Systems Biology

Protocols in Current Issue
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0 Q&A 2305 Views Jan 20, 2021

Magnetic resonance spectroscopy (MRS) can be used to measure in vivo concentrations of neurometabolites. This information can be used to identify neurotransmitter involvement in healthy (e.g., perceptual and cognitive processes) and unhealthy brain function (e.g., neurological and psychiatric illnesses). The standard approach for analyzing MRS data is to combine spectral transients acquired over a ~10 min scan to yield a single estimate that reflects the average metabolite concentration during that period. The temporal resolution of metabolite measurements is sacrificed in this manner to achieve a sufficient signal-to-noise ratio to produce a reliable estimate. Here we introduce two analyses that can be used to increase the temporal resolution of neurometabolite estimates produced from MRS measurements. The first analysis uses a sliding window approach to create a smoothed trace of neurometabolite concentration for each MRS scan. The second analysis combines transients across participants, rather than time, producing a single “group trace” with the highest possible temporal resolution achievable with the data. These analyses advance MRS beyond the current “static” application by allowing researchers to measure dynamic changes in neurometabolite concentration and expanding the types of questions that the technique can be used to address.

1 Q&A 5761 Views Jul 20, 2020
Macrophages are highly plastic immune cells that are capable of adopting a wide array of functional phenotypes in response to environmental stimuli. The changes in macrophage function are often supported and regulated by changes in cellular metabolism. Capturing a comprehensive picture of metabolism is vital for understanding the role of metabolic rewiring in the immune response. Here we present a method for systematically quantifying the abundance of metabolites and lipids in primary murine bone marrow derived macrophages (BMDMs). This method simultaneously extracts polar metabolites and lipids from BMDMs using a rapid two-phase extraction procedure. The polar metabolite fraction and lipid fraction are subsequently analyzed by separate liquid chromatography-mass spectrometry (LC-MS) methods for optimized coverage and quantification. This allows for a comprehensive characterization of cellular metabolism that can be used to understand the impact of a variety of environmental stimuli on macrophage metabolism and function.
0 Q&A 4225 Views Mar 20, 2020
Acclimation of leaf traits to fluctuating environments is a key mechanism to maximize fitness. One of the most important strategies in acclimation to changing light is to maintain efficient utilization of nitrogen in the photosynthetic apparatus by continuous modifications of between-leaf distribution along the canopy depth and within-leaf partitioning between photosynthetic functions according to local light availability. Between-leaf nitrogen distribution has been intensively studied over the last three decades, where proportional coordination between nitrogen concentration and light gradient was considered optimal in terms of maximizing canopy photosynthesis, without taking other canopy structural and physiological factors into account. We proposed a mechanistic model of protein turnover dynamics in different photosynthetic functions, which can be parameterized using leaves grown under different levels of constant light. By integrating this dynamic model into a multi-layer canopy model, constructed using data collected from a greenhouse experiment, it allowed us to test in silico the degree of optimality in photosynthetic nitrogen use for maximizing canopy carbon assimilation under given light environments.
0 Q&A 6934 Views Nov 20, 2019
Cancer is a disease characterized by altered metabolism, and there has been renewed interest in understanding the metabolism of tumors. Even though nutrient availability is a critical determinant of tumor metabolism, there has been little systematic study of the nutrients directly available to cancer cells in the tumor microenvironment. Previous work characterizing the metabolites present in the tumor interstitial fluid has been restricted to the measurement of a small number of nutrients such as glucose and lactate in a limited number of samples. Here we adapt a centrifugation-based method of tumor interstitial fluid isolation readily applicable to a number of sample types and a mass spectrometry-based method for the absolute quantitation of many metabolites in interstitial fluid samples. In this method, tumor interstitial fluid (TIF) is analyzed by liquid chromatography-mass spectrometry (LC/MS) using both isotope dilution and external standard calibration to derive absolute concentrations of targeted metabolites present in interstitial fluid. The use of isotope dilution allows for accurate absolute quantitation of metabolites, as other methods of quantitation are inadequate for determining nutrient concentrations in biological fluids due to matrix effects that alter the apparent concentration of metabolites depending on the composition of the fluid in which they are contained. This method therefore can be applied to measure the absolute concentrations of many metabolites in interstitial fluid from diverse tumor types, as well as most other biological fluids, allowing for characterization of nutrient levels in the microenvironment of solid tumors.
0 Q&A 3859 Views Oct 20, 2019
The accurate determination of metabolite distribution in subcellular compartments is still challenging in plant science. Various methodologies, such as fluorescence resonance energy transfer-based technology, nuclear magnetic resonance spectroscopy and protoplast fractionation allow the study of metabolite compartmentation. However, large changes in metabolite levels occur during such procedures. Therefore, the non-aqueous fractionation (NAF) technique is currently the best method for the study of in-vivo metabolite distribution. Our protocol presents a detailed workflow including the NAF procedure and quantification of compartment-specific markers for three subcellular compartments: ADP glucose pyrophosphorylase (AGPase) as plastidic marker, phosphoenolpyruvate carboxylase (PEPC) as cytosolic marker, and nitrate and acid invertase as vacuolar markers.
0 Q&A 12154 Views Nov 20, 2018
Short-Chain Fatty Acids (SCFAs) are a product of the fermentation of resistant starches and dietary fibers by the gut microbiota. The most important SCFA are acetate (C2), propionate (C3) and butyrate (C4). These metabolites are formed and absorbed in the colon and then transported through the hepatic vein to the liver. SCFAs are more concentrated in the intestinal lumen than in the serum. Butyrate is largely consumed in the gut epithelium, propionate in the liver and acetate in the periphery. SCFAs act on many cells including components of the immune system and epithelial cells by two main mechanisms: activation of G-protein coupled receptors (GPCRs) and inhibition of histone deacetylase. Considering the association between changes in SCFA concentrations and the development of diseases, methods to quantify these acids in different biological samples are important. In this study, we describe a protocol using gas chromatography to quantify SCFAs in the serum, feces and colonic luminal content. Separation of compounds was performed using a DB-23 column (60 m x 0.25 mm internal diameter [i.d.]) coated with a 0.15 µm thick layer of 80.2% 1-methylnaphatalene. This method has a good linear range (15-10,000 µg/ml). The precision (relative standard deviation [RSD]) is less than 15.0% and the accuracy (error relative [ER]) is within ± 15.0%. The extraction efficiency was higher than 97.0%. Therefore, this is cost effective and reproducible method for SCFA measurement in feces and serum.
0 Q&A 5701 Views Sep 20, 2018
Stable-isotope labeled metabolic analysis is an essential methodology to characterize metabolic regulation during biological processes. However, the method using stable-isotope-labeled tracer (e.g., 13C-glucose) in live animal is only beginning to be developed. Here, we contribute a qualitative metabolic labeling experiment protocol in Drosophila melanogaster using stable-isotope-labeled 13C-glucose tracer followed by liquid chromatography-mass spectrometry (LC-MS) analysis. Detailed experimental setup, data acquisition and analysis are provided to facilitate the application of in vivo metabolic labeling analysis that might be applied in a wide range of biological studies.
0 Q&A 7557 Views Feb 5, 2018
Modern triple quadrupole mass spectrometers provide the ability to detect and quantify a large number of metabolites using tandem mass spectrometry (MS/MS). Liquid chromatography (LC) is advantageous, as it does not require derivatization procedures and a large diversity in physiochemical characteristics of analytes can be accommodated through a variety of column chemistries. Recently, the comprehensive optimization of LC-MS metabolomics using design of experiments (COLMeD) approach has been described and used by our group to develop robust LC-MS workflows (Rhoades and Weljie, 2016). The optimized LC-MS/MS method described here has been utilized extensively for metabolomics analysis of polar metabolites. Typically, tissue or biofluid samples are extracted using a modified Bligh-Dyer protocol (Bligh and Dyer, 1959; Tambellini et al., 2013). The protocol described herein describes this workflow using targeted polar metabolite multiple reaction monitoring (MRM) from tissues and biofluids via ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). This workflow has been utilized extensively for chronometabolic analysis (Krishnaiah et al., 2017), with applications generalized to other types of analyses as well (Sengupta et al., 2017; Sivanand et al., 2017).
0 Q&A 10706 Views Jan 20, 2018
Increased antibiotic resistance of plants and human pathogens and continuous use of chemical fertilizers has pushed microbiologists to explore new microbial sources as potential antagonists. In this study, eight strains of Pseudomonas aurantiaca and Pseudomonas chlororaphis, have been isolated from different plant sources and screened for their antagonistic and plant growth promoting potential (Shahid et al., 2017). All strains were compared with reference strain PB-St2 and their secondary metabolites were isolated by the use of solvent partitioning and subjected to LC/ESI/MS for confirmation of compounds. The ESI-mass spectra obtained were used to characterize the surfactants ionization behavior and [M + H]+ and [M + Na]+ ions were monitored for phenazines, derivatives of lahorenoic acid and cyclic lipopeptide (WLIP). LC-MS and HPLC methods were developed to see the elution of dominant metabolites in a single run to avoid the labor and separate methods of detection for all compounds. The method was found suitable and distinctively separated the compounds at different retention times in gradient flow. This method can be helpful to explore the metabolome of Pseudomonas sp. overall and in identification and quantification of strain specific metabolites.
0 Q&A 8867 Views Sep 20, 2017
Metabolomic is an emerging field of system biology. Lipidomic, a branch of metabolomic, aims to characterize lipophilic metabolites in biological systems. Caenorhabditis elegans (C. elegans) is a genetically tractable and versatile animal model for novel discovery of lipid metabolism. In addition, C. elegans embryo is simple and homogeneous. Here, we demonstrate detailed procedures of C. elegans culture, embryo isolation, lipid extraction and metabolomic data analysis.

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