Histological chorioamnionitis (HCA) is an intrauterine inflammatory condition that increases the ... more Histological chorioamnionitis (HCA) is an intrauterine inflammatory condition that increases the risk for preterm birth, death, and disability because of persistent systemic and localized inflammation. The immunological mechanisms sustaining this response in the preterm newborn remain unclear. We sought to determine the consequences of HCA exposure on the fetal CD4+ T lymphocyte exometabolome. We cultured naive CD4+ T lymphocytes from HCA-positive and -negative preterm infants matched for gestational age, sex, race, prenatal steroid exposure, and delivery mode. We collected conditioned media samples before and after a 6-h in vitro activation of naive CD4+ T lymphocytes with soluble staphylococcal enterotoxin B and anti-CD28. We analyzed samples by ultraperformance liquid chromatography ion mobility–mass spectrometry. We determined the impact of HCA on the CD4+ T lymphocyte exometabolome and identified potential biomarker metabolites by multivariate statistical analyses. We discovere...
An understanding of how cells respond to perturbation is essential for biological applications; h... more An understanding of how cells respond to perturbation is essential for biological applications; however, most approaches for profiling cellular response are limited in scope to pre-established targets. Global analysis of molecular mechanism will advance our understanding of the complex networks constituting cellular perturbation and lead to advancements in areas, such as infectious disease pathogenesis, developmental biology, pathophysiology, pharmacology, and toxicology. We have developed a high-throughput multi-omics platform for comprehensive, de novo characterization of cellular mechanisms of action. Platform validation using cisplatin as a test compound demonstrates quantification of over 10,000 unique, significant molecular changes in less than 30 days. These data provide excellent coverage of known cisplatin-induced molecular changes and previously unrecognized insights into cisplatin resistance. This proof-of-principle study demonstrates the value of this platform as a resou...
Untargeted multiomics data sets are obtained for samples in systems, synthetic, and chemical biol... more Untargeted multiomics data sets are obtained for samples in systems, synthetic, and chemical biology by integrating chromatographic separations with ion mobility-mass spectrometry (IM-MS) analysis. The data sets are interrogated using bioinformatics strategies to organize the data for identification prioritization. The use of big data approaches for data mining of massive data sets in systems-wide analyses is presented. Untargeted biological data across multiomics dimensions are obtained using a variety of chromatography strategies with structural MS. Separation timescales for different techniques and the resulting data deluge when combined with IM-MS are presented. Data mining self-organizing map strategies are used to rapidly filter the data, highlighting those features describing uniqueness to the query. Examples are provided in longitudinal analyses in synthetic biology and human liver exposure to acetaminophen, and in chemical biology, natural product discovery from bacterial b...
MALDI-imaging mass spectrometry correlates detailed analyte information with spatial distribution... more MALDI-imaging mass spectrometry correlates detailed analyte information with spatial distribution in biological tissue sections. By combining post-ionization ion mobility separation with mass analysis, difficult peak assignments arising from spectrum congestion are alleviated.
Imaging mass spectrometry (MS) analysis allows scientists the ability to obtain spatial and chemi... more Imaging mass spectrometry (MS) analysis allows scientists the ability to obtain spatial and chemical information of analytes on a wide variety of surfaces. The ability to image biological analytes is an important tool in many areas of life science research, including: the ability to map pharmaceutical drugs in targeted tissue, to spatially determine the expression profile of specific proteins in healthy vs. diseased tissue states, and to rapidly interrogate biomolecular microarrays. However, there are several avenues for improving the imaging MS experiment for biological samples. Three significant directions this work addresses include: (1) reducing chemical noise and increasing analyte identification by developing sample preparation methodologies, (2) improving the analytical figures of merit (i.e., spatial resolution, analysis time) by implementing a spatially dynamic optical system, and (3) increasing both mass spectral resolution and ion detection sensitivity by modifying a comm...
A metabolic system is composed of inherently interconnected metabolic precursors, intermediates, ... more A metabolic system is composed of inherently interconnected metabolic precursors, intermediates, and products. The analysis of untargeted metabolomics data has conventionally been performed through the use of comparative statistics or multivariate statistical analysis-based approaches; however, each falls short in representing the related nature of metabolic perturbations. Herein, we describe a complementary method for the analysis of large metabolite inventories using a data-driven approach based upon a self-organizing map algorithm. This workflow allows for the unsupervised clustering, and subsequent prioritization of, correlated features through Gestalt comparisons of metabolic heat maps. We describe this methodology in detail, including a comparison to conventional metabolomics approaches, and demonstrate the application of this method to the analysis of the metabolic repercussions of prolonged cocaine exposure in rat sera profiles.
Here we report the use of a microfluidic system to assess the differential metabolomics of murine... more Here we report the use of a microfluidic system to assess the differential metabolomics of murine embryos cultured with endometrial cells-conditioned media (CM). Groups of 10, 1-cell murine B6C3F1 × B6D2F1 embryos were cultured in the microfluidic device. To produce CM, mouse uterine epithelial cells were cultured in potassium simplex optimized medium (KSOM) for 24 h. Media samples were collected from devices after 5 days of culture with KSOM (control) and CM, analyzed by reverse phase liquid chromatography and untargeted positive ion mode mass spectrometry analysis. Blastocyst rates were significantly higher (p < 0.05) in CM (71.8%) compared to control media (54.6%). We observed significant upregulation of 341 compounds and downregulation of 214 compounds in spent media from CM devices when compared to control. Out of these, 353 compounds were identified showing a significant increased abundance of metabolites involved in key metabolic pathways (e.g., arginine, proline and pyrim...
Journal of the American Society for Mass Spectrometry, Dec 13, 2016
Metabolites are building blocks of cellular function. These species are involved in enzyme-cataly... more Metabolites are building blocks of cellular function. These species are involved in enzyme-catalyzed chemical reactions and are essential for cellular function. Upstream biological disruptions result in a series of metabolomic changes and, as such, the metabolome holds a wealth of information that is thought to be most predictive of phenotype. Uncovering this knowledge is a work in progress. The field of metabolomics is still maturing; the community has leveraged proteomics experience when applicable and developed a range of sample preparation and instrument methodology along with myriad data processing and analysis approaches. Research focuses have now shifted toward a fundamental understanding of the biology responsible for metabolomic changes. There are several types of metabolomics experiments including both targeted and untargeted analyses. While untargeted, hypothesis generating workflows exhibit many valuable attributes, challenges inherent to the approach remain. This Critic...
Understanding blood-brain barrier responses to inflammatory stimulation (such as lipopolysacchari... more Understanding blood-brain barrier responses to inflammatory stimulation (such as lipopolysaccharide mimicking a systemic infection or a cytokine cocktail that could be the result of local or systemic inflammation) is essential to understanding the effect of inflammatory stimulation on the brain. It is through the filter of the blood-brain barrier that the brain responds to outside influences, and the blood-brain barrier is a critical point of failure in neuroinflammation. It is important to note that this interaction is not a static response, but one that evolves over time. While current models have provided invaluable information regarding the interaction between cytokine stimulation, the blood-brain barrier, and the brain, these approaches-whether in vivo or in vitro-have often been only snapshots of this complex web of interactions. We utilize new advances in microfluidics, organs-on-chips, and metabolomics to examine the complex relationship of inflammation and its effects on bl...
Nuclear receptors are transcription factors that bind lipids, an event that induces a structural ... more Nuclear receptors are transcription factors that bind lipids, an event that induces a structural conformation of the receptor that favors interaction with transcriptional coactivators. The nuclear receptor steroidogenic factor-1 (SF-1, NR5A1) binds the signaling phosphoinositides PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3), and our previous crystal structures showed how the phosphoinositide headgroups regulate SF-1 function. However, what role the acyl chains play in regulating SF-1 structure remains unaddressed. Here, we used X-ray crystallography with in vitro binding and functional assays to examine how the acyl chains of PIP3 regulate human SF-1 ligand-binding domain structure and function. Altering acyl chain length and unsaturation regulates apparent binding of all tested phosphoinositides to SF-1. Mass spectrometry–based lipidomics data suggest C16 and C18 phospholipids preferentially associate with SF-1 expressed ectopically in bacteria. We then solved the 2.5 Å crystal structur...
The development of miniature surrogate human organs, coupled with highly sensitive mass spectrome... more The development of miniature surrogate human organs, coupled with highly sensitive mass spectrometry technologies, could one day revolutionize the way new drugs and toxic agents are studied. “By developing this ‘homo minutus,’ we are stepping beyond the need for animal or Petri dish testing: There are huge benefits in developing drug and toxicity analysis systems that can mimic the response of actual human organs,” said Rashi Iyer, a senior scientist at Los Alamos National Laboratory. ATHENA, the Advanced Tissue-engineered Human Ectypal Network Analyzer project team, is nearing the full integration of four human organ constructs — liver, heart, lung and kidney — each organ component is about the size of a smartphone screen, and the whole ATHENA “body” of interconnected organs will fit neatly on a desk. A new video available on the Los Alamos National Laboratory YouTube channel updates the ATHENA project as it begins to integrate the various organ systems into a single system (link t...
Innovative techniques for gene editing have enabled accurate animal models of human diseases to b... more Innovative techniques for gene editing have enabled accurate animal models of human diseases to be established. In order for these methods to be successfully adopted in the scientific community, the optimization of procedures used for breeding genetically altered mice is required. Among these, the in vitro fertilization (IVF) procedure is still suboptimal and the culture methods do not guarantee the development of competent embryos. Critical aspects in traditional in vitro embryo culture protocols include the use of mineral oil and the stress induced by repetitive handling of the embryos. A new microfluidic system was designed to allow for efficient in vitro culture of mouse embryos. Harmful fluidic stress and plastic toxicity were excluded by completing the industry gold standard Mouse Embryo Assay. The potential competence of the embryos developed in the device was quantified in terms of blastocyst rate, outgrowth assay, energy substrate metabolism and expression of genes related ...
Histological chorioamnionitis (HCA) is an intrauterine inflammatory condition that increases the ... more Histological chorioamnionitis (HCA) is an intrauterine inflammatory condition that increases the risk for preterm birth, death, and disability because of persistent systemic and localized inflammation. The immunological mechanisms sustaining this response in the preterm newborn remain unclear. We sought to determine the consequences of HCA exposure on the fetal CD4+ T lymphocyte exometabolome. We cultured naive CD4+ T lymphocytes from HCA-positive and -negative preterm infants matched for gestational age, sex, race, prenatal steroid exposure, and delivery mode. We collected conditioned media samples before and after a 6-h in vitro activation of naive CD4+ T lymphocytes with soluble staphylococcal enterotoxin B and anti-CD28. We analyzed samples by ultraperformance liquid chromatography ion mobility–mass spectrometry. We determined the impact of HCA on the CD4+ T lymphocyte exometabolome and identified potential biomarker metabolites by multivariate statistical analyses. We discovere...
An understanding of how cells respond to perturbation is essential for biological applications; h... more An understanding of how cells respond to perturbation is essential for biological applications; however, most approaches for profiling cellular response are limited in scope to pre-established targets. Global analysis of molecular mechanism will advance our understanding of the complex networks constituting cellular perturbation and lead to advancements in areas, such as infectious disease pathogenesis, developmental biology, pathophysiology, pharmacology, and toxicology. We have developed a high-throughput multi-omics platform for comprehensive, de novo characterization of cellular mechanisms of action. Platform validation using cisplatin as a test compound demonstrates quantification of over 10,000 unique, significant molecular changes in less than 30 days. These data provide excellent coverage of known cisplatin-induced molecular changes and previously unrecognized insights into cisplatin resistance. This proof-of-principle study demonstrates the value of this platform as a resou...
Untargeted multiomics data sets are obtained for samples in systems, synthetic, and chemical biol... more Untargeted multiomics data sets are obtained for samples in systems, synthetic, and chemical biology by integrating chromatographic separations with ion mobility-mass spectrometry (IM-MS) analysis. The data sets are interrogated using bioinformatics strategies to organize the data for identification prioritization. The use of big data approaches for data mining of massive data sets in systems-wide analyses is presented. Untargeted biological data across multiomics dimensions are obtained using a variety of chromatography strategies with structural MS. Separation timescales for different techniques and the resulting data deluge when combined with IM-MS are presented. Data mining self-organizing map strategies are used to rapidly filter the data, highlighting those features describing uniqueness to the query. Examples are provided in longitudinal analyses in synthetic biology and human liver exposure to acetaminophen, and in chemical biology, natural product discovery from bacterial b...
MALDI-imaging mass spectrometry correlates detailed analyte information with spatial distribution... more MALDI-imaging mass spectrometry correlates detailed analyte information with spatial distribution in biological tissue sections. By combining post-ionization ion mobility separation with mass analysis, difficult peak assignments arising from spectrum congestion are alleviated.
Imaging mass spectrometry (MS) analysis allows scientists the ability to obtain spatial and chemi... more Imaging mass spectrometry (MS) analysis allows scientists the ability to obtain spatial and chemical information of analytes on a wide variety of surfaces. The ability to image biological analytes is an important tool in many areas of life science research, including: the ability to map pharmaceutical drugs in targeted tissue, to spatially determine the expression profile of specific proteins in healthy vs. diseased tissue states, and to rapidly interrogate biomolecular microarrays. However, there are several avenues for improving the imaging MS experiment for biological samples. Three significant directions this work addresses include: (1) reducing chemical noise and increasing analyte identification by developing sample preparation methodologies, (2) improving the analytical figures of merit (i.e., spatial resolution, analysis time) by implementing a spatially dynamic optical system, and (3) increasing both mass spectral resolution and ion detection sensitivity by modifying a comm...
A metabolic system is composed of inherently interconnected metabolic precursors, intermediates, ... more A metabolic system is composed of inherently interconnected metabolic precursors, intermediates, and products. The analysis of untargeted metabolomics data has conventionally been performed through the use of comparative statistics or multivariate statistical analysis-based approaches; however, each falls short in representing the related nature of metabolic perturbations. Herein, we describe a complementary method for the analysis of large metabolite inventories using a data-driven approach based upon a self-organizing map algorithm. This workflow allows for the unsupervised clustering, and subsequent prioritization of, correlated features through Gestalt comparisons of metabolic heat maps. We describe this methodology in detail, including a comparison to conventional metabolomics approaches, and demonstrate the application of this method to the analysis of the metabolic repercussions of prolonged cocaine exposure in rat sera profiles.
Here we report the use of a microfluidic system to assess the differential metabolomics of murine... more Here we report the use of a microfluidic system to assess the differential metabolomics of murine embryos cultured with endometrial cells-conditioned media (CM). Groups of 10, 1-cell murine B6C3F1 × B6D2F1 embryos were cultured in the microfluidic device. To produce CM, mouse uterine epithelial cells were cultured in potassium simplex optimized medium (KSOM) for 24 h. Media samples were collected from devices after 5 days of culture with KSOM (control) and CM, analyzed by reverse phase liquid chromatography and untargeted positive ion mode mass spectrometry analysis. Blastocyst rates were significantly higher (p < 0.05) in CM (71.8%) compared to control media (54.6%). We observed significant upregulation of 341 compounds and downregulation of 214 compounds in spent media from CM devices when compared to control. Out of these, 353 compounds were identified showing a significant increased abundance of metabolites involved in key metabolic pathways (e.g., arginine, proline and pyrim...
Journal of the American Society for Mass Spectrometry, Dec 13, 2016
Metabolites are building blocks of cellular function. These species are involved in enzyme-cataly... more Metabolites are building blocks of cellular function. These species are involved in enzyme-catalyzed chemical reactions and are essential for cellular function. Upstream biological disruptions result in a series of metabolomic changes and, as such, the metabolome holds a wealth of information that is thought to be most predictive of phenotype. Uncovering this knowledge is a work in progress. The field of metabolomics is still maturing; the community has leveraged proteomics experience when applicable and developed a range of sample preparation and instrument methodology along with myriad data processing and analysis approaches. Research focuses have now shifted toward a fundamental understanding of the biology responsible for metabolomic changes. There are several types of metabolomics experiments including both targeted and untargeted analyses. While untargeted, hypothesis generating workflows exhibit many valuable attributes, challenges inherent to the approach remain. This Critic...
Understanding blood-brain barrier responses to inflammatory stimulation (such as lipopolysacchari... more Understanding blood-brain barrier responses to inflammatory stimulation (such as lipopolysaccharide mimicking a systemic infection or a cytokine cocktail that could be the result of local or systemic inflammation) is essential to understanding the effect of inflammatory stimulation on the brain. It is through the filter of the blood-brain barrier that the brain responds to outside influences, and the blood-brain barrier is a critical point of failure in neuroinflammation. It is important to note that this interaction is not a static response, but one that evolves over time. While current models have provided invaluable information regarding the interaction between cytokine stimulation, the blood-brain barrier, and the brain, these approaches-whether in vivo or in vitro-have often been only snapshots of this complex web of interactions. We utilize new advances in microfluidics, organs-on-chips, and metabolomics to examine the complex relationship of inflammation and its effects on bl...
Nuclear receptors are transcription factors that bind lipids, an event that induces a structural ... more Nuclear receptors are transcription factors that bind lipids, an event that induces a structural conformation of the receptor that favors interaction with transcriptional coactivators. The nuclear receptor steroidogenic factor-1 (SF-1, NR5A1) binds the signaling phosphoinositides PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3), and our previous crystal structures showed how the phosphoinositide headgroups regulate SF-1 function. However, what role the acyl chains play in regulating SF-1 structure remains unaddressed. Here, we used X-ray crystallography with in vitro binding and functional assays to examine how the acyl chains of PIP3 regulate human SF-1 ligand-binding domain structure and function. Altering acyl chain length and unsaturation regulates apparent binding of all tested phosphoinositides to SF-1. Mass spectrometry–based lipidomics data suggest C16 and C18 phospholipids preferentially associate with SF-1 expressed ectopically in bacteria. We then solved the 2.5 Å crystal structur...
The development of miniature surrogate human organs, coupled with highly sensitive mass spectrome... more The development of miniature surrogate human organs, coupled with highly sensitive mass spectrometry technologies, could one day revolutionize the way new drugs and toxic agents are studied. “By developing this ‘homo minutus,’ we are stepping beyond the need for animal or Petri dish testing: There are huge benefits in developing drug and toxicity analysis systems that can mimic the response of actual human organs,” said Rashi Iyer, a senior scientist at Los Alamos National Laboratory. ATHENA, the Advanced Tissue-engineered Human Ectypal Network Analyzer project team, is nearing the full integration of four human organ constructs — liver, heart, lung and kidney — each organ component is about the size of a smartphone screen, and the whole ATHENA “body” of interconnected organs will fit neatly on a desk. A new video available on the Los Alamos National Laboratory YouTube channel updates the ATHENA project as it begins to integrate the various organ systems into a single system (link t...
Innovative techniques for gene editing have enabled accurate animal models of human diseases to b... more Innovative techniques for gene editing have enabled accurate animal models of human diseases to be established. In order for these methods to be successfully adopted in the scientific community, the optimization of procedures used for breeding genetically altered mice is required. Among these, the in vitro fertilization (IVF) procedure is still suboptimal and the culture methods do not guarantee the development of competent embryos. Critical aspects in traditional in vitro embryo culture protocols include the use of mineral oil and the stress induced by repetitive handling of the embryos. A new microfluidic system was designed to allow for efficient in vitro culture of mouse embryos. Harmful fluidic stress and plastic toxicity were excluded by completing the industry gold standard Mouse Embryo Assay. The potential competence of the embryos developed in the device was quantified in terms of blastocyst rate, outgrowth assay, energy substrate metabolism and expression of genes related ...
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