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  • 1
    Language: English
    In: Nature communications, 2017-09-13, Vol.8 (1), p.526-10
    Description: Wolbachia are intracellular maternally inherited bacteria that can spread through insect populations and block virus transmission by mosquitoes, providing an important approach to dengue control. To better understand the mechanisms of virus inhibition, we here perform proteomic quantification of the effects of Wolbachia in Aedes aegypti mosquito cells and midgut. Perturbations are observed in vesicular trafficking, lipid metabolism and in the endoplasmic reticulum that could impact viral entry and replication. Wolbachia-infected cells display a differential cholesterol profile, including elevated levels of esterified cholesterol, that is consistent with perturbed intracellular cholesterol trafficking. Cyclodextrins have been shown to reverse lipid accumulation defects in cells with disrupted cholesterol homeostasis. Treatment of Wolbachia-infected Ae. aegypti cells with 2-hydroxypropyl-β-cyclodextrin restores dengue replication in Wolbachia-carrying cells, suggesting dengue is inhibited in Wolbachia-infected cells by localised cholesterol accumulation. These results demonstrate parallels between the cellular Wolbachia viral inhibition phenotype and lipid storage genetic disorders. Wolbachia infection of mosquitoes can block dengue virus infection and is tested in field trials, but the mechanism of action is unclear. Using proteomics, Geoghegan et al. here identify effects of Wolbachia on cholesterol homeostasis and dengue virus replication in Aedes aegypti.
    Subject(s): Accumulation ; Aedes - metabolism ; Aedes - microbiology ; Aedes - virology ; Aedes aegypti ; Animals ; Aquatic insects ; Bacteria ; Blocking ; Cholesterol ; Cholesterol - metabolism ; Cyclodextrin ; Cyclodextrins ; Cytoplasmic Vesicles - metabolism ; Dengue ; Dengue - transmission ; Dengue - virology ; Dengue fever ; Dengue Virus - physiology ; Disease control ; Endoplasmic reticulum ; Esterification ; Genetic disorders ; Homeostasis ; Humans ; Infections ; Inhibition ; Insects ; Intracellular ; Lipid metabolism ; Lipids ; Metabolism ; Midgut ; Mosquitoes ; Proteomics ; Replication ; Vector-borne diseases ; Viral diseases ; Viruses ; Wolbachia ; Wolbachia - physiology
    ISSN: 2041-1723
    E-ISSN: 2041-1723
    Source: Nature Open Access
    Source: PubMed Central
    Source: DOAJ Directory of Open Access Journals - Not for CDI Discovery
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  • 2
    Language: English
    In: Science (American Association for the Advancement of Science), 2015-07-17, Vol.349 (6245), p.309-312
    Description: Morphinan alkaloids from the opium poppy are used for pain relief. The direction of metabolites to morphinan biosynthesis requires isomerization of (S)- to (R)-reticuline. Characterization of high-reticuline poppy mutants revealed a genetic locus, designated STORR [(S)- to (R)-reticuline] that encodes both cytochrome P450 and oxidoreductase modules, the latter belonging to the aldo-keto reductase family. Metabolite analysis of mutant alleles and heterologous expression demonstrate that the P450 module is responsible for the conversion of (S)-reticuline to 1,2-dehydroreticuline, whereas the oxidoreductase module converts 1,2-dehydroreticuline to (R)-reticuline rather than functioning as a P450 redox partner. Proteomic analysis confirmed that these two modules are contained on a single polypeptide in vivo. This modular assembly implies a selection pressure favoring substrate channeling. The fusion protein STORR may enable microbial-based morphinan production.
    Subject(s): Biosynthesis ; Enzymes ; Metabolites ; Modules ; Morphine ; Narcotics ; Pathways ; Poppies ; Proteins ; REPORTS
    ISSN: 0036-8075
    E-ISSN: 1095-9203
    Source: JSTOR Life Sciences
    Source: Academic Search Ultimate
    Source: Alma/SFX Local Collection
    Source: Get It Now
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  • 3
    Language: English
    In: Nature communications, 2018-02-22, Vol.9 (1), p.756-12
    Description: Thermobia domestica belongs to an ancient group of insects and has a remarkable ability to digest crystalline cellulose without microbial assistance. By investigating the digestive proteome of Thermobia, we have identified over 20 members of an uncharacterized family of lytic polysaccharide monooxygenases (LPMOs). We show that this LPMO family spans across several clades of the Tree of Life, is of ancient origin, and was recruited by early arthropods with possible roles in remodeling endogenous chitin scaffolds during development and metamorphosis. Based on our in-depth characterization of Thermobia's LPMOs, we propose that diversification of these enzymes toward cellulose digestion might have endowed ancestral insects with an effective biochemical apparatus for biomass degradation, allowing the early colonization of land during the Paleozoic Era. The vital role of LPMOs in modern agricultural pests and disease vectors offers new opportunities to help tackle global challenges in food security and the control of infectious diseases.
    Subject(s): Animals ; Arthropods ; Arthropods - enzymology ; Arthropods - genetics ; Arthropods - growth & development ; Biochemistry ; Biochemistry, Molecular Biology ; Biodegradation ; Biodegradation, Environmental ; Biomass ; Cellulose ; Cellulose - metabolism ; Chitin ; Chitin - metabolism ; Colonization ; Crystalline cellulose ; Digestion ; Environmental degradation ; Evolution, Molecular ; Food security ; Genes, Insect ; Infectious diseases ; Insect Proteins - chemistry ; Insect Proteins - genetics ; Insect Proteins - metabolism ; Insecta - enzymology ; Insecta - genetics ; Insecta - growth & development ; Insects ; Life Sciences ; Metamorphosis ; Microorganisms ; Mixed Function Oxygenases - chemistry ; Mixed Function Oxygenases - genetics ; Mixed Function Oxygenases - metabolism ; Models, Molecular ; Paleozoic ; Pests ; Phylogeny ; Polysaccharides - metabolism ; Proteomes ; Proteomics ; Thermobia ; Vectors
    ISSN: 2041-1723
    E-ISSN: 2041-1723
    Source: Nature Open Access
    Source: PubMed Central
    Source: DOAJ Directory of Open Access Journals - Not for CDI Discovery
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  • 4
    Language: English
    In: Molecular cancer, 2015-01-27, Vol.14 (1), p.13-13
    Description: Voltage-gated Na(+) channels (VGSCs) are heteromeric protein complexes containing pore-forming α subunits and smaller, non-pore-forming β subunits. VGSCs are classically expressed in electrically excitable cells, e.g. neurons. VGSCs are also expressed in tumour cells, including breast cancer (BCa) cells, where they enhance cellular migration and invasion. However, despite extensive work defining in detail the molecular mechanisms underlying the expression of VGSCs and their pro-invasive role in cancer cells, there has been a notable lack of clinically relevant in vivo data exploring their value as potential therapeutic targets. We have previously reported that the VGSC-blocking antiepileptic drug phenytoin inhibits the migration and invasion of metastatic MDA-MB-231 cells in vitro. The purpose of the present study was to establish whether VGSCs might be viable therapeutic targets by testing the effect of phenytoin on tumour growth and metastasis in vivo. We found that expression of Nav1.5, previously detected in MDA-MB-231 cells in vitro, was retained on cells in orthotopic xenografts. Treatment with phenytoin, at a dose equivalent to that used to treat epilepsy (60 mg/kg; daily), significantly reduced tumour growth, without affecting animal weight. Phenytoin also reduced cancer cell proliferation in vivo and invasion into surrounding mammary tissue. Finally, phenytoin significantly reduced metastasis to the liver, lungs and spleen. This is the first study showing that phenytoin reduces breast tumour growth and metastasis in vivo. We propose that pharmacologically targeting VGSCs, by repurposing antiepileptic or antiarrhythmic drugs, should be further studied as a potentially novel anti-cancer therapy.
    Subject(s): Animals ; Anticonvulsants - administration & dosage ; Anticonvulsants - pharmacology ; Antiepileptic ; Antineoplastic Agents - administration & dosage ; Antineoplastic Agents - pharmacology ; Apoptosis - drug effects ; Breast cancer ; Breast Neoplasms - drug therapy ; Breast Neoplasms - metabolism ; Breast Neoplasms - pathology ; Cell Line, Tumor ; Cell Proliferation - drug effects ; Disease Models, Animal ; Female ; Growth ; Humans ; Liver ; Metastasis ; Mice ; Neoplasm Metastasis ; Neovascularization, Pathologic - drug therapy ; Neovascularization, Pathologic - metabolism ; Phenytoin ; Phenytoin - administration & dosage ; Phenytoin - pharmacology ; Short Communication ; Sodium Channel Blockers - administration & dosage ; Sodium Channel Blockers - pharmacology ; Sodium Channels - metabolism ; Tumor Burden - drug effects ; Voltage-gated Na+ channel ; Xenograft Model Antitumor Assays
    ISSN: 1476-4598
    E-ISSN: 1476-4598
    Source: BioMedCentral Open Access
    Source: Academic Search Ultimate
    Source: PubMed Central
    Source: DOAJ Directory of Open Access Journals - Not for CDI Discovery
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  • 5
    Language: English
    In: Scientific reports, 2017-05-24, Vol.7 (1), p.2356-10
    Description: Microbial communities metabolize plant biomass using secreted enzymes; however, identifying extracellular proteins tightly bound to insoluble lignocellulose in these microbiomes presents a challenge, as the rigorous extraction required to elute these proteins also lyses the microbes associated with the plant biomass releasing intracellular proteins that contaminate the metasecretome. Here we describe a technique for targeting the extracellular proteome, which was used to compare the metasecretome and meta-surface-proteome of two lignocellulose-degrading communities grown on wheat straw and rice straw. A combination of mass spectrometry-based proteomics coupled with metatranscriptomics enabled the identification of a unique secretome pool from these lignocellulose-degrading communities. This method enabled us to efficiently discriminate the extracellular proteins from the intracellular proteins by improving detection of actively secreted and transmembrane proteins. In addition to the expected carbohydrate active enzymes, our new method reveals a large number of unknown proteins, supporting the notion that there are major gaps in our understanding of how microbial communities degrade lignocellulosic substrates.
    Subject(s): Biomass ; Enzymes ; Intracellular ; Lignin - metabolism ; Lignocellulose ; Mass Spectrometry ; Mass spectroscopy ; Membrane proteins ; Metagenome - genetics ; Metagenomics - methods ; Microbiota ; Oryza - growth & development ; Oryza - microbiology ; Plant communities ; Proteins ; Proteome - genetics ; Proteome - metabolism ; Proteomics ; Proteomics - methods ; Secretome ; Straw ; Transcriptome - genetics ; Triticum - growth & development ; Triticum - microbiology
    ISSN: 2045-2322
    E-ISSN: 2045-2322
    Source: Nature Open Access
    Source: Academic Search Ultimate
    Source: PubMed Central
    Source: DOAJ Directory of Open Access Journals - Not for CDI Discovery
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  • 6
    Language: English
    In: Molecular & cellular proteomics, 2019-07, Vol.18 (7), p.1271-1284
    Description: A comprehensive, quantified identification of the mRNA-binding and whole cell proteomes in the three main Leishmania lifecycle stages, the first such comparison in kinetoplastid parasites, demonstrates trans-regulator RBPs select distinct, specific mRNA target pools in a stage-regulated manner despite equivalent, constitutive transcript levels available. Results further indicate that in L. mexicana parasites, mRNA levels are not a strong predictor of whole cell expression or RNA binding potential of encoded proteins. Included are the first proteomes from the human-infective metacyclic promastigote stage. [Display omitted] Highlights •Comprehensive, quantified mRNA-binding and whole cell proteomes of the three main Leishmania spp. lifecycle stages.•Decisive evidence that trans-regulator RBPs can selectively bind distinct mRNA target pools in a stage-regulated manner despite equivalent transcript levels.•Quantified evidence that in L. mexicana parasites, mRNA levels are not a strong predictor of whole cell expression or RNA binding potential of encoded proteins.•Use of Histone H4 as the first marker of procyclic stage cells in Leishmania spp. Leishmania parasite infections, termed the leishmaniases, cause significant global infectious disease burden. The lifecycle of the parasite embodies three main stages that require precise coordination of gene regulation to survive environmental shifts between sandfly and mammalian hosts. Constitutive transcription in kinetoplastid parasites means that gene regulation is overwhelmingly reliant on post-transcriptional mechanisms, yet strikingly few Leishmania trans-regulators are known. Using optimized crosslinking and deep, quantified mass spectrometry, we present a comprehensive analysis of 1400 mRNA binding proteins (mRBPs) and whole cell proteomes from the three main Leishmania lifecycle stages. Supporting the validity, although the crosslinked RBPome is magnitudes more enriched, the protein identities of the crosslinked and non-crosslinked RBPomes were nearly identical. Moreover, multiple candidate RBPs were endogenously tagged and found to associate with discrete mRNA target pools in a stage-specific manner. Results indicate that in L. mexicana parasites, mRNA levels are not a strong predictor of the whole cell expression or RNA binding potential of encoded proteins. Evidence includes a low correlation between transcript and corresponding protein expression and stage-specific variation in protein expression versus RNA binding potential. Unsurprisingly, RNA binding protein enrichment correlates strongly with relative replication efficiency of the specific lifecycle stage. Our study is the first to quantitatively define and compare the mRBPome of multiple stages in kinetoplastid parasites. It provides novel, in-depth insight into the trans-regulatory mRNA:Protein (mRNP) complexes that drive Leishmania parasite lifecycle progression.
    Subject(s): Animals ; Biochemical Research Methods ; Biochemistry & Molecular Biology ; Gene Ontology ; kinetoplastid ; Label-free quantification ; Leishmania ; Leishmania mexicana - genetics ; Life Cycle Stages ; Life Sciences & Biomedicine ; Mice, Inbred BALB C ; Microbes ; Microbiology ; Molecular biology ; mRNA binding proteome ; mRNP ; mRNP/trans-regulator ; Parasite ; Parasites - genetics ; Pathogens ; Principal Component Analysis ; Protein Cross-linking ; Protein Identification ; Proteome ; Proteome - metabolism ; Proteomics ; Protozoan Proteins - metabolism ; RBPome ; RBPome/mRNA binding proteome ; Reproducibility of Results ; Ribonucleoproteins ; RNA, Messenger - genetics ; RNA, Messenger - metabolism ; RNA-Binding Proteins - metabolism ; Science & Technology ; trans-regulator ; Transcriptome - genetics
    ISSN: 1535-9476
    E-ISSN: 1535-9484
    Source: HighWire Press (Free Journals)
    Source: Web of Science - Science Citation Index Expanded - 2019〈img src="http://exlibris-pub.s3.amazonaws.com/fromwos-v2.jpg" /〉
    Source: PubMed Central
    Source: Alma/SFX Local Collection
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  • 7
    Language: English
    In: PloS one, 2017, Vol.12 (2), p.e0172588-e0172588
    Description: Salmonella effector protein SseJ is secreted by Salmonella into the host cell cytoplasm where it can then modify host cell processes. Whilst host cell small GTPase RhoA has previously been shown to activate the acyl-transferase activity of SseJ we show here an un-described effect of SseJ protein production upon microtubule dynamism. SseJ prevents microtubule collapse and this is independent of SseJ's acyl-transferase activity. We speculate that the effects of SseJ on microtubules would be mediated via its known interactions with the small GTPases of the Rho family.
    Subject(s): Acyltransferases - genetics ; Acyltransferases - metabolism ; Amino Acid Sequence ; Animals ; Bacteria ; Bacterial infections ; Bacterial Proteins - genetics ; Bacterial Proteins - metabolism ; Binding Sites ; Biology ; Biology and Life Sciences ; Cell Line ; Cloning ; Cytoplasm ; Defects ; Deoxyribonucleic acid ; DNA ; Effector cells ; Epithelial Cells - microbiology ; Epithelial Cells - ultrastructure ; Gene Expression Regulation ; Genes ; Genetic aspects ; Genomic Islands ; Genomic Library ; Guanosine triphosphatases ; Host-Pathogen Interactions ; Humans ; Immunoprecipitation ; Kidney - microbiology ; Kidney - pathology ; Macrophages - microbiology ; Macrophages - ultrastructure ; Medicine and Health Sciences ; Microtubules ; Microtubules - microbiology ; Microtubules - ultrastructure ; Pathogens ; Protein Binding ; Proteins ; Rats ; Research and Analysis Methods ; rho GTP-Binding Proteins - genetics ; rho GTP-Binding Proteins - metabolism ; RhoA protein ; Saccharomyces cerevisiae - genetics ; Saccharomyces cerevisiae - metabolism ; Salmonella ; Salmonella typhimurium - genetics ; Salmonella typhimurium - metabolism ; Salmonella typhimurium - pathogenicity ; Sequence Alignment ; Signal Transduction
    ISSN: 1932-6203
    E-ISSN: 1932-6203
    Source: Academic Search Ultimate
    Source: PubMed Central
    Source: DOAJ Directory of Open Access Journals - Not for CDI Discovery
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  • 8
    Language: English
    In: Nucleic acids research, 2020-06-04, Vol.48 (10), p.5511-5526
    Description: RNA binding proteins (RBPs) are the primary gene regulators in kinetoplastids as transcriptional control is nearly absent, making Leishmania an exceptional model for investigating methylation of non-histone substrates. Arginine methylation is an evolutionarily conserved protein modification catalyzed by Protein aRginine Methyl Transferases (PRMTs). The chromatin modifier PRMT7 is the only Type III PRMT found in higher eukaryotes and a restricted number of unicellular eukaryotes. In Leishmania major, PRMT7 is a cytoplasmic protein implicit in pathogenesis with unknown substrates. Using comparative methyl-SILAC proteomics for the first time in protozoa, we identified 40 putative targets, including 17 RBPs hypomethylated upon PRMT7 knockout. PRMT7 can modify Alba3 and RBP16 trans-regulators (mammalian RPP25 and YBX2 homologs, respectively) as direct substrates in vitro. The absence of PRMT7 levels in vivo selectively reduces Alba3 mRNA-binding capacity to specific target transcripts and can impact the relative stability of RBP16 in the cytoplasm. RNA immunoprecipitation analyses demonstrate PRMT7-dependent methylation promotes Alba3 association with select target transcripts and thus indirectly stabilizes mRNA of a known virulence factor, δ-amastin surface antigen. These results highlight a novel role for PRMT7-mediated arginine methylation of RBP substrates, suggesting a regulatory pathway controlling gene expression and virulence in Leishmania. This work introduces Leishmania PRMTs as epigenetic regulators of mRNA metabolism with mechanistic insight into the functional manipulation of RBPs by methylation.
    Subject(s): AcademicSubjects ; Gene Expression Regulation ; Leishmania major - enzymology ; Leishmania major - genetics ; Methylation ; Molecular Biology ; Narese ; Protein Stability ; Protein-Arginine N-Methyltransferases - metabolism ; Protozoan Proteins - metabolism ; RNA-Binding Proteins - metabolism ; SCI00010
    ISSN: 0305-1048
    E-ISSN: 1362-4962
    Source: PubMed Central
    Source: DOAJ Directory of Open Access Journals - Not for CDI Discovery
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  • 9
    Language: English
    In: Biotechnology for biofuels, 2018, Vol.11 (1), p.59-59
    Description: Lignocellulose forms the structural framework of woody plant biomass and represents the most abundant carbon source in the biosphere. Turnover of woody biomass is a critical component of the global carbon cycle, and the enzymes involved are of increasing industrial importance as industry moves away from fossil fuels to renewable carbon resources. Shipworms are marine bivalve molluscs that digest wood and play a key role in global carbon cycling by processing plant biomass in the oceans. Previous studies suggest that wood digestion in shipworms is dominated by enzymes produced by endosymbiotic bacteria found in the animal's gills, while little is known about the identity and function of endogenous enzymes produced by shipworms. Using a combination of meta-transcriptomic, proteomic, imaging and biochemical analyses, we reveal a complex digestive system dominated by uncharacterized enzymes that are secreted by a specialized digestive gland and that accumulate in the cecum, where wood digestion occurs. Using a combination of transcriptomics, proteomics, and microscopy, we show that the digestive proteome of the shipworm is mostly composed of enzymes produced by the animal itself, with a small but significant contribution from symbiotic bacteria. The digestive proteome is dominated by a novel 300 kDa multi-domain glycoside hydrolase that functions in the hydrolysis of β-1,4-glucans, the most abundant polymers in wood. These studies allow an unprecedented level of insight into an unusual and ecologically important process for wood recycling in the marine environment, and open up new biotechnological opportunities in the mobilization of sugars from lignocellulosic biomass.
    Subject(s): Carbon cycle (Biogeochemistry) ; Cellulose ; Digestive organs ; Enzymes ; Physiological aspects ; Properties
    ISSN: 1754-6834
    E-ISSN: 1754-6834
    Source: BioMedCentral Open Access
    Source: Academic Search Ultimate
    Source: PubMed Central
    Source: DOAJ Directory of Open Access Journals - Not for CDI Discovery
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  • 10
    Language: English
    In: Biotechnology for biofuels, 2018, Vol.11 (1), p.166-166
    Description: Lignocellulose is one of the most abundant forms of fixed carbon in the biosphere. Current industrial approaches to the degradation of lignocellulose employ enzyme mixtures, usually from a single fungal species, which are only effective in hydrolyzing polysaccharides following biomass pre-treatments. While the enzymatic mechanisms of lignocellulose degradation have been characterized in detail in individual microbial species, the microbial communities that efficiently breakdown plant materials in nature are species rich and secrete a myriad of enzymes to perform "community-level" metabolism of lignocellulose. Single-species approaches are, therefore, likely to miss important aspects of lignocellulose degradation that will be central to optimizing commercial processes. Here, we investigated the microbial degradation of wheat straw in liquid cultures that had been inoculated with wheat straw compost. Samples taken at selected time points were subjected to multi-omics analysis with the aim of identifying new microbial mechanisms for lignocellulose degradation that could be applied in industrial pre-treatment of feedstocks. Phylogenetic composition of the community, based on sequenced bacterial and eukaryotic ribosomal genes, showed a gradual decrease in complexity and diversity over time due to microbial enrichment. Taxonomic affiliation of bacterial species showed dominance of and and high relative abundance of genera , and . The eukaryotic members of the community were enriched in peritrich ciliates from genus that thrived in the liquid cultures compared to fungal species that were present in low abundance. A targeted metasecretome approach combined with metatranscriptomics analysis, identified 1127 proteins and showed the presence of numerous carbohydrate-active enzymes extracted from the biomass-bound fractions and from the culture supernatant. This revealed a wide array of hydrolytic cellulases, hemicellulases and carbohydrate-binding modules involved in lignocellulose degradation. The expression of these activities correlated to the changes in the biomass composition observed by FTIR and ssNMR measurements. A combination of mass spectrometry-based proteomics coupled with metatranscriptomics has enabled the identification of a large number of lignocellulose degrading enzymes that can now be further explored for the development of improved enzyme cocktails for the treatment of plant-based feedstocks. In addition to the expected carbohydrate-active enzymes, our studies reveal a large number of unknown proteins, some of which may play a crucial role in community-based lignocellulose degradation.
    Subject(s): Abundance ; Analysis ; Biodegradation ; Biodiversity ; Biomass ; Biosphere ; Carbohydrates ; CAZy ; Cell culture ; Cellulose ; Chemical properties ; Ciliates ; Composition ; Composts ; Degradation ; Ecosystems ; Enzymes ; Fungi ; Gene expression ; Genera ; Genomics ; Hemicellulases ; Hydrolases ; Lead ; Lignin ; Lignocellulose ; Mass spectrometry ; Mass spectroscopy ; Metabolism ; Metasecretome ; Microbial activity ; Microbial degradation ; Microorganisms ; Phylogeny ; Physiological aspects ; Plant communities ; Polysaccharides ; Pretreatment ; Principal components analysis ; Proteins ; Proteomics ; Relative abundance ; Ribosomal DNA ; Saccharides ; Species ; Species diversity ; Straw ; Usage ; Wheat
    ISSN: 1754-6834
    E-ISSN: 1754-6834
    Source: BioMedCentral Open Access
    Source: Academic Search Ultimate
    Source: PubMed Central
    Source: DOAJ Directory of Open Access Journals - Not for CDI Discovery
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