Use of food to soothe infant distress has been linked to greater weight in observational studies. We used ecological momentary assessment to capture detailed patterns of food to soothe and evaluate if a responsive parenting intervention reduced parents’ use of food to soothe.n = 157) were surveyed for two 5–8 day bursts at infant ages 3 and 8 weeks. Surveys were sent via text message every 4 h between 10:00 AM-10:00 PM, with 2 surveys sent at 8:00 AM asking about nighttime hours. Infant fusses and feeds were reported for each 4-h interval. Food to soothe was defined as “Fed First” and “Not Fed First” in response to a fussy event. Use of food to soothe was modeled using random-intercept logistic regression.Primiparous mother-newborn dyads were randomized to a responsive parenting intervention designed for obesity prevention or a safety control group. Responsive parenting curriculum included guidance on using alternative soothing strategies , rather than feeding, as the first response to infant fussiness. After the initial intervention visit 3 weeks after delivery, mothers . More responsive parenting mothers reported using a responsive parenting intervention strategy first, before feeding, than controls at ages 3 and 8 weeks . At both ages combined, fewer fusses from responsive parenting infants were soothed best by feeding compared to controls . For both study groups combined, parents had greater odds of having Fed First during the nighttime compared to the daytime at both ages .The control group had greater odds of having Fed First, compared to the responsive parenting group at ages 3 and 8 weeks (3 weeks: OR = 1.9; 95% CI = 1.4–2.7; INSIGHT’s responsive parenting intervention reduced use of food to soothe and increased use of alternative soothing strategies in response to infant fussiness. Education on responsive parenting behaviors around fussing and feeding during early infancy has the potential to improve later self-regulation and weight gain trajectory.NCT01167270. Registered July 21, 2010. During infancy, parental feeding practices shape infant eating behaviors by determining what, when, how, and how much infants are fed . During In contrast, parental use of responsive feeding supports the development of infant appetite regulation . ResponsThe measurement of food to soothe has typically relied on retrospective reports of maternal use of food to soothe in different general contexts . Recall Intervention Nurses Start Infants Growing on Healthy Trajectories (INSIGHT) study is a randomized, clinical trial designed to prevent rapid infant weight gain and overweight during early childhood [The hildhood . INSIGHThildhood , 21. INShildhood  and feedhildhood . For then = 157), which is 56.3% of the 279 total participants that were randomized and received the first nurse home visit. Because a number of participants found the EMA portion of the study burdensome, the collection of EMA data was discontinued in the interest of long-term retention of participants. This study was approved by the Human Subjects Protection Office at Penn State College of Medicine prior to enrollment of the first participant.Mothers were recruited by research staff from the maternity ward of one hospital  shortly after giving birth. Enrollment occurred from January 2012 to March 2014. The eligibility criteria included singleton infants that were full-term (≥37 weeks gestation) and ≥ 2500 g at delivery. Mothers had to be primiparous, English-speaking, and ≥ 20 years of age. Two weeks after delivery, enrolled mothers were randomized into a responsive parenting or control group, stratified by intended feeding mode (breast or formula) and birth weight for gestational age (<50th or ≥ 50th). Additional details on the INSIGHT protocol are published elsewhere . A subsaThe INSIGHT responsive parenting curriculum taught parents to respond promptly, contingently, and in developmentally appropriate ways to infant cues across 4 behavioral states: drowsy, sleepy, fussy, and alert/calm . The conOne focus of the INSIGHT curriculum was to promote infant self-regulation of food intake by using responsive feeding techniques. A detailed description of the feeding-specific messages can be found elsewhere . BrieflyDemographic information, such as race/ethnicity, income, and marital status were collected at enrollment. Data on maternal age, infant sex, birth weight and length, and gestational age at birth were extracted from medical records.Mothers completed the EMA survey when infants were ages 3 and 8 weeks. These timepoints were chosen, given that 3 weeks was close to the start of the intervention and immediately followed the first nurse home visit. The 8-week timepoint was multiple weeks later and at an age when infant crying peaks. In addition, at 8 weeks, the majority of mothers had not yet returned to work full-time and were able to answer surveys on their infants’ behaviors throughout the day. Mothers were given a study-specific smartphone (LG Fathom VS750) programmed with software (Mobile Survey Development Toolkit) customized for this study. The smartphones beeped 5 times per day, every day, for 5–8 days, until 25 assessments were completed. Participants answered each beep by responding to a series of questions shown in Fig. The participants were asked to carry the study smartphones with them throughout the day, in order to answer the questions immediately after each beep. If the questions were not answered within 1 h following a given beep, it was considered a missed response. At each beep, participants provided information on soothing strategies used for a maximum of 3 fussy bouts that occurred in the previous 4 h. If an infant fussed more than 3 times in the prior 4 h, soothing strategy data were only collected on the first 3 fussy bouts  Fed First or 2) Not Fed First. Fed First occurred if mothers reported feeding as the first strategy to soothe/calm a given fussy bout  Fed Later or 2) Not Fed. Fed Later occurred if mothers reported feeding as the second or later strategy used to soothe/calm a given fussy bout . If mothers reported using rub or pat, shush/white noise, pacifier, or TV/music, this was considered a “low-level” soothing strategy (e.g. did not involve picking the infant up). Last, we considered changing the infant’s diaper as a biological need, rather than a soothing strategy; therefore, “change diaper” was removed post hoc from the response options of first soothing strategies. If mothers reported “change diaper” as the first soothing strategy used, then the second reported soothing strategy was considered to be the first soothing strategy used.If mothers reported rub or pat, swaddle, bounce/rock/walk/swing, shush/white noise, or pacifier as the first soothing strategy used, this was considered a responsive parenting strategy. If mothers reported using one of the other soothing strategies first , this was considered as Infant fussiness was quantified as the number of infant fusses reported during each 4-h interval. At each cell phone beep, mothers reported how many times their infant fussed in the previous last 4 h  in the generalized estimating equations framework to examine factors related to the number of infant fusses and feeds at a descriptive level. A repeated measures GLM was fitted to compare the numbers of responses across these factors.Two repeated-measures GLMs (one for feeding and one for fussing) grouped by mother-infant dyad examined the relationship between infant age and the number of times infants fussed or were fed over a four-hour interval, without including time of day or study group. These models accounted for repeated measures within the same mother-infant dyad to account for within-dyad dependencies. A second pair of models examined the relationship between time of day (e.g. 6:00 PM – 10:00 PM) and fussing/feeding behavior to examine diurnal patterns, without including study group or infant age. A third pair of models examined the relationship between study group and fussing/feeding behavior, without regard to time of day or infant age. Next, a series of GLMs were used to test the relationships of these same predictors  on the proportion of fusses that needed to be soothed and the number of soothing strategies used for each fuss. A Poisson link function was not used for models including count data, as the distribution of these data were relatively normal. To ensure this was appropriate, models were also run using a Poisson link function and no differences in the pattern of results were found.A second series of repeated-measures GLMs was used to test the influence of infant age, time of day, and study group on the use of food to soothe. For these models, time of day was reduced to two categories: daytime (6:00 AM – 10:00 PM) and nighttime (10:00 PM – 6:00 AM). The duration for nighttime was chosen to most closely resemble when parents were asleep. We fitted these models to answer the question if infants were fed as the first soothing strategy (coded as a dichotomous variable: Fed First/Not Fed First). An initial test for a three-way interaction was not significant, so the three-way interaction was removed from the model. Of the possible two-way interactions, study group by infant age and infant age by time of day were significant. We then tested for the main effects of study group and time of day within each age group.not feed first. These bouts were further divided into those in which infants were Fed Later vs. Not Fed in response to this fussy bout. Instances where mothers fed first were not included. This model followed a similar procedure as the preceding model: tests of the higher-level interactions were performed first and removed if non-significant. In this case, only the infant age by time of day interaction was retained.Next, we examined fussy bouts in which mothers did To examine the relationship of infant fussiness on the use of food to soothe, we repeated the previous two models while including the number of fussy bouts during each time period and feeding mode (predominantly breastfed or not predominantly breastfed) as a predictor. Study group, infant age, and time of day were tested as possible moderators using interaction terms. Again, higher-level interactions were tested first and removed if not significant. Infant age and time of day, but not study group, were significant moderators.proc genmod in SAS Version 9.4 , with statistical significance defined a priori with a cutoff of p < .05. For binary outcomes we used a logistic link function. Results are reported as mean ± standard deviation or odds ratios (OR) with 95% confidence intervals (CI) for logistic analyses.GLMs used p > 0.05).Participants were predominantly White, married, and college educated Table . About 7p < 0.01). Infants were fed 2.0 ± 0.8 times/4-h interval at 3 weeks and 1.8 ± 0.8 times/4-h interval at 8 weeks (p < 0.01). The number of fusses and feeds within a 4-h interval differed by time of day (p < 0.01). Averaged across the two infant ages, the most fusses and feeds occurred between 6:00 PM – 10:00 PM , while the least occurred between 2:00 AM – 6:00 AM , compared to all other time points within a day (p < 0.05). The number of fusses and feeds within a given 4-h interval did not differ by study group (p > 0.05).Mothers reported that infants fussed 2.4 ± 1.3 times/4-h interval at 3 weeks and 2.2 ± 1.3 times/4-h interval at 8 weeks (p > 0.05 for both). Among fusses that needed to be soothed, mothers reported using an average of 2.5 ± 1.0 (range: 0–6.5) different soothing strategies, which did not differ by study group (p > 0.05). Mothers reported using more soothing strategies between 6:00 PM – 10:00 PM (2.7 ± 1.0) and 10:00 PM – 2:00 AM (2.6 ± 1.2), than between 2:00 AM – 6:00 AM (2.4 ± 1.2) (p < 0.05).Mothers reported that 87.3 ± 14.6% of all fusses needed to be soothed, which did not differ by study group or infant age (p < 0.01). Overall, feeding was the most common strategy used first. Half (50.7%) of all fusses from control infants and 37.8% from responsive parenting infants were soothed by feeding first. The next most common strategy used first was bounce/rock/walk/swing  while play TV/music and put in bed with me were rarely used (0.4–0.6% of fusses).The distribution for the proportion of soothing strategies used first differed by study group . More responsive parenting mothers reported using a responsive parenting intervention strategy first, before feeding, than control mothers at 3 weeks  and 8 weeks . Similarly, during the nighttime (10:00 PM – 6:00 AM), significantly more responsive parenting mothers reported using a “low-level” soothing strategy before feeding or a “high-level” soothing strategy at 3 weeks , but not 8 weeks .There was a significant study group by infant age interaction on the outcome of Fed First vs. Not Fed First (p < 0.01). The next best soothing strategy was bounce/rock/walk/swing . Soothing strategies that mothers reported rarely worked best included play TV/music  and put in bed with me .Feeding was the soothing strategy that mothers reported worked best. When examining this by study group, fewer fusses from responsive parenting group infants (49.5%), then controls (61.0%), were soothed best by feeding (p = 0.03). At ages 3 and 8 weeks, the odds of having Fed First were greater during the nighttime than during the daytime, with this effect increasing over time (Table p = 0.28).There was a significant time of day by infant age interaction on Fed First vs. Not Fed First . There were no study group differences in Fed Later, compared to Not Fed (p = 0.48). Figure Next, among the subset of fussy bouts where mothers did p = 0.01). Parents had greater odds of having Fed First during periods when infants fussed less, with this effect increasing over time (Table p = 0.04). Parents had greater odds of having Fed First during periods when infants fussed less, with this effect being greater in the nighttime than in the daytime (Table p > 0.05); however, a main effect of infant fussiness revealed that overall, parents had greater odds of having Fed Later , compared to Not Fed, during periods when infants fussed less.The association between infant fussiness and being Fed First differed by infant age (me Table . The relme Table . There wp = 0.38). Feeding mode moderated the intervention effect on Fed First vs. Not Fed First (p = 0.04). Among predominantly breastfed infants, the control group had greater odds of having Fed First, compared to the responsive parenting group (Table p = 0.03). For predominantly breastfed infants, there were greater odds of having Fed First during the nighttime, compared to the daytime, while for infants not predominantly breastfed, the use of Fed First did not differ by time of day (Table p = 0.33) or the relationship of study group, time of day, or infant age on Fed Later vs. Not Fed (p > 0.05).At 2 weeks of age, 60.9% of infants were predominantly breastfed while 39.1% were not, which did not differ by study group , rather than how infants were fed (at the breast vs. bottle), and future studies should look at how infants are fed on mothers use of food to soothe. Further, feeding mode for these analyses was quantified when infants were 2 weeks of age. It is possible that predominantly breastfed infants at 2 weeks of age were no longer predominantly breastfed at 8 weeks of age when the EMA survey on using food to soothe was completed.We also showed (in agreement with earlier findings from self-reported data ) that feAt the 3-week assessment point, more responsive parenting mothers used a “low-level” soothing strategy (e.g. did not involve picking the infant up), before a “high-level” strategy or feeding, during the night. “High-level” soothing strategies are thought to be non-adaptive since infants cannot recreate these strategies on their own . The repA greater use of food to soothe has been shown among mothers who perceive their infants as having a more negative or fussy temperament –8. To buOne of the greatest strengths of this analysis was the EMA study design; yet, this method has limitations. The intensive nature of collecting survey data every 4 h, across multiple days, increased participant burden. At the time of data collection, a separate mobile device with EMA software was given to mothers to be carried with them throughout the day. Mothers reported that carrying this extra device with them and the frequent ringing was demanding. Consequently, the EMA data was collected in just over half of the INSIGHT sample. With the recent developments in technology, EMA software is now available for personal mobile devices and platforms such as REDCap , 32. We Overall, the INSIGHT responsive parenting intervention was successful at reducing maternal use of feeding as the first response to infant distress early in life. The responsive parenting intervention also reduced the frequency in which feeding was considered the most effective strategy at soothing infant fussiness. Educating parents on responsive parenting behaviors at the start of infancy can effectively modify parenting practices around infant fussing and feeding. Clinical guidance and intervention messaging should consider these findings and continue to encourage mothers to try alternative soothing strategies before feeding when infants show signs of hunger. Early infancy is an opportune time to establish this behavior given the frequency at which fussing and feeding naturally occur. The use of alternative soothing strategies, rather than feeding first, has the potential to improve infants’ self-regulation and weight gain trajectory. To continue this work, future analyses should test if reducing food to soothe during infancy improves weight status and obesogenic eating behaviors, such as emotional eating, in later childhood.


Body tactile stimulation has a positive effect upon highly social animals, such as mammals and cleaner-client coral-reef fish, by relieving stress and improving health. Conversely, some tactile contacts are naturally detrimental, such as those resulted from aggressive interactions. To study whether positive responses from tactile stimulation are generalized among vertebrates, we tested its effect on stress response and aggressive behavior in a territorial fish species, Nile tilapia. We developed an apparatus made of a row of sticks bordered by silicone bristles that was positioned in the middle of the aquarium, and through which fish had to pass to access food, thus receiving tactile stimulation. Isolated fish experienced tactile stimulation for 7 days, and  were assigned to 2 types of stressors: non-social (confinement) or social (aggressive interaction). Each of them had a corresponding control treatment without tactile stimulation. Although fish spontaneously crossed the apparatus, we did not observe a decrease in plasma cortisol levels immediately after stressor application as a response to the use of the apparatus, either for social or non-social treatment. However, tactile stimulation reduced aggressive interaction in the social treatment, showing a positive effect on a territorial fish species, and pointing to a way to improve welfare. Piglets2, lambs3 and dairy cows4 experience lower heart rates, and cattle5 show lower cortisol levels when scratched and stroked by humans. Such positive effects can also be artificially achieved. In a cleaner-client fish interaction, for instance, Soares et al.6 observed that the tactile stimulation performed by a coral-reef cleaner fish reduces the stress level in the client fish Ctenochaetus striatus. The authors developed an apparatus composed of a cleaner fish model that mechanically rubbed the client fish, thus confirming that positive effects can be achieved even by inanimate objects.Tactile stimulation (like scratches and touches) is the mechanical contact between two or more individuals of the same or different animal species that are perceived either as a positive or a negative interaction. The positive effect of tactile stimulation on mammals, including humans, is known when physical contact is performed as massage therapy. In this interaction, the tactile stimulation relieves stress7 and cleaner-client interactions among coral-reef fish8, are part of the natural behavior of these animals. Both types of interaction are adaptive, respectively, to keep social groups together by bonding individuals7, and to keep the cooperation in the cleaner-client fish mutualism. Conversely, several types of tactile contact are naturally detrimental, such as agonistic physical interactions, which may cause injuries, pain and social stress9 to individuals. Therefore, the tactile stimulation could be a negative stimulus for species whose natural behavior is characterized by territorial defense and social hierarchy, such as cichlid fish.Most positive responses to tactile stimulation are associated with animals’ natural behavioral repertoire. Physical contact between individuals, such as allo-grooming in primates10, overpopulation11, confinement12, and grading13. Furthermore, there are other conditions which may lead to aggressive interactions among them increasing the stress levels and detrimental effects on their health14, including increased probability of mortality15.Many cichlid species are commonly found in fish farming activities, being frequently subjected to stressful situations, such as net catching16. Although stress is considered an adaptive response to environmental challenges17, if the stressor is intense or lingering, animals may undergo a chronic stress state (distress), characterized by high cortisol level and marked by depression of the immune system18, decrease in growth rates19, and impairment of reproduction14. Distress also causes neuron death20 with consequent reductions in the cognitive ability. All those effects are undesirable, both for fish culture and for fish welfare. As most handling methods for husbandry fish in rearing environments are still unavoidable, finding ways to relieve any kind of distress is important to promote animal welfare. Anecdotal information about fish pets indicates that, even for cichlids, tactile stimulation could be positive, since fish seem to choose being touched or petted by human owners (see ref.21). Empiric data, however, is unavailable. In fact, some degree of positive tactile stimulation probably occurs during cichlids courtship behavior, when male and female touch each other’s body several times by a less intense biting, quivering and lateral undulations22. In this moment, tactile stimulation cannot be negative, otherwise male and female would avoid each other and mating would be impaired, being disadvantageous to Darwinian fitness. In this study, we tested whether tactile stimulation has a positive effect on the cichlid fish Nile tilapia, Oreochromis niloticus (L.), a territorial species highly farmed in the world, and a widespread model to study mechanisms underlying fish behavior. We tested the effect of tactile stimulation on Nile tilapia that underwent confinement , often applied in fish farming12, and aggressive interactions , which is part of the cichid’s natural behavior, but can be exacerbated under some rearing conditions23. We developed an apparatus consisting in a row of sticks with silicone bristles by their sides  leads to an increased circulating glucocorticoid levels (as cortisol)des Fig.  placed ides Fig. . Fish hades Fig. , thus re24, to the best of our knowledge, a very modest number when compared to the knowledge about mammals. Thus, studies about the functions and effects involved with tactile stimulation in fish can help us understand the evolution of this mechanism in vertebrates.There are only two other studies conerning the positive effects of tactile stimulation on fish = 6.24, p = 0.00003; Fig. vs absence of food: F = 41.58, p < 0.0001; among days: F = 3.85, p = 0.001; no statistic interaction: F = 1.37, p = 0.22; Fig. We assumed that the apparatus would fit our goal whether fish passed through it spontaneously. We compared the number of crossings through the apparatus (20 min/day) both in the presence and absence of food, considering spontaneity the act of crossing the apparatus in the absence of food. Fish went through the apparatus under both conditions, although less frequently in the absence than in the presence of food  = 1.29, p = 0.26; Fig.  = 4.11, p = 0.052), and a significant one within treatments  = 7.73, p = 0.01). Planned comparisons showed that the number of crossings was different before stressor between treatments  = 6.36, p = 0.017), and it was similar after stressor  = 0.63, p = 0.43). Fish crossed the center of the aquarium more frequently after the stressor in the treatment without tactile stimulation  = 7.68, p = 0.009), and with similar frequency in the treatment with tactile stimulation  = 1.34, p = 0.25).To test whether fish would use the apparatus as a way to alleviate stress, we compared the number of crossings through it right after the first stressing session with the mean number of crossings from the 7 previous days. ANOVA main results showed no significant interaction  = 0.04, p = 0.84; Fig.  = 0.22, p = 0.64); but we found difference within treatments  = 66.41, p < 0.0001). Planned comparisons showed similar number of crossings between treatments before  = 0.06, p = 0.79) and after stressor  = 0.26, p = 0.61). Fish went 4 times more often through the apparatus or crossed the center of the aquarium than before stressor in treatment with tactile stimulation  = 34.91, p < 0.0001) and treatment without tactile stimulation  = 31.53, p < 0.0001).After aggressive interactions, there was no significant difference in ANOVA interaction between and within treatments  = 0.39, p = 0.53; within treatments: F = 2.73, p = 0.11; between treatments: F = 0.39, p = 0.53; Fig.  = 0.19, p = 0.66) and after stressor  = 0.23, p = 0.63) between treatments. Cortisol levels were also similar before and after stressor in treatments with  = 2.61, p = 0.12) and without  = 0.52, p = 0.47) tactile stimulation. Furthermore, a significant difference was found for cortisol levels after aggressive contests  = 5.77, p = 0.03; Fig.  = 0.05, p = 0.82). Planned comparisons showed that cortisol level was similar before  = 2.49, p = 0.13) and after stressor  = 2.05, p = 0.17) between treatments. Cortisol level was lower before stressor than after stressor in treatment with tactile stimulation  = 14.62, p = 0.001) and was similar in treatment without tactile stimulation  = 0.18, p = 0.67).Plasma cortisol level was not reduced by tactile stimulation in the confinement experiment. It was similar between treatments with and without tactile stimulation, as well as within sampling days , or between the mean of crossings on the 7 days before stress and cortisol levels . We did not find a correlation between crossings and cortisol levels after aggressive interactions as well .We did not find a significant correlation between the number of displays and cortisol levels after social stress, either for the treatment with tactile stimulation  or for the treatment without tactile stimulation . However, there was a positive correlation between the number of attacks and cortisol after stress following tactile stimulation , whereas a non-significant correlation was observed in the treatment without tactile stimulation .In this study we successfully created a protocol for providing fish with tactile stimulation. Such stimulation did not change cortisol levels on Nile tilapia immediately after stressor application, but it was effective in decreasing aggressiveness, thus probably bringing about positive effects on fish’s welfare.We considered the apparatus efficient in providing fish with body tactile stimulation because they spontaneously went through it after training. In both treatments, (with and without tactile stimulation) food stimulated fish locomotion, but the frequency of crossings before and after feeding indicates that fish use the stimulator apparatus irrespective of food presence. In the treatment without tactile stimulation, fish were able to cross the center of the aquarium since the first day of observation, whereas in treatment with tactile stimulation they hardly crossed through the apparatus on the first day; the number of crossings increased gradually up to the third day of experiment. This shows that some learning processes was required to deal with the new structure inside the aquarium since the apparatus was initially avoided but, after that, the stimulator did not affect the fish’s freedom in swimming and exploring their environment. In addition, tactile stimulation seemed to be soft enough to avoid body injuries or scale loss, which would be a gateway for pathogens. Therefore, our protocol is suitable to test several hypotheses regarding tactile stimulation in fish.25) and show strong motivation to access preferred items in the environment, while avoiding non-preferred ones26. Therefore, we expected there would be an increase in the number of crossing through the apparatus after fish were subjected to stressors, thus indicating that body tactile stimulation could alleviate stress in fish. Even though it was not observed after confinement, it was evident after social stress and, at first, it could be interpreted as a motivational behavior for receiving tactile stimulation. However, there was also an increase in crossing the center of the aquarium in the treatment without tactile stimulation, at the same intensity; thus, crossing through the apparatus was a consequence of fish’s increased locomotion after fighting. This behavior did not seem to be a stereotyped activity because the high rate of locomotion was not rhythmically repetitive25. Nevertheless, it seemed to be a type of displacement behavior observed in fish after aggressive interactions27. Since in our study there were no elements in the environment to redirect aggression27, motivation to fight could be turned into swimming activity.After a stressful experience, animals tend to escape from the detrimental stimulus. It is supposed that animals can also search for ways to alleviate stress , since fish’s locomotion was similar in both the treatments with and without tactile stimulation. However, it is known that body tactile stimulation increases serotonin levels in humans and other mammals1, and serotonin does present an inhibitory effect on the aggressive behavior in fish32 and other vertebrates. Then, we infer that tactile stimulation may reduce aggressiveness by increasing serotonin levels, although this mechanism should yet be tested.Greater amount of physical activity increases the levels of endorphin22. In this moment, aggressiveness shoud be reduced to allow mating.An animal’s response to a determined stimulus is based on a capacity evolutionary selected for such response. Thereby, the natural response to some positive effect from tactile stimulation probably was selected in social fish species, particularly for courtship behaviorCtenochaetus striatus6, a fish species that naturally receives frequent tactile stimulation from the cleaner fish Labroides dimidiatus8. 2. The effect exists, but longer stimulation would be required. 3. Cortisol levels were already very high, due to social isolation. In fact, the animals presented high levels of cortisol in contrast to basal levels reported to GIFT Nile tilapia23, which could be explained by social isolation34. Fish could be already stressed therefore it precluded the observation of significant increment on plasma cortisol after non-social stress. Although a higher cortisol level was shown after social stress, there was no difference from the treatment without tactile stimulation, thus suggesting there was an effect of social isolation on the fish response which was not counteracted by tactile stimulation. 4. Blood sampling occurred during cortisol peak (see ref.35). As we designed a study to be compared to that by Soares et al.6, we sampled blood right after the stressor application. Thus, we had no data regarding the possible effect of tactile stimulation on cortisol levels after peak elapsed time. In this sense, it is important to evaluate the effect of tactile stimulation during the stress-recovering stage from social interaction (see ref.36).Despite the association found between tactile stimulation and aggressive interaction, stress was not reduced, which suggested some possible non-exclusive explanations: 1. tactile stimulation does not reduce stress in this territorial fish species because positive body tactile stimulation is not associated to its natural repertoire. For instance, stress is reduced in 16. Moreover, cortisol is not always the best physiological marker for stress in highly social species37. Then, a higher set of physiological indicators, as well gene expression in specific brain areas could be necessary to evaluate the stress in a broader sense38. Thus, we have to take in account that the cortisol solely brought a limitation for understanding the entire scenario related to Nile tilapia’s stress response to tactile stimulation. An associated pattern with a positive correlation between the number of attacks and cortisol levels was found after tactile stimulation, which was non-significant in the treatment without tactile stimulation (dissociated pattern). Some differences in the social environment can cause differences between associated/dissociated hormones and behavior patterns in cichlids39. Therefore, tactile stimulation possibly affects the HPI axis, associating it with social stress.Although cortisol level is the main stress indicator, it can also be involved in other functions, for instance, in inflammatory/immune responses to mechanical damages on tissues, and other functions that requires a metabolic changing40, petting or stroking40. Such interactions show both positive and negative effects on the animals. Certain studies focus on the affection and bonds that are created between humans and animals43, so they do not eliminate the social context of human contact, which may influence the animal’s perception. In this study, tactile stimulation was isolated from the human presence, therefore highlighting the potential of tactile stimulation alone for reducing aggressiveness in Nile tilapia, which may lead to improved welfare.Studies carried out with domestic mammals are usually combined with human presence, and stimulation is provided by touchAdult male Nile tilapia specimens from the Aquaculture Center of UNESP (CAUNESP) in Jaboticabal, SP, Brazil, were kept in outdoor ponds at the Laboratory of Animal Behavior, UNESP, São José do Rio Preto. They were selected for the study and taken to the laboratory where they were acclimated for 20 days in polyethylene water tanks  at 27 °C and light regime from 7:00 a.m. to 7:00 p.m. Fish were fed with food for tropical fish  twice a day (9:00 a.m. and 3:00 p.m.). Water quality was maintained using biological filters (400 L/h) and constant aeration.Nile tilapia males were individually assigned to one aquarium equipped with an apparatus developed to provide them with body tactile stimulation Fig. . Fish weWe developed an apparatus made of a rectangular polyvinyl chloride (PVC) pipe structure, filled with vertical sticks bordered by silicone bristles Fig. . We chosIsolated fish individuals were adjusted in the aquaria for 3 days before introducing the apparatus. During those days, fish were fed with dry shrimp at 9:00 a.m. and 3:00 p.m., by using the feeder. The same was done in the treatment without tactile stimulation. Animals that did not eat during this period were withdrawn from the study.th day, fish were subjected to stressors twice, the first record being made to quantify the number of crossings after stress, and the second one to quantify cortisol right after stress, so that it was possible to compare data with those by Soares et al.6. Blood samples were obtained on the 3rd day for evaluating the baseline plasma cortisol levels and on the 11th day for checking the effect of tactile stimulation on stress response. The same procedure was repeated in the treatments without tactile stimulation. This protocol was the baseline for testing both social (aggressive interaction) and non-social (confinement) stress, which were run independently. The protocol timeline is summarized in Table After the adjustments, the apparatus was introduced in the center of the aquarium and remained there for 7 days. Fish were video-recorded daily to quantify the number of crossings through the apparatus; 5 min before, 10 min during and 5 min after the introduction of the feeder. Records started at 8:00 a.m. and at 2:00 p.m. On the 11th day, fish were subjected to the confinement stress test, which consisted of reducing in 90% the aquarium space using an opaque plate, thus confining the animal to one end of the aquarium for 30 min . This stressor has already been tested for Nile tilapia45. After the confinement, the plate was removed, and fish were video-recorded (20 min) to quantify the number of crossings through the apparatus. As the crossings could be affected by fish locomotion in the aquarium, we recorded the number of times fish crossed through the center of the aquarium in the treatment without tactile stimulation. The stressor was applied again after 6 hours (in the afternoon), followed by blood sampling for plasma cortisol assay. In the treatment without tactile stimulation, the animals underwent the same procedure described, yet without the presence of the apparatus in the aquaria. Fifteen isolated males were tested in each treatment both with and without tactile stimulation.In the morning of the 11th day, fish were subjected to the aggressive interaction test. Two isolated individuals were removed from their aquaria and were paired in a new aquarium (40 × 30 × 40 cm ca. 48 L) to avoid prior residence effect46. Two fish of the same treatment and with similar size were paired as they underwent the same procedures (absence or presence of tactile stimulation). Aggressive interaction was video-recorded for 30 min, then, each fish was returned to its original aquarium and was video-recorded (20 min) to quantity the number of crossings through the apparatus. As in the non-social experiment, we also recorded the number of times fish crossed through the center of the aquarium in the treatment without tactile stimulation. Fish were paired again in the afternoon for aggressive interaction video-recording (30 min), and a blood sampling for plasma cortisol assay was performed. Different individuals (from the first encounter) were paired to avoid the effect of the previous experience47. In the treatment without tactile stimulation the animals underwent the same procedure, yet without the insertion of the apparatus. Fourteen isolated males were tested in each treatment, both with and without tactile stimulation.In the morning of the 1150. Aggressive behavior was labeled as attacks or displays. Attacks are the interactions with physical contact and associated with high energy expenditure ; on the other hand, displays are aggressive interactions without physical contact and associated with less energy expenditure51 .In the social stress experiment, fish were individually identified by a red visible implanted elastomer (VIE tags), inserted under 2 or 3 scales on each side of the body. Aggressive interactions were quantified according to refs52. The second is the hypothalamic–pituitary–interrenal tissue (HPI) axis52. In HPI axis the corticotrophin releasing hormone (CRH) released from the hypothalamus stimulates the anterior pituitary gland to release adrenocorticotrophic hormone (ACTH) that, in its turn, stimulate the interrenal tissue to increase the glucocorticoid hormones releasing, being cortisol the main corticosteroid in teleost fishes52. This increment in cortisol levels in response to a stressor is a primary indicator of stress levels in fishes16, although some caution should be considered when using only one marker.In fish stress is marked by an activation of two main axes. The first is the hypothalamus-sympathetic-chromaffin cells axis, which release the neurohormones norepinephrine and epinephrine, whose trigger a fast response to increased energy demand−1), a fish blood sample was collected from the caudal vein by hypodermic needles and heparinized syringes. The blood sampling of each fish lasted less than 2 minutes to avoid interference of manipulation in the results18. Blood was centrifuged at 3,000 rpm for 10 min and the plasma was frozen at −20 °C for further cortisol assays. Cortisol was evaluated through ELISA - Enzyme Linked Immunosorbent Assay, using commercial kits . The intra-assay CV = 3.32%, 6.51% and 4.62% in each plate, and the inter-assay CV = 6.81%.After being anesthetized by immersion in Benzocaine (0.09 g.L−1), weighed, measured and tagged with an elastomer. Fish mean (±S.E.) standard length and weight were respectively: Non-social experiment – Treatment with tactile stimulation: 11.44 cm ± 0.92 cm; 47.26 g ± 11.15 g (N = 15); Treatment without tactile stimulation: 11.74 cm ± 0.90 cm; 51.33 g ± 10.62 g (N = 15). Social stress experiment: – Treatment with tactile stimulation: 11.42 cm ± 0.52 cm; 51.83 g ± 8.76 g (N = 14); Treatment without tactile stimulation: 10.97 cm ± 0.41 cm; 48.20 g ± 6.55 g (N = 14). We did not find significant differences in the biometric data between treatments . At the end of the experiment all fish were killed (Benzocaine 0.18 g.L−1) and opened for gonadal inspection. Adult male Nile tilapia present developed testicles filled with semen, thus showing an opaque white color53. We confirmed macroscopically that all individuals were adult males.Before isolation, animals were anesthetized by immersion in Benzocaine  coated with blue plastic to avoid visual contact with animals in neighboring aquaria. Video-recording was made with cameras placed above the aquaria, which sent the records to a computer in an adjacent room. The photoperiod was set to 12 L:12D (from 7:00 a.m. to 7:00 p.m.) and temperature to 27 °C. Water quality was monitored by commercial kits and electronic devices and given by mean ± SE: Ammonia (0.025 ± 0.019 ppm); Nitrite (0.125 ± 0.072 ppm); pH (7.36 ± 0.11).54. The data were, then, checked for normality by Kolmogorov-Smirnov test and homoscedasticity by Fmax55. When necessary, data were transformed by log (x + 1) to fit parametric assumptions. In both social and non-social experiments, a mixed-model ANOVA was used to compare between (with tactile stimulations vs without tactile stimulation) and repeated data for the following variables: number of crossings/min through the center of the aquarium in periods with and without food); number of crossings/min before and after stress test in both treatments; plasma cortisol concentrations on the 3rd and 11th days. Tukey-HSD post hoc test was applied when necessary. Planned comparisons were used to contrast between and within treatments. T-test was used to compare the number of attacks and displays between treatments. Correlations between the number of crossings and the cortisol levels were checked by Pearson’s correlation test. Statistical significance was set at p ≤ 0.05.Data were tested for outliers by Grubbs test and those found were replaced by the meanThis study followed the Animal Behavior Society guidelines for animal usage in research (2012) and was in accordance with the Ethical Principles on Animal Experimentation adopted by the National Council for the Control of Animal Experimentation (CONCEA/Brazil). It was approved by the Committee on Ethics in Animal Use, IBILCE, UNESP, São José do Rio Preto, permit #129/2016.


Most patients of established retinitis pigmentosa (RP) have subnormal peripheral vision and heavily rely on central vision for their daily activities. Central visual acuity is dependent on photoreceptor survival at the macula. Identification of structural changes that precede visual loss is essential. The aim of this study was to correlate the Spectral Domain-Optical Coherence Tomography (SD-OCT) characteristics with visual acuity in patients with typical RP.This was a retrospective, observational case series of 224 eyes of 113 RP patients conducted a tertiary eye care center. SD-OCT imaging was done for all eyes. Central retinal thickness (CRT), photoreceptor outer segment length (PROS), foveal outer segment pigment epithelial thickness (FOSPET) and ellipsoid zone (EZ) extent were measured. A new variable, FOSPET-PROS ratio (FPR), obtained by dividing FOSPET by PROS is defined and correlated to corrected distance visual acuity (CDVA) in logMAR using linear regression.p = 0.048) and r = 0.842 (p = 0.0001), respectively. FOSPET (mean = 71.15 ± 13.8 μm) and PROS (mean = 44.85 ± 12.5 μm) did not show a significant correlation to CDVA, independent of FPR.Out of 113 patients, 71 were males and 42 females. Mean age of the patients was 35.4 ± 15.1 years. Mean CDVA was 0.33 ± 0.25 logMAR with no difference between the genders. Mean CRT (218.74 ± 83.5 μm) and FPR (1.63 ± 0.22) significantly correlated to CDVA with a correlation coefficient of r = − 0.139 (Retinal microstructural changes on SD-OCT, especially the FPR, can be used as a surrogate marker to monitor disease progression in the central retina in degenerative diseases like RP. Retinitis pigmentosa (RP) is a complex hereditary disease characterized by progressive degeneration of photoreceptors . WorldwiPatients typically present with visual impairment involving night and peripheral vision with gradual deterioration of the central visual acuity. Photoreceptor survival in central retina correlates closely with visual function in these patients . TherefoOptical coherence tomography (OCT) is a well-established method of analyzing in vivo retinal architecture  and has < 1.0 logMAR were included. Pedigree charts were included for the analysis whenever available.This was a retrospective, observational, cross-sectional study of 224 eyes of 113 patients with RP, evaluated at a tertiary eye care center between 2011 and 2014 after approval from the Ethics Committee and Research Board of Narayana Nethralaya Eye Institute, Bangalore, India. The diagnosis of RP was based on a history of night vision problem, impairment in peripheral visual fields, reduction in electroretinogram rod and cone amplitudes using ISCEV  standard full-field electroretinograms 13]SD-OCT scans were acquired using the Spectral–domain Heidelberg retinal angiograph + optical coherence tomography (SD-HRA + OCT)© Heidelberg Engineering. The OCT component of the machine is a spectral/Fourier domain OCT with 5 μm axial resolution and an image acquisition speed of 40,000 scans/second. It employs a broad band 870 nm super luminescent diode source. Sixteen OCT scans were averaged to reduce noise. An experienced operator certified by the DARC  acquired all images through a dilated pupil. Two independent observers (PBC and AKK) did the measurements in horizontal and vertical scans through the fovea by first magnifying the OCT scan 400% Fig. a and theExtent of the preserved EZ line was also measured in horizontal and vertical scans, centered at the fovea Fig. . All meaTo assess the inter-observer repeatability of the measurements of preserved EZ extent and measurements of CRT, PROS and FOSPET on SD-OCT images, the methods described by Bland and Altman were used . The meaNormal: CDVA = logMAR 0 in both eyes.Mild visual impairment: logMAR 0 < CDVA < logMAR 0.477.Moderate visual impairment: logMAR 0.477 < CDVA < logMAR 1. Data was analyzed by Shapiro Wilk test to test for normality. To determine the correlation between CDVA and the retinal microstructure on the SD-OCT, a linear mixed model (LMM) analysis was performed with CDVA as a dependent variable and SD-OCT values as covariates, as well as their interaction terms. All data were analyzed using the statistical software package SPSS version 22.0 .To ascertain the mean CDVA and across pedigree groups as well as for statistical analysis, Snellen’s visual acuity was converted to logMAR units and patients were categorized into 3 subgroups based on their visual acuity as follows:Overall, 224 eyes of 113 patients were included in the study. These included both eyes of 111 patients and one eye in two patients. The other eye of these two patients were excluded due to a macular hole and post traumatic phthisical eye. 71 patients were male and 42 were females. Mean age of patients was 35.4 years (range 8–72 years). Mean CDVA was 0.33 logMAR  with a range of 0.0–1.0 .n = 122) had normal CDVA, 26.3% (n = 59) had mild visual impairment and 19.2% (n = 43) had moderate visual impairment. Pedigree charts were available for 72.5% (n = 82) patients. Most common inheritance pattern was the Simplex variant, seen in 34.5% (n = 39) followed by autosomal recessive (AR) , autosomal dominant (AD)  and X-linked . Mean CDVA across pedigree groups did not show a significant difference   , PROS = 44.85 μm (range = 20–68 μm) and FOSPET = 71.15 μm (range = 45 – 95 μm). A new variable, termed the FOSPET-PROS ratio (FPR), was derived by dividing the FOSPET value by the PROS value. Mean FPR was 1.63 (range = 1.30–2.23). Mean values of CRT, PROS, FOSPET and FPR across visual acuity groups are given in Table 2 = 0.874, p < 0.001, but not to CDVA. None of these three measurements of the outer retina showed any significant correlation to preserved EZ extent.Mean extent of the preserved EZ in the horizontal and vertical direction was 2856.4 μm (range = 518–8799 μm) and 2446.3 μm (range = 528–7914 μm), respectively, and strongly correlated to each other with rp = 0.44), none of the other variables were significantly different. The mean difference in CRT, PROS and FOSPET between Observer 1 and 2 were + 4 μm, + 3.5 μm and + 4.4 μm, respectively, and there was no significant bias between the observers . The bias for the measurements of preserved EZ in the SD-OCT images was 10.4 μm and 11.2 μm in horizontal and vertical scan respectively with no significant bias between the observers .Mean values of CDVA, CRT, PROS, FOSPET, FPR, and the horizontal and vertical extent of the preserved EZ in different pedigree subgroups are shown in Table In recent years, several authors , 17 haveThe earliest histopathological change in the photoreceptors of eyes with RP is a shortening of the photoreceptor outer segments  while thFoveal thinning, probably secondary to photoreceptor loss, as measured on OCT has been shown to correlate with visual acuity in RP , 27. In There are two possible explanations for the strong correlation of FPR with CDVA. The height of the RPE-Bruch’s complex may correlate with the height of photoreceptor outer segments, measured as PROS on SD-OCT. Instead of absolute values, a ratio may be a more accurate reflection of shortening of the photoreceptor outer segments, i.e. PROS shortening with reference to the RPE. Moreover, the RPE-Bruch’s complex is itself thickened and may reflect an additional pathology in the disease process, i.e. PROS thinning and RPE-Bruch’s complex thickening occur as the disease progresses despite a clinically normal appearing macula. Indeed, some histopathological reports mention the presence of acidic mucosal substances in the inter photoreceptor matrix space  and wideThe CDVA, PROS, FOSPET and FPR showed a trend toward progressive decline when analyzed across pedigree subgroups with simplex or sporadic cases being the best and X-linked, the worst Table ; howeverOur study is an attempt to identify microstructural changes at the macula on SD-OCT that precede functional loss. Most patients of established RP have subnormal peripheral vision and heavily rely on central vision for their daily activities. Central visual acuity is dependent on photoreceptor survival at the macula. Identification of structural changes that precede visual loss is essential. To the best of our knowledge this is the first study that has quantitatively measured three vertical foveal parameters on OCT, the CRT, PROS and FOSPET, and their correlation with CDVA in RP patients. We have also measured the FOSPET: PROS ratio (FPR) and demonstrated that the FPR has the best correlation with CDVA compared to the other vertical foveal parameters and strongly believe that it is a potential marker of disease progression. Further research and establishment of a normative database of FPR might help us in early detection of progression of the disease, much before drop in visual acuity. Further, it might also help in monitoring treatment response. Studies are required to establish the role of the RPE-Bruch’s complex in patients with RP and the interaction of transverse outer retinal structures such as ELM, EZ and IZ with vertical measurements to clearly delineate the true measure and predictor of CDVA, contrast sensitivity and color vision in retinal degenerative disorders. It is possible that these SD-OCT measurements denote different aspects of photoreceptor functions. Identifying these markers can also help to formulate the inclusion and exclusion criteria for clinical trials in RP.The limitations of our study are the use of Snellen’s visual acuity for measurement of CDVA, lack of documentation of transverse structural changes and their correlation to CDVA. We did not have the genetic mutational data for all patients and did not use other functional measures of central vision or a control group.There is a continuous need to detect micro structural changes in degenerative diseases like RP in its earliest stage when therapy may be most useful. To identify surrogate end points to monitor response to therapeutic intervention before true functional loss has occurred is the need of the hour.


Infants and young children with severe acute malnutrition (SAM) are treated with empiric broad‐spectrum antimicrobials. Parenteral ceftriaxone is currently a second‐line agent for invasive infection. Oral metronidazole principally targets small intestinal bacterial overgrowth. Children with SAM may have altered drug absorption, distribution, metabolism, and elimination. Population pharmacokinetics of ceftriaxone and metronidazole were studied, with the aim of recommending optimal dosing. Eighty‐one patients with SAM (aged 2–45 months) provided 234 postdose pharmacokinetic samples for total ceftriaxone, metronidazole, and hydroxymetronidazole. Ceftriaxone protein binding was also measured in 190 of these samples. A three‐compartment model adequately described free ceftriaxone, with a Michaelis–Menten model for concentration and albumin‐dependent protein binding. A one‐compartment model was used for both metronidazole and hydroxymetronidazole, with only 1% of hydroxymetronidazole predicted to be formed during first‐pass. Simulations showed 80 mg/kg once daily of ceftriaxone and 12.5 mg/kg twice daily of metronidazole were sufficient to reach therapeutic targets. WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC?☑ There are no published pharmacokinetic data on ceftriaxone in infants and young children with severe acute malnutrition (SAM). A small study on oral metronidazole has been conducted, but no information on its major active metabolite is available. Changes in body composition and metabolism may affect the distribution and elimination of these agents. Inappropriate dosing may result in reduced efficacy, antimicrobial resistance, or increased adverse effects.WHAT QUESTION DID THIS STUDY ADDRESS?☑ What dose of ceftriaxone and metronidazole should be used in children with SAM who are sick and hospitalized?WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE?☑ A dose of 80 mg/kg of ceftriaxone (rather than 50 mg/kg) is predicted to maximize target attainment. Twice daily metronidazole at 12.5 mg/kg (rather than 7.5 mg/kg three times daily) may provide a good compromise between achieving circulating target concentrations, maintaining adequate small intestinal concentrations, and reducing caregiver time. Reduced serum albumin increased the unbound fraction of ceftriaxone. Edema significantly affected the disposition of both drugs, with weight‐based dosing leading to higher exposures, although this does not appear to warrant dose reduction.HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE?☑ Our data inform optimal dosing in this vulnerable group of children. Careful prospective study design enables pharmacokinetic parameter estimation of two drugs using only three postdose samples.In Sub‐Saharan Africa, infants and young children with complicated severe acute malnutrition (SAM) requiring hospital admission have a 12 to > 30% inpatient case fatality,Ceftriaxone, a broad‐spectrum third‐generation cephalosporin, is a potentially useful first‐line treatment for children with SAM. It is inexpensive and, due to its long half‐life, only requires once‐daily administration, which is important to overstretched healthcare settings where children with SAM are usually managed. When assessing its likely antimicrobial efficacy, the free concentration needs to be considered, since ceftriaxone protein binding is nonlinear, with higher total concentrations being associated with larger fraction unbound. Its nonlinear concentration effect is further compounded by increased free fraction with decreasing plasma proteins, most notably albumin. Models describing nonlinear ceftriaxone protein binding have been developed in special populations such those as in intensive care.In addition to sepsis, small intestinal bacterial overgrowth and intestinal parasitosis are implicated in malabsorption, diarrhea, and reduced nutritional recovery in children with SAM. Empiric oral metronidazole, a nitroimidazole antibacterial active against a wide range of bacteria including anaerobes, is advocated by some experts,http://clinicaltrials.gov NCT03174236), which will compare ceftriaxone (±metronidazole) with standard‐of‐care antimicrobials (±metronidazole) in a 2 × 2 factorial design for outcomes including mortality and nutritional recovery. An important risk associated with unrestricted ceftriaxone use is selection for extended spectrum beta lactamase (ESBL) and resistance to other antibiotics classes. It will therefore be particularly important for the study of real‐world resistance development to be done in the context of optimally efficacious ceftriaxone dosing.Here we report a study on ceftriaxone and metronidazole pharmacokinetics in order to define appropriate dosing for the sick, hospitalized infants and young children with SAM. This study forms the initial stage of the First Line Antimicrobials in Complicated Severe Acute Malnutrition (FLACSAM) trial . For the simultaneous modeling of metronidazole and hydroxymetronidazole, a one‐compartment disposition model was adequate for both, and adding a zero order lag to the first order absorption gave a significant improvement in fit (P < 0.05). Allometric scalinga priori along with a fixed sigmoidal maturation function on clearance based on glomerular filtration maturation for ceftriaxoneP < 0.001). A low extraction ratio of around 1% indicated most hydroxymetronidazole is formed from systemic metronidazole. Both models terminated with successful covariance steps, and model parameters are reported in TableSupplementary Materials.A three‐compartment model provided a slightly better fit than a two‐compartment model (⊿ OFV −7.4) to total ceftriaxone, with unbound measurements predicted assuming instantaneous binding with a Michaelis–Menten model  was associated with decreased apparent weight on volume parameters in the allometric model with any grade of edema of 26%. For metronidazole the effect of edema was less pronounced, with the largest OFV drop occurring with an edema score of 2 or more leading to a decrease in apparent weight of 17%.P < 0.001) associated with age‐corrected serum creatinine.P < 0.001) improve the fit when added to the Bmax parameter in the Michaelis–Menten model, with lower serum albumin being associated with higher fraction unbound. FiguresSupplementary Materials.Free ceftriaxone clearance was significantly P < 0.00 associatft > MIC) for ceftriaxone free concentration over the first 24 hours was simulated for 50, 80, and 100 mg/kg once daily dosing  of 22.9 mg/L is similar to the value (28.7 mg/L) that was recently reported by Schleibinger et al.Comparing our model parameters with other studies is complicated by many reporting parameters for total ceftriaxone and a lack of standardization of age and weight (or size) scaling.ft > MIC values.ft > MIC or AUC:MIC) with ascending MIC values, along with important percentiles from the simulations. This therefore shows not just the simulated typical patient, but, for example, the 5th percentile shows the minimum target attainment expected in 95% of patients. We would suggest that presenting antimicrobial simulation data in this way  against ascending MIC values. Our reasons for this are twofold: first, such plots tend to give measures of spread of target attainment across the population; second, the preferred target may not be fixed, with recent work showing that different populations may require different  Figures and 4 prft > MIC, which is the index usually found to correlate with antimicrobial activity,Figureft > MIC of at least 50% of the dosing interval, and typically Enterobacteriaciae with MICs below 1 mg/L, were considered susceptible, although 2 mg/L is now a more common breakpoint, with MIC >8 mg/L indicating the need to test for the presence of ESBL.By parameterizing the model in terms of free ceftriaxone, simulations of free concentrations were therefore straightforward, and the FigureSupplementary Materials. Including hydroxymetronidazole was important, since it is known to have antimicrobial activity in its own right.FigureIncluding metronidazole formation from first‐pass and circulating concentrations gave a 70.8 point lower objective function value than when only circulating concentrations were allowed to form metabolite, and hence it was retained despite the small fraction (around 1%) formed in this way. In the standard parent metabolite model, allowing formation from circulation and first‐pass is not uniquely identifiable since the formation rate cannot be separated from the fraction metabolized.8 have been proposed, and could potentially be used to justify once‐daily dosing along with the use of a loading dose, when treating small intestinal overgrowth it could be argued that local upper gastrointestinal tract concentrations maybe more important for efficacy. Taking our estimated absorption rate constant and assuming a gut lumen volume of 100 mL (dose taken with food/drink) a 12.5 mg/kg dose administered to an 8‐kg child would leave a local concentration of 7.3 mg/L prior to the next dose. Twice‐daily dosing would also give acceptable AUC and ft > MIC target attainment . Parents of eligible patients were approached for informed consent.A three‐center open‐label phase II PK study was undertaken in children aged 2–59 months with severe acute malnutrition (SAM). For children aged 6–59 months, SAM was defined as kwashiorkor  or a middle upper arm circumference (MUAC) less than 11.5 cm, or weight‐for‐height Z‐score of less than –3. For children aged 1–5 months SAM was defined as kwashiorkor or MUAC less than 11 cm or weight‐for‐height Z score less than –3. Infants with weight less than 2.5 kg were excluded. The study received favorable ethical review from the Kenya Medical Research Institute Review Committee, Nairobi, the Oxford Tropical Research Ethics Committee, Oxford, UK, and the Kenyan Expert Committee on Clinical Trials of the Pharmacy & Poisons Board, and is registered with Enrolled patients received ceftriaxone 50 mg/kg (or 100 mg/kg if meningitis suspected) 24 hourly by intravenous bolus , and metronidazole 7.5 mg/kg 8 hourly by oral suspension . PK samples were taken up to 72 hours following the first dose. A baseline PK sample was taken and then the first metronidazole dose followed by the first ceftriaxone dose. To minimize study invasiveness, only three further PK samples were taken. Patients were randomized to one of three possible early, middle, and late samples as follows: Early: 5 minutes, 30 minutes, or 1 hour post first dose; Middle: 2 hours, 4 hours, or 8 hours post first dose; Late: 24 hours, 48 hours, or 72 hours post first dose with exact dosing and sampling time recorded to the nearest minute.et al.Samples were analyzed for total ceftriaxone, metronidazole, and hydroxymetronidazole. Free ceftriaxone was also measured in a subset of samples, with preference given to the early and late timepoints. Full details on the assay were recently published by Ongas CU) was predicted from total concentration (CT) in the central compartment, with bound concentration (CB) assumed to follow Michaelis–Menten kinetics with instantaneous binding:Bmax and Kd are the estimated Michaelis–Menten parameters, and CT=CU+CB. The unbound concentration was therefore derived from the Michaelis–Menten equation above:Unbound ceftriaxone was first analyzed alone to derive a base structural disposition model, with one‐, two‐, and three‐compartment disposition models tested. It was assumed that bound drug is unavailable for elimination, distribution, or pharmacological effect, so when total concentrations were added, free concentration  scaled by measured hematocrit according to the expression reported by Price et al.QHP is hepatic plasma flow, w is body weight in kg, and h is the hematocrit. The fraction transformed to hydroxymetronidazole during first‐pass was assumed to be 1 minus the extraction ratio (ER) and hepatic clearance of circulating metronidazole to hydroxymetronidazole was given by the following:CLI, was an estimated parameter. Clearance of metronidazole by other routes was assumed to be linear, as was clearance of hydroxymetronidazole.Metronidazole and hydroxymetronidazole were simultaneously fitted using the well‐stirred liver model, using a similar model to previous study on oseltamivir carboxylate formation from oseltamivir.a priori. Furthermore, a maturation function for renal clearance based on a previous studyet al.CLI.Biological prior information in the form of allometric scaling with exponents of 0.75, 1, and –0.25 for all clearance volume and absorption rate parameters, respectively, was added Bmax, age corrected serum creatinine on free ceftriaxone clearance and hydroxymetronidazole clearance, and edema to scale weight in the allometric models. The likelihood ratio test was used to determine covariate inclusion, with an improvement in –2 log likelihood of > 3.84 considered significant. Model diagnostics included plots of observations vs. population predictions, conditional weighted residuals (CWRES) vs. time and prediction, and individual profile plots. Simulation properties were tested with a visual predictive check (VPC). Parameter stability was investigated using a nonparametric bootstrap. Modeling was undertaken using NONMEM v. 7.3Further covariates tested in the following order were: albumin and total plasma protein on ft > MIC for MIC values in the range 0.25–8 mg/L. The 5th, 10th, 20th, and 50th percentiles were presented to show the minimum ft > MIC for 95, 90, 80, and 50% of patients, respectively. A target of 0.5 for ft > MIC with a MIC cutoff of 2 mg/L (the current EUCAST resistance breakpoint) was considered. For metronidazole, a pharmacodynamic target of area under the curve to MIC ratio AUC(0‐24)/MIC of 708 and the ft > MIC of 8 mg/L were investigated.A simulated population of 1,000 subjects with demographics sampled from the study population was generated and the ceftriaxone model was used to simulate the The FLACSAM trial, including this PK study, is funded by the United Kingdom Medical Research Council/Department for International Development/Wellcome Trust Joint Global Health Trials Scheme [MR/M007367/1]. J.F.S. was supported by a United Kingdom Medical Research Council Fellowship (grant MR/M008665/1) and at institution level by the National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London. J.A.B is supported by the United Kingdom Medical Research Council/Department for International Development/Wellcome Trust Joint Global Health Trials Scheme [MR/M007367/1] and the Bill & Melinda Gates Foundation for the Childhood Acute Illness & Nutrition Network (CHAIN) [OPP1131320].The authors declare no competing interests for this work.J.F.S. and J.A.B. wrote the article; J.A.B., M.O.O., B.O., J.F.S., and K.K. designed the research; C.A.O., R.A., N.M., N.K., M.T., C.M., L.M., V.L.B., S.M., M.O.O., and K.K. performed the research; J.F.S., S.M., M.O.O., and J.A.B. analyzed the data.Supplementary MaterialClick here for additional data file.


The relationship between thyroid nodules (TNs) and adiposity is controversial. This paper describes a cross-sectional investigation performed to determine the existence of any such relationship. To assess adiposity, body mass index (BMI) and visceral fat area (VFA) were utilized.Between January 1, 2017 and March 3, 2019. Three thousand five hundred thirty four healthy people were examined using thyroid ultrasonography, visceral fat and anthropometric measurements, laboratory tests and questionnaire interview. Binary logistic regression analyses were used.2 and 39.67% (1402/3534) had VFA ≥ 100 cm2. After adjustment to address confounders, BMI-based overweight and obesity levels only correlated with higher risk TNs when used as a continuous variable , while VFA was both a continuous variable  and a categorical variable  associated with significantly elevated risk of TNs. Analyzing the subgroups, BMI ≥ 25 kg/m2  was significantly correlated with TN risk in individuals with TG ≥ 1.7 mmol/L. VFA ≥ 100 cm2 correlated with the TN risk irrespective of age  and in the following subgroups: women , FBG ≥ 6.1 mmol/L , and TG ≥ 1.7 mmol/L .Of the 3534 healthy subjects, 58.69% (2074/3534) of the subjects had TNs. A total of 55.91% (1976/3534) had BMI ≥ 25 kg/mAdiposity correlates with TNs. To assess TN risk in Chinese individuals, VFA is better than BMI. Among the most prevalent diseases in the population are thyroid nodules (TNs), the incidence of which is rising along in parallel with technological progress in treatments and diagnosis, lifestyle changes and environmental pollution. Many reports have stated that between 4 and 7% of TNs are diagnosed by palpation and 19 and 67% are found using high-resolution ultrasound . DelimitThe association between TNs and adiposity have been investigated by many studies , 3–5. ThAdipose tissue distribution, and particularly visceral fat area (VFA), can be accurately quantified using computed tomography, magnetic resonance imaging, and bioelectrical impedance analysis –16. It iWe aimed to bring additional clarity to the association of TNs with adiposity, using BMI and VFA as the measurement of adiposity. BMI and VFA were compared to determine the better indicator of TNs risk.The study was carried out among healthy residents in Beijing, at Beijing Rehabilitation Hospital between January 1, 2017 and March 1, 2019. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Beijing Rehabilitation Hospital, Capital Medical University. The inclusion criteria included: (1) age over 25 years and under 90 years; (2) history of regular check up at the medical center in the past; and (3) meeting the requirements of the SENIEUR Protocol on the definition of healthy adults . The excAfter obtaining written informed consent, the eligible participants were asked to fast overnight and were interviewed by trained personnel using a structured questionnaire about thyroid disease and other conditions, smoking and high salt intake status and demographic characteristics. A similar questionnaire was used by Bin Song et al. . Partici2, following the adult obesity criteria established by the Japan Society for the Study of Obesity [2 and obesity as BMI ≥ 30 kg/m2, following the World Health Organization criteria [A TN was defined as a small lesion differentiated from the rest of the thyroid parenchyma, possessing a solid part, with or without the presence of a cystic part . Ultraso Obesity . BMI wascriteria . Normal P < 0.05 was regarded as statistically significant. Forest plots were drawn using R software, version 3.6.0 .Data analysis was performed using the Statistical Package for the Social Sciences, version 22 . Percentage and counts were used to report categorical data. Continuous data were described using mean and standard deviation (SD), or median and interquartile range (IR) if the data did not follow a normal distribution. The Kolmogorov-Smirnov test was used to determine whether the data were normally distributed. The chi-square test and unpaired t-test were used to assess the significance of the proportions or mean differences among individuals with or without TNs, respectively. Odd ratios (ORs) and 95% confidence intervals (95% CIs) were calculated using binary logistic regression analyses, in order to determine associations of TNs with BMI and VFA. Twelve probable risk factors, that is, TC, HDL-C, LDL-C, TG, FBG, UA, DBP, SDP, high salt intake and smoking status, age and gender were considered using adjusted logistic regression models. Subgroups analyses were performed using VFA and BMI as categorical variables and dividing the participants by four probable risk factors: gender, age (< 50 or ≥ 50 years), FBG (< 6.1 or ≥ 6.1 mmol/L), and TG (< 1.7 or ≥ 1.7 mmol/L). P = 0.000). Of the 3534 healthy subjects, mean BMI was 25.78 ± 3.75 kg/m2 and mean VFA was 91.27 ± 39.42 cm2; 55.91% (1976/3534) of these subjects had BMI ≥ 25 kg/m2, and 39.67% (1402/3534) had VFA ≥ 100 cm2. The individuals with TNs were significantly older than those without TNs  and had a significantly higher proportion of females , higher BMI , higher VFA , higher SBP , and higher proportion of VFA ≥ 100 cm2 . Individuals with or without TNs were similar in terms of DBP, UA, TG, LDL-C, HDL-C, TC, smoking status, and high salt intake status  and 143 subjects with previous thyroid diseases, 3534 healthy subjects  were included in the analysis , maintaining its statistical association with TNs after adjusting the model for gender, age, smoking, high salt intake status, SBP, DBP, UA, FBG, TG, LDL-C, HDL-C and TC . The categorical variable BMI was not significantly associated with TNs in either the unadjusted or the adjusted model: the OR for individuals with BMI ≥ 25 kg/m2 relative to those with BMI < 25 kg/m2 was 1.080  in the unadjusted model and the OR in adjusted model was 1.101 . The continuous variable VFA was significantly related to higher risk of TNs in the unadjusted model  and also in the adjusted model . The categorical variable VFA maintained its significant relation with TNs in both unadjusted  and adjusted  models.Figure 2 was associated with higher TN risk significantly only in individuals with TG ≥ 1.7 mmol/L , while VFA ≥ 100 cm2 significantly correlated with increased risk of TNs in women , individuals < 50 years old , individuals ≥50 years old , individuals with FBG ≥ 6.1 mmol/L  and individuals with TG ≥ 1.7 mmol/L . Only VFA ≥ 100 cm2 (P for interaction = 0.000) had a statistically significant interaction effect with gender.Potential relations of TNs with VFA or BMI, both considered as categorical variables, were explored in various subgroups of study participants Fig. . BMI ≥ 2This study suggests that adiposity correlates with TNs. It is possible that adiposity was associated with insulin resistance and increased production of insulin and insulin-like growth factors, which in turn was associated with thyroid disorders –26. MoreIn the present study, elevated VFA is more strongly associated with higher risk of TNs than it is associated with BMI. BMI was associated with higher TNs risk only as a continuous variable, but not when defined as a categorical variable. This is similar to findings from previous studies. Bin Song et al. reported that waist circumference was superior to BMI for assessing risk of TNs in Chinese subjects. Weimin Xu et al. found there is a stronger relationship among TNs and body surface area than among TNs and BMI, both of which can be used to better indicate the risk of TNs in presence of obesity and overweight. This lack of accuracy in the predictability or as an indicator of BMI for TNs risk may be due to the fact that it is a nonspecific measurement method for adiposity, merging bone and abdominal adipose tissue and muscle mass measurements .VFA was associated with higher TN risk both as a categorical and as a continuous variable, suggesting a more important role of VFA in the predictability of TNs risk. Many researchers have found that accumulation of visceral fat can promote lipid synthesis because of the presence of increased amounts of free fatty acids, inducing metabolic syndrome through glycogen heterogenesis . The mai2 was significantly related to TN risk in almost all groups, while BMI ≥ 25 kg/m2 was only significantly related to increased risk of TN in the TG ≥ 1.7 mmol/L group. This supports the notion that VFA ≥ 100 cm2 is more accurate for indicating TN risk than a general obesity measurement such as BMI.Subgroup analysis demonstrated that VFA ≥ 100 cm2 than in men with VFA ≥ 100 cm2. There have been many studies reporting the association between TNs and women. Ju-Yeon found that healthy Korean women who were < 160 cm and ≥ 60 kg were at risk of TNs [We found that the prevalence of TNs in women was higher than that of men. Subgroup analysis also resulted in the finding of a higher risk of TNs in women with VFA ≥ 100 cmk of TNs . Xu founk of TNs . Howeverk of TNs . Anotherk of TNs . The exaThe current study has some limitations: 1) it was a cross-sectional study and did not reflect any temporal impact; 2) the numbers of subjects in our study were modest; 3) we did not test thyroid function and therefore could not take them as cofounders when analyzing the data; and 4) this was a single-center study; therefore, the results may not be generalizable. Nevertheless, this study is the first report for the relationship between VFA and TNs; we set strict inclusion and exclusion criteria, our study subjects were healthy individuals without thyroid surgery or treatment that may affect the function of thyroid and the excretion of iodine, and we used binary logistic regression analysis to control confounding factors when analyzing the association of TNs with BMI and VFA. Therefore, the present findings remain valuable. In the future, a multicenter study with a larger sample size, a more detailed collection of biochemical indicators, a comprehensive questionnaire, and a long follow-up period may provide more valuable data.Adiposity is related with TNs. Elevated VFA is more strongly associated with higher risk of TNs than it is associated with BMI, possibly serving as a potential indicator for TNs risk. Maintaining normal VFA may be helpful to avoid the appearance of TNs, especially in women.


AS) present severe intellectual disability, lack of speech, ataxia, seizures, abnormal electroencephalography (EEG), and a characteristic behavioral phenotype. Around 10% of patients with a clinical diagnosis of AS (AS‐like) do not have an identifiable molecular defect. Some of these patients harbor alternative genetic defects that present overlapping features with AS.Patients affected by Angelman syndrome , which is a rare congenital syndrome. Clinical reevaluation of the patient confirmed the presence of characteristic clinical features of CSS, many of them overlapping with AS.We identified a novel heterozygous CSS should be added to the expanding list of differential diagnoses for AS.Taking into account the novel finding reported in this study, we consider that  Around 10% of patients with a clinical diagnosis of AS are not molecularly confirmed  is a neurodevelopmental disorder characterized by severe intellectual disability, lack of speech, ataxia of gait, seizures, a characteristic electroencephalography (EEG), and a unique behavior that includes any combination of happy demeanor, easily excitable personality, frequent laughter, and stereotypes Bird, . AS is cSMARCE1 (OMIM 603111) splicing variant in a patient with an Angelman‐like phenotype.Genomic approaches such as array comparative genomic hybridization and whole‐exome sequencing have already been useful to identify alternative genes responsible for other heterogeneous genetic diseases such as Rett, Kleefstra, and Smith–Magenis syndromes . In addition, subtelomeric MLPA (SALSA MLPA P070), Autism MLPA (SALSA MLPA P343‐C1) and 60K array‐based comparative genomic hybridization (aCGH) were performed with normal results.The proband is a 14‐year‐old boy who was born at term to non‐consanguineous parents following a normal pregnancy. The patient had sucking difficulties during the neonatal period. At the age of 3 months, he suffered from seizures and at 6 months, he was found to be hypotonic. He presented global developmental delay: He sat unsupported at 12 months, walked independently at 27 months, and speaks only three words. Dentition was delayed. At 2 years old, neurological examination detected severe intellectual disability, ataxia of gait, receptive and non‐verbal communication skills higher than verbal ones and frequent drooling. He presented an abnormal electroencephalogram (EEG), although it was not the characteristic found in AS patients. The behavioral phenotype included happy demeanor, easily excitable personality, hyperactivity, attention deficit, stereotypies, attraction to water, aggressiveness, and autistic features. A clinical suspicion of AS was raised which was not confirmed molecularly. AS testing included methylation PCR of the 15q11.2‐q13 region, 2.3Trio whole‐exome sequencing of the patient and his parents was performed using the SureSelect Human All Exon V5+UTR kit . Sequencing was performed on an Illumina Hiseq2000 platform  producing 2x100nt paired‐end reads at the National Centre of Genomic Analysis . High‐quality reads were aligned to the GRCh37 decoy reference genome used by the 1000 genomes project (hs37d5) using the GEM3 aligner and variants identified following GATK Best Practices . Primers amplifying the region, including exons 3, 4, 5, 6, and 7 of SMARCE1 gene (NCBI RefSeq NM_003079.4), were designed in order to analyze mRNA splicing.RNA was extracted from isolated peripheral blood buffy coat stored at −196°C in liquid nitrogen using the Biostic Blood Total RNA Isolation Kit Sample , and cDNA synthesis was carried out using the PrimeScript3SMARCE1 gene (NCBI RefSeq NM_003079.4) after filtering the data according to a dominant de novo model of inheritance, a population allele frequency of <1/1,000 and a predicted impact on the protein. Variants in the SMARCE1 gene are known to cause Coffin–Siris syndrome , which is a rare congenital syndrome affecting many organs, characterized by moderate to severe intellectual disability  encoding subunits of the switch/sucrose non‐fermenting (SWI/SNF) ATP remodeling complex cause CSS  in the G>T in th et al.,  reportedThe patient described here shows almost all the consistent and frequent clinical features associated to AS (Williams, Driscoll, & Dagli, SMARCE1 pathogenic variant showed the presence of clinical features associated to CSS but not to AS (Table Clinical reevaluation of the patient after the identification of the AS Table . CSS patSMARCE1 missense pathogenic variants have been reported (Zarate et al., SMARCE1, who had a diagnosis of AS‐like and who presents some clinical features characteristic of AS, which have not been previously associated to CSS. Taking into account these results, we believe that CSS should be added to the expanding list of differential diagnoses for AS, probably accounting for some of the molecularly undiagnosed AS‐like patients.To date, only six individuals with The increasing use of exome sequencing in diagnostic laboratories will allow the analysis of all those genes that are involved in severe neurodevelopment disorders in patients who present AS clinical features, improving the diagnostic rate, and providing knowledge of the phenotypic spectrum of AS‐like causative genes, among them those responsible for Coffin–Siris syndrome.The authors declare no conflict of interest.