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Right ventricular (RV) failure remains the strongest determinant of survival in pulmonary hypertension (PH). We aimed to identify relevant mechanisms, beyond pressure overload, associated with maladaptive RV hypertrophy in PH. To separate the effect of pressure overload from other potential mechanisms, we developed in pigs two experimental models of PH (M1, by pulmonary vein banding and M2, by aorto-pulmonary shunting) and compared them with a model of pure pressure overload (M3, pulmonary artery banding) and a sham-operated group. Animals were assessed at 1 and 8 months by right heart catheterization, cardiac magnetic resonance and blood sampling, and myocardial tissue was analyzed. Plasma unbiased proteomic and metabolomic data were compared among groups and integrated by an interaction network analysis. A total of 33 pigs completed follow-up (M1, n = 8; M2, n = 6; M3, n = 10; and M0, n = 9). M1 and M2 animals developed PH and reduced RV systolic function, whereas animals in M3 showed increased RV systolic pressure but maintained normal function. Significant plasma arginine and histidine deficiency and complement system activation were observed in both PH models (M1&M2), with additional alterations to taurine and purine pathways in M2. Changes in lipid metabolism were very remarkable, particularly the elevation of free fatty acids in M2. In the integrative analysis, arginine-histidine-purines deficiency, complement activation, and fatty acid accumulation were significantly associated with maladaptive RV hypertrophy. Our study integrating imaging and omics in large-animal experimental models demonstrates that, beyond pressure overload, metabolic alterations play a relevant role in RV dysfunction in PH.
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Modelos Animais de Doenças , Hipertensão Pulmonar , Hipertrofia Ventricular Direita , Metabolômica , Proteômica , Animais , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/fisiopatologia , Hipertensão Pulmonar/diagnóstico por imagem , Hipertrofia Ventricular Direita/metabolismo , Hipertrofia Ventricular Direita/fisiopatologia , Hipertrofia Ventricular Direita/diagnóstico por imagem , Função Ventricular Direita , Remodelação Ventricular , Sus scrofa , Suínos , MasculinoRESUMO
This study aimed to elucidate the genetic causes underlying the juvenile parkinsonism (JP) diagnosed in a girl with several family members diagnosed with spinocerebellar ataxia type 2 (SCA2). To achieve this, whole-exome sequencing, analysis of CAG repeats, RNA sequencing analysis on fibroblasts, and metabolite identification were performed. As a result, a homozygous missense mutation SNP T>C (rs2254562) in synaptojamin 1 (SYNJ1), which has been implicated in the regulation of membrane trafficking in the synaptic vesicles, was identified. Additionally, we observed overexpression of L1 cell adhesion molecule (L1CAM), Cdc37, GPX1, and GPX4 and lower expression of ceruloplasmin in the patient compared to the control. We also found changes in sphingolipid, inositol, and inositol phosphate metabolism. These findings help to clarify the mechanisms of JP and suggest that the etiology of JP in the patient may be multifactorial. This is the first report of the rs2254562 mutation in the SYNJ gene identified in a JP patient with seizures and cognitive impairment.
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Transtornos Parkinsonianos , Humanos , Feminino , Transtornos Parkinsonianos/genética , Mutação de Sentido Incorreto , Sequenciamento do Exoma , Linhagem , Polimorfismo de Nucleotídeo Único , Proteínas do Tecido Nervoso/genética , Criança , MultiômicaRESUMO
Lipids are involved in many biological processes and their study is constantly increasing. To identify a lipid among thousand requires of reliable methods and techniques. Ion Mobility (IM) can be coupled with Mass Spectrometry (MS) to increase analytical selectivity in lipid analysis of lipids. IM-MS has experienced an enormous development in several aspects, including instrumentation, sensitivity, amount of information collected and lipid identification capabilities. This review summarizes the latest developments in IM-MS analyses for lipidomics and focuses on the current acquisition modes in IM-MS, the approaches for the pre-treatment of the acquired data and the subsequent data analysis. Methods and tools for the calculation of Collision Cross Section (CCS) values of analytes are also reviewed. CCS values are commonly studied to support the identification of lipids, providing a quasi-orthogonal property that increases the confidence level in the annotation of compounds and can be matched in CCS databases. The information contained in this review might be of help to new users of IM-MS to decide the adequate instrumentation and software to perform IM-MS experiments for lipid analyses, but also for other experienced researchers that can reconsider their routines and protocols.
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Lipidômica , Lipídeos , Bases de Dados Factuais , Espectrometria de Mobilidade Iônica/métodos , Lipídeos/análise , Espectrometria de Massas/métodosRESUMO
Ataxia in children is a common clinical sign of numerous neurological disorders consisting of impaired coordination of voluntary muscle movement. Its most common form, cerebellar ataxia, describes a heterogeneous array of neurologic conditions with uncountable causes broadly divided as acquired or genetic. Numerous genetic disorders are associated with chronic progressive ataxia, which complicates clinical management, particularly on the diagnostic stage. Advances in omics technologies enable improvements in clinical practice and research, so we proposed a multi-omics approach to aid in the genetic diagnosis and molecular elucidation of an undiagnosed infantile condition of chronic progressive cerebellar ataxia. Using whole-exome sequencing, RNA-seq, and untargeted metabolomics, we identified three clinically relevant mutations (rs141471029, rs191582628 and rs398124292) and an altered metabolic profile in our patient. Two POLR1C diagnostic variants already classified as pathogenic were found, and a diagnosis of hypomyelinating leukodystrophy was achieved. A mutation on the MMACHC gene, known to be associated with methylmalonic aciduria and homocystinuria cblC type, was also found. Additionally, preliminary metabolome analysis revealed alterations in our patient's amino acid, fatty acid and carbohydrate metabolism. Our findings provided a definitive genetic diagnosis reinforcing the association between POLR1C mutations and hypomyelinating leukodystrophy and highlighted the relevance of multi-omics approaches to the disease.
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Ataxia Cerebelar/diagnóstico , RNA Polimerases Dirigidas por DNA/genética , Genoma/genética , Oxirredutases/genética , Transcriptoma/genética , Adolescente , Adulto , Ataxia Cerebelar/genética , Ataxia Cerebelar/patologia , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Metaboloma/genética , Mutação/genética , Linhagem , RNA-Seq , Deficiência de Vitamina B 12/genética , Sequenciamento do Exoma/métodos , Adulto JovemRESUMO
A breadboard approach for electrophoretic separations with contactless conductivity detection is presented. This is based on miniature off-the-shelf components such as syringe pumps, valves, and pressure controllers which could be set up in a very compact overall arrangement. It has a high flexibility for different tasks at hand, and the common operations of hydrodynamic injection and capillary flushing are automated. For demonstration of the versatility of the proposition, several very diverse configurations and modes of electrophoresis were successfully implemented, namely, standard capillary zone electrophoresis, pressure assisted zone electrophoresis, the simultaneous separation of cations and anions by dual-capillary zone electrophoresis, the separation of cationic amino acids by isotachophoresis, as well as the separation of small carboxylic acids by gradient elution moving boundary electrophoresis. The system also allows fast separations, as demonstrated by the analysis of six inorganic cations within 35 s. The approach addresses respective limitations of either conventional capillary electrophoresis instruments as well as electrophoretic lab-on-chip devices, while maintaining a performance in terms of detection limits and reproducibility comparable to standard instrumentation.
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A CE with contactless conductivity detection methodology using a novel background electrolyte for the separation and determination of 17 metal cations (Cs+ , Rb+ , K+ , Ca2+ , Na+ , Mg2+ , Mn2+ , Sr2+ , Li+ , Ba2+ , Fe2+ , Pb2+ , Cd2+ , Zn2+ , Co2+ , Cu2+ and Ni2+ ) and ammonium has been investigated. The buffer, based on lactic acid and ß-alanine, was experimentally compared with other two commonly used electrolytes, showing important improvements, such as shorter analysis times (<11 min), better electrophoretic resolutions and higher detectabilities for certain analytes, such as Fe2+ and Pb2+ . The inclusion of other additives such as 18-Crown-6 and α-hydroxyisobutyric acid was studied in order to obtain the best separation of the analytes of interest. The optimised method was applied to the analysis of 11 water-based pen inks and the determination of their metal composition. The methodology was demonstrated for the comparison and differentiation of pen inks.
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Cátions/análise , Eletroforese Capilar/métodos , Tinta , Condutividade Elétrica , Limite de Detecção , Modelos Lineares , Metais , Reprodutibilidade dos TestesAssuntos
Metabolômica , Animais , Cromatografia Gasosa , Eletroforese Capilar , Humanos , Espectrometria de MassasRESUMO
A novel micro-injector for capillary electrophoresis for the handling of samples with volumes down to as little as 300 nL was designed and built in our laboratory for analyses in which the available volume is a limitation. The sample is placed into a small cavity located directly in front of the separation capillary, and the injection is then carried out automatically by controlled pressurization of the chamber with compressed air. The system also allows automated flushing of the injection chamber as well as of the capillary. In a trial with a capillary electrophoresis system with contactless conductivity detector, employing a capillary of 25 µm diameter, the results showed good stability of migration times and peak areas. To illustrate the technique, the fast separation of five inorganic cations (Na(+) , K(+) , NH4 (+) , Ca(2+) , and Mg(2+) ) was set up. This could be achieved in less than 3 min, with good limits of detection (10 µM) and linear ranges (between about 10 and 1000 µM). The system was demonstrated for the determination of the inorganic cations in porewater samples of a lake sediment core.
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Eletroforese Capilar/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Cátions/análise , Desenho de Equipamento , Sedimentos Geológicos/química , Lagos/química , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos Testes , Água/químicaRESUMO
Scientists often need to build dedicated computer-controlled experimental systems. For this purpose, it is becoming common to employ open-source microcontroller platforms, such as the Arduino. These boards and associated integrated software development environments provide affordable yet powerful solutions for the implementation of hardware control of transducers and acquisition of signals from detectors and sensors. It is, however, a challenge to write programs that allow interactive use of such arrangements from a personal computer. This task is particularly complex if some of the included hardware components are connected directly to the computer and not via the microcontroller. A graphical user interface framework, Instrumentino, was therefore developed to allow the creation of control programs for complex systems with minimal programming effort. By writing a single code file, a powerful custom user interface is generated, which enables the automatic running of elaborate operation sequences and observation of acquired experimental data in real time. The framework, which is written in Python, allows extension by users, and is made available as an open source project.
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A simple and inexpensive method for the identification of four substituted amphetamines, namely, 3,4-methylenedioxy methamphetamine (MDMA), methamphetamine (MA), 3,4-methylenedioxy amphetamine (MDA) and 3,4-methylenedioxy-N-ethylamphetamine (MDEA) was developed using an in-house constructed semi-automated portable capillary electrophoresis instrument (CE) with capacitively coupled contactless conductivity detection (C(4)D). Arginine 10mM adjusted to pH4.5 with acetic acid was found to be the optimal background electrolyte for the CE-C(4)D determination of these compounds. The best detection limits achieved with and without a sample preconcentration process were 10ppb and 500ppb, respectively. Substituted amphetamines were found in different seized illicit club drug tablets and urine samples collected from different suspected users. Good agreement between results from CE-C(4)D and those with the confirmation method (GC-MS) was achieved, with correlation coefficients for the two pairs of data of more than 0.99.
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Anfetaminas/isolamento & purificação , Estimulantes do Sistema Nervoso Central/isolamento & purificação , Drogas Ilícitas/isolamento & purificação , Anfetaminas/química , Estimulantes do Sistema Nervoso Central/química , Condutividade Elétrica , Eletroforese Capilar/métodos , Humanos , Drogas Ilícitas/química , Comprimidos/químicaRESUMO
Over the last few years, there has been an increase in the reports of drug-facilitated crimes. The list of drugs associated with these crimes is extensive and benzodiazepines constitute one of the groups of substances more commonly used. The sedative properties, which characterize benzodiazepines, are enhanced when such drugs are combined with alcohol, being more attractive for committing these types of crimes. In this work, a capillary electrophoresis method was applied to the analysis of 63 different samples of club drinks spiked with benzodiazepine tablets. The resulting electropherograms were processed and analyzed with the chemometric multivariate techniques: principal component analysis (PCA) and soft independent modeling of class analogies (SIMCA) classification. The PCA results allowed a clear differentiation of each drug class in a 3D plot. In addition, the SIMCA classification model (5% significance level) showed that eight out of nine test samples were automatically assigned by software to their proper sample class. The conflicting sample was correctly classified in the Coomans' plot (95% confidence). This novel approach based on the comparison of electrophoretic profiles of spiked drinks by chemometric tools allows determining the benzodiazepine used for drink spiking without the use of drug standards. Moreover, it provides an opportunity for the forensic laboratories to incorporate the identification capability provided by the electrophoretic fingerprinting of benzodiazepine solutions in existing or new databases.
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Benzodiazepinas/química , Benzodiazepinas/classificação , Ciências Forenses/métodos , Bebidas Alcoólicas/análise , Bebidas Gaseificadas/análise , Análise de Componente Principal , Comprimidos/químicaRESUMO
Consumer fireworks are a heterogeneous group of pyrotechnic items widely used by citizens around the world. There are a wide number of forensic cases related to consumer fireworks that require knowing their chemical composition and variety of designs to conduct accurate and comprehensive analyses. In this research paper, a selection of six consumer firework types (firecracker, rocket, pyrotechnic fountain, pyrotechnic battery, sparkler, and smoke bomb) is physically described and their anionic compositions are determined. Preblast (fuses and charges) samples and postblast residues of the different consumer fireworks were analyzed by CE in order to determine their anionic composition. Different types of chemical compositions in fuses and pyrotechnic charges were determined, although they were not related to any type of item. Additionally, several discrepancies were found between the analytical results and the declared item compositions. Regarding postblast residues, a huge variety of anions were identified and attributed to some unconsumed starting materials and different chemical reactions occurring during combustion.
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Ânions/análise , Eletroforese Capilar/métodos , Explosões , Ciências Forenses/métodos , Substâncias ExplosivasRESUMO
Acid sphingomyelinase deficiency is a neurodegenerative lysosomal storage disorder caused by mutations in the sphingomyelin-degrading enzyme acid sphingomyelinase (ASM) gene. Upregulated neuroinflammation has been well-characterized in an ASM knockout mouse model of acid sphingomyelinase deficiency disease, but lipid mediator pathways involved in 'mediating' inflammation and inflammation-resolution have yet to be characterized. In this study, we 1) measured free (bioactive) and esterified (inactive) lipid mediators involved in inflammation and inflammation resolution in cerebellum and neuronal cultures of ASM knockout (ASMko) mice and wildtype (WT) controls, and 2) quantified the esterification of labeled pro-resolving free d11-14(15)-epoxyeicosatrienoic acid in cultured neurons from ASMko and WT mice. We found elevated concentrations of esterified pro-resolving lipid mediators and hydroxyeicosatrienoic acids typically destined for pro-resolving lipid mediator synthesis (e.g. lipoxins) in the cerebellum and neurons of ASMko mice compared to controls. Free d11-14(15)-epoxyeicosatrienoic acid esterification within neurons of ASMko mice was significantly elevated compared to WT. Our findings show evidence of increased inactivation of free pro-resolving lipid mediators through esterification in ASMko mice, suggesting impaired resolution as a new pathway underlying ASM deficiency pathogenesis.
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Doença de Niemann-Pick Tipo A , Doenças de Niemann-Pick , Animais , Camundongos , Encéfalo/metabolismo , Esterificação , Inflamação/metabolismo , Camundongos Knockout , Neurônios/metabolismo , Doença de Niemann-Pick Tipo A/genética , Doença de Niemann-Pick Tipo A/metabolismo , Doença de Niemann-Pick Tipo A/patologia , Doenças de Niemann-Pick/metabolismo , Doenças de Niemann-Pick/patologia , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/metabolismo , Esfingomielinas/metabolismoRESUMO
A portable capillary electrophoresis instrument featuring an automated, robust, valve-based injection system was developed. This significantly facilitates operation in the field compared to previous injection approaches. These generally required delicate manual operations which are difficult to perform outside the laboratory environment. The novel system relies on pressurized air for solution delivery and a micromembrane pump for sample aspiration. Contactless conductivity detection was employed for its versatility and low power requirement. The instrument has a compact design, with all components arranged in a briefcase with dimensions of 45 × 35 × 15 cm (w × d × h) and a weight of about 8 kg. It can operate continuously for 9 h in the battery-powered mode. Depending on the task at hand, the injection system allows easy optimization for high separation efficiency, for fast separations, or for low limits of detection. To illustrate these features, the separation of four anions within 16 s is demonstrated as well as the determination of nitrite below 1 µM. The determination of phosphate at a sewage treatment plant was carried out to demonstrate a field application.
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In this work, a new purpose-made portable CE instrument with a contactless conductivity detector was used for the determination of degradation products of nitrogen mustards in different water samples. The capillary was coated with poly(1-vinylpyrrolidone-co-2-dimethylaminoethyl methacrylate) to avoid analyte-wall interactions. The coating procedure was studied to obtain the best repeatability of the migration time of the analytes. Four different coating procedures were compared; flushing the capillary with the copolymer at 100 psi for 2 min at 60°C provided the best RSD values (<4%). The analytical method was also optimized. The use of 20 mM of MES adjusted to pH 6.0 with His as running buffer allowed a good baseline separation of the three analytes in different water samples without matrix interferences. The method permitted the detection of the three degradation products down to 5 µM.
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Substâncias para a Guerra Química/análise , Eletroforese Capilar/instrumentação , Mecloretamina/análise , Poluentes Químicos da Água/análise , Desenho de Equipamento , Limite de Detecção , Ácidos Polimetacrílicos/química , Pirrolidinonas/química , Rios/químicaRESUMO
Peanut is recognized as a potent food allergen producing one of the most frequent food allergies. This fact has originated the publication of an elevated number of scientific reports dealing with peanut allergens and, especially, the prevalence of peanut allergy. For this reason, the information available on peanut allergens is increasing and the debate about peanut allergy is always renewed. This article reviews the information currently available on peanut allergens and on the techniques used for their chemical characterization. Moreover, a general overview on the current biotechnological approaches used to reduce or eliminate peanut allergens is also provided.
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Alérgenos/imunologia , Arachis/química , Hipersensibilidade a Amendoim , Proteínas de Plantas/imunologia , Alérgenos/química , Animais , Biotecnologia/métodos , Humanos , Hipersensibilidade a Amendoim/imunologia , Hipersensibilidade a Amendoim/prevenção & controle , Proteínas de Plantas/análise , Proteínas de Plantas/químicaRESUMO
In recent years, scopolamine has become a drug of common use for recreational and predatory purposes and several ways of administration have been devised. A method for the rapid analysis of suspicious samples was developed, using a portable capillary electrophoresis with contactless conductivity detection. The method allows the separation of scopolamine from atropine which has a similar structure and is present along with scopolamine in some samples. The method was demonstrated to be useful for the fast analysis of several types of evidential items which have recently been reported to have been abused with fatal consequences or employed for criminal purposes. An infusion of Datura stramonium L., in which scopolamine and atropine naturally coexist, was analyzed for being frequently consumed for recreational purposes. A spiked moisturizing cream and six spiked alcoholic beverages were also analyzed. In spite of the complexity of the specimens, the sample pre-treatment methods developed were simple and fast.
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Adjuvantes Anestésicos/química , Condutividade Elétrica , Eletroforese Capilar/métodos , Escopolamina/química , Detecção do Abuso de Substâncias/métodos , Crime , Toxicologia Forense , HumanosRESUMO
It is increasingly evident that a more detailed molecular structure analysis of isomeric lipids is critical to better understand their roles in biological processes. The occurrence of isomeric interference complicates conventional tandem mass spectrometry (MS/MS)-based determination, necessitating the development of more specialised methodologies to separate lipid isomers. The present review examines and discusses recent lipidomic studies based on ion mobility spectrometry combined with mass spectrometry (IMS-MS). Selected examples of the separation and elucidation of structural and stereoisomers of lipids are described based on their ion mobility behaviour. These include fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, and sterol lipids. Recent approaches for specific applications to improve isomeric lipid structural information using direct infusion, coupling imaging, or liquid chromatographic separation workflows prior to IMS-MS are also discussed, including: 1) strategies to improve ion mobility shifts; 2) advanced tandem MS methods based on activation of lipid ions with electrons or photons, or gas-phase ion-molecule reactions; and 3) the use of chemical derivatisation techniques for lipid characterisation.
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ATPase Inhibitory Factor 1 (IF1) regulates the activity of mitochondrial ATP synthase. The expression of IF1 in differentiated human and mouse cells is highly variable. In intestinal cells, the overexpression of IF1 protects against colon inflammation. Herein, we have developed a conditional IF1-knockout mouse model in intestinal epithelium to investigate the role of IF1 in mitochondrial function and tissue homeostasis. The results show that IF1-ablated mice have increased ATP synthase/hydrolase activities, leading to profound mitochondrial dysfunction and a pro-inflammatory phenotype that impairs the permeability of the intestinal barrier compromising mouse survival upon inflammation. Deletion of IF1 prevents the formation of oligomeric assemblies of ATP synthase and alters cristae structure and the electron transport chain. Moreover, lack of IF1 promotes an intramitochondrial Ca2+ overload in vivo, minimizing the threshold to Ca2+-induced permeability transition (mPT). Removal of IF1 in cell lines also prevents the formation of oligomeric assemblies of ATP synthase, minimizing the threshold to Ca2+-induced mPT. Metabolomic analyses of mice serum and colon tissue highlight that IF1 ablation promotes the activation of de novo purine and salvage pathways. Mechanistically, lack of IF1 in cell lines increases ATP synthase/hydrolase activities and installs futile ATP hydrolysis in mitochondria, resulting in the activation of purine metabolism and in the accumulation of adenosine, both in culture medium and in mice serum. Adenosine, through ADORA2B receptors, promotes an autoimmune phenotype in mice, stressing the role of the IF1/ATP synthase axis in tissue immune responses. Overall, the results highlight that IF1 is required for ATP synthase oligomerization and that it acts as a brake to prevent ATP hydrolysis under in vivo phosphorylating conditions in intestinal cells.
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Adenosina , Inflamação , Proteínas Mitocondriais , Animais , Humanos , Camundongos , Trifosfato de Adenosina , Diferenciação Celular , Camundongos Knockout , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Proteínas Mitocondriais/metabolismo , Proteína Inibidora de ATPaseRESUMO
The reprogramming of cellular metabolism of immune cells is an essential process in the regulation of antifungal immune responses. In particular, glucose metabolism has been shown to be required for protective immunity against infection with Aspergillus fumigatus. However, given the intricate cross talk between multiple metabolic networks and signals, it is likely that cellular metabolic pathways other than glycolysis are also relevant during fungal infection. In this study, we demonstrate that glutamine metabolism is required for the activation of macrophage effector functions against A. fumigatus. Glutamine metabolism was found to be upregulated early after fungal infection and glutamine depletion or the pharmacological inhibition of enzymes involved in its metabolism impaired phagocytosis and the production of both proinflammatory and T-cell-derived cytokines. In an in vivo model, inhibition of glutaminase increased susceptibility to experimental aspergillosis, as revealed by the increased fungal burden and inflammatory pathology, and the defective cytokine production in the lungs. Moreover, genetic variants in glutamine metabolism genes were found to regulate cytokine production in response to A. fumigatus stimulation. Taken together, our results demonstrate that glutamine metabolism represents an important component of the immunometabolic response of macrophages against A. fumigatus both in vitro and in vivo. IMPORTANCE The fungal pathogen Aspergillus fumigatus can cause severe and life-threatening forms of infection in immunocompromised patients. The reprogramming of cellular metabolism is essential for innate immune cells to mount effective antifungal responses. In this study, we report the pivotal contribution of glutaminolysis to the host defense against A. fumigatus. Glutamine metabolism was essential both in vitro as well as in in vivo models of infection, and genetic variants in human glutamine metabolism genes regulated cytokine production in response to fungal stimulation. This work highlights the relevance of glutaminolysis to the pathogenesis of aspergillosis and supports a role for interindividual genetic variation influencing glutamine metabolism in susceptibility to infection.