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.