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At least 127 records · Page 7

Impact of Fatty-Acid Labeling of Bacillus subtilis Membranes on the Cellular Lipidome and Proteome

Developing cultivation methods that yield chemically and isotopically defined fatty acid (FA) compositions within bacterial cytoplasmic membranes establishes an in vivo experimental platform to study membrane biophysics and cell membrane regulation using novel approaches. Yet before fully realizing the potential of this method, it is prudent to understand the systemic changes in cells induced by the labeling procedure itself. In this work, analysis of cellular membrane compositions was paired with proteomics to assess how the proteome changes in response to the directed incorporation of exogenous FAs into the membrane of Bacillus subtilis. Key findings from this analysis include an alteration in lipid headgroup distribution, with an increase in phosphatidylglycerol lipids and decrease in phosphatidylethanolamine lipids, possibly providing a fluidizing effect on the cell membrane in response to the induced change in membrane composition. Changes in the abundance of enzymes involved in FA biosynthesis and degradation are observed; along with changes in abundance of cell wall enzymes and isoprenoid lipid production. The observed changes may influence membrane organization, and indeed the well-known lipid raft-associated protein flotillin was found to be substantially down-regulated in the labeled cells – as was the actin-like protein MreB. Taken as a whole, this study provides a greater depth of understanding for this important cell membrane experimental platform and presents a number of new connections to be explored in regard to modulating cell membrane FA composition and its effects on lipid headgroup and raft/cytoskeletal associated proteins.

59 BASIC BIOLOGICAL SCIENCES↗

Morphologic and histochemical studies of bone cells from SL-3 rats

Previous studies of rat bone following space flight indicate a significant reduction in new bone formation as a result of hypogravity. In the present study of animals from SL-3 flight, the cellular activity of the bone forming cells, the osteoblasts, was investigated. Measurements of alkaline and acid phosphatase, Golgi activity, secretory granule size, and lysosomal activity, all indicated very little difference between flight and flight-simulated controls. However, there was a tendency for osteoblasts in compact bone of flight animals to show a smaller cytoplasmic volume compared to non-flight controls. If, as in previous studies, a significant reduction in bone formation occurred, it could be due to a normal level of procollagen degradation within these smaller osteoblasts, resulting in less collagen secretion per cell.

Doty, S. B.↗

Is Water a Universal Solvent for Life?

There are strong reasons to believe that the laws, principles and constraints of physics and chemistry are universal. It is much less clear how this universality translates into our understanding of the origins of life. Conventionally, discussions of this topic focus on chemistry that must be sufficiently rich to seed life. Although this is clearly a prerequisite for the emergence of living systems, I propose to focus instead on self-organization of matter into functional structures capable of reproduction, evolution and responding to environmental changes. In biology, most essential functions are largely mediated by noncovalent interactions (interactions that do not involve making or breaking chemical bonds). Forming chemical bonds is only a small part of what living systems do. There are specific implications of this point of view for universality. I will concentrate on one of these implications. Strength of non-covalent interactions must be properly tuned. If they were too weak, the system would exhibit undesired, uncontrolled response to natural fluctuations of physical and chemical parameters. If they were too strong kinetics of biological processes would be slow and energetics costly. This balance, however, is not a natural property of complex chemical systems. Instead, it has to be achieved with the aid of an appropriate solvent for life. In particular, potential solvents for life must be characterized by a high dielectric constant to ensure solubility of polar species and sufficient flexibility of biological structures stabilized by electrostatic interactions. Among these solvents, water exhibits a remarkable trait that it also promotes solvophobic (hydrophobic) interactions between non-polar species, typically manifested by a tendency of these species to aggregate and minimize their contacts with the aqueous solvent. Hydrophobic interactions are responsible, at least in part, for many self-organization phenomena in biological systems, such as the formation of cellular boundary structures or protein folding. Strengths of electrostatic and hydrophobic interactions are similar and can be balanced over a wide range of temperatures, which considerably increases the repertoire of interactions that can be used to modulate biological functions. Some properties of water, e.g. its chemical activity against polymerization reactions, are considered as unfavorable to life. In actuality, this might be a favorable trait because life requires a balance between constructive and destructive processes. For example, molecules synthesized in response to specific conditions must be degraded once these conditions change. Otherwise regulation of biological processes would be virtually impossible. Water might not be the only liquid with favorable properties for supporting life. It has been proposed that formamide, which might be present elsewhere in the universe in sufficient quantities to warrant interest, could be a potential alternative to water for the origin of life. However, this will remain highly hypothetical until it is demonstrated in further studies on its physical, chemical and biological properties it is capable of mediating self-organization of matter and providing proper balance between different types of non-covalent interactions.

Pohorill, Andrew↗

Effect of Ex Vivo Ionizing Radiation on Static and Fatigue Properties of Mouse Vertebral Bodies

For a variety of medical and scientific reasons, human bones can be exposed to a wide range of ionizing radiation levels. In vivo radiation therapy (0.05 kGy) is used in cancer treatment, and ex vivo irradiation (25-35 kGy) is used to sterilize bone allografts. Ionizing radiation in these applications has been shown to increase risk of fracture, decrease bone quality and degrade collagen integrity. Past studies have investigated the deleterious effects of radiation on cortical or trabecular bone specimens individually, but to date no studies have examined whole bones containing both cortical and trabecular tissue. Furthermore, a clear relationship between the dose and the mechanical and biochemical response of bone's extracellular matrix has yet to be established for doses ranging from cancer therapy to allograft sterilization (0.05-35 kGy). To gain insight into these issues, we conducted an ex vivo radiation study to investigate non-cellular (i.e. matrix) effects of ionizing radiation dose on vertebral whole bone mechanical properties, over a range of radiation doses (0.05-35 kGy), with a focus on any radiation-induced changes in collagen. With underlying mechanisms of action in mind, we hypothesized that any induced reductions in mechanical properties would be associated with changes in collagen integrity. METHODS: 20-week old female mice were euthanized and the lumbar spine was dissected using IACUC approved protocols. The lumbar vertebrae (L1- S1) were extracted from the spine via cuts through adjacent intervertebral discs, and the endplates, posterior processes, surrounding musculature, and soft tissues were removed (approx. 1.5mm diameter, approx. 2mm height). Specimens were randomly assigned to one of five groups for ex vivo radiation exposure: x-ray irradiation at 0.05, 1, 17, or 35 kGy, or a 0 kGy control. Following irradiation, the vertebrae were imaged using microcomputed tomography (micro-CT) and then subjected to either monotonic compressive loading to failure or uniform cyclic compressive loading. During cyclic testing, samples were loaded in force control to a force level that corresponded to a strain of 0.46%, as determined in advance by a linearly elastic micro-CT-based finite element analysis for each specimen. Tests were stopped at imminent fracture, defined as a rapid increase in strain. The main outcome for the monotonic test was the strength (maximum force); for cyclic testing it was the fatigue life (log of the number of cycles of loading at imminent failure). A fluorometric assay was used on the S1 vertebrae to measure the number of non-enzymatic collagen crosslinks[4]. A one-way ANOVA was performed on mechanical properties and collagen crosslinks; means were compared with controls using Dunnett's method, with a Tukey-Kramer post-hoc analysis when significance was found (p < or = 0.05). RESULTS: Compared to the unirradiated control group, the concentration of non-enzymatic collagen crosslinks was significantly increased for all irradiated groups (p < 0.0001), and being higher by at least 50% (Figure 1a). By contrast, the radiation effects on the collagen were only evident at the higher doses. For irradiation exposures of 17 kGy or more, strength decreased substantially as the radiation level was increased, but no effect was evident below 17 kGy (Figure 1b). There was no significant change in the stiffness or maximum displacement for any radiation dose (p>0.05). The finite element analysis prescribed force level for cyclic loading exceeded the measured (monotonic) strength of the 17 and 35 kGy irradiated groups (mean +/- SD, 20.6 +/- 5.6 N; 13.2 +/- 3.7 N, respectively) and therefore these groups were eliminated from the fatigue study. The fatigue life for the 0.05 and 1 kGy groups were similar to each other and were not statistically significantly different from the control group (Figure 1c).

radiation↗

Isoform-specific functions of synaptopodin-2 variants in cytoskeleton stabilization and autophagy regulation in muscle under mechanical stress

Highlights: • Muscle cells express different synaptopodin-2 isoforms with individual functions. • Reduced SYNPO2 expression results in enhanced vulnerability of myofibrils. • SYNPO2e recruites BAG3 to mechanically stressed myofibrillar areas. • SYNPO2 is an organizer and regulator of BAG3-containing proteostasis systems. • BAG3 forms functionally distinct protein complexes with different SYNPO2 isoforms. Protein homeostasis (proteostasis) in multicellular organisms depends on the maintenance of force-bearing and force-generating cellular structures. Within myofibrillar Z-discs of striated muscle, isoforms of synaptopodin-2 (SYNPO2/myopodin) act as adapter proteins that are engaged in proteostasis of the actin-crosslinking protein filamin C (FLNc) under mechanical stress. SYNPO2 directly binds F-actin, FLNc and α-actinin and thus contributes to the architectural features of the actin cytoskeleton. By its association with autophagy mediating proteins, i.e. BAG3 and VPS18, SYNPO2 is also engaged in protein quality control and helps to target mechanical unfolded and damaged FLNc for degradation. Here we show that deficiency of all SYNPO2-isoforms in myotubes leads to decreased myofibrillar stability and deregulated autophagy under mechanical stress. In addition, isoform-specific proteostasis functions were revealed. The PDZ-domain containing variant SYNPO2b and the shorter, PDZ-less isoform SYNPO2e both localize to Z-discs. Yet, SYNPO2e is less stably associated with the Z-disc than SYNPO2b, and is dynamically transferred into FLNc-containing myofibrillar lesions under mechanical stress. SYNPO2e also recruits BAG3 into these lesions via interaction with the WW domain of BAG3. Our data provide evidence for a role of myofibrillar lesions as a transient quality control compartment essential to prevent and repair contraction-induced myofibril damage in muscle and indicate an important coordinating activity for SYNPO2 therein.

60 APPLIED LIFE SCIENCES↗

Enzymes as parts in need of replacement - and how to extend their working life

Enzymes catalyze reactions in vivo at different rates and each enzyme molecule has a lifetime limit before it is degraded and replaced to enable catalysis to continue. Considering these rates together as a unitless ratio of catalytic cycles until replacement (CCR) provides a new quantitative tool to assess the replacement schedule of and energy investment into enzymes as they relate to function. Here, we outline the challenges of determining CCRs and new approaches to overcome them and then assess the CCRs of selected enzymes in bacteria and plants to reveal a range of seven orders of magnitude for this ratio. Modifying CCRs in plants holds promise to lower cellular costs, to tailor enzymes for particular environments, and to breed enzyme improvements for crop productivity.

Catalytic rate↗

Molecular Mechanisms Regulating Muscle Fiber Composition Under Microgravity

The overall goal of this project is to reveal the molecular mechanisms underlying the selective and debilitating atrophy of specific skeletal muscle fiber types that accompanies sustained conditions of microgravity. Since little is currently known about the regulation of fiber-specific gene expression programs in mammalian muscle, elucidation of the basic mechanisms of fiber diversification is a necessary prerequisite to the generation of therapeutic strategies for attenuation of muscle atrophy on earth or in space. Vertebrate skeletal muscle development involves the fusion of undifferentiated mononucleated myoblasts to form multinucleated myofibers, with a concomitant activation of muscle-specific genes encoding proteins that form the force-generating contractile apparatus. The regulatory circuitry controlling skeletal muscle gene expression has been well studied in a number of vertebrate animal systems. The goal of this project has been to achieve a similar level of understanding of the mechanisms underlying the further specification of muscles into different fiber types, and the role played by innervation and physical activity in the maintenance and adaptation of different fiber phenotypes into adulthood. Our recent research on the genetic basis of fiber specificity has focused on the emergence of mature fiber types and have implicated a group of transcriptional regulatory proteins, known as E proteins, in the control of fiber specificity. The restriction of E proteins to selected muscle fiber types is an attractive hypothetical mechanism for the generation of muscle fiber-specific patterns of gene expression. To date our results support a model wherein different E proteins are selectively expressed in muscle cells to determine fiber-restricted gene expression. These studies are a first step to define the molecular mechanisms responsible for the shifts in fiber type under conditions of microgravity, and to determine the potential importance of E proteins as upstream targets for the effects of weightlessness. In the past year we have determined that the expression of E Proteins is restricted to specific fiber types by post-transcriptional mechanisms. By far, the most prevalent mechanism of cellular control for achieving post-transcriptional regulation of gene expression is selective proteolysis -through the ubiquitin -proteasome pathway. Steady-state levels of HEB message are similar in all fast and slow skeletal muscle fiber types, yet the protein is restricted to Type IIX fibers. HEB appears to be a nodal point for regulating fiber-specific transcription, as expression of the transcription factor is regulated at the post-transcriptional level. It is not clear at present whether the regulation is at the level of protein synthesis or degradation. We are now poised to evaluate the biological role of ubiquitination in fiber specific-gene expression by controlling the post-transcriptional expression of E Proteins. The use of metabolic labelling and pharmacological inhibitors of the ubiquitin pathway will be used to identify the mode of regulation of the Type IIX expression pattern. The potential role of specific kinases in effecting the restriction of HEB expression will be examined by using both inhibitors and activators. The results of these studies will provide the necessary information to evaluate the biological role of E proteins in controlling fiber type transitions, and in potentially attenuating the atrophic effects of microgravity conditions. We have also recently shown that ectopic expression of the HEB protein transactivates the Type IIX-specific skeletal a-actin reporter. The 218 bp skeletal a-actin promoter drives transgene expression solely in mature Type IIX fibers. A mouse also carrying the transgene MLCI/HEB (which ectopically expresses the E Protein HEB in Type IIB fibers) forces expression of the skeletal a-actin reporter gene in Type IIB fibers. We can now dissect the composition of this fiber-specific cis-element. The skeletal a-actin promoter is quite compact and has been extensively characterized in vitro for activity and binding factors. The single E box may act as a binding target of myogenic factor/HEB heterodimer to allow for IIX expression. The HEB transcription factor may recognize either the precise flanking sequences of the E Box, or perhaps interacting with other proteins bound nearby, and activating expression in Type IIX fibers. This E box will be both ablated, and alternatively, as ablation may well destroy any muscle-specific transcriptional activity, flanking sequences substituted with those surrounding the E box (El) of the myogenin promoter. Modification of fiber-specific transgene expression will be tested in transgenic mice. The results of these studies will provide basic information on the regulatory circuitry underlying fiber specificity, and will form the basis for building appropriate transgenic regulatory cassettes to effect fiber transitions in subsequent experimental manipulations on unweighted muscles.

Rosenthal, Nadia A.↗

Femoral Head Bone Loss Following Short and Long-Duration Spaceflight

Exposure to mechanical unloading during spaceflight is known to have significant effects on the musculoskeletal system. Our ongoing studies with the mouse bone model have identified the failure of normal stem cell-based tissue regeneration, in addition to tissue degeneration, as a significant concern for long-duration spaceflight, especially in the mesenchymal and hematopoietic tissue lineages. The 30-day BionM1 and the 37-day Rodent Research 1 (RR1) missions enabled the possibility of studying these effects in long-duration microgravity experiments. We hypothesized that the inhibition of stem cell-based tissue regeneration in short-duration spaceflight would continue during long-duration spaceflight and furthermore would result in significant tissue alterations. MicroCT analysis of BionM1 femurs revealed 31 decrease in bone volume ratio, a 14 decrease in trabecular thickness, and a 20 decrease in trabecular number in the femoral head of space-flown mice. Furthermore, high-resolution MicroCT and immunohistochemical analysis of spaceflight tissues revealed a severe disruption of the epiphyseal boundary, resulting in endochondral ossification of the femoral head and perforation of articular cartilage by bone. This suggests that spaceflight in microgravity may cause rapid induction of an aging-like phenotype with signs of osteoarthritic disease in the hip joint. However, mice from RR1 exhibited significant bone loss in the femoral head but did not exhibit the severe aging and disease-like phenotype observed during BionM1. This may be due to increased physical activity in the RH hardware. Immunohistochemical analysis of the epiphyseal plate and investigation of cellular proliferation and differentiation pathways within the marrow compartment and whole bone tissue is currently being conducted to determine alterations in stem cell-based tissue regeneration between these experiments. Our results show that the observed inhibition of stem cell-based tissue regeneration persists during long-duration spaceflight. Furthermore, spaceflight femurs from BionM1 indicate onset of an accelerated aging-like phenotype with signs of osteoarthritic disease shown by disruption of the epiphyseal boundary and endochondral ossification. These effects are likely caused by a failure of stem cells to regenerate degraded tissues and may have significant implications for bone and cartilage health following extensive periods of mechanical unloading during long-duration spaceflight.

stem cells↗

Femoral Head Bone Loss Following Short and Long-Duration Spaceflight

Exposure to mechanical unloading during spaceflight is known to have significant effects on the musculoskeletal system. Our ongoing studies with the mouse bone model have identified the failure of normal stem cell-based tissue regeneration, in addition to tissue degeneration, as a significant concern for long-duration spaceflight, especially in the mesenchymal and hematopoietic tissue lineages. The 30-day BionM1 and the 37-day Rodent Research 1 (RR1) missions enabled the possibility of studying these effects in long-duration microgravity experiments. We hypothesized that the inhibition of stem cell-based tissue regeneration in short-duration spaceflight would continue during long-duration spaceflight and furthermore would result in significant tissue alterations. MicroCT analysis of BionM1 femurs revealed 31% decrease in bone volume ratio, a 14% decrease in trabecular thickness, and a 20% decrease in trabecular number in the femoral head of space-flown mice. Furthermore, high-resolution MicroCT and immunohistochemical analysis of spaceflight tissues revealed a severe disruption of the epiphyseal boundary, resulting in endochondral ossification of the femoral head and perforation of articular cartilage by bone. This suggests that spaceflight in microgravity may cause rapid induction of an aging-like phenotype with signs of osteoarthritic disease in the hip joint. However, mice from RR1 exhibited significant bone loss in the femoral head but did not exhibit the severe aging and disease-like phenotype observed during BionM1.This may be due to increased physical activity in the RH hardware. Immunohistochemical analysis of the epiphyseal plate and investigation of cellular proliferation and differentiation pathways within the marrow compartment and whole bone tissue is currently being conducted to determine alterations in stem cell-based tissue regeneration between these experiments. Our results show that the observed inhibition of stem cell-based tissue regeneration persists during long-duration spaceflight. Furthermore, spaceflight femurs from BionM1 indicate onset of an accelerated aging-like phenotype with signs of osteoarthritic disease shown by disruption of the epiphyseal boundary and endochondral ossification. These effects are likely caused by a failure of stem cells to regenerate degraded tissues and may have significant implications for bone and cartilage health following extensive periods of mechanical unloading during long-duration spaceflight.

microgravity↗

Identification And Survival Of Bacteriohopanepolyol In A Hot Spring Microbial Mat

The polar lipids of a hot spring microbial mat located in Yellowstone National Park were examined for the presence of bacteriohopanepolvols (BHP). BHP are a group of molecules consisting of a hopanoid (peotacyclic triterpene) linked via a n-alkyl polyhydroxylated chain to a variety of polar end groups. BHP have been isolated in varying amounts from phylogenetically diverse eubacterial groups including cyanobacteria, methanotrophs and the Rhodospirillaceae. The hopanoids are excellent biomarkers and have been detected in sedimentary rocks as old as 1.7 bya. In order to interpret the ancient organic record, it is important to understand the abundance, source and fate of such biomarker compounds in microbial mats. A 40 sq cm mat section was taken from a 52 to 55 C site in the effluent channel of Octopus Spring and was sampled vertically over approximately 16 mm. The first 5-6 mm was sectioned into a top green layer (310 mg dry weight) and several subjacent, deep orange layers (240 and 250 mg, respectively). The lower 10 mm of the mat was sectioned into two gelatinous orange layers containing a siliceous gritty material (260 and 440 mg) which increased with depth, and a bottom layer composed almost exclusively of siliceous sinter (4.1 g). The progressive decrease in total organic carbon from 45% in the top green layer to only 4% in the bottom layer reflects the observed increase in siliceous deposition. GC-MS analysis of the phospholipid and glycolipid fatty acids yielded predominantly saturated normal chain acids, n-15 to n-18, and iso-branched acids, i-15 to i-17. Small amounts of unsaturated fatty acids (16:1, two positional isomers of 18:1, and two cyclopropyl acids, C(sub 17) and C(sub 19)) were present mainly in the top layer. Esterified fatty acid which is a good index for intact cellular membrane, i.e. viable organisms, was highest in the top two layers (203 and 231 micro g/mg total lipid, respectively) and gradually decreased to 66 micro g/mg total lipid in the bottom layer. Small amounts of BHP were present in all six layers, however in this case, BHP was lowest in the top green and subjacent deep-orange layers (118 and 172 micro g/mg total lipid, respectively) and increased with depth reaching almost 400 micro g/mg in the bottom two layers. This data suggest that BHP are survivina the initial phase of mat degradation and may be preferentially enriched in any organic record of such thermal environments. The relatively low level of BHP in the top layer also suggests that cyanobacteria may not be the major source of BHP in this mat. Since Chloroflexus a major component of the deep-orange layer has been reported to lack BHP, this material may prove a valuable biomarker for some other mat inhabitant. Further isotopic characterization of this BHP should help resolve this finding.

Janke, Linda L.↗

Is Water Necessary for Life?

The universality of water as the solvent for life is usually justified by its role in supporting the rich organic chemistry. It has been pointed out, however, that even richer synthetic chemistry is possible in other organic solvents. Does it mean that water is not necessary for life? Here, other, essential criteria for solvent for life that have not been sufficiently considered are discussed. In biological systems, complex molecules are not only constantly synthesized but also degraded. Solvent-mediated degradation is essential for regulating cell content, preventing overcrowding and allowing for recycling organic material. Achieving a balance between synthetic and degradative processes is facile in water, but not in many other organic liquids. Thus, the so-called “water paradox” according to which water is both necessary to life and toxic to biopolymer synthesis might not be paradoxical at all. The machinery of life is based on non-covalent interactions that do not involve making or breaking chemical bonds. Their strength needs to be properly tuned. If they are too weak, there might be undesired response to natural fluctuations of physical or chemical parameters. If they are too strong, the kinetics and energetics of cellular processes could adversely influenced. The solvent must allow for balancing these interactions, which provides strong, universal constraints on the medium for life. Water influences non-covalent interactions mainly by two mechanisms. First, it reduces strong, electrostatic interactions between molecules, chemical groups or atoms carrying electric charge or dipole. Second, it induces the hydrophobic effect, the tendency to remove non-polar (hydrophobic) molecules and groups from direct contact with aqueous solution and, instead, interact with each other. In living systems, the hydrophobic effect is largely responsible for self-organization of molecules to more complex structures, such as aggregation of lipid molecules to form biological membranes and protein folding. Water exists as stable liquid in a large temperature range, and the hydrophobic effects are a consequence of the temperature insensitivity of essential properties of its liquid state. In summary, water accomplishes an amazing feat – it reduces strong interactions between dissolved species and simultaneously increases the strength of weak interactions, bringing all of them to the right range. Once we consider not only synthetic capabilities but also other required traits of the solvent for life, no viable alternative to water is currently known.

Life↗

Effect of salinity on the preservation of biomarkers in hypersaline microbial mat kerogens

Kerogen is the residue that remains after the minerals and organic compounds have been extracted from a rock using acid and organic solvents, respectively [1]. It is not only the largest organic carbon sink in the geological record but the best-preserved fraction due to its recalcitrance and ability to retain syngenetic source information during deposition [2]. Due to the complex, macromolecular, cross-linked polymeric structure of kerogen, it is difficult to analyse intact, often requiring breakdown into smaller subunits through thermal (pyrolysis) or chemical treatment (chemolysis) [3]. Saline and hypersaline environments host diverse microbial communities, which can trap and bind organic matrices such as cellular components (e.g., extracellular polymeric substances, cell wall, peptidoglycan, sheath material, etc.) with sediments, forming layered, laminated structures called microbial mats. Here we present depth profiles of two hypersaline cores through a microbial mat with differing total salinities. We looked at the composition of organic compounds released from kerogen during high temperature, hydrogen-assisted, pyrolysis to understand the effect of salinity on the composition and timing of their incorporation [4]. Field Site: The Exportadora del Sal S. A. (ESSA) saltern of Guerrero Negro (Baja California Sur, Mexico) lies within a typical sabhka environment adjacent to Laguna Ojo de Liebre. Note the spatial extent of the 13 major ponds of the ESSA system. The GN ponds thrive under well-monitored and stable conditions with steady state accretion and degradation of mat layers (around 0.5–1.4 cm per year). Organic mats typically attain up to 10 cm in thickness, depending on the location, which represents about 60 years of growth since these ponds were established. Sedimentation rate is rapid in these non-lithifying and non-mineralising mats, 1–1.5 cm per year. This environment is ideal for investigating in situ lipid preservation and diagenesis without the influence of constantly changing physical parameters. Methods: P4n5 and P5AB cores were collected as ~9 cm and 6.5 cm fresh cores, respectively, in June 2001 and September 2010, respectively from the ESSA saltern. The P4n5 core (9-9.1% salinity) was taken from Pond 4 near 5 and sub-divided into 10 layers. The P5AB core (11.2% salinity) was taken from Pond 5A near 5B and sub-divided into 8 layers. Lyophilised microbial mat powders were solvent-extracted using a modified Bligh-Dyer method and analysed by gas chromatography-mass spectrometry (GC-MS). Additionally, a subset of layers—layer 4 and layer 7—which represented active biomass and sediment-processed, respectively, were subjected to mild-acid methanolysis. The respective residues from conventional solvent extraction and acid methanolysis were loaded with a molybdenum sulfide catalyst and placed into a stainless-steel reactor. Samples were run on the hydropyrolysis set up (heated to 500 °C with 13-15 MPa of H2 pressure), extracted, and analysed by GC-MS. Results: Molecular profiles from the two ponds were significantly different and reflect the contributions of different photosynthetic and respiratory microbial communities to preserved organic matter. For example, the higher salinity P5AB core showed evidence of greater archaeal inputs (similarly observed in lab culture experiments [5]) as well as potential kerogen-bound carotenoids/carotenoid rearranged products or fragments. A hydrophobic emulsion formed during the acid methanolysis processing of layer 4 from both P4n5 and P5AB and layer 7 of P5AB. Hydropyrolysis of this hydrophobic residues released a greater abundance of polycyclic lipids relative to the pre-extracted control. Implications and Future Work: Catalytic hydropyrolysis of kerogen can rapidly generate abundant saturated pyrolysate products from the bound biomarker pool without altering the structures or stereochemistries of the products [6]. Chemical processing of solvent-extractable residues indicated that cellular matrices such as extracellular polymeric substances (EPS) may play a role in the sequestration of polycyclic lipid biomarkers such as steranes and hopanes. Their higher relative abundance compared to the pre-extracted control in the higher salinity layers and cores preliminarily aligns with this hypothesis, although further experiments are required to confirm this. It has been well-documented that EPS plays an important role in mineralisation, specifically carbonate formation, in microbial ecosystems. EPS can enhance calcium carbonate precipitation by providing diffusion-limited sites that create alkalinity gradients in response to microbial processes [7]. It has been experimentally demonstrated that salinity influences the total amount of EPS (both loosely- and tightly-bound) which increases with increasing salinity [8]. The higher salinity at Guerrero Negro switches the microbial population from filamentous Microcoleus to being dominated by Phormidium, Oscillatoria, and unicellular cyanobacteria. We demonstrated that lipid binding into kerogen via strong covalent linkages occurs at the very earliest stages of sedimentary diagenesis. We are currently investigating the influence of salinity on organic preservation through experimental and modelling approaches. Understanding how key biomarkers transform into preserved organic matter (i.e., from precursor biolipids to bound geolipids) in brine ecosystems will aid the search for organic biosignatures on other planetary bodies, especially Icy Moons and modern Mars.

hypersaline↗

SNF1-related protein kinase 1 represses Arabidopsis growth through post-translational modification of E2Fa in response to energy stress

Cellular sugar starvation and/or energy deprivation serves as an important signaling cue for the live cells to trigger the necessary stress adaptation response. When exposed to cellular energy stress (ES) conditions, the plants reconfigure metabolic pathways and rebalance energy status while restricting vegetative organ growth. Despite the vital importance of this ES-induced growth restriction, the regulatory mechanism underlying the response remains largely elusive in plants.

59 BASIC BIOLOGICAL SCIENCES↗

Direct Air Capture of CO 2 and Delivery to Photobioreactors for Algal Biofuel Production (Final Report)

A mobile DAC system was designed and constructed to pair with photobioreactors growing algae for biofuel production. The DAC system was designed as a versatile research system, rather than a compact production unit. The system was constructed and mounted on a mobile skid to facilitate transportation to the algae production site. Within the DAC system, CO 2 was captured using amine-loaded monoliths that allow for high CO 2 uptake with low pressure drop. The CO 2 is collected using a Global Thermostat patented temperature/vacuum swing adsorption (TVSA) process. Amine sorbents and process conditions were optimized to produce 10 to >20 g CO 2 .h-1. The stability of the amine sorbents was also studied, with sorbent modifications made to improve stability to degradation by oxidation. An Algenol-developed Spirulina strain (Arthrospira platensis AB2293) was selected as the production cyanobacterial strain. AB2293 cultured was inoculum for outdoor production following PBR installation by Algenol. The PBR system was composed of three independent PBRs, with each PBR composed of four hanging bags internally recirculated by a liquid turnover pump. The PBRs were operated outdoors in Atlanta, GA, and integrated with the DAC system. Algae were grown with similar productivity using DAC-CO 2 as algae grown using pure CO 2 obtained commercially (Airgas). Throughout the experimental duration, no discoloration was observed, and cellular morphology was consistent between the two experimental treatments. An LCA including lifecycle greenhouse gas emissions, full life cycle inventory of the Algenol system and the DAC system and integrated DAC+PBR system was developed. Lifecycle greenhouse gas emissions were calculated for capture of carbon dioxide using input from Global Thermostat and the National Renewable Energy Laboratory. Three scenarios for energy provision were evaluated: a natural gas combined heat and power system sized to meet the electricity requirement, a natural gas combined heat and power system sized to meet the process heat requirements, and a system without on-site power that procures the electricity from the grid. In all three cases, as expected, the major contributor to the emissions is the energy consumption associated with the desorption step of the DAC process. The LCA quantified the reduced potential energy and greenhouse gas emissions of heat and mass integration of DAC and Algenol compared to unintegrated DAC and Algenol systems. A life cycle assessment of the role of sorbent productivity and lifetime was also developed. The development of more robust, oxidation resistant DAC sorbents may enable small reductions in energy requirements and in lifecycle greenhouse gas emissions and other environmental impacts. NREL performed techno-economic analysis (TEA) to identify the integration scenario most likely to achieve a 15% cost reduction target versus the baseline. Heat and mass integration of DAC and the PBR is critical to minimizing the MFSP. The baseline case utilizes no heat and mass integration, and the DAC system provides 100% of the CO 2 required by the photobioreactors (20 tonnes/hr), operating for 12 hours/day capturing 40 tonnes CO 2 /operating hour. The minimum fuel selling price (MFSP) of ethanol calculated from the baseline case was $10.68/gal ethanol. This corresponds with a targeted MFSP of $9.07/gal ethanol (or 15% reduction). This target was achieved by integration Option 2a with the greatest cost reduction of 17.8% (or $8.78/gal) and integration Option 2b with a cost reduction of 16.4% (or $8.93/gal). Reductions in MFSP are attributed to two primary process considerations: (a) CO 2 storage at night reduces the capital expenses associated with DAC (i.e., increasing on-stream time); and (b) distributed DAC scenarios (DAC-PBR integration Options 2a and 2b) make use of boiler and DAC CHP flue gas CO 2 (free). Direct air capture on-stream time was one of the largest contributors to MFSP reduction.

09 BIOMASS FUELS↗

p14 ARF forms meso-scale assemblies upon phase separation with NPM1

NPM1 is an abundant nucleolar chaperone that, in addition to facilitating ribosome biogenesis, contributes to nucleolar stress responses and tumor suppression through its regulation of the p14 Alternative Reading Frame tumor suppressor protein (p14 ARF ). Oncogenic stress induces p14 ARF to inhibit MDM2, stabilize p53 and arrest the cell cycle. Under non-stress conditions, NPM1 stabilizes p14 ARF in nucleoli, preventing its degradation and blocking p53 activation. However, the mechanisms underlying the regulation of p14 ARF by NPM1 are unclear because the structural features of the p14 ARF -NPM1 complex were elusive. Here we show that p14 ARF assembles into a gel-like meso-scale network upon phase separation with NPM1. This assembly is mediated by intermolecular contacts formed by hydrophobic residues in an α-helix and β-strands within a partially folded N-terminal portion of p14 ARF . These hydrophobic interactions promote phase separation with NPM1, enhance p14 ARF nucleolar partitioning, restrict NPM1 diffusion within condensates and nucleoli, and reduce cellular proliferation. Our structural analysis provides insights into the multifaceted chaperone function of NPM1 in nucleoli by mechanistically linking the nucleolar localization of p14 ARF to its partial folding and meso-scale assembly upon phase separation with NPM1.

59 BASIC BIOLOGICAL SCIENCES↗

The oleaginous yeast Cutaneotrichosporon oleaginosum modifies corn stover alkali lignin

The current paradigm in synthetic biology for lignin bioconversion platforms includes primarily bacteria and filamentous fungi. Yeast are notoriously understudied for their role in lignin degradation and utilization, despite their ubiquity in saprophytic microbial communities. A few publications report lignin-modifying yeasts, but investigations to date have relied on model aromatic compounds or lignin-containing substrates replete with other carbon sources. In this work, we use a suite of analytical tools to evaluate interactions between corn stover-extracted lignin and the oleaginous yeast Cutaneotrichosporon oleaginosum. Notably, 2D-NMR analysis showed a significant decrease in the H-lignin component as well as resinol (β-β) and phenylcoumaran (β-5) linkages. Using super-resolution fluorescence microscopy, we demonstrated that this yeast may uptake polymeric lignin and/or undertakes interactions at the cellular envelope. To explore mechanisms of lignin modification, transport, and aromatics catabolism, extensive secretomics and proteomics analyses were conducted. Compared to carbon-limited glucose and “No Carbon” controls, several putative laccases, quinone reductases, superoxide dismutases, and glyoxal/oxalate oxidases were upregulated in the lignin condition. Excitingly, two ferric reductases and an oxalate exchanger were only observed in the lignin condition. These results indicate that C. oleaginosum may perform extracellular quinone redox cycling to generate lignin-modifying reactive oxygen species. These findings enhance our understanding of yeast-lignin interactions and provide valuable insights for validation studies and metabolic engineering.

09 BIOMASS FUELS↗

Soil aminopeptidase induction is unaffected by inorganic nitrogen availability

Soil microbes release a variety of aminopeptidase enzymes to degrade proteins into their constituent amino acid monomers, thereby increasing amino acid availability in the near-cell environment. In this work we investigated how inorganic N availability controls the substrate induction response of aminopeptidase enzymes by conducting protein amendment experiments via gelatin addition to fertilized and unfertilized soil from a long-term N addition study. Protein addition triggered a measurable increase in enzyme activity, which we interpreted as an induction response, in all five of the aminopeptidase enzymes we investigated. The magnitude of these induction responses did not differ by fertilization regime even when unamended aminopeptidase activity differed between long-term fertilization treatments. Though soil microbes are thought to release aminopeptidase enzymes as a means of cellular N acquisition at steady-state concentrations, our results suggest that they use the same enzymes to access protein-derived C as an energetic resource at non-steady-state conditions, such as those triggered by the substrate induction response. The substrate induction response of aminopeptidase enzymes is therefore best viewed as a means of microbial competition for the substantial energetic resources available in protein subsidies.

60 APPLIED LIFE SCIENCES↗

Tunable, biodegradable grafting-from glycopolypeptide bottlebrush polymers

The cellular glycocalyx and extracellular matrix are rich in glycoproteins and proteoglycans that play essential physical and biochemical roles in all life. Synthetic mimics of these natural bottlebrush polymers have wide applications in biomedicine, yet preparation has been challenged by their high grafting and glycosylation densities. Using one-pot dual-catalysis polymerization of glycan-bearing α-amino acid N-carboxyanhydrides, we report grafting-from glycopolypeptide brushes. The materials are chemically and conformationally tunable where backbone and sidechain lengths were precisely altered, grafting density modulated up to 100%, and glycan density and identity tuned by monomer feed ratios. The glycobrushes are composed entirely of sugars and amino acids, are non-toxic to cells, and are degradable by natural proteases. Inspired by native lipid-anchored proteoglycans, cholesterol-modified glycobrushes were displayed on the surface of live human cells. Our materials overcome long-standing challenges in glycobrush polymer synthesis and offer new opportunities to examine glycan presentation and multivalency from chemically defined scaffolds.

36 MATERIALS SCIENCE↗