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At least 793 records · Page 44

Evaluation of 1,2-diacyl-3-acetyl triacylglycerol production in Yarrowia lipolytica

Plants produce many high-value oleochemical molecules. While oil-crop agriculture is performed at industrial scales, suitable land is not available to meet global oleochemical demand. Worse, establishing new oil-crop farms often comes with the environmental cost of tropical deforestation. The field of metabolic engineering offers tools to transplant oleochemical metabolism into tractable hosts while simultaneously providing access to molecules produced by non-agricultural plants. Here, we evaluate strategies for rewiring metabolism in the oleaginous yeast Yarrowia lipolytica to synthesize a foreign lipid, 3-acetyl-1,2-diacyl-sn-glycerol (acTAG). Oils made up of acTAG have a reduced viscosity and melting point relative to traditional triacylglycerol oils making them attractive as low-grade diesels, lubricants, and emulsifiers. Furthermore, this manuscript describes a metabolic engineering study that established acTAG production at g/L scale, exploration of the impact of lipid bodies on acTAG titer, and a techno-economic analysis that establishes the performance benchmarks required for microbial acTAG production to be economically feasible.

59 BASIC BIOLOGICAL SCIENCES↗

A Dual-Gated Structures for Lossless Ion Manipulations-Ion Mobility Orbitrap Mass Spectrometry Platform for Combined Ultra-High-Resolution Molecular Analysis

High-resolution ion mobility spectrometry-mass spectrometry (HR-IMS-MS) instruments have enormously advanced the ability to characterize complex biological mixtures. Unfortunately, HR-IMS and HR-MS measurements are typically performed independently due to mismatches in analysis time scales. Here we overcome this limitation by using a dual-gated ion injection approach to couple an 11-meter path length structures for lossless ion manipulations (SLIM) module to a Q-Exactive Plus Orbitrap MS. The dual-gate setup was implemented by placing one ion gate before the SLIM module and a second ion gate after. The dual-gated ion injection approach allowed the new SLIM-Orbitrap platform to simultaneously perform an 11-meter SLIM separation, Orbitrap mass analysis using the highest selectable mass resolution setting (up to 140k), and high-energy collision induced dissociation (HCD) in ~25 minutes over an $m/z$ range of ~1500 amu. The SLIM-Orbitrap was initially characterized using a mixture of standard phosphazene cations and demonstrated an average SLIM CCS resolving power (Rp CCS ) of ~218 and SLIM peak capacity of ~156 while simultaneously obtaining high mass resolutions. SLIM-Orbitrap analysis with fragmentation was then performed on mixtures of standard peptides and two reverse peptides (SDGRG 1+ , GRGDS 1+ , Rp CCS = 305) to demonstrate the utility of combined HR-IMS-MS/MS measurements for peptide identification. Our new HR-IMS-MS/MS capability was further demonstrated by analyzing a complex lipid mixture and showcasing SLIM separations on isobaric lipids. In conclusion, this new SLIM-Orbitrap platform demonstrates a critical new capability for proteomics and lipidomics applications, and the high-resolution multimodal data obtainable with this system establishes the foundation for reference-free identification of unknown ion structures.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Quantifying Phospholipids in Organic Samples Using a Hydrophilic Interaction Liquid Chromatography–Inductively Coupled Plasma High-Resolution Mass Spectrometry (HILIC-ICP-HRMS) Method

Here, in this study, a novel method using hydrophilic interaction liquid chromatography (HILIC) coupled with inductively coupled plasma high-resolution mass spectrometry (ICP-HRMS) was introduced for the quantification of phospholipids in oil samples. The method employed a bridged ethyl hybrid (BEH) stationary phase HILIC column with a tetrahydrofuran (THF)/water mobile phase, enhancing the solubility and detection of phospholipids. During the study, a gradient/matrix effect on ICP-HRMS sensitivity was observed and successfully compensated for experimentally, ensuring reliable quantification results. This approach has proven effective for a wide range of different oil samples including vegetable oils, animal fats, and phospholipid supplements. Notably, this method allowed the direct quantification of phospholipids in oil samples, bypassing the need for prior sample preparation methods, such as solid phase extraction (SPE), thereby streamlining the analytical process. The precision, accuracy, and reduced need for extensive sample preparation offered by this method mark a significant advancement in lipids analysis. Its robustness and broad applicability have substantial implications for industries such as food and renewable energy production, where both efficient and accurate lipid identification and quantification are crucial.

09 BIOMASS FUELS↗

Neuronal Plasma Membranes as Supramolecular Assemblies for Biological Memory

Biological memory is the ability to develop, retain, and retrieve information over time. Currently, it is widely accepted that memories are stored in synapses (i.e., connections between brain cells throughout the brain) through a process known as synaptic plasticity, which leads to either long-term potentiation (LTP) or long-term depression (LTD). However, the strengthening (LTP) and weakening (LTD) of synapses involve post-translational modifications to neural networks requiring de novo gene expression, a lengthy and energetically expensive process. Recently, we observed that lipid bilayers in the absence of peptides/proteins are capable of LTP, not unlike what has been observed in mammals and birds. As such, this finding has prompted us to postulate that the lipid bilayer provides a good model for understanding the molecular basis of biological memory. Here, in this article, we discuss the status, challenges, and opportunities of neuronal plasma membranes as structures for biological memory and learning, therapeutic targets for various brain disorders, and platforms for neural network developments.

59 BASIC BIOLOGICAL SCIENCES↗

Subventricular Accumulation of Cu in the Aging Mouse Brain Does Not Associate with Anticipated Increases in Markers of Oxidative Stress

Natural aging is associated with mild memory loss and cognitive decline, and age is the greatest risk factor for neurodegenerative diseases, such as Alzheimer’s disease. There is substantial evidence that oxidative stress is a major contributor to both natural aging and neurodegenerative disease, and coincidently, levels of redox active metals such as Fe and Cu are known to be elevated later in life. Recently, a pronounced age-related increase in Cu content has been reported to occur in mice and rats around a vital regulatory brain region, the subventricular zone of lateral ventricles. In our study herein, we have characterized lateral ventricle Cu content in a unique murine model of accelerated aging, senescence accelerated mouse-prone 8 (SAMP8) mice. Our results confirm an age-related increase in ventricle Cu content, consistent with the studies by others in wild-type mice and rats. Specifically, we observed Cu content to increase over the time frame 1 to 5 months and 5 to 9 months, but interestingly, no significant increase occurred between 9 and 12 months (although brain Cu content at 12 months was significantly elevated relative to 1 and 5 month-old animals). Despite the magnitude of Cu increase observed within the cells that comprise the subventricular zone of lateral ventricles (average 3 mM Cu, with isolated subcellular concentrations of 17 mM), we did not detect spectroscopic markers of thiol oxidation, protein aggregation, or lipid oxidation. The lack of evidence for oxidative stress in ex vivo animal tissue is in contrast to in vitro studies demonstrating that thiol, protein, and lipid oxidation is pronounced at these Cu concentrations. We suggest that our findings most likely indicate that the Cu ions in this brain region are sequestered in an unreactive form, possibly extended chains of Cu-thiolate complexes, which do not readily redox cycle in the aqueous cytosol. In conclusion, these results also appear to partially challenge the long-held view that age-related increases in brain metal content drive oxidative stress as we did not observe a concomitant association between age-related Cu increase and markers of oxidative stress, nor did we observe a net increase in Cu content between mice aged 9 and 12 months.

X-ray fluorescence↗

Anaerobic Storage and Conversion of Microalgal Biomass to Manage Seasonal Variation in Cultivation

Seasonal variation in microalgal biomass production is a well-known challenge when optimizing economics for algal fuel conversion, especially given the fluctuation in biomass production between winter and summer. Wet storage offers significant potential for cost and energy savings compared to dewatering and dry storage. This study demonstrates the feasibility of preserving harvested Scenedesmus acutus biomass through wet anaerobic storage for use in biochemical conversion. Anaerobic storage effectively preserved biomass with minimal degradation of carbohydrates and preservation of lipids and proteins. Screening experiments identified optimal pretreatment conditions for stored biomass. Scale-up of pretreatment enabled fermentation of the hydrolysate to butyric acid and indicated no observable difference in conversion between unstored and stored biomass. Lipid extraction improved by a relative 12% for stored biomass. These results suggest that wet anaerobic storage can effectively manage seasonal variation in biomass production and is compatible with biochemical approaches for biofuel production.

29 ENERGY PLANNING, POLICY, AND ECONOMY↗

Comparing Liquid Vortex Capture & the Rapid Droplet Sampling Interface for Single Cell Mass Spectrometry

High-throughput single-cell mass spectrometry is a rapidly evolving field that requires innovative sampling and ionization techniques to balance speed, sensitivity, and reliability for metabolomic and lipidomic analyses. This study provides a comparative analysis of two cutting-edge ionization platforms for single-cell analysis: Liquid Vortex Capture (LVC) and Rapid Droplet Sampling Interface (RDSI). The performance was benchmarked by testing pharmaceuticals, EquiSPLASH, and single-cell experiments. RDSI demonstrated up to 100-fold improvements in sensitivity for drugs and lipids such as propranolol, amiodarone, atorvastatin, and phosphocholines in water and phosphate-buffered solutions. This was attributed to its low-flow rate operation (3 μL/min) and reduced dilution. Conversely, LVC excelled in handling higher liquid volumes with greater reproducibility due to its higher solvent flow rate (200 μL/min), enabling increased dilution, solubility, and cleaning. Single-cell uptake of atorvastatin incubated for 10 min, or amiodarone incubated for 24 h in HepG2 cells, similarly revealed up to 85-fold enhancement in sensitivity by RDSI for drugs and lipids. These findings highlight the potential of RDSI for enhancing sensitivity in single-cell drug monitoring and lipidomics.

Cahill, John [ORNL] (ORCID:0000000298664010)↗

Graphene-based sensing of oxygen transport through pulmonary membranes

Lipid-protein complexes are the basis of pulmonary surfactants covering the respiratory surface and mediating gas exchange in lungs. Cardiolipin is a mitochondrial lipid overexpressed in mammalian lungs infected by bacterial pneumonia. In addition, increased oxygen supply (hyperoxia) is a pathological factor also critical in bacterial pneumonia. In this paper we fabricate a micrometer-size graphene-based sensor to measure oxygen permeation through pulmonary membranes. Combining oxygen sensing, X-ray scattering, and Atomic Force Microscopy, we show that mammalian pulmonary membranes suffer a structural transformation induced by cardiolipin. We observe that cardiolipin promotes the formation of periodic protein–free inter–membrane contacts with rhombohedral symmetry. Membrane contacts, or stalks, promote a significant increase in oxygen gas permeation which may bear significance for alveoli gas exchange imbalance in pneumonia.

36 MATERIALS SCIENCE↗

Ligand dependent interaction between PC-TP and PPARδ mitigates diet-induced hepatic steatosis in male mice

Phosphatidylcholine transfer protein (PC-TP; synonym StarD2) is a soluble lipid-binding protein that transports phosphatidylcholine (PC) between cellular membranes. To better understand the protective metabolic effects associated with hepatic PC-TP, we generated a hepatocyte-specific PC-TP knockdown (L-Pctp -/- ) in male mice, which gains less weight and accumulates less liver fat compared to wild-type mice when challenged with a high-fat diet. Hepatic deletion of PC-TP also reduced adipose tissue mass and decreases levels of triglycerides and phospholipids in skeletal muscle, liver and plasma. Gene expression analysis suggest that the observed metabolic changes are related to transcriptional activity of peroxisome proliferative activating receptor (PPAR) family members. An in-cell protein complementation screen between lipid transfer proteins and PPARs uncovered a direct interaction between PC-TP and PPARδ that was not observed for other PPARs. We confirmed the PC-TP– PPARδ interaction in Huh7 hepatocytes, where it was found to repress PPARδ-mediated transactivation. Mutations of PC-TP residues implicated in PC binding and transfer reduce the PC-TP-PPARδ interaction and relieve PC-TP-mediated PPARδ repression. Reduction of exogenously supplied methionine and choline reduces the interaction while serum starvation enhances the interaction in cultured hepatocytes. Together our data points to a ligand sensitive PC-TP– PPARδ interaction that suppresses PPAR activity.

59 BASIC BIOLOGICAL SCIENCES↗

Coupling sensor to enzyme in the voltage sensing phosphatase

Voltage-sensing phosphatases (VSPs) dephosphorylate phosphoinositide (PIP) signaling lipids in response to membrane depolarization. VSPs possess an S4-containing voltage sensor domain (VSD), resembling that of voltage-gated cation channels, and a lipid phosphatase domain (PD). The mechanism by which voltage turns on enzyme activity is unclear. Structural analysis and modeling suggest several sites of VSD-PD interaction that could couple voltage sensing to catalysis. Voltage clamp fluorometry reveals voltage-driven rearrangements in three sites implicated earlier in enzyme activation—the VSD-PD linker, gating loop and R loop—as well as the N-terminal domain, which has not yet been explored. N-terminus mutations perturb both rearrangements in the other segments and enzyme activity. Our results provide a model for a dynamic assembly by which S4 controls the catalytic site.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Structural basis of undecaprenyl phosphate glycosylation leading to polymyxin resistance in Gram-negative bacteria

In Gram-negative bacteria, the enzymatic modification of Lipid A with aminoarabinose (L-Ara4N) leads to resistance against polymyxin antibiotics and cationic antimicrobial peptides. ArnC, an integral membrane glycosyltransferase, attaches a formylated form of aminoarabinose to the lipid undecaprenyl phosphate, enabling its association with the bacterial inner membrane. Here, we present cryo-electron microscopy structures of ArnC from S. enterica in apo and nucleotide-bound conformations. These structures reveal a conformational transition that takes place upon binding of the partial donor substrate. Using coarse-grained and atomistic simulations, we provide insights into substrate coordination before and during catalysis, and we propose a catalytic mechanism that may operate on all similar metal-dependent polyprenyl phosphate glycosyltransferases. The reported structures provide a new target for drug design aiming to combat polymyxin resistance.

Ashraf, Khuram U. [Rutgers University, Newark, NJ ↗

Neuroinflammation is dependent on sex and ovarian hormone presence following acute woodsmoke exposure

Woodsmoke (WS) exposure is associated with significant health-related sequelae. Different populations can potentially exhibit varying susceptibility, based on endocrine phenotypes, to WS and investigating neurological impacts following inhaled WS is a growing area of research. In this study, a whole-body inhalation chamber was used to expose both male and female C57BL/6 mice (n = 8 per group) to either control filtered air (FA) or acute WS (0.861 ± 0.210 mg/m 3 ) for 4 h/d for 2 days. Neuroinflammatory and lipid-based biological markers were then assessed. In a second set of studies, female mice were divided into two groups: one group was ovariectomized (OVX) to simulate an ovarian hormone-deficient state (surgical menopause), and the other underwent Sham surgery as controls, to mechanistically assess the impact of ovarian hormone presence on neuroinflammation following FA and acute WS exposure to simulate an acute wildfire episode. There was a statistically significant impact of sex (P ≤ 0.05) and statistically significant interactions between sex and treatment in IL-1β, CXCL-1, TGF-β, and IL-6 brain relative gene expression. Hippocampal and cortex genes also exhibited significant changes in acute WS-exposed Sham and OVX mice, particularly in TGF-β (hippocampus) and CCL-2 and CXCL-1 (cortex). Cortex GFAP optical density (OD) showed a notable elevation in male mice exposed to acute WS, compared to the control FA. Sham and OVX females demonstrated differential GFAP expression, depending on brain region. Overall, targeted lipidomics in phosphatidylcholine (PC) and phosphatidylethanolamine (PE) serum and brain lipids demonstrated more significant changes between control FA and acute WS exposure in female mice, compared to males. In summary, male and female mice show distinct neuroinflammatory markers in response to acute WS exposure. Furthermore, ovarian hormone deficiency may impact the neuroinflammatory response following an acute WS event.

59 BASIC BIOLOGICAL SCIENCES↗

Neutrons reveal the dynamics of leaf thylakoids in living plants

The study is the first known exploration of photosynthetic membranes dynamics in living plants by high resolution quasielastic neutron scattering spectroscopy. We investigated the mobility and flexibility of thylakoid membranes in common duckweed (Landoltia punctata) and identified dynamics across various length scales corresponding to individual membranes and membranes stack. We employed classical models typically used to study lipid bilayers to characterize the undulation modes and rigidity of the membranes and reveal how structural variations influence the observed complex dynamics. Our findings show that the stacks of thylakoids in duckweed behave as rigid systems, exhibiting an effective bending coefficient in the lower range associated with surfactant membranes. In contrast, the single thylakoid leaflets display greater apparent flexibility and are well situated within the bi-continuous surfactant phase dynamics. While our observations enhance the understanding of the intricate architecture and mobility of photosynthetic cellular machinery, they also highlight the limitations of applying ideal lipid membranes models to describe complex biological systems. This work opens more questions and the need for further investigations across extended length and time scales, as well as the importance of rigorous sample preparation and experimental control.

Applied physics↗

A multi-omic characterization of temperature stress in a halotolerant Scenedesmus strain for algal biotechnology

Microalgae efficiently convert sunlight into lipids and carbohydrates, offering bio-based alternatives for energy and chemical production. Improving algal productivity and robustness against abiotic stress requires a systems level characterization enabled by functional genomics. Here, we characterize a halotolerant microalga Scenedesmus sp. NREL 46B-D3 demonstrating peak growth near 25 °C that reaches 30 g/m 2 /day and the highest biomass accumulation capacity post cell division reported to date for a halotolerant strain. Functional genomics analysis revealed that genes involved in lipid production, ion channels and antiporters are expanded and expressed. Exposure to temperature stress shifts fatty acid metabolism and increases amino acids synthesis. Co-expression analysis shows that many fatty acid biosynthesis genes are overexpressed with specific transcription factors under cold stress. These and other genes involved in the metabolic and regulatory response to temperature stress can be further explored for strain improvement.

09 BIOMASS FUELS↗

Insights into pathological mechanisms and interventions revealed by analyzing a mathematical model for cone metabolism

This work analyzes a mathematical model for the metabolic dynamics of a cone photoreceptor, which is the first model to account for energy generation from fatty acids oxidation of shed photoreceptor outer segments (POS). Multiple parameter bifurcation analysis shows that joint variations in external glucose, the efficiency of glucose transporter 1 (GLUT1), lipid utilization for POS renewal, and oxidation of fatty acids affect the cone’s metabolic vitality and its capability to adapt under glucose-deficient conditions. The analysis further reveals that when glucose is scarce, cone viability cannot be sustained by only fueling energy production in the mitochondria, but it also requires supporting anabolic processes to create lipids necessary for cell maintenance and repair. In silico experiments are used to investigate how the duration of glucose deprivation impacts the cell without and with a potential GLUT1 or oxidation of fatty acids intervention as well as a dual intervention. The results show that for prolonged duration of glucose deprivation, the cone metabolic system does not recover with higher oxidation of fatty acids and requires greater effectiveness of GLUT1 to recover. Finally, time-varying global sensitivity analysis (GSA) is applied to assess the sensitivity of the model outputs of interest to changes and uncertainty in the parameters at specific times. The results reveal a critical temporal window where there would be more flexibility for interventions to rescue a cone cell from the detrimental consequences of glucose shortage.

59 BASIC BIOLOGICAL SCIENCES↗

Tocopherol controls D1 amino acid oxidation by oxygen radicals in Photosystem II

Photosystem II (PSII) is an intrinsic membrane protein complex that functions as a light-driven water:plastoquinone oxidoreductase in oxygenic photosynthesis. Electron transport in PSII is associated with formation of reactive oxygen species (ROS) responsible for oxidative modifications of PSII proteins. In this study, oxidative modifications of the D1 and D2 proteins by the superoxide anion (O 2 •- ) and the hydroxyl (HO•) radicals were studied in WT and a tocopherol cyclase (vte1) mutant, which is deficient in the lipid-soluble antioxidant α-tocopherol. In the absence of this antioxidant, high-resolution tandem mass spectrometry was used to identify oxidation of D1: 130 E to hydroxyglutamic acid by O 2 •- at the Pheo D1 site. Additionally, D1: 246 Y was modified to either tyrosine hydroperoxide or dihydroxyphenylalanine by O 2 •- and HO • , respectively, in the vicinity of the nonheme iron. We propose that α-tocopherol is localized near PheoD1 and the nonheme iron, with its chromanol head exposed to the lipid–water interface. This helps to prevent oxidative modification of the amino acid’s hydrogen that is bonded to PheoD1 and the nonheme iron (via bicarbonate), and thus protects electron transport in PSII from ROS damage.

59 BASIC BIOLOGICAL SCIENCES↗

Nanoscale dynamics of Dynamin 1 helices reveals squeeze-twist deformation mode critical for membrane fission

Dynamin 1 (Dyn1) GTPase, a principal driver of membrane fission during synaptic endocytosis, self-assembles into short mechanoactive helices cleaving the necks of endocytic vesicles. While structural information about Dyn1 helix is abundant, little is known about the nanoscale dynamics of the helical scaffolding at the moment of fission, complicating mechanistic understanding of Dyn1 action. To address the role of the helix dynamics in fission, we used High-Speed Atomic Force Microscopy (HS-AFM) and fluorescence microscopy to track and compare the spatiotemporal characteristics of the helices formed by wild-type Dyn1 and its K44A mutant impaired in GTP hydrolysis on minimal lipid membrane templates. In the absence of nucleotide, membrane-bound WT Dyn1 and K44A Dyn1 self-assembled into tubular protein scaffolding of similar diameter encaging the lipid bilayer. In both cases, the GTP addition caused scaffold constriction coupled with formation of 20 to 30 nm nanogaps in the protein coverage. While both proteins reached scaffold diameters characteristic for membrane superconstriction causing fission, the fission was detected only with WT Dyn1. We associated the fission activity with the dynamic evolution of the nanogaps: K44A Dyn1 gaps were static, while WT Dyn1 gaps actively evolved via repetitive nonaxisymmetric constriction-bending deformations caused by localized GTP hydrolysis. Modeling of the deformations implicated filament twist as an additional deformation mode which combines with superconstriction to facilitate membrane fission. Our results thus show that the dynamics of the Dyn1 helical scaffold goes beyond radial constriction and involves nonaxisymmetric deformations, where filament twist emerges as a critical driver of membrane fission.

59 BASIC BIOLOGICAL SCIENCES↗

Oxidative stress is a shared characteristic of ME/CFS and Long COVID

Over 65 million individuals worldwide are estimated to have Long COVID (LC), a complex multisystemic condition marked by fatigue, post-exertional malaise, and other symptoms resembling myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). With no clinically approved treatments or reliable diagnostic markers, there is an urgent need to define the molecular underpinnings of these conditions. By studying bioenergetic characteristics of peripheral blood lymphocytes in 25 healthy controls, 27 ME/CFS, and 20 LC donors, we find both ME/CFS and LC donors exhibit signs of elevated oxidative stress, especially in the memory subset. Using a combination of flow cytometry, RNA-seq, mass spectrometry, and systems chemistry analysis, we observed aberrations in reactive oxygen species (ROS) clearance pathways including elevated glutathione levels, decreases in mitochondrial superoxide dismutase protein levels, and glutathione peroxidase 4–mediated lipid oxidative damage. Strikingly, these redox pathways changes show sex-specific trends. While ME/CFS females exhibit higher total ROS and mitochondrial calcium levels, males have normal ROS levels, with pronounced mitochondrial lipid oxidative damage. In females, these higher ROS levels correlate with T cell hyperproliferation, consistent with the known role of elevated ROS in initiating proliferation. This hyperproliferation can be attenuated by metformin, suggesting this Food and Drug Administration (FDA)-approved drug as a possible treatment, as also suggested by a recent clinical study of LC patients. Moreover, these results suggest a shared mechanistic basis for the systemic phenotypes of ME/CFS and LC, which can be detected by quantitative blood cell measurements, and that effective, patient-tailored drugs might be discovered using standard lymphocyte stimulation assays.

ME/CFS↗