RESUMO
Elevated glucose metabolism in immune cells represents a hallmark feature of many inflammatory diseases, such as sepsis. However, the role of individual glucose metabolic pathways during immune cell activation and inflammation remains incompletely understood. Here, we demonstrate a previously unrecognized anti-inflammatory function of the O-linked ß-N-acetylglucosamine (O-GlcNAc) signaling associated with the hexosamine biosynthesis pathway (HBP). Despite elevated activities of glycolysis and the pentose phosphate pathway, activation of macrophages with lipopolysaccharide (LPS) resulted in attenuated HBP activity and protein O-GlcNAcylation. Deletion of O-GlcNAc transferase (OGT), a key enzyme for protein O-GlcNAcylation, led to enhanced innate immune activation and exacerbated septic inflammation. Mechanistically, OGT-mediated O-GlcNAcylation of the serine-threonine kinase RIPK3 on threonine 467 (T467) prevented RIPK3-RIPK1 hetero- and RIPK3-RIPK3 homo-interaction and inhibited downstream innate immunity and necroptosis signaling. Thus, our study identifies an immuno-metabolic crosstalk essential for fine-tuning innate immune cell activation and highlights the importance of glucose metabolism in septic inflammation.
Assuntos
Apoptose/fisiologia , Inflamação/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Necrose/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Linhagem Celular , Glucose/metabolismo , Humanos , Imunidade Inata/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Serina/metabolismo , Transdução de Sinais/fisiologia , Treonina/metabolismoRESUMO
Metabolomics commonly relies on using one-dimensional (1D) 1H NMR spectroscopy or liquid chromatography-mass spectrometry (LC-MS) to derive scientific insights from large collections of biological samples. NMR and MS approaches to metabolomics require, among other issues, a data processing pipeline. Quantitative assessment of the performance of these software platforms is challenged by a lack of standardized data sets with "known" outcomes. To resolve this issue, we created a novel simulated LC-MS data set with known peak locations and intensities, defined metabolite differences between groups (i.e., fold change > 2, coefficient of variation ≤ 25%), and different amounts of added Gaussian noise (0, 5, or 10%) and missing features (0, 10, or 20%). This data set was developed to improve benchmarking of existing LC-MS metabolomics software and to validate the updated version of our MVAPACK software, which added gas chromatography-MS and LC-MS functionality to its existing 1D and two-dimensional NMR data processing capabilities. We also included two experimental LC-MS data sets acquired from a standard mixture andMycobacterium smegmatiscell lysates since a simulated data set alone may not capture all the unique characteristics and variability of real spectra needed to assess software performance properly. Our simulated and experimental LC-MS data sets were processed with the MS-DIAL and XCMSOnline software packages and our MVAPACK toolkit to showcase the utility of our data sets to benchmark MVAPACK against community standards. Our results demonstrate the enhanced objectivity and clarity of software assessment that can be achieved when both simulated and experimental data are employed since distinctly different software performances were observed with the simulated and experimental LC-MS data sets. We also demonstrate that the performance of MVAPACK is equivalent to or exceeds existing LC-MS software programs while providing a single platform for processing and analyzing both NMR and MS data sets.
Assuntos
Espectrometria de Massas , Metabolômica , Software , Metabolômica/métodos , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Espectrometria de Massa com Cromatografia LíquidaRESUMO
Viral infections alter host cell metabolism and homeostasis; however, the mechanisms that regulate these processes have only begun to be elucidated. We report here that Zika virus (ZIKV) infection activates the antioxidant nuclear factor erythroid 2-related factor 2 (Nrf2), which precedes oxidative stress. Downregulation of Nrf2 or inhibition of glutathione (GSH) synthesis resulted in significantly increased viral replication. Interestingly, 6-amino-nicotinamide (6-AN), a nicotinamide analog commonly used as an inhibitor of the pentose phosphate pathway (PPP), decreased viral replication by over 1,000-fold. This inhibition was neither recapitulated by the knockdown of PPP enzymes, glucose 6-phosphate dehydrogenase (G6PD), or 6-phosphogluconate dehydrogenase (6PGD), nor prevented by supplementation with ribose 5-phosphate. Instead, our metabolomics and metabolic phenotype studies support a mechanism in which 6-AN depletes cells of NAD(H) and impairs NAD(H)-dependent glycolytic steps resulting in inhibition of viral replication. The inhibitory effect of 6-AN was rescued with precursors of the salvage pathway but not with those of other NAD+ biosynthesis pathways. Inhibition of glycolysis reduced viral protein levels, which were recovered transiently. This transient recovery in viral protein synthesis was prevented when oxidative metabolism was inhibited by blockage of the mitochondrial pyruvate carrier, fatty acid oxidation, or glutaminolysis, demonstrating a compensatory role of mitochondrial metabolism in ZIKV replication. These results establish an antagonistic role for the host cell Nrf2/GSH/NADPH-dependent antioxidant response against ZIKV and demonstrate the dependency of ZIKV replication on NAD(H). Importantly, our work suggests the potential use of NAD(H) antimetabolite therapy against the viral infection. IMPORTANCE Zika virus (ZIKV) is a major public health concern of international proportions. While the incidence of ZIKV infections has declined substantially in recent years, the potential for the reemergence or reintroduction remains high. Although viral infection alters host cell metabolism and homeostasis to promote its replication, deciphering the mechanism(s) involved in these processes is important for identifying therapeutic targets. The present work reveals the complexities of host cell redox regulation and metabolic dependency of ZIKV replication. An antagonistic effect of the Nrf2/GSH/NADP(H)-dependent antioxidant response against ZIKV infection and an essential role of NAD(H) metabolism and glycolysis for viral replication are established for the first time. These findings highlight the potential use of NAD(H) antimetabolites to counter ZIKV infection and pathogenesis.
Assuntos
Interações entre Hospedeiro e Microrganismos , Fator 2 Relacionado a NF-E2 , Replicação Viral , Infecção por Zika virus , Zika virus , Humanos , Antioxidantes/metabolismo , Antioxidantes/uso terapêutico , NAD/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Oxirredução , Proteínas Virais/metabolismo , Replicação Viral/fisiologia , Zika virus/fisiologia , Infecção por Zika virus/tratamento farmacológico , Infecção por Zika virus/prevenção & controle , Infecção por Zika virus/virologia , Oxirredutases/genética , Técnicas de Silenciamento de Genes , Células Cultivadas , Interações entre Hospedeiro e Microrganismos/fisiologiaRESUMO
BACKGROUND: The National Cancer Institute issued a Request for Information (RFI; NOT-CA-23-007) in October 2022, soliciting input on using and reusing metabolomics data. This RFI aimed to gather input on best practices for metabolomics data storage, management, and use/reuse. AIM OF REVIEW: The nuclear magnetic resonance (NMR) Interest Group within the Metabolomics Association of North America (MANA) prepared a set of recommendations regarding the deposition, archiving, use, and reuse of NMR-based and, to a lesser extent, mass spectrometry (MS)-based metabolomics datasets. These recommendations were built on the collective experiences of metabolomics researchers within MANA who are generating, handling, and analyzing diverse metabolomics datasets spanning experimental (sample handling and preparation, NMR/MS metabolomics data acquisition, processing, and spectral analyses) to computational (automation of spectral processing, univariate and multivariate statistical analysis, metabolite prediction and identification, multi-omics data integration, etc.) studies. KEY SCIENTIFIC CONCEPTS OF REVIEW: We provide a synopsis of our collective view regarding the use and reuse of metabolomics data and articulate several recommendations regarding best practices, which are aimed at encouraging researchers to strengthen efforts toward maximizing the utility of metabolomics data, multi-omics data integration, and enhancing the overall scientific impact of metabolomics studies.
Assuntos
Imageamento por Ressonância Magnética , Metabolômica , Metabolômica/métodos , Espectroscopia de Ressonância Magnética/métodos , Espectrometria de Massas/métodos , AutomaçãoRESUMO
DJ-1 is a homodimeric protein that is centrally involved in various human diseases including Parkinson disease (PD). DJ-1 protects against oxidative damage and mitochondrial dysfunction through a homeostatic control of reactive oxygen species (ROS). DJ-1 pathology results from a loss of function, where ROS readily oxidizes a highly conserved and functionally essential cysteine (C106). The over-oxidation of DJ-1 C106 leads to a dynamically destabilized and biologically inactivated protein. An analysis of the structural stability of DJ-1 as a function of oxidative state and temperature may provide further insights into the role the protein plays in PD progression. NMR spectroscopy, circular dichroism, analytical ultracentrifugation sedimentation equilibrium, and molecular dynamics simulations were utilized to investigate the structure and dynamics of the reduced, oxidized (C106-SO2 - ), and over-oxidized (C106-SO3 - ) forms of DJ-1 for temperatures ranging from 5°C to 37°C. The three oxidative states of DJ-1 exhibited distinct temperature-dependent structural changes. A cold-induced aggregation occurred for the three DJ-1 oxidative states by 5°C, where the over-oxidized state aggregated at significantly higher temperatures than both the oxidized and reduced forms. Only the oxidized and over-oxidized forms of DJ-1 exhibited a mix state containing both folded and partially denatured protein that likely preserved secondary structure content. The relative amount of this denatured form of DJ-1 increased as the temperature was lowered, consistent with a cold-denaturation. Notably, the cold-induced aggregation and denaturation for the DJ-1 oxidative states were completely reversible. The dramatic changes in the structural stability of DJ-1 as a function of oxidative state and temperature are relevant to its role in PD and its functional response to oxidative stress.
Assuntos
Doença de Parkinson , Humanos , Temperatura , Espécies Reativas de Oxigênio , Doença de Parkinson/metabolismo , Estrutura Secundária de Proteína , Estresse Oxidativo/fisiologiaRESUMO
The process of identifying and quantifying metabolites in complex mixtures plays a critical role in metabolomics studies to obtain an informative interpretation of underlying biological processes. Manual approaches are time-consuming and heavily reliant on the knowledge and assessment of nuclear magnetic resonance (NMR) experts. We propose a shifting-corrected regularized regression method, which identifies and quantifies metabolites in a mixture automatically. A detailed algorithm is also proposed to implement the proposed method. Using a novel weight function, the proposed method is able to detect and correct peak shifting errors caused by fluctuations in experimental procedures. Simulation studies show that the proposed method performs better with regard to the identification and quantification of metabolites in a complex mixture. We also demonstrate real data applications of our method using experimental and biological NMR mixtures.
Assuntos
Imageamento por Ressonância Magnética , Metabolômica , Humanos , Espectroscopia de Prótons por Ressonância Magnética , Espectroscopia de Ressonância Magnética/métodos , Metabolômica/métodos , AlgoritmosRESUMO
Metabolomics aims to achieve a global quantitation of the pool of metabolites within a biological system. Importantly, metabolite concentrations serve as a sensitive marker of both genomic and phenotypic changes in response to both internal and external stimuli. NMR spectroscopy greatly aids in the understanding of both in vitro and in vivo physiological systems and in the identification of diagnostic and therapeutic biomarkers. Accordingly, NMR is widely utilized in metabolomics and fluxomics studies due to its limited requirements for sample preparation and chromatography, its non-destructive and quantitative nature, its utility in the structural elucidation of unknown compounds, and, importantly, its versatility in the analysis of in vitro, in vivo, and ex vivo samples. This review provides an overview of the strengths and limitations of in vitro and in vivo experiments for translational research and discusses how ex vivo studies may overcome these weaknesses to facilitate the extrapolation of in vitro insights to an in vivo system. The application of NMR-based metabolomics to ex vivo samples, tissues, and biofluids can provide essential information that is close to a living system (in vivo) with sensitivity and resolution comparable to those of in vitro studies. The success of this extrapolation process is critically dependent on high-quality and reproducible data. Thus, the incorporation of robust quality assurance and quality control checks into the experimental design and execution of NMR-based metabolomics experiments will ensure the successful extrapolation of ex vivo studies to benefit translational medicine.
Assuntos
Imageamento por Ressonância Magnética , Metabolômica , Espectroscopia de Ressonância Magnética/métodos , Imageamento por Ressonância Magnética/métodos , Metabolômica/métodos , Biomarcadores/análise , Pesquisa Translacional BiomédicaRESUMO
Organelles display characteristic morphologies that are intimately tied to their cellular function, but how organelles are shaped is poorly understood. The endoplasmic reticulum is particularly intriguing, as it comprises morphologically distinct domains, including a dynamic network of interconnected membrane tubules. Several membrane proteins have been implicated in network formation, but how exactly they mediate network formation and whether they are all required are unclear. Here we reconstitute a dynamic tubular membrane network with purified endoplasmic reticulum proteins. Proteoliposomes containing the membrane-fusing GTPase Sey1p (refs 6, 7) and the curvature-stabilizing protein Yop1p (refs 8, 9) from Saccharomyces cerevisiae form a tubular network upon addition of GTP. The tubules rapidly fragment when GTP hydrolysis of Sey1p is inhibited, indicating that network maintenance requires continuous membrane fusion and that Yop1p favours the generation of highly curved membrane structures. Sey1p also forms networks with other curvature-stabilizing proteins, including reticulon and receptor expression-enhancing proteins (REEPs) from different species. Atlastin, the vertebrate orthologue of Sey1p, forms a GTP-hydrolysis-dependent network on its own, serving as both a fusion and curvature-stabilizing protein. Our results show that organelle shape can be generated by a surprisingly small set of proteins and represents an energy-dependent steady state between formation and disassembly.
Assuntos
Retículo Endoplasmático/química , Retículo Endoplasmático/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/ultraestrutura , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacologia , Hidrólise/efeitos dos fármacos , Fusão de Membrana , Proteínas de Membrana Transportadoras/ultraestrutura , Proteolipídeos/química , Proteolipídeos/metabolismo , Proteolipídeos/ultraestrutura , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Proteínas de Transporte Vesicular/ultraestruturaRESUMO
Metabolomics samples like human urine or serum contain upwards of a few thousand metabolites, but individual analytical techniques can only characterize a few hundred metabolites at best. The uncertainty in metabolite identification commonly encountered in untargeted metabolomics adds to this low coverage problem. A multiplatform (multiple analytical techniques) approach can improve upon the number of metabolites reliably detected and correctly assigned. This can be further improved by applying synergistic sample preparation along with the use of combinatorial or sequential non-destructive and destructive techniques. Similarly, peak detection and metabolite identification strategies that employ multiple probabilistic approaches have led to better annotation decisions. Applying these techniques also addresses the issues of reproducibility found in single platform methods. Nevertheless, the analysis of large data sets from disparate analytical techniques presents unique challenges. While the general data processing workflow is similar across multiple platforms, many software packages are only fully capable of processing data types from a single analytical instrument. Traditional statistical methods such as principal component analysis were not designed to handle multiple, distinct data sets. Instead, multivariate analysis requires multiblock or other model types for understanding the contribution from multiple instruments. This review summarizes the advantages, limitations, and recent achievements of a multiplatform approach to untargeted metabolomics.
Assuntos
Metaboloma , Metabolômica , Humanos , Reprodutibilidade dos Testes , Metabolômica/métodos , Análise Multivariada , Análise de Componente PrincipalRESUMO
Staphylococcus aureus is a common source of hospital-acquired bacterial infections, where the emergence of antibiotic resistance is a serious human health concern. Most investigations into S. aureus virulence and antibiotic resistance have relied on in vitro cultivation conditions and optimized media formulations. However, S. aureus can survive and adapt to a hostile host environment or antibiotic treatments by rapidly adjusting its metabolic activity. To assess this metabolic response, S. aureus strains susceptible and nonsusceptible to daptomycin were cultivated in medium supplemented with 55% serum to more closely approximate in vivo conditions. Growth analyses, MIC testing, and NMR-based metabolomics determined that serum decreased daptomycin susceptibility and altered metabolism in S. aureus. Both S. aureus strains exhibited altered amino acid biosynthesis and catabolism, enhanced fermentation, and a modified salt tolerance response. The observation that growth conditions defined an adaptive metabolic response to antibiotics by S. aureus may be a critical consideration for designing an effective drug discovery study.
Assuntos
Daptomicina , Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Antibacterianos/farmacologia , Daptomicina/metabolismo , Daptomicina/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/metabolismoRESUMO
The mechanism of action (MoA) of a clickable fatty acid analogue 8-(2-cyclobuten-1-yl)octanoic acid (DA-CB) has been investigated for the first time. Proteomics, metabolomics, and lipidomics were combined with a network analysis to investigate the MoA of DA-CB against Mycobacterium smegmatis (Msm). The metabolomics results showed that DA-CB has a general MoA related to that of ethionamide (ETH), a mycolic acid inhibitor that targets enoyl-ACP reductase (InhA), but DA-CB likely inhibits a step downstream from InhA. Our combined multi-omics approach showed that DA-CB appears to disrupt the pathway leading to the biosynthesis of mycolic acids, an essential mycobacterial fatty acid for both Msm and Mycobacterium tuberculosis (Mtb). DA-CB decreased keto-meromycolic acid biosynthesis. This intermediate is essential in the formation of mature mycolic acid, which is a key component of the mycobacterial cell wall in a process that is catalyzed by the essential polyketide synthase Pks13 and the associated ligase FadD32. The multi-omics analysis revealed further collateral alterations in bacterial metabolism, including the overproduction of shorter carbon chain hydroxy fatty acids and branched chain fatty acids, alterations in pyrimidine metabolism, and a predominate downregulation of proteins involved in fatty acid biosynthesis. Overall, the results with DA-CB suggest the exploration of this and related compounds as a new class of tuberculosis (TB) therapeutics. Furthermore, the clickable nature of DA-CB may be leveraged to trace the cellular fate of the modified fatty acid or any derived metabolite or biosynthetic intermediate.
Assuntos
Mycobacterium tuberculosis , Ácidos Micólicos , Ácidos Micólicos/metabolismo , Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/metabolismo , Mycobacterium smegmatis/metabolismo , Ácidos Graxos/metabolismo , Antituberculosos/farmacologia , Antituberculosos/metabolismoRESUMO
The accuracy and ease of metabolite assignments from a complex mixture are expected to be facilitated by employing a multispectral approach. The two-dimensional (2D) 1H-13C heteronuclear single quantum coherence (HSQC) and 2D 1H-1H-total correlation spectroscopy (TOCSY) are the experiments commonly used for metabolite assignments. The 2D 1H-13C HSQC-TOCSY and 2D 1H-13C heteronuclear multiple-bond correlation (HMBC) are routinely used by natural products chemists but have seen minimal usage in metabolomics despite the unique information, the nearly complete 1H-1H and 1H-13C and spin systems provided by these experiments that may improve the accuracy and reliability of metabolite assignments. The use of a 13C-labeled feedstock such as glucose is a routine practice in metabolomics to improve sensitivity and to emphasize the detection of specific metabolites but causes severe artifacts and an increase in spectral complexity in the HMBC experiment. To address this issue, the standard HMBC pulse sequence was modified to include carbon decoupling. Nonuniform sampling was also employed for rapid data collection. A dataset of reference 2D 1H-13C HMBC spectra was collected for 94 common metabolites. 13C-13C spin connectivity was then obtained by generating a covariance pseudo-spectrum from the carbon-decoupled HMBC and the 1H-13C HSQC-TOCSY spectra. The resulting 13C-13C pseudo-spectrum provides a connectivity map of the entire carbon backbone that uniquely describes each metabolite and would enable automated metabolite identification.
Assuntos
Artefatos , Metabolômica , Espectroscopia de Ressonância Magnética/métodos , Isótopos de Carbono , Reprodutibilidade dos Testes , Metabolômica/métodosRESUMO
The cochaperone protein DNAJA1 (HSP40) is downregulated four-fold in pancreatic cancer cells. The impact of DNAJA1 expression on pancreatic ductal adenocarcinoma (PDAC) progression remains unclear. The metabolic impacts of increased DNAJA1 expression were evaluated using a combination of untargeted metabolomics, stable isotope-resolved metabolomics (SIRM), confocal microscopy, flow cytometry, and cell-based assays. Differential Warburg glycolysis, an increase in redox currency, and alterations in amino acid levels were observed in both overexpression cell lines. DNAJA1 overexpression also led to mitochondrial fusion, an increase in the expression of Bcl-2, a modest protection from redox-induced cell death, a loss of structural integrity due to the loss of actin fibers, and an increase in cell invasiveness in BxPC-3. These differences were more pronounced in BxPC-3, which contains a loss-of-function mutation in the tumor-suppressing gene SMAD4. These findings suggest a proto-oncogenic role of DNAJA1 in PDAC progression and suggest DNAJA1 may function synergistically with other proteins with altered activities in pancreatic cancer cell lines.
Assuntos
Carcinoma Ductal Pancreático , Proteínas de Choque Térmico HSP40/genética , Neoplasias Pancreáticas , Apoptose , Carcinoma Ductal Pancreático/genética , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pancreáticas/genética , FenótipoRESUMO
Staphylococcus aureus causes acute and chronic infections resulting in significant morbidity. Urease, an enzyme that generates NH3 and CO2 from urea, is key to pH homeostasis in bacterial pathogens under acidic stress and nitrogen limitation. However, the function of urease in S. aureus niche colonization and nitrogen metabolism has not been extensively studied. We discovered that urease is essential for pH homeostasis and viability in urea-rich environments under weak acid stress. The regulation of urease transcription by CcpA, Agr, and CodY was identified in this study, implying a complex network that controls urease expression in response to changes in metabolic flux. In addition, it was determined that the endogenous urea derived from arginine is not a significant contributor to the intracellular nitrogen pool in non-acidic conditions. Furthermore, we found that during a murine chronic renal infection, urease facilitates S. aureus persistence by promoting bacterial fitness in the low-pH, urea-rich kidney. Overall, our study establishes that urease in S. aureus is not only a primary component of the acid response network but also an important factor required for persistent murine renal infections.
Assuntos
Staphylococcus aureus/metabolismo , Urease/metabolismo , Urease/fisiologia , Ácidos/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Feminino , Homeostase/fisiologia , Concentração de Íons de Hidrogênio , Rim/microbiologia , Nefropatias/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nitrogênio/metabolismo , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/patogenicidade , Ureia/metabolismo , Urease/genéticaRESUMO
Preeclampsia is a spontaneously occurring pregnancy complication diagnosed by new-onset hypertension and end-organ dysfunction with or without proteinuria. This pregnancy-specific syndrome contributes to maternal morbidity and mortality and can have detrimental effects on fetal outcomes. Preeclampsia is also linked to increased risk of maternal cardiovascular disease throughout life. Despite intense investigation of this disorder, few treatment options are available. The aim of this study was to investigate the potential therapeutic effects of maternal l-citrulline supplementation on pregnancy-specific vascular dysfunction in the male C57BL/6J × female C57BL/6J C1q-/- preeclampsia-like mouse model. l-Citrulline is a nonessential amino acid that is converted to l-arginine to promote smooth muscle and blood vessel relaxation and improve nitric oxide (NO)-mediated vascular function. To model a preeclampsia-like pregnancy, female C57BL/6J mice were mated to C1q-/- male mice, and a subset of dams was supplemented with l-citrulline throughout pregnancy. Blood pressure, systemic vascular glycocalyx, and ex vivo vascular function were investigated in late pregnancy, and postpartum at 6 and 10 mo of age. Main findings show that l-citrulline reduced blood pressure, increased vascular glycocalyx volume, and rescued ex-vivo vascular function at gestation day 17.5 in this preeclampsia-like model. The vascular benefit of l-citrulline also extended postpartum, with improved vascular function and glycocalyx measures at 6 and 10 mo of age. l-Citrulline-mediated vascular improvements appear, in part, attributable to NO pathway signaling. Taken together, l-citrulline supplementation during pregnancy appears to have beneficial effects on maternal vascular health, which may have translational implications for improved maternal cardiovascular health.
Assuntos
Citrulina/farmacologia , Fenômenos Fisiológicos da Nutrição Materna/efeitos dos fármacos , Parto/efeitos dos fármacos , Pré-Eclâmpsia/tratamento farmacológico , Animais , Arginina/sangue , Pressão Sanguínea/efeitos dos fármacos , Citrulina/sangue , Feminino , Camundongos Endogâmicos C57BL , Placenta/metabolismo , Pré-Eclâmpsia/fisiopatologia , GravidezRESUMO
Osteochondral lesions of the talus (OLT) are often associated with ankle pain and dysfunction. They can occur after ankle trauma, such as sprains or fractures, but they usually present as a continued ankle pain after the initial injury has resolved. Chronic ankle ligament instability and subsequent microtrauma may lead to insidious development of an OLT. Medial-sided lesions are more common (67%) than lateral-sided lesions. For acute lesions that are nondisplaced, nonoperative management is initially performed, with a 4-6 week period of immobilization and protected weight bearing. Symptomatic improvement results in more than 50% of patients by 3 months. Acute osteochondral talus fractures, which have a bone fragment thickness greater than 3 mm with displacement will benefit from early surgical intervention. These injuries should undergo primary repair via internal fixation with bioabsorbable compression screws 3.0 mm or smaller using at least 2 points of fixation. Acute lesions that are too small for fixation can be treated with morselization and reimplantation of the cartilage fragments. If OLTs are persistently symptomatic following an appropriate course of nonoperative treatment, various reparative and restorative surgical options may be considered on the basis of diameter, surface area, depth, and location of the lesion. A small subset of symptomatic osteochondral lesions of the talus involve subchondral pathology with intact overlying articular cartilage; in these cases, retrograde drilling into the cystic lesion can be employed to induce underlying bony healing. Cancellous bone graft augmentation may be used for subchondral cysts with volume greater than 100 mm3 or with those with a depth of more than 10 mm. Debridement, curettage, and bone marrow stimulation is a reparative technique that may be considered in lesions demonstrating a diameter less than 10 mm, with surface area less than 100 mm2, and a depth less than 5 mm. This technique is commonly performed arthroscopically using curettes and an arthroscopic shaver to remove surrounding unstable cartilage. A microfracture awl of 1 mm or less is used to puncture the subchondral bone with 3-4 mm of spacing between to induce punctate bleeding. Initial (<5 year) results are good to excellent in 80% of cases, with some deterioration of improvement over time. Factors contributing to poor results include surface area greater than 1.5 cm2, overall osteochondral lesion depth over 7.8 mm, smoking history, age over 40, and uncontained lesions. Lesions greater than 1.29 cm2, cystic lesions, and lesions that have failed prior treatment are potential candidates for osteochondral autograft transplantation. The autograft is typically harvested from the lateral femoral condyle of the ipsilateral knee with an optimal plug depth and diameter of 12-15 mm. Transplantation often involves open technique and may even require malleolar osteotomy for perpendicular access to the defect, as well as visualization of a flush, congruent graft fit. Good to excellent outcomes have been reported in up 87.4% of cases with the most common complication being donor site morbidity in up to 15% of cases. Failure rates increased significantly in lesions larger than 225 mm2. Scaffold-based therapies, such as matrix-associated chondrocyte implantation, can be employed in primary or revision settings in lesions larger than 1 cm2, including uncontained shoulder lesions with or without cysts. Lesions with greater than 4 mm of bone loss following debridement may require bone grafting to augment with the scaffold. This technique requires an initial procedure for chondrocyte harvest and a secondary procedure for transplantation of the scaffold. Outcomes have been good to excellent in up to 93% of cases; however, this technique requires a two-stage procedure and can be cost-prohibitive. Particulated juvenile cartilage is a restorative technique that employs cartilage allograft from juvenile donors. The cartilage is placed into the defect and secured with fibrin glue in a single-stage procedure. Studies have shown favorable outcomes in 92% of cases, with lesions between 10 and 15 mm in diameter, but increased failure rates and poorer outcomes in lesions larger than 15 mm. This may be an alternative option for contained lesions between 10 and 15 mm in diameter. Osteochondral allograft plugs are an option for larger contained lesions (>1.5 cm in diameter) and in patients with knee osteoarthritis (OA) and concern for donor site morbidity. Furthermore, bulk osteochondral allograft from a size-matched talus can also be used for even larger, unstable/uncontained shoulder lesions. An anterior approach is often employed and fixation is achieved via placement of countersunk headless compression screws. Failure of the aforementioned options associated with persistent pain or progressive OA would then lend consideration to ankle arthroplasty versus ankle arthrodesis.
Assuntos
Traumatismos do Tornozelo , Cartilagem Articular , Tálus , Traumatismos do Tornozelo/cirurgia , Transplante Ósseo , Cartilagem Articular/cirurgia , Humanos , Tálus/cirurgia , Transplante AutólogoRESUMO
Gadolinium is a paramagnetic relaxation enhancement (PRE) agent that accelerates the relaxation of metabolite nuclei. In this study, we noted the ability of gadolinium to improve the sensitivity of two-dimensional, non-uniform sampled NMR spectral data collected from metabolomics samples. In time-equivalent experiments, the addition of gadolinium increased the mean signal intensity measurement and the signal-to-noise ratio for metabolite resonances in both standard and plasma samples. Gadolinium led to highly linear intensity measurements that correlated with metabolite concentrations. In the presence of gadolinium, we were able to detect a broad array of metabolites with a lower limit of detection and quantification in the low micromolar range. We also observed an increase in the repeatability of intensity measurements upon the addition of gadolinium. The results of this study suggest that the addition of a gadolinium-based PRE agent to metabolite samples can improve NMR-based metabolomics.
Assuntos
Gadolínio/química , Imageamento por Ressonância Magnética/métodos , Metabolômica/métodos , Aumento da Imagem/métodos , Espectroscopia de Ressonância Magnética/métodos , Razão Sinal-RuídoRESUMO
Stable isotopes are routinely employed by NMR metabolomics to highlight specific metabolic processes and to monitor pathway flux. 13C-carbon and 15N-nitrogen labeled nutrients are convenient sources of isotope tracers and are commonly added as supplements to a variety of biological systems ranging from cell cultures to animal models. Unlike 13C and 15N, 31P-phosphorus is a naturally abundant and NMR active isotope that does not require an external supplemental source. To date, 31P NMR has seen limited usage in metabolomics because of a lack of reference spectra, difficulties in sample preparation, and an absence of two-dimensional (2D) NMR experiments, but 31P NMR has the potential of expanding the coverage of the metabolome by detecting phosphorus-containing metabolites. Phosphorylated metabolites regulate key cellular processes, serve as a surrogate for intracellular pH conditions, and provide a measure of a cell's metabolic energy and redox state, among other processes. Thus, incorporating 31P NMR into a metabolomics investigation will enable the detection of these key cellular processes. To facilitate the application of 31P NMR in metabolomics, we present a unified protocol that allows for the simultaneous and efficient detection of 1H-, 13C-, 15N-, and 31P-labeled metabolites. The protocol includes the application of a 2D 1H-31P HSQC-TOCSY experiment to detect 31P-labeled metabolites from heterogeneous biological mixtures, methods for sample preparation to detect 1H-, 13C-, 15N-, and 31P-labeled metabolites from a single NMR sample, and a data set of one-dimensional (1D) 31P NMR and 2D 1H-31P HSQC-TOCSY spectra of 38 common phosphorus-containing metabolites to assist in metabolite assignments.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Metabolômica/métodos , Fósforo/química , Escherichia coli/química , Mycobacterium smegmatis/químicaRESUMO
Preeclampsia is a spontaneously occurring, pregnancy-specific syndrome that is clinically diagnosed by new onset hypertension and proteinuria. Epidemiological evidence describes an association between a history of preeclampsia and increased risk for cardiovascular disease in later life; however, the mechanism(s) driving this relationship are unclear. Our study aims to leverage a novel preeclampsia-like mouse model, the C1q-/- model, to help elucidate the acute and persistent vascular changes during and following a preeclampsia-like pregnancy. Female C57BL/6J mice were mated to C1q-/- male mice to model a preeclampsia-like pregnancy ("PE-like"), and the maternal cardiovascular phenotype (blood pressure, renal function, systemic glycocalyx, and ex vivo vascular function) was assessed in late pregnancy and postpartum at 6 and 10 mo of age. Uncomplicated, normotensive pregnancies (female C57BL/6J bred to male C57BL/6J mice) served as age-matched controls. In pregnancy, PE-like dams exhibited increased systolic and diastolic pressure during mid- and late gestation, renal dysfunction, fetal growth restriction, and reduced placental efficiency. Ex vivo wire myography studies of mesenteric arteries revealed severe pregnancy-specific endothelial-dependent and -independent vascular dysfunction. At 3 and 7 mo postpartum (6 and 10 mo old, respectively), hypertension resolved in PE-like dams, whereas mild vascular dysfunction persisted at 3 mo postpartum. In conclusion, the female C57BL/6J-by-male C57BL/6J C1q-/- model recapitulates many aspects of the human preeclampsia syndrome in a low-risk, wild-type female mouse. The pregnancy-specific phenotype results in systemic maternal endothelial-dependent and -independent vascular dysfunction that persists postpartum.