Delineation of RAID1, the RACK1 interaction domain located within the unique N-terminal region of the cAMP-specific phosphodiesterase, PDE4D5

Background The cyclic AMP specific phosphodiesterase, PDE4D5 interacts with the β-propeller protein RACK1 to form a signaling scaffold complex in cells. Two-hybrid analysis of truncation and mutant constructs of the unique N-terminal region of the cAMP-specific phosphodiesterase, PDE4D5 were used to define a domain conferring interaction with the signaling scaffold protein, RACK1. Results Truncation and mutagenesis approaches showed that the RACK1-interacting domain on PDE4D5 comprised a cluster of residues provided by Asn-22/Pro-23/Trp-24/Asn-26 together with a series of hydrophobic amino acids, namely Leu-29, Val-30, Leu-33, Leu-37 and Leu-38 in a 'Leu-Xaa-Xaa-Xaa-Leu' repeat. This was done by 2-hybrid analyses and then confirmed in biochemical pull down analyses using GST-RACK1 and mutant PDE4D5 forms expressed in COS cells. Mutation of Arg-34, to alanine, in PDE4D5 attenuated its interaction with RACK1 both in 2-hybrid screens and in pull down analyses. A 38-mer peptide, whose sequence reflected residues 12 through 49 of PDE4D5, bound to RACK1 with similar affinity to native PDE4D5 itself (Ka circa 6 nM). Conclusions The RACK1 Interaction Domain on PDE4D5, that we here call RAID1, is proposed to form an amphipathic helical structure that we suggest may interact with the C-terminal β-propeller blades of RACK1 in a manner akin to the interaction of the helical G-γ signal transducing protein with the β-propeller protein, G-β

In Vivo Metal Substitutions in Metal Sequestering Subcellular Compartments: X-Ray Mapping in Cryosections

Qualitative digital X-ray mapping techniques were employed to determine the distributions of essential and non-essential elements in three invertebrate models : (1) Pb, Zn, Cd, Cu, Fe in thin cryosections of the hepatopancreas of the terrestrial isopod, Oniscus asellus; (2) Pb, Zn, Cd, Ca in thin cryosections of the chloragogenous tissue of the earthworm, Lumbricus rubellus; and (3) As in air-dried smears and thin cryosections of chloragogen in L. rubellus. Four general conclusions were drawn from the results of these studies: (a) non-essential elements can accumulate, distribute and be compartmentalized because they, or the organo-complexes that they form, act as mimics of essential elements with which they share to a greater or lesser extent certain chemical affinities; (b) thermodynamic considerations notwithstanding, the influence of biological factors on the sequestration and fates of certain elements (e.g., arsenic) is profound through modifications of redox states and organo-compound formation; (c) X-ray mapping, combined with anhydrous preparative procedures, yields unbiased information concerning the relative spatial distributions of several elements in structurally heterogeneous sampling fields , although the morphological characterization of (occasionally unsuspected) subcellular compartments may be constrained by the intrinsic quality of the preparation; and (d) X-ray microanalysis yields codistribution data, when integrated with biochemical information from other sources, which give strong pointers to the identity of binding ligands and of the valence state of sequestered cations

Bulk mineralogical characterisation of oilfield reservoir rocks and sandstones using DRIFTS and partial least squares analysis

The feasibility of applying Partial Least Squares (PLS) to the Diffuse Reflectance Infrared Fourier Transform Spectroscopy (DRIFTS) spectra of mineral mixtures, quarry sandstones and oilfield reservoir rocks has been investigated and shown considerable potential for accurate and precise mineralogical analysis. Rapid spectrum acquisition and data processing together with small sample size requirements are key advantages of the PLS–DRIFTS method. A PLS model was created from the DRIFTS spectra of mixtures of seven mineral standards chosen to represent the most frequently encountered minerals in sandstone-type rocks; quartz, dolomite, montmorillonite, illite, kaolinite, chlorite and albite. The PLS–DRIFTS model was able to quantify the mineral components of independent mixtures with an absolute error of 1 wt.% for all the minerals (concentration range 0–30 wt.%) with the exception of quartz which exhibited an absolute error of 3 wt.% (concentration range 50–90 wt.%). The results provided by applying this PLS–DRIFTS model to several sandstone-type quarry rocks and a suite of oilfield reservoir rocks were considerably better than anticipated even though the model did not describe all the mineral components present in the samples nor the entire variance of constituent mineral components (e.g. crystallinity). The model was not able to differentiate between montmorillonite and illite probably due to the similarity of the DRIFTS spectra of these minerals, but it was able to quantify the combined (montmorillonite + illite) concentrations to within 1 wt.%. The model over-predicted the concentration of albite in the quarry rocks due to the presence of K-feldspar, which has a similar DRIFTS spectrum and was not included in the model. However, the model accurately predicted the total (albite and K-feldspar) concentration to within 4 wt.%. A separate PLS–DRIFTS model constructed using the DRIFTS spectra of the oilfield reservoir rocks showed that the carbonate components, calcite and dolomite could be differentiated and quantified to within 5.0 and 3.6 wt.%, respectively. This feasibility study confirmed the strong potential of combining DRIFTS with a multivariate statistical approach such as PLS and it is clear that more sophisticated models, that incorporates and describes a higher percentage of the variance in unknowns, would further improve the predictions

Vascular smooth muscle cells enhance immune/vascular interplay in a 3-cell model of vascular inflammation

Atherosclerosis is a serious cardiovascular disease that is characterised by the development of atheroma, which are lipid-laden plaques that build up within arterial walls due to chronic inflammatory processes. These lesions are fundamentally attributed to a complex cellular crosstalk between vascular smooth muscle cells (VSMCs), vascular endothelial cells (VECs) and central immune cells, such as macrophages (Mɸs), which promote vascular inflammation. The presence of VSMCs exerts both positive and negative effects during atheroma development, which can be attributed to their phenotypic plasticity. Understanding the interactions between these key cell types during the development of vascular inflammation and atheroma will enhance the scope for new therapeutic interventions. This study aims to determine the importance of VSMCs for shaping the extracellular cytokine/chemokine profile and transcriptional responses of VECs (human coronary artery endothelial cells; HCAECs) to activated lipopolysaccharide (LPS)-stimulated THP1 Mɸs, in a 3-cell model of human vascular inflammation. It is evident that within the presence of VSMCs, enhanced cytokine production was associated with up-regulation of genes associated with vascular inflammation t. Results demonstrate that the presence of VSMCs in co-culture experiments enhanced cytokine production (including CXCL1/GROα, IL-6, IL-8 and CCL2/MCP1) and inflammatory gene expression (including genes involved in JAK/STAT, Jun and NFκB signalling) in HCAECs co-cultured with LPS-stimulated THP1 Mɸs. Our results highlight the importance of VSMCs in immune/endothelial cell interplay and indicate that 3-cell, rather than 2-cell co-culture, may be more appropriate for the study of cellular crosstalk between immune and vascular compartments in response to inflammatory and atherogenic stimuli

The Lore of Low Methane Livestock:Co-Producing Technology and Animals for Reduced Climate Change Impact

Methane emissions from sheep and cattle production have gained increasing profile in the context of climate change. Policy and scientific research communities have suggested a number of technological approaches to mitigate these emissions. This paper uses the concept of co-production as an analytical framework to understand farmers’ evaluation of a 'good animal’. It examines how technology and sheep and beef cattle are co-produced in the context of concerns about the climate change impact of methane. Drawing on 42 semi-structured interviews, this paper demonstrates that methane emissions are viewed as a natural and integral part of sheep and beef cattle by farmers, rather than as a pollutant. Sheep and beef cattle farmers in the UK are found to be an extremely heterogeneous group that need to be understood in their specific social, environmental and consumer contexts. Some are more amenable to appropriating methane reducing measures than others, but largely because animals are already co-constructed from the natural and the technical for reasons of increased production efficiency

Soil microbial communities in diverse agroecosystems exposed to the herbicide glyphosate

© 2020 American Society for Microbiology. Despite glyphosate\u27s wide use for weed control in agriculture, questions remain about the herbicide\u27s effect on soil microbial communities. The existing scientific literature contains conflicting results, from no observable effect of glyphosate to the enrichment of agricultural pathogens such as Fusarium spp. We conducted a comprehensive field-based study to compare the microbial communities on the roots of plants that received a foliar application of glyphosate to adjacent plants that did not. The 2-year study was conducted in Beltsville, MD, and Stoneville, MS, with corn and soybean crops grown in a variety of organic and conventional farming systems. By sequencing environmental metabarcode amplicons, the prokaryotic and fungal communities were described, along with chemical and physical properties of the soil. Sections of corn and soybean roots were plated to screen for the presence of plant pathogens. Geography, farming system, and season were significant factors determining the composition of fungal and prokaryotic communities. Plots treated with glyphosate did not differ from untreated plots in overall microbial community composition after controlling for other factors. We did not detect an effect of glyphosate treatment on the relative abundance of organisms such as Fusarium spp

Capture Hi-C identifies a novel causal gene, IL20RA, in the pan-autoimmune genetic susceptibility region 6q23

BACKGROUND: The identification of causal genes from genome-wide association studies (GWAS) is the next important step for the translation of genetic findings into biologically meaningful mechanisms of disease and potential therapeutic targets. Using novel chromatin interaction detection techniques and allele specific assays in T and B cell lines, we provide compelling evidence that redefines causal genes at the 6q23 locus, one of the most important loci that confers autoimmunity risk. RESULTS: Although the function of disease-associated non-coding single nucleotide polymorphisms (SNPs) at 6q23 is unknown, the association is generally assigned to TNFAIP3, the closest gene. However, the DNA fragment containing the associated SNPs interacts through chromatin looping not only with TNFAIP3, but also with IL20RA, located 680 kb upstream. The risk allele of the most likely causal SNP, rs6927172, is correlated with both a higher frequency of interactions and increased expression of IL20RA, along with a stronger binding of both the NFκB transcription factor and chromatin marks characteristic of active enhancers in T-cells. CONCLUSIONS: Our results highlight the importance of gene assignment for translating GWAS findings into biologically meaningful mechanisms of disease and potential therapeutic targets; indeed, monoclonal antibody therapy targeting IL-20 is effective in the treatment of rheumatoid arthritis and psoriasis, both with strong GWAS associations to this region

Metagenomics Reveals Bacterial and Archaeal Adaptation to Urban Land-Use : N Catabolism, Methanogenesis, and Nutrient Acquisition

Urbanization results in the systemic conversion of land-use, driving habitat and biodiversity loss. The "urban convergence hypothesis" posits that urbanization represents a merging of habitat characteristics, in turn driving physiological and functional responses within the biotic community. To test this hypothesis, we sampled five cities (Baltimore, MD, United States; Helsinki and Lahti, Finland; Budapest, Hungary; Potchefstroom, South Africa) across four different biomes. Within each city, we sampled four land-use categories that represented a gradient of increasing disturbance and management (from least intervention to highest disturbance: reference, remnant, turf/lawn, and ruderal). Previously, we used amplicon sequencing that targeted bacteria/archaea (16S rRNA) and fungi (ITS) and reported convergence in the archaeal community. Here, we applied shotgun metagenomic sequencing and QPCR of functional genes to the same soil DNA extracts to test convergence in microbial function. Our results suggest that urban land-use drives changes in gene abundance related to both the soil N and C metabolism. Our updated analysis found taxonomic convergence in both the archaeal and bacterial community (16S amplicon data). Convergence of the archaea was driven by increased abundance of ammonia oxidizing archaea and genes for ammonia oxidation (QPCR and shotgun metagenomics). The proliferation of ammonia-oxidizers under turf and ruderal land-use likely also contributes to the previously documented convergence of soil mineral N pools. We also found a higher relative abundance of methanogens (amplicon sequencing), a higher relative abundance of gene sequences putatively identified as Ni-Fe hydrogenase and nickel uptake (shotgun metagenomics) under urban land-use; and a convergence of gene sequences putatively identified as contributing to the nickel transport function under urban turf sites. High levels of disturbance lead to a higher relative abundance of gene sequences putatively identified as multiple antibiotic resistance protein marA and multidrug efflux pump mexD, but did not lead to an overall convergence in antibiotic resistance gene sequences.Peer reviewe

Community perspectives on the benefits and risks of technologically enhanced communicable disease surveillance systems: a report on four community juries

BACKGROUND:Outbreaks of infectious disease cause serious and costly health and social problems. Two new technologies - pathogen whole genome sequencing (WGS) and Big Data analytics - promise to improve our capacity to detect and control outbreaks earlier, saving lives and resources. However, routinely using these technologies to capture more detailed and specific personal information could be perceived as intrusive and a threat to privacy. METHOD:Four community juries were convened in two demographically different Sydney municipalities and two regional cities in New South Wales, Australia (western Sydney, Wollongong, Tamworth, eastern Sydney) to elicit the views of well-informed community members on the acceptability and legitimacy of: making pathogen WGS and linked administrative data available for public health researchusing this information in concert with data linkage and machine learning to enhance communicable disease surveillance systems Fifty participants of diverse backgrounds, mixed genders and ages were recruited by random-digit-dialling and topic-blinded social-media advertising. Each jury was presented with balanced factual evidence supporting different expert perspectives on the potential benefits and costs of technologically enhanced public health research and communicable disease surveillance and given the opportunity to question experts. RESULTS:Almost all jurors supported data linkage and WGS on routinely collected patient isolates for the purposes of public health research, provided standard de-identification practices were applied. However, allowing this information to be operationalised as a syndromic surveillance system was highly contentious with three juries voting in favour, and one against by narrow margins. For those in favour, support depended on several conditions related to system oversight and security being met. Those against were concerned about loss of privacy and did not trust Australian governments to run secure and effective systems. CONCLUSIONS:Participants across all four events strongly supported the introduction of data linkage and pathogenomics to public health research under current research governance structures. Combining pathogen WGS with event-based data surveillance systems, however, is likely to be controversial because of a lack of public trust, even when the potential public health benefits are clear. Any suggestion of private sector involvement or commercialisation of WGS or surveillance data was unanimously rejected.Chris Degeling, Stacy M. Carter, Antoine M. van Oijen, Jeremy McAnulty, Vitali Sintchenko, Annette Braunack-Mayer ... et al

Global-change effects on early-stage decomposition processes in tidal wetlands – implications from a global survey using standardized litter

Tidal wetlands, such as tidal marshes and mangroves, are hotspots for carbon sequestration. The preservation of organic matter (OM) is a critical process by which tidal wetlands exert influence over the global carbon cycle and at the same time gain elevation to keep pace with sea-level rise (SLR). The present study assessed the effects of temperature and relative sea level on the decomposition rate and stabilization of OM in tidal wetlands worldwide, utilizing commercially available standardized litter. While effects on decomposition rate per se were minor, we show strong negative effects of temperature and relative sea level on stabilization, as based on the fraction of labile, rapidly hydrolyzable OM that becomes stabilized during deployment. Across study sites, OM stabilization was 29 % lower in low, more frequently flooded vs. high, less frequently flooded zones. Stabilization declined by ∼ 75 % over the studied temperature gradient from 10.9 to 28.5 ∘C. Additionally, data from the Plum Island long-term ecological research site in Massachusetts, USA, show a pronounced reduction in OM stabilization by > 70 % in response to simulated coastal eutrophication, confirming the potentially high sensitivity of OM stabilization to global change. We therefore provide evidence that rising temperature, accelerated SLR, and coastal eutrophication may decrease the future capacity of tidal wetlands to sequester carbon by affecting the initial transformations of recent OM inputs to soil OM