féon 481 et al., 2018; Flaminio et al., 2021; Dubaić et al., 2022). The fact that Megachile sculpturalis has 482 only been recorded once in the five years of the project may indicate that it is reaching either the 483 northern or western limits of its range in North America, or it could indicate the low population 484 densities typical of the early stages of colonization (Dubaić et al., 2022). Increased monitoring 485 effort is needed to assess its status and potential impact. With outreach to engage public interest, 486 the Minnesota Bee Atlas iNaturalist project may be able to produce accurate and up to date 487 distribution maps for Megachile sculpturalis, allowing biologists to determine its spread in the 488 state. 489 490 One limitation of iNaturalist is that observations do not reflect relative abundance. Larger bees 491 comprise the majority of observations, both non-research and research-grade, with over half of 492 non-research grade observations from the families Apidae and Megachilidae. Among the larger 493 bees, a subset of more easily identified bees, bumble bees and honey bees, make up 85% of 494 research-grade observations. This is consistent with other opportunistic participatory science 495 programs, which either focus on bumble bees exclusively or broad bee groupings (Beckham & 496 Atkinson, 2017; Maher, Manco & Ings, 2019; Flaminio et al., 2021; Griffin et al., 2021). In 497 strong contrast, sweep netting collections in this region show high abundances of bees from the 498 family Halictidae (Lane et al., 2020; Evans et al., 2022). For many other bee groups, and 457 conservation concern. Manuscript to be reviewed Manuscript to be reviewed 457 conservation concern. The ecological associations and patterns of abundance discovered by the 458 Minnesota Bee Atlas can inform management decisions to improve pollinator conservation 459 actions and recovery of endangered species. 460 461 iNaturalist 462 There are strengths and limitations to using iNaturalist to study bees. One clear strength is the 463 large number of observers, which increases the chances of finding rare species (Donnelly et al., 464 2014; Wilson et al., 2020), especially bumble bees, which were most frequently photographed 465 and identified in our project. Many bumble bee species are becoming less abundant and 466 experiencing reductions in their geographic ranges, making information about their status 467 particularly important for conservation efforts (Goulson, Lye & Darvill, 2008; Hatfield et al., 468 2015; Beckham & Atkinson, 2017). New location information for B. affinis is important for 469 recovery plans for this endangered bee (U.S. Fish and Wildlife Service, 2021). The iNaturalist 470 records of B. frigidus and B. nevadensis, which were not found in the more structured surveys, 471 also illustrate the utility of the large number of observers and widespread observations on the 472 platform. 473 474 A second strength of iNaturalist is that observations are rapidly available, making the platform 475 useful for monitoring adventive species that can be quickly identified to research grade. 476 Previously documented in neighboring states (Parys, Tripodi & Sampson, 2015), Megachile 477 sculpturalis, an introduced species with an expanding range, was recorded for the first time in 478 Minnesota in the first year of the Bee Atlas project. Although it was only recorded once in the 479 Minnesota Bee Atlas iNaturalist project, it is a large and easily recognized bee, and opportunistic 480 participatory science platforms have been important to monitoring its spread in Europe (Le Féon 481 et al., 2018; Flaminio et al., 2021; Dubaić et al., 2022). The fact that Megachile sculpturalis has 482 only been recorded once in the five years of the project may indicate that it is reaching either the 483 northern or western limits of its range in North America, or it could indicate the low population 484 densities typical of the early stages of colonization (Dubaić et al., 2022). Increased monitoring 485 effort is needed to assess its status and potential impact. Manuscript to be reviewed Bombus 438 impatiens, Bombus auricomus (Robertson, 1903), and possibly B. affinis were associated with 439 developed land cover. An alternative CCA using presence-absence data instead of abundance 440 data for bumble bees is available in Appendix 4 to address possible aggregation effects from nest 441 proximity. PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed With outreach to engage public interest, 486 the Minnesota Bee Atlas iNaturalist project may be able to produce accurate and up to date 487 distribution maps for Megachile sculpturalis, allowing biologists to determine its spread in the 488 state. 489 490 One limitation of iNaturalist is that observations do not reflect relative abundance. Larger bees 491 comprise the majority of observations, both non-research and research-grade, with over half of 492 non-research grade observations from the families Apidae and Megachilidae. Among the larger 493 bees, a subset of more easily identified bees, bumble bees and honey bees, make up 85% of 494 research-grade observations. This is consistent with other opportunistic participatory science 495 programs, which either focus on bumble bees exclusively or broad bee groupings (Beckham & 496 Atkinson, 2017; Maher, Manco & Ings, 2019; Flaminio et al., 2021; Griffin et al., 2021). In 497 strong contrast, sweep netting collections in this region show high abundances of bees from the 498 family Halictidae (Lane et al., 2020; Evans et al., 2022). For many other bee groups, and 499 especially smaller species, existing identification methods require expert examination of physica 500 specimens to assign species-level identifications (Le Féon et al., 2016; Woodard et al., 2020; 501 Flaminio et al., 2021). If iNaturalist records are used to describe the structure of the bee 502 community, one should keep in mind that some groups of bees are likely to be overlooked 457 conservation concern. The ecological associations and patterns of abundance discovered by the 458 Minnesota Bee Atlas can inform management decisions to improve pollinator conservation 459 actions and recovery of endangered species. 460 461 iNaturalist 462 There are strengths and limitations to using iNaturalist to study bees. One clear strength is the 463 large number of observers, which increases the chances of finding rare species (Donnelly et al., 464 2014; Wilson et al., 2020), especially bumble bees, which were most frequently photographed 465 and identified in our project. Many bumble bee species are becoming less abundant and 466 experiencing reductions in their geographic ranges, making information about their status 467 particularly important for conservation efforts (Goulson, Lye & Darvill, 2008; Hatfield et al., 468 2015; Beckham & Atkinson, 2017). New location information for B. affinis is important for 469 recovery plans for this endangered bee (U.S. Fish and Wildlife Service, 2021). The iNaturalist 470 records of B. frigidus and B. Manuscript to be reviewed The ecological associations and patterns of abundance discovered by the 458 Minnesota Bee Atlas can inform management decisions to improve pollinator conservation 459 actions and recovery of endangered species. Manuscript to be reviewed Alternatively, the availability of resin plants as a nesting 542 resource may not limit distribution. Westerfeld, Weslien and Widenfalk (2018) found that tunnel 543 nesting bee nest abundance could be predicted by both nest substrate and food plant availability, 544 but to different degrees for pollen generalists and specialists. Heriades carinata is considered 545 polylectic, but Megachile campanulae has been associated with plants in the genus Campanula, 546 and their distribution may be predicted more by food resources. This bee was associated with 547 developed land cover in our study, and high abundance of the weedy plant Campanula 548 rapunculoides in developed areas could be a driver in their nesting success. In another case, the 503 because of their small size, nondescript coloring, or habitat specialization. However, the 504 likelihood of identification may be improved with training to improve photo quality and 505 advancements in artificial intelligence. 518 519 Clarifying distributions allows us to start associating bees with climates and habitats, as well as 520 providing baseline data for future comparisons. By using standardized, repeatable methods to 521 survey the whole state simultaneously, we were able to compare nest frequency and explore 522 ecological province and landcover associations. Province associations could be due to climatic or 523 plant community differences. For example, both factors may influence the distribution of 524 Megachile relativa. This species can have lower supercooling points than Megachile rotundata, 525 which allowed Megachile relativa to survive winter outdoors in Alberta, Canada (Krunic & Salt, 526 1971) and may contribute to its northern distribution and association with the LMF in this study. 527 The LMF plant community could also contribute to this observed association. The LMF is 528 characterized by broad areas of conifer forest, mixed hardwood and conifer forests, and conifer 529 bogs and swamps (Hanson & Hargrave, 1996). Despite our finding of no association of 530 Megachile relativa with forested land cover, previous observations showed that this species 531 preferred nest sites at woodland edges in Wisconsin (Medler & Koerber, 1958). Other bee 532 species showed associations that were counter to our expectations based on current knowledge of 533 their biology. We expected the bee species that use resin for nest construction, Heriades carinata 534 and Megachile campanulae, to nest more frequently in the LMF due to the dominance of many 535 resin-producing trees in the LMF, including Pinus, Abies, Picea, and Populus spp. Manuscript to be reviewed Other bee 532 species showed associations that were counter to our expectations based on current knowledge of 533 their biology. We expected the bee species that use resin for nest construction, Heriades carinata 534 and Megachile campanulae, to nest more frequently in the LMF due to the dominance of many 535 resin-producing trees in the LMF, including Pinus, Abies, Picea, and Populus spp. (Minnesota 536 Department of Natural Resources, 2022) and accounts of conifer resin use in Heriades and 537 Megachile campanulae (Medler & Lussenhop, 1968; Maciel de Almeida Correia, 1977, MacIvor 538 & Salehi, 2014). However, we found that Megachile campanulae was absent from the LMF and 539 that Heriades carinata nested more frequently in the PP and EBF than the LMF and was 540 associated with grassland land cover. The plant communities of the PP and EBF may contain 541 acceptable resin sources for these bees. Alternatively, the availability of resin plants as a nesting 542 resource may not limit distribution. Westerfeld, Weslien and Widenfalk (2018) found that tunnel 543 nesting bee nest abundance could be predicted by both nest substrate and food plant availability, 544 but to different degrees for pollen generalists and specialists. Heriades carinata is considered 545 polylectic, but Megachile campanulae has been associated with plants in the genus Campanula, 546 and their distribution may be predicted more by food resources. This bee was associated with 547 developed land cover in our study, and high abundance of the weedy plant Campanula 548 rapunculoides in developed areas could be a driver in their nesting success. In another case, the 503 because of their small size, nondescript coloring, or habitat specialization. However, the 504 likelihood of identification may be improved with training to improve photo quality and 505 advancements in artificial intelligence. 506 507 Tunnel-nesting bees 508 Nest traps and stem bundles combined with iNaturalist observations enhanced our understanding 509 of species distributions in Minnesota for 32 tunnel-nesting species. For 16 species, our project 510 expanded the known geographic extent of their distribution in the state compared to the UMN 511 Insect Collection. We documented that the ranges of five cleptoparasitic bee species mimicked 512 that of their hosts but with a smaller geographic spread. This may indicate the range in which the 513 host bees have a large enough population to support these parasitic bees (Sheffield et al., 2013). Manuscript to be reviewed 514 The collection of four new species records for the state along with rarely collected species is 515 consistent with Westphal et al. (2008), who found numerous species in nest-traps in Europe that 516 were not recorded with any other sampling methods. It may also reflect our expansion of 517 collection efforts over the whole state or possible recent changes in species’ ranges. 518 519 Clarifying distributions allows us to start associating bees with climates and habitats, as well as 520 providing baseline data for future comparisons. By using standardized, repeatable methods to 521 survey the whole state simultaneously, we were able to compare nest frequency and explore 522 ecological province and landcover associations. Province associations could be due to climatic or 523 plant community differences. For example, both factors may influence the distribution of 524 Megachile relativa. This species can have lower supercooling points than Megachile rotundata, 525 which allowed Megachile relativa to survive winter outdoors in Alberta, Canada (Krunic & Salt, 526 1971) and may contribute to its northern distribution and association with the LMF in this study. 527 The LMF plant community could also contribute to this observed association. The LMF is 528 characterized by broad areas of conifer forest, mixed hardwood and conifer forests, and conifer 529 bogs and swamps (Hanson & Hargrave, 1996). Despite our finding of no association of 530 Megachile relativa with forested land cover, previous observations showed that this species 531 preferred nest sites at woodland edges in Wisconsin (Medler & Koerber, 1958). Other bee 532 species showed associations that were counter to our expectations based on current knowledge of 533 their biology. We expected the bee species that use resin for nest construction, Heriades carinata 534 and Megachile campanulae, to nest more frequently in the LMF due to the dominance of many 535 resin-producing trees in the LMF, including Pinus, Abies, Picea, and Populus spp. (Minnesota 536 Department of Natural Resources, 2022) and accounts of conifer resin use in Heriades and 537 Megachile campanulae (Medler & Lussenhop, 1968; Maciel de Almeida Correia, 1977, MacIvor 538 & Salehi, 2014). However, we found that Megachile campanulae was absent from the LMF and 539 that Heriades carinata nested more frequently in the PP and EBF than the LMF and was 540 associated with grassland land cover. The plant communities of the PP and EBF may contain 541 acceptable resin sources for these bees. 461 iNaturalist 474 A second strength of iNaturalist is that observations are rapidly available, making the platform 475 useful for monitoring adventive species that can be quickly identified to research grade. PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed 503 because of their small size, nondescript coloring, or habitat specialization. However, the 504 likelihood of identification may be improved with training to improve photo quality and 505 advancements in artificial intelligence. 506 507 Tunnel-nesting bees 508 Nest traps and stem bundles combined with iNaturalist observations enhanced our understanding 509 of species distributions in Minnesota for 32 tunnel-nesting species. For 16 species, our project 510 expanded the known geographic extent of their distribution in the state compared to the UMN 511 Insect Collection. We documented that the ranges of five cleptoparasitic bee species mimicked 512 that of their hosts but with a smaller geographic spread. This may indicate the range in which the 513 host bees have a large enough population to support these parasitic bees (Sheffield et al., 2013). 514 The collection of four new species records for the state along with rarely collected species is 515 consistent with Westphal et al. (2008), who found numerous species in nest-traps in Europe that 516 were not recorded with any other sampling methods. It may also reflect our expansion of 517 collection efforts over the whole state or possible recent changes in species’ ranges. 518 519 Clarifying distributions allows us to start associating bees with climates and habitats, as well as 520 providing baseline data for future comparisons. By using standardized, repeatable methods to 521 survey the whole state simultaneously, we were able to compare nest frequency and explore 522 ecological province and landcover associations. Province associations could be due to climatic or 523 plant community differences. For example, both factors may influence the distribution of 524 Megachile relativa. This species can have lower supercooling points than Megachile rotundata, 525 which allowed Megachile relativa to survive winter outdoors in Alberta, Canada (Krunic & Salt, 526 1971) and may contribute to its northern distribution and association with the LMF in this study. 527 The LMF plant community could also contribute to this observed association. The LMF is 528 characterized by broad areas of conifer forest, mixed hardwood and conifer forests, and conifer 529 bogs and swamps (Hanson & Hargrave, 1996). Despite our finding of no association of 530 Megachile relativa with forested land cover, previous observations showed that this species 531 preferred nest sites at woodland edges in Wisconsin (Medler & Koerber, 1958). Manuscript to be reviewed 549 province associations we found differ from past records. We found significantly higher nest 550 frequency for Megachile pugnata in the EBF and PP, while specimens in the UMN Insect 551 Collection were predominantly in the EBF and LMF. This discrepancy could be due to different 552 collecting efforts or could reflect previous landscapes or distributions (Gardner & Spivak, 2014). 553 We found an association of Megachile pugnata with grassland land cover, which could explain 554 their higher frequency in the PP and the EBF. 555 556 Five of the nine tunnel-nesting bee species tested in this study showed no association in the land 557 cover analysis. This may indicate that a single broad land cover category does not capture the 558 habitat elements to which many tunnel-nesting bees are responsive. In addition, it should be 559 noted that the distributions of O. lignaria and Megachile rotundata may be influenced by human 560 management, including commercial sales, in addition to climatic differences and plant 561 communities. 562 563 Although nest-traps have been shown to be a reliable and unbiased way to assess ecological 564 association of tunnel-nesting bee species (Staab et al., 2018), nest-traps typically only sample a 565 portion of the tunnel-nesting bee community (Westphal et al., 2008; Prendergast et al., 2020). 566 Several factors may have contributed to the non-detection of tunnel-nesting bee species in this 567 study, which should not be interpreted as absence. It is possible that species may utilize nest- 568 traps less frequently in areas with more suitable natural nesting substrates (Westphal et al., 2008; 569 Carper & Bowers, 2017), which is a complicating factor for this sampling method. However, in 570 this study, overall bee nest frequency was statistically similar across all ecological provinces, 571 forested or otherwise (Table 3). Some nests produced no identifiable offspring due to parasitism 572 or other causes. These nests were left out of all analyses. As we saw in this study, some tunnel- 573 nesting bee species in Minnesota may have more than one generation per year. Species emerging 574 before our fall nest trap collection would not be captured if they did not re-nest in the traps. Manuscript to be reviewed Rare 575 species take more effort to detect, and even with our full coverage of the state, three years of 576 sampling, and focus on natural habitats, we may have sampled too small a proportion of bees to 577 reliably find some rare species, or species that prefer rare habitats. Solid wood traps may not be 578 an acceptable or preferred nest substrate for some tunnel-nesting bee species. Although Osmia 579 and Megachile are often considered tunnel-nesting genera, a proportion of species in both genera 580 nest in the ground, and we would not have expected them in this study (Cane, Griswold & 581 Parker, 2007; Sheffield et al., 2011; Rightmyer, Griswold & Brady, 2013). Similarly, bees in the 582 genus Ceratina Latreille are obligate stem excavators and would not be expected (Rehan & 583 Richards, 2010; Vickruck et al., 2011). Two species that we collected rarely in the Bee Atlas, 584 Hylaeus mesillae and Anthophora terminalis (Cresson, 1869), were common in UMN Insect 585 Collection records, suggesting that wood block nest-traps are a less effective sampling method 586 for these species. Anthophora terminalis is known from fallen or rotting wood substrates 587 (Cockerell, 1903; Sladen, 1919; Medler, 1964), as are Megachile frigida Smith, 1853 and Osmia 588 bucephela Cresson, 1864 (Stephen, 1956; Krombein, 1967) which we did not collect. Pithy or 589 hollow stems of many plant species are also used as nest substrates (Satyshur & Evans, 2021) 590 and might be preferred by some bees. Our stem bundles did not produce enough bee nests to 591 distinguish any preference between plant stem species but did produce two bee species not 592 collected in our wood nest-traps: Megachile brevis and Hoplitis albifrons. Hoplitis species and 593 Hylaeus messillae are frequently found in stems (Parker & Bohart 1966; Medler & Lussenhop 562 563 Although nest-traps have been shown to be a reliable and unbiased way to assess ecological 564 association of tunnel-nesting bee species (Staab et al., 2018), nest-traps typically only sample a 565 portion of the tunnel-nesting bee community (Westphal et al., 2008; Prendergast et al., 2020). 566 Several factors may have contributed to the non-detection of tunnel-nesting bee species in this 567 study, which should not be interpreted as absence. Manuscript to be reviewed It is possible that species may utilize nest- 568 traps less frequently in areas with more suitable natural nesting substrates (Westphal et al., 2008; 569 Carper & Bowers, 2017), which is a complicating factor for this sampling method. However, in 570 this study, overall bee nest frequency was statistically similar across all ecological provinces, 571 forested or otherwise (Table 3). Some nests produced no identifiable offspring due to parasitism 572 or other causes. These nests were left out of all analyses. As we saw in this study, some tunnel- 573 nesting bee species in Minnesota may have more than one generation per year. Species emerging 574 before our fall nest trap collection would not be captured if they did not re-nest in the traps. Rare 575 species take more effort to detect, and even with our full coverage of the state, three years of 576 sampling, and focus on natural habitats, we may have sampled too small a proportion of bees to 577 reliably find some rare species, or species that prefer rare habitats. Solid wood traps may not be 578 an acceptable or preferred nest substrate for some tunnel-nesting bee species. Although Osmia 579 and Megachile are often considered tunnel-nesting genera, a proportion of species in both genera 580 nest in the ground, and we would not have expected them in this study (Cane, Griswold & 581 Parker, 2007; Sheffield et al., 2011; Rightmyer, Griswold & Brady, 2013). Similarly, bees in the 582 genus Ceratina Latreille are obligate stem excavators and would not be expected (Rehan & 583 Richards, 2010; Vickruck et al., 2011). Two species that we collected rarely in the Bee Atlas, 584 Hylaeus mesillae and Anthophora terminalis (Cresson, 1869), were common in UMN Insect 585 Collection records, suggesting that wood block nest-traps are a less effective sampling method 586 for these species. Anthophora terminalis is known from fallen or rotting wood substrates 587 (Cockerell, 1903; Sladen, 1919; Medler, 1964), as are Megachile frigida Smith, 1853 and Osmia 588 bucephela Cresson, 1864 (Stephen, 1956; Krombein, 1967) which we did not collect. Pithy or 589 hollow stems of many plant species are also used as nest substrates (Satyshur & Evans, 2021) 590 and might be preferred by some bees. Manuscript to be reviewed (Minnesota 536 Department of Natural Resources, 2022) and accounts of conifer resin use in Heriades and 537 Megachile campanulae (Medler & Lussenhop, 1968; Maciel de Almeida Correia, 1977, MacIvor 538 & Salehi, 2014). However, we found that Megachile campanulae was absent from the LMF and 539 that Heriades carinata nested more frequently in the PP and EBF than the LMF and was 540 associated with grassland land cover. The plant communities of the PP and EBF may contain 541 acceptable resin sources for these bees. Alternatively, the availability of resin plants as a nesting 542 resource may not limit distribution. Westerfeld, Weslien and Widenfalk (2018) found that tunnel 543 nesting bee nest abundance could be predicted by both nest substrate and food plant availability, 544 but to different degrees for pollen generalists and specialists. Heriades carinata is considered 545 polylectic, but Megachile campanulae has been associated with plants in the genus Campanula, 546 and their distribution may be predicted more by food resources. This bee was associated with 547 developed land cover in our study, and high abundance of the weedy plant Campanula 548 rapunculoides in developed areas could be a driver in their nesting success. In another case, the PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed Our stem bundles did not produce enough bee nests to 591 distinguish any preference between plant stem species but did produce two bee species not 592 collected in our wood nest-traps: Megachile brevis and Hoplitis albifrons. Hoplitis species and 593 Hylaeus messillae are frequently found in stems (Parker & Bohart, 1966; Medler & Lussenhop, 594 1968) but were rare in this study. Megachile brevis is known from a wide variety of substrates PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed A more targeted study, returning to known 600 collection areas and looking for species that have not been recorded in Minnesota in recent years 601 is warranted. 602 603 In addition to distribution data, we collected data on nesting phenology, which returned a date 604 range when stem nesting bee species are likely to complete nesting and indicated the relative 605 seasonality of species. Volunteer observations also allowed us to catch Megachile relativa 606 emerging both mid-summer and the following spring. This agrees with the bivoltine life cycle for 607 Megachile relativa found in Wisconsin (Medler & Koerber, 1958) and expands the known range 608 of bivoltinism into Minnesota. It is important to remember that the phenology event volunteers 609 recorded was nest plugs, which are made after a nest is completed. Therefore, the bee’s active 610 period likely begins several weeks earlier. Despite this, in 10 of the 17 species we have data for, 611 nest plugs were observed several days to several weeks earlier than the range of collection dates 612 for the same species in the UMN Insect Collection (Fig. 5). This could be due to the large 613 increase in records and full season of data collection made possible by participatory science 614 (Soroye, Ahmed & Kerr, 2018; Dubaić et al., 2022). Another possibility is that earlier recorded 615 activity periods are the result of advancing phenology with climate change. Bartomeus et al. 616 (2011) compared collection dates of museum specimens collected between 1880 and 2010 for 10 617 bee species in northeastern North America, including two of the species in this project. They 618 found an average phenological advance of 10.4 days. The phenological data we have recorded 619 helps define these bees’ temporal habitats and lays the groundwork for assessing changes. 620 621 Bumble Bees 622 The bumble bee surveys of the Minnesota Bee Atlas project used consistent survey effort across 623 routes, providing the opportunity to examine patterns of bumble bee abundances and species 624 associations with land use, all of which have been difficult to do from museum collections or 625 biodiversity portal observations alone. We have reliable information on ranges of Minnesota 626 bumble bees due to numerous records of bumble bee species courtesy of the Bumble Bees of 627 North America database (Richardson, 2021). Our surveys not only confirm ranges, such as the 628 northern distributions of B. ternarius, B. Manuscript to be reviewed 595 including dead stems, ground, leaves and under dried cattle dung (Michener, 1953). Some 596 Minnesota species not found in this study, such as Megachile montivaga Cresson, 1878 (Orr, 597 Portman & Griswold, 2015) and Osmia atriventris Cresson, 1864 (Fye, 1965) are also known 598 from stems. Future studies of tunnel-nesting bees are likely to sample a larger proportion of the 599 community by using both wood and stem substrates. A more targeted study, returning to known 600 collection areas and looking for species that have not been recorded in Minnesota in recent years 601 is warranted. 602 603 In addition to distribution data, we collected data on nesting phenology, which returned a date 604 range when stem nesting bee species are likely to complete nesting and indicated the relative 605 seasonality of species. Volunteer observations also allowed us to catch Megachile relativa 606 emerging both mid-summer and the following spring. This agrees with the bivoltine life cycle for 607 Megachile relativa found in Wisconsin (Medler & Koerber, 1958) and expands the known range 608 of bivoltinism into Minnesota. It is important to remember that the phenology event volunteers 609 recorded was nest plugs, which are made after a nest is completed. Therefore, the bee’s active 610 period likely begins several weeks earlier. Despite this, in 10 of the 17 species we have data for, 611 nest plugs were observed several days to several weeks earlier than the range of collection dates 612 for the same species in the UMN Insect Collection (Fig. 5). This could be due to the large 613 increase in records and full season of data collection made possible by participatory science 614 (Soroye, Ahmed & Kerr, 2018; Dubaić et al., 2022). Another possibility is that earlier recorded 615 activity periods are the result of advancing phenology with climate change. Bartomeus et al. 616 (2011) compared collection dates of museum specimens collected between 1880 and 2010 for 10 617 bee species in northeastern North America, including two of the species in this project. They 618 found an average phenological advance of 10.4 days. The phenological data we have recorded 619 helps define these bees’ temporal habitats and lays the groundwork for assessing changes. Manuscript to be reviewed terricola, B. borealis, B. flavidus, and B. perplexus, but 629 also provide insight into bumble bee community structure. For example, although B. griseocollis 630 is present throughout the state, they are the dominant bumble bee community members in only 631 two of the three examined ecological provinces (PP and EBF). Further exploration could reveal 632 specific ecological drivers of this pattern. Although we identified many of the submitted 633 photographs for B. vagans and B. sandersoni to species level to create maps showing their 634 distributions, B. vagans had to be combined with B. sandersoni for comparisons of abundance 635 and habitat associations, because many observations could not be distinguished. Future volunteer 636 surveys may be able to distinguish these species as the quality of cameras available to volunteers 637 increases. Minnesota bumble bees not found on survey routes include B. frigidus, Bombus huntii 638 Greene 1860, Bombus variabilis (Cresson, 1872), Bombus ashtoni (Cresson, 1864) (sometimes 639 considered to be conspecific with Bombus bohemicus Seidl, 1837), Bombus fraternus (Smith, 602 603 In addition to distribution data, we collected data on nesting phenology, which returned a date 604 range when stem nesting bee species are likely to complete nesting and indicated the relative 605 seasonality of species. Volunteer observations also allowed us to catch Megachile relativa 606 emerging both mid-summer and the following spring. This agrees with the bivoltine life cycle for 607 Megachile relativa found in Wisconsin (Medler & Koerber, 1958) and expands the known range 608 of bivoltinism into Minnesota. It is important to remember that the phenology event volunteers 609 recorded was nest plugs, which are made after a nest is completed. Therefore, the bee’s active 610 period likely begins several weeks earlier. Despite this, in 10 of the 17 species we have data for, 611 nest plugs were observed several days to several weeks earlier than the range of collection dates 612 for the same species in the UMN Insect Collection (Fig. 5). This could be due to the large 613 increase in records and full season of data collection made possible by participatory science 614 (Soroye, Ahmed & Kerr, 2018; Dubaić et al., 2022). Another possibility is that earlier recorded 615 activity periods are the result of advancing phenology with climate change. Bartomeus et al. Manuscript to be reviewed 620 621 Bumble Bees 622 The bumble bee surveys of the Minnesota Bee Atlas project used consistent survey effort across 623 routes, providing the opportunity to examine patterns of bumble bee abundances and species 624 associations with land use, all of which have been difficult to do from museum collections or 625 biodiversity portal observations alone. We have reliable information on ranges of Minnesota 626 bumble bees due to numerous records of bumble bee species courtesy of the Bumble Bees of 627 North America database (Richardson, 2021). Our surveys not only confirm ranges, such as the 628 northern distributions of B. ternarius, B. terricola, B. borealis, B. flavidus, and B. perplexus, but 629 also provide insight into bumble bee community structure. For example, although B. griseocollis 630 is present throughout the state, they are the dominant bumble bee community members in only 631 two of the three examined ecological provinces (PP and EBF). Further exploration could reveal 632 specific ecological drivers of this pattern. Although we identified many of the submitted 633 photographs for B. vagans and B. sandersoni to species level to create maps showing their 634 distributions, B. vagans had to be combined with B. sandersoni for comparisons of abundance 635 and habitat associations, because many observations could not be distinguished. Future volunteer 636 surveys may be able to distinguish these species as the quality of cameras available to volunteers 637 increases. Minnesota bumble bees not found on survey routes include B. frigidus, Bombus huntii 638 Greene 1860, Bombus variabilis (Cresson, 1872), Bombus ashtoni (Cresson, 1864) (sometimes 639 considered to be conspecific with Bombus bohemicus Seidl, 1837), Bombus fraternus (Smith, 595 including dead stems, ground, leaves and under dried cattle dung (Michener, 1953). Some 596 Minnesota species not found in this study, such as Megachile montivaga Cresson, 1878 (Orr, 597 Portman & Griswold, 2015) and Osmia atriventris Cresson, 1864 (Fye, 1965) are also known 598 from stems. Future studies of tunnel-nesting bees are likely to sample a larger proportion of the 599 community by using both wood and stem substrates. A more targeted study, returning to known 600 collection areas and looking for species that have not been recorded in Minnesota in recent years 601 is warranted. Manuscript to be reviewed 602 603 In addition to distribution data, we collected data on nesting phenology, which returned a date 604 range when stem nesting bee species are likely to complete nesting and indicated the relative 605 seasonality of species. Volunteer observations also allowed us to catch Megachile relativa 606 emerging both mid-summer and the following spring. This agrees with the bivoltine life cycle for 607 Megachile relativa found in Wisconsin (Medler & Koerber, 1958) and expands the known range 608 of bivoltinism into Minnesota. It is important to remember that the phenology event volunteers 609 recorded was nest plugs, which are made after a nest is completed. Therefore, the bee’s active 610 period likely begins several weeks earlier. Despite this, in 10 of the 17 species we have data for, 611 nest plugs were observed several days to several weeks earlier than the range of collection dates 612 for the same species in the UMN Insect Collection (Fig. 5). This could be due to the large 613 increase in records and full season of data collection made possible by participatory science 614 (Soroye, Ahmed & Kerr, 2018; Dubaić et al., 2022). Another possibility is that earlier recorded 615 activity periods are the result of advancing phenology with climate change. Bartomeus et al. 616 (2011) compared collection dates of museum specimens collected between 1880 and 2010 for 10 617 bee species in northeastern North America, including two of the species in this project. They 618 found an average phenological advance of 10.4 days. The phenological data we have recorded 619 helps define these bees’ temporal habitats and lays the groundwork for assessing changes. 620 621 Bumble Bees 622 The bumble bee surveys of the Minnesota Bee Atlas project used consistent survey effort across 623 routes providing the opportunity to examine patterns of bumble bee abundances and species 595 including dead stems, ground, leaves and under dried cattle dung (Michener, 1953). Some 596 Minnesota species not found in this study, such as Megachile montivaga Cresson, 1878 (Orr, 597 Portman & Griswold, 2015) and Osmia atriventris Cresson, 1864 (Fye, 1965) are also known 598 from stems. Future studies of tunnel-nesting bees are likely to sample a larger proportion of the 599 community by using both wood and stem substrates. Manuscript to be reviewed Since these prairie habitats make up less than 1% of the 662 PP (Lark et al., 2018), increasing floral availability and abundance along the extensive amount of 663 roadside habitat in the PP could provide support to bumble bees in these isolated prairie remains, 664 particularly along roads with lower traffic to reduce risks from vehicle collisions and road 665 pollution (Keilsohn et al., 2018; Shephard et al., 2022). ), y p y , g 641 barely extend into Minnesota, or because they are not usually found on roadsides. 642 643 Bumble bee abundance information gathered by the bumble bee surveys provides important 644 baseline information and informs management decisions to support bumble bees. Many studies 645 of bumble bee decline rely on relative rather than absolute abundances of bumble bees (Colla & 646 Packer, 2008; Koch, 2011; Cameron et al., 2011). While this approach helps us understand shifts 647 in communities, it does not answer questions about broad trends in abundance, a key 648 conservation concern. Even with consistent survey effort, we do not expect counts of bumble 649 bees on flowers to reflect true population sizes at a particular site due to possible aggregation 650 effects from floral abundance or nest proximity (Harder 1986; Hines & Hendrix 2005; Geib et al. 651 2015). However, we do expect to get metrics that can be repeated across a broad geographical 652 and temporal scale to detect bumble bee abundance patterns. The observed lower bumble bee 653 abundance in the PP could indicate lower bumble bee abundance in that ecological province 654 overall, could indicate differences in the attractiveness of roadside habitat to foraging bumble 655 bees between ecological provinces due to concentration or dilution effects with varying floral 656 abundance in non-roadside habitats, or could be an artifact of the smaller number of routes that 657 were run in this ecological province. Our volunteers did not gather information on the floral 658 cover at survey sites, but volunteers in the PP more frequently reported difficulty finding areas 659 with flowers along their assigned routes. A recent study in the same area in restored prairies 660 found abundant bumble bee populations, indicating that the PP is not depauperate of bumble 661 bees across habitats (Lane et al., 2020). Manuscript to be reviewed 640 1854), and B. nevadensis. This is likely because these species are extremely rare, their ranges 641 barely extend into Minnesota, or because they are not usually found on roadsides. 642 640 1854), and B. nevadensis. This is likely because these species are extremely rare, their ranges 641 barely extend into Minnesota, or because they are not usually found on roadsides. 642 643 Bumble bee abundance information gathered by the bumble bee surveys provides important 644 baseline information and informs management decisions to support bumble bees. Many studies 645 of bumble bee decline rely on relative rather than absolute abundances of bumble bees (Colla & 646 Packer, 2008; Koch, 2011; Cameron et al., 2011). While this approach helps us understand shifts 647 in communities, it does not answer questions about broad trends in abundance, a key 648 conservation concern. Even with consistent survey effort, we do not expect counts of bumble 649 bees on flowers to reflect true population sizes at a particular site due to possible aggregation 650 effects from floral abundance or nest proximity (Harder 1986; Hines & Hendrix 2005; Geib et al. 651 2015). However, we do expect to get metrics that can be repeated across a broad geographical 652 and temporal scale to detect bumble bee abundance patterns. The observed lower bumble bee 653 abundance in the PP could indicate lower bumble bee abundance in that ecological province 654 overall, could indicate differences in the attractiveness of roadside habitat to foraging bumble 655 bees between ecological provinces due to concentration or dilution effects with varying floral 656 abundance in non-roadside habitats, or could be an artifact of the smaller number of routes that 657 were run in this ecological province. Our volunteers did not gather information on the floral 658 cover at survey sites, but volunteers in the PP more frequently reported difficulty finding areas 659 with flowers along their assigned routes. A recent study in the same area in restored prairies 660 found abundant bumble bee populations, indicating that the PP is not depauperate of bumble 661 bees across habitats (Lane et al., 2020). Manuscript to be reviewed 616 (2011) compared collection dates of museum specimens collected between 1880 and 2010 for 10 617 bee species in northeastern North America, including two of the species in this project. They 618 found an average phenological advance of 10.4 days. The phenological data we have recorded 619 helps define these bees’ temporal habitats and lays the groundwork for assessing changes. 620 622 The bumble bee surveys of the Minnesota Bee Atlas project used consistent survey effort across 623 routes, providing the opportunity to examine patterns of bumble bee abundances and species 624 associations with land use, all of which have been difficult to do from museum collections or 625 biodiversity portal observations alone. We have reliable information on ranges of Minnesota 626 bumble bees due to numerous records of bumble bee species courtesy of the Bumble Bees of 627 North America database (Richardson, 2021). Our surveys not only confirm ranges, such as the 628 northern distributions of B. ternarius, B. terricola, B. borealis, B. flavidus, and B. perplexus, but 629 also provide insight into bumble bee community structure. For example, although B. griseocollis 630 is present throughout the state, they are the dominant bumble bee community members in only 631 two of the three examined ecological provinces (PP and EBF). Further exploration could reveal 632 specific ecological drivers of this pattern. Although we identified many of the submitted 633 photographs for B. vagans and B. sandersoni to species level to create maps showing their 634 distributions, B. vagans had to be combined with B. sandersoni for comparisons of abundance 635 and habitat associations, because many observations could not be distinguished. Future volunteer 636 surveys may be able to distinguish these species as the quality of cameras available to volunteers 637 increases. Minnesota bumble bees not found on survey routes include B. frigidus, Bombus huntii 638 Greene 1860, Bombus variabilis (Cresson, 1872), Bombus ashtoni (Cresson, 1864) (sometimes 639 considered to be conspecific with Bombus bohemicus Seidl, 1837), Bombus fraternus (Smith, PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed Since these prairie habitats make up less than 1% of the 662 PP (Lark et al., 2018), increasing floral availability and abundance along the extensive amount of 663 roadside habitat in the PP could provide support to bumble bees in these isolated prairie remains, 664 particularly along roads with lower traffic to reduce risks from vehicle collisions and road 665 pollution (Keilsohn et al., 2018; Shephard et al., 2022). 666 667 Association of bumble bee species with surrounding land cover can help assess habitat needs of 668 different bumble bee species. While our survey routes were limited to roadside habitats, the 669 predominant land uses surrounding our survey routes varied, providing an opportunity to 670 examine the influence of land use on bumble bees. Many of the associations we found are similar 671 to those found in an examination of land cover and the probability of bumble bee occurrence in 672 Vermont (Richardson et al., 2019). We both found B. vagans group and B. terricola to be 673 positively associated with forested land cover, B. fervidus, B. griseocollis, and B. bimaculatus to 674 be positively associated with grassland land covers, and B. impatiens to be positively associated 675 with developed land cover. Our study included several species not present in the Vermont 676 survey. The positive association of B. auricomus and possibly B. affinis with developed land 677 cover have not been previously reported to our knowledge. 678 679 Most recent records for B. affinis have been contributed by the public and are associated with 680 urban areas in Minnesota, Wisconsin, Iowa, and Illinois (U.S. Fish and Wildlife Service, 2021). 681 It is not clear whether this phenomenon is due to more people in urban areas looking for rare 682 species and contributing records to public monitoring or whether B. affinis is associated with 683 developed areas. Since our survey routes were spread throughout the state across a wide range of 684 habitats, our finding of a possible association between B. affinis and developed land cover 685 indicates that the phenomenon may not be entirely due to increased participation in monitoring in 679 Most recent records for B. affinis have been contributed by the public and are associated with 680 urban areas in Minnesota, Wisconsin, Iowa, and Illinois (U.S. Fish and Wildlife Service, 2021). Manuscript to be reviewed Providing a variety of 711 stem and wood nesting substrates mimicking natural density may support nesting. Interpreting 712 our findings from bumble bee abundance patterns, we found a need for increased floral 713 availability in roadside habitat in the PP ecological province to support bumble bees, which 714 could also support other pollinators. 715 716 The baseline data we provided can be compared with future surveys using comparable methods 717 to examine trends in populations of tunnel-nesting bees and bumble bees, with the understanding 718 that the distributions we have documented have been influenced by current land use and climate 719 as well as historic land use changes These comparisons can help assess the impact of subsequent 692 Conclusions 693 Through four field seasons and participation from over 2,500 members of the public, the 694 Minnesota Bee Atlas used uniform methods to survey bees across Minnesota. Our findings 695 include 1) documentation of rare and endangered bees of conservation concern, 2) extension of 696 known ranges for tunnel-nesting species, 3) bee associations with ecological provinces, 4) 697 nesting phenology data for tunnel-nesting species, 5) state-wide abundance patterns for bumble 698 bees in roadside habitats, and 6) habitat associations for bumble bee and tunnel-nesting bee 699 species. In addition, we documented new state records and gathered baseline, replicable data on 700 tunnel-nesting bees and bumble bees across the state. An added benefit of our program is the 701 increased awareness of pollinator conservation among our volunteers, who continue to contribute 702 to other participatory science projects, submit thousands of iNaturalist records, and lead their 703 own outreach efforts. Our findings support several habitat management recommendations. 704 Broad-scale land use changes have occurred over the last 150 years leading to reduction of 705 natural habitat to less than 2% across all ecological provinces due to conversion to cropland and 706 managed forests (Wendt, 1988), impacting both nesting and foraging habitats for bees (Benton, 707 Vickery & Wilson, 2003; Holzschuh, Steffan-Dewenter & Tscharntke, 2010; Le Féon et al., 708 2010). With similar abundances of tunnel-nesting bees in the prairie and two forested ecological 709 provinces, and with a variety of habitat associations among species, a broad range of regions and 710 habitats are suitable targets for tunnel-nesting bee habitat enhancement. Providing a variety of 711 stem and wood nesting substrates mimicking natural density may support nesting. Manuscript to be reviewed 681 It is not clear whether this phenomenon is due to more people in urban areas looking for rare 682 species and contributing records to public monitoring or whether B. affinis is associated with 683 developed areas. Since our survey routes were spread throughout the state across a wide range of 684 habitats, our finding of a possible association between B. affinis and developed land cover 685 indicates that the phenomenon may not be entirely due to increased participation in monitoring in PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed Interpreting 712 our findings from bumble bee abundance patterns, we found a need for increased floral 713 availability in roadside habitat in the PP ecological province to support bumble bees, which 714 could also support other pollinators. 715 716 The baseline data we provided can be compared with future surveys using comparable methods 717 to examine trends in populations of tunnel-nesting bees and bumble bees, with the understanding 718 that the distributions we have documented have been influenced by current land use and climate 719 as well as historic land use changes. These comparisons can help assess the impact of subsequent 720 pollinator conservation efforts as well as long-term stressors such as climate change. We 721 recommend the following improvements to survey methods: 1) Publicizing information about 722 Megachile scupturalis and other easily identified introduced species and engaging iNaturalist 723 users in tracking their spread in the state, 2) using stem substrates in conjunction with wood 724 substrates for nest-traps to increase the number of species captured, 3) targeted nest-trap surveys 725 in regions and habitats that were underrepresented in this project, 4) the inclusion of a wider 726 variety of habitat types in surveys to improve assessment of the bumble bee community, and 5) 727 additional participant training to assess habitat in survey locations to help identify habitat 728 improvements needed to support bumble bees in different regions. 729 730 Overall, the Bee Atlas project shows the strength of involving the public in scientific research to 731 cover the geographic range of a state with methods that enable comparison of relative and Manuscript to be reviewed 686 urban areas. Historically, B. affinis nests have been noted to be associated with urban areas, and 687 have been found near houses (Medler, 1963). The possible association of a federally protected 688 endangered species with developed land has important implications for conservation strategies, 689 which often take advantage of publicly owned land. Conservation efforts on private, multi-use 690 property have additional complications (Kamal, GrodziEska-Jurczak & Brown, 2015). 691 686 urban areas. Historically, B. affinis nests have been noted to be associated with urban areas, and 687 have been found near houses (Medler, 1963). The possible association of a federally protected 688 endangered species with developed land has important implications for conservation strategies, 689 which often take advantage of publicly owned land. Conservation efforts on private, multi-use 690 property have additional complications (Kamal, GrodziEska-Jurczak & Brown, 2015). 691 692 Conclusions 693 Through four field seasons and participation from over 2,500 members of the public, the 694 Minnesota Bee Atlas used uniform methods to survey bees across Minnesota. Our findings 695 include 1) documentation of rare and endangered bees of conservation concern, 2) extension of 696 known ranges for tunnel-nesting species, 3) bee associations with ecological provinces, 4) 697 nesting phenology data for tunnel-nesting species, 5) state-wide abundance patterns for bumble 698 bees in roadside habitats, and 6) habitat associations for bumble bee and tunnel-nesting bee 699 species. In addition, we documented new state records and gathered baseline, replicable data on 700 tunnel-nesting bees and bumble bees across the state. An added benefit of our program is the 701 increased awareness of pollinator conservation among our volunteers, who continue to contribute 702 to other participatory science projects, submit thousands of iNaturalist records, and lead their 703 own outreach efforts. Our findings support several habitat management recommendations. 704 Broad-scale land use changes have occurred over the last 150 years leading to reduction of 705 natural habitat to less than 2% across all ecological provinces due to conversion to cropland and 706 managed forests (Wendt, 1988), impacting both nesting and foraging habitats for bees (Benton, 707 Vickery & Wilson, 2003; Holzschuh, Steffan-Dewenter & Tscharntke, 2010; Le Féon et al., 708 2010). With similar abundances of tunnel-nesting bees in the prairie and two forested ecological 709 provinces, and with a variety of habitat associations among species, a broad range of regions and 710 habitats are suitable targets for tunnel-nesting bee habitat enhancement. 737 Acknowledgements We thank V. Scott and A. Rose 741 the Bees' Needs project of Colorado for initial conversations and initial plug material photos. 742 Thank you to Jason Gibbs for confirming identifications for new state records, Ryan Oram fo 743 verifying representatives of Hylaeus, Michael Orr and Sam Droege for confirming identificat 744 of Anthophora terminalis, John Luhmen for identifying Ichneumonid specimens and providin 745 consultation on Chalcidoidea identification, and Jorge González and Mike Gates for confirmi 746 Kocourekia cf. debilis identification. The iNaturalist identifications from Zach Portman, John 747 Ascher, Joel Neylon, Tony Ernst, and Brian Dagley were invaluable for data quality on that 748 platform and we thank them for their time and efforts. Thank you to Clarence Lehman for 749 consultation and editing, and Robin Thomson for curation of the University of Minnesota Ins 750 Collection. The feedback of five anonymous reviewers helped polish the manuscript. 751 752 753 754 755 756 References 757 Andrus R, Droege S, Griswold T. 2020a. Draft guide to the Anthophora of North America. 758 Available at https://www.discoverlife.org/mp/20q?guide=Anthophora_female (accessed 759 December 1, 2019). 760 761 Andrus R, Droege S, Griswold T. 2020b. Hylaeus female. Available at 762 https://www.discoverlife.org/mp/20q?guide=Hylaeus_female (accessed December 1, 2019). 763 764 Andrus R, Droege S, Griswold T. 2020c. Draft guide to the male Osmia of eastern North 765 America. Available at https://www.discoverlife.org/mp/20q?guide=Osmia_male (accessed 766 December 1, 2019). 767 768 Appenfeller LR, Lloyd S, Szendrei Z. 2020. Citizen science improves our understanding of th 769 impact of soil management on wild pollinator abundance in agroecosystems. PLOS ONE 770 15:e0230007. DOI: 10.1371/journal.pone.0230007. 771 772 Arduser M. 2018. Hylaeus species of the tallgrass prairie and greater midwest. [unpublished 773 work, Revised May 7, 2018]. 774 775 Bartomeus I, Ascher JS, Wagner D, Danforth BN, Colla S, Kornbluth S, Winfree R. 2011. 776 Climate-associated phenological advances in bee pollinators and bee-pollinated plants. 738 Thank you to all our volunteers who hosted nest blocks, adopted bumble bee survey routes, 739 submitted pictures to iNaturalist, and helped with nest block construction, bee rearing and 740 pinning in the lab. We could not have done this without you. We thank V. Scott and A. Rose of 741 the Bees' Needs project of Colorado for initial conversations and initial plug material photos. 737 Acknowledgements 737 Acknowledgements 738 Thank you to all our volunteers who hosted nest blocks, adopted bumble bee survey routes, 739 submitted pictures to iNaturalist, and helped with nest block construction, bee rearing and 740 pinning in the lab. We could not have done this without you. We thank V. Scott and A. Rose o 741 the Bees' Needs project of Colorado for initial conversations and initial plug material photos. 742 Thank you to Jason Gibbs for confirming identifications for new state records, Ryan Oram for 743 verifying representatives of Hylaeus, Michael Orr and Sam Droege for confirming identificatio 744 of Anthophora terminalis, John Luhmen for identifying Ichneumonid specimens and providing 745 consultation on Chalcidoidea identification, and Jorge González and Mike Gates for confirmin 746 Kocourekia cf. debilis identification. The iNaturalist identifications from Zach Portman, John 747 Ascher, Joel Neylon, Tony Ernst, and Brian Dagley were invaluable for data quality on that 748 platform and we thank them for their time and efforts. Thank you to Clarence Lehman for 749 consultation and editing, and Robin Thomson for curation of the University of Minnesota Inse 750 Collection. The feedback of five anonymous reviewers helped polish the manuscript. 751 752 753 754 755 756 References 757 Andrus R, Droege S, Griswold T. 2020a. Draft guide to the Anthophora of North America. 758 Available at https://www.discoverlife.org/mp/20q?guide=Anthophora_female (accessed 759 December 1, 2019). 760 761 Andrus R, Droege S, Griswold T. 2020b. Hylaeus female. Available at 762 https://www.discoverlife.org/mp/20q?guide=Hylaeus_female (accessed December 1, 2019). 763 764 Andrus R, Droege S, Griswold T. 2020c. Draft guide to the male Osmia of eastern North 765 America. Available at https://www.discoverlife.org/mp/20q?guide=Osmia_male (accessed 766 December 1, 2019). 767 768 Appenfeller LR, Lloyd S, Szendrei Z. 2020. Citizen science improves our understanding of the 769 impact of soil management on wild pollinator abundance in agroecosystems. PLOS ONE 770 15:e0230007. DOI: 10.1371/journal.pone.0230007. 771 772 Arduser M. 2018. Hylaeus species of the tallgrass prairie and greater midwest. [unpublished 773 work, Revised May 7, 2018]. 774 775 Bartomeus I, Ascher JS, Wagner D, Danforth BN, Colla S, Kornbluth S, Winfree R. 2011. 776 Climate-associated phenological advances in bee pollinators and bee-pollinated plants. 737 Acknowledgements 738 Thank you to all our volunteers who hosted nest blocks, adopted bumble bee survey routes, 739 submitted pictures to iNaturalist, and helped with nest block construction, bee rearing and 740 pinning in the lab. We could not have done this without you. Manuscript to be reviewed 732 absolute abundance in different habitats and to document species that have n 733 using other methods. Coupled with professional experts, trained volunteers p 734 information that University researchers alone would have been unable to col 735 value of public participation in bee research and monitoring. 736 737 Acknowledgements 738 Thank you to all our volunteers who hosted nest blocks, adopted bumble bee 739 submitted pictures to iNaturalist, and helped with nest block construction, be 740 pinning in the lab. We could not have done this without you. We thank V. Sc 741 the Bees' Needs project of Colorado for initial conversations and initial plug 742 Thank you to Jason Gibbs for confirming identifications for new state record 743 verifying representatives of Hylaeus, Michael Orr and Sam Droege for confi 744 of Anthophora terminalis, John Luhmen for identifying Ichneumonid specim 745 consultation on Chalcidoidea identification, and Jorge González and Mike G 746 Kocourekia cf. debilis identification. The iNaturalist identifications from Zac 747 Ascher, Joel Neylon, Tony Ernst, and Brian Dagley were invaluable for data 748 platform and we thank them for their time and efforts. Thank you to Clarenc 749 consultation and editing, and Robin Thomson for curation of the University 750 Collection. The feedback of five anonymous reviewers helped polish the ma 751 752 753 754 755 756 References 757 Andrus R, Droege S, Griswold T. 2020a. Draft guide to the Anthophora of No 758 Available at https://www.discoverlife.org/mp/20q?guide=Anthophora_female 759 December 1, 2019). 760 761 Andrus R, Droege S, Griswold T. 2020b. Hylaeus female. Available at 762 https://www.discoverlife.org/mp/20q?guide=Hylaeus_female (accessed Dec 763 764 Andrus R, Droege S, Griswold T. 2020c. Draft guide to the male Osmia of ea 765 America Available at https://www discoverlife org/mp/20q?guide=Osmia m 732 absolute abundance in different habitats and to doc 733 using other methods. Coupled with professional ex 734 information that University researchers alone wou 735 value of public participation in bee research and m 736 737 Acknowledgements 738 Thank you to all our volunteers who hosted nest b 739 submitted pictures to iNaturalist, and helped with n 740 pinning in the lab. We could not have done this wi 741 the Bees' Needs project of Colorado for initial con 742 Thank you to Jason Gibbs for confirming identific 743 verifying representatives of Hylaeus, Michael Orr 744 of Anthophora terminalis, John Luhmen for identi 745 consultation on Chalcidoidea identification, and Jo 746 Kocourekia cf. debilis identification. 692 Conclusions 693 Through four field seasons and participation from over 2,500 members of the public, the 694 Minnesota Bee Atlas used uniform methods to survey bees across Minnesota. Our findings 695 include 1) documentation of rare and endangered bees of conservation concern, 2) extension of 696 known ranges for tunnel-nesting species, 3) bee associations with ecological provinces, 4) 697 nesting phenology data for tunnel-nesting species, 5) state-wide abundance patterns for bumble 698 bees in roadside habitats, and 6) habitat associations for bumble bee and tunnel-nesting bee 699 species. In addition, we documented new state records and gathered baseline, replicable data on 700 tunnel-nesting bees and bumble bees across the state. An added benefit of our program is the 701 increased awareness of pollinator conservation among our volunteers, who continue to contribute 702 to other participatory science projects, submit thousands of iNaturalist records, and lead their 703 own outreach efforts. Our findings support several habitat management recommendations. 704 Broad-scale land use changes have occurred over the last 150 years leading to reduction of 705 natural habitat to less than 2% across all ecological provinces due to conversion to cropland and 706 managed forests (Wendt, 1988), impacting both nesting and foraging habitats for bees (Benton, 707 Vickery & Wilson, 2003; Holzschuh, Steffan-Dewenter & Tscharntke, 2010; Le Féon et al., 708 2010). With similar abundances of tunnel-nesting bees in the prairie and two forested ecological 709 provinces, and with a variety of habitat associations among species, a broad range of regions and 710 habitats are suitable targets for tunnel-nesting bee habitat enhancement. Providing a variety of 711 stem and wood nesting substrates mimicking natural density may support nesting. Interpreting 712 our findings from bumble bee abundance patterns, we found a need for increased floral 713 availability in roadside habitat in the PP ecological province to support bumble bees, which 714 could also support other pollinators. 730 Overall, the Bee Atlas project shows the strength of involving the public in scientific research to 731 cover the geographic range of a state with methods that enable comparison of relative and PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed The iNatural 747 Ascher, Joel Neylon, Tony Ernst, and Brian Dagle 748 platform and we thank them for their time and effo 749 consultation and editing, and Robin Thomson for c 750 Collection. The feedback of five anonymous revie 751 752 753 754 755 756 References 757 Andrus R, Droege S, Griswold T. 2020a. Draft guid 758 Available at https://www.discoverlife.org/mp/20q?g 759 December 1, 2019). 760 761 Andrus R, Droege S, Griswold T. 2020b. Hylaeus 762 https://www.discoverlife.org/mp/20q?guide=Hylae 763 764 Andrus R, Droege S, Griswold T. 2020c. Draft guid 732 absolute abundance in different habitats and to document species that have not been discovered 733 using other methods. Coupled with professional experts, trained volunteers provided vital 734 information that University researchers alone would have been unable to collect, showing the 735 value of public participation in bee research and monitoring. 736 732 absolute abundance in different habitats and to document species that have not been discovered 733 using other methods. Coupled with professional experts, trained volunteers provided vital 734 information that University researchers alone would have been unable to collect, showing the 735 value of public participation in bee research and monitoring. 736 Manuscript to be reviewed 777 Proceedings of the National Academy of Sciences 108:20645-20649. DOI: 778 10.1073/pnas.1115559108. 779 780 Bates D, Mächler M, Bolker B, Walker S. 2015. Fitting linear mixed-effects models using lm 781 Journal of Statistical Software 67:1348. DOI: 10.18637/jss.v067.i01. 782 783 Beckham JL, Atkinson S. 2017. An updated understanding of Texas bumble bee (Hymeno 784 Apidae) species presence and potential distributions in Texas, USA. PeerJ 5:e3612. DOI: 785 10.7717/peerj.3612. 786 787 Benton TG, Vickery JA, Wilson JD. 2003. Farmland biodiversity: is habitat heterogeneity th 788 key? Trends in Ecology & Evolution 18:182-188. DOI: 10.1016/S0169-5347(03)00011-9. 789 790 ter Braak CJF. 1986. Canonical correspondence analysis: A new eigenvector technique fo 791 multivariate direct gradient analysis. Ecology 67:1167-1179. DOI: 10.2307/1938672. 792 793 Brooks M E, Kristensen K, Benthem K J ,van, Magnusson A, Berg C W, Nielsen A, Skaug 794 Mächler M, Bolker B M. 2017. glmmTMB balances speed and flexibility among packages f 795 zero-inflated generalized linear mixed modeling. The R Journal 9:378. DOI: 10.32614/RJ-2 796 066. 797 798 Burkle LA, Marlin JC, Knight TM. 2013. Plant-pollinator interactions over 120 years: Loss o 799 species, co-occurrence, and function. Science 339:1611-1615. DOI: 10.1126/science.123 800 801 Cameron SA, Lozier JD, Strange JP, Koch JB, Cordes N, Solter LF, Griswold TL. 2011. Pa 802 of widespread decline in North American bumble bees. Proceedings of the National Acade 803 Sciences 108:662-667. DOI: 10.1073/pnas.1014743108. 804 805 Cane JH, Griswold T, Parker FD. 2007. Substrates and materials used for nesting by Nort 806 American Osmia bees (Hymenoptera: Apiformes: Megachilidae). Annals of the Entomolog 807 Society of America 100:350-358. DOI: 10.1603/0013-8746(2007)100[350:SAMUFN]2.0.C 808 809 Cao H-X, Salle JL, Fornoff F, Guo P-F, Zhu C-D. 2017. Notes on Kocourekia Boucek 810 (Hymenoptera: Eulophidae: Tetrastichinae) with description of a new species from China 777 Proceedings of the National Academy of Sciences 108:20645-20649. DOI: 778 10.1073/pnas.1115559108. 779 777 Proceedings of the National Academy of Sciences 108:20645-20649. DOI: 778 10.1073/pnas.1115559108. 780 Bates D, Mächler M, Bolker B, Walker S. 2015. Fitting linear mixed-effects models using lme4. 781 Journal of Statistical Software 67:1348. DOI: 10.18637/jss.v067.i01. 782 783 Beckham JL, Atkinson S. 2017. An updated understanding of Texas bumble bee (Hymenoptera: 784 Apidae) species presence and potential distributions in Texas, USA. PeerJ 5:e3612. DOI: 785 10.7717/peerj.3612. 786 787 Benton TG, Vickery JA, Wilson JD. 2003. Farmland biodiversity: is habitat heterogeneity the 788 key? Trends in Ecology & Evolution 18:182-188. DOI: 10.1016/S0169-5347(03)00011-9. 789 789 790 ter Braak CJF. 1986. 737 Acknowledgements 742 Thank you to Jason Gibbs for confirming identifications for new state records, Ryan Oram for 743 verifying representatives of Hylaeus, Michael Orr and Sam Droege for confirming identification 744 of Anthophora terminalis, John Luhmen for identifying Ichneumonid specimens and providing 745 consultation on Chalcidoidea identification, and Jorge González and Mike Gates for confirming 746 Kocourekia cf. debilis identification. The iNaturalist identifications from Zach Portman, John 747 Ascher, Joel Neylon, Tony Ernst, and Brian Dagley were invaluable for data quality on that 748 platform and we thank them for their time and efforts. Thank you to Clarence Lehman for 749 consultation and editing, and Robin Thomson for curation of the University of Minnesota Insect 750 Collection. The feedback of five anonymous reviewers helped polish the manuscript. 751 756 References 757 Andrus R, Droege S, Griswold T. 2020a. Draft guide to the Anthophora of North America. 758 Available at https://www.discoverlife.org/mp/20q?guide=Anthophora_female (accessed 759 December 1, 2019). 760 761 Andrus R, Droege S, Griswold T. 2020b. Hylaeus female. Available at 762 https://www.discoverlife.org/mp/20q?guide=Hylaeus_female (accessed December 1, 2019). 763 764 Andrus R, Droege S, Griswold T. 2020c. Draft guide to the male Osmia of eastern North 765 America. Available at https://www.discoverlife.org/mp/20q?guide=Osmia_male (accessed 766 December 1, 2019). 767 768 Appenfeller LR, Lloyd S, Szendrei Z. 2020. Citizen science improves our understanding of the 769 impact of soil management on wild pollinator abundance in agroecosystems. PLOS ONE 770 15:e0230007. DOI: 10.1371/journal.pone.0230007. 771 772 Arduser M. 2018. Hylaeus species of the tallgrass prairie and greater midwest. [unpublished 773 work, Revised May 7, 2018]. 774 775 Bartomeus I, Ascher JS, Wagner D, Danforth BN, Colla S, Kornbluth S, Winfree R. 2011. 776 Climate-associated phenological advances in bee pollinators and bee-pollinated plants. 771 772 Arduser M. 2018. Hylaeus species of the tallgrass prairie and greater midwest. [unpublished 773 work, Revised May 7, 2018]. 774 771 772 Arduser M. 2018. Hylaeus species of the tallgrass prairie and greater midwest. [unpublished 773 work, Revised May 7, 2018]. 774 775 Bartomeus I, Ascher JS, Wagner D, Danforth BN, Colla S, Kornbluth S, Winfree R. 2011. 776 Climate-associated phenological advances in bee pollinators and bee-pollinated plants. 775 Bartomeus I, Ascher JS, Wagner D, Danforth BN, Colla S, Kornbluth S, Winfree R. 2011. 776 Climate-associated phenological advances in bee pollinators and bee-pollinated plants. PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed Manuscript to be reviewed 824 Colla SR, Packer L. 2008. Evidence for decline in eastern North American bumblebees 825 (Hymenoptera: Apidae), with special focus on Bombus affinis Cresson. Biodiversity and 826 Conservation 17:1379-1391. DOI: 10.1007/s10531-008-9340-5. 825 (Hymenoptera: Apidae), with special focus on Bombus affinis Cresson. Biodiversity and 825 (Hymenoptera: Apidae), with special focus on Bombus affinis Cresson. Biodiversity and 826 Conservation 17:1379-1391 DOI: 10 1007/s10531-008-9340-5 828 Dewitz J. 2019. National Land Cover Database (NLCD) 2016 Products (ver. 2.0, July 2020): U.S. 829 Geological Survey data release. 830 830 831 Donnelly A, Crowe O, Regan E, Begley S, Caffarra A. 2014. The role of citizen science in 832 monitoring biodiversity in Ireland. International Journal of Biometeorology 58:1237-1249. DOI: 833 10.1007/s00484-013-0717-0. 834 835 Dubaić JB, Lanner J, Rohrbach C, Meimberg H, Wyatt F, Čačija M, Galešić M, Ješovnik A, 836 Samurović K, Plećaš M, Raičević J, Ćetković A. 2022. Towards a real-time tracking of an 837 expanding alien bee species in southeast Europe through citizen science and floral host 838 monitoring. Environmental Research Communications 4:085001. DOI: 10.1088/2515- 839 7620/ac8398. 841 Evans E, Ascher J, Cariveau DP, Spivak M. 2022. A century of change for prairie bees and their 842 floral associations. Prairie Naturalist Special Issue 1:78-102. 843 844 Flaminio S, Ranalli R, Zavatta L, Galloni M, Bortolotti L. 2021. Beewatching: A project for 845 monitoring bees through photos. Insects 12:841. DOI: 10.3390/insects12090841. 846 847 Fye RE. 1965. Biology of Apoidea taken in trap nests in northwestern Ontario (Hymenoptera). 848 The Canadian Entomologist 97:863-877. DOI: 10.4039/Ent97863-8. 849 849 850 Ganzevoort W, van den Born RJG. 2021. Counting bees: Learning outcomes from participation 851 in the Dutch National Bee Survey. Sustainability 13:4703. DOI: 10.3390/su13094703. 852 853 Gardner JD, Spivak M. 2014. A survey and historical comparison of the Megachilidae 854 (Hymenoptera: Apoidea) of Itasca State Park, Minnesota. Annals of the Entomological Society of 855 America 107:983-993. DOI: 10.1603/AN14023. 857 Gaston KJ, Fuller RA. 2009. The sizes of species’ geographic ranges. Journal of Applied 858 Ecology 46:139. DOI: https://doi.org/10.1111/j.1365-2664.2008.01596.x. 857 Gaston KJ, Fuller RA. 2009. The sizes of species’ geographic ranges. Journal of Applied 858 Ecology 46:139 DOI: https://doi org/10 1111/j 1365-2664 2008 01596 x 860 Gathmann A, Tscharntke T. 2002. Foraging ranges of solitary bees. Journal of Animal Ecology 861 71:757-764. 862 son GAP, Huber JT, Woolley JB. 1997. Annotated keys to the genera of Nearctic alcidoidea (Hymenoptera). NRC Research Press. DOI: 10.1139/9780660166698. Manuscript to be reviewed Canonical correspondence analysis: A new eigenvector technique for 791 multivariate direct gradient analysis. Ecology 67:1167-1179. DOI: 10.2307/1938672. 792 792 793 Brooks M E, Kristensen K, Benthem K J ,van, Magnusson A, Berg C W, Nielsen A, Skaug H J, 794 Mächler M, Bolker B M. 2017. glmmTMB balances speed and flexibility among packages for 795 zero-inflated generalized linear mixed modeling. The R Journal 9:378. DOI: 10.32614/RJ-2017- 796 066. 797 797 798 Burkle LA, Marlin JC, Knight TM. 2013. Plant-pollinator interactions over 120 years: Loss of 799 species, co-occurrence, and function. Science 339:1611-1615. DOI: 10.1126/science.1232728. 800 801 Cameron SA, Lozier JD, Strange JP, Koch JB, Cordes N, Solter LF, Griswold TL. 2011. Patterns 802 of widespread decline in North American bumble bees. Proceedings of the National Academy of 803 Sciences 108:662-667. DOI: 10.1073/pnas.1014743108. 804 805 Cane JH, Griswold T, Parker FD. 2007. Substrates and materials used for nesting by North 806 American Osmia bees (Hymenoptera: Apiformes: Megachilidae). Annals of the Entomological 807 Society of America 100:350-358. DOI: 10.1603/0013-8746(2007)100[350:SAMUFN]2.0.CO;2. 808 809 Cao H-X, Salle JL, Fornoff F, Guo P-F, Zhu C-D. 2017. Notes on Kocourekia Boucek 810 (Hymenoptera: Eulophidae: Tetrastichinae) with description of a new species from China. 811 Zootaxa 4317:391. DOI: 10.11646/zootaxa.4317.2.13. 8 813 Cardoso P, Erwin TL, Borges PAV, New TR. 2011. The seven impediments in invertebrate 814 conservation and how to overcome them. Biological Conservation 144:2647-2655. DOI: 815 10.1016/j.biocon.2011.07.024. 817 Carper AL, Bowers MD. 2017. The conservation value of woody debris for cavity-nesting bees on 818 Boulder County Open Space. Final Report. Available at https://assets.bouldercounty.gov/wp- 819 content/uploads/2018/01/woody-debris-for-bees.pdf (accessed December 1, 2020). 820 820 821 Cockerell WP. 1903. The nesting of a carpenter bee. Birds and Nature in Natural Colors 14:127- 822 128. 823 PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed 863 Gibson GAP, Huber JT, Woolley JB. 1997. Annotated keys to the genera of Nearctic 864 Chalcidoidea (Hymenoptera). NRC Research Press. DOI: 10.1139/9780660166698. 865 866 Geib JC, Strange JP, Galen, C. 2015. Bumble bee nest abundance, foraging distance, and 867 host-plant reproduction: implications for management and conservation. Ecological Applications 868 25: 768-778. https://doi.org/10.1890/14-0151.831. 869 PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed 870 Golick DA, Ellis MD. 2006. An update on the distribution and diversity of Bombus in Nebraska 871 (Hymenoptera: Apidae). Journal of the Kansas Entomological Society 79:341-347. DOI: 872 10.2317/0508.5.1. 870 Golick DA, Ellis MD. 2006. An update on the distribution and diversity of Bombus in Nebraska 871 (Hymenoptera: Apidae). Journal of the Kansas Entomological Society 79:341-347. DOI: 872 10 2317/0508 5 1 8 3 874 Goulson D, Lye G, Darvill B. 2008. The decline and conservation of bumblebees. Annual Review 875 of Entomology 53:191-208. DOI: 10.1146/annurev.ento.53.103106.093454. 8 6 876 877 Griffin B, LaTora AG, Bhattarai U, Braman SK. 2021. Knowledge gleaned from the first Great 878 Georgia Pollinator Census. Journal of Entomological Science 57:39-63. DOI: 10.18474/JES21- 879 05. 880 880 881 Griswold T, Ikerd H, Droege S, Pascarella JB, Pickering J. 2020. Draft guide to the female Osmia 882 of eastern North America. Available at 883 https://www.discoverlife.org/mp/20q?guide=Osmia_female (accessed December 1, 2019). 884 885 Hagen M, Wikelski M, Kissling WD. 2011. Space use of bumblebees (Bombus spp.) revealed by 886 radio-tracking. PLoS ONE 6:1-10. DOI: 10.1371/journal.pone.0019997. 887 887 888 Hanson DS, Hargrave B. 1996. Development of a multilevel ecological classification system for 889 the state of Minnesota. Environmental Monitoring and Assessment 39:75-84. DOI: 890 10.1007/BF00396137. 891 891 892 Harder LD. 1986. Influences on the density and dispersion of bumble bee nests (Hymenoptera: 893 Apidae). Ecography 9: 99-103. 894 895 Harmon-Threatt A. 2020. Influence of nesting characteristics on health of wild bee communities. 896 Annual Review of Entomology 65:39-56. DOI: 10.1146/annurev-ento-011019-024955. 897 898 Hatfield R, Colla S, Jepsen S, Richardson LL, Thorp R, Foltz Jordan S. 2015. IUCN 899 Assessments for North American Bombus spp. December 2014, 1356. Available at 900 https://xerces.org/sites/default/files/publications/14-065.pdf (Accessed January 22, 2022). 901 902 Heraty J. 2008. Identification atlas of the Vespidae (Hymenoptera, Aculeata) of the northeastern 903 nearctic region - Edited by M. Buck, S.A. Marshall and D.K.B. Cheung. Systematic Entomology 904 33:579-580. DOI: 10.1111/j.1365-3113.2008.00431.x. 905 906 Hines HM, Hendrix SD. 2005. Bumble bee (Hymenoptera: Apidae) diversity and abundance in 907 tallgrass prairie patches: effects of local and landscape floral resources. Environmental 908 Entomology 34:1477-1484. 909 910 Hollister JW, Gonzalez ML, Paul JF, August, PV, Copeland JL. 2004. Assessing the accuracy o 911 National Land Cover Dataset area estimates at multiple spatial extents. Photogrammetric 912 Engineering & Remote Sensing, 4:405-414. https://doi.org/10.14358/PERS.70.4.405. 913 914 Holzschuh A, Steffan-Dewenter I, Tscharntke T. 2010. Manuscript to be reviewed How do landscape composition and 915 configuration, organic farming and fallow strips affect the diversity of bees, wasps and their 916 parasitoids? Journal of Animal Ecology 79:491-500. 917 898 Hatfield R, Colla S, Jepsen S, Richardson LL, Thorp R, Foltz Jordan S. 2015. IUCN 899 Assessments for North American Bombus spp. December 2014, 1356. Available at 900 https://xerces.org/sites/default/files/publications/14-065.pdf (Accessed January 22, 2022). 901 898 Hatfield R, Colla S, Jepsen S, Richardson LL, Thorp R, Foltz Jordan S. 2015. IUCN 899 Assessments for North American Bombus spp. December 2014, 1356. Available at 900 https://xerces org/sites/default/files/publications/14 065 pdf (Accessed January 22 2022) 900 https://xerces.org/sites/default/files/publications/14-065.pdf (Accessed January 22, 2022). 901 902 Heraty J. 2008. Identification atlas of the Vespidae (Hymenoptera, Aculeata) of the northeastern 903 nearctic region - Edited by M. Buck, S.A. Marshall and D.K.B. Cheung. Systematic Entomology 904 33:579-580. DOI: 10.1111/j.1365-3113.2008.00431.x. 905 904 33:579-580. DOI: 10.1111/j.1365-3113.2008.00431.x. 905 906 Hines HM, Hendrix SD. 2005. Bumble bee (Hymenoptera: Apidae) diversity and abundance in 907 tallgrass prairie patches: effects of local and landscape floral resources. Environmental 908 Entomology 34:1477-1484. 909 910 Hollister JW, Gonzalez ML, Paul JF, August, PV, Copeland JL. 2004. Assessing the accuracy of 911 National Land Cover Dataset area estimates at multiple spatial extents. Photogrammetric 912 Engineering & Remote Sensing, 4:405-414. https://doi.org/10.14358/PERS.70.4.405. 913 914 Holzschuh A, Steffan-Dewenter I, Tscharntke T. 2010. How do landscape composition and 915 configuration, organic farming and fallow strips affect the diversity of bees, wasps and their 916 parasitoids? Journal of Animal Ecology 79:491-500. 917 906 Hines HM, Hendrix SD. 2005. Bumble bee (Hymenoptera: Apidae) diversity and abundance in 907 tallgrass prairie patches: effects of local and landscape floral resources. Environmental 908 Entomology 34:1477-1484. 909 906 Hines HM, Hendrix SD. 2005. Bumble bee (Hymenoptera: Apidae) diversity and abundance in 907 tallgrass prairie patches: effects of local and landscape floral resources. Environmental 908 Entomology 34:1477-1484. 909 910 Hollister JW, Gonzalez ML, Paul JF, August, PV, Copeland JL. 2004. Assessing the accuracy of 911 National Land Cover Dataset area estimates at multiple spatial extents. Photogrammetric 912 Engineering & Remote Sensing, 4:405-414. https://doi.org/10.14358/PERS.70.4.405. 914 Holzschuh A, Steffan-Dewenter I, Tscharntke T. 2010. How do landscape composition and 915 configuration, organic farming and fallow strips affect the diversity of bees, wasps and their 916 parasitoids? Journal of Animal Ecology 79:491-500. 917 PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed 918 Kamal S, Grodzińska-Jurczak M, Brown G. 2015. Conservation on private land: a review of 919 global strategies with a proposed classification system. Journal of Environmental Planning and 920 Management 58:576-597. DOI: 10.1080/09640568.2013.875463. 921 918 Kamal S, Grodzińska-Jurczak M, Brown G. 2015. Conservation on private land: a review of 919 global strategies with a proposed classification system. Journal of Environmental Planning and 920 Management 58:576-597. DOI: 10.1080/09640568.2013.875463. 921 918 Kamal S, Grodzińska Jurczak M, Brown G. 2015. Conservation on private land: a review of 919 global strategies with a proposed classification system. Journal of Environmental Planning and 920 Management 58:576-597. DOI: 10.1080/09640568.2013.875463. , , p 919 global strategies with a proposed classification system. Journal of Environmental Planning and 920 Management 58:576 597 DOI: 10 1080/09640568 2013 875463 921 922 Keilsohn W, Narango DL, Tallamy DW. 2018. Roadside habitat impacts insect traffic mortality. 923 Journal of Insect Conservation 22:183–188 https://doi.org/10.1007/s10841-018-0051-2. 924 925 Klein A-M, Vaissière BE, Cane JH, Steffan-Dewenter I, Cunningham SA, Kremen C, Tscharntke 926 T. 2007. Importance of pollinators in changing landscapes for world crops. Proceedings of the 927 Royal Society B: Biological Sciences 274:303-313. DOI: 10.1098/rspb.2006.3721. 928 928 929 Koch JB. 2011. The decline and conservation status of North American bumble bees. Master’s 930 Thesis, Utah State University. 931 932 Koffler S, Barbiéri C, Ghilardi-Lopes NP, Leocadio JN, Albertini B, Francoy TM, Saraiva AM. 933 2021. A Buzz for Sustainability and Conservation: The growing potential of citizen science 934 studies on bees. Sustainability 13:959. DOI: 10.3390/su13020959. 935 935 936 Kremen C, Ullman KS, Thorp RW. 2011. Evaluating the quality of citizen-scientist data on 937 pollinator communities. Conservation Biology 25:607-617. DOI: 10.1111/j.1523- 938 1739.2011.01657.x. 939 940 Krombein KV. 1967. Trap-nesting wasps and bees: life histories, nests, and associates. 941 Washington: Smithsonian Press. 942 942 943 Krunic MD, Salt RW. 1971. Seasonal changes in glycerol content and supercooling points of 944 Megachile rotundata (F.) and M. relativa Cress. Canadian Journal of Zoology 49:663-666. DOI: 945 10.1139/z71-104. 946 947 Lane IG, Herron-Sweet CR, Portman ZM, Cariveau DP. 2020. Floral resource diversity drives 948 bee community diversity in prairie restorations along an agricultural landscape gradient. Journal 949 of Applied Ecology 57:2010-2018. DOI: 10.1111/1365-2664.13694. 950 , , , y 948 bee community diversity in prairie restorations along an agricultural landscape gradient. Journal 949 of Applied Ecology 57:2010-2018. DOI: 10.1111/1365-2664.13694. 950 951 Lanterman J, Reeher P, Mitchell RJ, Goodell K. 2019. Manuscript to be reviewed 965 Requier F, Vaissière BE. 2016. An expert-assisted citizen science program involving agricultural 966 high schools provides national patterns on bee species assemblages. Journal of Insect 967 Conservation 20:905-918. DOI: 10.1007/s10841-016-9927-1. 965 Requier F, Vaissière BE. 2016. An expert assisted citizen science program involving agricultural 966 high schools provides national patterns on bee species assemblages. Journal of Insect 967 Conservation 20:905 918 DOI: 10 1007/s10841 016 9927 1 966 high schools provides national patterns on bee species assemblages. Journal of Insect 969 Le Féon V, Schermann-Legionnet A, Delettre Y, Aviron S, Billeter R, Bugter R, Hendrickx F, 970 Burel F. 2010. Intensification of agriculture, landscape composition and wild bee communities: A 971 large scale study in four European countries. Agriculture, Ecosystems & Environment 137:143- 972 150. DOI: 10.1016/j.agee.2010.01.015. 969 Le Féon V, Schermann-Legionnet A, Delettre Y, Aviron S, Billeter R, Bugter R, Hendrickx F, 970 Burel F. 2010. Intensification of agriculture, landscape composition and wild bee communities: A 971 large scale study in four European countries. Agriculture, Ecosystems & Environment 137:143- 972 150. DOI: 10.1016/j.agee.2010.01.015. 974 Lebuhn G, Droege S, Connor EF, Gemmill-Herren B, Potts SG, Minckley RL, Griswold T, Jean 975 R, Kula E, Roubik DW, Cane J, Wright KW, Frankie G, Parker F. 2013. Detecting insect 976 pollinator declines on regional and global scales. Conservation Biology: The Journal of the 977 Society for Conservation Biology 27:113-120. DOI: 10.1111/j.1523-1739.2012.01962.x. 978 979 Legendre P, Legendre L. 2012. Numerical ecology. Amsterdam; Boston, Oxford: Elsevier. 980 981 Lenth RV, Bolker B, Buerkner P, Giné-Vázquez I, Herve M, Jung M, Love J, Miguez F, Riebl H, 982 Singmann H. 2023. emmeans: Estimated Marginal Means, aka Least-Squares Means. 983 981 Lenth RV, Bolker B, Buerkner P, Giné-Vázquez I, Herve M, Jung M, Love J, Miguez F, Riebl H, 982 Singmann H. 2023. emmeans: Estimated Marginal Means, aka Least-Squares Means. 984 Lye GC, Osborne JL, Park KJ, Goulson D. 2012. Using citizen science to monitor Bombus 985 populations in the UK: Nesting ecology and relative abundance in the urban environment. 986 Journal of Insect Conservation 16:697-707. DOI: 10.1007/s10841-011-9450-3. 987 98 988 Maciel de Almeida Correia M de L. 1977. Sur l'origine des résines employées par Heriades 989 truncorum L. (Hymenoptera, Megachilidae) pour la construction de ses nids. Apidologie 8:101- 990 109. 991 992 MacIvor JS, Packer L. 2016. The bees among us: Modelling occupancy of solitary bees. PLOS 993 ONE 11:e0164764. DOI: 10.1371/journal.pone.0164764. Manuscript to be reviewed Habitat preference and phenology of nest 952 seeking and foraging spring bumble bee queens in Northeastern North America (Hymenoptera: 953 Apidae: Bombus). The American Midland Naturalist 182:131-159. DOI: 10.1674/0003-0031- 954 182.2.131. 955 956 Lark TJ, Larson B, Schelly I, Batish S, Gibbs HK. 2019. Accelerated conversion of native prairie 957 to cropland in Minnesota. Environmental Conservation 46:155–162. DOI: 958 10.1017/S0376892918000437. 959 960 Le Féon V, Aubert M, Genoud D, Andrieu-Ponel V, Westrich P, Geslin B. 2018. Range 961 expansion of the Asian native giant resin bee Megachile sculpturalis (Hymenoptera, Apoidea, 962 Megachilidae) in France. Ecology and Evolution 8:1534-1542. DOI: 10.1002/ece3.3758. 963 964 Le Féon V, Henry M, Guilbaud L, Coiffait-Gombault C, Dufrêne E, Kolodziejczyk E, Kuhlmann M 951 Lanterman J, Reeher P, Mitchell RJ, Goodell K. 2019. Habitat preference and phenology of nest 952 seeking and foraging spring bumble bee queens in Northeastern North America (Hymenoptera: 953 Apidae: Bombus). The American Midland Naturalist 182:131-159. DOI: 10.1674/0003-0031- 954 182.2.131. 956 Lark TJ, Larson B, Schelly I, Batish S, Gibbs HK. 2019. Accelerated conversion of native prairie 957 to cropland in Minnesota. Environmental Conservation 46:155–162. DOI: 958 10.1017/S0376892918000437. 960 Le Féon V, Aubert M, Genoud D, Andrieu-Ponel V, Westrich P, Geslin B. 2018. Range 961 expansion of the Asian native giant resin bee Megachile sculpturalis (Hymenoptera, Apoidea, 962 Megachilidae) in France. Ecology and Evolution 8:1534-1542. DOI: 10.1002/ece3.3758. 963 963 964 Le Féon V, Henry M, Guilbaud L, Coiffait-Gombault C, Dufrêne E, Kolodziejczyk E, Kuhlmann M, PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed 994 995 Maher S, Manco F, Ings TC. 2019. Using citizen science to examine the nesting ecology of 996 ground-nesting bees. Ecosphere 10. DOI: 10.1002/ecs2.2911. 942. 99 998 McFarland KP, Richardson LL, Zahendra S. 2015.Vermont Bumblebee Atlas. Vermont Center 999 for Ecostudies - Vermont Atlas of Life. Available at https://val.vtecostudies.org/projects/bumble- 1000 bee-atlas/ (accessed November 28, 2022). 1001 1002 Medler JT. 1963. Bumblebees of Wisconsin: (hymenoptera: apidae). Wisconsin: Agricultural 1003 Experiment Station, University of Wisconsin. 1004 1004 1005 Medler JT. 1964. A Note on Megachile (Sayapis) pugnata pugnata Say in trap-nests in 1006 Wisconsin (Hymenoptera: Megachilidae). The Canadian Entomologist 96:9183921. DOI: 1007 10.4039/Ent96918-6. 1008 1009 Medler JT, Koerber TW. 1958. Biology of Megachile relativa Cresson (Hymenoptera, 1010 Megachilidae) in trap-nests in Wisconsin. Annals of the Entomological Society of America 1011 51:337-344. DOI: 10.1093/aesa/51.4.337. 1012 PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed 1060 1061 Parker FD, Bohart RM. 1966. Host-parasite associations in some twig-nesting Hymenoptera 1062 from western North America. Pan-Pacific Entomologist 42:91-98. 1061 Parker FD, Bohart RM. 1966. Host-parasite associations in some twig-nesting Hymenoptera 1062 from western North America Pan Pacific Entomologist 42:91 98 1064 Parys KA, Tripodi AD, Sampson BJ. 2015. The giant resin bee, Megachile sculpturalis Smith: 1065 New distributional records for the mid- and gulf-south USA. Biodiversity Data Journal:e6733. 1066 DOI: 10.3897/BDJ.3.e6733. 1067 1068 Portman ZM, Dolan C. 2022. Documenting Bombus nevadensis in Minnesota, with some notes 1069 on discerning it from B. auricomus (Hymenoptera: Apidae). :2022.05.23.493105. DOI: 1070 10.1101/2022.05.23.493105. 1071 1072 Portman ZM, Gardner J, Lane I, Gergets N, Petersen J, Ascher JS, Arduser M, Evans 1073 E, Boyd C, Thomson R, Cariveau D. 2023. A checklist of the bees (Hymenoptera: 1074 Apoidea) of Minnesota. Zootaxa 5304(1) 1-95. DOI: 10.11646/ZOOTAXA.5304.1.1. 1075 1076 Potts SG, Vulliamy B, Dafni A, Ne’eman G, Willmer P. 2003. Linking bees and flowers: How do 1077 floral communities structure pollinator communities? Ecology 84:2628-2642. DOI: 10.1890/02- 1078 0136. 1079 1080 Prendergast KS, Menz MHM, Dixon KW, Bateman PW. 2020. The relative performance of 1081 sampling methods for native bees: an empirical test and review of the literature. Ecosphere 11. 1082 DOI: 10.1002/ecs2.3076. 1083 1084 R Core Team. 2022. R: A language and environment for statistical computing. R Foundation for 1085 Statistical Computing, Vienna, Austria. URL https://www.R-project.org/. 1086 1087 Rao S, Strange JP. 2012. Bumble bee (Hymenoptera: Apidae) foraging distance and colony 1088 density associated with a late-season mass flowering crop. Environmental Entomology 41:905- 1089 915. DOI: 10.1603/EN11316. 1090 1089 915. DOI: 10.1603/EN11316. 1090 1091 Rehan SM, Richards MH. 2010. Nesting biology and subsociality in Ceratina calcarata 1092 (Hymenoptera: Apidae). The Canadian Entomologist 142:65-74. DOI: 10.4039/n09-056. 1093 1094 Richardson LL. 2021. Bumble Bees of North America. Available at 1095 https://www.leifrichardson.org/bbna.html (Accessed January 22, 2022). 1096 1097 Richardson LL, McFarland KP, Zahendra S, Hardy S. 2019. Bumble bee (Bombus) distribution 1098 and diversity in Vermont, USA: a century of change. Journal of Insect Conservation 23:45-62. 1099 DOI: 10.1007/s10841-018-0113-5. 1100 1101 Rightmyer MG, Griswold T, Brady SG. 2013. Phylogeny and systematics of the bee genus 1102 Osmia (Hymenoptera: Megachilidae) with emphasis on North American Melanosmia: 1103 subgenera, synonymies and nesting biology revisited. Systematic Entomology 38:561-576. DOI 1104 10.1111/syen.12013. 1105 1090 1091 Rehan SM, Richards MH. 2010. Nesting biology and subsociality in Ceratina calcarata 1092 (Hymenoptera: Apidae). Manuscript to be reviewed 1013 Medler JT, Lussenhop JF. 1968. Leafcutter bees of Wisconsin. Research Division, College of 1014 Agricultural and Life Sciences, University of Wisconsin. 1015 1015 1016 Michener CD. 1953. The biology of a leafcutter bee (Megachile brevis) and its associates. 1017 University of Kansas Science Bulletin 35:98. 1018 1019 Minnesota Department of Natural Resources. 2022. Laurentian Mixed Forest Province. 1020 Available at https://www.dnr.state.mn.us/ecs/212/index.html (accessed December 1, 2022). 1021 1022 Minnesota Department of Natural Resources. 2023. Ecological Classification System. Available 1023 at https://www.dnr.state.mn.us/ecs/index.html (accessed August 14, 2023). 1024 1025 Mitchell TB. 1962. Bees of the Eastern United States. North Carolina Agricultural Experiment 1026 Station. 1027 0 1028 Nelson RA, Droege S. 2020a. Guide to the female Megachile of eastern North America. 1029 Available at https://www.discoverlife.org/mp/20q?guide=Megachile_female (accessed 1030 December 1, 2019). 1031 1032 Nelson RA, Droege S. 2020b. Guide to the male Megachile of eastern North America. Available 1033 at https://www.discoverlife.org/mp/20q?guide=Megachile_male (accessed December 1, 2019). 1034 1035 Ogilvie JE, Forrest JR. 2017. Interactions between bee foraging and floral resource phenology 1036 shape bee populations and communities. Current Opinion in Insect Science 21:75-82. DOI: 1037 10.1016/j.cois.2017.05.015. 1038 1038 1039 Oksanen J, Blanchet FG, Friendly M, Kindt R, Legendre P, McGlinn D, Minchin P, 1040 O’Hara RB, Simpson G, Solymos P, Stevens MHH, Szöcs E, Wagner H. 2020. Vegan 1041 community ecology package version 2.5-7. November 2020. 1042 1042 1043 Ollerton J, Winfree R, Tarrant S. 2011. How many flowering plants are pollinated by animals? 1044 Oikos 120:321-326. DOI: 10.1111/j.1600-0706.2010.18644.x. 1045 1045 1046 Olsen K, Holm TE, Pape T, Simonsen TJ. 2020. Natural history museum collection and citizen 1047 science data show advancing phenology of Danish hoverflies (Insecta: Diptera, Syrphidae) with 1048 increasing annual temperature. PLOS ONE 15:e0232980. DOI: 10.1371/journal.pone.0232980. 1049 1050 Orr M, Andrus R, Droege S, Griswold T. 2020.Guide to the male Anthophora of North America. 1051 Available at https://www.discoverlife.org/mp/20q?guide=Anthophora_male (accessed December 1052 1, 2019). 1053 1054 Orr MC, Portman ZM, Griswold TL. 2015. Megachile (Megachile) montivaga (Hymenoptera: 1055 Megachilidae) nesting in live thistle (Asteraceae: Cirsium). Journal of Melittology:1. DOI: 1056 10.17161/jom.v0i48.4847. 1057 1057 1058 Palmer MW. 1993. Putting things in even better order: The advantages of canonical 1059 correspondence analysis. Ecology 74:2215-2230. DOI: 10.2307/1939575. PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed The Canadian Entomologist 142:65-74. DOI: 10.4039/n09-056. 1093 1094 Richardson LL. 2021. Bumble Bees of North America. Available at 1095 https://www.leifrichardson.org/bbna.html (Accessed January 22, 2022). 1096 1097 Richardson LL, McFarland KP, Zahendra S, Hardy S. 2019. Bumble bee (Bombus) distribution 1098 and diversity in Vermont, USA: a century of change. Journal of Insect Conservation 23:45-62. 1099 DOI: 10.1007/s10841-018-0113-5. 1100 1101 Rightmyer MG, Griswold T, Brady SG. 2013. Phylogeny and systematics of the bee genus 1102 Osmia (Hymenoptera: Megachilidae) with emphasis on North American Melanosmia: 1103 subgenera, synonymies and nesting biology revisited. Systematic Entomology 38:561-576. DOI: 1104 10.1111/syen.12013. 1105 096 1097 Richardson LL, McFarland KP, Zahendra S, Hardy S. 2019. Bumble bee (Bombus) distribution 1098 and diversity in Vermont, USA: a century of change. Journal of Insect Conservation 23:45-62. 1099 DOI: 10.1007/s10841-018-0113-5. PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed 1106 Rose AP, Scott VL, Bowers MD. 2015. The Bees’ Needs: using citizen scientists to explore 1107 backyard native bee and wasp diversity. Informal Learning Review 132:335. 1108 1106 Rose AP, Scott VL, Bowers MD. 2015. The Bees’ Needs: using citizen scie g p 1107 backyard native bee and wasp diversity. Informal Learning Review 132:335. 1108 1109 RStudio Team. 2022. RStudio: Integrated Development for R. RStudio, PBC, Boston, MA URL 1110 http://www.rstudio.com/. 1111 1112 Russo L. 2016. Positive and negative impacts of non-native bee species around the world. 1113 Insects 7:69. DOI: 10.3390/insects7040069. 1114 1115 Sandhouse GA. 1939. The North American bees of the genus Osmia (Hymenoptera: Apoidea). 1116 Washington, D.C.: The Entomological Society of Washington. 1117 1118 Satyshur C, Evans E. 2021. How to create habitat for stem-nesting bees. University of 1119 Minnesota Extension. Available at https://beelab.umn.edu/create-nesting-habitat. 1120 1121 Satyshur C, Evans T, Forsberg B, Blair R. 2021. Minnesota state records for Osmia georgica, 1122 Megachile inimica, and Megachile frugalis (Hymenoptera, Megachilidae), including a new nest 1123 description for Megachile frugalis compared with other species in the subgenus Sayapis. The 1124 Great Lakes Entomologist 53. 1125 1126 Satyshur C, Evans T, Forsberg B, Gibbs J. 2022. First records of Stelis permaculata Cockerell 1127 (Hymenoptera: Megachilidae) in Minnesota, United States of America and Manitoba, Canada. 1128 The Great Lakes Entomologist 54. 1129 1130 Sheffield CS, Heron JM. 2019. The bees of British Columbia (Hymenoptera: Apoidea, 1131 Apiformes). Journal of the Entomological Society of British Columbia 115:44-85. 1132 1130 Sheffield CS, Heron JM. 2019. The bees of British Columbia (Hymenoptera: Apoidea, 1131 Apiformes) Journal of the Entomological Society of British Columbia 115:44 85 1133 Sheffield CS, Pindar A, Packer L, Kevan PG. 2013. The potential of cleptoparasitic bees as 1134 indicator taxa for assessing bee communities. Apidologie 44:501-510. DOI: 10.1007/s13592- 1135 013-0200-2. 1136 1137 Sheffield C, Ratti C, Packer L, Griswold T. 2011. Leafcutter and mason bees of the genus 1138 Megachile Latreille (Hymenoptera: Megachilidae) in Canada and Alaska. Canadian Journal of 1139 Arthropod Identification 18:1-107. DOI: 10.3752/cjai.2011.18. 1140 0 1141 Shephard AM, Agnew L, Herdtle A, Mitchell TS, Borer ET, Snell‐Rood EC. Traffic patterns, 1142 more than adjacent land use, influence element content of roadside forbs for insect pollinators. 1143 Ecological Solutions and Evidence, 3 (4). https://doi.org/10.1002/2688-8319.12195. 1144 1145 Sladen FWL. 1919. Notes on the Canadian representative of British species of bees. The 1146 Canadian Entomologist 51:124-130. DOI: 10.4039/Ent51124-6. Manuscript to be reviewed 1147 1148 Soroye P, Ahmed N, Kerr JT. 2018. Opportunistic citizen science data transform understanding 1149 of species distributions, phenology, and diversity gradients for global change research. Global 1150 Change Biology 24:5281-5291. DOI: 10.1111/gcb.14358. 1151 PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed 1152 Staab M, Pufal G, Tscharntke T, Klein A. 2018. Trap nests for bees and wasps to analyse 1153 trophic interactions in changing environments: A systematic overview and user guide. Methods 1154 in Ecology and Evolution 9:2226-2239. DOI: 10.1111/2041-210X.13070. , , , p p y 1153 trophic interactions in changing environments: A systematic overview and user guide. Methods 1154 in Ecology and Evolution 9:2226-2239 DOI: 10 1111/2041-210X 13070 1153 trophic interactions in changing environments: A systematic overview and user guide. Methods 1156 Steffan-Dewenter I, Münzenberg U, Bürger C, Thies C, Tscharntke T. 2002. Scale-dependent 1157 effects of landscape context on three pollinator guilds. Ecology 83:1421-1432. DOI: 1158 10.2307/3071954. 1159 1156 Steffan-Dewenter I, Münzenberg U, Bürger C, Thies C, Tscharntke T. 2002. Scale-dependent 1157 effects of landscape context on three pollinator guilds. Ecology 83:1421-1432. DOI: 1158 10.2307/3071954. 1159 1160 Stephen WP. 1956. Notes on the biologies of Megachile irigida Smith and M. Inermis 1161 Provancher (Hymenoptera: Megachlidae). The Pan-Pacific Entomologist 32. 1162 1160 Stephen WP. 1956. Notes on the biologies of Megachile irigida Smith and M. Inermis 1161 Provancher (Hymenoptera: Megachlidae). The Pan-Pacific Entomologist 32. 1162 1163 Triplehorn CA. 2005. Borror and DeLong’s introduction to the study of insects. Australia: 1164 Thomson, Brooks/Cole. 1165 1165 1166 U.S. Fish and Wildlife Service. 2021. Recovery Plan for the Rusty Patched Bumble Bee 1167 (Bombus affinis). Midwest Regional Office, Bloomington, MN. 1168 1168 1169 USGS Patuxent Wildlife Research Center. 2017. North American Breeding Bird Survey. 1170 Available at https://www.pwrc.usgs.gov/bbs/RouteMap/Map.cfm (Accessed January 1, 2015). 1171 1172 Vickruck JL, Rehan SM, Sheffield CS, Richards MH. 2011. Nesting biology and DNA barcode 1173 analysis of Ceratina dupla and C. mikmaqi, and comparisons with C. calcarata (Hymenoptera: 1174 Apidae: Xylocopinae). The Canadian Entomologist 143:254-262. DOI: 10.4039/n11-006. 1175 1176 van der Wal R, Anderson H, Robinson A, Sharma N, Mellish C, Roberts S, Darvill B, 1177 Siddharthan A. 2015. Mapping species distributions: A comparison of skilled naturalist and lay 1178 citizen science recording. Ambio 44:584-600. DOI: 10.1007/s13280-015-0709-x. 1179 1178 citizen science recording. Ambio 44:584-600. DOI: 10.1007/s13280-015-0709-x. 1179 1180 Wendt, KM and Coffin, BA. 1988. Natural vegetation of Minnesota at the time of 1181 the Public Land Survey, 1847-1907. Biological Report No. 1. Minnesota Department of Natura 1182 Resources, St. Paul, Minnesota. 1183 1184 Westerfelt P, Weslien J, Widenfalk O. 2018. 1199 Woodard SH, Federman S, James RR, Danforth BN, Griswold TL, Inouye D, 1200 McFrederick QS, Morandin L, Paul DL, Sellers E, Strange JP, Vaughan M, Williams 1201 NM, Branstetter MG, Burns CT, Cane J, Cariveau AB, Cariveau DP, Childers A, 1202 Childers C, Cox-Foster DL, Evans EC, Graham KK, Hackett K, Huntzinger KT, Irwin 1203 RE, Jha S, Lawson S, Liang C, López-Uribe MM, Melathopoulos A, Moylett HMC, Otto 1204 CRV, Ponisio LC, Richardson LL, Rose R, Singh R, Wehling W. 2020. Towards a U.S. 1205 national program for monitoring native bees. Biological Conservation 252:108821. DOI: 1206 10.1016/j.biocon.2020.108821. Manuscript to be reviewed Population patterns in relation to food and nestin 1185 resource for two cavity-nesting bee species in young boreal forest stands. Forest Ecology and 1186 Management 430:629-638. DOI: 10.1016/j.foreco.2018.08.053. 1187 1188 Westphal C, Bommarco R, Carré G, Lamborn E, Morison N, Petanidou T, Potts SG, 1189 Roberts SPM, Szentgyörgyi H, Tscheulin T, Vaissière BE, Woyciechowski M, Biesmeijer 1190 JC, Kunin WE, Settele J, Steffan-Dewenter I. 2008. Measuring bee diversity in different 1191 European habitats and biogeographical regions. Ecological Monographs 78:653-671. 1192 DOI: https://doi.org/10.1890/07- 1292.1. 1193 1194 Wilson JS, Pan AD, General DEM, Koch JB. 2020. More eyes on the prize: an 1195 observation of a very rare, threatened species of Philippine bumble bee, Bombus 1196 irisanensis, on iNaturalist and the importance of citizen science in conservation biology. 1197 Journal of Insect Conservation 24:727-729. DOI: 10.1007/s10841-020-00233-3. 1198 1180 Wendt, KM and Coffin, BA. 1988. Natural vegetation of Minnesota at the time of 1180 Wendt, KM and Coffin, BA. 1988. Natural vegetation of Minnesota at the time of 1181 the Public Land Survey, 1847-1907. Biological Report No. 1. Minnesota Department of Natural 1182 Resources, St. Paul, Minnesota. 1180 Wendt, KM and Coffin, BA. 1988. Natural vegetation of Minnesota at the time of 1181 the Public Land Survey, 1847-1907. Biological Report No. 1. Minnesota Department of Natural 1182 Resources, St. Paul, Minnesota. 1183 , , g 1181 the Public Land Survey, 1847-1907. Biological Report No. 1. Minnesota Department of Natural 1182 Resources, St. Paul, Minnesota. 1181 the Public Land Survey, 1847-1907. Biological Report No. 1. Minnesota Department of Natural 1182 Resources, St. Paul, Minnesota. 1184 Westerfelt P, Weslien J, Widenfalk O. 2018. Population patterns in relation to food and nesting 1185 resource for two cavity-nesting bee species in young boreal forest stands. Forest Ecology and 1186 Management 430:629-638. DOI: 10.1016/j.foreco.2018.08.053. 1188 Westphal C, Bommarco R, Carré G, Lamborn E, Morison N, Petanidou T, Potts SG, 1189 Roberts SPM, Szentgyörgyi H, Tscheulin T, Vaissière BE, Woyciechowski M, Biesmeijer 1190 JC, Kunin WE, Settele J, Steffan-Dewenter I. 2008. Measuring bee diversity in different 1191 European habitats and biogeographical regions. Ecological Monographs 78:653-671. 1192 DOI: https://doi.org/10.1890/07- 1292.1. 1193 PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed 1199 Woodard SH, Federman S, James RR, Danforth BN, Griswold TL, Inouye D, 1200 McFrederick QS, Morandin L, Paul DL, Sellers E, Strange JP, Vaughan M, Williams 1201 NM, Branstetter MG, Burns CT, Cane J, Cariveau AB, Cariveau DP, Childers A, 1202 Childers C, Cox-Foster DL, Evans EC, Graham KK, Hackett K, Huntzinger KT, Irwin 1203 RE, Jha S, Lawson S, Liang C, López-Uribe MM, Melathopoulos A, Moylett HMC, Otto 1204 CRV, Ponisio LC, Richardson LL, Rose R, Singh R, Wehling W. 2020. Towards a U.S. 1205 national program for monitoring native bees. Biological Conservation 252:108821. DOI: 1206 10.1016/j.biocon.2020.108821. PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed Locations of Minnesota Bee Atlas observations. Locations of Minnesota Bee Atlas observations. Observations include research grade iNaturalist observations of bees between 29 July 2005 and 9 March 2021, nest traps and stem bundles monitored from 2016 to 2019, and bumble bee routes surveyed from 2016 to 2020. Observations took place across Minnesota’s four ecological provinces. Maps in this study were created using Esri ArcGIS Online with MN DNR layer: Ecological Sections of Minnesota; and Esri layers: United States State Boundaries 2018, World Ocean Reference (English), Ocean/World_Ocean_Base. Provinces and Territories of Canada. PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Figure 2 Species distribution maps of tunnel nesting bees in the genera Heriades, Hylaeus, Osmia and Stelis found from the Minnesota Bee Atlas nest traps. Data from nest traps and bundles (2016-2019) are shown as bee nests per trap, with traps grouped within 1 km locations and accounting for diﬀerent numbers of traps per location. For clarity, trap locations with no nests of a species are not shown. Additional locations depicted are research-grade iNaturalist observations through October 2020 and specimens from a 2019 version of the UMN Insect Collection database, overlaid over Minnesota’s four major ecological provinces. If UMN Insect Collection specimens did not have associated latitude and longitude, we used the location description to estimate the most accurate position possible. We chose the approximate center of geographic areas such as cities and state parks. If only county location was available, we placed the specimen in the county center and identiﬁed the records as such. Locations of cleptoparasitic bees are nests of their hosts from which they emerged. 1 Cleptoparasite on Heriades. PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed Figure 3 Species distribution maps of tunnel nesting bees in the genera Anthophora, Hoplitis, Megachile and Coelioxys, found from the Minnesota Bee Atlas nest traps. Manuscript to be reviewed PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Species distribution maps of tunnel nesting bees in the genera Anthophora, Hoplitis, Megachile and Coelioxys, found from the Minnesota Bee Atlas nest traps. Nest traps and bundle data (2016-2019), shown as bee nests per trap and grouped as in Figure 2. Also shown are research-grade iNaturalist observations through October 2020 and specimens from a 2019 version of the UMN Insect Collection database, overlaid over Minnesota’s four major ecological provinces. UMN Insect Collection specimens were assigned locations as in Figure 2. Locations of cleptoparasitic bees are nests of their hosts from which they emerged. 1 Cleptoparasite on M. pugnata. 2 Cleptoparasite on M. relativa. 3 Cleptoparasite on M. campanulae. PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed Manuscript to be reviewed PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed Figure 5 Phenology of tunnel-nesting bee nest completion. Figure 4 Ordination showing the relationship of land cover to tunnel-nesting bee presence and bumble bee abundance. The location of each point relative to the arrows indicates the land cover variable associated with that species (Palmer 1993). Arrow length indicates the importance of the habitat variable in predicting the variability in the model (ter Braak 1986). Arrow direction indicates the strength of correlation with the axes with a small angle between arrow and axis indicating high correlation. A. Redundancy analysis (RDA) axes 1 and 2 show the relationship of tunnel-nesting bees to land cover within 250 m of nest trap locations. B. Constrained correspondence analysis (CCA) axes 1 and 2 show the relationship of bumble bee species to land cover within 2 km of survey locations. Axis 1 eigenvalue=0.60, F=66.32, p<0.001, axis 2 eigenvalue=0.10, F=9.62, p<0.001. PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed Phenology of tunnel-nesting bee nest completion. We calculated nest completion date ranges, equal to the last empty tunnel date until the ﬁrst full plug date, for all nests with observation quality rated "medium" or "high". Each day in the nest completion date range was assigned equal probability. These probabilities were summed over all nests with suﬃcient quality observations and the median value was determined, indicating the date where nests were equally likely to be completed before or after. We also calculated the 0.25 and 0.75 quartile values, which bound the central 50% when most nests were likely completed. Because bees may be active for several weeks before nests are completed and plugged, we want to emphasize the beginning of the period and indicate the earliest 25% of ranges with light shading. The genus Osmia is shaded in blue, Heriades in gray, Megachile in green. Each species name is followed by parenthesis in which we list the number of nests used to calculate phenology from Minnesota Bee Atlas nest traps from 2016-2018, then the number of UMN insect collections specimens. * Indicates species with more than one generation per year in Minnesota. PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Figure 6 Species distribution maps for bumble bee species found during Minnesota Bee Atlas surveys with maximum average abundances between 1 and 25 bees per route per year. The Atlas observations are overlaid over Minnesota’s four major ecological provinces. We summarized survey information as the total abundance per species per route per year and displayed the average abundance per route per year for routes that were sampled over multiple years. A. Species with maximum abundances of 10 or fewer. B. Species with maximum abundances between 11 and 25. Additional records displayed are from iNaturalist from 2014 to 2020, Bumble Bee Watch from 2010 to 2022, and specimen-based Minnesota records from the Bumble Bees of North America database from 1889 to 2020 (Richardson 2021). * Species abundances for B. sandersoni are likely lower due to exclusion of records that could not be distinguished between B. vagans and B. sandersoni. PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed Manuscript to be reviewed PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed Manuscript to be reviewed Figure 7 Species distribution maps for bumble bees found during Minnesota Bee Atlas surveys with maximum average abundances per route per year between 25 and 180. These observations are overlaid over Minnesota’s four major ecological provinces. Additional records displayed are from iNaturalist from 2014 to 2020, Bumble Bee Watch from 2010 to 2022, and specimen-based Minnesota records from the Bumble Bees of North America database from 1889 to 2020 (Richardson 2021). * Species abundances for B. vagans are likely lower than expected due to exclusion of records that could not be distinguished between B. vagans and B. sandersoni. PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Figure 8 Bumble bee abundance across the Eastern Broadleaf Forest, Laurentian Mixed Forest, and Prairie Parkland ecological provinces. Bumble bee abundance is shown as the average abundance per route per year for routes with three completed survey dates within a year. A single route from the TAP ecological province was combined with routes from the PP ecological province due to the low sample size in this province and ecological similarity. PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed Table 1(on next page) Manuscript to be reviewed PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed Manuscript to be reviewed Table 2(on next page) Table 2(on next page) Table 1(on next page) Bumble bee survey routes. Volunteers adopted routes and completed surveys (three route runs with ﬁve 10-minute observations per route run) along routes between 2016 and 2020 across the Prairie Parkland (PP), Laurentian Mixed Forest (LMF), and Eastern Broadleaf Forest (EBF). Only one route was adopted in the Tallgrass Aspen Parklands province. This route is included in totals for the Prairie Parkland for routes adopted but did not have any completed surveys. Land cover was determined within 2 km of routes using the 2016 National Land Cover Database (NLCD) (Dewitz 2019) veriﬁed by examining aerial photographs. PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) 1 Ecological province Routes adopted Total complete surveys Surveys in 2016, 2017, 2018, 2019, or 2020 Dominant, secondary land covers Prairie Parkland 6 6 on 4 routes 0, 2, 1, 2, 1 crops, wetlands Laurentian Mixed Forest 18 28 on 14 routes 2, 6, 6, 6, 8 wetlands, forest Eastern Broadleaf Forest 21 45 on 19 routes 5, 8, 10, 11, 11 crops, forest Overall 45 79 on 37 routes 7, 16, 17, 19, 20 2 PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed Table 3(on next page) Table 2(on next page) Number of tunnels in trap nests that produced tunnel-nesting bee species in the four ecological provinces of Minnesota. Between 2016 and 2019 volunteers placed 69 nest traps in the Laurentian Mixed Forest (LMF), 224 traps in the Eastern Broadleaf Forest (EBF), 87 traps in the Prairie Parkland (PP), and two traps in the Tallgrass Aspen Parkland (TAP). PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed Species Total tunnels PP EBF LMF TAP Native/Introduced Anthophora terminalis 1 1 native Hylaeus annulatus 5 3 2 native Hylaeus leptocephalus 8 5 3 introduced (Russo, 2016) Hylaeus mesillae (group) 6 1 5 native Hylaeus nelumbonis 1 1 native Hylaeus sp.(modesta/sp.A) 3 3 Hylaeus verticalis 4 2 2 native Coelioxys alternata* 8 3 4 1 native, on M. pugnata Coelioxys modesta 30 2 28 native, on M. campanulae Coelioxys moesta 11 1 2 8 native, on M. relativa Heriades carinata 375 117 221 36 1 native Heriades leavitti 5 5 native Heriades variolosa 22 18 4 native Megachile brevis b 1 1 native Megachile campanulae 128 34 94 native Megachile centuncularis 3 3 ~ introduced (Sheffield et al., 2011) Megachile frugalis 1 1 native Megachile inermis 27 3 15 9 native Megachile inimica 5 2 3 native Megachile lapponica 1 1 native Megachile mendica 10 5 5 native Megachile pugnata 151 62 79 9 1 native Megachile relativa 132 11 57 62 2 native Megachile rotundata 36 14 20 2 introduced (Russo, 2016) Osmia albiventris 2 2 native Osmia georgica 1 1 native Osmia lignaria 484 43 245 195 1 native Osmia pumila 173 1 172 native Osmia tersula 77 5 9 61 2 native Stelis coarctatus 42 4 33 5 native, on H. carinata Stelis permaculata 3 3 native, on H. carinata Hoplitis albifrons b 1 1 native Total nests 1757 345 1009 396 7 Cleptoparasitic species: number of nests parasitized. b Species only found in stem bundles 1 PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Table 3(on next page) Table 3(on next page) Results of linear mixed eﬀects models of the inﬂuence of ecological provinces on frequency of tunnel-nests. Species presented are a subset of all species collected representing those collected from more than 10% of nest blocks, representing species in the genera Heriades, Osmia, and Megachile. Signiﬁcant results are indicated in bold. Means and standard errors are calculated from the raw data. Post hoc tests are the results of estimated marginal means comparisons. EBF=Eastern Broadleaf Forest, PP=Prairie Parklands, LMF=Laurentian Mixed Forest PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed Manuscript to be reviewed Table 4(on next page) Table 4(on next page) Manuscript to be reviewed mean nest frequency per block +/- s.e. Post hoc tests species EBF PP LMF X2 df p-value direction p-value <0.05 EBF = PP 0.2439 EBF > LMF 0.0352 H. carinata 1.02 ± 0.12 1.34 ± 0.21 0.52 ± 0.20 6.05 2 PP > LMF 0.0152 9.22 2 <0.01 EBF > PP 0.0447 EBF = LMF 0.1113 O. lignaria 1.13 ± 0.23 0.5 ± 0.25 2.9 ± 0.70 LMF > PP 0.0027 O. pumila 0.79 ± 0.12 0.01 ± 0.01 NA 6.03 1 <0.01 EBF > PP 0.0001 52.84 2 <0.01 EBF = PP 0.7153 LMF > EBF <.0001 O. tersula 0.04 ± 0.02 0.06 ± 0.03 0.90 ± 0.20 LMF > PP <.0001 M. campanulae 0.42 0.05 0.38 0.12 NA 0.40 2 1 EBF = PP 0.53 8.66 2 <0.05 EBF = PP 0.1205 EBF > LMF 0.0475 M. pugnata 0.37 ± 0.08 0.72 ± 0.19 0.12 ± 0.06 PP > LMF 0.0043 9.26 2 <0.001 EBF = PP 0.3295 LMF > EBF 0.0126 M. relativa 0.27 ± 0.09 0.13 ± 0.05 0.91 ± 0.30 LMF > PP 0.0047 2.27 2 0.3216 EBF = PP 0.3008 EBF = LMF 0.4346 Overall nesting 4.2 ± 1.2 3.6 ± 1.4 4.9 ± 1.5 LMF = PP 0.1380 PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Table 4(on next page) Biplot scores for constraining variables of land cover related to presence of tunnel- nesting bee species or bumble bee species abundance. The forest category combines deciduous, mixed, and evergreen forest. All levels of development were combined into the category. The grassland category includes grasslands/herbaceous and pasture/hay. The wetland category includes woody wetlands and emergent herbaceous wetlands. Correlations with absolute values ≥ 0.5 are bolded. PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed Tunnel-nesting bees RDA1 RDA2 RDA3 Tunnel-nesting bees RDA1 RDA2 RDA3 g developed -0.07 -.99 -0.02 forest 0.82 0.25 -0.51 grassland -0.73 0.42 -0.53 Bumble bees CCA1 CCA2 CCA3 CCA4 developed 0.57 0.62 0.41 0.34 wetland -0.83 0.41 -0.18 -0.30 forest -0.76 0.35 0.50 -0.11 grassland 0.67 -0.24 0.03 -0.71 1 1 PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) 2 a Bombus vagans group includes B. vagans (Smith, 1854) and B. sandersoni (Franklin, 1913). 3 b Categorized with IUCN status vulnerable or critically endangered (Hatfield, 2015) 3 b Categorized with IUCN status vulnerable or critically endangered (Hatfield, 2015) 2 a Bombus vagans group includes B. vagans (Smith, 1854) and B. sandersoni (Fra Manuscript to be reviewed Table 6(on next page) Table 6(on next page) Table 5(on next page) Table 5(on next page) Bumble bee species total abundance and abundance within three ecological provinces. Eastern Broadleaf Forest (EBF), Laurentian Mixed Forest (LMF), and Prairie Parkland (PP). Species are ordered from greatest to least total abundance. PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Manuscript to be reviewed 1 Bombus species Total EBF LMF PP ternarius (Say, 1873) 2069 602 1466 1 impatiens (Cresson, 1863) 1975 1781 140 54 vagans groupa 1904 756 1116 32 bimaculatus (Cresson, 1863) 1257 1109 71 77 griseocollis (DeGeer, 1773) 977 733 142 102 borealis (Kirby, 1837) 252 68 173 11 auricomus (Robertson, 1903) 145 116 7 22 rufocinctus (Cresson, 1863) 143 122 21 0 fervidusb (Fabricius, 1798) 131 103 14 14 terricolab (Kirby, 1837) 103 34 69 0 perplexus (Cresson, 1863) 71 28 43 0 citrinus (Smith, 1854) 42 20 20 2 flavidus (Eversmann, 1892) 36 20 16 0 pensylvanicusb (DeGeer, 1773) 22 20 0 2 affinisb (Cresson, 1863) 18 17 1 0 insularis (Smith, 1861) 2 1 1 0 1 PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) Table 6(on next page) Results of linear mixed eﬀects model of inﬂuence of ecological provinces on overall bumble bee abundance. Bee abundances are log-transformed. Signiﬁcant results are indicated in bold. Post hoc tests are the results of estimated marginal means comparisons. EBF=Eastern Broadleaf Forest, PP=Prairie Parklands, LMF=Laurentian Mixed Forest PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023) 1 2 3 Post hoc tests Fixed effects X2 df p-value β + 95 9 CI direction p-value Ecological province 12.03 2 <0.01 EBF LMF PP 4.28 (3.75-4.69) 4.40 (4.02-4.79) 3.02 (2.29-3.74) EBF = LMF EBF > PP LMF > PP 0.88 <0.01 <0.01 Year 3.26 4 0.52 2016 2017 2018 2019 2020 3.84 (3.37-4.31) 3.83 (3.47-4.19) 3.81 (3.45-4.17) 4.04 (3.69-4.39) 3.99 (3.64-4.33) Random effects Variance + SD Route Residual 0.40 + 0.63 0.17 + 0.41 Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed 1 2 3 Post hoc tests Fixed effects X2 df p-value β + 95 9 CI direction p-value Ecological province 12.03 2 <0.01 EBF LMF PP 4.28 (3.75-4.69) 4.40 (4.02-4.79) 3.02 (2.29-3.74) EBF = LMF EBF > PP LMF > PP 0.88 <0.01 <0.01 Year 3.26 4 0.52 2016 2017 2018 2019 2020 3.84 (3.37-4.31) 3.83 (3.47-4.19) 3.81 (3.45-4.17) 4.04 (3.69-4.39) 3.99 (3.64-4.33) Random effects Variance + SD Route Residual 0.40 + 0.63 0.17 + 0.41 1 2 3 Post hoc tests Fixed effects X2 df p-value β + 95 9 CI direction p-value Ecological province 12.03 2 <0.01 EBF LMF PP 4.28 (3.75-4.69) 4.40 (4.02-4.79) 3.02 (2.29-3.74) EBF = LMF EBF > PP LMF > PP 0.88 <0.01 <0.01 Year 3.26 4 0.52 2016 2017 2018 2019 2020 3.84 (3.37-4.31) 3.83 (3.47-4.19) 3.81 (3.45-4.17) 4.04 (3.69-4.39) 3.99 (3.64-4.33) Random effects Variance + SD Route Residual 0.40 + 0.63 0.17 + 0.41 PeerJ reviewing PDF \| (2022:12:80802:2:0:NEW 21 Aug 2023)
https://openalex.org/W4220702735	https://www.nature.com/articles/s41378-022-00363-5.pdf	English	null	Origami-inspired folding assembly of dielectric elastomers for programmable soft robots	Microsystems & nanoengineering	2,022	cc-by	9,068	© The Author(s) 2022 OpenAccessThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,whichpermitsuse,sharing,adaptation,distributionandreproduction 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/. A R T I C L E O p e n A c c e s s Abstract Origami has become an optimal methodological choice for creating complex three-dimensional (3D) structures and soft robots. The simple and low-cost origami-inspired folding assembly provides a new method for developing 3D soft robots, which is ideal for future intelligent robotic systems. Here, we present a series of materials, structural designs, and fabrication methods for developing independent, electrically controlled origami 3D soft robots for walking and soft manipulators. The 3D soft robots are based on soft actuators, which are multilayer structures with a dielectric elastomer (DE) ﬁlm as the deformation layer and a laser-cut PET ﬁlm as the supporting ﬂexible frame. The triangular and rectangular design of the soft actuators allows them to be easily assembled into crawling soft robots and pyramidal- and square-shaped 3D structures. The crawling robot exhibits very stable crawling behaviors and can carry loads while walking. Inspired by origami folding, the pyramidal and square-shaped 3D soft robots exhibit programmable out-of-plane deformations and easy switching between two-dimensional (2D) and 3D structures. The electrically controllable origami deformation allows the 3D soft robots to be used as soft manipulators for grasping and precisely locking 3D objects. This work proves that origami-inspired fold-based assembly of DE actuators is a good reference for the development of soft actuators and future intelligent multifunctional soft robots. Origami-inspired folding assembly of dielectric elastomers for programmable soft robots Yanhua Sun1,2, Dengfeng Li3, Mengge Wu1,3, Yale Yang1,2, Jingyou Su3, Tszhung Wong3, Kangming Xu2, Ying Li2, Lu Li2✉, Xinge Yu 3✉and Junsheng Yu 1✉ Sun et al. Microsystems & Nanoengineering (2022) 8:37 https://doi.org/10.1038/s41378-022-00363-5 Sun et al. Microsystems & Nanoengineering (2022) 8:37 https://doi.org/10.1038/s41378-022-00363-5 Microsystems & Nanoengineering www.nature.com/micronano Introduction as an operator in medical robotic systems during clinical surgeries8–10. In addition, soft actuators have been widely used as manipulators in large demission robots11. In practice, to increase their maneuverability for robotic walking, medical manipulation, and three-dimensional (3D) object grasping, it is crucial to build soft robots with ﬂexible and variable 3D structures. Soft robots have irreplaceable advantages in mechanical and biomedical engineering1–4. Because of their soft bodies, soft robots can adapt their body shape to complex physical environments and walk through narrow passages that rigid robots cannot5,6. The soft nature of their bodies also prevents sharp injuries to objects they touch, allowing them to enter the human body for drug transport7 or act Currently, 3D soft robots are typically manufactured through 3D printing and assembly with small actua- tors12,13. Compared to 3D shapes, 2D shapes are more space-efﬁcient in terms of their spatial dimension14. Thus, origami-inspired 3D soft robot construction, derived from an inherent simpliﬁed and low-cost folding-based assembly technique, is a good strategy due to its ability to perform out-of-plane deformations for 3D structure construction and to switch between 2D and 3D15–19. The detailed advantages of origami robots are as follows: (i) Soft, simple preparation process, and low cost. Compared with traditional robots, origami robots have a more 1State Key Laboratory of Electronic Thin Films and Integrated Devices, School of Optoelectronic Science and Engineering, University of Electronic Science and Technology of China (UESTC), Chengdu 610054, People’s Republic of China 2Chongqing Key Laboratory of Materials Surface & Interface Science, Chongqing Co-Innovation Center for Micro/Nano Optoelectronic Materials and Devices, Micro/Nano Optoelectronic Materials and Devices International Science and Technology Cooperation Base of China, School of Materials Science and Engineering, Chongqing University of Arts and Sciences, Chongqing 402160, People’s Republic of China Full list of author information is available at the end of the article Correspondence: Lu Li (lli@cqwu.edu.cn) or Xinge Yu (xingeyu@cityu.edu.hk) or Junsheng Yu (jsyu@uestc.edu.cn) © The Author(s) 2022 ( ) OpenAccessThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,whichpermitsuse,sharing,adaptation,distributionandreproduction 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/. Page 2 of 11 Sun et al. Microsystems & Nanoengineering (2022) 8:37 controlled automatically33,34. When combined with intelligent sensing systems35–38, soft and durable robotic systems can assist humans with long-term tasks through human–machine interactions39–43. Electrical actuation allows robots to be precisely controlled in various envir- onments as long as driving programs are established. At present, nearly all functional intelligent robots are elec- trically driven due to the advantages of electrical actuation in terms of handling precision. In recent years, an increasing number of soft robots have been powered by electrical energy based on electrothermal44, piezo- electric45, and dielectric32 principles. Programmable electrical actuation, such as independent leg control of multilegged robots46 and segmented control of single- body robots47, enables soft robots to move and function in various ways. In contrast, dielectric elastomers have rarely been combined with origami design for electrically actu- ated paper-folding robots. For the ﬁrst time, we intro- duced VHB4910 elastomers into origami-inspired robots because of their outstanding advantages, such as their quick response time and superior resilience. streamlined structure, eliminating many complex trans- mission gear structures and requiring less production material. (ii) High degree of collapse and space efﬁciency. Origami robots have less transportation and storage requirements since they are capable of converting from two- to three-dimensional shapes. (iii) Scalability and various applications. The diversity of the origami method enables robots to be highly scalable in terms of structure and functionality. However, more novel designs and research on origami robots are urgently needed to achieve complex functionality. Scientists have developed different types of origami- inspired soft robots with a variety of materials and actuation methods, each with speciﬁc mechanical manipulation functions and movement styles20. © The Author(s) 2022 For instance, a battery-free miniature origami robotic arm based on origami actuation was developed with shape memory alloys and has been used for arm orientation control and object grasping21. 2D nanomaterials, such as MXene22 and graphene23,24, have been used as functional layers in soft actuators and robots, with light ﬁelds typi- cally used as an actuation method for 3D structural control, programmable actuation, movements, and var- ious artistic displays. A new triple-layer dual-chip actuator based on photothermal actuation was successfully used to assemble a fast crawling soft robot and a powerful mechanical clamp25. In addition, 3D structures fabricated by the kirigami technique in phase-change liquid crystal elastomers are a new type of robotic technology, with light beams serving as the actuation source to control motion and steer the movement direction in 2D26. For the active folding assembly to interchange between 2D and 3D and repeatably deform, the actuator must be a soft deforma- tion material. In addition to the materials mentioned above, dielectric elastomers are an excellent choice due to their relatively large actuation force with large deforma- tions27,28. Moreover, dielectric elastomers are actuated by electrical energy, which is convenient for future integra- tion with robotic systems. q p p In this work, we combined origami technology and electronically controlled actuators to develop program- mable 3D soft robots that can reversibly switch between 2D and 3D structures. These soft robots rely on a variety of programming controls to assemble multiple origami structures and to perform functions such as walking, grasping, and locking objects. These electrical actuators are composed of a pre-stretched dielectric elastomer with conductive carbon grease on both sides, which functions as a stretchable conductive electrode, and a laser-cut polyethylene terephthalate (PET) ﬁlm, which functions as a ﬂexible support frame. By designing the shape of the PET frame, a wide range of 3D origami assemblies can be produced in a cost-effective and easy- to-process manner. These origami-inspired soft robots based on electronically controlled dielectric elastomers perform well in terms of movement, assembly, and function, serving as good models for future 3D soft robot construction. Soft robots should be developed with intelligent sensing systems. A class of actuators that combine tensile and torsional deformation to achieve sensing and various motions has been investigated29. Humidity-driven ﬁber muscles detect changes in external humidity while twist- ing and stretching30. © The Author(s) 2022 Twisted elastomeric ﬁbers ﬁtted with carbon nanotube sheaths and contact clasps for sensing can monitor resistance signals during electrothermally driven twisting31. In addition, a spiral ﬁber crawling robot, which simulates the musculoskeletal structure of a human arm, can detect body deformations while crawling with resistive strain sensors32. These ﬁndings have been used to develop intelligent textiles and soft robots that can perceive, interact with and adapt to environmental sti- muli. Future intelligent robotic systems will inevitably be Results and discussion Figure 1 shows the fabrication and actuation principles of the origami-inspired soft actuators. As shown in Fig. 1a, the planar 2D and spatial 3D structures of the origami- inspired soft robot can be easily switched by four soft actuators. The soft actuator consists of a dielectric elas- tomer (DE), which acts as the active deformation layer, and a laser-cut PET ﬁlm, which acts as the ﬂexible sup- port frame. To fabricate the soft actuator, a VHB4910 DE ﬁlm was ﬁrst prestretched to 400% × 400% with a self- designed, precisely adjustable stretching tool (Fig. 1b and Fig. S1). The thickness of the DE ﬁlm was reduced from 0.93 to 0.04 mm (Fig. S2). The stretched DE ﬁlm was ﬁxed Sun et al. Microsystems & Nanoengineering (2022) 8:37 Page 3 of 11 to an acrylic frame, and a laser-cut 0.1 mm-thick PET ﬁlm with a circular hole was pasted on it as a ﬂexible frame. Then, two laser-cut 0.25 mm thick PET ﬁlms with speciﬁc semicircular radii were pasted on the other side of the DE ﬁlm in the same position as the reinforcement fram apply an actuation voltage to the DE ﬁlm, condu carbon grease was painted on both sides of the m round area, which functioned as the actuation re a c 11.5 12.0 12.5 13.0 13.5 60 90 120 150 180 Experimental data Linear fitting (degree) R (mm) 115° 144° 106° 130° 76° 94° 150° 137° 112° d e OFF State ON State V R 1 cm Pre-stretch VHB4910 Fix with acrylic frame The actuator Remove retainer clip Attach PET substrate Paint carbon grease Attach PET reinforcement Paint carbon grease b 3D , spatial 2D , planar Origami Fig. 1 Fabrication and actuation principles of origami soft actuators with dielectric elastomers. a Schematic diagram of the origami-ins soft robots. b Flow chart of the fabrication process for the soft actuator. After release, an actuator with a certain initial bending angle was obta c Schematic diagram of the actuation principle and the expanded layered structure of the soft actuator with a dielectric layer (VHB4910), a reinforcement layer (PET ﬁlm with a thickness of 0.25 mm), and a ﬂexible substrate (PET ﬁlm with a thickness of 0.1 mm). d Relationship between semicircular radius of the actuation region with the dielectric layer and the bending angle. Results and discussion After cutting the DE ﬁlm along the outer edge of the PET ﬁlm and releasing it, a soft actuator with an original saddle shape was acquired48. A thin wire was placed at the edge of the carbon grease electrode, and the soft actuator was actuated by a voltage source. After cutting the DE ﬁlm along the outer edge of the PET ﬁlm and releasing it, a soft actuator with an original saddle shape was acquired48. When a voltage is applied to the actuator, charge accumulates on the ﬂexible electrode surfaces on both sides of the ﬁlm, as shown in Fig. 1c. When the accu- mulation reaches a certain threshold, the electrostatic force on the positive and negative electrode surfaces squeezes the middle DE ﬁlm layer, causing it to expand in all directions. As the DE ﬁlm expands in the actuation area, the shape of the actuator is no longer in equilibrium, resulting in bending and braking effects49. The braking effect is not only voltage-dependent but also related to the thickness and elastic modulus of the elastomer material. Prestretching can greatly reduce the thickness of the DE ﬁlm, allowing for braking deformation with a low actua- tion voltage. In this work, a VHB4910 elastomer ﬁlm (3 M, USA) was used as the actuator due to its high tensile rate, low elastic modulus, and low cost50. As shown in Fig. 1b, e, the actuator exhibits an original bending angle in its natural state. When an external vol- tage is applied, the actuator tends to straighten its body, reducing the angle between the actuator and the hor- izontal line, which we deﬁne as the bending angle of the actuator. The original bending angle is crucial for con- structing 3D origami soft robots. For example, a 90° soft actuator can be used to build square 3D origami robots, while a 120° soft actuator is the best choice for building pyramidal 3D origami robots. The original bending angle could be adjusted by the area of the actuation region. Therefore, we investigated the relationship between the bending angle and the semicircular radius of the actuation region. A set of soft actuators with two semicircular radii ranging from 11.5 to 13.5 mm spaced 6 mm apart were fabricated. The obtained soft actuators with different original bending angles are shown in Fig. 1e. The linear relationship between the bending degree and the radius is summarized in Fig. 1d. Results and discussion e Optical images of actuators with different semicir radii in the actuation region with different bending angles Pre-stretch VHB4910 Fix with acrylic frame The actuator Remove retainer clip Attach PET substrate Paint carbon grease Attach PET reinforcement Paint carbon grease a 3D , spatial 2D , planar Origami b The actuator Paint carbon grease 115° 144° 106° 130° 76° 94° 150° 137° 112° e 1 cm e c c OFF State ON State V 11.5 12.0 12.5 13.0 13.5 60 90 120 150 180 Experimental data Linear fitting (degree) R (mm) d R d Fig. 1 Fabrication and actuation principles of origami soft actuators with dielectric elastomers. a Schematic diagram of the origami-inspired soft robots. b Flow chart of the fabrication process for the soft actuator. After release, an actuator with a certain initial bending angle was obtained. c Schematic diagram of the actuation principle and the expanded layered structure of the soft actuator with a dielectric layer (VHB4910), a reinforcement layer (PET ﬁlm with a thickness of 0.25 mm), and a ﬂexible substrate (PET ﬁlm with a thickness of 0.1 mm). d Relationship between the semicircular radius of the actuation region with the dielectric layer and the bending angle. e Optical images of actuators with different semicircular radii in the actuation region with different bending angles to an acrylic frame, and a laser-cut 0.1 mm-thick PET ﬁlm with a circular hole was pasted on it as a ﬂexible frame. Then, two laser-cut 0.25 mm thick PET ﬁlms with speciﬁc semicircular radii were pasted on the other side of the DE ﬁlm in the same position as the reinforcement frame. To apply an actuation voltage to the DE ﬁlm, conductive carbon grease was painted on both sides of the middle round area, which functioned as the actuation region. Page 4 of 11 Page 4 of 11 Sun et al. Microsystems & Nanoengineering (2022) 8:37 Sun et al. Microsystems & Nanoengineering (2022) 8:37 Sun et al. Microsystems & Nanoengineering (2022) 8:37 tended to straighten during the actuation process. Therefore, we deﬁned this 120° soft actuator as the tri- angular actuator. Similarly, we used a 90° soft actuator with an 11.75 mm radius as a rectangular actuator for the 3D square soft robot. Results and discussion The actuation and deformation behaviors of these two soft actuators were critical for the performance of the 3D soft robots, and the electrical test results are summarized in Fig. 2. After the triangular soft actuator was connected to the power supply via thin wires, the actuation voltage was gradually increased from 0 to 5.52 kV in steps of 0.5 kV. The results show that the triangular soft actuator deformed to a horizontal state at 5.52 kV with a bending angle of only 3° (Fig. 2a). The relationship between the bending angle and the actuation voltage is shown in Fig. 2e. The triangular soft actuator also exhibited excellent cycling consistency. The cycling test results in Fig. 2b, f and Movie S1 show that the soft actuators maintained their original shape after 100 cycles. This indicates that soft actuators and 3D soft robots with DE ﬁlms have a stable and reproducible performance during repeated use. For the rectangular actuator, an excellent deformation performance was also achieved, as shown in Fig. 2c–f and Movie S2. Under an actuation voltage of 4.09 kV, the rectangular soft actuator deformed to a horizontal state with a bending angle of only 1°. After 100 cycles, there was no signiﬁcant difference in the shape of the actuator. We also tested the life cycle of six straight- edge DE actuators with 90° angles and found that the robots fully recovered to their initial angle within the ﬁrst 500 cycles. As the number of cycles increased, the recovery characteristics of the DE actuators worsened due to the bending fatigue of the PET substrates. After 5000 cycles, these DE actuators only recovered to an angle of 60°, while they were expected to recover to 90°. In addi- tion, the mechanical properties of both actuators were investigated by using micromechanical sensors to mea- sure the actuator’s force at different voltages (Fig. S4). The result showed that a 3.61 mN force was generated when the triangular soft actuator was actuated by a voltage of 5.52 kV. The rectangular actuator only exhibited a force of 2.16 mN under a voltage of 4.09 kV due to the relatively small change in the bending angle. A thin wire was placed at the edge of the carbon grease electrode, and the soft actuator was actuated by a voltage source. Results and discussion A soft actuator with a radius of 11.75 mm was bent at approximately 90°, while an actuator with a radius of 12.5 mm was bent at approxi- mately 120°. By adjusting the radius of the actuation region, soft actuators with speciﬁc bending angles could be easily acquired. This result provides strong support for the subsequent assembly experiments with crawling robots and 3D folding assemblies with origami-inspired soft robots. In nature, many animals crawl or walk by deforming and actuating their bodies or joints. The soft actuators in this work are well suited for use as an artiﬁcial muscle in a crawling robot. Therefore, we designed a 3D crawling soft robot with rectangular actuators and studied its walking behavior (Fig. 3). The soft crawling robot has a square body and rectangular bipeds at both ends. Figure S5 shows the planar structure design, which included two actuation regions with radii of 12.5 mm. The original bending angle for the two feet of this soft robot was 120°. The lengths of the feet and the main body were 40 and 60 mm, respectively. Figure 3a shows the crawling To construct the 3D soft robots, we chose soft actuators with bending angles of 90° and 120° as examples. Figure S3 presents the planar structure design for these two soft actuators. To create a 3D pyramid soft robot, we used a 120° soft actuator with a triangular actuation side and a 12.5 mm-radius actuation region; the triangular side Page 5 of 11 Sun et al. Microsystems & Nanoengineering (2022) 8:37 0 50 100 150 (degree) Time (s) Triangular Rectangular 0 10 740 750 760 0 1 2 3 4 5 6 0 30 60 90 120 (degree) Voltage (kV) Triangular Rectangular a f 117° 1 cycle 116° 100 cycle 92° 0.00 kV 88° 1.03 kV 77° 2.02 kV 49° 3.04 kV 9° 4.00 kV 1° 4.09 kV 0.00 kV 3.02 kV 118° 2.05 kV 110° 97° 4.01 kV 70° 5.02 kV 28° 5.52 kV 3° c 1 cm 1 cm 1 cm 92° 1 cycle 91° 100 cycle 1 cm b d 1 cycle 100 cycle e Triangular Rectangular Fig. 2 Study of triangular and rectangular soft actuators. a Photos of the 12.5 mm-radius triangular actuator under different actuation voltages. b Photos of the triangular actuator after the ﬁrst and 100th actuation cycles. Results and discussion c Optical images of the 11.75 mm-radius rectangular actuator under different actuation voltages. d Photos of the rectangular actuator after the ﬁrst and 100th actuation cycles. e The relationship between the bending angle and the input voltage for the triangular (blue) and rectangular (orange) actuators. f The stability during 100 actuation cycles for the triangular (blue) and rectangular (orange) actuators a 117° 1 cycle 116° 100 cycle 0.00 kV 3.02 kV 118° 2.05 kV 110° 97° 4.01 kV 70° 5.02 kV 28° 5.52 kV 3° 1 cm 1 cm b Triangular 117° 1 cycle 116° 100 cycle 1 cm b a 0.00 kV 3.02 kV 118° 2.05 kV 110° 97° 4.01 kV 70° 5.02 kV 28° 5.52 kV 3° 1 cm Triangular b 92° 0.00 kV 88° 1.03 kV 77° 2.02 kV 49° 3.04 kV 9° 4.00 kV 1° 4.09 kV c 1 cm Rectangular d 92° 1 cycle 91° 100 cycle 1 cm d c 0 1 2 3 4 5 6 0 30 60 90 120 (degree) Voltage (kV) Triangular Rectangular e f 0 50 100 150 (degree) Time (s) Triangular Rectangular 0 10 740 750 760 f 1 cycle 100 cycle e (degree) Voltage (kV) Fig. 2 Study of triangular and rectangular soft actuators. a Photos of the 12.5 mm-radius triangular actuator under different actuation voltages. b Photos of the triangular actuator after the ﬁrst and 100th actuation cycles. c Optical images of the 11.75 mm-radius rectangular actuator under different actuation voltages. d Photos of the rectangular actuator after the ﬁrst and 100th actuation cycles. e The relationship between the bending angle and the input voltage for the triangular (blue) and rectangular (orange) actuators. f The stability during 100 actuation cycles for the triangular (blue) and rectangular (orange) actuators foot was stopped, causing it to contract, and the soft robot leaned forward, shifting its center of gravity forward again. Finally, the voltage on the rear foot was released, and the rear foot contracted and returned to its initial state because of the increased friction generated by the front foot due to the forward shift of the center of gravity. Therefore, the robot’s movement was highly dependent on the interface friction. The amount of friction generated on the surface affected the crawling displacement of the soft robot. Results and discussion To further investigate the effect of rough surfaces on crawling, sandpapers with various grit sizes (P1500, P1000, and P600) were used to study the crawling speed. The soft robot was actuated by a square-wave voltage with a frequency of 0.29 Hz and duty cycle of behaviors of the soft robot at each step, including the actuator’s switching state, the force direction, and the displacement direction. By actuating the front and back feet separately, the soft robot can move forward on the sandpaper. Each walking cycle can be separated into four steps (Fig. 3a and Fig. S6). In the ﬁrst step, the front foot was actuated against the ground to unfold, and the soft robot tilted backward. Next, after the front foot com- pletely unfolded, the rear foot actuated. Due to the backward shift in the center of gravity, the rear foot produced more friction force when it contacted the ground quickly, causing the soft robot to jump and crawl forward. Then, the robot’s step stabilized, and its center of gravity balanced. In the third step, the voltage on the front Sun et al. Microsystems & Nanoengineering (2022) 8:37 Page 6 of 11 a Step 1 Step 2 Step 3 Step 4 Step 5 P1500 sandpaper P1000 P600 1 cm Volt Off On Volt On On Volt Off Off Volt On Off Volt Off Off f1 S S S f2 f2 f1 f2 f1 f2 f1 4.3 mm 10.2 mm 26.4 mm 4.09 3.28 1.71 0 1 2 3 4 Speed (mm/s) Voltage (kV) 0 30 60 90 120 Time (s) Front foot Back foot 0 40 80 120 Distance (mm) Time (s) Separate actuation Simultaneous actuation 3 4 5 0 1 2 3 0 5 10 15 20 25 0 1 2 3 4 20 25 30 35 40 Height (mm) Time (s) Front foot Back foot e c b d f a P600 1g weights 2g weights P600 1 cm Step 1 Step 2 Step 3 Step 4 Step 5 P1500 sandpaper P1000 P600 1 cm Volt Off On Volt On On Volt Off Off Volt On Off Volt Off Off f1 S S S f2 f2 f1 f2 f1 f2 f1 4.3 mm 10.2 mm 26.4 mm 12.2 mm 6.2 mm 3.97 mm/s 2.80 mm/s (degree) Fig. 3 Crawling behaviors of the origami-inspired soft robot. Results and discussion a Analysis of the soft robot’s behaviors at each step of the crawling process, including the actuator’s switching state, the force direction, and the displacement direction. The soft robots walked on sandpaper with different grits, including P1500, P1000, and P600. b Angle variation of the soft robot’s front and rear feet with time during one motion cycle. c Height variation of the soft robot’s front and rear foot joints with time during one motion cycle. d Displacement variation with time for the soft robot under separate and simultaneous actuation. e Crawling speed of the soft robot under different actuation voltages. f Crawling behaviors of the soft robot with different loads a 0 30 60 90 120 Time (s) Front foot Back foot 0 1 2 3 0 1 2 3 4 20 25 30 35 40 Height (mm) Time (s) Front foot Back foot c b (degree) 4.09 3.28 1.71 0 1 2 3 4 Speed (mm/s) Voltage (kV) 0 40 80 120 Distance (mm) Time (s) Separate actuation Simultaneous actuation 3 4 5 0 5 10 15 20 25 e d Speed (mm/s) 0 40 80 120 Distance (mm) Time (s) Separate actuation Simultaneous actuation 0 5 10 15 20 25 e d b d e f f P600 1g weights 2g weights P600 1 cm 12.2 mm 6.2 mm 3.97 mm/s 2.80 mm/s Fig. 3 Crawling behaviors of the origami-inspired soft robot. a Analysis of the soft robot’s behaviors at each step of the crawling process, including the actuator’s switching state, the force direction, and the displacement direction. The soft robots walked on sandpaper with different grits including P1500, P1000, and P600. b Angle variation of the soft robot’s front and rear feet with time during one motion cycle. c Height variation o the soft robot’s front and rear foot joints with time during one motion cycle. d Displacement variation with time for the soft robot under separate and simultaneous actuation. e Crawling speed of the soft robot under different actuation voltages. f Crawling behaviors of the soft robot with different loads f P600 1g weights 2g weights P600 1 cm 3.97 mm/s 2.80 mm/s Fig. 3 Crawling behaviors of the origami-inspired soft robot. a Analysis of the soft robot’s behaviors at each step of the crawling process, including the actuator’s switching state, the force direction, and the displacement direction. Results and discussion a Expanded multilayered diagrams of the 3D pyramid and square folding assemblies. b Programmable unfolding process of the pyramid-shaped soft robot. c Programmable unfolding process of the square-shaped soft robot a Carbon grease (0.1 mm) PET reinfor (0.25 m PET substrate VHB 4910 ela Carbon grease (0.1 mm) (i) Carbo a Carbon grease (0.1 mm) PET reinforcement (0.25 mm) PET substrate (0.1 mm) VHB 4910 elastomer Carbon grease (0.1 mm) (i) (ii) Carbon grease (0.1 mm) Carbon grease (0.1 mm) PET reinforcement (0.25 mm) PET substrate (0.1 mm) VHB 4910 elastomer (ii) Carbon grease (0.1 mm) Carbon grease (0.1 mm) b 1 cm b 1 3 4 4 3 2 3 2 3 c c 1 cm 1 3 2 4 3 4 1 3 2 Fig. 4 3D folding assembly of the origami-inspired soft robot. a Expanded multilayered diagrams of the 3D pyramid and square folding assemblies. b Programmable unfolding process of the pyramid-shaped soft robot. c Programmable unfolding process of the square-shaped soft robot stimulate the crawling ability of the soft robot, we used a higher actuation voltage of 5.5 kV. The results (Fig. S12) demonstrated that the walking speed ﬁrst increased and then decreased as the power-off frequency increased due to the response time requirement of bipedal charging and discharging. As the frequency increased, the speed of bipedal actuation accelerated. Considering that the power-off time affects the stride angle during contraction, the robots’ pace per step decreased (Fig. S11) when the power-off time was less than the time required for bipedal contraction, reducing the crawling speed. According to Tables S1 and S2, the crawling speed of the robots in this paper was at the same level (~mm/s) as in previous reports. Table S2 compares the soft robot performance of existing DE actuator-powered robots. The clear advantage of our soft robots is their space efﬁciency and scalability due to the origami assembly. that (Fig. S10 and Movie S5) the maximum speed reached 5.12 mm/s for a tilt angle of 20°, which was greater than the speeds of 3.20 and 3.88 mm/s achieved for tilt angles of 10° and 30° and greater than the maximum speed of 4.09 mm/s achieved on sandpaper at the same voltage (5 kV) and frequency (0.29 Hz). Results and discussion The soft robots walked on sandpaper with different grits, including P1500, P1000, and P600. b Angle variation of the soft robot’s front and rear feet with time during one motion cycle. c Height variation of the soft robot’s front and rear foot joints with time during one motion cycle. d Displacement variation with time for the soft robot under separate and simultaneous actuation. e Crawling speed of the soft robot under different actuation voltages. f Crawling behaviors of the soft robot with different loads 28.6%. The bandwidth of soft actuators made from VHB4910 elastomers is usually less than 10 Hz due to the viscoelasticity of VHB elastomers. The results show that the average crawling speed of the robot on P600 sandpaper (4.09 mm/s) was nearly 30 times higher than that on P1500 sandpaper (0.15 mm/s) (Fig. 3a and Movie S3), which indicates that soft robots crawl better on rough surfaces. In addition, we measured the angle and height variations of the front and rear feet of the robot as it crawled on P600 sandpaper during one motion cycle at an input voltage of 5 kV. In this intermittent separate actuation method, the center of gravity was moved by changing the height of the robot’s feet separately; thus, the front and rear feet generated different friction forces and crawled to one side during actuation. To verify the superiority of this actuation method, the crawling robot was also actuated simultaneously at the same voltage for comparison (Fig. 3d). When the front and rear feet were actuated simultaneously, it was difﬁcult to achieve stable motion in one direction, and the resulting movement was hesitation in one place (Fig. S7 and Movie S4). In addition to the rough sandpaper, we tested the dynamic properties of soft robots crawling on zigzag surfaces. As shown in Fig. S8, zigzag surfaces with tilt angles of 10°, 20°, and 30° were built by stacking 300 pieces of 0.9 mm-thick acrylic sheets with different zigzag serration widths. It was found Sun et al. Microsystems & Nanoengineering (2022) 8:37 Page 7 of 11 1 2 3 4 1 2 3 4 a b c Carbon grease (0.1 mm) PET reinforcement (0.25 mm) PET substrate (0.1 mm) VHB 4910 elastomer Carbon grease (0.1 mm) (i) (ii) Carbon grease (0.1 mm) Carbon grease (0.1 mm) 1 cm 1 cm Fig. 4 3D folding assembly of the origami-inspired soft robot. Results and discussion f A square-shaped soft robot captures a rolling ball and then locks it 1 2 3 4 5 6 a 2 cm a 0 5 10 15 Maximum loading weight (g) Sandpaper (P) None 400 240 120 6.0 10.0 13.5 14.5 d 2 cm Sandpaper Sandpaper b 0 5 10 15 Maximum loading weight (g) Sandpaper (P) None 400 240 120 6.0 10.0 13.5 14.5 d c 2 cm Without sandpaper P120 sandpaper 14.5g 2 cm 6.0g 2 cm Sandpaper Sandpaper b c b d c 1 2 3 4 5 6 e 1 cm e f 3 2 1 6 5 4 f 1 cm Fig. 5 Demonstrations of the grasping and locking functions of the origami-inspired 3D soft robot acting on static and dynamic objects a A pyramid-shaped soft gripper captures a static ball and transports it to a beaker. b Sandpaper placed on the inside of the soft gripper. c Sandpape placed on the inside of the soft gripper. d Maximum gripping weight of the soft gripper with different grit sandpapers. e A square-shaped soft robo captures a falling ball and locks it in a box f A square shaped soft robot captures a rolling ball and then locks it Fig. 5 Demonstrations of the grasping and locking functions of the origami-inspired 3D soft robot acting on static and dynamic objects. a A pyramid-shaped soft gripper captures a static ball and transports it to a beaker. b Sandpaper placed on the inside of the soft gripper. c Sandpaper placed on the inside of the soft gripper. d Maximum gripping weight of the soft gripper with different grit sandpapers. e A square-shaped soft robot captures a falling ball and locks it in a box. f A square-shaped soft robot captures a rolling ball and then locks it difﬁcult to fold into an ideal closed-form than the shorter edges. Thus, a larger radius was needed to increase the tensile stress when folding. As shown in Fig. S14, a ske- leton radius of 12.5 mm was chosen to allow the bottom of the long side to fold naturally, and the voltage required to unfold was increased from 4.1 to 5 kV, allowing a complete 3D square to be assembled (Movie S10). square-shaped soft robots. The corresponding planar structure designs are presented in Figs. S13 and S14. As shown in Fig. Results and discussion As shown in Movie S5, the speed of the robot on the zigzag surface with a tilt angle of 10° was lower due to pronounced surface slip- page. On the other hand, the 30° angle of inclination formed a wide serration, which hindered bipedal actua- tion and reduced the speed. p The actuation voltage also affected the crawling speed of the soft robot. On P600 sandpaper, the soft robot crawled at speeds of 1.71, 3.28, and 4.09 mm/s at actuation voltages of 3, 4, and 5 kV (Movie S6). The soft robot clearly exhibited a larger deformation angle at higher voltages, with one step moving a longer distance. Soft robots can not only crawl on rough surfaces but also carry cargo. The motion status of a robot with 1 and 2 g loads is shown in Fig. 3f and Movie S7. The soft robot weighs 2.94 g and could carry up to 2.00 g while crawling, although its speed decreased from 4.09 to 2.80 mm/s. We also investigated the effect of different power on/off frequencies on the walking state of the robots. To Considering the signiﬁcant deformation and large actuation forces of DE-based soft actuators, they are ideal for developing complex 3D soft robots. Inspired by ori- gami technology, these soft actuators were used to create two origami 3D soft robots with different shapes that can switch between 2D and 3D structures. Figure 4a shows expanded multilayered diagrams of the 3D pyramid- and Page 8 of 11 Sun et al. Microsystems & Nanoengineering (2022) 8:37 1 2 3 4 5 6 3 2 1 6 5 4 2 cm Sandpaper Sandpaper 1 2 3 4 5 6 a e f 2 cm b 0 5 10 15 Maximum loading weight (g) Sandpaper (P) None 400 240 120 6.0 10.0 13.5 14.5 d c 2 cm Without sandpaper P120 sandpaper 14.5g 2 cm 6.0g 1 cm 1 cm Fig. 5 Demonstrations of the grasping and locking functions of the origami-inspired 3D soft robot acting on static and dynamic objects. a A pyramid-shaped soft gripper captures a static ball and transports it to a beaker. b Sandpaper placed on the inside of the soft gripper. c Sandpaper placed on the inside of the soft gripper. d Maximum gripping weight of the soft gripper with different grit sandpapers. e A square-shaped soft robot captures a falling ball and locks it in a box. Results and discussion 4b, the original shape of the origami- inspired 3D soft robot is a standard pyramid, and the pyramid-shaped 3D structure consists of four triangular actuators. Each triangular face of the pyramid can be controlled independently to open into a 2D planar structure (Movie S9). Similarly, as shown in Fig. 4c, the square-shaped 3D soft robot consists of ﬁve rectangular actuators that can be programmably actuated. Unlike the pyramid, the planar structure of the rectangular robot was not centrosymmetric, and it had a longer side that needed to withstand more gravity. The longer edges were more Soft robots for object manipulation are another important application in the ﬁeld of soft robotics. The pyramid robot was transformed into a pyramid-shaped soft gripper (Fig. 5a). The back side of the pyramid-shaped gripper was attached to a rolled PET stick. The pyramid-shaped soft gripper (2.44 g) could transfer spheres (2.73 g) from a petri Page 9 of 11 Sun et al. Microsystems & Nanoengineering (2022) 8:37 dish to a beaker with a ﬁnger-like grasping process. As shown in Movie S11, the soft gripper opened quickly after the actuation voltage was applied. Then, after the power was turned off, it took 2–3 s for the “ﬁnger” to completely close. The robot could pick up ping pong balls with weights up to 6.0 g. We improved the gripping ability of the soft gripper by placing rough sandpaper on the inside of the gripper. The weight of the ping pong ball was continuously increased by ﬁlling it with water, and the gripping experiment was per- formed in weight steps of 0.5 g. As expected (Fig. 5c, d and Movie S12), the gripping ability improved due to the roughness of the sandpaper: 10.0 g for P400, 13.5 g for P240, and 14.5 g for P120. In addition to the gripper-shaped manipulator, the square-shaped soft robot can be fabricated as a box to capture static and dynamic objects (Movie S13). The ball in Fig. 5e fell vertically, and the ball in Fig. 5f was rolled horizontally from the right side. Figure 5e illustrates the entire process of locking a falling ball. The top lid of the box was independently actuated and opened; after the ball fell into the square, the lid closed and locked the ball after stopping the actuation voltage. Fabrication of origami robots In this work, we developed DE-based soft actuators with stable folding and unfolding functions and designed and fabricated 3D soft robots based on 3D origami folding. The soft actuator consists of a VHB4910 elastomer, which acts as the dielectric layer, and a PET ﬁlm, which acts as the ﬂexible substrate and reinforcement layer. The relationship between the semicircular radius of the actuation region and the original bending angle of the soft actuator was inves- tigated. A triangular soft actuator with a 120° bending angle was suitable for assembling 3D pyramid-shaped soft robots, while a rectangular actuator with a 90° bending angle was used to construct a crawling soft robot and a square- shaped 3D soft robot. The stable structure of the soft actuator after 100 cycles ensured structural stability during 3D construction and durability during long-term applica- tions of the 3D soft robots. For example, a crawling robot with rectangular soft actuators demonstrated a stable crawling ability on different grit papers and can carry cargo while walking in a speciﬁc direction. The origami-inspired 3D pyramid- and square-shaped soft robots had stable The origami robots were designed with CAD software, and the PET ﬂexible frame and reinforcement layers were made with a laser cutter. The robots were fabricated by the same process as the soft actuators, but a wire was connected to each actuation region to serve as the positive pole. The negative pole was connected to the actuation region with carbon grease (AMKE G-660A) before it was adhered to the soft substrate. Finally, a wire was used as the common negative electrode. Actuation and deformation tests The fabricated soft actuators were connected to a high- voltage DC power supply. When a certain voltage was applied, the soft actuator straightened or deformed. During the cycling test, the triangular and rectangular actuators were continuously charged and discharged to fully deform and return to their initial states at voltages of 5.5 and 4.1 kV, respectively. The cycles were repeated 100 times. All defor- mation processes were recorded with a camera (SONY). Movement characterization Two high-voltage DC power supplies were used as actuation sources to control the front and back feet of the robot. The whole crawling process was recorded using a camera (SONY). Materials and methods Fabrication of the soft actuator First, the PET ﬂexible ﬁlms, including 0.1 mm-thick substrate layers and 0.25 mm-thick reinforcement layers, were cut into speciﬁc shapes with a laser cutting machine (Mintron MC-3020) based on a pattern designed in AutoCAD. The VHB4910 elastomer (3 M, 60 mm × 60 mm) was stretched to 400% × 400% using a pre- stretching tool. The prestretched ﬁlm was ﬁxed using an acrylic ﬁxation frame. Then, the PET ﬂexible substrates and reinforcement layers with preconnected electrodes were ﬁxed to the center of the actuator. The skeletonized area of the PET ﬁlm was coated with carbon grease (AMKE G-660A). Finally, the soft actuator was obtained after it was cut and removed from the ﬁxation frame. g This work is sponsored by the Regional Joint Fund of the National Science Foundation of China (Grant No. U21A20492), the National Key R&D Program of China (Grant No. 2018YFB0407102), the City University of Hong Kong (Grant Nos. Results and discussion Figure 5f demonstrates the full process by which the square-shaped soft robot captured and locked a small ball that rolled in from the side. The long side of the rectangle was actuated independently, and it unfolded rapidly (2 s) with an applied voltage of 5 kV. After the ball rolled into the rectangular box from the right side, the long side was closed to lock the ball. The whole process took only 8 s (Movie S14). These results demonstrate the unlimited potential of origami-inspired soft robots with dielectric elastomer actuators, which have considerable advantages for multifunctional ﬁeld applications in the ﬁeld of soft robotics. structures and could grasp and lock 3D objects. The 3D design of the assembly could also be more complex and versatile, and the DE actuator, with its advantages of a fast response time, light weight, and high durability, offers new possibilities for the development of origami soft robots. This work provides a good method for the structural and functional design of origami soft robots. Author details 1 22. Cai, G., Ciou, J.-H., Liu, Y., Jiang, Y. & Lee, P. S. Leaf-inspired multiresponsive MXene-based actuator for programmable smart devices. Sci. Adv. 5, eaaw7956 (2019). 1State Key Laboratory of Electronic Thin Films and Integrated Devices, School of Optoelectronic Science and Engineering, University of Electronic Science and Technology of China (UESTC), Chengdu 610054, People’s Republic of China. 2Chongqing Key Laboratory of Materials Surface & Interface Science, Chongqing Co-Innovation Center for Micro/Nano Optoelectronic Materials and Devices, Micro/Nano Optoelectronic Materials and Devices International Science and Technology Cooperation Base of China, School of Materials Science and Engineering, Chongqing University of Arts and Sciences, Chongqing 402160, People’s Republic of China. 3Department of Biomedical 23. Mu, J. et al. Origami-inspired active graphene-based paper for programmable instant self-folding walking devices. Sci. Adv. 1, e1500533 (2015). 24. Ling, Y. et al. Laser‐induced graphene for electrothermally controlled, mechanically guided, 3D assembly and human–soft actuators interaction. Adv. Mater. 32, 1908475 (2020). 25. Li, J. et al. Photothermal actuators: Photothermal bimorph actuators with in- built cooler for light mills, frequency switches, and soft robots. Adv. Funct. Mater. 29, 1970184 (2019). Engineering, City University of Hong Kong, Hong Kong SAR 999077, People’s Republic of China 26. Cheng, Y., Lu, H., Lee, X., Zeng, H. & Priimagi, A. Soft actuators: Kirigami‐based light‐induced shape‐morphing and locomotion. Adv. Mater. 32, 2070047 (2020). References 32. Liu, Z. et al. Somatosensitive ﬁlm soft crawling robots driven by artiﬁcial muscle for load carrying and multi-terrain locomotion. Mater. Horiz. 8, 1783–1794 (2021). 32. Liu, Z. et al. Somatosensitive ﬁlm soft crawling robots driven by artiﬁcial muscle for load carrying and multi-terrain locomotion. Mater. Horiz. 8, 1783–1794 (2021). 1. Cianchetti, M., Laschi, C., Menciassi, A. & Dario, P. Biomedical applications of soft robotics. Nat. Rev. Mater. 3, 143–153 (2018). 1. Cianchetti, M., Laschi, C., Menciassi, A. & Dario, P. Biomedical applications of soft robotics. Nat. Rev. Mater. 3, 143–153 (2018). 2. Hann, S. Y., Cui, H., Nowicki, M. & Zhang, L. G. 4D printing soft robotics for biomedical applications. Addit. Manuf. 36, 101567 (2020). 33. Chen, S., Pang, Y., Cao, Y., Tan, X. & Cao, C. Soft robotic manipulation system capable of stiffness variation and dexterous operation for safe human- machine interactions. Adv. Mater. Technol. 6, 2100084 (2021). 2. Hann, S. Y., Cui, H., Nowicki, M. & Zhang, L. G. 4D printing soft robotics for biomedical applications. Addit. Manuf. 36, 101567 (2020). biomedical applications. Addit. Manuf. 36, 1015 3. Zhalmuratova, D. & Chung, H.-J. Reinforced gels and elastomers for biome- dical and soft robotics applications. ACS Appl. Polym. Mater. 2, 1073–1091 (2020). 34. Dou, W. et al. Soft robotic manipulators: Designs, actuation, stiffness tuning, and sensing. Adv. Mater. Technol. 6, 2100018 (2021). 35. Li, D., Yao, K., Gao, Z., Liu, Y. & Yu, X. Recent progress of skin-integrated electronics for intelligent sensing. Light.: Adv. Manuf. 2, 4 (2021). 4. Rich, S. I., Wood, R. J. & Majidi, C. Untethered soft robotics. Nat. Electron. 1, 102–112 (2018). 5. Zhakypov, Z., Mori, K., Hosoda, K. & Paik, J. Designing minimal and scalable insect-inspired multi-locomotion millirobots. Nature 571, 381–386 (2019). 36. Wang, L. et al. A metal-electrode-free, fully integrated, soft triboelectric senso array for self-powered tactile sensing. Microsyst. Nanoeng. 6, 59 (2020). 37. Liu, H. et al. An epidermal sEMG tattoo-like patch as a new human-machine i t f f ti t ith l f i Mi t N 6 16 (2020) 37. Liu, H. et al. An epidermal sEMG tattoo-like patch as a new human-machine interface for patients with loss of voice. Microsyst. Nanoeng. 6, 16 (2020). 6. Li, G. et al. Self-powered soft robot in the Mariana Trench. Nature 591, 66–71 (2021). 7. Fusco, S. et al. An integrated microrobotic platform for on-demand, targeted therapeutic interventions. Adv. Mater. Received: 26 November 2021 Revised: 24 January 2022 Accepted: 13 February 2022 Received: 26 November 2021 Revised: 24 January 2022 Accepted: 13 February 2022 Received: 26 November 2021 Revised: 24 January 2022 Accepted: 13 February 2022 30. Jia, T. et al. Moisture sensitive smart yarns and textiles from self‐balanced silk ﬁber muscles. Adv. Funct. Mater. 29, 1808241 (2019). 31. Wang, R. et al. Tensile and torsional elastomer ﬁber artiﬁcial muscle by entropic elasticity with thermo-piezoresistive sensing of strain and rotation by a single electric signal. Mater. Horiz. 7, 3305–3315 (2020). Acknowledgements h k d This work is sponsored by the Regional Joint Fund of the National Science Foundation of China (Grant No. U21A20492), the National Key R&D Program of China (Grant No. 2018YFB0407102), the City University of Hong Kong (Grant Nos. Page 10 of 11 Sun et al. Microsystems & Nanoengineering (2022) 8:37 Sun et al. Microsystems & Nanoengineering (2022) 8:37 Sun et al. Microsystems & Nanoengineering (2022) 8:37 9667221, 9680322), the Research Grants Council of the Hong Kong Special Administrative Region (Grant No. 21210820, 11213721), the Shenzhen Science and Technology Innovation Commission (Grant No. JCYJ20200109110201713), the Natural Science Foundation of Chongqing Municipality (Grant No. cstc2019jcyjjqX0021), the Science and Technology Innovation Leading Talents Program of Chongqing Municipality (No:T04040012) and Science and Technology of Sichuan Province (Grant No. 2020YFH0181), the National Natural Science Foundation of China (NSFC) (Grant Nos. U21A20492, 62122002). This work was also sponsored by the Sichuan Province Key Laboratory of Display Science and Technology, and Qiantang Science & Technology Innovation Center. M.W. thanks the Academic Support Program for PhD students supported by UESTC. 15. Rus, D. & Tolley, M. T. Design, fabrication and control of origami robots. Nat. Rev. Mater. 3, 101–112 (2018). 16. Rus, D. & Sung, C. Spotlight on origami robots. Sci. Robot. 3, eaat0938 (2018). 17. Kotikian, A. et al. Untethered soft robotic matter with passive control of shape morphing and propulsion. Sci. Robot. 4, eaax7044 (2019). morphing and propulsion. Sci. Robot. 4, eaax7044 (2019). 18. Miao, L. et al. 3D temporary‐magnetized soft robotic structures for enhanced energy harvesting. Adv. Mater. 33, 2102691 (2021). energy harvesting. Adv. Mater. 33, 2102691 (2021). 19. Kim, B. H. et al. Three-dimensional electronic microﬂiers inspired by wind- dispersed seeds. Nature 597, 503–510 (2021). 20. Ryu, J. et al. Paper robotics: Self‐folding, gripping, and locomotion. Adv. Mater. Technol. 5, 1901054 (2020). 21. Boyvat, M., Koh, J.-S. & Wood, R. J. Addressable wireless actuation for multijoint folding robots and devices. Sci. Robot. 2, eaan1544 (2017). Conﬂict of interest h h d l The authors declare no competing interests. 27. Shintake, J., Cacucciolo, V., Shea, H. & Floreano, D. Soft biomimetic ﬁsh robot made of dielectric elastomer actuators. Soft Robot. 5, 466–474 (2018). Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41378-022-00363-5. Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41378-022-00363-5. 28. Gu, G., Zou, J., Zhao, R., Zhao, X. & Zhu, X. Soft wall-climbing robots. Sci. Robot. 3, eaat2874 (2018). 29. Zou, M. et al. Progresses in tensile, torsional, and multifunctional soft actuators. Adv. Funct. Mater. 31, 2007437 (2021). Sun et al. Microsystems & Nanoengineering (2022) 8:37 49. Qiu, Y., Zhang, E., Plamthottam, R. & Pei, Q. Dielectric elastomer artiﬁcial muscle: Materials innovations and device explorations. Acc. Chem. Res. 52, 316–325 (2019). 47. Li, D. et al. Bioinspired ultrathin piecewise controllable soft robots. Adv. Mater. Technol. 6, 2001095 (2021). 48. Lu, X., Wang, K. & Hu, T. Development of an annelid-like peristaltic crawling soft robot using dielectric elastomer actuators. Bioinspiration Biomim. 15, 046012 (2020). 49. Qiu, Y., Zhang, E., Plamthottam, R. & Pei, Q. Dielectric elastomer artiﬁcial muscle: Materials innovations and device explorations. Acc. Chem. Res. 52, 316–325 (2019). 50. Chen, D. & Pei, Q. Electronic muscles and skins: A review of soft sensors and actuators. Chem. Rev. 117, 11239–11268 (2017). 50. Chen, D. & Pei, Q. Electronic muscles and skins: A review of soft sensors and actuators. Chem. Rev. 117, 11239–11268 (2017). 49. Qiu, Y., Zhang, E., Plamthottam, R. & Pei, Q. Dielectric elastomer artiﬁcial muscle: Materials innovations and device explorations. Acc. Chem. Res. 52, 316–325 (2019). 50. Chen, D. & Pei, Q. Electronic muscles and skins: A review of soft sensors and actuators. Chem. Rev. 117, 11239–11268 (2017). References 26, 952–957 (2013). 38. Li, D. et al. Miniaturization of mechanical actuators in skin-integrated elec- tronics for haptic interfaces. Microsyst. Nanoeng. 7, 85 (2021). 8. Le, H. M., Do, T. N. & Phee, S. J. A survey on actuators-driven surgical robots. Sens. Actuators A: Phys. 247, 323–354 (2016). 39. Wu, M. et al. Self-powered skin electronics for energy harvesting and healthcare monitoring. Mater. Today Energy 21, 100786 (2021). g y gy 40. Liu, Y. et al. Epidermal electronics for respiration monitoring via thermo- sensitive measuring. Mater. Today Phys. 13, 100199 (2020). 9. Ranzani, T., Gerboni, G., Cianchetti, M. & Menciassi, A. A bioinspired soft manipulator for minimally invasive surgery. Bioinspiration Biomim. 10, 035008 (2015). 41. Yu, X. et al. Skin-integrated wireless haptic interfaces for virtual and aug- mented reality. Nature 575, 473–479 (2019). 10. Arezzo, A. et al. Total mesorectal excision using a soft and ﬂexible robotic arm: A feasibility study in cadaver models. Surgical Endosc. 31, 264–273 (2016). 42. Song, E. et al. Miniaturized electromechanical devices for the characterization of the biomechanics of deep tissue. Nat. Biomed. Eng. 5, 759–771 (2021). 11. Mintchev, S., Salerno, M., Cherpillod, A., Scaduto, S. & Paik, J. A portable three- degrees-of-freedom force feedback origami robot for human-robot interac- tions. Nat. Mach. Intell. 1, 584–593 (2019). 43. Wong, T. H. et al. Tattoo-like epidermal electronics as skin sensors for human machine interfaces. Soft Sci. 1, 10 (2021). 12. Zhao, W. et al. Vat photopolymerization 3D printing of advanced soft sensors and actuators: From architecture to function. Adv. Mater. Technol. 6, 2001218 (2021). 44. Wang, C. et al. Adaptive soft robots: Soft ultrathin electronics innervated adaptive fully soft robots. Adv. Mater. 30, 1870087 (2018). 45. Saikumar, J. et al. Inducing different severities of traumatic brain injury in Drosophila using a piezoelectric actuator. Nat. Protoc. 16, 263–282 (2020). 13. Ning, X. et al. Mechanically active materials in three-dimensional mesos- tructures. Sci. Adv. 4, eaat8313 (2018). 14. Morgan, J., Magleby, S. P. & Howell, L. L. An approach to designing origami- adapted aerospace mechanisms. J. Mech. Des. 138, 052301 (2016). 46. He, Q. et al. Electrically controlled liquid crystal elastomer-based soft tubular actuator with multimodal actuation. Sci. Adv. 5, eaax5746 (2019). 46. He, Q. et al. Electrically controlled liquid crystal elastomer-based soft tubular actuator with multimodal actuation. Sci. Adv. 5, eaax5746 (2019). Page 11 of 11
https://openalex.org/W4392503863	https://link.springer.com/content/pdf/10.1007/s11063-024-11558-4.pdf	English	null	HF-YOLO: Advanced Pedestrian Detection Model with Feature Fusion and Imbalance Resolution	Neural processing letters/Neural Processing Letters	2,024	cc-by	8,994	Lihu Pan1 · Jianzhong Diao1 · Zhengkui Wang2 · Shouxin Peng1 · Cunhui Zhao3 Accepted: 11 February 2024 / Published online: 6 March 2024 © The Author(s) 2024 Jianzhong Diao, Zhengkui Wang, Shouxin Peng and Cunhui Zhao have contributed equally to this work. Neural Processing Letters (2024) 56:90 https://doi.org/10.1007/s11063-024-11558-4 Neural Processing Letters (2024) 56:90 https://doi.org/10.1007/s11063-024-11558-4 1 Introduction Pedestrian detection is a widely studied object detection problem that ﬁnds extensive appli- cations in domains such as intelligent video surveillance [1], intelligent transportation [2], and autonomous driving systems [3, 4]. It also serves as a fundamental technology support- ing tasks like pedestrian pose estimation [5, 6] and pedestrian re-identiﬁcation [7, 8]. The accuracy of pedestrian detection algorithms directly impacts the performance of these tasks. In real-world, the diversity in pedestrian poses, varying scales, and environmental factors, including occlusions, present signiﬁcant challenges to detection algorithms, necessitating robust and precise solutions. Traditional pedestrian detection methods, such as Viola–Jones [9], histogram of oriented gradients (HOG) [10], and scale-invariant feature transform (SIFT) [11], rely on manually designedfeaturesandtemplatematchingtechniques.Whilethesemethodsarestraightforward to implement, their performance and generalization capacity are limited by the constraints of handcrafted feature engineering, often resulting in suboptimal outcomes when faced with complex scenarios. In recent years, considerable progress has been made in object detection techniques. State-of-the-art algorithms, such as multi-anchor faster R-CNN [12], multiscale attention fusion [13], and multimodaldetectors [14], leverage convolutionalneuralnetworks (CNNs) to enhance pedestrian detection performance by improving accuracy. Among these approaches, the YOLO (you only look once) series has consistently maintained a prominent position, striking a favorable trade-off between detection precision and processing speed. However, it is noteworthy that YOLO models exhibit certain limitations that hinder their performance in pedestrian detection. Speciﬁcally, these models face challenges in effectively handling the diverse scales of pedestrians and addressing the imbalance between positive and negative samples. Primarily, the inherent ﬂexibility and variability in human poses give rise to pedestrians appearing at various scales within images (Fig.1a). Consequently, accurately detecting pedestrians across these varying scales becomes a complex task. Furthermore, scenarios characterized by dense pedestrian crowds introduce additional difﬁculties due to occlusions occurring between individuals (Fig.1b). Such occlusions obstruct the complete visibility of pedestrians, further complicating their accurate detection by YOLO models. In addition to scale and occlusion challenges, the imbalance between positive and negative sam- ples during the target regression process adversely impacts the precision of object localization by YOLO models. This imbalance skews the learning process towards the dominant class, resulting in suboptimal performance when localizing pedestrians. Hence, the task of effec- tively addressing these limitations and devising resilient solutions continues to be a pivotal and captivating area of research. Abstract Pedestrian detection is crucial for various applications, including intelligent transportation and video surveillance systems. Although recent research has advanced pedestrian detec- tion models like the YOLO series, they still face limitations in handling diverse pedestrian scales, leading to performance challenges. To address these issues, we propose HF-YOLO, an advanced pedestrian detection model. HF-YOLO tackles the complexities of pedestrian detection in complex scenes by addressing scale variations and occlusions among pedestri- ans. In the feature fusion stage, our algorithm leverages both shallow localization information and deep semantic information. This involves fusing P2 layer features and adding a high- resolution detection layer, signiﬁcantly improving the detection of small-scale pedestrians and occluded instances. To enhance feature representation, HF-YOLO incorporates the HardSwish activation function, introducing more non-linear factors and strengthening the model’s ability to represent complex and discriminative features. Additionally, to address regression imbalance, a balance factor is introduced to the CIoU loss function. This mod- iﬁcation effectively resolves the imbalance problem and enhances pedestrian localization accuracy. Experimental results demonstrate the effectiveness of our proposed algorithm. HF- YOLO achieves notable improvements, including a 3.52% increase in average precision, 1 School of Computer Science and Technology, Taiyuan University of Science and Technology, 63 Waliu Rd, Taiyuan 030024, Shanxi, China 2 ICT Cluster, Singapore Institute of Technology, 10 Dover Drive, Singapore 139651, Singapore 3 Jingying Shuzhi Technology Co.,Ltd., 103 Changzhi Rd, Taiyuan 030012, Shanxi, China 3 Jingying Shuzhi Technology Co.,Ltd., 103 Changzhi Rd, Taiyuan 030012, Shanxi, China 123 123 123 L. Pan et al. Page 2 of 20 90 a 1.35% boost in accuracy, and a 4.83% enhancement in recall. Moreover, the algorithm maintainsreal-timeperformancewithadetectiontimeof8.5ms,meetingthestringentrequire- ments of real-time applications. Keywords Pedestrian detection · Object detection · Activation function · YOLO · Loss function 2 Related Work Currently, object detection algorithms can be broadly classiﬁed into two major categories: Anchor-based algorithms and Anchor-free algorithms. Anchor-based algorithms encompass both two-stage approaches (e.g., RCNN [15], Fast R-CNN [16], Faster R-CNN [17]) and one- stage approaches (e.g., SSD [18] and YOLO series [19–21]). In Anchor-based algorithms, a set of predeﬁned anchor boxes are generated on the image at different scales and aspect ratios. These anchor boxes serve as reference regions for detecting objects. Convolutional neural networks are employed to extract features from the image, followed by classiﬁcation and regression operations performed on each anchor box. Finally, a non-maximum suppression algorithm is applied to ﬁlter out redundant detection boxes and obtain the ﬁnal detection results. In contrast, Anchor-free methods, such as CenterNet [22] and FCOS [23], adopt a different paradigm. These approaches treat objects as single points during model construction. After feature extraction, speciﬁc points (e.g., center points) are selected as key representations for object detection. The classiﬁcation and regression tasks are decoupled into separate branches, allowing for independent prediction of object presence and spatial localization. Subsequently, a non-maximum suppression technique is applied to eliminate overlapping detections and produce the ﬁnal detection results. While Anchor-free methods alleviate the need for anchor box generation, they often require a larger number of candidate points during detection. Con- sequently, these methods necessitate more extensive training data and sophisticated training strategies to achieve optimal detection performance in various scenarios. The development of object detection has progressed from traditional methods to the era of deep learning-based detection. Early object detection algorithms relied on handcrafted image feature descriptors tailored for speciﬁc tasks, aiding in discerning target positions or categories. However, these manually crafted features lacked effective image representa- tions, necessitating the design of more intricate feature representations to obtain high-quality image features. As detection techniques advanced, methods rooted in deep learning emerged, leveraging convolutional neural networks (CNNs) and akin methodologies to acquire robust and sophisticated feature representations. Yu et al. extracted multiple features from input images and an image database. They constructed multiple hypergraph Laplacian operators and formulated sparse codes [24]. Simultaneously, they preserved the locality of the obtained sparse codes by employing manifold learning on the hypergraph. Cao et al. introduced a dis- criminative region of interest (RoI) pooling scheme that samples from various sub-regions of a proposal and performs adaptive weighting to obtain discriminative features [25]. Yu et al. 1 Introduction 123 123 90 Page 4 of 20 L. Pan et al. 90 1 Introduction Resolving these challenges holds paramount importance for advancing the ﬁeld of pedestrian detection and enabling the development of more accurate and reliable systems. 123 123 123 Page 3 of 20 90 HF-YOLO: Advanced Pedestrian Detection... Fig. 1 a and b showcase the scenarios of pedestrians in real-world environments. In Figure (a), the red and green boxes depict pedestrians of varying scales. In Figure (b), the blue and orange boxes illustrate instances of occlusion. (Color ﬁgure online) Fig. 1 a and b showcase the scenarios of pedestrians in real-world environments. In Figure (a), the red and green boxes depict pedestrians of varying scales. In Figure (b), the blue and orange boxes illustrate instances of occlusion. (Color ﬁgure online) To overcome these challenges, this paper presents a groundbreaking model called HF- YOLO, which addresses the limitations in pedestrian detection performance. Our model introduces innovative techniques to enhance the accuracy of detecting pedestrians with small scalesandocclusions.Itachievesthisbyleveragingfeaturefusionacrossmultiplehierarchical levels, enabling the integration of high-resolution features to capture both high-level semantic information and low-level localization cues. Additionally, a dedicated small object detection layer is introduced to improve the accuracy of detecting pedestrians with varying scales and occlusions. The contributions of our work can be summarized as follows: • We propose HF-YOLO, a novel pedestrian detection model that incorporate the HardSwish activation function within the convolutional blocks of our model, enhanc- ing its feature representation capability and introducing greater non-linearity. • We employ feature fusion across multiple hierarchical levels. This novel approach effectively tackles the challenges associated with small-scale pedestrian detection and occlusions. • We employ feature fusion across multiple hierarchical levels. This novel approach effectively tackles the challenges associated with small-scale pedestrian detection and occlusions. • To address the issue of imbalanced high- and low-quality samples in the regression loss function, we introduce a balancing factor. This factor redirects the model’s focus towards accurate regression for high-quality samples, overcoming the bias introduced by the larger number of low-quality samples. • Extensive evaluations are conducted, comparing our proposed model against six base- lines. The results demonstrate the superior performance of our model, surpassing existing approaches in terms of detection accuracy and reliability. The paper’s structure is as follows: In Sect.2, we review prior research on pedestrian detection. Section3 provides a detailed explanation of the HF-YOLO model. In Sect.4, we present the dataset used and discuss the experimental results. Finally, in Sect.5, we conclude the paper. 123 2 Related Work designed a novel ﬁne-grained image recognition framework [26], introducing a feature selection module to address noise and high dimensionality in features. They incorporated weight vectors with sparse constraints and an improved "RELU" operator. The feature selec- tion model achieved higher accuracy and a larger compression ratio. Woo et al. optimized the network from both channel and spatial perspectives, further enhancing the model’s feature extraction effectiveness in both dimensions [27]. Lv et al. proposed the RTDETR model, which utilizes Transformers to process the features extracted from the last layer of the back- bone, enhancing the model’s ability to differentiate features among various objects [28]. While signiﬁcant advancements have been made in the ﬁeld of general object detection algorithms, their direct application to pedestrian detection has proven to be unsatisfactory [29]. Zhang et al. proposed a novel occlusion-aware R-CNN detection algorithm [30], build- ing upon the faster R-CNN framework. This approach incorporates an occlusion processing unit, which effectively captures ﬁve distinct partial features of pedestrians. These part-level 123 Page 5 of 20 HF-YOLO: Advanced Pedestrian Detection... 90 features are subsequently combined with the global features of the target, employing a weighted summation technique to obtain a comprehensive pedestrian detection outcome. Liu et al. introduced a density prediction module and an adaptive non-maximum suppression (NMS) method in order to address the challenges associated with pedestrian detection [31]. The adaptive NMS method dynamically adjusts the NMS threshold based on the density of the objects being detected. In scenarios with dense pedestrian presence, the NMS threshold is heightened to ensure a high recall rate. Conversely, in cases with sparser object distribu- tions, the NMS threshold is lowered to alleviate the issue of redundant detection boxes. This adaptive approach effectively resolves the predicament of losing highly overlapped targets or generating false positives due to a ﬁxed threshold. Chu et al. proposed a multi-instance prediction method to handle severe overlap among multiple targets [32]. Instead of predicting a single instance, their method predicts a set of highly overlapped instances for each proposal box. This approach reduces the false negative rate by designing a loss function that mini- mizes the distance between predicted boxes and ground truth boxes, supervising the learning of instance set prediction. Xia et al. argued that incorporating multi-scale information can improve the robustness of the network without losing information [33]. 2 Related Work They introduced multi-scale dilation residual modules into the backbone network to increase the receptive ﬁeld and capture more global and higher-levelsemanticfeatures.Lietal.utilizedanimprovedRes2Netasthebackbonenetwork to enhance the model’s multi-scale representation capability for pedestrians [34]. Addressing the limited ability of a single feature extraction block to extract semantic information at different levels, Wang et al. proposed Three ResNet Blocks [35]. This module integrates three different basic blocks, each of which extracts pedestrian information, to enhance the information ﬂow in the network structure and improve the accuracy of detection results. These related works demonstrate the efforts made to overcome the limitations and chal- lenges in pedestrian detection. The proposed methods introduce novel techniques, such as occlusion processing, adaptive NMS, and multi-instance prediction, to enhance the perfor- mance of pedestrian detection models. Additionally, advancements in backbone networks, such as Res2Net [36], further contribute to the improvement of multi-scale representation and feature extraction capabilities. 3.1 Model Overview The YOLO series models are a prominent category of one-stage object detection meth- ods. These models combine the tasks of object classiﬁcation and localization regression by utilizing anchor boxes. This integration allows YOLO models to achieve high efﬁciency, ﬂex- ibility, and good generalization performance, making them highly popular in both academia and industry.Wang et al. proposed a real-time detection model called YOLOv7 [37]. It is an advanced version of the YOLO series of real-time object detection models. Building on the success of its predecessor, further optimization has been introduced to improve its detection performance. To address the challenges associated with pedestrian detection in complex environments, we propose the HF-YOLO model, which builds upon the foundation of YOLOv7. The HF- YOLO architecture, illustrated in Fig.2, comprises three essential components: Backbone, Neck, and Head. 123 L. Pan et al. 90 Page 6 of 20 90 Page 6 of 20 90 Fig. 2 The HF-YOLO model structure mainly consists of three parts: backbone (feature extraction), neck (feature fusion), and head (detection) Fig. 2 The HF-YOLO model structure mainly consists of three parts: backbone (feature extraction), neck (feature fusion), and head (detection) Fig. 2 The HF-YOLO model structure mainly consists of three parts: backbone (feature extraction), neck (feature fusion), and head (detection) The backbone component incorporates encapsulated convolutional blocks (CBH), Max- Pooling, ELAN, and SPP modules for efﬁcient feature extraction, as depicted in Fig.3. CBH is deﬁned as a sequence of operations: The backbone component incorporates encapsulated convolutional blocks (CBH), Max- Pooling, ELAN, and SPP modules for efﬁcient feature extraction, as depicted in Fig.3. CBH is deﬁned as a sequence of operations: (1) Xout = HardSwish(BN(Conv(Xin))) (1) where Xin and Xout respectively represent the feature maps of the input and output. apply a 3×3 convolution operation to Xin, followed by batch normalization (BN), and subsequently an activation function (HardSwish). Speciﬁcally, the ELAN module combines concepts from VoVNet [38] and CSPNet [39], utilizing a gradient path strategy to control the shortest and longest gradient paths in each layer. This enables different computational units to learn diverse information, maximizing parameter utilization efﬁciency. Additionally, the ELAN module ensures stable model learn- ing by directly propagating information to update the weights of each computational unit, thereby mitigating degradation issues during training. Its gradient path design strategy pro- Page 7 of 20 90 HF-YOLO: Advanced Pedestrian Detection... Fig. 3.1 Model Overview 3 CBH consists of the operations convolution (Conv), batch normalization (BN), and the HardSwish activation function. The ELAN module employs a multi-branch structure, incorporating residual connections after each convolutional operation. This approach alleviates the issue of gradient vanishing associated with the increase in model depth. SPP module is overall composed of two parallel branch structures. The ﬁrst part ﬁrst undergoes CBH operation, then goes through max pooling with kernel sizes of 5×5, 9×9, and 13×13, followed by a residual connection. The second part consists of a residual connection with CBH operation. Finally, these two parts are concatenated to obtain the output Fig. 3 CBH consists of the operations convolution (Conv), batch normalization (BN), and the HardSwish activation function. The ELAN module employs a multi-branch structure, incorporating residual connections after each convolutional operation. This approach alleviates the issue of gradient vanishing associated with the increase in model depth. SPP module is overall composed of two parallel branch structures. The ﬁrst part ﬁrst undergoes CBH operation, then goes through max pooling with kernel sizes of 5×5, 9×9, and 13×13, followed by a residual connection. The second part consists of a residual connection with CBH operation. Finally, these two parts are concatenated to obtain the output motes efﬁcient parameter utilization, enabling the network to achieve higher accuracy without the need for additional complex architectures. Spatial pyramid pooling (SPP) can generate ﬁxed-size outputs, effectively addressing the issue of repetitive feature extraction in convolutional neural networks, while also reducing computational costs. Following the backbone, the neck component focuses on feature fusion and informa- tion propagation. It integrates various techniques, such as feature concatenation, to combine features from different levels of the backbone. This facilitates the integration of low-level localization information and high-level semantic information, empowering the model to effectively handle pedestrians of diverse scales and occlusion levels. The Head of the HF-YOLO model consists of four detection layers, which perform object detectiononfeaturemapswithsizesof20×20,40×40,80×80,and160×160,respectively. These detection layers enable the model to capture pedestrians at different scales and generate precise detection results. By combining these components, the HF-YOLO model addresses the challenges of pedes- trian detection by enhancing feature extraction, facilitating feature fusion, and enabling accurate detection across various scales. 3.2 Optimizing Feature Representation with the HardSwish Activation Function In convolutional neural networks, the output of each layer is obtained by applying a linear transformation to the input from the previous layer. However, without an activation function, the network’s output remains a linear combination of the inputs, regardless of its depth. Activation functions play a crucial role in introducing non-linearity to the data, enabling the network to capture complex patterns and effectively represent non-linear mappings between input and output domains. By incorporating non-linear transformations, activation functions enhance the expressive power of the network, facilitating the learning of intricate and abstract representations. This, in turn, enables the network to effectively model and extract features from high-dimensional data. 123 123 L. Pan et al. 90 Page 8 of 20 90 Page 8 of 20 90 Fig. 4 The plotting of the Leaky ReLU and HardSwish activation functions with it’s non-monotonic bump for x less than 0 Fig. 4 The plotting of the Leaky ReLU and HardSwish activation functions with it’s non-monotonic bump for x less than 0 In model architectures, it is common to use Conv-BatchNorm-LeakyReLU (CBL) con- volutional blocks, where convolutional operations (Conv), batch normalization (BN), and activation functions (LeakyReLU) are encapsulated together. The inclusion of activation functions introduces non-linearity, allowing the model to learn and represent intricate fea- tures. The LeakyReLU activation function is an adaptation of ReLU that introduces a small slope for negative values, addressing the issue of vanishing gradients encountered in ReLU: LeakReLU(x) = x, x ≥0 0.1x, x < 0 (2) (2) However, due to its near-linear behavior, LeakyReLU may not be optimal for detecting complex patterns in intricate data. The HardSwish activation function is deﬁned as: However, due to its near-linear behavior, LeakyReLU may not be optimal for detecting complex patterns in intricate data. The HardSwish activation function is deﬁned as: HardSwish(x) = ⎧ ⎨ ⎩ 0, x ≤−3 x, x ≥3 x(x+3) 6 , otherwise (3) (3) The most prominent distinction between Hard-Swish and Leaky ReLU is the non- monotonic concavity when x is less than 0. As shown in Fig.4, the HardSwish [40] activation function undergoes a smoothing process, which enhances its smoothness and continuity. This characteristic facilitates gradient computation and optimization, mitigating problems such as gradient explosion and vanishing gradients. Consequently, it contributes to an accel- erated convergence rate of the model. 3.2 Optimizing Feature Representation with the HardSwish Activation Function Compared to LeakyReLU, HardSwish introduces a stronger non-linearity while maintaining computational efﬁciency, thereby amplifying the representational capacity of neural networks. Consequently, replacing the activation func- tion with HardSwish leads to the construction of the Conv-BatchNorm-HardSwish (CBH) convolutional block, which is used to build the ELAN and SPP modules. HF-YOLO: Advanced Pedestrian Detection... Page 9 of 20 90 90 Fig. 5 The features of {P2,P3,P4,P5} extracted from the backbone were fused from top to bottom Fig. 5 The features of {P2,P3,P4,P5} extracted from the backbone were fused from top to bottom 3.3 Fusion of High-Resolution Features The detection of pedestrians in an image presents challenges due to the inherent variability in their distances from the camera, resulting in diverse scales. This scale variability can lead to scale imbalance and impact the performance of detection algorithms. Additionally, occluded pedestrians often lack discriminative features, which can result in false positives or false negatives during the detection process. Detection algorithms leverage shallower feature maps forprecisespatialinformationanddeeperfeaturemapsforsemanticcontext.Byfusingfeature maps from different layers, it becomes possible to exploit the complementary characteristics of both shallow and deep features. This fusion strategy enables the algorithm to capture richer contextual information, achieve multi-scale receptive ﬁelds, enhance detection capacity for objects of varying scales and occluded targets, and ultimately improve object localization precision. During the feature extraction process, successive down-sampling operations are employed to derive informative feature representations. As the feature map’s resolution decreases, it encapsulates higher-level semantic information pertinent to the targets. However, challenges arise, particularly in scenarios involving occluded pedestrians, where multiple individuals might share a common feature representation. Consequently, this can lead to the exclusion of occluded targets, hampering detection accuracy. To bolster HF-YOLO’s detection performance in handling diverse object scales and occluded pedestrians, we enhance the feature fusion module, illustrated in Fig. 5. Initially, we augment the C3 layer features using operations like convolution and upsampling. These augmented features are then fused with the P2 layer features extracted from the backbone network, leveraging the Concatenation (Concat) operation. This fusion yields C2, C3, C4, with its formulation outlined as follows: C4 = Cat[U(C BH(P5)), C BH(P4)] (4) C3 = Cat[U(C BH(E(C4)), C BH(P3)] (5) C2 = Cat[U(C BH(E(C3)), C BH(P2)] (6) (4) ( ) (5) (6) (6) where Cat concatenates the feature maps along the channel dimension, C BH represents the Conv_BN_HardSwish operation, adjusting the number of channels. U enlarges the feature map to twice its original size. E represents the feature extraction operation. This fusion facilitates the propagation of high-level semantic information. where Cat concatenates the feature maps along the channel dimension, C BH represents the Conv_BN_HardSwish operation, adjusting the number of channels. U enlarges the feature map to twice its original size. E represents the feature extraction operation. This fusion facilitates the propagation of high-level semantic information. 90 Page 10 of 20 L. Pan et al. 90 Furthermore, we employ a bottom-up fusion process to propagate the localization infor- mation from the lower layers to the higher layers. 3.3 Fusion of High-Resolution Features This process generates N2, N3, N4, N5, where the features from the lower layers carry important positional details. Its expression is as follows: N2 = E(C2) (7) N3 = E(Cat[C BH(N2), C3]) (8) N4 = E(Cat[C BH(N3), C3]) (9) N5 = E(Cat[C BH(N4), P5]) (10) (10) Following two iterations of feature fusion operations, the amalgamation of high-level seman- tic information and low-level localization details serves to bolster the efﬁcacy of feature extraction for occluded targets. Following two iterations of feature fusion operations, the amalgamation of high-level seman- tic information and low-level localization details serves to bolster the efﬁcacy of feature extraction for occluded targets. Based on the fused feature maps, we construct a detection layer with a dimension of 160 × 160 to enhance the detection capability, particularly for small-sized targets. This increase in spatial resolution aids in capturing ﬁner details and improving localization accuracy. The fusion process described above is visually depicted in Fig.5. α is a weight parameter deﬁned as follows: α is a weight parameter deﬁned as follows: α is a weight parameter deﬁned as follows: α = v (1 −IoU) + v (14) (14) In comparison to other regression loss functions, CIoU loss has demonstrated notable advancements in terms of convergence speed and detection accuracy. However, one inherent limitation of CIoU loss is its neglect of the inherent imbalance within the regression samples. During the training process, there is often a scarcity of high-quality samples accompanied by an abundance of low-quality samples. As a result, the contribution of gradients derived from the former category to the overall regression gradient is diminished To overcome this In comparison to other regression loss functions, CIoU loss has demonstrated notable advancements in terms of convergence speed and detection accuracy. However, one inherent limitation of CIoU loss is its neglect of the inherent imbalance within the regression samples. In comparison to other regression loss functions, CIoU loss has demonstrated notable advancements in terms of convergence speed and detection accuracy. However, one inherent limitation of CIoU loss is its neglect of the inherent imbalance within the regression samples. During the training process, there is often a scarcity of high-quality samples accompanied by an abundance of low-quality samples. As a result, the contribution of gradients derived from the former category to the overall regression gradient is diminished. To overcome this challenge and further enhance the precision of pedestrian target localization, we propose the utilization of F-CIoU. F-CIoU introduces the IoU as a balancing factor to weigh the CIoU loss, as exempliﬁed by Equation 5, where β is assigned a value of 0.5. By incorporating F-CIoU, higher-quality samples with larger IoU values incur greater losses and correspondingly higher weights. Consequently, the model places increased emphasis on the regression of high-quality sam- ples, thereby promoting superior localization accuracy. The utilization of F-CIoU addresses the issue of imbalance in the regression samples, enabling the model to better optimize its performance by giving appropriate attention to different sample categories based on their quality. L F−C IoU = IoU β 1 −IoU + ρ2 b, bgt c2 + av (15) (15) Following the aforementioned enhancements, we have derived the HF-YOLO model. The HF-YOLO model integrates the proposed improvements, including the novel feature fusion module, the utilization of the HardSwish activation function, and the introduction of the F-CIoU loss function. 4 Experiment In this section, we will provide an overview of the dataset used and the experimental setup employed in our study. We will then introduce the evaluation metrics utilized to assess the performance of the proposed HF-YOLO model. Additionally, we will present the experi- mental evaluations conducted on activation functions and the bounding box regression loss function. Subsequently, we will compare the results obtained from our proposed model with existing models through comparative analysis. Finally, we will discuss the ablation experi- ments performed to investigate the individual contributions of different components in the HF-YOLO model. α is a weight parameter deﬁned as follows: These enhancements collectively contribute to the improved performance of the HF-YOLO model in handling objects of various scales and occluded pedestrians, ultimately leading to enhanced object detection accuracy. 3.4 Bounding Box Regression Loss Function The bounding box regression loss function plays a crucial role in object detection algorithms as it encompasses accurate localization of objects. By comparing the predicted bounding boxes with the ground truth boxes, the bounding box regression loss value is computed, allowing the model to continuously optimize and improve the accuracy of object localization. In object detection models, the complete IoU (CIoU) [41]metric is widely utilized for com- puting regression losses. The CIoU metric incorporates three essential geometric factors—the overlapping area, distance, and aspect ratio of the bounding box—into the loss calculation. This comprehensive metric provides a more accurate measure of the spatial discrepancy between predicted and ground truth bounding boxes. By considering these geometric fac- tors, CIoU facilitates a more precise evaluation of the localization accuracy, enabling the optimization of object detection models for improved precision and robustness. pp g p g This comprehensive metric provides a more accurate measure of the spatial discrepancy between predicted and ground truth bounding boxes. By considering these geometric fac- tors, CIoU facilitates a more precise evaluation of the localization accuracy, enabling the optimization of object detection models for improved precision and robustness. p j p p The formula for CIoU is as follows: The formula for CIoU is as follows: LC IoU = 1 −IoU + ρ2 b, bgt c2 + αv (11) (11) Where IoU represents the intersection over union of bounding boxes A and B, which is calculated using the following formula: Where IoU represents the intersection over union of bounding boxes A and B, which is calculated using the following formula: IoU = \|A ∩B\| \|A ∪B\| (12) (12) where b represents the predicted bounding box’s center coordinates, bgt represents the ground truth bounding box’s center coordinates, ρ2(b, bgt) represents the squared distance between the center points of the two boxes, c2 represents the squared diagonal length of the minimum enclosing rectangle of the two boxes. v is a parameter that measures the aspect ratio, w and h represent the width and height of the predicted box, and hgt and hgt represent the width and height of the ground truth box, deﬁned as follows: v = 4 π2 arctan wgt hgt −arctan w h 2 (13) (13) 123 123 Page 11 of 20 90 HF-YOLO: Advanced Pedestrian Detection... 90 α is a weight parameter deﬁned as follows: 4.1 Experimental Setup The experimental setup in this study is described in Table 1. In this study, we utilized the SGD optimizer for training the HF-YOLO model. The model was conﬁgured with an input image size of 640×640 pixels. The batch size is 64. The initial learning rate was set to 0.001„ and the cosine annealing algorithm is used to update the learning rate. The momentum factor 123 L. Pan et al. 90 Page 12 of 20 90 90 90 Page 12 of 20 L. Pan et al. Table 1 Experimental environment Name Version Operating System Ubuntu20.0 CPU Intel® Xeon® Glod 5230 CPU @2.20 GHz GPU NVIDIA GeForce RTX A4000 Python 3.8 Pytorch 1.11.0 CUDA CUDA11.3 of 0.9 was employed during optimization. The total training duration comprised 300 epochs, during which the model was iteratively updated and ﬁne-tuned using the training data. of 0.9 was employed during optimization. The total training duration comprised 300 epochs, during which the model was iteratively updated and ﬁne-tuned using the training data. 4.2 Datasets The dataset used in this study consisted of two main sources: the CrowdHuman dataset and the WiderPerson dataset. The CrowdHuman dataset [42] is composed of images primarily obtained from Google search. It comprises complex scenes with dense pedestrian crowds. The training set of this dataset contains approximately 470,000 instances, with an average of around 23 people per image. The annotation information includes three types: full body, visual body, and head. For this study, the full body annotations were selected for training the model. The WiderPerson dataset [43] is speciﬁcally designed for outdoor pedestrian detection. It consists of 13,382 images, and the annotations include ﬁve categories: pedestrian, cyclist, partially visible person, crowd, and ignore region. In this study, the ﬁrst three categories (pedestrian, cyclist, partially visible person) were combined and treated as the "person" class. To create the dataset for our experiments, we extracted a total of 16,000 images from the CrowdHuman and WiderPerson datasets. These images were then partitioned into three sub- sets: a training set, a validation set, and a testing set, following an 8:1:1 ratio. All experimental results were obtained in the test set. 4.4.1 Experimental Comparison of Activation Functions To ascertain the effectiveness of the HardSwish activation function, a comparative evalua- tion was performed, contrasting it with alternative activation functions, including SiLU [44], GELU [45], and LeakyReLU. The results of the experiments, presented in Table 2, unequivo- cally establish the superiority of HardSwish in terms of performance. These ﬁndings provide compelling evidence of its ability to introduce a higher level of non-linearity to the model, thereby augmenting its capacity to represent intricate patterns and complex relationships in the data. 4.3 Evaluation Metrics The performance evaluation of the model in this study encompasses four key metrics: preci- sion, recall, mean Average Precision (mAP), and detection time. Precision quantiﬁes the accuracy of positive predictions, serving as a measure of false detections. It is computed using the formula: Precision = T P T P + F P (16) (16) where TP represents the number of correctly detected objects by the model, and FP denotes the count of objects falsely detected by the model. where TP represents the number of correctly detected objects by the model, and FP denotes the count of objects falsely detected by the model. Recall, on the other hand, gauges the probability of identifying positive samples within the predicted results, indicating the extent of missed detections. The calculation is given by: Recall = T P T P + F N (17) (17) 123 123 Page 13 of 20 90 HF-YOLO: Advanced Pedestrian Detection... 90 Table 2 Comparison of experimental results of activation function Activation function mAP@0.5 (%) Precision (%) Recall (%) SiLU 77.83 85.55 65.79 GELU 77.93 83.73 66.14 LeakyReLU 78.08 84.90 66.88 HardSwish 78.70 86.59 66.03 Bold font is designated to signify the optimal result pertaining to the respective evaluation metric where FN represents the number of positive samples overlooked by the model. The average precision (AP) is determined by computing the area under the precision- recall curve, which provides an assessment of the detection quality for each class. The mean average precision (mAP) corresponds to the average AP values across multiple classes and is derived as follows: AP = 1 0 Pecision(Recall)dRecall (18) m AP = 1 m m i=1 APi (19) AP = 1 0 Pecision(Recall)dRecall (18) m AP = 1 m m i=1 APi (19) (18) (19) 4.4.2 Comparative Experiment of Bounding Bbox Regression Loss Function ToassesstheeffectivenessoftheF-CIoUlossfunction,acomparativeanalysiswasconducted, comparing it with alternative loss functions, including CIoU, SIoU [46], and WiseIoU [47]. The experimental results, presented in Table 3, yield valuable insights. The SIoU loss incorporates an additional angle loss component to address the issue of predicted boxes exhibiting undesired wandering behavior during training. As a result, it achieves a modest improvement of 0.41% in recall compared to the CIoU loss. However, this improvement comes at the cost of a slight decline in both average precision and precision measures. The WiseIoU loss, which introduces an attention-based regression loss, aims to enhance object detection performance. However, when compared to the CIoU loss, it does not yield any substantial improvement in model performance. In contrast, the F-CIoU loss, by incorporating an IoU weighting factor, effectively miti- gates the issue of regression imbalance. It outperforms the original loss function by achieving 123 90 Page 14 of 20 90 90 L. Pan et al. Table 3 Comparative experimental results of boundary box regression loss function Loss function mAP@0.5 (%) Precision (%) Recall (%) CIoU 80.72 86.21 68.59 SIoU 80.57 85.66 69.00 WiseIoU 80.12 85.45 67.77 F-CIoU 81.60 86.25 71.71 Bold font is designated to signify the optimal result pertaining to the respective evaluation metric Table 4 Experimental results in comparison with other models Model mAP@0.5(%) Precision(%) Recall(%) Times(ms) SSD 65.30 72.24 66.99 13.0 RTDETR 80.10 83.06 69.09 22.9 YOLOv3tiny 67.40 75.80 58.30 3.3 YOLOv4tiny 70.93 82.49 61.53 4.1 YOLOv5s 79.50 85.45 67.23 8.0 YOLOv6n 79.45 84.43 68.86 7.0 YOLOv7tiny 78.08 84.90 66.88 7.7 YOLOv8n 79.78 84.81 68.69 7.0 Ours 81.60 86.25 71.71 8.5 Bold font is designated to signify the optimal result pertaining to the respective evaluation metric Table 4 Experimental results in comparison with other models a notable improvement of 0.88% in mean average precision (mAP) and a signiﬁcant 3.12% increase in recall. These results clearly demonstrate the effectiveness of the F-CIoU loss in optimizing the model’s performance by addressing the regression imbalance and improving detection accuracy. a notable improvement of 0.88% in mean average precision (mAP) and a signiﬁcant 3.12% increase in recall. These results clearly demonstrate the effectiveness of the F-CIoU loss in optimizing the model’s performance by addressing the regression imbalance and improving detection accuracy. 4.4.3 Experimental Results in Comparison with Other Models The HF-YOLO model, incorporating the proposed enhancements, was evaluated against sev- eral state-of-the-art detection models, including SSD, RTDETR, YOLOv3tiny, YOLOv4tiny, YOLOv5s, YOLOv6n, YOLOv7tiny, and YOLOv8n, in a series of comparative experiments. The HF-YOLO model, incorporating the proposed enhancements, was evaluated against sev- eral state-of-the-art detection models, including SSD, RTDETR, YOLOv3tiny, YOLOv4tiny, YOLOv5s, YOLOv6n, YOLOv7tiny, and YOLOv8n, in a series of comparative experiments. Table 4. presents compelling evidence supporting the superiority of our HF-YOLO model across various crucial metrics such as mean average precision (mAP), precision, and recall. The results showcase a clear advancement compared to all other models examined. Speciﬁ- cally, HF-YOLO demonstrates superior performance over SSD and RTDETR, registering an impressive increase of 16.3% in detection accuracy compared to SSD and 1.5% compared to RTDETR. Moreover, our model achieves a reduction in detection time by 4.5ms in contrast to SSD and 14.4ms compared to RTDETR. Although YOLOv3tiny and YOLOv4tiny exhibit the quickest detection times, they compromise on mAP, precision, and recall metrics. Meanwhile, our HF-YOLO model, while maintaining a comparable detection time to YOLOv5s, YOLOv6s, YOLOv7tiny, and YOLOv8n,signiﬁcantlyoutperformsthemacrossallotherevaluatedmetrics.Theseoutcomes underscore the practical applicability and efﬁcacy of our proposed model in real-world object detection scenarios. 123 123 HF-YOLO: Advanced Pedestrian Detection... Page 15 of 20 90 Page 15 of 20 90 Table 5 Ablation experiment design Model HardSwish Detection Layer F-CIoU YOLOv7tiny ✗ ✗ ✗ 1⃝ ✓ ✗ ✗ 2⃝ ✓ ✓ ✗ 3⃝ ✓ ✓ ✓ Table 6 Results of ablation experiment Model mAP@0.5 (%) Precision (%) Recall (%) Times (ms) YOLOv7tiny 78.08 84.90 66.88 7.7 1⃝ 78.70 86.59 66.03 7.7 2⃝ 80.72 86.21 68.59 8.5 3⃝ 81.60 86.25 71.71 8.5 Bold font is designated to signify the optimal result pertaining to the respective evaluation metric Table 6 Results of ablation experiment 4.4.4 Results and Analysis of Ablation Experiments To evaluate the efﬁcacy of each module improvement, a series of ablation experiments were conducted to analyze the impact of different modules. The ablation experiment design, out- lined in Table 5, utilized YOLOv7tiny as the baseline model. The following improvements were investigated: Improvement 1⃝: Replacement of LeakyReLU with HardSwish activation function. • Improvement 1⃝: Replacement of LeakyReLU with HardSwish activation function Improvement 2⃝: Fusion of high-resolution features and addition of small object detection layer. layer. • Improvement 3⃝: Modiﬁcation of regression loss function from CIoU to F-CIoU. • Improvement 3⃝: Modiﬁcation of regression loss function from CIoU to F-CIoU. By conducting these ablation experiments and comparing the results with the baseline model, the effectiveness of each improvement can be assessed. The experimental results, presented in Table 6, demonstrate the outcomes of the conducted ablation experiments. In experiment 1⃝, involving the replacement of the activation function, the introduction of HardSwish resulted in increased non-linearity, leading to improved feature representation capabilities. Compared to the original model, there was a noticeable enhancement of 0.62% in mean average precision (mAP) and 1.69% in Precision. The detection time remained consistent at 7.7ms, indicating the effectiveness of the HardSwish activation function in improving model performance. Building upon the ﬁndings of Experiment 1⃝, Experiment 2⃝introduced additional detec- tion layers, enhancing the model’s ability to detect pedestrians of varying scales. This led to a signiﬁcant improvement of 2.64% in mAP and 1.71% in Recall, with the detection time increasing by 0.8ms to reach 8.5ms, compared to the original model. Experiment 3⃝focused on improving the regression loss function. By doing so, the detec- tion rate of high-quality samples was enhanced, resulting in a substantial increase in Recall to 71.71%. The mAP was elevated to 81.60%, and the Precision reached 86.25%. The detection time remained consistent at 8.5 ms. 123 L. Pan et al. 90 Page 16 of 20 90 Pag 90 Fig. 6 Comparison of mAP between HF-YOLO and YOLOv7tiny Fig. 6 Comparison of mAP between HF-YOLO and YOLOv7tiny Fig. 6 Comparison of mAP between HF-YOLO and YOLOv7tiny The mAP comparison between HF-YOLO and YOLOv7tiny is presented in Fig. 6, where it can be observed that HF-YOLO exhibits higher accuracy, achieving an improvement of 3.52 in mAP. 4.4.5 Visualization of Detection Results Figure7servesasavisualvalidationoftheproposedalgorithm’sefﬁcacy,displayingdetection results for SSD, RTDETR, YOLOv3tiny, YOLOv4tiny, YOLOv5s, YOLOv6s, YOLOV7tiny, YOLOv8n, and HF-YOLO algorithms. The purpose of this experiment is to showcase the superior performance of the HF-YOLO algorithm. The results indicates a signiﬁcant difference between our proposed model and others. In the ﬁrst set of images, SSD, YOLOv3tiny, YOLOv6s, and YOLOv8n display conspicuous shortcomings in detecting small targets. Moreover, excluding our improved algorithm, other detection outcomes suffer from imprecise localization. Moving to the second and third sets of images, depicting more complex scenes with partially occluded pedestrians, our algorithm consistently exhibits commendable detection capabilities. Relative to the results of YOLOv7tiny, our enhanced algorithm notably diminishes false positives and elevates the accuracy of targetlocalization. Furthermore, itconsistently achieves precise target detection even in complex scenes. 5 Conclusion In conclusion, this paper presents HF-YOLO, an advanced pedestrian detection model that effectively addresses the challenges associated with pedestrian detection, including scale vari- ations and occlusion. By leveraging feature fusion from shallow and deep layers, HF-YOLO enhances the model’s detection performance, resulting in improved accuracy and robustness. Moreover, to tackle the issue of imbalance in bounding box regression, HF-YOLO incorpo- rates a balancing factor that prioritizes the accurate localization of high-quality samples. This 123 Page 17 of 20 90 Page 17 of 20 90 HF-YOLO: Advanced Pedestrian Detection... In contrast to the detection outcomes of SSD, RTDETR, and other algorithms within t the HF-YOLO algorithm consistently demonstrates accurate detection of pedestrian targets, ency in scenarios characterized by both small-scale targets and occlusions Fig. 7 In contrast to the detection outcomes of SSD, RTDETR, and other algorithms within the YOLO series, the HF-YOLO algorithm consistently demonstrates accurate detection of pedestrian targets, exhibiting proﬁciency in scenarios characterized by both small-scale targets and occlusions 123 Page 18 of 20 L. Pan et al. 90 ensures that the model focuses on optimizing the detection of relevant objects and improves overall detection efﬁcacy. Experimental evaluations conducted in this study provide com- pelling evidence of the effectiveness of the proposed HF-YOLO model. It outperforms the baseline model, demonstrating signiﬁcant improvements in detection accuracy and overall performance. The results highlight the potential of HF-YOLO as an advanced pedestrian detection solution, with practical applications in real-world scenarios. ensures that the model focuses on optimizing the detection of relevant objects and improves overall detection efﬁcacy. Experimental evaluations conducted in this study provide com- pelling evidence of the effectiveness of the proposed HF-YOLO model. It outperforms the baseline model, demonstrating signiﬁcant improvements in detection accuracy and overall performance. The results highlight the potential of HF-YOLO as an advanced pedestrian detection solution, with practical applications in real-world scenarios. 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. References 1. Maqsood M, Yasmin S, Gillani S et al (2023) An efﬁcient deep learning-assisted person re-identiﬁcation solution for intelligent video surveillance in smart cities. Front Comp Sci 17(4):174329 1. Maqsood M, Yasmin S, Gillani S et al (2023) An efﬁcient deep learning-assisted person re-identiﬁcation solution for intelligent video surveillance in smart cities. Front Comp Sci 17(4):174329 2. El Hamdani S, Benamar N, Younis M (2020) Pedestrian support in intelligent transportation systems: challenges, solutions and open issues. Transp Res part C Emerg Technol 121:102856. https://doi.org/10. 1016/j.trc.2020.102856 2. El Hamdani S, Benamar N, Younis M (2020) Pedestrian support in intelligent transportation systems: challenges, solutions and open issues. Transp Res part C Emerg Technol 121:102856. https://doi.org/10. 1016/j.trc.2020.102856 j 3. Lee S, Lee S, Seong H et al (2023) Fallen person detection for autonomous driving. Expert Syst Appl 213:119242. https://doi.org/10.1016/j.eswa.2022.119242 j 3. Lee S, Lee S, Seong H et al (2023) Fallen person detection for autonomous driving. Expert Syst Appl 213:119242. https://doi.org/10.1016/j.eswa.2022.119242 4. Wang K, Li G, Chen J et al (2020) The adaptability and challenges of autonomous vehicles to pedestrian in urban china. Accid Anal Prev 145:105692. https://doi.org/10.1016/j.aap.2020.105692 4. Wang K, Li G, Chen J et al (2020) The adaptability and challenges of autonomous vehicles to pedestrians in urban china. Accid Anal Prev 145:105692. https://doi.org/10.1016/j.aap.2020.105692 Hariyono J, Jo KH (2017) Detection of pedestrian crossing road: a study on pedestrian pose recognition. 5. Hariyono J, Jo KH (2017) Detection of pedestrian crossing road: a study on pedestrian pose Neurocomputing 234:144–153. https://doi.org/10.1016/j.neucom.2016.12.050 6. Lee S, Rim J, Jeong B, et al (2023) Human pose estimation in extremely low-light conditions. In: Pro- ceedings of the IEEE/CVF conference on computer vision and pattern recognition, pp 704–714 6. Lee S, Rim J, Jeong B, et al (2023) Human pose estimation in extremely low-light conditions. In: Pro- ceedings of the IEEE/CVF conference on computer vision and pattern recognition, pp 704–714 7. Wong PKY, Luo H, Wang M et al (2021) Recognition of pedestrian trajectories and attributes with computer vision and deep learning techniques. Adv Eng Inf 49:101356. https://doi.org/10.1016/j.aei. 2021.101356 7. Wong PKY, Luo H, Wang M et al (2021) Recognition of pedestrian trajectories and attributes with computer vision and deep learning techniques. Adv Eng Inf 49:101356. https://doi.org/10.1016/j.aei. 2021.101356 8. Feng J, Wu A, Zheng WS (2023) Shape-erased feature learning for visible-infrared person re- identiﬁcation. In: 2023 IEEE/CVF conference on computer vision and pattern recognition (CVPR). IEEE, pp 22752–22761 8. 5 Conclusion 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/. References Feng J, Wu A, Zheng WS (2023) Shape-erased feature learning for visible-infrared person re- identiﬁcation. In: 2023 IEEE/CVF conference on computer vision and pattern recognition (CVPR). IEEE, pp 22752–22761 pp 9. PaulV,MichaelJ(2001)Rapidobjectdetectionusingaboostedcascadeofsimplefeatures.In:Proceedings of the 2001 IEEE computer society conference on computer vision and pattern recognition. CVPR 2001, pp I–I 9. PaulV,MichaelJ(2001)Rapidobjectdetectionusingaboostedcascadeofsimplefeatures.In:Proceedings of the 2001 IEEE computer society conference on computer vision and pattern recognition. CVPR 2001, pp I–I pp 10. Dalal N, Triggs B (2005) Histograms of oriented gradients for human detection. In: 2005 IEEE computer society conference on computer vision and pattern recognition (CVPR’05), pp 886–893 pp 10. Dalal N, Triggs B (2005) Histograms of oriented gradients for human detection. In: 2005 IEEE computer society conference on computer vision and pattern recognition (CVPR’05), pp 886–893 y p p g pp 11. Lowe DG (2004) Distinctive image features from scale-invariant keypoints. Int J Comput Vis 60:91–110. https://doi.org/10.1016/j.infrared.2021.103694 11. Lowe DG (2004) Distinctive image features from https://doi.org/10.1016/j.infrared.2021.103694 11. Lowe DG (2004) Distinctive image features from scale-invariant keypoints. Int J Comput Vis 60:91–110. https://doi.org/10.1016/j.infrared.2021.103694 12. Dai X, Hu J, Zhang H et al (2021) Multi-task faster R-CNN for nighttime pedestrian detection and dista estimation. Infrared Phys Technol 115:103694. https://doi.org/10.1016/j.infrared.2021.103694 13. Xue Y, Ju Z, Li Y et al (2021) MAF-YOLO: multi-modal attention fusion based yolo for pedest detection. Infrared Phys Technol 118:103906. https://doi.org/10.1016/j.infrared.2021.103906 y p g j 14. Jain DK, Zhao X, González-Almagro G et al (2023) Multimodal pedestrian detection using metaheuristics with deep convolutional neural network in crowded scenes. Inf Fusion 95:401–414 15. Girshick R, Donahue J, Darrell T, et al (2014) Rich feature hierarchies for accurate object detection and semantic segmentation. In: Proceedings of the IEEE conference on computer vision and pattern recognition, pp 580–587 15. Girshick R, Donahue J, Darrell T, et al (2014) Rich feature hierarchies for accurate object detection and semantic segmentation. In: Proceedings of the IEEE conference on computer vision and pattern recognition, pp 580–587 g pp 16. Girshick R (2015) Fast R-CNN. In: Proceedings of the IEEE international conference on computer vision, pp 1440–1448 g pp 16. Girshick R (2015) Fast R-CNN. In: Proceedings of the IEEE international conference on computer vision, pp 1440–1448 123 HF-YOLO: Advanced Pedestrian Detection... Page 19 of 20 90 17. Ren S, He K, Girshick RB et al (2015) Faster R-CNN: towards real-time object detection with region proposal networks. IEEE Trans Pattern Anal Mach Intell 39:1137–1149. https://doi.org/10.1109/tpami. 2016.2577031 18. References Liu W, Anguelov D, Erhan D, et al (2016) Ssd: Single shot multibox detector. In: Computer vision–ECCV 2016: 14th European conference, Amsterdam, The Netherlands, October 11-14, 2016, Proceedings, Part I 14, pp 21–37 pp 19. Redmon J, Divvala S, Girshick R, et al (2016) You only look once: uniﬁed, real-time object detection. In: Proceedings of the IEEE conference on computer vision and pattern recognition, pp 779–788 20. Redmon J, Farhadi A (2017) Yolo9000: better, faster, stronger. In: Proceedings of the IEEE conference on computer vision and pattern recognition, pp 7263–7271 21. Redmon J, Farhadi A (2018) Yolov3: an incremental improvement. Preprint at https://arXiv.org/abs/1804. 02767 22. Zhou X, Wang D, Krähenbühl P (2019) Objects as points. Preprint at https://arXiv.org/abs/1904.0785 22. Zhou X, Wang D, Krähenbühl P (2019) Objects as points. Preprint at https://arXiv.org/abs/1904.07850 23. Tian Z, Shen C, Chen H et al (2020) FCOS: a simple and strong anchor-free object detector. IEEE Trans Pattern Anal Mach Intell 44(4):1922–1933 23. Tian Z, Shen C, Chen H et al (2020) FCOS: a simple and strong anchor-free object detector. IEEE Tr Pattern Anal Mach Intell 44(4):1922–1933 24. Yu J, Rui Y, Tao D (2014) Click prediction for web image reranking using multimodal sparse coding. IEEE Trans Image Process 23(5):2019–2032 25. Cao J, Cholakkal H, Anwer RM, et al (2020) D2det: Towards high quality object detection and instance segmentation. In: Proceedings of the IEEE/CVF conference on computer vision and pattern recognition, pp 11485–11494 26. Yu J, Tan M, Zhang H et al (2019) Hierarchical deep click feature prediction for ﬁne-grained image recognition. IEEE Trans Pattern Anal Mach Intell 44(2):563–578 27. Woo S, Park J, Lee JY, et al (2018) Cbam: Convolutional block European conference on computer vision (ECCV), pp 3–19 27. Woo S, Park J, Lee JY, et al (2018) Cbam: Convolutional block attention module. In: Proceedings of the European conference on computer vision (ECCV), pp 3–19 28. Lv W, Xu S, Zhao Y, et al (2023) Detrs beat yolos on real-time object detection. Preprint at https://ar org/abs/2304.08069 29. Zhang L, Lin L, Liang X, et al (2016) Is faster R-CNN doing well for pedestrian detection? In: Computer vision–ECCV 2016: 14th European conference, Amsterdam, The Netherlands, October 11–14, 2016, Proceedings, Part II 14, Springer, pp 443–457 Proceedings, Part II 14, Springer, pp 443–457 30. Zhang S, Wen L, Bian X, et al (2018) Occlusion-aware R-CNN: Detecting pedestrians in a crowd. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. References Proceedings of the European conference on computer vision (ECCV), pp 637–653 31. Liu S, Huang D, Wang Y (2019) Adaptive NMS: reﬁning pedestrian detection in a crowd. In: Proceedi of the IEEE/CVF conference on computer vision and pattern recognition, pp 6459–6468 p p g pp 32. Chu X, Zheng A, Zhang X, et al (2020) Detection in crowded scenes: one proposal, multiple predictions. In: Proceedingsof theIEEE/CVFconferenceon computer vision and pattern recognition,pp 12214–12223 g p p g pp 33. Xia H, Ma J, Ou J et al (2021) Pedestrian detection algorithm based on multi-scale feature extraction and attention feature fusion. Digital Signal Process 121:103311. https://doi.org/10.1016/j.dsp.2021.103311 34. Li Q, Qiang H, Li J (2021) Conditional random ﬁelds as message passing mechanism in anchor-free network for multi-scale pedestrian detection. Inf Sci 550:1–12. https://doi.org/10.1016/j.ins.2020.10.049 35. Wang M, Ma H, Liu S et al (2023) A novel small-scale pedestrian detection method base on residual block group of CenterNet. Comput Stand Interfaces 84:103702. https://doi.org/10.1016/j.csi.2022.103702 36. Gao S, Cheng MM, Zhao K et al (2019) Res2Net: a new multi-scale backbone architecture. IEEE Tr Pattern Anal Mach Intell 43(2):652–662 37. Wang CY, Bochkovskiy A, Liao HYM (2023) Yolov7: Trainable bag-of-freebies sets new state-of-the- art for real-time object detectors. In: Proceedings of the IEEE/CVF conference on computer vision and pattern recognition, pp 7464–7475 38. Lee Y, won Hwang J, Lee S, et al (2019) An energy and GPU-computation efﬁcient backbone network for real-time object detection. In: Proceedings of the IEEE/CVF conference on computer vision and pattern recognition workshops, pp 0–0 39. Wang CY, Liao HYM, Wu YH, et al (2020) Cspnet: a new backbone that can enhance learning capabil- ity of CNN. In: Proceedings of the IEEE/CVF conference on computer vision and pattern recognition workshops, pp 390–391 40. Howard A, Sandler M, Chu G, et al (2019) Searching for mobilenetv3. In: Proceedings of the IEEE/CVF international conference on computer vision, pp 1314–1324 41. Zheng Z, Wang P, Ren D et al (2021) Enhancing geometric factors in model learning and inference for object detection and instance segmentation. IEEE Trans Cybern 52(8):8574–8586 42. Shao S, Zhao Z, Li B, et al (2018) Crowdhuman: a benchmark for detecting human in a crowd. Preprint at https://arXiv.org/abs/1805.00123 43. Zhang S, Xie Y, Wan J et al (2019) Widerperson: a diverse dataset for dense pedestrian detection in the wild. 44. Elfwing S, Uchibe E, Doya K (2018) Sigmoid-weighted linear units for neural network function appr imation in reinforcement learning. Neural Netw 107:3–11. https://doi.org/10.1016/j.neunet.2017.12.0 g p g j 45. Hendrycks D, Gimpel. K (2016) Bridging nonlinearities and stochastic regularizers with gaussian error linear units. Preprint at https://arxiv.org/abs/1606.08415 References IEEE Trans Multimed 22(2):380–393 123 123 123 90 Page 20 of 20 90 Page 20 of 2 90 L. Pan et al. 44. Elfwing S, Uchibe E, Doya K (2018) Sigmoid-weighted linear units for neural network function approx- imation in reinforcement learning. Neural Netw 107:3–11. https://doi.org/10.1016/j.neunet.2017.12.012 45. Hendrycks D, Gimpel. K (2016) Bridging nonlinearities and stochastic regularizers with gaussian error linear units. Preprint at https://arxiv.org/abs/1606.08415 p p g 46. Gevorgyan Z (2022) Siou Loss: More powerful learning for bounding box regression. Preprint at https:// arxiv.org/abs/2205.12740 p p g 46. Gevorgyan Z (2022) Siou Loss: More powerful learning for bounding box regression. Preprint at https:// arxiv.org/abs/2205.12740 g 47. Tong Z, Chen Y, Xu Z, et al (2023) Wise-iou: Bounding box regression loss with dynamic focusing mechanism. Preprint at https://arxiv.org/abs/2301.10051 g 47. Tong Z, Chen Y, Xu Z, et al (2023) Wise-iou: Bounding box regression loss with dynamic focus mechanism. Preprint at https://arxiv.org/abs/2301.10051 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. 123
https://openalex.org/W4230846825	https://ojs.lib.unideb.hu/IJEMS/article/download/5264/4969	English	null	Speed Variation along an Imposed Linear Trajectory, for Robotic Arms Motion	International journal of engineering and management sciences	2,019	cc-by	4,599	International Journal of Engineering and Management Sciences (IJEMS) Vol. 4. (2019). No. 1 DOI: 10.21791/IJEMS.2019.1.61. International Journal of Engineering and Management Sciences (IJEMS) Vol. 4. (2019). No. 1 DOI: 10.21791/IJEMS.2019.1.61. Speed Variation along an Imposed Linear Trajectory, for Robotic Arms Motion L. M. MATICA1, Z. KOVENDI2, E. GERGELY3, L. COROIU4 Faculty of Electrical Engineering and Information Technologies, University of Oradea; Universitatii str., no.1, 410087, Oradea, Romania. Universitatii str., no.1, 410087, Oradea, Romania. e-mail: lmatica@uoradea.ro1, zkovendi@uoradea.ro2, egergely@uoradea.ro3, lcoroiu@uoradea.ro4 e-mail: lmatica@uoradea.ro1, zkovendi@uoradea.ro2, egergely@uoradea.ro3, lcoroiu@uoradea.ro4 Abstract. The paper describes a method of speed (velocity) computation, named mixt profile, during a motion upon an imposed linear trajectory. The method assures an accurate positioning at the end of motion (movement), in a well determinate time lapse. The described method is linked with position vector computation, about a robotic arm. Keywords: cinematics of robotic arm, vectors and versors, waypoints, acceleration or deceleration of movement, axle steps. 1. About movement of an robotic arm. is ,: (2) ) , ( ) , ( )) ( , ( ) , ( 1 1 1 i i i i i i i i i i OX Rot l OX Trans t d OZ Trans OZ Rot A          (2) , where the variable parameter is ) (t di ; it is the distance between origin 1  i O and origin i O along axle 1  i OZ (the others parameters are identical as it was defined in rel.1). i O , where the variable parameter is ) (t di ; it is the distance between origin 1  i O and origin i O along axle 1  i OZ (the others parameters are identical as it was defined in rel.1). , p ; g g g 1  i OZ (the others parameters are identical as it was defined in rel.1). For any robotic arm, those two formulas (rel.1 and rel.2) make easy the determination of the direct kinematics. It is only a mathematic problem to determine the inverse kinematics formulas. For any robotic arm, those two formulas (rel.1 and rel.2) make easy the determination of the direct kinematics. It is only a mathematic problem to determine the inverse kinematics formulas. 2. Example of kinematics analyze. 1. About movement of an robotic arm. named i C , the Cartesian coordinate system indexed i is obtained from Cartesian coordinate system indexed 1  i by homogeny transformations defined with formula that we wrote: For a rotation d.c.c. named i C , the Cartesian coordinate system indexed i is obtained from Cartesian coordinate system indexed 1  i by homogeny transformations defined with formula that we wrote: ) , ( ) , ( ) , ( ) ) ( , ( 1 1 1 i i i i i i i i i i i OX Rot l OX Trans a OZ Trans t OZ Rot A            (1) (1) (1) The formula is described by the algorithm:  the parameter ) (t i is the variable angle of the rotation i C d.c.c. around axle 1  i OZ ; it has different values at different moments of time, it a variable parameter;  -the constant angle i has a different from zero value if axle 1  i OX is not parallel with axle i OX ; it express the rotation (around the axle 1  i OZ ) of axle 1  i OX to obtain the axle i OX ; it depend of the robotic arm construction structure;  -the constant distances ia and il has a different from zero value if origin 1  i O is not identical with origin i O (its define translations);  -the constant angle i  has a different from zero value if the axle 1  i OZ is not parallel with axle i OZ ; it express the rotation (around the axle i OX ) of axle 1  i OZ to obtain the axle i OZ ; it depend of the robotic arm construction structure. The formula that we wrote for a translation d.c.c. 1. About movement of an robotic arm. About movement command of a robotic arm [1; 2; 3], it is necessary to define direct and inverse cinematics. A cinematics analyze example of a robotic arm, type RRRRRR, is illustrated in fig.1. Fig.1. Cinematics analyze of an robotic arm, type RRRRRR. Fig.1. Cinematics analyze of an robotic arm, type RRRRRR. The notations in fig.1 define several Cartesian coordinate systems with its axles: OXi ; OYi ; OZi and its origins: Oi (i=1..6). The motion of the robotic arm is determinated by six rotation driving cinematics couples (d.c.c.) named: Ci (index i goes from 1 to 6; i=1..6); the variable parameters of d.c.c. are named: Ɵi (i=1..6) ; there are constant parameters of the robotic arm named: d1 ; a2 ; d4 ; d6 . The orientation of 495 International Journal of Engineering and Management Sciences (IJEMS) Vol. 4. (2019). No. 1 DOI: 10.21791/IJEMS.2019.1.61. International Journal of Engineering and Management Sciences (IJEMS) Vol. 4. (2019). No. 1 DOI: 10.21791/IJEMS.2019.1.61. the robotic are is defined by the versors (a versor is a vectors with module equal to 1 value): ; n ; oa (the versors are identical with the sense and direction of axles OX6; OY6 and OZ6 [3] (the index 6 Cartesian coordinate system has the origin in the tool point of the robotic arm (according with the Denawitt-Hartenberg convention). According to the Denawitt-Hartenberg convention, a Cartesian coordinate system, indexed i, is obtained by homogeneous transformations, from previous one, indexed i-1. Those homogeneous transformation are (always in this order): the robotic are is defined by the versors (a versor is a vectors with module equal to 1 value): ; n ; oa (the versors are identical with the sense and direction of axles OX6; OY6 and OZ6 [3] (the index 6 Cartesian coordinate system has the origin in the tool point of the robotic arm (according with the Denawitt-Hartenberg convention). According to the Denawitt-Hartenberg convention, a Cartesian coordinate system, indexed i, is obtained by homogeneous transformations, from previous one, indexed i-1. Those homogeneous transformation are (always in this order): 1) rotation around OZi-1 axle; 2) translation along OZi-1 axle; 3) translation along OXi axle; 4) rotation around OXi axle. 1) rotation around OZi-1 axle; 2) translation along OZi-1 axle; 3) translation along OXi axle; 4) rotation around OXi axle. 4) rotation around OXi axle. For a rotation d.c.c. The described algorithm may be applied for every d.c.c. [3]. The described algorithm may be applied for every d.c.c. [3]. 2. Example of kinematics analyze. kinematics analyze. 496 About C1 d.c.c of the RRRRRR robotic arm, fig.1, the direct kinematics formula is: 496 About C1 d.c.c of the RRRRRR robotic arm, fig.1, the direct kinematics formula is: 496 International Journal of Engineering and Management Sciences (IJEMS) Vol. 4. (2019). No. 1 DOI: 10.21791/IJEMS.2019.1.61. International Journal of Engineering and Management Sciences (IJEMS) Vol. 4. (2019). No. 1 DOI: 10.21791/IJEMS.2019.1.61. ) 2 , ( ) , ( ) 2 ) ( , ( 1 1 0 1 0        OX Rot a OZ Trans t OZ Rot (3) (3) The direct kinematic determine the + 2  rotation angle around axle 0 OZ , the 1a translation upon axle 0 OZ and the 2   rotation angle around axle 1 OX in concordance with the graphical explications, presented in fig.3: Fig.3. Graphical explication concerning C1 d.c.c. Fig.3. Graphical explication concerning C1 d.c.c. 3. The location matrix of a robotic arm. According with Denawitt-Hartenberg convention, the matrix G6 is the neutral matrix for multiplication; the relation between location matrix index 0 and index 6 is: 6 5 5 4 4 3 3 2 2 1 1 0 6 6 5 5 4 4 3 3 2 2 1 1 0 6 6 0 A A A A A A G A A A A A A G T Go                6 5 5 4 4 3 3 2 2 1 1 0 6 6 5 5 4 4 3 3 2 2 1 1 0 6 6 0 A A A A A A G A A A A A A G T Go                (11) (11) The inverse kinematics (as a result of direct kinematics) compute the d.c.c. parameters, named ) (t i , starting with location matrix values. 3. The location matrix of a robotic arm. The robotic arm position (location) is defined by the location matrix. It contains axles components of position vector, named p (it defines the position of tool centre point, TCP). This matrix contains axles components of orientation versors: ; n ; oa (orientation versors have the module equal to 1, its describe only the orientation (about the robotic arm);) fig.9. Fig.9. The position and orientations vectors that define the location matrix of an robotic arm. Fig.9. The position and orientations vectors that define the location matrix of an robotic arm. 497 International Journal of Engineering and Management Sciences (IJEMS) Vol. 4. (2019). No. 1 DOI: 10.21791/IJEMS.2019.1.61. So, the location matrix of the robotic arm, contains the axle components, indexed x; y ; z (along the three axles: OX; OY; OZ) of orientation versors and position vector:              1 0 0 0 z z z z y y y y x x x x i p a o n p a o n p a o n G (9) (9) The relation between location matrix indexed i (refering to index i Cartesian coordinate system), and ocation matrix indexed i-1, (refering to index i Cartesian coordinate system) is: The relation between location matrix indexed i (refering to index i Cartesian coordinate system), and location matrix indexed i-1, (refering to index i Cartesian coordinate system) is: i i i i G A G    1 1 (10) i i i i G A G    1 1 (10) , where matrixs i-1Ai are defined by direct kinematics analyze, for example rel.3-8. 4. Acceleration, motion on trajectory and deceleration. For example, the formulas for compute position vector components of robotic arm type RRRRRR (fig.1), (notations Si; i=1..6, means sine of Ɵi angle and Ci means cosine of same angle, the others notation are identical with those explained), are: ) ( ) ( ) ( 4 6 5 3 2 1 3 2 1 2 2 1 6 5 4 1 6 5 4 3 2 1 3 2 1 d d C C C S S S S a C S d S S C d S C C S S S C S px                           (12) ) ( ) ( ) ( 4 6 5 3 2 1 3 2 1 2 2 1 6 5 4 1 6 5 4 3 2 1 3 2 1 d d C S C C C C C a C C d S S S d S C C S C S C C py                             (13) 1 2 2 4 6 5 3 2 3 2 6 5 4 3 2 3 2 ) ( ) ( ) ( d a S d d C S C C S d S C C C S S pz                   (14) (12) (13) (14) 498 International Journal of Engineering and Management Sciences (IJEMS) Vol. 4. (2019). No. 1 DOI: 10.21791/IJEMS.2019.1.61. International Journal of Engineering and Management Sciences (IJEMS) Vol. 4. (2019). No. 1 DOI: 10.21791/IJEMS.2019.1.61. 4. Acceleration, motion on trajectory and deceleration. The speed (velocity) profile of a motion is important for different reasons. Those reasons may be: speed of motion; time of motion; precision of motion; precision for reach the final motion point. A linear motion trajectory, for the robotic arm, contains several intermediary positions, named waypoints. If the trajectory type is not required, intermediary points may be determined according to several conditions; for example it ensures no jerking. Another condition could be a certain programed speed. About a robotic arm, if the trajectory is imposed (linear or circular), it is computed location matrices for intermediary points (waypoints). Considering inverse kinematics, the commands for every d.c.c. of the robotic arm are computed, starting with every location matrix that composes the trajectory. 4. Acceleration, motion on trajectory and deceleration. The motion of a robotic arm may contain three stages: The motion of a robotic arm may contain three stages: The motion of a robotic arm may contain three stages: 1)-the acceleration from zero motion speed to the imposed motion speed; 1)-the acceleration from zero motion speed to the imposed motion speed; 2)-the motion with imposed motion speed (constant); 2)-the motion with imposed motion speed (constant); 2)-the motion with imposed motion speed (constant); Commonly, acceleration to the imposed speed depends on the speed profile that was selected (trapezoidal or parabolic), fig.3 and fig.4 (those graphics consider continuous time) Fig.4. Parabolic profile. Fig.3. Trapezoidal profile (of speed). Fig.3. Trapezoidal profile (of speed). Fig.4. Parabolic profile. Fig.3. Trapezoidal profile (of speed). Fig.4. Parabolic profile. Fig.4. Parabolic profile. This paper describes another method about deceleration stage; the method describes another speed profile, named mixt profile of speed, fig.5. Fig.5. Mixt profile. 4. Acceleration and Deceleration Stages for Mixt Profile of Velocity Fig.5. Mixt profile. 4. Acceleration and Deceleration Stages for Mixt Profile of Velocity 4. Acceleration and Deceleration Stages for Mixt Profile of Velocity The acceleration variation depends of the maximum acceleration possible, on a sample period of time (in a numerical computation system with numerical processor). The numerical process of command computation, about a robotic arm movement, is a discrete one. The variation of robotic arm position, movements speed, acceleration and deceleration values depend of a discrete variable defined by relation: T k  , where T is the sampling period of time, and k is the number of the sample periods of time considered (for example, the variable had the value T  10 after ten sampling periods of time from the start of movement). In the computation described in this paper, it is considered the value of maximum possible acceleration in a sample period of time, named amax . About this new method, the acceleration stage for mixt speed profile, is described by the relation: (15) max 0 ) ( a k v kT v    499 International Journal of Engineering and Management Sciences (IJEMS) Vol. 4. (2019). No. 1 DOI: 10.21791/IJEMS.2019.1.61. International Journal of Engineering and Management Sciences (IJEMS) Vol. 4. (2019). No. 1 DOI: 10.21791/IJEMS.2019.1.61. The initial value of movement speed is zero, it results 0 0  v . After every sampling period of time, the pozition varies with the values max ) ( a k T kT v T     ; this variation may be computed for every component of the pozition vector, considering the maximum possible acceleration along every axle, named aM (instead of amax) M a T k   . The initial value of movement speed is zero, it results 0 0  v . After every sampling period of time, the pozition varies with the values max ) ( a k T kT v T     ; this variation may be computed for every component of the pozition vector, considering the maximum possible acceleration along every axle, named aM (instead of amax) M a T k   . 4. Acceleration and Deceleration Stages for Mixt Profile of Velocity Considering the initial pozition of the robotic arm defined by those components of pozition vector: x p ,0 ; y p ,0 ; y p ,0 after k sampling periods of time during the acceleration, the components of the position vector (indexed k), have the values: M x x k a T k p p     ,0 , M y y k a T k p p     ,0 , (16) M z z k a T k p p     ,0 , M x x k a T k p p     ,0 , M y y k a T k p p     ,0 , M z z k a T k p p     ,0 , M x x k a T k p p     ,0 , (16) M y y k a T k p p     ,0 , Considering the maximum value of motion speed, named vM , the acceleration ends when M M a T k v    , this special value of k, named kA , may be computed. The maximum value of motion speed defines the number of sampling periods of time for the acceleration, named kA: M M A a T v k   / (17) (17) M M A a T v k   / The value of kA must be an integer value (the value of kA must be adapted of this rounding, it is the next bigger integer value of the computed value). The value of kA must be an integer value (the value of kA must be adapted of this rounding, it is the next bigger integer value of the computed value). It is a different situation for a movement with a programed speed, named vP . 4. Acceleration and Deceleration Stages for Mixt Profile of Velocity Considering its component vP,x ; vP,y ; vP,z ; the described method compute different values for each axle; the kA value is determined by the maximum component (considering each component of programed speed, as previous described in rel.10): M z P y P x P A a T v v v k   /) ; ; max( , , , M z P y P x P a T v v v  /) ; ; , , , (18) (18) M z P y P x P A a T v v v k   /) ; ; max( , , , During the acceleration stage of motion, the position vector components are computed according withprevious considerations (usually, the components of initial speed, v0,x ; v0,y ; v0,z is zero): A x x P x x k k v v T k p p /) ( ,0 , ,0 ,      A k k .. 1 ,  A y y P y y k k v v T k p p /) ( ,0 , ,0 ,      (19) A z P z z k k vz v T k p p /) ( , ,0 ,      (19) A z P k vz v T k /) ( ,     In the previous relation k is an integer value, and goes from 1 to kA. The method may be applied for any values of initial speed of motion. 4. Acceleration and Deceleration Stages for Mixt Profile of Velocity It result the logical conclusion: the movement of a robotic arm, with a programed speed is described by the relations (where k starts from kA and goes till is necessary the deceleration stage, defined by the value named kD): x P x k x k v T p p , , ,1     y P y k y k v T p p , , ,1     z P z k z k v T p p , , ,1     x P x k x k v T p p , , ,1     (20) y P y k y k v T p p , , ,1     z P z k z k v T p p , , ,1     500 International Journal of Engineering and Management Sciences (IJEMS) Vol. 4. (2019). No. 1 DOI: 10.21791/IJEMS.2019.1.61. International Journal of Engineering and Management Sciences (IJEMS) Vol. 4. (2019). No. 1 DOI: 10.21791/IJEMS.2019.1.61. The start of deceleration stage is defined by the value kD. The speed variation is described by the relationship, where aD is the deceleration: (21) 2 ) ( ) ( k b v kT a T v kT v P D P       D k k ... 1 ,  2 ) ( ) ( k b v kT a T v kT v P D P       D k k ... 1 ,  In the previous relation, the value aD is a variable value and b is a constant value adapted of desired characteristics about robotic arm motion. The deceleration decreases at the motion end. Considering the condition: 2 0 D P k b v    and components of speed for each axle, it results the number of sampling period of time for the deceleration, named kD: In the previous relation, the value aD is a variable value and b is a constant value adapted of desired characteristics about robotic arm motion. The deceleration decreases at the motion end. 4. Acceleration and Deceleration Stages for Mixt Profile of Velocity Considering the condition: 2 0 D P k b v    and components of speed for each axle, it results the number of sampling period of time for the deceleration, named kD: b v v v k z P y P x P D ) ; ; max( , , ,  (22) b v v v k z P y P x P D ) ; ; max( , , ,  (22) The resulting speed profile is named mixt profile, fig.5 (the graphic considers continuous time). The described method ensures a better precision about stop point proximity. Typically, for a displacement with precise positioning at the end of a robotic arm, it can’t be specified the time needed; the mixt profile of speed specifies exactly the time needed for the motion with precise positioning at the end. The described method, named mixt profile (of speed), was implemented at a flexible welding cellule (for the manufacture of mining machinery), and the agreed motion characteristics (with the beneficiary) were ok. For example, considering a linear trajectory and same orientation of robotic arm (along the movement), considering the values of programed speed: vP = 2 5 mm/s; vP,x = 3mm/s; vP,y = 4mm/s; vP,z = 5mm/s; aM = 25mm/s2 and T = 2 10s; an example of computation determines 20  A k of sampling periods of time for acceleration process: For example, considering a linear trajectory and same orientation of robotic arm (along the movement), considering the values of programed speed: vP = 2 5 mm/s; vP,x = 3mm/s; vP,y = 4mm/s; vP,z = 5mm/s; aM = 25mm/s2 and T = 2 10s; an example of computation determines 20  A k of sampling periods of time for acceleration process: 20 ) / 25 10 /( / ) 5;4;3 max( 2 2     s mm s s mm k A (23) (23) 20 ) / 25 10 /( / ) 5;4;3 max( 2 2     s mm s s mm k A ter the determination of kA, the computation about the waypoints of the linear trajectory g After the determination of kA, the computation about the waypoints of the linear trajectory gets typical. 4. Acceleration and Deceleration Stages for Mixt Profile of Velocity Applying the difference analyzing algorithm[3] (about movement on a linear trajectory) the speed for each axle differs with values: δvx = 3/20 mm/s; δvy = 4/20 mm/s; δvz = 5/20 mm/s; for each sampling period of time, and the position differs with values: δpx =  2 10 3/20 mm; δpy =  2 10 4/20mm; δpz =  2 10 5/20mm. The number of sampling period of time for deceleration, considering b = 5mm/900s, is: The number of sampling period of time for deceleration, considering b = 5mm/900s, is: The number of sampling period of time for deceleration, considering b = 5mm/900s, is: 30 900 / 5 / ) 5;4;3 max(   s mm s mm kD (24) 30 900 / 5 / ) 5;4;3 max(   s mm s mm kD (24) The computation of waypoints coordinates (during the deceleration) involves speed values: 2 2 2 , , 900 3 3 ) ( k k k v v kT v D x P x P x       D k k ... 1 ,  2 2 2 , , 900 4 4 ) ( k k k v v kT v D y P y P y       (25) 2 2 2 , , 900 5 5 ) ( k k k v v kT v D y P z P z       (25) 501 International Journal of Engineering and Management Sciences (IJEMS) Vol. 4. (2019). No. 1 DOI: 10.21791/IJEMS.2019.1.61. International Journal of Engineering and Management Sciences (IJEMS) Vol. 4. (2019). No. 1 DOI: 10.21791/IJEMS.2019.1.61. For each sampling period of time, the position differs with values: For each sampling period of time, the position differs with values: D k k ... 1 ,  px = ) (kT v T x  D k k ... 1 ,  δpy = ) (kT v T y  (26) δpz = ) (kT v T z  (26) The previous example considered a linear trajectory. The previous example considered a linear trajectory. A circular trajectory imposes the computation of waypoints on spherical coordinates (applying the difference analyzing algorithm) and conversion on Cartesian coordinates of those values. A circular trajectory imposes the computation of waypoints on spherical coordinates (applying the difference analyzing algorithm) and conversion on Cartesian coordinates of those values. 5. Conclusions The advantages of mixt profile (of motion velocity) are: the best precision to reach the end point of motion, minimum time of acceleration up to programed motion speed and exact determination of motion time. The method may have others diverse applications, about motion on a linear or circular trajectory; for example about turning or milling process. The method may have others diverse applications, about motion on a linear or circular trajectory; for example about turning or milling process. References [1] L.M.Matica – H. Oros (2017) Speed Computation for Robotic arms Motion Followed by Accurate Positioning. International Journal of Computers, Communications & Control,http://univagora.ro/jour/index.php/ijccc/article/download/2785/1061 , accessed January 2018. [2] L.M. Matica – Z. Kovendi (2011) Structure Analysis for an Robotic arm. Journal of Computer Science and Control Systems. Conference on Engineering of Modern Electric Systems 2011, Oradea. 4 (1) p. 89. [3] T. Horsch – B.Juttler: Cartesian Spline Interpolation for Robotic arms. University of Technology, Departament of mathematics, Darmstadt, Germany (http://www.ag.jku.at/pubs/csi98.pdf) 502
https://openalex.org/W4206220338	https://s3.ca-central-1.amazonaws.com/assets.jmir.org/assets/preprints/preprint-26168-accepted.pdf	English	null	Rapid Design and Delivery of an Experience-Based Co-designed Mobile App to Support the Mental Health Needs of Health Care Workers Affected by the COVID-19 Pandemic: Impact Evaluation Protocol (Preprint)	null	2,020	cc-by	11,820	JMIR Preprints JMIR Preprints An Impact Evaluation protocol for the rapid design and delivery of an experience-based co-designed mobile app to support the mental health needs of healthcare workers impacted by COVID-19 Submitted to: JMIR Research Protocols on: December 02, 2020 Disclaimer: © The authors. All rights reserved. This is a privileged document currently under peer-review/community review. Authors have provided JMIR Publications with an exclusive license to publish this preprint on it's website for review purposes only. While the final peer-reviewed paper may be licensed under a CC BY license on publication, at this stage authors and publisher expressively prohibit redistribution of this draft paper other than for review purposes. Disclaimer: © The authors. All rights reserved. This is a privileged document currently under peer-review/community review. Authors have provided JMIR Publications with an exclusive license to publish this preprint on it's website for review purposes only. While the final peer-reviewed paper may be licensed under a CC BY license on publication, at this stage authors and publisher expressively prohibit redistribution of this draft paper other than for review purposes. Disclaimer: © The authors. All rights reserved. This is a privileged document currently under peer-review/community review. Authors have provided JMIR Publications with an exclusive license to publish this preprint on it's website for review purposes only. While the final peer-reviewed paper may be licensed under a CC BY license on publication, at this stage authors and publisher expressively prohibit redistribution of this draft paper other than for review purposes. [unpublished, peer-reviewed preprint] https://preprints.jmir.org/preprint/26168 Lewis et al JMIR Preprints https://preprints.jmir.org/preprint/26168 Table of Contents Research Program The Department of General Practice, Melbourne Medical School The University of Melbourne 1Integrated Mental Health Research Program The Department of General Practice, Melbourne Medical School The University of Melbourne Melbourne AU 2The Centre for Digital Transformation of Health The University of Melbourne Melbourne AU 3Deprtment of Medicine The University of Melbourne Melbourne AU 4Phoenix Australia Department of Psychiatry The University of Melbourne Melbourne AU 5Centre for Workplace Leadership Faculty of Business and Economics The University of Melbourne Melbourne AU 6Deprtment of Medicine The University of Melbourne Parkville AU 7Department of Cardiology Royal Melbourne Hospital Parkville AU 1Integrated Mental Health Research Program The Department of General Practice, Melbourne Medical School The U Melbourne AU 1Integrated Mental Health Research Program The Department of General Practice, Melbourne Medical School The University Melbourne AU 2The Centre for Digital Transformation of Health The University of Melbourne Melbourne AU 3Deprtment of Medicine The University of Melbourne Melbourne AU 4Phoenix Australia Department of Psychiatry The University of Melbourne Melbourne AU 5Centre for Workplace Leadership Faculty of Business and Economics The University of Melbourne Melbourne AU 6Deprtment of Medicine The University of Melbourne Parkville AU 7Department of Cardiology Royal Melbourne Hospital Parkville AU Corresponding Author: Luke Burchill MBBS, PhD Deprtment of Medicine The University of Melbourne Royal Melbourne Hospital 4th Floor, Clinical Sciences Building Parkville AU Table of Contents [unpublished, peer-reviewed preprint] https://preprints.jmir.org/preprint/26168 JMIR Preprints Lewis et al An Impact Evaluation protocol for the rapid design and delivery of an experience-based co-designed mobile app to support the mental health needs of healthcare workers impacted by COVID-19 Matthew Lewis1 BAPSC, PhD; Victoria J Palmer1, 2 BA, PhD; Aneta Kotevski3 BMEDSC, PhD; Konstancja Densley1 MSc; Meaghan L O'Donnell4 BSc, MPsych, PhD; Caroline Johnson1 MBBS, FRACGP, GCUT, PhD; Franz Wohlgezogen5 PhD; Kathleen Gray2 MLS, MEnvSci, PhD; Kate Robins-Browne1 MBBS, FRACGP, PhD; Luke Burchill6, 7 MBBS, PhD 1Integrated Mental Health Research Program The Department of General Practice, Melbourne Medical School The University of Melbourne Melbourne AU 2The Centre for Digital Transformation of Health The University of Melbourne Melbourne AU 3Deprtment of Medicine The University of Melbourne Melbourne AU 4Phoenix Australia Department of Psychiatry The University of Melbourne Melbourne AU 5Centre for Workplace Leadership Faculty of Business and Economics The University of Melbourne Melbourne AU 6Deprtment of Medicine The University of Melbourne Parkville AU 7Department of Cardiology Royal Melbourne Hospital Parkville AU Corresponding Author: Luke Burchill MBBS, PhD Deprtment of Medicine The University of Melbourne Royal Melbourne Hospital 4th Floor, Clinical Sciences Building Parkville AU Abstract Background: The COVID-19 pandemic has highlighted the central importance of health care workers’ (HCWs) mental health and wellbeing for the successful function of the health care system. Few targeted digital tools exist to support HCWs’ mental health and none appear to have been co-designed with end users. Melbourne AU 2The Centre for Digital Transformation of Health The University of Melbourne Melbourne AU 3Deprtment of Medicine The University of Melbourne Melbourne AU 4Phoenix Australia Department of Psychiatry The University of Melbourne Melbourne AU 5Centre for Workplace Leadership Faculty of Business and Economics The University of Melbourne Melbourne AU 6Deprtment of Medicine The University of Melbourne Parkville AU 7Department of Cardiology Royal Melbourne Hospital Parkville AU Abstract Background: The COVID-19 pandemic has highlighted the central importance of health care workers’ (HCWs) mental health and wellbeing for the successful function of the health care system. Few targeted digital tools exist to support HCWs’ mental health and none appear to have been co-designed with end users. Objective: RMHive is being developed as a mobile app to support the mental health challenges being posed by COVID-19 to HCWs using experience-based co-design (EBCD) processes. We present the Impact Evaluation protocol for the rapid design and delivery of the RMHive mobile app. Methods: The Impact Evaluation will adopt a mixed-methods approach. Qualitative data from photo interviews undertaken with HCWs exploring needs and experiences, and semi-structured interviews conducted with governance stakeholders during design development and implementation will be integrated with quantitative user analytics data and user generated demographic and mental health data entered into the app. Analyses will address three evaluation questions related to: (1) engagement with and use of the mobile app; (2) implementation and integration; and (3) the quantifiable and qualitative impacts on individual mental health. The mobile app design and development will be described using the mobile health (mHealth) evidence reporting and assessment (mERA) guidelines. Implementation of the app will be evaluated using Normalisation Process Theory (NPT) as a framework to analyse qualitative data combined with text and video analysis from semi-structured interviews. Mental health impacts will be assessed using the Patient Health Questionnaire (PHQ4) total score and subscale scores for the Patient Health Questionnaire (PHQ2) for depression and Generalised Anxiety Scale (GAD2) for anxiety. The PHQ4 will be completed at download (baseline), then at 14 and 28 days. Results: The anticipated use period of the app is an average of 30 days. The rapid design will occur over four months using EBCD approaches to collect qualitative data and develop app content. The Impact Evaluation will monitor outcome data for up to 12 weeks following the Minimal Viable Product release. The study received funding and institutional ethics approvals in June, 2020. Outcome data is expected to be available in March, 2021 and the Impact Evaluation published mid 2021. https://preprints.jmir.org/preprint/26168 [unpublished, peer-reviewed preprint] JMIR Preprints Lewis et al Conclusions: The Impact Evaluation will examine the rapid design, development and implementation of the RMHive app and the mental health and wellbeing outcomes for HCWs. Evaluation outcomes will provide guidance for the integration of EBCD in rapid design and implementation processes. Abstract Outcomes will inform future development and roll out of the app programmatically to support the mental health needs of HCWs more widely. (JMIR Preprints 02/12/2020:26168) DOI: https://doi.org/10.2196/preprints.261 (JMIR Preprints 02/12/2020:26168) DOI: https://doi.org/10.2196/preprints.26168 ( p ) DOI: https://doi.org/10.2196/preprints.26168 https://preprints.jmir.org/preprint/26168 Preprint Settings 1) Would you like to publish your submitted manuscript as preprint? 1) Would you like to publish your submitted manuscript as preprint? Please make my preprint PDF available to anyone at any time (recommended). Please make my preprint PDF available to anyone at any time (recommended). Please make my preprint PDF available only to logged-in users; I understand that my title and abstract will remain visible to all users. Only make the preprint title and abstract visible. No I do not wish to publish my submitted manuscript as a preprint Please make my preprint PDF available only to logged-in users; I understand that my title and abstract will remain visible to all users. Only make the preprint title and abstract visible. 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Authors Matthew Lewis 1, Victoria J Palmer 1,5 , Aneta Kotevski 2, Konstancja Densley 1, Meaghan L O’Donnell 3, Caroline Johnson 1, Franz Wohlgezogen 4, Kathleen Gray 5, Kate Robins-Browne 1, Luke Burchill 2,6 1. Integrated Mental Health Research Program, The Department of General Practice, Melbourne Medical School, The University of Melbourne 2. Department of Medicine, The University of Melbourne. 3. Phoenix Australia, Department of Psychiatry, The University of Melbourne 4. Centre for Workplace Leadership, Faculty of Business and Economics, The University of Melbourne 5. The Centre for Digital Transformation of Health, The University of Melbourne 6. Department of Cardiology, Royal Melbourne Hospital Corresponding Author Associate Professor Luke Burchill The University of Melbourne, Department of Medicine Royal Melbourne Hospital 4th Floor, Clinical Sciences Building Parkville, VIC 3050 T: +61 3 8344 7161 F: +61 3 9347 1863 E: blj@unimelb.edu.au https://preprints.jmir.org/preprint/26168 b.edu.au Original Manuscript Original Manuscript [unpublished, peer-reviewed preprint] https://preprints.jmir.org/preprint/26168 Protocol title Lewis et al JMIR Preprints Methods The Impact Evaluation adopts a mixed-method design. Qualitative data from photo interviews undertaken with up to 30 HCWs exploring needs and experiences, and semi- structured interviews conducted with up to 30 governance stakeholders will be integrated with qualitative and quantitative user analytics data (using Think Aloud methods) and user generated demographic and mental health data entered into the app. Analyses will address three evaluation questions related to: (1) engagement with and use of the mobile app; (2) implementation and integration of the app; and (3) the quantifiable and qualitative impacts on individual mental health outcomes. The mobile app design and development will be described using the mobile health (mHealth) evidence reporting and assessment (mERA) guidelines. Implementation of the app will be evaluated using Normalisation Process Theory (NPT) as a framework to analyse qualitative data from interviews combined with text and video analysis from semi-structured interviews using a Think Aloud approach. Mental health impacts will be assessed using the Patient Health Questionnaire (PHQ4) total score and subscale scores for the Patient Health Questionnaire (PHQ2) for depression and Generalised Anxiety Scale (GAD2) for anxiety. The PHQ4 will be completed at download (baseline), then at 14 and 28 days. Objectives RMHive is being led and developed by HCWs as a mobile app to support the mental health challenges being posed by COVID-19 to HCWs using experience-based co-design (EBCD) processes. We present the Impact Evaluation protocol for the rapid design and delivery of the RMHive mobile app. Background The COVID-19 pandemic has highlighted the central importance of health care workers’ (HCWs) mental health and wellbeing for the successful function of the health care system. Few targeted digital tools exist to support hospital HCWs’ mental health and none appear to have been led and co-designed by HCWs. Corresponding Author 1 [unpublished, peer-reviewed preprint] https://preprints.jmir.org/preprint/26168 JMIR Preprints Lewis et al Results The anticipated use period of the app is an average of 30 days. The rapid design will occur over four months using EBCD to collect qualitative data and develop app content. The Impact Evaluation will monitor outcome data for up to 12 weeks following the Minimal Viable Product release hospital wide for all health care workers to use. The study received 2 [unpublished, peer-reviewed preprint] 2 [unpublished, peer-reviewed preprint] https://preprints.jmir.org/preprint/26168 https://preprints.jmir.org/preprint/26168 JMIR Preprints Lewis et al funding and institutional ethics approvals in June, 2020. Outcome data is expected to be available in March 2021 and the Impact Evaluation published mid-2021. https://preprints.jmir.org/preprint/26168 Conclusions The Impact Evaluation will examine the rapid design, development and implementation of the RMHive app and its impact on the mental health outcomes for HCWs. Findings from the Impact Evaluation will provide guidance for the integration of EBCD in rapid design and implementation processes. The Evaluation will also inform the future development and roll out of the app to support the mental health needs of hospital based HCWs more widely. Key words: Mental Health; Mobile Applications; COVID-19; Health Personnel; Experience- Based Co-Design; Impact Evaluation; Digital Interventions 3 [unpublished, peer-reviewed preprint] https://preprints.jmir.org/preprint/26168 JMIR Preprints Lewis et al Background The mental health and wellbeing of health care workers (HCWs) should be a major public health priority [1] both during COVID-19 and beyond to support the successful functioning of the health care system. HCWs, particularly nurses and doctors, experience significant mental health challenges and these have been exacerbated through the early stages of the COVID-19 pandemic with high rates of depression (23.2%), anxiety (22.8%) and insomnia (38.9%) reported. [2] Similarly high rates of clinically significant anxiety (45%), depression (38%), and post-traumatic stress disorder (19%) have been observed during and following significant viral outbreaks and pandemics. [3] Despite the recognised impact of pandemics on the mental health and wellbeing of HCWs, few digital solutions have been developed and delivered to support hospital based HCWs mental health and wellbeing. A mobile app could provide a readily available, evidence based support for stress management, mental health and wellbeing to hospital based HCWs through the COVID-19 pandemic. By using Experience-based Co-Design approaches, this app could deliver appropriate supports across professional groups. HCWs are often reluctant to seek appropriate and timely mental health support. [4] Perceived stigma and career impact, time challenges and a work culture that values stoicism are recognised as contributing barriers to help-seeking. [4-6] Without support, all HCWs are at increased risk of major mental health complications. [4, 7] Targeted approaches to maintain and improve HCW mental health are needed that can address barriers to help- seeking and meet HCWs ‘where they are,’ and ‘according to their professional needs.’ A HCW co-designed mobile app offering mental health support may have potential as a readily accessible, cost effective, evidence based, and scalable tool to achieve these goals. [8] The more that a mobile app can integrate the lived experiences of HCWs through the use of co-design processes, the more likely it will respond to their needs and ideally lead to increased uptake and engagement. While digital health interventions and mobile app development utilise Human Centred Design principles (HCD) to meet user needs, [9] few embed and follow an Experience-Based Co-Design (EBCD) approach. EBCD offers power sharing and approaches that can extend HCD methods further than developing deep insights to enable users to actively shape and be co-designers of solutions. Background [10] EBCD has typically been used in healthcare quality improvement as a method for staff, patients and carers to 4 [unpublished, peer-reviewed preprint] 4 [unpublished, peer-reviewed preprint] https://preprints.jmir.org/preprint/26168 JMIR Preprints Lewis et al collaborate on the design of solutions drawing on narrative and story-based approaches through interview, film or other visual methods such as digital stories, coupled with facilitated co-design using design thinking and participatory approaches. The integration of the lived experience of end users via EBCD processes will ensure that outcomes are targeted and co-designed with those most likely to be impacted by an issue, change to process, or an intervention. [11, 12] EBCD provides an avenue to more deeply embed end users’ experiences as the primary driver of change processes and provides a commitment to shared power arrangements and decision-making not currently addressed by the adoption of Human Centred Design (HCD) principles alone.[10] The explicit utilisation of EBCD as a way to embed lived-experience will ideally result in greater uptake of the mobile app and increased engagement. Several digital mental health support responses for HCWs have been developed to address the impact of COVID-19, [13] and while some are HCW led, peer designed, and delivered, we have been unable to identify any that have been co-designed by and for HCWs themselves. Addressing this gap may be part of what is required to improve overall uptake, engagement and use of mobile apps by individuals. The RMHive will attempt to do this by embedding HCWs in the design, development and implementation of a mobile app using EBCD. Integrating the lived experience of HCWs in the design and development of mobile apps is important to support their mental health needs. [12] Equally important is a systematic evaluation of the development process and outcomes that can be used to support replication of mobile apps or digital interventions. This includes outlining the underlying theoretical frameworks, clear overviews of the design, development, and implementation and documentation of impact. [8, 14] Existing evaluations focus on the development and implementation of mobile apps largely from a technological perspective but can take a more limited evaluation of the wider contextual, organisational and individual factors that influence uptake and impact on outcomes. [15] A broader Impact Evaluation approach allows for socio- technical digital health perspectives to be considered in the context of intersecting factors and causal attributions across the design, development and implementation continuum for programmatic scaling up. Background [16] adaptive This protocol describes the Impact Evaluation of the RMHive mobile app, being led by HCWs as an EBCD intervention for HCWs with mental health needs arising from the coronavirus pandemic. Using EBCD, HCWs from different professional groupings will be co-design partners across all phases of research from ideation to implementation. RMHive is 5 [unpublished, peer-reviewed preprint] 5 [unpublished, peer-reviewed preprint] https://preprints.jmir.org/preprint/26168 JMIR Preprints Lewis et al not being developed as a clinical or therapeutic tool. The Impact Evaluation will integrate data from the design and development and the wider implementation of RMHive and lead to the formulation of a theory of change that can explain both positive and negative impacts to guide future delivery of the mobile app across other health services and hospital networks. Objectives A rapid design and development cycle will be adopted using EBCD to identify experiences and create an app prototype within 3-4 months to support the mental health needs of hospital based HCWs working in the COVID-19 context. The approach adopted is analogous to rapid prototyping in design thinking, [17] through the use of rapid co-design processes. The Impact Evaluation addresses three questions using quantitative and qualitative data that will be collected across all stages: 1) What is HCWs’ engagement with RMHive? (including use patterns, perceptions of content, and overall level of engagement)? 2) What contextual, socio-technical, organisational and individual features support or hinder implementation?, and 3) What are the identifiable impacts on individual HCWs' mental health through app adoption, implementation and use? This protocol will describe the planned design, development and implementation of the RMHive mobile app using EBCD. It will describe the data to be collected to inform the Impact Evaluation and the proposed analysis plan. Setting and locations RMHive will undergo development, beta-testing and implementation with hospital based HCWs across Royal Melbourne Hospital (RMH). RMH is a large tertiary referral hospital that includes two major campuses; an acute care city campus and a second inner city campus providing sub-acute services including aged care and which have been heavily impacted by COVID-19. RMH is the largest provider of mental health services in Victoria with services spanning the northern and western suburbs of Melbourne. RMH is a part of the Melbourne Health network who in 2018/2019 Melbourne Health reported 10,000 staff and volunteers. In 2019, there were 79,799 presentations to the emergency department and 105,493 inpatient 6 [unpublished, peer-reviewed preprint] 6 [unpublished, peer-reviewed preprint] https://preprints.jmir.org/preprint/26168 https://preprints.jmir.org/preprint/26168 JMIR Preprints Lewis et al admissions [18]. Design, development and beta-testing will be conducted with the emergency and cardiology departments as two settings impacted by the COVID-19 pandemic. Following completion of the beta-test phases and refinements arising from this process, RMHive will be implemented across RMH and made available to all HCWs hospital wide. While it is recognised that there may be context and profession specific needs that remain unaddressed from this preliminary rapid design process, a decision was made to roll out the app hospital wide to explore use patterns and gather feedback to inform future iterations and refinements of the app. Ethics approval for this study has been provided by the University of Melbourne Human Research Ethics Committee (Ethics ID #2056866.2). Overview of the RMHive design, development and implementation process The project will adopt a rapid design, development, and implementation process using EBCD to progress from HCWs needs identification and content creation through to app design and development, and beta-testing to a minimally viable product (MVP) release. An overview of the Design, Development and Implementation Framework is shown in Figure 1. Figure 1: Planned design, development and implementation framework for RMHive Figure 1: Planned design, development and implementation framework for RMHive Figure 1: Planned design, development and implementation framework for RMHive 7 [unpublished, peer-reviewed preprint] https://preprints.jmir.org/preprint/26168 JMIR Preprints Lewis et al Project and governance team j g An important governance principle for the research team will be adaptiveness to ensure that the design and implementation process is flexible enough to adjust to complex, unpredictable changes that may occur while undertaking the research (e.g. due to emerging events throughout the COVID-19 pandemic). An adaptive governance framework will be adopted to support the design, development and implementation of RMHive [19]. Adaptive governance provides a structured approach for decision making in complex and dynamic systems while paying attention to the needs of grass roots community members. [20] Key to adaptive governance is shared decision-making arrangements which complements the collaborative and power-sharing goals of the EBCD approach. When applied to RMHive, adaptive governance is being used to ensure HCWs from diverse professional backgrounds including clinical (nurses, doctors, psychologists, primary care specialists) and non-clinical workers (administrative staff, environmental service assistants) are continually involved in shared decision making and feedback across all phases of research and according to changing HCW community needs. This extends to providing mechanisms of accountability of the research team in the EBCD processes. An important governance principle for the research team will be adaptiveness to ensure that the design and implementation process is flexible enough to adjust to complex, unpredictable changes that may occur while undertaking the research (e.g. due to emerging events throughout the COVID-19 pandemic). An adaptive governance framework will be adopted to support the design, development and implementation of RMHive [19]. Adaptive governance provides a structured approach for decision making in complex and dynamic systems while paying attention to the needs of grass roots community members. [20] Key to adaptive governance is shared decision-making arrangements which complements the collaborative and power-sharing goals of the EBCD approach. Overview of the RMHive design, development and implementation process When applied to RMHive, adaptive governance is being used to ensure HCWs from diverse professional backgrounds including clinical (nurses, doctors, psychologists, primary care specialists) and non-clinical workers (administrative staff, environmental service assistants) are continually involved in shared decision making and feedback across all phases of research and according to changing HCW community needs. This extends to providing mechanisms of accountability of the research team in the EBCD processes. RMHive app co-design and implementation will be managed by a HCW-led project team and include HCWs in multiple project governance roles and as advisory committee members. HCW members of the project team and advisors will be invited to participate through posters and flyers posted in staff common rooms and drawn from critical care emergency and cardiology units for the first rapid design phases. It is anticipated that HCWs will most commonly be nurses and physicians as these are the two largest professional groups in the hospital. The HCW advisory group will provide input and feedback and share decision- making throughout the project. The adaptive governance framework supports feedback loops that ensure HCW input is reviewed on a continuing basis with decision making shared by HCWs on the project team meeting every 2 weeks. The project team will be multidisciplinary and incorporate experts in EBCD, implementation science, evaluation, applied ethics, mental health clinical care, project governance, mental health care research and delivery, mobile app development, digital health interventions and evaluation, and creative content production. 8 [unpublished, peer-reviewed preprint] 8 [unpublished, peer-reviewed preprint] https://preprints.jmir.org/preprint/26168 https://preprints.jmir.org/preprint/26168 JMIR Preprints Lewis et al Preparatory phase Prior to commencement of the co-design work, scoping interviews were conducted during the initial wave of COVID-19 in early 2020 with a small but diverse group of HCWs (N=6). This was to understand the experiences and needs of HCWs in working through the pandemic. Semi-structured interviews included scoping questions co-developed by HCWs and researchers leading the study (available upon request). HCWs reported being overwhelmed with written communication (guidelines, clinical directives, news reports) and expressed a clear preference for video rather than text based information. Using these experiences, the project team generated ideas for video-based content that reflected HCWs’ experiences but which also responded to their needs. These were: 1) mental preparation and coping strategies; 2) remaining connected through hearing other experiences of working on the frontline; and 3) having ‘time out’ / a break from the ‘dark bubble’ they were living in. https://preprints.jmir.org/preprint/26168 Overview of the RMHive design, development and implementation process The broad content themes were pitched to HCW members of the implementation team and led to video content to be developed within the app. Needs analysis and understanding context Leveraging the expertise of EBCD researchers working on the project team, HCWs selected photo interviews (photo-elicitation) as a relevant method for understanding HCW mental health needs and lived-experiences. We have previously shown photo elicitation to be a relevant participatory visual method that can be successfully applied across different contexts and research settings [21]. Photo-interview methods enable a narrative approach to identifying needs and understanding context [21] and can assist people to articulate complex concepts, discuss sensitive topics and provide an insight into the inner worlds of feelings, emotions and thoughts. [22] Up to 30 HCWs from the emergency and cardiology departments will be invited to participate in photo interviews [21]The choice for these departments was based on the professional groups representing the largest proportion of hospital staff. The photo interview method will involve HCWs being asked to provide the research team with a series of 3-5 digital images produced on smartphones reflecting their experience of COVID-19 including challenges associated with working on the frontline as well as experiences or behaviours that are helping them to meet these challenges The images will inform a semi-structured telephone interview to draw out key “touchpoints” related to HCWs experiences during the pandemic and the impacts on mental health. A touchpoint is a term used in EBCD to refer to the places, and the way that a person comes in touch with a 9 [unpublished, peer-reviewed preprint] 9 [unpublished, peer-reviewed preprint] https://preprints.jmir.org/preprint/26168 JMIR Preprints Lewis et al particular service, or organisation, or how an issue touches them directly (e.g. the subjective world of the person). [23] The touchpoints will be further explored in a deliberative workshop to scope content areas to be developed within the app. The workshop discussion will be analysed to identify one or two key touchpoints that will inform an additional co- design workshop to be conducted by the app developers, Curve Tomorrow (explained below). Early in the RMHive development process, a series of interviews will be conducted with a diverse range of hospital managers and leaders within RMH (N=30) to gain insights into existing mental health programs, systems, organisational context and services for HCWs’ mental health. Overview of the RMHive design, development and implementation process It is likely that this group will be involved in initiatives for mental health and this interview data will provide further contextual data to inform the implementation of RMHive and its Impact Evaluation. Content creation Video content will be developed by a University-based video producer working with a team of externally based creatives including film makers, animators and script writers based on the expressed needs and specific questions posed by HCWs in the preparatory phase. This has resulted in the creative team receiving HCW approval for four planned video series. The first series of videos will feature health experts answering questions posed by HCWs working on the frontline during the COVID-19 pandemic. The second series will include animated videos developed in conjunction with Phoenix Australia, national experts in trauma informed care, that focus on resilience and coping strategies (an issue that was raised by HCWs in the preparatory phase scoping interviews). Series 3 will include short videos based on themes identified by HCWs and which take a humorous tone based on HCW feedback that they needed a mental break from the daily challenges of COVID-19. The final series will be short films that use the audio taken from the photo interviews that address HCWs need to connect with others and film makers will creatively reflect on their experiences. HCWs identified the experience of discussing their work lives and challenges as cathartic and the content creation team has engaged eight film-makers who will be asked to respond to each audio interview with a short film in an attempt to maintain a creative dialogue between the HCWs and creative team. Closing the communication loop, HCWs will then respond to the short film with a written reflection. The films and responses are both planned to appear in the app. A team of mental health clinicians will develop information about clinical support pathways and curate existing digital and face to face mental health support services and tools to be 10 [unpublished, peer-reviewed preprint] 10 [unpublished, peer-reviewed preprint] https://preprints.jmir.org/preprint/26168 JMIR Preprints Lewis et al included in the RMHive app. These resources will match the touchpoints identified in photo interviews with HCWs. The goal is to provide targeted pathways for mental health support depending on the needs of users, in a simple, private and focused way in response to need. HCW engagement and feedback in the preparatory phase scoping interviews of the study identified that options to self-monitor mental health would be valuable. This self-monitoring is intended as a reflective point for individuals and to provide personalised engagement opportunities in the app for the user. Content creation The mental health team determined that the Kessler Psychological Distress Scale (K10), [24] a measure of distress commonly used by general practitioners (GPs) and in mental health settings, would be a useful tool to monitor mental health within the app. Self-monitoring mental health using mobile apps is an established effective method to increase emotional self- awareness. [25] Four general health and wellbeing questions will also be provided to prompt self-reflection regarding whether they are on track or not across four domains: mood, relationships, physical health, and productivity. In the absence of an evidence-based self- tracking tool, the four general health questions were co-developed through consensus discussions with the project lead (LB) and mental health stream leads (MO'D, CJ), and the HCW advisory group. The goal was to provide a simple, self-monitoring check-in option that could be completed as often as a user chose. In addition to self-monitoring tools, the app will include the 4 item Patient Health Questionnaire (PHQ4) as a measure of symptom burden, functional impairment and disability. The PHQ4 comprises the Patient Health Questionnaire-2 (PHQ2) for depression and the Generalised Anxiety Scale-2 (GAD2) as brief, validated subscales. [26] These questionnaires will help to establish a baseline of HCWs mental health need (e.g. Impact Evaluation question 1) and be used to identify any changes to this during and post app use (e.g. Impact Evaluation question 3). App architecture RMHive will be designed to be a stand-alone app and will not be integrated into existing data repositories, human resource records or electronic medical records. The RMHive app will be developed using the Ionic app development framework with a Rails backend and will function on iOS and Android operating systems. Hosting will be via the secure cloud application platform Heroku. Data entered within the app will be anonymous to the research team and only de-identified data will be provided to the research team for analysis. In the Terms of Use, app users will be asked to provide consent for the research team to make contact for Think Aloud interviews and the impact evaluation data collection. It is planned that RMHive will be incorporated as a standalone resource into the wellbeing programs and policy response at RMH as one of a suite of support options for HCWs. Industry best practice standards for Personal Health Information and Data Security will be followed. Data will be kept secure using industry-standard encryption over the wire and at rest. Regulations to host data in Australia will be followed and data security measures will comply with the Open Web Application Security Project Healthcare guidelines, Australian Privacy Act, ISO AS/NZ 27001, ISO AS/NZ 27017, ISO AS/NZ 27018 and SOC2. The outcome paper will present a more detailed architecture in accordance with guidelines for reporting of health interventions using mobile phones. [29] RMHive development, beta-testing and refinement p g A co-design workshop will be conducted by the app developer and industry partner, Curve Tomorrow, using Human Centred Design principles and building on the data collected from the preparatory scoping interviews, needs analysis and content creation stages. This workshop will lead to a clickable prototype of RMHive that will be used to gather additional user feedback prior to the beta-test version of the app. Curve Tomorrow will gather phone feedback from HCWs about the clickable prototype to generate the beta-test app version. The 11 [unpublished, peer-reviewed preprint] 11 [unpublished, peer-reviewed preprint] https://preprints.jmir.org/preprint/26168 https://preprints.jmir.org/preprint/26168 JMIR Preprints Lewis et al beta-test version will be released to one hospital unit (the Emergency Department) and feedback from users will inform the refinements that are needed for the implementation of the MVP for HCW use hospital wide release. Think- Aloud interviews [27] will be conducted with a sub-sample of users through the beta- test to inform refinements, the identification of bugs and required fixes. Think Aloud interviews are commonly used to test new products and technologies, as simulated situations and enable a user to think out loud their thoughts and feelings as they use the app in real time. [28] The Think Aloud interviews will be audio and video recorded and outcomes summarised for beta test changes and reported in the Impact Evaluation. Key themes related to usability, and perception of content will be explored. An opt-in process to participate in Think Aloud interviews will be employed. Participants and eligibility HCWs in emergency and cardiology will participate in the preparatory phase and adaptive governance structures and frameworks, and the planned design and development processes. Up to 30 HCWs will provide photo-interview data and take part in deliberative workshops (including subsequent developer conducted co-design workshops) and up to 15 HCWs will participate in Think Aloud interviews to inform the beta-test. Governance interviews will be completed by up to 30 managers and leaders through the hospital. For wider release and implementation, all HCWs at RMH will be able to access the app and download it for use. Users will be provided an access code. In terms of understanding use and engagement with the app, 20% of HCWs who use the app for at least 7 to 30 days will be invited to participate in a Think Aloud interview. For the identification of mental health need and impacts of the app use on HCWs mental health, only data collected from the MVP stage will be included. Implementation The RMHive app will be implemented for HCW use hospital wide. An implementation 12 [unpublished, peer-reviewed preprint] https://preprints.jmir.org/preprint/26168 https://preprints.jmir.org/preprint/26168 JMIR Preprints Lewis et al strategy will be developed to guide the wider hospital release and the effectiveness of this strategy in supporting engagement, adoption, integration, and future sustainability of the app into individual work and organisational culture will be reported on in the Impact Evaluation. A twelve-week Impact Evaluation period has been set following the release of the MVP to assess the uptake, engagement, use patterns, and impacts on mental health. This includes an assessment of the implementation enablers and challenges, and integration of data from design and development stages that will inform the three Impact Evaluation questions. Harms As the RMHive app development process will involve discussions and content about mental health and work challenges, there may be some risks to participant wellbeing. A protocolised participant distress response has been developed to support the needs of participants, and study communications will provide avenues to national and institutional support services. While RMHive is being developed to support HCW mental health, the MVP is not a clinical or triaging tool, and will not be developed to treat clinically determined symptoms of mental illness for this stage. Participants will be informed of this and will be encouraged both within the app and in study communications to seek further professional help if they feel their mental health and wellbeing needs warrant it. Some of the messages and advice within the app will be tailored to mental health screening outcomes (PHQ2 and GAD2) and provide directions to seek assistance where this is warranted. Recommended actions for K10 results for self-monitoring will be included to guide access to professional support services. Support information will be made available in study communications through the research and development process and within the app. Sample size Up to 10 000 HCWs at RMH will have access to the RMHive app. Our sample size for app users is calculated on an anticipated minimum rate of 10% of total staff downloading RMHive (n=1000). Of this, 50% may go on to use the app (n=500) and of this, a further 50% are anticipated to cease using the app within the first five days (n=250). Of the 250 remaining users beyond five days, an additional 30% are anticipated to continue using the app for 7 days or more (n=175) and a further 50% are likely to use the app for the full 30 day use period is expected (n=87). These figures are based on download and use patterns reported in other health related mobile apps. [30] 13 [unpublished, peer-reviewed preprint] 13 [unpublished, peer-reviewed preprint] https://preprints.jmir.org/preprint/26168 https://preprints.jmir.org/preprint/26168 JMIR Preprints Lewis et al Impact Evaluation procedure Conceptual framework The RMHive development process will use EBCD concepts to embed the lived experience of HCWs within the app content and features, and adaptive governance frameworks to share power arrangements and decision-making in the co-design and implementation. This means that HCWs are embedded at all stages and processes. It is anticipated that the integration of HCW perspectives using an EBCD and adaptive governance approach will increase the engagement and ongoing use of the RMHive app and subsequently lead to sustainability for organisational use. Additionally, developmental processes that seek to engage and understand organisational governance efforts will increase the ability for RMHive to be presented as a key component of the health and wellbeing strategy across the organisation. The success of this integration and support from governance stakeholders will be evaluated with attention to the adaptive governance framework underpinning the research collaboration. [19] Impact Evaluation questions https://preprints.jmir.org/preprint/26168 Impact Evaluation questions The three Impact Evaluation questions are outlined again in Table 1 alongside the data sources to inform these questions, and the reporting or analytical framework proposed to 14 s://preprints.jmir.org/preprint/26168 [unpublished, peer-review 14 [unpublished, peer-reviewed preprint] 14 [unpublished, peer-reviewed preprint] https://preprints.jmir.org/preprint/26168 https://preprints.jmir.org/preprint/26168 JMIR Preprints Lewis et al answer the question. Further explanation of the planned analyses for data sources are then described. 15 [unpublished, peer-reviewed preprint] https://preprints.jmir.org/preprint/26168 JMIR Preprints Lewis et al Table 1. Impact Evaluation questions, data sources and planned analysis Impact Evaluation question Data sources Analytical Focus and/or Framework 1) What is HCWs’ engagement with RMHive? (including use patterns, perceptions of content, and overall level of engagement)? - Change log from beta-test to Minimally Viable Product (MVP). - User demographics and mental health baseline measures (PHQ4, PHQ2, GAD2, K10 self-monitoring and general health self-tracking questionnaires). - App analytics data (bounce rates and patterns of use including total time using, and content use). - Qualitative Think Aloud semi- structured interview text and video data from beta-test and implementation phase. - Descriptive overview of beta-test engagement and content changes. - Descriptive statistics on user demographics (age, gender, profession), mental health baseline scores and averages, K10 first completion averages. - Number of app downloads, use patterns, content accessed. Patterns of content engaged with, number of videos watched, time spent on content (where possible). - Thematic analysis of Think Aloud interviews text content examining (a) user perceptions of the app and content; and, (b) video analysis for app usability and feature engagement (attention to facial gestures, body language and user workflow). This data will be considered against the touchpoints identified in the photo-interviews and deliberative workshops to evaluate the question of whether the app meets HCWs needs. 2) What contextual, socio-technical, organisational and individual features support or hinder implementation? - Qualitative governance interview data with leaders in the hospital setting. - Touchpoints that emerged from the photo-interviews and deliberative workshops during design and development that were related to contextual, socio-technical, organisational and individual barriers and - Normalisation Process Theory using the four NPT constructs to code interview, mapping and brief survey data according to: coherence (understanding of the problem – how people make sense of HCW mental health needs and wellbeing, and the role of a mobile app in providing support), cognitive participation (engagement – how is the new technology driven forward who buys in Table 1. Analytical Focus and/or Framework Analytical Focus and/or Framework Impact Evaluation Data sources Impact Evaluation question Data sources facilitators for implementation. Review of available mental health and wellbeing programs at the hospital. - Qualitative Think Aloud semi- structured interview text and video analysis. - Web-based implementation survey of team leaders, managers and other appropriate staff distributed via hospital contacts. ) h h d h d l to this and how is practice sustained), collective action (integration of new technology, skill-set fit, integration of new technology, what work is done to operationalise and contextually execute new technology) and reflexive monitoring (how do groups and individuals start to assess whether new approach or practice is working and what reconfigurations are undertaken by them to embed change). facilitators for implementation. Review of available mental health and wellbeing programs at the hospital. - Qualitative Think Aloud semi- structured interview text and video analysis. - Qualitative Think Aloud semi- structured interview text and video analysis. - Web-based implementation survey of team leaders, managers and other appropriate staff distributed via hospital contacts. Identification of themes at the different levels in qualitative interview data and deliberative workshop related to what supports or hinders app implementation and integration, these will also be mapped to NPT where appropriate. Summary findings from brief survey of managers and team leaders regarding the implementation of the mobile app. Summary findings from brief survey of managers and team leaders regarding the implementation of the mobile app. 3) What are the identifiable impacts on individual HCW’s mental health through app adoption, implementation and use? - User demographics and mental health post app use measures for depression, anxiety and overall PHQ4 mental health. - Self-monitoring data using K10 for psychological distress - General health self-tracking questions: physical activity, relationships, productivity and wellbeing. - Age, gender, professional role where available. Pre PHQ2, GAD2 and overall PHQ4 scores compared with post app use scores (defined as 30 days or last mental health entry on screening questionnaires). - Age, gender, professional role where available. Pre PHQ2, GAD2 and overall PHQ4 scores compared with post app use scores (defined as 30 days or last mental health entry on screening questionnaires). - User demographics and mental health post app use measures for depression, anxiety and overall PHQ4 mental health. - Self-monitoring data using K10 for psychological distress - K10 self-monitoring scores first time of app use and last user completion. Impact Evaluation questions Impact Evaluation questions, data sources an Impact Evaluation question Data sources 1) What is HCWs’ engagement with RMHive? (including use patterns, perceptions of content, and overall level of engagement)? - Change log from beta-test to Minimally Viable Product (MVP). - User demographics and mental health baseline measures (PHQ4, PHQ2, GAD2, K10 self-monitoring and general health self-tracking questionnaires). - App analytics data (bounce rates and patterns of use including total time using, and content use). - Qualitative Think Aloud semi- structured interview text and video data from beta-test and implementation phase. 2) What contextual, socio-technical, organisational and individual features support or hinder implementation? - Qualitative governance interview data with leaders in the hospital setting. - Touchpoints that emerged from the photo-interviews and deliberative workshops during design and development that were related to contextual, socio-technical, organisational and individual barriers and - Number of app downloads, use patterns, content accessed. Patterns of content engaged with, number of videos watched, time spent on content (where possible). - App analytics data (bounce rates and patterns of use including total time using, and content use). Thematic analysis of Think Aloud interviews text content examining (a) user perceptions of the app and content; and, (b) video analysis for app usability and feature engagement (attention to facial gestures, body language and user workflow). This data will be considered against the touchpoints identified in the photo-interviews and deliberative workshops to evaluate the question of whether the app meets HCWs needs. - Qualitative Think Aloud semi- structured interview text and video data from beta-test and implementation phase. - Qualitative Think Aloud semi- structured interview text and video data from beta-test and implementation phase. - Touchpoints that emerged from the photo-interviews and deliberative workshops during design and development that were related to contextual, socio-technical, organisational and individual barriers and 16 [unpublished, peer-reviewed preprint] https://preprints.jmir.org/preprint/26168 https://preprints.jmir.org/preprint/26168 JMIR Preprints Lewis et al Impact Evaluation question Data sources facilitators for implementation. Review of available mental health and wellbeing programs at the hospital. - Qualitative Think Aloud semi- structured interview text and video analysis. - Web-based implementation survey of team leaders, managers and other appropriate staff distributed via hospital contacts. 3) What are the identifiable impacts on individual HCW’s mental health through app adoption, implementation and use? - User demographics and mental health post app use measures for depression, anxiety and overall PHQ4 mental health. Impact Evaluation questions - Self-monitoring data using K10 for psychological distress - General health self-tracking questions: physical activity, relationships, productivity and llb i https://preprints.jmir.org/preprint/26168 Analytical Focus and/or Framework - First and last entry of self-tracking general health questions. - Case studies of patterns for K10 and the four general questions will be possible for further exploration of user mental 17 [unpublished, peer-reviewed preprint] 17 [unpublished, peer-reviewed preprint] https://preprints.jmir.org/preprint/26168 JMIR Preprints Lewis et al Impact Evaluation question Data sources Analytical Focus and/or Framework health patterns over time if relevant. JMIR Preprints Lewis et al 18 [unpublished, peer-reviewed preprint] https://preprints.jmir.org/preprint/26168 JMIR Preprints Lewis et al Data analysis plan Use patterns (Impact Evaluation question 1 and 3) Use patterns (Impact Evaluation question 1 and 3) Participation rates (e.g. total app downloads and bounce rates) and demographic summaries of age, gender and profession will be provided using descriptive statistics. User analytics will be described in terms of time using the app; engagement with video content (yes or no responses to whether content was helpful or not); frequency of accessing individual elements of the app; time spent watching video content; links to mental health support services; and number of uses within the evaluation period. The presentation of the technological aspect of RMHive development and implementation will follow the mobile health (mHealth) evidence reporting and assessment (mERA) reporting guidelines. [29] The user analytics overview is presented in supplementary Table 1. Engagement with the app, perceptions of content and meeting mental health needs (Impact Evaluation question 1) The photo interview and deliberative workshop text will be thematically analysed using Braun and Clarke’s approach, a theoretically flexible analysis method for qualitative data that draws out common patterns from the data that relate to the research question/s. [31] This will enable themes related to mental health need from the needs analysis to be noted and considered against themes that may emerge from the discussion of use and content in the Think Aloud interviews through the beta-test and implementation of the MVP. Video analysis will examine body language, facial expressions and discomfort or comfort in use of the app to explore engagement with the app. Voice tone will be considered for user engagement. Contextual, socio-technical, organisational and individual factors affecting implementation (Impact Evaluation question 2) The dynamic influence of contextual, socio-technical organisational and individual factors and their impact on the implementation of and engagement with RMHive will be assessed by using Normalisation Process Theory (NPT) [32] and the mERA reporting guidelines. [29] NPT is an implementation science theoretical framework that is used to evaluate the success of implementation through a focus on actions rather than beliefs or intentions. Data analysis plan [32] NPT is comprised of 4 key constructs: Coherence which relates to the level of understanding people have about an intervention and the ways they make sense of new practices or technologies; 19 [unpublished, peer-reviewed preprint] 19 [unpublished, peer-reviewed preprint] https://preprints.jmir.org/preprint/26168 JMIR Preprints Lewis et al Cognitive Participation describing the level of engagement and commitment people have to an intervention and ways they start to embed or sustain a new practice or technology; Collective Action that explores how well an intervention integrates with an organisation’s goals and activities, socio-technical workflows and compatibility with existing practices; and Reflexive Monitoring that relates to engagement in the appraisal and monitoring of the intervention and outcomes, including the extent to which individuals and groups reconfigure their practice to sustain new practices or technologies [33] The NPT framework will be applied specifically to governance interview content, the brief implementation survey and to assess the implementation strategies developed for MVP release. If there are relevant contextual or organisational themes identified from photo-interviews and the beta-test phase Think Aloud interviews, these will be coded to the NPT constructs also to support this analysis. Implementation leads (managers, team leaders and HCW members of the project team) will be provided with a link to an online based survey to identify awareness of RMHive, use and any notable barriers or challenges that have been experienced. The facilitators and barriers of RMHive uptake will be examined at an individual level through engagement with app users, and at an institutional level through ongoing governance interviews with respondents in management roles within RMH and the broader socio-cultural context. mERA will be used to describe further technical implementation. [29] Impacts on mental health (Impact Evaluation question 3) Impacts on mental health (Impact Evaluation question 3) The Impact Evaluation will examine the profile of HCWs using RMHive, how RMHive is being used by HCWs through user analytics, and how the self-reported mental health of HCWs changes over the evaluation period. On using the app for the first time, RMHive users will be prompted to establish a user profile and enter baseline data including the PHQ 4 including the PHQ2 and GAD2 subscales. The RMHive app will capture broad demographics including age range; gender; whether the person is in a leadership position, and broad professional group (allied health; medical; nursing; administrative; environmental services; other). Additionally, users will be prompted to enter subjective general health ratings of their mood; physical health; productivity; and relationships on a 3 point scale (on track; neutral; not on track). Descriptive statistics will be used to summarise socio-demographic and professional characteristics of participants, mental health responses, and subjective ratings collected at baseline and last completed measure. For continuous data with a skewed distribution, medians and quartiles will be used instead. 20 [unpublished, peer-reviewed preprint] 20 [unpublished, peer-reviewed preprint] https://preprints.jmir.org/preprint/26168 https://preprints.jmir.org/preprint/26168 JMIR Preprints Lewis et al The PHQ4 [26] will be the primary study outcome as an indicator of symptom burden, functional impairment and disability and RMHive users will be prompted to complete the PHQ4 at baseline, day 14 and day 28. The PHQ4 consists of the 2 item Generalized Anxiety Disorder scale (GAD2) anxiety subscale and the 2 item Patient Health Questionnaire (PHQ2) depression subscale. The PHQ2 assesses the presence of symptoms of depression over the last two weeks using a four-point Likert scale (0=Not at all, 1=Several days, 2=More than half the days, 3=Nearly every day). Total scores are calculated by summing the two items, and range between zero and 6. The GAD2 assesses the presence of generalised anxiety symptoms over the past two weeks using a four-point Likert scale (0=Not at all, 1=Several days, 2=More than half the days, 3=Nearly every day). GAD2 has been determined to also indicate post-traumatic stress. Scores above 3 on each subscale will indicate symptoms of depression or anxiety [26, 34, 35]. The two subscales results will be reported individually, and then summed to generate a PHQ4 score which can range from 0 to 12, with higher scores indicating an increased likelihood of underlying depressive or anxiety disorder. Impacts on mental health (Impact Evaluation question 3) Users will be provided with access to the 10 standard item Kessler Psychological Distress Scale (K10) for self-monitoring upon completing their profile in the RMHive app. Respondents are asked to indicate how often in the past four weeks they have experienced certain symptoms (e.g., nervousness, hopelessness, fatigue, agitation, and depressed mood), using a five-point Likert scale (where 1=‘not at all’ and 5=‘all the time’). The total K10 score is the sum of the 10 items, ranging from 10 to 50, where higher K10 scores indicate greater higher psychological distress. If one item on the K10 is missing a response, the missing values will be substituted with the mean response of the completed items, otherwise the total score will be coded as missing. Users will be able to self-monitor their emotional state at any time and will receive reminder prompts on day 2, day 5 and then weekly through app use. Completion of the K10 will be optional throughout the study and users will be asked about the benefits or otherwise of having access to the K10 for self-monitoring in Think Aloud interviews for the wider release. K10 results will be reported using first and last completion of the measure by users. Sub-analyses will be explored based on developing user case studies to examine over time outcomes and self-monitoring trajectories. Completion of the K10 will be optional throughout the study and users will be asked about the benefits or otherwise of having access to the K10 for self-monitoring in Think Aloud interviews for the wider release. K10 results will be reported using first and last completion of the measure by users. Sub-analyses will be explored based on developing user case studies to examine over time outcomes and self-monitoring trajectories. Primary analysis will involve repeated measures ANCOVA of the PHQ4 and subscale scores from baseline to day 14 and day 28. For users where there is only baseline data, this will be used to inform the question of overall mental health need of HCWs. For users who enter data at baseline, and again at both or either day 14 and day 28 these time points will be reported as baseline, middle and post use. For users with only two completed PHQ4 scores these will be 21 view 21 [unpublished, peer-reviewed preprint] 21 [unpublished, peer-reviewed preprint] https://preprints.jmir.org/preprint/26168 https://preprints.jmir.org/preprint/26168 JMIR Preprints Lewis et al reported as pre and post. Impacts on mental health (Impact Evaluation question 3) Analysis will progress will existing data at each time point. For the secondary analysis, linear regression will be used to estimate the difference in mean change from baseline in the mean K10 emotional state tracking and last use of K10. Data handling De-identified data with unique record identifiers for each participant will be extracted from the data collection system in the form of comma-separated value (CSV) data files. All research data will be stored in a de-identified format and will only be accessible to named research team members involved in the analysis process approved by ethics. Data transfers from the app to the evaluation team will be conducted weekly through the beta-test and following the MVP release as CSV files. The project manager will then download the CSV data files. The project manager will then save the dummy-coded files to the central password- protected University system where they will be stored securely and backed up regularly. Aggregate user analytics will be extracted using Firebase and queries analysed through Google Analytics and exported to CSV files for reporting and analysis. The data manager will then import the CSV files into Stata 15 [36] for data processing and statistical analysis. Data will be checked to identify and where possible resolve errors prior to analyses being conducted. Steps will include labelling the variables and values, creating composite variables and creating the total scores according to the instrument’s guidelines. Datasets will be merged using the unique identifier (UID) generated for each participant. De-identified data will be stored on password protected university servers with access limited to the research team for future use in accordance with the National Statement on Ethical Conduct in Human Research. [37] Discussion The adverse impact of pandemics on the mental health of HCWs has been well established [2, 3] and prior research has established that mental health supports that address the needs of HCWs are required. [13] To date, the majority of mental health support tools for HCWs addressing pandemics have not incorporated the lived-experience of end users in their development or implementation. [13] This may be a reason for the limited uptake and engagement with mobile apps. Mental health support tools developed and deployed in a compressed timeframe and without an understanding of the lived experience of HCWs and their mental health needs carry an increased risk of not being delivered in a format that is readily accessible, desired, or ultimately, used by HCWs. RMHive seeks to address these risks by using EBCD to support clinicians and researchers working together in a process of shared decision making and co-design leading to an app that centres the lived experience of HCWs as a basis for responding to their mental health needs. [10] In doing so, this extends the HCD approach further to ensure active co-design by people with lived-experience and shared power. The RMHive app is further supported by an Impact Evaluation that will provide critical insights into the contextual, socio-technical, organisational and individual factors that contribute to its implementation, engagement, and use. The Impact Evaluation [38] will expand current digital health frameworks by providing new insights into how EBCD processes inform the design, development and implementation of an app directed toward addressing HCW mental health needs. In keeping with the Impact Evaluation method, a theory of change will be produced from the evaluation to inform the future roll out and wider use of the app as a possible mental health and wellbeing intervention or support program. The impact of RMHive on HCW mental health outcomes will also be assessed. It is recognised that in this evaluation we are only focusing on near impacts of the RMHive app within the implementation context, and it is possible that the evaluation timeframe may not be sufficient for longer-term individual and organisational level changes to be observed. To our knowledge, RMHive is the first mobile-app developed using EBCD to support the mental health of HCWs in response to a pandemic. Results The RMHive program of work received funding in June 2020 and institutional ethics approval on the 9th of June, 2020. Governance structures and committees were implemented in June and data collection commenced in July. The Impact Evaluation will continue from design, development and implementation up to mid-February 2021. It is anticipated that study outcomes will be published mid-2021. 22 [unpublished, peer-reviewed preprint] https://preprints.jmir.org/preprint/26168 https://preprints.jmir.org/preprint/26168 JMIR Preprints Lewis et al scaled in response to major events such as a global pandemic. https://preprints.jmir.org/preprint/26168 Discussion It is hoped that RMHive will be a valuable support through the COVID-19 pandemic for HCWs who are experiencing increased challenges to their mental health and wellbeing. The Impact Evaluation outcomes will provide a valuable addition to local and international efforts to support HCWs mental health through the deployment of digital mental health tools that can be rapidly co-designed and 23 [unpublished, peer-reviewed preprint] 23 [unpublished, peer-reviewed preprint] https://preprints.jmir.org/preprint/26168 Lewis et al JMIR Preprints scaled in response to major events such as a global pandemic. 24 [unpublished, peer-reviewed preprint] https://preprints.jmir.org/preprint/26168 JMIR Preprints Lewis et al Acknowledgements The authors would like to acknowledge Ms Roxanne Kritharidis and Ms Maria Potiriadis for providing research support to this study. The COVID Digital Asset (COVIDDA) program of work encompasses the RMHive app and is supported by a healthcare worker advisory group and an implementation group comprised of stream leads and health care worker representatives from the hospital. Additional support was provided by the Executive Steering Committee and the Digital & Mental Health Advisory Group. Funding source This research was funded by a grant from the Australian Government Department of Health, the Peter Doherty Philanthropic Advisory Group, Royal Melbourne Hospital and The University of Melbourne. The funders had no input into the preparation of this manuscript and will not be involved in analyses for its outcomes. https://preprints.jmir.org/preprint/26168 https://preprints.jmir.org/preprint/26168 Conflicts of interest No conflicts of interest have been identified. 25 [unpublished, peer-reviewed preprint] https://preprints.jmir.org/preprint/26168 JMIR Preprints Lewis et al Abbreviations COVIDDA COVID Digital Asset CSV comma-separated value EBCD experience-based co-design GAD2 Generalised Anxiety Scale 2 item HCD Human Centred Design HCWs Health Care Worker K10 Kessler Psychological Distress Scale mERA mobile health (mHealth) evidence reporting and assessment MVP minimally viable product NPT Normalisation Process Theory PHQ4 Patient Health Questionnaire 4 item PHQ2 Patient Health Questionnaire 2 item RMH Royal Melbourne Hospital UID unique identifier 26 [unpublished, peer-reviewed preprint] https://preprints.jmir.org/preprint/26168 Lewis et al JMIR Preprints References 1. Salazar de Pablo G, Vaquerizo-Serrano J, Catalan A, Arango C, Moreno C, Ferre F, et al. Impact of coronavirus syndromes on physical and mental health of health care workers: Systematic review and meta-analysis. J Affect Disord. 2020 Oct 1;275:48-57. PMID: 32658823. doi: 10.1016/j.jad.2020.06.022. 2. Pappa S, Ntella V, Giannakas T, Giannakoulis VG, Papoutsi E, Katsaounou P. Prevalence of depression, anxiety, and insomnia among healthcare workers during the COVID-19 pandemic: A systematic review and meta-analysis. Brain Behav Immun. 2020 Aug;88:901-7. PMID: 32437915. doi: 10.1016/j.bbi.2020.05.026. 3. Ricci-Cabello I, Gangannagaripalli J, Mounce LTA, Valderas JM. Identifying Factors Leading to Harm in English General Practices: A Mixed-Methods Study Based on Patient Experiences Integrating Structural Equation Modeling and Qualitative Content Analysis. J Patient Saf. 2020 Mar 13. PMID: 32175959. doi: 10.1097/PTS.0000000000000669. 4. Petrie K, Crawford J, Baker STE, Dean K, Robinson J, Veness BG, et al. Interventions to reduce symptoms of common mental disorders and suicidal ideation in physicians: a systematic review and meta-analysis. The Lancet Psychiatry. 2019 2019/03/01/;6(3):225-34. doi: https://doi.org/10.1016/S2215-0366(18)30509-1. 5. Clough BA, March S, Leane S, Ireland MJ. What prevents doctors from seeking help for stress and burnout? A mixed-methods investigation among metropolitan and regional- based australian doctors. J Clin Psychol. 2019 Mar;75(3):418-32. PMID: 30431644. doi: 10.1002/jclp.22707. 6. Jones N, Whybrow D, Coetzee R. UK military doctors; stigma, mental health and help- seeking: a comparative cohort study. J R Army Med Corps. 2018 Aug;164(4):259-66. PMID: 29523754. doi: 10.1136/jramc-2018-000928. 7. Milner AJ, Maheen H, Bismark MM, Spittal MJ. Suicide by health professionals: a retrospective mortality study in Australia, 2001–2012. 2016;205(6):260-5. doi: 10.5694/mja15.01044. 8. Lagan S, Aquino P, Emerson MR, Fortuna K, Walker R, Torous J. Actionable health app evaluation: translating expert frameworks into objective metrics. NPJ Digit Med. 2020;3:100. PMID: 32821855. doi: 10.1038/s41746-020-00312-4. 9. Giacomin J. What Is Human Centred Design? The Design Journal. 2014 2014/12/01;17(4):606-23. doi: 10.2752/175630614X14056185480186. 10. van der Bijl-Brouwer M, Dorst K. Advancing the strategic impact of human-centred design. Design Studies. 2017 2017/11/01/;53:1-23. doi: https://doi.org/10.1016/j.destud.2017.06.003. 11. Donetto S, Pierri P, Tsianakas V, Robert G. Experience-based Co-design and Healthcare Improvement: Realizing Participatory Design in the Public Sector. The Design Journal. 2015 2015/06/01;18(2):227-48. doi: 10.2752/175630615X14212498964312. 27 [unpublished, peer-reviewed preprint] https://preprints.jmir.org/preprint/26168 JMIR Preprints Lewis et al 12. Palmer VJ, Chondros P, Piper D, Callander R, Weavell W, Godbee K, et al. References The CORE study protocol: a stepped wedge cluster randomised controlled trial to test a co-design technique to optimise psychosocial recovery outcomes for people affected by mental illness in the community mental health setting. BMJ open. 2015;5(3):e006688. doi: 10.1136/bmjopen-2014-006688. 12. 13. Drissi N, Ouhbi S, Marques G, de la Torre Díez I, Ghogho M, Janati Idrissi MA. A Systematic Literature Review on e-Mental Health Solutions to Assist Health Care Workers During COVID-19. Telemedicine and e-Health. 2020. doi: 10.1089/tmj.2020.0287. 14. Van Ameringen M, Turna J, Khalesi Z, Pullia K, Patterson B. There is an app for that! The current state of mobile applications (apps) for DSM-5 obsessive-compulsive disorder, posttraumatic stress disorder, anxiety and mood disorders. Depression and Anxiety. 2017;34(6):526-39. doi: 10.1002/da.22657. 15. Moshi MR, Tooher R, Merlin T. Suitability of current evaluation frameworks for use in the health technology assessment of mobile medical applications: a systematic review. International Journal of Technology Assessment in Health Care. 2018;34(5):464-75. doi: 10.1017/S026646231800051X. 16. White H. A Contribution to Current Debates in Impact Evaluation. Evaluation. 2010;16(2):153-64. doi: 10.1177/1356389010361562. 17. Chasanidou D, Gasparini AA, Lee E, editors. Design Thinking Methods and Tools for Innovation. 2015; Cham: Springer International Publishing. ISBN:978-3-319-20885-5 18. Melbourne Health. 2018/19 Annual Report. Melbourne, Victoria. Melbourne Health. 2019. 19. Chaffin BC, Gosnell H, A CB. A decade of adaptive governance scholarship: synthesis and future directions. Ecology and Society. 2014;19(3):56. doi: 10.5751/ES-06824-190356. 20. Janssen M, van der Voort H. Agile and adaptive governance in crisis response: Lessons from the COVID-19 pandemic. International Journal of Information Management. 2020 2020/12/01/;55:1-7. doi: 10.1016/j.ijinfomgt.2020.102180. 21. Glaw X, Inder K, Kable A, Hazelton M. Visual Methodologies in Qualitative Research: Autophotography and Photo Elicitation Applied to Mental Health Research. International Journal of Qualitative Methods. 2017 2017/12/01;16(1):1-8. doi: 10.1177/1609406917748215. 22. Palmer VJ, Furler J. A room with a view: a metaphor analysis of Vietnamese women’s representations of living with depression using photo elicitation. Visual Studies. 2018 2018/07/03;33(3):251-63. doi: 10.1080/1472586X.2018.1525309. 23. Bate P, Robert G. Experience-based design: from redesigning the system around the patient to co-designing services with the patient. Qual Saf Health Care. 2006 Oct;15(5):307- 10. PMID: 17074863. doi: 10.1136/qshc.2005.016527. 28 [unpublished, peer-reviewed preprint] https://preprints.jmir.org/preprint/26168 JMIR Preprints Lewis et al 24. Kessler RC, Barker PR, Colpe LJ, Epstein JF, Gfroerer JC, Hiripi E, et al. Screening for serious mental illness in the general population. Arch Gen Psychiatry. 2003 Feb;60(2):184-9. PMID: 12578436. doi: 10.1001/archpsyc.60.2.184. 25. 36. StataCorp. Stata Statistical Software: Release 15. . College Station, TX: StataCorp LLC.; 2017. 38. Bamberger M. Introduction to Mixed Methods in Impact Evaluation. Impact Evaluation Notes. 2012;3:1-38. References Kauer SD, Reid SC, Crooke AHD, Khor A, Hearps SJC, Jorm AF, et al. Self-monitoring using mobile phones in the early stages of adolescent depression: randomized controlled trial. Journal of Medical Internet Research. 2012;14(3):e67. PMID: 22732135. doi: 10.2196/jmir.1858. 26. Kroenke K, Spitzer RL, Williams JB, Löwe B. An ultra-brief screening scale for anxiety and depression: the PHQ-4. Psychosomatics. 2009 Nov-Dec;50(6):613-21. PMID: 19996233. doi: 10.1176/appi.psy.50.6.613. 27. Ericsson KA, Simon HA. Protocol analysis: Verbal reports as data. Cambridge, Massachusetts, United States: The MIT Press; 1984. ISBN: 9780262050470. 28. Yen P-Y, Bakken S. Review of health information technology usability study methodologies. Journal of the American Medical Informatics Association. 2011;19(3):413- 22. doi: 10.1136/amiajnl-2010-000020. 29. Agarwal S, LeFevre AE, Lee J, L'Engle K, Mehl G, Sinha C, et al. Guidelines for reporting of health interventions using mobile phones: mobile health (mHealth) evidence reporting and assessment (mERA) checklist. BMJ. 2016 Mar 17;352:i1174. PMID: 26988021. doi: 10.1136/bmj.i1174. 30. Bauer M, Glenn T, Geddes J, Gitlin M, Grof P, Kessing LV, et al. Smartphones in mental health: a critical review of background issues, current status and future concerns. Int J Bipolar Disord. 2020 Jan 10;8(1):1-19. PMID: 31919635. doi: 10.1186/s40345-019-0164-x. 31. Braun V, Clarke V. Using thematic analysis in psychology. Qualitative Research in Psychology. 2006 2006/01/01;3(2):77-101. doi: 10.1191/1478088706qp063oa. 32. May CR, Cummings A, Girling M, Bracher M, Mair FS, May CM, et al. Using Normalization Process Theory in feasibility studies and process evaluations of complex healthcare interventions: a systematic review. Implementation Science. 2018 2018/06/07;13(80):1-27. doi: 10.1186/s13012-018-0758-1. 33. May CR, Finch T, Ballini L, MacFarlane A, Mair F, Murray E, et al. Evaluating complex interventions and health technologies using normalization process theory: development of a simplified approach and web-enabled toolkit. BMC Health Services Research. 2011 2011/09/30;11(245):1-11. doi: 10.1186/1472-6963-11-245. 34. Kroenke K, Spitzer RL, Williams JB, Monahan PO, Löwe B. Anxiety disorders in primary care: prevalence, impairment, comorbidity, and detection. Ann Intern Med. 2007 Mar 6;146(5):317-25. PMID: 17339617. doi: 10.7326/0003-4819-146-5-200703060-00004. 35. Kroenke K, Spitzer RL, Williams JBW. The Patient Health Questionnaire-2: Validity of a Two-Item Depression Screener. 2003;41(11):1284-92. PMID: 00005650-200311000-00008. doi: 10.1097/01.Mlr.0000093487.78664.3c. 29 [unpublished, peer-reviewed preprint] https://preprints.jmir.org/preprint/26168 JMIR Preprints Lewis et al 36. StataCorp. Stata Statistical Software: Release 15. . College Station, TX: StataCorp LLC.; 2017. 37. National Health and Medical Research Council, Australian Research Council, Universities Australia. National Statement on Ethical Conduct in Human Research. Canberra: Commonwealth of Australia, 2007 (updated 2018). 37. 37. National Health and Medical Research Council, Australian Research Council, Universities Australia. National Statement on Ethical Conduct in Human Research. Canberra: Commonwealth of Australia, 2007 (updated 2018). https://preprints.jmir.org/preprint/26168 References National Health and Medical Research Council, Australian Research Council, Universities Australia. National Statement on Ethical Conduct in Human Research. Canberra: Commonwealth of Australia, 2007 (updated 2018). 38. Bamberger M. Introduction to Mixed Methods in Impact Evaluation. Impact Evaluation Notes. 2012;3:1-38. 38. Bamberger M. Introduction to Mixed Methods in Impact Evaluation. Impact Evaluation Notes. 2012;3:1-38. 30 [unpublished, peer-reviewed preprint] https://preprints.jmir.org/preprint/26168 JMIR Preprints Lewis et al https://preprints.jmir.org/preprint/26168 https://preprints.jmir.org/preprint/26168 Supplementary Files [unpublished, peer-reviewed preprint] https://preprints.jmir.org/preprint/26168 JMIR Preprints Lewis et al Figures Figures https://preprints.jmir.org/preprint/26168 Figures [unpublished, peer-reviewed preprint] https://preprints.jmir.org/preprint/26168 JMIR Preprints Lewis et al Planned design, development and implementation framework for RMHive. Planned design, development and implementation framework for RMHive. Planned design, development and implementation framework for RMHive. https://preprints.jmir.org/preprint/26168 [unpublished, peer-reviewed preprint] JMIR Preprints Lewis et al https://preprints.jmir.org/preprint/26168 a Appendixes https://preprints.jmir.org/preprint/26168 Supplementary Table 1. Overview of user analytics plan. URL: https://asset.jmir.pub/assets/87f06e64bf29c80e52e9a9ae9901d025.docx Multimedia Appendixes [unpublished, peer-reviewed preprint] https://preprints.jmir.org/preprint/26168 Lewis et al JMIR Preprints [unpublished, peer-reviewed preprint] https://preprints.jmir.org/preprint/26168
https://openalex.org/W3161870900	https://www.nature.com/articles/s41598-021-90194-7.pdf	English	null	A functional requirement for sex-determination M/m locus region lncRNA genes in Aedes aegypti female larvae	Scientific reports	2,021	cc-by	8,770	A functional requirement for sex‑determination M/m locus region lncRNA genes in Aedes aegypti female larvae Keshava Mysore1,2, Limb K. Hapairai1,2, Ping Li1,2, Joseph B. Roethele1,2, Longhua Sun1,2, Jessica Igiede1,2, Joi K. Misenti1,2 & Molly Duman‑Scheel1,2* OPEN Although many putative long non-coding RNA (lncRNA) genes have been identified in insect genomes, few of these genes have been functionally validated. A screen for female-specific larvicides that facilitate Aedes aegypti male sex separation uncovered multiple interfering RNAs with target sites in lncRNA genes located in the M/m locus region, including loci within or tightly linked to the sex determination locus. Larval consumption of a Saccharomyces cerevisiae (yeast) strain engineered to express interfering RNA corresponding to lncRNA transcripts resulted in significant female death, yet had no impact on male survival or fitness. Incorporation of the yeast larvicides into mass culturing protocols facilitated scaled production and separation of fit adult males, indicating that yeast larvicides could benefit mosquito population control strategies that rely on mass releases of male mosquitoes. These studies functionally verified a female-specific developmental requirement for M/m locus region lncRNA genes, suggesting that sexually antagonistic lncRNA genes found within this highly repetitive pericentromeric DNA sequence may be contributing to the evolution of A. aegypti sex chromosomes. Female mosquitoes differ from males in morphological, physiological, and behavioral traits, such as blood feeding behavior, that promote the spread of disease-causing pathogens. Although genes that regulate female-specific traits may represent novel targets for vector control, a majority of these genes have not yet been functionally characterized in mosquitoes, including Aedes aegypti, the primary vector for arboviruses that cause Zika, chi- kungunya, yellow fever, and dengue1. Moreover, population-based strategies for mosquito control, including the sterile insect technique and the incompatible insect technique, are often dependent on the mass release of adult male mosquitoes2. The identification of genes with female-specific functions could also permit the elucida- tion of genetically-based effective, affordable, and scalable mosquito sex-sorting technology that can be readily deployed worldwide, which would facilitate global implementation of emerging population-based mosquito control strategies2. g A. aegypti sex determination is regulated by a non-recombining Y-chromosome-like male determining M locus (Supplementary Fig. S1), which has a pericentromeric location on chromosome one3,4 and contains the male-determining factor Nix5. Males, which possess one copy of the chromosome bearing the M locus and one which lacks it, have an M/m genotype, while females, which lack the male determining locus, are m/m6. Although the A. aegypti M and m sex chromosomes are homomorphic, the sex-differentiated region extends to a ~ 100 Mb region that surrounds the ~ 1.5 Mb M locus7. www.nature.com/scientificreports www.nature.com/scientificreports A functional requirement for sex‑determination M/m locus region lncRNA genes in Aedes aegypti female larvae Keshava Mysore1,2, Limb K. Hapairai1,2, Ping Li1,2, Joseph B. Roethele1,2, Longhua Sun1,2, Jessica Igiede1,2, Joi K. Misenti1,2 & Molly Duman‑Scheel1,2* OPEN Rare recombination events in the M locus region, in which recombination is typically suppressed, result in sex ratio distortion8,9. These distortions suggest that clusters of loci which cause sex-specific effects reside within the sex-determining region and are gained or lost through crossover events, causing sex-specific lethality8,9. The factors, which may include genes that are vital for development or which are sexually antagonistic, may be shaping the stable boundaries of non-recombining sex chromosomes during A. aegypti sex chromosome evolution9, but the identities of these loci are unknown. g gyp Although characterization and sequencing of the M/m locus had been thwarted by the repetitive nature of DNA located in this region, recent innovations in sequencing technology generated an improved and re-anno- tated genome assembly that facilitated better estimation of the M/m locus7. The improved sequence revealed the 1Department of Medical and Molecular Genetics, Indiana University School of Medicine, Raclin‑Carmichael Hall, 1234 Notre Dame Ave., South Bend, IN 46617, USA. 2University of Notre Dame Eck Institute for Global Health, Notre Dame, IN, USA. email: mscheel@nd.edu Scientific Reports \| (2021) 11:10657 \| https://doi.org/10.1038/s41598-021-90194-7 www.nature.com/scientificreports/ Figure 1. siRNAs that induce significant female-specific lethality. Significant female-specific larval mortality resulted from soaking treatments with the indicated siRNAs ( = p < 0.05, = p < 0.01, and * = p < 0.001, Chi- square). No significant differences (p > 0.05, Chi-square) were observed in male survival following treatments with these siRNAs, and a control siRNA had no significant impact (p > 0.05, Chi-square) on survival of males or females. Data are represented as mean survival based on adult emergence, (n = 40 larvae/treatment), and error bars denote SEM. Figure 1. siRNAs that induce significant female-specific lethality. Significant female-specific larval mortality resulted from soaking treatments with the indicated siRNAs (* = p < 0.05, = p < 0.01, and * = p < 0.001, Chi- square). No significant differences (p > 0.05, Chi-square) were observed in male survival following treatments with these siRNAs, and a control siRNA had no significant impact (p > 0.05, Chi-square) on survival of males or females. Data are represented as mean survival based on adult emergence, (n = 40 larvae/treatment), and error bars denote SEM. presence of many putative long non-coding RNA (lncRNA) genes in the M/m locus sex-determining region7,10. lncRNAs are a class of non-coding transcripts that are > 200 nucleotides in length which are not translated into proteins11. A functional requirement for sex‑determination M/m locus region lncRNA genes in Aedes aegypti female larvae Keshava Mysore1,2, Limb K. Hapairai1,2, Ping Li1,2, Joseph B. Roethele1,2, Longhua Sun1,2, Jessica Igiede1,2, Joi K. Misenti1,2 & Molly Duman‑Scheel1,2* OPEN lncRNAs regulate a wide array of cellular activities, such as the recruitment of chromatin modifiers and transcription factors, the regulation of chromosome looping, microRNA sequestration, and translational control12. Although thousands of putative lncRNAs have been annotated in insect genomes13, including A. aegypti10,14–16, very few have been functionally validated as lncRNAs. Two of the most well-characterized insect lncRNAs, Drosophila melanogaster roX1 and roX2, function as components of the Male Specific Lethal (MSL) complex, which regulates dosage compensation by upregulating X-linked gene expression in male fruit flies17,18. Mammals regulate dosage compensation through random inactivation of one X chromosome, a process that is regulated by X inactive specific transcript (Xist), an lncRNA which promotes chromatin silencing19,20. Given the critical sex-specific roles of these lncRNAs, it was hypothesized that A. aegypti M/m locus region lncRNA loci encode functional transcripts that have evolved sex-specific functions.i p pi In this investigation, a small interfering RNA (siRNA) screen for female-specific larval lethal genes uncovered multiple lncRNA genes located at or tightly linked to the M/m locus, a chromosomal location herein referred to as the M/m locus region (Supplementary Fig. S1). Silencing several of these transcripts with yeast interfering RNA technology revealed a female-specific requirement for M/m locus region lncRNAs in larvae that could be exploited for the development of scalable male sex separation strategies. These findings suggest that lncRNA genes in the M/m locus region may be contributing to sex chromosome evolution in A. aegypti. Results and discussioni Results and discussion A screen identifies siRNAs that induce female‑specific larval mortality. Recent high-throughput screens in which first instar (L1) larvae were briefly soaked in siRNAs led to the discovery of hundreds of pro- tein-coding larval lethal genes and a new class of RNAi-based mosquito insecticides21–23. Given that the M/m locus is believed to be tightly linked to developmental genes that confer sex-specific lethal effects in A. aegypti8,9, lncRNA loci located both within, as well as flanking the M/m locus, were evaluated in a female larval lethal soak- ing screen that employed a similar strategy. These studies permitted functional assessment of the hypothesis that silencing A. aegypti M/m locus region lncRNA genes during larval development would induce female-specific lethality. A total of 50 siRNAs corresponding to lncRNA genes in and flanking the M/m locus, which is referred to herein as the M/m locus region (Supplementary Fig. S1), were screened (Supplementary Tables S1, S2, S3, S4).h Results and discussion A screen identifies siRNAs that induce female‑specific larval mortality. Recent high-throughput screens in which first instar (L1) larvae were briefly soaked in siRNAs led to the discovery of hundreds of pro- tein-coding larval lethal genes and a new class of RNAi-based mosquito insecticides21–23. Given that the M/m locus is believed to be tightly linked to developmental genes that confer sex-specific lethal effects in A. aegypti8,9, lncRNA loci located both within, as well as flanking the M/m locus, were evaluated in a female larval lethal soak- ing screen that employed a similar strategy. These studies permitted functional assessment of the hypothesis that silencing A. aegypti M/m locus region lncRNA genes during larval development would induce female-specific lethality. A total of 50 siRNAs corresponding to lncRNA genes in and flanking the M/m locus, which is referred to herein as the M/m locus region (Supplementary Fig. S1), were screened (Supplementary Tables S1, S2, S3, S4). The soaking screen uncovered a total of 19 siRNAs (Supplementary Tables S1 and S2) corresponding to M/m locus region lncRNAs that induced significant female-specific mortality and had no significant impact on male survival (Fig. 1). These siRNAs corresponded to target sites in 25 M/m locus region lncRNA genes, the identifica- tion numbers and chromosomal locations of which are provided in Supplementary Fig. S1 and Supplementary Table S3. Some of the siRNAs corresponded to target sites in singular M/m locus region lncRNA genes (Sup- plementary Table S1). www.nature.com/scientificreports/ These genes (Sup- plementary Table S4) may lack sex-specific functions or could be active during different stages of the life cycle. It is also possible that siRNAs targeting different sites in these same genes could produce more effective silencing and yield female-specific killing. However, given the overall success of the screen, which had already identified multiple female-specific larvicides (Fig. 1), evaluation of additional siRNAs and further characterization of these particular lncRNA genes (Supplementary Table S4) were not pursued at this time. Generation of a female‑specific yeast larvicide that targets M/m locus region lncRNA genes. In recent years, S. cerevisiae has been developed as a system for inexpensive and scalable manufacture of larvicidal interfering RNAs23. The yeast can also be used as a delivery system for RNAi larvicides to mosqui- toes, in which effective gene silencing is observed in larvae that consume the larvicides in the form of inactivated yeast tablets21–23. Yeast RNAi larvicide technology, which could also potentially facilitate scaled culturing and sex separation of male mosquitoes, was therefore used for further characterization of several lncRNAs identi- fied in the screen. siRNA 478, which induced significant levels of female mortality, but which did not have a significant impact on male survival (Fig. 1), was down-selected for these studies. A yeast strain designed to express a short hairpin RNA (shRNA) corresponding to the siRNA 478 target site was generated. shRNA expres- sion was confirmed in this strain, as well as a control interfering RNA strain, through PCR amplification of cDNA corresponding to the 3’ end of each hairpin and the terminator sequence that had been prepared from total RNA extractions from each strain (Supplementary Fig. S2). Dried inactivated yeast prepared from each of these strains was fed to larvae throughout larval development. Treatment with the yeast larvicide, but not con- trol interfering RNA yeast, resulted in significantly higher male:female ratios among the surviving mosquitoes (Fig. 2a; p < 0.001). Yeast larvicide #478 was therefore characterized in further detail.i ( g ; p ) When larvae were reared on yeast larvicide #478, although no significant impact on male survival was observed, only 10 ± 2% of expected adult females emerged (p < 0.001), with the bulk of these animals dying as fourth instar larvae (Supplementary Fig. S2b). Yeast larvicide #478 targets three M/m locus region loci: AAEL020379, AAEL020813, and AAEL022952 (Supplementary Table S2). www.nature.com/scientificreports/ the female-specific larvicidal siRNAs identified in the screen corresponded to target sites identically conserved in multiple lncRNA genes, at least one of which resides in the M/m locus region (Supplementary Table S2).hii the female-specific larvicidal siRNAs identified in the screen corresponded to target sites identically conserved in multiple lncRNA genes, at least one of which resides in the M/m locus region (Supplementary Table S2).hii p g , g ( pp y ) The lncRNA genes identified in the female-specific lethal screen were located throughout the M/m locus region (Supplementary Table S3). A majority (17 of 25) of the lncRNA genes are intergenic, though several (8 of 25) are intragenic (Supplementary Table S3). None of the lncRNA genes identified in the screen have known orthologs reported in Vectorbase10, potentially because few lncRNA genes have been annotated in other mos- quito species. Alternatively, a recent comparative analysis of the genomes of several Anopheles species revealed that female-biased protein-coding genes evolve more rapidly in sequence, expression, and genic turnover than male-biased protein-coding genes; this is an atypical pattern that is proposed to have resulted from sex-specific life history challenges, such as blood feeding, that are encountered by female mosquitoes24, and which could also apply to lncRNA genes. Several of the genes were located within the M locus in a region that was not thought to be found in female (genotype m/m) mosquitoes (Supplementary Fig. S1). For example, a perfect match for the siRNA 470 target sequence is only known to reside in AAEL026346, which lies between the two male-specific M locus genes myo-sex (AaegL5_1: 151,955,864–152,241,832) and Nix (AaegL5_1:152,616,641–152,718,167). Although siRNA 470 and the other siRNAs identified in the screen are not known to have identical target sites in mature transcripts that correspond to genes other than those noted in Supplementary Tables S1, S2, and S3, it is possible that the female-specific phenotypes observed could result, at least in part, from off-site targeting. Moreover, it is also possible that these siRNAs specifically target genes located in the known gap in the sequence at the sex determination locus, a region which has not yet been successfully sequenced but is believed to be highly repetitive7. g y p Finally, 31 of the siRNAs screened had identically conserved target sites in M/m locus region lncRNA genes, but had no significant impact on female or male larval survival (Supplementary Table S4). Results and discussioni Due to the highly repetitive nature of DNA located in this pericentric region10, several of g pp y g pp y The soaking screen uncovered a total of 19 siRNAs (Supplementary Tables S1 and S2) corresponding to M/m locus region lncRNAs that induced significant female-specific mortality and had no significant impact on male survival (Fig. 1). These siRNAs corresponded to target sites in 25 M/m locus region lncRNA genes, the identifica- tion numbers and chromosomal locations of which are provided in Supplementary Fig. S1 and Supplementary Table S3. Some of the siRNAs corresponded to target sites in singular M/m locus region lncRNA genes (Sup- plementary Table S1). Due to the highly repetitive nature of DNA located in this pericentric region10, several of https://doi.org/10.1038/s41598-021-90194-7 Scientific Reports \| (2021) 11:10657 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ The sequences of these genes (both exons and an intron) are identical7,10 and correspond to a single transcript that is detected throughout larval development (Fig. 2b), is expressed at comparable levels in male and female larvae just prior to the time of death (Fig. 2c, p > 0.05), and which is silenced though treatment with yeast larvicide #478 (Fig. 2d; 71.1 ± 7.9% reduction in transcript levels with respect to larvae reared on control interfering RNA yeast, p < 0.001). Scaled production of adult male mosquitoes. Male mosquitoes released en masse for control strate- gies such as the incompatible insect technique (IIT) and sterile insect technique (SIT) must successfully compete with wild-type males in areas in which they are mass-released2,25–27. It is therefore critical that yeast larvicides used for sex separation are specific to females and do not have undesired impacts on adult males. To examine if the impact of yeast larvicide #478 is specific to female larvae, life history traits were assessed in adult male mos- quitoes that had been reared on the larvicide during larval development. Treatment with yeast larvicide #478 did not significantly impact the capacity of males to mate (Fig. 3a). The number of eggs laid (fertility) by wild-type females that mated with males treated with the larvicide, as well as the percentage of larvae that hatched from these eggs (fecundity) did not significantly differ from control male matings (Fig. 3b).i gg y gi yf g g Mass rearing facilities utilize special larval diets that are optimized to produce fit male mosquitoes28. It is therefore helpful if yeast larvicides are compatible with these diets. Dried inactivated nutritional yeast is often a component of such diets28, suggesting that the nutritional yeast component could be replaced with female-specific yeast larvicides. To assess whether use of the larvicides would facilitate scaled production of males, a larval diet employed at mass-rearing mosquito facilities28 was modified by replacing the nutritional yeast component of the diet with dried inactivated yeast larvicide #478. The modified diet was tested on mosquitoes grown in mass- rearing trays containing 200 larvae/L of water. With respect to the control interfering RNA diet, larvicide #478 Scientific Reports \| (2021) 11:10657 \| https://doi.org/10.1038/s41598-021-90194-7 www.nature.com/scientificreports/ Figure 2. A female-specific lethal yeast interfering RNA larvicide targeting lncRNA genes. Significant female larval mortality resulted from oral consumption of yeast interfering RNA larvicide strain #478 [(a), * = p < 0.001, Chi-square]. www.nature.com/scientificreports/ Silencing of these lncRNA targets of larvicide #478 (d) was confirmed through qRT-PCR (* = p < 0.001 vs. target gene levels in control interfering RNA-fed larvae, Student’s t-test; error bars denote standard deviation). Figure 2. A female-specific lethal yeast interfering RNA larvicide targeting lncRNA genes. Significant female larval mortality resulted from oral consumption of yeast interfering RNA larvicide strain #478 [(a), * = p < 0.001, Chi-square]. No significant death was observed in males following treatments with larvicide [(a); p > 0.05, Chi-square], and a control interfering RNA strain did not significantly impact survival of male or female larvae [(a) p > 0.05, Chi-square; data are represented as mean survival based on adult emergence following treatment of 180 total larvae, and error bars denote SEM]. (b–d) display quantification of the identical AAEL020379, AAEL020813, and AAEL022952 transcripts, with mRNA levels normalized to levels of the rpS17 housekeeping gene; error bars denote standard deviations. The #478 larvicide target transcripts are expressed throughout larval development in untreated first (L1), second (L2), third (L3), and fourth (L4) instar larvae (b), with no significant differences in expression levels noted between the various larval stages (p > 0.05, ANOVA; the expression levels are shown relative to L4). No significant differences in transcript levels were noted between third instar male and female larvae [(c), t-test, p > 0.05]. Silencing of these lncRNA targets of larvicide #478 (d) was confirmed through qRT-PCR (* = p < 0.001 vs. target gene levels in control interfering RNA-fed larvae, Student’s t-test; error bars denote standard deviation). induced significant female mortality, resulting in 5 male:1 female ratios in emerging adults (Fig. 3c). The fitness of male survivors, which was ascertained through measurements of wing lengths, a proxy for body size and fitness, was not significantly different than males raised on the standard mass-rearing diet (Fig. 3d), providing further evidence that the larvicide is lethal to females, but does not impact male mosquitoes. Although the yeast larvicides characterized here do not eliminate all females and could not be used in a stand-alone capacity, replacing nutritional yeast with the larvicidal yeast could further improve the efficacy of existing sex separation technologies2 or immensely reduce labor associated with hand separation strategies. Yeast interfering RNA technology, which could be implemented in remote and resource-limited locations, would likely benefit mass-rearing facilities worldwide. www.nature.com/scientificreports/ No significant death was observed in males following treatments with larvicide [(a); p > 0.05, Chi-square], and a control interfering RNA strain did not significantly impact survival of male or female larvae [(a) p > 0.05, Chi-square; data are represented as mean survival based on adult emergence following treatment of 180 total larvae, and error bars denote SEM]. (b–d) display quantification of the identical AAEL020379, AAEL020813, and AAEL022952 transcripts, with mRNA levels normalized to levels of the rpS17 housekeeping gene; error bars denote standard deviations. The #478 larvicide target transcripts are expressed throughout larval development in untreated first (L1), second (L2), third (L3), and fourth (L4) instar larvae (b), with no significant differences in expression levels noted between the various larval stages (p > 0.05, ANOVA; the expression levels are shown relative to L4). No significant differences in transcript levels were noted between third instar male and female larvae [(c), t-test, p > 0.05]. Silencing of these lncRNA targets of larvicide #478 (d) was confirmed through qRT-PCR (* = p < 0.001 vs. target gene levels in control interfering RNA-fed larvae, Student’s t-test; error bars denote standard deviation). Figure 2. A female-specific lethal yeast interfering RNA larvicide targeting lncRNA genes. Significant Figure 2. A female-specific lethal yeast interfering RNA larvicide targeting lncRNA genes. Significant female larval mortality resulted from oral consumption of yeast interfering RNA larvicide strain #478 [(a), * = p < 0.001, Chi-square]. No significant death was observed in males following treatments with larvicide [(a); p > 0.05, Chi-square], and a control interfering RNA strain did not significantly impact survival of male or female larvae [(a) p > 0.05, Chi-square; data are represented as mean survival based on adult emergence following treatment of 180 total larvae, and error bars denote SEM]. (b–d) display quantification of the identical AAEL020379, AAEL020813, and AAEL022952 transcripts, with mRNA levels normalized to levels of the rpS17 housekeeping gene; error bars denote standard deviations. The #478 larvicide target transcripts are expressed throughout larval development in untreated first (L1), second (L2), third (L3), and fourth (L4) instar larvae (b), with no significant differences in expression levels noted between the various larval stages (p > 0.05, ANOVA; the expression levels are shown relative to L4). No significant differences in transcript levels were noted between third instar male and female larvae [(c), t-test, p > 0.05]. www.nature.com/scientificreports/ Yeast larvicide #478 does not significantly impact male mating capacity [(a), p > 0.05, Student’s t-test], the number of eggs laid by females that mated with these males [(b), p > 0.05, Student’s t-test], or the percentage of larvae that hatched from these eggs [(b), p > 0.05, Student’s t-test]; results were compiled from 41 matings with #478-treated males and 72 matings with males treated with control interfering RNA yeast). Incorporation of the yeast larvicide into a larval diet used for mass-rearing (MR; n = 1200 total larvae per treatment) resulted in significant female mortality [(c), * = p < 0.001, Chi-square] with no significant impact on male survival [(c), p > 0.05, Chi- square] or fitness [(d), p > 0.05, Student’s t-test; n = 83 control diet male wings, n = 40 #478-treated male wings]. Error bars denote SEM in all panels. chromosomes. These initial lncRNA studies have elucidated key findings that may help shape our understanding of sex chromosome evolution.h The evolution of sex chromosomes is believed to occur in several stages29–31. Initially, a homologous pair of autosomes acquires sex-determining loci, forming a proto-Y chromosome bearing a male fertility locus (M) and a dominant female suppressor (SuF), as well as a proto-X chromosome carrying a female fertility locus (F) and a male sterility locus (m). Suppressed recombination in the sex-determining region evolves and eventually spreads over a larger portion of the proto-sex chromosomes. The A. aegypti homomorphic sex chromosomes appear to have evolved into proto-sex chromosomes bearing a sex determining M/m region3,4 which contains a male-determining factor, Nix5, that is present on the proto-Y chromosome. Nix regulates male-specific splicing of another chromosome 1 gene, doublesex (dsx), permitting expression of the male-specific splice form of dsx32 rather than the female splice form which is important for ovary development and fertility33. A sex-differentiated region of suppressed recombination has also evolved and is believed to have extended ~ 100 Mb beyond the M/m locus7,34,35.h The suppression of recombination on sex chromosomes permits accumulation of transposable elements and other non-coding sequences, as well as chromosomal rearrangements and the acquisition of sexually antagonistic genes with different alleles that differentially benefit either males or females29–31. Further loss of recombination between these genes and the sex-determination locus is expected to follow, eventually resulting in evolution of heteromorphic X and Y chromosomes31. www.nature.com/scientificreports/ Moreover, the use of yeast interfering RNA larvicides would circumvent a need to further genetically manipulate existing mosquito strains that have already been developed for popula- tion control strategies, for which regulatory permits may have already been attained or might need to be acquired. Conclusions and potential implications for understanding the evolution of sex chromosomes in A. aegypti. In summary, these studies functionally verified a female larval requirement for multiple lncRNA genes located at the M/m locus region (Figs. 1, 2, Supplementary Fig. S1). In multiple instances, silenc- ing lncRNA genes resulted in significantly increased male:female ratios that resulted from female lethality, with- out any significant impact on male survival or fitness (Figs. 1, 2, 3). The complete phased structure of the male M locus and the female m locus have not yet been determined, and a ~ 163 kb gap in the sequence remains7. Com- pletion of the entire phased sequence will undoubtedly facilitate further interpretation and a more sophisticated understanding of these lncRNA screen data. Nevertheless, as predicted by Matthews et al.7, the availability of the existing M locus assembly has provided the opportunity to study the evolution of A. aegypti homomorphic sex Scientific Reports \| (2021) 11:10657 \| https://doi.org/10.1038/s41598-021-90194-7 www.nature.com/scientificreports/ Figure 3. Yeast interfering RNA larvicide technology can be used for scaled production of males. Yeast larvicide #478 does not significantly impact male mating capacity [(a), p > 0.05, Student’s t-test], the number of eggs laid by females that mated with these males [(b), p > 0.05, Student’s t-test], or the percentage of larvae that hatched from these eggs [(b), p > 0.05, Student’s t-test]; results were compiled from 41 matings with #478-treated males and 72 matings with males treated with control interfering RNA yeast). Incorporation of the yeast larvicide into a larval diet used for mass-rearing (MR; n = 1200 total larvae per treatment) resulted in significant female mortality [(c), * = p < 0.001, Chi-square] with no significant impact on male survival [(c), p > 0.05, Chi- square] or fitness [(d), p > 0.05, Student’s t-test; n = 83 control diet male wings, n = 40 #478-treated male wings]. Error bars denote SEM in all panels. Figure 3. Yeast interfering RNA larvicide technology can be used for scaled production of males. Methods M it Mosquito rearing. The A. aegypti Liverpool-IB12 (LVP-IB12) strain used in this investigation was reared as described40 in an insectary maintained at 26° C, ~ 80% relative humidity, and with a 12 h light/12 h dark cycle with 1 h crepuscular periods at the beginning and end of each cycle. Sheep blood meals (purchased from HemoStat Laboratories, Dixon, CA) were provided to adult females using a Hemotek artificial membrane feed- ing system (Hemotek Limited, Blackburn, UK). Larval soaking screen. 50 custom siRNAs corresponding to target sequences in annotated lncRNA genes located in the M/m locus region on chromosome one (Supplementary Tables S1, S2, S3, S4) were selected using the Integrated DNA Technologies (IDT) Custom Dicer-Substrate siRNA (DsiRNA) tool41. In several cases, this custom siRNA design tool identified target sites that were conserved in multiple lncRNA target genes (Supple- mentary Table S2); these siRNAs were also screened to ascertain their potential use in sex-separation strategies. Custom siRNAs, as well as a control siRNA with no known target in mosquitoes42, were purchased from IDT (Coralville, IA). For the screen, larval soaking experiments were performed (per the methodology of Singh et al.43) in duplicate with 20 L1 larvae soaked in 20 ul of control or experimental siRNA at a concentration of 0.5 ug/ul for 4 h. Following soaking, larvae were reared as described44 in accordance with the WHO45 guidelines for larvicide testing. Corrections for control larval death in this assay, as well as other larvicide assays conducted in this study, were not necessary, as control mortality rates were negligible. The Chi-square test was used to test deviations from the expected survival of male and female mosquitoes (which was set at 1 male:1 female based on assessment of the sexes of untreated larvae from this strain that were reared in the insectary as described). Production and evaluation of yeast interfering RNA larvicides. Previously described methods for larvicidal yeast preparation21 were used to generate the yeast strains used in this investigation. A more detailed overview of this methodology was provided in a recent methods chapter44. In summary, a custom shRNA expression cassette corresponding to the siRNA #478 target sequence (Supplementary Tables S1, S2) was syn- thesized by Invitrogen (Carlsbad, CA) and cloned into the pRS426 GPD shuttle vector46. Following restriction digestion and sequencing to confirm the inserts, the plasmids were transformed into S. www.nature.com/scientificreports/ that functional lncRNA genes that are required in female larvae are located in this region. Given that retrotrans- posons can contribute to both the origin and diversification of lncRNAs36, one could speculate that accumula- tion of retrotransposons in A. aegypti has also contributed to the origin and diversification of M/m locus region lncRNA genes that evolved female-specific functions. It is predicted that these genes may eventually contribute to the formation of heteromorphic A. aegypti sex chromosomes and lead to genetic degeneration and reduced size of the Y chromosome29–31.i lncRNAs regulate a wide array of cellular activities that could contribute to sex-specific gene expression during sexually dimorphic development or differentiation, including the regulation of chromatin modifiers12. Although A. aegypti is not yet believed to possess dosage compensation, recent studies suggest that the region of non-recombination between M and m chromosomes is more extensive than previously believed7,34,35, suggesting that the evolution of such dosage compensation mechanisms could eventually initiate in A. aegypti. Interest- ingly, centromeric repeats in Saccharmocyes pombe produce dsRNA that targets formation and maintenance of heterochromatin through RNA interference (RNAi)37, which occurs through sequence-specific targeting of histone modifications regulated by small RNA silencing38. Woolcock et al.39 demonstrated that RNAi proteins interact with ncRNAs and retrotransposon long terminal repeats. The authors39 speculate that similar mecha- nisms could operate in other eukaryotes. Future studies will consider if lncRNAs regulate heterochromatin at the A. aegypti sex determination locus and elucidate the sex-specific molecular functions of lncRNAs in A. aegypti and other species of mosquitoes. Yeast interfering RNA technology, which may benefit efforts to mass produce male mosquitoes for emerging mosquito control programs, will likewise enhance future laboratory studies aimed at dissecting the molecular functions of mosquito lncRNAs during sex-specific development and differentiation. www.nature.com/scientificreports/ Highly repetitive DNA, which comprises > 70% of the M locus and includes long terminal repeat retrotransposons7, has accumulated in A. aegypti. This investigation has revealed Scientific Reports \| (2021) 11:10657 \| https://doi.org/10.1038/s41598-021-90194-7 www.nature.com/scientificreports/ Methods M it The analyses of lncRNA expression in different larval stages included two different pools of 10 untreated larvae of each of the following larval instars : L1, L2 L3, and L4. Total RNA from the separate pools was isolated, processed, and analyzed as described above. qRT-PCR results were standardized by setting the expression level of L4 larvae to one. Data were analyzed through ANOVA followed by Tukey’s post hoc test. Analysis of lncRNA expression in untreated individual male vs. female larvae. Following preparation of total RNA from individual L3 larvae using the methodology described above, genomic DNA was isolated from each individual larva using the TRIzol reagent according to the manufacturer’s instructions. Each individual was then sexed through amplification of this genomic DNA using standard PCR assays in conjunction with the follow- ing primers, which correspond to the M locus male-specific myosin heavy chain gene myo-sex (AAEL021838): forward: 5′-CGCTTTCTGGGGAAAAGGG-3′ and reverse: 5′-CTTTGGAGGCCTTGTCCTGT-3′. Following confirmation of the sex of each larva, expression of the identical AAEL020379, AAEL020813, and AAEL022952 (#478 target) transcripts was performed as detailed above in individual males (n = 6) and individual females (n = 7). Data were statistically evaluated with the Student’s t-test. Verification of target gene silencing. For verification of #478 yeast larvicide target gene silencing (identical tran- scripts AAEL020379, AAEL020813, and AAEL022952), total RNA was extracted as described above from two different pools of 20 larvae that had been reared on #478 or control yeast. qRT-PCR was then performed as detailed above using rpS17 expression as the internal standard for data normalization. Results were expressed as fold-differences in the #478-larvicide treated larvae compared to larvae that had fed on control interfering RNA yeast. Data were statistically evaluated with the Student’s t-test. Mass rearing experiments. 200 LVP-IB12 first instar (L1) larvae, which had been hatched from eggs placed under vacuum for 1 h to synchronize hatching, were distributed in 34 × 25 × 4 cm trays (1426B, Bio- quip, Rancho Dominquez, CA) containing 1 L of distilled water. The larvae were reared on yeast + liver pow- der + shrimp (YSL) diet, which was developed based on the mass rearing diet of Zhang et al.28. Methods M it cerevisiae CEN.PK strain yeast (genotype MATa/α ura3-52/ura3-52 trp1-289/trp1-289 leu2-3_112/leu2-3_112 his3 Δ1/his3 Δ1 MAL2-8C/ MAL2-8C SUC2/SUC247), and transformants were selected by growth on minimal media lacking uracil. shRNA expression in these strains was confirmed in each of two biological replicate trials performed as described48. In summary, cDNA prepared from total RNA extracted from the yeast was used as template in PCR amplifications performed with a forward primer corresponding to the 3’ end of each hairpin construct and a reverse primer corresponding to the terminator sequence in each construct. Primer sequences were as follows: Control shRNA Forward 5′-ACGCTAACATCTATCAGTGC-3′ (specific to control shRNA), #478 shRNA forward 5′-TTTATA CTAATTCCAGACATTAGTC-3′ (specific to #478 shRNA), and reverse primer 5′-TCCTTCCTTTTCGGTTAG AGC-3′ (which matches the terminator sequence in all three strains). PCR products were visualized with eth- idium bromide staining following gel electrophoresis. The original image of this gel (Supplementary Fig. S1) was labeled using Adobe Photoshop 2021 software. g pt For larvicide assays, dried inactivated yeast interfering RNA was prepared from the control and #478 strains as described44. As discussed in Hapairai et al.21, larval bioassays that conformed to the WHO45 guidelines for larvicide testing were performed in small container trials conducted as described44. In the small container assays, 20 newly hatched L1 larvae were placed in 500 ml plastic cups containing 50 ml of distilled water and a yeast tablet, which was sufficient to permit ad libitum yeast consumption throughout development. Adult emergence rates and the sexes of these adults were recorded. Control and larvicidal yeast tablets were evaluated in nine replicate container trials for each treatment. The Chi-square test was used to test for deviations from 1:1 male to female ratios, female and male survival for each treatment. Scientific Reports \| (2021) 11:10657 \| https://doi.org/10.1038/s41598-021-90194-7 www.nature.com/scientificreports/ Evaluation of male life history traits. The number of eggs produced per female (fecundity) and eggs produced per female that generated first instar larvae (fertility) were assessed as described by Hill et al.49 in individual females that had been mated to individual male survivors which fed on #478 or control interfering RNA yeast, or bovine liver powder (MP Biomedicals, Santa Ana, CA; also a control diet). Methods M it The number of fertile females, which served as evidence of successful mating with the treated male survivors, was also recorded; for females that did not lay eggs, mating success or failure was further assessed through dissection of the sper- matheca to discern the presence or absence of sperm. Results from the indicated numbers of matings combined from multiple biological replicate trials were assessed: #478-treated males (n = 41 matings), male mosquitoes fed with a standard larval laboratory diet of bovine liver powder40 (n = 75 matings), and males that fed on con- trol yeast interfering RNA strain tablets (n = 72 matings). Data were analyzed through ANOVA as described33. Following mass-rearing trials (see below), male fitness was assessed through evaluation of wing lengths in sur- viving males, which were measured from the apical notch to the axillary margin, excluding the wing fringe as described33. Wing lengths of males combined from each of two replicate trials (see further information about experimental setup below) were compared using one-way ANOVA. qRT‑PCR. qRT-PCR assays were performed using previously described methods50. In summary, total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA) from pooled or individual larvae (see further details for each specific experiment below). Total RNA was DNase treated using the DNA-free kit (Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts) according to the recommendations of the manufacturer. cDNA was prepared according to the manufacturer’s instructions using the High Capacity RNA to cDNA Kit (Applied Biosystems, Foster City, CA). Real-time quantification was performed using the Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) in conjunction with an Applied Biosystems Step One Plus Real-Time PCR System. The following primer sets were used for quantification of the identical transcripts encoded by the yeast larvicide #478 target genes AAEL020379, AAEL020813, and AAEL022952: #478 forward 5′-GAAAAACGCAGT TGCGGACT-3′ and #478 reverse 5′-TGCACTTAACCTACAATGCTACA-3′. As previ- ously described50, primers for the housekeeping gene rpS17, which was used as the internal standard for data normalization in all qRT-PCR assays performed in this investigation were: forward 5′-AGACAACTACGTGCC GGAAG-3′and reverse 5′-TTGGTGACCTGGACAACGATG-3′. All PCR reactions were performed in 3–6 rep- licate wells in each of two biological replicate experiments. Results from qRT-PCR were quantified by standard- izing reactions to rpS17 levels using the ΔΔCt method as described50. Larval stage‑specific analysis of lncRNA expression in untreated larvae. Methods M it This diet, which was used as a control in the present studies, consisted of 1000 mg brewer’s yeast (Spring Valley, Bentonville, AR), 250 mg of bovine liver powder (MP Biomedicals, Santa Ana, CA), and 150 mg of ground baby shrimp (Tetra GMBH, Melle, Germany) provided ad libitum. The dry components were mixed by hand into 10 ml of distilled water, producing a slurry that was stored at 4 °C. For sex-sorting experiments, the slurry was prepared without brewer’s yeast and provided to the larvae ad libitum; yeast interfering RNA tablets were fed to the larvae as fol- lows: two tablets at L1, two tablets at L2, two tablets at L3, and four tablets at L4. The tablets were resuspended in distilled water and mixed with the liver powder-shrimp slurry, and the combined mixture was fed to larvae. Larval trays were examined daily for pupae, which were removed on the day of pupation, manually sorted by sex, and counted. https://doi.org/10.1038/s41598-021-90194-7 Scientific Reports \| (2021) 11:10657 \| www.nature.com/scientificreports/ Sex ratio distortion and mortality were evaluated in three biological replicate trials, each with two replicate trays of larvae fed on YSL diets prepared with the brewer’s yeast or #478 yeast. The Chi-square test was used to test deviations from 1:1 male to female ratios for each treatment. Mortality levels for males and females for each treatment were compared according to the WHO45 guidelines using one-way ANOVA followed by Tukey’s post hoc multiple comparisons on data that had been arcsin transformed. Male fitness was assessed through measure- ment of wing lengths as described above (n = 83 control diet males; n = 40 #478-treated males). Ethics statement. No human subjects or vertebrate animals were used in this investigation. References VectorBase: An updated bioinformatics resource for invertebrate vectors and other organisms related with human diseases. Nucleic Acids Res. 43, D707-713. https://doi.org/10.1093/nar/gku1117 (2015). y p g y 10. Giraldo-Calderon, G. I. et al. VectorBase: An updated bioinformatics resource for invertebrate vectors and other organisms re with human diseases. Nucleic Acids Res. 43, D707-713. https://doi.org/10.1093/nar/gku1117 (2015). 1. Kapranov, P. et al. RNA maps reveal new RNA classes and a possible function for pervasive transcription. Science 316, 1484–1488 https://doi.org/10.1126/science.1138341 (2007).f p g 2. Neguembor, M. V., Jothi, M. & Gabellini, D. Long noncoding RNAs, emerging players in muscle differentiation and disease. Skele Muscle 4, 8. https://doi.org/10.1186/2044-5040-4-8 (2014).i p g ( ) 13. Legeai, F. & Derrien, T. Identification of long non-coding RNAs in insects genomes. Curr. Opin. Insect Sci. 7, 37–44 (2015).i gi g g g p ( ) 14. Etebari, K., Asad, S., Zhang, G. & Asgari, S. Identification of Aedes aegypti long intergenic non-coding RNAs and their associ with Wolbachia and dengue virus infection. PLoS Negl. Trop. Dis. 10, e0005069. https://doi.org/10.1371/journal.pntd.000 (2016). 15. Nene, V. et al. Genome sequence of Aedes aegypti, a major arbovirus vector. Science 316, 1718–1723. https://doi.org/10.1126/scien ce.1138878 (2007).i 16. Azlan, A., Obeidat, S. M., Yunus, M. A. & Azzam, G. Systematic identification and characterization of Aedes aegypti long noncoding RNAs (lncRNAs). Sci Rep 9, 12147. https://doi.org/10.1038/s41598-019-47506-9 (2019).hi 7. Smith, E. R. et al. The Drosophila MSL complex acetylates histone H4 at lysine 16, a chromatin modification linked to dosage compensation. Mol. Cell Biol. 20, 312–318. https://doi.org/10.1128/mcb.20.1.312-318.2000 (2000). p p g 8. Deng, X. & Meller, V. H. roX RNAs are required for increased expression of X-linked genes in Drosophila melanogaster males Genetics 174, 1859–1866. https://doi.org/10.1534/genetics.106.064568 (2006). p g g 9. Augui, S., Nora, E. P. & Heard, E. Regulation of X-chromosome inactivation by the X-inactivation centre. Nat. Rev. Genet. 12 429–442. https://doi.org/10.1038/nrg2987 (2011). p g g 0. Lee, J. T. & Bartolomei, M. S. X-inactivation, imprinting, and long noncoding RNAs in health and disease. Cell 152, 1308–1323 https://doi.org/10.1016/j.cell.2013.02.016 (2013). p g j 1. Hapairai, L. K. et al. Lure-and-kill yeast interfering RNA larvicides targeting neural genes in the human disease vector mosquito Aedes aegypti. Sci. Rep. 7, 13223. https://doi.org/10.1038/s41598-017-13566-y (2017). gyp p g y 2. Mysore, K. et al. Yeast interfering RNA larvicides targeting neural genes induce high rates of Anopheles larval mortality. Malar. J 16, 461. https://doi.org/10.1186/s12936-017-2112-5 (2017). p g ( ) 3. Duman-Scheel, M. Data availability y All data generated or analyzed during this study are included in this article. Yeast strains and corresponding plasmids generated in this study are available subject to completion of a material transfer agreement with Indiana University and pending procurement of any required import permits by the requesting party. Received: 31 December 2020; Accepted: 30 April 2021 References 1. Duman-Scheel, M. & Syed, Z. Developmental neurogenetics of sexual dimorphism in Aedes aegypti. Front. Ecol. Evol. 3, 61. https:// doi.org/10.3389/fevo.2015.00061 (2015).fi 1. Duman-Scheel, M. & Syed, Z. Developmental neurogenetics of sexual dimorphism in Aedes aegypti. Front. Ecol. Evol. 3, 61. https:// doi.org/10.3389/fevo.2015.00061 (2015).fi 1. Duman-Scheel, M. & Syed, Z. Developmental neurogenetics of sexual dimorphism in Aedes aegypti. Front. Ecol. Evol. 3, 61. https:// doi.org/10.3389/fevo.2015.00061 (2015).fi g 2. Papathanos, P. A. et al. A perspective on the need and current status of efficient sex separation methods for mosquito ge control. Parasit Vectors 11, 654. https://doi.org/10.1186/s13071-018-3222-9 (2018). , p g ( ) 3. McClelland, G. A. H. Sex-linkage in Aedes aegypti. Trans. R. Soc. Trop. Med. Hyg. 56,4 (1962). g 3. McClelland, G. A. H. Sex-linkage in Aedes aegypti. Trans. R. Soc. Trop. Med. Hyg. 56,4 (1962). 3. McClelland, G. A. H. Sex-linkage in Aedes aegypti. Trans. R. Soc. Trop. Med. Hyg. 56,4 (1962). 4. Newton, M. E., Wood, R. J. & Southern, D. I. Cytological mapping of the M and D loci in the mosquito, Aedes aegypti (L.). Genetica 48, 137–143 (1978). 5. Hall, A. B. et al. Sex determination. A male-determining factor in the mosquito Aedes aegypti. Science 348, 1268–1270. https:// doi.org/10.1126/science.aaa2850 (2015). 5. Hall, A. B. et al. Sex determination. A male-determining factor in the mosquito Aedes aegypti. Science 348, 1268–1270. https:/ doi.org/10.1126/science.aaa2850 (2015). g 6. Motara, M. A. & Rai, K. S. Giemsa C-banding patterns in Aedes (Stegomyia) mosquitoes. Chromosoma 70(1), 51–58 (1978). M h B J l I d f f A d f b l N h 6. Motara, M. A. & Rai, K. S. Giemsa C-banding patterns in Aed 7. Matthews, B. J. et al. Improved reference genome of Aedes aegypti informs arbovirus vector control. Nature 563, 501–507. https:// doi.org/10.1038/s41586-018-0692-z (2018). 7. Matthews, B. J. et al. Improved reference genome of Aedes aegypti informs arbovirus vector control. Nature 563, 501–507. htt doi.org/10.1038/s41586-018-0692-z (2018). g 8. Wood, R. J. Lethal genes on the sex chromosomes concealed in a population of the mosquito Aedes aegypti L.. Genetica 46, 49–66 (1990). 9. Krzywinska, E. et al. The sex locus is tightly linked to factors conferring sex-specific lethal effects in the mosquito Aedes aeg Heredity (Edinb.) 117, 408–416. https://doi.org/10.1038/hdy.2016.57 (2016). y ( ) p g y ( ) 10. Giraldo-Calderon, G. I. et al. www.nature.com/scientificreports/ 4 M K t l P ti d f t hRNA d li t f il i i it l M th d M l Bi l 44. Mysore, K. et al. Preparation and use of a yeast shRNA delivery system for gene silencing in mosquito larvae. Methods Mol. Biol. 213–231, 2019. https://doi.org/10.1007/978-1-4939-8775-7_15 (1858). p g ( ) 45. WHO. Guidelines for Laboratory and Field Testing of Mosquito Larvicides (World Health Organization, Geneva, 2005). b ll & k f h ll d f h l d ff 46. Mumberg, D., Muller, R. & Funk, M. Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene 156, 119–122 (1995). 47. van Dijken, J. P. et al. An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains. Enzyme Microb. Technol. 26, 706–714 (2000). y 8. Mysore, K. et al. Characterization of a dual-action adulticidal and larvicidal interfering RNA pesticide targeting the Shaker gene of multiple disease vector mosquitoes. PLoS Negl Trop Dis 14, e0008479. https://doi.org/10.1371/journal.pntd.0008479 (2020).l 49. Hill, C. L., Sharma, A., Shouche, Y. & Severson, D. W. Dynamics of midgut microflora and dengue virus impact on life history traits in Aedes aegypti. Acta Trop. 140, 151–157. https://doi.org/10.1016/j.actatropica.2014.07.015 (2014). 50. Morlais, I., Mori, A., Schneider, J. R. & Severson, D. W. A targeted approach to the identification of candidate genes determining susceptibility to Plasmodium gallinaceum in Aedes aegypti. Mol. Genet. Genomics 269, 753–764. https://doi.org/10.1007/s00438- 003-0882-7 (2003). Acknowledgementsh g Thanks to Na Wei and Chien-Wei Wang for suggestions on yeast production, to Rachel Wiltshire for helpfu iscussion of the data, and to Zhiyong Xi for recommendations on preparation of the larval mass rearing diet This work was funded by NIH-NIAID Award 1 R21 AI144256-01 to MDS. Thanks to Na Wei and Chien Wei Wang for suggestions on yeast production, to Rachel Wiltshire for helpful discussion of the data, and to Zhiyong Xi for recommendations on preparation of the larval mass rearing diet. This work was funded by NIH-NIAID Award 1 R21 AI144256-01 to MDS. Author contributions M.D.S. conceived of the study. K.M., L.K.H., P.L., J.R., and M.D.S. developed methodology. L.K.H., P.L., J.R., L.S., and J.I. validated the work. K.M., L.H., P.L., J.R., L.S., J.I., J.K.M. and M.D.S. performed the investigation. K.M., L.K.H., and M.D.S. were responsible for visualization of the data, drafting and editing of the manuscript, which was approved by all authors. K.M. and M.D.S. supervised the research and administered the project. M.D.S. acquired funding for this research. References Saccharomyces cerevisiae (baker’s yeast) as an interfering RNA expression and delivery system. Curr. Drug Targets 20, 942–952. https://doi.org/10.2174/1389450120666181126123538 (2019). g p g 4. Papa, F. et al. Rapid evolution of female-biased genes among four species of Anopheles malaria mosquitoes. Genome Res. 27 1536–1548. https://doi.org/10.1101/gr.217216.116 (2017). 25. Papathanos, P. A. et al. Sex separation strategies: Past experience and new approaches. Malar. J. 8(Suppl 2), S5. https://doi.org/10. 1186/1475-2875-8-S2-S5 (2009). 26. Gilles, J. R. et al. Towards mosquito sterile insect technique programmes: Exploring genetic, molecular, mechanical and behav- ioural methods of sex separation in mosquitoes. Acta Trop. 132(Suppl), S178-187. https://doi.org/10.1016/j.actatropica.2013.08. 015 (2014).i 7. Harris, A. F. et al. Successful suppression of a field mosquito population by sustained release of engineered male mosquitoes. Nat Biotechnol. 30, 828–830. https://doi.org/10.1038/nbt.2350 (2012). p g 28. Zhang, D. et al. Establishment of a medium-scale mosquito facility: optimization of the larval mass-rearing unit for Aedes albopictus (Diptera: Culicidae). Parasit. Vectors 10, 569. https://doi.org/10.1186/s13071-017-2511-z (2017). 9. Charlesworth, B. Model for evolution of Y chromosomes and dosage compensation. Proc. Natl. Acad. Sci. USA 75, 5618–5622 https://doi.org/10.1073/pnas.75.11.5618 (1978).h p g p 0. Charlesworth, B. The evolution of sex chromosomes. Science 251, 1030–1033. https://doi.org/10.1126/science.1998119 (1991). https://doi.org/10.1038/s41598-021-90194-7 Scientific Reports \| (2021) 11:10657 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ 31. Charlesworth, D., Charlesworth, B. & Marais, G. Steps in the evolution of heteromorphic sex chromosomes. Heredity (Edinb.) 95, 118–128. https://doi.org/10.1038/sj.hdy.6800697 (2005).fil p g j y 2. Aryan, A. et al. Nix alone is sufficient to convert female Aedes aegypti into fertile males and myo-sex is needed for male flight. Proc Natl Acad Sci U S A 117, 17702–17709. https://doi.org/10.1073/pnas.2001132117 (2020). 3. Mysore, K. et al. siRNA-mediated silencing of doublesex during female development of the dengue vector mosquito Aedes aegypti PLoS Negl. Trop. Dis. 9, e0004213. https://doi.org/10.1371/journal.pntd.0004213 (2015). g g p. Dis. 9, e0004213. https://doi.org/10.1371/journal.pntd.0004213 4. Juneja, P. et al. Assembly of the genome of the disease vector Aedes aegypti onto a genetic linkage map allows mapping of genes affecting disease transmission. PLoS Negl. Trop. Dis. 8, e2652. https://doi.org/10.1371/journal.pntd.0002652 (2014).f f g g p p g j p 5. Fontaine, A. et al. Extensive genetic differentiation between homomorphic sex chromosomes in the mosquito vector, Aedes aegypti Genome Biol. Evol. 9, 2322–2335. https://doi.org/10.1093/gbe/evx171 (2017).l p g g 6. Ganesh, S. & Svoboda, P. Retrotransposon-associated long non-coding RNAs in mice and men. Pflugers Arch. 468, 1049–1060 https://doi.org/10.1007/s00424-016-1818-5 (2016). https://doi.org/10.1007/s00424-016-1818-5 (2016). p g 7. Volpe, T. A. et al. Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. Science 297, 1833–1837 https://doi.org/10.1126/science.1074973 (2002). p g 8. Reinhart, B. J. & Bartel, D. P. Small RNAs correspond to centromere heterochromatic repeats. Science 297, 1831. https://doi.org/ 10.1126/science.1077183 (2002). 9. Woolcock, K. J., Gaidatzis, D., Punga, T. & Buhler, M. Dicer associates with chromatin to repress genome activity in Schizosac- charomyces pombe. Nat. Struct. Mol. Biol. 18, 94–99. https://doi.org/10.1038/nsmb.1935 (2011). 0. Clemons, A., Mori, A., Haugen, M., Severson, D. W. & Duman-Scheel, M. Culturing and egg collection of Aedes aegypti. Cold Spring Harb Protoc 2010, pdb prot 5507. https://doi.org/10.1101/pdb.prot5507 (2010). 1. Integrated DNA Technologies. Custom Dicer-Substrate siRNA (DsiRNA), https://www.idtdna.com/site/order/designtool/index/ DSIRNA_CUSTOM (2017). 2. Tomchaney, M. et al. Examination of the genetic basis for sexual dimorphism in the Aedes aegypti (dengue vector mosquito) pupa brain. Biol. Sex Differ. 5, 10. https://doi.org/10.1186/s13293-014-0010-x (2014). ff p g 3. Singh, A. D., Wong, S., Ryan, C. P. & Whyard, S. Oral delivery of double-stranded RNA in larvae of the yellow fever mosquito g g y y y y q Aedes aegypti: implications for pest mosquito control. J. Insect Sci. 13, 69. https://doi.org/10.1673/031.013.6901 (2013). Competing interests p g M.D.S. is inventor on U.S. patent application number 62/751,052. The application did not impact her interpreta- tion of the data or decision to publish it. All other authors declare that they have no 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 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. 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https://openalex.org/W2952976465	https://jyx.jyu.fi/bitstream/123456789/51944/1/husalovesalainenuniquenesshsvv4.pdf	English	null	Shape identification in inverse medium scattering problems with a single far-field pattern	arXiv (Cornell University)	2,015	public-domain	8,420	This is an electronic reprint of the original article. This reprint may differ from the original in pagination and typographic detail. Author(s): Title: Year: Version: Please cite the original version: Shape identification in inverse medium scattering problems with a single far-field pattern Hu, Guanghui; Salo, Mikko; Vesalainen, Esa Hu, G., Salo, M., & Vesalainen, E. (2016). Shape identification in inverse medium scattering problems with a single far-field pattern. SIAM Journal on Mathematical Analysis, 48(1), 152-165. https://doi.org/10.1137/15M1032958 2016 This is an electronic reprint of the original article. This reprint may differ from the original in pagination and typographic detail. Author(s): Title: Year: Version: Please cite the original version: Shape identification in inverse medium scattering problems with a single far-field pattern Hu, Guanghui; Salo, Mikko; Vesalainen, Esa Hu, G., Salo, M., & Vesalainen, E. (2016). Shape identification in inverse medium scattering problems with a single far-field pattern. SIAM Journal on Mathematical Analysis, 48(1), 152-165. https://doi.org/10.1137/15M1032958 2016 Abstract Consider time-harmonic acoustic scattering from a bounded penetrable obstacle D ⊂RN embedded in a homogeneous background medium. The index of refraction characterizing the material inside D is supposed to be H¨older continuous near the corners. If D ⊂R2 is a convex polygon, we prove that its shape and location can be uniquely determined by the far-ﬁeld pattern incited by a single incident wave at a ﬁxed frequency. In dimensions N ≥3, the uniqueness applies to penetrable scatterers of rectangular type with additional assumptions on the smoothness of the contrast. Our arguments are motivated by recent studies on the absence of non- scattering wavenumbers in domains with corners. As a byproduct, we show that the smoothness conditions in previous corner scattering results are only required near the corners. Guanghui Hu∗, Mikko Salo†, Esa V. Vesalainen‡ Guanghui Hu∗, Mikko Salo†, Esa V. Vesalainen‡ ‡University of Jyvaskyla, Department of Mathematics and Statistics, P.O. Box 35 (MaD), FI-4001 University of Jyvaskyla, Finland. Email: esavesalainen@gmail.com ∗Weierstrass Institute, Mohrenstr. 39, 10117 Berlin, Germany. Email: hu@wias-berlin.de †University of Jyvaskyla, Department of Mathematics and Statistics, P.O. Box 35 (MaD), FI-40014 University of Jyvaskyla, Finland. Email: mikko.j.salo@jyu.fi ‡U i it f J k l D t t f M th ti d St ti ti P O B 35 (M D) FI 40014 ∗Weierstrass Institute, Mohrenstr. 39, 10117 Berlin, Germany. Email: hu@wias-berlin.de †University of Jyvaskyla, Department of Mathematics and Statistics, P.O. Box 35 (MaD), FI-40014 University of Jyvaskyla, Finland. Email: mikko.j.salo@jyu.fi ‡University of Jyvaskyla, Department of Mathematics and Statistics, P.O. Box 35 (MaD), FI-40014 University of Jyvaskyla, Finland. Email: esavesalainen@gmail.com This is an electronic reprint of the original article. This reprint may differ from the original in pagination and typographic detail. Author(s): Title: Year: Version: Please cite the original version: Shape identification in inverse medium scattering problems with a single far-field pattern Hu, Guanghui; Salo, Mikko; Vesalainen, Esa Hu, G., Salo, M., & Vesalainen, E. (2016). Shape identification in inverse medium scattering problems with a single far-field pattern. SIAM Journal on Mathematical Analysis, 48(1), 152-165. https://doi.org/10.1137/15M1032958 2016 This is an electronic reprint of the original article. This reprint may differ from the original in pagination and typographic detail. Author(s): Title: Year: Version: Please cite the original version: Shape identification in inverse medium scattering problems with a single far-field pattern Hu, Guanghui; Salo, Mikko; Vesalainen, Esa Hu, G., Salo, M., & Vesalainen, E. (2016). Shape identification in inverse medium scattering problems with a single far-field pattern. SIAM Journal on Mathematical Analysis, 48(1), 152-165. https://doi.org/10.1137/15M1032958 2016 This is an electronic reprint of the original article. This reprint may differ from the original in pagination and typographic detail. the original version: Hu, G., Salo, M., & Vesalainen, E. (2016). Shape identification in inverse medium scattering problems with a single far-field pattern. SIAM Journal on Mathematical Analysis, 48(1), 152-165. https://doi.org/10.1137/15M1032958 All material supplied via JYX is protected by copyright and other intellectual property rights, and duplication or sale of all or part of any of the repository collections is not permitted, except that material may be duplicated by you for your research use or educational purposes in electronic or print form. You must obtain permission for any other use. Electronic or print copies may not be offered, whether for sale or otherwise to anyone who is not an authorised user. 1 Introduction and main results Assume a time-harmonic incident wave is incident onto a bounded penetrable obstacle D ⊂RN (N ≥2) embedded in a homogeneous medium. The incident ﬁeld uin may be any non-trivial solution in L2 loc(RN) of the Helmholtz equation ∆uin + k2uin = 0 in RN, where k > 0 is the wavenumber. For instance, the incident wave is allowed to be a plane wave exp(ikx · d) with incident direction d ∈SN−1 := {x ∈RN : \|x\| = 1}, or a Herglotz wave of the form where k > 0 is the wavenumber. For instance, the incident wave is allowed to be a plane wave exp(ikx · d) with incident direction d ∈SN−1 := {x ∈RN : \|x\| = 1}, or a Herglotz wave of the form uin(x) = Z SN−1 exp(ikx · d) g(d) ds(d), g ∈L2(SN−1). ∗Weierstrass Institute, Mohrenstr. 39, 10117 Berlin, Germany. Email: hu@wias-berlin.de †University of Jyvaskyla, Department of Mathematics and Statistics, P.O. Box 35 (MaD), FI-40014 University of Jyvaskyla, Finland. Email: mikko.j.salo@jyu.fi ‡University of Jyvaskyla, Department of Mathematics and Statistics, P.O. Box 35 (MaD), FI-40014 University of Jyvaskyla, Finland. Email: esavesalainen@gmail.com ∗Weierstrass Institute, Mohrenstr. 39, 10117 Berlin, Germany. Email: hu@wias-berlin.de 1 In this paper we suppose the scatterer D to be a convex polygon in R2 or a convex polyhedron in RN. The physical properties of the inhomogeneous medium D can be characterized by the refractive index function (or potential) q(x). Without loss of gener- ality we suppose q(x) = 1 for x ∈De = RN\D due to the homogeneity of the background medium. Denote by u = uin + usc the total ﬁeld generated by uin, where usc is the outgoing scattered ﬁeld which satisﬁes the Helmholtz equation (∆+ k2)usc = 0 in De and the Sommerfeld radiation condition lim \|x\|→∞\|x\| N−1 2 ∂usc ∂\|x\| −ikusc = 0, (1.1) (1.1) uniformly in all directions. The propagation of the total wave is governed by the Helmholtz equation 2 N uniformly in all directions. The propagation of the total wave is governed by the Helmholtz equation 2 N ∆u(x) + k2q(x)u(x) = 0 in RN. 1 Introduction and main results (1.2) (1.2) Across the interface ∂D, we assume the continuity of the total ﬁeld and its normal derivative (already implicitly contained in the formulation (1.2)), i.e., Across the interface ∂D, we assume the continuity of the total ﬁeld and its normal derivative (already implicitly contained in the formulation (1.2)), i.e., u+ = u−, ∂νu+ = ∂νu− on ∂D. (1.3) (1.3) Here the superscripts (·)± stand for the limits taken from outside and inside, respectively, and ν ∈SN−1 is the unit normal on ∂D pointing into De. The unique solvability of the scattering problem (1.1)–(1.3) in H2 loc(RN) is well-known if q ∈L∞(RN) (see e.g. [CK98, Chapter 8] or [Ki11, Chapter 6]). In particular, the Sommerfeld radiation condition (1.1) leads to the asymptotic expansion usc(x) = eik\|x\| \|x\|(N−1)/2 u∞(ˆx) + O 1 \|x\|N/2 , \|x\| →+∞, (1.4) (1.4) uniformly in all directions ˆx := x/\|x\|, x ∈RN. The function u∞(ˆx) is a real-analytic function deﬁned on SN−1 and is referred to as the far-ﬁeld pattern or the scattering amplitude for uin. The vector ˆx ∈SN−1 is the observation direction of the far ﬁeld. p This paper concerns the uniqueness in recovering the boundary ∂D (or equivalently, the convex hull of the support of the contrast q −1) from the far-ﬁeld pattern generated by one incident wave at a ﬁxed frequency. The study on global uniqueness with a single incident plane or point source wave is usually diﬃcult and challenging. For sound-soft or sound-hard obstacles, such uniqueness results have been obtained within the class of polyhedral or polygonal scatterers; see e.g., [AR05,CY03,EY06,EY08,HL14,LZ06]. The proofs rely heavily on the reﬂection principles for the Helmholtz equation with respect to a Dirichlet or Neumann hyperplane and on properties of the incident wave (for instance, plane waves do not decay at inﬁnity and point source waves are singular). However, the approach of using reﬂection principles does not apply to penetrable scatterers due to the lack of ”reﬂectible” (Dirichlet or Neumann) boundary conditions for the Helmholtz equation. To the best of our knowledge, uniqueness with one incident wave is still unknown within the class of non-convex polyhedral obstacles of impedance type. 2 Earlier uniqueness results on shape identiﬁcation in inverse medium scattering were derived by sending plane waves with inﬁnitely many directions at a ﬁxed frequency (see e.g., [EH11, Is08, Is90, KG08, Ki93]), which results in overdetermined inverse problems. 1 Introduction and main results Uniqueness with a single far-ﬁeld pattern has been veriﬁed in two cases: D is a ball (not necessarily centered at the origin) and q ≡q0 ̸= 1 is a constant in D [HLL15], or D is a convex polygon or polyhedron and q is real-analytic on D satisfying \|q −1\| > 0 on ∂D [EH15]. The unique determination of a variable index of refraction q in RN from knowledge of the far-ﬁeld patterns of all incident plane waves at ﬁxed frequency, or by measuring the Dirichlet-to-Neumann map of the Helmholtz equation, has also been intensively studied. We refer to [SU87,HN87,Na88] and the survey [Uh14] for results for N ≥3 and to recent results [Bu08,BIY15] for N = 2. The purpose of this article is to remove the real-analyticity assumption made in [E- H15] on the refractive index. To do this, we employ a diﬀerent method that is motivated by the recent studies [BPS14, PSV14] on the absence of non-scattering wavenumbers in corner domains. This method relies on the construction of suitable complex geo- metrical optics (CGO) solutions to the Helmholtz equation. Recall that k is called a non-scattering wavenumber if there is a nontrivial incident wave whose far-ﬁeld pattern vanishes identically. If k is a non-scattering wavenumber, the functions w = uin\|D and u\|D solve the interior transmission eigenvalue problem ∆w + k2w = 0, ∆u + k2qu = 0 in D, w = u, ∂νw = ∂νu on ∂D. (1.5) (1.5) Thus each non-scattering wavenumber is an interior transmission eigenvalue (see the survey [CH13]). On the other hand, if k2 is an interior transmission eigenvalue and if the non-trivial solution w of (1.5) has a real-analytic extension from D to RN, then k2 is also a non-scattering wavenumber. This implies that, when k2 is a non-scattering wavenumber, the Cauchy data of the total ﬁeld u on ∂D coincide with the Cauchy data of a real-analytic function which satisﬁes the Helmholtz equation in a neighborhood of D. A similar phenomenon can be observed around a corner point, if two distinct convex polygons or polyhedra generate the same far-ﬁeld pattern. Therefore, the argument for proving the absence of non-scattering wavenumber can be used for justifying uniqueness in determining the shape of a penetrable scatterer. Thus each non-scattering wavenumber is an interior transmission eigenvalue (see the survey [CH13]). (b) N ≥3 and X = Hs,p for some s, p with 1 < p ≤2 and s > N/p. Theorems 1.1 and 1.2 are valid for H¨older or Sobolev potentials and avoid the real- analyticity assumption required in [EH15]. The results in dimensions N ≥3 are conﬁned to penetrable scatterers of rectangular type. It is still open how to prove Theorem 1.2 for general convex polyhedra with H¨older continuous contrasts. We remark that the above results remain valid for a large class of incident waves which do not vanish identically in a neighborhood of the scatterer. For instance, uin is also allowed to be a spherical point source emitted from some source position located in De. Our technique improves the regularity conditions of the corner scattering results of [BPS14, PSV14]. Namely, regularity is only required in a small neighborhood of the corner point, and otherwise the contrasts are only required to be L∞. We state the results on the absence of non-scattering wavenumbers as follows. Throughout the paper we write Br := {x ∈RN : \|x\| < r} for r > 0. Theorem 1.3. Let q ∈L∞(R2), and let W ⊂R2 be a closed sector with angle < π and with vertex at O. Suppose that q ≡1 in W e, that q −1 is compactly supported, and that q\|W∩Br is Cα for some α > 0 and r > 0. Finally, assume that q(O) ̸= 1. Then, with q as the contrast, for any incoming wave uin ̸≡0, the far-ﬁeld pattern u∞can not vanish identically. Theorem 1.4. Let q ∈L∞(RN). Suppose that q ≡1 in W e, that q −1 is compactly supported, and that q\|W∩Br has regularity X for some r > 0, where one of the following conditions holds: (a) N = 3, W = [0, ∞[3, and X = Cα for some α > 1/4. (b) N ≥3, W = [0, ∞[N, and X = Hs,p for some s, p with 1 < p ≤2 and s > N/p. (a) N = 3, W = [0, ∞[3, and X = Cα for some α > 1/4. (b) N ≥3, W = [0, ∞[N, and X = Hs,p for some s, p with 1 < p ≤2 and s > N/p. Finally, assume that q(O) ̸= 1. Then, with q as the contrast, for any incoming wave uin ̸≡0, the far-ﬁeld pattern u∞can not vanish identically. The subsequent Section 2 is devoted to the proofs of the two-dimensional results, i.e., Theorems 1.1 and 1.3. 1 Introduction and main results (b) N ≥3 and X = Hs,p for some s, p with 1 < p ≤2 and s > N/p. (a) N = 3 and X = Cα for some α > 1/4. (b) N ≥3 and X = Hs,p for some s, p with 1 < p ≤2 and s > N/p. 1 Introduction and main results On the other hand, if k2 is an interior transmission eigenvalue and if the non-trivial solution w of (1.5) has a real-analytic extension from D to RN, then k2 is also a non-scattering wavenumber. This implies that, when k2 is a non-scattering wavenumber, the Cauchy data of the total ﬁeld u on ∂D coincide with the Cauchy data of a real-analytic function which satisﬁes the Helmholtz equation in a neighborhood of D. A similar phenomenon can be observed around a corner point, if two distinct convex polygons or polyhedra generate the same far-ﬁeld pattern. Therefore, the argument for proving the absence of non-scattering wavenumber can be used for justifying uniqueness in determining the shape of a penetrable scatterer. Let Dj for j = 1, 2 be two penetrable scatterers with contrasts qj. Denote by u∞ j the far-ﬁeld pattern of the scattered ﬁeld caused by a ﬁxed incoming wave uin incident onto Dj with ﬁxed wavenumber k > 0. The ﬁrst uniqueness result is in two dimensions, and applies to convex polygons. Theorem 1.1. Let Dj ⊂R2 for j = 1, 2 be bounded convex polygons. Assume that qj ∈L∞(R2) are contrasts such that qj ≡1 in De j, and each vertex of Dj has some neighborhood Uj such that qj\|Dj∩Uj is Cα for some α > 0. Furthermore, assume that qj(O) ̸= 1 for each vertex O of Dj. Then the relation u∞ 1 = u∞ 2 on S1 implies that D1 = D2. The next result applies in dimensions N ≥3 but requires that the scatterers are closed rectangular boxes, i.e. sets of the form [0, a1] × · · · × [0, aN] for some aj > 0 up 3 to rotations and translations. We write Hs,p for the fractional Lp Sobolev space with smoothness index s. Theorem 1.2. Let Dj ⊂RN for j = 1, 2 be two rectangular boxes. Assume that qj ∈L∞(RN) are contrasts such that qj ≡1 in De j, and each corner of Dj has some neighborhood Uj such that qj\|Dj∩Uj has regularity X as speciﬁed below. Furthermore, assume that qj(O) ̸= 1 for each corner O of Dj. Then the relation u∞ 1 = u∞ 2 on SN−1 implies that D1 = D2, provided that one of the following assumptions holds. (a) N = 3 and X = Cα for some α > 1/4. (b) N ≥3 and X = Hs,p for some s, p with 1 < p ≤2 and s > N/p. The unique determination of a rectangular box in any dimension N ≥3, Theorem 1.2, will be proved in Section 3. The result of Theorem 1.4 on non- scattering wavenumbers can be derived by using the same argument as in Theorem 1.3 and we omit its proof. 4 2 Proofs in two dimensions Denote by (r, ϕ) the polar coordinates in R2, and by BR the disk centered at the origin O with radius R > 0. For ϕ0 ∈(0, π/2), deﬁne W ⊂R2 as the inﬁnite sector between the half-lines Γ± := {(r, ϕ) : ϕ = ±ϕ0}. The closure of W will be denoted by W, which is a closed cone in R2. Set (see Figure 1) SR = W ∩BR, Γ± R = Γ± ∩BR, SR = W ∩BR, Se R = BR\SR. SR = W ∩BR, Γ± R = Γ± ∩BR, SR = W ∩BR, Se R = BR\SR. Figure 1: Geometrical settings. Figure 1: Geometrical settings. The following two lemmas are the essential ingredients in the proofs. The ﬁrst one concerns the construction of suitable Complex Geometrical Optics (CGO) solutions to the Schr¨odinger equation in R2. For convenience we employ the common notation ⟨x⟩:= (1 + \|x\|2)1/2 throughout the paper. Lemma 2.1. Let ˜q ∈L∞(R2) satisfy ˜q ≡1 in R2\W and ⟨·⟩β (˜q −1) ∈Cα(W) for ome α > 0 and β > 5/3. If ρ ∈C2 satisﬁes ρ·ρ = −k2 and \|Im (ρ)\| is suﬃciently large, hen there exists a solution of the Helmholtz equation Lemma 2.1. Let ˜q ∈L∞(R2) satisfy ˜q ≡1 in R2\W and ⟨·⟩β (˜q −1) ∈Cα(W) for some α > 0 and β > 5/3. If ρ ∈C2 satisﬁes ρ·ρ = −k2 and \|Im (ρ)\| is suﬃciently large, then there exists a solution of the Helmholtz equation ∆u(x) + k2˜q(x)u(x) = 0 in R2 (2.1) (2.1) of the form u = e−ρ·x(1 + ψ(x)), (2.2) (2.2) where ψ satisﬁes ∥ψ∥L6(R2) = O(\|Im (ρ)\|−1/3−δ) as \|ρ\| →∞, (2.3) (2.3) subject to the transmission conditions subject to the transmission conditions subject to the transmission conditions v1 = v2, ∂νv1 = ∂νv2 on Γ± R . (2.4) ≡0 in BR if q(O) ̸= 1. v1 = v2, ∂νv1 = ∂νv2 on Γ± R . (2.4) (2.4) v1 = v2, ∂νv1 = ∂νv2 on Γ± R . (2.4) Then we have v1 = v2 ≡0 in BR if q(O) ̸= 1. v1 = v2, ∂νv1 = ∂νv2 on Γ± R . Then we have v1 = v2 ≡0 in BR if q(O) ̸= 1. From Lemma 2.2 it follows that the Cauchy data of non-trivial solutions of the Schr¨odinger equations with constant and piecewise H¨older continuous potentials cannot coincide on two intersecting lines, if the potentials involved do not coincide on the inter- section. The same result was veriﬁed in [EH15] but restricted to real-analytic potentials. Making use of classical corner regularity results for the Laplace equation in the plane (see e.g., [MNP00, Chapter 1.2], [Gr92, Chapter 2] or [Da88, Example 16.12]), the approach of Taylor expansion [EH15] can be generalized only to inﬁnitely smooth potentials on SR. Hence, the above Lemma 2.2 has signiﬁcantly relaxed the regularity assumption used in [EH15]. Below we carry out the proof of Lemma 2.2 which is valid only when the corner of SR is convex, i.e., ϕ0 < π/2. Proof of Lemma 2.2. We shall follow the approach from [PSV14, Section 4] but modiﬁed to be applicable to a polygonal convex cone with ﬁnite height. For clarity we divide the proof into three steps. Step 1. Establish an orthogonality identity with an exponentially decaying remainder term. Set w = v1 −v2. Then w ∈H2(BR), and we have ∆w + k2qw = k2(q −1)v1 in BR, w = ∂νw = 0 on Γ± R . (2.5) (2.5) Extending q from BR/2 to R2 in a suitable way, we get a new potential ˜q ∈L∞(R2) satisfying ˜q\|W ∈Cα(W) such that Extending q from BR/2 to R2 in a suitable way, we get a new potential ˜q ∈L∞(R2) satisfying ˜q\|W ∈Cα(W) such that ˜q = q in SR/2 , ˜q ≡1 in (W\SR) ∪(R2\W). Clearly ˜q fulﬁlls the assumptions in Lemma 2.1. Set β := π/2 −ϕ0 > 0. for some δ > 0. for some δ > 0. Lemma 2.1 is the special case N = 2 of Lemma 3.1 in [PSV14], the proof of which was based on the uniform Sobolev estimates of Kenig, Ruiz and Sogge [KRS87]. Relying on the construction of CGO solutions of the form (2.2), we next verify a result for the transmission problem between the Schr¨odinger equations with constant and piecewise H¨older continuous potentials in a ﬁnite polygonal cone. 5 Lemma 2.2. Suppose q ∈L∞(BR) satisﬁes q\|SR ∈Cα(SR) with some α > 0, and q ≡1 in Se R. Let v1, v2 ∈H2(BR) be solutions to ∆v1(x) + k2v1(x) = 0, ∆v2(x) + k2q(x)v2(x) = 0 in BR subject to the transmission conditions For any ϕ with ϕ ∈] −β/2, β/2 [, let ω = (cos ϕ, sin ϕ) ∈S1 and let ω⊥ ± be the two vectors orthogonal to ω, i.e., ω⊥ ± = ±(−sin ϕ, cos ϕ). For τ > 0, introduce the parameter-dependent vectors ρτ,ϕ,± ∈C2 as follows ρτ,ϕ,± = τω + i (τ 2 + k2)1/2ω⊥ ±. ρτ,ϕ,± = τω + i (τ 2 + k2)1/2ω⊥ ±. Obviously, ρτ,ϕ,± · ρτ,ϕ,± = −k2 and \|ρτ,ϕ,±\| ∼ √ 2τ as τ →∞. By Lemma 2.1, we may construct solutions to the Schr¨odinger equation (2.1) of the form Obviously, ρτ,ϕ,± · ρτ,ϕ,± = −k2 and \|ρτ,ϕ,±\| ∼ √ 2τ as τ →∞. By Lemma 2.1, we may construct solutions to the Schr¨odinger equation (2.1) of the form u(x) = uτ,ϕ,±(x) = exp(−ρτ,ϕ,± · x) (1 + ψτ,ϕ,±(x)) in R2, (2.6) (2.6) 6 provided τ > 0 is suﬃciently large. Applying Green’s formula and using (2.5) yields 0 = Z SR/2 (∆u + k2˜qu)w dx = Z SR/2 (∆u + k2qu)w dx = Z SR/2 (∆w + k2qw)u dx + Z ∂(SR/2) (∂νu w −∂νw u) ds = k2 Z SR/2 (q −1)v1 u dx + Z ΛR/2 (∂νu w −∂νw u) ds (2.7) SR/2 = Z SR/2 (∆w + k2qw)u dx + Z ∂(SR/2) (∂νu w −∂νw u) ds = k2 Z SR/2 (q −1)v1 u dx + Z ΛR/2 (∂νu w −∂νw u) ds (2.7) (2.7) with ΛR/2 := {\|x\| = R/2} ∩W. Since the constructed CGO solutions decay in W\{O}, we shall prove that the integral over ΛR/2 in (2.7) converges to zero exponentially fast as τ →∞. Figure 2: Conﬁgurations of ΛR/2 and Dϵ,R in the proof of Lemma 2.2. Figure 2: Conﬁgurations of ΛR/2 and Dϵ,R in the proof of Lemma 2.2. For 0 < ϵ < min{β/2, R/2}, deﬁne a neighborhood of ΛR/2 by (see Figure 2) Dϵ,R := {(r, ϕ) : R/2 −ϵ < r < R/2 + ϵ, \|ϕ\| < ϕ0 + ϵ}. Then, there exists δ0 = δ0(ϵ, R) > 0 such that For 0 < ϵ < min{β/2, R/2}, deﬁne a neighborhood of ΛR/2 by (see Figure 2) Dϵ,R := {(r, ϕ) : R/2 −ϵ < r < R/2 + ϵ, \|ϕ\| < ϕ0 + ϵ}. subject to the transmission conditions Then, there exists δ0 = δ0(ϵ, R) > 0 such that Re (ρτ,ϕ,± · x) = τ(ω · x) ≥τ δ0 > 0 for all x ∈Dϵ,R, ϕ ∈] −β/2, β/2 [. Re (ρτ,ϕ,± · x) = τ(ω · x) ≥τ δ0 > 0 for all x ∈Dϵ,R, ϕ ∈] −β/2, β/2 [. This together with the estimates of ∥ψτ,ϕ,±∥L6(R2) (see (2.3)) implies the exponential decay of the L2-norm of uτ,ϕ,± over L2(Dϵ,R), i.e., ∥uτ,ϕ,±∥L2(Dϵ,R) = O(e−τδ0) as τ →∞. On the other hand, since uτ,ϕ,± solves the Schr¨odinger equation in R2, the standard elliptic interior regularity estimate allows us to estimate for ϵ′ < ϵ that ∥uτ,ϕ,±∥H2(Dϵ′,R) ≤C ∥uτ,ϕ,±∥L2(Dϵ,R) ≤Ce−τδ0. 7 7 Applying the Cauchy-Schwarz inequality and using the trace lemma, we may estimate the last term on the right-hand side of (2.7) as follows Z ΛR/2 ((∂νu) w −(∂νw) u) ds ≤ C ∥u∥H3/2(ΛR/2) ∥w∥H3/2(ΛR/2) ≤ C ∥u∥H2(Dϵ′,R) ∥v1∥H2(BR) + ∥v2∥H2(BR) . ≤ C ∥u∥H3/2(ΛR/2) ∥w∥H3/2(ΛR/2) ≤ C ∥u∥H2(Dϵ′,R) ∥v1∥H2(BR) + ∥v2∥H2(BR) . Combining (2.7) with the previous two inequalities, we get the following orthogonality identity over SR/2 with an exponentially decaying remainder term Combining (2.7) with the previous two inequalities, we get the following orthogonality identity over SR/2 with an exponentially decaying remainder term Z SR/2 (q −1)v1 uτ,ϕ,± dx = O(e−τδ0) as τ →∞, ϕ ∈] −β/2, β/2 [. (2.8) (2.8) Step 2. Reduction to Laplace transforms. Assume that v1 ̸≡0. Since v1 is a solution of the Helmholtz equation in BR, the lowest order nontrivial homogeneous polynomial H(x) in the Taylor expansion of v1 around the origin is a harmonic function (see [BPS14, Lemma 2.4]). Without loss of generality, we assume H is of order n for some n ≥0, i.e., v1(x) = H(x) + K(x), K(x) = O(\|x\|n+1) as \|x\| →0. (2.9) (2.9) Deﬁne F to be the Laplace transform of H in W, F(z) := Z W exp(−z · x) H(x) dx, (2.10) (2.10) for z ∈C2 such that Re (z) · (1, 0) > cos(β/2). subject to the transmission conditions On the other hand, since the cone W remains invariant under the transform x →\|ρ\|x and H is a homogeneous polynomial, it is easy to check that On the other hand, since the cone W remains invariant under the transform x →\|ρ\|x nd H is a homogeneous polynomial, it is easy to check that F(ρ) = \|ρ\|−n−2 F(ρ/\|ρ\|). (2.13) F(ρ) = \|ρ\|−n−2 F(ρ/\|ρ\|). (2.13) Consequently, taking τ →∞in (2.12) gives F((ω + iω⊥ ±)/ √ 2) = 0. Moreover, the homogeneity of F shown as in (2.13) yields Consequently, taking τ →∞in (2.12) gives F((ω + iω⊥ ±)/ √ 2) = 0. Moreover, the homogeneity of F shown as in (2.13) yields F(τ(ω + iω⊥ ±)) = 0 for all τ > 0, ϕ ∈] −β/2, β/2 [. (2.14) (2.14) This implies the vanishing of the Laplace transform of χW H at z = τ(ω + iω⊥ ±) for all τ > 0 and ϕ ∈] −β/2, β/2 [. Step 3. End of the proof. Repeating the arguments of [PSV14, Section 5], one can deduce from (2.14), taking both signs ±, that H ≡0. This implies that v1 ≡0 in BR. As a consequence, the Cauchy data of v2 on Γ± R vanish due to the transmission conditions (2.4). Finally, we get v2 ≡0 by the unique continuation of solutions to the Schr¨odinger equation. This ﬁnishes the proof of Lemma 2.2. We are now ready to prove Theorems 1.1 and 1.3 for general incident waves, including point source waves. Proof of Theorem 1.1. Since u∞ 1 (ˆx) = u∞ 2 (ˆx) for all ˆx ∈S1, applying Rellich’s lemma we know that usc 1 = usc 2 in R2\(D1 ∪D2). Thus Proof of Theorem 1.1. Since u∞ 1 (ˆx) = u∞ 2 (ˆx) for all ˆx ∈S1, applying Rellich’s lemma we know that usc 1 = usc 2 in R2\(D1 ∪D2). Thus u1(x) = u2(x) (2.15) (2.15) for all x ∈R2\(D1 ∪D2). for all x ∈R2\(D1 ∪D2). for all x ∈R2\(D1 ∪D2). \( ) If ∂D1 ̸= ∂D2, without loss of generality we may assume there exists a corner O ∈R2 of ∂D2 such that O /∈D1. We suppose further that this corner point coincides with the origin and we pick a ﬁxed number R > 0 such that BR ⊂De 1. subject to the transmission conditions Taking z = ρ = ρτ,ϕ,± and splitting F(ρ) into two terms, we see F(ρ) = Z SR/2 exp(−ρ · x) H(x) dx + Z W\SR/2 exp(−ρ · x) H(x) dx = Z SR/2 exp(−ρ · x) H(x) dx + O(e−τδ1) (2.11) F(ρ) = Z SR/2 exp(−ρ · x) H(x) dx + Z W\SR/2 exp(−ρ · x) H(x) dx Z = Z SR/2 exp(−ρ · x) H(x) dx + O(e−τδ1) (2.11) (2.11) as τ →∞for some δ1 = δ1(R, ϕ0) > 0. By the assumption q(O) ̸= 1, we may set η := q(O) −1 ̸= 0. Inserting (2.6) and (2.9) into (2.8) and then combining the resulting expression with (2.11) gives as τ →∞for some δ1 = δ1(R, ϕ0) > 0. By the assumption q(O) ̸= 1, we may set η := q(O) −1 ̸= 0. Inserting (2.6) and (2.9) into (2.8) and then combining the resulting expression with (2.11) gives F(ρ) = Z SR/2 exp(−ρ · x) η H(x) −(q(x) −1)(H(x) + K(x))(1 + ψ(x)) dx + O(e−τδ2) as τ →∞, with δ2 := min{δ0, δ1}. Making use of [BPS14, Lemma 3.6], we can estimate the integral on the right hand of the previous identity by (see e.g., [PSV14, Section 4]) as τ →∞, with δ2 := min{δ0, δ1}. Making use of [BPS14, Lemma 3.6], we can estimate the integral on the right hand of the previous identity by (see e.g., [PSV14, Section 4]) Z SR/2 exp(−ρ · x) η H(x) −(q(x) −1)(H(x) + K(x))(1 + ψ(x)) dx Z SR/2 exp(−ρ · x) η H(x) −(q(x) −1)(H(x) + K(x))(1 + ψ(x)) dx = Z SR/2 exp(−ρ · x) {(q(O) −q(x))H(x) −(q(x) −1)[K(x) + ψ(x)(H(x) + K(x))]} dx ≤ C τ −n−2−δ Z SR/2 exp(−ρ · x) η H(x) −(q(x) −1)(H(x) + K(x))(1 + ψ(x)) dx = Z SR/2 exp(−ρ · x) {(q(O) −q(x))H(x) −(q(x) −1)[K(x) + ψ(x)(H(x) + K(x))]} dx ≤ C τ −n−2−δ = Z SR/2 exp(−ρ · x) {(q(O) −q(x))H(x) −(q(x) −1)[K(x) + ψ(x)(H(x) + K(x))]} dx ≤ C τ −n−2−δ Z SR/2 ≤ C τ −n−2−δ ≤ C τ −n−2−δ 8 8 for some δ > 0. Therefore, we arrive at η F(ρ) ≤C τ −n−2−δ (2.12) (2.12) for all ϕ ∈] −β/2, β/2 [ and τ > 0 suﬃciently large. for all ϕ ∈] −β/2, β/2 [ and τ > 0 suﬃciently large. subject to the transmission conditions Since D2 is a convex polygon, rotating coordinate axes if necessary, we may assume that D2 ∩BR = {(r, ϕ) : \|ϕ\| < ϕ0} for some ϕ0 ∈(0, π/2); see Figure 3. From (2.15), it follows that If ∂D1 ̸= ∂D2, without loss of generality we may assume there exists a corner O ∈R2 of ∂D2 such that O /∈D1. We suppose further that this corner point coincides with the origin and we pick a ﬁxed number R > 0 such that BR ⊂De 1. Since D2 is a convex polygon, rotating coordinate axes if necessary, we may assume that D2 ∩BR = {(r, ϕ) : \|ϕ\| < ϕ0} for some ϕ0 ∈(0, π/2); see Figure 3. From (2.15), it follows that u− 1 = u+ 1 = u+ 2 = u− 2 , ∂νu− 1 = ∂νu+ 1 = ∂νu+ 2 = ∂νu− 2 on ∂D2 ∩BR, where the superscripts (·)−, (·)+ stand for the limits taken from D2 and De 2, respectively. On the other hand, the function u1 satisﬁes the Helmholtz equation with the wave number k2 in BR, while u2 fulﬁlls the Schr¨odinger equation where the superscripts (·)−, (·)+ stand for the limits taken from D2 and De 2, respectively. On the other hand, the function u1 satisﬁes the Helmholtz equation with the wave number k2 in BR, while u2 fulﬁlls the Schr¨odinger equation ∆u2 + k2q2u2 = 0 in BR. Since q2(O) ̸= 1, applying Lemma 2.2 leads to u1 = u2 ≡0 in BR. Moreover, by unique continuation we obtain u1 = u2 ≡0 in R2. This implies that the scattered ﬁelds satisfy usc 1 = usc 2 = −uin in all of R2. Hence uin ≡0 in R2\(D1 ∪D2), but since uin solves the free Helmholtz equation, unique continuation implies uin ≡0. This contradiction gives D1 = D2. D1 = D2. 9 O D1 D2 Figure 3: Two distinct convex penetrable scatterers D1 and D2 of polygonal-type. Figure 3: Two distinct convex penetrable scatterers D1 and D2 of polygonal-type. Proof of Theorem 1.3. Let us consider the incident wave uin ̸≡0 with the total wave u, so that we have ∆uin(x) + k2uin(x) = 0 and ∆u(x) + k2q(x)u(x) = 0 in R2. ∆uin(x) + k2uin(x) = 0 and ∆u(x) + k2q(x)u(x) = 0 in R2. subject to the transmission conditions If for this incident wave u∞≡0, then Rellich’s lemma tells us that u ≡uin in R2 \ W, and Lemma 2.2 applied with v1 = uin and v2 = u gives uin ≡0 in BR. By unique continuation we get uin ≡0, which is a contradiction. 3 Proofs in higher dimensions We ﬁrst present the proof under the assumption (a) of Theorem 1.2, that is, Dj ⊂R3 is a rectangular box and the potential is Cα near the corners with α > 1/4. Let W = [0, ∞[3. We will make use of the following result concerning complex geometrical optics solutions. Recall the notation ⟨x⟩:= (1 + \|x\|2)1/2. Lemma 3.1. Let eq ≡1 in R3 \ W and ⟨·⟩β (eq −1) ∈Cα(W) for some α > 1/4 and β > 9/4. If ρ ∈C3 satisﬁes ρ·ρ = −k2 and \|Im (ρ)\| is suﬃciently large, then there exists a solution of the Helmholtz equation ∆u(x) + k2eq(x)u(x) = 0 in R3 of the form of the form of the form u = e−ρ·x(1 + ψ(x)), where ψ satisﬁes where ψ satisﬁes ∥ψ∥L4(R3) = O(\|Im (ρ)\|−3/4−δ), as \|ρ\| →∞, for some δ > 0. for some δ > 0. 10 The proof of the above lemma is also based on the uniform Sobolev estimates of Kenig, Ruiz and Sogge [KRS87]. In order to avoid repeating the arguments presented in [PSV14], we shall verify Lemma 3.1 by indicating the changes necessary to the proof of [PSV14, Theorem 3.1]. For this purpose we need to know into which Sobolev spaces the characteristic function of a cube belongs. Below we will write χQ for the charac- teristic function of a set Q, and denote by C∞ c (R3) the space of smooth functions with compact support. Lemma 3.2. Let Q ⊂R3 be a closed cube. Then χQ ∈Hτ,p(R3) Lemma 3.2. Let Q ⊂R3 be a closed cube. Then χQ ∈Hτ,p(R3) Lemma 3.2. Let Q ⊂R3 be a closed cube. Then χQ ∈Hτ,p(R3) for τ ∈[0, 1/2[ and p ∈]1, 2]. for τ ∈[0, 1/2[ and p ∈]1, 2]. Proof. Without loss of generality, we consider Q = [−1, 1]3. Since χQ ∈L1(R3), the Fourier transform c χQ of χQ is continuous. For ξ = (ξ1, ξ2, ξ3) ∈R3 with ξ1ξ2ξ3 ̸= 0, the transform c χQ takes the explicit form Q Q transform c χQ takes the explicit form c χC(ξ) = 23 sin ξ1 sin ξ2 sin ξ3 ξ1ξ2ξ3 . Thus, we may estimate ∥χQ∥2 Hτ,2(R3) = Z R3 ⟨ξ⟩2τ \|c χC(ξ)\|2 dξ ≤C Z R3 ⟨ξ⟩2τ ⟨ξ1⟩−2 ⟨ξ2⟩−2 ⟨ξ3⟩−2 dξ. The last integral is ﬁnite when τ < 1/2. Thus, χQ ∈Hτ,2(R3) for τ ∈[0, 1/2[. Next, let ψ be a ﬁxed function in C∞ c (R3) satisfying ψχQ = χQ. By H¨older’s in equality, for any k ∈{0, 1}, f ∈Hk,2(R3) and ﬁxed p ∈]1, 2], ∥ψf∥Hk,p(R3) ≤C ∥ψf∥Hk,2(R3) ≤C ∥f∥Hk,2(R3) . ∥ψf∥Hk,p(R3) ≤C ∥ψf∥Hk,2(R3) ≤C ∥f∥Hk,2(R3) . the mapping f 7→ψf maps Hs,2(R3) into Hs,p(R3) for s R3) for all τ ∈[0, 1/2[ and p ∈]1, 2]. ∥ψf∥Hk,p(R3) ≤C ∥ψf∥Hk,2(R3) ≤C ∥f∥Hk,2(R3) . By interpolation, the mapping f 7→ψf maps Hs,2(R3) into Hs,p(R3) for s ∈]0, 1[, and thus χQ ∈Hτ,p(R3) for all τ ∈[0, 1/2[ and p ∈]1, 2]. ∥ψf∥Hk,p(R3) ≤C ∥ψf∥Hk,2(R3) ≤C ∥f∥Hk,2(R3) . of the form By interpolation, the mapping f 7→ψf maps Hs,2(R3) into Hs,p(R3) for s ∈]0, 1[, and thus χQ ∈Hτ,p(R3) for all τ ∈[0, 1/2[ and p ∈]1, 2]. The construction of CGO solutions for a cube is proved as follows. Proof of Lemma 3.1. We may assume that α < 1/2. The proof of the complex geometric optics construction in [PSV14] is mostly independent of the shape of W. For W = [0, ∞[3 we only need to check that V := χW (1−˜q) has the pointwise Sobolev multiplier property of Proposition 3.4 in [PSV14], i.e., we need to check that ∥V f∥Hα−ε,4/3(R3) ≤C ∥f∥Hα−ε,4(R3) for some constant C > 0 for arbitrarily small ﬁxed ε > 0. The desired multiplier property in turn follows immediately, if we can show that (cf. [PSV14, Proposition 3.7]) ⟨·⟩−γ χ[0,∞[3 ∈Hτ,p(R3) (3.1) ⟨·⟩−γ χ[0,∞[3 ∈Hτ,p(R3) (3.1) 11 for p ∈]1, 2], τ ∈[0, 1/2[ and γ ∈]3/p, ∞[. Given β1, β2 ∈[0, ∞[ with β1 < β2, we set Λ = [0, β2]3 \ [0, β1[3. Applying Lemma 3.2 we know that the function χΛ belongs to Hτ,p(R3). This leads to the relation (3.1) by changing variables and scaling the Sobolev norm; see the proof for Proposition 3.7 in [PSV14]. To continue the proof of Theorem 1.2 under the assumption (a), we again introduce some notation. Let SR = W ∩BR, Se R = BR \ SR, and ΓR = ∂W ∩BR. As in two dimensions, we will employ a result of the following type. Lemma 3.3. Let q ∈L∞(BR) satisfy q\|SR ∈Cα(SR), where α > 1/4, and q ≡1 in Se R. Let v1, v2 ∈H2(BR) be solutions to ∆v1(x) + k2v1(x) = 0 and ∆v2(x) + k2q(x)v2(x) = 0 in BR, ∆v1(x) + k2v1(x) = 0 and ∆v2(x) + k2q(x)v2(x) = 0 in BR, subject to the transmission conditions subject to the transmission conditions subject to the transmission conditions v1 = v2, ∂νv1 = ∂νv2 on ΓR. 0 in BR, if q(O) ̸= 1. v1 = v2, ∂νv1 = ∂νv2 on ΓR. n we have v1 = v2 ≡0 in BR, if q(O) ̸= 1. v1 = v2, ∂νv1 = ∂νv2 on ΓR. Then we have v1 = v2 ≡0 in BR, if q(O) ̸= 1. Then we have v1 = v2 ≡0 in BR, if q(O) ̸= 1. Proof. We carry over the proof of Lemma 2.2 to three dimensions. Set w = v1 −v2. Then we have w = ∂νw = 0 on ΓR and Proof. We carry over the proof of Lemma 2.2 to three dimensions. Set w = v1 −v2 Then we have w = ∂νw = 0 on ΓR and ∆w(x) + k2q(x)w(x) = k2(q(x) −1)v1(x) in BR. Extending q from BR/2 to R3, we can obtain a new potential eq ∈Cα(W) such that eq = q in SR/2 and that eq −1 satisﬁes the smoothness conditions required by Lemma 3.1. Next, write β = π/3 and a = (3−1/2, 3−1/2, 3−1/2). Choose τ ∈R+ and ω, ω⊥∈R3 with \|ω\| = ω⊥ = 1, ω · ω⊥= 0 and ω · a > cos(β/2). We parameterize the CGO solutions with the complex vector ρ = ρτ,ω,ω⊥= τω + i(τ 2 −k2)1/2ω⊥. Provided that τ is suﬃciently large, Lemma 3.1 gives solutions u(x) = uτ,ω,ω⊥(x) = e−ρ·x(1 + ψ(x)) to the Helmholtz equation ∆u(x) + k2eq(x)u(x) = 0 in R3. Furthermore, the remainder ψ has the L4-estimate ψ L4(R3) < C τ −3/4−δ for some δ > 0. Arguing as before in the two-dimensional case, we get to the Helmholtz equation ∆u(x) + k2eq(x)u(x) = 0 in R3. Furthermore, the remainder ψ has the L4-estimate ψ L4(R3) < C τ −3/4−δ for some δ > 0. Arguing as before in the two-dimensional case, we get 0 = k2 Z SR/2 (q −1)v1u dx + Z ΛR/2 ((∂νu) w −u ∂νw) ds, where ΛR/2 = W ∩∂BR/2. The selection of the parameters β, a and ω ensures the decay of the integral over ΛR/2, Z ΛR/2 ((∂νu)w −u∂νw) ds = O(e−τδ0) as τ →∞ Z ΛR/2 ((∂νu)w −u∂νw) ds = O(e−τδ0) as τ →∞ 12 for some δ0 > 0. Thus, we again get the orthogonality relation Z SR/2 (q −1)v1u dx = O(e−τδ0) as τ →∞ for any given admissible ω and ω⊥. subject to the transmission conditions Denote by H the lowest degree nontrivial homogeneous polynomial in the Taylor expansion of v1 around the origin, and consider the the Laplace transform F(z) of H (see (2.10)) for z ∈C3 such that Re (z) · a > cos(β/2). If H has degree n, similarly as before we obtain F(ρ) = O(τ −n−3−δ) as τ →∞, if q(O) ̸= 1. This estimate involves using the H¨older inequality so that the L4-norm of ψ appears. In the other direction, by homogeneity, we have F(ρ) = \|ρ\|−n−3 F ρ \|ρ\| . Taking τ →∞gives then F(τω + iτω⊥) = 0 for all τ ∈R+ and all admissible ω and ω⊥. From [BPS14, Theorem 2.5] we obtain the conclusion H(x) ≡0, which implies v1 = v2 ≡0 in BR. Proof of Theorem 1.2 under assumption (a). The result follows by the same arguments as in the proof of Theorem 1.1, except that Lemma 3.3 is used instead of Lemma 2.2. Next we indicate how to prove Theorem 1.2 under the assumption (b). Now we let W = [0, ∞[N where N ≥3. The required complex geometrical optics solutions were constructed in [BPS14] and they are given by the following result. Lemma 3.4. Let eq ≡1 in RN \ (W ∩BR) for some R > 0, and let eq \|W∩BR be in Hs,p where 1 < p ≤2 and s > N/p. Let also D ⊂RN be a bounded open set, and let 2 ≤r < ∞. If ρ ∈CN satisﬁes ρ · ρ = 0 and \|Im (ρ)\| is suﬃciently large, then there exists a solution of the Helmholtz equation ∆u(x) + k2eq(x)u(x) = 0 in D of the form u = e−ρ·x(1 + ψ(x)), where ψ satisﬁes ∥ψ∥Lr(D) = O(\|Im (ρ)\|−1), as \|ρ\| →∞. Proof. We can write eq = 1−χKϕ for some cube K = [0, a]N and for some ϕ ∈Hs,p c (RN) by the conditions on eq and the Sobolev extension theorem on Lipschitz domains. Writing m = χKϕ, the equation that we need to solve is Proof. We can write eq = 1−χKϕ for some cube K = [0, a]N and for some ϕ ∈Hs,p c (RN) by the conditions on eq and the Sobolev extension theorem on Lipschitz domains. subject to the transmission conditions It is enough to use Lemma 3.4 to prove an analogue of Lemma 3.3, and then argue as in the proof of Theorem 1.1. subject to the transmission conditions Writing m = χKϕ, the equation that we need to solve is m = χKϕ, the equation that we need to solve is (∆+ k2(1 −m))u = 0 in D. The result would then follow from [BPS14, Theorem 2.3], except that this theorem was proved under the condition ϕ ∈C∞instead of ϕ ∈Hs,p. The result would then follow from [BPS14, Theorem 2.3], except that this theorem was proved under the condition ϕ ∈C∞instead of ϕ ∈Hs,p. 13 Inspecting the proof in [BPS14] we see that it is enough that the function Inspecting the proof in [BPS14] we see that it is enough that the function Q = −k2(1 −m)ΦD, where ΦD ∈C∞ c (RN) satisﬁes ΦD = 1 near D, satisﬁes [BPS14, formula (34)], i.e. tha one has d Q ∈d B1 r,1 and ∥Qg∥d B1 r,1 ≤CQ∥g∥\ B−1 r,∞ (3.2) (3.2) where the spaces are as in [BPS14]. Now we can write Q = Q1 + Q2 where Q1 = −k2ΦD ∈C∞ c (RN) and Q2 = fχK where where the spaces are as in [BPS14]. Now we can write Q = Q1 + Q2 where Q1 = −k2ΦD ∈C∞ c (RN) and Q2 = fχK where f = k2ϕΦD, f = k2ϕΦD, so that f ∈Hs,p c (RN). We use [BPS14, Lemma 4.3 and preceding discussion] to conclude that when supp(q) ⊂BR, one has ∥q∥d B1 r,1 ≤CR∥ˆq∥Lr, ∥qg∥d B1 r,1 ≤2R2∥ˆq∥L1∥g∥\ B−1 r,∞, ∥χKg∥d B1 r,1 ≤Cr∥g∥d B1 r,1. ∥χKg∥d B1 r,1 ≤Cr∥g∥d B1 r,1. It follows that (3.2) will be satisﬁed if the function f deﬁned above satisﬁes ˆf ∈ L1 ∩Lr. Since f ∈L1 c(RN), we have ˆf ∈L∞(RN) and it is enough to check that ˆf ∈L1. But if (ψj(ξ))∞ j=0 is a Littlewood–Paley partition of unity and if 1 ≤p ≤2, we obtain by the H¨older and Hausdorﬀ–Young inequalities that Z RN \| ˆf(ξ)\| dξ = ∞ X j=0 Z RN ψj(ξ)\| ˆf(ξ)\| dξ ≤C ∞ X j=0 2jN/p∥ψj(ξ) ˆf(ξ)∥Lp′ ≤C ∞ X j=0 2jN/p∥ψj(D)f∥Lp ≤C∥f∥BN/p p,1 where ψj(D) is the Fourier multiplier with symbol ψj(ξ) and the last norm is a Besov norm. Since Hs,p ⊂Bs p,∞⊂BN/p p,1 for s > N/p, we get ˆf ∈L1 as required. This shows that (3.2) is satisﬁed, which concludes the proof. Proof of Theorem 1.2 under assumption (b). 4 Concluding remarks In two dimensions, we have veriﬁed the uniqueness in identifying a convex penetrable scatterer of polygonal type with a single far-ﬁeld pattern, provided the refractive index is discontinuous at the corner points but Cα-H¨older continuous inside near the corners. 14 In higher dimensions, the uniqueness applies to convex polyhedra with additional as- sumptions on the geometrical shape (i.e., boxes) and on the smoothness of the contrast. In this study, the smoothness assumption is required only near the corner points. Our future eﬀorts will be devoted to the uniqueness proof in 3D for convex polyhedra with general Cα-H¨older (α > 0) continuous potentials. Since the CGO solutions can be constructed with plenty of generality, the 3D proof essentially requires to evaluate the Laplace transform of a harmonic homogeneous polynomial over a general three- dimensional corner domain and then to prove the vanishing of this polynomial through novel techniques. Another possible approach would be to analyze the corner and edge singularities of an elliptic equation with analytical Cauchy data in weighted H¨older spaces. Further results will be presented in a forthcoming publication. Acknowledgment The ﬁrst author would like to acknowledge the support from the German Research Foun- dation (DFG) under Grant No. HU 2111/1-2. The second author was partly supported by an ERC Starting Grant (grant agreement no. 307023) and CNRS. The second and third authors were also supported by the Academy of Finland (Centre of Excellence in Inverse Problems Research), and they would like to thank the Institut Henri Poincar´e Program on Inverse Problems in 2015 where part of this work was carried out. References [AR05] G. Alessandrini and L. Rondi, Determining a sound-soft polyhedral scatterer by a single far-ﬁeld measurement, Proc. Amer. Math. Soc. , 133 (2005): 1685– 1691 (Corrigendum: arXiv: math/0601406v1). [BIY15] E. Bl˚asten, O. Yu. Imanuvilov, M. Yamamoto, Stability and uniqueness for a two-dimensional inverse boundary value problem for less regular potentials, Inverse Probl. Imaging (to appear). [BPS14] E. Bl˚asten, L. P¨aiv¨arinta and J. Sylvester, Corners always scatter, Comm. Math. Phys., 331 (2014): 725–753. [Bu08] A. L. Bukhgeim, Recovering a potential from Cauchy data in the two dimen- sional case, J. Inverse Ill-Posed Probl., 16 (2008): 19–33. [CH13] F. Cakoni and H. Haddar, Transmission eigenvalues in inverse scattering theo- ry, in Inverse Problems and Applications: Inside Out II (edited by G. Uhlman- n), MSRI Publications volume 60, Cambridge University Press, 2013. [CY03] J. Cheng and M. Yamamoto, Uniqueness in an inverse scattering problem with non-trapping polygonal obstacles with at most two incoming waves, Inverse Problems, 19 (2003): 1361-1384 (Corrigendum: Inverse Problems 21 (2005): 1193). 15 [CK98] D. Colton and R. Kress, Inverse Acoustic and Electromagnetic Scattering The- ory, volume 93 of Applied Mathematical Sciences, Springer, New York, 1998. [Da88] M. Dauge, Elliptic Boundary Value Problems in Corner Domains, Lecture Notes in Mathematics, Vol. 1341, Springer-Verlag, Berlin, 1988. [EY06] J. Elschner and M. Yamamoto, Uniqueness in determining polygonal sound- hard obstacles with a single incoming wave, Inverse Problems, 22 (2006): 355- 364. [EY08] J. Elschner and M. Yamamoto, Uniqueness in determining polyhedral sound- hard obstacles with a single incoming wave, Inverse Problems, 24 (2008): 035004. [EH11] J. Elschner and G. Hu, Uniqueness in inverse transmission scattering problems for multilayered obstacles, Inverse Problems and Imaging, 5 (2011): 793–813. [EH15] J. Elschner and G. Hu, Corners and edges always scatter, Inverse Problems, 31 (2015): 015003. [Gr92] P. Grisvard, Singularities in Boundary Value Problems, Research Notes in Applied Mathematics, volume 22, Masson, Paris, 1992. [HN87] G.M. Henkin, R.G. Novikov, The ∂-equation in the multidimensional inverse scattering problem, Russian Math. Surveys, 42 (1987): 109–180. [HLL15] G. Hu, J. Li and H. Liu, Uniqueness in determining refractive indices by formally determined far-ﬁeld data, Appl. Anal., 94 (2015): 1259–1269. [HL14] G. Hu and X. Liu, Unique determination of balls and polyhedral scatterers with a single point source wave, Inverse Problems, 30 (2014): 065010 [Is08] V. Isakov, On uniqueness of domains and general transmission conditions in inverse scattering, Comm. Math. Phys., 280 (2008): 843–858. [Is90] V. References Isakov, On uniqueness in the inverse transmission scattering problem, Com- m. Partial Diﬀerential Equations, 15 (1990): 1565–1587. [KRS87] C. E. Kenig, A. Ruiz, C. D. Sogge, Uniform Sobolev inequalities and unique continuation for second order constant coeﬃcient diﬀerential operators, Duke Math. J., 55 (1987): 329–347. [Ki11] A. Kirsch, An introduction to the mathematical theory of inverse problems, 2nd edition, volume 120 of Applied Mathematical Sciences, Springer, 2011. [KG08] A. Kirsch and N. Grinberg, The Factorization Method for Inverse Problem- s, volume 36 of Oxford Lecture Series in Mathematics and its Applications, Oxford University Press, Oxford, 2008. [Ki93] A. Kirsch and R. Kress, Uniqueness in inverse obstacle scattering, Inverse Problems, 9 (1993): 285-299. [LZ06] H. Liu and J. Zou, Uniqueness in an inverse obstacle scattering problem for both sound-hard and sound-soft polyhedral scatterers, Inverse Problems 22, (2006): 515-524. 16 [MNP00] V. G. Maz’ya, S. A. Nazarov and B. A. Plamenevskii, Asymptotic Theory of Elliptic Boundary Value Problems in Singularly Perturbed Domains Volume I, Operator Theory: Advances and Applications Vol. 111, Birkh¨auser-Verlag, Basel, 2000. Translated from the German by Georg Heinig and Christian Posthoﬀ. [Na88] A. Nachman, Reconstructions from boundary measurements, Ann. of Math., 128 (1988): 531–576. [PSV14] L. P¨aiv¨arinta, M. Salo and E. V. Vesalainen, Strictly convex corners scatter, arXiv:1404.2513. [SU87] J. Sylvester and G. Uhlmann, A global uniqueness theorem for an inverse boundary value problem, Ann. of Math., 125 (1987): 153–169. [Uh14] G. Uhlmann, Inverse problems: seeing the unseen, Bull. Math. Sci., 4 (2014): 209–279. 17
https://openalex.org/W4384829280	https://www.researchsquare.com/article/rs-3185730/latest.pdf	English	null	Elderly Abuse and Neglect in Long Term Care Facilities in America : A systematic Review	Research Square (Research Square)	2,023	cc-by	7,194	Systematic Review Keywords: physical abuse, sexual abuse, abandonment, self-neglect, emotional abuse, ¦nancial or material exploitation, neglect Posted Date: July 20th, 2023 Title: Elderly Abuse and Neglect in Long Term Care Facilities in America : A systematic Review Title: Elderly Abuse and Neglect in Long Term Care Facilities in America : A systematic Review Affiliation: The Wright Center For Graduate Study ; The Wright Center For Community Health DOI: https://doi.org/10.21203/rs.3.rs-3185730/v1 DOI: https://doi.org/10.21203/rs.3.rs-3185730/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License ABSTRACT Hypothesis: Elderly abuse and neglect occurs as a result of lack of relevant personnel training, poor remuneration, and depression among caregivers. To test the hypothesis, overall findings analyzed confirm if they were in agreement or not. Background: Reports of cases of elderly abuse and neglect have been on the increase. To add on to that, not enough studies exist that can be of help in guiding policy-makers to formulate solutions and offer answers as to why this problem is on the increase solutions and offer answers as to why this problem is on the increase. Method: These researchers conducted a review of existing literature about elderly abuse in order to better understand the risk factors and causes of elderly abuse and neglect. Information was mined from various databases that contained information relevant to this review. Results: Results of findings showed that reports of abuse cases were on the rise, especially among the elderly patients; worse still for those with a secondary chronic illness like dementia and Parkinson’s Disease. Women reported more cases of abuse compared to those reported by men. By demographics, abuse was found to be much higher among minority groups like the African Americans and Asians Americans. Some elderly patients in the studies experienced concurrent types or forms of abuse. Those with severe forms of cognitive impairments reported the highest cases of abuse and self-neglect. Conclusion: Better remuneration, continuous training to caregivers about aging and better healthcare skills are necessary to help to end this scourge. This study concludes that despite great efforts made by some institutions to end abuse and neglect, more publicity, more studies or research and more funds are required in order to build a sufficient body of knowledge that can be relied upon by the relevant policy-makers. Keywords: physical abuse, sexual abuse, abandonment, self-neglect, emotional abuse, financial or material exploitation, neglect INTRODUCTION Elder abuse and neglect in community settings has in the recent past received more attention because of the rising number of cases of abuse. According to available literature, about 2 million cases of elderly abuse and neglect are reported annually (Cornell University, 2014). This literature suggests that elder abuse is a complex phenomenon, which may or may not involve a malicious perpetrator but that which may present severe health issues that include physical injury and death. Physical abuse, sexual exploitation, emotional abuse, and financial exploitation of the elderly and the vulnerable qualify as forms of abuse (Cooper & Livingston, 2016). Despite fuzzy literature estimations, we simply do not know for certain the number of elderly people that are suffering from abuse and neglect. Abuse may go unnoticed or be missed by the professionals that work with older Americans because of lack adequate training or resources for detecting it. The elderly in some instances may be reluctant to report abuse themselves because of fear of retaliation, or lack of physical and or cognitive ability to report, or because they do not want to get the abuser in trouble. In most cases the perpetrators tend to be of the male gender, a close family member like their child or spouse, have a history of substance abuse, have mental or physical health problems, have a history of trouble with the law, are financially unstable, or are experiencing major stresses like depression (Laumann, Leitsch, and Waite, 2008). The abuse itself may be in the form of intentional or unintentional neglect in the hands of an able person or by the elders themselves through what is referred to as self-neglect (Fulmer, 2012). These researchers have grandparents or elderly relatives who are almost entirely dependent on the family in every aspect of their life. What scares them is the rise in the number of elderly people who have been victims of abuse, exploitation, and neglect, especially in the hands of close relatives and professional caregivers. What if they were not there to provide for them and the elderly relatives were then placed in the hands of someone that would later turn out to be abusive or neglectful? What if they ended up in one of those nursing homes where reports indicate that abuse and neglect cases happened or continue to happen (U.S. Government Accountability Office, 2016)? INTRODUCTION It is because of this reason; the rise in the number of abused elderly, that these researchers sought to do a review of literature about cases of abuse and neglect in homes and long-term healthcare facilities. This will help shine the light on a subject that they consider to be the most abhorrent of any form of abuse outside of child abuse. A National Research Council study findings pointed to the fact that no efforts had been made yet to come up with , to evaluate or even to carry out interventions that had a scientifically grounded hypothesis as pertains to causes of elderly maltreatment. In addition, there exists a scant body of literature on abuse, its prevalence, and interventions used to curb these acts (National Research Council, 2003, p.121). So this review seeks to add to the available literature and body of knowledge on a topic that has for a long time been ignored or avoided; this research also contributes to the field of gerontology by providing a source of reference for students, teachers and healthcare providers just in case they encounter cases of abuse and are in need of answers to questions covered by this review. METHODOLOGY The researcher evaluated data that was extracted by utilizing the following checklist. The researcher evaluated data that was extracted by utilizing the following checklist. 1. Was the target population sample chosen using a clear inclusion and exclusion criteria? 1. Was the target population sample chosen using a clear inclusion and exclusion criteria? 2. Were data collection instruments in the studies standardized ? 1. Was the target population sample chosen using a clear inclusion and exclusion criteria? 2. Were data collection instruments in the studies standardized ? 2. Were data collection instruments in the studies standardized ? 3. Was the abuse measure instrument valid and reliable? 3. Was the abuse measure instrument valid and reliable? 4. Were features of sampling design factored into the final data analysis by appropriate and relevant weighting ? To determine the depth and quality of existing literature about elderly abuse and neglect, a thorough review of healthcare literature was conducted and each publication was rated. The literature inclusion criteria were: Published research , written in English and reporting on abuse of people aged over 65 years.The inclusion and exclusion criteria is detailed in a section below. The researcher conducted an electronic search of five databases; PubMed, Cumulative Index to Nursing and Allied Health Literature(CINAHL), Excerpta Medica dataBASE (EMBASE), and TRIP medical database. What is Elderly Abuse and Neglect? For the purpose of this review, the phrase “elderly abuse and neglect” is used as an all inclusive term as listed in most of the jurisdictions in the United States found through perusal of adult literature archived in the National Library of Medicine ( NLM). In broad terms, the NLM defines elder abuse as any form of physical , nutritional and emotional maltreatment of an older person by a family member or by a professional caregiver at home or at an institutional setting. In an attempt to offer a more clearer definition of elder abuse and neglect, professionals in social studies agree to a broad categorization of what entails abuse. In some studies, aftermath and results of the abusive acts and and how such acts are perceived in society have been the main focus. Hudson and Carlson (1998) set forth what is considered to be a more clearer definition of elder abuse and neglect offering comparisons of competing perspectives as viewed by the general public and those of a group of professionals in social studies. Both perspectives identify psychological, physical, social, and financial exploitation as the most common forms that abuse may take. In addition, the context in which the abusive acts happen has an effect on the perception and approach by the two groups. Most definitions of abuse are derived from an analysis of existing State and Federal definitions of elder abuse, neglect and exploitation. The National Center on Elder Abuse defines seven different types of elder abuse: physical abuse; sexual abuse; emotional abuse; financial exploitation; neglect; abandonment; and self-neglect. Prevalence of some of these is summarized in Bath and North East Somerset Council figure of 2012 (Figure 1). Note: Data of forms of abuse sourced from Bath and North East Somerset Council (2012) Note: Data of forms of abuse sourced from Bath and North East Somerset Council (2012) Data Extraction These databases were searched from the time when Elder abuse was first described in the medical literature to the most recent publication on the same topic (Murphy, Waa, Jaffer & Sauter, 2013). The databases were perused using a combination of keywords such as: abuse and neglect, aged population, abused elderly, elderly abuse, and elderly exploitation. To add to this, a manual lookup of references of publications dated prior to 2010 and a reference search of elderly abuse literature reviews and annotations was conducted. All selected literature for this research were published in peer reviewed journals and contained primary data on elderly abuse and neglect. To cover all bases additional research was conducted in books, book chapters, conference presentations, un-published articles, and magazine articles. This was done just in case any findings to the contrary existed outside of peer reviewed and other open access journals. Single case reports were eliminated from consideration in this report. The articles were critically reviewed and assigned a rating based on the methodology of research used, validity and reliability of the findings and relevance. The rating method used in this research emphasized the credibility of evidence and validity and reliability as provided for by the Center for Evidence Based Medicine at The University of Oxford (Center for Evidence Based Medicine, 2009). In general the research and literature review followed the steps as shown in the Literature Review Process Figure (Figure 2). Physical abuse Any action by a caregiver towards the elderly under their care that may result in bodily injury, physical harm, or even impairment. Emotional abuse Infliction of , pain, anguish or distress through verbal or non-verbal actions directed towards the victim. Financial or material exploitation Illegal or improper use of an elder's finances, valuables, or property. Refusal, or failure of a caregiver to provide for or fulfill any part of a person’s obligations to an elderly person that is under their care. Sexual Abuse xual contact of any kind with an elderly person without their permission or consent Sexual contact of any kind with an elderly person without their permission or consent. Abandonment Desertion of an elderly person by a caregiver or one who has physical custody of the person that had assumed role and responsibility of providing for them. Self-neglect These are personal actions or behaviors that may threaten the safety and health of the same individual (elder). Emotional abuse Inclusion and Exclusion Criteria Inclusion Criteria ● Studies presenting primary data, and with more than two study subjects ● Studies that compared abused and non abused elderly outcomes ● Studies that were published in a peer-reviewed journal and less than 10 years ● Studies of patients over the age of 65 years. ● Studies that were in English ● Studies that were in English Exclusion Criteria ● Studies which did not meet the selection criteria and were not primary studies ● Studies which did not meet the selection criteria and were not primary studies ● Studies that had less than two study subjects ● Studies that did not do a comparison between abused and non abused elderly adults ● Studies that were not published in a peer-reviewed journal and less than 10 years ● Studies that did not do a comparison between abused and non abused elderly ● Studies that were not published in a peer-reviewed journal and less than 10 years ● Studies of patients under the age of 65 years old ● Studies of patients under the age of 65 years old ● Studies that were not written in English ● Studies that were not written in English 8. Comments This section contained a comment on design and summary topic 6. Quality of Study 6. Quality of Study 7. Magnitude of Benefit This section briefly summarizes how strong a benefit is of the study: small, medium, large, or none. How the Evidence Table was Created The evidence table was designed to include 8 columns that provides a summary describing the study, methodology , significance, size of sample, quality of the study and statistical significance of the published research. These columns are briefly described below. 1. Condition 1. Condition This section describes the type of disease the study addresses 2. Study Design This column names the study methodology used. 3. Author, Year This section identifies researchers and year of the study publication . 4. N This section noted the total number of people sampled for the study . 5. Statistically Significance This column names the study methodology used. This column names the study methodology used. This section noted the total number of people sampled for the study . 5. Statistically Significance Results here were noted as being statistically significant if the study's authors reported that statistical significance, was present. 6. Quality of Study Demarcated by numerical score between 0 and 5; this was assigned as an estimated measure of study design quality (0 being weakest and 5 being strongest). Jadad Score Calculation was utilized 6. Quality of Study Demarcated by numerical score between 0 and 5; this was assigned as an estimated measure of study design quality (0 being weakest and 5 being strongest). Jadad Score Calculation was utilized Prevalence of Abuse and Neglect This research identified 28 studies done in the United States and outside that had information relating to cases of elderly and neglect , predisposing factors for abuse, and relevant data on outcomes for the said victims. Of the abuse types mentioned above , psychological abuse seemed to form the greatest portion of the abuse perpetrated against the elderly adults. It was estimated to form between 30 percent and 60 percent of all prevalence cases of abuse in general. The other forms of abuse accounted for a small portion of the cases and the numbers ranged between 4 percent and 24 percent (Cooper, Manela, Katona, Livingston, 2008). In this research it was found that, of 200 elderly abuse cases reported, over 50 percent of the victims suffered from some cognitive and physical impairment . In one study it was documented that 380 caregivers for elderly patients admitted to the fact that they had in one way or the other abused or neglected an elderly patient under their care (Burgess & Phillips, 2006). In another research that comprised over 4000 elderly patients living in an assisted living community , 20 percent of men and 14 percent of women suffered self-neglect form of abuse (Lee, Kolomer, 2007). In a different survey study of about 100 providers for elderly patients with dementia, about fifteen percent of their patients reported as having been a victim of financial exploitation ( Southern, 2013). Some elderly patients, especially those living in community settings reported experiencing different forms of abuse simultaneously. For instance, in one study of about 125 elderly patients, 30 percent of those that reported abuse had endured multiple forms of abuse and neglect simultaneously (Wiglesworth et.al., 2010). Some studies showed abuse directed towards the caregiver as a retaliation to abuse by the latter. The abuse by patients mostly took the form of verbal and psychological abuse. A small percentage involved physical abuse (Wiglesworth et.al., 2010). In another study of 510 elderly participants, it was found that abuse and neglect led to increased cases of depression as reported among the elderly (Gil,Kislaya,Santos,Nunes,Nicolau & Fernandes , 2017). Existence of depression secondary to abuse resulted in increased intensity and occurrence of abuse. It had a cumulative effect regardless of the fact that abuse and neglect resulted in the cases of depression. Prevalence of Abuse and Neglect From research evidence it was noted that abuse was most prevalent in nursing homes and long-term care facilities that included rehabilitation centers. There was a study done that comprised 800 households where patients were receiving long-term healthcare and 13 percent of those sampled reported rampant incidents elderly abuse (Post et.al., 2010). Adding to this, elder-to-elder abuse in these care facilities was surprisingly commonplace (Lach et.al., 2009). In elder-to elder abuse, it was found that a majority of the victims, just like in other common forms of abuse, were either cognitively or physically impaired (Shinoda-Tagawa et.al., 2004). Risk Factors for Abuse Considering the complexity of abuse , the researcher decided to categorize the risk factors into two main groups. They are victim risk factors and caregiver risk factors. Victim Risk Factors Older patients, especially those with physical or cognitive impairment tend to display aggression and frustration; this may serve as a recipe for conflict between the patient and the care-provider that may have led to the many cases of abuse reported and which in most cases had been detrimental to the health of the victims. This is especially true among patients with Alzheimer's as the disease predisposes them to frustration and aggressive behavior. Self neglect was found to be prevalent among those patients with severe cases of physical and cognitive impairment. The more impaired a person is , the more likely they are to be victims of abuse by caregivers. However, this does not mean that the elderly patients without physical or cognitive impairment were any safer (Yan & Kwok, 2011). Caregiver Risk Factors Caregiver Risk Factors Results show that most perpetrators of abuse are either close family members or paid professional healthcare givers. Others may include friends, nursing home workers and even physicians. Stress and fatigue were singled out as major risk factors for abuse. High ratio of patient-to-caregiver, and burdensome work hours have been blamed for the rising cases of abuse. Most nursing homes have seen an influx of elderly patients seeking their services ; little has been done in terms of expanding the workforce to cater for the surge (Yan, 2011). The US Census Bureau figure (Figure 3) illustrates the growth in population numbers of the elderly. Life circumstances may also lead elderly to take custody of their grandchildren. In a study conducted among such parents, cases of self neglect were high. These grandparents view the care of their grandchildren to be of utmost importance and sometimes neglect taking care of themselves. They take care of them because of the sense of fulfillment they derive out of it. To these parents self-care is most of the time misunderstood to mean just physical health while they disregard other aspects of their health (Lee & Kolomer , 2017). Other factors like depression and substance abuse have been cited as factors responsible for this vice ( Compton, Flanagan & Gregg, 1997). Poor training on crisis management as regards the elderly population is also another reason. Victim Risk Factors Cases of poor relationships with the patients and lack of skills to handle patients of different backgrounds were found to have triggered occurrences of conflicts that in most cases led to conflict and then abuse (Cooper, Manela, Katona, Livingston, 2008). Lastly poor compensation and remuneration for services provided were also seen as a source of major stress that predisposes elderly patients to acts of abuse and neglect. provided were also seen as a source of major stress that predisposes elderly patients to acts of abuse and neglect. Genetic predisposition to a person committing abuse has also been a subject of debate in recent times. A study evaluated 151 publications that analyzed biomarkers such as cortisol and epinephrine as a basis for determining why there existed a high number of abuse cases among certain communities like those in the Yan & Kwok (2011) study. The studies showed an increased amount of cortisol level among abusers. Same studies on epinephrine and other biomarkers were inconclusive (Allen et al., 2017). Does Demography Play a Role ? Scant information was available on demography and this made it difficult to draw a clear conclusion as to whether abuse and neglect of the elderly followed a racial, social or ethnic trend. However, in one research review and as noted above, the findings suggested that cases of exploitation, especially monetary, were commonly directed against patients with physical and cognitive impairment ( Yan & Kwok, 2011). Acts of abuse were more prevalent among African Americans as compared to other races. Other minorities like the Asian Americans also reported cases of abuse with numbers that were higher than those of other communities (Beach, Schulz, Castle & Rosen, 2010). In another review , the findings of the study showed that there existed scant research in the area of elderly abuse among minorities and historically underserved groups. It found that most of the research in existence explained abuse cases as being a result of culture and beliefs as practices among these minorities. The review called for more research and more funds for the same; and research that will widen the scope beyond cultural aspects and views as regards to abuse and neglect. It encourages an examination of such factors as social economic status, access to healthcare facilities, geographical location, and historical effects of social and racial discrimination (Jervis, Hamby, Beach, Williams, Maholmes, & Castille, 2016). In light of these findings, this researcher also believes that more studies should be conducted to help shed more light on why these discrepancies exist as you move from one community to another. Are there cultural beliefs and practices alone to blame for the findings? Are there other confounders that explain this phenomenon? Are there differences in the way abuse is perceived and carried out as one moves from one community to the next? Better solutions will be found against this problem if and when the answers to the questions above are provided. DISCUSSION Upon review, findings emphasized the need for a better approach and understanding of abuse and neglect cases elderly patients faced in nursing homes and other healthcare facilities. Even though the reviewed studies show different trends in terms of prevalence, all of them show a spike in the number of abuse cases in healthcare facilities across the United States. This calls more elaborate national studies that will provide a better understanding of this scourge; with relevant data that is both representative and applicable to all cases of abuse that are reported in the United States. relevant data that is both representative and applicable to all cases of abuse that are reported in the United States. Most of the studies examined for this research and listed in the Evidence table (Table 1), centered on abuse occurring among patients with some form of chronic illness like Alzheimer's or those with a physical handicap. In the study it emerged that very few studies exist dedicated to a specific form of abuse like exploitation or elder-to-elder abuse (Cooper, Manela, Katona, Most of the studies examined for this research and listed in the Evidence table (Table 1), centered on abuse occurring among patients with some form of chronic illness like Alzheimer's or those with a physical handicap. In the study it emerged that very few studies exist dedicated to a specific form of abuse like exploitation or elder-to-elder abuse (Cooper, Manela, Katona, Livingston, 2008). Also very little research existed that evaluated abuse cases among minorities and underserved communities (Jervis, Hamby, Beach, Williams, Maholmes, & Castille, 2016). Providers and patients alike would benefit from more studies that widened their scope to include data from all demographics; they will utilize the findings in formulating more pointed strategies that will then translate to reduced cases of abuse and neglect of elderly patients. Older patients who are isolated and whose sole source of contact is through the caregivers are at an increased risk of abuse. A Council on the Aging (COTA) study found that the number of people who are socially will increase twice its current size in 20 years. These trends as noted above are illustrated in summary in Figure 3. The study goes on to say that the numbers may increase even more than this projection considering that more and more people are living longer and thus the number of seniors is likely to increase (Pate , 2014). DISCUSSION A National Institute of Justice research study findings confirmed an existence of a connection between social isolation and increased incidents of abuse and neglect of the elderly. It's however safe to note that these researchers weren’t sure whether cases were as a result of pure circumstance or as a result of perpetrators actively isolating the elders from their contacts or relatives in order to minimize the risk of being reported (Park & Mulford 2017). All in all, they don't have contact with friends or relatives that can advocate for their needs and protection and are left at the mercy of their caregivers. Even though there is gender disparity in abuse cases whereby women formed a bigger percentage of the victims, other surveys’ findings could not conclusively link this fact to instances of abuse (Friedman et.al, 2011). As noted above , substance abuse, stress, depression and poor training formed the core of character of most abusers (Yan & Kwok, 2011). Evidence based research was also scant. Learning from such research, especially when it comes directly from those practicing in the field, offers a first hand experience that is more reliable and which can be used by agencies to draw policy. Further research and review is therefore of great need to help enhance the current approaches utilized in curbing this problem. This review highlights significant gaps of knowledge on multicultural perspectives. Caregivers lacking such requisite knowledge tend to have problems when dealing with elderly patients especially if they come from a different background from the caregiver (Wiglesworth et.al., 2010). American society is increasingly growing more diverse and this presents complexities and challenges more so in healthcare settings where communication and physical interaction is key to successful outcomes. Knowledge and understanding of the major cultural perspective that forms the melting pot that is American society will go a long way in reducing conflicts. This trend of migration is projected to continue and educating caregivers or providers on issues pertaining to multiculturalism specific to aging will help to reduce cases of abuse and neglect across the country; some of the abuse cases reported have been as result of lack of this particular knowledge. The knowledge on diversity helps one to be more sensitive and respectful when it comes to dealing with those that are different . Community organizations , colleges and This review highlights significant gaps of knowledge on multicultural perspectives. DISCUSSION Caregivers lacking such requisite knowledge tend to have problems when dealing with elderly patients especially if they come from a different background from the caregiver (Wiglesworth et.al., 2010). American society is increasingly growing more diverse and this presents This review highlights significant gaps of knowledge on multicultural perspectives. complexities and challenges more so in healthcare settings where communication and physical interaction is key to successful outcomes. Knowledge and understanding of the major cultural perspective that forms the melting pot that is American society will go a long way in reducing conflicts. This trend of migration is projected to continue and educating caregivers or providers on issues pertaining to multiculturalism specific to aging will help to reduce cases of abuse and neglect across the country; some of the abuse cases reported have been as result of lack of this particular knowledge. The knowledge on diversity helps one to be more sensitive and respectful when it comes to dealing with those that are different . Community organizations , colleges and schools should be encouraged to participate in this effort. Companies should offer grants and scholarships that will support research in this area (Mosqueda & Dong, 2011). This review further highlights the need for policy driven initiatives by the federal and local government that is geared towards education and training of personnel about abuse, its detection and its prevention and then providing a guide to documenting and reporting. Such initiatives will need to inculcate study findings especially those specific to the elderly population in nursing homes and long term care facilities. Georgia Anetzberger (2000), stresses the need for enhanced education and training of personnel as the best way to help fight abuse and neglect of elderly patients.Its findings demonstrated that training helped equip the caregivers with better communication skills and a better understanding of the patients they took care of. Another study published in the European Journal of Public Medicine asserts that different strategies can be implemented to reduce or eliminate cases of elderly abuse. Such steps include but are not limited to awareness campaigns, better screening for abuse, integration into educational curriculums, and caregiver training (Yon, Ramiro-Gonzalez, Mikton, Huber, Sethi, 2018). The findings of this study have been referenced by major health organizations including World Health Organization (WHO) and the American Red Cross. DISCUSSION Knowledge about screening instruments that are currently in use like the Elder Abuse Index and Elder Abuse Suspicion Index that inculcate social and physical evidence in their results with proven validity and reliability has been shown to help in the management and reduction of elderly abuse and neglect (Fulmer, Strauss & Russell , 2012). It is therefore safe to say that training in general provides a better understanding of this scourge and then guides in documentation and reporting procedures as required by various community oversight bodies and federal agencies. documentation and reporting procedures as required by various community oversight bodies and federal agencies. The community has a big role to play. Citizens, relatives, law enforcement, social workers and everyone that is a stakeholder in ensuring the welfare of elderly patients, should take a proactive approach in making sure that the elderly among us are treated with utmost respect and care. Any instances of abuse no matter how small should be of concern to just about everyone. One day if we live long enough, we would also love for someone to stand up for us and be encouraging to those that care for us, to be more compassionate and understanding. Limitations of the Study Different surveys that the researcher interrogated presented challenges and limitations that could sway validity and reliability of the conclusion of this research. To begin, many of the studies in this review were cross-sectional studies.The summaries of the main studies are noted in Table 1. Because of this the causal factors identified in the studies pointed more to correlation as opposed to causality. Other studies in the survey were retrospective, a type of study that is very susceptible to recall bias. When it comes to prevalence, varied results as displayed across the studies used in this review raised questions and doubts as to the statistical significance of the findings and how representative the data was while some did not indicate the statistical significance of their studies. These differences could have been because of the varied use of methodologies and criteria for determining what qualified to be called abuse and what didn't. For example, in some studies the act of abuse had to be verified, in others the patients or employees were relied upon to provide data or report abuse and in other the researchers just went by suspected abuse or signs of abuse as displayed by victims. Most of the studies used in this review and included in the evidence table (Table 1) utilized highly valid and reliable instruments. However, a small number of the studies had not accounted for confounding factors and how such factors may have influenced the overall findings. With this in mind, the researcher minimized the impact of these inadequacies, by drawing conclusions using data from studies that were considered to have been done correctly and with reliable and valid data. CONCLUSION Elderly abuse and neglect is a growing problem facing many communities in the United States. With rapid increase in population as illustrated by Pate (2014) , the problem is more likely than not to continue increasing. This is more the reason for enhanced efforts where emphasis should be placed in trying to find a long-term solution. Caregivers, family members , government agencies and private entities have a role to play. Jervis, Hamby, Beach, Williams, Maholmes, & Castille (2016) calls for more research especially among minorities and underserved communities. More research that focuses on elderly abuse and neglect will help seal the gaps that exist and create a rich source to help understand elderly abuse and neglect; it will provide enough data that will be useful in formulating policies aimed at curtailing the menace. It can be noted that abuse in general has received a lot of attention and awareness. This has forced the federal and state agencies to institute strict laws and guidelines for handling abuse cases with stiff penalties as punishment to those that fail to comply. Despite these efforts, a large section of caregivers are still in need of adequate training necessary for proper handling and caring of elderly patients. Such training can be done in collaboration with various colleges and schools with funding sourced from private and government agencies supportive of the cause (Mosqueda, Dong, 2011). Current evidence supports the multifactorial etiology of elder abuse involving risk factors within the elder person, the perpetrator, their relationship and the environment. Lack of consistency in this field limits the potency of evidence derived from different studies and further research is required to test the strength and independence of the analyzed risk factors. Ultimately, it is hoped that this will lead to the development of a practical screening instrument to be used by health professionals, as well as informing them when developing new interventions (Wiglesworth et.al., 2010). Future Direction Future studies more specific to the various forms of abuse should be done. This will provide more pointed data relevant to specific forms of abuse and help draw remedies or solutions that are more specific. For instance, if a study or studies were done that evaluated financial exploitation form abuse, if patients were subjected to such acts, those in charge will have a source of reference for data and suggestions that will help provide answers and solutions; these studies are currently lacking (Jervis, Hamby, Beach, Williams, Maholmes, & Castille, 2016). The researchers view that the overall findings of this review are in agreement with the hypothesis of the study that states, “ Elderly abuse and neglect occurs as a result of lack of relevant personnel training, poor remuneration, and depression among caregivers.” TABLES AND FIGURES Table 1 : Elder Abuse and Neglect Publication Evidence Table Table 1 : Elder Abuse and Neglect Publication Evidence Table Condition Study Design Author, Year N Statistical Significance Quality of Study Magnitude of Benefit Comments Elderly Abuse Review Cooper, Manela, Katona, Livingston, 2008 86 Not reported 5 Large Most comprehensive study on screening Elderly Abuse Among minorities Systematic Review Jervis, Hamby, Beach, Williams, Maholmes, & Castille, 2016 123 Not reported 4 Medium Review of research in existence that covers abuse cases among minorities Elder and Grandchil dren Qualitativ e Inquiry Lee, Kolomer, 2017 24 Not reported 4 Medium Grandparent caregiving challenges. Elderly Abuse Review Southern, 2013 100 Not reported 4 Medium Perception of abuse by Alzheimer's society in London Elderly Abuse Case Control Wiglesworth et.al., 2010 125 Significant 5 Large Investigate characteristics of caregivers that are associated with mistreatment Elderly Abuse Case Control Post et.al., 2010 800 Significant 5 Large types and patterns of elder abuse by paid caregivers Elderly Abuse Case Control Yan & Kwok, 2011 122 Significant 5 Large Examined the prevalence and risk factors for elder abuse in older Chinese. Elder Abuse Survey Beach, Schulz, Castle & Rosen, 2010 620 N/A 5 Large ExamineD racial differences in the prevalence of financial exploitation. Future Direction Elder Abuse Survey Beach, Schulz, Castle & Rosen, 2018 903 N/A 5 Large Association between perceived social support, social network Elder Abuse Effects Cross Sectional Gil,Kislaya,Santo s,Nunes,Nicolau & Fernandes , 2017 510 Significant 4 Large Exploring the Correlates to Depression in Elder Abuse Victims Biologic al perspecti ve of Elder abuse Review Allen,et al.,2017 151 Significant 5 Large Psychobiologic al burden of care/ dementia patients Elder abuse preventi on Systematic review Yon,Ramiro-Gon zalez, Mikton, Huber, Sethi, 2018. 55 Significant 5 Large Prevalence of elder abuse in institutional settings. Figure 1 : Prevalence of Different Forms of Abuse Figure Note: Data of forms of abuse sourced from Bath and North East Somerset Council Note: Data of forms of abuse sourced from Bath and North East Somerset Council (2012) Figure 2: Literature Review Process Figure Figure 2: Literature Review Process Figure Note: Figure created by the author of this review (2019) Note: Figure created by the author of this review (2019) Figure 3: Elder Population Trends Figure Figure 3: Elder Population Trends Figure Note: Data for elder population trends from US Census Bureau (2013) Note: Data for elder population trends from US Census Bureau (2013) Note: Data for elder population trends from US Census Bureau (2013) REFERENCES Allen P., Curran E., Duggan A., Cryan, J., Ní Chorcoráin, et al. (2017). A systematic review of the psychobiological burden of informal caregiving for care recipients with dementia: Focus on cognitive and biological markers of chronic stress. Neurosci Biobehav Rev 73: 123- 164. Anetzberger G.J. , Palmisano B.R. , Sanders M., Bass D., Dayton C., & Eckert S.,(2000). A model intervention for elder abuse and dementia. Gerontologist . 2000 ; 40 ( 4 ): 492- 7 . Beach, S. R., Schulz, R., & Sneed, R. (2018). Associations Between Social Support, Social Networks, and Financial Exploitation in Older Adults. Journal of Applied Gerontology, 37(8), 990–1011. Beach R., Schulz R., Castle G., & Rosen J. (2010). Financial exploitation and psychological mistreatment among older adults: differences between African Americans and non–African Americans in a population-based survey.Gerontologist . 2010 ; 50 ( 6 ): 744 -57 . Burgess W. , Phillips L., (2006). Sexual abuse, trauma, and dementia in the elderly: a retrospective study of 284 cases .Vict Offender . 1 ( 2 ): 193-204. Centre for Evidence Based Medicine (2009). Critical appraisal. Retrieved 12th of December , 2018 from http://www.cebm.net/index.aspx?o=1157. Compton A. , Flanagan P , Gregg W . (1997). Elder abuse in people with dementia in Northern Ireland: prevalence and predictors in cases referred to a psychiatry of old age service . Int J Geriatr Psychiatry . 12 ( 6 ): 632-5. Cooper C. , Manela M. , Katona C. , Livingston G . (2008). Screening for elder abuse in dementia in the LASER-AD study: prevalence, correlates and validation of instruments . Int J Geriatr Psychiatry ; 23 ( 3 ): 283-8. Friedman L., Avila S., Tanouye K., (2011). A case control study of severe physical abuse of older adults. J Am Geriatr Soc, 59 , pp. 417-422. Fulmer T., Strauss S., Russell S., (2012).Screening for elder mistreatment in dental and medical clinics. Gerodontology, 29 (2012), pp. 96-105. Gil P.,Kislaya I., Santos J., Nunes B., Nicolau R., Fernandes A. (2017). Elder abuse in Portugal: Findings from the first national prevalence study. Journal of Elder Abuse & Neglect, 27, 174–195. Hudson, M. F., Armachain, W. D., Beasley, C. M., & Carlson, J. R. (1998). Elder abuse: Two Native American views. The Gerontologist, 38, 538-548. Hudson, M. F., Armachain, W. D., Beasley, C. M., & Carlson, J. R. (1998). Elder abuse: Two Native American views. The Gerontologist, 38, 538-548. Jervis, L. REFERENCES L., Hamby, S., Beach, S. R., Williams, M. L., Maholmes, V., & Castille, D. M. (2016). Elder mistreatment in underserved populations: Opportunities and challenges to developing a contemporary program of research. Journal of elder abuse & neglect, 28(4-5), 301-319. Lachs M. , Bachman R. , Williams S. , & O’Leary R . (2009). Resident-to-resident elder mistreatment and police contact in nursing homes: findings from a population-based cohort. J Am Geriatr Soc ; 55 ( 6 ): 840-5. Laumann, E. O., Leitsch, S. A., & Waite, L. J. (2008). Elder mistreatment in the United States: Prevalence estimates from a nationally representative study. Journal of Gerontology, 63, 48-54. Lee, M., & Kolomer, S. (2017). If I Don ’t Take Care of Myself, I Can ’t Take Care of Them: Exploring Caregiving Grandmothers ’ Experiences of a 9-Session Self-Care Curriculum. Journal of Gerontology. Lee, M., & Kolomer, S. (2007). Design of an Assessment of Caregivers’ Impulsive Feelings to Commit Elder Abuse. Research on Social Work Practice, 17(6), 729–735. Mosqueda L. , & Dong X.Q., (2011) . Elder abuse and self-neglect: “I don’t care anything about going to the doctor, to be honest. JAMA ; 306 ( 5 ): 532-40 . Murphy K., Waa S., Jaffer H., & Sauter A.,(2013). A Literature Review of Findings in Physical Elder Abuse. Retrieved on January 12th, 2019 from https://www.sciencedirect.com/science/article/pii/S0846537112001192#bib11 National Research Council, Panel to Review Risk and Prevalence of Elder Abuse and Neglect. (2003). Elder mistreatment: Abuse, neglect, and exploitation in an aging America. Washington, DC: National Academies Press. Park, Y., & Mulford, C., (2017). Social Support Can Diminish Negative Effects of Elder Abuse. National Institute of Justice (NIJ). Washington. Pate , A., (2014). Social Isolation: Its impact on the mental health and wellbeing of older Victorians. COTA for older Australians. Post, L., Page, C., Conner, T., Prokhorov, A., Yu Fang, & Biroscak, B. J. (2010). Elder Abuse in Long-Term Care: Types, Patterns, and Risk Factors. Research on Aging, 32(3), 323–348. Shinoda-Tagawa T , Leonard R , Pontikas J , McDonough J.E, Allen D , & Dreyer P. I. (2004) Resident-to-resident violent incidents in nursing homes. JAMA . 291 ( 5 ): 591-8. Southern, A. (2013). Mistreatment and abuse of people with dementia. Alzheimer’s Society. London. Wiglesworth A. , Mosqueda L. , Mulnard R. , Liao S. , Gibbs L. , & Fitzgerald W., (2010). Screening for abuse and neglect of people with dementia. REFERENCES J Am Geriatr Soc . 2010 ; 58 (3): 493-500. Yan E., & Kwok T.,(2011). Abuse of older Chinese with dementia by family caregivers: an inquiry into the role of caregiver burden. Int J Geriatr Psychiatry ; 26 ( 5 ): 527 – 35 . Yon Y., Ramiro-Gonzalez M., Mikton C., Huber M., & Sethi D., (2018). The prevalence of elder abuse in institutional settings: a systematic review and meta-analysis. European Journal of Public Health.\ Canfell K, Egger S, Velentzis LS, Brown JD, O'Connell DL, et al. (2015) Factors related to vaccine uptake by young adult women in the catch-up phase of the National HPV Vaccination Program in Australia: Results from an observational study. Vaccine 33(20): 2387-2394.
https://openalex.org/W3120772509	https://europepmc.org/articles/pmc7794475?pdf=render	English	null	Evaluation of the chemical defense fluids of Macrotermes carbonarius and Globitermes sulphureus as possible household repellents and insecticides	Scientific reports	2,021	cc-by	11,128	Evaluation of the chemical defense fluids of Macrotermes carbonarius and Globitermes sulphureus as possible household repellents and insecticides S. Appalasamy1,2, M. H. Alia Diyana2, N. Arumugam2 & J. G. Boon3 The use of chemical insecticides has had many adverse effects. This study reports a novel perspective on the application of insect-based compounds to repel and eradicate other insects in a controlled environment. In this work, defense fluid was shown to be a repellent and insecticide against termites and cockroaches and was analyzed using gas chromatography-mass spectrometry (GC– MS). Globitermes sulphureus extract at 20 mg/ml showed the highest repellency for seven days against Macrotermes gilvus and for thirty days against Periplaneta americana. In terms of toxicity, G. sulphureus extract had a low LC50 compared to M. carbonarius extract against M. gilvus. Gas chromatography–mass spectrometry analysis of the M. carbonarius extract indicated the presence of six insecticidal and two repellent compounds in the extract, whereas the G. sulphureus extract contained five insecticidal and three repellent compounds. The most obvious finding was that G. sulphureus defense fluid had higher potential as a natural repellent and termiticide than the M. carbonarius extract. Both defense fluids can play a role as alternatives in the search for new, sustainable, natural repellents and termiticides. Our results demonstrate the potential use of termite defense fluid for pest management, providing repellent and insecticidal activities comparable to those of other green repellent and termiticidal commercial products. A termite infestation could be silent, but termites are known as destructive urban pests that cause structural damage by infesting wooden and timber structures, leading to economic loss. Despite the negative perception that humans have of termites as pests, termites play a vital role in the maintenance of soil organic matter in natural habitats and in agroecosystems1–3 in both forests and urban environments. Termites are also an important food source for various other insects owing to their vast abundance in a range of habitats, with their main predators being ants4,5. Upon facing such strong predation from ants, termites have evolved a defense system involving the presence of specialized soldier termites amid sterile ones. Termites have both mechanical and chemical defense mechanisms4,6. However, termite defense mechanisms are one of the least explored and reported subjects. This intriguing insect defense mechanism could reveal the interrelationship and coevolution of compounds and termite species. The earliest insect defense mechanism was reported in4, and various defense mechanisms in termites were listed, including sensory organs, physical defenses, and chemical secretion. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Materials and method Ethics declaration. All applicable international and national guidelines for the care and use of animals were followed in this study. All procedures performed involving animals (insects) were conducted in accordance with the ethical standards of the institution or practice at which the studies were conducted. Species collection and identification. This study was conducted in Kelantan state, Malaysia. The soldier castes of M. carbonarius and G. sulphureus were collected from mounds around Rantau Panjang and Jeli, Kelan- tan. The specimens were brought back to the postgraduate laboratory of the Faculty of Earth Science, Universiti Malaysia Kelantan, Jeli Campus, and maintained at room temperature (23 °C) with relative humidity greater than 50% in dark conditions. Identification of the termite soldier morphology was performed according to keys provided in21 with the aid of a MOTIC 2500 5.0 MP Live Resolution (MOTIC, Hong Kong, China) camera attached to a stereomicroscope. Extraction of defense fluid. The defense fluid extraction protocol was adapted from22 with modifica- tion of the extraction duration. Soldier termites were rinsed with distilled water and then air-dried. Then, both species were dissected, weighed, homogenized and extracted in methanol for 24 h of extraction (HPLC grade; MERCK, Selangor, Malaysia). The extract was filtered by using a muslin cloth and WHATMAN Filter Paper No. 1 (SIGMA ALDRICH, Darmstadt, Germany). Then, the solution was dried using a rotary evaporator (IKA, Staufen, Germany) at 60 °C. The extract was weighed and stored at − 20 °C until further use. Repellency against the termite Macrotermes gilvus. This method was modified from23. The termite species Macrotermes gilvus was chosen to test the repellency of the extracted defense fluid. Macrotermes gilvus is the most abundant pest species that attacks the structural buildings in Kelantan10. A colony of M. gilvus was collected from mounds around Jeli, Kelantan. The boxes (28 L) were filled with a mixture of sand. The defense fluid extract was diluted to five concentrations using methanol (0, 1, 5, 15, and 20 mg/ml). The concentrations were used in every assay in this study. Half of the sand area was mixed with the extract. Thirty termite individuals (25 workers, 3 minor soldiers, 2 major soldiers) were released into the box24. The fungal combs were provided in small quantities (2 g), and hyphal growth was monitored and removed every 12 h. www.nature.com/scientificreports/ and irritant and have congealing effects on the secretion released into the wound11. This proves that the existence of chemical compounds in the defense fluid has a particular repellent and toxic effect on other insect species.hh lf The introduction of insect-based products as natural pesticides in industry is especially common. These prod- ucts utilize pheromones from semiochemicals produced via animal communication12. In Malaysia, pheromone traps are currently used as one of the products in the integrated pest management program for Lepidoptera and Coleoptera13. At present, chemical-based products are still in use, despite the introduction of biopesticides. The drawbacks of chemical-based products are their negative effects on safety, public health and the environment, including sublethal effects on nontarget species, such as the effects on the foraging patterns of honey bees14,15. In contrast, the production of plant-based pesticides is challenging. The utilization of essential oils as natural pesticides, such as oils from Mentha spp. and Lavanda spp. that help in the inhibition of acetylcholinesterase, is affected by the low quality, productivity and factor dependence (i.e., soil acidity) of these oils16–18. Thus, the essential oil composition can change along with changes in various factors. p g g g Natural pesticide product development is more focused on agricultural pests than on urban pests because of the economic benefits. This can be identified through the trends of publications over the years regarding the biological control of termites19. For example, one of the studies described the lack of success in the biological control of termites, as Nasutitermes species have evolved to resist diseases using biochemical and immunologi- cal strategies20. Hence, this research was conducted to study the potential of termite defense fluids as natural pesticides by utilizing chemical communication. Defense fluids of Macrotermes carbonarius and Globitermes sulphureus were used in this study. These species were selected due to their high abundance in rural areas of Kelantan10. In addition, both species are known as defense fluid producers and consist of a high number of soldier castes that can be found in mounds or colonies7. Evaluation of the chemical defense fluids of Macrotermes carbonarius and Globitermes sulphureus as possible household repellents and insecticides However, for chemical secretion, only a few species of termites, such as Macrotermes carbonarius (Blattodea: Termitidae) and Globitermes sulphureus, and their chemical compounds of interest were reported as of June 20107. hl p p p J The termite defense fluid helps to protect their colony by secreting acetate-derived compounds (from fatty acid metabolism) found in Macrotermes and Globitermes6. In a related study, the terpenoid hydrocarbon β-selinene excreted by Reticulitermes speratus helped fend off the Asian needle ant Brachyponera chinensis and ponerine ant Myopias amblyops8. Similarly, M. carbonarius and G. sulphureus soldiers can defend themselves from other insects by secreting defense fluid9,10. In a previous study, termites from the family Mastotermitidae and several from the subfamily Macrotermitinae produced quinone mono- and sesquiterpenes with macrocyclic lactones that are toxic 1Institute of Food Security and Sustainable Agriculture (IFSSA), Universiti Malaysia Kelantan, Jeli Campus, 17600 Jeli, Kelantan, Malaysia. 2Faculty of Earth Science, Universiti Malaysia Kelantan, Jeli Campus, 17600 Jeli, Kelantan, Malaysia. 3Faculty of Bioengineering and Technology, Universiti Malaysia Kelantan, Jeli Campus, 17600 Jeli, Kelantan, Malaysia. email: suganthi.a@umk.edu.my \| https://doi.org/10.1038/s41598-020-80018-5 Scientific Reports \| (2021) 11:153 www.nature.com/scientificreports/ Materials and method The mortality data were recorded daily at 20:00 h. The commercial product Chlorpyrifos (DYNA-PEST, Kuala Lum- pur, Malaysia) was used as a positive control, and methanol was used as a negative control. Toxicity against the cockroach Periplaneta americana. For the toxicity test of the cockroach P. amer- icana, the method was adapted from26,27. Test cockroaches were starved for three days before the toxicity test. Five concentrations of extract were mixed with crushed cat food (POWERCAT, Selangor, Malaysia) and fed to the cockroaches. Commercial cockroach insecticide (BAYGON, Wisconsin, US) was diluted in methanol at 20 mg/ml and mixed with crushed cat food as a positive control. The data for cockroach mortality were recorded every hour on the first day and every 12 h subsequently for 30 days. The cockroaches were considered dead when no movement was observed after they were touched with forceps. The experiment was performed in triplicate. Compound separation. The compound separation procedure was divided into two parts: thin layer chro- matography (TLC) and gas chromatography–mass spectrometry analysis (GC–MS). The extract was fraction- ated and analyzed according to28. Silica gel F254 MERCK (MERCK, Selangor, Malaysia) and two mobile phases were used. The mobile phases were chloroform/methanol/water (98:2:1, v/v/v) and hexane/diethyl ether/acetic acid (8/5/0.2, v/v/v). The fractioned components were visualized by charring the plate at 200 °C under a flame for M. carbonarius and viewing under 254 nm UV light in a UV Viewing Chamber (UVITEC, England, United Kingdom) for G. sulphureus. The standards used were lauric acid methyl ester (SIGMA-ALDRICH, Missouri, USA), pentadecanoic acid (SIGMA-ALDRICH, Missouri, USA), octadecanoic acid (SIGMA-ALDRICH, Mis- souri, USA), and tridecane (SIGMA-ALDRICH, Missouri, USA). The TLC results were used to visualize and preconfirm the presence of compounds. For GC–MS, termite defense fluid extracts were diluted and analyzed using GC–MS-QP2010 (SHIMADZU, Columbia, USA) with a SLB5MS column. The solvent delay was 2 min, injector temperature was 280 °C, MS quadrupole temperature was 150 °C, MS source temperature was 230 °C, and transfer line temperature was 300 °C, with splitless mode injection and a helium flow rate of 0.75 mL/min. The extracts were diluted in methanol and injected into the GC–MS instrument. The analytical standard was spiked at 1 ppm. The standards used were as described in the first part. Compound identification was performed using mass spectrometric detectors and the NIST17 MS library database. Data analysis. Materials and method The positive control used in this study was a commercial natural insect repellent, WATSON Insect Repellent (WATSON, Hong Kong, China), due to the unavailability of termite chemical repellents. Each treatment was prepared in three replicates, and observations were recorded daily at 20:00 h. The repellency data were recorded for seven days for M. gilvus. Repellency against the cockroach Periplaneta americana. The repellency assay was modified from the protocol reported in24,25. Adult Periplaneta americana cockroaches were purchased from the Vector Control Research Unit, School of Biological Sciences, Universiti Sains Malaysia (USM), Penang Island. The repellency chamber was set up by using two boxes (45 L), and a 30 mm diameter acrylic tube was used as the connector. Both chambers were supplied with an egg carton for hiding, and cat food, water, and wood shavings were placed on the chamber floor. Food and water were provided ad libitum. All of the conditions were prepared according to26. Both chambers were covered using muslin cloths and secured with Velcro tape without a lid. The solution was applied on the surface of the treated box. Then, the treated surface was dried for 10 min. Ten cockroaches were released in both areas. The boxes were kept for 12 h under light conditions (approximately 71 lx) and 12 h under dark conditions. Commercial naphthalene (GANSO, Johor, Malaysia) was used as a positive control, and methanol was used as a negative control. The test was carried out for 30 days in triplicate, and data were recorded by opening the muslin cloth and observing the number of cockroaches in each chamber daily at 22:00 h. https://doi.org/10.1038/s41598-020-80018-5 Scientific Reports \| (2021) 11:153 \| www.nature.com/scientificreports/ Table 1. Scale used to estimate the class for mean percentage repellency. Class Repellency rate (%) Interpretation 0 > 0.01–< 0.1 Nonrepellent I 0.1–20 Very weakly repellent II 20.1–40 Moderately repellent III 40.1–60 Average repellency IV 60.1–80 Fairly repellent V 80.1–100 Very repellent Table 1. Scale used to estimate the class for mean percentage repellency. Toxicity against the termite Macrotermes gilvus. The method was modified from23,24 and tested on the termite M. gilvus. Filter paper was soaked with the extract at five concentrations. The fungal comb was not provided as it was a force-feeding-based design. The arena was supplied with a plate of water at 70–80% humid- ity. Triplicates were examined for every concentration, and the experiment was set up using a 7.5 ml box. Materials and method The repellent activity was corrected to the percentage of repellency (PR) using the formula described in29, as shown in Eq. (1). The PR was classified as in Table 1. Then, the PR and percentage of toxicity against time were analyzed by ANOVA according to days for the longevity of the extracts and Duncan’s test as a post hoc test at an alpha level of 0.05. The statistical analysis (ANOVA and Duncan’s test) was conducted using SPSS Statistics Version 23 (IBM, NY, US). The results are expressed as the mean ± standard deviation. (1) PR = [Nc −Nt] [Nc + Nt] × 100 (1) where Nc is the number of insects in the control group, and Nt is the number of insects in the treatment group where Nc is the number of insects in the control group, and Nt is the number of insects in the treatment group. Result Repellency effect and efficiency against termites. Globitermes sulphureus produces a higher volume of defense fluid (2.93 g for 100 soldiers) than M. carbonarius (0.68 g for 100 soldiers). Almost all concentrations of the defense fluid extracts of M. carbonarius and G. sulphureus showed insect pest repellent activity, and the fluid was categorized as a class IV to class V repellent (Table 2). Macrotermes carbonarius extract at a concentra- tion of 1 mg/ml showed the lowest mean percentage of repellency (68.57%) compared to G. sulphureus (83.05%). The highest mean percentage repellency was observed for extracts of both species at 20 mg/ml, with values of 98.00% and 97.62%, respectively. At 0 mg/ml, the percentage of repellency was zero, and this value was used for data normalization for other concentrations. Repellency activities per day observed for M. carbonarius and G. sulphureus extracts against pest termite M. gilvus are tabulated in Table 2. The minimum repellency of the M. carbonarius extract (1 mg/ml) was 42%, https://doi.org/10.1038/s41598-020-80018-5 Scientific Reports \| (2021) 11:153 \| www.nature.com/scientificreports/ Table 2. Mean percentage repellency with the standard error and repellency class of Macrotermes carbonarius and Globitermes sulphureus extracts against Macrotermes gilvus. Different letters in the same row indicate a significant difference (p < 0.05, Duncan’s test). Mean PR = mean percentage of repellency. Mean of three replicates (n = 30 termites per replicate). Repellency class = class 0 (> 0.01 to < 0.1%), nonrepellent; class I (0.1 to 20%), very weakly repellent; class II (20.1–40%), moderately repellent; class III (40.1–60%), averagely repellency; class IV (60.1 to 80%), fairly repellent; class V (80.1–100%), very repellent. Day Percentage of repellency (%) 1 5 15 20 Positive control Concentration of G. Result Both extracts showed low repellent activity at the beginning of the experiment (day 1), and the repelling intensity increased from day 2 until day 4. The repellency of both extracts against M. gilvus began to decline from day 5 until day 7. However, both extracts at 15 and 20 mg/ml showed an increasing trend of repellency after four days of exposure, reaching 100.00% repellency against pest termites. These results were comparable to those of the positive control. Overall, these results indicated that even at low concentrations, M. carbonarius and G. sulphureus extracts showed repel- lency activities against pest termites. Repellency effect and efficiency on cockroach. The defense fluids of M. carbonarius and G. sulphureus soldier termites were tested on another pest, the American cockroach P. americana (Table 3). The repellent activ- ity of M. carbonarius extract achieved only class II and III values (28.61% and 56.83%), including no repellence effect even at the highest concentration, 20 mg/ml. The highest percentage repellency (78.56%, class IV) of the G. sulphureus extract was observed at a concentration of 20 mg/ml.hli p g The results for M. carbonarius defense fluid at 1, 5, and 15 mg/ml showed significantly increasing repellency for 25 days (Table 3). However, from day 25 until day 30, the 1 and 5 mg/ml extracts showed a decline in repel- lence activities. In contrast, the highest concentration of M. carbonarius extract showed the opposite repellent activity trend, with 14.67% repellency at day 1 to no repellency at day 30 (− 27.00%). The percentage repellency for G. sulphureus extracts is also shown in Table 3. The percentage repellency of the 1, 5, and 15 mg/ml extracts increased at the beginning of the experiment and peaked after 20 days, and the extracts at 20 mg/ml showed increasing repellent activity. The highest performance (98.67%) was observed after 30 days. At 1 mg/ml, the extracts showed repellency for only five days, after which no further activity was observed. Insecticidal effect of both extracts on termites. The LC50 results showed a significant effect on ter- mites, and no insecticidal activity was recorded against P. americana. As observed in Table 4, the LC50 of the G. sulphureus extract was 16.917 mg/ml, which was lower than that of the M. carbonarius extract. This result may be related to the repellent activity of M. gilvus. The feed weight after mortality-inducing treatment of M. gilvus are presented in Table 5. Result Mean PR = mean percentage of repellency. Mean of three replicates (n = 30 termites per replicate). Repellency class = class 0 (> 0.01 to < 0.1%), nonrepellent; class I (0.1 to 20%), very weakly repellent; class II (20.1–40%), moderately repellent; class III (40.1–60%), averagely repellency; class IV (60.1 to 80%), fairly repellent; class V (80.1–100%), very repellent. Table 2. Mean percentage repellency with the standard error and repellency class of Macrotermes carbonarius and Globitermes sulphureus extracts against Macrotermes gilvus. Different letters in the same row indicate a significant difference (p < 0.05, Duncan’s test). Mean PR = mean percentage of repellency. Mean of three replicates (n = 30 termites per replicate). Repellency class = class 0 (> 0.01 to < 0.1%), nonrepellent; class I (0.1 to 20%), very weakly repellent; class II (20.1–40%), moderately repellent; class III (40.1–60%), averagely repellency; class IV (60.1 to 80%), fairly repellent; class V (80.1–100%), very repellent. Table 2. Mean percentage repellency with the standard error and repellency class of Macrotermes carbonarius and Globitermes sulphureus extracts against Macrotermes gilvus. Different letters in the same row indicate a significant difference (p < 0.05, Duncan’s test). Mean PR = mean percentage of repellency. Mean of three replicates (n = 30 termites per replicate). Repellency class = class 0 (> 0.01 to < 0.1%), nonrepellent; class I (0.1 to 20%), very weakly repellent; class II (20.1–40%), moderately repellent; class III (40.1–60%), averagely repellency; class IV (60.1 to 80%), fairly repellent; class V (80.1–100%), very repellent. whereas for G. sulphureus, it was 31.67% after exposure to pest termites for seven days. Both extracts showed low repellent activity at the beginning of the experiment (day 1), and the repelling intensity increased from day 2 until day 4. The repellency of both extracts against M. gilvus began to decline from day 5 until day 7. However, both extracts at 15 and 20 mg/ml showed an increasing trend of repellency after four days of exposure, reaching 100.00% repellency against pest termites. These results were comparable to those of the positive control. Overall, these results indicated that even at low concentrations, M. carbonarius and G. sulphureus extracts showed repel- lency activities against pest termites. whereas for G. sulphureus, it was 31.67% after exposure to pest termites for seven days. Result sulphureus extract (mg/ml) 1 93.33 ± 1.67a 91.67 ± 1.67ab 91.67 ± 1.67b 85.00 ± 2.87c 88.33 ± 1.67e 2 91.67 ± 1.67a 98.33 ± 1.67b 96.67 ± 3.33b 100.00 ± 0.00c 100 ± 0.00e 3 91.67 ± 1.67a 98.33 ± 1.67b 96.67 ± 3.33c 98.33 ± 1.67d 100 ± 0.00e 4 93.33 ± 1.67a 95.00 ± 2.89b 98.83 ± 1.67c 100.00 ± 0.00d 100 ± 0.00e 5 86.67 ± 1.67a 88.33 ± 1.67ab 98.83 ± 1.67b 100.00 ± 0.00d 100 ± 0.00e 6 38.33 ± ± 1.67a 80.00 ± 2.89b 91.67 ± 1.67c 100.00 ± 0.00d 100 ± 0.00e 7 31.67 ± 1.67a 78.33 ± 1.67ab 81.67 ± 1.67b 100.00 ± 0.00c 100 ± 0.00d Mean PR (%) 83.05 90.48 93.81 97.62 98.33 Repellency Class V V V V V Concentration of M. carbonarius extract (mg/ml) 1 56.00 ± 1.00ab 86.67 ± 1.67b 88.33 ± 1.67c 98.33 ± 1.67d 88.33 ± 1.67e 2 75.67± 2.33ab 100.00 ± 0.00b 89.67 ± 0.33c 91.67 ± 1.67d 100 ± 0.00e 3 81.67 ± 1.67ab 88.33 ± 1.67bc 98.33 ± 1.67c 100.00 ± 0.00d 100 ± 0.00e 4 85.00 ± 2.89ab 93.33 ± 1.67bc 90.00 ± 2.89c 97.67 ± 1.45d 100 ± 0.00e 5 82.00 ± 1.52ab 96.67 ± 1.67bc 100.00 ± 0.00 c 98.33 ± 1.67d 100 ± 0.00e 6 72.33 ± 1.45ab 88.33 ± 1.67bc 100.00 ± 0.00 c 100.00 ± 0.00d 100 ± 0.00e 7 42.00 ± 1.53a 93.33 ± 1.67ab 93.33 ± 1.67b 100.00 ± 0.00c 100 ± 0.00 d Mean PR (%) 68.57 91.90 93.33 98.00 98.33 Repellency Class IV V V V V Table 2. Mean percentage repellency with the standard error and repellency class of Mac and Globitermes sulphureus extracts against Macrotermes gilvus. Different letters in the sam Day Percentage of repellency (%) 1 5 15 20 Positive control Concentration of G. Result sulphureus extract (mg/ml) 1 93.33 ± 1.67a 91.67 ± 1.67ab 91.67 ± 1.67b 85.00 ± 2.87c 88.33 ± 1.67e 2 91.67 ± 1.67a 98.33 ± 1.67b 96.67 ± 3.33b 100.00 ± 0.00c 100 ± 0.00e 3 91.67 ± 1.67a 98.33 ± 1.67b 96.67 ± 3.33c 98.33 ± 1.67d 100 ± 0.00e 4 93.33 ± 1.67a 95.00 ± 2.89b 98.83 ± 1.67c 100.00 ± 0.00d 100 ± 0.00e 5 86.67 ± 1.67a 88.33 ± 1.67ab 98.83 ± 1.67b 100.00 ± 0.00d 100 ± 0.00e 6 38.33 ± ± 1.67a 80.00 ± 2.89b 91.67 ± 1.67c 100.00 ± 0.00d 100 ± 0.00e 7 31.67 ± 1.67a 78.33 ± 1.67ab 81.67 ± 1.67b 100.00 ± 0.00c 100 ± 0.00d Mean PR (%) 83.05 90.48 93.81 97.62 98.33 Repellency Class V V V V V Concentration of M. carbonarius extract (mg/ml) 1 56.00 ± 1.00ab 86.67 ± 1.67b 88.33 ± 1.67c 98.33 ± 1.67d 88.33 ± 1.67e 2 75.67± 2.33ab 100.00 ± 0.00b 89.67 ± 0.33c 91.67 ± 1.67d 100 ± 0.00e 3 81.67 ± 1.67ab 88.33 ± 1.67bc 98.33 ± 1.67c 100.00 ± 0.00d 100 ± 0.00e 4 85.00 ± 2.89ab 93.33 ± 1.67bc 90.00 ± 2.89c 97.67 ± 1.45d 100 ± 0.00e 5 82.00 ± 1.52ab 96.67 ± 1.67bc 100.00 ± 0.00 c 98.33 ± 1.67d 100 ± 0.00e 6 72.33 ± 1.45ab 88.33 ± 1.67bc 100.00 ± 0.00 c 100.00 ± 0.00d 100 ± 0.00e 7 42.00 ± 1.53a 93.33 ± 1.67ab 93.33 ± 1.67b 100.00 ± 0.00c 100 ± 0.00 d Mean PR (%) 68.57 91.90 93.33 98.00 98.33 Repellency Class IV V V V V Table 2. Mean percentage repellency with the standard error and repellency class of Macrotermes carbonarius and Globitermes sulphureus extracts against Macrotermes gilvus. Different letters in the same row indicate a significant difference (p < 0.05, Duncan’s test). Mean PR = mean percentage of repellency. Mean of three replicates (n = 30 termites per replicate). Repellency class = class 0 (> 0.01 to < 0.1%), nonrepellent; class I (0.1 to 20%), very weakly repellent; class II (20.1–40%), moderately repellent; class III (40.1–60%), averagely repellency; class IV (60.1 to 80%), fairly repellent; class V (80.1–100%), very repellent. Table 2. Mean percentage repellency with the standard error and repellency class of Macrotermes carbonarius and Globitermes sulphureus extracts against Macrotermes gilvus. Different letters in the same row indicate a significant difference (p < 0.05, Duncan’s test). Result The weight of the filter paper that was treated with M. carbonarius extract at 1 mg/ ml increased from 0.024 g and decreased gradually. At 20 mg/ml, the feed weight increased, in contrast with the repellent activity of the M. carbonarius extract. Likewise, the mean feeding of P. americana on filter paper showed a decreasing trend from 0.075 g to 0.015 g as the concentration of the G. sulphureus extract increased. https://doi.org/10.1038/s41598-020-80018-5 Scientific Reports \| (2021) 11:153 \| www.nature.com/scientificreports/ Table 3. Mean percentage repellency with the standard error and repellency class of Macrotermes carbonarius and Globitermes sulphureus extracts against Periplaneta americana. Different letters in the same row indicate a significant difference (p < 0.05, Duncan’s test). Mean PR = mean percentage of repellency. Mean of three replicates (n = 30 termites per replicate). Repellency class = class 0 (> 0.01 to < 0.1%), nonrepellent; class I (0.1–20%), very weakly repellent; class II (20.1–40%), moderately repellent; class III (40.1–60%), averagely repellency; class IV (60.1–80%), fairly repellent; class V (80.1–100%), very repellent. Day Percentage of repellency (%) 1 5 15 20 Positive control Concentration of M. carbonarius extract (mg/ml) 5 − 19.33 ± 1.76a 26.67 ± 2.40b 49.33 ± 1.33c 14.67 ± 1.76d 44.00 ± 2.31e 10 7.33 ± 1.76ab 24.67 ± 1.76b 69.33 ± 2.40c 30.67 ± 1.33d 36.67 ± 2.44e 15 13.33 ± 2.67a 41.33 ± 1.33b 45.33 ± 1.33c − 19.33 ± 1.33d 6.00 ± 1.15e 20 52.67 ± 1.76a 22.67 ± 2.67b 49.00 ± 2.52c − 19.33 ± 0.67d 71.00 ± 2.51e 25 20.00 ± 2.31a 30.67 ± 1.20b 62.67 ± 2.67c − 14.33 ± 0.88d 66.67 ± 3.53e 30 11.33 ± 2.40ab 22.67 ± 2.67b 65.33 ± 1.16c − 27.00 ± 1.20d − 5.33 ± 1.33e Mean PR (%) 16.44 28.61 56.83 0.00 98.33 Repellency Class Nonrepellent II III Nonrepellent V Concentration of G. Result sulphureus extract (mg/ml) 5 10.67 ± 1.33a 32.00 ± 1.15b 64.67 ± 1.76c 32.00 ± 2.00d 44.00 ± 2.31e 10 − 5.00 ± 1.53a 52.67 ± 1.76ab 71.33 ± 0.67b 82.00 ± 3.06c 36.67 ± 3.33d 15 − 24.00 ± 2.31a 55.33 ± 0.67b 78.67 ± 1.33c 87.00 ± 1.00d 6.00 ± 1.15e 20 − 21.33 ± 1.33a 42.00 ± 1.15b 64.33 ± 2.33c 89.67 ± 1.20d 71.00 ± 1.00e 25 − 24.00 ± 4.00a 26.67 ± 1.33b 66.00 ± 2.00c 84.00 ± 0.00d 66.67 ± 3.53e 30 − 31.33 ± 0.67a 34.00 ± 1.15b 67.00 ± 1.00c 98.67 ± 1.33d − 5.33 ± 1.33e Mean PR (%) 0.00 40.33 68.89 78.56 98.33 Repellency Class Nonrepellent III IV IV V Table 3. Mean percentage repellency with the standard error and repellency class of Macrotermes carbonarius and Globitermes sulphureus extracts against Periplaneta americana. Different letters in the same row indicate a significant difference (p < 0.05, Duncan’s test). Mean PR = mean percentage of repellency. Mean of three replicates (n = 30 termites per replicate). Repellency class = class 0 (> 0.01 to < 0.1%), nonrepellent; class I (0.1–20%), very weakly repellent; class II (20.1–40%), moderately repellent; class III (40.1–60%), averagely repellency; class IV (60.1–80%), fairly repellent; class V (80.1–100%), very repellent. Table 3. Mean percentage repellency with the standard error and repellency class of Macrotermes carbonarius and Globitermes sulphureus extracts against Periplaneta americana. Different letters in the same row indicate a significant difference (p < 0.05, Duncan’s test). Mean PR = mean percentage of repellency. Mean of three replicates (n = 30 termites per replicate). Repellency class = class 0 (> 0.01 to < 0.1%), nonrepellent; class I (0.1–20%), very weakly repellent; class II (20.1–40%), moderately repellent; class III (40.1–60%), averagely repellency; class IV (60.1–80%), fairly repellent; class V (80.1–100%), very repellent. Table 4. Half-lethal dose concentration (LC50) of the two different extracts against Macrotermes gilvus. Treatment n p LC50 (mg/ml) Upper and lower boundaries M. carbonarius extract 90 0.039 18.875 17.910–19.627 G. sulphureus extract 90 0.042 16.917 15.554–20.39 Table 4. Half-lethal dose concentration (LC50) of the two different extracts against Macrotermes gilvus. Treatment n p LC50 (mg/ml) Upper and lower boundaries M. carbonarius extract 90 0.039 18.875 17.910–19.627 G. sulphureus extract 90 0.042 16.917 15.554–20.39 Table 4. Half-lethal dose concentration (LC50) of the two different extracts against Macrotermes gilvus. Result carbonarius extract (mg/ml) 1 0.00 ± 0.00a 0.00 ± 0.00b 0.00 ± 0.00c 0.00 ± 0.00d 4.44 ± 0.43e 2 0.00 ± 0.00a 0.00 ± 0.00b 0.00 ± 0.00c 3.33 ± 0.11d 6.67 ± 0.29e 3 1.11 ± 0.37a 1.11 ± 0.04b 2.22 ± 0.07c 1.11 ± 0.04d 7.78 ± 0.08e 4 1.11 ± 0.37a 1.11 ± 0.35b 1.11 ± 0.04c 2.22 ± 0.07d 22.22 ± 0.07e 5 0.00 ± 0.00a 1.11 ± 0.04b 3.33 ± 0.11c 8.89 ± 0.40d 23.33 ± 0.15e 6 0.00 ± 0.00a 1.11 ± 0.04b 2.22 ± 0.07c 8.89 ± 0.40d 13.33 ± 0.11e 7 0.00 ± 0.00a 1.11 ± 0.00b 0.00 ± 0.00c 6.67 ± 0.11d 11.11 ± 0.04e Concentration of G. sulphureus extract (mg/ml) 1 0.00 ± 0.00a 1.11 ± 0.33b 0.00 ± 0.00c 0.00 ± 0.00d 2.22 ± 0.33e 2 0.00 ± 0.00ab 3.33 ± 0.58b 8.89 ± 1.46c 1.11 ± 0.33d 4.44 ± 0.33e 3 1.11 ± 0.33ab 8.89 ± 0.67b 8.89 ± 0.33c 15.56 ± 1.20cd 7.78 ± 0.67e 4 1.11 ± 0.33ab 5.56 ± 0.33b 11.11 ± 0.67c 17.78 ± 1.20d 24.44 ± 0.67de 5 0.00 ± 0.00ab 3.33 ± 1.00bc 4.44 ± 0.67cd 17.78 ± 2.40d 28.89 ± 2.40de 6 0.00 ± 0.00a 0.00 ± 0.00b 0.00 ± 0.00c 4.44 ± 0.89d 14.44 ± 0.33e 7 0.00 ± 0.00a 0.00 ± 0.00b 0.00 ± 0.00c 6.67 ± 0.00d 11.11 ± 2.00e Table 6. Mean mortality percentage for seven days using Macrotermes carbonarius and Globitermes sulphureus extracts against Macrotermes gilvus at five different concentrations. Different letters in the same row indicate a significant difference (p < 0.05, Duncan’s test). Day Percentage of mortality per day (%) 0 1 5 15 20 Concentration of M. Result carbonarius extract (mg/ml) 1 0.00 ± 0.00a 0.00 ± 0.00b 0.00 ± 0.00c 0.00 ± 0.00d 4.44 ± 0.43e 2 0.00 ± 0.00a 0.00 ± 0.00b 0.00 ± 0.00c 3.33 ± 0.11d 6.67 ± 0.29e 3 1.11 ± 0.37a 1.11 ± 0.04b 2.22 ± 0.07c 1.11 ± 0.04d 7.78 ± 0.08e 4 1.11 ± 0.37a 1.11 ± 0.35b 1.11 ± 0.04c 2.22 ± 0.07d 22.22 ± 0.07e 5 0.00 ± 0.00a 1.11 ± 0.04b 3.33 ± 0.11c 8.89 ± 0.40d 23.33 ± 0.15e 6 0.00 ± 0.00a 1.11 ± 0.04b 2.22 ± 0.07c 8.89 ± 0.40d 13.33 ± 0.11e 7 0.00 ± 0.00a 1.11 ± 0.00b 0.00 ± 0.00c 6.67 ± 0.11d 11.11 ± 0.04e Concentration of G. sulphureus extract (mg/ml) 1 0.00 ± 0.00a 1.11 ± 0.33b 0.00 ± 0.00c 0.00 ± 0.00d 2.22 ± 0.33e 2 0.00 ± 0.00ab 3.33 ± 0.58b 8.89 ± 1.46c 1.11 ± 0.33d 4.44 ± 0.33e 3 1.11 ± 0.33ab 8.89 ± 0.67b 8.89 ± 0.33c 15.56 ± 1.20cd 7.78 ± 0.67e 4 1.11 ± 0.33ab 5.56 ± 0.33b 11.11 ± 0.67c 17.78 ± 1.20d 24.44 ± 0.67de 5 0.00 ± 0.00ab 3.33 ± 1.00bc 4.44 ± 0.67cd 17.78 ± 2.40d 28.89 ± 2.40de 6 0.00 ± 0.00a 0.00 ± 0.00b 0.00 ± 0.00c 4.44 ± 0.89d 14.44 ± 0.33e 7 0.00 ± 0.00a 0.00 ± 0.00b 0.00 ± 0.00c 6.67 ± 0.00d 11.11 ± 2.00e Table 6. Mean mortality percentage for seven days using Macrotermes carbonarius and Globitermes sulphureus extracts against Macrotermes gilvus at five different concentrations. Different letters in the same row indicate a significant difference (p < 0.05, Duncan’s test). Table 7. Retention factor determined by TLC for Macrotermes carbonarius defense fluid, calculated from plate a and compared to the retention factor of the standard in plate b. Compound Plate Band Retention factor (Rf) Compounds identified – a 1 0.11 Unknown – a 2 0.27 Unknown – a 3 0.42 Unknown Lauric acid methyl ester a (Extract) 4 0.70 Pentadecanoic acid b (Standard) 1 0.69 Table 7. Retention factor determined by TLC for Macrotermes carbonarius defense fluid, calculated from plate a and compared to the retention factor of the standard in plate b. Table 7. Retention factor determined by TLC for Macrotermes carbonarius defense fluid, calculated from plate a and compared to the retention factor of the standard in plate b. Result Concentration (mg/ml) Mean feeding on filter paper (g) Type of defense fluid extract M. carbonarius G. sulphureus 1 0.024 ± 0.002 0.075 ± 0.025 5 0.016 ± 0.001 0.028 ± 0.001 15 0.015 ± 0.001 0.021 ± 0.001 20 0.103 ± 0.002 0.015 ± 0.002 Table 5. Weight of feed consumed by Macrotermes gilvus after mortality-inducing treatment with the two types of extracts. Table 5. Weight of feed consumed by Macrotermes gilvus after mortality-inducing treatment with the two types of extracts. Table 5. Weight of feed consumed by Macrotermes gilvus after mortality-inducing treatment with the two types of extracts. Insecticidal efficiency of the extracts against termites. An increased percentage of termite mortal- ity per day was detected when the concentrations of the extract increased (Table 6). However, termite mortality with M. carbonarius extracts at 15 and 20 mg/ml was different from that at other concentrations. A significant pattern of toxicity was observed for the G. sulphureus extract (Table 6). Extracts at all concentrations showed toxicity from day 3 until day 7, with 20 mg/ml causing the highest mortality. Both extracts showed the highest mortality on days 4 and 5 after treatments were administered. In addition, the G. sulphureus extract showed high mortality on days 4 and 5 compared to the M. carbonarius extract. Compound profiling of both extracts. Four compounds from the M. carbonarius extract were obtained with retention factors of 0.11, 0.27, 0.42, and 0.70 by TLC (Fig. 1, Table 7). One of the compounds in band 4 was identified as lauric acid methyl ester after comparison with a standard in plate b. Thus, the results suggest that https://doi.org/10.1038/s41598-020-80018-5 Scientific Reports \| (2021) 11:153 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. Thin layer chromatography (TLC) result of the M. carbonarius defense fluid extract shows four different compounds in plate a and the standard band (red circle) in plate b. Figure 1. Thin layer chromatography (TLC) result of the M. carbonarius defense fluid extract shows four different compounds in plate a and the standard band (red circle) in plate b. Table 6. Mean mortality percentage for seven days using Macrotermes carbonarius and Globitermes sulphureus extracts against Macrotermes gilvus at five different concentrations. Different letters in the same row indicate a significant difference (p < 0.05, Duncan’s test). Day Percentage of mortality per day (%) 0 1 5 15 20 Concentration of M. Result Retention factor determined by TLC for Globitermes sulphureus defense fluid, calculated from plate a and compared to the retention factor of the standard in plate b. Table 9. Retention factor determined by TLC for Globitermes sulphureus defense fluid, calculated from plate a and compared to the retention factor of the standard in plate b. Table 9. Retention factor determined by TLC for Globitermes sulphureus defense fluid, calculated from plate a and compared to the retention factor of the standard in plate b. Figure 2 and Table 9 demonstrate the compound separation on thin layer chromatography (TLC) for the G. sulphureus extract. In this experiment, two compounds were spotted on plate a after the extract was developed. The compounds were observed at retention factors of 0.47 and 0.43, and one of the compounds was identified as pentadecanoic acid.h p Eight compounds were discovered in this study by GC–MS analysis (Table 10). The highest concentration was recorded for octadecanoic acid (16.24 mg/l), and methyl stearate (0.67 mg/l) was the compound with the lowest concentration. Result at least four compounds are responsible for the repellent and insecticidal activity of the extract against termites and cockroaches. A total of six major compounds from M. carbonarius were recognized using GC–MS (Table 8). The compound with the highest concentration was octadecanoic acid (20.95 mg/l), and that with the lowest concentration was lauric acid methyl ester (0.76 mg/l). https://doi.org/10.1038/s41598-020-80018-5 Scientific Reports \| (2021) 11:153 \| www.nature.com/scientificreports/ p / Figure 2. Thin layer chromatography (TLC) results of G. sulphureus defense fluid extract in plate a and standard band in plate b under 254 nm UV light in a UV Viewing Chamber (UVITEC, England, United Kingdom). The red circle indicates a band obtained on both plates. Figure 2. Thin layer chromatography (TLC) results of G. sulphureus defense fluid extract in plate a and standard band in plate b under 254 nm UV light in a UV Viewing Chamber (UVITEC, England, United Kingdom). The red circle indicates a band obtained on both plates. Table 8. Compound profile of Macrotermes carbonarius defense fluid determined by GC–MS, with retention time, similarity, concentration and compound activity. Peak Retention time (RT) Compound name Similarity Concentration in 100 mg/l 1 6.908 Furanone 95 9.76 2 9.781 Lauric acid methyl ester 91 0.76 3 11.860 Hydroquinone 91 19.61 4 12.490 Pentadecanoic acid 94 15.0 6 13.476 Cis-Vaccenic acid 84 11.16 7 13.583 Octadecanoic acid 90 20.95 Table 8. Compound profile of Macrotermes carbonarius defense fluid determined by GC–MS, with retentio time, similarity, concentration and compound activity. Table 8. Compound profile of Macrotermes carbonarius defense fluid determined by GC–MS, with retention time, similarity, concentration and compound activity. Table 9. Retention factor determined by TLC for Globitermes sulphureus defense fluid, calculated from plate a and compared to the retention factor of the standard in plate b. Compound Plate Band Retention factor (Rf) Compounds identified Tridecane a (Extract) 1 0.45 Unknown b (Standard) 1 0.30 Pentadecanoic acid a (Extract) 2 0.47 Pentadecanoic acid b (Standard) 2 0.50 Table 9. Retention factor determined by TLC for Globitermes sulphureus defense fluid, calculated from plate a and compared to the retention factor of the standard in plate b. Compound Plate Band Retention factor (Rf) Compounds identified Tridecane a (Extract) 1 0.45 Unknown b (Standard) 1 0.30 Pentadecanoic acid a (Extract) 2 0.47 Pentadecanoic acid b (Standard) 2 0.50 Table 9. Discussionh sulphureus extract is likely related to the defense mechanism of the carpenter ant Camponotus spp. (Hymenoptera: Formi- cidae), which is similar to G. sulphureus. It releases polyacetate-derived aromatic compounds during autothysis to kill or repel enemies37,38. It is therefore likely that the repellent activity of G. sulphureus defense fluid is the same as that of Camponotus spp.h The study shows that both extracts tested in this study share the same trend of repellency activity, but the longevity and efficiency were concentration-dependent for both extracts. The extracts from this study showed the same trend for the dose-dependent behavioral response when insects were exposed to pheromones39. Our findings on M. gilvus repellency at least hint that the defense fluid has the ability to act as a natural repellent, as the species exhibits the lowest susceptibility to baiting or other chemical pesticides40.l y g Overall, the repellent activities of M. carbonarius and G. sulphureus defense fluid against the American cock- roach were lower than those against M. gilvus. This difference caused by the differences in function, method, and reception in terms of chemical communication for cockroaches and termites. Chemical communication mechanisms of cockroaches involve mainly sex pheromones and aggregation41. Furthermore, cockroach protec- tion mechanisms are much more advanced in the presence of nocifensive behavior that is stimulated by sensory stimuli for potential injury to insects42. This finding explains the low response of cockroaches to the repellence. This condition may also be due to the differences in the interpretation of chemical cues between both species. For instance, male moths are more strongly attracted to pheromone traps baited with a blend of synthetic phero- mones mixed with some plant-related volatiles than to pheromones alone43. The same response was observed in a study on P. americana antennae44.l y Generally, the repellent activity of M. carbonarius extract was influenced by extract concentration. However, at the highest concentration, no repellent activity was observed due to the occurrence of odor generalization. This is because components of alarm pheromones cause more generalization than botanical compounds in insects with olfactory receptors45. At low concentrations, repellent activity is present due to low stimulus loads, and olfactory receptors (ORs) are more narrowly changed46–48. This explains the response of the American cockroach toward M. carbonarius extract. In addition, the G. sulphureus extract exhibited better performance than the M. carbon- arius extract due to the differences in specific compounds in the G. sulphureus extract. Discussionh The yield of defense fluid was influenced by insect size and method of defense against threats. Globitermes sulphureus is smaller in size and does not have dimorphic soldiers that act as major and minor soldiers dur- ing defense, unlike M. carbonarius. Thus, a high volume of defense fluid production is crucial for the chemical defense mechanism of G. sulphureus. Small soldiers such as nasutoids usually rely mainly on the chemical weapons produced by their large front gland, whereas large-jawed soldiers produce less defensive secretion6,30,31. Scientific Reports \| (2021) 11:153 \| https://doi.org/10.1038/s41598-020-80018-5 www.nature.com/scientificreports/ Table 10. Compound profile of Globitermes sulphureus defense fluid determined by GC–MS, with time, similarity, concentration and compound activity. Peak Retention time (RT) Compound name Similarity Concentration (mg/l) in 78 mg/l 1 8.180 Tridecane 95 1.87 2 9.785 Phenol, 3,5-bis(1,1-dimethylethyl)- 91 1.62 4 12.304 Hexadecanoic acid, methyl ester 91 6.14 6 12.493 Pentadecanoic acid 94 11.32 7 13.392 Methyl stearate 88 0.63 8 13.586 Octadecanoic acid 90 16.24 Table 10. Compound profile of Globitermes sulphureus defense fluid determined by GC–MS, with retention time, similarity, concentration and compound activity. Termites also produce significantly more defense fluid than other social insects, such as ants, due to differences in the mechanism of action32,33.h These chemical cues or pheromones are required for various purposes, such as for the recognition of biological processes and caste identification. The presence of M. carbonarius and G. sulphureus extracts enabled M. gilvus to recognize them as isolated chemical cues other than their own. In nature, the presence of different cuticular compounds in one-piece nesting termites causes avoidance, and they become aggressive34. Specific species, such as M. gilvus, respond to the situation with some level of alarm or avoidance even at a different distances from their colonies35. In the present study, the presence of active compounds such as quinone in the M. carbonarius extract could also influence repellency at the highest concentration6.hfh l p y g The G. sulphureus extracts were also effective at lower concentrations. This outcome is contrary to the study of Benth plants, in which treatment with Derris elliptica (Fabales: Fabaceae) extract at 5 mg/ml led to a 6.6% reduction in M. gilvus attack after seven days36. This finding suggested that in addition to existing botanical insecticides, termite defense fluid has potential applications as a natural repellent. The efficiency of G. www.nature.com/scientificreports/ same substance can act as a repellent or attractant depending on the conditions used in the bioassay58,59. These findings may help us to understand the importance of concentration and the compound involved in the food preferences of M. gilvus. p g For mortality of termites per day, at high concentrations of M. carbonarius extracts, the mortality corre- sponded to the highest consumption of filter paper (0.103 g). Continuous feeding caused a sudden increase in termite mortality. Thus, the compound identity, concentration and behavioral context of semiochemicals need to be taken into account as tools for insect control60. The G. sulphureus extract had a mortality effect that was associated with the percentage repellency. The toxicity was not only caused by ingestion but also starvation due to the high repellency effect. This result is consistent with studies conducted by other researchers23. described that the strong repellency of a toxic plant led to slow death of G. sulphureus and Coptotermes gestroi due to starvation, while close contact with the extract led to the termites becoming disoriented and eventually dying. Therefore, as both studies used extracts to study the toxicity, the similarity is reasonable.h y y y The results highlighted the compounds from the two species. In this study, hydroquinone was shown to be related to the repellent activity (Table 8). Another study showed that hydroquinone isolated from Formosan sub- terranean termites, Coptotermes formansus, repelled the same species, and no increase in tunneling activity was observed in the sand tested61. In contrast, in this study, hydroquinone had an alternate function as an attractant at certain concentrations, in addition to its insecticidal effect. This compound plays a role as a phagostimulant that attracts termites such as M. gilvus to the feeding site52,62. The function of this compound is consistent with a previ- ous study, which may explain the sudden increase in the consumption of filter paper by M. gilvus at a 20 mg/ml extract concentration. The compound had the second-highest concentration in the extract, and advanced testing is required for further confirmation. The insecticidal activity was also contributed to by other compounds with insecticidal properties, which were observed in a previous study63. Other compounds, namely, pentadecanoic acid and furanone, were believed to be responsible for the repellency activity, as shown in a previous study, but no detailed investigation was performed in this study64,65. www.nature.com/scientificreports/ These compounds were believed to contribute to the LC50.l g p yh p Repellent constituents of G. sulphureus are influenced by pentadecanoic acid, hexadecanoic acid methyl ester and tridecane66. Tridecane is widely acknowledged since it was included in integrated pest management as a semiochemical pesticide for the same species67. Phenol and hexadecanoic acid methyl ester have dual functions, as repellent and insecticide, which is consistent with the previous study. This compound may also contribute to the efficiency of the defense fluid extract as a repellent and insecticide, but further detailed investigations are needed63,68–71. The insecticidal activity of this extract was contributed to by octadecanoic acid and stearic acid, leading to a low LC50 of this extract (16.92 mg/ml), as inferred in a previous study. A study described in72 proved that a formulation of fatty acid methyl ester (FAME) that consists of methyl stearate showed larvicidal activity against C. quinquefasciatus. The previous study showed that the insecticidal activity of this compound occurred through ingestion, which is in consistent with and validated the result of the present study. O l d d h h l d d ff f h d d d Our results demonstrated that the similarity and differences of the compounds compared to a previous study give rise to different effects of repellency and toxicity. The different functions of the two extracts led to different results and performances. Both defense fluid extracts showed repellent and insecticidal effects. These extracts were also identified as termiticides. However, G. sulphureus defense fluid has more potential as a natural repel- lent and insecticide than M. carbonarius defense fluid. Even though the chemical identities of individual extract components were determined, their specific insecticidal and repellent activities await determination. This may be considered a promising aspect of new effective potential repellents and insecticides that use termite chemical communication. Further work is certainly required to unravel the complexities of the synergistic activity of the compounds, the behavioral context, and field application. Received: 26 August 2020; Accepted: 9 December 2020 References e e e ces 1. Batalha, L. S., Silva Filho, D. F. & Martius, C. Using termite nests as a source of organic matter in agrosilvicultural production systems in Amazonia. Scientia Agricola 52, 318–325 (1995). 1. Batalha, L. S., Silva Filho, D. F. & Martius, C. Using termite nests as a source of organic matter in agrosilvicultural production systems in Amazonia. Scientia Agricola 52, 318–325 (1995). y g 2. Ayuke, F. O. et al. Soil fertility management: Impacts on soil macrofauna, soil aggregation and soil organic matter allocation. Appl. Soil. Ecol. 48, 53–62 (2011). y g 2. Ayuke, F. O. et al. Soil fertility management: Impacts on soil macrofauna, soil aggregation and soil organic matter allocation. Appl. Soil. Ecol. 48, 53–62 (2011). ( ) 3. Jouquet, P., Chaudhary, E. & Kumar, A. R. V. Sustainable use of termite activity in agro-ecosystems with reference to earthworms A review. Agron. Sustain. Dev. 38, 3 (2018).h g 4. Deligne, J., Quennedey, A. & Blum, M. S. The enemies and defense mechanisms of termites. In Social Insects (ed. Hermann, H. R.) 1–76 (Academic Press, Cambridge, 1981). 4. Deligne, J., Quennedey, A. & Blum, M. S. The enemies and defense mechanisms of termites. In Social Insects (ed. Hermann, H. R.) 1–76 (Academic Press, Cambridge, 1981). 5. Grasse, P. P. Termitologia, Tome III (Masson, Paris, 1986). g 6. Prestwich, G. D. Defense mechanisms of termites. Annu. Rev. Entomol. 29(1), 201–232 (1984). h h h l d f h f l h l ( 6. Prestwich, G. D. Defense mechanisms of termites. Annu. Rev. Entomol. 29(1), 201–232 (1984). 6. Prestwich, G. D. Defense mechanisms of termites. Annu. Rev 7. Chuah, C. H. Chemical Weapons and Defense Mechanism of Malaysian Termites. In Chemistry in Malaysia 4–11 (Institut Kimia Malaysia, 2010).fi 8. Iida, M. & Akino, T. Defensive effect of soldier-specific secretion by Reticulitermes speratus (Isoptera: Rhinotermitidae) on the facultative termitophagous ponerine ant, Brachyponera chinensis (Hymenoptera: Ponerinae). Appl. Entomol. Zool. 51, 111–116 (2016). 8. Iida, M. & Akino, T. Defensive effect of soldier-specific secretion by Reticulitermes speratus (Isoptera: Rhinotermitidae) on the facultative termitophagous ponerine ant, Brachyponera chinensis (Hymenoptera: Ponerinae). Appl. Entomol. Zool. 51, 111–116 (2016). ( ) 9. Kori, N. S. M. & Arumugam, N. Termites of Agropark, Universiti Malaysia Kelantan, Jeli Campus: Diversity and pest composition. J. Trop. Resour. Sustain. Sci. 5, 104–108 (2017).i 9. Kori, N. S. M. & Arumugam, N. Discussionh This can be observed from the stable repellent activity of the extract at 20 mg/ml. The efficiency of G. sulphureus defense fluid extract as a repellent against the American cockroach for 30 days makes it a promising alternative as an insecticide.h p g y p g Macrotermes carbonarius extract has a higher LC50 than G. sulphureus extract. This may be explained by the presence of toxic compounds such as quinone. This compound has been reported as a toxic, ubiquitous defensive compound found in arthropods such as the red flour beetle Tribolium castaneum (Coleoptera: Ten- ebrionidae)49–53. The reason for the low LC50 of G. sulphureus defense fluid is its aggressive and toxic effects. When threatened, this insect ruptures a large gland and release the thick, yellow fluid that entangles ants or other insects that are trying to invade their nest54,55. The defense fluids of other species, such as Neocapritermes taracua (Termitidae: Termitinae) and Ruptitermes, also showed to repellent and death-inducing effects32,56.i p p gf In this study, the weight of filter paper consumed by M. gilvus was connected to the percentage of repel- lency for both extracts. On the other hand, the highest concentration of M. caronarius showed high attraction of termites, as determined by filter paper consumption. This result is consistent with those of previous studies. The presence of hydroquinone stimulated termite feeding, as in the Darwin termite, Mastotermes darwiniensis (Blattodea: Mastotermitidae), and Coptotermes acinaciformis (Blattodea: Rhinotermitidae), which consumed significantly more wood in the presence of hydroquinone57. Another possible explanation for this is that the Scientific Reports \| (2021) 11:153 \| https://doi.org/10.1038/s41598-020-80018-5 www.nature.com/scientificreports/ Received: 26 August 2020; Accepted: 9 December 2020 www.nature.com/scientificreports/ www.nature.com/scientificreports/ 15. Desneux, N., Decourtye, A. & Delpuech, J.-M. The sublethal effects of pesticides on beneficial arthropods. Ann. Rev. Entomol. 52(1), 81–106 (2007). 16. Rattan, R. Mechanism of action of insecticidal secondary m 7. Regnault-Roger, C., Vincent, C. & Arnason, J. T. Essential oils in insect control: Low-risk products in a high-stakes world. Annu Rev. Entomol. 57, 405–424 (2012). 8. Pavela, R. & Benelli, G. Essential oils as ecofriendly biopesticides? Challenges and constraints. Trends Plant Sci. 21(12), 1000–1007 (2016). ( ) 19. Chouvenc, T., Su, N. & Kenneth, G. J. Fifty years of attempted biological control of termites—Analysis of a failure. Biol. Control 59(2), 69–82 (2011). 20. Meikle, W. G. et al. Evaluation of an entomopathogenic fungus, Paecilomyces fumosoroseus (Wize) Brown and Smith (Deuteromy- cota: Hyphomycetes) obtained from Formosan subterranean termites (Isop, Rhinotermitidae). J. Appl. Entomol. 129(6), 315–322 (2005).h ( ) 21. Tho, Y. P. Termites of Peninsular Malaysia (Forest Research Institute Malaysia, Selangor, 1992). h 22. Krasulova, J. et al. Chemistry and anatomy of the frontal gland in soldiers of the sand termite Psammotermes hybostoma. J. Chem. Ecol. 38(5), 557–565 (2012). 3. Bakaruddin, N. H., Dieng, H., Sulaiman, S. F. & Ab Majid, A. H. Evaluation of the toxicity and repellency of tropical plant extrac against subterranean termites, Globitermes sulphureus and Coptotermes gestroi. Inf. Process. Agric. 5(3), 298–307 (2018). 4. Lee, C. C. & Lee, C. Y. A laboratory maintenance regime for a fungus-growing termite Macrotermes gilvus (Blattodea: Termitidae) J. Econ. Entomol. 108(3), 1243–1250 (2015). 25. Zibaee, I. & Pooya, B. K. Evaluation of repellent activity of two essential oils and their mixed formulation against cockroaches (Dictyoptera: Blattidae, Blattellidae) in Iran. J. Entomol. Zool. Stud. 4, 106 (2016).fi 6. OECD. Guidance Document on Assays for Testing the Efficacy of Baits Against Cockroaches Health and Safety Publications (OECD Environment, Paris, 2013). ) 7. Syed, R., Manzoor, F., Adalat, R., Abdul-Sattar, A. & Syed, A. Laboratory evaluation of toxicity of insecticide formulations from different classes against American cockroach (Dictyoptera: Blattidae). J. Arthropod-Borne Dis. 8(1), 21–34 (2014). f 28. Ohta, M., Matsuura, F., Henderson, G. & Laine, R. A. Novel free ceramides as components of the soldier defense gland of the Formosan subterranean termite (Coptotermes formosanus). J. Lipid Res. 48(3), 656–664 (2007). 29. McDonald, L. L., Guy, R. H. & Speirs, R. D. www.nature.com/scientificreports/ Preliminary evaluation of new candidate materials as toxicants, repellents, and attract- ants against stored-product insects-1 (Agriculture Research Service, 1970).hth g p g 30. Johnson, R. A., Thomas, R. J., Wood, T. G. & Swift, M. J. The inoculation of the fungus comb in newly founded colonies of s species of the Macrotermitinae (Isoptera) from Nigeria. J. Nat. Hist. 15(5), 751–756 (2007). p p g 31. de Mello, A. P., Azevedo, N. R., da Silva, A. M. B. & Gusmão, M. A. B. Chemical composition and variability of the defensive secretion in Nasutitermes corniger (Motschulsky, 1885) in urban area in the Brazilian semiarid region. Entomotropica 31, 82–90 (2016). 32. Bordereau, C., Robert, A., Van Tuyen, V. & Peppuy, A. Suicidal defensive behaviour by frontal gland dehiscence in Globitermes sulphureus Haviland soldiers (Isoptera). Insectes Soc. 44(3), 289–297 (1997).i p p 33. Touchard, A., Dejean, A., Escoubas, P. & Orivel, J. Intraspecific variations in the venom peptidome of the ant Odontomachus haematodus (Formicidae: Ponerinae) from French Guiana. J. Hymenoptera Res. 47, 87–101 (2015).i 4. Aguilera-Olivares, D., Burgos-Lefimil, C., Melendez, W., Flores-Prado, L. & Niemeyer, H. M. Chemical basis of nestmate recogni tion in a defense context in a one-piece nesting termite. Chemoecology 26(5), 163–172 (2016).i 5. Kuswanto, E., Ahmad, I., Putra, R. E. & Harahap, I. S. Two novel volatile compounds as the key for intraspecific colony recognition in Macrotermes gilvus (Isoptera: Termitidae). J. Entomol. 12(2), 87–94 (2015).i 6. Ismanto, A. & Baedowi, A. Efikasi ekstrak akar tuba dalam mengendalikan rayap tanah Macrotermes gilvus hagen pada pertanaman kayu putih. Jurnal Ecogreen. 5(1), 57–62 (2019).h y g 7. Jones, T. H. et al. The chemistry of exploding ants, Camponotus spp. (cylindricus complex). J. Chem. Ecol. 30(8), 1479–1492 (2004) 37. Jones, T. H. et al. The chemistry of exploding ants, Camponotus spp. (cylindricus complex). J. Chem. Ecol. 30(8), 1479 1492 (2 38. Laciny, A. et al. Colobopsis explodens sp. n., model species for studies on “exploding ants” (Hymenoptera, Formicidae), biological notes and first illustrations of males of the Colobopsis cylindrica group. ZooKeys 751, 1–40 (2018). 38. Laciny, A. et al. Colobopsis explodens sp. n., model species for studies on “exploding ants” (Hymenopter biological notes and first illustrations of males of the Colobopsis cylindrica group. ZooKeys 751, 1–40 (2018) i 39. Kuwahara, Y. Chemical Ecology of Astigmatid Mites (Cambridge University Press, Cambridge, 2004). 0. Iqbal, N. & Saeed, S. www.nature.com/scientificreports/ Toxicity of six new chemical insecticides against the termite, Microtermes mycophagus D. (Isoptera: Termitidae Macrotermitinae). Pak. J. Zool. 45(3), 709–713 (2013). q , , y g , y p g ( p Macrotermitinae). Pak. J. Zool. 45(3), 709–713 (2013). 41 Lih M & Ri l C Ki i i i i l h d b h k h i l lif B h E l 20 46 53 (2009) 1. Lihoreau, M. & Rivault, C. Kin recognition via cuticular hydrocarbons shapes cockroach social life. Behav. Ecol. 20, 46–53 (2009) 2 E l S & Lib F N i i P h i h k h P i l i F Ph i l h //d i /10 3389 2. Emanuel, S. & Libersat, F. Nociceptive Pathway in the cockroach Periplaneta americana. Front. Physiol. https://doi.org/10.3389 fphys.2019.01100 (2019). 3. Deisig, N., Dupuy, F., Anton, S. & Renou, M. Responses to pheromones in a complex odor world: Sensory processing and behavior Insects. 5(2), 399–422 (2014).i 44. Nishino, H. et al. Spatial receptive fields for odor localization. Curr. Biol. 28(4), 600–608 (2018). i 45. Sandoz, J. C., Pham, D. M., Renou, M. & Wadhams, L. Asymmetrical generalisation between pheromonal and floral odou appetitive olfactory conditioning of the honey bee (Apis mellifera L.). J. Comp. Physiol. 187, 559–568 (2001). h l Th l l b f d d h h l l ( ) 6. Kreher, S. A., Kwon, J. Y. & Carlson, J. R. The molecular basis of odor coding in the Drosophila larva. Neuron 46(3), 445–456 (2005). 7. Grosjean, Y. et al. An olfactory receptor for food-derived odours promotes male courtship in Drosophila. Nature 478(7368), 236 (2011).i 48. Vosshall, L. B. & Hansson, B. S. A unified nomenclature system for the insect olfactory coreceptor. Chem. Senses 36(6), 497–498 (2011). 9. Peschke, K. & Eisner, T. Defensive secretion of the tenebrionid beetle, Blaps mucronata: Physical and chemical determinants o effectiveness. J. Comp. Physiol. 161(3), 377–388 (1987).f f p y 0. Dettner, K. Solvent-dependent variablity of effectiveness of quinone-defensive systems of Oxytelinae beetles (Coleoptera: Staphyli- nidae). Entomologia Generalis. 15, 275–292 (1991). p yf nidae). Entomologia Generalis. 15, 275–292 (1991). g 51. Roth, L. & Eisner, T. Chemical defenses of arthropods. Ann. Rev. Entomol. 7, 107–136 (2003).l g oth, L. & Eisner, T. Chemical defenses of arthropods. Ann. Rev. En 2. Li, J. et al. References Termites of Agropark, Universiti Malaysia Kelantan, Jeli Campus: Diversity and pest composition J. Trop. Resour. Sustain. Sci. 5, 104–108 (2017).i p 0. Alia-Diyana, M. H., Appalasamy, S. & Arumugam, N. Termite species and structural pest identification in selected rural areas o K l M l IOP C f S E h d E S h //d / / / / / ( ) 10. Alia-Diyana, M. H., Appalasamy, S. & Arumugam, N. Termite species and structural pest identification in selected rural areas of Kelantan, Malaysia. IOP Conf. Ser. Earth and Environ. Sci. https://doi.org/10.1088/1755-1315/549/1/012053 (2020). 10. Alia-Diyana, M. H., Appalasamy, S. & Arumugam, N. Termite species and structural pest identification in selected rural areas of Kelantan, Malaysia. IOP Conf. Ser. Earth and Environ. Sci. https://doi.org/10.1088/1755-1315/549/1/012053 (2020). y f p g 1. Sillam-Dussès, D. et al. Comparative Study of the Labial Gland Secretion in Termites (Isoptera). PLoS ONE 7(10), e46431 (2012) , p y ( p ) ( ), ( ) 12. Wyatt, T. D. Pheromones and other chemical communication in animals. In Encyclopedia of Neuroscience (ed. Squire, L. R.) 611–616 (Academic Press, Oxford, 2017). p y ( p ) ( ) ( ) 2. Wyatt, T. D. Pheromones and other chemical communication in animals. In Encyclopedia of Neuroscience (ed. Squire, L. R.) 611–616 (Academic Press, Oxford, 2017). 12. Wyatt, T. D. Pheromones and other chemical communication in animals. In Encyclopedia of Neuroscience (ed. Squire, L. R.) 611–616 (Academic Press, Oxford, 2017). 13. Ahmad, N. & Kamarudin, N. Pheromone Trapping in Controlling Key Insect Pests: Progress and Prospects (Malaysia Palm Oil Board, Kajang 2016) ( ) 3. Ahmad, N. & Kamarudin, N. Pheromone Trapping in Controlling Key Insect Pests: Progress and Prospects (Malaysia Palm Oil Board Kajang, 2016). https://doi.org/10.1038/s41598-020-80018-5 Scientific Reports \| (2021) 11:153 \| www.nature.com/scientificreports/ Antitermitic and antifungal activities of eugenol and its congeners from the flower buds of Syzgium aromaticum (clove) Ind. Crops Prod. 77, 780–786 (2015).l p 71. Zhang, Z., Yang, T., Zhang, Y., Wang, L. & Xie, Y. Fumigant toxicity of monoterpenes against fruitfly, Drosophila melanogaster. Ind. Crops Prod. 81, 147–151 (2016).hl p 72. Silva, L. N. D. et al. The influence of fatty acid methyl esters (FAMEs) in the biochemistry and the Na+/K+-ATPase activity of Culex quinquefasciatus Larvae. J. Membr. Biol. 249(4), 459–467 (2016). p 72. Silva, L. N. D. et al. The influence of fatty acid methyl esters (FAMEs) in the biochemistry and the Na+/K+-ATPase activity of Culex quinquefasciatus Larvae. J. Membr. Biol. 249(4), 459–467 (2016). Competing interests h p g The authors declare no competing interests. p g The authors declare no competing interests. Acknowledgements g The work was supported by the Ministry of Higher Education Malaysia with FRGS Grant (R/FRGS/ A08.00/01330A/002/2016/000375). The research was carried out in the Faculty of Earth Science, Universiti g The work was supported by the Ministry of Higher Education Malaysia with FRGS Grant (R/FRGS/ A08 00/01330A/002/2016/000375) The research was carried out in the Faculty of Earth Science Universiti The work was supported by the Ministry of Higher Education Malaysia with FRGS Grant (R/FRGS/ A08.00/01330A/002/2016/000375). The research was carried out in the Faculty of Earth Science, Universiti Malaysia Kelantan Jeli Campus, and GC-MS analysis was carried out in the centralized laboratory, Universiti Malaysia Terengganu. A08.00/01330A/002/2016/000375). The research was carried out in the Faculty of Earth Science, Universiti Malaysia Kelantan Jeli Campus, and GC-MS analysis was carried out in the centralized laboratory, Universiti Malaysia Terengganu. y Malaysia Terengganu. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 58. Hasyim, A., Istianto, M. & de Kogel, W. Male fruit fly, Bactrocera tau (Diptera; Tephritidae) attractants from Elsholtzia pubescens Bth. Asian J. Plant Sci. 6(1), 181–183 (2007). 9. Chen, Z. Y. et al. Insecticidal and repellent activity of essential oil from Amomum villosum Lour. and its main compounds against two stored-product insects. Int. J. Food Prop. 21(1), 2265–2275 (2018). p p 60. Reisenman, C. E., Lei, H. & Guerenstein, P. G. Neuroethology of olfactory-guided behavior and its potential application in the control of harmful insects. Front. Physiol. 7, 271–271 (2016). y 1. Raina, A. K., Bland, J. M. & Osbrink, W. Hydroquinone is not a phagostimulant for the Formosan subterranean termite. J. Chem Ecol. 31(3), 509–517 (2005).h ( ) ( ) 62. Bagnères, A.-G. & Hanus, R. Communication and social regulation in termites. In Social Recognition in Invertebrates: The Kn and the Unknowns (eds Aquiloni, L. & Tricarico, E.) 193–248 (Springer, Cham, 2015). l l d f h l d f fl d d f 63. Alia Diyana, M. H., Appalasamy, S., Arumugam, N. & Boon, J. G. A study of a termite chemical defense fluid compound of Mac- rotermes carbonarius. IOP Conf. Ser. Earth Environ. Sci. https://doi.org/10.1088/1755-1315/269/1/012009 (2019). l ( l f l bl d f f p g 4. Environmental Protection Agency. Furanone. Prevention P A T S (National Center for Environmental Publications and Informa- tion, 1993).i 65. Igwe, O. U. & Udofia, D. E. Secondary metabolites of the cuticular abdominal glands of variegated grasshopper (Zonocerus var- iegatus L.). Int. J. Spectrosc. 2015, 1–6 (2015). g p 6. Neoh, K. B., Yeap, B.-K., Tsunoda, K., Yoshimura, T. & Lee, C.-Y. Do termites avoid carcasses? Behavioral responses depend on the nature of the carcasses. PLoS ONE 7(4), 36375 (2012).f 7. Blassioli-Moraes, M. C., Laumann, R. A., Michereff, M. F. F. & Borges, M. Semiochemicals for integrated pest management. In Sustainable Agrochemistry: A Compendium of Technologies (ed. Vaz, S., Jr.) 85–112 (Springer, Cham, 2019).f 68. de Melo, A. R. et al. Toxicity of different fatty acids and methyl esters on Culex quinquefasciatus larvae. Ecotoxicol. Environ. Saf. 154, 1–5 (2018). ( ) 69. Xie, Y., Wang, K., Huang, Q. & Lei, C. Evaluation toxicity of monoterpenes to subterranean termite, Reticulitermes chinensis Snyder. Ind. Crops Prod. 53, 163–166 (2014).l p 0. Xie, Y. et al. www.nature.com/scientificreports/ Odoriferous defensive stink gland transcriptome to identify novel genes necessary for quinone synthesis in the red flou beetle Tribolium castaneum. PLoS Genet. 9(7), 1003596–1003596 (2013). 52. Li, J. et al. Odoriferous defensive stink gland transcriptome to identify novel gen beetle Tribolium castaneum. PLoS Genet. 9(7), 1003596–1003596 (2013). Tribolium castaneum. PLoS Genet. 9(7), 1003596–1003596 (2013) 53. Delattre, O. et al. Complex alarm strategy in the most basal termite species. Behav. Ecol. Sociobiol. 69(12), 1945–1955 (2015). 54 Prestwich G D & Chen D Soldier defense secretions of Trinervitermes bettonianus (Isoptera Nasutitermitinae): Chemical vari- 53. Delattre, O. et al. Complex alarm strategy in the most basal termite species. Behav. Ecol. Sociobiol. 69(12), 1945–1955 (2015). ttre, O. et al. Complex alarm strategy in the most basal termite sp , p gy p ( ), ( ) 4. Prestwich, G. D. & Chen, D. Soldier defense secretions of Trinervitermes bettonianus (Isoptera, Nasutitermitinae): Chemical vari ation in allopatric populations. J. Chem. Ecol. 7(1), 147–157 (1981). p p p 5. Piper, R. Extraordinary Animals: An Encyclopedia of Curious and Unusual Animals (Greenwood Publishing Group, Westport 2007). 56. Costa-Leonardo, A. A new interpretation of the defense glands of neotropical Ruptitermes (Isoptera, Termitidae, Apicotermitinae). Sociobiology 44, 391–402 (2004). gy 7. Reinhard, J., Lacey, M. & Lenz, M. Application of the natural phagostimulant hydroquinone in bait systems for termite manage ment (Isoptera). Sociobiology 39(2), 213–230 (2002). https://doi.org/10.1038/s41598-020-80018-5 Scientific Reports \| (2021) 11:153 \| Author contributions A.D.M.H., A.N., J.G.B., and A.S. plan and design the experiments. A.S. and A.D.M.H. performed the experiments, analyzed the data and wrote the manuscript. All authors read and approved the manuscript. 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https://openalex.org/W3010582361	https://europepmc.org/articles/pmc7142422?pdf=render	English	null	Impact of ANCA-Associated Vasculitis on Outcomes of Hospitalizations for Goodpasture’s Syndrome in the United States: Nationwide Inpatient Sample 2003–2014	Medicina	2,020	cc-by	5,120	Impact of ANCA-Associated Vasculitis on Outcomes of Hospitalizations for Goodpasture’s Syndrome in the United States: Nationwide Inpatient Sample 2003–2014 Charat Thongprayoon 1, Wisit Kaewput 2 , Boonphiphop Boonpheng 3, Patompong Ungprasert 4, Tarun Bathini 5, Narat Srivali 6, Saraschandra Vallabhajosyula 7 , Jorge L. Castaneda 8, Divya Monga 8, Swetha R. Kanduri 8, Juan Medaura 8 and Wisit Cheungpasitporn 8,* 1 Division of Nephrology and Hypertension, Department of Medicine, Mayo Clinic, Rochester, MN 55905, USA; charat.thongprayoon@gmail.com 1 Division of Nephrology and Hypertension, Department of Medicine, Mayo Clinic, Rochester, MN 5590 USA; charat.thongprayoon@gmail.com 1 Division of Nephrology and Hypertension, Department of Medicine, Mayo Clinic, Rochester, MN 55905, USA; charat.thongprayoon@gmail.com gp y g 2 Department of Military and Community Medicine, Phramongkutklao College of Medicine, Bangkok 10400, Thailand; wisitnephro@gmail.com 2 Department of Military and Community Medicine, Phramongkutklao College of Medicine, Bangkok 10400, Thailand; wisitnephro@gmail.com 3 Department of Medicine, David Geﬀen School of Medicine, University of California, Los Angeles, CA 90095, USA; boonpipop.b@gmail.com 3 Department of Medicine, David Geﬀen School of Medicine, University of California, Los Angeles, CA 90095, USA; boonpipop.b@gmail.com nical Epidemiology Unit, Department of Research and Development, Faculty of Medicine, Siriraj Hospital, ahidol University, Bangkok 10700, Thailand; p.ungprasert@gmail.com 5 Department of Internal Medicine, University of Arizona, Tucson, AZ 85721, USA; tarunjacobb@gmail.c 6 Department of Internal Medicine, University of Arizona, Tucson, AZ 85721, USA; tarunjacobb@gmail.com Department of Internal Medicine, St. Agnes Hospital, Baltimore, MD 21229, USA; nsrivali@gmail.com D f C di l M di i M Cli i R h MN 55905 USA 5 Department of Internal Medicine, University of Arizona, Tucson, AZ 85721, USA; tarunjacobb@gmail.com 6 Department of Internal Medicine, St. Agnes Hospital, Baltimore, MD 21229, USA; nsrivali@gmail.com 7 Department of Cardiovascular Medicine, Mayo Clinic, Rochester, MN 55905, USA; V ll bh j l S h d @ d Department of Internal Medicine, University of Arizona, Tucson, AZ 85721, USA; tarunjacobb@gmail.c 6 Department of Internal Medicine, St. Agnes Hospital, Baltimore, MD 21229, USA; nsrivali@gmail.com 7 6 Department of Internal Medicine, St. Agnes Hospital, Baltimore, MD 21229, USA; nsrivali@gmail.com 7 Department of Cardiovascular Medicine, Mayo Clinic, Rochester, MN 55905, USA; Vallabhajosyula Saraschandra@mayo edu 7 Department of Cardiovascular Medicine, Mayo Clinic, Rochester, MN 55905, USA; Vallabhajosyula.Saraschandra@mayo.edu 8 Division of Nephrology, Department of Medicine, University of Mississippi Medical Center, Jackson, MS 39216, USA; jcastaneda@umc.edu (J.L.C.); dmonga@umc.edu (D.M.); skanduri@umc.edu (S.R.K.); jmedaura@umc.edu (J.M.) * Correspondence: wcheungpasitporn@gmail.com medicina medicina medicina Keywords: Goodpasture syndrome; ANCA; anti-GBM disease; vasculitis; hospitalization; outcomes 1. Introduction Although it has been shown that patients with coexistence of ANCA and GS have severe kidney injury with rapidly progressive glomerulonephritis (RPGN) [4,22,23], the impacts of ANCA vasculitis on mortality and resource utilization among patients with GS are unclear. Thus, we conducted this study using the 2003–2014 National Inpatient Sample (NIS) database to assess the impacts of coexistence of ANCA vasculitis on inpatient mortality, and resource utilization among patients with GS in the United States. Studies have demonstrated that the coexistence of ANCA and GS is recognized to occur at a much higher frequency than expected by chance alone, given the rarity of the individual diseases [3,15–17]. This phenomenon was first reported within a few years of the first description of ANCA [3,16,17], and it is clear that the autoantibodies are antigenically distinct and that double positivity is not caused by a cross-reactive anomaly [4,18]. It is postulated that the renal involvement in ANCA vasculitis leads to the exposure of antigens from the basement membrane and the formation of anti-glomerular basement membrane (anti-GBM) antibodies [4,18]. The coexistence of ANCAs (mostly myeloperoxidase (MPO-ANCAs)) with anti-GBM antibodies has been increasingly recognized, with a reported prevalence of 20% to 40% [4,13,19–28]. With this common coexistence, it is therefore recommended that ANCA and anti-GBM should be analyzed in parallel in patients with renal disease, especially those with extrarenal/pulmonary manifestations [3]. Although it has been shown that patients with coexistence of ANCA and GS have severe kidney injury with rapidly progressive glomerulonephritis (RPGN) [4,22,23], the impacts of ANCA vasculitis on mortality and resource utilization among patients with GS are unclear. Thus, we conducted this study using the 2003–2014 National Inpatient Sample (NIS) database to assess the impacts of coexistence of ANCA vasculitis on inpatient mortality, and resource utilization among patients with GS in the United States. Received: 4 February 2020; Accepted: 25 February 2020; Published: 1 March 2020 Received: 4 February 2020; Accepted: 25 February 2020; Published: 1 March 2020 Abstract: Background and objectives: Goodpasture’s syndrome (GS) is a rare, life-threatening autoimmune disease. Although the coexistence of anti-neutrophil cytoplasmic antibody (ANCA) with Goodpasture’s syndrome has been recognized, the impacts of ANCA vasculitis on mortality and resource utilization among patients with GS are unclear. Materials and Methods: We used the National Inpatient Sample to identify hospitalized patients with a principal diagnosis of GS from 2003 to 2014 in the database. The predictor of interest was the presence of ANCA-associated vasculitis. We tested the differences concerning in-hospital treatment and outcomes between GS patients with and without ANCA-associated vasculitis using logistic regression analysis with adjustment for other clinical characteristics. Results: A total of 964 patients were primarily admitted to hospital for GS. Of these, 84 (8.7%)hadaconcurrentdiagnosisofANCA-associatedvasculitis. HemoptysiswasmoreprevalentinGS patients with ANCA-associated vasculitis. During hospitalization, GS patients with ANCA-associated required non-significantly more mechanical ventilation and non-invasive ventilation support, but non-significantly less renal replacement therapy and plasmapheresis than those with GS alone. There was no significant difference in in-hospital outcomes, including organ failure and mortality, between GS patients with and without ANCA-associated vasculitis. Conclusions: Our study demonstrated no significant differences between resource utilization and in-hospital mortality among hospitalized patients with coexistence of ANCA vasculitis and GS, compared to those with GS alone. ywords: Goodpasture syndrome; ANCA; anti-GBM disease; vasculitis; hospitalization; outcomes www.mdpi.com/journal/medicina Medicina 2020, 56, 103; doi:10.3390/medicina56030103 Medicina 2020, 56, 103 2 of 8 2.1. Data Source The 2003–2014 National Inpatient Sample (NIS) was used to conduct this cohort study. The NIS is the publically available, inpatient, all-payer database in the United States. This database was developed and maintained by the Healthcare Cost and Utilization Project (HCUP) under the sponsorship of the Agency for Healthcare Research and Quality (AHRQ). The dataset contained more than 7 million hospitalizations annually, which was obtained from a 20% stratiﬁed sample of over 4000 non-federal acute care hospitals in more than 40 states of the United States. A survey procedure using discharge weight provided by HCUP-NIS was used to generate national estimates for 95% of hospitalizations nationwide [29]. This dataset included codes for principal and secondary diagnosis as well as codes for procedures performed during the hospitalization. The HCUP-NIS does not capture individual patients. The database captures all information for a given admission. Institutional Review Board approval was not required due to the publicly available nature of this de-identiﬁed database (https://hcup-us.ahrq.gov/DUA/dua_508/DUA508version.jsp#hipaa). These data are available to other authors via the HCUP-NIS database with the AHRQ. 1. Introduction Goodpasture’s syndrome (GS) is a rare but potentially life-threatening autoimmune disease [1–6]. The syndrome is usually mediated by autoantibodies directed against the non-collagenous domain of the alpha 3 chain of collagen type IV in the basement membranes of the kidneys and lungs, resulting in glomerulonephritisandalveolarhemorrhage[1,3,4,7–14]. Interestingly, GShasbeenreportedconcurrently with several other renal diseases, including anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitis, IgA nephropathy, fibrillary glomerulonephritis, and membranous nephropathy [3,7–11]. This process may explain the association of GS with other disorders that disrupt or modify the structure of the basement membranes of the kidneys (such as ANCA vasculitis and lithotripsy) or lungs (such as smoking), and the induction of humoral responses following T cell-mediated glomerular injury [1,3,4]. Goodpasture’s syndrome (GS) is a rare but potentially life-threatening autoimmune disease [1–6]. The syndrome is usually mediated by autoantibodies directed against the non-collagenous domain of the alpha 3 chain of collagen type IV in the basement membranes of the kidneys and lungs, resulting in glomerulonephritisandalveolarhemorrhage[1,3,4,7–14]. Interestingly, GShasbeenreportedconcurrently with several other renal diseases, including anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitis, IgA nephropathy, fibrillary glomerulonephritis, and membranous nephropathy [3,7–11]. This process may explain the association of GS with other disorders that disrupt or modify the structure of the basement membranes of the kidneys (such as ANCA vasculitis and lithotripsy) or lungs (such as smoking), and the induction of humoral responses following T cell-mediated glomerular injury [1,3,4]. Studies have demonstrated that the coexistence of ANCA and GS is recognized to occur at a much higher frequency than expected by chance alone, given the rarity of the individual diseases [3,15–17]. This phenomenon was first reported within a few years of the first description of ANCA [3,16,17], and it is clear that the autoantibodies are antigenically distinct and that double positivity is not caused by a cross-reactive anomaly [4,18]. It is postulated that the renal involvement in ANCA vasculitis leads to the exposure of antigens from the basement membrane and the formation of anti-glomerular basement membrane (anti-GBM) antibodies [4,18]. The coexistence of ANCAs (mostly myeloperoxidase (MPO-ANCAs)) with anti-GBM antibodies has been increasingly recognized, with a reported prevalence of 20% to 40% [4,13,19–28]. With this common coexistence, it is therefore recommended that ANCA and anti-GBM should be analyzed in parallel in patients with renal disease, especially those with extrarenal/pulmonary manifestations [3]. 2.2. Study Population All patients with a principal diagnosis of GS for the hospitalization based on International Classiﬁcation of Diseases, Ninth Revision (ICD-9) diagnosis code of 446.21 were included. 3 of 8 Medicina 2020, 56, 103 2.3. Predictor of Interest The predictor of interest was the presence of anti-neutrophil cytoplasmic antibody (ANCA)- associated vasculitis. ANCA-associated vasculitis was identiﬁed using the ICD-9 diagnosis codes of 446.0 (microscopic polyangiitis (MPA)) and 446.4 (granulomatosis polyangiitis (GPA) with and without eosinophilia). 2.5. Statistical Analysis Continuous variables were summarized as mean ± standard deviation. Categorical variables were summarized as percentages. The diﬀerences within in-hospital treatment and outcomes between GS patients with and without ANCA-associated vasculitis were tested using logistic regression analysis. The odds ratio was adjusted for age, sex, race, smoking, the presence of hemoptysis, and the use of plasmapheresis. A two-tailed p-value of less than 0.05 was considered statistically signiﬁcant. All analyses were performed using SPSS (version 22.0, IBM Corporation, Armonk, NY, USA). 2.4. In-Hospital Treatment and Outcome of Interest Treatments and outcomes during hospitalization were identiﬁed based on ICD-9 codes (Table S1). Treatments of interest included invasive mechanical ventilation, non-invasive ventilation, and renal replacement therapy. Outcomes of interest included respiratory failure, circulatory failure, renal failure, hematologic failure, sepsis, and in-hospital mortality. ANCA = anti-neutrophil cytoplasmic antibody. 3.2. In-Hospital Treatments Table 2 shows in-hospital treatment among GS patients. GS patients with ANCA-associated vasculitis required non-signiﬁcantly more mechanical ventilation (odds ratio (OR) 1.48; 95% conﬁdence interval (CI) 0.87–2.52), and non-invasive ventilation support (OR 1.94; 0.86–4.35) but less renal replacement therapy (OR 0.67; 95% CI 0.42–1.17) than GS alone. Table 2. In-hospital treatment among patients with Goodpasture’s syndrome. Table 2. In-hospital treatment among patients with Goodpasture’s syndrome. Treatment Goodpasture’s Syndrome Alone Goodpasture’s Syndrome and ANCA Mechanical ventilation 18% 25% Unadjusted OR 1 (ref) 1.50 (0.89–2.53) Adjusted OR 1 (ref) 1.48 (0.87–2.52) Non-invasive ventilation 5% 10% Unadjusted OR 1 (ref) 2.15 (0.98–4.76) Adjusted OR 1 (ref) 1.94 (0.86–4.35) Renal replacement therapy 53% 42% Unadjusted OR 1 (ref) 0.64 (0.41–1.01) Adjusted OR 1 (ref) 0.67 (0.42–1.07) Adjusted for age, sex, race, smoking, hemoptysis, and plasmapheresis. OR = odds ratio. nt Goodpasture’s Syndrome Alone Goodpasture’s Syndrome and ANCA GS patients with GPA required more mechanical ventilation than GS patients alone (OR 1.88; 95% CI 1.00–3.54). In contrast, GS patients with MPA required more non-invasive ventilation (OR 3.34; 95% CI 1.19–9.41) but less renal replacement therapy (OR 0.40; 95% CI 0.18–0.89) than GS patients alone. 3.1. Clinical Characteristics A total of 964 patients were primarily admitted to hospital for GS. Of these, 84 (8.7%) had a concurrent diagnosis of ANCA-associated vasculitis, whereas 880 (91.2%) had GS alone without ANCA-associated vasculitis. Among patients with a concurrent diagnosis of ANCA-associated vasculitis, 54 (64%) had granulomatosis polyangiitis and 30 (36%) had microscopic polyangiitis. The mean age of patients was 54 ± 21 years, and 47% were female. Table 1 shows the clinical characteristics based on the presence or absence of ANCA-associated vasculitis in GS patients. GS patients with ANCA-associated vasculitis were older (mean age 57 vs. 54 years; p = 0.13), more likely to be male (54% vs. 47%; p = 0.23), more likely to have hemoptysis (42% vs. 27%; p < 0.01), but less likely to receive plasmapheresis (32% vs. 40%; p = 0.18) than GS patients alone. Table 1. Clinical characteristics. Characteristics Total Goodpasture’s Syndrome Alone Goodpasture’s Syndrome and ANCA p-Value Number 964 880 84 Age (years) 54 ± 21 54 ± 21 57 ± 19 0.13 ≤39 27% 28% 20% 0.01 40–49 9% 10% 6% 50–59 15% 14% 24% 60–69 21% 21% 13% ≥70 28% 28% 37% Male 47% 47% 54% 0.23 Caucasian 65% 65% 64% 0.96 Smoking 10% 10% 8% 0.62 Hemoptysis 28% 27% 42% <0.01 Plasmapheresis 39% 40% 32% 0.18 ANCA = anti-neutrophil cytoplasmic antibody. Table 1. Clinical characteristics. 4 of 8 Medicina 2020, 56, 103 Adjusted for age, sex, race, smoking, hemoptysis and plasmapheresis. 4. Discussion In this study, we demonstrated that hospitalized patients with coexistence of ANCA vasculitis and GS were more likely to have hemoptysis than those with GS alone. Patients with the coexistence of ANCA and GS required non-significantly more mechanical ventilation and non-invasive ventilation support, but non-significantly less renal replacement therapy and plasmapheresis than those with GS alone. There was no significant difference in in-hospital outcomes, including organ failure and mortality between GS patients with and without ANCA-associated vasculitis. There was no significant difference between in-hospital mortality among hospitalized patients with coexistence of ANCA vasculitis with GS and those with GS alone. Our study found a difference in age distribution among patients with coexistence of ANCA vasculitis with GS compared to those with GS alone. While there was a higher percentage of patients with GS alone aged ≤39 years old and aged 60–69 years old, there were higher percentages of patients with coexistence of ANCA vasculitis and GS aged 50–59 years old and ≥70 years old. This is likely because ANCA vasculitis is most prevalent in patients >50 years old [30], with the peak age between 65 and 74 years old [31], while it is known that GS has a bimodal age distribution in ages 20 to 30 years old and 60 to 70 years old [1,19,22,32,33]. Previous studies have demonstrated the prevalence of ANCA positivity among GS patients of 20% to 40% [4,13,19–28,34]. A perinuclear fluorescent pattern (P-ANCA) with anti-myeloperoxidase reactivity predominates in GS patients with reported frequencies of 66% to 81% [7,17,35–37]. In contrast, circulating anti-GBM antibodies in ANCA-positive patients has been detected in 8% to 14% of patients [17,22,35]. Although there are limited data on types of ANCA among patients with GS in our database, we successfully identified phenotypes of patients with coexistence of ANCA vasculitis (including GPA and MPA) and GS by diagnosis codes. Although previous reports have shown that some patients with coexistence of ANCA vasculitis and GS have features typical for GPA or MPA [4,22,23], the prevalence of coexistence of GPA or MPA phenotypes with GS was unknown. In this study, we demonstrated that 8.7% of patients hospitalized for GS had coexistence of ANCA vasculitis (either GPA or MPA phenotypes). Patients with the coexistence of ANCA vasculitis with GS usually present with severe kidney and lung disease requiring aggressive immunosuppressive treatment and plasmapheresis [25]. 3.3. Outcomes The presence of ANCA-associated vasculitis was associated with non-signiﬁcantly increased risks of respiratory failure (OR 1.42; 95% CI 0.88–2.29), circulatory failure (OR 1.21; 95% CI 0.46–3.17), renal failure (OR 1.47; 95% CI 0.89–2.43), non-signiﬁcantly decreased risks of hematologic failure (OR 0.68; 95% CI 0.30–1.52), sepsis (OR 0.75; 95% CI 0.26–2.16), and in-hospital mortality (OR 0.71; 95% CI 0.29–1.74) in GS patients, as shown in Table 3. There was no association between ANCA-associated vasculitis and in-hospital mortality in both patients aged <65 or ≥65 years. Table 3. Outcomes of patients with Goodpasture’s syndrome. Outcomes Goodpasture’s Syndrome Alone Goodpasture’s Syndrome and ANCA Respiratory failure 29% 38% Unadjusted OR 1 (ref) 1.54 (0.97–2.45) Adjusted OR 1 (ref) 1.42 (0.88–2.29) Circulatory failure 6% 5% Unadjusted OR 1 (ref) 1.10 (0.42–2.84) Adjusted OR 1 (ref) 1.21 (0.46–3.17) Renal failure 61% 70% Unadjusted OR 1 (ref) 1.50 (0.92–2.44) Adjusted OR 1 (ref) 1.47 (0.89–2.43) Hematologic failure 14% 8% Unadjusted OR 1 (ref) 0.58 (0.26–1.28) Adjusted OR 1 (ref) 0.68 (0.30–1.52) Sepsis 7% 5% Unadjusted OR 1 (ref) 0.71 (0.25–2.00) Adjusted OR 1 (ref) 0.75 (0.26–2.16) In-hospital mortality 8% 7% Unadjusted OR 1 (ref) 0.92 (0.39–2.19) Adjusted OR 1 (ref) 0.71 (0.29–1.74) Adjusted for age, sex, race, smoking, hemoptysis and plasmapheresis. Table 3. Outcomes of patients with Goodpasture’s syndrome. Table 3. Outcomes of patients with Goodpasture’s syndrome. Table 3. Outcomes of patients with Goodpasture’s syndrome. Goodpasture’s Syndrome Alone Goodpasture’s Syndrome and ANCA 5 of 8 Medicina 2020, 56, 103 The rates of organ failure and in-hospital mortality in GS patients with GPA and in GS patients with MPA were comparable to GS patients alone. 4. Discussion The ﬁndings of our study, using a database of hospitalized patients in the United States, also conﬁrmed that patients with coexistence of ANCA vasculitis with GS had a greater rate of hemoptysis than those with GS alone. However, our study demonstrated no signiﬁcant diﬀerences between resource utilization (plasmapheresis, mechanical ventilator, non-invasive ventilator, and renal replacement therapy) among hospitalized patients with coexistence of ANCA vasculitis and GS, compared to those with GS alone. There has been conflicting data on the prognostic significance of ANCA positivity [7,27,35,36,38,39]. In a large cohort of 221 Chinese patients with GS, the serum level of anti-GBM antibodies and the coexistence of ANCA were independent predictors for mortality [27]. In a recent study of 43 patients with GS that referred to two departments in England over a 20-year period, the coexistence of ANCA positivity was associated with one-year patient mortality [40]. In contrast, Rutgers et al. [7] compared outcomes of 46 MPO-ANCA-positive patients, 10 patients with coexistence of ANCA with the anti-GBM-antibody, and 13 patients with only the anti-GBM-antibody. In this study from the Netherlands, the investigators reported no significant impact of the coexistence of ANCA positivity on one-year mortality [7]. In this study, we utilized the United States’ inpatient hospitalization data from the NIS database and demonstrated no significant difference between in-hospital mortality among hospitalized patients with coexistence of ANCA vasculitis and GS and those with GS alone. Patients with coexistence of ANCA vasculitis and GS usually present with rapidly progressive glomerulonephritis (RPGN), have severe renal involvement at diagnosis, and are similar to patients with GS alone but more severe than in those with ANCA vasculitis alone [7,19]. While patients with coexistence Medicina 2020, 56, 103 6 of 8 of ANCA vasculitis and GS may have crescentic lesions that tend to vary in a range of activity versus chronicity, and crescents in GS alone are usually at the same stage of activity, studies have demonstrated that short-term renal survival is poor and comparable among patients with coexistence of ANCA vasculitis and GS and those with GS alone, despite standard immunosuppression regimens [1,10,24,27]. Our study confirmed that hospitalized patients with coexistence of ANCA vasculitis and GS had similar rates of renal failure and renal replacement therapy when compared to those with GS alone. Data on the impact coexistence of ANCA on the long-term renal prognosis of patients with GS were unclear [25,40]. 4. Discussion While some data suggest that patients with coexistence of ANCA vasculitis and GS present late and, thus, are dialysis-dependent at diagnosis [7,19,40], a recent study of 37 patients with coexistence of ANCA vasculitis and GS at four centers in Europe demonstrated that GS patients with coexisting ANCA vasculitis had a greater tendency to recover renal function from being dialysis-dependent after treatment, compared to patients with GS alone [25]. Data on outpatient follow-up were limited in the database. Thus, future large multicenter studies with long-term follow-up are needed to evaluate the impact coexistence of ANCA on the long-term renal prognosis of patients with GS. p g p g p There are several limitations in this study. Firstly, although the use of the NIS database allows us to evaluate the impacts of ANCA vasculitis on mortality, and resource utilization among patients with GS, possible inaccuracies in ICD-9 CM coding (especially ICD-9 CM coding for GPA and MPA) may confound results. Secondly, given the administrative nature of the dataset, the data on medications, such as immunosuppressants, was limited in this study. Consequently, we could not assess the potential eﬀects of immunosuppressants, such as cyclophosphamide treatment, on hospital outcomes of patients with coexistence of ANCA vasculitis and GS. Thirdly, this is an analysis of an inpatient U.S. database, and this limits the generalizability to the patient population in other countries. Fourthly, kidney biopsy laboratory data (including anti-GBM-antibody titer and types of ANCA), and clinical courses prior to hospitalization are lacking in the database. It has been shown that ANCA may be detected before the onset of GS, suggesting that ANCA-induced glomerular injury could be a trigger for the development of GS [18]. Due to the limitation of the database, future studies are needed to assess if the duration of the ANCAs preceding the development of GS aﬀects the outcomes of GS patients with coexisting ANCA vasculitis. 5. Conclusions In summary, there are no signiﬁcant diﬀerences between resource utilization and in-hospital mortality among hospitalized patients with coexistence of ANCA vasculitis with GS, compared to those with GS alone. Supplementary Materials: The following are available online at http://www.mdpi.com/1010-660X/56/3/103/s1, Table S1: ICD-9 code for treatments and outcomes. Author Contributions: Conceptualization, C.T., W.K., P.U., T.B., S.V., J.L.C., J.M. and W.C.; Data curation, C.T., W.K. and B.B.; Formal analysis, C.T. and W.K.; Investigation, C.T., W.K. and W.C.; Methodology, W.K., N.S. and W.C.; Project administration, C.T., B.B. and N.S.; Resources, B.B. and N.S.; Software, W.K.; Supervision, P.U., T.B., S.V., J.L.C., D.M., S.R.K., J.M. and W.C.; Validation, W.C.; Visualization, W.K., P.U., S.V. and W.C.; Writing—original draft, C.T. and W.C.; Writing—review & editing, C.T., W.K., B.B., P.U., T.B., S.V., N.S., J.L.C., D.M., S.R.K., J.M. and W.C. All authors have read and agreed to the published version of the manuscript. Author Contributions: Conceptualization, C.T., W.K., P.U., T.B., S.V., J.L.C., J.M. and W.C.; Data curation, C.T., W.K. and B.B.; Formal analysis, C.T. and W.K.; Investigation, C.T., W.K. and W.C.; Methodology, W.K., N.S. and W.C.; Project administration, C.T., B.B. and N.S.; Resources, B.B. and N.S.; Software, W.K.; Supervision, P.U., T.B., S.V., J.L.C., D.M., S.R.K., J.M. and W.C.; Validation, W.C.; Visualization, W.K., P.U., S.V. and W.C.; Writing—original draft, C.T. and W.C.; Writing—review & editing, C.T., W.K., B.B., P.U., T.B., S.V., N.S., J.L.C., D.M., S.R.K., J.M. and W.C. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Funding: This research received no external funding. Conﬂicts of Interest: The authors have declared that no competing interests exist. Conﬂicts of Interest: The authors have declared that no competing interests exist. Conﬂicts of Interest: The authors have declared that no competing interests exist. References 1. Greco, A.; Rizzo, M.I.; De Virgilio, A.; Gallo, A.; Fusconi, M.; Pagliuca, G. Goodpasture’s syndrome: A clinical update. Autoimmun. Rev. 2015, 14, 246–253. [CrossRef] 2. Moroni, G.; Ponticelli, C. Rapidly progressive crescentic glomerulonephritis: Early treatment is a must. Autoimmun. Rev. 2014, 13, 723–729. [CrossRef] [PubMed] 2. Moroni, G.; Ponticelli, C. Rapidly progressive crescentic glomerulonephritis: Early treatment is a must. Autoimmun. Rev. 2014, 13, 723–729. [CrossRef] [PubMed] 7 of 8 Medicina 2020, 56, 103 3. Gulati, K.; McAdoo, S.P. Anti-Glomerular Basement Membrane Disease. Rheum Dis. Clin. N. Am. 2018, 44, 651–673. [CrossRef] [PubMed] 4. Hellmark, T.; Segelmark, M. Diagnosis and classiﬁcation of Goodpasture’s disease (anti-GBM). J. Autoimmun. 2014, 48–49, 108–112. [CrossRef] [PubMed] 5. Sinico, R.A.; Radice, A.; Corace, C.; Sabadini, E.; Bollini, B. Anti-glomerular basement membrane antibodies in the diagnosis of Goodpasture syndrome: A comparison of diﬀerent assays. Nephrol. Dial. Transpl. 2006, 21, 397–401. [CrossRef] 6. Kaewput, W.; Thongprayoon, C.; Boonpheng, B.; Ungprasert, P.; Bathini, T.; Chewcharat, A. Inpatient Burden and Mortality of Goodpasture’s Syndrome in the United States: Nationwide Inpatient Sample 2003–2014. J. Clin. Med. 2020, 9, 455. [CrossRef] 7. Rutgers, A.; Slot, M.; van Paassen, P.; van Breda Vriesman, P.; Heeringa, P.; Tervaert, J.W. Coexistence of anti-glomerular basement membrane antibodies and myeloperoxidase-ANCAs in crescentic glomerulonephritis. Am. J. Kidney Dis. 2005, 46, 253–262. [CrossRef] 8. Jia, X.Y.; Hu, S.Y.; Chen, J.L.; Qu, Z.; Liu, G.; Cui, Z. The clinical and immunological features of patients with combined anti-glomerular basement membrane disease and membranous nephropathy. Kidney Int. 2014, 85, 945–952. [CrossRef] 9. Cheungpasitporn, W.; Zacharek, C.C.; Fervenza, F.C.; Cornell, L.D.; Sethi, S.; Herrera Hernandez, L.P. Rapidly progressive glomerulonephritis due to coexistent anti-glomerular basement membrane disease and ﬁbrillary glomerulonephritis. Clin. Kidney J. 2016, 9, 97–101. [CrossRef] 10. File, I.; Pucsok, K.; Trinn, C.; Ujhelyi, L.; Balla, J.; Matyus, J. Clinical consequence and signiﬁcance of anti-neutrophil cytoplasmic antibody positivity in anti-glomerular basement membrane disease. Orv. Hetil. 2013, 154, 1696–1701. [CrossRef] 11. Holmes, D. Glomerular disease: MN linked to improved prognosis in anti-GBM disease. Nat. Rev. Nephrol. 2013, 9, 626. [CrossRef] [PubMed] 12. Booth, A.; Harper, L.; Hammad, T.; Bacon, P.; Griﬃth, M.; Levy, J. Prospective study of TNFalpha blockade with inﬂiximab in anti-neutrophil cytoplasmic antibody-associated systemic vasculitis. Journal of the American Society of Nephrology. Clin. J. Am. Soc. Nephrol. 2004, 15, 717–721. [CrossRef] [PubMed] 13. Van Daalen, E.E.; Jennette, J.C.; McAdoo, S.P.; Pusey, C.D.; Alba, M.A.; Poulton, C.J. References Predicting Out Patients with Anti-GBM Glomerulonephritis. Clin. J. Am. Soc. Nephrol. 2018, 13, 63–72. [CrossRef] 14. Angioi, A.; Cheungpasitporn, W.; Sethi, S.; De Vriese, A.S.; Lepori, N.; Schwab, T.R. Familial antiglomerular basement membrane disease in zero human leukocyte antigen mismatch siblings. Clin. Nephrol. 2017, 88, 277–283. [CrossRef] [PubMed] 15. McAdoo, S.P.; Pusey, C.D. Anti-Glomerular Basement Membrane Disease. Clin. J. Am. Soc. Nephrol. 2017, 12, 1162–1172. [CrossRef] [PubMed] 16. O’Donoghue, D.J.; Short, C.D.; Brenchley, P.E.; Lawler, W.; Ballardie, F.W. Sequential development of systemic vasculitis with anti-neutrophil cytoplasmic antibodies complicating anti-glomerular basement membrane disease. Clin. Nephrol. 1989, 32, 251–255. 17. Jayne, D.R.; Marshall, P.D.; Jones, S.J.; Lockwood, C.M. Autoantibodies to GBM and neutrophil cytoplasm in rapidly progressive glomerulonephritis. Kidney Int. 1990, 37, 965–970. [CrossRef] 18. Olson, S.W.; Arbogast, C.B.; Baker, T.P.; Owshalimpur, D.; Oliver, D.K.; Abbott, K.C. Asymptomatic autoantibodies associate with future anti-glomerular basement membrane disease. J. Am. Soc. Nephrol. 2011, 22, 1946–1952. [CrossRef] 19. Henderson, S.R.; Salama, A.D. Diagnostic and management challenges in Goodpasture’s (anti-glomerular basement membrane) disease. Nephrol. Dial. Transpl. 2018, 33, 196–202. [CrossRef] 20. Dez, J.; Taylor, D.; Thein, H.; Yehia, M. Incidence and features of dual anti-GBM-positive and ANCA-positive patients. Nephrology 2011, 16, 725–729. 21. Verburgh, C.A.; Bruijn, J.A.; Daha, M.R.; van Es, L.A. Sequential development of anti-GBM nephritis and ANCA-associated Pauci-immune glomerulonephritis. Am. J. Kidney Dis. 1999, 34, 344–348. [CrossRef] 22. Hellmark, T.; Niles, J.L.; Collins, A.B.; McCluskey, R.T.; Brunmark, C. Comparison of anti-GBM antibodies in sera with or without ANCA. J. Am. Soc. Nephrol. 1997, 8, 376–385. [PubMed] 23. Chen, M.; Cui, Z.; Zhao, M.H. ANCA-associated vasculitis and anti-GBM disease: The experience in China. Nephrol. Dial. Transpl. 2010, 25, 2062–2065. [CrossRef] [PubMed] Medicina 2020, 56, 103 8 of 8 24. Huart, A.; Josse, A.G.; Chauveau, D.; Korach, J.M.; Heshmati, F.; Bauvin, E. Outcomes of patients with Goodpasture syndrome: A nationwide cohort-based study from the French Society of Hemapheresis. J. Autoimmun. 2016, 73, 24–29. [CrossRef] [PubMed] 25. McAdoo, S.P.; Tanna, A.; Hruskova, Z.; Holm, L.; Weiner, M.; Arulkumaran, N. Patients double-seropositive for ANCA and anti-GBM antibodies have varied renal survival, frequency of relapse, and outcomes compared to single-seropositive patients. Kidney Int. 2017, 92, 693–702. [CrossRef] [PubMed] 26. Hirayama, K.; Yamagata, K.; Kobayashi, M.; Koyama, A. Anti-glomerular basement membrane antibody disease in Japan: Part of the nationwide rapidly progressive glomerulonephritis survey in Japan. Clin. Exp. Nephrol. 2008, 12, 339–347. [CrossRef] 27. References Cui, Z.; Zhao, J.; Jia, X.Y.; Zhu, S.N.; Jin, Q.Z.; Cheng, X.Y. Anti-glomerular basement membrane disease: Outcomes of diﬀerent therapeutic regimens in a large single-center Chinese cohort study. Medicine 2011, 90, 303–311. [CrossRef] 28. Levy, J.B.; Turner, A.N.; Rees, A.J.; Pusey, C.D. Long-term outcome of anti-glomerular basement membrane antibody disease treated with plasma exchange and immunosuppression. Ann. Intern. Med. 2001, 134, 1033–1042. [CrossRef] 29. Simon, N.; Coiteux, V.; Bruno, B.; Taque, S.; Charbonnier, A.; Souchet, L. Dose adaptation of the drugs used for hematopoietic stem-cell transplantation in patients with comorbidity: Obesity, chronic renal disease or hepatopathy: Guidelines from the Francophone Society of Bone Marrow Transplantation and Cellular Therapy (SFGM-TC)]. Bulletin du Cancer. Consensus Development Conference Practice Guideline Review. Bull. Cancer 2017, 104, S99–S105. 30. Jennette, J.C.; Nachman, P.H. ANCA Glomerulonephritis and Vasculitis. Clin. J. Am. Soc. Nephrol. 2017, 12, 1680–1691. [CrossRef] 31. Jeﬀerson, J.A. Treating elderly patients with ANCA-associated vasculitis. Clin. J. Am. Soc. Nephrol. 2015, 10, 1110–1113. [CrossRef] [PubMed] 32. Shiferaw, B.; Miro, V.; Smith, C.; Akella, J.; Chua, W.; Kim, Z. Goodpasture’s Disease: An Uncommon Disease With an Atypical Clinical Course. J. Clin. Med. Res. 2016, 8, 52–55. [CrossRef] [PubMed] 33. Savage, C.O.; Pusey, C.D.; Bowman, C.; Rees, A.J.; Lockwood, C.M. Antiglomerular basement membrane antibody mediated disease in the British Isles 1980–1984. Br. Med. J. 1986, 292, 301–304. [CrossRef] [PubMed] 34. Weber, M.F.; Andrassy, K.; Pullig, O.; Koderisch, J.; Netzer, K. Antineutrophil-cytoplasmic antibodies and antiglomerular basement membrane antibodies in Goodpasture’s syndrome and in Wegener’s granulomatosis. J. Am. Soc. Nephrol. 1992, 2, 1227–1234. 35. Levy, J.B.; Hammad, T.; Coulthart, A.; Dougan, T.; Pusey, C.D. Clinical features and outcome of patients with both ANCA and anti-GBM antibodies. Kidney Int. 2004, 66, 1535–1540. [CrossRef] [PubMed] 36. Bosch, X.; Mirapeix, E.; Font, J.; Borrellas, X.; Rodriguez, R.; Lopez-Soto, A. Prognostic implication of anti-neutrophil cytoplasmic autoantibodies with myeloperoxidase speciﬁcity in anti-glomerular basement membrane disease. Clin. Nephrol. 1991, 36, 107–113. 37. Yu, J.T.; Li, J.N.; Wang, J.; Jia, X.Y.; Cui, Z.; Zhao, M.H. Deglycosylation of myeloperoxidase uncovers its novel antigenicity. Kidney Int. 2017, 91, 1410–1419. [CrossRef] 38. Yang, R.; Hellmark, T.; Zhao, J.; Cui, Z.; Segelmark, M.; Zhao, M.H. Antigen and epitope speciﬁcity of anti-glomerular basement membrane antibodies in patients with goodpasture disease with or without anti-neutrophil cytoplasmic antibodies. J. Am. Soc. Nephrol. 2007, 18, 1338–1343. [CrossRef] 39. Segelmark, M.; Hellmark, T.; Wieslander, J. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). References The prognostic signiﬁcance in Goodpasture’s disease of speciﬁcity, titre and aﬃnity of anti-glomerular-basement-membrane antibodies. Nephron. Clin. Pract. 2003, 94, c59–c68. [CrossRef] 40. Alchi, B.; Griﬃths, M.; Sivalingam, M.; Jayne, D.; Farrington, K. Predictors of renal and patient outcomes in anti-GBM disease: Clinicopathologic analysis of a two-centre cohort. Nephrol. Dial. Transpl. 2015, 30, 814–821. [CrossRef] © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W2131474215	https://zenodo.org/records/290681/files/Experimental%20Investigation%20of%20Discrete%20Multitone.pdf	English	null	Experimental Investigation of Discrete Multitone Transmission in the Presence of Optical Noise and Chromatic Dispersion	Optical Fiber Communication Conference	2,014	cc-by	2,566	1. Introduction Discrete Multitone Transmission (DMT) with intensity modulation (IM) and direct detection (DD) has recently emerged as promising candidate for 100 GbE interconnects [3]. As variant of orthogonal frequency division multiplexing (OFDM) with real valued time signal, it also offers a potentially low-cost approach for N x 100Gb/s dense wavelength division multiplexing (DWDM) inter-data center interconnects over distances beyond 40 km. While conventional DMT easily can compensate high values of chromatic dispersion (CD), the DD of a standard double sideband (DSB) signal transforms the optical channel into a highly frequency selective fading channel for large amounts of CD, thereby limiting the usable bandwidth when operated in the 1.55µm wavelength range (as required for DWDM transmission). The standard approach that allows even long-haul transmission with DMT systems [1] uses a single sideband (SSB) signal to prevent the power fading problem. A frequency gap equal to the bandwidth of the electrical signal is necessary between the carrier and the first modulated subcarrier to avoid signal- signal beat distortions in that frequency region. Drawbacks of this approach are the increased complexity of the setup due to the need for a single sideband filter or optical I-Q-modulator as well as higher bandwidth requirements of the components. Milion [2] did an early experimental demonstration of the transmission of a DSB DMT system with bit and power loading, achieving 19 Gb/s over a 25 km PON link. A later experimental study by Yan [3] showed 100 Gb/s over a distance of 10 km and 60 Gb/s over 40 km. For the application of the technique to optically amplified links that allow longer distances, Paul [4] suggested a simplified ‘on-off’ loading for 42.8 Gb/s over a 80 km span with optical pre-amplification, showing by means of simulations that loading can be an adequate alternative to the ‘gap approach’. Also, in a detailed simulative study [5], various DMT variants were compared with optimized bit (BL) and power loading (PL). p p g In this paper, we experimentally demonstrate the optical transmission of 56 Gb/s DSB DMT (including 7% FEC overhead) without any explicit frequency gap over a dispersive and optically amplified link of up to 80 km of SSMF at 1.55µm. To the best of our knowledge this is the highest data rate for such a system achieved over this distance. Experimental Investigation of Discrete Multitone Transmission in the Presence of Optical Noise and Chromatic Dispersion Annika Dochhan1, Laia Nadal2, Helmut Griesser3, Michael Eiselt1, Michela Svaluto Moreolo2, and Jörg-Peter Elbers3 1 ADVA Optical Networking SE, Maerzenquelle 1-3, 98617 Meiningen, Germany 2 CTTC, Av. Carl Friedrich Gauss 7, Castelldefels, Spain 3 ADVA Optical Networking SE, Fraunhoferstr. 9a, 82152 Martinsried, Germany ADochhan@advaoptical.com Annika Dochhan1, Laia Nadal2, Helmut Griesser3, Michael Eiselt1, Michela Svaluto Moreolo2, and Jörg-Peter Elbers3 1 ADVA Optical Networking SE, Maerzenquelle 1-3, 98617 Meiningen, Germany 2 CTTC, Av. Carl Friedrich Gauss 7, Castelldefels, Spain 3 ADVA Optical Networking SE, Fraunhoferstr. 9a, 82152 Martinsried, Germany ADochhan@advaoptical.com Abstract: Enabled by channel adaptive bit and power loading, we experimentally demonstrate discrete multitone transmission at 56Gb/s with simple intensity modulation and direct detection and achieve 50 km reach in the 1.55µm window. OCIS codes: (060.2330) Fiber optics communications; (060.4080) Modulation 1. Introduction The required optical bandwidth is around 45 GHz, thus offering the prospect of operation over DWDM links with 50 GHz channel grid. Combining 2 or 8 optical carriers to a superchannel, multiple 100 Gb/s or 400Gb/s signals can be transmitted. 2. Experimental setup The experimental setup is shown in Figure 1. The electrical DMT signal was generated offline using Python routines and stored inside the memory of a digital to analog converter (DAC) with 64 GS/s. It was amplified by a linear driver and modulated onto the optical carrier at 192.5 THz by a Mach-Zehnder-Modulator (MZM). The MZM was biased at the power quadrature point, leading to a strong carrier, but reduced subcarrier-subcarrier intermodulation products. The DAC exhibits a bandwidth of ~ 13 GHz and 64 GS/s while the driver and the MZM are suited for 30 GHz and 40 GHz bandwidth signals, respectively. Subsequently the optical signal was transmitted over a single span of standard single mode fiber (SSMF) with 50.5 or 82.1 km length and a loss of 0.2 dB/km. The launch power into the SSMF was set to 5 dBm. At the receiver, after optional additive noise loading to control the OSNR, the signal was optically pre-amplified by an Erbium-doped fiber amplifier (EDFA). After noise filtering with a 100 GHz demultiplexer, the signal was directly detected with a 50 GHz bandwidth PIN photo diode and captured by an 80 GS/s real time oscilloscope with 29.4 GHz bandwidth. Offline processing was again performed using Python. Due to component availability, the devices used in this setup have a bandwidth well beyond 20 GHz. However, since the bandwidth requirement is mainly determined by the DAC and the DD optical channel, optoelectronic devices with a bandwidth below 20 GHz could have been used, resulting in a more cost-effective design. demultiplexer, the signal was directly detected with a 50 GHz bandwidth PIN photo diode and captured by an 80 GS/s real time oscilloscope with 29.4 GHz bandwidth. Offline processing was again performed using Python. Due to component availability, the devices used in this setup have a bandwidth well beyond 20 GHz. However, since the bandwidth requirement is mainly determined by the DAC and the DD optical channel, optoelectronic devices with a bandwidth below 20 GHz could have been used, resulting in a more cost-effective design. Fig. 1: Experimental setup and signal processing steps for the DMT transmission system under investigation. Fig. 1: Experimental setup and signal processing steps for the DMT transmission system under investigation. Fig. 1 also shows the main DSP building blocks, which are needed in order to obtain the DMT signal to be transmitted from a random data sequence dk. 2. Experimental setup The BL and PL distributions were determined by sending a probe signal with a uniform 16-QAM bit loading for all subcarriers, estimating the signal-to-noise ratio (SNR) at the receiver side and finally applying the Levin-Campello loading algorithm [6]. For symbol synchronization and channel estimation five training symbols (TS) are inserted. Limited by the DAC memory, the total length of a DMT frame is 124 symbols (including TS). We used a 2048-point IFFT, yielding 1023 usable carriers (no data is transmitted on the central subcarrier) to obtain a real-valued baseband signal. Out of these, at maximum 852 carriers are actually modulated to introduce some oversampling to support aliasing filtering. A cyclic prefix of 1/64 was used, leading to data rates of 56 Gb/s to 112 Gb/s (including TS). The DMT time signal was clipped with a clipping ratio of 12 dB to reduce the peak-to-average power ratio. At the receiver side the symbols can be properly de- mapped for Monte Carlo type error counting after applying resampling, synchronization (a variant of the Schmidl- Cox algorithm), FFT, removal of all overhead, and one tap equalization with decision directed channel estimation. 3. Results In a first measurement, the system was operated without optical fiber to assess the back-to-back OSNR performance of the modulation format. Fig. 2 (left) shows the bit error ratio (BER) versus the optical signal-to-noise ratio (OSNR). For an error correcting threshold of 4e-3 (assuming a hard decision forward error correcting (HD-FEC) scheme with 7% overhead), error free performance requires 31 dB OSNR for 56 Gb/s and 40 dB for 96 Gb/s. A rate of 112 Gb/s shows an error floor above the HD-FEC threshold. Using a soft decision FEC (SD-FEC, threshold 1.9e- 2) this gross data rate would be still feasible at the cost of a higher overhead and thus reduced net bit rate. The right side of Fig. 2 shows the BL distribution with the estimated SNR as an inset. 26 30 34 38 42 46 50 54 1e-2 1e-3 1e-4 1e-5 HD-FEC limit 4e-3 SD-FEC limit 1.9e-2 OSNR [dB] Bit Error Ratio (BER) 112 Gb/s 96 Gb/s 86 Gb/s 76 Gb/s 56 Gb/s 0 5 10 15 20 25 0 2 4 6 8 f [GHz] No. of Bits Fig. 2: OSNR sensitivity of the DMT system for back-to-back (left), the result of the bitloading for 56 Gb/s (right), and the estimated SNR (inset) that is the input for the bit and power loading algorithm. 0 5 10 15 20 25 -25 -20 -15 -10 -5 0 f [GHz] Normalized SNR [dB] 26 30 34 38 42 46 50 54 1e-2 1e-3 1e-4 1e-5 HD-FEC limit 4e-3 SD-FEC limit 1.9e-2 OSNR [dB] Bit Error Ratio (BER) 112 Gb/s 96 Gb/s 86 Gb/s 76 Gb/s 56 Gb/s 0 5 10 15 20 25 0 2 4 6 8 f [GHz] No. of Bits 0 5 10 15 20 25 -25 -20 -15 -10 -5 0 f [GHz] Normalized SNR [dB] Fig. 2: OSNR sensitivity of the DMT system for back-to-back (left), the result of the bitloading for 56 Gb/s (right), and the estimated SNR (inset) that is the input for the bit and power loading algorithm. After the transmission over 50.5 km of SSMF (see Fig. 3, left) a data rate of 56 Gb/s requires an increased OSNR of 37 dB and 76 Gb/s is no longer feasible with HD-FEC. An SD-FEC, however, would allow error-free operation at an OSNR of 40 dB. 3. Results The chromatic fiber dispersion leads to significant power fading in the frequency region of interest, as is visible from the SNR estimation in the inset of Fig. 3 (right), and the BL algorithm has to assign more After the transmission over 50.5 km of SSMF (see Fig. 3, left) a data rate of 56 Gb/s requires an increased OSNR of 37 dB and 76 Gb/s is no longer feasible with HD-FEC. An SD-FEC, however, would allow error-free operation at an OSNR of 40 dB. The chromatic fiber dispersion leads to significant power fading in the frequency region of interest, as is visible from the SNR estimation in the inset of Fig. 3 (right), and the BL algorithm has to assign more bits to lower subcarriers as can be seen in Fig. 3 (right). The resulting use of constellations up to 128-QAM is the main reason for the loss in OSNR sensitivity compared to back-to-back. 26 30 34 38 42 46 50 54 1e-2 1e-3 1e-4 1e-5 HD-FEC limit 4e-3 SD-FEC limit 1.9e-2 OSNR [dB] Bit Error Ratio (BER) 96 Gb/s 86 Gb/s 76 Gb/s 56 Gb/s 0 5 10 15 20 25 0 2 4 6 8 f [GHz] No. of Bits Fig. 3: OSNR sensitivity of the DMT system after transmission over 50.5 km of SSMF (left), the result of the bitloading for 56 Gb/s (right), and the estimated SNR (inset). 0 5 10 15 20 25 -25 -20 -15 -10 -5 0 f [GHz] Normalized SNR [dB] 0 5 10 15 20 25 0 2 4 6 8 f [GHz] No. of Bits 0 5 10 15 20 25 -25 -20 -15 -10 -5 0 f [GHz] Normalized SNR [dB] 26 30 34 38 42 46 50 54 1e-2 1e-3 1e-4 1e-5 HD-FEC limit 4e-3 SD-FEC limit 1.9e-2 OSNR [dB] Bit Error Ratio (BER) 96 Gb/s 86 Gb/s 76 Gb/s 56 Gb/s [ ] [ ] Fig. 3: OSNR sensitivity of the DMT system after transmission over 50.5 km of SSMF (left), the result of the bitloading for 56 Gb/s (right), and the estimated SNR (inset). [ ] [ ] R sensitivity of the DMT system after transmission over 50.5 km of SSMF (left), the result of the bitloading for 56 Gb/s (right), and d SNR (inset). 4. Conclusions Using discrete multitone modulation, we demonstrated 56 Gb/s optical transmission in the 1.55µm window over up to 80 km of SSMF without optical dispersion compensation with an error rate below the threshold of a 7% overhead hard decision FEC. This performance is achieved by employing a per-subcarrier bit and power loading that is adapted to the dispersive channel with optical noise. By avoiding the frequency gap, we believe that the concept is suitable for DWDM systems with a 50 GHz grid. Combining 2 or 8 DMT signals to optical superchannels, it allows to transmit multiple 100 or 400Gb/s signals over high capacity DWDM links as used for high capacity inter- datacenter interconnects. 3. Results 30 32 34 36 38 40 1e-2 1e-3 HD-FEC limit 4e-3 SD-FEC limit 1.9e-2 OSNR [dB] Bit Error Ratio (BER) 86 Gb/s 76 Gb/s 56 Gb/s 0 5 10 15 20 25 0 2 4 6 8 f [GHz] No. of Bits Fig. 4: OSNR sensitivity of the DMT system after transmission over 82.1 km of SSMF (left), the result of the bitloading for 56 Gb/s (right), and the estimated SNR (inset). 0 5 10 15 20 25 -25 -20 -15 -10 -5 0 f [GHz] Normalized SNR [dB] 0 5 10 15 20 25 0 2 4 6 8 f [GHz] No. of Bits 0 5 10 15 20 25 -25 -20 -15 -10 -5 0 f [GHz] Normalized SNR [dB] 30 32 34 36 38 40 1e-2 1e-3 HD-FEC limit 4e-3 SD-FEC limit 1.9e-2 OSNR [dB] Bit Error Ratio (BER) 86 Gb/s 76 Gb/s 56 Gb/s 34 36 OSNR [dB] [ ] [ ] Fig. 4: OSNR sensitivity of the DMT system after transmission over 82.1 km of SSMF (left), the result of the bitloading for 56 Gb/s (right), and the estimated SNR (inset). In a third experiment the fiber span was increased to 80.1 km. Fig. 4 (left) shows the results for data rates of 56, 76, and 86 Gb/s. For a data rate of 56 Gb/s, the required OSNR of 38 dB is only slightly increased compared to the 50 km transmission, whereas for 76 Gb/s only an SD-FEC would allow error-free transmission. The BL shows a similar pattern as for the 50 km span with even more frequent fading, slightly increasing the number of high order constellations. In a third experiment the fiber span was increased to 80.1 km. Fig. 4 (left) shows the results for data rates of 56, 76, and 86 Gb/s. For a data rate of 56 Gb/s, the required OSNR of 38 dB is only slightly increased compared to the 50 km transmission, whereas for 76 Gb/s only an SD-FEC would allow error-free transmission. The BL shows a similar pattern as for the 50 km span with even more frequent fading, slightly increasing the number of high order constellations. 5. Acknowledgements g The results were obtained in the framework of the SASER-ADVAntage-NET project, partly funded by the German ministry of education and research (BMBF) under contract 16BP12400, the FP7 EU-Japan project STRAUSS (GA 608528), and the project FARO (TEC2012-38119), the FPI research scholarship grants BES-2010-031072 and EEBB-I-13-06102 funded by the Spanish MINECO. [5] D.J.F. Barros and J.M. Kahn, JLT, Vol. 28, No. 12 (2010), pp. 1811-20 [6] J. M. Cioffi, "Data Transmission Theory," course text for EE379C (http://www.stanford.edu/group/cioffi/). [4] H. Paul and K.-D. Kammeyer, ECOC’09, paper P3.11 6. References [1] B.J.C. Schmidt, A.J. Lowery, J. Armstrong, JLT, Vol. 26, No. 1, (2008), pp. 196-203 [2] C. Milion et al., ECOC’09, paper 7.5.4. [3] W. Yan et al., OFC’13, OM3H.1 [4] H. Paul and K.-D. Kammeyer, ECOC’09, paper P3.11 [5] D.J.F. Barros and J.M. Kahn, JLT, Vol. 28, No. 12 (2010), pp. 1811-20 [6] J. M. Cioffi, "Data Transmission Theory," course text for EE379C (http://www.stanford.edu/group/cioffi/). [2] C. Milion et al., ECOC’09, paper 7.5.4.
https://openalex.org/W4366574457	https://zenodo.org/records/7852965/files/ibad_V03i02001.pdf	Turkish	null	MEDIA AND WOMEN; A RESEARCH ON GRADUATE THESES WITH THE WORDS GENDER, WOMEN AND SERIES IN THEIR NAME	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	6,297	Journal of Communication Science Researches - IBAD ISSN: 2757-8496, May 2023 Volume 3 Issue 2, p.95-107 MEDYA VE KADIN; İSMİNDE TOPLUMSAL CİNSİYET, KADIN VE DİZİ KELİMELERİ GEÇEN LİSANSÜSTÜ TEZLER ÜZERİNE BİR ARAŞTIRMA Canan YAVUZ Çanakkale Onsekiz Mart Üniversitesi, Türkiye canan_98yvz@outlook.com https://orcid.org/0000-0002-3792-3912 Hicran Özlem ILGIN Çanakkale Onsekiz Mart Üniversitesi, Türkiye hicranilgin@comu.edu.tr https://orcid.org/0000-0002-0549-0710 Atıf Yavuz, C. & Ilgın, H. Ö. (2023). Medya ve Kadın; İsminde Toplumsal Cinsiyet, Kadın ve Dizi Kelimeleri Geçen Lisansüstü Tezler Üzerine Bir Araştırma. Journal of Communication Science Researches, 3 (2), 95-107. Journal of Communication Science Researches - IBAD ISSN: 2757-8496, May 2023 Volume 3 Issue 2, p.95-107 ÖZ Kitle iletişim araçlarından televizyon, dünyada en yaygın biçimde kullanılan araçlardan biridir. Televizyon yayın formatlarından olan televizyon dizileri ise dünyada olduğu gibi Türkiye’de de sıklıkla izlerkitlelerce tercih edilen bir formattır. Televizyon dizileri yapım olarak toplumsal birçok olayı konu alan geniş bir çerçeveye sahiptir. Bu noktadan hareketle toplumsal cinsiyet üzerine değerlendirmelerin yapılabildiği televizyon dizileri akademinin çalışma alanlarından birini oluşturmaktadır. Dizilerde yer alan karakterlerin kadın ve erkek toplumsal cinsiyet rolleri üzerine değerlendirmelerini oluşturan bu senaryolar üzerine yazılan lisansüstü tezler bu çalışmanın konusunu oluşturmaktadır. Bu araştırmada isminde toplumsal cinsiyet, kadın ve dizi kelimeleri geçen lisansüstü tezlerin incelenmesi amaçlanmıştır. Bu araştırma toplumsal cinsiyet, kadın ve medyayı içeren lisansüstü tezlerin ortaya koyulması ve benzer çalışmalara kaynak oluşturması bakımından önemlidir. Tez adında “toplumsal cinsiyet, kadın ve dizi” kelimeleri geçen lisansüstü tezlerin incelendiği benzer bir çalışma olmaması bu çalışmanın özgünlüğünü göstermektedir. Araştırmanın örneklemini 9 yüksek lisans tezi oluşturmaktadır. Bu araştırmada Yüksek Öğretim Kurumu Ulusal Tez Merkezi veri tabanında yer alan tezler incelenmiştir. Tezler doküman analizi tekniği ile incelenmiştir. Tezlerde; tez yılı, anabilim dalı, tez türü, üniversitesi, yöntemi, yazarın cinsiyeti ve tez bulgularına ilişkin ayrıntılar incelenmiştir. İncelemeler sonucunda toplumsal cinsiyet, kadın ve dizi konularını içeren lisansüstü tezlerin çoğunlukta kadınlar tarafından yazıldığı sonucuna ulaşılmıştır. Tez adında “toplumsal cinsiyet, kadın ve dizi” kelimeleri geçen lisansüstü tezlerin 2008- 2021 yılları arasında yazıldığı bulgusuna ulaşılmıştır. Anahtar Kelimeler: Kadın, Toplumsal Cinsiyet, Dizi, Lisansüstü Tezler, Kadın Araştırmaları. Submit Date: 24.02.2023, Acceptance Date: 21.04.2023, DOI NO: 10.5281/zenodo.7852965 Research Article - This article was checked by iThenticate Copyright © Journal of Communication Science Researches MEDYA VE KADIN; İSMİNDE TOPLUMSAL CİNSİYET, KADIN VE DİZİ KELİMELERİ GEÇEN LİSANSÜSTÜ TEZLER ÜZERİNE BİR ARAŞTIRMA Canan YAVUZ Çanakkale Onsekiz Mart Üniversitesi, Türkiye canan_98yvz@outlook.com https://orcid.org/0000-0002-3792-3912 Canan YAVUZ Çanakkale Onsekiz Mart Üniversitesi, Türkiye canan_98yvz@outlook.com https://orcid.org/0000-0002-3792-3912 Hicran Özlem ILGIN Çanakkale Onsekiz Mart Üniversitesi, Türkiye hicranilgin@comu.edu.tr https://orcid.org/0000-0002-0549-0710 Atıf Yavuz, C. & Ilgın, H. Ö. (2023). Medya ve Kadın; İsminde Toplumsal Cinsiyet, Kadın ve Dizi Kelimeleri Geçen Lisansüstü Tezler Üzerine Bir Araştırma. Journal of Communication Science Researches, 3 (2), 95-107. Submit Date: 24.02.2023, Acceptance Date: 21.04.2023, DOI NO: 10.5281/zenodo.7852965 Research Article - This article was checked by iThenticate Copyright © Journal of Communication Science Researches GİRİŞ Ş Toplum içerisinde ve toplumlar arasında iletişimin sağlanması için çeşitli iletişim araçları vardır ve bu araçlar çağ değişimlerinde farklılıklar göstermektedir (Yengin & Bayrak, 2022: 41). Televizyon bu iletişim araçlarından bir tanesidir. Televizyon insanların gününün belli bir zaman dilimini ayırdıkları ve çevreden haberdar oldukları önemli bir iletişim aracıdır. Tüm dünyada ortak nokta olan televizyon ile insanlar haberler, diziler, reklamlar vb. içerikler aracılığıyla birbirinden haberdar olurlar. Televizyon cinsiyet özelliklerini yansıtan önemli bir araçtır. İzleyici kitlesi cinsiyete özgü davranışların hangi cinsiyete ait olduğunu film, çizgi roman, reklam gibi aracılardan öğrenmektedir (Yılmaz & Uluyağcı, 2007: 147). Kadınlar televizyon izleyicisi olarak önemli bir kitledir. Ev kadınlarının gündüz saatlerinde evde bulunması, televizyon kanallarının yayın akışını belirlemesinde etkili olmaktadır. Bu yüzden kadınların ilgisini çekecek eğlence programları gündüz kuşağında yer almaktadır (Etiler & Zengin, 2015: 138). Televizyon kanallarının içeriklerini üretirken kadınların ilgisini çekecek konulara yönelmesi, izleyici kitlesini arttırması ve reyting elde etmesi amacını taşımaktadır. Kadınların televizyonda yansıtılma biçimi dizi, film ve reklam gibi yapımlarda farklılık göstermektedir. Özdemir’e (2010) göre; reklamlarda kadınlar anne rolü çerçevesinde mutfak işleri ve çocuk bakımı rolleriyle yansıtılırken, erkekler baba rolü çerçevesinde aileyi geçindirmekle sorumlu kişi olarak yansıtılır. Televizyon dizilerinde toplumsal değerler yeniden şekillendirilerek televizyona taşınmaktadır (Ilgın, 2020: 54). Televizyon dizileri çekildiği ülkenin, içinde bulunduğu toplumun yansımasıdır. Yapımcılar televizyon sunularını oluştururken toplumsal değerlerden etkilenir. Kadının değeri toplumlara göre değişiklik göstermektedir. Eski Türk Devletleri’nde kadın kıymetli bir varlık olarak görülmektedir. Kadın ailede söz hakkına sahiptir. Kadın ekonomide ve sosyal hayatta yer almaktadır. Türk devlet geleneğine göre anne ailenin ikinci temsilcisidir. Anne, babanın akraba soyundan önce gelir ve kadının ailedeki konumunu küçültecek davranışlardan uzak durulur (Acar, 2019: 397). İçinde bulunduğumuz yüzyılda kadınlar toplumda erkekler ile eşit haklara sahip olabilmek için mücadelelerini sürdürmektedir. Gelişen teknoloji ağında televizyon dizileri tüm dünyada topluma erişebilmekte ve kadına yönelik düşüncelerin şekillenmesinde önemli bir köprü görevi gördüğü düşünülmektedir. Bu bağlamda televizyon dizilerindeki kadın rolü kadınların toplumsal yaşamdaki durumu noktasında önemlidir. ABSTRACT Television is one of the most widely used tools in the world. Television series, one of the broadcast formats, is frequently preferred by audiences in Turkey as well as in the world. Television series have a wide framework that covers many social events as productions. From this point of view, television series 95 Journal of Communication Science Researches - IBAD ISSN: 2757-8496, May 2023 Volume 3 Issue 2, p.95-107 Journal of Communication Science Researches - IBAD ISSN: 2757-8496, May 2023 Volume 3 Issue 2, p.95-107 in which evaluations on gender can be made constitute one of the study areas of the academy. The postgraduate theses written on these scenarios, which constitute the evaluations of the characters in the TV series on gender roles of men and women, are the subject of this study. The current study aims to examine the postgraduate theses that have the words gender, woman and TV series in their titles. This research is significant in terms of revealing postgraduate theses involving gender, women and media and creating a source for similar studies. The fact that there is no similar study examining the postgraduate theses with the words "gender, women and TV series" in the thesis name shows the originality of this study. The sample of the study consists of 9 master's theses, and theses in the database of the Council of Higher Education National Thesis Center were examined. These were examined using document analysis technique. In theses, the thesis year, department, thesis type, university, method, gender of the author and thesis findings were examined. As a result of the examinations, the postgraduate theses on gender, women and TV series were mostly written by women. The findings show that the postgraduate theses with the words "gender, woman and serial" in the thesis title were written between 2008-2021. Keywords: Women, Gender, Series, Graduate Theses, Women's Studies. Toplumsal Cinsiyet, Kadın ve Televizyon Dizileri Üzerine p y , y Cinsiyet biyolojik olarak kadın ve erkek olmayı ifade ederken toplumsal cinsiyet, toplumun bireye yüklediği kalıplaşmış erkek ve kadın davranışlarını ifade eder (Şakrak, 2020: 422). Toplumsal cinsiyet, toplumun yarattığı kadın-erkek rolleri ve bu rollerin cinsiyetler tarafından kazanılmasıdır (Ecevit, 2003: 83). Kadın ve erkeklere yüklenen rol ve görevler toplumsal cinsiyet olarak ifade edilir. “Kadın ve erkeklerin biyolojik olarak doğuştan getirdiği farklılık, üstünlükler ve zayıflıklar vardır. Bu nedenle birinin diğeri ile biyolojik açıdan yarışması ya da karşılaştırılması söz konusu değildir. İki cins de biyolojik olarak kendine özgüdür ve özeldir” (Ünal vd., 2017: 228). Toplumsal cinsiyet ile toplumun 96 Journal of Communication Science Researches - IBAD ISSN: 2757-8496, May 2023 Volume 3 Issue 2, p.95-107 Journal of Communication Science Researches - IBAD ISSN: 2757-8496, May 2023 Volume 3 Issue 2, p.95-107 insanları algılama ve insanlardan beklediği davranış şekilleri arasında ilişki vardır (Dumanlı, 2011: 133). Kadınların çocuk bakımı ve ev işleri görevi, erkeklerin ev dışı çalışma hayatı ve evin ekonomik giderlerinin karşılanması görevi gibi sorumluluklar toplumsal cinsiyetin gerektirdiği rollerdir (Özdemir, 2010: 102). Toplumsal cinsiyetin özünde iki cinsiyet arasında sosyal eşitsizlik yer alır (Kutlu, 2010: 42). Toplum erkeğe sınırsız özgürlük atfederken kadını kalıplar içerisinde yaşamaya mahkûm eder. Aslında toplumsal cinsiyet rolleri hem kadın hem de erkekler için geçerli ve uygulanması beklenen davranışlardır. Fakat ataerkil toplumlarda toplumsal cinsiyet rollerinin uygulanması genellikle kadınlar için geçerli görülmektedir. Televizyon 21. yüzyılın en çok dikkat çeken icatlarından biridir. Günümüzde televizyon, diğer kitle iletişim araçlarına göre daha önemlidir (Ilgın, 2020). İnsanlar kitle iletişim araçları içerisinde bulunan ilgi duydukları konuların yer aldığı programları takip ederler (Arklan & Taşdemir, 2008: 74). Kitle iletişim araçları cinsiyete özgü kalıp yargı ve önyargıları meşru kılmada önemlidir (Dökmen, 2015: 133). Kitle iletişim araçlarında ilgi çekmek için kadınlar tercih edilmektedir. Bu sebeple kitle iletişim araçlarında kadınlar daha çok yer almaktadır (Ilgaz Büyükbaykal, 2012: 21). Televizyonda kadın, aile içerisindeki varlığı ile erkek, aile dışındaki rolleri ile karşımıza çıkmaktadır. Bu durum televizyondaki cinsiyetçi yaklaşımı göstermektedir (Kutlu, 2010: 55). Kadınlar yerli dizilerde yabancı dizilere göre daha muhafazakâr olarak gösterilmektedir. Dizilerde Türk toplum değerlerine uygun olarak aktarılan kadının, ideal anne ve eş olması üzerinde durulmaktadır (Ilgaz Büyükbaykal, 2012: 24). Dizilerde genellikle ideal kadın vurgusu yapılmaktadır. İdeal kadının geleneksel değerleri taşıması, anne, iş kadını, ev kadını gibi görevlerini yerine getirmesi beklenir (Güzel, 2014: 191). Dizilerde erkekler baskın, saldırgan, rasyonel, uyumlu bireyler olarak yansıtılırken, kadınlar arzu edilen, evcil olarak yansıtılmaktadır (Craig, 1992). Medya erkekliğin inşasına katkıda bulunmaktadır. Submit Date: 24.02.2023, Acceptance Date: 21.04.2023, DOI NO: 10.5281/zenodo.7852965 Research Article - This article was checked by iThenticate Copyright © Journal of Communication Science Researches Toplumsal Cinsiyet, Kadın ve Televizyon Dizileri Üzerine Medyada erkeklik şiddet içeren ve ev içi işlerle teması olmayan bir durum olarak yansıtılır (Kellner, 2008). Ayrıca televizyon dizilerindeki şiddet söylem ve gösterimleri toplumda şiddetin etkinliğini arttırmasına sebep olabilmektedir. Şiddet bir döngüdür. Şiddet şiddeti doğurur (Görgün Baran vd., 2017: 108). Bu bağlamda televizyonda yansıtılan şiddet toplumda şiddetin oluşmasına sebep olabilir. Dizi, film ve diğer programlar izleyiciyi etkiler. Bu etki sonucunda izleyici kitle etkilendiği detayları kendi hayatına katmaya çalışır böylelikle toplumda değişimler meydana gelebilir (Kutlu, 2010: 50). Kadına yönelik şiddetin aktarılmasında medya doğru bir yol izlememektedir. Kadına yönelik şiddeti konu alan haberlere üçüncü sayfada yer verilirken, aile içi şiddeti konu alan haberlere de adli vaka veya magazinsel haber şeklinde yer verilmektedir (Görgün Baran vd., 2017: 111). Kadına karşı olumsuz tutumları kapsayan bu haberlerin medyada aktarılma ve kategorize edilme şeklinde problem vardır. Haberlerin sunuş biçimi kadına yönelik şiddeti özendirmekte ve tetiklemektedir. Medya, spor veya siyasetle uğraşan erkeklerin başarılarına odaklanırken kadınların görünüşüne odaklanır (Ellemers, 2018: 284). Kadının medyadaki temsili, medyanın dünyaya bakışını yansıtır. Bu bakış toplumun kadını algılama şekline yön verir (Tanrıöver, 2007: 154). Medyada kadınların ikincil sınıf vatandaş konumunda yansıtılması; medyada cinsiyetçi tavrın sürmesi kadın sorunlarının artmasına sebep olmakta ve toplumu olumsuz etkilemektedir (Güzel, 2014: 192). Bu bağlamda medyada kadının yansıtılma şeklinin toplumun bakış açısını yansıttığı ve toplumun kadına yönelik tutumun oluşmasında etkili olduğu söylenebilir. Medya sektöründe çalışan kadın ve erkekler arasında eşitsizlik olduğu görülmektedir. Medya sektöründe çalışan kadın muhabirlerin sayısında yaşın ilerlemesi ile azalma görülürken bu durum erkek muhabirler için aynı değildir (Mendes & Carter, 2008: 1709). Kadının fiziksel özellikleri, yaşı, ilgi çekiciliği medyada kadının yer almasında önemli bir etkendir. Kadın haber sunucuları, kadın dizi ve film başrolleri, reklamlarda yer alan kadınlar genellikle fiziksel özellikleri ile ön plana çıkmaktadır. Kadının toplumdaki konumu zaman içerisinde değişime uğramıştır. Bu değişime sebep olan faktörlerden biri de medyadır. Medya kadının toplumdaki konumunun hızla değişmesinde, kadına yönelik bakışın şekillenmesinde önem arz etmektedir (Akçalı & İnceoğlu, 2020: 164). Mendes ve Carter’e (2008) göre cinsiyet ve iletişim araştırmacılarının kadınlara odaklanması 20. Yüzyılın sonunda 97 Submit Date: 24.02.2023, Acceptance Date: 21.04.2023, DOI NO: 10.5281/zenodo.7852965 Research Article - This article was checked by iThenticate Copyright © Journal of Communication Science Researches Journal of Communication Science Researches - IBAD ISSN: 2757-8496, May 2023 Volume 3 Issue 2, p.95-107 Journal of Communication Science Researches - IBAD ISSN: 2757-8496, May 2023 Volume 3 Issue 2, p.95-107 gerçekleşmiştir (Mendes & Carter 2008: 1702). Toplumsal Cinsiyet, Kadın ve Televizyon Dizileri Üzerine Medyada kadının sunumu izleyicinin dikkatini çekmek ve izleyici sayısını arttırmak üzerine kurgulanmaktadır. Bu yapı ataerkil düzenin toplumda sürdürülmesine neden olmaktadır (Güdekli, 2016: 63). “Medyanın toplumsal temsiller aracılığıyla ürettiği içerikler, toplumların değer yargıları, inanışları ve politik yaklaşımlarıyla yakından ilişkilidir” (Pınarbaşı, 2022: 180). Medya, toplumun aynası niteliğindedir. Medyada yansıtılan ögeler toplumun değerlerini ifade etmektedir. Türkiye’de kadın araştırmalarını sürdüren üniversitelerin uygulama ve araştırma birimleri bulunmaktadır. Ayrıca üniversitelerde kadın ve aile araştırmaları bilim dalında yüksek lisans ve doktora düzeyinde eğitim verilmektedir (Ilgın & Yavuz, 2022: 16). Toplumsal cinsiyete yönelik gerçekleştirilen araştırmalarda, toplumdaki cinsiyetle ilgili yargıların, kadını negatif şekilde etkilediği ayrıca kadının toplumda ikincil sınıfta olması fikrini güçlendirdiği ve ayrımcılığın sürdürülmesine sebep olduğu vurgulanmaktadır (Saraç, 2013: 27). Türkiye’de toplumsal cinsiyetin dizilere yansıması konusunda akademik çalışmalar yapılmaktadır. Özellikle son yıllarda bu araştırma alanı popülerlik kazanmıştır. Medya ve toplumsal cinsiyet ilişkisi üzerine yapılan akademik çalışmalarda genel olarak kadınlar toplumda bağımsızlığını elde edemeyen, engellere karşı mücadele etmeyi tercih etmeyen, düşük motivasyonlu, ikna edilmesi kolay, uzlaşma taraftarı ve fedakâr kişiler olarak yer almaktadır (Akçalı & İnceoğlu, 2020: 165). Toplumsal cinsiyetin bir yansıması olan televizyon dizileri, toplumda; toplumsal cinsiyetin değişmesi zor kurallarını güçlendirmektedir. Bu bağlamda toplum üzerinde etkisi yüksek olarak kabul edilen dizilerin oluşturulması ve yayına sunulması aşamasında denetimler gerçekleştirilmeli ve dizilerde kadına karşı duruş topluma olumsuz örnek oluşturmayacak şekilde düzenlenmelidir. YÖNTEM Bu araştırmada aynı anda isminde “kadın, toplumsal cinsiyet ve dizi” kelimeleri geçen lisansüstü tezlerin incelenmesi amaçlanmaktadır. Araştırmanın örneklemini amaçlı örneklem tekniği ile seçilen 9 lisansüstü tez oluşturmaktadır. Araştırma verilerine YÖK Ulusal Tez Merkezi veri tabanında yer alan lisansüstü tezler aracılığıyla ulaşılmıştır. Birinci aşamada YÖK Ulusal Tez Merkezi veri tabanında gelişmiş tarama seçeneği kullanılarak isminde kadın, toplumsal cinsiyet ve dizi kelimeleri geçen tüm lisansüstü tezler taranmış, bu tarama sonucunda 9 lisansüstü teze ulaşılmıştır. İkinci aşamada ise örneklemi oluşturan tezler derinlemesine şekilde incelenmiş ve inceleme sonuçları bulgular kısmına eklenmiştir. Tezlerde; tez yılı, anabilim dalı, tez türü, üniversitesi, yöntemi, yazarın cinsiyeti ve tez bulgularına ilişkin ayrıntılar incelenmiştir. Araştırmada nitel araştırma yöntemlerinden biri olan doküman analizi tekniği kullanılmıştır. Doküman analizi tekniği, yazılı belgelerin detaylarına inilmesi ve ulaşılan detayların analiz edilmesini amaçlayan nitel araştırma yöntemlerinden biridir (Kıral, 2020: 4). Bu araştırmanın örneklemini oluşturan 9 lisansüstü tezin incelenmesi noktasında en uygun yöntem olarak doküman analizi tekniği tercih edilmiştir. Bu teknikle her tez kendi içerisinde detaylı bir şekilde incelenmiş, yapılan incelemeler sonucunda genel çerçevede tezler değerlendirilmiştir. Doküman analizi tekniği belgelerin incelenmesini amaçlayan çalışmalarda tercih edilen yaygın bir yöntem olması sebebiyle bu araştırmanın yöntemi olarak tercih edilmiştir. 98 Submit Date: 24.02.2023, Acceptance Date: 21.04.2023, DOI NO: 10.5281/zenodo.7852965 Research Article - This article was checked by iThenticate Copyright © Journal of Communication Science Researches Journal of Communication Science Researches - IBAD ISSN: 2757-8496, May 2023 Volume 3 Issue 2, p.95-107 Journal of Communication Science Researches - IBAD ISSN: 2757-8496, May 2023 Volume 3 Issue 2, p.95-107 BULGULAR Tablo 1. Birinci Tez 1. Tez Adı: Toplumsal Cinsiyet Bağlamında Kadına Yönelik Cinsel Şiddet Tahakküm Distopyası: ‘‘Damızlık Kızın Öyküsü’’ Adlı Dizi Örneği. Yıl: Bilim Dalı: Türü: Üniversite: Yöntem: Yazarın Cinsiyeti: 2021 Toplumsal Cinsiyet ve Kadın Çalışmaları Anabilim Dalı Yüksek Lisans Tezi Trakya Üniversitesi Nitel Erkek BULGULAR Tablo 1. Birinci Tez Tablo 1, İncelenen birinci yüksek lisans tezi 2021 yılında Trakya Üniversitesi Toplumsal Cinsiyet ve Kadın Çalışmaları Anabilim Dalı’nda nitel araştırma yöntemi kullanılarak yazılmıştır. Tez yazarının cinsiyeti erkektir. Tablo 1, İncelenen birinci yüksek lisans tezi 2021 yılında Trakya Üniversitesi Toplumsal Cinsiyet ve Kadın Çalışmaları Anabilim Dalı’nda nitel araştırma yöntemi kullanılarak yazılmıştır. Tez yazarının cinsiyeti erkektir. Tablo 1, İncelenen birinci yüksek lisans tezi 2021 yılında Trakya Üniversitesi Toplumsal Cinsiyet ve Kadın Çalışmaları Anabilim Dalı’nda nitel araştırma yöntemi kullanılarak yazılmıştır. Tez yazarının cinsiyeti erkektir. Tezde toplumsal cinsiyet bağlamında kadına yönelik cinsel şiddet, Damızlık Kızın Öyküsü dizisinde sahne analizi ve eleştirel söylem analizi yöntemiyle incelenmiştir. Submit Date: 24.02.2023, Acceptance Date: 21.04.2023, DOI NO: 10.5281/zenodo.7852965 Research Article - This article was checked by iThenticate Copyright © Journal of Communication Science Researches YÖNTEM Meşru Tecavüz, Çalınan Hazlar, Jezebel, Kürtaj, Kadınlar Arası Hiyerarşik Şiddet, Damızlık Kızların İç Dünyası temaları çerçevesinde dizi incelenmiştir. “Damızlık Kızın Öyküsü dizisindeki Gilead hükümetinin kadına yönelik işlediği cinsel şiddet analiz edilerek, bu şiddetin gelecekte hangi noktaya evrilebileceğine dikkat çekmiştir” (Altun, 2021: 131). Tablo 2. İkinci Tez 2. Tez Adı: Toplumsal Cinsiyet Bağlamında Türk Dizilerinde Femme Fatale Kadın Temsilleri: Zalim İstanbul ve Yasak Elma Dizilerindeki Femme Fatale Kadın Karakterlerin İncelenmesi. Yıl: Bilim Dalı: Türü: Üniversite: Yöntem: Yazarın Cinsiyeti: 2021 Gazetecilik Anabilim Dalı Yüksek Lisans Tezi Atatürk Üniversitesi Nitel Kadın Tablo 2, İncelenen ikinci yüksek lisans tezi 2021 yılında Atatürk Üniversitesi Gazetecilik Anabilim Dalı’nda nitel araştırma yöntemi kullanılarak yazılmıştır. Tez yazarının cinsiyeti kadındır. Tablo 2, İncelenen ikinci yüksek lisans tezi 2021 yılında Atatürk Üniversitesi Gazetecilik Anabilim Dalı’nda nitel araştırma yöntemi kullanılarak yazılmıştır. Tez yazarının cinsiyeti kadındır. Tablo 2, İncelenen ikinci yüksek lisans tezi 2021 yılında Atatürk Üniversitesi Gazetecilik Anabilim Dalı’nda nitel araştırma yöntemi kullanılarak yazılmıştır. Tez yazarının cinsiyeti kadındır. Tezde eleştirel söylem analizi yöntemi kullanılarak Zalim İstanbul ve Yasak Elma adlı diziler incelenmiştir. Yapılan incelemeler sonucunda “dizilerdeki femme fatale kadın temsillerinin ataerkil toplum yapısı için bir kırılma noktası oluşturmalarına rağmen bu karakterlerin geleneksel kodların dışına çıktıkları gerekçesiyle cezalandırıldıkları, erkek karşısında kalıcı bir başarı elde edemedikleri ve böylelikle ataerkil değerlerin sarsılmasının önüne geçildiği sonucuna varılmıştır” (Kanca, 2021). 99 Submit Date: 24.02.2023, Acceptance Date: 21.04.2023, DOI NO: 10.5281/zenodo.7852965 Research Article - This article was checked by iThenticate Copyright © Journal of Communication Science Researches Tablo 3. Üçüncü Tez 3. Tez Adı: Türk Dizilerinde Toplumsal Değişim ve Toplumsal Cinsiyet Açısından Kadının Sunumu: 1975 ve 2008 Yıllarında Yayınlanan Aşk-ı Memnu Dizisi Örneği. Yıl: Bilim Dalı: Türü: Üniversite: Yöntem: Yazarın Cinsiyeti: 2020 Medya ve İletişim Anabilim Dalı Yüksek Lisans Tezi Beykent Üniversitesi Nitel Kadın Tablo 3, İncelenen üçüncü yüksek lisans tezi 2020 yılında Beykent Üniversitesi Medya ve İletişim Anabilim Dalı’nda nitel araştırma yöntemi kullanılarak yazılmıştır. Tez yazarının cinsiyeti kadındır. Tezde 1975 ve 2008 yılında yayınlanan Aşk-ı Memnu dizisinin kadın karakterleri söylem analizi yöntemiyle incelenmiştir. İki dizide de kadının benzer şekilde yansıtıldığı bulgusuna ulaşılmıştır. Tezde diziler arasındaki benzerlik ve farklılıklar üzerinde durulmuştur. 1975 ve 2008 yılında yayınlanan iki dizide kadınlar benzer şekilde aktarılmıştır. Kadınlar erkeklere bağlı şekilde hareket eden bireyler olarak aktarılmıştır. Kadınların evlilik yoluyla varlık sahibi olma çabaları olduğu bulgusuna ulaşılmıştır. YÖNTEM Dizilerde geleneksel olarak kadına yüklenen toplumsal rollerin kadınlar tarafından yerine getirilmediği konusu üzerinde durulmuş, 1975 ve 2008 Aşk-ı Memnu dizileri karşılaştırıldığında müstehcen sahnelerin 2008 yılı Aşk-ı Memnu dizisinde açıkça aktarıldığı, 1975 yılı Aşk-ı Memnu dizisinde müstehcen sahnelerin gösterilmediği tespitinde bulunulmuştur. Tezde karşılaştırılan Aşk-ı Memnu dizilerinde kadına karşı toplumsal cinsiyet algısının korunduğu sonucuna ulaşılmıştır. Tablo 4. Dördüncü Tez 4. Tez Adı: Toplumsal Cinsiyet Bağlamında Kadının Medyada Temsili: ‘Kadın’ ve ‘Ufak Tefek Cinayetler’ Dizilerindeki Kadın Karakterler. Yıl: Bilim Dalı: Türü: Üniversite: Yöntem: Yazarın Cinsiyeti: 2019 Gazetecilik Anabilim Dalı Yüksek Lisans Tezi Atatürk Üniversitesi Nitel Kadın Tablo 4, İncelenen dördüncü yüksek lisans tezi 2019 yılında Atatürk Üniversitesi Gazetecilik Anabilim Dalı’nda nitel araştırma yöntemi kullanılarak yazılmıştır. Tez yazarının cinsiyeti kadındır. Tezde eleştirel söylem analizi yöntemiyle Kadın ve Ufak Tefek Cinayetler adlı diziler incelenmiştir. Geleneksel ve modern kadın temsiline yönelik karşılaştırmalar yapılmış diziler arasındaki benzer ve farklı yönler ortaya koyulmuştur. İncelemeler sonucunda her iki dizide de kadın temsilinin ataerkil kalıplar içerisinde kaldığı sonucuna ulaşılmıştır. 100 Submit Date: 24.02.2023, Acceptance Date: 21.04.2023, DOI NO: 10.5281/zenodo.7852965 Research Article - This article was checked by iThenticate Copyright © Journal of Communication Science Researches Journal of Communication Science Researches - IBAD ISSN: 2757-8496, May 2023 Volume 3 Issue 2, p.95-107 Tablo 5. Beşinci Tez 5. Tez Adı: Türkiye’de Yayınlanan Hint Dizilerinde Üretilen Toplumsal Cinsiyet Rolleri ve Bu Rollerin Kadınlar Tarafından Alımlama Biçimleri: Gaziantep Örneği. Yıl: Bilim Dalı: Türü: Üniversite: Yöntem: Yazarın Cinsiyeti: 2018 İletişim ve Toplumsal Dönüşüm Anabilim Dalı Yüksek Lisans Tezi Gaziantep Üniversitesi Nitel Kadın Tablo 5, İncelenen beşinci yüksek lisans tezi 2018 yılında Gaziantep Üniversitesi İletişim ve Toplumsal Dönüşüm Anabilim Dalı’nda nitel araştırma yöntemi kullanılarak yazılmıştır. Tez yazarının cinsiyeti kadındır. Tablo 5, İncelenen beşinci yüksek lisans tezi 2018 yılında Gaziantep Üniversitesi İletişim ve Toplumsal Dönüşüm Anabilim Dalı’nda nitel araştırma yöntemi kullanılarak yazılmıştır. Tez yazarının cinsiyeti kadındır. Tezde görüşme yöntemi kullanılarak kolayda örnekleme tekniğiyle seçilmiş Gaziantep’in Ulaş Mahallesi’nde yaşayan Şırnaklı Kürt kadınlara Bir Garip Aşk adlı diziyle ilgili sorular yöneltilmiştir. Kadınların Bir Garip Aşk adlı dizide yansıtılan toplumsal cinsiyet rollerini algılama biçimleri üzerinde durulmuştur. İncelemeler sonucunda bu dizinin kadınların gündelik hayatını değiştirmede etkili olduğu, dizinin kadının çaresizliğinin yeniden üretilmesine sebep olduğu ve kadınların bu dizideki kültürel ögelere yakınlık duyup kendi hayatlarına bu ögeleri yansıttıkları sonucuna ulaşılmıştır. Tablo 6. Altıncı Tez 6. Tez Adı: Uyarlama Dizilerdeki Kültürel Farkların Küyerelleşme ve Toplumsal Cinsiyet Rolleri Bağlamında Temsili: “Desperate Housewıves,” / “Umutsuz Ev Kadınları” Örneği. Submit Date: 24.02.2023, Acceptance Date: 21.04.2023, DOI NO: 10.5281/zenodo.7852965 Research Article - This article was checked by iThenticate Copyright © Journal of Communication Science Researches YÖNTEM Yıl: Bilim Dalı: Türü: Üniversite: Yöntem: Yazarın Cinsiyeti: 2014 Medya ve Kültürel Çalışmalar Anabilim Dalı Yüksek Lisans Tezi İstanbul Arel Üniversitesi Nitel Kadın Tablo 6, İncelenen altıncı yüksek lisans tezi 2014 yılında İstanbul Arel Üniversitesi Medya ve Kültürel Çalışmalar Anabilim Dalı’nda nitel araştırma yöntemi kullanılarak yazılmıştır. Tez yazarının cinsiyeti k d d Tablo 6, İncelenen altıncı yüksek lisans tezi 2014 yılında İstanbul Arel Üniversitesi Medya ve Kültürel Çalışmalar Anabilim Dalı’nda nitel araştırma yöntemi kullanılarak yazılmıştır. Tez yazarının cinsiyeti kadındır. Tablo 6, İncelenen altıncı yüksek lisans tezi 2014 yılında İstanbul Arel Üniversitesi Medya ve Kültürel Çalışmalar Anabilim Dalı’nda nitel araştırma yöntemi kullanılarak yazılmıştır. Tez yazarının cinsiyeti kadındır. Tablo 6, İncelenen altıncı yüksek lisans tezi 2014 yılında İstanbul Arel Üniversitesi Medya ve Kültürel Çalışmalar Anabilim Dalı’nda nitel araştırma yöntemi kullanılarak yazılmıştır. Tez yazarının cinsiyeti kadındır. Tezde Desperate Housewıves adlı yabancı dizi ile bu dizinin Türkiye uyarlaması olan Umutsuz Ev Kadınları adlı dizi karşılaştırılmıştır. Tezde toplumsal cinsiyet bağlamında kadına yönelik bir inceleme yapılmış olup karşılaştırılan iki dizide de kadının ikinci sınıf konumunda olduğu sonucuna ulaşılmıştır. Ayrıca incelemeler sonucunda bekar kadınların çalışma oranının yüksek olduğu, evli kadınların ise çalışma oranının düşük olduğuna ve her iki dizide de evli kadınların ihtiyaçlarının eşi tarafından 101 Submit Date: 24.02.2023, Acceptance Date: 21.04.2023, DOI NO: 10.5281/zenodo.7852965 Research Article - This article was checked by iThenticate Copyright © Journal of Communication Science Researches Journal of Communication Science Researches - IBAD ISSN: 2757-8496, May 2023 Volume 3 Issue 2, p.95-107 karşılandığına dikkat çekilmiştir. İki dizi arasında kültürel farklar olmasına karşın dizilerdeki en önemli benzer nokta olarak kadının toplumda ikincil planda olması üzerinde durulmuştur. Tablo 7. Yedinci Tez 7. Tez Adı: Televizyon Dizilerinde Toplumsal Cinsiyet Açısından Kadının Sunumu: Huzur Sokağı Dizisi Kadın Karakterleri. Yıl: Bilim Dalı: Türü: Üniversite: Yöntem: Yazarın Cinsiyeti: 2013 Medya ve Kültürel Çalışmalar Anabilim Dalı Yüksek Lisans Tezi İstanbul Arel Üniversitesi Nitel Kadın Tablo 7, İncelenen yedinci yüksek lisans tezi 2013 yılında İstanbul Arel Üniversitesi Medya ve Kültürel Çalışmalar Anabilim Dalı’nda nitel araştırma yöntemi kullanılarak yazılmıştır. Tez yazarının cinsiyeti kadındır. Tablo 7, İncelenen yedinci yüksek lisans tezi 2013 yılında İstanbul Arel Üniversitesi Medya ve Kültürel Çalışmalar Anabilim Dalı’nda nitel araştırma yöntemi kullanılarak yazılmıştır. Tez yazarının cinsiyeti kadındır. Tablo 7, İncelenen yedinci yüksek lisans tezi 2013 yılında İstanbul Arel Üniversitesi Medya ve Kültürel Çalışmalar Anabilim Dalı’nda nitel araştırma yöntemi kullanılarak yazılmıştır. Tez yazarının cinsiyeti kadındır. Tezde söylem analizi yöntemiyle Huzur Sokağı adlı dizideki kadın karakterler incelenmiştir. Submit Date: 24.02.2023, Acceptance Date: 21.04.2023, DOI NO: 10.5281/zenodo.7852965 Research Article - This article was checked by iThenticate Copyright © Journal of Communication Science Researches YÖNTEM Kadının geleneksel kalıplar içerisinde kaldığı, kadının toplumdaki yerinin öncelikli olarak ideal eş, ideal anne statüsü olduğu sonucuna ulaşılmıştır. Tablo 8. Sekizinci Tez 8. Tez Adı: Televizyon Dizilerinde Toplumsal Cinsiyet Açısından Kadının Sunumu: Kanal D’de Yayınlanan Yaprak Dökümü Dizisinde Kadın Karakterler. Yıl: Bilim Dalı: Türü: Üniversite: Yöntem: Yazarın Cinsiyeti: 2010 Radyo Televizyon Sinema Anabilim Dalı Yüksek Lisans Tezi İstanbul Üniversitesi Nitel Kadın Tablo 8, İncelenen sekizinci yüksek lisans tezi 2010 yılında İstanbul Üniversitesi Radyo Televizyon Sinema Anabilim Dalı’nda nitel araştırma yöntemi kullanılarak yazılmıştır. Tez yazarının cinsiyeti kadındır. Tablo 8, İncelenen sekizinci yüksek lisans tezi 2010 yılında İstanbul Üniversitesi Radyo Televizyon Sinema Anabilim Dalı’nda nitel araştırma yöntemi kullanılarak yazılmıştır. Tez yazarının cinsiyeti kadındır. Tezde söylem analizi yöntemiyle Yaprak Dökümü adlı dizideki kadın karakterler incelenmiştir. Hikayesini Yaprak Dökümü kitabından alan bu dizide kadının geleneksel roller içerisinde kaldığı vurgusu yapılmıştır. “Cumhuriyet’in ilanından sonra yazılan bu kitapta kadına karşı geliştirilen tezlerin, kadın lehine pek çok değişimin yaşandığı günümüz Türkiye’sinde korunuyor olduğu sonucuna” ulaşılmıştır (Kutlu, 2010). 102 Submit Date: 24.02.2023, Acceptance Date: 21.04.2023, DOI NO: 10.5281/zenodo.7852965 Research Article - This article was checked by iThenticate Copyright © Journal of Communication Science Researches Journal of Communication Science Researches - IBAD ISSN: 2757-8496, May 2023 Volume 3 Issue 2, p.95-107 Tablo 9. Dokuzuncu Tez 9. Tez Adı: Toplumsal Cinsiyet Kalıplarının Yerli Televizyon Dizilerindeki Kadın Karakterlere Yansımaları: Binbir Gece Örneği. Yıl: Bilim Dalı: Türü: Üniversite: Yöntem: Yazarın Cinsiyeti: 2008 İletişim Anabilim Dalı Yüksek Lisans Tezi Kocaeli Üniversitesi Nitel Kadın Tablo 9, İncelenen dokuzuncu yüksek lisans tezi 2008 yılında Kocaeli Üniversitesi İletişim Anabilim Dalı’nda nitel araştırma yöntemi kullanılarak yazılmıştır. Tez yazarının cinsiyeti kadındır. Tablo 9, İncelenen dokuzuncu yüksek lisans tezi 2008 yılında Kocaeli Üniversitesi İletişim Anabilim Dalı’nda nitel araştırma yöntemi kullanılarak yazılmıştır. Tez yazarının cinsiyeti kadındır. Tezde görüşme yöntemi kullanılarak tesadüfi bir şekilde seçilen 20 kadına Binbir Gece adlı diziyle ilgili sorular yöneltilmiştir. İncelemeler sonucunda “izleyici gözüyle yapılan yorumlarda geleneksel rollerde yaşanan değişim tespit edilmiş ve gelenekselliğin modernizm tarafından stilize edildiği” sonucuna ulaşılmıştır (Dönmez, 2008). Genel çerçevede tezlerin yazıldığı üniversiteler, anabilim dalları ve yazarların cinsiyetleri incelendiğinde; incelenen 9 tezin yazıldığı bilim dalları şu şekildedir: Medya ve Kültürel Çalışmalar Anabilim Dalı ve Gazetecilik Anabilim Dalı’nda ikişer tez, Medya ve İletişim, Toplumsal Cinsiyet ve Kadın Çalışmaları, İletişim, Radyo Televizyon Sinema ve İletişim ve Toplumsal Dönüşüm Anabilim Dalı’nda birer kez tez yazılmıştır. İncelenen 9 tezin tümü yüksek lisans tezi olup örnekleme uygun doktora tezine ulaşılmamıştır. Submit Date: 24.02.2023, Acceptance Date: 21.04.2023, DOI NO: 10.5281/zenodo.7852965 Submit Date: 24.02.2023, Acceptance Date: 21.04.2023, DOI NO: 10.5281/zenodo.7852965 Research Article - This article was checked by iThenticate Copyright © Journal of Communication Science Researches 103 Submit Date: 24.02.2023, Acceptance Date: 21.04.2023, DOI NO: 10.5281/zenodo.7852965 Research Article - This article was checked by iThenticate Copyright © Journal of Communication Science Researches YÖNTEM Journal of Communication Science Researches - IBAD ISSN: 2757-8496, May 2023 Volume 3 Issue 2, p.95-107 Distopya 1 Damızlık Kızın Öyküsü 1 Femme Fatale 1 Feminizm 1 Hegemonya ve İdeoloji 1 Kültürel Çalışmalar 1 Alımlama Çalışmaları 1 Tablo 10, Tezlerde kullanılan anahtar kelimeler incelendiğinde “toplumsal cinsiyet” kelimesi yedi kez, “kadın” ve “yerli dizi” kelimeleri üç kez kullanılmıştır. “Toplumsal cinsiyet” kelimesi bu tezlerde en çok tercih edilen anahtar kelimedir. İncelenen dokuz tezin sekizinde anahtar kelime yer alırken bir tezde anahtar kelime yer almamaktadır. Tezlerde kullanılan tüm anahtar kelimeler ve kullanım sayısı Tablo 10’da belirtildiği şekildedir. Tablo 10, Tezlerde kullanılan anahtar kelimeler incelendiğinde “toplumsal cinsiyet” kelimesi yedi kez, “kadın” ve “yerli dizi” kelimeleri üç kez kullanılmıştır. “Toplumsal cinsiyet” kelimesi bu tezlerde en çok tercih edilen anahtar kelimedir. İncelenen dokuz tezin sekizinde anahtar kelime yer alırken bir tezde anahtar kelime yer almamaktadır. Tezlerde kullanılan tüm anahtar kelimeler ve kullanım sayısı Tablo 10’da belirtildiği şekildedir. KAYNAKÇA Acar, H. (2019). Türk Kültür ve Devlet Geleneğinde Kadın. İnsan ve İnsan, 6 (21), 395-411. Akçalı, E. & İnceoğlu, İ. (2020). Kadınlık ve Erkekliğin Değişmeyen Halleri: Televizyon Dizilerinde Toplumsal Cinsiyet. Fe Dergi, 12 (2), 161-173. YÖNTEM İncelenen 9 tezin yazıldığı üniversiteler şu şekildedir: İstanbul Arel Üniversitesi ve Atatürk Üniversitesi’nde iki tez, Beykent Üniversitesi, Trakya Üniversitesi, İstanbul Üniversitesi, Gaziantep Üniversitesi ve Kocaeli Üniversitesi’nde bir tez yazılmıştır. İncelenen 9 tezin tümünde nitel araştırma yöntemi kullanılmıştır. Tez yazarlarının cinsiyetleri incelendiğinde ise 8 tezin yazarının kadın, 1 tezin yazarının erkek olduğu sonucu ortaya çıkmıştır. Submit Date: 24.02.2023, Acceptance Date: 21.04.2023, DOI NO: 10.5281/zenodo.7852965 Research Article - This article was checked by iThenticate Copyright © Journal of Communication Science Researches 103 Tablo 10. Tezlerde Kullanılan Anahtar Kelimeler Anahtar Kelimeler Frekans Toplumsal Cinsiyet 8 Kadın 3 Yerli Diziler 3 Televizyon Dizisi 2 Televizyon 2 Kadın Temsili 2 Küreselleşme 1 Yerelleşme 1 Küyerelleşme 1 Kültürel Farklılıklar 1 Toplumsal Değişim 1 Cinsel Şiddet 1 Medya 1 Tablo 10. Tezlerde Kullanılan Anahtar Kelimeler Anahtar Kelimeler Frekans Toplumsal Cinsiyet 8 Kadın 3 Yerli Diziler 3 Televizyon Dizisi 2 Televizyon 2 Kadın Temsili 2 Küreselleşme 1 Yerelleşme 1 Küyerelleşme 1 Kültürel Farklılıklar 1 Toplumsal Değişim 1 Cinsel Şiddet 1 Medya 1 Tablo 10. Tezlerde Kullanılan Anahtar Kelimeler Anahtar Kelimeler Frekans Toplumsal Cinsiyet 8 Kadın 3 Yerli Diziler 3 Televizyon Dizisi 2 Televizyon 2 Kadın Temsili 2 Küreselleşme 1 Yerelleşme 1 Küyerelleşme 1 Kültürel Farklılıklar 1 Toplumsal Değişim 1 Cinsel Şiddet 1 Medya 1 103 Journal of Communication Science Researches - IBAD ISSN: 2757-8496, May 2023 Volume 3 Issue 2, p.95-107 Distopya 1 Damızlık Kızın Öyküsü 1 Femme Fatale 1 Feminizm 1 Hegemonya ve İdeoloji 1 Kültürel Çalışmalar 1 Alımlama Çalışmaları 1 Tablo 10, Tezlerde kullanılan anahtar kelimeler incelendiğinde “toplumsal cinsiyet” kelimesi yedi kez, “kadın” ve “yerli dizi” kelimeleri üç kez kullanılmıştır. “Toplumsal cinsiyet” kelimesi bu tezlerde en çok tercih edilen anahtar kelimedir. İncelenen dokuz tezin sekizinde anahtar kelime yer alırken bir tezde anahtar kelime yer almamaktadır. Tezlerde kullanılan tüm anahtar kelimeler ve kullanım sayısı Tablo 10’da belirtildiği şekildedir. Journal of Communication Science Researches - IBAD ISSN: 2757-8496, May 2023 Volume 3 Issue 2, p.95-107 Distopya 1 Damızlık Kızın Öyküsü 1 Femme Fatale 1 Feminizm 1 Hegemonya ve İdeoloji 1 Kültürel Çalışmalar 1 Alımlama Çalışmaları 1 Tablo 10, Tezlerde kullanılan anahtar kelimeler incelendiğinde “toplumsal cinsiyet” kelimesi yedi kez, “kadın” ve “yerli dizi” kelimeleri üç kez kullanılmıştır. “Toplumsal cinsiyet” kelimesi bu tezlerde en çok tercih edilen anahtar kelimedir. İncelenen dokuz tezin sekizinde anahtar kelime yer alırken bir tezde anahtar kelime yer almamaktadır. Tezlerde kullanılan tüm anahtar kelimeler ve kullanım sayısı Tablo 10’da belirtildiği şekildedir. Akçalı, E. & İnceoğlu, İ. (2020). Kadınlık ve Erkekliğin Değişmeyen Halleri: Televizyon Dizilerinde Toplumsal Cinsiyet. Fe Dergi, 12 (2), 161-173. SONUÇ Ç Bu çalışmada tez adında kadın, toplumsal cinsiyet ve dizi kelimeleri geçen lisansüstü tezler doküman analizi tekniği kullanılarak incelenmiştir. Araştırmanın amacına uygun 9 teze ulaşılmıştır. Ulaşılan bu 9 tezin tümü yüksek lisans tezidir. Bu bağlamda tez adında toplumsal cinsiyet, kadın ve dizi kelimeleri geçen doktora tezinin yazılmadığı bulgusuna ulaşılmıştır. İncelenen tezlerin çoğunlukta kadınlar tarafından yazıldığı sonucuna ulaşılmıştır. İncelenen 8 tezin yazarının kadın, 1 tezin yazarının ise erkek olduğu ortaya koyulmuştur. İncelenen 9 tezin 7’sinde dizilerin toplumsal cinsiyet bağlamında analiz edilip kadın karakterlerin incelendiği, 2’sinde ise dizilerin toplumda algılanma biçimi üzerinde durulduğu bulgusuna ulaşılmıştır. İncelenen tezler Trakya Üniversitesi, Atatürk Üniversitesi, Beykent Üniversitesi, Gaziantep Üniversitesi, İstanbul Arel Üniversitesi, İstanbul Üniversitesi, Kocaeli Üniversitesi’nde yazılmıştır. Tezlerin Toplumsal Cinsiyet ve Kadın Çalışmaları, Gazetecilik, Medya ve İletişim, İletişim ve Toplumsal Dönüşüm, Medya ve Kültürel Çalışmalar, Radyo Televizyon Sinema, İletişim Anabilim dallarında yazıldığı görülmektedir. Bu bağlamda incelenen 9 tezin tümünün Sosyal Bilimler alanında yazıldığı sonucuna ulaşılmıştır. İncelenen tezlerin 2008 yılı itibariyle yazıldığı görülmektedir. 2008, 2010, 2013, 2014, 2018, 2019, 2020, 2021 yılları incelenen tezlerin yazıldığı yıllar olarak karşımıza çıkmaktadır. Bu verilerden yola çıkarak toplumsal cinsiyet, kadın ve dizi konularını içeren lisansüstü tezlerin son 15 yıl içerisinde yazılmış olduğu görülmektedir. Bu veriler yakın tarihte medya, kadın ve toplumsal cinsiyete yönelik araştırmaların gerçekleştirildiğinin bir kanıtıdır. Tezlerde kullanılan anahtar kelimeler incelendiğince “toplumsal cinsiyet” kelimesi en çok tercih edilen anahtar kelime olarak karşımıza çıkmaktadır. Bu çalışma toplumsal cinsiyet, kadın ve medyayı içeren lisansüstü araştırmaların ortaya koyulması ve benzer çalışmalara kaynak oluşturması bakımından önemlidir. Tez adında “toplumsal cinsiyet, kadın ve dizi” kelimeleri geçen lisansüstü tezlerin incelendiği benzer bir çalışma olmadığından gerçekleştirilen bu çalışma özgün bir çalışmadır. Toplumsal cinsiyet, kadın ve medya araştırmaları kadının geleceği için önemlidir. Toplumsal cinsiyet eşitsizliğinin önüne geçilmesi bakımında televizyon dizilerinde kadının sunumuna dikkat edilmesi ve kadının medyanın her alanında metalaşmamış, erkekten bağımsız bir birey olarak yer alması ve yansıtılması gerekmektedir. Kadının geleceği noktasında dizilerde yansıtılan kadın imgesi toplumda kadına yönelik algının oluşmasında oldukça önemlidir. Bu bağlamda kadının medyadaki konumunun ortaya koyulması toplumun bilinçlenmesi ve kadın araştırmalarına yönelik literatürün genişlemesi açısından önemlidir. Gerçekleştirilen bu araştırmada aynı anda toplumsal cinsiyet, kadın ve medya konularını içeren lisansüstü tezlerin kısıtlı sayıda olduğu sonucuna ulaşılmıştır. Kadının geleceği açısından medya ve kadına yönelik gerçekleştirilen lisansüstü araştırmalar desteklenmeli ve teşvik edilmelidir. 104 Journal of Communication Science Researches - IBAD ISSN: 2757-8496, May 2023 Volume 3 Issue 2, p.95-107 Altun, M. (2021). Submit Date: 24.02.2023, Acceptance Date: 21.04.2023, DOI NO: 10.5281/zenodo.7852965 Research Article - This article was checked by iThenticate Copyright © Journal of Communication Science Researches 105 SONUÇ 105 Journal of Communication Science Researches - IBAD ISSN: 2757-8496, May 2023 Volume 3 Issue 2, p.95-107 Journal of Communication Science Researches - IBAD ISSN: 2757-8496, May 2023 Volume 3 Issue 2, p.95-107 Kaçar. Ö. (2007). Toplumsal Cinsiyet ve Kadının Konumu: Türkiye'de Yakın Zamanlardaki Değişimi Anlamak (Tez No. 209028) [Yayınlanmamış yüksek lisans tezi, Afyon Kocatepe Üniversitesi. Ulusal Tez Merkezi]. (Kaçar, 2007) Kanca, L. (2021). Toplumsal Cinsiyet Bağlamında Türk Dizilerinde Femme Fatale Kadın Temsilleri: Yasak Elma ve Zalim İstanbul Dizilerindeki Femme Fatale Kadın Karakterlerin İncelenmesi (Tez No. 696003) [Yayınlanmamış yüksek lisans tezi, Atatürk Üniversitesi. Ulusal Tez Merkezi]. (Kanca, 2021) Kellner, D. (2008). Guys and Guns Amok: Domestic Terrorism and School Shootings from the Oklahoma City Bombings to the Virginia Tech Massacre. Boulder. CO: Paradigm. Kıral, B. (2020). Nitel Bir Veri Analizi Yöntemi Olarak Doküman Analizi. Siirt Üniversitesi Sosyal Bilimler Enstitüsü Dergisi, 8 (15), 170-189. Kutlu, T. (2010). Televizyon Dizilerinde Toplumsal Cinsiyet Açısından Kadının Sunumu: Kanal D’de Yayınlanan Yaprak Dökümü Dizisinde Kadın Karakterler (Tez No. 257901) [Yayınlanmamış yüksek lisans tezi, İstanbul Üniversitesi. Ulusal Tez Merkezi]. (Kutlu, 2010) Mendes, K. & Carter, C. (2008). Feminist And Gender Studies: A Critical Overview. Sociological Compass, 2(6), 1701- 1718. Özdemir, M. (2010). Türkiye’deki Reklamlarda Toplumsal Cinsiyet ve Sunumu. Millî Folklor, 22 (88), 101-111. Pınarbaşı, G. (2022). Diktatör Filmi ve Bodyguard Dizisindeki İslamofobik Söylemler. Medya ve Din Araştırmaları Dergisi (Mediad), 5 (1), 177-200. Saraç, S. (2013). Toplumsal Cinsiyet. L. Gültekin, G. Güneş, C. Ertung ve A. Şimşek (Ed.), Toplumsal Cinsiyet ve Yansımaları, Ankara: Atılım Üniversitesi Yayınları (s. 27-31) Şakrak, B. E. (2020). Dizi Filmlerde Toplumsal Cinsiyet Bağlamında Kadının Temsili Örnek İnceleme: “Kadın” Dizisi. Avrasya Uluslararası Araştırmalar Dergisi, 8 (22), 420-434. Şen, R. (2018). Türkiye'de Yayınlanan Hint Dizilerinde Üretilen Toplumsal Cinsiyet Rolleri ve Bu Rollerin Kadınlar Tarafından Alımlama Biçimleri: Gaziantep Örneği (Tez No. 576177) [Yayınlanmamış yüksek lisans tezi, Gaziantep Üniversitesi. Ulusal Tez Merkezi]. (Şen, 2018) Şentürk, N. (2019). Toplumsal Cinsiyet Bağlamında Kadının Medyada Temsili: ‘Kadın’ ve ‘Ufak Tefek Cinayetler’ Dizilerindeki Kadın Karakterler (Tez No. 594867) [Yayınlanmamış yüksek lisans tezi, Atatürk Üniversitesi. Ulusal Tez Merkezi]. (Şentürk, 2019) Tanrıöver, H. U. (2007). Medyada Kadınların Temsil Biçimleri ve Kadın Hakları İhlalleri. S. Alankuş (Der.), Kadın Odaklı Habercilik, İstanbul: IPS İletişim Vakfı Yayınları (s. 151-168) Uğur, D. (2020). Türk Dizilerinde Toplumsal Değişim ve Toplumsal Cinsiyet Açısından Kadının Sunumu: 1975 ve 2008 Yıllarında Yayınlanan Aşk-I Memnu Dizisi Örneği (Tez No. 664464) [Yayınlanmamış yüksek lisans tezi, Beykent Üniversitesi. Ulusal Tez Merkezi]. Submit Date: 24.02.2023, Acceptance Date: 21.04.2023, DOI NO: 10.5281/zenodo.7852965 Research Article - This article was checked by iThenticate Copyright © Journal of Communication Science Researches SONUÇ Toplumsal Cinsiyet Bağlamında Kadına Yönelik Cinsel Şiddet Tahakküm Distopyası: ‘‘Damızlık Kızın Öyküsü’’ Adlı Dizi Örneği (Tez No. 684043) [Yayınlanmamış yüksek lisans tezi, Trakya Üniversitesi. Ulusal Tez Merkezi]. (Altun, 2021) Baltacı, B. (2012). Televizyon Dizilerinde Kadın Temsili Öyle Bir Geçer Zaman Ki Örneği (Tez No. 323448) [Yayınlanmamış yüksek lisans tezi, Erciyes Üniversitesi. Ulusal Tez Merkezi]. (Baltacı, 2012) raig, S. (Ed.) (1992). Men, Masculinity, and the Media. Thousand Oaks, CA: Sage Publications. Çavuşoğlu, Ç. (2014). Uyarlama Dizilerdeki Kültürel Farkların Küyerelleşme ve Toplumsal Cinsiyet Rolleri Bağlamında Temsili: Desperate Housewives / Umutsuz Ev Kadınları Örneği (Tez No. 375375) [Yayınlanmamış yüksek lisans tezi, İstanbul Arel Üniversitesi. Ulusal Tez Merkezi]. (Çavuşoğlu, 2014) Dökmen, Z. Y. (2015). Toplumsal Cinsiyet: Sosyal Psikolojik Açıklamalar, İstanbul: Remzi Kitabevi. Dönmez, N. (2008). Toplumsal Cinsiyet Kalıplarının Yerli Televizyon Dizilerindeki Kadın Karakterlere Yansımaları: Binbir Gece Örneği Karakterler (Tez No. 229467) [Yayınlanmamış yüksek lisans tezi, Kocaeli Üniversitesi. Ulusal Tez Merkezi]. (Dönmez, 2008) Dumanlı, D. (2011). Reklamlarda Toplumsal Cinsiyet Kavramı ve Kadın İmgesinin Kullanımı: Bir İçerik Analizi. Yalova Üniversitesi Sosyal Bilimler Dergisi, 1(2), 132-149. Ecevit, Y. (2003). Toplumsal Cinsiyetle Yoksulluk İlişkisi Nasıl Kurulabilir? Bu İlişki Nasıl Çalışılabilir?. C. Ü. Tıp Fakültesi Dergisi, 25(4), 83-8. Ellemers, N. (2018) Gender Stereotypes. Annu. Rev. Psychol. (69), 275–298. Ertingü Sevmiş, O. (2013). Televizyon Dizilerinde Toplumsal Cinsiyet Açısından Kadının Sunumu: Huzur Sokağı Dizisi Kadın Karakterleri (Tez No. 375407) [Yayınlanmamış yüksek lisans tezi, İstanbul Arel Üniversitesi. Ulusal Tez Merkezi]. (Ertingü Sevmiş, 2013) Etiler, N. & Zengin, Ü. (2016). Televizyon Kanallarındaki Gündüz Programlarında Kadın Sağlığı ve Toplumsal Cinsiyete Bakışın Değerlendirilmesi. Turkish Journal of Public Health, 13 (2), 137-146. Görgün Baran, A., Tuba Sarıtaş, C. & Şahin Kütük, B. (2017). Analysis Of News On Violence Against Women In Media In Terms Of Content And Presentatıon: Beyazgazete.Com Case. Istanbul Journal Of Sociological Studies, (55), 107-132. Güdekli, A. (2016). Küresel Erkek (Lik) ve Medya. Literatürk Academia. Güzel, E. (2014). Toplumsal Cinsiyete Dayalı Ayrımcılık ve Medyanın Rolü. Global Media Journal: Tr Edition 4(8), Spring, 185-199. Ilgaz Büyükbaykal, C. (2012). Medyada Kadın Olgusu. İstanbul Üniversitesi İletişim Fakültesi Dergisi, 0 (28), 19-30. Ilgın, H. Ö. & Yavuz, C. (2022, July 1-3). Kadın Araştırmaları; İsminde Kadın Kelimesi Geçen Lisansüstü Tezler Üzerine Bibliyometrik Analiz. H. Çiftçi, Ç. Tanyel Başar (Ed.), 8th Internatıonal "Communıcatıon In The New World" Congress (s. 6-17). Ilgın, H. Ö. (2020). Televizyon Dizilerinde Kent İmajı. Eğitim Yayınevi. Ünal, F., Tarhan, S. & Çürükvelioğlu Köksal, E. (2017). Toplumsal Cinsiyet Algısını Yordamada Cinsiyet, Sınıf, Bölüm ve Toplumsal Cinsiyet Oluşumunun Rolü. Bartın University Journal of Faculty of Education, 6 (1), 227-236. Journal of Communication Science Researches - IBAD ISSN: 2757-8496, May 2023 Volume 3 Issue 2, p.95-107 Yengin, D. & Bayrak, T. (2022). Yeni Medya Kuramları 101. Der Yayınları SONUÇ (Uğur, 2020) Uluyağcı, C. & Yılmaz, R. A. (2007). Televizyon Reklamlarında Çocuğa İlişkin Toplumsal Cinsiyet Rollerinin Sunumu. Galatasaray Üniversitesi İletişim Dergisi, (6), 141-157. 106 Journal of Communication Science Researches - IBAD ISSN: 2757-8496, May 2023 Volume 3 Issue 2, p.95-107 Ünal, F., Tarhan, S. & Çürükvelioğlu Köksal, E. (2017). Toplumsal Cinsiyet Algısını Yordamada Cinsiyet, Sınıf, Bölüm ve Toplumsal Cinsiyet Oluşumunun Rolü. Bartın University Journal of Faculty of Education, 6 (1), 227-236. Yengin, D. & Bayrak, T. (2022). Yeni Medya Kuramları 101. Der Yayınları Journal of Communication Science Researches - IBAD ISSN: 2757-8496, May 2023 Volume 3 Issue 2, p.95-107 Ünal, F., Tarhan, S. & Çürükvelioğlu Köksal, E. (2017). Toplumsal Cinsiyet Algısını Yordamada Cinsiyet, Sınıf, Bölüm ve Toplumsal Cinsiyet Oluşumunun Rolü. Bartın University Journal of Faculty of Education, 6 (1), 227-236. Yengin, D. & Bayrak, T. (2022). Yeni Medya Kuramları 101. Der Yayınları Journal of Communication Science Researches - IBAD ISSN: 2757-8496, May 2023 Volume 3 Issue 2, p.95-107 Ünal, F., Tarhan, S. & Çürükvelioğlu Köksal, E. (2017). Toplumsal Cinsiyet Algısını Yordamada Cinsiyet, Sınıf, Bölüm ve Toplumsal Cinsiyet Oluşumunun Rolü. Bartın University Journal of Faculty of Education, 6 (1), 227-236. Yengin, D. & Bayrak, T. (2022). Yeni Medya Kuramları 101. Der Yayınları Yengin, D. & Bayrak, T. (2022). Yeni Medya Kuramları 101. Der Yayınları 107
https://openalex.org/W4391254578	https://pressto.amu.edu.pl/index.php/cph/article/download/41069/34446	Polish	null	Uwagi o polskim państwie podziemnym w latach 1863- 1864	Czasopismo Prawno-Historyczne	2,001	cc-by	8,240	CZASOPISMO PRAWNO-HISTORYCZNE Tom LIII — 2001 — Zeszyt 1 CZASOPISMO PRAWNO-HISTORYCZNE Tom LIII — 2001 — Zeszyt 1 MAKSYMILIAN STANULEWICZ (Poznań) MAKSYMILIAN STANULEWICZ (Poznań) 1 A. Giller, Historia powstania narodu polskiego w 1861-1864, t. I, wyd. 2, Paryż 1864 2 Ibidem, s. 64. 3 Ibidem, s. 88. Uwagi o polskim państwie podziemnym w latach 1863-1864 Niniejszy tekst jest próbą zebrania i podsumowania opinii, a także pew nych, ustalonych przez dotychczasową naukę, faktów, dotyczących struktury i form działania tzw. Organizacji Narodowej z okresu powstania stycznio wego. Jest to zadanie o tyle trudne, że wokół charakteru tej organizacji od kilkudziesięciu lat toczą się spory. Jedni badacze nazywają ją polskim pań stwem podziemnym, inni odmawiają jej tego miana, powołując się zresztą na różne argumenty. Zadaniem tego artykułu jest nie tyle ostateczna odpowiedź na pytanie o cechy państwowości powstania styczniowego, ile konfrontacja istniejących w nauce i literaturze poglądów na tę kwestię, a także wskazanie cech charakterystycznych omawianej struktury. Koncepcja określenia Organizacji Narodowej mianem podziemnego pań stwa polskiego pojawiła się po raz pierwszy w pracy jednego ze współtwór ców struktury organizacyjnej władz powstańczych Agatona Gillera1. Stwier dził on, że Organizacja Narodowa zmieniła się „w tajemne państwo polskie, które funkcjonowało regularnie i należy do najpiękniejszych objawów ruchu narodowego - [nie miała ona - M.S.] podobnej do siebie w dziejach świata, ani co do rozmiarów, ani co do form”2. W innym miejscu Giller wskazywał, iż „Polacy pozbawieni bytu politycznego stworzyli tajemny byt polityczny [...] dali dziejom nieznany widok państwa podziemnego. [...] Naprzeciw or ganizacji niejezdniczej [naród polski - M. S.] postawił również spójną orga nizację narodową polską”3. Podobnie, w odniesieniu do organizacji litewskiej, wypowiadał się jej czo łowy działacz, naczelnik cywilny województwa grodzieńskiego Apollo Hoff meister. Pisał on, w kilkadziesiąt lat po powstaniu, że naród polski umiał 232 Maksymilian Stanulewicz utworzyć swój Rząd Narodowy, któremu byt on posłuszny [...] a Rząd ten przetrwał dwa lata. Rząd w rządzie - w podobnych warunkach dotąd histo ria świata na żadnej karcie nie zapisała4. Już wcześniej, bo w 1863 r. Mi chaił Katkow stwierdził na łamach Moskowskich Wiedmosti nr 113, że takiej podwójnej władzy jaka istnieje obecnie w Polsce, nigdy i nigdzie nie było5. W kilkadziesiąt lat później, na lamach tego samego czasopisma, badacz dzie jów Rosji L. Tichomirow zanotował, że Rząd Narodowy zyskał charakter ja kiegoś faktycznego rządu, strony wojującej [...] a Wydział Litewski Rządu stał się współwłaścicielem olbrzymiego terenu6. Pojęcie państwa podziemnego, tajemnego czy też konspiracyjnego prze wijało się w pracach historyków przez następne sto lat. Przed II wojną po sługiwali się tym pojęciem Józef Piłsudski i Edward Maliszewski, Adam Sze- lągowski i Józef Grabiec - Dąbrowski, Tomasz Kędzierski i Eugeniusz Przy byszewski7. y y p 9 S. Kieniewicz, Powstanie Styczniowe, Warszawa 1972; D. Fajnhauz, Litwa u schył ku powstania styczniowego, „Teki Historyczne”, t. XIX, 1988-1989; idem, 1863. Litwa i Bia łoruś, Warszawa 1999; idem Niektóre zagadnienia powstania styczniowego na Kowieńszczyź- nie, Kwart. Hist., 1962, z. 4; F. Ramotowska, Rząd Narodowy Polski 1863-1864, Warsza wa 1978; taże, Narodziny tajemnego państwa polskiego 1859-1862, Warszawa 1990; eadem, Tajemne państwo polskie 1863-1864, Warszawa 1999. 4 Ruch społeczno-polityczny na Ukrainie w 1863-1864, Kijów 1964, s. 444. 5 Podaję za F. Ramatowską, Legitymizm władz polskich w okresach powstań i ruchów wolnościowych (1830-1863). Miscellanea Historico-Archivistica t. IV, 1994, s. 72. 6 Cyt. za D. Fajnhauz, 1863. Litwa i Białoruś, Warszawa 1999, s. 237. 7 J. Piłsudski, Rok 1863, Warszawa 1989, s. 37; E. Maliszewska, Organizacja po wstania styczniowego, Warszawa 1925, s. 38 i n.; A. Szelągowski, Powstanie Komitetu Cen tralnego [w:] Polska, jej dzieje i kultura, t. III, Warszawa 1927; J. Grabiec [Dąbrowski], Rok 1863, Poznań 1923; T. Kędzierski, Powstanie styczniowe. Geneza. Przebieg. Udział praw ników, Warszawa 1936, s. 3 i n.; E. Przybyszewski, Pisma, Warszawa 1961, s. 102 i 159. 8 E. Przybyszewski, op. cit., s. 102. 9 S. Kieniewicz, Powstanie Styczniowe, Warszawa 1972; D. Fajnhauz, Litwa u schył ku powstania styczniowego, „Teki Historyczne”, t. XIX, 1988-1989; idem, 1863. Litwa i Bia łoruś, Warszawa 1999; idem Niektóre zagadnienia powstania styczniowego na Kowieńszczyź- nie, Kwart. Hist., 1962, z. 4; F. Ramotowska, Rząd Narodowy Polski 1863-1864, Warsza wa 1978; taże, Narodziny tajemnego państwa polskiego 1859-1862, Warszawa 1990; eadem, Tajemne państwo polskie 1863-1864, Warszawa 1999. 4 Ruch społeczno-polityczny na Ukrainie w 1863-1864, Kijów 1964, s. 444. 5 Podaję za F. Ramatowską, Legitymizm władz polskich w okresach powstań i ruchów wolnościowych (1830-1863). Miscellanea Historico-Archivistica t. IV, 1994, s. 72. 6 Cyt. za D. Fajnhauz, 1863. Litwa i Białoruś, Warszawa 1999, s. 237. 7 J. Piłsudski, Rok 1863, Warszawa 1989, s. 37; E. Maliszewska, Organizacja po wstania styczniowego, Warszawa 1925, s. 38 i n.; A. Szelągowski, Powstanie Komitetu Cen tralnego [w:] Polska, jej dzieje i kultura, t. III, Warszawa 1927; J. Grabiec [Dąbrowski], Rok 1863, Poznań 1923; T. Kędzierski, Powstanie styczniowe. Geneza. Przebieg. Udział praw ników, Warszawa 1936, s. 3 i n.; E. Przybyszewski, Pisma, Warszawa 1961, s. 102 i 159. 8 E. Przybyszewski, op. cit., s. 102. 10 S. Salmonowicz w recenzji pracy Z. Golby Rozwój władz Królestwa Polskiego w okresie powstania listopadowego, CPH, t. XXIV, z. 1, 1972; idem. List do Redakcji, CPH, t. XXXV, z. 1. 1983, s. 368-369; W. Sobociński, Dyskusja nad IV t. Historii państwa i pra wa Polski, CPH, XX, z. 2, 1968, s. 95-114; idem, Dzieje Rządów Narodowych Polski z lat 1863-1864 (rec. pracy F. Ramotowskiej), CPH, t. XXXIII, 1981, z. 2; idem, O władzy naczel nej w powstaniu styczniowym, Przegląd Historyczny, t. LXXII, 1981, z. 2. 11 S. Salmonowicz, Z. Golba, „Rozwój...", s. 249 (petitem). 12 Idem, List..., s. 369. 13 W. Sobociński, O władzy..., s. 289. Uwagi o polskim państwie podziemnym w latach 1863-1864 Ten ostatni, dając pierwszą marksistowską interpretację powsta nia, pisał: „Rozpoczęła się szeroka praca [...] z [...] myślą wytworzenia pod kierunkiem moralnego Rządu Narodowego, tajnego państwa polskiego, które podkopując stopniowo podstawy istnienia rządu najezdniczego, zmierzałoby konsekwentnie do zupełnego wyparcia go z kraju”8. W historiografii powojennej, mimo znacznego uszczerbku w źródłach, po wstały znaczące monografie dotyczące powstania styczniowego, takie jak Ste fana Kieniewicza Powstanie Styczniowe, Dawida Fajnhauza prace o Litwie w powstaniu czy swoista trylogia Franciszki Ramotowskiej9. Wszystkie one posługują się pojęciem polskiego państwa podziemnego, często pogłębionym ustrojowo, jednak bez interpretacji teoretyczno - prawnej. Dla tej ostatniej znaczenie miały uwagi Franciszki Ramotowskiej oraz Władysława Sobocińskiego i Stanisława Salmonowicza, wyartykułowane za równo podczas ich wieloletniej dyskusji jaka toczyła się na łamach CPH, na stępnie nad IV t. Historii państwa i prawa Polski (1848/63-1918), następ nie nad pracą Zdzisława Gołby dotyczącą władz narodowych w powstaniu li- UWAGI O POLSKIM PAŃSTWIE PODZIEMNYM W LATACH 1863-1864 233 stopadowym, oraz w dwóch recenzjach pracy F. Ramotowskiej, o Rządzie Narodowym lat 1863 - 1864, pióra W. Sobocińskiego10. stopadowym, oraz w dwóch recenzjach pracy F. Ramotowskiej, o Rządzie Narodowym lat 1863 - 1864, pióra W. Sobocińskiego10. We wspomnianej dyskusji S. Salmonowicz po raz pierwszy wyraźnie sfor mułował potrzebę badań nad państwem podziemnym w powstaniu stycznio wym, którą wówczas „poparli profesorowie J. Bardach i J. Mazurkiewicz”11. W Liście do Redakcji CPH z 1982 prof. S. Salmonowicz stwierdzał: „Polski historyk prawa nie może zbyt pochopnie, by nie rzec formalistycznie, wyrzu cać poza nawias, rozważań historyczno - prawnych tych form organizacyj nych narodu polskiego walczącego o odzyskanie (bądź kontynuację) bytu pań stwowego, które wykształciły się w XIX-XX w. i stanowią ważki element polskiej tradycji państwowej”12. W odpowiedzi na powyższe postulaty W. Sobociński jako pierwszy wy sunął argument o nieodpowiedniości posługiwania się terminem polskie pań stwo podziemne w odniesieniu do władz narodowych w okresie powstania styczniowego. Pisał on m.in. „Przyjmując, że problematyka powstańcza nale ży przede wszystkim do dziejów polityki, ideologii i walki zbrojnej, należy wyjść od ogólnego pojęcia ruchu [narodowego], podczas gdy państwo i pra wo mogą, ale nie muszą być produktem tego ruchu”13. Generalnie argumen tację W. Sobocińskiego można uszeregować w kilka punktów: 1. Organizacja Narodowa w powstaniu styczniowym nazywana jest pol skim państwem podziemnym w wyniku nieporozumienia, polegającego na przejęciu tej nazwy przez historiografię powojenną, z dziejów II wojny świa towej, jako że, nazwa ta nie pojawia się w żadnym dokumencie władz po wstańczych. 13 W. Sobociński, O władzy..., s. 289. Uwagi o polskim państwie podziemnym w latach 1863-1864 Nawet jeśli nazwa taka występuje (dekrety uwłaszczeniowe), to ich twórcy mieli na myśli państwo przyszłe, wywalczone w drodze powsta nia. 2. W organizacji tajnej, o nieznanym składzie osobowym organów naczel nych, nie mogły wykształcić się formy charakterystyczne dla państwa w po rozumieniu doktryny prawnej, zaś akty władz powstania miały raczej charak ter prawa wewnątrz organizacyjnego. p g yj g 3. Określenie państwo podziemne jest tożsame z pojęciem zorganizowa nej konspiracji, czyli Organizacji Narodowej i w odniesieniu do niej stanowi twór językowy nieco sztuczny. Wychodząc z marksistowskiego punktu wi dzenia na państwo jako aparat przymusu, poprzez który klasa panująca reali zuje swoją wolę, W. Sobociński wskazywał, że organizacja nie spełniała te- 3. Określenie państwo podziemne jest tożsame z pojęciem zorganizowa nej konspiracji, czyli Organizacji Narodowej i w odniesieniu do niej stanowi twór językowy nieco sztuczny. Wychodząc z marksistowskiego punktu wi dzenia na państwo jako aparat przymusu, poprzez który klasa panująca reali zuje swoją wolę, W. Sobociński wskazywał, że organizacja nie spełniała te- 234 Maksymilian Stanulewicz go kryterium, służąc odzyskaniu niepodległości, a nie realizacji zadań pań stwa. Gdyby przyjąć istnienie pod władzą RN jakiegoś państwa, zauważał W. Sobociński, to musiałoby ono rozciągać się na te wszystkie tereny, które pod legały jego władzy, a więc nie tylko Litwę, Białoruś i Ukrainę czy Inflanty, ale - w okresie dwuwładzy - również na terytoria państw zachodnich. go kryterium, służąc odzyskaniu niepodległości, a nie realizacji zadań pań stwa. Gdyby przyjąć istnienie pod władzą RN jakiegoś państwa, zauważał W. Sobociński, to musiałoby ono rozciągać się na te wszystkie tereny, które pod legały jego władzy, a więc nie tylko Litwę, Białoruś i Ukrainę czy Inflanty, ale - w okresie dwuwładzy - również na terytoria państw zachodnich. 4. Władze powstania styczniowego „kopiowały”, zdaniem W. Sobociń skiego, ustrój państwowy z myślą, że w przyszłości stanie się on podstawą ustroju niepodległego państwa, istnienie zaś Rządu Narodowego jest dowo dem na funkcjonowanie hierarchii władz, której nie należy utożsamiać z pań stwem, „gdyż władze tak lub inaczej nazywane są we wszystkich organiza cjach i związkach, nawet najbardziej prywatnych”. 5. Organizacja powstańcza była o tyle wyjątkowa, że korzystała z oficjal nego systemu administracji Królestwa Polskiego, jednak, sprawując skutecz nie swe funkcje, pozostała do końca konspiracją, bowiem jak pisał O. Awej- de słabość powstania kazała działać tajemnie, być spiskiem . 14 W. Sobociński, O władzy..., s. 291, 302-305; tenże, Dzieje rządów..., 130-131, 138-142 15 F. Ramotowska, Dziedzictwo ideowe Konstytucji 3 Maja w powstaniu styczniowym, „Mi scellanea Historico - Archivistica”, t. V, 1995, s. 74 i n., taże, Tajemne państwo polskie, s. 11 16 F. Ramotowska, Legitymizm władz polskich..., s. 64 i n., taże, Narodziny tajemnego państwa..., s. 7 i n. F. Ramotowska, Dziedzictwo ideowe Konstytucji 3 Maja w powstaniu styczniowym, „Mi nea Historico - Archivistica”, t. V, 1995, s. 74 i n., taże, Tajemne państwo polskie, s. 11 14 W. Sobociński, O władzy..., s. 291, 302-305; tenże, Dzieje rządów..., 130-131, 138-142 6 F. Ramotowska, Legitymizm władz polskich..., s. 64 i n., taże, Narodziny tajemnego wa..., s. 7 i n. Uwagi o polskim państwie podziemnym w latach 1863-1864 14 Przytoczone powyżej argumenty skłoniły Władysława Sobocińskiego do stwierdzenia, że pojęcie polskie państwo podziemne, zarówno w odniesieniu do powstania styczniowego jak i II wojny światowej, jest kategorią politycz ną nie prawną, a którą należy stasować z umiarem, na określenie silnej, ogól nopolskiej struktury spiskowej, połączonej z ruchem oporu w różnych for mach, przede wszystkim walki zbrojnej, w celu stworzenia przyszłej admini stracji państwowej i restytucji państwowości. Takie stwierdzenie, w odniesieniu do powstania styczniowego wywołało polemikę ze strony Franciszki Ramotowskiej, które dała wyraz w kolejnych swoich publikacjach. Za punkt wyjścia przyjęła ona pojęcie suwerenności narodu, mające swo je źródło w Konstytucji 3 maja15. To właśnie to pojęcie dało początek okre śleniu władz narodowych w powstaniu styczniowym, które sprawowały wła dzę nad samoorganizującym się społeczeństwem w sposób tajny, w formie zakonspirowanej struktury administracyjnej. Rząd Narodowy i jego struktury przeciwstawiały się władzy zaborczej, która była bojkotowana przez społe czeństwo. Jak wskazywała F. Ramotowska, faktu rozbiorów naród polski nie akceptował, traktując rządy zaborcze jako nielegalne i najezdnicze. Szczegól nie w okresie powstania styczniowego, gdy naród nie mógł się wypowiedzieć poprzez reprezentację narodową, wyrazem braku akceptacji dla porządku za borczego był czyn zbrojny i bojkot administracji rozbiorców, głównie jednak rosyjskiej16. F. Ramotowska pisała: „Przy całym [...] zróżnicowaniu postaw 235 UWAGI O POLSKIM PAŃSTWIE PODZIEMNYM W LATACH 1863-1864 [...] niwelowanym wspólną wolą odzyskania odzyskania niepodległości, na ród jako całość zaświadczył w dobie powstania styczniowego, że czuje się rzeczywistym suwerenem w swym kraju, a pobudzony silnym pragnieniem wolności [...] stał się jedynie prawowitym źródłem, władzy legalnej oraz jej działalności legislacyjnej, jedyną tejże władzy rękojmią oraz czynnikiem pań- stwowotwórczym, a ściślej państwowoodtwórczym”17. Z celami tak sformułowanymi wiązały się czynności Organizacji Narodo wej, która stanowiła konstrukcję wyzwalającego się państwa polskiego. Zda niem F. Ramotowskiej, na państwowość tę składały się następujące elemen ty: 1. naród zamieszkujący określone terytorium; 2. własny rząd i administra cja; 3. skarb i siły zbrojne; 4. służba zagraniczna. Zatem polskie państwo pod ziemne w powstaniu styczniowym to twór, „którego zespół cech [...] kwali fikował [...] do rangi państwowości, nadawał [...] charakter państwa, funkcjo nującego w specyficznych warunkach”18. Odmienne akcenty rozłożył, definiując państwo podziemne z lat 1863-1864 w t. III Historii państw i prawa Polski, Zdzisław Stankiewicz19. Podkreślając, że musiało być one budowane na zasadach konspiracji, Z. Stan kiewicz wskazywał, iż z punktu widzenia prawa międzynarodowego, Organi zacja Narodowa państwem nie była. 17 F. Ramotowska, Narodziny..., s. 10. 18 Ibidem, s. 5 19 Z. Stankiewicz, Polskie państwo podziemne w powstaniu styczniowym [w:] Historia państwa i prawa Polski, t. III, Od rozbiorów do uwłaszczenia, s. 431 i n. 20 Sytuacja w Europie była już i tak napięta z powodu wchodzącej w decydującą fazę woj ny secesyjnej w USA. Konfederatów (CSA) popierały (choć nieoficjalnie) zarówno Francja jak i Anglia, zaś Unię (USA) Rosja, obawiająca się konfliktu z wymienionymi państwami na tle sprawy polskiej. Spowodowało to naturalną zbieżność interesów Rządu Narodowego i rządu CSA na arenie międzynarodowej, a także nawiązanie pewnej współpracy między nimi. 21 Z. Stankiewicz, op. cit., s. 432-433. 21 Z. Stankiewicz, op. cit., s. 432-433. p p 20 Sytuacja w Europie była już i tak napięta z powodu wchodzącej w decydującą fazę woj ny secesyjnej w USA. Konfederatów (CSA) popierały (choć nieoficjalnie) zarówno Francja jak i Anglia, zaś Unię (USA) Rosja, obawiająca się konfliktu z wymienionymi państwami na tle sprawy polskiej. Spowodowało to naturalną zbieżność interesów Rządu Narodowego i rządu CSA na arenie międzynarodowej, a także nawiązanie pewnej współpracy między nimi. 17 F. Ramotowska, Narodziny..., s. 10. Z. Stankiewicz, Polskie państwo podziemne w powstaniu styczniowym [w:] Historia wa i prawa Polski, t. III, Od rozbiorów do uwłaszczenia, s. 431 i n. 21 Z. Stankiewicz, op. cit., s. 432-433. 22 S. Salmonowicz, Polskie Państwo Podziemne. Z dziejów walki cywilnej 1939-1945, Warszawa 1994. s. 18-19. 23 S. Salmonowicz, M. Ney-Krwawicz, G. Górski, Polskie Państwo Podziemne, Warszawa 1999. s. 18. 24 G. Górski, Administracja Polski Podziemnej w latach 1939-1945, Toruń, 1995, s. VI-VIII. 25 S. Salmonowicz, M. Ney-Krwawicz, G. Górski, op. cit., s. 17-18. Uwagi o polskim państwie podziemnym w latach 1863-1864 Rząd Narodowy nigdy nie mógł liczyć na to, co stanowiło idéw fix jego dyplomacji, a mianowicie na uznanie po wstańców za stronę wojującą. Stanowiło by to bowiem naruszenie zasad Kon gresu Wiedeńskiego, tworząc preludium do nowej wojny europejskiej20. Po siadając atrybuty państwa (rząd w stolicy, siły zbrojne, sądownictwo, admi nistracja terenowa, prasa), pwostańcza Organizacja Narodowa nie mogła roz winąć tych form działalności ani osiągnąć podstawowego celu, czyli odzyska nia niepodległości. Państwo podziemne w powstaniu styczniowym było, zda niem Z. Stankiewicza, „organizacją narodu walczącego o niepodległość i prze kształcenia polityczne; do wykrystalizowania się ostatecznego kształtu ustro jowego mogło dojść jedynie w razie zwycięstwa powstania”21. Stąd też wielokrotnie podkreślana w literaturze odmienność dwóch pol skich państw podziemnych, tego z powstania styczniowego i tego z lat 1939-1945. Ta ostatnia konstrukcja ustrojowa może mieć znaczenie dla roz ważań nad Organizacją Narodową w powstaniu styczniowym jako pewien mo del teoretyczny. W pracy pt. Polskie Państwo Podziemne. Z dziejów walki 236 Maksymilian Stanulewicz cywilnej 1939-1945. S. Salmonowicz pisał iż państwo podziemne to: „zespół struktur prawnopaństwowych, organizacyjnych i obywatelskich, które miały zapewnić konstytucyjną ciągłość działania państwa polskiego na jego wła snym terytorium”22. Definicja powyższa, akcentująca legalizm takiej kon strukcji i ciągłość bytu państwowego na okupowanym terytorium państwa, nie odpowiada zbytnio realiom powstania styczniowego, wobec tego nie może być traktowana jako ujęcie modelowe. Jednak w kolejnej swojej pracy S. Sal monowicz stwierdził, że nie ulega wątpliwości, iż organizatorzy Polskiego Państwa Podziemnego sięgali generalnie także do tradycji narodowej, do wzorów struktur Rządu Narodowego okresu powstania styczniowego23. Inną definicję państwa podziemnego, nie tyle legalną co opisową, zawarł w swojej pracy pt. Administracja Polski Podziemnej w latach 1939-1945 Grzegorz Górski24. Pisał on o państwie podziemnym z lat 1939-1045 nastę pująco: „Było to państwo, które dysponowało własnym wykonawczym aparatem administracyjnym we wszystkich dziedzinach życia. Aparatem przygotowa nym do natychmiastowego przejęcia władzy w okresie planowanego powsta nia zbrojnego i wyzwolenia kraju spod okupacji. Było to państwo, posiada jące realną siłę zbrojną i dysponujące tzw. przymusem państwowym, pozwa lającym egzekwować w sposób fizyczny jego wolę. Było to państwo, w któ rym funkcjonował wymiar sprawiedliwości i którego orzeczenia były z całą bezwzględnością wykonywane. 26 F. Ramotowska, Rząd Narodowy Polski 1863-1864, Warszawa 1978, s. 351 i n.; ta- że, Legitymizm władz polskich w okresach powstań i ruchów wolnościowych (1830-1863), „Mi scellanea Historico - Archivistica”, t. IV, 1994, s. 58 i n. 27 Prasa tajna z lat 1861-1865, cz. 1, Wroclaw 1966, s. 200 i n.; ks. K. Mikoszewski, Pamiętniki moje. Opracowanie, przedmowa i przypisy R. Bender, Warszawa 1987, s. 55 i n.; S. Kieniewicz, Powstanie styczniowe, Warszawa 1972, s. 310-320. 28 Prasa tajna, cz. 1, s. 202. Uwagi o polskim państwie podziemnym w latach 1863-1864 Było to zatem państwo, które realnie i istot nie wpływało na życie dużej częci narodu.” Jak wynika z przytoczonych powyżej prób definicji, samo pojęcie pań stwo podziemne weszło do nauk historycznoprawnych jako pojęcie ustrojowe w odniesieniu do lat 1939-1945, wyrażając fakt, że „obok jawnych struktur polskiej administracji działających czasowo poza krajem, istniały w konspi racji na terytorium kraju [...] struktury państwa polskiego, które wykonywa ły wiele funkcji państowych i opierały się na przepisach polskiego prawa kon stytucyjnego, administracyjnego, karnego itd.”25 Tymczasem charakter prawny Organizacji Narodowej lat 1862-1864 mo że, wobec braku elementów legalizmu i podstaw konstytucyjnych swojej dzia łalności, wzbudzać wiele kontrowersji. Jeśli przyjąć definicję państwa jako tworu politycznego, obejmującego swym działaniem pewien obszar i ogól członków społeczeństwa zamieszku jących na tym obszarze oraz spełniającego właściwe sobie cele za pomocą UWAGI O POLSKIM PAŃSTWIE PODZIEMNYM W LATACH 1863-1864 237 środków materialnych i instrumentów zarządzania, to można stwierdzić, że organizacja powstańcza spełnia wszystkie kryteria uznania jej za państwo. Składają się bowiem na nią, z jednej strony tkanka państwa w postaci orga nów władzy naczelnej i terenowej, a z drugiej strony właśnie środki material ne, czyli np. skarbowość powstańcza. To wszystko zaś działało na określo nym terytorium, zamieszkałym przez społeczeństwo poddające się - często dobrowolnie - władzy organów powstańczych, mimo, że dysponowały one środkami przymusu. Nie przesądzając sprawy ostatecznie, należy wskazać, iż pewne znaczenie ma tutaj fakt rzeczywistego sprawowania władzy przez RN, który skutecznie kierował walką powstańczą, będąc przy tym uznawany przez większość społeczeństwa26. Można stwierdzić, jak czynił to W. Sobociński, że państwo podziemne w omawianym okresie było raczej formą spisku niż kategorią prawnoustrojową. Należy jednak pamiętać o tym, iż organizacja taj na musi działać poza legalnymi strukturami, tym bardziej, jeśli jest alterna tywna dla istniejącego porządku. Dlatego też wydaje się, że zbyt daleko idą ce są argumenty wskazujące na ścisłe powiązanie państwowości z prawem pozytywnym jako kryterium uznania za państwo. Dobitnym przykładem jest tutaj, zasłużone w propagowaniu idei i programu Organizacji Narodowej pi smo, redagowane przez ks. Karola Mikoszewskiego (ks. „Syktus”), pt. Glos Kapłana Polskiego, które pozostawało w jawnej opozycji do abp Zygmunta Felińskiego27. Mikoszewski, członek KCN (od XII 1862 r.), wybitny kazno dzieja kościoła św. Aleksandra, nadał temu organowi prasowemu, charakter tuby propagandowej Komitetu, poprzez którą objaśniał najważniejsze zasady działania ruchu narodowego. W pierwszym, nie datowanym numerze z czerw ca 1862 r. Uwagi o polskim państwie podziemnym w latach 1863-1864 znajdujemy następujący, wiele mówiący passus: Gdy prawo ma ludźmi kierować do osiągnięcia powszechnej pomyślności, nie może być ustanawiane przez kogokolwiek tylko przez samą społeczność lub też tego, kogo społeczność do tego upoważni. Wybór panującego bezpośrednio nale ży do narodu i nikt oprócz narodu [...] nie ma prawa przywłaszczać sobie władzy nad innymi28. Źródłem zatem władzy w państwie jest tylko naród, który mając prawo wybierania sobie władzy, ma prawo sądzić władzę, zmie niać ją [i] [...] pozbyć się jej. Oznaczało to przyznanie społeczeństwu prawa oporu, które jakkolwiek [...] nie jest pisane, lecz wrodzone, złożone w ser cach naszych. Treść tych wywodów służyła przede wszystkim wskazaniu na nielegalność rządów zaborczych w Polsce, podkreśleniu prawa narodu pol skiego do wyłonienia władz narodowych i przelania na nie kompetencji wład- środków materialnych i instrumentów zarządzania, to można stwierdzić, że organizacja powstańcza spełnia wszystkie kryteria uznania jej za państwo. Składają się bowiem na nią, z jednej strony tkanka państwa w postaci orga nów władzy naczelnej i terenowej, a z drugiej strony właśnie środki material ne, czyli np. skarbowość powstańcza. To wszystko zaś działało na określo nym terytorium, zamieszkałym przez społeczeństwo poddające się - często dobrowolnie - władzy organów powstańczych, mimo, że dysponowały one środkami przymusu. Nie przesądzając sprawy ostatecznie, należy wskazać, iż pewne znaczenie ma tutaj fakt rzeczywistego sprawowania władzy przez RN, który skutecznie kierował walką powstańczą, będąc przy tym uznawany przez większość społeczeństwa26. Można stwierdzić, jak czynił to W. Sobociński, że państwo podziemne w omawianym okresie było raczej formą spisku niż kategorią prawnoustrojową. Należy jednak pamiętać o tym, iż organizacja taj na musi działać poza legalnymi strukturami, tym bardziej, jeśli jest alterna tywna dla istniejącego porządku. Dlatego też wydaje się, że zbyt daleko idą ce są argumenty wskazujące na ścisłe powiązanie państwowości z prawem pozytywnym jako kryterium uznania za państwo. Dobitnym przykładem jest tutaj, zasłużone w propagowaniu idei i programu Organizacji Narodowej pi smo, redagowane przez ks. Karola Mikoszewskiego (ks. „Syktus”), pt. Glos Kapłana Polskiego, które pozostawało w jawnej opozycji do abp Zygmunta Felińskiego27. Mikoszewski, członek KCN (od XII 1862 r.), wybitny kazno dzieja kościoła św. Aleksandra, nadał temu organowi prasowemu, charakter tuby propagandowej Komitetu, poprzez którą objaśniał najważniejsze zasady działania ruchu narodowego. W pierwszym, nie datowanym numerze z czerw ca 1862 r. znajdujemy następujący, wiele mówiący passus: Gdy prawo ma ludźmi kierować do osiągnięcia powszechnej pomyślności, nie może być ustanawiane przez kogokolwiek tylko przez samą społeczność lub też tego, kogo społeczność do tego upoważni. 29 Jak pisał bowiem Zygmunt Ziembiński: „... jeśli się szuka podstawy dla uznawania nor my za prawnie obowiązującą, to przede wszystkim wskazuje się na to, że została ona ustano wiona na podstawie normy udzielającej do tego kompetencji... normy kompetencyjnej sformuło wanej w przepisach konstytucji. Wszakże jeśli jest to konstytucja suwerenna uchwalona, a nie oktrojowana, to dla uznania mocy obowiązującej takiego aktu należy odwoływać się do innych czynników... Można odwołać się do tak czy inaczej pojmowanej efektywności społecznej danej konstytucji, czy też do jakiegoś, całkowicie swoistego rodzaju kompetencji ideologicznej.” [w:] Z. Ziembiński, O zawiłościach z pojmowaniem kompetencji, PiP, nr 4, 1991. p j p j 30 S. Kieniewicz, Powstanie styczniowe, Warszawa 1972; idem. Sprawa włościańska w powstaniu styczniowym, Wrocław 1953; tenże, Między ugodą a rewolucją, Warszawa 1961; F. Ramotowska, Narodziny tajemnego państwa polskiego, Warszawa 1978; taże Tajemne państwo polskie 1863-1864, Warszawa 1999; taże Jak powstały władze narodowe w 1863 r., „Miscellanea Historico - Archivistica”, t. V, 1995, s. 153 i n.,: eadem, Legitymizm władz pol skich...: Powstanie styczniowe 1863-1864. Wrzenie. Bój. Europa, Wizje., pod red. S. Kalemb- ki, Warszawa 1990; D. Fajnhauz, 1863 Litwa i Białoruś, Warszawa 1999; E. Halicz. Kwe stia chłopska w Królestwie w dobie powstania styczniowego, Warszawa 1955; I. Koberdo- wa, Polityka czartoryszczyzny w okresie powstania styczniowego, Warszawa 1957; J. Nowak, I. Szelembaum, Warszawscy Czerwoni w przededniu powstania styczniowego, Rocz. War szawski, R. VI, 1967. 31 F. Ramotowska, Dziedzictwo ideowe Konstytucji 3 Maja w Powstaniu Styczniowym, „Miscellanea Historico - Archivistica”, t. V, 1995, s. 67 i n., eadem, Legitymizm władz pol skich..., s. 66 i n., taże, Jak powstawały władze..., s. 164-168; Z. Stankiewicz, Polskie pań stwo podziemne, s. 432-433. 32 Por. np. odezwy KCN i RN: do Żydów (9 IX 1862 r.), Litwinów (29 I 1863 r.), Rusi nów (7 II 1863 r.); do Niemców zamieszkałych w Polsce (26 III 1863 r.) [w:] Dokumenty KCN i RN, pod red. S. Kieniewicza i E. Halicza, Wrocław 1968, s. 21, 43, 44 i 72. 33 J.K. Janowski, Pamiętniki o powstaniu styczniowym, t. I-III, Lwów 1923 - Warsza wa 1931; L. Mierosławski, Dokumenta do dziejów organizacji jedneralnej powstania naro dowego w latach 1863 i 1864, Paryż, 1864; A. Giller, Historia powstania narodu polskiego w 1863 i 1864 r., t. I-IV, Paryż 1864; W. Przyborowski, Historia dwóch lat 1861-1862, t. I-V, Kraków 1892-1893; idem. Dzieje 1864 roku przez autora „Dwóch lat", t. I-II, Kraków 1897-1898; idem, Ostatnie chwile powstania styczniowego, t. I-IV, Poznań, 1887-1889; A. Szelągowski, Powstanie KCN [w:] Polska jej dzieje i kultura, t. III, Warszawa 1927; F. Ra- motowska, Rząd Narodowy Polski 1863-1864, Warszawa 1978; eadem, Narodziny tajemne go państwa polskiego, s. 149 i n.; idem. Tajemne państwo polskie, s. 15 i n.; S. Kieniewicz, Powstanie styczniowe, s. 257 i n.; idem. Geneza KCN, Zeszyty Naukowe UJ, Prace Hist., 1969, z. 26; J. Nowak, I. Szelembaum, Warszawscy Czerwoni w przededniu powstania stycznio wego, Rocz. Warszaw., R. VI, 1967. Uwagi o polskim państwie podziemnym w latach 1863-1864 Wybór panującego bezpośrednio nale ży do narodu i nikt oprócz narodu [...] nie ma prawa przywłaszczać sobie władzy nad innymi28. Źródłem zatem władzy w państwie jest tylko naród, który mając prawo wybierania sobie władzy, ma prawo sądzić władzę, zmie niać ją [i] [...] pozbyć się jej. Oznaczało to przyznanie społeczeństwu prawa oporu, które jakkolwiek [...] nie jest pisane, lecz wrodzone, złożone w ser cach naszych. Treść tych wywodów służyła przede wszystkim wskazaniu na nielegalność rządów zaborczych w Polsce, podkreśleniu prawa narodu pol skiego do wyłonienia władz narodowych i przelania na nie kompetencji wład- 238 Maksymilian Stanulewicz czych, wyrażała prawo do niepodległego i podmiotowego bytu państwa i na rodu polskiego. Tutaj zatem należy szukać uzasadnienia kompetencji prawo dawczych, w jakie był wyposażony Komitet Centralny, a następnie Rząd Na rodowy. Jest to ze wszech miar uzasadnienie akcjologiczne, odwołujące się do systemu ocen, a nie do normy w rozumieniu prawno-pozytywnym29. Założenia ustrojowe jakie przyświecały twórcom Organizacji Narodowej, można rozpatrywać na dwócj płaszczyznach: 1. ideologicznej, rozumianej ja ko koncepcje (myśli) polityczne, na której opierało państwo podziemne swój byt; 2. instrumentów zarządzania, a więc struktury administracji powstańczej, składu, kompetencji oraz wzajemnych relacji jej organów. Jeśli chodzi o sfe rę ideologiczną, to należy pamiętać, że myśl ustrojowa powstania stycznio wego kształtowała się w ogniu nieustannych walk politycznych pomiędzy stronnictwami Białych i Czerwonych. Spięcia te zawsze dotyczyły kwestii ak tualnych, takich jak sposób przeprowadzenia uwłaszczenia, charakter powsta nia zbrojnego (rewolucja ludowa czy demonstracja zbrojna) czy kierunki po lityki zagranicznej RN30. Spór o to, jaka ma być Polska, rozumiana jako państwo w granicach sprzed 1772 r., pozostawiono do czasu odzyskania peł nej niepodległości. Praktycznie istniała zgoda co do tego, że przyszłe pań stwo polskie ma być republiką, a nie monarchią. Myśl monarchistyczna była wtedy w społeczeństwie polskim niezbyt popularna, szczególnie po śmierci Adama Jerzego ks. Czartoryskiego w 1861 r., co świadczy o wielkich prze mianach mentalnych i strukturalnych w narodzie od czasu ostatniego powsta nia31. Samo państwo podziemne miało charakter demokratyczny, opierając się na szerokiej bazie społecznej. O jego istnieniu decydował konsensus sił UWAGI O POLSKIM PAŃSTWIE PODZIEMNYM W LATACH 1863-1864 239 społecznych, a idea niepodległości została tu sprzęgnięta z zasadą wolności i równości obywatelskiej, bez względu na przynależność narodową, pocho dzenie społeczne i wyznawaną religię32. 34 Sytuację komplikuje fakt, że w KCN i rządzie Majewskiego (VI-VIII 1863 r.) istniały komisje obok wydziałów. W przypadku KCN była to Komisja Interesów Duchownych i Opie ki, a przypadku rządu Majewskiego Komisja Kwalifikacyjna. Wydaje się, że w tych przypad kach mamy do czynienia z zespołem osób, powołanych przez organ rządowy do wykonania ści śle określonych zadań (w pierwszym chodziło o kontakty z duchowieństwem, w drugim o przy gotowanie projektów aktów prawnych). Liczba wydziałów i komisji była różna i tworzono je w zależności od zakresu spraw, pozostających do załatwienia. Komórki resortowe obejmowały zadania z zakresu: policji, stosunków zagranicznych, skarbu, wojny i prasy. W początkach po wstania szczególnie skomplikowana była kwestia Warszawy jako stolicy państwa podziemnego. Obok Wydziału Miejskiego, który zajmował się sprawami organizacji powstańczej w Warszawie, w ramach KCN istniał jeszcze Wydział Interesów m. st. Warszawy. Ten ostatni był jednym z pię ciu wydziałów na jakie dzielił się KCN, tymczasem Wydział Miejski, był instytucją kontrolną w stosunku do organizacji warszawskiej i nie wchodził w skład KCN, choć mu podlegał, tym samym podlegając, przynajmniej teoretycznie, Wydziałowi Interesów. Ten jednak nie rozwinął szerszej działalności, za to Wydział Miejski realnie sprawował kontrolę nad miastem. 35 Istotne miejsce w dziajach Organizacji Narodowej zajmowały dyktatury. Zauważyć nale ży, że obie miały charakter krótkotrwały i głównie wojskowy. Dyktatura oznaczała skupienie władzy w jednym ręku w momentach najtrudniejszych. Dlatego też zamierzano podporządkować dyktatorowi rządy cywilne. Zamiary te pozostały jednak w sferze projektów. Idea jednoosobo wego kierownictwa powstania będzie pojawiać się, bądź, w przypadku przesileń politycznych (Langiewicz, Mierosławski) bądź też w okresie końcowym powstania, kiedy okoliczności fak tycznie uniemożliwiły funkcjonowanie rządu kolegialnego (nieformalna dyktatura Traugutta); F. Ramotowska, Rząd Narodowy Polski, s. 356 i n.; E. Halicz, U źródeł dyktatury Traugut- Uwagi o polskim państwie podziemnym w latach 1863-1864 p y ą g ę U podstaw rozwoju administracji powstańczej i jej struktury legły zasad nicze pytania: decentralizacja czy centralizacja, jawność czy tajność, wybór czy nominacja, organizacja cywlna czy wojskowa, wreszcie oparcie się na strukturach wiejskich czy miejskich. Twórcy tajemnego państwa polskiego w latach 1863-1864, często wybierali metodę pośrednią, dyktowaną warun kami obiektywnymi. Z jednej strony było to dążenie do centralizacji i pod porządkowania organizacji terenowej (województwa, miasta wojewódzkie) bezpośrednio Rządowi Narodowemu, z drugiej zaś próby wprowadzenia w ży cie pewnych form decentralizacji na poziomie województwa i powierzenia w nich pełni władzy np. komisarzom pełnomocnym. Co więcej, w okresie po wstania współistniały dwa piony administracji - cywilny i wojskowy, nieza leżnie od siebie, choć początkowo administracja cywilna została podporząd kowana wojskowej. Rząd Narodowy pozostał tajnym, a urzędnicy pochodzi li z nominacji a nie z wyboru. j y Tak ukształtowana administracja znajdowała oparcie zarówno na wsi jak i w mieście, co spowoduje, iż inaczej została uregulowana struktura municy palna (szczególnie Organizacji Warszawskiej) a inaczej organizacja gminna. Skłania to do wniosku, że kolejne ekipy rządzące organizowały swą pracę od powiednio do sytuacji politycznej i wojskowej powstania, założeń programo wych, umiejętności własnych oraz warunków działania. Na czele struktury administracyjnej powstania stał organ o charakterze ko legialnym, posiadający nieograniconą władzę, mający kompetencje tak w za kresie wojskowym jak i cywilnym33. Bez względu na jego nazwę, zakres atry- bucji był zgodny z ideą centralizmu, tj. skupienia władzy w jednym organie. Nosił on różne nazwy: Komitet Centralny Narodowy, Tymczasowy Rząd Na rodowy, Rząd Narodowy. Decyzje w jego łonie podejmowano większością głosów, zaś władza była sprawowana przez szefów komórek resortowych, bę- 240 Maksymilian Stanulewicz dących zarazem członkami kolegium rządowego z tytułami dyrektorów. Ma my zatem tutaj przykład rządu w którym administracja należała do szefów komórek resortowych, podległych kolegium rządowemu. Dopiero za Trau gutta ustali się wewnętrzna struktura rządu, ze stanowiskiemprezesa współ pracującego, na zasadach nadrzędności z szefami resortów. Centralne organy zarządu resortowego państwa podziemnego nosiły nazwy: wydziałów (w KCN i TRN do kwietnia 1863 r. oraz w RN od maja 1863 r. do kwiet nia 1864 r.) i komisji (kwiecień 1863). Chwiejna ówczesna terminologia nie widziała różnicy pomiędzy kolegialnym organem administracji państwowej, zbliżonym do ministerstwa, jakim była komisja, a wydziałem wyodrębionym ze względu na zakres działania z pewnej całości. Ta ustawiczna zmiana na zewnictwa (wydział, komisja, znów wydział) motywowana była zmianą rela cji wewnątrz Rządu34. 34 Sytuację komplikuje fakt, że w KCN i rządzie Majewskiego (VI-VIII 1863 r.) istniały komisje obok wydziałów. W przypadku KCN była to Komisja Interesów Duchownych i Opie ki, a przypadku rządu Majewskiego Komisja Kwalifikacyjna. Wydaje się, że w tych przypad kach mamy do czynienia z zespołem osób, powołanych przez organ rządowy do wykonania ści śle określonych zadań (w pierwszym chodziło o kontakty z duchowieństwem, w drugim o przy gotowanie projektów aktów prawnych). Liczba wydziałów i komisji była różna i tworzono je w zależności od zakresu spraw, pozostających do załatwienia. Komórki resortowe obejmowały zadania z zakresu: policji, stosunków zagranicznych, skarbu, wojny i prasy. W początkach po wstania szczególnie skomplikowana była kwestia Warszawy jako stolicy państwa podziemnego. Obok Wydziału Miejskiego, który zajmował się sprawami organizacji powstańczej w Warszawie, w ramach KCN istniał jeszcze Wydział Interesów m. st. Warszawy. Ten ostatni był jednym z pię ciu wydziałów na jakie dzielił się KCN, tymczasem Wydział Miejski, był instytucją kontrolną w stosunku do organizacji warszawskiej i nie wchodził w skład KCN, choć mu podlegał, tym samym podlegając, przynajmniej teoretycznie, Wydziałowi Interesów. Ten jednak nie rozwinął szerszej działalności, za to Wydział Miejski realnie sprawował kontrolę nad miastem. 35 Istotne miejsce w dziajach Organizacji Narodowej zajmowały dyktatury. Zauważyć nale ży, że obie miały charakter krótkotrwały i głównie wojskowy. Dyktatura oznaczała skupienie władzy w jednym ręku w momentach najtrudniejszych. Dlatego też zamierzano podporządkować dyktatorowi rządy cywilne. Zamiary te pozostały jednak w sferze projektów. Idea jednoosobo wego kierownictwa powstania będzie pojawiać się, bądź, w przypadku przesileń politycznych (Langiewicz, Mierosławski) bądź też w okresie końcowym powstania, kiedy okoliczności fak tycznie uniemożliwiły funkcjonowanie rządu kolegialnego (nieformalna dyktatura Traugutta); F. Ramotowska, Rząd Narodowy Polski, s. 356 i n.; E. Halicz, U źródeł dyktatury Traugut- Uwagi o polskim państwie podziemnym w latach 1863-1864 Na przełomie maja i kwietnia 1863 r., w koalicyjnym rządzie Agatona Gillera, ostatecznie ustaliła się nazwa wydział, których kom petencje były poszerzone a tryb działania bardiej autonomiczny. Wcześniej bowiem ani wydziały w KCN ani komisje w TRN nie mogły podejmować samodzielnie postanowień, gdyż decyzje scentralizowane były w samym rzą dzie i firmowane jego aktualną nazwą. Rozszerzenie zakresu spraw wymaga jących pilnych rozstrzygnięc ze strony władzy centralnej, spowodowało przy jęcie przez RN decyzji o pewnym usamodzielnieniu się wydziałów, czyli de centralizacji wewnętrznej. Postanowienia zapadły w wydziale mogły być prze zeń odtąd firmowane nazwą wydziału, jednak podlegały kontroli kolegium rządowego. Taki model wewnętrznej organizacji rządu utrzymał się do koń ca powstania35. dących zarazem członkami kolegium rządowego z tytułami dyrektorów. Ma my zatem tutaj przykład rządu w którym administracja należała do szefów komórek resortowych, podległych kolegium rządowemu. Dopiero za Trau gutta ustali się wewnętrzna struktura rządu, ze stanowiskiemprezesa współ pracującego, na zasadach nadrzędności z szefami resortów. Centralne organy zarządu resortowego państwa podziemnego nosiły nazwy: wydziałów (w KCN i TRN do kwietnia 1863 r. oraz w RN od maja 1863 r. do kwiet nia 1864 r.) i komisji (kwiecień 1863). Chwiejna ówczesna terminologia nie widziała różnicy pomiędzy kolegialnym organem administracji państwowej, zbliżonym do ministerstwa, jakim była komisja, a wydziałem wyodrębionym ze względu na zakres działania z pewnej całości. Ta ustawiczna zmiana na zewnictwa (wydział, komisja, znów wydział) motywowana była zmianą rela cji wewnątrz Rządu34. Na przełomie maja i kwietnia 1863 r., w koalicyjnym rządzie Agatona Gillera, ostatecznie ustaliła się nazwa wydział, których kom petencje były poszerzone a tryb działania bardiej autonomiczny. Wcześniej bowiem ani wydziały w KCN ani komisje w TRN nie mogły podejmować samodzielnie postanowień, gdyż decyzje scentralizowane były w samym rzą dzie i firmowane jego aktualną nazwą. Rozszerzenie zakresu spraw wymaga jących pilnych rozstrzygnięc ze strony władzy centralnej, spowodowało przy jęcie przez RN decyzji o pewnym usamodzielnieniu się wydziałów, czyli de centralizacji wewnętrznej. Postanowienia zapadły w wydziale mogły być prze zeń odtąd firmowane nazwą wydziału, jednak podlegały kontroli kolegium rządowego. Taki model wewnętrznej organizacji rządu utrzymał się do koń ca powstania35. 241 UWAGI O POLSKIM PAŃSTWIE PODZIEMNYM W LATACH 1863-1864 Aspirując do objęcia swoją władzą wszystkich ziem przedrozbiorowej Rzeczypospolitej, Rząd Narodowy sprawował swoją władzę na terenach Li twy, Rusi oraz Galicji i zaboru pruskiego, bądź przez autonomiczne komite ty i wydziały wykonawcze tych prowincji (jak w przypadku Litwy, Rusi i za boru pruskiego, a od maja do listopada 1863 r. 37 W. Czartoryski, Pamiętniki, Warszawa 1960; J. Feldman, Mocarstwa wobec po wstania styczniowego, Kraków 1929; A. Lewak, Idee przewodnie polskiej polityki zagranicz nej 1863-1864, t. I-II, Warszawa 1937-1963, S. Kieniewicz, Prace nad historią dyploma cji okresu powstania styczniowego, Kwartalnik Historyczny, LXVI, 1959, z. 3. ta. Z dziejów wojny i polityki. Księga pamiątkowa ku uczczeniu 7O-tej rocznicy urodzin prof. Janusza Wolińskiego, Warszawa 1964; M. Dubiecki, Romuald Traugutt, Warszawa 1924; J. Jarzębowski, Traugutt, Warszawa 1938; B. Merwin, Pisma wojskowe Mariana Langiewicza, Warszawa 1920; T. Szarota, Dyktatura Langiewicza a przystąpienie Bia łych do powstania, Przegląd Historyczny, t. LIV, 1963; M. Żychowski, Ludwik Mierosław ski 1818-1878, Warszawa 1963; H. Rzadkowska, Marian Langiewicz, Warszawa 1967; M. Langiewicz, Pisma, pod red. H. Rzadkowskiej, Kraków 1971. 36 D. Fajnhauz, Niektóre zagadnienia powstania styczniowego na Kowieńszczyźnie, Kwartalnik Historyczny LXIX, 1962, nr 4; tenże, Litwa u schyłku powstania styczniowego, Te ki Historyczne, t. XIX, 1988-1989; idem, 1863 Litwa i Białoruś, s. 113 i n.; W. Kordowicz. Konstanty Kalinowski, Warszawa 1959; P. Łossowski, Z. Młynarski, Rosjanie, Białoru sini i Ukraińcy w powstaniu styczniowym, Wrocław 1959; R. Bender, O. Rafał Kalinowski. Powstaniec i zakonnik, Warszawa 1977; J. Gieysztor, Pamiętniki z lat 1857-1865, Kraków 1987; J. Łukaszewski, Zabór pruski w czasie powstania styczniowego 1863-1864, Drezno - Jassy 1870; A. Guttry, Pamiętniki, Poznań 1891-1894; Z. Grot, Rok 1863 w zaborze pru skim, Poznań 1963; F.H. Gentzen, Zabór pruski w powstaniu styczniowym, Kwartalnik Hi storyczny, t. LXII, z. 2, 1955, idem, Grosspolen in Januaraufstand 1863/1864, Berlin 1958; F. Rowita - Gawroński, Rok 1863 na Rusi, t. I-II, Lwów 1903; J. Stella - Sawic ki, Galicja w powstaniu styczniowym, Lwów 1909, S. Kieniewicz, Udział Galicji w po wstaniu styczniowym, Przegląd Historyczny, t. XXXIV, 1937/1938; W. Tokarz, Kraków w po czątkach powstania styczniowego i wyprawa na Miechów, t. I-II, Kraków 1913; Kraków w po wstaniu styczniowym, Kraków 1968; O. Beiersdorf, Walka polityczna Czerwonych i Bia łych na terenie Krakowa w okresie powstania styczniowego, „Małopolskie Studia Historycz ne”, 1962, nr 3/4. ta. Z dziejów wojny i polityki. Księga pamiątkowa ku uczczeniu 7O-tej rocznicy urodzin prof. Janusza Wolińskiego, Warszawa 1964; M. Dubiecki, Romuald Traugutt, Warszawa 1924; J. Jarzębowski, Traugutt, Warszawa 1938; B. Merwin, Pisma wojskowe Mariana Langiewicza, Warszawa 1920; T. Szarota, Dyktatura Langiewicza a przystąpienie Bia łych do powstania, Przegląd Historyczny, t. LIV, 1963; M. Żychowski, Ludwik Mierosław ski 1818-1878, Warszawa 1963; H. Rzadkowska, Marian Langiewicz, Warszawa 1967; M. Langiewicz, Pisma, pod red. H. Rzadkowskiej, Kraków 1971. 36 D. Fajnhauz, Niektóre zagadnienia powstania styczniowego na Kowieńszczyźnie, Kwartalnik Historyczny LXIX, 1962, nr 4; tenże, Litwa u schyłku powstania styczniowego, Te ki Historyczne, t. XIX, 1988-1989; idem, 1863 Litwa i Białoruś, s. 113 i n.; W. Kordowicz. Konstanty Kalinowski, Warszawa 1959; P. Łossowski, Z. Młynarski, Rosjanie, Białoru sini i Ukraińcy w powstaniu styczniowym, Wrocław 1959; R. Bender, O. Rafał Kalinowski. Powstaniec i zakonnik, Warszawa 1977; J. Gieysztor, Pamiętniki z lat 1857-1865, Kraków 1987; J. Łukaszewski, Zabór pruski w czasie powstania styczniowego 1863-1864, Drezno - Jassy 1870; A. Guttry, Pamiętniki, Poznań 1891-1894; Z. Grot, Rok 1863 w zaborze pru skim, Poznań 1963; F.H. Gentzen, Zabór pruski w powstaniu styczniowym, Kwartalnik Hi storyczny, t. LXII, z. 2, 1955, idem, Grosspolen in Januaraufstand 1863/1864, Berlin 1958; F. Rowita - Gawroński, Rok 1863 na Rusi, t. I-II, Lwów 1903; J. Stella - Sawic ki, Galicja w powstaniu styczniowym, Lwów 1909, S. Kieniewicz, Udział Galicji w po wstaniu styczniowym, Przegląd Historyczny, t. XXXIV, 1937/1938; W. Tokarz, Kraków w po czątkach powstania styczniowego i wyprawa na Miechów, t. I-II, Kraków 1913; Kraków w po wstaniu styczniowym, Kraków 1968; O. Beiersdorf, Walka polityczna Czerwonych i Bia łych na terenie Krakowa w okresie powstania styczniowego, „Małopolskie Studia Historycz ne”, 1962, nr 3/4. 38 E. Maliszewski, Organizacja powstania styczniowego, Warszawa 1925; F. Koper- nicki, Pamiętniki z powstania w województwie kaliskim z 1863 r., Warszawa 1958; H. Ja błoński, Struktura cywilnej organizacji prowincjonalnej w Królestwie Polskim, Przegląd Hi storyczny, t. XXXIV, 1937/1938; E. Halicz, Rys ogólny działań rządowych w Królestwie Pol skim za rok 1863, Studia Historyczne, nr 15, 1967; J. Tomczyk, Organizacja cywilno-wojsko- wa powstania styczniowego na Podlasiu, „Rocznik Lubelski”, R. VI, 1963; S. Chankowski, Powstanie styczniowe w Augustowskiem, Warszawa 1972, W. Dąbkowski, Powstanie stycz niowe w Puszczy Kozienieckiej, Warszawa 1974; W. Saletra, Kilka uwag nad taktyką i admi nistracją powstania styczniowego w świetle memoriałów pik Ludwika Zwierzdowskiego, Studia Kieleckie, R. 17, 1990-1991; R. Szwed, Powstanie styczniowe w Zagłębiu Dąbrowskim, War szawa 1978; idem, Powstanie styczniowe w Radomskiem, Radomsko 1995; Powstanie stycznio we. Sprawy, regiony, ludzie, pod red. W. Śladkowskiego, Annales UMCS, Lublin 1994; F. Ramotowska, Organizacja parafialna w powstaniu styczniowym (akty normatywne) [w:] Na przełomie stuleci. Naród - Kościół - Państwo w XIX wieku. Księga pamiątkowa dedykowana Prof. Ryszardowi Benderowi, red. M. Piotrowski, Lublin 1997. 40 J. Chądzyński, Wspomnienia powstańca polskiego z lat 1863-1864, Warszawa 1963; K. Kalita, Wspomnienia krwawych walk, Lwów 1913; J. Oxiński, Wspomnienia z powstania pol skiego 1863-1864, Warszawa 1965; Roman Rogiński, powstaniec 1863 r. Zeznania i wspomnie nia, Warszawa 1983; F. Erlach, Partyzantka w Polscew 1863 r., Warszawa 1960; L. Rataj czyk, Polska wojna partyzancka 1863-1864, Warszawa 1966; W. Caban, Działalność powstań- 39 W momencie wprowadzenia gmin i przyjęcia organizacji parafialnej, okręg pośredniczył pomiędzy parafią a powiatem, zgodnie jednak z reformą z 6 VIII 1863 r. gminy zostały zniesio ne i najniższym szczeblem administracji została parafia. Prof. Ryszardowi Benderowi, red. M. Piotrowski, Lublin 1997. Uwagi o polskim państwie podziemnym w latach 1863-1864 w Galicji) bądź ławy główne i organizatorów (od lutego 1864 w Galicji). Ponadto w kontaktach z tymi za borami Rząd Narodowy posługiwał się komisarzami pełnomocnymi i nadzwy czajnymi36. Jeśli chodzi o politykę i służbę zagraniczną to RN prowadził ją w opar ciu i za pośrednictwem komisarzy pełnomocnych i nadzwyczajnych (takich jak Władysław ks. Czartoryski) oraz agencji instalowanych w krajach zachod nich37. Administracja państwa podziemnego lat 1863-1864 opierała się na kon cepcji Agotona Gillera, oznaczającej podkopywanie administracji zaborczej i stopniowe pozbawienie jej wpływu na społeczeństwo, aż do całkowitego przejęcia zarządu nad krajem. Spowodowało to sytuację, w której doszło do konfrontacji dwóch administracji: polskiej, tajnej która rozporządzała słaby mi siłami materialnymi ale była uznawana przez większość społeczeństwa za jedyną legalną, oraz rosyjskiej, zaborczej która swe panowanie zawdzięczała sile policji i armii. Skutkiem tego było korzystanie z oficjalnie funkcjonują- 242 Maksymilian Stanulewicz cych organów zaborczych w realizacji celów powstania, co było o tyle uła twione, że urzędy zaborcze były obsadzone Polakami38. y y y Struktura terenowa administracji powstańczej była wielopoziomowa. Ob szar Królestwa został podzielony przez władze powstańcze na 8 województw i 39 powiatów podzielonych na okręgi. Na tych trzech poziomach operowa ło tajemne państwo polskie. Dopiero w czerwcu wprowadzono gminy i wy korzystano kościelne parafie. Cała ta struktura miała od swego początku, u podstaw, konspiracyjny system dziesiątkowy, co spowodowało, że nastąpi ło niewyraźne przejście ze struktur opartych na więzi osobistej (dziesiątka) do struktury terytorialnej (okręg)39. Na czele każdej z tych jednostek podziału stał urzędnik zwany naczelni kiem, rozumianym tutaj jako zwierzchnik danej struktury, któremu przysługi wały określone uprawnienia, wynikające z tego, że był on reprezentantem rzą du. Jedynie w przypadku okręgu występował nie naczelnik, a przełożony zwa ny okręgowym. W ramach tego samego podziału terytorialnego utworzono odrębną admi nistrację wojskową, której początkowo podlegała administracja cywilna. Pro blem wzajemnych relacji pomiędzy tymi władzami został rozwiązany dwoja ko. Obok regulacji prawnych rozgraniczających zakres kompetencji obu tych władz, RN wprowadził - jako arbitra - komisarza wojskowego Rządu dla roz strzygania konfliktów między władzami wojskowymi i cywilnymi. Jesienią 1863 r. wprowadzono komisarza pełnomocnego RN na województwo, łączą cego kompetencje wojskowe i cywilne, znosząc pozostałych urzędników szczebla wojewódzkiego40. 243 UWAGI O POLSKIM PAŃSTWIE PODZIEMNYM W LATACH 1863-1864 Posiadło również tajemne państwo polskie własny wymiar sprawiedliwo ści. Był on oparty na dwóch pionach, tj. wojskowym i cywilnym. [ ] y 42 J. Adamus, Przegląd źródeł powstańczego prawa wojskowego (1863 r.), WPP, 1932, s. 254-276; T. Kędzierski, Powstanie styczniowe. Geneza, przebieg, udział prawników. War szawa 1936; H. Cederbaum, Powstanie Styczniowe. Wyroki Audytorium Polowego z lat 1863-1864, Warszawa 1917; L. Hochberg, Kodeks Karny Wojskowy z roku 1863, WPP, nr 3/53; L. Czubiński, Przestępstwa przeciw karności w Kodeksie RN z 20 XI 1863 r. WPP, nr 4/1961; J.P. Haraschin, Organizacja sądownictw i prokuratury w Wojsku Polskim, [w:] Roz prawy i studia UJ, Kraków 1961; J. Ziewiński, Setna rocznica KKW z 20 XI 1863 r., „Pra wo i Życie”, nr 24 (171), (1963). cza płk Bogdana Boziny w woj. sandomierskim i krakowskim, Kieleckie Studia Historyczne, t. 8, 1990; idem, Z dziejów powstania styczniowego w rejonie Gór Świętokrzyskich, Warszawa - Kra ków, 1989; E. Halicz, O potrzebie studiów z dziedziny wojskowo-historycznej powstania stycz niowego, Biuletyn WAP, Studia Historyczne, I, 1958; idem, Powstanie styczniowe - polską woj ną partyzancką, „Żołnierz Polski”, nr 3, 1963; Obok Orla znak Pogoni. Z dziejów powstania stycz niowego na Bialoctocczyźnie, pod red. Z. Kosztyły, Białystok 1985; Studia i Materiały z Dzie jów Powstania Styczniowego, t. 1-4, Warszawa - Pułtusk - Ciechanów - Mława, 1994. 43 F. Ramotowska, Powstanie tajemnego państwa..., s. 248; o znaczeniu pieczęci jako symbolu władzy narodowej patrz: I. Sztakelberg, Pieczęcie powstańcze 1863-1864, Warsza wa 1988; H. Eile, Symbol siły, siła symbolu, Kwartalnik Literacko Naukowy, R. 13, 1936, nr 3. j y g 41 Kodeks karny wojskowy z 30 VII 1863 r. i K.K.W. z 20 XI 1863 r. [w:] Dokumenty Wy działu Wojny RN, pod red. S. Kieniewicza i I. Millera, Wrocław 1974, nr 35, 39, s. 83 i n. 113 i n.; Dekret wprowadzający Trybunaty Rewolucyjne i Dekret o ustanowieniu prawa kar nego w sprawach przestępstw politycznych, obydwa z 2 VI 1863 r., oraz ich nowelizacja z 2 VII t. r. [w:] Dokumenty KCN i RN, nr 132, 133, 166, s. 129-130, 172-174. Uwagi o polskim państwie podziemnym w latach 1863-1864 Ten ostat ni odnosił się do urzędników tak narodowych jak i zaborczych, choć podle gały mu także osoby niezwiązane z aparatem urzędniczym walczących stron. Początkowo orzeczenie wydawane przez organy administracji powstańczej w ramach dwóch pionów, opierały się na zasadach doraźności. Dopiero la tem 1863 r. uregulowano zasady działania zarówno sądownictw wojskowego jak i urzędniczego41. Sądownictwo wojskowe opierało się odtąd na jednoli tych sądach wojskowych, złożonych z oficerów, czyli sądach wojennych, któ rym podlegali przede wszystkim, należący jakimkolwiek bądź tytułem do skła du armii polskiej. Sądownictwo urzędnicze opierało się na instytucji Trybu nałów Rewolucyjnych, funkcjonujących początkowo na szczeblu powiatu, a po nowelizacji z 2 VII 1863 r., na szczeblu województwa. Na jej podsta wie, trybunały podlegać miały trzem, nowopowołanym Sądom Najwyższym, odrębnie dla Korony, Litwy i Rusi42. Jak wynika z powyżej przytoczonej charakterystyki, Organizacja Narodo wa w powstaniu styczniowym, dysponowała wszystkimi atrybutami państwa w rozumieniu dzisiejszym, z wyjątkiem uznania międzynarodowego i szero ko rozumianej suwerenności. Widomym znakiem władzy Rządu Narodowe go były jego pieczęcie. F. Ramotowska pisała: „Zasadnicze [ich - M.S.] zna czenie [...] polegało na nadawaniu [...] dokumentom cech urzędowych prawo mocnych. Odgrywały one ponadto ogromną rolę jako symbol państwowości polskiej, symbol niezależności od reżimu zaborczego, symbol nadziei [...] Okazanie dokumentu opatrzonego pieczęcią władzy narodowej będzie wywo ływano głębokie poruszenie i posłuch u wszystkich Polaków”43. Tzw. pieczęć 244 Maksymilian Stanulewicz główna, trójdzielna, z Ortem, Pogonią i Archaniołem Gabrielem, była sym bolem jedności państwowej Korony, Litwy i Rusi, nawiązując do ideologii Rzeczypospolitej przedrozbiorowej i do tradycji wcześniejszych zrywów na rodowowyzwoleńczych. Podkreślając ciągłość państwowości polskiej, władze powstańcze umieszczały na swoich pieczęciach i herbach symbol korony, nie jako elementu tradycji monarchistycznej, ale znak suwerenności i tradycyjny szczegół godła państwa polskiego. O znaczeniu pieczęci tak wypowiadał się Marian Langiewicz: Czyż istniał Rząd centralny? Na to pytanie do dziś dnia odpowiedzi dać nie umiem; dla mnie Rząd... istniał tylko jako pieczątka, a pieczątka jest nieśmiertelną, jej rozmnażanie się jest nieograniczonem [...] boć cała hierarchia Rządu tajną była i ciągle zagrożoną więzieniem [...]44 Na koniec sięgnijmy jeszcze raz do przemyśleń wybitnego patrioty i współtwórcy Organizacji Narodowej, Agatona Gillera: „Ponieważ istnienie narodu polskiego tajemniczo tylko może być zasilane pod rządami obcymi [...] więc naturalnym następstwem jest [...] rząd własny, tajemny. Władzy ta jemnej słuchano podczas powstania dobrowolnie, mord polityczny nie był tak powszechny jak mordowania moskiewskie, które dotąd są tajemnymi”45. 44 List do Ludwika Bulewskiego z 23 IV 1863 r. [w:] Pisma wojskowe Mariana Langie wicza, s. 50. 45 A. Giller, Historia powstania..., t. I, s. 28 i 81. Uwagi o polskim państwie podziemnym w latach 1863-1864 główna, trójdzielna, z Ortem, Pogonią i Archaniołem Gabrielem, była sym bolem jedności państwowej Korony, Litwy i Rusi, nawiązując do ideologii Rzeczypospolitej przedrozbiorowej i do tradycji wcześniejszych zrywów na rodowowyzwoleńczych. Podkreślając ciągłość państwowości polskiej, władze powstańcze umieszczały na swoich pieczęciach i herbach symbol korony, nie jako elementu tradycji monarchistycznej, ale znak suwerenności i tradycyjny szczegół godła państwa polskiego. O znaczeniu pieczęci tak wypowiadał się Marian Langiewicz: Czyż istniał Rząd centralny? Na to pytanie do dziś dnia odpowiedzi dać nie umiem; dla mnie Rząd... istniał tylko jako pieczątka, a pieczątka jest nieśmiertelną, jej rozmnażanie się jest nieograniczonem [...] boć cała hierarchia Rządu tajną była i ciągle zagrożoną więzieniem [...]44 główna, trójdzielna, z Ortem, Pogonią i Archaniołem Gabrielem, była sym bolem jedności państwowej Korony, Litwy i Rusi, nawiązując do ideologii Rzeczypospolitej przedrozbiorowej i do tradycji wcześniejszych zrywów na rodowowyzwoleńczych. Podkreślając ciągłość państwowości polskiej, władze powstańcze umieszczały na swoich pieczęciach i herbach symbol korony, nie jako elementu tradycji monarchistycznej, ale znak suwerenności i tradycyjny szczegół godła państwa polskiego. O znaczeniu pieczęci tak wypowiadał się Marian Langiewicz: Czyż istniał Rząd centralny? Na to pytanie do dziś dnia odpowiedzi dać nie umiem; dla mnie Rząd... istniał tylko jako pieczątka, a pieczątka jest nieśmiertelną, jej rozmnażanie się jest nieograniczonem [...] boć cała hierarchia Rządu tajną była i ciągle zagrożoną więzieniem [...]44 ą j ą y ąg g ą ę Na koniec sięgnijmy jeszcze raz do przemyśleń wybitnego patrioty i współtwórcy Organizacji Narodowej, Agatona Gillera: „Ponieważ istnienie narodu polskiego tajemniczo tylko może być zasilane pod rządami obcymi [...] więc naturalnym następstwem jest [...] rząd własny, tajemny. Władzy ta jemnej słuchano podczas powstania dobrowolnie, mord polityczny nie był tak powszechny jak mordowania moskiewskie, które dotąd są tajemnymi”45.
https://openalex.org/W4285169905	https://link.springer.com/content/pdf/10.1007/978-981-19-2456-9_115.pdf	English	null	A Multi-modal Time Series Intelligent Prediction Model	Lecture notes in electrical engineering	2,022	cc-by	3,025	A Multi-modal Time Series Intelligent Prediction Model Qingyu Xian(B) and Wenxuan Liang International College, Beijing University of Posts and Telecommunications, Beijing, China xianqingy321@163.com International College, Beijing University of Posts and Telecommunications, Beijing, China xianqingy321@163.com Abstract. The power load prediction can ensure the power supply and dispatch, which will be useful for market participants to plan and make strategic decisions to enhance reliability, save operation and maintenance costs. Short-term load data series have obvious approximate periodicity, while long-term load data series show variability and dynamic features. In addition, time series data of various modalities, such as market reports and production management data, could play a role in load prediction. One kind of multi-modal CNN-BiLSTM architecture is proposed to predict short-term and long-term load data, which have an improved shared parameter convolutional network to learn feature representation and an improved attention-based BiLSTM mechanism, which could model the dynamic features of multimodal on time series data. Experimental results on multimodal dataset show that, compared with other baseline systems, this model has some advantages in the prediction accuracy. Keywords: Power load prediction · Multi-modal · CNN-BiLSTM · Attention-based BiLSTM © The Author(s) 2022 Z. Qian et al. (Eds.): WCNA 2021, LNEE 942, pp. 1150–1157, 2022. https://doi.org/10.1007/978-981-19-2456-9_115 1 Introduction Load prediction is a key link in power supply planning, as well as a basic feature and important calculation basis for intelligent power supply planning. In addition to tradi- tional machine learning models, deep neural networks, as the most popular intelligent research framework at present, have been widely implied by researchers in the active distribution network load prediction research. Active distribution network load predic- tion data can be regarded as time series data, which means it could be classiﬁed by chronological order. Time series analysis method describes and interprets phenomena that change over time to derive various predictive decisions. Deep learning neural net- works can automatically learn arbitrarily complex mapping from input to output, and support multiple inputs and outputs [1]. It provides many ways for time series prediction tasks, such as automatic learning of time dependence or trends and seasonality automatic processing of data based on time structure. Although deep neural networks can approximate any complex function arbitrarily and perform good non-linear modelling of a variety of data, in the historical data used in the active distribution network load prediction, the short-term load data sequence has A Multi-modal Time Series Intelligent Prediction Model 1151 obvious approximate period characteristics, and the long-term load data sequence shows the variability and rich dynamic characteristics. Besides, with the development of the Internet and big data technology, it will improve the performance of active distribution network load prediction by importing some kinds of time series data, such as market reports and production management data and other modalities. LSTM (Long Short- Term Memory) and other RNN (recurrent neural network) structures could not effective in predicting the difference between peak hours and minimum power consumption times, and usually requires higher computational cost. This paper proposes a multi-modal CNN-BiLSTM (Convolutional Neural Network- Bidirectional Long Short-Term Memory) architecture, which have an improved shared parameter parallel convolutional network to learn feature representations for short-term load data sequences, and an improved bidirectional attention LSTM network. The model presents the dynamic changing characteristics of data affected by some disturbances with the text features, such as temperature and holidays. On the 24 months of load and market report data set, the method is compared with the convolutional neural network and the bidirectional long short-term memory neural network. The experimental results show that the model has some advantages on the computational speed and accuracy. 1 Introduction The rest of this paper includes: The part II introduces the characteristics of the load sequence data and the variables that may affect the prediction. The third part introduces the multi-modal deep learning. The fourth part details the structure of the proposed multi-modal. The experimental and evaluation results are given in the ﬁfth part and the last one is the summary. 2.2 Load Feature Prediction As a type of time series data, load prediction can also be implemented using neural network technology. In monthly and quarterly time series, time series prediction based on neural network has more obvious advantages than traditional statistical methods and artiﬁcial judgment methods compared with traditional statistical time series methods [7]. Mbamalu et al. believe that load prediction is an autoregressive process, and use iterative re-weighted least squares to estimate model parameters [8]. Based on the combination prediction model of neural network, by learning the weights of different prediction models in the combination, the variable weight coefﬁcient combination prediction model is shown in Eq. 1. yij = K t=1 wt(i, j) ftij + etij (1) (1) Where yij is the actual load of month i in year j, ftij is the predicted value of month i in year j of the ﬁrst method, etij = yij −ftij and w = Min n i=1 12 j=1 yij −g f1ij, f2ij, . . . , fKij 2. j Since there is a relatively complicated non-linear relationship between the actual prediction input and the ﬁnal output, a three-layer forward neural network is used to ﬁt an arbitrary function. Through the continuous iteration of the network and the update of the gradient back propagation, the ﬁnal reasonable parameters are obtained. And by these parameters, the combined predicted value of any predicted input value is realized. The load forecasting results by Autoregressive Integrated Moving Average and Seasonal AutoregressiveIntegratedMovingAverageshowedthatobtained9.13%and4.36%mean absolute percentage error respectively. With deep learning Long Short-Term Memory model, it will reduce to 2% [9]. 2.1 Load Feature Extraction The load types can be distinguished according to the reaction guidance mechanism and the non-reaction guidance mechanism, which are respectively controllable load and uncontrollable load. The load type is divided into friendly load and non-friendly load. The load prediction model can be constructed by analysing the active load characteristics and energy storage characteristics including friendly load and according to the constraint conditions[2].Anothermethodistousethebottom-uppredictionmethod[3],inthesmall area divided according to certain properties, ﬁrst perform load prediction, and ﬁnally superimpose the obtained load demand curve to obtain a complete load prediction result. For example, a large amount of data can be processed in parallel through the cloud computing platform, the maximum entropy algorithm can be used to classify the data, the abnormal data and the available data can be distinguished, and the local weighted linear regression model can be combined with the Map-Reduce model framework to realize the active conﬁguration of cloud computing [4]. The Spark platform is used to divide all the obtained data and compute them in parallel to speed up the processing of big data. First, the data is pre-processed through feature extraction, and the input that meets the requirements of the model is obtained, which input into the multivariate L2-Boosting for training and learning and get the ﬁnal regression model [5]. The grey prediction method is also a common method of load prediction, which added secondary smoothing processing through historical data to Q. Xian and W. Liang 1152 eliminate the interference factors of historical data with Markov chain and grey theory to predict the residual sequence and the sign of the future residual together to revise the results [6]. 3 Multi-modal Deep Learning Deep neural networks have been widely used on single modal data such as text, images or audio, which included a variety of supervised and unsupervised deep feature learning model architectures [10]. Multi-modal deep learning refers to training new deep network applications to learn the features of multiple modes. For example, in emotion recognition technology, the voice and text information fusion can improve the effect of emotion recognition [3]. Establishing a private domain network (for visual information and audio information in short videos to extract individual features) and a public domain network (for acquiring joint features) could solve the problem of short video classiﬁcation [8]. The principle of multi-modal feature learning is, if there are multiple modalities at the same time, one of the modalities can be learned better than a single modal in-depth feature. It can also be learned by sharing representations between multiple modalities to A Multi-modal Time Series Intelligent Prediction Model 1153 further improve the accuracy index on speciﬁc tasks. Researchers have begun to carry out research in various ﬁelds for multi-modal model, such as multi-modal model based on fuzzy cognitive maps [5], which ﬁrst extract a subset from the complete data and trained separately on each subset, then used fuzzy cognitive maps for modelling and prediction, and ﬁnally the output was fused from each subset by the information granulation. The time series data is widely available, such as holidays, weather and other data, which can be used to jointly predict the city’s trafﬁc conditions [6]. Firstly, the holiday and weather feature information were extracted, and the Prophet algorithm is selected to predict the trafﬁc ﬂow characteristics during the holidays with one DCRNN network to predict the trafﬁc ﬂow on the combination of road network structure data and ﬂow data. Besides, image and time series data are indispensable in the automatic driving system. The time series refers to the speed series and steering wheel angle series. The multi- modal network serving the autonomous driving system includes CNN, RNN, horizontal control network and vertical control network. The time series data is input into the RNN network for processing, and the image data is input into the CNN network for feature extraction. The extracted features are input into the horizontal and vertical control network respectively. Finally, the predicted value of the steering wheel and speed is obtained to guide the steering wheel angle and the speed. 4 An Improved Multi-modal CNN-LSTM Prediction Model Although classic time series prediction algorithms can be used for load prediction, the ﬂuctuation of load does not only depend on historical time series data. Due to the diversiﬁcation of intelligent load management requirements, it is manifested as a multi- modal data form in time series. Shared parameter parallel convolutional network Shared parameter parallel convolutional network Short/long-term load data series Short/long-term temperature data series Holiday data series Market report data sequence Attention Layer Bidirectional long short- term memory neural network Short-term load data series prediction Long-term load data series prediction Fig. 1. Multi-modal CNN-BiLSTM network structure. Fig. 1. Multi-modal CNN-BiLSTM network structure. This paper proposes a multi-modal convolutional neural network-long short term memory neural network prediction method on load data and its primary structure is shown in Fig. 1. For short-term load data series, introduce data such as temperature and holidays, and use an improved shared parameter parallel convolutional network to Q. Xian and W. Liang 1154 learn feature representation; and use an improved two-way attention mechanism long and short-term memory neural network, combined with medium and long-term load sequences and effects. The relevant text data is introduced in this model for its dynamic change features. In the multi-modal convolutional neural network-bidirectional long and short term memoryneuralnetworkstructureinFig.1,twoparallelconvolutionalneuralnetworksare used to extract features from the original historical load and other modal data sequences such as temperature and text. These convolutional neural networks share parameters. The ﬁrst convolutional layer includes two convolution kernels with sizes 44 and 55. The number of convolution kernels is 64, and then a shared connection is used. The structure is to extract some of the convolution kernels from the previous layer of convolution kernels to form the current layer of convolution kernels. The fully connected output needs to be sent to the attention layer, trained according to the attention mechanism, and output to the BiLSTM network. The size of the hidden state is 64. The ﬁnal output is the short-term load data sequence and the long-term load data sequence. Input data Pooling shared-parameter layer 44 convoluon kernels, 64 ﬁlters 33 convoluon kernels, 16 ﬁlters 11 convoluon kernels, 256 ﬁlters 55 convoluon kernels, 64 ﬁlters 44 convoluon kernels, 16 ﬁlters 11 convoluon kernels, 256 ﬁlters Full Connecon layer Fig. 2. Shared-parameter convolutional neural network structure. Fig. 2. Shared-parameter convolutional neural network structure. 5 Experiments and Results In this section, we introduce the experimental evaluation methods and results of the baseline system and the above-mentioned improved methods on existing data sets. The data set contains unit hour load data of a city in North China for about 2 years, local daily maximum temperature, minimum temperature, average temperature and precipitation data, local public holiday date data, and local quarterly market operation information report data within 2 years. The entities and their types in the maximum and minimum temperatures, holiday information, and text are represented as vectors of length 128. The load value is divided into short-term load data series and long-term load data series according to the time period. The former contains the load data series within a quarter, and the latter contains the load data series greater than one quarter. Use these data to predict the unit hour load value on a speciﬁed time series period. The evaluation index is the mean absolute percentage error (MAPE) based on the short-term load data series and the long-term load data series prediction and its A Multi-modal Time Series Intelligent Prediction Model 1155 A Multi-modal Time Series Intelligent Prediction Model 1155 1155 calculation method is shown in Eq. 2. calculation method is shown in Eq. 2. MAPE = 1 N N k=1 v (k) −v(k) v(k) × 100% (2) (2) Where N represents the total number of samples in the test set, v(k) represents the actual value, and v (k) represents the predicted value. Fig. 3. MAPE results of short-term load prediction. Fig. 3. MAPE results of short-term load prediction. The baseline system adopts weighted least squares method WLS, autoregressive moving average ARMA, seasonal autoregressive integrated moving average SARIMA and CNN-LSTM architectures, and divides a total of 731 days * 24 h of data into training data and veriﬁcation data in chronological order And the test data, the ratio is 4:2:4. Under the four baseline systems and the multi-modal CNN-BiLSTM model, the average absolute percentage error MAPE results and the average error MAE results of short-term load data series prediction and long-term load data series prediction are obtained, as shown in Fig. 3 and Fig. 4, respectively. The ﬁgure shows that the multi- modal CNN-BiLSTM method has certain advantages for short-term load data sequence prediction and long-term load data sequence prediction on the training set and testing dataset. 6 Conclustion Load prediction has the characteristics of time trend. There are obvious differences in load in different seasons. Precise prediction is helpful for efﬁcient decision-making and reasonable planning. This paper proposes a multi-modal convolutional neural network- bidirectional long and short-term memory neural network architecture, which uses a parallel convolutional network with shared parameters and a bidirectional attention mechanism. The long-term and short-term memory neural network processes load data, temperature data and text data. The multi-modal data sequence, etc., can predict the short-term load data sequence and the long-term load data sequence. The experimental results verify that the network structure can achieve a certain improvement in prediction accuracy compared with other baseline systems. 5 Experiments and Results Compared with the CNN-LSTM architecture, it has a certain error reduction. Especially in the long-term load data series prediction, it has higher prediction accuracy than the short-term load data series. Fig. 4. MAPE results of long-term load prediction. Fig. 4. MAPE results of long-term load prediction. 1156 Q. Xian and W. Liang 1156 Q. Xian and W. Liang 1156 References 1. Brownlee, J.: Deep learning for time series forecasting: predict the future with MLPs, CNNs and LSTMs in Python. Machine Learning Mastery (2018) 2. Zhuang, H., Zhang, J.: Coordinated voltage control based on model prediction in active distribution networks. Electric Power, 12 (2016) 3. Kuzle, I., Bosnjak, D., Pandzic, H.: Comparison of load growth prediction methods in distri- bution network planning. In: CIRED 2009–20th International Conference and Exhibition on Electricity Distribution-Part 1, pp. 1–4. IET (2009) 4. Zhang, S., Liu, J., Zhao, B., Cao, J.: Cloud computing-based analysis on residential electricity consumption behavior. Power Syst. Technol. 37(6), 1542–1546 (2013) 5. Du, D., Xie, J., Fu, Z.: Short-term power load forecasting based on spark platform and improved parallel ridge regression algorithm. In: 2018 37th Chinese Control Conference (CCC), pp. 8951–8956. IEEE (2018) 6. Niu, Y., Wang, Z.Y., Wang, H.J., Sun, Y., Li, X.: Application of improved grey model for mid and long-term power demand forecasting. J. Northeast Dianli Univ. (Nat. Sci. Ed.), 2 (2009) 6. Niu, Y., Wang, Z.Y., Wang, H.J., Sun, Y., Li, X.: Application of improved grey model for mid and long-term power demand forecasting. J. Northeast Dianli Univ. (Nat. Sci. Ed.), 2 (2009) 7. Nelson, M., Hill, T., Remus, W., O’Connor, M.: Time series forecasting using neural networks: h ld th d t b d li d ﬁt? J F t 18(5) 359 367 (1999) 7. Nelson, M., Hill, T., Remus, W., O’Connor, M.: Time series forecasting using neural networks: should the data be deseasonalized ﬁrst? J. Forecast. 18(5), 359–367 (1999) 8. Mbamalu, G.A.N., El-Hawary, M.E.: Load forecasting via suboptimal seasonal autoregressive models and iteratively reweighted least squares estimation. IEEE Trans. Power Syst. 8(1), 343–348 (1993) 9. Nguyen, H., Hansen, C.K.: Short-term electricity load forecasting with Time Series Anal- ysis. In: 2017 IEEE International Conference on Prognostics and Health Management (ICPHM), pp. 214–221. IEEE (2017) 10. Ngiam, J., Khosla, A., Kim, M., Nam, J., Lee, H., Ng, A.Y.: Multimodal deep learning. In: ICML (2011) A Multi-modal Time Series Intelligent Prediction Model A Multi-modal Time Series Intelligent Prediction Model 1157 Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), 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 chapter are included in the chapter’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter’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.
https://openalex.org/W2064559752	https://europepmc.org/articles/pmc1847682?pdf=render	English	null	Description of industrial pollution in Spain	BMC public health	2,007	cc-by	9,671	BioMed Central BioMed Central Description of industrial pollution in Spain García-Pérez* - jgarcia@isciii.es; Elena Boldo - eiboldo@isciii.es; Rebeca Ramis - rramis@isciii.es; Email: Javier García-Pérez* - jgarcia@isciii.es; Elena Boldo - eiboldo@isciii.es; Rebeca Ramis - rramis@isciii.es; Marina Pollán - mpollan@isciii.es; Beatriz Pérez-Gómez - bperez@isciii.es; Nuria Aragonés - naragones@isciii.es; Gonzalo López- Abente - glabente@isciii.es mpollan@isciii.es; Beatriz Pérez-Gómez - bperez@isciii.es; Nuria Aragonés - naragones@isciii.es; Gonzalo López- e@isciii.es * Corresponding author Published: 21 March 2007 BMC Public Health 2007, 7:40 doi:10.1186/1471-2458-7-40 Received: 30 August 2006 Accepted: 21 March 2007 This article is available from: http://www.biomedcentral.com/1471-2458/7/40 © 2007 García-Pérez et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativeco which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited Published: 21 March 2007 BMC Public Health 2007, 7:40 doi:10.1186/1471-2458-7-40 R A This article is available from: http://www.biomedcentral.com/1471-2458/7/40 © 2007 García-Pérez et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Com which permits unrestricted use, distribution, and reproduction in any medium, Received: 30 August 2006 Accepted: 21 March 2007 Received: 30 August 2006 Accepted: 21 March 2007 BMC Public Health 2007, 7:40 doi:10.1186/1471-2458-7-40 ; 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. BMC Public Health Open Access Methods f All information on industrial pollution discharged in 2001 was drawn from EPER-Spain records. This informa- tion is public, and the records are accessible as a relational database, from the European Commission server [12]. In every case where the geographic WGS84-projection coor- dinates for the pertinent industrial installation could be imported and incorporated into a Geographic Informa- tion System (GIS), these are shown against the entry con- cerned. The data were obtained and processed to include all the information necessary for analysis purposes, in three data files, namely, emissions to air, direct to water and indirect to water (via sewage treatment plants). Although there are 4,949 installations affected by the IPPC Act in Spain, the EPER-Spain included only 1,437, which released pollut- ants that have exceeded the reporting thresholds in 2001. Data were not published for the remaining installations (3,512) because they did not exceed the reporting thresh- olds for the pollutants, and consequently they are not considered in this study. The data were obtained and processed to include all the information necessary for analysis purposes, in three data files, namely, emissions to air, direct to water and indirect to water (via sewage treatment plants). Although there are 4,949 installations affected by the IPPC Act in Spain, the EPER-Spain included only 1,437, which released pollut- ants that have exceeded the reporting thresholds in 2001. In January 2000, the European Council gave a favourable opinion on the implementation of a European Pollutant Emission Register (EPER) [11]. Under the terms of this project, all Member States are required to report to the Commission on emissions to air, soil and water from all agricultural or industrial facilities engaging in one or more activities listed in Annex I to Council Directive 96/61/EC. Industrial activities classified in the EPER fall into the fol- lowing 6 categories: 1) Energy industries; 2) Production and processing of metals; 3) Mineral industry; 4) Chemi- cal industry and chemical installations; 5) Waste manage- ment; and 6) Other activities (which include paper and board production, manufacture of fibres or textiles, tan- ning of hides and skins, slaughterhouses, intensive poul- try or pig rearing, installations using organic solvents, and the production of carbon or graphite). The Directive envisages the reporting of 50 pollutant emissions in excess of a given threshold. Abstract Background: Toxic substances released into the environment (to both air and water) by many types of industries might be related with the occurrence of some malignant tumours and other diseases. The publication of the EPER (European Pollutant Emission Register) Spanish data allows to investigate the presence of geographical mortality patterns related to industrial pollution. The aim of this paper is to describe industrial air and water pollution in Spain in 2001, broken down by activity group and specific pollutant, and to plot maps depicting emissions of carcinogenic substances. Methods: All information on industrial pollution discharge in 2001 was drawn from EPER-Spain public records provided by the European Commission server. We described the distribution of the number of industries and amounts discharged for each pollutant, as well as emission by pollutant group and the industrial activities associated with each pollutant. Maps of Spain were drawn up, with UTM coordinates being used to plot pollutant foci, and circles with an area proportional to the emission to depict pollution emission values. Results: The EPER-Spain contained information on 1,437 industrial installations. The industrial plants that discharge pollutant substances into air and water above the pollutant-specific EPER threshold were mainly situated in the Autonomous Regions of Aragon, Andalusia and Catalonia and in Catalonia, the Basque Country and Andalusia respectively. Pollution released in 2001 into air approached 158 million Mt. Emissions into water were over 8 million Mt. Conclusion: A few single industrial plants are responsible for the highest percentage of emissions, thus rendering monitoring of their possible health impact on the surrounding population that much simpler. Among European countries Spain is the leading polluter in almost one third of all EPER- registered pollutant substances released into the air and ranks among the top three leading polluters in two-thirds of all such substances. Information obtained through publication of EPER data means that the possible consequences of reported pollutant foci on the health of neighbouring populations can now be studied. Page 1 of 13 (page number not for citation purposes) BMC Public Health 2007, 7:40 http://www.biomedcentral.com/1471-2458/7/40 http://www.biomedcentral.com/1471-2458/7/40 Background aid of EPER information, the relationship between indus- trial pollution and public health consequences in Europe can thus be studied by analyzing the influence of spatial distribution of emissions on geographic morbidity and mortality patterns. g Toxic substances, which are released constantly into the environment (to both air and water) by many types of industrial activities, include a long list of products and pollutants that until now have never been quantified in Spain. Evidence as to the health risk posed by residing in the vicinity of such polluting industries is limited, with cancer and congenital malformations being the most widely studied health problems in the international liter- ature [1-6]. The geographic patterns displayed by certain tumour sites in "small-area" mortality studies in Spain [7- 9] suggest that there are environmental factors, closely associated with the territory, which may play an impor- tant role in tumour aetiology. It has recently been sug- gested that exposures deriving from the process of industrial and economic development might have some influence on the appearance of haematological tumours in Spain [10]. Methods f Industrial plants shown by the EPER to discharge pollutant substances indirectly into water (via sewage treatment plants) in Spain, and associated industrial activities The industrial plants were mostly situated in Catalonia, 50 (31%), the Basque Country, 31 (19%) and Andalusia, 18 (11%). Industrial activities having the highest number of associated plants were: 'Manufacture of food products and beverages (in slaughterhouses, plants for the produc- tion of milk and other animal or vegetable raw materials)' (33 industries); 'Surface treatment of metals and plastics (metal industries and metal ore roasting or sintering installations. Installations for the production of ferrous and non-ferrous metals)' (29 industries); and 'Manufac- ture of basic organic chemical products' (24 industries). Industrial plants shown by the EPER to discharge pollutant substances indirectly into water (via sewage treatment plants) in Spain, and associated industrial activities The industrial plants were mostly situated in Catalonia, 50 (31%), the Basque Country, 31 (19%) and Andalusia, 18 (11%). Industrial activities having the highest number of associated plants were: 'Manufacture of food products and beverages (in slaughterhouses, plants for the produc- tion of milk and other animal or vegetable raw materials)' (33 industries); 'Surface treatment of metals and plastics (metal industries and metal ore roasting or sintering installations. Installations for the production of ferrous and non-ferrous metals)' (29 industries); and 'Manufac- ture of basic organic chemical products' (24 industries). Based on this material, we described the distribution of the number of industries and amounts discharged for each pollutant, as well as emission by pollutant group and the industrial activities associated with each pollutant. Maps of Spain were drawn up, with UTM coordinates being used to plot pollutant foci, and circles with an area proportional to the emission to depict pollution emission values. In order to plot the maps, pollutants classified as group-1, -2A and -2B carcinogens by the International Agency for Research on Cancer (IARC) were selected [13]. Lastly, we compared Spain's percentage emissions to those of European Union countries, focusing on air pol- lutants for which Spain had some type of emission regis- tered. Table 1 lists the individual air pollutants and the indus- trial activities associated with these. Table 2 shows pollution released in 2001, with a break- down in quantitative terms for each of the three pollution groups (emissions to air, direct to water and indirect to water) and by pollutant group. Results At the date of study, the EPER-Spain contained informa- tion on 1,437 plants or industrial installations. Registry data showed that a total of 1,250 plants released pollutant substances to air, 133 direct to water and 164 indirect to water, and that some of these plants discharged sub- stances into both air and water. Industrial plants shown by the EPER to discharge pollutant substances into air in Spain, and associated industrial activities Table 3 gives a detailed description of industrial air pollu- tion in Spain in 2001, as reflected by the EPER, with a breakdown by specific pollutant. While the rows show information relating to the pollutants, the columns reflect the statutory reporting thresholds for the respective pol- lutant emissions, information for Spain as a whole (total emissions, number of plants that release substances, and mean emission per plant) and information on the Auton- omous Regions that registered the highest emission values for each of the pollutants (total emissions, number of plants, and mean emission per plant). The thresholds in the first column of pollutants provide a crude idea of the relative toxicity or importance of the substance. Tables 4 and 5 display the same information as Table 3, but with reference to direct and indirect emissions to water respec- tively. Most of the reported industries were situated in the Autonomous Regions of Aragon, 425 (34%), Andalusia, 208 (17%) and Catalonia, 190 (15%). Industrial activities registering the greatest number of plants were: 'Manure management. Installations devoted to the rearing of poul- try and pigs' (723 industries); 'Manufacture of plaster, asphalt, concrete, cement, glass, fibres, bricks, tiles or ceramic products (mineral product industry involving fuel combustion)' (136 industries); and 'Enteric fermenta- tion. Installations devoted to the rearing of poultry and pigs' (75 industries). Methods f The results refer to total emissions for Spain, highlighting the Autonomous Regions with the highest emissions for the respective pol- lutant groups and their percentages relative to the overall figure. Methods f The information available allows for different types of industrial activities to be identified and, in addition, contains abundant data on emissions of the pollutant substances to air, water or soil, including the amount released annually. Data were not published for the remaining installations (3,512) because they did not exceed the reporting thresh- olds for the pollutants, and consequently they are not considered in this study. Pollutants/Substances were classified into the following groups: 1) Environmental themes: methane, carbon monoxide, carbon dioxide, hydrofluorocarbons, nitrous oxide, ammonia, non-methane volatile organic compounds (NMVOC), nitrogen dioxide, perfluorocarbons, sulphur hexafluoride, sulphur dioxide, nitrogen and phosphorus. 2) Metals and compounds: arsenic, cadmium, chromium, copper, mercury, nickel, lead and zinc. 2) Metals and compounds: arsenic, cadmium, chromium, copper, mercury, nickel, lead and zinc. 3) Chlorinated organic substances: dichloroethane-1,2, dichloromethane, chloroalkanes, hexachlorobenzene, hexachlorobutadiene, hexachlorocyclohexane, halogen- ated organic compounds, dioxins and furans, pentachlo- rophenol, tetrachloroethylene, tetrachloromethane, trichlorobenzenes, trichloroethane-1,1,1, trichloroethyl- ene and trichloromethane. 3) Chlorinated organic substances: dichloroethane-1,2, dichloromethane, chloroalkanes, hexachlorobenzene, hexachlorobutadiene, hexachlorocyclohexane, halogen- ated organic compounds, dioxins and furans, pentachlo- rophenol, tetrachloroethylene, tetrachloromethane, trichlorobenzenes, trichloroethane-1,1,1, trichloroethyl- ene and trichloromethane. In February 2004, EPER data on Spain (for 2001) were published. This register traces its origin to the Integrated Pollution Prevention and Control (IPPC) Act 16/2002 of 1 July, governing the authorization of activities falling into 11 categories. The EPER includes all IPPC industrial plants that have exceeded the reporting thresholds for one or more of the pollutants included in EU Decision 2000/ 479/CE. The aim of this paper was to describe industrial air and water pollution in Spain, broken down by activity group and specific pollutant, and to plot maps depicting emissions of carcinogenic substances. Lastly, the situation in Spain as regards emission of pollutant substances to air is compared to that of other European countries. With the 4) Other organic compounds: benzene, toluene, ethyl- benzene, xylenes, brominated diphenylether, organotin- compounds, polycyclic aromatic hydrocarbons (PAH), phenols and total organic carbon. Page 2 of 13 (page number not for citation purposes) http://www.biomedcentral.com/1471-2458/7/40 BMC Public Health 2007, 7:40 5) Other compounds: chlorides, chlorine and inorganic compounds, cyanides, fluorides, fluorine and inorganic compounds, hydrogen cyanide and PM10. 5) Other compounds: chlorides, chlorine and inorganic compounds, cyanides, fluorides, fluorine and inorganic compounds, hydrogen cyanide and PM10. Industrial plants shown by the EPER to discharge pollutant substances directly into water in Spain, and associated industrial activities Insofar as the pollutant groups were concerned, Table 2 shows that air received the most group-1 pollutants (envi- ronmental themes), with the high emissions of carbon dioxide, sulphur dioxide, nitrogen dioxide and carbon monoxide being especially noteworthy in terms of mass (Table 3). In contrast to air, water – both directly and indi- rectly – received more group-5 pollutants (Other com- pounds) (Table 2), chlorides in particular (Tables 4 and 5). The industrial plants registered were mainly situated in Catalonia, 26 (20%), the Basque Country, 23 (18%) and Andalusia, 22 (17%). Industrial activities having the high- est number of associated plants were: 'Manufacture of paper, pulp and paper products' (25 industries); 'Manu- facture of basic organic chemical products' (19 indus- tries); and 'Manufacture of basic inorganic chemical products or fertilizers' (14 industries). 5). Page 3 of 13 (page number not for citation purposes) Page 3 of 13 (page number not for citation purposes) http://www.biomedcentral.com/1471-2458/7/40 BMC Public Health 2007, 7:40 Table 1: Description of industrial air pollution, by pollutant and associated industrial activity (2001). AIR POLLUTANTS MAIN ASSOCIATED ACTIVITY1 ACTIVITY GROUP GROUP 1: ENVIRONMENTAL THEMES Methane Landfills (solid waste disposal on land). Installations for the disposal of non-hazardous waste (>50 Mt/day) and landfills (>10 Mt/day) Waste management Carbon monoxide; Perfluorocarbons Primary and secondary metal production (ferrous and non-ferrous) or sinter plants (metal industry and metal ore roasting or sintering installations) Production and processing of metals Carbon dioxide; Sulphur dioxide; Nitrogen dioxide Combustion installations >300 MW Energy industries Hydrofluorocarbons Manufacture of basic organic chemicals Chemical industry and chemical installations Nitrous oxide Manufacture of basic inorganic chemicals and fertilizers Chemical industry and chemical installations Ammonia Manure management. Industrial plants shown by the EPER to discharge pollutant substances directly into water in Spain, and associated industrial activities Installations for poultry (>40000), pigs (>2000) or sows (>750) Other activities Non-methane volatile organic compounds Petroleum product processing (mineral oil and gas refineries) Energy industries GROUP 2: METALS AND COMPOUNDS Arsenic; Cadmium; Nickel Combustion installations >300 MW Energy industries Chromium; Copper; Lead; Zinc Characteristic processes in the manufacture of metals (ferrous and non-ferrous) and metal product (metal industry and metal ore roasting or sintering installations) Production and processing of metals Mercury Manufacture of basic inorganic chemicals and fertilizers Chemical industry and chemical installations GROUP 3: CHLORINATED ORGANIC SUBSTANCES Dichloroethane-1,2; Tetrachloromethane Manufacture of basic organic chemicals Chemical industry and chemical installations Dichloromethane; Trichloromethane Manufacture of pharmaceutical products (solvent use) Chemical industry and chemical installations Hexachlorobenzene; Tetrachloroethylene Characteristic processes in the manufacture of metals (ferrous and non-ferrous) and metal product (metal industry and metal ore roasting or sintering installations) Production and processing of metals Dioxins and furans Combustion installations >300 MW Energy industries Trichloroethylene Paint application (solvent use). Installations for surface treatment or products using organic solvents (>200 Mt/year) Other activities GROUP 4: OTHER ORGANIC COMPOUNDS Benzene; Polycyclic aromatic hydrocarbons Petroleum product processing (mineral oil and gas refineries) Energy industries GROUP 5: OTHER COMPOUNDS Chlorine and inorganic compounds; Fluorine and inorganic compounds; PM10 Combustion installations >300 MW Energy industries Hydrogen cyanide Characteristic processes in the manufacture of metals (ferrous and non-ferrous) and metal product (metal industry and metal ore roasting or sintering installations) Production and processing of metals 1 Industrial activity that generates the highest emissions for each pollutant. Description of industrial air pollution, by pollutant and associated industrial activity (2001). http://www.biomedcentral.com/1471-2458/7/40 BMC Public Health 2007, 7:40 Figure 1 shows the geographic distribution of the foci or industrial plants for some carcinogenic pollutants. In the f i ll t t th IARC h l ifi d i b Discussion EPER provides data on emissions of key pollutants to air d t f j E i d t i l f iliti Th Table 2: Description of pollution released by industrial plants in Spain (2001), by pollutant group. Industrial plants shown by the EPER to discharge pollutant substances directly into water in Spain, and associated industrial activities AIR POLLUTION TOTAL SPAIN REGION WITH HIGHEST VALUE Emission (Mt/year) Region Emission (Mt/year) % Group 1: Environmental themes 157,585,773 Asturias 24,103,456 (15%) Group 2: Metals and compounds 1,048 Basque Country 496 (47%) Group 3: Chlorinated organic substances 240 Basque Country 77 (32%) Group 4: Other organic compounds 218 Andalusia 109 (50%) Group 5: Other compounds 47,887 Castile & Leon 11,560 (24%) WATER POLLUTION (DIRECT) TOTAL SPAIN REGION WITH HIGHEST VALUE Emission (Mt/year) Region Emission (Mt/year) % Group 1: Environmental themes 5,790 Andalusia 1,925 (33%) Group 2: Metals and compounds 167 Cantabria 99 (59%) Group 3: Chlorinated organic substances 320 Andalusia 101 (31%) Group 4: Other organic compounds 31,470 Cantabria 11,930 (37%) Group 5: Other compounds 7,696,422 Basque Country 6,517,962 (85%) WATER POLLUTION (INDIRECT) WATER POLLUTION (DIRECT) TOTAL SPAIN REGION WITH HIGHEST VALUE Emission (Mt/year) Region Emission (Mt/year) % Group 1: Environmental themes 5,790 Andalusia 1,925 (33%) Group 2: Metals and compounds 167 Cantabria 99 (59%) Group 3: Chlorinated organic substances 320 Andalusia 101 (31%) Group 4: Other organic compounds 31,470 Cantabria 11,930 (37%) Group 5: Other compounds 7,696,422 Basque Country 6,517,962 (85%) WATER POLLUTION (DIRECT) WATER POLLUTION (INDIRECT) TOTAL SPAIN REGION WITH HIGHEST VALUE Emission (Mt/year) Region Emission (Mt/year) % Group 1: Environmental themes 8,458 Andalusia 7,257 (86%) Group 2: Metals and compounds 38 Basque Country 20 (53%) Group 3: Chlorinated organic substances 87 Basque Country 72 (82%) Group 4: Other organic compounds 53,348 Andalusia 37,558 (70%) Group 5: Other compounds 366,784 Andalusia 312,204 (85%) WATER POLLUTION (INDIRECT) Figure 1 shows the geographic distribution of the foci or industrial plants for some carcinogenic pollutants. In the case of air pollutants, the IARC has classified arsenic, ben- zene, cadmium and chromium as carcinogens (group 1), trichloroethylene as a probable carcinogen (group 2A), and dichloromethane as a possible carcinogen (group 2B). In the case of pollutants discharged direct to water, lead and nickel, both of which have been classified as pos- sible carcinogens by the IARC, are shown [13]. Industrial plants shown by the EPER to discharge pollutant substances directly into water in Spain, and associated industrial activities AIR POLLUTION TOTAL SPAIN REGION WITH HIGHEST VALUE Emission (Mt/year) Region Emission (Mt/year) % Group 1: Environmental themes 157,585,773 Asturias 24,103,456 (15%) Group 2: Metals and compounds 1,048 Basque Country 496 (47%) Group 3: Chlorinated organic substances 240 Basque Country 77 (32%) Group 4: Other organic compounds 218 Andalusia 109 (50%) Group 5: Other compounds 47,887 Castile & Leon 11,560 (24%) WATER POLLUTION (DIRECT) TOTAL SPAIN REGION WITH HIGHEST VALUE Emission (Mt/year) Region Emission (Mt/year) % Group 1: Environmental themes 5,790 Andalusia 1,925 (33%) Group 2: Metals and compounds 167 Cantabria 99 (59%) Group 3: Chlorinated organic substances 320 Andalusia 101 (31%) Group 4: Other organic compounds 31,470 Cantabria 11,930 (37%) Group 5: Other compounds 7,696,422 Basque Country 6,517,962 (85%) WATER POLLUTION (INDIRECT) TOTAL SPAIN REGION WITH HIGHEST VALUE Emission (Mt/year) Region Emission (Mt/year) % Group 1: Environmental themes 8,458 Andalusia 7,257 (86%) Group 2: Metals and compounds 38 Basque Country 20 (53%) Group 3: Chlorinated organic substances 87 Basque Country 72 (82%) Group 4: Other organic compounds 53,348 Andalusia 37,558 (70%) Group 5: Other compounds 366,784 Andalusia 312,204 (85%) Table 2: Description of pollution released by industrial plants in Spain (2001), by pollutant group. Discussion EPER provides data on emissions of key pollutants to air and water from major European industrial facilities. The first set of emissions data, published in February 2004, should be used with caution because of several limita- tions. One of them is the non-registered industrial plants and/or unquantified emissions, due to the fact that indus- tries are still in the phase of adaptation to EPER regula- tions and the emission reporting will not become compulsory until 2007. Therefore, data were gathered in 2001 from industries participating in EPER in a voluntary way. In view of the EPER's novelty, the completeness or data quality of this register is not well-known, though given the IPPC's regulatory stance with respect to indus- trial activity, sufficient quality for the proposed descrip- tion can be reasonably assumed. However, the 'picture' derived from our study could change in part if complete- ness and accuracy data were improved. Finally, Figure 2 sets out the percentage emissions recorded by the EPER-Europe of pollutant substances released into the air in the European Union (for 2001). With respect to Spain, note should be taken of the per- centage emissions of hexachlorobenzene substances (Spain accounting for 100% of reported emissions), and chromium, nickel and zinc (Spain accounting for around 40% of reported European emissions). Page 5 of 13 (page number not for citation purposes) http://www.biomedcentral.com/1471-2458/7/40 BMC Public Health 2007, 7:40 On the other hand, it should be noted that reported emis- sion data can be obtained by monitoring or modelling. There are three possible codes to indicate the emission determination method for the reported emission data: Table 3: Description of pollution discharged into the air by industrial plants in Spain (2001), by specific pollutant. Discussion POLLUTANTS TOTAL SPAIN AUTONOMOUS REGIONS WITH HIGHEST EMISSIONS SUBSTANCES THRESHOL D (Mt/year)1 EMISSION (Mt/year)2 PLANTS3 MEAN EMISSION PER PLANT (Mt/ year)4 REGION EMISSION (Mt/year)2 PLA NTS3 MEAN EMISSION PER PLANT (Mt/ year)4 GROUP 1: ENVIRONMENTAL THEMES Methane 100 78670 58 1356 Aragon 19048 6 3175 Carbon monoxide 500 243834 56 4354 Asturias 119754 5 23951 Carbon dioxide 100000 155211000 153 1014451 Asturias 23823000 13 1832538 Hydrofluorocarbons 0.1 276 5 55 Catalonia 193 4 48 Nitrous oxide 10 6208 37 168 Castile-La Mancha 1494 6 249 Ammonia 10 24049 815 30 Aragon 10015 399 25 Non-methane volatile organic compounds 100 63781 60 1063 Andalusia 16810 7 2401 Nitrogen dioxide 100 801301 217 3693 Catalonia 394548 33 11956 Perfluorocarbons 0.1 30 4 8 Galicia 18 2 9 Sulphur dioxide 150 1156625 156 7414 Galicia 412414 12 34368 GROUP 2: METALS AND COMPOUNDS Arsenic 0.02 6 33 0.17 Andalusia 2 8 0.20 Cadmium 0.01 5 46 0.11 Basque Country 1 13 0.10 Chromium 0.10 80 40 2 Basque Country 44 11 4 Copper 0.10 29 28 1 Andalusia 15 6 2 Mercury 0.01 3 34 0.08 Catalonia 0.5 8 0.06 Nickel 0.05 171 96 2 Andalusia 42 18 2 Lead 0.20 100 50 2 Basque Country 53 14 4 Zinc 0.20 655 43 15 Basque Country 368 15 25 GROUP 3: CHLORINATED ORGANIC SUBSTANCES Dichloroethane-1,2 1 3 1 3 Catalonia 3 1 3 Dichloromethane 1 61 4 15 Madrid Region 29 1 29 Hexachlorobenzene 0.01 0.03 2 0.02 Extremadura 0.02 1 0.02 Dioxins and furans 0.000001 0.0002 11 0.00002 Basque Country 0.0001 2 0.00005 Tetrachloroethylene 2 72 1 72 Basque Country 72 1 72 Tetrachloromethane 0.10 0.50 1 0.50 Catalonia 0.50 1 0.50 Trichloroethylene 2 81 3 27 Castile & Leon 62 1 62 Trichloromethane 0.50 23 2 12 Andalusia 18 1 18 GROUP 4: OTHER ORGANIC COMPOUNDS Benzene 1 197 20 10 Andalusia 97 5 19 Polyciclic aromatic hydrocarbons 0.05 21 16 1 Andalusia 12 3 4 GROUP 5: OTHER COMPOUNDS Chlorine and inorganic compounds 10 2217 44 50 Andalusia 1582 13 122 Fluorine and inorganic compounds 5 2091 35 60 Andalusia 1122 15 75 HCN 0.2 4 6 0.6 Basque Country 4 6 0.6 PM10 50 43575 119 366 Castile & Leon 11547 10 1155 1 Established air emission threshold or limit value (in Mt/year) for each pollutant. Discussion Should any plant exceed this threshold, it must report the release of the pollutant (voluntarily) 2 Amount of pollutant (in Mt/year) released by industrial plants 3 Number of industrial plants that discharge said pollutant 4 Mean emission = Emission/Plants Table 3: Description of pollution discharged into the air by industrial plants in Spain (2001), by specific pollutant. n of pollution discharged into the air by industrial plants in Spain (2001), by specific pollutant. There are three possible codes to indicate the emission determination method for the reported emission data: There are three possible codes to indicate the emission determination method for the reported emission data: On the other hand, it should be noted that reported emis- sion data can be obtained by monitoring or modelling. Page 6 of 13 (page number not for citation purposes) BMC Public Health 2007, 7:40 http://www.biomedcentral.com/1471-2458/7/40 Table 4: Description of pollution directly discharged to water by industrial plants in Spain (2001), by specific pollutant. Discussion POLLUTANTS TOTAL SPAIN AUTONOMOUS REGIONS WITH HIGHEST EMISSIONS SUBSTANCES THRESHOLD (Mt/year)1 EMISSION (Mt/year)2 PLANTS3 MEAN EMISSION PER PLANT (Mt/year)4 REGION EMISSION (Mt/year)2 PLANTS3 MEAN EMISSION PER PLANT (Mt/year)4 GROUP 1: ENVIRONMENTAL THEMES Nitrogen 50 5255 18 292 Andalusia 1759 4 440 Phosphorus 5 535 25 21 Aragon 175 3 58 GROUP 2: METALS AND COMPOUNDS Arsenic 0.005 0.412 11 0.037 Catalonia 0.161 3 0.054 Cadmium 0.005 2 16 0.106 Andalusia 1 8 0.133 Chromium 0.050 7 24 0.293 Catalonia 2 7 0.336 Copper 0.050 9 28 0.309 Basque Country 3 5 0.690 Mercury 0.001 0.281 17 0.017 Cantabria 0.102 2 0.051 Nickel 0.020 9 33 0.275 Aragon 3 2 2 Lead 0.020 3 18 0.187 Andalusia 1 5 0.208 Zinc 0.100 137 37 4 Cantabria 97 6 16 GROUP 3: CHLORINATED ORGANIC SUBSTANCES Dichloroethane-1,2 0.010 0.332 2 0.166 Catalonia 0.308 1 0.308 Chloroalkanes 0.001 0.186 2 0.093 Cantabria 0.161 1 0.161 Hexachlorobutadien e 0.001 0.001 1 0.001 Catalonia 0.001 1 0.001 Halogenated organic compounds 1 319 16 20 Andalusia 100 3 33 GROUP 4: OTHER ORGANIC COMPOUNDS Benzene, toluene, ethylbenzene, xylenes 0.200 64 4 16 Cantabria 47 1 47 Polycyclic aromatic hydrocarbons 0.005 7 6 1 Cantabria 5 2 2 Phenols 0.020 24 17 1 Andalusia 17 4 4 Total organic carbon 50 31375 55 570 Cantabria 11878 6 1980 GROUP 5: OTHER COMPOUNDS Chlorides 2000 7696120 11 699647 Basque Country 6517950 3 2172650 Cyanides 0.050 153 5 31 Asturias 151 1 151 Fluorides 2 149 15 10 Asturias 60 2 30 scription of pollution directly discharged to water by industrial plants in Spain (2001), by specific pollutant. Page 7 of 13 (page number not for citation purposes) BMC Public Health 2007, 7:40 http://www.biomedcentral.com/1471-2458/7/40 Code M: is used when the emissions of a facility are derived from direct monitoring results for specific proc- esses at the facility, based on actual measurements of pol- lutant concentrations for a given discharge route. Code C: emission data are based on calculations using nationally or internationally agreed estimation methods (like as fuel used, production rate) and emission factors, which are representative for the industrial sectors. Further- Table 5: Description of pollution indirectly discharged to water by industrial plants in Spain (2001), by specific pollutant. Discussion Page 8 of 13 (page number not for citation purposes) http://www.biomedcentral.com/1471-2458/7/40 BMC Public Health 2007, 7:40 phic distribution of industrial foci, by specific pollutant 1 Total industries: 33 Total emission: 5.6 Mt Autonomous regions Andalusia: 1.6 Mt Basque Country: 1.5 Mt Asturias: 0.5 Mt Castile-La Mancha: 0.4 Mt Castile & Leon: 0.4 Mt C. Valenciana: 0.3 Mt Galicia: 0.3 Mt Cantabria: 0.2 Mt Murcia Region: 0.2 Mt Extremadura: 0.1 Mt Catalonia: 0.03 Mt Total industries: 33 Total emission: 5.6 Mt Autonomous regions Andalusia: 1.6 Mt Basque Country: 1.5 Mt Asturias: 0.5 Mt Castile-La Mancha: 0.4 Mt Castile & Leon: 0.4 Mt C. Valenciana: 0.3 Mt Galicia: 0.3 Mt Cantabria: 0.2 Mt Murcia Region: 0.2 Mt Extremadura: 0.1 Mt Catalonia: 0.03 Mt Total industries: 46 Total emission: 4.9 Mt Autonomous regions Basque Country: 1.4 Mt Cantabria: 0.8 Mt Castile-La Mancha: 0.7 Mt C. Valenciana: 0.4 Mt Murcia Region: 0.3 Mt Andalusia: 0.3 Mt Galicia: 0.3 Mt Asturias: 0.2 Mt Catalonia: 0.2 Mt Extremadura: 0.1 Mt Castile & Leon: 0.1 Mt Canary Islands: 0.01 Mt Total industries: 46 Total emission: 4.9 Mt Autonomous regions Basque Country: 1.4 Mt Cantabria: 0.8 Mt Castile-La Mancha: 0.7 Mt C. Valenciana: 0.4 Mt Murcia Region: 0.3 Mt Andalusia: 0.3 Mt Galicia: 0.3 Mt Asturias: 0.2 Mt Catalonia: 0.2 Mt Extremadura: 0.1 Mt Castile & Leon: 0.1 Mt Canary Islands: 0.01 Mt Total industries: 33 Total emission: 9.1 Mt Autonomous regions Aragon: 3.3 Mt Catalonia: 1.2 Mt Asturias: 1.1 Mt Andalusia: 1.1 Mt Basque Country: 1.0 Mt Cantabria: 0.7 Mt Castile & Leon: 0.3 Mt C. Valenciana: 0.2 Mt Galicia: 0.1 Mt Total industries: 33 Total emission: 9.1 Mt Autonomous regions Aragon: 3.3 Mt Catalonia: 1.2 Mt Asturias: 1.1 Mt Andalusia: 1.1 Mt Basque Country: 1.0 Mt Cantabria: 0.7 Mt Castile & Leon: 0.3 Mt C. Valenciana: 0.2 Mt Galicia: 0.1 Mt Total industries: 3 Total emission: 81.3 Mt Autonomous regions Castile & Leon: 62.4 Mt Andalusia: 13.1 Mt Catalonia: 5.8 Mt Total industries: 3 Total emission: 81.3 Mt Autonomous regions Castile & Leon: 62.4 Mt Andalusia: 13.1 Mt Catalonia: 5.8 Mt Total industries: 20 Total emission: 196.5 Mt Autonomous regions Andalusia: 97.3 Mt Asturias: 55.9 Mt Basque Country: 18.2 Mt C. Valenciana: 9.3 Mt Aragon: 8.0 Mt Canary Islands: 6.6 Mt Catalonia: 1.3 Mt Total industries: 20 Total emission: 196.5 Mt Autonomous regions Andalusia: 97.3 Mt Asturias: 55.9 Mt Basque Country: 18.2 Mt C. Discussion Valenciana: 1.0 Mt Murcia region: 0.8 Mt Galicia: 0.6 Mt Castile & Leon: 0.4 Mt Total industries: 33 Total emission: 9.1 Mt Autonomous regions Aragon: 3.3 Mt Catalonia: 1.2 Mt Asturias: 1.1 Mt Andalusia: 1.1 Mt Basque Country: 1.0 Mt Cantabria: 0.7 Mt Castile & Leon: 0.3 Mt C. Valenciana: 0.2 Mt Galicia: 0.1 Mt Total industries: 33 Total emission: 9.1 Mt Autonomous regions Aragon: 3.3 Mt Catalonia: 1.2 Mt Asturias: 1.1 Mt Andalusia: 1.1 Mt Basque Country: 1.0 Mt Cantabria: 0.7 Mt Castile & Leon: 0.3 Mt C. Valenciana: 0.2 Mt Galicia: 0.1 Mt Total industries: 3 Total emission: 81.3 Mt Autonomous regions Castile & Leon: 62.4 Mt Andalusia: 13.1 Mt Catalonia: 5.8 Mt Total industries: 3 Total emission: 81.3 Mt Autonomous regions Castile & Leon: 62.4 Mt Andalusia: 13.1 Mt Catalonia: 5.8 Mt Total industries: 4 Total emission: 60.6 Mt Autonomous regions Madrid Region: 28.8 Mt Catalonia: 22.3 Mt Asturias: 18.2 Mt Total industries: 4 Total emission: 60.6 Mt Autonomous regions Madrid Region: 28.8 Mt Catalonia: 22.3 Mt Asturias: 18.2 Mt Total industries: 46 Total emission: 4.9 Mt Autonomous regions Basque Country: 1.4 Mt Cantabria: 0.8 Mt Castile-La Mancha: 0.7 Mt C. Valenciana: 0.4 Mt Murcia Region: 0.3 Mt Andalusia: 0.3 Mt Galicia: 0.3 Mt Asturias: 0.2 Mt Catalonia: 0.2 Mt Extremadura: 0.1 Mt Castile & Leon: 0.1 Mt Canary Islands: 0.01 Mt Total industries: 46 Total emission: 4.9 Mt Autonomous regions Basque Country: 1.4 Mt Cantabria: 0.8 Mt Castile-La Mancha: 0.7 Mt C. Valenciana: 0.4 Mt Murcia Region: 0.3 Mt Andalusia: 0.3 Mt Galicia: 0.3 Mt Asturias: 0.2 Mt Catalonia: 0.2 Mt Extremadura: 0.1 Mt Castile & Leon: 0.1 Mt Canary Islands: 0.01 Mt Total industries: 40 Total emission: 80.3 Mt Autonomous regions Basque Country: 43.8 Mt Andalusia: 23.3 Mt Asturias: 2.7 Mt Extremadura: 2.0 Mt Castile-La Mancha: 1.8 Mt Catalonia: 1.5 Mt Cantabria: 1.3 Mt Aragon: 1.2 Mt C. Valenciana: 1.0 Mt Murcia region: 0.8 Mt Galicia: 0.6 Mt Castile & Leon: 0.4 Mt Total industries: 40 Total emission: 80.3 Mt Autonomous regions Basque Country: 43.8 Mt Andalusia: 23.3 Mt Asturias: 2.7 Mt Extremadura: 2.0 Mt Castile-La Mancha: 1.8 Mt Catalonia: 1.5 Mt Cantabria: 1.3 Mt Aragon: 1.2 Mt C. Valenciana: 1.0 Mt Murcia region: 0.8 Mt Galicia: 0.6 Mt Castile & Leon: 0.4 Mt Total industries: 46 Total emission: 4.9 Mt Autonomous regions Basque Country: 1.4 Mt Cantabria: 0.8 Mt Castile-La Mancha: 0.7 Mt C. Discussion Valenciana: 0.4 Mt Murcia Region: 0.3 Mt Andalusia: 0.3 Mt Galicia: 0.3 Mt Asturias: 0.2 Mt Catalonia: 0.2 Mt Extremadura: 0.1 Mt Castile & Leon: 0.1 Mt Canary Islands: 0.01 Mt Total industries: 46 Total emission: 4.9 Mt Autonomous regions Basque Country: 1.4 Mt Cantabria: 0.8 Mt Castile-La Mancha: 0.7 Mt C. Valenciana: 0.4 Mt Murcia Region: 0.3 Mt Andalusia: 0.3 Mt Galicia: 0.3 Mt Asturias: 0.2 Mt Catalonia: 0.2 Mt Extremadura: 0.1 Mt Castile & Leon: 0.1 Mt Canary Islands: 0.01 Mt Total industries: 3 Total emission: 81.3 Mt Autonomous regions Castile & Leon: 62.4 Mt Andalusia: 13.1 Mt Catalonia: 5.8 Mt Total industries: 3 Total emission: 81.3 Mt Autonomous regions Castile & Leon: 62.4 Mt Andalusia: 13.1 Mt Catalonia: 5.8 Mt Total industries: 3 Total emission: 81.3 Mt Autonomous regions Castile & Leon: 62.4 Mt Andalusia: 13.1 Mt Catalonia: 5.8 Mt Total industries: 3 Total emission: 81.3 Mt Autonomous regions Castile & Leon: 62.4 Mt Andalusia: 13.1 Mt Catalonia: 5.8 Mt Total industries: 4 Total emission: 60.6 Mt Autonomous regions Madrid Region: 28.8 Mt Catalonia: 22.3 Mt Asturias: 18.2 Mt Total industries: 4 Total emission: 60.6 Mt Autonomous regions Madrid Region: 28.8 Mt Catalonia: 22.3 Mt Asturias: 18.2 Mt Geographic distribution of industrial foci, by specific pollutant Figure 1 Geographic distribution of industrial foci, by specific pollutant. Total industries: 18 Total emission: 3.4 Mt Autonomous regions Andalusia: 1.0 Mt Asturias: 0.7 Mt Castile & Leon: 0.5 Mt Cantabria: 0.3 Mt Catalonia: 0.3 Mt Basque Country: 0.2 Mt C. Valenciana: 0.2 Mt Navarra: 0.1 Mt Total industries: 18 Total emission: 3.4 Mt Autonomous regions Andalusia: 1.0 Mt Asturias: 0.7 Mt Castile & Leon: 0.5 Mt Cantabria: 0.3 Mt Catalonia: 0.3 Mt Basque Country: 0.2 Mt C. Valenciana: 0.2 Mt Navarra: 0.1 Mt Total industries: 33 Total emission: 9.1 Mt Autonomous regions Aragon: 3.3 Mt Catalonia: 1.2 Mt Asturias: 1.1 Mt Andalusia: 1.1 Mt Basque Country: 1.0 Mt Cantabria: 0.7 Mt Castile & Leon: 0.3 Mt C. Valenciana: 0.2 Mt Galicia: 0.1 Mt Total industries: 33 Total emission: 9.1 Mt Autonomous regions Aragon: 3.3 Mt Catalonia: 1.2 Mt Asturias: 1.1 Mt Andalusia: 1.1 Mt Basque Country: 1.0 Mt Cantabria: 0.7 Mt Castile & Leon: 0.3 Mt C. Discussion POLLUTANTS TOTAL SPAIN AUTONOMOUS REGIONS WITH HIGHEST EMISSIONS SUBSTANCES THRESHOLD (Mt/year)1 EMISSION (Mt/year)2 PLANTS3 MEAN EMISSION PER PLANT (Mt/year)4 REGION EMISSION (Mt/year)2 PLANTS3 MEAN EMISSION PER PLANT (Mt/year)4 GROUP 1: ENVIRONMENTAL THEMES Nitrogen 50 6580 11 598 Andalusia 5673 3 1891 Phosphorus 5 1877 26 72 Andalusia 1584 7 226 GROUP 2: METALS AND COMPOUNDS Arsenic 0.005 0.162 4 0.041 Galicia 0.130 1 0.130 Cadmium 0.005 0.370 15 0.025 Cantabria 0.150 1 0.150 Chromium 0.050 5 19 0.261 Basque Country 3 5 0.579 Copper 0.050 4 18 0.221 Basque Country 2 6 0.251 Mercury 0.001 0.078 5 0.016 Basque Country 0.047 2 0.023 Nickel 0.020 8 39 0.206 Aragon 3 2 2 Lead 0.020 2 15 0.141 Cantabria 0.882 1 0.882 Zinc 0.100 18 25 0.738 Basque Country 14 8 2 GROUP 3: CHLORINATED ORGANIC SUBSTANCES Dichloroethane-1,2 0.010 0.023 1 0.023 Catalonia 0.023 1 0.023 Dichloromethane 0.010 0.212 1 0.212 Catalonia 0.212 1 0.212 Chloroalkanes 0.001 0.016 2 0.008 Catalonia 0.016 2 0.008 Halogenated organic compounds 1 87 7 12 Basque Country 72 3 24 GROUP 4: OTHER ORGANIC COMPOUNDS Benzene, toluene, ethylbenzene, xylenes 0.200 10 3 3 Catalonia 9 2 5 Organotin- compounds 0.050 0.154 1 0.154 Andalusia 0.154 1 0.154 Polycyclic aromatic hydrocarbons 0.005 2 7 0.265 Catalonia 2 3 0.502 Phenols 0.020 4 21 0.184 Basque Country 2 3 0.505 Total organic carbon 50 53332 65 820 Andalusia 37557 11 3414 GROUP 5: OTHER COMPOUNDS Chlorides 2000 366750 8 45844 Andalusia 312200 2 156100 Cyanides 0.050 4 4 1 Catalonia 4 2 2 Fluorides 2 30 4 7 Basque Country 26 3 9 ption of pollution indirectly discharged to water by industrial plants in Spain (2001), by specific pollutant. Table 5: Description of pollution indirectly discharged to water by industrial plants in Spain (2001), by spe GROUP 3: CHLORINATED ORGANIC SUBSTANCES Code C: emission data are based on calculations using nationally or internationally agreed estimation methods (like as fuel used, production rate) and emission factors, which are representative for the industrial sectors. Further- Code M: is used when the emissions of a facility are derived from direct monitoring results for specific proc- esses at the facility, based on actual measurements of pol- lutant concentrations for a given discharge route. Discussion Valenciana: 9.3 Mt Aragon: 8.0 Mt Canary Islands: 6.6 Mt Catalonia: 1.3 Mt Total industries: 33 Total emission: 5.6 Mt Autonomous regions Andalusia: 1.6 Mt Basque Country: 1.5 Mt Asturias: 0.5 Mt Castile-La Mancha: 0.4 Mt Castile & Leon: 0.4 Mt C. Valenciana: 0.3 Mt Galicia: 0.3 Mt Cantabria: 0.2 Mt Murcia Region: 0.2 Mt Extremadura: 0.1 Mt Catalonia: 0.03 Mt Total industries: 33 Total emission: 5.6 Mt Autonomous regions Andalusia: 1.6 Mt Basque Country: 1.5 Mt Asturias: 0.5 Mt Castile-La Mancha: 0.4 Mt Castile & Leon: 0.4 Mt C. Valenciana: 0.3 Mt Galicia: 0.3 Mt Cantabria: 0.2 Mt Murcia Region: 0.2 Mt Extremadura: 0.1 Mt Catalonia: 0.03 Mt Geographic distribution of industrial foci, by specific pollutant Figure 1 Geographic distribution of industrial foci, by specific pollutant. Total industries: 18 Total emission: 3.4 Mt Autonomous regions Andalusia: 1.0 Mt Asturias: 0.7 Mt Castile & Leon: 0.5 Mt Cantabria: 0.3 Mt Catalonia: 0.3 Mt Basque Country: 0.2 Mt C. Valenciana: 0.2 Mt Navarra: 0.1 Mt Total industries: 18 Total emission: 3.4 Mt Autonomous regions Andalusia: 1.0 Mt Asturias: 0.7 Mt Castile & Leon: 0.5 Mt Cantabria: 0.3 Mt Catalonia: 0.3 Mt Basque Country: 0.2 Mt C. Valenciana: 0.2 Mt Navarra: 0.1 Mt Total industries: 46 Total emission: 4.9 Mt Autonomous regions Basque Country: 1.4 Mt Cantabria: 0.8 Mt Castile-La Mancha: 0.7 Mt C. Valenciana: 0.4 Mt Murcia Region: 0.3 Mt Andalusia: 0.3 Mt Galicia: 0.3 Mt Asturias: 0.2 Mt Catalonia: 0.2 Mt Extremadura: 0.1 Mt Castile & Leon: 0.1 Mt Canary Islands: 0.01 Mt Total industries: 46 Total emission: 4.9 Mt Autonomous regions Basque Country: 1.4 Mt Cantabria: 0.8 Mt Castile-La Mancha: 0.7 Mt C. Valenciana: 0.4 Mt Murcia Region: 0.3 Mt Andalusia: 0.3 Mt Galicia: 0.3 Mt Asturias: 0.2 Mt Catalonia: 0.2 Mt Extremadura: 0.1 Mt Castile & Leon: 0.1 Mt Canary Islands: 0.01 Mt Total industries: 40 Total emission: 80.3 Mt Autonomous regions Basque Country: 43.8 Mt Andalusia: 23.3 Mt Asturias: 2.7 Mt Extremadura: 2.0 Mt Castile-La Mancha: 1.8 Mt Catalonia: 1.5 Mt Cantabria: 1.3 Mt Aragon: 1.2 Mt C. Valenciana: 1.0 Mt Murcia region: 0.8 Mt Galicia: 0.6 Mt Castile & Leon: 0.4 Mt Total industries: 40 Total emission: 80.3 Mt Autonomous regions Basque Country: 43.8 Mt Andalusia: 23.3 Mt Asturias: 2.7 Mt Extremadura: 2.0 Mt Castile-La Mancha: 1.8 Mt Catalonia: 1.5 Mt Cantabria: 1.3 Mt Aragon: 1.2 Mt C. Discussion Valenciana: 0.2 Mt Galicia: 0.1 Mt Total industries: 18 Total emission: 3.4 Mt Autonomous regions Andalusia: 1.0 Mt Asturias: 0.7 Mt Castile & Leon: 0.5 Mt Cantabria: 0.3 Mt Catalonia: 0.3 Mt Basque Country: 0.2 Mt C. Valenciana: 0.2 Mt Navarra: 0.1 Mt Total industries: 18 Total emission: 3.4 Mt Autonomous regions Andalusia: 1.0 Mt Asturias: 0.7 Mt Castile & Leon: 0.5 Mt Cantabria: 0.3 Mt Catalonia: 0.3 Mt Basque Country: 0.2 Mt C. Valenciana: 0.2 Mt Navarra: 0.1 Mt Geographic distribution of industrial foci, by specific pollutant Figure 1 Geographic distribution of industrial foci, by specific pollutant. Total industries: 18 Total emission: 3.4 Mt Autonomous regions Andalusia: 1.0 Mt Asturias: 0.7 Mt Castile & Leon: 0.5 Mt Cantabria: 0.3 Mt Catalonia: 0.3 Mt Basque Country: 0.2 Mt C. Valenciana: 0.2 Mt Navarra: 0.1 Mt Total industries: 18 Total emission: 3.4 Mt Autonomous regions Andalusia: 1.0 Mt Asturias: 0.7 Mt Castile & Leon: 0.5 Mt Cantabria: 0.3 Mt Catalonia: 0.3 Mt Basque Country: 0.2 Mt C. Valenciana: 0.2 Mt Navarra: 0.1 Mt Total industries: 33 Total emission: 9.1 Mt Autonomous regions Aragon: 3.3 Mt Catalonia: 1.2 Mt Asturias: 1.1 Mt Andalusia: 1.1 Mt Basque Country: 1.0 Mt Cantabria: 0.7 Mt Castile & Leon: 0.3 Mt C. Valenciana: 0.2 Mt Galicia: 0.1 Mt Total industries: 33 Total emission: 9.1 Mt Autonomous regions Aragon: 3.3 Mt Catalonia: 1.2 Mt Asturias: 1.1 Mt Andalusia: 1.1 Mt Basque Country: 1.0 Mt Cantabria: 0.7 Mt Castile & Leon: 0.3 Mt C. Valenciana: 0.2 Mt Galicia: 0.1 Mt Geographic distribution of industrial foci, by specific pollutant Figure 1 Geographic distribution of industrial foci, by specific pollutant. Page 9 of 13 (page number not for citation purposes) BMC Public Health 2007, 7:40 http://www.biomedcentral.com/1471-2458/7/40 missions of pollutant substances released to air in the European Union missions of pollutant substances released to air in the European Union. %EMISSION COUNTRIES 20 40 60 80 Austria Belgium Denmark Finland France Germany Greece Ireland Italy Luxembourg Netherlands Portugal Spain Sweden U. Kingdom Ammonia 20 40 60 80 Arsenic 20 40 60 80 Benzene 20 40 60 80 Cadmium 20 40 60 80 Carbon dioxide 20 40 60 80 Carbon monoxide 20 40 60 80 Chroline 20 40 60 80 Chromium Austria Belgium Denmark Finland France Germany Greece Ireland Italy Luxembourg Netherlands Portugal Spain Sweden U. http://www.biomedcentral.com/1471-2458/7/40 http://www.biomedcentral.com/1471-2458/7/40 http://www.biomedcentral.com/1471-2458/7/40 http://www.biomedcentral.com/1471-2458/7/40 BMC Public Health 2007, 7:40 more, this code is used when the emission calculation methods is obtained from published references [14,15]. more, this code is used when the emission calculation methods is obtained from published references [14,15]. Analysis of the comparative percentage emissions of pol- lutant substances released to air in the 15 European Union countries shows that Spain features as a polluter in 32 substances. In percentage terms it ranks: as the leading polluter in 10 of these (arsenic, copper, chromium, nitro- gen dioxide, hexachlorobenzene, hydrofluorocarbons, nickel, sulphur oxides, PM10 and zinc); as the second- leading polluter in 3 more (ammonia, cadmium and flu- orine); and as the third-leading polluter and in a further 7 (non-methane volatile organic compounds, dioxins and furans, polycyclic aromatic hydrofluorocarbons, lead, tet- rachloroethylene, trichloroethylene and trichlorometh- ane). According to data released by EPER in 2004, Spain would be the leading polluter in almost one third of all EPER-registered pollutant substances released into the air and ranks among the top three leading polluters in two- thirds of all such substances. It should be noted that this situation can reflect differences in reporting between countries. Code E: emission data are based on non-standardised esti- mations derived from best assumptions or expert judge- ment. In this first approach, analysis of the EPER 2001 pollution data reveals that industrial air pollution is more intense for substances grouped under: "Environmental themes" in Andalusia, Aragon, Asturias, Castile la Mancha, Catalo- nia and Galicia; "Metals and compounds" in Andalusia, Catalonia and the Basque Country; "Chlorinated organic substances" in Andalusia, Castile & Leon, Catalonia, Madrid Autonomous Region, Extremadura and the Basque Country; "Other organic compounds" in Andalu- sia; and "Other compounds" in Andalusia, Castile & Leon and the Basque Country. On the other hand, industrial pollution discharged directly into water proved more intense for substances grouped under: "Environmental themes" in Andalusia and Aragon; "Metals and compounds" in Andalusia, Aragon, Cantabria, Catalonia and the Basque Country; "Chlorinated organic substances" in Andalusia, Cantabria and Catalonia; "Other organic compounds" in Andalusia and Cantabria; and "Other compounds" in Asturias and the Basque Country. With regard to release of substances to air, in which Spain is pre-eminent vis-à-vis the remaining European coun- tries, hexachlorobenzene & a secondary product formed during the manufacture of other chemical substances – has been classified by the IARC as a possible carcinogen to human beings (group 2B) [13]. http://www.biomedcentral.com/1471-2458/7/40 Its principal health effects stem from ingesting products highly contaminated with this substance [16]. Zinc, whose principal exposure occurs when eating food, drinking water or breathing air pol- luted with this compound [17], has not been classified by the IARC in terms of carcinogenicity [13], but can none- theless cause a number of disorders. The IARC has con- cluded that some nickel compounds are carcinogenic to humans (group 1) and that metallic nickel is possibly car- cinogenic to humans (group 2B) [13]. Its most harmful effects are seen when large amounts of compounds of this substance are inhaled [18]. The IARC has decided that chromium compounds (VI) are carcinogenic to humans (group 1) [13] and may increase the risk of contracting lung cancer. The principal health effects follow on from inhaling high levels of this compound [19]. Some studies conducted in Spain have found evidence of risk posed to the population living near industries that release some of these compounds [20-22]. Lastly, industrial pollution discharged indirectly into water (via sewage treatment plants) was more intense for substances grouped under: "Environmental themes" in Andalusia; "Metals and compounds" in Aragon, Cantab- ria, Galicia and the Basque Country; "Chlorinated organic substances" in Catalonia and Basque Country; "Other organic compounds" in Andalusia, Catalonia and the Basque Country; and "Other compounds" in Andalusia, Catalonia and the Basque Country. In Spain, a total of 655 towns have at least one EPER reg- istered facility (excluding farms) in their administrative limit. A total of 215 towns have at least one pollutant facility located within two kilometres from their town centroid (centre point of town) with a population esti- mated at 500,000 inhabitants. These figures have been calculated after a thorough quality control of the facility UTM coordinates provided by EPER. The EPER contains data on the main pollutant emissions to air and water reported by over 10000 medium- and large-sized industrial installations in 17 European coun- tries. Online information searches can be made via the EPER web page, according to type of industrial plant, industrial activity, area, year and pollutant. It is a user- friendly register, from which, not only tables, but also crude data on pollutant emissions and interactive maps can be obtained. Page 11 of 13 (page number not for citation purposes) Discussion Kingdom Copper Dichloroethane-1,2 Dichloromethane Dinitrogenoxide Dioxins and furans Fluorine Hexachlorobenzene Hydrofluorocarbons Austria Belgium Denmark Finland France Germany Greece Ireland Italy Luxembourg Netherlands Portugal Spain Sweden U. Kingdom Hydrogen cyanide Lead Mercury Methane Nickel Nitrogen oxides NMVOC PAH Austria Belgium Denmark Finland France Germany Greece Ireland Italy Luxembourg Netherlands Portugal Spain Sweden U. Kingdom 20 40 60 80 Perfluorocarbons 20 40 60 80 PM10 20 40 60 80 Sulphur oxides 20 40 60 80 Tetrachloroethylene 20 40 60 80 Tetrachloromethane 20 40 60 80 Trichloroethylene 20 40 60 80 Trichloromethane 20 40 60 80 Zinc Percentage emissions of pollutant substances released to air in the European Union Figure 2 Percentage emissions of pollutant substances released to air in the European Union. Percentage emissions of pollutant substances released to air in the European Union Figure 2 Percentage emissions of pollutant substances released to air in the European Union. Page 10 of 13 (page number not for citation purposes) References 1. Benedetti M, Iavarone I, Comba P, Lavarone I: Cancer risk associ- ated with residential proximity to industrial sites: a review. Arch Environ Health 2001, 56:342-349. 1. Benedetti M, Iavarone I, Comba P, Lavarone I: Cancer risk associ- ated with residential proximity to industrial sites: a review. Arch Environ Health 2001, 56:342-349. Some authors reported statistically significant associa- tions between lung cancer risk and residential proximity to smelters, complex industrial areas, and other emission sources. There was some evidence that leukaemia and lymphomas occurred in the neighbourhoods that con- tained industrial sites [1,4,5] The modelling of distance effect to sources of pollution under isotropic or aniso- tropic assumptions is complex and there are very limited examples in the literature. To date, most pollution source studies concentrate on incidence or mortality of a single disease [26,27]. 2. Dolk H, Vrijheid M: The impact of environmental pollution on congenital anomalies. Br Med Bull 2003, 68:25-45. 2. Dolk H, Vrijheid M: The impact of environmental pollution on congenital anomalies. Br Med Bull 2003, 68:25-45. congenital anomalies. Br Med Bull 2003, 68:25-45. 3. Gottlieb MS, Shear CL, Seale DB: Lung cancer mortality and res- idential proximity to industry. Environ Health Perspect 1982, 45:157-164. congenital anomalies. Br Med Bull 2003, 68:25 45. 3. Gottlieb MS, Shear CL, Seale DB: Lung cancer mortality and res- idential proximity to industry. Environ Health Perspect 1982, 45:157-164. 4. Johnson KC, Pan S, Fry R, Mao Y: Residential proximity to indus- trial plants and non-Hodgkin lymphoma. Epidemiology 2003, 14:687-693. 4. Johnson KC, Pan S, Fry R, Mao Y: Residential proximity to indus- trial plants and non-Hodgkin lymphoma. Epidemiology 2003, 14:687-693. 5. Linos A, Blair A, Gibson RW, Everett G, Van Lier S, Cantor KP, Schu- man L, Burmeister L: Leukemia and non-Hodgkin's lymphoma and residential proximity to industrial plants. Arch Environ Health 1991, 46:70-74. 6. Sans S, Elliott P, Kleinschmidt I, Shaddick G, Pattenden S, Walls P, Grundy C, Dolk H: Cancer incidence and mortality near the Baglan Bay petrochemical works, South Wales. Occup Environ Med 1995, 52:217-224. The EPER is scheduled to be upgraded and replaced by the European Pollutant Release and Transfer Register (PRTR), which will include more comprehensive information on industrial pollution from 91 substances and 65 industrial activities, as well as information on waste management by industrial installations. Authors' contributions JGP and GLA conceived the idea and JGP wrote the man- uscript. EB contributed to manuscript writing. EB, RR, MP, BPG, NA and GLA revised the manuscript for important intellectual content. All authors contributed to the final version of the manuscript. In relation to public health, there has been growing inter- est in the development of useful statistical methods for detection of patterns of health events linked with pollu- tion sources in recent years. The information obtained through EPER and PRTR would allow to study the conse- quences of pollutant foci in population health applying focused clustering methods [25,26]. Raised incidence of the health outcomes in the target population living near to the source or directional preference related to a domi- nant wind direction may provide evidence of such a link [27]. Acknowledgements This study was supported by FIS 040041 and by the Spanish Network for Cooperative Research in Epidemiology and Public Health (RCESP-FIS C03/ 09). Competing interests The author(s) declare that they have no competing inter- ests. http://www.biomedcentral.com/1471-2458/7/40 This tool enables useful information to be generated in a public health/environmental pollution As it can be observed from the EPER, a few single indus- trial plants are responsible for the highest percentage of emissions (Tables 3, 4 and 5). Identification of a small number of high emission plants should elicit implemen- tation of adequately designed health studies in their sur- rounding. Page 11 of 13 (page number not for citation purposes) http://www.biomedcentral.com/1471-2458/7/40 http://www.biomedcentral.com/1471-2458/7/40 BMC Public Health 2007, 7:40 proximity of one or more industries on cancer- and all- cause mortality observed in the surrounding towns and villages. context, with similar limitations than other registries as, for instance, Toxic Release Inventory (TRI register) in US [23,24]. It should be noted here that the 2001-based data used for this study were in fact published in February 2004, and that in the last two years there may have been corrections by industries to the emission reports submit- ted for said year. This gives rise to slight discrepancies between the information reported in this paper and that shown on the EPER-Spain web page. context, with similar limitations than other registries as, for instance, Toxic Release Inventory (TRI register) in US [23,24]. It should be noted here that the 2001-based data used for this study were in fact published in February 2004, and that in the last two years there may have been corrections by industries to the emission reports submit- ted for said year. This gives rise to slight discrepancies between the information reported in this paper and that shown on the EPER-Spain web page. References It will also compile pollutionre- ports from a range of sources, such as road traffic, avia- tion, shipping and agriculture. The reports will be annual (rather than triennial as envisaged under the EPER), with the first becoming available in 2007. As from 2009, the PRTR will be accessible by Internet and will have replaced the EPER. 7. Benach J, Yasui Y, Borrell C, Rosa E, Pasarin MI, Benach N, Espanol E, Martinez JM, Daponte A: Examining geographic patterns of mortality: the atlas of mortality in small areas in Spain (1987-1995). Eur J Public Health 2003, 13:115-123. ( ) J 8. Lopez-Abente G, Hernandez-Barrera V, Pollan M, Aragones N, Perez- Gomez B: Municipal pleural cancer mortality in Spain. Occup Environ Med 2005, 62:195-199. 9. 9. Lopez-Abente G, Aragones N, Ramis R, Hernandez-Barrera V, Perez- Gomez B, Escolar A, Pollan M: Municipal distribution of bladder cancer mortality in Spain: Possible role of mining and indus- try. BMC Public Health 2006, 6:17. y 10. Ramis PR, Garcia-Perez J, Pollan M, Aragones N, Perez-Gomez B, Lopez-Abente G: Modelling of municipal mortality due to hae- matological neoplasias in Spain. J Epidemiol Community Health 2007, 61:165-171. 11. COMMUNITIES COMMISSIONOFTHEEUROPEAN: COMMIS- SION DECISION of 17 July 2000 on the implementation of a European pollutant emission register (EPER) according to Article 15 of Council Directive 96/61/EC concerning inte- grated pollution prevention and control (IPPC). Official Journal of the European Communities 2000, 192:36-43. Conclusion Wiley; 2003. y 27. Lawson AB: On the analysis of mortality events associated with a prespecified fixed point. J R Stat Soc Ser A Stat Soc 1993, 156:363-377. Conclusion Information obtained through publication of EPER data means that the possible consequences of reported pollut- ant foci on the health of neighbouring populations can now be studied, by analyzing geographic mortality pat- terns of different tumours with reference to the industrial emission of pollutants labelled as carcinogens. This will, in turn, make it possible to quantify the effect exerted by 12. EPER 2006 [http://eper.ec.europa.eu/eper/]. 12. EPER 2006 [http://eper.ec.europa.eu/eper/]. 13. IARC: IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. 2006 [http://monographs.iarc.fr/ENG/Mono graphs/index.php]. 13. IARC: IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. 2006 [http://monographs.iarc.fr/ENG/Mono graphs/index.php]. 14. UNECE: Task Force on Emission Inventories and Projections. 2004 [http://tfeip-secretariat.org/unece.htm]. 14. UNECE: Task Force on Emission Inventories and Projections. 2004 [http://tfeip-secretariat.org/unece.htm]. [ p p g ] 15. EMEP-CORINAIR: Emission Inventory Guidebook-2005. 2006 [http://reports.eea.europa.eu/EMEPCORINAIR4/en/page002.html]. [ p p g ] 15. EMEP-CORINAIR: Emission Inventory Guidebook-2005. 2006 [http://reports.eea.europa.eu/EMEPCORINAIR4/en/page002.html]. Page 12 of 13 (page number not for citation purposes) Page 12 of 13 (page number not for citation purposes) BMC Public Health 2007, 7:40 http://www.biomedcentral.com/1471-2458/7/40 http://www.biomedcentral.com/1471-2458/7/40 16. Agency for Toxic Substances & Disease Registry (ATSDR): ToxFAQs for Hexachlorobenzene. 2006 [http:// www.atsdr.cdc.gov/tfacts90.html]. g ] 17. Agency for Toxic Substances & Disease Registry (ATSDR): ToxFAQs for Zinc. 2006 [http://www.atsdr.cdc.gov/tfacts60.html]. p g 18. Agency for Toxic Substances & Disease Registry (ATSDR): ToxFAQs for Nickel . 2006 [http://www.atsdr.cdc.gov/ tfacts15.html]. ] 19. Agency for Toxic Substances & Disease Registry (ATSDR): ToxFAQs for Chromium. 2006 [http://www.atsdr.cdc.gov/ tfacts7.html]. 20. Grimalt JO, Sunyer J, Moreno V, Amaral OC, Sala M, Rosell A, Anto JM, Albaiges J: Risk excess of soft-tissue sarcoma and thyroid cancer in a community exposed to airborne organochlorin- ated compound mixtures with a high hexachlorobenzene content. Int J Cancer 1994, 56:200-203. J 21. Nadal M, Schuhmacher M, Domingo JL: Metal pollution of soils and vegetation in an area with petrochemical industry. Sci Total Environ 2004, 321:59-69. 22. Sala M, Sunyer J, Otero R, Santiago-Silva M, Camps C, Grimalt J: Organochlorine in the serum of inhabitants living near an electrochemical factory. Occup Environ Med 1999, 56:152-158. y p 23. Toxic Release Inventory (TRI) Explorer 2006 [http:// www.epa.gov/triexplorer/]. y ( ) p [ p www.epa.gov/triexplorer/]. 24. SCORECARD 2006 [http://www.scorecard.org/general/tri/ tri_data.html]. p g p ] 24. SCORECARD 2006 [http://www.scorecard.org/general/tri/ tri_data.html]. 25. Elliot P, Wakefield JC, Best NG, Briggs DJ: Spatial Epidemiology. Methods and Applications. Oxford Medical Publications; 2000. pp 26. Lawson AB, Browne WJ, Vidal Rodeiro CL: Disease Mapping with WinBUGS and MLwiN. Pre-publication history The pre-publication history for this paper can be accessed here: The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2458/7/40/prepub http://www.biomedcentral.com/1471-2458/7/40/prepub Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." 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Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge Page 13 of 13 (page number not for citation purposes)
https://openalex.org/W2109073389	https://repozytorium.uwb.edu.pl/jspui/bitstream/11320/1101/3/M._Karwowski%2c_J._Uszy%c5%84ska-Jarmoc%2c_Creativity_The_Show_Must_Go_On.pdf	English	null	Creativity: The Show Must Go On	Creat!vity	2,014	cc-by-sa	2,944	INTRODUCTION As creativity researchers are deeply en- gaged in, and strongly motivated to study the complex phenomena of creativity, they also need much more space than before to share their ideas, theories, research findings or The great majority of all works devoted to creativity – including articles published in this first issue of the very first volume of Creativity: Theories – Research – Applications (CTRA) The great majority of all works devoted to creativity – including articles published in this first issue of the very first volume of Creativity: Theories – Research – Applications (CTRA) – start with a kind of invocation, calling creativity the fuel of economic and cultural growth, the source of flow and well-being, and the goal of functioning for parents, teachers, man- agers, politicians, and indeed everyone else. Even if such claims are heavily ideological, they do reflect a very special and specific attitude creativity researchers share in common – start with a kind of invocation, calling creativity the fuel of economic and cultural growth, the source of flow and well-being, and the goal of functioning for parents, teachers, man- agers, politicians, and indeed everyone else. Even if such claims are heavily ideological, they do reflect a very special and specific attitude creativity researchers share in common – fascination or even love of their research topic. As creativity researchers are deeply en- gaged in, and strongly motivated to study the complex phenomena of creativity, they also need much more space than before to share their ideas, theories, research findings or even speculations. By creating this journal we aim at widening the number of possible outputs for creativity researchers and scholars interested in related phenomena. In this inaugural editorial we sketch our goals and plans, and invite potential contributors to con- sider publishing with us. – fascination or even love of their research topic. As creativity researchers are deeply en- gaged in, and strongly motivated to study the complex phenomena of creativity, they also need much more space than before to share their ideas, theories, research findings or even speculations. By creating this journal we aim at widening the number of possible outputs for creativity researchers and scholars interested in related phenomena. In this inaugural editorial we sketch our goals and plans, and invite potential contributors to con- sider publishing with us. Keywords: Creativity literature Paradigms in creativity research Education for creativity ISSN: DOI: 10.15290/ctra.2014.01.01.01 Creativity: The Show Must Go On Maciej Karwowski Academy of Special Education, Poland E-mail address: maciek.karwowski@gmail.com Janina Uszyńska-Jarmoc University of Bialystok, Poland E-mail address: j.uszynska@uwb.edu.pl A B S T R A C T In this opening editorial to the new peer-reviewed journal entitled “Creativity: Theories - Research - Applications,” we present our vision, mission and the philosophy of this new journal in the field of creativity studies. As creativity re- searchers and editors, we start by identifying several gaps in the existing creativity literature and propose how a new jour- nal could fill them. We conclude with an invitation to scholars worldwide to participate in the creation of this new, fascinat- ing project. 4 Vol. 1, Issue 1, 2014 INTRODUCTION The great majority of all works devoted to creativity – including articles published in this first issue of the very first volume of Creativity: Theories – Research – Applications (CTRA) – start with a kind of invocation, calling creativity the fuel of economic and cultural growth, the source of flow and well-being, and the goal of functioning for parents, teachers, man- agers, politicians, and indeed everyone else. Even if such claims are heavily ideological, they do reflect a very special and specific attitude creativity researchers share in common – fascination or even love of their research topic. As creativity researchers are deeply en- gaged in, and strongly motivated to study the complex phenomena of creativity, they also need much more space than before to share their ideas, theories, research findings or even speculations. By creating this journal we aim at widening the number of possible outputs for creativity researchers and scholars interested in related phenomena. In this inaugural editorial we sketch our goals and plans, and invite potential contributors to con- sider publishing with us. The great majority of all works devoted to creativity – including articles published in this first issue of the very first volume of Creativity: Theories – Research – Applications (CTRA) – start with a kind of invocation, calling creativity the fuel of economic and cultural growth, the source of flow and well-being, and the goal of functioning for parents, teachers, man- agers, politicians, and indeed everyone else. Even if such claims are heavily ideological, they do reflect a very special and specific attitude creativity researchers share in common – fascination or even love of their research topic. Why a new journal? Five building blocks In the last few decades, the field of creativity studies has been growing rapidly. Decades ago scholars involved in exploring the antecedents, correlates, conditions and conse- quences of creative thinking and functioning published their works in two - now classic - journals: the Journal of Creative Behavior and the Creativity Research Journal. In recent years at least five new, dedicated journals have appeared: starting with the APA Division Creativity: The Show Must Go On / CREATIVITY 1(1) 2014 5 10’s Psychology of Aesthetics, Creativity, and the Arts, and including Thinking Skills and Creativity, Creativity and the Innovation Management, International Journal of Creativity and Problem Solving, Creativity and Human Development, as well as Creativity and Lei- sure: An Interdisciplinary and Cross-Cultural Journal. If we add to these titles several cre- ativity-related journals open to creativity research, but focused for instance, on art or gift- edness (Gifted Child Quarterly, High Ability Studies, Empirical Studies in the Arts, Imagi- nation, Cognition and Personality, among others), the number becomes hard to ignore. Thus, readers may reasonably doubt and ask whether another journal is needed at all. Our role is not to convince the unconvinced, but here we would like to clarify and present our beliefs and arguments that guide the creation of this journal. We see at least five dif- ferent building-blocks for this journal. First, the great majority of all works published in creativity journals are quantitative in nature. The research is usually correlational or experimental, less often meta-analytical or historiometrical, but very rarely based on qualitative methods. We feel that space for cre- ativity researchers who use qualitative methods is especially needed and we do hope that CTRA will provide such space. Second, the main creativity periodicals rarely include theoretical articles. We see this as a serious obstacle and believe that CTRA may provide space for advanced, even con- troversial theoretical discussions about the main aspects of creativity theories, and gener- ate constructive dialogue even about more speculative ideas. Third, although creativity as a human activity and creativity research in general have no boundaries, in fact there is wide differentiation of creativity studies across the world. These different perceptions and paradigms in studying creativity have relatively rarely been taken into consideration (see Kaufman & Sternberg, 2006). Why a new journal? Five building blocks Some time ago, while editing a special issue of the International Journal of Creativity and Problem Solving (see Karwowski & Glăveanu, 2013; Glăveanu & Karwowski, 2013) we realized how many in- teresting studies on creativity are conducted in different parts of the world, and how rarely these studies are published in mainstream creativity journals. Thus, we do believe that CTRA will form a platform for sharing theoretical ideas, research results, and good prac- tices of scholars from different parts of the world, not necessarily from just Western Eu- rope and North America. Fourth, creativity journals devote the majority of their space to presenting research pa- pers. Theoretical papers are in the great minority, while there are almost no articles that deal with the practical applications of creative thinking in art, school or family. We hope that CTRA will provide a forum for researchers, and practitioners to share their ideas Maciej Karwowski, Janina Uszyńska-Jarmoc / CREATIVITY 1(1) 2014 6 about the applications of creativity, as well as their own creative products. Fifth, all creativity journals available on the market are in fact written from the perspec- tive of one (rarely two) scholarly discipline – mainly psychology. Creativity, however, is not only a psychological phenomenon. Hence, we will devote our time and energy to en- courage scholars from other fields, such as sociology, education, art, anthropology, or linguistics to share their ideas and present them in our pages. We are aware of the dan- gers of eclecticism, but we also believe that only a real dialogue between scholars from different fields and specializations can push the science of creativity further. What are we looking for? Every editor dreams about high-quality, top-tier papers. So do we. But we are also realis- tic, and fully aware that expecting that a new journal will receive a number of revolution- ary submissions is naive. We do believe and hope that in the near future leading creativi- ty scholars will consider CTRA as an output for their best work. Although we invite all scholars to share their research and theoretical ideas with us, at this point we should highlight three important matters: openness to the new generation of researchers, no harm in presenting null findings, and encouragement of replications. Openness to the new generation. First and foremost, we would like to explicitly state that we especially warmly invite young scholars and graduate students to share their ide- as with an international audience. We all know how competitive the contemporary world of science is and how difficult it is to publish a paper in a peer-reviewed journal. We offer fast turn-around and extremely competent referees-just take a look at our Editorial Board. No harm in null findings. Our drawers are full of non-significant results without any real chance of getting to get published. Although null findings are always problematic, we are open to publishing highly-powered and well-developed studies, even when the result is inconsistent with the expectations, and not significant. We will encourage our reviewers to focus on the quality of the study itself, not the p value. We do believe that literature brings overestimated effect sizes due to the file drawer problem, and we think it is worth publish- ing well-designed studies irrespective of the result. Encouragement of replications. The recent movement toward replications in psycholo- gy and the more general discussion about the quality of research in the field of psycholo- gy of creativity (Makel, 2014) deals with an extremely important problem that we would also like to address. Our editorial policy emphasizes that replications, both direct and conceptual, of important findings established in the psychology of creativity are welcome and will be considered for publication. Of course these three elements do not limit our expectations, hopes and plans. Creativity: The Show Must Go On / CREATIVITY 1(1) 2014 7 We do welcome studies from all levels and aspects of creativity: from very mini-c (Kaufman & Beghetto, 2009) to eminent creativity, from case studies to massive quantita- tive works. What are we looking for? We are open to theoretical papers and works that describe applications, pro- grams and interventions. We plan to publish interviews with renowned creators and crea- tivity researchers, as well as book reviews. As openness is the main characteristic of vir- tually all creative people, we admit it very seriously – we are really open to all kinds of in- teresting and high-quality works devoted to creativity. Call for commentaries Before we present this issue and the papers included in it, we would like to focus our readers’ attention on the more specific call for commentaries. We are starting this new journal with a very specific first part, entitled Theories. It is comprised of three papers written by Vlad Petre Glăveanu from Aalborg University, Denmark, Izabela Lebuda from the Academy of Special Education, Poland, as well as Michael Chruszczewski from the University of Warsaw, Poland. These three papers share something in common – all are thought-provoking, controversial at certain points, and posted in the main as “position- papers” presenting the Authors’ personal ideas and experiences in the field. We invite all interested scholars to share their opinions and comments regarding any of these three papers. In the near future we plan to publish special issues related to the condition of the psychology of creativity and the question of whether it is really a field in crisis (see Glăveanu’s paper). It is also our intention to publish a special issue dealing with the sta- tus of research on big-C creativity in the creativity literature, especially the costs associat- ed with studying higher level creative achievement and its role for psychology and educa- tion (see Lebuda’s paper). Another special issue we are planning will be devoted to the old, but still controversial topic of the associations between creativity and mental illness (see Chruszczewski’s paper). We welcome all commentaries of no more than 1,500 words (including abstract, references, possible tables and figures) submitted by the end of the October 2014. This issue This issue consists of eleven papers written by scholars who mainly represent Poland, but also Denmark and Romania (Vlad Petre Glăveanu who is opening this issue is a Ro- manian scholar working in Denmark). Part I –Theories – is composed of the aforemen- tioned articles by Glăveanu (“The psychology of creativity: A critical reading”), Lebuda (“Big C Research – The Big Challenge? Reflections from research into eminent creativity in the light of the investment theory of creativity”) and Chruszczewski (“The creative side of mood disorders”). These papers are not void of controversies, but we do see their Maciej Karwowski, Janina Uszyńska-Jarmoc / CREATIVITY 1(1) 2014 8 great potential for evoking a fruitful and constructive discussion based on the ideas pre- sented there. Part II – Research – is composed of four papers, and presents empirical studies conducted within qualitative (Chmielińska & Modrzejewska-Świgulska) or quanti- tative paradigms (Nowacki; Szen-Ziemiańska; Pufal-Struzik & Szewczyk). Bartłomiej Nowacki discusses the timely problem of creativity styles and describes the first steps to- ward adapting an instrument for measuring different styles of creativity in accordance with Galenson’s theoretical model. Joanna Szen-Ziemiańska, places her research within the psychology of science and discusses the structure of self-beliefs concerning scientific work and their consequences for creative activity and achievement in science. Aleksan- dra Chmielińska and Monika Modrzejewska-Świgulska present the results of qualitative discussions with teachers concerning the barriers to innovative activity in- and out-of-the school. Irena Pufal-Struzik and Agnieszka Szewczyk focus on the relationship between adolescents’ creative attitudes and their perception of family functioning. Part III – Appli- cations – includes four articles which mainly deal with art and showing how creativity works in different settings. Anna Boguszewska attempts to build bridges between famous artists’ styles of work and their educational consequences for teaching and didactic activi- ty in a broad sense. Similarly, Ewa Tomaszewska presents her experiences with the ap- plication of art-based activity realized in a theatre setting, as a way of enriching the crea- tive activity of children. Beata Sokolowska-Smyl, analyzes the role of early childhood ex- periences for the creativity of one of the most creative Polish painters of XX century – Zdzislaw Beksinski. Finally, Czeslaw Dziekanowski closes this issue with an excerpt from his prose – an example of creative work in a creativity journal. This issue The work on this issue took us longer than we had initially expected, but it would not have been possible without the great enthusiasm of our authors and the priceless assis- tance of our reviewers – members of our Editorial Board. We also turned to external, ad hoc experts for comments. We appreciate their help and the quality of the comments we received. Beginnings are frightening, but also intriguing. We believe we are beginning a new initi- ative that will turn into a fascinating adventure. We would be happy if you were to take part in this adventure together with us. Enjoy reading this issue, but also remember our invitation to create this journal with us. Editors Editors Creativity: The Show Must Go On / CREATIVITY 1(1) 2014 9 REFERENCES Glăveanu, V. P. & Karwowski, M. (2013). Joining the Debate: Creativity seen from Eastern and Central Europe. International Journal of Creativity and Problem Solving, 23, 5-11. Karwowski, M. & Glăveanu, V. P. (Eds.) (2013). Creativity in Central and Eastern- European perspectives. Special issue of International Journal of Creativity and Prob- lem Solving, 23. Kaufman, J. C. & Beghetto, R. A. (2009). Beyond big and little: The four C model of crea- tivity. Review of General Psychology, 13, 1-12. Kaufman, J. C. & Sternberg, R. J. (Eds.) (2006). The International Handbook of Creativi- ty. New York, NY: Cambridge University Press. ty. New York, NY: Cambridge University Press. Makel, M. C. (2014). The empirical march: Making science better at self-correction. Makel, M. C. (2014). The empirical march: Making science better at self-correction. Psychology of Aesthetics, Creativity, and the Arts, 8, 2-7. Psychology of Aesthetics, Creativity, and the Arts, 8, 2-7. Corresponding author at: Maciej Karwowski, Department of Educational Sciences, Aca- demy of Special Education, 40 Szczesliwicka St., 02-353 Warsaw, Poland. E-mail: maciek.karwowski@gmail.com Corresponding author at: Maciej Karwowski, Department of Educational Sciences, Aca- demy of Special Education, 40 Szczesliwicka St., 02-353 Warsaw, Poland. E-mail: maciek.karwowski@gmail.com Corresponding author at: Janina Uszyńska-Jarmoc, Faculty of Pedagogy and Psycho- logy, University of Bialystok, 20 Świerkowa St., 15-328 Bialystok, Poland. E-mail: j.uszynska@uwb.edu.pl Corresponding author at: Janina Uszyńska-Jarmoc, Faculty of Pedagogy and Psycho- logy, University of Bialystok, 20 Świerkowa St., 15-328 Bialystok, Poland. E-mail: j.uszynska@uwb.edu.pl Corresponding author at: Janina Uszyńska-Jarmoc, Faculty of Pedagogy and Psycho- logy, University of Bialystok, 20 Świerkowa St., 15-328 Bialystok, Poland. E-mail: j.uszynska@uwb.edu.pl
https://openalex.org/W3102964490	https://scipost.org/10.21468/SciPostPhys.9.2.025/pdf	English	null	On the low-energy description for tunnel-coupled one-dimensional Bose gases	SciPost physics	2,020	cc-by	14,835	On the low-energy description for tunnel-coupled one-dimensional Bose gases Yuri D. van Nieuwkerk1,⋆and Fabian H. L. Essler1 1 The Rudolf Peierls Centre for Theoretical Physics, Oxford University, Oxford OX1 3PU, UK On the low-energy description for tunnel-coupled one-dimensional Bose gases Yuri D. van Nieuwkerk1,⋆and Fabian H. L. Essler1 1 The Rudolf Peierls Centre for Theoretical Physics, Oxford University, Oxford OX1 3PU, UK Abstract We consider a model of two tunnel-coupled one-dimensional Bose gases with hard-wall boundary conditions. Bosonizing the model and retaining only the most relevant inter- actions leads to a decoupled theory consisting of a quantum sine-Gordon model and a free boson, describing respectively the antisymmetric and symmetric combinations of the phase ﬁelds. We go beyond this description by retaining the perturbation with the next smallest scaling dimension. This perturbation carries conformal spin and couples the two sectors. We carry out a detailed investigation of the effects of this coupling on the non-equilibrium dynamics of the model. We focus in particular on the role played by spatial inhomogeneities in the initial state in a quantum quench setup. Copyright Y. D. van Nieuwkerk and F. H. L Essler. This work is licensed under the Creative Commons Attribution 4.0 International License. Published by the SciPost Foundation. Received 19-03-2020 Accepted 31-07-2020 Published 25-08-2020 Check for updates doi:10.21468/SciPostPhys.9.2.025 Contents 1 Introduction 2 2 Tunnel-coupled Bose gases in a hard-wall box 4 2.1 Low-energy effective theory 4 2.2 Time-of-ﬂight measurements 6 2.3 Mode expansions for the two-component Luttinger liquid 7 3 Self-consistent time-dependent harmonic approximation 8 3.1 Gaussian initial states 9 3.2 Equations of motion 10 3.3 Self-consistent expectation values 12 3.3.1 One-point functions 12 3.3.2 Two-point functions 12 3.4 Full distribution functions 12 4 Results for experimentally relevant initial states 14 4.1 Choice of initial state 14 4.2 Experimental parameters 15 4.3 Time evolution 15 4.3.1 No coupling between symmetric and antisymmetric sectors (σ = 0) 15 1 Copyright Y. D. van Nieuwkerk and F. H. L Essler. This work is licensed under the Creative Commons Attribution 4.0 International License. Published by the SciPost Foundation. Received 19-03-2020 Accepted 31-07-2020 Published 25-08-2020 Check for updates doi:10.21468/SciPostPhys.9.2.025 Received 19-03-2020 Accepted 31-07-2020 Published 25-08-2020 Check for updates doi:10.21468/SciPostPhys.9.2.025 Copyright Y. D. van Nieuwkerk and F. H. L Essler. This work is licensed under the Creative Commons Attribution 4.0 International License. Published by the SciPost Foundation. SciPost Phys. 9, 025 (2020) On the low-energy description for tunnel-coupled one-dimensional Bose gases Yuri D. van Nieuwkerk1,⋆and Fabian H. L. Essler1 Yuri D. van Nieuwkerk1,⋆and Fabian H. L. Essler1 1 The Rudolf Peierls Centre for Theoretical Physics, Oxford University, Oxford OX1 3PU, UK ⋆yuri.vannieuwkerk@physics.ox.ac.uk 1 Introduction The study of one-dimensional quantum many-body systems out of equilibrium has seen great progress in the past decades. Long-standing questions concerning the equilibration of observ- ables, spreading of correlations and entanglement, and the emergence of statistical mechan- ics from microscopics have been successfully tackled using a range of innovative theoretical ideas [1–9], whilst spectacular advances in the ability to realize archetypical one-dimensional quantum many-body sytems using cold atoms [10–14] have made it possible to test many of these theoretical developments using tabletop experiments [15–20]. However, such ex- perimental engineering of quantum many-body Hamiltonians relies on certain assumptions to make the experiments map onto a model of physical interest. These assumptions often include having a low energy density, at which an effective low-energy theory holds, and translational invariance, which can generally simplify the problem and speciﬁcally play an important role in the integrability of the low-energy theory. When studying non-equilibrium problems in ﬁnite quantum many-body systems, these two assumptions are sometimes brought into question. We here study a situation where both the successes and challenges described above are clearly present: we consider pairs of tunnel-coupled, elongated Bose gases, as realized in the Vienna experiments [13,14,17–19,21–24]. An interesting feature of these experiments is that in certain limits, density measurements after matter-wave interference [13, 25] correspond to projective von Neumann measurements of the relative phase ﬁeld [26]. This allows for the reconstruction of full distribution functions of quantum mechanical observables [21–23], which is of considerable theoretical interest [27–44] in general. In the case at hand, situations without tunnel-coupling can be modelled by a two-component Luttinger liquid [45–47]. This description in terms of a quadratic quantum critical model has yielded theoretical results for the full ﬂuctuation statistics of the relative phase ﬁeld [28, 48, 49] which show a satisfying match with experimental results [17,19]. Our interest lies in the effect of a ﬁnite tunnel barrier between the gases [14,50–52]. This introduces a relevant perturbation and at sufﬁciently low energies leads to a decoupled theory of a Luttinger liquid describing the symmetric combination of Bose gas phases (“symmetric sector”) and a sine-Gordon model [53] describing the relative phase (“antisymmetric sector”). Contents 1 Introduction 2 2 Tunnel-coupled Bose gases in a hard-wall box 4 2.1 Low-energy effective theory 4 2.2 Time-of-ﬂight measurements 6 2.3 Mode expansions for the two-component Luttinger liquid 7 3 Self-consistent time-dependent harmonic approximation 8 3.1 Gaussian initial states 9 3.2 Equations of motion 10 3.3 Self-consistent expectation values 12 3.3.1 One-point functions 12 3.3.2 Two-point functions 12 3.4 Full distribution functions 12 4.1 Choice of initial state 4.2 Experimental parameters 1 SciPost Phys. 9, 025 (2020) SciPost Phys. 9, 025 (2020) 4.3.2 Finite coupling between sectors (σ > 0) and homogeneous initial con- ditions 15 4.3.3 Finite coupling between sectors (σ > 0) and inhomogeneous initial conditions 18 4.3.4 Distribution functions of the density after time of ﬂight 19 5 Conclusion 21 A Tensors occurring in HSCH(t) 22 References 22 1 Introduction The sine-Gordon model is of great theoretical importance as it is an exactly solvable, Lorentz invariant quantum ﬁeld theory that exhibits a rich range of physical phenomena like dynamical mass generation and topological excitations and moreover has important applications to elec- tronic degrees of freedom in solids [54]. Its behaviour out of equilibrium has received a lot of attention in the past decade. To be able to study dynamics, the very weakly interacting limit is 2 SciPost Phys. 9, 025 (2020) amenable to a simple harmonic approximation [55–57], while the free fermion point can also be used to obtain exact results [55]. Integrability-based methods were used in Refs. [58–61] to study quenches from “integrable” initial states, whereas semiclassical methods [62, 63] were applied to the study of the time-dependence of one and two-point functions as well as the prob- ability distribution of the phase. The truncated conformal space approach [64] was employed in Ref. [65] to analyse the time evolution of two and four-point functions after a quantum quench. A ﬁrst litmus test for the experimental realization of the sine-Gordon model using split Bose gas experiments was performed in an equilibrium situation: high order equilibir- ium correlation functions extracted from projective phase measurements in the classical limit have been found to agree well with classical ﬁeld simulations [23]. For non-equilibrium ini- tial conditions, however, experimental studies [24, 66, 67] have shown puzzling behaviour: when preparing two elongated Bose gases with an initial phase difference, applying a tunnel- coupling between them sets Josephson oscillations of density and phase in motion. These oscillations show a rapid damping, accompanied by a narrowing of the distribution function of the phase. To date, no satisfying theoretical explanation of this damping is known [68]. The damping seems incompatible with a description in terms of a translationally invariant sine-Gordon model, which fails to provide a mechanism for the observed strong and rapid damping in both a self-consistent harmonic treatment [69] and in a combination of truncated Wigner and truncated conformal space approaches [70]. In this work, we go beyond previous studies of the low-energy physics in two importan ways: 1. We take into account the next most relevant perturbation at low energies. This pertur- bation induces an interaction between the symmetric and antisymmetric sectors. 1. We take into account the next most relevant perturbation at low energies. 1 Introduction This pertur- bation induces an interaction between the symmetric and antisymmetric sectors. 2. We drop the assumption of translational invariance. To this end we place the model in a hard-wall box geometry and consider inhomogeneous initial conditions. 2. We drop the assumption of translational invariance. To this end we place the model in a hard-wall box geometry and consider inhomogeneous initial conditions. We stress that our focus is on the vicinity of the scaling regime, which is approximately de- scribed by a sine-Gordon model as discussed above. This means that we consider energy densities that are small compared to the cutoff of the ﬁeld theory, which can be taken as the inverse coherence length of the underlying Bose gas. In addition the energy density is of the same order as the mass scale induced by the tunnel-coupling between the Bose gases. Trans- lating these requirements into parameters relevant to the existing experiments gives an energy scale of roughly 5nK. This is lower than typical energy scales realized in current experimental set-ups, which operate above 18nK, but sufﬁciently close to make this regime interesting in light of possible future experiments. Our strategy is to treat the resulting perturbed sine-Gordon model in the self-consistent time-dependent harmonic approximation (SCTDHA) as described in [69]. In that paper we have benchmarked the approximation using the dynamics of the zero modes in the antisym- metric sector only. It is the expectation value of these modes that displays the Josephson oscillations, making their dynamics vital to the problem at hand. Comparison to numerically exact results for this simpliﬁed problem indicated that the SCTDHA offers reliable results for early times corresponding to ∼3 density-phase oscillation periods. Based on those ﬁndings, we apply the approximation in the current work to the ﬁrst few oscillation periods in the pres- ence of sector coupling. We consider the dynamics after initializing the system in a state in which the sectors are uncorrelated and observe how the new coupling term causes correlations between the two sectors to develop over time. In addition to this, energy starts to oscillate between the sec- tors. Depending on the initial density proﬁle imprinted on the gas, Josephson oscillations of density and phase are affected by the presence of the additional term, showing modulations 3 SciPost Phys. 1 Introduction 9, 025 (2020) of the amplitude that differ from the ones observed in the SCTDHA treatment of isolated sine- Gordon dynamics [69]. However, the observed effects are rather weak. This means that the presence of the box potential and the new sector-coupling term are insufﬁcient to explain the experimentally observed damping phenomenon. At the same time, our results indicate that the simulation of a sine-Gordon model using the hard-wall box potential setup described here should not be severely restricted by the presence of the additional coupling term, which has only mild effects. This paper is organized as follows. In Sec. 2, we introduce the low-energy effective theory in a box geometry, the additional interaction term and the observable relevant for experiment. We also establish some notational conventions. In Sec. 3, we recapitulate the self-consistent time-dependent harmonic approximation as well as the framework to compute observables and some important distribution functions. In Sec. 4, we apply our formalism to an initial state which is commonly used in the literature, and present results on energy ﬂow and growth of correlations between the sectors, along with the effect on Josephson oscillations, due to the additional interaction term. Sec. 5 summarizes our conclusions and discusses questions for further study. 2 Tunnel-coupled Bose gases in a hard-wall box An appropriate model for the experiments carried out by the Vienna group is an interacting Bose gas conﬁned in three-dimensional space by a tight harmonic potential in the z-direction, a double-well potential V⊥(y) in the y-direction and a shallow harmonic potential in the x- direction. We will refer to the x-direction as longitudinal, and to the remaining directions as transverse. To simplify the problem, we take the longitudinal potential to be an inﬁnite square well V\|\|(x) = ¨ 0 if 0 < x < L, ∞ otherwise. (1) (1) (1) Just like a shallow harmonic potential this breaks translational invariance in the longitudinal direction, but it has the additional advantage to be considerably simpler to analyze. Our starting point is thus the following Hamiltonian Just like a shallow harmonic potential this breaks translational invariance in the longitudinal direction, but it has the additional advantage to be considerably simpler to analyze. Our starting point is thus the following Hamiltonian H3d = Z d x d y dz § Ψ†(x, y,z) −∇2 2m + V\|\|(x) + V⊥(y) + mω2 z 2 z2 Ψ(x, y,z) +c Ψ†(x, y,z) 2Ψ(x, y,z) 2ª , (2) +c Ψ†(x, y,z) 2Ψ(x, y,z) 2ª , (2) (2) where Ψ(x, y,z) are complex Bose ﬁelds obeying the usual bosonic commutation relations. 2.1 Low-energy effective theory In situations where the transverse potentials are sufﬁciently tight, the dynamics in the y- and z-directions can be integrated out, in a way analogous to Ref. [71]. Details of this procedure will be reported elsewhere [72]. Projecting to the lowest two states of the transverse potential, and taking appropriate linear combinations of these, we obtain a Hamiltonian for two species 4 4 4 SciPost Phys. 9, 025 (2020) of bosons, Ψ1,2, which are approximately localized in wells 1 and 2: of bosons, Ψ1,2, which are approximately localized in wells 1 and 2: of bosons, Ψ1,2, which are approximately localized in wells 1 and 2: H1d = Z L 0 d x X j=1,2 1 2m∂xΨ† j (x)∂xΨj(x) + X j,k,l,m=1,2 Γjklm Ψ† j (x)Ψ† k(x)Ψl(x)Ψm(x) − T⊥Ψ† 1(x)Ψ2(x) + h.c. . (3) (3) Here the Bose ﬁelds Ψi(x) have commutation relations Ψi(x),Ψ† j (x′) = δi,jδ(x −x′). The two Bose gases are coupled by a tunnelling term as well as contact interactions. The corre- sponding coupling constants Γjklm follow from the details of the low-energy projection [72]. For our purposes, we will assume the diagonal elements to be equal to the usual Lieb-Liniger interaction constant, Γj j j j = g ∀j. Hard-wall boundary conditions are imposed by restricting our problem to states \|Φ〉where the density at the boundary has a vanishing eigenvalue: Here the Bose ﬁelds Ψi(x) have commutation relations Ψi(x),Ψ† j (x′) = δi,jδ(x −x′). The two Bose gases are coupled by a tunnelling term as well as contact interactions. The corre- sponding coupling constants Γjklm follow from the details of the low-energy projection [72]. For our purposes, we will assume the diagonal elements to be equal to the usual Lieb-Liniger interaction constant, Γj j j j = g ∀j. Hard-wall boundary conditions are imposed by restricting our problem to states \|Φ〉where the density at the boundary has a vanishing eigenvalue: Ψ† j (L)Ψj(L)\|Φ〉= Ψ† j (0)Ψj(0)\|Φ〉= 0. (4) (4) (4) The one-dimensional model (3) gives an accurate description of the full theory H3d at energies that are small compared to the energy E⊥,2 of the second excited state of the transverse con- ﬁning potential. In the actual experiments this is a large energy scale. The physics of interest occurs at energies that are small compared to v/ξ ≪E⊥,2, where ξ is the coherence length and v the speed of sound. 2.1 Low-energy effective theory 9, 025 (2020) 020) For weak interactions, the sound velocity v and Luttinger parameter K are related to the pa- rameters in the Lieb-Liniger model in a simple way [73] For weak interactions, the sound velocity v and Luttinger parameter K are related to the pa- rameters in the Lieb-Liniger model in a simple way [73] v = ρ0 m pγ 1 − pγ 2π 1/2 , K = π 2pγ 1 − pγ 2π −1/2 . (10) (10) Here γ = mg/ρ0 is the dimensionless interaction strength and ρ0 the average density of each of the two Bose gases. In the next step we take into account the tunnelling term in (3) as well as “off-diagonal” in- teraction terms proportional to Γi jkl with not all indices being equal. These introduce relevant perturbations (in the renormalization group sense) with respect to the critical Hamiltonian (6). Inserting the bosonization identity (5) and assuming Γ to be real, permutation symmetric and symmetric under 1 ↔2, we ﬁnd that the perturbations with the lowest scaling dimensions can be written in the form H⊥= −2t⊥ Z L 0 d x [ρ0 + σΠs(x)]cosφa(x), (11) (11) where t⊥and σ depend on the microscopic parameters in (3). Importantly, the two terms in (11) get generated independently and we will therefore treat t⊥and σ as independent phe- nomenological parameters in the following. The Hamiltonian Hs+Ha+H⊥should be viewed as the result of integrating out high energy degrees of freedom in a renormalization group sense. As t⊥grows much faster than t⊥σ under the renormalization group it would be unphysical to consider very large values of σ. We have therefore restricted the numerical analyses reported below to the range 0 ≤σ ≤2. In addition to (11) there are other perturbations with higher scaling dimensions. Their systematic derivation as well as an analysis of their effects will be presented elsewhere [72]. In the case σ = 0 the full low-energy theory decouples into sym- metric and antisymmetric sectors H = Hs + H′ a, where H′ a is the Hamiltonian of a quantum sine-Gordon model [53] H′ a = v 2π Z L 0 d x 1 K (∂xθa(x))2 + K (∂xφa(x))2 −2t⊥ρ0 Z L 0 d x cosφa(x). (12) (12) The non-equilibrium dynamics of this model was analyzed for the translationally invariant case in the framework of a SCTDHA in our recent work [69]. 2.1 Low-energy effective theory The additional σ-term in (11) couples the sine-Gordon model to the Luttinger liquid Hamiltonian Hs. In the following we extend the analysis [69] to H = Ha + Hs + H⊥. (13) (13) 2.1 Low-energy effective theory This enables us to make a second low-energy projection by employing bosonization [45] The one-dimensional model (3) gives an accurate description of the full theory H3d at energies that are small compared to the energy E⊥,2 of the second excited state of the transverse con- ﬁning potential. In the actual experiments this is a large energy scale. The physics of interest occurs at energies that are small compared to v/ξ ≪E⊥,2, where ξ is the coherence length and v the speed of sound. This enables us to make a second low-energy projection by employing bosonization [45] Ψ† j (x) ∼ q ρ0 + ∂xθj/π e−iφj(x) ∞ X n=−∞ Bne2ni(xπρ0+θj). (5) (5) This provides a low-energy description of (3) in terms of phase ﬁelds φj and θj with a cutoff length scale set by the coherence length of the gases, which for weak interactions is given by ξ = π/mv (the sound velocity v is deﬁned below). The hard-wall condition is encoded in the boundary conditions of the θ-ﬁelds in a way that is described in Sec. 2.3. Let us ﬁrst consider the case where interactions and tunnelling between the two gases are absent, meaning that both T⊥and the non-diagonal elements of Γ are zero. This leaves us with two Lieb-Liniger models in a hard-wall box, with interaction strength g. Under the mapping (5), the low-energy physics of this model maps to a pair of Luttinger liquids H j = v 2π Z L 0 d x 1 K ∂xθj(x) 2 + K ∂xφj(x) 2 , j = s, a. (6) (6) (6) Here we have deﬁned (anti)symmetric combinations of the phase ﬁelds by φs/a = φ1 ± φ2, ∂xθs/a = ∂xθ1 ± ∂xθ2 2 . (7) These ﬁelds are compact φ = φ + 2π, θ = θ + π and fulﬁll commutation relations ∂xθj(x),φl(y) = iπδj,lδ(x −y). (8) φs/a = φ1 ± φ2, ∂xθs/a = ∂xθ1 ± ∂xθ2 2 . (7) (7) These ﬁelds are compact φ = φ + 2π, θ = θ + π and fulﬁll commutation relations are compact φ = φ + 2π, θ = θ + π and fulﬁll commutation relations ∂xθj(x),φl(y) = iπδj,lδ(x −y). (8) ∂xθj(x),φl(y) = iπδj,lδ(x −y). (8) This implies that the canonically conjugate ﬁelds to φj are given by Πj(x) ≡ ∂xθj(x) π . (9) (9) 5 SciPost Phys. 2.2 Time-of-ﬂight measurements (17) (17) 2.3 Mode expansions for the two-component Luttinger liquid 2.2 Time-of-ﬂight measurements In the Vienna experiments [13,14,17–19,23,24,74,75] measurements are performed by turn- ing off the trapping potential at some time t0, letting the gas expand freely and imaging the three-dimensional boson density after a time-of-ﬂight t1. The outcome of each such “single- shot” measurement is determined by the eigenvalues e i 2 ϕa,s(x,t) of the bosonic vertex operators e i 2 φa,s(x,t0) [26, 28]. As shown in [26], the result of a single measurement of the boson den- sity after a time-of-ﬂight t1 in the regime relevant for the Vienna experiments can be well approximated by ϱtof(x,⃗r, t1, t0) ≃ρ0 f (⃗r, t1) 2 × Z d x′ G(x −x′, t1) ei m 2t1 ⃗r·⃗de i 2(ϕs(x′,t0)+ϕa(x′,t0)) + e−i m 2t1 ⃗r·⃗de i 2(ϕs(x′,t0)−ϕa(x′,t0)) 2 . (14) (14) 6 SciPost Phys. 9, 025 (2020) Here ⃗d is the distance between the minima of the double well, x, x′ and ⃗r = (y,z) respectively denote longitudinal and transverse coordinates, and G(x, t) is the Green’s function for a free particle G(x, t) = s m 2πit exp i m 2t x2 . (15) (15) The function f (⃗r, t) is an overall envelope whose precise from follows from the details of the trapping potential. By measuring ϱtof, the system collapses to a simultaneous eigenstate of all e i 2 φa,s(x,t). The outcome of such measurements can be simulated if one has access to distri- bution functions of the corresponding eigenvalues e i 2 ϕa,s(x,t). Such distribution functions will be computed in Sec. 3.4. In principle, the observable (14) also contains small contributions from the density ﬁelds Πa,s(x) [26]. In order to treat these, the above description of a projec- tive measurement has to be preceded by a diagonalization of the full observable, which now contains non-commuting ﬁelds. We do not pursue this further here because these effects are expected to be small in the regime where our low-energy approximation applies. Experiments typically report results related to the quantity Experiments typically report results related to the quantity R(x0,⃗r, t1, t0) = Z x0+ℓ/2 x0−ℓ/2 d x ϱtof(x,⃗r, t1, t0) (16) = ρ0 f (⃗r, t1) 2 Z x0+ℓ/2 x0−ℓ/2 d x \|g+(x)\|2 + \|g−(x)\|2 + 2Re g+(x)g∗ −(x)ei m⃗r·⃗d t1 , (16) where we have deﬁned where we have deﬁned g±(x) = Z d x′ G(x −x′, t1)e i 2(ϕs(x′,t0)±ϕa(x′,t0)). 2.3 Mode expansions for the two-component Luttinger liquid The free boson Hamiltonians Ha,s are diagonalized by the mode expansions (see e.g. [73]) The free boson Hamiltonians Ha,s are diagonalized by the mode expansions (see e.g. [73]) θj(x) = θj,0 + πx L δNj + i X q>0 πK qL 1/2 sinqx bj,q −b† j,q , (18) φj(x) = φj,0 + X q>0 π qK L 1/2 cosqx bj,q + b† j,q , (19) (18) φj(x) = φj,0 + X q>0 π qK L 1/2 cosqx bj,q + b† j,q , (19) (19) where q = πn L , n ∈Z, bq, b† k = δq,k and δNj,φl,0 = iδj,l. The zero modes δNj have integer eigenvalues. The Hamiltonians then take the form where q = πn L , n ∈Z, bq, b† k = δq,k and δNj,φl,0 = iδj,l. The zero modes δNj have integer eigenvalues. The Hamiltonians then take the form H j = vπ 2LK δN 2 j + X q>0 vq b† j,qbj,q, j = a,s. (20) (20) Going back to Eq. (5), we see that the hard-wall condition (4) is guaranteed by choosing the c-number θ0 such that Going back to Eq. (5), we see that the hard-wall condition (4) is guaranteed by choosing the c-number θ0 such that θ(0) = θ0 /∈Z. (21) θ(0) = θ0 /∈Z. (21) It turns out to be useful in what follows to rewrite the mode-expansions in the form It turns out to be useful in what follows to rewrite the mode-expansions in the form φl(x, t) = X ν u(l) ν (x) bν(t) + b† ν(t) , (22) ∂xθl(x, t)/π = X ν w(l) ν (x) bν(t) −b† ν(t) , l = a,s. (23) (22) (23) 7 SciPost Phys. 9, 025 (2020) Here we have introduced a multi-index ν = (l,q) that runs over all positive momenta q ≥0 and the two sectors l = a,s and we have deﬁned u(l) (j,q)(x) = δj,l ( π qK L 1/2 cosqx, if q ̸= 0, 1 2 q 1 K if q = 0, (24) w(l) (j,q)(x) = δj,l ( i qK πL 1/2 cosqx, if q ̸= 0, i L p K if q = 0, (25) bj,0 = p Kφj,0 −i 2 v t 1 K δNj. (26) (24) (25) (26) 3 Self-consistent time-dependent harmonic approximation (35) (35) One subtlety associated with the SCTDHA concerns the zero mode φa,0. The spectrum of φa,0 originally reﬂected the compact nature of the phase ﬁeld φa(x) = φa(x) + 2π. The latter feature is lost in the SCTDHA, where ﬂuctuations are assumed to be small but the ﬁelds themselves take arbitrary real values. One subtlety associated with the SCTDHA concerns the zero mode φa,0. The spectrum of φa,0 originally reﬂected the compact nature of the phase ﬁeld φa(x) = φa(x) + 2π. The latter feature is lost in the SCTDHA, where ﬂuctuations are assumed to be small but the ﬁelds themselves take arbitrary real values. 3 Self-consistent time-dependent harmonic approximation Our aim is to determine the non-equilibrium evolution after a quantum quench: the system is prepared in a density matrix ρ(0) that does not commute with the Hamiltonian (13). We moreover take the density matrix to be Gaussian for simplicity. The ensuing time evolution is described in the Schrödinger picture via the time evolving density matrix ρ(t) = e−iHtρ(0)eiHt. (27) (27) As our Hamiltonian of interest (13) is not solvable we resort to an analysis by means of a SCTDHA [43, 69, 76–78]. Below we generalize the analysis of [69] to include the nonlin- ear interaction between the symmetric and antisymmetric sectors. The SCTDHA amounts to replacing the exact time evolution operator with e−iHt −→USCH(t) = Te−i R t 0 HSCH(τ)dτ, (28) (28) where where HSCH(t) = Ha + Hs + Z d x f (x, t) + φa(x)g(1)(x, t) + Πs(x)g(2)(x, t) + φ2 a(x)h(1)(x, t) + φa(x)Πs(x)h(2)(x, t) . (29) (29) Here the functions g(1,2)(x, t) and h(1,2)(x, t) are determined self-consistently. In order to derive (29) we decompose the ﬁelds into their space and time dependent expectation values and their ﬂuctuations φl(x, t) = 〈φl(x, t)〉+ χl(x, t), (30) Πl(x, t) = 〈Πl(x, t)〉+ πl(x, t), l = a,s. (31) (30) (30) (31) Substituting this decomposition into the interaction part of the Hamiltonian (11) gives H⊥= −2t⊥ Z L 0 d x [ρ0 + σ 〈Πs〉+ σπs][cos〈φa〉cosχa −sin〈φa〉sinχa]. (32) (32) In the next step we expand the Hamiltonian to quadratic order in ﬂuctuations following [69], which gives H⊥≈−2t⊥ Z d x ρ0 + σ πs −1 2 (ρ0 + σ 〈Πs〉)χ2 a −σ 〈χa πs〉χa cos〈φa〉 (33) − (ρ0 + σ (πs + 〈Πs〉))χa −σ 2 〈χa πs〉χ2 a sin〈φa〉 e−1 2〈χ2 a〉+ const. (33) 8 SciPost Phys. 9, 025 (2020) After re-expressing this in terms of the original ﬁelds φa and Πs, we arrive at Eq. (29), where the functions h(j)(x, t) and g(j)(x, t) are determined self-consistently by h(1)(x, t) = ReF(x, t)/2, h(2)(x, t) = σImF(x, t), g(1)(x, t) = ImF(x, t) −2〈φa(x, t)〉h(1)(x, t) −〈Πs(x, t)〉h(2)(x, t), g(2)(x, t) = −σReF(x, t) −〈φa(x, t)〉h(2)(x, t). (34) (34) Here we have deﬁned two functions Here we have deﬁned two functions F(x, t) = 2t⊥Tr USCH(t)ρ(0)U† SCH(t)eiφa(x) , F(x, t) = 2t⊥Tr USCH(t)ρ(0)U† SCH(t)eiφa(x) (ρ0 + σΠs(x)) . 3.1 Gaussian initial states In order to investigate the effects of the σ-term that couples the symmetric and antisymmetric sectors we want to start from a factorized state and study how correlations develop over time. An important requirement is related to our use of the SCTDHA: its accuracy strongly depends on the initial state obeying Wick’s theorem. These two considerations lead us to consider the same class of initial states previously used in the literature [46–49] ρ(0) = ρa(0) ⊗ρs(0), (36) (36) where ρa(0) = \|V, r,ϕ〉aa〈V, r,ϕ\| is a Gaussian pure state where ρa(0) = \|V, r,ϕ〉aa〈V, r,ϕ\| is a Gaussian pure state where ρa(0) = \|V, r,ϕ〉aa〈V, r,ϕ\| is a Gaussian pure state \|V, r,ϕ〉a = N exp X pq Vp sech r T pq b† a,q + X p,q,k 1 2 b† a,p (tanh r)pq eiϕqk b† a,k ! \|0〉a. (37) \|V, r,ϕ〉a = N exp X pq Vp sech r T pq b† a,q + X p,q,k 1 2 b† a,p (tanh r)pq eiϕqk b† a,k ! \|0〉a. (37) (37) It is useful to deﬁne new annihilation operators αa,k satisfying αa,k \|V, r,ϕ〉a = 0, (38) (38) which are related to the b-operators via the canonical transformation ba,q = X k (cosh r)qk αa,k + Vk + sinh reiϕ qk α† a,k + V ∗ k . (39) (39) In previous works it has been assumed that the symmetric sector is initialized in a thermal state [49]. We will follow this assumption, but in order to study the effects of spatial inhomogeneity we take our initial state to be given by a “displaced” thermal density matrix ρs = D(R) e−βHs Tr e−βHs D†(R), (40) (40) where the displacement operators are deﬁned via where the displacement operators are deﬁned via where the displacement operators are deﬁned via here the displacement operators are deﬁned via D†(R)bj,kD(R) = bj,k + R j,k, j = a,s. (41) (41) 9 SciPost Phys. 9, 025 (2020) This suggests the deﬁnition of displaced annihilation operators αs,k via a constant shift bs,k = αs,k + Rs,k, (42) (42) so that 〈αs,k〉= 0, (43) (43) on the initial state. Since ρs(0) satisﬁes Wick’s theorem, it is then completely ﬁxed by the vector Rs,k along with connected two-point functions of the ﬁelds. Using the mode expansion of Hs from Eq. 3.1 Gaussian initial states (20) we simply ﬁnd bosonic occupation numbers for q > 0, ¬ b† s,qbs,k ¶ c = δq,k eβvq −1 ≡n(s,q), (44) (44) the anomalous expectation values 〈bs,qbs,q′〉c being zero. For the zero mode, the only expec- tation values on ρs(0) that we will need are the anomalous expectation values 〈bs,qbs,q′〉c being zero. For the zero mode, the only expec- tation values on ρs(0) that we will need are 〈δN 2 s 〉c = P n e−β vπ 2K L n2n2 P n e−β vπ 2K L n2 , 〈δNs〉= 0, (45) (45) where the second identity implies ImRs,0(0) = 0. As will become clear in the next section, ex- pectation values involving the ﬁeld φs,0 will never be required for the computation of physical quantities. where the second identity implies ImRs,0(0) = 0. As will become clear in the next section, ex- pectation values involving the ﬁeld φs,0 will never be required for the computation of physical quantities. 3.2 Equations of motion To this end, we write the SCTDHA Hamiltonian in the generic form HSCH(t) = b† νAνµ(t)bµ + 1 2 b† νB† νµ(t)b† µ + bνBνµ(t)bµ + C(t) + Dν(t) bν + b† ν + Eν(t) bν −b† ν . (50) (50) The matrices A, B and vectors D, E depend on the self-consistency functions g(1,2) and h(1,2), cf. Eqs. (34), and are given in Appendix A. Inserting the expansion (46) into the Heisenberg equation of motion, i d dt bν(t) = USCH(t)[bν, HSCH(t)] U† SCH(t), (51) (51) yields a system of coupled, ﬁrst order differential equations i˙Rν(t) = Aνµ(t)Rµ(t) + B† νµ(t)R∗ µ(t) + Dν(t) −Eν(t) i˙Sνµ(t) = Aνλ(t)Sλµ(t) + B† νλ(t)Tλµ(t) (52) −i ˙Tνµ(t) = A∗ νλ(t)Tλµ(t) + BT νλ(t)Sλµ(t). (52) This system of ODE’s is nonlinear: as a result of the self-consistency functions (34) on which the tensors A, B, D and E depend, these tensors are themselves functions of R,S and T, which therefore enter the system (52) in nonlinear combinations. To simplify some of the following equations we introduce linear combinations Qνµ(t) = Sνµ(t) + Tνµ(t), Qνµ(t) = Sνµ(t) −Tνµ(t). (53) (53) In terms of these functions mode expansions of the time evolved ﬁelds take the form φa(x, t) = X ν u(a) ν (x) 2ReRν(t) + X µ Qνµ(t)αµ + Q∗ νµ(t)α† µ , (54) Πl(x, t) = X ν w(l) ν (x) 2iImRν(t) + X µ Qνµ(t)αµ −Q ∗ νµ(t)α† µ . (55) (54) (55) The functions (35) can then be computed using Wick’s theorem for the α-operators, based on the above expressions. This closes the system of ODE’s (52). The zero mode in the symmetric sector φs,0 reﬂects the compact nature of the phase ﬁeld φs and therefore needs to be treated separately from the ﬁnite momentum modes. We therefore deﬁne a ﬁeld f φs(x) ≡φs(x) −φs,0, (56) (56) which time evolves as f φs(x, t) = X ν̸=(s,0) u(s) ν (x) 2ReRν(t) + X µ Qνµ(t)αµ + Q∗ νµ(t)α† µ . (57) (57) Importantly the zero mode φs,0 does not get generated under Heisenberg time evolution of other ﬁelds. This is easily checked by inspection of the Hamiltonian (13) which is seen to not involve φs,0. This in turn implies that the zero mode cannot appear on the rhs of the Heisenberg equation of motion (51). 3.2 Equations of motion The SCTDHA allows for a closed-form expression of the equations of motion. We will work in the Heisenberg picture from here onwards. The SCTDHA guarantees that time evolving annihilation operators can always be written as bν(t) = Rν(t) + Sνµ(t)αµ + T ∗ νµ(t)α† µ, (46) (46) where αµ are a set of bosonic creation and annihilation operators. We choose these to be given by where αµ are a set of bosonic creation and annihilation operators. We choose these to be given by αν = ¨ αa,k if ν = (a, k) αs,k if ν = (s, k), (47) (47) where the αa,k are deﬁned in (39) and the αs,k in (42). For (46) to be a canonical transforma- tion we require SS† −T ∗T T = 1, ST † −T ∗ST = 0. (48) (48) The initial conditions on R,S and T are given by The initial conditions on R,S and T are given by Rµ(0) = ¨P q(cosh r)pq Vq + (sinh reiϕ)pq V ∗ q if µ = (a, p), 0 else, Sν,µ(0) =      (cosh r)pq if ν = (a, p), µ = (a,q), δpq if ν = (s, p), µ = (s,q), 0 else, (49) T ∗ ν,µ(0) = ¨ (sinh reiϕ)pq if ν = (a, p), µ = (a,q), 0 else. (49) T ∗(0) = ¨ (sinh reiϕ)pq if ν = (a, p), µ = (a,q), 10 SciPost Phys. 9, 025 (2020) We note that the αµ’s satisfy Wick’s theorem on the initial state, along with αµ = 0 for all µ. We note that the αµ’s satisfy Wick’s theorem on the initial state, along with αµ = 0 for all µ. The time evolution of any operator is then encoded in the time-dependence of the tensors We note that the αµ’s satisfy Wick’s theorem on the initial state, along with αµ = 0 for all µ. The time evolution of any operator is then encoded in the time-dependence of the tensors R,S and T, which we will now determine. To this end, we write the SCTDHA Hamiltonian in e o e a e αµ s sa s y c s eo e o e a s a e, a o g αµ 0 o a µ. The time evolution of any operator is then encoded in the time-dependence of the tensors R,S and T, which we will now determine. 3.3.2 Two-point functions Comparing the deﬁnitions from Section 3.1 to the initial conditions (49), we ﬁnd that for any ν,µ, Comparing the deﬁnitions from Section 3.1 to the initial conditions (49), we ﬁnd that for any ν,µ, gν,µ = ¬ α† ναµ ¶ = ¬ ανα† µ ¶ −δν,µ = δν,µ ¨ 0 if ν ∈{(a,q),(s,0)} n(s,q) if ν ∈{(s,q)\|q ̸= 0}. (63) (63) If we deﬁne P(s) 0 to be the projector on the symmetric zero modes, along with its complement ˜1 = 1 −P(s) 0 , we then ﬁnd the following connected two-point functions φj(x, t)φl(y, t) c = u(j)(x) 2Re(Q∗gQT) + Q˜1Q† + 〈δN 2 s0〉 K ImQP(s) 0 ImQT u(l)(y), φj(x, t)Πl(y, t) c = −u(j)(x) 2iIm(QgQ †) + Q˜1Q † + i 〈δN 2 s0〉 K ImQP(s) 0 ReQ T w(l)(y). (64) In the above, indices on all matrices and vectors have been suppressed for conciseness. If we want to consider the ﬁeld f φs instead of φs, we need leave out the symmetric zero mode term. This leads, for instance, to ¬ f φs(x, t)Πl(y, t) ¶ c = u(j)(x) P(s) 0 −1 × × 2iIm(QgQ †) + Q˜1Q † + i 〈δN 2 s0〉 K ImQP(s) 0 ReQ T w(l)(y), (65) nd analogous modiﬁcations for ¬ f φs(x, t)f φs(y, t) ¶ c and ¬ f φs(x, t)φa(y, t) ¶ c. (65) and analogous modiﬁcations for ¬ f φs(x, t)f φs(y, t) ¶ c and ¬ f φs(x, t)φa(y, t) ¶ c. 3.3 Self-consistent expectation values 3.3.1 One-point functions 3.2 Equations of motion Since we can express the zero mode at t = 0 as φs,0 = α(s,0) + α† (s,0) / p 4K, (58) (58) 11 SciPost Phys. 9, 025 (2020) we conclude that this linear combination of α-operators does not appear in the sums over modes in (54,55) except in the expansion for φs(x, t), where it occurs in the term with ν = (s,0). This directly leads to ReQν,(s,0)(t) = 0 ∀ν ̸= (s,0), ImQν,(s,0)(t) = 0 ∀ν. (59) (59) 3.3.1 One-point functions As all relevant one-point functions of αν and δNs are zero we have ¬ f φs(x, t) ¶ = 2 X ν̸=(s,0) u(s) ν (x)ReRν(t), (60) 〈φa(x, t)〉= 2 X ν u(a) ν (x)ReRν(t), (61) 〈Πl(x, t)〉= 2i X ν w(l) ν (x)ImRν(t). (62) (60) (61) (62) 3.3.2 Two-point functions 3.3.2 Two-point functions 3.4 Full distribution functions 3.4 Full distribution functions Individual measurement outcomes in interference experiments of interest [24] are fully deter- mined by the eigenvalues ϕa and f ϕs of the phase ﬁelds φa and f φs [26], cf. Eq. (14). To model the outcomes of such measurements we therefore require the time-dependent distribution 12 SciPost Phys. 9, 025 (2020) functions for ϕa and f ϕs. These can be determined in the framework of the SCTDHA [43,69]. For the case at hand, we ﬁrst expand the eigenvalues of the phase ﬁelds as Fourier series, f ϕs(x, t) = X µ̸=(s,0) u(s) µ (x)fµ,t, ϕa(x, t) = X µ u(a) µ (x)fµ,t. (66) (66) Here we have again used our multi-index notations µ = (j,q), where j = a,s labels the sector and q the momentum. Each measurement selects a particular set of Fourier coefﬁcients and we denote the averages over many measurements by Here we have again used our multi-index notations µ = (j,q), where j = a,s labels the sector and q the momentum. Each measurement selects a particular set of Fourier coefﬁcients and we denote the averages over many measurements by fµ,t, fµ,t fν,t etc. (67) (67) The mean values for the Fourier coefﬁcients can be read off from the one-point functions calculated earlier, cf. Eqs. (60,61) The mean values for the Fourier coefﬁcients can be read off from the one-point functions calculated earlier, cf. Eqs. (60,61) fµ,t = 2ReRµ(t). (68) (68) The object of interest is then the time-dependent joint probability distribution P of Fourier coefﬁcients {fµ}. Within the SCTDHA all cumulants of φa,s other than the variance vanish, so that this probability distribution is Gaussian The object of interest is then the time-dependent joint probability distribution P of Fourier coefﬁcients {fµ}. Within the SCTDHA all cumulants of φa,s other than the variance vanish, so that this probability distribution is Gaussian P({fµ}, t) = 1 (2π)N/2 1 p det M(t) exp −1 2 X µ,ν fµ −fµ,t M−1 µν (t) fν −fν,t . (69) (69) Here N is the total number of Fourier modes retained in (66). Noting that φj(x, t)φl(y, t) c = u(j) µ (x) fµ,t fν,t −fµ,t fν,t u(l) ν (y), j, l ∈{a,s} (70) (70) and comparing to Eq. 4.1 Choice of initial state We now specialize to an initial state that is often used in the literature, see e.g. [46–49]. In [46], a quasi-classical argument is used to conjecture how the state of a pair of elongated Bose gases follows from the splitting process of a single gas. It is reasoned that when splitting a gas, each particle has an equal probability to end up in well 1 or in well 2. The relative particle number resulting from this Poisson process is thus a stochastic variable with mean zero and variance proportional to the particle density. Assuming short-range correlations, one arrives at 〈Πa(x,0)Πa(y,0)〉c = ηρ0 2 δξ(x −y), (76) (76) with η a phenomenological parameter which we will set to 1. Following [49], the delta func- tion above is understood as a ﬂat sum over plane waves running up to momentum π/ξ. To reproduce this initial two-point function, it sufﬁces to use the initial state (37), with r a real and diagonal matrix and ϕ = 0. The resulting initial condition on Q, with η a phenomenological parameter which we will set to 1. Following [49], the delta func- tion above is understood as a ﬂat sum over plane waves running up to momentum π/ξ. To reproduce this initial two-point function, it sufﬁces to use the initial state (37), with r a real and diagonal matrix and ϕ = 0. The resulting initial condition on Q, Q(0)(a,j)(a,k) = δjke−rj j, (77) (77) then leads to 〈Πa(x,0)Πa(y,0)〉c = K L2 e−2r00 + X j>0 qK πL cos qj x cos qj y e−2rj j. (78) (78) Comparing Eqs. (76) and (49), we can thus read off Comparing Eqs. (76) and (49), we can thus read off Comparing Eqs. (76) and (49), we can thus read off e−2rjk = δjk ¨ Lηρ0 2K if q = 0, πηρ0 qK if q > 0, (79) (79) for the antisymmetric sector. for the antisymmetric sector. For the symmetric sector, we again follow Refs. [47–49]: the above quasi-classical splitting argument applies to the relative degrees of freedom, leaving the symmetric combinations of densities and phases unaltered. In [49], the symmetric sector is therefore taken to be in a ﬁnite temperature equilibrium state. We adhere to this conjecture here and use the thermal density matrix described in Section 3.1, thereby ﬁxing the initial conditions for both T and S in conjunction with the above discussion. 3.4 Full distribution functions (64), we can directly read off the covariance matrix as well: M(t) = 2Re(QgQ†) + QQ† + 〈δN 2 s0〉 K ImQP(s) 0 ImQT. (71) (71) Having obtained a time-dependent probability distribution for the coefﬁcients {fµ,t}, we can directly model experiments: we draw coefﬁcients {fµ,t} from the distribution (69), recon- struct the corresponding eigenvalues (66), and insert these in the time-of-ﬂight density (14) to compute the measured density proﬁle. We note that in the notations used above the set {fµ} contains the non-physical Fourier coefﬁcient f(s,0). This quantity does not enter the observable (14), and can simply be discarded, whenever a set of coefﬁcients is drawn from P {fµ}, t . µ By repeating the above procedure for modelling a measurement many times over we can reconstruct the full distribution function of any observable that depends only on the phase ﬁelds φa,s. In what follows, we will focus on the “interference term” in the spatially integrated density after time-of-ﬂight Rtof(x0,⃗r, t1, t0) deﬁned in (16). The eigenvalues of this observable are proportional to Iℓ {fµ}, x0, t0, t1 = 1 ℓ Z x0+ℓ/2 x0−ℓ/2 d x g+(x)g∗ −(x), (72) (72) where g±(x) are deﬁned in (17) and are related to the coefﬁcients fµ via (66). Motivated by the experimental data analyses of Refs [21,22,68] we parametrize the interference term (72) as Iℓ {fµ}, x0, t0, t1 = Cℓ(x0, t0, t1,{fµ})eiΦℓ(x0,t0,t1,{fµ}). (73) (73) 13 SciPost Phys. 9, 025 (2020) By drawing many sets {fµ} of coefﬁcients from the distribution function P {fµ}, t and plot- ting the resulting values of Φℓor Cℓin a normalized histogram, we converge to probability distributions PΦℓ,Cℓfor these quantities. These distribution functions can formally be written as as as PΦℓ(α, t0, t1) = Y µ Z dfµ δ α −ArgIℓ {fµ}, x0, t0, t1 P {fµ}, t0 , (74) PCℓ(γ, t0, t1) = Y µ Z dfµ δ γ −AbsIℓ {fµ}, x0, t0, t1 P {fµ}, t0 . (75) (74) (75) 4.1 Choice of initial state Finally, the initial conditions for R can be used to enforce various initial proﬁles on the density and phase ﬁelds in both sectors, which we will explore in Sec. 4.3 below. 14 SciPost Phys. 9, 025 (2020) 4.2 Experimental parameters We ﬁx the parameters for our plots by following Ref. [21]: the one-dimensional density is taken to be ρ0 = 45µm−1, the coherence length is ξ = ħhπ/mv = π × 0.42µm, the sound velocity is given by v ≈1.738 · 10−3 m/s and the Luttinger parameter in our conventions is K ≈28. We take the one-dimensional box size as large as we can achieve for a given value of the cutoff length scale, which amounts to L = 80µm. This is comparable to the size reported in [21]. In all ﬁgures, time is measured in units of the traversal time [4], ttr = L/2v, which is the time it takes for a light cone to reach the edge of the system from the centre of the box. We work at a temperature of 5nK throughout. This ensures that we are well in the scaling regime, at an energy density that is 1/8 times the cutoff energy εc = vπ/ξ, with ξ the coherence length. We have chosen t⊥= 15Hz, which guarantees that the gap is of the same order as the temperature for the above parameters. The only exception to this choice is Fig. 2, where we take t⊥≈1.17Hz following Ref. [69], to enable a comparison with the case of periodic boundary conditions as presented in that paper. 4.3.1 No coupling between symmetric and antisymmetric sectors (σ = 0) ¬ f φs(x,0) ¶ = 0 = 〈Πs(x,0)〉 (81) (81) and σ = 0. This will serve as our benchmark, as it most closely resembles the translationally invariant scenario described in [69] in which the (anti)symmetric sectors remain uncorrelated. It is characterized by Josephson oscillations between density and phase, see Fig. 1(a), with a phase variance that initially grows, and then shows oscillating behavior, see Fig. 1(b). To connect with our previous work [69] we include a comparison between results from that paper, where periodic boundary conditions were used, and the results derived for a box geometry in the present paper. Fig. 2 shows that the two geometries give extremely similar results in the centre of the trap for times below the traversal time, whereas deviations do occur after this time. It should also be noted that in [69] and Fig. 2, results are presented for smaller tunnel couplings (t⊥≈1.17Hz) than in the rest of this paper. The reason for choosing these values in [69] was that for a relatively shallow ﬁeld potential, the inharmonicity of the cosine in the sine-Gordon model manifests itself more strongly, making deviations from the purely quadratic theory more apparent. For the purposes of this paper, however, it is more interesting to look at relatively large tunnel-couplings (t⊥= 15Hz, see Sec. 4.2), as this enhances the coupling between the sectors in which we are interested. 4.3 Time evolution We now consider time evolution under the SCTDHA Hamiltonian (29), with the initial condi- tion described in Sec. 4.1. Throughout, we choose R(0) such that 〈φa(x,0)〉= 0.2, 〈Πa(x,0)〉= 0. (80) (80) The one-point functions f φs(x,0) and 〈Πs(x,0)〉will be given different spatial proﬁles, to investigate the effects of broken translational invariance. The one-point functions f φs(x,0) and 〈Πs(x,0)〉will be given different spatial proﬁles, to investigate the effects of broken translational invariance. 4.3.2 Finite coupling between sectors (σ > 0) and homogeneous initial conditions We next investigate different values of the coupling constant σ, and the resulting mixing be- tween the sectors. Fig. 3 shows results for σ = 0,1/2,1,3/2,2, starting from completely ﬂat proﬁles, as in Eqs. (80), (81). When increasing σ, the phase oscillations remain essentially 15 SciPost Phys. 9, 025 (2020) 0.0 0.5 1.0 1.5 2.0 t/ttr. −0.3 −0.2 −0.1 0.0 0.1 0.2 ⟨φa(x0, t)⟩ ∼⟨Πa(x0, t)⟩ 0.0 0.5 1.0 1.5 2.0 t/ttr. 0.025 0.050 0.075 0.100 0.125 0.150 0.175 φ2 a(x0, t) c (a) (b) Figure 1: (a) Josephson oscillations of relative density (arbitrary units) and phase (radians) at the centre of the gas, x0 = L/2. (b) initial growth and oscillations of the variance of the relative phase. The initial phase and density proﬁles are chosen ac- cording to Eqs. (80,81) and coupling between the sectors is absent in these pictures, meaning σ = 0. 0.0 0.5 1.0 1.5 2.0 t/ttr. 0.025 0.050 0.075 0.100 0.125 0.150 0.175 φ2 a(x0, t) c (b) 0.0 0.5 1.0 1.5 2.0 t/ttr. −0.3 −0.2 −0.1 0.0 0.1 0.2 ⟨φa(x0, t)⟩ ∼⟨Πa(x0, t)⟩ (a) (b) (a) Figure 1: (a) Josephson oscillations of relative density (arbitrary units) and phase (radians) at the centre of the gas, x0 = L/2. (b) initial growth and oscillations of the variance of the relative phase. The initial phase and density proﬁles are chosen ac- cording to Eqs. (80,81) and coupling between the sectors is absent in these pictures, meaning σ = 0. 0 1 2 3 4 5 6 t/ttr. −0.3 −0.2 −0.1 0.0 0.1 0.2 ⟨φa(x0, t)⟩Box ⟨φa(x0, t)⟩PBC 0.0 0.5 1.0 1.5 2.0 t/ttr. 0.0 0.2 0.4 0.6 φ2 a(x0, t) c,Box φ2 a(x0, t) c,PBC (a) (b) Figure 2: Comparison between results for box boundary conditions (blue) and peri- odic boundary conditions (red). The curves are in perfect agreement until the traver- sal time ttr = L/2v, after which deviations occur. (a) Josephson oscillations of phase (radians) at the centre of the gas, x0 = L/2. (b) initial growth and subsequent oscil- lations in the variance of the relative phase. 0 1 2 3 4 5 6 t/ttr. −0.3 −0.2 −0.1 0.0 0.1 0.2 ⟨φa(x0, t)⟩Box ⟨φa(x0, t)⟩PBC (a) 0.0 0.5 1.0 1.5 2.0 t/ttr. 4.3.2 Finite coupling between sectors (σ > 0) and homogeneous initial conditions (b) a somewhat stronger effect is the development of correlations between φa,s, where the normalized covariance from Eq. (82) is displayed, for x0 = L/2. 0.0 0.5 1.0 1.5 2.0 t/ttr. 0.05 0.10 0.15 φ2 a(x0, t) c \|σ=0 φ2 a(x0, t) c \|σ=2 Figure 4: Variance of the relative phase, for σ = 0 (blue) and σ = 2 (red). A slight increase in the variance is visible for the larger value of σ for times below ttr = L/2v. 0.0 0.5 1.0 1.5 2.0 t/ttr. 0.05 0.10 0.15 φ2 a(x0, t) c \|σ=0 φ2 a(x0, t) c \|σ=2 Figure 4: Variance of the relative phase, for σ = 0 (blue) and σ = 2 (red). A slight increase in the variance is visible for the larger value of σ for times below ttr = L/2v. To this end we deﬁne the following quantities To this end we deﬁne the following quantities ea,0(t) = 〈Ha〉 L , ea,⊥(t) = −2t⊥ρ0 L Z L 0 d x 〈cosφa(x)〉, esG(t) = ea,0(t) + ea,⊥(t), eint(t) = −2t⊥σ L Z L 0 d x 〈Πs(x)cosφa(x)〉, es(t) = eint(t) + 〈Hs(t)〉/L. (83) (83) We note that the total energy density, which is given by esG(t) + eint(t) + 〈Hs〉/L, is indepen- dent of time, as required for a closed quantum system. Since we are interested in the time dependence of the various energy densities we subtract their values in the initial state and consider We note that the total energy density, which is given by esG(t) + eint(t) + 〈Hs〉/L, is indepen- dent of time, as required for a closed quantum system. Since we are interested in the time dependence of the various energy densities we subtract their values in the initial state and consider ∆ej(t) ≡ej(t) −ej(0). (84) (84) To quantify the effects of the σ-coupling on the ﬂow of energy from and to the sine-Gordon model we show ∆eSG(t) in Fig. 5. To ascertain which fraction of the energy change is due to the kinetic and interaction parts of the sine-Gordon model we also show ∆ea(t) and ∆e⊥,a(t) in Fig. 5(a). We observe that the change in ∆eSG(t) is very small, as signiﬁcantly larger changes in ∆ea(t) and ∆e⊥,a(t) largely compensate each other. In Fig. 4.3.2 Finite coupling between sectors (σ > 0) and homogeneous initial conditions 0.0 0.2 0.4 0.6 φ2 a(x0, t) c,Box φ2 a(x0, t) c,PBC (b) (b) (a) Figure 2: Comparison between results for box boundary conditions (blue) and peri- odic boundary conditions (red). The curves are in perfect agreement until the traver- sal time ttr = L/2v, after which deviations occur. (a) Josephson oscillations of phase (radians) at the centre of the gas, x0 = L/2. (b) initial growth and subsequent oscil- lations in the variance of the relative phase. unchanged. A stronger effect is visible in the covariance between φa and f φs, however. To quantify this, we deﬁne C(x, t) ≡ f φs(x, t)φa(x, t) c Ç f φs(x, t)f φs(x, t) c φa(x, t)φa(x, t) c . (82) (82) As can be seen in Fig. 3(b), the covariance C(x, t) increases to appreciable values as σ is increased. We also note that for larger values of σ, the variance of the relative phase increases somewhat for times below the traversal time, see Fig. 4. It is also instructive to consider the energy ﬂow between different terms in the Hamiltonia 16 SciPost Phys. 9, 025 (202 0.0 0.5 1.0 1.5 2.0 t/ttr. −0.2 −0.1 0.0 0.1 0.2 ⟨φa(x0, t)⟩ σ = 0 σ = 0.5 σ = 1 σ = 1.5 σ = 2 0.0 0.5 1.0 1.5 2.0 t/ttr. −0.02 −0.01 0.00 0.01 0.02 C(x0, t) (a) (b) Figure 3: (a) time evolution of the phase in the antisymmetric sector at the box centre x0 = L/2. Curves are displayed for different values of σ, with a ﬂat initial density proﬁle 〈Πs(x)〉= 0. A change of σ has no appreciable effect on this observable. (b) a somewhat stronger effect is the development of correlations between φa,s, where the normalized covariance from Eq. (82) is displayed, for x0 = L/2. SciPost Phys. 9, 025 (2020) 0.0 0.5 1.0 1.5 2.0 t/ttr. −0.2 −0.1 0.0 0.1 0.2 ⟨φa(x0, t)⟩ σ = 0 σ = 0.5 σ = 1 σ = 1.5 σ = 2 (a) 0.0 0.5 1.0 1.5 2.0 t/ttr. −0.02 −0.01 0.00 0.01 0.02 C(x0, t) (b) (b) (a) Figure 3: (a) time evolution of the phase in the antisymmetric sector at the box centre x0 = L/2. Curves are displayed for different values of σ, with a ﬂat initial density proﬁle 〈Πs(x)〉= 0. A change of σ has no appreciable effect on this observable. 4.3.2 Finite coupling between sectors (σ > 0) and homogeneous initial conditions 5(b) we show how much of the energy from the sine-Gordon model ∆eSG(t) ends up in the new interaction term eint(t) and how much goes to 〈Hs(t)〉/L. To quantify the effects of the σ-coupling on the ﬂow of energy from and to the sine-Gordon model we show ∆eSG(t) in Fig. 5. To ascertain which fraction of the energy change is due to the kinetic and interaction parts of the sine-Gordon model we also show ∆ea(t) and ∆e⊥,a(t) in Fig. 5(a). We observe that the change in ∆eSG(t) is very small, as signiﬁcantly larger changes in ∆ea(t) and ∆e⊥,a(t) largely compensate each other. In Fig. 5(b) we show how much of the energy from the sine-Gordon model ∆eSG(t) ends up in the new interaction term eint(t) and how much goes to 〈Hs(t)〉/L. 17 SciPost Phys. 9, 025 (202 0.0 0.5 1.0 1.5 2.0 t/ttr. −0.6 −0.4 −0.2 0.0 0.2 0.4 0.6 ∆esG(t)/er × 103 ∆es(t)/er × 103 ∆ea(t)/er ∆e⊥,a(t)/er 0.0 0.2 0.4 0.6 0.8 1.0 t/ttr. −0.0003 −0.0002 −0.0001 0.0000 0.0001 0.0002 ∆esG(t)/er ∆⟨Hs(t)⟩/Ler ∆eint(t)/er ∆etot(t)/er (a) (b) Figure 5: Energy ﬂow between the different terms in Eqs. (83), as a ratio with the reference scale er = 〈Hs(0)〉/L. SciPost Phys. 9, 025 (2020) 0.0 0.5 1.0 1.5 2.0 t/ttr. −0.6 −0.4 −0.2 0.0 0.2 0.4 0.6 ∆esG(t)/er × 103 ∆es(t)/er × 103 ∆ea(t)/er ∆e⊥,a(t)/er (a) 0.0 0.2 0.4 0.6 0.8 1.0 t/ttr. −0.0003 −0.0002 −0.0001 0.0000 0.0001 0.0002 ∆esG(t)/er ∆⟨Hs(t)⟩/Ler ∆eint(t)/er ∆etot(t)/er (b) (b) (a) Figure 5: Energy ﬂow between the different terms in Eqs. (83), as a ratio with the reference scale er = 〈Hs(0)〉/L. 4.3.3 Finite coupling between sectors (σ > 0) and inhomogeneous initial conditions As a next step, we investigate the effect of initial density proﬁles 〈Πs(x)〉that are spatially inhomogeneous. These proﬁles will evolve in time as is shown in Fig. 6 (a,b). The proﬁles x/L 0.0 0.2 0.4 0.6 0.8 1.0 t/ttr 0.0 0.2 0.4 0.6 0.8 1.0 ⟨ρ0 + Πs(x)⟩/ρ0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 x/L 0.0 0.2 0.4 0.6 0.8 1.0 t/ttr 0.0 0.2 0.4 0.6 0.8 1.0 ⟨ρ0 + Πs(x)⟩/ρ0 0.6 0.8 1.0 1.2 1.4 (a) (b) Figure 6: Examples of the time evolution of the density proﬁle for σ = 0. The initial proﬁle in (a) is symmetric around the origin, while the one in (b) is not. x/L 0.0 0.2 0.4 0.6 0.8 1.0 t/ttr 0.0 0.2 0.4 0.6 0.8 1.0 ⟨ρ0 + Πs(x)⟩/ρ0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 (a) x/L 0.0 0.2 0.4 0.6 0.8 1.0 t/ttr 0.0 0.2 0.4 0.6 0.8 1.0 ⟨ρ0 + Πs(x)⟩/ρ0 0.6 0.8 1.0 1.2 1.4 (b) (b) (a) Figure 6: Examples of the time evolution of the density proﬁle for σ = 0. The initial proﬁle in (a) is symmetric around the origin, while the one in (b) is not. 〈φa(x)〉and 〈Πa(x)〉are strongly affected by the strength of the σ-coupling to the inhomo- geneous proﬁle 〈Πs(x)〉and develop inhomogeneities as a consequence. This is illustrated in Figs. 7(a,b) and has repercussions for the Josephson oscillations. The latter now display spatial variations, which are caused by the oscillation frequency becoming σ- and position-dependent due to the presence of the space-dependent Πs(x)-ﬁeld in the interaction term. This local and σ-dependent frequency is illustrated in Fig. 8. The spatial average of the phase, which is equal to the zero mode φa0, does not show any σ-dependence in its Josephson frequency, see Figs. 9,10. In this case, however, a σ-dependent modulation in the amplitudes is visible: the oscil- lations at different points in the box move out of phase due to the spatially varying frequency mentioned above. This leads to a decrease in the spatial average. For an inhomogeneous pro- ﬁle of 〈Πs(x,0)〉, the covariance grows in time, in resemblance with the homogeneous case. This happens to an extent that is roughly proportional to σ. The same can be said of the en- ergy ﬂow between the (anti)symmetric sectors, as shown in Fig. 11. 4.3.3 Finite coupling between sectors (σ > 0) and inhomogeneous initial conditions The effects of the sector coupling term become stronger when we increase σ, but in the window of applicability of our bosonization approach the effects remain small. 〈φa(x)〉and 〈Πa(x)〉are strongly affected by the strength of the σ-coupling to the inhomo- geneous proﬁle 〈Πs(x)〉and develop inhomogeneities as a consequence. This is illustrated in Figs. 7(a,b) and has repercussions for the Josephson oscillations. The latter now display spatial variations, which are caused by the oscillation frequency becoming σ- and position-dependent due to the presence of the space-dependent Πs(x)-ﬁeld in the interaction term. This local and σ-dependent frequency is illustrated in Fig. 8. The spatial average of the phase, which is equal to the zero mode φa0, does not show any σ-dependence in its Josephson frequency, see Figs. 9,10. In this case, however, a σ-dependent modulation in the amplitudes is visible: the oscil- lations at different points in the box move out of phase due to the spatially varying frequency mentioned above. This leads to a decrease in the spatial average. For an inhomogeneous pro- ﬁle of 〈Πs(x,0)〉, the covariance grows in time, in resemblance with the homogeneous case. This happens to an extent that is roughly proportional to σ. The same can be said of the en- ergy ﬂow between the (anti)symmetric sectors, as shown in Fig. 11. The effects of the sector coupling term become stronger when we increase σ, but in the window of applicability of our bosonization approach the effects remain small. 18 SciPost Phys. 9, 025 (2020) x/L 0.0 0.2 0.4 0.6 0.8 1.0 t/ttr 0.0 0.2 0.4 0.6 0.8 1.0 ⟨φa(x)⟩ −1.0 −0.5 0.0 0.5 1.0 x/L 0.0 0.2 0.4 0.6 0.8 1.0 t/ttr 0.0 0.2 0.4 0.6 0.8 1.0 ⟨Πa(x)⟩/ρ0 −0.10 −0.05 0.00 0.05 0.10 (a) (b) Figure 7: (a) The time and position dependence of 〈φa(x)〉corresponding to the same initial condition as Fig. 6(a) with σ = 2. We see that the initially ﬂat proﬁle develops inhomogeneities due to the sector coupling. (b) the same as panel (a), but showing 〈Πa(x)〉. x/L 0.0 0.2 0.4 0.6 0.8 1.0 t/ttr 0.0 0.2 0.4 0.6 0.8 1.0 ⟨Πa(x)⟩/ρ0 −0.10 −0.05 0.00 0.05 0.10 (b) x/L 0.0 0.2 0.4 0.6 0.8 1.0 t/ttr 0.0 0.2 0.4 0.6 0.8 1.0 ⟨φa(x)⟩ −1.0 −0.5 0.0 0.5 1.0 (a) (b) (a) Figure 7: (a) The time and position dependence of 〈φa(x)〉corresponding to the same initial condition as Fig. 4.3.3 Finite coupling between sectors (σ > 0) and inhomogeneous initial conditions 6(a) with σ = 2. We see that the initially ﬂat proﬁle develops inhomogeneities due to the sector coupling. (b) the same as panel (a), but showing 〈Πa(x)〉. 0.0 0.5 1.0 1.5 2.0 t/ttr. −0.2 −0.1 0.0 0.1 0.2 ⟨φa(x0, t)⟩ σ = 0 σ = 0.5 σ = 1 σ = 1.5 σ = 2 0.0 0.5 1.0 1.5 2.0 t/ttr. −0.2 −0.1 0.0 0.1 0.2 ⟨φa(x0, t)⟩ σ = 0 σ = 0.5 σ = 1 σ = 1.5 σ = 2 (a) (b) Figure 8: Time dependence of the relative phase in the centre of the box for the same initial conditions as Fig. 6(a) and (b), respectively. 0.0 0.5 1.0 1.5 2.0 t/ttr. −0.2 −0.1 0.0 0.1 0.2 ⟨φa(x0, t)⟩ σ = 0 σ = 0.5 σ = 1 σ = 1.5 σ = 2 0.0 0.5 1.0 1.5 2.0 t/ttr. −0.2 −0.1 0.0 0.1 0.2 ⟨φa(x0, t)⟩ σ = 0 σ = 0.5 σ = 1 σ = 1.5 σ = 2 (a) (b) Figure 8: Time dependence of the relative phase in the centre of the box for the same initial conditions as Fig. 6(a) and (b), respectively. 0.0 0.5 1.0 1.5 2.0 t/ttr. −0.2 −0.1 0.0 0.1 0.2 ⟨φa,0⟩ σ = 0 σ = 0.5 σ = 1 σ = 1.5 σ = 2 0.0 0.5 1.0 1.5 2.0 t/ttr. −0.02 −0.01 0.00 0.01 0.02 C(x0, t) Figure 9: Time dependence of the space-averaged relative phase and the covariance C(x0, t) (82) in the centre of the box for the proﬁles shown in panel (a) of Fig. 6. 0.0 0.5 1.0 1.5 2.0 t/ttr. −0.2 −0.1 0.0 0.1 0.2 ⟨φa(x0, t)⟩ σ = 0 σ = 0.5 σ = 1 σ = 1.5 σ = 2 (b) 0.0 0.5 1.0 1.5 2.0 t/ttr. −0.2 −0.1 0.0 0.1 0.2 ⟨φa(x0, t)⟩ σ = 0 σ = 0.5 σ = 1 σ = 1.5 σ = 2 (a) (b) (a) Figure 8: Time dependence of the relative phase in the centre of the box for the same initial conditions as Fig. 6(a) and (b), respectively. 0.0 0.5 1.0 1.5 2.0 t/ttr. −0.2 −0.1 0.0 0.1 0.2 ⟨φa,0⟩ σ = 0 σ = 0.5 σ = 1 σ = 1.5 σ = 2 0.0 0.5 1.0 1.5 2.0 t/ttr. 4.3.3 Finite coupling between sectors (σ > 0) and inhomogeneous initial conditions −0.02 −0.01 0.00 0.01 0.02 C(x0, t) Figure 9: Time dependence of the space-averaged relative phase and the covariance C(x0, t) (82) in the centre of the box for the proﬁles shown in panel (a) of Fig. 6. 0.0 0.5 1.0 1.5 2.0 t/ttr. −0.02 −0.01 0.00 0.01 0.02 C(x0, t) 0.0 0.5 1.0 1.5 2.0 t/ttr. −0.2 −0.1 0.0 0.1 0.2 ⟨φa,0⟩ σ = 0 σ = 0.5 σ = 1 σ = 1.5 σ = 2 Figure 9: Time dependence of the space-averaged relative phase and the covariance C(x0, t) (82) in the centre of the box for the proﬁles shown in panel (a) of Fig. 6. 4.3.4 Distribution functions of the density after time of ﬂight As described in Sec. 3.4, our formalism allows the construction of distribution functions for the measured density after time-of-ﬂight expansion. As a proof of principle we present such distribution functions in Fig. 12, for the observables Φℓand Cℓdeﬁned in Eq. (73). 19 SciPost Phys. 9, 025 (2020) 0.0 0.5 1.0 1.5 2.0 t/ttr. −0.2 −0.1 0.0 0.1 0.2 ⟨φa,0⟩ σ = 0 σ = 0.5 σ = 1 σ = 1.5 σ = 2 0.0 0.5 1.0 1.5 2.0 t/ttr. −0.02 −0.01 0.00 0.01 C(x0, t) Figure 10: Time dependence of the space-averaged relative phase and the covariance C(x0, t) (82) in the centre of the box for the proﬁles shown in panel (b) of Fig. 6. SciPost Phys. 9, 025 (2020) 0.0 0.5 1.0 1.5 2.0 t/ttr. −0.2 −0.1 0.0 0.1 0.2 ⟨φa,0⟩ σ = 0 σ = 0.5 σ = 1 σ = 1.5 σ = 2 0.0 0.5 1.0 1.5 2.0 t/ttr. −0.02 −0.01 0.00 0.01 C(x0, t) Figure 10: Time dependence of the space-averaged relative phase and the covariance C(x0, t) (82) in the centre of the box for the proﬁles shown in panel (b) of Fig. 6. 0.0 0.5 1.0 1.5 2.0 t/ttr. −0.02 −0.01 0.00 0.01 0.02 ∆esG(t)/er × 10 ∆es(t)/er × 10 ∆ea(t)/er ∆e⊥,a(t)/er 0.0 0.5 1.0 1.5 2.0 t/ttr. −0.02 −0.01 0.00 0.01 0.02 ∆esG(t)/er × 10 ∆es(t)/er × 10 ∆ea(t)/er ∆e⊥,a(t)/er Figure 11: Energy ﬂow between different terms in Eqs. (83), as a ratio with the reference scale er = 〈Hs(0)〉/L. Results are shown for the density proﬁle from Fig. 6(a), with σ = 1 (left panel) and σ = 2 (right panel). 0.0 0.5 1.0 1.5 2.0 t/ttr. −0.02 −0.01 0.00 0.01 C(x0, t) 0.0 0.5 1.0 1.5 2.0 t/ttr. −0.2 −0.1 0.0 0.1 0.2 ⟨φa,0⟩ σ = 0 σ = 0.5 σ = 1 σ = 1.5 σ = 2 Figure 10: Time dependence of the space-averaged relative phase and the covariance C(x0, t) (82) in the centre of the box for the proﬁles shown in panel (b) of Fig. 6. 0.0 0.5 1.0 1.5 2.0 t/ttr. −0.02 −0.01 0.00 0.01 0.02 ∆esG(t)/er × 10 ∆es(t)/er × 10 ∆ea(t)/er ∆e⊥,a(t)/er 0.0 0.5 1.0 1.5 2.0 t/ttr. −0.02 −0.01 0.00 0.01 0.02 ∆esG(t)/er × 10 ∆es(t)/er × 10 ∆ea(t)/er ∆e⊥,a(t)/er Figure 11: Energy ﬂow between different terms in Eqs. (83), as a ratio with the reference scale er = 〈Hs(0)〉/L. 5 Conclusion We have extended the theory for non-equilibrium dynamics in pairs of elongated, tunnel- coupled Bose gases using a self-consistent time-dependent harmonic approximation (SCTDHA) in the low-energy scaling limit (energy scales ≲5nK). In contrast to earlier works, we have studied the effect of a relevant perturbation which couples the (anti)symmetric sectors de- scribing (anti)symmetric combinations of the two Bose gas phases. On top of this, we have dropped the assumption of translational invariance by placing the system in a box and by imposing inhomogeneous initial density proﬁles. Starting from an initial state in which these sectors are uncorrelated, the coupling of the sectors under time evolution leads to a number of new but weak effects. First of all we ob- serve the development of correlations between the sectors over time. This effect is present for all initial states we have considered, but the covariance between the sectors never reaches more than a few percent of the geometric mean of the variances. Second, the spreading of correlations is accompanied by a small transfer of energy between the sectors. And ﬁnally, the presence of the coupling term makes the dynamics in the antisymmetric sector susceptible to the breaking of translational invariance in the symmetric sector. The well-known Josephson oscillations of relative density and phase are modulated when taking an inhomogeneous ini- tial density proﬁle of the symmetric sector. This shows that the role of the trapping potential, which creates strong inhomogeneities, may play a more important role in experiment than was previously assumed. However, the model presented here does not capture the puzzling damping phenomenon observed recently [24, 66, 67]. This is not surprising given that our box potential is very different from the quadratic potential used in experiment. In future ex- periments, however, the box potential is likely to be used, which adds to the relevance of the calculations presented here. p We conclude that (i) the new term coupling the (anti)symmetric sectors leads to very weak effects. This means that the simulation of a sine-Gordon model using the setup described in this paper should not be severely hampered by the presence of this term. (ii) we have shown that it is possible to treat states with broken translational invariance in the SCTDHA formalism as presented in [69]. Combined with the sector coupling, we ﬁnd that inhomogeneities in the density can have weak but nontrivial effects on the amplitude of Josephson oscillations. 5 Conclusion This means that the trapping potential is likely to have an effect on the dynamics probed in experiment. In a forthcoming paper, we will present a study of the projected Hamiltonian (3) in a microscopic analysis that takes a quadratic longitudinal potential into account. It would be interesting to combine such a microscopic approach with low-energy effective ﬁeld theory calculations in the presence of such a quadratic trapping potential. This could be done by combining a SCTDHA with recent results [79, 80] for inhomogeneous Luttinger liquids [81–84]. However, the calculations using the box potential presented here may gain additional relevance when more experiments using a box potential, such as Refs. [85,86], are performed. 4.3.4 Distribution functions of the density after time of ﬂight Results are shown for the density proﬁle from Fig. 6(a), with σ = 1 (left panel) and σ = 2 (right panel). 0.0 0.5 1.0 1.5 2.0 t/ttr. −0.02 −0.01 0.00 0.01 0.02 ∆esG(t)/er × 10 ∆es(t)/er × 10 ∆ea(t)/er ∆e⊥,a(t)/er 0.0 0.5 1.0 1.5 2.0 t/ttr. −0.02 −0.01 0.00 0.01 0.02 ∆esG(t)/er × 10 ∆es(t)/er × 10 ∆ea(t)/er ∆e⊥,a(t)/er Figure 11: Energy ﬂow between different terms in Eqs. (83), as a ratio with the reference scale er = 〈Hs(0)〉/L. Results are shown for the density proﬁle from Fig. 6(a), with σ = 1 (left panel) and σ = 2 (right panel). (a) (b) Figure 12: Distribution functions PΦℓ(α, t, t1) (a) and PCℓ(γ, t, t1) for the observables Φℓand Cℓdeﬁned in Eq. (73). We choose a time of ﬂight t1 = 15ms and integration length ℓ= 20µm. The density proﬁle used for these plots is homogeneous, with σ = 1. (b) (a) (a) (b) Figure 12: Distribution functions PΦℓ(α, t, t1) (a) and PCℓ(γ, t, t1) for the observables Φℓand Cℓdeﬁned in Eq. (73). We choose a time of ﬂight t1 = 15ms and integration length ℓ= 20µm. The density proﬁle used for these plots is homogeneous, with σ = 1. 20 SciPost Phys. 9, 025 (2020) A Tensors occurring in HSCH(t) The tensors occurring in HSCH(t) as written in Eq. (50) are given by g SCH( ) q ( ) g y A =             ... ... ... ... vq δq,k + 2∆(1) q,k(t)u(a) a,q(0)u(a) a,k(0) ∆(2) q,k(t)u(a)∗ a,q (0)w(s) s,k(0) ... ... ... ... ... ... ... ... ∆(2) q,k(t)u(a) a,k(0)w(s)∗ s,q (0) vq δq,k ... ... ... ...             , B =             ... ... ... ... −vπ L δq,kδq,0 + 2∆(1) q,k(t)u(a) a,q(0)u(a) a,k(0) ∆(2) q,k(t)u(a) a,q(0)w(s) s,k(0) ... ... ... ... ... ... ... ... ∆(2) q,k(t)u(a) a,k(0)w(s) s,q(0) −vπ L δq,kδq,0 ... ... ... ...             , D =            ... Γ (1) q (t)u(a) a,q(0) ... ... 0 ...            , E =            ... 0 ... ... Γ (2) q (t)w(s) s,q(0) ...            . (85) (85) The momentum indices q, k run within the blocks demarcated by solid lines, whereas the sector indices j = a,s change from one block to the other. The functions occurring above are deﬁned via The momentum indices q, k run within the blocks demarcated by solid lines, whereas the sector indices j = a,s change from one block to the other. The functions occurring above are deﬁned via Γ (i) q (t) = Z L 0 d x g(i)(x, t)cos(qx), (86) ∆(i) q,k(t) = Z L 0 d x h(i)(x, t)cos(qx)cos(kx) = 1 2 h(i) q+k(t) + h(i) \|q−k\|(t) (87) h(i) q (t) = Z L 0 d x h(i)(x, t)cos(qx). (88) (86) ∆(i) q,k(t) = Z L 0 d x h(i)(x, t)cos(qx)cos(kx) = 1 2 h(i) q+k(t) + h(i) \|q−k\|(t) (87) Z L (87) h(i) q (t) = Z L 0 d x h(i)(x, t)cos(qx). (88) (88) erences Acknowledgements We are grateful to Jörg Schmiedmayer, Thomas Schweigler and Marine Pigneur for stimu- lating discussions and to the Erwin Schrödinger International Institute for Mathematics and Physics for hospitality and support during the programme on Quantum Paths. This work was supported by the EPSRC under grant EP/S020527/1 and YDvN is supported by the Merton College Buckee Scholarship and the VSB, Muller and Prins Bernhard Foundations. 21 SciPost Phys. 9, 025 (2020) A Tensors occurring in HSCH(t) References [1] M. Rigol, V. Dunjko, V. Yurovsky and M. Olshanii, Relaxation in a completely inte- grable many-body quantum system: an ab initio study of the dynamics of the highly excited states of 1D lattice hard-core bosons, Phys. Rev. Lett. 98, 050405 (2007), doi:10.1103/PhysRevLett.98.050405. [2] P. Calabrese and J. Cardy, Quantum quenches in extended systems, J. Stat. Mech. P06008 (2007), doi:10.1088/1742-5468/2007/06/P06008. [2] P. Calabrese and J. Cardy, Quantum quenches in extended systems, J. Stat. Mech. P06008 (2007), doi:10.1088/1742-5468/2007/06/P06008. 22 SciPost Phys. 9, 025 (2020) [3] A. Polkovnikov, K. Sengupta, A. Silva and M. Vengalattore, Colloquium: Nonequilib- rium dynamics of closed interacting quantum systems, Rev. Mod. Phys. 83, 863 (2011), doi:10.1103/RevModPhys.83.863. [4] F. H. L. Essler and M. Fagotti, Quench dynamics and relaxation in isolated in- tegrable quantum spin chains, J. Stat. Mech. 064002 (2016), doi:10.1088/1742- 5468/2016/06/064002. [5] L. Vidmar and M. Rigol, Generalized Gibbs ensemble in integrable lattice models, J. Stat. Mech. 064007 (2016), doi:10.1088/1742-5468/2016/06/064007. [6] L. D’Alessio, Y. Kafri, A. Polkovnikov and M. Rigol, From quantum chaos and eigenstate thermalization to statistical mechanics and thermodynamics, Adv. Phys. 65, 239 (2016), doi:10.1080/00018732.2016.1198134. [7] C. Gogolin and J. Eisert, Equilibration, thermalisation, and the emergence of sta- tistical mechanics in closed quantum systems, Rep. Prog. Phys. 79, 056001 (2016), doi:10.1088/0034-4885/79/5/056001. [8] P. Calabrese and J. Cardy, Quantum quenches in 1+1 dimensional conformal ﬁeld theories, J. Stat. Mech. 064003 (2016), doi:10.1088/1742-5468/2016/06/064003. [9] P. Calabrese, Entanglement and thermodynamics in non-equilibrium isolated quantum sys- tems, Phys. A: Stat. Mech. Appl. 504, 31 (2018), doi:10.1016/j.physa.2017.10.011. [10] A. Görlitz et al., Realization of Bose-Einstein condensates in lower dimensions, Phys. Rev. Lett. 87, 130402 (2001), doi:10.1103/PhysRevLett.87.130402. [11] M. Greiner, I. Bloch, O. Mandel, T. W. Hänsch and T. Esslinger, Bose–Einstein condensates in 1D- and 2D optical lattices, Appl. Phys. B 73, 769 (2001), doi:10.1007/s003400100744. [12] T. Kinoshita, T. Wenger and D. S. Weiss, Observation of a one-dimensional Tonks-Girardeau gas, Science 305, 1125 (2004), doi:10.1126/science.1100700. [13] T. Schumm, S. Hofferberth, L. M. Andersson, S. Wildermuth, S. Groth, I. Bar-Joseph, J. Schmiedmayer and P. Krüger, Matter-wave interferometry in a double well on an atom chip, Nat. Phys. 1, 57 (2005), doi:10.1038/nphys125. [14] S. Hofferberth, I. Lesanovsky, B. Fischer, T. Schumm and J. Schmiedmayer, Non- equilibrium coherence dynamics in one-dimensional Bose gases, Nature 449, 324 (2007), doi:10.1038/nature06149. [15] S. Trotzky, Y.-A. Chen, A. Flesch, I. P. McCulloch, U. Schollwöck, J. Eisert and I. References Bloch, Probing the relaxation towards equilibrium in an isolated strongly correlated one- dimensional Bose gas, Nat. Phys. 8, 325 (2012), doi:10.1038/nphys2232. [16] M. Cheneau et al., Light-cone-like spreading of correlations in a quantum many-body sys- tem, Nature 481, 484 (2012), doi:10.1038/nature10748. [17] M. Gring et al., Relaxation and prethermalization in an isolated quantum system, Science 337, 1318 (2012), doi:10.1126/science.1224953. [18] T. Langen, R. Geiger, M. Kuhnert, B. Rauer and J. Schmiedmayer, Local emergence of thermal correlations in an isolated quantum many-body system, Nat. Phys. 9, 640 (2013), doi:10.1038/nphys2739. 23 SciPost Phys. 9, 025 (2020) [19] T. Langen et al., Experimental observation of a generalized Gibbs ensemble, Science 348, 207 (2015), doi:10.1126/science.1257026. [20] A. M. Kaufman, M. E. Tai, A. Lukin, M. Rispoli, R. Schittko, P. M. Preiss and M. Greiner, Quantum thermalization through entanglement in an isolated many-body system, Science 353, 794 (2016), doi:10.1126/science.aaf6725. [21] M. Kuhnert, R. Geiger, T. Langen, M. Gring, B. Rauer, T. Kitagawa, E. Demler, D. Adu Smith and J. Schmiedmayer, Multimode dynamics and emergence of a characteristic length scale in a one-dimensional quantum system, Phys. Rev. Lett. 110, 090405 (2013), doi:10.1103/PhysRevLett.110.090405. [22] D. A. Smith, M. Gring, T. Langen, M. Kuhnert, B. Rauer, R. Geiger, T. Kitagawa, I. Mazets, E. Demler and J. Schmiedmayer, Prethermalization revealed by the relaxation dynam- ics of full distribution functions, New J. Phys. 15, 075011 (2013), doi:10.1088/1367- 2630/15/7/075011. [23] T. Schweigler et al., Experimental characterization of a quantum many-body system via higher-order correlations, Nature 545, 323 (2017), doi:10.1038/nature22310. [24] M. Pigneur, T. Berrada, M. Bonneau, T. Schumm, E. Demler and J. Schmiedmayer, Relax- ation to a phase-locked equilibrium state in a one-dimensional bosonic Josephson junction, Phys. Rev. Lett. 120, 173601 (2018), doi:10.1103/PhysRevLett.120.173601. [25] M. R. Andrews, C. G. Townsend, H.-J. Miesner, D. S. Durfee, D. M. Kurn and W. Ket- terle, Observation of interference between two Bose condensates, Science 275, 637 (1997), doi:10.1126/science.275.5300.637. [26] Y. D. van Nieuwkerk, J. Schmiedmayer and F. Essler, Projective phase measurements in one- dimensional Bose gases, SciPost Phys. 5, 046 (2018), doi:10.21468/SciPostPhys.5.5.046. [27] R. W. Cherng and E. Demler, Quantum noise analysis of spin systems realized with cold atoms, New J. Phys. 9, 7 (2007), doi:10.1088/1367-2630/9/1/007. [28] A. Imambekov, V. Gritsev, and E. Demler, Fundamental noise in matter interferometers (2007), arXiv:cond-mat/0703766. [29] A. Lamacraft and P. Fendley, Order parameter statistics in the critical quantum ising chain, Phys. Rev. Lett. 100, 165706 (2008), doi:10.1103/PhysRevLett.100.165706. [30] D. References A. Ivanov and A. G. Abanov, Characterizing correlations with full counting statis- tics: Classical Ising and quantum XY spin chains, Phys. Rev. E 87, 022114 (2013), doi:10.1103/PhysRevE.87.022114. [31] Y. Shi and I. Klich, Full counting statistics and the Edgeworth series for matrix product states, J. Stat. Mech.: Theory Exp. 05001 (2013), doi:10.1088/1742-5468/2013/05/P05001. [32] V. Eisler, Universality in the full counting statistics of trapped fermions, Phys. Rev. Lett. 111, 080402 (2013), doi:10.1103/PhysRevLett.111.080402. [33] I. Klich, A note on the full counting statistics of paired fermions, J. Stat. Mech.: Theory Exp. P11006 (2014), doi:10.1088/1742-5468/2014/11/P11006. [34] J.-M. Stéphan and F. Pollmann, Full counting statistics in the Haldane-Shastry chain, Phys. Rev. B 95, 035119 (2017), doi:10.1103/PhysRevB.95.035119. 24 SciPost Phys. 9, 025 (2020) [35] M. Collura, F. H. L. Essler and S. Groha, Full counting statistics in the spin-1/2 Heisen- berg XXZ chain, J. Phys. A: Math. Theor. 50, 414002 (2017), doi:10.1088/1751- 8121/aa87dd. [36] K. Najaﬁand M. A. Rajabpour, Full counting statistics of the subsystem energy for free fermions and quantum spin chains, Phys. Rev. B 96, 235109 (2017), doi:10.1103/PhysRevB.96.235109. [37] S. Humeniuk and H. P. Büchler, Full counting statistics for interacting fermions with de- terminantal quantum monte carlo simulations, Phys. Rev. Lett. 119, 236401 (2017), doi:10.1103/PhysRevLett.119.236401. [38] I. Lovas, B. Dóra, E. Demler and G. Zaránd, Full counting statistics of time-of-ﬂight images, Phys. Rev. A 95, 053621 (2017), doi:10.1103/PhysRevA.95.053621. [39] A. Bastianello, L. Piroli and P. Calabrese, Exact local correlations and full counting statis- tics for arbitrary states of the one-dimensional interacting Bose gas, Phys. Rev. Lett. 120, 190601 (2018), doi:10.1103/PhysRevLett.120.190601. [40] S. Groha, F. Essler and P. Calabrese, Full counting statistics in the transverse ﬁeld Ising chain, SciPost Phys. 4, 043 (2018), doi:10.21468/SciPostPhys.4.6.043. [41] M. Arzamasovs and D. M. Gangardt, Full counting statistics and large de- viations in a thermal 1D Bose gas, Phys. Rev. Lett. 122, 120401 (2019), doi:10.1103/PhysRevLett.122.120401. [42] M. Collura, Relaxation of the order-parameter statistics in the Ising quantum chain, SciPost Phys. 7, 072 (2019), doi:10.21468/SciPostPhys.7.6.072. [43] M. Collura and F. H. L. Essler, How order melts after quantum quenches, Phys. Rev. B 101, 041110 (2020), doi:10.1103/PhysRevB.101.041110. [44] P. Calabrese, M. Collura, G. Di Giulio and S. Murciano, Full counting statistics in the gapped XXZ spin chain, Europhys. Lett. 129, 60007 (2020), doi:10.1209/0295- 5075/129/60007. [45] F. D. M. Haldane, Effective harmonic-ﬂuid approach to low-energy proper- ties of one-dimensional quantum ﬂuids, Phys. Rev. Lett. 47, 1840 (1981), doi:10.1103/PhysRevLett.47.1840. [46] R. References Bistritzer and E. Altman, Intrinsic dephasing in one-dimensional ultracold atom inter- ferometers, Proc. Natl. Acad. Sci. 104, 9955 (2007), doi:10.1073/pnas.0608910104. [47] A. A. Burkov, M. D. Lukin and E. Demler, Decoherence dynamics in low- dimensional cold atom interferometers, Phys. Rev. Lett. 98, 200404 (2007), doi:10.1103/PhysRevLett.98.200404. [48] T. Kitagawa, S. Pielawa, A. Imambekov, J. Schmiedmayer, V. Gritsev and E. Demler, Ramsey interference in one-dimensional systems: the full distribution function of fringe contrast as a probe of many-body dynamics, Phys. Rev. Lett. 104, 255302 (2010), doi:10.1103/PhysRevLett.104.255302. [49] T. Kitagawa, A. Imambekov, J. Schmiedmayer and E. Demler, The dynamics and prethermalization of one-dimensional quantum systems probed through the full dis- tributions of quantum noise, New J. Phys. 13, 073018 (2011), doi:10.1088/1367- 2630/13/7/073018. 25 SciPost Phys. 9, 025 (2020) [50] M. Albiez, R. Gati, J. Fölling, S. Hunsmann, M. Cristiani and M. K. Oberthaler, Direct observation of tunneling and nonlinear self-trapping in a single bosonic josephson junction, Phys. Rev. Lett. 95, 010402 (2005), doi:10.1103/PhysRevLett.95.010402. [51] R. Gati, M. Albiez, J. Fölling, B. Hemmerling and M. K. Oberthaler, Realization of a single Josephson junction for Bose–Einstein condensates, Appl. Phys. B 82, 207 (2006), doi:10.1007/s00340-005-2059-z. [52] S. Levy, E. Lahoud, I. Shomroni and J. Steinhauer, The a.c. and d.c. Josephson effects in a Bose–Einstein condensate, Nature 449, 579 (2007), doi:10.1038/nature06186. [53] V. Gritsev, A. Polkovnikov and E. Demler, Linear response theory for a pair of cou- pled one-dimensional condensates of interacting atoms, Phys. Rev. B 75, 174511 (2007), doi:10.1103/PhysRevB.75.174511. [54] F. H. L. Essler and R. M. Konik, Application of massive integrable quantum ﬁeld theories to problems in condensed matter physics, in “From ﬁelds to strings: cir- cumnavigating theoretical physics”, World scientiﬁc, ISBN 9789812389558 (2005), doi:10.1142/9789812775344_0020. [55] A. Iucci and M. A. Cazalilla, Quantum quench dynamics of the sine-Gordon model in some solvable limits, New J. Phys. 12, 055019 (2010), doi:10.1088/1367-2630/12/5/055019. [56] L. Foini and T. Giamarchi, Nonequilibrium dynamics of coupled Luttinger liquids, Phys. Rev. A 91, 023627 (2015), doi:10.1103/PhysRevA.91.023627. [57] L. Foini and T. Giamarchi, Relaxation dynamics of two coherently coupled one-dimensional bosonic gases, Eur. Phys. J. Spec. Top. 226, 2763 (2017), doi:10.1140/epjst/e2016- 60383-x. [58] B. Bertini, D. Schuricht and F. H. L. Essler, Quantum quench in the sine-Gordon model, J. Stat. Mech.: Theory Exp. P10035 (2014), doi:10.1088/1742-5468/2014/10/P10035. [59] A. C. Cubero and D. Schuricht, Quantum quench in the attractive regime of the sine-Gordon model, J. Stat. Mech.: Theory Exp. 103106 (2017), doi:10.1088/1742-5468/aa8c2e. [60] D. References X. Horváth and G. Takács, Overlaps after quantum quenches in the sine-Gordon model, Phys. Lett. B 771, 539 (2017), doi:10.1016/j.physletb.2017.05.087. [61] D. X. Horváth, M. Kormos and G. Takács, Overlap singularity and time evolu- tion in integrable quantum ﬁeld theory, J. High Energ. Phys. 08, 170 (2018), doi:10.1007/JHEP08(2018)170. [62] M. Kormos and G. Zaránd, Quantum quenches in the sine-Gordon model: A semiclassical approach, Phys. Rev. E 93, 062101 (2016), doi:10.1103/PhysRevE.93.062101. [63] C. Pa¸scu Moca, M. Kormos and G. Zaránd, Hybrid semiclassical theory of quan- tum quenches in one-dimensional systems, Phys. Rev. Lett. 119, 100603 (2017), doi:10.1103/PhysRevLett.119.100603. [64] A. J. A. James, R. M. Konik, P. Lecheminant, N. J. Robinson and A. M. Tsvelik, Non- perturbative methodologies for low-dimensional strongly-correlated systems: From non- abelian bosonization to truncated spectrum methods, Rep. Prog. Phys. 81, 046002 (2018), doi:10.1088/1361-6633/aa91ea. 26 SciPost Phys. 9, 025 (2020) [65] I. Kukuljan, S. Sotiriadis and G. Takacs, Correlation functions of the quantum sine- Gordon model in and out of equilibrium, Phys. Rev. Lett. 121, 110402 (2018), doi:10.1103/PhysRevLett.121.110402. [66] M. Pigneur, Relaxation to a phase-locked equilibrium state in a one-dimensional bosonic Josephson junction, Ph.D. thesis (2019). [67] T. Schweigler, Correlations and dynamics of tunnel-coupled one-dimensional Bose gases, Ph.D. thesis (2019), arXiv:1908.00422. [68] M. Pigneur and J. Schmiedmayer, Analytical pendulum model for a bosonic Josephson junction, Phys. Rev. A 98, 063632 (2018), doi:10.1103/PhysRevA.98.063632. [69] Y. D. van Nieuwkerk and F. H. L. Essler, Self-consistent time-dependent harmonic approx- imation for the sine-Gordon model out of equilibrium, J. Stat. Mech. 084012 (2019), doi:10.1088/1742-5468/ab3579. [70] D. X. Horváth, I. Lovas, M. Kormos, G. Takács and G. Zaránd, Nonequilibrium time evolu- tion and rephasing in the quantum sine-Gordon model, Phys. Rev. A 100, 013613 (2019), doi:10.1103/PhysRevA.100.013613. [71] M. Olshanii, Atomic scattering in the presence of an external conﬁnement and a gas of impenetrable bosons, Phys. Rev. Lett. 81, 938 (1998), doi:10.1103/PhysRevLett.81.938. [72] Y. D. van Nieuwkerk and F. H. L. Essler, in preparation. 2] Y. D. van Nieuwkerk and F. H. L. Essler, in preparation. [73] M. A. Cazalilla, Bosonizing one-dimensional cold atomic gases, J. Phys. B: At. Mol. Opt. Phys. 37, S1 (2004), doi:10.1088/0953-4075/37/7/051. [74] S. Hofferberth, I. Lesanovsky, T. Schumm, A. Imambekov, V. Gritsev, E. Demler and J. Schmiedmayer, Probing quantum and thermal noise in an interacting many-body system, Nat. Phys. 4, 489 (2008), doi:10.1038/nphys941. [75] J.-F. Schaff, T. Langen and J. Schmiedmayer, Interferometry with atoms, Riv. References Nuovo Ci- mento 509 (2014), doi:10.1393/ncr/i2014-10105-7. [76] D. Boyanovsky, F. Cooper, H. J. de Vega and P. Sodano, Evolution of inhomogeneous condensates: Self-consistent variational approach, Phys. Rev. D 58, 025007 (1998), doi:10.1103/PhysRevD.58.025007. [77] S. Sotiriadis and J. Cardy, Quantum quench in interacting ﬁeld theory: A self-consistent approximation, Phys. Rev. B 81, 134305 (2010), doi:10.1103/PhysRevB.81.134305. [78] A. Lerose, B. Žunkoviˇc, J. Marino, A. Gambassi and A. Silva, Impact of nonequilibrium ﬂuc- tuations on prethermal dynamical phase transitions in long-range interacting spin chains, Phys. Rev. B 99, 045128 (2019), doi:10.1103/PhysRevB.99.045128. [79] J. Dubail, J.-M. Stéphan, J. Viti and P. Calabrese, Conformal ﬁeld theory for inhomogeneous one-dimensional quantum systems: the example of non-interacting Fermi gases, SciPost Phys. 2, 002 (2017), doi:10.21468/SciPostPhys.2.1.002. [80] Y. Brun and J. Dubail, The inhomogeneous Gaussian free ﬁeld, with application to ground state correlations of trapped 1d Bose gases, SciPost Phys. 4, 037 (2018), doi:10.21468/SciPostPhys.4.6.037. [81] D. M. Gangardt and G. V. Shlyapnikov, Stability and phase coherence of trapped 1D Bose Gases, Phys. Rev. Lett. 90, 010401 (2003), doi:10.1103/PhysRevLett.90.010401. 27 SciPost Phys. 9, 025 (2020) [82] M. Olshanii and V. Dunjko, Short-distance correlation properties of the lieb-liniger system and momentum distributions of trapped one-dimensional atomic gases, Phys. Rev. Lett. 91, 090401 (2003), doi:10.1103/PhysRevLett.91.090401. [83] T. K. Ghosh, Quantized hydrodynamic theory of bosons in quasi-one-dimensional harmonic trap, Int. J. Mod. Phys. B 20, 5443 (2006), doi:10.1142/S0217979206035783. [84] R. Citro, S. De Palo, E. Orignac, P. Pedri and M.-L. Chiofalo, Luttinger hydrodynamics of conﬁned one-dimensional Bose gases with dipolar interactions, New J. Phys. 10, 045011 (2008), doi:10.1088/1367-2630/10/4/045011. [85] B. Rauer, S. Erne, T. Schweigler, F. Cataldini, M. Tajik and J. Schmiedmayer, Re- currences in an isolated quantum many-body system, Science 360, 307 (2018), doi:10.1126/science.aan7938. [86] M. Tajik, B. Rauer, T. Schweigler, F. Cataldini, J. Sabino, F. S. Møller, S.-C. Ji, I. E. Mazets and J. Schmiedmayer, Designing arbitrary one-dimensional potentials on an atom chip, Opt. Express 27, 33474 (2019), doi:10.1364/OE.27.033474. 28 28
https://openalex.org/W3004184418	https://elib.sfu-kras.ru/bitstream/2311/142340/1/recurrent_and_multi-layer_neural_networks_playing_.pdf	English	null	Recurrent and multi-layer neural networks playing Even-Odd”: reflection against regression	IOP conference series. Materials science and engineering	2,020	cc-by	4,318	This content was downloaded from IP address 181.214.34.73 on 29/01/2020 at 17:09 IOP Conference Series: Materials Science and Engineering IOP Conference Series: Materials Science and Engineering Recurrent and multi-layer neural networks playing Even-Odd”: reflection against regression To cite this article: S Bartsev and G Markova 2020 IOP Conf. Ser.: Mater. Sci. Eng. 734 012109 View the article online for updates and enhancements. This content was downloaded from IP address 181.214.34.73 on 29/01/2020 at 17:09 MIST: Aerospace 2019 IOP Conf. Series: Materials Science and Engineering 734 (2020) 012109 IOP Publishing doi:10.1088/1757-899X/734/1/012109 Conf. Series: Materials Science and Engineering 734 (2020) 012109 doi:10.1088/1757-899X/734/1/0121 Recurrent and multi-layer neural networks playing "Even- Odd": reflection against regression S Bartsev1,2,3 and G Markova2 1Institute of Biophysics SB RAS, Federal Research Center, Krasnoyarsk Scientific Center SB RAS, 50, Akademgorodok, Krasnoyarsk, 660036, Russian Federation 2Siberian Federal University,79 Svobodny pr., Krasnoyarsk, 660041, Russian Federation S Bartsev1,2,3 and G Markova2 1Institute of Biophysics SB RAS, Federal Research Center, Krasnoyarsk Scientific Center SB RAS, 50, Akademgorodok, Krasnoyarsk, 660036, Russian Federation 2Siberian Federal University,79 Svobodny pr., Krasnoyarsk, 660041, Russian Federation 3E-mail: bartsev@yandex.ru Abstract. Reflection understood as an internal representation of the external world by the subject is the key property of consciousness. In a refined form this property is manifested in reflective games. To win a reflective game a player has to use reflection of strictly one rank higher than the opponent. So it can be assumed that there are only two game modes - when only one player uses reflection and wins and when both players use reflection but one of them chooses incorrect reflection rank. The option of random move selection is not considered since firstly, starting the game for a draw is strange, and secondly, it is technically impossible to make random moves without a special device. Experiments with recurrent neural networks playing with each other showed that the entire set of game patterns (time series of the game score) is split into two sharply different groups that can be associated with two modes mentioned above. Experiments, in which a multilayer neural network, which is basically incapable of reflection, played against a recurrent neural network, showed that a recurrent neural network has a clear advantage winning confidently in more than 90% of the games. At the same time game patterns demonstrate splitting into two sharply different groups as was observed in experiments with the game of two recurrent neural networks and in the reflexive game of living people. Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. Published under licence by IOP Publishing Ltd 1 1. Introduction The nature of consciousness is the one of the fundamental problems of science and philosophy. The long-term goal of cognitive neurobiology is to identify the neural correlates that underlie cognitive phenomena, and to determine which neurological events correlate with one or another state and content of consciousness. This approach is based on the concept of “neural correlates of consciousness” (NCC) [1, 2]. Orientation of NCC concept to the identification of minimal neural structures that accompany some conscious experience gives rise to move away from studying the complex functions of the human brain [3-8] to the study of individual phenomena of consciousness realized by simple systems. According to V. Lefebvre [9] subjectivity (consciousness, mind) is associated with the presence of an internal representation of the external world including the image of the subject itself. Lefebvre called this type of inner representation "reflection." Reflection is possible to be the key attribute of consciousness. 1 Obviously the most effective approach is to study such behavior which in addition to reflection contains the minimum contribution of other cognitive functions, such as logical reasoning, pattern recognition, memory, etc. Reflexive games almost completely meet these requirements. In the course of the previous research [10] two groups of similar patterns of “Even-Odd” reflexive game dynamics was revealed. Significant difference observed in these patterns indicates qualitatively different dynamic patterns of neuron excitations of the playing networks. We have made an assumption the difference is the manifestation of two game modes - when only one player uses reflection and wins and when both players use reflection, but one of them chooses incorrect reflection rank. Let’s consider it in more detail. From obvious considerations it is clear that reflection is a kind of interaction of external signals with the internal representation of the external world and the intentions of the subject, which, ultimately is also a combination of signals generated by the neural network (NN) itself. Therefore, recurrent neural networks (RNNs) seem to be a natural candidate for modeling reflection. A steady win in any reflective game including the simplest one – "even-odd" is a sign of a player using a reflection that is exactly one rank higher than the playmate reflection level. In the study of reflection it seems important to find out how the “hardware” characteristics of a RNN affect the quality of the playing and therefore the ability to the reflection of the desired rank. 1. Introduction However, when two RNNs are playing some complex situations may arise when both neural networks turn to reflection, but of different ranks. To study the simplest case, it would be useful to turn to a system that can reveal patterns, but cannot possess reflection, in principle. As such a system, multilayer (feed-forward) neural networks (MNNs) can be used. But if the processing of temporary patterns is natural for the RNN then for the MNN the temporal pattern of the opponent’s moves can be set using the shift register in which the next opponent’s move is entered in the left place and the content of the register is shifted by one position. Unlike RNN where reflection may occur, in the case of MNN we are guaranteed to deal only with non-linear regression - the MNN goal is to predict the next move of the playmate on the base of the found regression dependence. Starting a reflective game between the RNN and the MNN we can evaluate the effectiveness of reflection and regression for this type of non-cooperative interaction. In addition if reflection does not occur in every RNN then one should expect splitting of all game patterns into two typical groups as was observed in [10]. So the aim of the work is to compare the capabilities of systems potentially capable of reflective information processing and systems without reflection in the situation of non-cooperative strategic interactions. 3. Results A comparison of the playing abilities of the RNN and the MNN showed that with the same number of neurons (10) and the depth of memory (5 moves to the past) the MNNs as a whole demonstrate a significantly worse playing quality than the RNNs. It is important to note about one feature of the used RNN. The fact is that information about the opponent’s move received at the current step of the game will only be available to other RNN neurons in the next step, then the response of internal neurons will only be available to output neurons in the second step, and therefore, the RNN reaction to this opponent’s move can only appear on third step after the move. Then it turns out the RNN used in the work wins by planning the opponent's move two steps forward. p In contrast to the RNN the state of the shift register is accessible to the MNN immediately after the opponent moves, and the calculations for all layers occur in one clock cycle. Then "honestly" we need to introduce a temporary delay in the submission of information about the playmate's move to the shift register. The share of MNN wins in the game with RNN depending on the value of the time delay is shown in table 1. Table 1. The share of MNN wins in the game with RNN depending on the value of the time delay. Delay 0 1 2 MNN win percentage 10.9% 8.8% 5.4% 1. The share of MNN wins in the game with RNN depending on the value of the time delay. White's nonparametric T-test showed that the difference between the quality of the game for different delay values differ at the 5% significance level. At the same time the appearance of the curves of RNN-MNN game patterns looks quite variable (figure 1B), but visually they are not essentially different from the game patterns of RNN-RNN games (figure 1A). Before a more detailed comparison of the RNN and MNN playing abilities the dependency of the RNN playing abilities on their parameters — the number of neurons and the depth of error propagation to the past — were studied (figure 2). Each point on the graph is the result of averaging from 1,500 to 3,000 games (depending on the severity of the result). 2. Methods and materials We used fully-connected RNN and three-layer MNN with different numbers of neurons. In addition we used different depths of error propagation for the RNN, and the length of the shift register displaying the previous moves of the playmate for the MNN. NN played with each other in "even-odd" game. Let’s recall the rules of the game. Each player secretly selects “0” or “1”, and then their choice is presented and summed. An "even" amount means that one player receives one point, while an "odd" amount means that the point is received by another player. "Even-odd" is an example of a reflexive game in which there is no fixed winning strategy, and a draw strategy is a random choice. If players cannot choose randomly or try to avoid a draw the game becomes non-trivial: the one who better predicts opponent's moves becomes the winner. The functioning and training of the used RNN is described in details in the previous work [10]. In order to single out only the effect of the structure on the playing abilities of NNs, the transition function of the MNN was chosen the same as for the RNN: n i j n j ij n i n i n i n i A w a            , 1 , (1) (1) 2 2 MIST: Aerospace 2019 MIST: Aerospace 2019 IOP Conf. Series: Materials Science and Engineering 734 (2020) 012109 IOP Publishing doi:10.1088/1757-899X/734/1/012109 Conf. Series: Materials Science and Engineering 734 (2020) 012109 doi:10.1088/1757-899X/734/1/0121 where n i  is the output signal of the i-th neuron at the n-th moment of time; wij is the matrix of weight coefficients (for a RNN this matrix is single, and for a layered network there are several of them); Ani is the input signal arriving at the i-th neuron at the n-th moment of time. where n i  is the output signal of the i-th neuron at the n-th moment of time; wij is the matrix of weight coefficients (for a RNN this matrix is single, and for a layered network there are several of them); Ani is the input signal arriving at the i-th neuron at the n-th moment of time. Information about the move of the partner is fed through two input neurons of the RNN and to the first place of the MNN shift register. 2. Methods and materials The ratio of signals of two output neurons determines the move of the neural network - “0” or “1”. The objective functions for the NNs differ because one NN wins when its move coincides with the move of the second NN, and the second NN wins when it makes a move different from the opponent’s move:         2 4 2 3 1 ) 2 1( 2 2 1 , move move n H n n n i         and         2 4 2 3 2 1 )1 1( 2 1 , move move n H n n n i         . (2) (2) The synapses of the RNN and the MNN were modified after each move according to the well- known algorithm of back propagation of the error. The synapses of the RNN and the MNN were modified after each move according to the well- known algorithm of back propagation of the error. Open soft, ‘Lazarus’ (https://www.lazarus-ide.org/) was used for the simulation. For statistic analyses of experimental data, MS Excel (Fourier transform) and open soft ‘Tanagra’ (http://eric.univ- lyon2.fr/~ricco/tanagra/en/tanagra.html) (cluster analyses) were used. 3. Results The gain of NN is the deviation of score from the case of equal playmates when the expected score is zero. Each game was of 500 moves. The reference RNN was of 10 neurons and the error propagation depth was 5 clock cycles. These 3 MIST: Aerospace 2019 IOP Publishing IOP Conf. Series: Materials Science and Engineering 734 (2020) 012109 doi:10.1088/1757-899X/734/1/012109 parameters happened historically – a minimal neural network that successfully played against a human had such parameters [11]. Figure 1. Examples of game patterns of two recurrent NNs (A) and recurrent and multilayer NNs (B). -20 -10 0 10 20 30 40 50 1 51 101 151 201 251 301 351 401 451 501 Move number Game score -100 -80 -60 -40 -20 0 20 40 1 51 101 151 201 251 301 351 401 451 501 Move number Game score А) B) -20 -10 0 10 20 30 40 50 1 51 101 151 201 251 301 351 401 451 501 Move number Game score А) Game score Move number Move number Figure 1. Examples of game patterns of two recurrent NNs (A) and recurrent and multilayer NNs (B). Figure 1. Examples of game patterns of two recurrent NNs (A) and recurrent and multilayer NNs (B) Figure 1. Examples of game patterns of two recurrent NNs (A) and recurrent and multilayer NNs (B). Figure 2. Dependence of the quality of NN playing on the number of neurons (A), and the depth of error propagation (B). -30 -20 -10 0 10 20 30 40 50 60 1 5 9 13 error propagation depth reference neural network gain -110 -90 -70 -50 -30 -10 10 30 50 70 90 110 130 4 9 14 19 number of neurons reference neural network gain А) B) -30 -20 -10 0 10 20 30 40 50 60 1 5 9 13 error propagation depth reference neural network gain B) -110 -90 -70 -50 -30 -10 10 30 50 70 90 110 130 4 9 14 19 number of neurons reference neural network gain А) reference neural network gain number of neurons number of neurons error propagation depth Figure 2. Dependence of the quality of NN playing on the number of neurons (A), and the depth of error propagation (B). Figure 2. Dependence of the quality of NN playing on the number of neurons (A), and the depth of error propagation (B). 3. Results In principle the results presented were not unexpected, because if we associate success in a reflective game with the presence of reflection of the corresponding rank (one more than the opponent's one) then a larger number of neurons allows to organize better reflection – reflection need brains. And the optimum of the depth of error propagation can be explained: for effective game in the situation when a partner can quickly change strategies extremely deep back propagation of errors into the past reduces the possibility of fast response to changes in the opponent's strategy. y g gy The results of RNN against MNN game (reflection against regression) turned out to be quite unexpected (figure 3). MNN with three layers (4; 4; 2) and a shift register length of 5 was initially played against RNN. Formally, the number of neurons of this MNN coincided with the number of neurons of the referent RNN, and the memory depth coincided with the depth of error propagation. However, it turned out that RNNs triumphed even with a minimum number of neurons - 4 (two input 4 MIST: Aerospace 2019 IOP Conf. Series: Materials Science and Engineering 734 (2020) 012109 IOP Publishing doi:10.1088/1757-899X/734/1/012109 MIST: Aerospace 2019 MIST: Aerospace 2019 IOP Conf. Series: Materials Science and Engineering 734 (2020) 012109 IOP Publishing doi:10.1088/1757-899X/734/1/012109 IOP Conf. Series: Materials Science and Engineering 734 (2020) 012109 doi:10.1088/1757-899X/734/1/012109 Conf. Series: Materials Science and Engineering 734 (2020) 012109 doi:10.1088/1757-899X/734/1/0121 and two output neurons) (figure 3A). The dependence on the depth of error propagation is also quite unusual - RNN loses only at a depth of 1, i.e. when only the synapses of the output neurons change (figure 3B). It turned out that the MNN can outperform four neuron RNN with a propagation depth of error 2, if the number of MNN neurons is increased, for example, in the second layer (figure 3C). and two output neurons) (figure 3A). The dependence on the depth of error propagation is also quite unusual - RNN loses only at a depth of 1, i.e. when only the synapses of the output neurons change (figure 3B). It turned out that the MNN can outperform four neuron RNN with a propagation depth of error 2, if the number of MNN neurons is increased, for example, in the second layer (figure 3C). Figure 3. 3. Results Dependences of the gain of the referent RNN on the number of neurons (A) in it, the depth of error propagation (B), and on the number of neurons in the second layer of MNN (C) (the RNN was of 4 neurons and the error propagation depth was 2). -250 -200 -150 -100 -50 0 50 100 150 0 2 4 6 8 error propagation depth recurrent neural network gain 90 110 130 150 170 190 3 5 7 9 11 number of neurons recurrent neural network gain -40 -20 0 20 40 60 80 100 120 140 3 5 7 9 11 13 15 the number of second layer neurons recurrent neural network gain А) B) C) -250 -200 -150 -100 -50 0 50 100 150 0 2 4 6 8 error propagation depth recurrent neural network gain B) -40 -20 0 20 40 60 80 100 120 140 3 5 7 9 11 13 15 the number of second layer neurons recurrent neural network gain C) 90 110 130 150 170 190 3 5 7 9 11 number of neurons recurrent neural network gain А) the number of second layer neurons Figure 3. Dependences of the gain of the referent RNN on the number of neurons (A) in it, the depth of error propagation (B), and on the number of neurons in the second layer of MNN (C) (the RNN was of 4 neurons and the error propagation depth was 2). Figure 3. Dependences of the gain of the referent RNN on the number of neurons (A) in it, the depth of error propagation (B), and on the number of neurons in the second layer of MNN (C) (the RNN was of 4 neurons and the error propagation depth was 2). The analysis of patterns repeatability was carried out for an RNN with 10 neurons and an error propagation depth of 5. MNN was of "5; 5; 2" structure and the shift register length of 5. Fast Fourier transform showed a wide variety of frequency and phase spectra obtained by processing game patterns. At the same time despite the variety of frequency spectra they are not very expressive (figure 4) in comparison with phase spectra (figure 5). 3. Results 0 500 1000 1500 2000 2500 1 101 201 0 500 1000 1500 2000 2500 3000 3500 1 101 201 0 500 1000 1500 2000 2500 3000 3500 1 101 201 0 500 1000 1500 2000 2500 1 101 201 Figure 4. Some examples of frequency spectra of game patterns obtained using the Fourier transform. Figure 4. Some examples of frequency spectra of game patterns obtained using the Fourier transform. Figure 4. Some examples of frequency spectra of game patterns obtained using the Fourier transform. 5 5 IOP Publishing MIST: Aerospace 2019 IOP Conf. Series: Materials Science and Engineering 734 (2020) 012109 doi:10.1088/1757-899X/734/1/012109 -4 -3 -2 -1 0 1 2 3 4 1 101 201 -4 -3 -2 -1 0 1 2 3 4 1 101 201 -4 -3 -2 -1 0 1 2 3 4 1 101 201 -4 -3 -2 -1 0 1 2 3 4 1 101 201 Figure 5. Some examples of phase changes in game patterns obtained by the Fourier transform. igure 5. Some examples of phase changes in game patterns obtained by the Fourier transform. Cluster analysis (hierarchical clustering, Ward's criterion) as in the case of RNN-RNN game [10] showed a similar splitting into two sharply different groups of game patterns (figure6). Cluster analysis (hierarchical clustering, Ward's criterion) as in the case of RNN-RNN game [10] showed a similar splitting into two sharply different groups of game patterns (figure6). Cluster analysis (hierarchical clustering, Ward's criterion) as in the case of RNN-RNN game [10] showed a similar splitting into two sharply different groups of game patterns (figure6). Figure 6. Hierarchical proximity tree of frequency (A) and phase (B) spectra of the Fourier transform of game patterns between recurrent (left pair) and recurrent and multilayer (right pair) NNs. А) Б) А) Б) B) B) А) Б) B) Figure 6. Hierarchical proximity tree of frequency (A) and phase (B) spectra of the Fourier transform of game patterns between recurrent (left pair) and recurrent and multilayer (right pair) NNs. The results obtained in this work are similar to obtained before [10], and as was already noted these results are in a good agreement with the results of a massive study of decision-making in non- cooperative strategic interactions [12]. 3. Results The study revealed the presence of two types of strategies in people: 1) "won - repeated, lost - changed", characteristic of most persons; 2) a strategy based on knowledge of the “typical person” strategy described above. The second strategy is to take the position of the opponent (it is a reflective action) that allows the player to prevail over the first strategy. Based on this work we can assume obtained cardinal difference in game patterns is associated with the presence or absence of reflection inside players. According to the results of clustering the phase spectra of game patterns, it can be seen (figure 6B, right pair) that one of the groups of patterns makes up a small fraction commensurate with the share of MNN wins. Presumably these are the cases when the RNN was not able to perform the reflection mode. 6 6 MIST: Aerospace 2019 MIST: Aerospace 2019 MIST: Aerospace 2019 IOP Conf. Series: Materials Science and Engineering 734 (2020) 012109 IOP Publishing doi:10.1088/1757-899X/734/1/012109 p IOP Conf. Series: Materials Science and Engineering 734 (2020) 012109 g doi:10.1088/1757-899X/734/1/012109 4. Conclusion In reflective games a steady gain implies the winning player has a reflection of one rank higher than opponent's one. In favor of the assumption that in the most of the analyzed game patterns the winnings were not the result of the random success, but the result of a “conscious” (reflexive) choice of the move, says the splitting of all game patterns into two groups. In the case of random outcomes of the game such clustering seems to be impossible. It was shown the increase in the number of neurons causes the increase of the effectiveness of RNN. This effect seems quite understandable - for reflection, bigger brains are needed. But regarding the depth of error propagation, i.e. the degree of influence of past experience on the current choice situation is different - there is an optimal depth of error propagation. This effect also can be naturally explained. Indeed, in the case of a rapidly changing strategy of opponent, relying on old patterns leads to loss. And finally, it has been demonstrated regression cannot effectively replace reflection in non- cooperative strategic interactions, which include reflexive games. From which it follows that the development of a truly powerful artificial intelligence is impossible without the reproduction of reflexive processes in intellectual systems. References [1] Crick F and Koch C 1990 Towards a neurobiological theory of consciousness. Seminars in Neuroscience 2 263–75 [1] Crick F and Koch C 1990 Towards a neurobiological theory of consciousness. Seminars in Neuroscience 2 263–75 [2] Crick F and Koch C 2003 Framework for consciousness Nature Neuroscience 6(2) 119– ] Dehaene S 2009 Conscious and Nonconscious Processes: Distinct Forms of Evid Accumulation? Seminaire Poincare XII pp 89-114 pp [4] Dehaene S and Naccache L 2001 Towards a cognitive neuroscience of consciousness: basic evidence and a workspace framework. Cognition 79 1-37 pp [4] Dehaene S and Naccache L 2001 Towards a cognitive neuroscience of consciousness: basic evidence and a workspace framework. Cognition 79 1-37 p g [5] Dehaene S and Changeux J-P 2011 Experimental and Theoretical Approaches to Conscious Processing. Neuron 70 200–27 [6] Kiefer M and Pulvermüller F 2012 Conceptual representations in mind and brain: Theoretical developments, current evidence and future directions Сortex 48 805–25 [7] Mehta N and Mashour G 2013 General and specific consciousness: a first-order representationalist approach. Frontiers in Psychology: Consciousness Research 4 407–10 [8] Tononi G, Boly M, Massimini M and Koch C 2016 Integrated information theory: from consciousness to its physical substrate Nature Reviews: Neuroscience 17 450–61 [9] Lefebvre V 2003 Reflexion p 496 [9] Lefebvre V 2003 Reflexion p 496 f p [10] Dolgova T and Bartsev S 2019 Neural networks playing ‘matching pennies’ with each other: reproducibility of game dynamics IOP Conf. Ser.: Mater. Sci. Eng. 537 042002 p y g y f g [11] Bartsev S and Okhonin V 1991 Self-learning neural networks playing "Two coins" Neurocomputers and attention II (Manchester Univ.Press) pp 453 – 8 [12] Wang Z, Xu B and Zhou H-J 2014 Social cycling and conditional responses in the Rock-Paper- Scissors game Scientific reports 4 5830 7 7
https://openalex.org/W3010363312	https://europepmc.org/articles/pmc7085031?pdf=render	English	null	Investigation of Biogenic Passivating Layers on Corroded Iron	Materials	2,020	cc-by	8,914	Received: 12 February 2020; Accepted: 3 March 2020; Published: 6 March 2020 Abstract: This study evaluates mechanisms of biogenic mineral formation induced by bacterial iron reduction for the stabilization of corroded iron. As an example, the Desulﬁtobacterium hafniense strain TCE1 was employed to treat corroded coupons presenting urban natural atmospheric corrosion, and spectroscopic investigations were performed on the samples’ cross-sections to evaluate the corrosion stratigraphy. The treated samples presented a protective continuous layer of iron phosphates (vivianite Fe2+3(PO4)2·8H2O and barbosalite Fe2+Fe3+2(PO4)2(OH)2), which covered 92% of the surface and was associated with a decrease in the thickness of the original corrosion layer. The results allow us to better understand the conversion of reactive corrosion products into stable biogenic minerals, as well as to identify important criteria for the design of a green alternative treatment for the stabilization of corroded iron. Keywords: iron corrosion; SEM; Raman spectroscopy; biogenic minerals; bacterial iron reduction; cultural heritage; conservation-restoration; corrosion stabilization Investigation of Biogenic Passivating Layers on Corroded Iron Lucrezia Comensoli 1,2,†, Monica Albini 1,2,‡,§, Wafa Kooli 1,2,§, Julien Maillard 3 Tiziana Lombardo 4, Pilar Junier 1 and Edith Joseph 2,5,* 1 Laboratory of Microbiology, Institute of Biology, University of Neuchâtel, 2000 Neuchâtel, Switzerland; lucrezia.comensoli@empa.ch (L.C.); monica.albini@cnr.it (M.A.); wafam.kooli@gmail.com (W.K.); pilar.junier@unine.ch (P.J.) 1 Laboratory of Microbiology, Institute of Biology, University of Neuchâtel, 2000 Neuchâtel, Switzerland; lucrezia.comensoli@empa.ch (L.C.); monica.albini@cnr.it (M.A.); wafam.kooli@gmail.com (W.K.); pilar.junier@unine.ch (P.J.) p j 2 Laboratory of Technologies for Heritage Materials, Institute of Chemistry, University of Neuchâtel, 2000 Neuchâtel, Switzerland 3 Laboratory for Environmental Biotechnology, ENAC-IIE-LBE, Ecole Polytechnique Fédérale de Lausanne 1015 Lausanne, Switzerland; julien.maillard@epﬂ.ch 4 Laboratory of conservation research, Sammlungszentrum, Swiss national museum, Lindenmoosstrasse 1, 8910 Aﬀoltern am Albis, Switzerland; Tiziana.Lombardo@nationalmuseum.ch 4 Laboratory of conservation research, Sammlungszentrum, Swiss national museum, Lindenmoosstrasse 1, 8910 Aﬀoltern am Albis, Switzerland; Tiziana.Lombardo@nationalmuseum.ch 5 Haute Ecole Arc Conservation-Restauration, HES-SO, 2000 Neuchâtel, Switzerland 5 Haute Ecole Arc Conservation-Restauration, HES-SO, 2000 Neuchâtel, Switzerland , , , * Correspondence: edith.joseph@unine.ch † Present address: Laboratory for Mechanical Systems Engineering, Swiss Federal Laboratories for Materials Science and Technology, 8600 Dübendorf, Switzerland. ‡ Present address: Institute for the study of Nanostructured Materials, Italian National council of Research (CNR-ISMN), 7-00185 Rome, Italy. § These authors contributed equally to this work. § These authors contributed equally to this work. Received: 12 February 2020; Accepted: 3 March 2020; Published: 6 March 2020 ceived: 12 February 2020; Accepted: 3 March 2020; Published: 6 March 2020 materials materials materials 1. Introduction Also, a large quantity of generated waste needs to be processed afterward for safe disposal. Second, electrolytic reduction allows an increase in the porosity of the corrosion layer and thus enhances the diﬀusion of harmful salts from the objects [13]. However, this method is restricted to large marine ﬁnds, as there is a signiﬁcant loss of the surface and a lack of control over the amount of salts extracted and the corrosion products reduced during hydrogen bubbling [3]. Finally, plasma treatment is usually applied as a pre-treatment, as it creates cracks and ﬁssures that will facilitate the diﬀusion of chloride ions during a successive alkaline bath [14]. For all these reasons, there is a general consensus that, to date, an eﬃcient and durable protective system to control iron corrosion on archaeological artifacts does not exist [15]. Microbes are frequently considered detrimental to metallic surfaces, as they are associated with the process of microbial induced corrosion (MIC) [16–18]. However, given the large metabolic diversity found within the microbial world, an increasing number of studies focus on the exploitation of microbial processes to inhibit corrosion. Experimental evidence suggests that a bioﬁlm of aerobic bacteria attached to copper surfaces in freshwater and seawater reduces the copper corrosion rate by decreasing the oxygen content nearby [15]. In addition, several studies reported the protective behavior of speciﬁc bioﬁlms on submerged carbon steel pipelines [19–22]. In particular, the formation of iron phosphates as a protective layer was accomplished through the exploitation of bacterial biomineralization processes. For instance, electrochemical measurements demonstrated that the presence of a biogenic layer of vivianite (Fe2+3(PO4)2·8H2O) produced by Geobacter sulfurreducens on the surface of carbon steel coupons had a protective eﬀect against corrosion [23]. In the examples cited above, most of the bio-based approaches were developed for the protection of bare iron surfaces before their exposure to outdoor environments. As part of our research topic, we investigated the potential of microbes for the stabilization of already corroded iron (archaeological objects and outdoor surfaces) by converting part of the reactive corrosion layer into more stable biogenic minerals (Figure 1). In particular, we studied diﬀerent bacterial species, Shewanella loihica, Desulﬁtobacterium hafniense and Aeromonas spp., for their ability to produce biogenic iron minerals on corroded steel coupons [24–28]. Hence, vivianite and siderite were produced by S. loihica on costal-exposed coupons, while D. hafniense and Aeromonas spp. 1. Introduction Cast iron and steel objects corrode rapidly when they are exposed outdoors, and complex layers composed of iron oxides and iron hydroxides are formed over time [1–3]. In the presence of moisture, these corrosion layers adsorb water and incorporate particulate matter and pollution agents from the atmosphere, which instigate further corrosion processes. Generally, in addition to iron oxides and iron hydroxides, iron sulfates are also frequently reported compounds within an atmospheric corrosion layer, especially in polluted and urban areas [4,5]. Near coastal areas, chlorine is a problematic element. Iron items exposed to such surroundings are susceptible to chloride-promoted corrosion, leading to chemical as well as mechanical damage to these objects [2,3,5]. Chloride ions diﬀused into the objects Materials 2020, 13, 1176; doi:10.3390/ma13051176 www.mdpi.com/journal/materials www.mdpi.com/journal/materials 2 of 13 Materials 2020, 13, 1176 as counter-ions to iron(II) ions are produced by the oxidation of the metal [6]. Iron oxyhydroxides are then produced, including chloride-bearing akaganéite FeO0.833(OH)1.167Cl0.167, and another ferrous hydroxychloride β–Fe2(OH)3Cl [6,7]. In the presence of such hygroscopic compounds, iron corrosion starts at low relative humidity (RH) values, down to 15% RH, and dramatically increases above 35% RH, leading to the fragmentation and disintegration of the objects [8]. Hence, outdoor iron objects that are exposed to these environments are subjected to severe degradation and would be damaged or lost without maintenance and appropriate conservation strategies. Therefore, several conservation and restoration methods are currently performed to stabilize the corrosion layers on outdoor iron-based structures. Organic coatings, such as waxes, resins, and oils, are applied to protect iron surfaces, acting as a physical barrier against atmospheric agents [3,9,10]. Nevertheless, these protective systems often need frequent maintenance to avoid failures of the coating ﬁlm and exposure of the metal substrate to further oxidation [11]. Another approach employed is the use of corrosion inhibitors. However, most of these are hazardous compounds such as chromates, benzoates or nitrites, which have to be carefully manipulated [3]. Concerning archaeological iron artifacts, three methods are used to stabilize their corrosion layer. First, to remove the chloride ions from the corrosion layer, the object is immersed in anoxic and alkaline aqueous solutions [3,12]. Nevertheless, this method is based on osmotic diﬀusion, which is a slow process, and thus the solution needs to be replaced regularly when the chloride ions’ concentration stops increasing or stays low (below 20 ppm). 2.2. Bacterial Treatment 2.2. Bacterial Treatment 2.2. Bacterial Treatment The D. hafniense strain TCE1 (DSMZ-German Collection of Microorganisms and Cell Culture GmbH 12704) was used for this study. This bacterium was selected as it can use a variety of electron acceptors, especially halogenated organic compounds and metals [29,30]. In addition, a previous study revealed that this strain was able to reduce Fe3+-citrate in the presence of 0.2% and 0.3% NaCl, and that it was more efficient in terms of production of iron phosphates on the surface of corroded iron coupons [25,26]. Bacterial pre-incubation was performed in the dark at 30 °C under agitation in a standard mineral medium under anoxic conditions in 500 mL serum bottles, until reaching an optical density (OD600) of 0.1−0.15, as previously described [31]. Quantities of 45 mM of lactate and 20 mM of fumarate were added as an electron donor and acceptor, respectively, as well as a buffer solution containing phosphates and carbonates (4 mM K2HPO4 and 1 mM NaH2PO4; 54 mM NaHCO3 and 6 mM NH4HCO3) to maintain the pH at 7.3. To avoid the corrosion of iron coupons during treatment that would lead to a misinterpretation of the results obtained, passivating conditions were achieved by replacing O2 with a mix of N2/CO2 (80%/20%) and by adding Na2S as a reducing agent. The treatment of the coupons was then performed as previously described [25,26]. Before treatment, the coupons were sterilized by spraying them with ethanol 70 % (wt/wt in deionized water) and exposure to UV radiation (20 minutes on each face). The samples were then placed into 50 mL serum bottles, and autoclaving was performed under anoxic conditions (as defined in pre-incubation). Next, 20 mL of bacterial solution or culture medium (abiotic control) was added. After 7 days of incubation, the coupons were taken out of the treatment solution and sterilized as above (no more bacteria or culture media were present on the treated surfaces). All of the experiments were performed in triplicates, and the results presented here were identical for each set of samples. The D. hafniense strain TCE1 (DSMZ-German Collection of Microorganisms and Cell Culture GmbH 12704) was used for this study. This bacterium was selected as it can use a variety of electron acceptors, especially halogenated organic compounds and metals [29,30]. 2.2. Bacterial Treatment 2.2. Bacterial Treatment In addition, a previous study revealed that this strain was able to reduce Fe3+-citrate in the presence of 0.2% and 0.3% NaCl, and that it was more eﬃcient in terms of production of iron phosphates on the surface of corroded iron coupons [25,26]. Bacterial pre-incubation was performed in the dark at 30 ◦C under agitation in a standard mineral medium under anoxic conditions in 500 mL serum bottles, until reaching an optical density (OD600) of 0.1−0.15, as previously described [31]. Quantities of 45 mM of lactate and 20 mM of fumarate were added as an electron donor and acceptor, respectively, as well as a buﬀer solution containing phosphates and carbonates (4 mM K2HPO4 and 1 mM NaH2PO4; 54 mM NaHCO3 and 6 mM NH4HCO3) to maintain the pH at 7.3. To avoid the corrosion of iron coupons during treatment that would lead to a misinterpretation of the results obtained, passivating conditions were achieved by replacing O2 with a mix of N2/CO2 (80%/20%) and by adding Na2S as a reducing agent. The treatment of the coupons was then performed as previously described [25,26]. Before treatment, the coupons were sterilized by spraying them with ethanol 70% (wt/wt in deionized water) and exposure to UV radiation (20 minutes on each face). The samples were then placed into 50 mL serum bottles, and autoclaving was performed under anoxic conditions (as deﬁned in pre-incubation). Next, 20 mL of bacterial solution or culture medium (abiotic control) was added. After 7 days of incubation, the coupons were taken out of the treatment solution and sterilized as above (no more bacteria or culture media were present on the treated surfaces). All of the experiments were performed in triplicates, and the results presented here were identical for each set of samples. 2.1. Description of Samples 2.1. Description of Samples p f p Samples of 12.5 × 25 × 2–3 mm were obtained from a steel plate with a natural urban corrosion layer mainly composed of lepidocrocite and goethite [25,26]. The plate was exposed for about 10 years in an urban environment (Zürich, Switzerland). Samples of 12.5 × 25 × 2–3 mm were obtained from a steel plate with a natural urban corrosion layer mainly composed of lepidocrocite and goethite [25,26]. The plate was exposed for about 10 years in an urban environment (Zürich, Switzerland). p f p Samples of 12.5 × 25 × 2–3 mm were obtained from a steel plate with a natural urban corrosion layer mainly composed of lepidocrocite and goethite [25,26]. The plate was exposed for about 10 years in an urban environment (Zürich, Switzerland). Samples of 12.5 × 25 × 2–3 mm were obtained from a steel plate with a natural urban corrosion layer mainly composed of lepidocrocite and goethite [25,26]. The plate was exposed for about 10 years in an urban environment (Zürich, Switzerland). 1. Introduction induced the formation of vivianite and siderite on urban-exposed coupons [26–28]. In order to better understand the biomineralization process involved, corroded coupons exposed in an urban environment and treated with D. hafniense have been investigated through the present stratigraphic study. 3 of 13 Materials 2020, 13, 1176 Figure 1. (a) Schematic cross-section of a corroded iron coupon submitted to bacterial treatment, showing microbial-induced modifications occurring; (b) the analytical methodology performed. Figure 1. (a) Schematic cross-section of a corroded iron coupon submitted to bacterial treatment, showing microbial-induced modiﬁcations occurring; (b) the analytical methodology performed. 2 M t i l d M th d Figure 1. (a) Schematic cross-section of a corroded iron coupon submitted to bacterial treatment, showing microbial-induced modifications occurring; (b) the analytical methodology performed. Figure 1. (a) Schematic cross-section of a corroded iron coupon submitted to bacterial treatment, showing microbial-induced modiﬁcations occurring; (b) the analytical methodology performed. 2.3.3. Raman Spectroscopy Raman spectroscopy was also performed to study the molecular composition of the corrosion layer before and after bacterial treatment. The analyses were carried out with a Horiba-Jobin Yvon Labram Aramis microscope equipped with an Nd:YAG (neodymium-doped yttrium aluminum garnet; Nd:Y3Al5O12) laser of 532 nm at a power lower than 1 mW. Single-point measurements were carried out with the following conditions: spectral range 100–1600 cm−1, 400 µm hole, 200 µm slit, and 10 accumulations of 10 s. Raman mapping was performed on selected areas with the same conditions and a step size of 2.5 µm in the x and y directions. Spectrum correction (automatic baseline correction) and chemical maps were elaborated using LabSpec Raman spectroscopy software suite (version 6, HORIBA France SAS, Villeneuve d’Ascq, France). The identiﬁcation of the compounds present was based on literature records and a reference spectra library compiled by the authors. 2.3. Analytical Techniques 2.3. Analytical Techniques After treatment, the coupons were sampled and cold-embedded in methacrylate resin using the EpoFix Kit (Struers GmbH - Zweigniederlassung Schweiz, Birmensdorf, Switzerland). Cross- polishing was performed using successive silicon carbide abrasive papers (250, 500, and 1000 grit) and Micro-Mesh abrasive cloths (1800 2400 3200 3600 4000 6000 8000 and 12 000 grades) The After treatment, the coupons were sampled and cold-embedded in methacrylate resin using the EpoFix Kit (Struers GmbH—Zweigniederlassung Schweiz, Birmensdorf, Switzerland). Cross-polishing was performed using successive silicon carbide abrasive papers (250, 500, and 1000 grit) and Micro-Mesh 4 of 13 Materials 2020, 13, 1176 abrasive cloths (1800, 2400, 3200, 3600, 4000, 6000, 8000, and 12,000 grades). The cross-sectioned samples were then analyzed with optical and scanning electron microscopy, as well as with Raman spectroscopy. abrasive cloths (1800, 2400, 3200, 3600, 4000, 6000, 8000, and 12,000 grades). The cross-sectioned samples were then analyzed with optical and scanning electron microscopy, as well as with Raman spectroscopy. 2 3 1 O ti l Mi 2.3.1. Optical Microscopy Microscopic observations were performed under a Polyvar MET optical microscope (Leica Microsystems (Schweiz) AG, <br>Verkaufsgesellschaft, Heerbrugg, Switzerland) to characterize the corrosion layer and the biogenic crystals formed. An estimation of the conversion percentage of the original corrosion layer into biogenic crystals was extrapolated with Axio Vision LE® software (version 4.8.2.0, Carl Zeiss MicroImaging GmbH, Iéna, Germany). 2.3.2. Scanning Electron Microscopy Scanning Electron Microscopy coupled with Energy Dispersive Spectroscopy (SEM–EDS) mapping was carried out to evaluate the elemental composition as well as the distribution of the corrosion products and biogenic minerals. Coupons were mounted onto stubs using carbon conductive tape to ensure electrical conductivity and were directly analyzed using a environmental scanning electron microscope Philips XL30 ESEM FEG (Thermo Fisher Scientiﬁc, Hillsboro, Oregon, USA) equipped with an energy-dispersive X-ray analyzer. Backscattered electron images were acquired at an acceleration potential of 10 to 25 keV and a working distance of 10 mm. For elemental mapping, a resolution of 64 × 50 pixels and a dwell of 1000 were employed. 3.1. Structure, Thickness and Continuity of the Corrosion Layer Microscopic observations of the untreated samples revealed a corrosion layer with brown, red and orange-colored compounds (Figure 2a). The corrosion layer of abiotic control coupons had a comparable thickness and color to the untreated coupons (Figure 2b,c). On the outer part of this layer, some blue spots were also detected. However, these did not form a continuous layer (Figure 2c). The formation of these blue compounds on the abiotic control coupons was probably due to an interaction between the iron corrosion products and the buﬀer solution containing phosphates and carbonates present in the culture medium. On the contrary, after bacterial treatment, the surface color of the iron coupons drastically changed. Indeed, the original corrosion layer disappeared almost completely, and a continuous layer of blue biogenic crystals was observed instead (Figure 2d). 5 of 13 5 of 13 Materials 2020, 13, 1176 M i l 13 FOR Figure 2. Optical microscope images of untreated (a), two different zones of the abiotic control (b,c), and bacterially treated (d) iron coupons. Figure 2. Optical microscope images of untreated (a), two diﬀerent zones of the abiotic control (b,c), and bacterially treated (d) iron coupons. Figure 2. Optical microscope images of untreated (a), two different zones of the abiotic control (b,c), and bacterially treated (d) iron coupons. Figure 2. Optical microscope images of untreated (a), two diﬀerent zones of the abiotic control (b,c), and bacterially treated (d) iron coupons. Even if the thickness of a layer of naturally formed corrosion products is uneven, an overall decrease in the corrosion thickness was observed in the coupons treated with bacteria (Figure 3a). In fact, the mean value of the thickness of the corrosion layer decreased from nearly 28 µm (untreated coupons) to about 7 µm on the treated coupons (Figure 3a). The corrosion thickness decrease is due to the dissolutive microbial reduction of the iron phases composing the original corrosion layer. As a result, part of the iron oxyhydroxides is converted into iron biogenic minerals. The results demonstrated that this specific microbial process did convert a part of the original corrosion layer into reduced iron compounds, as least within the 7-day treatment duration. In fact, it is worth mentioning that treatment duration is a key element to assess and that the metal substrate could eventually become corroded if the growing conditions are not carefully set. El l f h 3.2. Elemental Composition of the Corrosion Layer 3.2. Elemental Composition of the Corrosion Layer Elemental mapping revealed that the corrosion layer of untreated coupons was mainly composed of Fe and O (Figure 4a). The same layer (numbered 1) was observed on the abiotic control coupons (Figure 4b). However, an upper layer (numbered 2) mainly composed of S was also detected. In addition, an outermost layer (numbered 3) composed of Fe, O, and P, was detected and corresponded to the areas with blue spots (Figure 4b). Regarding the treated coupons, the same stratigraphy as for the abiotic control was observed, with layer 2, composed of S, superimposed by layer 3, which is rich in Fe, O and P, corresponding to the area covered by the biogenic crystals (Figure 4c). In this case, no discontinuity within layer 3 was observed (Figure 4c). The presence of a sulfur- rich layer under the biogenic crystals is an interesting observation. In fact, a previous study showed the formation of elemental sulfur (S8) and partially oxidized mackinawite (Fe2+/Fe3+S) on the surface of coupons used as an abiotic control, but not when incubated with bacteria [26]. Analyzing the cross- sectioned samples, the current study demonstrates that a layer mainly composed of S is also present on the bacterially treated coupons, and is localized between the remaining original corrosion layer and the biogenic minerals. This sulfur-rich layer is probably the result of an abiotic reaction between Na2S added to ensure anoxic conditions and the reactive corroded surface of the iron coupons. Since this layer is located underneath the biogenic crystals, it can be assumed that it was produced first. It is worth mentioning that the formation of such a layer is already reported during iron corrosion in anoxic environments [32]. Even if the effect of sulfur on the corrosion process of iron is still under evaluation, experimental evidence suggests that elemental sulfur could speed up the corrosion rate of iron objects [33,34]. Indeed, this compound is known to be highly reactive, oxidizing organic and inorganic material regardless of the oxygen content [34]. In the presence of humidity, iron and steel surfaces exposed to elemental sulfur are corroded by an electrochemical reaction involving the reduction of elemental sulfur coupled with the oxidation of iron [33,34]. El l f h 3.2. Elemental Composition of the Corrosion Layer Hence, the sulfur-rich layer d t t d h d l li d d th th bi i l if l t l lf ld b d t i t l Elemental mapping revealed that the corrosion layer of untreated coupons was mainly composed of Fe and O (Figure 4a). The same layer (numbered 1) was observed on the abiotic control coupons (Figure 4b). However, an upper layer (numbered 2) mainly composed of S was also detected. In addition, an outermost layer (numbered 3) composed of Fe, O, and P, was detected and corresponded to the areas with blue spots (Figure 4b). Regarding the treated coupons, the same stratigraphy as for the abiotic control was observed, with layer 2, composed of S, superimposed by layer 3, which is rich in Fe, O and P, corresponding to the area covered by the biogenic crystals (Figure 4c). In this case, no discontinuity within layer 3 was observed (Figure 4c). The presence of a sulfur-rich layer under the biogenic crystals is an interesting observation. In fact, a previous study showed the formation of elemental sulfur (S8) and partially oxidized mackinawite (Fe2+/Fe3+S) on the surface of coupons used as an abiotic control, but not when incubated with bacteria [26]. Analyzing the cross-sectioned samples, the current study demonstrates that a layer mainly composed of S is also present on the bacterially treated coupons, and is localized between the remaining original corrosion layer and the biogenic minerals. This sulfur-rich layer is probably the result of an abiotic reaction between Na2S added to ensure anoxic conditions and the reactive corroded surface of the iron coupons. Since this layer is located underneath the biogenic crystals, it can be assumed that it was produced ﬁrst. It is worth mentioning that the formation of such a layer is already reported during iron corrosion in anoxic environments [32]. Even if the eﬀect of sulfur on the corrosion process of iron is still under evaluation, experimental evidence suggests that elemental sulfur could speed up the corrosion rate of iron objects [33,34]. Indeed, this compound is known to be highly reactive, oxidizing organic and inorganic material regardless of the oxygen content [34]. In the presence of humidity, iron and steel surfaces exposed to elemental sulfur are corroded by an electrochemical reaction involving the reduction of elemental sulfur coupled with the oxidation of iron [33,34]. 3.1. Structure, Thickness and Continuity of the Corrosion Layer Materials 2020, 13, 1176 6 of 13 Figure 3. Efficiency of the treatment in terms of thickness and surface covering: (a) a graphic representation of the thickness of the corrosion layer of untreated, abiotic control, and bacterially treated iron coupons; (b) an estimation of the surface covered by iron phosphates by microscopic observation of the cross-sections Figure 3. Eﬃciency of the treatment in terms of thickness and surface covering: (a) a graphic representation of the thickness of the corrosion layer of untreated, abiotic control, and bacterially treated iron coupons; (b) an estimation of the surface covered by iron phosphates by microscopic observation of the cross-sections. Figure 3. Efficiency of the treatment in terms of thickness and surface covering: (a) a graphic representation of the thickness of the corrosion layer of untreated, abiotic control, and bacterially treated iron coupons; (b) an estimation of the surface covered by iron phosphates by microscopic observation of the cross sections Figure 3. Eﬃciency of the treatment in terms of thickness and surface covering: (a) a graphic representation of the thickness of the corrosion layer of untreated, abiotic control, and bacterially treated iron coupons; (b) an estimation of the surface covered by iron phosphates by microscopic observation of the cross-sections. 3.1. Structure, Thickness and Continuity of the Corrosion Layer Even if the thickness of a layer of naturally formed corrosion products is uneven, an overall decrease in the corrosion thickness was observed in the coupons treated with bacteria (Figure 3a). In fact, the mean value of the thickness of the corrosion layer decreased from nearly 28 µm (untreated coupons) to about 7 µm on the treated coupons (Figure 3a). The corrosion thickness decrease is due to the dissolutive microbial reduction of the iron phases composing the original corrosion layer. As a result, part of the iron oxyhydroxides is converted into iron biogenic minerals. The results demonstrated that this speciﬁc microbial process did convert a part of the original corrosion layer into reduced iron compounds, as least within the 7-day treatment duration. In fact, it is worth mentioning that treatment duration is a key element to assess and that the metal substrate could eventually become corroded if the growing conditions are not carefully set. Another important feature observed was the continuity of the newly formed biogenic layer. In fact, in order to inhibit corrosion, this layer has to completely cover the remaining original corrosion layer, avoiding further contact of the metal core with atmospheric oxygen and moisture [3]. Microscopic observations of the cross-sections confirmed that after 7 days of incubation, about 92% of the analyzed surface was covered by biogenic crystals (iron phosphates), while only 55% of the original corrosion layer was covered by blue spots on the abiotic control coupons (Figure 3b). Thus, it can be concluded that bacteria are needed to produce a uniform and continuous layer of biogenic minerals and that an abiotic reduction is not enough to achieve comparable results. Another important feature observed was the continuity of the newly formed biogenic layer. In fact, in order to inhibit corrosion, this layer has to completely cover the remaining original corrosion layer, avoiding further contact of the metal core with atmospheric oxygen and moisture [3]. Microscopic observations of the cross-sections conﬁrmed that after 7 days of incubation, about 92% of the analyzed surface was covered by biogenic crystals (iron phosphates), while only 55% of the original corrosion layer was covered by blue spots on the abiotic control coupons (Figure 3b). Thus, it can be concluded that bacteria are needed to produce a uniform and continuous layer of biogenic minerals and that an abiotic reduction is not enough to achieve comparable results. El l f h 3.2. Elemental Composition of the Corrosion Layer Hence, the sulfur-rich layer detected here and localized underneath the biogenic layer, if elemental sulfur, could be detrimental for 7 of 13 Materials 2020, 13, 1176 the objects, and has to be avoided. In conclusion, for future improvements, Na2S should be replaced by other reducing agents containing less sulfur, such as cysteine [35] or titanium(III) citrate [36]. replaced by other reducing agents containing less sulfur, such as cysteine [35] or titanium(III) citrate [36] As previously reported, the formation of biogenic iron minerals containing phosphorus can be attributed to the microbial reduction of iron phases, and the reaction of Fe2+ ions with phosphates (PO43−) added in the buﬀered bacterial medium [26]. [36]. As previously reported, the formation of biogenic iron minerals containing phosphorus can be attributed to the microbial reduction of iron phases, and the reaction of Fe2+ ions with phosphates (PO 3−) added in the buffered bacterial medium [26] Figure 4. SEM–EDS characterization of (a) untreated, (b) abiotic control and (c) bacterially treated iron coupons. On the left, images of backscattered electrons with a square box indicating the analyzed area and numbers indicating the different layers present: 0 = bulk metal; 1 = original corrosion layer; 2 = newly formed S-rich layer; 3 = biogenic layer. On the right, elemental mapping showing the presence of iron, oxygen, sulfur, and phosphorus in the different layers. Figure 4. SEM–EDS characterization of (a) untreated, (b) abiotic control and (c) bacterially treated iron coupons. On the left, images of backscattered electrons with a square box indicating the analyzed area and numbers indicating the diﬀerent layers present: 0 = bulk metal; 1 = original corrosion layer; 2 = newly formed S-rich layer; 3 = biogenic layer. On the right, elemental mapping showing the presence of iron, oxygen, sulfur, and phosphorus in the diﬀerent layers. Figure 4. SEM–EDS characterization of (a) untreated, (b) abiotic control and (c) bacterially treated iron coupons. On the left, images of backscattered electrons with a square box indicating the analyzed area and numbers indicating the different layers present: 0 = bulk metal; 1 = original corrosion layer; 2 = newly formed S-rich layer; 3 = biogenic layer. On the right, elemental mapping showing the presence of iron, oxygen, sulfur, and phosphorus in the different layers. Figure 4. SEM–EDS characterization of (a) untreated, (b) abiotic control and (c) bacterially treated iron coupons. El l f h 3.2. Elemental Composition of the Corrosion Layer On the left, images of backscattered electrons with a square box indicating the analyzed area and numbers indicating the diﬀerent layers present: 0 = bulk metal; 1 = original corrosion layer; 2 = newly formed S-rich layer; 3 = biogenic layer. On the right, elemental mapping showing the presence of iron, oxygen, sulfur, and phosphorus in the diﬀerent layers. 3 3 Molecular Composition of the Corrosion Layer 3.3. Molecular Composition of the Corrosion Layer o e u a o po i io of e o o io aye Single points, as well as areas, were analyzed with Raman spectroscopy, allowing for the identification of lepidocrocite and goethite as the main compounds in the corrosion layer of untreated coupons (Figure 5). Single points, as well as areas, were analyzed with Raman spectroscopy, allowing for the identiﬁcation of lepidocrocite and goethite as the main compounds in the corrosion layer of untreated coupons (Figure 5). 8 of 13 8 of 13 Materials 2020, 13, 1176 Materials 2020, 13, x FOR P Figure 5. Molecular mapping performed by Raman spectroscopy on untreated coupons. (a) From left to right, a microscopic image with a square box indicating the analyzed area and chemical maps of goethite (Go) and lepidocrocite (Le). (b) Representative Raman spectrum of a mixture of lepidocrocite and goethite with the corresponding spectral regions selected for the elaboration of the respective chemical maps of lepidocrocite (region labeled “Le” with orange dashed lines) and goethite (region labeled “Go” with green dashed lines). Figure 5. Molecular mapping performed by Raman spectroscopy on untreated coupons. (a) From left to right, a microscopic image with a square box indicating the analyzed area and chemical maps of goethite (Go) and lepidocrocite (Le). (b) Representative Raman spectrum of a mixture of lepidocrocite and goethite with the corresponding spectral regions selected for the elaboration of the respective chemical maps of lepidocrocite (region labeled “Le” with orange dashed lines) and goethite (region labeled “Go” with green dashed lines). Figure 5. Molecular mapping performed by Raman spectroscopy on untreated coupons. (a) From left to right, a microscopic image with a square box indicating the analyzed area and chemical maps of goethite (Go) and lepidocrocite (Le). (b) Representative Raman spectrum of a mixture of lepidocrocite and goethite with the corresponding spectral regions selected for the elaboration of the respective chemical maps of lepidocrocite (region labeled “Le” with orange dashed lines) and goethite (region labeled “Go” with green dashed lines). Figure 5. Molecular mapping performed by Raman spectroscopy on untreated coupons. (a) From left to right, a microscopic image with a square box indicating the analyzed area and chemical maps of goethite (Go) and lepidocrocite (Le). 3 3 Molecular Composition of the Corrosion Layer 3.3. Molecular Composition of the Corrosion Layer (b) Representative Raman spectrum of a mixture of lepidocrocite and goethite with the corresponding spectral regions selected for the elaboration of the respective chemical maps of lepidocrocite (region labeled “Le” with orange dashed lines) and goethite (region labeled “Go” with green dashed lines). On the abiotic control, lepidocrocite and goethite were detected, but also siderite and vivianite (Figure 6). Siderite was probably produced as a consequence of the interaction between iron and the carbonaceous sources present in the culture medium (lactate, fumarate, or carbonated buffer) [35]. The formation of vivianite (an Fe2+ phosphate) on the abiotic control coupons can be the result of the interaction of the phosphorus buffer with Fe2+ ions. The latter can either be present in the original corrosion layer or be produced when the Fe3+ phases of the original layer are abiotically reduced by Na2S. In fact, the reduction of iron in the abiotic control has already been documented in similar conditions [14] On the abiotic control, lepidocrocite and goethite were detected, but also siderite and vivianite (Figure 6). Siderite was probably produced as a consequence of the interaction between iron and the carbonaceous sources present in the culture medium (lactate, fumarate, or carbonated buﬀer) [35]. The formation of vivianite (an Fe2+ phosphate) on the abiotic control coupons can be the result of the interaction of the phosphorus buﬀer with Fe2+ ions. The latter can either be present in the original corrosion layer or be produced when the Fe3+ phases of the original layer are abiotically reduced by Na2S. In fact, the reduction of iron in the abiotic control has already been documented in similar conditions [14]. 9 of 13 of 13 Materials 2020, 13, 1176 Materials 2020, 13, x FOR Figure 6. Raman characterization of abiotic control coupons: (a) Microscopic image of the analyzed area with identified compounds indicated as siderite (Si), vivianite (Vi), and a mixture of goethite (Go) and lepidocrocite (Le). Representative Raman spectra of the different analyzed regions where (b) siderite (Si), (c) vivianite (Vi) and (d) a mix of goethite and lepidocrocite were identified (Le/Go), Figure 6. Raman characterization of abiotic control coupons: (a) Microscopic image of the analyzed area with identiﬁed compounds indicated as siderite (Si), vivianite (Vi), and a mixture of goethite (Go) and lepidocrocite (Le). 3 3 Molecular Composition of the Corrosion Layer 3.3. Molecular Composition of the Corrosion Layer Representative Raman spectra of the diﬀerent analyzed regions where (b) siderite (Si), (c) vivianite (Vi) and (d) a mix of goethite and lepidocrocite were identiﬁed (Le/Go), respectively. Figure 6. Raman characterization of abiotic control coupons: (a) Microscopic image of the analyzed area with identified compounds indicated as siderite (Si), vivianite (Vi), and a mixture of goethite (Go) and lepidocrocite (Le). Representative Raman spectra of the different analyzed regions where (b) siderite (Si), (c) vivianite (Vi) and (d) a mix of goethite and lepidocrocite were identified (Le/Go), Figure 6. Raman characterization of abiotic control coupons: (a) Microscopic image of the analyzed area with identiﬁed compounds indicated as siderite (Si), vivianite (Vi), and a mixture of goethite (Go) and lepidocrocite (Le). Representative Raman spectra of the diﬀerent analyzed regions where (b) siderite (Si), (c) vivianite (Vi) and (d) a mix of goethite and lepidocrocite were identiﬁed (Le/Go), respectively. respectively. In the samples treated with bacteria, lepidocrocite and goethite were not detected. In fact, after treatment, the thickness of the original corrosion layer drastically decreased, making its detection difficult by Raman spectroscopy (spatial resolution of about 1 µm). As observed above during SEM– EDS analyses, these iron corrosion compounds have been converted into biogenic crystals through microbial reduction. These newly formed minerals were identified as a mixture of two different iron phosphates. The most abundant was vivianite (Figure 7). The vivianite spectrum displayed an intense vibrational band at 949 cm−1 and two less intense vibrational bands at 1014 and 1052 cm−1, typical of the P-O stretching mode [37]. In order to localize this compound, its characteristic Raman shift at 949 cm−1 was employed for chemical mapping (Figure 7). Interestingly, another iron phosphate compound identified as barbosalite Fe2+Fe3+2(PO4)2(OH)2 was also detected. The same bands related to the P-O stretching mode were present but with different relative intensities. For barbosalite, the main Raman shift was at 1015 cm−1 (Figure 7). Since barbosalite contains both Fe2+ and Fe3+ ions, its formation could be the consequence of the interaction between Fe3+ ions present in the original corrosion layer, Fe2+ ions already present or produced from microbial iron reduction, and the PO43− ions contained in the buffer solution. Finally, Raman measurements did not allow the detection of the sulfur-rich inner layer revealed by SEM–EDS. 3 3 Molecular Composition of the Corrosion Layer 3.3. Molecular Composition of the Corrosion Layer This could be explained by the low thickness of this l b bl b l th ti l l ti li it f R t ( b t 1 ) In the samples treated with bacteria, lepidocrocite and goethite were not detected. In fact, after treatment, the thickness of the original corrosion layer drastically decreased, making its detection diﬃcult by Raman spectroscopy (spatial resolution of about 1 µm). As observed above during SEM–EDS analyses, these iron corrosion compounds have been converted into biogenic crystals through microbial reduction. These newly formed minerals were identiﬁed as a mixture of two diﬀerent iron phosphates. The most abundant was vivianite (Figure 7). The vivianite spectrum displayed an intense vibrational band at 949 cm−1 and two less intense vibrational bands at 1014 and 1052 cm−1, typical of the P-O stretching mode [37]. In order to localize this compound, its characteristic Raman shift at 949 cm−1 was employed for chemical mapping (Figure 7). Interestingly, another iron phosphate compound identiﬁed as barbosalite Fe2+Fe3+2(PO4)2(OH)2 was also detected. The same bands related to the P-O stretching mode were present but with diﬀerent relative intensities. For barbosalite, the main Raman shift was at 1015 cm−1 (Figure 7). Since barbosalite contains both Fe2+ and Fe3+ ions, its formation could be the consequence of the interaction between Fe3+ ions present in the original corrosion layer, Fe2+ ions already present or produced from microbial iron reduction, and the PO43−ions contained in the buﬀer solution. Finally, Raman measurements did not allow the detection of the sulfur-rich inner layer revealed by SEM–EDS. This could be explained by the low thickness of this layer, probably below the spatial resolution limit of Raman spectroscopy (about 1 µm). 10 of 13 10 of 13 Materials 2020, 13, 1176 Materials 2020, 13, x FOR Figure 7. Molecular mapping performed by Raman spectroscopy on bacterially treated iron coupons: (a) image of secondary electrons with a square box indicating the area analyzed by Raman spectoscopy, chemical maps (b) of vivianite (Vi, blue) and (c) of barbosalite (Ba, pink), and (d) Raman spectra of vivianite and barbosalite with the corresponding spectral regions selected for the elaboration of the respective chemical maps of vivianite (indicated with blue dashed lines) and barbosalite (indicated with magenta dashed lines). Figure 7. 3 3 Molecular Composition of the Corrosion Layer 3.3. Molecular Composition of the Corrosion Layer Molecular mapping performed by Raman spectroscopy on bacterially treated iron coupons: (a) image of secondary electrons with a square box indicating the area analyzed by Raman spectoscopy, chemical maps (b) of vivianite (Vi, blue) and (c) of barbosalite (Ba, pink), and (d) Raman spectra of vivianite and barbosalite with the corresponding spectral regions selected for the elaboration of the respective chemical maps of vivianite (indicated with blue dashed lines) and barbosalite (indicated with magenta dashed lines). Figure 7. Molecular mapping performed by Raman spectroscopy on bacterially treated iron coupons: (a) image of secondary electrons with a square box indicating the area analyzed by Raman spectoscopy, chemical maps (b) of vivianite (Vi, blue) and (c) of barbosalite (Ba, pink), and (d) Raman spectra of vivianite and barbosalite with the corresponding spectral regions selected for the elaboration of the respective chemical maps of vivianite (indicated with blue dashed lines) and barbosalite (indicated with magenta dashed lines) Figure 7. Molecular mapping performed by Raman spectroscopy on bacterially treated iron coupons: (a) image of secondary electrons with a square box indicating the area analyzed by Raman spectoscopy, chemical maps (b) of vivianite (Vi, blue) and (c) of barbosalite (Ba, pink), and (d) Raman spectra of vivianite and barbosalite with the corresponding spectral regions selected for the elaboration of the respective chemical maps of vivianite (indicated with blue dashed lines) and barbosalite (indicated with magenta dashed lines). Figure 7. Molecular mapping performed by Raman spectroscopy on bacterially treated iron coupons: (a) image of secondary electrons with a square box indicating the area analyzed by Raman spectoscopy, chemical maps (b) of vivianite (Vi, blue) and (c) of barbosalite (Ba, pink), and (d) Raman spectra of vivianite and barbosalite with the corresponding spectral regions selected for the elaboration of the respective chemical maps of vivianite (indicated with blue dashed lines) and barbosalite (indicated with magenta dashed lines) Figure 7. Molecular mapping performed by Raman spectroscopy on bacterially treated iron coupons: (a) image of secondary electrons with a square box indicating the area analyzed by Raman spectoscopy, chemical maps (b) of vivianite (Vi, blue) and (c) of barbosalite (Ba, pink), and (d) Raman spectra of vivianite and barbosalite with the corresponding spectral regions selected for the elaboration of the respective chemical maps of vivianite (indicated with blue dashed lines) and barbosalite (indicated with magenta dashed lines). 4 Conclusions 4. Conclusions 4. Conclusions The development of new and ecologically friendly strategies to protect outdoor iron surfaces has a clear economical, as well as ecological, interest. We demonstrated the potential of bacteria as an alternative for the development of innovative and green methods to protect iron artifacts from detrimental corrosion [24–28]. Here, the obtained results allow us to better understand how reactive corrosion layers are converted into biogenic iron phosphates with Desulfitobacterium hafniense. These biogenic minerals covered almost all of the remaining original corrosion layer. This layer could act as a barrier isolating the unstable iron corrosion products that would eventually still be present The development of new and ecologically friendly strategies to protect outdoor iron surfaces has a clear economical, as well as ecological, interest. We demonstrated the potential of bacteria as an alternative for the development of innovative and green methods to protect iron artifacts from detrimental corrosion [24–28]. Here, the obtained results allow us to better understand how reactive corrosion layers are converted into biogenic iron phosphates with Desulﬁtobacterium hafniense. These biogenic minerals covered almost all of the remaining original corrosion layer. This layer could act as a barrier isolating the unstable iron corrosion products that would eventually still be 11 of 13 Materials 2020, 13, 1176 present underneath from the exposure to atmospheric oxygen and moisture that could lead to further corrosion. Vivianite is in fact a stable, poorly soluble, and non-oxidizing Fe2+ mineral [22]. However, great attention has to be drawn to the composition of the bacterial solution applied, which not only drives the type of biogenic minerals produced, but also potentially contaminates the corrosion layer with undesired compounds that are able to instigate further corrosion, such as sulfur-containing compounds. In fact, through the stratigraphy study carried out here, a sulfur-rich layer was detected below the biogenic iron phosphates. This layer was not detected with surface analyses of the coupons, and only stratigraphic investigations allowed us to conclude that careful attention has to be paid to the culture medium composition in order to produce a stable vivianite layer that would passivate the iron surface. Our study on cross-sectioned samples further improved the evaluation of the depth eﬃciency of the proposed bacterial treatment, as well as demonstrated the formation of biogenic vivianite as an adherent, even, and uniform layer. These features are important criteria when designing new protective systems to provide long-term inhibition of corrosion on iron surfaces. 4 Conclusions 4. Conclusions Author Contributions: Conceptualization, E.J.; methodology, L.C., M.A. and W.K.; validation, L.C., J.M., T.L., P.J. and E.J.; investigation, L.C., M.A., W.K., and T.L.; resources, J.M.; data curation, L.C.; writing—original draft preparation, L.C. and E.J.; writing—review and editing, L.C., M.A., W.K., J.M., T.L., P.J. and E.J.; visualization, L.C.; supervision, P.J. and E.J.; project administration, E.J.; funding acquisition, E.J. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Swiss National Science Foundation, Ambizione, grant number PZ00P2_142514, 2013–2016 and professorship grant number PP00P2_163653, 2016–2020. Funding: This research was funded by the Swiss National Science Foundation, Ambizione, grant number PZ00P2_142514, 2013–2016 and professorship grant number PP00P2_163653, 2016–2020. Funding: This research was funded by the Swiss National Science Foundation, Ambizione, grant num PZ00P2_142514, 2013–2016 and professorship grant number PP00P2_163653, 2016–2020. Acknowledgments: The authors are also grateful to the research conservation laboratory of the Swiss Nati Museum for providing the steel plate used in the experiments. Acknowledgments: The authors are also grateful to the research conservation laboratory of the Swiss National Museum for providing the steel plate used in the experiments. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. References 1. Yamashita, M.; Miyuki, H.; Matsuda, Y.; Nagano, H.; Misawa, T. The long term growth of the protective rust layer formed on weathering steel by atmospheric corrosion during a quarter of a century. Corros. Sci. 1994, 36, 283–299. [CrossRef] 2. Tamura, H. The role of rusts in corrosion and corrosion protection of iron and steel. Corros. Sci. 2008, 50, 1872–1883. [CrossRef] 3. Scott, D.A.; Eggert, G. Iron and Steel in Art; Archetype Publications Ltd.: London, UK, 2009. 3. Scott, D.A.; Eggert, G. Iron and Steel in Art; Archetype Publications Ltd.: London, UK, 2009. 4. Narain, S.; Jain, K.K. Iron Artifacts: History, Metallurgy, Corrosion and Conservation; Agam Kala Prakashan: Delhi, India, 2009. 4. Narain, S.; Jain, K.K. Iron Artifacts: History, Metallurgy, Corrosion and Conservation; Agam Kala Prakashan: Delhi, India, 2009. 5. Moore, J.D. Long-term corrosion processes of iron and steel shipwrecks in the marine environment: A review of current knowledge. J. Marit. Archaeol. 2015, 10, 191–204. [CrossRef] 6 R S N ﬀD B ll t G l t L Dill P D t i ti f i h l i l t f t Mi R 5. Moore, J.D. Long-term corrosion processes of iron and steel shipwrecks in the marine environment: A review of current knowledge. J. Marit. Archaeol. 2015, 10, 191–204. [CrossRef] 7. Ståhl, K.; Nielsen, K.; Jiang, J.; Lebech, B.; Hanson, J.C.; Norby, P.; van Lanschot, J. On the akaganéite crystal structure, phase transformations and possible role in post-excavational corrosion of iron artifacts. Corros. Sci. 2003, 45, 2563–2575. [CrossRef] 7. Ståhl, K.; Nielsen, K.; Jiang, J.; Lebech, B.; Hanson, J.C.; Norby, P.; van Lanschot, J. On the akaganéite crystal structure, phase transformations and possible role in post-excavational corrosion of iron artifacts. Corros. Sci. 2003, 45, 2563–2575. [CrossRef] 8. Rimmer, M.; Watkinson, D.; Wang, Q. The impact of chloride desalination on the corrosion rate of archaeological iron. Stud. Conserv. 2013, 58, 326–337. [CrossRef] 9. Dillmann, P.; Watkinson, D.; Angelini, E.; Adriaens, A. Introduction: Conservation versus laboratory investigation in the preservation of metallic heritage artefacts. In Corrosion and Conservation of Cultural Heritage Metallic Artefacts, 1st ed.; Woodhead Publishing Limited on Behalf of the European Federation of Corrosion: Cambridge, UK, 2013. Jegdi´c, B.; Bajat, J.B.; Popi´c, J.P.; Miškovi´c-Stankovi´c, V. Corrosion stability of polyester coatings on stee pretreated with diﬀerent iron–phosphate coatings. Prog. Org. Coat. 2011, 70, 127–133. [CrossRef] 11. Zheng, S.; Li, J. Author Contributions: Conceptualization, E.J.; methodology, L.C., M.A. and W.K.; validation, L.C., J.M., T.L., P.J. and E.J.; investigation, L.C., M.A., W.K., and T.L.; resources, J.M.; data curation, L.C.; writing—original draft preparation, L.C. and E.J.; writing—review and editing, L.C., M.A., W.K., J.M., T.L., P.J. and E.J.; visualization, L.C.; supervision, P.J. and E.J.; project administration, E.J.; funding acquisition, E.J. All authors have read and agreed to the published version of the manuscript. References Inorganic–organic sol gel hybrid coatings for corrosion protection of metals. J. Sol-Gel Sci. Technol. 2010, 54, 174–187. [CrossRef] Materials 2020, 13, 1176 12 of 13 12. Selwyn, L. Overview of archaeological iron: The corrosion problem, key factors aﬀecting treatment, and gaps in current knowledge. In Metal 04: Proceedings of the International Conference on Metals Conservation; Ashton, J., Hallan, D., Eds.; National Museum of Australia: Canberra, Australia, 2004; pp. 294–306. 3. Carlin, W.; Keith, D.; Rodriguez, J. Less is more: Measure of chloride removal rate from wrought iron artif during electrolysis. Stud. Conserv. 2001, 46, 68–76. 14. Schmidt-Ott, K.; Boissonnas, V. Low-pressure hydrogen plasma: An assessment of its application on archaeological iron. Stud. Conserv. 2002, 47, 81–87. 15. Zarasvand, K.A.; Rai, V.R. Microorganisms: Induction and inhibition of corrosion in metals. Int. Biodeterior. Biodegrad. 2014, 87, 66–74. [CrossRef] 16. Iverson, W.P. Microbial corrosion of metals. Adv. Appl. Microbiol. 1987, 32, 1–36. [CrossRef] 17. Little, B.; Wagner, P.; Mansfeld, F. Microbiologically inﬂuenced corrosion of metals and alloys. Int. Mater. Rev. 1991, 36, 253–272. [CrossRef] 18. Videla, H.A.; Herrera, L.K. Microbiologically inﬂuenced corrosion: Looking to the future. Int. Microbiol. 2005, 8, 169. 19. Volkland, H.-P.; Harms, H.; Müller, B.; Repphun, G.; Wanner, O.; Zehnder, A.J. Bacterial phosphating of mild (unalloyed) steel. Appl. Environ. Microbiol. 2000, 66, 4389–4395. [CrossRef] 0. Mansfeld, F.; Hsu, H.; Örnek, D.; Wood, T.K.; Syrett, B. Corrosion control using regenerative bioﬁlm aluminum 2024 and brass in diﬀerent media. J. Electrochem. Soc. 2002, 149, B130–B138. [CrossRef] 21. Zuo, R.; Örnek, D.; Syrett, B.; Green, R.; Hsu, C.-H.; Mansfeld, F.; Wood, T.K. Inhibiting mild steel corrosion from sulfate-reducing bacteria using antimicrobial-producing bioﬁlms in Three-Mile-Island process water. Appl. Microbiol. Biotechnol. 2004, 64, 275–283. [CrossRef] 22. Chongdar, S.; Gunasekaran, G.; Kumar, P. Corrosion inhibition of mild steel by aerobic bioﬁlm. Electrochim. Acta 2005, 50, 4655–4665. [CrossRef] 23. Cote, C.; Rosas, O.; Basséguy, R. Geobacter sulfurreducens: An iron reducing bacterium that can protect carbon steel against corrosion? Corros. Sci. 2015, 94, 104–113. [CrossRef] 24. Comensoli, L.; Bindschedler, S.; Junier, P.; Joseph, E. Iron and fungal physiology: A review of biotechnological opportunities. Adv. Appl. Microbiol. 2017, 98, 31–60. [CrossRef] 25. Joseph, E.; Bindschedler, S.; Albini, M.; Comensoli, L.; Kooli, W.; Mathys, L. Microorganisms for Safeguarding Cultural Heritage. In The Fungal Community: Its Organization and Role in the Ecosystem, 4th ed.; Dighton, J., White, J.F., Eds.; CRC Press: Boca Raton, FL, USA, 2017; pp. 509–518. 26. References Comensoli, L.; Maillard, J.; Albini, M.; Sandoz, F.; Junier, P.; Joseph, E. Use of bacteria to stabilize archaeological iron. Appl. Environ. Microbiol. 2017, 83, e03478-16. [CrossRef] [PubMed] 27. Kooli, W.M.; Comensoli, L.; Maillard, J.; Albini, M.; Gelb, A.; Junier, P.; Joseph, E. Bacterial iron reduction and biogenic mineral formation for the stabilisation of corroded iron objects. Sci. Rep. 2018, 8, 764. [CrossRef] [PubMed] 28. Kooli, W.M.; Junier, T.; Shakya, M.; Monachon, M.; Davenport, K.W.; Vaideeswaran, K.; Vernudachi, A.; Marozau, I.; Monrouzeau, T.; Gleasner, C.D. Remedial treatment of corroded iron objects by environmental Aeromonas isolates. Appl. Environ. Microbiol. 2019, 85, e02042-18. [CrossRef] [PubMed] 29. Villemur, R.; Lanthier, M.; Beaudet, R.; Lépine, F. The Desulﬁtobacterium genus. FEMS Microbiol. Rev. 2006, 30, 706–733. [CrossRef] 29. Villemur, R.; Lanthier, M.; Beaudet, R.; Lépine, F. The Desulﬁtobacterium genus. FEMS Microbiol. Rev. 2006, 30, 706–733. [CrossRef] 30. Futagami, T.; Furukawa, K. The Genus Desulﬁtobacterium. In Organohalide-Respiring Bacteria; Adrian, L., p g 30, 706–733. [CrossRef] 30. Futagami, T.; Furukawa, K. The Genus Desulﬁtobacterium. In Organohalide-Respiring Bacteria; Adrian, L., Löﬄer F E Eds ; Springer: Berlin/Heidelberg Germany 2016; pp 173 207 [CrossRef] Futagami, T.; Furukawa, K. The Genus Desulﬁtobacterium. In Organohalide-Respiring Bacteria; Adrian, L Löﬄer, F.E., Eds.; Springer: Berlin/Heidelberg, Germany, 2016; pp. 173–207. [CrossRef] 31. Prat, L.; Maillard, J.; Grimaud, R.; Holliger, C. Physiological Adaptation of Desulﬁtobacterium hafniense Strain TCE1 to Tetrachloroethene Respiration. Appl. Environ. Microbiol. 2011, 77, 3853–3859. [CrossRef] 32. Bellot-Gurlet, L.; Neﬀ, D.; Reguer, S.; Monnier, J.; Saheb, M.; Dillmann, P. Raman studies of corrosion layers formed on archaeological irons in various media. J. Nano Res. 2009, 8, 147–156. [CrossRef] 33. Schmitt, G. Eﬀect of elemental sulfur on corrosion in sour gas systems. Corrosion 1991, 47, 285–308. [CrossRef] 33. Schmitt, G. Eﬀect of elemental sulfur on corrosion in sour gas systems. Corrosion 1991, 47, 285–308. [CrossRef] 34. Leonard, S.E.; Carroll, K.S. Chemical ‘omics’ approaches for understanding protein cysteine oxidation in 34. Leonard, S.E.; Carroll, K.S. Chemical ‘omics’ approaches for understanding protein cysteine oxidation in biology. Curr. Opin. Chem. Biol. 2011, 15, 88–102. [CrossRef] 35. Legrand, L.; Savoye, S.; Chausse, A.; Messina, R. Study of oxidation products formed on iron in solutions containing bicarbonate/carbonate. Electrochim. Acta 2000, 46, 111–117. [CrossRef] 13 of 13 Materials 2020, 13, 1176 36. Zehnder, A.; Wuhrmann, K. Titanium (III) citrate as a nontoxic oxidation-reduction buﬀering system for the culture of obligate anaerobes. Science 1976, 194, 1165–1166. [CrossRef] 37. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). 37. Frost, R.L.; Martens, W.; Williams, P.A.; Kloprogge, J.T. Raman and infrared spectroscopic study of the vivianite-group phosphates vivianite, baricite and bobierrite. Mineral. Mag. 2002, 66, 1063. [CrossRef] References Frost, R.L.; Martens, W.; Williams, P.A.; Kloprogge, J.T. Raman and infrared spectroscopic study of the vivianite-group phosphates vivianite, baricite and bobierrite. Mineral. Mag. 2002, 66, 1063. [CrossRef] © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W2896659754	https://arpi.unipi.it/bitstream/11568/937780/2/PhysRevD.100.072002.pdf	English	null	Improving Cleaning and Disinfection of High-Touch Surfaces in Intensive Care during Carbapenem-Resistant Acinetobacter baumannii Endemo-Epidemic Situations	International journal of environmental research and public health/International journal of environmental research and public health	2,018	cc-by	6,480	International Journal of Environmental Research and Public Health International Journal of Environmental Research and Public Health Improving Cleaning and Disinfection of High-Touch Surfaces in Intensive Care during Carbapenem-Resistant Acinetobacter baumannii Endemo-Epidemic Situations Beatrice Casini 1,, Anna Righi 1, Nunzio De Feo 2, Michele Totaro 1, Serena Giorgi 1, Lavinia Zezza 1, Paola Valentini 1, Enrico Tagliaferri 3, Anna Laura Costa 1, Simona Barnini 4, Angelo Baggiani 1, Pietro Luigi Lopalco 1, Paolo Malacarne 2 and Gaetano Pierpaolo Privitera 1 1 Department of Translational Research, N.T.M.S., University of Pisa, 56123 Pisa, Italy; righianna@gmail.com (A.R.); micheleto@hotmail.it (M.T.); giorgiserena@yahoo.it (S.G.); laviniazezza@alice.it (L.Z.); paola.valentini@dps.unipi.it (P.V.); alauracosta@alice.it (A.L.C.); angelo.baggiani@med.unipi.it (A.B.); pierluigi.lopalco@unipi.it (P.L.L.); gaetano.privitera@med.unipi.it (G.P.P.) 1 Department of Translational Research, N.T.M.S., University of Pisa, 56123 Pisa, Italy; righianna@gmail.com (A.R.); micheleto@hotmail.it (M.T.); giorgiserena@yahoo.it (S.G.); laviniazezza@alice.it (L.Z.); paola.valentini@dps.unipi.it (P.V.); alauracosta@alice.it (A.L.C.); angelo.baggiani@med.unipi.it (A.B.); pierluigi.lopalco@unipi.it (P.L.L.); gaetano.privitera@med.unipi.it (G.P.P.) 2 Anesthesia and Intensive Care Unit PS, University Hospital, 56124 Pisa, Italy; n.defeo@med.unipi.it (N.D.F.) pmalacarne@hotmail.com (P.M.) 3 Infectious Disease Unit, University Hospital, 56124 Pisa, Italy; tagliaferrienrico@alice.it 4 Unit of Microbiology, Azienda Ospedaliero Universitaria Pisana, 56124 Pisa, Italy; s.barnini@ao-pisa.toscana.it Correspondence: beatrice.casini@med.unipi.it Keywords: high-touch surfaces; carbapenem-resistant A. baumannii; pre-impregnated wipes; outsourced cleaning services 1. Introduction High-touch surfaces are recognized as a possible reservoir of infectious agents and their contamination can pose a risk also for the spread of multi-resistant organisms [1–4], hence they are recommended to be cleaned and disinfected on a more frequent schedule than minimal touch surfaces [5]. Environmental cleaning and disinfection (C&D) are important components of a comprehensive strategy in order to control healthcare-associated infections [6–9], especially in wards such as Intensive Care Unit (ICU) where patients are compromised. Studies evaluating improved cleaning interventions have reported that approximately 5–30% of surfaces remain potentially contaminated due to the inability of existing detergent and disinfectants formulations to disrupt bioﬁlms [10]. To achieve higher rates of effectiveness in the ﬁeld, new C&D strategies should be evaluated. The ready-to-use wipes are increasingly used in health care settings, although different antimicrobial wipes have shown a variable effectiveness in removing microbial bioburden from inanimate surfaces and in reducing the pathogens transfer between surfaces [11]. As reported by Sattar et al. [12], the use of wipes containing 0.5% accelerated H2O2 or sodium hypochlorite solution <3% were effective in removing both Acinetbacter baumannii and Staphylococcus aureus (at least 7 log10 CFU reduction). Kenters et al., evaluated the effectiveness of different cleaning-disinfectant wipes with different composition demonstrating for all the type of wipes a log10 reduction higher than 5 with a 5-min exposure time on Klebsiella pneumoniae OXA-48, A. baumannii and VRE outbreak strains [13]. The aim of this study was to evaluate the effectiveness on the ﬁeld of pre-impregnated wipes (Modiﬁed Operative Protocol, MOP) to reduce environmental bacterial burden and to maintain a disinfection activity on high-touch surfaces as an alternative to the currently used Standard Operative Protocol (SOP) in a 12-bed ICU during a carbapenem-resistant A. baumannii (CRAB) endemo-epidemic situation. Received: 4 September 2018; Accepted: 17 October 2018; Published: 19 October 2018 Abstract: Aims: High-touch surfaces cleaning and disinfection require the adoption of effective and proper executed protocols, especially during carbapenem-resistant Acinetobacter baumannii (CRAB) endemo-epidemic situations. We evaluated the effectiveness and residual disinfectant activity of disposable pre-impregnated wipes (Modiﬁed Operative Protocol, MOP) in reducing environmental bioburden versus a two-step Standard Operative Protocol (SOP) in a 12-bed Intensive Care Unit. Methods: Five high-touch surfaces were cleaned and disinfected either according to the SOP (alcohol-based cleaning and chlorine-based disinfection) or using quaternary ammonium compounds-based disposable wipes (MOP). Sampling was performed before each procedure and at 0.5, 2.5, 4.5 and 6.5 h after (560 sites). Total viable count (TVC) was evaluated according to Italian hygiene standard (<50 CFU/24 cm2). Clinical and environmental CRAB strains isolated were genotyped. Results: On non-electromedical surfaces the difference between TVC before procedure and at each of the following times was signiﬁcant only for the MOP (p < 0.05, Wilcoxon test). Using the MOP, only 7.4% (10/135) of sites showed TVC >50 CFU/24 cm2 (hygiene failures) versus 18.9% (25/132) after SOP (p < 0.05, Fisher’s Exact test). On infusion pumps a higher number of hygiene failures was observed after the SOP (7/44, 15.9%) compared with the MOP (4/45, 8.9%). Genotyping highlighted a common source of infection. Conclusion: On high-touch surfaces, the use of disposable wipes by in-house auxiliary nurses may represent a more effective alternative to standard cleaning and disinfection procedure performed by outsourced cleaning services, showing effectiveness in reducing microbial contamination and residual disinfection activity up to 6.5 h. Int. J. Environ. Res. Public Health 2018, 15, 2305; doi:10.3390/ijerph15102305 www.mdpi.com/journal/ijerph 2 of 9 Int. J. Environ. Res. Public Health 2018, 15, 2305 2. Materials and Methods Setting: The study was performed in a 12-bed ICU at a university Italian hospital. During the study, from 1 March to 30 April 2016, 82 patients (mean age 66.7 ± 16.4 years) were admitted to the ICU and eight were already hospitalized (case mix: 21 trauma, 14 emergency surgery, eight elective surgery, 47 general medicine), 41 of which ventilated for at least 24 h. The bed turnaround was 6.6 and the average occupancy 88%. In this hospital, cleaning services was outsourced and according to the SOP, housekeeping staff using disposable clothes, applied an alcohol-based detergent (Keradet, Kiehl, Odelzhausen, Germany) followed by a chlorine-based disinfectant (Antisapril 2%, Angelini, Rome, Italy, active chlorine 540 mg/L) on furniture surfaces except electromedical devices when in use. Monitors and pumps were sanitized only at the patient discharge. During the study, according to the MOP, on two units disposable wipes impregnate with cationic surfactant tensioactives, quaternary ammonium compounds and biguanide (Clinell Universal Wipes, GAMA, Watford, UK) were applied by in-house auxiliary nurses, trained about the proper use of wipes. According to manufacturer’s instructions, a “one wipe, one surface, one direction” approach was adopted. The two units treated with the MOP were compared with two of the units managed with the SOP. Sampling procedure: Five inanimate surfaces for each patient unit were chosen to determine the bacterial bioburden before and after both the SOP and the MOP. In Figure 1, the patient units selected and include in the study and C&D procedures applied in each unit are reported. In order to evaluate the temporal trend of microbial contamination [14,15], samples were obtained immediately before and at 0.5, 2.5, 4.5 and 6.5 h after each C&D procedure (overall 560 samples). 3 of 9 3 of 8 Int. J. Environ. Res. Public Health 2018, 15, 2305 Int J Environ Res Public Health 2018 15 x Figure 1. Patient units selected for the study (2 in each opposite side of the open-space, functionally separated) and cleaning and disinfection procedures applied in each units (SOP: Standard Operative Protocol, MOP: Modified Operative Protocol). Figure 1. Patient units selected for the study (2 in each opposite side of the open-space, functionally separated) and cleaning and disinfection procedures applied in each units (SOP: Standard Operative Protocol, MOP: Modiﬁed Operative Protocol). Figure 1. 2. Materials and Methods Patient units selected for the study (2 in each opposite side of the open-space, functionally separated) and cleaning and disinfection procedures applied in each units (SOP: Standard Operative Protocol, MOP: Modified Operative Protocol). Figure 1. Patient units selected for the study (2 in each opposite side of the open-space, functionally separated) and cleaning and disinfection procedures applied in each units (SOP: Standard Operative Protocol, MOP: Modiﬁed Operative Protocol). According to ISO 14698-1, 55-mm diameter Rodac plates containing Plate Count Agar, PCA, with neutralizers (VWR International PBI, Radnor, PA, USA) were used for TVC enumeration and Violet Red Bile Dextrose Agar, VRBD, (Oxoid, Basingstoke, UK) for Gram-negative bacteria qualitative evaluation Contact plates were incubated aerobically at 37 °C for 48 h According to ISO 14698-1, 55-mm diameter Rodac plates containing Plate Count Agar, PCA, with neutralizers (VWR International PBI, Radnor, PA, USA) were used for TVC enumeration and Violet Red Bile Dextrose Agar, VRBD, (Oxoid, Basingstoke, UK) for Gram-negative bacteria qualitative evaluation. Contact plates were incubated aerobically at 37 ◦C for 48 h. qualitative evaluation. Contact plates were incubated aerobically at 37 C for 48 h. Suspect Acinetobacter spp. or Klebsiella spp. were subcultured on chromID™ mSuperCARBA (bioMérieux, Marcy l’Etoile, France) and identified by API/ID32 Strep Miniature System (bioMérieux). The total microbial load and the presence of pathogens were evaluated according to the Suspect Acinetobacter spp. or Klebsiella spp. were subcultured on chromID™mSuperCARBA (bioMérieux, Marcy l’Etoile, France) and identiﬁed by API/ID32 Strep Miniature System (bioMérieux). The total microbial load and the presence of pathogens were evaluated according to the hygienic standards proposed by the Italian National Guidelines (for the ICU: <50 CFU/plate 24 cm2 and qualitative evaluation. Contact plates were incubated aerobically at 37 C for 48 h. Suspect Acinetobacter spp. or Klebsiella spp. were subcultured on chromID™ mSuperCARBA (bioMérieux, Marcy l’Etoile, France) and identified by API/ID32 Strep Miniature System (bioMérieux) Suspect Acinetobacter spp. or Klebsiella spp. were subcultured on chromID™mSuperCARBA (bioMérieux, Marcy l’Etoile, France) and identiﬁed by API/ID32 Strep Miniature System (bioMérieux). (bioMérieux, Marcy l Etoile, France) and identified by API/ID32 Strep Miniature System (bioMérieux). 2. Materials and Methods The total microbial load and the presence of pathogens were evaluated according to the hygienic standards proposed by the Italian National Guidelines (for the ICU: <50 CFU/plate -24 cm2- and absence of pathogens) [16] The total microbial load and the presence of pathogens were evaluated according to the hygienic standards proposed by the Italian National Guidelines (for the ICU: <50 CFU/plate -24 cm2- and absence of pathogens) [16]. and absence of pathogens) [16]. In the ICU, a systematic surveillance for CRAB colonization/infection was performed through weekly rectal swabs and/or bronchial aspirate sampling. Clinical and environmental CRAB strains were genotyped in order to assess the source of nosocomial colonization/infection, comparing the genomic profile according to the PFGE Typing Protocol recommended for A. baumannii [17,18]. In the ICU, a systematic surveillance for CRAB colonization/infection was performed through weekly rectal swabs and/or bronchial aspirate sampling. Clinical and environmental CRAB strains were genotyped in order to assess the source of nosocomial colonization/infection, comparing the genomic proﬁle according to the PFGE Typing Protocol recommended for A. baumannii [17,18]. genomic profile according to the PFGE Typing Protocol recommended for A. baumannii [17,18]. Statistical analysis: For each C&D procedure, we compared TVC at baseline and at each of the following times using Wilcoxon rank-sum test and the number of hygiene failures (environmental samples with TVC > 50 CFU/24 cm2) using McNemar’s test with continuity correction. We compared the number of hygiene failures between the two protocols with the Fisher’s Exact test. (Epi Info version 7.2, CDC, GA, USA). Statistical analysis: For each C&D procedure, we compared TVC at baseline and at each of the following times using Wilcoxon rank-sum test and the number of hygiene failures (environmental samples with TVC > 50 CFU/24 cm2) using McNemar’s test with continuity correction. We compared the number of hygiene failures between the two protocols with the Fisher’s Exact test. (Epi Info version 7.2, CDC, GA, USA). 3. Results 3. Results After 0.5 h from the C6D, the initial average TVC detected on all non-electromedical high-touch surfaces (bed rails, overbed table, worktop) was reduced from 34 CFU/24 cm2 (SD ± 44 CFU/24 cm2) to 21 CFU/24 cm2 (SD ± 31 CFU/24 cm2) after the SOP, and from 52 CFU/24 cm2 (SD ± 63 CFU/24 cm2) to 15 CFU/24 cm2 (SD ± 24 CFU/24 cm2) after the MOP. The percentage decrease was −38.2% (Wilcoxon test p = 0 3192) and 71 2% Wilcoxon test p = 0 0005) respectively After 0.5 h from the C&D, the initial average TVC detected on all non-electromedical high-touch surfaces (bed rails, overbed table, worktop) was reduced from 34 CFU/24 cm2 (SD ± 44 CFU/24 cm2) to 21 CFU/24 cm2 (SD ± 31 CFU/24 cm2) after the SOP, and from 52 CFU/24 cm2 (SD ± 63 CFU/24 cm2) to 15 CFU/24 cm2 (SD ± 24 CFU/24 cm2) after the MOP. The percentage decrease was −38.2% (Wilcoxon test, p = 0.3192) and −71.2%, (Wilcoxon test, p = 0.0005) respectively. (Wilcoxon test, p = 0.3192) and −71.2%, Wilcoxon test, p = 0.0005) respectively. In Figure 2, the Total Viable Counts (TVCs) trend and the percentage reduction of the values before and after the application of the two different procedures on non-electromedical surfaces are reported In Figure 2, the Total Viable Counts (TVCs) trend and the percentage reduction of the values before and after the application of the two different procedures on non-electromedical surfaces are reported. Int. J. Environ. Res. Public Health 2018, 15, 2305 4 of 9 Figure 2. Trend of the Total Viable Counts (TVCs) and percentage reduction of the values before and after the application of the cleaning and disinfection procedures on non-electromedical surfaces: (A) Average TVCs before and after 0.5 h the two procedures (B) Temporal trend of TVCs during the 6.5 h of sampling (C) Percentage reductions of the TVCs. Note: The dashed line represents the targeted bacterial burden proposed by the Italian Workers Compensation Authority (TVC < 50 CFU/plate, plate equal to 24 cm2). Figure 2. Trend of the Total Viable Counts (TVCs) and percentage reduction of the values before and after the application of the cleaning and disinfection procedures on non-electromedical surfaces: (A) Average TVCs before and after 0.5 h the two procedures (B) Temporal trend of TVCs during the 6.5 h of sampling (C) Percentage reductions of the TVCs. 3. Results 3. Results Similarly the difference between the number of the hygiene failures immediately before the According to the hygienic standard proposed by the national guidelines (TCV < 50 CFU/24 cm2), before the procedures a comparable hygienic level on all the surfaces was observed, since no signiﬁcant differences resulted between the hygiene failures found on surfaces subsequently treated with the SOP (9/33 failures) and those dealt with the MOP (13/36) (p = 0.60, Fisher’s Exact test). Similarly, the difference between the number of the hygiene failures immediately before the procedure and at each of the subsequent times was statistical significant only for the MOP (p <0 .05, McNemar’s test with continuity correction). Considering only sites with hygiene failures at baseline using the MOP 11/13 surfaces (84 6%) After the SOP on non-electromedical surfaces, hygiene failures were 25 out of 132 (18.9%) versus 10 out of 135 after the MOP (7.4%), (p < 0.05, Fisher’s Exact test). In Table the hygiene failures and the pathogens found on each type of high-touch surface are reported. Considering only sites with hygiene failures at baseline, using the MOP 11/13 surfaces (84.6%) met the hygienic standard at all the following times, while applying the SOP only 3/9 (33.3%) (p < 0.05, Fisher’s Exact test). The ability to preserve clean surfaces with basal TVC < 50 CFU/24 cm2 was greater for the MOP but not significantly (13/20 for the wipe protocol 11/24 for the SOP) Similarly, the difference between the number of the hygiene failures immediately before the procedure and at each of the subsequent times was statistical signiﬁcant only for the MOP (p < 0.05, McNemar’s test with continuity correction). greater for the MOP but not significantly (13/20 for the wipe protocol, 11/24 for the SOP). On monitors, TVCs resulted <50 CFU/24 cm2 in almost all (111/112) of the sampled sites and no pathogens were isolated. On pumps under the MOP only the TVC at 2.5 h was significantly lower than the baseline one (Wilcoxon test), while under the SOP, the TVCs at 2.5 h and at 6.5 h were significantly higher than the basal ones The McNemar’s test with continuity correction pointed out Considering only sites with hygiene failures at baseline, using the MOP 11/13 surfaces (84.6%) met the hygienic standard at all the following times, while applying the SOP only 3/9 (33.3%) (p < 0.05, Fisher’s Exact test). 3. Results 3. Results Note: The dashed line represents the targeted bacterial burden proposed by the Italian Workers Compensation Authority (TVC < 50 CFU/plate, plate equal to 24 cm2). Figure 2. Trend of the Total Viable Counts (TVCs) and percentage reduction of the values before and after the application of the cleaning and disinfection procedures on non-electromedical surfaces: (A) Average TVCs before and after 0.5 h the two procedures (B) Temporal trend of TVCs during the 6.5 h of sampling (C) Percentage reductions of the TVCs. Note: The dashed line represents the targeted bacterial burden proposed by the Italian Workers Compensation Authority (TVC < 50 CFU/plate, plate equal to 24 cm2). Figure 2. Trend of the Total Viable Counts (TVCs) and percentage reduction of the values before and after the application of the cleaning and disinfection procedures on non-electromedical surfaces: (A) Average TVCs before and after 0.5 h the two procedures (B) Temporal trend of TVCs during the 6.5 h of sampling (C) Percentage reductions of the TVCs. Note: The dashed line represents the targeted bacterial burden proposed by the Italian Workers Compensation Authority (TVC < 50 CFU/plate, plate equal to 24 cm2). After the SOP on non-electromedical surfaces, hygiene failures were 25 out of 132 (18.9%) versus 10 out of 135 after the MOP (7.4%), (p < 0.05, Fisher’s Exact test). In Table the hygiene failures and the pathogens found on each type of high-touch surface are reported. Similarly the difference between the number of the hygiene failures immediately before the According to the hygienic standard proposed by the national guidelines (TCV < 50 CFU/24 cm2), before the procedures a comparable hygienic level on all the surfaces was observed, since no signiﬁcant differences resulted between the hygiene failures found on surfaces subsequently treated with the SOP (9/33 failures) and those dealt with the MOP (13/36) (p = 0.60, Fisher’s Exact test). After the SOP on non-electromedical surfaces, hygiene failures were 25 out of 132 (18.9%) versus 10 out of 135 after the MOP (7.4%), (p < 0.05, Fisher’s Exact test). In Table the hygiene failures and the pathogens found on each type of high-touch surface are reported. 3. Results 3. Results The ability to preserve clean surfaces with basal TVC < 50 CFU/24 cm2 was greater for the MOP but not signiﬁcantly (13/20 for the wipe protocol, 11/24 for the SOP). significantly higher than the basal ones. The McNemar s test with continuity correction pointed out no significant difference either for the MOP or for the SOP (Table 1). Since there was no significant difference between the hygiene failures before the procedure (1/12 for the MOP, 0/11 for the SOP), we compared the total hygiene failures at the subsequent times performing the Fisher’s Exact test: the hygiene failures were higher (not significantly) for the SOP: 4/45 (8 9%) for the MOP and 7/44 (15 9%) for the SOP Considering only the hygiene failures at 6 5 h On monitors, TVCs resulted <50 CFU/24 cm2 in almost all (111/112) of the sampled sites and no pathogens were isolated. On pumps under the MOP only the TVC at 2.5 h was signiﬁcantly lower than the baseline one (Wilcoxon test), while under the SOP, the TVCs at 2.5 h and at 6.5 h were signiﬁcantly higher than the basal ones. The McNemar’s test with continuity correction pointed out no signiﬁcant difference either for the MOP or for the SOP (Table 1). 5 of 9 Int. J. Environ. Res. Public Health 2018, 15, 2305 Table 1. Hygiene failures before and after the cleaning and disinfection procedures on non-electromedical surfaces and on the infusion pumps. Note: t0 = before cleaning and disinfection, t1 = 30 min after, t2 = 2.5 h after, t3 = 4.5 h after, t4 = 6.5 h after. Grey boxes indicate the hygiene failures (>50 CFU/24 cm2); Bed in bold italics indicates a bed occupied by a CRAB positive patient; the hash symbol (#) indicates environmental site positive for CRAB and the asterisk symbol () indicates patient transferred to another ward. 4. Discussion In order to assess the environmental hygiene level in hospital, as visual cleanliness does not always correlate with microbiological cleanliness, and in the presence of microbial contamination, microbiological limit considered safe on high-touch sites are not always standardized. Dancer et al., proposed as hygiene standards, <1 CFU/cm2 for pathogens and a <5 CFU/cm2 as a starting point and <2.5 CFU/cm2 as a more suitable goal although with no distinction between different care settings (low-risk or high-risk patients) [19]. These standards were aimed at hospitals using detergent cleaning and double sided dipslides for sampling. For intensive care units the Italian Workers Compensation Authority [16] suggests the use of contact plate and proposes as standard <50 CFU/plate (24 cm2) and the absence of pathogens (S. aureus, Pseudomonas aeruginosa, enterobacteria, Aspergillus spp.). To clean and disinfect environmental surfaces that necessitate low-level disinfection, the housekeeping staff traditionally uses a two-step method (detergent and subsequent disinfectant) with disposable or reusable cloths. To avoid contamination spread, reusable cloths should be adequately cleaned and disinfected [3]. Disposable cloths could solve this potential weakness. Pre-impregnated disposable cloths cleaning and disinfecting in one step could make the sanitization process faster and easier with consequent increase in cleaning staff compliance, although environmental impact due to waste disposal and cost-effectiveness should be considered [20]. Furthermore, since wipes showed their efﬁcacy to remove and retain Gram negative bacteria, they might be an appropriate choice in health-care settings where CRAB is endemic [11–13]. In our study, only the pre-impregnated wipe protocol was effective in reducing signiﬁcantly TVC or hygiene failures on non-electromedical surfaces from baseline up to 6.5 h, showing a long residual disinfection activity and a better performance when compared to the standard operative protocol, especially when the surfaces were highly contaminated (TVC > 50 CFU/24 cm2). On electromedical-devices, such as the monitors, although not routinely sanitized, almost all TVC values were compliant with the standard and no pathogens were isolated. However, on infusion pumps was observed a higher number of hygiene failures after the SOP, although the difference between the two protocols was not as evident as on non-electromedical surfaces, probably because of the fewer sampled sites. Even if not always signiﬁcant, data showed a greater effectiveness of pre-impregnated wipes compared to the SOP. Carbapenem-Resistant A. baumannii Strains During the studied period, four patients resulted colonized/infected with CRAB, one of which in contact precautions in a single room due to sepsis and respiratory-gastrointestinal colonization by CRAB as well as respiratory-gastrointestinal colonization by KPC-producing K. pneumoniae. The single room was included only once in the monitoring to increase the recovery of CRAB from surfaces. Overall 23 on 100 environmental samples were positive (23%) in the high-touch area surrounding three of the four patients (Table 1). In particular, in the single room, 15/25 high-touch surfaces resulted positive for CRAB, but none for K. pneumoniae. No statistically signiﬁcant differences were found between the different protocols and considering the temporal trend of each of them. The clinical CRAB strains were genetically similar to the environmental ones (similarity >95%), suggesting that the clonal spread of CRAB in the ICU played an important role in the endemic/epidemic situation. 3. Results 3. Results J. Environ. Res. Public Health 2018, 15, 2305 Since there was no signiﬁcant difference between the hygiene failures before the procedure (1/12 for the MOP, 0/11 for the SOP), we compared the total hygiene failures at the subsequent times performing the Fisher’s Exact test: the hygiene failures were higher (not signiﬁcantly) for the SOP: 4/45 (8.9%) for the MOP and 7/44 (15.9%) for the SOP. Considering only the hygiene failures at 6.5 h, the difference between the two protocols was signiﬁcant (0/11 for the MOP versus 5/11 for the SOP). 3. Results 3. Results Wipe Protocol Times Bedrails Overbed Table Worktop Day 1 Day 2 Day 3 Day 4 Day 5 Day 1 Day 2 Day 3 Day 4 Day 5 Day 1 Day 2 Day 3 Day 4 Day 5 Bed 1 Bed 2 Bed 1 Bed 2 Bed 1 Bed 2 Bed 1 Bed 2 Bed 3 Bed 1 Bed 2 Bed 3 Bed 1 Bed 2 Bed 1 Bed 2 Bed 1 Bed 2 Bed 1 Bed 2 Bed 3 Bed 1 Bed 2 Bed 3 Bed 1 Bed 2 Bed1 Bed 2 Bed1 Bed 2 Bed 1 Bed 2 Bed 3 Bed 1 Bed 2 Bed 3 t0 13 11 33 68 0 1 18 9 23 0 3 165 # 9 45 1 4 0 1 108 63 64 0 6 259 # 22 37 159 16 126 19 99 137 96 38 70 161 # t1 0 0 0 16 0 5 12 52 0 0 0 20 # 0 7 0 6 29 2 10 4 0 0 84 9 # 102 38 59 6 4 3 3 4 11 6 22 29 # t2 3 3 0 19 1 5 10 2 4 8 1 # 3 15 11 1 3 * 25 24 0 0 31 21 # 1 176 39 13 106 * 4 24 0 55 0 40 t3 103 73 9 6 31 * 7 22 9 0 18 44 # 9 17 3 0 3 * 6 3 0 1 39 44 # 1 23 9 14 8 * 13 3 0 6 9 27 # t4 88 23 0 0 0 * 2 7 2 22 3 34 0 9 0 1 0 * 3 4 1 0 1 34 # 26 18 28 6 8 * 29 16 10 3 8 41 Standard Protocol Bedrails Overbed Table Worktop Day 1 Day 2 Day 3 Day 4 Day 5 Day 1 Day 2 Day 3 Day 4 Day 5 Day 1 Day 2 Day 3 Day 4 Day 5 Bed 3 Bed 4 Bed 3 Bed 4 Bed 3 Bed 4 Bed 4 Bed 5 Bed 6 Bed 4 Bed 5 Bed 3 Bed 4 Bed 3 Bed 4 Bed 3 Bed 4 Bed 4 Bed 5 Bed 6 Bed 4 Bed 5 Bed 3 Bed 4 Bed 3 Bed 4 Bed 3 Bed 4 Bed 4 Bed 5 Bed 6 Bed 4 Bed 5 t0 0 0 89 27 1 1 4 10 1 0 0 49 60 0 0 56 19 41 40 52 7 95 27 73 4 48 15 25 219 56 # 70 4 31 t1 3 10 0 1 9 26 45 54 # 151 # 6 7 45 79 5 5 6 22 51 18 56 6 10 2 4 10 10 9 13 1 11 10 17 0 t2 33 8 82 6 3 23 16 35 15 2 2 3 118 22 46 13 7 15 31 70 23 24 127 19 15 75 1 74 11 44 # 38 28 12 t3 193 29 25 28 100 45 9 12 16 4 10 247 20 5 12 51 99 29 16 21 15 5 24 10 10 33 37 87 63 25 15 # 13 9 t4 45 4 24 15 6 19 1 9 1 2 8 5 28 78 55 73 10 15 20 76 19 12 61 8 19 9 51 143 1 66 # 27 40 5 Wipe Protocol Standard Protocol Infusion Pumps Infusion Pumps Day 1 Day 2 Day 3 Day 4 Day 5 Day 1 Day 2 Day 3 Day 4 Day 5 Bed 1 Bed 2 Bed 1 Bed 2 Bed 1 Bed 2 Bed 1 Bed 2 Bed 3 Bed 1 Bed 2 Bed 3 Bed 3 Bed 4 Bed 3 Bed 4 Bed 3 Bed 4 Bed 4 Bed 5 Bed 6 Bed 4 Bed 5 t0 99 29 2 1 2 3 6 4 0 24 17 8 1 6 0 0 0 1 18 13 23 18 0 t1 0 58 0 0 0 1 0 6 72 8 13 50 0 1 0 0 1 0 1 0 1 11 1 t2 17 0 0 2 1 * 2 0 4 0 10 1 # 11 64 1 23 1 9 11 82 34 # 26 1 t3 10 55 0 0 1 * 3 0 3 0 17 7 # 2 2 0 10 45 4 5 13 # 38 15 2 t4 47 0 1 1 0 * 2 1 1 0 7 6 # 7 75 4 19 54 83 129 29 135 1 6 6 of 9 Int. Our Study has Some Limitations Firstly, we did not supervise the housekeeping staff who performed the SOP, but this activity was conducted by the hospital executive director of the contract. We do not really know if they strictly complied with the terms of the contract, even though no penalty was applied. Housekeepers generally receive little or no training [8]; this lack of competence can lead to inadequate environmental cleaning, regardless of the products in use. Moreover, outsourced cleaning services are associated with worse patient perception of cleanliness [22]. On the other hand, in-house auxiliary nurses were trained about the proper use of wipes. Secondly, evaluating the CRAB-surface contamination, we did not observe a reduction of the CRAB-positive sites, probably due to the small size of data. Sattar et al., showed a >5 log10 reduction in A. baumannii applying wipes with similar active ingredients [23]. The effectiveness of these wipe against CRAB should be further investigated. 4. Discussion It is known that the physical structure of the wipe inﬂuences the capacity to pick up and hold soils, microbes, and particles [21], for this reason the higher proportion of removal demonstrated by the wipes protocol compared to the SOP may be due to physical effect alone of the material of which the wipes are made. 7 of 9 Int. J. Environ. Res. Public Health 2018, 15, 2305 The recovery on environmental surfaces (ﬁve isolated on the infusion pumps, four on the worktops and two on the bedrails), of CRAB strains genetically similar to clinical strains suggested the importance of high-touch sites as a source of nosocomial colonization/infection, stressing the importance of an appropriate high-touch near-patient sites C&D. 5. Conclusions Hospitals are encouraged to develop programs to optimize the thoroughness of high-touch surface cleaning in high-risk areas, especially when CRAB is epidemic or endemic as strongly recommended by WHO [9]. However is very difﬁcult to implement cleaning and disinfection strategies when there is extensive outsourcing of hospital cleaning services. Contracted-out services are considered too inﬂexible to deal with changing circumstances, including unscheduled cleaning or changes in products and protocols. The use of disposable wipes by in-house auxiliary nurses on near-patient inanimate surfaces may represent a more effective alternative to the two-step procedures performed by outsourced cleaning services in reducing the microbial contamination. Auxiliary nurses have a greater awareness of the crucial rule of cleaning and disinfection in infection prevention and they could be trained about the proper use of wipes. Few studies investigated the rate of recontamination over time on frequently touched sites after use of cleaning wipes [14] or disinfectants [15,23] and only one [24] evaluates the temporal trend speciﬁcally using wipes designed for one-step C&D as performed in our study. Author Contributions: B.C., G.P.P. and P.M. conceived and designed the experiments. A.R., S.G., L.Z., M.T., P.V., N.D.F., E.T., S.B., A.B. and A.L.C. performed the experiments and wrote the paper. P.L.L. analyzed the d Author Contributions: B.C., G.P.P. and P.M. conceived and designed the experiments. A.R., S.G., L.Z., M.T., B.C., P.V., N.D.F., E.T., S.B., A.B. and A.L.C. performed the experiments and wrote the paper. P.L.L. analyzed the data. Funding: This research received no external funding. Funding: This research received no external funding. Acknowledgments: The study was not funded. The authors wish to thank Benedetta Tuvo for technical assistance during various stages of this investigation and the ICU auxiliary nurses for their support. Acknowledgments: The study was not funded. The authors wish to thank Benedetta Tuvo for technical assistance during various stages of this investigation and the ICU auxiliary nurses for their support. Conﬂicts of Interest: The authors declare no conﬂicts of interest. Conﬂicts of Interest: The authors declare no conﬂicts of interest. References [CrossRef] [PubMed] 9. World Health Organization. Guidelines for the Prevention and Control of Carbapenem-Resistant Enterobacteriaceae, Acinetobacter Baumannii and Pseudomonas Aeruginosa in Health Care Facilities. Available online: http://www.who.int/infection-prevention/publications/guidelines-cre/en/ (accessed on 1 November 2017). 10. Vickery, K.; Deva, A.; Jacombs, A.; Allan, J.; Valente, P.; Gosbell, I.B. Presence of bioﬁlm containing viable multiresistant organisms despite terminal cleaning on clinical surfaces in an intensive care unit. J. Hosp. Infect. 2012, 80, 52–55. [CrossRef] [PubMed] 11. Ramm, L.; Siani, H.; Wesgate, R.; Maillard, J.-Y. Pathogen transfer and high variability in pathogen removal by detergent wipes. Am. J. Infect. Control 2015, 43, 724–728. [CrossRef] [PubMed] 12. Sattar, S.A.; Bradley, C.; Kibbee, R.; Wesgate, R.; Wilkinson, M.A.C.; Sharpe, T.; Maillard, J.Y. Disinfectant wipes are appropriate to control microbial bioburden from surfaces: Use of a new ASTM standard test protocol to demonstrate efﬁcacy. J. Hosp. Infect. 2015, 91, 319–325. [CrossRef] [PubMed] 13. Kenters, N.; Huijskens, E.G.W.; de Wit, S.C.J.; van Rosmalen, J.; Voss, A. Effectiveness of cleaning-disinfection wipes and sprays against multidrug-resistant outbreak strains. Am. J. Infect. Control 2017, 45, e69–e73. [CrossRef] [PubMed] 14. Bogusz, A.; Stewart, M.; Hunter, J.; Yip, B.; Reid, D.; Robertson, C.; Dancer, S.J. How quickly do hospital surfaces become contaminated after detergent cleaning? Healthc Infect. 2013, 18, 3–9. [CrossRef] 14. Bogusz, A.; Stewart, M.; Hunter, J.; Yip, B.; Reid, D.; Robertson, C.; Dancer, S.J. How quickly do hospital surfaces become contaminated after detergent cleaning? Healthc Infect. 2013, 18, 3–9. [CrossRef] 15. Attaway, H.H.; Fairey, S.; Steed, L.L.; Salgado, C.D.; Michels, H.T.; Schmidt, M.G. Intrinsic bacterial burden associated with intensive care unit hospital beds: Effects of disinfection on population recovery and 15. Attaway, H.H.; Fairey, S.; Steed, L.L.; Salgado, C.D.; Michels, H.T.; Schmidt, M.G. Intrinsic bacterial burden associated with intensive care unit hospital beds: Effects of disinfection on population recovery and mitigation of potential infection risk. Am. J. Infect. Control 2012, 40, 907–912. [CrossRef] [PubMed] 16. Italian Workers Compensation Authority, INAIL. LINEE GUIDA SUGLI STANDARD DI SICUREZZA E DI IGIENE NEL REPARTO OPERATORIO. Available online: https://www.inail.it/cs/internet/docs/linee- guida-igiene-reparto-operatorio.pdf?section=attivita (accessed on 1 December 2009). 17. Bannerman, T.L.; Hancock, G.A.; Tenover, F.C.; Miller, J.M. Pulsed-ﬁeld gel electrophoresis as a replacement for bacteriophage typing of Staphylococcus aureus. J. Clin. Microbiol. 1995, 33, 551–555. [PubMed] 18. Seifert, H.; Dolzani, L.; Bressan, R.; van der Reijden, T.; van Strijen, B.; Stefanik, D.; Heersma, H.; Dijkshoorn, L. Standardization and Interlaboratory Reproducibility Assessment of Pulsed-Field Gel Electrophoresis-Generated Fingerprints of Acinetobacter baumannii. References 1. Otter, J.A.; Yezli, S.; French, G.L. The Role Played by Contaminated Surfaces in the Transmission of Nosocomial Pathogens. Infect. Control Hosp. Epidemiol. 2011, 32, 687–699. [CrossRef] [PubMed] 2. Otter, J.A.; Yezli, S.; Salkeld, J.A.; French, G.L. Evidence that contaminated surfaces contribute to the transmission of hospital pathogens and an overview of strategies to address contaminated surfaces in hospital settings. Am. J. Infect. Control 2013, 41, S6–S11. [CrossRef] [PubMed] p g f 3. Rutala, W.A.; Weber, D.J.; HICPAC. Guideline for Disinfection and Sterilization in Healthcare Facilities, Centers for Disease Control, US; HICPAC: Atlanta, GA, USA, 2008. 4. Donskey, C.J. Does improving surface cleaning and disinfection reduce health care-associated infections? Am. J. Infect. Control 2013, 41, S12–S19. [CrossRef] [PubMed] 4. Donskey, C.J. Does improving surface cleaning and disinfection reduce health care-associated infections? Am. J. Infect. Control 2013, 41, S12–S19. [CrossRef] [PubMed] 8 of 9 Int. J. Environ. Res. Public Health 2018, 15, 2305 5. Centers for Disease Control and Prevention. Guidelines for environmental infection control in health-care facilities: Recommendations of CDC and the Healthcare Infection Control Practices Advisory Committee (HICPAC). MMWR 2003, 52, 1–48. 6. White, L.F.; Dancer, S.J.; Robertson, C.; McDonald, J. Are hygiene standards useful in assessing in risk? Am. J. Infect. Control 2008, 36, 381–384. [CrossRef] [PubMed] J f 7. Wilson, A.P.R.; Livermore, D.M.; Otter, J.A.; Warren, R.E.; Jenks, P.; Enoch, D.A.; Newsholme, W.; Oppenheim, B.; Leanord, A.; McNulty, C.; et al. Prevention and control of multi-drug-resistant Gram-negative bacteria: Recommendations from a Joint Working Party. J. Hosp. Infect. 2016, 92, S1–S44. [CrossRef] [PubMed] 8. Dancer, S.J. Controlling Hospital-Acquired Infection: Focus on the Role of the Environment and New Technologies for Decontamination. Clin. Microbiol. Rev. 2014, 27, 665–690. [CrossRef] [PubMed] 7. Wilson, A.P.R.; Livermore, D.M.; Otter, J.A.; Warren, R.E.; Jenks, P.; Enoch, D.A.; Newsholme, W.; Oppenheim, B.; Leanord, A.; McNulty, C.; et al. Prevention and control of multi-drug-resistant Gram-negative bacteria: Recommendations from a Joint Working Party. J. Hosp. Infect. 2016, 92, S1–S44. [CrossRef] [PubMed] pp , ; , ; y, ; g g bacteria: Recommendations from a Joint Working Party. J. Hosp. Infect. 2016, 92, S1–S44. [CrossRef] [PubMed] 8. Dancer, S.J. Controlling Hospital-Acquired Infection: Focus on the Role of the Environment and New Technologies for Decontamination. Clin. Microbiol. Rev. 2014, 27, 665–690. [CrossRef] [PubMed] 8. Dancer, S.J. Controlling Hospital-Acquired Infection: Focus on the Role of the Environment and New Technologies for Decontamination. Clin. Microbiol. Rev. 2014, 27, 665–690. 23. Aldeyab, M.A.; McElnay, J.C.; Elshibly, S.M.; Hughes, C.M.; McDowell, D.A.; McMahon, M.A.S.; Scott, M.G.; Kearney, M.P. Evaluation of the Efﬁcacy of a Conventional Cleaning Regimen in Removing Methicillin-Resistant Staphylococcus aureus From Contaminated Surfaces in an Intensive Care Unit. Infect. Control Hosp. Epidemiol. 2009, 30, 304–306. [CrossRef] [PubMed] © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). 24. Stewart, M.; Bogusz, A.; Hunter, J.; Devanny, I.; Yip, B.; Reid, D.; Robertson, C.; Dancer, S.J. Evaluating Use of Neutral Electrolyzed Water for Cleaning Near-Patient Surfaces. Infect. Control Hosp. Epidemiol. 2014, 35, 1505–1510. [CrossRef] [PubMed] References J. Clin. Microbiol. 2005, 43, 4328–4335. [CrossRef] [PubMed] 19. Dancer, S.J. How do we assess hospital cleaning? A proposal for microbiological standards for surface hygiene in hospitals. J. Hosp. Infect. 2004, 56, 10–15. [CrossRef] [PubMed] 20. Wiemken, T.L.; Curran, D.R.; Pacholski, E.B.; Kelley, R.R.; Abdelfattah, R.R.; Carrico, R.M.; Ramirez, J.A. The value of ready-to-use disinfectant wipes: Compliance, employee time, and costs. Am. J. Infect. Control 2014, 42, 329–330. [CrossRef] [PubMed] 21. Sattar, S.A.; Maillard, J.Y. The crucial role of wiping in decontamination of high-touch environmental surfaces: Review of current status and directions for the future. Am. J. Infect. Control 2013, 41, S97–S104. [CrossRef] [PubMed] 22. Toffolutti, V.; Reeves, A.; McKee, M.; Stuckler, D. Outsourcing cleaning services increases MRSA incidence: Evidence from 126 English acute trusts. Soc. Sci. Med. 2017, 174, 64–69. [CrossRef] [PubMed] 9 of 9 Int. J. Environ. Res. Public Health 2018, 15, 2305 23. Aldeyab, M.A.; McElnay, J.C.; Elshibly, S.M.; Hughes, C.M.; McDowell, D.A.; McMahon, M.A.S.; Scott, M.G.; Kearney, M.P. Evaluation of the Efﬁcacy of a Conventional Cleaning Regimen in Removing Methicillin-Resistant Staphylococcus aureus From Contaminated Surfaces in an Intensive Care Unit. Infect. Control Hosp. Epidemiol. 2009, 30, 304–306. [CrossRef] [PubMed] 24. Stewart, M.; Bogusz, A.; Hunter, J.; Devanny, I.; Yip, B.; Reid, D.; Robertson, C.; Dancer, S.J. Evaluating Use of Neutral Electrolyzed Water for Cleaning Near-Patient Surfaces. Infect. Control Hosp. Epidemiol. 2014, 35, 1505–1510. [CrossRef] [PubMed] © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W3098129087	https://link.springer.com/content/pdf/10.1140/epjc/s10052-020-7931-9.pdf	Latin	null	S-wave single heavy baryons with spin-3/2 at finite temperature	European physical journal. C, Particles and fields	2,020	cc-by	10,853	S-wave single heavy baryons with spin-3/2 at ﬁnite temperature The CDF collabration reported the ﬁrst observation of b and ∗ b baryons later [7]. The D0 collabration declared the observation of b [8] and it was conﬁrmed by CDF in a short time [9]. The observation of ground and excited states of c were proclaimed by Belle and BABAR collabrations [10,11]. ∗ c observed by Belle in 2008 [12] and discovery of ∗ b was reported by CMS and LHCb collaborations [13,14]. S-wave single heavy baryons with spin-3/2 at ﬁnite temperature K. Azizi1,2,a, A. Türkan3 1 Department of Physics, University of Tehran, North Karegar Avenue, Tehran 14395-547, Iran 2 Department of Physics, Doˇgu¸s University, Acıbadem-Kadıköy, 34722 Istanbul, Turkey 3 Department of Natural and Mathematical Sciences, Özyeˇgin University, Çekmeköy, 34794 Istanbul, Turkey K. Azizi1,2,a, A. Türkan3 K. Azizi1,2,a, A. Türkan3 1 Department of Physics, University of Tehran, North Karegar Avenue, Tehran 14395-547, Iran 2 Department of Physics, Doˇgu¸s University, Acıbadem-Kadıköy, 34722 Istanbul, Turkey 3 Department of Natural and Mathematical Sciences, Özyeˇgin University, Çekmeköy, 34794 Istanbul, Turkey Received: 24 October 2019 / Accepted: 15 April 2020 / Published online: 15 May 2020 © The Author(s) 2020 Abstract Thethermalbehaviorofthespectroscopicparam- eters of the S-wave single heavy baryons ∗ Q, ∗ Q and ∗ Q with spin-3/2 are investigated in QCD at ﬁnite temperature. for the heavy quark is utilized to classify the single heavy baryons [1–3]. In this case, for the two light quarks, the total ﬂavor-spin wave function has to be symmetric because their color wave function is antisymmetric. Hence there are two different representations for the S-wave heavy baryons (3 ⊗3 = 3 ⊕6): antisymmetric 3 or symmetric 6. The antitriplet (3) of baryons contain only spin-1/2 states while the sextet (6) of baryons contain both spin-1/2 and spin-3/2 states. In this study, we investigate the thermal properties of the single heavy bottom/charmed spin-3/2 sextet states: The members for charmed baryons are shown in Fig. 1. eters of the S-wave single heavy baryons Q, Q and Q with spin-3/2 are investigated in QCD at ﬁnite temperature. We analyze the variations of the mass and residue of these baryons taking into consideration the contributions of QCD thermal condensates up to dimension eight in Wilson expan- sion. At ﬁnite temperature, due to the breakdown of the Lorentz invariance by the choice of reference frame and pres- ence of an extra O(3) symmetry, some new four-dimensional operators come out in the form of the fermionic and glu- onic parts of the energy momentum tensor that are taken into account in the calculations. Our analyses show that at lower temperatures, the parameters of baryons under consid- eration are not affected by the medium. These parameters, however, show rapid variations with respect to temperature at higher temperatures near to a pseudo-critical temperature, after which the baryons are melted. Regular Article - Theoretical Physics Regular Article - Theoretical Physics a e-mail: kazem.azizi@ut.ac.ir (corresponding author) S-wave single heavy baryons with spin-3/2 at ﬁnite temperature The results of the masses and residues at T →0 limit are compatible with the avail- able experimental data and predictions of other theoretical studies. Experimentally, the 1 2 + antitriplet (+ c , + c , 0 c) states, the 1 2 + sextet (c, c, ′ c) baryons and the 3 2 + sextet (∗ c, ∗ c , ∗ c) resonances have been observed in the charmed sector while the only b, (∗) b , (∗) b and b have been dis- covered in the bottom picture [4]. Some history of discoveries are in order: In 2006 the CDF collabration reported observa- tion of b [5] and ∗ c discovered by the Babar collaboration [6]. The CDF collabration reported the ﬁrst observation of b and ∗ b baryons later [7]. The D0 collabration declared the observation of b [8] and it was conﬁrmed by CDF in a short time [9]. The observation of ground and excited states of c were proclaimed by Belle and BABAR collabrations [10,11]. ∗ c observed by Belle in 2008 [12] and discovery of ∗ b was reported by CMS and LHCb collaborations [13,14]. On the other hand, various theoretical studies in vacuum have been utilized to investigate the spectroscopic parame- ters of single heavy baryons. In 1982 Shuryak primarily cal- culated the heavy baryon masses via QCD sum rule [15]. Capstick and Isgur [16] examined the heavy baryon sys- tems in a quark potential model. Bagan et al. [17] investi- gated the heavy baryons by taking into account the separa- tion of negative and positive parity contributions. Grozin and Yakovlev [18] evaluated the masses of Q and (∗) Q using the heavy quark effective theory (HQET). Charmed baryons wereinvestigatedinChiralperturbationtheorybySavageand also results were extended for b-baryons in the same study Experimentally, the 1 2 + antitriplet (+ c , + c , 0 c) states, the 1 2 + sextet (c, c, ′ c) baryons and the 3 2 + sextet (∗ c, ∗ c , ∗ c) resonances have been observed in the charmed sector while the only b, (∗) b , (∗) b and b have been dis- covered in the bottom picture [4]. Some history of discoveries are in order: In 2006 the CDF collabration reported observa- tion of b [5] and ∗ c discovered by the Babar collaboration [6]. Eur. Phys. J. C (2020) 80:425 https://doi.org/10.1140/epjc/s10052-020-7931-9 1 Introduction The mass spectra of heavy baryons were studied by the help of the motivated relativistic quark model by Ebert [42]. Kim et al. investigated the single heavy baryon mass based on the self-consistent Chiral quark soliton model [43]. Finally, Azizi and Er studied the in-medium properties of spin-3/2 heavy baryons in nuclear matter using QCD sum rule in a dense medium [44]. Fig. 1 The sextet representation of single charmed baryons with total spin-3/2. The same picture is valid for bottom baryons with the replace- ment c →b Theoretical investigations of spectroscopic parameters of the single heavy baryons at ﬁnite temperature will help us better understand and analyze the results of heavy-ion colli- sion experiments and gain valuable information on the inter- nal structures of these baryons, behavior of these baryons near to a pseudo-critical temperature, possible phase transi- tion/ crossover [45,46] to/with quark gluon plasma (QGP) (adopted as a new phase of matter ) as well as the pertur- bative and nonperturbative dynamics of QCD. At extreme temperatures, two different possibilities can be considered: crossover and phase transition. Many Lattice calculations predict that crossover occurs at Tpc ≈155 MeV [47,48]. For the QGP phase transition, we need greater temperature values and there is no unique temperature to the phase transi- tion of QGP. At short distances, to describe the strong inter- action QCD is a suitable theory. However, the calculations of hadronicparameters includingnonperturbativeeffects (occur in low energy scale) usually need some nonperturbative phe- nomenological models. Many phenomenological models are available in the literature: QCD sum rule is one of the pow- erful ones among them. This method ﬁrstly suggested by Shifman, Vainshtein and Zakharov to investigate the vacuum properties of mesons [49,50] and then Ioffe [51] applied this method for baryons. The thermal version of the QCD sum rule was extended by Bochkarev and Shaposhnikov [52]. In addition to the vacuum expectation values of quark and gluon condensates, their thermal forms and some new operators appear in the thermal version. [19]. Roncaglia et al. [20,21] estimated the heavy baryon masses with one/two heavy quark/quarks in the framework of Feynman-Hellman theorem. Jenkins [22] studied the masses of heavy baryons in the 1/mQ and 1/Nc expansions. The 1/m corrections to heavy baryon masses were calculated by Dai et al. [23] in the framework of the HQET. 1 Introduction With the rising number of experimental data on charmed and bottom baryons, the interest in the investigation of heavy baryonshasincreased,considerably.Beforegivingthedetails oftheexperimentalstudiesonheavybaryons,itwouldbeuse- ful to give some theoretical information. The Quark Model is one of the most successful tools to classify the mesons and baryons. The traditional single heavy baryons (Qqq) consist of one heavy (Q = b or c) and two light quarks (q = u, d or s). The mass of heavy quark is very large com- pared to the light quark masses and the light degrees of free- dom form a diquark qq, which orbits the nearly static heavy Q quark. Therefore, inﬁnitely heavy mass limit (mQ →∞) On the other hand, various theoretical studies in vacuum have been utilized to investigate the spectroscopic parame- ters of single heavy baryons. In 1982 Shuryak primarily cal- culated the heavy baryon masses via QCD sum rule [15]. Capstick and Isgur [16] examined the heavy baryon sys- tems in a quark potential model. Bagan et al. [17] investi- gated the heavy baryons by taking into account the separa- tion of negative and positive parity contributions. Grozin and Yakovlev [18] evaluated the masses of Q and (∗) Q using the heavy quark effective theory (HQET). Charmed baryons wereinvestigatedinChiralperturbationtheorybySavageand also results were extended for b-baryons in the same study 12 3 3 Eur. Phys. J. C (2020) 80 :425 425 Page 2 of 12 425 Page 2 of 12 Fig. 1 The sextet representation of single charmed baryons with total spin-3/2. The same picture is valid for bottom baryons with the replace- ment c →b the NLO perturbative corrections for the static properties of heavy baryons. The mass of Q and (∗) Q baryons were cal- culated by Zhang and Huang [36] via QCD sum rule tak- ing into account operators up to dimension six. Using the coupled channel formalism, Gerasyuta and Matskevich cal- culated the S-wave bottom baryons masses [37]. Karliner et al. [38] investigated the b-baryons in the quark model. In two-point and light cone QCD sum rule methods Aliev et al. studied the mass and magnetic moments of single heavy baryons with spin-3/2 [39]. Lewis and Shyn [40] predicted the bottom baryon masses based on a 2 + 1 ﬂavor dynamical lattice QCD simulation. The spin-3/2+ heavy and doubly heavy baryon states [41] were investigated by subtracting the contributions from the corresponding negative parity by Wang. 2 Calculations s uμ(q, s)¯uν(q, s) = − ̸q + m BSH gμν −1 3γμγν −2 qμqν 3m2 BSH + qμγν −qνγμ 3m BSH . (4) uμ(q, s)¯uν(q, s) In this section, QCD sum rules for the spectroscopic param- eters of the spin-3/2 ∗ Q, ∗ Q and ∗ Q baryons are obtained at ﬁnite temperature. To this end, we start with the following two-point thermal correlation function: = − ̸q + m BSH gμν −1 3γμγν −2 qμqν 3m2 BSH + qμγν −qνγμ 3m BSH . (4) −2 qμqν 3m2 BSH + qμγν −qνγμ 3m BSH . (4) (4) μν(q, T ) = i d4x eiq·x⟨ \|T {Jμ(x) ¯Jν(0)}\| ⟩, (1) (1) By using the above behest, we recast the physical side as Phys μν (q, T ) = λ2 BSH (T )(̸q + m BSH ) q2 −m2 BSH gμν −1 3γμγν −2 qμqν 3m2 BSH + qμγν −qνγμ 3m BSH + · · · , (5) where q is the four-momentum of the chosen baryon, is the ground state of the hot medium, T denotes the time-ordering operator and Jμ(x) is the interpolating current of the single heavy baryon, BSH. (5) As the standard procedures of the QCD sum rule, the cor- relation function given above can be calculated at different contexts. At large distances, it is evaluated in terms of the hadronic parameters such as the mass and residue of hadron. As the standard procedures of the QCD sum rule, the cor- relation function given above can be calculated at different contexts. At large distances, it is evaluated in terms of the hadronic parameters such as the mass and residue of hadron. We call it the physical or hadronic representation of the cor- relator. The same correlator can be expressed in terms of the quark, gluon and mixed condensates by the help of the OPE at q2 << 0 region. The computations in this way contain short distance effects. This representation, is generally called the OPE or QCD side of the correlation function. Finally, we match the two windows and compare the coefﬁcients of the same Lorentz structures from both sides. To remove the unwanted contributions coming from the higher states and continuum, Borel transformation as well as continuum sub- traction, supplied by the quark-hadron duality assumption at ﬁnite temperature, are performed. 2 Calculations These procedures bring some auxiliary parameters, which we ﬁx them before making any numerical estimations on the physical quantities. where λ2 BSH (T ) = λBSH (T )¯λBSH (T ). It should also be spec- iﬁed that the interpolating current Jμ(x) couples to both the spin-1/2 and spin-3/2 states. In this study, we only consider the contribution of spin-3/2 heavy baryons and we need to comb out the pollution of spin-1/2 state. These unwanted contributions can be eliminated in two different ways: (1) For spin-3/2 state, it should be introduced a projection operator which destroys the spin-1/2 contributions, (2) By a speciﬁc ordering of the Dirac matrices and remove the terms corre- sponding to the spin-1/2 particles (for more details see for instance [53]). The contribution of the spin-1/2 states can be traced using where λ2 BSH (T ) = λBSH (T )¯λBSH (T ). It should also be spec- iﬁed that the interpolating current Jμ(x) couples to both the spin-1/2 and spin-3/2 states. In this study, we only consider the contribution of spin-3/2 heavy baryons and we need to comb out the pollution of spin-1/2 state. These unwanted contributions can be eliminated in two different ways: (1) For spin-3/2 state, it should be introduced a projection operator which destroys the spin-1/2 contributions, (2) By a speciﬁc ordering of the Dirac matrices and remove the terms corre- sponding to the spin-1/2 particles (for more details see for instance [53]). The contribution of the spin-1/2 states can be traced using ⟨ \|Jμ(0)\|1 2(q)⟩= κ1qμ + κ2γμ u(q), (6) (6) where κ1 and κ2 are some constants. By applying the condi- tion Jμγ μ = 0 (for more details see [54]), we get κ1 in terms of κ2. Hence, To obtain the physical side of the correlator, a complete set of intermediate state with the same quantum numbers and quark content as the chosen current is inserted between the interpolating currents in correlation function. This is fol- lowed by the integral over four-x, which leads to ⟨ \|Jμ(0)\|1 2(q)⟩= κ2 γμ −4 m 1 2 qμ u(q). (7) (7) As is seen from Eq. (7), the pollution coming from spin-1/2 resonances are commensurate to either qμ or γμ. 1 Introduction where m BSH (T ) is the temperature-dependent mass of the ground state of BSH. The matrix element ⟨ \|Jμ(0)\| BSH(q, s)⟩is deﬁned in terms of the temperature dependent residue, λBSH (T ), as operators coming from OPE due to breaking of the Lorentz invariance by the choice of the thermal rest frame, conden- sates up to dimension eight are considered. The article is arranged in the following form. In Sect. 2, the in-medium sum rules for the mass and residues of the ∗ Q, ∗ Q and ∗ Q single heavy baryons are obtained. In Sect. 3 the numerical analysis for the spectroscopic parameters under considera- tion is performed. The last section includes the summary and our concluding remarks. ⟨ \|Jμ(0)\|BSH(q, s)⟩= λBSH (T )uμ(q, s), (3) (3) where uμ(q, s) is the Rarita–Schwinger spinor. The ﬁnal form of the physical side can be obtained by inserting Eq. (3) into Eq. (2) and summing over the spins of the BSH. The summation over Rarita–Schwinger spinors is performed using where uμ(q, s) is the Rarita–Schwinger spinor. The ﬁnal form of the physical side can be obtained by inserting Eq. (3) into Eq. (2) and summing over the spins of the BSH. The summation over Rarita–Schwinger spinors is performed using 1 Introduction QCD sum rule for heavy baryons at leading order in 1/mQ and at next to the leading order in αs were evaluated by Groote et al. [24]. Wang et al. [25] improved the analysis for the Q and Q baryon masses to order QC D/mQ from QCD sum rule. Mathur et al. [26] predicted the mass spectrum of charmed and bot- tom baryons from Lattice QCD. Wang and Huang [27] stud- ied the mass, coupling constant, and Isgur-Wise function for ground-state heavy baryons within the framework of HQET by taking into account both the two and three-point correla- tion functions. Ebert et al. computed heavy baryon masses in the heavy-quark light-diquark approximation in the frame- work of constituent quark model [28]. Garcilazo et al. [29] solved exactly the three quark problem via Faddeev method in momentum space. Zang and Huang [30] calculated the charm and bottom baryon masses up to operator dimension six in operator product expansion (OPE) by the help of the QCD sum rule approach. The mass and residue of ∗ c and ∗ b with spin parity 3/2+ were studied by Wang via QCD sum rule [31]. A quark model was applied to the spectrum of baryons containing one heavy baryon by Roberts and Pervin [32]. Bottom baryon spectra were investigated using Faddeev method in momentum space by Valcarce et al. [33]. Liu et al. [34] performed a systematic study of the masses of bottom baryons up to 1/mQ in HQET. Groote et al. [35] computed In this study, we investigate the temperature effects on the spectroscopic parameters of the ground state sextet baryons including single heavy quark and with spin-3/2 via thermal QCD sum rule method. Taking into account the additional 123 Eur. Phys. J. C (2020) 80 :425 Page 3 of 12 425 12 425 425 operators coming from OPE due to breaking of the Lorentz invariance by the choice of the thermal rest frame, conden- sates up to dimension eight are considered. The article is arranged in the following form. In Sect. 2, the in-medium sum rules for the mass and residues of the ∗ Q, ∗ Q and ∗ Q single heavy baryons are obtained. In Sect. 3 the numerical analysis for the spectroscopic parameters under considera- tion is performed. The last section includes the summary and our concluding remarks. Phys μν (q, T ) 2 Calculations To go further in the calculations, the thermal light quark propagator in coordinate space is selected as (see also [58,59]) where M2 is the Borel parameter and dots denote the con- tributions of other structures as well as the higher states and continuum. Si j q (x) = i ̸ x 2π2x4 δi j − mq 4π2x2 δi j −⟨¯qq⟩T 12 δi j −x2 192m2 0⟨¯qq⟩T 1 −i mq 6 ̸ x δi j + i 3 ̸ x mq 16 ⟨¯qq⟩T −1 12⟨uμ f μνuν⟩ +1 3 u · x ̸u⟨uμ f μνuν⟩ δi j −igsλi j A 32π2x2 G A μν ̸ xσ μν + σ μν ̸ x −i x2 ̸ xg2 s ⟨¯qq⟩2 T 7776 δi j −x4⟨¯qq⟩T ⟨g2 s G2⟩T 27648 + · · · , (12) The next step is to calculate the OPE side of the correlation function. In deep Euclidean region, the correlation function is evaluated in terms of the quark and gluon degrees of free- dom by the help of Wilson expansion. To achieve this goal, the basic point it to choose a suitable interpolating current deﬁning the particles under study. The interpolating current for spin-3/2 BSH in a compact form can be written as [55–57] Jμ(x) = A ϵabc qaT 1 (x)Cγμqb 2(x) Qc(x) + qaT 2 (x)CγμQb(x) qc 1(x) + QaT (x)Cγμqb 1(x) qc 2(x) , (9) (9) (12) where A is the normalization constant, ϵabc is the anti- symmetric Levi–Civita tensor, a, b, c are color indices, q1(2) denotes the light quark (u, d or s), Q is the bottom (b) or charm (c) quark and C is the charge conjugation operator. The normalization constant A and the q1(2) quark for the considered baryons are given in Table 1. which includes the thermal quark and gluon condensates (⟨¯qq⟩T and ⟨g2 s G2⟩T ), gluon ﬁelds in thermal bath, mixed condensate (m2 0⟨¯qq⟩T = ⟨¯qgsσGq⟩) as well as new opera- tors containing the energy momentum tensor, μν. 2 Calculations To remove these contributions, the Dirac matrices are ordered as γμ ̸qγν and terms proportional to qμ or qν, also those beginning with γμ orendingwithγν aresettozero.Finally,thecleanphysical side of the correlator, in the Borel scheme, is obtained as Phys μν (q, T ) = −⟨ \|Jμ(0)\|BSH(q, s)⟩⟨BSH(q, s)\|J † ν (0)\| ⟩ q2 −m2 BSH (T ) (2) + contribution of higher states and continuum, 123 3 425 Page 4 of 12 Eur. Phys. J. C (2020) 80 :425 Table 1 The light quark ﬂavors for the single heavy baryons with spin- 3/2 and the value of normalization constant A ∗+(++) b(c) ∗0(+) b(c) ∗−(0) b(c) ∗0(+) b(c) ∗−(0) b(c) ∗−(0) b(c) A √1/3 √2/3 √1/3 √2/3 √2/3 √1/3 q1 u u d u d s q2 u d d s s s ˆBPhys μν (q, T ) = λ2 BSH (T )e−m2 BSH (T )/M2 ̸qgμν +λ2 BSH (T )m BSH e−m2 BSH (T )/M2 gμν + · · · , (8) Some extra contractions arise because of the identical par- ticles in the case of q1 = q2 = q, and the correlator is obtained as Table 1 The light quark ﬂavors for the single heavy baryons with spin- 3/2 and the value of normalization constant A Table 1 The light quark ﬂavors for the single heavy baryons with spin- 3/2 and the value of normalization constant A ∗+(++) b(c) ∗0(+) b(c) ∗−(0) b(c) ∗0(+) b(c) ∗−(0) b(c) ∗−(0) b(c) A √1/3 √2/3 √1/3 √2/3 √2/3 √1/3 q1 u u d u d s q2 u d d s s s O P E μν (q, T ) = i 3ϵabcϵa′b′c′ d4xeiq·x 2Scc′ Q Tr[Sbb′ q γνSaa′ q γμ] +2Scc′ q Tr[Sbb′ Q γνSaa′ q γμ] +2Scc′ q Tr[Sbb′ q γνSaa′ Q γμ] +4Sca′ Q γνSab′ q γμSbc′ q + 4Sca′ q γνSab′ q γμSbc′ Q +4Sca′ q γνSab′ Q γμSbc′ q } , (11 ˆBPhys μν (q, T ) = λ2 BSH (T )e−m2 BSH (T )/M2 ̸qgμν +λ2 BSH (T )m BSH e−m2 BSH (T )/M2 gμν + · · · , (8) (11) (8) where Si j q(Q) = CSi jT q(Q)C. 2 Calculations The fermionic part f μν appears explicitly in the light-quark propagator, while the gluonic part of the energy-momentum tensor g λσ appears in the expansion of the trace of two-gluon ﬁeld strength tensor in heat bath [62]: O P E 1(2) = ∞ smin ds ρO P E 1(2) (s, T ) s −q2 + nonpert 1(2) , (21) (21) where smin = (mq1+mq2+mQ)2, ρO P E 1(2) (s, T ) is the spectral density obtained via the imaginary part of the perturbative correlation function (pert in the following equation stands for the perturbative contributions) ρO P E 1(2) (s, T ) = 1 π Im[O P E,pert 1(2) ], (22) (22) and nonpert 1(2) represents the contributions coming from all the nonperturbative effects. In this step, our main aim is to calcu- late the spectral densities, corresponding to the perturbative effects in the present study, as well as the nonperturbative contributions to the QCD side. To this end, the explicit forms of the heavy and light quark propagators are inserted into Eqs. (10) and (11). The next step is to perform the standard but lengthy calculations: These calculations contain Fourier integrals appearing in different forms, Borel transformation as well as continuum subtraction. By matching the coefﬁ- cients of the selected structures from both the physical and OPE sides of the correlation function, we ﬁnd the desired sum rules: and nonpert 1(2) represents the contributions coming from all the nonperturbative effects. In this step, our main aim is to calcu- late the spectral densities, corresponding to the perturbative effects in the present study, as well as the nonperturbative contributions to the QCD side. To this end, the explicit forms of the heavy and light quark propagators are inserted into Eqs. (10) and (11). The next step is to perform the standard but lengthy calculations: These calculations contain Fourier integrals appearing in different forms, Borel transformation as well as continuum subtraction. By matching the coefﬁ- cients of the selected structures from both the physical and OPE sides of the correlation function, we ﬁnd the desired sum rules: ⟨TrcGαβGμν⟩= 1 24(gαμgβν −gανgβμ)⟨G2⟩T +1 6 gαμgβν −gανgβμ −2(uαuμgβν −uαuνgβμ −uβuμgαν + uβuνgαμ) ×⟨uλg λσuσ⟩. 2 Calculations MS Alike to the physical part, the correlation function on the OPE side is expanded in terms of the Lorentz structures as Alike to the physical part, the correlation function on the OPE side is expanded in terms of the Lorentz structures as ⟨¯qq⟩T ⟨0\|¯qq\|0⟩= (A1e T 0.025[GeV ] + 1.015), (14) O P E μν (q, T ) = O P E 1 ̸qgμν + O P E 2 gμν +other structures, (20) (14) (20) and where O P E 1(2) is the coefﬁcient of the selected Lorentz struc- ture. These functions can be expressed by the help of follow- ing dispersion integral: ⟨¯ss⟩T ⟨0\|¯ss\|0⟩= (A2e T 0.019[GeV ] + 1.002), (15) (15) where A1 = −6.534 × 10−4 and A2 = −2.169 × 10−5. As we previously mentioned, because of the choice of the thermal rest frame in Wilson expansion, the Lorentz invari- ance is broken. To restore that the four-velocity vector of the medium uμ = (1, 0, 0, 0) is introduced, which implies u2 = 1 and q · u = q0. In the rest frame of heat bath, ⟨uμ f,g μν uν⟩= ⟨u f,gu⟩= ⟨ f,g 00 ⟩= ⟨ f,g⟩, as well. In thermal version, as also mentioned above, new operators representing the fermionic and gluonic parts of the energy- momentum tensor arises in OPE. The fermionic part f μν appears explicitly in the light-quark propagator, while the gluonic part of the energy-momentum tensor g λσ appears in the expansion of the trace of two-gluon ﬁeld strength tensor in heat bath [62]: where A1 = −6.534 × 10−4 and A2 = −2.169 × 10−5. As we previously mentioned, because of the choice of the thermal rest frame in Wilson expansion, the Lorentz invari- ance is broken. To restore that the four-velocity vector of the medium uμ = (1, 0, 0, 0) is introduced, which implies u2 = 1 and q · u = q0. In the rest frame of heat bath, ⟨uμ f,g μν uν⟩= ⟨u f,gu⟩= ⟨ f,g 00 ⟩= ⟨ f,g⟩, as well. In thermal version, as also mentioned above, new operators representing the fermionic and gluonic parts of the energy- momentum tensor arises in OPE. 2 Calculations For the heavy quark, the following propagator including the thermal gluon condensate and gluon ﬁelds in hot medium is used [60]: By inserting the explicit form of the interpolating current into the correlator and contracting all heavy and light quark ﬁelds via Wick’s theorem, we get the corelation function in the case of q1 ̸= q2 in terms of the thermal light(heavy) quark propagators, Sq(Q), as Si j Q(x) = i d4ke−ik·x (2π)4 ̸k + mQ k2 −m2 Q δi j − gsGαβ i j 4 σ αβ(̸k + mQ) + (̸k + mQ)σ αβ (k2 −m2 Q)2 +mQ 12 k2 + mQ ̸k (k2 −m2 Q)4 ⟨g2 s G2⟩T δi j + · · · . (13) Si j Q(x) = i d4ke−ik·x (2π)4 ̸k + mQ k2 −m2 Q δi j − gsGαβ i j 4 σ αβ(̸k + mQ) + (̸k + mQ)σ αβ (k2 −m2 Q)2 O P E μν (q, T ) = −2i 3 ϵabcϵa′b′c′ d4xeiq·x Scc′ Q Tr[Sba′ q2 γνSab′ q1 γμ] +Scc′ q1 Tr[Sba′ Q γνSab′ q2 γμ] +Scc′ q2 Tr[Sba′ q1 γνSab′ Q γμ] + Sca′ Q γνSbb′ q2 γμSac′ q1 +Scb′ Q γνSaa′ q1 γμSbc′ q2 +Scb′ q1 γνSaa′ q2 γμSbc′ Q + Sca′ q1 γνSbb′ Q γμSac′ q2 +Sca′ q2 γνSbb′ q1 γμSac′ Q + Scb′ q2 γνSaa′ Q γμSbc′ q1 } . (10 +mQ 12 k2 + mQ ̸k (k2 −m2 Q)4 ⟨g2 s G2⟩T δi j + · · · . (13) (13) In Eqs. (12) and (13), mq(Q) denotes the light(heavy) quark mass. The thermal quark condensates, ⟨¯qq⟩T (for q = u, d) and ⟨¯ss⟩T are parameterized in terms of the vacuum condensates, ⟨0\|¯qq\|0⟩and⟨0\|¯ss\|0⟩.Forthesequantities,weusethefollow- ingparametrizationsintermsoftemperature,whicharebased (10) 12 3 Eur. Phys. J. C (2020) 80 :425 Page 5 of 12 425 Page 5 of 12 425 g−2 s (T ) = 11 8π2 ln 2πT MS + 51 88π2 ln 2 ln 2πT MS , (19) on the lattice QCD predictions [61]. Note that in this study the temperature dependence of these quantities are given up to a temperature T = 300 MeV. However, we parameterize them up to Tpc ≈155 MeV, which is considered as the pseudo- critical temperature for the crossover phase transition at zero chemical potential. We get, (19) where, MS ≃Tpc/1.14. where, MS ≃Tpc/1.14. 2 Calculations where stands for the unit-step function, L(s, z) = s z(1 − z) −m2 b z and β = z −1. 2 Calculations They are obtained as β ⟨ ⟩⟨ ⟩ × 2m2 b + β 5M2 + 8q2 0 [L(s0, z)] ρO P E 1 (s, T ) = −1 96π4β 1 0 dz z m2 b + sβ × z 3m2 b(z + 1) −12mbmu + sβ(7z + 3) −12md(mbz −2muβ) [L(s, z)], (26) +e− m2 b M2 π2 m2 0 72M2 ⟨¯dd⟩[2mbmdmu + M2(md −6mu)] +⟨¯uu⟩[2mbmdmu + M2(mu −6md)] +e− m2 b M2 π2 m2 0 72M2 ⟨¯dd⟩[2mbmdmu + M2(md −6mu)] +⟨¯uu⟩[2mbmdmu + M2(mu −6md)] 1 +e− m2 b M2 π2 m2 0 72M2 ⟨¯dd⟩[2mbmdmu + M2(md −6mu)] × z 3m2 b(z + 1) −12mbmu + sβ(7z + 3) −12md(mbz −2muβ) [L(s, z)], (26 and ˆBnonpert 1 = −1 1152π4 s0(T ) smin ds 1 0 dz × −96π2 ⟨¯dd⟩ 2z(−2mb + md + 2mu) −3mdz2 +md −4mu + ⟨¯uu⟩ −4mbz + 4βmd + mu(2 −3z)z + mu +g2 s ⟨G2⟩ (43 −6z)z + 2 +2⟨ugu⟩[z(21z + 23) + 15] +256π2β⟨u f u⟩(5z −1) [L(s, z)] + 1 0 dze m2 b M2β g2 s −1 1152π4M2β2 m2 bz⟨G2⟩ × z(2mb(md + mu) + M2) −4mdmu + 1 13824π2M6β3 ⟨¯dd⟩ ⟨G2⟩ −16m4 bmdz +8m3 bβ(mdmu + 2M2z) −4m2 bM2β 5md(3z + 1) −4mu + 8mbM2β 2mdmu(3z −2) + M2(z(10z −3) −3) +119md M4β3 + 8M2β2⟨ugu⟩ 6m2 bmd −12mbM2 + mdβ 31M2 −8q2 0 + ⟨¯uu⟩ ⟨G2⟩ −16m4 bmuz +8m3 bβ(mdmu + 2M2z) + 4m2 bM2β 4md −5(3muz + mu) + 8mbM2β 2mdmu(3z −2) + M2(z(10z −3) −3) +119mu M4β3 + 8M2β2⟨ugu⟩ 6m2 bmu −12mbM2 +muβ 31M2 −8q2 0 + 1 663552π4M6β3 × m2 b⟨G2⟩2g2 s 32m2 bz + M2β(187z + 16) +4⟨G2⟩ 64π2⟨u f u⟩ 8m4 bz −2m3 bβ(md + mu) −2m2 bβ(M2(z −5) −32q2 0z) −4mbM2β(3z −2)(md + mu) −55M4β3 −m2 bg2 s ⟨ugu⟩ 16m2 bz + M2β(85z −16) 123 +⟨¯uu⟩[2mbmdmu + M2(mu −6md)] − 1 972M4 ⟨¯uu⟩ 27π2⟨¯dd⟩ 3m2 bmdmu −8mbM2(md + mu) +2M2(3mdmu + 8M2) − 1 972M4 ⟨¯uu⟩ 27π2⟨¯dd⟩ 3m2 bmdmu −8mbM2(md + mu) +2M2(3mdmu + 8M2) and ˆBnonpert 1 +4M2g2 s ⟨¯uu⟩ mb(md + mu) + M2 +4M2g2 s ⟨¯uu⟩ mb(md + mu) + M2 +4M2g2 s ⟨¯uu⟩ mb(md + mu) + M2 + 1 6912M2 ⟨¯dd⟩ 13md M2⟨G2⟩g2 s + 52md M2g2 s ⟨ugu⟩ +512π2⟨u f u⟩ 3m2 bmd −4mbM2 −4md M2 + 2q2 0 +⟨¯uu⟩ ⟨G2⟩g2 s 16m2 b(md + mu) + M2(32mb + 16md + 35mu) + 76mu M2g2 s ⟨ugu⟩ +512π2⟨u f u⟩ 3m2 bmu −4mbM2 −12mu M2 + 2q2 0 + 1 162M8 3π2m2 0⟨¯dd⟩⟨¯uu⟩ 3m4 bmdmu −5m3 bM2(md + mu) +4M2g2 s ⟨¯uu⟩ mb(md + mu) + M2 + 1 6912M2 ⟨¯dd⟩ 13md M2⟨G2⟩g2 s + 52md M2g2 s ⟨ugu⟩ +512π2⟨u f u⟩ 3m2 bmd −4mbM2 −4md M2 + 2q2 0 +⟨¯uu⟩ ⟨G2⟩g2 s 16m2 b(md + mu) + M2(32mb + 16md + 35mu) + 76mu M2g2 s ⟨ugu⟩ +512π2⟨u f u⟩ 3m2 bmu −4mbM2 −12mu M2 + 2q2 0 + 1 162M8 3π2m2 0⟨¯dd⟩⟨¯uu⟩ 3m4 bmdmu −5m3 bM2(md + mu) + 1 6912M2 ⟨¯dd⟩ 13md M2⟨G2⟩g2 s + 52md M2g2 s ⟨ugu⟩ +512π2⟨u f u⟩ 3m2 bmd −4mbM2 −4md M2 + 2q2 0 +⟨¯uu⟩ ⟨G2⟩g2 s 16m2 b(md + mu) + M2(32mb + 16md + 35mu) + 76mu M2g2 s ⟨ugu⟩ +512π2⟨u f u⟩ 3m2 bmu −4mbM2 −12mu M2 + 2q2 0 + 1 162M8 3π2m2 0⟨¯dd⟩⟨¯uu⟩ 3m4 bmdmu −5m3 bM2(md + mu) +3m2 bM2(4M2 −mdmu) +3M4(4M2 −mdmu) −M4⟨u f u⟩ × M2⟨G2⟩g2 s + 4M2g2 s ⟨ugu⟩ +3m2 bM2(4M2 −mdmu) +3M4(4M2 −mdmu) −M4⟨u f u⟩ × M2⟨G2⟩g2 s + 4M2g2 s ⟨ugu⟩ +16π2⟨u f u⟩ 3m2 b + 8M2 + 16q2 0 , (27) (27) where stands for the unit-step function, L(s, z) = s z(1 − z) −m2 b z and β = z −1. 2 Calculations (16) (16) The temperature dependent gluon condensate ⟨G2⟩T is parameterized in terms of the vacuum gluon condensate ⟨0\|G2\|0⟩[61] as: λ2 BSH (T )e−m2 BSH (T )/M2 = ˆBO P E 1 , (23) (23) δ⟨αs π G2⟩T = −8 9[δT μ μ (T ) −muδ⟨¯uu⟩T −mdδ⟨¯dd⟩T −msδ⟨¯ss⟩T ], (17) and λ2 BSH (T )m BSH (T )e−m2 BSH (T )/M2 = ˆBO P E 2 , (24) (24) (17) where the functions ˆBO P E 1(2) denote the O P E 1(2) in Borel scheme and are given as wherethevacuumsubtractedvaluesoftheconsiderquantities are used as δf (T ) ≡f (T ) −f (0) and δT μ μ (T ) = ε(T ) − 3p(T ): ε(T ) is the energy density and p(T ) is the pressure. Taking into account the recent Lattice calculations [63,64] we get the ﬁt function of δT μ μ (T ) as ˆBO P E 1(2) = s0(T ) smin dsρO P E 1(2) (s, T )e−s/M2 + ˆBnonpert 1(2) , (2 (25) δT μ μ (T ) T 4 = (0.020 × e T 0.034[GeV ] + 0.115). (18) with s0(T ) being the temperature-dependent continuum threshold. We will use the above sum rules to extract the values of the mass and residue of the baryons under consid- eation as well as their thermal behavior in next section. (18) For the temperature-dependent strong coupling [65,66] we utilize 123 3 Eur. Phys. J. C (2020) 80 :425 425 Page 6 of 12 As examples, we would like to present the explicit forms of the ρO P E 1 (s, T ) and ˆBnonpert 1 for the ∗ b baryon. 2 Calculations They are obtained as ρO P E 1 (s, T ) = −1 96π4β 1 0 dz z m2 b + sβ × z 3m2 b(z + 1) −12mbmu + sβ(7z + 3) −12md(mbz −2muβ) [L(s, z)], (26 and ˆBnonpert 1 = −1 1152π4 s0(T ) smin ds 1 0 dz × −96π2 ⟨¯dd⟩ 2z(−2mb + md + 2mu) −3mdz2 +md −4mu + ⟨¯uu⟩ −4mbz + 4βmd + mu(2 −3z)z + mu +g2 s ⟨G2⟩ (43 −6z)z + 2 +2⟨ugu⟩[z(21z + 23) + 15] +256π2β⟨u f u⟩(5z −1) [L(s, z)] + 1 0 dze m2 b M2β g2 s −1 1152π4M2β2 m2 bz⟨G2⟩ × z(2mb(md + mu) + M2) −4mdmu + 1 13824π2M6β3 ⟨¯dd⟩ ⟨G2⟩ −16m4 bmdz +8m3 bβ(mdmu + 2M2z) −4m2 bM2β 5md(3z + 1) −4mu + 8mbM2β 2mdmu(3z −2) + M2(z(10z −3) −3) +119md M4β3 + 8M2β2⟨ugu⟩ 6m2 bmd −12mbM2 + mdβ 31M2 −8q2 0 + ⟨¯uu⟩ ⟨G2⟩ −16m4 bmuz +8m3 bβ(mdmu + 2M2z) + 4m2 bM2β 4md −5(3muz + mu) + 8mbM2β 2mdmu(3z −2) + M2(z(10z −3) −3) +119mu M4β3 + 8M2β2⟨ugu⟩ 6m2 bmu −12mbM2 +muβ 31M2 −8q2 0 + 1 663552π4M6β3 × m2 b⟨G2⟩2g2 s 32m2 bz + M2β(187z + 16) +4⟨G2⟩ 64π2⟨u f u⟩ 8m4 bz −2m3 bβ(md + mu) −2m2 bβ(M2(z −5) −32q2 0z) −4mbM2β(3z −2)(md + mu) −55M4β3 −m2 bg2 s ⟨ugu⟩ 16m2 bz + M2β(85z −16) 1 −3072π2M2β2⟨u f u⟩⟨ugu⟩ × 2m2 b + β 5M2 + 8q2 0 [L(s0, z)] As examples, we would like to present the explicit forms of the ρO P E 1 (s, T ) and ˆBnonpert 1 for the ∗ b baryon. 3 Numerical results The main contribution in nonperturbative part belongs to the quark condensate, ⟨qq⟩. (29) which, we are going to use them in our numerical compu- tations. The next problem is to obtain the parametrization of s0(T ) as a function of temperature. This function shall reduce to the vacuum threshold, s0, at zero temperature. We parameterize it as s0(T ) = s0 f (T ), (30) (30) s0(T ) = s0 f (T ), such that at T →0 limit, f (T ) →1. Hence, we should ﬁrst determine s0 based on the standard prescriptions of the method, afterwards we will extract the function f (T ) from the calculations. Besides the continuum threshold in vacuum the sum rules obtained in previous section include another auxiliary param- eter, Borel parameter M2, which should also be ﬁxed. We need to determine the working regions of s0 and M2 such that the physical quantities under consideration show mild dependence on these parameters. The continuum threshold s0 is not totally free but it is related to the energy of the ﬁrst excited state in the same channel. Thanks to the exper- iments that have provided many new results not only on the ground states but also on the excited states of some single heavy baryons, recently [70]. In view of PDG, we see that the excited states generally have energies about 300 MeV above the ground states masses. In choosing the working window for the s0, we also look after the pole dominance and OPE convergence in our sum rules. These considerations leads to the window: To obtain M2 max, we utilize the condition of the pole dom- inance as PC = O P E 1(2) (M2, s0) O P E 1(2) (M2, ∞) ≥1 2. (32) (32) As a result, we get the working region of the Borel param- eter as M2 ∈[6, 10] GeV 2. We plot, as an example, a 3D graphic of the mass of ∗ b baryon as functions of M2 and s0 at T = 0 in Fig. 3. As is seen the mass shows good stabil- ity against the variations of the auxiliary parameters in the selected windows. Now, we proceed to ﬁnd the function f (T ) and the tem- perature dependent mass m BSH (T ) and residue λBSH (T ) of the single heavy spin-3/2 baryons. 3 Numerical results In this section, we analyze the obtained sum rules for the masses and residues. They includes some input parameters suchastheheavyandlightquarkmasses,m2 0,quarkandgluon condensates in vacuum and energy of the quasi-particle in medium, q0. Their numerical values are presented in Table 2. In addition, we also need to have the gluonic and fermionic parts of the energy density. Based on the lattice QCD results on the thermal behavior of the energy-momentum tensor given in [63], their parametrizations, up to the pseudo-critical point under consideration in the present study, are obtained as ⟨ f ⟩ T 4 = (0.009 × e T 0.0402[GeV ] + 0.024), (28) ⟨ f ⟩ T 4 = (0.009 × e T 0.0402[GeV ] + 0.024), ⟨ f ⟩ T 4 = (0.009 × e T 0.0402[GeV ] + 0.024), (28) 12 3 Page 7 of 12 425 Eur. Phys. J. C (2020) 80 :425 Table 2 Input parameters used in calculations [61,67–70] Parameter Numeric value q ∗ b 0 ; q∗c 0 (5832.1 ± 1.9) MeV; (2518.48 ± 0.20) MeV q ∗ b 0 ; q∗ c 0 (5949 ± 1.9) MeV; (2646.32 ± 0.31) MeV q ∗ b 0 ; q∗ c 0 (6.08 ± 0.40) GeV; (2765.9 ± 2.0) MeV mu ; md (2.2+0.5 −0.4) MeV; (4.7+0.7 −0.3) MeV ms (95+9 −3) MeV mb ; mc (4.18+0.04 −0.03) GeV; (1.275+0.025 −0.035) GeV m2 0; (0.8 ± 0.2) GeV2 ⟨0\|qq\|0⟩(q = u, d) −(272(5) MeV)3 ⟨0\|ss\|0⟩ −(296(11) MeV)3 ⟨0 \| 1 π αsG2 \| 0⟩ 0.028(3) GeV4 Table 2 Input parameters used in calculations [61,67–70] ⟨g⟩ T 4 = (0.091 × e T 0.047[GeV ] −0.731), (29) ⟨g⟩ higher the dimension of the nonperturbative operator the lower its contribution is satisﬁed. Our calculations show that the operators having eight dimensions, the higher dimen- sion that we include into the analyses, constitute only one percent of the total contribution at lower value of M2, i.e. 8,O P E 1(2) (M2 min, s0)/total,O P E 1(2) (M2 min, s0) ≃0.01. Figure 2 shows the perturbative and nonperturbative contributions to total OPE as well as the contributions of different nonpertur- bative operators with various mass dimensions, separately. This ﬁgure depicts a nice convergence of the OPE in our cal- culations. As it is clear, the perturbative contribution domi- nates over nonperturbative contributions and it is about 53% of the total at M2 min = 6 GeV 2. 425 Page 8 of 12 Fig. 2 Up: Contributions of perturbative and nonperturbative parts to total OPE. Down: Contributions of various operators with different dimensions to nonperturbative part: ⟨¯qq⟩(dimension 3), ⟨G2⟩+ ⟨u f (g)u⟩(dimension 4), ⟨qGq⟩(dimension 5) , ⟨qq⟩2 (dimension 6), ⟨qq⟩⟨G2⟩+ ⟨qq⟩⟨u f (g)u⟩ (dimension 7), ⟨G2⟩2 + ⟨u f (g)u⟩2(dimension 8) Fig. 2 Up: Contributions of perturbative and nonperturbative parts to total OPE. Down: Contributions of various operators with different dimensions to nonperturbative part: ⟨¯qq⟩(dimension 3), ⟨G2⟩+ ⟨u f (g)u⟩(dimension 4), ⟨qGq⟩(dimension 5) , ⟨qq⟩2 (dimension 6), ⟨qq⟩⟨G2⟩+ ⟨qq⟩⟨u f (g)u⟩ (dimension 7), ⟨G2⟩2 + ⟨u f (g)u⟩2(dimension 8) Fig. 3 The mass of the ∗ b baryon as functions of M2 and s0 at T = 0 Fig. 3 The mass of the ∗ b baryon as functions of M2 and s0 at T = 0 perature but after that, they start to decrease with increas- ing the temperature. Our analyses show that the charmed baryons present similar behavior, as well. The points that the stability starts to break down for mass and residue are T ∼= 0.14 GeV and T ∼= 0.13 GeV, respectively. After these points the mass and residue starts to diminish. The mass and residue fall substantially near to the pseudo-critical temper- ature. The amount of decrements at Tpc are 75% (66–71%) for the mass of bottom (charmed) and 71–80% (42–50%) for the residue of bottom (charmed) baryons, respectively com- pared to their vacuum values. These behavior of baryons can be interpreted as substantial melting of the heavy baryons near to the pseudo-critical temperature. At the end of this section, we would like to present our results for the masses of the single heavy spin-3/2 baryons at T →0 limit. This is done in Table 3. For comparison, we also present the existing theoretical predictions in the literature and experimental data in the same table. With a quick glance in this table, we see that our predictions, within the errors, are overall consistent with other theoretical predictions made using different methods and approaches. Our predictions are also well consistent with the existing experimental data for ﬁve members within the presented uncertainties. ∗ b baryon is only missing member, which has not been discovered in the experiment. We hope that, our result together with other Fig. 3 Numerical results To this end, we use the two sum rules in Eqs. (23) and (24) and one extra equation obtained by applying the derivative with respect to d d(−1 M2 ) to both sides of Eq. (23). Simultaneous solving of the resultant three equations with the aim of obtaining the three mentioned unknowns gives the function f (T ) as Now, we proceed to ﬁnd the function f (T ) and the tem- perature dependent mass m BSH (T ) and residue λBSH (T ) of the single heavy spin-3/2 baryons. To this end, we use the two sum rules in Eqs. (23) and (24) and one extra equation obtained by applying the derivative with respect to d d(−1 M2 ) to [m BSH + 0.3]2 GeV 2 ≤s0 ≤[m BSH + 0.5]2 GeV 2. (31) (31) The upper and lower limits of the Borel parameter are ﬁxed consider the criteria of the QCD sum rule method. To ﬁnd the lower limit, we apply the criterion of the OPE conver- gence at the chosen window for the continuum threshold. To this end, we demand that the perturbative part exceeds the total nonperturbative contributions and the slogan of the The upper and lower limits of the Borel parameter are ﬁxed consider the criteria of the QCD sum rule method. To ﬁnd the lower limit, we apply the criterion of the OPE conver- gence at the chosen window for the continuum threshold. To this end, we demand that the perturbative part exceeds the total nonperturbative contributions and the slogan of the M both sides of Eq. (23). Simultaneous solving of the resultant three equations with the aim of obtaining the three mentioned unknowns gives the function f (T ) as f (T ) = 1 −0.96 T Tpc 9 . (33) (33) 12 3 425 Page 8 of 12 Eur. Phys. J. C (2020) 80 :425 Fig. 2 Up: Contributions of perturbative and nonperturbative parts to total OPE. Down: Contributions of various operators with different dimensions to nonperturbative part: ⟨¯qq⟩(dimension 3), ⟨G2⟩+ ⟨u f (g)u⟩(dimension 4), ⟨qGq⟩(dimension 5) , ⟨qq⟩2 (dimension 6), ⟨qq⟩⟨G2⟩+ ⟨qq⟩⟨u f (g)u⟩ (dimension 7), ⟨G2⟩2 + ⟨u f (g)u⟩2(dimension 8) Fig. 3 Numerical results 3 The mass of the ∗ b baryon as functions of M2 and s0 at T = 0 In the following, we proceed to discuss the thermal behav- ior of the masses and residues under study as the main goal of the present work. In this context, as examples, we plot the m(T )/m(0) and λ(T )/λ(0) for the bottom members as functions of T/Tpc and M2 in Fig. 4 at average value of the vacuum continuum threshold. This ﬁgure shows that the spectroscopic parameters of the ∗ b, ∗ b and ∗ b baryons are stable against the changes in temperature until a certain tem- perature but after that, they start to decrease with increas- ing the temperature. Our analyses show that the charmed baryons present similar behavior, as well. The points that the stability starts to break down for mass and residue are T ∼= 0.14 GeV and T ∼= 0.13 GeV, respectively. After these points the mass and residue starts to diminish. The mass and residue fall substantially near to the pseudo-critical temper- ature. The amount of decrements at Tpc are 75% (66–71%) for the mass of bottom (charmed) and 71–80% (42–50%) for the residue of bottom (charmed) baryons, respectively com- pared to their vacuum values. These behavior of baryons can be interpreted as substantial melting of the heavy baryons near to the pseudo-critical temperature. At the end of this section, we would like to present our results for the masses of the single heavy spin-3/2 baryons at T →0 limit. This is done in Table 3. For comparison, we also present the existing theoretical predictions in the literature and experimental data in the same table. With a quick glance in this table, we see that our predictions, within the errors, are overall consistent with other theoretical predictions made using different methods and approaches. Our predictions are also well consistent with the existing experimental data for ﬁve members within the presented uncertainties. ∗ b baryon is only missing member, which has not been discovered in the experiment. We hope that, our result together with other Eur. Phys. J. C (2020) 80 :425 425 Page 8 of 12 425 Page 8 of 12 3 The mass of the ∗ b baryon as functions of M2 and s0 at T = 0 In the following, we proceed to discuss the thermal behav- ior of the masses and residues under study as the main goal of the present work. In this context, as examples, we plot the m(T )/m(0) and λ(T )/λ(0) for the bottom members as functions of T/Tpc and M2 in Fig. 4 at average value of the vacuum continuum threshold. This ﬁgure shows that the spectroscopic parameters of the ∗ b, ∗ b and ∗ b baryons are stable against the changes in temperature until a certain tem- 12 123 3 Page 9 of 12 425 Page 9 of 12 425 Eur. Phys. J. C (2020) 80 :425 Eur. Phys. J. C (2020) 80 :425 Page 9 of 12 42 (a) (b) (c) (d) Eur. Phys. J. C (2020) 80 :425 Page 9 of 12 425 (a) (b) (c) (d) (e) (f) Fig. 4 The mass (right) and residue (left) of the bottom baryons as functions of M2 and T/Tpc (a) (b) (b) (a) (c) (d) (e) (f) (d) (c) (f) (e) Fig. 4 The mass (right) and residue (left) of the bottom baryons as functions of M2 and T/Tpc 3 425 Page 10 of 12 Eur. Phys. J. 425 Page 8 of 12 The decre- ments order in the mass and residue of the considered baryons near to the pseudo-critical temperature are obtained as (66– 75)% and (42–80)%, respectively, representing substantial melting of the heavy baryons near to the pseudo-critical tem- perature. In the literature, there are no other studies on the thermal behavior of single heavy baryons to make a compres- sion with our predictions. However, there are some studies on the temperature dependence of the masses of light baryons, In Refs. [58,71] the authors investigated the light octet and decuplet baryons using the thermal QCD sum rule, but con- sidering a pseudo-critical temperature of Tpc = 197 MeV. They obtained that the shifts in the masses of the considered baryons are overall about 80%. The pole mass of the octet and decuplet baryons were also evaluated in Ref. [72] via the chiral perturbation theory. The authors observed that a 20% mass shift occurs around the temperature T ⋍150 MeV, where the freeze-out in the relativistic heavy-ion collision is expected to be formed. Using the many-body techniques at ﬁnitetemperature,allbaryonicstatesoftheoctetanddecuplet ﬂavors were examined in Ref. [73]. They obtained that the baryon masses decrease with the temperature and there are theoretical predictions will help experimental group in the course of search for this particle. 425 Page 8 of 12 C (2020) 80 :425 Table 3 The vacuum mass comparison of the single heavy spin-3/2 baryons in GeV with existing theoretical predictions and experimental data (Exp [4]) m∗ b m∗c m∗ b m∗c m∗ b m∗c Present work 6.08+0.10 −0.15 2.75+0.08 −0.26 5.88+0.11 −0.11 2.56+0.08 −0.07 5.95+0.12 −0.13 2.65+0.08 −0.07 [16] – – 5.805 2.495 – – [17] – – 5.4 ∼6.2 2.15 ∼2.92 – – [19] – 2.768 – 2.518 – – [20,21] 6.090 ± 0.050 2.770 ± 0.030 5.850 ± 0.040 2.520 ± 0.020 5.980 ± 0.040 2.650 ± 0.020 [22] 6.083 2.760 5.840 – 5.966 – [23] – – 5.84 ± 0.09 2.55 ± 0.08 – – [25] – – 5.82 ± 0.13 2.59 ± 0.20 – – [26] 6.060 2.752 5.871 2.538 5.959 2.680 [28] 6.088 2.768 5.834 2.518 5.963 2.654 [30,36] 6.00 ± 0.16 2.74 ± 0.23 5.81 ± 0.19 2.56 ± 0.24 5.94 ± 0.17 2.64 ± 0.22 [31] 6.06 ± 0.13 2.76 ± 0.10 – – – – [33] 6.079 2.767 5.829 2.502 5.961 2.642 [34] 6.063+0.083 −0.082 2.790+0.109 −0.105 5.835+0.082 −0.077 2.534+0.096 −0.081 5.929+0.083 −0.079 2.634+0.102 −0.094 [37] – – 5.829 – – – [38] 6.082 – – – 5.959 ± 0.004 – [39] 6.08 ± 0.40 2.72 ± 0.20 5.85 ± 0.35 2.51 ± 0.15 5.97 ± 0.40 2.66 ± 0.18 [40] 6.044 ± 0.018 – 5.842 ± 0.026 – 5.950 ± 0.021 – [41] 6.17 ± 0.15 2.79 ± 0.19 5.85 ± 0.20 2.48 ± 0.25 6.02 ± 0.17 2.65 ± 0.20 [42] 6.088 2.768 5.834 2.519 5.963 2.649 [43] 6.073 – 5.834 – 5.954 – Exp [4] – 2.7659 ± 0.0020 5.83032 ± 0.00027 2.51848 ± 0.00020 5.9523 ± 0.0009 2.64638 ± 0.00021 Table 3 The vacuum mass comparison of the single heavy spin-3/2 baryons in GeV with existing theoretical predictions and experimental data (Exp [4]) mparison of the single heavy spin-3/2 baryons in GeV with existing theoretical predictions and experimental data comparison of the single heavy spin-3/2 baryons in GeV with existing theoretical predictions and experimental data that the spectroscopic parameters remain unchanged up to a certain temperature, after which they start to diminish con- siderably near to the pseudo-critical temperature. 4 Summary and concluding remarks In this study, we have performed two-point thermal QCD sum rule analyses for ∗ Q, ∗ Q and ∗ Q single heavy baryons which are the members of the spin-3/2 sextet family. In the OPE,operatorsuptodimensioneightweretakenintoaccount which lead to a good OPE convergence as well as pole dom- inance. We included the thermal effects by two ways: We replaced the vacuum condensates by their thermal versions and considered the extra operators, appearing in the forms of the fermionic and gluonic parts of the energy momentum ten- sor due to the restoration of the Lorentz invariance. We ﬁxed the auxiliary parameters entering the calculations by the stan- dard prescriptions of the method. By simultaneous solving of the two sum rules obtained together with an extra equation derived from one of the sum rules, we found three unknowns: Thermal continuum threshold, temperature-dependent mass and temperature-dependent residue. We discussed the ther- mal behavior of the mass and residue for all the bottom and charmed baryon members having the spin-3/2. We observed 123 123 Page 11 of 12 425 Page 11 of 12 425 Eur. Phys. J. C (2020) 80 :425 strong dependencies on the melting (or deconﬁnement) tem- peraturedependingontheﬂavorcontentofthebaryons.Inthe framework of the thermal QCD sum rule, the masses of the decuplet baryons were also investigated in Ref. [74]. Accord- ing to this study, the masses of the decuplet baryons show very little temperature dependence below T = 0.11 GeV and the melting or hadron-quark phase transition occurs at a tem- perature T ≥0.11 GeV. Our results indicate that this point is T = 0.14 GeV for heavy baryons, after which the masses start to decrease with the increasing of the temperature and the dependence of the masses on temperature near to the critical temperature is very strong. These information on the behavior of the masses of different baryons may help experi- mental groups in the analyses of the results of the in-medium and heavy ion collision experiments, despite the statistical hadronization model claims that any thermal modiﬁcation of masses is negligibly small at pseudo-critical temperature and the in-medium mass shifts at Tpc would be excluded. 6. B. Aubert et al., (BABAR Collaboration), Phys. Rev. Lett. 97,232001 (2006) 7. T. Aaltonen et. al., (CDF Collaboration), Phys. Rev. Lett. 99,202001 (2007) 8. V. Abazov et. al., (D0 Collaboration), Phys. Rev. Lett. 99,052001 (2007) 9. T. Aaltonen et. al., (CDF Collaboration), Phys. Rev. Lett. 99,052002 (2007) 10. 4 Summary and concluding remarks C 54, 231 (2008) 31. Z.G. Wang, Eur. Phys. J. C 54, 231 (2008) 32. W. Roberts, M. Pervin, Int. J. Mod. Phys. A 32. W. Roberts, M. Pervin, Int. J. Mod. Phys. A 23, 2817 (2008) Open Access This article is licensed under a Creative Commons Attri- bution 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, pro- vide 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 indi- cated 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 permit- ted use, you will need to obtain permission directly from the copy- right holder. To view a copy of this licence, visit http://creativecomm ons.org/licenses/by/4.0/. 33. A. Valcarce, H. Garcilazo, J. Vijande, Eur. Phys. J. A 37, 217 (2008) 34. X. Liu, H.X. Chen, Y.R. Liu, A. Hosaka, S.L. Zhu, Phys. Rev. D 77, 014031 (2008) 35. S. Groote, J.G. Korner, A.A. Pivovarov, Eur. Phys. J. C 58, 355 (2008) 36. J.R. Zhang, M.Q. Huang, Phys. Rev. D 77, 094002 (20 37. S.M. Gerasyuta, E.E. Matskevich, Int. J. Mod. Phys. E 18, 1785 (2009) 38. M. Karliner, B. Keren-Zura, H.J. Lipkin, J.L. Rosner, Annals Phys. 324, 2 (2009) 39. T.M. Aliev, K. Azizi, A. Ozpineci, Nucl. Phys. B 808, 137 (2009) 40. R. Lewis, R.M. Woloshyn, Phys. Rev. D 79, 014502 (2009) 41. Z.G. Wang, Eur. Phys. J. C 68, 459 (2010) g y Funded by SCOAP3. 42. D. Ebert, R.N. Faustov, V.O. Galkin, Phys. Rev. D 84, 014025 (2011) 43. J.Y. Kim, H.C. Kim, G.S. Yang, Phys. Rev. D 98, 054004 (2018) 44. K. Azizi, N. Er, Nucl. Phys. A 970, 422 (2018) 45. Y. Aoki, G. Endrodi, Z. Fodor, S.D. Katz, K.K. Szabo, Nature 443, 675–678 (2006) 4 Summary and concluding remarks R. Chistov et al., (Belle Collaboration), Phys. Rev. Lett. 97,162001 (2006) 11. B. Aubert et al., (BABAR Collaboration), Phys. Rev. D 77,012002 (2008) 12. T. Lesiak et al., (Belle Collaboration), Phys. Ltt. B 665, 9 (2008) 12. T. Lesiak et al., (Belle Collaboration), Phys. Ltt. B 665 13. S. Chatrchyan et al., (CMS Collaboration), Phys. Rev. Lett. 108, 252002 (2012) 14. R. Aij et al., (LHCb Collaboration), JHEP 1605, 161 (2016) 15. E.V. Shuryak, Nucl. Phys. B 198, 83 (1982) 15. E.V. Shuryak, Nucl. Phys. B 198, 83 (1982) 16. S. Capstick, N. Isgur, Phys. Rev. D 34, 2809 (1986) 17. E. Bagan, M. Chabab, H.G. Dosch, S. Narison, Phys. Lett. B 278, 367 (1992) ( ) 18. A.G. Grozin, O.I. Yakovlev, Phys. Lett. B 285, 254 (1992) 18. A.G. Grozin, O.I. Yakovlev, Phys. Lett. B 285, 25 19. M.J. Savage, Phys. Lett. B 359, 189 (1995) 20. R. Roncaglia, D.B. Lichtenberg, E. Predazzi, Phys. Rev. D 52, 1722 (1995) We extracted the values of the masses for both the bot- tom and charmed baryons at T →0 limit and compared with the predictions of other phenomenological models and experimental data. The obtained results are well consistent with existing experimental data. Our result on the mass of ∗ b baryon as the only undiscovered member together with other predictions may help the experimental group to hunt this particle and measure its parameters. 21. R. Roncaglia, A. Dzierba, D.B. Lichtenberg, E. Predazzi, Phys. Rev. D 51, 1248 (1995) 22. E. Jenkins, Phys. Rev. D 54, 4515 (1996) 23. Y.B. Dai, C.S. Huang, C. Liu, C.D. Lu, Phys. Lett. B 371, 99 (1996) S. Groote, J.G. Körner, O.I. Yakovlev, Phys. Rev. D 55, 3016 25. D.W. Wang, M.Q. Huang, C.Z. Li, Phys. Rev. D 65, 094036 (2002) 26. N. Mathur, R. Lewis, R.M. Woloshyn, Phys. Rev. D 66, 014502 (2002) 27. D.W. Wang, M.Q. Huang, Phys. Rev. D 67, 074025 (2003) Data Availability Statement This manuscript has no associated data or the data will not be deposited. [Authors’ comment: All the numerical and mathematical data have been included in the paper and we have no other data regarding this paper.] 28. D. Ebert, R.N. Faustov, V.O. Galkin, Phys. Rev. D 72, 034026 (2005) 29. H. Garcilazo, J. Vijande, A. Valcarce, J. Phys. G 34, 961 (2007) 30. J.R. Zhang, M.Q. Huang, Phys. Rev. D 78, 094 31. Z.G. Wang, Eur. Phys. J. 5. D. Acosta et al., CDF Collaboration. Phys. Rev. Lett. 96, 202201 (2006) 2. N. Isgur, M.B. Wise, Phys. Rev. Lett. 66, Phys. 2015, 794243 (2015) 72. P.F. Bedaque, Phys. Lett. B 387, 1 (1996) 72. P.F. Bedaque, Phys. Lett. B 387, 1 (1996) 60. L.J. Reinders, H. Rubinstein, S. Yazaki, Phys. Rep. 127, 1 (1985) 73. J.M. Torres-Rincon, B. Sintes, J. Aichelin, Phys. Rev. C 91, 065206 (2015) 61. P. Gubler, D. Satow, Prog. Part. Nucl. Phys. 106, 1 (2019). [arXiv:1812 00385 [hep ph]] References M. Tanabashi et al., Phys. Rev. D 98, 030001 (2018) 59. K. Azizi, A. Türkan, E. Veli Veliev, H. Sundu, Adv. High Energy Phys. 2015, 794243 (2015) 71. K. Azizi, G. Bozkir, Eur. Phys. J. C 76, 521 (2016) 65. O. Kaczmarek, F. Karsch, F. Zantow, P. Petreczky, Phys. Rev. D 70, 074505 (2004) References 46. M. Cheng et al., Phys. Rev. D 74, 054507 (2006) 1. N. Isgur, M.B. Wise, Phys. Lett. B 232, 113 (1989) 47. T. Bhattacharya et al., Phys. Rev. Let. (PRL) 113, 082001 (2014) g y 2. N. Isgur, M.B. Wise, Phys. Rev. Lett. 66, 1130 (1991) 48. A. Bazavov et al., Phys. Rev. D 95, 054504 (2017) 3. H. Georgi, Phys. Lett. B 240, 447 (1990) 49. M.A. Shifman, A.I. Vainstein, V.I. Zakharov, Nucl. Phys. B 147, 385 (1979) 4. M. Tanabashi et al., Particle Data Group. Phys. Rev. D 98, 030001 (2018) 4. M. Tanabashi et al., Particle Data Group. Phys. Rev. D 98, 030001 (2018) 50. M.A. Shifman, A.I. Vainstein, V.I. Zakharov, Nucl. Phys. B 147, 448 (1979) 5. D. Acosta et al., CDF Collaboration. Phys. Rev. Lett. 96, 202201 (2006) 5. D. Acosta et al., CDF Collaboration. Phys. Rev. Lett. 96, 202201 (2006) 51. B.L. Ioffe, Nucl. Phys. B 188, 317 (1981) 12 3 425 Page 12 of 12 Eur. Phys. J. C (2020) 80 :425 65. O. Kaczmarek, F. Karsch, F. Zantow, P. Petreczky, Phys. Rev. D 70, 074505 (2004) 66. K. Morita, S.H. Lee, Phys. Rev. C 77, 064904 (2008) 67. V.M. Belyaev, B.L. Ioffe, Sov. Phys. JETP 57, 716 (1983) 68. H.G. Dosch, M. Jamin, S. Narison, Phys. Lett. B 220, 251 (1989) 69. B.L. Ioffe, Prog. Part. Nucl. Phys. 56, 232 (2006) 70. M. Tanabashi et al., Phys. Rev. D 98, 030001 (2018) 71. K. Azizi, G. Bozkir, Eur. Phys. J. C 76, 521 (2016) 72. P.F. Bedaque, Phys. Lett. B 387, 1 (1996) 73. J.M. Torres-Rincon, B. Sintes, J. Aichelin, Phys. Rev. C 91, 065206 (2015) 74. Y. Xu, Y. Liu, M. Huang, Commun. Theor. Phys. 63, 209 (2015) 52. A.I. Bochkarev, M.E. Shaposhnikov, Nucl. Phys. B 268, 220 (1986) 65. O. Kaczmarek, F. Karsch, F. Zantow, P. Petreczky, Phys. Rev. D 70, 074505 (2004) 53. T.M. Aliev, M. Savci, Phys. Rev. D 90(116006), 11 (2014) 54. K. G. Savvidy, (2005) [arXiv:1005.3455 [hep-th]] 66. K. Morita, S.H. Lee, Phys. Rev. C 77, 064904 (2008) 55. T.M. Aliev, K. Azizi, M. Savci, Phys. Rev. D 82, 096006 (2010) 56. T.M. Aliev, K. Azizi, M. Savci, Phys. Lett. B 681, 240 (2009) 57. F.X. Lee, Phys. Rev. D 57, 1801 (1998) 69. B.L. Ioffe, Prog. Part. Nucl. Phys. 56, 232 (2006) 58. K. Azizi, G. Kaya, J. Phys. G 43(5), 055002 (2016) 70. 66. K. Morita, S.H. Lee, Phys. Rev. C 77, 064904 (2008) 65. O. Kaczmarek, F. Karsch, F. Zantow, P. Petreczky, Phys. Rev. D 70, 074505 (2004) 66. K. Morita, S.H. Lee, Phys. Rev. C 77, 064904 (2008) 67. V.M. Belyaev, B.L. Ioffe, Sov. Phys. JETP 57, 716 (1983) 68. H.G. Dosch, M. Jamin, S. Narison, Phys. Lett. B 220, 251 (1989) 69. B.L. Ioffe, Prog. Part. Nucl. Phys. 56, 232 (2006) 70. M. Tanabashi et al., Phys. Rev. D 98, 030001 (2018) 71. K. Azizi, G. Bozkir, Eur. Phys. J. C 76, 521 (2016) 72. P.F. Bedaque, Phys. Lett. B 387, 1 (1996) 73. J.M. Torres-Rincon, B. Sintes, J. Aichelin, Phys. Rev. C 91, 065206 (2015) 74. Y. Xu, Y. Liu, M. Huang, Commun. Theor. Phys. 63, 209 (2015) [arXiv:1812.00385 [hep-ph]] 74. Y. Xu, Y. Liu, M. Huang, Commun. Theor. Phys. 63, 209 (2015) 62. S. Mallik, Phys. Lett. B 416, 373 (1998) 63. A. Bazavov et al., Phys. Rev. D 90, 094503 (2014) 64. S. Borsanyi et al., Phys. Lett. B 730, 99–104 (2014) 123 123
https://openalex.org/W4385446495	https://link.springer.com/content/pdf/10.1007/s12247-023-09743-4.pdf	English	null	Oral Dissolving Film of Rivastigmine: Optimization Using Factorial Design	Journal of pharmaceutical innovation	2,023	cc-by	8,842	Keywords Alzheimer’s disease · Rivastigmine · Geriatric patients · Oral dissolving film · Patient compliance · 32 Factorial design Keywords Alzheimer’s disease · Rivastigmine · Geriatric patients · Oral dissolving film · Patient compliance · 32 Factorial design Journal of Pharmaceutical Innovation (2023) 18:1892–1907 https://doi.org/10.1007/s12247-023-09743-4 Journal of Pharmaceutical Innovation (2023) 18:1892–1907 https://doi.org/10.1007/s12247-023-09743-4 ORIGINAL ARTICLE ORIGINAL ARTICLE Oral Dissolving Film of Rivastigmine: Optimization Using Factorial Design Dalia A. Farghaly1 · Samar A. Afifi2 · Ahmed A. Aboelwafa3 · Magdy I. Mohamed3 Accepted: 17 May 2023 / Published online: 1 August 2023 © The Author(s) 2023 3 Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Kasr El‑Aini St., Cairo 11562, Egypt Abstract Purpose Due to impairments in memory and judgment, it is difficult for dementia patients to understand why they need medicine. Moreover, they often have swallowing difficulties. In this investigation, an oral dissolving film of rivastigmine tartrate (RT-ODF) was developed, offering a unique and convenient formulation for dementia patients. Purpose Due to impairments in memory and judgment, it is difficult for dementia patients to understand why they need medicine. Moreover, they often have swallowing difficulties. In this investigation, an oral dissolving film of rivastigmine tartrate (RT-ODF) was developed, offering a unique and convenient formulation for dementia patients. Methods RT-ODF was developed using a solvent-casting technique. Sodium alginate and sodium carboxymethyl cellulose were used as film-forming polymers, and glycerol was used as a plasticizer. A full factorial design (32) was employed to estimate the impact of two factors at three levels: polymer concentration (1, 1.5, and 2% w/v) and plasticizer concentration (30, 40, and 50% w/v) on the responses, i.e., the tensile strength (TS), the disintegration time (DT), and the quantity of drug released (Q10 min). f Methods RT-ODF was developed using a solvent-casting technique. Sodium alginate and sodium carboxymethyl cellulose were used as film-forming polymers, and glycerol was used as a plasticizer. A full factorial design (32) was employed to estimate the impact of two factors at three levels: polymer concentration (1, 1.5, and 2% w/v) and plasticizer concentration (30, 40, and 50% w/v) on the responses, i.e., the tensile strength (TS), the disintegration time (DT), and the quantity of drug released (Q10 min). Results The optimized formula (A1) that had the highest desirability value (0.923) exhibited the lowest tensile strength (3.67 ± 0.72 MPa), the shortest disintegration time (20 ± 2.0 s), and the highest percentage of drug released after 10 min (97.12 ± 2.01%). It was composed of 1% w/v sodium alginate (ALG-Na) and plasticized with 30% w/v glycerol. The phar- macokinetic study revealed that the RT-ODFs enhanced the drug’s bioavailability by 1.91-fold relative to the reference product (Exelon® capsule).i Conclusion Oral dissolving films of rivastigmine tartrate could be a promising approach to promote drug bioavailability and convenience for geriatric patients. 2 Reference Laboratory of Egyptian Drug Authority (EDA), 51 Wezaret El‑Zeraa St., Dokki, Giza 35521, Egypt 1 Executive Office of Egyptian Pharmacopoeia, Egyptian Drug Authority (EDA), Al Haram, Giza 3514037, Egypt * Dalia A. Farghaly dodi23121@yahoo.com Introduction suppressing both acetylcholinesterase and butyrylcholinest- erase enzymes. RT is available as an oral solution, capsules, and transdermal patches [4]. The oral bioavailability of RT is mainly altered by first-pass metabolism. Besides, it is associ- ated with the most frequent adverse effects such as nausea and abdominal pain [5]. Rivastigmine tartrate (RT) is indicated for the sympto- matic treatment of mild-to-moderate Alzheimer’s disease (AD) [1, 2]; this disease is characterized by fatal memory deterioration that is attributed to a significant deficiency of acetylcholine in the brain [3]. RT is an acetylcholinesterase inhibitor that promotes acetylcholine levels in the brain by Dementia patients may not be capable of taking their medications orally because of dysphagia, a major and often healthcare problem in geriatrics [6]. Moreover, most car- egivers for dementia patients often encounter resistance to care in the early and middle stages of the disease because memory loss makes it difficult for patients to comprehend the need for medication. Helping a patient with dementia to take medication safely and easily can be challenging for him and his caregiver [7].i * Dalia A. Farghaly dodi23121@yahoo.com 1 Executive Office of Egyptian Pharmacopoeia, Egyptian Drug Authority (EDA), Al Haram, Giza 3514037, Egypt Fast-dissolving oral films (ODFs) represent an alterna- tive for geriatric patients with dementia as a convenient way to administer when the film placed on the tongue would be Vol:1 :.(123456789 3 Journal of Pharmaceutical Innovation (2023) 18:1892–1907 1893 (CMC) was supplied by El Nasr Pharmaceutical Chemi- cals Company (ADWIC) (Qaliubiya, Egypt). Glycerol was purchased from Alpha Chemika (Mumbai, India). Tween 80 was obtained from MP Biomedicals (Santa Ana, CA, USA). Potassium dihydrogen phosphate was supplied by Dae-Jung Chemicals and Metals Company (Korea). Ace- tonitrile (HPLC grade) was supplied by Scharlau (Spain/ European Union). Erythrosine (E127) (Eurocert Erythros- ine 311807B20) was kindly donated by Minapharm Phar- maceuticals (Cairo, Egypt). Peppermint oil was donated by Medizen Pharmaceutical Industries (Alexandria, Egypt). Methanol (HPLC grade) was supplied from Fisher Scientific (UK). Glacial acetic acid, sodium hydroxide, and n-hexane were supplied from PioChem (Giza, Egypt). 1-Butanol was supplied from El Nasr Pharmaceutical Chemicals Company (ADWIC) (Qaliubiya, Egypt). DI water was collected from the Milli-Q-Millipore water system. All reagents were of analytical grade. The reference product was Exelon® cap- sule (3 mg) (EXP: 2022; LOT: BPF44) (Novartis Pharma AG, Basel, Switzerland). Formulation Design of RT‑ODFs In this investigation, we have developed fast-dissolving oral films (ODFs) using a solvent-casting technique. Sodium algi- nate (ALG-Na) and sodium carboxymethyl cellulose (CMC) polymers were chosen depending on their good film-forming characteristics after preliminary studies were done (data was not shown). Glycerol was used as a proper plasticizer based on its ability to provide mechanical strength and flexibility to the film. Tween 80 is used as a surfactant having solubilizing and wetting properties, and sucralose was used as a sweetener. Preparation of RT‑Loaded ODFs (RT‑ODFs) RT-ODFs were developed by a solvent-casting technique [13]. In brief, a precisely weighed polymer was dissolved in DI water with continuous stirring until it became trans- parent. Subsequently, a solution of the desired amounts of the other ingredients was added to the resulting solution of polymer and was left aside for 24 h to remove any air bub- bles. Then, the solution was cast into a petri dish (lubricate the petri dish with castor oil) and then dried into a film in the open air for 48 h. The films were cut into dimensions 2 × 2 cm2, containing rivastigmine 3 mg (equivalent to 4.8 mg RT), and stored in aluminum foils. Finally, the in vivo study was employed using albino male rabbits comparing the pharmacokinetics of RT from the optimized formula with that of the reference product (Exelon® capsule). Introduction immediately hydrated by saliva and in a few seconds disin- tegrate and dissolve to release the drug for trans-mucosal absorption. Fast-dissolving films offer a unique approach for dosing medications, especially for geriatric patients who suffer from swallowing difficulties, providing dose accuracy, avoid- ing the first-pass metabolism, and enhancing bioavailability, thus overcoming issues of poor patient compliance [8, 9].i ODFs are prepared with a film-forming polymer with the addition of a suitable plasticizer and other excipients aimed to optimize physicomechanical properties in terms of homogeneous texture, smooth surface and ductility, and disintegration time. The disintegration time of ODFs is valuable, as it has a significant impact on drug release and thereafter bioavailability [10]. The reported data from the literature indicate that the selec- tion of polymer has a significant effect where an increase in polymer concentration results in disintegration time prolonga- tion. Moreover, the plasticizer concentration affects the tensile strength of the film. In general, plasticizers are molecules that may interpose between the polymer chains allowing them to tilt and rotate freely, imparting higher flexibility. This is com- monly observed in the form of films with high elongation and low tensile strength [11]. Differential Scanning Calorimetry (DSC) DSC measurements of RT, polymers, glycerol, and the film matrix were performed using a DSC131 EVO (SETARAM Inc., France) in the heating zone of − 25 to 300 °C under a dry nitrogen gas purge and at a heating rate of 10 °C/min. The thermograms were built using CALISTO software v.149 [12]. The factorial design has played a valuable role in pharma- ceutical formulations. It is valuable in investigating and under- standing the impact of formulation variables on the character- istics of ODFs. A full factorial design (32) was implemented using Design-Expert® software v. 11. In this design, the con- centration of polymer (F1) and plasticizer (F2) was chosen as independent variables, while the dependent variables were the tensile strength (TS) (R1), the disintegration time (DT) (R2), and the % RT released after 10 min (Q10 min). Optimization of RT‑ODFs The two polymers that exhibited the shortest disintegration time and the lowest values of tensile strength were chosen to prepare RT-ODFs to be evaluated. Uniformity of Drug Content The drug content of each film was performed by dissolving the film in 100 mL of phosphate buffer pH = 6.8 with stirring using a magnetic stirrer for 1 h. This solution was filtered through a 0.45-µm membrane filter and then assayed using a validated HPLC method [4]. The results were presented as the mean of three values [10]. The assay was performed on an HPLC UltiMate 3000 system (Dionex Softron GmbH, Germany) equipped with a UV detector. Samples were injected onto a Kromasil 100 C18 (250 × 4.6 mm, 5 μm) col- umn, delivered at a flow rate of 1 mL/min, and maintained at 25 °C. The mobile phase was composed of a mixture of 0.02 M potassium dihydrogen orthophosphate buffer (pH = 3.0) with o-phosphoric acid and acetonitrile at a ratio of 70:30 (v/v), and the injection volume was 20 μL. RT was quantified at 218 nm, and a standard calibration curve was plotted with a correlation coefficient (R2) of 0.9995. Weight Three RT-ODFs of each formula were individually weighed on an electronic balance (AND HR 202, Tokyo, Japan) to estimate the average weight [9]. Film Thickness The thickness of RT-ODF was measured by a micrometer screw gauge (Messwelk 0.01 mm, Germany). The thickness was tested at three different locations of each strip, and the average value was reported [12]. Surface pH In vitro disintegration time of the RT-ODFs was determined in a beaker of 25 mL distilled water at 37 ± 0.5 °C swirling every 10 s. The disintegration time is defined as the time required for the film to disintegrate into small pieces. The test was repeated three times [10]. The pH was determined by moistening the RT-ODF with 1 mL of distilled water and kept for 1 min, then touching the pH electrode with its surface allowing equilibration for 1 min. Surface pH was measured using a pH meter (Jenway-3510, Staffordshire, UK) in triplicate [13]. Materials Rivastigmine tartrate (RT) was gifted from Eva Pharma (Cairo, Egypt). Sodium alginate (ALG-Na) was supplied by Sigma-Aldrich (UK). Sodium carboxymethylcellulose RT-ODFs were developed according to a full factorial design (32) to examine the impact of formulation variables 1 3 1894 Journal of Pharmaceutical Innovation (2023) 18:1892–1907 they were kept in a desiccator for 3 consecutive days, and then, the weight was recorded again. Percentage moisture loss was estimated according to the following formula [14]: on the ODF characteristics applying Design-Expert® soft- ware v. 11. In this design, two factors were evaluated, each at three levels; the experimental trials were performed at nine prob- able combinations. The independent variables investigated were the concentration of polymer (F1) and the concen- tration of plasticizer (F2), whereas the chosen dependent variables were the tensile strength (TS) (R1), the in vitro disintegration time (DT) (R2), and the % RT released after 10 min (Q10 min) (R3) as shown in Table 1. % Moisture loss = Initial weight −Final weight Initial weight × 100 Table 1 Full factorial design for optimization of RT-ODFs Percentage Moisture Loss The mechanical characteristics of the developed films including tensile strength (TS) and percent elongation at (% E) break were determined using Zwick 1425 material test- ing machine (Germany). The measurements were done in triplicate for each formula [10]. The percentage moisture loss test was performed to ensure the physical stability and integrity of the film. The weight of six films of each formula was assessed accurately using an electronic balance (AND HR 202, Tokyo, Japan); then, Table 1 Full factorial design for optimization of RT-ODFs l factorial design for n of RT-ODFs Factors (independent variables) Levels F1: concentration of polymer Low (1%) Medium (1.5%) High (2%) F2: concentration of plasticizer Low (30%) Medium (40%) High (50%) Responses (dependent variables) Desirability constraints R1: TS (MPa) Minimize R2: DT (s) Minimize R3: Q10 min (%) Maximize 1 3 Journal of Pharmaceutical Innovation (2023) 18:1892–1907 1895 In Vitro Dissolution Profile of RT‑ODFs p < 0.05. The “Graph Pad InStat Demo v.” software was adopted for analysis [17, 18]. The dissolution test of the RT-ODFs was performed using a USP Dissolution Apparatus I (Basket Apparatus) (Hanson SR8 plus, Chatsworth, CA, USA) at a rotation speed of 100 rpm in 900 mL of phosphate buffer pH = 6.8 maintained at 37 ± 0.5 °C [15, 16]. Samples of RT-ODFs, equivalently containing 4.8 mg (2 × 2 cm2) of RT, were placed in baskets. Three milliliters of samples was with- drawn and replaced with a fresh medium. RT was assayed using HPLC as described before. All experiments were performed in triplicate. The % drug released was calcu- lated using the standard calibration curve of RT in phos- phate buffer pH = 6.8. The linearity of the standard cali- bration curve was appropriately in the range of 0.5–5 µg/ mL (Y = 0.3247 X, R2 = 0.9995). The graphs were plotted to compare the % drug released versus time. Statistical Analysis All determinations were done in triplicate, and the values were expressed as mean ± SD. Data analysis was done by applying one-way analysis of variance (ANOVA) followed by Tukey test. A value of p < 0.05 was considered signifi- cant. The “Graph Pad InStat Demo v.” software was used for this purpose. Scanning Electron Microscopy (SEM) The surface morphology of the optimized RT-ODF was examined using a JEOL JSM-5200 scanning electron microscope (Tokyo, Japan) operating at 25 kV. The sam- ples were coated under vacuum with gold under an argon atmosphere before the examination. The micrographs were observed to study the morphological and surface charac- teristics of RT and the RT-ODF [12]. Before dose administration, rabbits fasted overnight for 10 h. Following drug administration, serial aliquots of blood samples were withdrawn at periodic time intervals of 0, 0.08, 0.17, 0.25, 0.5, 1, 2, 4, and 6 h. Blood samples were collected in heparinized tubes, and plasma was immediately harvested by centrifugation at 3000 rpm for 15 min at room temperature using a centrifuge (Uni- versal 16, Hettich, Germany). Separated plasma was directly aspirated and transferred into plastic tubes and was stored at − 20 °C till analyzed. Pharmacokinetic Study The study was conducted to determine the pharmacokinetics of RT after oral administration of the optimized ODF formula compared to that of the reference product Exelon® 3 mg cap- sule. The experimental procedure and the in vivo study protocol were reviewed and approved by the Research Ethics Commit- tee (REC) at the Faculty of Pharmacy, Cairo University, Egypt, on 29 October 2018 (REC Approval No. is PI (2286)). Twelve adult New Zealand male white rabbits, weighing 3.0 ± 0.5 kg, were housed individually and had free access to food and water. A single dose of the drug was given to New Zealand male rabbits using a two-way crossover design with a wash- out period of 7 days between the two doses. During the first phase, rabbits received either the reference product (Exelon® capsule) or the optimized formula. In the second phase, rabbits, that administered the optimized formula first, received the reference product or vice versa [19]. Twelve rabbits were randomly divided into two groups each contain- ing six rabbits. The first group received the reference product Exelon® capsule. Five milliliters of water was given to the rabbits to facilitate swallowing the dose. The second group received the selected RT-ODF formula, which was placed above the rabbit’s tongue and allowed to dissolve in saliva. Blood samples (2 mL) were obtained from the retro-orbital plexus of rabbits [20, 21]. Stability Studies According to ICH guidelines, stability studies were con- ducted at 40 ± 2 °C with relative humidity RH 75 ± 5% to investigate the impact of temperature and relative humidity on the drug in the optimized formula. The films were envel- oped in an aluminum wrap in a glass container. The container was stored for 90 days. After the storage time, the films were evaluated concerning their appearance, disintegration time in addition to their drug content, and in vitro drug release. Extraction Procedure Extraction was performed by adding 100 µL of 1 M NaOH and 3 mL of 1-butanol/n-hexane (2: 98, v/v) to 1 mL of plasma. After centrifugation at 6000 rpm, the whole organic layer was separated. Then, 600 µL of 0.1% acetic acid was added. The mixture was vortex-mixed and centrifuged. The aqueous phase was analyzed for RT [22]. Statistical analysis of data was employed using paired sample t-test. Any variation was considered significant at 1 3 1896 Journal of Pharmaceutical Innovation (2023) 18:1892–1907 Factorial Design Analysis In an attempt to improve the developed formulae, factorial design is a valuable tool to reduce the number of experi- ments and investigate the relationship between investigated factors and responses of interest [26]. It was found that the polymer and plasticizer concentrations were the most effec- tive factors in the previous literature that affected the disin- tegration time and drug release. Depending on the results of preliminary trials, ALG-Na and CMC were chosen to be included in the factorial design. Nine experimental runs were assigned, A1–A9 and C1–C9 for ALG-Na and CMC polymers, respectively. The responses of the experimental runs are shown in Tables 3 and 4. High‑Performance Liquid Chromatography (HPLC) Analysis of RT in Plasma CMC had lower tensile stress values and disintegrated more rapidly than those prepared by carrageenan and polyvinyl alcohol (PVA). Data was not shown here. Plasma samples were analyzed for RT using a validated HPLC method. An Agilent 1260 Infinity II HPLC apparatus (Santa Clara, CA, USA) consisted of Quat-pump, 1260 Vial- sampler, and a fluorescence detector (1260 FLD Spectra). Data were collected and analyzed by OpenLAB software (v. A.01.05; Agilent Technologies). Samples of RT in plasma were injected onto a Sep-Tech ST150-10-C18 (250 × 4.6 mm) column and delivered at a flow rate of 1 mL/min at room temperature (25 °C). The mobile phase was a mix- ture of 0.02 M potassium dihydrogen orthophosphate buffer (pH = 3.0) with o-phosphoric acid and acetonitrile at a ratio of 70: 30 (v/v), the injection volume was 100 μL, and the detection of RT was carried out at an excitation wavelength (λexc) of 220 nm and emission wavelength (λem) of 293 nm [23]. The % drug released was calculated using a standard calibration curve of RT in plasma. The linearity of the stand- ard calibration curve was found in the range of 15–480 ng/ mL (Y = 0.0359 X, R2 = 0.998]. The graphs were plotted to compare the % drug released versus time. Preparation of RT‑ODFs The solvent-casting technique is the most frequent approach for the manufacturing of ODFs due to the ease of the tech- nique and its low cost [26]. In this casting study, we selected hydrophilic polymers that produce homogenous and flexible films, easily removable from the casting support. Glycerol was selected as a plasticizer as it developed transparent homogenous and flexible preparation with good mechani- cal strength. The compositions of RT-ODFs are depicted in Table 2. Pharmacokinetic Analysis Non-compartmental pharmacokinetic analysis was carried out using WinNonlin® 5.2 (Pharsight Corporation) [24]. The maximum plasma concentration (Cmax, ng/mL), the time taken to reach maximum plasma concentration (Tmax, h), the area under the plasma concentration–time curve (AUC0–∞, ng h/mL), the t1/2 (h) which was calculated as 0.693/Ke, the mean residence time (MRT, h), and the elimination rate constant (Ke, h−1) after oral administration were determined. The data were expressed as the mean ± standard deviation. Data were analyzed using one-way ANOVA adopting the “Graph Pad InStat Demo v.” software. The significance of the difference will be determined at the 95% confidence limit (α = 0.05). Differential Scanning Calorimetry (DSC) The DSC thermograms of RT, polymers, glycerol, and the film matrix are shown in Fig. 1. DSC test was used to evalu- ate drug compatibility with formula components. When the endothermic peak of the drug was shifted, it might indicate the interaction of drug molecules with other excipients [25]. DSC showed no interaction between RT and ALG-Na in the optimized formulae (A1). Effect of Formulation Variables on Tensile Strength (TS) In the pre-formulation studies, attention was given to the selection of a proper polymer at a suitable concentration to prepare a blank film that had good ductility and flexibility and could disintegrate fast [12]. The preliminary screening of familiar polymers in the literature revealed that ALG-Na, carrageenan, polyvinyl alcohol (PVA), and CMC had good film-forming capacity at certain concentrations. Obtained films were homogenous, smooth, non-sticky, and transpar- ent. The disintegration time and tensile strength of the film are the most important characteristics for evaluating the ODFs [12]. It was clear that films made by ALG-Na and Tensile strength is defined as the maximum tension force that a material can withstand during stretching before tearing up. Hence, it is important to optimize the polymeric blends to afford enough tensile strength to endure the stress along with the production, packaging, transport, and handling pro- cess [25, 26]. From Tables 3 and 4, it was found that the tensile strength of formulae A1–A9 ranged from 3.67 ± 0.72 to 5.83 ± 0.68 MPa, whereas the tensile strength of formulae 3 3 Journal of Pharmaceutical Innovation (2023) 18:1892–1907 1897 C1–C9 was in the range of 7.07 ± 0.46 to 11.37 ± 0.25 MPa. The influence of the independent variables on the tensile strength of RT-ODFs is demonstrated in Fig. 2. It was revealed that the concentration of polymer and plasticizer significantly affected the tensile strength of the prepared formulae (p = 0.0002). Regarding the polymer concentration it was found the TS value of the film which was in an agreement with Alhayali et al. (2019). The lowest tensile strength was obtained as the polymer concentration was 1% w/v. On the other hand, plasticizer content showed no signifi- cant effect on the TS values of ALG-Na formulae. However, in the case of CMC formulae, TS was affected by plasticizer concentration It was found that increasing the Fig. 1 DSC thermograms of RT, polymers, glycerol, and the film matrix Fig. 1 DSC thermograms of RT, polymers, glycerol, and the film matrix Fig. 1 DSC thermograms of RT, polymers, glycerol, and the film matrix C1–C9 was in the range of 7.07 ± 0.46 to 11.37 ± 0.25 MPa. The influence of the independent variables on the tensile strength of RT-ODFs is demonstrated in Fig. 2. C1–C9 was in the range of 7.07 ± 0.46 to 11.37 ± 0.25 MPa. Effect of Formulation Variables on Tensile Strength (TS) The influence of the independent variables on the tensile strength of RT-ODFs is demonstrated in Fig. 2. the TS value of the film which was in an agreement with Alhayali et al. (2019). The lowest tensile strength was obtained as the polymer concentration was 1% w/v.i On the other hand, plasticizer content showed no signifi- cant effect on the TS values of ALG-Na formulae.f It was revealed that the concentration of polymer and plasticizer significantly affected the tensile strength of the prepared formulae (p = 0.0002). f However, in the case of CMC formulae, TS was affected by plasticizer concentration. It was found that increasing the plasticizer content decreased the TS value because small Regarding the polymer concentration, it was found that reducing the concentration of the polymer decreased Table 2 Composition of RT-ODFs Formula code Ingredients RT (mg/film) ALG-Na (mg) CMC (mg) Glycerol (mg) Tween 80 (mg) Sucralose (mg) Peppermint oil (mg) Coloring agent (mg) Water (mL) A1 4.8 100 - 30 10 100 80 10 10 A2 4.8 150 - 45 10 100 80 10 10 A3 4.8 200 - 60 10 100 80 10 10 A4 4.8 100 - 40 10 100 80 10 10 A5 4.8 150 - 60 10 100 80 10 10 A6 4.8 200 - 80 10 100 80 10 10 A7 4.8 100 - 50 10 100 80 10 10 A8 4.8 150 - 75 10 100 80 10 10 A9 4.8 200 - 100 10 100 80 10 10 C1 4.8 - 100 30 10 100 80 10 10 C2 4.8 - 150 45 10 100 80 10 10 C3 4.8 - 200 60 10 100 80 10 10 C4 4.8 - 100 40 10 100 80 10 10 C5 4.8 - 150 60 10 100 80 10 10 C6 4.8 - 200 80 10 100 80 10 10 C7 4.8 - 100 50 10 100 80 10 10 C8 4.8 - 150 75 10 100 80 10 10 C9 4.8 - 200 100 10 100 80 10 10 Table 2 Composition of RT-ODFs 1 Journal of Pharmaceutical Innovation (2023) 18:1892–1907 1898 Table 3 Experimental runs, independent variables, and measured responses of the full factorial experimental design of RT-ODFs using ALG-Na polymer. Effect of Formulation Variables on the Amount of Drug Released After 10 min (Q10 min) Disintegration is considered one of the most critical quality aspects to guarantee patient compliance and acceptance of the ODFs [12]. Tables 3 and 4 show that the DT of formu- lae prepared with ALG-Na polymer ranged from 20 ± 2.0 to 99 ± 1.2 s, whereas the DT of formulae prepared with CMC was in the range of 24.33 ± 1.16 to 147.70 ± 2.52 s. The disintegration time of some formulae exceeded the recom- mended FDA limit (30 s) [29]. The effect of the independent variables on the DT of RT-ODFs is shown in Fig. 3. The percentage of the drug released from formulae prepared with ALG-Na polymer after 10 min (Q10 min) ranged from 66.46 ± 4.01 to 97.12 ± 2.01% as shown in Table 3. Whereas for formulae prepared with CMC, the Q10 min ranged from 49.48 ± 4.15 to 85.09 ± 4.93% as depicted in Table 4. The effect of the two independent variables on the Q10 min of RT-ODFs is demonstrated in Fig. 4. It was clear that the polymer and plasticizer concentration had significant impacts on the DT of the prepared formulae (p < 0.0001). The results revealed that both the polymer and plasti- cizer concentrations exhibited significant impacts on the Q10 min of the developed formulae (p < 0.0001).f The results suggested that increasing the polymer concen- tration resulted in increasing the DT of the prepared formu- lae. This could be attributed to high polymer concentration The amount of drug released was directly affected by the polymer concentration. It was found that the release of the drug increased at the lowest polymer concentration Table 4 Experimental runs, independent variables, and measured responses of the full factorial experimental design of RT-ODFs using CMC polymer. Effect of Formulation Variables on Tensile Strength (TS) Data represent the mean value ± SD (n = 3) Formula code F1: polymer concentration F2: plasticizer concentration R1: tensile strength (MPa) R2: disintegration time (s) R3: Q10 min (%) A1 Low Low 3.67 ± 0.72 20 ± 2.0 97.12 ± 2.01 A2 Medium Low 5.59 ± 0.45 24 ± 0.6 79.98 ± 4.95 A3 High Low 5.35 ± 0.31 93 ± 2.9 78.84 ± 0.87 A4 Low Medium 4.57 ± 0.38 24 ± 0.6 95.41 ± 3.45 A5 Medium Medium 5.02 ± 0.24 31 ± 1.7 72.74 ± 2.43 A6 High Medium 5.83 ± 0.68 91 ± 1.2 68.56 ± 1.51 A7 Low High 4.85 ± 0.71 25 ± 0.6 91.41 ± 3.47 A8 Medium High 5.63 ± 0.12 46 ± 1.2 70.65 ± 3.45 A9 High High 5.67 ± 0.08 99 ± 1.2 66.46 ± 4.01 molecules of plasticizer could simply incorporate into poly- mer molecular chains, resulting in more free space and thus enhancing the mobility of the polymer chains. This in turn reduces the glass transition temperature (Tg) and eventually improves the elasticity of the film [27, 28]. which constituted a viscous gel that retard the penetration of the disintegration medium thereby prolonging the disin- tegration time [28, 30]. It was obvious that the disintegration time was dependent on the plasticizer concentration. This means that an increase in the plasticizer content resulted in increasing the disinte- gration time. Effect of Formulation Variables on the Amount of Drug Released After 10 min (Q10 min) Data represent the mean value ± SD (n = 3) Formula code F1: polymer concentration F2: plasticizer concentration R1: tensile strength (MPa) R2: disintegration time (s) R3: Q10 min (%) C1 Low Low 9.50 ± 0.70 24.33 ± 1.16 85.09 ± 4.93 C2 Medium Low 11.00 ± 1.31 30.33 ± 1.53 64.31 ± 2.49 C3 High Low 7.93 ± 0.38 91.67 ± 2.08 61.07 ± 0.88 C4 Low Medium 8.20 ± 0.85 40.33 ± 1.53 71.51 ± 2.35 C5 Medium Medium 10.47 ± 0.81 91.00 ± 1.73 50.57 ± 2.19 C6 High Medium 11.37 ± 0.25 100.30 ± 1.53 51.95 ± 2.27 C7 Low High 7.07 ± 0.46 42.33 ± 2.08 75.11 ± 2.34 C8 Medium High 8.70 ± 0.20 101.70 ± 2.89 52.08 ± 2.17 C9 High High 8.93 ± 0.06 147.70 ± 2.52 49.48 ± 4.15 1 3 Journal of Pharmaceutical Innovation (2023) 18:1892–1907 1899 Fig. 2 Effect of polymer concentration on the tensile strength of RT- ODFs using ALG-Na polymer desired to dissolve the polymer in addition to the hydro- philic polymer created pores for the drug to diffuse out of the film thus promoting the release of the drug [9]. i On the other hand, the dissolution of the drug decreased at higher polymer concentrations. This might be due to the creation of a gel-like barrier surrounding the drug and hence, retaining it inside the polymer matrix leading to a slower drug release as reported in the literature for fast- dissolving oral films [9, 30–32]. i The amount of drug released decreased with increased plasticizer concentration which was in agreement with the data mentioned by Maher et al. [32]. Optimization of RT‑ODFs Generally, the purpose of optimization of any pharmaceuti- cal formula is to specify the levels of variables needed to get a good quality product. RT-ODFs were optimized for the responses R1 (TS), R2 (DT), and R3 (Q10 min). The tar- get was to minimize TS and DT in addition to maximiz- ing the Q10 min. The optimal values of the variables were Fig. 2 Effect of polymer concentration on the tensile strength of RT- ODFs using ALG-Na polymer (1% w/v) due to the increased dissolution at low polymer concentration leading to a minimum quantity of fluid Fig. 3 Effect of formulation variables on the tensile strength of RT-ODFs using CMC polymer Fig. 3 Effect of formulation variables on the tensile strength of RT-ODFs using CMC polymer Fig. 3 Effect of formulation variables on the tensile strength of RT-ODFs using CMC polymer 1 3 Journal of Pharmaceutical Innovation (2023) 18:1892–1907 Journal of Pharmaceutical Innovation (2023) 18:1892–1907 1900 Fig. 4 Effect of formulation variables on the disintegration time of RT-ODFs using ALG-Na polymer Fig. 4 Effect of formulation variables on the disintegration time of RT-ODFs using ALG-Na polymer in vivo study as it exhibited the lowest tensile strength (3.67 ± 0.72 MPa), the shortest disintegration time (20 ± 2.0 s), and the highest percentage of drug released after 10 min (97.12 ± 2.01%). The optimized formula (A1) was composed of 1% w/v ALG-Na polymer and plasticized with 30% w/v glycerol. developed by numerical optimization using the Design- Expert® software v. 11 depending on the desirability cri- terion. It was obvious that the formulae A1 and C1 had the highest desirability values, 0.923 and 0.847, respectively, as shown in Tables 5 and 6. Hence, A1 was selected for further Table 5 Optimization of RT-ODFs using ALG-Na polymer Number Polymer concentration Plasticizer concentration Tensile strength Disintegration time Q10 min Desirability 1 Low (1%) Low (30%) 4.361 20.000 99.304 0.923 Selected 2 Low (1%) Medium (40%) 4.361 24.333 93.714 0.847 3 Low (1%) High (50%) 4.361 25.333 90.922 0.810 4 Medium (1.5%) Low (30%) 5.412 24.333 78.353 0.576 5 Medium (1.5%) Medium (40%) 5.412 31.000 72.763 0.464 6 Medium (1.5%) High (50%) 5.412 45.667 69.971 0.369 7 High (2%) Low (30%) 5.618 93.333 76.006 0.190 8 High (2%) Medium (40%) 5.618 90.667 70.416 0.176 9 High (2%) High (50%) 5.618 99.333 67.624 0.049 Table 5 Optimization of RT-ODFs using ALG-Na polymer Journal of Pharmaceutical Innovation (2023) 18:1892–1907 1901 Table 6 Optimization of RT-ODFs using CMC polymer Number Polymer concentration Plasticizer concentration Tensile strength Disintegration time Q10 min Desirability 1 Low (1%) Low (30%) 9.500 24.333 85.043 0.847 Selected 2 Low (1%) High (50%) 7.067 42.333 73.777 0.765 3 Low (1%) Medium (40%) 8.200 40.333 72.897 0.732 4 Medium (1.5%) Low (30%) 11.000 30.333 63.458 0.537 5 High (2%) Low (30%) 7.933 91.667 61.973 0.463 6 Medium (1.5%) High (50%) 8.767 101.667 52.193 0.292 7 Medium (1.5%) Medium (40%) 10.467 91.000 51.313 0.273 8 High (2%) Medium (40%) 11.367 100.333 49.827 0.207 9 High (2%) High (50%) 8.933 147.667 50.707 0.075 Table 6 Optimization of RT-ODFs using CMC polymer formulae. It was observed that there is a significant increase in the film thickness (p < 0.0001) as the polymer concentra- tion increased. Weight The weight of formulae of A1–A9 was found in the range of 74.10 ± 0.56 to 121.87 ± 4.08 mg while the formulae C1–C9 ranged from 76.20 ± 0.62 to 122.17 ± 1.75 mg as shown in Tables 7 and 8, indicating the uniformity in the distribution of drug as well as excipients. Determination of Physicochemical Parameters of RT‑ODFs RT-ODFs were evaluated according to the following param- eters: weight variation, thickness, surface pH, moisture loss, and drug content. Surface pH The surface pH of ODFs is one of the indicators of patient compliance. It should be close to saliva pH to avoid any irritability to the oral mucosa. The surface pH of the for- mulae A1–A9 was found to be in the range of 5.47 ± 0.01 to 5.86 ± 0.03 while the formulae C1–C9 ranged from 5.81 ± 0.02 to 6.74 ± 0.01 which was within acceptable lim- its as depicted in Tables 7 and 8. Film Thickness The formulae A1–A9 showed a moisture loss in the range of 1.15 ± 0.46 to 2.30 ± 0.33% while C1–C9 showed a moisture loss ranging from 0.98 ± 0.15 to 2.15 ± 0.29% as shown in Tables 7 and 8, which indicates good physical stability and integrity of the films. Thickness values of the formulae A1–A9 were found to be 0.11 ± 0.01 to 0.25 ± 0.01 mm while the formulae C1–C9 ranged from 0.12 ± 0.01 to 0.21 ± 0.01 mm as shown in Tables 7 and 8, indicating uniform cast of respective Table 7 Data of evaluation parameters of RT-ODFs prepared with ALG-Na. Data represent the mean value ± SD (n = 3) and moisture loss data (n = 6) Formula code Thickness (mm) Elongation (%) Weight variation (mg) Drug content (%) Surface pH Moisture loss (%) A1 0.11 ± 0.01 68.67 ± 26.1 74.10 ± 0.56 104.52 ± 1.23 5.55 ± 0.03 1.80 ± 0.22 A2 0.13 ± 0.01 65.67 ± 24.54 97.87 ± 0.59 96.42 ± 1.28 5.47 ± 0.01 1.35 ± 0.25 A3 0.22 ± 0.02 60.33 ± 17.39 113.87 ± 0.40 102.89 ± 0.40 5.86 ± 0.03 1.32 ± 0.15 A4 0.12 ± 0.01 69.33 ± 11.93 87.67 ± 1.05 95.35 ± 1.37 5.61 ± 0.02 2.30 ± 0.33 A5 0.14 ± 0.00 63.00 ± 17.06 99.43 ± 0.32 102.90 ± 2.19 5.47 ± 0.01 1.15 ± 0.46 A6 0.22 ± 0.01 68.33 ± 26.58 120.97 ± 1.07 101.23 ± 1.97 5.77 ± 0.02 1.51 ± 0.25 A7 0.15 ± 0.01 57.50 ± 9.19 91.07 ± 0.84 97.97 ± 2.21 5.47 ± 0.02 1.16 ± 0.28 A8 0.20 ± 0.01 70.00 ± 12.17 109.53 ± 0.40 95.54 ± 1.65 5.67 ± 0.02 1.66 ± 0.16 A9 0.25 ± 0.01 60.67 ± 14.01 121.87 ± 4.08 108.12 ± 1.22 5.73 ± 0.04 1.35 ± 0.19 1 Journal of Pharmaceutical Innovation (2023) 18:1892–1907 1902 Uniformity of Drug Content Scanning Electron Microscopy (SEM) Table 8 Data of evaluation parameters of RT-ODFs prepared with CMC. Film Thickness Data represent the mean value ± SD (n = 3) and moisture loss data (n = 6) Formula code Thickness (mm) Elongation (%) Weight variation (mg) Drug content (%) Surface pH Moisture loss (%) C1 0.12 ± 0.01 135.33 ± 25.58 81.17 ± 7.22 104.37 ± 2.00 5.81 ± 0.02 1.37 ± 0.28 C2 0.14 ± 0.01 84.33 ± 21.73 98.77 ± 0.25 99.13 ± 2.48 5.83 ± 0.03 1.41 ± 0.20 C3 0.18 ± 0.01 101.67 ± 29.74 104.07 ± 2.40 97.22 ± 2.90 6.53 ± 0.03 2.05 ± 0.27 C4 0.13 ± 0.01 210.00 ± 20.00 76.20 ± 0.62 97.27 ± 2.69 6.74 ± 0.01 1.62 ± 0.48 C5 0.13 ± 0.01 118.67 ± 35.39 99.93 ± 7.23 106.48 ± 1.05 6.43 ± 0.01 1.45 ± 0.23 C6 0.21 ± 0.01 133.33 ± 25.66 120.17 ± 6.21 108.91 ± 1.11 6.16 ± 0.02 0.98 ± 0.15 C7 0.13 ± 0.01 181.33 ± 25.15 87.93 ± 2.08 108.19 ± 2.39 6.43 ± 0.02 2.15 ± 0.29 C8 0.16 ± 0.01 178.67 ± 30.07 103.77 ± 7.50 96.23 ± 1.08 6.61 ± 0.01 1.41 ± 0.18 C9 0.21 ± 0.01 122.00 ± 27.40 122.17 ± 1.75 100.46 ± 1.06 6.22 ± 0.02 1.18 ± 0.25 Uniformity of Drug Content The drug content of the formulae A1–A9 ranged from 95.35 ± 1.37 to 108.12 ± 1.22% while the formulae C1–C9 were found to be in the range of 96.23 ± 1.08 to 108.91 ± 1.11% as shown in Tables 7 and 8. All formulae had drug content in the range of 90 to 110% of the limit given in the USP [33], which indicated the uniform and even distribution of the drug. The SEM images showed that pure drug appears as irregular rod- like-shaped particles as shown in Fig. 5, whereas the SEM image of the optimized film and its cross-sectional image indicated that rough surfaces were observed due to the presence of irregular rod-like-shaped drug particles that were distributed uniformly throughout the film without any aggregation (Fig. 5) [30]. Fig. 5 Effect of formulation variables on the disintegration time of RT-ODFs using CMC polymer Fig. 5 Effect of formulation variables on the disintegration time of RT-ODFs using CMC polymer 1903 Journal of Pharmaceutical Innovation (2023) 18:1892–1907 Table 9 Effect of storage on the properties of the optimized formulae. Data represent the mean value ± SD (n = 3) Parameter Fresh A1 Stored A1 Fresh C1 Stored C1 I. Physical stability Appearance No change in color and elasticity Disintegration time (s) 20.00 ± 2.00 22.00 ± 1.00 24.33 ± 1.16 23.00 ± 2.00 Weight variation (mg) 74.10 ± 0.56 75.58 ± 1.20 81.17 ± 7.22 83.34 ± 5.80 II. Chemical stability Drug content (%) 104.52 ± 1.23 102.00 ± 1.20 104.37 ± 2.00 101.20 ± 1.60 Q10 min (%) 97.12 ± 2.01 92.78 ± 2.00 85.09 ± 4.93 76.28 ± 3.40 Journal of Pharmaceutical Innovation (2023) 18:1892–1907 1904 Journal of Pharmaceutical Innovation (2023) 18:1892–1907 Fig. 7 Effect of formulation variables on the Q10 min of RT-ODFs using CMC polymer Fig. 7 Effect of formulation variables on the Q10 min of RT-ODFs using CMC polymer Fig. 7 Effect of formulation variables on the Q10 min of RT-ODFs using CMC polymer Fig. 7 Effect of formulation variables on the Q10 min of RT-ODFs using CMC polymer (Exelon® capsule) is shown in Figs. 6 and 7. Pharmacoki- netic parameters including Cmax, Tmax, AUC0-6, AUC0-∞, t1/2, MRT, and Ke are depicted in Table 10. interval after dosing, increasing the bioavailability by 1.91-fold relative to that of the reference oral capsule (Fig. 9). This might be due to the large exposed surface area of the film allowing fast disintegration and instant release of the drug. Moreover, the absorption of a frac- tion of the drug across the oral mucosa might bypass the first-pass hepatic metabolism and promote bioavailability. The results clearly showed a significant increase (p < 0.0001) in the mean Cmax (177.99 ± 25.35 ng/mL) and AUC0-∞ values (283.97 ± 42.50 ng h/mL) after oral admin- istration of RT-ODF achieving a higher rate and extent of drug absorption than the mean Cmax (53.48 ± 2.84 ng/mL) and the AUC0-∞ values (148.50 ± 9.40 ng h/mL) of the refer- ence product (Exelon® capsule) (Fig. 8). Table 10 Pharmacokinetics of RT-ODFs (A1) and Exelon® follow- ing oral administration. Data represent the mean value ± SD (n = 6) Pharmacokinetic parameters RT-ODF (A1) Exelon® Cmax (ng/mL) 177.99 ± 25.35 53.48 ± 2.84 Tmax (h) 0.17 ± 0.00 0.25 ± 0.00 AUC0-6 (ng h/mL) 245.27 ± 26.21 109.97 ± 7.23 AUC0-∞ (ng h/mL) 283.97 ± 42.50 148.50 ± 9.40 t1/2 (h) 2.38 ± 0.45 3.28 ± 0.08 MRT (h) 2.70 ± 0.38 4.32 ± 0.10 Ke (h−1) 0.30 ± 0.06 0.21 ± 0.00 Table 10 Pharmacokinetics of RT-ODFs (A1) and Exelon® follow- ing oral administration. Data represent the mean value ± SD (n = 6) Similarly, the Tmax and MRT values of the RT-ODF showed a significant variation when compared to the refer- ence product (Exelon® capsule) (p < 0.0001), indicating that the various dosage forms had different impacts on the maximum time needed for the drug to reach the maximum concentration as well as its residence time in the body. The pharmacokinetic study revealed that the RT-ODFs modified the pharmacokinetic profile of the drug. Stability Studies The results of stability studies of the optimized RT-ODFs for 90 days are summarized in Table 9. The optimized formulae A1 and C1 showed no significant variation in weight, disintegration time, drug content, and the amount of drug released after 10 min (Q10 min), indicating good physicochemical stability. RT was analyzed by applying a validated HPLC method using a fluorescence detector to detect small amounts of the drug in plasma samples. For the pharmacokinetic study, the plasma concentration–time profile of RT following oral administration of RT-ODFs (A1) and the reference product Fig. 6 Effect of formulation variables on the Q10 min of RT-ODFs using ALG-Na polymer Fig. 6 Effect of formulation variables on the Q10 min of RT-ODFs using ALG-Na polymer Fig. 6 Effect of formulation variables on the Q10 min of RT-ODFs using ALG-Na polymer 1 3 1 3 Journal of Pharmaceutical Innovation (2023) 18:1892–1907 The drug was more rapidly absorbed from the fast-dissolving films and achieved higher plasma concentration in a short 1 3 1 3 Journal of Pharmaceutical Innovation (2023) 18:1892–1907 1905 Fig. 8 Scanning electron micrographs of a pure rivastigmine tartrate (RT), b, c RT-ODF, and d its cross-sectional image Fig. 8 Scanning electron micrographs of a pure rivastigmine tartrate (RT), b, c RT-ODF, and d its cross-sectional image Conclusion In this study, the RT-ODFs were well developed using ALG-Na and CMC as film-forming polymers and plas- ticized with glycerol. The physicomechanical properties of the formulae including the tensile strength (TS) in addition to the disintegration time were affected by the concentration of polymer and plasticizer. The optimized formula showed excellent mechanical properties, short dis- integration time, and high dissolution. The higher values of pharmacokinetic parameters indicated the superiority of fast-dissolving oral films in the enhancement of the Fig. 9 Time versus mean plasma concentration profiles of RT following oral adminis- tration of RT-ODFs (A1) and Exelon.® in rabbits (n = 6) Fig. 9 Time versus mean plasma concentration profiles of RT following oral adminis- tration of RT-ODFs (A1) and Exelon.® in rabbits (n = 6) Fig. 9 Time versus mean plasma concentration profiles of RT following oral adminis- tration of RT-ODFs (A1) and Exelon.® in rabbits (n = 6) addition to the disintegration time were affected by the concentration of polymer and plasticizer. The optimized formula showed excellent mechanical properties, short dis- integration time, and high dissolution. The higher values of pharmacokinetic parameters indicated the superiority of fast-dissolving oral films in the enhancement of the Ethics Approval 8. Soni G, Yadav KS. Fast-dissolving films of sumatriptan suc- cinate: factorial design to optimize in vitro dispersion time. J Pharm Innov. 2015;10:166–74. https://doi.org/10.1007/ s12247-015-9217-6. The experimental procedure and the in vivo study proto- col were reviewed and approved by the Research Ethics Committee (REC) at the Faculty of Pharmacy, Cairo Uni- versity, Egypt, on 29 October 2018 (REC Approval No. is PI (2286)). 9. Ullas D, Miriam S, Jivaji P, Kumar L, Kalidas R. Rapidly dis- solving felodipine nanoparticle strips: formulation using design of experiment and characterisation. J Drug Deliv Sci Technol. 2020;60:102053. https://doi.org/10.1016/j.jddst.2020.102053 10. Elmeshad AN, El Hagrasy AS. Characterization and optimi- zation of orodispersible mosapride film formulations. AAPS PharmSciTech. 2011;12:1384–92. https://doi.org/10.1208/ s12249-011-9713-z. Funding Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyp- tian Knowledge Bank (EKB). 11. Lim H, Hoag SW. Plasticizer effects on physical-mechanical properties of solvent cast Soluplus® films. AAPS PharmSciTech. 2013;14:903–10. https://doi.org/10.1208/s12249-013-9971-z. Data Availability The data that support the findings of this study are available from the corresponding author, [D.A.F.], upon reasonable request. 12. Xu LL, Shi LL, Cao QR, Xu WJ, Cao Y, et al. Formulation and in vitro characterization of novel sildenafil citrate-loaded polyvinyl alcohol-polyethylene glycol graft copolymer-based orally dissolving films. Int J Pharm. 2014;473:398–406. https:// doi.org/10.1016/j.ijpharm.2014.07.037. Conclusion In this study, the RT-ODFs were well developed using ALG-Na and CMC as film-forming polymers and plas- ticized with glycerol. The physicomechanical properties of the formulae including the tensile strength (TS) in 1 3 1906 Journal of Pharmaceutical Innovation (2023) 18:1892–1907 pharmacokinetics, and biodistribution. J Drug Deliv Sci Technol. 2018;43:129–40. https://doi.org/10.1016/j.jddst.2017.09.021. pharmacokinetics, and biodistribution. J Drug Deliv Sci Technol. 2018;43:129–40. https://doi.org/10.1016/j.jddst.2017.09.021. pharmacokinetics, and biodistribution. J Drug Deliv Sci Technol. 2018;43:129–40. https://doi.org/10.1016/j.jddst.2017.09.021. bioavailability of the drug. The large surface area of the film, the fast disintegration of the film, and the enhanced drug dissolution in the oral cavity were the reasons for promoting bioavailability. In conclusion, RT-ODFs could potentially introduce a fast and easily administered dosage form for the treatment of dementia, providing improved patient compliance. p g j j 5. Palle S, Neerati P. Enhancement of oral bioavailability of riv- astigmine with quercetin nanoparticles by inhibiting CYP3A4 and esterases. Pharmacol Reports. 2017;69:365–70. https://doi. org/10.1016/j.pharep.2016.12.002. 5. Palle S, Neerati P. Enhancement of oral bioavailability of riv- astigmine with quercetin nanoparticles by inhibiting CYP3A4 and esterases. Pharmacol Reports. 2017;69:365–70. https://doi. org/10.1016/j.pharep.2016.12.002. 6. Quinn HL, Hughes CM, Donnelly RF. Novel methods of drug administration for the treatment and care of older patients. Int J Pharm. 2016;512:366–73. https://doi.org/10.1016/j.ijpharm. 2016.01.050. 7. Lindauer A, Sexson K, Harvath TA. Medication management for people with dementia. Am J Nurs. 2017;117(2):60–4. https:// doi.org/10.1097/01.NAJ.0000512300.41511.9d.i Declarations Conflict of Interest The authors declare no competing interests. 13. Chevala NT, Matangi S, Chevala NPK, Mohammed G, Seethamraju SMK, et al. Design and development of fluoxetine hydrochloride oro flash films to alleviate major depressive disorder. J Pharm Investig. 2015;45:493–501. https://doi.org/10.1007/s40005-015-0198-8. Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, 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/. 14. Bala R, Sharma S. Formulation optimization and evaluation of fast dissolving film of aprepitant by using design of experiment. Bull Fac Pharmacy Cairo Univ. 2018;56:159–68. https://doi.org/ 10.1016/j.bfopcu.2018.04.002. 15. Abdelbary A, Bendas ER, Ramadan AA, Mostafa DA. Phar- maceutical and pharmacokinetic evaluation of a novel fast dis- solving film formulation of flupentixol dihydrochloride. AAPS PharmSciTech. 2014;15:1603–10. https://doi.org/10.1208/ s12249-014-0186-8. 16. Lakshmi PK, Sreekanth J, Sridharan A. Formulation development of fast releasing oral thin films of levocetrizine dihydrochloride with Eudragit® Epo and optimization through Taguchi orthogonal experimental design. Asian J Pharm. 2011;5:84–92. https://doi. org/10.22377/ajp.v5i2.89 References g jp 17. Kokare CK, Tagalpallewar AA, Aragade PS, Bagul US, Bacchav RK, et al. Formulation, evaluation and optimization of asenap- ine maleate fast mouth dissolving film. J Pharm Sci Pharmacol. 2016;2:194–207. https://doi.org/10.1166/jpsp.2015.1062.i 1. Joshi SA, Chavhan SS, Sawant KK. Rivastigmine-loaded PLGA and PBCA nanoparticles: preparation, optimization, characteriza- tion, in vitro and pharmacodynamic studies. Eur J Pharm Biop- harm. 2010;76:189–99. https://doi.org/10.1016/j.ejpb.2010.07.007. 1. Joshi SA, Chavhan SS, Sawant KK. Rivastigmine-loaded PLGA and PBCA nanoparticles: preparation, optimization, characteriza- tion, in vitro and pharmacodynamic studies. Eur J Pharm Biop- harm. 2010;76:189–99. https://doi.org/10.1016/j.ejpb.2010.07.007. p g jp p 18. Kanth NP, Prasad G, Kumar VB. Oral dissolving films of chlor- pheniramine maleate. Int J Pharm Sci Res. 2014;5(5):1859– 73. https://doi.org/10.13040/IJPSR.0975-8232.5(5).1859-73 2. Yang ZZ, Zhang YQ, Wang ZZ, Wu K, Lou JN, et al. Enhanced brain distribution and pharmacodynamics of rivastigmine by liposomes following intranasal administration. Int J Pharm. 2013;452:344–54. https://doi.org/10.1016/j.ijpharm.2013.05.009. 2. Yang ZZ, Zhang YQ, Wang ZZ, Wu K, Lou JN, et al. Enhanced brain distribution and pharmacodynamics of rivastigmine by liposomes following intranasal administration. Int J Pharm. 2013;452:344–54. https://doi.org/10.1016/j.ijpharm.2013.05.009. 19. Aljimaee YHM, El-Helw ARM, Ahmed OAA, El-Say KM. Development and optimization of carvedilol orodispersible tab- lets: enhancement of pharmacokinetic parameters in rabbits. Drug Des Devel Ther. 2015;9:1379–92. https://doi.org/10.2147/DDDT. S80294. 3. Williams BR, Nazarians A, Gill MA. A review of rivastigmine: a reversible cholinesterase inhibitor. Clin Ther. 2003;25(6):1634– 53. https://doi.org/10.1016/S0149-2918(03)80160-1. 20. Fouad SA, Shamma RN, Basalious EB, El-Nabarawi MA, Tayel SA. Novel instantly-soluble transmucosal matrix (ISTM) using dual mechanism solubilizer for sublingual and nasal delivery of 4. Abouhussein DMN, Khattab A, Bayoumi NA, Mahmoud AF, Sakr TM. Brain targeted rivastigmine mucoadhesive thermosensitive in situ gel: optimization, in vitro evaluation, radiolabeling, in vivo 1 3 1907 Journal of Pharmaceutical Innovation (2023) 18:1892–1907 dapoxetine hydrochloride: in vitro/ in vivo evaluation. Int J Pharm. 2016;505:212–22. https://doi.org/10.1016/j.ijpharm.2016.04.006. endurance and tensile properties. Int J Pharm. 2020;589:119876; https://doi.org/10.1016/j.ijpharm.2020.119876i p g j jp 21. Zayed GM, Rasoul SA, Ibrahim MA, Saddik MS, Alshora DH. In vitro and in vivo characterization of domperidone-loaded fast dissolving buccal films. Saudi Pharm J. 2020;28:266–73. https:// doi.org/10.1016/j.jsps.2020.01.005. 28. Hosny KM, El-say KM, Ahmed OA. Optimized sildenafil citrate fast orodissolvable film: a promising formula for overcoming the barriers hindering erectile dysfunction treatment. Drug Deliv. 2014;23(1):1–7. https://doi.org/10.3109/10717544.2014.916763. 29. Food Drug and Administration FDA. Guidance for industry: orally disintegrating tablets. 2023. https://www.fda.gov/regulatory- information/search-fda-guidancedocuments/orally-disintegrating- tablets. Accessed 04 Feb 2023. 22. Amini H, Ahmadiani A. References High-performance liquid chromato- graphic determination of rivastigmine in human plasma for application in pharmacokinetic studies. Iran J Pharm Res. 2010;9(2):115–21; PMID: 24363716; PMCID: PMC3862057 23. Kapil R, Dhawan S, Beg S, Singh B. Buccoadhesive films for once-a-day administration of rivastigmine: systematic formulation development and pharmacokinetic evaluation. Drug Dev Ind Pharm. 2013;39:466–80. https://doi.org/10.3109/03639045.2012.665926.i 30. Singh H, Singla YP, Narang RS, Pandita D, Singh S, et al. Frovatriptan loaded hydroxy propyl methyl cellulose/treated chitosan based com- posite fast dissolving sublingual films for management of migraine. J Drug Deliv Sci Technol. 2018;47:230–9. https://doi.org/10.1016/j. jddst.2018.06.018.i p g 24. Zhao Y, Quan P, Fang L. Preparation of an oral thin film containing meclizine hydrochloride: in vitro and in vivo evaluation. Int J Pharm. 2015;496:314–22. https://doi.org/10.1016/j.ijpharm.2015.10.008.f 31. Alhayali A, Vuddanda PR, Velaga S. Silodosin oral films: develop- ment, physic-mechanical properties and in vitro dissolution studies in simulated saliva. J Drug Deliv Sci Technol. 2019;53:101122. https:// doi.org/10.1016/j.jddst.2019.06.019. 25. Kazemi Z, Taghizadeh SM, Keshavarz ST, Lahootifard F. Effect of composition on mechanical and physicochemical properties of mucoadhesive buccal films containing buprenorphine hydrochlo- ride: from design of experiments to optimal formulation. J Drug Deliv Sci Technol. 2020;56:101578; https://doi.org/10.1016/j. jddst.2020.101578 32. Maher EM, Ali AMA, Salem HF, Abdelrahman AA. In vitro/in vivo evaluation of an optimized fast dissolving oral film contain- ing olanzapine co-amorphous dispersion with selected carboxylic acids. Drug Deliv. 2016;23:3088–100. https://doi.org/10.3109/ 10717544.2016.1153746. 26. Shamma R, Elkasabgy N. Design of freeze-dried Soluplus/poly- vinyl alcohol-based film for the oral delivery of an insoluble drug for the pediatric use. Drug Deliv. 2016;23:489–99. https://doi.org/ 10.3109/10717544.2014.921944. 33. Rockville MD. U. S. Pharmacopeia and National Formulary USP 44-NF 39. United States Pharmacopeial Convention. 2021. 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. 27. Takeuchi Y, Ikeda N, Tahara K, Takeuchi H. Mechanical char- acteristics of orally disintegrating films: comparison of folding 1 3 1 3
W4240600106.txt	https://www.researchsquare.com/article/rs-591162/latest.pdf	en		Zinc Systematics Quantify Crustal Thickness Control on Fractionating Assemblages of Arc Magmas	Research Square (Research Square)	2,021	cc-by	9,953	Zinc Systematics Quantify Crustal Thickness Control on Fractionating Assemblages of Arc Magmas M. Chiaradia (  Massimo.Chiaradia@unige.ch ) University of Geneva Research Article Keywords: Zinc, chemical, Earth Sciences, magmatic arc rocks, minerals, calc-alkaline Posted Date: June 10th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-591162/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Scientific Reports on July 19th, 2021. See the published version at https://doi.org/10.1038/s41598-021-94290-6. Page 1/19 Abstract Understanding the processes leading to the broad chemical variability of arc magmas is an essential, yet not fully elucidated, issue in Earth Sciences. Here, I show that Zn-MgO-SiO2 systematics of magmatic arc rocks correlate significantly with arc thickness. Because Zn-MgO-SiO2 systematics are mostly controlled by fractionation of different mineral phases this suggests a systematic change in the proportions of fractionating mineral assemblages depending on arc thickness. Using a mass balance model with a Monte Carlo approach, I show that Zn-MgO-SiO2 systematics can be quantitatively explained by a continuous transition from plagioclase-dominated fractionating assemblages in thin arcs to amphibole-garnet-magnetitedominated assemblages in increasingly thicker arcs. Such a systematic change results from the increase of average depth of magma differentiation that is ultimately controlled by arc thickness. Results presented have implications on the causes of different geochemical trends in arcs, the role of arcs as H2O filters, and their association with major porphyry deposits. Introduction Arc magmas are the building blocks of the continental crust 1 and are associated with the formation of economic mineral deposits2–4 as well as with catastrophic eruptions impacting human lives 5. They are also key to understand the recycling of elements and volatiles from the Earth crust and surface to the deep mantle because they represent the output of such interaction in the mantle wedge 6. In recent years various studies have shown systematic correlations of major and trace elements of arc magmas with the crustal thickness of the overriding plate7–11. However, it is not clear to what extent such correlations are controlled by mantle wedge or by intracrustal processes. For instance, it has been shown that the development of distinct tholeiitic and calc-alkaline trends in arc magmas depends on crustal thickness 12, but this has been interpreted either as the result of crustal processes7,13 or of different degrees of partial melting of the mantle wedge 14. The latter would be due to a different thermal structure of the mantle wedge that is controlled by the thickness of the overriding plate crust and lithosphere 15. Least evolved rocks of arcs have also been shown to display systematic correlations of their major and trace element contents with crustal thickness 10,11. This too has been interpreted as the result of differential partial melting degrees of the mantle wedge depending on the different depths of mantle wedge melting 10,11, which are ultimately controlled by the thickness of the overriding plate crust. In this study I use Zn systematics to investigate the role of fractional crystallization in producing different geochemical signatures and evolutionary trends in arc magmas and their relationship with the crustal thickness of the overriding plate. Zn is a lithophile element that has received an increased attention in magmatic processes during the last years because it is used as a reference element (e.g., Zn/Fe ratios) for tracing the oxidation state of the mantle 16, it allows investigation of mantle melting processes 17, and because Zn isotopes may elucidate processes of magma differentiation 18, slab contributions 19, and planetary evolution 20. Zinc is incorporated to different extents into the crystal lattice of all main minerals crystallizing from arc magmas (olivine, pyroxene, amphibole, plagioclase, magnetite, and garnet) 17. This property, together with systematics of major elements like SiO2 and MgO, which are also sensitive to fractionation of major mineral phases, may be used to reveal and quantify changes in the assemblages fractionating during arc magma evolution and to better understand the processes through which crustal thickness determines the development of different geochemical signatures and trends in arc magmas. The advantage of using Zn over other elements, typically used to evaluate fractionation of mineral assemblages in arc magmas (e.g., REE, Sr, Y, Cu), is that Zn is not incorporated to high extent into accessory minerals (zircon, titanite, apatite, garnet, not even sulfide minerals: refs. 21,22) that may strongly bias the systematics of these elements. Here, I show that arcs display Zn-MgO and MgO-SiO2 trends that systematically change with crustal thickness. Using a Monte Carlo-based modelling of fractionating assemblages in the SiO2-MgO-Zn tridimensional space I show that the systematic changes in the Zn-MgO and MgO-SiO2 trends are controlled by fractionating assemblages that shift from plagioclase-dominated in thin arcs to amphibole-garnet-magnetite-dominated in thick arcs, and quantify the proportions of fractionating minerals. The amphibole plus garnet proportions on one hand and plagioclase proportions on the other also correlate with the tholeiitic versus calc-alkaline features of arc magmas showing that there is a gradual transition between two geochemical and mineralogical extremes, which is ultimately controlled by the different depths at which average magma differentiation occurs in arcs of different thickness. Methods Arc crustal thickness Average crustal thicknesses of arcs and associated uncertainties were taken from Zellmer (2008), except the thickness of Tonga 61, Kermadec 62, Aleutians 63, and Northern Andes64. The Tonga crustal thickness here taken corresponds to the maximum crustal thickness of ref. 61 because arc magmatism occurs in coincidence with the thickest part of the Tonga arc (Fig. 9 in ref. 61). Crustal thicknesses have been calculated by ref. 60 using the global crustal model at 2x2 degrees, CRUST 2.0, administered by the US Geological Survey and the Institute for Geophysics and Planetary Physics at the University of California 65, which is an updated version of CRUST 5.1, a global crustal model at 5x5 degrees 66.The model is based on seismic refraction data published up to 1995 and a detailed compilation of sediment thickness. The crustal thicknesses of ref. 60 are within the ranges of crustal thicknesses reported in previous studies67,68 with which they show good linear correlations (r = 0.70 with respect to crustal thicknesses of ref. 67 , and r = 0.74 with respect to crustal thicknesses of ref. 68). Oceanic crust thickness is from ref. 69. Page 2/19 Data collection and treatment Geochemical data of bulk volcanic rocks (N = 42600) from 21 recent arcs were collected from the Georoc database (http://georoc.mpchmainz.gwdg.de/georoc/; Supplementary Data File 1). The Central Volcanic Zone of the Andes was excluded because of the very large thickness of the crust that biases the correlations between geochemical parameters and arc thickness (see also ref. 10). The Izu Bonin arc was excluded because of the extensive occurrence of boninitic rocks that are formed through different processes than typical arc basalts in the other arcs. As in ref. 7, to reduce the bias induced by outliers and to extract information on general trends, median values of Zn, SiO2, and MgO for subpopulations corresponding to bins of 0.5 wt% MgO were calculated (Supplementary Data File 1). When less than 10 data were available for one of the investigated elements within the 0.5 wt.% MgO bin, the MgO interval was extended to a higher value (e.g., 1 or 1.5 wt.%) to incorporate more values of that element. For comparison with arc systematics, data were also collected for MORB (https://www.earthchem.org/; N ~ 24000) and for Hawaii (http://georoc.mpchmainz.gwdg.de/georoc/; N ~ 5000), and treated in the same way as for arcs. Linear correlations for the median arc values in the Zn-MgO and MgO-SiO2 spaces were detected for the arc products going from the most primitive rocks to variably evolved rocks, depending on the arc (Supplementary Figures S1-2). Statistically significant linear correlations in the Zn-MgO space were detected down to variably low MgO values ~ 0–4 wt.% (Supplementary Figure S1 and Table 1), after which, in most arcs, Zn drops to low values. Statistically non-significant correlations are highlighted in Table 1. The linear correlations considered in the MgO-SiO2 space were those regarding the early to intermediate stages of magmatic evolution (e.g., for MgO > 2–5 wt.%). Below MgO values in the 2–5 wt.% range, depending on the arc, the more differentiated median values displayed a visible change in the slope with respect to the less evolved median values (Supplementary Figure S2). Modelling the linear trends in the tridimensional Zn-MgO-SiO2 space by fractional crystallization The linear Zn-MgO and MgO-SiO2 trends were modelled in the Zn-MgO-SiO2 space for a pure fractional crystallization process using simple mass balance equations (Supplementary Note 1 in Supplementary Information) in which the starting composition of the parental melt (Supplementary Table S2) was changed by subtracting variable proportions of 6 typical fractionating minerals in arc and MOR magmas (olivine, clinopyroxene, amphibole, plagioclase, magnetite, garnet). The objective of the model was to constrain the statistically most probable fractionating mineral assemblage (in terms of mineral proportions) and the extent of fractionation (residual melt fraction). For each arc the composition of the most primitive median value was taken as that of the parental melt (Supplementary Table S2 and Supplementary Figures S1-S2). Sometimes, when the median values showed some scatter on the parent side, the parent compositions were allowed to vary within a tight range ( < ± 0.2 wt% for SiO2 and MgO, ≤ 4 ppm for Zn; Supplementary Table S2). The target values were the compositions of the most evolved median values (i.e., the points with the lowest MgO content) of the individual linear MgO-SiO2 and Zn-MgO trends, i.e., the last points of these trends on the low MgO side before the change in the slopes (Supplementary Figure S2; target compositions in Supplementary Table S2). The target compositions were also allowed to vary within < ± 0.2 wt% for SiO2 and MgO and ≤ 4 ppm for Zn (Supplementary Table S2). The model was run using a Monte Carlo approach, i.e., besides the small ranges of parent and target compositions discussed above, also the mineral compositions were allowed to vary randomly within given ranges (Supplementary Tables S3-S4). MgO and SiO2 mineral composition ranges were derived from experimental petrology and were typical of those minerals fractionating in basaltic to andesitic magmas (Supplementary Table S3). The Zn contents in those same minerals were instead modelled using a range of Zn KD values for those minerals crystallizing from basaltic to andesitic magmas (Supplementary Tables S1 and S3). The model was initially run (5 million simulations) leaving a large degree of freedom for the proportions of all potential fractionating minerals (i.e., 00.7 for plagioclase, amphibole, clinopyroxene, olivine, 0-0.3 for garnet and 0-0.15 for magnetite) and for the residual melt fraction (20–70%). The 5 million simulation runs were carried out for these broad ranges between 15 and 20 times for each arc to identify the most recurrent proportions of minerals and residual melt fractions. After this initial step, the ranges of fractionating minerals were narrowed down progressively to narrower ranges around the median values obtained in step 1 until a variably high number of successful solutions were obtained (depending on the arc) with a normal distribution (i.e., average ~ mean value: Supplementary Table S2) of all parameters involved (mineral proportions, residual melt fraction). Then, 5 million simulation runs, corresponding to the conditions (mineral proportions and residual melt fraction ranges) returning the highest number of solutions, were repeated five times for each arc and an average value with associated standard deviation was calculated from those 5 repeats (Supplementary Table S4; Supplementary Data File 2). It is important to highlight that the model was run in order to obey simultaneously to the constraints imposed by both the Zn-MgO and MgO-SiO2 trends (in a tridimensional MgO-SiO2-Zn space). This reduces significantly the number of successful solutions but at the same time provides more stringent constraints on mineral proportions and residual melt fractions. Modelling of Zn contents in basaltic melts from partial mantle melting The Zn content of melt from mantle melting was calculated for both batch and fractional melting (Fig. 6b) using the following equations, respectively: Page 3/19 is the Zn concentration in the melt (in ppm), where is the Zn concentration of the mantle (55 ppm or 30 ppm in the calculations), is the bulk partition coefficient for Zn between mantle lherzolite and melt, and F is the melt fraction. More than 5000 simulations were run using the above equations and allowing random variation of F between 0.05 and 0.3 and of 0.5 and 0.7. The range of F encompasses potential melt fractions in the sub-arc and sub-MOR mantle 70 whereas the range of between is a common range of values based on olivine, clinopyroxene and orthopyroxene partition coefficients between melt and peridotite 17 and typical proportions of these minerals melting from a lherzolite. Modelling the H2O solubility of silicate melt during fractional crystallization at different pressures Variations of H2O solubility and excess H2O with changing residual melt fraction (100 to 20%) at 4 different pressures (0.2, 0.4, 0.6, 0.8 GPa) of magma fractionation were modelled from the parametrization of 58 based on the model of ref. 49 (see Supplementary Note 2 in Supplementary Information). The different curves are the result of 500000 simulations for ranges of initial H2O content of 3.9–4.1 wt.% and pressures ranges of 0.19–0.21, 0.39–0.41, 0.59–0.61 and 0.79–0.81 GPa. The H2O is considered to behave as completely incompatible in the model. Results Data collection and treatment Geochemical data of bulk volcanic rocks from 21 modern arcs (Supplementary Data File 1) were collected from the Georoc database (http://georoc.mpch-mainz.gwdg.de/georoc/) and treated according to the method described by ref. 7 and detailed in the Methods section. As in ref. 7, to reduce the bias induced by outliers and to extract information on general trends, median values of Zn, SiO2, and MgO for subpopulations corresponding to bins of 0.5 wt% MgO were calculated (Supplementary Data File 1). When less than 10 data were available for one of the investigated elements within the 0.5 wt.% MgO bin, the MgO interval was extended to a higher value (e.g., 1 or 1.5 wt.%) to incorporate more values of that element. For comparison with arc systematics, data were also collected for the mid-ocean ridge (MOR) environment (https://www.earthchem.org/), a typical oceanic island basalt magmatic sequence like Hawaii (http://georoc.mpch-mainz.gwdg.de/georoc/), and treated in the same way as for arcs. Median values of zinc contents of magmas from different arcs follow distinct trends with geochemical differentiation (i.e., with decreasing MgO: Supplementary Figure S1). The averages of the median values of the 7 arcs (Bismark-New Britain, Kurile, Kermadec, Mariana, New Hebrides, South Sandwich, Tonga) emplaced in intraoceanic arcs with thin crust thickness (< 20 km) display a systematic increase of Zn contents with decreasing MgO from an initial value of ~ 70 ppm up to 90–100 ppm Zn at MgO values between 1 and 2 wt.%, after which Zn decreases to values of 40–50 ppm for the most evolved rocks (Figs. 1a and Supplementary Figure S1). However, several thin arcs do not show such a decrease because they do not evolve to rocks differentiated enough (Supplementary Figure S1). Average values of zinc median contents of magmas emplaced in thick arcs (> 30 km: Mexico, Aleutians, Cascades, NVZ), in contrast, display an overall slight decline with decreasing MgO down to MgO values ~ 1.5 wt.%, after which Zn decreases along a steeper trend in the most differentiated rocks to an average value of ~ 40 ppm (Figs. 1a and Supplementary Figure S1). Zn trends in magmas of intermediate thickness arcs display an intermediate behavior (Supplementary Figure S1). The most differentiated magmas of the Lesser Antilles arc display an increase in Zn contents rather than a decrease like all other arcs (Supplementary Figure S1). Page 4/19 Table 1 Summary of the values of the regression slopes (MgO vs. SiO2 and Zn vs. MgO) and associated statistical values. Bold and italics indicate poor statistical correlations (see Supplementary Figure S1). Arc Crust thickness (a) error (a) Type according to crust thickness (b) slope MgOSiO2 error (c) r2 (c) slope ZnMgO error (c) r2 (c) Average of median Fe2O3tot at MgO 4–6 wt.% (d) error (d) Average of median Sr/Y at MgO 4–6 wt.% (e) error (e) S. Sandwich 11.8 0.1 < 20 km -1.7719 0.2444 0.913 -5.0157 0.7201 0.802 10.6 0.3 7.3 0.7 Mariana 14.5 1 < 20 km -1.9778 0.2066 0.884 -4.6166 0.52011 0.840 11 0.3 14.1 2.3 Kermadec 15 3 < 20 km -1.3980 0.3308 0.749 -3.9468 0.6076 0.710 11 0.5 10.5 2.9 New Hebrid. 15.6 0.2 < 20 km -1.6128 0.4708 0.516 -2.1136 0.8971 0.270 11.2 0.9 29.6 3.8 Kuriles 18.3 0.9 < 20 km -1.5281 0.2560 0.798 -2.0405 0.7744 0.332 9.2 0.2 15.3 0.9 Tonga 20 0 < 20 km -1.4867 0.2237 0.786 -2.6703 0.6144 0.486 11.1 0.3 11.9 1.8 BismarkNB 22.5 6.5 < 20 km -1.7614 0.2099 0.865 -0.7489 0.4575 0.124 9.8 0.3 27.9 7.9 Ryukyu 24.5 3.4 20–30 km -1.2420 0.3479 0.614 3.2332 1.4993 0.215 9.6 1 14.6 1.4 Kamchatka 24.6 5.4 20–30 km -1.1359 0.3028 0.501 -0.7217 0.5025 0.108 9.2 0.3 19.3 1.7 Lesser Antilles 24.7 0.7 20–30 km -1.0392 0.2039 0.743 1.4182 0.4301 0.420 9.3 0.2 16.3 4.8 Aeolian 24.9 1 20–30 km -0.9813 0.1454 0.820 2.2710 0.5283 0.627 8.5 0.1 30.8 2 Sulawesi 27.4 2.2 20–30 km -0.9439 0.4256 0.451 -1.1683 0.5830 0.334 10.3 0.5 20.2 1.8 Luzon 27.8 4.5 20–30 km -1.0296 0.0941 0.916 -1.0374 1.5086 0.044 9 0.5 25.9 3.4 Central America 28 7 20–30 km -0.8691 0.0470 0.961 -0.8611 0.4467 0.179 9.6 0.3 31 2.8 Aegean 28.2 0.6 20–30 km -0.7484 0.1090 0.825 -2.1850 0.5792 0.587 7.5 0.8 24 3.9 New Zealand 28.6 3.2 20–30 km -0.7093 0.1482 0.638 2.8065 0.5898 0.618 7.8 0.3 na na Honshu 29.2 2.6 > 30 km -1.4021 0.1133 0.922 -0.2739 0.3475 0.029 9.7 0.3 na na Mexico 30.3 5.5 > 30 km -0.6281 0.0410 0.933 1.2997 0.3705 0.370 7.6 0.5 27.4 2 Aleutians 37.5 2.5 > 30 km -0.8012 0.0885 0.911 2.6022 0.5834 0.434 9.4 0.2 18.3 3.9 (a) From 60, except Tonga thickness, which is from 61, Kermadec thickness, which is from 62, Aleutians thickness which is from 63, and Northern Andes thickness which is from 64. The Tonga crustal thickness here taken corresponds to the maximum crustal thickness of 61 because arc magmatism occurs in coincidence with the thickest part of the Tonga arc (Fig. 9 in 61). Crustal thicknesses have been calculated by 60 using the global crustal model at 2x2 degrees, CRUST 2.0, administered by the US Geological Survey and the Institute for Geophysics and Planetary Physics at the University of California 65, which is an updated version of CRUST 5.1, a global crustal model at 5x5 degrees 66.The model is based on seismic refraction data published up to 1995 and a detailed compilation of sediment thickness. The crustal thicknesses of 60 are within the ranges of crustal thicknesses reported in previous studies 67,68 with which they show good linear correlations (r = 0.70 with respect to crustal thicknesses of 67, and r = 0.74 with respect to crustal thicknesses of 68). Oceanic crust thickness is from . (b) Attribution to a crust thickness type takes into account the 1σ uncertainty and the geochemical systematics: for instance, Bismark/New Britain has average crust thickness slightly above 20 km, but taking into account the 1σ uncertainty minimum values are largely < 20 km and geochemical trends are more typical of arcs < 20 km thick. This subdivision is purely semantic and does not change the mathematical correlations. (c) errors and correlation coefficients r2 of the regressions were calculated using the "LINEST" function in Excel. (d) From 7 (e) From 8 Page 5/19 Arc Crust thickness (a) error (a) Type according to crust thickness (b) slope MgOSiO2 error (c) r2 (c) slope ZnMgO error (c) r2 (c) Average of median Fe2O3tot at MgO 4–6 wt.% (d) error (d) Average of median Sr/Y at MgO 4–6 wt.% (e) error (e) Cascades 38.8 1.9 > 30 km -0.9349 0.0950 0.898 0.2498 0.4216 0.019 8.4 0.5 30.3 1.6 NVZ 42 9.8 > 30 km -0.4906 0.0236 0.971 3.7100 0.6348 0.698 7.6 0.2 40.1 0.9 MOR 6.5 0.5 < 20 km -2.3322 0.3155 0.785 -10.076 0.6704 0.922 14.3 1.7 4.1 1.5 (a) From 60, except Tonga thickness, which is from 61, Kermadec thickness, which is from 62, Aleutians thickness which is from 63, and Northern Andes thickness which is from 64. The Tonga crustal thickness here taken corresponds to the maximum crustal thickness of 61 because arc magmatism occurs in coincidence with the thickest part of the Tonga arc (Fig. 9 in 61). Crustal thicknesses have been calculated by 60 using the global crustal model at 2x2 degrees, CRUST 2.0, administered by the US Geological Survey and the Institute for Geophysics and Planetary Physics at the University of California 65, which is an updated version of CRUST 5.1, a global crustal model at 5x5 degrees 66.The model is based on seismic refraction data published up to 1995 and a detailed compilation of sediment thickness. The crustal thicknesses of 60 are within the ranges of crustal thicknesses reported in previous studies 67,68 with which they show good linear correlations (r = 0.70 with respect to crustal thicknesses of 67, and r = 0.74 with respect to crustal thicknesses of 68). Oceanic crust thickness is from . (b) Attribution to a crust thickness type takes into account the 1σ uncertainty and the geochemical systematics: for instance, Bismark/New Britain has average crust thickness slightly above 20 km, but taking into account the 1σ uncertainty minimum values are largely < 20 km and geochemical trends are more typical of arcs < 20 km thick. This subdivision is purely semantic and does not change the mathematical correlations. (c) errors and correlation coefficients r2 of the regressions were calculated using the "LINEST" function in Excel. (d) From 7 (e) From 8 The trend of MOR magmatic rocks is characterized by a steeper negative slope than the average trend of the thin arcs in the Zn-MgO space (Fig. 1a). The most primitive MOR basalts have similar median Zn contents as the most primitive thin and thick arc basalts (~ 75 ppm) and grow to a median Zn content of ~ 170 ppm at ~ 2 wt.% MgO, that is significantly higher than the average value of thin arcs (Fig. 1a), after which Zn contents drop to a median value of ~ 100 ppm with further differentiation. In order to quantify the differences of the Zn-MgO trends in different arcs, slopes of the linear regressions between Zn and MgO were calculated for the early differentiation segments, i.e., excluding the median values corresponding to the strong Zn decreases (or increase for the Lesser Antilles) in the most differentiated rocks of all arcs (Table 1 and Supplementary Figure S1). Thicker arcs are characterized by positive Zn-MgO slopes, whereas thin arcs are characterized by negative Zn-MgO slopes and intermediate thickness arcs have intermediate slope values (Fig. 1b). The slopes so calculated display a statistically significant correlation with the thickness of the corresponding arc crust (Fig. 1b). Several arcs, however, do not display statistically significant correlations between Zn and MgO (e.g., Bismark-New Britain, Ryukyu, Cascades, Luzon, Honshu, Central America: Table 1) and are represented by a shaded symbol in Fig. 1b. Also MgO-SiO2 trends of the early evolutionary paths of arc magmas (i.e., before a break in the slope of the trend to a significantly shallower slope for MgO contents below a variable arc-dependent threshold of 2–5 wt.%; Supplementary Figure S2) display different slopes, steeper in thin arcs and shallower in thick arcs (Fig. 2a, Supplementary Figure S2, and Table 1). MOR magmatic rocks display an overall steeper slope than thin arcs during early evolution in the MgO-SiO2 space (Fig. 2a). The values of the slopes defined by the early to intermediate differentiation trend of arc and MOR magmas in the MgO-SiO2 space display again significant correlations with the crust thickness of the corresponding arc and of MOR (Fig. 2b). Although there might be some degree of arbitrariness in the choice of the point at which the slope breaks in the MgO-SiO2 space (especially for some arcs: Supplementary Figure S2), changing the break point to higher MgO values does not significantly change the slope of arcs in such a way to affect the correlation of Fig. 2b. In fact, inter-arc changes of such slopes are much larger than the small changes that arise from a different choice of the break point within a specific arc. Crustal thickness control on fractionating mineral assemblages The data reduction of the large dataset used in this study leads to results (median values) that statistically represent the most common values within a population with a normal distribution like that for the elements here considered within the ~ 0.5 wt.% MgO bins. This means that the interpretation of these results is forcedly a simplification of the processes occurring in arcs, as indicated by the large distribution clouds of single rock analyses compared to the median values calculated from them for the MgO bins within each arc (Supplementary Figures S1-S2). Therefore, the processes interpreted on such a basis are first-order processes and do not exclude, within each specific arc, the occurrence of additional second or lower order processes (e.g., ref. 23). Such processes might be important in some arcs, where correlations of median values of elements are not statistically significant (e.g., Fig. 1b and Table 1). Page 6/19 The different trends displayed by magmas of distinct arcs (and MOR) in the Zn-MgO and MgO-SiO2 spaces (Figs. 1–2 and Supplementary Figures S1S2) must be, to their greatest extent, the result of differentiation processes occurring within the crust because of the large SiO2 and MgO ranges that they encompass (Supplementary Figures S1-S2). A variety of studies agree in considering differentiation of arc magmas as the complex result of various processes, including fractional crystallization24,25, recharge/mixing13,26,27, partial melting 28 and assimilation of host rocks 29. Arguably, fractional crystallization (and to some extent partial melting, which is the opposite process of fractional crystallization) can be considered as the main process responsible for the large SiO2 and MgO variability observed in arc magmas 25. Superimposed on this, mixing/recharge are also universal processes occurring in arcs that tend to homogenize the signals of fractional crystallization 27. Figure 1 shows that Zn displays different degrees of increase or decrease during magmatic differentiation that depend on the thickness of the arc crust. Because primitive basalts have very similar values (70–80 ppm) both in arcs of different thickness and in MOR (Fig. 1a) and because the continental (72 ppm: ref. 30) and oceanic crust (~ 75 ppm: Fig. 1a) Zn contents also fall within this range, wholesale assimilation of crustal lithologies (either oceanic or continental) concomitant or not with fractional crystallization cannot explain the Zn systematics observed in arcs and MOR magmas. Partial melting of crustal lithologies producing SiO2-rich melts could be a significant process in arcs, especially at higher depths (and therefore in thicker arcs) because of thermal constraints 31. Mixing of basalt with such SiO2-rich and either Zn-poor or Zn-rich partial melts would be needed to explain the decreasing and increasing trends of Zn with MgO in thick and thin arcs, respectively. However, almost all arcs show a kink in both the Zn-MgO and MgO-SiO2 trends suggesting that mixing with a SiO2-rich and Zn-poor or Zn-rich crustal melts is not a viable explanation either. Mixing certainly occurs in arcs (there is ample petrographic and mineral chemistry evidence for that) and is probably also a cause of the scatter of single rock analyses around the kinked trends in the Zn-MgO and MgO-SiO2 spaces (Supplementary Figures S1S2). However, mixing in the crust must involve end-member magmas whose Zn-MgO-SiO2 systematics are controlled by fractionating or restitic minerals. Therefore, although treating the different trends observed in arcs in the Zn-MgO-SiO2 space as the result of fractional crystallization is an approximation, these trends ultimately tell us what are the mineral phases that are involved in producing their different slopes through combined fractional crystallization, partial melting and mixing processes. Thus, for simplicity I will model the distinct Zn-MgO-SiO2 trends of arcs as the dominant result of fractional crystallization during which Zn behavior shifts from incompatible to compatible for magma differentiation occurring within an increasing crustal thickness (Fig. 1a). Such a behavior should be discussed considering the partition coefficients of Zn between melt and the main minerals fractionating in arc (and MOR) magmas (i.e., olivine, plagioclase, amphibole, clinopyroxene, garnet and magnetite). A compilation of KD values from the literature (Supplementary Table S1 and Fig. 3) suggests that, among the potential fractionating phases during the early and intermediate stages of arc magma differentiation, magnetite is the one for which Zn has the highest affinity, compared to pyroxenes and particularly to plagioclase, which has very low KD values for Zn. Zn is slightly incompatible in olivine in equilibrium with basalt but becomes compatible in this mineral when the latter crystallizes from basaltic andesite and andesite melt (Fig. 3). In contrast Zn is compatible in magnetite already crystallizing from basalt and its compatibility strongly increases with magmatic differentiation (Fig. 3). Zn has KD values slightly < 1 for garnet in equilibrium with basaltic to andesitic melts 32 (Fig. 3 and Supplementary Table S1). At intermediate stages of magmatic evolution (andesite, dacite) Zn becomes increasingly compatible in amphibole, clinopyroxene (Fig. 3) and biotite (KD ~18 in dacitic melts: Supplementary Table S1). The onset, in the most evolved stages of arc magmas, of crystallization of biotite and magnetite, plus other accessory mineral phases (e.g., ilmenite) for which Zn has a strong affinity, is likely responsible for the strong Zn decreases in most arcs below 2–4 wt.% MgO. Only some thin arcs do not display such a decrease (South Sandwich, Kermadec, New Hebrides, Kurile), perhaps because not enough differentiated rocks occur in the databases of these arcs. Zn contents display a strong increase in the most differentiated rocks of the Lesser Antilles arc. Although these trends in the most differentiated rocks may be of interest for those specific arcs, their discussion is beyond the scope of this work which considers the Zn-MgO trends of the early to intermediate differentiating magmas, excluding the most differentiated rocks. Modelling trends in the MgO-SiO2 and MgO-Zn spaces In order to quantify the relationship of Zn-MgO-SiO2 systematics with fractionating mineral assemblage of arcs with different thickness, mass balance calculations using a Monte Carlo approach (see Methods and Supplementary Tables S2-S3) have been used to model simultaneously the Zn-MgO and MgO-SiO2 trends of the different arcs (and of MOR magmas) through fractionation of the main phenocrystic minerals occurring in mafic to intermediate arc and MOR magmas (olivine, amphibole, clinopyroxene, plagioclase, magnetite, garnet). This corresponds to reproducing the trends through fractionation of mineral assemblages in the tridimensional SiO2-MgO-Zn space. This approach provides stringent constraints to the combination of mineral proportions and residual melt fractions that satisfy simultaneously the Zn-MgO and MgO-SiO2 trends. The model was run using a home-made RStudio script (see Methods and the examples of Supplementary Data File 1). The solutions of the simulations returned the combinations of bulk fractionating mineral assemblages and residual melt fractions able to reproduce the end point in the tridimensional SiO2-MgO-Zn space of the trends of each arc starting from an appropriate parental composition in the same tridimensional space (Supplementary Figures S1-S2). Overall, results of the Monte Carlo simulations of fractionation processes applied to Zn-MgO-SiO2 systematics (Supplementary Table S4) show that the fractionating mineral assemblages gradually shift from plagioclase-dominated in thin arcs to amphibole-, magnetite-, and garnet-dominated in increasingly thicker arcs (Fig. 4). Clinopyroxene and olivine do not significantly correlate with crustal thickness, suggesting that these minerals act as Page 7/19 buffers in the fractionating assemblages. High proportions of both olivine and plagioclase in fractionating magmas of thin arcs are needed to explain on one hand the steep decrease of MgO at low SiO2 values (olivine effect, because of SiO2/MgO < 1 in olivine: Supplementary Table S5) and on the other the broadly incompatible behavior of Zn (plagioclase effect, due to the very low KD values of Zn in plagioclase: Fig. 3). In contrast the high proportions of clinopyroxene, amphibole and garnet and the low proportions of plagioclase in fractionating magmas in thick arcs are consistent with both the shallower decrease of MgO with SiO2 (due to the high SiO2/MgO values in all these minerals, between 3 and 4: Supplementary Table S5) and with the slightly compatible behavior of Zn (due to the KD values of Zn in these minerals around or slightly > 1: Fig. 3). These results agree with petrographic observations that phenocrysts in relatively primitive thin arc rocks (e.g., Mariana, South Sandwich: refs. 23,33,34) consist of olivine, plagioclase and pyroxenes (with virtually no amphibole), whereas relatively primitive rocks and cumulates of thick arcs (e.g., Ecuador, Mexico, Cascades, Central Andes: refs. 35–38) contain variable amounts of amphibole. They also support and quantify the suggestion that extensive cryptic amphibole (and garnet) fractionation may occur in arcs 39, especially in increasingly thicker ones. Crust thickness control on fractionating assemblages The systematic correlations of the changing proportions of fractionating amphibole, garnet, magnetite, olivine and plagioclase with changing crustal thickness are consistent with experimental petrology results carried out on hydrous basaltic to andesitic melts fractionating at different pressures40– 47 . These results show that plagioclase, clinopyroxene and olivine are the main minerals crystallizing at the liquidus of hydrous mafic melts at low pressures (e.g., < 0.1–0.3 GPa depending on H2O content) whereas amphibole, garnet, clinopyroxene, garnet, magnetite crystallize at or near the liquidus of hydrous mafic melts at high pressures (> 0.8 GPa). The data presented and discussed here suggest that there is a gradual and continuous crustal thickness-controlled change in the proportions of fractionating minerals between the above two end-member assemblages that results in the systematic changes of SiO2-MgO-Zn trends of arc magmas. The preferential fractionation of amphibole and garnet in thicker arcs is unlikely to result only from higher H2O contents in the primitive basalts of thicker arcs10,11,14, considering that the H2O contents of primitive arc basalts are broadly similar for all arcs independent of their thickness 48. In addition, the occurrence of variable amounts of garnet required for the thicker arcs by the modelling here presented indicates pressures of crystallization of at least 0.8 GPa even in H2O-rich magmas 42. A thicker crust will result, as suggested by the data above discussed, in an average magma evolution at deeper levels 8 which will increase H2O contents in the residual magma more significantly than magma evolution at shallower levels already in the early fractionation stages (Supplementary Note 2 in Supplementary Information), because of the strong pressure dependency of H2O solubility in silicate melts49. This will, in turn, further stabilize the fractionation of amphibole and garnet from relatively unevolved basaltic andesite and andesite magmas at mid- to lower crustal levels41,42. If a systematic H2O enrichment in primary basalts of thick arcs does occur11,14, this would further enhance amphibole and garnet fractionation in thick arcs. The variable crustal thickness-controlled proportions of the fractionating mineral assemblages obtained by modelling Zn-MgO-SiO2 arc systematics also correlate with the median Fe2O3tot values at 4–6 wt.% MgO of arcs (Fig. 5), which are a measure of the tholeiitic versus calc-alkaline character of arc magmas7,50. Overall, these data suggest that first order processes of differentiation observed in arc magmas and the generation of a continuous transition from tholeiitic to calc-alkaline character are the result of pressure-dependent stability of different fractionating mineral phases (see also ref. 25 ), which is ultimately controlled by crustal thickness. A thicker crust results in an average evolution of arc magmas at deeper crustal levels and, therefore, is characterized by fractionation of higher-pressure assemblages (olivine, clinopyroxene, amphibole, garnet, magnetite) from the hydrous basalts typical of the arc environment. This leads to the development of a typical calc-alkaline trend in associated arc magmas (Fig. 5). In contrast, a thinner crust results in an average shallower crustal evolution of arc magmas characterized by the fractionation of the assemblage olivine, plagioclase, pyroxenes from such hydrous basalts. This leads to the development of tholeiitic trends in associated arc magmas (Fig. 5). Nonetheless, it is important to highlight that the results presented here suggest that there is a continuum between these two extremes without a neat subdivision between calc-alkaline and tholeiitic trends, but rather a gradual transition, that is controlled by the role that crustal thickness of the arc has on the proportions of fractionating minerals. It is significant that MOR magmas, which are almost anhydrous 51, fall on the continuation of the trends of Figs. 1b and 2b suggesting that Zn-MgO and MgO-SiO2 systematics seem to be insensitive to the largely different H2O contents of primitive basalts in MOR (~ 0.1–0.2 wt.%: ref. 51) and thin arcs (~ 4 wt.%: Plank et al., 2013) and that crustal thickness seems to be the main controlling factor on the different Zn-MgO-SiO2 systematics of magmas in these distinct settings. Discussion The results above discussed have several implications for large-scale processes associated with arc magmatism, encompassing Zn contents in the mantle, the crust role in modulating the H2O flux from mantle to Earth surface, and the formation of porphyry Cu deposits. The most primitive basalts from both thick and thin arcs and from MOR have similar Zn contents (~ 75–80 ppm: Fig. 1a). This suggests that, like Cu 52 , arc basalts and MORB derive from a similar mantle in terms of Zn contents and that Zn, like Cu 53, is not significantly enriched in the mantle wedge Page 8/19 by subduction-related processes. A possibility allowing for a Zn flux from the subducted slab would be that the mantle wedge be depleted in Zn compared to MOR mantle (Fig. 6a). However, this seems to be inconsistent with available data suggesting similar Zn contents for both Primitive and Depleted Mantle (~ 55 ppm: https://earthorg/GERM) and even higher Zn contents in Enriched Mantle types (> 100 ppm:ref. 17). Alternatively, differential partial melting in subduction versus MOR settings (Methods) could possibly be compensated by a Zn flux from the slab in the subduction setting if mantle wedge-derived basalts were Zn-depleted with respect to MORBs. However, also this seems to be unlikely. In fact, using available partition coefficients for olivine, clinopyroxene and orthopyroxene 17, that allow calculating the bulk partition coefficient between basaltic melt and the above mentioned lherzolite minerals, it results that Zn is only slightly incompatible during mantle melting (see Methods). It follows that the 75–80 ppm content in a primary mantle-derived basalt is quite insensitive to the melt fraction, using either batch melt or fractional melt models (Fig. 6b). In contrast to the similar Zn contents of primitive basaltic rocks in arcs and MOR, primitive Hawaii basalts display a significant Zn enrichment (> 100 ppm), which is consistent with their derivation from an enriched mantle source 22; 54 compared to that sourcing basalts in arcs and MOR (Fig. 1a). The results of this work also indicate that the suggested role of amphibole as a crustal filter of mantle-derived water is modulated by the crustal thickness of the arc: in other words, not all arcs act as a crustal sponge for H2O. In fact, results here presented suggest that intermediate magmas in the thinnest arcs carry towards the surface > 99% of the average initial ~ 4 wt.% H2O content 48 of their basaltic parent (because very little H2O is lost to the trivial amounts of amphibole crystallizing in thin crust: Fig. 7 and Supplementary Table S5). This corresponds to theoretical H2O concentrations of such intermediate magmas of ~ 10 wt.% (assuming that they correspond to ~ 0.4 residual melt fraction as suggested by the model here presented: Supplementary Table S5). In order to solubilize this H2O concentration in an intermediate silicate melt a depth of at least ~ 20 km (~ 0.6 GPa) is needed49 (Fig. 8), which is thicker than that of the thinnest intraoceanic arcs (Table 1). Therefore, magmas in such thin arcs exsolve and loose water since the early stages of differentiation because they cannot evolve at depths high enough to allow them to retain in solution the H2O they carry from the mantle during differentiation (Fig. 8; Supplementary Note 2 in Supplementary Information). Thus, the mantle-derived H2O is nearly completely fluxed towards the surface in thin arcs (Fig. 8). In contrast, primitive basalts in thick continental arcs can lose up to ~ 10% of their initial H2O (also ~ 4 wt.%: ref. 48) to amphibole crystallized in the crust (Fig. 7 and Supplementary Table S5). This implies the formation of progressively more abundant amphibole-rich cumulates in the crust of increasingly thicker arcs. Therefore, only in thick arcs a significant portion of mantle-derived H2O is, at least temporarily, locked in amphibole-rich cumulates. Such structurally-bound H2O may subsequently be released during ascent of magma incorporating cumulate amphibole 38 or during metamorphism to promote dehydration-assisted crustal melting or source fluids that may be at the origin of different types of ore deposits 55. The data here presented and discussed also provide quantitative explanations for the link between various geochemical systematics of arc magmas (e.g., Cu and Sr/Y: refs. 7–9) and arc thickness-controlled fractionation of different mineral assemblages. The systematic loss of Cu in intermediate magmas of increasingly thicker arcs could be the result of continuous iron depletion in this setting 56 due to fractionation not only of magnetite 7 and garnet 56, as previously suggested, but also of abundant amphibole (Figs. 5 and 9a) as recently suggested by 57: fractionation of all these minerals drive the evolution of thick arc magmas into the calc-alkaline field. A dominant proportion of amphibole in the fractionating assemblage of thick arc magmas (Fig. 4a-b) is also consistent with the dominant spoon-shaped REE patterns of thick arc magmas. Also the progressively higher Sr/Y values in intermediate magmas of increasingly thick arcs8,9 can be quantitatively explained by the increasing amounts of fractionating amphibole plus garnet (Fig. 9b) and decreasing amounts of fractionating plagioclase in thicker arcs. All these inter-correlations suggest an overarching role played by crustal thickness-controlled differential fractionation of amphibole plus garnet, plagioclase and magnetite in the development of variable Sr/Y and calcalkaline signatures in arc magmas. Despite the apparent negative effects of the thick arc setting on H2O and Cu contents of residual thick arc magmas discussed above, the latter are the most fertile for the formation of porphyry Cu deposits because they compensate such losses with a deeper crust evolution of the magmas 58. This leads to significant magma volume accumulation through time in the lower to mid-crust, with such magma being characterized by high H2O concentrations 58 because of the pressure dependency of H2O solubility in silicate magmas49 (Fig. 8; Supplementary Note 2 in Supplementary Information). In fact, in the thickest arc settings, residual intermediate magmas, despite losing ~ 10% of the initial H2O to amphibole crystallizing in the crust (Fig. 7), still hold ~ 90% of the average initial mantle-derived ~ 4 wt.% H2O content 48. This corresponds to H2O concentrations between ~ 7 and ~ 11 wt% in the residual melts (for residual melt fractions between 30 and 50% as calculated in the model here presented: Supplementary Table S5 and Fig. 8). These H2O contents are soluble in intermediate melts at depths between 12 and 22 km49 which are largely within the crustal thickness of thick arcs. Such large amounts of H2O can be subsequently released when thick arc intermediate magmas ascend to shallower levels where they can form porphyry Cu deposits 58. These data could finally explain the association of Au-rich porphyry Cu-Au deposits with thinner island arcs (e.g., Indonesia, Papua New Guinea, Philippines: ref. 4) which would be favored by the early H2O exsolution typical of thin arc magmas (Fig. 8). Such early fluid exsolution, during the magma differentiation process, allows Au partitioning into the fluid phase before magmatic sulfide saturation, which would strongly deplete Au in the residual magma and make Au-rich porphyry mineralization impossible 59. Declarations Data availability statement: All data needed to evaluate the conclusions in the paper are present in the paper and/or in the Supplementary Information. Page 9/19 Author contributions: MC designed the work, wrote the manuscript and drafted the figures. Funding: This study was funded by the Swiss National Foundation (grant N. 200021_169032). Competing interests: The author declares no competing interests. Data Set 1: Datasets for individual arcs, MORB and Hawaii and calculated median values for MgO bins of ~0.5 wt.%. Data Set 2: Dataset of Monte Carlo simulations. References 1. Gazel, E. et al. Continental crust generated in oceanic arcs. Nature Geoscience8, 321–327 (2015). 2. Sillitoe, R. H. A Plate Tectonic Model for the Origin of Porphyry Copper Deposits. Economic Geology67, 184–197 (1972). 3. Wilkinson, J. J. Triggers for the formation of porphyry ore deposits in magmatic arcs. Nature Geoscience6, 917–925 (2013). 4. Chiaradia, M. Gold endowments of porphyry deposits controlled by precipitation efficiency. Nature Communications11, 248 (2020). 5. Sheldrake, T., Caricchi, L. & Scutari, M. Tectonic Controls on Global Variations of Large-Magnitude Explosive Eruptions in Volcanic Arcs. Front. Earth Sci.8, (2020). 6. Zellmer, G. F., Edmonds, M. & Straub, S. M. Volatiles in subduction zone magmatism. Geological Society, London, Special Publications410, 1–17 (2015). 7. Chiaradia, M. Copper enrichment in arc magmas controlled by overriding plate thickness. Nature Geoscience7, 43–46 (2014). 8. Chiaradia, M. Crustal thickness control on Sr/Y signatures of recent arc magmas: an Earth scale perspective. Scientific Reports5, 8115 (2015). 9. Profeta, L. et al. Quantifying crustal thickness over time in magmatic arcs. Scientific Reports5, 17786 (2015). 10. Turner, S. J. & Langmuir, C. H. The global chemical systematics of arc front stratovolcanoes: Evaluating the role of crustal processes. Earth and Planetary Science Letters422, 182–193 (2015). 11. Turner, S. J. & Langmuir, C. H. What processes control the chemical compositions of arc front stratovolcanoes? Geochemistry, Geophysics, Geosystems16, 1865–1893 (2015). 12. Coulon, C. & Thorpe, R. S. Role of continental crust in petrogenesis of orogenic volcanic associations. Tectonophysics77, 79–93 (1981). 13. Lee, C.-T. A., Lee, T. C. & Wu, C.-T. Modeling the compositional evolution of recharging, evacuating, and fractionating (REFC) magma chambers: Implications for differentiation of arc magmas. Geochimica et Cosmochimica Acta143, 8–22 (2014). 14. Chin, E. J., Shimizu, K., Bybee, G. M. & Erdman, M. E. On the development of the calc-alkaline and tholeiitic magma series: A deep crustal cumulate perspective. Earth and Planetary Science Letters482, 277–287 (2018). 15. Perrin, A., Goes, S., Prytulak, J., Rondenay, S. & Davies, D. R. Mantle wedge temperatures and their potential relation to volcanic arc location. Earth and Planetary Science Letters501, 67–77 (2018). 16. Lee, C.-T. A. et al. The redox state of arc mantle using Zn/Fe systematics. Nature468, 681–685 (2010). 17. Le Roux, V., Dasgupta, R. & Lee, C.-T. A. Mineralogical heterogeneities in the Earth’s mantle: Constraints from Mn, Co, Ni and Zn partitioning during partial melting. Earth and Planetary Science Letters307, 395–408 (2011). 18. Chen, H., Savage, P. S., Teng, F.-Z., Helz, R. T. & Moynier, F. Zinc isotope fractionation during magmatic differentiation and the isotopic composition of the bulk Earth. Earth and Planetary Science Letters369–370, 34–42 (2013). 19. Pons, M.-L., Debret, B., Bouilhol, P., Delacour, A. & Williams, H. Zinc isotope evidence for sulfate-rich fluid transfer across subduction zones. Nature Communications7, 13794 (2016). 20. Paniello, R. C., Day, J. M. D. & Moynier, F. Zinc isotopic evidence for the origin of the Moon. Nature490, 376–379 (2012). 21. Shimazaki, H. & MacLean, W. H. An experimental study on the partition of zinc and lead between the silicate and sulfide liquids. Mineral. Deposita11, 125–132 (1976). 22. Le Roux, V., Lee, C.-T. A. & Turner, S. J. Zn/Fe systematics in mafic and ultramafic systems: Implications for detecting major element heterogeneities in the Earth’s mantle. Geochimica et Cosmochimica Acta74, 2779–2796 (2010). 23. Pearce, J. A., Baker, P. E., Harvey, P. K. & Luff, I. W. Geochemical Evidence for Subduction Fluxes, Mantle Melting and Fractional Crystallization Beneath the South Sandwich Island Arc. J Petrology36, 1073–1109 (1995). 24. Nandedkar, R. H., Ulmer, P. & Müntener, O. Fractional crystallization of primitive, hydrous arc magmas: an experimental study at 0.7 GPa. Contrib Mineral Petrol167, 1–27 (2014). 25. Lee, C.-T. A. & Bachmann, O. How important is the role of crystal fractionation in making intermediate magmas? Insights from Zr and P systematics. Earth and Planetary Science Letters393, 266–274 (2014). 26. Spera, F. J. & Bohrson, W. A. Energy-Constrained Open-System Magmatic Processes I: General Model and Energy-Constrained Assimilation and Fractional Crystallization (EC-AFC) Formulation. J Petrology42, 999–1018 (2001). Page 10/19 27. Reubi, O. & Blundy, J. A dearth of intermediate melts at subduction zone volcanoes and the petrogenesis of arc andesites. Nature461, 1269–1273 (2009). 28. Petford, N. & Gallagher, K. Partial melting of mafic (amphibolitic) lower crust by periodic influx of basaltic magma. Earth and Planetary Science Letters193, 483–499 (2001). 29. Spera, F. J. & Bohrson, W. A. Energy-constrained open-system magmatic processes 3. Energy-Constrained Recharge, Assimilation, and Fractional Crystallization (EC-RAFC). Geochemistry, Geophysics, Geosystems3, 1–20 (2002). 30. Holland, H. D., Rudnick, R. L. & Turekian, K. K. The Crust. (Elsevier, 2005). 31. Annen, C., Blundy, J. D. & Sparks, R. S. J. The Genesis of Intermediate and Silicic Magmas in Deep Crustal Hot Zones. Journal of Petrology47, 505–539 (2006). 32. Pertermann, M., Hirschmann, M. M., Hametner, K., Günther, D. & Schmidt, M. W. Experimental determination of trace element partitioning between garnet and silica-rich liquid during anhydrous partial melting of MORB-like eclogite. Geochemistry, Geophysics, Geosystems5, (2004). 33. Woodhead, J. D. The Origin of Geochemical Variations in Mariana Lavas: A General Model for Petrogenesis in Intra-Oceanic Island Arcs? J Petrology29, 805–830 (1988). 34. Meijer, A. & Reagan, M. Petrology and geochemistry of the island of Sarigan in the Mariana arc; calc-alkaline volcanism in an oceanic setting. Contr. Mineral. and Petrol.77, 337–354 (1981). 35. Chiaradia, M. Adakite-like magmas from fractional crystallization and melting-assimilation of mafic lower crust (Eocene Macuchi arc, Western Cordillera, Ecuador). Chemical Geology265, 468–487 (2009). 36. Dessimoz, M., Müntener, O. & Ulmer, P. A case for hornblende dominated fractionation of arc magmas: the Chelan Complex (Washington Cascades). Contrib Mineral Petrol163, 567–589 (2012). 37. Luhr, J. F. & Carmichael, I. S. E. Jorullo Volcano, Michoacán, Mexico (1759–1774): The earliest stages of fractionation in calc-alkaline magmas. Contr. Mineral. and Petrol.90, 142–161 (1985). 38. Velázquez Santana, L. C., McLeod, C. L., Blakemore, D., Shaulis, B. & Hill, T. Bolivian hornblendite cumulates: Insights into the depths of Central Andean arc magmatic systems. Lithos370–371, 105618 (2020). 39. Davidson, J., Turner, S., Handley, H., Macpherson, C. & Dosseto, A. Amphibole “sponge” in arc crust? Geology35, 787–790 (2007). 40. Moore, G. & Carmichael, I. S. E. The hydrous phase equilibria (to 3 kbar) of an andesite and basaltic andesite from western Mexico: constraints on water content and conditions of phenocryst growth. Contrib Mineral Petrol130, 304–319 (1998). 41. Müntener, O., Kelemen, P. B. & Grove, T. L. The role of H2O during crystallization of primitive arc magmas under uppermost mantle conditions and genesis of igneous pyroxenites: an experimental study. Contrib Mineral Petrol141, 643–658 (2001). 42. Alonso-Perez, R., Müntener, O. & Ulmer, P. Igneous garnet and amphibole fractionation in the roots of island arcs: experimental constraints on andesitic liquids. Contributions to Mineralogy and Petrology157, 541–558 (2009). 43. Sisson, T. W. & Grove, T. L. Experimental investigations of the role of H2O in calc-alkaline differentiation and subduction zone magmatism. Contr. Mineral. and Petrol.113, 143–166 (1993). 44. Pichavant, M. & Macdonald, R. Crystallization of primitive basaltic magmas at crustal pressures and genesis of the calc-alkaline igneous suite: experimental evidence from St Vincent, Lesser Antilles arc. Contrib Mineral Petrol154, 535–558 (2007). 45. Loucks, R. R. Distinctive composition of copper-ore-forming arcmagmas. Australian Journal of Earth Sciences61, 5–16 (2014). 46. Barclay, J. & Carmichael, I. S. E. A Hornblende Basalt from Western Mexico: Water-saturated Phase Relations Constrain a Pressure–Temperature Window of Eruptibility. J Petrology45, 485–506 (2004). 47. Feig, S. T., Koepke, J. & Snow, J. E. Effect of water on tholeiitic basalt phase equilibria: an experimental study under oxidizing conditions. Contrib Mineral Petrol152, 611–638 (2006). 48. Plank, T., Kelley, K. A., Zimmer, M. M., Hauri, E. H. & Wallace, P. J. Why do mafic arc magmas contain ∼4wt% water on average? Earth and Planetary Science Letters364, 168–179 (2013). 49. S. Newman & J. B. Lowenstern. VolatileCalc: a silicate melt–H2O–CO2solution modelwritten in Visual Basic for excel. Computers & Geosciences28, 597–604 (2002). 50. Zimmer, M. M. et al. The Role of Water in Generating the Calc-alkaline Trend: New Volatile Data for Aleutian Magmas and a New Tholeiitic Index. J Petrology51, 2411–2444 (2010). 51. Sobolev, A. V. & Chaussidon, M. H2O concentrations in primary melts from supra-subduction zones and mid-ocean ridges: Implications for H2O storage and recycling in the mantle. Earth and Planetary Science Letters137, 45–55 (1996). 52. Lee, C.-T. A. et al. Copper Systematics in Arc Magmas and Implications for Crust-Mantle Differentiation. Science336, 64–68 (2012). 53. Rezeau, H. & Jagoutz, O. The importance of H2O in arc magmas for the formation of porphyry Cu deposits. Ore Geology Reviews126, 103744 (2020). 54. Weis, D., Garcia, M. O., Rhodes, J. M., Jellinek, M. & Scoates, J. S. Role of the deep mantle in generating the compositional asymmetry of the Hawaiian mantle plume. Nature Geoscience4, 831–838 (2011). 55. Yardley, B. W. D. & Bodnar, R. J. Fluids in the Continental Crust. GeochemPersp3, 1–127 (2014). Page 11/19 56. Lee, C.-T. A. & Tang, M. How to make porphyry copper deposits. Earth and Planetary Science Letters529, 115868 (2020). 57. Barber, N. D., Edmonds, M., Jenner, F., Audétat, A. & Williams, H. Amphibole Control on Copper Systematics in Arcs: Insights from the Analysis of Global Datasets. Geochimica et Cosmochimica Acta (2021) doi:10.1016/j.gca.2021.05.034. 58. Chiaradia, M. & Caricchi, L. Stochastic modelling of deep magmatic controls on porphyry copper deposit endowment. Scientific Reports7, 44523 (2017). 59. Park, J.-W., Campbell, I. H., Chiaradia, M., Hao, H. & Lee, C.-T. A. Crustal magmatic controls on the formation of porphyry copper deposits. Nature Reviews - Earth & Environmentin press, (2021). 60. Zellmer, G. F. Some first-order observations on magma transfer from mantle wedge to upper crust at volcanic arcs. Geological Society, London, Special Publications304, 15–31 (2008). 61. Contreras‐Reyes, E. et al. Deep seismic structure of the Tonga subduction zone: Implications for mantle hydration, tectonic erosion, and arc magmatism. Journal of Geophysical Research: Solid Earth116, (2011). 62. Turner, S. & Hawkesworth, C. Constraints on flux rates and mantle dynamics beneath island arcs from Tonga–Kermadec lava geochemistry. Nature389, 568–573 (1997). 63. Janiszewski, H. A., Abers, G. A., Shillington, D. J. & Calkins, J. A. Crustal structure along the Aleutian island arc: New insights from receiver functions constrained by active-source data. Geochemistry, Geophysics, Geosystems14, 2977–2992 (2013). 64. Aranda, N. & Assumpç?o, M. Crustal thickness in the northern Andes from teleseismic pP and sS precursors. in 13th International Congress of the Brazilian Geophysical Society & EXPOGEF, Rio de Janeiro, Brazil, 26?29 August 2013 1781–1785 (Brazilian Geophysical Society, 2013). doi:10.1190/sbgf2013-366. 65. Bassin, C. The Current Limits of resolution for surface wave tomography in North America. Eos, Transactions American Geophysical Union (2000) doi:null. 66. Mooney, W. D., Laske, G. & Masters, T. G. CRUST 5.1: A global crustal model at 5° × 5°. Journal of Geophysical Research: Solid Earth103, 727–747 (1998). 67. Mantle, G. W. & Collins, W. J. Quantifying crustal thickness variations in evolving orogens: Correlation between arc basalt composition and Moho depth. Geology36, 87–90 (2008). 68. Plank, T. & Langmuir, C. H. An evaluation of the global variations in the major element chemistry of arc basalts. Earth and Planetary Science Letters90, 349–370 (1988). 69. Minshull, T. A. Oceanic Crust. in Encyclopedia of Physical Science and Technology (Third Edition) (ed. Meyers, R. A.) 91–98 (Academic Press, 2003). doi:10.1016/B0-12-227410-5/00508-1. 70. Kelley, K. A. et al. Mantle melting as a function of water content beneath back-arc basins. Journal of Geophysical Research: Solid Earth111, (2006). Figures Page 12/19 Figure 1 Zn-MgO systematics of arc, MOR and Hawaii magmatic rocks. (a) Zn-MgO plot of the averages of median values of all thin (<20 km) and all thick (>30 km) arcs compared to median values of MOR and Hawaii magmas for MgO bins ~0.5 wt.% (data reported in Supplementary Data File 1); (b) plot of the Zn-MgO slopes of individual arcs and MOR magmatic rocks versus crustal thickness (data reported in Table 1). The light red symbols denote poor statistical significance for the Zn-MgO regressions (see Table 1). Page 13/19 Figure 2 MgO-SiO2 systematics of arc and MOR magmatic rocks. (a) MgO-SiO2 plot of the averages of median values of all thin (<20 km) and all thick (>30 km) arcs compared to median values of MOR magmas for MgO bins ~0.5 wt.% (data reported in Supplementary Data File 1); (b) plot of the MgO-SiO2 slopes of the early evolutionary parts (MgO>2-5 wt.% depending on the arc) of individual arcs and MOR magmatic rocks versus crustal thickness (data reported in Table 1). Page 14/19 Figure 3 Zn partition coefficient (KD) values for mineral-melts of different compositions The KD values are reported as median values (coloured ticks) and their 20th and 80th percentile (upper and lower boundaries of the coloured fields). Source data are reported in Supplementary Table S1. Page 15/19 Figure 4 Results of Monte Carlo modelling of fractionating mineral proportions. (a) Proportion of amphibole fractionating in arc magmas with respect to arc thickness; (b) proportion of amphibole (Amph) + garnet (gar) fractionating in arc magmas with respect to arc thickness; (c) proportion of plagioclase fractionating in arc magmas with respect to arc thickness; (d) proportion of magnetite fractionating in arc magmas with respect to arc thickness. Data are reported in Supplementary Table S4. Page 16/19 Figure 5 Proportions of mineral fractions versus tholeiitic character. (a) Proportions of fractionating amphibole + garnet versus the tholeiitic index (as defined by 7; (b) proportions of fractionating plagioclase versus the tholeiitic index (as defined by 7. Different circle sizes highlight different arc crust thicknesses. Figure 6 Zn contents of mantle-derived melts. (a) Zn contents of batch melting mantle-derived melts starting from mantles with different initial Zn contents (30 and 55 ppm); (b) Zn contents of batch melting and fractional melting mantle-derived melts starting from a mantle with 55 ppm Zn. Page 17/19 Figure 7 H2O stored in amphibole crystallized within the crust versus crustal thickness. H2O is the percentage of the initial H2O (~4 wt.%: 48 in primitive arc basalts. Data from Supplementary Table S5. Figure 8 Variations of H2O solubility (a) and excess H2O (b) with changing residual melt fraction at 4 different pressures of magma fractionation (see Supplementary Note 2 in Supplementary Information). The starting H2O content at all pressures is 3.9-4.1 wt.% and the H2O is considered to behave as completely incompatible. The pressure and melt composition dependency of H2O solubility in silicate melts are from the parametrization of ref. 58 based on the model of ref. 49. The different curves are the result of >1000 simulations for ranges of initial H2O content of 3.9-4.1 wt.% and pressures ranges of 0.19-0.21, 0.39-0.41, 0.59-0.61 and 0.79-0.81 GPa. The kinks of the curves in Figure 8a indicate the point at which H2O saturation occurs Page 18/19 (i.e., when on the right hand plot the curves raise above 0 wt.% excess H2O). In Figure 8b negative values indicate H2O-undersaturated conditions whereas positive ones indicate H2O-saturated conditions. At 0.2 GPa the magma is already saturated without any crystallization if it has an initial H2O content between 3.9 and 4.1 wt.%. Figure 9 Proportions of fractionating minerals versus Cu and Sr/Y. (a) Proportions of fractionating amphibole versus Cu at 4-6 wt.% MgO (as defined by 7; (a) proportions of fractionating plagioclase versus Sr/Y at 4-6 wt.% MgO (as defined by 8). Different circle sizes highlight different arc crust thicknesses. Bismark-New Britain and New Hebrides are excluded from the plot b because their high Sr/Y values likely result from slab melting 8. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SupplementaryMaterial.pdf Page 19/19
https://openalex.org/W259784655	https://molmed.biomedcentral.com/track/pdf/10.1007/BF03402218	English	null	iNOS Expression In Dystrophinopathies Can Be Reduced By Somatic Gene Transfer of Dystrophin or Utrophin	Molecular medicine	2,001	cc-by	10,047	Address correspondence and reprint requests to: James M. Wilson, M.D., Ph.D. Institute for Human Gene Therapy, 204 Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104-4268, USA. Phone: (215) 898-3000. Fax: (215) 898-6588. E-mail: wilsonjm@mail.med.upenn.edu iNOS Expression In Dystrophinopathies Can Be Reduced By Somatic Gene Transfer of Dystrophin or Utrophin Jean-Pierre Louboutin,1 Karl Rouger,2 Jonathon M. Tinsley,3 Jeff Halldorson,1 and James M. Wilson.1 1Institute for Human Gene Therapy and Department of Molecular and Cellular Engineering, Wistar Institute, University of Pennsylvania, Philadelphia, PA 19104, USA Jean-Pierre Louboutin,1 Karl Rouger,2 Jonathon M. Tinsley,3 Jeff Halldorson,1 and James M. Wilson.1 1Institute for Human Gene Therapy and Department of Molecular and Cellular Engineering, Wistar Institute, University of Pennsylvania, Philadelphia, PA 19104, USA 2UMR 533 Inserm-Hotel Dieu-44000-Nantes Cedex–France 3Department of Human Anatomy and Genetics, Oxford University, Oxford, UK OX1 3QU; Present Address: MRC Mammalian Genetics Unit, Harwell, UK Contributed by J.M. Wilson. Accepted January 15, 2001. Jean-Pierre Louboutin,1 Karl Rouger,2 Jonathon M. Tinsley,3 Jeff Halldorson,1 and James M. Wilson.1 1Institute for Human Gene Therapy and Department of Molecular and Cellular Engineering, Wistar Institute, University of Pennsylvania, Philadelphia, PA 19104, USA Jean-Pierre Louboutin,1 Karl Rouger,2 Jonathon M. Tinsley,3 Jeff Halldorson,1 and James M. Wilson.1 1Institute for Human Gene Therapy and Department of Molecular and Cellular Engineering, Wistar Institute, University of Pennsylvania, Philadelphia, PA 19104, USA 2UMR 533 Inserm-Hotel Dieu-44000-Nantes Cedex–France 3Department of Human Anatomy and Genetics, Oxford University, Oxford, UK OX1 3QU; Present Address: MRC Mammalian Genetics Unit, Harwell, UK Contributed by J.M. Wilson. Accepted January 15, 2001. 3Department of Human Anatomy and Genetics, Oxford University, Oxford, UK OX1 3QU; Present Address: MRC Mammalian Genetics Unit, Harwell, UK Abstract NADPH-d reactivity was increased and mainly localized in regenerating muscle ﬁbers. In mdx mice quadriceps, iNOS expression was mainly observed in regenerating muscle ﬁbers, but not prior to 4 weeks postnatal, and was still present 8 weeks after birth. The expression of dys- trophin and the overexpression of utrophin using adenovirus-mediated constructs reduced the number of iNOS-positive ﬁbers in mdx quadriceps muscles. The cor- rection of some pathology in mdx by dystrophin expres- sion or utrophin overexpression was independent of the presence of nNOS. Background: Nitric oxide (NO) is an inorganic gas pro- duced by a family of NO synthase (NOS) proteins. The presence and the distribution of inducible-NOS (NOS II or iNOS), and NADPH-diaphorase (NADPH-d), a marker for NOS catalytic activity, were determined in muscle sec- tions from control, DMD, and BMD patients. p Materials and Methods: NADPH-d reactivity, iNOS- and nNOS (NOS I)-immunolocalization were studied in mus- cles from mdx mice before and after somatic gene transfer of dystrophin or utrophin. Results: In control patients, few ﬁbers (2%) demon- strated focal accumulation of iNOS in sarcolemma. In DMD patients, a strong iNOS immunoreactivity was ob- served in some necrotic muscle ﬁbers as well as in some mononuclear cells, and regenerating muscle ﬁbers had dif- fusely positive iNOS immunoreactivity. In DMD patients, Conclusions: These results suggest that iNOS could play a role in the physiopathology of DMD and that the abnormal expression of iNOS could be corrected by gene therapy. Keywords: iNOS, DMD, dystrophin, utrophin, gene therapy. ally very low or absent. NO formed by iNOS is in- volved in nonspeciﬁc immune responses (8). Molecular Medicine 7(5): 355–364, 2001 © 2001 The Picower Institute Press Molecular Medicine 7(5): 355–364, 2001 © 2001 The Picower Institute Press Antibodies For immunocytochemistry, we used antibodies: anti-iNOS (mouse anti-iNOS, Transduction Labora- tories, KY; rabbit anti-iNOS, Calbiochem, CA; rab- bit anti-iNOS, Santa Cruz Laboratories, Santa Cruz, CA), anti-developmental myosin heavy chain (mouse anti-d-MHC), anti-dystrophin (mouse anti- dys 2) (Novocastra, UK), anti-complement mem- brane attack complex C5b-9 (mouse anti-C5b-9, Dako, CA), anti-p65 subunit of NFB (goat or rabbit anti-p65), anti-nNOS (rabbit anti-nNOS) (Santa Cruz, CA) and anti-myc (mouse anti-myc) (InVitro- gen, CA). All antibodies were diluted 1:100, except the anti-dys 2 and anti-d-MHC antibodies, which were diluted 1:20. Morphological Analysis, Histochemistry, and Immunocytochemistry General Characterization and Histochemistry Muscle specimens were frozen in isopentane cooled in liq- uid nitrogen. Transverse cryostat sections (10-m thick:Frigocut 2800; Reichert-Jung, Nussloch, Germany) were stained by hematoxylin and eosin. Fiber type classiﬁcation was made in serial sections stained with myoﬁbrillar adenosine triphosphatase (ATPase) (29). mdx Mice The histochemical staining for NADPH- diaphorase (NADPH-d) is a marker for NOS catalytic activity (6,10,12). NADPH-d localizes with nNOS in the muscle ﬁbers (12), and NADPH-d negative cell lines become NADPH-d positive after they are trans- fected with nNOS, iNOS or eNOS cDNAs (5–7). Sev- eral other enzymes besides NOS also demonstrate NADPH-d activity but unlike NOS, they are inacti- vated by paraformaldehyde ﬁxation (20). Homozygous mdx mice were obtained from the Jackson Laboratory (Bar Harbor, ME). All animals were kept in the animal care unit of the Wistar Institute according to animal care guidelines. mdx mice were studied at different times (14,21,28, 42,56, and 70 days postnatal). Quadriceps, Tibialis Anterior (TA), Extensor Digitorum Longus (EDL), Soleus (SOL), and diaphragm (DIA) muscles were studied. y p y ( ) Studies have recently shown that nNOS com- plexes with dystrophin and is absent from skeletal muscle sarcolemma in Duchenne muscular dystro- phy (DMD) and Becker muscular dystrophy (BMD), as well as in mdx mice (21,22). Moreover, the inter- action of nNOS with the post-synaptic density pro- tein PSD-95 and 1-syntrophin has been shown to be mediated by PDZ domains (23). However, some NOS is located within muscle mitochondria. iNOS expression was demonstrated in a number of neu- rodegenerative and neurologic inﬂammatory dis- eases (24–26), but only in one muscle disease (i.e., inclusion body myopathy (27). In this disorder, Yang et al. demonstrated iNOS immunoreactivity, colocalized with nNOS immunoreactivity within the vacuoles or in vacuole-free cytoplasm. Because of its putative implication in the physiopathology of the affections, it seemed interesting to study if iNOS was expressed in other neuromuscular disorders than inclusion body myopathy. No data are actually available concerning the expression of iNOS in muscle tissue from DMD patients. Moreover, if the involvement of iNOS has been shown in experi- mental crush injury (28), no in situ study of the ex- pression of iNOS has been performed in degenerat- ing/regenerating muscles, and the situation has not been examined in models of muscular dystrophies. The aim of the present study was to determine the distribution of iNOS in muscle tissue from DMD and BMD patients, as well as in the muscles from mdx mice. Moreover, we have studied the conse- quences of the expression of dystrophin and over- expression of utrophin following adenovirus- mediated gene transfer on the expression of iNOS and nNOS in mdx mice. Introduction muscles from non-weak control subjects (n 5) ob- tained during surgical intervention. The biopsy specimens of DMD/BMD were considered as “pre- existing pathological specimens” obtained for diag- nosis purpose and did not require informed consent. Informed consent was obtained from control sub- jects, and the Ethical Committee of the Institution approved the study. Introduction Nitric oxide (NO) is an inorganic gas that derives from L-arginine as the product of a complex enzy- matic reaction catalyzed by a family of three NO syn- thase (NOS) proteins (1). NO mediates a variety of biological functions, including intracellular signal transduction, neurotransmission, and vasodilatation (2–4). Two of the NOS are constitutively expressed and are low-input Ca2 activated enzymes whose physiological function is signal transduction. NOS I (nNOS) is prominent in neurons but also occurs in skeletal muscle ﬁbers, in epithelial cells of the lung, and in secretory cells of some endocrine glands (5,6). NOS III (eNOS) is present in all vascular en- dothelial cells (7). NOS II (iNOS), originally discov- ered in cytokine-induced macrophages, is a largely inducible calcium-independent NOS, which is expressed at highest levels in immunologically activated cells. In resting cells, iNOS levels are usu- NO is involved in the regulation of contractile re- sponses of muscle ﬁbers (9–11) and NOS activity is found in skeletal muscle tissue (9,12–15), as well as in skeletal muscle cell lines (16). nNOS is localized to the sarcolemma, with enrichment at the neuro- muscular endplates, myotendinous junctions, and costameres (9,10,17). nNOS is expressed at higher levels in muscles composed of type II muscle ﬁbers, particularly in rodents (9), and in both types I and II muscle ﬁbers in primates (12). Conﬂicting results ex- ist concerning the expression of iNOS in skeletal muscle. In one study, iNOS immunoreactivity was found in the particulate fraction of skeletal muscle (10), whereas another group failed to demonstrate iNOS immunoreactive bands on Western im- munoblots of ADP-sepharose-fractionated protein ex- tracts from skeletal muscle under control conditions (15). Following immunohistochemistry using anti- bodies against iNOS, Gath et al. reported a spotty distribution throughout the sarcoplasm in guinea-pig skeletal muscle (10). However, Young et al. (18) and Thompson et al. (15) reported that no speciﬁc im- munostaining was observed in guinea-pig and mouse Molecular Medicine, Volume 7, Number 4, 2001 356 skeletal muscles, respectively. Expression of iNOS was also noted in macrophages, endothelium, and to a lower extent, in myocytes during inﬂammatory con- ditions in vivo (i.e., LPS administration), which inten- siﬁed iNOS expression and immunostaining (10,15). It has been demonstrated that iNOS was induced in C2C12 cells by combination of cytokines (16). eNOS was also found in vascular endothelium of skeletal muscle tissue (19). NADPH-diaphorase (NADPH-d) Staining After a 10-min ﬁxation in 4% formaldehyde, the sections were washed in PBS then incubated for 30 min at 37C in a solution containing 1 mM - NADPH (Sigma, St-Louis, MO) and 0.3 mM nitro- blue tetrazolium (NBT), plus 1% formaldehyde. Sections from control and DMD/BMD patients were incubated together. Afterward, the sections were rinsed in PBS and ﬁnally mounted in glycerol jelly. To test the speciﬁcity of the reaction, tissue was in- cubated in the above reaction medium without the NADPH. J. Louboutin et al.: iNOS in DMD J. Louboutin et al.: iNOS in DMD 357 Immunocytochemistry Ten m-thick serial cryostat sections were ﬁxed for 3 min in acetone (except for dystrophin, myc, d-MHC single labeling). Blocking was performed by 1-hour incubation with 10% of goat serum or donkey serum, depending on the secondary antibody. For im- munoﬂuorescence, sections were incubated during 1 hour with the primary antibody at room temperature then washed with PBS, and incubated with either of the following:1) 1:100 diluted ﬂuorescein-labeled goat anti-mouse IgG (Sigma, St Louis, MO); 2) 1:100 diluted ﬂuorescein or rhodamin-labeled donkey anti- mouse IgG, (Jackson ImmunoResearch, ME); 3) 1:100 diluted ﬂuorescein or rhodamin-labeled don- key anti-rabbit IgG (Jackson ImmunoResearch, ME); or 4) 1:100 diluted ﬂuorescein or rhodamin-labeled donkey anti-goat IgG (Jackson ImmunoResearch, ME. Double immunostaining was also performed ac- cording to a modiﬁed protocol, with each incubation followed by extensive washing in PBS. Specimens were ﬁnally examined under ﬂuorescence microscope Nikon Microphot-FXA. For immunoperoxidase, sec- tions were incubated with 3% H2O2 to inactivate en- dogenous peroxidases and then were blocked with 10% donkey or goat serum. Sections were then rinsed in PBS and incubated for 1 hour at room temperature in primary antibody in PBS. After rinsing, im- munoreactions were visualized by a biotinylated anti-rabbit IgG or anti-mouse IgG (Sigma, St Louis, MO) diluted 1:100 in PBS for 30 min, an Extravidin peroxidase conjugate (Sigma, St Louis, MO) diluted 1:200 in PBS during 30 min, and an AEC staining system (Sigma, St Louis, MO). Specimens were ex- amined under a microscope Nikon Microphot-FXA. In all cases, following incubation, each specimen un- derwent extensive washing with PBS. As negative controls, we used preincubation by PBS, omission of the primary antibody and substitution of the primary antibody by an isotype-matched nonimmune control antibody. Mouse IgG1 and IgG2a (Dako), rabbit IgG (Dako), and goat IgG (Dako) were used as controls. The three types of controls mentioned were per- formed for each experiment on the same day. iNOS-, dystrophin-, myc-positive ﬁbers were counted in cross sections of muscle appropriately stained, and the number of each was calculated as a percentage of at least 300 ﬁbers from 5 different regions. Regions were selected on a random basis within a section of the muscle, using an image analyser (Phase Three Imaging Systems). Immunoblotting Sample Preparations from Muscle Biopsies for Immunoblotting The isolation method of the cytoso- lic fraction involved the preparation of muscular ho- mogenate in buffer (pH 7.2) containing 20 mM MOPS, 100 mM KCl, and subsequent differential centrifugations. All of these procedures were carried out at 04C. Muscle tissue (250–400 mg) was minced and homogenized in the above buffer by several strokes of the homogenizer. Homogenization was with a Polytron 20ST (Kinematica, Lucerne, Switzerland) for 30–35 sec at 20,000 rpm. The crude homogenate was centrifuged at 1000 g for 10 min. The resulting supernatant fraction was removed and the 1000 g pellet was washed twice with the ho- mogenization buffer. The combined 1000 g super- natant fractions were then centrifuged at 8000 g for 1 hr to separate sarcolemma, mitochondria, Golgi complex, and endoplasmic reticulum from the cy- tosolic fraction. The resultant supernatant fraction was stored immediately at 20C. SDS-polyacrylamide Gel Electrophoresis and Western Blotting The expression of iNOS protein was studied by Western blot analysis in isolated cytosolic fraction of skeletal muscle. Cytosolic proteins were pre- pared as described below and quantitated with a bicinchoninic acid protein assay reagent kit (Pierce Chemical, Rockford, IL). Western blot analysis was done by separating proteins (45 g) in reduc- ing conditions with 8% SDS-polyacrylamide gel electrophoresis and electrophoretically transfer- ring the products to nitrocellulose membranes (Bio-Rad, Paris, France). The efﬁciency of transfer was monitored by ponceau S staining of the nitro- cellulose ﬁlter. Prestained molecular weight markers were from Bio-Rad. Nonspeciﬁc binding to the membrane was blocked by 5% nonfat dry milk in PBS-0.1% (w/v) Tween 20 (Sigma, St. Louis, MO) for 1 hour at room temperature. Optimal concentra- tion of antibodies was determined in separate ex- periment. Blots were washed in PBS-0.1% (w/v) Tween 20 and then incubated overnight at 4C with monoclonal anti-iNOS antibody (1:1000 dilution, Transduction Laboratories, KY). Membranes were washed an additional three times in PBS containing 0.1% Tween 20 and 1% milk protein. Then, the bound antibodies were revealed, depending on sec- ondary antibodies,with either nitroblue tetrazolium Morphometry Immunocytochemistry Materials and Methods Muscle Biopsy Skeletal muscle biopsy samples were from the quadriceps muscles of patients presenting with DMD (n 6) and BMD (n 2) and from quadriceps Vital Staining Evans blue was purchased from Sigma (St Louis, MO). The dye was dissolved in PBS (0.15 M NaCl, 10 mM phosphate buffer, pH7), sterilized by passage through membrane ﬁlters, and kept at 4C. Dye solu- tion was injected intravenously through the tail vein (1 mg dye/0.1 mL/10 g body weight), and 6 hours af- ter injection, mice were sacriﬁced. Quadriceps mus- cles were frozen immediately in isopentane cooled in liquid nitrogen. Ten m-thick cryostat sections were mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA). By ﬂuorescence microscopy analysis, Evans blue staining showed a bright red emission. All sections were examined un- der a microscope Nikon Microphot-FXA. Molecular Medicine, Volume 7, Number 4, 2001 358 Fig. 1. Expression of iNOS in DMD muscle. (A)Western blot analysis of human quadriceps muscle extract for iNOS. Proteins were separated in reducing conditions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS) and transferred to nitrocellulose membrane. The blot was incubated with an antibody speciﬁc for iNOS and developed as described in Methods. Prestained molecular standards were shown on the left. Lane 1, positive control: mouse macrophage lysate prepared from the RAW 264.7 cell line, which were stimulated with IFN and LPS for 12 hr; lanes 3, 5, samples from patients with DMD; lane 7, patient with BMD; lanes 2, 4, 6, samples from control normal subjects. All lanes contain 45 g of total protein. No speciﬁc signal was detected on the muscle samples from control normal subjects whereas a signal was observed on the muscle samples from patients with DMD/BMD. (B) Transverse serial cryostat chloride (NBT 0.41 mM)/5-bromo-4-chloro-3- indolyl phosphate (BCIP 0.38 mM) (Promega) in 200 mM Tris-HCl, pH9.5, containing 10 mM MgCl2 as substrate, and peroxidase substrate kit AEC (Vector Laboratories, Burlingame, CA). chloride (NBT 0.41 mM)/5-bromo-4-chloro-3- indolyl phosphate (BCIP 0.38 mM) (Promega) in 200 mM Tris-HCl, pH9.5, containing 10 mM MgCl2 as substrate, and peroxidase substrate kit AEC (Vector Laboratories, Burlingame, CA). Gene Transfer E1 E3 deleted adenovirus vectors containing the hCMVie promoter driving either a 5.4 Kb 13-48 truncated dystrophin gene (DYS group), a 6.0 Kb truncated dystrophin-homologue (utrophin) gene (UTR group), or LacZ were injected to transduce the quadriceps of 5-day-old mdx mice. A myc TAG was incorporated into the utrophin transgene, enabling us to distinguish the transgene from the endogenous encoded protein. To serve as a vector control, a group of age-matched mdx mice was injected with Ad-LacZ. In each group, the contralateral quadriceps muscle was not injected with vector to serve as an internal control. Quadriceps muscles were harvested at day 42 postnatal. Fig. 1. Expression of iNOS in DMD muscle. (A)Western blot analysis of human quadriceps muscle extract for iNOS. Proteins were separated in reducing conditions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS) and transferred to nitrocellulose membrane. The blot was incubated with an antibody speciﬁc for iNOS and developed as described in Methods. Prestained molecular standards were shown on the left. Lane 1, positive control: mouse macrophage lysate prepared from the RAW 264.7 cell line, which were stimulated with IFN and LPS for 12 hr; lanes 3, 5, samples from patients with DMD; lane 7, patient with BMD; lanes 2, 4, 6, samples from control normal subjects. All lanes contain 45 g of total protein. No speciﬁc signal was detected on the muscle samples from control normal subjects whereas a signal was observed on the muscle samples from patients with DMD/BMD. (B) Transverse serial cryostat sections (10-m thick) of a deltoid muscle from control (left col- umn) and DMD (right column) patients stained by HE (upper row) and immunostained by an antibody anti-iNOS (lower row). Muscle ﬁbers from control patient did not exhibit immunoreactiv- ity for iNOS whereas a cytoplasmic staining for iNOS was observed in DMD muscle ﬁbers. No immunostaining of connec- tive tissue or vessels was seen in DMD. (C) NADPH-d reactivity in the muscle of control and DMD patients. Transverse serial cryostat sections (10-m thick) of a deltoid muscle from control (left column) and DMD (right column) patients stained by HE (upper row) and for NADPH-d reactivity (lower row). NADPH-d reactivity was lower in control patients compared to DMD patients. In DMD patients, NADPH-d reactivity was predomi- nantly found in the sarcoplasm where it showed a spotty dis- tribution. No reactivity was shown in the connective tissue. Some vessels exhibited NADPH-reactivity. Bar: 80 m. iNOS Is Expressed in DMD Muscle The presence of iNOS protein was investigated by Western blot analysis in freshly isolated cytosolic fractions of skeletal muscles from control subjects and DMD/BMD patients. Western blot of samples separated under reducing conditions revealed a band of 65 kDa in muscle samples from DMD patients (Figure 1A). No band was seen when ex- tracts from normal skeletal muscle were incubated under the same conditions. iNOS immunoreactivity was not detected on cryostat sections from muscle of control subjects, whereas cytoplasmic immuno- staining for iNOS was observed in cryostat sections from DMD/BMD patients. Immunoreactivity for iNOS was not detected in vessels or in the con- junctive tissue (Figure 1B). The percentage of iNOS-positive ﬁbers was different from one patient to another; however, iNOS-positive ﬁbers were more frequently observed in DMD than in BMD (2–4% in BMD patients; 6–18% in DMD patients). iNOS immunoreactivity was also rarely observed in small mononucleated cells invading muscle ﬁbers (data not shown). Fig. 1. Expression of iNOS in DMD muscle. (A)Western blot analysis of human quadriceps muscle extract for iNOS. Proteins were separated in reducing conditions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS) and transferred to nitrocellulose membrane. The blot was incubated with an antibody speciﬁc for iNOS and developed as described in Methods. Prestained molecular standards were shown on the left. Lane 1, positive control: mouse macrophage lysate prepared from the RAW 264.7 cell line, which were stimulated with IFN and LPS for 12 hr; lanes 3, 5, samples from patients with DMD; lane 7, patient with BMD; lanes 2, 4, 6, samples from control normal subjects. All lanes contain 45 g of total protein. No speciﬁc signal was detected on the muscle samples from control normal subjects whereas a signal was observed on the muscle samples from patients with DMD/BMD. (B) Transverse serial cryostat sections (10-m thick) of a deltoid muscle from control (left col- umn) and DMD (right column) patients stained by HE (upper row) and immunostained by an antibody anti-iNOS (lower row). Muscle ﬁbers from control patient did not exhibit immunoreactiv- ity for iNOS whereas a cytoplasmic staining for iNOS was observed in DMD muscle ﬁbers. No immunostaining of connec- tive tissue or vessels was seen in DMD. (C) NADPH-d reactivity in the muscle of control and DMD patients. Statistical Analysis Statistical comparison of results was performed using the Wilcoxon signed-rank test for paired data and the Mann-Whitney U test for nonpaired data. A p value 0.05 was considered signiﬁcant. iNOS Is Expressed in DMD Muscle Transverse serial cryostat sections (10-m thick) of a deltoid muscle from control (left column) and DMD (right column) patients stained by HE (upper row) and for NADPH-d reactivity (lower row). NADPH-d Fig. 1. Expression of iNOS in DMD muscle. (A)Western blot analysis of human quadriceps muscle extract for iNOS. Proteins were separated in reducing conditions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS) and transferred to nitrocellulose membrane. The blot was incubated with an antibody speciﬁc for iNOS and developed as described in Methods. Prestained molecular standards were shown on the left. Lane 1, positive control: mouse macrophage lysate prepared from the RAW 264.7 cell line, which were stimulated with IFN and LPS for 12 hr; lanes 3, 5, samples from patients with DMD; lane 7, patient with BMD; lanes 2, 4, 6, samples from control normal subjects. All lanes contain 45 g of total protein. No speciﬁc signal was detected on the muscle samples from control normal subjects whereas a signal was observed on the muscle samples from patients with DMD/BMD. (B) Transverse serial cryostat sections (10-m thick) of a deltoid muscle from control (left col- umn) and DMD (right column) patients stained by HE (upper row) and immunostained by an antibody anti-iNOS (lower row). Muscle ﬁbers from control patient did not exhibit immunoreactiv- ity for iNOS whereas a cytoplasmic staining for iNOS was observed in DMD muscle ﬁbers. No immunostaining of connec- tive tissue or vessels was seen in DMD. (C) NADPH-d reactivity in the muscle of control and DMD patients. Transverse serial cryostat sections (10-m thick) of a deltoid muscle from control (left column) and DMD (right column) patients stained by HE (upper row) and for NADPH-d reactivity (lower row). NADPH-d J. Louboutin et al.: iNOS in DMD 359 Fig. 2. Distribution of iNOS and NADPH-d in DMD muscle. Transverse serial cryostat sections (10-m thick) of a deltoid muscle from DMD patient immunostained by iNOS antibody (ﬁrst column, upper row; third column, ﬁrst row), developmental myosin heavy chain antibody, d-MHC (ﬁrst column, second row; fourth column lower row), complement membrane attack complex C5b-C9, MAC (second column, lower row; third column, second row), stained for NADPH-d reactivity (second and fourth columns, ﬁrst row) and succinate dehydrogenase activity (second and fourth columns, second row). Double immunoﬂuorescence staining was realized using dual ﬁlters (ﬁrst and fourth columns, lower row). First column: iNOS-positive ﬁbers colocalized with muscle ﬁbers expressing d-MHC. Relationship Between iNOS Immunostaining and NADPH-d Reactivity in DMD particularly clear for the normal-sized or hypertro- phied ﬁbers (Figure 3). iNOS Is Expressed in DMD Muscle Second column: whenever the SDH activity is low in the small ﬁbers double-immunostained for iNOS and d-MHC, NADPH-d reactivity is higher in these ﬁbers. No immunoreactivity was shown for MAC. Third column: Some necrotic ﬁbers, immunostained by anti-MAC antibody, were stained with anti-iNOS antibody. Fourth column: NADPH-d reactivity and SDH activity were not present in degenerating muscle ﬁbers. Note that some mononuclear cells were reactive for NADPH-d. Few ﬁbers were d-MHC positive. Fig. 2. Distribution of iNOS and NADPH-d in DMD muscle. Transverse serial cryostat sections (10-m thick) of a deltoid muscle from DMD patient immunostained by iNOS antibody (ﬁrst column, upper row; third column, ﬁrst row), developmental myosin heavy chain antibody, d-MHC (ﬁrst column, second row; fourth column lower row), complement membrane attack complex C5b-C9, MAC (second column, lower row; third column, second row), stained for NADPH-d reactivity (second and fourth columns, ﬁrst row) and succinate dehydrogenase activity (second and fourth columns, second row). Double immunoﬂuorescence staining was realized using dual ﬁlters (ﬁrst and fourth columns, lower row). First column: iNOS-positive ﬁbers colocalized with muscle ﬁbers expressing d-MHC. Second column: whenever the SDH activity is low in the small ﬁbers double-immunostained for iNOS and d-MHC, NADPH-d reactivity is higher in these ﬁbers. No immunoreactivity was shown for MAC. Third column: Some necrotic ﬁbers, immunostained by anti-MAC antibody, were stained with anti-iNOS antibody. Fourth column: NADPH-d reactivity and SDH activity were not present in degenerating muscle ﬁbers. Note that some mononuclear cells were reactive for NADPH-d. Few ﬁbers were d-MHC positive. Expression of iNOS in mdx Mouse Muscle Fibers Fixation in 4% formaldehyde dissolved in PBS and in- cubation in solution containing 1% formaldehyde re- sulted in a weaker NADPH-d staining of muscle ﬁbers, both in control and DMD patients (data not shown). Low NADPH-d reactivity was present in the sar- coplasm of muscle ﬁbers from control patients relative to DMD patients for which NADPH-d reactivity was higher and predominantly found in the sarcoplasm, where it showed a spotty distribution (Figure 1C). NADPH-d reactivity was absent from necrotic muscle ﬁbers (Figure 2), but iNOS was detected in necrotic ﬁbers; regenerating muscle ﬁbers exhibited a diffuse NADPH-d sarcoplasmic staining as well as an iNOS cytoplasmic immunostaining (Figure 2). In contrast, regenerating muscle ﬁbers were not concurrently stained by another enzymatic staining (SDH staining) (Figure 2). Some mononucleated cells exhibited NADPH-d reactivity. NADPH-d reactivity was found in the walls of some vessels, but was absent in con- nective tissue. In BMD patients, NADPH-d reactivity was observed in small-sized ﬁbers (data not shown). We investigated the expression of iNOS in muscle ﬁbers of mdx mouse. Expression of iNOS was observed in the quadriceps muscle of the mdx mouse, Fig. 3. Immunolocalization of NF-B, iNOS and NADPH-d in DMD muscle. (A) Transverse cryostat sections (10 m-thick) of a deltoid muscle from DMD patient stained for HE (upper row, left column), NADPH-d reactivity (upper row, right column), immunostained by anti-iNOS antibody (lower row, left column) and by anti-p65 subunit of NF-B (lower row, right column). Most of the iNOS-positive muscle ﬁbers exhib- ited an immunoreaction for p65. However, all the p65-positive ﬁbers were not iNOS-positive. Fig. 3. Immunolocalization of NF-B, iNOS and NADPH-d in DMD muscle. (A) Transverse cryostat sections (10 m-thick) of a deltoid muscle from DMD patient stained for HE (upper row, left column), NADPH-d reactivity (upper row, right column), immunostained by anti-iNOS antibody (lower row, left column) and by anti-p65 subunit of NF-B (lower row, right column). Most of the iNOS-positive muscle ﬁbers exhib- ited an immunoreaction for p65. However, all the p65-positive ﬁbers were not iNOS-positive. Colocalization of iNOS and p65 NF-B Subunit in Muscle Fibers of DMD Colocalization of iNOS and p65 NF-B Subunit in Muscle Fibers of DMD (C) Transverse cryostat sections (10 m-thick) of a quadriceps muscle from mdx mouse stained for HE (upper row, left column), NADPH-d reactivity (upper row, right column), im- munostained by anti-iNOS antibody and stained for Evans blue (lower row, right column) and immunostained for d-MHC and stained for Evans blue (lower row, left column). Necrotic ﬁbers (indicated by double arrows) were NADPH-d negative and the NADPH-d staining was more intense in small-sized centronucle- ated muscle ﬁbers (single arrows). Evans blue stained degenerat- ing muscle ﬁbers. An immunostaining for iNOS was observed in the small sized ﬁbers, as well as partly in degenerating ﬁbers. Small-sized centronucleated muscle ﬁbers were immunostained by anti d-MHC antibody. Fig. 4. Expression of iNOS in mdx muscles. (A) Histogram of repartition of iNOS-positive ﬁbers in the quadriceps muscle of mdx mouse. No iNOS-positive ﬁber was observed before 4 weeks. Standard deviations bars are not indicated on the histogram be- cause standard deviations were very small. (B) Transverse cryostat sections (10 m-thick) of a Tibialis Anterior (TA, ﬁrst row, left col- umn), Soleus (SOL, ﬁrst row, right column), Extensor Digitorum Longus (EDL, second row, left column) and diaphragm (DIA, sec- ond row, right column) muscles from 42 days-old mdx mouse im- munostained by anti-iNOS antibody. All the muscles had iNOS- positive muscle ﬁbers. Control 1 (third row, left column) represents immunostaining of mdx TA muscle where the primary antibody has been substituted by a non-immune IgG. Control 2 (third row, right column) is a section of a C57BL10 mouse TA muscle immunostained for iNOS. Same results concerning the con- trols were observed in other muscles (EDL, DIA, SOL) of mdx and C57BL10 mice. (C) Transverse cryostat sections (10 m-thick) of a quadriceps muscle from mdx mouse stained for HE (upper row, left column), NADPH-d reactivity (upper row, right column), im- munostained by anti-iNOS antibody and stained for Evans blue (lower row, right column) and immunostained for d-MHC and stained for Evans blue (lower row, left column). Necrotic ﬁbers (indicated by double arrows) were NADPH-d negative and the NADPH-d staining was more intense in small-sized centronucle- ated muscle ﬁbers (single arrows). Evans blue stained degenerat- ing muscle ﬁbers. An immunostaining for iNOS was observed in the small sized ﬁbers, as well as partly in degenerating ﬁbers. Small-sized centronucleated muscle ﬁbers were immunostained by anti d-MHC antibody. Fig. 4. Expression of iNOS in mdx muscles. Colocalization of iNOS and p65 NF-B Subunit in Muscle Fibers of DMD (B) Transverse cryostat sections (10 m-thick) of a Tibialis Anterior (TA, ﬁrst row, left col- umn), Soleus (SOL, ﬁrst row, right column), Extensor Digitorum Longus (EDL, second row, left column) and diaphragm (DIA, sec- ond row, right column) muscles from 42 days-old mdx mouse im- munostained by anti-iNOS antibody. All the muscles had iNOS- positive muscle ﬁbers. Control 1 (third row, left column) represents immunostaining of mdx TA muscle where the primary antibody has been substituted by a non-immune IgG. Control 2 (third row, right column) is a section of a C57BL10 mouse TA muscle immunostained for iNOS. Same results concerning the con- trols were observed in other muscles (EDL, DIA, SOL) of mdx and C57BL10 mice. (C) Transverse cryostat sections (10 m-thick) of a quadriceps muscle from mdx mouse stained for HE (upper row, left column), NADPH-d reactivity (upper row, right column), im- munostained by anti-iNOS antibody and stained for Evans blue (lower row, right column) and immunostained for d-MHC and stained for Evans blue (lower row, left column). Necrotic ﬁbers (indicated by double arrows) were NADPH-d negative and the NADPH-d staining was more intense in small-sized centronucle- ated muscle ﬁbers (single arrows). Evans blue stained degenerat- ing muscle ﬁbers. An immunostaining for iNOS was observed in the small sized ﬁbers, as well as partly in degenerating ﬁbers. Small-sized centronucleated muscle ﬁbers were immunostained by anti d-MHC antibody. Fig. 4. Expression of iNOS in mdx muscles. (A) Histogram of repartition of iNOS-positive ﬁbers in the quadriceps muscle of mdx mouse. No iNOS-positive ﬁber was observed before 4 weeks. Standard deviations bars are not indicated on the histogram be- cause standard deviations were very small. (B) Transverse cryostat sections (10 m-thick) of a Tibialis Anterior (TA, ﬁrst row, left col- umn), Soleus (SOL, ﬁrst row, right column), Extensor Digitorum Longus (EDL, second row, left column) and diaphragm (DIA, sec- ond row, right column) muscles from 42 days-old mdx mouse im- munostained by anti-iNOS antibody. All the muscles had iNOS- positive muscle ﬁbers. Control 1 (third row, left column) represents immunostaining of mdx TA muscle where the primary antibody has been substituted by a non-immune IgG. Control 2 (third row, right column) is a section of a C57BL10 mouse TA muscle immunostained for iNOS. Same results concerning the con- trols were observed in other muscles (EDL, DIA, SOL) of mdx and C57BL10 mice. Colocalization of iNOS and p65 NF-B Subunit in Muscle Fibers of DMD Colocalization of iNOS and p65 NF-B Subunit in Muscle Fibers of DMD Most iNOS-positive ﬁbers exhibited immunoreac- tivity for the p65 NF-B subunit, although all p65- positive ﬁbers were not iNOS-positive; this was Molecular Medicine, Volume 7, Number 4, 2001 360 Expression of Dystrophin or Overexpression of Utrophin in mdx Mouse Reduces the Number of iNOS-positive Fibers and similar studies on tissue from congenic control animals were negative. Expression of iNOS was ob- served 4 weeks after birth, in mdx mice, and it re- mained present 2 months after birth (Figure 4A). The expression of iNOS was observed in different mus- cles of mdx mouse: TA, EDL, SOL, and DIA (Figure 4B). Systemic administration of Evans blue was used as a stain for degenerating muscle ﬁbers. Evans blue- positive ﬁbers were partly immunostained by iNOS antibody, but most of the iNOS positive ﬁbers were regenerating developmental-myosin-positive ﬁbers. The majority of iNOS- positive ﬁbers were also reac- tive for NADPH-d (Figure 4C). and similar studies on tissue from congenic control animals were negative. Expression of iNOS was ob- served 4 weeks after birth, in mdx mice, and it re- mained present 2 months after birth (Figure 4A). The expression of iNOS was observed in different mus- cles of mdx mouse: TA, EDL, SOL, and DIA (Figure 4B). Systemic administration of Evans blue was used as a stain for degenerating muscle ﬁbers. Evans blue- positive ﬁbers were partly immunostained by iNOS antibody, but most of the iNOS positive ﬁbers were regenerating developmental-myosin-positive ﬁbers. The majority of iNOS- positive ﬁbers were also reac- tive for NADPH-d (Figure 4C). Few dystrophin-positive ﬁbers (less than 1%, corre- sponding probably to the revertant ﬁbers) were ob- served at 42 days postnatal in the quadriceps mus- cles of mdx mice injected at 5 days postnatal with Ad-LacZ, a control vector. Because utrophin is spon- taneously expressed in mdx, a myc TAG was incor- porated into the utrophin transgene, enabling us to distinguish the overexpressed transgene from the endogenous encoded protein. mdx mice in- jected into the quadriceps at 5 days postnatal with Fig. 4. Expression of iNOS in mdx muscles. (A) Histogram of repartition of iNOS-positive ﬁbers in the quadriceps muscle of mdx mouse. No iNOS-positive ﬁber was observed before 4 weeks. Standard deviations bars are not indicated on the histogram be- cause standard deviations were very small. Colocalization of iNOS and p65 NF-B Subunit in Muscle Fibers of DMD (A) Histogram of repartition of iNOS-positive ﬁbers in the quadriceps muscle of mdx mouse. No iNOS-positive ﬁber was observed before 4 weeks. Standard deviations bars are not indicated on the histogram be- cause standard deviations were very small. (B) Transverse cryostat sections (10 m-thick) of a Tibialis Anterior (TA, ﬁrst row, left col- umn), Soleus (SOL, ﬁrst row, right column), Extensor Digitorum Longus (EDL, second row, left column) and diaphragm (DIA, sec- ond row, right column) muscles from 42 days-old mdx mouse im- munostained by anti-iNOS antibody. All the muscles had iNOS- positive muscle ﬁbers. Control 1 (third row, left column) represents immunostaining of mdx TA muscle where the primary antibody has been substituted by a non-immune IgG. Control 2 (third row, right column) is a section of a C57BL10 mouse TA muscle immunostained for iNOS. Same results concerning the con- trols were observed in other muscles (EDL, DIA, SOL) of mdx and C57BL10 mice. (C) Transverse cryostat sections (10 m-thick) of a quadriceps muscle from mdx mouse stained for HE (upper row, left column), NADPH-d reactivity (upper row, right column), im- munostained by anti-iNOS antibody and stained for Evans blue (lower row, right column) and immunostained for d-MHC and stained for Evans blue (lower row, left column). Necrotic ﬁbers (indicated by double arrows) were NADPH-d negative and the NADPH-d staining was more intense in small-sized centronucle- ated muscle ﬁbers (single arrows). Evans blue stained degenerat- ing muscle ﬁbers. An immunostaining for iNOS was observed in the small sized ﬁbers, as well as partly in degenerating ﬁbers. Small-sized centronucleated muscle ﬁbers were immunostained by anti d-MHC antibody. Fig. 4. Expression of iNOS in mdx muscles. (A) Histogram of repartition of iNOS-positive ﬁbers in the quadriceps muscle of mdx mouse. No iNOS-positive ﬁber was observed before 4 weeks. d d d i i b i di d h hi b J. Louboutin et al.: iNOS in DMD 361 Ad-dystrophin showed 60% of dystrophin-positive ﬁbers when analyzed 42 days postnatal (data not shown). Quadriceps muscles of mdx mice injected 5 days postnatal with Ad-utrophin construct contain- ing a myc TAG showed 44% myc-positive ﬁbers at 42 days postnatal (data not shown). No immunostain- ing for dystrophin or myc was observed in the contralateral quadriceps muscles. Discussion iNOS in DMD It is not surprising to observe iNOS-positive mononu- clear cells in DMD. These cells are probably acti- vated macrophages because iNOS was originally discovered in cytokine-induced macrophages (8,30), and activated macrophages are known to be present in increased number in DMD muscle. Activated macrophages may release cytokines such as tumor necrosis factor alpha (TNF) and interleukin 1 (IL-1). iNOS immunoreactivity observed in the cytoplasm of some necrotic muscle ﬁbers could have resulted from invading macrophages into these ﬁbers. Such an immunoreactivity has not been observed in non- necrotic muscle ﬁbers. More interesting, iNOS im- munoreactivity was observed in the cytoplasm of re- generating muscle ﬁbers in DMD, and NADPH-d reactivity and iNOS immunoreactivity were colocal- ized in these ﬁbers. NO was proposed as a messen- ger molecule for myoblast fusion (31). iNOS may be induced by one or more cytokines derived from skeletal myoblasts and myotubes (16). Correction of mdx Muscle Pathology by Dystrophin Expression or Utrophin Overexpression Is Independent of the Presence of nNOS As previously reported, nNOS is not expressed in mdx muscles, which contrasts with the control ani- mals where colocalization of dystrophin and nNOS is observed in the quadriceps muscles at the sar- colemmal level (Figure 5). Expression of dystrophin or the overexpression of utrophin after gene transfer corrected some of the pathology of mdx muscle; how- Fig. 5. Study of the expression of nNOS before and after somatic gene transfer of dystrophin or utrophin in the quadriceps of mdx mouse. Transverse serial cryostat sections (10-m thick) of a quadriceps muscle from mdx mouse stained for HE (upper row), double-immunostained by anti-nNOS antibody (lower row), and by anti-dystrophin antibody (second row, three ﬁrst columns), or by anti-myc antibody (second row, fourth column). First column: C57BL10 mouse (C57BL10C: C57BL10 control). Second column: mdx mouse aged 42 days. Third column: mdx mouse aged 42 days after injection of adenovirus-mini-dystrophin gene at 5 days postnatal. Fourth column: mdx mouse aged 42 days after injection of adenovirus-utrophin gene at 5 days postnatal. First column: there was a colocalization of dystrophin and nNOS in control mouse. Second column: no dystrophin and no nNOS was detected in mdx mouse. HE demonstrated necrotic ﬁbers (star), and centronucleated ﬁbers. Third column: adenovirus-mediated gene transfer of dystrophin corrected partially the morphological phenotype (less centronucleated muscle ﬁbers, less necrotic ﬁbers, less variation of the size of the ﬁbers). There was no expression of nNOS. Fourth column: adenovirus-mediated gene transfer of utrophin: there were less necrotic and centronucleated muscle ﬁbers and the sizes of the muscle ﬁbers exhibited less variability. No expression of nNOS was demonstrated. Fig. 5. Study of the expression of nNOS before and after somatic gene transfer of dystrophin or utrophin in the quadriceps of mdx mouse. Transverse serial cryostat sections (10-m thick) of a quadriceps muscle from mdx mouse stained for HE (upper row), double-immunostained by anti-nNOS antibody (lower row), and by anti-dystrophin antibody (second row, three ﬁrst columns), or by anti-myc antibody (second row, fourth column). First column: C57BL10 mouse (C57BL10C: C57BL10 control). Second column: mdx mouse aged 42 days. Third column: mdx mouse aged 42 days after injection of adenovirus-mini-dystrophin gene at 5 days postnatal. Fourth column: mdx mouse aged 42 days after injection of adenovirus-utrophin gene at 5 days postnatal. First column: there was a colocalization of dystrophin and nNOS in control mouse. Second column: no dystrophin and no nNOS was detected in mdx mouse. Colocalization of iNOS and p65 NF-B Subunit in Muscle Fibers of DMD The percentage of iNOS-positive ﬁbers in the contralateral quadriceps muscles in the Ad-dystrophin group was 23.7%, whereas this percentage was of 9.5% in the injected quadriceps muscles (p 0.02). In the UTR group, the percentage of iNOS-positive cells in the contralateral quadriceps muscles was 22%, whereas this percentage was 10.5% in the injected quadri- ceps muscles (p 0.05). ever, it was not accompanied by nNOS sarcolemmal expression as measured by immunohistochemistry (Figure 5). NADPH-d Staining in DMD NADPH-d staining is a marker for NOS activity. In the present study, we have shown that NADPH-d reactivity was low in control patients, whereas in DMD/BMD patients, a strong NADPH-d reactivity was demonstrated in the cytoplasm of non-necrotic muscle ﬁbers, particularly in regenerating muscle ﬁbers. Because iNOS immunostaining was not ob- served in all NADPH-reactive muscle ﬁbers, the punctuated NADPH-d reactivity observed in the cytoplasm of some non-necrotic muscle ﬁbers may re- ﬂect the expression of other NOS than iNOS. It is in- teresting to note that an aberrant translocation of nNOS from sarcolemma to cytosol has been shown in DMD and mdx muscles. However, the isoforms of NOS are more diverse than originally assumed, and a more comprehensive classiﬁcation is likely to evolve. Potential Role of NO Secretion NO produced by iNOS in activated macrophages ex- erts direct cytostatic and cytotoxic actions on target cells, owing to its capacity to combine with iron- containing enzymes involved in both the respiratory cycle and pathway for the synthesis of DNA. The formation of potent oxidants (hydroxyl radical and nitrogen dioxide) mediate the cytotoxic effects of NO and induce the inhibition of DNA synthesis, as well as the inhibition of mitochondrial respiratory enzymes (1,2,4). NO has been demonstrated to re- versibly inhibit cytochrome C oxidase in skeletal muscle respiration (39), which decreases ATP pro- duction, and leads to respiratory muscle dysfunction (40). Although already cytotoxic, NO can also react with other free radicals to generate molecules, such as peroxynitrite, which enhance its cytotoxicity. When produced in an excessive amount and for an extended period, NO can also be cytotoxic for host cells. In this way, it is possible that excessive and prolonged production of NO by iNOS in inﬁltrating inﬂammatory cells modulates the mitochondrial res- piration in surrounding myocytes, thus exerting muscle dysfunction. p y Numerous enzymes use NADPH-d as a cofactor and some electron-transferring enzymes that use either NADH or NADPH as a substrate (32). Sufﬁ- cient information has accumulated to suggest that the occurrence of NOS in muscle ﬁbers cannot be unequivocally demonstrated by the standard NADPH-d staining (12). Thus, directly correlating NADPH-d histochemistry with the presence of NOS may be misleading because NADPH-d activity nei- ther indicates the existence of a speciﬁc NOS iso- form nor clearly discriminates between NOS and other enzymes, which may possess NADPH-d activ- ity (18). However, unlike NOS, these enzymes are inactivated by paraformaldehyde ﬁxation (20). In the present study, the ﬁxation in formaldehyde re- sulted in a signiﬁcantly weaker NADPH-d staining of muscle ﬁbers, in both control and DMD patients. In this way, we conﬁrm recent compiled data that indicate the NADPH-d of NOS can be selectively disclosed by adding formaldehyde or 0.5 mM potassium permanganate to the incubation medium (12,33,34). iNOS expression may be adaptive in that pro- duction of NO may lead to enhanced oxygen and substrate delivery to vital tissues during pro- longed periods of disease. NO may act as a mes- senger molecule for myoblast fusion (31); on the other hand, NO can also induce apoptosis in skele- tal myoblasts (41). This reﬂects the dual nature of NO, which is both cytotoxic and potentially protective. Molecular Medicine, Volume 7, Number 4, 2001 362 The present study clearly demonstrates that iNOS may be induced in regenerating muscle ﬁbers of DMD patients. However, further studies are needed to determine if iNOS expression is a gen- eral phenomenon of muscle regeneration and to precise the routes of iNOS induction. In the present study, we have shown a colocalization of p65-im- munoreactive ﬁbers and iNOS-positive ﬁbers. The induction of iNOS by NF-B is a well-known phe- nomenon but has not been shown in vivo yet (35). Although the role of cytokines derived from macrophages remains unproven, an induction of iNOS by cytokines that include IFN, IL-1, and TNF has been reported in different tissues (30,36–38). NADPH-d Staining in DMD Correction of mdx Muscle Pathology by Dystrophin Expression or Utrophin Overexpression Is Independent of the Presence of nNOS HE demonstrated necrotic ﬁbers (star), and centronucleated ﬁbers. Third column: adenovirus-mediated gene transfer of dystrophin corrected partially the morphological phenotype (less centronucleated muscle ﬁbers, less necrotic ﬁbers, less variation of the size of the ﬁbers). There was no expression of nNOS. Fourth column: adenovirus-mediated gene transfer of utrophin: there were less necrotic and centronucleated muscle ﬁbers and the sizes of the muscle ﬁbers exhibited less variability. No expression of nNOS was demonstrated. Molecular Medicine, Volume 7, Number 4, 2001 References Bredt D, Glatt C, Hwang P, Fotuhi M, Dawson T, Snyder S. (1991) Nitric oxide synthase protein and mRNA are discretely localized in neuronal populations of the mammalian CNS together with NADPH diaphorase. Neuron 7: 615–624. 6. Dawson T, Bredt D, Fotuhi M, Hwang P, Snyder S. (1991) Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues. Proc. Natl. Acad. Sci. USA 88: 7797–7801. 7. Lamas S, Marsden P, Li G, Tempst P, Michel T. (1992) En- dothelial nitric oxide synthase: molecular cloning and charac- terization of a distinct constitutive enzyme isoform. Proc. Natl. Acad. Sci. USA 89: 6348–6352. 8. Hibbs Jr. J, Vavrin Z, Taintor R. (1987) L-arginine is required for expression of the activated macrophage effector mecha- nism causing selective metabolic inhibition in target cells. J. Immunol. 138: 550–565. 9. Kobzik L, Reid M, Bredt D, Stamler J. (1994) Nitric oxide in skeletal muscle. Nature 372: 546–548. 10. Gath I, Closs E, Godtel-Armbrust U, Schmitt S, Nakane M, Wessler I, Forstermann U. (1996) Inducible NO synthase II and neuronal NO synthase I are constitutively expressed in different structures of guinea pig skeletal muscle: implica- tions for contractile function. FASEB J. 10: 1614–1620. 11. Murrant C, Woodley N, Barclay J. (1994) Effect of nitroprus- side and endothelium-derived products on slow-twitch skeletal muscle function in vitro. Can. J. Physiol. & Pharmacol. 72: 1089–1093. 12. Grozdanovic Z, Nakos G, Dahrmann G, Mayer B, Gossrau R. (1995) Species-independent expression of nitric oxide syn- thase in the sarcolemma region of visceral and somatic stri- ated muscle ﬁbers. Cell & Tissue Res. 281: 493–499. 13. Balon T, Nadler J. (1994) Nitric oxide release is present from incubated skeletal muscle preparations. J. Appl. Physiology. 77: 2519–2521. 14. Nakane M, Schmidt H, Pollock J, Forstermann U, Murad F. (1993) Cloned human brain nitric oxide synthase is highly expressed in skeletal muscle. FEBS Letters. 316: 175–180. 15. Thompson M, Becker L, Bryant D, Williams G, Levin D, Margraf L, Giroir B. (1996) Expression of the inducible nitric oxide synthase gene in diaphragm and skeletal muscle. J. Appl. Physiol. 81: 2415–2420. Recent reports have shown that iNOS could be immunolocalized to the neuromuscular junction in DMD (52) and that iNOS activity was signiﬁcantly increased in the mdx mouse heart (53). References p y p ( ) The overexpression of utrophin and expression of dystrophin corrected some pathological abnor- malities of mdx muscle, but these corrections were not accompanied by nNOS restoration. It has been shown that the expression of a minigene lacking exons 17–48 of dystrophin in transgenic mdx mice fails to recruit nNOS to sarcolemma and is associ- ated with a very mild phenotype (Becker type) (22). However, different results have been reported, prob- ably owing to mouse variations in the level of the 17-48 transgene expression (49). Restoration of nNOS at the sarcolemma level of muscle ﬁbers from TA of mdx mice has been reported using a plasmid DNA encoding the human 6.3kB minidystrophin gene lacking exons 17–48 (50). However, the num- ber of transduced ﬁbers and the eventual correction of the phenotype were not well described. In our study, we used a human minidystrophin gene lack- ing exons 13–48. It is improbable that the difference between the two studies could be related to a differ- ent level of the transgene expression because more than 50% of the ﬁbers were transduced in our study (the level of transduction is usually lower when us- ing a plasmid). The speciﬁc muscle studied can also be important to consider because, in the mouse, nNOS occurs mainly at high levels in fast-twich muscle ﬁbers and TA is composed at 99% of such ﬁbers. This contrasts the quadriceps, which are com- posed of more than only fast-twich muscle ﬁbers. Chao et al. (22) also found that nNOS does not asso- ciate with utrophin-containing complexes, which is in agreement with our results. Regarding the partial correction of the morphological phenotype after dy- strophin expression or utrophin overexpression without expression of nNOS, our ﬁndings support the idea that nNOS by itself does not inﬂuence the extent of the pathologic damage, and that the loss of nNOS activity from the sarcolemma could be one of several factors that collectively produce the pathol- ogy observed in dystrophinopathies (49,51). 1. Moncada S, Higgs A. (1993) The L-arginine-nitric oxide path- way. N. Engl. J. Med. 329: 2002–2012. 2. Marletta M. (1993) Nitric oxide synthase structure and mech- anism. J. Biol. Chem. 268: 12231–12234. 3. Snyder S. (1992) Nitric oxide: ﬁrst in a new class of neuro- transmitters. Science 257: 494–496. 4. Bredt D, Snyder S. (1994) Nitric oxide: a physiologic mes- senger molecule. Ann. Rev. Biochem. 63: 175–195. 5. Acknowledgments The contribution of the Vector and Cell Morphology Cores was greatly appreciated. This work was sup- ported by the Association Française contre les Myopathies (AFM), the NIH (P01 AR/NS43648-05 and P30 DK47757-07) and Genovo, Inc. a company Dr. Wilson founded and holds equity in. The present study showed a reduction of the iNOS- positive muscle ﬁbers in the quadriceps of mdx mouse after expression of dystrophin, or overexpres- sion of utrophin. These results are in agreement with previous data showing a reduction of the necrotic and regenerating muscle ﬁbers in mdx mouse after such gene transfer (unpublished personal results) and conﬁrm previously published ones (44–48). Mechanisms of NOS Induction Induction of NOS is a complex process that re- quires a number of signaling pathways. The activa- tion of macrophages by LPS and IFN gamma (IFN), either singly or in combination, results in the induction of iNOS (1). This induction, which is inhibited by glucocorticoids, yields sustained pro- duction of NO, which diffuses to target cells such as tumor cells. At these cells, NO combines with iron-sulfur centers in key enzymes of both the res- piratory cycle and the pathway for the synthesis of DNA. Regulation of iNOS occurs at the level of gene transcription and is relatively slow but leads to long-lasting and signiﬁcant increases in NO pro- duction. In contrast, nNOS and eNOS, constitu- tively expressed at the level of transcription, are regulated by various soluble ligands; NO concen- trations produced by this route are usually rela- tively low (1). The physiological signiﬁcance of NO formation in the sarcolemma of normal striated muscles is not precisely known. It was proposed that NO may interact with the cGMP signal transduction pathway to control resting muscle and with reactive oxygen intermediates in actively contracting muscle to mod- ulate force development. However, by inhibiting the ryanodine receptor calcium release channel (42), NO may decrease calcium release from sarcoplasmic reticulum, depress the contractile force, and decrease the efﬁciency of excitation contraction coupling J. Louboutin et al.: iNOS in DMD 363 (19,43). NO also increases muscle metabolism by regulating glucose uptake (13). dystrophin or utrophin can reduce the expression of iNOS in mdx mice quadriceps muscle. Consequences of Dystrophin Expression and Utrophin Overexpression After Gene Transfer Molecular Medicine, Volume 7, Number 4, 2001 364 36. Nussler A, Di Silvio M, Billiar T, Hoffman R, Geller D, Selby R, Madariaga J, Simmons R. (1992) Stimulation of the nitric oxide synthase pathway in human hepatocytes by cytokines and endotoxin. J. Exp. Med. 176: 261–264. 18. Young H, O’Brien A, Furness J, Ciampoli D, Hardwick J, McCabe T, Narayanasami R, Masters B, WR. T. (1997) Rela- tionships between NADPH diaphorase staining and neu- ronal, endothelial, and inducible nitric oxide synthase and cytochrome P450 reductase immunoreactivities in guinea-pig tissues. Histochem. & Cell Biol. 107: 19–29. 37. Liew F, Li Y, Millott S. (1990) Tumor necrosis factor-alpha synergizes with IFN-gamma in mediating killing of Leishma- nia major through the induction of nitric oxide. J. Immunol. 145: 4306–4310. 19. Kobzik L, Stringer B, Balligand J, Reid M, Stamler J. (1995) Endothelial type nitric oxide synthase in skeletal muscle ﬁbers: mitochondrial relationships. Biochem. & Biophys. Res. Comm. 211: 375–381. 38. Curran R, Billiar T, Stuehr D, Ochoa J, Harbrecht B, Flint S, Simmons R. (1990) Multiple cytokines are required to induce hepatocyte nitric oxide production and inhibit total protein synthesis. Ann. Surg. 212: 462–469. 20. Matsumoto T, Nakane M, Pollock J, Kuk J, Forstermann U. (1993) A correlation between soluble brain nitric oxide syn- thase and NADPH-diaphorase activity is only seen after exposure of the tissue to ﬁxative. Neurosci. Letters 155(1): 61–64, 1993 155: 61–64. 39. Cleeter M, Cooper J, Darley-Usmar V, Moncada S, Schapira A. (1994) Reversible inhibition of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, by nitric oxide. Implications for neurodegenerative diseases. FEBS Letters 345: 50–54. 21. Brenman J, Chao D, Xia H, Aldape K, Bredt D. (1995) Nitric oxide synthase complexed with dystrophin and absent from skeletal muscle sarcolemma in Duchenne muscular dystro- phy. Cell 82: 743–752. 40. Schweizer M, Richter C. (1994) Nitric oxide potently and re- versibly deenergizes mitochondria at low oxygen tension. Biochem. & Biophys.l Res. Comm. 204: 169–175. 22. Chao D, Gorospe J, Brenman J, Rafael J, Peters M, Froehner S, Hoffman E, Chamberlain J, Bredt D. (1996) Selective loss of sarcolemmal nitric oxide synthase in Becker muscular dystrophy. J. Exp. Med. 184: 609–618. 41. Stangel M, Zettl U, Mix E, Zielasek J, Toyka K, Hartung H, Gold R. (1996) H2O2 and nitric oxide-mediated oxidative stress induce apoptosis in rat skeletal muscle myoblasts. J. Neuropathol. & Exp. Neurol. 55: 36–43. 23. Molecular Medicine, Volume 7, Number 4, 2001 (1998) Involvement of nitric oxide system in experimental muscle crush injury. J. Clin. Invest. 101: 1325–1333. 48. Wakeﬁeld PM, Tinsley JM, Wood MJ, Gilbert R, Karpati G, Davies KE. (2000) Prevention of the dystrophic phenotype in dystrophin/utrophin-deﬁcient muscle following adenovirus- mediated transfer of a utrophin minigene. Gene Ther. 7: 201–204. 29. Brooke M, Kaiser K. (1969) Some comments on the histo- chemical characterization of muscle adenosine triphos- phatase. J. Histochem. & Cytochem. 17: 431–432. 30. Xie Q, Cho H, Calaycay J, Mumford R, Swiderek K, Lee T, Ding A, Troso T, Nathan C. (1992) Cloning and characteriza- tion of inducible nitric oxide synthase from mouse macrophages. Science 256: 225–228. 49. Crosbie R, Straub V, Yun H, Lee J, Rafael J, Chamberlain J, Dawson V, Dawson T, Campbell K. (1998) mdx muscle pathol- ogy is independent of nNOS perturbation. Hum. Mol. Gen. 7: 823–829. 31. Lee K, Baek M, Moon K, Song W, Chung C, Ha D, Kang M. (1994) Nitric oxide as a messenger molecule for myoblast fu- sion. J. Bio. Chem. 269: 14371–14374. 50. Decrouy A, Renaud J, Lunde J, Dickson G, Jasmin B. (1998) Mini- and full-length dystrophin gene transfer induces the recovery of nitric oxide synthase at the sarcolemma of mdx4cv skeletal muscle ﬁbers. Gene Ther. 5: 59–64. 32. Webb E. (1992) Enzyme nomenclature 1992: recommenda- tions of the nomenclature committee of the International Union of Biochemistry and Molecular Biology. New York: Academic Press. 51. Chao D, Silvagno F, Bredt D. (1998) Muscular dystrophy in mdx mice despite lack of neuronal nitric oxide synthase. J. Neurochem. 71: 784–789. 33. Nakos G, Gossrau R. (1994) When NADPH diaphorase (NADPHd) works in the presence of formaldehyde, the enzyme appears to visualize selectively cells with constitutive nitric oxide synthase (NOS). Acta Histochemica. 96: 335–343. 52. Yang C, Alvarez R, Engel W, Haun C, Askanas V. (1997) Immunolocalization of nitric oxide synthases at the postsy- naptic domain of human and rat neuromuscular junctions— light and electron microscopic studies. Exp. Neurol. 148: 34-44. 34. Grozdanovic Z, Nakos G, Mayer B, Gossrau R. (1995) A mod- iﬁed method allows for correlation between NADPH- diaphorase histochemistry and immunohistochemistry for the demonstration of neuronal nitric oxide synthase (nNOS). Folia Histochemica et Cytobiologica 33: 11–18. 53. Bia B, Cassidy P, Young M, Rafael J, Leighton B, Davies K, Radda G, Clarke K. Molecular Medicine, Volume 7, Number 4, 2001 Brenman J, Chao D, Gee S, McGee A, Craven S, Santillano D, Wu Z, Huang F, Xia H, Peters M, Froehner S, Bredt D. (1996) Interaction of nitric oxide synthase with the postsynaptic density protein PSD-95 and alpha1-syntrophin mediated by PDZ domains. Cell 84(5): 757–767. 42. Meszaros L, Minarovic I, Zahradnikova A. (1996) Inhibition of the skeletal muscle ryanodine receptor calcium release channel by nitric oxide. FEBS Letters 380: 49–52. 43. Reid M. (1996) Reactive oxygen and nitric oxide in skeletal muscle. News Physiol. Sci. 11: 114–119. 24. Koprowski H, Zheng Y, Heber-Katz E, Fraser N, Rorke L, Fu Z, Hanlon C, Dietzschold B. (1993) In vivo expression of inducible nitric oxide synthase in experimentally induced neurologic diseases. Proc. Natl. Acad. Sci. USA 90: 3024–3027. 44. Ragot T, Vincent N, Chafey P, Vigne E, Gilgenkrantz H, Couton D, Cartaud J, Briand P, Kaplan JC, Perricaudet M, Kahn A. (1993) Efﬁcient adenovirus-mediated transfer of a human minidystrophin gene to skeletal muscle of mdx mice. Nature 361: 647–650. 25. Bo L, Dawson T, Wesselingh S, Mork S, Choi S, Kong P, Hanley D, Trapp B. (1994) Induction of nitric oxide synthase in demyelinating regions of multiple sclerosis brains. Ann. Neurol. 36: 778–786. 45. Vincent N, Ragot, T., Gilgenkrantz, H., Couton, D., Chafey, P., Gregoire, A., Briand, P., Kaplan, J.C., Perricaudet, M., and Kahn, A. (1993) Long term correction of mouse dystrophic degenration by adenovirus transfer of a mini-dystrophin gene. Nature Genet. 5: 130–134. 26. Adamson D, Wildemann B, Sasaki M, Glass J, McArthur J, Christov V, Dawson T, Dawson V. (1996) Immunologic NO synthase: elevation in severe AIDS dementia and induction by HIV-1 gp41. Science 274: 1917–1921. 46. Acsadi G, Lochmuller H, Jani A, Huard J, Massie B, Prescott S, Simoneau M, Petrof BJ, Karpati G. (1996) Dystrophin expression in muscles of mdx mice after adenovirus- mediated in vivo gene transfer. Hum. Gene Ther. 7: 129–140. 27. Yang C, Alvarez R, Engel W, Heller S, Askanas V. (1998) Nitric oxide-induced oxidative stress in autosomal recessive and dominant inclusion-body myopathies. Brain 121: 1089–1097. 47. Gilbert R, Nalbantoglu J, Petrof BJ, Ebihara S, Guibinga GH, Tinsley JM, Kamen A, Massie B, Davies KE, Karpati G. (1999) Adenovirus-mediated utrophin gene transfer miti- gates the dystrophic phenotype of mdx mouse muscles. Hum. Gene Ther. 10: 1299–1310. 28. Rubinstein I, Abassi Z, Coleman R, Milman F, Winaver J, Better OS. References The present results demonstrated the following: 1) iNOS could be involved in the physiopathology of dys- trophinopathies, and 2) somatic gene transfer of pp y 16. Williams G, Brown T, Becker L, Prager M, Giroir B. (1994) Cytokine-induced expression of nitric oxide synthase in C2C12 skeletal muscle myocytes. Am. J. Physiol. 267: R1020– 1025. 17. Chang W, Iannaccone S, Lau K, Masters B, McCabe T, McMillan K, Padre R, Spencer M, Tidball J, Stull J. (1996) Neuronal nitric oxide synthase and dystrophin-deﬁcient muscular dystrophy. Proc. Natl. Acad. Sci. USA 93: 9142–9147. Molecular Medicine, Volume 7, Number 4, 2001 Molecular Medicine, Volume 7, Number 4, 2001 (1999) Decreased myocardial nNOS, increased iNOS and abnormal ECGs in mouse models of Duchenne muscular dystrophy. J. Mol. & Cellular Cardiol. 31: 1857–1862. 35. Barnes P, Adcock I. (1997) NF-kappa B: a pivotal role in asthma and a new target for therapy. Trends Pharmacol. Sci. 18: 46–50.
https://openalex.org/W2771772968	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/1AA0DA8005C8A7ECD6DFD0352D0FF39B/S0959270917000491a.pdf/div-class-title-updated-breeding-distribution-and-population-status-of-jankowski-s-bunting-span-class-italic-emberiza-jankowskii-span-in-china-div.pdf	English	null	Updated breeding distribution and population status of Jankowski’s Bunting <i>Emberiza jankowskii</i> in China	Bird conservation international	2,017	cc-by	5,764	Summary Since 2010, Jankowski’s Bunting Emberiza jankowskii has been listed as ‘Endangered’ on the IUCN Red List of Threatened Species. However, because no comprehensive surveys had been conducted, it was not known whether undiscovered populations existed elsewhere, so the population status of the species could not be assessed accurately. The aim of this study was to assess the breeding distri- bution and population size of Jankowski’s Bunting in China. Fifty sites in Inner Mongolia, and Jilin, Heilongjiang, Liaoning and Hebei Provinces were surveyed to locate suitable habitat and breeding populations of Jankowski’s Bunting. The surveyed sites included historical breeding distribution areas, wintering sites, and regions adjacent to historical breeding distribution areas. We confirmed that Jankowski’s Bunting has disappeared from most of its former breeding distributions, with the exceptions of Dagang, Xiergen and Tumiji. Additionally, 13 new breeding sites were discovered in Inner Mongolia. All currently known populations breed in Mongolian steppe-vegetation zones, with shrubs dominated by the natural Siberian apricot Prunus sibirica, indicating that this type of habitat is crucial for the survival of the species. Based on remote sensing, the suitable breeding habitat for Jankowski’s Bunting is estimated to be approximately 280 km2. The population size of Jankowski’s Bunting could range between 9,800 and 12,500 individuals, which is much higher than the numbers estimated in previous reports that were based on partial surveys. The suitable habitat remaining in Inner Mongolia would highly benefit from the implementation of the National Key Public Forest Protection Project. The population size of Jankowski’s Bunting is larger than previously estimated, but it is still threatened by habitat degradation and fragmentation, and our survey results reinforce the need for more research. The status of Jankowski’s Bunting in China still meets the IUCN criteria B2ab for an ‘Endangered’ species. Bird Conservation International (2018) 28:643–652. © BirdLife International, 2017. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. doi:10.1017/S0959270917000491 Bird Conservation International (2018) 28:643–652. © BirdLife International, 2017. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. doi:10.1017/S0959270917000491 Bird Conservation International (2018) 28:643–652. © BirdLife International, 2017. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. doi:10.1017/S0959270917000491 Updated breeding distribution and population status of Jankowski’s Bunting Emberiza jankowskii in China ZHENG HAN, LI-SHI ZHANG, BO QIN, LIN WANG, YU LIU, VIVIAN WING KAN FU, YUN-LEI JIANG, JIANPING FU and HAI-TAO WANG ZHENG HAN, LI-SHI ZHANG, BO QIN, LIN WANG, YU LIU, VIVIAN WING KAN FU, YUN-LEI JIANG, JIANPING FU and HAI-TAO WANG Introduction Geographical range size is a basic criterion for determining if a species faces a heightened risk of extinction (IUCN 2001). With large-scale changes in land use, it is generally easier to eliminate a species with a narrow range than a species with a large range (Sekercioglu et al. 2008). The distri- bution of Jankowski’s Bunting Emberiza jankowskii is restricted to China, Russia and North Korea, and it has been listed as ‘Endangered’ on the IUCN Red List of Threatened Species since 2010 due to its small distribution and rapid population decline (BirdLife International 2012). The main threat to this species appears to be the conversion of its habitat into agricultural land, pas- ture and sometimes even forestry (BirdLife International 2016). Nevertheless, recent information https://doi.org/10.1017/S0959270917000491 Published online by Cambridge University Press 644 Z. Han et al. on its population status is lacking and scattered in the literature (e.g. Jiang et al. 2008, Wang et al. 2010, BirdLife International 2016). Historically, Jankowski’s Bunting bred in Inner Mongolia, Heilongjiang, western and eastern Jilin Province in China, extreme north-eastern North Korea and southern Primorye in Russia (BirdLife International 2001). However, the range of Jankowski’s Bunting has been drastically contracting for many years. Before the early 1970s, Jankowski’s Bunting previously bred in the extreme southern regions of the Russian Far East (Vorob’ev 1954, Litvinenko and Shibaev 1966, Panov 1973), but there has been no record of the species in Russia for more than 40 years. Similarly, there has been no information reported on the species in North Korea for many years (BirdLife International 2001). ( ) In China, Jankowski’s Bunting has disappeared from a number of breeding sites, such as Dongning (site 32 in Figure 1), Tumen (site 52) and Tuquan (site 14). A small population at Dagang in Jilin was reported in 2010 (Wang et al. 2010). Gao (2002) reported that a small popula- tion bred at Tumuji and Maanshan of Zhalaite Qi in Inner Mongolia, though fewer than 30 indi- viduals were detected (Wang et al. 2010). Breeding populations at Xiergen and Xinjiamu of Keerqinyouyizhong Qi in Inner Mongolia were found in 2008, and the population sizes reportedly fluctuated around approximately 50 individuals (Wang et al. 2010). Jankowski’s Bunting breeds in a variety of open habitats at low elevations. Historically, it bred in three different habitat types, defined by their plant assemblages. Introduction The first habitat consisted of low hills, meadows or coastal plains with xerophilous grass species and scattered young oak bushes; this habitat was found in eastern Jilin Province and the surrounding areas (Yamashina 1957, Stresemann and Portenko 1981). The second consisted of steppe-like habitats in Xianghai that were composed of mixed shrubs of Prunus sibirica, Ulmus macrocarpa and Ulmus pumila (Gao 2002); this also included afforested habitats with young Pinus sylvestris mongolica and Populus spp. in Changling (Zhao et al. 1994). The third habitat consisted of Mongolian steppes or low hills dominated by grass species, such as Filifolium sibiricum, Stipa baicalensis and Spodiopogon sibiricus, interspersed with Prunus sibirica (Gao 2002, Gao et al. 2003). Currently, known populations are all found in the Mongolian steppes. Such habitat specialisation limits the distribution range of Jankowski’s Bunting and makes it vulnerable to habitat loss caused by human activities. For example, the habitat in Inner Mongolia is increasingly used for pasture and crops, so the bunting’s population is likely to decrease even further (BirdLife International 2001). Jankowski’s Bunting has vanished from most of its historic breeding sites during the past 40 years (Gao 2002, Jiang et al. 2008, Wang et al. 2010). This bird was once abundant in the border areas between Jilin Province and Inner Mongolia, and some sites had encounter rates higher than 10 birds per hour. Fu and Chen (1966) suggested the areas surrounding the Daxingan Mountains (Greater Khingan Mountains) should support many Jankowski’s Buntings given the similar veg- etation and climatic elements. With small, discrete sites (i.e. Xiergen and Xinjiamu) found as suit- able habitats in 2008 (Wang et al. 2010), we suspect that undiscovered populations may exist elsewhere. However, no systematic surveys have been conducted, so the population status and any trends related to this species cannot be accurately assessed (BirdLife International 2016). Therefore, we developed this study with the objective of assessing the current breeding distribu- tion and population size of Jankowski’s Bunting within China, which would allow us to provide a more accurate assessment of this species’ conservation status. Methods Field investigations Field investigations The breeding habitat of Jankowski’s Bunting was surveyed between late April and early September, from 2011 to 2016. The survey covered Inner Mongolia and Jilin, Heilongjiang, Liaoning and Hebei Provinces (Fig. 1), which included historical breeding distribution areas, wintering sites, and regions adjacent to historical breeding distribution areas. https://doi.org/10.1017/S0959270917000491 Published online by Cambridge University Press Updated status of Jankowski’s Bunting on China Updated status of Jankowski’s Bunting on China 645 Figure 1. The current and historical breeding distribution of Jankowski’s Bunting Emberiza jankowskii in China. Figure 1. The current and historical breeding distribution of Jankowski’s Bunting Emberiza jankowskii in China. (1) Yakeshi City; (2) Chenbaerhu Qi; (3) Xinbaerhuyou Qi; (4) Xinbaerhuzuo Qi; (5) Hailaer City; (6) Ewenkezuzizhi Qi; (7) Zhalantun City; (8) Keerqinyouyiqian Qi; (9) Zhalaite Qi; (10) Dongwuzhumuqin Qi; (11) Zhaodong City; (12) Zhenlai County; (13) Wulanhaote City; (14) Tuquan County; (15) Taonan City; (16) Keerqinyouyizhong Qi; (17) Baicheng City; (18) Da’an City; (19) Shuangcheng City; (20) Zhalute Qi; (21) Songyuan City; (22) Qianguoerluosi County; (23) Xiwuzhumuqin Qi; (24) Qian’an County; (25) Tongyu County; (26) Alukeerqin Qi; (27) Muling City; (28) Nong’an County; (29) Xilinhaote City; (30) Balinzuo Qi; (31) Changling County; (32) Dongning County; (33) Dunhua City; (34) Keerqinzouyizhong Qi; (35) Balinyou Qi; (36) Linxi County; (37) Keshiketeng Qi; (38) Kailu County; (39) Gongzhuling City; (40) Shuangliao City; (41) Tongliao City; (42) Lishu County; (43) Keerqinzuoyihou Qi; (44) Naiman Qi; (45) Huichun City; (46) Wengniute Qi; (47) Longjing City; (48) Antu County; (49) Kulun Qi; (50) Yanji City; (51) Zhenglan Qi; (52) Tumen City; (53) Aohan Qi; (54) Helong City; (55) Zhangwu County; (56) Chifeng City; (57) Faku County; (58) Duolun County; (59) Weichang County; (60) Fuxinmengguzuzizhi County; (61) Jianping County; (62) Beipiao City; (63) Kalaqin Qi; (64) Fuxin City; (65) Heishan County; (66) Chaoyang County; (67) Yi County. Breeding Site: a) Bayanhua; b) Tianshan; c) Xinmin; d) Kundu; e) Saihantala; f) Bayantala; g) Xiangshan; h) Daolaodu; i) Lubei; j) Wulijimuren; k) Gahaitu; l) Xiergen; m) Bayanhushu; n) Eergetu; o) Tumuji; p) Dagang. o) Tumuji; p) Dagang. First, we visited local forestry bureaus or consulted with local residents for information on the distribution of habitats with similar vegetation structure to the historical breeding sites of the species reported in the literature (Yamashina 1957, Stresemann and Portenko 1981, Zhao et al. 1994, Gao 2002, Gao et al. 2003). Field investigations If detailed information on potential habitats (such as location, patch size, natural or artificial) was obtained, we visited the sites to verify whether Jankowski’s Buntings were breeding there. A site was classified as a breeding ground https://doi.org/10.1017/S0959270917000491 Published online by Cambridge University Press 646 Z. Han et al. strictly based on the presence of Jankowski’s Bunting (determined via call or nest), and each site was checked several times in different breeding seasons. Once a breeding site was identi- fied, we conducted an extended survey centred on the site. If a region had a similar vegetation structure as that of historical breeding sites but no Jankowski’s Bunting were found during the first investigation, we repeated the survey at that site at least once more in subsequent years. Precise locations of breeding sites were recorded using a hand-held GPS (Garmin eTrex Summit HC, Garmin Int. Inc., Kansas). For small suitable habitats, such as Dagang and Eertuge, which are surrounded by farmland or forest, the boundary of the patch was distinct, we directly measured the patch size using GPS. Range size estimation In China, the distribution of Jankowski’s Bunting is currently restricted to Mongolian steppes, where Siberian apricot Prunus sibirica and grass density are important vegetation characteris- tics that could be helpful as reference signatures for classification of remote sensing-derived images. Therefore, we applied visual interpretation of Landsat imagery, a widely used method in land-cover and vegetation mapping (Wilson and Sader 2002, Lillesand et al. 2014), to evalu- ate breeding areas of Jankowski’s Bunting. First, we acquired Landsat 7 Enhanced TM plus (ETM+) and panchromatic Landsat 8 OLI satellite images (path/row 121/28, 121/29) from May and September of 2014, with 30-m and 15-m resolution, respectively. After georeferencing, original DN values of these images were converted into reflectance ratios, and panchromatic and multispectral data were merged in ENVI 5.0 to yield a pan-sharpened false colour compos- ite for high-resolution image interpretation (Jiang et al. 2004, Thomas et al. 2008). Then we performed a Gaussian contrast transformation during the image enhancement process, along with different RGB band combinations (bands 4, 3 and 2; bands 3, 4 and 5; bands 5, 4 and 1), to further delineate vegetation cover and highlight forest, shrub and grass. These steps were repeated as many times as required to assign a final classification. Additionally, we obtained a GIS map of land-use cover (1:100,000 scale; 2012) from the National Science & Technology Infrastructure Center (http://www.geodata.cn) to help discern differences in plant structure at a coarser level, and we combined field GPS sites and trajectories of line transects to create ancillary datasets that improved the accuracy and quality of the final land-cover classification. The above work was carried out in ENVI 5.0 (ENVI 2011), and habitat areas were calculated in ArcGIS 10.1 (ESRI 2012). Population size estimation We estimated the population density of Jankowski’s Bunting using line transects, a commonly used method in bird abundance assessments (Bibby et al. 2000). Bird surveys were conducted from late May to early June in 2014 and 2016, covering currently known breeding ranges. A total of 99 line transects, covering a total of 474.13 km, were established in 2014. In 2016, we resurveyed another six line transects, where bird density fluctuated heavily due to habitat degradation caused by grazing. We established paralleled line transects in intervals of 500 to 1,000 m at each patch, the lengths of which were roughly determined in proportion to the actual patch size. All surveys were completed by six experienced observers who used binocu- lars for visual observations. All surveys were carried out between 05h00 and 09h00 and 15h00–18h00 and only when light and weather conditions were favourable for observation. The observers moved at a speed of 1–1.5 km hr-1 and stopped occasionally to watch and listen. Since Jankowski’s Buntings already bred by the time of the surveys, the males were usually singing on perches, and the females were incubating and were not easily found. At each patch, we recorded the number of male individuals detected by sight or call within a fixed distance of 0.05 km on either side of the central transect line, and the population size was estimated by multiplying the number of observations by two (Jiang et al. 2008). In addition to males https://doi.org/10.1017/S0959270917000491 Published online by Cambridge University Press 647 Updated status of Jankowski’s Bunting on China that were seen or heard, occasional flocks with three or more Jankowski’s Buntings were also recorded during monitoring visits. that were seen or heard, occasional flocks with three or more Jankowski’s Buntings were also recorded during monitoring visits. To estimate the population size, we first estimated bird density in each patch as follows: D=n/2ωL, where D is population density, n is the count number, L is the length of the transect, and ω is 0.05 km (Buckland et al. 2008). The number of birds in the population was then calculated by multiplying bird density (mean ± standard deviation) by the total size (A) of the breeding area: N =AD. We reported the results of our investigation at the town level. A schematic plot on the survey sites and distribution ranges of Jankowski’s Bunting was created in ArcGIS 10.1. Population size estimation To clearly indicate the current distribution boundary of the Jankowski’s Bunting, the 17 pre- viously surveyed counties/cities that were identified as unsuitable for the species were also shown on the map. Discussion Our results show that the population size of Jankowski’s Bunting in China ranges between 9,800 and 12,500 individuals, and the suitable breeding area covers approximately 280 km2, which is mainly located in north-eastern Inner Mongolia but is scattered across different counties. The currently known populations in China are the most significant known globally, especially when the limited information from other historical distribution regions is considered. Previously, the total population of Jankowski’s Bunting was anecdotally estimated to be fewer than 500 pairs (e.g. Wang and Li 2008). Brazil (2009) assumed that there were approxi- mately 100–10,000 breeding pairs, 50–1,000 individuals in migration and 50–1,000 individu- als wintering in China. Having discovered 13 new breeding sites during this survey, declines in the population and the breeding range may be less serious than previously suggested by non-systematic surveys and casual sightings by birdwatchers. However, the situation cannot be considered optimistic. Our findings showed that the distribution range of Jankowski’s Bunting has been substantially reduced from its historical breeding range. In fact, several sites no longer appear to support the species, such as Xinjiamu and Maanshan. The main reason for this decline is habitat loss due to agricultural development, human colonisation, tree planting and overgrazing (Zhao et al. 1994, Gao 2002, Jiang et al. 2008, Wang et al. 2010, BirdLife International 2012). The current breeding distribution of Jankowski’s Bunting is highly scattered (Figure 2). Habitat isolation and fragmentation may drive population declines. For example, Tumuji, Dagang and Eergetu were isolated from other breeding sites, and the remaining population in Dagang has undergone a dramatic decline from about 60 pairs in 1999 to about 10 pairs in 2010. The population in Tumuji has remained relatively constant at about three (ranging from one to five) pairs (Wang et al. 2010). Zhalute Qi and Alukeerqin Qi are newly discovered breeding areas during this study, and they support major popula- tions of Jankowski’s Bunting. However, suitable habitat is severely fragmented here. This fragmentation may affect population persistence by reducing connectivity and restricting dispersal across breeding sites (Fahrig and Merriam 1994). For example, the population severely fluctuates in Xiangshan, where habitat is heavily fragmented, and some isolated habitat patches were recorded as unoccupied during our survey. Jankowski’s Bunting was historically described as a breeding and wintering bird in western Jilin (Fu and Chen 1966), and we also found this species wintering at its current breeding sites in Inner Mongolia. Results In the study, we confirmed that Jankowski’s Bunting has disappeared from most of its historical breeding regions in China (Figure 1), which included Dongning of Heilongjiang, Yanbian, Siping, Taonan, Tongyu and Songyuan of Jilin, and Tuquan of Inner Mongolia. For the remain- ing populations identified prior to 2010, no breeding individuals were detected at Xinjiamu or Maanshan, and the population at Tumiji was unstable (only one pair was observed in 2012, and no birds were recorded in 2014); however, populations at Dagang and Xiergen still persisted. Encouragingly, 13 new breeding localities were discovered in Inner Mongolia during this survey (Figure 2). Figure 2. Breeding habitat distribution of Jankowski’s Bunting (up to 2014) derived from Landsat imagery. The upper-left indicates its entire breeding range in China. Suitable habitats are shown in Zhalute Qi (right) and Alukeerqin Qi (lower left). Among the 16 towns, 13 of them were discovered as new breeding sites for the Jankowski’s Bunting, with the exception of Tumuji, Dagang and Xiergen. Figure 2. Breeding habitat distribution of Jankowski’s Bunting (up to 2014) derived from Landsat imagery. The upper-left indicates its entire breeding range in China. Suitable habitats are shown in Zhalute Qi (right) and Alukeerqin Qi (lower left). Among the 16 towns, 13 of them were discovered as new breeding sites for the Jankowski’s Bunting, with the exception of Tumuji, Dagang and Xiergen. https://doi.org/10.1017/S0959270917000491 Published online by Cambridge University Press 648 Z. Han et al. The area of suitable breeding habitat for Jankowski’s Bunting is approximately 280 km2, as determined by remote sensing (Figure 2). The estimated population size in China ranges between 9,800 and 12,500 individuals. The currently known populations of Jankowski’s Bunting are scattered within the scope of 16 towns in six counties/cities. The population and habitat sizes at Zhalute Qi were the largest, followed by Alukeerqin Qi and Keerqinyouyizhong Qi (Table 1). One pair was recorded in 2012, but no birds were recorded in 2014. Only a small flock of Jankowski’s Bunting exists there. Discussion Higher and dense grasses, especially dry grasses, not only provide nesting materials but also provide shelter for the nests and incubating individuals (Gao 2002). The nearby plant assemblages generally consist of Stipa baicalensis and Spodiopogon sibiricus, which are favoured by livestock as well. In addition to causing physical disturbance, grazing generally reduces the availability of plant biomass and alters plant composition, which may reduce the availability of specific food items (i.e. grass seed and insects) for Jankowski’s Bunting. Grazing also reduces overall vegetation height and cover and increases the risk of predation associated with inter-patch movements, reducing the species’ survival. Other human activities, such as digging up medicinal plants and picking the fruits of Siberian apricot, changed vegetation structure and increased nest-robbing probability (Jiang et al. 2008). Fruit-pickers and shepherds, with poor conservation awareness, usually remove the nests out of curiosity. All currently known Jankowski’s Bunting populations breed in Mongolian steppe-vegetation zones with shrubs dominated by natural Siberian apricots and its suitable breeding habitat is only about 280 km2. Among all breeding areas surveyed, only two of the known sites (Tumuji and Alukeerqin) are officially protected as National Nature Reserves. Most remnant habitats highly benefit from the implementation of the National Key Public Forest Protection Project, which fences protected areas and excludes these habitats from grazing and other agricultural activities. However, the project will be terminated at several sites in 2017, according to China’s State Forestry Administration (http://www.forestry.gov.cn/main/4818/content-797309.html). At other sites, this project was not being strictly implemented, which resulted in portions of suitable breeding habitats being destroyed by overgrazing. In sites where fences have been gradually demolished, such as Saihantala of Alukeerqin Qi and Xiangshan, Daolaodu and Wulijimuren of Zhalute Qi, the local populations declined rapidly. Breeding populations at Xinjiamu and Maanshan have vanished due to habitat degradation. In fact, degraded habitats could be restored if grazing can be managed effectively, which could be done by reducing grazing intensity or by local administrations establishing protected areas. g Third, scattered and small populations are unstable, and the population fluctuates more severely in smaller patches. For example, the population at Tumuji, which is located at the fringe of the Jankowski’s Bunting distribution, varied between one and five pairs from 2001 to 2010 (Wang et al. 2010). One pair was recorded in 2012, but no individuals were recorded during this study. Discussion Thus, as a poor Table 1. The population status of Jankowski’s Bunting in China. County Habitat area (km2) Population density Population Size Transect line (km) Bird counts Zhalute Qi 161.92 39.35 ± 4.04 6372 ± 654 248.39 940 Alukeerqin Qi 95.94 46.50 ± 7.59 4460 ± 728 190.71 775 Keerqinyouyizhong Qi 4.83 60.79 ± 7.85 294 ± 38 16.36 93 Zhenlai 14.68 3.56 c. 50 16.83 6 Keerqinyouyiqian Qi 0.31 * c. 20 1.82 26 Zhalaite Qi 2.4 ** c. 2 Only a small flock of Jankowski’s Bunting exists there. Table 1. The population status of Jankowski’s Bunting in China. https://doi.org/10.1017/S0959270917000491 Published online by Cambridge University Press 649 Updated status of Jankowski’s Bunting on China disperser, Jankowski’s Bunting is more vulnerable to fragmentation. Genetic studies would be necessary to examine the impact of fragmentation on each of these populations. disperser, Jankowski’s Bunting is more vulnerable to fragmentation. Genetic studies would be necessary to examine the impact of fragmentation on each of these populations. The population of Jankowski’s Bunting continues to be vulnerable to a number of threats. First, low reproductive success would result in population declines. Fledging success is a reli- able index of recruitment at the population level (Weatherhead and Dufour 2000); however, the reproductive success of Jankowski’s Bunting in Dagang was very low (23% in Gahaitu, Lubei of Zhalute Qi and Taohai; unpubl. data). Several causes have been identified, including interspecific parasitism by cuckoos, nest abandonment due to grazing disturbance, nest- robbing, predation and other anthropogenic disturbances. Other environmental factors, such as drought and wildfires, also pose severe threats to the breeding and survival of this species (Jiang et al. 2008). ( g ) Second, the suitable breeding habitats of Jankowski’s Bunting are vulnerable to degradation due to grazing. Jankowski’s Bunting is strictly selective in its breeding habitat (Gao et al. 2003). The occurrences of Siberian apricot and higher and dense grasses were crucial factors in its selec- tion of nesting sites (Gao et al. 2003). Its nest is placed amongst sparse grass on the ground, sometimes at the base of a small tree, and is lined with grass and sometimes horse hair (Fu and Chen 1966). Siberian apricot provides perches for the buntings, which may play an important role in territory defence and predator detection (Tong et al. 2002, Bai et al. 2003). https://doi.org/10.1017/S0959270917000491 Published online by Cambridge University Press Discussion At Eergetu of Keerqinyouyiqian Qi, approximately 40 breeding individuals were found centred on a small habitat patch (less than 35 ha) in 2014, but by 2015, the population declined to approximately 20 individuals due to disturbance from raising domestic fowl. The distance to (and from) other breeding sites poses another barrier, as the breeding population https://doi.org/10.1017/S0959270917000491 Published online by Cambridge University Press 650 Z. Han et al. cannot disperse easily to colonise suitable neighbouring habitats. At Xiergen, the population was found to be stable within a fenced, small area (about 33 ha), but this was not the case for the surrounding areas. cannot disperse easily to colonise suitable neighbouring habitats. At Xiergen, the population was found to be stable within a fenced, small area (about 33 ha), but this was not the case for the surrounding areas. Duse to lack of current information, the population status of Jankowski’s Bunting in Russia and North Korea remains unclear, but Litvinenko (1989) reported that the bunting declined from being a locally common breeding bird in the mid-1960s to apparent extinction in Russia. Jankowski’s Bunting is currently breeding close to the Mongolian border, suggesting the pos- sible presence of a nearby, previously unknown breeding population. One male Jankowski’s Bunting was recorded in south-eastern Mongolia (46.8°N 116.2°E) in September 2013 by a local photographer. However, no Jankowski’s Buntings were observed in June 2015 in eastern Mongolia, and the population size is likely to be small if populations do exist within the Numrug Protected Area (Muzika et al. 2015). More surveys should be conducted to ascertain whether unknown breeding populations exist in Mongolia, Russia and North Korea, to pro- vide a more reliable assessment of the status of the species. In China, Jankowski’s Bunting was historically described as a “locally common” breeding and wintering bird in Dongning, Tumen and western Jilin (Yamashina 1957, Fu and Chen 1966). Wintering populations or individuals were occasionally found in winter in several regions of Inner Mongolia, Liaoning, Hebei and Beijing (Seys and Licent 1933, Morrison 1948, Cheng 1987, Huang et al. 1989). We also found Jankowski’s Bunting wintering at its current breeding sites in Inner Mongolia. Additionally, the species was recently found in Beijing (Xing 2016), suggesting that it is capable of dispersing to areas far outside of its known breeding range. Discussion It is important to study its dispersal behaviour (colour-ringing or data-logger studies), which would determine whether young birds are able to colonise distant habitat islands. This will also help to determine if winter habitat requirements are limiting the populations of Jankowski’s Bunting. g In any case, according to the IUCN Red List Categories and Criteria (IUCN 2001), the current status of Jankowski’s Bunting in China still meets criteria B2ab for an ‘Endangered’ species, as the area of occupancy is less than 500 km2, the current population is confined to Inner Mongolia (B2a), and the number of subpopulations in Dagang, Tumuji, and Eertuge is continuously decreas- ing (B2b). Conservation efforts are urgently needed based on the continued loss and degradation of the species’ habitat and the fact that only two of the known sites have any form of official protection. New protected areas and/or nature reserves should be considered to protect the main breeding populations in Alukeerqin Qi and Zhalute Qi. Ecological studies should be conducted at the breeding sites to clarify habitat requirements during the breeding season, while detailed man- agement regimes need to be established to manage the remaining habitat. Surveys targeted at identifying the dispersal patterns of Jankowski’s Bunting would help determine its dispersal routes and wintering grounds. Genetic studies would also be necessary to examine the impact of fragmentation on each subpopulation. Finally, it is also critical to develop a conservation manage- ment plan for this species. References IUCN (2001) Red List categories and crite- ria. Version 3.1. Gland, Switzerland and Cambridge, UK: IUCN Species Survival Commission. Bai, H. S., Gao, W. and Zhou, D. W. (2003) The breeding habits of the Jankowski’s Bunting (Emberiza jankowskii). J. Northeast Normal Univ. 38: 36–40. Jiang, H., Strittholt, J. R., Frost, P. A. and Slosser, N. C. (2004) The classification of late seral forests in the Pacific Northwest, USA using Landsat ETM+ imagery. Remote Sens Environ. 91: 320–331. Bibby, C. J., Burgess, N. D., Hill, D. A. and Mustoe, S. H. (2000) Bird census techniques. London: Academic Press. BirdLife International (2001) Threatened birds of Asia: The BirdLife International Red Data Book. Cambridge, UK: BirdLife International. Jiang, Y. L., Gao, W., Lei, F. M., Wan, D. M., Zhao, J. and Wang, H. T. (2008) Nesting biology and population dynamics of endan- gered Jankowski’s Bunting Emberiza jankowskii in Western Jilin, China. Bird Conserv Internatn. 18: 153–163. BirdLife International (2012) Emberiza jankowskii. The IUCN Red List of Threatened Species 2012: e.T22720905A38107539. http:// dx.doi.org/10.2305/IUCN.UK.2012 1.RLTS. T22720905A38107539.en. Downloaded on 04 January 2016. Lillesand, T., Kiefer, R. W. and Chipman, J. (2014) Remote sensing and image interpretation. London, UK: John Wiley & Sons. BirdLife International (2016) Emberiza jankowskii. The IUCN Red List of Threatened Species 2016: e.T22720905A94689303. http:// dx.doi.org/10.2305/IUCN.UK.2016-3.RLTS. T22720905A94689303.en Litvinenko, N. M. (1989) [Rufous-backed Bunting Emberiza jankowskii Taczanowski, 1888]. Pp. 172–173 in P. A. Ler, ed. [Rare vertebrates of the Soviet Far East and their protection]. Leningrad: Nauka. (In Russian). Brazil, M. (2009) Birds of East Asia: eastern China, Taiwan, Korea, Japan, eastern Russia. London, UK: Christopher Helm. Litvinenko, N. M. and Shibaev, J. W. (1966) Zur Brutökologie von Emberiza jankowskii Taczanowski. J. Orn. 107: 346–351. Buckland, S. T., Marsden, S. J. and Green, R. E. (2008) Estimating bird abundance: making methods work. Bird Conserv Internatn. 18: S91–S108. Morrison, A. (1948) Emberiza jankowskii from near Peking. Ibis 90: 132. Muzika, Y., Fu, V., Townshend, T. and Yu, Y. T. (2015) Ornithological survey in Dornod province, eastern Mongolia. Birding Asia 24: 54–63. Cheng, T. H. (1987) A synopsis of the avi- fauna of China. Beijing: Science Press. ENVI (2011) Envi 5.0 User’s Guide. Boulder, Colorado: Exelis Visual Information Solutions. Panov, E. N. (1973) [The birds of South Ussuriland]. Novosibirsk: Nauka. (In Russian). ESRI (2012) ArcGIS Desktop and Spatial Analyst Extension: Release 10.1. Redlands, CA: Environmental Systems Research Institute. Sekercioglu, C. H., Schneider, S. H., Fay, J. Updated status of Jankowski’s Bunting on China 651 Acknowledgements This work was supported by the State Bureau of Forestry (Special Investigation Project on Jankowski’s Bunting), the National Natural Science Foundation of China (No. 31172109, 31670398), the Jilin Provincial Science and Technology Department (No. 20150101067JC), and the Fundamental Research Funds for the Central Universities (2412016KJ043). We thank the “National Earth System Science Data Sharing Infrastructure”, National Science & Technology Infrastructure of China (http://www.geodata.cn) for geographical data support. We thank Gui-quan Xiang, Zhi-jie Yang, Tuo Wang, Ji-Yuan Yao, Hui-Juan Tao, Bo Zhang, Jiang-ping YU, Le-yong Chen, Hong-wei Xu, and Yang-yang Yu for their assistance in the field. We thank Pang Chun Chiu, Terry Townshend, Dave Buckingham and anonymous reviewers for their helpful remarks on this paper. https://doi.org/10.1017/S0959270917000491 Published online by Cambridge University Press Updated status of Jankowski’s Bunting on China References P., and Loarie, S. R. (2008) Climate change, ele- vational range shifts, and bird extinctions. Conserv Biol. 22: 140–150. Fahrig, L. and Merriam, G. (1994) Conservation of fragmented populations. Conserv. Biol. 8: 50–59. Seys, G. and Licent, E. (1933) La collection d’oiseaux du Musee Hoangho Paiho de Tien Tsin. Publ.. Mus. Hoangho Paiho de Tien Tsin No. 19. Fu, T. S. and Chen, P. (1966) The distribution and breeding habitats of Emberiza jankowskii. Acta Zool. Sinica 18: 197–198. Stresemann, E. and Portenko, L. A. (1981) Atlas der Verbreitung Palaearktischer Vögell, 9. Berlin: Akademie-Verlag. Gao, W. (2002) Ecology in Jankowski’s Bunting. Jilin: Science and Technology Press. Thomas, C., Ranchin, T., Wald, L. and Chanussot, J. (2008) Synthesis of multispectral images to high spatial resolution: A critical review of fusion methods based on remote sensing physics. IEEE T Geosci Remote 46: 1301–1312. Gao, W., Wang, H. T. and Sun, D. T. (2003) The habitat and nest-site selection of Jankowski’s Bunting. Acta Ecol. Sincia 23: 665–672. Huang, M. P. (1989) Fauna Liaoningica (Aves). Liaoning: Science and Technology Press. https://doi.org/10.1017/S0959270917000491 Published online by Cambridge University Press 652 Z. Han et al. Wilson, E. H. and Sader, S. A. (2002) Detection of forest harvest type using multiple dates of Landsat TM imagery. Remote Sens Environ. 80: 385–396. Tong, F. C., Xiao, Y. H., Bai, H. S., Sun, D. T., Gao, W. and Wang, Q. L. (2002) Breeding ecology of Jankowski’s Bunting in the dry grassland in Baicheng, Jilin Province. Acta Ecol. Sincia 22: 1485–1490. Xing, C., Huang, M., Holt, P., Townshend, T. and Wielstra, B. (2016) First Jankowski’s Buntings Emberiza jankowskii wintering in Beijing for 75 years. Birding Asia 25: 62–68. Vorob’ev, K. A. (1954) [The birds of Ussuriland]. Moscow: Academy of Sciences of the USSR. (In Russian). Wang, H. T., Jiang, Y. L. and Gao, W. (2010) Jankowski’s Bunting (Emberiza jankowskii): current status and conservation. Chinese Birds 1: 251–258. Yamashina, Y. (1957) Notes on Emberiza jankowskii Taczanowski with special refer- ence to its speciation. J. Fac. Sci. Hokkaido Univ. Ser. VI, Zool. 13: 164–171. Wang, R. Q. and Li, F. (2008) The Jankowski’s Bunting on the edge. Forest and Humankind 10: 90–95. Zhao, Z. J., Nickel, H. and Groh, G. (1994) Vorkommen und Gesang der Jankowskiammer (Emberiza jankowskii) in der chinesischen Provinz Jilin. J. Orn. 135: 617–620. Weatherhead, P. J. and Dufour, K. W. YU LIU, HAI-TAO WANG Jilin Provincial Engineering Laboratory of Avian Ecology and Conservation Genetics, Northeast Normal University, Changchun 130024, China. ZHENG HAN, LIN WANG School of Life Sciences, Jilin Provincial Key Laboratory of Animal Resource Conservation and Utilization, Northeast Normal University, Changchun 130024, China. References (2000) Fledging success as an index of recruitment in red-winged blackbirds. Auk 117: 627–633. LI-SHI ZHANG, YUN-LEI JIANG Animal’s Scientific and Technological Institute, Agricultural University of Jilin, Xincheng Street 2888, Changchun 130118, China. Animal’s Scientific and Technological Institute, Agricultural University of Jilin, Xincheng Street 2888, Changchun 130118, China. BO QIN Jilin Momoge National Nature Reserve Administration, Zhenlai 132000, China. VIVIAN WING KAN FU The Hong Kong Bird Watching Society, 7 C, V Ga Building, 532 Castle Peak Road, Lai Chi Kok, Kowloon, Hong Kong. JIANPING FU China Biodiversity Conservation and Green Development Foundation, Beijing 100089, China. *Author for correspondence; e-mail: wanght402@nenu.edu.cn Received 24 November 2016; revision accepted 24 September 2017; Published online 14 December 2017 BO QIN VIVIAN WING KAN FU The Hong Kong Bird Watching Society, 7 C, V Ga Building, 532 Castle Peak Road, Lai Chi Kok, Kowloon, Hong Kong. https://doi.org/10.1017/S0959270917000491 Published online by Cambridge University Press
https://openalex.org/W4283456632	https://link.springer.com/content/pdf/10.1007/s00192-022-05277-4.pdf	English	null	Systematic review exploring the relationship between sexual abuse and lower urinary tract symptoms	International urogynecology journal	2,022	cc-by	12,639	Abstract Abstract Introduction and hypothesis Patients presenting with lower urinary tract symptoms (LUTS) may report a history of sexual abuse (SA), and survivors of SA may report LUTS; however, the nature of the relationship is poorly understood. The aim of this review is to systematically evaluate studies that explore LUT dysfunction in survivors of SA. Methods A systematic literature search of six databases, Cochrane Database of Systematic Reviews, MEDLINE, EMBASE, CINAHL, AMED, and PsycINFO, was performed. The last search date was June 2021 (PROSPERO CRD42019122080). Studies reporting the prevalence and symptoms of LUTS in patients who have experienced SA were included. The literature was appraised according to the PRISMA statement. The quality of the studies was assessed. Results Out of 272 papers retrieved, 18 publications met the inclusion criteria: studies exploring LUTS in SA survivors (n=2), SA in patients attending clinics for their LUTs (n=8), and cross-sectional studies (n=8). SA prevalence ranged between 1.3% and 49.6%. A history of SA was associated with psychosocial stressors, depression, and anxiety. LUTS included urinary storage symptoms, voiding difficulties, voluntary holding of urine and urinary tract infections. Most studies were of moderate quality. Assessment of SA and LUTS lacked standardisation. Conclusions The review highlights the need for a holistic assessment of patients presenting with LUTS. Although most of the studies were rated as being of ‘moderate’ quality, the evidence suggests the need to provide a “safe space” in clinic for patients to share sensitive information about trauma. Any such disclosure should be followed up with further assessment. Keywords Childhood sexual abuse · Childhood trauma · Post-traumatic stress disorder · Lower urinary tract symptoms · Trauma Keywords Childhood sexual abuse · Childhood trauma · Post-traumatic stress disorder · Lower urinary tract symptoms · Trauma Keywords Childhood sexual abuse · Childhood trauma · Post-traumatic stress disorder · Lower urinary tr International Urogynecology Journal (2023) 34:635–653 https://doi.org/10.1007/s00192-022-05277-4 International Urogynecology Journal (2023) 34:635–653 https://doi.org/10.1007/s00192-022-05277-4 REVIEW ARTICLE 4 Department of Urology, Academic Hospital Pitié‑Salpêtrière, Medical School, Sorbonne University, AP–HP, 47–83, Bd de l’Hôpital, 75651 Paris cedex 13, France * Caroline Selai c.selai@ucl.ac.uk Systematic review exploring the relationship between sexual abuse and lower urinary tract symptoms Caroline Selai1,2 · Michael S. Elmalem1,3 · Emmanuel Chartier‑Kastler4 · Natalia Sassoon1 · S Maria Francisca Rocha1 · Larisa Klitsinari1 · Jalesh N. Panicker2,3 Received: 9 March 2022 / Accepted: 3 June 2022 / Published online: 25 June 2022 © The Author(s) 2022 1 UCL Department of Clinical and Movement Neurosciences, Queen Square Institute of Neurology, Queen Square, London WC1N 3BG, UK * Caroline Selai c.selai@ucl.ac.uk Introduction The following key words were used: “sexual dysfunction” OR “sexual abuse” OR “adult sexual abuse” OR “sexual trauma” OR “child- hood sexual abuse” OR “CSA” OR “sexual maltreatment” OR “rape” OR “sexual offences” OR “sexual harassment” OR “sexual harm” OR “urinary tract” OR “urologist” OR “urological dysfunction” OR “urological symptoms” OR “LUTS” OR “lower urinary tract symptoms” OR “lower urinary tract problems” OR “uroneurology” OR “ure- thral” OR “genitourinary” OR “urinary frequency” OR “urgency” OR “urinary infection” AND “treatment” OR “management” OR “symptoms”. reported, including anxiety, anger, depression, re-victimi- sation, self-mutilation, sexual difficulties, substance abuse, suicidality, impairment of self-concept, interpersonal problems, obsessions and compulsions, dissociation and post-traumatic stress responses to somatisation character- ised by medically unexplained symptoms [7–11]. Somatisation, functional neurological symptoms and other medically unexplained symptoms can lead to repeated consul- tations and help-seeking behaviour, which can have significant financial implications in terms of use of health care resources and receipt of financial assistance [12]. Abuse occurring in childhood before the age of 17 (CSA) can result in multiple long-term consequences such as depression, anxiety, poor physical health and risky health behaviours [13]. Furthermore, CSA has been found to be significantly associated with poor outcomes when treating conversion disorders/functional neu- rological disorder [14]. Urological symptoms are likely to be common amongst survivors of SA. A Dutch study suggested that 2.1% of men and 13% of women seeking urological care may report SA [15]. Many of the physical and psychological sequelae of CSA were found to persist into adulthood [16] and up to one-third of patients attending a gynaecology clinic had experienced CSA [17, 18]. Victims of CSA younger than 6 years old most com- monly reported urinary tract infections, daytime incontinence and nocturnal enuresis [19]. SA is likely to be underreported and in the Dutch study, only 15% of participants with a history of SA had disclosed this to their urologist [15]. In a study across five Nordic countries, most women did not disclose a history of SA to their gynaecologist [17]. Seventy percent of Dutch urolo- gists enquired about SA when taking the medical history [20]; however, enquiry rates may vary across specialities and different health care settings. Abstracts were imported into bibliography management software (EndNote X8; Thomson Reuters, PA, USA) and were independently evaluated by two reviewers (NS and SH). Introduction from being a complete stranger to someone familiar to the victim [2] and acts can be committed in private or in public spaces. The prevalence of SA is largely underestimated; however, the results of a recent survey suggests that 1 in 5 women and 1 in 59 men have been exposed to an attempted or completed act of rape during their lifetime [2]. Rates of childhood sexual abuse (CSA) can vary: between 2% and 62% of females and between 3% and 16% of males [3]. The reason for underreporting by victims are manifold, and can include feelings of shame, fear and guilt, a risk of retaliation by the perpetrator [4] and a lack of awareness that forced sexual acts constitute SA [5]. Attempted or executed sexual abuse (SA) conducted without consent from the victim can involve penetrative or non-penetra- tive acts and non-contact [1]. The perpetrator of abuse can range 1 UCL Department of Clinical and Movement Neurosciences, Queen Square Institute of Neurology, Queen Square, London WC1N 3BG, UK Abuse can have a profound impact on victims, rang- ing from reduced global functioning levels to lengthened trauma-related symptoms and an increased risk of devel- oping substance abuse [6]. Both male and female victims can report increased rates of depression, anxiety, suicidal ideation and post-traumatic stress disorder (PTSD) [7]. Multiple physical and psychological sequelae have been 2 Department of Uro‑Neurology, The National Hospital for Neurology and Neurosurgery, Queen Square, London, UK 3 UCL Department of Brain Repair and Rehabilitation, Queen Square Institute of Neurology, Queen Square, London, UK (0123 1 3456789) 3 International Urogynecology Journal (2023) 34:635–653 636 with the International Prospective Register of Systematic Reviews (PROSPERO; CRD42019122080). A literature search was performed in December 2018 and updated in June 2021 for studies published in the English language without date restrictions in the following databases: Cochrane Database of Systematic Reviews, MEDLINE, EMBASE, CINAHL, AMED, and PsycINFO. The same search strategy (i.e. keywords and inclusion and exclusion criteria) was used for all the databases. Results The PRISMA flow diagram is presented in Fig. 1. A total of 272 studies were retrieved, and 18 studies met the inclu- sion criteria: studies exploring LUTS in SA survivors (n=2), studies exploring SA in patients attending clinics for their LUTS (n=8), and large cross-sectional studies evaluating different health issues including SA and LUTS Introduction Studies relevant to the review reporting the preva- lence and symptoms of LUTS in male and female patients who have experienced SA were included, whereas experi- mental studies in animals and studies primarily assessing interstitial cystitis, bladder pain syndrome and pain were excluded. The results of the two reviewers were compared and consensus was achieved by discussion; unresolved dif- ferences were reviewed independently (JNP). Accepted abstracts were retrieved in full text and assessed by the two reviewers (NS and SH), and the fol- lowing variables were assessed: setting and nature of cohort, definition of SA, assessment of SA, nature of abuse, other types of abuse, nature of LUTS, assessment of LUTS, diagnostic LUTS test and findings, and other co-morbidities. The quality of the studies and risk of bias were assessed using the assessment tool for quantitative studies by the Effective Public Health Practice Project (EPHPP) [22]. Each section was rated by the two review- ers and any discrepancies between scores were discussed and reconciled. A recent systematic review and meta-analysis of 38 stud- ies has demonstrated a significant association between a history of sexual assault and developing different gynae- cological disorders such as pelvic pain, dyspareunia, dys- menorrhea, abnormal menstrual bleeding and urinary incontinence later in life [21]; however, lower urinary tract dysfunction was not specifically evaluated. i The relationship between SA and LUT dysfunction, how- ever, has been poorly understood. The purpose of this sys- tematic review was to evaluate the reported prevalence of SA, pattern of lower urinary tract symptoms (LUTS) and explore possible associations between SA and LUT dysfunction. Studies exploring SA in patients attending clinics for their LUTS Table 2 summarises the results of these studies [23, 24, 26–29, 37, 40]. Four studies used validated scales to assess LUTS: UDI-6 [23, 24, 26, 29], IIQ-7 [24, 26, 29], OABq-SF Studies exploring LUTS in survivors of sexual assault Table 1 summarises the results of two studies [25, 39]. SA was assessed using a non-validated questionnaire includ- ing questions about inappropriate unwanted sexual behav- iours experienced before the age of 16 [25] or not reported [39]. LUTS were assessed using either a non-validated [25] or validated (Amsterdam Hyperactive Pelvic Floor Scale Women) [39] questionnaire. Materials and methods The systematic review conformed to the Preferred Report- ing Items for Systematic Review and Meta-Analysis (PRISMA) statement and the protocol was registered 1 3 International Urogynecology Journal (2023) 34:635–653 637 (n=8). The majority of studies were prospective question- naire-based cross-sectional studies (n=13; see Tables 1, 2 and 3). One study was a case–control study [23] and one was longitudinal [24]. The other studies were retrospec- tive, cross-sectional in nature (n=3). Fourteen studies were conducted in the US [23–36], 2 in Germany [37, 38], 1 in the Netherlands [39] and 1 in Hong Kong [40]. [23] or a battery of questionnaires (ICIQ-UI, ICIQ-OAB, OABq, USS) [29]. Four studies used non-validated scales or other methods [27, 28, 37, 40]. The prevalence of reported SA ranged from 1.3% [40] to 49.6% [26]. Rates of trauma were significantly higher in patients with LUTS than in control subjects in six studies [23, 24, 26, 29, 37, 40]. SA was assessed using validated scales in three studies: Childhood Traumatic Events Scale and Recent Traumatic Events Scale [29], Modified Abuse Assessment Screen [28], Behavioral Risk Factor Surveil- lance Scheme BRFSS-ACE Module [23], a non-validated questionnaire [37], a modified previous survey [27], and by a single question [26–28]. The definition of SA differed according to study and included forced sexual activity [27, 40], childhood traumatic events occurring prior to age 17 [29], unwanted sexual activity [28], unwanted sexual touch- ing, forced unwanted sexual touching and forced sex during childhood [23]. A precise definition—complete sexual pen- etration of the vagina, mouth or rectum without a women’s consent, involving the use of force or threat of harm—was used in only one study ([24]. SA was not defined in two studies [26, 37]. Large cross‑sectional studies evaluating different health issues including SA and LUTS Table 3 summarises the results of these studies [30–36, 38]. SA was assessed using different methods and only one study used a validated questionnaire, The Childhood Traumatic Events Scale [36]. The prevalence of SA varied greatly between studies, from 9% [33] to 52.5% [38]. A total of 11.4% reported CSA and 39.2% reported an unwanted first sexual experience [35]. The prevalence of CSA was 21.6% and SA in adolescence/adulthood was reported to be 19.5% [30]; 25% (n=127) of women and 8% of men (n=38) reported traumatic sexual experience [36]. LUTS were assessed using validated questionnaires in only three stud- ies: OABq-SF, PFDI-20; POPDI-6, UDI-6 [31], UDI-6 [32], the LUTS tool and the PFDI-20 [36]. Duplicates removed (n = 16) Records excluded (n = 65) Studies retrieved from search (n = 272) Studies meeting inclusion/exclusion criteria (n = 18) Full-text studies reviewed (n = 83) Records excluded (n = 173) Records screened by title and abstract (n = 256) Fig. 1 Preferred Reporting Items for Systematic Review and Meta- Analysis flow diagram Studies retrieved from search (n = 272) Duplicates removed (n = 16) Records screened by title and abstract (n = 256) Records excluded (n = 173) Assessment of quality of included studies Full-text studies reviewed (n = 83) Using the EPHPP assessment tool, the quality of five studies were rated “weak” [24, 25, 28, 32, 38], 12 studies were rated “moderate” [23, 26, 27, 29–31, 33, 35–37, 39, 40] and only one study was rated “strong” (Fig. 2) [34]. Records excluded (n = 65) Studies meeting inclusion/exclusion criteria (n = 18) Discussion In this review we present a synthesis of 18 studies that explore LUTS in survivors of SA. The wide prevalence of abuse across studies reflects differences in the cohorts Fig. 1 Preferred Reporting Items for Systematic Review and Meta- Analysis flow diagram 1 3 638 International Urogynecology Journal (2023) 34:635–653 Table 1 Observational studies exploring lower urinary tract symptoms (LUTS) amongst sexual abuse survivors (n=2) CSA childhood sexual abuse, FSFI Female Sexual Function Index, LUTS lower urinary tract symptoms, AHPFS-W hyperactive pelvic floor scale-women, PTSD post-traumatic stress disorder, SA sexual abuse Study Setting and cohort Assessment and comple- tion rate Definition of SA Other forms of abuse assessed? LUT diagnostic tests Findings Other comorbidities reported Davila et al. [25] Prospective, cross- sectional, question- naire-based study on childhood SA survivors (n=58; mean age 41.5 years) attending support therapy groups (women only). Controls (n=51; mean age 48 years) attending a general gynaecology clinic without urological com- plaints or SA history CSA and LUTS: non-val- idated 52-item question- naire. CSA survivors: female (n=58). Controls: female (n=51). Comple- tion rate not reported None No None A total of 72% of CSA survivors vs 22% of controls reported urinary incontinence. Signifi- cantly more problems in all (4/4) aspects of stress incontinence (lose with exertion, lose small spurts, lose standing, able to stop flow volun- tarily), in (3/8) aspects of urge incontinence (urgency before loss, strong urge with loss, strong urge with loss, leak before reaching toi- let) and in (4/6) aspects of voiding dysfunc- tion (difficulty starting stream, slow stream, dribbling/fullness, hold urine until painful) were reported by abuse survivors compared with controls Diabetes, kidney disease, lumbar disc disease, neu- rological disease, history of stroke. Significantly more in the abuse survivor group than in controls reported emotional problems, psychiatric conditions, difficulties achieving orgasm and dys- pareunia Postma et al. [39] Prospective, cross-sec- tional, questionnaire- based study. Cross-sec- tional study; 89 young women aged 18–25 years victimised by rape in adolescence; 114 non- victimised age-matched controls. Rape victims: mean age: 20.9 years. Controls: mean age: 20.8 years Sexual functioning: Dutch version of the FSFI. LUTS: AHPFS-W. Discussion Com- pletion rate not reported Involuntary (attempted or completed) penetration of the victim’s vagina or anus by penis, tongue, fingers, or object, or the victim’s mouth by penis No None Victims 2.7 times more likely to have pelvic floor dysfunction (including LUTS, pro- voked vulvodynia) than non-victimised controls PTSD, irritable bowel syn- drome, rectal problems, stress. Rape victims had 2.4 times greater likeli- hood of sexual dysfunc- tion than controls Table 1 Observational studies exploring lower urinary tract symptoms (LUTS) amongst sexual abuse survivors (n=2) Study Setting and cohort Assessment and comple- tion rate Definition of SA Other forms of abuse assessed? LUT diagnostic tests Findings Other comorbidities reported Davila et al. [25] Prospective, cross- sectional, question- naire-based study on childhood SA survivors (n=58; mean age 41.5 years) attending support therapy groups (women only). Controls (n=51; mean age 48 years) attending a general gynaecology clinic without urological com- plaints or SA history CSA and LUTS: non-val- idated 52-item question- naire. CSA survivors: female (n=58). Controls: female (n=51). Comple- tion rate not reported None No None A total of 72% of CSA survivors vs 22% of controls reported urinary incontinence. Signifi- cantly more problems in all (4/4) aspects of stress incontinence (lose with exertion, lose small spurts, lose standing, able to stop flow volun- tarily), in (3/8) aspects of urge incontinence (urgency before loss, strong urge with loss, strong urge with loss, leak before reaching toi- let) and in (4/6) aspects of voiding dysfunc- tion (difficulty starting stream, slow stream, dribbling/fullness, hold urine until painful) were reported by abuse survivors compared with controls Diabetes, kidney disease, lumbar disc disease, neu- rological disease, history of stroke. Significantly more in the abuse survivor group than in controls reported emotional problems, psychiatric conditions, difficulties achieving orgasm and dys- pareunia Postma et al. [39] Prospective, cross-sec- tional, questionnaire- based study. Cross-sec- tional study; 89 young women aged 18–25 years victimised by rape i d l 114 Sexual functioning: Dutch version of the FSFI. LUTS: AHPFS-W. Com- pletion rate not reported Involuntary (attempted or completed) penetration of the victim’s vagina or anus by penis, tongue, fingers, or object, or the victim’s mouth by penis No None Victims 2.7 times more likely to have pelvic floor dysfunction (including LUTS, pro- voked vulvodynia) than non-victimised controls PTSD, irritable bowel syn- drome, rectal problems, stress. Discussion 121 women attend- ing primary care clinic and referred to specialised LUTS clinic (mean age = 50 years) vs 1,298 controls (mean age 48 years) Sexual trauma ques- tion: “have you ever been forced to have sex against your will?” LUTS: UDI-6, IIQ-7. Completion rates not mentioned Not fully provided, “sexual trauma” Emotional abuse, physical abuse None Rates of sexual trauma were significantly higher for women in the LUTS clinic (49.6%) (n=60/121) than for those in the primary care clinic (20.1%) (n=285/1,298). Rates of psychiatric comorbidity were significantly higher for women in the LUTS clinic (64.5%) than for those in the primary care clinic (25.9%). IIQ-7 scores significantly higher in patients with both psychiatric comorbidities and sexual trauma than scores of patients with neither Psychiatric comorbidi- ties Lai et al. [29] Prospective, cross-sectional, questionnaire-based study. Patients (both men and women) in early 50s with OAB (n=51) and age-matched healthy controls (n=30). Mean age OAB=53.4 years; mean age controls= 54.2years Childhood traumatic events: Childhood Traumatic Events Scale and Recent Traumatic Events Scale. LUT: ICIQ- UI, ICIQ-OAB, OAB-q, UDI-6, IIQ- 7, USS. Comple- tion rates were not mentioned Not fully provided. Childhood traumatic events occurring prior to age 17 Death of a fam- ily member or a close friend, parental divorce or separation, victim of violence, been extremely ill or injured Clinical assessment of OAB based on AUA guidelines; patients with a positive cul- ture or a post-void residual ≥150 ml were excluded Childhood sexual trauma more preva- lent in OAB patients than in controls, 29.4% (n=15) vs 6.7% (n=2) Childhood trauma associated with worse bladder pain, non- urologic pain, poorer mood, higher anxi- ety, higher somatic symptom burden and higher psychological stress Table 2 Observational studies exploring sexual abuse amongst patients attending clinics for lower urinary tract symptoms (LUTS; n = 8) Study Setting and cohort Assessment and com- pletion rate Definition of SA Other forms of abuse assessed? LUT diagnostic tests Findings Other comorbidities reported Klausner et al. [26] Prospective, cross- sectional, question- naire-based study. 121 women attend- ing primary care clinic and referred to specialised LUTS clinic (mean age = 50 years) vs 1,298 controls (mean age 48 years) Sexual trauma ques- tion: “have you ever been forced to have sex against your will?” LUTS: UDI-6, IIQ-7. Discussion Rape victims had 2.4 times greater likeli- hood of sexual dysfunc- tion than controls None 1 3 639 International Urogynecology Journal (2023) 34:635–653 with neither Lai et al. [29] Prospective, cross-sectional, questionnaire-based study. Patients (both men and women) in early 50s with OAB (n=51) and age-matched healthy controls (n=30). Mean age OAB=53.4 years; mean age controls= 54.2years Childhood traumatic events: Childhood Traumatic Events Scale and Recent Traumatic Events Scale. LUT: ICIQ- UI, ICIQ-OAB, OAB-q, UDI-6, IIQ- 7, USS. Comple- tion rates were not mentioned Not fully provided. Childhood traumatic events occurring prior to age 17 Death of a fam- ily member or a close friend, parental divorce or separation, victim of violence, been extremely ill or injured Clinical assessment of OAB based on AUA guidelines; patients with a positive cul- ture or a post-void residual ≥150 ml were excluded Childhood sexual trauma more preva- lent in OAB patients than in controls, 29.4% (n=15) vs 6.7% (n=2) Childhood trauma associated with worse bladder pain, non- urologic pain, poorer mood, higher anxi- ety, higher somatic symptom burden and higher psychological stress Table 2 Observational studies exploring sexual abuse amongst patients attending clinics for lower urinary tract symptoms (LUTS; n = 8) Study Setting and cohort Assessment and com- pletion rate Definition of SA Other forms of abuse assessed? LUT diagnostic tests Findings Other comorbidities reported Klausner et al. [26] Prospective, cross- sectional, question- naire-based study. 121 women attend- ing primary care clinic and referred to specialised LUTS clinic (mean age = 50 years) vs 1,298 controls (mean age 48 years) Sexual trauma ques- tion: “have you ever been forced to have sex against your will?” LUTS: UDI-6, IIQ-7. Completion rates not mentioned Not fully provided, “sexual trauma” Emotional abuse, physical abuse None Rates of sexual trauma were significantly higher for women in the LUTS clinic (49.6%) (n=60/121) than for those in the primary care clinic (20.1%) (n=285/1,298). Rates of psychiatric comorbidity were significantly higher for women in the Psychiatric comorbidi- ties Table 2 Observational studies exploring sexual abuse amongst patients attending clinics for lower urinary tract symptoms (LUTS; n = 8) Study Setting and cohort Assessment and com- pletion rate Definition of SA Other forms of abuse assessed? LUT diagnostic tests Findings Other comorbidities reported Klausner et al. [26] Prospective, cross- sectional, question- naire-based study. Discussion Completion rates not mentioned Not fully provided, “sexual trauma” Emotional abuse, physical abuse None Rates of sexual trauma were significantly higher for women in the LUTS clinic (49.6%) (n=60/121) than for those in the primary care clinic (20.1%) (n=285/1,298). Rates of psychiatric comorbidity were significantly higher for women in the LUTS clinic (64.5%) than for those in the primary care clinic (25.9%). IIQ-7 scores significantly higher in patients with both psychiatric comorbidities and sexual trauma than scores of patients with neither Psychiatric comorbidi- ties Lai et al. [29] Prospective, cross-sectional, questionnaire-based study. Patients (both men and women) in early 50s with OAB (n=51) and age-matched healthy controls (n=30). Mean age OAB=53.4 years; mean age controls= 54.2years Childhood traumatic events: Childhood Traumatic Events Scale and Recent Traumatic Events Scale. LUT: ICIQ- UI, ICIQ-OAB, OAB-q, UDI-6, IIQ- 7, USS. Comple- tion rates were not mentioned Not fully provided. Childhood traumatic events occurring prior to age 17 Death of a fam- ily member or a close friend, parental divorce or separation, victim of violence, been extremely ill or injured Clinical assessment of OAB based on AUA guidelines; patients with a positive cul- ture or a post-void residual ≥150 ml were excluded Childhood sexual trauma more preva- lent in OAB patients than in controls, 29.4% (n=15) vs 6.7% (n=2) Childhood trauma associated with worse bladder pain, non- urologic pain, poorer mood, higher anxi- ety, higher somatic symptom burden and higher psychological stress Lai et al. [29] 1 International Urogynecology Journal (2023) 34:635–653 640 40 International Urogynecology Jo ( ) Setting and cohort Assessment and com- pletion rate Definition of SA Other forms of abuse assessed? LUT diagnostic tests Findings Other comorbidities reported t al. [37] Prospective, cross- sectional, question- naire-based study. 243 women divided into three groups: OAB (n = 85; mean age = 56.3); SUI, (n = 101; mean age = 54.6) and control (n= 57; mean age = 46.4) Anonymous, non- validated ques- tionnaire about bladder function and physical/sexual violence. Response rate: 69.4% Not provided Physical abuse N/A 30.6% (n=26) of women with OAB had previously been physically or sexually abused, 17.8% (n=18) of women with stress incontinence and 17.5% (n=10) of the control group had the same history. 85 (34.9%) had OAB, 101 (41.5%) had SUI and the remain- der (57, 23.4%) had no LUTS Not reported dorf et al. Discussion [27] Prospective, observa- tional study of 190 women attending a urogynaecology subspecialty clinic. Mean/median age not reported SA: HITS screen for IPV, and other 30 unvalidated items (demographics, overall health, rela- tionship duration, IPV history and more). Response rate: 75% Not provided Physical abuse N/A 29 women (20%) reported history of forced sex; 4 women (2.8%) positive HITS screen; 39 (27.5%) responded positively to at least one of the HITS items. Lifetime prevalence of physical abuse in 10 women (7%). 23% reported a history of abuse in their family. No statistically sig- nificant associations between a positive HITS score and any of the urogynaecol- ogy symptoms Not reported 3 641 International Urogynecology Journal (2023) 34:635–653 gy gy ( ) (continued) Setting and cohort Assessment and com- pletion rate Definition of SA Other forms of abuse assessed? LUT diagnostic tests Findings Other comorbidities reported y et al. [24] A cross-sectional analysis of a nation- wide cohort study. A longitudinal study of 1,702 female veterans (mean age = 31.1years) SA: questions modified from past national surveys as well as studies on violence in women and female veterans. LUT: computer- assisted telephone interview: the UDI and IIQ-7 (short form). Response rate (48%): 1,702 completed baseline interview. Asked to participate: n=3,538 Completed sexual penetration of the vagina, mouth or rectum without a women’s consent, involving the use of force or threat of harm No No Overall, 375 partici- pants (22%) reported overactive bladder, 27% reported prior sexual assault. Female veterans with depression, anxiety or PTSD had two to three times greater odds of OAB symptoms than those without these mental health problems. Increased urinary symptom and functional impact were associ- ated with mental health symptoms and SA Post-traumatic stress disorder (19%), anxi- ety (21%), depression (10%) d Pun [40] Prospective, cross- sectional question- naire-based study. Chinese women 18–70 years (mean age=56 years) presenting with urinary symptoms to the Urogynecology Clinic and General Gynecology Clinic, Hong Kong SA: Modified Abuse Assessment Screen. LUT: ICS defini- tions and physi- cal examination. Response rate was 96.2%. Not fully provided “forced sexual activity” Domestic violence, verbal abuse, physi- cal abuse No 1.3% reported SA. Abuse survivors (n=17): 41.2% OAB symptoms, 53% mixed UI, 5.9% SUI 5.9%. Discussion No abuse cohort (n=208): 13.9% OAB symp- toms, 75% mixed UI, 5.9% SUI 11.1% Not reported ( ) Study Setting and cohort Assessment and com- pletion rate Definition of SA Other forms of abuse assessed? LUT diagnostic tests Findings Other comorbidities reported Bradley et al. [24] A cross-sectional analysis of a nation- wide cohort study. A longitudinal study of 1,702 female veterans (mean age = 31.1years) SA: questions modified from past national surveys as well as studies on violence in women and female veterans. LUT: computer- assisted telephone interview: the UDI and IIQ-7 (short form). Response rate (48%): 1,702 completed baseline interview. Asked to participate: n=3,538 Completed sexual penetration of the vagina, mouth or rectum without a women’s consent, involving the use of force or threat of harm No No Overall, 375 partici- pants (22%) reported overactive bladder, 27% reported prior sexual assault. Female veterans with depression, anxiety or PTSD had two to three times greater odds of OAB symptoms than those without these mental health problems. Increased urinary symptom and functional impact were associ- ated with mental health symptoms and SA Post-traumatic stress disorder (19%), anxi- ety (21%), depression (10%) Ma and Pun [40] Prospective, cross- sectional question- naire-based study. Chinese women 18–70 years (mean age=56 years) presenting with urinary symptoms to the Urogynecology Clinic and General Gynecology Clinic, Hong Kong SA: Modified Abuse Assessment Screen. LUT: ICS defini- tions and physi- cal examination. Response rate was 96.2%. Not fully provided “forced sexual activity” Domestic violence, verbal abuse, physi- cal abuse No 1.3% reported SA. Abuse survivors (n=17): 41.2% OAB symptoms, 53% mixed UI, 5.9% SUI 5.9%. No abuse cohort (n=208): 13.9% OAB symp- toms, 75% mixed UI, 5.9% SUI 11.1% Not reported Ma and Pun [40] 1 3 International Urogynecology Journal (2023) 34:635–653 642 Table 2 (continued) Study Setting and cohort Assessment and com- pletion rate Definition of SA Other forms of abuse assessed? LUT diagnostic tests Findings Other comorbidities reported Cichowski et al. [28] Retrospective chart review and ques- tionnaire study of 1,899 prospectively recruited female patients (mean age = 54.7) presenting to a urogynaecology clinic SA: yes/no question enquiring about a history of SA. LUT: interviews using a standardised, phy- sician-administered intake questionnaire. Standardised pelvic examination by the attending physician including the POPQ. Discussion Response rate: 66% were asked about a history of SA Any unwanted sexual activity at any point in time No No SA prevalence 17% (n=213). Comparing those with and those without a history of SA, there was no significant difference in prevalence of SUI (57.2% vs 62.4%) and OAB symptoms (50.8% vs 55.9%) Depression, anxiety, current use of antide- pressants/anxiolytics and tobacco use Komesu et al. [23] Case–control study, women recruited from general gynae- cology and primary care clinics. Mean age of 57 years. 322 participants enrolled SA: three questions on the BRFSS-ACE Module. LUT: OAB case inclu- sion: presence of urinary urgency, usually accompa- nied by frequency and nocturia, with or without urgency incontinence and without other obvi- ous causes. OAB group received the UDI-6 and OABq-SF. Response rate: 322 partici- pants enrolled/427 screened (75%) Received unwanted sexual touch- ing, forced into unwanted sexual touching; forced to have sex during childhood The BRFSS-ACE Module incudes abuse (sexual/ emotional/physical) and household chal- lenges domains POPQ examinations ruling out prolapse; urine dipsticks examination ruling out acute urinary tract infections LUT findings, odds of high ACE occur- rence increased two-fold in OAB. 34% (n=31) of OAB cases versus 22% (n=20) of controls reported sexual ACEs Compared with controls OAB cases had higher median ACEs (3 vs 1). OAB cases: significantly more fibromyalgia, history of substance abuse, comorbidities (Charlson Comorbid- ity Index), Depression and Anxiety, OABq symptom score and UDI-6 than controls ACE adverse childhood experience, BRFSS-ACE Behavioural Risk Factor Surveillance System Adverse Childhood Experience Module, CRADI-8 ColoRectal-Anal Distress Inventory, FSFI Female Sexual Function Index, HITS Hurt–Insult–Threaten–Scream, ICIQ-OAB International Consultation on Incontinence Questionnaire Overactive Bladder Module, ICIQ-UI International Consultation on Incontinence Questionnaire Urinary Incontinence Short Form, ICS International Continence Society, IIQ-7 Incontinence Impact Questionnaire-7, IPV intimate partner violence, N/A not available, OAB overactive bladder, OABq Overactive Bladder symptom and health-related quality of life questionnaire, OABq-SF Overactive Bladder Questionnaire-Short Form, POPQ pelvic organ prolapse quantification, PTSD post-traumatic stress disorder, SA sexual abuse, UDI-6 Urogenital Distress Inventory-6, USS Indevus Urgency Severity Scale Komesu et al. [23] 643 International Urogynecology Journal (2023) 34:635–653 Table 3 Large cross-sectional studies evaluating different health issues including sexual abuse and lower urinary tract symptoms (LUTS; n = 8) Study Setting and cohort Assessment and com- pletion rate Definition of SA Other forms of abuse assessed? LUT diagnostic tests Findings Other comorbidities reported Mark et al. Discussion [38] Cross-sectional survey of 730 women (mean age=35.7) presenting to 6 gynaecological and 7 general clinics in Germany SA: questions on physi- cal and sexual abuse in different periods of life adapted from a previous survey. LUT: non-validated Ques- tionnaire (enquiring about gynaecological symptoms including dysmenorrhoea, vagi- nal infections, adnexi- tis, urinary tract infections, menstrual cycle changes and pelvic pain. Response rate 37.6% Not fully provided Physical abuse No Lifetime prevalence of SA and IPV 52.5% and 28.3% respec- tively. Urinary tract infections (43.7% sel- dom, 20.4% frequent/ chronic). Urinary tract infections were significantly associ- ated with (frequent/ chronic) major physical violence (57.5%), (frequent/ chronic) major sexual violence (45.6%) and (frequent/chronic) intimate partner violence (40.5%) Dysmenorrhoea, (changes in the men- strual cycle, pelvic pain independent of menstrual bleeding, vaginal infections and adnexitis rent health issues including sexual abuse and lower urinary tract symptoms (LUTS; n = 8) Mark et al. [38] 1 3 International Urogynecology Journal (2023) 34:635–653 644 Setting and cohort Assessment and com- pletion rate Definition of SA Other forms of abuse assessed? LUT diagnostic tests Findings Other comorbidities reported [30] Analyses were based on data from the Boston Area Community Health survey, a community-based epidemiological study of many different uro- logical symptoms and risk factors compris- ing a cross-sectional random sample of community-dwelling adults. 5,506 adults, (aged 30–79 years, of which 2,301 men) SA and LUTS assessed using self-adminis- tered (non-validated) questionnaire. Includ- ing questions about urinary frequency, urgency and nocturia. Composite measures were created which were “different from the current Interna- tional Continence Society definitions”. Response rate not reported Sexual abuse defined as any of the following (unwanted) experi- ences (and the perpe- trator was an adult): exposed sex organs of their body to victim (only included the definition of child- hood sexual abuse), threatened to have sex, touched respond- ent’s sex organs, made respondent touch their sex organs, forced respondent to have sex, or other sexual experiences Physical and emotional abuse No Prevalence of CSA 21.6% (number not reported) and sexual abuse experienced in adolescence/adult- hood 19.5 % (number not reported). Urinary frequency, urgency and nocturia positively linked to sexual, physical and emotional abuse. Prevalence of child- hood sexual abuse significantly associ- ated with urinary frequency (42.6%, odds ratio 1.74), with urgency (18.1%, odds ratio 1.95) and nocturia (32.8%, odds ratio 1.31). Discussion LUT: Overactive Blad- der Questionnaire (OABq-SF), PFDI-20, UDI-6, CRADI-8. Of 380, 338 (89%) answered questions about bullying or abuse Not provided No No Prevalence of SA: n=90 women (24%). Prevalence of bul- lying: n=94 women (24.7%). Women with a history of both SA and bullying had increased overall PFDI-20, POPDI, and UDI-6 scores. CRADI scores increased in patients with a history of SA. OABq-6 and OABq-13 were not significantly different between the groups History of abuse or bullying or both more likely to suffer from depression, anxiety, IBS, migraines, fibro- myalgia and constipa- tion, increased cigarette use. History of abuse and bullying group: increased overall pain and vulvar pain Bradley et al. [32] 1-year prospective cohort study of female veterans. Eligible women identified through the Defense Man- power Data Center and recruited by mail and telephone. Participants: n=1,107. Median age 29 (range 20–67) SA: lifetime history of sexual assault assessed using ques- tions modified from past national surveys and studies (e.g. National Violence Against Women Prevention Research). LUT: items from UDI-6. Response rate: of 1,702 who completed baseline interview, 1,107 completed the Year 1 interview (65%) Sexual penetration of the vagina, mouth, or rectum without con- sent, involving force or threat of harm No No At baseline: OAB was identified in 242 (22%), and 287 (25.9%) reported life- time sexual assault. At 1-year follow- up: 8dence 10.5% and remission rate 36.9%. New onset OAB occurred more often in women with lifetime sexual assault (16% vs 9%, baseline anxiety [21% vs 9%], post-traumatic stress disorder [19% vs 9%]) At baseline: depres- sion (9.2%), anxiety (19.7%), PTSD (17%) International Urogynecology Journal (2023) 34:635–653 tudy Setting and cohort Assessment and com- pletion rate Definition of SA Other forms of abuse assessed? LUT diagnostic tests Findings Other comorbidities reported ault et al. [31] Retrospective chart review of consecutive new female patients in their 40s and 50s presenting to a women’s urology cen- tre; (n=380; mean age 50 years) divided into four groups according to history: (1) abuse and bullying; (2) bul- lying but not abuse; (3) abuse but not bul- lying; (4) neither SA: unvalidated questionnaires about: sexual health. LUT: Overactive Blad- der Questionnaire (OABq-SF), PFDI-20, UDI-6, CRADI-8. Of 380, 338 (89%) answered questions about bullying or abuse Not provided No No Prevalence of SA: n=90 women (24%). Prevalence of bul- lying: n=94 women (24.7%). Discussion Preva- lence of adolescent/ adult sexual abuse significantly associ- ated with urinary frequency (40.8%, odds ratio 1.56), with urgency (19.0%, odds ratio 2.09) and noc- turia (33.0% , odds ratio 1.31) Not reported ( ) dy Setting and cohort Assessment and com- pletion rate Definition of SA Other forms of abuse assessed? LUT diagnostic tests Findings Other comorbidities reported k et al. [30] Analyses were based on data from the Boston Area Community Health survey, a community-based epidemiological study of many different uro- logical symptoms and risk factors compris- ing a cross-sectional random sample of community-dwelling adults. 5,506 adults, (aged 30–79 years, of which 2,301 men) SA and LUTS assessed using self-adminis- tered (non-validated) questionnaire. Includ- ing questions about urinary frequency, urgency and nocturia. Composite measures were created which were “different from the current Interna- tional Continence Society definitions”. Response rate not reported Sexual abuse defined as any of the following (unwanted) experi- ences (and the perpe- trator was an adult): exposed sex organs of their body to victim (only included the definition of child- hood sexual abuse), threatened to have sex, touched respond- ent’s sex organs, made respondent touch their sex organs, forced respondent to have sex, or other sexual experiences Physical and emotional abuse No Prevalence of CSA 21.6% (number not reported) and sexual abuse experienced in adolescence/adult- hood 19.5 % (number not reported). Urinary frequency, urgency and nocturia positively linked to sexual, physical and emotional abuse. Prevalence of child- hood sexual abuse significantly associ- ated with urinary frequency (42.6%, odds ratio 1.74), with urgency (18.1%, odds ratio 1.95) and nocturia (32.8%, odds ratio 1.31). Preva- lence of adolescent/ adult sexual abuse significantly associ- ated with urinary frequency (40.8%, odds ratio 1.56), with urgency (19.0%, odds ratio 2.09) and noc- turia (33.0% , odds ratio 1.31) Not reported Link et al. [30] 1 3 645 ternational Urogynecology Journal (2023) 34:635–653 Study Setting and cohort Assessment and com- pletion rate Definition of SA Other forms of abuse assessed? LUT diagnostic tests Findings Other comorbidities reported Nault et al. [31] Retrospective chart review of consecutive new female patients in their 40s and 50s presenting to a women’s urology cen- tre; (n=380; mean age 50 years) divided into four groups according to history: (1) abuse and bullying; (2) bul- lying but not abuse; (3) abuse but not bul- lying; (4) neither SA: unvalidated questionnaires about: sexual health. Discussion Women with a history of both SA and bullying had increased overall PFDI-20, POPDI, and UDI-6 scores. CRADI scores increased in patients with a history of SA. OABq-6 and OABq-13 were not significantly different between the groups History of abuse or bullying or both more likely to suffer from depression, anxiety, IBS, migraines, fibro- myalgia and constipa- tion, increased cigarette use. History of abuse and bullying group: increased overall pain and vulvar pain radley et al. [32] 1-year prospective cohort study of female veterans. Eligible women identified through the Defense Man- power Data Center and recruited by mail and telephone. Participants: n=1,107. Median age 29 (range 20–67) SA: lifetime history of sexual assault assessed using ques- tions modified from past national surveys and studies (e.g. National Violence Against Women Prevention Research). LUT: items from UDI-6. Response rate: of 1,702 who completed baseline interview, 1,107 completed the Year 1 interview (65%) Sexual penetration of the vagina, mouth, or rectum without con- sent, involving force or threat of harm No No At baseline: OAB was identified in 242 (22%), and 287 (25.9%) reported life- time sexual assault. At 1-year follow- up: 8dence 10.5% and remission rate 36.9%. New onset OAB occurred more often in women with lifetime sexual assault (16% vs 9%, baseline anxiety [21% vs 9%], post-traumatic stress disorder [19% vs 9%]) At baseline: depres- sion (9.2%), anxiety (19.7%), PTSD (17%) No No Table 3 (continued) Study Setting and cohort Assessment and com- pletion rate Definition of SA Other f assesse Nault et al. [31] Retrospective chart review of consecutive new female patients in their 40s and 50s presenting to a women’s urology cen- tre; (n=380; mean age 50 years) divided into four groups according to history: (1) abuse and bullying; (2) bul- lying but not abuse; (3) abuse but not bul- lying; (4) neither SA: unvalidated questionnaires about: sexual health. LUT: Overactive Blad- der Questionnaire (OABq-SF), PFDI-20, UDI-6, CRADI-8. Of 380, 338 (89%) answered questions about bullying or abuse Not provided No Bradley et al. [32] 1-year prospective cohort study of female veterans. Eligible women identified through the Defense Man- power Data Center and recruited by mail and telephone. Participants: n=1,107. Median age 29 (range 20–67) SA: lifetime history of sexual assault assessed using ques- tions modified from past national surveys and studies (e.g. National Violence Against Women Prevention Research). LUT: items from UDI-6. Discussion Response rate: of 1,702 who completed baseline interview, 1,107 completed the Year 1 interview (65%) Sexual penetration of the vagina, mouth, or rectum without con- sent, involving force or threat of harm No No Nault et al. [31] 1 3 International Urogynecology Journal (2023) 34:635–653 646 (continued) Setting and cohort Assessment and com- pletion rate Definition of SA Other forms of abuse assessed? LUT diagnostic tests Findings Other comorbidities reported et al. [33] Cross-sectional data from National Social Life, Health, and Aging Project, a national area proba- bility sample of older community-dwelling adults (n=1,551 older women; mean age = 69) SA: assessed using the question: “anyone ever made you have sex by using force or threatening to harm to you or someone close to you?” LUT: struc- tured-item questions previously used in epidemiological stud- ies of older women. Response rate: 75.5% overall-weighted response rate Any lifetime sexual assault Past-year physical abuse; past-year emotional abuse N/A 9% (n=99) reported sexual assault. Urinary incontinence and other urinary problems reported by 42% and 17% respectively; 42% of sexually active women reported vagi- nal symptoms with intercourse. Lifetime history of sexual assault not associ- ated with urinary symptoms 42% of sexually active women reported vaginal symptoms with intercourse. 23% reported emotional abuse and 1% reported physical abuse t al. 34] Cross-sectional, multi-ethnic cohort study (n=1,999 women; aged 40–80 years; mean age 60.2) enrolled in an integrated health care system SA:. assessed using the question: “has anyone ever touched sexual parts of your body after you said or showed that you did not want them to, or without your consent?” LUT: structured, inter- viewer-administered questionnaire items previously validated against a detailed bladder diary. Response rate: 71.7% consented to partici- pate Not fully provided Lifetime exposure to physical abuse by an intimate partner, emotional abuse by an intimate partner N/A 19.7% (n=382) reported sexual assault. 45% reported weekly urinary incon- tinence of any type; 34.5% reported fre- quent nocturia. 23% stress-type inconti- nence, 23% urgency- type incontinence, and 35% nocturia. Sexual assault was associated with an increased odds of any bothersome incon- tinence but not any nocturia outcomes Emotional IPV associ- ated with increased odds for all urinary symptoms; physical IPV as not associated with any incontinence outcomes, however associated with an increased odds of fre- quent nocturia. Discussion Women with a history of PTSD and depression had increased odds of reporting all urinary symptoms assessed 1 Table 3 (continued) Study Setting and cohort Assessment and com- pletion rate Definition of SA Other forms of abuse assessed? LUT diagnostic tests Findings Other comorbidities reported Gibson et al. [33] Cross-sectional data from National Social Life, Health, and Aging Project, a national area proba- bility sample of older community-dwelling adults (n=1,551 older women; mean age = 69) SA: assessed using the question: “anyone ever made you have sex by using force or threatening to harm to you or someone close to you?” LUT: struc- tured-item questions previously used in epidemiological stud- ies of older women. Response rate: 75.5% overall-weighted response rate Any lifetime sexual assault Past-year physical abuse; past-year emotional abuse N/A 9% (n=99) reported sexual assault. Urinary incontinence and other urinary problems reported by 42% and 17% respectively; 42% of sexually active women reported vagi- nal symptoms with intercourse. Lifetime history of sexual assault not associ- ated with urinary symptoms 42% of sexually active women reported vaginal symptoms with intercourse. 23% reported emotional abuse and 1% reported physical abuse Boyd et al. 34] Cross-sectional, multi-ethnic cohort study (n=1,999 women; aged 40–80 years; mean age 60.2) enrolled in an integrated health care system SA:. assessed using the question: “has anyone ever touched sexual parts of your body after you said or showed that you did not want them to, or without your consent?” LUT: structured, inter- viewer-administered questionnaire items previously validated against a detailed bladder diary. Response rate: 71.7% consented to partici- pate Not fully provided Lifetime exposure to physical abuse by an intimate partner, emotional abuse by an intimate partner N/A 19.7% (n=382) reported sexual assault. 45% reported weekly urinary incon- tinence of any type; 34.5% reported fre- quent nocturia. 23% stress-type inconti- nence, 23% urgency- type incontinence, and 35% nocturia. Sexual assault was associated with an increased odds of any bothersome incon- tinence but not any nocturia outcomes Emotional IPV associ- ated with increased odds for all urinary symptoms; physical IPV as not associated with any incontinence outcomes, however associated with an increased odds of fre- quent nocturia. Discussion Women with a history of PTSD and depression had increased odds of reporting all urinary symptoms assessed 647 International Urogynecology Journal (2023) 34:635–653 ( ) udy Setting and cohort Assessment and com- pletion rate Definition of SA Other forms of abuse assessed? LUT diagnostic tests Findings Other comorbidities reported alchandani et al. [35] Cross-sectional analysis of nationally repre- sentative observa- tional data from the National Social Life, Health and Aging Project (n= 1,745 women; mean age 71 years) SA: childhood sexual abuse. One question: “Before you were 12 or 13 years old, did anyone touch you sexually?” LUT: structured questions adapted from other epidemiological stud- ies of older women. Response rate: 79% Childhood sexual abuse (being touched sexu- ally before the age of 12 or 13) No N/A 11.4% (n=183) reported child- hood SA and 39.2% (n=684) reported an unwanted first sexual experience. After adjusting for age, race/ethnic- ity and education, women with a history of childhood abuse had increased odds of reporting other urinary problems, i.e. voiding difficul- ties (16.1% vs 27%) but not UI (40.4% vs 45.2%) Difficulty with ADLs and IADLs and cur- rent emotional abuse by family or friends and by their partner. Women with a his- tory of unwanted first sexual experience had increased odds of reporting difficulty with mobility Lalchandani et al. [35] 1 3 International Urogynecology Journal (2023) 34:635–653 648 8 1 3 Study Setting and cohort Assessment and com- pletion rate Definition of SA Other forms of abuse assessed? LUT diagnostic tests Findings Other comorbidities reported Geynisman-Tan et al. [36] Secondary analysis of baseline data obtained from the Symptoms of Lower Urinary Tract Research Net- work Observational Cohort Study (US). n=1,064 (of which 519 men; median age 58.8) SA: CTES includes sex- ual and other trauma occurring before age 17. LUT: LUTS tool and PFDI-20 (short form). Completion rate: 95% Traumatic sexual experience, e.g. rape or molestation Childhood traumatic events N/A 25% (n=127) of women and 8% of men (n=38) reported traumatic sexual experience in child- hood. Childhood sexual trauma was significantly associ- ated with greater incontinence severity (adjusted mean differ- ence 4.5 points, 95% confidence interval 1.11–7.88, p=0.009). Depression, anxiety and perceived stress, geni- tourinary pain, bowel symptoms, physical functioning, mobility and sleep disturbance. Discussion Approximately half of the effect of Childhood Traumatic Experi- ences impact score on overall LUTS severity was direct, whereas the other half mediated through the association 1 ( ) Study Setting and cohort Assessment and com- pletion rate Definition of SA Other forms of abuse assessed? LUT diagnostic tests Findings Other comorbidities reported Geynisman-Tan et al. [36] Secondary analysis of baseline data obtained from the Symptoms of Lower Urinary Tract Research Net- work Observational Cohort Study (US). n=1,064 (of which 519 men; median age 58.8) SA: CTES includes sex- ual and other trauma occurring before age 17. LUT: LUTS tool and PFDI-20 (short form). Completion rate: 95% Traumatic sexual experience, e.g. rape or molestation Childhood traumatic events N/A 25% (n=127) of women and 8% of men (n=38) reported traumatic sexual experience in child- hood. Childhood sexual trauma was significantly associ- ated with greater incontinence severity (adjusted mean differ- ence 4.5 points, 95% confidence interval 1.11–7.88, p=0.009). 69% reported at least one childhood traumatic event on the CTES, and 60% of those reported two or more traumas. In women, the number of traumas was asso- ciated with worsen- ing PFDI scores, with each additional trauma endorsed increasing the average PFDI score by 4.2 Depression, anxiety and perceived stress, geni- tourinary pain, bowel symptoms, physical functioning, mobility and sleep disturbance. Approximately half of the effect of Childhood Traumatic Experi- ences impact score on overall LUTS severity was direct, whereas the other half mediated through the association between trauma and patient’s mental health, i.e. anxiety, depression and perceived stress ADLs activities of daily living, BRFSS-ACE Behavioural Risk Factor Surveillance System Adverse Childhood Experience Module, CRADI-8 ColoRectal-Anal Distress Inventory, CTES Child- hood Traumatic Events Scale, FSFI Female Sexual Function Index, HITS Hurt–Insult–Threaten–Scream, IADLs independent activities of daily living, ICIQ-OAB International Consultation on Incontinence Questionnaire Overactive Bladder Module, ICIQ-UI International Consultation on Incontinence Questionnaire Urinary Incontinence Short Form, IIQ-7 Incontinence Impact Questionnaire-7, IPV intimate partner violence, LUT lower urinary tract, OABq Overactive Bladder symptom and health-related quality of life questionnaire, OABq-SF Overactive Bladder Ques- tionnaire-Short Form, PFDI-20 Pelvic Floor Distress Inventory-20, PTSD post-traumatic stress disorder, SA sexual abuse, UDI-6 Urogenital Distress Inventory-6, USS Indevus Urgency Severity Scale ( ) Study Setting and cohort Assessment and com- pletion rate Definition of SA Other forms of abuse assessed? LUT diagnostic tests Findings Other comorbidities reported Geynisman-Tan et al. Discussion Fur- thermore, only four studies used a validated scale to assess SA [23, 29, 36, 40], which limited the extent to which the nature, length and severity of abuse could be assessed. The wide prevalence range of SA reported in the studies, from 1.3% [40] to 49.6% [28] may not accurately reflect the true prevalence of SA in patients reporting with LUTS; however, it is somewhat in keeping with the prevalence reported in other cohorts without LUTS [41, 42]. factors of shame and stigma were possible factors respon- sible for underreporting [40]. Other reasons could include recall bias, disquiet in a public hospital setting, wording of questions about SA and concerns regarding confidentiality. i Lower urinary tract symptoms were variably assessed and urinary storage problems such as urinary incontinence, frequency and nocturia were reported most often. Some patients were reporting incontinence in the context of hold- ing the urine too long until it became painful [25]. Urody- namics testing was not performed in any of the studies. The cause of urinary incontinence was unclear and inclusion of validated questionnaires and possibly urodynamics in future studies would help to understand whether incontinence was due to overactive bladder, stress incontinence or mixed. Establishing dysfunction such as bladder hypersensitivity and/or detrusor overactivity would be critical when tailoring therapeutic strategies for managing these symptoms [44]. Voiding difficulties were less often reported and symptoms reported were pain with urination, hesitancy, slow stream, dribbling, holding urine until painful, incomplete bladder emptying, weak urinary stream and straining to begin urina- tion [25, 33]. Questionnaires such as the UDI-6 do specifi- cally enquire about voiding difficulties; however, only the total score was reported in studies. Urinary retention was not reported and post-void residual volumes were not measured in any of the studies; therefore, the extent of incomplete bladder emptying could not be assessed. Although trauma features in the history of patients presenting with idiopathic urinary retention in men and women [45–47], none of the studies in this review specifically explored urinary retention related to sexual trauma.f Because of the sensitive nature of SA, there were lim- its to the extent to which patients could be approached by health care professionals about possible SA. Discussion [36] Secondary analysis of baseline data obtained from the Symptoms of Lower Urinary Tract Research Net- work Observational Cohort Study (US). n=1,064 (of which 519 men; median age 58.8) SA: CTES includes sex- ual and other trauma occurring before age 17. LUT: LUTS tool and PFDI-20 (short form). Completion rate: 95% Traumatic sexual experience, e.g. rape or molestation Childhood traumatic events N/A 25% (n=127) of women and 8% of men (n=38) reported traumatic sexual experience in child- hood. Childhood sexual trauma was significantly associ- ated with greater incontinence severity (adjusted mean differ- ence 4.5 points, 95% confidence interval 1.11–7.88, p=0.009). 69% reported at least one childhood traumatic event on the CTES, and 60% of those reported two or more traumas. In women, the number of traumas was asso- ciated with worsen- ing PFDI scores, with each additional trauma endorsed increasing the average PFDI score by 4.2 Depression, anxiety and perceived stress, geni- tourinary pain, bowel symptoms, physical functioning, mobility and sleep disturbance. Approximately half of the effect of Childhood Traumatic Experi- ences impact score on overall LUTS severity was direct, whereas the other half mediated through the association between trauma and patient’s mental health, i.e. anxiety, depression and perceived stress ADLs activities of daily living, BRFSS-ACE Behavioural Risk Factor Surveillance System Adverse Childhood Experience Module, CRADI-8 ColoRectal-Anal Distress Inventory, CTES Child- hood Traumatic Events Scale, FSFI Female Sexual Function Index, HITS Hurt–Insult–Threaten–Scream, IADLs independent activities of daily living, ICIQ-OAB International Consultation on Incontinence Questionnaire Overactive Bladder Module, ICIQ-UI International Consultation on Incontinence Questionnaire Urinary Incontinence Short Form, IIQ-7 Incontinence Impact Questionnaire-7, IPV intimate partner violence, LUT lower urinary tract, OABq Overactive Bladder symptom and health-related quality of life questionnaire, OABq-SF Overactive Bladder Ques- ionnaire-Short Form, PFDI-20 Pelvic Floor Distress Inventory-20, PTSD post-traumatic stress disorder, SA sexual abuse, UDI-6 Urogenital Distress Inventory-6, USS Indevus Urgency Severity Scale 1 International Urogynecology Journal (2023) 34:635–653 649 studied and heterogeneity in definitions and study designs used. Most studies defined SA broadly as forced or unwanted sexual activity, ranging from the broadest, “unwanted sexual touching” [23] to the narrowest, “complete sexual penetra- tion of the vagina, mouth or rectum without a women’s con- sent, involving the use of force or threat of harm” [24]. Fig. 2 Assessment of quality of included studies using the Effective Public Health Practice Project tool Discussion Some clinical teams, acknowledging the challenges, are highlighting the need for a multi-disciplinary approach [67]. Notably, duloxetine, a serotonin and norepinephrine reuptake inhibitor (SNRI), that is well established in the treatment of depression and anxiety, has been used with success in the management of both OAB and stress urinary incontinence (SUI) [68, 69]. Whether other types of abuse contribute to the occurrence of LUT dysfunction is unclear, as an association between emotional abuse and voiding difficulties [35] and urinary incontinence [33] have been reported. Limitations to study designs precluded any meaningful exploration of the association of these different types of abuse with the occurrence of LUTS. The association between trauma and functional somatic syndromes is well established [48, 49] and the stressor response occurring following trauma has been shown to result in physiological changes in body and brain functions that can persist through life and predispose indi- viduals to a range of physical and psychological sequelae.i There were some limitations to this review. Few stud- ies were relevant to the topic, and the overall quality was “moderate”. In the absence of an operational definition for SA, the cohorts differed between studies. Furthermore, a standardised assessment was lacking and therefore the extent of details about types of abuse and their frequency, relationship to the perpetrator, time-frame of abuse, age and impact on childhood development were often missing. A challenge for any research in this area is recall bias, and the wording used in the enquiry about SA and also the setting differed between studies. The extent of rapport and trust between health care professionals and the participants was not assessed; however, these would be critical when exploring such a sensitive topic. Bias in sampling resulting from poor response rates amongst participants approached was not addressed in any of the studies. The assessment of LUTS also differed considerably between studies and therefore the true extent and pattern of LUT dysfunction could not be assessed. Nonetheless, it can be concluded that there exists an association between SA and urinary storage and voiding symptoms. g p y p y g q The age at which SA occurs is also significant; SA occur- ring during critical developmental periods has been shown to result in profound endocrinological and immunological consequences that may have long-term effects on an indi- vidual’s ability to react and respond to illness [50]. Discussion Only 66% of women with pelvic floor disorders were asked about SA [21], whereas in a study exploring physical and SA in patients with an overactive bladder, only women who were not accompanied by a male were approached because of concerns regarding safety [37]. Clinicians would have been reluctant to enquire about SA owing to assumptions that patients may react negatively when questioned [43], lack of familiarity with how to enquire and/or uncertainty about how to proceed if a patient were to disclose SA [20]. In a survey of survivors, more than 70% of abused respondents favourably considered the idea of screening for SA in uro- logical practice [15]. However, patients may not be readily prepared to engage, and over 20% of participants in a study exploring interpersonal trauma and genitourinary dysfunc- tion did not disclose information about sexual assault, more commonly African American and non-partnered women [33]. In a study of Chinese women, which reported the high- est response rate of 96%, only 1.3% reported SA and cultural Sexual trauma may be one of different types of abuses suf- fered by individuals, and in these studies emotional and physi- cal abuse [23, 26], violence [29], physical abuse [27, 37], and domestic violence, verbal and physical abuse [40] were reported. 1 3 Fig. 2 Assessment of quality of included studies using the Effective Public Health Practice Project tool 1 1 9 13 1 5 12 6 2 3 16 10 5 3 10 2 1 3 17 6 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Overall Withdrawals & dropouts Data Collecon Methods Blinding Confounders Study Design Selecon Bias Strong Moderate Weak N/A 1 3 1 1 9 13 1 5 12 6 2 3 16 10 5 3 10 2 1 3 17 6 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Overall Withdrawals & dropouts Data Collecon Methods Blinding Confounders Study Design Selecon Bias Strong Moderate Weak N/A 1 1 9 13 1 5 12 6 2 3 16 10 5 3 10 2 1 3 17 6 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Overall Withdrawals & dropouts Data Collecon Methods Blinding Confounders Study Design Selecon Bias Strong Moderate Weak N/A 1 3 650 International Urogynecology Journal (2023) 34:635–653 linked to SA; however, this needs to be further explored. Discussion Somatic problems such as musculoskeletal pain, ear, nose, and throat symptoms, abdominal pain and gastrointestinal symptoms, fatigue, and dizziness have been found to be more common in adults with a history of childhood trauma than in non- traumatised counterparts [10]. These subjective, medically unexplained physical health problems often persist and pre- sent as functional somatic syndromes such as fibromyalgia, chronic fatigue/pain, and irritable bowel syndrome [51]. A recent study found that complex PTSD symptoms mediate the association between childhood maltreatment and trauma and physical health problems. Complex PTSD is associ- ated with a number of psychological sequelae, including hypervigilance, anxiety, agitation, dissociation [52], anger, aggression, self-harm [53], dysregulation in emotion pro- cessing, self-organisation (including bodily integrity), rela- tional functioning [54], and psychological interventions that effectively treat symptoms may additionally reduce the risk of physical health problems [55]. Urological symptoms such as OAB are associated with a number of psychiatric condi- tions such as depression, anxiety and CSA [56]. One major limitation of the review is the low quality and low level of evidence of these 18 studies. Also, the EPHPP does not explore characteristics from each study design that other quality tools can do, such as the Newcastle–Ottawa Scale [70]. There is a need for further research to explain the relation between SA and LUTS. Further, as the stud- ies included in this review were too heterogeneous, a meta- analysis was not performed. It is likely, however, that there are different mechanisms responsible for LUTS in survivors of SA. Physical trauma to the perineum and pelvis [57, 58] can result in damage to the regional anatomy. Studies have shown an associa- tion between LUTS and anxiety, depression [59–62] and PTSD [63]. Neurobiological mechanisms implicate corti- cotrophin-releasing factor and serotonergic and dopamin- ergic systems in the pathogenesis of mood disorders and PTSD, and possible links with LUTS. There is a possibil- ity that adverse life events may lead to neurobiological and physiological changes that increase the risk of both mood disorders and somatic disorders, but that the risk factors may be different [64]. Discussion Somatisation may be an adaptive response to psychological distress [65] and although spe- cific symptoms linked to SA have not been consistently identified [66], it is plausible that LUTS may be associated with complex PTSD and a manifestation of somatisation Treatment options, which should take a multi-discipli- nary approach, were outside the scope of this review, but, drawing on the current published evidence of treatments for PTSD and complex PTSD, we hypothesise that a proportion of these patients may be helped by trauma-focussed cog- nitive behavioural therapy and/or other psychotherapeutic interventions. Declarations 13. Neumann DA, Houskamp BM, Pollock VE, Briere J. The long- term sequelae of childhood sexual abuse in women: a meta-ana- lytic review. Child Maltreat. 1996;1(1):6–16. https://doi.org/10. 1177/1077559596001001002. Conflicts of interest J.N.P has received honoraria from Novartis, Coloplast, Allergan, Idorsia and Wellspect and royalties for book edit- ing from Cambridge University Press. He has no disclosures related to this work. E.C.-K. has received honoraria from Coloplast, Aller- gan, Medtronic, Convatec, B Braun, Boston Scientific, Promedon, Uromems. He has no disclosures related to this work. 14. Van der Feltz-Cornelis CM, Allen SF, Eck V, van der Sluijs JF. Childhood sexual abuse predicts treatment outcome in conver- sion disorder/functional neurological disorder. An observational longitudinal study. Brain Behav. 2020;10(3):e01558. 15. Beck JJH, Bekker MD, van Driel MF, Roshani H, Putter H, Pelger RCM, et al. Prevalence of sexual abuse among patients seeking general urological care. J Sex Med. 2011;8(10):2733–8. Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, 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/. 16. Warlick CA, Mathews R, Gerson AC. Keeping childhood sexual abuse on the urologic radar screen. Urology. 2005;66(6):1143–9.f 17. Wijma B, Schei B, Swahnberg K, Hilden M, Offerdal K, Pikarinen U, et al. Emotional, physical, and sexual abuse in patients visit- ing gynaecology clinics: a Nordic cross-sectional study. Lancet. 2003;361(9375):2107–13. 18. Kaliray P, Drife J. Childhood sexual abuse and subsequent gynae- cological conditions. Obstet Gynaecol. 2004;6(4):209–14. 19. Ellsworth P, Merguerian P, Copening M. Sexual abuse. J Urol. 1995;153:773–6. 20. Beck J, Bekker M, Van Driel M, et al. Female sexual abuse evalu- ation in the urological practice: results of a Dutch survey. J Sex Med. 2010;7:1464–8. Conclusion The review highlights the need to provide a holistic assess- ment of patients presenting with LUTS that includes stand- ardised screening for SA in a “safe space” for patients to share sensitive information, and screening for concurrent 1 3 3 International Urogynecology Journal (2023) 34:635–653 651 inter-related factors such as trauma, affective symptoms and somatisation which can impact LUTS. Well-designed studies are required to explore what impact such an assessment may have on the management of LUTS. sexual assault trauma syndrome. Women Health. 1991;17(1):1– 19. https://doi.org/10.1300/J013v17n01_01. 7. Paolucci EO, Genuis ML, Violato C. A meta-analysis of the published research on the effects of child sexual abuse. J Psy- chol. 2001;135(1):17–36. https://doi.org/10.1080/0022398010 9603677. 8. Subica AM. Psychiatric and physical sequelae of childhood physi- cal and sexual abuse and forced sexual trauma among individuals with serious mental illness. J Trauma Stress. 2013;26(5):588–96.f Acknowledgements J.N. Panicker is supported in part by funding from the United Kingdom’s Department of Health NIHR Biomedical Research Centres funding scheme. 9. Leserman J. Sexual abuse history: prevalence, health effects, mediators, and psychological treatment. Psychosom Med. 2005;67(6):906–15. Author contributions C. Selai: protocol/project development, data col- lection or management, data analysis, manuscript writing/editing; M. Elmalem: protocol/project development, data collection or manage- ment, data analysis, manuscript writing/editing; E. Chartier-Kästler: protocol/project development, data collection or management, manu- script writing/editing; N. Sassoon: data collection or management, data analysis; S. Hewitt: data collection or management, data analysis; M.F. Rocha: data collection or management, data analysis; L. Klitsinari: data collection or management, data analysis; J.N. Panicker: protocol/ project development, data collection or management, data analysis, manuscript writing/editing. 10. Nelson S, Baldwin N, Taylor J. Mental health problems and medi- cally unexplained physical symptoms in adult survivors of child- hood sexual abuse: an integrative literature review. J Psychiatr Ment Health Nurs. 2012;19(3):211–20. 11. Walker JL, Carey PD, Mohr N, Stein DJ, Seedat S. Gender dif- ferences in the prevalence of childhood sexual abuse and in the development of pediatric PTSD. Arch Womens Ment Health. 2004;7(2):111–21. 12. Allanson J, Bass C, Wade DT. Characteristics of patients with persistent severe disability and medically unexplained neuro- logical symptoms: a pilot study. J Neurol Neurosurg Psychiatry. 2002;73(3):307–9. References Ho GWK, Karatzias T, Vallières F, Bondjers K, Shevlin M, Cloitre M, et al. Complex PTSD symptoms mediate the associa- tion between childhood trauma and physical health problems. J Psychosom Res. 2021;142:110358. 35. Lalchandani P, Lisha N, Gibson C, Huang AJ. Early life sexual trauma and later life genitourinary dysfunction and functional dis- ability in women. J Gen Intern Med. 2020;35(11):3210–7. 56. Dolat M, Klausner A. UROPSYCHIATRY: the relationship between overactive bladder and psychiatric disorders. Curr Bladder Dysfunct Rep. 2012;8:69–76. 36. Geynisman-Tan J, Helmuth M, Smith AR, Lai HH, Amundsen CL, Bradley CS, et al. Prevalence of childhood trauma and its associa- tion with lower urinary tract symptoms in women and men in the LURN study. Neurourol Urodyn. 2021;40(2):632–41. 57. Kim K, Nam J, Choi B, et al. The association of lower uri- nary tract symptoms with incidental falls and fear of falling in later life: The Community Health Survey. Neurourol Urodyn. 2017;37:775–84. 37. Jundt K, Scheer I, Schiessl B, Pohl K, Haertl K, Peschers UM. Physical and sexual abuse in patients with overactive bladder: is there an association? Int Urogynecol J Pelvic Floor Dysfunct. 2007;18(4):449–53. 58. Owusu Sekyere E, Hardcastle T, Sathiram R, et al. Overview of lower urinary tract symptoms post-trauma intensive care unit admission. Afr J Urol. 2020;26:1–7. 38. Mark H, Bitzker K, Klapp BF, Rauchfuss M. Gynaecological symptoms associated with physical and sexual violence. J Psy- chosom Obstet Gynaecol. 2008;29(3):164–72.l 59. Coyne KS, Wein AJ, Tubaro A, Sexton CC, Thompson CL, Kopp ZS, et al. The burden of lower urinary tract symptoms: evaluating the effect of LUTS on health-related quality of life, anxiety and depression: EpiLUTS. BJU Int. 2009;103(Suppl 3):4–11. 39. Postma R, Bicanic I, van der Vaart H, Laan E. Pelvic floor muscle problems mediate sexual problems in young adult rape victims. J Sex Med. 2013;10(8):1978–87. 60. Yang YJ, Koh JS, Ko HJ, Cho KJ, Kim JC, Lee S-J, et al. The influence of depression, anxiety and somatization on the clini- cal symptoms and treatment response in patients with symptoms of lower urinary tract symptoms suggestive of benign prostatic hyperplasia. J Korean Med Sci. 2014 Aug;29(8):1145–51. 40. Ma WSP, Pun TC. Prevalence of domestic violence in Hong Kong Chinese women presenting with urinary symptoms. PLoS One. 2016;11(7):e0159367. 41. Pereda N, Guilera G, Forns M, Gómez-Benito J. The prevalence of child sexual abuse in community and student samples: a meta- analysis. Clin Psychol Rev. 2009;29(4):328–38. 61. References 21. Hassam T, Kelso E, Chowdary P, Yisma E, Mol BW, Han A. Sexual assault as a risk factor for gynaecological morbidity: an exploratory systematic review and meta-analysis. Eur J Obstet Gynecol Reprod Biol. 2020;255:222–30. 1. Negriff S, Schneiderman JU, Smith C, Schreyer JK, Trickett PK. Characterizing the sexual abuse experiences of young adolescents. Child Abuse Negl. 2014;38(2):261–70. 22. Thomas BH, Ciliska D, Dobbins M, Micucci S. A process for systematically reviewing the literature: providing the research evi- dence for public health nursing interventions. Worldviews Evid- Based Nurs. 2004;1(3):176–84. 2. Breiding MJ. Prevalence and characteristics of sexual violence, stalking, and intimate partner violence victimization—National Intimate Partner and Sexual Violence Survey, United States, 2011. Am J Public Health. 2015;105(4):e11–e12. 23. Komesu YM, Petersen TR, Krantz TE, Ninivaggio CS, Jepp- son PC, Meriwether KV, et al. Adverse childhood experi- ences in women with overactive bladder or interstitial cystitis/ bladder pain syndrome. Female Pelvic Med Reconstr Surg. 2021;27(1):e208–14. 3. Johnson CF. Child sexual abuse. Lancet. 2004;364(9432):462–70. 4. Sable MR, Danis F, Mauzy DL, Gallagher SK. Barriers to report- ing sexual assault for women and men: perspectives of college students. J Am Coll Heal. 2006;55(3):157–62.i 24. Bradley CS, Nygaard IE, Torner JC, Hillis SL, Johnson S, Sadler AG. Overactive bladder and mental health symptoms in recently deployed female veterans. J Urol. 2014;191(5):1327–32. 5. Holmes WC. Men’s self-definitions of abusive childhood sexual experiences, and potentially related risky behavioral and psychi- atric outcomes. Child Abuse Negl. 2008;32(1):83–97.. 25. Davila GW, Bernier F, Franco J, Kopka SL. Bladder dysfunction in sexual abuse survivors. J Urol. 2003;170(2 Pt 1):476–9. g ; ( ) 6. Ruch LO, Amedeo SR, Leon JJ, Gartrell JW. Repeated sexual victimization and trauma change during the acute phase of the g 6. Ruch LO, Amedeo SR, Leon JJ, Gartrell JW. Repeated sexual victimization and trauma change during the acute phase of the 1 3 International Urogynecology Journal (2023) 34:635–653 652 45. Williams GE, Johnson AM. Recurrent urinary retention due to emotional factors; report of a case. Psychosom Med. 1956;18(1):77–80. 26. Klausner AP, Ibanez D, King AB, Willis D, Herrick B, Wolfe L, et al. The influence of psychiatric comorbidities and sexual trauma on lower urinary tract symptoms in female veterans. J Urol. 2009;182(6):2785–90. 46. Von Heyden B, Steinert R, Bothe HW, Hertle L. Sacral neuro- modulation for urinary retention caused by sexual abuse. Psy- chosom Med. 2001;63(3):505–8. 27. Lutgendorf MA, Snipes MA, O’Boyle AL. References Prevalence and pre- dictors of intimate partner violence in a military urogynecology clinic. Mil Med. 2017;182(3):e1634–8. 47. Selai C, Pakzad M, Simeoni S, Joyce E, Petrochilos P, Panicker J. Psychological co-morbidities in young women presenting with chronic urinary retention. 2019. Available from: https:// www.ics.org/2019/abstract/705 28. Cichowski SB, Dunivan GC, Komesu YM, Rogers RG. Sexual abuse history and pelvic floor disorders in women. South Med J. 2013;106(12):675–8. 29. Lai HH, Morgan CD, Vetter J, Andriole GL. Impact of childhood and recent traumatic events on the clinical presentation of overac- tive bladder. Neurourol Urodyn. 2016;35(8):1017–23. 48. Kugler BB, Bloom M, Kaercher LB, Truax TV, Storch EA. Somatic symptoms in traumatized children and adolescents. Child Psychiatry Hum Dev. 2012;43(5):661–73. 49. Bonvanie IJ, van Gils A, Janssens KAM, Rosmalen JGM. Sex- ual abuse predicts functional somatic symptoms: an adolescent population study. Child Abuse Negl. 2015;46:1–7. 30. Link CL, Lutfey KE, Steers WD, McKinlay JB. Is abuse causally related to urologic symptoms? Results from the Boston Area Com- munity Health (BACH) Survey. Eur Urol. 2007;52(2):397–406. 50. Danese A, McEwen BS. Adverse childhood experiences, allosta- sis, allostatic load, and age-related disease. Physiol Behav. 2012;106(1):29–39. 31. Nault T, Gupta P, Ehlert M, Dove-Medows E, Seltzer M, Car- rico DJ, et al. Does a history of bullying and abuse predict lower urinary tract symptoms, chronic pain, and sexual dysfunction? Int Urol Nephrol. 2016;48(11):1783–8. 51. Mayou R, Farmer A. Functional somatic symptoms and syn- dromes. BMJ. 2002;325(7358):265–8. 32. Bradley CS, Nygaard IE, Hillis SL, Torner JC, Sadler AG. Lon- gitudinal associations between mental health conditions and overactive bladder in women veterans. Am J Obstet Gynecol. 2017;217(4):430.e1–8. 52. Herman J. Complex PTSD: a syndrome in survivors of pro- longed and repeated trauma. J Trauma Stress. 1992;5:377–91. 53. Dyer KFW, Dorahy MJ, Hamilton G, Corry M, Shannon M, MacSherry A, et al. Anger, aggression, and self-harm in PTSD and complex PTSD. J Clin Psychol. 2009;65(10):1099–114. 33. Gibson CJ, Lisha NE, Walter LC, Huang AJ. Interpersonal trauma and aging-related genitourinary dysfunction in a national sample of older women. Am J Obstet Gynecol. 2019;220(1):94.e1–7. 54. Ford JD. Complex PTSD: research directions for nosology/ assessment, treatment, and public health. Eur J Psychotrauma- tol. 2015;6:27584. y 34. Boyd BAJ, Gibson CJ, Van Den Eeden SK, McCaw B, Subak LL, Thom D, et al. Interpersonal trauma as a marker of risk for urinary tract dysfunction in midlife and older women. Obstet Gynecol. 2020;135(1):106–12. 55. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. References Lung-Cheng Huang C, Ho C-H, Weng S-F, Hsu Y-W, Wang J-J, Wu M-P. The association of healthcare seeking behavior for anxiety and depression among patients with lower urinary tract symptoms: a nationwide population-based study. Psychiatry Res. 2015;226(1):247–51. 42. Stoltenborgh M, van Ijzendoorn MH, Euser EM, Bakermans- Kranenburg MJ. A global perspective on child sexual abuse: meta-analysis of prevalence around the world. Child Maltreat. 2011;16(2):79–101. 62. Vrijens D, Drossaerts J, van Koeveringe G, Van Kerrebroeck P, van Os J, Leue C. Affective symptoms and the overactive blad- der—a systematic review. J Psychosom Res. 2015;78(2):95–108. 43. Peschers UM, Mont JD, Jundt K, Pfürtner M, Dugan E, Kindermann G. Prevalence of sexual abuse among women seeking gynecologic care in Germany. Obstet Gynecol. 2003;101(1):103–8. 63. Breyer BN, Cohen BE, Bertenthal D, Rosen RC, Neylan TC, Seal KH. Lower urinary tract dysfunction in male Iraq and Afghanistan war veterans: association with mental health disorders: a popula- tion-based cohort study. Urology. 2014;83(2):312–9. 44. Peyronnet B, Mironska E, Chapple C, Cardozo L, Oelke M, Dmo- chowski R, et al. A comprehensive review of overactive blad- der pathophysiology: on the way to tailored treatment. Eur Urol. 2019;75(6):988–1000. 64. Epperson CN, Duffy KA, Johnson RL, Sammel MD, New- man DK. Enduring impact of childhood adversity on lower 1 3 International Urogynecology Journal (2023) 34:635–653 653 urinary tract symptoms in adult women. Neurourol Urodyn. 2020;39(5):1472–81. 69. Cardozo L, Lange R, Voss S, Beardsworth A, Manning M, Vik- trup L, et al. Short- and long-term efficacy and safety of dulox- etine in women with predominant stress urinary incontinence. Curr Med Res Opin. 2010;26(2):253–61. 65. Bae SM, Kang JM, Chang HY, Han W, Lee SH. PTSD corre- lates with somatization in sexually abused children: Type of abuse moderates the effect of PTSD on somatization. PLoS One. 2018;13(6):e0199138. 70. Wells G, Brodsky L, O’Connell D, Shea B, Henry D, Mayank S, Tugwell P. XI Cochrane Colloquium: evidence, health care and culture. Barcelona, Spain; 2003. Evaluation of the Newcastle- Ottawa Scale (NOS): an assessment tool for evaluating the quality of non-randomized studies. http://www.citeulike.org/user/SRCMe thodsLibrary/article/12919189. 66. Iloson C, Moller A, Sundfeldt A, Bernhardsson S. Symptoms within somatisation after sexual abuse among women: a scoping review. Acta Obstet Gynecol Scand. 2021;100:758–67. 67. Cour F, Robain G, Claudon B, Chartier-Kästler E. Childhood sexual abuse: how important is the diagnosis to understand and manage sexual, anorectal and lower urinary tract symptoms. Prog Urol. 2013;23(9):780–92. References 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. 68. Schagen van Leeuwen JH, Lange RR, Jonasson AF, Chen WJ, Viktrup L. Efficacy and safety of duloxetine in elderly women with stress urinary incontinence or stress-predominant mixed uri- nary incontinence. Maturitas. 2008;60(2):138–47. 1 3
https://openalex.org/W4295708845	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0273487&type=printable	English	null	Combining aerial photos and LiDAR data to detect canopy cover change in urban forests	PloS one	2,022	cc-by	6,423	PLOS ONE RESEARCH ARTICLE a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Kathleen CouplandID1, David Hamilton2, Verena C. GriessID1,3 1 Faculty of Forestry, Department of Forest Resources Management, University of British Columbia, Forest Sciences Centre, Vancouver, Canada, 2 College of Forestry, Oregon State University, Corvallis, OR, United States of America, 3 Department of Environmental System Sciences, Institute of Terrestrial Ecosystems, Eidgeno¨ssische Technische Hochschule Zu¨rich, Universita¨tstrasse, Zu¨rich, Switzerland kathleen.coupland@ubc.ca a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Editor: Joe McFadden, University of California Santa Barbara, UNITED STATES Editor: Joe McFadden, University of California Santa Barbara, UNITED STATES Received: January 26, 2022 Accepted: August 10, 2022 Published: September 14, 2022 Editor: Joe McFadden, University of California Santa Barbara, UNITED STATES Received: January 26, 2022 Accepted: August 10, 2022 Published: September 14, 2022 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0273487 Abstract The advancement and accessibility of high-resolution remotely sensed data has made it fea- sible to detect tree canopy cover (TCC) changes over small spatial scales. However, the short history of these high-resolution collection techniques presents challenges when assessing canopy changes over longer time scales (> 50 years). This research shows how using high-resolution LiDAR data in conjunction with historical aerial photos can overcome this limitation. We used the University of British Columbia’s Point Grey campus in Vancou- ver, Canada, as a case study, using both historical aerial photographs from 1949 and 2015 LiDAR data. TCC was summed in 0.05 ha analysis polygons for both the LiDAR and aerial photo data, allowing for TCC comparison across the two different data types. Methods were validated using 2015 aerial photos, the means (Δ 0.24) and a TOST test indicated that the methods were statistically equivalent (±5.38% TCC). This research concludes the methods outlined is suitable for small scale TCC change detection over long time frames when incon- sistent data types are available between the two time periods. Combining aerial photos and LiDAR data to detect canopy cover change in urban forests Kathleen CouplandID1*, David Hamilton2, Verena C. GriessID1,3 OPEN ACCESS Citation: Coupland K, Hamilton D, Griess VC (2022) Combining aerial photos and LiDAR data to detect canopy cover change in urban forests. PLoS ONE 17(9): e0273487. https://doi.org/10.1371/ journal.pone.0273487 Citation: Coupland K, Hamilton D, Griess VC (2022) Combining aerial photos and LiDAR data to detect canopy cover change in urban forests. PLoS ONE 17(9): e0273487. https://doi.org/10.1371/ journal.pone.0273487 PLOS ONE PLOS ONE Introduction There is a growing body of research examining urban environments, including the benefits provided by urban forests and trees [1–4]. Urban forests offer many benefits to community members including, better quality of life [2,5], health benefits [4], psychological benefits, aes- thetic benefits, and mitigation of pollutants [6]. They provide recreational space and allow peo- ple to experience nature within local settings [6]. Copyright: © 2022 Coupland 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. Despite being of great value, urban forests are constantly under threat of development as densification and urban expansion continue [1]. Continuing urbanization has raised concerns about the loss of urban tree canopy cover (TCC) on global and regional scales [7,8]. A study looking at 2002 to 2009 across the United States showed that there was an average TCC decline of 0.2% per 6 years [8]. The results of this study have been further corroborated by a 2018 study that examined TCC changes across all of the United States and showed that 23 states had statistically significant declining TCC and 22 others showed a declining trend, although the decline was not statistically significant [9]. Monitoring urban TCC is important to making Data Availability Statement: 2015 LiDAR data are available at the UBC Abacus data network (https:// hdl.handle.net/11272.1/AB2/KET75X). 1949 UBC geography air photos are available in the collection archive (https://gic.geog.ubc.ca/resources/air- PLOS ONE \| https://doi.org/10.1371/journal.pone.0273487 September 14, 2022 1 / 13 PLOS ONE Combining aerial photos and LiDAR data to detect canopy cover change photo-collection-and-services/). Photos to request: BC792:6, BC728:81, BC792:7, BC728:5, BC728:7, BC737:64, BC737:63, BC737:61, BC728:9. 2015 images were retrieved from Google Earth Pro 7.3. (May 19, 2015) Metro Vancouver Regional District, B.C, Canada. 49˚15’38.21"N, 123˚14’15.90"W (centroid coordinates). No labels, Historical Imagery. The authors confirm that they had no special privileges in accessing these datasets which other interested researchers would not have. informed management and development decisions to ensure that the benefits of urban forests are retained. Beyond identifying TCC changes, mapping these changes allows for spatially explicit visualization of urban development and its impacts on the urban forests [3], allowing for targeted decision making to ensure that development does not unduly impact the urban forest in already adversely impacted areas. Introduction This mapping can also aid in the identification of areas that are at risk of deforestation or in need of reforestation or afforestation, aiding future development and management decisions. photo-collection-and-services/). Photos to request: BC792:6, BC728:81, BC792:7, BC728:5, BC728:7, BC737:64, BC737:63, BC737:61, BC728:9. 2015 images were retrieved from Google Earth Pro 7.3. (May 19, 2015) Metro Vancouver Regional District, B.C, Canada. 49˚15’38.21"N, 123˚14’15.90"W (centroid coordinates). No labels, Historical Imagery. The authors confirm that they had no special privileges in accessing these datasets which other interested researchers would not have. TCC is the percentage of an area covered by tree canopies and is the most common mea- surement for assessing urban forests, in part because it is easily understood by members of the public and it is a simple proxy to measure the amount of urban forest [3,7]. Urban forests are defined as “forest stands and trees with amenity values situated in or near urban areas” [10]. Various approaches and data sources have been used to estimate TCC, including field sam- pling, aerial photography interpretation, satellite imagery, Light Detection and Ranging (LiDAR) from both manned and unmanned machines [11]. Each of these methods have differ- ences in costs, resolution, and time ranges. Remotely sensed data has increased in use for TCC measurements because it is often more cost effective than field surveys and sampling [12]. Change detection over time requires a minimum of two data sets from different periods of time regardless of the methodology used to determine and detect the changes [13]. Funding: KC - Award # 6372 from the University of British Columbia. Award is funding for a PhD. 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. Examination of time periods outside of new multispectral and point cloud data collection methods traditionally rely on access to historical aerial photos or manually collected field data [14–16]. The accessibility of aerial photos in many major urbanized areas and the ability to re- analyze the images using modern technology make it the predominate choice for historical TCC assessments [1,8]. Air photo interpretation has been used to determine urban TCC through using digitally orthorectified images due to its simplicity and availability of low-cost data [3]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0273487 September 14, 2022 Introduction The numerous advances in multispectral and point cloud data access, collection, and storage is occurring concurrently with increased computer processing improving canopy clas- sification speed and fidelity [17,18]. This has precipitated contemporary remotely sensed TCC assessments to trend away from single spectrum aerial photo data towards these newer data types. Landsat satellites despite providing the longest continuous series of remotely sensed global images beginning in 1972, is limited by its short legacy and is unable to examine changes over time scales that are available from other non-satellite sources. Although 50 years is a long time- scale in some industries (e.g. medicine, education, technology development) forestry often uses planning horizons spanning 100–300 years. Having data spanning the entirety of these planning horizons is often impossible and forest managers rely on data modeling to extrapo- late forest changes and growth for decision making processes. Combining data sets and types would allow for TCC to be examined over longer time frames, reducing the need for extrapola- tion. For data continuity, many TCC change assessments use 1972 or 73 as the earliest year of study as this is when Landsat started delivering imagery [19–21]. Landsat is further restricted from small scale use because of the large (30x30m) pixel size. MODIS and RapidEye are other commonly used satellite scanners that are used for TCC and face similar issues as Landsat [22– 24]. Starting in 1999, MODIS produces images with spatial resolutions ranging from 250 m to 1 km. RapidEye is a group of 5 satellites launched in 2008 and has a spectral resolution as fine as 5m. While RapidEye has finer a finer spatial scale than Landsat it is still limited by its recent initiation date. Additionally, RapidEye, much like other commercial satellites (including those with finer scale, e.g. Worldview, Planet, etc.) have financial costs associated with data access, limiting accessibility. Unlike satellite scanners (Landsat, MODIS, RapidEye), laser scanners have advanced to allow for spatial resolutions of 10 cm (or less), allowing for examinations of individual tree PLOS ONE \| https://doi.org/10.1371/journal.pone.0273487 September 14, 2022 2 / 13 PLOS ONE Combining aerial photos and LiDAR data to detect canopy cover change crowns, shapes and forms [25]. LiDAR, like all active sensors produce images without influ- ence of shadows or light variations, which are common error sources in satellite and aerial imagery [26]. Study area The University of British Columbia’s Vancouver Campus (UBC) (Canada) was selected as the study site based on data availability. A comparably large collection of historical aerial photos dating back to 1949 was available, as well as high resolution LiDAR data obtained in 2015. The study area is situated at the Western most tip of Vancouver’s Point Grey Peninsula. The histor- ically dominant tree species are Tsuga heterophylla, Thuja plicata, and Pseudotsuga menziesii. While the historical dominant species are still present in large quantities, there are also many non-native and planted species that occupy part of the urban forest. In addition to changes in the urban forest UBC has seen increases in enrollment and associated development to handle the growing population. In the last 60 to 70 years, student enrollment has grown almost tenfold from 7,960 to over 61,000. This increase in population has driven a demand for additional housing, leading to accelerated urbanization and drastic changes in TCC. The study area is constrained by the flight area of the LiDAR survey, which covered a total of 855.1 ha (Fig 1). Introduction LiDAR’s resolution abilities and lack of light interference make it optimal for the examination of tree canopies in urban areas where there are an intimate mixture of trees and human development. However, LiDAR has only recently been able to achieve such high reso- lutions and as adoption has increased the cost of collection has decreased making it more feasible. Presently, in the assessment of TCC detection there is a dichotomy occurring between research examining small-scale spatial changes over short time periods (< 50 years) [8,9], and large-scale spatial change examining timescales within 1973 –present [27–29]. However, there are limited studies that have tried to combine canopy data types to allow for longer period (> 50 years) examinations of TCC on small scales. This study aims to determine the validity of combining historical aerial photos with LiDAR data as a method for detecting small-scale changes in urban TCC over time frames outside of LiDAR history. Methods TCC for aerial photos was determined using the Tree Cover Mapping tool and methods from the USGS tree cover mapping tool manual [32]. The Tree Cover Mapping tool allows users to map canopy cover using visual interpretation of high-resolution photo imagery. Using a sys- tematic grid, the user can estimate the TCC of each sample unit. A sample unit size of 0.05 ha was selected. The sample unit size was selected to stay consistent with previous research by Coupland et al. [34]. A calibration overlay of 10x10 dots was used to ensure accuracy and con- sistency of TCC values. Dots that intersected with tree canopies were summed, with each dot representing 1% canopy cover, to determine the canopy cover for each sample unit. Shape, size, texture and association was used to reduce the inclusion of shrubs or trees less than 5 m. Aerial photo interpretation was done by an single interpreter, with input available from other researchers familiar with aerial photos if object association was doubted. A TCC percentage was assigned to each of the 17 113 analysis polygons of 0.05 ha area. LiDAR TCC was calculated using the R-package “Forest Tools” [33], and followed the forest cover classification methods from Coupland et al.’s research connecting urban TCC with for- estry learning objectives [34]. Forest tools delineated tree canopies from CHM produced from high-density point clouds by identifying local maxima and creating a window radius to look for the surrounding local minimum. The radius size is dependent upon the height of the local maximum and canopy edges are delineated using the minima surrounding each maximum. As such, individual canopies are outlined and canopy polygons are created. The same 0.05 ha grid used in the aerial photo analysis was overlaid onto the LiDAR derived canopy polygons. The canopy polygons for each 0.05 ha section of the grid was summed and converted to a TCC percent. Data collection and processing Map of UBC showing the study extent and location of 342 randomly selected polygons used in the validation process. Base map layer credit: Esri, HERE, Garmin, Intermap, increment P Corp., Gebco, USGS, FAO, NPS, NRCAN, GeoBase, ING (ArcGIS Licence 10.8.1). https://doi.org/10.1371/journal.pone.0273487.g001 Fig 1. Map of UBC showing the study extent and location of 342 randomly selected polygons used in the validation process. Base map layer credit: Esri, HERE, Garmin, Intermap, increment P Corp., Gebco, USGS, FAO, NPS, NRCAN, GeoBase, ING (ArcGIS Licence 10.8.1). https://doi.org/10.1371/journal.pone.0273487.g001 https://doi.org/10.1371/journal.pone.0273487.g001 exceeding the minimum resolution requirements for single tree canopy assessment [30]. The data was pre-processed; buildings were removed, and a canopy height model was created. Buildings were removed using ESRI’s “Classify LAS Buildings”. The canopy height model used a minimum height of 5m to remove shrubs and small plants [31] and had a 1m resolution. Validation To ensure validity of comparing the 1949 aerial photos with LiDAR data, 2015 aerial photos were compared with the 2015 LiDAR data and examined for equivalency. We randomly selected 2% (342 polygons) of the 0.05 ha polygons randomly for validation (Fig 1). TCC for the 2015 aerial photos was determined using the same methods as for the analysis of 1949 aerial photos. Descriptive statistics, including a comparison of the mean and quantile-quantile (Q-Q) plot were completed. A Shapiro-Wilk test, a Wilcoxon-Signed rank test and a two-one- sided t-tests (TOST) equivalency test was performed to determine the validity of comparing the TCC between the two data sources. All statistical tests assumed α = 0.1 and β = 0.8. Data collection and processing Aerial Photos– 1949 and 2015. Historical aerial photos of the study area were acquired from the Air Photo collection at UBC’s Geographic Information Centre. Photos were scanned man- ually at a resolution of 1200x1200 dpi and imported into ArcMap 10.6.1. Ground resolution of the photos was calculated at 11.64cm which is sufficient to examine TCC. All aerial photos were from April 1949. A spring month was selected because leaf-on seasonal photos are easier for aerial photo interpretation of TCC [3]. White borders were manually cropped off the images. Photos were georeferenced and rectified in ArcMap using ground control points and 2019 Google Earth imagery. A minimum of 10 ground control points were used, and a maxi- mum root mean square error of 2m per image. A total of 10 georeferenced images were stitched together to create one image using ArcMap that covered the entire study area. In cases where images overlapped the image closest to vertical were used. 2015 georeferenced aerial photos were exported from Google Earth’s historical imagery and imported into ArcMap. The 2015 images were resampled to have the same cell size as the 1949 images decreasing the reso- lution by about one third. They were stitched together and cropped to the study area. LiDAR. The LiDAR data for this study was collected on May 20th 2015 spanning the study area was used for this analysis (University of British Columbia. Campus and Community Planning 2015). The dataset has a point spacing of 0.143m and a point density of 49.05 pts/m2, LiDAR. The LiDAR data for this study was collected on May 20th 2015 spanning the study area was used for this analysis (University of British Columbia. Campus and Community Planning 2015). The dataset has a point spacing of 0.143m and a point density of 49.05 pts/m2, LiDAR. The LiDAR data for this study was collected on May 20th 2015 spanning the study area was used for this analysis (University of British Columbia. Campus and Community Planning 2015). The dataset has a point spacing of 0.143m and a point density of 49.05 pts/m2, 3 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0273487 September 14, 2022 PLOS ONE Combining aerial photos and LiDAR data to detect canopy cover change 4 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0273487 September 14, 2022 PLOS ONE Combining aerial photos and LiDAR data to detect canopy cover change Fig 1. Validation results The mean TCC percent for the validation polygons were 54.94 for the 2015 aerial photos and 54.70 for the 2015 LiDAR data (Fig 2). The Q-Q plots were visually examined for normality (Fig 3). The Q-Q plots showed heavy-tails indicating a non-normal distribution with values at 5 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0273487 September 14, 2022 PLOS ONE Combining aerial photos and LiDAR data to detect canopy cover change Fig 2. Comparison of the means with 99% confidence interval between 2015 canopy cover using the USGS tree cover mapping tool and methods (AerialPhotoCC) and LiDAR analysis (LidarCC). https://doi.org/10.1371/journal.pone.0273487.g002 Fig 2. Comparison of the means with 99% confidence interval between 2015 canopy cover using the USGS tree cover mapping tool and methods (AerialPhotoCC) and LiDAR analysis (LidarCC). https://doi org/10 1371/journal pone 0273487 g002 Fig 2. Comparison of the means with 99% confidence interval between 2015 canopy cover using the USGS tree cover mapping tool and methods (AerialPhotoCC) and LiDAR analysis (LidarCC). https://doi.org/10.1371/journal.pone.0273487.g002 both extremes (<10% and >90%) being more common. However, both Q-Q plots followed identical patterns indicating that the results from both methods picked up the same data trend of heavy-tails. The Shapiro-Wilk test showed that the data for both the 2015 aerial photos and the 2015 LiDAR analysis were not normally distributed (p < 0.000), corroborating the visual analysis of the Q-Q plots. The non-parametric Wilcoxon-Signed rank test was used to test if the means canopy cover values from the 2015 aerial photo’s canopy cover and the LiDAR data we differ- ent. We found that p = 0.238, suggesting the means were not significantly different. However, lack of statistical differences does not equate to statistical similarity. To further test for equivalency a TOST equivalence test using H01: -Δ < μ1 - μ2 > Δ. Where Δ is 6% TCC and μ1- μ2 is the difference between the 2015 areal photo TCC and the 2015 LiDAR TCC. The TOST test based on Welch’s t-test showed that the overserved effect size (d = 0.01) was significantly within the equivalent bounds of dcohen = ±0.14, t(681.85) = -1.75 and p = 0.04. These results indicate that the methods are similar are within a range ±5.38% TCC and were deemed equivalent. Validation results We concluded that the methods used produced statistically equivalent results, allowing a comparison between the two data sources validating the methods and consequently allowing for comparison of TCC between the 1949 aerial photos and the 2015 LiDAR data. Canopy cover change results TCC analysis of the 1949 aerial photos showed a mean TCC of 58.8% and the 2015 LiDAR showed a mean TCC of 49.9%. Since the 2015 aerial photo analysis and the 2015 LiDAR analy- sis were statistically equivalent, the differences between the 1949 aerial photo analysis and the 2015 LiDAR analysis represent realized changes in the TCC between the two time periods. A differences map (Fig 4) was created, with red and green showing deforestation and reforesta- tion respectively, yellow indicated no change. These differences can be divided into three gen- eral trend areas: developed pre-1949, modern development, and coastal. The location of these three trend areas are roughly indicated in Fig 4. 6 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0273487 September 14, 2022 PLOS ONE Combining aerial photos and LiDAR data to detect canopy cover change Fig 3. Quantile-quantile normality plots for the 2015 aerial photo canopy covers and the 2015 LiDAR analysis. The plots are “heavy-tailed” indicating that the data is not normally distributed. https://doi.org/10.1371/journal.pone.0273487.g003 Fig 3. Quantile-quantile normality plots for the 2015 aerial photo canopy covers and the 2015 LiDAR analysis. The plots are “heavy-tailed” indicating that the data is not normally distributed. Fig 3. Quantile-quantile normality plots for the 2015 aerial photo canopy covers and the 2015 LiDAR analysis. The plots are “heavy-tailed” indicating that the data is not normally distributed. https://doi.org/10.1371/journal.pone.0273487.g003 https://doi.org/10.1371/journal.pone.0273487.g003 https://doi.org/10.1371/journal.pone.0273487.g003 Discussion TCC changes were successfully detected for the 64-year period through combining 1949 aerial photo and 2015 LiDAR data derived canopy covers. Two different data types were used and we demonstrated that urban TCC can be compared using the methods presented despite dif- ferences in data types. This allowed examining TCC over a longer time period. Being able to compare TCC using two different data types potentially expands the timeframes available for other studies examining changes in urban forests over time. TCC in areas that would naturally be forested have tree cover ranges from 45–65%, which is consistent with the mean values from both 1949 and 2015 [35]. The non-normal distribution 7 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0273487 September 14, 2022 PLOS ONE Combining aerial photos and LiDAR data to detect canopy cover change Fig 4. Map showing the differences in forest cover at UBC’s campus between 1949 and 2015. Red indicates canopy reduction and green indicates an increase in canopy cover. The blue outline denotes the coastal area, black is the post 1949 (or modern) development and purple is the pre-1949 development area. Base map layer credit: Esri, HERE, Garmin, Intermap, increment P Corp., Gebco, USGS, FAO, NPS, NRCAN, GeoBase, ING (ArcGIS Licence 10.8.1). https://doi.org/10.1371/journal.pone.0273487.g004 Fig 4. Map showing the differences in forest cover at UBC’s campus between 1949 and 2015. Red indicates canopy reduction and green indicates an increase in canopy cover. The blue outline denotes the coastal area, black is the post 1949 (or modern) development and purple is the pre-1949 development area. Base map layer credit: Esri, HERE, Garmin, Intermap, increment P Corp., Gebco, USGS, FAO, NPS, NRCAN, GeoBase, ING (ArcGIS Licence 10.8.1). https://doi.org/10.1371/journal.pone.0273487.g004 https://doi.org/10.1371/journal.pone.0273487.g004 8 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0273487 September 14, 2022 PLOS ONE Combining aerial photos and LiDAR data to detect canopy cover change of the data and the heavy tails on the Q-Q plots was expected. Both ends of the Q-Q plots were heavy indicating that the extreme canopy covers (<10% and >90%) were more common than mean values. This seems realistic for an urban setting where there will be patchiness in built- up areas and areas of high canopy cover, such as parks. This study removed all features classified as trees that were less than 5 m. Discussion This height restriction was implemented to reduce the false classification of shrubs and is consistent with the FAO method of differentiating between trees and shrubs in classification processes [31], and was not validated further. However, it is likely that some small trees that have a height potential >5 m have been excluded in this research and some shrubs taller than 5 m have been included. The net impact of these inclusions and exclusions were deemed negligible due to the large data set size. An additional limitation in this research is the inability to ensure equality between the 1949 and 2015 aerial photos. The 2015 aerial photo cell size was adjusted to match the 1949 photos, but other attributes were not edited. Reduction of the cell size ensures that the overall photo resolution is the same but, cannot correct for differences in shadows, and color, leading to differences in TCC identification between the 2015 and 1949. These differ- ences were therefor not accounted for in the validation process. Ideally, this research would have been able to validate the 1949 data directly with 1949 LiDAR data. Given this limitation, the two different years of aerial photos were made as equal as possible and future studies using these methods could provide further corroboration of the results from this research. Finally, this research found statistical equivalence within ±5.38% TCC. While statistically significant, this margin of error could have implications for practitioners. Urban forestry practitioners are often asked by municipalities to manage TCC within ranges of 1–2% and propose plans to ensure these goals are reached. From this perspective an error of ±5.38% TCC is significant. However, this research highlights an approach to monitoring long-term TCC trends that would otherwise be impossible with shorter data sets. To reduce error, we recommend that TCC change detection studies always be conducted with the best available data and only use two data types when other options are unavailable. PLOS ONE \| https://doi.org/10.1371/journal.pone.0273487 September 14, 2022 Patterns of cover change In the coastal areas, there was an increase in TCC on the western side of UBC and a decrease on the northern side. The reduction in TCC on the northern coastal area is likely due to cliff erosion. The erosion of the Point Grey cliffs has been a concern for UBC since the initial devel- opment of the campus in the late 1910’s. Studies were conducted in 2002 and 2004 assessing the stability of the slopes [36] with a joint committee between UBC, the University Endow- ment Lands Administration, the British Columbia Ministry of Transportation and Infrastruc- ture, and Metro Vancouver to conduct further studies in 2018 [37]. One of the purposes in collecting the 2015 LiDAR data of UBC was to accurately map the Point Grey cliffs to allow for comparisons of the slope with future data [38]. The western coastal area had an increase in TCC from 1949–2015. This coastal area has undergone changes that has also affected the tidal currents and patterns. In 1935 the North Arm Jetty, which now runs along the southern and western coast of UBC underwent major expansion. This jetty acts as a breakwater to calm the waters allowing for log booming [39]. The North Arm Jetty has undergone several additional changes including the addition of a wood debris processing facility in the mid-1960s [40]. Although the expansion of the jetty occurred 16 years before the time frame of this study, the effects of dampening currents would likely have long lasting impacts. Calmer waters reduce erosion and allow for the gathering of sedimentation and slow expansion of the coastline outwards. Over time, this could lead to an PLOS ONE \| https://doi.org/10.1371/journal.pone.0273487 September 14, 2022 9 / 13 PLOS ONE Combining aerial photos and LiDAR data to detect canopy cover change increase in land available for tree growth causing an increase in TCC, matching the patterns seen at UBC. In the areas developed pre-1949 there is an increase in TCC (Fig 4). The pre-1949 develop- ment is composed of educational buildings and residential single homes. Building types and development age are likely influencing the TCC gain [1,41,42]. Single-family homes have a positive correlation with TCC [41] and there is a lag between development and maximum can- opy size [42]. This occurs because the urban forest canopy has time to recover from destructive development activities. Acknowledgments The authors would like to thank UBC geography for allowing access to their air photo collec- tion, and UBC community and campus planning for access to the campus wide LiDAR data. Patterns of cover change This pattern is consistent with Berland’s [1] findings that showed TCC increased with the age of urban development. Since 1949, areas of historical development have had time to recover. The final area identified was the modern development area (Fig 4) in the southern portion of the study area. This area has undergone development within the last 15 years (initial plans adopted in 2005) [43]. Berland [1] found that TCC decreased with intensity of urbanization with conversion to urban land use causing the most substantial immediate loss of TCC. There is an immediate loss followed by a period of recovery. Since much of the development is recent, it is likely that the recovery period for TCC has not been realized. Future research could expand the study area to capture all of the Metro Vancouver regional district to deter- mine if the patters detected hold consistent across a larger region. Conclusion This research increases the ability to look at TCC changes when there are inconsistent data types available to researchers. This case-study was able to show that historical aerial photos can be used in conjunction with modern LiDAR data to examine TCC changes at small scales over times exceeding many remotely sensed data sources. Comparing between 2015 aerial phots and 2015 LiDAR data identified means of 54.94 and 54.70 respectively. Further statistical anal- ysis identified no difference with each 0.05ha polygon having an equivalency of ±5.38% TCC. Concluding that the analysis of the two different data types lead to comparable results allowed for the analysis to be conducted between modern (2015) LiDAR data and historical (1949) aerial photos, and subsequent comparisons of the TCC. Future research at different locations with different data resolutions would be able to refine the data requirements for the methods uses. However, the data and methods used in this study indicate the ability to examine TCC in new ways through combining data types, expanding the ability to examine TCC over time- frames larger than is captured by a single data source. PLOS ONE \| https://doi.org/10.1371/journal.pone.0273487 September 14, 2022 References 1. Berland A. Long-term urbanization effects on tree canopy cover along an urban-rural gradient. Urban Ecosyst. 2012; 15: 721–738. https://doi.org/10.1007/s11252-012-0224-9 2. Canetti A, Garrastazu MC, Mattos PP de, Braz EM, Pellico Netto S. Understanding multi-temporal urban forest cover using high resolution images. Urban For Urban Green. 2018; 29: 106–112. https:// doi.org/10.1016/j.ufug.2017.10.020 3. Walton JT, Nowak DJ, Greenfield EJ. Assessing urban forest canopy cover using airborne or satellite imagery. Arboriculture and Urban Forestry. 2008. pp. 334–340. Available: www.discover-aai.com/ software/products/IMAGINE_Subpixel_. 4. Wolf KL, Lam ST, McKeen JK, Richardson GRA, Bosch M van den, Bardekjian AC. Urban trees and human health: A scoping review. Int J Environ Res Public Health. 2020; 17: 1–30. https://doi.org/10. 3390/ijerph17124371 PMID: 32570770 5. Nowak DJ, Dwyer JF. Understanding the benefits and costs of urban forest ecosystems. Urban and Community Forestry in the Northeast. Springer Netherlands; 2007. pp. 25–46. https://doi.org/10.1007/ 978-1-4020-4289-8_2 6. Tyrva¨inen L, Pauleit S, Seeland K, De Vries S. Benefits and uses of urban forests and trees. Urban For- ests and Trees: A Reference Book. Springer Berlin Heidelberg; 2005. pp. 81–114. https://doi.org/10. 1007/3-540-27684-X_5 7. Richardson JJ, Moskal LM. Uncertainty in urban forest canopy assessment: Lessons from seattle, WA, USA. Urban For Urban Green. 2014; 13: 152–157. https://doi.org/10.1016/j.ufug.2013.07.003 8. Nowak DJ, Greenfield EJ. Tree and impervious cover change in U.S. cities. Urban For Urban Green. 2012; 11: 21–30. https://doi.org/10.1016/j.ufug.2011.11.005 9. Nowak DJ, Greenfield EJ. Declining urban and community tree cover in the United States. Urban For Urban Green. 2018; 32: 32–55. https://doi.org/10.1016/j.ufug.2018.03.006 10. Andersen F, Konijnendijk CC, Randrup TB. Higher education on urban forestry in Europe: An overview. Forestry. 2002; 75: 501–511. https://doi.org/10.1093/forestry/75.5.501 11. Ucar Z, Bettinger P, Merry K, Siry J, Bowker JM, Akbulut R. A comparison of two sampling approaches for assessing the urban forest canopy cover from aerial photography. Urban For Urban Green. 2016; 16: 221–230. https://doi.org/10.1016/j.ufug.2016.03.001 12. Lee JH, Ko Y, McPherson EG. The feasibility of remotely sensed data to estimate urban tree dimen- sions and biomass. Urban For Urban Green. 2016; 16: 208–220. https://doi.org/10.1016/j.ufug.2016. 02.010 13. Chen J, Gong P, He C, Pu R, Shi P. Land-use/land-cover change detection using improved change- vector analysis. Photogramm Eng Remote Sensing. 2003; 69: 369–379. https://doi.org/10.14358/ PERS.69.4.369 14. Wasige JE, Groen TA, Smaling E, Jetten V. Monitoring basin-scale land cover changes in Kagera Basin of Lake Victoria using: Ancillary data and remote sensing. Int J Appl Earth Obs Geoinf. 2012; 21: 32–42. https://doi.org/10.1016/j.jag.2012.08.005 15. Author Contributions Conceptualization: Kathleen Coupland. Conceptualization: Kathleen Coupland. Conceptualization: Kathleen Coupland. Data curation: Kathleen Coupland. Formal analysis: Kathleen Coupland. Methodology: Kathleen Coupland. Supervision: Verena C. Griess. Validation: Kathleen Coupland. Data curation: Kathleen Coupland. Formal analysis: Kathleen Coupland. 10 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0273487 September 14, 2022 PLOS ONE Combining aerial photos and LiDAR data to detect canopy cover change Visualization: Kathleen Coupland. Writing – original draft: Kathleen Coupland, David Hamilton. Writing – review & editing: Kathleen Coupland, David Hamilton, Verena C. Griess. Writing – review & editing: Kathleen Coupland, David Hamilton, Verena C. Griess. References Mallinis G, Emmanoloudis D, Giannakopoulos V, Maris F, Koutsias N. Mapping and interpreting histori- cal land cover/land use changes in a Natura 2000 site using earth observational data: The case of Nes- tos delta, Greece. Appl Geogr. 2011; 31: 312–320. https://doi.org/10.1016/j.apgeog.2010.07.002 16. Gerard F, Petit S, Smith G, Thomson A, Brown N, Manchester S, et al. Land cover change in Europe between 1950 and 2000 determined employing aerial photography. Prog Phys Geogr. 2010; 34: 183– 205. https://doi.org/10.1177/0309133309360141 17. Awad MM. Forest mapping: a comparison between hyperspectral and multispectral images and tech- nologies. J For Res. 2018; 29: 1395–1405. https://doi.org/10.1007/s11676-017-0528-y 18. Upadhyay V, Kumar A. Hyperspectral Remote Sensing of Forests: Technological advancements, Opportunities and Challenges. Earth Science Informatics. Springer Verlag; 2018. pp. 487–524. https:// doi.org/10.1007/s12145-018-0345-7 19. Vogeler JC, Braaten JD, Slesak RA, Falkowski MJ. Extracting the full value of the Landsat archive: Inter-sensor harmonization for the mapping of Minnesota forest canopy cover (1973–2015). Remote Sens Environ. 2018; 209: 363–374. https://doi.org/10.1016/j.rse.2018.02.046 PLOS ONE \| https://doi.org/10.1371/journal.pone.0273487 September 14, 2022 11 / 13 PLOS ONE Combining aerial photos and LiDAR data to detect canopy cover change 20. Ahmed OS, Franklin SE, Wulder MA, White JC. Characterizing stand-level forest canopy cover and height using Landsat time series, samples of airborne LiDAR, and the Random Forest algorithm. ISPRS J Photogramm Remote Sens. 2015; 101: 89–101. https://doi.org/10.1016/j.isprsjprs.2014.11. 007 21. Habtamu T, Casper IM, Joel OB, Abubeker H, Ayana A, Yared M. Evaluation of land use land cover changes using remote sensing Landsat images and pastoralists perceptions on range cover changes in Borana rangelands, Southern Ethiopia. Int J Biodivers Conserv. 2018; 10: 1–11. https://doi.org/10. 5897/ijbc2017.1123 22. Nguyen LH, Joshi DR, Clay DE, Henebry GM. Characterizing land cover/land use from multiple years of Landsat and MODIS time series: A novel approach using land surface phenology modeling and random forest classifier. Remote Sens Environ. 2020; 238: 111017. https://doi.org/10.1016/j.rse.2018.12.016 23. Xulu S, Peerbhay K, Gebreslasie M, Ismail R. Drought influence on forest plantations in Zululand, South Africa, using MODIS time series and climate data. Forests. 2018; 9: 528. https://doi.org/10.3390/ f9090528 24. Latifi H, Dahms T, Beudert B, Heurich M, Ku¨bert C, Dech S. Synthetic RapidEye data used for the detection of area-based spruce tree mortality induced by bark beetles. GIScience Remote Sens. 2018; 55: 839–859. https://doi.org/10.1080/15481603.2018.1458463 25. Jung SE, Kwak DA, Park T, Lee WK, Yoo S. Estimating crown variables of individual trees using air- borne and terrestrial laser scanners. References Remote Sens. 2011; 3: 2346–2363. https://doi.org/10.3390/ rs3112346 26. Wehr A, Lohr U. Airborne laser scanning—An introduction and overview. ISPRS J Photogramm Remote Sens. 1999; 54: 68–82. https://doi.org/10.1016/S0924-2716(99)00011-8 27. Hansen MC, DeFries RS, Townshend JRG, Carroll M, Dimiceli C, Sohlberg RA. Global percent tree cover at a spatial resolution of 500 meters: First results of the MODIS vegetation continuous fields algo- rithm. Earth Interact. 2003; 7: 1–15. https://doi.org/10.1175/1087-3562(2003)007<0001:gptcaa>2.0. co;2 28. Homer CG, Dewitz JA, Yang Jin, S. L, Danielson P, Xian G, Coulston J, et al. Completion of the 2011 national land cover database for the conterminous United States: representing a decade of land cover change information. Photogramm Eng Remote Sensing. 2018; 81: 345–354. https://doi.org/10.14358/ PERS.81.5.345 29. Soares C, Prı´ncipe A, Ko¨bel M, Nunes A, Branquinho C, Pinho P. Tracking tree canopy cover changes in space and time in high nature value farmland to prioritize reforestation efforts. Int J Remote Sens. 2018; 39: 4714–4726. https://doi.org/10.1080/01431161.2018.1475777 30. Gaulton R, Malthus TJ. LiDAR mapping of canopy gaps in continuous cover forests: A comparison of canopy height model and point cloud based techniques. Int J Remote Sens. 2010; 31: 1193–1211. https://doi.org/10.1080/01431160903380565 31. Di Gregorio A, Jansen J. Land Cover Classification System (LCCS): Classification Concepts and User Manual. FAO. 2000. Available: https://agris.fao.org/agris-search/search.do?recordID=SO2005100463. 32. Cotillon SE, Mathis ML. Tree Cover Mapping Tool—Documentation and user manual. Open-File Rep. Reston, VA; 2016. https://doi.org/10.3133/OFR20161067 33. Plowright A. Canopy analysis in R using Forest Tools. 2018 [cited 19 Apr 2021] pp. 1–9. Available: https://cran.r-project.org/web/packages/ForestTools/vignettes/treetopAnalysis.html. 34. Coupland K, Magalhães J, Griess VC. Connecting Forestry Learning Objectives to Urban Forest Types. J For. 2021 [cited 21 Jan 2022]. https://doi.org/10.1093/JOFORE/FVAB053 35. Wells RW. DEVELOPMENTAL TRENDS OF STAND STRUCTURE AND TREE MORTALITY IN COASTAL WESTERN HEMLOCK FORESTS. 1996. Available: https://summit.sfu.ca/item/10544. 36. Lee P, Coster A, Liu B. Stability of the UBC Point Grey Cliffs and the Effects of Vegetation on Slope Sta- bility. 2016. p. 15. Available: https://web.viu.ca/earle/geol312/point-grey-erosion.htm. 37. Harris M. Electoral Area A Director’s Update. 2018. Available: http://www.metrovancouver.org/boards/ search/. 38. UBC. University of British Columbia Point Grey Campus Lidar, 2015. 2015. 39. Hajer MA. The Fraser’s North Arm. Inpired 55+ Lifestyle Magazine. 2009. Available: https://www. seniorlivingmag.com/frasers-north-arm/. Accessed 19 Apr 2021. 40. Page N. Iona Beach Regional Park: Strategies for Maintaining Native Ecological Communities ii. 2011 41. Troy AR, Grove JM, O’Neil-Dunne JPM, Pickett STA, Cadenasso ML. Predicting opportunities for greening and patterns of vegetation on private urban lands. Environ Manage. 2007; 40: 394–412. PLOS ONE \| https://doi.org/10.1371/journal.pone.0273487 September 14, 2022 42. Grove JM, Troy AR, O’Neil-Dunne JPM, Burch WR, Cadenasso ML, Pickett STA. Characterization of households and its implications for the vegetation of urban ecosystems. Ecosystems. 2006; 9: 578– 597. https://doi.org/10.1007/s10021-006-0116-z 43. UBC. Wesbrook Place Neighbourhood Plan. 2005 [cited 21 Apr 2021]. Available: https://planning.ubc. ca/sites/default/files/2020-04/PLANS_UBC_WesbrookPlaceNP.pdf. Combining aerial photos and LiDAR data to detect canopy cover change PLOS ONE \| https://doi.org/10.1371/journal.pone.0273487 September 14, 2022 References https://doi.org/10.1007/s00267-006-0112-2 PMID: 17602257 12 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0273487 September 14, 2022 PLOS ONE Combining aerial photos and LiDAR data to detect canopy cover change 13 / 13
https://openalex.org/W2470959842	https://europepmc.org/articles/pmc4926224?pdf=render	English	null	Propylene/propane permeation properties of ethyl cellulose (EC) mixed matrix membranes fabricated by incorporation of nanoporous graphene nanosheets	Scientific reports	2,016	cc-by	6,964	Propylene/propane permeation properties of ethyl cellulose (EC) mixed matrix membranes fabricated by incorporation of nanoporous graphene nanosheets Bingbing Yuan1,, Haixiang Sun1,2,, Tao Wang2, Yanyan Xu2, Peng Li1, Ying Kong1 & Q. Jason Niu1 received: 24 September 2015 accepted: 06 June 2016 Published: 29 June 2016 Nanopore containing graphene nanosheets were synthesized by graphene oxide and a reducing agent using a facile hydrothermal treatment in sodium hydroxide media. The as-prepared nanoporous graphene was incorporated into ethyl cellulose (EC) to prepare the mixed matrix membranes (MMMs) for C3H6/C3H8 separation. Transmission electron microscopy (TEM) photograph and X-ray photoelectron spectroscopy (XPS) analysis of nanoporous graphene nanosheets indicated that the structure of nano- pore was irregular and the oxygen-containing groups in the surface were limited. More importantly, the as-prepared MMMs presented better separation performance than that of pristine EC membrane due to simultaneous enhancement of C3H6 permeability and ideal selectivity. The ideal selectivity of the MMMs with 1.125 wt‰ nanoporous graphene content for C3H6/C3H8 increased from 3.45 to 10.42 and the permeability of C3H6 increased from 57.9 Barrer to 89.95 Barrer as compared with the pristine membrane. The presumed facilitated mechanism was that the high specific surface area of nanoporous graphene in polymer matrix increased the length of the tortuous pathway formed by nanopores for the gas diffusion as compared with the pristine graphene nanosheets, and generated a rigidified interface between the EC chains and fillers, thus enhanced the diffusivity selectivity. Therefore, it is expected that nanoporous graphene would be effective material for the C3H6/C3H8 separation. The separation of low-carbon olefin/paraffin mixtures is one of the most challenging tasks in the petrochemical industry due to their similar molecular sizes and physical properties. Currently, the separation is mainly achieved by fractional distillation at cryogenic temperature1, and large capital investment and high energy consumption involved in this conventional technology stimulates the researchers to find a more cost-effective separation pro- cess2. As an alternatively energy-saving approach, membrane separation technology has a great potential in the petrochemical industry3–5. Mixed matrix membranes (MMMs) that are composed by blending inorganic particles into the polymer matrix are promising approaches; they combine the easy processing polymeric membranes with superior separation performance of inorganic nanoparticles6. These advantages also provide an opportunity to overcome the individual deficiencies of inorganic materials and polymers, offering attractive solutions for indus- trial applications. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports \| 6:28509 \| DOI: 10.1038/srep28509 www.nature.com/scientificreports/ www.nature.com/scientificreports/ propane using cellulose acetate-silica nanocomposite membranes, and the selectivity for C3H6/C3H8 was 6.12 and the permeability of C3H6 was 0.098 Barrer with a nanocomposite membrane that contains 30 wt% silica particles under 2 bar feed absolute pressure and 35 °C temperature. Koros et al.13 found that mixed matrix membranes, fab- ricated by 6FDA-DAM polyimide and ZIF-8, had an ideal selectivity of 31.0 for C3H6/C3H8, and a permeability of 56.2 Barrer for C3H6 with 48.0 wt% ZIF-8 loading, which were 150% and 258% higher than the pure 6FDA-DAM membrane respectively for selectivity and permeability.i p y y p y As mentioned above, most available literatures focused on the spherical and square shape nanofillers to enhance the gas separation performance. It is inevitable to generate the ‘sieve-in-a-cage morphology’ between these nanofiller materials and polymer interface due to the low aspect ratio of the spherical and square shape nanofillers and the weak interfacial adhesion. Such voids decrease the selectivity of the MMMs, even the per- meability is increased7,16. In contract, the graphene nanosheets are intrinsically more compatible with polymers as compared to other quadrate or sphere molecule sieves because of its high aspect ratio (>1000), easy surface functionalization, and high thermal and mechanical properties17,18. Moreover, the high specific surface area fill- ers in the polymer matrix increase the length of the tortuous pathway for gas diffusion and reduce the mobil- ity of polymer chains. This advantage will restrict the diffusion of larger molecules, and favor the diffusion of small molecules with less resistance, thus improving gas diffusivity selectivity19–24. On the other hand, Checchetto et al.25 studied the gas transport performance of nanocomposite membrane that was composed of polyethylene with dispersed graphite nanoplatelets (GNPS), and the results indicated that GNPS with nominal content of 5 wt% inclusions reduced the permeability by approximately a factor of two as compared with that of pure poly- mer membrane. This was due to the fact that the defect-free graphene and its derivatives like graphene oxide were theoretically gas impermeable, and such ultrathin two dimensional structure hindered gas diffusion, which was consistent with the theoretical compute studies that a perfect graphene was impermeable to gases even as small as He26,27. Therefore, it is useful to fabricate nanopores in the graphene nanosheets to further enhance the tortuous pathway of small gas molecule diffusion and hinder the big gas molecule diffusion in the polymer matrix. www.nature.com/scientificreports/ p y gf g gf p y For the reasons discussed above, ethyl cellulose (EC) is selected in this study as the polymer matrix based on its comparatively large free volume and relatively low glass transition temperatures, which might provide high gas permeability and a superb C3H6 diffusion coefficient. Furthermore, massive oxygen functional groups on the sur- face of graphene nanosheets generate better interfacial bonding strength with EC polymer28–30. To be specific, the nanoporous graphene was synthesized by sodium hydroxide using a facile hydrothermal treatment, and then the MMMs were fabricated with the addition of nanoporous graphene into EC polymer through solution blending method. In order to prove the nanopores in the surface of graphene nanosheets are beneficial for the enhancement of C3H6/C3H8 separation performance, light reduction graphene oxide (L-rGO) containing oxygen-containing groups such as carboxyl, hydroxyl group and reduction graphene oxide (rGO) were synthesized and incorpo- rated into EC polymer to prepare MMMs for C3H6/C3H8 permeation. The morphology and microstructure of the as-prepared graphene material were confirmed, and the physical properties of MMMs in terms of microstructure, crystallization, tensile property and thermal stability (see Supplementary Information Table S1 and Fig. S3) were investigated as well. Moreover, for nanoporous graphene MMMs, the effects of feed pressure on the C3H6/C3H8 permeation performance were systematically examined and evaluated (see Supplementary Information Fig. S4 and Fig. S5). Results and Discussion Characterization of the L-rGO, rGO and nanoporous rGO nanosheets. Different methods of treat- ment on GO nanosheets may result various morphologies, and these changes can be directly observed through TEM characterization. Figure 1 shows the TEM images of L-rGO, rGO and nanoporous rGO nanosheets. As shown in Fig. 1(a), L-rGO presents relatively smooth nanosheets in comparison with rGO and nanoporous rGO nanosheets, since that trace amount of NaBH4 is inadequate for the chemical reduction of the vast oxygen groups on the GO nanosheets. However, intensive deoxygenation processes can be completed with abundant NaBH4 reduction and NaOH hydrothermal treatment, as shown in Fig. 1(b,c). The nanopores with irregular shapes are clearly distributed on the nanoporous rGO nanosheets, indicating that substantial decarbonisation and violent deoxygenation process had occurred during the hydrothermal treatment. In contrast, the rGO nanosheets prac- tically exhibit no obvious nanopores and merely wrinkled texture, due to the deletion of oxygen groups31. The nanopores on the surface of rGO nanosheets may increase the specific surface area and the length of the tortuous pathway for the gas diffusion in the EC polymer matrix. In addition, the graphene nanosheets with high-aspect ratio may also contribute to the polymer and graphene nanosheets in the formation of MMMs with excellent permeability performance and mechanical properties. p y p p p Variation on the morphologies of graphene nanosheets can also be indirectly measured by the Raman spectra. As observed in Fig. 2, from L-rGO to rGO, and then nanoporous rGO nanosheets, it is clearly observed that the intensity of D band gradually increases as compared with G band, and the intensity of G band marginally decreases32,33. During the chemical reduction process, the average size of sp2 regions in the graphene were gradu- ally weakened and resulted in a defected surface. After that, these defected sites increasingly expanded and finally generated nanopores in the intensive hydrothermal treatment process. Therefore, an increased ID/IG intensity ratio gradually emerges from light chemical reduction (L-rGO) to chemical reduction (rGO), and then hydro- thermal treatment (nanoporous rGO). Combined with the results of TEM images, it can be concluded that nano- porous rGO nanosheets would contain large amount of nanopores as compared with L-rGO and rGO nanosheets. p g p p The micro-structure of graphene nanosheets were also characterized by FTIR spectroscopy. As shown in Fig. Propylene/propane permeation properties of ethyl cellulose (EC) mixed matrix membranes fabricated by incorporation of nanoporous graphene nanosheets Bingbing Yuan1,, Haixiang Sun1,2,, Tao Wang2, Yanyan Xu2, Peng Li1, Ying Kong1 & Q. Jason Niu1 Correspondingly, there are several inorganic materials such as zeolites, carbon molecular sieves (CMS), carbon nanotubes (CNTs), C60, metal-organic frameworks (MOFs), and covalent organic frameworks (COFs) that have been amalgamated into the polymer matrix to prepare the MMMs for gas separation, and sub- sequently achieved enhanced CO2/CH4, O2/N2, H2/CO2 and CO2/N2 selectivity7–12. q y 2 4 2 2 2 2 2 2 y In recent years, a few studies have been reported about employing mixed matrix platform to the C3H6/C3H8 system using nanofillers, and most nanofillers are spherical shape like silica materials and C60, or square zeolitic like imidazolate framework ZIF-8 13–15. For example, Naghsh and Sadeghi14 studied the separation of propylene/ 1State Key Laboratory of Heavy Oil Processing, China University of Petroleum (East China), Qingdao 266580, P.R. China. 2College of Science, China University of Petroleum (East China), Qingdao 266580, P.R. China. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to H.S. (email: sunhaixiang@upc.edu.cn) or Q.J.N. (email: qjniu@upc.edu.cn) Scientific Reports \| 6:28509 \| DOI: 10.1038/srep28509 1 Results and Discussion 3(a), the peaks at around 1072, 1552, and 3401 cm−1 wavenumber are ascribed to the C–O, C=C and O–H groups in the L-rGO with a relative higher intensity in comparison with the rGO and nanoporous rGO nano- sheets34,35. On the other hand, the appearance of asymmetric bands for the alkyl groups at 2917 and 2837 cm−1 Scientific Reports \| 6:28509 \| DOI: 10.1038/srep28509 2 www.nature.com/scientificreports/ Figure 1. TEM images of the L-rGO nanosheets prepared under GO and NaBH4 mass ratio 1:2 at 80 °C for 0.5 h (a); the rGO nanosheets prepared under GO and NaBH4 mass ratio 1:25 at 80 °C for 2 h (b) and the nanoporous rGO nanosheets prepared using hydrothermal treatment (c). Figure 1. TEM images of the L-rGO nanosheets prepared under GO and NaBH4 mass ratio 1:2 at 80 °C for 0.5 h (a); the rGO nanosheets prepared under GO and NaBH4 mass ratio 1:25 at 80 °C for 2 h (b) and the nanoporous rGO nanosheets prepared using hydrothermal treatment (c). Figure 2. The Raman images of L-rGO nanosheets (a); rGO nanosheets (b) and nanoporous rGO nanosheets (c). Figure 2. The Raman images of L-rGO nanosheets (a); rGO nanosheets (b) and nanoporous rGO nanosheets (c). n rGO and nanoporous rGO results from the greatly decrease of the oxygen functional groups, indicating the ompletion of chemical reduction process.if In order to further confirm the difference of micro-structure of L-rGO, rGO and nanoporous rGO nanosheets, XPS measurement was conducted (Fig. 4). As shown in Fig. 4a, the C1s XPS spectrum of L-rGO clearly indicates a considerable degree of oxidation with four components which correspond to carbon atoms in different oxygen functional groups: the non-oxygenated ring C, the C in C–O bonds, the carbonyl C, and the carboxylate carbon (O–C=O)36. Moreover, L-rGO has a much lower peak intensity as compared with the C1s spectrum of rGO (Fig. 4b) and nanoporous rGO (Fig. 4c), despite possess the same oxygen functionalities. Figure 4d shows the direct atomic ratios (C1s/O1s) of L-rGO, rGO and nanoporous rGO, which also demonstrates the decrease of oxygen functionalities intensities. In addition, it is observed that rGO nanosheets exhibit lower oxygen functional intensity than that of nanoporous rGO, which are consistent with the results of FTIR. Scientific Reports \| 6:28509 \| DOI: 10.1038/srep28509 3 www.nature.com/scientificreports/ Figure 3. FTIR spectra of L-rGO nanosheets (a); rGO nanosheets (b) and nanoporous rGO nanosheets (c). Figure 3. Results and Discussion FTIR spectra of L-rGO nanosheets (a); rGO nanosheets (b) and nanoporous rGO nanosheets (c). Figure 4. XPS spectra of L-rGO nanosheets (a), rGO nanosheets (b), nanoporous rGO nanosheets (c) and their XPS scan spectra (d). Figure 4. XPS spectra of L-rGO nanosheets (a), rGO nanosheets (b), nanoporous rGO nanosheets (c) and their XPS scan spectra (d). Figure 5 shows the XRD patterns of L-rGO, rGO and nanoporous rGO nanosheets. The typical peak at 2θ = 11.6° is attributed to the (002) plane of GO and 2θ = 23.2°, and is the characteristic of the parallel graphene layers, indicating that L-rGO, rGO and nanoporous rGO sheets are present both the consistent layers and the size of crystallite37. Furthermore, morphology and micro-structure confirm that three graphene nanosheets exhibit different oxygen-containing functional groups and nanopores, that is, L-rGO nanosheets with large amounts of oxygen functional groups in the surface, rGO nanosheets with predominant aromatic rings structure in the sur- face, and nanoporous rGO nanosheets with aromatic rings structure and nanopores in the surface. Meanwhile, the consistent layers and the size of crystallite also show that three graphene nanosheets are no difference except oxygen functional groups and nanopores. Scientific Reports \| 6:28509 \| DOI: 10.1038/srep28509 4 www.nature.com/scientificreports/ tificreports/ Figure 5. XRD patterns of L-rGO nanosheets (a); rGO nanosheets (b) and nanoporous rGO nanosheets (c). Figure 5. XRD patterns of L-rGO nanosheets (a); rGO nanosheets (b) and nanoporous rGO nanosheets (c). Figure 6. XRD patterns of pure EC membrane (a); L-rGO MMMs (b); rGO MMMs (c) and nanoporous rGO MMMs (d). Figure 6. XRD patterns of pure EC membrane (a); L-rGO MMMs (b); rGO MMMs (c) and nanoporous rGO MMMs (d). Membrane Characterization. XRD is performed to investigate the effects of graphene nanosheets on the EC polymer chains. As shown in Fig. 6, the representative diffraction peaks of L-rGO, rGO and nanoporous rGO nanosheets were disappeared. Moreover, Fig. 6 also provides that the graphene nanosheets have a slight impact on the diffraction patterns of the EC polymer, especially on the variation of interlayer distance. Diffraction peaks at 2θ (°) of 7.93 and 20.56 are the characteristic of cholesteric liquid crystallinity. Scientific Reports \| 6:28509 \| DOI: 10.1038/srep28509 Results and Discussion The small angle peak at 2θ (°) of 20.56 attributes to the interlayer distance of the ordered chains of the EC polymer, with the second peak reflecting the interchain distance38,39, therefore, graphene nanosheets have no influence on the interlayer and interchain distance of EC polymer matrix. On the other hand, the diffraction peak at 2θ (°) of 7.93 in L-rGO, rGO and nan- oporous rGO nanosheets has an increased intensity as compared with that of pure EC, indicating an enhanced crystalline arrangement of EC chains. In this case, the incorporation of nanofillers into the EC polymer matrix generates a rigid interface between EC chains and graphene nanosheets, which hinders the mobility of macro- molecular segments resulted from an increased chains crystallinity40,41, and eventually decreases the free volumes among polymer chains.h g p y The cross-section morphologies of three MMMs are presented in Fig. 7. It can be observed that the PI ultra- filtration membrane presents a larger finger-like pore structure, which is unfavorable for the permeation of gas. Furthermore, the morphology of membrane has no significant change among EC membrane and MMMs, indicat- ing a superb compatibility between graphene nanosheets and EC polymer42,43. Magnified images of cross-section morphologies are shown in Fig. 7(b,d,f,h). It is evident that no nanofillers are distinguished and MMMs are relatively flatter than that of EC, which indicates that graphene nanosheets are well dispersed in EC polymer matrix. According to Fig. 6, incorporation of nanofillers into the EC polymer matrix hinders the mobility of macromolecular segments and increases its chains crystallinity, thus graphene nanosheets can act as a nucleating agent in the polymer membranes44–46. The excellent dispersion and compatibility was observed, since graphene nanosheets with high aspect ratio can increase the interfacial area between EC polymer chains and nanosheets, resulting in a better compatibility and dispersion in polymer matrix. Scientific Reports \| 6:28509 \| DOI: 10.1038/srep28509 5 www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 7. SEM images of cross-section of pure EC membrane (a,b), L-rGO MMMs (c,d), rGO MMMs ( nanoporous rGO MMMs (g,h). Figure 7. SEM images of cross-section of pure EC membrane (a,b), L-rGO MMMs (c,d), rGO MMMs (e,f) and nanoporous rGO MMMs (g,h). The TEM image of nanoporous rGO MMMs solution further verifies the nanoscale morphology of nano- porous rGO nanosheets. As observed in Fig. Results and Discussion 8, a relatively ambiguous morphology of nanoporous graphene nanosheets was observed in nanoporous rGO in MMMs, probably due to the existence of EC chains. Moreover, Scientific Reports \| 6:28509 \| DOI: 10.1038/srep28509 6 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 8. TEM image of nanoporous rGO nanosheets in the MMMs solution. Figure 8. TEM image of nanoporous rGO nanosheets in the MMMs solution. Figure 9. Effect of graphene nanosheets content in the EC polymer matrix on the C3H6/C3H8 ideal selectivity: (a) L-rGO MMMs; (b) rGO MMMs; (c) nanoporous rGO MMMs. (Feed pressure at 0.1 MPa, temperature at 298 K). Figure 9. Effect of graphene nanosheets content in the EC polymer matrix on the C3H6/C3H8 ideal selectivity: (a) L-rGO MMMs; (b) rGO MMMs; (c) nanoporous rGO MMMs. (Feed pressure at 0.1 MPa, temperature at 298 K). it can be clearly seen that there are plenty of nanopores in the nanoporous rGO nanosheets, and these nanopores are facile channels for the small gas molecules to permeate. These phenomena are complementary with the TEM images of nanoporous rGO nanosheets described in the earlier section. Membrane permeation performance. In order to investigate the structure of graphene nanosheets and their loading content on the membrane permeation performances, three kinds of MMMs are fabricated with different nanofiller loading. Figures 9 and 10 present the correlation between the ideal selectivity and permea- bility with different loading of graphene nanosheets in the EC polymer matrix. As shown in Figs 9 and 10, C3H6 permeability, C3H6/C3H8 ideal selectivity gradually increase with the increase of graphene nanosheets content from 0 to 1.125 wt‰, while C3H8 permeability decreases with the content of graphene nanosheets increase from 0 to 0.375 wt‰ and then remain unchanged after 0.0375 wt‰ of graphene nanosheets loading. However, when the loading of the graphene nanosheets in the EC polymer matrix increases to 1.5 wt‰, both C3H6 permeability and C3H6/C3H8 ideal selectivity decrease, indicating that the graphene nanosheets are theoretically impermeable to all atoms and molecules, and greater loading in the MMMs could cause the agglomeration of the graphene nanosheets, resulting in the decrease of the effective surface area47–50. With 1.125 wt‰ of L-rGO, rGO and nan- oporous rGO nanosheets loading in the EC polymer matrix, the ideal selectivity of the MMMs could reach as high as 5.74, 7.29 and 10.42 with C3H6 permeability of 66.05, 76.34 and 89.95 Barrer, respectively. Results and Discussion Meanwhile, it is clearly showed that the ideal selectivity of pure EC polymer membrane is 3.45 and the permeability of C3H6 and C3H8 is 57.9 and 16.78 Barrer, which is consistence with the related literature51. The notable increment involved in both C3H6 permeability of 1.55-fold and C3H6/C3H8 ideal selectivity of 3.02-fold confirm the fact that the nan- opores blended into the MMMs enhance the C3H6 permeability as compared with the pure EC membrane gas permeation performance. However, the blending of graphene nanosheets into the EC polymer matrix hinders the C3H8 permeability. Among three kinds of MMMs, nanoporous graphene MMMs have C3H6/C3H8 ideal selectivity and C3H6 permeability advantages over that of L-rGO and rGO MMMs, which indicates that oxygen-containing functional groups decrease the C3H6 permeability. A reasonable explanation of the gas separation mechanism is Scientific Reports \| 6:28509 \| DOI: 10.1038/srep28509 7 www.nature.com/scientificreports/ Figure 10. Effect of three graphene nanosheets content in EC polymer matrix on the permeability of C3H6 and C3H8: (a) L-rGO MMMs; (b) rGO MMMs; (c) nanoporous rGO MMMs. (Feed pressure at 0.1 MPa, temperature at 298 K). Figure 10. Effect of three graphene nanosheets content in EC polymer matrix on the permeability of C3H6 and C3H8: (a) L-rGO MMMs; (b) rGO MMMs; (c) nanoporous rGO MMMs. (Feed pressure at 0.1 MPa, temperature at 298 K). Figure 11. Diffusion coefficient (a) and solubility coefficient (b) of C3H6 and C3H8 in the membranes. (Feed pressure at 0.1 MPa, temperature at 298 K). igure 11. Diffusion coefficient (a) and solubility coefficient (b) of C3H6 and C3H8 in the membranes. (Feed ressure at 0.1 MPa, temperature at 298 K). proposed as shown in Fig. S6, and 1.125 wt‰ of nanofiller loading is chosen as the optimal loading in MMMs for the further discussion of gas permeation properties.f To further explore the effect of nanopore in the graphene nanosheets on the gas permeation performance, the diffusivity coefficient (D) and solubility coefficient (S) of the membranes with the optimal 1.125 wt‰ of nanofiller loading are compared in Fig. 11. As shown in Fig. 11(a), it is found that C3H6/C3H8 diffusivity selectivity of L-rGO MMMs, rGO MMMs and nanoporous rGO MMMs are higher than that of pure EC membrane. It is notable that the nanoporous rGO MMMs has the highest C3H6/C3H8 diffusivity selectivity among L-rGO MMMs and rGO MMMs. Scientific Reports \| 6:28509 \| DOI: 10.1038/srep28509 Methods i l Materials. EC (Mw = 200,000) was purchased from Shanghai Reagent Corporation (China). Graphite oxide was obtained from Nanjing XFNANO Materials Tech Co. Ltd. (China). Sodium borohydride (NaBH4), sodium hydroxide (NaOH), sodium dodecylbenzene sulfonates (SDBS), hydrochloric acid (HCl 36~38%), acetone and all other types of reactants were bought from Shanghai Chemical Co., Ltd. (China). The propylene (purity > 99.5%) and propane (purity > 99.9%) were purchased from KODI Gas Chemical Industry Co. Ltd. (Foshan, China). Fabrication of LGO, rGO and nanoporous rGO nanosheets. In this case, GO colloidal solution (0.1 g in 150 mL deionized water) was obtained using a mild ultrasonic exfoliation method for 2 h. The above colloidal solution was centrifuged to remove the impurities under 8000 r min−1 for 10 min. The rGO and L-rGO nano- sheets were obtained through chemical reduction with the mass ratio of GO and NaBH4 1:25 at 80 °C for 2 h and 1:2 at 80 °C for 0.5 h respectively. The reduction solutions were subsequently collected based on filtration, flushing with deionized water and ethanol, and then the resulting products were stored in ethanol at a given concentration (0.012 g in 15 mL ethanol).h g The nanoporous rGO nanosheets were synthesized with the treatment of sodium hydroxide under hydrother- mal condition. Firstly, GO colloidal solution (0.1 g in 150 mL deionized water) obtained from an mild ultrasonic exfoliation method for 2 h was chemically reduced to L-rGO using the GO to NaBH4 mass ratio of 1:2 at 80 °C for 0.5 h, and then the above L-rGO was collected by filtration. Subsequently, 10 mL SDBS (0.086 mol L−1) and 20 mL NaOH (12.5 mol L−1) were successively added dropwise to form a 60 mL solution. After stirred for 0.5 h, the reaction solution was decanted into a 100 mL Teflon-lined stainless steel autoclave and conducted the hydro- thermal treatment at 180 °C for 3 h. Eventually, the reaction solution was cooled to ambient temperature, and the resulting solution was consecutively filtered and washed with sufficient deionized water (five times) and ethanol, and the nanoporous rGO nanosheets were placed in ethanol at a given concentration (0.012 g in 15 mL ethanol). Fabrication of MMMs. A series of different concentration of L-rGO, rGO and nanoporous rGO ethanol solutions were prepared via sonication for 1 h. Results and Discussion The lack of nanopores in the nanosheets hinders the further enhancement of C3H6 diffusivity coefficient, even L-rGO nanosheets and rGO nanosheets generate a rigid interface with EC polymer chains and increase the tortuous pathway of the gas diffusion to enhance the diffusivity selectivity. We can conclude that the nanopores in the nanosheets further increase the tortuous pathway of the C3H6 diffusion with less resistance but restrain the diffusion of C3H8 molecules.fi f Figure 11(b) shows the gas solubility coefficients and C3H6/C3H8 solubility selectivity of L-rGO MMMs, rGO MMMs and nanoporous rGO MMMs with the optimal 1.125 wt‰ of nanofiller loading. The solubility of C3H6 and C3H8 in the polymer membranes depends on their relative condensability, characterized by critical tempera- ture, the interaction between polymer and gases, the fraction and amount of free volumes in glassy polymers. The critical temperatures of C3H6 and C3H8 are in the following order: C3H6(364.76 K) < C3H8(369.8 K). It is believed that higher condensability has the higher solubility of gas in the polymer matrix52–55, however, C3H6 exhibits greater solubility than C3H8. This is thanks to the high electron cloud on the double bond with high polarity and flat structure, resulting in more affinity of C3H6 to generate interaction with polymer, and thus enhancing the C3H6 solubility. Moreover, the incorporation of graphene nanosheets increases the density of polar groups such as -COOH and -OH in the MMMs. As a result, it creates polar spaces between the interface of nanofillers and polymer. This formation of polar spaces would result in the enhancement in the solubility coefficient of conden- sable gases56.hf g The diffusivity selectivity increases by 5.24-fold in comparison with 2.66-fold for L-rGO MMMs and 2.93-fold for rGO MMMs, although the loading of nanoporous graphene nanosheets in the polymer matrix cannot effi- ciently increase the solubility selectivity of the gases due to the decrease of the free volume of the glassy polymer. Scientific Reports \| 6:28509 \| DOI: 10.1038/srep28509 8 www.nature.com/scientificreports/ The incorporation of nanoporous graphene nanosheets into the EC polymer matrix mainly affects the diffusivity selectivity of C3H6/C3H8 molecule to enhance the ideal selectivity of MMMs, due to the existence of nanopores and the formation of rigid interface. The incorporation of nanoporous graphene nanosheets into the EC polymer matrix mainly affects the diffusivity selectivity of C3H6/C3H8 molecule to enhance the ideal selectivity of MMMs, due to the existence of nanopores and the formation of rigid interface. Methods i l Dried EC (in a vacuum situation at 40 °C for 24 h) was dissolved in methylbenzene to form a solution with the concentration of 19.7 wt%. Under a nitrogen atmosphere and at ambi- ent temperature, the above dispensed graphene ethanol solutions were then added dropwise to the EC solutions with stirring, The resulting casting solution had a mass ratio of graphene to EC of 0.375, 0.75, 1.125 and 1.5 wt‰ respectively. After 24 h dissolution, the casting solutions were filtered with stainless steel filter, and still degassing for 24 h. The above casting solution was then cast onto the polyimide (PI) ultrafiltration supported membrane using a micrometer film applicator under RH 40% at 298 K. The final membranes were subsequently preserved for gas separation tests. All membrane thicknesses were approximately 15–20 μm. Results and Discussion g In summary, nanoporous rGO nanosheets were successfully fabricated by sodium hydroxide using a facile hydrothermal treatment. The morphology and micro-structure of the nanoporous rGO material were confirmed by TEM and XPS measurement. Nanoporous rGO nanosheets were incorporated into the EC polymer matrix to prepare the MMMs for the C3H6/C3H8 permeation. The permselectivity of nanoporous rGO MMMs exhibits a significant increase with the simultaneous enhancement of diffusivity selectivity and solubility selectivity, espe- cially the 5.24-fold increase of the diffusivity selectivity. This result indicates that nanoporous rGO nanosheets are superior in terms of enhancing the diffusivity selectivity of C3H6 gas molecule. The ideal selectivity for C3H6/C3H8 and the permeability coefficient for C3H6 exhibit a significant increase of 3.02-fold and 1.55-fold in comparison with the pristine EC membrane, respectively. This study shows that nanoporous rGO nanosheets can be effective nanofiller in the polymer matrix to enhance the C3H6/C3H8 permeability. g ,i pfi p gy g , 3. Stern, S. A. Polymers for gas separation: the next decade. J. Membr. Sci. 94, 1–65 (1994).i 4. Burns, R. L. & Koros, W. J. Defining the challenges for C3H6/C3H8 separation using polymeric membranes. J. Membr. Sci. 211 299–309 (2003). References 1. Baker, R. W. Future directions of membrane gas separation technology. Ind. Eng. Chem. Res. 41, 1393–1411 (20 2 Eldridge, R B Olefin/paraffin separation technology: a review Ind Eng Chem Res 32, 2208–2212 (1993) References References 1. Baker, R. W. Future directions of membrane gas separation technology. Ind. Eng. Chem. Res. 41, 1393–1411 (200 g ,i pfi p gy g , 3. Stern, S. A. Polymers for gas separation: the next decade. J. Membr. Sci. 94, 1–65 (1994).i gifi gy g 3. Stern, S. A. Polymers for gas separation: the next decade. J. Membr. Sci. 94, 1–65 (1994).i y g p 4. Burns, R. L. & Koros, W. J. Defining the challenges for C3H6/C3H8 separation using polymeric membranes. J. Membr. Sci. 211, 299–309 (2003). 5. Ravanchi, M. T., Kaghazchi, T. & Kargari, A. Application of membrane separation processes in petrochemical industry: a review. Desalination 235, 199–244 (2009). ( ) 6. Moore, T. T., Mahajan, R., Vu, D. Q. & Koros, W. J. Hybrid membrane materials comprising organic polymers with rigid dispersed phases. AIChE J. 50, 311–321 (2004).f 7. Mahajan, R., Burns, R., Schaeffer, M. & Koros, W. J. Challenges in forming successful mixed matrix membranes with rigid polymeric materials. J. Appl. Polym. Sci. 86, 881–890 (2002). 8. Tin, P. S., Chung, T. S., Jiang, L. & Kulprathipanja, S. Carbon–zeolite composite membranes for gas separation. Carbon 43, 2025–2027 (2005). 9. Clarizia, G., Algieri, C. & Driolia, E. Filler-polymer combination: a route to modify gas transport properties of a polym membrane. Polymer 45, 5671–5681 (2004).i 9. Clarizia, G., Algieri, C. & Driolia, E. Filler-polymer combination: a route to modify gas transport properties of a polymeric membrane. Polymer 45, 5671–5681 (2004).i y 10. Ismail, A. F., Kusworo, T. D. & Mustafa, A. Enhanced gas permeation performance of polyethersulfone mixed matrix hollow fiber membranes using novel Dynasylan Ameo silane agent. J. Membr. Sci. 319, 306–312 (2008). 10. Ismail, A. F., Kusworo, T. D. & Mustafa, A. Enhanced gas permeation performance of polyethersulfone mixed matrix hollow fiber membranes using novel Dynasylan Ameo silane agent. J. Membr. Sci. 319, 306–312 (2008). g y y g 11. Zhang, Y., Musselman, I. H., Ferraris, J. P. & Balkus, Jr. K. J. Gas permeability properties of mixed-matrix Matrimid membranes containing a carbon aerogel: A material with both micropores and mesopores. Ind. Eng. Chem. Res. 47, 2794–2802 (2008). 12 Perez E V Balkus Jr K J Ferraris J P & Musselman I H Mixed matrix membranes containing MOF 5 for gas separations 11. Zhang, Y., Musselman, I. H., Ferraris, J. P. & Balkus, Jr. K. J. References Polymer nanosieve membranes for CO2-capture applications. N 21. Galve, A. et al. Combination of ordered mesoporous silica MCM-41 and layered titanosilicate JDF-L1 fillers for 6FDA-based copolyimide mixed matrix membranes. J. Membr. Sci. 431, 163–170 (2013). p y 2. Galve, A. et al. Copolyimide mixed matrix membranes with oriented microporous titanosilicate JDF-L1 sheet particles. J. Membr Sci. 370, 131–140 (2011). 3. Aroon, M. A., Ismail, A. F., Matsuura, T. & Montazer-Rahmati, M. M. Performance studies of mixed matrix membranes for gas separation: A review. Sep. Purif. Technol. 75, 229–242 (2010).̀ p p f 4. Bertelle, S., Gupta, T., Roizard, D., Vallières, C. & Favre, E. Study of polymer-carbon mixed matrix membranes for CO2 separation from flue gas. Desalination 199, 401–402 (2006). l g 5. Checchetto, R., Miotello, A., Nicolais, L. & Carotenuto, G. Gas transport through nanocomposite membrane composed by polyethylene with dispersed graphite nanoplatelets. J. Membr. Sci. 463, 196–204 (2014). p y y p g p p 26. Leenaerts, O., Partoens, B. & Peeters, F. M. Graphene: A perfect nanoballoon. Appl. Phys. Lett. 93, 193107 (2008). 27. Bunch, J. S. et al. Impermeable atomic membranes from graphene sheets. Nano Lett. 8, 2458–2462 (2008). 28. Zhang, W. D., Shen, L., Phang, I. Y. & Liu, T. Carbon nanotubes reinforced Nylon-6 composite prepared by simple melt- compounding. Macromolecules 37, 256–259 (2004). 29. Paiva, M. C. et al. Mechanical and morphological characterization of polymer-carbon nanocomposites from functionalized carbon nanotubes. Carbon 42, 2849–2854 (2004). 0. Liu, L., Barber, A. H., Nuriel, S. & Wagner, H. D. Mechanical properties of functionalized single-walled carbon-nanotube/poly(viny alcohol) nanocomposites. Adv. Funct. Mater. 15, 975–980 (2005). p 31. Chen, I.-W. P., Jhou, S.-H. S. & Chen, Y.-W. Preparation of high-quality graphene sheets and their applications in highly conductive papers and a high-performance electromechanical actuator. J. Mater. Chem. C. 1, 5970–5975 (2013). g 32. Wang, Y. et al. Raman studies of monolayer graphene: The substrate effect. J. Phys. Chem. C. 112, 10637–10640 (2008). S k h S l S h f h b d h h l d f f l d h d C 33. Stankovich, S. et al. Synthesis of graphene-based nanosheets via chemical reduction of exfoliated graphite oxide. Carb 1558–1565 (2007).f 34. Stankovich, S., Piner, R. D., Nguyen, S. T. & Ruoff, R. S. Synthesis and exfoliation of isocyanate-treated graphene oxide nanoplatelets. Carbon 44, 3342–3347 (2006). 5. Fernández-Merino, M. J. et al. References Vitamin C is an ideal substitute for hydrazine in the reduction of graphene oxide suspensions. J. Phys Chem. C. 114, 6426–6432 (2010).it 6. Yang, D. et al. Chemical analysis of graphene oxide films after heat and chemical treatments by X-ray photoelectron and Micro 36. Yang, D. et al. Chemical analysis of graphene oxide films after heat and chemical treatments by X-ray photoelectron and Micro- Raman spectroscopy Carbon 47, 145–152 (2009) 6. Yang, D. et al. Chemical analysis of graphene oxide films after heat and chemical treatments by X ray photoelectron and Micro Raman spectroscopy. Carbon 47, 145–152 (2009). g y g pi Raman spectroscopy. Carbon 47, 145–152 (2009). p py 37. Dervishi, E. et al. Large-scale graphene production by RF-cCVD method. Chem. Commun. 27, 4061–4063 (2009). 38 B L l T d i ff h l i f h l ll l i l d 38. Bruno, L. et al. Temperature and time effects on the structural properties of a non-aqueous ethyl cellulose topical drug del system. Carbohyd. Polym. 86, 644–651 (2011). 39. Bruno, L., Kasapis, S. & Heng, P. W. S. Effect of hydration on the structure of non-aqueous ethyl cellulose/propylene g dicaprylate gels. Int. J. Biol. Macromol. 50, 385–392 (2012).i dicaprylate gels. Int. J. Biol. Macromol. 50, 385–392 (2012). 40. Xu, J.-Z., Zhong. G.-J., Hsiao, B. S., Fu, Q. & Li, Z.-M. Low-dimensional carbonaceous nanofiller induced polymer crystallization. Prog. Polym. Sci. 39, 555–593 (2014). p y g 0. Xu, J.-Z., Zhong. G.-J., Hsiao, B. S., Fu, Q. & Li, Z.-M. Low-dimensional carbonaceous nanofiller induced polymer crystallization Prog. Polym. Sci. 39, 555–593 (2014). g y ( ) 41. Ismail, A. F. et al. Gas separation performance of polyethersulfone/ multi-walled carbon nanotubes mixed matrix membranes. Sep. Purif. Technol. 80, 20–31 (2011). l f l h b d l lf b h h d f f 2. Wu, H., Tang, B. & Wu, P. Development of novel SiO2-GO nanohybrid/ polysulfone membrane with enhanced performance J. Membr. Sci. 45, 194–102 (2014). 3. May, P., Khan, U., O’Neill, A. & Coleman, J. N. Approaching the theoretical limit for reinforcing polymers with graphene. J. Mater Chem. 22, 1278–1282 (2012). ( ) 44. Cheng, S., Chen, X., Hsuan, Y. G. & Li, C. Y. Reduced graphene oxide induced polyethylene crystallization in solution and composites. Macromolecules 45, 993–1000 (2011). p 5. Xu, J.-Z. et al. Graphene oxide nanosheet induced intrachain conformational ordering in a semicrystalline polymer. References Gas permeability properties of mixed-matrix Matrimid membranes containing a carbon aerogel: A material with both micropores and mesopores. Ind. Eng. Chem. Res. 47, 2794–2802 (2008). g g p p g , ( ) 2. Perez, E. V., Balkus, Jr. K. J., Ferraris, J. P. & Musselman, I. H. Mixed-matrix membranes containing MOF-5 for gas separations J. Membr. Sci. 328, 165–173 (2009). J , ( ) 13. Zhang, C., Dai, Y., Johnson, J. R., Karvan, O. & Koros, W. J. High performance ZIF-8/6FDA-DAM mixed matrix membrane for propylene/propane separation. J. Membr. Sci. 389, 34–42 (2012). ( ) 13. Zhang, C., Dai, Y., Johnson, J. R., Karvan, O. & Koros, W. J. High performance ZIF-8/6FDA-DAM mixed matrix membrane for propylene/propane separation. J. Membr. Sci. 389, 34–42 (2012). 3. Zhang, C., Dai, Y., Johnson, J. R., Karvan, O. & Koros, W. J. High performance ZIF-8/6FDA-DAM mixed matrix membrane fo propylene/propane separation. J. Membr. Sci. 389, 34–42 (2012). Scientific Reports \| 6:28509 \| DOI: 10.1038/srep28509 9 www.nature.com/scientificreports/ 14. Naghsh, M., Sadeghi, M., Moheb, A., Chenar, M. P. & Mohagheghian, M. Separation of ethylene/ethane and propylene/propan cellulose acetate-silica nanocomposite membranes. J. Membr. Sci. 423–424, 97–106 (2012).i 15. Sun, H. et al. Preparation and characterization of C60-filled ethyl cellulose mixed-matrix membranes for gas separation of propy propane. Chem. Eng. Technol. 37, 611–619 (2014). p p g , ( ) 16. Chung, T.-S., Jiang, L. Y., Li, Y. & Kulprathipanja, S. Mixed matrix membranes (MMMs) comprising organic polymers with dispersed inorganic fillers for gas separation. Prog. Polym. Sci. 32, 483–507 (2007). p gi g p g y Bai, H., Li, C., Wang, X. & Shi, G. A pH-sensitive graphene oxide co g p g p p y g 18. Li, H. et al. Ultrathin, molecular-sieving graphene oxide membranes for selective hydrogen separation. Science 342, 95–98 (20 g p g p p y g 18. Li, H. et al. Ultrathin, molecular-sieving graphene oxide membranes for selective hydrogen 19. Zulhairun, A. K. & Ismail, A. F. The role of layered silicate loadings and their dispersion states on the gas separation performance of mixed matrix membrane. J. Membr. Sci. 468, 20–30 (2014). 19. Zulhairun, A. K. & Ismail, A. F. The role of layered silicate loadings and their dispersion states on the gas separatio mixed matrix membrane. J. Membr. Sci. 468, 20–30 (2014). 20. Du, N. et al. Polymer nanosieve membranes for CO2-capture applications. Nat. Mater. 10, 372–375 (2011).i al. Acknowledgementsh g The authors gratefully acknowledge the financial support from the National Natural Science Foundation of China (No. 21406268), the Shandong Provincial Natural Science Foundation (No. ZR2014BM005) and the Fundamental Research Funds for the Central Universities (No. 14CX05034A). Author Contributions H.S. and B.Y. designed the research plan and synthesized the nanoporous graphene nanosheets using the facile hydrothermal treatment. T.W. and Y.X. conducted the fabrication of MMMs and C3H6/C3H8 separation experiments. Q.J.N., P.L. and Y.K. performed the characterization of graphene nanosheets and MMMs. H.S. and B.Y. analyzed the data and interpreted the results. B.Y. and H.S. drafted the manuscript, and all authors revised the manuscript. References Scientific Reports \| 6:28509 \| DOI: 10.1038/srep28509 10 www.nature.com/scientificreports/ Scientific Reports \| 6:28509 \| DOI: 10.1038/srep28509 References J. Phys. Chem Lett. 3, 530–535 (2012).fiiii 46. Wang, M.-J. Effect of polymer-filler and filler-filler interactions on dynamic properties of filled vulcanizates. Rubber. Chem. Technol. 71, 520–589 (1998). 47. Zhao, Q. et al. Bio-inspired polyelectrolyte complex/graphene oxide nanocomposite membranes with enhanced tensile strength and ultra-low gas permeability. Polym. Chem. 4, 4298–4302 (2013). g p y y 8. Tsai, M.-H., Tseng, I.-H., Liao, Y.-F. & Chiang, J.-C. Transparent polyimide nanocomposites with improved moisture barrier using graphene. Polym. Int. 62, 1302–1309 (2013). g y 49. Scherillo, G. et al. Tailoring assembly of reduced graphene oxide nanosheets to control gas barrier properties of natural rubber nanocomposites. L. ACS Appl. Mater. Interfaces 6, 2230–2234 (2014). 50. Koolivand, H. et al. Functionalized graphene oxide/polyimide nanocomposites as highly CO2-selective membranes. J. Polym. Res. 21, 599 (2014). ( ) 1. Sridhar, S. & Khan, A. A. Simulation studies for the separation of propylene and propane by ethyl cellulose membrane. J. Membr. Sci 159, 209–219 (1999). 52. Lin, H., Van Wagner, E., Raharjo, R., Freeman, B. D. & Roman, I. High-performance polymer membranes for natural-gas sweetening. Adv. Mater. (Weinheim, Ger.) 18, 39–44 (2006). 53. Du, N., Park, H. B., Dal-Cin, M. M. & Guiver, M. D. Advances in high permeability polymeric membrane materials for CO2 separations. Energy Environ. Sci. 5, 7306–7322 (2012). p gy 54. Lin, H. et al. Dehydration of natural gas using membranes. Part I: Composite membranes. J. Membr. Sci. 70–81, 413–414 (201 p gy 4. Lin, H. et al. Dehydration of natural gas using membranes. Part I: Composite membranes. J. Membr. Sci. 70–81, 413–414 (2012). l k f d l d bb l h l 4. Lin, H. et al. Dehydration of natural gas using membranes. Part I: Composite membranes. J. Membr. Sci. 70 81, 413 414 (2012). 5. Matteucci, S., Yampolskii, Y., Freeman, B. D. & Pinnau, I. Transport of gases and vapors in glassy and rubbery polymers. John Wiley & Sons: New York (2006). y g g p 55. Matteucci, S., Yampolskii, Y., Freeman, B. D. & Pinnau, I. Transport of gases and vapors in glassy and rubbery polymers. John Wiley & Sons: New York (2006). 6. Sadeghia, M. et al. Gas permeation properties of ethylene vinyl acetate-silica nanocomposite membranes. J. Membr. Sci. 322 423–428 (2008). 56. Sadeghia, M. et al. Gas permeation properties of ethylene vinyl acetate-silica nanocomposite membranes. J. Membr. Sci. 423–428 (2008). Additional Information Supplementary information accompanies this paper at http://www.nature.com/srepi Competing financial interests: The authors declare no competing financial interests. How to cite this article: Yuan, B. et al. Propylene/propane permeation properties of ethyl cellulose (EC) mixed matrix membranes fabricated by incorporation of nanoporous graphene nanosheets. Sci. Rep. 6, 28509; doi: 10 1038/srep28509 (2016) How to cite this article: Yuan, B. et al. Propylene/propane permeation properties of ethyl cellulose (EC) mixed matrix membranes fabricated by incorporation of nanoporous graphene nanosheets. Sci. Rep. 6, 28509; doi: 10.1038/srep28509 (2016). This work is licensed under a Creative Commons Attribution 4.0 International License. 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https://openalex.org/W2145739904	https://europepmc.org/articles/pmc4071640?pdf=render	English	null	Slow gait speed â€“ an indicator of lower cerebral vasoreactivity in type 2 diabetes mellitus	Frontiers in aging neuroscience	2,014	cc-by	8,073	ORIGINAL RESEARCH ARTICLE published: 26 June 2014 Keywords: diabetes, gait, vasoreactivity, vasomotor, metabolic INTRODUCTION Reviewed by: Reviewed by: Richard Camicioli, McGill University, Canada Research design and methods: We studied 61 adults with diabetes (65 ± 8 years) and 67 without diabetes (67 ± 9 years) but with similar distribution of cardiovascular risk factors. Preferred gait speed was calculated from a 75 m walk. Global and regional perfusion, vasoreactivity and vasodilation reserve were measured using 3-D continuous arterial spin labeling MRI at 3 Tesla during normo-, hyper- and hypocapnia and normalized for end-tidal CO2. Franziska Matthäus, University of Heidelberg, Germany Correspondence: Azizah J. Jor’dan, Syncope and Falls in the Elderly Laboratory, Division of Gerontology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, 185 Pilgrim Road, Palmer 117, Boston, MA 02215, USA e-mail: ajjordan@bidmc.harvard.edu Results: Diabetic participants had slower gait speed as compared to non-diabetic par- ticipants (1.05 ± 0.15 m/s vs. 1.14 ± 0.14 m/s, p < 0.001). Lower global vasoreactivity (r 2adj = 0.13, p = 0.007), or lower global vasodilation reserve (r 2adj = 0.33, p < 0.001), was associated with slower walking in the diabetic group independently of age, BMI and hematocrit concentration. For every 1 mL/100 g/min/mmHg less vasodilation reserve, for example, gait speed was 0.05 m/s slower. Similar relationships between vasodilation reserve and gait speed were also observed regionally within the cerebellum, frontal, tempo- ral, parietal, and occipital lobes (r 2adj = 0.27–0.33, p < 0.0001). In contrast, vasoreactivity outcomes were not associated with walking speed in non-diabetic participants, despite similar vasoreactivity ranges across groups. Conclusion: In the diabetic group only, lower global vasoreactivity was associated with slower walking speed. Slower walking in older diabetic adults may thus hallmark reduced vasomotor reserve and thus the inability to increase perfusion in response to greater metabolic demands during walking. Keywords: diabetes, gait, vasoreactivity, vasomotor, metabolic Azizah J. Jor’dan1, Brad Manor 1,2 and Vera Novak 3 1 Syncope and Falls in the Elderly Laboratory, Division of Gerontology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA 2 Institute for Aging Research, Hebrew SeniorLife, Harvard Medical School, Boston, MA, USA 3 Department of Neurology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA 2 Institute for Aging Research, Hebrew SeniorLife, Harvard Medical School, Boston, MA, USA 3 Department of Neurology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA Objective: Gait speed is an important predictor of health that is negatively affected by aging and type 2 diabetes. Diabetes has been linked to reduced vasoreactivity, i.e., the capacity to regulate cerebral blood ﬂow in response to CO2 challenges. This study aimed to determine the relationship between cerebral vasoreactivity and gait speed in older adults with and without diabetes. Edited by: Philip P. Foster, The University of Texas Health Science Center at Houston, USA Reviewed by: Richard Camicioli, McGill University, Canada Franziska Matthäus, University of Heidelberg, Germany Correspondence: Azizah J. Jor’dan, Syncope and Falls in the Elderly Laboratory, Division of Gerontology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, 185 Pilgrim Road, Palmer 117, Boston, MA 02215, USA e-mail: ajjordan@bidmc.harvard.edu Philip P. Foster, The University of Texas Health Science Center at Houston, USA Philip P. Foster, The University of Texas Health Science Center at Houston, USA Frontiers in Aging Neuroscience INTRODUCTION Slowing of gait may thus reﬂect an early manifestation of underly- ing abnormalities in vasoreactivity and perfusion adaptation to the metabolic demands of walking. However, the relationship between brain vascular health and walking has not yet been established. Gait speed is predictive of mobility, morbidity, and mortality in older adults (Guralnik et al., 1995; Studenski et al., 2011). Vasore- activity is an important cerebrovascular control mechanism used to maintain brain perfusion during increased metabolic demands (Bullock et al., 1985; Schroeder, 1988) such as walking, and can be clinically quantiﬁed by the vasodilation responses to hypercap- nia (Low et al., 1999; Lavi et al., 2006). In healthy older adults, blood ﬂow velocities in the middle cerebral artery territory, which supplies numerous brain regions involved in locomotor control, increased proportionally to walking speed (Novak et al., 2007). In a population-based study comprising community-dwelling older adults both with and without risk factors for falls (e.g., diabetes, stroke, use of walking aids, etc.), slower walkers exhibited lower vasoreactivity within the middle cerebral artery territory as mea- sured by Transcranial Doppler ultrasound (Sorond et al., 2010). Type 2 diabetes accelerates brain aging (Biessels et al., 2002; Last et al., 2007) and has also been linked with microvascular dis- ease and altered cerebral blood ﬂow regulation (Allet et al., 2008; Várkuti et al., 2011) and vasoreactivity (Novak et al., 2011). Dia- betes is associated with reduced gait speed and related functional decline (Volpato et al., 2010). In older adults, gait characteristics have been linked to gray matter atrophy and white matter hyperin- tensities (Rosano et al., 2007a,b; Callisaya et al., 2013). Moreover, gray matter atrophy appears to have a stronger effect on locomotor control in those with type 2 diabetes as compared those without, suggesting that the control of walking may be more dependent upon supraspinal control within this population (Manor et al., June 2014 \| Volume 6 \| Article 135 \| 1 Frontiers in Aging Neuroscience www.frontiersin.org www.frontiersin.org Diabetes, vasoreactivity, and gait speed Jor’dan et al. drug or alcohol abuse. MRI exclusion criteria were incompatible metal implants, pacemakers, arterial stents, claustrophobia and morbid obesity (i.e., BMI > 40). 2012). This study therefore aimed to determine the relationship between vasoreactivity and gait speed in older adults with and without type 2 diabetes. PARTICIPANTS This secondary analysis was completed on prospectively col- lected data from community-dwelling older adults originally recruited via local advertisement. We analyzed records from three completed projects spanning March 2003–July 2012: Cerebral vasoregulation in the elderly with stroke (March 2003–April 2005); Cerebral perfusion and cognitive decline in type 2 diabetes (Jan- uary 2006–December 2009); and Cerebromicrovascular disease in elderly with diabetes (August 2009–July 2012). Grant numbers are provided in the study funding section. INTRODUCTION We hypothesized that lower global and regional vasoreactivity would be associated with slower gait speed in older adults, particularly in those with type 2 diabetes. PROTOCOL Participants completed medical history, autonomic symptoms, and physical activity questionnaires. A study physician completed physical, neurological, and ophthalmologic examinations. None of the study participants had active foot ulcers during the study. A study nurse completed a fasting blood draw and recorded vital signs, anthropometric and adiposity measures. Participants also completed a comprehensive cognitive exam, autonomic testing, perfusion MRI of the brain and a gait assessment. For this study, we focused analyses on gait and MRI-based measures of cerebral perfusion and vasoreactivity. Magnetic resonance imaging (MRI) Brain imaging was completed in a 3T GE HDx MRI scan- ner (GE Medical Systems, Milwaukee, WI, USA) within the Center for Advanced MR Imaging at the BIDMC. 3D spi- ral continuous arterial spin labeling (CASL) MRI was used to quantify cerebral perfusion (Alsop and Detre, 1998; Detre et al., 1998; Floyd et al., 2003) during normocapnia, hypocap- nia, and hypercapnia. Vasoreactivity was assessed as perfusion responses to vasodilation during hypercapnia and vasocon- striction to hypocapnia (Kety and Schmidt, 1948), as a non- invasive reliable method of assessing the integrity of cerebral vasculature (Fujishima et al., 1971; Yen et al., 2002). Speciﬁ- cally, two-minute scans were acquired during normal breath- ing (i.e., baseline normocapnia; end tidal CO2 concentration 33–38 mmHg), hyperventilation (i.e., hypocapnia; participants hyperventilated to reduce CO2 to a target of 25 mmHg), and rebreathing (i.e., hypercapnia; participants breathed a mix- ture of 5% CO2 and 95% air to increase CO2 to a target of 45 mmHg). Participants were originally screened by medical history and physical, neurological, and laboratory examinations. Research protocols were conducted in accordance with the ethical standards of the Beth Israel Deaconess Medical Center (BIDMC) Clinical Research Center and all participants signed an informed consent, as approved by the Institutional Review board at BIDMC. The diabetic group included men and women aged 50–85 years with a physician diagnosis and treatment of type 2 diabetes mel- litus with oral agents and/or combinations with insulin for at least one year. Diabetes treatments included insulin, oral glucose- control agents (sulfonylurea, second generation agents), their combinations and diet. Non-diabetic participants had no history of metabolic disorder and were recruited to match the age and gender characteristics of the diabetic group (Table 1). Exclusion criteria for the current analysis were history of stroke, myocardial infarction, clinically signiﬁcant arrhythmia or other cardiac disease, nephropathy, severe hypertension (i.e., systolic BP > 200, diastolic BP > 110 mm Hg or the use of three or more antihypertensive medications), seizure disorder, kidney or liver transplant, renal disease, any other neurological or systemic disor- der (aside from peripheral neuropathy), and current recreational Respiratory rate, tidal volume and end-tidal CO2 values were measured during each scan using an infrared end-tidal volume gas monitor (Capnomac Ultima, General Electric, Fairﬁeld, CT, USA) attached to a face-mask. Blood pressure and heart rate were also recorded at one-minute intervals using an upper-arm automatic blood pressure cuff and ﬁnger photoplethysmogram. Frontiers in Aging Neuroscience Walking test i A 12-min walk was completed along a 75 m course on an 80 m × 4 m indoor hallway. Participants were instructed to walk at preferred speed (i.e., a pace they deemed as comfortable or nor- mal), which has excellent test–retest reliability, even in those with severe diabetic complications (Steffen et al., 2002; Manor et al., 2008). The time taken to complete each 75 m length and total distance were recorded. For the present analysis, we only exam- ined data from the ﬁrst hallway length (i.e., the ﬁrst 75 m of the trial) in order to minimize potential confounders of turning and fatigue. Assistive devices were not used for ambulation. A rat- ing of perceived exertion was asked of the participant before the start of the walk and once the walk was completed. Rating of per- ceived exertion ranged from 0 (no exertion) to 10 (very, very strong exertion). Collectively, these three studies recruited 447 participants who signed informed consent (212 non-diabetics, 151 diabetics, 84 stroke). 213 participants (103 non-diabetics, 69 diabetics, 41 stroke) were excluded at that time for the following reasons: (1) ineligible after the screening visit (n = 117); (2) withdrew consent (n = 31); (3) lost to follow-up (n = 13); (4) study termi- nated (n = 52) for reasons related to exclusion criteria or other reasons such as lack of permission from primary care provider, no transcranial Doppler insonation window, unstable/untreated hypertension, high BMI, cerebral palsy, claustrophobia, atrial ﬁb- rillation, inappropriate behavior during screening, metal implant, abdominal pain due to kidney stone, or entered a nursing home. For the present analysis, we excluded an additional 43 stroke records that met the exclusion criteria for the current analyses, 34 records that did not have complete datasets, and 29 records from subjects who completed more than one of the above-mentioned studies. In each of the latter cases, the most recent record was kept. Thus, records from a total of 128 subjects were included in the present analysis. Magnetic resonance imaging (MRI) June 2014 \| Volume 6 \| Article 135 \| 2 www.frontiersin.org www.frontiersin.org www.frontiersin.org Diabetes, vasoreactivity, and gait speed Jor’dan et al. Table 1 \| Demographic characteristics of the non-diabetic and diabetic groups. Non-diabetic group Diabetic group p N 67 61 Age (years) 67 ± 9 65 ± 8 NS Sex (women, %) 59 49 NS Body Mass Index (kg/m2) 25.6 ± 4 29.1 ± 5 < 0.0001 Mini-Mental State Exam (1–30) 28.2 ± 1.8 28.2 ± 1.8 NS Diabetes duration (years) – 12.7 ± 9 – Systolic blood pressure (mmHg) 130.6 ± 10.8 133.5 ± 8.5 NS Diastolic blood pressure (mmHg) 68.5 ± 8.4 71 ± 7.6 NS Hypertension (yes/no) 20/47 38/23 0.0003 Peripheral neuropathy (%) 18 51 0.001 Hyperlipidemia (yes/no) 7/60 34/27 < 0.0001 Gait speed (m/s) 1.14 ± 0.14 1.05 ± 0.15 0.0004 Rating of perceived exertion (1–10) 1.49 ± 1.43 2.17 ± 2.13 0.0386 Global gray matter (cm3) 639 ± 82 620 ± 62 NS Global white matter (cm3) 436 ± 56 424 ± 52 NS Global white matter hyperintensities (cm3) 11 ± 7 13 ± 7 NS Global vasoreactivity (mL/100g/min/mmHg) 0.98 ± 0.09 1.10 ± 0.09 NS Global vasodilation reserve (mL/100g/min/mmHg) 0.35 ± 1.7 0.44 ± 1.7 NS Global vasoconstriction reserve (mL/100g/min/mmHg) 1.5 ± 3.2 1.4 ± 2.5 NS Hemoglobin A1c (%) 5.7 ± 0.3 7.3 ± 1.3 < 0.0001 Hematocrit (%) 40.4 ± 3.7 39.3 ± 3.7 NS Fasting glucose (mg/dL) 84.7 ± 12.3 121.7 ± 43.1 < 0.0001 Total cholesterol (mg/dl) 194 ± 36 166 ± 38.8 < 0.0001 Cholesterol-to-HDL ratio 3.4 ± 0.9 3.4 ± 1.2 NS Triglycerides (mg/dl) 130.2 ± 70 146 ± 94.6 NS Data = means ± SD unless otherwise indicated. p = between-group comparisons. NS = non-signiﬁcant. Table 1 \| Demographic characteristics of the non-diabetic and diabetic groups. N Data = means ± SD unless otherwise indicated. p = between-group comparisons. NS = non-signiﬁcant. Perfusion images were acquired using a custom 3D CASL sequence (TR/TE = 10.476/2.46 ms, Label duration = 1.45 s, post- label delay = 1.525 s, with 64 × 64 matrix in the axial plane and 40 slices with thickness = 4.5 mm, seven spiral interleaves and the bandwidth = 125 kHz). Images were averaged over each condition to maximize signal-to-noise ratio. using the Statistical Parametric Mapping software package (SPM, Wellcome Department of Imaging Neuroscience, University Col- lege, London, UK). Perfusion analyses P f i d Perfusion and vasoreactivity were calculated in ﬁve regions- of-interest: the cerebellum, frontal, temporal, parietal, and occipital lobe. Within each region, perfusion was normal- ized for tissue volume and thus expressed in mL/100 g/min. Four perfusion measures were calculated for each region: base- line perfusion during normal breathing, cerebral vasoreactivity, Gait speed Averagegaitspeed(m/s)wascomputedfromtheﬁrst75mof walk- ing by dividing distance by time. This valid and reliable outcome predicts future health status and functional decline in numerous older adult populations (Quach et al.,2011; Studenski et al.,2011). Frontiers in Aging Neuroscience Magnetic resonance imaging (MRI) This “normalization” module was employed to stereotactically normalize structural images to a standard space deﬁned by ideal template image(s). The registered perfusion image was then overlaid on the segmented anatomical regions to obtain regional perfusion measurements. Generated maps of gray mat- ter and white matter were segmented based upon the LONI Probabilistic Brain Atlas (Shattuck et al., 2008) and was used to calculate global volumes. All image segmentations were completed using Interactive Data Language (IDL, Research Systems, Boul- der, CO, USA) and MATLAB (MathWorks, Natick, MA, USA) software. A T1-weighted MP-RAGE structural imaging sequence was completed and used for registration of CASL images. Imaging parameters were: TE/TR = 3.3/8.1 ms, ﬂip angle of 10◦, 1–3 mm slice thickness, 24 cm × 19 cm ﬁeld of view (FOV), 256 × 192 matrix size. THE EFFECTS OF DIABETES ON GAIT SPEED The diabetic group had slower preferred gait speed as compared to the non-diabetic group (1.05 ± 0.15 m/s vs. 1.14 ± 0.14 m/s, p < 0.001; Table 1). This group difference remained signiﬁcant (p = 0.007) after adjusting for age, gender, and BMI. We examined the effects of diabetes on both perfusion measures and gait speed usingANCOVA. For perfusion measures, the model effect was group and covariates included age, hematocrit (Hct) concentration and hypertension. Hct was included because it is inversely correlated with blood viscosity and is higher in men than women (Wells and Merrill,1962; Kameneva et al.,1999; Zeng et al., 2000). Hypertension was included as a covariate because it affects small blood vessels of the body and may therefore alter cerebral blood ﬂow regulation (Alexander, 1995; Hajjar et al., 2010). For gait speed, the model effect was group and covariates included age, gender and BMI. Across all participants, those with higher BMI had slower gait speed (r2adj = 0.04, p = 0.01). Speciﬁcally, within the diabetic group, those with higher fasting glucose had slower gait speed (r2adj = 0.13, p = 0.003). Gait speed was not correlated with the participant’s rating of perceived exertion, HbA1c levels or diabetes diagnosis duration. The diabetic group had a higher change in rating of perceived exertion (i.e., difference from the start of walk from the end of the walk) compared to the non-diabetic group (2.17 ± 2.13 vs. 1.49 ± 1.43, p = 0.039). Linear least-square regression analyses were used to test the hypotheses that (1) those with lower vasoreactivity demon- strate slower preferred gait speed, and (2) this association between vasoreactivity and gait speed is stronger (as reﬂected in the correlation coefﬁcient, r2adj) in older adults with dia- betes as compared to those without diabetes. The dependent variable was gait speed. Model effects included perfusion out- come, group (non-diabetic, diabetic), and their interaction. Separate models were performed for each global and regional perfusion and vasoreactivity outcome. Age, BMI, and Hct con- centration were included as covariates. Signiﬁcance level was set to p = 0.05 for each global perfusion and vasoreactivity outcome. The Bonferroni-adjusted signiﬁcance level for multi- ple comparisons (p = 0.01) was used to determine signiﬁcance of models examining outcomes within each of the ﬁve brain regions-of-interest. Image analysis A rigid-body model (Collignon et al., 1995; Wells et al., 1996) was used for registration of the MP-RAGE image on CASL images June 2014 \| Volume 6 \| Article 135 \| 3 Frontiers in Aging Neuroscience www.frontiersin.org www.frontiersin.org www.frontiersin.org Diabetes, vasoreactivity, and gait speed Jor’dan et al. vasodilation reserve, and vasoconstriction reserve. Each out- come was computed globally and within each brain region-of- interest. (p < 0.0001). The prevalence of hypertension and peripheral neu- ropathy was also higher in the diabetic group as compared to the non-diabetic group (62% vs. 30%, p < 0.001 and 51% vs. 18%, p < 0.001, respectively). Participants with diabetes had greater HbA1c and serum glucose levels, but lower total cholesterol as compared to the non-diabetic group. Blood Hct concentration was similar between groups, but overall, higher in males as com- pared to females (42% vs. 38%, p < 0.001). Groups did not differ in global gray matter, white matter or white matter hyperintensity volumes (see Table 1). Perfusion values were normalized to each subject’s average CO2 level during this condition. Vasoreactivity measures were calculated as previously described (Last et al., 2007; Hajjar et al., 2010; Novak et al., 2011). Brieﬂy, vasoreactivity was deﬁned as the slope of the best-ﬁt line produced by linear regression of per- fusion and CO2 values across the three conditions (i.e., normal breathing, CO2 rebreathing, and hyperventilation). Vasodilation reservewasdeﬁnedastheincreaseinperfusionfrombaselinetothe rebreathing condition, normalized to the change in CO2 between these two conditions. Vasoconstriction reserve was deﬁned as the decrease in perfusion from baseline to the hyperventilation condition, normalized to the change in CO2 between these two conditions. Baseline perfusion and cerebral vasoreactivity The diabetic and non-diabetic groups had similar global and regional perfusion at baseline after normalizing for baseline CO2 levels and adjusting for age, Hct concentration and the presence of hypertension. Global and regional vasoreactivity, as well as vasodi- lation and vasoconstriction reserve, were also similar between groups (Table 1). STATISTICAL ANALYSIS All analyses were performed using JMP software (SAS Institute, Cary, NC, USA). Descriptive statistics were used to summa- rize all variables. Outcomes have been expressed as either the mean ± SD or categorical (yes/no) for each group. Student’s t, Fisher’s Exact and Chi-squared tests were used to compare group demographics. Cerebral vasoreactivity d l Least square models revealed that global vasoreactivity was related to gait speed, but that this relationship was dependent upon group (F1,96 = 5.48, p = 0.024). This group by vasoreactivity inter- action was independent of age, BMI, and Hct levels. Post hoc testing indicated that within the diabetic group, those with lower global vasoreactivity walked more slowly (r2adj = 0.13, p = 0.007; Figures 1A,B). In the non-diabetic group, however, global vasore- activity was not correlated with gait speed (Figure 1C). A trend towards a similar interaction was also observed between frontal lobe vasoreactivity and group (F1,95 = 4.32, p = 0.04); that is, in thediabeticgrouponly,thosewithlowerfrontallobevasoreactivity tended to walk slower (r2adj = 0.13, p = 0.007). Yet, this inter- action was not signiﬁcant based upon the Bonferroni-adjusted signiﬁcance level (p = 0.01). Vasodilation reserve Groups were matched by age and gender and had a similar cardiovascular risk factors (e.g., blood pressure, triglycerides, car- diovascular disease history), yet the diabetic group had higher BMI Least square models revealed a signiﬁcant relationship between global vasodilation reserve and gait speed, but that this relationship was also dependent upon group (F1,97 = 12, June 2014 \| Volume 6 \| Article 135 \| 4 Frontiers in Aging Neuroscience Frontiers in Aging Neuroscience www.frontiersin.org www.frontiersin.org Diabetes, vasoreactivity, and gait speed Jor’dan et al. FIGURE 1 \| (A) Reconstructed anatomical (i.e., MP-RAGE) and perfusion maps for two participants with type 2 diabetes mellitus. The top row represents a participant with diabetes that has high global vasoreactivity and fast gait speed (see Diabetic Participant 1 in A). The bottom row represents a participant with diabetes that has low global vasoreactivity and slow gait speed (see Diabetic Participant 2 in A). (B) The relationship between global vasoreactivity and gait speed in the diabetic group. (C) The relationship between global vasoreactivity and gait speed in the non-diabetic group. Vasoreactivity was calculated as the change in perfusion from hypocapnia (hyperventilation) to hypercapnia (CO2 rebreathing) conditions, normalized to the change in CO2 values. Best ﬁt – red solid line; Conﬁdence Intervals – red dotted lines; Gait speed mean – blue dotted line; unit – mL/100g/min/mmHg. p < 0.001). This signiﬁcant interaction between group and vasodilation reserve was independent of age, BMI, and Hct levels. Post-hoc testing revealed that within the diabetic group only, those with lower global vasodilation reserve walked more slowly (r2adj = 0.33, p < 0.0001; Figure 2A). Similar interactions were present between group and vasodi- lation reserve within each brain region-of-interest (cerebellum: F1,94 = 13, p < 0.001; frontal lobe: F1,96 = 8.49, p = 0.005; tem- poral lobe: F1,96 = 17.1, p < 0.001; parietal lobe: F1,95 = 8.72, p = 0.004; occipital lobe: F1,95 = 8.99, p = 0.004). In each case, within the diabetic group only, those with lower vasodi- lation reserve walked slower (Least square: r2adj = 0.27–0.33, p ≤0.001; Table 2). In the non-diabetic group, neither global nor regional vasodilation reserve was correlated with gait speed (Figure 2B). FIGURE 2 \| Relationship between global vasodilation reserve and gait speed. (A) Diabetic group (B) Non-diabetic group. Best ﬁt – red solid line; Conﬁdence bounds – red dotted lines; Gait speed mean – blue dotted line. Vasoconstriction reserve Global and regional vasoconstriction was not related to gait speed in either group. Global and regional vasoconstriction was not related to gait speed in either group. Baseline perfusion Global or regional baseline perfusion was not related to gait speed within either group. Previous research in older adults has linked slow gait speed to impaired “neurovascular coupling,” or the change in cerebral blood ﬂow in response to the performance of a cognitive task (Girouard and Iadecola, 2006; Iadecola and Nedergaard, 2007; Sorond et al., 2011). For example, Sorond et al. (2011) investigated the association between gait speed and neurovascular coupling as quantiﬁed by the change in blood ﬂow velocity within the mid- dle cerebral artery (using Transcranial Doppler Ultrasonography) in response to performance of the n-back cognitive task. Those with impaired neurovascular coupling walked more slowly. They also reported an interaction between neurovascular coupling and white matter hyperintensity burden, such that the presence of white matter hyperintensities was associated with reduced gait speed, except in those individuals with relatively strong neurovas- cular coupling. Previous work by Novak et al. (2007, 2011) further demonstrated that lower vasoreactivity is linked to reduced gait speed independently of white matter hyperintensities speciﬁcally within older adults with type 2 diabetes. Therefore, neurovascular coupling appears to one mechanism that links vascular changes to neuronal activity, and is therefore essential for the preserva- tion of functional outcomes. This notion is in line with the “brain reserve” hypothesis (Bullock et al., 1985; Stern, 2002) and may help explain the results of the current study. In other words, while diabetes was associated with reduced gait speed overall, those diabetic participants with greater vasoreactivity (or vasodi- lation reserve) tended to walk at similar speeds as non-diabetic controls. Vasodilation reserve FIGURE 1 \| (A) Reconstructed anatomical (i.e., MP-RAGE) and perfusion maps for two participants with type 2 diabetes mellitus. The top row represents a participant with diabetes that has high global vasoreactivity and fast gait speed (see Diabetic Participant 1 in A). The bottom row represents a participant with diabetes that has low global vasoreactivity and slow gait speed (see Diabetic Participant 2 in A). (B) The relationship between global vasoreactivity and gait speed in the diabetic group. (C) The relationship between global vasoreactivity and gait speed in the non-diabetic group. Vasoreactivity was calculated as the change in perfusion from hypocapnia (hyperventilation) to hypercapnia (CO2 rebreathing) conditions, normalized to the change in CO2 values. Best ﬁt – red solid line; Conﬁdence Intervals – red dotted lines; Gait speed mean – blue dotted line; *unit – mL/100g/min/mmHg. FIGURE 2 \| Relationship between global vasodilation reserve and gait speed. (A) Diabetic group (B) Non-diabetic group. Best ﬁt – red solid line; Conﬁdence bounds – red dotted lines; Gait speed mean – blue dotted line. June 2014 \| Volume 6 \| Article 135 \| 5 Frontiers in Aging Neuroscience www.frontiersin.org Diabetes, vasoreactivity, and gait speed Jor’dan et al. Table 2 \| Vasodilation reserve and gait speed relationship in the diabetic group. M r 2adj p Gait speed (m/s) Vasodilation reserve (mL/100g/min/mmHg) 1.05 ± 0.02 Global 0.42 ± 0.2 0.33 <0.0001 Cerebellum 0.62 ± 0.2 0.33 <0.0001 Frontal 0.31 ± 0.3 0.27 <0.0001 Temporal 0.48 ± 0.2 0.33 <0.0001 Parietal 0.32 ± 0.3 0.30 <0.0001 Occipital 0.42 ± 0.3 0.29 <0.0001 Data are least square means (M) ± SE, r2adj and p value adjusted for age, BMI, and Hct. Table 2 \| Vasodilation reserve and gait speed relationship in the diabetic group. or HbA1c. Furthermore, as can be observed in Figure 2A, sev- eral participants with diabetes that walked the slowest appeared to have abnormal responses to the hypercapnia condition (i.e., no change or decreased perfusion). For these individuals, this response may function as a compensatory response to ensure adequate perfusion even during resting conditions (Novak et al., 2006). Additional covariates Secondary analyses were performed to determine if within the diabetic group, the observed relationships between cerebral blood ﬂow regulation outcomes and gait speed were inﬂuenced by the participant’s height, weight, rating of perceived exertion, the burden of white matter hyperintensities, or the prevalence of hypertension or peripheral neuropathy. In each case, relationships between cerebral blood ﬂow regulation and gait speed remained signiﬁcant after adjusting for potential covariance associated with these factors. Frontiers in Aging Neuroscience June 2014 \| Volume 6 \| Article 135 \| 6 DISCUSSION This study has shown that within the diabetic group, those with lower global vasoreactivity walked more slowly. Our results fur- ther indicate that within this group, vasodilation reserve, or the capacity to increase cerebral perfusion speciﬁcally in response to hypercapnia, was linked to gait speed, which is an over- all measure of health in older adults. This relationship was observed both globally and within each brain region-of-interest (i.e., cerebellum, frontal lobe, temporal lobe, parietal lobe, and occipital lobe). Speciﬁcally, for every 1 mL/100 g/min/mmHg less global vasodilation reserve, gait speed was 0.05 m/s slower in the diabetic group. These relationships were independent of age, BMI, Hct, and additional covariates (i.e., height, weight, rating of perceived exertion, white matter hyperin- tensities, and the prevalence of hypertension or peripheral neuropathy). Walking is a complex act that requires the coordination of locomotor, cardiovascular, and autonomic systems. The lack of relationship between cerebral vasoreactivity and gait speed in those without diabetes is supported by the notion that gait is largely autonomous and governed primarily by supraspinal ele- ments of the motor control system under normal or healthy conditions (Stoffregen et al., 2000; Manor et al., 2010; Kloter et al., 2011). In those with diabetes, however, the capacity to Both groups presented with average walking speeds that were slower than published norms; i.e., 1.2–1.4 m/s for healthy adults over 50 years of age (Bohannon, 1997). Diabetic participants walked 0.09 ± 0.15 m/s more slowly than those without diabetes, which reﬂects a clinically signiﬁcant difference between groups (Kwon et al., 2009). In the diabetic group, walking speed was cor- related with fasting glucose levels, but not with diabetes duration June 2014 \| Volume 6 \| Article 135 \| 6 www.frontiersin.org www.frontiersin.org www.frontiersin.org Diabetes, vasoreactivity, and gait speed Jor’dan et al. while participants were lying supine and not during walking. Although these regional perfusion measures may be lost, future studies are warranted to utilize wireless cerebral blood ﬂow mea- surement tools (e.g., portable TCD or functional near-infrared spectroscopy) to examine the effects of diabetes on cerebral per- fusion when walking at different speeds. Moreover, this is a cross-sectional study and thus, observed relationships between low vasoreactivity and slow gait speed does not necessarily imply a causal link between the two. DISCUSSION As such, prospective studies are needed to determine potential mechanisms underlying the observed relationship between vasoreactivity and gait speed in those with diabetes, the predictive value of vasoreactivity as a clin- ical tool, and the potential for therapies targeting cerebral blood ﬂow regulation to improve functional outcome in this vulnerable population. modulate cerebral perfusion between conditions of hyper- and hypocapnia (i.e., vasoreactivity, a widely used prognosis of metabolic cerebral blood ﬂow regulation) was associated with gait speed. These results suggest that in diabetic patients, the regulation of walking speed is dependent upon cerebral ele- ments related to the locomotor control system. This notion is supported by research demonstrating that walking requires adjust- ments of the cardiovascular and cerebrovascular systems that are coordinated to increase blood pressure and cerebral blood ﬂow velocities in order to meet metabolic demands (Novak et al., 2007; Perrey, 2013). Therefore, those diabetic participants with reduced vasoreactivity may have a diminished ability to increase perfusion in response to the metabolic demand associated with walking. g The relationship between vasoreactivity and gait speed that was observed in the diabetic group, but not in the non-diabetic group might also be explained by the complex effects of dia- betes on cerebral vasculature and metabolism. Diabetes accelerates aging in the brain (Launer, 2006) and alters vascular reactiv- ity through the combined effects of central insulin resistance on microvasculature, brain metabolism, glucose utilization, and neuronal survival. Central insulin plays an important role as a neuromodulator in key processes such as cognition (Shemesh et al., 2012; Freiherr et al., 2013), energy homeostasis, and glu- cose utilization during activity (e.g., walking). Cerebral insulin may directly modulate neuron–astrocyte signaling through neu- rovascular coupling and autonomic control of vascular tone and thus enable better regulation of local and regional perfusion (Lok et al., 2007) and neuronal activity in response to various stim- uli (Amir and Shechter, 1987; Cranston et al., 1998; Kim et al., 2006; Muniyappa et al., 2007) including walking. Type 2 dia- betes decreases insulin sensitivity in the brain, insulin transport through the blood–brain barrier, and insulin receptor’s sensi- tivity, and it alters glucose metabolism and energy utilization (Plum et al., 2005, 2006; Hallschmid et al., 2007; Freiherr et al., 2013). ACKNOWLEDGMENTS h k d This work was conducted with support from a National Insti- tute on Aging (NIA) T32 (5T32AG023480) fellowship awarded to Azizah J. Jor’dan, a KL2 Medical Research Investigator Training (MeRIT) award (1KL2RR025757-04) and NIA career develop- ment grant (1-K01-AG044543-01A1) awarded to Brad Manor, the Harvard Clinical and Translational Science Center (NIH Award KL2 RR 025757), and grants from the National Institute of Dia- betes and Digestive and Kidney Diseases (5R21-DK-084463-02) and the NIA (1R01-AG-0287601-A2) awarded to Vera Novak. The content is solely the responsibility of the authors and does not necessarily represent the ofﬁcial views of Harvard Cata- lyst, Harvard University and its afﬁliated academic health care centers, the National Center for Research Resources, or the NIH. AUTHOR CONTRIBUTIONS h ’d l d h Azizah J. Jor’dan analyzed the data, performed statistical analyses andwrotethemanuscript. BradManoroversawstatisticalanalyses, data interpretation and contributed to manuscript preparation. Vera Novak designed the study, conducted experiments, and over- saw all aspects of the study, data interpretation and manuscript preparation. DISCUSSION Glucotoxicity and endothelial dysfunction associated with chronic hyperglycemia further affect perfusion, vasoreactivity, and metabolism (Makimattila andYki-Jarvinen,2002; Brownlee,2005; Kilpatrick et al., 2010) and contribute to neuronal loss (Manschot et al., 2006, 2007; Last et al., 2007). Therefore, inadequate insulin delivery to brain tissue combined with altered energy metabolism may affect neuronal activity in multiple regions, but in partic- ular the motor and cognitive networks that have high demands on energy (Gunning-Dixon and Raz, 2000). Diabetes may there- fore especially alter neuronal activity and energy utilization during complex tasks like walking which require coordination of neu- ronal activity in numerous brain regions. As such, even if the same amount of blood ﬂow is delivered to the neurons, energy utiliza- tion may be reduced in diabetic as compared to non-diabetic brain, leading to reduced neuronal activity and function, such as walking speed. Frontiers in Aging Neuroscience REFERENCES Brain structural change and gait decline: a longitudinal population-based study. J. Am. Geriatr. Soc. 61, 1074–1079. doi: 10.1111/jgs.12331 Last, D., Alsop, D. C., Abduljalil, A. M., Marquis, R. P., de Bazelaire, C., Hu, K., et al. (2007). Global and regional effects of type 2 diabetes on brain tissue volumes and cerebral vasoreactivity. Diabetes Care 30, 1193–1199. doi: 10.2337/ dc06-2052 Brain structural change and gait decline: a longitudinal population-based study. J. Am. Geriatr. Soc. 61, 1074–1079. doi: 10.1111/jgs.12331 Chamberlin, M. E., Fulwider B. D., Sanders, S. L., and Medeiros, J. M. (2005). Does fear of falling inﬂuence spatial and temporal gait parameters in elderly persons beyond changes associated with normal aging? J. Gerontol. A Biol. Sci. Med. Sci. 60, 1163–1167. doi: 10.1093/gerona/60.9.1163 Launer, L. J. (2006). Diabetes and brain aging: epidemiologic evidence. Curr. Diab. Rep. 5, 59–63. doi: 10.1007/s11892-005-0069-61 Collignon, A., Maes, F., Delaere, D., Vancermeulen, D., Suetens, P., and Marchal, G. (1995). “Automated multi-modality image registration based on information theory,” in Information Processing in Medical Imaging, eds Y. Bizais, C. Barillot, and R. Di Paola (Dordrecht: Kluwer Academic Publishers), 263–274. Lavi, S., Gaitini, D., Milloul, V., and Jacob, G. (2006). Impaired cerebral CO2 vasoreactivity: association with endothelial dysfunction. Am. J. Physiol. Heart Circ. Physiol. 291, H1856–H1861. doi: 10.1152/ajpheart.00014.2006 Lok, J., Gupta, P., Guo, S., Kim, W. J., Whalen, M. J., van Leyen, K., et al. (2007). Cell–cell signaling in the neurovascular unit. Neurochem. Res. 32, 2032–2045. doi: 10.1007/s11064-007-9342-9 Cranston, I., Marsden, P., Matyka, K., Evans, M., Lomas, J., Sonksen, P., et al. (1998). Regional differences in cerebral blood ﬂow and glucose utilization in diabetic man: the effect of insulin. J. Cereb. Blood Flow Metab. 18, 130–140. doi: 10.1097/00004647–199802000-00002 Low, P.A., Novak,V., Spies, J. M., Novak, P., and Petty, G.W. (1999). Cerebrovascular regulation in the postural orthostatic tachycardia syndrome (POTS). Am. J. Med. Sci. 317, 124–133. doi: 10.1097/00000441-199902000-00007 Detre, J. A., Alsop, D. C., Vives, L. R., Maccotta, L., Teener, J. W., and Raps, E. C. (1998). Noninvasive MRI evaluation of cerebral blood ﬂow in cerebrovascular disease. Neurology 50, 633–641. doi: 10.1212/wnl.50.3.633 Makimattila, S., and Yki-Jarvinen, H. (2002). Endothelial dysfunction in human diabetes. Curr. Diab. Rep. 2, 26–36. doi: 10.1007/s11892-002- 0054-x Floyd, T. F., Ratcliffe, S. J., Wang, J., Resch, B., and Detre, J. A. (2003). Precision of the CASL-perfusion MRI technique for the measurement of cerebral blood ﬂow with in whole brain and vascular territories. J. Magn. Reson. REFERENCES Imaging 18, 649–655. doi: 10.1002/jmri.10416 Manor, B., Doherty, A., and Li, L. (2008). The reliability of physical perfor- mance measures in peripheral neuropathy. Gait Posture 28, 343–346. doi: 10.1016/j.gaitpost.2008.01.004 Manor, B., Hu, K., Zhao, P., Selim, M., Alsop, D., Novak, P., et al. (2010). Altered control of postural sway following cerebral infarction: a cross-sectional analysis. Neurology 76, 458–464. doi: 10.1212/WNL.0b013e3181cef647 Freiherr, J., Hallschmid, M., Frey,W. H., Brunner,Y. F., Chapman C. D., Holscher, C., et al. (2013). Intranasal insulin as a treatment for Alzheimer’s disease: a review of basicresearchandclinicalevidence. CNSDrugs 27,505–514. doi: 10.1007/s40263- 013-0076-8 Manor, B., Newton, E., Abduljalil, A., and Novak, V. (2012). The relationship between brain volume and walking outcomes in older adults with and without diabetic peripheral neuropathy. Diabetes Care 35, 1907–1912. doi: 10.2337/dc11- 2463 Fujishima, M., Scheinberg, P., Busto, R., and Reinmuth, O. M. (1971). The relation between cerebral oxygen consumption and cerebral vascular reactivity to carbon dioxide. Stroke 2, 251–257. doi: 10.1161/01.str.2.3.251 Girouard, H., and Iadecola, C. (2006). Neurovascular coupling in the normal brain and in hypertension, stroke, and Alzheimer disease. J. Appl. Physiol. 100, 328–335. doi: 10.1152/japplphysiol.00966.2005 Manschot, S. M., Biessels, G. J., deValk, H.,Algra,A., Rutten, G. E., van der Grond, J., et al. (2007). Metabolic and vascular determinants of impaired cognitive perfor- mance and abnormalities on brain magnetic resonance imaging in patients with type 2 diabetes. Diabetologia 50, 2388–2397. doi: 10.1007/s00125-007-0792-z Gunning-Dixon, F. M., and Raz, N. (2000). The cognitive correlates of white matter abnormalities in normal aging: a quantitative review. Neuropsychology 14, 224– 232. doi: 10.1037//0894-4105.14.2.224 pe 2 diabetes. Diabetologia 50, 2388–2397. doi: 10.1007/s00125-007 Manschot, S. M., Brands, A. M., van der Grond, J., Kessels, R. P., Algra, A., Kappelle, L. J., et al. (2006). Brain magnetic resonance imaging correlates of impaired cognition in patients with type 2 diabetes. Diabetes Metab. Res. Rev. 55, 1106– 1113. doi: 10.2337/diabetes.55.04.06.db05-1323 Guralnik, J. M., Ferrucci, L., Simonsick, E. M., Salive, M. E., and Wallace, R. B. (1995). Lower-extremity function in persons over the age of 70 years as a predictor of subsequent disability. N. Engl. J. Med. 332, 556–561. doi: 10.1056/nejm199503023320902 Muniyappa, R., Montagnani, M., Koh, K. K., and Quon, M. J. (2007). Cardiovascular actions of insulin. Endocr. Rev. 28, 463–491. doi: 10.1210/er.2007-0006 Hajjar, I., Zhao, P., Alsop, D., and Novak, V. (2010). Hypertension and cere- bral vasoreactivity: a continuous arterial spin labeling magnetic resonance imaging study. Hypertension 56, 859–864. doi: 10.1161/hypertensionaha.110. REFERENCES Alexander, R. W. (1995). Hypertension and the pathogenesis of atherosclerosis: oxidative stress and the mediation of arterial inﬂammatory response: a new perspective. Hypertension 25, 155–161. doi: 10.1161/01.hyp.25.2.155 Allet, L., Armand, S., Golay, A., Monnin, D., de Bie, R. A., and de Bruin, E. D. (2008). Gait characteristics of diabetic patients: a systematic review. Diabetes Metab. Res. Rev. 24, 173–191. doi: 10.1002/dmrr.809 Alsop, D. C., and Detre, J. A. (1998). Multisection cerebral blood ﬂow MR imaging with continuous arterial spin labeling. Radiology 208, 410–416. Amir, S., and Shechter,Y. (1987). Centrally mediated hypoglycemic effect of insulin: apparent involvement of speciﬁc insulin receptors. Brain Res. 418, 152–156. doi: 10.1016/0006-8993(87)90972-3 Bendall, M. J., Bassey, E. J., and Pearson, M. B. (1989). Factors affecting walking speed of elderly people. Age Ageing 18, 327–332. doi: 10.1093/ageing/18.5.327 While our study controlled for numerous variables associated with gait speed, it did not control for other associated variables, such as muscular strength or fear of falling (Bendall et al., 1989; Chamberlin et al., 2005). The current study has the advantage of investigating regional perfusion in response to CO2 challenges using 3-D CASL MRI; however, the measures were recorded Biessels, G. J., van der Heide, L. P., Kamal, A., Bleys, R. L., and Gispen, W. H. (2002). Ageing and diabetes: implications for brain function. Eur. J. Pharmacol. 441, 1–14. doi: 10.1016/S0014-2999(02)01486-3 Bohannon, R. W. (1997). Comfortable and maximum walking speed of adults age 20–79 years: reference values and determinants. Age Ageing 26, 15–19. doi: 10.1093/ageing/26.1.15 June 2014 \| Volume 6 \| Article 135 \| 7 www.frontiersin.org Diabetes, vasoreactivity, and gait speed Jor’dan et al. Kloter, E., Wirz, M., and Dietz, V. (2011). Locomotion in stroke subjects: interactions between unaffected an affected sides. Brain 134, 721–731. doi: 10.1093/brain/awq370 Brownlee, M. (2005). The pathobiology of diabetic complications. Diabetes Metab. Res. Rev. 54, 441–452. doi: 10.2337/diabetes.54.6.1615 Bullock,R.,Mendelow,A. D.,Bone,I.,Patterson,J.,Macleod,W. N.,andAllardice,G. Bullock,R.,Mendelow,A. D.,Bone,I.,Patterson,J.,Macleod,W. N.,andAllardice,G. (1985). Cerebral blood ﬂow and CO2 responsiveness as an indicator of collateral reserve capacity in patients with carotid arterial disease. Br. J. Surg. 72, 348–351. doi: 10.1002/bjs.1800720506 Kwon, S., Perera, S., Pahor, M., Katula, J. A., King, A. C., Groessl, E. J., et al. (2009). What is a meaningful change in physical performance? Findings from a clinical trial in older adults (the LIFE-P study). J. Nutr. Health Aging 13, 538–544. doi: 10.1007/s12603-009-0104-z Callisaya, M., Beare, R., Pham, T., Blizzard, L., Thrift, A. G., Chen, J., et al. (2013). REFERENCES 160002 Novak, V., Hu, K., Vyas, M., and Lipsitz, L. A. (2007). Cardiolocomotor coupling in young and elderly people. J. Gerontol. A Biol. Sci. Med. Sci. 62, 86–92. doi: 10.1093/gerona/62.1.86 Novak, V., Last, D., Alsop, D., Abduljalil, A. M., Hu, K., Lepicovsky, L., et al. (2006). Cerebral blood ﬂow velocity and periventricular white matter hyper- intensities in type 2 diabetes. Diabetes Care 29, 1529–1534. doi: 10.2337/ dc06-0261 Hallschmid, M., Benedict, C., Born, J., and Kern, W. (2007). Targeting metabolic and cognitive pathways of the CNS by intranasal insulin administration. Expert Opin. Drug Deliv. 4, 319–322. doi: 10.1517/17425247.4.4.319 Novak, V., Zhao, P., Manor, B., Sejdic, E., Alsop, D., Abduljalil, A., et al. (2011). Adhesion molecules, altered vasoreactivity, and brain atrophy in type 2 diabetes. Diabetes Care 34, 2438–2441. doi: 10.2337/dc11-0969 Iadecola, C., and Nedergaard, M. (2007). Glial regulation of the cerebral microvasculature. Nat. Neurosci. 10, 1369–1376. doi: 10.1038/nn2003 Kameneva, M. V., Watach, M. J., and Borovetz, H. S. (1999). Gender difference in rheologicpropertiesof bloodandriskof cardiovasculardiseases. Clin. Hemorheol. Microcirc. 21, 357–363. Perrey, S. (2013). Promoting motor function by exercising the brain. Brain Sci. 3, 101–122. doi: 10.3390/brainsci3010101 Plum, L., Belgardt, B. F., and Bruning, J. C. (2006). Central insulin action in energy and glucose homeostasis. J. Clin. Invest. 116, 1761–1766. doi: 10.1172/ jci29063 Kety, S. S., and Schmidt, C. F. (1948). The effects of altered arterial tensions of carbon dioxide and oxygen on cerebral blood ﬂow and cerebral oxygen con- sumption of normal young men. J. Clin. Investig. 27, 484–492. doi: 10.1172/ jci101995 Plum, L., Schubert, M., and Bruning, J. C. (2005). The role of insulin receptor signaling in the brain. Trends Endocrinol. Metab. 16, 59–65. doi: 10.1016/j.tem.2005.01.008 Kilpatrick, E. S., Rigby, A. S., and Atkin, S. L. (2010). For debate. Glucose variability and diabetes complication risk: we need to know the answer. Diabet. Med. 27, 868–871. doi: 10.1111/j.1464-5491.2010.02929.x Quach, L., Galica, A. M., Jones, R. N., Procter-Gray, E., Manor, B., Hannan, M. T., et al. (2011). The nonlinear relationship between gait speed and falls: the maintenance of balance, independent living, intellect, and zest in the elderly of Boston study. J. Am. Geriatr. Soc. 59, 1069–1073. doi: 10.1111/j.1532-5415.2011. 03408.x Kim, J. A., Montagnani, M., Koh, K. K., and Quon, M. J. (2006). Reciprocal relationships between insulin resistance and endothelial dysfunction: molec- ular and pathophysiological mechanisms. Circulation 113, 1888–1904. REFERENCES doi: 10.1161/circulationaha.105.563213 June 2014 \| Volume 6 \| Article 135 \| 8 Frontiers in Aging Neuroscience www.frontiersin.org Diabetes, vasoreactivity, and gait speed Jor’dan et al. Rosano, C., Aizenstein, H. J., Studenski, S., and Newman, A. B. (2007a). A regions-of-interest volumetric analysis of mobility limitations in community- dwelling older adults. J. Gerontol. A Biol. Sci. Med. Sci. 62, 1048–1055. doi: 10.1093/gerona/62.9.1048 Várkuti, B., Cavusoglu, M., Kullik, A., Schifﬂer, R., Yilmaz, O., Rosenstiel, W., et al. (2011). Quantifying the link between anatomical connectivity, gray matter volume and regional cerebral blood ﬂow: an integrative MRI study. PLoS ONE 6:e14801. doi: 10.1371/journal.pone.0014801 Rosano, C., Brach, J., Studenski, S., Longstreth, W. T. Jr., and Newman, A. B. (2007b). Gait variability is associated with subclinical brain vascular abnor- malities in high-functioning older adults. Neuroepidemiology 29, 193–200. doi: 10.1159/000111582 Volpato,S.,Maraldi,C.,and Renato,F. (2010). Type 2 diabetes and risk for functional decline and disability in older persons. Curr. Diabetes Rev. 6, 134–143. doi: 10.2174/157339910791162961 Wells, R. E., and Merrill, E. W. (1962). Inﬂuence of ﬂow properties of blood upon viscosity–hematocrit relationships. J. Clin. Invest. 41, 159–1598. doi: 10.1172/JCI104617 Schroeder, T. (1988). Hemodynamic signiﬁcance of internal carotid artery disease. Acta Neurol. Scand. 77, 353–372. doi: 10.1111/j.1600-0404.1988.tb05921.x upon viscosity–hematocrit relationships. J. Clin. Invest. 41, 159–1598. doi: 10.1172/JCI104617 Shattuck, D. W., Mirza, M., Adisetivo, V., Hojatkashani, C., Salamon, G., Narr, K. L., et al. (2008). Construction of a 3D probabilistic atlas of human cortical structures. Neuroimage 39, 1064–1080. doi: 10.1016/j.neuroimage.2007.09.031 Wells,W. M.,Viola, P.,Atsumi, H., Nakajima, S., and Kikinis, R. (1996). Multi-modal volume registration by maximization of mutual information. Med. Image Anal. 1, 35–51. doi: 10.1016/S1361–8415(01)80004-9 Yen, Y. F., Field, A. S., and Martin, E. M. (2002). Test–retest reproducibility of quantitative CBF measurements using FAIR perfusion MRI and acetazolamide challenge. Magn. Reson. Med. 47, 921–928. doi: 10.1002/mrm.10140 Shemesh, E., Rudich, A., Harman-Boehm, I., and Cukierman-Yaffe, T. (2012). Effect of intranasal insulin on cognitive function: a systematic review. J. Clin. Endocrinol. Metab. 97, 366–376. doi: 10.1210/jc.2011–1802 Sorond, F. A., Galica, A., Serrador, J. M., Kiely, D. K., Iloputaife, I., Cup- ples, L. A., et al. (2010). Cerebrovascular hemodynamics, gait, and falls in an elderly population. Neurology 74, 1627–1633. doi: 10.1212/WNL.0b013e31 81df0982 Zeng, S. M.,Yankowitz, J.,Widness, J.A., and Strauss, R. G. (2000). Etiology of differ- ences in hematocrit between males and females: sequence-based polymorphisms in erythropoietin and its receptor. J. Gend. Specif. Med. 4, 35–40. June 2014 \| Volume 6 \| Article 135 \| 9 REFERENCES Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Sorond, F. A., Kiely, D. K., Galica, A., Moscufo, N., Serrador, J. M., Ikoputaife, I., et al. (2011). Neurovascular coupling is impaired in slow walkers: the mobilize Boston study. Ann. Neurol. 70, 213–220. doi: 10.1002/ana.22433 Steffen, T. M., Hacker, T. A., and Mollinger, L. (2002). Age- and gender-related test performance in community-dwelling elderly people: Six-Minute Walk Test, Berg Balance Scale, Timed Up & Go, and gait speeds. Phys. Ther. 82, 128–137. Received: 07 April 2014; accepted: 09 June 2014; published online: 26 June 2014. Received: 07 April 2014; accepted: 09 June 2014; published online: 26 June 2014. Citation: Jor’dan AJ, Manor B and Novak V (2014) Slow gait speed – an indicator of Stern, Y. (2002). What is cognitive reserve? Theory and research applica- tion of the reserve concept. J. Int. Neuropsychol. Soc. 8, 448–460. doi: 10.1017/S1355617702813248 lower cerebral vasoreactivity in type 2 diabetes mellitus. Front. Aging Neurosci. 6:135. doi: 10.3389/fnagi.2014.00135 This article was submitted to the journal Frontiers in Aging Neuroscience. Stoffregen, T. A., Pagulayan, R. J., Bardy, B. G., and Hettinger, L. J. (2000). Modulat- ing postural control to facilitate visual performance. Hum. Mov. Sci. 19, 22–30. doi: 10.1016/s0167-9457(00)00009–9 Copyright © 2014 Jor’dan, Manor and Novak. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, dis- tribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Copyright © 2014 Jor’dan, Manor and Novak. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, dis- tribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Studenski, S., Perera, S., Patel, K., Rosano, C., Faulkner, K., Inzitari, M., et al. (2011). Gait speed and survival in older adults. J. Am. Med. Assoc. 305, 50–58. Frontiers in Aging Neuroscience Received: 07 April 2014; accepted: 09 June 2014; published online: 26 June 2014. Citation: Jor’dan AJ, Manor B and Novak V (2014) Slow gait speed – an indicator of lower cerebral vasoreactivity in type 2 diabetes mellitus. Front. Aging Neurosci. 6:135. doi: 10.3389/fnagi.2014.00135 This article was submitted to the journal Frontiers in Aging Neuroscience. Copyright © 2014 Jor’dan, Manor and Novak. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, dis- tribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. REFERENCES doi: 10.1001/jama.2010.1923 June 2014 \| Volume 6 \| Article 135 \| 9 Frontiers in Aging Neuroscience Frontiers in Aging Neuroscience www.frontiersin.org
https://openalex.org/W1520086199	http://www.globalizationandhealth.com/content/pdf/1744-8603-2-6.pdf	English	null	Globalization and Health	Oxford University Press eBooks	2,006	cc-by	8,449	Abstract Background: Antimicrobial resistance is an under-appreciated threat to public health in nations around the globe. With globalization booming, it is important to understand international patterns of resistance. If countries already experience similar patterns of resistance, it may be too late to worry about international spread. If large countries or groups of countries that are likely to leap ahead in their integration with the rest of the world – China being the standout case – have high and distinctive patterns of resistance, then a coordinated response could substantially help to control the spread of resistance. The literature to date provides only limited evidence on these issues. Methods: We study the recent patterns of antibiotic resistance in three geographically separated, and culturally and economically distinct countries – China, Kuwait and the United States – to gauge the range and depth of this global health threat, and its potential for growth as globalization expands. Our primary measures are the prevalence of resistance of specific bacteria to specific antibiotics. We also propose and illustrate methods for aggregating specific "bug-drug" data. We use these aggregate measures to summarize the resistance pattern for each country and to study the extent of correlation between countries' patterns of drug resistance. Results: We find that China has the highest level of antibiotic resistance, followed by Kuwait and the U.S. In a study of resistance patterns of several most common bacteria in China in 1999 and 2001, the mean prevalence of resistance among hospital-acquired infections was as high as 41% (with a range from 23% to 77%) and that among community- acquired infections was 26% (with a range from 15% to 39%). China also has the most rapid growth rate of resistance (22% average growth in a study spanning 1994 to 2000). Kuwait is second (17% average growth in a period from 1999 to 2003), and the U.S. the lowest (6% from 1999 to 2002). Patterns of resistance across the three countries are not highly correlated; the most correlated were China and Kuwait, followed by Kuwait and the U.S., and the least correlated pair was China and the U.S. Conclusion: Antimicrobial resistance is a serious and growing problem in all three countries. To date, there is not strong international convergence in the countries' resistance patterns. This finding may change with the greater international travel that will accompany globalization. BioMed Central BioMed Central Published: 07 April 2006 Published: 07 April 2006 Globalization and Health 2006, 2:6 doi:10.1186/1744-8603-2-6 This article is available from: http://www.globalizationandhealth.com/content/2/1/6 This article is available from: http://www.globalizationandhealth © 2006 Zhang 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. Globalization and Health Open Access Received: 04 September 2005 Accepted: 07 April 2006 Received: 04 September 2005 Accepted: 07 April 2006 Ruifang Zhang1, Karen Eggleston2, Vincent Rotimi3 and g Zhang1, Karen Eggleston2, Vincent Rotimi3 and Address: 1Goldman Sachs International, Global Investment Research, London, UK, 2Tufts University Economics Department, Medford, MA 02155, USA, 3Department of Microbiology, Faculty of Medicine, Kuwait University, Kuwait and 4Harvard University Kennedy School of Government, Cambridge, MA, USA Email: Ruifang Zhang - ruifang.zhang@gs.com; Karen Eggleston* - karen.eggleston@tufts.edu; Vincent Rotimi - vincent@HSC.EDU.KW; Richard J Zeckhauser - richard_zeckhauser@harvard.edu mail: Ruifang Zhang - ruifang.zhang@gs.com; Karen Eggleston* - karen.eggleston@tufts.edu; Vincent Rotimi - vince chard J Zeckhauser - richard_zeckhauser@harvard.edu * Corresponding author http://www.globalizationandhealth.com/content/2/1/6 http://www.globalizationandhealth.com/content/2/1/6 http://www.globalizationandhealth.com/content/2/1/6 Globalization and Health 2006, 2:6 In 1942, the first U.S. patient with streptococcal infection was miraculously cured with a small dose of penicillin. Sixty years later, penicillin-resistant Streptococcus is wide- spread. Such antimicrobial resistance threatens the health of many throughout the world, since both old and new infectious diseases remain a formidable public health threat. In 1942, the first U.S. patient with streptococcal infection was miraculously cured with a small dose of penicillin. Sixty years later, penicillin-resistant Streptococcus is wide- spread. Such antimicrobial resistance threatens the health of many throughout the world, since both old and new infectious diseases remain a formidable public health threat. some critical mass in the face of invading antimicrobials, treatment outcome is compromised; this phenomenon is referred as antimicrobial resistance (AMR) [3-9]. This paper focuses on antibiotic resistance, a major form of AMR. Resistance mechanisms may develop over months or years [6]. Once established, a single resistance mechanism can often allow a bacterium to resist multiple drugs. It remains unclear whether resistance is reversible, and thus whether drug effectiveness is a renewable or non-renewa- ble resource [10-15]. Drug resistance raises the cost of treatment for infectious diseases, sometimes manifold, as well as increasing morbidity and mortality from such dis- eases [16-23]. Among the issues that merit further scrutiny for under- standing the possible spread of antimicrobial resistance, few are as salient as the impact of globalization. Clearly the movement of people and goods around the globe con- tributes to transmission of disease [1,2]. To what extent drug resistance and globalization are similarly related remains unclear. The breakout of Severe Acute Respiratory Syndrome (SARS) in the spring of 2003 illustrates how an infectious disease with limited therapeutic options can spread rapidly across national borders. With globalization booming, it is important to understand international pat- terns of resistance. If countries already experience similar patterns of resistance, it may be too late to worry about international spread. If large countries or groups of coun- tries that are likely to leap ahead in their integration with the rest of the world – China being the standout case – have high and distinctive patterns of resistance, then a coordinated response could help substantially to control the spread of resistance. The literature to date provides only limited evidence on these issues. The greatest long-term threat of AMR is that resistant strains erode drug efficacy over time. http://www.globalizationandhealth.com/content/2/1/6 The development of drug-resistant Staphylococci aureus (SAU) well illustrates the see-saw battle between pathogens and drugs. SAU is a bacterium that harmlessly lives in the human body but can cause infections on wounds or lesions. After the clini- cal application of penicillin in the 1940s, SAU soon adapted to the treatment mechanism of penicillin, and by the 1950s, almost half of SAU strains had become resist- ant to penicillin. A new antibiotic, methicillin, was devel- oped in the 1960s. Yet by the late 1970s, methicillin- resistant SAU, i.e. MRSA, again became widespread. Today MRSA has become a major infectious culprit that can only be effectively treated with vancomycin, one of the few last killers of superbugs. Unfortunately, in 1996, a Japanese hospital reported the first case of vancomycin-resistant SAU (VRSA) during surgery on a four-month-old boy. The U.S., France and Hong Kong subsequently all reported VRSA incidents. A few years later in 2000, linezolid was launched as a new antibiotic to combat both MRSA and VRSA. But only one year later, Boston researchers reported the first case of linezolid-resistant MRSA in an 85-year-old man undergoing peritoneal dialysis. After failing to con- tain his MRSA by linezolid, researchers tried five antibiot- ics (ampicillin, azithromycin, gentamicin, levofloxacin, and quinupristin-dalfopristin) but the unlucky man even- tually died from the uncontrollable infection [24]. We study the pattern of antibiotic resistance in specific countries to gauge the range and depth of this global health threat. China and the U.S. stand out as good choices for study. Both are world economic powerhouses increasingly responding to the forces of economic globali- zation. In addition, both are major consumers of antibiot- ics, with the U.S. also being a leading source of new antibiotics. On the other hand, it would also be interest- ing to compare patterns of antibiotic resistance in smaller countries that stand relatively distant from these two. Accordingly, we compare the experiences of the U.S. and China with new data on the resistance experience of Kuwait. The first section gives brief background on antibiotic resistance and its costs. We then turn to a detailed compar- ison of surveillance data from China, Kuwait, and the U.S. We conclude with a plea for more research and attention on this critical issue for health and globalization. Resistant pathogens within a hospital or specific commu- nity can spread to a nation at large or across national boundaries. http://www.globalizationandhealth.com/content/2/1/6 Thus, for example, rapidly increasing travel and migration within China probably contributes to the growth of that nation's resistance problem. It may also spur the spread of China's resistance problems overseas as globalization greatly increases travel from and to that nation (see Figure 1). Abstract Future research on the determinants of drug resistance patterns, and their international convergence or divergence, should be a priority. Page 1 of 14 (page number not for citation purposes) (page number not for citation purposes) http://www.globalizationandhealth.com/content/2/1/6 http://www.globalizationandhealth.com/content/2/1/6 http://www.globalizationandhealth.com/content/2/1/6 Background: The challenge of antimicrobial resistance According to laws of Darwinian evolution, antimicrobial use creates a selection pressure on microorganisms: weak ones are killed, but stronger ones might adapt and survive. When pathogenic microorganisms can multiply beyond Page 2 of 14 (page number not for citation purposes) Globalization and Health 2006, 2:6 Methods using standard year-on-year growth calculations. Where appropriate, we smooth variance in small-sample data series by using three-year running averages. We collected data on drug resistance in China, the U.S. and Kuwait, drawing from published studies, reports from national surveillance systems, and previously unpub- lished data from a large hospital in Kuwait. Such data must be viewed with caution. Differences between coun- tries arise not only from genuine differences in preva- lence, but also from differences in sampling strategies, laboratory processing, and standards for defining a "resist- ant" strain. Moreover, within-country comparisons across time are biased by measurement error, particularly for small samples. However, analysis of the currently availa- ble data does yield some evidence and may help to raise awareness and efforts to improve the data and methods for addressing the problem. We also develop methods to aggregate specific "bug-drug" data to summarize the resistance pattern for each country. These measures weight resistance rates by (1) the isolation frequency for each bacterium (that is, the proportion of a particular bacterium among all bacteria studied); and, where possible, by (2) the proportion of resistant cases hospital- versus community-acquired; and (3) the fre- quency with which each drug is used to treat infections caused by each bacterium. (For most calculations, meas- ure (3) is not available.) Finally, we compare and contrast each country's resistance experience and, using the subset of data comparable across the three countries, examine correlations in patterns of resistance. Our primary measure is the prevalence of resistance by a specific bacterium to a specific drug. The prevalence is cal- culated as the number of resistant isolates divided by the number of total isolates collected, multiplied by 100. We compute growth rates of resistance to specific bacteria These methods represent preliminary steps to gauge whether patterns of antibiotic resistance converge over time amongst countries that currently have little popula- Page 3 of 14 (page number not for citation purposes) Travel to and from China has increased tremendously over the past decade Figure 1 Travel to and from China has increased tremendously over the past decade. 0 20000 40000 60000 80000 100000 120000 19941995199619971998199920002001200220032004 0 5000 10000 15000 20000 25000 30000 35000 Thousands persons Thousands persons Arrivals of foreigners to China (left axis) Departures of Chinese residents to overseas (right axis) Source: CEIC Data Inc Thousands persons Thousands persons Arrivals of foreigners to China (left axis) Departures of Chinese residents to overseas (right axis) 19941995199619971998199920002001200220032004 19941995199619971998199920002001200220032004 Source: CEIC Data Inc Travel to and from China has increased tremendously over the past decade Figure 1 Travel to and from China has increased tremendously over the past decade. http://www.globalizationandhealth.com/content/2/1/6 http://www.globalizationandhealth.com/content/2/1/6 Globalization and Health 2006, 2:6 Table 1: Resistance prevalence of ten common bacteria to Ciprofloxacin in China, 1994–2000 unit: % Rank Bacter. 1994 1995 1996 1997 1998 1999 2000 Average Resistance* Average Growth Rate* 1 Escherichia coli (ECO) 53 49 60 61 60 63 62 59 3 2 Pseudomonas aeruginosa (PAE) 9 10 7 18 13 17 18 13 17 3 Klebsiella pneumoniae (KPN) 2 4 7 8 14 17 18 10 40 4 Staphylococci epidermidis (SEP) 22 33 34 35 41 40 46 36 9 5 Staphylococci aureus (SAU) MRS A 47 65 74 88 83 78 76 76 7 MSS A 8 18 10 5 8 20 14 11 8 6 Enterococcus faecalis (EFA) 25 34 28 34 32 45 45 34 9 7 Enterobacter cloacae (ECL) 12 9 13 14 22 31 30 18 26 8 Acinetobacter baumannii (ABA) 7 7 19 20 23 31 37 20 29 9 Citrobacter freundii (CFR) 10 21 20 17 22 26 26 20 10 10 Proteus mirabilis (PMI) 8 2 13 2 5 14 12 7 10 Mea n 28 15 Med ian 20 10 * Based on three-year running averages. ** Staphylococci aureus (SAU) is further grouped as methicillin susceptible staphylococci aureus (MSSA) and methicillin resistant staphylococci aureus (MRSA). Table 1: Resistance prevalence of ten common bacteria to Ciprofloxacin in China, 1994–2000 * Based on three-year running averages. * Based on three-year running averages. ** Staphylococci aureus (SAU) is further grouped as methicillin susceptible staphylococci aureus (MSSA) and methicillin resistant staphylococci (MRSA) ng averages. U) is further grouped as methicillin susceptible staphylococci aureus (MSSA) and methicillin resistant staphylococci tion interchange. Future research would benefit from bet- ter surveillance of resistance, more comparable data reporting, data on antibiotic utilization, and further meth- odological advances in clinically- and policy-relevant aggregation of "bug-drug" data. ria such as ECO and MRSA have high proportions (60– 80%) of resistant strains, whereas the prevalence of resist- ant strains for others such as PMI is quite low. Thousands persons Almost all but MSSA and PMI have shown considerable growth in resistance over the study period, resulting in an average annual growth rate of about 15%. Results China For the seven Page 4 of 14 (page number not for citation purposes) Page 4 of 14 (page number not for citation purposes) Page 5 of 14 (page number not for citation purposes) Table 2: Resistance patterns of the seven most common bacteria for Hospital-acquired Infections (HAI) a mon bacteria for Hospital-acquired Infections (HAI) and Community-acquired Infections (CAI), China 200 on and Health 2006, 2:6 http://www.globalizationandhealth.com/content/2 unit: % Antibiotic(s) SAU (n = 176) SEP (n = 84) ECO (n = 308) ECL (n = 78) PAE (n = 232) KPN (n = 215) ABA (n = 191) γ HAI (37) CAI (139) HAI (14) CAI (70) HAI (44) CAI (264) HAI (27) CAI (51) HAI (95) CAI (137) HAI (48) CAI (167) HAI (46) CAI (145) HAIγH CAIγC Methicillin 89 30 43 27 n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a 11 5 Ampicillin 100 82 86 67 89 80 100 90 n/a n/a 54 66 n/a n/a 38 35 Amoxicillin 89 27 29 6 84 81 100 94 n/a n/a 90 95 48 50 38 31 Ceftizoxime 87 28 14 7 32 25 96 86 n/a n/a 33 26 96 92 24 16 Cefaclor 87 31 21 10 32 26 89 78 n/a n/a 33 25 65 57 23 15 Cefuroxime 89 29 22 4 32 25 74 47 n/a n/a 29 23 57 41 22 12 Cefprozil. Results China Another series of studies by the China Bacterial Resistance Surveillance Study Group focused on resistance preva- lence among different patient types, i.e. those with hospi- tal-acquired infections (HAI) versus community-acquired infections (CAI) [26,27]. We construct two measures to compare HAI and CAI resistance prevalence. First, by aggregating the seven bacteria, we get a measure γ indexed on the nineteen drugs. γ is calculated by multiplying the resistance rate of each bacterium by its isolation frequency and proportion among HAI (or CAI) infections, and then summing across bacteria. The measure is reported in the last two columns of Table 2 and graphed in Figure 2. Sec- ond, by aggregating the drugs, we obtain a measure indexed on bacteria. However, because we lack data on how often each drug is used, the best we can do is report the simple average for all drugs (implicitly assuming each drug is used with equal frequency). We name this measure Mean Resistance, shown in the last row in Table 2 and graphed in Figure 3. In 1988, the World Health Organization West Pacific Regional Office set up two antimicrobial resistance sur- veillance centers in Beijing and Shanghai. Meanwhile, China's Ministry of Health also established the China Nosocomial Infection Surveillance (CNIS) program, which monitors hospital-acquired infections. Unfortu- nately, most of the surveillance programs in China focus on urban hospitals. We lack data on urban communities and for the rural majority. Nevertheless, the available data allows us to piece together a picture of the extent of anti- microbial resistance in the most populous country in the world. To examine AMR development in China, we use annual data from a seven-year (1994–2000) study by China's National Center for Antimicrobial Resistance, which reports resistance levels of ten most prevalent bacteria to a common antibiotic, ciprofloxacin (Table 1) [25]. With small sample sizes, the annual measured percentage of isolates found to be resistant varies considerably; to smooth the random variation attributable to small sam- ple size, we use three-year running averages. Some bacte- Both measures reinforce the finding that infections acquired in a hospital are often more drug resistant than other (community-acquired) infections. Results China Cefaclor Cefuroxime Levofloxacin Ceftazidime Ceftriaxone Moxifloxacin Cefotaxime Gatifloxacin Methicillin Imipenem Meropenem HAI CAI unit: % Hospital-acquired infections (HAI) are more resistant than community-acquired infections (CAI) to a wide range of antibiotics in China Figure 2 Hospital-acquired infections (HAI) are more resistant than community-acquired infections (CAI) to a wide range of antibiotics in China. Ampicillin Amoxicillin Sparfloxacin Ciprofloxacin Ofloxacin Gentamicin Ceftizoxime Cefprozil. Cefaclor Cefuroxime Levofloxacin Ceftazidime Ceftriaxone Moxifloxacin Cefotaxime Gatifloxacin Methicillin Imipenem Meropenem HAI CAI Hospital-acquired infections (HAI) are more resistant than community-acquired infections (CAI) to a wide range of antibiotics in China Figure 2 Hospital-acquired infections (HAI) are more resistant than community-acquired infections (CAI) to a wide range of antibiotics in China. Hospital-acquired infections (HAI) are more resistant than community-acquired infections (CAI) to a wide range of antibiotics in China Figure 2 Hospital-acquired infections (HAI) are more resistant than community-acquired infections (CAI) to a wide range of antibiotics in China moniae (SPN) from 1997 to 2002 to examine prevalence and trends (Table 3). The average growth rate of resistance for this bacterium was 8%, lower than the 15% number for China. Interestingly, unlike the upward resistance trend in China, SPN resistance declined in the last two years of the study period in the US, following an initial rise. Such data should not be interpreted to mean that actual prevalence is permanently declining, since meas- urement issues engender considerable year-to-year varia- tion in the sample prevalence. moniae (SPN) from 1997 to 2002 to examine prevalence and trends (Table 3). The average growth rate of resistance for this bacterium was 8%, lower than the 15% number for China. Interestingly, unlike the upward resistance trend in China, SPN resistance declined in the last two years of the study period in the US, following an initial rise. Such data should not be interpreted to mean that actual prevalence is permanently declining, since meas- urement issues engender considerable year-to-year varia- tion in the sample prevalence. bacteria, the mean resistance rate of HAI is on average 1.5 times that of CAI in China. For the nineteen drugs, the aggregate measure of resistance for HAI, γH, is on average 1.9 times that for CAI, γC. This pattern is most extreme for infections caused by SAU, where resistance of HAI is two- to three- times that of CAI, depending on which measure is used. Results China (T-tests of the difference between two groups indi- cate a p-value of less than 0.01 for the γ's and less than 0.09 for the mean resistance). Moreover, the prevalence of drug resistance for both kinds of infections is quite high. Mean resistance of HAI is 41% and that of CAI is 28%. The US NNIS program provides data for inpatients and outpatients. Further, among inpatients, the NNIS differ- entiates between those in and not in the ICU. For almost every bug-drug pair, resistance prevalence is highest among ICU patients, followed by non-ICU inpatients, with the lowest prevalence among outpatients (Table 4 and Figure 4). This pattern seems consistent with clinical reality, since patients in ICUs are more likely to have a weak immune system, either because of prolonged treat- Page 6 of 14 (page number not for citation purposes) Results China 87 26 21 4 34 25 78 61 n/a n/a 33 23 94 86 24 15 Ceftazidime 92 37 50 13 5 7 59 28 11 14 21 4 30 15 19 8 Cefotaxime 84 28 21 6 0 7 44 26 41 26 4 5 28 16 15 8 Ceftriaxone 89 28 21 3 9 8 48 29 40 25 6 5 33 15 18 8 Imipenem 76 21 21 1 2 0 0 2 2 3 0 1 2 1 8 2 Meropenem 78 21 14 1 2 0 0 0 2 2 0 1 2 2 8 2 Ciprofloxacin 87 35 36 30 75 53 63 33 26 13 19 14 26 17 29 18 Ofloxacin 78 30 36 30 75 55 59 31 17 15 15 14 22 17 27 18 Levofloxacin 46 7 29 10 68 52 33 20 22 15 10 11 13 12 21 13 Sparfloxacin 89 39 50 40 75 56 63 33 43 31 25 16 15 14 32 21 Moxifloxacin 5 2 14 3 64 43 22 18 43 27 4 8 13 15 17 12 Gatifloxacin 30 1 14 4 36 25 7 6 23 17 6 6 15 14 13 7 Gentamicin 87 31 36 21 43 38 30 24 37 29 27 16 35 21 25 16 Mean Resistance 77 28 30 15 42 34 54 39 26 18 23 20 35 28 http://www.globalizationandhealth.com/content/2/1/6 http://www.globalizationandhealth.com/content/2/1/6 Globalization and Health 2006, 2:6 Hospital-acquired infections (HAI) are more resistant than community-acquired infections (CAI) to a wide range of antibiotics in China Figure 2 Hospital-acquired infections (HAI) are more resistant than community-acquired infections (CAI) to a wide range of antibiotics in China. 0 5 10 15 20 25 30 35 40 Ampicillin Amoxicillin Sparfloxacin Ciprofloxacin Ofloxacin Gentamicin Ceftizoxime Cefprozil. Cefaclor Cefuroxime Levofloxacin Ceftazidime Ceftriaxone Moxifloxacin Cefotaxime Gatifloxacin Methicillin Imipenem Meropenem HAI CAI unit: % Hospital-acquired infections (HAI) are more resistant than community-acquired infections (CAI) to a wide range of antibiotics in China Figure 2 Hospital-acquired infections (HAI) are more resistant than community-acquired infections (CAI) to a wide range of antibiotics i Chi 0 5 10 15 20 25 30 35 40 Ampicillin Amoxicillin Sparfloxacin Ciprofloxacin Ofloxacin Gentamicin Ceftizoxime Cefprozil. United States Fairly comprehensive data on resistance trends in the U.S. come from the National Nosocomial Infections Surveil- lance System (NNIS) for hospital-based resistance, and the U.S. Active Bacterial Core Surveillance (ABC) project, which surveys a population of 16 million to 25 million community residents in 9 states each year [28-30]. We use data from an ABC program that surveys Streptococcus pneu- Page 6 of 14 (page number not for citation purposes) Page 6 of 14 (page number not for citation purposes) http://www.globalizationandhealth.com/content/2/1/6 http://www.globalizationandhealth.com/content/2/1/6 Globalization and Health 2006, 2:6 The Seven most common bacteria show higher resistance among hospital-acquired infections (HAI) than community-acquired infections (CAI) in China Figure 3 The Seven most common bacteria show higher resistance among hospital-acquired infections (HAI) than community-acquired infections (CAI) in China. 0 10 20 30 40 50 60 70 80 90 SAUECLECOABASEPPAEKPN HAI CAI unit: % SAUECLECOABASEPPAEKPN SAUECLECOABASEPPAEKPN The Seven infections Figure 3 Th S The Seven most common bacteria show higher resistance among hospital acquired infections (HAI) than community acquired infections (CAI) in China Figure 3 The Seven most common bacteria show higher resistance among hospital-acquired infections (HAI) than community-acquired infections (CAI) in China. for hospital-acquired infections is 41% and that for com- munity-acquired infections is 28%. ment or their own compromised conditions; moreover, many are catheterized, offering a conduit for bacteria. ment or their own compromised conditions; moreover, many are catheterized, offering a conduit for bacteria. Compared with China, the U.S. exhibits more moderate differences in resistance prevalence among different patients. The average prevalence of resistance for ICU, other inpatients, and outpatients in the U.S. are 20%, 17% and 13%, respectively; in China, average resistance Pooling all patients together (Table 5), we find the preva- lence of resistance and its growth to be 17% and 7% respectively, consistent with our previous observation that the U.S. seems to have both lower resistance prevalence and less dramatic increase in resistance than China does. Table 3: Non-susceptibilities of Streptococcus pneumoniae (SPN) in U.S. communities, 1997–2002 Unit: % Antibiotic 1997 1998 1999 2000 2001 2002 Average Resistance Average Growth Rate Penicillin 25 24 27 28 26 21 25 2 Cefotaxime 13 14 17 18 16 12 15 -1 Erythromycin 15 15 21 22 19 17 18 4 TMP/Sulfa 29 29 32 32 30 25 30 -3 Levofloxacin n/a 0.2 0.2 0.3 0.7 0.5 0.4 39 Vancomycin 0 0 0 0 0 0 18 8 Table 3: Non-susceptibilities of Streptococcus pneumoniae (SPN) in U.S. communities, 1997–2002 Globalization and Health 2006, 2:6 http://www.globalizationandhealth.com/content/2/1/6 http://www.globalizationandhealth.com/content/2/1/6 Table 4: Resistance prevalence for selected drug-bug pairs by patient type, U.S. 1999–2002 unit: % Pair Bacterium (resistant to) → drug ICU patients non-ICU inpatients Outpatients A PAE → Ciprofloxacin/ ofloxacin 32 25 23 B PAE → Levofloxacin 37 28 25 C PAE → Imipenem 18 12 9 D PAE → Ceftazidime 13 8 5 E PAE → Piperacillin 16 11 6 F SAU → Methicillin 47 38 23 G Enterococcus spp → Vancomycin 13 11 4 H ECO → Cef3* 1 1 0 I ECO → Quinolone** 5 4 2 J KPN → Cef3 6 5 2 K Enterobacter spp → Cef3 26 21 10 L Enterobacter spp → Carbapenum 1 1 1 M CNS → Methicillin 75 63 46 N Pneumococcus → Penicillin 18 17 17 O Pneumococcus → Cef3 7 8 6 Mean 21 17 12 Cef3 (3rd generation cephalosporin) = ceftazidime, cefotaxime or ceftriaxone; Quinolone = ciprofloxacin, ofloxacin or levofloxacin. Table 4: Resistance prevalence for selected drug-bug pairs by patient type, U.S. 1999–2002 Cef3 (3rd generation cephalosporin) = ceftazidime, cefotaxime or ceftriaxone; *Quinolone = ciprofloxacin, ofloxacin or levofloxacin. ered data on antimicrobial resistance among isolates of eight different bacterial diseases over the most recent five years. The data is based on surveillance from a single large Kuwait There is considerably less detailed data on antibiotic resistance for Kuwait than for China or the U.S. We gath- Table 5: Resistance prevalence of eight common bacteria, U.S. (all patients pooled), 1999–2002 unit: % Bacterium Resistant to antibiotic(s) 1999 2000 2001 2002 Average Resistance Average Growth Rate PAE Ciprofloxacin /ofloxacin 23 25 28 29 26 8 Levofloxacin 29 30 31 30 30 1 Imipenem 12 12 15 13 13 4 Ceftazidime 8 8 9 9 9 4 Piperacillin 10 10 11 12 11 6 SAU (MRSA) Methicillin 32 35 38 39 36 7 Enterococcus spp Vancomycin 11 8 10 10 10 -1 ECO Cef3 1 1 1 1 1 0 Quinolone 2 3 4 5 4 36 KPN Cef3 4 4 4 5 4 8 Enterobacter spp Cef3 19 19 18 19 19 0 Carbapenum 1 1 1 1 1 0 CNS Methicillin 60 61 62 63 62 2 Pneumococcu s spp Penicillin 14 16 19 19 17 11 Cef3 5 8 7 7 7 16 Mean: 17 7 able 5: Resistance prevalence of eight common bacteria, U.S. (all patients pooled), 1999–2002 Globalization and Health 2006, 2:6 http://www.globalizationandhealth.com/content/2/1/6 ICU patients have the highest resistance rates in selected drug-bug pairs, followed by non-ICU inpatients and outpatients, U.S. 1999–2002 Figure 4 ICU patients have the highest resistance rates in selected drug-bug pairs, followed by non-ICU inpatients and outpatients, U.S. 1999–2002. http://www.globalizationandhealth.com/content/2/1/6 Globalization and Health 2006, 2:6 ICU patients have the highest resistance rates in selected drug-bug pairs, followed by non-ICU inpatients and outpatients, U.S 1999–2002 Figure 4 ICU patients have the highest resistance rates in selected drug-bug pairs, followed by non-ICU inpatients and outpatients, U.S 1999–2002. ghest resistance rates in selected drug-bug pairs, followed by non-ICU inpatients and outpatients, U.S. ghest resistance rates in selected drug-bug pairs, followed by non-ICU inpatients and outpatients, U.S. ICU patients have the highest resistance rates in selected drug bug pairs, followed by non ICU inpatients and outpatients, U.S. 1999 2002 Figure 4 ICU patients have the highest resistance rates in selected drug-bug pairs, followed by non-ICU inpatients and outpatients, U.S. 1999–2002. lower than it is in the other two countries against similar quinolone drugs (Table 5 and Table 10). This is probably because ECO resistance may have virtually reached equi- librium in China by the beginning of the study period; hence it didn't grow much in subsequent years. Kuwait teaching hospital, Mubarak Al-Kabeer Hospital, which serves a catchment area representing about 60% of Kuwait's population. We report that data for the first time here and in a companion paper [31] (see Tables 6, 7, 8, 9).The average resistance level for all surveyed bacteria was about 27% from 1999 to 2003 (Table 10), higher than the 17% for the U.S. and about the same as the 28% China. As for the other two countries, resistance appears to be growing in Kuwait. teaching hospital, Mubarak Al-Kabeer Hospital, which serves a catchment area representing about 60% of Kuwait's population. We report that data for the first time here and in a companion paper [31] (see Tables 6, 7, 8, 9).The average resistance level for all surveyed bacteria was about 27% from 1999 to 2003 (Table 10), higher than the 17% for the U.S. and about the same as the 28% China. As for the other two countries, resistance appears to be growing in Kuwait. That resistance does not grow without bound highlights the importance of comparing the current prevalence of resistance in the three countries. After all, the prevalence of resistance reflects the risk of a drug-resistant infection for any given patient. A low rate of growth is small conso- lation if patients already face a high baseline risk of a acquiring an expensive, debilitating and even potentially untreatable "superbug" infection. Page 9 of 14 (page number not for citation purposes) Discussion: Comparing antibiotic resistance in China, the U.S. and Kuwait In China, resistance rates exhibit a clear and rapid upward trend. In the U.S., resistance currently appears to grow at a more leisurely pace. Kuwait seems to be somewhere in between. It is important to note that the pace of growth may depend on the whether resistance to a particular anti- biotic has reached a potential equilibrium. As shown in the previous data, the 3% resistance growth rate of ECO against Ciprofloxacin in China (Table 1), is considerably The prevalence of resistance also substantially differs across countries, although as noted previously, surveil- lance data is far from ideal in capturing the true scope of the problem. As shown in Table 11, using the data cur- rently available, China has far higher prevalence of resist- Page 9 of 14 (page number not for citation purposes) Page 9 of 14 (page number not for citation purposes) http://www.globalizationandhealth.com/content/2/1/6 http://www.globalizationandhealth.com/content/2/1/6 Globalization and Health 2006, 2:6 Table 6: Resistance trend in isolates of Salmonella spp. over 5 years in Kuwait Antibiotic Percentage (%) of resistant isolates in: 1999 (n = 216) 2000 (n = 215) 2001 (n = 129) 2002 (n = 167) 2003 (n = 165) Amikacin 0 0 0 0 0 Ampicillin 6 12 7 25 26 Amoxicillin- clavulanate 5 10 7 2 0 Cefotaxime 0 1 0 1 0 Ceftriaxone 0 1 0 2 0 Cefuroxime 1 1 0 27 41 Cephalexin 2 10 37 57 50 Chloramphenicol 8 21 0 18 18 Ciprofloxacin 0 0 14 10 16 TMP/SMX 8 8 10 20 20 Gentamicin 6 1 0 42 42 Imipenem 0 0 0 0 0 Meropenem 0 0 0 0 0 Piperacillin 6 13 13 23 25 Piperacillin/ tazobactam 0 0 0 0 0 No ESBL-producing strain has been isolated so far Table 6: Resistance trend in isolates of Salmonella spp. over 5 years in Kuwait No ESBL-producing strain has been isolated so far No ESBL-producing strain has been isolated so far ant bacteria: MRSA, penicillin resistant SPN (PRSP) and vancomycin-resistant Enterococcus spp (VRE) [32-34]. Interestingly, each country has its own most problematic resistance culprit. For China, MRSA is the biggest threat, where resistance among hospital-acquired infections reaches almost 90%, the highest among the five countries. For the U.S., VRE is high. VRE growth in the U.S. can be traced to the late 1980s and is probably among the highest in the world. For Kuwait, PRSP is considerable. Discussion: Comparing antibiotic resistance in China, the U.S. and Kuwait Both Tai- wan and Japan are also troubled by at least one of these three resistant bacteria. ance for all the bacteria studied. For example, in China resistance of SPN to one of the oldest antibiotics, erythro- mycin, reaches 73%, while the figure for Kuwait is only 23%. A challenge for the U.S. is the exceptionally high level of Vancomycin-Resistant Enterococcus spp (VRE). In the U.S., 53% of Shigella spp are resistant to Trimetho- prim/Sulfamethoxazole (TMP/SMX), in contrast to 0% in both of the other countries. These examples suggest that severity of resistance may be correlated with volume of usage. Vancomycin is less affordable in both China and Kuwait, presumably resulting in less usage in those coun- tries. Resistance correlations Table 12 compares the three countries with Japan and Tai- wan regarding prevalence of three important drug-resist- How similar or different are resistance patterns in differ- ent countries? Does transmission travel across national Table 7: Resistance trend in isolates of Streptococcus pneumoniae over a 5-year period in Kuwait Antibiotics Percentage (%) of resistant isolates in: 1999 (n = 78) 2000 (n = 61) 2001 (n = 73) 2002 (n = 66) 2003 (n = 90) Cefotaxime 0 0 4 5 6 Ceftriaxone 0 0 3 5 4 Cefuroxime 0 0 8 9 41 Cephalexin 0 0 NT NT NT Chloramphenicol 3 5 25 5 0 Erythromycin 16 20 23 26 30 Imipenem 0 0 0 0 0 Penicillin 32 38 46 52 54 Teicoplanin 0 0 0 0 0 Vancomycin 0 0 0 0 0 NT = not tested Table 7: Resistance trend in isolates of Streptococcus pneumoniae over a 5-year period in Kuwait http://www.globalizationandhealth.com/content/2/1/6 http://www.globalizationandhealth.com/content/2/1/6 Globalization and Health 2006, 2:6 Table 8: Percentage of Enterococcus species resistant to often-tested antibiotics over 5 years in Kuwait Antibiotic Percentage (%) of resistant isolates in: 1999 (n = 370) 2000 (n = 335) 2001 (n = 322) 2002 (n = 248) 2003 (n = 212) Ampicillin 1 1 3 2 0 Erythromycin 59 78 77 75 92 Gentamicin 26 36 61 52 98 Nitrofurantoin 2 2 2 36 86 Norfloxacin 36 47 47 NT NT Penicillin 16 38 35 53 85 Teicoplanin 0 0 0 1 0 Vancomycin 1 0 0 2 0 NT = not tested Table 8: Percentage of Enterococcus species resistant to often-tested antibiotics over 5 years in Kuwait example, resistance rates in China are much more strongly correlated with those in Kuwait than those in the U.S. This correlation pattern suggests that at least in the short run, resistance in a country is more likely to be determined by endogenous factors (such as strictness of practices for pre- scribing drugs). In the long run, the frequency and magni- tude of contacts among nations with different resistance problems is likely to be critical. Because Kuwait and China are relatively isolated countries, it is less surprising that their antibiotic resistance problems show domestic char- acters. However, as we expect them to be opening more to the world, particularly China, the problem may worsen when these countries can increasingly export and import antibiotic resistance. Resistance correlations SAU Average Resistanc e Average Growth Average Annual Resistance 13 8 5 31 45 65 37 8 27 17 Table 10: Average Resistance Levels of Major Bacteria in Kuwait, 1999–2003 whether countries can control their own resistance prob- lems, and also avoid importing the problem from abroad. past decade and continues to show upward momentum in recent years. The original U.S. NNIS reported resistance rates to either one of the Cef3 drugs, i.e. ceftazidime, cefotaxime or ceftriaxone. We assume the same rates for each drug. Based on surveillance of ICU patients * TMP/SMX = Trimethoprim/Sulfamethoxazole Resistance correlations China, the most populous country in the world and an economy with the highest growth, is par- ticularly likely to exacerbate the problem. As illustrated in Figure 1, the number of Chinese departures to overseas destinations has been growing at increasing rates in the borders as humans do? If so, do countries' resistance pat- terns converge? To begin to examine this issue, we con- struct coefficients of resistance correlation among China, U.S. and Kuwait. We rank resistance rates for 24 bug-drug pairs and define perfect correlation as each bug-drug pair displaying the same resistance rank. Perfect negative cor- relation exists if the ranks in two countries go in precisely the opposite order. Table 13 reports the correlation coeffi- cient for each pair of countries. The statistic by definition is bounded between -1 and 1, where -1 means perfect dis- agreement while 1 means perfect agreement. Thus the big- ger the statistic, the more correlated two countries' resistance patterns are. Of course, methods for aggregation and comparing pat- terns of resistance across countries and over time should be improved, and applied more fruitfully with better data from increased local and global surveillance. But even this preliminary analysis reveals some interesting patterns. For Table 9: Percentage of Staphylococcus aureus resistant to often-tested antibiotics over 5 years in Kuwait Antibiotic Percentage (%) of resistant isolates in: 1999 (n = 648) 2000 (n = 595) 2001 (n = 484) 2002 (n = 420) 2003 (n = 286) Ampicillin 96 100 98 96 98 Amoxicillin-clavulanic acid 6 33 27 22 29 Cephalexin 33 30 25 36 34 Ciprofloxacin 10 35 30 45 50 Clindamycin 18 24 20 20 27 Cloxacillin 23 24 9 22 17 Erythromycin 38 34 26 28 27 Fusidic acid NA 20 19 64 27 Gentamicin 25 21 16 24 27 Methicillin 23 24 9 22 17 Penicillin 95 95 99 96 99 Teicoplanin 0 0 0 0 0 TMP/SMX 24 27 31 18 94 Vancomycin 0 0 0 0 0 Table 9: Percentage of Staphylococcus aureus resistant to often-tested antibiotics over 5 years in Kuwait e of Staphylococcus aureus resistant to often-tested antibiotics over 5 years in Kuwait Page 11 of 14 (page number not for citation purposes) http://www.globalizationandhealth.com/content/2/1/6 http://www.globalizationandhealth.com/content/2/1/6 Globalization and Health 2006, 2:6 Table 10: Average Resistance Levels of Major Bacteria in Kuwait, 1999–2003 unit: % ECO KPN PAE SPN Shigella spp. Salmonella spp. Enterococ cus spp. p *** TMP/SMX = Trimethoprim/Sulfamethoxazole Conclusion No doubt, there are also complex interactions with levels of economic well- being. Drugs become more affordable as countries become richer, but they are likely to be given out more carefully, particularly since concerns about resistance also increase. The critical question for policy is We have outlined the nature of the antimicrobial resist- ance problem as an important health and cost issue for three quite disparate nations, and by inference for a broad swath of the world's population. Surprisingly, this issue Table 11: Resistance rates in China, U.S. and Kuwait, hospital surveillance data for 2001 From Tables 1,2,3,8 and 9; Unit: % Bacterium(a) Antibiotic(s) Pair China U.S. Kuwait SAU Methicillin A 37 38 9 SPN Erythromycin B 73 19 23 Cefotaxime C 0 16 4 Enterococcus spp Vancomycin D 4 10 0 ECO Ceftazidime E 9 1* 5 Cefotaxime F 18 1* 1 Ceftriaxone G 21 1* 1 Ciprofloxacin/ Ofloxacin H 56 3 26 PAE Ceftazidime I 17 9 27 Ciprofloxacin/ Ofloxacin J 27 28 31 KPN Ceftazidime K 9 4* 14 Cefotaxime L 17 4* 13 Ceftriaxone M 20 4* 13 Ciprofloxacin N 18 12[27] 18 Salmonella spp Amoxicillin- clavulanate O 10 4 7 Ceftriaxone P 5 1 0 Ciprofloxacin Q 0 0.4 10 TMP/SMX* R 0 3 0 Gentamicin S 10 2 0 Shigella spp Amoxicillin- clavulanate T 35 2 20 Ceftriaxone U 6 0 0 Ciprofloxacin V 6 0 0 TMP/SMX W 0 53 0 Gentamicin X 18 0.2 0 Average 17 7 9 * The original U.S. NNIS reported resistance rates to either one of the Cef3 drugs, i.e. ceftazidime, cefotaxime or ceftriaxone. We assume the same rates for each drug. Table 11: Resistance rates in China, U.S. and Kuwait, hospital surveillance data for 2001 Page 12 of 14 (page number not for citation purposes) http://www.globalizationandhealth.com/content/2/1/6 http://www.globalizationandhealth.com/content/2/1/6 Globalization and Health 2006, 2:6 Table 12: MRSA, PRSP & VRE in Selected Countries Table 12: MRSA, PRSP & VRE in Selected Countries Unit: % MRSA (HAI only) PRSP VRE China 89 (2001) 27 (2001) 0 (2001) U.S. 16 (2001) 26 (2001) 0.3 (1989), 8 (1993), 12.8 (2001) in ICU Kuwait 9 (2001) 46 (2001) 0 (2001) Japan [33] 60–80% (1999) 11–40 (1999) n/a Taiwan [34] n/a 69 (2000) 2 (2000) tions about how to coordinate an effective international response [35]. Conclusion virtually never receives prominent attention at the national or international level, despite its scope and potentially devastating impact on global public health in the coming decades. Future research should develop better methods of data aggregation, explore the patterns of drug resistance across more countries, analyze the determinants of transmission of drug resistance across national boundaries, and assess how those determinants are progressing. Individuals eve- rywhere would benefit if far greater attention were paid to the problem of antimicrobial resistance. We examined antimicrobial resistance data for China, Kuwait, and the United States. In each country, we looked at specific infectious agents and their resistance to partic- ular antibiotics or other antimicrobials. Though an upward trend of resistance is found broadly, the patterns of correlation between countries' resistance rates suggest predominantly independent profiles. But we would expect greater convergence as globalization increases con- tacts between different nations' populations, raising ques- References 1. Kimball AM, Arima Y, Hodges RH: Trade related infections: far- ther, faster, quieter. Globalization and Health 2005, 1:3. 1. Kimball AM, Arima Y, Hodges RH: Trade related infections: far- ther, faster, quieter. Globalization and Health 2005, 1:3. q 2. Hodges RH, Kimball AM: The global diet: trade and novel infec- tions. Globalization and Health 2005, 1:4. 2. Hodges RH, Kimball AM: The global diet: trade and novel infec- tions. Globalization and Health 2005, 1:4. 28. National Nosocomial Infections Surveillance System 2001 [http://www.cdc.gov/ncidod/hip/SURVEILL/nnis_pubs.HTM.]. report 3. Wilson KH, Blitchington RB: Human colonic biota studied by ribosomal DNA sequence analysis. Appl Environ Microbiol 62(7):2273-2278. 29. National Antimicrobial Resistance Monitoring System for Enteric Bacteria 2002 [http://www.cdc.gov/narms/]. report 30. Active Bacterial Core Surveillance 2001 [http://www.cdc.gov/ ncidod/dbmd/abcs/reports.htm.]. report 4. Institute of Medicine: Emerging infections: microbial threats to health in the United States. National Academy Press 1994. 31. Eggleston KE, Rotimi V, Ghayda A, Zhang R, Zeckhauser RJ: An eco- nomic analysis of antimicrobial resistance in Kuwait. J Antimi- crobial Chemotherapy . Forthcoming health in the United States. National Academy Press 1994. 5. Wilson KH, Blitchington RB: Human colonic biota studied by ribosomal DNA sequence analysis. Appl Environ Microbiol 62(7):2273-8. 32. Neuhauser MM: Antibiotic resistance among Gram-negative bacilli in US intensive care units: implications for fluoroqui- nolone use. JAMA 2003, 289(7):885-888. ( ) 6. Davies J: Bacteria on the rampage. Nature 1996, 383:219-220. J p g 7. Field HJ, Coen DM: Pathogenicity of herpes simplex virus mutants containing drug resistance mutations in the viral DNA polymerase gene. J Virol 1986, 60(1):286-289. J ( ) 33. Sun Z: Antimicrobial resistance and clinical interventions. Adverse Drug Reaction J 2003, 3:151-155. p y g J ( ) 8. Muder RR, Brennen C, Wagener MM, et al.: Temporal shifts in traits of Vibrio cholerae strains isolated from hospitalized patients in Calcutta: a 3-year (1993–1995) analysis. J Clin Microbiol 1996, 34:2537-2543. g J 34. McDonald LC, Lauderdale T, et al.: The status of antimicrobial resistance in Taiwan among Gram-positive pathogens: the Taiwan Surveillance of Antimicrobial Resistance (TSAR) programme 2000. Int J Antimicrob Agents 2004, 23(4):362-370. 35. Fidler DP: Globalization, international law, and emerging infectious diseases. Emerg Infect Dis 1996, 2(2):77-84. 34. McDonald LC, Lauderdale T, et al.: The status of antimicrobial resistance in Taiwan among Gram-positive pathogens: the Taiwan Surveillance of Antimicrobial Resistance (TSAR) programme 2000. Int J Antimicrob Agents 2004, 23(4):362-370. 9. http://www.globalizationandhealth.com/content/2/1/6 http://www.globalizationandhealth.com/content/2/1/6 Globalization and Health 2006, 2:6 22. Authors' contributions 22. Haley RW, Morgan WM, Culver DH, et al.: Update from the SENIC project. Hospital infection control: Recent progress and opportunities under prospective payment. Am J Infect Con- trol 1985, 13:97-108. RFZ assembled the data, carried out the analysis and drafted the manuscript. KE and RJZ conceived of the study, participated in its design and coordination, and helped to draft the manuscript. VR provided the Kuwait data and helped to draft the manuscript. All authors read and approved the manuscript. 23. Duckworth GJ: Diagnosis and management of methicillin- resistant Staphylococcus aureus infection. British Medical Jour- nal 1993, 307:1049-1052. 24. Tsiodras S, Gold H, et al.: Linezolid resistance in a clinical isolate of Staphylococcus aureus. Lancet 2001, 358:207-208. 25. Chen H, Ma Y, National Antimicrobial Resistance Surveillance Center, et al.: The Changing Patterns of Ciprofloxacin Resist- ant Bacteria Strains Isolated from Fifty Hospitals in China from 1994 to 2000. Chin J Infect Chemotherapy 2002, 2(4):43-45. Competing interests The author(s) declare that they have no competing inter- ests. Table 13: Ranks of resistance rates in China, U.S. and Kuwait, 2001(Rank correlations at bottom of table) Bacterium(a) Antibiotic(s) China U.S. Kuwait SAU Methicillin 3 2 11 SPN Erythromycin 1 4 4 Cefotaxime 21 5 14 Enterococcus spp Vancomycin 20 7 17 ECO Ceftazidime 15 17 13 Cefotaxime 8 18 15 Ceftriaxone 6 19 16 Ciprofloxacin/Ofloxacin 2 13 3 PAE Ceftazidime 11 8 2 Ciprofloxacin/Ofloxacin 5 3 1 KPN Ceftazidime 16 9 7 Cefotaxime 12 10 8 Ceftriaxone 7 11 9 Ciprofloxacin 9 6 6 Salmonella spp Amoxicillin-clavulanate 13 12 12 Ceftriaxone 19 20 18 Ciprofloxacin 22 21 10 TMP/SMX 23 14 19 Gentamicin 14 15 20 Shigella spp Amoxicillin-clavulanate 4 16 5 Ceftriaxone 17 23 21 Ciprofloxacin 18 24 22 TMP/SMX 24 1 23 Gentamicin 10 22 24 Correlation Coefficients CHN_US: 0.18 US_KW: 0.46 CHN_KW: 0.60 ce rates in China, U.S. and Kuwait, 2001(Rank correlations at bottom of table) able 13: Ranks of resistance rates in China, U.S. and Kuwait, 2001(Rank correlations at bottom of table) Acknowledgements g The authors gratefully acknowledge financial support from the Kuwait g The authors gratefully acknowledge financial support from the Kuwait J py ( ) 26. Li J, Li G, Wang J: Surveillance on gram-positive bacteria iso- lated from patients with hospital acquired infections or com- munity acquired infections. Natl Med J China 2003, 83(5):365-374. (in Chinese) The authors gratefully acknowledge financial support from the Kuwait Foundation for the Advancement of Sciences through the John F. Kennedy Foundation for the Advancement of Sciences through the John F. Kennedy School of Government at Harvard University. g J y School of Government at Harvard University. School of Government at Harvard University. School of Government at Harvard University. ( ) ( ) 27. Li J, Li G, Wang J: Surveillance on gram-positive bacteria iso- lated from patients with hospital acquired infections or com- munity acquired infections. Natl Med J China 2003, 83(12):1035-1045. (in Chinese) References Publish with BioMed Central and every scientist can read your work free of charge References Bisognano C, et al.: Increased expression of fibronectin-binding proteins by fluoroquinolone-resistant Staphylococcus aureus exposed to subinhibitory levels of ciprofloxacin. Anti- microb Agents Chemother 1997, 41(5):906-913. p g J g ( ) 35. Fidler DP: Globalization, international law, and emerging infectious diseases. Emerg Infect Dis 1996, 2(2):77-84. g ( ) 10. World Health Organization: WHO Global Strategy for Contain- ment of Antimicrobial Resistance. Geneva 2001. 11. Arason VA, Kristinsson KG, et al.: Do antimicrobials increase the carriage rate of penicillin resistant pneumococci in children? Cross sectional prevalence study. BMJ 1996, 313:387-91. p y J 12. Seppala H, Klaukka T, et al.: The effect of changes in the con- sumption of macrolide antibiotics on erythromycin resist- ance in group A streptococci in Finland. Finnish Study Group for Antimicrobial Resistance. N Engl J Med 1997, 337:441-6. g J 13. Levy SB, Fitzgerald GB, Macone AB: Spread of Antibiotic-Resist- ant Plasmids from Chicken to Chicken and from Chicken to Man. Nature 1976, 260(5546):40-42. ( ) 14. Levy SB, Fitzgerald GB, Macone AB: Changes in Intestinal Flora of Farm Personnel after Introduction of a Tetracycline-Supple- mented Feed on a Farm. N Engl J Med 1976, 295:583-588. g J 15. Witte W: Selective pressure by antibiotic use in livestock. Int J Antimicrob Agents 2000, 16(Suppl 1):S19-24. Review Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral 16. Breathnach AS, de Ruiter A, Holdsworth GM, et al.: An outbreak of multi-drug-resistant tuberculosis in a London teaching hos- pital. J Hosp Infect 1998, 39(2):111-117. Publish with BioMed Central and every scientist can read your work free of charge p J p ( ) 17. Hannan MM, Azadian BS, Gazzard BG, et al.: Hospital infection control in an era of HIV infection and multi-drug resistant tuberculosis. J Hosp Infect 2000, 44(1):5-11. J p ( ) 18. Martin MA: Methicillin-resistant Staphylococcus aureus: the persistent resistant nosocomial pathogen. Curr Clin Top Infect Dis 1994, 14:170-191. 19. Wang F, Zhu D, et al.: Surveillance of bacterial resistance in Shanghai. Chin J Infect Chemother 2002, 2(1):1-9. 20. Cox RA, Conquest C, Malaghan C, et al.: A major outbreak of methicillin-resistant Staphylococcus aureus caused by new phage-type (EMRSA-16). J Hosp Infect 1995, 29:87-106. 21. Astagneau P, et al.: Cost of antimicrobial treatment for nosoco- mial infections based on a French prevalence survey. J Hosp Infect 1999, 42:303-312.
https://openalex.org/W2114961148	https://hal.science/hal-02612981/document	English	null	A New Snake Skull from the Paleocene of Bolivia Sheds Light on the Evolution of Macrostomatans	PloS one	2,013	cc-by	7,292	A New Snake Skull from the Paleocene of Bolivia Sheds Light on the Evolution of Macrostomatans Agustin Scanferla, Hussam Zaher, Fernando Novas, Christian de Muizon, Ricardo Céspedes in Scanferla, Hussam Zaher, Fernando Novas, Christian de Muizon, Ricardo Céspedes To cite this version: Agustin Scanferla, Hussam Zaher, Fernando Novas, Christian de Muizon, Ricardo Céspedes. A New Snake Skull from the Paleocene of Bolivia Sheds Light on the Evolution of Macrostomatans. PLoS ONE, 2013, 8 (3), pp.e57583. 10.1371/journal.pone.0057583. hal-02612981 Abstract Macrostomatan snakes, one of the most diverse extant clades of squamates, display an impressive arsenal of cranial features to consume a vast array of preys. In the absence of indisputable fossil representatives of this clade with well-preserved skulls, the mode and timing of these extraordinary morphological novelties remain obscure. Here, we report the discovery of Kataria anisodonta n. gen. n. sp., a macrostomatan snake recovered in the Early Palaeocene locality of Tiupampa, Bolivia. The holotype consists of a partial, minute skull that exhibits a combination of booid and caenophidian characters, being the presence of an anisodont dentition and diastema in the maxilla the most distinctive trait. Phylogenetic analysis places Kataria basal to the Caenophidia+Tropidophiidae, and represents along with bolyeriids a distinctive clade of derived macrostomatans. The discovery of Kataria highlights the morphological diversity in the maxilla among derived macrostomatans, demonstrating the relevance of maxillary transformations in the evolution of this clade. Kataria represents the oldest macrostomatan skull recovered, revealing that the diversification of macrostomatans was well under way in early Tertiary times. This record also reinforces the importance of Gondwanan territories in the history of snakes, not only in the origin of the entire group but also in the evolution of ingroup clades. tation: Scanferla A, Zaher H, Novas FE, de Muizon C, Ce´spedes R (2013) A New Snake Skull from the Paleocene of Bolivia Sheds Lig crostomatans. PLoS ONE 8(3): e57583. doi:10.1371/journal.pone.0057583 aher H, Novas FE, de Muizon C, Ce´spedes R (2013) A New Snake Skull from the Paleocene of Bolivia Sheds Light on the Evolution o NE 8(3): e57583. doi:10.1371/journal.pone.0057583 Editor: Richard J. Butler, Ludwig-Maximilians-Universita¨t Mu¨nchen, Germany Editor: Richard J. Butler, Ludwig-Maximilians-Universita¨t Mu¨nchen, Germany Received September 21, 2012; Accepted January 21, 2013; Published March 1, 2013 Received September 21, 2012; Accepted January 21, 2013; Published March 1, 2013 Copyright: 2013 Scanferla et al. This is an open-access article distributed under the terms of the Creative Comm unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: 2013 Scanferla 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. Abstract Funding: Research was funded by Museo de Historia Natural de Cochabamba ‘‘Alcide dOrbigny’’, National Geographic Society (grant 7163-01), Conicet (grant PIP 5153), Agencia Nacional de Promocio´n Cientı´fica y Tecnolo´gica (grants PICT 13803 and 53). HZ is supported by grants from Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (2011/50206-9) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (303545/2010-0 and 565046/2010-1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: agustin_scanferla@yahoo.com.ar in several independent molecular phylogenies, precludes the establishment of a clear evolutionary scenario and suggests that additional evidence is needed to clarify this issue. Meanwhile, we prefer to consider here a monophyletic Macrostomata, as suggested by all previous morphological analyses [2,4,5,6,7], and corroborated by the most exhaustive morphology-based phylogeny of Squamata [15]. According to these preferred phylogenetic proposals, Macrostomata includes the basal forms Xenopeltidae and Loxocemidae, and two more derived subclades that display distinct morphologies and ecological requirements: 1) a group formed by the ungaliophiines, erycines, pythonines, and boines; and 2) a group including bolyeriids, tropidophiids, and caenophi- dians (acrochordids and colubroideans). Despite their current impressive diversity and cosmopolitan distribution, nearly all that we know about the evolution of the macrostomatan cranial bauplan comes from studies focused in recent forms [11,16,17]. Indeed, due to the lack of well-preserved cranial fossil remains, little is known about the origin and diversification of their highly specialized cranial features. HAL Id: hal-02612981 https://hal.science/hal-02612981v1 Submitted on 19 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. Agustı´n Scanferla1, Hussam Zaher2, Fernando E. Novas3, Christian de Muizon4, Ricardo Ce´spedes5 1 Consejo Nacional de Investigaciones Cientı´ficas Y Te´cnicas, Instituto de Bio y Geociencias del NOA, Museo de Ciencias Naturales de Salta, Salta, Argentina, 2 Museu de Zoologia, Universidade de Sa˜o Paulo, Sa˜o Paulo, Brasil, 3 Laboratorio de Anatomı´a Comparada y Evolucio´n de los Vertebrados, Museo Argentino de Ciencias Naturales ‘‘Bernardino Rivadavia’’, Buenos Aires, Argentina, 4 UMR 7207 CNRS, CP 38, De´partement Histoire de la Terre, Paris, France, 5 Museo de Historia Natural ‘‘Alcide DOrbigny’’, Cochabamba, Bolivia March 2013 \| Volume 8 \| Issue 3 \| e57583 Phylogenetic Analysis The character-taxon matrix used in the phylogenetic analysis is mainly based on a published phylogenetic analysis [7], with the addition of Kataria and 2 new characters. Thus, the reported analysis results in a data matrix of 156 characters scored across 23 taxa (Text S1, Dataset S1). All characters were treated as unordered, as in the original phylogenetic analysis [7]. Locality and horizon. Tiupampa locality, Mizque province of the department of Cochabamba, Bolivia. Medium-grained sandstones of the middle levels of Santa Lucı´a Formation, Early Paleocene (Danian [22]). Diagnosis. A small, derived macrostomatan snake that can be distinguished from all other members of Serpentes by the following combination of characters: an elongated vomer with a reduced contribution to the vomeronasal fenestra; one foramen piercing the cavity housing Jacobson’s organ; maxilla with 21 tooth positions and the posterior most tooth separated by a diastema from the others; ectopterygoid with a small medial process and a ventral articular surface with the pterygoid; a broad choanal process of the palatine; optic fenestra formed by both frontal and parietal. Diagnosis. A small, derived macrostomatan snake that can be distinguished from all other members of Serpentes by the following combination of characters: an elongated vomer with a reduced contribution to the vomeronasal fenestra; one foramen piercing the cavity housing Jacobson’s organ; maxilla with 21 tooth positions and the posterior most tooth separated by a diastema from the others; ectopterygoid with a small medial process and a ventral articular surface with the pterygoid; a broad choanal process of the palatine; optic fenestra formed by both frontal and parietal. We analysed our dataset using TNT [18,19] with a heuristic search of 1000 replicates of Wagner trees followed by tree bisection-reconnection (TBR) branch swapping. All characters were equally weighted. Zero length branches were collapsed if they lack support under any of the most parsimonious reconstructions. Also, two alternative support measures (Bremer support [20] and bootstrap resampling) were used to evaluate the robustness of the nodes of the obtained most parsimonious trees (see Text S1). Additionally, the morphological dataset was analyzed with the 13 extant terminal taxa constrained with a backbone formed by the topology derived from the molecular analysis performed by Wiens and colleagues [21]. This analysis served to test the effect of a molecular topology on our dataset and, more specifically, on the phylogenetic position of Kataria. New Paleocene Macrostomatan Snake from Bolivia this relevant clade of snakes. It further provides a relevant calibration point to discuss the evolutionary timing of advanced terms of macrostomatan snakes. Kataria anisodonta Scanferla, Zaher, Novas, Muizon and Ce´spedes sp. nov. urn:lsid:zoobank.org:act:EEF2F3CF-6CBA- 447A-953B-7539F3C90388. Etymology. The generic name derives from the Aymara word ‘‘Katari’’, a winged mythological snake of South American Andean people. The specific name refers to the particular maxillary dentition, combining the Greek words ‘‘aniso’’ (hetero- Ontogenetic Stage of the Specimen Estimation of the ontogenetic stage of a fossil snake skull is problematic because there are few studies on postnatal ontogeny in snakes. Those that do exist all focus on allometric variations in skull elements [23,24,25]. These works, however, do not provide strong grounds to assess the ontogenetic stage of Kataria because they are based on quantitative characters with no descriptions of useful discrete features. At a first glance, the tiny size (Table 1) and poorly developed parietal table and sagittal crest appear to indicate that the holotypic specimen of Kataria represents a juvenile postnatal ontogenetic stage. However, adult specimens of several small-sized macrostomatan taxa (e.g. Lichanura trivirgata, Apostolepis spp.), approach Kataria in their size and parietal morphology. Hence, somatically mature small-bodied snakes exhibit skull morphology similar to juveniles of larger taxa. Methods All necessary permits were obtained for the described study from Comite´ de Preservacio´n del Patrimonio Departamental (Cochabamba department, Bolivia), which complied with all relevant regulations. geneous) and ‘‘donta’’ (tooth). geneous) and ‘‘donta’’ (tooth). Holotype. MHNC 13323 (Museo de Historia Natural de Cochabamba ‘‘Alcides Dorbigny, Cochabamba, Bolivia), an articulated incomplete skull consisting of a left vomer, incomplete left septomaxilla, left maxilla, left ectopterygoid, left palatine, the anterior tip of the left pterygoid, left postorbital, both frontals, parietal, and parasphenoid rostrum (Fig. 1). Nomenclatural Acts The electronic edition of this article conforms to the require- ments of the amended International Code of Zoological Nomen- clature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix ‘‘http://zoobank.org/’’. The LSID for this publication is: urn:lsid:zoobank.org:pub:30F0320E-11D7-450A-B108- 24EA5D56827C. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS. Based on personal observations, ontogenetic transformations in the postorbital bone may be useful in distinguishing ontogenetic stages in macrostomatan snakes. Like those of several adult macrostomatan skulls examined (see Text S1), the postorbital of Kataria displays a distinct thickening of the postorbital shaft, and the posterior process of the dorsal head is enlarged. We consider that these anatomical traits present in the postorbital bone, together with the advanced state of ossification observed in this minute skull, suggest an adult postnatal ontogenetic stage for the holotype. 24EA5D56827C. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS. Phylogenetic Analysis The nine fossil taxa in the dataset were pruned (using ‘‘pruntax’’ command in TNT) and allowed to insert freely into their optimal positions during the constrained analysis. The molecular tree was constructed using the ‘‘edit’’ command in TNT. The constrained analysis was performed using a heuristic tree search with the molecular tree enforced as a backbone with ‘‘force’’ and ‘‘cons’’ commands in TNT. Introduction In recent years, the discovery of new and nearly complete fossil specimens as well as the reanalysis of previously known materials has dramatically improved our knowledge about the evolution of snakes [1,2,3,4,5,6,7,8]. Such renewed interest on ophidian evolution is based primarily on the study of fossil exemplars that preserved skull elements, an unusual situation for this group of squamates. However, their phylogenetic position fails to offer any clue about the evolution of the more advanced extant snake clades. Macrostomata constitutes the most diverse group of snakes today, including nearly all of the extant species [9]. This clade is characterized by a suite of exceptionally distinct skull traits responsible for an increased gape size that permits the ingestion of large preys [10,11,12]. Despite these numerous uniquely derived features, recent molecular studies [13,14] suggested that this diverse group constitutes a polyphyletic lineage, with tropidophiids clustering outside macrostomatans as the sister-group of Anilius, whereas remaining macrostomatans form a monophyletic unit that includes the rest of ‘‘anilioid’’ alethinophidians. These phyloge- netic hypotheses suggest that macrostomatan traits might have been lost or appeared independently twice within alethinophidian snakes. However, the unstable position of uropeltids (including Anomochilus and Cylindrophis) within macrostomatan snakes, shown Here we report a new fossil snake from Paleocene beds of Bolivia that emerges in our analysis as the sister-group of the clade formed by bolyeriids, tropidophiids, and caenophidians. The new fossil preserves the most complete and oldest macrostomatan skull found so far, filling an important gap in the evolutionary history of 1 March 2013 \| Volume 8 \| Issue 3 \| e57583 PLOS ONE \| www.plosone.org New Paleocene Macrostomatan Snake from Bolivia March 2013 \| Volume 8 \| Issue 3 \| e57583 PLOS ONE \| www.plosone.org New Paleocene Macrostomatan Snake from Bolivia The tip of the rear tooth is broken; however, the preserved portion is larger than the two teeth positioned just anterior to the toothless gap. The enamel surface of this tooth is not satisfactorily preserved, but it is possible to confirm that a groove is lacking (Fig. 2D). The left ectopterygoid is present and articulates with the maxilla (Fig. 1C– D). This bone bears a short shaft (Table 1), in constrast with the elongated shape present in tropidophiids and caenophidians. The anterior part, which has an angulated lateral margin, bears a small medial process similar to that present in many macrostomatan snakes. This region of the ectopterygoid overlaps the dorsomedial surface of the posterior tip of the maxilla. The caudal end of the ectopterygoid has a small, flattened region that articulates with the pterygoid when the latter is present. We here interpret that this region must have overlapped the pterygoid on its dorsal surface as in bolyeriids, tropidophiids and caenophidians. In macrostomatan snakes, the posterior region of the maxilla and the anterior head of the ectopterygoid form a horizontal overlapping joint. In Kataria, this articulation and the posterior articular surface of the ectopterygoid that contacts the pterygoid are in the same plane, supporting the dorsal ectopterygoid-pterygoid contact interpreted above. The general shape of the palatine bone resembles Anilius and tropidophiids, being characterized by a prominent dentiger- ous process, a broad-based choanal process, and a small laterally projected maxillary process pierced by a foramen that corresponds to the maxillary branch of trigeminal nerve (Fig. 1D). The ventral view reveals seven tooth positions, and preserved teeth are morphological similar to the marginal (maxillary) teeth. Un- fortunately, the poorly preserved region of the palatine-pterygoid contact avoids any interpretation of the nature of this articulation. A small fragment corresponds to the anteriormost part of the palatine ramus of the pterygoid. This fragment retains four tooth positions and one of these reveals the base of a tooth. The tooth sockets and the preserved fragmentary tooth on the anterior fragment are smaller than the more posterior palatine tooth sockets, suggesting that the gradual transition in the size of palatal teeth known in many booids [28] was absent in Kataria. The maxilla of Kataria is the element of the palatomaxillary apparatus that displays most apomorphic and intriguing traits. This bone is elongated (Table 1) and has a slightly recurved shape as in many colubroideans (Fig. New Paleocene Macrostomatan Snake from Bolivia New Paleocene Macrostomatan Snake from Bolivia Figure 1. The skull of Kataria anisodonta (MHNC 13323). Photographs and half-tone drawings in (A) left lateral, (B) right lateral, (C) dorsal and (D) ventral views. Dotted areas indicate matrix. chp, choanal process; ec, ectopterygoid; fr, frontal; ip, interchoanal process; mx, maxilla; mxp, maxillary process; op, optic foramen; p, parietal; pf, prefrontal; pl, palatine; plp, palatine process; po, postorbital; ps, parasphenoid; pt, pterygoid; sm, septomaxilla; v, vomer. doi:10.1371/journal.pone.0057583.g001 Figure 1. The skull of Kataria anisodonta (MHNC 13323). Photographs and half-tone drawings in (A) left lateral, (B) right lateral, (C) dorsal and (D) ventral views. Dotted areas indicate matrix. chp, choanal process; ec, ectopterygoid; fr, frontal; ip, interchoanal process; mx, maxilla; mxp, maxillary process; op, optic foramen; p, parietal; pf, prefrontal; pl, palatine; plp, palatine process; po, postorbital; ps, parasphenoid; pt, pterygoid; sm, septomaxilla; v, vomer. doi:10.1371/journal.pone.0057583.g001 present in Anilius and many macrostomatans except caenophidians [26]. A single foramen pierces the posterior wall of the cavity housing Jacobson’s organ, and the sidewall of this structure is formed largely by the septomaxilla rather than the vomer, a condition shared also with non-caenophidian snakes [26]. Contrary to the plesiomorphic condition present in basal macrostomatans, the vomeronasal cupola of Kataria is closed medially by an extensive medial contact between vomer and septomaxilla (Fig. 1B). The lateral flange of the septomaxilla is present, but somewhat crushed, and shows a broad base. The septomaxilla projects caudally to the posterior border of the vomer to form a postero-dorsally ascending flange that reaches the frontal medially. The posterior tip of the ascending flange of the septomaxilla is clearly rounded, and there are no traces of articular structures that link this bone with the frontal subolfactory process. A slight dorsal displacement of the snout complex gives the impression of a septomaxilla-frontal contact (Fig. 2). However, suspension of the snout unit probably occurred through soft tissues and/or the nasal bone (not preserved in Kataria) instead of the septomaxilla. is flat and ventro-dorsally thin with respect to the rest of the bone (Fig. 3C), and its dental (ventral) region is smooth and without traces of tooth sockets or interdental ridges [27]. A flexion can be seen in dorsal view starting just at the level of the last tooth. This trait is present in all opistoglyphous colubroids examined. New Paleocene Macrostomatan Snake from Bolivia 3C), differing from the condition present in other macrostomatans where the anterior third of the maxilla curves medially. The rounded anterior tip indicates no close association with the premaxilla, suggesting a loose, ligamen- tous connection between these elements. As in tropidophiids and caenophidians, the lateral surface lacks the lateral foramina present in many booids. The medial (palatine) process is situated in the middle region, between the 9th and 12th tooth positions. Although the prefrontal and palatine bones obscure part of its morphology, it is possible to verify the absence of the foramen for the passage of the maxillary branch of the trigeminal nerve, a derived condition shared with caenophidian snakes. The ectopterygoid process is weakly expressed as a small ventromedial projection, located at the level of the space between the rear tooth and the rest of the tooth row. There are 21 tooth positions, distributed in two clearly defined sections (Fig. 3C). The anterior tooth row has 20 tooth positions occupied by elongate, needle- shaped recurved teeth. These teeth gradually diminish towards the posterior region, in contrast with the condition present in rear- fanged colubroids where teeth are similar in size. Notably, there is one tooth separated from the rest of the tooth row by a conspicuous, toothless gap. The maxilla in this toothless region The preserved left prefrontal of Kataria is in articulation with the frontal, but slightly rotated medially (Fig. 1A–C). As in boines, bolyeriids and tropidophiids, the lachrymal duct is open ventrally (Fig. 2B). Also, there is no indication of the typical boid dorsal lappet. On its lateral side, the prefrontal exhibits a short lateral lamina, and this bone retains only a posterior contact with the maxilla. Although the anteriormost region of the lateral lamina is incomplete, it is possible that this structure exhibited an anterior projection similar to the one present in Casarea [29]. The postorbital bone is well developed, being slightly displaced from its original position. The shape of the dorsal head resembles the forked condition present in tropidophiids, with the difference that its long anterior process was clearly in contact with the dorsolateral edge of the frontal bone. The longitude that shows this bone and the acute shape of the ventral tip indicate that the postorbital terminated well dorsal to the ectopterygoid-maxillary joint. Table 1. Selected measurements of Kataria anisodonta. March 2013 \| Volume 8 \| Issue 3 \| e57583 Results The type specimen consists of a small articulated skull, with some elements (e.g. snout bones) barely displaced. Its anatomy reveals booid traits in combination with some apomorphic features present in tropidophiids and caenophidian snakes. Systematic Paleontology Serpentes Linnaeus 1758. Alethinophidia Nopcsa 1923. Macrostomata Mu¨ller 1831. Systematic Paleontology Serpentes Linnaeus 1758. Alethinophidia Nopcsa 1923. Macrostomata Mu¨ller 1831. The preserved snout bones of Kataria retain a typical ‘‘booid’’ morphology. The vomer is remarkably long, with well-developed vertical and horizontal posterior laminae (Fig. 1B), condition PLOS ONE \| www.plosone.org March 2013 \| Volume 8 \| Issue 3 \| e57583 March 2013 \| Volume 8 \| Issue 3 \| e57583 2 New Paleocene Macrostomatan Snake from Bolivia PLOS ONE \| www.plosone.org 3 March 2013 \| Volume 8 \| Issue 3 \| e57583 March 2013 \| Volume 8 \| Issue 3 \| e57583 3 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org New Paleocene Macrostomatan Snake from Bolivia New Paleocene Macrostomatan Snake from Bolivia Maxilla length 4,6 Ectopterygoid length 2,2 Palatine length 3,1 Frontal length 1,75 Postorbital height 0,9 Measurements are in mm; refers to estimated value. doi:10.1371/journal.pone.0057583.t001 PLOS ONE \| www.plosone.org Table 1. Selected measurements of Kataria anisodonta. Maxilla length 4,6 Ectopterygoid length 2,2 Palatine length 3,1 Frontal length 1,75 Postorbital height 0,9 Measurements are in mm; *refers to estimated value. doi:10.1371/journal.pone.0057583.t001 PLOS ONE \| www plosone org Table 1. Selected measurements of Kataria anisodonta. March 2013 \| Volume 8 \| Issue 3 \| e57583 March 2013 \| Volume 8 \| Issue 3 \| e57583 4 New Paleocene Macrostomatan Snake from Bolivia Figure 2. Details of the holotype specimen of Kataria anisodonta. (A) frontal view of the partial skull; (B) dorsolateral view of the left orbit; (C) ventral view of the palatal region; (D) scanning electron microscope image of the rear maxillary region. chp, choanal process; dot, ductus for olfactory tract; ec, ectopterygoid; fr, frontal; ip, interchoanal process; mfr, medial frontal flange; mx, maxilla; mxp, maxillary process; op, optic foramen; p, parietal; pf, prefrontal; pl, palatine; plp, palatine process; po, postorbital; ps, parasphenoid; pt, pterygoid; sm, septomaxilla; v, vomer. doi:10.1371/journal.pone.0057583.g002 Figure 2. Details of the holotype specimen of Kataria anisodonta. (A) frontal view of the partial skull; (B) dorsolateral view of the left orbit; (C) ventral view of the palatal region; (D) scanning electron microscope image of the rear maxillary region. chp, choanal process; dot, ductus for olfactory tract; ec, ectopterygoid; fr, frontal; ip, interchoanal process; mfr, medial frontal flange; mx, maxilla; mxp, maxillary process; op, optic foramen; p, parietal; pf, prefrontal; pl, palatine; plp, palatine process; po, postorbital; ps, parasphenoid; pt, pterygoid; sm, septomaxilla; v, vomer. doi:10.1371/journal.pone.0057583.g002 The frontals are slender bones that show complete (fused) interolfactory pillars (Fig. 2A), a condition shared with caenophi- dians [17]. In Kataria, as in the vast majority of macrostomatan snakes, the optic foramen lies between the frontal and parietal, excluding the parasphenoid rostrum. As in tropidophiids and caenophidians, the parietal of Kataria is longitudinally short, without the posterior supratemporal processes present in boid snakes. This bone bears small postorbital processes, clasped by the forked dorsal head of the postorbital. The overall shape of the preserved portion of the parasphenoid in Kataria is very similar to that of bolyeriids. This structure is elongate and slender, with concave ventral surface. Anteriorly, the parasphenoidal rostrum forms a ventrally projecting, narrow-based interchoanal process (Fig. 1B). Tropidophiidae and Caenophidia. New Paleocene Macrostomatan Snake from Bolivia Our phylogenetic results also retrieve tropidophiids and caenophidians as sister taxa, in contrast to the basal alethinophidian position of the former in all recent molecular analyses [13,14,21,30,31,32]. Despite the incomplete- ness of Kataria and of several other fossils added to the analysis, the obtained cladogram exhibits rather strong support values for several nodes (see electronic supplementary material). As in more recent morphological analyses [2,4,5,6,7,15], our results recovered the following two monophyletic sister-groups of advanced macrostomatans (i.e. excluding Xenopeltis and Loxocemus): 1) a clade composed by Ungaliophiidae+Erycinae+Boinae+Pytho- ninae, with a weak bootstrap support of 61% but moderate Bremer value of 5; and 2) a well-supported clade composed by bolyeriids, Kataria, tropidophiids, and caenophidian snakes, which received a strong bootstrap support of 82% but a low Bremer value of 3. This last group is supported by three unambiguous synapomorphies, all of which represent traits of the palatomax- illary arch: internal articulation of palatine with pterygoid short (70-.0), ectopterygoid contact with the pterygoid is expanded March 2013 \| Volume 8 \| Issue 3 \| e57583 Discussion Our additional constrained analysis conducted to test the effect of a molecular topology in the phylogenetic dataset resulted in two most parsimonious topologies. The strict consensus tree (Fig. 5) shows that all fossil taxa, except Kataria, were nested inside the enforced clade formed by the extant taxa Anilius and Tropidophiidae. Kataria retained its position as the sister-group of the clade formed by Acrochordidae and Colubroides, showing that enforcing a molecular tree as Phylogenetic Analysis The tree obtained from the phylogenetic analysis of parsimony (Fig. 4) placed Kataria deeply nested within derived macrostoma- tans, more precisely as the sister group of a clade composed by March 2013 \| Volume 8 \| Issue 3 \| e57583 March 2013 \| Volume 8 \| Issue 3 \| e57583 PLOS ONE \| www.plosone.org 5 New Paleocene Macrostomatan Snake from Bolivia Figure 3. Lateral view of maxillary bones showing differences in tooth row morphology of macrostomatan snakes. (A) the boid Eunectes notaeus, (b) the bolyeriid Casarea dussumieri [44], (C) Kataria anisodonta and (D) the opistoglyphous colubroid Philodryas trilineatus. Not to scale. amx, anterior maxilla; ecp, ectopterygoid process; plp, palatine process; pmx, posterior maxilla; soo, suborbital ossification. doi:10.1371/journal.pone.0057583.g003 Figure 3. Lateral view of maxillary bones showing differences in tooth row morphology of macrostomatan snakes. (A) the boid Eunectes notaeus, (b) the bolyeriid Casarea dussumieri [44], (C) Kataria anisodonta and (D) the opistoglyphous colubroid Philodryas trilineatus. Not to scale. amx, anterior maxilla; ecp, ectopterygoid process; plp, palatine process; pmx, posterior maxilla; soo, suborbital ossification. doi:10.1371/journal.pone.0057583.g003 Figure 3. Lateral view of maxillary bones showing differences in tooth row morphology of macrostomatan snakes. (A) the boid Eunectes notaeus, (b) the bolyeriid Casarea dussumieri [44], (C) Kataria anisodonta and (D) the opistoglyphous colubroid Philodryas trilineatus. Not to scale. amx, anterior maxilla; ecp, ectopterygoid process; plp, palatine process; pmx, posterior maxilla; soo, suborbital ossification. doi:10.1371/journal.pone.0057583.g003 a backbone did not affect the position of Kataria as recovered in our previous unconstrained analysis. significantly on the dorsal surface of the pterygoid body (76-.1), and maxilla with posteromedial (ectopterygoid) expansions in the posterior region (156-.1) (see Text S1 for a list of apomorphies for each clade). Morphology The result of this developmental phenomenon is that the posterior region of the maxilla evolves independently, and exhibits conspicuous morphological differences with respect to the anterior maxillary region in postnatal individuals, especially in tooth morphology. The presence of a maxillary diastema and the distinct shape of the rear tooth in the maxilla of Kataria suggest that a similar developmental process occurred during the development of this bone in Kataria. Significantly, other derived macrostomatans show anatomical innovations in the maxilla beyond the plesio- morphic condition present in the rest of macrostomatans (Fig. 3). Within macrostomatan snakes, bolyeriids exhibit a maxilla divided in anterior and posterior parts by a transverse movable joint [29] while Colubroides display a tremendous variety of maxillary forms related to a venom-delivery system [11,34,35]. The peculiar maxillary shape of Kataria contrasts the conservative maxillary morphology of booids, indicating that the maxillary element might maxillary lamina, recognizing the independent posterior dental lamina as responsible for the formation of the rear-fanged morphology characteristic of the endoglyptodont colubroideans (sensu [33]). The result of this developmental phenomenon is that the posterior region of the maxilla evolves independently, and exhibits conspicuous morphological differences with respect to the anterior maxillary region in postnatal individuals, especially in tooth morphology. The presence of a maxillary diastema and the distinct shape of the rear tooth in the maxilla of Kataria suggest that a similar developmental process occurred during the development of this bone in Kataria. Significantly, other derived macrostomatans show anatomical innovations in the maxilla beyond the plesio- morphic condition present in the rest of macrostomatans (Fig. 3). Within macrostomatan snakes, bolyeriids exhibit a maxilla divided in anterior and posterior parts by a transverse movable joint [29] while Colubroides display a tremendous variety of maxillary forms related to a venom-delivery system [11,34,35]. The peculiar maxillary shape of Kataria contrasts the conservative maxillary morphology of booids, indicating that the maxillary element might Another feature of Kataria shared with tropidophiids, bolyeriids, and caenophidians, is the dorsal articulation between the ectopterygoid and pterygoid bones. The ectopterygoid bone has a crucial role in the highly derived feeding mechanisms of snakes [10,11]. In macrostomatans with a lateromedial form of intraoral transport (booids), the lateral or laterodorsal immobile contact between these bones results in the functioning of each (left and right) palatomaxillary arch functions as a consolidated unit. Morphology With the exception of a few differences, the maxillary morphology of Kataria resembles that observed in many rear- fanged Colubroides, characterized by a tooth row composed of two toothed zones divided by a diastema. In a recent study on the development and evolution of snake fangs, Vonk et al. [16] proposed that the rear-fang condition found within colubroidean snakes is the product of a developmental decoupling of the dental March 2013 \| Volume 8 \| Issue 3 \| e57583 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org March 2013 \| Volume 8 \| Issue 3 \| e57583 6 New Paleocene Macrostomatan Snake from Bolivia Figure 4. Phylogenetic relationships of Kataria anisodonta. Temporally calibrated cladogram of the most parsimonious tree obtained in this analysis. Thick gray lines indicate stratigraphic range of known taxa (dashed area indicates that these records are based on vertebral remains). Dashed lines represent ghost lineages implied by the stratigraphic distribution of fossils with respect to the phylogenetic relationships shown here (note the exceptionally abundant ghost lineages for Macrostomata). Ages of first appearance for taxa used in the calibrated phylogeny are given in electronic supplementary material. Al, Albian; Ce, Cenomanian; Tu, Turonian; Co, Coniacian; Sa, Santonian; Cam, Campanian; Ma, Maastrichtian; Pal, Paleocene; Eoc, Eocene; Oli, Oligocene; Mi, Miocene; Pl-Ple, Plio-Pleistocene. doi:10.1371/journal.pone.0057583.g004 Figure 4. Phylogenetic relationships of Kataria anisodonta. Temporally calibrated cladogram of the most parsimonious tree obtained in this analysis. Thick gray lines indicate stratigraphic range of known taxa (dashed area indicates that these records are based on vertebral remains). Dashed lines represent ghost lineages implied by the stratigraphic distribution of fossils with respect to the phylogenetic relationships shown here (note the exceptionally abundant ghost lineages for Macrostomata). Ages of first appearance for taxa used in the calibrated phylogeny are given in electronic supplementary material. Al, Albian; Ce, Cenomanian; Tu, Turonian; Co, Coniacian; Sa, Santonian; Cam, Campanian; Ma, Maastrichtian; Pal, Paleocene; Eoc, Eocene; Oli, Oligocene; Mi, Miocene; Pl-Ple, Plio-Pleistocene. doi:10.1371/journal.pone.0057583.g004 have played a relevant role in the early evolution of derived macrostomatans that was not necessarily associated with a venom delivery system. maxillary lamina, recognizing the independent posterior dental lamina as responsible for the formation of the rear-fanged morphology characteristic of the endoglyptodont colubroideans (sensu [33]). Morphology In contrast, many colubroideans (including rear-fanged species) display a medial form of intraoral transport, with a freely movable joint that allows the ectopterygoid to swing rostrally or caudally in the horizontal plane during the translation of the palatopterygoid bar. These movements are permitted by the loose condition of the articular capsule, which even allows a slight degree of dorso- ventral movements [36,37]. Thus, the palatopterygoid bar assumes the role of transporting (carrying) the prey during the March 2013 \| Volume 8 \| Issue 3 \| e57583 March 2013 \| Volume 8 \| Issue 3 \| e57583 7 PLOS ONE \| www.plosone.org New Paleocene Macrostomatan Snake from Bolivia Figure 5. Strict consensus tree resulted from the constrained analysis. doi:10.1371/journal.pone.0057583.g005 Figure 5. Strict consensus tree resulted from the constrained analysis. doi:10.1371/journal.pone.0057583.g005 highlight the biogeographic importance of southern continents in the evolution of snakes, which was also pointed out by other lines of evidence such as molecular phylogenetics [32,38]. intraoral transport of prey, which liberates the maxilla from an active role in prey intraoral transport [10]. Our analysis indicate that a medial form of intraoral transport appeared early in the history of derived macrostomatans, although myological studies in bolyeriids and tropidophiids indicate that the pterygoid muscula- ture that produces the complex movements of the palatomaxillary arch necessary to act as medial transporters in caenophidians are not yet present in these groups [34]. Numerous snake materials assigned to different groups of macrostomatans have been found in Cretaceous and Paleocene deposits around the world [39,40,41,42,43]. However, it is worth noting that these records are represented by fragmentary remains, most of which composed by isolated vertebrae. The fragmentary condition of these specimens precludes rigorous phylogenetic analyses. Recent published work about the genus Coniophis represents an illustrative example of problems in the use of fragmentary snake material to determine phylogenetic relation- ships. Coniophis was previously known only by vertebral material scattered around the world and was historically classified as an ‘‘anilioid’’ alethinophidian. Using new material including skull elements, Longrich and colleagues [8] tested the phylogenetic relationships of Coniophis using a cladistic analysis and discovered that Coniophis constitutes a basal snake (i.e. a stem Serpentes), not an alethinophidian. It is widely assumed that the most important evolutionary innovation of macrostomatan snakes is the increase of gape size to swallow large items of food [10,11,12]. Morphology In this respect, our results suggest two different pathways in the evolution of the palatomax- illary arch of macrostomatan snakes. Booid snakes bear a later- omedial intraoral transport and no conspicuous modification in maxillary bone morphology. In contrast, small-bodied derived macrostomatans that freed their maxilla and pterygoid bones from a tight articulation with the ectopterygoid experienced drastic modifications in their maxillary morphology. The new function in active prey ingestion played by the palato-pterygoid arch of derived macrostomatans triggered important changes in the maxilla, including its tooth row morphology. Kataria represents the earliest documented record of such changes in maxillary tooth row morphology. In light of these comments about the nature of the early fossil record of Macrostomata, Kataria emerges as the oldest calibration point for this entire clade of alethinophidian snakes tested through a resolved phylogenetic tree topology. Our temporally calibrated cladogram (Fig. 4) suggests that most cladogenetic events associated to the history of the clade Macrostomata, including the split between booids and derived macrostomatans, took place during Early Tertiary times at least. Also, the discovery of this new fossil snake indicates an unknown long history of the very distinctive families Bolyeriidae and Tropidophiidae, both repre- senting important pieces of evidence to discern the evolutionary events of most derived forms of macrostomatan snakes. References Longrich NR, Bhullar BAS, Gauthier JA (2012) A transitional snake from the Late Cretaceous period of North America. Nature 488: 205–208. (DOI 10.1038/nature11227). J p ( j ) 30. Vidal N, Hedges SB (2004) Molecular evidence for a terrestrial origin of snakes. Proc R Soc B 271: 226–229. (DOI 10.1098/rsbl.2003.0151). 9. Uetz P (2012) The Reptile Database. European Molecular Biology Laboratory, Heidelberg. Available: http://research.amnh.org/vz/herpetology/amphibian. Accessed 2012 January 25. 31. Vidal N, Delmas AS, Hedges SB (2007) The higher-level relationships of alethinophidian snakes inferred from seven nuclear and mitochondrial genes. In: Henderson RW, Powell R, editors. Biology of boas and pythons. Utah: Eagle Mountain Publishing. 27–33. 10. Cundall D, Greene HW (2000) Feeding in snakes. In: Schwenk K, editor. Feeding: Form, Function and Evolution in Tetrapod Vertebrates. San Diego: Academic Press. 293–333. 32. Vidal N, Rage J-C, Couloux A, Hedges SB (2009) Snakes (Serpentes). In: Hedges SB, Kumar S, editors. The Timetree of Life. New York: Oxford University Press. 390–397. 11. Cundall D, Irish F (2008) The snake skull. In: Gans C, Gaunt AS, Adler K, editors. Biology of the Reptilia, vol. 20, The skull of Lepidosauria. Ithaca: New York Society for the Study of Amphibians and Reptiles. 349–692. 33. Zaher H, Grazziotin FG, Cadle JE, Murphy JW, Moura-Leite JC, et al. (2009) Molecular phylogeny of advanced snakes (Serpentes, Caenophidia) with emphasis on South American xenodontines: a revised classification and description of new taxa. Pap Avul Zool 49: 115–153. (DOI 10.1590/S0031– 10492009001100001). 12. McDowell SB (2008) The skull of Serpentes. In: Gans C, Gaunt AS, Adler K, editors. Biology of the Reptilia, vol. 21, The Skull and Appendicular Locomotor Apparatus of Lepidosauria. Ithaca: New York Society for the Study of Amphibians and Reptiles. 467–620. ) 34. McDowell SB (1986) The architecture of the corner of the mouth of colubroid snakes. J Herpet 20: 353–407. 13. Vidal N, Hedges SB (2002) Higher-level relationships of snakes inferred from four nuclear and mitochondrial genes. C R Biologies 325: 977–985. (DOI S1631–0691(02)01510-X). J p 35. Jackson K (2003) The evolution of venom-delivery systems in snakes. Zool J Linn Soc 137: 337–354. (DOI 10.1046/j.1096–3642.2003.00052.x). 14. Wiens JJ, Kuczynski CA, Smith SA, Mulcahy DG, Sites JR, et al. (2008) Branch lengths, support, and congruence: Testing the phylogenomic approach with 20 nuclear loci in snakes. Syst Biol 57: 420–431. (DOI 10.1080/ 10635150802166053). 36. References 1. Caldwell MW, Lee MSY (1997) A snake with legs from the marine Cretaceous of the Middle East. Nature 386: 705–709. (DOI 10.1038/386705a0). 22. Gelfo JN, Goin FJ, Woodburne MO, de Muizon C (2009) Biochronological relationships of the earliest South American Paleogene mammalian faunas. Palaeontology 52 (1): 251–269. (DOI 10.1111/j.1475–4983.2008.00835.x). 2. Tchernov E, Rieppel O, Zaher H, Polcyn MJ, Jacobs IJ (2000) A new fossil snake with limbs. Science 287: 2010–2012. (DOI 10.1126/sci- ence.287.5460.2010). gy ( ) ( j ) 23. Rossman CE (1980) Ontogenetic changes in skull proportions of the diamond- back water snake, Nerodia rhombifera. Herpetologica 36: 42–46. 23. Rossman CE (1980) Ontogenetic changes in skull proportions o 24. Young BA (1989) Ontogenetic changes in the feeding system of the red-side garter snake, Thamnophis sirtalis parietalis. J Zool 218: 365–381. 3. Scanlon JD, Lee MSY (2000) The Pleistocene serpent Wonambi and the early evolution of snakes. Nature 403: 416–420. (DOI 10.1038/35000188). 25. Monteiro LR (1998) Ontogenetic changes in the skull of Corallus caninus (L. 1758) and Corallus enhydris (L. 1758) (Serpentes-Boidae), an allometric study. The Snake 28: 51–58. 4. Scanlon JD (2006) Skull of the large non-macrostomatan snake Yurlunggur from the Australian Oligo-Miocene. Nature 439: 839–842. (DOI 10.1038/ nature04137). 26. Groombridge B (1979) On the vomer in Acrochordidae (Reptilia: Serpentes), and its cladistic significance. J Zool 189: 559–567. 5. Apesteguı´a S, Zaher H (2006) A Cretaceous terrestrial snake with robust hindlimbs and a sacrum. Nature 440: 1037–1040. (DOI 10.1038/nature04413). 27. Zaher H, Rieppel O (1999) Tooth implantation and replacement in squamates, with special reference to mosasaur lizards and snakes. Am Mus Novit 3271: 1– 19. (DOI hdl.handle.net/2246/3047). 6. Wilson JA, Mohabey D, Peters S, Head JJ (2010) Predation upon hatchling sauropod dinosaurs by a new basal snake from the Late Cretaceous of India. PLoS Biol 8: 1–5. (DOI 10.1371/journal.pbio.1000322). 28. Mahler DL, Kearney M (2006) The palatal dentition in squamate reptiles: morphology, development, attachment, and replacement. Fieldiana (Zool) 108: 1–61. (DOI 10.3158/0015–0754(2006)108[1:TPDISR]2.0.CO;2). j p 7. Zaher H, Scanferla A (2012) The skull of the Upper Cretaceous snake Dinilysia patagonica Smith-Woodward, 1901, and its phylogenetic position revisited. Zool J Linn Soc 164: 194–238. (DOI 10.1111/j.1096–3642.2011.00755.x). 29. Maisano JA, Rieppel O (2007) The skull of the Round Island boa, Casarea dussumieri Schlegel, based on High-Resolution X-Ray computed tomography. J Morphol 268: 371–384. (DOI 10.1002/jmor.10519). 8. Biogeographic Implications and Evolutionary Timing of Derived Macrostomatans Recent discoveries of relevant fossil specimens in Mesozoic and Caenozoic strata have elucidated the central role of southern landmasses in the origin of snakes [2,3,4,5,6]. Moreover, the discovery of Kataria in South American bedrocks, together with the Neotropical distribution of extant tropidophiids and African (Seychelles archipelago) distribution of bolyeriids, suggest that the origin and early diversification of derived macrostomatans may also have taken place in Gondwanan terrains. These facts March 2013 \| Volume 8 \| Issue 3 \| e57583 PLOS ONE \| www.plosone.org 8 New Paleocene Macrostomatan Snake from Bolivia References Frazzetta TH (1966) Studies on the morphology and function of the skull in the Boidae (Serpentes). Part II. Morphology and function of the jaw apparatus in Python sebae and Python molurus. J Morphol 118: 217–296. (DOI 10.1002/ jmor.1051180206). 15. Gauthier JA, Kearney M, Maisano JA, Rieppel O, Behlke ADB (2012) Assembling the Squamate Tree of Life: Perspectives from the phenotype and the fossil record. Bull Peabody Mus Nat Hist 53 (1): 1–308. (DOI 10.3374/ 014.053.0101). 37. Young BA (1988) The arthrology of the head of the red-sided garter snake, Thamnophis sirtalis parietalis. Neth J Zool 38: 166–205. 38. Noonan BP, Chippindale PT (2006) Dispersal and vicariance: the complex biogeographic history of boid snakes. Mol Phylogenet Evol 40 (2): 347–358. (DOI 10.1016/j.ympev.2006.03.010). 16. Vonk FJ, Admiraal JF, Jackson K, Reshef R, de Bakker MAG, et al. (2008) Evolutionary origin and development of snake fangs. Nature 454: 630–633. (DOI 10.1038/nature07178). ( j y p ) 39. Rage J-C (1991) Squamates reptiles from the Early Paleocene of the Tiupampa area (Santa Lucı´a Formation), Bolivia. Rev Te´c YPFB 12 (3–4): 503–508. 17. Rieppel O (2007) The naso-frontal joint in snakes as revealed by high-resolution X-ray computed tomography of intact and complete skulls. Zool Anz 246: 177– 191. (DOI 10.1016/j.jcz.2007.04.001). ( ), ( ) 40. Rage J-C (1998) Fossil snakes from the Palaeocene of Sa˜o Jose´ de Itaboraı´,Brazil. Part I. Madtsoiidae, Aniliidae. Palaeovertebrata 27 (3–4): 109–144. 41. Rage J-C, Werner C (1999) Mid-Cretaceous (Cenomanian) snakes from Wadi Abu Hashim, Sudan: the earliest snake assemblage. Palaeontol Afr 35: 85–110. 18. Goloboff PA, Farris JS, Nixon K (2008) TNT: Tree Analysis Using New Technology, vers. 1.1 (Willi Hennig Society Edition). Available: http://www. zmuc.dk/public/phylogeny/tnt. Accessed 20 February 2011. 42. Auge´ M, Rage J-C (2006) Herpetofaunas from the Upper Paleocene and Lower Eocene of Morocco. Ann Pale´ont 92: 235–253. (DOI 10.1016/ j.bbr.2011.03.031). 19. Goloboff PA, Farris JS, Nixon K (2008) TNT, a free program for phylogenetic analysis. Cladistics 24: 774–786. (DOI 10.1111/j.1096–0031.2008.00217.x). 43. Head JJ, Bloch JI, Hastings AK, Bourque JR, Cadena EA, et al. (2009) Giant boid snake from the Palaeocene neotropics reveals hotter past equatorial temperatures. Nature 457: 715–718. (DOI 10.1038/nature07671). 20. Bremer K (1994) Branch support and tree stability. Cladistics r K (1994) Branch support and tree stability. Cladistics 10: 295–304. 21. Wiens JJ, Hutter CR, Mulcahy DG, Noonan BP, Townsend TM, et al. Author Contributions Conceived and designed the experiments: AS HZ. Performed the experiments: AS HZ. Analyzed the data: AS HZ. Contributed reagents/ materials/analysis tools: AS HZ FN CM RC. Wrote the paper: AS HZ FN CM. Text S1 Details of phylogenetic analyses and specimens examined. (DOC) Dataset S1 Nexus file containing the phylogenetic data matrix. Acknowledgments We thank Darrell Frost, Ivan Ineich, Julia´n Faivovich, Jorge Williams and Francisco L. Franco for loan of specimens. Gratitude is also due to Manuel Supporting Information Sosa for the skillful drawings and Camilo Arredondo for the photographs. Jack Conrad and an anonymous reviewer gave thorough and constructive criticisms. We are grateful to Diego Pol for his advice on the use of the T.N.T. program package. Finally, we are grateful to Anjan Bhullar for the revision of English grammar. The phylogenetic analysis was performed with the program TNT that is freely available through the Willi Hennig Society. Text S1 Details of phylogenetic analyses and specimens examined. References (2012) Resolving the phylogeny of lizards and snakes (Squamata) with extensive sampling of genes and species. Biol Lett 8 (6): 1043–1046. (DOI 10.1098/ rsbl.2012.0703). 44. Maisano JA, Rieppel O (2007) ‘‘Casarea dussumieri’’ (On-line), Digital Morphol- ogy. Available: http://digimorph.org/specimens/Casarea_dussumieri/. Ac- cessed 2012 March 10. March 2013 \| Volume 8 \| Issue 3 \| e57583 March 2013 \| Volume 8 \| Issue 3 \| e57583 9 PLOS ONE \| www.plosone.org
https://openalex.org/W1989409192	https://europepmc.org/articles/pmc4315064?pdf=render	English	null	Partial remission of acute myeloid leukemia complicating multiple myeloma following COAP chemotherapy: A case report	Oncology Letters	2,015	cc-by	3,291	ONCOLOGY LETTERS 9: 1303-1306, 2015 ONCOLOGY LETTERS 9: 1303-1306, 2015 DOI: 10.3892/ol.2015.2867 Abstract. A 77‑year‑old male was admitted to hospital after complaining of fever and a cough for three days. A diagnosis of multiple myeloma was confirmed following M protein identification and a bone marrow biopsy. The patient received chemotherapy regimens of bortezomib plus dexamethasone, cyclophosphamide, thalidomide and dexamethasone, and thalidomide and dexamethasone, and was prescribed thalido mide (100 mg/d) to be taken orally for maintenance therapy. After a further two years the patient was subsequently diag nosed with acute myeloid leukemia. Chemotherapy regimens of cytarabine, aclacinomycin and daunorubicin, homohar ringtonine and etoposide, and mitoxantrone and cytarabine resulted in no remission. Partial remission was obtained with a course of ifosfamide, vindesine, cytarabine and prednisone chemotherapy. This therapy may be an alternative treatment for secondary leukemia, particularly in elderly patients. have made a contribution to secondary malignancies following multiple myeloma (5). The therapies included oral alkylating therapy (6-13), myeloablative therapy (used in conjunction with ASCT) (10-12), radiotherapy (14-16) and lenalidomide (17-19). There are several ongoing studies are attempting to understand the underlying mechanisms. Several studies have shown that the rate of hematological malignancy after multiple myeloma was 0.5%-12.2%. Reece and Goswami reported that the rate of MDS/AML after diagnosis of multiple myeloma of initiation of lenalidomide was 2.6%. Treatment of patients with two hema tological cancers is difficult. The current study reports a case of MM complicated by AML. The patient received classic chemotherapy regimens including cytarabine, aclacinomycin, daunorubicin (CAG), mitoxantrone and cytarabine (NA) and homoharringtonine and etoposide (HE), however, did not respond to the treatment. A course of COAP chemotherapy was subsequently administered, which was selected as it is frequently used to treat AML and has been well‑tolerated by patients, with few side effects. Guthrie reported that fifteen AML patients had received chemotherapy with a combination of high-dose, continuous-infusion COAP (20). The results showed that the rate of complete remission was 47% and the rate of partial remission was 40%. The main toxicity was primarily myelosuppression and there were no toxicities such as hemorrhagic cystitis or central nervous system, hepatic or pulmonary toxicity. Written informed consent was obtained from the patient's family. Introduction Multiple myeloma (MM) survival rates have improved signifi cantly over the last 10 years (1‑3). For example, in subsequent 10‑year calendar periods, the median overall survival increased from 24.3 to 56.3 months (P=0.036) in patients ≤65 years old. With such improvements, a relatively new clinical chal lenge that has emerged is the risk of second malignancies. The association of acute leukemia [most frequently acute myeloid leukemia (AML)] and MM has often been reported, not only as a complication of chemotherapy, but also occurring in the absence of chemotherapy or simultaneously at the time of diagnosis (4). Prior studies have found that various therapies Partial remission of acute myeloid leukemia complicating multiple myeloma following COAP chemotherapy: A case report MAN SHEN1, WAN‑JUN SUN2, ZHONG‑XIA HUANG1, JIA‑JIA ZHANG1, NA AN1 and XIN LI1 1Department of Hematology and Oncology, Beijing Chaoyang Hospital, Capital Medical University, Beijing 100043; 2Department of Hematology, The Second Artillery General Hospital, Beijing 100088, P.R. China Received February 27, 2014; Accepted November 6, 2014 Case report A 77‑year‑old male was admitted to the Department of Hematology, Peking Union Medical College Hospital (Beijing, China) on June 21st, 2011, after presenting with fever and a cough persisting for three days. Following hospi talization, laboratory tests revealed a white blood cell count of 3,900/mm3 (normal range, 4,000‑10,000/mm3), a hemoglobin level of 128 g/l (normal range, 131‑172 g/l), a platelet count of 54,000/mm3 (normal range, 100,000‑300,000/mm3), and a serum creatinine level of 133 µmol/l (normal range, 53‑115 µmol/l). Further analysis revealed a blood microglobulin level of 4.53 mg/l (normal range, 1.0‑3.0 mg/l), a blood sedimentation rate of 21 mm/h (normal range, 0‑20 mm/h), and M protein levels of 6.78 g/l for IgG (normal range, 7.51‑15.6 g/l), 0.22 g/l for IgA (normal range, 0.82‑4.53 g/l) and 0.26 g/l for IgM (normal range, 0.46‑3.04 g/l). The serum free light chain (κ Correspondence to: Dr Xin Li, Department of Hematology and Oncology, Beijing Chaoyang Hospital, Capital Medical University, 5 Jingyuan Road, Beijing 100043, P.R. China E‑mail: lixin0628@sohu.com Correspondence to: Dr Xin Li, Department of Hematology and Oncology, Beijing Chaoyang Hospital, Capital Medical University, 5 Jingyuan Road, Beijing 100043, P.R. China E mail: li in0628@sohu com Correspondence to: Dr Xin Li, Department of Hematology and Oncology, Beijing Chaoyang Hospital, Capital Medical University, 5 Jingyuan Road, Beijing 100043, P.R. China il li i 0628@ h Key words: acute myeloid leukemia, multiple myeloma, partial remission, COAP SHEN et al: ACUTE MYELOID LEUKEMIA COMPLICATING MULTIPLE MYELOMA The hole‑like lesions of the right upper lobe had increased markedly in size, with massive pleural effusion on the right side. Anti‑inflammatory treatment was adjusted to sulp erazon (3.0 g, twice a day) combined with tigecycline (50 mg, twice a day). The patient's fever persisted throughout this period, and he subsequently succumbed to respiratory failure. and λ) levels were measured as 6.52 g/l (κ) and 2.16 g/l (λ), and the total amount of κ measured in the urine was 7.5 g in 24 h. Myeloma cells accounted for 12.5% of bone marrow cytology. A bone marrow biopsy showed elevated numbers of plasma cells (12.5%). Immunohistochemistry revealed that the patient was positive for CD38, CD138, κ and epithelial membrane antigen. CD20, CD3, CD79a, Mum‑1 were also positive, but scattered, and the positive rate of Ki‑67 was 60%. A bone scan revealed uneven uptake of radioactive tracer in the thoracolumbar region. A diagnosis of multiple myeloma κ type light chain (Durie‑Salmon stage II B, International Staging System stage III) was determined. The patient received a chemotherapeutic regimen of bortezomib (1.3 mg/m2, days 1, 4, 8 and 11) plus dexamethasone (20 mg, days 1, 4, 8 and 11) (PD) for three cycles, cyclophosphamide (500 mg, days 1‑4), thalido mide (100 mg/day, every day) and dexamethasone (20 mg, days 1‑4) (CTD) for one cycle, and thalidomide (100 mg/day, every day) and dexamethasone (20 mg, days 1‑4) (TD) for one cycle, sequentially, where one cycle comprised 21 days. This was followed by a course of thalidomide (100 mg/d) for mainte nance therapy. Blood analysis and tests of hepatorenal function and M protein levels were conducted every three months for two years, yielding normal results. In April 2013, tests revealed a leukocyte count of 20,830/mm3 (normal range, 4,000‑10,000/mm3) and a platelet count of 27,000/mm3 (normal range, 100,000‑300,000/mm3), while the hemoglobin level was measured at 126 g/l (normal range, 131‑172 g/l). Bone marrow examination showed clear signs of hyperplasia, with primitive granulocyte cells increasing from 1 to 74% and visible Auer's bodies. The rate of positive peroxidase staining was 98%, which differentiated the patient's diagnosis of AML from ALL. CD117, CD38, CD34, CD13, and HLA‑DR were all positive in the immune peripheral blood clas sification. No FLT3/ITD, NPM1, c‑kit/D816V and AML1‑ETO mutations were identified in the bone marrow by immunohis tochemistry. A diagnosis of AML‑M2 was determined. SHEN et al: ACUTE MYELOID LEUKEMIA COMPLICATING MULTIPLE MYELOMA 2 function was observed to be abnormal, with an elevated serum creatinine level of 110 µmol/l (normal range, 53‑115 µmol/l). After two years, serum creatinine levels had increased to 140‑150 µmol/l, while routine urine test results remained normal. In 2010, the patient was diagnosed with diabetes, cata racts and benign prostatic hyperplasia. Chemotherapy with NA (mitoxantrone, 4 mg, days 1‑3; cytarabine, 100 mg, days 1‑7) was accepted in July 2013 when bone marrow aspiration cytology results revealed that the high proportion of myeloblasts in the bone marrow (57%) had not yet returned to normal. On October 26th 2013, the patient began a chemotherapy cycle of ifosfamide, vindesine, cytarabine and prednisone (COAP) with ifosfamide, 0.5 g from d1 to d4 combined with vindesine, 4 mg, d1; cyta rabine, 100 mg and prednisone, 50 mg every other day from d1 to d7. On November 28th 2013, a re‑examination of the patient's bone marrow indicated partial remission, with primitive cells accounting for 14.7% of all nucleated cells (>50% decrease). During this intermittent chemotherapy, the patient exhibited stable vital signs, with no fever, no cough, no sputum, and no evident abnormalities in routine blood test results. The periodic review of IFE was negative, which revealed that the patient had achieved complete remission of MM. Since November 28th, the patient continued COAP chemotherapy for three cycles. During this chemotherapy cycle, the patient exhibited fever, pancytopenia, agranulocytosis and severe anemia as a result of immunosupression induced by the chemotherapy treatment. A lung CT scan revealed pulmonary consolidation in the right upper lobe and possible lung abscess. This was treated with maxipime (2.0 g, twice a day), meropenem (0.5 g, three times a day) combined with vancomycin (1.0 g, twice a day) and teicoplanin (200 mg, twice a day). Candida albicans, detected in pathological examination of the sputum, was treated with caspofungin antifungal therapy (50 mg, once a day). Although the T spot result was negative, the lesions in the right upper lobe combined with a continuing low‑grade fever led to a diagnosis of tuberculosis (TB). Isoniazid (0.3 g, once a day) and rifapentine (0.45 g, once a day) were administered orally to treat the TB. Thereafter, the patient's fever persisted, peaking once per day. One week later, a further lung CT revealed serious pneumonia, airway obstructive pulmonary consolidation and occupying lesions. SHEN et al: ACUTE MYELOID LEUKEMIA COMPLICATING MULTIPLE MYELOMA On April 29th 2013, after receiving the CAG regimen (cytarabine, 10 mg/m2 every 12 h, days 1‑14; aclarubicin, 10 mg, days 1‑8; G‑CSF, 200 µg/m2, days 1‑14) for one cycle as induction chemo therapy, no remission (NR) was observed in the bone marrow biopsy. The patient subsequently received an additional regimen of HE (homoharringtonine, 3 mg, days 1‑7; etoposide, 100 mg, days 1‑7). During the entire treatment period, the patient had a respiratory infection and pleural effusion, which ultimately improved following aggressive anti‑infection treatment. In July 2013, the patient began attending the Department of Hematology and Oncology at Beijing Chaoyang Hospital (West Branch, Beijing 100043, China) for treatment. Tests revealed that myeloblasts accounted for 79% (normal range, <5%) of bone marrow cytology, while immature plasma cells accounted for 2% (normal range, <5%). This indicated that the patient now had leukemia, but that the multiple myeloma was well controlled at the same time. M protein identification and immunofixation electrophoresis (IFE) examination results indi cated a polyclonal immunoglobulin, while M component was not detected. The patient's previous medical history included tuberculosis and hemoptysis in 1956, hypertension in 2006 and a coronary computed tomography angiogram in 2010 which revealed 70% coronary stenosis. Coronary angiography and stent therapy was recommended, but the patient refused. In 2006, renal Contributed equally Contributed equally Key words: acute myeloid leukemia, multiple myeloma, partial remission, COAP Key words: acute myeloid leukemia, multiple myeloma, partial remission, COAP SHEN et al: ACUTE MYELOID LEUKEMIA COMPLICATING MULTIPLE MYELOMA References and site‑specific standardized incidence ratio (SIR) and 95% confidence intervals (CI) for 2,012 SPM cases diagnosed within the 35‑year follow‑up. No significant overall risk of SPM was identified (SIR=0.98; 95% CI=0.94‑1.02). 1. Turesson I, Velez R, Kristinsson SY and Landgren O: Patterns of improved survival in patients with multiple myeloma in the twenty‑first century: a population‑based study. J Clin Oncol 28: 830‑834, 2010. Numerous risk factors are associated with SPM, including MM disease‑related factors, such as treatment and tumor microenvironment, in addition to host‑related processes, such as genetic and environmental factors (26). Early observations indicated that treatment‑related factors, such as from melphalan, may be the main cause of the increased incidence of myelodysplastic syndrome/acute leukemia in MM patients (13). Cyclophosphamide was found to be less leukemogenic than melphalan (27). In addition, maintenance therapy has been evaluated in relation to the risk of second malignancies in three recently reported multicenter random ized phase III trials (IFM 2005‑02, CALGB 100104, and MM‑015) (17‑19). In the IFM 2005‑02 and CALGB 100104 trials, 5.5 and 6.5% of lenalidomide‑treated patients devel oped second malignancies, compared with 1 and 2.5% in the respective control groups. 2. Kumar SK, Rajkumar SV, Dispenzieri A, et al: Improved survival in multiple myeloma and the impact of novel therapies. Blood 111: 2516‑2520, 2008. 3. Kristinsson SY, Landgren O, Dickman PW, et al: Patterns of survival in multiple myeloma: a population‑based study of patients diagnosed in Sweden from 1973 to 2003. J Clin Oncol 25: 1993‑1999, 2007. 4. Dunkley S, Gibson J, Iland H, Li C and Joshua D: Acute promy elocytic leukaemia complicating multiple myeloma: evidence of different cell lineages. Leuk Lymphoma 35: 623-626, 1999. 5. Morgan GJ, Davies FE, Gregory WM, et al: Long‑term follow‑up of MRC Myeloma IX trial: Survival outcomes with bisphosphonate and thalidomide treatment. Clin Cancer Res 19: 6030‑6038, 2013. 6. Bergsagel DE, Bailey AJ, Langley GR, et al: The chemotherapy on plasma-cell myeloma and the incidence of acute leukemia. N Engl J Med 301: 743-748, 1979. g 7. [No Authors Listed]: Acute leukaemia and other secondary neoplasms in patients treated with conventional chemotherapy for multiple myeloma. Eur J Haematol 65: 123-127, 2000. The current study reports one case of a patient who devel oped AML two years after being diagnosed with MM. This patient had received velcade and ifosfamide treatment for three cycles, and continued to take thalidomide for two years thereafter. References The cause of AML in this patient was unclear. Studies have reported that thalidomide may also potentiate solid SPMs (26,28). We therefore considered that thalidomide may be a cause of AML. Three clinical trials had shown that lenalidomide and thalidomide maintenance therapy could lead to a higher incidence of second primary malignancies, which is relevant to patients receiving melphalan (17-19). The patient in the present case had oral thalidomide as maintainance therapy for two years and, during this period, the patient was not adminstered any other drugs that could induce a tumor (28). The patient had taken thalidomide orally for two years prior to the diagnosis of AML and had received chemotherapy (PD, CTD, CAG and HE regimens) for seven cycles, however, complete remission was not achieved. After receiving the COAP chemotherapy regimen, the leukemia cells of the bone marrow decreased by >50% and the disease stabilized. A second cycle of COAP chemo therapy was predicted to produce CR, however, the patient subsequently acquired an obstructive pneumonia infection, which may have been associated with chemotherapy treat ment. Although vigorous anti‑infection treatments, including various antibiotics, antifungal agents and antituberculosis drugs, were administered, the patient succumbed to respira tory failure.i p y 8. Dong C and Hemminki K: Second primary neoplasms among 53,159 haematolymphoproliferative malignancy patients in Sweden, 1958-1996: a search for common mechanisms. Br J Cancer 85: 997-1005, 2001. 9. Hasskarl J, Ihorst G, De Pasquale D, et al: Association of multiple myeloma with different neoplasms: systematic analysis in consecutive patients with myeloma. Leuk Lymphoma 52: 247-259, 2011.i 10. Barlogie B, Tricot G, Haessler J, et al: Cytogenetically defined myelodysplasia after melphalan-based autotransplantation for multiple myeloma linked to poor hematopoietic stem-cell mobi lization: the Arkansas experience in more than 3,000 patients treated since 1989. Blood 111: 94-100, 2008. 11. Przepiorka D, Buadi F, McClune B, et al: Myelodysplastic syndrome after autologous peripheral blood stem cell transplantation for multiple myeloma. Bone Marrow Transplant 40: 759-764, 2007. p y p 12. Govindarajan R, Jagannath S, Flick JT, et al: Preceding standard therapy is the likely cause of MDS after autotransplants for multiple myeloma. Br J Haematol 95: 349-353, 1996. 13. Cuzick J, Erskine S, Edelman D and Galton DA: A comparison of the incidence of the myelodysplastic syndrome and acute myeloid leukaemia following melphalan and cyclophosphamide treatment for myelomatosis. A report to the Medical Research Council's working party on leukaemia in adults. Discussion A number of studies have reported an increased risk of second primary malignancies (SPM) following MM diagnosis, which are proposed to be associated with novel anti‑myeloma treatments (17‑19). The introduction of agents such as thalido mide (5), bortezomib (21,22) and lenalidomide (17,23,24) has improved MM survival rates over the last decade. However, accurate estimates of incidence and pathogenesis of second malignancies following MM are lacking. Razavi et al (25) evaluated the risk of SPM among 36,491 MM cases reported to the surveillance, epidemiology and end results program between 1973 and 2008. The authors calculated overall ONCOLOGY LETTERS 9: 1303-1306, 2015 3 References Br J Cancer 55: 523‑529, 1987. y 14. Featherstone C, Delaney G, Jacob S and Barton M: Estimating the optimal utilization rates of radiotherapy for hematologic malignancies from a review of the evidence: part II leukemia and myeloma. Cancer 103: 393-401, 2005. 15. Berrington de Gonzalez A, Curtis RE, Kry SF, et al: Proportion of second cancers attributable to radiotherapy treatment in adults: a cohort study in the US SEER cancer registries. Lancet Oncol 12: 353-360, 2011. 16. Berrington de Gonzalez A, Curtis RE, Gilbert E, et al: Second solid cancers after radiotherapy for breast cancer in SEER cancer registries. Br J Cancer 102: 220-226, 2010. g 17. Attal M and Cances‑Lauwers V: Maintenance treatment with lenalidomide after transplantation for myeloma: analysis of secondary malignancies within the IFM 2005‑02 trial. 13th International Myeloma Workshop; 2011; Paris, France. This study showed that the regimen of CA was an effica cious chemotherapy for MM combined with AML. However, the present study also indicated that the use of immunomodula tory drugs as a chemotherapy treatment may increase the risk of SPM development in older patients. Therefore, further studies are required to investigate the association between oral thalido mide and the development of AML. y p 18. McCarthy P and Anderson K: Phase III Intergroup study of lenalidomide versus placebo maintenance therapy following single autologous stem cell transplant for multiple myeloma CALGB ECOG BMT‑CTN 100104. 13th International Myeloma Workshop; 2011; Paris, France. 19. Palumbo AP, Catalano J and editors: Incidence of second primary malignancy in melphalan‑prednisone‑lenalidomide combination followed by lenalidomide maintenance in newly diagnosed multiple myeloma patients age 65 or older [abstract]. J Clin Oncol 29: 2011. Acknowledgements 20. Guthrie TH Jr: High-dose, continuous-infusion cyclophos phamide, cytarabine, vincristine, and prednisone for remission induction in refractory adult acute leukemia. Cancer 59: 1255‑1257, 1987. The authors would like to thank Dr Wan‑jun Sun for helpful suggestions and for stimulating discussions. SHEN et al: ACUTE MYELOID LEUKEMIA COMPLICATING MULTIPLE MYELOMA SHEN et al: ACUTE MYELOID LEUKEMIA COMPLICATING MULTIPLE MYELOMA 4 25. Razavi P, Rand KA, Cozen W, et al: Patterns of second primary malignancy risk in multiple myeloma patients before and after the introduction of novel therapeutics. Blood Cancer J 28: e121, 2013. 21. Nooka AK, Kaufman JL, Behera M, et al: Bortezomib‑containing induction regimens in transplant‑eligible myeloma patients: a meta‑analysis of phase 3 randomized clinical trials. Cancer 119: 4119‑4128, 2013. , 22. Richardson PG, Schlossman RL, Alsina M, et al: PANORAMA 2: panobinostat in combination with bortezomib and dexa methasone in patients with relapsed and bortezomib‑refractory myeloma. Blood 122: 2331‑2337, 2013. , 26. Thomas A, Mailankody S, Korde N, et al: Second malignancies after multiple myeloma: from 1960s to 2010s. Blood 119: 2731‑2737, 2012. 27. Greene MH, Harris EL, Gershenson DM, et al: Melphalan may be a more potent leukemogen than cyclophosphamide. Ann Intern Med 105: 360‑367, 1986. y 23. Gay F, Hayman SR, Lacy MQ, et al: Lenalidomide plus dexamethasone versus thalidomide plus dexamethasone in newly diagnosed multiple myeloma: a comparative analysis of 411 patients. Blood 115: 1343‑1350, 2010. 28. Usmani SZ, Sexton R, Hoering A, et al: Second malignancies in total therapy 2 and 3 for newly diagnosed multiple myeloma: influence of thalidomide and lenalidomide during maintenance. Blood 20: 1597‑1600 2012. p 24. Dimopoulos MA, Delforge M, Hájek R, et al: Lenalidomide, melphalan, and prednisone, followed by lenalidomide main tenance, improves health‑related quality of life in newly diagnosed multiple myeloma patients aged 65 years or older: results of a randomized phase III trial. Haematologica 98: 784‑788, 2013.
https://openalex.org/W2890948523	http://jos.hueuni.edu.vn/index.php/HUJOS-NS/article/download/4922/538	Vietnamese	null	NGHIÊN CỨU ẢNH HƯỞNG CỦA NGUỒN CARBON VÀ MỘT SỐ ELICITOR LÊN KHẢ NĂNG SINH TRƯỞNG CỦA TẾ BÀO HUYỀN PHÙ ĐINH LĂNG ( POLYSCIAS FRUTICOSA (L.) HARMS)	Tạp chí Khoa học Đại học Huế: Khoa học Tự nhiên/Tạp chí Khoa học Đại học Huế: Khoa học Tự nhiên (online)	2,018	cc-by-sa	4,122	ẢNH HƯỞNG CỦA NGUỒN CARBON VÀ MỘT SỐ ELICITOR LÊN KHẢ NĂNG SINH TRƯỞNG CỦA TẾ BÀO HUYỀN PHÙ ĐINH LĂNG (POLYSCIAS FRUTICOSA (L.) HARMS) Phan Thị Á Kim1,2, Nguyễn Thị Hà Ngân1, Lê Thị Anh Thư1, Lê Văn Tường Huân1* 1 Khoa Sinh học, Trường Đại học Khoa học, Đại học Huế, 77 Nguyễn Huệ, Huế, Việt Nam 2 Sở Khoa học và Công nghệ tỉnh Quảng Nam, 54 Hùng Vương, Quảng Nam, Việt Nam Tóm tắt. Đinh lăng (Polyscias fruticosa (L.) Harms) là một loài cây thuốc có giá trị, được dân gian sử dụng rộng rãi làm thuốc tăng cường sức khỏe. Trong nghiên cứu này, ảnh hưởng của nguồn carbon và một số loại elicitor (dịch chiết nấm men, salicylic acid và AgNO3) lên khả năng sinh trưởng của tế bào huyền phù đinh lăng đã được khảo sát. Kết quả cho thấy môi trường MS (Murashige and Skoog) lỏng có bổ sung α-naphthaleneacetic acid (NAA) 2 mg/L, Kinetin 0,5 mg/L và sucrose 3% là tốt nhất cho khả năng sinh trưởng của tế bào đinh lăng; sinh khối tế bào tươi đạt 7,50 g (0,40 g khô) sau 16 ngày nuôi cấy. Tất cả các loại elicitor sử dụng trong nghiên cứu đều ức chế sự sinh trưởng của tế bào huyền phù; nồng độ elicitor càng cao sinh khối tế bào càng giảm. Đây là điều kiện cần thiết để tăng sự tích lũy các hợp chất thứ cấp trong nuôi cấy tế bào huyền phù. Từ khóa: đinh lăng, elicitor, nguồn carbon, Polyscias fruticosa, tế bào huyền phù Nhận bài: 3–8–2018; Hoàn thành phản biện: 22–8–2018; Ngày nhận đăng: 30–8–2018 Tạp chí Khoa học Đại học Huế: Khoa học Tự nhiên; ISSN 1859–1388 Tạp chí Khoa học Đại học Huế: Khoa học Tự nhiên; ISSN 1859–1388 Tập 127, Số 1C, 2018, Tr. 85–94; DOI: 10.26459/hueuni-jns.v127i1C.4922 * Liên hệ: tuonghuanle@gmail.com * Liên hệ: tuonghuanle@gmail.com Nhận bài: 3–8–2018; Hoàn thành phản biện: 22–8–2018; Ngày nhận đăng: 30–8–2018 1 Đặt vấn đề Đinh lăng còn gọi là cây Gỏi cá, Nam dương lâm, có tên khoa học là Polyscias fruticosa (L.) Harms, thuộc họ Nhân sâm (Araliaceae), là một loài cây thuốc đã được đưa vào dược điển Việt Nam. Đinh lăng là loài được dân gian sử dụng rộng rãi làm thuốc tăng cường sức khỏe và hoạt huyết dưỡng não từ rất lâu [2]. Trong đinh lăng có các loại alkaloid, glucoside, saponin, flavonoid, tanin, vitamin B1 và các amino acid, trong đó lycine, cysteine và methionine là những amino acid không thể thay thế được trong cây. Đinh lăng chứa các hợp chất saponin tương tự như trong nhân sâm. Trong một số trường hợp, rễ củ đinh lăng được thay thế cho nhân sâm như một nguyên liệu dễ tìm ở Việt Nam [6]. Với nhiều tác dụng dược lý đã được chứng minh nên đinh lăng càng được sử dụng nhiều để làm thuốc. Tuy nhiên, nguồn nguyên liệu không đủ đáp ứng nhu cầu do thời gian thu hoạch khá lâu (ít nhất từ 3 năm trở lên, cây trồng càng lâu năm càng tốt), năng suất thường thấp, phụ thuộc rất lớn vào điều kiện đất đai, khí hậu, mùa vụ, chi phí nhân công và vật tư sản xuất. Vì vậy, những nghiên cứu thu nhận hợp chất thứ cấp bằng phương pháp nuôi cấy tế bào cây đinh lăng Nhận bài: 3–8–2018; Hoàn thành phản biện: 22–8–2018; Ngày nhận đăng: 30–8–2018 Phan Thị Á Kim và Cs. Tập 127, Số 1C, 2018 là lĩnh vực nghiên cứu rất được quan tâm, hứa hẹn tiềm năng to lớn, giúp giải quyết việc gia tăng sinh tổng hợp saponin. Bên cạnh đó, nhiều nghiên cứu cho thấy hàm lượng các chất có hoạt tính sinh học tích lũy trong tế bào thực vật nuôi cấy in vitro tương đương hoặc cao hơn nhiều lần so với tích lũy trong cây ngoài tự nhiên [10]. Thành phần môi trường, điều kiện nuôi cấy, các tiền chất hoặc các elicitor trong môi trường nuôi cấy tế bào có ảnh hưởng đến việc tăng hiệu suất tổng hợp các chất chuyển hóa thứ cấp, rút ngắn thời gian và giảm chi phí sản xuất so với cây ngoài tự nhiên. 1 Đặt vấn đề Hiện nay, các nghiên cứu về nhân giống in vitro cây đinh lăng [3, 8], nuôi cấy tế bào huyền phù [5] và nuôi cấy rễ tơ [1] để sản xuất saponin từ cây đinh lăng cũng đã được thực hiện. Tuy nhiên, các nghiên cứu về sử dụng elicitor trong quá trình nuôi cấy tế bào cây đinh lăng lại chưa được công bố. Bài báo này trình bày các kết quả về thiết lập nuôi cấy tế bào huyền phù từ mô callus đinh lăng (số liệu nuôi cấy callus chưa công bố), ảnh hưởng của nguồn carbon cũng như một số elicitor lên sinh trưởng của tế bào, làm cơ sở cho việc sản xuất một số hoạt chất có giá trị dược liệu sau này. Nuôi cấy tạo callus Rễ non của cây in vitro được nuôi cấy trên môi trường MS (Murashige và Skoog, 1962) có bổ sung NAA 2 mg/L kết hợp Kinetin (KIN) 0,5 mg/L, agar 0,8% và sucrose 3% để tạo callus. Callus được cấy chuyển sau mỗi 15 ngày (môi trường nuôi cấy như môi trường tạo callus). Môi trường nuôi cấy được điều chỉnh pH đến 5,8 và khử trùng ở 121°C (1 atm) trong 20 phút. Xác định sinh khối tế bào Tiến hành thu sinh khối tế bào từ ngày nuôi cấy thứ 2 đến ngày thứ 18 (2 ngày thu mẫu 1 lần) để xác định khối lượng tươi và khô của tế bào. Khối lượng tươi: Dịch tế bào huyền phù được lọc chân không, rửa sạch sinh khối bằng nước cất 2–3 lần, cân để xác định khối lượng tươi. Khối lượng khô: Khối lượng tươi của tế bào được sấy khô ở 50 °C đến khối lượng không đổi, cân để xác định khối lượng khô. Chỉ số sinh trưởng: được tính bằng tỷ lệ giữa khối lượng tươi sau một thời gian nuôi cấy (g) và khối lượng tươi lúc đưa vào nuôi cấy (g). Nuôi cấy tế bào huyền phù Để xác định khả năng sinh trưởng của tế bào huyền phù, 2 g callus 30 ngày tuổi được nuôi trong 50 mL môi trường cơ bản MS lỏng chứa NAA 2 mg/L kết hợp với KIN 0,5 mg/L và sucrose 3%. Khả năng sinh trưởng của tế bào được xác định qua khối lượng tươi, khối lượng khô và chỉ số sinh trưởng của tế bào. Để xác định ảnh hưởng của nguồn carbon lên sinh trưởng tế bào, sucrose được thay thế bằng đường maltose và fructose với các nồng độ 10–40 g/L. Ảnh hưởng của các elicitor lên sinh trưởng của tế bào huyền phù được xác định bằng cách bổ sung dịch chiết nấm men (YE) nồng độ 1–5 g/L, salicylic acid (SA) nồng độ 50–250 µM và AgNO3 10–100 µM vào môi trường nuôi cấy ở thời điểm ban đầu; sau đó đánh giá khả năng sinh trưởng của tế bào. Tế bào huyền phù được nuôi cấy trong bình 250 mL trên máy lắc với tốc độ 120 vòng/phút, nhiệt độ 25–27 °C, cường độ chiếu sáng 2.000 lux, thời gian chiếu sáng 16 giờ/ngày. 2.1 Nguyên liệu Nguyên liệu nghiên cứu sử dụng trong các thí nghiệm là callus có nguồn gốc từ rễ cây đinh lăng in vitro (Hình 1). Hình 1. Callus có nguồn gốc từ rễ đinh lăng sau 30 ngày nuôi cấy Hình 1. Callus có nguồn gốc từ rễ đinh lăng sau 30 ngày nuôi cấy 86 Tập 127, Số 1C, 2018 jos.hueuni.edu.vn Xử lý thống kê Các thí nghiệm được bố trí hoàn toàn ngẫu nhiên. Mỗi công thức thí nghiệm gồm 2 bình và mỗi thí nghiệm được lặp lại 3 lần để tính trung bình. Số liệu được xử lý bằng phương pháp thống kê sinh học, phân tích one-way ANOVA bằng Duncan’s test theo chương trình SPSS 22.0 với mức xác xuất có ý nghĩa p < 0,05. 87 Phan Thị Á Kim và Cs. Tập 127, Số 1C, 2018 3.1 Sinh trưởng của tế bào huyền phù Kết quả nghiên cứu cho thấy pha lag của tế bào đinh lăng rất dài (đến 6 ngày). Sau 6 ngày, tế bào bắt đầu vào pha log, sinh trưởng tương đối nhanh. Pha log kéo dài trong khoảng 10 ngày và sinh khối đạt cực đại ở ngày thứ 16 với 7,50 g khối lượng tươi (0,40 g khối lượng khô) tăng 3,75 lần so với lúc bắt đầu nuôi cấy. Sau pha log, tế bào chuyển sang pha suy vong, sinh khối tế bào chỉ còn 7,33 g khối lượng tươi (0,39 g khối lượng khô), không thấy được pha cân bằng do chỉ thoáng qua (Bảng 1, Hình 2, Hình 3). Lúc này, dịch tế bào từ màu vàng chuyển sang nâu có thể liên quan đến sự oxy hóa các sản phẩm phenol tiết ra trong quá trình nuôi cấy bởi các enzyme ngoại bào. Bảng 1. Tích lũy sinh khối của tế bào đinh lăng theo thời gian nuôi cấy Thời gian nuôi cấy (ngày) Khối lượng tế bào (g) Chỉ số sinh trưởng Tươi Khô 2 3,23e 0,27h 1,62 4 3,47de 0,29g 1,74 6 3,48de 0,29fg 1,74 8 4,09d 0,30f 2,04 10 5,03c 0,34e 2,52 12 5,67bc 0,35d 2,84 14 6,08b 0,38c 3,04 16 7,50a 0,40a 3,75 18 7,33b 0,39b 3,67 Ghi chú: Các chữ cái khác nhau trên cùng một cột chỉ sự sai khác có ý nghĩa thống kê của các trung bình mẫu Bảng 1. Tích lũy sinh khối của tế bào đinh lăng theo thời gian nuôi cấy Ghi chú: Các chữ cái khác nhau trên cùng một cột chỉ sự sai khác có ý nghĩa thống kê của các trung bình mẫu với p < 0,05 (Duncan’s test). Ghi chú này được sử dụng cho tất cả các bảng về sau. Ghi chú: Các chữ cái khác nhau trên cùng một cột chỉ sự sai khác có ý nghĩa thống kê của các trung bình mẫu với p < 0,05 (Duncan’s test). Ghi chú này được sử dụng cho tất cả các bảng về sau. Hình 2. Tế bào đinh lăng sau 16 ngày nuôi cấy lắc: trái: sinh khối tươi, phải: sinh khối khô sau khi được nghiền mịn Hình 2. 3.1 Sinh trưởng của tế bào huyền phù Tế bào đinh lăng sau 16 ngày nuôi cấy lắc: trái: sinh khối tươi, phải: sinh khối khô sau khi được nghiền mịn 88 Tập 127, Số 1C, 2018 jos.hueuni.edu.vn 1.62 1.74 1.74 2.04 2.52 2.84 3.04 3.75 3.67 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 2 4 6 8 10 12 14 16 18 Chỉ số sinh trưởng Thời gian nuôi cấy (ngày) Hình 3. Đường cong sinh trưởng của tế bào đinh lăng dựa theo chỉ số sinh trưởng Hình 3. Đường cong sinh trưởng của tế bào đinh lăng dựa theo chỉ số sinh trưởng Fructose Số liệu ảnh hưởng của fructose lên sinh trưởng của tế bào đinh lăng đánh giá ở 16 ngày tuổi được trình bày ở Bảng 2. Có thể thấy rằng fructose 10–40 g/L không thích hợp cho việc tích lũy sinh khối tế bào đinh lăng; tất cả các công thức thí nghiệm đều cho kết quả thấp hơn đối chứng là sucrose 3%. Khi thay thế sucrose bằng fructose, hàm lượng fructose sử dụng càng nhiều thì sinh khối tế bào càng giảm. Maltose Khả năng sinh trưởng của tế bào đinh lăng trong môi trường có bổ sung maltose 10–40 g/L sau 16 ngày nuôi cấy được trình bày ở Bảng 3. Số liệu cho thấy maltose 10–40 g/L không thích hợp cho việc tích lũy sinh khối của tế bào đinh lăng, thấp hơn so với mẫu đối chứng được nuôi ở môi trường có chứa sucrose 3%. Bảng 2. Ảnh hưởng của frucrose lên sinh trưởng của tế bào Nồng độ fructose (g/L) Khối lượng tế bào (g) Chỉ số sinh trưởng Tươi Khô ĐC 7,50a 0,40a 3,75 10 5,68 b 0,36ab 2,84 20 4,43c 0,33b 2,23 30 3,26d 0,29c 1,63 40 2,74d 0,28c 1,37 Bảng 2. Ảnh hưởng của frucrose lên sinh trưởng của tế bào 89 Tập 127, Số 1C, 2018 Phan Thị Á Kim và Cs. Bảng 3. Ảnh hưởng của maltose lên sinh trưởng của tế bào Nồng độ maltose (g/L) Khối lượng tế bào (g) Chỉ số sinh trưởng Tươi Khô ĐC 7,50a 0,40a 3,75 10 4,35c 0,26c 2,13 20 4,38c 0,29bc 2,19 30 4,75b 0,31ab 2,38 40 4,37c 0,27bc 2,19 Bảng 3. Ảnh hưởng của maltose lên sinh trưởng của tế bào Như vậy, qua nghiên cứu ảnh hưởng của nguồn carbon lên khả năng sinh trưởng của tế bào huyền phù thì sucrose là nguồn carbon thích hợp cho sự sinh trưởng của tế bào đinh lăng hơn fructose và maltose. Theo các tài liệu đã công bố, sucrose được xem là nguồn carbon thích hợp nhất cho sinh trưởng của tế bào thực vật; nồng độ thường dùng khoảng từ 20 g/L đến 70 g/L. Sucrose vừa là nguồn cung cấp năng lượng vừa là một thành phần nguyên liệu trong sinh tổng hợp các chất thứ cấp. Tốc độ tăng trưởng sinh khối của tế bào luôn luôn liên quan trực tiếp với sự tiêu thụ sucrose [4]. Salicylic acid Salicylic acid ở các nồng độ 50–250 µM được bổ sung vào môi trường lúc bắt đầu nuôi cấy để khảo sát khả năng sinh trưởng của tế bào đinh lăng sau 16 ngày. Có thể thấy trong các môi trường có nồng độ SA khác nhau thì khả năng sinh trưởng của nuôi cấy tế bào huyền phù đinh lăng là khác nhau (Bảng 5). Nồng độ SA càng tăng thì màu sinh khối thu được có màu nâu tăng dần và lượng sinh khối có màu xanh giảm dần do sự oxy hóa các sản phẩm phenol tiết ra trong quá trình nuôi cấy bởi các enzyme ngoại bào. Ở nồng độ SA 250 µM, sinh trưởng của tế bào huyền phù bị ức chế với sinh khối tế bào huyền phù thấp nhất (khối lượng tươi trung bình chỉ còn 2,91 g, khối lượng khô trung bình là 0,24 g). Dịch chiết nấm men Khả năng tích lũy sinh khối của tế bào đinh lăng trong môi trường nuôi cấy có bổ sung dịch chiết nấm men ở các nồng độ khác nhau được trình bày ở Bảng 4. Kết quả cho thấy nồng độ dịch chiết nấm men có ảnh hưởng lên sự tích lũy sinh khối tế bào. Tế bào đinh lăng sinh trưởng trong môi trường chứa YE chậm hơn so với đối chứng không xử lý elicitor. Khi nồng độ YE tăng từ 1 g/L đến 5 g/L, khối lượng tế bào giảm dần, thấp nhất ở 5 g/L đạt 1,93 g tươi (0,18 g khô). Như vậy, sự tích lũy sinh khối tế bào tỷ lệ nghịch với nồng độ YE bổ sung vào môi trường nuôi cấy; nồng độ YE càng cao thì sinh khối tích lũy càng giảm. Nói cách khác, dịch chiết nấm men ức chế sự tích lũy sinh khối của tế bào đinh lăng nuôi cấy huyền phù trong bình 250 mL. Khi bổ sung dịch chiết nấm men ở nồng độ 1–2 g/L thì sinh khối tế bào có màu nâu và một số vẫn còn màu xanh của callus đưa vào lúc đầu nuôi cấy lắc. Tuy nhiên, khi bổ sung nồng độ 3– 5 g/L thì sinh khối có màu nâu toàn bộ do sự oxy hóa các sản phẩm phenol tiết ra trong quá trình nuôi cấy bởi các enzyme ngoại bào. Ở các môi trường không bổ sung elicitor, khối lượng tươi trung bình đạt 4,77 g và khối lượng khô trung bình đạt 0,38 g. Sinh khối thu được các tế bào nhỏ mịn thì có màu vàng nhạt và dạng hạt thì có màu xanh, xuất hiện rễ tơ. Như vậy, sự tích lũy sinh khối tế bào trong môi trường chứa elicitor giảm so với mẫu đối chứng không bổ sung elicitor. 90 Tập 127, Số 1C, 2018 jos.hueuni.edu.vn Bảng 4. Ảnh hưởng của dịch chiết nấm men lên sinh trưởng của tế bào Nồng độ YE (g/L) Khối lượng tế bào (g) Chỉ số sinh trưởng Tươi Khô ĐC 4,77a 0,38a 2,39 1 2,65b 0,28b 1,32 2 2,00c 0,20c 1,00 3 1,95c 0,19c 0,98 4 1,94c 0,19c 0,97 5 1,93c 0,18c 0,97 Bảng 4. Ảnh hưởng của dịch chiết nấm men lên sinh trưởng của tế bào Salicylic acid AgNO3 Số liệu ở Bảng 6 cho thấy trong môi trường có nồng độ AgNO3 khác nhau thì khả năng sinh trưởng của tế bào huyền phù đinh lăng khác nhau. Tế bào đinh lăng được bổ sung AgNO3 10–100 µM sinh trưởng chậm hơn so với đối chứng. Sinh khối tế bào giảm khi tăng nồng độ AgNO3. Sinh khối tươi giảm 3,71–1,78 g (0,29–0,19 g khô) so với đối chứng 4,77 g (0,38 g khô). Nồng độ càng tăng thì tế bào có màu nâu đậm dần do sự oxy hóa các sản phẩm phenol tiết ra trong quá trình nuôi cấy bởi các enzyme ngoại bào. Bảng 5. Ảnh hưởng của salicylic acid lên sinh trưởng của tế bào Nồng độ SA (µM) Khối lượng tế bào (g) Chỉ số sinh trưởng Tươi Khô ĐC 4,77a 0,38a 2,39 50 3,57b 0,30b 1,78 100 3,53b 0,29bc 1,18 150 3,32bc 0,27cd 1,66 200 3,24c 0,25d 1,62 250 2,91d 0,24d 1,46 Bảng 5. Ảnh hưởng của salicylic acid lên sinh trưởng của tế bào 91 Tập 127, Số 1C, 2018 Phan Thị Á Kim và Cs. Bảng 6. Ảnh hưởng của AgNO3 lên sinh trưởng của tế bào Nồng độ AgNO3 (µM) Khối lượng tế bào (g) Chỉ số sinh trưởng Tươi Khô ĐC 4,77a 0,38a 2,39 10 3,71b 0,29b 1,86 25 3, 32b 0,26c 1,66 50 2,28c 0,23d 1,14 75 2,15cd 0,23d 1,08 100 1,78d 0,19e 0,89 Bảng 6. Ảnh hưởng của AgNO3 lên sinh trưởng của tế bào Như vậy, ảnh hưởng của AgNO3 lên khả năng sinh trưởng của tế bào đinh lăng cũng giống như ảnh hưởng của các elicitor khác: khi nồng độ tăng lên thì sự sinh trưởng của tế bào bị ức chế. Trong quá trình sản xuất hợp chất thứ cấp từ nuôi cấy tế bào thực vật, elicitor thông thường sẽ làm giảm quá trình sinh trưởng của tế bào đồng thời tăng cường sản xuất các hợp chất thứ cấp. Trong nghiên cứu của chúng tôi, trong môi trường có bổ sung elicitor, sinh trưởng của tế bào kém hơn so với đối chứng không bổ sung elicitor: nồng độ elicitor càng cao thì sự sinh trưởng của tế bào càng giảm. Kết quả này phù hợp với các nghiên cứu trước đây về sự ức chế của elicitor lên sinh trưởng của tế bào thực vật. AgNO3 Chẳng hạn, các elicitor như MeJA, Ag+, chitosan và dịch chiết từ nấm (polysaccharide) ở các nồng độ khác nhau đều ức chế sinh trưởng của tế bào cây thông đỏ bắc (Taxus chinensis) [11]. Thanh và cs. đã sử dụng MeJA để tổng hợp ginsenoside trong nuôi cấy huyền phù tế bào của nhân sâm trong biorector 5 L. Nhóm tác giả tiến hành thăm dò bổ sung MeJA ở các nồng độ 50–400 µM vào môi trường nuôi cấy; sau 25 ngày nuôi cấy thu được hàm lượng ginsenoside cao nhất ở nồng độ 200 µM, cao gấp 2,2 lần so với tế bào nuôi cấy không bổ sung MeJA. Trong khi đó, sinh khối tươi và sinh khối khô của tế bào giảm 1,06 và 1,10 lần so với tế bào nuôi cấy không bổ sung MeJA [9]. Kết quả nghiên cứu của Frankfater và cs. về ảnh hưởng của MeJA và SA lên sự tạo thành gossypol, 6-methoxygossypol và 6,6’-dimethoxygossypol trong nuôi cấy rễ tơ cây bông vải (Gossypium barbadense) cho thấy MeJA có tác dụng ức chế quá trình sinh trưởng của tế bào [7]. 4 Kết luận Môi trường MS lỏng có bổ sung NAA 2 mg/L và KIN 0,5 mg/L và sucrose 3% là tốt nhất cho khả năng sinh trưởng của tế bào đinh lăng; sinh khối tế bào tươi đạt 7,50 g (0,40 g khô) sau 16 ngày nuôi cấy. Tất cả các loại elicitor sử dụng trong nghiên cứu đều ức chế sự sinh trưởng của tế bào huyền phù: nồng độ elicitor càng cao sinh khối tế bào càng giảm. Đây là điều kiện cần thiết để tăng sự tích lũy các hợp chất thứ cấp trong tế bào. 92 Tập 127, Số 1C, 2018 jos.hueuni.edu.vn Tài liệu tham khảo 1. Nguyễn Trung Hậu, Trần Văn Minh (2015), Nuôi cấy mô lá đinh lăng (Polyscias fruticosa L. Harms) tạo rễ tơ và nhận biết hoạt chất saponin tích lũy. Tạp chí Khoa học Trường đại học An Giang 7(1), 75–83. 2. Phạm Hoàng Hộ (2003), Cây cỏ Việt Nam, quyển III, Nxb. Trẻ, TP. Hồ Chí Minh. 3. Hà Bích Hồng, Vũ Thị Thơm, Vũ Đức Lợi, Lê Anh Tuấn, Nguyễn Thanh Hải (2013), Bước đầu xây dựng quy trình nhân giống in vitro cây Đinh lăng lá nhỏ (Polyscias fruticosa (L.) Harms). Tạp chí Dược học, 450, 25–30. 4. Bùi Văn Lệ, Nguyễn Ngọc Hồng (2006), Ảnh hưởng của chất điều hòa tăng trưởng thực vật và đường saccharose lên dịch nuôi cấy huyền phù tế bào dừa cạn (Catharanthus roseus). Tạp chí Phát triển Khoa học và Công nghệ 9(6), 5–66. 5. Phạm Thị Tố Liên, Võ Thị Bạch Mai (2007), Bước đầu nghiên cứu sự tạo dịch treo tế bào cây Đinh lăng Polyscias fruticosa L. Harms. Tạp chí Phát triển Khoa học và Công nghệ 10(7), 11–16. 6. Đỗ Tất Lợi (1986), Những cây thuốc và vị thuốc Việt Nam, Nxb. Khoa học và Kỹ thuật Hà Nội. 7. Frankfater C. R., Dowd M. K., Triplett B. A. (2009), Effect of elicitors on the production of gossypol and methylated gossypol in cotton hairy roots. Plant Cell Tiss Organ Cult, 98, 341–349. 8. Sakr S. S., Melad S. S., El-Shamy M. A., Elhafez A. E. A. (2014), In vitro propagation of Polyscias fruticosa plant. International Journal of Plant & Soil Science, 3(10), 1254–1265. 9. Thanh N. T., Murthy H. N., Yu K. W., Hahn E. J., Peak K. Y. (2005), Methyl jasmonate elicitation enhanced synthesis of ginsenoside by cell suspension culture of Panax ginseng in 5-l balloon type bubble bioreactor. Appl Microbiol Biotechnol, 67, 197–201. 10. Vijaya S. N., Udayasri P. V. V., Aswani K. Y., Ravi B. B., Phani K. Y., Vijay V. M. (2010), Advancements in the production of secondary metabolites. Natural Products, 3, 112–123. 11. Zhang C. H., Mei X. G., Liu L., Yu L. J. (2000), Enhanced paclitaxel production induced by the combination of elicitors in cell suspension cultures of Taxus chinensis. Biotechnol Lett, 22, 1561–1564. 93 93 Tập 127, Số 1C, 2018 Phan Thị Á Kim và Cs. EFFECTS OF CARBON SOURCES AND ELICITORS ON GROWTH OF POLYSCIAS FRUTICOSA (L.) HARMS SUSPENSION CELLS Phan Thi A Kim1,2, Nguyen Thi Ha Ngan1, Le Thi Anh Thu1, Le Van Tuong Huan1* Phan Thi A Kim1,2, Nguyen Thi Ha Ngan1, Le Thi Anh Thu1, Le Van Tuong Huan1* 1 Department of Biology, University of Sciences, Hue University, 77 Nguyen Hue St., Hue, Vietnam 2 Department of Science and Technology, Quang Nam, 54 Hung Vuong St., Quang Nam, Vietnam Abstract. Polyscias fruticosa (L.) Harms is a valuable medicinal plant, widely used in folk medicine. In this study, the effects of carbon sources and elicitors (yeast extract, salicylic acid, and silver nitrate) on the growth of suspension cells were evaluated. The results indicated that the optimal medium for growth of the cells was liquid MS supplemented with 2 mg/L NAA, 0.5 mg/L Kinetin, and 3% sucrose, with fresh cell biomass reaching 7.50 g (0.40 g dry weight) after 16 days of culture. All elicitors used in this study inhibited the cell growth. These were necessary conditions for the accumulation of secondary substances in the suspension cell culture. Keywords: carbon source, elicitor, Polyscias fruticosa (L.) Harms, suspension cell 94
W4389482617.txt	https://www.frontiersin.org/articles/10.3389/fpls.2023.1333927/pdf?isPublishedV2=False	en		Editorial: Alternative fertilizer harnessing plant-microbe interactions (AFPMI) for improved soil and plantnutrient management	Frontiers in plant science	2,023	cc-by	3,802	Editorial 06 December 2023 DOI 10.3389/fpls.2023.1333927 TYPE PUBLISHED OPEN ACCESS EDITED AND REVIEWED BY Gianpiero Vigani, University of Turin, Italy CORRESPONDENCE Adil Mihoub adilmihoub15@yahoo.com Iftikhar Ahmad iftikharahmad@cuivehari.edu.pk Emanuele Radicetti rdcmnl@unife.it 06 November 2023 29 November 2023 PUBLISHED 06 December 2023 RECEIVED ACCEPTED CITATION Mihoub A, Ahmad I and Radicetti E (2023) Editorial: Alternative fertilizer harnessing plant-microbe interactions (AFPMI) for improved soil and plant nutrient management. Front. Plant Sci. 14:1333927. doi: 10.3389/fpls.2023.1333927 Editorial: Alternative fertilizer harnessing plant-microbe interactions (AFPMI) for improved soil and plant nutrient management Adil Mihoub 1, Iftikhar Ahmad 2* and Emanuele Radicetti 3* 1 Center for Scientiﬁc and Technical Research on Arid Regions (CRSTRA), Biophysical Environment Station, Touggourt, Algeria, 2 Department of Environmental Sciences, COMSATS University Islamabad, Vehari, Pakistan, 3 Department of Chemical, Pharmaceutical and Agricultural Sciences (DOCPAS), University of Ferrara, Ferrara, Italy KEYWORDS nutrient imbalances, bio-fertilizers, beneﬁcial microbes, climate change, ﬁeld crops, food security, soil health COPYRIGHT © 2023 Mihoub, Ahmad and Radicetti. 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. Frontiers in Plant Science Editorial on the Research Topic Alternative fertilizer harnessing plant-microbe interactions (AFPMI) for improved soil and plant nutrient management Introduction Worldwide, agriculture is the main source of food for people and is essential to maintaining food security (Jamal et al., 2023). However, there are many challenges in this sector, including the depletion of soil fertility and land degradation. These issues have led to a decrease in the amount of food available per person in various regions globally (Bouma, 2020). The sustainability of agroecosystems, preservation of biodiversity, and food security are threatened by land degradation and climate change (AbdelRahman, 2023; Kumawat et al., 2023). Improper agricultural land management has reduced 25% of total land worldwide and caused an annual soil loss of approximately 24 billion tons (Ejaz et al., 2023). This has serious implication for global food supply because degraded soil is less resilient and less productive (Mihoub et al., 2022a). It is estimated that worldwide food supply is expected to fall by 12% over the next 25 years, resulting in a 30% increase in food costs (AbdelRahman, 2023). A healthy soil that can provide balanced nutrients to plants is essential for increased productivity (BoukhalfaDeraoui et al., 2015a). Soil is composed of mineral components, organic matter, and microorganisms that have evolved over thousands and millions of years. Unfortunately, due to human activities and improper land management, this delicate balance is disrupted, resulting in rapid soil degradation within a few years (AbdelRahman, 2023). Climate change and depleting water and land resources have built up pressure on food supply and demand, which will be greatly increased by 2050 to feed 9.7 billion people worldwide (Becker and Fanzo, 2023). Unfortunately, this situation is further worsened by a hike in fertilizer prices and mismanagement of fertilizer allocation (Mihoub and Boukhalfa-Deraoui, 2014). 01 frontiersin.org Mihoub et al. 10.3389/fpls.2023.1333927 comprehensive approach not only helps protect the environment but also supports the long-term viability of agricultural systems. As we work towards reducing the effects of climate change, Alternative Fertilizers Harnessing Plant-Microbe Interactions serves as a tool for promoting soil wellbeing and ensuring food production for generations. Fertilizer prices have risen signiﬁcantly in many countries, particularly in Africa, Latin America, and Asia, and smallholder farmers do not have access to fertilizers (Hebebrand and Laborde Debucquet, 2023); thus, rising uncertainty and high fertilizer prices are already affecting food production prospects and the livelihood of farmers (BoukhalfaDeraoui et al., 2015b; Mihoub et al., 2016). Additional fertilizer production is not the only solution to this crisis, further initiatives must be made to encourage alternative fertilizer sources for sustainable soil management (SSM), sustainable crop production (SCP), and food security in changing climates, which signiﬁcantly help to support crop production and mitigate the negative impacts of biotic and abiotic stresses (Astapati and Nath, 2023; Kumar et al., 2023). These alternative fertilizer sources play a pivotal role in SSM and SCP, as they not only contribute to cost savings but also offer numerous environmental beneﬁts. By the use of organic fertilizers derived from waste materials not only helps manage and recycle urban waste ﬂuxes but also provides added value in economic terms related to nutrient contents (Khan et al., 2022). Furthermore, the use of bio-fertilizers enriched with beneﬁcial microbes enhances soil health and fertility (Mahmud et al., 2021). Harnessing plant-microbe interactions for alternative fertilization methods is a promising approach to enhance farm productivity and reduce the environmental impact of synthetic chemical fertilizers (Macdonald and Singh, 2014). The Research Topic, titled “Alternative Fertilizer Harnessing Plant-Microbe Interactions (AFPMI) for Improved Soil and Plant Nutrient Management,” explores various strategies to foster alternative fertilizer sources for sustainable soil management, crop production, and food security in changing climates. AFPMI aims to advocate for the use of manures, biochar, bio-fertilizers, nanofertilizers, and beneﬁcial microbes. The ultimate objective is to maximize crop yields by highlighting the crucial role of alternative fertilizer sources and technologies in soil nutrition. This approach will undoubtedly optimize resource consumption while minimizing detrimental effects on natural resources. Alternative fertilizer harnessing plantmicrobe interactions for improved crop productivity Alternative Fertilizer Harnessing Plant-Microbe Interactions (AFPMI) for Improved Soil and Plant Nutrient Management plays a vital role in improving plant productivity. This is especially important amid rapid climate change, where ensuring food security is a top priority. AFPMI optimizes crop nutrient utilization, ensuring higher crop yields, and crop resilience through the strategic application of organic and bio-fertilizers (Daraz et al., 2023; Salman et al., 2023; Ullah et al., 2023). By embracing the practices of Alternative Fertilizers Harnessing Plant-Microbe Interactions, we can effectively tackle the challenges presented by climate change and guarantee a sustainable and secure food supply for future generations. Alternative fertilizer harnessing plantmicrobe interactions: a solution to rising fertilizer cost and food security Among the major beneﬁts of AFPMI that make it crucial is that it minimizes further reliance on expensive chemical fertilizers. Due to the escalation in fertilizer prices and the consequences of climatic change, compliance with methods brought about by AFPMI indeed emerges as an attractive and cost-effective option for the farming community (Mihoub et al., 2022b; Mihoub et al., 2022c). AFPMI improves the efﬁciency of nutrient usage and hence reduces the cyclic use of costly chemical inputs. In this way, we can reduce the cost of production and promote sustainable agricultural practices (Khan et al., 2022). Food security is closely attached to the utilization of AFPMI and is especially concerned with challenges linked to climate change. Improved crop yield has been embraced through the adoption of AFPMI practices towards increased production levels required for food security cases (Ahmad et al., 2022; Jamal et al., 2023). AFPMI has tremendous beneﬁts concerning the improvement of soil fertility and acidity vis-à-vis the apparent effects of environmental change such as heavy metals, long droughts, and salt stress (Hamid et al., 2021; Tahir et al., 2021; Ahmad et al., 2022; Daraz et al., 2023). AFPMI techniques are used by farmers to protect food crops from nutrient deﬁciency, promote uniformity, and predictable food production (Daraz et al., 2021; Hussain et al., 2022). Further, AFPMI is a sustainable kind of agriculture that signiﬁcantly reduces the impact on the environment. This Alternative fertilizers harnessing plantmicrobe interactions and soil health Improving soil health is crucial with reference to climate change. One effective approach is to use Alternative Fertilizer Harnessing PlantMicrobe Interactions (AFPMI) which combine types of fertilizers like organic, chemical, nano, and bio fertilizers. This holistic method helps in development and proliferation of soil edaphon (Amin and Mihoub, 2021; Daraz et al., 2021; Tahir et al., 2021; Babar et al., 2022), which have a vital role in enhancing soil structure and fertility (Khan et al., 2022; Jamal et al., 2023). By adopting practices that involve Alternative Fertilizers Harnessing Plant-Microbe Interactions, farmers can establish soils that’re better equipped to endure harsh weather conditions and get high economic yields from crops. This resilience is crucial as agriculture faces the challenges posed by climate change. Research conducted by Krasilnikov et al. (2022) further emphasizes the signiﬁcance of AFPMI in adapting to these climate variations. Through AFPMI practices, farmers can ensure that their soil remains fertile and productive when faced with changing weather patterns. This Frontiers in Plant Science 02 frontiersin.org Mihoub et al. 10.3389/fpls.2023.1333927 food security. This study advocates the use of blend of biofertilizers, organic fertilizers, and chemical fertilizers to improve wheat yield. The combination of DAP+FYM+BA treatment not only improves wheat growth, biomass, and yield but also improves the quality of wheat grain (Asghar et al.). This research highlights the positive role of biofertilizers supplements as alternatives to conventional fertilizers to promote soil health, increase productivity, and food security while reducing the ecological footprint. This study reveals that the application of biofertilizers can help the strawberry plant overcome the negative effects of nitrogen deﬁciency (Garcı́a -Ló pez et al.). PGPRinoculation improved the economic yield of strawberries by enhancing the efﬁciency of photosystem-II and N-nutrition for plants in an N-deﬁcient environment. This approach conﬁrms that biofertilizers may be promising complementary tools to improve nitrogen-based chemical fertilization processes, thereby signiﬁcantly reducing the use of this agrochemical. This study (Ujvá ri et al.) reveals that microbial communities regulate soil fertility and are mainly dependent on crop species and cropping systems adapted by farmers (Ujvá ri et al.). The NP fertilization and maize genotypes played a vital role in reshaping the microbial communities in the maize rhizosphere at the plant emergence stage. Application of NP fertilization, maize hybrids, and biostimulants increased plant growth, early ﬂowering, and the economic yield of maize in ﬁeld conditions (Capo et al.). They also proposed alternative techniques, such as utilizing early-vigor genotypes or applying biostimulants to seeds, which can help reduce the negative impacts of abiotic stresses associated with early planting and result in faster crop development. These innovations may minimize or, in some circumstances, eliminate the need for starting fertilization. Two studies focus on the application of pristine biochar or its combination with ﬁshpond sediment and iron as an alternative or supplement source of nutrient management under nutrient or heavy metal stress. Mahmood et al. highlighted the signiﬁcance of using the optimal amounts of biochar and ﬁshpond sediments to improve the nutrient content and availability of soil. It provides valuable information on how biochar and ﬁshpond sediments can be used together to reduce phosphorus ﬁxation and prevent the loss of residual phosphorus. Ultimately, this approach helps to sustain food production. In another study, Algethami et al. found that application of iron-modiﬁed biochar (Fe-BC) improved the nutritional quality of soil and mitigated metal toxicity in wheat plants under heavy metal stress. The results showed that Fe-BC is a promising strategy for improving soil fertility and reducing the harmful effects of Cd and Pb contamination. This research has signiﬁcant implications for enhancing food security in stressful environments. approach helps in protecting ecosystems and ensuring food security (Nadia et al., 2023), thus fostering development towards a more resilient and safe food future. Alternative fertilizer harnessing plantmicrobe interactions and climate change adaptation As the Earth’s climate continues to undergo signiﬁcant changes, it is crucial to explore effective strategies for adapting to these shifts, particularly in the ﬁeld of agriculture. AFPMI practices are designed to help crops withstand the increasingly challenging environmental stressors brought about by climate change, such as rising temperatures and unpredictable precipitation patterns. By enhancing the resilience of crops, AFPMI practices enable them to better cope with heat and water scarcity, ultimately safeguarding agricultural productivity (Krasilnikov et al., 2022). AFPMI not only beneﬁts crop directly but also supports global initiatives to mitigate climate change. It contributes to the development of carbon-free agricultural farming, which could be achieved by reducing greenhouse gases in the atmosphere and increasing the passive pool of carbon in the soil through biochar (Wang et al., 2023). Such farming practices would be eco-friendlier and climate resilient. Articles and insights This Research Topic presents a collection of articles that provide insights into the various impacts of alternative fertilizer harnessing plant-microbe interactions (AFPMI) on sustainable agriculture. These research contributions cover important aspects of agricultural sustainability, including interventions in soil microbiota and the management of organic waste. This editorial outline the signiﬁcant ﬁndings of all the manuscript published in this Research Topic with a focus on their signiﬁcance to soil and plant health, crop yields, fertilizer costs, food security, and climate change. One of the articles discusses the long-term application of sheep manure fertilizer at a depth of 50–70 cm on tea plantations. This practice has been found to alleviate soil acidiﬁcation by improving soil pH, ammonium nitrogen content, nitrogen ﬁxation ability, and root activity. As a result, tea tree roots are better able to absorb nitrogen from the soil, leading to increased tea yield and quality (Jia et al.). This research not only contributes to sustainable agriculture but also reduces the need for chemical pesticides and promotes soil health. Biofertilizer or PGPR has a vital role in maintaining soil and plant health thus, ﬁve studies deal with either the use of sole biofertilizer or its co-inoculation with organic fertilizers. For instance, Ma et al. isolated the new bacterial strain MQR6T (Pantoea rhizosphaerae) from the rhizosphere of Acer truncatum Bunge. This strain can solubilize inorganic P produce indole acetic acid and siderophores. The inoculation of strain MQR6T with A. truncatum improves plant growth, biomass, and P-accumulation in plants. This strategy has the potential to reduce dependence on inorganic fertilizers, improve beneﬁt to cost ratio, thus contribute to Frontiers in Plant Science Concluding remarks and future perspectives Alternative fertilizer harnessing plant-microbe interactions (AFPMI) play a vital role in improving soil health, plant productivity, and climate resilience. We can achieve maximum 03 frontiersin.org Mihoub et al. 10.3389/fpls.2023.1333927 economic yields through judicious application of organic fertilizers (sheep manure, biochar), microbial-based fertilizers (biofertilizers), synergistic use of bio-synthetic fertilizers (PGPR + chemical fertilizers), adopting various agronomic practices (seed priming, crop genotypes) and cropping systems. These AFPMI practices not only improve soil fertility status and soil edaphon but also translate it into better crop stand, economic yields, and resistance to climate change. By utilizing agricultural technologies and adopting innovative practices, we have the potential to create a future where sustainable agriculture forms the basis of food security, despite the challenges posed by climate change. Acknowledgments We want to express our gratitude to Frontier in Plant Science for giving us the opportunity to be guest editors for the Research Topic “Alternative Fertilizer and Plant-Microbe Interactions for Improved Soil and Plant Nutrient Management.” Our warmest gratitude also goes to the authors, contributors and experts who have played a signiﬁcant role in making this Research Topic a valuable and engaging resource. Conﬂict of interest Author contributions 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. AM: Conceptualization, Validation, Visualization, Writing – original draft, Writing – review & editing. IA: Writing – original draft, Writing – review & editing. ER: Writing – original draft, Writing – review & editing. 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. Funding The author(s) declare that no ﬁnancial support was received for the research, authorship, and/or publication of this article. References AbdelRahman, M. A. E. (2023). An overview of land degradation, desertiﬁcation and sustainable land management using GIS and remote sensing applications. Rendiconti Lincei. Sci. Fisiche e Naturali 34, 1–42. doi: 10.1007/s12210-023-01155-3 Daraz, U., Li, Y., Sun, Q., Zhang, M., and Ahmad, I. (2021). Inoculation of Bacillus spp. modulate the soil bacterial communities and available nutrients in the rhizosphere of vetiver plant irrigated with acid mine drainage. Chemosphere 263, 128345. doi: 10.1016/j.chemosphere.2020.128345 Ahmad, I., Munsif, F., Mihoub, A., Jamal, A., Saeed, M. F., Babar, S., et al. (2022). Beneﬁcial effect of melatonin on growth and chlorophyll content in wheat (Triticum aestivum L.) grown under salt stress conditions. Gesunde Pﬂanzen 74 (4), 997–1009. doi: 10.1007/s10343-022-00684-5 Ejaz, N., Elhag, M., Bahrawi, J., Zhang, L., Gabriel, H. F., and Rahman, K. U. (2023). Soil erosion modelling and accumulation using RUSLE and remote sensing techniques: case study Wadi Baysh, Kingdom of Saudi Arabia. Sustainability 15 (4), 3218. doi: 10.3390/su15043218 Amin, A.E.E.A.Z., and Mihoub, A. (2021). Effect of sulfur-enriched biochar in combination with sulfur-oxidizing bacterium (Thiobacillus spp.) on release and distribution of phosphorus in high calcareous p-ﬁxing soils. J. Soil Sci. Plant Nutr. 21 (3), 2041–2047. doi: 10.1007/s42729-021-00500-5 Hamid, B., Zaman, M., Farooq, S., Fatima, S., Sayyed, R. Z., Baba, Z. A., et al. (2021). Bacterial plant biostimulants: a sustainable way towards improving growth, productivity, and health of crops. Sustainability 13 (5), 2856. doi: 10.3390/su13052856 Hebebrand, C., and Laborde Debucquet, D. (2023). “High fertilizer prices contribute to rising global food security concerns,” in The Russia-Ukraine conﬂict and global food security, (International Food Policy Research Institute (IFPRI)), 7, 38–42. Astapati, A. D., and Nath, S. (2023). The complex interplay between plant-microbe and virus interactions in sustainable agriculture: Harnessing phytomicrobiomes for enhanced soil health, designer plants, resource use efﬁciency, and food security. Crop Design 2, 100028. doi: 10.1016/j.cropd.2023.100028 Hussain, S., Khan, S. M., Jamal, A., Mihoub, A., Saeed, M. F., Khalid, M. S., et al. (2022). Improvement of growth, yield and biochemical properties of potato (Solanum tuberosum L.”Montreal” and “Red bull”) by foliar application of zinc under calcareous soil conditions. Gesunde Pﬂanzen 74 (3), 561–570. doi: 10.1007/s10343-022-00631-4 Babar, S., Jilani, G., Mihoub, A., Jamal, A., Ahmad, I., Chaudhary, A. N., et al. (2022). Bacterial redox cycling of manganese in calcareous soil enhances the nutrients bioavailability to wheat. J. Soil Sci. Plant Nutr. 21 (3), 1215–1223. doi: 10.1007/ s42729-021-00725-4 Jamal, A., Saeed, M. F., Mihoub, A., Hopkins, B. G., Ahmad, I., and Naeem, A. (2023). Integrated use of phosphorus fertilizer and farmyard manure improves wheat productivity by improving soil quality and P availability in calcareous soil under subhumid conditions. Front. Plant Sci. 14. doi: 10.3389/fpls.2023.1034421 Becker, S., and Fanzo, J. (2023). Population and food systems: what does the future hold? Population Environ. 45 (3), 20. doi: 10.1007/s11111-023-00431-6 Boukhalfa-Deraoui, N., Haniﬁ-Mekliche, L., Mekliche, A., Mihoub, A., and Daddibouhoun, M. (2015b). Effect of phosphorus application on durum wheat in alkaline sandy soil in arid condition of Southern Algeria. Asian J. Crop Sci. 7 (1), 61–71. doi: 10.3923/ajcs.2015.61.71 Khan, I., Amanullah, Jamal, A., Mihoub, A., Farooq, O., Farhan Saeed, M., et al. (2022). Partial substitution of chemical fertilizers with organic supplements increased wheat productivity and proﬁtability under limited and assured irrigation regimes. Agriculture 12 (11), 1754. doi: 10.3390/agriculture12111754 Boukhalfa-Deraoui, N., Haniﬁ-Mekliche, L., and Mihoub, A. (2015a). Effect of incubation period of phosphorus fertilizer on some properties of sandy soil with low calcareous content, Southern Algeria. Asian J. Agric. Res. 9 (3), 123–131. doi: 10.3923/ajar.2015.123.131 Krasilnikov, P., Taboada, M. A., and Amanullah, (2022). Fertilizer use, soil health and agricultural sustainability. Agriculture 12 (4), 462. doi: 10.3390/ agriculture12040462 Bouma, J. (2020). Soil security as a roadmap focusing soil contributions on sustainable development agendas. Soil Secur. 1, 100001. doi: 10.1016/ j.soisec.2020.100001 Kumar, U., Raj, S., Sreenikethanam, A., Maddheshiya, R., Kumari, S., Han, S., et al. (2023). Multi-omics approaches in plant–microbe interactions hold enormous promise for sustainable agriculture. Agronomy 13 (7), 1804. doi: 10.3390/agronomy13071804 Daraz, U., Ahmad, I., Li, Q. S., Zhu, B., Saeed, M. F., Li, Y., et al. (2023). Plant growth promoting rhizobacteria induced metal and salt stress tolerance in Brassica juncea through ion homeostasis. Ecotoxicology Environ. Saf. 267, 115657. doi: 10.1016/ j.ecoenv.2023.115657 Frontiers in Plant Science Kumawat, A., Yadav, D., Srivastava, P., Babu, S., Kumar, D., Singh, D., et al. (2023). Restoration of agroecosystems with conservation agriculture for food security to achieve sustainable development goals. Land Degradation Dev. 34, 3079–3097. doi: 10.1002/ldr.4677 04 frontiersin.org Mihoub et al. 10.3389/fpls.2023.1333927 Macdonald, C., and Singh, B. (2014). Harnessing plant-microbe interactions for enhancing farm productivity. Bioengineered 5 (1), 5–9. doi: 10.4161/bioe.25320 decreasing p adsorption in a calcareous sandy soil. Commun. Soil Sci. Plant Anal. 53 (19), 2596–2607. doi: 10.1080/00103624.2022.2072859 Mahmud, A. A., Upadhyay, S. K., Srivastava, A. K., and Bhojiya, A. A. (2021). Biofertilizers: A Nexus between soil fertility and crop productivity under abiotic stress. Curr. Res. Environ. Sustainability 3, 100063. doi: 10.1016/j.crsust.2021.100063 Nadia, Amanullah, Arif, M., and Muhammad, D. (2023). Improvement in wheat productivity with integrated management of beneﬁcial microbes along with organic and inorganic phosphorus sources. Agriculture 13 (6), 1118. doi: 10.3390/ agriculture13061118 Mihoub, A., Amin, A.E.E.A.Z., Motaghian, H. R., Saeed, M. F., and Naeem, A. (2022b). Citric acid (CA)–modiﬁed biochar improved available phosphorus concentration and its half-life in a P-fertilized calcareous sandy soil. J. Soil Sci. Plant Nutr. 21, 465–474. doi: 10.1007/s42729-021-00662-2 Salman, M., Inamullah, Jamal, A., Mihoub, A., Saeed, M. F., Radicetti, E., et al. (2023). Composting sugarcane ﬁlter mud with different sources differently beneﬁts sweet maize. Agronomy 13 (3), 748. doi: 10.3390/agronomy13030748 Mihoub, A., and Boukhalfa-Deraoui, N. (2014). Performance of different phosphorus fertilizer types on wheat grown in calcareous sandy soil of El-Menia, southern Algeria. Asian J. Crop Sci. 6 (4), 383–391. doi: 10.3923/ajcs.2014.383.391 Tahir, M., Imran, M., Nawaz, F., Shahid, M., Naeem, M. A., Ahmad, I., et al. (2021). Effects of Bacillus sp. MR-1/2 and magnetite nanoparticles on yield improvement of rice by urea fertilizer under different watering regimes. J. Appl. Microbiol. 131 (5), 2433–2447. doi: 10.1111/jam.15110 Ullah, J., Shah, S., Mihoub, A., Jamal, A., Saeed, M. F., Szé kely, Á ., et al. (2023). Assessing the effect of combining phosphorus fertilizers with crop residues on maize (Zea Mays L.) productivity and ﬁnancial beneﬁts. Gesunde Pﬂanzen 75, 1995–2008. doi: 10.1007/s10343-023-00829-0 Mihoub, A., Daddi Bouhoun, M., and Saker, M. L. (2016). Phosphorus adsorption isotherm: A key aspect for effective use and environmentally friendly management of phosphorus fertilizers in calcareous soils. Commun. Soil Sci. Plant Anal. 47 (16), 1920– 1929. doi: 10.1080/00103624.2016.1206923 Mihoub, A., Koull, N., Helimi, S., Elhafed Kherraze, M., Mokhtari, S., and Pulido Fernández, M. (2022a). Developing scoring functions for soil quality to assess land suitability for irrigated wheat in Southern Algeria. Soil Use Manage. 38 (1), 262–276. doi: 10.1111/sum.12770 Wang, L., Chen, D., and Zhu, L. (2023). Biochar carbon sequestration potential rectiﬁcation in soils: Synthesis effects of biochar on soil CO 2 , CH 4 and N 2 O emissions. Sci. Total Environ. 904, 167047. doi: 10.1016/j.scitotenv. 2023.167047 Mihoub, A., Naeem, A., Amin, A.E.E.A.Z., Jamal, A., and Saeed, M. F. (2022c). Pigeon manure tea improves phosphorus availability and wheat growth through Frontiers in Plant Science 05 frontiersin.org
https://openalex.org/W2555127608	http://shura.shu.ac.uk/14205/1/Bhowmik%20-%20Dataflow%20IR%20for%20memory%20efficient%20RIPL%20%28AM%29.pdf	English	null	A Dataflow IR for Memory Efficient RIPL Compilation to FPGAs	Lecture notes in computer science	2,016	public-domain	6,635	A dataflow IR for memory efficient RIPL compilation to FPGAs STEWART, Robert, MICHAELSON, Greg, BHOWMIK, Deepayan <http://orcid.org/0000-0003-1762-1578>, GARCIA, Paulo and WALLACE, Andy Available from Sheffield Hallam University Research Archive (SHURA) at: http://shura.shu.ac.uk/14205/ TEWART, Robert, MICHAELSON, Greg, BHOWMIK, Deepayan http://orcid.org/0000-0003-1762-1578>, GARCIA, Paulo and WALLACE, ndy Available from Sheffield Hallam University Research Archive (SHURA) at: http://shura.shu.ac.uk/14205/ Available from Sheffield Hallam University Research Archive (SHURA) at: http://shura.shu.ac.uk/14205/ This document is the author deposited version. You are advised to consult the publisher's version if you wish to cite from it. Published version STEWART, Robert, MICHAELSON, Greg, BHOWMIK, Deepayan, GARCIA, Paulo and WALLACE, Andy (2016). A dataflow IR for memory efficient RIPL compilation to FPGAs. In: CARRETERO, Jesus, GARCIA-BLAS, Javier, GERGEL, Victor, VOEVODIN, Vladimir, MEYEROV, Iosif, RICO-GALLEGO, Juan A., DIAZ-MARTIN, Juan C., ALONSO, Pedro, DURILLO, Juan, GARCIA SANCHEZ, Jose Daniel, LASTOVETSKY, Alexey L., MAROZZO, Fabrizio, LIU, Qin, BHUIYAN, Zakirul Alam, FURLINGER, Karl, WEIDENDORFER, Josef and GARCIA, Jose, (eds.) Algorithms and architectures for parallel processing : ICA3PP 2016 Collocated Workshops: SCDT, TAPEMS, BigTrust, UCER, DLMCS, Granada, Spain, December 14-16, 2016, Proceedings. Lecture Notes in Computer Science (10049). Springer, 174-188. Copyright and re-use policy See http://shura.shu.ac.uk/information.html Sheffield Hallam University Research Archive http://shura.shu.ac.uk A Dataﬂow IR for Memory Eﬃcient RIPL Compilation to FPGAs Robert Stewart1, Greg Michaelson1 Deepayan Bhowmik2, Paulo Garcia2, and Andy Wallace2 1 School of Mathematical and Computer Sciences, Heriot-Watt University, Edinburgh, UK 2 School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh, UK Abstract. Field programmable gate arrays (FPGAs) are fundamentally diﬀerent to ﬁxed processors architectures because their memory hierar- chies can be tailored to the needs of an algorithm. FPGA compilers for high level languages are not hindered by ﬁxed memory hierarchies. The constraint when compiling to FPGAs is the availability of resources. Abstract. Field programmable gate arrays (FPGAs) are fundamentally diﬀerent to ﬁxed processors architectures because their memory hierar- chies can be tailored to the needs of an algorithm. FPGA compilers for high level languages are not hindered by ﬁxed memory hierarchies. The constraint when compiling to FPGAs is the availability of resources. In this paper we describe how the dataﬂow intermediary of our declara- tive FPGA image processing DSL called RIPL3 enables us to constrain memory. We use ﬁve benchmarks to demonstrate that memory use with RIPL is comparable to the Vivado HLS OpenCV library without the need for language pragmas to guide hardware synthesis. The benchmarks also show that RIPL is more expressive than the Darkroom FPGA image processing language. In this paper we describe how the dataﬂow intermediary of our declara- tive FPGA image processing DSL called RIPL3 enables us to constrain memory. We use ﬁve benchmarks to demonstrate that memory use with RIPL is comparable to the Vivado HLS OpenCV library without the need for language pragmas to guide hardware synthesis. The benchmarks also show that RIPL is more expressive than the Darkroom FPGA image processing language. Keywords: domain speciﬁc languages, FPGAs, data locality 3 Rathlin Image Processing Language 1 Introduction 1.1 Memory Costs of High Level FPGA Languages 1.2 Data Locality Fixed memory architectures comprise very fast cache access, oﬀchip DDR mem- ory access, or slow disk storage. Each application must ﬁt into a ﬁxed mem- ory architecture representing a single large hierarchical memory space. A com- mon approach is to build data locality aware compilers [11], e.g. locality aware scheduling of OpenMP tasks on multicore CPUs [9] and mapping nested access patterns on GPUs [8]. Minimising cache misses involves proﬁling cache traces, moreover trading function inlining with executable size, and managing memory pressure. Minimising memory requirements is a particular problem for close to sensor real-time image processing on FPGAs, where hard choices must be made in trading oﬀmemory and processing. 1.1 Memory Costs of High Level FPGA Languages General Purpose Languages Programming with C++ for FPGAs often re- lies heavily on the programmer’s use of language pragmas to control how data structures should be implemented in hardware. For example when using Xilinx Vivado HLS [13], if a 3×3 window for applying a 2D ﬁlter is needed, the pro- grammer must use an array partition pragma to partition the 3×3 pixel window array into individual scalar elements, to avoid its implementation using BRAM. Image Processing Languages and Libraries Domain speciﬁc languages (DSLs) oﬀer potential for clearer syntax, stronger semantic checks, type-system- based guarantees and compiler optimisation for improved code execution. Com- pared to compiling C/C++ with HLS tools, DSLs can capture domain knowledge to abstract hardware templates that encapsulate common data access patterns that can more easily be analysed, e.g. for FIFO depth and bitwidth requirements. 2 A DSL may be an existing collection of language primitives ported to FP- GAs, e.g. the Vivado HLS support [10] for a subset of the OpenCV [2] library. OpenCV C++ library code is not synthesisable directly, instead OpenCV func- tion calls in existing software code must be replaced with corresponding function calls from the HLS library. In this restricted setting, it is not possible to use dy- namic memory allocation e.g. in the construction of image whose dimensions are decided at runtime. For good performance using this library, the programmer must use explicit pragmas to guide hardware generation. Alternatively, DSLs may be embedded within the programming model of an existing language, e.g. the Darkroom [5] FPGA image processing DSL is embedded within Terra [4]. Darkroom is compiled to line-buﬀered pipelines, with all intermediate values in local line-buﬀer storage. Images at each stage of computation are speciﬁed as pure functions from 2D coordinates to the values at those coordinates. Our RIPL DSL for FPGAs is implemented as a standalone language, i.e. it has its own syntax and its stream processing based programming model is not hindered by a programming model of any general purpose host language. The memory performance and expressivity of HLS OpenCV, Darkroom and RIPL is compared in Section 5. 1.3 FPGA Memory An important FPGA language implementation choice is whether on chip or oﬀ chip memory should be used to store data structures. Utilising oﬀchip memory is sometimes unavoidable depending on the data transforms an algorithm requires, e.g. transposing or rotating an image, both of which require an image frame buﬀer. However, frequently using oﬀchip memory from diﬀerent parts of FPGA circuits does not scale, because only one memory read from an on chip circuit can be performed in each clock cycle. This can sequentialise execution and hence hurt throughput performance. Moreover, oﬀchip memory access can take multiple clock cycles compared to latency-free LUT RAM or one cycle to access BRAM. On chip memory provides contention free local buﬀer access for diﬀerent parts of the application speciﬁc circuit, because it is distributed across the FPGA’s fabric. 3 Compilers of high level real-time languages should therefore prioritise wholly on chip memory implementations. However, the scarcity of BRAM introduces its own set of constraints for programming language designers to consider. Memory layout on FPGA chips is fundamentally diﬀerent to ﬁxed processor architectures. Instead of compiling a program to map eﬃciently to ﬁxed memory hierarchies, synthesis of high level languages builds a custom memory architec- ture on chip tailored for the needs of an algorithm. The constraint when compil- ing high level languages to FPGAs is the available resources, e.g. on chip memory ranges from 4Mb to 68Mb. The challenge for HLS compilers is therefore to min- imise memory use from algorithms expressed with high level software languages. Synthesis tools can choose to implement memory using registers, lookup tables (LUTs), or block RAM (BRAM). Unlike cache contention issues on multicore CPUs, there is no contention to access BRAM memory because it is distributed across the fabric of an FPGA. 2.1 Image Buﬀer Capacity The main FPGA resource for implementing memory is BRAM blocks. For exam- ple, the Z-7020 chip on the Zedboard has 140 36Kb BRAM blocks amounting to 4.4Mb. The XC7K480T chip on the Kintex-7 board has 1,910 18Kb blocks and 995 36Kb blocks amounting to 34Mb. The XC7VX1140T chip on the Virtex-7 board has 3,760 18Kb blocks and 1,880 36Kb blocks amounting to 68Mb. A single channel image pixel is 8 bits, or 1 byte. A 320×240 image with a single colour channel is 76, 800 bytes, a 512×512 image is 262,144 bytes, a 1024×768 image is 786,432 bytes, and a 1920×1080 image is 2,073,600 bytes. Storing entire image frame buﬀers on FPGAs does not scale. The cost of buﬀering entire image frames on chip is shown in Fig. 1. The Zedboard can store up to seven 320×240 frame buﬀers and just two 512×512 frame buﬀers. The Kintex-7 can store up to ﬁve 1024×768 buﬀers and two 1920×1080 buﬀers, and the Virtex-7 is able to store four 1920×1080 buﬀers. Localised pixel, row and region buﬀers should instead be generated by high level language compilers. 2.2 Eliminating Intermediate Buﬀers With Compiler Optimisation When compiling high level programs to FPGAs, it is important to eliminate intermediate data structures because on chip BRAM is a scarce resource. A motivating example is shown in Fig. 2. This C++ code applies a Sobel edge detection ﬁlter with a nested for loop, and then brightens the result with another nested for loop. It uses the OpenCV Mat class for two intermediate images, image2 and image3. The xGradient() and yGradient() functions are omitted for brevity. Whilst these intermediate image structures could be oﬄoaded to oﬀchip DDR memory, this would result in a latency of multiple clock cycles for every memory access, compared to a single cycle for on chip access. There is a need for Fig. 1: Storing Multiple Frame buﬀers with on chip FPGA Memory Fig. 1: Storing Multiple Frame buﬀers with on chip FPGA Memory FPGA language compilers to avoid wasteful memory resources on intermediate images image2 and image3. One data locality approach in data parallel language compilers is to start from an imperative language with loops, and fuse the successive loops over the input image1 into an expression tree in a single loop, to improve cache locality and on chip register locality e.g. [6,12]. For CPU or GPU scheduling, this expression tree can be duplicated to apply the same fused computation on image chunks in a data parallel fashion. However for pipelined FPGA scheduling, where diﬀerent computations are applied to separate regions of an image stream, a compiler would apply loop fusion optimisations, and then expression pipelining in the body of those loops to create hardware pipelines of ﬁne grained operator dataﬂow graphs. Fig. 3: RIPL Equivalent of the OpenCV C++ in Fig. 2 Fig. 3: RIPL Equivalent of the OpenCV C++ in Fig. 2 RIPL has a declarative non-terminating programming model that is con- strained for processing inﬁnite image stream, a programming model from which minimal memory costs can more easily be extracted. It represents a high pro- gramming abstraction when compared to direct hardware design with HDLs. We term RIPLs stream combinator primitives as algorithmic skeletons [3]. They capture the common pattern of many low and medium level image signal pro- cessing operations such as 1 dimensional (1D) and 2D ﬁlters, combining images, and global operations such as ﬁnding the maximum pixel value. Intermediate images in RIPL programs are transformed to streams that are shared through parallel hardware pipelines, rather than copying whole images for each pipeline phase to process. The RIPL implementation is available online4. 4 https://github.com/robstewart57/ripl 3 RIPL: An FPGA DSL for Maximising Data Locality We take a diﬀerent approach with RIPL. The language design is inspired by streaming libraries e.g. [7], which provides stream combinators like map, fold and sum. Composition of these RIPL primitives is a natural way of expressing pipelines of low and medium level image processing kernels. These pipelines are preserved during compilation and mapped into hardware as concurrent circuits. 5 Mat image2; /* Sobel filter / for(int y = 1; y < image1.rows - 1; y++) { for(int x = 1; x < image1.cols - 1; x++) { gx = xGradient(image1 , x, y); gy = yGradient(image1 , x, y); sum = abs(gx) + abs(gy); image2.at <uchar >(y,x) = sum; } } Mat image3; / brighten image2 / for(int y = 1; y < image2.rows - 1; y++) { for(int x = 1; x < image2.cols - 1; x++) { newPixel = image2.at <uchar >(y, x) + 50; image3.at <uchar >(y,x) = newPixel > 255 ? 255 : newPixel; } } gy = yGradient(image1 , x, y); sum = abs(gx) + abs(gy); Fig. 2: Intermediate Images using OpenCV’s Mat class Fig. 2: Intermediate Images using OpenCV’s Mat class image1 = imread 512 512; / Sobel filter / image2 = filter2D image1 (3 ,3) (\p1 p2 p3 p4 p5 p6 p7 p8 p9 -> abs ((p1 + (2 p2) + p3) - (p7 + (2* p8) + p9)) + abs ((p3 + (2p6) + p9) - (p1 + (2 p4) + p7))); image1 = imread 512 512; image1 = imread 512 512; g /* Sobel filter / / Sobel filter / f image2 = filter2D image1 (3 ,3) g g (\p1 p2 p3 p4 p5 p6 p7 p8 p9 -> (\p1 p2 p3 p4 p5 p6 p7 p8 p9 -> abs (( 1 + (2 2) + 3) ( 7 + (2* 8) + 9)) (\p1 p2 p3 p4 p5 p6 p7 p8 p9 > abs ((p1 + (2* p2) + p3) - (p7 + (2* p8) + p9)) p p p p p p p p p abs ((p1 + (2* p2) + p3) - (p7 + (2* p8) + p9 + abs ((p3 + (2p6) + p9) - (p1 + (2 p4) + p7))); /* brighten image2 / image3 = map image2 (\[ pixel] -> [min 255 (pixel + 50) ]); / brighten image2 / image3 = map image2 (\[ pixel] -> [min 255 (pixel + 50) ]); Fig. 3: RIPL Equivalent of the OpenCV C++ in Fig. 2 3.1 RIPL Skeletons RIPL skeletons abstract common data access patterns, to which the user sup- plies functions and values. They have been designed such that dataﬂow analysis can be performed on their composition, and to extract the minimal memory 6 imreadM,N : (M : Int) →(N : Int) →IR (M,N) mapM,N,A,B : IR (M,N) →([P]A →[P]B) →IR (M∗(B/A),N) mapM,N,A,B : IC (M,N) →([P]A →[P]B) →IC (M,N∗(B/A)) imapM,N,A : I(M,N) →([P]A →P) →I(M,N) convolveM,N,A,B : I(M,N) →(A, B) : (Int, Int) →[K](A∗B) →I(M,N) filter2DM,N,A,B : I(M,N) →(A, B) : (Int, Int) →([P](A∗B) →P) →I(M,N) zipWithM,N,A : I(M,N) →I(M,N) →([P]A →[P]A →[P]A) →I(M,N) zipWithScalarM,N,A : I(M,N) →P →(P →P →P) →I(M,N) zipWithV ectorM,N,A,B : I(M,N) →[P]A →([P]A →P →P) →I(M,N) unzipM,N,A : IR (M,N) →([P]A →P) →([P]A →P) →(IR (M/2,N), IR (M/2,N)) unzipM,N,A : IC (M,N) →([P]A →P) →([P]A →P) →(IC (M,N/2), IC (M,N/2)) scanM,N : I(M,N) →Int →(P →Int →Int) →I(M∗N) foldScalarM,N : I(M,N) →Int →(P →Int →Int) →Int foldV ectorM,N,A : I(M,N) →Int →(A : Int) →(P →[Int]A →[Int]A) →[Int]A transposeM,N : IR (M,N) →IC (M,N) transposeM,N : IC (M,N) →IR (M,N) convolveM,N,A,B : I(M,N) →(A, B) : (Int, Int) →[K](A∗B) →I(M,N) filter2DM,N,A,B : I(M,N) →(A, B) : (Int, Int) →([P](A∗B) →P) →I(M,N) zipWithM,N,A : I(M,N) →I(M,N) →([P]A →[P]A →[P]A) →I(M,N) zipWithScalarM,N,A : I(M,N) →P →(P →P →P) →I(M,N) zipWithV ectorM,N,A,B : I(M,N) →[P]A →([P]A →P →P) →I(M,N) unzipM,N,A : IR (M,N) →([P]A →P) →([P]A →P) →(IR (M/2,N), IR (M/2,N)) unzipM,N,A : IC (M,N) →([P]A →P) →([P]A →P) →(IC (M,N/2), IC (M,N/2)) scanM,N : I(M,N) →Int →(P →Int →Int) →I(M∗N) foldScalarM,N : I(M,N) →Int →(P →Int →Int) →Int foldV ectorM,N,A : I(M,N) →Int →(A : Int) →(P →[Int]A →[Int]A) →[Int]A transposeM,N : IR (M,N) →IC (M,N) transposeM,N : IC (M,N) →IR (M,N) Fig. 4: RIPL skeletons requirements of their use. The RIPL program in Fig. 3 broadly corresponds to the OpenCV C++ in Fig. 2, though RIPLs convolve and ﬁlter2D skeletons also mirrors edge pixels over image boundaries to apply the kernel function to edge pixels. When compiled to hardware, the image stream image1 is incrementally processed, ﬁrst by hardware logic that computes Sobel edge detection and then by logic that brightens each pixel in the stream. Skeleton API The RIPL skeletons are shown in Fig. 4, using a standard nota- tion for function type signatures, e.g. map is a skeleton that takes two arguments: an M×N image, and function from a vector of A pixels to a vector of B pixels. It returns an M×N image. Each skeleton is repeatedly applied over an image stream. Types in Fig. 4 are annotated with pixel major order, vector lengths and image dimensions. For example, [P]A is a vector of pixels of length A, so an argument in a function of the form λ[a, b] has an implicit type [P]2. An image IR (M,N) is M pixels wide and N pixels high, and whose pixels are in row (R) major order. RIPLs map and unzip skeletons are implicitly directional, sliding linearly either row wise or column wise over a one dimensional vector of 7 1 2 3 (a) traversing images with map 1 2 3 4 5 6 7 (b) traversing images with imap Fig. 5: Comparison of map with indexed map 7 1 2 3 (a) traversing images with map (a) traversing images with map (a) traversing images with map Fig. 5: Comparison of map with indexed map pixels. This meta information about stream order, image dimensions and vector lengths is not speciﬁed by the programmer; it is inferred by the RIPL compiler. Skeletons with Non-overlapping Sliding Windows The map skeleton slides over an image and applies the user deﬁned function with a non-overlapping 1D vector on each execution. The zipWith skeleton is similar, though it slides a vector window of the same length over two images in lock step. The map and zipWith skeletons are stateless and do not carry state between executions. Their memory costs are therefore solely determined by the length of the sliding vec- tor that they consume. Fig. 4: RIPL skeletons The zipWithScalar skeletons combines every pixel and a scalar value with a user deﬁned function, and similarly zipWithVector allows the programmer to use a random access vector to modify an image. As an example of non-overlapping sliding windows, the following RIPL as- signment combines two images using zipWith with a mean average combinator. The memory cost is 2 8 bit integers, one each for pixels p1 and p2 from images image1 and image2 respectively. image3 = zipWith image1 image2 (\[p1] [p2] -> [(p1+p2)/2]); Skeletons with Overlapping Sliding Windows The imap skeleton is use- ful for applying 1D ﬁlters to an image. It is an indexed map that slides over contiguously positioned pixels in a non-discrete fashion. The imap syntax diﬀers from map, because imap applies a function from a pixel position [.] to a new value for that position, using the current pixel value and its neighbouring pixels using +/-, e.g. [.-1] points to the pixel to the left of [.] in an IR image. The diﬀerence in how map and imap traverses an image is depicted in Fig. 5, which is labelled with repeated execution counts show the diﬀerence in their data pro- cessing rates of an image row. Fig. 6 shows the expression of a 1D blur ﬁlter in RIPL, along with its memory cost. The hardware implementation of this imap consumes pixels into a 3 element circular buﬀer, updating the mid point index for [.], before executing the user deﬁned blur function. RIPLs unzip skeleton is for splitting apart an image into two images, and shares the pixel position syntax with imap. The hardware memory generated from unzip is similar to imap, the diﬀerence being its scheduling – the hardware for unzip creates two image streams, which are produced by alternating the execution of the two user deﬁned functions. 8 index 0 1 2 [.] [.+1] [.-1] midpoint new pixel output pixel (\[.] -> ([.-1] + [.] + [.+1]) / 3) 1D blur ﬁlter in RIPL: new pixel (\[.] -> ([.-1] + [.] + [.+1]) / 3) 1D blur ﬁlter in RIPL: midpoint output pixel Fig. 6: Memory requirements of 1D blur with imap satisﬁed with a circular buﬀ Fig. 6: Memory requirements of 1D blur with imap satisﬁed with a circular buﬀer Fig. Fig. 4: RIPL skeletons 6: Memory requirements of 1D blur with imap satisﬁed with a circular buﬀer output processed stream pixel value being computed stream to be consumed buﬀered pixels Fig. 7: Memory Cost for convolve and ﬁlter2D output processed stream pixel value being computed stream to be consumed buﬀered pixels Fig. 7: Memory Cost for convolve and ﬁlter2D Fig. 7: Memory Cost for convolve and ﬁlter2D Skeletons for 2D Filters Many 2D ﬁlters can be implemented by combining the results of two 1D ﬁlters, one in the horizontal direction and one in the vertical direction. This implementation approach is possible in RIPL by applying a 1D horizontal ﬁlter with imap, transposing the result with transpose, then applying a vertical 1D ﬁlter with imap. However, this is a very memory costly composition, because transpose generates a frame buﬀer. For better stream data locality, RIPL has two 2D ﬁlters convolve and ﬁlter2D. The convolve skeletons modiﬁes each pixel by applying a convolution of its neighbours using a small user deﬁned M×N kernel. The following example applies 3×3 kernel to sharpen an image. image2 = convolve image1 (3,3) {0,-1,0,-1,5,-1,0,-1,0}; image2 = convolve image1 (3,3) {0,-1,0,-1,5,-1,0,-1,0}; The ﬁlter2D skeleton provides more expressivity than convolve. When using ﬁlter2D with a 3×3 window, the programmer is provided 9 pixel values that can be used in their own function body, as shown earlier in Fig. 3 which computes the approximate magnitude \|G\| = \|Gx\| + \|Gy\| for Sobel edge detection. The memory requirements for convolve and ﬁlter2D is shown in Fig. 7. This has the capacity to store two rows and a further three pixels. Stream based pro- cessing begins once one row and two pixels are streamed into the corresponding buﬀer, which is when the top left pixel can be processed. Stateful Skeletons Stateful programming is achieved with RIPL using two skeletons, foldScalar and foldVector. They apply user deﬁned reduction opera- tions on images or image regions. Reducing an image to a scalar value is done using foldScalar, e.g. ﬁnding the maximum pixel value. The scan skeleton is 9 Example Skeleton buﬀer size RIPL code M × N buﬀer size mapM,N,A,B A map image1 (λ[a, b, c] →...) n/a 3 imapM,N,A A + 1 imap image1 (λ[.] →([. −1] + [.] + [. Fig. 4: RIPL skeletons + 1])/3) n/a 4 zipW ithM,N,A A ∗2 zipWith image1 (λ[a, b] [c, d] →...) n/a 4 zipW ithScalarM,N,A A + 1 zipWithScalar image1 (λ[a, b] x →...) n/a 3 zipW ithV ectorM,N,A,B A + B + 1 zipWithVector image1 (λ[a, b] vect →...) n/a 3 + B unzipM,N,A A + 1 unzip image1 (λ[a, b] →...) (λ[c, d] →...) n/a 3 convolveM,N,A,B M ∗2 + 3 convolve image1 (3,3) kernel 512×512 1027 filter2DM,N,A,B M ∗2 + 3 ﬁlter2D image1 (3,3) (λ... →...) 512×512 1027 scanM,N 2 scan image1 0 (λ.. →..) n/a 2 foldScalarM,N 2 foldScalar image1 0 (λ.. →..) n/a 2 foldV ectorM,N,A A + 1 foldVector image1 255 0 (λ.. →..) n/a 256 transposeM,N M ∗N transpose image1 512 × 512 262144 Table 1: Memory Costs for RIPL Skeletons similar to foldScalar, but returns a stream of intermediate successive reduced values. Reducing an image to a vector is done using foldVector, e.g. computing a colour histogram with each bin initialised to 0. Maximum pixel and histogram calculations are expressed as: similar to foldScalar, but returns a stream of intermediate successive reduced values. Reducing an image to a vector is done using foldVector, e.g. computing a colour histogram with each bin initialised to 0. Maximum pixel and histogram calculations are expressed as: maxValue = foldScalar image1 0 (\p currMax -> max p currMax); histogram = foldVector image1 0 255 (\p hist -> hist[p]++); maxValue = foldScalar image1 0 (\p currMax -> max p currMax); histogram = foldVector image1 0 255 (\p hist -> hist[p]++); 4.1 Memory Costs for Computation RIPL programs are compiled to a dataﬂow intermediary of small computational actors and FIFOs. The memory costs for each RIPL skeleton in bytes is shown in Table 1. The map, imap, zipWith and unzip skeletons are implemented with either overlapping or non-overlapping sliding vectors, and hence their memory requirements are not determined by an image’s dimensions. These costs are calculated from their oﬀsets in stream access, analogous to array access oﬀset analysis in for loops in imperative languages. The map and zipWith memory costs are solely determined by the vector length of the λ argument in the user deﬁned function. The memory cost of zipWithScalar and zipWithVector is the stored scalar or vector, and the next incoming pixel value. The memory costs for imap are determined by the biggest X in [.+X] occurrences in the output expression, minus the biggest Y in [.-Y] occurrences. The memory cost for the foldScalar and scan skeletons is the folded scalar and the next pixel from the image stream. The foldVector skeleton’s memory re- quirements are determined by the programmer’s choice of output vector length which is folded through through each execution, and the next pixel from the image stream. The convolve and ﬁlter2D skeleton’s memory requirements are 10 image1 = imread 512 512; image2 = filter2D image1 (3,3) (\p1 p2 p3 p4 p5 p6 p7 p8 p9 -> abs ((p1 + (2p2) + p3) - (p7 + (2p8) + p9)) + abs ((p3 + (2p6) + p9) - (p1 + (2*p4) + p7))); image3 = imap image2 (\[.] -> ([.-2] + [.-1] + [.] + [.+1] + [.+2])/3); out image3; image1 image2 image3 ﬁlter2d imap 1 1 (a) Edge ﬁlter then 1D blur image1 = imread 512 512; maxPixel = foldScalar image1 0 (\p i -> max p i); normalisedImage = zipWithScalar image1 maxPixel (\p maxP -> if p > (maxP-100) then 255 else 0); out normalisedImage; image1 image2 zipWithScalar 512x512 1 233 foldScalar 1 (b) Image threshold Fig. 8: Memory costs for dataﬂow FIFOs image1 = imread 512 512; maxPixel = foldScalar image1 0 (\p i -> max p i); normalisedImage = zipWithScalar image1 maxPixel (\p maxP -> if p > (maxP-100) then 255 else 0); out normalisedImage; image1 image2 zipWithScalar 512x512 1 233 foldScalar 1 (b) Image threshold Fig. 8: Memory costs for dataﬂow FIFOs determined by the processed image’s width. 4.1 Memory Costs for Computation The most costly skeleton is trans- pose, because it requires an entire image to be stored in a buﬀer before being outputted with a transpose index. 4.2 Memory Costs for Communication An addition memory cost is the depth of dataﬂow wires to ensure deadlock free RIPL execution. Dataﬂow wires are derived by data dependencies in RIPL pro- grams, i.e. if the output of one skeleton is used as an input to another, then a FIFO point-to-point connection is created in hardware to support that data sharing. When the output of one RIPL skeleton is used in just one place in a program, only one output FIFO will be connected from the hardware implement- ing that skeleton, shown in Fig. 8a. In these cases, the required FIFO depth is determined by the vector length of the λ argument in the receiving skeleton. For example, if a map takes λ[a, b, c] then the required depth is 3. The overall memory cost for FIFOs in Fig. 8a is 2 8 bit integers. If the output image of one skeleton is used in multiple places, then depth re- quirements can increase, shown in Fig. 8b. This RIPL program ﬁnds the biggest pixel value of 233 with foldScalar, which is used to threshold the original im- age using zipWithScalar with a threshold of 233 −100. The generated hardware duplicates image image1 over two FIFOs, one to the dataﬂow actor for comput- ing the maximum value, and the other to threshold the image. Pixel tokens are transmitted to both FIFOs in lock step. Therefore in order for maxP to be com- puted, the actor executing foldScalar needs to receive all tokens, so the FIFO to the threshold actor needs capacity to buﬀer the entire 512×512 image for the maxP value to be computed. The overall memory cost for FIFOs in Fig. 8b is (2 + 512 × 512) 8 bit integers. 4.3 FPGA Memory Implementation Once RIPL programs are compiled to dataﬂow graphs, actor computation code and dataﬂow wires are compiled to HDL using an open source dataﬂow com- 11 piler [1], which makes choices about how to implement memory. For scalar in- teger values it uses FPGA slice registers used as memory. For small arrays that the RIPL compiler generates to support convolve, ﬁlter2D and foldVector, the dataﬂow compiler may also use slice registers depending on the overall mem- ory requirements of the complete hardware design. The beneﬁt of implementing these memories with slice registers is that the larger BRAM blocks are avail- able for other parts of an algorithm, and because BRAM access is one clock cycle whilst LUTs RAM can be accessed without any latency. BRAM is used to support larger arrays generated by the RIPL compiler to support convolve and ﬁlter2D on big images, and for transpose which needs an entire image buﬀer. FIFOs are compiled to HDL as generic memories, leaving the FPGA synthesis tools to choose how to implement them. Rendevous single token FIFOs will be implemented using registers or LUTs. Small FIFOs, e.g. to buﬀer a single row, are likely to be implemented with LUTs, whilst large FIFO depths, e.g. to support duplicating image streams in Fig. 8b, will likely be implemented using BRAM. 5.1 Expressivitiy However, when an image is used in multiple 12 hls::Mat<512,512, HLS_8UC1> img_0(rows,cols); hls::Mat<512,512, HLS_8UC1> img_1(rows,cols); hls::Mat<512,512, HLS_8UC1> img_2(rows,cols); hls::Mat<512,512, HLS_8UC1> img_3(rows,cols); // explicit depth for img_2 to prevent deadlock #pragma HLS stream depth=262144 variable=img 2 da // explicit depth for img_2 to prevent deadlock // convert AXI4 stream data to hls::mat format hls::AXIvideo2Mat(INPUT_STREAM1, img_0); // duplicate the img_0 stream hl D li t (i 0 i 1 i // duplicate the img_0 stream hls::Duplicate(img_0,img_1,img_2); hls::Duplicate(img_0,img_1,img_2); // find the maximum pixel of img_1 duplicate int maxP, minP; hls::Point p1,p2; hls::MinMaxLoc(img_1,&minP,&maxP,p1,p2); // threshold the img_2 duplicate using the max pixel - 50 // find the maximum pixel of img_1 duplicate int maxP, minP; 5.1 Expressivitiy We next compare RIPL with the Vivado HLS OpenCV library. A key diﬀerence is that RIPL supports used deﬁned functions to be expressed, whilst HLS OpenCV is a collection of predeﬁned functions. For example, the HLS OpenCV hls::Max function combines two images by retaining the brighter of the pixels at each point, which can be expressed using RIPL’s zipWith and max in the function body. Another example is RIPLs ﬁlter2D, which enables the programmer to deﬁne the mid pixel point with any function, whereas hls::Filter2D only supports convolution of a user deﬁned kernel, equivalent to RIPL’s convolve skeleton. Another diﬀerence between RIPL and HLS OpenCV is image sharing. When an image is used in two places in a RIPL program, the image stream is automati- cally duplicated and shared to both consuming skeletons. With the HLS OpenCV model, an equivalent program would deadlock because the ﬁrst function that uses the image will consume its pixels, emptying the FIFO. The hls::Duplicate function must be used explicitly to avoid this. OpenCV programming requires explicit dimension and bitwidth information for each image declaration. The hls :: Mat <> template class is used to ini- tialise an image, e.g. hls :: Mat < 512, 512, HLS 8UC1 > deﬁnes a 512×512 single channel image, using 8 unsigned bits per pixel. In contrast, the RIPL compiler infers the dimension of every image, by following dimension transfor- mations performed by skeletons through the implicit dataﬂow paths starting from imread, the only place where dimensions are explicit. The compiler also infers pixel bitwidths automatically, by calculating maximum upper bounds on bitwidth requirements as image data ﬂows through arithmetic operators. An- other diﬀerence is the inference of FIFO depths. The default FIFO depth in both RIPL and HLS OpenCV is 1. Fig. 9: Thresholding with HLS OpenCV using a maximum pixel value Fig. 9: Thresholding with HLS OpenCV using a maximum pixel value places the RIPL compiler increases the FIFOs automatically to frame buﬀers (Section 4.2). The HLS compiler does not make this inference, leaving the pro- grammer to use FPGA co-simulation to identify deadlocks. The user then pro- grammatically uses #pragma HLS stream depth =< N > to specify the FIFO depth to avoid deadlock. Fig. 9 shows a HLS OpenCV example that demon- strates the need for explicit image duplication and explicit FIFO depths. The RIPL compiler infers both of these properties automatically. places the RIPL compiler increases the FIFOs automatically to frame buﬀers (Section 4.2). The HLS compiler does not make this inference, leaving the pro- grammer to use FPGA co-simulation to identify deadlocks. The user then pro- grammatically uses #pragma HLS stream depth =< N > to specify the FIFO depth to avoid deadlock. Fig. 9 shows a HLS OpenCV example that demon- strates the need for explicit image duplication and explicit FIFO depths. The RIPL compiler infers both of these properties automatically. The ﬁnal diﬀerence is how image processing pipelines are constructed. Pipelined parallelism in RIPL is automatic. When two skeletons are composed in sequence over an input image, they will execute in parallel over diﬀerent regions of the image stream. In HLS OpenCV, the programmer must specify #pragma HLS dataﬂow above the function calls intended to be pipelined over the image stream. One similarity between HLS OpenCV and RIPL is the implementation of image data structures. An OpenCV image is a hls::Mat. The Vivado HLS FPGA implementation of hls::Mat images uses hls::stream internally, so OpenCV images on FPGAs are FIFOs, which is also true for RIPL. Hence random image access is not possible in either case. Darkroom can be used to express benchmarks 1 and 2 (), but not 3, 4 or 5 (). Global reductions are not supported, because Darkroom’s line buﬀers cannot be used to store values beyond a traversing a single line. Such a buﬀer is required to compute the maximum pixel value (3) and the histogram (4). RIPL is the only language of the three compared that supports image transposition, which again requires a frame buﬀer that uses 64 BRAMs. Fig. 9: Thresholding with HLS OpenCV using a maximum pixel value 13 Benchmark RIPL HLS OpenCV Darkroom BRAM LUTs BRAM LUTs 1 Image brighten 0 (0%) 118 (0%) 0 450 (0%) 2 Sobel 2D edge detection 1 (0%) 12273 (23%) 3 (1%) 713 (1%) 3 Threshold with max pixel 64 (45%) 280 (0%) 64 (45%) 9172 (1%) 4 Histogram normalisation 64 (45%) 799 (1%) 3 (1%) 2918 (5%) 5 Image transposition 64 (45%) 321 (0%) Table 2: Memory Implementation and Expressivity Results Table 2: Memory Implementation and Expressivity Results 5.2 Space Performance We use ﬁve benchmarks to compare the space performance of RIPL and OpenCV compiled to FPGAs using Vivado HLS. The benchmarks are 1) brighten each pixel in an image by 50, 2) 2D Sobel edge detection, 3) ﬁnd the maximum pixel maxPixel value then threshold the image with (maxPixel −50), 4) compute a sum histogram for an image then normalise the image using the histogram, and 5) transpose an image. All programs are compiled for the Xilinx Zedboard XC7Z020 for 512×512 single channel images. The memory use performance of Darkroom cannot be compared because the line buﬀer to Verilog compiler backend is not publicly available. The synthesis results in Table 2 are for RIPL and HLS OpenCV. RIPL and OpenCV occupy very similar memory resources for image brightening (1) and image thresholding (3). For Sobel edge detection (2), RIPL uses 2 BRAMs less than OpenCV by instead using more LUTs. Thresholding and histogram nor- malisation (4) in RIPL require a FIFO depth equal to the number of pixels in the image in RIPL’s hardware backend. To support 8 bit pixels, the synthesis tools use BRAMs in 32Kb mode, so storing a 512×512 image requires 64 BRAM blocks for these two benchmarks. The same is true for HLS OpenCV for thresholding, but not histogram normalisation. This is because of an optimisation built into hls::EqualizeHist(), which normalises frame N +1 using the histogram computed for the previous frame N. This results in more eﬃcient BRAM use compared to RIPL. We plan this optimisation for RIPL. 6 Conclusion Memory resources on FPGAs can be tailored to the needs of an algorithm, so FPGA compilers are not hindered by ﬁxed memory hierarchies such as those on CPUs and GPUs. They are however constrained by the limited amount of on chip BRAM and LUT memory resources. This paper describes the memory eﬃciency aspects of RIPL, our image processing DSL for FPGAs. RIPL is more concise than Vivado HLS OpenCV, because it automatically infers upper bounds on bitwidths and the required FIFO depths for image streams, and image streams are automatically duplicated when necessary. Despite these abstractions, RIPL 14 memory use is competitive on three of the four benchmarks expressible with the Vivado HLS OpenCV. RIPL is more expressive than the Darkroom image processing DSL, because Darkroom compiles to line buﬀers so does not support global image reductions. Future work will explore temporal video processing capabilities in RIPL, where new opportunities for dataﬂow analysis for data locality may arise. We also wish to explore the applicability of RIPL for FPGA acceleration of other stream based domains beyond image processing. References 1. Bezati, E.: High-level synthesis of dataﬂow programs for heterogeneous platforms. Ph.D. thesis, STI, EPFL, Switzerland (2015) , , , ( ) 2. Bradski, G.R., Kaehler, A.: Learning OpenCV - computer vision with the OpenCV library: software that sees. O’Reilly (2008) 3. Cole, M.: Algorithmic Skeletons: Structured Management of Parallel Computation. MIT Press, Cambridge, MA, USA (1991) 4. DeVito, Z., Hegarty, J., Aiken, A., Hanrahan, P., Vitek, J.: Terra: a multi-stage language for high-performance computing. In: ACM SIGPLAN Conference on Pro- gramming Language Design and Implementation, Seattle, WA, USA, June 16-19, 2013. pp. 105–116. ACM (2013) 5. Hegarty, J., Brunhaver, J., DeVito, Z., Ragan-Kelley, J., Cohen, N., Bell, S., Vasi- lyev, A., Horowitz, M., Hanrahan, P.: Darkroom: compiling high-level image pro- cessing code into hardware pipelines. ACM Trans. Graph. 33(4), 1–11 (2014) 6. Kennedy, K., McKinley, K.S.: Maximizing loop parallelism and improving data locality via loop fusion and distribution. In: Languages and Compilers for Parallel Computing, 6th International Workshop, Portland, Oregon, USA, August 12-14, 1993. Lecture Notes in Computer Science, vol. 768, pp. 301–320. Springer (1993) 7. Kiselyov, O.: Iteratee IO: Safe, Practical, Declarative Input Processing. In: 11th International Symposium on Functional and Logic Programming. Lecture Notes in Computer Science, vol. 7294, pp. 166–181 (2012) ( ) 8. Lee, H., Brown, K.J., Sujeeth, A.K., Rompf, T., Olukotun, K.: Locality-Aware Mapping of Nested Parallel Patterns on GPUs. In: 47th Annual IEEE/ACM In- ternational Symposium on Microarchitecture, MICRO 2014, Cambridge, United Kingdom, December 13-17, 2014. pp. 63–74. IEEE (2014) ( ) 9. Muddukrishna, A., Jonsson, P.A., Brorsson, M.: Locality-Aware Task Scheduling and Data Distribution for OpenMP Programs on NUMA Systems and Manycore Processors. Scientiﬁc Programming 2015, 981759:1–981759:16 (2015) 10. Stephen Neuendorﬀer, T.L., Wang, D.: Accelerating OpenCV Applications with Zynq-7000 All Programmable SoC using Vivado HLS Video Libraries. Tech. rep., Xilinx (June 2015) 11. Tate, A., et al.: Programming abstractions for data locality. In: Workshop on Pro- gramming Abstractions for Data Locality, Swiss National Supercomputing Center, Lugano, Switzerland (April 2014) 12. Wieser, V., Grelck, C., Haslinger, P., Guo, J., Korzeniowski, F., Bernecky, R., Moser, B., Scholz, S.: Combining high productivity and high performance in image processing using Single Assignment C on multi-core CPUs and many-core GPUs. J. Electronic Imaging 21(2) (2012) J. Electronic Imaging 21(2) (2012) ( ) ( ) 13. Xilinx: Implementing Memory Structures for Video Processing in the Vivado HLS Tool. Tech. rep., Xilinx (September 2012)
https://openalex.org/W2613802396	https://journals.ssau.ru/index.php/vestnik/article/download/3301/3203	Russian	null	Using the «virtual engine» software in automatic control system of gas turbine engine	Vestnik Samarskogo universiteta. Aèrokosmičeskaâ tehnika, tehnologii i mašinostroenie	2,016	cc-by	4,073	DOI: 10.18287/2541-7533-2016-15-4-47-56 УДК 621.452.3+004.9 УДК 621.452.3+004.9 УДК 621.452.3+004.9 DOI: 10.18287/2541-7533-2016-15-4-47-56 ПРИМЕНЕНИЕ ПРОГРАММНОГО ОБЕСПЕЧЕНИЯ «ВИРТУАЛЬНЫЙ ДВИГАТЕЛЬ» В СИСТЕМЕ АВТОМАТИЧЕСКОГО УПРАВЛЕНИЯ ГАЗОТУРБИННОГО ДВИГАТЕЛЯ Ф. Д. Гольберг О. С. Гуревич А. А. Петухов Ф. Д. Гольберг доктор технических наук, профессор, начальник сектора, Центральный институт авиационного моторостроения имени П.И. Баранова, Москва, fegolb@ciam.ru О. С. Гуревич доктор технических наук, профессор, заместитель генерального директора, начальник отделения, Центральный институт авиационного моторостроения имени П.И. Баранова, Москва, gurevich_os@ciam.ru А. А. Петухов младший научный сотрудник, Центральный институт авиационного моторостроения имени П.И. Баранова, Москва, petuhov-ctrl@ciam.ru Ф. Д. Гольберг доктор технических наук, профессор, начальник сектора, Центральный институт авиационного моторостроения имени П.И. Баранова, Москва, fegolb@ciam.ru О. С. Гуревич доктор технических наук, профессор, заместитель генерального директора, начальник отделения, Центральный институт авиационного моторостроения имени П.И. Баранова, Москва, gurevich_os@ciam.ru А. А. Петухов младший научный сотрудник, Центральный институт авиационного моторостроения имени П.И. Баранова, Москва, petuhov-ctrl@ciam.ru доктор технических наук, профессор, начальник сектора, Центральный институт авиационного моторостроения имени П.И. Баранова, Москва, fegolb@ciam.ru доктор технических наук, профессор, заместитель генерального директора, начальник отделения, Центральный институт авиационного моторостроения имени П.И. Баранова, Москва, gurevich_os@ciam.ru А. А. Петухов младший научный сотрудник, Центральный институт авиационного моторостроения имени П.И. Баранова, Москва, petuhov-ctrl@ciam.ru Разработан математический аппарат и программное обеспечение «виртуальный двигатель» для применения в цифровых системах автоматического управления (САУ) современных газотурбинных двигателей (ГТД). Программное обеспечение базируется на всережимной термогазодинамической математической модели двигателя, функционирующей в бортовых вычислителях в реальном масштабе времени. С помощью программного обеспечения «виртуальный двигатель» в САУ ГТД реализуются новые принципы управления двигателем по параметрам, непосредственно определяющим его основные характеристики, но недоступным для измерения. Реализуются также новые принципы повышения отказоустойчивости двигателя. Проведён синтез контуров управления двухконтурного турбореактивного двигателя (ТРДД) большой степени двухконтурности по определяемым расчётом критическим параметрам – тяге двигателя, температуре газа в камере сгорания, запасам газодинамической устойчивости. Разработана методика восстановления информации о параметрах потока воздуха на входе в двигатель и значений его регулирующих факторов двигателя при отказе информационных каналов. В результате проведённых расчётных исследований показано существенное повышение качества регулирования двигателя и повышение его ресурса при изменении характеристик ТРДД в процессе эксплуатации. Разработана методика идентификации в реальном масштабе времени бортовой термогазодинамической математической модели двигателя, основанная на принципе отрицательной обратной связи. Системы автоматического управления; газотурбинный двигатель; бортовая математическая модель. Цитирование: Гольберг Ф.Д., Гуревич О.С., Петухов А.А. Применение программного обеспечения «виртуальный двигатель» в системе автоматического управления газотурбинного двигателя // Вестник Самарского университета. Аэрокосмическая техника, технологии и машиностроение. 2016. Т. 15, № 4. С. 47-56. DOI: 10.18287/2541-7533- 2016-15-4-47-56 Авиационная и ракетно-космическая техника Авиационная и ракетно-космическая техника Введение В качестве одного из важнейших элементов перспективных газотурбинных двига- телей в России и за рубежом [1;2] рассматривается программное обеспечение его циф- ровой САУ «виртуальный двигатель», базирующееся на встроенной термогазодинами- ческой математической модели двигателя (БММД). Возможность использования в САУ ГТД бортовых моделей достаточно высокого уровня с сохранением возможности расчёта в реальном времени связана с ростом быстродействия электронных цифровых систем управления. Такие модели основаны на описании физических процессов в двигателе с помощью уравнений газовой динамики, термодинамики и механики в нестационарной форме. Разработанные в ЦИАМ термога- зодинамические математические модели этого типа позволяют рассчитывать характе- 47 Вестник Самарского университета. Аэрокосмическая техника, технологии и машиностроение Т. 15, № 4, 2016 г. ристики двигателя на установившихся и переходных режимах работы во всём диапа- зоне (от запуска до режима максимальной тяги) во всей области полётов. Проведён комплекс теоретических и экспериментальных исследований, связан- ных с построением САУ, в программном обеспечении которых содержатся термогазо- динамические математические модели ГТД, включивший в себя разработку: Д, р р у - алгоритмического обеспечения САУ, обеспечивающего с помощью БММД управление двигателем по параметрам, непосредственно определяющим его основные характеристики, но недоступным для измерения; - алгоритмического обеспечения САУ, обеспечивающего с помощью БММД управление двигателем по параметрам, непосредственно определяющим его основные характеристики, но недоступным для измерения; р р у р - методов компенсации отказов датчиков регулируемых параметров двигателя; - методов компенсации отказов датчиков параметров потока на входе в двигатель (температуры * ВХ T и давления * ВХ P воздуха); р у р - методов компенсации отказов датчиков регулируемых параметров двигателя; - методов компенсации отказов датчиков регулируемых параметров двигателя; - методов компенсации отказов датчиков параметров потока на входе в двигатель (температуры * ВХ T и давления * ВХ P воздуха); - методов идентификации БММД в процессе её функционирования на работаю- щем двигателе. - методов идентификации БММД в процессе её функционирования на работаю- щем двигателе. р математической модели ТРДД, предназначенной для функционирования в составе САУ На рис. 1 приведена расчётная схема ТРДД, принятая при разработке его термогазодинамической модели. На схеме отмечены входные и выходные сечения основных узлов, в которых производится расчёт параметров рабочего процесса в двигателе (температуры Ti, давления Pi, расхода Gi воздуха и газа): - входное устройство (ВХ); - наружный контур (НК); - компре - камера б - компрессор высокого (КВД) давления; - входное устройство (ВХ); - компрессор высокого (КВД) давления; - наружный контур (НК); - камера сгорания (КС); - наружный контур (НК); - камера сгорания (КС); - сопло наружного контура с реверсивным устройством (РУ); - турбина высокого (ТВД) давления; турбина высокого (ТВД) давления; - турбина низкого давления (ТНД); - турбина низкого давления (ТНД); у р ( ) - компрессор низкого (КНД) давления; - затурбинный диффузор и сопло внутрен- него контура. - затурбинный диффузор и сопло внутрен- него контура. - компрессор низкого (КНД) давления; - подпорная ступень (ПС) компрессора; - подпорная ступень (ПС) компрессора; - подпорная ступень (ПС) компрессора; Рис. 1. Расчётная схема ТРДД Рис. 1. Расчётная схема ТРДД 48 Авиационная и ракетно-космическая техника Рассчитываются также частоты вращения ni роторов и эксплуатационные параметры двигателя (тяга R, удельный расход топлива Cr и др.). Входными параметрами модели являются регулирующие факторы двигателя: расход топлива Gf в камере сгорания (КС), углы φ2 установки НА КВД, положения VаL и ValРС клапанов (КПКВД, КППС) перепуска воздуха из КВД и ПС, а также органов (СОХЛт) управления расходом воздуха, отбираемого из КВД на охлаждение турбин. Модель позволяет рассчитывать параметры двигателя на установившихся и переходных режимах его работы в полном диапазоне их изменения (от режима запуска до максимального режима) во всех условиях полёта самолёта. Обеспечение возможности проведения расчётов в реальном масштабе времени 15, № 4, 2016 г. Обеспечение возможности проведения расчётов в реальном масштабе времени В программах расчёта параметров ГТД с помощью полной термогазодинамической модели двигателя применены специальные методы численного решения уравнений модели, обеспечивающие выполнение расчётов на современных персональных компьютерах за время, более чем в 10 раз меньшее реального. К таким особенностям, в частности, относятся: решение системы уравнений модели без применения итераций путём атного численного расчёта всей системы на каждом шаге по времени; - решение системы уравнений модели без применения итераций путём однократного численного расчёта всей системы на каждом шаге по времени; б - преобразование уравнений, описывающих процессы в воздушных и газовых ёмкостях в обыкновенные дифференциальные уравнения в сосредоточенных параметрах, что исключает необходимость решения дифференциальных уравнений в частных производных; - преобразование уравнений, описывающих процессы в воздушных и газовых ёмкостях в обыкновенные дифференциальные уравнения в сосредоточенных параметрах, что исключает необходимость решения дифференциальных уравнений в частных производных; - величина ∆t шага интегрирования дифференциальных уравнений модели меня- ется в зависимости от рассчитываемого режима работы двигателя. Проведена оценка времени расчёта процессов в двигателе с помощью модифици- рованной таким образом программы расчёта бортовой модели при реализации про- граммы как на персональных компьютерах, так и на базе микроконтроллера, аналогич- ного применяемым в современном электронном регуляторе. Проведена оценка времени расчёта процессов в двигателе с помощью модифици- рованной таким образом программы расчёта бортовой модели при реализации про- граммы как на персональных компьютерах, так и на базе микроконтроллера, аналогич- ного применяемым в современном электронном регуляторе. При тестировании программы на двух персональных компьютерах с процессора- ми AMD Athlon(tm) 64 X2 Dual Core Processor 5600+; 2,91 ГГц (система 1) и Intel® Celeron CPU, 2,4 ГГц (система 2) получено: - время расчёта переходного процесса длительностью 500 с для системы 1 зани- мает 7,6 с. В него входят также затраты времени на вывод результатов расчёта в файлы данных. Без учёта этих затрат время расчёта составляет 5,6 с; - для системы 2 время расчёта такого же переходного процесса составляет 40,5 с и 30,6 с соответственно. Проведено также тестирование программы при введении её в микроконтроллер- ный модуль TE-STM32F103, построенный на базе 32-разрядного Cortex-М3 микро- контроллера STM32F103 (отечественный аналог – микроконтроллер 1986ВЕ91Т), име- ющего тактовую частоту 72 МГц, 512 Кбайт флэш-памяти и 64 Кбайта оперативного запоминающего устройства. По результатам тестирования величина загрузочного модуля программы во флеш- память микроконтроллера составила 54196 байта, а время решения всех уравнений мо- дели на одном шаге интегрирования, равном 7 мс, составляет 3,7 мс, что позволяет вы- полнять расчёты примерно в два раза быстрее реального времени. 49 Вестник Самарского университета. Аэрокосмическая техника, технологии и машиностроение Т. Управление с помощью бортовой модели по неизмеряемым параметрам Применение в САУ ГТД таких математических моделей позволяет принципиаль- но изменить способ регулирования двигателя путём перехода на управление по пара- метрам, непосредственно определяющим его основные характеристики, но недоступ- ным для измерения, таким как тяга R двигателя, удельный расход Cr топлива, темпера- тура T* Г газа в камере сгорания, запасы ΔКуi газодинамической устойчивости (ГДУ) и к.п.д. ηi компрессоров, коэффициент αКС избытка воздуха в камере сгорания и ряд дру- гих. Разработаны алгоритмы «виртуальных» регуляторов двигателя по параметрам, вычисляемым с помощью бортовой модели. На рис. 2 приведена структурная схема такой САУ для ТРДД типа ПД-14. Она со- держит регуляторы, воздействующие на расход GT топлива в КС, регуляторы НА КВД, а также клапаны отбора и перепуска воздуха из КВД и подпорной ступени ПС. По сравнению с САУ традиционного типа рассматриваемая система дополни- тельно содержит БММД с алгоритмами её идентификации, а также «виртуальные» кон- туры регулирования и ограничения, отличающиеся от «реальных» тем, что в качестве регулируемых в них используются параметры двигателя, определяемые расчётом в БММД. На рис. 3 показана структурная схема контуров САУ, воздействующих на расход GT топлива в КС с бортовой математической моделью двигателя и блоками формиро- вания программ управления по рассчитываемым параметрам и программ ограничения по измеряемым параметрам. Величины разности, определяемые по расчётным Xi мод и измеренным Xi изм параметрам, селектируются из условия минимального или макси- мального потребного расхода топлива. Выходной сигнал UGТ выбранного канала управления воздействует на дозатор топлива. Рис. 2.Структурная схема САУ ТРДД с БММД Рис. 2.Структурная схема САУ ТРДД с БММД 50 Авиационная и ракетно-космическая техника Рис. 3. Структурная схема контура регулирования расхода топлива Рис. 3. Структурная схема контура регулирования расхода топлива На установившихся режимах работы основными для управления двигателем яв- ляются программы, использующие параметры, рассчитываемые в БММД: тягу R, удельный расход топлива CR и температуру газа * Г T в КС. На переходных режимах работы двигателя дополнительно применяются програм- мы, обеспечивающие необходимую для выполняемой задачи полёта скорость измене- ния тяги   , ПР ЛАi R f t C  при ограничении параметров, определяющих устойчивость рабочего процесса и влияющих на надёжность и ресурс: запасов ГДУ i Ку  компрессо- ров, скорости * / Г dT dt изменения температуры газа, величины коэффициента избытка воздуха в КС и т.п. Структурная схема регулятора НА КВД представлена на рис. 4. Рис. 4 Структурная схема контура регулирования НА КВД Рис. 4 Структурная схема контура регулирования НА КВД 51 Вестник Самарского университета. Аэрокосмическая техника, технологии и машиностроение Т. 15, № 4, 2016 г. Управление с помощью бортовой модели по неизмеряемым параметрам Здесь также содержатся программы управления по рассчитываемым с помощью БММД и измеряемым параметрам рабочего процесса в двигателе. Здесь также содержатся программы управления по рассчитываемым с помощью БММД и измеряемым параметрам рабочего процесса в двигателе. Основными являются программы, обеспечивающие получение максимального значения КПД КВД ( * max КВД  ) при ограничении минимальной величины запасов ГДУ ( min КВД Ky  ). Положения КЛi L клапанов отбора воздуха на охлаждение турбин изменяет- ся в зависимости от расчётной величины * ВХi T – температуры газа на входе в соответ- ствующую турбину. Положение КЛ ПС L клапана перепуска воздуха из ПС осуществля- ется в зависимости от величины ПС Кy  запаса ГДУ ПС, рассчитанной в БММД. Тради- ционные программы управления органами механизации в зависимости от приведённых частот вращения роторов 1 К ПР n остаются как страхующие. Рассмотрим далее некоторые результаты проведённых расчётных исследований по оценке эффективности управления ТРДД с помощью программного обеспечения «виртуальный двигатель». Расчёты показывают, что та- кое управление может существен- но уменьшить чувствительность двигателя к ухудшению характе- ристик его узлов в процессе экс- плуатации. Для примера на рис. 5, 6 показано изменение параметров двигателя при управлении по рас- чётным параметрам (на рис. 5 – управление по тяге R и на рис. 6 – управление по КС  ) и традицион- ным программам (на рис. 5 – управление по К n и на рис. 6 – управление по * / Т К G P ), получен- ное при отклонении характеристик узлов двигателя от принятых в ка- честве номинальных или при вве- дении погрешностей датчиков САУ. Рис. 5. Дроссельные характеристики двигателя при управлении по параметрам nВ и R Рис. 6. Изменение αКС в процессе сброса при управлении по GT/P* K и αКС Расчёты показы кое управление може но уменьшить чувс двигателя к ухудше ристик его узлов в п плуатации. Для прим 6 показано изменени двигателя при управл чётным параметрам управление по тяге R управление по КС  ) ным программам (н управление по К n и управление по / Т G P ное при отклонении х узлов двигателя от пр честве номинальных дении погрешносте САУ. Как видно и управление по расч метрам позволило величину тяги и ре Рис. 5. Дроссельные характеристики двигателя при управлении по параметрам nВ и R Рис. 6. Изменение αКС в процессе сброса при управлении по GT/P* K и αКС Рис. 5. Дроссельные характеристики двигателя при управлении по параметрам nВ и R Рис. 5. Управление с помощью бортовой модели по неизмеряемым параметрам Дроссельные характеристики двигателя при управлении по параметрам nВ и R Рис. 5. Дроссельные характеристики двигателя при управлении по параметрам nВ и R Как видно из рисунков, управление по расчётным пара- метрам позволило восстановить величину тяги и режим работы камеры сгорания. Рис. 6. Изменение αКС в процессе сброса при управлении по GT/P* K и αКС камеры сгорания. Эффективность применения «виртуального» управления клапаном отбора воздуха на охлаждение турбин демонстрируют графики на рис. 7, 8. На рис. 7 приведено изменение параметров двигателя типа ПД–14 в диапазоне дроссельных режимов, где происходит срабатывание клапана отбора воздуха на охла- ждение турбин. Величина 1 К ПР n , при достижении которой осуществляется увеличение (уменьшение) отбираемого расхода воздуха, задана равной 85%. Как видно из графи- ков, эта величина 1 К ПР n на установившихся режимах работы двигателя соответствует величинам * Г ТВД T и * Г ТНД Т (Т31 и Т41 на графиках), равным примерно 1200 К и 830 К. 52 Авиационная и ракетно-космическая техника На рис. 8 показано изменение параметров двигателя в процессе приёмистости в диапазоне режимов МГ…МАХ. Видно, что в данном случае величина 1 К ПР n , равная 85%, при которой увеличивается расход воздуха, отбираемого на охлаждение турбин, соответствует существенно более высоким значениям температуры газа Т* Г ТВД (при- мерно 1550 К). Как показывают расчёты, при этом также наблюдается увеличение тем- пературы Т* Г ТНД до 1100 К. Такое увеличение температуры газа может привести и к существенному увеличению температуры лопаток турбин и уменьшению ресурса дви- гателя за счёт увеличения их малоцикловой усталости. При применении клапана отбора воздуха, изменение положения которого осу- ществляется по температуре Т* Г ТВД, рассчитываемой с помощью БММД, величины температур Т* Г ТВД, Т* Г ТНД, при которых увеличивается интенсивность охлаждения тур- бин, практически совпадают с их значениями на установившихся режимах работы дви- гателя. Рис. 7. Изменение параметров ТРДД на дроссельных режимах Рис. 8. Изменение параметров ТРДД в процессах приёмистости Рис. 7. Изменение параметров ТРДД на дроссельных режимах Рис. 7. Изменение параметров ТРДД на дроссельных режимах Рис. 8. Изменение параметров ТРДД в процессах приёмистости Рис. 8. Изменение параметров ТРДД в процессах приёмистости 53 Вестник Самарского университета. Аэрокосмическая техника, технологии и машиностроение Т. 15, № 4, 2016 г. Методы идентификации бортовой математической модели двигателя в процессе её функционирования в составе САУ Одной из задач, требующих решения при применении бортовых математических моделей двигателя, является проведение их идентификации в реальном масштабе вре- мени в связи с происходящим изменением характеристик узлов двигателя в процессе эксплуатации. Известные формальные методы идентификации разработаны для линей- ных математических моделей. Термогазодинамическая бортовая модель двигателя является существенно нели- нейной. Для её идентификации в реальном масштабе времени выполнена разработка новых методов, основанных на принципе обратной связи. Для их реализации формиру- ется эквивалент замкнутому контуру управления, в котором объектом управления яв- ляется БММД, а в качестве регулирующих факторов объекта управления используются параметры узлов двигателя: величины к.п.д. и приведённого расхода воздуха (газа) ло- паточных машин, коэффициенты восстановления давления и т.п. Регулируемыми параметрами здесь являются получаемые расчётом величины из- меряемых параметров двигателя ( В n , К n , * К P и * Т Т ), а уставками регуляторов – их изме- ренные величины. Алгоритмы динамики контура идентификации выбираются в классе ПИД регуляторов, параметры которых выбираются из условий обеспечения необходи- мой скорости выполнения процесса идентификации и устойчивости. Воздействуя на выбранные регулирующие факторы, система «управления» сводит к нулю разность между измеренными и расчётными значениями регулируемых параметров. При этом «регулирование» одного и того же измеряемого параметра может обеспечиваться одно- временным воздействием на несколько регулирующих факторов. При реализации такого алгоритма идентификации для конкретного двигателя надо путём расчётного эксперимента выбрать оптимальные соотношения между регу- лируемыми параметрами (Хi) и регулирующими факторами (ΔW), обеспечивающие наилучшую точность идентификации по неизмеряемым параметрам. Для этого предва- рительно необходимо определить коэффициенты влияния регулируемых (измеряемых) параметров на идентифицируемые неизмеряемые параметры при воздействии на раз- личные регулирующие факторы. На рис. 9 показаны процессы идентификации рассчитываемых параметров R, К Ky  , полученные с помощью рассматриваемого метода. Рис. 9. Погрешность расчёта параметров двигателя с помощью БММД Рис. 9. Погрешность расчёта параметров двигателя с помощью БММД 54 Авиационная и ракетно-космическая техника Для имитации процессов идентификации БММД в программу её расчёта встроен генератор случайных чисел, задающий различные наборы отклонений (в пределах ±5%) от принятых в качестве базовых характеристик основных узлов двигателя: характери- стик всех лопаточных машин, значений коэффициентов восстановления давления на различных участках проточной части двигателя и т.п. В результате идентификации ошибка расчёта неизмеряемых параметров двигате- ля может быть существенно уменьшена. По величине тяги R погрешность расчёта после проведения идентификации не превышает 1…5%, по величине К Ky  – 1 … 10%, по величине температуры * Г Т – 1 … 2% и по величине αКС – 1 … 3%. Восстановление информации при отказе датчиков ТВХ, РВХ и датчиков обратной связи USING THE «VIRTUAL ENGINE» SOFTWARE IN AUTOMATIC CONTROL SYSTEM OF GAS TURBINE ENGINE © 2016 F. D. Golberg Doctor of Science (Engineering), Professor, Head of Sector, Central institute of Aviation Motors, Moscow, Russian Federation, fegolb@ciam.ru O. S. Gurevich Doctor of Science (Engineering), Professor, Deputy Director General, Head of Department, Central institute of Aviation Motors, Moscow, Russian Federation, gurevich_os@ciam.ru A. A. Petukhov junior researcher, Central institute of Aviation Motors, Moscow, Russian Federation, petuhov-ctrl@ciam.ru Doctor of Science (Engineering), Professor, Head of Sector, Central institute of Aviation Motors, Moscow, Russian Federation, fegolb@ciam.ru Doctor of Science (Engineering), Professor, Head of Sector, Central institute of Aviation Motors, Moscow, Russian Federation, fegolb@ciam.ru Doctor of Science (Engineering), Professor, Head of Sector, Central institute of Aviation Motors, Moscow, Russian Federation, fegolb@ciam.ru Doctor of Science (Engineering), Professor, Deputy Director General, Head of Department, Central institute of Aviation Motors, Moscow, Russian Federation, gurevich_os@ciam.ru Doctor of Science (Engineering), Professor, Deputy Director General, Head of Department, Central institute of Aviation Motors, Moscow, Russian Federation, gurevich_os@ciam.ru A. A. Petukhov junior researcher, Central institute of Aviation Motors, Moscow, Russian Federation, petuhov-ctrl@ciam.ru The paper presents the mathematical tools and «virtual engine» software used in digital automatic control systems of modern gas turbine engines (GTE). The software is based on a real-time fully variable thermogasdynamic engine model operating in onboard computers. The new approach makes it possible to control the engine by the parameters inaccessible for measurement due to the «virtual engine» software in the gas turbine engine control system. The engine fault tolerance improves. We synthesized the control loops of a bypass turbojet engine with a high bypass ratio according to critical parameters (engine thrust, gas temperature in the combustor, stall margin) determined by calculations. We propose a methodology of restoring information on inlet airflow data and values of control factors in case of failure of information channels. The quality of engine regulation improved considerably. The lifetime of the engine increased essentially in the context of varying its characteristics in operation. We developed a methodology of real-time identification of an onboard engine thermogasdynamic simulation model based on the reverse feedback approach. The paper presents the mathematical tools and «virtual engine» software used in digital automatic control systems of modern gas turbine engines (GTE). The software is based on a real-time fully variable thermogasdynamic engine model operating in onboard computers. The new approach makes it possible to control the engine by the parameters inaccessible for measurement due to the «virtual engine» software in the gas turbine engine control system. Библиографический список 1. Culley D., Garg S., Hiller S.-J., Horn W., Kumar A., Mathews H.K., Moustapha H., Pfoertner H., Rosenfeld T., Rybarik P., Schadow K., Stiharu I., Viassolo D.E., Webster J. More Intelligent Gas Turbine Engines: RTO technical report. 2009. http://www.rto.nato.int/ g g p p 2. Гольберг Ф.Д., Гуревич О.С. САУ ГТД с бортовой математической моделью двигателя // Труды Международной научно-технической конференции «Новые рубежи авиационной науки» (ASTEC’07). М.: Центральный аэрогидродинамический институт, 2007. Citation: Golberg F.D., Gurevich O.S., Petukhov A.A. Using the «virtual engine» software in automatic control system of gas turbine engine. Vestnik of Samara University. Aerospace and Mechanical Engineering. 2016. V. 15, no. 4. P. 47-56. DOI: 10.18287/2541-7533-2016-15-4-47-56 при отказе датчиков ТВХ, РВХ и датчиков обратной связи Применение в составе САУ термогазодинамической модели двигателя позволяет восстанавливать информацию о величинах параметров потока воздуха на входе в дви- гатель   * * , ВХ ВХ Т Р и значений регулирующих факторов двигателя   , Т НА G  при отказе датчиков обратной связи. Принципы восстановления этой информации с помощью БММД аналогичны рас- смотренным выше. БММД имитирует объект регулирования, регулирующими факторами которого являются либо значения параметров * * , ВХ ВХ Т Р (в случае отказа соответствующих датчи- ков их измерения), либо регулирующие факторы двигателя Т G или НА  (при отказе со- ответствующих информационных каналов). В качестве параметров регулирования ис- пользуются рассчитываемые с помощью БММД величины измеряемых параметров двигателя ( В n , К n , * К P , * Т Т ), а в качестве уставок регуляторов – их измеряемые ве- личины. Как и ранее, алгоритмы динамики контуров компенсации выбираются в классе ПИД регуляторов. Процедура восстановления включается по сигналу отказа, поступающему из си- стемы контроля и диагностики САУ. Применение метода для восстановления информации в САУ ТРДД типа ПД-14 показало, что для достижения максимальной точности восстановления информации по параметру Т* ВХ целесообразно использовать в качестве «регулируемого» параметра ча- стоту вращения nК КВД, по параметру Р* ВХ – давление воздуха Р* К за КВД, а при вос- становлении информации по сигналу измерения расхода топлива GТ – частоту враще- ния nВ КНД. Время восстановления информации по параметрам Т* ВХ, Р* ВХ на всех ре- жимах работы двигателя не превышает 3…5 с. Точность восстановления информации с помощью БММД составляет 1…3 град по Т* ВХ и 0.01…0.03 ата по Р* ВХ. Д р Время восстановления информации по расходу топлива GТ в КС составляет 1…3 с., а погрешность – 1…2%. Время восстановления информации по расходу топлива GТ в КС составляет 1…3 с., а погрешность – 1…2%. Экспериментальная отработка системы управления с бортовой моделью была проведена на двигателе-демонстраторе, установленном на стенде У-7М ЦИАМ. Для управления двигателем используется демонстрационная электроприводная САУ с цифровым электронным регулятором типа FADEC, выполненным на базе промышленного компьютера. Программное обеспечение электронного регулятора включает в себя разработанную для этого двигателя бортовую поузловую динамическую математическую модель. 55 Вестник Самарского университета. Аэрокосмическая техника, технологии и машиностроение Т. 15, № 4, 2016 г. USING THE «VIRTUAL ENGINE» SOFTWARE IN AUTOMATIC CONTROL SYSTEM OF GAS TURBINE ENGINE The engine fault tolerance improves. We synthesized the control loops of a bypass turbojet engine with a high bypass ratio according to critical parameters (engine thrust, gas temperature in the combustor, stall margin) determined by calculations. p ( g , g p , g ) y We propose a methodology of restoring information on inlet airflow data and values of control factors in case of failure of information channels. The quality of engine regulation improved considerably. The lifetime of the engine increased essentially in the context of varying its characteristics in operation. We developed a methodology of real-time identification of an onboard engine thermogasdynamic simulation model based on the reverse feedback approach. Automatic control system; gas turbine engine; on-board mathematical model. References 1. Culley D., Garg S., Hiller S.-J., Horn W., Kumar A., Mathews H.K., Moustapha H., Pfoertner H., Rosenfeld T., Rybarik P., Schadow K., Stiharu I., Viassolo D.E., Webster J. More Intelligent Gas Turbine Engines: RTO technical report. 2009. Available at: http://www.rto.nato.int/ p 2. Golberg F.D., Gurevich O.S. ACS of GTE with on-board mathematical model of en- gine. Proc. International Conference «New Challenges in Aeronautics» (ASTEC’07). Mos- cow: Central Aerohydrodynamik Institute Publ., 2007. (In Russ.) 56
https://openalex.org/W4246684395	https://www.qeios.com/read/DAL0JG/pdf	English	null	Mycobacterium kansasii	Definitions	2,020	cc-by	66	Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Open Peer Review on Qeios Mycobacterium kansasii National Cancer Institute National Cancer Institute Qeios ID: DAL0JG · https://doi.org/10.32388/DAL0JG Source National Cancer Institute. Mycobacterium kansasii. NCI Thesaurus. Code C85543. A species of Mycobacterium that can cause tuberculosis and leprosy in mammals, but that is generally not dangerous to healthy people. Qeios ID: DAL0JG · https://doi.org/10.32388/DAL0JG 1/1
https://openalex.org/W1841750173	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0135995&type=printable	English	null	Leptospermum flavescens Constituent-LF1 Causes Cell Death through the Induction of Cell Cycle Arrest and Apoptosis in Human Lung Carcinoma Cells	PloS one	2,015	cc-by	8,666	RESEARCH ARTICLE Suerialoasan Navanesan1, Norhanom Abdul Wahab2, Sugumaran Manickam3, Kae Shin Sim1* a11111 1 Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia, 2 Biology Division, Centre for Foundation Studies in Science, University of Malaya, Kuala Lumpur, Malaysia, 3 Rimba Ilmu Botanic Garden, Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia OPEN ACCESS This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: © 2015 Navanesan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Leptospermum flavescens Constituent-LF1 Causes Cell Death through the Induction of Cell Cycle Arrest and Apoptosis in Human Lung Carcinoma Cells Suerialoasan Navanesan1, Norhanom Abdul Wahab2, Sugumaran Manickam3, Kae Shin Sim1* OPEN ACCESS Citation: Navanesan S, Abdul Wahab N, Manickam S, Sim KS (2015) Leptospermum flavescens Constituent-LF1 Causes Cell Death through the Induction of Cell Cycle Arrest and Apoptosis in Human Lung Carcinoma Cells. PLoS ONE 10(8): e0135995. doi:10.1371/journal.pone.0135995 Leptospermum flavescens Sm. (Myrtaceae), locally known as ‘Senna makki’ is a smallish tree that is widespread and recorded to naturally occur in the montane regions above 900 m a.s.l from Burma to Australia. Although the species is recorded to be used traditionally to treat various ailments, there is limited data on biological and chemical investigations of L. flavescens. The aim of the present study was to investigate and understand the ability of L. flavescens in inducing cell death in lung cancer cells. The cytotoxic potentials of the extrac- tion yields (methanol, hexane, ethyl acetate and water extracts as wells as a semi pure frac- tion, LF1) were evaluated against two human non-small cell lung carcinoma cell lines (A549 and NCI-H1299) using the MTT assay. LF1 showed the greatest cytotoxic effect against both cell lines with IC50 values of 7.12 ± 0.07 and 9.62 ± 0.50 μg/ml respectively. LF1 treated cells showed a sub-G1 region in the cell cycle analysis and also caused the presence of apoptotic morphologies in cells stained with acridine orange and ethidium bromide. Treat- ment with LF1 manifested an apoptotic population in cells that were evaluated using the Annexin V/ propidium iodide assay. Increasing dosage of LF1 caused a rise in the presence of activated caspase-3 enzymes in treated cells. Blockage of cell cycle progression was also observed in LF1-treated cells. These findings suggest that LF1 induces apoptosis and cell cycle arrest in treated lung cancer cells. Further studies are being conducted to isolate and identify the active compound as well to better understand the mechanism involved in inducing cell death. Editor: Yu-Jia Chang, Taipei Medicine University, TAIWAN Received: March 14, 2015 Accepted: July 28, 2015 Published: August 19, 2015 Copyright: © 2015 Navanesan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Received: March 14, 2015 Accepted: July 28, 2015 Published: August 19, 2015 Copyright: © 2015 Navanesan et al. * simkaeshin@um.edu.my Introduction Cancer has plagued mankind since the beginning of recorded history. It is a persistent ailment where more than a million people are diagnosed with this disease each year and has a considerably social impact and significant economic burden on our healthcare system. One form of this disease that is severely overlooked is lung cancer which accounts for 19.8% of all medically certified deaths due to cancer in Malaysia [1]. On a global perspective, new cancer diagnosis largely consists of lung cancer cases, whereby 1,350,000 or 12.4% of new cases are contributed from lung cancer [2]. Mutations in the respiratory epithelium gives rise to lung cancer which can be categorized into two broad groups, small cell lung cancer (SCLC) and non–small cell lung cancer (NSCLC). 15% of all lung cancer cases are SCLC which is a highly malignant form of tumour that originates from cells that exhibit neuroendocrine characteristics. The remaining 85% of cases fall into the NSCLC category which is broken down into 3 more groups based on their pathologic subtypes, adenocarcinoma, large cell carcinoma and squamous cell carcinoma [3]. Worldwide, NSCLC patients have a low survival rate; less than 20% of patients survived beyond 5 years after being diagnosed with the disease [4–6]. According to the World Health Organization (WHO), an increasing trend is expected in the number of new lung cancer cases in the years to come, especially in Asia. In fact, since 1985, the number of lung cancer cases has risen by 51%, mostly in females whereby a jump of 76% of new cases have occurred. Both of these incidences are based on a global increase in the usage of tobacco, which is found to be the principal risk factor for lung cancer and causes a large portion of all pulmonary carcinomas [7]. Non-smokers are also affected by this ever prominent disease, mainly through one or a combination of reasons including genetic factors [8], asbestos, air pol- lution [9], second-hand smoke and also radon exposure [10], which is the second major cause of lung cancer. The usage of standard chemotherapy may help to a certain extent but more often the survival benefits are at the expense of severe toxicity. Data Availability Statement: All relevant data are within the paper. Funding: This work was supported by research funding from University of Malaya Research Grant (UMRG), RG082-13BIO and Fundamental Research Grant Scheme (FRGS), FP006-2013A. 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. 1 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0135995 August 19, 2015 L. flavescens Induces Cell Death in Lung Carcinoma Cells PLOS ONE \| DOI:10.1371/journal.pone.0135995 August 19, 2015 Introduction Based on this reasoning, an ethi- cal imperative arises to seek non-toxic alternative to current chemotherapy drugs that is able to effectively combat and manage this disease and at the same time exclude severe toxicity and unwanted complications that are synonymous with current treatment. For this purpose, we turn our attention towards natural products, which is a huge repository of stereochemically complex molecules that appear to have a high specificity towards certain biological target. More than half of approved drugs available today have either been directly sourced from natural origins or are analogues to naturally occurring compounds [11]. In the current study, Leptospermum flavescens was chosen as the candidate for a natural product based treatment against NSCLC. L. flavescens is a medium sized shrub in the Myrta- ceae family with many twiggy angular branches and a reddish-brown bark. The flowers are sol- itary, numerous and white with a yellow tinge. L. flavescens has been used as a natural remedy by locals, either eaten raw or taken as a concoction brewed from fresh plants. In South East Asia, L. flavescens is used to treat constipation, lethargy, hypertension, diabetes and kidney pains [12]. The leaves are used to stimulate appetite and a decoction of the leaves is drunk for relief of stomach discomfort and dysmenorrhoea [13]. The volatile oil extracted from the leaves is used as a remedy to relieve bronchitis when inhaled and also used as an embrocation for rheumatism. Although L. flavescens is reported to be used in traditional medicine preparations, there is limited recorded data on the biological and chemical investigations of the species. At the time of writing, there has been no specific evaluation of the anti-tumour properties against lung cancer cells. Demuner et al. [14] studied the chemical composition as well as the antimi- crobial activities of the essential oil from this plant. The study showed that nerolidol was a major component of the extracted volatile oils with a higher antimicrobial activity against Gram-positive bacteria compared to Gram-negative bacteria. 2 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0135995 August 19, 2015 L. flavescens Induces Cell Death in Lung Carcinoma Cells The aim of this study was to evaluate the cytotoxic effect of L. flavescens against selected NSCLC. The ability of the active fraction to induce apoptosis and disrupt the cell cycle progres- sion was also assessed in this study. Cell lines and culture medium The A549 (ATCC CCL-185) and NCI-H1299 (ATCC CRL-5803) human non-small cell lung carcinoma cell lines and MRC-5 (ATCC CCL-171) normal human lung fibroblast cell line were purchased directly from American Type Culture Collection (ATCC, Manassas, VA, USA). The A549 and NCI-H1299 cells were maintained in a RPMI 1640 medium while MRC- 5 cells in an EMEM medium. All media were supplemented with 10% foetal bovine serum, 1% penicillin/streptomycin (100×) and 0.5% amphotericin B while addition of 1% sodium pyru- vate and 20% foetal bovine serum was supplemented for the EMEM medium. The cells were cultured at 37°C in a Shellab humidified CO2 incubator (Sheldon Manufacturing, Inc., Corne- lius, OR, USA). Extraction of extracts and LF1 The collected plant samples were washed, dried and ground into a fine powder. The dried and ground plant materials (1000.00 g) were extracted with methanol for three days at room tem- perature to obtain crude methanol extract (163.00 g, 16.3%). Upon reserving a small portion (10.00 g) for testing, the remainder (153.00 g) was subjected to fractionation with hexane to yield hexane-soluble extract (6.30 g, 4.12%) and hexane-insoluble residue. The residue was fur- ther partitioned with ethyl acetate and water (1:1, 100 ml: 100 ml) to obtain an ethyl acetate (30.00 g, 19.61%) and a water (29.40 g, 19.22%) extract. A semi-pure fraction (LF1; approx. 12.6 g) which was a white powder with slight greenish tinge was precipitated during methanol extraction, and was included in the current study together with the crude methanol and frac- tionated (hexane, ethyl acetate and water) extracts. All extracts and LF1 were weighed and stored in a fridge (4°C) before further experimentation was done. Prior to testing, all the extracts were dissolved in dimethyl sulfoxide (DMSO), with the exception of the water extract which was dissolved in distilled water to form the stock solution. Plant sample collection and identification Fresh plant sample of L. flavescens was collected from Genting Highlands, Pahang, Malaysia on April 2012. No specific permissions were required for collecting samples in this location. The area of collection is accessible to general public and not designated as a specific area where permission is needed for collection such as a nature park or a wildlife sanctuary. The species is also not an endangered plant or a protected species as it is generally found established on elevations of around 1200 m or higher throughout Malaysia. The plant was identified by Dr Sugumaran Manickam of Institute of Biological Sciences, Faculty of Science, University of Malaya, Malaysia with a voucher specimen number: KLU 47798. The voucher specimen was deposited at the herbarium of the Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia. Introduction The study managed to fulfill these objectives by showing that L. flavescens possess the capacities of eliminating the cancer cell in a controlled manner through the apoptotic pathway. Acridine Orange / Ethidium Bromide (AO/EB) double staining assay The cytotoxic extracts were assessed using the AO/EB staining technique as previously described by Ribble et al. [16]. The cells were seeded at a concentration of 100,000 cells per well in a six-well plate and incubated for 24 hours at 37°C before treatment with the active fraction LF1 at varying concentrations (5, 15 and 25 μg/ml). Following an incubation period of 24 hours, the cells were detached and pelleted. The supernatant was removed and the cells were subsequently stained with the prepared dye mixture (25 μl cold PBS and 2 μl EB/AO dye mixed in a 1:1 ratio). The stained cell suspension was transferred onto a clean glass slide and covered with a coverslip. The morphological changes relative to the untreated control was observed using the narrow blue excitation filter on an Olympus IX73 fluorescent microscope (Olympus Corporation, Shinjuku, Tokyo, JPN). The images were photographed at ×400 magnification. Active Caspase-3 detection assay LF1 induced apoptosis was ascertained by detecting the presence of activated caspase-3 in treated cells. The FITC Active Caspase-3 Apoptosis Kit (BD Biosciences, San Jose, CA, USA) was used to assess the presence of active caspase-3. The antibody used in this assays specifically recognize the active form of caspase-3 in human cells. The cells were plated at a concentration of 300,000 cells per dish on to a 60 mm culture dish and left for 24 hours at 37°C before addi- tion of treatment at predetermined concentrations. The cells were then left to incubate for another 24 hours before being harvested and stained according to the manufacturer’s protocol. The cells were then analysed using the BD FACS Canto II flow cytometer (BD Biosciences, San Jose, CA, USA) with a minimum of 10,000 events being acquired. Annexin V/ Propidium Iodide (PI) assay The Annexin V-FITC/ PI assay was conducted to further assess the effects of the cytotoxic frac- tion LF1 in inducing cell death through apoptosis. The cells were plated at a concentration of 300,000 cells per dish on a 60 mm culture dish and incubated for 24 hours at 37°C before being treated with a few different concentrations of LF1. In order to determine the induction of apo- ptosis, cells were harvested at 24, 48 and 72 hours post treatment. The cells were detached, washed with phosphate buffered saline (PBS) and stained with Annexin V-FITC conjugates and Propidium Iodide (PI) (BD Biosciences, San Jose, CA, USA). The cells were incubated for 15 minutes before they were analysed using a BD FACS Canto II flow cytometer (BD Biosci- ences, San Jose, CA, USA). A minimum of 10,000 events were collected and analysed. MTT cytotoxicity assay The cytotoxicity of extracts and LF1 were assessed using MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay as previously described by Mosmann [15] with 3 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0135995 August 19, 2015 L. flavescens Induces Cell Death in Lung Carcinoma Cells modifications. The cells were seeded at a concentration of 7,000 cells per well in a 96-well plate a day prior to treatment with the extracts and LF1 that were diluted to contain 0.5% DMSO. The treated cells were left to incubate for 72 hours in the CO2 incubator. Addition of the MTT solution (working concentration of 5 mg/ml) was followed by a further incubation period of 3 hours before dissolving the purple formazan crystals with DMSO. The absorbance values was measured using a Multiskan GO micro plate spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 570 nm with 650 nm as a reference wavelength. Cisplatin was used as positive reference standard. All assays were performed in triplicates. Morphological detection of cell death caused by LF1 using AO/EB double staining LF1 treated cells showed morphological features consistent with apoptosis. In comparison with the untreated cells which produced a nuclei uniformly stained green, LF1 treated A549 and NCI-H1299 cells showed bright patches of bright green fluorescent stains within the cells (Fig 1), indicative of chromatin condensation. Moreover, at concentration of 5 μg/ml of LF1, both cell lines produced protrusion out of the plasma membrane which could be characterized as membrane blebbing, one of the symptoms commonly associated with early apoptosis. With the increase in dosage of LF1 used, an elevation in the number of cells stained orange is also observed. Treatment with the highest concentration (25 μg/ml) of LF1 appears to cause the cells to be uniformly stained orange. The cells also tend to appear to have a decreased definition of the nuclear outline, possibly due to the rupturing of nuclear and plasma membrane. Cell cycle analysis The presence of cell cycle disruption caused by the treatment of the cytotoxic fraction LF1 was evaluated using BD Cycletest Plus DNA Reagent Kit (BD Biosciences, San Jose, CA, USA). The cells were plated at a concentration of 300,000 cells per dish on to a 60 mm culture dish and 4 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0135995 August 19, 2015 L. flavescens Induces Cell Death in Lung Carcinoma Cells left for 24 hours at 37°C before addition of treatment at predetermined concentrations. The cells were then left to incubate for another 24 hours before being harvested. The kit was then used for the isolation and staining of nuclei in treated cells. The PI stained nuclei was then ana- lysed using the BD FACS Canto II flow cytometer (BD Biosciences, San Jose, CA, USA). The DNA samples were acquired at a low flow rate to ensure the best resolution for the collected data. A minimum of 10,000 events were collected and analysed. The sub-G1 was gated and quantified using the FACS Diva software (Beckton-Dickinson, USA) while the cell cycle data was analysed using the ModFit software (Verity Software House, Topsham, ME, USA). Statistical analysis The IC50 values of cytotoxic activity were obtained by non-linear regression using GraphPad Prism 5 software (GraphPad Software Inc., USA). The software was also used to perform a Dunnett's Multiple Comparison Test on the results from the Annexin V-FITC/PI assay, cell cycle, sub-G1 analysis and active caspase-3 detection assay to determine if the variation from the untreated control is statistically significant. Cytotoxic activity of L. flavescens extracts and LF1 The cytotoxicity of the crude methanol extract, its fractions (hexane, ethyl acetate and water) and the LF1 fraction was assessed using the MTT cytotoxic assay described in the methodology section. The samples were tested on three different lung cells line (A549, NCI- H1299 and MRC-5). The results obtained were presented in Table 1. An extract is considered significantly cytotoxic should it produce an IC50 value 20 μg/ml. Based on this criteria, none of the crude and fractionated extracts managed to exert a significant cytotoxic affect against all three cell lines. The semi-pure fraction, LF1, managed to produce and IC50 value of 7.12 ± 0.07 and 9.62 ± 0.50 μg/ml against the population of A549 and NCI-H1299 respectively. The cells were also tested using an FDA approved drug for the treatment of lung cancer, cisplatin. An IC50 value of 8.70 ± 1.30 μg/ml for A549 and 21.41 ± 3.99 μg/ml against NCI-H1299 were obtained. Cisplatin seemed to be more toxic towards the normal fibroblast cell, MRC-5, compared to LF1 (1.27 ± 0.06 μg/ml against 6.66 ± 0.17 μg/ml). Assessing LF1-induced apoptosis using Annexin V-FITC /Propdium I did Incidence of apoptosis in cells exposed to LF1 was quantified using the Annexin V-FITC/Pro- pidium Iodide assay. Flow cytometric analysis of A549 and NCI-H1299 cells stained with Annexin V-FITC conjugates and PI expressed a significant (p < 0.05) increase in the early apo- ptotic and secondary necrotic cell population with the increasing concentration of LF1 used (Fig 3). With reference to the untreated control, the number of viable cells suffered a steep drop in population for both cell lines used; from 93.4 ± 1.9% to 40.7 ± 5.3% for A549 and 91.4 ± 2.3% to 44.6 ± 3.3% for NCI-H1299 (Fig 3). These observations were done with refer- ence to the untreated controls and the differences with the control were statistically significant (p < 0.05) in most cases. In order to distinguish between secondary necrotic cells and necrotic cells, a time course study was performed. Both cells lines showed an increase in early apoptotic cells over time that was then followed by an increase in double positively stained cells (Figs 4 and 5), indicating that the treated cells have undergone apoptosis. L. flavescens Induces Cell Death in Lung Carcinoma Cells Table 1. Cytotoxic activity (IC50 values) of extracts. Extracts/Fraction Cytotoxicity (IC50) in μg/ml A549 NCI-H1299 MRC-5 Methanol 96.38 ± 4.03 > 100 > 100 Hexane 40.91 ± 0.97 90.44 ± 1.55 69.11 ± 5.15 Ethyl Acetate 55.77 ± 2.59 77.10 ± 2.81 62.20 ± 1.45 Water > 100 > 100 > 100 LF1 7.12 ± 0.07 9.62 ± 0.50 6.66 ± 0.17 Cisplatin * 8.70 ± 1.30 21.41 ± 3.99 1.27 ± 0.06 *Positive reference standard. Values are expressed as mean ± standard deviation (n = 3). doi:10 1371/journal pone 0135995 t001 Table 1. Cytotoxic activity (IC50 values) of extracts. Table 1. Cytotoxic activity (IC50 values) of extracts. gated-population using the flow cytometer. As the concentration of LF1 is increased, the quan- tity of cells which produces a Sub-G1 peak in DNA frequency histogram also gradually increases (Fig 2). The increment is significantly (p<0.05) prominent upon treatment with 7.5 and 10 μg/ml of LF1. This situation is observed in both A549 and NCI-H1299 cell lines. The percentage of sub-G1 population increases from 1.20 ± 0.10% in untreated cells to 10.77 ± 0.97% in A549 cells treated with 10 μg/ml of LF1, while NCI-H1299 cells exposed to similar conditions increased from 2.05 ± 1.73% to 8.98 ± 1.59%. Sub-G1 population analysis of LF1 treated cells Quantification of the Sub-G1 population was done concurrently with the cell cycle analysis which utilized the PI dye. The results were expressed as percentage relative to the total acquired 5 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0135995 August 19, 2015 doi:10.1371/journal.pone.0135995.g001 Detecting the presence of active (cleaved) caspase-3 The activated caspase-3 binds covalently and irreversibly with the FITC-conjugated antibody and is expressed as percentage relative to the total acquired gated-population using the flow cytometer. As shown in Fig 6, an increasing presence of activated caspase-3 is observed with the increasing treatment of LF1 in both A549 and NCI-H1299, although a stronger effect is seen in the former (Fig 6). A shift towards the right is seen in the histogram as higher dosages of LF1 are used against both cell lines, indicating a rise in cleaved caspase-3 presence. Treat- ment with 5 μg/ml of LF1 did not seem to elicit significant caspase activation as compared to the untreated control on both cell lines. On the other hand, a significant increase (p < 0.05) from 0.13 ± 0.15% to 53.90 ± 2.36% of active caspase-3 signal was observed in A549 treated with 20 μg/ml of LF1 whilst NCI-H1299 cells experienced a smaller jump from 0.40 ± 0.26% to 20.47 ± 0.80% under the same conditions. 6 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0135995 August 19, 2015 L. flavescens Induces Cell Death in Lung Carcinoma Cells Fig 1. Morphological observation of LF1 treated A549 and NCI-H1299 using AO/EB staining at × 400 magnifications. A549 and NCI-H12 treated without (untreated control) and with LF1 at different concentrations. Green arrows indicate early apoptotic cells (chromatin condensation green); blue arrows are late apoptotic cells (chromatin condensation stained orange); purple arrows shows membrane blebbing; yellow arrows a which appear shrunken and red arrows indicate loss of membrane shape. The higher the concentration of LF1 used, the more aggressive pathw the induction of death in the cancer cells. doi:10.1371/journal.pone.0135995.g001 L. flavescens Induces Cell Death in Lung Ca Fig 1. Morphological observation of LF1 treated A549 and NCI-H1299 using AO/EB staining at × 400 magnifications. A549 and NCI-H1299 were treated without (untreated control) and with LF1 at different concentrations. Green arrows indicate early apoptotic cells (chromatin condensation stained green); blue arrows are late apoptotic cells (chromatin condensation stained orange); purple arrows shows membrane blebbing; yellow arrows are for cells which appear shrunken and red arrows indicate loss of membrane shape. The higher the concentration of LF1 used, the more aggressive pathway taken in the induction of death in the cancer cells. 7 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0135995 August 19, 2015 7 / 16 L. flavescens Induces Cell Death in Lung Carcinoma Cells Fig 2. Analysing the disruptive effect of LF1 on the cell cycle Blockage in the cell cycle as a result of treatment with LF1 was assessed by staining fixed and permeabilized cell with the PI dye. The DNA content which was in the G0/G1 phase gradually Fig 3. Effects of exposure to LF 1 for 24 hours on the externalization of PS in A549 and NCI-H1299 (Dose—dependent). Summary of the results for 2 hours treatment with LF1 was presented in a bar chart (A) for A549 and bar chart (B) for NCI-H1299. A significant increase in the early apoptotic and secondary necrotic cell population is observed with the increasing concentration of LF1 in both cell lines. doi:10.1371/journal.pone.0135995.g003 L. flavescens Induces Cell Death in Lung Carcinoma Ce Analysing the disruptive effect of LF1 on the cell cycle Blockage in the cell cycle as a result of treatment with LF1 was assessed by staining fixed and permeabilized cell with the PI dye. The DNA content which was in the G0/G1 phase gradually Fig 3. Effects of exposure to LF 1 for 24 hours on the externalization of PS in A549 and NCI-H1299 (Dose—dependent). Summary of the results for 24 hours treatment with LF1 was presented in a bar chart (A) for A549 and bar chart (B) for NCI-H1299. A significant increase in the early apoptotic and secondary necrotic cell population is observed with the increasing concentration of LF1 in both cell lines. doi:10.1371/journal.pone.0135995.g003 L. flavescens Induces Cell Death in Lung Carcinoma Cell Fi Eff f LF f h h li i f PS i A d NCI H (D d d ) S f h l f Fig 3. Effects of exposure to LF 1 for 24 hours on the externalization of PS in A549 and NCI-H1299 (Dose—de hours treatment with LF1 was presented in a bar chart (A) for A549 and bar chart (B) for NCI-H1299. A significant inc secondary necrotic cell population is observed with the increasing concentration of LF1 in both cell lines. Fig 3. Effects of exposure to LF 1 for 24 hours on the externalization of PS in A549 and NCI-H1299 (Dose—dependent). Summary of the results for 24 hours treatment with LF1 was presented in a bar chart (A) for A549 and bar chart (B) for NCI-H1299. A significant increase in the early apoptotic and secondary necrotic cell population is observed with the increasing concentration of LF1 in both cell lines. doi:10.1371/journal.pone.0135995.g003 Detecting the presence of active (cleaved) caspase-3 Sub G1 population of LF1 treated cells. A549 (A) and NCI-H1299 (B) cells were treated with 5, 7.5 and 10 μg/ml of LF1. The resultant sub-G1 population show an increase with the increase in concentration of LF1 used. doi:10.1371/journal.pone.0135995.g002 Fig 2. Sub G1 population of LF1 treated cells. A549 (A) and NCI-H1299 (B) cells were treated with 5, 7.5 and 10 μg/ml of LF1. The resultant sub-G1 population show an increase with the increase in concentration of LF1 used Fig 2. Sub G1 population of LF1 treated cells. A549 (A) and NCI-H1299 (B) cells were treated with 5, 7.5 and 10 μg/ml of LF1. The resultant sub-G1 population show an increase with the increase in concentration of LF1 used. Fig 2. Sub G1 population of LF1 treated cells. A549 (A) and NCI-H1299 (B) cells were treated with 5, 7.5 and 10 μg/ml of LF1. The resultant sub-G1 population show an increase with the increase in concentration of LF1 used. doi:10.1371/journal.pone.0135995.g002 PLOS ONE \| DOI:10.1371/journal.pone.0135995 August 19, 2015 8 / 16 L. flavescens Induces Cell Death in Lung Carcinoma Cells L. flavescens Induces Cell Death in Lung Carcinoma Cells Fig 4. Effects of exposure to LF1 on the externalization of PS in A549 at different time points (Time—dependent). Cells were treated with 10μg/ml of LF1 for 24 hours (A), 48 hours (B) and 72 hours (C). Summary of the results were presented in a bar chart (D). An increase in early apoptotic cells over time that was then followed by an increase in double positively stained cells indicate an apoptotic pathway was taken by LF1 treated cells. d i 10 1371/j l 0135995 004 Fig 4. Effects of exposure to LF1 on the externalization of PS in A549 at different time points (Time—dependent). Cells were treated with 10μg/ml of LF1 for 24 hours (A), 48 hours (B) and 72 hours (C). Summary of the results were presented in a bar chart (D). An increase in early apoptotic cells over time that was then followed by an increase in double positively stained cells indicate an apoptotic pathway was taken by LF1 treated cells. doi:10.1371/journal.pone.0135995.g004 increased with the increasing exposure to LF1 on both A549 and NCI-H1299 cell lines. In gen- eral, a reverse was observe in the S and G2/M phase population of both treated cell lines, whereby the number of events recorded depleted with the escalating dosage of LF1 used (Fig 7). These observations were done with reference to the untreated controls and the differences with the control were statistically significant (p < 0.05) in most cases. PLOS ONE \| DOI:10.1371/journal.pone.0135995 August 19, 2015 Analysing the disruptive effect of LF1 on the cell cycle Blockage in the cell cycle as a result of treatment with LF1 was assessed by staining fixed and permeabilized cell with the PI dye. The DNA content which was in the G0/G1 phase gradually PLOS ONE \| DOI:10.1371/journal.pone.0135995 August 19, 2015 9 / 16 L. flavescens Induces Cell Death in Lung Carcinoma Cells Fig 5. Effects of exposure to LF1 on the externalization of PS in NCI-H1299 at different time points (Time—dependent). Cells were treated with 15 μg/ ml of LF1 for 24 hours (A), 48 hours (B) and 72 hours (C). Summary of the results were presented in a bar chart (D). An increase in early apoptotic cells over time that was then followed by an increase in double positively stained cells indicate an apoptotic pathway was taken by LF1 treated cells. doi:10 1371/journal pone 0135995 g005 Fig 5. Effects of exposure to LF1 on the externalization of PS in NCI-H1299 at different time points (Time—dependent). Cells were treated with 15 μg/ ml of LF1 for 24 hours (A), 48 hours (B) and 72 hours (C). Summary of the results were presented in a bar chart (D). An increase in early apoptotic cells over time that was then followed by an increase in double positively stained cells indicate an apoptotic pathway was taken by LF1 treated cells. doi:10.1371/journal.pone.0135995.g005 promoting apoptotic cell death in damaged cells [18]. A normal human lung fibroblast cell line (MRC-5) was used to determine the effect on non-carcinoma cells. The criteria set forward by US National Cancer Institute (NCI) Plant Screening Program was that a compound is incu- bated with the cancer cells between 48 and 72 hours, it should produce an IC50 value less than 20 μg/ml for a crude extract and 4 μg/ml for a pure compound [19]. With reference to Table 1, the toxicity towards both the cancer cell lines tested indicates that there is a possibility that the fraction has utilized a p53 independent pathway in inducing cell death as a similar cytotoxic effect was present regardless of the presence of a functional p53 gene expression. Although LF1 may seem to be toxic towards the normal MRC-5 cell, the positive control used (cisplatin), which is a drug approved by US Food and Drug Administration (FDA) for usage in lung cancer treatment, exhibited a much more potent effect towards the normal fibro- blast cell. In fact, LF1 was five times less toxic towards the normal cells when compared to cis- platin. Based on these results, the ability of LF1 to induce cell death was further assessed. LF1 was further examined using the AO/EB double staining method. Discussion The alarming rise in incidence of cancer, especially lung cancer, encourages us to look for alter- native cures which are more effective in the fight against cancer but at the same time, harmless towards the patient. The purpose of this study was to explore the usage of Leptospermum flaves- cens to fulfill the above needs. The MTT assay is a mode of convenient quantitative measurement used in evaluating a population of cell’s response to external factors which may cause an increase or decrease in cell growth [17]. In this study, the crude methanol and fractionated (hexane, ethyl acetate and water) extracts as well as the semi-pure fraction LF1 were tested against three lung cells, two cancerous and one normal cell line. The comparable characteristic of the cancerous cells used is that A549 has a functional wild type p53 expression while NCI-H1299 is p53 deficient. The p53 functions by coordinately blocking cell proliferation, stimulating DNA repair and 10 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0135995 August 19, 2015 PLOS ONE \| DOI:10.1371/journal.pone.0135995 August 19, 2015 L. flavescens Induces Cell Death in Lung Carcinoma Cells Fig 6. Presence of active caspase-3 in LF1 treated A549 and NCI-H1299. (A) A549 and (B) NCI-H1299 cells were treated with 5, 10 and 20 μg/ml of LF1 for 24 hours. Summary of the results were presented in a bar chart (C). An increase in the presence of cleaved caspase-3 was detected in both cell lines in a dose-dependent manner, indicating an apoptotic pathway was taken by LF1 treated cells. doi:10 1371/journal pone 0135995 g006 Fig 6. Presence of active caspase-3 in LF1 treated A549 and NCI-H1299. (A) A549 and (B) NCI-H1299 cells were treated with 5, 10 and 20 μg/ml of LF1 for 24 hours. Summary of the results were presented in a bar chart (C). An increase in the presence of cleaved caspase-3 was detected in both cell lines in a dose-dependent manner, indicating an apoptotic pathway was taken by LF1 treated cells. doi:10.1371/journal.pone.0135995.g006 Fig 7. Effects of LF1 on cell cycle distribution in A549 NCI-H1299 cells. A549 (A) and NCI-H1299 (B) were incubated in absence (control) and presence of LF1 at 5, 7.5 and 10 μg/ml for 24 hours. Summary of results indicate an increase in G0/G1 population with increasing dosages of LF1 used. doi:10.1371/journal.pone.0135995.g007 Fig 6. Presence of active caspase-3 in LF1 treated A549 and NCI-H1299. (A) A549 and (B) NCI-H1299 cells were treated with 5, 10 and 20 μg/ml of LF1 for 24 hours. Summary of the results were presented in a bar chart (C). An increase in the presence of cleaved caspase-3 was detected in both cell lines in a dose-dependent manner, indicating an apoptotic pathway was taken by LF1 treated cells. Fig 6. Presence of active caspase-3 in LF1 treated A549 and NCI-H1299. (A) A549 and (B) NCI-H1299 cells were treated with 5, 10 and 20 μg/ml of LF1 for 24 hours. Summary of the results were presented in a bar chart (C). An increase in the presence of cleaved caspase-3 was detected in both cell lines in a dose-dependent manner, indicating an apoptotic pathway was taken by LF1 treated cells. doi:10.1371/journal.pone.0135995.g006 Fig 7. Effects of LF1 on cell cycle distribution in A549 NCI-H1299 cells. A549 (A) and NCI-H1299 (B) were incubated in absence (control) and presence of LF1 at 5, 7.5 and 10 μg/ml for 24 hours. Summary of results indicate an increase in G0/G1 population with increasing dosages of LF1 used. This combination of dyes is commonly applied in the identification and differentiation of live, apoptotic and necrotic cells. Cells are differentiated through the staining pattern. Viable cells are stained uni- formly green whereas early apoptotic cells have bright green patches, indicative of chromatin condensation. Late apoptotic cells have orange to red stained nuclei with condensed or PLOS ONE \| DOI:10.1371/journal.pone.0135995 August 19, 2015 11 / 16 L. flavescens Induces Cell Death in Lung Carcinoma Cells fragmented chromatin and necrotic cells have a uniformly stained orange to red nuclei [20]. Morphological observations commonly associated with apoptosis such as membrane blebbing and fragmented nuclei were used to classify the cells as apoptotic or otherwise [21]. By taking into account the nuclear morphology visualized through the fluorescent AO/EB stain (chromatin condensation, nuclear collapse) and the common light microscopy appear- ances (membrane blebbing, cell rounding and shrinkage) of these cells, a complete profile of an apoptotic cells is present. These observations point towards the interpretation that LF1 treated cells undergo apoptosis which will lead to the eventual death of the cells. The observations during treatment with the highest concentration vary considerably with the pattern seen in the other treatment groups. The uniformly stained orange nucleus suggests that at high concentration of LF1, the cells undergo necrosis instead of apoptosis. According to Sancho-Martinez et al. [22], the concentration of many cytotoxic molecules and stimuli, including different anticancer drugs, was the determinant in the manner of cell death, being either apoptosis or necrosis. The high concentration of the drug which caused necrosis in the treated cells could have been caused by general toxicity of foreign compound towards the cells. In short, the higher the concentration of LF1 used, the more aggressive pathway taken in the induction of death in the cancer cells. To further prove the hypothesis that LF1 is able to induce cell death in cancer cells through apoptosis, presence of a sub-G1 population in the DNA content frequency histogram was investigated. Cells that are or have undergone apoptosis should contain fractional DNA con- tent which translates into the presence of a hypo-diploid DNA population that lies before the diploid (2N) region in the DNA content frequency histogram that was analyzed through the flow cytometer [23,24]. The hypo-diploid DNA content is an outcome from the activation of endogenous endonucleases in apoptotic cells that in turn causes DNA fragmentation [25]. Relative to the untreated control, treatment with incremental dosages of LF1 resulted in an increment of a distinct sub-G1 population in the DNA frequency histogram (Fig 2), which in turn can be interpreted as an increase in apoptotic effects towards the treated cells. This obser- vation is consistent with the outcome from the MTT assay as well as AO/EB staining of LF1-treated cells. doi:10.1371/journal.pone.0135995.g007 Fig 7. Effects of LF1 on cell cycle distribution in A549 NCI-H1299 cells. A549 (A) and NCI-H1299 (B) were incubated in absence (control) and presence of LF1 at 5, 7.5 and 10 μg/ml for 24 hours. Summary of results indicate an increase in G0/G1 population with increasing dosages of LF1 used. doi:10.1371/journal.pone.0135995.g007 Fig 7. Effects of LF1 on cell cycle distribution in A549 NCI-H1299 cells. A549 (A) and NCI-H1299 (B) were incubated in absence (control) and presence of LF1 at 5, 7.5 and 10 μg/ml for 24 hours. Summary of results indicate an increase in G0/G1 population with increasing dosages of LF1 used. Fig 7. Effects of LF1 on cell cycle distribution in A549 NCI-H1299 cells. A549 (A) and NCI-H1299 (B) were incubated in absence (control) and presence of LF1 at 5, 7.5 and 10 μg/ml for 24 hours. Summary of results indicate an increase in G0/G1 population with increasing dosages of LF1 used. doi:10.1371/journal.pone.0135995.g007 doi:10.1371/journal.pone.0135995.g007 PLOS ONE \| DOI:10.1371/journal.pone.0135995 August 19, 2015 12 / 16 L. flavescens Induces Cell Death in Lung Carcinoma Cells route, consistent with the data from the previous assays (MTT assay, AO/EB staining and Sub G1 population). The cysteine-dependent aspartate driven proteases (caspases), which consist of several pro- teases, are major executors of apoptosis which are interconnected in a series to from a proteo- lytic cascade [28]. Caspases are typically divided into initiator caspases (caspase-2, -8, -9, and -10) which are activated upon interaction with adaptor protein, and the effector caspase (cas- pase-3, -6, and -7) that act directly on specific cellular substrate involved in the disablement of key proteins of the cell upon activation by the initiator caspases [29]. Among these enzymes, caspase-3 is a death protease that is frequently activated, triggering the specific cleavage of many key cellular proteins involved in apoptosis [28]. Activation of cas- pase-3 may occur through the intrinsic pathway which involves the release of cytochrome c and the initiator caspase-9 or via the extrinsic pathway that includes activation of death recep- tors as well as caspase-8 [29,30]. As such, caspase-3 is a perfect biomarker to affirm the role of LF1 in inducing apoptosis among treated cells as it is detectable in apoptotic cells regardless of the pathway taken. The results obtain reflect an increasing presence of the activated caspase-3 enzyme, suggest- ing and a rise in the apoptotic activity in LF1 treated cells. Moreover, the dose-dependent char- acteristic exhibited by LF1 in the activated caspase-3 quantification is consistent with the data from the other apoptotic assessment within this study. Increment of LF1 dosage showed a simi- lar trend of increased apoptotic activity in the Annexin V/PI assay and the sub-G1 quantification. Caspase-3 plays a central role proteolytic cleavage of several proteins and is responsible for the apoptosis-associated chromatin condensation (observed in the AO/EB staining), DNA fragmentation (present in the Sub-G1 analysis), and nuclear collapse of the effected cells [31]. The elevated presence of active caspase-3 serves as a biochemical validation of the ability of LF1 in inducing apoptosis in treated lung cancer cells. The growth development process in multicellular organisms is a complex process and need to be done in an appropriate and orderly fashion to avoid accumulated errors which may trans- late into unwanted situation, such as the formation of cancer cells. PLOS ONE \| DOI:10.1371/journal.pone.0135995 August 19, 2015 The sub-G1 population detection is a convenient assay in assessing the poten- tial apoptotic capabilities of LF1, but has to be validated with tests that are specific towards the apoptotic markers. This leads us to the next phase of this study; the evaluation of apoptosis using the Annexin V /Propidium Iodide staining method. Under normal physiological conditions, cells maintain an asymmetrical distribution of phospholipids such as phosphatidylserine (PS) between the inner and outer leaflet of the plasma membrane [26]. Apoptosis disrupts this asymmetric architecture which leads to the sustained presence of PS in the outer leaflet of the plasma membrane [27]. By utilizing the FITC-conjugated Annexin V, which has a specific affinity towards the PS region, this charac- teristic that is unique to the apoptotic process can be used to differentiate between viable and apoptotic cells. Moreover, the cell population can be further categorize into early apoptotic cells, secondary necrotic cells (late apoptotic cells) as well as necrotic cells through the concur- rent usage of PI as an exclusion dye. This assay allows for a quantitative analysis into the occur- rence of apoptosis in LF1-treated cells. Being an operator independent method, this method has the advantage of being more accurate as it is free from the user bias. Another advantage of this method is the increased statistical significance compared to more conventional methods such as microscopy assays, as it is possible to obtain a larger sample size (typically 10,000 or events). The increasing presence of early and late apoptotic cells with the progression of time and increasing concentrations of treatment suggest that LF1 managed to induce a controlled demo- lition at the cellular level via the apoptotic pathway, as opposed to the un-programmed necrotic PLOS ONE \| DOI:10.1371/journal.pone.0135995 August 19, 2015 13 / 16 Conclusion This study has shown that L. flavescens fraction, LF1 is capable of exerting cytotoxic effect on the tested lung carcinoma cells via the apoptotic pathway. The disruption of the cell cycle at the G0/G1 phase suggests LF1 may be targeting specific kinases or genes in the cells, channel- ling them into the apoptotic pathway. While it is still premature to suggest anti-tumour devel- opment from the constituents of L. flavescens, the potential of such an idea cannot be disregarded with the results obtained. Nevertheless, a better understanding on the exact mechanism of how L. flavescens exerts its cytotoxic activity would be crucial, thus requiring further investigations. L. flavescens Induces Cell Death in Lung Carcinoma Cells activation of this gene through cellular stress is known to inhibit the functionality of these kinases. Experimental data from the previous experimentation in this study has allowed us to rule out the p53 gene, which is usually associated with G0/G1 phase arrest, as a possible target of LF1. Based on these findings, the next step would be to evaluate if LF1 directly inhibits the activities of the kinases mentioned above or functionality of these kinases are prevented through the activation of the p16 gene. Author Contributions Conceived and designed the experiments: KSS. Performed the experiments: SN. Analyzed the data: SN NAW. Contributed reagents/materials/analysis tools: NAW SM. Wrote the paper: SN KSS NAW SM. PLOS ONE \| DOI:10.1371/journal.pone.0135995 August 19, 2015 For this purpose, the cell employs a regulatory network of genes and proteins which serves as a check and balance in the development process to avoid formation of mutant cells. When this system fails or is bypassed due to external factors, the cell requires stimulants to activate the system in order to eliminate the defective (cancer) cell. Certain chemotherapeutic drugs disrupt the developmental process in the cancer cell by halting the cell cycle at a particular stage which results in the suppression of tumour proliferation before activating the apoptotic pathway leading to the demise of the cancer cells [32]. Our study thus far shows that LF1 is able to induce apoptosis in cancer cells. The cell cycle analysis was performed in the hopes of determining if cell death caused by LF1 through apoptosis involved arresting the cell cycle at a specific stage [33]. By doing so, the spe- cific targets (genes or proteins) of LF1 can be narrowed and this in turns allows a better under- standing into the nature of the semi-pure fraction. The findings in this experiment suggest that LF1 managed to cause an arrest of the cell cycle at the G0/G1 phase in treated cells, thus preventing the transition from the G1 phase to the S phase leading to the demise of the cell. This argumentation also explains the decrease in the S and G2/M phase in both A549 and NCI-H1299 treated cells. As both the cell lines reacted simi- larly towards LF1, the role of the p53 gene can be ruled out by using the same line of reasoning mentioned in the MTT assay. Possible targets of LF1 which may lead to the cell cycle arrest at the G0/G1 phase includes cyclin D and cyclin E as well as their corresponding cyclin-dependent kinase (CdK), CdK 2 and CdK4/6 [34]. Moreover, the p16 gene could also be a possible target of LF1 as the PLOS ONE \| DOI:10.1371/journal.pone.0135995 August 19, 2015 14 / 16 References 1. Sachithanandan A, Badmanaban B. Screening for Lung cancer in Malaysia: Are we there yet? Med J Malaysia. 2012; 67(1):3–6. PMID: 22582540 2. Cruz CSD, Lynn TT, Richard AM. Lung cancer: epidemiology, etiology, and prevention. Clin Chest Med. 2011; 32(4): 605–644. doi: 10.1016/j.ccm.2011.09.001 PMID: 22054876 3. Bi X, Xia X, Mou T, Jiang B, Fan D, Wang P, et al. Anti-tumor activity of three ginsenoside derivatives in lung cancer is associated with Wnt/β-catenin signaling inhibition. Eur J Pharmacol. 2014; 742(0):145– 152. 4. Bartucci M, Svensson S, Romania P, Dattilo R, Patrizii M, Signore M, et al. Therapeutic targeting of Chk1 in NSCLC stem cells during chemotherapy. Cell Death Differ. 2011; 19(5):768–778. doi: 10.1038/ cdd.2011.170 PMID: 22117197 5. Collins LG, Haines C, Perkel R, Enck RE. Lung cancer: diagnosis and management. Am Fam Physi- can. 2007; 75(1):56–63. 6. Jemal A, Siegel R, Xu J, Ward E. Cancer statistics, 2010. CA Cancer J Clin. 2010; 60(5):277–300. doi: 10.3322/caac.20073 PMID: 20610543 7. Parkin DM, Pisani P, Lopez AD, Masuyer E. At least one in seven cases of cancer is caused by smok- ing. Global estimates for 1985. Int J Cancer. 1994; 59(4):494–504. PMID: 7960219 8. Gorlova OY, Weng SF, Zhang Y, Amos CI, Spitz MR. Aggregation of cancer among relatives of never- smoking lung cancer patients. Int J Cancer. 2007; 121(1):111–118. PMID: 17304511 9. Kabir Z, Bennett K, Clancy L. Lung cancer and urban air-pollution in Dublin: a temporal association? Ir Med J. 2007; 100(2): 367–369. PMID: 17432813 10. Catelinois O, Rogel A, Laurier D, Billon S, Hemon D, Verger P, Tirmarche M. Lung cancer attributable to indoor radon exposure in France: impact of the risk models and uncertainty analysis. Environ Health Perspect. 2006; 114(9):1361–1366. PMID: 16966089 11. Khazir J, Mir BA, Mir SA, Cowan D. Natural products as lead compounds in drug discovery. J Asian Nat Prod Res. 2013; 15(7):764–788. 12. Batugal PA, Kanniah J, Sy L, Oliver JT. Medicinal Plants Research in Asia-Volume I: The Framework and Project Workplans: Bioversity International; 2004. 13. Stark R. The book of aphrodisiacs: Stein and Day; 1981. 15 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0135995 August 19, 2015 L. flavescens Induces Cell Death in Lung Carcinoma Cells 14. Demuner AJ, Almeida Barbosa LC, Gonçalves Magalhaes C, Da Silva CJ, Alvares Maltha CR, Lelis Pinheiro A. Seasonal variation in the chemical composition and antimicrobial activity of volatile oils of three species of Leptospermum (Myrtaceae) grown in Brazil. References Molecules. 2011; 16(2):1181–1191. doi: 10.3390/molecules16021181 PMID: 21270734 15. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983; 65(1):55–63. 16. Ribble D, Goldstein NB, Norris DA, Shellman YG. A simple technique for quantifying apoptosis in 96- well plates. BMC Biotechnol. 2005; 5(1):12. 17. Van de Loosdrecht A, Beelen R, Ossenkoppele G, Broekhoven M, Langenhuijsen M. A tetrazolium- based colorimetric MTT assay to quantitate human monocyte mediated cytotoxicity against leukemic cells from cell lines and patients with acute myeloid leukemia. J Immunol Methods. 1994; 174 (1):311–320. 18. King RJB, Robins MW. Cancer biology: Pearson Education; 2006. 19. Mahavorasirikul W, Viyanant V, Chaijaroenkul W, Itharat A, Na-Bangchang K. Cytotoxic activity of Thai medicinal plants against human cholangiocarcinoma, laryngeal and hepatocarcinoma cells in vitro. BMC Complement Altern Med. 2010; 10(1):55. 20. Baskić D, Popović S, Ristić P, Arsenijević NN. Analysis of cycloheximide-induced apoptosis in human leukocytes: fluorescence microscopy using annexin V/propidium iodide versus acridin orange/ethidium bromide. Cell Biol Int. 2006; 30(11):924–932. PMID: 16895761 21. Ariffin SZ, Omar WW, Ariffin ZZ, Safian MF, Senafi S, Wahab RMA. Intrinsic anticarcinogenic effects of Piper sarmentosum ethanolic extract on a human hepatoma cell line. Cancer Cell Int. 2009; 9 (6):10.1186. 22. Sancho-Martinez SM, Piedrafita FJ, Cannata-Andia JB, Lopez-Novoa JM, Lopez-Hernandez FJ. Necrotic concentrations of cisplatin activate the apoptotic machinery but inhibit effector caspases and interfere with the execution of apoptosis. Toxicol Sci. 2011; 122(1):73–85. doi: 10.1093/toxsci/kfr098 PMID: 21527773 23. Vermes I, Haanen C, Reutelingsperger C. Flow cytometry of apoptotic cell death. J Immunol Methods. 2000; 243(1):167–190. 24. Kajstura M, Halicka HD, Pryjma J, Darzynkiewicz Z. Discontinuous fragmentation of nuclear DNA dur- ing apoptosis revealed by discrete “sub-G1” peaks on DNA content histograms. Cytometry A. 2007; 71 (3):125–131. PMID: 17252584 25. Arends M, Morris R, Wyllie A. Apoptosis. The role of the endonuclease. Am J Pathol. 1990; 136(3):593. PMID: 2156431 26. Higgins CF. Flip-flop: The transmembrane translocation of lipids. Cell. 1994; 79(3):393–395. PMID: 7954806 27. Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL, Henson PM. Exposure of phosphatidyl- serine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macro- phages. J Immunol. 1992; 148(7):2207–2216. PMID: 1545126 28. Porter AG, Jänicke RU. Emerging roles of caspase-3 in apoptosis. Cell Death Differ. 1999; 6(2): 99– 104. PMID: 10200555 29. Parrish AB, Freel CD, Kornbluth S. Cellular mechanisms controlling caspase activation and function. PLOS ONE \| DOI:10.1371/journal.pone.0135995 August 19, 2015 References Cold Spring Harb Perspect Biol. 2013; 5(6):a008672. doi: 10.1101/cshperspect.a008672 PMID: 23732469 30. Cullen SP, Martin SJ. Caspase activation pathways: some recent progress. Cell Death Differ. 1999; 16 (7): 935–938. 31. Jeruc J,Vizjak A, Rozman B, Ferluga D. Immunohistochemical expression of activated caspase-3 as a marker of apoptosis in glomeruli of human lupus nephritis. Am J Kidney Dis. 2006; 48(3): 410–418. PMID: 16931214 32. Hseu Y-C, Lee C-C, Chen Y-C, Senthil Kumar KJ, Chen C-S, Huang Y-C, et al. The anti-tumor activity of Antrodia salmonea in human promyelocytic leukemia (HL-60) cells is mediated via the induction of G1 cell-cycle arrest and apoptosis in vitro or in vivo. J Ethnopharmacol. 2014; 153(2):499–510. doi: 10. 1016/j.jep.2014.03.012 PMID: 24631961 33. Song G, Luo Q, Qin J, Wang L, Shi Y, Sun C. Effects of oxymatrine on proliferation and apoptosis in human hepatoma cells. Colloids Surf B Biointerfaces. 2006; 48(1):1–5. PMID: 16458489 34. Li L, Dai H-J, Ye M, Wang S-L, Xiao X-J, Zheng J, c Lycorine induces cell-cycle arrest in the G0/G1 phase in K562 cells via HDAC inhibition. Cancer Cell Int. 2012; 12(1):49. doi: 10.1186/1475-2867-12- 49 PMID: 23176676 16 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0135995 August 19, 2015
https://openalex.org/W4391326423	https://bmjpaedsopen.bmj.com/content/bmjpo/8/1/e002352.full.pdf	English	null	Mental Health Admissions to Paediatric Wards Study (MAPS): a protocol for the analysis of Hospital Episode Statistics (HES) data	BMJ paediatrics open	2,024	cc-by	4,302	To cite: Hudson LD, Ward J, Vázquez-Vázquez A, et al. Mental Health Admissions to Paediatric Wards Study (MAPS): a protocol for the analysis of Hospital Episode Statistics (HES) data. BMJ Paediatrics Open 2024;8:e002352. doi:10.1136/ bmjpo-2023-002352 Mental Health Admissions to Paediatric Wards Study (MAPS): a protocol for the analysis of Hospital Episode Statistics (HES) data ublished as 10.1136/bmjpo-2023-002352 on 29 January 2024. Downloaded from https://bmjpaedsopen.bmj.com on 24 October 2024 by guest. Protected by copyright. Lee Duncan Hudson,1,2 Joseph Ward,1 Adriana Vázquez-Vázquez ‍ ‍ ,1 Kate Settle,1 Francesca Cornaglia,3 Faith Gibson,1,2,4 Kirsty Phillips,1 Gabrielle Mathews,5 Helen Roberts,1 Damian Roland ‍ ‍ ,6,7 Dasha E Nicholls,8 Holly Elphinstone,1 Russell Viner ‍ ‍ 1 Lee Duncan Hudson,1,2 Joseph Ward,1 Adriana Vázquez-Vázquez ‍ ‍ ,1 Kate Settle,1 Francesca Cornaglia,3 Faith Gibson,1,2,4 Kirsty Phillips,1 , g , , y p , Gabrielle Mathews,5 Helen Roberts,1 Damian Roland ‍ ‍ ,6,7 Dasha E Nicholls,8 Holly Elphinstone,1 Russell Viner ‍ ‍ 1 ABSTRACT Introduction Children and young people (CYP) presenting with a mental health (MH) crisis are frequently admitted to general acute paediatric wards as a place of safety. Prior to the pandemic, a survey in England showed that CYP occupied 6% of general paediatric inpatient beds due to an MH crisis, and there have been longstanding concerns about the quality of care to support these patients in this setting. Mental Health Admissions to Paediatric Wards Study aims to generate a theory of change (ToC) model to improve the quality of care for CYP admitted to acute paediatric services after presenting in a MH crisis. M h d d l i 52 on 29 January 2024. Downloaded from https://bmjpaedsopen.bmj.com on 24 October 2024 by guest. copyright. ⇒HCPs from children’s wards are reporting that they are finding supporting CYP admitted with MH prob- lems challenging. ⇒HCPs from children’s wards are reporting that they are finding supporting CYP admitted with MH prob- lems challenging. HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY Ethics and dissemination WP1 received ethical approval (Ref 23/NW/0192). We will publish the overall synthesis of data and the final ToC to improve care of CYP with MH crisis admitted to general acute paediatric settings. As coproducers of the ToC, we will work with our stakeholder group to ensure wide dissemination of findings. Potential impacts will be on service development, new models of care, training and workforce planning. ⇒By producing a ToC approach, we expect to gener- ate a system map to identify transformation plans to share with policymakers, commissioners, service leads, and professionals. ⇒By producing a ToC approach, we expect to gener- ate a system map to identify transformation plans to share with policymakers, commissioners, service leads, and professionals. ⇒Our data and outputs will enable advocating for and improving cultural views on CYP with MH crises as part of the acute paediatric system. Received 23 October 2023 Accepted 25 November 2023 For numbered affiliations see end of article. © Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY. Published by BMJ. INTRODUCTION with MH problems was one of the main chal- lenges for acute children’s services.3 Open access Open access Protocol BMJ Paediatrics Open: first published as 10.1136/bmjpo-2023-002352 on 29 January 2024. Do copyright BMJ Paediatrics Open: first published as 10.1136/bmjpo-2023-002352 on 29 January 2024. Downloaded from https://bmjpaedsopen.bmj.com on 24 October 2024 by gue copyright. BMJ Paediatrics Open: first published as 10.113 BMJ Paediatrics Open: first published as 10.1136/bmjpo-2023-0 WHAT IS ALREADY KNOWN ON THIS TOPIC ⇒There is evidence that both the number of paediatric admissions and the severity of MH crisis in CYP have increased. WHAT THIS STUDY HOPES TO ADD Received 23 October 2023 Accepted 25 November 2023 Received 23 October 2023 Accepted 25 November 2023 ⇒This study will provide a comprehensive national analysis describing trends and characteristics of acute admissions due to MH problems in CYP. ⇒This study will provide the financial burden asso- ciated with MH admissions and an analysis of the community MH support and the local burden of admissions. Methods and analysis We will undertake a national (England), sequential, mixed methods study to inform a ToC framework alongside a stakeholder group consisting of patients, families/carers and healthcare professionals (HCPs). Our study consists of four work packages (WP) undertaken over 30 months. WP1 is limited to using national routine administrative data to identify and characterise trends in MH admissions in acute paediatric wards in England between 2015– 2022. ded from https://bmjpaedsopen.bmj.com on 24 October 2024 by guest. Protected by ⇒This study will generate a ToC model to positively impact on quality of care for CYP who are admitted to paediatric service because of a MH crisis. Open access BMJ Paediatrics Open: first published as 10.1136/bmjpo-2023-002352 on 29 January 2024. Downloaded from https://bmjpaedsopen.bmj.com on 24 October 2024 by guest. Protected by copyright. METHODS AND ANALYSIS Objectives The analysis of routine administrative data will be highly informative regarding national trends and characteristics of CYP admitted. The aims are to: The analysis of routine administrative data will be highly informative regarding national trends and characteristics Overall, MH admissions to acute paediatric wards are a long-standing issue that has been identified as a leading safety and quality concern for acute paediatric providers for some years.1 3 The Mental Health Admissions to Paedi- atric Wards Study aims to generate a theory of change (ToC) model to improve the quality of care for CYP admitted to acute paediatric services after presenting in an MH crisis. Our study consists of four work packages. Here, we describe work package 1 (WP1), which is limited to using national routine administrative data to identify and characterise trends in MH admissions in acute paedi- atric wards in England between 2015 and 2022. WP2 and 3 were described separately.10 y g informative regarding national trends and characteristi of CYP admitted. The aims are to: of CYP admitted. The aims are to: of CYP admitted. The aims are to: 1. Describe trends in MH admissions to acute inpatient services, attendances to accident & emergency (A&E) and outpatient appointments related to MH within England 2015–2022 (or the latest available data) in CYP aged 5–18. 29 January 2024. Downloaded from https://bmjpaedsopen.bmj.com on 24 October 2024 by guest. Protecte opyright. g 2. Understand the financial burden of MH admissions to acute paediatric wards among CYP aged 5–18 in En- gland. 3. Examine the role of primary and community health- care services in England. METHODS AND ANALYSIS inpatient Child and Adolescent Mental Health Services (CAMHS) services were not accessible, and during the third wave, admissions to acute wards appeared to peak.7 This mismatch of greater distress and reduced access led to increases in already unmet needs.8 We will use a ToC approach as our framework, which uses logic (quantitative) data and coproduction (qualitative) data to map change. This approach has been applied to a range of areas of health and social care improvement settings.11–13 There is a striking lack of information available to guide care and service delivery for the rapidly growing issue of CYP with MH problems being admitted and managed on acute paediatric wards. Evidence on interventions to avoid inpatient admissions for CYP presenting in MH crisis is poor and limited,9 meaning that CYP are likely to continue to need to be admitted in crisis, with paediatric wards a commonplace while awaiting assessment given the lack of direct access to a specialist MH ward form most CYP. We will undertake a national, sequential, mixed methods study to inform our ToC framework alongside a stakeholder group consisting of patients, families/carers and HCPs. Three work packages will deliver the types of evidence needed to inform our ToC (figure 1). Here we describe the study design for WP1. Children and young people (CYP) presenting with a mental health (MH) crisis are frequently admitted to general acute paediatric wards as a place of safety, despite not always having the resources or training.1 2 Before the pandemic, a survey carried out in 2019 with 60% of acute paediatric services in England found that 6% of general paediatric beds were occupied by CYP with MH problems.3 Moreover, data from London suggest that the management of CYP Children and young people (CYP) presenting with a mental health (MH) crisis are frequently admitted to general acute paediatric wards as a place of safety, despite not always having the resources or training.1 2 Before the pandemic, a survey carried out in 2019 with 60% of acute paediatric services in England found that 6% of general paediatric beds were occupied by CYP with MH problems.3 Moreover, data from London suggest that the management of CYP For numbered affiliations see end of article. © Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY. Published by BMJ. g The rise in MH problems among CYP during the COVID-19 pandemic has been also well described.4 5 Recent national data report that being at high risk of MH problems rose from 1 in 9 in 2017 to 1 in 6 by 2021, with a doubling of the proportion of CYP at risk of eating problems over that same period.6 During the first wave, acute services became ‘default providers’ where community or Correspondence to Dr Lee Duncan Hudson; l. hudson@ucl.ac.uk Correspondence to Dr Lee Duncan Hudson; l. hudson@ucl.ac.uk Hudson LD, et al. BMJ Paediatrics Open 2024;8:e002352. doi:10.1136/bmjpo-2023-002352 1 BMJ Paediatrics Open: first published as 10.1136/bmjpo-2023-002352 on 29 January 2024. Do copyright BMJ Paediatrics Open: first published as 10.1136/bmjpo-2023-002352 on 29 January 2024. Downloaded from https://bmjpaedsopen.b copyright. To do this, we will need to analyse secondary inpa- tient and outpatient MH care activity (held within MHDS and HES OPC), attendances to A&E due to MH concerns (data held within ECDS) and MH care activity within acute inpatient services (held within HES APC). We will describe trends over time, by age group, sex, markers of deprivation, prior MH healthcare activity and comorbid physical health problems and analyse geographic varia- tion in MH healthcare activity within England. p 4. Diagnostic coding within MHDS to identify MH outpa- tient activity not identified within HES OPC. We will link, clean and collapse data using established protocols, we have developed for HES analyses. We will then describe: We will link, clean and collapse data using established protocols, we have developed for HES analyses. We will then describe: 1. Burden and trends in healthcare activity related to MH disorders by sex, ethnicity, level of deprivation and geographic region, and examine variation at Trust level where numbers allow. 2. Burden and trends in acuity of MH-related inpatient admissions. This will be examined by examining re- peat admissions and readmissions and the numbers of admissions under the MH act. In addition to sociodemographic characteristics of CYP attending secondary healthcare due to MH concerns, we will describe prior attendance to A&E (data held within HEE ECDS and HES A+E), prior admissions to acute paediatric services (data held within HES APC), prior outpatient MH activity (data held within MHDS and HES OPC) and prior MH inpatient activity (data held within MHDS). 3. Burden and trends in admission source and discharge destination of healthcare activity related to MH disor- ders (ie, MH ‘tier 4’ inpatients unit, criminal justice system, etc). m https://bmjpaedsopen.bmj.com on 24 October 2024 by guest. Protected by BMJ Paediatrics Open: first published as 10.1136/bmjpo-2023-002352 on 29 January 2024. Downloaded from https://bmjpaedsopen.b copyright. We will identify healthcare activity (inpatient, outpa- tient, A&E) related to MH and physical disorders as follows: attendances for primary MH disorders in England for the 5 years 2017 to 2021/22 (or the latest available data). y The data subjects for this project are all CYP aged 5–18 who have been admitted to hospital, attended A&E or had planned outpatient activity for any cause (physical or MH complaints) in England between 2017 and 2022 (or the latest available data). 1. Primary diagnostic ICD-10 codes (DIAG_1) relating to mental and physical health disorders and provider codes for treating consultant (TREATSPEF and MAINSPEF) within finished consultant episodes within HES APC. 1. Primary diagnostic ICD-10 codes (DIAG_1) relating to mental and physical health disorders and provider codes for treating consultant (TREATSPEF and MAINSPEF) within finished consultant episodes within HES APC. p 2. Provider codes for treating consultant (TREATSPEF and MAINSPEF) for planned outpatient activity relat- ing to mental and physical health specialty within HES OPC. We will purchase pseudo-anonymised individual-level data from the following data sets: HES admitted patient care (APC), HES outpatient care (OPC), HES A&E, HES Emergency Care Dataset (ECDS) and Mental Health Dataset (MHDS). 3. ECDS codes for MH presentations linked to a same- day acute admission with a primary diagnosis consis- tent with a mental or physical health presentation. This approach is only possible for 2018 onwards and cannot be done for pre-2018 ED data as reasons for presentation are not available. These data will allow the objectives described above to be achieved as follows: Objective 1 of this project is to describe trends in health- care activity related to MH problems in secondary care in England between 2017 and 2022 (or the latest available data). To do this, we will need to analyse secondary inpa- tient and outpatient MH care activity (held within MHDS and HES OPC), attendances to A&E due to MH concerns (data held within ECDS) and MH care activity within acute inpatient services (held within HES APC). We will describe trends over time, by age group, sex, markers of deprivation, prior MH healthcare activity and comorbid physical health problems and analyse geographic varia- tion in MH healthcare activity within England. Objective 1 of this project is to describe trends in health- care activity related to MH problems in secondary care in England between 2017 and 2022 (or the latest available data). BMJ Paediatrics Open: first published as 10.1136/bmjpo-2023-002352 on 29 January 2024. Do copyright Open access BMJ Paediatrics Open: first published as 10.1136/bmjpo-2023-002352 on 29 January 2024. Downloaded from https://bmjpaedsopen.bmj.com on 24 October 2024 by guest. Protected by copyright. Study design We will use National Health Service (NHS) administra- tive data to characterise acute hospital admissions, outpa- tient attendances and emergency department (ED) m https://bmjpaedsopen.bmj.com on 24 October 2024 by guest. Protected by Figure 1 WP1 data flow diagram. CYP, Children and Young People; HES, Hospital Episodes Statistics; MH, mental health; NHS, National Health Service; UCL, University College London. ttps://bmjpaedsopen.bmj.com on 24 October 2024 by guest. Protected by Figure 1 WP1 data flow diagram. CYP, Children and Young People; HES, Hospital Episodes Statistics; MH, mental health; NHS, National Health Service; UCL, University College London. Hudson LD, et al. BMJ Paediatrics Open 2024;8:e002352. doi:10.1136/bmjpo-2023-002352 2 Data protection This study is registered under reference No Z6364106/2023/03/96 health research in line with UCL’s Data Protection Policy. This study is registered under reference No Z6364106/2023/03/96 health research in line with UCL’s Data Protection Policy. NHS England data will be stored and analysed entirely within the UCL data safe haven (DSH), which has been certified to the ISO27001 information security standard and conforms to the NHS Information Governance Toolkit. Access to these data will be limited to those UCL employees contributing to this project. Data will be kept within the European Economic Area. The data will be encrypted for transfer, and information compliance training for information security, freedom of information and data protection will be completed by all staff who have access to the data. Data will be fully anonymised prior to the analysis and then extracted from UCL DSH after analysis. 5. Burden and trends in healthcare activity related to MH disorders associated with other medical condi- tions and comorbidities, identified through primary and secondary diagnostic coding within HES. We will particularly examine the common chronic conditions (diabetes, asthma, epilepsy) as well as use broader definitions of medical comorbidity. published as 10.1136/bmjpo-2023-002352 on 29 January 2024. Downloaded from https://bmjpaedsopen.bmj.com on 24 October 2024 by guest. Protected by copyright. 6. Burden and trends in healthcare activity related to non-MH disorders within CYP identified as having healthcare activity related to MH disorders, including eating disorders, anxiety, depression and psychotic dis- orders—grouping used to define MH disorder to be determined. The data will not be linked with any record-level data. There will be no requirement nor attempt to reidentify individuals from the data. The data will not be made avail- able to any third parties other than those specified except in the form of aggregated outputs with small numbers suppressed in line with the HES analysis guide. Objective 2 of this study seeks to understand the financial impact of admissions to acute inpatient services due to MH problems. To do this, we need to first identify and group all hospitalisations in England in CYP due to MH concerns in acute inpatient services (objective 1). We will then use data within the National Cost Collection for NHS to estimate the financial burden associated with admitting CYP to an acute hospital inpatient unit for all causes and those related to MH admissions. Open access 4. Burden and trends in healthcare activity related to MH disorders by MH diagnosis (note diagnostic cod- ing within outpatients is limited). This will be limited to large groups, for example, eating disorder admis- sions, anxiety and depression, psychotic disorders— grouping to be determined. Figure 2 MAPS work package flow diagram. CAG, Confidentially Advisory Group; CYP, Children and Young People; ED, emergency department; HCP, Health Care Professionals; MAPS, Mental Health Admissions to Paediatric Wards Study; MH, mental health; ToC, theory of change; PPI, patient and public involvement. mjpaedsopen.bmj.com on 24 October 2024 by guest. Protected by Figure 2 MAPS work package flow diagram. CAG, Confidentially Advisory Group; CYP, Children and Young People; ED, emergency department; HCP, Health Care Professionals; MAPS, Mental Health Admissions to Paediatric Wards Study; MH, mental health; ToC, theory of change; PPI, patient and public involvement. Figure 2 MAPS work package flow diagram. CAG, Confidentially Advisory Group; CYP, Children and Young People; ED, emergency department; HCP, Health Care Professionals; MAPS, Mental Health Admissions to Paediatric Wards Study; MH, mental health; ToC, theory of change; PPI, patient and public involvement. 3 Hudson LD, et al. BMJ Paediatrics Open 2024;8:e002352. doi:10.1136/bmjpo-2023-002352 DISSEMINATION See WP4 of the study in figure 1. As coproducers of the ToC, we will work with our stakeholder group to ensure wide dissemination of findings to effect change. Potential impacts will be on service development, new models of care, training and workforce planning. RESEARCH ETHICS APPROVAL This study was approved by Northwest—Preston Research Ethics Committee (Ref 23/NW/0192). We will not directly inform each data subject of the specific data transfer. Information regarding data transfer will be on our project’s website for the public to see (https:// www.ucl.ac.uk/child-health/research/population-policy- and-practice-research-and-teaching-department/mental- health-admissions). We will be transparent with what data will be included and how it will be transferred, stored and analysed. Confidentiality advisory group approval was not required because the data requested are classified as pseudonymised data. aded from https://bmjpaedsopen.bmj.com on 24 October 2024 by guest. Protected by y p g Objective 3 of this study seeks to examine the role of primary and community healthcare services both before and after admission of CYP with MH conditions to and from paediatric wards. We will seek to use quality of community MH provision as a predictor for regional variation in the numbers of CYP admitted to acute inpatient units related to MH crises. We will do this by establishing the quality of community MH service provision using publicly available data, including staffing levels within general practice, and quality outcome framework indicators related to MH. We will also use data within the MHDS to assess community provi- sion of MH services, including proxy indicators including wait time from referral to the first appointment. For this indi- cator, we require data held within MHDS. 2352 on 29 January 2024. Downloaded from https://bmjpaedsopen.bmj.com on 24 October 2024 by guest. copyright. A summary of our study procedure and lawful basis under which we are processing the data is seen in figure 2. Patient We presented our research proposal to members of Think- 4Brum, which is the youth advisory group for Forward Thinking Birmingham, and the GOSH Young Persons’ Advi- sory Group for research as part of a PPIE initiative. Focus groups of 40 young people (aged <18) and parents were held to discuss the acceptability of the methods and the use of HES data without consent. Feedback from these groups showed CYP believe this is an important area to research and the use of patient-identifiable data without consent is accept- able within this study. Author affiliations 1Population, Policy and Practice Research Programme, UCL Great Ormond Street Institute of Child Health, London, UK 2Great Ormond Street Hospital for Children NHS Trust, London, UK 3Queen Mary University of London, London, UK 4University of Surrey, Guildford, UK 5CYP Transformation Team, NHS England and NHS Improvement London, London, UK 6SAPPHIRE Group, Population Health Sciences, Leicester University, Leicester, UK 7Paediatric Emergency Medicine Leicester Academic (PEMLA) Group, Children's Emergency Department, Leicester Royal Infirmary, Leicester, UK Author affiliations 1Population, Policy and Practice Research Programme, UCL Great Ormond Street Institute of Child Health, London, UK 2Great Ormond Street Hospital for Children NHS Trust, London, UK 3Queen Mary University of London, London, UK 4University of Surrey, Guildford, UK 5CYP Transformation Team, NHS England and NHS Improvement London, London, UK 6SAPPHIRE Group, Population Health Sciences, Leicester University, Leicester, UK 7Paediatric Emergency Medicine Leicester Academic (PEMLA) Group, Children's Emergency Department, Leicester Royal Infirmary, Leicester, UK Data protection We will then extrapolate this cost to a national estimate and describe changes in the financial burden of MH admission to acute inpatient paediatric services over time, and this varies by different parts of England. ORCID iDs Adriana Vázquez-Vázquez http://orcid.org/0000-0002-9269-741X Damian Roland http://orcid.org/0000-0001-9334-5144 Russell Viner http://orcid.org/0000-0003-3047-2247 11 Wilkinson H, Hills D, Penn A, et al. Building a system-based theory of change using Participatory systems mapping. Evaluation 2021;27:80–101. 12 Maini R, Mounier-Jack NS, Borghi J. How to and how not to develop a theory of change to evaluate a complex intervention-reflections on an experience in the Democratic Republic of Congo BMJ. BMJ Glob Health 2018;3:e000617. Provenance and peer review Not commissioned; internally peer-reviewed. Provenance and peer review Not commissioned; internally peer-reviewed. Open access This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See: https://creativecommons.org/ licenses/by/4.0/. 8 Dubicka B, Bullock T. Mental health services for children fail to meet soaring demand. BMJ 2017:j4254. 9 Clisu DA, Layther I, Dover D, et al. Alternatives to mental health admissions for children and adolescents experiencing mental health crises: A systematic review of the literature. Clin Child Psychol Psychiatry 2022;27:35–60. 23-002352 on 29 January 2024. Downloaded from https://bmjpaedsopen.bmj.com on 24 October 2024 by copyright. 10 Hudson L, Vázquez-Vázquez A, Gibson F, et al. n.d. Mental health admissions to Paediatric wards study (MAPS): protocol of a prospective study of mental health admissions to Paediatric wards in England using surveillance and qualitative methods. BMJ Paediatr Open 8Division of Psychiatry, Imperial College London, London, UK 8Division of Psychiatry, Imperial College London, London, UK 2 Review of acute care services for children and young people: healthy London partnership, London. 2017. Available: https:// www.healthylondon.org/resource/peer-reviewacute-care-services- children-young-people/ 2 Review of acute care services for children and young people: healthy London partnership, London. 2017. Available: https:// www.healthylondon.org/resource/peer-reviewacute-care-services- children-young-people/ Twitter Damian Roland @damian_roland Twitter Damian Roland @damian_roland Contributors RV, JW, FC and LH were major contributors to the development of the study protocol. AV-V drafted the manuscript. All authors contributed to revising the manuscript and approved the final version. 3 Royal College of Paediatrics and Child Health. A Snapshot of general Paediatric services and workforce in the UK. 2020. g 4 Ford T, John A, Gunnell D. Mental health of children and young people during pandemic. BMJ 2021;372:614. people during pandemic. BMJ 2021;372:614. Funding This study is supported by the National Institute for Health and Care Research (NIHR) grant/award number 135036. 5 Samji H, Wu J, Ladak A, et al. Review: mental health impacts of the COVID-19 pandemic on children and youth - a systematic review. Child Adolesc Ment Health 2022;27:173–89. Competing interests None declared. Competing interests None declared. 6 NHS Digitial. Mental health of children and young people in England, 2021: wave 2 follow up to the 2017 survey: NHS Digital, health and social care information centre. 2021. Available: https://files.digital. nhs.uk/97/B09EF8/mhcyp_2021_rep.pdf Patient and public involvement Patients and/or the public were involved in the design, or conduct, or reporting, or dissemination plans of this research. Refer to the Methods section for further details. nhs.uk/97/B09EF8/mhcyp_2021_rep.pdf 7 Hudson LD, Chapman S, Street KN, et al. Increased admissions to Paediatric wards with a primary mental health diagnosis: results of a survey of a network of eating disorder Paediatricians in England. Arch Dis Child 2022;107:309–10. 10.1136/archdischild-2021-322700 Available: https://doi.org/10.1136/archdischild-2021-322700 Patient consent for publication Not applicable. Patient consent for publication Not applicable. Open access Author affiliations 1 3Queen Mary University of London, London, UK 4University of Surrey, Guildford, UK 7Paediatric Emergency Medicine Leicester Academic (PEMLA) Group, Children's Emergency Department, Leicester Royal Infirmary, Leicester, UK g y ( ) p, Emergency Department, Leicester Royal Infirmary, Leicester, UK 4 Hudson LD, et al. BMJ Paediatrics Open 2024;8:e002352. doi:10.1136/bmjpo-2023-002352 Open access BMJ Paediatrics Open: first published as 10.1136/bmjpo-2023-002352 on 29 January 2024. Downloaded from https://bmjpaedsopen.bmj.com on 24 October 2024 by guest. Protected by copyright. REFERENCES 1 Royal College of Paediatrics and Child Health. Role of Paediatricians in supporting children and young people’s mental health – position statement. 2020. Available: https://www.rcpch.ac.uk/resources/role- paediatricians-supporting-children-youngpeoples-mental-health- position-statement 13 Breuer E, Lee L, De Silva M, et al. Using theory of change to design and evaluate public health interventions-a systematic review implementation science 11:63. Implement Sci 2016;11:63. Downloaded from https://bmjpaedsopen.bmj.com on 24 October 2024 by guest. Protected by Hudson LD, et al. BMJ Paediatrics Open 2024;8:e002352. doi:10.1136/bmjpo-2023-002352 5
https://openalex.org/W2592378268	https://europepmc.org/articles/pmc5345261?pdf=render	English	null	Effects of cold sensitivity in the extremities on circulating adiponectin levels and metabolic syndrome in women	BMC complementary and alternative medicine	2,017	cc-by	7,186	© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Park and Cha BMC Complementary and Alternative Medicine (2017) 17:150 DOI 10.1186/s12906-017-1658-7 Park and Cha BMC Complementary and Alternative Medicine (2017) 17:150 DOI 10.1186/s12906-017-1658-7 Open Access Open Access Effects of cold sensitivity in the extremities on circulating adiponectin levels and metabolic syndrome in women Ah Yeon Park and Seongwon Cha* Abstract Background: In adipose tissues, adipokine levels, including adiponectin and leptin, are involved in insulin sensitivity and are reciprocally induced by cold temperature stress. Thermogenic response in the extremities (hands and feet) against cold stress can be negatively related to fat mass accumulation, particularly in the abdomen. However, the relationship between the sensation of cold in the extremities and circulating levels of adipokines is not fully understood. Here, we investigated whether adipokine levels are associated with cold hypersensitivity in the hands and feet (CHHF), independent of body mass, and whether the CHHF is related to metabolic syndrome (MS). Methods: Associations of the CHHF with serum levels of adipokines and MS risk were evaluated in 1021 Koreans (372 men and 649 women), using a linear regression model while controlling for thermogenic factors and a logistic regression model, respectively. Results: The adiponectin levels were positively associated with the CHHF, particularly in women, irrespective of thermogenic factors, including body mass index (β = 1.23 μg/mL, 95% confidence interval [1.04–1.45]). Logistic regression analysis for MS risk via the CHHF showed that there was a significant inverse association in women (odds ratio = 0.449, 95% confidence interval [0.273–0.737]). Conclusions: In summary, our founding indicated that the CHHF could induce increased levels of circulating adiponectin and in turn reduce the MS risk in women. Despite complaints of feeling cold, these women could be at lower risk of cardiovascular disease. Keywords: Cold hypersensitivity in the hands and feet, High-molecular-weight adiponectin, Metabolic syndrome, Emotional cold stress, Extremities * Correspondence: scha@kiom.re.kr Mibyeong Research Center, Korea Institute of Oriental Medicine, 1672 Yuseongdae-ro, Yuseong-gu, Daejeon 34054, Republic of Korea Background remedies [particularly Korean red ginseng (KRG)] and acupuncture have been used to attempt to relieve hyper- sensitivity in the extremities [2, 3]. People exhibit differential cold sensitivity at the same environmental temperature, particularly in the extrem- ities, such as the hands and feet. Cold hypersensitivity in the hands and feet (CHHF), which can decrease quality of life, e.g., functional dyspepsia, is considered an im- portant factor in oriental medicine as a form of cold and heat pattern identification, in which the cold and heat refers to someone’s subjective feeling, as well as object- ive measures of body temperature in the context of warm or cool environments [1]. Treatments with herbal The thermogenic response to a homeothermic state in the extremities, e.g., cold temperature (or emotional stress) [4], varies according to an individual’s fat mass accumulation; hand temperature in obese individuals tends to be higher than that in normal weight individ- uals [5]. The skin temperature of the extremities, which is negatively correlated with that of the abdomen, can be affected by the abdominal heat retained under an insu- lating layer of subcutaneous fat since the core heat may be released through the extremities, in which the skin is not insulated by a substantial amount of fat [5]. In * Correspondence: scha@kiom.re.kr Mibyeong Research Center, Korea Institute of Oriental Medicine, 1672 Yuseongdae-ro, Yuseong-gu, Daejeon 34054, Republic of Korea Park and Cha BMC Complementary and Alternative Medicine (2017) 17:150 Page 2 of 9 density lipoprotein cholesterol (HDLC) of less than 40 mg/dL for men and less than 50 mg/dL for women; and (5) fasting blood glucose of 110 mg/dL or more or medication for hyperglycemia. addition, individuals with a higher body mass index (BMI) tend to have warmer hands, as shown in a twin study [6]. y Fat accumulation is associated with secretion of adipo- kines from adipose tissue, including increased leptin secretion and decreased adiponectin secretion [7]. These two adipokines affect thermogenesis during cold stress [8, 9]. That is, the adipose tissue is tightly connected with sympathetic nervous system activity through the response to cold-stress via reduced plasma levels of adi- ponectin (with no changes in leptin levels) [8, 10], whereas adipose tissue interacts with glucose metabol- ism during cold stress to facilitate diet-induced thermo- genesis and increase circulating adiponectin levels, which can be used in glucose metabolism (accompanied by decreased leptin levels) [9]. Background Thus, these findings sug- gest that adiponectin and leptin levels associated with fat accumulation may be related to cold sensation in the extremities. However, the relationship between CHHF and circulating adipokines has not been elucidated. Participants A total of 1282 participants (467 men and 815 women) were recruited from 20 oriental medicine clinics by the Korea medicine Data Center (KDC) from 2007 to 2009. All participants provided written informed consent to participate in the study, and the study was approved by the Institutional Review Board of the Korea Institute of Oriental Medicine. Participants with a history of cancer treatment, hypertension medication, diabetes medication, dyslipidemia medication, and/or unknown menopausal status were excluded. MS was defined according to the modified guidelines of the National Cholesterol Educa- tion Program Adult Treatment Panel III (NCEP ATP III) [15], which stipulated that at least three of the following five criteria had to be met: (1) abdominal obesity with a waist circumference of 90 cm or more for men and 80 cm or more for women; (2) systolic blood pressure of 130 mmHg or more, diastolic blood pressure (DBP) of 85 mmHg or more, or medication for hypertension; (3) triglycerides (TGs) of 150 mg/dL or more; (4) high- Anthropometric factors and biochemical analyses Anthropometric factors and biochemical analyses Blood pressure was measured manually at the upper part of left arm after sufficient relaxation using a standard sphygmomanometer. Waist-to-hip ratio (WHR) was de- fined as the waist circumference divided by the hip cir- cumference. Circumferences of the waist and hip were measured horizontally with a tape measure to the near- est 1 mm at the umbilical level and upper margin of the pubis, respectively. Blood samples were drawn from the participants in the morning after overnight fasting for at least 8 h. Biochemical analyses for glucose, TGs, and HDLC were performed by Seoul Clinical Laboratories (SCL, Seoul, Republic of Korea) based on standardized protocols (ADVIA1800; Siemens, USA). Serum samples were stored at −70 °C until analysis. Classification according to the sensation of cold in the extremities The sensation of cold in both hands and feet was assessed using a questionnaire that asked the partici- pants to rate the usual temperature of their extremities as warm, neutral, cold, and unknown. The questionnaire for CHHF has previously been used in heritability esti- mation with twin subjects and in the association with functional dyspepsia [1, 6]. Additionally, the reliability (a correlation coefficient of 0.609 via test-retest) and valid- ity (74.5% agreement and 0.487 kappa value, compared to a professional’s examination) of a seven item ques- tionnaire on cold and heat pattern identification, which includes the CHHF questionnaire, has been assessed [16]. The participants were then grouped by their re- sponses as follows: the non-CHHF group consisted of participants who felt warm in both their hands and feet; the CHHF group consisted of participants who felt cold in both their hands and feet; and the inter- mediate group consisted of participants who felt neu- tral in either their hands or feet or in both. The 118 participants who could not be classified into one of these three groups were excluded from the following analyses (final cohort: n = 1164, including 420 men and 744 women). Therefore, in this study, we hypothesized that adipo- kines, such as adiponectin and leptin (probably from ab- dominal fat), may be associated with CHHF because the abdominal fat is inversely related to warm temperature of extremities. Because CHHF is more common in women [6, 11] and is probably affected by body mass, the relationship was also assessed after controlling for sex and BMI. In addition, because the leptin to adipo- nectin ratio (LAR) and high-molecular-weight (HMW) adiponectin have been suggested to indicate insulin re- sistance and metabolic syndrome (MS) [12–14], we also examined the association between CHHF and MS. Statistical analysis Kruskal-Wallis tests were performed to compare the clinical characteristics between the three cold sensation groups. Linear regression analyses were performed to es- timate adiponectin levels (ln-transformed) versus CHHF (referenced by non-CHHF) according to the following adjustment models for confounding variables: Model 1 - Determination of serum adipokine levels Serum leptin (ng/mL) was measured by Seoul Clinical La- boratories using a radioimmunoassay method with 125I and double antibodies. Of the various isoforms of circulat- ing adiponectin, the HMW form is considered the most clinically relevant [17]. Therefore, the serum concentra- tion of HMW adiponectin (μg/mL) was determined using Park and Cha BMC Complementary and Alternative Medicine (2017) 17:150 Park and Cha BMC Complementary and Alternative Medicine (2017) 17:150 Page 3 of 9 Page 3 of 9 adjusted for age and/or sex (in women, menopause sta- tus); Model 2 - adjusted for model 1 covariates as well as diastolic blood pressure (DBP) and TGs (ln-trans- formed); and Model 3 - adjusted for Model 2 covariates as well as BMI. Logistic regression analyses were per- formed to estimate odds ratios (ORs) for MS and the five MS components versus the CHHF by adjustment for age, sex, and menopausal status. Subgroup analysis was performed for both linear and logistic regression analyses after dividing women by the median value of WHR. Results with a p value of less than 0.05 were considered significant. All statistical analyses were performed using R version 3.0.2 software (http://www.r- project.org/). a Quantikine Human HMW adiponectin/Acrp30 im- munoassay kit (R&D Systems, Minneapolis, MN, USA). This kit uses a quantitative sandwich enzyme immuno- assay technique to measure total HMW adiponectin con- centrations. Each serum sample was analyzed in duplicate (intra-assay coefficient of variation < 10% for all assays), and 62 participants were excluded who had more than a two-fold difference between two repeated measurements (final cohort: n = 1102; 396 men and 706 women). In addition, 81 participants with blank data (all leptin) were excluded. A total of 1021 participants were included in the final statistical analyses (372 men and 649 women). n-CHHF (cold hypersensitivity in the hands and feet): people feel that both hands and feet are warm Relationship between sensitivity to cold and MS On the basis of the association of HMW adiponectin with MS [14], our results provided evidence for the relationship between CHHF and HMW adiponectin. Therefore, we performed logistic regression analyses of the CHHF for MS and five MS components. The CHHF was associated protectively with MS (OR = 0.465, p = 1.01 × 10-4) and three components, i.e., low HDLC, high TGs, and large waist circumference (WC) (Table 4). After stratification according to sex, the association of the CHHF with MS was maintained in women, similar to the results for adipo- nectin (OR = 0.449, p = 1.54 × 10-3; Table 4). Among the five MS components, high TGs, high BP, and large WC were associated with CHHF in women. Interestingly, CHHF in men was associated with decreased large WC. These associational trends between CHHF and MS risk were consistent with the results presented in Table 1, except HDLC. A linear regression analysis was performed to deter- mine whether adipokine levels were associated with cold sensitivity after controlling for confounding factors that could influence thermogenesis (i.e., demographic factors [age and/or female menopausal status], vascular function [DBP], lipid fuel [TGs], and/or an anthropometric factor related to the accumulation of fat [BMI]). The associations of the CHHF (referenced by the non- CHHF) with adiponectin, leptin, and LAR are reported in Table 2. Adiponectin levels were associated with an increased sensitivity to cold, regardless of additional adjustments for DBP, TG, and BMI (Model 1: β = 1.33 μg/mL, p = 4.44 × 10-5; Model 2: β = 1.23 μg/mL, p = 3.28 × 10-3; Model 3: β = 1.18 μg/mL, p = 2.32 × 10−2), although the association was attenuated by the addition of confounding factors. When performing subgroup ana- lysis according to sex, the association for adiponectin levels was maintained and even enriched in women (Model 1: β = 1.40 μg/mL, p = 7.87 × 10-5; Model 2: β = 1.27 μg/mL, p = 3.17 × 10-3; Model 3: β = 1.23 μg/mL, p = 1.80 × 10-2). Characteristics of participants based on CHHF Characteristics of participants based on CHHF The characteristics of the 1021 participants classified into the non-CHHF, intermediate, and CHHF groups are presented in Table 1. Characteristics of participants based on CHHF Differences in cardiometabolic and Table 1 Characteristics of participants in the study Cold sensation in hands and feet Characteristics All non-CHHF Intermediate CHHF P value† Men N 372 122 173 77 Age (years) 48.9 (14.9) 49.7 (13.3) 48.8 (15.4) 47.6 (16.3) 0.774 Body mass index (kg/m2) 24.1 (3.46) 25.1 (3.08) 24.0 (3.24) 22.8 (4.01) 1.07 × 10-7 Waist circumference (cm) 87.3 (9.25) 90.4 (8.29) 87.2 (9.41) 82.8 (8.49) 1.31 × 10-7 Waist-to-hip ratio 0.933 (0.0618) 0.950 (0.0518) 0.933 (0.0627) 0.906 (0.0656) 1.31 × 10-5 Systolic blood pressure (mmHg) 124 (13.9) 123 (12.6) 123 (12.6) 124 (18.1) 0.992 Diastolic blood pressure (mmHg) 79.8 (10.4) 81.4 (10.3) 79.4 (9.16) 78.1 (12.8) 0.100 Fasting blood glucose (mg/dL) 104 (32.4) 104 (34.1) 104 (31.9) 101 (31.0) 0.254 Triglyceride (mg/dL) 147 (89.2) 151 (75.2) 145 (97.9) 144 (90.0) 0.109 HDL cholesterol (mg/dL) 42.4 (10.5) 41.0 (10.0) 42.6 (10.9) 44.0 (10.1) 0.0727 Women N 649 130 229 290 Age (years) 47.9 (15.8) 52.8 (16.4) 47.7 (16.1) 45.7 (14.7) 1.74 × 10-4 Body mass index (kg/m2) 23.1 (3.34) 24.5 (3.73) 23.6 (3.24) 22.0 (2.85) 2.07 × 10-14 Waist circumference (cm) 82.3 (9.93) 86.5 (10.2) 83.7 (9.39) 79.2 (9.27) 2.62 × 10-13 Waist-to-hip ratio 0.890 (0.0698) 0.917 (0.0686) 0.899 (0.0642) 0.870 (0.0693) 3.24 × 10-10 Systolic blood pressure (mmHg) 119 (14.9) 123 (14.3) 120 (15.0) 115 (14.3) 1.12 × 10-8 Diastolic blood pressure (mmHg) 76.1 (11.0) 79.7 (10.6) 77.2 (11.0) 73.6 (10.6) 2.51 × 10-8 Fasting blood glucose (mg/dL) 97.0 (23.7) 102 (34.7) 97.1 (22.9) 94.7 (17.0) 0.133 Triglyceride (mg/dL) 116 (75.3) 142 (91.9) 123 (74.8) 99.0 (62.3) 5.76 × 10-8 HDL cholesterol (mg/dL) 49.5 (11.9) 47.2 (12.2) 48.9 (11.3) 50.9 (12.1) 1.39 × 10-3 Values are presented as means (standard deviations) Non-CHHF (cold hypersensitivity in the hands and feet): people feel that both hands and feet are warm Intermediate: people feel that hands and/or feet are neutral CHHF: people feel that both hands and feet are cold †P value: Kruskal-Wallis test Table 1 Characteristics of participants in the study Park and Cha BMC Complementary and Alternative Medicine (2017) 17:150 Page 4 of 9 Page 4 of 9 Because the correlation between adipokine and cold sensitivity in the extremities could have been affected by differential adiposity among the three groups (non- CHHF, intermediate, and CHHF), the circulating adipo- kine levels were assessed in terms of abdominal body mass. Characteristics of participants based on CHHF The high- and low-WHR subgroups corresponded to women with a WHR higher or lower than the median value of WHR for the entire group, respectively. Although no associations were found between CHHF and adiponectin levels in low-WHR women, adiponectin levels were significantly different between the non- CHHF and CHHF groups in high-WHR women after controlling for DBP, TG, BMI, and menopause status (Table 3; β = 1.31 μg/mL, p = 2.00 × 10-2). These data showed that the levels of adiponectin were associated with cold sensitivity independent of body mass, even in the high-WHR group (Table 2). anthropometric traits among the three groups were not similar between men and women. BMI and abdominal traits showed differences in men, whereas all traits ex- cept fasting blood glucose levels in women were the highest in the non-CHHF group and the lowest in the CHHF group; the opposite trend was observed for HDLC. These trends were consistent with the inverse correlation between abdominal fat and cold sensitivity in the extremities, as reported previously [5]. anthropometric traits among the three groups were not similar between men and women. BMI and abdominal traits showed differences in men, whereas all traits ex- cept fasting blood glucose levels in women were the highest in the non-CHHF group and the lowest in the CHHF group; the opposite trend was observed for HDLC. These trends were consistent with the inverse correlation between abdominal fat and cold sensitivity in the extremities, as reported previously [5]. Relationship between sensitivity to cold and MS Additionally, the association between the CHHF and LAR showed a trend similar to that of adipo- nectin level, except an inverse relationship (β < 0) was observed, and the significant association signal in Model 1 (in all subjects: β = −1.38 μg/mL, p = 1.28 × 10-3; in women: β = −1.48 μg/mL, p = 2.03 × 10-3) was reduced in Models 2 and 3 (Table 2). This attenuation could be at- tributed to the lack of association signal for leptin since there was no synergy from the combination of adiponectin and leptin in the form of a ratio. Overall, the association of cold sensitivity with adiponectin levels was independent of thermogenic factors, including BMI. The relationship between CHHF and MS was also affected by abdominal body mass, as shown by the WHR subgroup analysis in women. As shown in Table 5, the association between CHHF and MS remained significant in high-WHR women (OR = 0.476, p = 1.66 × 10-2). Inter- estingly, the association with large WC was found in low-WHR women but not in high-WHR women. Signifi- cant MS risk in high-WHR women was associated with low HDLC and high TGs (low HDLC: OR = 0.498, p = 2.91 × 10-2; high TGs: OR = 0.357, p = 1.93 × 10-3). Relationship between circulating adipokine levels and sensitivity to cold From our analysis of the trends in adipokine serum levels, including adiponectin, leptin, and LAR, according to sensitivity to cold in the extremities, women showed significantly increased adiponectin levels and decreased LARs in the order of non-CHHF, intermediate, and CHHF groups, whereas men only exhibited differential levels of adiponectin between the non-CHHF and CHHF groups (Fig. 1). Leptin levels tended to decrease in women, although there were no associations between leptin levels and cold sensation. Discussion correlation between adiponectin levels and CHHF [10]. The results of this previous study suggested that normal sympathetic activity can induce uncoup- ling protein 1-dependent thermogenesis via de- creased adiponectin levels, but problems in sympathetic activity can reduce the thermogenic re- sponse through maintenance of adiponectin levels. However, in this study, we did not examine sympa- thetic activity; thus, additional studies are needed to confirm the relationships among CHHF, adiponectin, and sympathetic activity. correlation between adiponectin levels and CHHF [10]. The results of this previous study suggested that normal sympathetic activity can induce uncoup- ling protein 1-dependent thermogenesis via de- creased adiponectin levels, but problems in sympathetic activity can reduce the thermogenic re- sponse through maintenance of adiponectin levels. However, in this study, we did not examine sympa- thetic activity; thus, additional studies are needed to confirm the relationships among CHHF, adiponectin, and sympathetic activity. p y However, although women with high abdominal fat tend to have warm hands and feet [5], it is unclear why some high-WHR women show a propensity to for CHHF. A clinical study examining the effects of KRG (a potent vasodilator) on CHHF showed that 8- week treatment with KRG resulted in higher skin temperature in the extremities, lower CHHF severity based on visual analog scale assessment, and less parasympathetic activity from heart rate variability analysis [2]. Accordingly, owing to low sympathetic activity, individuals with CHHF exhibit greater re- sponses in the context of low temperatures and show excessive vasoconstriction in the extremities. A previous study on cold acclimation (8-week expos- ure) using apolipoprotein E and low-density lipopro- tein receptor-knockout mice also supported the concept that sympathetic activity may explain the CHHF is considered to be a latent Raynaud’s phenomenon (RP) with no color changes [6, 20]. A recent study with the Charleston Heart Study cohort has shown that RP is associated with increased mor- tality owing to cardiovascular disease (CVD). How- ever, women with CHHF showed lower MS risk (particularly low risk of hypo-HDL-cholesterolemia and hypertriglyceridemia), even in participants with high WHR. Discussion In this study, we found that the sensation of cold in the extremities was associated with increased levels of adi- ponectin (not leptin) and decreased risk of MS in women, independent of body mass. In addition, these trends were enriched in women with high WHR but Park and Cha BMC Complementary and Alternative Medicine (2017) 17:150 Page 5 of 9 Fig. 1 Comparison of adipokine levels according to cold sensitivity in extremities in each group. The p-values for different levels of adipokines among cold sensation groups were estimated using Kruskal-Wallis tests (error bars: standard errors). LAR, leptin-to-adiponectin ratio; CHHF, cold hypersensitivity in the hands and feet Fig. 1 Comparison of adipokine levels according to cold sensitivity in extremities in each group. The p-values for different levels of adipokines among cold sensation groups were estimated using Kruskal-Wallis tests (error bars: standard errors). LAR, leptin-to-adiponectin ratio; CHHF, cold hypersensitivity in the hands and feet Fig. 1 Comparison of adipokine levels according to cold sensitivity in extremities in each group. The p-values for different levels of adipokines among cold sensation groups were estimated using Kruskal-Wallis tests (error bars: standard errors). LAR, leptin-to-adiponectin ratio; CHHF, cold hypersensitivity in the hands and feet were absent in women with low WHR. In men, CHHF was only related to abdominal obesity. elevated glucose utilization through the action of adipo- nectin in WAT. This physiological cold-stress response may resemble the emotional cold-stress response in our study. That is, the increased levels of adiponectin may be induced by cold stress (emotionally mimicked), repre- sented as the CHHF in the context of low environmental temperature. Additionally, because the association be- tween the CHHF and adiponectin levels was observed only in women with a high median WHR, it is possible that the adipose tissue may have an important role in mediating the activity of adiponectin against cold stress. In addition, lower MS risk in CHHF women, even after Elevation of adiponectin levels by chronic cold expos- ure enhances the browning of white adipose tissue (WAT) for adaptive thermogenesis [18]. Circulating adiponectin after long-term cold-stress acclimation is in- volved in glucose metabolism in WAT (and possibly beige adipose tissue), and adaptive thermogenesis is induced by dietary intake rather than by a sympathetic response [9]. Discussion Collectively, previous studies have sug- gested that long-term acclimation to environmental low temperature leads to diet-induced thermogenesis by Park and Cha BMC Complementary and Alternative Medicine (2017) 17:150 Page 6 of 9 Table 2 Linear regression analysis of CHHF with adipokine levels Model 1 Model 2 Model 3 Traitsa Beta (95% CI) P value Beta (95% CI) P value Beta (95% CI) P value All Adiponectin (μg/mL) 1.33 (1.16, 1.53) 4.44 × 10-5 1.23 (1.07, 1.40) 3.28 × 10-3 1.18 (1.02, 1.35) 2.32 × 10-2 Leptin (ng/mL) −2.32 (−7.67, 1.42) 0.168 −1.49 (−5.06, 2.27) 0.521 1.10 (−3.19, 3.84) 0.59 LAR −1.38 (−1.68, −1.14) 1.28 × 10-3 −1.22 (−1.48, 1.00) 4.62 × 10-2 −1.12 (−1.37, 1.09) 0.262 Men Adiponectin (μg/mL) 1.26 (−1.02, 1.60) 0.0686 1.21 (−1.04, 1.52) 0.113 1.13 (−1.13, 1.43) 0.332 Leptin (ng/mL) 1.50 (−3.45, 7.80) 0.628 1.64 (−3.23, 8.66) 0.562 1.85 (−3.09, 10.5) 0.163 LAR −1.27 (−1.77, 1.10) 0.164 −1.21 (−1.67, 1.15) 0.268 −1.09 (−1.53, 1.29) 0.636 Women Adiponectin (μg/mL) 1.40 (1.19, 1.66) 7.87 × 10-5 1.27 (1.08, 1.50) 3.17 × 10-3 1.23 (1.04, 1.45) 1.80 × 10-2 Leptin (ng/mL) −4.38 (−22.2, 1.16) 0.0752 −2.35 (−2.51, 2.22) 0.311 −1.22 (−6.49, 4.39) 0.334 LAR −1.48 (−1.89, −1.15) 2.03 × 10-3 −1.26 (−1.61, 1.01) 0.0611 −1.16 (−1.49, 1.10) 0.233 aln-transformed: adiponectin and LAR Linear regression, adjusting (Model 1) for age, sex (in all), menopausal status (in women); (Model 2) model 1 covariates, diastolic blood pressure, triglycerides (ln-transformed); (Model 3) model 2 covariates, body mass index Abbreviations: CHHF cold hypersensitivity in the hands and feet, CI confidence interval, LAR leptin-to-adiponectin ratio Table 2 Linear regression analysis of CHHF with adipokine levels aln-transformed: adiponectin and LAR Linear regression, adjusting (Model 1) for age, sex (in all), menopausal status (in women); (Model 2) model 1 covariates, (ln-transformed); (Model 3) model 2 covariates, body mass index Abbreviations: CHHF cold hypersensitivity in the hands and feet, CI confidence interval, LAR leptin-to-adiponectin ratio aln-transformed: adiponectin and LAR Linear regression, adjusting (Model 1) for age, sex (in all), menopausal status (in women); (Model 2) model 1 covariates, dias (ln-transformed); (Model 3) model 2 covariates, body mass index Abbreviations: CHHF cold hypersensitivity in the hands and feet, CI confidence interval, LAR leptin-to-adiponectin ratio ln transformed: adiponectin and LAR Linear regression, adjusting (Model 1) for age, sex (in all), menopausal status (in women); (Model 2) model 1 covariates, diastolic blood pressure, triglycerides (ln-transformed); (Model 3) model 2 covariates, body mass index Abbreviations: CHHF cold hypersensitivity in the hands and feet, CI confidence interval, LAR leptin-to-adiponectin ratio stratification according to the median WHR, may be affected by increased adiponectin (and possibly by diet- induced thermogenesis) because this adipokine is known to improve insulin sensitivity [9, 19]. Table 3 Subgroup analysis for association between CHHF and adipokines after dividing women by the WHR median Low WHRb High WHRb Traitsa Beta (95% CI) P value Beta (95% CI) P value Adiponectin (μg/mL) 1.11 (−1.16, 1.43) 0.412 1.31 (1.05, 1.65) 2.00 × 10-2 Leptin (ng/mL) 1.74 (−11.9, 3.92) 0.571 1.05 (−15.0, 13.6) 0.971 LAR −1.08 (−1.60, 1.38) 0.709 −1.23 (−1.68, 1.11) 0.191 aln-transformed: adiponectin and LAR bWomen (n = 649) were divided into two subgroups via the WHR median: low WHR women (n = 326; 45 non-CHHF, 105 intermediate, and 176 CHHF) and high WHR women (n = 323; 85 non-CHHF, 124 intermediate, and 114 CHHF) Linear regression, adjusting for age, diastolic blood pressure, triglycerides (ln-transformed), menopausal status, and body mass index Abbreviations: CHHF cold hypersensitivity in the hands and feet, WHR waist-to-hip ratio, CI confidence interval, LAR, Leptin-to-adiponectin ratio usting for age, diastolic blood pressure, triglycerides (ln-transformed), menopausal status, and body mass index ld h iti it i th h d d f t WHR i t t hi ti CI fid i t l LAR L ti t di ti old hypersensitivity in the hands and feet, WHR waist-to-hip ratio, CI confidence interval, LAR, Leptin-to-adiponectin ratio vided into two subgroups via the WHR median: low WHR women (n = 326; 45 non-CHHF, 105 intermediate, and 176 CHHF) and high non-CHHF, 124 intermediate, and 114 CHHF) Discussion selected after excluding individuals with a medication history for CVD. Interestingly, participants who com- plained of CHHF with no CVD may have healthier blood vessels. One of limitations of the current study was that we based our analysis on a questionnaire to determine sen- sations of cold in the extremities. Therefore, it is difficult to determine whether the changes in adiponectin levels may be caused by emotional cold-stress acclimation, although subjective and objective estimations of finger temperature are similar [28]. Additional studies (with larger sample populations) based on measurements of body temperature and cardiometabolic traits through modulation of the environmental temperature (e.g., sea- sonal variation) are needed to investigate the link between physiological and emotional cold-stress for adipokine induction, because adipokines such as adi- ponectin and leptin are associated with changes in cardiometabolic traits [7, 13]. Another limitation was that this was a cross-sectional study, so we cannot know whether the differences in cardiometabolic traits between the three groups (shown in Table 1) induce changes in adiponectin levels or vice versa. To resolve this, it would be necessary to follow the changes in cardiometabolic traits and adipokine levels in a pro- spective cohort study. The gender difference in the association between CHHF with adiponectin levels and MS risk could be at- tributed to estrogen, given that the hormone is involved in the regulation of weight, insulin sensitivity, and body temperature [21–23]. In addition, a previous study has reported that high-level estrogen during the menstrual cycle is associated with less sensitivity to cold stress [24]. Estrogen also improves the peripheral blood circulation by increasing vasodilation [23]. In postmenopausal women, after estrogen levels are notably reduced [25], higher estrogen levels have been associated with higher insulin resistance and lower adiponectin levels [26, 27]. In the present study, high WHR women included a higher proportion of postmenopausal women (64.7%) than low WHR women did (27.0%). Therefore, higher levels of adiponectin in high WHR women may be asso- ciated with lower levels of estrogen, which in turn is associated with higher sensitivity of the extremities to cold stress. Discussion Therefore, CHHF may represent very- early-phase RP, although our study participants were Table 3 Subgroup analysis for association between CHHF and adipokines after dividing women by the WHR median Low WHRb High WHRb Traitsa Beta (95% CI) P value Beta (95% CI) P value Adiponectin (μg/mL) 1.11 (−1.16, 1.43) 0.412 1.31 (1.05, 1.65) 2.00 × 10-2 Leptin (ng/mL) 1.74 (−11.9, 3.92) 0.571 1.05 (−15.0, 13.6) 0.971 LAR −1.08 (−1.60, 1.38) 0.709 −1.23 (−1.68, 1.11) 0.191 aln-transformed: adiponectin and LAR bWomen (n = 649) were divided into two subgroups via the WHR median: low WHR women (n = 326; 45 non-CHHF, 105 intermediate, and 176 CHHF) and high WHR women (n = 323; 85 non-CHHF, 124 intermediate, and 114 CHHF) Linear regression, adjusting for age, diastolic blood pressure, triglycerides (ln-transformed), menopausal status, and body mass index Abbreviations: CHHF cold hypersensitivity in the hands and feet, WHR waist-to-hip ratio, CI confidence interval, LAR, Leptin-to-adiponectin ratio aln-transformed: adiponectin and LAR b Park and Cha BMC Complementary and Alternative Medicine (2017) 17:150 Page 7 of 9 Table 4 Logistic regression analysis of CHHF with MS and the five components All Men Women Traits OR (95% CI) P value OR (95% CI) P value OR (95% CI) P value MS 0.465 (0.315, 0.684) 1.01 × 10-4 0.690 (0.364, 1.31) 0.256 0.449 (0.273, 0.737) 1.54 × 10-3 Low HDLC 0.656 (0.461, 0.932) 1.86× 10-2 0.660 (0.367, 1.19) 0.166 0.688 (0.440, 1.07) 0.0994 High TGs 0.467 (0.315, 0.691) 1.40× 10-4 0.711 (0.389, 1.30) 0.268 0.422 (0.250, 0.711) 1.21× 10-3 High BP 0.712 (0.492, 1.03) 0.0723 1.28 (0.713, 2.31) 0.406 0.578 (0.356, 0.938) 2.65× 10-2 High FBG 0.791 (0.499, 1.25) 0.319 0.701 (0.337, 1.46) 0.340 0.957 (0.514, 1.78) 0.888 Large WC 0.298 (0.203, 0.436) 5.35 × 10-10 0.300 (0.154, 0.567) 2.12 × 10-4 0.343 (0.208, 0.563) 2.38 × 10-5 Logistic regression, adjusting for age, sex (in all), and menopausal status (in women) Abbreviations: CHHF cold hypersensitivity in the hands and feet, MS metabolic syndrome, OR odds ratio, CI confidence interval, HDLC, HDL cholesterol, TGs triglycerides; BP blood pressure; FBG, fasting blood glucose, WC waist circumference Table 4 Logistic regression analysis of CHHF with MS and the five components Logistic regression, adjusting for age, sex (in all), and menopausal status (in women) Abbreviations: CHHF cold hypersensitivity in the hands and feet, MS metabolic syndrome, OR odds ratio, CI confidence interval, HDLC, HDL cholesterol, TGs triglycerides; BP blood pressure; FBG, fasting blood glucose, WC waist circumference because we did not measure the estrogen in the studied population. Logistic regression, adjusting age and menopausal status Consent for publication Consent for publication Not applicable. 19. Lihn AS, Pedersen SB, Richelsen B. Adiponectin: action, regulation and association to insulin sensitivity. Obes Rev. 2005;6(1):13–21. Discussion However, we cannot know the exact relation- ship between CHHF and adiponectin and estrogen, Table 5 Subgroup analysis for the association between CHHF and MS risk after dividing women by WHR median Low WHRa High WHRa Traits OR (95% CI) P-value OR (95% CI) P-value MS 0.669 (0.245, 1.83) 0.435 0.476 (0.259, 0.874) 1.66 × 10-2 Low HDLC 1.32 (0.655, 2.66) 0.437 0.498 (0.266, 0.931) 2.91 × 10-2 High TGs 0.714 (0.274, 1.86) 0.492 0.357 (0.186, 0.685) 1.93 × 10-3 High BP 0.499 (0.218, 1.14) 0.0997 0.717 (0.392, 1.31) 0.281 High FBG 0.835 (0.241, 2.89) 0.777 1.16 (0.563, 2.37) 0.693 Large WC 0.400 (0.188, 0.853) 1.78 × 10-2 0.377 (0.142, 1.00) 0.0510 aWomen (n = 649) were divided into two subgroups via the WHR median: low WHR women (n = 326; 45 non-CHHF, 105 intermediate, and 176 CHHF) and high WHR women (n = 323; 85 non-CHHF, 124 intermediate, and 114 CHHF) Logistic regression, adjusting age and menopausal status Abbreviations: MS metabolic syndrome, WHR waist-to-hip ratio, OR odds ratio, CI confidence interval, HDLC HDL cholesterol, TGs triglycerides, BP blood pressure, FBG fasting blood glucose, WC waist circumference Logistic regression, adjusting age and menopausal status Abbreviations: MS metabolic syndrome, WHR waist-to-hip ratio, OR odds ratio, CI confidence interval, HDLC HDL cholesterol, TGs triglycerides, BP blood pressure, FBG fasting blood glucose, WC waist circumference Park and Cha BMC Complementary and Alternative Medicine (2017) 17:150 Page 8 of 9 Page 8 of 9 Page 8 of 9 Page 8 of 9 Conclusions 2. Park KS, Park KI, Kim JW, Yun YJ, Kim SH, Lee CH, Park JW, Lee JM. Efficacy and safety of Korean red ginseng for cold hypersensitivity in the hands and feet: a randomized, double-blind, placebo-controlled trial. J Ethnopharmacol. 2014;158(Pt A):25–32. In conclusion, the results of this study showed that CHHF was correlated with adiponectin levels, inde- pendent of body mass, particularly in women with high median WHR. Despite complaints of feeling cold, these women could be at lower risk of MS owing to increased levels of adiponectin and insulin sensitivity. 3. Seo JC, Lee HJ, Kwak MA, Park SH, Shin I, Yun WS, Park K. Acupuncture in subjects with cold hands sensation: study protocol for a randomized controlled trial. Trials. 2014;15:348. 3. Seo JC, Lee HJ, Kwak MA, Park SH, Shin I, Yun WS, Park K. Acupuncture in subjects with cold hands sensation: study protocol for a randomized controlled trial. Trials. 2014;15:348. 4. Levien TL. Advances in the treatment of Raynaud’s phenomenon. Vasc Health Risk Manag. 2010;6:167–77. 4. Levien TL. Advances in the treatment of Raynaud’s phenomenon. Vasc Health Risk Manag. 2010;6:167–77. 5. Savastano DM, Gorbach AM, Eden HS, Brady SM, Reynolds JC, Yanovski JA. Adiposity and human regional body temperature. Am J Clin Nutr. 2009; 90(5):1124–31. 5. Savastano DM, Gorbach AM, Eden HS, Brady SM, Reynolds JC, Yanovski JA. Adiposity and human regional body temperature. Am J Clin Nutr. 2009; 90(5):1124–31. Funding Thi 11. Kim H, Richardson C, Roberts J, Gren L, Lyon JL. Cold hands, warm heart. Lancet. 1998;351(9114):1492. This research was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (grant no. NRF-2014M3A9D7034335) and by the research program of Korea Institute of Oriental Medicine (grant no. K17092). The funders had no role in study design, data collection and analysis, interpretation of data, and preparation of the manuscript. 12. Oda N, Imamura S, Fujita T, Uchida Y, Inagaki K, Kakizawa H, Hayakawa N, Suzuki A, Takeda J, Horikawa Y, et al. The ratio of leptin to adiponectin can be used as an index of insulin resistance. Metab Clin Exp. 2008;57(2):268–73. 13. Yun JE, Won S, Mok Y, Cui W, Kimm H, Jee SH. Association of the leptin to high-molecular-weight adiponectin ratio with metabolic syndrome. Endocr J. 2011;58(9):807–15. Acknowledgements We would like to thank Editage (www.editage.co.kr) for English language editing. 10. Dong M, Yang X, Lim S, Cao Z, Honek J, Lu H, Zhang C, Seki T, Hosaka K, Wahlberg E, et al. Cold exposure promotes atherosclerotic plaque growth and instability via UCP1-dependent lipolysis. Cell Metab. 2013;18(1):118–29. Availability of data and materials The data that support the findings of this study are available from the Korea medicine Data Center (KDC) but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. Data are however available from the authors upon reasonable request and with permission of the KDC and the Institutional Data Access/Ethics Committee of Korea Institute of Oriental Medicine. 14. Eglit T, Lember M, Ringmets I, Rajasalu T. Gender differences in serum high-molecular-weight adiponectin levels in metabolic syndrome. Eur J Endocrino. 2013;168(3):385–91. 15. Expert Panel on Detection E, Treatment of High Blood Cholesterol in A. Executive summary of the third report of the National Cholesterol Education Program (NCEP) expert panel on detection, evaluation, and treatment of high blood cholesterol in adults (adult treatment panel III). JAMA. 2001;285(19):2486–97. Ethics approval and consent to participate 20. Park KS, Kim JW, Jo JY, Hwang DS, Lee CH, Jang JB, Lee KS, Yeo I, Lee JM. Effect of Korean red ginseng on cold hypersensitivity in the hands and feet study protocol for a randomized controlled trial. Trials. 2013;14:438. All participants provided written informed consent to participate in the study, and the study was approved by the Institutional Review Board of the Korea Institute of Oriental Medicine. 21. Mayes JS, Watson GH. Direct effects of sex steroid hormones on adipose tissues and obesity. Obes Rev. 2004;5(4):197–216. Abbreviations 6. Hur YM, Chae JH, Chung KW, Kim JJ, Jeong HU, Kim JW, Seo SY, Kim KS. Feeling of cold hands and feet is a highly heritable phenotype. Twin Res Hum Genet. 2012;15(2):166–9. 95% CI: 95% confidence interval; BMI: Body mass index; CHHF: Cold hypersensitivity in the hands and feet; CVD: Cardiovascular disease; DBP: Diastolic blood pressure; HDLC: HDL cholesterol; HMW adiponectin: High-molecular-weight adiponectin; KDC: Korea medicine Data Center; KRG: Korean red ginseng; LAR: Leptin-to-adiponectin ratio; MS: Metabolic syndrome; OR: Odds ratio; RP: Raynaud’s phenomenon; TG: Triglyceride; WAT: White adipose tissue; WC: Waist circumference; WHR: Waist-to-hip ratio 7. Yadav A, Kataria MA, Saini V, Yadav A. Role of leptin and adiponectin in insulin resistance. Clin Chim Acta. 2013;417:80–4. 8. Iwen KA, Wenzel ET, Ott V, Perwitz N, Wellhoner P, Lehnert H, Dodt C, Klein J. Cold-induced alteration of adipokine profile in humans. Metab Clin Exp. 2011;60(3):430–7. 9. Lee P, Smith S, Linderman J, Courville AB, Brychta RJ, Dieckmann W, Werner CD, Chen KY, Celi FS. Temperature-acclimated brown adipose tissue modulates insulin sensitivity in humans. Diabetes. 2014;63(11):3686–98. Publisher’s note 22. Gupte AA, Pownall HJ, Hamilton DJ. Estrogen: an emerging regulator of insulin action and mitochondrial function. J Diabetes Res. 2015;2015:916585. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 23. Charkoudian N, Stachenfeld N. Sex hormone effects on autonomic mechanisms of thermoregulation in humans. Auton Neurosci. 2016;196: 75–80. Received: 4 August 2016 Accepted: 3 March 2017 Received: 4 August 2016 Accepted: 3 March 2017 Received: 4 August 2016 Accepted: 3 March 2017 24. Hellstrom B, Lundberg U. Pain perception to the cold pressor test during the menstrual cycle in relation to estrogen levels and a comparison with men. Integr Physiol Behav Sci. 2000;35(2):132–41. Authors’ contributions AYP designed and performed the experiments and wrote the manuscript; SC conceived and designed the study, performed the statistical analyses, interpreted the data, and wrote the manuscript. Both authors read and approved the final manuscript. 16. Yoon Y, Kim H, Lee Y, Yoo J, Lee S. Developing an optimized cold/heat questionnaire. Integrative Med Res. 2015;4(4):225–30. 17. Lara-Castro C, Luo N, Wallace P, Klein RL, Garvey WT. Adiponectin multimeric complexes and the metabolic syndrome trait cluster. Diabetes. 2006;55(1):249–59. Competing interests 18. Hui X, Gu P, Zhang J, Nie T, Pan Y, Wu D, Feng T, Zhong C, Wang Y, Lam KS, et al. Adiponectin enhances cold-induced browning of subcutaneous adipose tissue via promoting M2 macrophage proliferation. Cell Metab. 2015;22(2):279–90. The authors declare that they have no competing interests. Park and Cha BMC Complementary and Alternative Medicine (2017) 17:150 26. Kalish GM, Barrett-Connor E, Laughlin GA, Gulanski BI, Postmenopausal Estrogen/Progestin Intervention T. Association of endogenous sex hormones and insulin resistance among postmenopausal women: results from the Postmenopausal Estrogen/Progestin Intervention Trial. J Clin Endocrinol Metab. 2003;88(4):1646–52. 26. Kalish GM, Barrett-Connor E, Laughlin GA, Gulanski BI, Postmenopausal Estrogen/Progestin Intervention T. Association of endogenous sex hormones and insulin resistance among postmenopausal women: results from the Postmenopausal Estrogen/Progestin Intervention Trial. J Clin Endocrinol Metab. 2003;88(4):1646–52. 27. Laughlin GA, Barrett-Connor E, May S. Sex-specific determinants of serum adiponectin in older adults: the role of endogenous sex hormones. Int J Obes (Lond). 2007;31(3):457–65. 28. Polunina A, Gugleta K, Kochkorov A, Katamay R, Flammer J, Orgul S. Relationship between peripheral blood flow in extremities and choroidal circulation. Klin Monbl Augenheilkd. 2011;228(4):302–5. 27. Laughlin GA, Barrett-Connor E, May S. Sex-specific determinants of serum adiponectin in older adults: the role of endogenous sex hormones. Int J Obes (Lond). 2007;31(3):457–65. 28. Polunina A, Gugleta K, Kochkorov A, Katamay R, Flammer J, Orgul S. Relationship between peripheral blood flow in extremities and choroidal circulation. Klin Monbl Augenheilkd. 2011;228(4):302–5. References References 1. Bae KH, Lee JA, Park KH, Yoo JH, Lee Y, Lee S. Cold hypersensitivity in the hands and feet may be associated with functional dyspepsia: results of a multicenter survey study. Evid Based Complement Alternat Med. 2016;2016: 8948690. 1. Bae KH, Lee JA, Park KH, Yoo JH, Lee Y, Lee S. Cold hypersensitivity in the hands and feet may be associated with functional dyspepsia: results of a multicenter survey study. Evid Based Complement Alternat Med. 2016;2016: 8948690. 25. Pasquali R, Vicennati V, Bertazzo D, Casimirri F, Pascal G, Tortelli O, Labate AM. Determinants of sex hormone-binding globulin blood concentrations in premenopausal and postmenopausal women with different estrogen status. Virgilio-Menopause-Health Group. Metab Clin Exp. 1997;46(1):5–9. Page 9 of 9 Park and Cha BMC Complementary and Alternative Medicine (2017) 17:150 Park and Cha BMC Complementary and Alternative Medicine (2017) 17:150 • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step:
https://openalex.org/W4254377344	https://www.qeios.com/read/ZSE62A/pdf	English	null	Prophylactic Mastectomy Patient	Definitions	2,020	cc-by	50	Qeios · Definition, February 2, 2020 Open Peer Review on Qeios Open Peer Review on Qeios Prophylactic Mastectomy Patient National Cancer Institute National Cancer Institute Qeios ID: ZSE62A · https://doi.org/10.32388/ZSE62A Source National Cancer Institute. Prophylactic Mastectomy Patient. NCI Thesaurus. Code C147117. C147117. An individual who is undergoing prophylactic mastectomy. 1/1
https://openalex.org/W4385706941	https://www.nature.com/articles/s41431-023-01437-2.pdf	English	null	Novel homozygous variants in PRORP expand the genotypic spectrum of combined oxidative phosphorylation deficiency 54	European journal of human genetics	2,023	cc-by	4,215	1Division of Evolution, Infection and Genomics, School of Biological Sciences, University of Manchester, Manchester M13 9PL, UK. 2Manchester Centre for Genomic Medicine, St Mary’s Hospital, Manchester University NHS Foundation Trust, Manchester M13 9WL, UK. 3Wellcome Centre for Mitochondrial Research, Clinical and Translational Research Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne NE2 4HH, UK. 4Department of Applied Sciences, Faculty of Health & Life Sciences, Northumbria University, Newcastle upon Tyne, UK. 5Department of Molecular Neuroscience, UCL Queen Square Institute of Neurology, London WC1N 3BG, UK. 6Department of Biology, Faculty of Science, Shahid Chamran University of Ahvaz, Ahvaz, Iran. 7Narges Medical Genetics and Prenatal Diagnosis Laboratory, Kianpars, Ahvaz, Iran. 8NHS Highly Specialised Service for Rare Mitochondrial Disorders, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne NE1 4LP, UK. 9Department of Pediatric Neurology, Golestan Medical, Educational, and Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. 10Genetic Department, Hadassah Hebrew University Hospital, Jerusalem, Israel. 11Pediatric Neurology, Hadassah Hebrew University Hospital, Jerusalem, Israel. 12UC Davis Medical Center MIND Institute, 2825 50th Street, Sacramento, CA 95817, USA. 13Biosciences Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne NE2 4HH, UK. ✉email: william.newman@manchester.ac.uk; rokeefe@manchester.ac.uk BRIEF COMMUNICATION OPEN Novel homozygous variants in PRORP expand the genotypic spectrum of combined oxidative phosphorylation deﬁciency 54 Thomas B. Smith 1,2, Alessandro Rea 1,2, Huw B. Thomas1,2, Kyle Thompson3, Monika Oláhová3,4, Reza Marooﬁan 5, Mina Zamani6,7, Langping He8, Saeid Sadeghian 9, Hamid Galehdari7, Nava Shaul Lotan10, Tal Gilboa 11, Kristin C. Herman12, Thomas J. McCorvie 13, Wyatt W. Yue13, Henry Houlden 5, Robert W. Taylor3,8, William G. Newman 1,2✉and Raymond T. O’Keefe 1✉ © The Author(s) 2023 © The Author(s) 2023 Biallelic hypomorphic variants in PRORP have been recently described as causing the autosomal recessive disorder combined oxidative phosphorylation deﬁciency type 54 (COXPD54). COXPD54 encompasses a phenotypic spectrum of sensorineural hearing loss and ovarian insufﬁciency (Perrault syndrome) to leukodystrophy. Here, we report three additional families with homozygous missense PRORP variants with pleiotropic phenotypes. Each missense variant altered a highly conserved residue within the metallonuclease domain. In vitro mitochondrial tRNA processing assays with recombinant TRMT10C, SDR5C1 and PRORP indicated two COXPD54-associated PRORP variants, c.1159A>G (p.Thr387Ala) and c.1241C>T (p.Ala414Val), decreased pre-tRNAIle cleavage, consistent with both variants impacting tRNA processing. No signiﬁcant decrease in tRNA processing was observed with PRORP c.1093T>C (p.Tyr365His), which was identiﬁed in an individual with leukodystrophy. These data provide independent evidence that PRORP variants are associated with COXPD54 and that the assessment of 5′ leader mitochondrial tRNA processing is a valuable assay for the functional analysis and clinical interpretation of novel PRORP variants. European Journal of Human Genetics (2023) 31:1190–1194; https://doi.org/10.1038/s41431-023-01437-2 www.nature.com/ejhg Received: 21 April 2023 Revised: 7 July 2023 Accepted: 18 July 2023 Published online: 9 August 2023 INTRODUCTION Bi ll li i i maturation by catalysing endonucleolytic cleavage of the 5′ leader sequence from polycistronic mitochondrial RNA transcripts [4]. maturation by catalysing endonucleolytic cleavage of the 5′ leader sequence from polycistronic mitochondrial RNA transcripts [4]. Here, we present three further unrelated families with homo- zygous, missense PRORP variants. Consistent with the previously reported cases, the variants are within the functional metallonu- clease domain and alter highly conserved residues [1]. The affected individuals have phenotypes overlapping with the previous reports and provide evidence that the clinical severity correlates with the degree of mitochondrial tRNA processing deﬁcit. These ﬁndings provide independent supportive evidence for the association of biallelic PRORP variants with defective mitochondrial tRNA processing, resulting in a phenotypic spec- trum encompassing Perrault syndrome and COXPD54. Biallelic variants in Protein Only RNase P Catalytic Subunit (PRORP) have recently been associated with a newly deﬁned syndrome, combined oxidative phosphorylation deﬁciency 54 (COXPD54) (MIM #619737), encompassing a phenotypic spectrum of Perrault syndrome and leukodystrophy [1]. Perrault syndrome (MIM #233400) is a rare, autosomal recessive, clinically and genetically heterogeneous disorder characterised by bilateral sensorineural hearing loss (SNHL) in both sexes and primary ovarian insufﬁciency in 46, XX karyotype females [2, 3]. The individuals with biallelic PRORP variants, reported to date, presented with pleiotropic phenotypes ranging in clinical severity, with PRORP variant protein resulting in reduced mitochondrial tRNA processing in vitro [1]. With the small number of reported affected individuals with COXPD54, genotype–phenotype correlations have yet to be established and independently replicated. PRORP (MRPP3) is one of the three subunits constituting the human mitochondrial RNase P (mtRNase P) complex with MRPP1 (TRMT10C) and MRPP2/SDR5C1 (HSD17B10). This multimeric complex aids mitochondrial tRNA Here, we present three further unrelated families with homo- zygous, missense PRORP variants. Consistent with the previously reported cases, the variants are within the functional metallonu- clease domain and alter highly conserved residues [1]. The affected individuals have phenotypes overlapping with the previous reports and provide evidence that the clinical severity correlates with the degree of mitochondrial tRNA processing deﬁcit. These ﬁndings provide independent supportive evidence for the association of biallelic PRORP variants with defective mitochondrial tRNA processing, resulting in a phenotypic spec- trum encompassing Perrault syndrome and COXPD54. Variant identiﬁcation methodology Variant identiﬁcation methodology Variant identiﬁcation methodology gy Biallelic variants in PRORP were identiﬁed by exome sequence analysis in all three families (see Supplementary information). Segregation analysis Received: 21 April 2023 Revised: 7 July 2023 Accepted: 18 July 2023 Published online: 9 August 2023 DISCUSSION Biallelic PRORP variants have been associated with diverse, overlapping pleiotropic phenotypes, with variable defects in mitochondrial tRNA processing [1]. The aim of this work was to independently conﬁrm that novel PRORP variants identiﬁed in additional families are pathogenic and associated with the COXPD54 phenotypic spectrum. yp yg p y g The proband from family F2 is a 7-year-old American male of Mexican background. He presented with severe to profound bilateral SNHL (Supplementary Fig. S1C), undergoing assessment aged 3 years, exhibiting global developmental delay, spastic diplegia and truncal hypotonia. He is also both nonverbal and non-ambulatory. Brain MRI displayed no white matter lesions. Trio exome sequencing of the proband revealed a homozygous variant in PRORP (NM_014672.4:c.1159A>G (p.Thr387Ala)), with no other candidate variants identiﬁed. Both parents are heterozygous for this variant and unaffected, whilst his healthy younger sibling is homozygous for the reference allele. p yp p Because all novel variants were located within the functional metallonuclease domain (Fig. 1C), we investigated the ability of mtRNase P complexes with PRORP variants to conduct 5′ leader processing of pre-tRNAIle. mtRNase P complexes with the novel PRORP (p.Thr387Ala and p.Ala414Val) variants signiﬁcantly decreased the intensity of the cleavage product compared to wildtype, indicating the function of the mtRNase P complex was impaired. The defect in 5′ leader processing was more pronounced with the variant from the F3 proband with a more severe phenotype. Fibroblasts from the affected individual in F3 demonstrated deﬁciencies in complex I activity (Supplementary Fig. S2B), highlighting a respiratory chain defect. yg The proband from family F3 is the daughter of a consangui- neous couple of Arab Muslim background from Israel. She was affected by isovaleric acidemia detected by newborn screening and homozygous for the IVD: c.941C>T;p.(Ala314Val) hypomorphic variant [5, 6]. The proband did not pass the neonatal hearing screening test on the right ear; however, a formal hearing test was not performed. She presented at 3.5 months with episodes of lactic academia (up to 11.5 mmol/l, normal range 2–4 mmol/l), preceded by a febrile infection, and was additionally diagnosed with atrial septal defect and severe pulmonary hypertension. She had repeatedly high lactate levels (2.6–6.4 mmol/l). She was affected by hypotonia, developmental delay and severe failure to thrive. Aged 1 year, she suffered a lactic acidosis crisis (8.4 mmol/l) with status epilepticus, after which she experienced epilepsy, extrapyramidal movement disorder and loss of developmental skills. She died at age 19 months. T.B. Smith et al. 1191 comparison to wildtype PRORP (Fig. 2). The percentage decreases in relative intensity were 29% and 59%, respectively. The p.Tyr365His PRORP variant had little impact on 5′ leader processing, with a modest 4% decrease (p = 0.9330). was undertaken using Sanger sequencing for all unaffected and affected relatives where available. Details of respiratory chain activity assays, immunoblotting and mitochondrial tRNA processing assays are also outlined in detail in the Supplementary information. p g p We also independently compared the novel p.Thr387Ala and p.Ala414Val variants to a disease-associated PRORP variant to assess the reproducibility of the tRNA processing assay [1]. The p.Arg421Cys and p.Asn412Ser PRORP variants were evaluated in the tRNA processing assay because they altered the cleavage of pre-tRNAIle to variable degrees [1]. The novel p.Thr387Ala and p.Ala414Val PRORP variants signiﬁcantly reduced cleavage of the mtRNase P complex by 25% and 52% (p = 0.0153 and <0.0001), whilst the p.Arg421Cys and p.Asn412Ser variants diminished cleavage of the mtRNase P complex by 13% and 75% (p = 0.2873 and <0.0001) respectively, comparable to values in the previous report [1]. The variants p.Thr387Ala and p.Ala414Val were subsequently classiﬁed according to ACMG guidelines as likely pathogenic, whereas p.Tyr365His was classiﬁed as a variant of uncertain signiﬁcance (VUS), as submitted to ClinVar. RESULTS The three probands presented with diverse clinical phenotypes outlined below. Additional clinical, in vitro and protein modelling data is available in the Supplementary information. pp y Family F1 is a consanguineous family with a 28-year-old female proband from Iran who presented with mild intellectual impair- ment, gait abnormality, behavioural problems and hypothyroid- ism. There is no evidence of SNHL or ovarian insufﬁciency; however, brain MRI demonstrates cerebellar atrophy and multi- focal leukoencephalopathy (Supplementary Fig. S1A). Menstrua- tion is irregular (once every 4–6 months), and she has bilateral polycystic ovaries. Biochemical tests of ovarian function revealed hormone levels were within follicular phase reference values (Supplementary Fig. S1B). Exome sequencing revealed a homo- zygous missense PRORP (NM_014672.4:c.1093T>C (p.Tyr365His)) variant in the proband. The parents of the proband are heterozygous and her two clinically unaffected siblings, a brother and sister, are wildtype and heterozygous, respectively (Fig. 1A). The proband from family F2 is a 7-year-old American male of Mexican background. He presented with severe to profound bilateral SNHL (Supplementary Fig. S1C), undergoing assessment aged 3 years, exhibiting global developmental delay, spastic diplegia and truncal hypotonia. He is also both nonverbal and non-ambulatory. Brain MRI displayed no white matter lesions. Trio exome sequencing of the proband revealed a homozygous variant in PRORP (NM_014672.4:c.1159A>G (p.Thr387Ala)), with no other candidate variants identiﬁed. Both parents are heterozygous for this variant and unaffected, whilst his healthy younger sibling is homozygous for the reference allele pp y Family F1 is a consanguineous family with a 28-year-old female proband from Iran who presented with mild intellectual impair- ment, gait abnormality, behavioural problems and hypothyroid- ism. There is no evidence of SNHL or ovarian insufﬁciency; however, brain MRI demonstrates cerebellar atrophy and multi- focal leukoencephalopathy (Supplementary Fig. S1A). Menstrua- tion is irregular (once every 4–6 months), and she has bilateral polycystic ovaries. Biochemical tests of ovarian function revealed hormone levels were within follicular phase reference values (Supplementary Fig. S1B). Exome sequencing revealed a homo- zygous missense PRORP (NM_014672.4:c.1093T>C (p.Tyr365His)) variant in the proband. The parents of the proband are heterozygous and her two clinically unaffected siblings, a brother and sister, are wildtype and heterozygous, respectively (Fig. 1A). European Journal of Human Genetics (2023) 31:1190 – 1194 DISCUSSION After incubation with pre-tRNAIle, mtRNase P complex with the p.Thr387Ala (F2) or p.Ala414Val (F3) PRORP variants signiﬁcantly diminished the levels of 5′ cleavage product (p = 0.0121 and <0.0001 respectively) in European Journal of Human Genetics (2023) 31:1190 – 1194 ORP family pedigrees, conservation of PRORP variant residues across a selection of species and PRORP variant lo es for families F1–F3, each with homozygous PRORP variants in the proband. B Position of variant residues highlighted in elow the sequence alignments represent level of conservation across species, with * (asterisk) indicating full conservation strongly similar properties, . (period) indicating weakly similar properties and no symbol indicating no conservation. V tal Omega alignment software. C Schematic illustrating the location of all established PRORP variants to date. Novel var n orange hilst pre io sl p blished ariants are in bl e Created sing DOG (Domain Graph) soft are [9] MTS mitoc T.B. Smith et al. T.B. Smith et al. T.B. Smith et al. 1192 1192 Fig. 1 PRORP family pedigrees, conservation of PRORP variant residues across a selection of species and PRORP variant locations A Pedigrees for families F1–F3, each with homozygous PRORP variants in the proband. B Position of variant residues highlighted in orange Symbols below the sequence alignments represent level of conservation across species, with * (asterisk) indicating full conservation, : (colon indicating strongly similar properties, . (period) indicating weakly similar properties and no symbol indicating no conservation. Visualise using Clustal Omega alignment software. C Schematic illustrating the location of all established PRORP variants to date. Novel variants ar coloured in orange, whilst previously published variants are in blue. Created using DOG (Domain Graph) software [9]. MTS mitochondria targeting sequence, PPR pentatricopeptide repeat, CD central domain. Fig. 1 PRORP family pedigrees, conservation of PRORP variant residues across a selection of species and PRORP variant locations. A Pedigrees for families F1–F3, each with homozygous PRORP variants in the proband. B Position of variant residues highlighted in orange. Symbols below the sequence alignments represent level of conservation across species, with * (asterisk) indicating full conservation, : (colon) indicating strongly similar properties, . (period) indicating weakly similar properties and no symbol indicating no conservation. Visualised using Clustal Omega alignment software. C Schematic illustrating the location of all established PRORP variants to date. Novel variants are coloured in orange, whilst previously published variants are in blue. Created using DOG (Domain Graph) software [9]. DISCUSSION Brain MRI scans revealed cerebrospinal space dilation and diffuse restrictive changes in the perirolandic region and basal ganglia (Supplementary Fig. S1D). Exome sequencing identiﬁed the homozygous PRORP (NM_014672.4: c.1241C>T (p.Ala414Val)) variant. Both parents are heterozygous for the variant. In contrast, mtRNase P complexes with PRORP p.Tyr365His did not signiﬁcantly reduce pre-tRNAIle 5′ end cleavage. There are potential explanations for this ﬁnding. For example, protein modelling demonstrates that amino acid 365 is not located in the vicinity of the active site or any interaction regions (Supplementary Fig. S4). Furthermore, residues surrounding the amino acid are less conserved than the residues proximate to other disease-associated variants (Fig. 1). These observations indicate this region of the protein may be less essential for regular endonucleolytic function. The p.Tyr365His variant may also marginally reduce mtRNase P processing efﬁcacy to levels which are difﬁcult to detect experimentally but could still diminish processing in speciﬁc tissues. It is also possible that a different pathogenic mechanism is responsible for this phenotype, or that this variant does not cause COXPD54. With the discovery of additional affected families, comparisons can begin to be made between PRORP variants and associated phenotypes (Supplementary Table S3). The proband with the p.Tyr365His PRORP VUS has a similar presentation to the individuals in the initial discovery study with the p.Arg421Cys PRORP variant [1]. Affected individuals in both families presented with leukodystrophy, mild learning disabilities and behavioural abnormalities, with no hearing impairment or ovarian insufﬁ- ciency. Both variants also had little impact on mitochondrial tRNA processing. The probands from F3 in this study (PRORP p.Ala414Val) and P3 in the discovery study (PRORP p.Arg445Gln;p.- Ser400Ilefs6) were also comparable, presenting with severe childhood-onset features, including lactic acidosis, hypotonia, yg All three novel variants alter highly conserved residues (Fig. 1B) and are predicted deleterious by in silico analyses (Supplementary Table S2). The three variants are also absent from gnomAD, providing supportive evidence for pathogenicity [7]. To assess whether the novel PRORP variants altered mitochon- drial tRNA processing, we puriﬁed recombinant PRORP with the disease-associated variants and tested the endonucleolytic activity of the mtRNase P complex in the presence of pre-tRNAIle in vitro (Supplementary Fig. S3). DISCUSSION MTS mitochondrial targeting sequence, PPR pentatricopeptide repeat, CD central domain. European Journal of Human Genetics (2023) 31:1190 – 1194 T.B. Smith et al. Fig. 2 Functional assessment of PRORP variants in tRNA processing assays with mtRNase P. A Cleavage of the pre-tRNAIle 5′ leader sequence by mtRNase P containing wildtype (WT) or variant PRORP. The relative intensity of the pre-tRNAIle cleavage product was quantiﬁed with error bars representing the standard error of the mean. N = 4, p < 0.05, ***p < 0.0001, one-way ANOVA with Dunnett’s multiple comparisons test, comparing wildtype to variants. B Comparison of mtRNase P activity to previously published PRORP variants. N = 5, p < 0.05, ***p < 0.0001, one-way ANOVA with Dunnett’s multiple comparisons test, comparing wildtype to variants. 11 Fig. 2 Functional assessment of PRORP variants in tRNA processing assays with mtRNase P. A Cleavage of the pre-tRNAIle 5′ leader sequence by mtRNase P containing wildtype (WT) or variant PRORP. The relative intensity of the pre-tRNAIle cleavage product was quantiﬁed with error bars representing the standard error of the mean. N = 4, p < 0.05, ***p < 0.0001, one-way ANOVA with Dunnett’s multiple comparisons test, comparing wildtype to variants. B Comparison of mtRNase P activity to previously published PRORP variants. N = 5, p < 0.05, ***p < 0.0001, one-way ANOVA with Dunnett’s multiple comparisons test, comparing wildtype to variants. 11 1193 Fig. 2 Functional assessment of PRORP variants in tRNA processing assays with mtRNase P. A Cleavage of the pre-tRNAIle 5′ leader sequence by mtRNase P containing wildtype (WT) or variant PRORP. The relative intensity of the pre-tRNAIle cleavage product was quantiﬁed with error bars representing the standard error of the mean. N = 4, p < 0.05, ***p < 0.0001, one-way ANOVA with Dunnett’s multiple comparisons test, comparing wildtype to variants. B Comparison of mtRNase P activity to previously published PRORP variants. N = 5, p < 0.05, ****p < 0.0001, one-way ANOVA with Dunnett’s multiple comparisons test, comparing wildtype to variants. mechanisms of pathology may elucidate how variants with limited effect on mt-tRNA processing (p.Tyr365His) may cause disease. mechanisms of pathology may elucidate how variants with limited effect on mt-tRNA processing (p.Tyr365His) may cause disease. and seizures with white matter changes. Both missense variants reduced the levels of cleaved pre-tRNAIle in vitro, suggesting a relationship of increased phenotypic severity correlated with diminished mtRNase P function. DATA AVAILABILITY h b The p.Ala414Val PRORP variant reported here is in close proximity to the variant p.Asn412Ser reported previously [1], with both variants resulting in a signiﬁcant decrease in cleavage product. Interestingly, the phenotype in individual P2 in the previous report [1] of isolated SNHL was less severe than that observed in F3 in this study. This difference in phenotypic severity could be because the F3 proband is homozygous for the p.Ala414Val variant, whereas individual P2 was compound heterozygous for p.Asn412Ser and the less deleterious c.1301C>A (p.Ala434Asp) PRORP variant. The PRORP variants were submitted to ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) (GenBank: NM_014672.4; accession numbers SCV002820061–SCV002820063). European Journal of Human Genetics (2023) 31:1190 – 1194 REFERENCES 1. Hochberg I, Demain LAM, Richer J, Thompson K, Urquhart JE, Rea A, et al. Bi-allelic variants in the mitochondrial RNase P subunit PRORP cause mitochondrial tRNA processing defects and pleiotropic multisystem presentations. Am J Hum Genet. 2021;108:2195–204. 2. Perrault M, Klotz B, Housset E. Two cases of Turner syndrome with deaf-mutism in two sisters. Bull Mem Soc Med Hop Paris. 1951;67:79–84. Utilising the ClinGen scoring criteria for gene-disease validity [8], the initial PRORP discovery paper scored 9.75, which is concordant with moderate evidence for disease association. With the addition of our families, the score was increased to 12, upgrading the gene-disease association to strong. A third independent report is required for deﬁnitive conﬁrmation that PRORP variants cause disease. 3. Faridi R, Rea A, Fenollar-Ferrer C, O’Keefe RT, Gu S, Munir Z, et al. New insights into Perrault syndrome, a clinically and genetically heterogeneous disorder. Hum Genet. 2022;141:805–19. 3. Faridi R, Rea A, Fenollar-Ferrer C, O’Keefe RT, Gu S, Munir Z, et al. New insights into Perrault syndrome, a clinically and genetically heterogeneous disorder. Hum Genet. 2022;141:805–19. 4. Holzmann J, Frank P, Löfﬂer E, Bennett KL, Gerner C, Rossmanith W. RNase P without RNA: identiﬁcation and functional reconstitution of the human mito- chondrial tRNA processing enzyme. Cell. 2008;135:462–74. 4. Holzmann J, Frank P, Löfﬂer E, Bennett KL, Gerner C, Rossmanith W. RNase P without RNA: identiﬁcation and functional reconstitution of the human mito- chondrial tRNA processing enzyme. Cell. 2008;135:462–74. 5. Mohsen AW, Anderson BD, Volchenboum SL, Battaile KP, Tiffany K, Roberts D, et al. Characterization of molecular defects in isovaleryl-CoA dehydrogenase in patients with isovaleric acidemia. Biochemistry. 1998;37:10325–35. 5. Mohsen AW, Anderson BD, Volchenboum SL, Battaile KP, Tiffany K, Roberts D, et al. Characterization of molecular defects in isovaleryl-CoA dehydrogenase in patients with isovaleric acidemia. Biochemistry. 1998;37:10325–35. In summary, these data provide the ﬁrst independent con- ﬁrmation that biallelic PRORP missense variants can reduce mitochondrial tRNA processing in vitro and are associated with variable, overlapping pleiotropic phenotypes consistent with COXPD54. A possible association between phenotypic severity and mitochondrial tRNA processing deﬁcit is starting to emerge, and further cases need to be deﬁned to determine if robust genotype–phenotype correlations exist. Investigating alternate 6. Ensenauer R, Vockley J, Willard JM, Huey JC, Sass JO, Edland SD, et al. A common mutation is associated with a mild, potentially asymptomatic phenotype in patients with isovaleric acidemia diagnosed by newborn screening. Am J Hum Genet. AUTHOR CONTRIBUTIONS Correspondence and requests for materials should be addressed to William G. Newman or Raymond T. O’Keefe. TBS, WGN and RTO contributed to the conception and design of the study. RWT, WGN and RTO participated in the project supervision. RM, MZ, LH, SS, HG, NSL, TG, KCH and HH were involved in patients’ medical care and interpretation of genetic analyses. TBS, AR, HBT, KT and MO contributed to functional work. TJM and WWY oversaw modelling predictions. All authors contributed to the acquisition and/or analysis of data. TBS, RWT, WGN and RTO wrote the manuscript. All authors revised it critically and approved the ﬁnal version. Reprints and permission information is available at http://www.nature.com/ reprints ETHICS APPROVAL AND CONSENT TO PARTICIPATE 8. Rehm HL, Berg JS, Brooks LD, Bustamante CD, Evans JP, Landrum MJ, et al. ClinGen–the clinical genome resource. N Engl J Med. 2015;372:2235–42. All individuals (or their legal guardians) provided written informed consent in accordance with local regulations. Ethical approval for this study was granted by the All individuals (or their legal guardians) provided written informed consent in accordance with local regulations. Ethical approval for this study was granted by the 9. Ren J, Wen L, Gao X, Jin C, Xue Y, Yao X. DOG 1.0: illustrator of protein domain structures. Cell Res. 2009;19:271–73. National Health Service Ethics Committee (16/WA/0017) and University of Manchester. We would like to thank the families for their participation. Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41431-023-01437-2. Reprints and permission information is available at http://www.nature.com/ reprints reprints Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. European Journal of Human Genetics (2023) 31:1190 – 1194 REFERENCES 2004;75:1136–42. 6. Ensenauer R, Vockley J, Willard JM, Huey JC, Sass JO, Edland SD, et al. A common mutation is associated with a mild, potentially asymptomatic phenotype in patients with isovaleric acidemia diagnosed by newborn screening. Am J Hum Genet. 2004;75:1136–42. 7. Karczewski KJ, Francioli LC, Tiao G, Cummings BB, Alföldi J, Wang Q, et al. The mutational constraint spectrum quantiﬁed from variation in 141,456 humans. Nature. 2020;581:434–43. 7. Karczewski KJ, Francioli LC, Tiao G, Cummings BB, Alföldi J, Wang Q, et al. The mutational constraint spectrum quantiﬁed from variation in 141,456 humans. Nature. 2020;581:434–43. European Journal of Human Genetics (2023) 31:1190 – 1194 T.B. Smith et al. 1194 FUNDING This study was supported by the Medical Research Council (MR/W019027/1), the Royal National Institute for the Deaf/Masonic Charitable Foundation (S60_Newman) and the NIHR Manchester Biomedical Research Centre (NIHR 203308). RWT is supported by the Wellcome Centre for Mitochondrial Research (203105/Z/16/Z), the Mitochondrial Disease Patient Cohort (UK) (G0800674), the Medical Research Council International Centre for Genomic Medicine in Neuromuscular Disease (MR/S005021/ 1), the Lily Foundation, Mito Foundation, the Pathological Society, the UK NIHR Biomedical Research Centre for Ageing and Age-related disease award to the Newcastle upon Tyne Foundation Hospitals NHS Trust and the UK NHS Highly Specialised Service for Rare Mitochondrial Disorders of Adults and Children. MO is funded by Mito Foundation, The Lily Foundation and The Pathological Society. 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/. ADDITIONAL INFORMATION We would like to thank the families for their participation. We would like to thank the families for their participation. COMPETING INTERESTS © The Author(s) 2023 The authors declare no competing interests. European Journal of Human Genetics (2023) 31:1190 – 1194
https://openalex.org/W3125100093	https://www.mdpi.com/1420-3049/24/12/2297/pdf?version=1561102276	English	null	Bridged Nucleic Acids Reloaded	null	2,019	cc-by	12,477	Alfonso Soler-Bistué 1 , Angeles Zorreguieta 2 and Marcelo E. Tolmasky 3,* 1 Instituto de Investigaciones Biotecnológicas Dr. Rodolfo A. Ugalde, Instituto Tecnológico de Chascomús, CONICET, Universidad Nacional de San Martín, San Martín 1650, Argentina; asoler@iib.unsam.edu.ar 2 Fundación Instituto Leloir, IIBBA-CONICET, Buenos Aires C1405BWE, Argentina; azorreguieta@leloir.org.ar 3 Center for Applied Biotechnology Studies, Department of Biological Science, California State University Fullerton, Fullerton, CA 92834-6850, USA 1 Instituto de Investigaciones Biotecnológicas Dr. Rodolfo A. Ugalde, Instituto Tecnológico de Chascomús, CONICET, Universidad Nacional de San Martín, San Martín 1650, Argentina; asoler@iib.unsam.edu.ar 2 Fundación Instituto Leloir, IIBBA-CONICET, Buenos Aires C1405BWE, Argentina; azorreguieta@leloir.org.ar 3 Center for Applied Biotechnology Studies, Department of Biological Science, California State University Fullerton, Fullerton, CA 92834-6850, USA * Correspondence: mtolmasky@fullerton.edu; Tel.: +1-657-278-5263 * Correspondence: mtolmasky@fullerton.edu; Tel.: +1-657-278-5263 Academic Editor: Josef Jampilek Academic Editor: Josef Jampilek p Received: 31 May 2019; Accepted: 18 June 2019; Published: 21 June 2019 Received: 31 May 2019; Accepted: 18 June 2019; Published: 21 June 2019 ved: 31 May 2019; Accepted: 18 June 2019; Published: Abstract: Oligonucleotides are key compounds widely used for research, diagnostics, and therapeutics. The rapid increase in oligonucleotide-based applications, together with the progress in nucleic acids research, has led to the design of nucleotide analogs that, when part of these oligomers, enhance their eﬃciency, bioavailability, or stability. One of the most useful nucleotide analogs is the ﬁrst-generation bridged nucleic acids (BNA), also known as locked nucleic acids (LNA), which were used in combination with ribonucleotides, deoxyribonucleotides, or other analogs to construct oligomers with diverse applications. However, there is still room to improve their eﬃciency, bioavailability, stability, and, importantly, toxicity. A second-generation BNA, BNANC (2′-O,4′-aminoethylene bridged nucleic acid), has been recently made available. Oligomers containing these analogs not only showed less toxicity when compared to LNA-containing compounds but, in some cases, also exhibited higher speciﬁcity. Although there are still few applications where BNANC-containing compounds have been researched, the promising results warrant more eﬀort in incorporating these analogs for other applications. Furthermore, newer BNA compounds will be introduced in the near future, oﬀering great hope to oligonucleotide-based ﬁelds of research and applications. Keywords: oligonucleotides; bridged nucleic acids; locked nucleic acids; antisense; antibiotic resistance; hypercholesterolemia; myotonic dystrophy; CRISPR; Cas9; hematologic malignancies molecules Review Molecules 2019, 24, 2297; doi:10.3390/molecules24122297 molecules molecules 1. Oligonucleotides and Analogs Examples of these compounds are morpholino phosphoroamidates [19], peptide nucleic acids [20,21], nucleotides with modified nucleobases [22], guanidinium-linked oligomers [23], and locked nucleic acids (LNA) [24] (Figure 1). Exhaustive listings and a description of oligonucleotide analogs and their applications can be found in recent reviews [3,6,16,23,25–27]. A derivative of LNA, 2′-O,4′-aminoethylene bridged nucleic acid (BNA), also known as 2′,4′-BNANC (BNANC) [28–31] (Figure 1) has been recently introduced and other derivatives have followed or are in development. As a consequence, LNA is considered the earliest generation of bridged nucleic acids (BNA). This review will focus on the properties and applications of BNANC-containing compounds. Figure 1. Chemical structures of nucleotide analogs. Figure 1. Chemical structures of nucleotide analogs. Fi 1 Ch i l f l id l Figure 1. Chemical structures of nucleotide analogs. 1. Oligonucleotides and Analogs Oligonucleotides are short oligomers composed of ribonucleotides or deoxyribonucleotides. They have multiple uses in basic research, diagnostics, and therapeutics. Their most basic and widespread use is in primer-based techniques, which are used in the most diverse kinds of biological research or development projects, such as polymerase chain reaction (PCR), library construction, single nucleotide polymorphisms detection, gene silencing, tiling arrays, and many others. Besides these applications, oligonucleotides were also found to be immensely useful in the development of gene-silencing techniques. Several approaches were attempted to reduce the undesirable expression of genes, utilizing a variety of strategies, the vast majority of which have in common the utilization of antisense oligonucleotides [1–3]. Many of these compounds are known with diﬀerent names descriptive of their mechanism of action, like external guide sequences [4,5], ribozymes [6,7], aptamers [8,9], short interfering RNA [10–12], and microRNA [13,14]. These compounds interfere with gene expression by steric hindrance of transcription or translation, or by inducing enzymatic cleavage of the target mRNA [3,4,11,13]. The rapid increase in the techniques and applications for which these compounds are integral components, together with the progress in nucleic acid research, led to the design of analogs that are more appropriate to enhance eﬃciency and achieve speciﬁc objectives in each case. Molecules 2019, 24, 2297; doi:10.3390/molecules24122297 www.mdpi.com/journal/molecules www.mdpi.com/journal/molecules 2 of 17 2 of 17 Molecules 2019, 24, 2297 Molecules 2019, 24, x FOR In general, nucleic acid analogs should a) have higher aﬃnity per nucleotide unit than the cognate sequence, without changing the structure of duplexes, b) be resistant to nucleases, and c) have low toxicity. The ﬁrst oligonucleotide analogs consisted of relatively minor modiﬁcations to the natural counterparts, such as the replacement of an oxygen atom by sulfur (phosphorothioate) [15,16], methyl (methylphosphonate) [17], or amino groups (phosphoramidate) (Figure 1). The value of using nucleotide analogs was illustrated by the ﬁrst FDA approved antisense drug, fomivirsen, a 21-nucleotide oligomer composed of phosphorothioate units designed for the treatment of cytomegalovirus retinitis [10,18]. Subsequently, more modiﬁcations were introduced to the nucleotide molecule, such as adding chemical groups to the 2′ position of the ribose as in 2′-O-methyl compounds (Figure 1) or making more drastic structural changes replacing or modifying the ribose, substituting the nature of the bonds, or modifying the charge of the oligonucleotide, obtaining neutral or cationic derivatives. 1. Oligonucleotides and Analogs Examples of these compounds are morpholino phosphoroamidates [19], peptide nucleic acids [20,21], nucleotides with modiﬁed nucleobases [22], guanidinium-linked oligomers [23], and locked nucleic acids (LNA) [24] (Figure 1). Exhaustive listings and a description of oligonucleotide analogs and their applications can be found in recent reviews [3,6,16,23,25–27]. A derivative of LNA, 2′-O,4′-aminoethylene bridged nucleic acid (BNA), also known as 2′,4′-BNANC (BNANC) [28–31] (Figure 1) has been recently introduced and other derivatives have followed or are in development. As a consequence, LNA is considered the earliest generation of bridged nucleic acids (BNA). This review will focus on the properties and applications of BNANC-containing compounds. objectives in each case. In general, nucleic acid analogs should a) have higher affinity per nucleotide unit than the cognate sequence, without changing the structure of duplexes, b) be resistant to nucleases, and c) have low toxicity. The first oligonucleotide analogs consisted of relatively minor modifications to the natural counterparts, such as the replacement of an oxygen atom by sulfur (phosphorothioate) [15,16], methyl (methylphosphonate) [17], or amino groups (phosphoramidate) (Figure 1). The value of using nucleotide analogs was illustrated by the first FDA approved antisense drug, fomivirsen, a 21-nucleotide oligomer composed of phosphorothioate units designed for the treatment of cytomegalovirus retinitis [10,18]. Subsequently, more modifications were introduced to the nucleotide molecule, such as adding chemical groups to the 2′ position of the ribose as in 2′-O- methyl compounds (Figure 1) or making more drastic structural changes replacing or modifying the ribose, substituting the nature of the bonds, or modifying the charge of the oligonucleotide, obtaining neutral or cationic derivatives. Examples of these compounds are morpholino phosphoroamidates [19], peptide nucleic acids [20,21], nucleotides with modified nucleobases [22], guanidinium-linked oligomers [23], and locked nucleic acids (LNA) [24] (Figure 1). Exhaustive listings and a description of oligonucleotide analogs and their applications can be found in recent reviews [3,6,16,23,25–27]. A derivative of LNA, 2′-O,4′-aminoethylene bridged nucleic acid (BNA), also known as 2′,4′-BNANC (BNANC) [28–31] (Figure 1) has been recently introduced and other derivatives have followed or are in development. As a consequence, LNA is considered the earliest generation of bridged nucleic acids (BNA). This review will focus on the properties and applications of BNANC-containing compounds. In general, nucleic acid analogs should a) have higher aﬃnity per nucleotide unit than the cognate sequence, without changing the structure of duplexes, b) be resistant to nucleases, and c) have low toxicity. 1. Oligonucleotides and Analogs The ﬁrst oligonucleotide analogs consisted of relatively minor modiﬁcations to the natural counterparts, such as the replacement of an oxygen atom by sulfur (phosphorothioate) [15,16], methyl (methylphosphonate) [17], or amino groups (phosphoramidate) (Figure 1). The value of using nucleotide analogs was illustrated by the ﬁrst FDA approved antisense drug, fomivirsen, a 21-nucleotide oligomer composed of phosphorothioate units designed for the treatment of cytomegalovirus retinitis [10,18]. Subsequently, more modiﬁcations were introduced to the nucleotide molecule, such as adding chemical groups to the 2′ position of the ribose as in 2′-O-methyl compounds (Figure 1) or making more drastic structural changes replacing or modifying the ribose, substituting the nature of the bonds, or modifying the charge of the oligonucleotide, obtaining neutral or cationic derivatives. Examples of these compounds are morpholino phosphoroamidates [19], peptide nucleic acids [20,21], nucleotides with modiﬁed nucleobases [22], guanidinium-linked oligomers [23], and locked nucleic acids (LNA) [24] (Figure 1). Exhaustive listings and a description of oligonucleotide analogs and their applications can be found in recent reviews [3,6,16,23,25–27]. A derivative of LNA, 2′-O,4′-aminoethylene bridged nucleic acid (BNA), also known as 2′,4′-BNANC (BNANC) [28–31] (Figure 1) has been recently introduced and other derivatives have followed or are in development. As a consequence, LNA is considered the earliest generation of bridged nucleic acids (BNA). This review will focus on the properties and applications of BNANC-containing compounds. objectives in each case. In general, nucleic acid analogs should a) have higher affinity per nucleotide unit than the cognate sequence, without changing the structure of duplexes, b) be resistant to nucleases, and c) have low toxicity. The first oligonucleotide analogs consisted of relatively minor modifications to the natural counterparts, such as the replacement of an oxygen atom by sulfur (phosphorothioate) [15,16], methyl (methylphosphonate) [17], or amino groups (phosphoramidate) (Figure 1). The value of using nucleotide analogs was illustrated by the first FDA approved antisense drug, fomivirsen, a 21-nucleotide oligomer composed of phosphorothioate units designed for the treatment of cytomegalovirus retinitis [10,18]. Subsequently, more modifications were introduced to the nucleotide molecule, such as adding chemical groups to the 2′ position of the ribose as in 2′-O- methyl compounds (Figure 1) or making more drastic structural changes replacing or modifying the ribose, substituting the nature of the bonds, or modifying the charge of the oligonucleotide, obtaining neutral or cationic derivatives. Fi 2. LNA—A Brief Overview However, although internalization by diverse pathways and reasonable levels of activity were reported in the case of eukaryotic cells [68–72], the levels of internalization into bacterial cells seems not to be enough for productive inhibition of gene expression [66]. LNA-containing oligomers could be delivered to cultured eukaryotic cells by transfection reagents [73–76]. Other strategies to facilitate uptake, such as conjugation to cell penetrating peptides (CPP), which are usually cationic, has been challenging and reports of their utilization to silence gene expression are scarce. Turner et al. [77] reported the attachment of a CPP to an LNA/2′-O-methyl oligonucleotide; cell uptake of this compound was signiﬁcantly increased with respect to the naked antisense, but levels of inhibition of gene expression were disappointing. An LNA/DNA gapmer designed to target the amikacin resistance aac(6′)-Ib gene mRNA and elicit cleavage by the endogenous RNase P [4] was covalently bound to a CPP. The compound produced a modest reduction in the levels of resistance to amikacin in a clinical Acinetobacter baumannii isolate [78]. LNA-containing compounds were also delivered inside target cells using nanoparticles [79]. An 8-nucleotide LNA oligomer complementary to the oncogenic miR21 included in micelles could be delivered inside cancer cells and induced apoptosis [79]. Furthermore, these assays showed tumor growth inhibition in an animal model [79]. the 4′-carbon (2′-O,4′-methylene-β-d-ribofuranosyl nucleotides) [32–37]. They are characterized by reduced ﬂexibility of the ribose residue and exist in a locked N-type conformation, which favors the formation of stable duplexes with DNA or RNA [34]. Numerous structural and thermal stability studies on complexes formed by LNA oligomers and complementary DNA or RNA oligonucleotides showed higher melting temperatures and speciﬁcity when compared to the unmodiﬁed isosequential compounds [32,34,37–39]. LNA-containing oligomers are usually synthesized as chimeras containing a combination of ribonucleotide/deoxyribonucleotide or other nucleotide analogs and LNA residues [40–44]. The incorporation of LNA in oligonucleotides leads to melting temperature increases of 3–9 ◦C per residue [45]. Most LNA-containing chimeras can be classiﬁed into two kinds, gapmers and mixmers. Gapmers consist of oligomers where the LNA residues are located at the ends of the compound. Mixmers include the LNA and the other residues interspersed in diﬀerent conﬁgurations throughout the sequence. LNA-containing oligomers have been used for diagnostics and other applications, as probes or primers for hybridization, ampliﬁcation, mutagenesis, sequencing, and SNP genotyping [46–53], in addition to gene repair [54] and antisense drugs [13,41,42,55]. Fi 2. LNA—A Brief Overview While the increased binding capacity is advantageous in many antisense applications, it can also be detrimental due to the formation of duplex structures that cannot be recognized as substrates by the enzymes recruited to degrade the target molecule. The high aﬃnity of LNA-containing oligonucleotides can also result in toxic eﬀects due to unspeciﬁc oﬀ-target binding. Fortunately, recent studies showed that LNA-containing oligomers were innocuous in primates [56] and relatively safe in humans [57–59]. Furthermore, these compounds failed to show genotoxicity [60]. However, these results are far from deﬁnitive; other studies showed hepatoxicity [61–64]. Although the toxicity determinations of LNA-containing oligomers are encouraging, case-by-case studies will decide the possibility of development as therapies for human disease. At the moment, numerous potential drugs based on this nucleic acid analog are already in clinical trials (e.g., Miravirsen, MRG-106, and ISTH0036) [10,11]. An obstacle in the development of LNA-containing oligomers as drugs, particularly in silencing prokaryotic genes, is the low or null uptake by bacterial cells. Gymnotic uptake of LNA-containing oligomers was shown in eukaryotic and prokaryotic cells [65–67]. However, although internalization by diverse pathways and reasonable levels of activity were reported in the case of eukaryotic cells [68–72], the levels of internalization into bacterial cells seems not to be enough for productive inhibition of gene expression [66]. LNA-containing oligomers could be delivered to cultured eukaryotic cells by transfection reagents [73–76]. Other strategies to facilitate uptake, such as conjugation to cell penetrating peptides (CPP), which are usually cationic, has been challenging and reports of their utilization to silence gene expression are scarce. Turner et al. [77] reported the attachment of a CPP to an LNA/2′-O-methyl oligonucleotide; cell uptake of this compound was signiﬁcantly increased with respect to the naked antisense, but levels of inhibition of gene expression were disappointing. An LNA/DNA gapmer designed to target the amikacin resistance aac(6′)-Ib gene mRNA and elicit cleavage by the endogenous RNase P [4] was covalently bound to a CPP. The compound produced a modest reduction in the levels of resistance to amikacin in a clinical Acinetobacter baumannii isolate [78]. LNA-containing compounds were also delivered inside target cells using nanoparticles [79]. An 8-nucleotide LNA oligomer complementary to the oncogenic miR21 included in micelles could be delivered inside cancer cells and induced apoptosis [79]. Furthermore, these assays showed tumor growth inhibition in an animal model [79]. In summary, LNA-containing compounds show great promise as therapeutic agents [42,80,81]. Fi 2. LNA—A Brief Overview H h i d d i ll i hil k i h i bi l i l i i In summary, LNA-containing compounds show great promise as therapeutic agents [42,80,81]. However, more research is needed to improve cell penetration while keeping their biological activity and reduced toxicity. Comprehensive descriptions of properties and applications of LNA containing oligomers have been recently published [82–86]. Fi 2. LNA—A Brief Overview LNA compounds, ﬁrst introduced in the late 1990s, are bicyclic nucleotide analogs in which the furanose ring is modiﬁed by the introduction of a methylene group linking the 2′-oxygen and 3 of 17 Molecules 2019, 24, 2297 the 4′-carbon (2′-O,4′-methylene-β-d-ribofuranosyl nucleotides) [32–37]. They are characterized by reduced ﬂexibility of the ribose residue and exist in a locked N-type conformation, which favors the formation of stable duplexes with DNA or RNA [34]. Numerous structural and thermal stability studies on complexes formed by LNA oligomers and complementary DNA or RNA oligonucleotides showed higher melting temperatures and speciﬁcity when compared to the unmodiﬁed isosequential compounds [32,34,37–39]. LNA-containing oligomers are usually synthesized as chimeras containing a combination of ribonucleotide/deoxyribonucleotide or other nucleotide analogs and LNA residues [40–44]. The incorporation of LNA in oligonucleotides leads to melting temperature increases of 3–9 ◦C per residue [45]. Most LNA-containing chimeras can be classiﬁed into two kinds, gapmers and mixmers. Gapmers consist of oligomers where the LNA residues are located at the ends of the compound. Mixmers include the LNA and the other residues interspersed in diﬀerent conﬁgurations throughout the sequence. LNA-containing oligomers have been used for diagnostics and other applications, as probes or primers for hybridization, ampliﬁcation, mutagenesis, sequencing, and SNP genotyping [46–53], in addition to gene repair [54] and antisense drugs [13,41,42,55]. While the increased binding capacity is advantageous in many antisense applications, it can also be detrimental due to the formation of duplex structures that cannot be recognized as substrates by the enzymes recruited to degrade the target molecule. The high aﬃnity of LNA-containing oligonucleotides can also result in toxic eﬀects due to unspeciﬁc oﬀ-target binding. Fortunately, recent studies showed that LNA-containing oligomers were innocuous in primates [56] and relatively safe in humans [57–59]. Furthermore, these compounds failed to show genotoxicity [60]. However, these results are far from deﬁnitive; other studies showed hepatoxicity [61–64]. Although the toxicity determinations of LNA-containing oligomers are encouraging, case-by-case studies will decide the possibility of development as therapies for human disease. At the moment, numerous potential drugs based on this nucleic acid analog are already in clinical trials (e.g., Miravirsen, MRG-106, and ISTH0036) [10,11]. An obstacle in the development of LNA-containing oligomers as drugs, particularly in silencing prokaryotic genes, is the low or null uptake by bacterial cells. Gymnotic uptake of LNA-containing oligomers was shown in eukaryotic and prokaryotic cells [65–67]. 3. BNANC The success and advantages of LNA-containing oligomers for diverse applications stimulated the search for similar compounds, improving their properties. Many derivatives were recently 4 of 17 Molecules 2019, 24, 2297 introduced, like BNANC with diﬀerent substitutions at the N atom (of which a methyl group is the most commonly used to date) (Figure 1) [87], 2′-O,4′-C-ethylene-bridged nucleic acid (ENA) [88], 2′-O,4′-C-methylenoxymethylene-bridged nucleic acid [89], or unlocked nucleic acids (UNA) [90,91]. The bridge in the diﬀerent BNA compounds can have a diﬀerent number of members in the ring, the most widely used to date being BNANC, a six-member ring [30,31]. Oligonucleotides containing BNANC are more resistant to nucleases and less toxic than isosequential compounds containing LNA residues, they show high thermal stability and water solubility, and elicit RNase H degradation of a target RNA [29,62,87,92]. Twelve-mer oligodeoxynucleotides containing a variable number of BNANC or LNA residues showed a similar increase in melting temperature (Tm) per modiﬁed residue (4.7 to 7.0 ◦C) in comparisons of duplex stabilities with complementary single-stranded RNA (ssRNA). However, this was not the case for duplexes with complementary single-stranded DNA (ssDNA) [29]. Increasing the number of BNANC residues in the oligonucleotides did not result in a signiﬁcant Tm increase (−1.0 to 1.8 ◦C) as was the case when the analog used was LNA (1.3 to 3.0 ◦C) [29]. While the Tm values of the unsubstituted oligodeoxynucleotide in duplex with complementary ssRNA was about 5 ◦C higher than with complementary ssRNA, the Tm values for substituted oligonucleotides were higher for duplexes with complementary ssRNA. Furthermore, the values were higher for oligonucleotides substituted with BNANC than those that included LNA residues [29]. These results indicate that BNANC residues conferred a higher RNA selective binding aﬃnity to the antisense oligomers. BNANC-containing oligonucleotides also showed higher mismatch discrimination properties as well as more stable triplexes than LNA-containing oligomers [29,93]. BNANC-containing oligonucleotides have the potential to be utilized in diverse applications. In this review, we will focus on their utilization for diverse uses. The diﬀerent applications of the compounds reviewed in this article are summarized in Table 1. Table 1. BNANC applications. Organism Function or Disease Target Chemical Nature of Oligonucleotide Sequence of Active Oligomer Reference A. 3. BNANC baumannii Resistance to aminoglycosides aac(6′)-Ib BNANC/DNA conjugated to (RXR)4XB 1 (RXR)4XB-Cys-SMCC-C6 amino-cTgctGcgtAacaTc [94] Cell lines Myotonic dystrophy type 1 DMPK 2 BNANC/DNA gapmer CGGAGcggttgtgaaCTGGC [95] Murine, human cell lines, and mice Hypercholesterolemia PCSK9 3 BNANC/DNA mixmer CCaggCCTaTgagggTgCCg [92] Human gene Hematologic malignancies DNTM3A 4 BNANC/DNA mixmers cgccaAgcgGctcatgtt cgccAAgcagctcAtgtt cgccAAgtgGctcAtgtt cgccaAggggCtcatgtt cgccAAgctgCtcAtgtt [96] Human gene CRISPR-Cas9 speciﬁcity WAS 5 crRNA with BNANC substitutions uggauggagGAAugaggagu [97] Human gene CRISPR-Cas9 speciﬁcity EXM1 6 crRNA with BNANC substitutions gaguccgagcaGAAgaagaa [97] 1 R, arginine; X, 6-aminohexanoic acid; B, β-alanine; 2 Dystrophia myotonica protein kinase; 3 Proprotein convertase subtilisin/kexin type 9; 4 Human DNA methyltransferase 3A; 5 Gene responsible for Wiskott-Aldrich Syndrome; 6 Homeobox protein EMX1. 1 R, arginine; X, 6-aminohexanoic acid; B, β-alanine; 2 Dystrophia myotonica protein kinase; 3 Proprotein convertase subtilisin/kexin type 9; 4 Human DNA methyltransferase 3A; 5 Gene responsible for Wiskott-Aldrich Syndrome; 6 Homeobox protein EMX1. 3.1. Antisense Inhibition of Resistance to Amikacin by a BNANC-Containing Oligomer 3.1. Antisense Inhibition of Resistance to Amikacin by a BNANC-Containing Oligomer The inhibition of the expression of genes coding for antimicrobial resistance enzymes by diverse antisense mechanisms is the object of intense investigation, as a way to deal with the growing multiresistance problem [4,98–101]. An active antisense molecule could be combined as an adjuvant to the cognate antibiotic to treat resistant infections. The concept of treating resistant infections with combinations of antibiotic/inhibitor of resistance has reached the stage of human use in the case of β-lactam antibiotics that are administered in combination with β-lactamase inhibitors [99,102,103]. However, combinations of other kinds of antibiotics with inhibitors of resistance are still in experimental 5 of 17 Molecules 2019, 24, 2297 stages [99,104]. In particular, antisense inhibition of antibiotic resistance genes was explored using oligomers of diﬀerent natures that interfere with the expression of resistance by diﬀerent mechanisms [41, 42,78,98,101,105–108]. The aac(6′)-Ib gene codes for an acetyltransferase responsible for the resistance to amikacin and other aminoglycosides found in the vast majority of AAC(6′)-I-producing Gram-negative clinical isolates [104,109]. An antisense oligodeoxynucleotide complementary to a duplicated sequence located at the translation initiation location of the aac(6′)-Ib allele found in a clinical Acinetobacter baumannii isolate [110] inhibited translation in vitro [94]. An isosequential 15-residue antisense mixmer, including four BNANC and 11 deoxynucleotide residues, was covalently bound to the permeabilizing peptide (RXR)4XB (R, arginine; X, 6-aminohexanoic acid; B, β-alanine) to generate a compound resistant to nucleases and capable of penetrating the Gram-negative envelope to reach the cytosol. This BNANC-containing mixmer, designated CPPBD4, successfully inhibited growth in a liquid culture containing amikacin. Furthermore, a combination of CPPBD4/amikacin reduced the mortality of Galleria mellonella infected with amikacin-resistant A. baumannii to levels comparable to those of the uninfected controls [94]. BNANC-containing oligomers were also researched as elicitors of RNase P-mediated speciﬁc mRNA degradation, an antisense methodology known as external guide sequence (EGS) technology [4,111]. In this approach, antisense oligomers, known as EGSs, interact with the target mRNA, forming a structure that is recognized as a substrate by the endogenous RNase P. Then, the enzyme cleaves the mRNA, preventing its translation [5]. Early experiments showed that EGS molecules eﬃciently reduced the expression of aac(6′)-Ib and levels of resistance to amikacin [108]. Analysis of various nuclease-resistant oligonucleotide analogs indicated that DNA/LNA hybrid oligomers were eﬃcient EGSs that reversed aac(6′)-Ib-mediated resistance to amikacin in a hyperpermeable Escherichia coli strain [42]. 3.1. Antisense Inhibition of Resistance to Amikacin by a BNANC-Containing Oligomer Furthermore, conjugation of a selected DNA/LNA hybrid oligomer to the cell permeabilizing peptide (RXR)4XB reduced the levels of resistance to amikacin of an aac(6′)-Ib-containing Acinetobacter baumannii clinical isolate [41,78]. Assessment of isosequential hybrid oligomers containing BNANC in the place of the LNA residues showed that they failed to elicit cleavage of the mRNA at levels comparable to those found when testing LNA/DNAs [41]. 3.3. Reduction of Cholesterol Levels by BNANC Mixmers in Hypercholesterolemic Mice Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a role in the maintenance of cholesterol balance [118]. Gain-of-function mutations in this gene are associated with an increase in low-density lipoprotein cholesterol levels (i.e., autosomal dominant hypercholesterolemia), a known risk factor for coronary heart disease [119]. Conversely, loss-of-function mutations are responsible for low plasma low-density lipoprotein cholesterol (LDL-C) levels and reduced incidence of cardiovascular disease [120]. These phenotypes are the consequence of the ability of PCSK9 to interact with the LDL receptor (LDLR). The complex PCSK9–LDLR is transported from the cell’s surface to the endosome, where LDLR is degraded [121]. The involvement of PCSK9 in the modulation of LDL-C levels led to attempts to suppress its synthesis or activity that resulted in the development of drugs that have been approved by the FDA [122–126]. In particular, the inhibition of the expression of PCSK9 by antisense oligonucleotide analogs was attempted by several research groups. A 2′-O-methoxyethyl-modiﬁed phosphorothioate oligonucleotide analog modestly reduced hepatic PCSK9 mRNA and LDL-C in treated mice [127]. In another study, an LNA-containing gapmer was found to be more eﬃcient at lowering PCSK9 mRNA and LDL-C while increasing LDLR levels [74]. However, LNA-containing gapmers usually show high hepatotoxicity. BNANC-containing oligomers could be a better option to design compounds that show high eﬃciency without the high levels of toxicity observed when the analog used is LNA. Yamamoto et al. [92] tested LNA- and BNANC-containing antisense mixmers in cultured cells as well as in mice. Cells transfected with mixmers containing one or the other analog showed a dose-dependent reduction of PCSK9 mRNA and increase of LDLR protein levels. Also, the administration of these compounds to atherogenic diet-fed mice biweekly for six weeks resulted in a reduction of PCSK9 mRNA and LDL-C as well as an increase in high-density lipoprotein cholesterol. However, the comparison also showed that the animals treated with the BNANC-containing antisense mixmer experienced a reduction of LDL-C after a shorter time and tolerance was higher. These comparative studies identiﬁed BNANC-containing antisense compounds as better candidates than LNA-containing compounds. 3.4. Diagnostics of Hematologic Malignancies by the Detection of Somatic Mutations Using BNANC Mixmers DNA methylation, an epigenetic modiﬁcation, is involved in critical cellular processes, such as replication and transcription. Hypomethylation is correlated to some human cancers [128]. 3.2. Reversion of Splicing and Reduction of RNA Foci in Myotonic Dystrophy Cells by BNANC Gapmers 3.2. Reversion of Splicing and Reduction of RNA Foci in Myotonic Dystrophy Cells by BNANC Gapmers Myotonic dystrophy type 1 patients may suﬀer from skeletal muscle weakening and wasting, abnormalities in heart function, cataracts, breathing problems, speech and swallowing disorders, and other impairing symptoms [112]. The molecular basis of this condition is the presence of a CUG repeat expansion within the DMPK gene 3′-untranslated region [113,114]. This kind of genetic modiﬁcation, in which a group of nucleotides that in healthy genes is repeated a variable number of times, exists in an abnormally high number of repeat-characterized diseases, known as microsatellite expansion disorders [115,116]. The DMPK gene anomaly causes the mRNA to remain in the nucleus and to form foci structures, resulting in defects in developmentally regulated alternative splicing [117]. Removal of the toxic RNA species using antisense oligomers that induce RNase H cleavage is being intensely researched as a therapeutic strategy. Early work by researchers at IONIS Pharmaceuticals utilizing a gapmer consisting of a short DNA stretch ﬂanked by 2′-O-methoxyethyl residues designed to induce cleavage of the toxic RNA in muscle cells showed encouraging splicing changes, but stopped short of the goals of the trial [95]. The potency of these compounds can be substantially increased by replacing the 2′-O-methoxyethyl analogs with LNA residues [62]. However, antisense LNA-containing gapmers showed high hepatotoxicity [62–64]. The potency and toxicity of BNANC-containing gapmers as compared to isosequential LNA-containing gapmers was recently determined [95]. Both compounds were nearly equally eﬃcient in inducing the cleavage of the CUG repeat expansion-containing mRNA in cells transformed with a plasmid designed to express this RNA species. However, comparison of the toxicity of both antisense compounds showed that the LNA-containing gapmer induced an increase in caspases, the apoptosis eﬀector proteins, that was not observed with BNANC-containing gapmers [95]. Interestingly, a comparison of the region targeted showed that a gapmer complementary 6 of 17 Molecules 2019, 24, 2297 to a non-repetitive region of the RNA was more speciﬁc in eliciting degradation of the toxic RNA than a gapmer complementary to a segment containing the CUG repeats (Table 1) [95]. These studies indicate that the use of BNANC-containing gapmers could open new venues for developing therapies against myotonic dystrophy type 1. 3.3. Reduction of Cholesterol Levels by BNANC Mixmers in Hypercholesterolemic Mice 3.5. Incorporation of BNANC Residues to crRNA as an Enhancer of Cas9 Endonuclease Speciﬁcity Bacteria have evolved numerous strategies to defend against the presence of foreign genetic material, such as bacteriophage genomes, transposons, and plasmids. They include restriction-modiﬁcation systems, abortive infections and adsorption blockage, and surface exclusion [145–147]. While these systems are relatively unspeciﬁc, the latest discovered defense system, known as “clustered regularly interspaced short palindromic repeats” (CRISPR) is speciﬁc and, in a surprisingly analogous mode to vertebrate immune systems, it requires previous exposure to the foreign genetic material to create a memory record that can elicit a quick response in future exposure events [148]. In this system, small guide RNA molecules (crRNAs) guide the sequence-speciﬁc cleavage of foreign nucleic acids [149]. However, diﬀerences in molecular mechanisms permitted the classiﬁcation in two classes, class 1 and 2, which depend on a multiprotein complex or a single protein, respectively [149]. Each class is further divided into three types. In particular, the class 2, type II system, which depends on the endonuclease Cas9 (Figure 2), has been developed as a tool for numerous applications, such as the generation of mutants, gene editing, bacterial species identiﬁcation and typing, antibacterial agents, genome-wide screening in mammalian cells, regulation of gene expression (through the use of dCas9, a derivative that lost the cleavage activity), and others [148–152]. Molecules 2019, 24, x FOR PEER REVIEW 8 of 17 containing BNANC residues at positions 12–14 isosequential to the EXM1 crRNA (Table 1) specifically guided cleavage of the wild type target. Similarly, a crRNA containing BNANC residues at positions 10–12 (Table 1) guided cleavage of the wild type and only one of the modified targets. These results showed a significant enhancement of specificity in vitro when three nucleotides were replaced by BNANC residues. Interestingly, BNANC-containing crRNA molecules are compatible with Cas9 variants with improved specificity. Following these encouraging results, experiments to find out if the enhanced specificity observed in the in vitro experiments described above also occurs in vivo were carried out. Cas9-producing U2OS and HeLa cells were transfected with the unmodified and modified crRNA molecules, targeting the WAS and EXM1 genes. The levels of cleavage at the target and off-target sites were consistent with those observed in vitro. This work established that BNANC- containing crRNAs could be a venue for improving Cas9 cleavage specificity [97]. Figure 2. Molecular mechanism of Cas9-mediated digestion of a target DNA. The crRNA and tracrRNA are shown in red and blue, respectively. 3.3. Reduction of Cholesterol Levels by BNANC Mixmers in Hypercholesterolemic Mice The human DNA methyltransferase 3A (DNMT3A) is a 130 kDa protein that includes three domains, one of which is the S-adenosyl methionine-dependent methyltransferase that recognizes and binds DNA to catalyze the transfer of a methyl group to the C5 position of cytosine from S-adenosyl methionine [129]. The nuclear DNMT3A molecules can exist in oligomeric form as dimers, tetramers, and larger structures held by interactions of binding interfaces in the methyltransferase domain [130]. Numerous hematologic malignancies are characterized by the presence of somatic mutations in the DNA methyltransferase 3A (DNMT3A) gene [128]. Mutations on this gene are found in up to 35% of cases of diﬀerent myeloid malignancies [131–137]. The detection of mutations in this gene could, therefore, be a component of a group of tests to predict a higher risk of myeloid malignancies [138,139]. Techniques used to detect DNMT3A mutations include DNA sequencing, high-resolution DNA melting, restriction fragment length polymorphism, and denaturing high-performance liquid chromatography [140–144]. A microsphere-based suspension assay that utilizes oligonucleotide analogs, LNA- or BNANC-containing, as probes speciﬁc for wild type or mutant alleles was more eﬃcient than direct sequencing [96]. In this study, LNA- or BNANC-containing oligonucleotides speciﬁc for the wild type or four mutations within the codon R882 were coupled 7 of 17 Molecules 2019, 24, 2297 to ﬂuorescently labeled microspheres, which were used in hybridization assays against DNMT3A amplicons. The utilization of LNA- or BNANC-containing analogs facilitated the design of sequences that can discriminate between sequences that diﬀered in a single nucleotide. A comparison of isosequential oligonucleotides that diﬀer in the nature of the analog residues showed that BNANC-containing compounds showed the highest sensitivity, as well as speciﬁcity. These researchers concluded that BNANC-containing probes, coupled with ﬂuorescently labeled microspheres, are suitable reagents to detect DNMT3A R888 mutations [96]. 3.5. Incorporation of BNANC Residues to crRNA as an Enhancer of Cas9 Endonuclease Speciﬁcity 3.5. Incorporation of BNANC Residues to crRNA as an Enhancer of Cas9 Endonuclease Speciﬁcity The arrows indicate the point of cleavage on each Figure 2. Molecular mechanism of Cas9-mediated digestion of a target DNA. The crRNA and tracrRNA are shown in red and blue, respectively. The arrows indicate the point of cleavage on each target DNA strand. Figure 2. Molecular mechanism of Cas9-mediated digestion of a target DNA. The crRNA and tracrRNA are shown in red and blue, respectively. The arrows indicate the point of cleavage on each Figure 2. Molecular mechanism of Cas9-mediated digestion of a target DNA. The crRNA and tracrRNA are shown in red and blue, respectively. The arrows indicate the point of cleavage on each target DNA strand. target DNA strand. 4. Final Remarks In the past few years, oligonucleotides have been used for an increasing number of applications in basic science, as well as in clinics and other settings. In particular, applications related to human health include detection and diagnostics of pathologies caused by mutations, silencing of undesirable genes responsible for numerous genetic conditions, or interference with the expression of bacterial or viral genes to fight infection. The increased utilization of these compounds requires the constant search for improvement of their properties The key aspects that enhance the usability and efficiency The Cas9 system relies on two RNA molecules, a crRNA that includes a 20-nucleotide sequence complementary to the target DNA and a trans-acting molecule (tracrRNA) that acts as bridge between the crRNA and the Cas9 endonuclease (Figure 2) [153–155]. Experiments in which the tracrRNA and crRNA are covalently bound, forming a single guide RNA (sgRNA) showed that sgRNAs form viable complexes that result in target cleavage [155]. The Cas9 protein ﬁrst recognizes protospacer-adjacent motif (PAM) sequences on the target DNA. The role of the PAM sequences is to help in distinguishing self from foreign DNA. Then, a crRNA 20-nucleotides region pairs with the target DNA, forming the multicomponent complex that causes Cas9 double-stranded blunt-ended cleavage [153,156,157]. 8 of 17 8 of 17 Molecules 2019, 24, 2297 The Cas9 system has been widely adapted for gene editing and other applications [152,158–161]. An important consideration in the use of CRISPR-Cas9 as a tool is its speciﬁcity. While mutations in the PAM sequences usually interfere with cleavage of the target, mutations within the target sequence that interacts with crRNA (or sgRNA) can be tolerated, permitting digestion at oﬀ-target locations [162,163]. 3.5. Incorporation of BNANC Residues to crRNA as an Enhancer of Cas9 Endonuclease Speciﬁcity Since enhancing the speciﬁcity of cleavage is such an important aspect for numerous applications of CRISPR-Cas9, it is not surprising that several approaches have been tested to reduce oﬀ-target action [97,164–166]. The vast majority of these studies focused on the Cas9 protein [165,167–170]. Few others concentrated on modiﬁcations to the crRNA [97,170–172]. Cromwell et al. recently took advantage of the enhanced mismatch discrimination of BNANC-containing oligomers to design crRNA molecules that elicit cleavage speciﬁcity by the cas9 endonuclease [97]. Two crRNA molecules directed to the Wiskott–Aldrich syndrome (WAS) [173] and the homeobox EMX1 [174] genes that are known to also aﬀect oﬀ-target sites were used to test the eﬀect of replacing from one to four nucleotides with BNANC residues. In vitro cleavage assays were carried, out using as the target the wild type WAS or EMX1 sequences and ﬁve sequences including one, two, or three nucleotide substitutions for each of the genes. These wild type and modiﬁed target sequences were incubated in the presence of the endonuclease and crRNA or the isosequential variants, including one to eight BNANC residue replacements. While the crRNA molecules directed cleavage of the wild type and all of the modiﬁed targets, a crRNA containing BNANC residues at positions 12–14 isosequential to the EXM1 crRNA (Table 1) speciﬁcally guided cleavage of the wild type target. Similarly, a crRNA containing BNANC residues at positions 10–12 (Table 1) guided cleavage of the wild type and only one of the modiﬁed targets. These results showed a signiﬁcant enhancement of speciﬁcity in vitro when three nucleotides were replaced by BNANC residues. Interestingly, BNANC-containing crRNA molecules are compatible with Cas9 variants with improved speciﬁcity. Following these encouraging results, experiments to ﬁnd out if the enhanced speciﬁcity observed in the in vitro experiments described above also occurs in vivo were carried out. Cas9-producing U2OS and HeLa cells were transfected with the unmodiﬁed and modiﬁed crRNA molecules, targeting the WAS and EXM1 genes. The levels of cleavage at the target and oﬀ-target sites were consistent with those observed in vitro. This work established that BNANC-containing crRNAs could be a venue for improving Cas9 cleavage speciﬁcity [97]. References 1. Bennett, C.F.; Swayze, E.E. RNA targeting therapeutics: Molecular mechanisms of antisense oligonucleotides as a therapeutic platform. Annu. Rev. Pharmacol. Toxicol. 2010, 50, 259–293. [CrossRef] [PubMed] 2. Thomason, M.K.; Storz, G. Bacterial antisense RNAs: How many are there, and what are they doing? Annu. Rev. Genet. 2010, 44, 167–188. [CrossRef] [PubMed] 3. Rasmussen, L.C.; Sperling-Petersen, H.U.; Mortensen, K.K. Hitting bacteria at the heart of the central dogma: Sequence-speciﬁc inhibition. Microb. Cell Fact. 2007, 6, 24. [CrossRef] 4. Davies-Sala, C.; Soler-Bistue, A.; Bonomo, R.A.; Zorreguieta, A.; Tolmasky, M.E. External guide sequence technology: A path to development of novel antimicrobial therapeutics. Ann. N.Y. Acad. Sci. 2015, 1354, 98–110. [CrossRef] [PubMed] 5. Forster, A.C.; Altman, S. External guide sequences for an RNA enzyme. Science 1990, 249, 783–786. [CrossRef] [PubMed] 6. Burnett, J.C.; Rossi, J.J. RNA-based therapeutics: Current progress and future prospects. Chem. Biol. 2012, 19, 60–71. [CrossRef] [PubMed] 7. Li, M.J.; Kim, J.; Li, S.; Zaia, J.; Yee, J.K.; Anderson, J.; Akkina, R.; Rossi, J.J. Long-term inhibition of HIV-1 infection in primary hematopoietic cells by lentiviral vector delivery of a triple combination of anti-HIV shRNA, anti-CCR5 ribozyme, and a nucleolar-localizing TAR decoy. Mol. Ther. 2005, 12, 900–909. [CrossRef] Ni, X.; Castanares, M.; Mukherjee, A.; Lupold, S.E. Nucleic acid aptamers: Clinical applications and promising new horizons. Curr. Med. Chem. 2011, 18, 4206–4214. [CrossRef] p g 9. Ellington, A.D.; Szostak, J.W. In vitro selection of RNA molecules that bind speciﬁc ligands. Nature 1990, 346, 818–822. [CrossRef] 10. Crooke, S.T.; Witztum, J.L.; Bennett, C.F.; Baker, B.F. RNA-Targeted Therapeutics. Cell Metab. 2018, 27, 714–739. [CrossRef] 11. Setten, R.L.; Rossi, J.J.; Han, S.P. The current state and future directions of RNAi-based therapeutics. Nat. Rev. Drug Discov. 2019, 18, 421–446. [CrossRef] [PubMed] 12. Caplen, N.J.; Parrish, S.; Imani, F.; Fire, A.; Morgan, R.A. Speciﬁc inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc. Natl. Acad. Sci. USA 2001, 98, 9742–9747. [CrossRef] [PubMed] 13. van Rooij, E.; Kauppinen, S. Development of microRNA therapeutics is coming of age. EMBO Mol. Med. 2014, 6, 851–864. [CrossRef] [PubMed] van Rooij, E.; Sutherland, L.B.; Qi, X.; Richardson, J.A.; Hill, J.; Olson, E.N. Control of stress-dependen cardiac growth and gene expression by a microRNA. Science 2007, 316, 575–579. [CrossRef] 15. Eckstein, F. Nucleoside phosphorothioates. J. Am. Chem. Soc. 1966, 88, 4292–4294. [CrossRef] 16. Eckstein, F. Phosphorothioates, essential components of therapeutic oligonucleotides. Nucleic Acid Ther. 2014, 24, 374–387. [CrossRef] [PubMed] 17. Author Contributions: All authors contributed equally to writing this article. Author Contributions: All authors contributed equally to writing this article. Funding: Authors’ work cited in this review article was funded by Public Health Service Grant 2R15AI047 rom the National Institutes of Health. Funding: Authors’ work cited in this review article was funded by Public Health Service Grant 2R15AI047115 from the National Institutes of Health. Acknowledgments: Authors’ work cited in this review article was funded by Public Health Service Grant 2R15AI047115 from the National Institutes of Health. AZ and ASB are Career members of the Consejo Nacional del Investigaciones Cientíﬁcas y Tecnológicas (CONICET). Acknowledgments: Authors’ work cited in this review article was funded by Public Health Service Grant 2R15AI047115 from the National Institutes of Health. AZ and ASB are Career members of the Consejo Nacional del Investigaciones Cientíﬁcas y Tecnológicas (CONICET). Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the writing of the manuscript, or in the decision to publish the results. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the writing of the manuscript, or in the decision to publish the results. 4. Final Remarks In the past few years, oligonucleotides have been used for an increasing number of applications in basic science, as well as in clinics and other settings. In particular, applications related to human health include detection and diagnostics of pathologies caused by mutations, silencing of undesirable genes responsible for numerous genetic conditions, or interference with the expression of bacterial or viral genes to ﬁght infection. The increased utilization of these compounds requires the constant search for improvement of their properties. The key aspects that enhance the usability and eﬃciency of oligonucleotides are their stability, speciﬁcity, and bioavailability. While the latter has been dealt with by conjugating the compounds to permeabilizing peptides, using transfecting agents, packaging them into liposomes, and combining them with nanoparticles, stability and speciﬁcity were dramatically improved by the substitution of ribonucleotide or deoxyribonucleotide residues with analogs. The success observed by these chemical modiﬁcations incentivized the search for new and varying nucleotide analogs that enhanced the resistance to nucleases, speciﬁcity, aﬃnity, and activity of the oligomers. The ﬁrst-generation BNA, known as LNA, has been used for several applications with great success. However, despite the promising results achieved with LNA-containing oligomers, toxicity has been an impediment for their development as therapeutic agents. Conversely, oligonucleotide analogs, including the second generation of BNA compounds, BNANC, show lower toxicity while preserving and, in some cases, improving on the LNA-containing oligomers. Furthermore, next-generation BNA compounds are in the pipeline, and some are or will soon be available for experimentation [87,175]. The success experienced using these nucleotide analogs, and the vigorous eﬀorts to continue developing 9 of 17 Molecules 2019, 24, 2297 next-generation variants oﬀer promising alternatives to continue the development of new and improved applications of oligonucleotides for research as well as diagnostics and therapeutics. next-generation variants oﬀer promising alternatives to continue the development of new and improved applications of oligonucleotides for research as well as diagnostics and therapeutics. References Ts’o, P.O.; Miller, P.S.; Aurelian, L.; Murakami, A.; Agris, C.; Blake, K.R.; Lin, S.B.; Lee, B.L.; Smith, C.C. An approach to chemotherapy based on base sequence information and nucleic acid chemistry. Ann. N.Y. Acad. Sci. 1987, 507, 220–241. [CrossRef] 18. Geary, R.S.; Henry, S.P.; Grillone, L.R. Fomivirsen: Clinical pharmacology and potential drug interactions. Clin. Pharmacokinet. 2002, 41, 255–260. [CrossRef] 10 of 17 Molecules 2019, 24, 2297 19. Mellbye, B.L.; Weller, D.D.; Hassinger, J.N.; Reeves, M.D.; Lovejoy, C.E.; Iversen, P.L.; Geller, B.L. Cationic phosphorodiamidate morpholino oligomers eﬃciently prevent growth of Escherichia coli in vitro and in vivo. J. Antimicrob. Chemother. 2010, 65, 98–106. [CrossRef] 20. Gupta, A.; Mishra, A.; Puri, N. Peptide nucleic acids: Advanced tools for biomedical applicat Biotechnol. 2017, 259, 148–159. [CrossRef] 21. Nielsen, P.E. Peptide nucleic acids (PNA) in chemical biology and drug discovery. Chem. Biodivers. 2010, 7, 786–804. [CrossRef] 22. Le, B.T.; Hornum, M.; Sharma, P.K.; Nielsen, P.; Veedu, R.N. Nucleobase-modiﬁed antisense oligonucleotides containing 5-(phenyltriazol)-2 ′-deoxyuridine nucleotides induce exon-skipping in vitro. RSC Adv. 2017, 7, 54542–54545. [CrossRef] 23. Meng, M.; Ducho, C. Oligonucleotide analogues with cationic backbone linkages. Beilstein J. Org. Chem. 2018, 14, 1293–1308. [CrossRef] 24. Petersen, M.; Wengel, J. LNA: A versatile tool for therapeutics and genomics. Trends Biotechnol. 2003, 21, 74–81. [CrossRef] 25. Sharma, V.K.; Rungta, P.; Prasad, A.K. Nucleic acid therapeutics: Basic concepts and recent developments. RSC Adv. 2014, 4, 16618–16631. [CrossRef] 6. Sully, E.K.; Geller, B.L. Antisense antimicrobial therapeutics. Curr. Opin. Microbiol. 2016, 33, 47–55. [Cross 26. Sully, E.K.; Geller, B.L. Antisense antimicrobial therapeutics. Curr. Opin. Microbiol. 2016, 33, 47 55. [CrossRef] 27. Iversen, P.L. Phosphorodiamidate morpholino oligomers: Favorable properties for sequence-speciﬁc gene inactivation. Curr. Opin. Mol. Ther. 2001, 3, 235–238. 28. Miyashita, K.; Rahman, S.M.; Seki, S.; Obika, S.; Imanishi, T. N-Methyl substituted 2′,4′- BNANC: A highly nuclease-resistant nucleic acid analogue with high-aﬃnity RNA selective hybridization. Chem. Commun. 2007, 3765–3767. [CrossRef] 29. Rahman, S.M.; Seki, S.; Obika, S.; Yoshikawa, H.; Miyashita, K.; Imanishi, T. Design, synthesis, and properties of 2′,4′-BNA(NC): A bridged nucleic acid analogue. J. Am. Chem. Soc. 2008, 130, 4886–4896. [CrossRef] 30. Rahman, S.M.; Seki, S.; Utsuki, K.; Obika, S.; Miyashita, K.; Imanishi, T. 2′,4′-BNA(NC): A novel bridged nucleic acid analogue with excellent hybridizing and nuclease resistance proﬁles. Nucleosides Nucleotides Nucleic Acids 2007, 26, 1625–1628. [CrossRef] 31. Fujisaka, A.; Hari, Y.; Takuma, H.; Rahman, S.M.A.; Yoshikawa, H.; Pang, J.; Imanishi, T.; Obika, S. References Eﬀective syntheses of 2′,4′-BNA(NC) monomers bearing adenine, guanine, thymine, and 5-methylcytosine, and the properties of oligonucleotides fully modiﬁed with 2′,4′-BNA(NC). Bioorg. Med. Chem. 2019, 27, 1728–1741. [CrossRef] 32. Koshkin, A.A.; Nielsen, P.; Meldgaard, M.; Rajwanshi, V.K.; Singh, S.K.; Wengel, J. LNA (locked nucleic acid): An RNA mimic forming exceedingly stable LNA: LNA duplexes. J. Am. Chem. Soc. 1998, 120, 13252–13253. [CrossRef] 33. Koshkin, A.A.; Rajwanshi, V.K.; Wengel, J. Novel convenient syntheses of LNA [2.2.1] bicyclo nucleosides. Tetrahedron Lett. 1998, 39, 4381–4384. [CrossRef] 34. Koshkin, A.A.; Singh, S.K.; Nielsen, P.; Rajwanshi, V.K.; Kumar, R.; Meldgaard, M.; Olsen, C.E.; Wengel, J. LNA (Locked Nucleic Acids): Synthesis of the adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil bicyclonucleoside monomers, oligomerisation, and unprecedented nucleic acid recognition. Tetrahedron 1998, 54, 3607–3630. [CrossRef] 35. Singh, S.K.; Kumar, R.; Wengel, J. Synthesis of novel bicyclo [2.2.1] ribonucleosides: 2′-amino- and 2′-th monomeric nucleosides. J. Org. Chem. 1998, 63, 6078–6079. [CrossRef] 36. Wang, G.; Gunic, E.; Girardet, J.L.; Stoisavljevic, V. Conformationally locked nucleosides. Synthesis and hybridization properties of oligodeoxynucleotides containing 2′,4′-C-bridged 2′-deoxynucleosides. Bioorg. Med. Chem. Lett. 1999, 9, 1147–1150. [CrossRef] 37. Obika, S.; Nanbu, D.; Hari, Y.; Andoh, J.; Morio, K.; Doi, T.; Imanishi, T. Stability and structural features of the duplexes containing nucleoside analogues with a ﬁxed N-type conformation, 2′-O,4′-C-methyleneribonucleosides. Tetrahedron Lett. 1998, 39, 5401–5404. [CrossRef] 38. Veedu, R.N.; Wengel, J. Locked nucleic acids: Promising nucleic acid analogs for therapeutic applications. Chem. Biodivers. 2010, 7, 536–542. [CrossRef] 39. Bhattacharyya, J.; Maiti, S.; Muhuri, S.; Nakano, S.; Miyoshi, D.; Sugimoto, N. Eﬀect of locked nucleic acid modiﬁcations on the thermal stability of noncanonical DNA structure. Biochemistry 2011, 50, 7414–7425. [CrossRef] 11 of 17 Molecules 2019, 24, 2297 40. Braasch, D.A.; Corey, D.R. Locked nucleic acid (LNA): Fine-tuning the recognition of DNA and RNA. Chem. Biol. 2001, 8, 1–7. [CrossRef] 41. Jackson, A.; Jani, S.; Sala, C.D.; Soler-Bistue, A.J.; Zorreguieta, A.; Tolmasky, M.E. Assessment of conﬁgurations and chemistries of bridged nucleic acids-containing oligomers as external guide sequences: A methodology for inhibition of expression of antibiotic resistance genes. Biol. Methods Protoc. 2016, 1, bpw001. [CrossRef] 42. Soler Bistue, A.J.; Martin, F.A.; Vozza, N.; Ha, H.; Joaquin, J.C.; Zorreguieta, A.; Tolmasky, M.E. Inhibition of aac(6′)-Ib-mediated amikacin resistance by nuclease-resistant external guide sequences in bacteria. Proc. Natl. Acad. Sci. USA 2009, 106, 13230–13235. [CrossRef] 43. Arzumanov, A.; Stetsenko, D.A.; Malakhov, A.D.; Reichelt, S.; Sorensen, M.D.; Babu, B.R.; Wengel, J.; Gait, M.J. References A structure-activity study of the inhibition of HIV-1 Tat-dependent trans-activation by mixmer 2′-O-methyl oligoribonucleotides containing locked nucleic acid (LNA), alpha-L-LNA, or 2′-thio-LNA residues. Oligonucleotides 2003, 13, 435–453. [CrossRef] 44. Arzumanov, A.; Walsh, A.P.; Rajwanshi, V.K.; Kumar, R.; Wengel, J.; Gait, M.J. Inhibition of HIV-1 Tat-dependent trans activation by steric block chimeric 2′-O-methyl/LNA oligoribonucleotides. Biochemistry 2001, 40, 14645–14654. [CrossRef] 45. Singh, S.K.; Wengel, J. Universality of LNA-mediated high-aﬃnity nucleic acid recognition. Chem. Commun. 1998, 1247–1248. [CrossRef] 46. Stenvang, J.; Silahtaroglu, A.N.; Lindow, M.; Elmen, J.; Kauppinen, S. The utility of LNA in microRNA-based cancer diagnostics and therapeutics. Semin. Cancer Biol. 2008, 18, 89–102. [CrossRef] 47. Mouritzen, P.; Nielsen, A.T.; Pfundheller, H.M.; Choleva, Y.; Kongsbak, L.; Moller, S. Single nucleotide polymorphism genotyping using locked nucleic acid (LNA). Expert Rev. Mol. Diagn. 2003, 3, 27–38. [CrossRef] 48. Jakobsen, U.; Vogel, S. Mismatch discrimination of lipidated DNA and LNA-probes (LiNAs) in hybridization-controlled liposome assembly. Org. Biomol. Chem. 2016, 14, 6985–6995. [CrossRef] 49. Simon, L.; Probst, A.V. High-aﬃnity LNA-DNA mixmer probes for detection of chromosome-speciﬁc polymorphisms of 5S rDNA repeats in Arabidopsis thaliana. Methods Mol. Biol. 2018, 1675, 481–491. 50. Ikenaga, M.; Katsuragi, S.; Handa, Y.; Katsumata, H.; Chishaki, N.; Kawauchi, T.; Sakai, M. Improvements in bacterial primers to enhance selective SSU rRNA gene ampliﬁcation of plant-associated bacteria by applying the LNA oligonucleotide-PCR clamping technique. Microbes Environ. 2018, 33, 340–344. [CrossRef] g p g q 51. Levin, J.D.; Fiala, D.; Samala, M.F.; Kahn, J.D.; Peterson, R.J. Position-dependent eﬀects of locked nucleic acid (LNA) on DNA sequencing and PCR primers Nucleic Acids Res 2006 34 e142 [CrossRef] 51. Levin, J.D.; Fiala, D.; Samala, M.F.; Kahn, J.D.; Peterson, R.J. Position-dependent eﬀects of locked nuc (LNA) on DNA sequencing and PCR primers. Nucleic Acids Res. 2006, 34, e142. [CrossRef] 52. Pakpour, N.; Cheung, K.W.; Souvannaseng, L.; Concordet, J.P.; Luckhart, S. Transfection and mutagenesis of target genes in mosquito cells by locked nucleic acid-modiﬁed oligonucleotides. J. Vis. Exp. 2010, 2355. [CrossRef] 53. van Ravesteyn, T.W.; Dekker, M.; Fish, A.; Sixma, T.K.; Wolters, A.; Dekker, R.J.; Te Riele, H.P. LNA modiﬁcation of single-stranded DNA oligonucleotides allows subtle gene modiﬁcation in mismatch-repair-proﬁcient cells. Proc. Natl. Acad. Sci. USA 2016, 113, 4122–4127. [CrossRef] 54. Parekh-Olmedo, H.; Drury, M.; Kmiec, E.B. Targeted nucleotide exchange in Saccharomyces cerevisiae directed by short oligonucleotides containing locked nucleic acids. Chem. Biol. 2002, 9, 1073–1084. [CrossRef] 55. References Wojtkowiak-Szlachcic, A.; Taylor, K.; Stepniak-Konieczna, E.; Sznajder, L.J.; Mykowska, A.; Sroka, J.; Thornton, C.A.; Sobczak, K. Short antisense-locked nucleic acids (all-LNAs) correct alternative splicing abnormalities in myotonic dystrophy. Nucleic Acids Res. 2015, 43, 3318–3331. [CrossRef] 56. Elmen, J.; Lindow, M.; Schutz, S.; Lawrence, M.; Petri, A.; Obad, S.; Lindholm, M.; Hedtjarn, M.; Hansen, H.F.; Berger, U.; et al. LNA-mediated microRNA silencing in non-human primates. Nature 2008, 452, 896–899. [CrossRef] 57. Janssen, H.L.; Reesink, H.W.; Lawitz, E.J.; Zeuzem, S.; Rodriguez-Torres, M.; Patel, K.; van der Meer, A.J.; Patick, A.K.; Chen, A.; Zhou, Y.; et al. Treatment of HCV infection by targeting microRNA. N. Engl. J. Med. 2013, 368, 1685–1694. [CrossRef] 58. van der Ree, M.H.; de Vree, J.M.; Stelma, F.; Willemse, S.; van der Valk, M.; Rietdijk, S.; Molenkamp, R.; Schinkel, J.; van Nuenen, A.C.; Beuers, U.; et al. Safety, tolerability, and antiviral eﬀect of RG-101 in patients with chronic hepatitis C: A phase 1B, double-blind, randomised controlled trial. Lancet 2017, 389, 709–717. [CrossRef] 12 of 17 Molecules 2019, 24, 2297 59. van der Ree, M.H.; van der Meer, A.J.; de Bruijne, J.; Maan, R.; van Vliet, A.; Welzel, T.M.; Zeuzem, S.; Lawitz, E.J.; Rodriguez-Torres, M.; Kupcova, V.; et al. Long-term safety and eﬃcacy of microRNA-targeted therapy in chronic hepatitis C patients. Antivir. Res. 2014, 111, 53–59. [CrossRef] 60. Guerard, M.; Andreas, Z.; Erich, K.; Christine, M.; Martina, M.B.; Christian, W.; Franz, S.; Thomas, S.; Yann, T. Locked nucleic acid (LNA): Based single-stranded oligonucleotides are not genotoxic. Environ. Mol. Mutagen. 2017, 58, 112–121. [CrossRef] 61. Bianchini, D.; Omlin, A.; Pezaro, C.; Lorente, D.; Ferraldeschi, R.; Mukherji, D.; Crespo, M.; Figueiredo, I.; Miranda, S.; Riisnaes, R.; et al. First-in-human Phase I study of EZN-4176, a locked nucleic acid antisense oligonucleotide to exon 4 of the androgen receptor mRNA in patients with castration-resistant prostate cancer. Br. J. Cancer 2013, 109, 2579–2586. [CrossRef] 62. Swayze, E.E.; Siwkowski, A.M.; Wancewicz, E.V.; Migawa, M.T.; Wyrzykiewicz, T.K.; Hung, G.; Monia, B.P.; Bennett, C.F. Antisense oligonucleotides containing locked nucleic acid improve potency but cause signiﬁcant hepatotoxicity in animals. Nucleic Acids Res. 2007, 35, 687–700. [CrossRef] 63. Kakiuchi-Kiyota, S.; Koza-Taylor, P.H.; Mantena, S.R.; Nelms, L.F.; Enayetallah, A.E.; Hollingshead, B.D.; Burdick, A.D.; Reed, L.A.; Warneke, J.A.; Whiteley, L.O.; et al. Comparison of hepatic transcription proﬁles of locked ribonucleic acid antisense oligonucleotides: Evidence of distinct pathways contributing to non-target mediated toxicity in mice. Toxicol. Sci. 2014, 138, 234–248. [CrossRef] 64. References Kasuya, T.; Hori, S.; Watanabe, A.; Nakajima, M.; Gahara, Y.; Rokushima, M.; Yanagimoto, T.; Kugimiya, A. Ribonuclease H1-dependent hepatotoxicity caused by locked nucleic acid-modiﬁed gapmer antisense oligonucleotides. Sci. Rep. 2016, 6, 30377. [CrossRef] 65. Stein, C.A.; Hansen, J.B.; Lai, J.; Wu, S.; Voskresenskiy, A.; Hog, A.; Worm, J.; Hedtjarn, M.; Souleimanian, N.; Miller, P.; et al. Eﬃcient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents. Nucleic Acids Res. 2010, 38, e3. [CrossRef] 66. Traglia, G.M.; Sala, C.D.; Fuxman Bass, J.I.; Soler-Bistue, A.J.; Zorreguieta, A.; Ramirez, M.S.; Tolmasky, M.E. Internalization of locked nucleic acids/DNA hybrid oligomers into Escherichia coli. Biores. Open Access 2012, 1, 260–263. [CrossRef] 67. Soifer, H.S.; Koch, T.; Lai, J.; Hansen, B.; Hoeg, A.; Oerum, H.; Stein, C.A. Silencing of gene expression by gymnotic delivery of antisense oligonucleotides. Methods Mol. Biol. 2012, 815, 333–346. 68. Mettlen, M.; Pucadyil, T.; Ramachandran, R.; Schmid, S.L. Dissecting dynamin’s role in clathrin-mediated endocytosis. Biochem. Soc. Trans. 2009, 37, 1022–1026. [CrossRef] 9. Lajoie, P.; Nabi, I.R. Lipid rafts, caveolae, and their endocytosis. Int. Rev. Cell Mol. Biol. 2010, 282, 135– 70. Howes, M.T.; Mayor, S.; Parton, R.G. Molecules, mechanisms, and cellular roles of clathrin-independent endocytosis. Curr. Opin. Cell Biol. 2010, 22, 519–527. [CrossRef] 71. Juliano, R.L. The delivery of therapeutic oligonucleotides. Nucleic Acids Res. 2016, 44, 6518–6548. [CrossRef] 72 J h L L hi M C h ll i d li d li l i f h i RNA 71. Juliano, R.L. The delivery of therapeutic oligonucleotides. Nucleic Acids Res. 2016, 44, 6518–6548. [CrossRef] 72. Johannes, L.; Lucchino, M. Current challenges in delivery and cytosolic translocation of therapeutic RNAs. Nucleic Acid Ther. 2018, 28, 178–193. [CrossRef] 72. Johannes, L.; Lucchino, M. Current challenges in delivery and cytosolic translocation of therapeutic RNAs. Nucleic Acid Ther. 2018, 28, 178–193. [CrossRef] 73. Fabani, M.M.; Gait, M.J. miR-122 targeting with LNA/2′-O-methyl oligonucleotide mixmers, peptide nucleic acids (PNA), and PNA-peptide conjugates. RNA 2008, 14, 336–346. [CrossRef] 74. Gupta, N.; Fisker, N.; Asselin, M.C.; Lindholm, M.; Rosenbohm, C.; Orum, H.; Elmen, J.; Seidah, N.G.; Straarup, E.M. A locked nucleic acid antisense oligonucleotide (LNA) silences PCSK9 and enhances LDLR expression in vitro and in vivo. PLoS ONE 2010, 5, e10682. [CrossRef] p 75. Beane, R.; Gabillet, S.; Montaillier, C.; Arar, K.; Corey, D.R. Recognition of chromosomal DNA inside cells by locked nucleic acids. Biochemistry 2008, 47, 13147–13149. [CrossRef] 76. References Grunweller, A.; Wyszko, E.; Bieber, B.; Jahnel, R.; Erdmann, V.A.; Kurreck, J. Comparison of diﬀerent antisense strategies in mammalian cells using locked nucleic acids, 2′-O-methyl RNA, phosphorothioates and small interfering RNA. Nucleic Acids Res. 2003, 31, 3185–3193. [CrossRef] 77. Turner, J.J.; Jones, S.; Fabani, M.M.; Ivanova, G.; Arzumanov, A.A.; Gait, M.J. RNA targeting with peptide conjugates of oligonucleotides, siRNA and PNA. Blood Cells Mol. Dis. 2007, 38, 1–7. [CrossRef] 78. Jani, S.; Jackson, A.; Davies-Sala, C.; Chiem, K.; Soler-Bistue, A.; Zorreguieta, A.; Tolmasky, M.E. Assessment of external guide sequences’ (EGS) eﬃciency as inducers of RNase P-mediated cleavage of mRNA target molecules. Methods Mol. Biol. 2018, 1737, 89–98. 79. Yin, H.; Wang, H.; Li, Z.; Shu, D.; Guo, P. RNA micelles for systemic delivery of anti-miRNA for cancer targeting and inhibition without ligand. ACS Nano 2018, 13, 706–717. [CrossRef] 13 of 17 Molecules 2019, 24, 2297 80. Elmen, J.; Zhang, H.Y.; Zuber, B.; Ljungberg, K.; Wahren, B.; Wahlestedt, C.; Liang, Z. Locked nucleic acid containing antisense oligonucleotides enhance inhibition of HIV-1 genome dimerization and inhibit virus replication. FEBS Lett. 2004, 578, 285–290. [CrossRef] 81. Kurreck, J.; Wyszko, E.; Gillen, C.; Erdmann, V.A. Design of antisense oligonucleotides stabilized by nucleic acids. Nucleic Acids Res. 2002, 30, 1911–1918. [CrossRef] 82. Kaur, H.; Babu, B.R.; Maiti, S. Perspectives on chemistry and therapeutic applications of Locked Nucleic Acid (LNA). Chem. Rev. 2007, 107, 4672–4697. [CrossRef] 83. Hagedorn, P.H.; Persson, R.; Funder, E.D.; Albaek, N.; Diemer, S.L.; Hansen, D.J.; Moller, M.R.; Papargyri, N.; Christiansen, H.; Hansen, B.R.; et al. Locked nucleic acid: Modality, diversity, and drug discovery. Drug Discov. Today 2018, 23, 101–114. [CrossRef] 84. Grunweller, A.; Hartmann, R.K. Locked nucleic acid oligonucleotides: The next generation of antisense agents? Biodrugs 2007, 21, 235–243. [CrossRef] 85. Sadewasser, A.; Dietzel, E.; Michel, S.; Kluver, M.; Helfer, M.; Thelemann, T.; Klar, R.; Eickmann, M.; Becker, S.; Jaschinski, F. Anti-Niemann Pick C1 single-stranded oligonucleotides with locked nucleic acids potently reduce ebola virus infection in vitro. Mol. Ther. Nucleic Acids 2019, 16, 686–697. [CrossRef] 86. Hillebrand, F.; Ostermann, P.N.; Muller, L.; Degrandi, D.; Erkelenz, S.; Widera, M.; Pfeﬀer, K.; Schaal, H. Gymnotic delivery of LNA mixmers targeting viral SREs induces HIV-1 mRNA degradation. Int. J. Mol. Sci. 2019, 20, 1088. [CrossRef] 87. Kim, S.; Linse, K.; Retes, P.; Castro, P.; Castro, M. Bridged nucleic acids (BNAs) as molecular tools. J. Biochem. Mol. Biol. Res. 2015, 1, 67–71. 88. References In Frontiers in Clinical Drug Research-Anti Infectives; Atta-ur-Rhaman, Ed.; Bentham Books: Sharjah, United Arab Emirates, 2017; Volume 1, pp. 1–27. 100. Guerrier-Takada, C.; Salavati, R.; Altman, S. Phenotypic conversion of drug-resistant bacteria sensitivity. Proc. Natl. Acad. Sci. USA 1997, 94, 8468–8472. [CrossRef] 101. Ayhan, D.H.; Tamer, Y.T.; Akbar, M.; Bailey, S.M.; Wong, M.; Daly, S.M.; Greenberg, D.E.; Toprak, E. Sequence-speciﬁc targeting of bacterial resistance genes increases antibiotic eﬃcacy. PLoS Biol. 2016, 14, e1002552. [CrossRef] 102. Ju, L.C.; Cheng, Z.; Fast, W.; Bonomo, R.A.; Crowder, M.W. The continuing challenge of metallo-beta-lactamase inhibition: Mechanism matters. Trends Pharmacol. Sci. 2018, 39, 635–647. [CrossRef] 103. van den Akker, F.; Bonomo, R.A. Exploring additional dimensions of complexity in inhibitor design for serine beta-lactamases: Mechanistic and intra- and inter-molecular chemistry approaches. Front. Microbiol. 2018, 9, 622. [CrossRef] 104. Ramirez, M.S.; Tolmasky, M.E. Aminoglycoside modifying enzymes. Drug Resist. Updates 2010, 13, 151–171. [CrossRef] 105. Meng, J.; He, G.; Wang, H.; Jia, M.; Ma, X.; Da, F.; Wang, N.; Hou, Z.; Xue, X.; Li, M.; et al. Reversion of antibiotic resistance by inhibiting mecA in clinical methicillin-resistant Staphylococci by antisense phosphorothioate oligonucleotide. J. Antibiot. (Tokyo) 2015, 68, 158–164. [CrossRef] 106. Sully, E.K.; Geller, B.L.; Li, L.; Moody, C.M.; Bailey, S.M.; Moore, A.L.; Wong, M.; Nordmann, P.; Daly, S.M.; Sturge, C.R.; et al. Peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) restores carbapenem susceptibility to NDM-1-positive pathogens in vitro and in vivo. J. Antimicrob. Chemother. 2016, 72, 782–790. 107. Sarno, R.; Ha, H.; Weinsetel, N.; Tolmasky, M.E. Inhibition of aminoglycoside 6′-N-acetyltransferase type Ib-mediated amikacin resistance by antisense oligodeoxynucleotides. Antimicrob. Agents Chemother. 2003, 47, 3296–3304. [CrossRef] 108. Soler Bistue, A.J.; Ha, H.; Sarno, R.; Don, M.; Zorreguieta, A.; Tolmasky, M.E. External guide sequences targeting the aac(6′)-Ib mRNA induce inhibition of amikacin resistance. Antimicrob. Agents Chemother. 2007, 51, 1918–1925. [CrossRef] 109. Vakulenko, S.B.; Mobashery, S. Versatility of aminoglycosides and prospects for their future. Clin. Microbiol. Rev. 2003, 16, 430–450. [CrossRef] 110. Arivett, B.A.; Fiester, S.E.; Ream, D.C.; Centron, D.; Ramirez, M.S.; Tolmasky, M.E.; Actis, L.A. Draft genome of the multidrug-resistant Acinetobacter baumannii strain A155 clinical isolate. Genome Announc. 2015, 3, e00212-15. [CrossRef] 111. Lundblad, E.W.; Altman, S. Inhibition of gene expression by RNase P. New Biotechnol. 2010, 27, 212–221. [CrossRef] 112. Bird, T.D. Myotonic Dystrophy Type 1. In GeneReviews; Adam, M.P., Ardinger, H.H., Pagon, R.A., Wallace, S.E., Bean, L.J.H., Stephens, K., Amemiya, A., Eds.; University of Washington: Seattle, WA, USA, 1993. 113. References Morita, K.; Koizumi, M. Synthesis of ENA nucleotides and ENA oligonucleotides. Curr. Protoc. Nucleic Acid Chem. 2018, 72, 4.79.01–4.79.21. [CrossRef] 89. Mitsuoka, Y.; Kodama, T.; Ohnishi, R.; Hari, Y.; Imanishi, T.; Obika, S. A bridged nucleic acid, 2′,4′-BNA COC: Synthesis of fully modiﬁed oligonucleotides bearing thymine, 5-methylcytosine, adenine and guanine 2′,4′-BNA COC monomers and RNA-selective nucleic-acid recognition. Nucleic Acids Res. 2009, 37, 1225–1238. [CrossRef] 90. Kotkowiak, W.; Wengel, J.; Scotton, C.J.; Pasternak, A. Improved RE31 analogues containing modiﬁed nucleic acid monomers: Thermodynamic, structural, and biological eﬀects. J. Med. Chem. 2019, 62, 2499–2507. [CrossRef] 91. Ferino, A.; Miglietta, G.; Picco, R.; Vogel, S.; Wengel, J.; Xodo, L.E. MicroRNA therapeutics: Design of single-stranded miR-216b mimics to target KRAS in pancreatic cancer cells. RNA Biol. 2018, 15, 1273–1285. [CrossRef] 92. Yamamoto, T.; Harada-Shiba, M.; Nakatani, M.; Wada, S.; Yasuhara, H.; Narukawa, K.; Sasaki, K.; Shibata, M.A.; Torigoe, H.; Yamaoka, T.; et al. Cholesterol-lowering action of BNA-based antisense oligonucleotides targeting PCSK9 in atherogenic diet-induced hypercholesterolemic mice. Mol. Ther. Nucleic Acids 2012, 1, e22. [CrossRef] 93. Rahman, S.M.; Seki, S.; Obika, S.; Haitani, S.; Miyashita, K.; Imanishi, T. Highly stable pyrimidine-motif triplex formation at physiological pH values by a bridged nucleic acid analogue. Angew. Chem. Int. Ed. Engl. 2007, 46, 4306–4309. [CrossRef] 94. Lopez, C.; Arivett, B.A.; Actis, L.A.; Tolmasky, M.E. Inhibition of AAC(6′)-Ib-mediated resistance to amikacin in Acinetobacter baumannii by an antisense peptide-conjugated 2′,4′-bridged nucleic acid-NC-DNA hybrid oligomer. Antimicrob. Agents Chemother. 2015, 59, 5798–5803. [CrossRef] 95. Manning, K.S.; Rao, A.N.; Castro, M.; Cooper, T.A. BNA(NC) gapmers revert splicing and reduce RNA foci with low toxicity in myotonic dystrophy cells. ACS Chem. Biol. 2017, 12, 2503–2509. [CrossRef] 96. Shivarov, V.; Ivanova, M.; Naumova, E. Rapid detection of DNMT3A R882 mutations in hematologic malignancies using a novel bead-based suspension assay with BNA(NC) probes. PLoS ONE 2014, 9, e99769. [CrossRef] 97. Cromwell, C.R.; Sung, K.; Park, J.; Krysler, A.R.; Jovel, J.; Kim, S.K.; Hubbard, B.P. Incorporation of bridged nucleic acids into CRISPR RNAs improves Cas9 endonuclease speciﬁcity. Nat. Commun. 2018, 9, 1448. [CrossRef] 98. Good, L.; Nielsen, P.E. Antisense inhibition of gene expression in bacteria by PNA targeted to mRNA. Nat. Biotechnol. 1998, 16, 355–358. [CrossRef] Molecules 2019, 24, 2297 14 of 17 99. Tolmasky, M.E. Strategies to prolong the useful life of existing antibiotics and help overcoming the antibiotic resistance crisis. References Therapeutic RNAi targeting PCSK9 acutely lowers plasma cholesterol in rodents and LDL cholesterol in nonhuman primates. Proc. Natl. Acad. Sci. USA 2008, 105, 11915–11920. [CrossRef] [ ] 126. Chaudhary, R.; Garg, J.; Shah, N.; Sumner, A. PCSK9 inhibitors: A new era of lipid lowering therapy. World J. Cardiol. 2017, 9, 76–91. [CrossRef] 127. Graham, M.J.; Lemonidis, K.M.; Whipple, C.P.; Subramaniam, A.; Monia, B.P.; Crooke, S.T.; Crooke, R.M. Antisense inhibition of proprotein convertase subtilisin/kexin type 9 reduces serum LDL in hyperlipidemic mice. J. Lipid Res. 2007, 48, 763–767. [CrossRef] 128. Brunetti, L.; Gundry, M.C.; Goodell, M.A. DNMT3A in Leukemia. Cold Spring Harb. Perspect. Med. 2017, 7, a030320. [CrossRef] 129. Suetake, I.; Mishima, Y.; Kimura, H.; Lee, Y.H.; Goto, Y.; Takeshima, H.; Ikegami, T.; Tajima, S. Characterization of DNA-binding activity in the N-terminal domain of the DNA methyltransferase Dnmt3a. Biochem. J. 2011, 437, 141–148. [CrossRef] 130. Jurkowska, R.Z.; Rajavelu, A.; Anspach, N.; Urbanke, C.; Jankevicius, G.; Ragozin, S.; Nellen, W.; Jeltsch, A. Oligomerization and binding of the Dnmt3a DNA methyltransferase to parallel DNA molecules: Heterochromatic localization and role of Dnmt3L. J. Biol. Chem. 2011, 286, 24200–24207. [CrossRef] 31. Shivarov, V.; Gueorguieva, R.; Stoimenov, A.; Tiu, R. DNMT3A mutation is a poor prognosis biomarke AML: Results of a meta-analysis of 4500 AML patients. Leuk. Res. 2013, 37, 1445–1450. [CrossRef] 132. Itzykson, R.; Kosmider, O.; Fenaux, P. Somatic mutations and epigenetic abnormalities in myelodysplastic syndromes. Best Pract. Res. Clin. Haematol. 2013, 26, 355–364. [CrossRef] 133. Jankowska, A.M.; Makishima, H.; Tiu, R.V.; Szpurka, H.; Huang, Y.; Traina, F.; Visconte, V.; Sugimoto, Y.; Prince, C.; O’Keefe, C.; et al. Mutational spectrum analysis of chronic myelomonocytic leukemia includes genes associated with epigenetic regulation: UTX, EZH2, and DNMT3A. Blood 2011, 118, 3932–3941. [CrossRef] 134. Vannucchi, A.M.; Lasho, T.L.; Guglielmelli, P.; Biamonte, F.; Pardanani, A.; Pereira, A.; Finke, C.; Score, J.; Gangat, N.; Mannarelli, C.; et al. Mutations and prognosis in primary myeloﬁbrosis. Leukemia 2013, 27, 1861–1869. [CrossRef] 135. Traina, F.; Visconte, V.; Jankowska, A.M.; Makishima, H.; O’Keefe, C.L.; Elson, P.; Han, Y.; Hsieh, F.H.; Sekeres, M.A.; Mali, R.S.; et al. Single nucleotide polymorphism array lesions, TET2, DNMT3A, ASXL1 and CBL mutations are present in systemic mastocytosis. PLoS ONE 2012, 7, e43090. [CrossRef] 136. Grossmann, V.; Haferlach, C.; Weissmann, S.; Roller, A.; Schindela, S.; Poetzinger, F.; Stadler, K.; Bellos, F.; Kern, W.; Haferlach, T.; et al. References Brook, J.D.; McCurrach, M.E.; Harley, H.G.; Buckler, A.J.; Church, D.; Aburatani, H.; Hunter, K.; Stanton, V.P.; Thirion, J.P.; Hudson, T.; et al. Molecular basis of myotonic dystrophy: Expansion of a trinucleotide (CTG) repeat at the 3′ end of a transcript encoding a protein kinase family member. Cell 1992, 68, 799–808. [CrossRef] 114. Mahadevan, M.; Tsilﬁdis, C.; Sabourin, L.; Shutler, G.; Amemiya, C.; Jansen, G.; Neville, C.; Narang, M.; Barcelo, J.; O’Hoy, K.; et al. Myotonic dystrophy mutation: An unstable CTG repeat in the 3′ untranslated region of the gene. Science 1992, 255, 1253–1255. [CrossRef] 115. Brouwer, J.R.; Willemsen, R.; Oostra, B.A. Microsatellite repeat instability and neurological disease. Bioessays 2009, 31, 71–83. [CrossRef] 116. Lee, J.E.; Cooper, T.A. Pathogenic mechanisms of myotonic dystrophy. Biochem. Soc. Trans. 2009, 37, 1281–1286. [CrossRef] 117. Thornton, C.A. Myotonic dystrophy. Neurol. Clin. 2014, 32, 705–719. [CrossRef] 118. Blanchard, V.; Khantalin, I.; Ramin-Mangata, S.; Chemello, K.; Nativel, B.; Lambert, G. PCSK9: From biology to clinical applications. Pathology 2019, 51, 177–183. [CrossRef] 119. Abifadel, M.; Varret, M.; Rabes, J.P.; Allard, D.; Ouguerram, K.; Devillers, M.; Cruaud, C.; Benjannet, S.; Wickham, L.; Erlich, D.; et al. Mutations in PCSK9 cause autosomal dominant hypercholesterolemia. Nat. Genet. 2003, 34, 154–156. [CrossRef] 15 of 17 Molecules 2019, 24, 2297 120. Hallman, D.M.; Srinivasan, S.R.; Chen, W.; Boerwinkle, E.; Berenson, G.S. Relation of PCSK9 mutations to serum low-density lipoprotein cholesterol in childhood and adulthood (from The Bogalusa Heart Study). Am. J. Cardiol. 2007, 100, 69–72. [CrossRef] 21. Lambert, G.; Charlton, F.; Rye, K.A.; Piper, D.E. Molecular basis of PCSK9 function. Atherosclerosis 2009, 1–7. [CrossRef] 122. Cameron, J.; Ranheim, T.; Kulseth, M.A.; Leren, T.P.; Berge, K.E. Berberine decreases PCSK9 expression in HepG2 cells. Atherosclerosis 2008, 201, 266–273. [CrossRef] 123. Kong, W.J.; Wei, J.; Zuo, Z.Y.; Wang, Y.M.; Song, D.Q.; You, X.F.; Zhao, L.X.; Pan, H.N.; Jiang, J.D. Combination of simvastatin with berberine improves the lipid-lowering eﬃcacy. Metabolism 2008, 57, 1029–1037. [CrossRef] 124. Chan, J.C.; Piper, D.E.; Cao, Q.; Liu, D.; King, C.; Wang, W.; Tang, J.; Liu, Q.; Higbee, J.; Xia, Z.; et al. A proprotein convertase subtilisin/kexin type 9 neutralizing antibody reduces serum cholesterol in mice and nonhuman primates. Proc. Natl. Acad. Sci. USA 2009, 106, 9820–9825. [CrossRef] 125. Frank-Kamenetsky, M.; Grefhorst, A.; Anderson, N.N.; Racie, T.S.; Bramlage, B.; Akinc, A.; Butler, D.; Charisse, K.; Dorkin, R.; Fan, Y.; et al. References Remarkable mechanisms in microbes to resist phage infections. Annu. Rev. Virol. 2014, 1, 307–331. [CrossRef] 147. Arutyunov, D.; Frost, L.S. F conjugation: Back to the beginning. Plasmid 2013, 70, 18–32. [CrossRef] 148. Ishino, Y.; Krupovic, M.; Forterre, P. History of CRISPR-Cas from encounter with a mysterious repeated sequence to genome editing technology. J. Bacteriol. 2018, 200. [CrossRef] 149. Hille, F.; Charpentier, E. CRISPR-Cas: Biology, mechanisms and relevance. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2016, 371, 20150496. [CrossRef] 150. Strich, J.R.; Chertow, D.S. CRISPR-Cas Biology and Its Application to Infectious Diseases. J. Clin. Microbiol. 2019, 57, e01307-18. [CrossRef] 151. Wright, A.V.; Nunez, J.K.; Doudna, J.A. Biology and applications of CRISPR systems: Harnessing nature’s toolbox for genome engineering. Cell 2016, 164, 29–44. [CrossRef] 152. Yu, J.S.L.; Yusa, K. Genome-wide CRISPR-Cas9 screening in mammalian cells. Methods 2019, in press. [CrossRef] 153. Jinek, M.; Chylinski, K.; Fonfara, I.; Hauer, M.; Doudna, J.A.; Charpentier, E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 2012, 337, 816–821. [CrossRef] 153. Jinek, M.; Chylinski, K.; Fonfara, I.; Hauer, M.; Doudna, J.A.; Charpentier, E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 2012, 337, 816–821. [CrossRef] 154. Mali, P.; Esvelt, K.M.; Church, G.M. Cas9 as a versatile tool for engineering biology. Nat. Methods 2013, 10, 154. Mali, P.; Esvelt, K.M.; Church, G.M. Cas9 as a versatile tool for engineering biology. Nat. Methods 2013, 10, 957–963. [CrossRef] 155. Cong, L.; Ran, F.A.; Cox, D.; Lin, S.; Barretto, R.; Habib, N.; Hsu, P.D.; Wu, X.; Jiang, W.; Marraﬃni, L.A.; et al. Multiplex genome engineering using CRISPR/Cas systems. Science 2013, 339, 819–823. [CrossRef] 156. Gasiunas, G.; Barrangou, R.; Horvath, P.; Siksnys, V. Cas9-crRNA ribonucleoprotein complex mediates speciﬁc DNA cleavage for adaptive immunity in bacteria. Proc. Natl. Acad. Sci. USA 2012, 109, E2579–E2586. [CrossRef] 157. Sternberg, S.H.; LaFrance, B.; Kaplan, M.; Doudna, J.A. Conformational control of DNA target cleavage by CRISPR-Cas9. Nature 2015, 527, 110–113. [CrossRef] 158. Sander, J.D.; Joung, J.K. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 2014, 32, 347–355. [CrossRef] 159. Joung, J.; Konermann, S.; Gootenberg, J.S.; Abudayyeh, O.O.; Platt, R.J.; Brigham, M.D.; Sanjana, N.E.; Zhang, F. Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening. Nat. Protoc. 2017, 12, 828–863. [CrossRef] 160. Farhat, S.; Jain, N.; Singh, N.; Sreevathsa, R.; Dash, P.; Rai, R.; Yadav, S.; Kumar, P.; Sarkar, A.; Jain, A.; et al. References The molecular proﬁle of adult T-cell acute lymphoblastic leukemia: Mutations in RUNX1 and DNMT3A are associated with poor prognosis in T-ALL. Genes Chromosom. Cancer 2013, 52, 410–422. [CrossRef] 137. Gaidzik, V.I.; Schlenk, R.F.; Paschka, P.; Stolzle, A.; Spath, D.; Kuendgen, A.; von Lilienfeld-Toal, M.; Brugger, W.; Derigs, H.G.; Kremers, S.; et al. Clinical impact of DNMT3A mutations in younger adult patients with acute myeloid leukemia: Results of the AML Study Group (AMLSG). Blood 2013, 121, 4769–4777. [CrossRef] 138. Yuan, X.Q.; Peng, L.; Zeng, W.J.; Jiang, B.Y.; Li, G.C.; Chen, X.P. DNMT3A R882 Mutations Predict a Poor Prognosis in AML: A Meta-Analysis From 4474 Patients. Medicine (Baltimore) 2016, 95, e3519. [CrossRef] 16 of 17 Molecules 2019, 24, 2297 16 of 17 139. Kihara, R.; Nagata, Y.; Kiyoi, H.; Kato, T.; Yamamoto, E.; Suzuki, K.; Chen, F.; Asou, N.; Ohtake, S.; Miyawaki, S.; et al. Comprehensive analysis of genetic alterations and their prognostic impacts in adult acute myeloid leukemia patients. Leukemia 2014, 28, 1586–1595. [CrossRef] 140. Snetsinger, B.; Ferrone, C.K.; Rauh, M.J. Targeted, amplicon-based, next-generation sequencing to age-related clonal hematopoiesis. Methods Mol. Biol. 2019, in press. 141. Luthra, R.; Patel, K.P.; Reddy, N.G.; Haghshenas, V.; Routbort, M.J.; Harmon, M.A.; Barkoh, B.A.; Kanagal-Shamanna, R.; Ravandi, F.; Cortes, J.E.; et al. Next-generation sequencing-based multigene mutational screening for acute myeloid leukemia using MiSeq: Applicability for diagnostics and disease monitoring. Haematologica 2014, 99, 465–473. [CrossRef] 142. Marcucci, G.; Metzeler, K.H.; Schwind, S.; Becker, H.; Maharry, K.; Mrozek, K.; Radmacher, M.D.; Kohlschmidt, J.; Nicolet, D.; Whitman, S.P.; et al. Age-related prognostic impact of diﬀerent types of DNMT3A mutations in adults with primary cytogenetically normal acute myeloid leukemia. J. Clin. Oncol. 2012, 30, 742–750. [CrossRef] 143. Brewin, J.N.; Horne, G.A.; Bisling, K.E.; Stewart, H.J.; Chevassut, T.J. Rapid detection of DNMT3A R882 codon mutations allows early identiﬁcation of poor risk patients with acute myeloid leukemia. Leuk. Lymphoma 2013, 54, 1336–1339. [CrossRef] 144. Singh, R.R.; Bains, A.; Patel, K.P.; Rahimi, H.; Barkoh, B.A.; Paladugu, A.; Bisrat, T.; Ravandi-Kashani, F.; Cortes, J.E.; Kantarjian, H.M.; et al. Detection of high-frequency and novel DNMT3A mutations in acute myeloid leukemia by high-resolution melting curve analysis. J. Mol. Diagn. 2012, 14, 336–345. [CrossRef] 145. Shabbir, M.A.; Hao, H.; Shabbir, M.Z.; Wu, Q.; Sattar, A.; Yuan, Z. Bacteria vs. bacteriophages: Parallel evolution of immune arsenals. Front. Microbiol. 2016, 7, 1292. [CrossRef] 146. Dy, R.L.; Richter, C.; Salmond, G.P.; Fineran, P.C. References CRISPR-cas 9 directed genome engineering for enhancing salt stress tolerance in rice. Semin. Cell Dev. Biol. 2019, in press. [CrossRef] 17 of 17 17 of 17 Molecules 2019, 24, 2297 161. Kaushik, I.; Ramachandran, S.; Srivastava, S.K. CRISPR-Cas9: A multifaceted therapeutic strategy for cancer treatment. Semin. Cell Dev. Biol. 2019, in press. [CrossRef] 162. O’Geen, H.; Henry, I.M.; Bhakta, M.S.; Meckler, J.F.; Segal, D.J. A genome-wide analysis of Cas9 binding speciﬁcity using ChIP-seq and targeted sequence capture. Nucleic Acids Res. 2015, 43, 3389–3404. [CrossRef] 162. O’Geen, H.; Henry, I.M.; Bhakta, M.S.; Meckler, J.F.; Segal, D.J. A genome-wide analysis of Cas9 binding speciﬁcity using ChIP-seq and targeted sequence capture. Nucleic Acids Res. 2015, 43, 3389–3404. [CrossRef] 163. O’Geen, H.; Yu, A.S.; Segal, D.J. How speciﬁc is CRISPR/Cas9 really? Curr. Opin. Chem. Biol. 2015, 29, 72–78. [C R f] speciﬁcity using ChIP-seq and targeted sequence capture. Nucleic Acids Res. 2015, 43, 3389–3404. [CrossRef] 163. O’Geen, H.; Yu, A.S.; Segal, D.J. How speciﬁc is CRISPR/Cas9 really? Curr. Opin. Chem. Biol. 2015, 29, 72–78. [CrossRef] 163. O’Geen, H.; Yu, A.S.; Segal, D.J. How speciﬁc is CRISPR/Cas9 really? Curr. Opin. Chem. Biol. 2015, 29, 72–78. [CrossRef] 164. Molla, K.A.; Yang, Y. CRISPR/Cas-mediated base editing: Technical considerations and practical applications. Trends Biotechnol. 2019, in press. [CrossRef] 165. Tadic, V.; Josipovic, G.; Zoldos, V.; Vojta, A. CRISPR/Cas9-based epigenome editing: An overview of dCas9-based tools with special emphasis on oﬀ-target activity. Methods 2019, in press. 166. Kimberland, M.L.; Hou, W.; Alfonso-Pecchio, A.; Wilson, S.; Rao, Y.; Zhang, S.; Lu, Q. Strategies for controlling CRISPR/Cas9 oﬀ-target eﬀects and biological variations in mammalian genome editing experiments. J. Biotechnol. 2018, 284, 91–101. [CrossRef] 167. Guilinger, J.P.; Thompson, D.B.; Liu, D.R. Fusion of catalytically inactive Cas9 to FokI nuclease improves the speciﬁcity of genome modiﬁcation. Nat. Biotechnol. 2014, 32, 577–582. [CrossRef] 168. Slaymaker, I.M.; Gao, L.; Zetsche, B.; Scott, D.A.; Yan, W.X.; Zhang, F. Rationally engineered Cas9 nucleases with improved speciﬁcity. Science 2016, 351, 84–88. [CrossRef] 169. Kleinstiver, B.P.; Pattanayak, V.; Prew, M.S.; Tsai, S.Q.; Nguyen, N.T.; Zheng, Z.; Joung, J.K. High-ﬁdelity CRISPR-Cas9 nucleases with no detectable genome-wide oﬀ-target eﬀects. Nature 2016, 529, 490–495. [CrossRef] 170. Tycko, J.; Myer, V.E.; Hsu, P.D. Methods for optimizing CRISPR-Cas9 genome editing speciﬁcity. Mol. Cell 2016, 63, 355–370. [CrossRef] 171. O’Reilly, D.; Kartje, Z.J.; Ageely, E.A.; Malek-Adamian, E.; Habibian, M.; Schoﬁeld, A.; Barkau, C.L.; Rohilla, K.J.; DeRossett, L.B.; Weigle, A.T.; et al. References Extensive CRISPR RNA modiﬁcation reveals chemical compatibility and structure-activity relationships for Cas9 biochemical activity. Nucleic Acids Res. 2019, 47, 546–558. [CrossRef] 172. Hendel, A.; Bak, R.O.; Clark, J.T.; Kennedy, A.B.; Ryan, D.E.; Roy, S.; Steinfeld, I.; Lunstad, B.D.; Kaiser, R.J.; Wilkens, A.B.; et al. Chemically modiﬁed guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat. Biotechnol. 2015, 33, 985–989. [CrossRef] 173. Xiao, J.; Deng, Y.M.; Liu, X.R.; Cao, J.P.; Zhou, M.; Tang, Y.L.; Xiong, W.H.; Jiang, Z.S.; Tang, Z.H.; Liu, L.S. PCSK9: A new participant in lipophagy in regulating atherosclerosis? Clin. Chim. Acta 2019, 495, 358–364. [CrossRef] 174. Briata, P.; Di Blas, E.; Gulisano, M.; Mallamaci, A.; Iannone, R.; Boncinelli, E.; Corte, G. EMX1 homeoprotein is expressed in cell nuclei of the developing cerebral cortex and in the axons of the olfactory sensory neurons. Mech. Dev. 1996, 57, 169–180. [CrossRef] 175. Uehara, T.; Choong, C.J.; Nakamori, M.; Hayakawa, H.; Nishiyama, K.; Kasahara, Y.; Baba, K.; Nagata, T.; Yokota, T.; Tsuda, H.; et al. Amido-bridged nucleic acid (AmNA)-modiﬁed antisense oligonucleotides targeting alpha-synuclein as a novel therapy for Parkinson’s disease. Sci. Rep. 2019, 9, 7567. [CrossRef] © 2019 by the authors. Licensee MDPI, Basel, Switzerland. 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https://openalex.org/W3214678434	https://zenodo.org/records/5654870/files/Development%20of%20a%20method%20for%20maintaining%20the%20performance%20of%20drilling%20fluids%20during%20transportation%20by%20Platform%20Supply%20Vessel.pdf	English	null	Development of a method for maintaining the performance of drilling fluids during transportation by Platform Supply Vessel	Technology audit and production reserves	2,021	cc-by	5,699	SYSTEMS AND CONTROL PROCESSES SYSTEMS AND CONTROL PROCESSES UDC 621.526 DOI: 10.15587/2706-5448.2021.239437 Article type «Original research» UDC 621.526 DOI: 10.15587/2706-5448.2021.239437 Article type «Original research» How to cite Maryanov, D. (2021). Development of a method for maintaining the performance of drilling fluids during transportation by Platform Supply Vessel. Technology Audit and Production Reserves, 5 (2 (61)), 15–20. doi: http://doi.org/10.15587/2706-5448.2021.239437 Maryanov, D. (2021). Development of a method for maintaining the performance of drilling fluids during transportation by Platform Supply Vessel. Technology Audit and Production Reserves, 5 (2 (61)), 15–20. doi: http://doi.org/10.15587/2706-5448.2021.239437 The functional properties of drilling fluids include the provision of lubrication, cooling and anti-corrosion action on the drilling tool [2, 3]. Drilling fluid is a complex multicomponent dispersed system, the dispersed medium of which is a lubricant of petroleum origin, and various organic and inorganic compounds are used as the dispersed phase, the specific gravity of which exceeds the specific gravity of the lubricant [4, 5]. Depending on the type and amount of these compounds, drilling fluids have different physi- cal (primarily density and viscosity) and rheological (pri- marily shear resistance and fluidity) characteristics [6, 7]. In the process of transportation of drilling fluids, due to the action of gravitational forces on organic and inorganic compounds, a latent change in their dispersion over the volume of the fluid occurs. This leads to stratification of the drilling fluid and stratification of its density along the height, as well as to the formation of sediments at the Denys Maryanov © The Author(s) 2021 This is an open access article under the Creative Commons CC BY license © The Author(s) 2021 This is an open access article under the Creative Commons CC BY license Received date: 23.04.2021 Accepted date: 31.05.2021 Published date: 23.09.2021 DEVELOPMENT OF A METHOD FOR MAINTAINING THE PERFORMANCE OF DRILLING FLUIDS DURING TRANSPORTATION BY PLATFORM SUPPLY VESSEL Denys Maryanov TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 5/2(61), 2021 Denys Maryanov The object of research is the process of transportation of drilling fluid used for lubrication and cooling of drill- ing equipment of offshore oil production platforms. The subject of the study is the stratification of the density of the drilling fluid along the height of the cargo tank in which it is transported. The technology of transportation of drilling fluid on the Platform Supply Vessel is considered. A problematic point in ensuring this process is that during the transportation of drilling fluids, due to the action of gravitational forces on organic and inorganic compounds in their volume, there is a latent change in their dispersion over the volume of the fluid. This leads to the stratification and stratification of the fluid density along the height, as well as to the formation of sedi- ments at the bottom of the cargo tanks, in which the drilling fluid is transported. The study is aimed at developing a technology that maintains a constant value of the density of the drilling fluid along the depth of the tank in which it is transported. The studies were carried out in the vessel system for transporting drilling fluid of a specialized marine vessel of the Platform Supply Vessel type with a deadweight of 5850 tons. It is experimentally established that for a transportation time of 6–36 hours, the density stratification of the drilling fluid is 3.04–32.04 %. As a method that ensures the minimum stratification of the density of the drilling fluid during its transportation, it is proposed to use an additional X-shaped circulation of the drilling fluid in the volume of adjacent cargo tanks. Studies have confirmed that the density stratification over a time period of 6–36 hours decreases to a range of 2.30–9.01 %. The complex use of additional X-shaped circulation and simultaneous air supply to the bottom of the cargo tank provides a density stratification value of 0.73–2.93 %. The proposed technology was tested on a specialized seagoing vessel of the Platform Supply Vessel type with a deadweight of 5850 tons and can be used on offshore vessels that ensure the operation of offshore oil production platforms. ff p f ff p p f Keywords: Platform Supply Vessel, drilling fluid, transportation system, density stratification. INFORMATION AND CONTROL SYSTEMS: SYSTEMS AND CONTROL PROCESSES ISSN 2664-9969 3. Determine the parameters for ensuring forced cir- culation of the drilling fluid. bottom of the cargo tanks in which it is transported. In this case, situations are possible when the amount of high-density sediment does not allow for pumping the drilling fluid through the pipelines of the system and pump- ing it out (transferring) to the oil production platform. Removal of sediment manually or mechanically belongs to the category of work unusual for the vessel’s crew. Their implementation is associated with an increase in the sail- ing time of the vessel, leads to losses of thermal [8] and mechanical energy [9], therefore, increases financial costs and reduces the economic performance of PSV. 4. Propose parameters for maintaining the rheological characteristics of the drilling fluid during its transportation. 4. Research of existing solutions to the problem To maintain and restore the rheological characteris- tics (in particular, viscosity and density) of technical fluids, their hydrodynamic, ultrasonic and chemical treatment are used [11, 12]. Thus, maintaining the operational characteristics of drill- ing fluids during their transportation by PSV-class sea ves- sels is an urgent applied scientific and technical problem. The hydrodynamic effect on the fluid flow is carried out by creating an increased pressure and increasing the speed of its movement [13, 14]. In this case, due to the fragmentation of the elements of the dispersed phase, their dispersion and uniformity of the entire volume of the liquid increase [15, 16]. 2. The object of research and its technological audit The object of research is the process of transportation of drilling fluid used for lubrication and cooling of drill- ing equipment of offshore oil production platforms. The subject of research is the stratification of the density of the drilling fluid along the height of the cargo tank in which it is transported. During ultrasonic treatment in a fluid flow, there is a pulse increase and decrease in pressure in the local volumes of the fluid. This, as well as the provision of turbulent motion, leads to an increase in the homogeneity of the liquid and the prevention of sediment of heavy components that make up its composition [17, 18]. Maintaining the rheological characteristics (viscosity, dispersity, density) of drilling fluids ensures reliable opera- tion of the equipment and the continuity of the process of production and transportation of hydrocarbons. In most cases, these issues are considered in relation to continental fields, for conditions that do not take into account the specifics of the transportation and transfer of drilling fluids to offshore or ocean drilling platforms [10]. Maintaining and restoring the rheological characteristics of technical fluids is also possible by chemical treatment. In this case, additional reagents are introduced into the volume of the liquid, which, due to intermolecular interac- tions, ensure the maintenance of the homogeneity of the liquid at the required level during the periods of storage and transportation [19, 20]. The above methods for solving the problem of maintaining the rheological characteristics of technical fluids are used for fuels and oils used in power plants of vessels of sea and river transport. An optimal solution to the important problem of maintaining the physical and rheological cha racteristics of drilling fluids during their transportation by vessels of the PSV class has not yet been found. The vector of scientific research aimed at optimizing the operational characteristics of working substances used in vessel power plants is aimed at studying the properties of marine heavy fuels and motor oils [4, 6]. The issues of ensuring the functional properties and performance of technical fluids, such as drilling fluids, are practically not studied in relation to the marine industry. The rules for their transportation have no confirmed practical recom- mendations. Latent deterioration of their rheological cha racteristics cannot always be determined, evaluated and eliminated by the vessel’s crew. 5. Methods of research To determine the technology that ensures the mainte- nance of a constant value of the density of the drilling fluid along the depth of the tank in which it is transported, experimental studies were carried out on the PSV sea ves- sel with a deadweight of 5850 tons. Vessels of the PSV type belong to the medium-speed class (their maximum speed, as a rule, does not exceed 12–13 knots), therefore the duration of their passage (and, accordingly, the time of transportation of the drilling fluid) from the port to the oil platform is 2–5 days. In addition, there may be cases where some operational and technological reasons necessitate drifting or parking near drilling platforms. This time interval contributes to the gradual deposition of heavy components, which are alloyed in the fluids, and increases the stratification of the density of the drilling fluid along the depth of the cargo tank in which it is located [1, 20]. 1. Introduction Every year all countries with oil reserves increase its production. This is due to the constant growth in the consumption of thermal, mechanical and electrical energy, the source of which is liquid fuel. Oil fields are not only onshore. There are offshore fields, the main of which are the Persian, Venezuelan and Guinean Gulfs, as well as the North Sea. The development, production and transportation of oil in offshore areas (in comparison with continental sources) is characterized by an increased complexity of the technological process, as well as the use of special engi- neering structures (drilling platforms) and special purpose marine vessels – Platform Supply Vessel (PSV). These vessels provide delivery of water, fuel, oil, food, necessary equipment and special technical fluids (drilling fluids) from the shore to the offshore drilling platform [1]. TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 5/2(61), 2021 15 TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 5/2(61), 2021 Table 1 luid transportation system was modernized by: Change in the density of the drilling fluid (r, kg/m3) along the depth of the cargo tank, depending on the time and method of its transportation – installation of additional pipelines that ensured X- shaped circulation of the fluid in the tanks; Transport without changing the system design Measurement level Transportation time, hours 0 6 12 18 24 30 36 on a surface 1232 1218 1205 1188 1173 1135 1105 0.3h 1232 1230 1222 1205 1189 1178 1152 0.6h 1233 1248 1265 1285 1301 1345 1406 0.9h 1235 1255 1282 1312 1342 1385 1463 Transportation with additional X-shaped circulation on a surface 1272 1235 1175 1122 1068 1028 1002 0.3h 1232 1220 1215 1202 1182 1179 1176 0.6h 1233 1226 1222 1208 1196 1194 1185 0.9h 1233 1242 1246 1246 1249 1258 1262 Transportation with additional X-shaped circulation and air supply to the volume of the cargo tank on a surface 1233 1232 1231 1228 1227 1226 1226 0.3h 1233 1232 1232 1232 1231 1231 1230 0.6h 1235 1236 1242 1245 1248 1251 1253 0.9h 1236 1241 1248 1251 1258 1262 1263 – installation of aerodynamic inserts in the lower part of the cargo tank, through which air was supplied. The changes made to the system are shown in Fig. 1. – installation of aerodynamic inserts in the lower part of the cargo tank, through which air was supplied. The changes made to the system are shown in Fig. 1. a b Fig. 1. Schematic diagram of the inclusion of additional units in the vessel drilling fluid transportation system: a – ensuring the X-shaped circulation of the drilling fluid in the tanks; b – ensuring the X-shaped circulation of the drilling fluid in the tanks with simultaneous air supply a a b Graphical dependencies that show the change in the density of the drilling fluid over time for different condi- tions of its transportation are shown in Fig. 2. b Fig. 1. Schematic diagram of the inclusion of additional units in the vessel drilling fluid transportation system: a – ensuring the X-shaped circulation of the drilling fluid in the tanks; b – ensuring the X-shaped circulation of the drilling fluid in the tanks with simultaneous air supply The results, which are shown in Table 1 and Fig. 2 indicate that the decrease/increase in the density of the drilling fluid occurs exponentially. 6. Research results Density was taken as the control parameter by which the dispersed state of the drilling fluid was estimated. Its determination was carried out by an Anton Paar DMA35 Tag&Log electronic hydrometer from LEMIS Baltic (Lat- via-Germany), which allows measurements in the range of 650–1630 kg/m3 with an accuracy of ±1 kg/m3 while simultaneously monitoring the temperature of the mea- sured samples [21, 22]. Dr r r r = − ⋅ h h h 0 9 100 . %, where rh – density of the drilling fluid on the surface of the cargo tank, kg/m3; r0.9h – density of the drilling fluid at a depth of 90 %, kg/m3 (for a transportation time of 6–36 h, it is Dr = 3.04–32.04 %). Samples for determining the density of the drilling fluid were taken at various points of the tank, corresponding to 30, 60 and 90 % of its depth. For the experiment, the drilling fluid was transported under the following conditions: In the case of modernization of the system, the density stratification was: – with additional X-shaped circulation – Dr = 2.30–9.01 % 1) two tanks without changing the design of the system; 1) two tanks without changing the design of the system; 2) two tanks, in which the X-shaped circulation of the drilling fluid was additionally provided – Fig. 1, a; – with additional X-shaped circulation – Dr = 2.30–9.01 %; – with additional X-shaped circulation and simultaneous air supply – Dr = 0.73–2.93 %. – with additional X-shaped circulation – Dr = 2.30–9.01 %; – with additional X-shaped circulation and simultaneous air supply – Dr = 0.73–2.93 %. 3) two tanks, in which X-shaped circulation of the drilling fluid and forced air supply were additionally pro- vided – Fig. 1, b. The effectiveness of the proposed methods can be esti- mated from Fig. 2 as the area bounded by the exponents of the maximum increase rmax + and maximum decrease rmax − in density over a selected period of time. The time of the PSV’s ocean passage from the port to the drilling platform was 44 hours, which made it possible to sample the drilling fluid for all experimental conditions within 36 hours with an interval between density measure- ments of 6 hours. The research results are shown in Table 1. INFORMATION AND CONTROL SYSTEMS: SYSTEMS AND CONTROL PROCESSES INFORMATION AND CONTROL SYSTEMS: SYSTEMS AND CONTROL PROCESSES of sediment from heavy components, the vessel’s drilling fluid transportation system was modernized by: Table 1 The maximum increase in density rmax + (and the corresponding weighting of the drilling fluid) corresponds to the lower part of the cargo tank (90 % depth). The maximum decrease in density rmax − corresponds to the layer of drilling fluid that is on the surface of the cargo tank. The changes made to the system were carried out by the vessel’s crew and agreed with the technical depart- ment of the shipping company. The complexity of the work performed did not exceed 10 hours. The work was carried out during the ballast passage of the vessel. The changes made to the system were carried out by the vessel’s crew and agreed with the technical depart- ment of the shipping company. The complexity of the work performed did not exceed 10 hours. The work was carried out during the ballast passage of the vessel. The highest values rmax + and rmax − corresponds to the transportation of the drilling fluid without changing the design of the system. In this case, the stratification of the density of the drilling fluid along the depth of the cargo tank: TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 5/2(61), 2021 3. The aim and objectives of research The aim of research is to develop a technology that maintains a constant value of the density of the drilling fluid along the depth of the tank in which it is transported. This will provide: – maintaining the operational characteristics of the drilling fluid; – minimization of sediment formation of heavy com- ponents with which the drilling fluid is doped; – reduction of energy costs for the process of pumping d illi fl id f th PSV t th d illi l tf – reduction of energy costs for the process of pumping drilling fluid from the PSV to the drilling platform; – maintenance of the technical condition of the vessel drilling fluid transportation system. – reduction of energy costs for the process of pumping drilling fluid from the PSV to the drilling platform; maintenance of the technical condition of the vessel drilling fluid from the PSV to the drilling platform; – maintenance of the technical condition of the vessel drilling fluid transportation system. The fluid was transported in six tanks of the same type, located symmetrically on the left and right sides. The system provides for technological operations for taking on board and delivering drilling fluid to the oil platform, which are provided by cargo pumps. In order to minimize the process of stratification of the density of the drill- ing fluid, to prevent its stratification and the formation This aim can be achieved by solving the following objectives: 1. Monitor the density of the drilling fluid along the height of the cargo tank. 2. Consider ways to modernize the vessel’s drilling fluid storage and transportation system. TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 5/2(61), 2021 16 ISSN 2664-9969 Table 2 Criterion for assessing the stratification of the density of the drilling fluid along the depth of the cargo tank, depending on the time and method of its transportation Transportation method Transportation time, hours 6 12 18 24 30 36 1 120 342 603 879 1257 1824 2 96 216 312 438 537 597 3 36 78 120 162 201 219 Note: 1 – without changing the design of the system; 2 – transportation with additional X-shaped circulation; 3 – transportation with additional X-shaped circulation and air supply to the volume of the cargo tank Criterion for assessing the stratification of the density of the drilling fluid along the depth of the cargo tank, depending on the time and method of its transportation Transportation method Transportation time, hours 6 12 18 24 30 36 1 120 342 603 879 1257 1824 2 96 216 312 438 537 597 3 36 78 120 162 201 219 INFORMATION AND CONTROL SYSTEMS: SYSTEMS AND CONTROL PROCESSES ISSN 2664-9969 INFORMATION AND CONTROL SYSTEMS: SYSTEMS AND CONTROL PROCESSES Fig. 3. Criterion for assessing the stratification of the density of the drilling fluid depending on time for different transportation conditions: 1 – without changing the design of the system; 2 – additional X-shaped circulation; 3 – additional X-shaped circulation and forced air supply to the bottom of the cargo tank of the cargo tank and are a criterion for assessing the density stratification) are given in Table 2. The area of these trapeziums has the dimension (kg⋅h/m3), however, further (in the text and in Table 2) only numerical va lues are indicated. The increase in area corresponds to an increase in density stratification along the depth of the cargo tank and the formation of sludge of heavy compo- nents of the drilling fluid. The results of Table 2 are summarized in the form of a nomogram shown in Fig. 3. a b c Fig. 2. Changes in the density of the drilling fluid under different conditions of its transportation: a – transportation without changing design of the system; b – with additional X-shaped circulation; c – w additional X-shaped circulation and simultaneous air supply; 1 – den of the drilling fluid on the surface of the tank; 2, 3, 4 – at a leve corresponding to 30 %, 60 %, 90 % of the depth of the tank a Fig. 3. Criterion for assessing the stratification of the density of the drilling fluid depending on time for different transportation conditions: 1 – without changing the design of the system; 2 – additional X-shaped circulation; 3 – additional X-shaped circulation and forced air supply to the bottom of the cargo tank The nomograms shown in Fig. 3 confirm the effective- ness of the proposed methods for maintaining the density of the drilling fluid. None of the options can completely prevent the process of settling of heavy components that make up the drilling fluid. Moreover, their use contributes to a 3.6–10.9-fold decrease in density stratification. The nomograms shown in Fig. 3 confirm the effective- ness of the proposed methods for maintaining the density of the drilling fluid. None of the options can completely prevent the process of settling of heavy components that make up the drilling fluid. Moreover, their use contributes to a 3.6–10.9-fold decrease in density stratification. 7. SWOT analysis of research results Strengths. The strengths of this study are that the pro- posed method provides the minimum values of the density stratification of the drilling fluid along the depth of the tank in which it is transported. It also prevents the drill- ing fluid from stratifying and the formation of sediment of heavy components included in its composition. Maintaining the required density of the drilling fluid minimizes the time it takes to pump it from the vessel to the drilling platform and reduces energy costs for this operation. b Weaknesses. The weaknesses of this study are related to the fact that to use the proposed method, it is necessary to re-equip the vessel drilling fluid transportation system. Such works can be performed only by agreement with the vessel owner, and in some cases with the Register, which controls the operation of the sea vessel and its power plant. c Opportunities. Application of the proposed technology for maintaining the rheological characteristics of technical fluids is possible in various facilities of the technological complex to ensure oil production (for example, onshore bases providing production and temporary storage of drill- ing fluid, as well as in transport organizations carrying out the transportation of drilling fluid from onshore bases to the PSV vessel). c Fig. 2. Changes in the density of the drilling fluid under different conditions of its transportation: a – transportation without changing the design of the system; b – with additional X-shaped circulation; c – with additional X-shaped circulation and simultaneous air supply; 1 – density of the drilling fluid on the surface of the tank; 2, 3, 4 – at a level corresponding to 30 %, 60 %, 90 % of the depth of the tank Threats. The option of maintaining the rheological characteristics of technical fluids proposed in this work is of an applied nature and is based on practical research. For the analytical calculation of the optimal processing time for the drilling fluid, which ensures the minimum stratification of its density with the simultaneous mini- mum energy consumption for the processing process, it is necessary to develop a mathematical model and perform mathematical modeling [24, 25]. 6. Research results These areas (S0 6, S6 12, S12 18, S18 24, S24 30, S30 36) can be calcu- lated as the areas of the corresponding trapezoids [4, 23]. The values of the trapezoid areas (which characterize the density stratification of the drilling fluid along the depth TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 5/2(61), 2021 17 References The power required to ensure the functioning of such a system does not significantly affect the energy performance of the vessel power plant, which is characterized by increased values and a large safety factor for specialized vessels that ensure the operation of drilling platforms. 3. It is determined that the modernized system, which provides the minimum level of density stratification of the drilling fluid during its transportation on PSV-type vessels, includes a mobile circulation pump and an air compressor, pipelines and fittings. It can also be installed on a specialized vessel in accordance with the techno- logical scheme directly by the vessel’s crew. The power required to ensure the functioning of such a system does not significantly affect the energy performance of the vessel power plant, which is characterized by increased values and a large safety factor for specialized vessels that ensure the operation of drilling platforms. 11. Gnap, J., Senko, ., Marienka, P. (2020). Time Availability of the Public Terminal of Intermodal Transport ilina with a Selected European Maritime Port. Na e More, 67 (3), 217–225. doi: http://doi.org/10.17818/nm/2020/3.5 12. Kisel’, A. G., Purtov, E. D., Vyborov, S. S., Belan, D. Yu., Grechishnikov, V. A. (2019). Studying the function of the lifespan of carbide tools during turning of workpieces made of alloy 12H18N10T from the cooling properties of the cutting fluids. Journal of Physics: Conference Series, 1260, 062011. doi: http://doi.org/10.1088/1742-6596/1260/6/062011 13. Kondratenko, Y. P., Topalov, А. M., Kozlov, O. V. (2019). Simulation of the Initial Stability of the Floating Dock for the List and Trim Stabilization Tasks. Problems of the Re- gional Energetics, 1-2 (41), 12–24. doi: http://doi.org/10.5281/ zenodo.3239200 p g p 4. The following technological solutions are proposed: additional X-shaped circulation of the drilling fluid, as well as X-shaped circulation with simultaneous air supply to the bottom of the cargo tank. These solutions provide a 1.3–3.6 fold (in the first case) and 4.2–10.9 fold (in the second case) decrease in density stratification – the most important rheological characteristic of the drilling fluid. This prevents the formation of sediment of heavy compo- nents of the drilling fluid, reduces the energy load on the vessel’s power plant during pumping of the drilling fluid to the drilling platform. It also reduces the pumping time of the drilling fluid and helps to ensure the technological process of oil production. 14. Javadian, S., Sadrpoor, S. M. (2020). INFORMATION AND CONTROL SYSTEMS: SYSTEMS AND CONTROL PROCESSES responding to 30 %, 60 %, 90 % of the depth of the tank. It has been experimentally established that for a trans- portation time of 6–36 h, the density stratification of the drilling fluid is 3.04–32.04 %. 8. Conclusions Note: 1 – without changing the design of the system; 2 – transportation with additional X-shaped circulation; 3 – transportation with additional X-shaped circulation and air supply to the volume of the cargo tank Note: 1 – without changing the design of the system; 2 – transportation with additional X-shaped circulation; 3 – transportation with additional X-shaped circulation and air supply to the volume of the cargo tank 1. It is found that to monitor the density of the drilling fluid, it is necessary to perform measurements at levels cor- 1. It is found that to monitor the density of the drilling fluid, it is necessary to perform measurements at levels cor- TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 5/2(61), 2021 18 ISSN 2664-9969 References doi: http://doi.org/ 10.3844/ajassp.2016.200.208 In this case (in the case of transportation of the drilling fluid from the shore to the drilling platform in 6–36 h), the density stratification is: – option a – 2.30–9.01 %; – option a – 2.30–9.01 %; 7. Sagin, S. V., Solodovnikov, V. G. (2017). Estimation of Ope rational Properties of Lubricant Coolant Liquids by Optical Methods. International Journal of Applied Engineering Research, 12 (19), 8380–8391. – option b – 0.73–2.93 %. – option b – 0.73–2.93 %. The decrease in density stratification for these trans- portation options is explained by the artificial creation of a laminar flow of the drilling fluid (due to its forced circulation between adjacent cargo tanks). As well as its local turbulent currents (due to additional air supply to the lower part of the cargo tanks). The cross movement of the drilling fluid (which is carried out both under the action of gravitational forces and with the help of additio nally installed circulation pumps) and air can contribute to the occurrence of the cavitation phenomenon. This leads to a forceful effect on organic and inorganic compounds in the volume of the drilling fluid, which prevents their sedimentation and maintains them in fluid. 8. Budashko, V., Obniavko, T., Onishchenko, O., Dovidenko, Y., Ungarov, D. (2020). Main Problems of Creating Energy-efficient Positioning Systems for Multipurpose Sea Vessels. 2020 IEEE 6th International Conference on Methods and Systems of Navi- gation and Motion Control (MSNMC), 106–109. doi: http:// doi.org/10.1109/msnmc50359.2020.9255514 9. Kuropyatnyk, O. A., Sagin, S. V. (2019). Exhaust Gas Re- circulation as a Major Technique Designed to Reduce NOх Emissions from Marine Diesel Engines. Na e More, 66 (1), 1–9. doi: http://doi.org/10.17818/nm/2019/1.1 10. He, J. F., Zhang, P. Y., Yin, Q. L., Yin, K., Liu, H. P. (2015). Study of drilling muds on the anti-erosion property of a fluidic amplifier in directional drilling. Frattura Ed Integrit Strutturale, 9 (34), 564–573. doi: http://doi.org/10.3221/igf-esis.34.62 3. It is determined that the modernized system, which provides the minimum level of density stratification of the drilling fluid during its transportation on PSV-type vessels, includes a mobile circulation pump and an air compressor, pipelines and fittings. It can also be installed on a specialized vessel in accordance with the techno- logical scheme directly by the vessel’s crew. References 1. Karianskyi, S. A., Maryanov, D. M. (2020). Features of trans- portation of high-density technical liquids by marine specialized vessels. Scientific research of the SCO countries: synergy and integration. Beijing, 2, 150–153. Thus, heavy components of the drilling fluid, settling to the bottom of the tank, subsequently complicate the process of pumping it out. This can lead to breakdown or failure of cargo pumps, inability to pump drilling fluid onto the drilling platform and further disruption of the oil production process. 2. Lipin, A. A., Kharlamov, Y. P., Timonin, V. V. (2013). Circulation system of a pneumatic drill with central drilling mud removal. Journal of Mining Science, 49 (2), 248–253. doi: http://doi.org/ 10.1134/s1062739149020068 3. Liang, Y., Ju, X., Li, A., Li, C., Dai, Z., Ma, L. (2020). The Process of High-Data-Rate Mud Pulse Signal in Logging While Drilling System. Mathematical Problems in Engineering, 2020, 1–11. doi: http://doi.org/10.1155/2020/3207087 p p 2. It is shown that in order to reduce the stratification of the drilling fluid density, it is advisable to modernize the vessel’s storage and transportation system. The paper proposes the following upgrade options: 4. Zablotsky, Y. V., Sagin, S. V. (2016). Enhancing Fuel Efficiency and Environmental Specifications of a Marine Diesel When using Fuel Additives. Indian Journal of Science and Techno logy, 9 (46), 353–362. doi: http://doi.org/10.17485/ijst/2016/ v9i46/107516 a) the use of additional X-shaped circulation of the drilling fluid in the volume of adjacent cargo tanks (while pumping the drilling fluid between the lower and upper parts of the tanks); a) the use of additional X-shaped circulation of the drilling fluid in the volume of adjacent cargo tanks (while pumping the drilling fluid between the lower and upper parts of the tanks); 5. Lahoida, A., Boryn, V., Sementsov, G., Sheketa, V. (2020). De- velopment of an automated system of control over a drilling mud pressure at the inlet to a well. Eastern-European Journal of Enterprise Technologies, 4 (2 (106)), 82–94. doi: http://doi.org/ 10.15587/1729-4061.2020.209844 b) the complex use of additional X-shaped circula- tion and simultaneous air supply to the bottom of the cargo tanks. 6. Sagin, S. V., Semenov, O. V. (2016). Motor Oil Viscosity Strati- fication in Friction Units of Marine Diesel Motors. American Journal of Applied Sciences, 13 (2), 200–208. TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 5/2(61), 2021 References Demulsification of water in oil emulsion by surface modified SiO2 nanoparticle. Journal of Petroleum Science and Engineering, 184, 106547. doi: http:// doi.org/10.1016/j.petrol.2019.106547 15. Sagin, S. V., Solodovnikov, V. G. (2015). Cavitation Treat- ment of High-Viscosity Marine Fuels for Medium-Speed Diesel Engines. Modern Applied Science, 9 (5), 269–278. doi: http:// doi.org/10.5539/mas.v9n5p269 16. Shettigar, R. R., Misra, N. M., Patel, K. (2018). CTAB grafted PAM gel and its application in drilling fluid. Journal of Pe- troleum Science and Engineering, 160, 129–135. doi: http:// doi.org/10.1016/j.petrol.2017.10.040 TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 5/2(61), 2021 19 Denis Maryanov, Postgraduate Student, Department of Vessel Po wer Plants, National University Odessa Maritime Academy, Odessa, Ukraine, e-mail: denismaryanovv@gmail.com, ORCID: https://orcid.org/ 0000-0002-1355-5844 TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 5/2(61), 2021 INFORMATION AND CONTROL SYSTEMS: SYSTEMS AND CONTROL PROCESSES ISSN 2664-9969 fluids on the operation of frictional components. Russian En- gineering Research, 16 (9), 1–7. fluids on the operation of frictional components. Russian En- gineering Research, 16 (9), 1–7. 17. Shahbazov, E. (2015). Development and application of na nodrilling muds. Scientific Israel-Technological Advantages, 17 (2), 160–171. 23. Li, X., Zhang, J., Tang, X., Mao, G., Wang, P. (2020). Study on wellbore temperature of riserless mud recovery system by CFD approach and numerical calculation. Petroleum, 6 (2), 163–169. doi: http://doi.org/10.1016/j.petlm.2019.06.006 18. Sagin, S. V., Kuropyatnyk, O. A. (2018). The Use of Exhaust Gas Recirculation for Ensuring the Environmental Perfor- mance of Marine Diesel Engines. Na e More, 65 (2), 78–86. doi: http://doi.org/10.17818/nm/2018/2.3 24. Likhanov, V. A., Lopatin, O. P. (2020). Dynamics of soot formation and burnout in a gas diesel cylinder. IOP Confe rence Series: Materials Science and Engineering, 862, 062033. doi: http://doi.org/10.1088/1757-899x/862/6/062033 19. Petcovic, M., Zubcic, M., Krcum, M., Pavic, I. (2021) Wind Assisted Ship Propulsion Technologies – Can they Help in Emissions Reduction? Na e more, 68 (2), 102–109. doi: http:// doi.org/10.17818/nm/2021/2.6 25. Wanderley Neto, A. O., da Silva, V. L., Rodrigues, D. V., Ri- beiro, L. S., Nunes da Silva, D. N., Oliveira Freitas, J. C. (2020). A novel oil-in-water microemulsion as a cementation flushing fluid for removing non-aqueous filter cake. Journal of Petro- leum Science and Engineering, 184, 106536. doi: http://doi.org/ 10.1016/j.petrol.2019.106536 20. Mahmoudpour, M., Pourafshary, P. (2021). Investigation of the effect of engineered water/nanofluid hybrid injection on enhanced oil recovery mechanisms in carbonate reservoirs. Journal of Petroleum Science and Engineering, 196, 107662. doi: http://doi.org/10.1016/j.petrol.2020.107662 21. Sagin, S. V., Semenov, O. V. (2016). Marine Slow-Speed Die- sel Engine Diagnosis with View to Cylinder Oil Specifica- tion. American Journal of Applied Sciences, 13 (5), 618–627. doi: http://doi.org/10.3844/ajassp.2016.618.627 22. Popovskii, Yu. M., Sagin, S. V., Khanmamedov, S. A., Grebe- nyuk, M. N., Teregerya, V. V. (1996). Designing, calculation, testing and reliability of machines: influence of anisotropic TECHNOLOGY AUDIT AND PRODUCTION RESERVES — № 5/2(61), 2021 20
W4226175529.txt	https://journals.wlb-stuttgart.de/ojs/index.php/zwlg/article/download/2430/2501	de		Rezension von: Köster, Roman, Hugo Boss, 1924–1945	Zeitschrift für württembergische Landesgeschichte	2,022	cc-by	998	590 Buchbesprechungen 20. Jahrhunderts. Als 1953 in der Bundesrepublik die allgemeine Fahrtschreiberpflicht für LKW ab 7,5 Tonnen und Busse eingeführt wurde, brachen in Villingen „goldene Zeiten“ an, die noch eine neue Dimension erreichten, als die EWG 1969/70 die Einbaupflicht für alle Mitgliedsländer einführte. Zugleich partizipierte Kienzle äußerst erfolgreich am zweiten technisch-industriellen Großtrend des 20. Jahrhunderts – der elektronischen Datenverarbeitung – und trieb die Entwicklung der MDT, dem Bindeglied zwischen den unerschwinglichen Großrechnern und den mechanischen Büromaschinen mit eigenen Entwicklungen erfolgreich voran. Diese Erfolgsgeschichte schildert Armin Müller recht spannend und – Voraussetzung für eine moderne und gelungene Unternehmensgeschichte – im Kontext der wirtschaftlichen, technischen und gesellschaftspolitischen Entwicklung. Ausführlich stellt er die Produktgeschichte dar. Er geht nicht nur auf die verschiedenen technisch-organisatorischen Bereiche der Firmenentwicklung ein, sondern begreift das Unternehmen, wie es Toni Pierenkemper in seiner grundlegenden Einführung „Unternehmensgeschichte. Eine Einführung in ihre Methoden und Ergebnisse“ (2000) von einer Unternehmensgeschichte fordert, auch als soziales Gebilde, in dem Unternehmensführung und Management mit den Arbeitern und Angestellten ein Geflecht bilden, dessen Funktionsfähigkeit ein wichtiger Faktor für den Erfolg eines Unternehmens ist. Letztlich konnte Kienzle wie die gesamte deutsche Computerindustrie nicht gegen die amerikanisch-asiatische Konkurrenz bestehen. Unter dem wirtschaftlichen Druck entwickelte sich das Familienunternehmen zur Konzerntochter: 1981/82 wurde Kienzle Teil der Mannesmann AG, dann als Folge der bis dahin größten Firmenübernahme in der Geschichte – der feindlichen Übernahme Mannesmanns durch den Mobilfunkhersteller Vodafone – der Siemens AG und 2007 schließlich der Continental AG. „Bis heute steht insbesondere das Villinger Werk Continental AG für das industrielle Erbe Kienzles. Mit rund 1.300 Mitarbeitern ist es weiterhin größter industrieller Arbeitgeber der Region“, stellt Armin Müller gegen Ende seiner Firmengeschichte fest. Etlichen dieser Mitarbeiter ist der Markenname Kienzle kein Begriff mehr. Dies sollte sich mit Armin Müllers kenntnisreicher Firmengeschichte ändern. Petra Garski-Hoffmann Roman Köster: Hugo Boss, 1924–1945. Die Geschichte einer Kleiderfabrik zwischen Weimarer Republik und „Drittem Reich“ (Schriftenreihe zur Zeitschrift für Unternehmensgeschichte 23). München: Verlag C. H. Beck 2011. 117 S. ISBN 978-3-406-61992-2. A 29,90 Die Firma Hugo Boss in Metzingen hat sich in den letzten 40 Jahren von einer lokalen Kleiderfabrik zu einem weltweit agierenden Trendsetter der Bekleidungsindustrie entwickelt. Dies ist in erster Linie das Verdienst von Uwe und Jochen Holy, der beiden Enkel des Firmengründers Hugo Boss. Wurden in den Jahrzehnten von der Gründung 1924 bis nach dem Zweiten Weltkrieg vor allem Arbeitskleidung und Uniformen hergestellt, so konzentrierten die Brüder Holy die Produktion auf modische Herrenbekleidung. Vor allem aufgrund eines erfolgreichen Marketings entwickelte sich der Betrieb in den 1970er und 1980er Jahren zu einem der führenden Unternehmen in diesem Bereich. Ein wichtiger Absatzmarkt war der Fabrikverkauf der Firma in Metzingen, der zugleich den Grundstein für die Outletcity Metzingen legte. Im Bewusstsein der Zeitgenossen heute ist die Firma Hugo Boss ein Weltkonzern, weshalb alles, was dieses Unternehmen betrifft, eine hohe mediale Aufmerksamkeit erfährt. Dabei darf allerdings nicht übersehen werden, dass das Unternehmen bis in Zeitschrift für Württembergische Landesgeschichte 72 (2013). © Kommission für geschichtliche Landeskunde in Baden-Württemberg und Württembergischer Geschichts- und Altertumsverein e.V. ISSN 0044-3786 Kulturgeschichte, Literatur- und Musikgeschichte 591 die 1960er Jahre eine kleinere mittelständische Kleiderfabrik war, die noch nicht einmal in Metzingen zu den großen Unternehmen zählte. Dementsprechend spielte die Firma in der Zeit des Nationalsozialismus auch keine herausgehobene Rolle, wie der Eindruck in den Medien immer wieder erweckt wird. Roman Köster gibt im vorliegenden Band zunächst einen Überblick über den Untersuchungsgegenstand, den Forschungsstand und die Quellenlage, wobei deutlich wird, dass durch den Verlust der Firmenunterlagen zu diesem Thema nur eine schmale Quellenbasis vorhanden ist. Der Verfasser versucht dies durch eine stärkere „Kontextualisierung“ der Unternehmensgeschichte auszugleichen. Der Band schildert zunächst die Grundlinien der Entwicklung der Bekleidungsindustrie bis 1933, den Lebensgang von Hugo Boss und die Gründung der Firma Hugo Boss und ihre Entwicklung bis nach der Weltwirtschaftskrise. In einem zweiten Abschnitt werden die Rahmenbedingungen der Bekleidungs- und Uniformherstellung im Dritten Reich und die Entwicklung des Unternehmens zwischen 1933 und 1945 dargestellt. Dabei wird erkennbar, dass das Unternehmen durch die Uniformproduktion für NS-Organisationen und die Wehrmacht vom Nationalsozialismus profitiert hat. Dies gilt vor allem für die ersten Jahre des Zweiten Weltkriegs. Der dritte Abschnitt des Bandes widmet sich der Zwangsarbeit bei der Firma Hugo Boss, wobei der Verfasser aufgrund der etwas besseren Quellenlage ein detailliertes Bild von den Lebensumständen der bei Hugo Boss beschäftigten, vor allem aus Frankreich und Polen stammenden Zwangsarbeiter zeichnet. Das Verhalten der Unternehmensführung war gekennzeichnet von einem Nebeneinander von Härte, Zwang und Fürsorge. Ein letzter Abschnitt ist dem Entnazifizierungsverfahren gegen Hugo Boss und der Nachkriegsproduktion gewidmet. In seinem Resümee kommt Roman Köster zu dem Ergebnis, dass die Firma zwar nachweislich vom Nationalsozialismus profitiert habe, es jedoch keine ursächliche Verknüpfung zwischen der damaligen Uniformproduktion und dem Aufstieg des Unternehmens seit den 1970er Jahren gebe. Durch eine abwägende Interpretation der Quellen erhält der Leser ein differenziertes Bild vom Unternehmen in der Zeit des Nationalsozialismus. Weshalb jedoch Aussagen der Töchter von Hugo Boss, sie hätten nach dem Zweiten Weltkrieg noch Kontakt zu den Kindern der jüdischen Familie Herold gehabt (S. 63), in Zweifel gezogen werden müssen, erschließt sich dem Rezensenten nicht. Der ansonsten kompetent recherchierte Band arbeitet die Geschichte des Unternehmens Hugo Boss von der Gründung bis nach dem Zweiten Weltkrieg auf, soweit dies die Quellenlage zulässt, und stellt damit einen wichtigen Beitrag zur Wirtschaftsgeschichte im deutschen Südwesten dar. Rolf Bidlingmaier Kulturgeschichte, Literatur- und Musikgeschichte Aus der Werkstatt Diebold Laubers. Hg. von Christoph Fasbender (Kulturtopographie des alemannischen Raums 3). Berlin/Boston: Walter de Gruyter GmbH & Co. KG 2012. VI, 384 S. ISBN 978-3-11-026206-3. Geb. A 99,95 Der Sammelband enthält 14 Studien, die sich hauptsächlich der „textlichen Qualität“ sowie „der noch ganz unerforschten redaktionellen Mühewaltung“ der Hagenauer Werkstatt des Diebold Lauber und der dort entstandenen Textzeugen widmen wollen (Covertext). Schon in der Einleitung geht der Herausgeber Christoph Fasbender auf die Problematik interdisziplinärer Forschung – in diesem Fall zwischen Kunstgeschichte und Germanistik – ein, die die Arbeiten zu den spätmittelalterlichen Bilderhandschriften, zu denen auch dieje- Zeitschrift für Württembergische Landesgeschichte 72 (2013). © Kommission für geschichtliche Landeskunde in Baden-Württemberg und Württembergischer Geschichts- und Altertumsverein e.V. ISSN 0044-3786
https://openalex.org/W3045789408	https://journals.us.edu.pl/index.php/flit/article/download/9612/7440	Italian	null	Rassegna: Napoli di Herling. Preparato da Annagrazia Carriero, Ivan de Matteo	Fabrica Litterarum Polono-Italica	2,020	cc-by-sa	848	www.fabricalitterarum.com ISSN 2658-185X DOI: http://doi.org/10.31261/FLPI.2020.02.20 2020, nr 1 (2), s. 225–226 www.fabricalitterarum.com ISSN 2658-185X DOI: http://doi.org/10.31261/FLPI.2020.02.20 2020, nr 1 (2), s. 225–226 www.fabricalitterarum.com ISSN 2658-185X DOI: http://doi.org/10.31261/FLPI.2020.02.20 KRONIKA 225 Fabrica Litterarum Polono-Italica \| 2020, nr 1 (2) 225 Fabrica Litterarum Polono-Italica \| 2020, nr 1 (2) Rassegna: Napoli di Herling Il 2019 è stato dichiarato l’anno di Gustaw Herling-Grudziński, scrittore polacco che per più della metà della vita ha fatto di Napoli la propria casa. Per questo il Comune di Napoli, insieme con l’Istituto di Cultura Polacca di Roma, l’Università “L’Orientale” di Napoli, l’Istituto Italiano per gli Studi Storici e l’Università “Suor Orsola Benincasa” di Napoli, hanno voluto rendergli omaggio con un festival a lui interamente dedicato, che si è svolto dal 24 al 26 ottobre. Il festival si è aperto alle ore 17:00 del 24 ottobre con i saluti di Anna Maria Anders, ambasciatore della Repubblica di Polonia in Italia, figlia del generale Władysław Anders, per il quale Grudziński combatté la famosa battaglia di Monte Cassino del 1944, del sindaco di Napoli Luigi de Magistris, di Gaetano Daniele, assessore alla Cultura e al Turismo del Comune di Napoli, di Natalino Irti, presidente dell’Istituto Italiano per gli Studi Storici, di Elda Morlicchio, rettrice dell’“L’Orientale”, Lucio d’Alessandro, rettore del “Suor Orsola Benincasa”, e di Dario dal Verme, console onorario della Repubblica di Polonia a Napoli. A seguire, la presentazione del Meridiano Mondadori “Etica e Let- teratura”, che raccoglie le principali opere dello scrittore: Un mondo a parte, Diario scritto di notte e Racconti, introdotto e moderato da Emma Giammattei, alla quale sono intervenuti Krystyna Jaworska, Marta Herling, Goffredo Fofi e Silvio Perrella. Alle ore 20:00 della stessa giornata Lech Dzierżanowski ha letto “L’amicizia fra Ro- man Palester e Gustaw Herling nelle loro lettere”, aprendo il concerto “La musica ab- batte le frontiere”, in collaborazione con l’Istituto di Musica e Danza di Varsavia. I bra- ni di Karol Szymanowski, Dmitrij Šostakovič, Roman Palester, Mieczysław Weinberg, Grażyna Bacewicz e Ryszard Sielicki sono stati interpretati da Agnieszka Marucha (violino), Rafał Zambrzycki-Payne (violino) e Jakub Tchorzewski (pianoforte). A fine concerto l’Istituto di Cultura Polacca di Roma ha offerto agli ospiti una cena a buffet. 225 Fabrica Litterarum Polono-Italica \| 2020, nr 1 (2) Rassegna: Napoli di Herling Il giorno successivo, venerdì 25, presso il complesso monumentale di San Domenico Maggiore, si è aperto il convegno “L’Europa di Herling”, alla presen- za di illustri docenti, tra cui il professor Luigi Marinelli, moderatore della prima sessione del giorno, Krystyna Jaworska, Luca Bernardini e Laura Quercioli Mincer, che nei loro interventi hanno parlato di Europa, di colpe del passato, di religione ebraica e di molto altro. È stato inoltre presentato il libro L’autobiografia italiana nei racconti di Gustaw Herling-Grudziński di Alessandro Ajres, professore di lingua e letteratura polacca all’Università degli Studi di Torino. Alle ore 20:00, presso la Basilica di Santa Chiara, l’attore Toni Servillo ha de- liziato i fortunati presenti con la lettura di Requiem per il Campanaro di Gustaw Herling, tradotto da Vera Verdiani, adattato per l’occasione da Stefano de Matteis. La storia narra di un bambino di origini ebraiche che, dopo la morte dei genitori in un rogo causato dai soldati nazisti, viene accolto dai francescani. L’ultima giornata, sabato 26 ottobre, si è aperta con la lettura delle traduzioni di alcune pagine del Diario scritto di notte, a cura degli studenti che seguono i corsi di lingua e letteratura polacca in tredici Atenei italiani. Tre studenti prescelti da ciascuna cattedra di polonistica si sono cimentati nella traduzione di una pagina del Diario di Herling, che hanno poi letto di fronte ad altri studenti e docenti in una sessione dei lavori moderata dal professor Andrea Ceccherelli dell’Università degli Studi di Bologna. La giornata è proseguita all’Istituto Italiano degli Studi Storici in via Benedetto Croce (dedicata al filosofo, suocero di Herling-Grudziński) con le testimonianze di amici e parenti dello scrittore che ne hanno delineato un ritratto più intimo e familiare. A questa sessione, ovviamente moderata da Marta Herling, figlia dello scrittore, sono intervenuti Anna Bernhardt, Édith de la Héronnière, Elena de Varda, Wojciech Karpiński, Zdzisław Kudelski, Titti Marrone, Józef Opalski, Silvio Perrella, Piero Sinatti e Vera Verdiani. Mariusz Wilk, purtroppo, non ha potuto es- sere presente. Da non dimenticare, inoltre, l’inaugurazione della mostra documen- taria “Gustav Herling – ritratto napoletano”, aperta al pubblico fino al 15 novembre. Infine, a conclusione del festival, è stato proiettato il film del 1995 Diario scritto sotto il vulcano del regista polacco Andrzej Titkow. 225 I tre giorni del festival dedicato a un autore di profonda cultura e dai multiformi interessi, a uno scrittore poliedrico (come è stato definito da alcuni relatori), purtroppo lasciato a lungo nell’ombra a causa di questioni spesso puramente politiche, sono stati forse il modo più umile e affettuoso per chiedere perdono, ma anche per offrire alle future generazioni la possibilità di conoscere non solo un uomo, ma anche, attraverso le sue opere, un passato per molto tempo trascurato, se non di proposito nascosto. La nota è stata preparata da: Annagrazia Carriero, Ivan de Matteo La nota è stata preparata da: Annagrazia Carriero, Ivan de Matteo
https://openalex.org/W3196784785	https://hal-insu.archives-ouvertes.fr/insu-03331205/file/g49067.pdf	English	null	Fjord network in Namibia: A snapshot into the dynamics of the late Paleozoic glaciation	Geology	2,021	cc-by	5,948	To cite this version: Pierre Dietrich, Neil P Griﬀis, Daniel P Le Heron, Isabel P Montañez, Christoph Kettler, et al.. Fjord network in Namibia: A snapshot into the dynamics of the late Paleozoic glaciation. Geology, 2021, 49 (12), pp.1521-1526. 10.1130/G49067.1. insu-03331205 Fjord network in Namibia: A snapshot into the dynamics of the late Paleozoic glaciation Pierre Dietrich, Neil P Griﬀis, Daniel P Le Heron, Isabel P Montañez, Christoph Kettler, Cécile Robin, François Guillocheau Distributed under a Creative Commons Attribution 4.0 International License ABSTRACT infer the pace of ice-margin fluctuations and the paleo-ice thickness that once occupied these fjords. Furthermore, we estimate glacio- isostatic adjustment following deglaciation. This work highlights the dynamic nature of the LPIA in southwestern Gondwana and the potential for an under-appreciated deep-time carbon and sediment sink that, if valid, would have had direct climate implications through the impact of Carboniferous–Permian atmo spheric CO2. Fjords are glacially carved estuaries that profoundly influence ice-sheet stability by draining and ablating ice. Although abundant on modern high-latitude continental shelves, fjord-network morphologies have never been identified in Earth’s pre-Cenozoic glacial epochs, hindering our ability to constrain ancient ice-sheet dynamics. We show that U-shaped valleys in northwestern Namibia cut during the late Paleozoic ice age (LPIA, ca. 300 Ma), Earth’s penultimate icehouse, represent intact fjord-network morphologies. This preserved glacial morphology and its sedimentary fill permit a reconstruction of paleo-ice thicknesses, glacial dynamics, and resulting glacio-isostatic adjustment. Glaciation in this region was initially characterized by an acme phase, which saw an extensive ice sheet (1.7 km thick) covering the region, followed by a waning phase characterized by 100-m-thick, topographically constrained outlet glaciers that shrank, leading to glacial demise. Our findings demonstrate that both a large ice sheet and highland glaciers existed over northwestern Namibia at different times during the LPIA. The fjords likely played a pivotal role in glacier dynamics and climate regulation, serving as hotspots for organic carbon sequestration. Aside from the present-day arid climate, northwestern Namibia exhibits a geomorphology virtually unchanged since the LPIA, permitting unique insight into this icehouse. HAL Id: insu-03331205 https://insu.hal.science/insu-03331205v1 Submitted on 1 Sep 2021 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License RESULTS: FJORD NETWORK IN NAMIBIA The Kaokoland region of northwest ern Namibia is characterized by a prominent bimodal topography where staircase-like pla teaus (at ∼500 m, ∼1000 m, and ∼1500 m) separated by NNW-SSE–oriented escarpments are deeply dissected by a network of valleys in which modern rivers flow (Fig. 1A). These valleys are 1–5 km in width and 80–130 km in length and have steep, subvertical flanks defin ing U-shaped cross-profiles (Figs. 1B and 1C) whose depths range between 400 and 1200 m; interfluves occur up to 1.7 km above the thalweg of the Kunene Valley (Fig. 1A). These valleys are carved within hard Archean to Proterozoic lithologies of the Pan-African and Congo craton basement (Goscombe and Gray, 2008). Fjord network in Namibia: A snapshot into the dynamics of the late Paleozoic glaciation Pierre Dietrich1,2, Neil P. Griffis3,4, Daniel P. Le Heron5, Isabel P. Montañez3, Christoph Kettler5 François Guillocheau1 1Géosciences Rennes, Université de Rennes, CNRS, UMR 6118, 35000 Rennes, France 2Department of Geology, University of Johannesburg, Johannesburg 2006, South Africa 3Department of Earth and Planetary Sciences, University of California, Davis, California 95616, USA 4Berkeley Geochronology Center, Berkeley, California 94709, USA 5Department für Geologie, Universität Wien, Althanstraße 14, 1190 Wien, Austria François Guillocheau 1Géosciences Rennes, Université de Rennes, CNRS, UMR 6118, 35000 Rennes, France 2Department of Geology, University of Johannesburg, Johannesburg 2006, South Africa 3Department of Earth and Planetary Sciences, University of California, Davis, California 95616, USA 4Berkeley Geochronology Center, Berkeley, California 94709, USA 5Department für Geologie Universität Wien Althanstraße 14 1190 Wien Austria epartment für Geologie, Universität Wien, Althanstraße 14, 1190 Wien, Austria CITATION: Dietrich, P., et al., 2021, Fjord network in Namibia: A snapshot into the dynamics of the late Paleozoic glaciation: Geology, v. 49, p. X https://doi.org/10.1130/G49067.1 Manuscript received 20 October 2020 Revised manuscript received 19 March 2021 Manuscript accepted 21 June 2021 © 2021 The Authors. Gold Open Access: This paper is published under the terms of the CC-BY license. CITATION: Dietrich, P., et al., 2021, Fjord network in Namibia: A snapshot into the dynamics of the late Paleozoic glaciation: Geology, v. 49, p. X https://doi org/10 1130/G49067 1 Downloaded from http://pubs.geoscienceworld.org/gsa/geology/article-pdf/doi/10.1130/G49067.1/5393367/g49067.pdf by CNRS INSU user Geological Society of America \| GEOLOGY \| Volume XX \| Number XX \| www.gsapubs.org Downloaded from http://pubs.geoscienceworld.org/gsa/geology/article-pdf/doi/10.1130/G49067.1/5393367/g49067.pdf by CNRS INSU user INTRODUCTION (A) Digital elevation model of Kaokoland (northwestern Namibia) highlighting glacial valleys (white dashed lines). Circle labeled “B & C” represents geographic position of panels B and C. (B) U-shaped Gomatum valley. Note subvertical valley walls at background above modern alluvial fans, and Karoo Supergroup outliers. Valley is ∼2 km wide. (C) Topographic transect of U-shaped Gomatum valley, showing position of sedimentary and Etendeka Volcanics successions. (D) Longitudinal profile of Hoarusib valley and its interfluve (dashed line), and Karoo and Etendeka outliers. Note bedrock structures (Goscombe and Gray, 2008). v.e.—vertical exaggeration. Background topography is from SRTM3 (USGS 2006), Shuttle Radar Topographic Mission, 3 Arc Second scene SRTM_n47w053_n54w074, Filled Finished B 2.0, Global Land Cover Facility, University of Maryland, College Park, Maryland, February 2000) available at https://www2.jpl.nasa.gov/srtm/. Map was generated using GlobalMapper® GIS software. B C A B B C C D Fi 1 (A) Di it l l ti d l f K k l d ( th t N ibi ) hi hli hti l i l ll ( hit d h d li ) Ci l l b l d “B D Figure 1. (A) Digital elevation model of Kaokoland (northwestern Namibia) highlighting glacial valleys (white dashed lines). Circle labeled “B & C” represents geographic position of panels B and C. (B) U-shaped Gomatum valley. Note subvertical valley walls at background above modern alluvial fans, and Karoo Supergroup outliers. Valley is ∼2 km wide. (C) Topographic transect of U-shaped Gomatum valley, showing position of sedimentary and Etendeka Volcanics successions. (D) Longitudinal profile of Hoarusib valley and its interfluve (dashed line), and Karoo and Etendeka outliers. Note bedrock structures (Goscombe and Gray, 2008). v.e.—vertical exaggeration. Background topography is from SRTM3 (USGS 2006), Shuttle Radar Topographic Mission, 3 Arc Second scene SRTM_n47w053_n54w074, Filled Finished B 2.0, Global Land Cover Facility, University of Maryland, College Park, Maryland, February 2000) available at https://www2.jpl.nasa.gov/srtm/. Map was generated using GlobalMapper® GIS software. Figure 1. (A) Digital elevation model of Kaokoland (northwestern Namibia) highlighting glacial valleys (white dashed lines). Circle labeled “B & C” represents geographic position of panels B and C. (B) U-shaped Gomatum valley. Note subvertical valley walls at background above modern alluvial fans, and Karoo Supergroup outliers. Valley is ∼2 km wide. (C) Topographic transect of U-shaped Gomatum valley, showing position of sedimentary and Etendeka Volcanics successions. (D) Longitudinal profile of Hoarusib valley and its interfluve (dashed line), and Karoo and Etendeka outliers. INTRODUCTION stratigraphic relationships and geological map ping (e.g., Kneller et al., 2004; Tedesco et al., 2016, and references therein), while the exis tence of paleo-fjord networks have not been rig orously established. The scarcity of ancient fjord morphologies seemingly reflects the intrinsic transient nature of these large-scale geomor phological features (Bianchi et al., 2020), ren dering them prone to erosion over geological time scales. In turn, our ability to fully embrace large-scale dynamics of deep-time ice sheets and to assess their sensitivity to climate change is hindered. Fjords are long, deep, and narrow glacially carved estuaries that were occupied by outlet glaciers. They play a dramatic role in ice-sheet stability (i.e., drainage and ablation), are sensi tive to climate change during icehouse periods, and act as important sediment sinks (Syvitski et al., 1987; Bennett, 2003; Kessler et al., 2008; Briner et al., 2009; Moon et al., 2018). Despite occupying just 0.1% of Earth’s modern oceans, fjords account for >10% of Earth’s organic car bon burial and may thus significantly impact the global carbon cycle (Smith et al., 2015). Fjord morphology and the sedimentary infill more over play an instrumental role in assessing ice- sheet dynamics and climate change (Eilertsen et al., 2011; Steer et al., 2012; Normandeau et al., 2019; Bianchi et al., 2020). Despite their present-day abundance across high-latitude continental shelves, fjords from pre-Cenozoic glacial epochs have mostly been inferred from The valley floors display abundant hard- bed glacial erosion features such as striae, scratches, grooves, and crescentic gouges (Fig. 2A) superimposed on whalebacks and roches moutonnées (see also Martin, 1953, 1981; Martin and Schalk, 1959). Scratches and striations are also developed on subvertical walls that flank the U-shaped valleys as well as on the subvertical westernmost (Purros; Fig. 1) We present a geomorphic and sedimento logic analysis of a pristine paleo-fjord network across northwestern Namibia, which formed during Earths’ penultimate and long-lived icehouse, the late Paleozoic ice age (LPIA, 360–260 Ma; Montañez and Poulsen, 2013). Based on the geomorphology of the paleo-fjord network and its glaciogenic sediment infill, we al., 2021, Fjord network in Namibia: A snapshot into the dynamics of the late Paleozoic glaciation: Geology, v. 49, p. X 1 Geological Society of America \| GEOLOGY \| Volume XX \| Number XX \| www.gsapubs.org Downloaded from http://pubs.geoscienceworld.org/gsa/geology/article-pdf/doi/10.1130/G49067.1/5393367/g49067.pdf by CNRS INSU user A B C D Figure 1. INTRODUCTION (C) Discontinuous and deformed ridge of poorly sorted conglomerate; 18°30′00′′S, 12°49′10′′E, view toward west. Note hammer near top for scale. (D) Striated vertical wall against which Karoo Supergroup strata abut in background; 18°47′56′′S, 13°01′49′′E, view toward northwest. (E) Marginal moraine plastered on valley wall, encompass ing large erratic boulder; 18°47′53′′S, 13°01′45′′E, view toward northwest. (F) Rhythmites and sand-mud couplets; 18°29′59′′S, 12°49′05′′E, view toward south. (G) Lonestone encompassed within sand-sized, rippled sediments; 18°29′59′′S, 12°49′05′′E, view toward south. (H) Intertidal dep ositional environment (breached wave ripples) reworking underlying ice- contact fan; 18°29′59′′S, 12°49′05′′E, view toward south. Figure 2. Facies. (A) Striae and crescentic gouges superimposed on roches moutonnées; 18°11′00′′S, 12°45′40′′E, vertical view. (B) Striated boulder bed covering roches mou tonnées; 18°10′46′′S, 12°45′45′′E, vertical view. (C) Discontinuous and deformed ridge of poorly sorted conglomerate; 18°30′00′′S, 12°49′10′′E, view toward west. Note hammer near top for scale. (D) Striated vertical wall against which Karoo Supergroup strata abut in background; 18°47′56′′S, 13°01′49′′E, view toward northwest. (E) Marginal moraine plastered on valley wall, encompass ing large erratic boulder; 18°47′53′′S, 13°01′45′′E, view toward northwest. (F) Rhythmites and sand-mud couplets; 18°29′59′′S, 12°49′05′′E, view toward south. (G) Lonestone encompassed within sand-sized, rippled sediments; 18°29′59′′S, 12°49′05′′E, view toward south. (H) Intertidal dep ositional environment (breached wave ripples) reworking underlying ice- contact fan; 18°29′59′′S, 12°49′05′′E, view toward south. Figure 2. Facies. (A) Striae and crescentic gouges superimposed on roches moutonnées; 18°11′00′′S, 12°45′40′′E, vertical view. (B) Striated boulder bed covering roches mou tonnées; 18°10′46′′S, 12°45′45′′E, vertical view. (C) Discontinuous and deformed ridge of poorly sorted conglomerate; 18°30′00′′S, 12°49′10′′E, view toward west. Note hammer near top for scale. (D) Striated vertical wall against which Karoo Supergroup strata abut in background; 18°47′56′′S, 13°01′49′′E, view toward northwest. (E) Marginal moraine plastered on valley wall, encompass ing large erratic boulder; 18°47′53′′S, 13°01′45′′E, view toward northwest. (F) Rhythmites and sand-mud couplets; 18°29′59′′S, 12°49′05′′E, view toward south. (G) Lonestone encompassed within sand-sized, rippled sediments; 18°29′59′′S, 12°49′05′′E, view toward south. (H) Intertidal dep ositional environment (breached wave ripples) reworking underlying ice- contact fan; 18°29′59′′S, 12°49′05′′E, view toward south. C D D C E E F F (F) Rhythmites and sand-mud couplets; 18°29′59′′S, 12°49′05′′E, view toward south. (G) Lonestone encompassed within sand-sized, rippled sediments; 18°29′59′′S, 12°49′05′′E, view toward south. (H) Intertidal dep ositional environment (breached wave ripples) reworking underlying ice- contact fan; 18°29′59′′S, 12°49′05′′E, view toward south. INTRODUCTION Note bedrock structures (Goscombe and Gray, 2008). v.e.—vertical exaggeration. Background topography is from SRTM3 (USGS 2006), Shuttle Radar Topographic Mission, 3 Arc Second scene SRTM_n47w053_n54w074, Filled Finished B 2.0, Global Land Cover Facility, University of Maryland, College Park, Maryland, February 2000) available at https://www2.jpl.nasa.gov/srtm/. Map was generated using GlobalMapper® GIS software. eted, and/or striated clasts and discontinuous ridges, patches, and lenses of poorly sorted conglomerates commonly affected by syn- sedimentary ductile deformation (Figs. 1D and 2C), interpreted as ice-contact morainal banks or ridges that experienced glaciotectonic folding (Dowdeswell et al., 2015). Impor tantly, glaciogenic sediments encompassing numerous and large (1–3 m) glacial erratics are plastered to the sides of these U-shaped valleys and along the escarpment, in associa tion with scratched and striated valley walls (Figs. 2D and 2E). Glaciogenic deposits occur consistently at a height of 100 m above the valley bottom, which are interpreted as mar ginal moraines. escarpment, indicating westward and north ward paleo-ice flows, respectively (Fig. 1A). These valleys ubiquitously preserve remnants of partly eroded glaciogenic sediments of the Dwyka Group, forming the base of the Karoo Supergroup (Figs. 1B, 1C, and 2), whose age in the (restored) neighboring Paraná Basin of Brazil and Aranos and Karasburg Basins of Namibia is bracketed between ca. 299 Ma and ca. 296 Ma (Griffis et al., 2021). These glacio genic sediments consist here of boulder beds (Fig. 2B) encompassing numerous exotic, fac Sediments immediately covering the coarse glaciogenic deposits, infilling the valleys (Figs. 1C and 3) and abutting against the stri ated valley walls (Figs. 1B and 2D), are made up of a 5–20-m-thick shallowing-upward sequence of medium-grained sandstones with rhyth mic climbing ripples and sand-mud couplets (Fig. 2F) interstratified with diamictite layers www.gsapubs.org \| Volume XX \| Number XX \| GEOLOGY \| Geological Society of America Figure 2. Facies. (A) Striae and crescentic gouges superimposed on roches moutonnées; 18°11′00′′S, 12°45′40′′E, vertical view. (B) Striated boulder bed covering roches mou tonnées; 18°10′46′′S, 12°45′45′′E, vertical view. (C) Discontinuous and deformed ridge of poorly sorted conglomerate; 18°30′00′′S, 12°49′10′′E, view toward west. Note hammer near top for scale. (D) Striated vertical wall against which Karoo Supergroup strata abut in background; 18°47′56′′S, 13°01′49′′E, view toward northwest. (E) Marginal moraine plastered on valley wall, encompass ing large erratic boulder; 18°47′53′′S, 13°01′45′′E, view toward northwest. (F) Rhythmites and sand-mud couplets; 18°29′59′′S, 12°49′05′′E, view toward south. INTRODUCTION (G) Lonestone encompassed within sand-sized, rippled sediments; 18°29′59′′S, 12°49′05′′E, view toward south. (H) Intertidal dep ositional environment (breached wave ripples) reworking underlying ice- contact fan; 18°29′59′′S, 12°49′05′′E, view toward south. B A E D C F G H A B B A Geological Society of America \| GEOLOGY \| Volume XX \| Number XX \| www gsapubs org 3 characterized by scattered outsized clasts and highlighted by impact structures reflecting ice- rafted debris (Fig. 2G). We interpret this suc cession to represent ice-proximal subaqueous fan deposits (Dowdeswell et al., 2015), imply ing a glaciomarine depositional environment. Facies characteristic of intertidal processes (truncated, leveled, and breached wave ripples; Fig. 2H) were deposited in a postglacial con text, 20 m above the striated floors, well below valley interfluves (Figs. 1D and 3). The fjords were subsequently drowned and accumulated non-glaciogenic sedimentary units of the Perm ian–Cretaceous Karoo Supergroup, ultimately culminating with the Etendeka basalt flows at 130 Ma, whose remnants occur in valley axes topping both the sedimentary succession and valley interfluves (Fig. 1). The presented glaciogenic sediments and geo morphic features preserved within and along the sides of these U-shaped valleys and escarpments indicate that they were occupied by ice masses during the LPIA. Ice retreat was accompanied by postglacial marine incursion that therefore turned the valleys into fjords; the observed valley network therefore corresponds to the original fjord network, here mapped for the first time in Figure 1A. Figure 2. Facies. (A) Striae and crescentic gouges superimposed on roches moutonnées; 18°11′00′′S, 12°45′40′′E, vertical view. (B) Striated boulder bed covering roches mou tonnées; 18°10′46′′S, 12°45′45′′E, vertical view. (C) Discontinuous and deformed ridge of poorly sorted conglomerate; 18°30′00′′S, 12°49′10′′E, view toward west. Note hammer near top for scale. (D) Striated vertical wall against which Karoo Supergroup strata abut in background; 18°47′56′′S, 13°01′49′′E, view toward northwest. (E) Marginal moraine plastered on valley wall, encompass ing large erratic boulder; 18°47′53′′S, 13°01′45′′E, view toward northwest. (F) Rhythmites and sand-mud couplets; 18°29′59′′S, 12°49′05′′E, view toward south. (G) Lonestone encompassed within sand-sized, rippled sediments; 18°29′59′′S, 12°49′05′′E, view toward south. (H) Intertidal dep ositional environment (breached wave ripples) reworking underlying ice- contact fan; 18°29′59′′S, 12°49′05′′E, view toward south. E D C F G H Figure 2. Facies. (A) Striae and crescentic gouges superimposed on roches moutonnées; 18°11′00′′S, 12°45′40′′E, vertical view. (B) Striated boulder bed covering roches mou tonnées; 18°10′46′′S, 12°45′45′′E, vertical view. Downloaded from http://pubs.geoscienceworld.org/gsa/geology/article-pdf/doi/10.1130/G49067.1/5393367/g49067.pdf by CNRS INSU user INTRODUCTION As indicated by such an ice thickness, topo graphically constrained, westward-flowing out let glaciers occupied the valleys, and some (such as the glaciers that occupied the Gomatum and Hoarusib valleys; Fig. 1) bifurcated or fed into a bigger, northward-flowing ice stream (Fig. 4). These outlet glaciers are therefore regarded as having drained a residual ice sheet located to the east of the study area, possibly over the Otavi Range (Fig. 1A) or the Owambo Basin further east (Miller, 1997). Our findings therefore dem onstrate that both a large ice sheet and highland glaciers existed over northwestern Namibia dur ing particular intervals of the icehouse. As is the case for the Quaternary period, however, for which only the last cycle is preserved in the sedimentary and geomorphic record, this acme- waning succession may be representative of only an ultimate ice growth and decay cycle, and per haps the most important one, of a period that encompassed several cycles that were erased by subsequent ice advance. Figure 3. Stratigraphic log of lower fjord infill from the Hoarusib valley (from Karoo Supergroup outliers of Fig. 1D), depo sitional environments, and relative sea-level changes. c—clay; s—silt; vf—very fine-grained sandstone; f— fine-grained sandstones; m—medium-grained sand stone; c—coarse-grained sandstone; vc—very coarse-grained sandstone; g—gravel. INTRODUCTION G H H G characterized by scattered outsized clasts and highlighted by impact structures reflecting ice- rafted debris (Fig. 2G). We interpret this suc cession to represent ice-proximal subaqueous fan deposits (Dowdeswell et al., 2015), imply ing a glaciomarine depositional environment. Facies characteristic of intertidal processes (truncated, leveled, and breached wave ripples; Fig. 2H) were deposited in a postglacial con text, 20 m above the striated floors, well below valley interfluves (Figs. 1D and 3). The fjords were subsequently drowned and accumulated non-glaciogenic sedimentary units of the Perm ian–Cretaceous Karoo Supergroup, ultimately culminating with the Etendeka basalt flows at 130 Ma, whose remnants occur in valley axes topping both the sedimentary succession and valley interfluves (Fig. 1). text, 20 m above the striated floors, well below valley interfluves (Figs. 1D and 3). The fjords were subsequently drowned and accumulated non-glaciogenic sedimentary units of the Perm ian–Cretaceous Karoo Supergroup, ultimately culminating with the Etendeka basalt flows at 130 Ma, whose remnants occur in valley axes topping both the sedimentary succession and valley interfluves (Fig. 1). The presented glaciogenic sediments and geo morphic features preserved within and along the sides of these U-shaped valleys and escarpments indicate that they were occupied by ice masses during the LPIA. Ice retreat was accompanied by postglacial marine incursion that therefore turned the valleys into fjords; the observed valley network therefore corresponds to the original fjord network, here mapped for the first time in Figure 1A. Downloaded from http://pubs.geoscienceworld.org/gsa/geology/article-pdf/doi/10.1130/G49067.1/5393367/g49067.pdf by CNRS INSU user Figure 3. Stratigraphic log of lower fjord infill from the Hoarusib valley (from Karoo Supergroup outliers of Fig. 1D), depo sitional environments, and relative sea-level changes. c—clay; s—silt; vf—very fine-grained sandstone; f— fine-grained sandstones; m—medium-grained sand stone; c—coarse-grained sandstone; vc—very coarse-grained sandstone; g—gravel. an early glaciation acme phase (Fig. 4; Martin and Schalk, 1959; Staiger et al., 2005; Kessler et al., 2008; Steer et al., 2012 ; Livingstone et al., 2017), with ice of at least 1.7 km thick flow ing likely westward toward southeastern Brazil. During the subsequent waning phase, an esti mated ice thickness of 100 m existed within the valley, as extrapolated directly from the eleva tion difference between the valley bottom and the highest observed marginal moraine sedi ments plastered on the valley flanks (Figs. 1 and 3). Implications for Global Climate Change p g The Namibian paleo-fjords have major impli cations for understanding the turnover from the late Paleozoic icehouse to a permanent green house state. Delineating the extent and dynamics of ice masses in northwestern Namibia provides more realistic boundary conditions for the scale of glaciation in this region of Gondwana. In par ticular, the reconstructed paleo-landscape that requires an ice sheet during the acme followed by upland glaciation through the demise of the LPIA could well be explained by the insertion of fjords that promoted dramatic ice-mass loss through drainage and ablation (Bennett, 2003; Briner et al., 2009), in turn triggering abrupt climate change and enhanced ice-sheet sensitiv ity to climate change, ultimately leading to ice shrinkage (Kessler et al., 2008). Importantly, the Namibian paleo-fjord network together with its South American (Tedesco et al., 2016, and refer ences therein) and South African (Visser, 1987) counterparts could have facilitated the deposi tion and long-term sequestration of organic car bon analogous to Quaternary fjords (Smith et al., 2015). If this was the case, then burial of large amounts of glacially derived organic material may have contributed to a 10 m.y. nadir in atmo spheric CO2 in the earliest Permian that defines a paradox, given the loss of major carbon sinks prior to the close of the Carboniferous (Richey et al., 2020). The potential for paleo-fjords of southwestern Gondwana to have played an important role in atmospheric pCO2 and climate regulation is thus a worthwhile area of further study and carbon-cycle modeling. Boulton, G.S., 1990, Sedimentary and sea level changes during glacial cycles and their con trol on glaciomarine facies architecture, in Dowdeswell, J.A., and Scourse, J.D., eds., Glaci marine Environments: Processes and Sediments: Geological Society [London] Special Publica tion 53, p. 15–52, https://doi.org/10.1144/GSL .SP.1990.053.01.02. Briner, J.P., Bini, A.C., and Anderson, R.S., 2009, Rapid early Holocene retreat of a Laurentide out let glacier through an Arctic fjord: Nature Geosci ence, v. 2, p. 496–499, https://doi.org/10.1038/ ngeo556. g Dietrich, P., Ghienne, J.-F., Lajeunesse, P., Norman deau, A., Deschamps, R., and Razin, P., 2018, Deglacial sequences and glacio-isostatic adjust ment: Quaternary compared with Ordovician gla ciations, in Le Heron, D.P., et al., eds., Glaciated Margins: The Sedimentary and Geophysical Ar chive: Geological Society [London] Special Pub lication 475, p. 149–179, https://doi.org/10.1144/ SP475.9. INTERPRETATION: RECONSTRUCTED LPIA DYNAMICS AND ICE THICKNESSES IN NORTHWESTERN NAMIBIA of the dynamics of the LPIA ice masses (Fig. 4). The deep (as deep as 1.7 km) fjord incisions and the presence of numerous and large glacial erratics (e.g., Fig. 2E) transported by ice across major drainage divides imply the presence of an ice sheet largely overflowing the valleys during The glacial (fjord) geomorphology and its associated sedimentary infill record a snapshot Figure 4. Three-dimen sional model of late Paleozoic ice age dynam ics on Kaokoland, northwestern Namibia. RSL—relative sea level; GIA—glacio-isostatic adjustment. Throughout this acme-waning cycle, during ongoing deglaciation and ice-margin retreat, the postglacial sea invaded the valleys and turned them into fjords. Ice-contact fans and deformed morainal banks indicate that sedimentation took place at the front of retreating glaciers dur ing periods of episodic ice-margin stillstands or minor readvance (Fig. 4). Throughout this deglacial cycle, ice-contact and glaciomarine sedimentation was strictly confined within the topographic depression (the fjords) in a setting likely devoid of tectonic subsidence. Figure 4. Three-dimen sional model of late Paleozoic ice age dynam ics on Kaokoland, northwestern Namibia. RSL—relative sea level; GIA—glacio-isostatic adjustment. Considering such a succession of deglacial events, the sedimentary succession observed infilling the fjords is therefore thought to archive the glacio-isostatic adjustment (Boulton, 1990). Given that glacio-isostatic adjustment oper ates over a short (104 yr) time scale, i.e., well shorter than the temporal resolution available for such a deep-time record, the unravelling of this process is made by analogy between the fjordal deglacial sedimentary succession show cased here and a Quaternary deglacial succes sion (Dietrich et al., 2018). Thus, we posit that the basal, 20–40-m-thick shallowing-upward parasequence (glaciomarine capped by inter tidal facies) is interpreted as a response to fall ing relative sea level resulting from the glacio- isostatic adjustment. The subsequent drowning (offshore deposits upon intertidal deposits) www.gsapubs.org \| Volume XX \| Number XX \| GEOLOGY \| Geological Society of America Downloaded from http://pubs.geoscienceworld.org/gsa/geology/article-pdf/doi/10.1130/G49067.1/5393367/g49067.pdf by CNRS INSU user therefore likely corresponds to an eustatic rise during which non-glaciogenic sedimentation occurred (Fig. 3). The glacial dynamic that we envisage is largely comparable to the evolution of post–Last Glacial Maximum ice masses over Canada, Norway, and Greenland, whose post- acme recession saw the confinement of outlet glaciers into fjords, which after glacial demise were invaded by postglacial seas (e.g., Syvitski et al., 1987; Dietrich et al., 2018). ing post-orogenesis exhumation and peneplana tion (Krob et al., 2020). DISCUSSION AND IMPLICATIONS A Preserved LPIA Glacial Landscape: A Fjord Network Superimposed on Preexisting Plateaus and Escarpments DISCUSSION AND IMPLICATIONS A Preserved LPIA Glacial Landscape: A Fjord Network Superimposed on Preexisting Plateaus and Escarpments DISCUSSION AND IMPLICATIONS A Preserved LPIA Glacial Landscape: A Fjord Network Superimposed on Preexisting Plateaus and Escarpments g j g Baby, G., Guillocheau, F., Braun, J., Robin, C., and Dall’Asta, M., 2020, Solid sedimentation rates history of the Southern African continental mar gins: Implications for the uplift history of the South African Plateau: Terra Nova, v. 32, p. 53– 65, https://doi.org/10.1111/ter.12435. The preservation of glaciogenic deposits and erosion features indicates that the U-shaped val ley network forms an intact relict geomorphic landscape inherited from the LPIA and com plements temporally contemporaneous, though smaller, glacial landscapes and paleo-fjords preserved and exhumed (or still sealed) across southwestern Gondwana (e.g., Visser, 1987; Assine et al., 2018; Le Heron et al., 2019; Fall gatter and Paim, 2019). The presence of incised valleys furthermore implies that the plateaus and intervening escarpments were in place when the glacial topography was carved (Fig. 1D), i.e., before the Atlantic (Cretaceous) rifting. Ther mochronological studies (Krob et al., 2020) indicate that little denudation occurred after the LPIA prior to the deposition of the Cretaceous Etendeka basalts, whose remnants are preserved on valley interfluves (Fig. 1C). Therefore, mod ern valley depths and the whole of the Kaoko land landscape, although uplifted (Baby et al., 2020), are virtually unchanged since LPIA times (Martin, 1953). The observed network of valleys, troughs, and escarpments that currently charac terizes the Kaokoland therefore corresponds to an extensive, ∼50,000 km2 preserved glacial landscape (Fig. 1A). Our showcased example is unique because it represents the sole example of a pristine fjord network and glacial landscape yet described for a pre-Cenozoic glacial epoch. Furthermore, our work highlights the compat ibility of both large ice sheets and highland gla ciation across the LPIA in a single location (cf. Isbell et al., 2012). Bennett, M.R., 2003, Ice streams as the arteries of an ice sheet: Their mechanics, stability and signifi cance: Earth-Science Reviews, v. 61, p. 309–339, https://doi.org/10.1016/S0012-8252(02)00130-7. Bianchi, T.S., et al., 2020, Fjords as aquatic critical zones (ACZs): Earth-Science Re views, v. 203, 103145, https://doi.org/10.1016/ j.earscirev.2020.103145. INTERPRETATION: RECONSTRUCTED LPIA DYNAMICS AND ICE THICKNESSES IN NORTHWESTERN NAMIBIA A differential response of the Congo craton and the Kaoko orogen to vertical tectonic forces that promoted the initia tion of Karoo-aged basins over southwestern Gondwana (Pysklywec and Quintas, 1999) is tentatively invoked to explain this topographic rejuvenation. Alternatively, or complementarily, glacial erosion itself through isostatic uplift may have generated the mountainous relief required to create fjords (Medvedev et al., 2008). After the LPIA and until 130 Ma, this paleo-fjord network was progressively buried by the Karoo sediments and Etendeka volcanics, and subse quently exhumed until the present (Krob et al., 2020; Margirier et al., 2019; Baby et al., 2020). Thus, preservation of a pristine paleo-fjord net work, in spite of 130 m.y. of uplift and exhuma tion, is remarkable. Determining the reasons for this exceptional preservation will be a driver of future research. acknowledge funding from the U.S. National Science Foundation (grant EAR-1729882). Julia Tedesco and Michael Blum are thanked for their thorough and con structive reviews that greatly led to the improvement of the paper. Implications for Global Climate Change Dowdeswell, J.A., Hogan, K.A., Arnold, N.S., Mug ford, R.I., Wells, M., Hirst, J.P.P., and Decalf, C., 2015, Sediment-rich meltwater plumes and ice-proximal fans at the margins of modern and ancient tidewater glaciers: Observations and modeling: Sedimentology, v. 62, p. 1665–1692, https://doi.org/10.1111/sed.12198. Eilertsen, R.S., Corner, G.D., Aasheim, O., and Hansen, L., 2011, Facies characteristics and architecture related to palaeodepth of Ho locene fjord-delta sediments: Sedimentolo gy, v. 58, p. 1784–1809, https://doi.org/10.1111/ j.1365-3091.2011.01239.x. In the absence of recent (middle to late Paleozoic) major tectonic events antecedent to the LPIA in this region, the preexistence of plateaus and escarpments exploited by glacial erosion that carved fjords is interpreted as fol lows. The NNW-SSE–oriented escarpments correspond to basement sutures delineating the Congo craton to the west and segments of the Kaoko (Pan-African) orogen to the east (Gos combe and Gray, 2008). Because these plateaus and escarpments had already formed prior to the LPIA, we suggest that this existing topography resulted from substantial rejuvenation of the Kaoko orogenic structures after 200 Ma dur Fallgatter, C., and Paim, P.S.G., 2019, On the origin of the Itararé Group basal nonconformity and its implications for the Late Paleozoic glaciation in the Paraná Basin, Brazil: Palaeogeography, Pal aeoclimatology, Palaeoecology, v. 531, 108225, https://doi.org/10.1016/j.palaeo.2017.02.039. p g j p Goscombe, B.D., and Gray, D.R., 2008, Structure and strain variation at mid-crustal levels in a transpressional orogen: A review of Kaoko Belt structure and the character of West Gondwana amalgamation and dispersal: Gondwana Re search, v. 13, p. 45–85, https://doi.org/10.1016/ j.gr.2007.07.002. Downloaded from http://pubs.geoscienceworld.org/gsa/geology/article-pdf/doi/10.1130/G49067.1/5393367/g49067.pdf by CNRS INSU user REFERENCES CITED Assine, M.L., de Santa Ana, H., Veroslavsky, G., and Vesely, F.F., 2018, Exhumed subglacial landscape in Uruguay: Erosional landforms, depositional environments, and paleo-ice flow in the context of the late Paleozoic Gondwanan glaciation: Sedimentary Geology, v. 369, p. 1–12, https:// doi.org/10.1016/j.sedgeo.2018.03.011. ACKNOWLEDGMENTS P. Dietrich and D. Le Heron acknowledge funding from the South Africa–Austria joint project of the National Research Foundation (NRF) of South Africa and the Österreichischer Austauschdienst (OEAD project ZA 08/2019). I. Montañez and N. Griffis Geological Society of America \| GEOLOGY \| Volume XX \| Number XX \| www.gsapubs.org Downloaded from http://pubs.geoscienceworld.org/gsa/geology/article-pdf/doi/10.1130/G49067.1/5393367/g49067.pdf by CNRS INSU user Planetary Science Letters, v. 527, 115779, https:// doi.org/10.1016/j.epsl.2019.115779. Planetary Science Letters, v. 527, 115779, https:// doi.org/10.1016/j.epsl.2019.115779. Griffis, N., et al., 2021, High-latitude ice and climate control on sediment supply across SW Gondwana during the late Carboniferous and early Permian: Geological Society of America Bulletin, https:// doi.org/10.1130/B35852.1 (in press). Pysklywec, R.N., and Quintas, M.C.L., 1999, A man tle flow mechanism for the late Paleozoic subsid ence of the Paraná Basin: Journal of Geophysical Research, v. 105, p. 16,359–16,370, https://doi .org/10.1029/2000JB900080. Martin, H., 1953, Notes on the Dwyka Succession and on some Pre-Dwyka Valleys in South West Africa: Transactions of the Geological Society of South Africa, v. 56, p. 37–41. Isbell, J.L., Henry, L.C., Gulbranson, E.L., Limarino, C.O., Fraiser, M.L., Koch, Z.J., Ciccioli, P.L., and Dineen, A.A., 2012, Glacial paradoxes during the late Paleozoic ice age: Evaluating the equi librium line altitude as a control on glaciation: Gondwana Research, v. 22, p. 1–19, https://doi .org/10.1016/ j.gr.2011.11.005. Richey, J.D., Montañez, I.P., Goddéris, Y., Looy, C.V., Griffis, N.P., and DiMichele, W.A., 2020, Influ ence of temporally varying weatherability on CO2- climate coupling and ecosystem change in the late Paleozoic: Climate of the Past, v. 16, p. 1759– 1775, https://doi.org/10.5194/cp-16-1759-2020. p Martin, H., 1981, The Late Paleozoic Dwyka Group of the South Kalahari Basin in Namibia and Bo tswana and the subglacial valleys of the Kaok oveld in Namibia, in Hambrey, M.J. and Har land, W.B. eds., Earth’s Pre-Pleistocene Glacial Record: Cambridge, UK, Cambridge University Press, p. 61–66. Smith, R.W., Bianchi, T.S., Allison, M., Savage, C., and Galy, V., 2015, High rates of organic carbon burial in fjord sediments globally: Nature Geosci ence, v. 8, p. 450–453, https://doi.org/10.1038/ ngeo2421. Le Heron, D.P., Dietrich, P., Busfield, M.E., Kettler, C., Bermanschläger, S., and Grasemann, B., 2019, Scratching the surface: Footprint of a late Carboniferous ice sheet: Geology, v. 47, p. 1034– 1038, https://doi.org/10.1130/G46590.1. Martin, H., and Schalk, K., 1959, Gletscherschliffe an der Wand eines U-Tales im nördlichen Ka okofeld, Südwestafrika: Geologische Rund schau, v. 46, p. 571–575, https://doi.org/10.1007/ BF01803042. ACKNOWLEDGMENTS g Staiger, J.K.W., Gosse, J.C., Johnson, J.V., Fastook, J., Gray, J.T., Stockli, D.F., Stockli, L., and Fin kel, R., 2005, Quaternary relief generation by polythermal glacier ice: Earth Surface Processes and Landforms, v. 30, p. 1145–1159, https://doi .org/10.1002/esp.1267. Kessler, M.A., Anderson, R.S., and Briner, J.P., 2008, Fjord insertion into continental margins driven by topographic steering of ice: Nature Geosci ence, v. 1, p. 365–369, https://doi.org/10.1038/ ngeo201. Medvedev, S., Hartz, E.H., and Podladchikov, Y.Y., 2008, Vertical motions of the fjord regions of cen tral East Greenland: Impact of glacial erosion, deposition, and isostasy: Geology, v. 36, p. 539– 542, https://doi.org/10.1130/G24638A.1. Steer, P., Huismans, R.S., Valla, P.G., Gac, S., and Her man, F., 2012, Bimodal Plio-Quaternary glacial erosion of fjords and low-relief surfaces in Scan dinavia: Nature Geoscience, v. 5, p. 635–639, https://doi.org/10.1038/ngeo1549. Kneller, B., Milana, J.P., Buckee, C., and al Ja’aidi, O., 2004, A depositional record of deglaciation in a paleofjord (Late Carboniferous [Pennsylvanian] of San Juan Province, Argentina): The role of catastrophic sedimentation: Geological Society of America Bulletin, v. 116, p. 348–367, https:// doi.org/10.1130/B25242.1. Miller, R.M., 1997, The Owambo Basin of northern Namibia, in Selley, R.C., ed., Sedimentary Basins of the World, Volume 3: African Basins: Amster dam, Elsevier Science, p. 237–268, https://doi .org/10.1016/S1874-5997(97)80014-7. Syvitski, J.P.M., Burrell, D.C., and Skei, J.M., 1987, Fjords: Processes and Products: New York, Springer-Verlag, 379 p., https://doi .org/10.1007/978-1-4612-4632-9. g Montañez, I.P., and Poulsen, C.J., 2013, The Late Paleozoic Ice Age: An evolving paradigm: An nual Review of Earth and Planetary Scienc es, v. 41, p. 629–656, https://doi.org/10.1146/ annurev.earth.031208.100118. Krob, F.C., Eldracher, D.P., Glasmacher, U.A., Husch, S., Salomon, E., Hackspacher, P.C., and Titus, N.P., 2020, Late Neoproterozoic-to-recent long- term t–T-evolution of the Kaoko and Damara belts in NW Namibia: International Journal of Earth Sciences, v. 109, p. 537–567, https://doi .org/10.1007/s00531-020-01819-7. Tedesco, J., Cagliari, J., Coitinho, J.d.R., Lopes, R.d.C., and Lavina, E.L.C., 2016, Late Paleo zoic paleofjord in the southernmost Parana Basin (Brazil): Geomorphology and sedimentary fill: Geomorphology, v. 269, p. 203–214, https://doi .org/10.1016/j.geomorph.2016.06.035.l Moon, T., Sutherland, D.A., Carroll, D., Felikson, D., Kehrl, L., and Straneo, F., 2018, Subsurface ice berg melt key to Greenland fjord freshwater bud get: Nature Geoscience, v. 11, p. 49–54, https:// doi.org/10.1038/s41561-017-0018-z. g Livingstone, S.J., Chu, W., Ely, J.C., and Kingslake, J., 2017, Paleofluvial and subglacial channel net works beneath Humboldt Glacier, Greenland: Ge ology, v. 45, p. 551–554, https://doi.org/10.1130/ G38860.1. ACKNOWLEDGMENTS Visser, J.N.J., 1987, The influence of topography on the Permo-Carboniferous glaciation in the Karoo Basin and adjoining areas, southern Africa, in McKenzie, G.D., ed., Gondwana Six: Stratigraphy, Sedimen tology, and Paleontology: American Geophysical Union Geophysical Monograph 41, p. 123–129, https://doi.org/10.1029/GM041p0123. Normandeau, A., Dietrich, P., Hughes Clarke, J., Van Wychen, W., Lajeunesse, P., Burgess, D., and Ghienne, J.-F., 2019, Retreat pattern of gla ciers controls the occurrence of turbidity cur rents on high-latitude fjord deltas (Eastern Baf fin Island): Journal of Geophysical Research: Earth Surface, v. 124, p. 1559–1571, https://doi .org/10.1029/2018JF004970. Margirier, A., Braun, J., Gautheron, C., Carcaillet, J., Schwartz, S., Pinna Jamme, R., and Stanley, J., 2019, Climate control on Early Cenozoic denu dation of the Namibian margin as deduced from new thermochronological constraints: Earth and Printed in USA Printed in USA www.gsapubs.org \| Volume XX \| Number XX \| GEOLOGY \| Geological Society of America Downloaded from http://pubs.geoscienceworld.org/gsa/geology/article-pdf/doi/10.1130/G49067.1/5393367/g49067.pdf by CNRS INSU user

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