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Highlights • Neonatal handling affects the developmental course of cannabinoid receptors in rat brain. • In adult handled rats, [3H]CP55,940 binding levels are reduced in CPu, NAc and BLA. • Dorsal hippocampal [3H]CP55,940 binding levels are reduced only in female handled rats. • Handling increases adolescent and decreases adult [3H]CP55,940 binding levels in mPFC., Neonatal handling is an experimental model of early life experience associated with resilience in later life challenges, altering the ability of animals to respond to stress. The endocannabinoid system of the brain modulates the neuroendocrine and behavioral effects of stress, while this system is also capable of being modulated by stress exposure itself. The present study has addressed the question of whether neonatal handling in rats could affect cannabinoid receptors, in an age- and sex-dependent manner, using in situ hybridization and receptor binding techniques. Different effects of neonatal handling were observed in adolescent and adult brain on CB1 receptor mRNA and [3H]CP55,940 binding levels, which in some cases were sexually dimorphic. Neonatal handling interfered in the developmental trajectories of CB1 receptor mRNA levels in striatum and amygdaloid nuclei, as well as of [3H]CP55,940 binding levels in almost all regions studied. Adult handled rats showed reduced [3H]CP55,940 binding levels in the prefrontal cortex, striatum, nucleus accumbens and basolateral amygdala, while binding levels in prefrontal cortex of adolescent handled rats were increased. Finally, handling resulted in decreases in female [3H]CP55,940 binding levels in the striatum, nucleus accumbens, CA3 and DG of dorsal hippocampus and basolateral amygdala. Our results suggest that a brief and repeated maternal separation during the neonatal period induces changes on cannabinoid receptors differently manifested between adolescence and adulthood, male and female brain, which could be correlated to their stress response.

The level of rescue of clock function in genetically arrhythmic Drosophila melanogaster hosts using interspecific clock gene transformation was used to study the putative intermolecular coevolution between interacting clock proteins. Among them PER and TIM are the two important negative regulators of the circadian clock feedback loop. We transformed either the D. pseudoobscura per or tim transgenes into the corresponding arrhythmic D. melanogaster mutant (per01 or tim01) and observed >50% rhythmicity but the period of activity rhythm was either longer (D. pseudoobscura-per) or shorter than 24 h (D. pseudoobscura-tim) compared to controls. By introducing both transgenes simultaneously into double mutants, we observed that the period of the activity rhythm was rescued by the pair of hemizygous transgenes (~24 h). These flies also showed a more optimal level of temperature compensation for the period. Under LD 12:12 these flies have a D. pseudoobscura like activity profile with the absence of morning anticipation as well as a very prominent earlier evening peak of activity rhythm. These observation are consistent with the view that TIM and PER form a heterospecific coevolved module at least for the circadian period of activity rhythms. However the strength of rhythmicity was reduced by having both transgenes present, so while evidence for a coevolution between PER and TIM is observed for some characters it is not for others., Highlights • To understand the mechanism of intermolecular coevolution between period and its partner timeless, transgenes from D. pseudoobscura were introduced into D. melanogaster null mutants. • The single D. melanogaster transformants containing either per or tim from D. pseudoobscura displayed less than 50% rhythmicity and the period of locomotor activity rhythms was also either very long (26-31h for perps) or very short (21-22h for timps). • The two heterospecific transgenes together in the double mutant D. melanogaster flies could not show a significant increase in levels of rhythmicity however a significant improvement in the average period of locomotor activity was obtained (~24h). • Temperature compensation in the period of locomotor activity was also improved in these double tranformants. The Western blots and qPCR confirmed the expression of these transgenes.

Objectives Activation of the bile acid (BA) receptors farnesoid X receptor (FXR) or G protein-coupled bile acid receptor (GPBAR1; TGR5) improves metabolic homeostasis. In this study, we aim to determine the impact of pharmacological activation of bile acid receptors by INT-767 on reversal of diet-induced metabolic disorders, and the relative contribution of FXR vs. TGR5 to INT-767's effects on metabolic parameters. Methods Wild-type (WT), Tgr5−/−, Fxr−/−, Apoe−/− and Shp−/− mice were used to investigate whether and how BA receptor activation by INT-767, a semisynthetic agonist for both FXR and TGR5, could reverse diet-induced metabolic disorders. Results INT-767 reversed HFD-induced obesity dependent on activation of both TGR5 and FXR and also reversed the development of atherosclerosis and non-alcoholic fatty liver disease (NAFLD). Mechanistically, INT-767 improved hypercholesterolemia by activation of FXR and induced thermogenic genes via activation of TGR5 and/or FXR. Furthermore, INT-767 inhibited several lipogenic genes and de novo lipogenesis in the liver via activation of FXR. We identified peroxisome proliferation-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (CEBPα) as novel FXR-regulated genes. FXR inhibited PPARγ expression by inducing small heterodimer partner (SHP) whereas the inhibition of CEBPα by FXR was SHP-independent. Conclusions BA receptor activation can reverse obesity, NAFLD, and atherosclerosis by specific activation of FXR or TGR5. Our data suggest that, compared to activation of FXR or TGR5 only, dual activation of both FXR and TGR5 is a more attractive strategy for treatment of common metabolic disorders., Highlights • Bile acid receptor activation improves lipid homeostasis primarily via activation of FXR. • Bile acid receptor activation reverses DIO and induces energy expenditure. • Bile acid receptor activation reverses diet-induced NAFLD and atherosclerosis. • Bile acid receptor activation inhibits hepatic lipogenesis via activation of FXR. • Activation of FXR inhibits PPARγ dependent on SHP and CEBPα independent of SHP.

Protein-based vaccines offer a safer alternative to live-attenuated or inactivated vaccines but have limited immunogenicity. The identification of adjuvants that augment immunogenicity, specifically in a manner that is durable and antigen-specific, is therefore critical for advanced development. In this study, we use the filovirus virus-like particle (VLP) as a model protein-based vaccine in order to evaluate the impact of four candidate vaccine adjuvants on enhancing long term protection from Ebola virus challenge. Adjuvants tested include poly-ICLC (Hiltonol), MPLA, CpG 2395, and alhydrogel. We compared and contrasted antibody responses, neutralizing antibody responses, effector T cell responses, and T follicular helper (Tfh) cell frequencies with each adjuvant's impact on durable protection. We demonstrate that in this system, the most effective adjuvant elicits a Th1-skewed antibody response and strong CD4 T cell responses, including an increase in Tfh frequency. Using immune-deficient animals and adoptive transfer of serum and cells from vaccinated animals into naïve animals, we further demonstrate that serum and CD4 T cells play a critical role in conferring protection within effective vaccination regimens. These studies inform on the requirements of long term immune protection, which can potentially be used to guide screening of clinical-grade adjuvants for vaccine clinical development., Highlights • Adjuvants can prolong the protection afforded by protein-based vaccines and impact adaptive immune responses • Enhanced CD4 T cell responses, helper and effector, correlate with duration of protection • Durable protection from ma-EBOV is associated with Tfh frequency, Th1 antibody titers, and effector CD4 T cells Protein-based vaccines are extremely safe, but they sometimes require the addition of adjuvants to enhance immunogenicity. In this study, we compared the impact of multiple adjuvants on immunogenicity, focusing on the duration of vaccine-mediated protection in mice. We then looked at how each adjuvant impacted the immune response in order to identify correlates of that long lasting immunity. The most effective adjuvant/vaccine combinations elicited multifunctional CD4 T cell responses and a Th1-skewed antibody response. By transferring antigen-experienced CD4 T cells and serum into naïve animals, we demonstrated that both CD4 T cells and serum were critical for durable vaccine-mediated protection.