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    Insect Antennal Morphology: The Evolution of Diverse Solutions to Odorant Perception
    Elgar, MA ; Zhang, D ; Wang, Q ; Wittwer, B ; Hieu, TP ; Johnson, TL ; Freelance, CB ; Coquilleau, M (Yale University, 2018-12-01)
    Chemical communication involves the production, transmission, and perception of odors. Most adult insects rely on chemical signals and cues to locate food resources, oviposition sites or reproductive partners and, consequently, numerous odors provide a vital source of information. Insects detect these odors with receptors mostly located on the antennae, and the diverse shapes and sizes of these antennae (and sensilla) are both astonishing and puzzling: what selective pressures are responsible for these different solutions to the same problem - to perceive signals and cues? This review describes the selection pressures derived from chemical communication that are responsible for shaping the diversity of insect antennal morphology. In particular, we highlight new technologies and techniques that offer exciting opportunities for addressing this surprisingly neglected and yet crucial component of chemical communication.
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    Discovery of food identity markers by metabolomics and machine learning technology
    Erban, A ; Fehrle, I ; Martinez-Seidel, F ; Brigante, F ; Mas, AL ; Baroni, V ; Wunderlin, D ; Kopka, J (NATURE PORTFOLIO, 2019-07-04)
    Verification of food authenticity establishes consumer trust in food ingredients and components of processed food. Next to genetic or protein markers, chemicals are unique identifiers of food components. Non-targeted metabolomics is ideally suited to screen food markers when coupled to efficient data analysis. This study explored feasibility of random forest (RF) machine learning, specifically its inherent feature extraction for non-targeted metabolic marker discovery. The distinction of chia, linseed, and sesame that have gained attention as "superfoods" served as test case. Chemical fractions of non-processed seeds and of wheat cookies with seed ingredients were profiled. RF technology classified original seeds unambiguously but appeared overdesigned for material with unique secondary metabolites, like sesamol or rosmarinic acid in the Lamiaceae, chia. Most unique metabolites were diluted or lost during cookie production but RF technology classified the presence of the seed ingredients in cookies with 6.7% overall error and revealed food processing markers, like 4-hydroxybenzaldehyde for chia and succinic acid monomethylester for linseed additions. RF based feature extraction was adequate for difficult classifications but marker selection should not be without human supervision. Combination with alternative data analysis technologies is advised and further testing of a wide range of seeds and food processing methods.
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    Separation and Paired Proteome Profiling of Plant Chloroplast and Cytoplasmic Ribosomes
    Firmino, AAP ; Gorka, M ; Graf, A ; Skirycz, A ; Martinez-Seidel, F ; Zander, K ; Kopka, J ; Beine-Golovchuk, O (MDPI, 2020-07)
    Conventional preparation methods of plant ribosomes fail to resolve non-translating chloroplast or cytoplasmic ribosome subunits from translating fractions. We established preparation of these ribosome complexes from Arabidopsis thaliana leaf, root, and seed tissues by optimized sucrose density gradient centrifugation of protease protected plant extracts. The method co-purified non-translating 30S and 40S ribosome subunits separated non-translating 50S from 60S subunits, and resolved assembled monosomes from low oligomeric polysomes. Combining ribosome fractionation with microfluidic rRNA analysis and proteomics, we characterized the rRNA and ribosomal protein (RP) composition. The identity of cytoplasmic and chloroplast ribosome complexes and the presence of ribosome biogenesis factors in the 60S-80S sedimentation interval were verified. In vivo cross-linking of leaf tissue stabilized ribosome biogenesis complexes, but induced polysome run-off. Omitting cross-linking, the established paired fractionation and proteome analysis monitored relative abundances of plant chloroplast and cytoplasmic ribosome fractions and enabled analysis of RP composition and ribosome associated proteins including transiently associated biogenesis factors.
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    Systematic Review of Plant Ribosome Heterogeneity and Specialization
    Martinez-Seidel, F ; Beine-Golovchuk, O ; Hsieh, Y-C ; Kopka, J (FRONTIERS MEDIA SA, 2020-06-25)
    Plants dedicate a high amount of energy and resources to the production of ribosomes. Historically, these multi-protein ribosome complexes have been considered static protein synthesis machines that are not subject to extensive regulation but only read mRNA and produce polypeptides accordingly. New and increasing evidence across various model organisms demonstrated the heterogeneous nature of ribosomes. This heterogeneity can constitute specialized ribosomes that regulate mRNA translation and control protein synthesis. A prominent example of ribosome heterogeneity is seen in the model plant, Arabidopsis thaliana, which, due to genome duplications, has multiple paralogs of each ribosomal protein (RP) gene. We support the notion of plant evolution directing high RP paralog divergence toward functional heterogeneity, underpinned in part by a vast resource of ribosome mutants that suggest specialization extends beyond the pleiotropic effects of single structural RPs or RP paralogs. Thus, Arabidopsis is a highly suitable model to study this phenomenon. Arabidopsis enables reverse genetics approaches that could provide evidence of ribosome specialization. In this review, we critically assess evidence of plant ribosome specialization and highlight steps along ribosome biogenesis in which heterogeneity may arise, filling the knowledge gaps in plant science by providing advanced insights from the human or yeast fields. We propose a data analysis pipeline that infers the heterogeneity of ribosome complexes and deviations from canonical structural compositions linked to stress events. This analysis pipeline can be extrapolated and enhanced by combination with other high-throughput methodologies, such as proteomics. Technologies, such as kinetic mass spectrometry and ribosome profiling, will be necessary to resolve the temporal and spatial aspects of translational regulation while the functional features of ribosomal subpopulations will become clear with the combination of reverse genetics and systems biology approaches.
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    Unravelling the Metabolic and Hormonal Machinery During Key Steps of Somatic Embryogenesis: A Case Study in Coffee
    Awada, R ; Campa, C ; Gibault, E ; Dechamp, E ; Georget, F ; Lepelley, M ; Abdallah, C ; Erban, A ; Martinez-Seidel, F ; Kopka, J ; Legendre, L ; Leran, S ; Conejero, G ; Verdeil, J-L ; Crouzillat, D ; Breton, D ; Bertrand, B ; Etienne, H (MDPI, 2019-10)
    Somatic embryogenesis (SE) is one of the most promising processes for large-scale dissemination of elite varieties. However, for many plant species, optimizing SE protocols still relies on a trial-and-error approach. Using coffee as a model plant, we report here the first global analysis of metabolome and hormone dynamics aiming to unravel mechanisms regulating cell fate and totipotency. Sampling from leaf explant dedifferentiation until embryo development covered 15 key stages. An in-depth statistical analysis performed on 104 metabolites revealed that massive re-configuration of metabolic pathways induced SE. During initial dedifferentiation, a sharp decrease in phenolic compounds and caffeine levels was also observed while auxins, cytokinins and ethylene levels were at their highest. Totipotency reached its highest expression during the callus stages when a shut-off in hormonal and metabolic pathways related to sugar and energetic substance hydrolysis was evidenced. Abscisic acid, leucine, maltotriose, myo-inositol, proline, tricarboxylic acid cycle metabolites and zeatin appeared as key metabolic markers of the embryogenic capacity. Combining metabolomics with multiphoton microscopy led to the identification of chlorogenic acids as markers of embryo redifferentiation. The present analysis shows that metabolite fingerprints are signatures of cell fate and represent a starting point for optimizing SE protocols in a rational way.
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    Visualization of poly(ADP-ribose) bound to PARG reveals inherent balance between exo- and endo-glycohydrolase activities
    Barkauskaite, E ; Brassington, A ; Tan, ES ; Warwicker, J ; Dunstan, MS ; Banos, B ; Lafite, P ; Ahel, M ; Mitchison, TJ ; Ahel, I ; Leys, D (NATURE PUBLISHING GROUP, 2013-08)
    Poly-ADP-ribosylation is a post-translational modification that regulates processes involved in genome stability. Breakdown of the poly(ADP-ribose) (PAR) polymer is catalysed by poly(ADP-ribose) glycohydrolase (PARG), whose endo-glycohydrolase activity generates PAR fragments. Here we present the crystal structure of PARG incorporating the PAR substrate. The two terminal ADP-ribose units of the polymeric substrate are bound in exo-mode. Biochemical and modelling studies reveal that PARG acts predominantly as an exo-glycohydrolase. This preference is linked to Phe902 (human numbering), which is responsible for low-affinity binding of the substrate in endo-mode. Our data reveal the mechanism of poly-ADP-ribosylation reversal, with ADP-ribose as the dominant product, and suggest that the release of apoptotic PAR fragments occurs at unusual PAR/PARG ratios.
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    The transcriptional regulator CprK detects chlorination by combining direct and indirect readout mechanisms
    Kemp, LR ; Dunstan, MS ; Fisher, K ; Warwicker, J ; Leys, D (ROYAL SOC, 2013-04-19)
    The transcriptional regulator CprK controls the expression of the reductive dehalogenase CprA in organohalide-respiring bacteria. Desulfitobacterium hafniense CprA catalyses the reductive dechlorination of the terminal electron acceptor o-chlorophenol acetic acid, generating the phenol acetic acid product. It has been shown that CprK has ability to distinguish between the chlorinated CprA substrate and the de-halogenated end product, with an estimated an estimated 10(4)-fold difference in affinity. Using a green fluorescent protein GFPUV-based transcriptional reporter system, we establish that CprK can sense o-chlorophenol acetic acid at the nanomolar level, whereas phenol acetic acid leads to transcriptional activation only when approaching micromolar levels. A structure-activity relationship study, using a range of o-chlorophenol acetic-acid-related compounds and key CprK mutants, combined with pKa calculations on the effector binding site, suggests that the sensitive detection of chlorination is achieved through a combination of direct and indirect readout mechanisms. Both the physical presence of the bulky chloride substituent as well as the accompanying electronic effects lowering the inherent phenol pKa are required for high affinity. Indeed, transcriptional activation by CprK appears strictly dependent on establishing a phenolate-K133 salt bridge interaction, rather than on the presence of a halogen atom per se. As K133 is strictly conserved within the CprK family, our data suggest that physiological function and future applications in biosensing are probably restricted to phenolic compounds.
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    NUMT Confounding Biases Mitochondrial Heteroplasmy Calls in Favor of the Reference Allele
    Maude, H ; Davidson, M ; Charitakis, N ; Diaz, L ; Bowers, WHT ; Gradovich, E ; Andrew, T ; Huntley, D (FRONTIERS MEDIA SA, 2019-09-25)
    Homology between mitochondrial DNA (mtDNA) and nuclear DNA of mitochondrial origin (nuMTs) causes confounding when aligning short sequence reads to the reference human genome, as the true sequence origin cannot be determined. Using a systematic in silico approach, we here report the impact of all potential mitochondrial variants on alignment accuracy and variant calling. A total of 49,707 possible mutations were introduced across the 16,569 bp reference mitochondrial genome (16,569 × 3 alternative alleles), one variant at-at-time. The resulting in silico fragmentation and alignment to the entire reference genome (GRCh38) revealed preferential mapping of mutated mitochondrial fragments to nuclear loci, as variants increased loci similarity to nuMTs, for a total of 807, 362, and 41 variants at 333, 144, and 27 positions when using 100, 150, and 300 bp single-end fragments. We subsequently modeled these affected variants at 50% heteroplasmy and carried out variant calling, observing bias in the reported allele frequencies in favor of the reference allele. Four variants (chrM:6023A, chrM:4456T, chrM:5147A, and chrM:7521A) including a possible hypertension factor, chrM:4456T, caused 100% loss of coverage at the mutated position (with all 100 bp single-end fragments aligning to homologous, nuclear positions instead of chrM), rendering these variants undetectable when aligning to the entire reference genome. Furthermore, four mitochondrial variants reported to be pathogenic were found to cause significant loss of coverage and select haplogroup-defining SNPs were shown to exacerbate the loss of coverage caused by surrounding variants. Increased fragment length and use of paired-end reads both improved alignment accuracy.
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    YCu(TeO3)2(NO3)(H2O)3: a novel layered tellurite
    Mills, SJ ; Dunstan, MA ; Christy, AG (INT UNION CRYSTALLOGRAPHY, 2016-08)
    A new hydrated yttrium copper tellurite nitrate, yttrium(III) copper(II) bis-[trioxidotellurate(IV)] nitrate trihydrate, has been synthesized hydro-thermally in a Teflon-lined autoclave and structurally determined using synchrotron radiation. The new phase is the first example containing yttrium, copper and tellurium in one structure. Its crystal structure is unique, with relatively strongly bound layers extending parallel to (020), defined by YO8, CuO4 and TeO3 polyhedra, while the NO3 (-) anions and one third of the water mol-ecules lie between those layers. The structural unit consists of [Cu2(TeO3)4](4-) loop-branched chains of {Cu⋯Te⋯Cu⋯Te} squares running parallel to [001], which are linked further into layers only through Y(O,H2O)8 polyhedra. Weak 'secondary' Te bonds and O-H⋯O hydrogen-bonding inter-actions, involving water mol-ecules and layer O atoms, link the layers and inter-layer species. IR spectroscopic data are also presented.
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    Insights into the Vertical Stratification of Microbial Ecological Roles across the Deepest Seawater Column on Earth.
    Xue, C-X ; Liu, J ; Lea-Smith, DJ ; Rowley, G ; Lin, H ; Zheng, Y ; Zhu, X-Y ; Liang, J ; Ahmad, W ; Todd, JD ; Zhang, X-H (MDPI AG, 2020-08-27)
    The Earth's oceans are a huge body of water with physicochemical properties and microbial community profiles that change with depth, which in turn influences their biogeochemical cycling potential. The differences between microbial communities and their functional potential in surface to hadopelagic water samples are only beginning to be explored. Here, we used metagenomics to investigate the microbial communities and their potential to drive biogeochemical cycling in seven different water layers down the vertical profile of the Challenger Deep (0-10,500 m) in the Mariana Trench, the deepest natural point in the Earth's oceans. We recovered 726 metagenome-assembled genomes (MAGs) affiliated to 27 phyla. Overall, biodiversity increased in line with increased depth. In addition, the genome size of MAGs at ≥4000 m layers was slightly larger compared to those at 0-2000 m. As expected, surface waters were the main source of primary production, predominantly from Cyanobacteria. Intriguingly, microbes conducting an unusual form of nitrogen metabolism were identified in the deepest waters (>10,000 m), as demonstrated by an enrichment of genes encoding proteins involved in dissimilatory nitrate to ammonia conversion (DNRA), nitrogen fixation and urea transport. These likely facilitate the survival of ammonia-oxidizing archaea α lineage, which are typically present in environments with a high ammonia concentration. In addition, the microbial potential for oxidative phosphorylation and the glyoxylate shunt was enhanced in >10,000 m waters. This study provides novel insights into how microbial communities and their genetic potential for biogeochemical cycling differs through the Challenger deep water column, and into the unique adaptive lifestyle of microbes in the Earth's deepest seawater.