Reprogramming of trunk neural crest to a cranial crest-like identity alters their transcriptome and developmental potential.

Neural crest cells along the body axis of avian embryos differ in their developmental potential, such that the cranial neural crest forms cartilage and bone whereas the trunk neural crest is unable to do so. Previous studies have identified a cranial crest-specific subcircuit that can imbue the trunk neural crest with the ability to form cartilage after grafting to the head. Here, we examine transcriptional and cell fate changes that accompany this reprogramming. First, we examined whether reprogrammed trunk neural crest maintain the ability to form cartilage in their endogenous environment in the absence of cues from the head. The results show that some reprogrammed cells contribute to normal trunk neural crest derivatives, whereas others migrate ectopically to the forming vertebrae and express cartilage markers, thus mimicking heterotypically transplanted cranial crest cells. We find that reprogrammed trunk neural crest upregulated more than 3000 genes in common with cranial neural crest, including numerous transcriptional regulators. In contrast, many trunk neural crest genes are downregulated. Together, our findings show that reprogramming trunk neural crest with cranial crest subcircuit genes alters their gene regulatory program and developmental potential to be more cranial crest-like.

Ribosome heterogeneity results in leader sequence-mediated regulation of protein synthesis in Francisella tularensis.

Although ribosomes are generally examined in aggregate, ribosomes can be heterogenous in composition. Evidence is accumulating that changes in ribosome composition may result in altered function, such that ribosome heterogeneity may provide a mechanism to regulate protein synthesis. Ribosome heterogeneity in the human pathogen Francisella tularensis results from incorporation of one of three homologs of bS21, a small ribosomal subunit protein demonstrated to regulate protein synthesis in other bacteria. Loss of one homolog, bS21-2, results in genome-wide post-transcriptional changes in protein abundance. This suggests that bS21-2 can, either directly or indirectly, lead to preferential translation of particular mRNAs. Here, we examine the potential of bS21-2 to function in a leader sequence-dependent manner and to function indirectly, via Hfq. We found that the 5´ untranslated region (UTR) of some bS21-2-responsive genes, including key virulence genes, is sufficient to alter translation in cells lacking bS21-2. We further identify features of a 5´ UTR that allow responsiveness to bS21-2. These include an imperfect Shine-Dalgarno sequence and a particular six nucleotide sequence. Our results are consistent with a model in which a bS21 homolog increases the efficiency of translation initiation through interactions with specific leader sequences. With respect to bS21-2 indirectly regulating translation via the RNA-binding protein Hfq, we found that Hfq controls transcript abundance rather than protein synthesis, impacting virulence gene expression via a distinct mechanism. Together, we determined that ribosome composition in F. tularensis regulates translation in a leader sequence-dependent manner, a regulatory mechanism which may be used in other bacteria. IMPORTANCE Ribosome heterogeneity is common in bacteria, and there is mounting evidence that ribosome composition plays a regulatory role in protein synthesis. However, mechanisms of ribosome-driven gene regulation are not well understood. In the human pathogen Francisella tularensis, which encodes multiple homologs for the ribosomal protein bS21, loss of one homolog impacts protein synthesis and virulence. Here, we explore the mechanism behind bS21-mediated changes in protein synthesis, finding that they can be linked to altered translation initiation and are dependent on specific sequences in the leaders of transcripts. Our data support a model in which ribosome composition regulates gene expression through translation, a strategy that may be conserved in diverse organisms with various sources of ribosome heterogeneity.

Molecular Modeling Predicts Novel Antibody Escape Mutations in the Respiratory Syncytial Virus Fusion Glycoprotein.

Monoclonal antibodies are increasingly used for the prevention and/or treatment of viral infections. One caveat of their use is the ability of viruses to evolve resistance to antibody binding and neutralization. Computational strategies to identify viral mutations that may disrupt antibody binding would leverage the wealth of viral genomic sequence data to monitor for potential antibody-resistant mutations. The respiratory syncytial virus is an important pathogen for which monoclonal antibodies against the fusion (F) protein are used to prevent severe disease in high-risk infants. In this study, we used an approach that combines molecular dynamics simulations with FoldX to estimate changes in free energy in F protein folding and binding to the motavizumab antibody upon each possible amino acid change. We systematically selected 8 predicted escape mutations and tested them in an infectious clone. Consistent with our F protein stability predictions, replication-effective viruses were observed for each selected mutation. Six of the eight variants showed increased resistance to neutralization by motavizumab. Flow cytometry was used to validate the estimated (model-predicted) effects on antibody binding to F. Using surface plasmon resonance, we determined that changes in the on-rate of motavizumab binding were associated with the reduced affinity for two novel escape mutations. Our study empirically validated the accuracy of our molecular modeling approach and emphasized the role of biophysical protein modeling in predicting viral resistance to antibody-based therapeutics that can be used to monitor the emergence of resistant viruses and to design improved therapeutic antibodies. IMPORTANCE Respiratory syncytial virus (RSV) causes severe disease in young infants, particularly those with heart or lung diseases or born prematurely. Because no vaccine is currently available, monoclonal antibodies are used to prevent severe RSV disease in high-risk infants. While it is known that RSV evolves to avoid recognition by antibodies, screening tools that can predict which changes to the virus may lead to antibody resistance are greatly needed.

Prenatal exposures to per- and polyfluoroalkyl substances and epigenetic aging in umbilical cord blood: The Healthy Start study.

BACKGROUND: Per- and polyfluoroalkyl substances (PFAS) are ubiquitous, environmentally persistent chemicals, and prenatal exposures have been associated with adverse child health outcomes. Prenatal PFAS exposure may lead to epigenetic age acceleration (EAA), defined as the discrepancy between an individual's chronologic and epigenetic or biological age. OBJECTIVES: We estimated associations of maternal serum PFAS concentrations with EAA in umbilical cord blood DNA methylation using linear regression, and a multivariable exposure-response function of the PFAS mixture using Bayesian kernel machine regression. METHODS: Five PFAS were quantified in maternal serum (median: 27 weeks of gestation) among 577 mother-infant dyads from a prospective cohort. Cord blood DNA methylation data were assessed with the Illumina HumanMethylation450 array. EAA was calculated as the residuals from regressing gestational age on epigenetic age, calculated using a cord-blood specific epigenetic clock. Linear regression tested for associations between each maternal PFAS concentration with EAA. Bayesian kernel machine regression with hierarchical selection estimated an exposure-response function for the PFAS mixture. RESULTS: In single pollutant models we observed an inverse relationship between perfluorodecanoate (PFDA) and EAA (-0.148 weeks per log-unit increase, 95% CI: -0.283, -0.013). Mixture analysis with hierarchical selection between perfluoroalkyl carboxylates and sulfonates indicated the carboxylates had the highest group posterior inclusion probability (PIP), or relative importance. Within this group, PFDA had the highest conditional PIP. Univariate predictor-response functions indicated PFDA and perfluorononanoate were inversely associated with EAA, while perfluorohexane sulfonate had a positive association with EAA. CONCLUSIONS: Maternal mid-pregnancy serum concentrations of PFDA were negatively associated with EAA in cord blood, suggesting a pathway by which prenatal PFAS exposures may affect infant development. No significant associations were observed with other PFAS. Mixture models suggested opposite directions of association between perfluoroalkyl sulfonates and carboxylates. Future studies are needed to determine the importance of neonatal EAA for later child health outcomes.

SEMplMe: a tool for integrating DNA methylation effects in transcription factor binding affinity predictions.

MOTIVATION: Aberrant DNA methylation in transcription factor binding sites has been shown to lead to anomalous gene regulation that is strongly associated with human disease. However, the majority of methylation-sensitive positions within transcription factor binding sites remain unknown. Here we introduce SEMplMe, a computational tool to generate predictions of the effect of methylation on transcription factor binding strength in every position within a transcription factor's motif. RESULTS: SEMplMe uses ChIP-seq and whole genome bisulfite sequencing to predict effects of methylation within binding sites. SEMplMe validates known methylation sensitive and insensitive positions within a binding motif, identifies cell type specific transcription factor binding driven by methylation, and outperforms SELEX-based predictions for CTCF. These predictions can be used to identify aberrant sites of DNA methylation contributing to human disease. AVAILABILITY AND IMPLEMENTATION: SEMplMe is available from https://github.com/Boyle-Lab/SEMplMe .

Predicting the effects of SNPs on transcription factor binding affinity.

MOTIVATION: Genome-wide association studies have revealed that 88% of disease-associated single-nucleotide polymorphisms (SNPs) reside in noncoding regions. However, noncoding SNPs remain understudied, partly because they are challenging to prioritize for experimental validation. To address this deficiency, we developed the SNP effect matrix pipeline (SEMpl). RESULTS: SEMpl estimates transcription factor-binding affinity by observing differences in chromatin immunoprecipitation followed by deep sequencing signal intensity for SNPs within functional transcription factor-binding sites (TFBSs) genome-wide. By cataloging the effects of every possible mutation within the TFBS motif, SEMpl can predict the consequences of SNPs to transcription factor binding. This knowledge can be used to identify potential disease-causing regulatory loci. AVAILABILITY AND IMPLEMENTATION: SEMpl is available from https://github.com/Boyle-Lab/SEM_CPP. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Hnf4a Is Required for the Development of Cdh6-Expressing Progenitors into Proximal Tubules in the Mouse Kidney.

BACKGROUND: Hepatocyte NF 4α (Hnf4a) is a major regulator of renal proximal tubule (PT) development. In humans, a mutation in HNF4A impairs PT functions and is associated with Fanconi renotubular syndrome (FRTS). In mice, mosaic deletion of Hnf4a in the developing kidney reduces the population of PT cells, leading to FRTS-like symptoms. The molecular mechanisms underlying the role of Hnf4a in PT development remain unclear. METHODS: The gene deletion tool Osr2Cre removed Hnf4a in developing nephrons in mice, generating a novel model for FRTS. Immunofluorescence analysis characterized the mutant phenotype, and lineage analysis tested whether Cadherin-6 (Cdh6)-expressing cells are PT progenitors. Genome-wide mapping of Hnf4a binding sites and differential gene analysis of Hnf4a mutant kidneys identified direct target genes of Hnf4a. RESULTS: Deletion of Hnf4a with Osr2Cre led to the complete loss of mature PT cells, lethal to the Hnf4a mutant mice. Cdh6high, lotus tetragonolobus lectin-low (LTLlow) cells serve as PT progenitors and demonstrate higher proliferation than Cdh6low, LTLhigh differentiated PT cells. Additionally, Hnf4a is required for PT progenitors to differentiate into mature PT cells. Genomic analyses revealed that Hnf4a directly regulates the expression of genes involved in transmembrane transport and metabolism. CONCLUSIONS: Hnf4a promotes the differentiation of PT progenitors into mature PT cells by regulating the expression of genes associated with reabsorption, the major function of PT cells.

Fingolimod phosphate inhibits astrocyte inflammatory activity in mucolipidosis IV.

Mucolipidosis IV (MLIV) is an orphan neurodevelopmental disease that causes severe neurologic dysfunction and loss of vision. Currently there is no therapy for MLIV. It is caused by loss of function of the lysosomal channel mucolipin-1, also known as TRPML1. Knockout of the Mcoln1 gene in a mouse model mirrors clinical and neuropathologic signs in humans. Using this model, we previously observed robust activation of microglia and astrocytes in early symptomatic stages of disease. Here we investigate the consequence of mucolipin-1 loss on astrocyte inflammatory activation in vivo and in vitro and apply a pharmacologic approach to restore Mcoln1-/- astrocyte homeostasis using a clinically approved immunomodulator, fingolimod. We found that Mcoln1-/- mice over-express numerous pro-inflammatory cytokines, some of which were also over-expressed in astrocyte cultures. Changes in the cytokine profile in Mcoln1-/- astrocytes are concomitant with changes in phospho-protein signaling, including activation of PI3K/Akt and MAPK pathways. Fingolimod promotes cytokine homeostasis, down-regulates signaling within the PI3K/Akt and MAPK pathways and restores the lysosomal compartment in Mcoln1-/- astrocytes. These data suggest that fingolimod is a promising candidate for preclinical evaluation in our MLIV mouse model, which, in case of success, can be rapidly translated into clinical trial.

Mining the Unknown: Assigning Function to Noncoding Single Nucleotide Polymorphisms.

One of the formative goals of genetics research is to understand how genetic variation leads to phenotypic differences and human disease. Genome-wide association studies (GWASs) bring us closer to this goal by linking variation with disease faster than ever before. Despite this, GWASs alone are unable to pinpoint disease-causing single nucleotide polymorphisms (SNPs). Noncoding SNPs, which represent the majority of GWAS SNPs, present a particular challenge. To address this challenge, an array of computational tools designed to prioritize and predict the function of noncoding GWAS SNPs have been developed. However, fewer than 40% of GWAS publications from 2015 utilized these tools. We discuss several leading methods for annotating noncoding variants and how they can be integrated into research pipelines in hopes that they will be broadly applied in future GWAS analyses.

Decoy peptides derived from the extracellular domain of toll-like receptor 2 (TLR2) show anti-inflammatory properties.

Toll-like receptor 2 (TLR2) recognizes bacterial derived- and synthetic-lipopeptides after dimerization with TLR1 or TLR6. Hyper-activation of TLR2 has been described in several inflammatory diseases and the discovery of inhibitors of its pro-inflammatory activity represent potential starting points to develop therapeutics in such pathologies. We designed peptides derived from the TLR2 sequence comprising amino acid residues involved in ligand binding (Pam3CSK4) or heterodimerization (TLR2/TLR1) as pointed out by structural data.2 We identified several peptides (P13, P13(LL), P16, P16(LL)) which inhibited TLR2/1 signaling in HEK293-TLR2 cells (MAPK activation and NF-kB activity). Moreover, P13L and P16L decreased TNFα release in human primary PBMCs and mouse macrophages. The peptides were selective for TLR2/1 as they did not inhibit the activity of other TLRs tested. P13L and P16L inhibited the internalization of Pam3CSK4 fluorescently labeled in macrophages and the heterodimerization of TLR2 with TLR1 as demonstrated by immunoprecipitation studies. Our data demonstrate that peptides derived from the region comprising the leucine-rich repeats (LRR) 11 and 13 in the extracellular domain of TLR2 are good starting points to develop more potent anti-inflammatory peptides with TLR2 inhibitory activity.

Enhanced immunostimulatory activity of in silico discovered agonists of Toll-like receptor 2 (TLR2).

BACKGROUND: Emergent therapies in anticancer vaccination use Toll-like receptors (TLRs) agonists as dendritic cell (DC) vaccine adjuvants. DCs from the patient are isolated, stimulated with TLR agonists and tumor antigens ex vivo and then infused back into the patient. Although some TLR ligands have been tested in clinical trials, novel TLR agonists with improved immunomodulatory properties are essential to optimize treatment success. We report on the discovery of small-molecule TLR2 agonists, with favorable properties as synthetic adjuvants. METHODS: We performed a shape- and featured-based similarity virtual screening against a commercially available compound library. The selected virtual hits were experimentally tested in TLR2-reporter cells and their activity in phagocytes and DCs was characterized. A binding model of the compounds to TLR2 (docking studies) was proposed. RESULTS: Through a virtual screening approach against a library of three million compounds four virtual hits (AG1, AG2, AG3, AG4) were found to synergistically augment the NF-kB activation induced by the lipopeptide ligand Pam3CSK4 in luciferase reporter assays using HEK293-TLR2 cells. Biacore experiments indicated that AG1-AG4 are ago-allosteric modulators of TLR2 and AG2 bound TLR2 with high affinity (KD 0.8µM). The compounds induced TNF-α production in human peripheral blood mononuclear cells (PBMCs) and they activated DCs as indicated by IL-12 production and upregulation of CD83/CD86. CONCLUSIONS: Following a combined in silico/in vitro approach we have discovered TLR2-agonists (AG1-AG4) that activate human and mouse immune cells. GENERAL SIGNIFICANCE: We introduce four novel TLR2 ago-allosteric modulators that stimulate myeloid cell activity and constitute promising candidates as synthetic adjuvants.

Chromosome fusions triggered by noncoding RNA.

Chromosomal fusions are common in normal and cancer cells and can produce aberrant gene products that promote transformation. The mechanisms driving these fusions are poorly understood, but recurrent fusions are widespread. This suggests an underlying mechanism, and some authors have proposed a possible role for RNA in this process. The unicellular eukaryote Oxytricha trifallax displays an exorbitant capacity for natural genome editing, when it rewrites its germline genome to form a somatic epigenome. This developmental process provides a powerful model system to directly test the influence of small noncoding RNAs on chromosome fusion events during somatic differentiation. Here we show that small RNAs are capable of inducing chromosome fusions in 4 distinct cases (out of 4 tested), including one fusion of 3 chromosomes. We further show that these RNA-mediated chromosome fusions are heritable over multiple sexual generations and that transmission of the acquired fusion is associated with endogenous production of novel piRNA molecules that target the fused junction. We also demonstrate the capacity of a long noncoding RNA (lncRNA) to induce chromosome fusion of 2 distal germline loci. These results underscore the ability of short-lived, aberrant RNAs to act as drivers of chromosome fusion events that can be stably transmitted to future generations.

Expression, functionality, and localization of apurinic/apyrimidinic endonucleases in replicative and non-replicative forms of Trypanosoma cruzi.

Trypanosoma cruzi is the etiological agent of Chagas disease. The parasite has to overcome oxidative damage by ROS/RNS all along its life cycle to survive and to establish a chronic infection. We propose that T. cruzi is able to survive, among other mechanisms of detoxification, by repair of its damaged DNA through activation of the DNA base excision repair (BER) pathway. BER is highly conserved in eukaryotes with apurinic/apirimidinic endonucleases (APEs) playing a fundamental role. Previous results showed that T. cruzi exposed to hydrogen peroxide and peroxinitrite significantly decreases its viability when co-incubated with methoxyamine, an AP endonuclease inhibitor. In this work the localization, expression and functionality of two T. cruzi APEs (TcAP1, Homo sapiens APE1 orthologous and TcAP2, orthologous to Homo sapiens APE2 and to Schizosaccaromyces pombe Apn2p) were determined. These enzymes are present and active in the two replicative parasite forms (epimastigotes and amastigotes) as well as in the non-replicative, infective trypomastigotes. TcAP1 and TcAP2 are located in the nucleus of epimastigotes and their expression is constitutive. Epimastigote AP endonucleases as well as recombinant TcAP1 and TcAP2 are inhibited by methoxyamine. Overexpression of TcAP1 increases epimastigotes viability when they are exposed to acute ROS/RNS attack. This protective effect is more evident when parasites are submitted to persistent ROS/RNS exposition, mimicking nature conditions. Our results confirm that the BER pathway is involved in T. cruzi resistance to DNA oxidative damage and points to the participation of DNA AP endonucleases in parasite survival.

Manipulating the Maize (Zea mays) Microbiome.

Maize (Zea mays) is a multifaceted cereal grass used globally for nutrition, animal feed, food processing, and biofuels, and a model system in genetics research. Studying the maize microbiome sometimes requires its manipulation to identify the contributions of specific taxa and ecological traits (i.e., diversity, richness, network structure) to maize growth and physiology. Due to regulatory constraints on applying engineered microorganisms in field settings, greenhouse-based experimentation is often the first step for understanding the contribution of root-associated microbiota-whether natural or engineered-to plant phenotypes. In this protocol, we describe methods to inoculate maize with a specific microbiome as a tool for understanding the microbiota's influence on its host plant. The protocol involves removal of the native seed microbiome followed by inoculation of new microorganisms; separate protocols are provided for inoculations from pure culture, from soil slurry, or by mixing in live soil. These protocols cover the most common methods for manipulating the maize microbiome in soil-grown plants in the greenhouse. The methods outlined will ultimately result in rhizosphere microbial assemblages with varying degrees of microbial diversity, ranging from low diversity (individual strain and synthetic community [SynCom] inoculation) to high diversity (percent live inoculation), with the slurry inoculation method representing an "intermediate diversity" treatment.

Dealkylation of Poly(methyl methacrylate) by TiCl4 Vapor Phase Infiltration (VPI) and the Resulting Chemical and Thermophysical Properties of the Hybrid Material.

This study examines the chemical reaction pathways for vapor phase infiltration (VPI) of TiCl4 into poly(methyl methacrylate) (PMMA). VPI is a processing method that transforms organic polymers into organic-inorganic hybrid materials with new properties of interest for microelectronic patterning, technical textiles, and chemical separations. Understanding the fundamental chemical mechanisms of the VPI process is essential for establishing approaches to design the chemical structure and properties of these hybrid materials. While prior work has suggested that TiCl4 infiltration into PMMA does not disrupt the polymer's carbonyl bond, a clear reaction mechanism has yet to be proposed. Here, we present a detailed X-ray photoelectron spectroscopy study that presents evidence for a concerted reaction mechanism that involves TiCl4 coordinating with the PMMA's ester group to dealkylate the methyl side group, creating a chloromethane byproduct and primary chemical bonds between the organic and inorganic components of the hybrid material. Additional spectroscopy, quartz crystal microbalance gravimetry, and thermophysical and chemical property measurements of this material, including solubility studies and thermal expansion measurements, provide further evidence for this chemical reaction pathway and the subsequent creation of inorganic cross-links that network these TiOx-PMMA hybrid materials.

Differentially methylated regions interrogated for metastable epialleles associate with offspring adiposity.

Aim: Assess if cord blood differentially methylated regions (DMRs) representing human metastable epialleles (MEs) associate with offspring adiposity in 588 maternal-infant dyads from the Colorado Health Start Study.Materials & methods: DNA methylation was assessed via the Illumina 450K array (~439,500 CpG sites). Offspring adiposity was obtained via air displacement plethysmography. Linear regression modeled the association of DMRs potentially representing MEs with adiposity.Results & conclusion: We identified two potential MEs, ZFP57, which associated with infant adiposity change and B4GALNT4, which associated with infancy and childhood adiposity change. Nine DMRs annotating to genes that annotated to MEs associated with change in offspring adiposity (false discovery rate <0.05). Methylation of approximately 80% of DMRs identified associated with decreased change in adiposity.

[Box: see text].

Microbial short-chain fatty acids regulate drug seeking and transcriptional control in a model of cocaine seeking.

Cocaine use disorder represents a public health crisis with no FDA-approved medications for its treatment. A growing body of research has detailed the important connections between the brain and the resident population of bacteria in the gut, the gut microbiome, in psychiatric disease models. Acute depletion of gut bacteria results in enhanced reward in a mouse cocaine place preference model, and repletion of bacterially-derived short-chain fatty acid (SCFA) metabolites reverses this effect. However, the role of the gut microbiome and its metabolites in modulating cocaine-seeking behavior after prolonged abstinence is unknown. Given that relapse prevention is the most clinically challenging issue in treating substance use disorders, studies examining the effects of microbiome manipulations in relapse-relevant models are critical. Here, male Sprague-Dawley rats received either untreated water or antibiotics to deplete the gut microbiome and its metabolites. Rats were trained to self-administer cocaine and subjected to either within-session threshold testing to evaluate motivation for cocaine or 21 days of abstinence followed by a cue-induced cocaine-seeking task to model relapse behavior. Microbiome depletion did not affect cocaine acquisition on an fixed-ratio 1 schedule. However, microbiome-depleted rats exhibited significantly enhanced motivation for low dose cocaine on a within-session threshold task. Similarly, microbiome depletion increased cue-induced cocaine-seeking following prolonged abstinence and altered transcriptional regulation in the nucleus accumbens. In the absence of a normal microbiome, repletion of bacterially-derived SCFA metabolites reversed the behavioral and transcriptional changes associated with microbiome depletion. These findings suggest that gut bacteria, via their metabolites, are key regulators of drug-seeking behaviors, positioning the microbiome as a potential translational research target.

Repeated footshock stress induces an escalation of cocaine self-administration in male and female rats: Role of the cannabinoid receptor 1.

Stress is a significant contributor to the development and progression of substance use disorders (SUDs) and is problematic as it is unavoidable in daily life. Therefore, it is important to understand the neurobiological mechanisms that underlie the influence of stress on drug use. We have previously developed a model to examine the contribution of stress to drug-related behavior by administering a stressor, electric footshock stress, daily at the time of cocaine self-administration in rats resulting in an escalation of cocaine intake. This stress-induced escalation of cocaine intake involves neurobiological mediators of stress and reward such as cannabinoid signaling. However, all of this work has been conducted in male rats. Here we test the hypothesis that repeated daily stress can produce an escalation of cocaine in both male and female rats. We further hypothesize that cannabinoid receptor 1 (CB1R) signaling is recruited by repeated stress to influence cocaine intake in both male and female rats. Male and female Sprague-Dawley rats self-administered cocaine (0.5 mg/kg/inf, i.v.) during a modified short-access paradigm wherein the 2-hr access was separated into 4-30 min self-administration blocks separated by 4-5 min drug free period. Footshock stress produced a significant escalation of cocaine intake similarly in both male and female rats. Female stress-escalated rats did display greater time-out non-reinforced responding and greater "front-loading" behavior. In males, systemic administration of a CB1R inverse agonist/antagonist Rimonabant only attenuated cocaine intake in rats with a history of combined repeated stress and cocaine self-administration. However, in females, Rimonabant attenuated cocaine intake in the no stress control group but only at the highest dose of Rimonabant (3 mg/kg, i.p.) suggesting that females show a greater sensitivity to CB1R antagonism. However, female rats with a history of stress showed even greater sensitivity to CB1R antagonism as both doses of Rimonabant (1, 3 mg/kg) attenuated cocaine intake in stress-escalated rats similar to males. Altogether these data demonstrate that stress can produce significant changes in cocaine self-administration and suggests that repeated stress at the time of cocaine self-administration recruits CB1Rs to regulate cocaine-taking behavior across sexes.

Accelerated epigenetic age at birth and child emotional and behavioura development in early childhood: a meta-analysis of four prospective cohort studies in ECHO.

Background: 'Epigenetic clocks' have been developed to accurately predict chronologic gestational age and have been associated with child health outcomes in prior work.Methods: We meta-analysed results from four prospective U.S cohorts investigating the association between epigenetic age acceleration estimated using blood DNA methylation collected at birth and preschool age Childhood Behavior Checklist (CBCL) scores.Results: Epigenetic ageing was not significantly associated with CBCL total problem scores (ß = 0.33, 95% CI: -0.95, 0.28) and DSM-oriented pervasive development problem scores (ß = -0.23, 95% CI: -0.61, 0.15). No associations were observed for other DSM-oriented subscales.Conclusions: The meta-analysis results suggest that epigenetic gestational age acceleration is not associated with child emotional and behavioural functioning for preschool age group. These findings may relate to our study population, which includes two cohorts enriched for ASD and one preterm birth cohort.; future work should address the role of epigenetic age in child health in other study populations.Abbreviations: DNAm: DNA methylation; CBCL: Child Behavioral Checklist; ECHO: Environmental Influences on Child Health Outcomes; EARLI: Early Autism Risk Longitudinal Investigation; MARBLES: Markers of Autism Risk in Babies - Learning Early Signs; ELGAN: Extremely Low Gestational Age Newborns; ASD: autism spectrum disorder; BMI: body mass index; DSM: Diagnostic and Statistical Manual of Mental Disorders.

Herbivory Protection via Volatile Organic Compounds Is Influenced by Maize Genotype, Not Bacillus altitudinis-Enriched Bacterial Communities.

Belowground, plants interact with beneficial soil microbes such as plant growth-promoting rhizobacteria (PGPR). PGPR are rhizosphere bacteria that colonize roots and elicit beneficial effects in plants such as improved plant growth, pathogen resistance, abiotic stress tolerance, and herbivore protection. Treatment of plants with PGPR has been shown to trigger the emission of volatile organic compounds (VOCs). Volatile emissions can also be triggered by herbivory, termed herbivore-induced plant volatiles (HIPV), with important ramifications for chemical-mediated plant and insect interactions. Much of our current understanding of PGPR and herbivore-induced volatiles is based on studies using one plant genotype, yet domestication and modern breeding has led to the development of diverse germplasm with altered phenotypes and chemistry. In this study, we investigated if volatile emissions triggered by PGPR colonization and herbivory varies by maize genotype and microbial community assemblages. Six maize genotypes representing three decades of crop breeding and two heterotic groups were used, with four microbiome treatments: live or sterilized soil, with or without a Bacillus inoculant. Soil sterilization was used to delay microbiome establishment, resulting in low-diversity treatments. At planting, maize seeds were inoculated with PGPR Bacillus altitudinis AP-283 and grown under greenhouse conditions. Four weeks post planting, plants were subjected to feeding by third instar Helicoverpa zea (Lepidoptera: Noctuidae) larvae. Volatiles were collected using solid phase microextraction and analyzed with gas chromatography-mass spectrometry. Illumina NovaSeq 16S rRNA amplicon sequencing was carried out to characterize the rhizosphere microbiome. Maize genotype significantly influenced total volatile emissions, and relative abundance of volatile classes. We did not document a strong influence of microbe treatment on plant VOC emissions. However, inoculating plants with PGPR improved plant growth under sterile conditions. Taken together, our results suggest that genotypic variation is the dominant driver in HIPV composition and individual HIPV abundances, and any bacterial-mediated benefit is genotype and HIPV-specific. Therefore, understanding the interplay of these factors is necessary to fully harness microbially-mediated benefits and improve agricultural sustainability.