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At least 235 records · Page 13

Complex polar lipids of a hot spring cyanobacterial mat and its cultivated inhabitants

The complex polar lipids of the hot spring cyanobacterial mat in the 50 to 55 degrees C region of Octopus Spring, Yellowstone National Park, and of thermophilic bacteria cultivated from this or similar habitats, were compared in an attempt to understand the microbial sources of the major lipid biomarkers in this community. Intact complex lipids were analyzed directly by fast atom bombardment mass spectrometry (FAB-MS), two-dimensional thin-layer chromatography (TLC), and combined TLC-FAB-MS. FAB-MS and TLC gave qualitatively similar results, suggesting that the mat contains major lipids most like those of the cyanobacterial isolate we studied, Synechococcus sp. strain Y-7c-s. These include monoglycosyl, diglycosyl, and sulfoquinosovyl diglycerides (MG, DG, and SQ, respectively) and phosphatidyl glycerol (PG). Though Chloroflexus aurantiacus also contains MG, DG, and PG, the fatty acid chain lengths of mat MGs, DGs, and PGs resemble more those of cyanobacterial than green nonsulfur bacterial lipids. FAB-MS spectra of the lipids of nonphototrophic bacterial isolates were distinctively different from those of the mat and phototrophic isolates. The lipids of these nonphototrophic isolates were not detected in the mat, but most could be detected when added to mat samples. The mat also contains major glycolipids and aminophospholipids of unknown structure and origin. FAB-MS and TLC did not always give quantitatively similar results. In particular, PG and SQ may give disproportionately high FAB-MS responses.

Non-NASA Center↗

pH selectively regulates citric acid and lipid production in Yarrowia lipolytica W29 during nitrogen-limited growth on glucose

Yarrowia lipolytica has been used to produce both citric acid and lipid-based bioproducts at high titers. In this study, we found that pH differentially affects citric acid and lipid production in Y. lipolytica W29, with citric acid production enhanced at more neutral pH’s and lipid production enhanced at more acid pH’s. To determine the mechanism governing this pH-dependent switch between citric acid and lipid production, we profiled gene expression at different pH’s and found that the relative expression of multiple transporters is increased at neutral pH. These results suggest that this pH-dependent switch is mediated at the level of citric acid transport rather than changes in the expression of the enzymes involved in citric acid and lipid metabolism. In further support of this mechanism, thermodynamic calculations suggest that citric acid secretion is more energetically favorable at neutral pH’s, assuming the fully protonated acid is the substrate for secretion. Finally, collectively, these results provide new insights regarding citric acid and lipid production in Y. lipolytica and may offer new strategies for metabolic engineering and process design.

59 BASIC BIOLOGICAL SCIENCES↗

Lipid interactions and angle of approach to the HIV-1 viral membrane of broadly neutralizing antibody 10E8: Insights for vaccine and therapeutic design

Among broadly neutralizing antibodies to HIV, 10E8 exhibits greater neutralizing breadth than most. Consequently, this antibody is the focus of prophylactic/therapeutic development. The 10E8 epitope has been identified as the conserved membrane proximal external region (MPER) of gp41 subunit of the envelope (Env) viral glycoprotein and is a major vaccine target. However, the MPER is proximal to the viral membrane and may be laterally inserted into the membrane in the Env prefusion form. Nevertheless, 10E8 has not been reported to have significant lipid-binding reactivity. Here we report x-ray structures of lipid complexes with 10E8 and a scaffolded MPER construct and mutagenesis studies that provide evidence that the 10E8 epitope is composed of both MPER and lipid. 10E8 engages lipids through a specific lipid head group interaction site and a basic and polar surface on the light chain. In the model that we constructed, the MPER would then be essentially perpendicular to the virion membrane during 10E8 neutralization of HIV-1. As the viral membrane likely also plays a role in selecting for the germline antibody as well as size and residue composition of MPER antibody complementarity determining regions, the identification of lipid interaction sites and the MPER orientation with regard to the viral membrane surface during 10E8 engagement can be of great utility for immunogen and therapeutic design.

59 BASIC BIOLOGICAL SCIENCES↗

Controlling Protein Enrichment in Lipid Sponge Phase Droplets using SNAP–Tag Bioconjugation

All cells use organized lipid compartments to facilitate specific biological functions. Membrane-bound organelles create defined spatial environments that favor unique chemical reactions while isolating incompatible biological processes. Despite the fundamental role of cellular organelles, there is a scarcity of methods for preparing functional artificial lipid-based compartments. In this work, we demonstrate a robust bioconjugation system for sequestering proteins into zwitterionic lipid sponge phase droplets. Incorporation of benzylguanine (BG)-modified phospholipids that form stable covalent linkages with an O 6 -methylguanine DNA methyltransferase (SNAP-tag) fusion protein enables programmable control of protein capture. We show that this methodology can be used to anchor hydrophilic proteins at the lipid-aqueous interface, concentrating them within an accessible but protected chemical environment. SNAP-tag technology enables the integration of proteins that regulate complex biological functions in lipid sponge phase droplets, and should facilitate the development of advanced lipid-based artificial organelles.

59 BASIC BIOLOGICAL SCIENCES↗

Toward an understanding of lipid and starch accumulation in microalgae: A proteomic study of Neochloris oleoabundans cultivated under N-limited heterotrophic conditions

Neochloris oleoabundans is an oleaginous microalga of biotechnological and commercial interest. A proteomic study was performed to compare the abundance of proteins under two different culture conditions; i.e., preferential lipid accumulation and preferential carbohydrate accumulation. When N. oleoabundans was cultivated under long periods of nitrogen starvation, the acyl carrier protein and the protein biotin carboxylase from the lipid synthesis pathway were found to be upregulated. The central metabolism pathways were also found to be highly activated to redirect the carbon flow toward pyruvate dehydrogenase and ATP synthesis. Pyruvate dehydrogenase was upregulated to supply the precursors for lipid production. Furthermore, the pentose phosphate pathway, specifically the enzymes glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, were upregulated to supply reducing power in the form of NADPH for lipid synthesis and inorganic nitrogen assimilation. Carbohydrate synthesis-related enzymes that channel carbon to starch and sugar synthesis, such as UDP-glucose pyrophosphorylase and starch synthase, were upregulated when short durations of nitrogen limitation were applied to N. oleoabundans cultivation. However, ADP-glucose pyrophosphorylase was upregulated under preferential lipid accumulation conditions, indicating that under prolonged nitrogen starvation conditions, this enzyme potentially hydrolyzes starch chains to channel the carbon flow to lipid synthesis, which indicates a dual function of this protein.

59 BASIC BIOLOGICAL SCIENCES↗

Epidermal growth factor receptor mediated proliferation depends on increased lipid droplet density regulated via a negative regulatory loop with FOXO3/Sirtuin6

The proliferation of colon cancer cells is mediated in part by epidermal growth factor receptor (EGFR) signaling and requires sustained levels of cellular energy to meet its high metabolic needs. Intracellular lipid droplets (LDs) are a source of energy used for various cellular functions and they are elevated in density in human cancer, yet their regulation and function are not well understood. Here, in human colon cancer cells, EGF stimulates increases in LD density, which depends on EGFR expression and activation as well as the individual cellular capacity for lipid synthesis. Increases in LDs are blockaded by inhibition of PI3K/mTOR and PGE2 synthesis, supporting their dependency on select upstream pathways. In colon cancer cells, silencing of the FOXO3 transcription factor leads to down regulation of SIRT6, a negative regulator of lipid synthesis, and consequent increases in the LD coat protein PLIN2, revealing that increases in LDs depend on loss of FOXO3/SIRT6. Moreover, EGF stimulates loss of FOXO3/SIRT6, which is blockaded by the inhibition of upstream pathways as well as lipid synthesis, revealing existence of a negative regulatory loop between LDs and FOXO3/SIRT6. Elevated LDs are utilized by EGF treatment and their depletion through the inhibition of lipid synthesis or silencing of PLIN2 significantly attenuates proliferation. This novel mechanism of proliferative EGFR signaling leading to elevated LD density in colon cancer cells could potentially be therapeutically targeted for the treatment of tumor progression. - Highlights: • In colon cancer cells, EGFR activation leads to increases in LD density. • EGFR signaling includes PI3K/mTOR and PGE2 leading to lipid synthesis. • Increases in LDs are controlled by a negative regulatory loop with FOXO3/SIRT6. • EGFR mediated colon cancer cell proliferation depends on increased LD density.

60 APPLIED LIFE SCIENCES↗

Tetrandrine induces lipid accumulation through blockade of autophagy in a hepatic stellate cell line

Macroautophagy, or autophagy, is a cellular response in which unnecessary cytoplasmic components, including lipids and organelles, are self-degraded. Recent studies closely related autophagy to activation of hepatic stellate cells (HSCs), a process critical in the pathogenesis of liver fibrosis. During HSC activation, cytoplasmic lipid droplets (LDs) are degraded as autophagic cargo, and then cells express fibrogenic genes. Thus, inhibition of autophagy in HSCs is a potential therapeutic approach for attenuating liver fibrosis. We found that tetrandrine, a bisbenzylisoquinoline alkaloid isolated from Stephania tetrandra, induced lipid accumulation, a phenotype associated with quiescent HSCs, through blockade of autophagy in the rat-derived HSC line HSC-T6. Tetrandrine inhibited autophagic flux without affecting lysosomal function. A phenotypic comparison using siRNA knockdown suggested that tetrandrine may target regulators, involved in fusion between autophagosomes and lysosomes (e.g., syntaxin 17). Moreover, perilipin 1, an LD-coated protein, co-localized specifically with LC3, a marker protein for autophagosomes, in tetrandrine-treated HSC-T6 cells. This suggests a potential role for perilipin 1 in autophagy-mediated LD degradation in HSCs. Our results identified tetrandrine as a potential tool for prevention and treatment of HSC activation. - Highlights: • Autophagy is closely related to lipid degradation in hepatic stellate cells. • Tetrandrine (Tet) causes lipid accumulation via blockade of autophagy in HSC-T6 cells. • Tet blocked autophagy without affecting lysosomal function unlike bafilomycin A{sub 1}. • Perilipin 1 was specifically co-localized with LC3 in Tet-treated cells. • Perilipin 1 may play potential roles in autophagy-mediated lipid degradation.

60 APPLIED LIFE SCIENCES↗

Integrated thermal and biological conversion of microalgal proteins to lipids

Microalgal composition varies with cultivation strategy, and low-cost approaches often produce high-protein biomass. This presents challenges for biorefineries designed around static, lipid-rich feedstocks. In particular, hydrolysates from high-protein algae are nitrogen-rich and sugar-poor, limiting microbial conversion and reducing product yields. This study develops a sequential thermal conditioning and biological upgrading strategy to integrate high-protein hydrolysate processing within conventional lipid extraction and upgrading designs. Oxidative deconstruction was used to break down proteins into ammonium and short-chain carboxylates. Ammonium was subsequently removed to yield a nitrogen-depleted, carboxylate-rich medium suitable for microbial lipid production. Bioconversion trials with Cutaneotrichosporon oleaginosum showed lipid accumulation only from hydrolysates treated with both oxidative deconstruction and nitrogen removal, reaching 1.2 g/L lipids at 30 % intracellular content. This integrated approach enables protein-to-lipid conversion and improves flexibility to process variable algal feedstocks, advancing fuel-oriented microalgal biorefineries.

09 BIOMASS FUELS↗

Exploring the impact of nucleotide length on lipid nanoparticle structure and properties

Lipid nanoparticles (LNPs) are versatile carriers for nucleic acid (NA) therapeutics, including ASOs, siRNA, mRNA, and poly-IC. While lipid composition is known to influence LNP properties, the impact of NA length on morphology and internal structure is less understood, particularly during the stages of carrier–cargo assembly. Here, we examine NA length and lipid composition immediately after mixing using high-throughput SAXS, dynamic light scattering, and cryogenic electron microscopy. All LNPs form ordered NA/lipid compartments, with longer NAs promoting inverse hexagonal (H II ) phases and larger intercompartment distances. In contrast, short NAs, especially in formulations with SM102 ionizable lipid, favor lamellar phases. SAXS peak deconvolution quantifies ordered versus disordered phases via a Robustness of Ordered Phase factor, which correlates with particle size and encapsulation efficiency. Formulations with MC3 ionizable and DOPE helper lipids exhibit the most stable H II -phase packing, highlighting the role of helper-lipid curvature in compartment stabilization. Variations in NA compartmentalization indicate differences in payload capacity, offering a framework for rational LNP design across diverse nucleic acid cargos.

60 APPLIED LIFE SCIENCES↗

Amphiphilic Polypeptoids Rupture Vesicle Bilayers To Form Peptoid–Lipid Fragments Effective in Enhancing Hydrophobic Drug Delivery

Peptoids are highly biocompatible pseudopeptidic polyglycines with designable substituents on the nitrogen atoms. The therapeutic and drug-carrying potential of these materials requires a fundamental understanding of their interactions with lipid bilayers. Here, we use amphiphilic polypeptoids with up to 100 monomeric units where a significant fraction (26%) of the nitrogen atoms are functionalized with decyl groups (hydrophobes) that insert into the lipid bilayer through the hydrophobic effect. These hydrophobically modified polypeptoids (HMPs) insert their hydrophobes into lipid bilayers creating instabilities that lead to the rupture of vesicles. At low HMP concentrations, such rupture leads to the creation of large fragments which remarkably anchor to intact vesicles through the hydrophobic effect. At high HMP concentrations, all vesicles rupture to smaller HMP–lipid fragments of the order of 10 nm. We show that the technique for such nanoscale polymer–lipid fragments can be exploited to sustain highly hydrophobic drug species in solution. Using the kinase inhibitor, Sorafenib as a model drug, it is shown that HMP–lipid fragments containing the drug can efficiently enter a hepatocellular carcinoma cell line (Huh 7.5), indicating the use of such fragments as drug delivery nanocarriers.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗

Purple acid phosphatase2 stimulates a futile cycle of lipid synthesis and degradation, and mitigates the negative growth effects of triacylglycerol accumulation in vegetative tissues

Storage lipids (mostly triacylglycerols, TAGs) serve as an important energy and carbon reserve in plants and hyperaccumulation of TAG in vegetative tissues can cause negative effects on plant growth. Purple acid phosphatase2 (PAP2) was shown previously to affect carbon metabolism and boost plant growth. However, the effects of PAP2 on lipid metabolism remain unknown. Here, we demonstrated PAP2 as a factor that can stimulate a futile cycle of fatty acid (FA) synthesis and degradation and mitigate negative growth effects associated with TAG accumulation in vegetative tissues. Constitutive expression of PAP2 in Arabidopsis thaliana enhanced both lipid synthesis and degradation in leaves and led to substantial increase in seed oil yield. Suppressing lipid degradation in a PAP2-overexpressing line by disrupting sugar-dependent1 (SDP1), a predominant TAG lipase, significantly elevated vegetative TAG content and improved plant growth. Diverting FAs from membrane lipids to TAGs in PAP2-overexpressing plants by constitutively expressing phospholipid:diacylglycerol acyltransferase1 (PDAT1) increased TAG contents to up to 6% of dry weight in vegetative tissues without compromising biomass yield. Overall, these results highlight the potential of combining PAP2 with TAG-promoting factors to enhance carbon assimilation, FA synthesis and allocation to TAGs for optimized plant growth and storage lipid accumulation in vegetative tissues.

59 BASIC BIOLOGICAL SCIENCES↗

Refined dynamic structure factor of a lipid bilayer on scales comparable to its thickness

The structural inhomogeneity of a lipid bilayer is an obstacle to applying the classical Canham–Helfrich model to describe its dynamics on nanometer length scales. In this paper, a refined expression for the free energy of a single-component lipid bilayer is used to describe the dynamics of lipid density fluctuations. In particular, the expression with a term involving the gradient of the area per lipid [8] is used for the free energy per lipid. A refined expression has been derived for the dynamic structure factor of a free lipid bilayer in the hydrodynamic region. It leads to differences in the interpretation and values of the bilayer parameters in comparison with the standard model.

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗

Final Report for DE-FG02-04ER15626: P-type ATPases in Plants – Role of Lipid Flippases in Membrane Biogenesis

The long-range goal of the research is to understand the structure and biological functions of different P-type ATPases (ion pumps) in plant cells, and to use that knowledge to enhance the production of bioenergy from plants, or plant-research inspired technologies. Ptype ATPases include ion pumps that specifically transport H + , Ca 2 +, Zn 2+ , Cu 2+ , K + , or Na + , as well as at least one unusual subfamily that appears to function as lipid flippases, flipping specific lipids from one side of a membrane bilayer to the other. As a group, P-type ATPases are thought to consume more than 1/3 of the cellular ATP in typical eukaryotic cells. Recent research in the Harper lab focused on understanding the biochemical and biological functions of P-type ATPases that flip lipids. These flippases belong to the P4 subfamily of P-type ATPases. The activity of lipid flippases is thought to induce membrane curvature and/or create an asymmetry in which certain lipid head groups are preferential exposed to one surface or the other. In Arabidopsis thaliana there are 12 members of this family referred to as Aminophospholipid ATPase (ALA) 1 to ALA12. Using genetic knockouts, the Harper lab has established that this unusual subfamily of P-type ATPases are critical for plants to cope with even modest changes in temperature (e.g., down to 15°C, or up to 30°C). In addition, members of one subclade are critical for cell expansion, and loss of function mutants result in severe dwarfism. Other members of this same sub-clade are critical for pollen tube growth, and loss of function mutants are sterile under conditions of hot days and cold nights. While the cellular processes that depend on lipid flippases are still unclear, the genetic analysis of loss of function mutants clearly show they are of fundamental importance to plant growth and response to the environment.

09 BIOMASS FUELS↗

Lipids from High-Protein Algae Biomass

Biofuels from renewable and sustainable sources will be a critical component of reducing greenhouse gasses. To this end, in achieving maximum biomass productivity in outdoor ponds at a reasonable cost, algae must be grown at a maximal rate. This current algae cultivation strategy results in high-protein biomass because there is insufficient time for deplete-hold steps that increase lipids but also cost. Thus methods to convert high-protein (>50%) need to be developed for biofuels. The Consolidated Algae Processing (CAP) strategy has previously shown utility on high-carbohydrate algae biomass and has now been expanded to include methods aimed at converting the protein fraction to lipids. After acid-pretreatment, hydrolysate liquor is oxidatively treated to deaminate and convert soluble proteins and amino acids into four main carboxylate acids; formic, acetic, succinic, and propionic. These acids are readily and completely fermented into intracellular lipids using oleaginous yeast. Using a fed-batch fermentation strategy and no added nutrients, we achieved nearly 30% intracellular lipids and 1.3 g/L lipids demonstrating the potential of our expanded CAP process to generate additional lipids for conversion to biofuels as part of an algae-focused biorefinery.

AAD↗

Screening for Lipids From Marine Microalgae Using Nile Red

The fluorescent stain Nile Red has been used extensively for the quantification of lipids in phytoplankton, including microalgae, because it preferentially stains neutral lipids and it is economical and sensitive to use for screening purposes. Although its basic application has not changed for several decades, recent improvements have been made to improve its utility across applications. Here we describe additional refinements in its application and interpretation as a highthroughput method for the rapid quantification of neutral lipids in liquid cultures of marine phytoplankton. Specifically we address (1) interspecies comparisons, (2) fluorescence excitation and emission wavelengths, and (3) the time course of the Nile Red signal in the context of using bulk or cell-specific fluorescence to quantify neutral lipids oflive or preserved cells. Here, we show that with proper caution in its interpretation across species and physiological states the quantity of lipid in hundreds of small volume samples can be reliably assessed daily using a refined Nile Red protocol.

09 BIOMASS FUELS↗

Process design and economics for production of advanced biofuels from genetically modified lipid-producing sorghum

This study evaluates the potential for making advanced biofuels from genetically modified (GM) lipid–producing sorghum. A biodiesel coproduction process is developed to extract, purify, and upgrade lipids to diesel fuel while carbohydrates are utilized for making ethanol through acid thermal pretreatment, enzymatic hydrolysis, and fermentation. To assess the advantages of coproducing biodiesel from GM–sorghum, process economics are compared to a cellulosic ethanol biorefinery receiving non-GM sorghum. Minimum ethanol selling prices (MESP) that reach a breakeven point after 30 years of service life are calculated as an economic index to compare the two processes. Results indicate that biodiesel coproduction improves the economics by lowering the MESP from 3.08/gal for the ethanol-only process to 2.46/gal. Sensitivity analyses reveal that increasing sorghum’s lipid content, increasing the lipid extraction efficiency, and reducing the solvent-to-solids ratio in lipid extraction columns are the most important process parameters to further enhance technoeconomics. Analyses indicate that a lipid content above 13 wt% (dry basis) or a biomass price less than 65/Mg (dry basis) will result in a 2014 ethanol wholesale price of 2.25/gal for the coproduction process.

09 BIOMASS FUELS↗

Importance of phospholipid bilayer integrity in the analysis of protein–lipid interactions

Highlights: • We show long-term mechanical stabilization of solid supported bilayers. • Bilayer integrity is essential for the investigation of protein–lipid interactions. • Protein adsorption to a bilayer containing defects causes membrane destruction. - Abstract: The integrity of supported phospholipid bilayer membranes is of crucial importance for the investigation of lipid–protein interactions. Therefore we recorded the formation of supported membranes on SiO{sub 2} and mica by quartz crystal microbalance and controlled the integrity by atomic force microscopy. This study aims to analyze how membrane defects affect protein–lipid interactions. The experiments focused on a lipid mixture of POPC/DOPC/Chol/POPS/PI(4,5)P{sub 2} (37:20:20:20:3) and the binding of the peripheral membrane associated protein annexin A2. We found that formation of a continuous undisturbed bilayer is an indispensable precondition for a reliable determination and quantification of lipid–protein-interactions. If membrane defects were present, protein adsorption causes membrane disruption and lipid detachment on a support thus leading to false determination of binding constants. Our results obtained for PI(4,5)P{sub 2} and cholesterol containing supported membranes yield new knowledge to construct functional surfaces that may cover nanoporous substrates, form free standing membranes or may be used for lab-on-a-chip applications.

60 APPLIED LIFE SCIENCES↗

Saturation of SERCA's lipid annulus may protect against its thermal inactivation

The sarco(endo)plasmic reticulum Ca{sup 2+}-ATPase (SERCA) pumps are integral membrane proteins that catalyze the active transport of Ca{sup 2+} into the sarcoplasmic reticulum, thereby eliciting muscle relaxation. SERCA pumps are highly susceptible to oxidative damage, and cytoprotection of SERCA dampens thermal inactivation and is a viable therapeutic strategy in combating diseases where SERCA activity is impaired, such as muscular dystrophy. Here, we sought to determine whether increasing the percent of saturated fatty acids (SFA) within SERCA's lipid annulus through diet could protect SERCA pumps from thermal inactivation. Female Wistar rats were fed either a semi-purified control diet (AIN93G, 7% soybean oil by weight) or a modified AIN93G diet containing high SFA (20% lard by weight) for 17 weeks. Soleus muscles were extracted and SERCA lipid annulus and activity under thermal stress were analyzed. Our results show that SERCA's lipid annulus is abundant with short-chain (12–14 carbon) fatty acids, which corresponds well with SERCA's predicted bilayer thickness of 21 Å. Under control-fed conditions, SERCA's lipid annulus was already highly saturated (79%), and high-fat feeding did not increase this any further. High-fat feeding did not mitigate the reductions in SERCA activity seen with thermal stress; however, correlational analyses revealed significant and strong associations between % SFA and thermal stability of SERCA activity with greater %SFA being associated with lower thermal inactivation and greater % polyunsaturation and unsaturation index being associated with increased thermal inactivation. Altogether, these findings show that SERCA's lipid annulus may influence its susceptibility to oxidative damage, which could have implications in muscular dystrophy and age-related muscle wasting. - Highlights: • SERCA's lipid annulus in rat soleus was measured after immunoconcentration. • Short fatty acid chains surround SERCA and may ensure optimal hydrophobic matching. • SERCA's annulus is highly saturated in control-fed and high-fat fed rats. • Greater saturation associates with small levels of thermal inactivation. • Greater unsaturation associates with large levels of thermal inactivation.

60 APPLIED LIFE SCIENCES↗