Exercise Rescues Obesogenic-Related Genes in the Female Hypothalamic Arcuate Nucleus: A Potential Role of miR-211 Modulation.

Obesity is a major public health concern that is associated with negative health outcomes. Exercise and dietary restriction are commonly recommended to prevent or combat obesity. This study investigates how voluntary exercise mitigates abnormal gene expression in the hypothalamic arcuate nucleus (ARC) of diet-induced obese (DIO) rats. Using a transcriptomic approach, novel genes in the ARC affected by voluntary wheel running were assessed alongside physiology, pharmacology, and bioinformatics analysis to evaluate the role of miR-211 in reversing obesity. Exercise curbed weight gain and fat mass, and restored ARC gene expression. High-fat diet (HFD) consumption can dysregulate satiety/hunger mechanisms in the ARC. Transcriptional clusters revealed that running altered gene expression patterns, including inflammation and cellular structure genes. To uncover regulatory mechanisms governing gene expression in DIO attenuation, we explored miR-211, which is implicated in systemic inflammation. Exercise ameliorated DIO overexpression of miR-211, demonstrating its pivotal role in regulating inflammation in the ARC. Further, in vivo central administration of miR-211-mimic affected the expression of immunity and cell cycle-related genes. By cross-referencing exercise-affected and miR-211-regulated genes, potential candidates for obesity reduction through exercise were identified. This research suggests that exercise may rescue obesity through gene expression changes mediated partially through miR-211.

Embryonic heat conditioning in chicks induces transgenerational heat/immunological resilience via methylation on regulatory elements.

The question of whether behavioral traits are heritable is under debate. An obstacle in demonstrating transgenerational inheritance in mammals originates from the maternal environment's effect on offspring phenotype. Here, we used in ovo embryonic heat conditioning (EHC) of first-generation chicks, demonstrating heredity of both heat and immunological resilience, confirmed by a reduced fibril response in their untreated offspring to either heat or LPS challenge. Concordantly, transcriptome analysis confirmed that EHC induces changes in gene expression in the anterior preoptic hypothalamus (APH) that contribute to these phenotypes in the offspring. To study the association between epigenetic mechanisms and trait heritability, DNA-methylation patterns in the APH of offspring of control versus EHC fathers were evaluated. Genome-wide analysis revealed thousands of differentially methylated sites (DMSs), which were highly enriched in enhancers and CCCTC-binding factor (CTCF) sites. Overlap analysis revealed 110 differentially expressed genes that were associated with altered methylation, predominantly on enhancers. Gene-ontology analysis shows pathways associated with immune response, chaperone-mediated protein folding, and stress response. For the proof of concept, we focused on HSP25 and SOCS3, modulators of heat and immune responses, respectively. Chromosome conformational capture (3C) assay identified interactions between their promoters and methylated enhancers, with the strongest frequency on CTCF binding sites. Furthermore, gene expression corresponded with the differential methylation patterns, and presented increased CTCF binding in both hyper- and hypomethylated DMSs. Collectively, we demonstrate that EHC induces transgenerational thermal and immunological resilience traits. We propose that one of the mechanisms underlying inheritance depends on three-dimensional (3D) chromatin reorganization.

Early-life thermal stress mediates long-term alterations in hypothalamic microglia.

Stressful environmental events in early life have long-lasting consequences on later stress responses. We previously showed that heat conditioning of 3-day-old chicks during the critical period of heat-response development leads to heat vulnerability later in life. Here we assessed the role of early-life heat stress on the inflammatory response in the chick anterior hypothalamus (AH), focusing on hypothalamic microglia. We identified the microglial cell population in the chick AH using anti-KUL01 and anti-CD45 antibodies. Specific microglial features were also confirmed by expression of their signature genes. Under normal environmental conditions, hypothalamic microglia isolated from lipopolysaccharide (LPS)-injected chicks displayed a classical activated proinflammatory profile accompanied by a decreased homeostatic signature, demonstrating similarity of immune response with mammalian microglial cells. In accordance with our previous observations, conditioning of 3-day-old chicks under high ambient temperature decreased the number of newborn cells in the AH, among them microglial precursors. Although heat exposure did not affect microglial cell viability, it had a long-term impact on LPS-induced inflammatory response. Exposure to harsh heat led to heat vulnerability, and attenuated recruitment of peripheral monocytes and T cells into the AH following LPS challenge. Moreover, heat conditioning altered microglial reactivity, manifested as suppressed microglial activation in response to LPS. Innate immune memory developed by heat conditioning might underlie suppression of the microglial response to LPS challenge. We describe alterations in genome-wide CpG methylation profile of hypothalamic microglia, demonstrating probable epigenetic involvement in the reprogramming of microglial function, leading to heat-induced inflammatory cross-tolerance.

Early-life epigenetic changes along the corticotropin-releasing hormone (CRH) gene influence resilience or vulnerability to heat stress later in life.

Stressful events in early life might lead to stress resilience or vulnerability, depending on an adjustable stress-response set-point, which can be altered during postnatal sensory development and involves epigenetic regulation of corticotropin-releasing hormone (CRH). During the critical developmental period of thermal-control establishment in 3-day-old chicks, heat stress was found to affect both body temperature and expression of CRH in the hypothalamic paraventricular nucleus. Both increased during heat challenge in vulnerable chicks, whereas they decreased in resilient chicks. Our aim was to elucidate the epigenetic mechanism underlying the regulation of stress resilience or vulnerability. Accordingly, DNA CpG methylation (5mC) and hydroxymethylation (5hmC) at the CRH intron, which we found to serve as a repressor element, displayed low 5mc% alongside high 5hmc% in resilient chicks, and high 5mc% with low 5hmc% in vulnerable ones. RE1-silencing transcription factor (REST), which has a binding site on this intron, bound abundantly during acute heat stress and was nearly absent during moderate stress, restricting repression by the repressor element, and thus activating CRH gene transcription. Furthermore, REST assembled into a protein complex with TET3, which bound directly to the CRH gene. Finally, the adjacent histone recruited the histone acetylation enzyme GCN5 to this complex, which increased H3K27ac during harsh, but not moderate heat conditioning. We conclude that an epigenetic mechanism involving both post-translational histone modification and DNA methylation in a regulatory segment of CRH is involved in determining a resilient or vulnerable response to stress later in life.

Methyl CpG level at distal part of heat-shock protein promoter HSP70 exhibits epigenetic memory for heat stress by modulating recruitment of POU2F1-associated nucleosome-remodeling deacetylase (NuRD) complex.

Depending on its stringency, exposure to heat in early life leads to either resilience or vulnerability to heat stress later in life. We hypothesized that epigenetic alterations in genes belonging to the cell proteostasis pathways are attributed to long-term responses to heat stress. Epigenetic regulation of the mRNA expression of the molecular chaperone heat-shock protein (HSP) 70 (HSPA2) was evaluated in the chick hypothalamus during the critical period of thermal-control establishment on day 3 post-hatch and during heat challenge on day 10. Both the level and duration of HSP70 expression during heat challenge a week after heat conditioning were more pronounced in chicks conditioned under harsh versus mild temperature. Analyzing different segments of the promoter in vitro indicated that methylation of a distal part altered its transcriptional activity. In parallel, DNA-methylation level of this segment in vivo was higher in harsh- compared to mild-heat-conditioned chicks. Hypermethylation of the HSP70 promoter in high-temperature-conditioned chicks was accompanied by a reduction in both POU Class 2 Homeobox 1 (POU2F1) binding and recruitment of the nucleosome remodeling deacetylase (NuRD) chromatin-remodeling complex. As a result, histone H3 acetylation levels at the HSP70 promoter were higher in harsh-temperature-conditioned chicks than in their mild-heat-conditioned counterparts. These results suggest that methylation level of a distal part of the HSP70 promoter and POU2F1 recruitment may reflect heat-stress-related epigenetic memory and may be useful in differentiating between individuals that are resilient or vulnerable to stress.

Fear of clowns in hospitalized children: prospective experience.

Medical clowns (MC) have become an integral part of the pediatric staff of hospital wards. While several studies have demonstrated the huge benefits of MC, there are almost no data regarding fear of clowns, a known phenomenon that means an irrational fear of clowns. In the current study, we sought to examine the prevalence of fear of clowns in pediatrics wards, and to characterize the affected children. The clinical work of three certified MCs was prospectively assessed. Every child with fear of clowns was noted, data were retrieved from the medical records, and the parents/child completed a specific questionnaire with a research assistant. Fear of clowns was defined as crying, anxiety response or effort to avoid contact with the MCs in small children, while in older children, it was determined if the child either reported fear of MCs or made actions to avoid clowns' intervention. A total of 1160 children participated in the study. All were hospitalized in the department of pediatrics or the pediatric emergency medicine department at Carmel Medical Center, and were exposed to a MC intervention session. Of the 1160 children, 14 children experienced fear of clowns (1.2%). The average age of children who experienced fear of clowns was 3.5 years (range 1-15). Interestingly, most of the children demonstrating fear of clowns were girls (12 out of 14, 85.7%). We found no association between fear of clowns and specific diagnosis, fever, clinical appearance, religion, or ethnicity. CONCLUSION: The prevalence of fear of clowns in the general pediatric hospitalized population was 1.2%, with a significant predominance of girls (85.7%). Children who experienced significant fear of clowns also experienced significant fear of encountering or thinking about a MC visit. Fear of clowns can affect children at any age (range 1-15), any ethnicity, religion, or degree of illness. Further large scale studies are required to better understand this unique phenomenon of fear of clowns. What is Known: • Fear of clowns is a phenomenon known for more than several decades and related to the increased use of clowns as negative characters in horror movies and TV shows. • The increased use of medical clowns in hospital wards and corridors increases the significance of defining and characterizing this phenomenon in hospital wards. What is New: • The study is novel by giving new data related to the extent of fear of clowns in pediatrics wards and giving demographic characteristic of children experiencing fear of clowns.

Assistance of Medical Clowns Improves the Physical Examinations of Children Aged 2-6 Years.

BACKGROUND: A good physical exam is necessary to help pediatricians make the correct diagnosis and can save unnecessary imaging or invasive procedures. Distraction by medical clowns may create the optimal conditions for a proper physical examination. METHODS: Children aged 2-6 years who required physical examination in the pediatric emergency department were recruited and randomly assigned to one of two groups: physical exam by a pediatrician in the presence of caregivers vs. physical exam with the assistance of a medical clown. Outcome measures consisted of the level of child's discomfort, anxiety, and the quality of the physical examination. RESULTS: Ninety three children participated. Mean age was 3.3 ± 3.6 years (range 2-6). The duration of the physical exam was similar between the clown and control groups (4.6 ± 1.4 minutes vs. 4.5 ± 1.1 minutes (P = 0.64). The duration of discomfort was shorter in the clown group (0.2 ± 0.6 minutes) than the control group(1.6 ± 2.0 minutes, P = 0.001). In the medical clown group, 94% of pediatricians reported that the medical clown improved their ability to perform a complete physical examination. A trend of less hospitalization in the medical clown group was also noticed (11.3% in the medical clown group vs. 18.3% in the control group, P = 0.1); however, further study is required to verify this observation. CONCLUSIONS: Integration of a medical clown in physical examination improves the overall experience of the child and the caregivers and helps the pediatrician to perform a complete physical examination.

Overweight and CpG methylation of the Pomc promoter in offspring of high-fat-diet-fed dams are not "reprogrammed" by regular chow diet in rats.

This study aimed to determine whether epigenetic malprogramming induced by high-fat diet (HFD) has an obesogenic effect on nonmated and mated female rats and their offspring. Further, it aimed to reprogram offspring's epigenetic malprogramming and phenotype by providing normal diet after weaning. Body weight (BW) was measured, and plasma and hypothalamic arcuate nuclei were collected for analysis of hormones, mRNA, and DNA CpG methylation of the promoter of Pomc, a key factor in control of food intake. In nonmated females, HFD decreased Pomc/leptin ratio by ∼38%. This finding was associated with Pomc promoter hypermethylation. While heavier during pregnancy, during lactation HFD dams showed sharper BW decrease (2.5-fold) and loss of Pomc promoter hypermethylation. Moreover, their weight loss was correlated with demethylation (r=-0.707) and with gadd45b mRNA expression levels (r=0.905). Even though offspring of HFD dams ate standard chow from weaning, they displayed increased BW, Pomc promoter hypermethylation, and vulnerability to HFD challenge (3-fold kilocalorie intake increase). These findings demonstrate a long-term effect of maternal HFD on CpG methylation of the Pomc promoter in the offspring, which was not reprogrammed by standard chow from weaning. Further, the results suggest a possible mechanism of demethylation of the Pomc promoter following pregnancy and lactation.

Epigenetic Regulation is involved in Reversal of Obesity.

Epigenetic processes play a crucial role in mediating the impact of environmental energetic challenges, from overconsumption to starvation. Over-nutrition of energy-dense foods and sedentary lifestyles contribute to the development of obesity, characterized by excessive fat storage and impaired metabolic signaling, stemming from disrupted brain signaling. Conversely, dieting and physical activity facilitate body weight rebalancing and trigger adaptive neural responses. These adaptations involve the upregulation of neurogenesis, synaptic plasticity and optimized brain function and energy homeostasis, balanced hormone signaling, normal metabolism, and reduced inflammation. The transition of the brain from a maladaptive to an adaptive state is partially guided by epigenetic mechanisms. While epigenetic mechanisms underlying obesity-related brain changes have been described, their role in mediating the reversal of maladaptation/obesity through lifestyle interventions remains less explored. This review focuses on elucidating epigenetic mechanisms involved in hypothalamic adaptations induced by lifestyle interventions. Given that lifestyle interventions are widely prescribed and accessible approaches for weight loss and maintenance, it is our challenge to uncover epigenetic mechanisms moderating these hypothalamic-functional beneficial changes.

Embryonic manipulations shape life-long, heritable stress responses through complex epigenetic mechanisms: a review.

Enhancing an organism's likelihood of survival hinges on fostering a balanced and adaptable development of robust stress response systems. This critical process is significantly influenced by the embryonic environment, which plays a pivotal role in shaping neural circuits that define the stress response set-point. While certain embryonic conditions offer advantageous outcomes, others can lead to maladaptive responses. The establishment of this response set-point during embryonic development can exert life-long and inheritable effects on an organism's physiology and behavior. This review highlights the significance of multilevel epigenetic regulation and the intricate cross-talk among these layers in response to heat stress during the embryonic period, with a particular focus on insights gained from the avian model.

Paternal high-fat diet affects weight and DNA methylation of their offspring.

Obesity poses a public health threat, reaching epidemic proportions. Our hypothesis suggests that some of this epidemic stems from its transmission across generations via paternal epigenetic mechanisms. To investigate this possibility, we focused on examining the paternal transmission of CpG methylation. First-generation male Wistar rats were fed either a high-fat diet (HF) or chow and were mated with females fed chow. We collected sperm from these males. The resulting offspring were raised on a chow diet until day 35, after which they underwent a dietary challenge. Diet-induced obese (DIO) male rats passed on the obesogenic trait to both male and female offspring. We observed significant hypermethylation of the Pomc promoter in the sperm of HF-treated males and in the hypothalamic arcuate nucleus (Arc) of their offspring at weaning. However, these differences in Arc methylation decreased later in life. This hypermethylation is correlated with increased expression of DNMT3B. Further investigating genes in the Arc that might be involved in obesogenic transgenerational transmission, using reduced representation bisulfite sequencing (RRBS) we identified 77 differentially methylated regions (DMRs), highlighting pathways associated with neuronal development. These findings support paternal CpG methylation as a mechanism for transmitting obesogenic traits across generations.

Medical clowns improve sleep and shorten hospitalization duration in hospitalized children.

Intervention by medical clowns was proven to have a positive effect in reducing stress and anxiety, increasing cooperation and improving the child's experience prior to a medical procedure and during the various stages of hospitalization. Sleep has long been known to be essential for recovery from injury and sickness, improving immune functions, and there is an emerging understanding of the restorative role quality sleep has on health and diseases. Hospitalized children are more exposed to sleep disorders and sleep deprivation due to the hospitalized environment, anxiety, and illness. Different behavioral interventions to promote sleep were previously studied in hospitalized children, some showing potential benefits. In this study, we sought to examine the ability of medical clowns to positively impact the child's sleep during hospitalization. The study is an observational matching (case-control) interventional study which took place at the department of pediatrics in Carmel Medical Center. Forty-two hospitalized children ages 2-17 were included in two equal groups of intervention or control. Children in the control group were recruited based on a method of matching the chief complaint plus the medical diagnosis and age of the children in the intervention group in a 1:1 matching. The children's sleep parameters were objectively evaluated for two consecutive nights using an Actigraph device and subjectively by parent's questionnaire. Additional factors such as hospital length of stay and demographics were also monitored. The study group had an encounter with a medical clown (15-30 min) before bedtime on either the first or the second night, and the control group was not exposed to a medical clown at all. We then compared the data from both groups using unpaired t-tests. Hospitalized children exposed to a medical clown prior to bedtime (n = 21) and children not exposed to a medical clown (n = 21) were comparable in age and clinical characteristics. The study group had a significantly delayed wake-up time compared to the control group (06:59 ± 46 min vs. 07:26 ± 42 min, p < 0.05) (mean difference of 27 min). Night's duration (from bedtime to wake-up) was significantly longer in the study versus the control group (570 ± 76 vs. 500 ± 66.1 min, p < 0.05), a total mean increase of 70 min, and sleep efficiency were significantly increased (92.3 ± 4.6% vs. 87.9 ± 8.7%, p < 0.05). Within the clown group, when comparing nights with and without exposure to a medical clown, total sleep time was prolonged by a mean of 54 min on the night of the intervention (518 ± 74 min vs. 464 ± 59 min, p < 0.01), and the total wake time during the night were reduced (52 ± 27 min vs. 77 ± 61 min, P < 0.05), mean difference of 25 min), mainly by reduction of wake period after sleep onset (WASO) (42 ± 25 min vs. 66 ± 58 min, p < 0.05), mean difference of 24 min). Regarding general medical outcomes, hospital stay was significantly shorter in the clown group vs. control (104 ± 42 h vs. 128 ± 42 h, p < 0.05), a mean reduction of 23 h-nearly an entire day. An encounter with a medical clown before bedtime in hospitalized children positively affects sleep parameters, which may be of great importance for healing in general. The clown intervention was also shown to shorten the hospital stay. Larger scale studies are warranted to establish these findings.

Medical clowning in hospitalized children: a meta-analysis.

BACKGROUND: Medical clowning has been proven effective in reducing pain, anxiety, and stress in many sporadic, usually small-scale studies. Our meta-analysis aims to evaluate the efficiency of medical clowns in reducing pain and anxiety in hospitalized pediatric patients and their parents in different medical fields. METHODS: A thorough literature search was conducted from different databases, and only randomized controlled trials (RCTs) were included with children aged 0 to 18 years old. A total of 18 studies were included, and statistical analysis was performed on the combined data. RESULTS: A total of 912 children (14 studies) showed significantly reduced anxiety when procedures were performed with a medical clown compared with the controls (- 0.76 on anxiety score, P < 0.001). Preoperative anxiety was lower in 512 children (nine studies) with clown interventions than in the controls (- 0.78, P < 0.001). The pain scale was completed by 338 participants (six studies), indicating a trend toward reduced pain during procedures performed while the clown was acting compared to controls (- 0.49, P = 0.06). In addition, medical clown significantly (- 0.52, P = 0.001) reduced parental anxiety in 489 participants in ten studies; in six of the ten studies, with a total of 380 participants, medical clown significantly reduced parental preoperative anxiety (P = 0.02). CONCLUSION: Medical clowns have substantial positive and beneficial effects on reducing stress and anxiety in children and their families in various circumstances in pediatrics.

Dieting reverses histone methylation and hypothalamic AgRP regulation in obese rats.

Introduction: Although dieting is a key factor in improving physiological functions associated with obesity, the role by which histone methylation modulates satiety/hunger regulation of the hypothalamus through weight loss remains largely elusive. Canonically, H3K9me2 is a transcriptional repressive post-translational epigenetic modification that is involved in obesity, however, its role in the hypothalamic arcuate nucleus (ARC) has not been thoroughly explored. Here we explore the role that KDM4D, a specific demethylase of residue H3K9, plays in energy balance by directly modulating the expression of AgRP, a key neuropeptide that regulates hunger response. Methods: We used a rodent model of diet-induced obesity (DIO) to assess whether histone methylation malprogramming impairs energy balance control and how caloric restriction may reverse this phenotype. Using ChIP-qPCR, we assessed the repressive modification of H3K9me2 at the site of AgRP. To elucidate the functional role of KDM4D in reversing obesity via dieting, a pharmacological agent, JIB-04 was used to inhibit the action of KDM4D in vivo. Results: In DIO, downregulation of Kdm4d mRNA results in both enrichment of H3K9me2 on the AgRP promoter and transcriptional repression of AgRP. Because epigenetic modifications are dynamic, it is possible for some of these modifications to be reversed when external cues are altered. The reversal phenomenon was observed in calorically restricted rats, in which upregulation of Kdm4d mRNA resulted in demethylation of H3K9 on the AgRP promoter and transcriptional increase of AgRP. In order to verify that KDM4D is necessary to reverse obesity by dieting, we demonstrated that in vivo inhibition of KDM4D activity by pharmacological agent JIB-04 in naïve rats resulted in transcriptional repression of AgRP, decreasing orexigenic signaling, thus inhibiting hunger. Discussion: We propose that the action of KDM4D through the demethylation of H3K9 is critical in maintaining a stable epigenetic landscape of the AgRP promoter, and may offer a target to develop new treatments for obesity.

MiR-138 inhibits EZH2 methyltransferase expression and methylation of histone H3 at lysine 27, and affects thermotolerance acquisition.

Thermotolerance acquisition involves neuronal network remodeling and, hence, alteration in the repertoire of expressed proteins. We have previously demonstrated the role of histone H3 methylation at lysine 27 (H3K27) by EZH2 methyltransferase in the regulation of gene expression during the critical period for the establishment of thermal control in chicks. Here we describe another level of biological regulation, demonstrating the inhibitory role of microRNAs (miRNAs) in the regulation of EZH2 expression in thermoregulatory system development and functioning. During heat conditioning in the critical period for the establishment of thermal control, a decrease in expression of the EZH2-targeting miR-138 occurred simultaneously with an increase in EZH2 levels in the preoptic anterior hypothalamus. Intracranial injection of miR-138 during the critical period led to a transient reduction in EZH2 levels, which was accompanied by a decrease in H3K27 methylation. Injection of miR-138 followed by heat conditioning also abolished EZH2 induction during heat conditioning. Moreover, this miR-138-induced inhibition of EZH2 during the critical period resulted in a long-term effect on EZH2 expression. A week after the treatment, the EZH2 protein levels in conditioned and in nonconditioned chicks were different from those in their saline-injected counterparts and the directions of change were opposite to each other. Finally, miR-138 injection during the critical period disrupted the establishment of thermoregulation, manifested as a defective body temperature response to heat. These data demonstrate a role for miRNAs in regulating the expression of histone-modifying enzymes, and thus emphasize the multilevel regulation mechanism which includes both epigenetic and miRNA regulatory mechanisms in neuronal network organization during the critical period of sensory development.

Age-Specific Modulation of Prefrontal Cortex LTP by Glucocorticoid Receptors Following Brief Exposure to HFD.

The corticolimbic circuits in general and the medial prefrontal cortex in particular, undergo maturation during juvenility. It is thus expected that environmental challenges in forms of obesogenic diet can exert different effects in juvenile animals compared to adults. Further, the relationship between glucocorticoids and obesity has also been demonstrated in several studies. As a result, glucocorticoid receptor (GR) antagonists are currently being tested as potential anti-obesity agents. In the present study, we examined the effects of short-term exposure to high-fat diet (HFD) on prefrontal long-term potentiation (LTP) in both juvenile and adult rats, and the role of glucocorticoid receptors (GRs) in modulating these effects. We found HFD impaired prefrontal LTP in both juveniles and adults, but the effects of GR modulation were age- and diet-dependent. Specifically, GR antagonist RU-486 reversed the impairment of LTP in juvenile animals following HFD, and had no effect on control-diet animals. In adult animals, RU-486 has no effect on HFD-impaired LTP, but abolished LTP in control-diet animals. Furthermore, impairments in the prefrontal LTP following HFD are involved with an increase in the mPFC GR levels only in the juveniles. Further, we found that in vivo application of GR agonists into adult mPFC rescued HFD-induced impairment in LTP, suggesting that these receptors might represent strategic therapeutic targets to potentially combat obesity and metabolic related disorder.

Cross-tolerance: embryonic heat conditioning induces inflammatory resilience by affecting different layers of epigenetic mechanisms regulating IL6 expression later in life.

A stressor can induce resilience in another, different stressor, a phenomenon known as cross-tolerance. To learn if cross-tolerance is governed by epigenetic regulation, we used embryonic heat conditioning (EHC) in chicks, during the development of the hypothalamus, to increase the immunization response. Indeed, EHC induced a lifelong systemic antibody response to immunization, in addition to reduced hypothalamic IL6 inflammatory expression following LPS challenge. Since the outcome of EHC was long-term cross-tolerance with the immune system, we studied possible epigenetic mechanisms. We first analysed the methylation and hydroxymethylation patterns of IL6. We found reduced hydroxymethylation on IL6 intron 1 in the EHC group, a segment enriched with CpGs and NFkB-binding sites. Luciferase assay in cell lines expressing NFkB showed that IL6 intron 1 is indeed an enhancer. ChiP in the same segment against NFkB in the hypothalamus presented reduced binding to IL6 intron 1 in the EHC group, before and during LPS challenge. In parallel, EHC chicks' IL6 intron 1 presented increased H3K27me3, a repressive translational modification mediated by EZH2. This histone modification occurred during embryonic conditioning and persisted later in life. Moreover, we showed reduced expression of miR-26a, which inhibits EZH2 transcription, during conditioning along with increased EZH2 expression. We demonstrate that stress cross-tolerance, which was indicated by EHC-induced inflammatory resilience and displayed by attenuated inflammatory expression of IL6, is regulated by different epigenetic layers.

Saving Costs for Hospitals Through Medical Clowning: A Study of Hospital Staff Perspectives on the Impact of the Medical Clown.

This study explores the perspectives of doctors, nurses, and technicians on the medical clown. A total of 35 subjects were interviewed one on one. Interviews were transcribed and analyzed for common themes. Medical staff outlined novel limitations and views of future directions for the field of medical clowning. Most importantly, many previously unpublished benefits of medical clowns were described. These reported benefits included cost-saving measures for the hospital, increases in staff efficiency, better patient outcomes, and lower stress in medical staff. Given that most of the limitations on medical clowning are financial in nature, these findings have substantial implications for the future of the field. As medical clowning continues to grow, this study outlines the potential for various future research projects within this field of study.

Early-Life m6A RNA Demethylation by Fat Mass and Obesity-Associated Protein (FTO) Influences Resilience or Vulnerability to Heat Stress Later in Life.

Early life heat stress leads to either resilience or vulnerability to a similar stress later in life. We have previously shown that this tuning of the stress response depends on neural network organization in the preoptic anterior hypothalamus (PO/AH) thermal response center and is regulated by epigenetic mechanisms. Here, we expand our understanding of stress response establishment describing a role for epitranscriptomic regulation of the epigenetic machinery. Specifically, we explore the role of N6-methyladenosine (m6A) RNA methylation in long-term response to heat stress. Heat conditioning of 3-d-old chicks diminished m6A RNA methylation in the hypothalamus, simultaneously with an increase in the mRNA levels of the m6A demethylase, fat mass and obesity-associated protein (FTO). Moreover, a week later, methylation of two heat stress-related transcripts, histone 3 lysine 27 (H3K27) methyltransferase, enhancer of zeste homolog 2 (EZH2) and brain-derived neurotrophic factor (BDNF), were downregulated in harsh-heat-conditioned chicks. During heat challenge a week after conditioning, there was a reduction of m6A levels in mild-heat-conditioned chicks and an elevation in harsh-heat-conditioned ones. This increase in m6A modification was negatively correlated with the expression levels of both BDNF and EZH2 Antisense "knock-down" of FTO caused an elevation of global m6A RNA methylation, reduction of EZH2 and BDNF mRNA levels, and decrease in global H3K27 dimethylation as well as dimethyl H3K27 level along BDNF coding region, and, finally, led to heat vulnerability. These findings emphasize the multilevel regulation of gene expression, including both epigenetic and epitranscriptomic regulatory mechanisms, fine-tuning the neural network organization in a response to stress.

Embryonic Heat Conditioning Induces TET-Dependent Cross-Tolerance to Hypothalamic Inflammation Later in Life.

Early life encounters with stress can lead to long-lasting beneficial alterations in the response to various stressors, known as cross-tolerance. Embryonic heat conditioning (EHC) of chicks was previously shown to mediate resilience to heat stress later in life. Here we demonstrate that EHC can induce cross-tolerance with the immune system, attenuating hypothalamic inflammation. Inflammation in EHC chicks was manifested, following lipopolysaccharide (LPS) challenge on day 10 post-hatch, by reduced febrile response and reduced expression of LITAF and NFκB compared to controls, as well as nuclear localization and activation of NFκB in the hypothalamus. Since the cross-tolerance effect was long-lasting, we assumed that epigenetic mechanisms are involved. We focused on the role of ten-eleven translocation (TET) family enzymes, which are the mediators of active CpG demethylation. Here, TET transcription during early life stress was found to be necessary for stress resilience later in life. The expression of the TET family enzymes in the midbrain during conditioning increased in parallel to an elevation in concentration of their cofactor α-ketoglutarate. In-ovo inhibition of TET activity during EHC, by the α-ketoglutarate inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (BPTES), resulted in reduced total and locus specific CpG demethylation in 10-day-old chicks and reversed both thermal and inflammatory resilience. In addition, EHC attenuated the elevation in expression of the stress markers HSP70, CRHR1, and CRHR2, during heat challenge on day 10 post-hatch. This reduction in expression was reversed by BPTES. Similarly, the EHC-dependent reduction of inflammatory gene expression during LPS challenge was eliminated in BPTES-treated chicks. Thus, TET family enzymes and CpG demethylation are essential for the embryonic induction of stress cross-tolerance in the hypothalamus.