Predicting cortisol stress responses in older individuals: influence of serotonin receptor 1A gene (HTR1A) and stressful life events

Considerable variability in the activity of the hypothalamus-pituitary-adrenal (HPA) axis in response to stress has been found in quantitative genetic studies investigating healthy individuals suggesting that at least part of this variance is due to genetic factors. Since the HPA axis is regulated by a neuronal network including amygdala, hippocampus, prefrontal cortex as well as brainstem circuits, the investigation of candidate genes that impact neurotransmitter systems related to these brain regions might further elucidate the genetic underpinnings of the stress response. However, aside from genetic risk factors, past stressful life events might also result in long-term adjustments of HPA axis reactivity. Here, we investigated the effects of the − 1019 G/C polymorphism in the HTR1A gene encoding the serotonin (5-HT) receptor 1A (5-HT_1A) and stressful life events experienced during childhood and adolescence on changes in cortisol levels in response to the Trier Social Stress Test (TSST) in a sample of healthy older adults (N = 97). Regression analyses revealed a significant effect of HTR1A genotype with the G allele being associated with a less pronounced stress response. In addition, an inverse relationship between past stressful life events and cortisol release but no gene × environment interaction was detected. The results further underscore the crucial role of functional serotonergic genetic variation as well as stressful events during critical stages of development on the acute stress response later in life.     

An intragenic deletion in pilQ leads to nonpiliation of a Pseudomonas aeruginosa strain isolated from cystic fibrosis lung

Deficient motility is one of the characteristic hallmarks observed in Pseudomonas aeruginosa strains that chronically colonize the lungs of cystic fibrosis (CF) patients. Pseudomonas aeruginosa TB is a nonpiliated CF isolate known to be defective in twitching motility. Complementation confirmed a direct link of this phenotype to an intragenic out-of-frame deletion in pilQ (PA5040). Sequence alignment of pilQ derived from TB vs. PAO1 suggests that close direct repeats framing the deletion site may have triggered this mutation. This type of mutation could play a role in the emergence of pathoadaptive mutations of P. aeruginosa in the CF lung habitat.     

Three Types of Tyrosine Hydroxylase-Positive CNS Neurons Distinguished by Dopa Decarboxylase and VMAT2 Co-Expression

Sumary 1. We investigate here for the first time in primate brain the combinatorial expression of the three major functionally relevant proteins for catecholaminergic neurotransmission tyrosine hydroxylase (TH), aromatic acid acid decarboxylase (AADC), and the brain-specific isoform of the vesicular monoamine transporter, VMAT2, using highly specific antibodies and immunofluorescence with confocal microscopy to visualize combinatorial expression of these proteins.2. In addition to classical TH, AADC, and VMAT2-copositive catecholaminergic neurons, two unique kinds of TH-positive neurons were identified based on co-expression of AADC and VMAT2.3. TH and AADC co-positive, but VMAT2-negative neurons, are termed “nonexocytotic catecholaminergic TH neurons.” These were found in striatum, olfactory bulb, cerebral cortex, area postrema, nucleus tractus solitarius, and in the dorsal motor nucleus of the vagus.4. TH-positive neurons expressing neither AADC nor VMAT2 are termed “dopaergic TH neurons.” We identified these neurons in supraoptic, paraventricular and periventricular hypothalamic nuclei, thalamic paraventicular nucleus, habenula, parabrachial nucleus, cerebral cortex and spinal cord. We were unable to identify any dopaergic (TH-positive, AADC-negative) neurons that expressed VMAT2, suggesting that regulatory mechanisms exist for shutting off VMAT2 expression in neurons that fail to biosynthesize its substrates.5. In several cases, the corresponding TH phenotypes were identified in the adult rat, suggesting that this rodent is an appropriate experimental model for further investigation of these TH-positive neuronal cell groups in the adult central nervous system. Thus, no examples of TH and VMAT2 co-positive neurons lacking AADC expression were found in rodent adult nervous system.6. In conclusion, the adult mammalian nervous system contains in addition to classical catecholaminergic neurons, cells that can synthesize dopamine, but cannot transport and store it in synaptic vesicles, and neurons that can synthesize only L-dopa and lack VMAT2 expression. The presence of these additional populations of TH-positive neurons in the adult primate CNS has implications for functional catecholamine neurotransmission, its derangement in disease and drug abuse, and its rescue by gene therapeutic maneuvers in neurodegenerative diseases such as Parkinson's disease.     

Impact of CCR7 on priming and distribution of antiviral effector and memory CTL

The chemokine receptor CCR7 is a key factor in the coordinate migration of T cells and dendritic cells (DC) into and their localization within secondary lymphoid organs. In this study we investigated the impact of CCR7 on CD8(+) T cell responses by infecting CCR7(-/-) mice with lymphocytic choriomeningitis virus (LCMV). We found that the absence of CCR7 affects the magnitude of an antiviral CTL response during the acute phase, with reduced numbers of virus-specific CTL in all lymphoid and nonlymphoid organs tested. On the single cell level, CCR7-deficient CTL gained full effector function, such that antiviral protection in CCR7-deficient mice was complete, but delayed. Similarly, adoptive transfer experiments using DC from CCR7-deficient or competent mice for the priming of CCR7-positive or CCR7-negative CD8(+) T cells, respectively, revealed that ectopic positioning of DC and CTL outside organized T cell zones results in reduced priming efficacy. In the memory phase, CCR7-deficient mice maintained a stable LCMV-specific CTL population, predominantly in nonlymphoid organs, and rapidly mounted protective CTL responses against a challenge infection with a vaccinia virus recombinant for the gp33 epitope of LCMV. Taken together, the CCR7-dependent organization of the T cell zone does not appear to be a prerequisite for antiviral effector CTL differentiation and the sustenance of antiviral memory responses in lymphoid or peripheral tissues.     

Relaxation-optimised Hartmann–Hahn transfer using a specifically Tailored MOCCA-XY16 mixing sequence for carbonyl–carbonyl correlation spectroscopy in <Superscript>13</Superscript>C direct detection NMR experiments

Isotropic mixing sequences are one of the key methods to achieve efficient coherence transfer. Among them, the MOCCA-XY16, which keeps the magnetization longitudinal for a significant amount of time, is characterised by favourable relaxation properties. We show here that its adapted version is particularly suited for carbonyl–carbonyl correlations in ¹³C direct detection NMR experiments.     

Induced Variations in Brassinosteroid Genes Define Barley Height and Sturdiness,and Expand the Green Revolution Genetic Toolkit

Reduced plant height and culm robustness are quantitative characteristics important for assuring cereal crop yield and quality under adverse weather conditions. A very limited number of short-culm mutant alleles were introduced into commercial crop cultivars during the Green Revolution. We identified phenotypic traits, including sturdy culm, specific for deficiencies in brassinosteroid biosynthesis and signaling in semidwarf mutants of barley (Hordeum vulgare). This set of characteristic traits was explored to perform a phenotypic screen of near-isogenic short-culm mutant lines from the brachytic, breviaristatum, dense spike, erectoides, semibrachytic, semidwarf, and slender dwarf mutant groups. In silico mapping of brassinosteroid-related genes in the barley genome in combination with sequencing of barley mutant lines assigned more than 20 historic mutants to three brassinosteroid-biosynthesis genes (BRASSINOSTEROID-6-OXIDASE, CONSTITUTIVE PHOTOMORPHOGENIC DWARF, and DIMINUTO) and one brassinosteroid-signaling gene (BRASSINOSTEROID-INSENSITIVE1 [HvBRI1]). Analyses of F2 and M2 populations, allelic crosses, and modeling of nonsynonymous amino acid exchanges in protein crystal structures gave a further understanding of the control of barley plant architecture and sturdiness by brassinosteroid-related genes. Alternatives to the widely used but highly temperature-sensitive uzu1.a allele of HvBRI1 represent potential genetic building blocks for breeding strategies with sturdy and climate-tolerant barley cultivars.The introduction of dwarfing genes to increase culm sturdiness of cereal crops was crucial for the first Green Revolution (Hedden, 2003). The culms of tall cereal crops were not strong enough to support the heavy spikes of high-yielding cultivars, especially under high-nitrogen conditions. As a result, plants fell over, a process known as lodging. This caused losses in yield and grain-quality issues attributable to fungal infections, mycotoxin contamination, and preharvest germination (Rajkumara, 2008). Today, a second Green Revolution is on its way, to revolutionize the agricultural sector and to ensure food production for a growing world population. Concurrently, global climate change is expected to cause more frequent occurrences of extreme weather conditions, including thunderstorms with torrential rain and strong winds, thus promoting cereal culm breakage (Porter and Semenov, 2005; National Climate Assessment Development Advisory Committee, 2013). Accordingly, plant architectures that resist lodging remain a major crop-improvement goal and identification of genes that regulate culm length is required to enhance the genetic toolbox in order to facilitate efficient marker-assisted breeding. The mutations and the corresponding genes that enabled the Green Revolution in wheat (Triticum aestivum) and rice (Oryza sativa) have been identified (Hedden, 2003). They all relate to gibberellin metabolism and signal transduction. It is now known that other plant hormones such as brassinosteroids are also involved in the regulation of plant height. Knowledge of the molecular mechanisms underlying the effects of the two hormones on cell elongation and division has mainly come from studies in Arabidopsis (Arabidopsis thaliana; Bai et al., 2012). Mutant-based breeding strategies to fine-tune brassinosteroid metabolism and signaling pathways could improve lodging behavior in modern crops (Vriet et al., 2012) such as barley (Hordeum vulgare), which is the fourth most abundant cereal in both area and tonnage harvested (http://faostat.fao.org).A short-culm phenotype in crops is often accompanied by other phenotypic changes. Depending on the penetrance of such pleiotropic characters, but also the parental background and different scientific traditions and expertise, short-culmed barley mutants were historically divided into groups, such as brachytic (brh), breviaristatum (ari), dense spike (dsp), erectoides (ert), semibrachytic (uzu), semidwarf (sdw), or slender dwarf (sld; Franckowiak and Lundqvist, 2012). Subsequent mutant characterization was limited to intragroup screens and very few allelism tests between mutants from different groups have been reported (Franckowiak and Lundqvist, 2012). Although the total number of short-culm barley mutants exceeds 500 (Franckowiak and Lundqvist, 2012), very few have been characterized at the DNA level (Helliwell et al., 2001; Jia et al., 2009; Chandler and Harding, 2013; Houston et al., 2013). One of the first identified haplotypes was uzu barley (Chono et al., 2003). The Uzu1 gene encodes the brassinosteroid hormone receptor and is orthologous to the BRASSINOSTEROID-INSENSITIVE1 (BRI1) gene of Arabidopsis, a crucial promoter of plant growth (Li and Chory, 1997). The uzu1.a allele has been used in East Asia for over a century and is presently distributed in winter barley cultivars in Japan, the Korean peninsula, and China (Saisho et al., 2004). Its agronomic importance comes from the short and sturdy culm that provides lodging resistance, and an upright plant architecture that tolerates dense planting.Today, more than 50 different brassinosteroids have been identified in plants (Bajguz and Tretyn, 2003). Most are intermediates of the complex biosynthetic pathway (Shimada et al., 2001). Approximately nine genes code for the enzymes that participate in the biosynthetic pathway from episterol to brassinolide (Supplemental Fig. S1). Brassinosteroid deficiency is caused by down-regulation of these genes, but it can also be associated with brassinosteroid signaling. The first protein in the signaling network is the brassinosteroid receptor encoded by BRI1 (Li and Chory, 1997; Kim and Wang, 2010). In this work, we show how to visually identify brassinosteroid-mutant barley plants and we describe more than 20 relevant mutations in four genes of the brassinosteroid biosynthesis and signaling pathways that can be used in marker-assisted breeding strategies.     

Regulatory T cells selectively preserve immune privilege of self-antigens during viral central nervous system infection

Regulatory T cells (Tregs) are important for the attenuation of immune reactions. During viral CNS infections, however, an indiscriminate maintenance of CNS immune privilege through Treg-mediated negative regulation could prevent autoimmune sequelae but impair the control of viral replication. We analyzed in this study the impact of Tregs on the development of acute viral encephalomyelitis, T cell-mediated antiviral protection, and prevention of CNS autoimmunity following intranasal infection with the gliatropic mouse hepatitis virus strain A59. To assess the contribution of Tregs in vivo, we specifically depleted CD4(+)Foxp3(+) T cells in a diphtheria toxin-dependent manner. We found that depletion of Tregs had no impact on viral distribution and clearance and did not significantly alter virus-specific CD4(+) and CD8(+) T cell responses. However, Treg depletion led to a more severe CNS inflammation associated with neuronal damage. Dissection of the underlying immunopathological mechanisms revealed the elaborate Treg-dependent regulation of self-reactive CD4(+) T cell proliferation within the CNS-draining lymph node and downtuning of CXCR3 expression on T cells. Taken together, these results suggest that Tregs preserve CNS immune privilege through selective control of CNS-specific Th cells while keeping protective antiviral immunity fully operative.     

Water availability shapes edaphic and lithic cyanobacterial communities in the Atacama Desert

In the Atacama Desert, cyanobacteria grow on various substrates such as soils (edaphic) and quartz or granitoid stones (lithic). Both edaphic and lithic cyanobacterial communities have been described but no comparison between both communities of the same locality has yet been undertaken. In the present study, we compared both cyanobacterial communities along a precipitation gradient ranging from the arid National Park Pan de Azúcar (PA), which resembles a large fog oasis in the Atacama Desert extending to the semiarid Santa Gracia Natural Reserve (SG) further south, as well as along a precipitation gradient within PA. Various microscopic techniques, as well as culturing and partial 16S rRNA sequencing, were applied to identify 21 cyanobacterial species; the diversity was found to decline as precipitation levels decreased. Additionally, under increasing xeric stress, lithic community species composition showed higher divergence from the surrounding edaphic community, resulting in indigenous hypolithic and chasmoendolithic cyanobacterial communities. We conclude that rain and fog water, respectively, cause contrasting trends regarding cyanobacterial species richness in the edaphic and lithic microhabitats.     

CRHSP-24 phosphorylation is regulated by multiple signaling pathways in pancreatic acinar cells

Ca2+-regulated heat-stable protein of 24 kDa (CRHSP-24) is a serine phosphoprotein originally identified as a physiological substrate for the Ca2+-calmodulin regulated protein phosphatase calcineurin (PP2B). CRHSP-24 is a paralog of the brain-specific mRNA-binding protein PIPPin and was recently shown to interact with the STYX/dead phosphatase protein in developing spermatids (Wishart MJ and Dixon JE. Proc Natl Acad Sci USA 99: 2112-2117, 2002). Investigation of the effects of phorbol ester (12-o-tetradecanoylphorbol-13-acetate; TPA) and cAMP analogs in 32P-labeled pancreatic acini revealed that these agents acutely dephosphorylated CRHSP-24 by a Ca2+-independent mechanism. Indeed, cAMP- and TPA-mediated dephosphorylation of CRHSP-24 was fully inhibited by the PP1/PP2A inhibitor calyculin A, indicating that the protein is regulated by an additional phosphatase other than PP2B. Supporting this, CRHSP-24 dephosphorylation in response to the Ca2+-mobilizing hormone cholecystokinin was differentially inhibited by calyculin A and the PP2B-selective inhibitor cyclosporin A. Stimulation of acini with secretin, a secretagogue that signals through the cAMP pathway in acini, induced CRHSP-24 dephosphorylation in a concentration-dependent manner. Isoelectric focusing and immunoblotting indicated that elevated cellular Ca2+ dephosphorylated CRHSP-24 on at least three serine sites, whereas cAMP and TPA partially dephosphorylated the protein on at least two sites. The cAMP-mediated dephosphorylation of CRHSP-24 was inhibited by low concentrations of okadaic acid (10 nM) and fostriecin (1 microM), suggesting that CRHSP-24 is regulated by PP2A or PP4. Collectively, these data indicate that CRHSP-24 is regulated by diverse and physiologically relevant signaling pathways in acinar cells, including Ca2+, cAMP, and diacylglycerol.     

Unlocking the barley genome by chromosomal and comparative genomics

We used a novel approach that incorporated chromosome sorting, next-generation sequencing, array hybridization, and systematic exploitation of conserved synteny with model grasses to assign ~86% of the estimated ~32,000 barley (Hordeum vulgare) genes to individual chromosome arms. Using a series of bioinformatically constructed genome zippers that integrate gene indices of rice (Oryza sativa), sorghum (Sorghum bicolor), and Brachypodium distachyon in a conserved synteny model, we were able to assemble 21,766 barley genes in a putative linear order. We show that the barley (H) genome displays a mosaic of structural similarity to hexaploid bread wheat (Triticum aestivum) A, B, and D subgenomes and that orthologous genes in different grasses exhibit signatures of positive selection in different lineages. We present an ordered, information-rich scaffold of the barley genome that provides a valuable and robust framework for the development of novel strategies in cereal breeding.