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Maize (Zea mays) is a multifaceted cereal grass used globally for nutrition, animal feed, food processing, and biofuels, and a model system in genetics research. Studying the maize microbiome sometimes requires its manipulation to identify the contributions of specific taxa and ecological traits (i.e., diversity, richness, network structure) to maize growth and physiology. Due to regulatory constraints on applying engineered microorganisms in field settings, greenhouse-based experimentation is often the first step for understanding the contribution of root-associated microbiota-whether natural or engineered-to plant phenotypes. In this protocol, we describe methods to inoculate maize with a specific microbiome as a tool for understanding the microbiota's influence on its host plant. The protocol involves removal of the native seed microbiome followed by inoculation of new microorganisms; separate protocols are provided for inoculations from pure culture, from soil slurry, or by mixing in live soil. These protocols cover the most common methods for manipulating the maize microbiome in soil-grown plants in the greenhouse. The methods outlined will ultimately result in rhizosphere microbial assemblages with varying degrees of microbial diversity, ranging from low diversity (individual strain and synthetic community [SynCom] inoculation) to high diversity (percent live inoculation), with the slurry inoculation method representing an "intermediate diversity" treatment.
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This study examines the chemical reaction pathways for vapor phase infiltration (VPI) of TiCl4 into poly(methyl methacrylate) (PMMA). VPI is a processing method that transforms organic polymers into organic-inorganic hybrid materials with new properties of interest for microelectronic patterning, technical textiles, and chemical separations. Understanding the fundamental chemical mechanisms of the VPI process is essential for establishing approaches to design the chemical structure and properties of these hybrid materials. While prior work has suggested that TiCl4 infiltration into PMMA does not disrupt the polymer's carbonyl bond, a clear reaction mechanism has yet to be proposed. Here, we present a detailed X-ray photoelectron spectroscopy study that presents evidence for a concerted reaction mechanism that involves TiCl4 coordinating with the PMMA's ester group to dealkylate the methyl side group, creating a chloromethane byproduct and primary chemical bonds between the organic and inorganic components of the hybrid material. Additional spectroscopy, quartz crystal microbalance gravimetry, and thermophysical and chemical property measurements of this material, including solubility studies and thermal expansion measurements, provide further evidence for this chemical reaction pathway and the subsequent creation of inorganic cross-links that network these TiOx-PMMA hybrid materials.
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Cocaine use disorder represents a public health crisis with no FDA-approved medications for its treatment. A growing body of research has detailed the important connections between the brain and the resident population of bacteria in the gut, the gut microbiome, in psychiatric disease models. Acute depletion of gut bacteria results in enhanced reward in a mouse cocaine place preference model, and repletion of bacterially-derived short-chain fatty acid (SCFA) metabolites reverses this effect. However, the role of the gut microbiome and its metabolites in modulating cocaine-seeking behavior after prolonged abstinence is unknown. Given that relapse prevention is the most clinically challenging issue in treating substance use disorders, studies examining the effects of microbiome manipulations in relapse-relevant models are critical. Here, male Sprague-Dawley rats received either untreated water or antibiotics to deplete the gut microbiome and its metabolites. Rats were trained to self-administer cocaine and subjected to either within-session threshold testing to evaluate motivation for cocaine or 21 days of abstinence followed by a cue-induced cocaine-seeking task to model relapse behavior. Microbiome depletion did not affect cocaine acquisition on an fixed-ratio 1 schedule. However, microbiome-depleted rats exhibited significantly enhanced motivation for low dose cocaine on a within-session threshold task. Similarly, microbiome depletion increased cue-induced cocaine-seeking following prolonged abstinence and altered transcriptional regulation in the nucleus accumbens. In the absence of a normal microbiome, repletion of bacterially-derived SCFA metabolites reversed the behavioral and transcriptional changes associated with microbiome depletion. These findings suggest that gut bacteria, via their metabolites, are key regulators of drug-seeking behaviors, positioning the microbiome as a potential translational research target.
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Trastornos Relacionados con Cocaína , Cocaína , Ratones , Ratas , Masculino , Animales , Ratas Sprague-Dawley , Comportamiento de Búsqueda de Drogas , Trastornos Relacionados con Cocaína/metabolismo , Núcleo Accumbens , Recurrencia , Autoadministración , Señales (Psicología) , Extinción PsicológicaRESUMEN
Although ribosomes are generally examined in aggregate, ribosomes can be heterogenous in composition. Evidence is accumulating that changes in ribosome composition may result in altered function, such that ribosome heterogeneity may provide a mechanism to regulate protein synthesis. Ribosome heterogeneity in the human pathogen Francisella tularensis results from incorporation of one of three homologs of bS21, a small ribosomal subunit protein demonstrated to regulate protein synthesis in other bacteria. Loss of one homolog, bS21-2, results in genome-wide post-transcriptional changes in protein abundance. This suggests that bS21-2 can, either directly or indirectly, lead to preferential translation of particular mRNAs. Here, we examine the potential of bS21-2 to function in a leader sequence-dependent manner and to function indirectly, via Hfq. We found that the 5´ untranslated region (UTR) of some bS21-2-responsive genes, including key virulence genes, is sufficient to alter translation in cells lacking bS21-2. We further identify features of a 5´ UTR that allow responsiveness to bS21-2. These include an imperfect Shine-Dalgarno sequence and a particular six nucleotide sequence. Our results are consistent with a model in which a bS21 homolog increases the efficiency of translation initiation through interactions with specific leader sequences. With respect to bS21-2 indirectly regulating translation via the RNA-binding protein Hfq, we found that Hfq controls transcript abundance rather than protein synthesis, impacting virulence gene expression via a distinct mechanism. Together, we determined that ribosome composition in F. tularensis regulates translation in a leader sequence-dependent manner, a regulatory mechanism which may be used in other bacteria. IMPORTANCE Ribosome heterogeneity is common in bacteria, and there is mounting evidence that ribosome composition plays a regulatory role in protein synthesis. However, mechanisms of ribosome-driven gene regulation are not well understood. In the human pathogen Francisella tularensis, which encodes multiple homologs for the ribosomal protein bS21, loss of one homolog impacts protein synthesis and virulence. Here, we explore the mechanism behind bS21-mediated changes in protein synthesis, finding that they can be linked to altered translation initiation and are dependent on specific sequences in the leaders of transcripts. Our data support a model in which ribosome composition regulates gene expression through translation, a strategy that may be conserved in diverse organisms with various sources of ribosome heterogeneity.
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Francisella tularensis , Humanos , Francisella tularensis/genética , Ribosomas/genética , Proteínas Ribosómicas/genética , Regiones no Traducidas 5' , ARN Mensajero/genéticaRESUMEN
Background: 'Epigenetic clocks' have been developed to accurately predict chronologic gestational age and have been associated with child health outcomes in prior work.Methods: We meta-analysed results from four prospective U.S cohorts investigating the association between epigenetic age acceleration estimated using blood DNA methylation collected at birth and preschool age Childhood Behavior Checklist (CBCL) scores.Results: Epigenetic ageing was not significantly associated with CBCL total problem scores (ß = 0.33, 95% CI: -0.95, 0.28) and DSM-oriented pervasive development problem scores (ß = -0.23, 95% CI: -0.61, 0.15). No associations were observed for other DSM-oriented subscales.Conclusions: The meta-analysis results suggest that epigenetic gestational age acceleration is not associated with child emotional and behavioural functioning for preschool age group. These findings may relate to our study population, which includes two cohorts enriched for ASD and one preterm birth cohort.; future work should address the role of epigenetic age in child health in other study populations.Abbreviations: DNAm: DNA methylation; CBCL: Child Behavioral Checklist; ECHO: Environmental Influences on Child Health Outcomes; EARLI: Early Autism Risk Longitudinal Investigation; MARBLES: Markers of Autism Risk in Babies - Learning Early Signs; ELGAN: Extremely Low Gestational Age Newborns; ASD: autism spectrum disorder; BMI: body mass index; DSM: Diagnostic and Statistical Manual of Mental Disorders.
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Trastorno del Espectro Autista , Nacimiento Prematuro , Preescolar , Humanos , Recién Nacido , Metilación de ADN , Epigénesis Genética , Estudios ProspectivosRESUMEN
BACKGROUND: Per- and polyfluoroalkyl substances (PFAS) are ubiquitous, environmentally persistent chemicals, and prenatal exposures have been associated with adverse child health outcomes. Prenatal PFAS exposure may lead to epigenetic age acceleration (EAA), defined as the discrepancy between an individual's chronologic and epigenetic or biological age. OBJECTIVES: We estimated associations of maternal serum PFAS concentrations with EAA in umbilical cord blood DNA methylation using linear regression, and a multivariable exposure-response function of the PFAS mixture using Bayesian kernel machine regression. METHODS: Five PFAS were quantified in maternal serum (median: 27 weeks of gestation) among 577 mother-infant dyads from a prospective cohort. Cord blood DNA methylation data were assessed with the Illumina HumanMethylation450 array. EAA was calculated as the residuals from regressing gestational age on epigenetic age, calculated using a cord-blood specific epigenetic clock. Linear regression tested for associations between each maternal PFAS concentration with EAA. Bayesian kernel machine regression with hierarchical selection estimated an exposure-response function for the PFAS mixture. RESULTS: In single pollutant models we observed an inverse relationship between perfluorodecanoate (PFDA) and EAA (-0.148 weeks per log-unit increase, 95% CI: -0.283, -0.013). Mixture analysis with hierarchical selection between perfluoroalkyl carboxylates and sulfonates indicated the carboxylates had the highest group posterior inclusion probability (PIP), or relative importance. Within this group, PFDA had the highest conditional PIP. Univariate predictor-response functions indicated PFDA and perfluorononanoate were inversely associated with EAA, while perfluorohexane sulfonate had a positive association with EAA. CONCLUSIONS: Maternal mid-pregnancy serum concentrations of PFDA were negatively associated with EAA in cord blood, suggesting a pathway by which prenatal PFAS exposures may affect infant development. No significant associations were observed with other PFAS. Mixture models suggested opposite directions of association between perfluoroalkyl sulfonates and carboxylates. Future studies are needed to determine the importance of neonatal EAA for later child health outcomes.
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Ácidos Alcanesulfónicos , Contaminantes Ambientales , Fluorocarburos , Efectos Tardíos de la Exposición Prenatal , Lactante , Recién Nacido , Embarazo , Niño , Femenino , Humanos , Sangre Fetal , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Estudios Prospectivos , Teorema de Bayes , Alcanosulfonatos , Madres , Ácidos Carboxílicos , Epigénesis GenéticaRESUMEN
Neural crest cells along the body axis of avian embryos differ in their developmental potential, such that the cranial neural crest forms cartilage and bone whereas the trunk neural crest is unable to do so. Previous studies have identified a cranial crest-specific subcircuit that can imbue the trunk neural crest with the ability to form cartilage after grafting to the head. Here, we examine transcriptional and cell fate changes that accompany this reprogramming. First, we examined whether reprogrammed trunk neural crest maintain the ability to form cartilage in their endogenous environment in the absence of cues from the head. The results show that some reprogrammed cells contribute to normal trunk neural crest derivatives, whereas others migrate ectopically to the forming vertebrae and express cartilage markers, thus mimicking heterotypically transplanted cranial crest cells. We find that reprogrammed trunk neural crest upregulated more than 3000 genes in common with cranial neural crest, including numerous transcriptional regulators. In contrast, many trunk neural crest genes are downregulated. Together, our findings show that reprogramming trunk neural crest with cranial crest subcircuit genes alters their gene regulatory program and developmental potential to be more cranial crest-like.
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Cresta Neural , Transcriptoma , Diferenciación Celular , Cartílago , Huesos , Movimiento CelularRESUMEN
Stress is a significant contributor to the development and progression of substance use disorders (SUDs) and is problematic as it is unavoidable in daily life. Therefore, it is important to understand the neurobiological mechanisms that underlie the influence of stress on drug use. We have previously developed a model to examine the contribution of stress to drug-related behavior by administering a stressor, electric footshock stress, daily at the time of cocaine self-administration in rats resulting in an escalation of cocaine intake. This stress-induced escalation of cocaine intake involves neurobiological mediators of stress and reward such as cannabinoid signaling. However, all of this work has been conducted in male rats. Here we test the hypothesis that repeated daily stress can produce an escalation of cocaine in both male and female rats. We further hypothesize that cannabinoid receptor 1 (CB1R) signaling is recruited by repeated stress to influence cocaine intake in both male and female rats. Male and female Sprague-Dawley rats self-administered cocaine (0.5 mg/kg/inf, i.v.) during a modified short-access paradigm wherein the 2-hr access was separated into 4-30 min self-administration blocks separated by 4-5 min drug free period. Footshock stress produced a significant escalation of cocaine intake similarly in both male and female rats. Female stress-escalated rats did display greater time-out non-reinforced responding and greater "front-loading" behavior. In males, systemic administration of a CB1R inverse agonist/antagonist Rimonabant only attenuated cocaine intake in rats with a history of combined repeated stress and cocaine self-administration. However, in females, Rimonabant attenuated cocaine intake in the no stress control group but only at the highest dose of Rimonabant (3 mg/kg, i.p.) suggesting that females show a greater sensitivity to CB1R antagonism. However, female rats with a history of stress showed even greater sensitivity to CB1R antagonism as both doses of Rimonabant (1, 3 mg/kg) attenuated cocaine intake in stress-escalated rats similar to males. Altogether these data demonstrate that stress can produce significant changes in cocaine self-administration and suggests that repeated stress at the time of cocaine self-administration recruits CB1Rs to regulate cocaine-taking behavior across sexes.
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MOTIVATION: Aberrant DNA methylation in transcription factor binding sites has been shown to lead to anomalous gene regulation that is strongly associated with human disease. However, the majority of methylation-sensitive positions within transcription factor binding sites remain unknown. Here we introduce SEMplMe, a computational tool to generate predictions of the effect of methylation on transcription factor binding strength in every position within a transcription factor's motif. RESULTS: SEMplMe uses ChIP-seq and whole genome bisulfite sequencing to predict effects of methylation within binding sites. SEMplMe validates known methylation sensitive and insensitive positions within a binding motif, identifies cell type specific transcription factor binding driven by methylation, and outperforms SELEX-based predictions for CTCF. These predictions can be used to identify aberrant sites of DNA methylation contributing to human disease. AVAILABILITY AND IMPLEMENTATION: SEMplMe is available from https://github.com/Boyle-Lab/SEMplMe .
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Metilación de ADN , Factores de Transcripción , Sitios de Unión , Regulación de la Expresión Génica , Humanos , Unión Proteica , Factores de Transcripción/metabolismoRESUMEN
Monoclonal antibodies are increasingly used for the prevention and/or treatment of viral infections. One caveat of their use is the ability of viruses to evolve resistance to antibody binding and neutralization. Computational strategies to identify viral mutations that may disrupt antibody binding would leverage the wealth of viral genomic sequence data to monitor for potential antibody-resistant mutations. The respiratory syncytial virus is an important pathogen for which monoclonal antibodies against the fusion (F) protein are used to prevent severe disease in high-risk infants. In this study, we used an approach that combines molecular dynamics simulations with FoldX to estimate changes in free energy in F protein folding and binding to the motavizumab antibody upon each possible amino acid change. We systematically selected 8 predicted escape mutations and tested them in an infectious clone. Consistent with our F protein stability predictions, replication-effective viruses were observed for each selected mutation. Six of the eight variants showed increased resistance to neutralization by motavizumab. Flow cytometry was used to validate the estimated (model-predicted) effects on antibody binding to F. Using surface plasmon resonance, we determined that changes in the on-rate of motavizumab binding were associated with the reduced affinity for two novel escape mutations. Our study empirically validated the accuracy of our molecular modeling approach and emphasized the role of biophysical protein modeling in predicting viral resistance to antibody-based therapeutics that can be used to monitor the emergence of resistant viruses and to design improved therapeutic antibodies. IMPORTANCE Respiratory syncytial virus (RSV) causes severe disease in young infants, particularly those with heart or lung diseases or born prematurely. Because no vaccine is currently available, monoclonal antibodies are used to prevent severe RSV disease in high-risk infants. While it is known that RSV evolves to avoid recognition by antibodies, screening tools that can predict which changes to the virus may lead to antibody resistance are greatly needed.
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Modelos Moleculares , Mutación , Infecciones por Virus Sincitial Respiratorio , Virus Sincitial Respiratorio Humano , Proteínas Virales de Fusión , Anticuerpos Antivirales/metabolismo , Humanos , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitial Respiratorio Humano/genética , Virus Sincitial Respiratorio Humano/inmunología , Proteínas Virales de Fusión/genéticaRESUMEN
Belowground, plants interact with beneficial soil microbes such as plant growth-promoting rhizobacteria (PGPR). PGPR are rhizosphere bacteria that colonize roots and elicit beneficial effects in plants such as improved plant growth, pathogen resistance, abiotic stress tolerance, and herbivore protection. Treatment of plants with PGPR has been shown to trigger the emission of volatile organic compounds (VOCs). Volatile emissions can also be triggered by herbivory, termed herbivore-induced plant volatiles (HIPV), with important ramifications for chemical-mediated plant and insect interactions. Much of our current understanding of PGPR and herbivore-induced volatiles is based on studies using one plant genotype, yet domestication and modern breeding has led to the development of diverse germplasm with altered phenotypes and chemistry. In this study, we investigated if volatile emissions triggered by PGPR colonization and herbivory varies by maize genotype and microbial community assemblages. Six maize genotypes representing three decades of crop breeding and two heterotic groups were used, with four microbiome treatments: live or sterilized soil, with or without a Bacillus inoculant. Soil sterilization was used to delay microbiome establishment, resulting in low-diversity treatments. At planting, maize seeds were inoculated with PGPR Bacillus altitudinis AP-283 and grown under greenhouse conditions. Four weeks post planting, plants were subjected to feeding by third instar Helicoverpa zea (Lepidoptera: Noctuidae) larvae. Volatiles were collected using solid phase microextraction and analyzed with gas chromatography-mass spectrometry. Illumina NovaSeq 16S rRNA amplicon sequencing was carried out to characterize the rhizosphere microbiome. Maize genotype significantly influenced total volatile emissions, and relative abundance of volatile classes. We did not document a strong influence of microbe treatment on plant VOC emissions. However, inoculating plants with PGPR improved plant growth under sterile conditions. Taken together, our results suggest that genotypic variation is the dominant driver in HIPV composition and individual HIPV abundances, and any bacterial-mediated benefit is genotype and HIPV-specific. Therefore, understanding the interplay of these factors is necessary to fully harness microbially-mediated benefits and improve agricultural sustainability.
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The bystander effect reveals that people are less likely to help a person in need when others are present. We examined the impact of priming the concept of responsibility on the bystander effect in a field study. Lone pedestrians (N = 259) were randomly assigned to a two (Bystanders: none and three nonresponsive bystanders) by two (Shirt: blank shirt and shirt with "Be Responsible" written on the front) design. A researcher dropped eight pens approximately 15 ft from a lone pedestrian, while wearing one of the two shirts in the presence/absence of bystanders (confederates). The bystander effect was found: Pedestrians helped pick up pens more frequently in the no bystanders condition (59.05% helped) compared to the nonresponsive bystanders condition (41.67% helped). The responsibility prime tended to boost helping rates, but it did not significantly increase helping rates either as a main effect or as part of an interaction term. The bystander effect was replicated in a field setting, but priming the concept of responsibility did not appear to reduce it.
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Conducta de Ayuda , Responsabilidad Social , HumanosRESUMEN
BACKGROUND: Zebrafish are a foundational model organism for studying the spatio-temporal activity of genes and their regulatory sequences. A variety of approaches are currently available for editing genes and modifying gene expression in zebrafish, including RNAi, Cre/lox, and CRISPR-Cas9. However, the lac operator-repressor system, an E. coli lac operon component which has been adapted for use in many other species and is a valuable, flexible tool for inducible modulation of gene expression studies, has not been previously tested in zebrafish. RESULTS: Here we demonstrate that the lac operator-repressor system robustly decreases expression of firefly luciferase in cultured zebrafish fibroblast cells. Our work establishes the lac operator-repressor system as a promising tool for the manipulation of gene expression in whole zebrafish. CONCLUSION: Our results lay the groundwork for the development of lac-based reporter assays in zebrafish, and adds to the tools available for investigating dynamic gene expression in embryogenesis. We believe this work will catalyze the development of new reporter assay systems to investigate uncharacterized regulatory elements and their cell-type specific activities.
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UV irradiation induces pyrimidine dimers that block polymerases and disrupt the replisome. Restoring replication depends on the recF pathway proteins which process and maintain the replication fork DNA to allow the lesion to be repaired before replication resumes. Oxidative DNA lesions, such as those induced by hydrogen peroxide (H2O2), are often thought to require similar processing events, yet far less is known about how cells process oxidative damage during replication. Here we show that replication is not disrupted by H2O2-induced DNA damage in vivo. Following an initial inhibition, replication resumes in the absence of either lesion removal or RecF-processing. Restoring DNA synthesis depends on the presence of manganese in the medium, which we show is required for replication, but not repair to occur. The results demonstrate that replication is enzymatically inactivated, rather than physically disrupted by H2O2-induced DNA damage; indicate that inactivation is likely caused by oxidation of an iron-dependent replication or replication-associated protein that requires manganese to restore activity and synthesis; and address a long standing paradox as to why oxidative glycosylase mutants are defective in repair, yet not hypersensitive to H2O2. The oxygen-sensitive pausing may represent an adaptation that prevents replication from occurring under potentially lethal or mutagenic conditions.
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Proteínas de Escherichia coli , Escherichia coli , Daño del ADN , Reparación del ADN , Replicación del ADN , ADN Bacteriano , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Peróxido de Hidrógeno/farmacologíaRESUMEN
BACKGROUND: Hepatocyte NF 4α (Hnf4a) is a major regulator of renal proximal tubule (PT) development. In humans, a mutation in HNF4A impairs PT functions and is associated with Fanconi renotubular syndrome (FRTS). In mice, mosaic deletion of Hnf4a in the developing kidney reduces the population of PT cells, leading to FRTS-like symptoms. The molecular mechanisms underlying the role of Hnf4a in PT development remain unclear. METHODS: The gene deletion tool Osr2Cre removed Hnf4a in developing nephrons in mice, generating a novel model for FRTS. Immunofluorescence analysis characterized the mutant phenotype, and lineage analysis tested whether Cadherin-6 (Cdh6)-expressing cells are PT progenitors. Genome-wide mapping of Hnf4a binding sites and differential gene analysis of Hnf4a mutant kidneys identified direct target genes of Hnf4a. RESULTS: Deletion of Hnf4a with Osr2Cre led to the complete loss of mature PT cells, lethal to the Hnf4a mutant mice. Cdh6high, lotus tetragonolobus lectin-low (LTLlow) cells serve as PT progenitors and demonstrate higher proliferation than Cdh6low, LTLhigh differentiated PT cells. Additionally, Hnf4a is required for PT progenitors to differentiate into mature PT cells. Genomic analyses revealed that Hnf4a directly regulates the expression of genes involved in transmembrane transport and metabolism. CONCLUSIONS: Hnf4a promotes the differentiation of PT progenitors into mature PT cells by regulating the expression of genes associated with reabsorption, the major function of PT cells.
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Cadherinas/metabolismo , Factor Nuclear 4 del Hepatocito/genética , Factor Nuclear 4 del Hepatocito/metabolismo , Túbulos Renales Proximales/metabolismo , Lectinas/metabolismo , Células Madre/metabolismo , Animales , Cadherinas/genética , Diferenciación Celular/genética , Proliferación Celular , Modelos Animales de Enfermedad , Síndrome de Fanconi/genética , Femenino , Regulación de la Expresión Génica/genética , Ontología de Genes , Túbulos Renales Proximales/patología , Túbulos Renales Proximales/fisiopatología , Ratones , Ratones Noqueados , Fenotipo , Reabsorción Renal/genética , Células Madre/fisiologíaRESUMEN
Human respiratory syncytial virus (RSV) is the most common cause of viral bronchiolitis and pneumonia in infants and children worldwide. Inflammation induced by RSV infection is responsible for its hallmark manifestation of bronchiolitis and pneumonia. The cellular debris created through lytic cell death of infected cells is a potent initiator of this inflammation. Macrophages are known to play a pivotal role in the early innate immune and inflammatory response to viral pathogens. However, the lytic cell death mechanisms associated with RSV infection in macrophages remains unknown. Two distinct mechanisms involved in lytic cell death are pyroptosis and necroptosis. Our studies revealed that RSV induces lytic cell death in macrophages via both of these mechanisms, specifically through the ASC (Apoptosis-associated speck like protein containing a caspase recruitment domain)-NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3) inflammasome activation of both caspase-1 dependent pyroptosis and receptor-interacting serine/threonine-protein kinase 3 (RIPK3), as well as a mixed lineage kinase domain like pseudokinase (MLKL)-dependent necroptosis. In addition, we demonstrated an important role of reactive oxygen species (ROS) during lytic cell death of RSV-infected macrophages.
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Macrófagos/patología , Necroptosis , Piroptosis , Virus Sincitial Respiratorio Humano/patogenicidad , Proteínas Adaptadoras de Señalización CARD/metabolismo , Caspasa 1/metabolismo , Muerte Celular , Humanos , Inflamasomas/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Macrófagos/metabolismo , Macrófagos/virología , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteínas Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Células THP-1RESUMEN
INTRODUCTION: Autoantibody tests are commonly ordered when screening for rheumatic diseases. Rheumatoid factor (RF) and antinuclear antibody (ANA) have low positive predictive values in general practice. Overuse of diagnostic tests can result in an increase in unnecessary referrals, patient anxiety, and further costs. OBJECTIVE: The objective was to evaluate the utilization patterns, appropriateness, and associated costs of tests including ANA, extractable nuclear antibodies (ENA), anti-double stranded DNA (anti-dsDNA), RF, and HLA-B27 in patients referred to rheumatologists. METHODS: A review was conducted of consecutive referrals (accepted and rejected) using university rheumatologists' practices over one year. Inappropriate investigations, and associated costs were analyzed. Tests were considered appropriate if at least one criterion for a specific disease was provided. RESULTS: Of 638 referrals the most common reported reasons for referral were: spondyloarthropathies (SpA), rheumatoid arthritis (RA), and lupus (SLE). Prior to referral: 61% had undergone ANA testing at least once, ANA was repeated in one third; 19% had ENA and 21% had anti-dsDNA. 20% had ANA testing with no clinical indication. Half of ENA and anti-dsDNA testing was in the context of a negative ANA. RF was requested in 65% and in close to one third, there was no clinical suspicion of inflammatory arthritis. CONCLUSION: Despite the recommendations by CRA Choosing Wisely Campaign, at least 50% of laboratory investigations, including RF, ANA, ENA, and anti-dsDNA, are inappropriately ordered. More selective ordering of the above tests would lead to marked cost reduction.
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Enfermedades Reumáticas , Reumatólogos , Anticuerpos Antinucleares , Técnicas de Laboratorio Clínico , Humanos , Derivación y Consulta , Enfermedades Reumáticas/diagnósticoRESUMEN
MOTIVATION: Genome-wide association studies have revealed that 88% of disease-associated single-nucleotide polymorphisms (SNPs) reside in noncoding regions. However, noncoding SNPs remain understudied, partly because they are challenging to prioritize for experimental validation. To address this deficiency, we developed the SNP effect matrix pipeline (SEMpl). RESULTS: SEMpl estimates transcription factor-binding affinity by observing differences in chromatin immunoprecipitation followed by deep sequencing signal intensity for SNPs within functional transcription factor-binding sites (TFBSs) genome-wide. By cataloging the effects of every possible mutation within the TFBS motif, SEMpl can predict the consequences of SNPs to transcription factor binding. This knowledge can be used to identify potential disease-causing regulatory loci. AVAILABILITY AND IMPLEMENTATION: SEMpl is available from https://github.com/Boyle-Lab/SEM_CPP. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Estudio de Asociación del Genoma Completo , Polimorfismo de Nucleótido Simple , Sitios de Unión , Inmunoprecipitación de Cromatina , Unión Proteica , Factores de TranscripciónRESUMEN
Interspecific hybridization or barriers to hybridization may have contributed to the diversification of Antarctic icefishes (Channichthyidae), but data supporting these hypotheses is scarce. To understand the potential for hybridization and to investigate reproductive isolating mechanisms among icefish species, we performed in vitro fertilization experiments using eggs from a female blackfin icefish Chaenocephalus aceratus and sperm from a male of another genera, the ocellated icefish Chionodraco rastrospinosus. Sequencing of genomic and mitochondrial DNA confirmed the intergeneric hybrid nature of resulting embryos which successfully developed and hatched as active larvae at about four and a half months during the Antarctic winter. This result demonstrates the compatibility of gametes of these two species and the viability of resulting zygotes and larvae. Due to logistic constraints and the slow developmental rate of icefishes, we could not test for long-term hybrid viability, fertility, fitness, or hybrid breakdown. Analysis of our fishing records and available literature, however, suggests that the strongest barriers to hybridization among parapatric icefish species are likely to be behavioral and characterized by assortative mating and species-specific courtship and nesting behaviors. This conclusion suggests that, in long-lived fish species with late sexual maturity and high energetic investment in reproduction like icefishes, pre-mating barriers are energetically more efficient than post-mating barriers to prevent hybridization.
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Hibridación Genética , Perciformes/genética , Aislamiento Reproductivo , Animales , Ecosistema , Perciformes/fisiologíaRESUMEN
Toll-like receptor 2 (TLR2) is currently investigated as a potential therapeutic target in diseases with underlying inflammation like sepsis and arthritis. We reported the discovery, by virtual screening and biological testing, of eight TLR2 antagonists (AT1-AT8) which showed TLR2-inhibitory activity in human cells (Murgueitio et al., 2014). In this study, we have deepened in the mechanism of action and selectivity (TLR2/1 or TLR2/6) of those compounds in mouse primary cells and in vivo. The antagonists reduced, in a dose-dependent way the TNFα production (e.g. AT5 IC50 7.4 µM) and also reduced the nitric oxide (NO) formation in mouse bone marrow-derived macrophages (BMDM). Treatment of BMDM with the antagonists showed that downstream of TLR2, MAPKs phosphorylation and IkBα degradation was reduced. Notably, in a mouse model of tri-acylated lipopeptide (Pam3CSK4)-induced inflammation, AT5 attenuated the TNFα and IL-6 inflammatory response. Further, the effect of AT5 in the stimulation of BMDM by the endogenous alarmin HMGB1 was investigated. Our results indicate that AT4-AT7 and, particularly AT5 appear as good starting points for the development of inhibitors targeting TLR2 in inflammatory disorders.