A novel strategy for the isolation of luxl homologues: evidence for the widespread distribution of a LuxR:Luxl superfamily in enteric bacteria

The pheromone N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) regulates expression of bioluminescence in the marine bacterium Vibrio fischeri, the production of carbapenem antibiotic in Erwinia carotovora and exoenzymes in both E. carotovora and Pseudomonas aeruginosa. A characteristic feature of this regulatory mechanism in V. fischeri is that it is cell density-dependent, reflecting the need to accumulate sufficient pheromone to trigger the induction of gene expression. Using a lux plasmid-based bioluminescent sensor for OHHL, pheromone production by E. carotovora, Enterobacter agglomerans, Hafnia alvei, Rahnella aquatilis and Serratia marcescens has been demonstrated and shown also to be cell density-dependent. Production of OHHL implies the presence in these bacteria of a gene equivalent to luxl. Chromosomal banks from all five enteric bacteria have yielded clones capable of eliciting OHHL production when expressed in Escherichia coli. The luxl homologue from both E. carotovora (carl) and E. agglomerans (eagl) were characterized at the DNA sequence level and the deduced protein sequences have only 25% identity with the V. fischeri Luxl. Despite this, carl, eagl and luxl are shown to be biologically equivalent. An insertion mutant of eagl demonstrates that this gene is essential for OHHL production in E. agglomerans.     

Longitudinal investigation of neuroinflammation and metabolite profiles in the APPswe×PS1Δe9 transgenic mouse model of Alzheimer's disease

There is increasing evidence linking neuroinflammation to many neurological disorders including Alzheimer's disease (AD); however, its exact contribution to disease manifestation and/or progression is poorly understood. Therefore, there is a need to investigate neuroinflammation in both health and disease. Here, we investigate cognitive decline, neuroinflammatory and other pathophysiological changes in the APP_swe×PS1_Δe9 transgenic mouse model of AD. Transgenic (TG) mice were compared to C57BL/6 wild type (WT) mice at 6, 12 and 18 months of age. Neuroinflammation was investigated by [¹⁸F]DPA‐714 positron emission tomography and myo‐inositol levels using ¹H magnetic resonance spectroscopy (MRS) in vivo. Neuronal and cellular dysfunction was investigated by looking at N‐acetylaspartate (NAA), choline‐containing compounds, taurine and glutamate also using MRS. Cognitive decline was first observed at 12 m of age in the TG mice as assessed by working memory tests . A significant increase in [¹⁸F]DPA‐714 uptake was seen in the hippocampus and cortex of 18 m‐old TG mice when compared to age‐matched WT mice and 6 m‐old TG mice. No overall effect of gene was seen on metabolite levels; however, a significant reduction in NAA was observed in 18 m‐old TG mice when compared to WT. In addition, age resulted in a decrease in glutamate and an increase in choline levels. Therefore, we can conclude that increased neuroinflammation and cognitive decline are observed in TG animals, whereas NAA alterations occurring with age are exacerbated in the TG mice. These results support the role of neuroinflammation and metabolite alteration in AD and in ageing.

     

Influenza A viruses limit NLRP3‐NEK7‐complex formation and pyroptosis in human macrophages

Pyroptosis is a fulminant form of macrophage cell death, contributing to release of pro‐inflammatory cytokines. In humans, it depends on caspase 1/4‐activation of gasdermin D and is characterized by the release of cytoplasmic content. Pathogens apply strategies to avoid or antagonize this host response. We demonstrate here that a small accessory protein (PB1‐F2) of contemporary H5N1 and H3N2 influenza A viruses (IAV) curtails fulminant cell death of infected human macrophages. Infection of macrophages with a PB1‐F2‐deficient mutant of a contemporary IAV resulted in higher levels of caspase‐1 activation, cleavage of gasdermin D, and release of LDH and IL‐1β. Mechanistically, PB1‐F2 limits transition of NLRP3 from its auto‐repressed and closed confirmation into its active state. Consequently, interaction of a recently identified licensing kinase NEK7 with NLRP3 is diminished, which is required to initiate inflammasome assembly.     

SALT-OVERLY SENSITIVE5 Mediates Arabidopsis Seed Coat Mucilage Adherence and Organization through Pectins

Interactions between cell wall polymers are critical for establishing cell wall integrity and cell-cell adhesion. Here, we exploit the Arabidopsis (Arabidopsis thaliana) seed coat mucilage system to examine cell wall polymer interactions. On hydration, seeds release an adherent mucilage layer strongly attached to the seed in addition to a nonadherent layer that can be removed by gentle agitation. Rhamnogalacturonan I (RG I) is the primary component of adherent mucilage, with homogalacturonan, cellulose, and xyloglucan constituting minor components. Adherent mucilage contains rays composed of cellulose and pectin that extend above the center of each epidermal cell. CELLULOSE SYNTHASE5 (CESA5) and the arabinogalactan protein SALT-OVERLY SENSITIVE5 (SOS5) are required for mucilage adherence through unknown mechanisms. SOS5 has been suggested to mediate adherence by influencing cellulose biosynthesis. We, therefore, investigated the relationship between SOS5 and CESA5. cesa5-1 seeds show reduced cellulose, RG I, and ray size in adherent mucilage. In contrast, sos5-2 seeds have wild-type levels of cellulose but completely lack adherent RG I and rays. Thus, relative to each other, cesa5-1 has a greater effect on cellulose, whereas sos5-2 mainly affects pectin. The double mutant cesa5-1 sos5-2 has a much more severe loss of mucilage adherence, suggesting that SOS5 and CESA5 function independently. Double-mutant analyses with mutations in MUCILAGE MODIFIED2 and FLYING SAUCER1 that reduce mucilage release through pectin modification suggest that only SOS5 influences pectin-mediated adherence. Together, these findings suggest that SOS5 mediates adherence through pectins and does so independently of but in concert with cellulose synthesized by CESA5.Cellulosic cell walls are a defining feature of land plants. Primary cell walls are composed of three major classes of polysaccharides: cellulose, hemicelluloses, and pectins. In addition, approximately 10% of the primary cell wall is composed of protein (Burton et al., 2010). Cell walls provide mechanical support for the cell, and cell wall carbohydrates in the middle lamellae mediate cell-cell adhesion (Caffall and Mohnen, 2009). Current models of cell wall structure depict a cellulose-hemicellulose network embedded in an independent pectin gel (for review, see Albersheim et al., 2011). These components are believed to interact through both covalent and noncovalent bonds to provide structure and strength to the cell wall, although the relative importance of pectin and its interactions with the hemicellulose-cellulose network remain unclear (for review, see Cosgrove, 2005).Another gap in our understanding of cell wall structure and assembly is the role of arabinogalactan proteins (AGPs). AGPs are a family of evolutionarily conserved secreted proteins highly glycosylated with type II arabinogalactans, and they can be localized to the plasma membrane by a C-terminal glycophosphatidylinositol (GPI) lipid anchor (for review, see Schultz et al., 2000; Showalter, 2001; Johnson et al., 2003; Seifert and Roberts, 2007; Ellis et al., 2010). AGPs can be extensively modified in the cell wall; many glycosyl hydrolases can affect AGP function by cleaving their glycosyl side chains (Sekimata et al., 1989; Cheung et al., 1995; Wu et al., 1995; Kotake et al., 2005). The GPI anchor can also be cleaved, releasing the AGPs from the membrane into the cell wall (Schultz et al., 2000). Although their exact roles are still unclear, AGPs have been proposed to interact with cell wall polysaccharides, initiate intracellular signaling cascades, and influence a wide variety of biological processes (for review, see Seifert and Roberts, 2007; Ellis et al., 2010; Tan et al., 2013).Many fasciclin-like AGPs (FLAs), which contain at least one fasciclin domain (FAS) associated with protein-protein interactions, have been suggested to influence cellulose biosynthesis or organization (Seifert and Roberts, 2007; Li et al., 2010; MacMillan et al., 2010). FLA3 RNA interference lines have reduced intine cell wall biosynthesis and loss of Calcofluor white (a fluorescent dye specific for glycan molecules) staining in aborted pollen grains (Li et al., 2010). A fla11 fla12 double mutant was shown to have reduced cellulose deposition, altered cellulose microfibril angle, and reduced cell wall integrity (MacMillan et al., 2010). The fla11 fla12 double mutant also had reductions in arabinans, galactans, and rhamnose (MacMillan et al., 2010). FLA4/SALT-OVERLY SENSITIVE5 (SOS5) was identified in a screen for salt sensitivity in roots. The SOS5 gene encodes an FLA protein with a GPI anchor, two AGP-like domains, and two FAS domains (Shi et al., 2003). Plants homozygous for the loss-of-function conditional allele sos5-1 have thinner root cell walls that appear less organized (Shi et al., 2003). The presence of the two FAS domains has led to the suggestion that SOS5 may interact with other proteins, forming a network that strengthens the cell wall (Shi et al., 2003). SOS5 is involved in regulation of cell wall rheology through a pathway involving two Leu-rich repeat receptor-like kinases, FEI1 and FEI2 (Xu et al., 2008). SOS5 and FEI2 are also required for normal seed coat mucilage adherence and hypothesized to do so by influencing cellulose biosynthesis (Harpaz-Saad et al., 2011, 2012).Arabidopsis (Arabidopsis thaliana) seed coat mucilage is a powerful model for studying cell wall biosynthesis and polysaccharide interactions (Arsovski et al., 2010; Haughn and Western, 2012). Seed coat epidermal cells sequentially produce two distinct types of secondary cell walls with unique morphologies and properties (Western et al., 2000; Windsor et al., 2000). Between approximately 5 and 9 d approximate time of fertilization (DPA), seed coat epidermal cells synthesize mucilage and deposit it in the apoplast, creating a donut-shaped mucilage pocket that surrounds a central cytoplasmic column (Western et al., 2000, 2004; Haughn and Chaudhury, 2005). From 9 to 13 DPA, the cytoplasmic column is gradually replaced by a cellulose-rich, volcano-shaped secondary cell wall called the columella (Beeckman et al., 2000; Western et al., 2000; Windsor et al., 2000; Stork et al., 2010; Mendu et al., 2011).Seed mucilage is composed primarily of relatively unbranched rhamnogalacturonan I (RG I) with minor amounts of homogalacturonan (HG), cellulose, and hemicelluloses (for review, see Haughn and Western, 2012). When mucilage is hydrated, it expands rapidly from the apoplastic pocket, forming a halo that surrounds the seed. Mucilage separates into two fractions: a loose nonadherent fraction and an inner adherent fraction that can only be released by vigorous shaking, strong bases, or glycosidases (for review, see North et al., 2014). Galactans and arabinans are also present in mucilage, and their regulation by glycosidases is required for correct mucilage hydration (Dean et al., 2007; Macquet et al., 2007b; Arsovski et al., 2009). For example, β-XYLOSIDASE1 encodes a bifunctional β-d-xylosidase/α-l-arabinofuranosidase required for arabinan modification in mucilage, and β-xylosidase1 mutant seeds have a delayed mucilage release phenotype (Arsovski et al., 2009). MUCILAGE MODIFIED2 (MUM2) encodes a β-d-galactosidase, and mum2 seeds fail to release mucilage when hydrated in water (Dean et al., 2007; Macquet et al., 2007b). MUM2 is believed to modify RG I galactan side chains but may also affect the galactan component of other mucilage components (Dean et al., 2007; Macquet et al., 2007b). Galactans are capable of binding to cellulose in vitro and could affect mucilage hydration through pectin-cellulose interactions (Zykwinska et al., 2005, 2007a, 2007b; Dick-Pérez et al., 2011; Wang et al., 2012), although carbohydrate linkage analysis suggests that the galactan side chains are very short.Several studies indicate that seed mucilage extrusion and expansion are also influenced by methylesterification of HG. For example, both SUBTILISIN-LIKE SER PROTEASE1.7 and PECTIN METHYLESTERASE INHIBITOR6 are required for proper methyl esterification of mucilage (Rautengarten et al., 2008; Saez-Aguayo et al., 2013). Mutations in another gene, FLYING SAUCER1 (FLY1; a transmembrane E3 ubiquitin ligase), reduce the degree of pectin methylesterification in mucilage and cause increased mucilage adherence and defective mucilage extrusion (Voiniciuc et al., 2013). fly1 seeds have disc-like structures at the edge of the mucilage halo, which are outer primary cell wall fragments that detach from the columella during extrusion and are difficult to separate from the adherent mucilage (Voiniciuc et al., 2013).Recently, CELLULOSE SYNTHASE5 (CESA5) and SOS5 were proposed to facilitate cellulose-mediated mucilage adherence (Harpaz-Saad et al., 2011; Mendu et al., 2011; Sullivan et al., 2011). A simple hypothesis for the role of CESA5 in mucilage adherence is that it synthesizes cellulose, which interacts with the mucilage pectin to mediate adherence. Loss of CESA5 function results in a reduction of mucilage cellulose biosynthesis and a less adherent mucilage cell wall matrix (Mendu et al., 2011; Sullivan et al., 2011). The role of SOS5 in mucilage adherence is more difficult to explain. SOS5 null mutations cause a loss-of-adherence phenotype similar to cesa5-1 seeds, suggesting that SOS5 may regulate mucilage adherence by influencing CESA5 function (Harpaz-Saad et al., 2011). However, the mechanism through which SOS5 could influence CESA5 and/or cellulose biosynthesis is not clear.To better understand the role of SOS5 in mucilage adherence and its relationship to CESA5, we thoroughly investigated the seed coat epidermal cell phenotypes of the cesa5-1 and sos5-2 single mutants as well as those of the cesa5-1 sos5-2 double mutant. We also investigated how cellulose, SOS5, and pectin interact to mediate mucilage adherence by constructing double mutants with either cesa5-1 or sos5-2 together with either mum2-1 or fly1. Our results suggest that SOS5 mediates mucilage adherence independently of CESA5. Furthermore, compared with CESA5, SOS5 has a greater influence on mucilage pectin structure, suggesting that SOS5 mediates mucilage adherence through pectins, not cellulose.     

Epigenome-Wide DNA Methylation in Hearing Ability: New Mechanisms for an Old Problem

Epigenetic regulation of gene expression has been shown to change over time and may be associated with environmental exposures in common complex traits. Age-related hearing impairment is a complex disorder, known to be heritable, with heritability estimates of 57–70%. Epigenetic regulation might explain the observed difference in age of onset and magnitude of hearing impairment with age. Epigenetic epidemiology studies using unrelated samples can be limited in their ability to detect small effects, and recent epigenetic findings in twins underscore the power of this well matched study design. We investigated the association between venous blood DNA methylation epigenome-wide and hearing ability. Pure-tone audiometry (PTA) and Illumina HumanMethylation array data were obtained from female twin volunteers enrolled in the TwinsUK register. Two study groups were explored: first, an epigenome-wide association scan (EWAS) was performed in a discovery sample (n = 115 subjects, age range: 47–83 years, Illumina 27 k array), then replication of the top ten associated probes from the discovery EWAS was attempted in a second unrelated sample (n = 203, age range: 41–86 years, Illumina 450 k array). Finally, a set of monozygotic (MZ) twin pairs (n = 21 pairs) within the discovery sample (Illumina 27 k array) was investigated in more detail in an MZ discordance analysis. Hearing ability was strongly associated with DNA methylation levels in the promoter regions of several genes, including TCF25 (cg01161216, p = 6.6×10⁻⁶), FGFR1 (cg15791248, p = 5.7×10⁻⁵) and POLE (cg18877514, p = 6.3×10⁻⁵). Replication of these results in a second sample confirmed the presence of differential methylation at TCF25 (p(replication) = 6×10⁻⁵) and POLE (p(replication) = 0.016). In the MZ discordance analysis, twins'' intrapair difference in hearing ability correlated with DNA methylation differences at ACP6 (cg01377755, r = −0.75, p = 1.2×10⁻⁴) and MEF2D (cg08156349, r = −0.75, p = 1.4×10⁻⁴). Examination of gene expression in skin, suggests an influence of differential methylation on expression, which may account for the variation in hearing ability with age.     

IFITM3 Polymorphism rs12252-C Restricts Influenza A Viruses

The IFITM3 polymorphism rs12252-C, which encodes an IFITM3 isoform (Δ21 IFITM3) lacking 21 amino acids at the amino terminus, has been controversially associated with poor clinical outcomes in patients with H1N1 influenza A virus (IAV) infections. In vitro studies have shown that Δ21 IFITM3 loses its ability to restrict H1N1 IAV. Subsequent research has also revealed that tyrosine 20 is the key determinant for IFITM3 endocytic trafficking, which is essential for the efficient anti-viral activity of IFITM3. In contrast to previous studies, we demonstrated that both Δ21 IFITM3 and an IFITM3 variant (Y20A IFITM3), in which tyrosine 20 is substituted with alanine, strongly restricted entry mediated by IAV H1, H3, H5, and H7 proteins. Δ21 IFITM3 also efficiently suppressed replication of H1N1 and, to a lesser extent, H3N2 IAV. Δ21 IFITM3 and Y20A IFITM3 had broader subcellular distributions than full-length IFITM3 but an abundant amount of both IFITM3 variants still localized to late endosomes and lysosomes. Our data indicate that tyrosine 20 partially regulates the subcellular localization of IFITM3 but is not functionally essential for IFITM3-mediated IAV restriction. They also suggested that mechanisms, other than viral entry restriction, might contribute to variations in clinical outcomes of H1N1 influenza associated with rs12252-C.     

Phase II jaw movements and masseter muscle activity during chewing in Papio anubis

It was proposed that the power stroke in primates has two distinct periods of occlusal contact, each with a characteristic motion of the mandibular molars relative to the maxillary molars. The two movements are called phase I and phase II, and they occur sequentially in that order (Kay and Hiiemae [1974] Am J. Phys. Anthropol. 40:227-256, Kay and Hiiemae [1974] Prosimian Biology, Pittsburgh: University of Pittsburgh Press, p. 501-530). Phase I movement is said to be associated with shearing along a series of crests, producing planar phase I facets and crushing on surfaces on the basins of the molars. Phase I terminates in centric occlusion. Phase II movement is said to be associated with grinding along the same surfaces that were used for crushing at the termination of phase I. Hylander et al. ([1987] Am J. Phys. Anthropol. 72:287-312; see also Hiiemae [1984] Food Acquisition and Processing, London: Academic Press, p. 257-281; Hylander and Crompton [1980] Am J. Phys. Anthropol. 52:239-251, [1986] Arch. Oral. Biol. 31:841-848) analyzed data on macaques and suggested that phase II movement may not be nearly as significant for food breakdown as phase I movement simply because, based on the magnitude of mandibular bone strain patterns, adductor muscle and occlusal forces are likely negligible during movement out of centric occlusion. Our goal is to better understand the functional significance of phase II movement within the broader context of masticatory kinematics during the power stroke. We analyze vertical and transverse mandibular motion and relative activity of the masseter and temporalis muscles during phase I and II movements in Papio anubis. We test whether significant muscle activity and, by inference, occlusal force occurs during phase II movement. We find that during phase II movement, there is negligible force developed in the superficial and deep masseter and the anterior and posterior temporalis muscles. Furthermore, mandibular movements are small during phase II compared to phase I. These results suggest that grinding during phase II movement is of minimal importance for food breakdown, and that most food breakdown on phase II facets occurs primarily at the end of phase I movement (i.e., crushing during phase I movement). We note, however, that depending on the orientation of phase I facets, significant grinding also occurs along phase I facets during phase I.     

Cessation of neutrophil influx in C5a-induced acute experimental arthritis is associated with loss of chemoattractant activity from the joint space

Neutrophil emigration is a critical component of the inflammatory process and is generally thought to play a role in host defense as well as in the tissue injury that often accompanies inflammation. Most inflammatory reactions exhibit a sequence of emigrating cell types, thus clearly demonstrating that the neutrophil influx eventually ceases and that the neutrophils are then removed from the lesion. It has been our premise that in order to understand the processes that lead to the progressive inflammatory reactions that underly so many disease processes, it is important to determine the mechanism by which the "normal" inflammatory response resolves. The purpose of this study was to identify the time of cessation of neutrophil influx in experimental arthritis induced by the injection of C5 fragments (C5f) and to investigate mechanisms underlying the cessation process. The migration of i.v. delivery pulses to inflamed joints was assessed by lavage of the joint space and by external scintigraphy. We found no evidence for the development of inhibitory systems against chemotactic factors or "desensitization" of the inflamed site, because a second injection of C5f into joints which had been injected previously with C5f resulted in enhancement rather than inhibition of migration. Neither was evidence found for altered tissue barriers to migration or for desensitization of neutrophils as possible explanations for cessation of influx. The major mechanism appeared to be a loss of chemoattractant activity in the joint space between 2 h and 6 h after C5f injection which was detected by transfer into a fresh joint. Radiolabeled C5a des-Arg had a t1/2 of disappearance from the joint of less than 1 h, which suggested that the transferred chemoattractant must, in part, have been due to the generation of new chemotaxins by C5f injection. These observations suggest that continued generation of chemoattractants or failure of their subsequent removal may be mechanisms leading to persistent neutrophil influx in chronic inflammation.     

Effect of soluble epoxide hydrolase inhibition on epoxyeicosatrienoic acid metabolism in human blood vessels

We investigated the effects of soluble epoxide hydrolase (sEH) inhibition on epoxyeicosatrienoic acid (EET) metabolism in intact human blood vessels, including the human saphenous vein (HSV), coronary artery (HCA), and aorta (HA). When HSV segments were perfused with 2 micromol/l 14,15-[3H]EET for 4 h, >60% of radioactivity in the perfusion medium was converted to 14,15-dihydroxyeicosatrienoic acid (DHET). Similar results were obtained with endothelium-denuded vessels. 14,15-DHET was released from both the luminal and adventitial surfaces of the HSV. When HSVs were incubated with 14,15-[3H]EET under static (no flow) conditions, formation of 14,15-DHET was detected within 15 min and was inhibited by the selective sEH inhibitors N,N'-dicyclohexyl urea and N-cyclohexyl-N'-dodecanoic acid urea (CUDA). Similarly, CUDA inhibited the conversion of 11,12-[3H]EET to 11,12-DHET by the HSV. sEH inhibition enhanced the uptake of 14,15-[3H]EET and facilitated the formation of 10,11-epoxy-16:2, a beta-oxidation product. The HCA and HA converted 14,15-[3H]EET to DHET, and this also was inhibited by CUDA. These findings in intact human blood vessels indicate that conversion to DHET is the predominant pathway for 11,12- and 14,15-EET metabolism and that sEH inhibition can modulate EET metabolism in vascular tissue.     

Effects on Lignin Structure of Coumarate 3-Hydroxylase Downregulation in Poplar

The lignin structural ramifications of coumarate 3-hydroxylase (C3H) downregulation have not been addressed in hardwoods. Such information is required to accompany an assessment of the digestibility and bioenergy performance characteristics of poplar, in particular. Structurally rich 2D NMR methods were applied to the entire lignin fraction to delineate lignin p-hydroxyphenyl:guaiacyl:syringyl (H:G:S) levels and linkage distribution changes (and to compare with traditional degradative analyses). C3H downregulation reduced lignin levels by half and markedly increased the proportion of H units relative to the normally dominant G and S units. Relative stem H unit levels were up by ???100-fold to ???31?%, almost totally at the expense of G units; differences in the lignin interunit linkage distributions were more subtle. The H level in the most drastically C3H-downregulated transgenic poplar falls well beyond the H:G:S compositional bounds of normal angiosperms. The response observed here, in poplar, differs markedly from that reported for alfalfa where the S:G ratio remained almost constant even at substantial H levels, highlighting the often differing responses among plant species.