Sex, age and breed of the 16 horses used in this study.

<p>Sex, age and breed of the 16 horses used in this study.</p

Statistical models and results for the six outcome measures.

<p>Statistical models and results for the six outcome measures.</p

Are Eyes a Mirror of the Soul? What Eye Wrinkles Reveal about a Horse’s Emotional State

<div><p>Finding valid indicators of emotional states is one of the biggest challenges in animal welfare science. Here, we investigated in horses whether variation in the expression of eye wrinkles caused by contraction of the inner eyebrow raiser reflects emotional valence. By confronting horses with positive and negative conditions, we aimed to induce positive and negative emotional states, hypothesising that positive emotions would reduce whereas negative emotions would increase eye wrinkle expression. Sixteen horses were individually exposed in a balanced order to two positive (grooming, food anticipation) and two negative conditions (food competition, waving a plastic bag). Each condition lasted for 60 seconds and was preceded by a 60 second control phase. Throughout both phases, pictures of the eyes were taken, and for each horse four pictures per condition and phase were randomly selected. Pictures were scored in random order and by two experimenters blind to condition and phase for six outcome measures: qualitative impression, eyelid shape, markedness of the wrinkles, presence of eye white, number of wrinkles, and the angle between the line through the eyeball and the highest wrinkle. The angle decreased during grooming and increased during food competition compared to control phases, whereas the two phases did not differ during food anticipation and the plastic bag condition. No effects on the other outcome measures were detected. Taken together, we have defined a set of measures to assess eye wrinkle expression reliably, of which one measure was affected by the conditions the horses were exposed to. Variation in eye wrinkle expression might provide valuable information on horse welfare but further validation of specific measures across different conditions is needed.</p></div

Effect of the four conditions on eye wrinkle expression.

<p>The effect of the four conditions (Grooming (G), Food anticipation (FA), Food competition (FC), Plastic bag (PB)) on the six outcome measures is shown with respect to phase (Control (C), Treatment (T)). (a, b) <u>‘angle’, ‘number’</u>: boxplots with median (black line), interquartile range (box), 1.5 x interquartile range (whiskers). (c) <u>‘eye white’</u>: bar chart±binomial confidence interval. (d, e, f) ‘<u>qualitative assessment’, ‘eyelid shape’, ‘markedness’</u>: stacked bar charts with the three colours representing the three different scores. (d) <u>‘qualitative assessment’</u>: ‘no wrinkle’ (black), ‘weak’ (dark grey), ‘strong’ (light grey). (e) <u>‘eyelid shape’</u>: ‘round’ (black), ‘weakly pulled’ (dark grey), ‘strongly pulled’ (light grey). (f) <u>‘markedness’</u>: ‘no wrinkle’ (black), ‘weak’ (dark grey), ‘strong’ (light grey).</p

Measures for the assessment of eye wrinkle expression.

<p>(a) <u>Qualitative assessment</u>: Categories reflect the first subjective impression of the expression of eye wrinkles based on the number of wrinkles, their markedness, and the angle they are forming. ‘No wrinkle’: no wrinkle visible. ‘Weak’: the overall impression of the wrinkles is weak with, e.g., some weakly marked wrinkles forming a narrow angle. ‘Strong’: the overall impression of the wrinkles is strong with, e.g., several marked wrinkles forming a wide angle above the eyeball. (b) <u>Eyelid shape</u>: ‘Round’: smooth curve without any sign of the lid being pulled in the dorso-medial direction. ‘Weakly pulled’: curve is continuous but slightly pulled in the dorso-medial direction. The eye looks more angled. ‘Strongly pulled’: the lateral part of the lid is an almost straight line. (c) <u>Markedness</u>: the depth and width of the wrinkles is assessed. If the markedness differs between wrinkles, the most prominent wrinkle is assessed. ‘No wrinkle’: no wrinkle visible. ‘Weak’: wrinkles are flat and narrow lines. ‘Strong’: wrinkles are pronounced in depth and width. (d) <u>Eye white</u>: The sclera (eye white, also when brownish due to pigments) is assessed as visible (‘yes’) or not visible (‘no’). (e) <u>Number</u>: Only wrinkles above the eyelid and those of a minimum length of one third of the eyeball’s diameter are considered. A deep indent, often seen in older horses, is not considered as a wrinkle (as it is not caused by muscle contraction of the inner eyebrow raiser). Moreover, wrinkles originating on the eyelid (mostly one or two) are not counted. (f) <u>Angle</u>: The degree of the angle is measured on the intersection of the extension of a line drawn through the eyeball and the extension of the highest wrinkle. The line through the eyeball extends from the medial to the lateral corner of the eyeball. If the medial corner is not clearly defined, the line goes through the middle of the tear duct.</p

Model of DNA repair proteins at sites of DNA damage in the presence of the 4-protein complex.

<p>In the presence of ICP8 and helicase/primase, RPA can still coat ssDNA at sites of damage and recruit ATR/ATRIP. However, these four-proteins bind to the ss/dsDNA junction that would normally serve as the loading platform for the 9-1-1 complex and exclude it from binding the DNA. This serves to prevent all of the downstream proteins from being recruited to sites of DNA damage and effectively inhibits ATR signaling.</p

HSV-1 inhibition of ATR signaling requires ICP8 and UL8.

<p>(A) Vero cells were either mock-infected or infected with HSV-1 at an MOI of 10 PFU/cell. At 5 hours post infection cells were treated with HU for 1 hour or UV and allowed to recover for 1 hour. (B) Vero cells were either mock-infected or infected with HSV-1 at an MOI of 10 PFU/cell. Cells were treated with UV at the indicated time post infection and allowed to recover for 1 hour. (C) Vero cells were either mock-infected or infected with the indicated HSV-1 mutant viruses at an MOI of 10 PFU/cell. At 5 hours post infection cells were treated with UV and allowed to recover for one hour. All cell lysates were analyzed by Western blot with the indicated antibodies. The band marked with an asterisk (*) in the P-RPA-S33 blot corresponds to a non-specific band that does not cross react with antibodies to endogenous RPA and likely represents cross-reactivity with a viral protein.</p

ICP8 and UL8 are sufficient to inhibit ATR signaling.

<p>(A) U2OS or (B) Vero cells were transfected with ICP8, UL8, UL5, and UL52 (ICP8+H/P) or ICP8 and UL8 alone and then damaged with UV. Cells were fixed at 1 hour post damage and prepared for immunofluorescence as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003652#s4" target="_blank">materials and methods</a>. (C) Cells were treated as in A and stained for either P-RPA-S33 or P-RPA-S4/S8. At least 100 cells were counted between two independent experiments.</p

Essential ATR pathway proteins are excluded from the four-protein complex.

<p>Vero cells were transfected with ICP8, UL8, UL5, and UL52 and damaged with UV and allowed to recover for 1-RPA70 or Myc-TopBP1 were cotransfected with the viral proteins. To follow HA-Rad9 Vero cells stably expressing HA-Rad9 were transfected with the viral proteins prior to UV irradiation.</p

The four-protein complex localizes to sites of DNA damage.

<p>Vero cells were transfected with ICP8, UL8, UL5, and UL52 and BrdU was added at the time of transfection. Cells were treated with UV or HU at 24</p