RESUMEN
Bumble bees are an important group of insects that provide essential pollination services as a consequence of their foraging behaviors. These pollination services are driven, in part, by energetic exchanges between flowering plants and individual bees. Thus, it is important to examine bumble bee energy metabolism and explore how it might be influenced by external stressors contributing to declines in global pollinator populations. Two stressors that are commonly encountered by bees are insecticides, such as the neonicotinoids, and nutritional stress, resulting from deficits in pollen and nectar availability. Our study uses a metabolomic approach to examine the effects of neonicotinoid insecticide exposure on bumble bee metabolism, both alone and in combination with nutritional stress. We hypothesized that exposure to imidacloprid disrupts bumble bee energy metabolism, leading to changes in key metabolites involved in central carbon metabolism. We tested this by exposing Bombus impatiens workers to imidacloprid according to one of three exposure paradigms designed to explore how chronic versus more acute (early or late) imidacloprid exposure influences energy metabolite levels, then also subjecting them to artificial nectar starvation. The strongest effects of imidacloprid were observed when bees also experienced nectar starvation, suggesting a combinatorial effect of neonicotinoids and nutritional stress on bumble bee energy metabolism. Overall, this study provides important insights into the mechanisms underlying the impact of neonicotinoid insecticides on pollinators, and underscores the need for further investigation into the complex interactions between environmental stressors and energy metabolism.
Asunto(s)
Insecticidas , Nitrocompuestos , Abejas , Animales , Insecticidas/toxicidad , Néctar de las Plantas , Neonicotinoides/toxicidad , Polinización , Metabolismo EnergéticoRESUMEN
Cholesterol is an essential membrane structural component and steroid hormone precursor, and is involved in numerous signaling processes. Astrocytes regulate brain cholesterol homeostasis and they supply cholesterol to the needs of neurons. ATP-binding cassette transporter A1 (ABCA1) is the main cholesterol efflux transporter in astrocytes. Here we show dysregulated cholesterol homeostasis in astrocytes generated from human induced pluripotent stem cells (iPSCs) derived from males with fragile X syndrome (FXS), which is the most common cause of inherited intellectual disability. ABCA1 levels are reduced in FXS human and mouse astrocytes when compared with controls. Accumulation of cholesterol associates with increased desmosterol and polyunsaturated phospholipids in the lipidome of FXS mouse astrocytes. Abnormal astrocytic responses to cytokine exposure together with altered anti-inflammatory and cytokine profiles of human FXS astrocyte secretome suggest contribution of inflammatory factors to altered cholesterol homeostasis. Our results demonstrate changes of astrocytic lipid metabolism, which can critically regulate membrane properties and affect cholesterol transport in FXS astrocytes, providing target for therapy in FXS.
Asunto(s)
Síndrome del Cromosoma X Frágil , Células Madre Pluripotentes Inducidas , Masculino , Animales , Ratones , Humanos , Síndrome del Cromosoma X Frágil/genética , Astrocitos , Metabolismo de los Lípidos , Citocinas , HomeostasisRESUMEN
Ethiopian mustard (Brassica carinata) is an ancient crop with remarkable stress resilience and a desirable seed fatty acid profile for biofuel uses. Brassica carinata is one of six Brassica species that share three major genomes from three diploid species (AA, BB, and CC) that spontaneously hybridized in a pairwise manner to form three allotetraploid species (AABB, AACC, and BBCC). Of the genomes of these species, that of B. carinata is the least understood. Here, we report a chromosome scale 1.31-Gbp genome assembly with 156.9-fold sequencing coverage for B. carinata, completing the reference genomes comprising the classic Triangle of U, a classical theory of the evolutionary relationships among these six species. Our assembly provides insights into the hybridization event that led to the current B. carinata genome and the genomic features that gave rise to the superior agronomic traits of B. carinata. Notably, we identified an expansion of transcription factor networks and agronomically important gene families. Completion of the Triangle of U comparative genomics platform has allowed us to examine the dynamics of polyploid evolution and the role of subgenome dominance in the domestication and continuing agronomic improvement of B. carinata and other Brassica species.
Asunto(s)
Brassica , Brassica/genética , Tetraploidía , Genoma de Planta/genética , Poliploidía , DiploidiaRESUMEN
The filamentous fungus Neurospora crassa has historically been a model for understanding the relationship between genes and metabolism-auxotrophic mutants of N. crassa were used by Beadle and Tatum to develop the one-gene-one-enzyme hypothesis for which they earned the Nobel Prize in 1958. In the ensuing decades, several techniques have been developed for the systematic analysis of metabolites in N. crassa and other fungi. Untargeted and targeted approaches have been used, with a focus on secondary metabolites over primary metabolism. Here, we describe a pipeline for sample preparation, metabolite extraction, Liquid Chromatography-Mass Spectrometry (LC-MS), and data analysis that can be used for targeted metabolomics of primary metabolites in N. crassa. Liquid cultures are grown with shaking in a defined minimal medium and then collected using filtration. Samples are lyophilized for 2 days at -80°C, pulverized, and mixed with a solution to extract polar metabolites. The metabolites are separated and identified using LC-MS, with downstream analysis using Skyline interpretive software. Relative levels of hundreds of metabolites can be detected and compared across strains. © 2022 Wiley Periodicals LLC. Basic Protocol: Metabolite extraction and detection from Neurospora crassa cell cultures using Liquid Chromatography-Mass Spectrometry.
Asunto(s)
Neurospora crassa , Cromatografía Liquida/métodos , Metaboloma , Metabolómica/métodos , Espectrometría de Masas en TándemRESUMEN
A primary goal in biology is to understand the effects of multiple, interacting environmental stressors on organisms. Wild and domesticated bees are exposed to a wide variety of interacting biotic and abiotic stressors, with widespread declines in floral resources and agrochemical exposure being two of the most important. In this study, we used examinations of brain gene expression to explore the sublethal consequences of neonicotinoid pesticide exposure and pollen diet composition in nest-founding bumble bee queens. We demonstrate for the first time that pollen diet composition can influence the strength of bumble bee queen responses to pesticide exposure at the molecular level. Specifically, one pollen mixture in our study appeared to buffer bumble bee queens entirely against the effects of pesticide exposure, with respect to brain gene expression. Additionally, we detected unique effects of pollen diet and sustained (versus more temporary) pesticide exposure on queen gene expression. Our findings support the hypothesis that nutritional status can help buffer animals against the harmful effects of other stressors, including pesticides, and highlight the importance of using molecular approaches to explore sublethal consequences of stressors.
Asunto(s)
Plaguicidas , Animales , Abejas , Encéfalo , Dieta , Expresión Génica , Plaguicidas/análisis , Plaguicidas/toxicidad , Polen/químicaRESUMEN
The RAP (RNA-binding domain abundant in Apicomplexans) protein family has been identified in various organisms. Despite expansion of this protein family in apicomplexan parasites, their main biological functions remain unknown. In this study, we use inducible knockdown studies in the human malaria parasite, Plasmodium falciparum, to show that two RAP proteins, PF3D7_0105200 (PfRAP01) and PF3D7_1470600 (PfRAP21), are essential for parasite survival and localize to the mitochondrion. Using transcriptomics, metabolomics, and proteomics profiling experiments, we further demonstrate that these RAP proteins are involved in mitochondrial RNA metabolism. Using high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (eCLIP-seq), we validate that PfRAP01 and PfRAP21 are true RNA-binding proteins and interact specifically with mitochondrial rRNAs. Finally, mitochondrial enrichment experiments followed by deep sequencing of small RNAs demonstrate that PfRAP21 controls mitochondrial rRNA expression. Collectively, our results establish the role of these RAP proteins in mitoribosome activity and contribute to further understanding this protein family in malaria parasites.
Asunto(s)
Malaria Falciparum , Ribosomas Mitocondriales , Plasmodium falciparum , Proteínas Protozoarias , Proteínas de Unión al ARN , Genómica , Humanos , Malaria Falciparum/parasitología , Ribosomas Mitocondriales/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor that, upon activation by ligands, heterodimerizes with retinoid X receptor (RXR), binds to PPAR response elements (PPREs), and activates transcription of downstream genes. As PPARγ plays a central role in adipogenesis, fatty acid storage, and glucose metabolism, PPARγ-specific pharmaceuticals (e.g., thiazolidinediones) have been developed to treat Type II diabetes and obesity within human populations. However, to our knowledge, no prior studies have concurrently assessed the effects of PPARγ ligand exposure on genome-wide PPARγ binding as well as effects on the transcriptome and lipidome within human cells at biologically active, non-cytotoxic concentrations. In addition to quantifying concentration-dependent effects of ciglitazone (a reference PPARγ agonist) and GW 9662 (a reference PPARγ antagonist) on human hepatocarcinoma (HepG2) cell viability, PPARγ abundance in situ, and neutral lipids, HepG2 cells were exposed to either vehicle (0.1% DMSO), ciglitazone, or GW 9662 for up to 24 h, and then harvested for 1) chromatin immunoprecipitation-sequencing (ChIP-seq) to identify PPARγ-bound regions across the entire genome, 2) mRNA-sequencing (mRNA-seq) to identify potential impacts on the transcriptome, and 3) lipidomics to identify potential alterations in lipid profiles. Following exposure to ciglitazone and GW 9662, we found that PPARγ levels were not significantly different after 2-8 h of exposure. While ciglitazone and GW 9662 resulted in a concentration-dependent increase in neutral lipids, the magnitude and localization of PPARγ-bound regions across the genome (as identified by ChIP-seq) did not vary by treatment. However, mRNA-seq and lipidomics revealed that exposure of HepG2 cells to ciglitazone and GW 9662 resulted in significant, treatment-specific effects on the transcriptome and lipidome. Overall, our findings suggest that exposure of human cells to PPARγ ligands at biologically active, non-cytotoxic concentrations results in toxicity that may be driven by a combination of both PPARγ-dependent and PPARγ-independent mechanisms.
RESUMEN
Mechanistic understanding of the interaction of copper-based nanomaterials with crops is crucial for exploring their application in precision agriculture and their implications on plant health. We investigated the biological response of soybean (Glycine max) plants to the foliar application of copper hydroxide nanowires (CNWs) at realistic exposure concentrations. A commercial copper based-fungicide (Kocide), dissolved copper ions, and untreated controls were used for comparison to identify unique features at physiological, cellular, and molecular levels. After 32 d of exposure to CNW (0.36, 1.8, and 9 mg CNW/plant), the newly developed tissues accumulated significantly high levels of Cu (18-60 µg/g) compared to Kocide (10 µg/g); however, the rate of Cu translocation from the site of CNW treatment to other tissues was slower compared to other Cu treatments. Like Kocide, CNW exposure at medium and high doses altered Co, Mn, Zn, and Fe accumulation in the tissues and enhanced photosynthetic activities. The proteomic and metabolomic analyses of leaves from CNW-treated soybean plants suggest a dose-dependent response, resulting in the activation of major biological processes, including photosynthesis, energy production, fatty acid metabolism, lignin biosynthesis, and carbohydrate metabolism. In contrast to CNW treatments, Kocide exposure resulted in increased oxidative stress response and amino acid metabolism activation.
Asunto(s)
Cobre , Nanocables , Cobre/toxicidad , Fertilizantes , Hidróxidos , Proteómica , Glycine maxRESUMEN
Metabolite damage control is a critical but poorly defined aspect of cellular biochemistry, which likely involves many of the so far functionally uncharacterized protein domain (domains of unknown function; DUFs). We have determined the crystal structure of the human DUF89 protein product of the C6ORF211 gene to 1.85 Å. The crystal structure shows that the protein contains a core α-ß-α fold with an active site-bound metal ion and α-helical bundle N-terminal cap, which are both conserved features of subfamily III DUF89 domains. The biochemical activities of the human protein are conserved with those of a previously characterized budding yeast homolog, where an in vitro phosphatase activity is supported by divalent cations that include Co2+, Ni2+, Mn2+ or Mg2+. Full steady-state kinetics parameters of human DUF89 using a standard PNPP phosphatase assay revealed a six times higher catalytic efficiency in presence of Co2+ compared to Mg2+. The human enzyme targets a number of phosphate substrates similar to the budding yeast homolog, while it lacks a previously indicated methyltransferase activity. The highest activity on substrate was observed with fructose-1-phosphate, a potent glycating agent, and thus human DUF89 phosphatase activity may also play a role in limiting the buildup of phospho-glycan species and their related damaged metabolites.
Asunto(s)
Monoéster Fosfórico Hidrolasas/metabolismo , Proteína O-Metiltransferasa/metabolismo , Especificidad por Sustrato/fisiología , Sitios de Unión/fisiología , Catálisis , Humanos , Cinética , Metales/metabolismo , Polisacáridos/metabolismo , Conformación Proteica , Saccharomyces cerevisiae/metabolismoRESUMEN
Hibernators have adapted a physiological mechanism allowing them to undergo long periods of inactivity without experiencing bone loss. However, the biological mechanisms that prevent bone loss are unknown. Previous studies found meaningful changes, between active and hibernating marmots, in the endocannabinoid system of many tissues, including bone. Cannabinoid receptors (CB1 and CB2) have divergent localization in bone. CB1 is predominately found on sympathetic nerve terminals, while CB2 is more abundant on bone cells and their progenitors. This study aimed to determine the contribution of innervation on endocannabinoid regulation of bone properties in hibernating (during torpor) and non-hibernating yellow-bellied marmots. Neurectomy, a model for disuse osteoporosis, was performed unilaterally in both hibernating and active marmots. Endocannabinoid concentrations were measured in bone marrow, cortical, and trabecular regions from fourth metatarsals of both hindlimbs using microflow chromatography-tandem quadrupole mass spectrometry. Trabecular bone architectural properties of fifth metatarsals were evaluated using micro-computed tomography. There were ligand-specific increases with neurectomy in active, but not hibernating, marmots. Trabecular bone architectural properties were not affected by neurectomy during hibernation, but did show some minor negative changes in active marmots. These findings suggest protection from bone loss in hibernating rodents is peripherally rather than centrally regulated. Furthermore, findings suggest even active marmots with normal metabolism are partially protected from disuse induced bone loss compared to laboratory rodents. Understanding the mechanism hibernators use to maintain bone density may guide development for novel bone loss prevention therapies.
Asunto(s)
Endocannabinoides/metabolismo , Marmota/fisiología , Animales , Densidad Ósea , Resorción Ósea/metabolismo , Desnervación , Femenino , Hibernación/fisiología , Masculino , Marmota/metabolismoRESUMEN
Estrogen decline during menopause is associated with altered metabolism, weight gain and increased risk of cardiometabolic diseases. The gut microbiota also plays a role in the development of cardiometabolic dysfunction and is also subject to changes associated with age-related hormone changes. Phytoestrogens are plant-based estrogen mimics that have gained popularity as dietary supplements for the treatment or prevention of menopause-related symptoms. These compounds have the potential to both modulate and be metabolized by the gut microbiota. Hops (Humulus lupulus L.) contain potent phytoestrogen precursors, which rely on microbial biotransformation in the gut to estrogenic forms. We supplemented ovariectomized (OVX) or sham-operated (SHAM) C57BL/6 mice, with oral estradiol (E2), a flavonoid-rich extract from hops, or a placebo carrier oil, to observe effects on adiposity, inflammation, and gut bacteria composition. Hops extract (HE) and E2 protected against increased visceral adiposity and liver triglyceride accumulation in OVX animals. Surprisingly, we found no evidence of OVX having a significant impact on the overall gut bacterial community structure. We did find differences in the abundance of Akkermansia muciniphila, which was lower with HE treatment in the SHAM group relative to OVX E2 treatment and to placebo in the SHAM group.
Asunto(s)
Estrógenos/farmacología , Flavonoides/farmacología , Microbioma Gastrointestinal , Humulus/química , Extractos Vegetales/farmacología , Adiposidad/efectos de los fármacos , Akkermansia , Animales , Suplementos Dietéticos/microbiología , Estradiol/farmacología , Femenino , Flavanonas , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Ovariectomía , Fitoestrógenos/farmacología , Triglicéridos/metabolismo , Verrucomicrobia/efectos de los fármacos , Aumento de Peso/efectos de los fármacosRESUMEN
Honey bees are important insect pollinators used heavily in agriculture and can be found in diverse environments. Bees may encounter toxicants such as cadmium and selenate by foraging on plants growing in contaminated areas, which can result in negative health effects. Honey bees are known to have a simple and consistent microbiome that conveys many benefits to the host, and toxicant exposure may impact this symbiotic microbial community. We used 16S rRNA gene sequencing to assay the effects that sublethal cadmium and selenate treatments had over 7 days and found that both treatments significantly but subtly altered the composition of the bee microbiome. Next, we exposed bees to cadmium and selenate and then used untargeted liquid chromatography-mass spectrometry (LC-MS) metabolomics to show that chemical exposure changed the bees' metabolite profiles and that compounds which may be involved in detoxification, proteolysis, and lipolysis were more abundant in treatments. Finally, we exposed several strains of bee-associated bacteria in liquid culture and found that each strain removed cadmium from its medium but that only Lactobacillus Firm-5 microbes assimilated selenate, indicating the possibility that these microbes may reduce the metal and metalloid burden on their host. Overall, our report shows that metal and metalloid exposure can affect the honey bee microbiome and metabolome and that strains of bee-associated bacteria can bioaccumulate these toxicants.IMPORTANCE Bees are important insect pollinators that may encounter environmental pollution when foraging upon plants grown in contaminated areas. Despite the pervasiveness of pollution, little is known about the effects of these toxicants on honey bee metabolism and their symbiotic microbiomes. Here, we investigated the impact of selenate and cadmium exposure on the gut microbiome and metabolome of honey bees. We found that exposure to these chemicals subtly altered the overall composition of the bees' microbiome and metabolome and that exposure to toxicants may negatively impact both host and microbe. As the microbiome of animals can reduce mortality upon metal or metalloid challenge, we grew bee-associated bacteria in media spiked with selenate or cadmium. We show that some bacteria can remove these toxicants from their media in vitro and suggest that bacteria may reduce metal burden in their hosts.
Asunto(s)
Bacterias/metabolismo , Abejas/microbiología , Bioacumulación/fisiología , Cadmio/metabolismo , Microbioma Gastrointestinal/fisiología , Ácido Selénico/metabolismo , Animales , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , Abejas/efectos de los fármacos , Biodiversidad , Cadmio/toxicidad , Farmacorresistencia Bacteriana/efectos de los fármacos , Microbioma Gastrointestinal/efectos de los fármacos , Microbioma Gastrointestinal/genética , Lactobacillus/genética , Lactobacillus/metabolismo , Metaboloma , Plantas , Polinización , ARN Ribosómico 16S/metabolismo , Ácido Selénico/toxicidad , SimbiosisRESUMEN
Niclosamide is an antihelminthic drug used worldwide for the treatment of tapeworm infections. Recent drug repurposing screens have highlighted the broad bioactivity of niclosamide across diverse mechanisms of action. As a result, niclosamide is being evaluated for a range of alternative drug-repurposing applications, including the treatment of cancer, bacterial infections, and Zika virus. As new applications of niclosamide will require non-oral delivery routes that may lead to exposure in utero, it is important to understand the mechanism of niclosamide toxicity during early stages of embryonic development. Previously, we showed that niclosamide induces a concentration-dependent delay in epiboly progression in the absence of effects on oxidative phosphorylation - a well-established target for niclosamide. Therefore, the overall objective of this study was to further examine the mechanism of niclosamide-induced epiboly delay during zebrafish embryogenesis. Based on this study, we found that (1) niclosamide exposure during early zebrafish embryogenesis resulted in a decrease in yolk sac integrity with a concomitant decrease in the presence of yolk sac actin networks and increase in cell size; (2) within whole embryos, niclosamide exposure did not alter non-polar metabolites and lipids, but significantly altered amino acids specific to aminoacyl-tRNA biosynthesis; (3) niclosamide significantly altered transcripts related to translation, transcription, and mRNA processing pathways; and (4) niclosamide did not significantly alter levels of rRNA and tRNA. Overall, our findings suggest that niclosamide may be causing a systemic delay in embryonic development by disrupting the translation of maternally-supplied mRNAs, an effect that may be mediated through disruption of aminoacyl-tRNA biosynthesis.
Asunto(s)
Antihelmínticos/toxicidad , Embrión no Mamífero/efectos de los fármacos , Desarrollo Embrionario/efectos de los fármacos , Niclosamida/toxicidad , Pez Cebra/metabolismo , Animales , Línea Celular , Embrión no Mamífero/metabolismo , Humanos , Metabolómica , ARN/metabolismo , Saco Vitelino/efectos de los fármacos , Saco Vitelino/metabolismo , Pez Cebra/genética , CigotoRESUMEN
Triphenyl phosphate (TPHP) is a commonly used organophosphate flame retardant and plasticizer in the United States. Using zebrafish as a model, the overall objective of this study was to identify potential organs that might be targeted by TPHP during embryonic development. Based on mRNA-sequencing, TPHP exposure from 24 to 30 h post fertilization (hpf) and 24 to 48 hpf significantly affected the abundance of 305 and 274 transcripts, respectively, relative to vehicle (0.1% DMSO) controls. In addition to minor effects on cardiotoxicity- and nephrotoxicity-related pathways, ingenuity pathway analysis (IPA) of significantly affected transcripts within 30- and 48-hpf embryos revealed that hepatotoxicity-related pathways were strongly affected following exposure to TPHP-alone. Moreover, although pretreatment with fenretinide (a retinoic acid receptor agonist) mitigated TPHP-induced pericardial edema and liver enlargement at 72 and 128 hpf, respectively, IPA revealed that fenretinide was unable to block TPHP-induced effects on cardiotoxicity-, nephrotoxicity-, and hepatotoxicity-related pathways at 48 hpf, suggesting that TPHP-induced effects on the transcriptome were not associated with toxicity later in development. In addition, based on Oil Red O staining, we found that exposure to TPHP nearly abolished neutral lipids from the embryonic head and trunk and, based on metabolomics, significantly decreased the total abundance of metabolites-including betaine, a known osmoprotectant-at 48 and 72 hpf. Overall, our data suggest that, in addition to the heart, TPHP exposure during early development results in adverse effects on the liver, lipid utilization, and osmoregulation within embryonic zebrafish.
RESUMEN
Plant development, growth, and adaptation to stress are regulated by phytohormones, which can influence physiology even at low concentrations. Phytohormones are chemically grouped according to both structure and function as auxins, cytokinins, abscisic acid, jasmonates, salicylates, gibberellins, and brassinosteroids, among others. This chemical diversity and requirement for highly sensitive detection in complex matrices create unique challenges for comprehensive phytohormone analysis. Here, we present a robust and efficient quantitative UPLC-MS/MS assay for 17 phytohormones, including jasmonates, salicylates, abscisic acid, gibberellins, cytokinins, and auxins. Using this assay, 12 phytohormones were detected and quantified in sorghum plant tissue without the need for solid phase extraction (SPE) or liquid-liquid extraction. Variation of phytohormone profiles was explored in both root and leaf tissues between three genotypes, harvested at two different developmental time points. The results highlight the importance of tissue type, sampling time, and genetic factors when designing experiments that involve phytohormone analysis of sorghum. This research lays the groundwork for future studies, which can combine phytohormone profiling with other datasets such as transcriptome, soil microbiome, genome, and metabolome data, to provide important functional information about adaptation to stress and other environmental variables.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Reguladores del Crecimiento de las Plantas/análisis , Hojas de la Planta/química , Raíces de Plantas/química , Sorghum/química , Espectrometría de Masas en Tándem/métodosRESUMEN
Domoic acid (DA) is a marine neurotoxin produced by several species of Pseudo-nitzschia. DA causes severe neurological toxicity in humans and animals. To address the current analytical need to quantify low levels of DA in human and animal body fluids, a sensitive and selective high performance liquid chromatography-tandem mass spectrometry method was developed to measure DA in plasma and urine. This method was fully validated to accurately and precisely quantify DA between 0.31 and 16 ng/mL in plasma and between 7.8 and 1000 ng/mL in urine. Our group introduced the use of a novel internal standard, tetrahydrodomoic acid to control for matrix effects and other sources of variability. This validated method will be useful to assess DA concentrations in biological samples of human or animal origin after suspected DA exposure from contaminated food. It will also be applicable to sentinel programs and research studies to analyze body fluids with low levels of DA.
RESUMEN
Metabolomics is an "omics" approach to quantitatively measure a large set of metabolites. In this chapter, we describe an example method for performing liquid chromatography coupled to mass spectrometry (LC-MS)-based untargeted metabolomics on a cell extract from Vibrio cholerae.
Asunto(s)
Metaboloma , Metabolómica , Vibrio cholerae/metabolismo , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Metabolómica/métodos , Espectrometría de Masas en TándemRESUMEN
Hibernation is a naturally occurring model for studying diseases such as obesity and osteoporosis. Hibernators, marmots (Marmota flaviventris) among them, are able to nearly double their body mass by increasing fat stores prior to hibernation without the negative consequences of obesity. They are also physically inactive for extended periods of time without experiencing negative effects on the skeleton. The endocannabinoid system is involved in modulating neural signaling, circannual rhythms, behavior, appetite, thermogenesis, and bone and energy metabolism. These systems are also altered to maintain homeostasis during hibernation. This study aims to better understand the involvement of the endocannabinoid system in the regulation of physiological processes during hibernation by quantifying the seasonal variation of endocannabinoids and endocannabinoid-like ligands in both active and hibernating marmots. We hypothesized that there would be significant changes in endocannabinoid concentrations at the tissue level in marmots between active and hibernating states. Concentrations were measured in brain, serum, brown adipose tissue, white adipose tissue, bone marrow, cortical bone, and trabecular bone using microflow chromatography coupled with tandem quadrupole mass spectrometry. Significant changes were found, such as a 30-fold decrease in 2-arachidonoyl glycerol (2-AG) in cortical bone during hibernation. Many endocannabinoid and endocannabinoid-like ligands decreased in brown adipose tissue, white adipose tissue, and cortical bone, while several ligands increased in bone marrow. This result supports our hypothesis and suggests the possibility of a peripherally controlled shift in energy metabolism, reduction in bone metabolism, and suppression of the immune system during hibernation.
Asunto(s)
Ritmo Circadiano/fisiología , Endocannabinoides/análisis , Metabolismo Energético/fisiología , Hibernación/fisiología , Marmota/fisiología , Estaciones del Año , Tejido Adiposo/química , Animales , Temperatura Corporal , Médula Ósea/química , Huesos/química , Femenino , MasculinoRESUMEN
Background: Cannabis is a widely used drug in the United States, and the frequency of cannabis use in the human immunodeficiency virus (HIV)-infected population is disproportionately high. Previous human and macaque studies suggest that cannabis may have an impact on plasma viral load; however, the relationship between cannabis use and HIV-associated systemic inflammation and immune activation has not been well defined. Methods: The impact of cannabis use on peripheral immune cell frequency, activation, and function was assessed in 198 HIV-infected, antiretroviral-treated individuals by flow cytometry. Individuals were categorized into heavy, medium, or occasional cannabis users or noncannabis users based on the amount of the cannabis metabolite 11-nor-carboxy-tetrahydrocannabinol (THC-COOH) detected in plasma by mass spectrometry. Results: Heavy cannabis users had decreased frequencies of human leukocyte antigen (HLA)-DR+CD38+CD4+ and CD8+ T-cell frequencies, compared to frequencies of these cells in non-cannabis-using individuals. Heavy cannabis users had decreased frequencies of intermediate and nonclassical monocyte subsets, as well as decreased frequencies of interleukin 23- and tumor necrosis factor-α-producing antigen-presenting cells. Conclusions: While the clinical implications are unclear, our findings suggest that cannabis use is associated with a potentially beneficial reduction in systemic inflammation and immune activation in the context of antiretroviral-treated HIV infection.
Asunto(s)
Fármacos Anti-VIH/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Inmunidad Innata/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Abuso de Marihuana/inmunología , Terapia Antirretroviral Altamente Activa , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD8-positivos/efectos de los fármacos , Dronabinol/análogos & derivados , Dronabinol/sangre , Femenino , Citometría de Flujo , Humanos , Inflamación , Masculino , Persona de Mediana Edad , Monocitos/efectos de los fármacos , Carga Viral/efectos de los fármacosRESUMEN
Gram-negative bacterial pathogens of plants and animals employ type III secreted effectors to suppress innate immunity. Most characterized effectors work through modification of host proteins or transcriptional regulators, although a few are known to modify small molecule targets. The Xanthomonas type III secreted avirulence factor AvrRxo1 is a structural homolog of the zeta toxin family of sugar-nucleotide kinases that suppresses bacterial growth. AvrRxo1 was recently reported to phosphorylate the central metabolite and signaling molecule NAD in vitro, suggesting that the effector might enhance bacterial virulence on plants through manipulation of primary metabolic pathways. In this study, we determine that AvrRxo1 phosphorylates NAD in planta, and that its kinase catalytic sites are necessary for its toxic and resistance-triggering phenotypes. A global metabolomics approach was used to independently identify 3'-NADP as the sole detectable product of AvrRxo1 expression in yeast and bacteria, and NAD kinase activity was confirmed in vitro. 3'-NADP accumulated upon transient expression of AvrRxo1 in Nicotiana benthamiana and in rice leaves infected with avrRxo1-expressing strains of X. oryzae. Mutation of the catalytic aspartic acid residue D193 abolished AvrRxo1 kinase activity and several phenotypes of AvrRxo1, including toxicity in yeast, bacteria, and plants, suppression of the flg22-triggered ROS burst, and ability to trigger an R gene-mediated hypersensitive response. A mutation in the Walker A ATP-binding motif abolished the toxicity of AvrRxo1, but did not abolish the 3'-NADP production, virulence enhancement, ROS suppression, or HR-triggering phenotypes of AvrRxo1. These results demonstrate that a type III effector targets the central metabolite and redox carrier NAD in planta, and that this catalytic activity is required for toxicity and suppression of the ROS burst.