s2fieldsofstudy: Biology Psychology

text:
Coordination between Eye Movement and Whisking in Head-Fixed Mice Navigating a Plus Maze

Abstract Navigation through complex environments requires motor planning, motor preparation, and the coordination between multiple sensory–motor modalities. For example, the stepping motion when we walk is coordinated with motion of the torso, arms, head, and eyes. In rodents, movement of the animal through the environment is coordinated with whisking. Even head-fixed mice navigating a plus maze position their whiskers asymmetrically with the bilateral asymmetry signifying the upcoming turn direction. Here we report that, in addition to moving their whiskers, on every trial mice also move their eyes conjugately in the direction of the upcoming turn. Not only do mice move their eyes, but they coordinate saccadic eye movement with the asymmetric positioning of the whiskers. Our analysis shows that asymmetric positioning of whiskers predicted the turn direction that mice will make at an earlier stage than eye movement. Consistent with these results, our observations also revealed that whisker asymmetry increases before saccadic eye movement. Importantly, this work shows that when rodents plan for active behavior, their motor plans can involve both eye and whisker movement. We conclude that, when mice are engaged in and moving through complex real-world environments, their behavioral state can be read out in the movement of both their whiskers and eyes.

Introduction
One of the fundamental activities of the brain is to monitor and control the movement of the body. A simple everyday act like walking is associated with a sequence of movements that involve the body, limbs, head, and eyes (Bizzi et al., 1971;Grasso et al., 1998;Dietz et al., 2001;Dietz, 2002;Land, 2006;Foulsham, 2015). Despite the obvious importance of understanding the coordination between various sensory-motor elements engaged in planning and coordinating behaviors, the details of this coordination are still not completely clear.
In rodents, exploration of the environment is often linked to movement of the head and whiskers (Hartmann et al., 2003;Towal and Hartmann, 2006;Arkley et al., 2014;Sofroniew et al., 2014), with facial movements linked to activity observed in widespread areas of cortex (Musall et al., 2019;Steinmetz et al., 2019;Stringer et al., 2019). Eye movements, which are thought to have "just in time" and "look-ahead" function in many species (Land and Hayhoe, 2001;Land, 2006;Mennie et al., 2007;Land, 2009;Foulsham, 2015;Srivastava et al., 2018), have often been overlooked in rodents. Part of the reason for this neglect is related to the difficulty in measuring the motion of the small rodent eyes, especially in freely moving animals (Payne and Raymond, 2017;Meyer et al., 2018Meyer et al., , 2020. Additionally, rats and mice are nocturnal animals with a highly developed somatosensory system (Woolsey and van der Loos, 1970), where the relationship between whisking and navigation is observable more easily (Hartmann et al., 2003;Towal and Hartmann, 2006;Arkley et al., 2014;Sofroniew et al., 2014;Dominiak et al., 2019). But in the last decade there has been an evolution in our thinking as it has become increasingly obvious that in addition to using their tactile system, rodents can also use their visual system to guide their response to predators, and can and do move their eyes to guide navigation (Wallace et al., 2013;Yilmaz and Meister, 2013;Hoy et al., 2016;Meyer et al., 2020). Together, it is likely that even in rodents, eye movement is coordinated with actions in other sensory motor dimensions (i.e., for motor planning and for coordinating movement during navigation; Land, 2009).
Earlier work has shown that when mice plan their movement in a real-world floating maze, they begin to position their whiskers asymmetrically; the asymmetry reflects the direction that mice turn in as they navigate the maze . A key limitation of this earlier work, and of work in the rodent motor systems in general, has been our fixation on the highly specialized vibrissae system and whisking. The earlier work has not been clear about whether and how much mice coordinate the movement of their body with movement of both their eyes and whiskers. Furthermore, whether changes in the position of whiskers or eyes indeed reflect motor planning has not been examined, because it has not been feasible. Here we extended the work of Dominiak et al. (2019) by training mice to overcome their natural handedness preference and monitored both whisker and eye movement bilaterally. Our work shows that whisking is coordinated with the movement of the eyes, that mice move their eyes in a "look-ahead" fashion, and that both eye movement and whisking are related to turn direction.

Behavioral experiments
We performed all procedures in accordance with protocols for the care and use of laboratory animal approved by Charité-Universitätsmedizin Berlin and Berlin Landesamt für Gesundheit und Soziales (LaGeSo).

Surgery
Adult C57BL/6 male mice (n = 4), weighing 25-32 g were anesthetized with ketamine/xylazine (90 mg/kg/ 10 mg/kg). Lightweight aluminum head posts were affixed to the skull using Rely X and Jet Acrylic (Ortho-Jet) black cement Ebner et al., 2019). In the 2 d after surgery, analgesia was provided by buprenorphine and carprofen injections.

Airtrack
The Airtrack consists of the following three parts: (1) a square Plexiglas airtable with tiny, evenly spaced holes for holding the platform, a circular maze aloft; (2) a lightweight, circular maze that floats on the bed of air created; and (3) a pixy camera that tracks the position of the maze (Nashaat et al., 2016;Dominiak et al., 2019). Each hole of the airtable has a plastic ball bearing that moved to the mouth of the hole preventing airflow when the platform was not covering that particular hole. This reduced the hissing sound associated with the outflow of pressurized air. For the floating maze, Dominiak et al. (2019) used a platform that weighed 160 g and was 30 cm in diameter. In this study, we used a Styrofoam base with a milled out Plexiglas ring on the borders that reduced friction with the walls of the airtable. Our plus maze had a diameter of 22 cm and weighed 30 g. It had four 8-cm-long, 2.5-cmhigh identical opaque (black or red Plexiglas) lanes, each with the same texture engraved on the walls. One end of all lanes, toward the center of the maze, opened into a circular area, in which mice could turn and exit or enter lanes. The other end of the lane opened toward a lick port, which was attached to a linear actuator. A pair of LEDs, 7 cm apart, positioned directly in front of each eye of the mice, were set up on each side of the lick port and were used to indicate whether the trial was a right or left turn trial.
When mice were head fixed, their head post jutted over the walls of the lanes, but the head of the mouse was below the top of the wall. External visual cues were minimized by covering the setup with a black cloth.
Note, however, that the black/UV light (365-400 nm) used for tracking and fluorescing whiskers illuminated the setup.
The pixy camera positioned under the platform used a color code (Nashaat et al., 2016) to track the position, the direction of movement, and the speed of movement of the maze as the head-fixed mouse guided it along. When mice entered the correct lane, the pixy output was used to trigger a motor that lowered the lick port into position.

Plus maze task
Mice were trained to use LED cues to turn the maze in the cued direction (Fig. 1). A trial began when mice finished with the reward from the previous trial and were still at the end of a lane. One of two LEDs turned on, indicating the expected turn direction for the ensuing trial (Fig. 1A,B, Movie 1; generated using real time tracking of pupils). The light was left on until mice had propelled themselves backward, out of the lane, and had begun to turn in the Figure 1. Schematic of trials, and behavioral state estimates during whisker and eye movements. A, B, Schematic of the task. A trial starts with the animal at the end of the last rewarded lane. One of the two cue LEDs indicate the direction the animal should take next. The right LED indicates that for the trial mice had to move in a clockwise "right turn" direction and enter the adjacent lane where they wait for the reward tube to descend. The left LED indicated a left turn trial. C, Single right-turn and left-turn trials, showing whisker and eye movement. Mice move backward at their own pace, reach the center of the maze, and turn left (top) or right (bottom) until the reward LED turns off signaling that the mouse was at the mouth of the correct, rewarded lane. Mice learn to expect a reward at the end of the lane. Once the lick spout was in their reach, mice lick the reward. During this behavior, whiskers on two sides of the face (left-side whiskers, blue; right side whiskers, red) move in opposite directions for left (top) and right (bottom) turns. The side-to-side asymmetry becomes evident just as, or just before, mice begin to move backward in the lane. By comparison, the eyes (right eye, blue trace; left eye, red trace) move in a conjugate fashion on both sides of the face. The behavioral state was manually annotated: purple, end of lane; dark blue, backward movement; brown, right turns; orange, left turns; light blue, forward; red, expect reward; yellow, lick. Scale bars represent the amplitude of movement relative to the full amplitude of whisker motion (for whiskers) or relative to the width of the eye (for eyes) during the session. expected direction. When they were at the entrance to the correct adjacent lane, the LED was switched off. Note that there are no time constraints on the mice: they could move as fast or as slowly as they wanted, and they could make turns as many times as they wanted. Once a trial ended, mice could start the trial when they wanted and could go as fast or as slow as they wanted to.

Training
One week after surgery, mice were habituated to being handled, and to the Airtrack plus maze platform. In the first days of habituation, mice acclimated to having their head post handled by the experimenter and to short (up to several minutes) head fixation. In the course of the first week of habituation, the duration of head fixation was gradually increased from 5 to 40 min. In addition to head fixation, mice were habituated to having their whiskers painted. During whisker painting, mice were rewarded with sweetened condensed milk.
After a week of habituation, mice were water deprived and were trained to move the platform around themselves. At the beginning of this training phase, the experimenter manually nudged or guided the animal. Over the course of 14 consecutive days of training, mice learned to move the maze by themselves, and gradually increased the number of trials they performed. Note that there were no temporal constraints on mice; the trials were self-initiated, and each behavioral epoch and each trial could be as long or as short as mice made them.

Data acquisition
Video data from painted whiskers and the movement of the eyes were collected as mice performed the task (Fig.  1C). We manually annotated 113 trials taken from 21 sessions in four animals. These 113 trials were used to examine the effect of the behavioral states of the animal on whisker positions. To examine the relationship between behavioral states and eye positions, we visually validated the alignment between behavioral (whisker) videos and eye videos. After excluding the trials where behavioral and eye videos did not align properly with each other, we used 91 trials from 17 sessions recorded in four animals for the analysis of eye positions (Table 1).
To track the behavioral states and whisker positions, behavioral data were recorded at a resolution of 1200 pixels in width Â 700 pixels in height at 200 Hz with a camera (acA1920-155uc USB 3.0, Basler) and an f = 25 mm/F1.4 objective being set above the animal. The C2 whiskers on both sides were painted green (UV glow; https://www. uvglow.co.uk/), and illuminated by UV torches. Our goal was to capture whisking movement, which in mice can go up to ;20 Hz (Jin et al., 2004;Mitchinson et al., 2011;Sofroniew et al., 2014;Dominiak et al., 2019). A frame rate of 200 Hz is well above the Nyquist frequency and is sufficient for this. Note that our work follows on the work by Dominiak et al. (2019), which showed that tracking a single whisker on each side of the face was sufficient to determine both the setpoint and amplitude of whisker movement and that the tactile events, the very high-frequency slip stick events, do not explain the behaviors that are at the core of this work. Mice perform the behavior and move their whiskers in a similar manner after whiskers have been trimmed off.
Videos were acquired in the proprietary format from Matrox Imaging (https://www.matrox.com/) and later converted into the H.264 format. The acquisition and conversion were accomplished using ZR-view, a custom software (Robert Zollner, Eichenau, Germany).
For recording the pupils of the animal, two cameras (acA1300-200um USB 3.0, Basler), each with an f = 50 The numbers of trials with manual annotation of states (anno), with whisker tracking (whis) and with valid eye tracking (eyes) are shown. Because of validation after alignment of eye videos with behavior videos, the number of trials with eye tracking is smaller than the other types of trials.
Movie 1. Real-time eye tracking and behavior on two trials. A machine-learning algorithm was used to track eye position in real time and to illustrate how eye position changed with the position of the mouse in the maze. [View online] mm/F2.8 objective, were used. Two independent infrared light sources (model LIU850A, Thorlabs; https://www. thorlabs.com/) were directed toward the eyes. Video files of both pupils were acquired at a resolution of 192 pixels in width Â 200 pixels in height at 100 Hz using Pylon-PD (a custom software made by Eridian Systems, Berlin, Germany). An Atmel ATmega328P microcontroller was reprogrammed to generate synchronized 100 and 200 Hz trigger pulses for simultaneous frame acquisition from the three cameras (i.e., two pupil cameras and the behavioral camera). The Mega board (Arduino) that monitored Airtrack movement was also used to control the trial-based data acquisition. Trials were separated by two consecutive transistor-transistor logic (TTL) level pulses; the first TTL marked the end of a trial and the second one marked the start of a new trial. These TTL signals were used to trigger the start and the end of video acquisition.
Behavioral states in each trial were annotated manually using the behavioral video files, by marking the frames when state transitions occurred ( Fig. 1C), as follows: quietly sitting at end-of-lane, moving backward, turning, moving forward, waiting for reward, or licking. Entry into or exit from a lane were annotated by using the position of the nose in relation to the edges of the lanes. The frame on which the animal started moving continuously in one direction was defined as the onset of forward or backward movement.

Tracking whisker movement
Whisker positions were estimated using the custom Python script (videobatch; https://doi.org/10.5281/ zenodo.3407666). The top-view videos first underwent a maximum intensity projection using the videobatch script. Regions of interest (ROIs) for tracking were selected manually for the C2 whiskers on both sides of the face using the Fiji freehand selection tool Sehara et al., 2019). Using the Python script, pixels that belonged to a particular hue value were collected and the luma (brightness)-weighted average position was computed. For frames where the algorithm failed for any reason, values were dropped and were filled in later by linear interpolation.
The whisker positions tracked during each behavioral session were then converted to whisking angles. Using the fitting2d Python library, a circle was fit to the set of two-dimensional positions for each whisker, and the position at each time point was converted into the polar coordinates around the fitted circle.
The analysis of set point and amplitude of whisker motion in the Airtrack has been described in previous work by Dominiak et al. (2019). Here, taking into account this previously published work-and taking into account that (1) the motion of a whisker is determined by the motion of the whisker pad and the intrinsic muscles associated with individual whiskers; and (2) the set point and amplitude of whisker change in the course of single behavioral session in complex behavior state-dependent manner )-we used the angle corresponding to the session median position, defined as the provisional zero angle for each session. Deviations in whisker positions were represented as the percentage of the full range of deviation during each trial (i.e., between the trial minimum and the trial maximum angles).
Whisker asymmetry values were defined as the difference of normalized positions of the left whisker (w Left ) and the right whisker (w Right ). The value w Rightw Left was computed for a left-turning trial, whereas the value w Leftw Right was used for right-turning trials.

Tracking eye movements
DeepLabCut (version 2.1; Mathis et al., 2018;Nath et al., 2019) was used to track pupil positions from video frames. A deep-neural network model was trained to detect eight points on the edge of the pupil on each video frame. A single model was used for tracking pupils of both sides of the face of all animals in all sessions. In total, 284 frames from 28 sessions from different animals were manually annotated to train the network. For each video frame, the eight edge points of the eye detected by the deep-neural network were then fitted by an ellipse, using the fitting2d Python library. The position and the diameter of the fitted ellipse were considered to be those of the pupil.
Because the size of the eyes in the field of view of the video varied across behavioral sessions, the position of the pupil was normalized with respect to the size of the eye. For each eye during each behavioral session, the average eye shape was first delineated manually using the ROI Manager of ImageJ, based on the average-projection image of eye videos during the session. A parabola curve was then fitted to the trace corresponding to either the top or the bottom eyelid, using the fitting2d Python library. The two corners of the eye were then computed as the crossing points between the two fitted parabola curves. These defined the line segment representing the width of the eye. The position of the pupils was first projected onto the eye-width segment, and then was represented in terms of the fraction relative to the full width of the eye (hereafter called the normalized pupil position).
During analysis, pupil deviation values were defined as the average deviation of the normalized position of left and the right pupils in the direction of turn.

Detection of saccades
The first derivative (dX/dt) of normalized eye positions was computed to obtain the pupil speed. Sudden changes in eye position appeared as distinct spikes in the corresponding eye speed trace. Saccades were said to have occurred when the absolute values of eye speed for both eyes crossed a threshold value that was set empirically at 0.1% (relative to the full eye width) per frame. If a train of these high-speed events occurred within an interval of ,250 ms, it was considered to be a processing artifact and was discarded from the analysis.

Normalization of behavioral state durations
The duration for each behavioral state was normalized using interpolation. For each behavioral epoch, we first set up a normalized time base such that the time points 0 and 1 marked the start and the end of the epoch. The data points were resampled from the original time base (i.e., frames) to the normalized time base using interpolation.

Analysis using receiver operating characteristic curves
We used receiver operating characteristic (ROC) curves to examine whether eye movement, whisker asymmetry, or both could predict the turn direction (Green and Swets, 1966). We divided the backward-movement behavioral epoch into five distinct time bins. For each time bin, we set up a set of classifier models. As its input, each classifier model received either a whisker asymmetry value (i.e., the right whisker protraction subtracted by the left whisker protraction) or an eye position value (positive values indicating leftward deviations and negative values indicating rightward deviation). Each model had a certain prespecified threshold value. When input value was above the threshold value, the model determined that a left turn would occur. If the input value was below the threshold value, the model predicted a right turn. We supplied the set of models of different threshold values with behavioral data (either whisker asymmetry or eye positions). For each time bin, the probability of correct prediction for each model of the upcoming turn direction to the left, the "true positives," and the probability of predicting the upcoming turn direction incorrectly, the "false positives" (the model predicted to turn left, whereas the animal turned right in reality), were computed. The ROC curve for each time bin was then plotted based on these probabilities, using the set of models having different threshold values. The area-under-thecurve values generated by this analysis were compared with the shuffled data, and significance was assessed for each time bin, for whisker asymmetry and eye movement separately. Statistical comparison between the raw and shuffled data and between whisker-based and eyebased data in each time bin was performed using the Mann-Whitney U test. The Mann-Whitney U test with Bonferroni's correction was used for pairwise comparison between data belonging to different time bins.

Analysis using perceptrons
We used single-layer perceptrons to examine the contribution of eye movement and whisker asymmetry in predicting the turn direction (Minsky and Papert, 1969). For this analysis, we divided the backward-movement behavioral epoch into five distinct time bins and set up classifier models. Here, for each time bin, we laid out individual time points in a two-dimensional space of whisker asymmetry and eye positions, and a line was fitted to separate left-turning and right-turning points as much as possible. Thus, the classifier predicted that the left turn or right turn would occur based on which side of the line individual points were located. The fidelity of separation was defined as the fidelity of prediction, as follows: fidelity marked 1.0 if the line correctly separated the points into left-turning and right-turning ones, and 0.5 if each side of the line contained equal numbers of left-turning and right-turning points.
For each time bin, we first prepared two models; the full model was prepared based on the true data points, whereas in the null model, both whisker asymmetry and eye positions were shuffled in relation to the turning directions. To compute the unique contribution of either whisker asymmetry or eye positions to the separation fidelity of the full model, we prepared a partial model based on the dataset where only a single selected variable was shuffled, and computed the resulting decrease in the separation fidelity compared with the full model. Statistical comparisons were performed using the Kruskal-Wallis test with Bonferroni's correction by taking into account all of the four models of all the time bins.

Data availability
All the raw videos and behavioral state annotation data used in this study are deposited at GIN (https://dx.doi. org/10.12751/g-node.j9wxqe).

Whisking and eye position
We trained head-fixed mice to navigate on Airtrack, a real-world floating plus maze (Nashaat et al., 2016;Dominiak et al., 2019). In this task, mice started at the end of a lane, then backed out of the lane, and turned in the expected direction as indicated by an LED. If mice turned in the correct direction, entered a lane, and walked to the end of the lane, a lick spout descended for mice to lick. Mice have directional biases; they prefer turning to the left as they move backward and exit a lane. In the earlier work using these mazes, individual mice made left or right turns on every trial, almost exclusively .
To overcome this bias, as they navigate on Airtrack, we trained mice to attend to a go cue that indicated the expected turn direction for the trial (Fig. 1A,B). In the course of a session, mice had to turn left on some randomly selected trials and right on the other trials. Consistent with earlier work on these platforms, mice whisked when they were engaged in active behaviors, and they did not whisk much when they were inactive (Fig. 1C,D, Movie 1; Dominiak et al., 2019). In addition, whisking was asymmetric. In every trial, for a large fraction of each trial, as mice moved through the maze, they moved their right and left whiskers in opposite directions, retracting whiskers on one side and protracting them on the other side (Fig. 1C, top, left turn trial, bottom, right turn trial). The asymmetry of the whiskers reflected the direction of turn (Fig. 1C, top, left turning trial, bottom, right turning trial), with the right and left side C2 whiskers mirroring each other for the right and left turns. These observations suggested that mice position their whiskers asymmetrically as a part of their motor plan.
In addition to positioning the whiskers asymmetrically and whisking, mice also moved their eyes on every trial (Fig. 1C, Movie 1). They performed saccade-like, abrupt and intermittent fast eye movements when they were moving (i.e., when they were engaged in active behavior).
The movement/position traces of both eyes were similar (Fig. 1C, red and blue traces); mice moved their eyes conjugately, bilaterally, to the same extent, at approximately the same time, in the direction of the upcoming turn. Consequently, right and left turn trials elicited movement of both eyes, but the movement was in the opposite direction for the two turn directions. Once mice stopped moving-when they were expecting a reward, licking, or were waiting to begin the new trial-movement of the eyes diminished greatly.
To quantify the effects of the behavioral state of the animal on whisker and eye positions, we normalized the data for time (each trial and each behavioral epoch could have a different duration) and normalized whisker position and eye position data for the extent/amplitude of movement (Fig. 2). These normalized average traces confirm what the single-trial data show: the side-to-side whisker asymmetry ( Fig. 2A) and eye movement (Fig. 2B) were related to the behavioral state of the animal and the direction of turn in the maze. When right-turn trials were compared  The gray lines on the two left panels represent the AUC values when the whisker asymmetry or the eye position data were randomized within the backward period. The right panel shows the comparison of predictive performances between the whisker asymmetry and the eye position on different phases within the backward movement. Pairwise comparisons indicate that whisker asymmetrybased prediction (left) was significantly better for later time bins than for earlier ones, reaching significance midway through backward motion, as follows: bin #1 versus bin #2, p = 1.0000; bin #1 versus bin #3, p = 0.9037; bin #1 versus bin #4, ***p = 0.0007; bin #1 versus bin #5, ***p = 0.0002; bin #2 versus bin #3, p = 1.0000; bin #2 versus bin #4, *p = 0.0387; bin #2 versus bin #5, **p = 0.0016; bin #3 versus bin #4, *p = 0.0137; bin #3 versus bin #5, ***p = 0.0005; bin #4 versus bin #5, p = 0.3735; Mann-Whitney U test with Bonferroni's correction. Pairwise comparisons for eye movement (center) also indicate that late in the backward motion eye movement was a good predictor of upcoming turn direction, as follows: bin #1 versus bin #2, p = 1.0000; bin #1 versus bin #3, p = 0.2395; bin #1 versus bin #4, **p = 0.0032; bin #1 versus bin #5, ***p = 0.0001; bin #2 versus bin #3, p = 1.0000; bin #2 versus bin #4, *p = 0.0316; bin #2 versus bin #5, ***p = 0.0009; bin #3 versus bin #4, p = 0.4514; bin #3 versus bin #5, **p = 0.0033; bin #4 versus bin #5, *p = 0.0453; Mann-Whitney U test with Bonferroni's correction. Pairwise comparisons between whisker asymmetry versus eye positioning (right) indicate that whisker asymmetry is a better predictor of the upcoming turn direction in the early phases of backward movement period: bin #1, *p = 0.0293; bin #2, *p = 0.0269; bin #3, p = 0.2799; bin #4, *p = 0.0275; bin #5, p = 0.3560; Mann-Whitney U test. C, Two-dimensional distribution of whisker position and eye movement before left (red) and right (blue) turns. with left-turn trials, whisker asymmetry appeared as a mirror image, and eye movement traces appeared as inverted mirror images of each other. Whiskers on two sides of the face moved bilaterally but in the opposite directions, and at the same time mice moved their eyes conjugately in the direction of the upcoming turn. This pattern of whisking and eye movement were evident when mice were in an active state (i.e., when the animals were moving backward, forward, or turning), and the pattern was not evident when mice were standing still at the end of a lane, expecting reward or licking. When mice were active and moving backward or turning, in both right and left turning trials, there was significant whisker asymmetry and significant change in eye position compared with behavioral epochs where mice were just standing still (Fig. 2C,D). Together, these data indicate that whisker asymmetry and the movement of the eyes were active processes occurring in a behaviorally relevant manner as mice navigated the plus maze.

Predicting turn direction with whisking and eye movements
We used ROC curves to examine whether eye movement, whisker asymmetry, or both could predict the turn direction ( Fig. 3; see also Materials and Methods). When normalized for time (during the backward movement epoch) and binned, the ROC curves reveal that both the whisker asymmetry and eye position become increasingly stereotyped and increasingly predict the turn direction (Fig. 3A).
While both eye movement and whisker movement accurately predicted turn direction before the animal started to turn the maze, examination of the area under the curve (AUC) across behavioral sessions (n = 13 sessions taken from four animals, consisting of 77 backward movement epochs in total) showed that the AUC for models based on whisker asymmetry was significantly larger than the chance level from the very beginning of the backward-movement period (bin #1, **p = 0.0066; bin #2, **p = 0.0029; bin #3, ***p = 0.0000; bin #4, ***p = 0.0000; bin #5, ***p = 0.0000; Mann-Whitney U test; Fig. 3B, left). This implied that whisker asymmetry was a good predictor of the upcoming turn direction. Pairwise comparisons indicate that whisker asymmetrybased prediction was significantly better for later time bins than for earlier ones, reaching significance (p , 0.01, Mann-Whitney U test with Bonferroni's correction) midway through the backward motion (Fig. 3B, left).
Except for the beginning of the backward movement, eye position also predicted the upcoming turn direction (bin #1, p = 0.6444; bin #2, p = 0.1118; bin #3, **p = 0.0029; bin #4, ***p = 0.0000; bin #5, p = 0.0000; Mann-Whitney U test). As the animal moved closer to the turn, during or just after the mid-point of the backward motion, eye position became a good predictor of the upcoming turn direction (p , 0.05, Mann Whitney U test with Bonferroni's correction; Fig. 3B, middle).
We then asked whether having both whisker asymmetry and eye movement together contributes to a higher predictive power than having single behavioral variables alone. When histograms of whisker asymmetry and eye movement were plotted relative to each other over the five temporal bins as mice move backward in a lane, there was a marked separation between data points from rightturning and left-turning trials (Fig. 3C). The fidelity of separation for predicting the turn directions was examined for whisker and eye position. In later bins, the model using both eye and whisker movement data was significantly better at prediction, than the model based on the data where both of the input parameters were shuffled in relation to turning directions (bin #1, p = 0.3674; bin #2, *p = 0.0379; bin #3, **p = 0.0097; bin #4, ***p = 0.0000; bin #5, ***p = 0.0000; Kruskal-Wallis test with Bonferroni's correction; Fig. 3D, left). Shuffling the whisker asymmetry (Fig. 3D, middle) or eye positions (Fig. 3D, right) had no significant additional effect on the fidelity of the separation compared with the full model (p = 1.0 for all time bins, Kruskal-Wallis test with Bonferroni's correction).
Together, these analyses reveal that the information in whisker asymmetry and eye position, individually and together, were sufficient to predict the turn direction before the animal begins to turn. The results of our analysis also imply that whisker asymmetry and eye movement contain largely overlapping information in predicting the turn direction.
continued The histograms of whisker asymmetry and eye movement are plotted relative to each other over five temporal bins as mice move backward in a lane. The heights (densities) of the histograms were scaled for visualization, and time was normalized over the time periods during backward movement. Over time as the mice move backward and closer to the turn, there was a marked separation between data points from right-turning and left-turning trials. D, Fidelity of separation. The status of the separation was examined by drawing a line in the two-dimensional space that best separated the two sets of data points. The fidelity of separation (i.e., of predicting the left turning directions based on separation using that line) was computed. Left, The magenta line indicates the fidelity when true whisker and eye data were provided, and the gray line represents the results from the null models, where both whisker and eye data were shuffled relative to the direction of turn. Later bins during the backward period have significant fidelity in separation (bin #1, p = 0.3674, NS; bin #2, *p = 0.0379; bin #3, **p = 0.0097; bin #4, ***p = 0.0000; bin #5, ***p = 0.0000; Kruskal-Wallis test with Bonferroni's correction). Center and right: unique contribution of whisker asymmetry (center) and eye positions (right) to the fidelity of separation. Shuffling the whisker asymmetry or eye positions alone has no significant additional effect on the fidelity of the separation compared with the full model (NS, p = 1.0 for all time bins, Kruskal-Wallis test with Bonferroni's correction).

Saccadic eye movement and behavioral state
While the eye movement data from single trials shows that head-fixed mice moved their eyes in a stepwise fashion on each trial (Fig. 1), this fast movement of the eyes tended to be smoothed out in the average traces (Fig. 2). To examine the relationship between the rapid saccadic eye movements and behavioral state, we related behavioral states to saccades, which appeared as distinct spikes in the corresponding eye speed trace (Fig. 4A). Eye movements were counted as saccades if the following criteria were met: absolute value of eye speed for both eyes was above the threshold of an absolute eye speed of 0.1% of full eye-width per frame; and there was no additional spike of higher absolute speed related to eye movement within 250 ms of the saccade. The duration of each behavioral epoch (end of lane, backward movement, left or right turns, and forward movement) was normalized and divided into 10 equal bins, and the number of saccades that occurred within each time bin was counted. This analysis revealed that mice made saccades predominantly in the latter half of the backward movement, as they moved out of the lane. Saccades also occurred as mice turned and went forward into an adjacent lane (Fig. 4B). Consistent with the analysis of eye positions (Figs. 1C, 2), saccades were more likely to occur in the direction of the upcoming turn, rather than in the opposite direction. The distribution of saccades suggests that mice look in the direction of turn as they exit backward out of the lane, and again as they enter another lane. Note that, although saccades occurred primarily when the mice were close to a turn or turning, saccades also occurred when the animals were merely moving forward or backward in a lane.

Correlation between saccades and whisker asymmetry
In many species, eye movement is correlated with head movement (Land, 2006). In rodents, head movement has often been related to both eye and whisker movement (Hartmann et al., 2003;Towal and Hartmann, 2006;Meyer et al., 2018). But mice can move their whiskers and eyes independently of their heads, even when they are head fixed, so it was possible that, although movement of the head was prevented, eye and whisker movements could be coordinated. To examine the relationship between saccades and whisker movement, we plotted the level of asymmetry before and after saccadic eye movements as mice moved backward out of the lane (Fig. 5A). Our analysis revealed that as mice moved backward and moved their eyes, they also changed how they positioned their whiskers. Whisker asymmetry increased measurably and significantly, 100-200 ms before mice made saccades during their backward movement (Fig. 5B). These results suggest that mice coordinate eye movement with

Discussion
To behave means to plan and emit a sequence of actions (Tinbergen, 1955;Krakauer et al., 2017). On one hand, the behavior we used here was complex; mice were head fixed as they moved their entire body through a "real-world" maze, as they pushed and rotated the platform around themselves, and entered or exited lanes. On the other hand, the behavior was simple; mice were in an environment that they controlled. The environment had lanes, similar to the burrows that mice tend to live in, in their natural habitat. All that mice had to do was to keep track of a cue that indicated the direction to move in, to recognize an adjacent rewarding lane, and to enter it. There was no time pressure, no requirement for mice to discriminate one thing from another. The goal was just to forage for water by moving in the cued direction. In the course of this behavior, as part of their motor plan, mice moved their whiskers and eyes in anticipation, in preparation for a turn in the maze. The bilateral positioning of the whiskers and eyes predicted the turn direction mice expected to impose on the maze (Figs. 3, 6).
In this maze, mice moved their body, and, while they moved their body, they also moved their eyes and whiskers bilaterally. In fact, in the course of this behavior, mice even move their nose and reset the set point of their whiskers, presumably by moving their whisker pad . One key difference between the earlier work and this work is that here mice had to attend to cues, and move in one of two directions based on a cue. In the earlier work, mice mostly moved in their preferred direction, right or left, but not in both directions. Here we trained mice to overcome their bias by using directional cues, and we found that from trial to trial, the bilateral whisker movement reflected turn direction instead of an innate preference. We also found that the retraction and protraction of whiskers were coordinated with the direction of eye movement.
Eyes and whiskers move similarly in a look-ahead function. Eye movement could be used for assessing the distance to a wall or to an opening. Whiskers could be positioned to anticipate the upcoming turn. But whether mice innately coordinate these movements, or they first learn to coordinate the movement of eyes and whiskers/ face when they learn to navigate the maze, is not clear. Whether mice coordinate whisker and eye motion in their natural, freely behaving condition is also not known, in part because the entire behavioral repertoire that we have measured here is not easily or reliably measured in freely moving animals. We also do not know whether circuits for eye movement, whisker movement, motor planning, and body movement are all active simultaneously in adjacent parts of anterior frontal and sensorimotor cortices, and how activity in these circuits interacts with subcortical circuits to generate the sequence of movements.  . Coordination between saccadic movement and whisker asymmetry. A, Changes in whisker asymmetry in relation to the occurrence of saccades around the onset of movement during the backward movement period. The dashed line at zero, and the black triangle, mark saccade onset. On average, whisker asymmetry increased 100-200 ms before the time the eyes moved (n = 120 leftward saccade events and n = 94 rightward saccade events). The increased asymmetry was maintained for hundreds of milliseconds after each saccade. The correlograms based on the original data (magenta) and the data where the timings of saccade events were shuffled within the backward movement period in the trial (black) are shown. Solid lines indicate the median traces, and the shaded regions indicate the intervals between the 25th and 75th percentiles. B, Quantification of changes in whisker asymmetry. The median whisker asymmetry values of 100 ms bins were computed for each trace. The arrowhead indicates the saccade onset. Tukey box plots were generated based on the original data (magenta) and the shuffled data (gray). The distribution of values were compared in the bin-to-bin basis between the original and the shuffled datasets. ***p , 0.001, *p , 0.05, NS, p . 0.05; Mann-Whitney U test, n = 214 saccade events.

Eye movement in mice
Rodents use vision to locate objects and to avoid airborne (Morris, 1979) or ground-dwelling predators (Doncanster et al., 1990). Freely behaving mice move their eyes when they move their head; they move their head and eyes to monitor looming stimuli and to coordinate freezing or escaping behaviors (De Franceschi et al., 2016). One unique feature of freely moving rodents is that they move their eyes in both conjugate and disconjugate fashions (Sakatani and Isa, 2007;Wallace et al., 2013;Wang et al., 2015;Payne and Raymond, 2017;Meyer et al., 2018;Samonds et al., 2018). When rodents are head fixed, the frequency of eye movement is reduced and eye movement becomes almost completely conjugate (Wallace et al., 2013;Samonds et al., 2018).
Our work here confirms the earlier work, showing that head-fixed mice do indeed move their eyes, and they move their eyes conjugately. We extend the earlier work in two ways; first, we show that although mice are not instructed to move their eyes (Itokazu et al., 2018;Sato et al., 2019), mice move their eyes reliably, in a behaviorally relevant fashion just before and during turns. Mice moved their eyes in anticipation of the turn, in the direction of the turn on every trial. Second, we show that eye movement was embedded in the coordinated motion of the animal. When combined with earlier work, this work shows that when mice move, they move their eyes and whiskers in a coordinated fashion, in the same direction. In this floating real-world maze, movement of the eyes was part and parcel of the concerted movement of the face and body. Currently, we do not know whether mice move their eyes and whiskers in a similar fashion in virtual reality systems that have mazes, or virtual reality systems that have visual streaming built into them, but this should be an avenue of future investigations.

Sensory-motor coordination in the real-world environment
Simultaneous and continuous coordination of movement and sensation is part of the natural function of the brain (Welker, 1964;Wolpert and Ghahramani, 2000;Llinas, 2001;Wolpert and Landy, 2012;Musall et al., 2019;Stringer et al., 2019;Kaplan and Zimmer, 2020;Tantirigama et al., 2020). In their natural state, when animals move, they interact with their environment in multiple sensory-motor modalities; their limbs touch the floor, they look in the direction of the motion, their breathing changes, and, in the case of rodents, they move their whiskers. The movement of the animal changes the sensory scene for the animal, which in turn generates a new set of sensory stimuli (i.e., pressure on limbs, the novel texture under their skin, an updated stream of visual input, and tactile input from the whiskers) guiding mice around obstacles around and in front of them. These changing sets of stimuli elicit activity in the brain and are then used to reinforce the ongoing behavior or to modify the movement of the animal (Arkley et al., 2014;Kurnikova et al., 2017). As animals learn to navigate an environment, they can plan ahead, and anticipate what to expect before the actual sensory input occurs (Keller and Mrsic-Flogel, 2018).
It should be noted that head fixation is a key limitation of our work. When rodents are free to move, their eye movements are richer and more complex than in the head-fixed state (Wallace et al., 2013;Meyer et al., 2020). Consequently, the interaction between whisking and eye movements is expected to be much richer in the freely moving mouse. But it has not been possible to study this interaction in freely moving animals, so how the movement of each eye relates to the movement of whiskers on that side of the face in the freely moving animal, when eyes move disconjugately, is not known. Additionally, nothing is known about the interaction between the visual sensory-motor and vibrissal sensory motor systems as mice navigate their environment.
Another limitation of our work is the relatively small sample size. While this may have affected the statistical power in examining how much information is shared between whisker positioning and eye movement in predicting the planned motor behavior, it should be noted that, in the course of navigating the plus maze, mice position their whiskers asymmetrically and move their eyes in the direction of the upcoming turn on every trial. Whether the two behavioral variables encode information independent of each other would be the subject of future studies.
Our work sheds light on the innate strategy used by mice, and shows how they spontaneously, almost automatically, plan and coordinate the movement of their eyes, whiskers, and body. The floating plus maze we used here is likely to engage widespread cortical and subcortical circuits in a manner that is close to what the brain has evolved to do; that is, to move the animal through the world.


Humoral immune responses against SARS‐CoV‐2 in transplantation: Actionable biomarker or misplaced trust?

Measures of vaccine‐specific antibodies to SARS‐CoV‐2, as discussed by Werbel and Segev (Page 1316), may ignore responses to contemporaneous variants of concern and relevant cellular immune responses, thereby failing to provide the insights required to optimize clinical care.

Humoral immune responses against SARS-CoV-2 in transplantation: Actionable biomarker or misplaced trust?
In addition to a remarkable ability to spread and mutate, severe acute Boosting improves antibody quality in immune competent hosts via enhanced affinity maturation, providing antibodies that recognize a broader array of variants. 14 In immunocompromised hosts, incomplete responses to vaccination could produce greater humoral immune responses in the absence of enhanced affinity maturation. Such lower affinity antibodies may neutralize wildtype SARS-CoV-2 found in vaccines, but with lesser cross-reactivity to additional variants of concern (e.g., Omicron). Incomplete affinity maturation in SOT could relate to impaired T cell help, disrupted germinal center activity, or altered antigen presentation. 15 Thus, in the absence of a complete understanding the affin-

ity and breadth of neutralization of the antibody response, S-specific titers may not adequately characterize protection against SARS-CoV-2 variants.
Clinical laboratories are overwhelmed with requests for diagnostic assays during COVID. In a viewpoint paper by Werbel and Segev, measurement of antibody levels is advocated as an adjunct to clinical management. 16 They recommend quantitative FDA emergency use authorized anti-S1/RBD assays after full vaccination to assess antibody responses. As they note, there are few data supporting Mab use in vaccinated seronegative SOT recipients (or poor responders). The key question is whether antibody levels in SOT recipients are actionable biomarkers or simply an available assay, reassuring to the physician or patient but failing to reflect immune function?
AJT EDITORIAL With the appearance of Omicron BA.2, emerging antiviral therapies must be provided to those least likely to mount an effective immune response. Based on which tests? And is this the best use of a limited resource? In the immunodominant spike (S) protein, 5106 different amino acid replacements or substitutions have been identified as well as multiple deletions. 17 As variants emerged, natural antibodies (from prior infection), therapeutic monoclonal antibodies (targeting prior strains), and some vaccine-elicited antibodies have become less effective in preventing disease progression. 18 More than 85% of tested neutralizing antibodies are escaped by Omicron 19 ; nearly all are escaped by Omicron sublineage BA.2. 20 Thus, routine assays for SARS-CoV-2 antibody levels might erroneously suggest viral protection when none exists to emerging variants.
Antibody levels provide an imperfect foundation for decisions regarding disease prevention or treatment. Assays of neutralizing antibodies detect vaccine-specific antibodies but may not measure antibodies to contemporaneous variants of concern. Furthermore, maintenance of antibody levels sufficient to maintain lifelong protection against viral transmission is an unrealistic goal. Instead, we need to understand the precise functional correlates of immunity responsible for disease control. Emerging multiplexed antibody assays will interrogate antibody binding across variants and may provide information regarding the efficacy of vaccine-induced or monoclonal transmission blocking activity. As further vaccine variants emerge, new pan-variant vaccines will be required. Identification of the key correlates of immunity will provide SOT patients and clinicians the insights required to optimize clinical care in this and future pandemics.

D I SCLOS U R E
The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.


Synthesis and Investigation of the Analgesic Potential of Enantiomerically Pure Schiff Bases: A Mechanistic Approach

Schiff bases are a class of organic compounds with azomethine moiety, exhibiting a wide range of biological potentials. In this research, six chiral Schiff bases, three ‘S’ series (H1–H3) and three ‘R’ series (H4–H6), were synthesized. The reaction was neat, which means without a solvent, and occurred at room temperature with a high product yield. The synthesized compounds were evaluated for analgesic potential in vivo at doses of 12.5 and 25 mg/kg using acetic-acid-induced writhing assay, formalin test, tail immersion and hot plate models, followed by investigating the possible involvement of opioid receptors. The compounds H2 and H3 significantly (*** p < 0.001) reduced the writhing frequency, and H3 and H5 significantly (*** p < 0.001) reduced pain in both phases of the formalin test. The compounds H2 and H5 significantly (*** p < 0.001) increased latency at 90 min in tail immersion, while H2 significantly (*** p < 0.001) increased latency at 90 min in the hot plate test. The ‘S’ series Schiff bases, H1–H3, were found more potent than the ‘R’ series compounds, H4–H6. The possible involvement of opioid receptors was also surveyed utilizing naloxone in tail immersion and hot plate models, investigating the involvement of opioid receptors. The synthesized compounds could be used as alternative analgesic agents subjected to further evaluation in other animal models to confirm the observed biological potential.

Introduction
The term pain derives from the Latin and the Greek peone and poine, respectively, meaning "penalty" or "punishment" [1]. Pain is an "emotion" and the unpleasant sensory experience that is linked with potential or actual tissue damage [2]. The North American Nursing Diagnosis Association has defined pain as a condition in which the patient feels and reports severe discomforts and uncomfortable sensation (pain reporting may be descriptorencoded or verbal communication) [3]. A type of pain that usually abates rapidly is called acute pain. This type of pain is physiological and provides a warning sign about the disease and the unsafe conditions of a person as to something bad that has occurred in the body; mostly, it is nociceptive in nature but can be neuropathic too, and most associated etiologies are trauma, surgery, labor, acute illness and some special procedures [4].
A type of pain that exists for a longer period (from months to years) is called chronic pain and is different from acute pain in that it can be neuropathic, nociceptive or both [5]. Its further subtypes are: (i) pain that is associated with chronic disease (from secondary pain to osteoarthritis) and (ii) pain lacking a recognizable organic cause (e.g., fibromyalgia) [6]. Pain associated with the most severe situations, that is, cancer or malignancy, is simply named as cancer or malignant pain. This type of pain comprises both the acute and chronic

Pharmacological Activities 2.2.1. Acetic-Acid-Induced Writhing Method
The analgesic potential of the compounds H1-H6 was investigated by administering the doses of 12.5 and 25 mg/kg body weight into mice and its effect on pain was evaluated using the acetic-acid-induced writhing model. Writhing is a specific action in which the mice extend its abdomen and stretch hind limbs after injecting acetic acid to mice. The frequency of writhing was observed and calculated where compound H2 exhibited a significant decrease in the total writhes with the tested doses. At doses 12.5 and 25 mg/kg body weight, H1 showed 68.31% (17.87 ± 2.09, p < 0.01) and 75.08% (14.05 ± 2.11, p < 0.001) inhibitory effects, respectively, in comparison to that of the control group (56.39 ± 1.66), as presented in Figure 1. The Schiff bases were also effective at the tested doses. Among them, the most promising effects were observed for H3, which were found to be 68.49% (17.77 ± 1.89, p < 0.001) and 72.99% (15.23 ± 2.03, p < 0.001), corresponding to doses 12.5 and 25 mg/kg body weight (b.w). The standard diclofenac sodium at a dose of 10 mg/kg b.w showed 84.55% (8.71 ± 1.51) activity. Figure 1. Acetic-acid-induced analgesic activity of (H1-H6). Mean ± SEM, n = 6. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. the control group. Dunnett's multiple comparison tests was applied after a one-way ANOVA.
The 'S' series chiral Schiff bases (H1-H3) showed significantly reduction in the number of writhes. It was a similar case for the 'R' series Schiff bases (H4-H6), in which H5 exhibited a maximum effect, 70.12% (16.85 ± 2.05, n = 6) at 25 mg/kg b.w, followed by H4 at 68.47% (17.78 ± 1.36) at the same dose tested. The responses of 'R' series were comparable to those of the 'S' series compounds.

Formalin-Induced Paw-Licking Time
Pain induced by the formalin assay was also employed to investigate the analgesic effect of H1-H6 at the doses of 12.5 and 25 mg/kg b.w. The H1 exhibited the highest analgesic potential and lowered the pain by 49.40% (17.77 ± 1.07, p < 0.01) and 59.94% (14.07 ± 2.21, p < 0.001), correspondingly to the tested afore-mentioned doses in first phase, and 65.92% (20.95 ± 2.21, p < 0.05) and 73.95% (16.01 ± 1.42, p < 0.001) in the second phase, as compared to the control group (35.12 ± 1.22 and 61.47 ± 1.39), which is shown in Table 1. Animals pretreated with indomethacin (10 mg/kg) showed a considerable decrease in the paw-licking response of 76.10% (9.47 ± 1.37, p < 0.001) in the second phase, whereas a mild decline was found in the paw-licking time at 22.30% in the first phase.

Tail Immersion Method and Possible Involvement of Opioidergic System
The tail immersion model results of H1-H6 are shown in Table 2. The maximum analgesic effect was produced by H2, which was 65.96% (3.76 ± 0.049, p < 0.001) recorded after 90 min, whereas the latency period increased at the dose of 25 mg/kg b.w. The latency response was counted in seconds, where H3 expressed a significant upsurge in latency at the dose of 12.5 mg/kg b.w and 53.11% (2.73 ± 0.035, p < 0.01) and 59.37% (3.15 ± 0.028, p < 0.001, n = 6) at 25 mg/kg b.w, in comparison to the control group (1.28 ± 0.023).
Animals pretreated with naloxone substantially antagonized the analgesic potential of morphine and tramadol (centrally acting opioid analgesics), indicating the involvement of the opiodergic system in an analgesic response. However, pretreatment with naloxone causes partial decline in the analgesic response of H1-H6 in a dose-dependent manner in mice, indicating the partial involvement of opioid receptors (Table 3). Table 3. Involvement of the opioidergic system in the analgesic response using the tail immersion method.

Sample/Dose
Latency Response (s) The analgesic response of the synthesized Schiff bases (H1-H6) using the hot plat test was also estimated, as presented in Table 4. The extreme analgesic effect of H2 was observed at 90 min where latency was increased. The latency response counted in seconds was high for H2, which expressed a significant increase in latency at a dose of 12.5 and 25 mg/kg b.w, 61.32% (4.24 ± 0.039, p < 0.01) and 70.56% (5.57 ± 0.048, p < 0.001) correspondingly. For the control group, the recorded response was 1.28 ± 0.023.
Tramadol produced a noteworthy post-treatment potency, which was recorded as 83.79% (10.12 ± 0.038, p < 0.001, n = 6) after compared with the control group's response of 1.28 ± 0.023. Morphine also produced a high post-treatment potency response, which was found to be 84.68% (10.71 ± 0.022, p < 0.001) in comparison to the control group's response of 1.28 ± 0.023.
Animals pretreated with naloxone substantially antagonized the analgesic potential of morphine and tramadol (centrally acting opioid analgesics), indicating the involvement of the opiodergic system in analgesic response. However, pretreatment with naloxone causes a partial decline in the analgesic response of H1-H6 in a dose-dependent manner in mice, indicating the partial involvement of opioid receptors (Table 5).

Discussion
In this study, we synthesized six chiral Schiff bases belonging to the 'S' series (H1-H3) and 'R' series (H4-H6) in high yield using grindstone chemistry, which is an environmentally friendly procedure involving no catalysts or solvents.
Various pain models are used to evaluate analgesic potentials of natural and synthetic compounds. The most important and frequent in use among them are acetic-acid-induced writhing, the formalin pain test, tail flick test and the hot plate test in mice or rats [19]. Pain mechanism involve three main events due to noxious stimulation: transmission, transduction and modulation of the signals [20]. The writhing test has been suggested for the preliminary measurement of anti-nociceptive effects on potential substances [21].
In this test, nociceptors activation followed by an expected inflammation of the viscera are carried out by chemically noxious substances, such as glacial acetic acid (0.3-0.6%), 2-phenyl-1,4-benzoquinone and magnesium sulfate, which are administered to rodents (frequently mice) by the peritoneal cavity route [22]. Acetic acid causes the induction of pain in mice due to its localized inflammatory response of free arachidonic acid released from tissue phospholipids through COX-producing prostaglandin [23]. The post-injection period up to an hour includes writhing reactions such as stretching, twisting, extension of hind legs and contraction of the abdomen [24,25]. The acetic-acid-induced writhing test is suggested for peripherally acting drugs (antihistamine, meprobamate and chlorpromazine) and is thus employed as the preferred analgesic assay [26]. In this paper, all the tested compounds exhibited analgesic potential; however, among them, H2 from the 'S' series and H5 from the 'R' series were comparatively more potent. These compounds thus have the potential to be used as alternative analgesic drugs in the future, subjected to toxicological evaluation in different animal models.
Among the assays, the formalin test is considered as a satisfactory model to evaluate the analgesic drugs for their efficacies. This test is executed for acute and long lasting pain evaluation in the paws of mice or rats [22], which are sensitive towards centrally acting analgesic drugs [27]. The three main behavioral responses carried out by the formalin injection are phasic flexion, tonic flexion and licking of the injected limb after injecting intraplantar formalin in the paw of mice or rats [28]. The direct stimulation of nociceptors occurs in the first phase, such as C-fibre and low-threshold mechanoreceptors, including the upregulation of substance P; then, inflammatory phenomena occur involving the central sensitization of rodents within the dorsal horn neurons, including the upregulation of histamine, serotonin, bradykinin and prostaglandin [29]. A similar trend in efficacy was observed here also and, as representative compounds of each series, H2 and H5 were more potent than the other tested compounds.
For the evaluation of the centrally acting drugs, the tail immersion and hot plate methods are equally used and, in these tests, the nociception pathway is mostly involved. Tail emersion in hot water produces spinally mediated nociception, whereas the hot plate method is only selective for the supraspinally mediated nociception [30,31]. Thus, the initiation of the analgesic action of opioid agents involves the spinal and supraspinal receptors [32]. Our results show that the compounds H1-H6 inhibited nociception, which are associated with spinal and supraspinal opioid receptors in the central nervous system. The anti-nociceptive effect of compounds H1-H6 verified this hypothesis by naloxone antagonism and it was partially produced in the hot plate and tail flick models, whereas naloxone is a nonselective opioid receptor antagonist [33]. In both experimental models, naloxone reduced the morphine-induced latency time at the given concentration. The current findings suggest that the activity of the tramadol (standard opioid analgesic) is parallel with that of the tested samples, indicating the potential of these compounds to be used as analgesic drugs. In the same way, the analgesic action of the tested samples and tramadol was considerably exaggerated by the earlier administration of naloxone, thus expressing the involvement of the opioid receptors in their modes of action.
Animals pretreated with naloxone substantially antagonised the analgesic potential of morphine and tramadol (centrally acting opioid analgesics), indicating the involvement of the opiodergic system in analgesic response. However, pretreatment with naloxone causes a partial decline in the analgesic response of H1-H6 in a dose-dependent manner in mice using the tail immersion and hot plate methods, indicating the partial involvement of opioid receptors and other mechanisms, which requires further investigation to confirm the mechanisms involved in the management of pain observed in this study.
Taken all together, this current study testified the role of chiral Schiff bases (H1-H6) as analgesic agents, which may act as promising candidates, and this warrants particular consideration in the research and development of the management of pain.

Materials
All chemicals and solvents used in this work were of analytical grade, manufactured by Sigma Aldrich (Merck), Germany, and were obtained from the local market. The progress of the reaction was monitored by TLC (Thin-layer chromatography) on Merck 60F 254 silica-gelcoated plates. The structures of the synthesized compounds were confirmed by 1 H-NMR spectra (300 MHz) on Bruker Varian Mercury 300 MHz FT Spectrometer, in CDCl 3 , FT-IR.

General Procedure for the Synthesis and Characterization of Chiral Schiff Bases
Chiral Schiff bases were synthesized by treating commercially available aldehydes with chiral amine by grinding in a mortar and pestle for a period of 5-10 min at room temperature without the addition of any solvent or catalyst. The reaction progress was monitored by TLC. A paste was obtained upon grinding. After the completion of the reaction, water from the mixture was evaporated until dryness. The powder product obtained was finally recrystallized in absolute methanol [34]. The synthesized compounds were characterized through different spectroscopic techniques, including Proton Nuclear magnetic resonance spectroscopy ( 1 H NMR) and infrared Spectroscopy. The 1 H NMR was carried out in deuterated chloroform (CDCl3) on Bruker Avance II 400 MHz NMR Spectrometer using 300 MHz frequency for all compounds, except compound H1 which was run on 400 MHz frequency. The spectra were resolved through Topspin-4 (Bruker, London, UK) software. The IR spectroscopy was performed on Spectrum 3™ FT-IR Spectrometer.
As mentioned, all the synthesized compounds were successfully characterized through H NMR and IR Spectroscopic techniques. The technical details are described as follows:

Acute Toxicity Study
The synthesized Schiff bases (H1-H6) were evaluated for optimum dose determination for the in vivo studies. In two stages, different groups of animals were administered synthesized compounds orally. In stage I, animals received 6.25, 12.5, 30, 50, 75, 100 and 125 mg/kg doses, and 25, 50, 75, 100, 150, 200 and 250 mg/kg doses in stage II. The animals were observed continuously for 24 h after the dosing for toxicological symptoms such as lacrimation, tremors, convulsions, salivation, and loss of righting reflex, motor activity, sedation, hypnosis, muscle spasm and diarrhea. Animals were further observed for mortality up to 72 h and the tested compounds were found non-toxic in stage I and safe up to 125 mg/kg and, in stage II, were safe and non-toxic up to 250 mg/kg dose. Therefore, according to the OECD guidelines, the 12.5 mg/kg dose, which was 1/10th of 125 mg/kg, and 25 mg/kg dose, which was 1/10th of 250 mg/kg treated dose, were selected the suitable doses for further in vivo studies [36].

Acetic-Acid-Writhing Test
The analgesic potency of the synthesized chiral Schiff bases was carried out by the acetic-acid-induced writhing test. Mice were categorized into different groups, having 6 mice in each group. The control group received 2% Tween 80, and the tested compounds were administered at the 12.5 mg/kg and 25 mg/k p.o doses. Diclofenac sodium 10 mg/kg i.p served as the positive control. Acetic acid 0.1 mL (0.6%) was injected i.p to mice after a 15 min interval. The number of the writhes performed in each of the mouse was observed and noted for about 30 min by placing on a flat surface after the administration of the acetic acid [37]. The percentage inhibition of writhing was determined by the following formula:

Formalin Test
The formalin test was carried out in animals to assess the analgesic activity of the synthesized Schiff bases according to the standard procedure. Animals were randomly divided in different groups each having 6 mice and pre-treated (30 min) with normal saline (2% Tween 80); the synthesized compounds and indomethacin 10 mg/kg (standard drug) were injected intraperitoneally (i.p.) 15 min before the administration of formalin. The experimental groups received doses of 12.5 and 25 mg (p.o) per kg body weight of the synthesized compounds followed by the sub-cutaneous injection of formalin at 20 µL (2.5%) into the dorsal surface of the right hind paw of each animal [38]. The time of pain response (paw-licking time) for each animal group was observed and calculated in two periods, i.e., 0-5 min (direct activation of nociceptors) and 15-45 min (inflammatory-mediators-induced pain response), and compared with the control group animals [39].

Tail Immersion Test
The mice were used for checking the central analgesic potential of the synthesized Schiff bases. Mice were divided in different groups and the synthesized compounds at the doses of 12.5 and 25 mg as well as the normal saline (2% Tween 80) solution were administered to the animals at 30 min and morphine 2 mg/kg (standard; i.p) at 15 min before the test to the respective groups. The latency period (time of reaction measured) at 15, 30, 45, 60, 75 and 90 min after the administration of the test samples was recorded using a stopwatch (time measured for tail deflection by mice) and was calculated by immersing the tail of mice (1-2 cm) in hot water (temperature maintained at 55 ± 1 • C) [40].

Hot Plate Test
The Eddy and Leimbach method was used for assessing the analgesic activity of the synthesized Schiff bases with slight modifications in the hot plate model. Animals were divided in different groups each carrying 6 and placed on a hot plate at a temperature of 54 ± 2 • C and the time taken by animal for either jumping or paw licking were recorded. The latency time until animal discomfort (jumping, hind paw licking or hind paw lifting) 60 s cutoff time was recorded at different time intervals after the administration of normal saline (2% Tween 80 solution), synthetic compounds (12.5 and 25 mg/kg), tramadol (10 mg/kg) and morphine (2 mg/kg) [41].

Molecular-Level Mechanisms for Opioid Receptor Involvement
After the acetic-acid-induced writhing model, formalin test, tail emersion test and hot plate method, the synthesized compounds were subjected to mechanistic approach for the possible association of opioid receptors. In order to confirm the connection of opioid receptors, a previously reported procedure was followed [32]. In this assay, different groups of mice, each having 6 individuals, were pre-treated with an antagonist of non-selective opioid receptor; naloxone 2 mg/kg (i.p) was injected 30 min prior to the given dose of the pure compounds (12.5 mg/kg and 25 mg/kg p.o) and morphine (2 mg i.p), and were tested by using the tail immersion and hot plate tests, respectively.

Conclusions
In this research work, six chiral Schiff bases, three of 'S' series (H1-H3) and three of 'R' series (H4-H6), were synthesized and evaluated for analgesic potential in mice models. The synthesized Schiff bases showed promising analgesic responses in all the tested models in reducing the induced pain threshold, which suggests their partial involvement of the opioidergic system in their mode of actions. Thus, the synthesized compounds might be promising candidates that warrant particular consideration in the research and development of the management of pain. Further studies are needed to explore the exact mechanisms involved in the management of pain as these are preliminary results.


extfieldsofstudy: Medicine Biology

text:
Uncovering the universality of self-replication in protein aggregation and its link to disease

Fibrillar protein aggregates are a hallmark of a range of human disorders, from prion diseases to dementias, but are also encountered in several functional contexts. Yet, the fundamental links between protein assembly mechanisms and their functional or pathological roles have remained elusive. Here, we analyze the aggregation kinetics of a large set of proteins that self-assemble by a nucleated-growth mechanism, from those associated with disease, over those whose aggregates fulfill functional roles in biology, to those that aggregate only under artificial conditions. We find that, essentially, all such systems, regardless of their biological role, are capable of self-replication. However, for aggregates that have evolved to fulfill a structural role, the rate of self-replication is too low to be significant on the biologically relevant time scale. By contrast, all disease-related proteins are able to self-replicate quickly compared to the time scale of the associated disease. Our findings establish the ubiquity of self-replication and point to its potential importance across aggregation-related disorders.

Aggregation mechanisms
In the past, we have developed a range of integrated rate laws for variations on the mechanism of aggregation, for example due to differing reaction orders and saturation of particular processes.
Details on these mechanisms can be found in Meisl et al. (7). The majority of proteins in this work, including all for which only one concentration was available, were analysed with a model including primary nucleation, single step secondary nucleation and elongation. The proteins analysed with a different model are Aβ and α-synuclein.

Proteins
Here we briefly discuss each protein studied, including the biological role of aggregation and where the data were obtained. For most proteins, the data have not been previously analysed by fitting of integrated rate laws to obtain reaction rates. In those cases, i.e. when data was analysed for the first time in this work, the fits are shown in Figs. S1 to S22. For datasets which had been previously analysed (Aβ and some tau and IAPP datasets) please refer to the cited works for the details of the fits.

a-Synuclein
a-Synuclein in its amyloid fibril form is the major protein component of the Lewy bodies and neurites that are one of the hallmarks of Parkinson's disease (55). Gaspar et al. investigated the aggregation of full-length wild type a-synuclein (25). Data analysed in our study are those shown in figure 2c of that paper. The biologically relevant timescale is the length of Parkinson's disease. S2

Ab
Aggregation of both isoforms of the Ab peptide, Ab 40 and Ab 42 , is associated with Alzheimer's disease (AD) (16,24). The point mutations E22G, E22Q, and D23N are all associated with familial AD. The rate constants for Ab 40 are from Meisl et al. (24). The Ab 42 values, including all familial AD-associated mutants and the data and fits shown in Fig. 1 of the main text are from Yang et al. (54) (data and fits shown in figure 5 of that paper). The seeded data shown in Fig. 1 are from Weiffert et al. (52). The V18S+A21S Ab 42 is an artificial variant which was designed to try to abolish secondary nucleation, rate constants for this are determined in Thacker et al. (28). For Aβ42, while the aggregation at µM concentrations, under physiological slat concentrations, is dominated by secondary nucleation with a reaction order of 2, there appears to also be fragmentation of fibrils that proceeds at a much lower rate than secondary nucleation, as shown in Meisl et al. (56). However, when κ is calculated at low concentrations, one has to take into account the contribution from fragmentation as this process is expected to dominate over seocndary nucleation at sufficiently low concentrations, due to the fact that the fragmentation rate does not scale with monomer, unlike the secondary nucleation rate. We thus also evaluate κ due to fragmentation, and use this to construct the plots in Fig. 3. The relevant rate constants are given in table 3 under "Ab42 (fragmentation)". The biologically relevant timescale is the length of Alzheimer's disease (specifically average time from Braak stage 3 to Braak stage 6).

Actin
Actin is one of the major components of the eukaryotic cytoskeleton, and its conversion between monomeric (G-actin) and filamentous (F-actin) states facilitates the regulation of a multitude of cellular functions and properties, including cell shape and motility (57). Actin polymerisation is a distinct process from amyloid aggregation, with F-actin lacking the canonical cross-b structure S3 (58,59). Pure actin is only able to form polymers, and requires actin binding proteins (ABPs) to nucleate new F-actin fibres from existing F-actin surfaces, which we can equate to amyloid self-replication processes (60). Higgs et al. investigated the kinetics of actin polymerisation in the absence and presence of ABPs. The ABPs under study were the Arp2/3 complex, a weak cellular nucleating factor, and WA, which stimulates the Arp2/3 nucleation ability and consists of the C-terminal regions of Wiskott-Aldrich syndrome protein, N-WASP, and three isoforms of Scar, collectively termed the WASp/Scar protein family (40). Data analysed in our study were extracted from figure 9 (40). In the tables, aggregation under these conditions is referred to as "Actin + WAsp". The biologically relevant timescale is the length of cytoskeleton formation / rearrangement.

Apomyoglobin
The aggregation of apomyoglobin is not related to any human diseases, nor does it seem to play a role in vivo. The ability of apomyoglobin to form amyloid fibrils is therefore used as a model system and proof of the apparent universality of amyloid aggregation. Tryptophan residues at positions 7 and 14 were found to play an important role in the folding of apomyoglobin (61), and the double apomyoglobin mutant W7F/W14F (W7FW14F) was furthermore found to increase the aggregation propensity of the protein (62). Unlike the WT protein, the W7FW14F readily aggregates at physiological pH and temperature; Vilasi et al. therefore used this construct in their study (63). Data analysed in our study were extracted from figure 3 (63).
The curli fibrils are the major protein component of the extracellular matrix and thus play a key role in biofilm structuring. Andreasen et al. investigated the aggregation of the recombinantly S4 expressed mature form (residues 22-151) of CsgA (53). The data shown here, including the data and fits shown in Fig.1 of the main text, are from Andreasen et al. (53) (data and fits shown in figure 3a of that paper). The seeded data shown in Fig. 1 have not been published previously.
The biologically relevant timescale is the timescale of biofilm formation.

FapC
FapC is produced by Pseudomonas strains and, in its amyloid form, contributes to biofilm structuring and strengthening (44). The data shown here are from Andreasen et al. (53) (data and fits shown in figure 3b of that paper). The biologically relevant timescale is the timescale of biofilm formation.

Gadm1 (b-Parvalbumin)
b-Parvalbumin fibrils do not play a known functional or pathological role (65). Amyloid fibrils of b-parvalbumin are of interest because there is evidence suggesting that they are responsible for food allergies (65). The aggregation of Atlantic cod b-parvalbumin, Gadm1, was studied by Castellanos et al. (66). In this study, the aggregation behaviour of wild-type and two variants (I12C and C19S) of Gadm1 were investigated. These mutations were chosen, since residues I12 and C19 may play a role in amyloid assembly and calcium ion binding properties (66). Data analysed in our study were extracted from figure 3A (66).

Gelsolin
The aggregation of mutant gelsolin, with the D187N/Y mutation being the most common, is associated with gelsolin amyloid disease, or familial amyloidosis of Finnish type (FAF). Gelsolin is cleaved into several fragments, including 5 kDa and 8 kDa fragments, both containing the D187N/Y mutation. Both fragments are amyloidogenic, with the 8 kDa fragment comprising the majority of FAF-associated fibrils. Solomon et al. characterised the aggregation of these S5 two fragments with the D187N mutation (51). Data analysed in our study were extracted from figure 1D of that paper, where aggregation was performed at pH 7.4. The biologically relevant timescale is the length of Finnish gelsolin amyloidosis.

Huntingtin and polyQ peptides
Ectended CAG repeat sequences in trinucleotide (CAG) repeat expansion disorders result in the production of proteins with extended polyglutamine (polyQ) tracts, whose aggregation is characteristic of such diseases (67). Kakkar (67). Data analysed in our study were extracted from figures 1A and S1B-C (67). Kar et al. explored the effects of polyQ tract length on aggregation kinetics; we analysed their data on the N-terminal region of the protein ataxin-7 with a 30Q tract, and on SFQ 37 P 10 K 2 , a fragment of the Huntingtin N-terminal peptide (68). Data analysed in our study were extracted from figures 1C and 3B (68). The biologically relevant timescale is the length of Huntington's disease.

Islet amyloid polypetide precursor
The aggregation of islet amyloid polypeptide precursor (IAPP) is associated with Type II diabetes, in which aggregates of IAPP are found in the extracellular space of the islets of Langerhans (69). Pilkington et al. investigated the aggregation of the 37-residue peptide KCNTAT-CATQRLANFLVHSSNNFGAILSSTNVGSNTY with a disulphide bridge between residues 2 and 7 (69). Data analysed in our study were extracted from figure 1A (69). Additionally, both rates and reaction orders for IAPP aggregation were obtained by Rodriguez-Camargo et al. (26), also included here. The biologically relevant timescale is the timescale of Type 2 diabetes onset.

Insulin
The aggregation of insulin serves neither a functional purpose nor is it associated with pathology. Nielsen et al. studied the aggregation of bovine insulin induced by low pH (70). Data analysed in our study were extracted from figures 2A-B and 6 (70).

LacY
The aggregation of the membrane protein LacY serves neither a functional purpose nor is it associated with pathology. Stroobants et al. monitored the aggregation of the wild type LacY protein (71). Data analysed in our study were extracted from figure 3A (71).

Lysozyme
The aggregation of hen egg white lysozyme (hewL) serves neither a functional purpose nor is it associated with pathology, instead being used as a model system by which to study aggregation.
Hasecke et al. monitored the aggregation of wild-type hewL (72). Data analysed in our study were extracted from figure 2C (72).

Monellin
The aggregation of the plant protein monellin serves neither a functional purpose nor is it associated with pathology (73). Konno et al. studied the aggregation of the wild-type protein (73).
Data analysed in our study were extracted from figure 5A (73).

MSP2
Merozoite surface protein 2 (MSP2) is a 30 kDa protein found on the surface of the malariacausing Plasmodium falciparum (74,75). The structure of MSP2 in this physiological has not yet been fully established, however, some evidence suggests the presence of amyloid-S7 like oligomers, and the ability of MSP2 to form fibrils may play a role in immune recognition (74,76). Adda et al. studied the aggregation kinetics of MSP2 from the FC27 strain with a C-terminal hexa-His tag (76). Data analysed in our study were extracted from figure 5 (76).

Orb2
Aggregation of Orb2 into amyloid fibrils is important for its function in long-term memory formation in Drosophila melanogaster, and its analogues play similar roles in Aplysia californica and mice (77)(78)(79). In Drosophila melanogaster, Orb2 is present in two isoforms, the 551residue Orb2A and the predominant 704-residue Orb2B, which differ only at their N-termini.
investigated the aggregation of the first 87 residues of Orb2A (Orb2A87), whose metal binding properties may play a role in regulating aggregation (80). Data analysed in our study were extracted from figure 6A (80).

Phenol soluble modulins
Phenol soluble modulins (PSMs) are a family of peptides produced by Staphylococcus aureus with a range of functions, behaving as toxins, pro-inflammatory agents, or structural entities in biofilms (81). There are two classes of PSMs, aand b (82). PSMs of type b have been found to be consistently less cytotoxic than the a-type peptides, and are likely to play a key role in biofilm structuring (82)(83)(84)(85). PSMa1 fibrils have also been found to promote biofilm stability, whereas fibrillation of PSMa3 increases its toxicity, suggesting that its main role may be as a toxin against human cells (86)(87)(88). Data analysed in our study are those shown in Fig. 1 of Zaman et al. (89). The biologically relevant timescale is the timescale of biofilm formation.

Prion protein
Aggregation of prion protein (Prp) is responsible for prion disease and transmission. In vitro rate constants used here are from Sang et al. (20). The biologically relevant timescale is the length of prion disease. Prp is one of two systems for which we have determined the self-replication rate also in vivo (38): this was measured in 4 mouse lines which express different concentrations of Prp (resulting in different self-replication rates) and also have different survival times. This variation of self-replication rate, along with the relevant timescale, reflected in the diagonal region marked for "Prions in mice" in Fig. 3 of the main text.

Semenogelin
The deposition of aggregated semenogelin-1 (Sg1) protein in seminal vesicles is associated with

Sup35
The Saccharomyces cerevisiae Sup35 protein (Sup35p), in its soluble form, is a translation termination factor (105). Sup35p also exists in a transmissable amyloid form, wherein it functions as a yeast prion, being responsible for the [PSI + ] phenotype, increasing the phenotypic S10 diversity and thus conferring a survival advantage in changeable environments (106,107). The [PSI + ] phenotype can arise spontaneously and can be propagated to daughter cells via the cytoplasm, dependent on the presence of aggregated Sup35p seeds; self-replication of seeds is therefore crucial for its function (107)

Tau
The aggregation of tau is associated with a range of disorders, of which Alzheimer's disease is the most prevalent. The in vitro rates of tau(304-380) are from Rodriguez-Camargo et al. (27), the tau WT rates are from Kundel et al. (19). The tau datasets from Kundel et al. (19) use heparin to artificially induce primary nucleation, hence the primary rate constants and reaction orders observed in that experiment are not used here. As this means lambda is difficult to estimate, we show these tau data only in Fig. 3 (which is independent of lambda) and not Fig.   2 (this only contains the other tau dataset from Rodriguez-Camargo et al. (27)). While we can't estimate lambda for full length tau, the fact that no aggregation of full length tau is seen in the absence of an inducer of primary nucleation confirms that indeed secondary processes dominate over primary ones also in the full length system. Tau is one of two systems for which we have determined the self-replication rate also in vivo, see Meisl et al. (39): this was measured in humans with Alzheimer's disease. The relevant timescale is the length of Alzheimer's disease S11 (specifically average time from Braak stage 3 to Braak stage 6).

Ure2p
The Ure2 protein (Ure2p) is a yeast prion, which behaves similarly to Sup35p (107,110). Ure2p is responsible for the [URE3] phenotype in its prion form. In its soluble form, Ure2 is a regu-                           Table S3: Proteins shown in the main text, where a reaction order could be determined. When experiments were seeded or self-replication processes were too slow to be detected this is denoted by †. In those cases, upper bounds for λ or κ, were used instead, calculated as outlined in the Methods section. "Aβ42 (fragmentation)" simply records the estimate of the rate of fragmentation Aβ42 (see section on Aβ). The tau datasets from Kundel et al. (19) use heparin to artificially induce primary nucleation, hence the primary rate constants and reaction orders observed in that experiment are not used here (see section on tau). Some datasets had to be analysed with a model including saturation of secondary nucleation or elongation, the respective values for the saturation constants, K M and K E , respectively, are given in the "comments" column. For details on the models please refer to the cited papers as well as Meisl et al. (7).

Investigation of structure-mechanism correlation
In order to investigate whether any specific structural features could be identified that correlate with the propensity for self-replication to dominate over de novo formation, we used a recently developed method to identify sequence patterns, see Fig. S23. This analysis yields 36 different types of patterning score, for each of which we then computed the Pearson correlation coefficient, and its associated p-value for non-correlation, with our measure for dominance of self-replication, log(κ/λ). A weak correlation of log(κ/λ) with some patterning scores is observed, however, when the Bonferroni correction is applied to account for the fact that there are 36 different scores to correlate to, none of these correlations remain significant (p> 0.05).
While there are likely specific structural features that promote or prevent self-replication, our current dataset is likely not extensive enough to provide statistically significant insights and considerable additional work will be needed to elucidate these features. Figure S23: Z-scores from NARDINI analysis (45). Each column is a different protein (when data from multiple forms of the protein were available, the WT was chosen), each row a different patterning parameter, the colour denotes the z-score, which quantifies how much each patterning score differs from a random sequence, with blue and red indicating more and less uniform dispersion, respectively, than would be expected in a scrambled sequence. The data are ordered by increasing κ/λ ratio from left to right, i.e. proteins for which de novo formation dominates are on the left, those for which self-replication dominates are on the right.