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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 217 records · Page 12
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

National User Resource for Biological Accelerator Mass Spectrometry (Final Report)

The National User Resource for Biological Accelerator Mass Spectrometry (User Resource) will provide isotopic analysis (primarily radiocarbon or 14C) by accelerator mass spectrometry (AMS) for NIH- funded researchers across the United States and will be the only User Resource of its type in the United States. The User Resource will provide measurement capability and expertise to a research community that requires highly sensitive, quantitative isotope analyses. Since commissioning a new accelerator mass spectrometer in June 2014, we have measured over 4000 samples a year for collaborators and service users. The User Resource will enable us to continue to meet these research needs, as well as provide for new users whose research programs would benefit from AMS as a measurement tool. The User Resource’s forte will be ultra-high sensitivity quantitation of radiocarbon and selected other radioisotopes for research studies where isotopes are required. Radioisotope labeling studies have been and will continue to be an important tool for addressing many complex biomedical science problems. AMS is a specialized and unique type of mass spectrometry that provides absolute quantitation of radiocarbon and other relevant radioisotopes with extreme sensitivity, having limits of detection in real samples on the order of a few attomol/mg of sample at measurement precisions of ~3%. It is the only instrumental method capable of quantifying radioisotope-labeled agents routinely in real-world samples with such precision and sensitivity. The sensitivity of AMS allows for the quantification of radiolabeled metabolites in extremely complex matrices of cells and organisms at very low concentrations and in small samples. AMS allows studies to be conducted without perturbing metabolism leading to more relevant quantification of metabolic rates and pathways. In addition, it enables quantification of pharmacokinetic and metabolic properties of toxicants at environmentally relevant concentrations in model systems as well as the ability to quantify pharmacokinetics and other molecular endpoints directly in humans. Such quantitative assessments can 1) improve risk assessment for toxicants, 2) address safety and efficacy considerations for therapeutic entities, 3) deepen understanding of xenobiotic and intermediary metabolism, 4) help understand the interactions between critical molecular pathways, and 5) improve efforts to model and predict various metabolic and biological states. These capabilities have been applied in a number of areas including research in carcinogenesis, toxicology, nutrition, pharmacology/drug development and basic biological science. As a NIGMS National Resource the National User Resource for Biological Accelerator Mass Spectrometry will help NIH funded scientists achieve a deeper understanding of the etiology of human health concerns by (1) enabling the quantification of pharmacokinetics and other molecular endpoints directly in humans; (2) offering the ability to conduct quantitative studies using biologics such as proteins or lipids, and thereby reducing the amount of radioisotope usage in biomedical labs; and (3) enabling more relevant studies of metabolic pathways in health and disease through the use of much lower, more biologically-relevant, concentrations of metabolic substrates in cells and intact organisms. Such studies support NIGMS’s basic biomedical research areas that contribute to the understanding of fundamental cellular and physiological principles and enable research supported by the Biophysics, Biomedical Technology, and Computational Biosciences (BBCB); Genetics and Molecular, Cellular, and Developmental Biology (GMCDB); Pharmacology, Physiology, Biological Chemistry (PPBC) and Training, Workforce Development, and Diversity (TWD) Divisions. Over the next five years, our goals are to: 1. Improve the efficiency of operation for AMS measurements through installation of new interfaces to our AMS systems, technical modifications to improve gas accepting ion source efficiency and upgrading our data analysis software for improved ease of use and data reporting. 2. Increase the accessibility and visibility of ultra-sensitive 14C measurements for the biomedical research community by training of new investigators and expanding our national user base. 3. Provide high throughput, ultra-sensitive 14C analysis for the NIGMS and NIH user community.

47 OTHER INSTRUMENTATION↗
DOE OSTI2021

Utilizing Plant Synthetic Biology to Improve Human Health and Wellness

Plants offer a vast source of bioactive chemicals with the potential to improve human health through the prevention and treatment of disease. However, many potential therapeutics are produced in small amounts or in species that are difficult to cultivate. The rapidly evolving field of plant synthetic biology provides tools to capitalize on the inventive chemistry of plants by transferring metabolic pathways for therapeutics into far more tenable plants, increasing our ability to produce complex pharmaceuticals in well-studied plant systems. Plant synthetic biology also provides methods to enhance the ability to fortify crops with nutrients and nutraceuticals. In this review, we discuss (1) the potential of plant synthetic biology to improve human health by generating plants that produce pharmaceuticals, nutrients, and nutraceuticals and (2) the technological challenges hindering our ability to generate plants producing health-promoting small molecules.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Computational prediction of plant metabolic pathways

Uncovering genes encoding enzymes responsible for the biosynthesis of diverse plant metabolites is essential for metabolic engineering and production of plant metabolite-derived medicine. With the availability of multi-omics data for an ever-increasing number of plant species and the development of computational approaches, the metabolic pathways of many important plant compounds can be predicted, complementing a more traditional genetic and/or biochemical approach. In this work, we summarize recent progress in predicting plant metabolic pathways using genome, transcriptome, proteome, interactome, and/or metabolome data, and the utility of integrating these data with machine learning to further improve metabolic pathway predictions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Metabolic flux optimization of iterative pathways through orthogonal gene expression control: Application to the β-oxidation reversal

Balancing relative expression of pathway genes to minimize flux bottlenecks and metabolic burden is one of the key challenges in metabolic engineering. This is especially relevant for iterative pathways, such as reverse β-oxidation (rBOX) pathway, which require control of flux partition at multiple nodes to achieve efficient synthesis of target products. Here, we develop a plasmid-based inducible system for orthogonal control of gene expression (referred to as the TriO system) and demonstrate its utility in the rBOX pathway. Leveraging effortless construction of TriO vectors in a plug-and-play manner, we simultaneously explored the solution space for enzyme choice and relative expression levels. Remarkably, varying individual expression levels led to substantial change in product specificity ranging from no production to optimal performance of about 90% of the theoretical yield of the desired products. We obtained titers of 6.3 g/L butyrate, 2.2 g/L butanol and 4.0 g/L hexanoate from glycerol in E. coli, which exceed the best titers previously reported using equivalent enzyme combinations. Since a similar system behavior was observed with alternative termination routes and higher-order iterations, we envision our approach to be broadly applicable to other iterative pathways besides the rBOX. Here, considering that high throughput, automated strain construction using combinatorial promoter and RBS libraries remain out of reach for many researchers, especially in academia, tools like the TriO system could democratize the testing and evaluation of pathway designs by reducing cost, time and infrastructure requirements.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

MassIVE MSV000085948 - Utilization of syringyl lignin-derived compounds and production of 2-pyrone-4,6-dicarboxylic acid by Pseudomonas putida KT2440: Lignin is an abundant aromatic polymer formed in plants by coupling reactions of p-coumaryl, coniferyl, and sinapyl alcohols. Pseudomonas putida KT2440 (P. putida KT2440), a robust soil bacterium, naturally catabolizes many aromatic compounds derived from lignin as carbon and energy sources and has been extensively engineered to valorize guaiacyl (G) and p-hydroxyphenyl (H) lignin-derived aromatic species to valuable chemicals. However, only a few studies report the capacity of Pseudomonads to grow on syringyl (S) lignin-derived compounds. In this work, we demonstrate that P. putida KT2440 catabolizes syringate in the presence of an auxiliary energy source. Overexpression of vanAB, encoding a native Rieske non-heme iron monooxygenase, enabled catabolism of syringate and syringaldehyde without an additional carbon and energy source. In vitro biochemical characterization of VanAB confirmed that this enzyme O-demethylates syringate to 3-O-methylgallate and, subsequently, 3-O-methylgallate to gallate, albeit 4- and 55-fold less efficiently than vanillate, respectively. Transcriptomics and proteomics experiments demonstrate that the galADBC transcripts and enzymes required for gallate catabolism are induced when vanAB is overexpressed during cultivation in syringate. Further, we show that that the endogenous protocatechuate-3,4-dioxygenase PcaHG ring-opens gallate to produce 2-pyrone-4,6-dicarboxylic acid (PDC), a promising bio-based chemical. Constitutive overexpression of vanAB and pcaHG along with galA deletion enabled PDC production from SA at a 70% (mol/mol) yield. Furthermore, leveraging the LigAB dioxygenase from Sphingobium sp. SYK-6 and VanAB O-demethylase from Pseudomonas sp. HR199 enabled simultaneous conversion of monomers with S-, G-, and H-type functionality to PDC at 82% (mol/mol) yield. Overall, this study elucidates a native pathway for catabolism of syringyl lignin-derived compounds in P. putida KT2440 and demonstrates the potential of this robust host for biological funneling of substrates derived from all three canonical monolignols.

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Beckham, Gregg↗
NASA NTRSDate not supplied

Mitochondria Dysfunction Central in Driving Health Risks Associated with Spaceflight

Background: Determining the biological impact of spaceflight through novel approaches is essential to reduce the health risks to astronauts for long-term space missions. The current established health risks due to spaceflight are only reflecting known symptomatic and physiologic responses and do not reflect early onset of other potential diseases. There are many unknown variables which still need to be identified to fully understand the health impacts due to the environmental factors in space. Materials and Methods: One method to uncover potential novel biological mechanisms responsible for health risks in astronauts is by utilizing NASA’s GeneLab platform (genelab.nasa.gov). GeneLab is public repository that hosts multiple omics datasets generated from space biology experiments that include experiments flown in space, simulated cosmic radiation experiments, and simulated microgravity experiments. This presentation will provide an example of analysis and novel hypothesis generation that is being produced with GeneLab datasets. A comprehensive multi-omics approach was implemented correlating transcriptomics, proteomics, metabolomics, and methylation analysis. Results: We found that cells have stronger overall biological response than the tissues to spaceflight, with mitochondrial activity and innate immunity pathways being heavily impacted. NASA Twin Study results are consistent with a specific alteration in mitochondrial ATP production. Our results indicate that the space environment can directly induce mitochondrial damage, with mitochondrial dysfunctions being a cause for chronic inflammation and both being involved in the development of metabolic disorders that cause changes in lipid metabolism. We also found biological changes occurring during spaceflight with cell cycle, circadian rhythm and olfactory activity pathways can also influence and be influenced by alterations on mitochondrial activity

Afshin Beheshti↗
DOE OSTI2023

Evolutionary diversity of proton and water channels on the oxidizing side of photosystem II and their relevance to function

One of the reasons for the high efficiency and selectivity of biological catalysts arise from their ability to control the pathways of substrates and products using protein channels, and by modulating the transport in the channels using the interaction with the protein residues and the water/hydrogen-bonding network. Here, this process is clearly demonstrated in Photosystem II (PS II), where its light-driven water oxidation reaction catalyzed by the Mn 4 CaO 5 cluster occurs deep inside the protein complex and thus requires the transport of two water molecules to and four protons from the metal center to the bulk water. Based on the recent advances in structural studies of PS II from X-ray crystallography and cryo-electron microscopy, in this review we compare the channels that have been proposed to facilitate this mass transport in cyanobacteria, red and green algae, diatoms, and higher plants. The three major channels (O1, O4, and Cl1 channels) are present in all species investigated; however, some differences exist in the reported structures that arise from the different composition and arrangement of membrane extrinsic subunits between the species. Among the three channels, the Cl1 channel, including the proton gate, is the most conserved among all photosynthetic species. We also found at least one branch for the O1 channel in all organisms, extending all the way from Ca/O1 via the ‘water wheel’ to the lumen. However, the extending path after the water wheel varies between most species. The O4 channel is, like the Cl1 channel, highly conserved among all species while having different orientations at the end of the path near the bulk. The comparison suggests that the previously proposed functionality of the channels in T. vestitus (Ibrahim et al., Proc Natl Acad Sci USA 117:12624–12635, 2020; Hussein et al., Nat Commun 12:6531, 2021) is conserved through the species, i.e. the O1-like channel is used for substrate water intake, and the tighter Cl1 and O4 channels for proton release. The comparison does not eliminate the potential role of O4 channel as a water intake channel. However, the highly ordered hydrogen-bonded water wire connected to the Mn 4 CaO 5 cluster via the O4 may strongly suggest that it functions in proton release, especially during the S 0 → S 1 transition (Saito et al., Nat Commun 6:8488, 2015; Kern et al., Nature 563:421–425, 2018; Ibrahim et al., Proc Natl Acad Sci USA 117:12624–12635, 2020; Sakashita et al., Phys Chem Chem Phys 22:15831–15841, 2020; Hussein et al., Nat Commun 12:6531, 2021).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Metastable Clusters and Competitive Solvation Tune Ion Pairing at Liquid Interfaces

The balance of hydrophobic and hydrophilic interactions underlies emergent phenomena in complex multicomponent chemical systems. Here, we show that a supposedly ‘non–interacting’ nonpolar phase can be used to competitively solvate amphiphilic molecules at an oil/aqueous interface. This solvation, as probed by surface specific nonlinear spectroscopy and simulations, results in a molecularly thin corrugated phase boundary featuring metastable assemblies that alter the hydrogen bonding networks of water and the apparent ‘hard/soft’ descriptors used to describe ionic interactions. We show that competitive solvation enhances amphiphile mobility, opening up otherwise energetically inaccessible complexes that transiently interact with aqueous phase ions. These transient species impact ensemble binding affinities and may represent the molecular agents responsible for aspects of ionic transport and function. In conclusion, the result of this work highlights how seemingly unrelated nonpolar interactions feedback onto aqueous phase chemical phenomena, providing a pathway to tune phase separation and self-assembly to access new reaction pathways using interfaces for a range of chemical and biological systems.

Anions↗
DOE OSTI2022

TbasCO: trait-based comparative ‘omics identifies ecosystem-level and niche-differentiating adaptations of an engineered microbiome

A grand challenge in microbial ecology is disentangling the traits of individual populations within complex communities. Various cultivation-independent approaches have been used to infer traits based on the presence of marker genes. However, marker genes are not linked to traits with complete fidelity, nor do they capture important attributes, such as the timing of gene expression or coordination among traits. To address this, we present an approach for assessing the trait landscape of microbial communities by statistically defining a trait attribute as a shared transcriptional pattern across multiple organisms. Leveraging the KEGG pathway database as a trait library and the Enhanced Biological Phosphorus Removal (EBPR) model microbial ecosystem, we demonstrate that a majority (65%) of traits present in 10 or more genomes have niche-differentiating expression attributes. For example, while many genomes containing high-affinity phosphorus transporter pstABCS display a canonical attribute (e.g. up-regulation under phosphorus starvation), we identified another attribute shared by many genomes where transcription was highest under high phosphorus conditions. Taken together, we provide a novel framework for unravelling the functional dynamics of uncultivated microorganisms by assigning trait-attributes through genome-resolved time-series metatranscriptomics.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

ADP enhances the allosteric activation of eukaryotic elongation factor 2 kinase by calmodulin

Protein translation, one of the most energy-consumptive processes in a eukaryotic cell, requires robust regulation, especially under energy-deprived conditions. A critical component of this regulation is the suppression of translational elongation through reduced ribosome association of the GTPase eukaryotic elongation factor 2 (eEF-2) resulting from its specific phosphorylation by the calmodulin (CaM)-activated α–kinase eEF-2 kinase (eEF-2K). It has been suggested that the eEF-2K response to reduced cellular energy levels is indirect and mediated by the universal energy sensor AMP-activated protein kinase (AMPK) through direct stimulatory phosphorylation and/or downregulation of the eEF-2K-inhibitory nutrient-sensing mTOR pathway. Here, we provide structural, biochemical, and cell-biological evidence of a direct energy-sensing role of eEF-2K through its stimulation by ADP. A crystal structure of the nucleotide-bound complex between CaM and the functional core of eEF-2K phosphorylated at its primary stimulatory site (T348) reveals ADP bound at a unique pocket located on the face opposite that housing the kinase active site. Within this basic pocket (BP), created at the CaM/eEF-2K interface upon complex formation, ADP is stabilized through numerous interactions with both interacting partners. Biochemical analyses using wild-type eEF-2K and specific BP mutants indicate that ADP stabilizes CaM within the active complex, increasing the sensitivity of the kinase to CaM. Induction of energy stress through glycolysis inhibition results in significantly reduced enhancement of phosphorylated eEF-2 levels in cells expressing ADP-binding compromised BP mutants compared to cells expressing wild-type eEF-2K. These results suggest a direct energy-sensing role for eEF-2K through its cooperative interaction with CaM and ADP.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Engineered Cell Line Imaging Assay Differentiates Pathogenic from Non-Pathogenic Bacteria

Cell culture systems have greatly expanded our understanding of how bacterial pathogens target signaling pathways to manipulate the host and cause infection. Advances in genetic engineering have allowed for the creation of fluorescent protein readouts within signaling pathways, but these techniques have been underutilized in pathogen biology. Here, we genetically engineered a lung cell line with fluorescent reporters for extracellular signal-related kinase (ERK) and the downstream transcription factor FOS-related antigen 1 (Fra1) and evaluated signaling after inoculation with pathogenic and non-pathogenic bacteria. Cells were inoculated with 100 colony-forming units of Acinetobacter baylyi, Klebsiella pneumoniae, Pseudomonas aeruginosa, Streptococcus agalactiae, or Staphylococcus epidermidis and imaged in a multi-mode reader. The alamarBlue cell viability assay was used as a reference test and showed that pathogenic P. aeruginosa induced significant (p < 0.05) cell death after 8 h in both wild-type and engineered cell lines compared to non-pathogenic S. epidermidis. In engineered cells, we found that Fra1 signaling was disrupted in as little as 4 h after inoculation with bacterial pathogens compared to delayed disruption in signaling by non-pathogenic S. epidermidis. Overall, we demonstrate that low levels of pathogenic versus non-pathogenic bacteria can be rapidly and sensitively screened based on ERK-Fra1 signaling

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2000

Viral carcinogenesis: revelation of molecular mechanisms and etiology of human disease

The RNA and DNA tumor viruses have made fundamental contributions to two major areas of cancer research. Viruses were vital, first, to the discovery and analysis of cellular growth control pathways and the synthesis of current concepts of cancer biology and, second, to the recognition of the etiology of some human cancers. Transforming retroviruses carry oncogenes derived from cellular genes that are involved in mitogenic signalling and growth control. DNA tumor viruses encode oncogenes of viral origin that are essential for viral replication and cell transformation; viral oncoproteins complex with cellular proteins to stimulate cell cycle progression and led to the discovery of tumor suppressors. Viral systems support the concept that cancer development occurs by the accumulation of multiple cooperating events. Viruses are now accepted as bona fide etiologic factors of human cancer; these include hepatitis B virus, Epstein-Barr virus, human papillomaviruses, human T-cell leukemia virus type I and hepatitis C virus, plus several candidate human cancer viruses. It is estimated that 15% of all human tumors worldwide are caused by viruses. The infectious nature of viruses distinguishes them from all other cancer-causing factors; tumor viruses establish long-term persistent infections in humans, with cancer an accidental side effect of viral replication strategies. Viruses are usually not complete carcinogens, and the known human cancer viruses display different roles in transformation. Many years may pass between initial infection and tumor appearance and most infected individuals do not develop cancer, although immunocompromised individuals are at elevated risk of viral-associated cancers. Variable factors that influence viral carcinogenesis are reviewed, including possible synergy between viruses and environmental cofactors. The difficulties in establishing an etiologic role for a virus in human cancer are discussed, as well as the different approaches that proved viral links to cancer. Future directions for tumor virus studies are considered.

NASA Program Biomedical Research and Countermeasur↗
NASA NTRS2014

Biosentinel: Developing a Space Radiation Biosensor

Ionizing radiation presents a major challenge to human exploration and long-term residence in space. The deep-space radiation spectrum includes highly energetic particles that generate double strand breaks (DSBs), deleterious DNA lesions that are usually repaired without errors via homologous recombination (HR), a conserved pathway in all eukaryotes. While progress identifying and characterizing biological radiation effects using Earth-based facilities has been significant, no terrestrial source duplicates the unique space radiation environment.We are developing a biosensor-based nanosatellite to fly aboard NASAs Space Launch System Exploration Mission 1, expected to launch in 2017 and reach a 1AU (astronomic unit) heliocentric orbit. Our biosensor (called BioSentinel) uses the yeast S. cerevisiae to measure DSBs in response to ambient space radiation. The BioSentinel strain contains engineered genetic defects that prevent growth until and unless a radiation-induced DSB near a reporter gene activates the yeasts HR repair mechanisms. Thus, culture growth and metabolic activity directly indicate a successful DSB-and-repair event. In parallel, HR-defective and wild type strains will provide survival data. Desiccated cells will be carried within independent culture microwells, built into 96-well microfluidic cards. Each microwell set will be activated by media addition at different time points over 18 months, and cell growth will be tracked continuously via optical density. One reserve set will be activated only in the occurrence of a solar particle event. Biological measurements will be compared to data provided by onboard physical dosimeters and to Earth-based experiments.BioSentinel will conduct the first study of biological response to space radiation outside Low Earth Orbit in over 40 years. BioSentinel will thus address strategic knowledge gaps related to the biological effects of space radiation and will provide an adaptable platform to perform human-relevant measurements in multiple space environments. We hope that it can therefore be used on the ISS, on and around other planetary bodies as well as other exploration platforms as a self-contained system that will allow us to compare and calibrate different radiation environments.BioSentinels results will be critical for improving interpretation of the effects of space radiation exposure, and for reducing the risk associated with long-term human exploration.

space radiation↗
DOE OSTI2022

Rewiring yeast metabolism for producing 2,3-butanediol and two downstream applications: Techno-economic analysis and life cycle assessment of methyl ethyl ketone (MEK) and agricultural biostimulant production

Rising concerns for sustainability and global climate change have driven the development of sustainable pro- duction pathways for biofuels and chemicals from lignocellulosic biomass via integrated biological and chemical processes. We constructed an engineered Saccharomyces cerevisiae capable of producing 2,3-butanediol (2,3-BDO) from glucose without accumulating ethanol and glycerol, which hinder downstream processing of 2,3-BDO, through extensive metabolic reprogramming. Specifically, we introduced heterologous 2,3-BDO biosynthetic enzymes and deleted the major isozymes of ethanol and glycerol biosynthetic enzymes. In addition, we introduced an NAD + regenerating Pyruvate-Malate (PM) cycle and enhanced the NAD + regenerating capability of the PM cycle to resolve the redox imbalance from the deletion of ethanol and glycerol production pathways. The resulting engineered yeast produced 109.9 g/L of 2,3-BDO with a productivity of 1.0 g/L/h and a yield of 0.36 g/ g glucose in a fed-batch fermentation. We also conducted techno-economic analysis (TEA) and life cycle assessment (LCA) of the production of methyl ethyl ketone (MEK) through catalytic dehydration of 2,3-BDO. A TEA based on the experimental results indicated that the minimum product selling price (MPSP) was estimated to be $1.90/kg. Regarding cradle-to-grave LCA, 100-year global warming potential (GWP100) and fossil energy consumption (FEC) were found to be 0.37 kg CO 2 eq/kg and 3.1 MJ/kg, respectively. These results demonstrated the feasibility of cost-competitive and sustainable bio-based MEK production via yeast fermentation. In addition, we explored the possibility of using the fermentation broth containing 2,3-BDO as a biostimulant inducing drought tolerance in plants. As a result, the yeast 2,3-BDO fermentation broth can induce drought tolerance in Arabidopsis thaliana without a complicated purification process.

2,3-butanediol (2,3-BDO)↗
DOE OSTI2020

High-Throughput Large-Scale Targeted Proteomics Assays for Quantifying Pathway Proteins in Pseudomonas putida KT2440

Targeted proteomics is a mass spectrometry-based protein quantification technique with high sensitivity, accuracy, and reproducibility. As a key component in the multi-omics toolbox of systems biology, targeted liquid chromatography-selected reaction monitoring (LC-SRM) measurements are critical for enzyme and pathway identification and design in metabolic engineering. To fulfill the increasing need for analyzing large sample sets with faster turnaround time in systems biology, high-throughput LC-SRM is greatly needed. Even though nanoflow LC-SRM has better sensitivity, it lacks the speed offered by microflow LC-SRM. Recent advancements in mass spectrometry instrumentation significantly enhance the scan speed and sensitivity of LC-SRM, thereby creating opportunities for applying the high speed of microflow LC-SRM without losing peptide multiplexing power or sacrificing sensitivity. Here, we studied the performance of microflow LC-SRM relative to nanoflow LC-SRM by monitoring 339 peptides representing 132 enzymes in Pseudomonas putida KT2440 grown on various carbon sources. The results from the two LC-SRM platforms are highly correlated. In addition, the response curve study of 248 peptides demonstrates that microflow LC-SRM has comparable sensitivity for the majority of detected peptides and better mass spectrometry signal and chromatography stability than nanoflow LC-SRM.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

13 C-metabolic flux analysis of Clostridium ljungdahlii illuminates its core metabolism under mixotrophic culture conditions

Carbon dioxide-fixing acetogenic bacteria (acetogens) utilizing the Wood-Ljungdahl Pathway (WLP) play an important role in CO 2 fixation in the biosphere and in the development of biological processes – alone or in cocultures, under both autotrophic and mixotrophic conditions – for production of chemicals and fuels. To date, limited work has been reported in experimentally validating and quantifying reaction fluxes of their core metabolic pathways. Here, the core metabolic model of the acetogen Clostridium ljungdahlii was interrogated using 13 C-metabolic flux analysis ( 13 C-MFA), which required the development of a new defined culture medium. Autotrophic, heterotrophic, and mixotrophic growth in defined medium was possible by adding 1 mM methionine to replace yeast extract. Our 13 C-MFA found an incomplete TCA cycle and inactive core pathways/reactions, notably those of the oxidative pentose phosphate pathway, Entner-Doudoroff pathway, and malate dehydrogenase. 13 C-MFA during mixotrophic growth using the parallel tracers [1– 13 C]fructose, [1,2– 13 C]fructose, [1,2,3– 13 C]fructose, and [U– 13 C]asparagine found that externally supplied CO 2 contributed the majority of carbon consumed. All internally-produced CO 2 from the catabolism of asparagine and fructose was consumed by the WLP. While glycolysis of fructose was active, it was not a major contributor to overall production of ATP, NADH, and acetyl-CoA. Gluconeogenic reactions were active despite the availability of organic carbon. Asparagine was catabolized equally via conversion to threonine and subsequent cleavage to produce acetaldehyde and glycine, and via deamination to fumarate and then the anaplerotic conversion of malate to pyruvate. Both pathways for asparagine catabolism produced acetyl-CoA, either directly via pyruvate or indirectly via the WLP. Cofactor stoichiometry based on our data predicted an essentially zero flux through the ferredoxin-dependent transhydrogenase (Nfn) reaction. Instead, nearly all of NADPH generated from the hydrogenase reaction was consumed by the WLP. Reduced ferredoxin produced by the hydrogenase reaction and glycolysis was mostly used for ATP generation via the RNF/ATPase system, with the remainder consumed by the WLP. NADH produced by RNF/ATPase was entirely consumed via the WLP.

13C metabolic flux analysis↗
DOE OSTI2020

MONACO: accurate biological network alignment through optimal neighborhood matching between focal nodes

Motivation: Alignment of protein–protein interaction networks can be used for the unsupervised prediction of functional modules, such as protein complexes and signaling pathways, that are conserved across different species. To date, various algorithms have been proposed for biological network alignment, many of which attempt to incorporate topological similarity between the networks into the alignment process with the goal of constructing accurate and biologically meaningful alignments. Especially, random walk models have been shown to be effective for quantifying the global topological relatedness between nodes that belong to different networks by diffusing node-level similarity along the interaction edges. However, these schemes are not ideal for capturing the local topological similarity between nodes. Results: Here, we propose MONACO, a novel and versatile network alignment algorithm that finds highly accurate pairwise and multiple network alignments through the iterative optimal matching of ‘local’ neighborhoods around focal nodes. Extensive performance assessment based on real networks as well as synthetic networks, for which the ground truth is known, demonstrates that MONACO clearly and consistently outperforms all other state-of-the-art network alignment algorithms that we have tested, in terms of accuracy, coherence and topological quality of the aligned network regions. Furthermore, despite the sharply enhanced alignment accuracy, MONACO remains computationally efficient and it scales well with increasing size and number of networks.

97 MATHEMATICS AND COMPUTING↗