Stereoselective Synthesis and Antiproliferative Activities of Tetrafunctional Diterpene Steviol Derivatives.

A new family of diterpene-type aminotriol derivatives has been synthesised from stevioside in a stereoselective manner. The key intermediate spiro-epoxide was prepared through the methyl ester of the allilyc diol derived from steviol. The oxirane ring was opened with primary and secondary amines, providing a versatile library of aminotriols. The corresponding primary aminotriol was formed by palladium-catalysed hydrogenation, and an N,O-heterocyclic compound was synthesised in a regioselective reaction. All new compounds were characterised by 1D- and 2D-NMR techniques and HRMS measurements. In our in vitro investigations, we found that the aromatic N-substituted derivatives exhibited high inhibition of cell growth on human cancer cell lines (HeLa, SiHa, A2780, MCF-7 and MDA-MB-231). The antiproliferative activities were assayed by the MTT method. Furthermore, the introduction of an additional hydroxy group slightly increased the biological activity. The drug-likeness of the compounds was assessed by in silico and experimental physicochemical characterisations, completed by kinetic aqueous solubility and in vitro intestinal-specific parallel artificial membrane permeability assay (PAMPA-GI) measurements.

Interactions between Sleep and Emotions in Humans and Animal Models.

Recently, increased interest and efforts were observed in describing the possible interaction between sleep and emotions. Human and animal model studies addressed the implication of both sleep patterns and emotional processing in neurophysiology and neuropathology in suggesting a bidirectional interaction intimately modulated by complex mechanisms and factors. In this context, we aimed to discuss recent evidence and possible mechanisms implicated in this interaction, as provided by both human and animal models in studies. In addition, considering the affective component of brain physiological patterns, we aimed to find reasonable evidence in describing the two-way association between comorbid sleep impairments and psychiatric disorders. The main scientific literature databases (PubMed/Medline, Web of Science) were screened with keyword combinations for relevant content taking into consideration only English written papers and the inclusion and exclusion criteria, according to PRISMA guidelines. We found that a strong modulatory interaction between sleep processes and emotional states resides on the activity of several key brain structures, such as the amygdala, prefrontal cortex, hippocampus, and brainstem nuclei. In addition, evidence suggested that physiologically and behaviorally related mechanisms of sleep are intimately interacting with emotional perception and processing which could advise the key role of sleep in the unconscious character of emotional processes. However, further studies are needed to explain and correlate the functional analysis with causative and protective factors of sleep impairments and negative emotional modulation on neurophysiologic processing, mental health, and clinical contexts.

Repeated afternoon sleep recordings indicate first-night-effect-like adaptation process in family dogs.

The importance of dogs (Canis familiaris) in sleep research is primarily based on their comparability with humans. In spite of numerous differences, dogs' comparable sleep pattern, as well as several phenotypic similarities on both the behavioural and neural levels, make this species a most feasible model in many respects. Our aim was to investigate whether the so-called first-night effect, which in humans manifests as a marked macrostructure difference between the first and second sleep occasions, can be observed in family dogs. We used a non-invasive polysomnographic method to monitor and compare the characteristics of dogs' (N = 24) 3-hr-long afternoon naps on three occasions at the same location. We analysed how sleep macrostructure variables differed between the first, second and third occasions, considering also the effects of potential confounding variables such as the dogs' age and sleeping habits. Our findings indicate that first-night effect is present in dogs' sleep architecture, although its specifics somewhat deviate from the pattern observed in humans. Sleep macrostructure differences were mostly found between occasions 1 and 3; dogs slept more, had less wake after the first drowsiness episode, and reached drowsiness sleep earlier on occasion 3. Dogs, which had been reported to sleep rarely not at home, had an earlier non-rapid eye movement sleep, a shorter rapid eye movement sleep latency, and spent more time in rapid eye movement sleep on occasion 3, compared with occasion 1. Extending prior dog sleep data, these results help increase the validity of further sleep electroencephalography investigations in dogs.

Rapid eye movement density during REM sleep in dogs (Canis familiaris).

Dogs (Canis familiaris) are excellent models of human behavior as during domestication they have adapted to the same environment as humans. There have been many comparative studies on dog behavior; however, several easily measurable and analyzable psychophysiological variables that are widely used in humans are still largely unexplored in dogs. One such measure is rapid eye movement density (REMD) during REM sleep. The aim of this study was to test the viability of measuring REMD in dogs and to explore the relationship between the REMD and different variables (sex, age, body size, and REM sleep duration). Fifty family dogs of different breeds and ages (from 6 months to 15 years old) participated in a 3-h non-invasive polysomnography recording, and the data for 31 of them could be analyzed. The signal of the electro-oculogram (EOG) was used to detect the rapid eye movements during REM sleep, and REMD was calculated based on these data. The duration of REM sleep had a quadratic effect on REMD. Subjects' REMD increased with age, but only in male dogs with short REM sleep duration. Furthermore, in the case of dogs with short REM sleep, the interaction of body mass and REM sleep duration had a significant effect on REMD. No such effects were found in dogs with long REM duration. These results suggest that relationships may exist between REMD and several different variables.

Sleep macrostructure is modulated by positive and negative social experience in adult pet dogs.

The effects of emotionally valenced events on sleep physiology are well studied in humans and laboratory rodents. However, little is known about these effects in other species, despite the fact that several sleep characteristics differ across species and thus limit the generalizability of such findings. Here we studied the effect of positive and negative social experiences on sleep macrostructure in dogs, a species proven to be a good model of human social cognition. A non-invasive polysomnography method was used to collect data from pet dogs (n = 16) participating in 3-hour-long sleep occasions. Before sleep, dogs were exposed to emotionally positive or negative social interactions (PSI or NSI) in a within-subject design. PSI consisted of petting and ball play, while NSI was a mixture of separation, threatening approach and still face test. Sleep macrostructure was markedly different between pre-treatment conditions, with a shorter sleep latency after NSI and a redistribution of the time spent in the different sleep stages. Dogs' behaviour during pre-treatments was related to the macrostructural difference between the two occasions, and was further modulated by individual variability in personality. This result provides the first direct evidence that emotional stimuli affect subsequent sleep physiology in dogs.

The effect of oxytocin on human-directed social behaviour in dogs (Canis familiaris).

The oxytocin system has recently received increasing attention due to its effect on complex human behaviours. In parallel to this, over the past couple of decades, the human-analogue social behaviour of dogs has been intensively studied. Combining these two lines of research (e.g. studying the relationship between dog social behaviour and the oxytocin system) is a promising new research area. The present paper reviews the existing literature on how oxytocin is related to different aspects of human-directed social behaviour in dogs.

The effect of oxytocin on biological motion perception in dogs (Canis familiaris).

Recent studies have shown that the neuropeptide oxytocin is involved in the regulation of several complex human social behaviours. There is, however, little research on the effect of oxytocin on basic mechanisms underlying human sociality, such as the perception of biological motion. In the present study, we investigated the effect of oxytocin on biological motion perception in dogs (Canis familiaris), a species adapted to the human social environment and thus widely used to model many aspects of human social behaviour. In a within-subjects design, dogs (N = 39), after having received either oxytocin or placebo treatment, were presented with 2D projection of a moving point-light human figure and the inverted and scrambled version of the same movie. Heart rate (HR) and heart rate variability (HRV) were measured as physiological responses, and behavioural response was evaluated by observing dogs' looking time. Subjects were also rated on the personality traits of Neuroticism and Agreeableness by their owners. As expected, placebo-pretreated (control) dogs showed a spontaneous preference for the biological motion pattern; however, there was no such preference after oxytocin pretreatment. Furthermore, following the oxytocin pretreatment female subjects looked more at the moving point-light figure than males. The individual variations along the dimensions of Agreeableness and Neuroticism also modulated dogs' behaviour. Furthermore, HR and HRV measures were affected by oxytocin treatment and in turn played a role in subjects' looking behaviour. We discuss how these findings contribute to our understanding of the neurohormonal regulatory mechanisms of human (and non-human) social skills.

Oxytocin induces positive expectations about ambivalent stimuli (cognitive bias) in dogs.

Expectancy bias towards positive outcomes is a potential key to subjective well-being, and has been widely investigated in different species. Here we test whether oxytocin, suggested to play a role in human optimism and emotional processing, influences how dogs judge ambivalent situations (in a cognitive bias paradigm). Subjects first learned in a location discrimination task that a bowl either contained food (at the 'positive' location) or was empty (at the 'negative' location). Then, after receiving oxytocin or placebo nasal spray, they were presented with the bowl located halfway between the positive and negative positions in communicative or non-communicative contexts (N=4×16). A Positive Expectancy Score was calculated for each subject using the latency to approach this ambivalent location. Compared to placebo groups, subjects that received oxytocin pretreatment showed a positive expectation bias in both contexts, and this effect was more pronounced in the communicative context. Our study provides the first evidence for the impact of oxytocin on dogs' judgement bias and also shows that the social-communicative nature of the task situation modulates the effect of oxytocin.

Social learning by imitation in a reptile (Pogona vitticeps).

The ability to learn through imitation is thought to be the basis of cultural transmission and was long considered a distinctive characteristic of humans. There is now evidence that both mammals and birds are capable of imitation. However, nothing is known about these abilities in the third amniotic class-reptiles. Here, we use a bidirectional control procedure to show that a reptile species, the bearded dragon (Pogona vitticeps), is capable of social learning that cannot be explained by simple mechanisms such as local enhancement or goal emulation. Subjects in the experimental group opened a trap door to the side that had been demonstrated, while subjects in the ghost control group, who observed the door move without the intervention of a conspecific, were unsuccessful. This, together with differences in behaviour between experimental and control groups, provides compelling evidence that reptiles possess cognitive abilities that are comparable to those observed in mammals and birds and suggests that learning by imitation is likely to be based on ancient mechanisms.

The order of ostensive and referential signals affects dogs' responsiveness when interacting with a human.

Ostensive signals preceding referential cues are crucial in communication-based human knowledge acquisition processes. Since dogs are sensitive to both human ostensive and referential signals, here we investigate whether they also take into account the order of these signals and, in an object-choice task, respond to human pointing more readily when it is preceded by an ostensive cue indicating communicative intent. Adult pet dogs (n = 75) of different breeds were presented with different sequences of a three-step human action. In the relevant sequence (RS) condition, subjects were presented with an ostensive attention getter (verbal addressing and eye contact), followed by referential pointing at one of two identical targets and then a non-ostensive attention getter (clapping of hands). In the irrelevant sequence (IS) condition, the order of attention getters was swapped. We found that dogs chose the target indicated by pointing more frequently in the RS as compared to the IS condition. While dogs selected randomly between the target locations in the IS condition, they performed significantly better than chance in the RS condition. Based on a further control experiment (n = 22), it seems that this effect is not driven by the aversive or irrelevant nature of the non-ostensive cue. This suggests that dogs are sensitive to the order of signal sequences, and the exploitation of human referential pointing depends on the behaviour pattern in which the informing cue is embedded.

Humans rely on the same rules to assess emotional valence and intensity in conspecific and dog vocalizations.

Humans excel at assessing conspecific emotional valence and intensity, based solely on non-verbal vocal bursts that are also common in other mammals. It is not known, however, whether human listeners rely on similar acoustic cues to assess emotional content in conspecific and heterospecific vocalizations, and which acoustical parameters affect their performance. Here, for the first time, we directly compared the emotional valence and intensity perception of dog and human non-verbal vocalizations. We revealed similar relationships between acoustic features and emotional valence and intensity ratings of human and dog vocalizations: those with shorter call lengths were rated as more positive, whereas those with a higher pitch were rated as more intense. Our findings demonstrate that humans rate conspecific emotional vocalizations along basic acoustic rules, and that they apply similar rules when processing dog vocal expressions. This suggests that humans may utilize similar mental mechanisms for recognizing human and heterospecific vocal emotions.

Objective and subjective components of the first-night effect in young nightmare sufferers and healthy participants.

The first-night effect--marked differences between the first- and the second-night sleep spent in a laboratory--is a widely known phenomenon that accounts for the common practice of excluding the first-night sleep from any polysomnographic analysis. The extent to which the first-night effect is present in a participant, as well as its duration (1 or more nights), might have diagnostic value and should account for different protocols used for distinct patient groups. This study investigated the first-night effect on nightmare sufferers (NM; N = 12) and healthy controls (N = 15) using both objective (2-night-long polysomnography) and subjective (Groningen Sleep Quality Scale for the 2 nights spent in the laboratory and 1 regular night spent at home) methods. Differences were found in both the objective (sleep efficiency, wakefulness after sleep onset, sleep latency, Stage-1 duration, Stage-2 duration, slow-wave sleep duration, and REM duration) and subjective (self-rating) variables between the 2 nights and the 2 groups, with a more pronounced first-night effect in the case of the NM group. Furthermore, subjective sleep quality was strongly related to polysomnographic variables and did not differ among 1 regular night spent at home and the second night spent in the laboratory. The importance of these results is discussed from a diagnostic point of view.

A quick assessment tool for human-directed aggression in pet dogs.

Many test series have been developed to assess dog temperament and aggressive behavior, but most of them have been criticized for their relatively low predictive validity or being too long, stressful, and/or problematic to carry out. We aimed to develop a short and effective series of tests that corresponds with (a) the dog's bite history, and (b) owner evaluation of the dog's aggressive tendencies. Seventy-three pet dogs were divided into three groups by their biting history; non-biter, bit once, and multiple biter. All dogs were exposed to a short test series modeling five real-life situations: friendly greeting, take away bone, threatening approach, tug-of-war, and roll over. We found strong correlations between the in-test behavior and owner reports of dogs' aggressive tendencies towards strangers; however, the test results did not mirror the reported owner-directed aggressive tendencies. Three test situations (friendly greeting, take-away bone, threatening approach) proved to be effective in evoking specific behavioral differences according to dog biting history. Non-biters differed from biters, and there were also specific differences related to aggression and fear between the two biter groups. When a subsample of dogs was retested, the test revealed consistent results over time. We suggest that our test is adequate for a quick, general assessment of human-directed aggression in dogs, particularly to evaluate their tendency for aggressive behaviors towards strangers. Identifying important behavioral indicators of aggressive tendencies, this test can serve as a useful tool to study the genetic or neural correlates of human-directed aggression in dogs.

Dogs' social susceptibility is differentially affected by various dog-Human interactions. A study on family dogs, former shelter dogs and therapy dogs.

When pre-treated with social stimuli prior to testing, dogs are more susceptible to human influence in a food preference task. This means, after a positive social interaction they are more willing to choose the smaller amount of food indicated by the human, as opposed to their baseline preference for the bigger amount. In the current study we investigate if and how various forms of social interaction modulate choices in the same social susceptibility task, testing dogs with varying early life history (pet dogs, therapy dogs, former shelter dogs). In line with previous studies, dogs in general were found to be susceptible to human influence as reflected in the reduced number of "bigger" choices in the human influence, compared to baseline, trials. This was true not only for pet dogs with a normal life history, but also for dogs adopted from a shelter. Therapy dogs, however, did not uniformly change their preference for the bigger quantity of food in the human influence trials; they only did so if prior to testing they had been pre-treated with social stimuli by their owner (but not by a stranger). Pet dogs were also more influenced after pre-treatment with social stimuli by their owner compared to ignoring and separation; however after pre-treatment by a stranger their behaviour did not differ from ignoring and separation. Former shelter dogs on the other hand were equally influenced regardless of pre-treatment by owner versus stranger. In summary these results show that dogs' social susceptibility is modulated by both interactions immediately preceding the test as well as by long term social experiences.

Event-related potentials indicate differential neural reactivity to species and valence information in vocal stimuli in sleeping dogs.

Dogs live in a complex social environment where they regularly interact with conspecific and heterospecific partners. Awake dogs are able to process a variety of information based on vocalisations emitted by dogs and humans. Whether dogs are also able to process such information while asleep, is unknown. In the current explorative study, we investigated in N = 13 family dogs, neural response to conspecific and human emotional vocalisations. Data were recorded while dogs were asleep, using a fully non-invasive event-related potential (ERP) paradigm. A species (between 250-450 and 600-800 ms after stimulus onset) and a species by valence interaction (between 550 to 650 ms after stimulus onset) effect was observed during drowsiness. A valence (750-850 ms after stimulus onset) and a species x valence interaction (between 200 to 300 ms and 450 to 650 ms after stimulus onset) effect was also observed during non-REM specific at the Cz electrode. Although further research is needed, these results not only suggest that dogs neurally differentiate between differently valenced con- and heterospecific vocalisations, but they also provide the first evidence of complex vocal processing during sleep in dogs. Assessment and detection of ERPs during sleep in dogs appear feasible.

Owner-rated hyperactivity/impulsivity is associated with sleep efficiency in family dogs: a non-invasive EEG study.

Subjective sleep disturbances are reported by humans with attention-deficit/hyperactivity disorder (ADHD). However, no consistent objective findings related to sleep disturbances led to the removal of sleep problems from ADHD diagnostic criteria. Dogs have been used as a model for human ADHD with questionnaires validated for this purpose. Also, their sleep physiology can be measured by non-invasive methods similarly to humans. In the current study, we recorded spontaneous sleep EEG in family dogs during a laboratory session. We analyzed the association of sleep macrostructure and deep sleep (NREM) slow-wave activity (SWA) with a validated owner-rated ADHD questionnaire, assessing inattention (IA), hyperactivity/impulsivity (H/I) and total (T) scores. Higher H/I and T were associated with lower sleep efficiency and longer time awake after initial drowsiness and NREM. IA showed no associations with sleep variables. Further, no association was found between ADHD scores and SWA. Our results are in line with human studies in which poor sleep quality reported by ADHD subjects is associated with some objective EEG macrostructural parameters. This suggests that natural variation in dogs' H/I is useful to gain a deeper insight of ADHD neural mechanisms.

Methodological comparison of cancellation versus two-way choice spatial attention tests in humans and dogs.

Introduction: Behavioural problems in family dogs are amongst the leading reasons for relinquishment to shelters which adversely affects animal welfare. Recent research suggests that certain problematic behavioural patterns might be analogous to human psychiatric disorders. Veterinary diagnosis of such conditions, however, is scarce, probably due to the lack of appropriate measurement tools. The current study focuses on dog behaviour resembling the human hemispatial neglect condition, which manifests itself as a deficit in attention to and awareness of one side of the space. Methods: Healthy human subjects (N = 21) and adult family dogs (N = 23) were tested with tools aimed to measure spatial attention. Tests administered to humans included validated paper and pencil neuropsychological tools to assess hemispatial neglect (cancellation tasks), as well as the canine version of that task (visuo-spatial search task). Dogs were tested with the same visuo-spatial search task as well as a two-way choice task. Results: Results show that both in case of dogs and humans the visuo-spatial search task detects individual variation in subjects' side preferences. However, subjects' performance in the different tasks were not related.

Six facial prosodic expressions caregivers similarly display to infants and dogs.

Parents tend to use a specific communication style, including specific facial expressions, when speaking to their preverbal infants which has important implications for children's healthy development. In the present study, we investigated these facial prosodic features of caregivers with a novel method that compares infant-, dog- and adult-directed communication. We identified three novel facial displays in addition to the already described three facial expressions (i.e. the 'prosodic faces') that mothers and fathers are typically displaying when interacting with their 1-18 month-old infants and family dogs, but not when interacting with another adult. The so-called Special Happy expression proved to be the most frequent face type during infant- and dog-directed communication which always includes a Duchenne marker to convey an honest and intense happy emotion of the speaker. These results suggest that the 'prosodic faces' play an important role in both adult-infant and human-dog interactions and fulfil specific functions: to call and maintain the partner's attention, to foster emotionally positive interactions, and to strengthen social bonds. Our study highlights the relevance of future comparative studies on facial prosody and its potential contribution to healthy emotional and cognitive development of infants.

The influence of voice familiarity and linguistic content on dogs' ability to follow human voice direction.

Domestic dogs are well-known for their abilities to utilize human referential cues for problem solving, including following the direction of human voice. This study investigated whether dogs can locate hidden food relying only on the direction of human voice and whether familiarity with the speaker (owner/stranger) and the relevance of auditory signal features (ostensive addressing indicating the intent for communication to the receiver; linguistic content) affect performance. N = 35 dogs and their owners participated in four conditions in a two-way object choice task. Dogs were presented with referential auditory cues representing different combinations of three contextual parameters: the (I) 'familiarity with the human informant' (owner vs. stranger), the (II) communicative function of attention getter (ostensive addressing vs. non-ostensive cueing) and the (III) 'tone and content of the auditory cue' (high-pitched/potentially relevant vs. low-pitched/potentially irrelevant). Dogs also participated in a 'standard' pointing condition where a visual cue was provided. Significant differences were observed between conditions regarding correct choices and response latencies, suggesting that dogs' response to auditory signals are influenced by the combination of content and intonation of the message and the identity of the speaker. Dogs made correct choices the most frequently when context-relevant auditory information was provided by their owners and showed less success when auditory signals were coming from the experimenter. Correct choices in the 'Pointing' condition were similar to the experimenter auditory conditions, but less frequent compared to the owner condition with potentially relevant auditory information. This was paralleled by shorter response latencies in the owner condition compared to the experimenter conditions, although the two measures were not related. Subjects' performance in response to the owner- and experimenter-given auditory cues were interrelated, but unrelated to responses to pointing gestures, suggesting that dogs' ability to understand the referential nature of auditory cues and visual gestures partly arise from different socio-cognitive skills.

Dog brains are sensitive to infant- and dog-directed prosody.

When addressing preverbal infants and family dogs, people tend to use specific speech styles. While recent studies suggest acoustic parallels between infant- and dog-directed speech, it is unclear whether dogs, like infants, show enhanced neural sensitivity to prosodic aspects of speech directed to them. Using functional magnetic resonance imaging on awake unrestrained dogs we identify two non-primary auditory regions, one that involve the ventralmost part of the left caudal Sylvian gyrus and the temporal pole and the other at the transition of the left caudal and rostral Sylvian gyrus, which respond more to naturalistic dog- and/or infant-directed speech than to adult-directed speech, especially when speak by female speakers. This activity increase is driven by sensitivity to fundamental frequency mean and variance resulting in positive modulatory effects of these acoustic parameters in both aforementioned non-primary auditory regions. These findings show that the dog auditory cortex, similarly to that of human infants, is sensitive to the acoustic properties of speech directed to non-speaking partners. This increased neuronal responsiveness to exaggerated prosody may be one reason why dogs outperform other animals when processing speech.