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Abnormal blood coagulation is a major health problem and natural anticoagulants from blood-feeding organisms have been investigated as novel therapeutics. NAPc2, a potent nematode-derived inhibitor of coagulation, has an unusual mode of action that requires coagulation factor Xa but does not inhibit it. Molecular dynamics simulations of NAPc2 and factor Xa were generated to better understand NAPc2. The simulations suggest that parts of NAPc2 become more rigid upon binding factor Xa and reveal that two highly conserved residues form an internal salt bridge that stabilises the bound conformation. Clotting time assays with mutants confirmed the utility of the salt bridge and suggested that it is a conserved mechanism for stabilising the bound conformation of secondary structure-poor protease inhibitors.
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Anticoagulantes , Fator Xa , Simulação de Dinâmica Molecular , Ligação Proteica , Animais , Anticoagulantes/química , Anticoagulantes/farmacologia , Fator Xa/metabolismo , Fator Xa/química , Nematoides/metabolismo , Nematoides/efeitos dos fármacos , Humanos , Coagulação Sanguínea/efeitos dos fármacos , Proteínas de Helminto/química , Proteínas de Helminto/metabolismo , Proteínas de Helminto/genética , Sítios de LigaçãoRESUMO
This paper provides a comprehensive overview of the history of commercial poultry breeding, from domestication to the development of science and commercial breeding structures. The development of breeding goals over time, from mainly focusing on production to broad goals, including bird welfare and health, robustness, environmental impact, biological efficiency and reproduction, is detailed. The paper outlines current breeding goals, including traits (e.g., on foot and leg health, contact dermatitis, gait, cardiovascular health, robustness and livability), recording techniques, their genetic basis and how trait these antagonisms, for example, between welfare and production, are managed. Novel areas like genomic selection and gut health research and their current and potential impact on breeding are highlighted. The environmental impact differences of various genotypes are explained. A future outlook shows that balanced, holistic breeding will continue to enable affordable lean animal protein to feed the world, with a focus on the welfare of the birds and a diversity of choice for the various preferences and cultures across the world.
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Antimicrobial resistance has emerged as a global public health threat, and development of novel therapeutics for treating infections caused by multi-drug resistant bacteria is urgent. Staphylococcus aureus is a major human and animal pathogen, responsible for high levels of morbidity and mortality worldwide. The intracellular survival of S. aureus in macrophages contributes to immune evasion, dissemination, and resilience to antibiotic treatment. Here, we present a confocal fluorescence imaging assay for monitoring macrophage infection by green fluorescent protein (GFP)-tagged S. aureus as a front-line tool to identify antibiotic leads. The assay was employed in combination with nanoscaled chemical analyses to facilitate the discovery of a new, active rifamycin analogue. Our findings indicate a promising new approach for the identification of antimicrobial compounds with macrophage intracellular activity. The antibiotic identified here may represent a useful addition to our armory in tackling the silent pandemic of antimicrobial resistance.
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Rifamicinas , Infecções Estafilocócicas , Animais , Humanos , Staphylococcus aureus , Proteínas de Fluorescência Verde/genética , Rifamicinas/uso terapêutico , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Infecções Estafilocócicas/microbiologia , MacrófagosRESUMO
OBJECTIVES: The aim of this work was to establish national diagnostic reference levels (DRLs) in Ireland and compare these to existing European DRLs where available. This work surveyed all radiological facilities providing radiography, mammography, and dual-energy x-ray absorptiometry (DXA) services in Ireland. METHODS: A list of common procedures and clinical tasks was established. A national database of service providers was used to identify the appropriate medical radiological facilities providing these services. These facilities were issued with an online survey. National DRLs were set as the 75th percentile of the distribution of median values obtained. A national median dose was also established. The broad categorisation of equipment type was also considered. Where differences between DRLs established using different detector types were deemed statistically significant, equipment-specific national DRLs were established. RESULTS: National DRLs were established for 12 adult radiography projections. Equipment-specific (computed radiography and digital radiography) adult DRLs were established for four radiography projections. Paediatric DRLs were established for 11 radiography projections, including two based on clinical indications, for a range of paediatric weight categories. National DRLs were established for unilateral two-view mammography and breast tomosynthesis as well as for four DXA clinical indications and projections. All but one Irish DRL figure was found to be below or equal to European data. CONCLUSIONS: This work provided a unique opportunity to establish national DRLs based on census data for a range of procedures and clinical tasks across radiography, mammography and DXA and compare these with European levels. CLINICAL RELEVANCE STATEMENT: This work established national diagnostic reference levels (DRLs) based on census data for a range of procedures and clinical tasks across radiography, mammography and dual-energy x-ray absorptiometry. The establishment of national DRLs is an essential component in the optimisation of patient radiation dose. KEY POINTS: ⢠Diagnostic reference levels are easily measured quantities intended for use as an aid to optimise patient dose and to identify when levels of patient dose are unusually high. ⢠Data from all medical radiological facilities in Ireland was obtained to establish national diagnostic reference level (DRL) values and national median dose values in radiography, x-ray breast imaging and dual-energy x-ray absorptiometry (DXA) scanning and these were compared to existing European DRLs where available. ⢠National DRL values were established for the first time in breast tomosynthesis, DXA scanning, and paediatric radiography.
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Níveis de Referência de Diagnóstico , Mamografia , Adulto , Criança , Humanos , Absorciometria de Fóton , Irlanda/epidemiologia , Doses de Radiação , Valores de Referência , RadiografiaRESUMO
As one of the first families of marine natural products to undergo clinical trials, the didemnin depsipeptides have played a significant role in inspiring the discovery of marine drugs. Originally developed as anticancer therapeutics, the recent re-evaluation of these compounds including synthetically derived dehydrodidemnin B or Aplidine, has led to their advancement towards antiviral applications. While conventionally associated with production in colonial tunicates of the family Didemnidae, recent studies have identified their biosynthetic gene clusters from the marine-derived bacteria Tistrella mobilis. While these studies confirm the production of didemnin X/Y, the low titer and general lack of understanding of their biosynthesis in Tistrella currently prevents the development of effective microbial or synthetic biological approaches for their production. To this end, we conducted a survey of known species of Tistrella and report on their ability to produce the didemnin depsipeptides. These data were used to develop conditions to produce didemnin B at titers over 15 mg/L.
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Antineoplásicos , Depsipeptídeos , Antineoplásicos/química , Depsipeptídeos/química , Peptídeos Cíclicos/químicaRESUMO
This paper develops and expands upon social identity theory as an explanation for gambling among youth engaged in team sport. Analysing longitudinal data for over 4500 20-year-olds from the Growing Up in Ireland study, reveals that online gambling increased from 2.6 to 9.3% between 17 and 20 years in the cohort, with the increase driven by males. A statistically significant positive association is uncovered between playing team sports and regularly gambling, as well as online gambling behaviour, independent of socio-demographic and other risk factors for males but not for females. The findings provide support for a dose-response like effect for males, where a longer period of participation in team sports is associated with a higher likelihood of engaging in gambling behaviour compared to shorter periods. Implications of the findings for policy and practice are discussed.
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Jogo de Azar , Esportes , Masculino , Adolescente , Feminino , Humanos , Jogo de Azar/psicologia , Esportes de Equipe , Fatores de RiscoRESUMO
The oxytosis/ferroptosis regulated cell death pathway recapitulates many features of mitochondrial dysfunction associated with the aging brain and has emerged as a potential key mediator of neurodegeneration. It has thus been proposed that the oxytosis/ferroptosis pathway can be used to identify novel drug candidates for the treatment of age-associated neurodegenerative diseases that act by preserving mitochondrial function. Previously, we identified cannabinol (CBN) as a potent neuroprotector. Here, we demonstrate that not only does CBN protect nerve cells from oxytosis/ferroptosis in a manner that is dependent on mitochondria and it does so independently of cannabinoid receptors. Specifically, CBN directly targets mitochondria and preserves key mitochondrial functions including redox regulation, calcium uptake, membrane potential, bioenergetics, biogenesis, and modulation of fusion/fission dynamics that are disrupted following induction of oxytosis/ferroptosis. These protective effects of CBN are at least partly mediated by the promotion of endogenous antioxidant defenses and the activation of AMP-activated protein kinase (AMPK) signaling. Together, our data highlight the potential of mitochondrially-targeted compounds such as CBN as novel oxytotic/ferroptotic inhibitors to rescue mitochondrial dysfunction as well as opportunities for the discovery and development of future neurotherapeutics.
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Ferroptose , Canabinol/metabolismo , Canabinol/farmacologia , Morte Celular , Mitocôndrias/metabolismo , Receptores de Canabinoides/metabolismoRESUMO
Next-generation sequencing studies of saliva and dental plaque from subjects in both healthy and diseased states have identified bacteria belonging to the Rothia genus as ubiquitous members of the oral microbiota. To gain a deeper understanding of molecular mechanisms underlying the chemical ecology of this unexplored group, we applied a genome mining approach that targets functionally important biosynthetic gene clusters (BGCs). All 45 genomes that were mined, representing Rothia mucilaginosa, Rothia dentocariosa, and Rothia aeria, harbored a catechol-siderophore-like BGC. To explore siderophore production further, we grew the previously characterized R. mucilaginosa ATCC 25296 in liquid cultures, amended with glycerol, which led to the identification of the archetype siderophore enterobactin by using tandem liquid chromatography-mass spectrometry (LC-MS/MS), high-performance liquid chromatography (HPLC), and nuclear magnetic resonance (NMR) spectroscopy. Normally attributed to pathogenic gut bacteria, R. mucilaginosa is the first commensal oral bacterium found to produce enterobactin. Cocultivation studies including R. mucilaginosa or purified enterobactin revealed that enterobactin reduced growth of certain strains of cariogenic Streptococcus mutans and pathogenic strains of Staphylococcus aureus Commensal oral bacteria were either unaffected, reduced in growth, or induced to grow adjacent to enterobactin-producing R. mucilaginosa or the pure compound. Taken together with Rothia's known capacity to ferment a variety of carbohydrates and amino acids, our findings of enterobactin production add an additional level of explanation to R. mucilaginosa's prevalence in the oral cavity. Enterobactin is the strongest Fe(III) binding siderophore known, and its role in oral health requires further investigation.IMPORTANCE The communication language of the human oral microbiota is vastly underexplored. However, a few studies have shown that specialized small molecules encoded by BGCs have critical roles such as in colonization resistance against pathogens and quorum sensing. Here, by using a genome mining approach in combination with compound screening of growth cultures, we identified that the commensal oral community member R. mucilaginosa harbors a catecholate-siderophore BGC, which is responsible for the biosynthesis of enterobactin. The iron-scavenging role of enterobactin is known to have positive effects on the host's iron pool and negative effects on host immune function; however, its role in oral health remains unexplored. R. mucilaginosa was previously identified as an abundant community member in cystic fibrosis, where bacterial iron cycling plays a major role in virulence development. With respect to iron's broad biological importance, iron-chelating enterobactin may explain R. mucilaginosa's colonization success in both health and disease.
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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This report describes the first application of the novel NMR-based machine learning tool "Small Molecule Accurate Recognition Technology" (SMART 2.0) for mixture analysis and subsequent accelerated discovery and characterization of new natural products. The concept was applied to the extract of a filamentous marine cyanobacterium known to be a prolific producer of cytotoxic natural products. This environmental Symploca extract was roughly fractionated, and then prioritized and guided by cancer cell cytotoxicity, NMR-based SMART 2.0, and MS2-based molecular networking. This led to the isolation and rapid identification of a new chimeric swinholide-like macrolide, symplocolide A, as well as the annotation of swinholide A, samholides A-I, and several new derivatives. The planar structure of symplocolide A was confirmed to be a structural hybrid between swinholide A and luminaolide B by 1D/2D NMR and LC-MS2 analysis. A second example applies SMART 2.0 to the characterization of structurally novel cyclic peptides, and compares this approach to the recently appearing "atomic sort" method. This study exemplifies the revolutionary potential of combined traditional and deep learning-assisted analytical approaches to overcome longstanding challenges in natural products drug discovery.
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Produtos Biológicos/química , Aprendizado de Máquina , Redes Neurais de Computação , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/toxicidade , Linhagem Celular Tumoral , Quimioinformática , Cianobactérias/química , Humanos , Espectroscopia de Ressonância Magnética , Peptídeos Cíclicos/química , Peptídeos Cíclicos/isolamento & purificação , Peptídeos Cíclicos/toxicidadeRESUMO
Leptin is an important signaling hormone, mostly known for its role in energy expenditure and satiety. Furthermore, leptin plays a major role in other proteinopathies, such as cancer, marked hyperphagia, impaired immune function, and inflammation. In spite of its biological relevance in human health, there are no NMR resonance assignments of the human protein available, obscuring high-resolution characterization of the soluble protein and/or its conformational dynamics, suggested as being important for receptor interaction and biological activity. Here, we report the nearly complete backbone resonance assignments of human leptin. Chemical shift-based secondary structure prediction confirms that in solution leptin forms a four-helix bundle including a pierced lasso topology. The conformational dynamics, determined on several timescales, show that leptin is monomeric, has a rigid four-helix scaffold, and a dynamic domain, including a transiently formed helix. The dynamic domain is anchored to the helical scaffold by a secondary hydrophobic core, pinning down the long loops of leptin to the protein body, inducing motional restriction without a well-defined secondary or tertiary hydrogen bond stabilized structure. This dynamic region is well suited for and may be involved in functional allosteric dynamics upon receptor binding.
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Leptina/química , Leptina/metabolismo , Sítios de Ligação , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Dobramento de Proteína , Estrutura Secundária de ProteínaRESUMO
Marine cyanobacteria (blue-green algae) have been shown to possess an enormous capacity to produce structurally diverse natural products that exhibit a broad spectrum of potent biological activities, including cytotoxic, antifungal, antiparasitic, antiviral, and antibacterial activities. Using mass-spectrometry-guided fractionation together with molecular networking, cyanobacterial field collections from American Samoa and Palmyra Atoll yielded three new cyclic peptides, tutuilamides A-C. Their structures were established by spectroscopic techniques including 1D and 2D NMR, HR-MS, and chemical derivatization. Structure elucidation was facilitated by employing advanced NMR techniques including nonuniform sampling in combination with the 1,1-ADEQUATE experiment. These cyclic peptides are characterized by the presence of several unusual residues including 3-amino-6-hydroxy-2-piperidone and 2-amino-2-butenoic acid, together with a novel vinyl chloride-containing residue. Tutuilamides A-C show potent elastase inhibitory activity together with moderate potency in H-460 lung cancer cell cytotoxicity assays. The binding mode to elastase was analyzed by X-ray crystallography revealing a reversible binding mode similar to the natural product lyngbyastatin 7. The presence of an additional hydrogen bond with the amino acid backbone of the flexible side chain of tutuilamide A, compared to lyngbyastatin 7, facilitates its stabilization in the elastase binding pocket and possibly explains its enhanced inhibitory potency.
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Antineoplásicos/isolamento & purificação , Cianobactérias/química , Depsipeptídeos/isolamento & purificação , Inibidores Enzimáticos/isolamento & purificação , Neoplasias Pulmonares/tratamento farmacológico , Elastase Pancreática/antagonistas & inibidores , Peptídeos Cíclicos/isolamento & purificação , Aminoácidos/química , Aminobutiratos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Depsipeptídeos/química , Depsipeptídeos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/farmacologia , Humanos , Modelos Moleculares , Estrutura Molecular , Peptídeos Cíclicos/farmacologia , Piperidonas/química , Ligação Proteica , Espectrometria de Massas em Tandem , Cloreto de Vinil/químicaRESUMO
Natural product research continues to be a productive source of unusual chemistry, producing novel compounds for biomedical applications and, increasingly, sustainably providing commercially useful compounds [...].
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Produtos Biológicos/farmacologia , Biotecnologia/tendências , Avaliação Pré-Clínica de Medicamentos , Indústria Farmacêutica/tendências , Animais , HumanosRESUMO
The discovery of biologically active small molecules requires sifting through large amounts of data to identify unique or unusual arrangements of atoms. Here, we develop, test and evaluate an atom-based sort to identify novel features of secondary metabolites and demonstrate its use to evaluate novelty in marine microbial and sponge extracts. This study outlines an important ongoing advance towards the translation of autonomous systems to identify, and ultimately elucidate, atomic novelty within a complex mixture of small molecules.
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A lack of selectivity of classical agonists for the nicotinic acetylcholine receptors (nAChR) has prompted us to identify and develop a distinct scaffold of α7 nAChR-selective ligands. Noncanonical 2,4,6-substituted pyrimidine analogues were framed around compound 40 for a structure-activity relationship study. The new lead compounds activate selectively the α7 nAChRs with EC50's between 30 and 140 nM in a PNU-120596-dependent, cell-based calcium influx assay. After characterizing the expanded lead landscape, we ranked the compounds for rapid activation using Xenopus oocytes expressing human α7 nAChR with a two-electrode voltage clamp. This approach enabled us to define the molecular determinants governing rapid activation, agonist potency, and desensitization of α7 nAChRs after exposure to pyrimidine analogues, thereby distinguishing this subclass of noncanonical agonists from previously defined types of agonists (agonists, partial agonists, silent agonists, and ago-PAMs). By NMR, we analyzed pKa values for ionization of lead candidates, demonstrating distinctive modes of interaction for this landscape of ligands.
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Agonistas Nicotínicos/química , Agonistas Nicotínicos/farmacologia , Receptor Nicotínico de Acetilcolina alfa7/agonistas , Animais , Sítios de Ligação , Simulação por Computador , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Humanos , Isoxazóis/farmacologia , Espectroscopia de Ressonância Magnética , Neurotransmissores/metabolismo , Agonistas Nicotínicos/síntese química , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Compostos de Fenilureia/farmacologia , Relação Estrutura-Atividade , Xenopus laevis , Receptor Nicotínico de Acetilcolina alfa7/genética , Receptor Nicotínico de Acetilcolina alfa7/metabolismoRESUMO
Three decahydroisoquinoline alkaloids, lepadins I-K, were isolated from a specimen of Didemnum sp. collected in the Bahamas. The structures of the new compounds were assigned by an integrated analysis of MS, IR, and 1H, 13C, and 2D NMR spectra. Like previously reported lepadins, the structures of the new compounds contain a decahydroquinoline heterocyclic core in lepadin I, and a new variation, an octahydroquinoline in lepadin J, but differ from earlier reported compounds by acylation of the 3-hydroxyl group by a rare 3'-methylthioacrylate. The absolute configuration of lepadin I was solved by interpretation of NOE measurements, and exciton coupled circular dichroism (ECCD) of the corresponding N- p-bromobenzoyl derivative. The latter constitutes a general method for determination of absolute configuration of the entire lepadin family. The configuration of the remote side-chain secondary carbinol was solved by the modified Mosher's esters method. Lepadin I inhibited butyrylcholineesterase (BuChE, IC50 3.1 µM), but only weakly inhibited acetylcholineesterase (AChE) (10% at 100 µM).
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Alcaloides/química , Isoquinolinas/química , Quinolinas/química , Urocordados/química , Animais , Bahamas , Modelos Moleculares , Estrutura MolecularRESUMO
Viruses are underrepresented as targets in pharmacological screening efforts, given the difficulties of devising suitable cell-based and biochemical assays. In this study we found that a pre-fractionated organic extract of the Red Sea sponge Amphimedon chloros was able to inhibit the West Nile Virus NS3 protease (WNV NS3). Using liquid chromatographyâ»mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectroscopy, the identity of the bioactive compound was determined as a 3-alkylpyridinium with m/z = 190.16. Diffusion Ordered Spectroscopy (DOSY) NMR and NMR relaxation rate analysis suggest that the bioactive compound forms oligomers of up to 35 kDa. We observed that at 9.4 µg/mL there was up to 40â»70% inhibitory activity on WNV NS3 protease in orthogonal biochemical assays for solid phase extracts (SPE) of A. chloros. However, the LC-MS purified fragment was effective at inhibiting the protease up to 95% at an approximate amount of 2 µg/mL with negligible cytotoxicity to HeLa cells based on a High-Content Screening (HCS) cytological profiling strategy. To date, 3-alkylpyridinium type natural products have not been reported to show antiviral activity since the first characterization of halitoxin, or 3-alkylpyridinium, in 1978. This study provides the first account of a 3-alkylpyridinium complex that exhibits a proposed antiviral activity by inhibiting the NS3 protease. We suggest that the here-described compound can be further modified to increase its stability and tested in a cell-based assay to explore its full potential as a potential novel antiviral capable of inhibiting WNV replication.
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Antivirais/isolamento & purificação , Poríferos/química , Inibidores de Proteases/isolamento & purificação , Compostos de Piridínio/isolamento & purificação , Proteínas não Estruturais Virais/antagonistas & inibidores , Vírus do Nilo Ocidental/enzimologia , Animais , Antivirais/química , Antivirais/farmacologia , Cromatografia Gasosa-Espectrometria de Massas , Células HeLa , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Compostos de Piridínio/química , Compostos de Piridínio/farmacologia , Relação Estrutura-Atividade , Vírus do Nilo Ocidental/efeitos dos fármacosRESUMO
Various algorithms comparing 2D NMR spectra have been explored for their ability to dereplicate natural products as well as determine molecular structures. However, spectroscopic artefacts, solvent effects, and the interactive effect of functional group(s) on chemical shifts combine to hinder their effectiveness. Here, we leveraged Non-Uniform Sampling (NUS) 2D NMR techniques and deep Convolutional Neural Networks (CNNs) to create a tool, SMART, that can assist in natural products discovery efforts. First, an NUS heteronuclear single quantum coherence (HSQC) NMR pulse sequence was adapted to a state-of-the-art nuclear magnetic resonance (NMR) instrument, and data reconstruction methods were optimized, and second, a deep CNN with contrastive loss was trained on a database containing over 2,054 HSQC spectra as the training set. To demonstrate the utility of SMART, several newly isolated compounds were automatically located with their known analogues in the embedded clustering space, thereby streamlining the discovery pipeline for new natural products.