C Girault
email: ciska.girault@gmail.com

N Priymenko

M Helsly

C Duranton

F Gaunet

Dog behaviours in veterinary consultations: Part I. Effect of the owner's presence or absence

Keywords: Dog Behaviour, Owner Presence, Owner Absence, Stress, Veterinary Consultation

Dog behaviours in veterinary consultations: Part I.

Effect of the owner's presence or absence

Introduction

Improving animal welfare during veterinary consultations is a key concern of veterinarians, researchers and owners alike. Dogs frequently experience stress in these situations [START_REF] Lind | Assessing stress in dogs during a visit to the veterinary clinic: Correlations between dog behavior in standardized tests and assessments by veterinary staff and owners[END_REF][START_REF] Edwards | Fearful fido: Investigating dog experience in the veterinary context in an effort to reduce distress[END_REF] which can be assessed by monitoring behaviour [START_REF] Beerda | Manifestations of chronic and acute stress in dogs[END_REF][START_REF] Maximino | Measuring anxiety in zebrafish: a critical review[END_REF][START_REF] Koolhaas | Stress revisited: a critical evaluation of the stress concept[END_REF], for example, when entering the veterinary practice [START_REF] Stanford | Behavior of dogs entering a veterinary clinic[END_REF][START_REF] Mariti | Guardians' perceptions of dogs' welfare and behaviors related to visiting the veterinary clinic[END_REF], during time spent in the waiting room [START_REF] Mariti | The assessment of dog welfare in the waiting room of a veterinary clinic[END_REF][START_REF] Csoltova | Behavioral and physiological reactions in dogs to a veterinary examination: Owner-dog interactions improve canine well-being[END_REF][START_REF] Mariti | Guardians' perceptions of dogs' welfare and behaviors related to visiting the veterinary clinic[END_REF], and during the examination itself [START_REF] Döring | Fear-related behaviour of dogs in veterinary practice[END_REF][START_REF] Mariti | Guardians' perceptions of dogs' welfare and behaviors related to visiting the veterinary clinic[END_REF]. In a study by [START_REF] Döring | Fear-related behaviour of dogs in veterinary practice[END_REF], 80% of dogs showed stress-related behaviours on the examination table: 56.3% panted, 61.5% shivered, and 71.9% displayed avoidance behaviour. [START_REF] Glardon | Analyse du comportement des chiens et des chats pendant l'examen physique en cabinet vétérinaire[END_REF] found that around 25% of dogs could not be handled during the examination. Studies also report physiological signs of stress, such as increased plasma cortisol levels, pulse rates, and blood pressure [START_REF] Kallet | Comparison of blood pressure measurements obtained in dogs by use of indirect oscillometry in a veterinary clinic versus at home[END_REF][START_REF] Vonderen | Influence of veterinary care on the urinary corticoid: creatinine ratio in dogs[END_REF].

Chronic stress can cause impaired welfare which has negative effects on health, leading to reduced immune system performance [START_REF] Herbert | Stress and immunity in humans: a meta-analytic review[END_REF][START_REF] Stowell | Perceived stress and cellular immunity: When coping counts[END_REF][START_REF] Gimsa | Psychosocial stress and immunity -what can we learn from pig studies[END_REF], increased tumours [START_REF] Riley | Mouse mammary tumors: alteration of incidence as apparent function of stress[END_REF][START_REF] Dai | Chronic stress promotes cancer development[END_REF], and premature aging [START_REF] Epel | Accelerated telomere shortening in response to life stress[END_REF]. Acute stress, as expected in veterinary consultations, can lead to dysregulation of the stress system if it is extremely intense or recurrent [START_REF] Chrapusta | Effects of single and repeated footshock on dopamine release and metabolism in the brains of Fischer rats[END_REF][START_REF] De Kloet | Stress and the brain: from adaptation to disease[END_REF][START_REF] Vaessen | The dopaminergic response to acute stress in health and psychopathology: a systematic review[END_REF]. Stress also modifies the behaviour of dogs and may increase aggression. When faced with a threatening situation, dogs tend to react in three different ways: freeze; fight; and/or flight [START_REF] Bracha | Freeze, flight, fight, fright, faint: Adaptationist perspectives on the acute stress response spectrum[END_REF]. Aggression is dangerous for veterinarians and owners: in Australia in 2006, 48% of veterinarians declared that they had been bitten by a dog at work between one and five times in the previous 12 months [START_REF] Fritschi | Injury in Australian veterinarians[END_REF].

The ways in which dogs tend to react depend on their temperament [START_REF] Goodloe | Companion dog temperament traits[END_REF][START_REF] Serpell | Development and validation of a novel method for evaluating behavior and temperament in guide dogs[END_REF][START_REF] Svartberg | Shyness-boldness predicts performance in working dogs[END_REF][START_REF] Bray | Effects of maternal investment, temperament, and cognition on guide dog success[END_REF]; coping style [START_REF] Koolhaas | Coping styles in animals: current status in behavior and stress-physiology[END_REF][START_REF] Horváth | Three different coping styles in police dogs exposed to a short-term challenge[END_REF][START_REF] Diverio | Dogs' coping styles and dog-handler relationships influence avalanche search team performance[END_REF]; genetics [START_REF] Wilsson | The use of a behaviour test for selection of dogs for service and breeding. II. Heritability for tested parameters and effect of selection based on service dog characteristics[END_REF][START_REF] Saetre | The genetic contribution to canine personality[END_REF][START_REF] Meyer | Estimation of genetic parameters for behavior based on results of German Shepherd Dogs in Switzerland[END_REF][START_REF] Arvelius | Genetic analysis of a temperament test as a tool to select against everyday life fearfulness in Rough Collie[END_REF][START_REF] Persson | Human-directed social behaviour in dogs shows significant heritability[END_REF]; and previous experiences [START_REF] Seligman | Alleviation of learned helplessness in the dog[END_REF][START_REF] Döring | Fear-related behaviour of dogs in veterinary practice[END_REF][START_REF] Douglas | Environmental enrichment induces optimistic cognitive biases in pigs[END_REF]. According to [START_REF] Döring | Fear-related behaviour of dogs in veterinary practice[END_REF], even one past aversive experience increases stressrelated behaviour in dogs, thus modifying their behaviour at future visits to the veterinarian.

Many factors can be stressful for dogs in a veterinary practice [START_REF] Edwards | Fearful fido: Investigating dog experience in the veterinary context in an effort to reduce distress[END_REF]: transportation between home and the practice [START_REF] Beerda | Manifestations of chronic and acute stress in dogs[END_REF]; the novelty of the place [START_REF] Beerda | Manifestations of chronic and acute stress in dogs[END_REF]; the "white coat effect" [START_REF] Kallet | Comparison of blood pressure measurements obtained in dogs by use of indirect oscillometry in a veterinary clinic versus at home[END_REF][START_REF] Belew | Evaluation of the white-coat effect in cats[END_REF]; the presence of new people and animals [START_REF] Scotney | Environmental enrichment in veterinary practice[END_REF]; and unusual sounds and bustle [START_REF] Beerda | Manifestations of chronic and acute stress in dogs[END_REF][START_REF] Wells | The influence of auditory stimulation on the behaviour of dogs housed in a rescue shelter[END_REF]. Even smells such as those released by stressed people and animals can be stressful for dogs [START_REF] Graham | The influence of olfactory stimulation on the behaviour of dogs housed in a rescue shelter[END_REF][START_REF] Siniscalchi | Sniffing with the right nostril: lateralization of response to odour stimuli by dogs[END_REF][START_REF] Siniscalchi | The dog nose "KNOWS" fear: Asymmetric nostril use during sniffing at canine and human emotional stimuli[END_REF]. In addition, dogs can be fearful when entering a veterinary practice due to previous experiences [START_REF] Döring | Fear-related behaviour of dogs in veterinary practice[END_REF][START_REF] Ziv | The effects of using aversive training methods in dogs-A review[END_REF]. Veterinarians may also use gestures or postures that are stressful for dogs [START_REF] Mariti | Guardians' perceptions of dogs' welfare and behaviors related to visiting the veterinary clinic[END_REF][START_REF] Edwards | Fearful fido: Investigating dog experience in the veterinary context in an effort to reduce distress[END_REF], such as bending over them [START_REF] Vas | A friend or an enemy? Dogs' reaction to an unfamiliar person showing behavioural cues of threat and friendliness at different times[END_REF][START_REF] Győri | Friend or foe: Context dependent sensitivity to human behaviour in dogs[END_REF][START_REF] Mcgreevy | An overview of the dog-human dyad and ethograms within it[END_REF]; touching them [START_REF] Payne | Evidence of horsemanship and dogmanship and their application in veterinary contexts[END_REF]; placing them on the examination table [START_REF] Döring | Fear-related behaviour of dogs in veterinary practice[END_REF]; restraining them by force [START_REF] Beerda | Manifestations of chronic and acute stress in dogs[END_REF]; holding their collar or closing their mouth [START_REF] Kuhne | Emotions in dogs being petted by a familiar or unfamiliar person: Validating behavioural indicators of emotional states using heart rate variability[END_REF]; looking at them directly in the eyes [START_REF] Győri | Friend or foe: Context dependent sensitivity to human behaviour in dogs[END_REF]; or bringing their face close to the dog's head [START_REF] Rezac | Human behavior preceding dog bites to the face[END_REF]. As a result, veterinary practices may be fearful places for dogs.

Other stressful factors originate from the owner [START_REF] Lind | Assessing stress in dogs during a visit to the veterinary clinic: Correlations between dog behavior in standardized tests and assessments by veterinary staff and owners[END_REF]. Studies focused on dogowner attachment have shown that dogs can behave differently depending on whether their owner is present or absent [START_REF] Topál | Attachment behavior in dogs (Canis familiaris): a new application of Ainsworth's (1969) Strange Situation Test[END_REF]. In particular, when dogs are left in a novel place without a familiar caregiver, they show higher activity [START_REF] Tuber | Behavioral and glucocorticoid responses of adult domestic dogs (Canis familiaris) to companionship and social separation[END_REF]; higher glucocorticoid levels [START_REF] Tuber | Behavioral and glucocorticoid responses of adult domestic dogs (Canis familiaris) to companionship and social separation[END_REF][START_REF] Palestrini | Heart rate and behavioural responses of dogs in the Ainsworth's Strange Situation: A pilot study[END_REF]; higher heart rate levels [START_REF] Palestrini | Heart rate and behavioural responses of dogs in the Ainsworth's Strange Situation: A pilot study[END_REF]; and higher anxiety [START_REF] Prato-Previde | Is the dog-human relationship an attachment bond? An observational study using Ainsworth's strange situation[END_REF][START_REF] Palestrini | Heart rate and behavioural responses of dogs in the Ainsworth's Strange Situation: A pilot study[END_REF]Parthasrathy and Crowell-Davis, 2006), even if an unknown person is present (Parthasrathy and Crowell-Davis, 2006). Miklosi et al. (2003) showed that dogs looked at their owner when facing an unsolvable task, and [START_REF] Kerepesi | Dogs and their human companions: the effect of familiarity on dog-human interactions[END_REF] demonstrated that dogs moved closer to their owner but not towards other individuals, even familiar ones, in situations provoking anxiety or fear. Other studies have shown that dogs react in the same way as their owner when confronted with a strange object [START_REF] Merola | Social referencing in dog-owner dyads?[END_REF] or an unknown person [START_REF] Duranton | When facing an unfamiliar person, pet dogs present social referencing based on their owners' direction of movement alone[END_REF].

Owners can thus potentially modify the behaviour of their dog in a veterinary consultation. This hypothesis has been observed anecdotally by veterinarians in the field. Some believe that the very presence of owners can calm down their dog, whereas others maintain that dogs are easier to handle in the absence of their owner. Only a study by [START_REF] Stellato | Evaluation of associations between owner presence and indicators of fear in dogs during routine veterinary examinations[END_REF] focused on this question, and showed positive effects of owner presence on behavioural and physiological measures of fear in dogs during veterinary consultation.

In Part I of our study, we examined the effect of owner presence or absence on the behaviours of dogs during a veterinary consultation, regardless of the owners' actions. Based on the abovementioned literature, we expected the owner's presence to decrease stress-related behaviours of dogs during the veterinary examination and hence facilitate their handling by the veterinarian. In Part II of the study (see Helsly et al., under review), using the raw data from consultations with the owner present, we explored whether owners' actions affect dog behaviours by observing dog-owner dyads.

Materials and methods

Participants

A total of 32 dog-owner dyads were recruited. Four dogs were excluded due to the owner's withdrawal between the two appointments, another one due to aggressive behaviour and two due to protocol deviation. The final study included 25 owner-dog dyads. All participants were volunteers and were recruited via social media. The owners (all women) and dogs meeting the following criteria were selected: adult dogs between 12 months and 10 years old and unfamiliar with the researchers, measuring less than 70 cm at the withers in order to be easily lifted and examined on the table, in good general health, and nonaggressive towards humans to avoid the use of a muzzle that could modify their behaviour; owners not working as a veterinarian, assistant, or veterinary student.

Participant demographics are shown in Table 1.

Experimental procedure

The study protocol was approved by the ethical committee SSA (Science et Santé Animale) n°115 (SSA_2018_008) on 18 July 2018. The experiment took place in an examination room at the National Veterinary School of Toulouse (ENVT), France (Fig. 1).

All dogs underwent two videotaped veterinary consultations at an interval of five to seven weeks (Table 1), one in the presence of their owner and the other in their absence. Prior to each consultation, all owners were told how to behave with their dogs. The veterinary consultations were all carried out by the same two researchers: two female veterinary students, one in the role of the veterinarian (C. G.) and the other in the role of the assistant (M. H.). During the consultations, the researchers conducted the veterinary examination using as neutral a disposition as possible: the researchers stayed still and did not talk to the dogs, pet them, make eye contact with them, or punish them. The veterinary consultation was divided into three main phases: phase 1: exploration; phase 2: examination; and phase 3: greeting. We consider a "consultation" to include all events between the times when the dog entered and exited the examination room, whereas an "examination" includes only the phase where the dog was examined (see "Standardised protocol for the veterinary consultations" below). Dogs were randomly distributed into two groups using AB/BA crossover design: in the O/NoO group (n = 12), the owner was present for the first veterinary consultation and absent for the second, and vice versa in the NoO/O group (n = 13). Consultations were arranged by appointment according to the availability of owners.

Raw data collected for this study was also used in Part II (Helsly et al., under review).

Part II only focuses on data collected during consultations with the owner present. Four additional dogs were included in Part II but not in Part I because the dyads did not attend the second consultation with the owner absent.

Data collection and analysis

Consultations were videotaped from when the dog entered the consultation room until the end of the greeting phase. We used two cameras (a Canon Legria HF S21 and a Panasonic HC-WX970 with a Panasonic vW-W4907H wide-viewing angle) situated in two corners of the room and facing the examination table. The recorded videos from the two cameras were synchronised and assembled into a single video (Fig. 1).

The variables we studied, described below, differed depending on the phase of the consultation. A summary of all studied variables and their availability for the three phases is described in Appendix B: Supplementary material. Dog behaviour was analysed using the Solomon Coder beta 17.03.22 program1 and the behavioural repertoire adapted from the literature [START_REF] Beerda | Behavioural, saliva cortisol and heart rate responses to different types of stimuli in dogs[END_REF][START_REF] Mills | A triple blind placebo-controlled investigation into the assessment of the effect of Dog Appeasing Pheromone (DAP) on anxiety related behaviour of problem dogs in the veterinary clinic[END_REF][START_REF] Deldalle | Effects of 2 training methods on stress-related behaviors of the dog (Canis familiaris) and on the dog-owner relationship[END_REF][START_REF] Csoltova | Behavioral and physiological reactions in dogs to a veterinary examination: Owner-dog interactions improve canine well-being[END_REF]; see Table 2). Stress-related behaviours are described in Table 2. As the phase durations were variable, the durations of behaviours were converted into a time percentage (behaviour duration/phase duration) for all behaviours. Behavioural indices were further computed as detailed in Table 3. The Emotional State of the dog is a subjective rate assessing stress, scored by using a three-point scale defined as follows: relaxed, aroused, anxious, see definitions in Table 4. The dogs' apparent comfort in entering the room and the greeting intensity of the dogs towards their owner and of owners towards dogs were evaluated using a five-point scale defined in Table 5. The level of physical restraint was scored using a three-point scale defined as follows: low, medium, high. All definitions are given in Table 6. The success and difficulty of the manipulations were evaluated using a scale defined in Table 7.

Standardised protocol for the veterinary consultations

Owner: present condition Exploration phase (phase 1): The owner entered the room with her dog on a leash and sat on a chair (Fig. 2). The leash was dropped, and the dog explored the room freely for 2 minutes and 30 seconds. Neither the owner nor the researcher spontaneously interacted with the dog; the owner could nevertheless respond to the dog's requests (physical, verbal and visual interactions were allowed). The researchers asked questions similar to those asked in a standard veterinary consultation.

Examination phase (phase 2): The dog was put on the examination table by the researchers; the owner stood one metre away from the table in a designated spot, facing the dog. The veterinarian began a standardised veterinary examination following this predefined sequence: examination of eyes; ears; teeth; mouth mucosa; palpation of lymph nodes; examination of scapular skin fold; abdominal palpation; heart and lung auscultation; measurement of rectal temperature; and paw palpation. The assistant held the dog using a standardised restraint (Fig. 3): one hand on the chest and another on the base of the tail, using the minimal necessary strength to keep the dog sitting or standing on the table. If a particular manipulation failed because the dog was not cooperative for five seconds (for example, the dog struggled, resisted, or tried to escape), the veterinarian did not repeat the manipulation and continued with the next one. During the examination phase, the owner was only allowed to talk to or look at her dog (verbal and visual interactions were allowed). If the dog showed any sign of aggression toward the researchers or behaviour endangering them (for example, growling, showing teeth, or trying to bite), the procedure was terminated, and the dog was excluded from the study.

Greeting phase (phase 3): This phase started when the dog was on the floor and the owner in the room and lasted exactly 20 second. The dog was indeed taken down from the table by the researchers, and the leash was given back to the owner. The researchers then stepped aside and filled in forms in order to allow the owner and the dog to interact freely (physical, verbal and visual interactions were allowed).

Owner: absent condition

The procedure was the same as with 'Owner: present condition', except that the owner waited outside the room during the exploration and examination phases and only entered the room for the greeting phase. During the exploration phase, the owner accompanied the dog to the open door, gave the leash to the assistant and was free to interact with the dog in order to encourage the dog to enter the room. The dog explored the room freely for 2 minutes and 30 seconds while still on the leash. During this phase, the researchers did not interact with the dog and spoke in a neutral tone. The procedure of the examination phase was the same as described in 'Owner: present condition'. During the greeting phase, the dog was taken down from the table by the researchers. Then, the assistant went outside to fetch the owner, and the leash was given back to the owner when she came back in the room without specific instructions. The end of this phase was the same as described in 'Owner: present condition' after having given the leash back to the owner.

Interobserver agreement

Three assessors participated in the video analysis. The two researchers coded all the behaviours in all the videos: half of the behaviours were coded by one researcher and the other half by the second researcher. To assess the reproducibility of the behavioural analysis, a third assessor who was unaware of the study hypotheses and aims coded 30% of the behaviours in a random subset of 30% of the videos. Considering that a concordance, and not only a correlation, was needed to assess the interobserver reproducibility, Lin's concordance correlation test was used [START_REF] Lawrence | A concordance correlation coefficient to evaluate reproducibility[END_REF][START_REF] Barnhart | Overall concordance correlation coefficient for evaluating agreement among multiple observers[END_REF][START_REF] Barnhart | An overview on assessing agreement with continuous measurements[END_REF].

Interobserver agreement between the two assessors was determined by calculating 𝜌𝐶 values and rated according to [START_REF] Landis | The measurement of observer agreement for categorical data[END_REF] (𝜌𝐶 = 0 -0.2: slight agreement, 𝜌𝐶 = 0.21 -0.4: fair agreement, 𝜌𝐶 =0.41 -0.60: moderate agreement, 𝜌𝐶 = 0.61 -0.8: substantial agreement, 𝜌𝐶 > 0.81: excellent agreement). Lin's concordance correlation coefficients were excellent (𝜌𝐶>0.98) for whining, tail between the legs, and moving and gazing at the owner, and substantial for contact with the assistant (𝜌𝐶=0.71) and sniffing (𝜌𝐶=0.69). Physical restraint was evaluated by the assistant, the success and difficulty of the manipulations were assessed by the veterinarian, and the other scores were rated by the three assessors.

Statistical analysis

The exploration, examination, and greeting phases were analysed separately.

Comparisons were carried out for each behaviour or behavioural index with the owner absent or present conditions. A paired Student's t test was performed for the O/NoO and NoO/O groups using R software2 .

Results

On average, the exploration phase lasted 155.43 ± 9.54 seconds and the examination phase 142.53 ± 16.4 seconds. The greeting phase, standardised in the study, lasted 20 seconds.

The sample is described in Appendix A: Supplementary material.

Exploration phase (phase 1)

Entering the room Dogs appeared to enter the room more readily when the owner was present rather than absent, and the difference was significant (P<0.05; see Table 8, Video 1).

Emotional state

Dogs had a significantly lower score for emotional state during the exploration phase when the owner was present rather than absent (P< 0.05; see Table 8, Video 2).

Examination phase (phase 2)

Stress-related behaviour No differences were observed regarding the stress-related behaviours or Total Stress (defined in Table 3) during the examination phase in the presence or absence of the owners (P>0.05).

Behaviour towards the veterinarian and assistant

The dogs' contact with the veterinarian and/or assistant lasted significantly longer during the examination phase if the owner was present rather than absent (P< 0.05; Table 9).

Behaviour (gaze) towards the owner and/or door During the examination phase, dogs looked straight ahead toward the assigned place of the owner significantly more if the owner was present rather than absent. (P< 0.05; Table 9, Video 3). Furthermore, dogs looked at the door significantly less when their owner was present rather than absent (P< 0.05; Video 3).

Dogs' handling

No difference was observed regarding the restraint and the success and difficulty scores of manipulations in the presence or absence of the owners (P> 0.05).

Greeting phase (phase 3)

Intensity of greeting

When the dog and owner were reunited after the examination phase, dogs greeted their owners for a significantly shorter period of time (P< 0.05) and showed a lower reunion score (P< 0.05) if the owner was present rather than absent during the examination phase (Video 4).

Nevertheless, the scores of owner behaviour towards their dog were similar regardless of whether the owner was present or not in the previous phase (P> 0.05).

Door-directed gaze

Finally, dogs gazed at the door slightly less often when the owner was present rather than absent (P< 0.1; Table 10,Video 5).

Discussion

This experiment aimed at investigating whether the presence or absence of a dog's owner influences dogs' behaviours in veterinary consultations. Overall results suggest that allowing the owner to stay in the room during veterinary consultations is a better option for dogs' welfare: During the exploration phase, in the presence of their owner, dogs were more willing to enter the consultation room and appeared more relaxed. During the examination phase, dogs looked in direction of their owner in both condition: when their owner was present (standing in front of the dog), dogs looked straight ahead more often and at the door less often than in the absence of their owner. When the owner was absent (she had left the room through the door), the dogs looked at the door more often and less straight ahead than in the presence of the owner. Physical contacts engaged by dogs with the researchers lasted longer when their owners were present rather than absent. Our results also indicate that the presence or absence of the owner had no significant effect on the stress-related behaviour of the dog or the veterinarian's ability to handle the animal during the examination phase.

Finally, during the greeting phase, our results showed that the dogs exhibited less greeting behaviour and tended to look at the door less often if the owner had never left the room than if the owner had been absent. Only a study by [START_REF] Stellato | Evaluation of associations between owner presence and indicators of fear in dogs during routine veterinary examinations[END_REF] focused on the effect of the presence or absence of the owner on dog behaviour in veterinary practices, comparing two standardized veterinary consultation with owner present or absent. They showed that dogs had a lower rate of vocalisation, higher rate of yawning and lower mean axillary temperature in the presence of the owner. Thus, they encouraged owner to remain with their dog during routine veterinary examination. Note that we did not find such a difference for vocalisation and yawning, but we did find significant differences for other parameters. A study by [START_REF] Csoltova | Behavioral and physiological reactions in dogs to a veterinary examination: Owner-dog interactions improve canine well-being[END_REF] focused on the active or passive support of the owner during veterinary examinations. The authors compared behavioural and physiological measures of dogs during a veterinary examination in two conditions: the active presence of the owner (talking and petting), and the passive presence of the owner (sitting quietly next to the examination table). They found heart rate and internal temperature variations showing a beneficial effect of dog-owner interaction on the dogs' well-being but no significant behavioural changes. As mentioned in the introduction, owners can have various effects on the behaviour of their dog. Studies have shown that dogs adjust their behaviour to their owner's overall emotional body posture [START_REF] Vas | A friend or an enemy? Dogs' reaction to an unfamiliar person showing behavioural cues of threat and friendliness at different times[END_REF]Custance and Meyer 2012), to the owner's behaviours [START_REF] Millot | Olfactory and visual cues in the interaction systems between dogs and children[END_REF][START_REF] Merola | Social referencing in dog-owner dyads?[END_REF][START_REF] Horn | Domestic dogs (Canis familiaris) flexibly adjust their human-directed behavior to the actions of their human partners in a problem situation[END_REF][START_REF] Duranton | Canis sensitivus: Affiliation and dogs' sensitivity to others' behavior as the basis for synchronization with humans?[END_REF]; and to the owner's facial expressions [START_REF] Deputte | Do dogs understand human facial expressions[END_REF]. Other studies detailed in Part II have shown that physical contact does not have the same effect on dog behaviour as talking (Helsly et al., under review). The effect of the presence of the owner is thus hard to accurately predict, as it can depend on the owner's behaviour and mood.

In the present study, several elements can explain the absence of any significant differences in stress-related behaviours during the examination phase. First, physical contact between the owners and dogs was not allowed during the examination phase to control parameters not being studied. Yet the Csoltova study showed a beneficial effect of contact during veterinary examinations, and other studies detailed in Part II have shown that talking and physical contact do not have the same effect (Helsly et al., under review). Another element in our study involved the subjective evaluation of stress (emotional state). This evaluation showed higher levels of stress during the exploration phase (phase 1) when the owner was absent than when the owner was present. In contrast, during the examination phase (phase 2), no difference was found in the subjective evaluation by the judges (emotional state) or the video analyses (stress-related behaviours). A study by [START_REF] Firnkes | Appeasement signals used by dogs during dog-human communication[END_REF] demonstrated that some stress-related behaviours ("licking of lips" and "looking away") decrease even though the stressor intensity increases. We thus postulate that the dogs reached a high threshold of stress in the 'Owner: absent condition' that prevented them from displaying additional stress-related behaviours. Potentially, too many stressors may hide the display of stress-related behaviour at some point. Alternatively, the absence of any difference may show that dogs are not actually more stressed when the owner is absent during a veterinary examination. In the examination phase, dogs engaged in more physical contact with the researchers when their owner was present than when their owner was absent. This engagement with researchers suggests that in an interventionist situation, if we had imposed physical contact on the dogs to keep them on the table, the presence of the owner would act as a social reference for the dog towards the veterinarian and assistant. That is, the presence of the owner could help dogs to handle this difficulty. Even if no significant differences were shown in terms of stress-related behaviours during examination phase, other results suggest that the owner does play a role. For instance, dogs looked in the direction of their owner both in the absence and in the presence of the owner. This also highlights the importance of the owner's presence to help dogs to deal with the situation (e.g. social referencing in [START_REF] Merola | Social referencing in dog-owner dyads?[END_REF][START_REF] Duranton | When facing an unfamiliar person, pet dogs present social referencing based on their owners' direction of movement alone[END_REF]Salamon et al., 2020;and also Part II (Helsly et al., under review, for additional information). Furthermore, greeting behaviours during the two reunion conditions differed. Once dogs were placed back on the floor and the leash was given back to the owner, dogs showed more greetings when the owner came back than when the owner had never left the room. This is in accordance with studies showing that greeting behaviour is more marked when dogs spend a stressful time without their owner [START_REF] Konok | The behavior of the domestic dog (Canis familiaris) during separation from and reunion with the owner: A questionnaire and an experimental study[END_REF][START_REF] Rehn | The effect of time left alone at home on dog welfare[END_REF]. Dogs tended to look at the door more often during the greeting phase when the owner had come back than when the owner had never left. Dogs are known to look at a desirable object [START_REF] Gaunet | How do guide dogs of blind owners and pet dogs of sighted owners (Canis familiaris) ask their owners for food?[END_REF][START_REF] Gaunet | How do guide dogs and pet dogs (Canis familiaris) ask their owners for their toy and for playing?[END_REF][START_REF] Gaunet | Functionally referential and intentional communication in the domestic dog: effects of spatial and social contexts[END_REF], and thus they may have been more motivated to leave the room when their owner was absent, since the time they spent in the room was more stressful, in accordance with the emotional state evaluation during the exploration phase. Finally, the presence of the owner had no detrimental effect but some beneficial effects on dogs. Dogs were neither more stressed nor more difficult to handle during examination phase and appeared less stressed during the exploration phase. In conclusion, the presence of owners appears to be more beneficial than their absence during veterinary examinations.

Our sample size was small, the veterinary examination was minimally invasive; puppy, old dogs and dogs measuring more than 70cm were not included and aggressive dogs were excluded from our study for safety reasons. [START_REF] Glardon | Analyse du comportement des chiens et des chats pendant l'examen physique en cabinet vétérinaire[END_REF] estimated that 16% of dogs display aggressive behaviour during veterinary examinations. If these dogs had been included in the study, the results may have been different, since aggressive dogs are less tolerant of manipulations. Furthermore, the manipulations used in this study were standard but minimally invasive. Dogs' tolerance of manipulation would have differed if we had employed more invasive or painful procedures [START_REF] Holton | Development of a behaviourbased scale to measure acute pain in dogs[END_REF][START_REF] Hansen | Assessment of pain in dogs: veterinary clinical studies[END_REF], and the presence or absence of the owner could have had a different effect in these cases. Owner and researcher behaviour could have differed from a real-life scenario, as owners were not allowed to touch their dogs during the examination phase, and researchers kept a neutral disposition and did not initiate interaction toward dogs or respond to their requests for attention. In addition, the dogs involved in the current study were healthy and did not need any invasive manipulation.

Owners may be more stressed in a real veterinary examination and therefore have a different effect on dog behaviour than in the controlled experimental conditions. Furthermore, while it has been shown that dogs react differently to men and women, we were not able to study the gender effects of owners and researchers on the dogs' behaviours, as both the owners and researchers were all women [START_REF] Hennessy | Influence of male and female petters on plasma cortisol and behaviour: can human interaction reduce the stress of dogs in a public animal shelter[END_REF][START_REF] Wells | Male and female dogs respond differently to men and women[END_REF][START_REF] Deputte | Do dogs understand human facial expressions[END_REF]. In the present study, we focused on canine behaviour, although physiological measures such as plasma or salivary cortisol, heart rate, and infrared thermography can also be used. These measures could provide additional information about the stress experienced by dogs during veterinary consultations and help highlight the differences that cannot be observed by behaviour analysis alone, such as behaviours with high interindividual variation [START_REF] Firnkes | Appeasement signals used by dogs during dog-human communication[END_REF]. We thus encourage further studies to focus on neurochemical and physiological differences in conjunction with behavioural differences, to use a larger sample of dogs and to study gender effect.

Conclusions

In summary, despite some limitations (e.g. no male owners, no dog above 70cm, no old dogs nor puppies, no aggressive dogs, 25 dogs by condition, minimally invasive examinations, restricted owner-dog interactions), the current study shows that the presence of the owner in veterinary consultations (constituted by exploration, examination and greeting phases) may help to reduce the stress-related behaviours of dogs before veterinary examinations. During examination phase, dogs looked at their owners and appear to seek social information from owner, whereas when their owners were absent, they looked for them.

Behaviours like greeting their owners and door-related behaviours suggest that even if no significant differences were shown for stress-related behaviours in terms of the absence or presence of owners, dogs were less stressed during examination phase when their owner was present. Given these results, it seems more appropriate to allow owners to attend veterinary examinations with their dog, as only positive effects were observed in terms of the dogs' behaviour and well-being, in spite of the previously mentioned limitations.

Conflict of interest statement

None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper. Tail wagging Tail wagged below the spine but was not between the legs Tail between legs a Tail was between the rear limbs Tail low Tail was below the spine but was not wagging or between legs Tail high Tail was above the spine, whether wagging or not a Stress-related behaviour 772

Table 3

Behavioural indices calculated using several behaviours from Table 2. intentionally touched the assistant; Gaze V, The dog gazed with its head oriented towards the veterinarian; Gaze A, The dog gazed with its head oriented towards the assistant; Gaze VA,

The dog gazed with its head oriented towards the veterinarian and the assistant; Gaze O, The dog gazed with its head oriented towards the owner; Gaze Ad, The dog gazed with its head oriented straight ahead when on the table; Contact O, The dog intentionally touched the owner; Situation O, Half of the dog's body (head and chest) was situated less than 50 cm from the owner.

Table 4

Rated emotional states of dogs during exploration, examination, and greeting phases, their definition, and their score (from [START_REF] Mills | A triple blind placebo-controlled investigation into the assessment of the effect of Dog Appeasing Pheromone (DAP) on anxiety related behaviour of problem dogs in the veterinary clinic[END_REF]. 3). Results are expressed as the standard error of the mean. Non-significant results are not presented. Behav. Tow. Owner, Behaviour Towards Owner, Sum of percentages of time spent gazing at, having contact with, and seeking proximity to the owner (also see Table 3); Reunion / Dog, Rated greeting intensity by dog towards owner (1 = indifferent, 5 = very happy, jumps on the owner, seeks contact; also see Table 5); % time, Percentage of time (behaviour duration/phase duration); Gaze -Door, Percentage of time spent gazing at the door. Results are expressed as the standard error of the mean. Non-significant results are not presented.

Figure legends

  stress-related behaviours Scratching a / Sniffing a / Shivering a / Shaking a The dog scratched itself / The dog sniffed the ground or straight ahead / The dog trembled / The dog shook Low postures a The dog's tail was lowered, its ears faced backwards, or its legs were bent; at least two of these postures were exhibited Mouth (exclusive behaviours) Yawning a / Panting a / Licking a The dog yawned / The dog panted / The dog licked its mouth Vocalisations (exclusive behaviours) Whining a / Barking a The dog whined / The dog barked Gaze (exclusive behaviours) Gaze at a person The dog gazed with its head oriented towards the owner (Gaze O), the veterinarian (Gaze V), the assistant (Gaze A), or the veterinarian and the assistant (Gaze VA) Gaze at an object or thing The dog gazed with its head oriented towards the door (Gaze D) or straight ahead when on the table (Gaze Ad) dog's body (head and chest) was situated less than 50 cm from the owner (Situation O) or from the veterinarian and the assistant (Situation VA) Situation / something Half of the dog's body (head and chest) was situated less than 1 m from the door (Situation D), or the dog was not in one of the previous locations (Situation E) Movement (exclusive behaviours) Move The dog moved its four limbs with less than 1 second between the movement of each limb Contact (exclusive behaviours) Contact The dog intentionally touched the owner (Contact O), the veterinarian (Contact V), or the assistant (Contact A) Tail (exclusive behaviours)

%

  , Time percentage (behaviour duration/phase duration); Yawning, The dog yawned; Panting, The dog panted; Scratching, The dog scratched itself; Low posture, The dog's tail was lowered, its ears faced backwards, or its legs were bent, at least two of these postures were exhibited; Shaking, The dog shook; Sniffing, The dog sniffed the ground or straight ahead; Whining, The dog whined; Barking, The dog barked; Licking, The dog licked its mouth; Contact V, The dog intentionally touched the veterinarian; Contact A, The dog Index

Factor

  Rated scores of dogs when entering the examination room (exploration phase) and reuniting with the owner after the veterinary examination (greeting phase), along with the name of the factor and the signification of the score.

  Sum of percentages of time spent in contact with veterinarian (V) and/or assistant (A); Gaze O+Ad, Sum of percentages of time spent gazing at owner (O) when present or straight ahead (Ad) when absent; % time, Percentage of time (behaviour duration/phase duration; also see Table

Fig. 1 .

 1 Fig. 1. Image of the experimental room, with the two videos assembled in a single image.

Figure 1 Figure 2

 12 Figure 1

Table 1 765

 1 Characteristics of owners and dogs.

	766
	Dog Experimental	Interval between	Age	Sex	Breed	Age of
		group	visits (weeks)	(years)			owner
	1	NoO/O	5	3	FN	Mixed Shepherd	25-40
	2	NoO/O	5	1	MN	Mixed Retriever	25-40
	3	NoO/O	6	4	FN	Cavalier King Charles	41-60
	4	O/NoO	5	2.5	FN	Beauceron	25-40
	5	O/NoO	7	3	FN	Schapendoes	<25
	6	O/NoO	5	5	FE	Mixed Terrier	25-40
	7	O/NoO	6	5	FN	Mixed Terrier	>60 a
	8	O/NoO	6	5	ME	Mixed Terrier	>60 a
	9	O/NoO	6	7.5	MN	Labrador	41-60
	10	O/NoO	6	2.5	ME	Boxer	25-40
	11	NoO/O	5	5	MN	Mixed Terrier	25-40 a
	12	O/NoO	5	6	MN	Mini Australian Shepherd	25-40 a
	13	NoO/O	7	2.5	MN	Whippet	25-40
	14	O/NoO	5	2	ME	Boxer	41-60
	15	NoO/O	6	2	MN	French Bulldog	41-60
	16	O/NoO	5	1.5	ME	German Shepherd	41-60
	17	O/NoO	7	7	FN	Spitz	25-40
	18	O/NoO	6	2	FE	Boxer	41-60
	19	NoO/O	7	4	MN	Whippet	>60
	20	NoO/O	6	3.5	FN	Cotton Tulear	25-40
	21	NoO/O	5	3.5	MN	White Swiss Shepherd	25-40

Table 2 770

 2 Recorded dog behaviours during the examination and greeting phases and their definitions.

Table 6

 6 Rated levels of dog restraint performed by the assistant during the examination phase, along with their definition and score.

	Restraint	Definition	Score
		The assistant did not need to use force to keep the dog in the
	Low		1
		right position. The dog was voluntarily almost immobile.
		The assistant needed to increase her restraint of the dog to keep it
	Medium		2
		in the same position. The dog was agitated/moved frequently.
		The assistant had to hold the dog firmly to keep it on the
	High	examination table or help the veterinarian perform the clinical	3
		examination. The dog tried to escape.

Table 8

 8 Emotional state of dogs in a veterinary practice with the presence or absence of the owner during the exploration phase. Results are expressed as the standard error of the mean.Entering room, Rated apparent comfort in entering the room (1 = Dog has to be drawn to enter, 5 = Dog enters voluntarily, pulls on leash; also see Table5); Emotional State, Rated emotional states of dogs (1 = Relaxed: No or low frequency of movement, with no visual evidence of tension in the body, 2 = Aroused: Tense, with high frequency of movement, but no visual evidence of anxious behaviours, 3 = Tense, with licking, yawning, crying, agitation or observable fearful posture; also see Table4).

		Owner: present	Owner: absent			1-ß
	Behaviours (units)			t	P
		condition	condition			(%)
	Entering room (score)	3.24 ± 0.76	2.72 ± 1.27	-2.7	0.012	100
	Emotional State (score)	1.76 ± 0.63	2.22 ± 0.69	3.682 0.001	69.2

Table 9

 9 Behaviour of dogs in a veterinary practice with the presence or absence of the owner during the examination phase.

		Owner: present	Owner: absent
	Behaviours (units)	t	P	1-ß (%)
		condition	condition
	Behaviour towards veterinarian and/or assistant
	Contact -	Duration (%			12.2
		2.33 ± 4.91	1.34 ± 3.86	-2.187	0.039
	V+A	time)
	Behaviour towards owner and/or door
		Duration (%			96.9
	Gaze -Door	12.46 ± 9.56	25.15 ± 13.52	3.813	0.0008
		time)
		Duration (%
	Gaze O+Ad	37.32 ± 19.33	21.64 ± 12.17
		time)

Table 10

 10 Behaviour of dogs in a consultation room with the presence or absence of the owner during the greeting phase.

			Owner: present	Owner: absent
	Behaviours (units)			t		P	1-ß (%)
			condition	condition
	Behav. Tow.	Duration (%					83.3
	Owner	time)	129.24 ± 50.70	170.76 ± 49.70 3.455	0.002
	Reunion / Dog	Score	3.11 ± 1.29	4.24 ± 0.79	5.106	0.00003	96.2
		Duration (%					25.4
	Gaze -Door		8.12 ± 8.95	12.32 ± 13.50	1.818	0.081
		time)

See : Solomon Coder, András Péter, https://solomoncoder.com (accessed 10 September

2020).

See: The R Project for Statistical Computing. http://www.R-project.org. (accessed 24 May 2017)

Acknowledgements

We are grateful to Elodie Losserand who helped us during the study. We thank all the dog owners who voluntarily participated in this study. We are also grateful to Caniplex, Bagat-en-Quercy, France, for providing thank-you gifts to all the volunteers. Preliminary results were presented during a confidential veterinary thesis defence at the Ecole Nationale Vétérinaire de Toulouse on 13 December 2017.

Supplementary material

Supplementary data associated with this article can be found, in the online version, at doi: …

References Table 7

Rated manipulations performed by the veterinarian during the examination phase and the meaning of their success and difficulty score.

Manipulation

Definition Score and Value Three owners participated with more than one dog.

Appendix B

Summary of all studied variables and their availability for the three phases.


Cyrille Sabot
email: cyrille.sabot@univ-rouen.fr

Péter Kele
email: kele.peter@ttk.hu

Novel Approaches in Biomolecule Labeling 1

7

Selective functionalization of biomolecules such as proteins, nucleic acids, lipids or 8 carbohydrates is a focus of persistent interest due to their widespread use ranging from 9 basic chemical biology research in order to gain insight into biological processes, to the 10 most promising biomedical applications, including the development of diagnostics, or 11 targeted therapies. Although specific functionalization can be achieved via genetic engi-12 neering techniques such as fusion of fluorescent proteins or self-labelling tags (SNAP-tag, 13 CLIP-tag, HaloTag), chemical conjugation alternatives overcome some of their limitations 14 while providing new exciting perspectives.

15 Chemical ligation can be divided into two distinct classes of reaction: a) bioconjuga-16 tion that is based on the direct modification of native biomolecules, such as amino acid 17 side-chains of proteins which are intrinsically nucleophilic (mainly Cys, Lys); and b) 18 bioorthogonal chemistry which requires the prior metabolic, or genetic installation of a 19 biologically inert functional group into a biomolecule or biosystem. This chemical reporter 20 is subsequently detected with a complementary reactive group bearing a fluorophore, or 21 an affinity handle. Bioorthogonal reactions can advantageously take place inside living 22 systems whereas bioconjugation reactions are mostly limited to in vitro applications.

The choice of a chemical conjugation strategy has mainly been driven by its ability 24 to proceed rapidly, chemoselectivity, and with high yield under physiological conditions 25 to meet requirements of biomolecules. However, with recent developments, many other 26 attractive features can be taken into account when selecting a reaction. For example, ul-27 trafluorogenic ligation tools have dramatically improved the signal-to-noise ratio, and al-28 lowed live cells labeling experiments under "no wash" conditions. Besides, photoactiva-29 ble conjugations enable effective identification of weak, transient biomolecular interac-30 tions, or provide an unprecedented level of spatiotemporal control of biological processes 31 [START_REF] Li | Photo-controllable bioorthogonal chemistry for spatiotemporal control of bio-targets in living 120 systems[END_REF]. As another illustration, sophisticated conjugation technologies or platforms with ex-32 ceptional modularity are also available for dual labeling of proteins [START_REF] Canovas | Site-Specific Dual Labeling of Proteins 122 on Cysteine Residues with Chlorotetrazines[END_REF] or for the engi-33 neering of antibody-drug conjugates [START_REF] Maruani | A plug-and-play approach to antibody-124 based therapeutics via a chemoselective dual click strategy[END_REF]. This Special Issue of Biomolecules devoted to 34 "Novel Approaches in Biomolecule Labeling" provides detailed studies illustrating po-35 tential of biocompatible ligation strategies through a variety of applications.

37

The site-specific introduction of a bioorthogonal reporter into biomolecules such as 38 proteins or antibodies in the context of targeted delivery, provides an anchoring site for 39 subsequent easy attachment of any cargo of interest such as pharmacophores. For exam-40 ple, Tsao et al. [START_REF] Tsao | Site-Specific Fluorogenic Protein Labelling Agent for Bioconjugation[END_REF] reported the fluorogenic site-specific incorporation of an alkyne func-41 tional group onto a protein of interest, namely maltose binding protein (MBP), fused to a 42 small helical peptide tag (dC10a tag) presenting two Cys residues capable of reacting spe-43 cifically with novel dimaleimide-based conjugating linkers. These linkers consist of a cou-44 marin platform functionalized at each side by an alkyne group for subsequent cargo load-45 ing, and two maleimide units ensuring both bioconjugation and quenching of the couma-46 rin's fluorescence [START_REF] Renault | Covalent Modification of Biomolecules through Maleimide-Based Labeling 128 Strategies[END_REF]. Accordingly, the conjugation of the linker to the protein can advan-47 tageously be monitored by fluorescence under "no wash" conditions. While in the latter example, the fluorescence emission was triggered by a bioconju-49 gation reaction involving the consecutive addition of two Cys residues, Németh et al. [START_REF] Németh | A Bioorthogonally Applicable, Fluorogenic, Large Stokes-Shift Probe for Intracellu-130 lar Super-Resolution Imaging of Proteins[END_REF] 50 reported the design of novel bioorthogonally activatable fluorogenic probes. Such probes, 51 also termed as bioorthogonal smart probes [START_REF] Shieh | Design strategies for bioorthogonal smart probes[END_REF], are particularly interesting for labeling 52 specific proteins in biological systems while minimizing background fluorescence from 53 unreacted probe. However, most notable examples of fluorescence turn-on probes are 54 UV/blue excitable, whose low emission wavelengths can interfere with the autofluores-55 cence of biological samples. This present study addresses the lack of robust fluorogenic 56 probes that emit light in the orange, red region by creating a fluorogenic dye possessing 57 a large Stokes-shift. This was achieved by designing a coumarin dye having an extended 58 π-electron system due to a vinylpyridinium moiety at C3 position, and a two-in-one 59 bioorthogonal and fluorescence quencher phenyltetrazine handle at position C4. Notice-60 ably, this probe was found to be suitable for stimulated emission depletion (STED) super-61 resolution microscopy.

62

In the context of medical diagnostic imaging, radiolabeling of biomolecules consti-63 tutes an important alternative to aforementioned optical imaging approaches, by provid-64 ing deeper tissue penetrations. Metallic radionucleides require the use of bifunctional che-65 lators, which consist of a metal sequestration unit and a conjugatable function for covalent 66 attachment to the biomolecules of interest. Different bioconjugation strategies have been 67 developed, whose choice is guided by the preservation of biomolecules activity, in vivo 68 behavior (thermodynamic, kinetic, stability…), and synthetic accessibility. While the chal-69 lenge of large molecule primarily lies in generating homogeneous bioconjugates, the dif-70 ficulty with small molecules is associated with the preservation of their biological activity. 71 In this context, Poret et al. [START_REF] Poret | Development of Novel (111)-In-Labelled DOTA Urotensin II Analogues for Targeting the UT Receptor 135 Overexpressed in Solid Tumours[END_REF] developed two original 111 In radiolabeled cyclic oligopeptide 72 ligands (urotensin II, and urantide) targeting the urotensin receptor (UT), which is highly 73 expressed in several types of solid tumors including lung, prostate, and breast. Im-74 portantly, both of these radiolabeled urotensinergic ligands exhibited similar binding af-75 finities as the native peptides and activity demonstrating successful bioconjugation. 76 Flon and co-workers [START_REF] Flon | Fluorophore-Assisted Click Chemistry through Copper(I) 137 Complexation[END_REF] convince us that there is still room for further improvements 77 in CuAAC-based click-labeling schemes. Their smartly designed probe combines the rate 78 accelerating effects of bidentate azides with the fluorescent properties of copper-binding 79 azaphtalimide frames. The result is an unprecedented approach, where the novel az-80 idoethyl-azaphtalimide probe serves as the fluorescent label and the Cu(I)-complexing 81 ligand at the same time. This all-in-one chelating fluorescent azide probe indeed facili-82 tated the reaction rate compared to conventional Cu(I)-chelating ligands used in CuAAc 83 schemes and its applicability was demonstrated in the fluorescent labeling schemes of 84 short alkyne modified peptides in vitro and on alkyne modified cells, in cellulo.

85

UV-light based methods applied on live cells are often condemned for the cytotoxi-86 city of high energy irradiation. Pull-down assays aiming at tracking down protein-protein 87 interactions, however, benefited greatly from UV-activatable crosslinking probes capable 88 of capturing even weakly binding partners. In his paper, J-E. Hoffmann [START_REF] Hoffmann | Bifunctional Non-Canonical Amino Acids: Combining Photo-Crosslinking with Click Chemistry[END_REF] provides a 89 focused review on a small set of genetically encodable, bifunctional non-canonic amino 90 acids (ncAAs). On the one hand, such ncAAs are site specifically incorporated into pro-91 teins of interests, resulting in a precise installation of a minimally perturbing photo-acti-92 vatable cross-linker handle. Upon UV-irradiation any interaction partners in the close 93 proximity of the tagged protein is captured by covalent bond formation. On the other 94 hand, the bifunctional ncAAs also bear a function that allows for further highly specific 95 modulation of the POI carrying its interacting partner e.g., via click-chemistry. Such a sec-96 ond modification could result in the installation of a wide range of functionalities from 97 fluorescent labels to pull-down handles. The reviewed ncAAs demonstrate that having a 98 clickable group in each crosslinked assemblies of proteins could greatly facilitate the gen-99 eration of a wide variety of interactome fingerprints as a result of a combination of differ-100 ent codon and cell states.

The work of Krell and Wagenknecht [START_REF] Krell | Fluorogenic and Bioorthogonal Modification of RNA Using Photoclick Chemistry[END_REF] also sheds (UV) light on the beneficial ef-102 fects of a short burst of higher energy irradiation in the context of fluorogenic modification 103 of RNA using tetrazole-photoclick chemistry. Direct attachment of the bromoaryltetrazole 104 moiety to uracil furnished the conventional diaryltetrazole, which undergoes UV-B in-105 duced bioorthogonal photoclick reactions with various olefins. Their bromoaryltetrazole-106 modified uridine building block was readily incorporated via solid-phase synthesis into 107 two RNA strands either at an internal or a terminal position. While such photoclick reac-108 tions of tetrazoles are inherently fluorogenic, the resulting diarylpyrazolines are usually 109 weakly fluorescent. Reading on the manuscript, we can find out, how the fluorescent sig-110 nal can be boosted. Maleimide-dye conjugates are covalently attached to the tetrazole 111 modified RNA strands through photoclick reaction of the bromoaryltetrazole-uracil and 112 the maleimide. Efficient energy-transfer between the pyrazoline product and the append-113 ing strong red emitter dyes results in a ca. [START_REF] Hoffmann | Bifunctional Non-Canonical Amino Acids: Combining Photo-Crosslinking with Click Chemistry[END_REF]

  -fold fluorescence increase in the red channel. 114 Such photo-click reaction based methods with enhanced red fluorogenicity and excellent 115 spatiotemporal resolution may bring live cell RNA imaging to a new level. 116 117 Conflicts of Interest: The authors declare no conflict of interest.