Lysophosphatidic acid increases soluble ST2 expression in mouse lung and human bronchial epithelial cells

Lysophosphatidic acid (LPA), a naturally occurring bioactive lysophospholipid increases the expression of both pro-inflammatory and anti-inflammatory mediators in airway epithelial cells. Soluble ST2 (sST2), an anti-inflammatory mediator, has been known to function as a decoy receptor of interleukin (IL)-33 and attenuates endotoxin-induced inflammatory responses. Here, we show that LPA increased sST2 mRNA expression and protein release in a dose and time dependent manner in human bronchial epithelial cells (HBEpCs). LPA receptors antagonist and Gαi inhibitor, pertussis toxin, attenuated LPA-induced sST2 release. Inhibition of NF-κB or JNK pathway reduced LPA-induced sST2 release. LPA treatment decreased histone deacetylase 3 (HDAC3) expression and enhanced acetylation of histone H3 at lysine 9 that binds to the sST2 promoter region. Furthermore, limitation of intracellular LPA generation by the down-regulation of acetyl glycerol kinase attenuated exogenous LPA-induced histone H3 acetylation on sST2 promoter region, as well as sST2 gene expression. Treatment of HBEpCs with recombinant sST2 protein or sST2-rich cell culture media attenuated endotoxin-induced phosphorylation of PKC and airway epithelial barrier disruption. These results unravel a novel sST2 mediated signaling pathway that has physiological relevance to airway inflammation and remodeling.     

Mycorrhizal networks: common goods of plants shared under unequal terms of trade

Plants commonly live in a symbiotic association with arbuscular mycorrhizal fungi (AMF). They invest photosynthetic products to feed their fungal partners, which, in return, provide mineral nutrients foraged in the soil by their intricate hyphal networks. Intriguingly, AMF can link neighboring plants, forming common mycorrhizal networks (CMNs). What are the terms of trade in such CMNs between plants and their shared fungal partners? To address this question, we set up microcosms containing a pair of test plants, interlinked by a CMN of Glomus intraradices or Glomus mosseae. The plants were flax (Linum usitatissimum; a C₃ plant) and sorghum (Sorghum bicolor; a C₄ plant), which display distinctly different ¹³C/¹²C isotope compositions. This allowed us to differentially assess the carbon investment of the two plants into the CMN through stable isotope tracing. In parallel, we determined the plants’ “return of investment” (i.e. the acquisition of nutrients via CMN) using ¹⁵N and ³³P as tracers. Depending on the AMF species, we found a strong asymmetry in the terms of trade: flax invested little carbon but gained up to 94% of the nitrogen and phosphorus provided by the CMN, which highly facilitated growth, whereas the neighboring sorghum invested massive amounts of carbon with little return but was barely affected in growth. Overall biomass production in the mixed culture surpassed the mean of the two monocultures. Thus, CMNs may contribute to interplant facilitation and the productivity boosts often found with intercropping compared with conventional monocropping.Arbuscular mycorrhizal fungi (AMF) inhabit the soils of virtually all terrestrial ecosystems, forming symbiotic associations with most plants (Parniske, 2008; Smith and Read, 2008). The host plants incur substantial carbon costs to sustain this symbiosis (Jakobsen and Rosendahl, 1990), but in return, they obtain multiple benefits from the fungal partners, above all, the provision of mineral nutrients. AMF may supply up to 90% of the host plant’s nitrogen and phosphorus requirements (Smith and Read, 2008). Moreover, AMF are important determinants of plant community structure and ecosystem productivity (Grime et al., 1987; van der Heijden et al., 1998), and they represent a crucial asset for sustainable agriculture (Rooney et al., 2009). Typically, AMF exhibit little host specificity; a single individual may form a common mycorrhizal network (CMN) between several coexisting plant individuals, even from different species (Whitfield, 2007; Smith and Read, 2008; Bever et al., 2010). Such CMNs may be enlarged through hyphal fusion of conspecific AMF (Giovannetti et al., 2004). The functionality of CMNs formed by the fusion of two individual fungal networks by hyphal anastomoses has been demonstrated by tracing nutrient allocation between individual host plants upon the fusion of their associated CMNs (Mikkelsen et al., 2008).The potential role and importance of CMNs is most apparent in the case of mycoheterotrophic plants. These plants connect themselves to an existing CMN to receive both carbon and mineral nutrients (Bidartondo et al., 2002; Courty et al., 2011). There is an ongoing debate over whether carbon transfer through CMNs may also occur among autotrophic plants (Bever et al., 2010; Hodge et al., 2010). This is of a certain academic interest, but it may obscure a more general and obvious question arising from recent literature (Hodge et al., 2010; Hammer et al., 2011; Kiers et al., 2011; Smith and Smith, 2011; Fellbaum et al., 2012): What are the terms of trade between plants and their shared fungal partners? Put another way, what is the “investment” of a given plant into a CMN (in the currency of assimilated carbon), and what is the “return of investment” in terms of mineral nutrients provided by the CMN? Indeed, different cocultivated plants benefit differently from their CMN, depending on the AMF species involved, and these differences significantly affect plant coexistence (Zabinski et al., 2002; van der Heijden et al., 2003; Wagg et al., 2011). However, until now, the relationship between the carbon investment and the nutritional benefit of different plants engaged in a CMN has never been assessed.To address the terms of trade in a CMN experimentally, we established a model system consisting of two plant individuals growing side by side in compartmented microcosms (Fig. 1). The roots of the plants were confined to their respective “root hyphal compartments” (RHCs). In the treatments with AMF inoculation, however, the plants were able to connect through CMN in the “hyphal compartment” (HC) or in the “label-hyphal compartment” (LHC). We assessed the carbon investments of the single plants into the CMN through stable isotope tracing. To this end, we chose the C₃ plant flax (Linum usitatissimum) and the C₄ plant sorghum (Sorghum bicolor) for our experiments. Due to the different isotope fractionation during C₃ versus C₄ carbon fixation, these two species display distinctly different carbon isotope ratios (δ ¹³C approximately 33‰ for flax and approximately 14‰ for sorghum). This difference in the ¹³C signature of C₃ and C₄ plants has been widely used to track carbon flows in mycorrhizal symbioses (Allen and Allen, 1990; Fitter et al., 1998). The plants were grown either in “monocultures,” as a pair of identical plant species, or in a “mixed culture,” as a pair of different plant species. We used two different AMF species in the experiments for inoculation, Glomus intraradices and Glomus mosseae (recently renamed Glomus irregulare [Rhizophagus irregularis] and Funelliformis mosseae, respectively [Schüssler and Walker, 2010]). The chosen experimental setup allowed us to harvest the bulk of the CMN in the HC (Fig. 1) and to estimate the respective carbon investment of the two plants into the CMN through the analysis of the δ ¹³C of isolated AMF hyphae or, with higher precision, of the AMF-specific fatty acid C16:1ω5 (Olsson and Johnson, 2005). We estimated the return of investment with respect to nitrogen and phosphorus for each of the two plants using ¹⁵N and ³³P as tracers added to the LHC (Fig. 1). As a control, we also grew two monocultures and a mixed culture without any AMF inoculation.Open in a separate windowFigure 1.Compartmented microcosms to study the role of CMNs in monocultures and mixed culture. Microcosms, consisting of two plant individuals, were set up in compartmented containers subdivided by nylon mesh screens (25 and 65 μm, respectively, as indicated). Both types of screens are pervious for fungal hyphae but not for roots and allow the separation into two RHCs, a HC, and a LHC for supplying ¹⁵N and ³³P labels. The plants used were flax (F) and sorghum (S) either as a pair of conspecific plants (F:F, S:S) as a model of monoculture or in combination (F:S) as a model of a mixed culture.     

Characterization of the fungicidal activity of Calothrix elenkinii using chemical methods and microscopy

An investigation was directed towards biochemical characterization of cyanobacterium Calothrix elenkinii and analysis of the chemical nature and mode of action of its fungicidal metabolite(s) against oomycete Pythium debaryanum. Biochemical characterization of the culture in terms of carbohydrate utilization revealed the facultative nature of C. elenkinii. Unique antibiotic markers were also found for this strain. 16S rDNA sequencing of the strain revealed 98% similarity with Calothrix sp. PCC7101. The fungicidal activity was tested by disc diffusion assay of different fractions of the culture filtrate. A minimum inhibitory concentration of 10 microl was recorded for ethyl acetate fraction of the 7-weeks old culture filtrates. HPLC, followed by NMR spectral analysis demonstrated the presence of a substituted benzoic acid in the ethyl acetate fraction. Microscopic examination revealed distinct granulation, followed by disintegration of the hyphae of Pythium sp., indicating the presence of an active metabolite in the culture filtrates of Calothrix sp. The fungicidal activity of C. elenkinii can be attributed to the presence of 3-acetyl-2-hydroxy-6-methoxy-4-methyl benzoic acid. This is the first report of a benzoic acid derivative having fungicidal activity in cyanobacteria.     

The peptide-receptive transition state of MHC class I molecules: insight from structure and molecular dynamics

MHC class I (MHC-I) proteins of the adaptive immune system require antigenic peptides for maintenance of mature conformation and immune function via specific recognition by MHC-I-restricted CD8(+) T lymphocytes. New MHC-I molecules in the endoplasmic reticulum are held by chaperones in a peptide-receptive (PR) transition state pending release by tightly binding peptides. In this study, we show, by crystallographic, docking, and molecular dynamics methods, dramatic movement of a hinged unit containing a conserved 3(10) helix that flips from an exposed "open" position in the PR transition state to a "closed" position with buried hydrophobic side chains in the peptide-loaded mature molecule. Crystallography of hinged unit residues 46-53 of murine H-2L(d) MHC-I H chain, complexed with mAb 64-3-7, demonstrates solvent exposure of these residues in the PR conformation. Docking and molecular dynamics predict how this segment moves to help form the A and B pockets crucial for the tight peptide binding needed for stability of the mature peptide-loaded conformation, chaperone dissociation, and Ag presentation.     

Novel role for non-muscle myosin light chain kinase (MLCK) in hyperoxia-induced recruitment of cytoskeletal proteins, NADPH oxidase activation, and reactive oxygen species generation in lung endothelium

We recently demonstrated that hyperoxia (HO) activates lung endothelial cell NADPH oxidase and generates reactive oxygen species (ROS)/superoxide via Src-dependent tyrosine phosphorylation of p47(phox) and cortactin. Here, we demonstrate that the non-muscle ~214-kDa myosin light chain (MLC) kinase (nmMLCK) modulates the interaction between cortactin and p47(phox) that plays a role in the assembly and activation of endothelial NADPH oxidase. Overexpression of FLAG-tagged wild type MLCK in human pulmonary artery endothelial cells enhanced interaction and co-localization between cortactin and p47(phox) at the cell periphery and ROS production, whereas abrogation of MLCK using specific siRNA significantly inhibited the above. Furthermore, HO stimulated phosphorylation of MLC and recruitment of phosphorylated and non-phosphorylated cortactin, MLC, Src, and p47(phox) to caveolin-enriched microdomains (CEM), whereas silencing nmMLCK with siRNA blocked recruitment of these components to CEM and ROS generation. Exposure of nmMLCK(-/-) null mice to HO (72 h) reduced ROS production, lung inflammation, and pulmonary leak compared with control mice. These results suggest a novel role for nmMLCK in hyperoxia-induced recruitment of cytoskeletal proteins and NADPH oxidase components to CEM, ROS production, and lung injury.     

Initiation and progression of mechanical damage in the intervertebral disc under cyclic loading using continuum damage mechanics methodology: A finite element study

It is difficult to study the breakdown of disc tissue over several years of exposure to bending and lifting by experimental methods. There is also no finite element model that elucidates the failure mechanism due to repetitive loading of the lumbar motion segment. The aim of this study was to refine an already validated poro-elastic finite element model of lumbar motion segment to investigate the initiation and progression of mechanical damage in the disc under simple and complex cyclic loading conditions. Continuum damage mechanics methodology was incorporated into the finite element model to track the damage accumulation in the annulus in response to the repetitive loading. The analyses showed that the damage initiated at the posterior inner annulus adjacent to the endplates and propagated outwards towards its periphery under all loading conditions simulated. The damage accumulated preferentially in the posterior region of the annulus. The analyses also showed that the disc failure is unlikely to happen with repetitive bending in the absence of compressive load. Compressive cyclic loading with low peak load magnitude also did not create the failure of the disc. The finite element model results were consistent with the experimental and clinical observations in terms of the region of failure, magnitude of applied loads and the number of load cycles survived.     

Mycobacterium tuberculosis and dendritic cells: recognition, activation and functional implications

The highly complex nature of interactions of Mycobacterium tuberculosis with cells of the immune system has puzzled researchers the world-over in understanding the pathogenesis and immunology associated with tuberculosis (TB). This has contributed to the delay in development of effective vaccine(s) for TB. Several excellent studies have provided only a glimpse of the kind and degree of immune responses elicited following infection by mycobacteria. Preferred entry via respiratory route results in the capture of mycobacteria by alveolar macrophages that eventually become their long-term hosts. Since the pathogen is rarely cleared this has resulted in the human population serving as a large reservoir for mycobacteria. Owing to their unique ability to prime na?ve and memory T cells, dendritic cells (DCs) play important and indispensable roles in the initiation and maintenance of protective immune responses following infection. The kind of immune response initiated by DCs with respect to mycobacteria determines the character of immune responses mounted by the host against the pathogen. The profile of cytokines and chemokines secreted as a result of infection of DCs by mycobacteria further plays an important role in defining the course of infection. This minireview attempts to highlight key interactions of mycobacteria with dendritic cells. We discus the uptake of mycobacteria by DCs followed by DC activation and the spectrum of immune responses initiated by infected/activated DCs, followed by numerous ways the pathogen has devised to subvert protective responses.