Running Title; CRISPR analysis of an enhancer controlling ethanol intake.

Latina mothers, who have the highest fertility rate among all ethnic groups in the US, are often exposed to discrimination. The biological impacts of this discrimination are unknown. This study is the first to explore the relationship between discrimination and DNA methylation of stress regulatory genes in Latinas. Our sample was Latina women (n = 147) with a mean age of 27.6 years who were assessed at 24-32 weeks' gestation (T1) and 4-6 weeks postpartum (T2) and reside in the U.S. Blood was collected at T1, and the Everyday Discrimination Scale (EDS) was administered at T1 and T2. DNA Methylation at candidate gene regions was determined by bisulphite pyrosequencing. Associations between EDS and DNA methylation were assessed via zero-inflated Poisson models, adjusting for covariates and multipletest comparisons. Discrimination was negatively associated with methylation at CpG sites within the glucocorticoid receptor (NR3C1) and brain-derived neurotrophic factor (BDNF) genes that were consistent over time. In addition, discrimination was negatively associated with methylation of a CpG in the glucocorticoid binding protein (FKBP5) at T1 but not at T2. This study underscores the complex biological pathways between discrimination and epigenetic modification in Latina women that warrant further investigation to better understand the genetic and psychopathological impact of discrimination on Latino mothers and their families.

HIV-1 maturation inhibitors are a novel class of antiretroviral compounds that consist of two structurally distinct chemical classes: betulinic acid derivatives and the pyridone-based compound PF-46396. It is currently believed that both classes act by similar modes of action to generate aberrant noninfectious particles via inhibition of CA-SP1 cleavage during Gag proteolytic processing. In this study, we utilized a series of novel analogues with decreasing similarity to PF-46396 to determine the chemical groups within PF-46396 that contribute to antiviral activity, Gag binding, and the relationship between these essential properties. A spectrum of antiviral activity (active, intermediate, and inactive) was observed across the analogue series with respect to CA-SP1 cleavage and HIV-1 (NL4-3) replication kinetics in Jurkat T cells. We demonstrate that selected inactive analogues are incorporated into wild-type (WT) immature particles and that one inactive analogue is capable of interfering with PF-46396 inhibition of CA-SP1 cleavage. Mutations that confer PF-46396 resistance can impose a defective phenotype on HIV-1 that can be rescued in a compound-dependent manner. Some inactive analogues retained the capacity to rescue PF-46396-dependent mutants (SP1-A3V, SP1-A3T, and CA-P157S), implying that they can also interact with mutant Gag. The structure-activity relationships observed in this study demonstrate that (i) the tert-butyl group is essential for antiviral activity but is not an absolute requirement for Gag binding, (ii) the trifluoromethyl group is optimal but not essential for antiviral activity, and (iii) the 2-aminoindan group is important for antiviral activity and Gag binding but is not essential, as its replacement is tolerated.IMPORTANCE Combinations of antiretroviral drugs successfully treat HIV/AIDS patients; however, drug resistance problems make the development of new mechanistic drug classes an ongoing priority. HIV-1 maturation inhibitors are novel as they target the Gag protein, specifically by inhibiting CA-SP1 proteolytic cleavage. The lack of high-resolution structural information of the CA-SP1 target in Gag has hindered our understanding of the inhibitor-binding pocket and maturation inhibitor mode of action. Therefore, we utilized analogues of the maturation inhibitor PF-46396 as chemical tools to determine the chemical components of PF-46396 that contribute to antiviral activity and Gag binding and the relationship between these essential properties. This is the first study to report structure-activity relationships of the maturation inhibitor PF-46396. PF-46396 is chemically distinct from betulinic acid-derived maturation inhibitors; therefore, our data provide a foundation of knowledge that will aid our understanding of how structurally distinct maturation inhibitors act by similar modes of action.

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AbstractExcess maternal fat intake and obesity increase offspring susceptibility to conditions such as chronic anxiety and substance abuse. We hypothesised that these susceptibilities are established through environmentally modulated DNA-methylation (5mC/5hmC) changes in regions of the genome that modulate mood and consumptive behaviours. We explored the effects of environmental factors on 5mC/5hmC levels within the GAL5.1 enhancer that controls anxiety-related behaviours and alcohol intake. We first observed that 5mC/5hmC levels within the GAL5.1 enhancer differed significantly in different parts of the brain. Moreover, we noted that early life stress had no significant effect of 5mC/5hmC levels within GAL5.1. By contrast, we identified that allowing access of pregnant mothers to high-fat diet (>60% calories from fat) had a significant effect on 5mC/5hmC levels within GAL5.1 in hypothalamus and amygdala of resulting male offspring. Cell transfection based studies using GAL5.1 reporter plasmids showed that 5mC has a significant repressive effect on GAL5.1 activity and its response to known stimuli such as EGR1 expression and PKC agonism. Intriguingly, CRISPR driven disruption of GAL5.1 from the mouse genome, although having negligible effects on metabolism, significantly decreased intake of high fat diet suggesting that GAL5.1, in addition to being epigenetically modulated by high-fat diet, also actively contributes to the consumption of high-fat diet suggesting its involvement in an environmentally-influenced regulatory-loop. Furthermore, considering that GAL5.1 also controls alcohol preference and anxiety these studies may provide a first glimpse into an epigenetically controlled mechanism that links maternal high-fat diet with transgenerational susceptibility to alcohol abuse and anxiety.