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At least 307 records · Page 17

Shiga Toxin Induces Lipid Compression: A Mechanism for Generating Membrane Curvature

We report biomembranes are hard to compress laterally, and membrane area compressibility has not been associated with biological processes. Using X-ray surface scattering, we observed that bacterial Shiga toxin compresses lipid packing in a gel phase monolayer upon binding to its cellular receptor, the glycolipid Gb 3 . This toxin-induced reorganization of lipid packing reached beyond the immediate membrane patch that the protein was bound to, and linkers separating the Gb 3 carbohydrate and ceramide moieties modulated the toxin’s capacity to compress the membrane. Within a natural membrane, asymmetric compression of the toxin-bound leaflet could provide a mechanism to initiate narrow membrane bending, as observed upon toxin entry into cells. Such lipid compression and long-range membrane reorganization by glycolipid-binding proteins represent novel concepts in membrane biology that have direct implications for the construction of endocytic pits in clathrin-independent endocytosis.

36 MATERIALS SCIENCE↗

Upstream and Downstream Boundary Conditions Control the Physical and Biological Development of River Deltas

Relative sea level rise is depleting coastal land. Success in coastal restoration depends on understanding interactions between physical and biological processes that influence landscape change. We present results from flume experiments examining the coevolution of vegetation and delta channel networks. We show that channel mobility—controlled by upstream and downstream boundary conditions and delta size—controls vegetation colonization. The spatial patterns of plants in turn control the stability and spatial distribution of channels. Experiments with low–channel mobility develop large, dense plant patches that become denser over time and stabilize channels. Experiments with more mobile channels have sparse vegetation patterns that limit vegetation growth within existing patches, maintaining sparse, patchy vegetation. Thus, the ecogeomorphic development of deltas depends on the rate of channel lateral migration and avulsion, suggesting that upstream and downstream boundary conditions control both the physical and biological evolution of coastal landscapes.

54 ENVIRONMENTAL SCIENCES↗

Parameter Estimation and Uncertainty Quantification for Systems Biology Models

Mathematical models can provide quantitative insights into immunoreceptor signaling, and other biological processes, but require parameterization and uncertainty quantification before reliable predictions become possible. We review currently available methods and software tools to address these problems. We consider gradient-based and gradient-free methods for point estimation of parameter values, and methods of profile likelihood, bootstrapping, and Bayesian inference for uncertainty quantification. We consider recent and potential future applications of these methods to systems-level modeling of immune-related phenomena.

59 BASIC BIOLOGICAL SCIENCES↗

Single-Objective Airy Light-Sheet Imaging

Despite its massive potential, standard light-sheet imaging (LSI) faces key challenges, such as the incompatibility with common sample mounting techniques and low-resolution imaging. Single-objective LSI attempts to address these issues but often suffers from limited fields-of-view and throughput rates, or requires multiple optics that increase costs, alignment complexity, and losses. To overcome these challenges of standard single-objective LSI, we introduce single-objective Airy light-sheet imaging (SoALSI). SoALSI leverages the extraordinary self-acceleration properties of the Airy beam, achieving 5× higher imaging rates and enhanced imaging efficiency than standard single-objective LSI. Here, we demonstrate SoALSI’s versatility through rigorous contrast and resolution characterizations and by high-resolution imaging of diverse biological specimens, including malaria parasite-infected red blood cells and plant root tissue. SoALSI seamlessly integrates with any standard inverted microscope frame, enabling broader accessibility for the bioimaging community to explore biological processes in a wide range of specimens with enhanced resolution and imaging contrast.

airy beam↗

Sample Processing Instrumentation to Enable Lab-like Analysis of Lipids, Hydrocarbons, and Kerogen in situ

Introduction: Over the last decade, our team based out of NASA Ames Research Center has made advances in fluidic technologies to enable laboratory-grade processes in space environments. More recently, we have designed and developed two instruments that utilize sample processing steps to extract and purify organic molecules that are key targets in the search for life. The first instrument targets soluble lipids and hydrocarbons that have been preserved in soil or rock material for millions to billions of years called ExCALiBR (Extractor for Chemical Analysis of Lipid Biomarkers in Regolith). The second instrument called KAMELOT (Kerogen and Macromolecule Extractor Liberating Organics Thermolytically) targets kerogen or insoluble macromolecular material that reside in the residue left behind after solvent extraction. Together, these instruments can be coupled to existing flight sample acquisition and analytical instrumentation (e.g., mass spectrometers) to achieve our overall science goal of identifying molecular patterns and features that are diagnostic of how the organic matter was synthesized, either through biological or non-biological processes.

Mary Beth Wilhelm↗

Metaproteomics reveals insights into microbial structure, interactions, and dynamic regulation in defined communities as they respond to environmental disturbance

Abstract Background Microbe-microbe interactions between members of the plant rhizosphere are important but remain poorly understood. A more comprehensive understanding of the molecular mechanisms used by microbes to cooperate, compete, and persist has been challenging because of the complexity of natural ecosystems and the limited control over environmental factors. One strategy to address this challenge relies on studying complexity in a progressive manner, by first building a detailed understanding of relatively simple subsets of the community and then achieving high predictive power through combining different building blocks (e.g., hosts, community members) for different environments. Herein, we coupled this reductionist approach with high-resolution mass spectrometry-based metaproteomics to study molecular mechanisms driving community assembly, adaptation, and functionality for a defined community of ten taxonomically diverse bacterial members of Populus deltoides rhizosphere co-cultured either in a complex or defined medium. Results Metaproteomics showed this defined community assembled into distinct microbiomes based on growth media that eventually exhibit composition and functional stability over time. The community grown in two different media showed variation in composition, yet both were dominated by only a few microbial strains. Proteome-wide interrogation provided detailed insights into the functional behavior of each dominant member as they adjust to changing community compositions and environments. The emergence and persistence of select microbes in these communities were driven by specialization in strategies including motility, antibiotic production, altered metabolism, and dormancy. Protein-level interrogation identified post-translational modifications that provided additional insights into regulatory mechanisms influencing microbial adaptation in the changing environments. Conclusions This study provides high-resolution proteome-level insights into our understanding of microbe-microbe interactions and highlights specialized biological processes carried out by specific members of assembled microbiomes to compete and persist in changing environmental conditions. Emergent properties observed in these lower complexity communities can then be re-evaluated as more complex systems are studied and, when a particular property becomes less relevant, higher-order interactions can be identified.

59 BASIC BIOLOGICAL SCIENCES↗

Photocaged functional nucleic acids for spatiotemporal imaging in biology

Imaging of species in living organisms with high spatiotemporal resolution is essential for understanding biological processes. While functional nucleic acids (FNAs), such as catalytic nucleic acids and aptamers, have emerged as effective sensors for a wide range of molecules, photocaged control of these FNAs has played a key role in translating them into bioimaging agents with high spatiotemporal control. In this paper, we summarize methods and results of photocaged FNAs based on photolabile modifications, photoisomerization, and photothermal activation. Future directions, including strategies to improve the performance of these photocaged FNAs, are also described.

59 BASIC BIOLOGICAL SCIENCES↗

Thiol redox proteomics: Characterization of thiol-based post-translational modifications

Redox post-translational modifications on cysteine thiols (redox PTMs) have profound effects on protein structure and function, thus enabling regulation of various biological processes. Redox proteomics approaches aim to characterize the landscape of redox PTMs at the systems level. These approaches facilitate studies of condition-specific, dynamic processes implicating redox PTMs and have furthered our understanding of redox signaling and regulation. Mass spectrometry (MS) is a powerful tool for such analyses which has been demonstrated by significant advances in redox proteomics during the last decade. A group of well-established approaches involves the initial blocking of free thiols followed by selective reduction of oxidized PTMs and subsequent enrichment for downstream detection. Alternatively, novel chemoselective probe-based approaches have been developed for various redox PTMs. Direct detection of redox PTMs without any enrichment has also been demonstrated given the sensitivity of contemporary MS instruments. This review discusses the general principles behind different analytical strategies and covers recent advances in redox proteomics. Several applications of redox proteomics are also highlighted to illustrate how large-scale redox proteomics data can lead to novel biological insights.

59 BASIC BIOLOGICAL SCIENCES↗

Insights into the Lysine Acetylome of the Haloarchaeon Haloferax volcanii during Oxidative Stress by Quantitative SILAC-Based Proteomics

Oxidative stress adaptation strategies are important to cell function and are linked to cardiac, neurodegenerative disease, and cancer. Representatives of the Archaea domain are used as model organisms based on their extreme tolerance to oxidants and close evolutionary relationship with eukaryotes. A study of the halophilic archaeon Haloferax volcanii reveals lysine acetylation to be associated with oxidative stress responses. The strong oxidant hypochlorite: (i) stimulates an increase in lysine acetyltransferase HvPat2 to HvPat1 abundance ratios and (ii) selects for lysine deacetylase sir2 mutants. Here we report the dynamic occupancy of the lysine acetylome of glycerol-grown H. volcanii as it shifts in profile in response to hypochlorite. These findings are revealed by the: (1) quantitative multiplex proteomics of the SILAC-compatible parent and Δsir2 mutant strains and (2) label-free proteomics of H26 ‘wild type’ cells. The results show that lysine acetylation is associated with key biological processes including DNA topology, central metabolism, cobalamin biosynthesis, and translation. Lysine acetylation targets are found conserved across species. Moreover, lysine residues modified by acetylation and ubiquitin-like sampylation are identified suggesting post-translational modification (PTM) crosstalk. Overall, the results of this study expand the current knowledge of lysine acetylation in Archaea, with the long-term goal to provide a balanced evolutionary perspective of PTM systems in living organisms.

59 BASIC BIOLOGICAL SCIENCES↗

Identification of aberrant circular RNA expression and its potential clinical value in primary great saphenous vein varicosities

Highlights: • This study identified 232 significantly aberrant circRNAs in primary great saphenous vein varicosities (PGSVVs). • A circRNA-miRNA-gene co-expression network was created. • Hsa{sub c}irc{sub 0}006427, hsa{sub c}irc{sub 0}089810 and hsa{sub c}irc{sub 0}005267 might be potential biological markers for PGSVVs. This study aimed to identify aberrant circRNA expression in primary great saphenous vein varicosities (PGSVVs) and investigate its potential clinical significance. High-throughput sequencing was applied to analyze the differential expression of circRNAs in three paired vein samples of PGSVVs and a control group. Computational bioinformatics analyses, including gene ontology (GO) analysis and encyclopedia of genes and genomes (KEGG) pathway analysis, were performed to further enrich microRNA data. Real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to verify the selected aberrant circRNAs in all vein samples, and a receiver operating characteristic (ROC) curve was plotted to evaluate the diagnostic and potential clinical significance. A total of 232 circRNAs were significantly aberrantly expressed in PGSVVs, including 105 and 127 upregulated and downregulated circRNAs, respectively. The predicted top 10 downstream miRNAs of the significantly altered circRNAs are hsa-miR-103a-2-5p, hsa-miR-141-5p, hsa-miR-3692-5p, hsa-miR-4659a-3p, hsa-miR-4659b-3p, hsa-miR-4691-5p, hsa-miR-4778-3p, hsa-miR-6738-3p, hsa-miR-6792-3p and hsa-miR-6873-3p. GO analysis revealed that the most enriched and meaningful biological process, cellular component, and molecular function terms were related to the regulation of catabolic processes, the cytoplasm and ATP binding, respectively. The most enriched and meaningful pathways revealed by KEGG pathway analysis were related to “axon guidance” “vitamin digestion and absorption” and “NF-Kappa B signaling pathway”. Hsa{sub c}irc{sub 0}006427 (P = 0.007), hsa{sub c}irc{sub 0}089810 (P = 0.044) and hsa{sub c}irc{sub 0}005267 (P = 0.049) were significantly downregulated in the PGSVV group relative to their expression levels in the control group, and the area under the ROC curve was 0.9091, 0.8025 and 0.8148, respectively. This study demonstrated that circRNAs were differentially expressed in PGSVVs, and highlights the crucial roles of circRNAs in the pathogenesis of PGSVVs and indicates that hsa{sub c}irc{sub 0}006427, hsa{sub c}irc{sub 0}089810 and hsa{sub c}irc{sub 0}005267 might be potential diagnostic and clinical biological markers.

60 APPLIED LIFE SCIENCES↗

Inhibition of DNA methylation in P. soloecismus alters algae productivity

Eukaryotic organisms regulate the organization, structure, and accessibility of their genomes through chromatin remodeling that can be inherited as epigenetic modifications. These DNA and histone protein modifications are ultimately responsible for an organism’s molecular adaptation to the environment, resulting in distinctive phenotypes. Epigenetic manipulation of algae holds yet untapped potential for the optimization of biofuel production and bioproduct formation; however, epigenetic machinery and modes-of-action have not been well characterized in algae. We sought to determine the extent to which the biofuel platform species Picochlorum soloecismus utilizes DNA methylation to regulate its genome. We found candidate genes with domains for DNA methylation in the P. soloecismus genome. Whole-genome bisulfite sequencing revealed DNA methylation in all three cytosine contexts (CpG, CHH, and CHG). While global DNA methylation is low overall (~1.15%), it occurs in appreciable quantities (12.1%) in CpG dinucleotides in a bimodal distribution in all genomic contexts, though terminators contain the greatest number of CpG sites per kilobase. The P. soloecismus genome becomes hypomethylated during the growth cycle in response to nitrogen starvation. Algae cultures were treated daily across the growth cycle with 20 μM 5-aza-2'-deoxycytidine (5AZA) to inhibit propagation of DNA methylation in daughter cells. 5AZA treatment significantly increased optical density and forward and side scatter of cells across the growth cycle (16 days). This increase in cell size and complexity correlated with a significant increase (~66%) in lipid accumulation. Site specific CpG DNA methylation was significantly altered with 5AZA treatment over the time course, though nitrogen starvation itself induced significant hypomethylation in CpG contexts. Genes involved in several biological processes, including fatty acid synthesis, had altered methylation ratios in response to 5AZA; we hypothesize that these changes are potentially responsible for the phenotype of early induction of carbon storage as lipids. This is the first report to utilize epigenetic manipulation strategies to alter algal physiology and phenotype. Collectively, these data suggest these strategies can be utilized to fine-tune metabolic responses, alter growth, and enhance environmental adaption of microalgae for desired outcomes.

5-aza-2'-deoxycytidine↗

The Transformation and Export of Organic Carbon Across an Arctic River-Delta-Ocean Continuum

The Arctic Ocean is surrounded by land that feeds highly seasonal rivers with water enriched in high concentrations of dissolved and particulate organic carbon (DOC and POC). Explicit estimates of the flux of organic carbon across the land-ocean interface are difficult to quantify and many interdependent processes makes source attribution difficult. A high-resolution 3-D biogeochemical model was built for the lower Yukon River and coastal ocean to estimate biogeochemical cycling across the land-ocean continuum. The model solves for complex reactions related to organic carbon transformation, including mechanistic photodegradation and multi-reactivity microbial processing, DOC to POC flocculation, and phytoplankton dynamics. The baseline DOC and POC flux out of the delta from April to September 2019, was 977 and 536 Gg C (~80% of the annual total), but only 50% of the DOC and 25% of the POC exited the plume across the 10 m isobath. Microbial breakdown of DOC accounted for a net loss of 168 Gg C (17% of delta export) within the plume and photodegradation accounted for a net loss of 46.6 Gg C DOC (5% of delta export) in 2019. Flocculation decreased the total organic carbon flux by only 6.4 Gg C (~1%), while POC sinking accounted for 63.3 Gg C (10%) settling in the plume. The loss of chromophoric dissolved organic matter (CDOM) due to photodegradation increased the light available for phytoplankton growth throughout the coastal ocean, demonstrating the secondary effects that organic carbon reactions can have on biological processes and the net coastal carbon flux.

Land-Ocean Continuum↗

Comprehensive characterization of extracellular vesicles produced by environmental (Neff) and clinical (T4) strains of Acanthamoeba castellanii

We conducted a comprehensive comparative analysis of extracellular vesicles (EVs) from two Acanthamoeba castellanii strains, Neff (environmental) and T4 (clinical). Morphological analysis via transmission electron microscopy revealed slightly larger Neff EVs (average = 194.5 nm) compared to more polydisperse T4 EVs (average = 168.4 nm). Nanoparticle tracking analysis (NTA) and dynamic light scattering validated these differences. Proteomic analysis of the EVs identified 1,352 proteins, with 1,107 common, 161 exclusive in Neff, and 84 exclusively in T4 EVs. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) mapping revealed distinct molecular functions and biological processes and notably, the T4 EVs enrichment in serine proteases, aligned with its pathogenicity. Lipidomic analysis revealed a prevalence of unsaturated lipid species in Neff EVs, particularly triacylglycerols, phosphatidylethanolamines (PEs), and phosphatidylserine, while T4 EVs were enriched in diacylglycerols and diacylglyceryl trimethylhomoserine, phosphatidylcholine and less unsaturated PEs, suggesting differences in lipid metabolism and membrane permeability. Metabolomic analysis indicated Neff EVs enrichment in glycerolipid metabolism, glycolysis, and nucleotide synthesis, while T4 EVs, methionine metabolism. Furthermore, RNA-seq of EVs revealed differential transcript between the strains, with Neff EVs enriched in transcripts related to gluconeogenesis and translation, suggesting gene regulation and metabolic shift, while in the T4 EVs transcripts were associated with signal transduction and protein kinase activity, indicating rapid responses to environmental changes. In this novel study, data integration highlighted the differences in enzyme profiles, metabolic processes, and potential origins of EVs in the two strains shedding light on the diversity and complexity of A. castellanii EVs and having implications for understanding host-pathogen interactions and developing targeted interventions for Acanthamoeba-related diseases.

59 BASIC BIOLOGICAL SCIENCES↗

Proteomic studies of bone and skeletal health outcomes

Proteins are an essential part of essentially all biological processes, and there is enormous variation in protein forms and concentrations that is not reflected in DNA or RNA. Recently there have been rapid advances in the ability to measure protein sequence, modification and concentration, particularly with methods based in mass spectrometry. Global measures of proteins in tissues or in the circulation provide a broad assessment of the proteome that can be extremely useful for discovery, and targeted proteomic measures can yield specific and sensitive assessments of specific peptides and proteins. While most proteomic measures are directed at the detection of consensus peptide sequences, mass spectrometry based proteomic methods also allow a detailed examination of the peptide sequence differences that result from genetic variants and that may have important effects on protein function. In evaluating proteomic data, a number of analytical considerations are important, including an understanding of missing data, the challenge of multiple testing and replication, and the use of rapidly evolving methods in systems biology. While proteomics has not yet had a major impact in skeletal research, interesting recent research has used these approaches in the study of bone cell biology and the discovery of biomarkers of skeletal disorders. Proteomics can be expected to have an increasing influence in the study of bone biology and pathophysiology

Proteomic, Bone↗

The membrane bioreactor (MBR): A hybrid technology for bioregenerative wastewater treatment and resource recovery in space

Extraplanetary surface habitat life support systems (LSS) on the Moon and Mars, as well as long-duration space travel, will require novel capabilities to withstand anticipated unique, harsh conditions. In order to provide safe, habitable environments for the crew, water purification and waste processing systems will be required to treat all sources of water (condensate, Sabatier, urine, hygiene, fecal, food waste) in order to achieve the necessary levels of recovery needed to sustain life over the long-duration missions. The ability to recycle organic wastes creates an opportunity to recover critical elements (e.g., C, H, O, N, P) for subsequent food production, water purification, and atmospheric regeneration. Bioregenerative systems mimic functions of nature in engineered systems, or bioreactors, utilizing combination of prokaryotes, eukaryotes and archaea. While these systems are commonly used on Earth for wastewater treatment, bioreactors for space travel face additional challenges. Terrestrial bioreactors often rely on gravitational settling of dense flocs and granules for cell retention. For micro- or partial-gravity environments, density differential alone will not be adequate for cell retention; a gravity-independent means for cell retention is crucial. The membrane bioreactor represents the state of the art in wastewater treatment. This hybrid system combines biological processes with membrane filtration to achieve performance beyond what each can accomplish individually. The complete cell retention in an MBR allows for the decoupling of hydraulic retention time (HRT) and solids retention time (SRT), which result in a high-thruput, compact, treatment system. The Bioregenerative Water Technology Team at NASA Kennedy Space Center and the University of South Florida has developed a bioregenerative platform based on the hybrid MBR technology. The overall architecture is compact, modular, flexible, and adaptable to mission evolutions. The main subsystems of the bioregenerative architecture include: 1) Anaerobic membrane bioreactor (AnMBR): Also termed the Organic processor assembly (OPA), the function of the AnMBR is to treat organic wastes such as fecal and food wastes. These wastes are characterized by a concentration of suspended solids comprised of carbohydrates, proteins and lipids. The assigned function of the AnMBR is to break down and covert suspended solids to biogas (methane, hydrogen and carbon dioxide), reduce effluent chemical oxygen demand (COD), liberate organically-bound nutrients, and remove pathogenic organisms. 2) Phototrophic membrane bioreactor (PMBR): The PMBR is comprised of a co-culture of microalgae and bacteria. The assigned function of the PMBR is to polish the permeate of the AnMBR to further remove dissolved organic carbon, manage nutrients (nitrogen transformation, load dampening), and perform air revitalization. 3) Food processor assembly (FPA): The FPA is a food production platform (prokaryotic or eukaryotic), fueled by outputs from the AnMBR, or PMBR. For the presentation, we will describe each step of the bioregenerative architecture, and present performance data from extended trials treating analog and real metabolic wastes.

Bioreactor↗

Next Generation Life Support Project: Development of Advanced Technologies for Human Exploration Missions

Next Generation Life Support (NGLS) is one of several technology development projects sponsored by the National Aeronautics and Space Administration s Game Changing Development Program. NGLS is developing life support technologies (including water recovery, and space suit life support technologies) needed for humans to live and work productively in space. NGLS has three project tasks: Variable Oxygen Regulator (VOR), Rapid Cycle Amine (RCA) swing bed, and Alternative Water Processing. The selected technologies within each of these areas are focused on increasing affordability, reliability, and vehicle self sufficiency while decreasing mass and enabling long duration exploration. The RCA and VOR tasks are directed at key technology needs for the Portable Life Support System (PLSS) for an Exploration Extravehicular Mobility Unit (EMU), with focus on prototyping and integrated testing. The focus of the Rapid Cycle Amine (RCA) swing-bed ventilation task is to provide integrated carbon dioxide removal and humidity control that can be regenerated in real time during an EVA. The Variable Oxygen Regulator technology will significantly increase the number of pressure settings available to the space suit. Current spacesuit pressure regulators are limited to only two settings while the adjustability of the advanced regulator will be nearly continuous. The Alternative Water Processor efforts will result in the development of a system capable of recycling wastewater from sources expected in future exploration missions, including hygiene and laundry water, based on natural biological processes and membrane-based post treatment. The technologies will support a capability-driven architecture for extending human presence beyond low Earth orbit to potential destinations such as the Moon, near Earth asteroids and Mars.

Barta, Daniel J.↗

Characterizing the Number of Kinesin Motors Bound to Microtubules in the Gliding Motility Assay Using FLIC Microscopy [Book Chapter]

Intracellular transport by kinesin motors moving along their associated cytoskeletal filaments, microtubules, is essential to many biological processes. This active transport system can be reconstituted in vitro with the surface-adhered motors transporting the microtubules across a planar surface. In this geometry, the kinesin-microtubule system has been used to study active self-assembly, to power microdevices, and to perform analyte detection. Fundamental to these applications is the ability to characterize the interactions between the surface tethered motors and microtubules. Further, Fluorescence Interference Contrast (FLIC) microscopy can illuminate the height of the microtubule above a surface, which, at sufficiently low surface densities of kinesin, also reveals the number, locations, and dynamics of the bound motors.

59 BASIC BIOLOGICAL SCIENCES↗

The Promises, Challenges, and Opportunities of Omics for Studying the Plant Holobiont

Microorganisms are critical drivers of biological processes that contribute significantly to plant sustainability and productivity. In recent years, emerging research on plant holobiont theory and microbial invasion ecology has radically transformed how we study plant–microbe interactions. Over the last few years, we have witnessed an accelerating pace of advancements and breadth of questions answered using omic technologies. Herein, we discuss how current state-of-the-art genomics, transcriptomics, proteomics, and metabolomics techniques reliably transcend the task of studying plant–microbe interactions while acknowledging existing limitations impeding our understanding of plant holobionts.

59 BASIC BIOLOGICAL SCIENCES↗