RESUMEN
Immaturity of alveolar macrophages (AMs) around birth contributes to the susceptibility of newborns to lung disease. However, the molecular features differentiating neonatal and mature, adult AMs are poorly understood. In this study, we identify the unique transcriptomes and enhancer landscapes of neonatal and adult AMs in mice. Although the core AM signature was similar, murine adult AMs expressed higher levels of genes involved in lipid metabolism, whereas neonatal AMs expressed a largely proinflammatory gene profile. Open enhancer regions identified by an assay for transposase-accessible chromatin followed by high-throughput sequencing (ATAC-seq) contained motifs for nuclear receptors, MITF, and STAT in adult AMs and AP-1 and NF-κB in neonatal AMs. Intranasal LPS activated a similar innate immune response in both neonatal and adult mice, with higher basal expression of inflammatory genes in neonates. The lung microenvironment drove many of the distinguishing gene expression and open chromatin characteristics of neonatal and adult AMs. Neonatal mouse AMs retained high expression of some proinflammatory genes, suggesting that the differences in neonatal AMs result from both inherent cell properties and environmental influences.
Asunto(s)
Macrófagos Alveolares , FN-kappa B , Animales , Cromatina/genética , Cromatina/metabolismo , Pulmón/metabolismo , Ratones , FN-kappa B/metabolismo , Transcripción GenéticaRESUMEN
TET/JBP (ten-eleven translocation/base J binding protein) enzymes are iron(II)- and 2-oxo-glutarate-dependent dioxygenases that are found in all kingdoms of life and oxidize 5-methylpyrimidines on the polynucleotide level. Despite their prevalence, few examples have been biochemically characterized. Among those studied are the metazoan TET enzymes that oxidize 5-methylcytosine in DNA to hydroxy, formyl, and carboxy forms and the euglenozoa JBP dioxygenases that oxidize thymine in the first step of base J biosynthesis. Both enzymes have roles in epigenetic regulation. It has been hypothesized that all TET/JBPs have their ancestral origins in bacteriophages, but only eukaryotic orthologs have been described. Here we demonstrate the 5mC-dioxygenase activity of several phage TETs encoded within viral metagenomes. The clustering of these TETs in a phylogenetic tree correlates with the sequence specificity of their genomically cooccurring cytosine C5-methyltransferases, which install the methyl groups upon which TETs operate. The phage TETs favor Gp5mC dinucleotides over the 5mCpG sites targeted by the eukaryotic TETs and are found within gene clusters specifying complex cytosine modifications that may be important for DNA packaging and evasion of host restriction.
Asunto(s)
5-Metilcitosina/metabolismo , Bacteriófagos/metabolismo , ADN/metabolismo , Secuencia de Aminoácidos , Metilación de ADN , Dioxigenasas , Hidroxilación , Metagenómica , Motivos de Nucleótidos/genética , Oxidación-Reducción , FilogeniaRESUMEN
Isoprenoids are a large class of natural products with wide-ranging applications. Synthetic biology approaches to the manufacture of isoprenoids and their new-to-nature derivatives are limited due to the provision in nature of just two hemiterpene building blocks for isoprenoid biosynthesis. To address this limitation, artificial chemo-enzymatic pathways such as the alcohol-dependent hemiterpene (ADH) pathway serve to leverage consecutive kinases to convert exogenous alcohols into pyrophosphates that could be coupled to downstream isoprenoid biosynthesis. To be successful, each kinase in this pathway should be permissive of a broad range of substrates. For the first time, we have probed the promiscuity of the second enzyme in the ADH pathway-isopentenyl phosphate kinase from Thermoplasma acidophilum-towards a broad range of acceptor monophosphates. Subsequently, we evaluate the suitability of this enzyme to provide unnatural pyrophosphates and provide a critical first step in characterizing the rate-limiting steps in the artificial ADH pathway.
Asunto(s)
Hemiterpenos/síntesis química , Proteínas Quinasas/metabolismo , Especificidad por Sustrato , Terpenos/síntesis química , Thermoplasma/enzimología , Alcoholes , Difosfatos/metabolismo , Fosfatos/metabolismo , Biología Sintética/métodosRESUMEN
Asthma is a complex disease that is promoted by dysregulated immunity and the presence of many cytokine and lipid mediators. Despite this, there is a paucity of data demonstrating the combined effects of multiple mediators in asthma pathogenesis. Group 2 innate lymphoid cells (ILC2s) have recently been shown to play important roles in the initiation of allergic inflammation; however, it is unclear whether lipid mediators, such as cysteinyl leukotrienes (CysLTs), which are present in asthma, could further amplify the effects of IL-33 on ILC2 activation and lung inflammation. In this article, we show that airway challenges with the parent CysLT, leukotriene C4 (LTC4), given in combination with low-dose IL-33 to naive wild-type mice, led to synergistic increases in airway Th2 cytokines, eosinophilia, and peribronchial inflammation compared with IL-33 alone. Further, the numbers of proliferating and cytokine-producing lung ILC2s were increased after challenge with both LTC4 and IL-33. Levels of CysLT1R, CysLT2R, and candidate leukotriene E4 receptor P2Y12 mRNAs were increased in ILC2s. The synergistic effect of LTC4 with IL-33 was completely dependent upon CysLT1R, because CysLT1R-/- mice, but not CysLT2R-/- mice, had abrogated responses. Further, CysLTs directly potentiated IL-5 and IL-13 production from purified ILC2s stimulated with IL-33 and resulted in NFAT1 nuclear translocation. Finally, CysLT1R-/- mice had reduced lung eosinophils and ILC2 responses after exposure to the fungal allergen Alternaria alternata Thus, CysLT1R promotes LTC4- and Alternaria-induced ILC2 activation and lung inflammation. These findings suggest that multiple pathways likely exist in asthma to activate ILC2s and propagate inflammatory responses.
Asunto(s)
Inmunidad Innata , Interleucina-33/inmunología , Leucotrieno C4/metabolismo , Activación de Linfocitos , Linfocitos/inmunología , Neumonía/inmunología , Alérgenos/inmunología , Alternaria/inmunología , Animales , Asma/inmunología , Asma/fisiopatología , Citocinas/biosíntesis , Citocinas/inmunología , Citocinas/metabolismo , Eosinofilia/inmunología , Interleucina-33/administración & dosificación , Leucotrieno C4/inmunología , Pulmón/inmunología , Ratones , Neumonía/metabolismo , Receptores de Leucotrienos/administración & dosificación , Receptores de Leucotrienos/deficiencia , Receptores de Leucotrienos/genética , Receptores de Leucotrienos/inmunología , Receptores Purinérgicos P2Y12/genética , Receptores Purinérgicos P2Y12/inmunología , Células Th2/inmunologíaRESUMEN
Terpenes are the largest class of natural products with a wide range of applications including use as pharmaceuticals, fragrances, flavorings, and agricultural products. Terpenes are biosynthesized by the condensation of a variable number of isoprene units resulting in linear polyisoprene diphosphate units, which can then be cyclized by terpene synthases into a range of complex structures. While these cyclic structures have immense diversity and potential in different applications, their direct analysis in biological buffer systems requires intensive sample preparation steps such as salt cleanup, extraction with organic solvents, and chromatographic separations. Electrospray post-ionization can be used to circumvent many sample cleanup and desalting steps. SESI and IR-MALDESI are two examples of ionization methods that employ electrospray post-ionization at atmospheric pressure and temperature. By coupling the two techniques and doping the electrospray solvent with silver ions, olefinic terpenes of different classes and varying degrees of volatility were directly analyzed from a biological buffer system with no sample workup steps.
Asunto(s)
Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Terpenos/análisis , Alquenos/análisis , Tampones (Química) , Rayos Infrarrojos , Iones/análisis , Sesquiterpenos Monocíclicos , Sesquiterpenos/análisis , Espectrometría de Masa por Ionización de Electrospray/métodosRESUMEN
Perturbed biomechanical stimuli are thought to be critical for the pathogenesis of a number of congenital heart defects, including Hypoplastic Left Heart Syndrome (HLHS). While embryonic cardiomyocytes experience biomechanical stretch every heart beat, their molecular responses to biomechanical stimuli during heart development are poorly understood. We hypothesized that biomechanical stimuli activate specific signaling pathways that impact proliferation, gene expression and myocyte contraction. The objective of this study was to expose embryonic mouse cardiomyocytes (EMCM) to cyclic stretch and examine key molecular and phenotypic responses. Analysis of RNA-Sequencing data demonstrated that gene ontology groups associated with myofibril and cardiac development were significantly modulated. Stretch increased EMCM proliferation, size, cardiac gene expression, and myofibril protein levels. Stretch also repressed several components belonging to the Transforming Growth Factor-ß (Tgf-ß) signaling pathway. EMCMs undergoing cyclic stretch had decreased Tgf-ß expression, protein levels, and signaling. Furthermore, treatment of EMCMs with a Tgf-ß inhibitor resulted in increased EMCM size. Functionally, Tgf-ß signaling repressed EMCM proliferation and contractile function, as assayed via dynamic monolayer force microscopy (DMFM). Taken together, these data support the hypothesis that biomechanical stimuli play a vital role in normal cardiac development and for cardiac pathology, including HLHS.
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Embrión de Mamíferos/citología , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Transducción de Señal , Estrés Mecánico , Factor de Crecimiento Transformador beta/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Tamaño de la Célula , Regulación de la Expresión Génica/efectos de los fármacos , Ontología de Genes , Ratones , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Miofibrillas/metabolismo , Análisis de Secuencia de ARN , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Group 2 innate lymphoid cells (ILC2s) have recently been identified in human nasal polyps, but whether numbers of ILC2s differ by polyp endotype or are influenced by corticosteroid use is unknown. Here, we show that eosinophilic nasal polyps contained double the number of ILC2s vs. non-eosinophilic polyps. Polyp ILC2s were also reduced by 50% in patients treated with systemic corticosteroids. Further, using a fungal allergen challenge mouse model, we detected greatly reduced Th2 cytokine-producing and Ki-67+ proliferating lung ILC2s in mice receiving dexamethasone. Finally, ILC2 Annexin V staining revealed extensive apoptosis after corticosteroid treatment in vivo and in vitro. Thus, ILC2s are elevated in the eosinophilic nasal polyp endotype and systemic corticosteroid treatment correlated with reduced polyp ILC2s. Finally, allergen-challenged mice showed reduced ILC2s and increased ILC2 apoptosis after corticosteroid treatment suggesting that ILC2 may be responsive to corticosteroids in eosinophilic respiratory disease.
Asunto(s)
Dexametasona/farmacología , Linfocitos/clasificación , Metilprednisolona/farmacología , Pólipos Nasales/patología , Prednisona/farmacología , Adulto , Animales , Antiinflamatorios/administración & dosificación , Antiinflamatorios/farmacología , Dexametasona/administración & dosificación , Femenino , Humanos , Masculino , Metilprednisolona/administración & dosificación , Ratones , Pólipos Nasales/genética , Prednisona/administración & dosificación , Adulto JovenRESUMEN
BACKGROUND: Exposure to the fungal allergen Alternaria alternata as well as ryegrass pollen has been implicated in severe asthma symptoms during thunderstorms. We have previously shown that Alternaria extract induces innate type 2 lung inflammation in mice. We hypothesized that the innate eosinophilic response to Alternaria extract may enhance lung inflammation induced by ryegrass. METHODS: Mice were sensitized to ryegrass allergen and administered a single challenge with A. alternata extract before or after final ryegrass challenges. Levels of eosinophils, neutrophils, Th2 cells, innate lymphoid cells (ILC2), interleukin (IL)-5 and IL-13 in bronchoalveolar lavage (BAL) as well as inflammation and mucus were assessed. RESULTS: Mice receiving ryegrass sensitization and challenge developed an eosinophilic lung response. A single challenge with Alternaria extract given 3 days before or 3 days after ryegrass challenges resulted in increased eosinophils, peribronchial inflammation and mucus production in the airways compared with ryegrass-only challenges. Type 2 ILC2 and Th2 cell recruitment to the airways was increased after Alternaria extract exposure in ryegrass-challenged mice. Innate immune challenges with Alternaria extract induced BAL eosinophilia, Th2 cell recruitment as well as ILC2 expansion and proliferation. CONCLUSIONS: A single exposure to Alternaria extract in ryegrass-sensitized and -challenged mice enhances the type 2 lung inflammatory response, including airway eosinophilia, peribronchial infiltrate, and mucus production, possibly through Th2 cell recruitment and ILC2 expansion. If translated to humans, exposure to both grass pollen and Alternaria may be a potential cause of thunderstorm-related asthma.
Asunto(s)
Alternaria/inmunología , Alternariosis/inmunología , Asma/inmunología , Lolium/inmunología , Neumonía/inmunología , Alérgenos/inmunología , Alternariosis/complicaciones , Animales , Antígenos Fúngicos/inmunología , Antígenos de Plantas/administración & dosificación , Antígenos de Plantas/inmunología , Asma/complicaciones , Células Cultivadas , Citocinas/inmunología , Eosinófilos/inmunología , Femenino , Humanos , Inmunidad Innata , Inmunización , Ratones , Ratones Endogámicos BALB C , Neutrófilos/inmunología , Neumonía/complicaciones , Polen/inmunología , Pruebas de Función Respiratoria , Células Th2/inmunología , Tiempo (Meteorología)RESUMEN
BACKGROUND: Cysteinyl leukotrienes (CysLTs) contribute to asthma pathogenesis, in part through cysteinyl leukotriene receptor 1 (CysLT1R). Recently discovered lineage-negative type 2 innate lymphoid cells (ILC2s) potently produce IL-5 and IL-13. OBJECTIVES: We hypothesized that lung ILC2s might be activated by leukotrienes through CysLT1R. METHODS: ILC2s (Thy1.2(+) lineage-negative lymphocytes) and CysLT1R were detected in the lungs of wild-type, signal transducer and activator of transcription 6-deficient (STAT6(-/-)), and recombination-activating gene 2-deficient (RAG2(-/-)) mice by means of flow cytometry. T(H)2 cytokine levels were measured in purified lung ILC2s stimulated with leukotriene D4 (LTD4) in the presence or absence of the CysLT1R antagonist montelukast. Calcium influx was measured by using Fluo-4 intensity. Intranasal leukotriene C4, D4, and E4 were administered to naive mice, and levels of ILC2 IL-5 production were determined. Finally, LTD4 was coadministered with Alternaria species repetitively to RAG2(-/-) mice (with ILC2s) and IL-7 receptor-deficient mice (lack ILC2s), and total ILC2 numbers, proliferation (Ki-67(+)), and bronchoalveolar lavage fluid eosinophil numbers were measured. RESULTS: CysLT1R was expressed on lung ILC2s from wild-type, RAG2(-/-), and STAT6(-/-) naive and Alternaria species-challenged mice. In vitro LTD4 induced ILC2s to rapidly generate high levels of IL-5 and IL-13 within 6 hours of stimulation. Interestingly, LTD4, but not IL-33, induced high levels of IL-4 by ILC2s. LTD4 administered in vivo rapidly induced ILC2 IL-5 production that was significantly reduced by montelukast before treatment. Finally, LTD4 potentiated Alternaria species-induced eosinophilia, as well as ILC2 accumulation and proliferation. CONCLUSIONS: We present novel data that CysLT1R is expressed on ILC2s and LTD4 potently induces CysLT1R-dependent ILC2 production of IL-4, IL-5, and IL-13. Additionally, LTD4 potentiates Alternaria species-induced eosinophilia and ILC2 proliferation and accumulation.
Asunto(s)
Citocinas/biosíntesis , Pulmón/inmunología , Receptores de Leucotrienos/fisiología , Células Th2/inmunología , Alternaria/inmunología , Animales , Proteínas de Unión al ADN/fisiología , Femenino , Leucotrieno D4/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Factor de Transcripción STAT6/fisiologíaRESUMEN
Enzymatic modification of DNA nucleobases can coordinate gene expression, nuclease protection, or mutagenesis. We recently discovered a clade of phage-specific cytosine methyltransferase (MT) and 5-methylpyrimidine dioxygenase (5mYOX) enzymes that produce 5-hydroxymethylcytosine (5hmC) as a precursor for enzymatic hypermodifications on viral genomes. Here, we identify phage MT- and 5mYOX-associated glycosyltransferases (GTs) that catalyze linkage of diverse sugars to 5hmC nucleobase substrates. Metavirome mining revealed thousands of biosynthetic gene clusters containing enzymes with predicted roles in cytosine sugar hypermodification. We developed a platform for high-throughput screening of GT-containing pathways, relying on the Escherichia coli metabolome as a substrate pool. We successfully reconstituted several pathways and isolated diverse sugar modifications appended to cytosine, including mono-, di-, or tri-saccharides comprised of hexoses, N-acetylhexosamines, or heptose. These findings expand our knowledge of hypermodifications on nucleic acids and the origins of corresponding sugar-installing enzymes.
RESUMEN
OBJECTIVE: To evaluate the impact of a clinical decision support system (CDSS) to identify drug-related problems (DRPs) during community pharmacist medication reviews. DESIGN: Pilot 3-phase (group), open-label study. SETTING AND PARTICIPANTS: Two community pharmacies in Sarnia, Ontario, with pharmacists providing medication reviews to patients. STUDY PROCEDURES: Five pharmacists participated in three phases (groups). During Phase 1, pharmacists conducted medication reviews in 25 adult patients using the usual approaches. In Phase 2, pharmacists were trained to use a CDSS to identify DRPs, and then conducted medication reviews using the tool in a different group of 25 adult patients. In Phase 3, pharmacists conducted medication reviews without the aid of the CDSS in 25 additional adult patients. MAIN OUTCOME MEASURES: The primary outcome was recommendation to the primary care physician to alter pharmacotherapy based on medication review, assessed using mean number and frequency (yes/no) of recommendations by patient. Secondary outcomes included number of potential DRPs, actual DRPs, medication review duration time, pharmacist's perceptions of the CDSS and patient satisfaction with medication review. RESULTS: The mean number of recommendations to primary care physicians to alter pharmacotherapy per patient in Phases 1, 2 and 3 did not differ: 1.0 (SD = I.2) versus 1.5 (1.0) versus 1.5 (1.0), respectively; p = 0.223. The percentage of patients with a pharmacy recommendation sent to physicians across the phases, however, differed: 52% versus 80% versus 88%, respectively; p = 0.010, with more in Phases 2 and 3 compared to 1. There were more potential DRPs in group 2 compared to other groups. There were no differences in actual DRPs and medication review time. Pharmacists had positive attitudes about the CDSS. Patients were generally satisfied with their medication review. CONCLUSIONS: This small pilot study provides some preliminary evidence for performance and feasibility of a CDSS to identify DRPs that pharmacists will act on. Future research is recommended to validate these findings in a larger sample.
RESUMEN
Commercially synthesized genes are typically made using variations of homology-based cloning techniques, including polymerase cycling assembly from chemically synthesized microarray-derived oligonucleotides. Here, we apply Data-optimized Assembly Design (DAD) to the synthesis of hundreds of codon-optimized genes in both constitutive and inducible vectors using Golden Gate Assembly. Starting from oligonucleotide pools, we synthesize genes in three simple steps: (1) amplification of parts belonging to individual assemblies in parallel from a single pool; (2) Golden Gate Assembly of parts for each construct; and (3) transformation. We construct genes from receiving DNA to sequence confirmed isolates in as little as 4 days. By leveraging the ligation fidelity afforded by T4 DNA ligase, we expect to be able to construct a larger breadth of sequences not currently supported by homology-based methods, which require stability of extensive single-stranded DNA overhangs.
Asunto(s)
Oligonucleótidos , Biología Sintética , Oligonucleótidos/genética , Biología Sintética/métodos , ADN/genética , ADN de Cadena Simple/genética , Clonación Molecular , Vectores GenéticosRESUMEN
The mammalian Siglec receptor sialoadhesin (Siglec1, CD169) confers innate immunity against the encapsulated pathogen group B Streptococcus (GBS). Newborn lung macrophages have lower expression levels of sialoadhesin at birth compared with the postnatal period, increasing their susceptibility to GBS infection. In this study, we investigate the mechanisms regulating sialoadhesin expression in the newborn mouse lung. In both neonatal and adult mice, GBS lung infection reduced Siglec1 expression, potentially delaying acquisition of immunity in neonates. Suppression of Siglec1 expression required interactions between sialic acid on the GBS capsule and the inhibitory host receptor Siglec-E. The Siglec1 gene contains multiple STAT binding motifs, which could regulate expression of sialoadhesin downstream of innate immune signals. Although GBS infection reduced STAT1 expression in the lungs of wild-type newborn mice, we observed increased numbers of STAT1+ cells in Siglece-/- lungs. To test if innate immune activation could increase sialoadhesin at birth, we first demonstrated that treatment of neonatal lung macrophages ex vivo with inflammatory activators increased sialoadhesin expression. However, overcoming the low sialoadhesin expression at birth using in vivo prenatal exposures or treatments with inflammatory stimuli were not successful. The suppression of sialoadhesin expression by GBS-Siglec-E engagement may therefore contribute to disease pathogenesis in newborns and represent a challenging but potentially appealing therapeutic opportunity to augment immunity at birth.
Asunto(s)
Animales Recién Nacidos , Ratones Noqueados , Ácido N-Acetilneuramínico , Factor de Transcripción STAT1 , Lectina 1 Similar a Ig de Unión al Ácido Siálico , Infecciones Estreptocócicas , Streptococcus agalactiae , Animales , Ratones , Streptococcus agalactiae/inmunología , Ácido N-Acetilneuramínico/metabolismo , Lectina 1 Similar a Ig de Unión al Ácido Siálico/metabolismo , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/microbiología , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT1/genética , Inmunidad Innata , Ratones Endogámicos C57BL , Pulmón/inmunología , Pulmón/microbiología , Pulmón/metabolismo , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/metabolismo , Femenino , Macrófagos/inmunología , Macrófagos/metabolismo , Lectinas/metabolismo , Lectinas/genética , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismo , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/genética , Antígenos CD/metabolismo , Antígenos CD/genética , Antígenos de Diferenciación de Linfocitos BRESUMEN
The fungal allergen Alternaria alternata is associated with development of asthma, though the mechanisms underlying the allergenicity of Alternaria are largely unknown. The aim of this study was to identify whether the MAP kinase homologue Fus3 of Alternaria contributed to allergic airway responses. Wild-type (WT) and Fus3 deficient Alternaria extracts were given intranasal to mice. Extracts from Fus3 deficient Alternaria that had a functional copy of Fus3 introduced were also administered (CpFus3). Mice were challenged once and levels of BAL eosinophils and innate cytokines IL-33, thymic stromal lymphopoeitin (TSLP), and IL-25 (IL-17E) were assessed. Alternaria extracts or protease-inhibited extract were administered with (OVA) during sensitization prior to ovalbumin only challenges to determine extract adjuvant activity. Levels of BAL inflammatory cells, Th2 cytokines, and OX40-expressing Th2 cells as well as airway infiltration and mucus production were measured. WT Alternaria induced innate airway eosinophilia within 3 days. Mice given Fus3 deficient Alternaria were significantly impaired in developing airway eosinophilia that was largely restored by CpFus3. Further, BAL IL-33, TSLP, and Eotaxin-1 levels were reduced after challenge with Fus3 mutant extract compared with WT and CpFus3 extracts. WT and CpFus3 extracts demonstrated strong adjuvant activity in vivo as levels of BAL eosinophils, Th2 cytokines, and OX40-expressing Th2 cells as well as peribronchial inflammation and mucus production were induced. In contrast, the adjuvant activity of Fus3 extract or protease-inhibited WT extract was largely impaired. Finally, protease activity and Alt a1 levels were reduced in Fus3 mutant extract. Thus, Fus3 contributes to the Th2-sensitizing properties of Alternaria.
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Alérgenos/genética , Alternaria/genética , Alternaria/inmunología , Asma/etiología , Proteínas Fúngicas/genética , Proteínas Fúngicas/inmunología , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/inmunología , Alérgenos/metabolismo , Alternaria/patogenicidad , Animales , Asma/inmunología , Asma/microbiología , Modelos Animales de Enfermedad , Femenino , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Humanos , Inmunidad Innata , Pulmón/inmunología , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Mutación , Ovalbúmina/inmunología , Células Th2/inmunología , Células Th2/patologíaRESUMEN
Golden Gate Assembly is a flexible method of DNA assembly and cloning that permits the joining of multiple fragments in a single reaction through predefined connections. The method depends on cutting DNA using a Type IIS restriction enzyme, which cuts outside its recognition site and therefore can generate overhangs of any sequence while separating the recognition site from the generated fragment. By choosing compatible fusion sites, Golden Gate permits the joining of multiple DNA fragments in a defined order in a single reaction. Conventionally, this method has been used to join five to eight fragments in a single assembly round, with yield and accuracy dropping off rapidly for more complex assemblies. Recently, we demonstrated the application of comprehensive measurements of ligation fidelity and bias data using data-optimized assembly design (DAD) to enable a high degree of assembly accuracy for very complex assemblies with the simultaneous joining of as many as 52 fragments in one reaction. Here, we describe methods for applying DAD principles and online tools to evaluate the fidelity of existing fusion site sets and assembly standards, selecting new optimal sets, and adding fusion sites to existing assemblies. We further describe the application of DAD to divide known sequences at optimal points, including designing one-pot assemblies of small genomes. Using the T7 bacteriophage genome as an example, we present a protocol that includes removal of native Type IIS sites (domestication) simultaneously with parts generation by PCR. Finally, we present recommended cycling protocols for assemblies of medium to high complexity (12-36 fragments), methods for producing high-quality parts, examples highlighting the importance of DNA purity and fragment stoichiometric balance for optimal assembly outcomes, and methods for assessing assembly success. © 2023 New England Biolabs, Inc. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Assessing the fidelity of an overhang set using the NEBridge Ligase Fidelity Viewer Basic Protocol 2: Generating a high-fidelity overhang set using the NEBridge GetSet Tool Alternate Protocol 1: Expanding an existing overhang set using the NEBridge GetSet Tool Basic Protocol 3: Dividing a genomic sequence with optimal fusion sites using the NEBridge SplitSet Tool Basic Protocol 4: One-pot Golden Gate Assembly of 12 fragments into a destination plasmid Alternate Protocol 2: One-pot Golden Gate Assembly of 24+ fragments into a destination plasmid Basic Protocol 5: One-pot Golden Gate Assembly of the T7 bacteriophage genome from 12+ parts Support Protocol 1: Generation of high-purity amplicons for assembly Support Protocol 2: Cloning assembly parts into a holding vector Support Protocol 3: Quantifying DNA concentration using a Qubit 4 fluorometer Support Protocol 4: Visualizing large assemblies via TapeStation Support Protocol 5: Validating phage genome assemblies via ONT long-read sequencing.
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Bacteriófago T7 , Bacteriófagos , Ciclismo , Enzimas de Restricción del ADN , DomesticaciónRESUMEN
Innate lymphoid cells (ILC) promote lung inflammation in asthma through cytokine production. RNA-binding proteins (RBPs) are critical post-transcriptional regulators, although less is known about RBPs in ILC biology. Here, we demonstrate that RNA-binding motif 3 (RBM3) is highly expressed in lung ILCs and is further induced by alarmins TSLP and IL-33. Rbm3-/- and Rbm3-/-Rag2-/- mice exposed to asthma-associated Alternaria allergen develop enhanced eosinophilic lung inflammation and ILC activation. IL-33 stimulation studies in vivo and in vitro show that RBM3 suppressed lung ILC responses. Further, Rbm3-/- ILCs from bone marrow chimeric mice display increased ILC cytokine production suggesting an ILC-intrinsic suppressive function of RBM3. RNA-sequencing of Rbm3-/- lung ILCs demonstrates increased expression of type 2/17 cytokines and cysteinyl leukotriene 1 receptor (CysLT1R). Finally, Rbm3-/-Cyslt1r-/- mice show dependence on CysLT1R for accumulation of ST2+IL-17+ ILCs. Thus, RBM3 intrinsically regulates lung ILCs during allergen-induced type 2 inflammation that is partially dependent on CysLT1R.
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Asma , Neumonía , Alérgenos , Animales , Asma/metabolismo , Citocinas/metabolismo , Inmunidad Innata , Inflamación/metabolismo , Interleucina-33/genética , Interleucina-33/metabolismo , Pulmón/metabolismo , Linfocitos/metabolismo , Ratones , Neumonía/genética , Neumonía/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Receptores de LeucotrienosRESUMEN
Targeting host factors for anti-viral development offers several potential advantages over traditional countermeasures that include broad-spectrum activity and prevention of resistance. Characterization of host factors in animal models provides strong evidence of their involvement in disease pathogenesis, but the feasibility of performing high-throughput in vivo analyses on lists of genes is problematic. To begin addressing the challenges of screening candidate host factors in vivo, we combined advances in CRISPR-Cas9 genome editing with an immunocompromised mouse model used to study highly pathogenic viruses. Transgenic mice harboring a constitutively expressed Cas9 allele (Cas9 tg/tg ) with or without knockout of type I interferon receptors served to optimize in vivo delivery of CRISPR single-guide RNA (sgRNA) using Invivofectamine 3.0, a simple and easy-to-use lipid nanoparticle reagent. Invivofectamine 3.0-mediated liver-specific editing to remove activity of the critical Ebola virus host factor Niemann-Pick disease type C1 in an average of 74% of liver cells protected immunocompromised Cas9 tg/tg mice from lethal surrogate Ebola virus infection. We envision that immunocompromised Cas9 tg/tg mice combined with straightforward sgRNA in vivo delivery will enable efficient host factor loss-of-function screening in the liver and other organs to rapidly study their effects on viral pathogenesis and help initiate development of broad-spectrum, host-directed therapies against emerging pathogens.
Asunto(s)
Alérgenos/inmunología , Antígenos CD/metabolismo , Linfocitos/inmunología , Linfocitos/metabolismo , Rinitis Alérgica Perenne/inmunología , Rinitis Alérgica Perenne/metabolismo , Alérgenos/administración & dosificación , Humanos , Rinitis Alérgica , Familia de Moléculas Señalizadoras de la Activación LinfocitariaRESUMEN
Streptococcus agalactiae (Group B Streptococcus, GBS) is the most common neonatal pathogen. However, the cellular and molecular mechanisms for neonatal susceptibility to GBS pneumonia and sepsis are incompletely understood. Here we optimized a mouse model of GBS pneumonia to test the role of alveolar macrophage (ΑΜΦ) maturation in host vulnerability to disease. Compared with juvenile and adult mice, neonatal mice infected with GBS had increased mortality and persistence of lung injury. In addition, neonatal mice were defective in GBS phagocytosis and killing. ΑΜΦ depletion and disruption of ΑΜΦ differentiation in Csf2-/- mice both impaired GBS clearance. AMΦ engage the heavily sialylated GBS capsule via the cell surface Siglec receptors Sn and Siglec-E. Although both newborn and adult ΑΜΦ expressed Siglec-E, newborn ΑΜΦ expressed significantly lower levels of Sn. We propose that a developmental delay in Sn expression on ΑΜΦ may prevent effective killing and clearing of GBS from the newborn lung.
RESUMEN
Isoprenoids are constructed in nature using hemiterpene building blocks that are biosynthesized from lengthy enzymatic pathways with little opportunity to deploy precursor-directed biosynthesis. Here, an artificial alcohol-dependent hemiterpene biosynthetic pathway was designed and coupled to several isoprenoid biosynthetic systems, affording lycopene and a prenylated tryptophan in robust yields. This approach affords a potential route to diverse non-natural hemiterpenes and by extension isoprenoids modified with non-natural chemical functionality. Accordingly, the prototype chemo-enzymatic pathway is a critical first step toward the construction of engineered microbial strains for bioconversion of simple scalable building blocks into complex isoprenoid scaffolds.