Impact of Artificial Sputum Medium Formulation on Pseudomonas aeruginosa Secondary Metabolite Production.

In vitro culture media are being developed to understand how host site-specific nutrient profiles influence microbial pathogenicity and ecology. To mimic the cystic fibrosis (CF) lung environment, a variety of artificial sputum media (ASM) have been created. However, the composition of these ASM vary in the concentration of key nutrients, including amino acids, lipids, DNA, and mucin. In this work, we used feature-based molecular networking (FBMN) to perform comparative metabolomics of Pseudomonas aeruginosa, the predominant opportunistic pathogen infecting the lungs of people with CF, cultured in nine different ASM. We found that the concentration of aromatic amino acids and iron from mucin added to the media contributes to differences in the production of P. aeruginosa virulence-associated secondary metabolites. IMPORTANCE Different media formulations aiming to replicate in vivo infection environments contain different nutrients, which affects interpretation of experimental results. Inclusion of undefined components, such as commercial porcine gastric mucin (PGM), in an otherwise chemically defined medium can alter the nutrient content of the medium in unexpected ways and influence experimental outcomes.

Sex Differences in Nucleus Accumbens Transcriptome Profiles Associated with Susceptibility versus Resilience to Subchronic Variable Stress.

Depression and anxiety disorders are more prevalent in females, but the majority of research in animal models, the first step in finding new treatments, has focused predominantly on males. Here we report that exposure to subchronic variable stress (SCVS) induces depression-associated behaviors in female mice, whereas males are resilient as they do not develop these behavioral abnormalities. In concert with these different behavioral responses, transcriptional analysis of nucleus accumbens (NAc), a major brain reward region, by use of RNA sequencing (RNA-seq) revealed markedly different patterns of stress regulation of gene expression between the sexes. Among the genes displaying sex differences was DNA methyltransferase 3a (Dnmt3a), which shows a greater induction in females after SCVS. Interestingly, Dnmt3a expression levels were increased in the NAc of depressed humans, an effect seen in both males and females. Local overexpression of Dnmt3a in NAc rendered male mice more susceptible to SCVS, whereas Dnmt3a knock-out in this region rendered females more resilient, directly implicating this gene in stress responses. Associated with this enhanced resilience of female mice upon NAc knock-out of Dnmt3a was a partial shift of the NAc female transcriptome toward the male pattern after SCVS. These data indicate that males and females undergo different patterns of transcriptional regulation in response to stress and that a DNA methyltransferase in NAc contributes to sex differences in stress vulnerability. SIGNIFICANCE STATEMENT: Women have a higher incidence of depression than men. However, preclinical models, the first step in developing new diagnostics and therapeutics, have been performed mainly on male subjects. Using a stress-based animal model of depression that causes behavioral effects in females but not males, we demonstrate a sex-specific transcriptional profile in brain reward circuitry. This transcriptional profile can be altered by removal of an epigenetic mechanism, which normally suppresses DNA transcription, creating a hybrid male/female transcriptional pattern. Removal of this epigenetic mechanism also induces behavioral resilience to stress in females. These findings shed new light onto molecular factors controlling sex differences in stress response.

Activity-dependent Wnt 7 dendritic targeting in hippocampal neurons: plasticity- and tagging-related retrograde signaling mechanism?

Wnt proteins have emerged as transmembrane signaling molecules that regulate learning and memory as well as synaptic plasticity at central synapses (Inestrosa and Arenas (2010) Nat Rev Neurosci 11:77-86; Maguschak and Ressler (2011) J Neurosci 31:13057-13067; Tabatadze et al. (2012) Hippocampus 22: 1228-1241; Fortress et al. (2013) J Neurosci 33:12619-12626). For example, there is both a training-selective and Wnt isoform-specific increase in Wnt 7 levels in hippocampus seven days after spatial learning in rats (Tabatadze et al. (2012) Hippocampus 22: 1228-1241). Despite growing interest in Wnt signaling pathways in the adult brain, intracellular distribution and release of Wnt molecules from synaptic compartments as well as their influence on synaptic strength and connectivity remain less well understood. As a first step in such an analysis, we show here that Wnt 7 levels in primary hippocampal cells are elevated by potassium or glutamate activation in a time-dependent manner. Subsequent Wnt 7 elevation in dendrites suggests selective somato-dendritic trafficking followed by transport from dendrites to their spines. Wnt 7 elevation is also TTX-reversible, establishing that its elevation is indeed an activity-dependent process. A second stimulation given 6 h after the first significantly reduces Wnt 7 levels in dendrites 3 h later as compared to non-stimulated controls suggesting activity-dependent Wnt 7 release from dendrites and spines. In a related experiment designed to mimic the release of Wnt 7, exogenous recombinant Wnt 7 increased the number of active zones in presynaptic terminals as indexed by bassoon. This suggests the formation of new presynaptic release sites and/or presynaptic terminals. Wnt signaling inhibitor sFRP-1 completely blocked this Wnt 7-induced elevation of bassoon cluster number and cluster area. We suggest that Wnt 7 is a plasticity-related protein involved in the regulation of presynaptic plasticity via a retrograde signaling mechanism as previously proposed (Routtenberg (1999) Trends in Neuroscience 22:255-256). These findings provide support for this proposal, which offers a new perspective on the synaptic tagging mechanism (Redondo and Morris (2011) Nat Rev Neurosci 12:17-30).

Secondary metabolite profiling of Pseudomonas aeruginosa isolates reveals rare genomic traits.

Pseudomonas aeruginosa is a ubiquitous Gram-negative opportunistic pathogen with remarkable phylogenetic and phenotypic variabilities. In this work, we applied classical molecular networking analysis to secondary metabolite profiling data from seven Pseudomonas aeruginosa strains, including five clinical isolates from the lung secretions of people with cystic fibrosis (CF). We provide three vignettes illustrating how secondary metabolite profiling aids in the identification of rare genomics traits in P. aeruginosa. First, we describe the identification of a previously unreported class of acyl putrescines produced by isolate mFLRO1. Secondary analysis of publicly available metabolomics data revealed that acyl putrescines are produced by <5% of P. aeruginosa strains. Second, we show that isolate SH3A does not produce di-rhamnolipids. Whole-genome sequencing and comparative genomics revealed that SH3A cannot produce di-rhamnolipids because its genome belongs to clade 5 of the P. aeruginosa phylogenetic tree. Previous phylogenetic analysis of thousands of P. aeruginosa strains concluded that <1% of publicly available genome sequences contribute to this clade. Last, we show that isolate SH1B does not produce the phenazine pyocyanin or rhamnolipids because it has a one-base insertion frameshift mutation (678insC) in the gene rhlR, which disrupts rhl-driven quorum sensing. Secondary analysis of the tens of thousands of publicly available genomes in the National Center for Biotechnology Information (NCBI) and the Pseudomonas Genome Database revealed that this mutation was present in only four P. aeruginosa genomes. Taken together, this study highlights that secondary metabolite profiling combined with genomic analysis can identify rare genetic traits of P. aeruginosa isolates.IMPORTANCESecondary metabolite profiling of five Pseudomonas aeruginosa isolates from cystic fibrosis sputum captured three traits present in <1%-5% of publicly available data, pointing to how our current library of P. aeruginosa strains may not represent the diversity within this species or the genetic variance that occurs in the CF lung.

MBRA-2: a Modified Chemostat System to Culture Biofilms.

Culture-dependent approaches for investigating microbial ecology aim to model the nutrient content of specific environments by simplifying the system for high-resolution molecular analysis. These in vitro systems are enticing due to their increased throughput compared to animal models, flexibility in modulating nutrient content and community composition, scaling of culture volume to isolate biological molecules, and control of environmental parameters, such as temperature, humidity, and nutrient flow. However, different devices are used to investigate homogenous, planktonic microbial communities and heterogeneous biofilms. Here, we present the minibioreactor array 2 (MBRA-2) with media rails, a benchtop multireactor system derived from the MBRA system that enables researchers to use the same system to grow planktonic and biofilm cultures. We simplified flow through the system and reduced contamination, leakage, and time required for array assembly by designing and implementing a reusable media rail to replace the branched tubing traditionally used to convey media through chemostat arrays. Additionally, we altered the structure of the six-bioreactor strip to incorporate a removable lid to provide easy access to the bioreactor wells, enabling biofilm recovery and thorough cleaning for reuse. Using Pseudomonas aeruginosa, a model biofilm-producing organism, we show that the technical improvements of the MBRA-2 for biofilms growth does not disrupt the function of the bioreactor array. IMPORTANCE The MBRA-2 with media rails provides an accessible system for investigators to culture heterogenous, suspended biofilms under constant flow.

Chromatin-mediated alternative splicing regulates cocaine-reward behavior.

Neuronal alternative splicing is a key gene regulatory mechanism in the brain. However, the spliceosome machinery is insufficient to fully specify splicing complexity. In considering the role of the epigenome in activity-dependent alternative splicing, we and others find the histone modification H3K36me3 to be a putative splicing regulator. In this study, we found that mouse cocaine self-administration caused widespread differential alternative splicing, concomitant with the enrichment of H3K36me3 at differentially spliced junctions. Importantly, only targeted epigenetic editing can distinguish between a direct role of H3K36me3 in splicing and an indirect role via regulation of splice factor expression elsewhere on the genome. We targeted Srsf11, which was both alternatively spliced and H3K36me3 enriched in the brain following cocaine self-administration. Epigenetic editing of H3K36me3 at Srsf11 was sufficient to drive its alternative splicing and enhanced cocaine self-administration, establishing the direct causal relevance of H3K36me3 to alternative splicing of Srsf11 and to reward behavior.

Sustained hippocampal chromatin regulation in a mouse model of depression and antidepressant action.

To better understand the molecular mechanisms of depression and antidepressant action, we administered chronic social defeat stress followed by chronic imipramine (a tricyclic antidepressant) to mice and studied adaptations at the levels of gene expression and chromatin remodeling of five brain-derived neurotrophic factor (Bdnf) splice variant mRNAs (I-V) and their unique promoters in the hippocampus. Defeat stress induced lasting downregulation of Bdnf transcripts III and IV and robustly increased repressive histone methylation at their corresponding promoters. Chronic imipramine reversed this downregulation and increased histone acetylation at these promoters. This hyperacetylation by chronic imipramine was associated with a selective downregulation of histone deacetylase (Hdac) 5. Furthermore, viral-mediated HDAC5 overexpression in the hippocampus blocked imipramine's ability to reverse depression-like behavior. These experiments underscore an important role for histone remodeling in the pathophysiology and treatment of depression and highlight the therapeutic potential for histone methylation and deacetylation inhibitors in depression.

Circuit-specific hippocampal ΔFosB underlies resilience to stress-induced social avoidance.

Chronic stress is a key risk factor for mood disorders like depression, but the stress-induced changes in brain circuit function and gene expression underlying depression symptoms are not completely understood, hindering development of novel treatments. Because of its projections to brain regions regulating reward and anxiety, the ventral hippocampus is uniquely poised to translate the experience of stress into altered brain function and pathological mood, though the cellular and molecular mechanisms of this process are not fully understood. Here, we use a novel method of circuit-specific gene editing to show that the transcription factor ΔFosB drives projection-specific activity of ventral hippocampus glutamatergic neurons causing behaviorally diverse responses to stress. We establish molecular, cellular, and circuit-level mechanisms for depression- and anxiety-like behavior in response to stress and use circuit-specific gene expression profiling to uncover novel downstream targets as potential sites of therapeutic intervention in depression.

DeltaFosB in the nucleus accumbens regulates food-reinforced instrumental behavior and motivation.

Alterations in motivation have been implicated in the pathophysiology of several psychiatric disorders, including substance abuse and depression. Repeated exposure to drugs of abuse or stress is known to persistently induce the transcription factor deltaFosB in the nucleus accumbens (NAc) and dorsal striatum, effects hypothesized to contribute to neuroadaptations in dopamine-regulated signaling. Little is known, however, about the specific involvement of deltaFosB in dysregulation of appetitively motivated behaviors. We show here that inducible overexpression of deltaFosB in NAc and dorsal striatum of bitransgenic mice, or specifically in the NAc core of rats by use of viral-mediated gene transfer, enhanced food-reinforced instrumental performance and progressive ratio responding. Very similar behavioral effects were found after previous repeated exposure to cocaine, amphetamine, MDMA [(+)-3,4-methylenedioxymethamphetamine], or nicotine in rats. These results reveal the powerful regulation of motivational processes by deltaFosB, and provide evidence that drug-induced alterations in gene expression via induction of deltaFosB within the NAc core may play a critical role in the impact of motivational influences on instrumental behavior.

Oxidation of primary and secondary benzylic alcohols with hydrogen peroxide and tert-butyl hydroperoxide catalyzed by a "helmet" phthalocyaninato iron complex in the absence of added organic solvent.

The oxidation of four benzylic alcohols employing hydrogen peroxide and TBHP as oxidants, catalyzed by an iron(III) complex bearing a 14,28-[1,3-diiminoisoindolinato]phthalocyaninato (diiPc) ligand has been studied and found to proceed with good selectivity, high turnover numbers, and high turnover frequencies in the absence of organic solvents other than the substrates themselves.

Role for mTOR signaling and neuronal activity in morphine-induced adaptations in ventral tegmental area dopamine neurons.

While the abuse of opiate drugs continues to rise, the neuroadaptations that occur with long-term drug exposure remain poorly understood. We describe here a series of chronic morphine-induced adaptations in ventral tegmental area (VTA) dopamine neurons, which are mediated via downregulation of AKT-mTORC2 (mammalian target of rapamycin complex-2). Chronic opiates decrease the size of VTA dopamine neurons in rodents, an effect seen in humans as well, and concomitantly increase the excitability of the cells but decrease dopamine output to target regions. Chronic morphine decreases mTORC2 activity, and overexpression of Rictor, a component of mTORC2, prevents morphine-induced changes in cell morphology and activity. Further, local knockout of Rictor in VTA decreases DA soma size and reduces rewarding responses to morphine, consistent with the hypothesis that these adaptations represent a mechanism of reward tolerance. Together, these findings demonstrate a novel role for AKT-mTORC2 signaling in mediating neuroadaptations to opiate drugs of abuse.

Oligodendrocyte-myelin glycoprotein is a Nogo receptor ligand that inhibits neurite outgrowth.

The inhibitory activity associated with myelin is a major obstacle for successful axon regeneration in the adult mammalian central nervous system (CNS). In addition to myelin-associated glycoprotein (MAG) and Nogo-A, available evidence suggests the existence of additional inhibitors in CNS myelin. We show here that a glycosylphosphatidylinositol (GPI)-anchored CNS myelin protein, oligodendrocyte-myelin glycoprotein (OMgp), is a potent inhibitor of neurite outgrowth in cultured neurons. Like Nogo-A, OMgp contributes significantly to the inhibitory activity associated with CNS myelin. To further elucidate the mechanisms that mediate this inhibitory activity of OMgp, we screened an expression library and identified the Nogo receptor (NgR) as a high-affinity OMgp-binding protein. Cleavage of NgR and other GPI-linked proteins from the cell surface renders axons of dorsal root ganglia insensitive to OMgp. Introduction of exogenous NgR confers OMgp responsiveness to otherwise insensitive neurons. Thus, OMgp is an important inhibitor of neurite outgrowth that acts through NgR and its associated receptor complex. Interfering with the OMgp/NgR pathway may allow lesioned axons to regenerate after injury in vivo.