Astrocytic adenosine: from synapses to psychiatric disorders

Although it is considered to be the most complex organ in the body, the brain can be broadly classified into two major types of cells, neuronal cells and glial cells. Glia is a general term that encompasses multiple types of non-neuronal cells that function to maintain homeostasis, form myelin, and provide support and protection for neurons. Astrocytes, a major class of glial cell, have historically been viewed as passive support cells, but recently it has been discovered that astrocytes participate in signalling activities both with the vasculature and with neurons at the synapse. These cells have been shown to release d-serine, TNF-α, glutamate, atrial natriuretic peptide (ANP) and ATP among other signalling molecules. ATP and its metabolites are well established as important signalling molecules, and astrocytes represent a major source of ATP release in the nervous system. Novel molecular and genetic tools have recently shown that astrocytic release of ATP and other signalling molecules has a major impact on synaptic transmission. Via actions at the synapse, astrocytes have now been shown to regulate complex network signalling in the whole organism with impacts on respiration and the sleep–wake cycle. In addition, new roles for astrocytes are being uncovered in psychiatric disorders, and astrocyte signalling mechanisms represents an attractive target for novel therapeutic agents.     

Two sexes,one body: intra‐ and intersex covariation of gamete phenotypes in simultaneous hermaphrodites

By harboring male and female functions in the same genome and expressing them in every individual, simultaneous hermaphrodites may incur sexual conflict unless both sex functions can evolve phenotypic optima independently of each other. The first step toward understanding their capacity to do so lies in understanding whether sex functions are phenotypically correlated within individuals, but remarkably few data address this issue. We tested the potential for intra‐ and intersex covariation of gamete phenotypes to mediate sexual conflict in broadcast‐spawning hermaphrodites (the ascidians Ciona intestinalis and Pyura praeputialis), for which sex‐specific selection acts predominantly on sperm–egg interactions in the water column. In both species, gamete phenotypes covaried within and across sex functions, implying that selection may be unable to target them independently because its direct effects on male gametes translate into correlated effects on female gametes and vice versa. This alone does not preclude the evolution of a different phenotypic optimum for each sex function, but imposes the more restrictive requirement that selection – which ultimately sorts among whole individuals, not sex functions – aligns with the direction in which gamete phenotypes covary at this level.     

Proof of concept of microbiome-metabolome analysis and delayed gluten exposure on celiac disease autoimmunity in genetically at-risk infants

Celiac disease (CD) is a unique autoimmune disorder in which the genetic factors (DQ2/DQ8) and the environmental trigger (gluten) are known and necessary but not sufficient for its development. Other environmental components contributing to CD are poorly understood. Studies suggest that aspects of gluten intake might influence the risk of CD occurrence and timing of its onset, i.e., the amount and quality of ingested gluten, together with the pattern of infant feeding and the age at which gluten is introduced in the diet. In this study, we hypothesize that the intestinal microbiota as a whole rather than specific infections dictates the switch from tolerance to immune response in genetically susceptible individuals. Using a sample of infants genetically at risk of CD, we characterized the longitudinal changes in the microbial communities that colonize infants from birth to 24 months and the impact of two patterns of gluten introduction (early vs. late) on the gut microbiota and metabolome, and the switch from gluten tolerance to immune response, including onset of CD autoimmunity. We show that infants genetically susceptible to CD who are exposed to gluten early mount an immune response against gluten and develop CD autoimmunity more frequently than at-risk infants in which gluten exposure is delayed until 12 months of age. The data, while derived from a relatively small number of subjects, suggest differences between the developing microbiota of infants with genetic predisposition for CD and the microbiota from infants with a non-selected genetic background, with an overall lack of bacteria of the phylum Bacteriodetes along with a high abundance of Firmicutes and microbiota that do not resemble that of adults even at 2 years of age. Furthermore, metabolomics analysis reveals potential biomarkers for the prediction of CD. This study constitutes a definite proof-of-principle that these combined genomic and metabolomic approaches will be key to deciphering the role of the gut microbiota on CD onset.     

Camelina mutants resistant to acetolactate synthase inhibitor herbicides

Camelina (Camelina sativa L.) is a low-input oilseed crop of recent interest for sustainable biofuel production. As a relatively new crop in modern agriculture, considerable agronomic and regulatory problems need to be overcome. A common and troublesome problem is sensitivity to residues of acetolactate synthase (ALS) inhibitor herbicides in soils. To develop resistance to those residues, camelina seed were mutagenized by exposure to 0.3% ethyl methane sulfonate and screened at the M2 generation for increased resistance to imazethapyr and sulfosulfuron. Five lines with resistance were identified and characterized. Four mutants, identified in a screen for imazethapyr resistance (IM1, IM6, IM10, and IM18), appeared phenotypically identical and were controlled by the same co-dominant gene. One mutant identified in a screen for sulfosulfuron resistance was phenotypically different but also appears to be controlled by a single co-dominant gene. Further analysis with the IM1 and SM4 mutants confirmed they had increased resistance to imazethapyr, sulfosulfuron, and flucarbazone, with the resistance in the SM4 mutant being the highest. Compared to the wild type, doses of approximately 200 times more imazethapyr, 30 times more sulfosulfuron, and seven times as much flucarbazone were required to reduce plant growth by 50%. Sequence analysis of ALS genes from the SM4 line identified at least eight different genes or alleles. An allele associated with the highest levels of resistance was created by a single base substitution creating an amino acid shift previously found to cause ALS inhibitor resistance in yeast and tobacco.     

The Structure- and Metal-dependent Activity of Escherichia coli PgaB Provides Insight into the Partial De-N-acetylation of Poly-β-1,6-N-acetyl-D-glucosamine

Exopolysaccharides are required for the development and integrity of biofilms produced by a wide variety of bacteria. In Escherichia coli, partial de-N-acetylation of the exopolysaccharide poly-β-1,6-N-acetyl-d-glucosamine (PNAG) by the periplasmic protein PgaB is required for polysaccharide intercellular adhesin-dependent biofilm formation. To understand the molecular basis for PNAG de-N-acetylation, the structure of PgaB in complex with Ni²⁺ and Fe³⁺ have been determined to 1.9 and 2.1 Å resolution, respectively, and its activity on β-1,6-GlcNAc oligomers has been characterized. The structure of PgaB reveals two (β/α)_x barrel domains: a metal-binding de-N-acetylase that is a member of the family 4 carbohydrate esterases (CE4s) and a domain structurally similar to glycoside hydrolases. PgaB displays de-N-acetylase activity on β-1,6-GlcNAc oligomers but not on the β-1,4-(GlcNAc)₄ oligomer chitotetraose and is the first CE4 member to exhibit this substrate specificity. De-N-acetylation occurs in a length-dependent manor, and specificity is observed for the position of de-N-acetylation. A key aspartic acid involved in de-N-acetylation, normally seen in other CE4s, is missing in PgaB, suggesting that the activity of PgaB is attenuated to maintain the low levels of de-N-acetylation of PNAG observed in vivo. The metal dependence of PgaB is different from most CE4s, because PgaB shows increased rates of de-N-acetylation with Co²⁺ and Ni²⁺ under aerobic conditions, and Co²⁺, Ni²⁺ and Fe²⁺ under anaerobic conditions, but decreased activity with Zn²⁺. The work presented herein will guide inhibitor design to combat biofilm formation by E. coli and potentially a wide range of medically relevant bacteria producing polysaccharide intercellular adhesin-dependent biofilms.     

Dead Certain

Evolutionary psychologists have suggested that confidence and conservatism promoted aggression in our ancestral past, and that this may have been an adaptive strategy given the prevailing costs and benefits of conflict. However, in modern environments, where the costs and benefits of conflict can be very different owing to the involvement of mass armies, sophisticated technology, and remote leadership, evolved tendencies toward high levels of confidence and conservatism may continue to be a contributory cause of aggression despite leading to greater costs and fewer benefits. The purpose of this paper is to test whether confidence and conservatism are indeed associated with greater levels of aggression—in an explicitly political domain. We present the results of an experiment examining people’s levels of aggression in response to hypothetical international crises (a hostage crisis, a counter-insurgency campaign, and a coup). Levels of aggression (which range from concession to negotiation to military attack) were significantly predicted by subjects’ (1) confidence that their chosen policy would succeed, (2) score on a liberal-conservative scale, (3) political party affiliation, and (4) preference for the use of military force in real-world U.S. policy toward Iraq and Iran. We discuss the possible adaptive and maladaptive implications of confidence and conservatism for the prospects of war and peace in the modern world.     

Two-dimensional combinatorial screening identifies specific 6'-acylated kanamycin A- and 6'-acylated neamine-RNA hairpin interactions

Herein, we report the RNA hairpin loops from a six-nucleotide hairpin library that bind 6'-acylated kanamycin A (1) and 6'-acylated neamine (2) identified by two-dimensional combinatorial screening (2DCS). Hairpins selected to bind 1 have K(d)'s ranging from 235 to 1035 nM, with an average K(d) of 618 nM. For 2, the selected hairpins bind with K(d)'s ranging from 135 to 2300 nM, with an average K(d) of 1010 nM. The selected RNA hairpin-ligand interactions are also specific for the ligand that they were selected to bind compared with the other arrayed ligand. For example, the mixture of hairpins selected for 1 on average bind 33-fold more tightly to 1 than to 2, while the mixtures of hairpins selected for 2 on average bind 11-fold more tightly to 2 than to 1. Secondary structure prediction of the selected sequences was completed to determine the motifs that each ligand binds, and the hairpin loop preferences for 1 and 2 were computed. For 1, the preferred hairpin loops contain an adenine separated by at least two nucleotides from a cytosine, for example, ANNCNN (two-tailed p-value = 0.0010) and ANNNCN (two-tailed p-value <0.0001). For 2, the preferred hairpin loops contain both 5'GC and 5'CG steps (two-tailed p-value <0.0001). These results expand the information available on the RNA hairpin loops that bind small molecules and could prove useful for targeting RNA.     

Human immunodeficiency virus type 1 envelope gp120 induces a stop signal and virological synapse formation in noninfected CD4+ T cells

Human immunodeficiency virus type 1 (HIV-1)-infected T cells form a virological synapse with noninfected CD4⁺ T cells in order to efficiently transfer HIV-1 virions from cell to cell. The virological synapse is a specialized cellular junction that is similar in some respects to the immunological synapse involved in T-cell activation and effector functions mediated by the T-cell antigen receptor. The immunological synapse stops T-cell migration to allow a sustained interaction between T-cells and antigen-presenting cells. Here, we have asked whether HIV-1 envelope gp120 presented on a surface to mimic an HIV-1-infected cell also delivers a stop signal and if this is sufficient to induce a virological synapse. We demonstrate that HIV-1 gp120-presenting surfaces arrested the migration of primary activated CD4 T cells that occurs spontaneously in the presence of ICAM-1 and induced the formation of a virological synapse, which was characterized by segregated supramolecular structures with a central cluster of envelope surrounded by a ring of ICAM-1. The virological synapse was formed transiently, with the initiation of migration within 30 min. Thus, HIV-1 gp120-presenting surfaces induce a transient stop signal and supramolecular segregation in noninfected CD4⁺ T cells.     

The N-terminus of apolipoprotein A-V adopts a helix bundle molecular architecture

Previous studies of recombinant full-length human apolipoprotein A-V (apoA-V) provided evidence of the presence of two independently folded structural domains. Computer-assisted sequence analysis and limited proteolysis studies identified an N-terminal fragment as a candidate for one of the domains. C-Terminal truncation variants in this size range, apoA-V(1-146) and apoA-V(1-169), were expressed in Escherichia coli and isolated. Unlike full-length apoA-V or apoA-V(1-169), apoA-V(1-146) was soluble in neutral-pH buffer in the absence of lipid. Sedimentation equilibrium analysis yielded a weight-average molecular weight of 18811, indicating apoA-V(1-146) exists as a monomer in solution. Guanidine HCl denaturation experiments at pH 3.0 yielded a one-step native to unfolded transition that corresponds directly with the more stable component of the two-stage denaturation profile exhibited by full-length apoA-V. On the other hand, denaturation experiments conducted at pH 7.0 revealed a less stable structure. In a manner similar to that of known helix bundle apolipoproteins, apoA-V(1-146) induced a relatively small enhancement in 8-anilino-1-naphthalenesulfonic acid fluorescence intensity. Quenching studies with single-Trp apoA-V(1-146) variants revealed that a unique site predicted to reside on the nonpolar face of an amphipathic alpha-helix was protected from quenching by KI. Taken together, the data suggest the 146 N-terminal residues of human apoA-V adopt a helix bundle molecular architecture in the absence of lipid and, thus, likely exist as an independently folded structural domain within the context of the intact protein.