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At least 109 records · Page 6

Amino Acids Essential for the Assembly of Cellulose Synthase Complexes

Over 40 years ago, Andrew Staehelin's group showed that plant cellulose microfibrils are synthesized by protein complexes arranged in hexagonal arrays called 'particle rosettes'. Plant cellulose synthases (CESAs) differ from their bacterial ancestral forms by insertion of three unique sequences responsible for the assembly into compact multimeric units that aggregate further into rosettes. We used site-directed mutagenesis to replace amino acids within these sequences predicted to be essential for assembly and developed an in vivo method to determine the ability of mutated CesA1 transgenes to complement an Arabidopsis temperature-sensitive root-swelling1 (rsw1) mutant. Replacement of a Cys residue in the Class-Specific Region (CSR) or Pro417 and Arg453 of the Plant-Conserved Region (P-CR) rendered an AtCesA1 transgene unable to complement the rsw1 mutation. Despite an expected role for Arg457 in the trimerization of CESA proteins, AtCesA1 transgenes with Arg457Ala mutations were able to fully restore the wild-type phenotype in rsw1. Staehelin observed a third order of assembly in Micrasterias dendiculata, where hundreds of rosettes form large hexagonal arrays during the synthesis of secondary wall cellulose macrofibrils. We found by SEM of cytosolic faces of Micrasterias cell fragments that these arrays are associated with fields of regularly spaced slime secretion pore complexes.

arabidopsis↗

Effects of feruloyl-CoA 6'-hydroxylase 1 overexpression on lignin and cell wall characteristics in transgenic hybrid aspen

In plant cell walls, lignin, cellulose, and the hemicelluloses form intricate three-dimensional structures. Owing to its complexity, lignin often acts as a bottleneck for the efficient utilization of polysaccharide components as biochemicals and functional materials. A promising approach to mitigate and/or overcome lignin recalcitrance is the qualitative and quantitative modification of lignin by genetic engineering. Feruloyl-CoA 6'-hydroxylase (F6'H1) is a 2-oxoglutarate-dependent dioxygenase that catalyzes the conversion of feruloyl-CoA, one of the intermediates of the lignin biosynthetic pathway, into 6'-hydroxyferuloyl-CoA, the precursor of scopoletin (7-hydroxy-6-methoxycoumarin). In a previous study with Arabidopsis thaliana, we demonstrated that overexpression of F6'H1 under a xylem-preferential promoter led to scopoletin incorporation into the cell wall. This altered the chemical structure of lignin without affecting lignin content or saccharification efficiency. In the present study, the same F6'H1 construct was introduced into hybrid aspen (Populus tremula × tremuloides T89), a model woody plant, and its effects on plant morphology, lignin chemical structure, global gene expression, and phenolic metabolism were examined. The transgenic plants successfully overproduced scopoletin while exhibiting severe growth retardation, a phenotype not previously observed in Arabidopsis. Scopoletin accumulation was most pronounced in the secondary walls of tracheary elements and the compound middle lamella, with low levels in the fiber cell walls. Overexpression of F6'H1 also affected the metabolism of aromatics, including lignin precursors. Heteronuclear single-quantum coherence (HSQC) NMR spectroscopy revealed that scopoletin in cell walls was bound to lignin, leading to a reduction in lignin content and changes in its monomeric composition and molar mass distribution. Furthermore, the enzymatic saccharification efficiency of the transgenic cell walls was more than three times higher than that of the wild-type plants, even without pretreatment. Although addressing growth inhibition remains a priority, incorporating scopoletin into lignin demonstrates significant potential for improving woody biomass utilization.

59 BASIC BIOLOGICAL SCIENCES↗

ToF-SIMS imaging reveals that p -hydroxybenzoate groups specifically decorate the lignin of fibres in the xylem of poplar and willow

Esterified phenolics occur as γ-linked lignin acylations in many plant taxa but little is known about the distribution or function of such groups. In the family Salicaceae, p-hydroxybenzoate groups are present in the lignin of poplars (Populus spp.) and willows (Salix spp.). In this study, the distribution of cell wall-bound p-hydroxybenzoate was examined in different tissue and cell types of poplar and willow trees. This analysis showed that p-hydroxybenzoate groups were most prevalent in juvenile stems, and were elevated in tension wood and following nitrogen fertilisation. Closer examination of stem cross-sections using time-of-flight secondary-ion mass spectrometry (ToF-SIMS) directly showed that p-hydroxybenzoate groups occur predominantly in the cell walls of fibres and are largely absent from vessel elements. Furthermore, these results point to a role for ester-linked groups in modifying the syringyl-rich lignin of fibres, perhaps to strengthen the cell walls of these structural elements or to increase lignification rates.

59 BASIC BIOLOGICAL SCIENCES↗

On the magnetoaerothermal instability

A fundamental instability in MHD channel flow, hitherto unknown or unappreciated, is described. Lorentz force-driven secondary flow cells preferentially couple core temperature gradient modes into the near fields of the anode wall leading to a locally growing Lorentz force which eventually separates the anode boundary layer. The instability is described with both heuristic order-of-magnitude analyses and detailed three-dimensional, turbulent, MHD flow computations. This magnetoaerothermal instability will be manifested in commercial scale MHD generators of moderate MHD interaction parameter. Methods of control and prevention of the magnetoaerothermal instability exist.

Demetriades, S. T.↗

Monte-Carlo Simulations of Heavy Ions Track Structures and Applications

In space, astronauts are exposed to protons, high ]energy heavy (HZE) ions that have a high charge (Z) and energy (E), and secondary radiation, including neutrons and recoil nuclei produced by nuclear reactions in spacecraft walls or in tissue. The astronauts can only be partly shielded from these particles. Therefore, on travelling to Mars, it is estimated that every cell nucleus in an astronaut fs body would be hit by a proton or secondary electron (e.g., electrons of the target atoms ionized by the HZE ion) every few days and by an HZE ion about once a month. The risks related to these heavy ions are not well known and of concern for long duration space exploration missions. Medical ion therapy is another situation where human beings can be irradiated by heavy ions, usually to treat cancer. Heavy ions have a peculiar track structure characterized by high levels of energy ]deposition clustering, especially in near the track ends in the so ]called eBragg peak f region. In radiotherapy, these features of heavy ions can provide an improved dose conformation with respect to photons, also considering that the relative biological effectiveness (RBE) of therapeutic ions in the plateau region before the peak is sufficiently low. Therefore, several proton and carbon ion therapy facilities are under construction at this moment

Plante, Ianik↗

Evolutionary transition to the ectomycorrhizal habit in the genomes of a hyperdiverse lineage of mushroom-forming fungi

The ectomycorrhizal (ECM) symbiosis has independently evolved from diverse types of saprotrophic ancestors. In this study, we seek to identify genomic signatures of the transition to the ECM habit within the hyperdiverse Russulaceae. We present comparative analyses of the genomic architecture and the total and secreted gene repertoires of 18 species across the order Russulales, of which 13 are newly sequenced, including a representative of a saprotrophic member of Russulaceae, Gloeopeniophorella convolvens. The genomes of ECM Russulaceae are characterized by a loss of genes for plant cell wall-degrading enzymes (PCWDEs), an expansion of genome size through increased transposable element (TE) content, a reduction in secondary metabolism clusters, and an association of small secreted proteins (SSPs) with TE ‘nests’, or dense aggregations of TEs. Here, some PCWDEs have been retained or even expanded, mostly in a species-specific manner. The genome of G. convolvens possesses some characteristics of ECM genomes (e.g. loss of some PCWDEs, TE expansion, reduction in secondary metabolism clusters). Functional specialization in ECM decomposition may drive diversification. Accelerated gene evolution predates the evolution of the ECM habit, indicating that changes in genome architecture and gene content may be necessary to prime the evolutionary switch.

59 BASIC BIOLOGICAL SCIENCES↗

Molecular remodeling in Populus PdKOR RNAi roots profiled using LC-MS/MS proteomics

Plant endo-β-1,4-glucanases belonging to the Glycoside Hydrolase Family 9 have functional roles in cell wall biosynthesis and remodeling via endohydrolysis of (1→4)-β-D-glucosidic linkages. Modification of cell wall chemistry via RNAi-mediated downregulation of Populus deltoides KOR1 (PdKOR), a endo-β-1,4-glucanase gene, in Populus deltoides has been shown to have functional consequences for the composition of secondary metabolome and the ability of modified roots to interact with beneficial microbes. The molecular remodeling that underlies the observed differences at metabolic, physiological, and morphological levels in roots is not well understood. Here we used a LC-MS/MS-based proteome profiling approach to survey the molecular remodeling in root tissues of PdKOR and control plants. A total of 14316 peptides were identified and these mapped to 7139 P. deltoides proteins. Based on 90% sequence identity, the measured protein accessions represent 1187 functional protein groups. Analysis of GO categories and specific individual proteins showed differential expression of proteins relevant to plant-microbe interactions, cell wall chemistry, and metabolism. The new proteome dataset serves as a useful resource for deriving new hypotheses and empirical testing pertaining to functional roles of proteins and pathways in differential priming of plant roots to interactions with microbes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Further experiments on supersonic turbulent flow development in a square duct

The mean-flow structure of supersonic, turbulent, adiabatic-wall flow in a square duct is investigated experimentally over a development length x/D = 0-50 for a uniform flow, Mach 3.9 condition at the duct inlet. The results show that a secondary flow cell structure develops which is similar to that for the incompressible case. Development of the primary flow is influenced by the combined effects of the secondary flow and the streamwise adverse pressure gradient. Total pressure, axial mean velocity, and Mach number profiles are presented which show that the outer flow is sensitive primarily to the streamwise pressure gradient, while flow in the near-wall region is dominated by the secondary flow. Axial mean-velocity profiles plotted in terms of van Driest-scaled variables show that a well-defined log-law region exists in the near-wall layer. This region exists in the presence of a secondary flow which continuously modifies spanwise wall shear stress behavior along the length of the duct.

Davis, D. O.↗

The Inducible Accumulation of Cell Wall-Bound p -Hydroxybenzoates Is Involved in the Regulation of Gravitropic Response of Poplar

Lignin in Populus species is acylated with p-hydroxybenzoate. Monolignol p-hydroxybenzoyltransferase 1 (PHBMT1) mediates p-hydroxybenzoylation of sinapyl alcohol, eventually leading to the modification of syringyl lignin subunits. Angiosperm trees upon gravistimulation undergo the re-orientation of their growth along with the production of specialized secondary xylem, i.e., tension wood (TW), that generates tensile force to pull the inclined stem or leaning branch upward. Sporadic evidence suggests that angiosperm TW contains relatively a high percentage of syringyl lignin and lignin-bound p-hydroxybenzoate. However, whether such lignin modification plays a role in gravitropic response remains unclear. By imposing mechanical bending and/or gravitropic stimuli to the hybrid aspens in the wild type (WT), lignin p-hydroxybenzoate deficient, and p-hydroxybenzoate overproduction plants, we examined the responses of plants to gravitropic/mechanical stress and their cell wall composition changes. We revealed that mechanical bending or gravitropic stimulation not only induced the overproduction of crystalline cellulose fibers and increased the relative abundance of syringyl lignin, but also significantly induced the expression of PHBMT1 and the increased accumulation of p-hydroxybenzoates in TW. Furthermore, we found that although disturbing lignin-bound p-hydroxybenzoate accumulation in the PHBMT1 knockout and overexpression (OE) poplars did not affect the major chemical composition shifts of the cell walls in their TW as occurred in the WT plants, depletion of p-hydroxybenzoates intensified the gravitropic curving of the plantlets in response to gravistimulation, evident with the enhanced stem secant bending angle. By contrast, hyperaccumulation of p-hydroxybenzoates mitigated gravitropic response. These data suggest that PHBMT1-mediated lignin modification is involved in the regulation of poplar gravitropic response and, likely by compromising gravitropism and/or enhancing autotropism, negatively coordinates the action of TW cellulose fibers to control the poplar wood deformation and plant growth.

59 BASIC BIOLOGICAL SCIENCES↗

Space Exploration: A Risk for Neural Stem Cells

During spaceflights beyond low Earth orbit, astronauts are exposed to potentially carcinogenic and tissue damaging galactic cosmic rays, solar proton events, and secondary radiation that includes neutrons and recoil nuclei produced by nuclear reactions in spacecraft walls or in tissue (1). Such radiation risk may present a significant health risk for human exploration of the moon and Mars. Emerging evidence that generation of new neurons in the adult brain may be essential for learning, memory, and mood (2) and that radiation is deleterious to neurogenesis (3-5) underscores a previously unappreciated possible risk to the cognitive functions and emotional stability of astronauts exposed to radiation in space. Here we use a novel reporter mouse line to identify at-risk populations of stem and progenitor cells in the brain and find, unexpectedly, that quiescent stem-like cells (rather than their rapidly dividing progeny) in the hippocampus constitute the most vulnerable cell population. This finding raises concerns about the possible risks facing astronauts on long duration space missions.

Encinas, Juan M.↗

Editorial: Wood Development and Physiology in a Changing Climate

Wood of forest trees is a renewable, sustainable and easily workable material and has been widely used in construction, paper making, furniture, and as a feedstock for biofuels. Wood formation is the result of a highly ordered and finely regulated process. Secondary xylem (wood) and phloem are the inner and outer derivative products of the vascular cambium, respectively. Xylem is mainly composed of dead cells with thickened cell walls rich in cellulose, hemicelluloses, and lignin, which are responsible for providing mechanical support and conducting water and minerals for the trees. Phloem transports photoassimilates and signaling molecules, including phytohormones and peptides. Wood structure varies greatly between, and within, tree species. A thorough understanding of developmental processes and metabolic pathways during wood formation would provide a basis for improving wood biomass and modifying properties in forest trees.

54 ENVIRONMENTAL SCIENCES↗

Understanding the structure and composition of recalcitrant oligosaccharides in hydrolysate using high-throughput biotin-based glycome profiling and mass spectrometry

Novel Immunological and Mass Spectrometry Methods for Comprehensive Analysis of Recalcitrant Oligosaccharides in AFEX Pretreated Corn Stover. Lignocellulosic biomass is a sustainable alternative to fossil fuel and is extensively used for developing bio-based technologies to produce products such as food, feed, fuel, and chemicals. The key to these technologies is to develop cost competitive processes to convert complex carbohydrates present in plant cell wall to simple sugars such as glucose, xylose, and arabinose. Since lignocellulosic biomass is highly recalcitrant, it must undergo a combination of thermochemical treatment such as Ammonia Fiber Expansion (AFEX), dilute acid (DA), Ionic Liquid (IL) and biological treatment such as enzyme hydrolysis and microbial fermentation to produce desired products. However, when using commercial fungal enzymes during hydrolysis, only 75–85% of the soluble sugars generated are monomeric sugars, while the remaining 15–25% are soluble recalcitrant oligosaccharides that cannot be easily utilized by microorganisms. Previously, we successfully separated and purified the soluble recalcitrant oligosaccharides using a combination of charcoal and celite-based separation followed by size exclusion chromatography and studies their inhibitory properties on enzymes. We discovered that the oligosaccharides with higher degree of polymerization (DP) containing methylated uronic acid substitutions were more recalcitrant towards commercial enzyme mixtures than lower DP and neutral oligosaccharides. Here, we report the use of several complementary techniques that include glycome profiling using plant biomass glycan specific monoclonal antibodies (mAbs) to characterize sugar linkages in plant cell walls and enzymatic hydrolysate, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) using structurally-informative diagnostic peaks offered by negative ion post-secondary decay spectra, gas chromatography followed by mass spectrometry (GC–MS) to characterize oligosaccharide sugar linkages with and without derivatization. Since oligosaccharides (DP 4–20) are small, it is challenging to mobilize these molecules for mAbs binding and characterization. To overcome this problem, we have applied a new biotin-coupling based oligosaccharide immobilization method that successfully tagged most of the low DP soluble oligosaccharides on to a micro-plate surface followed by specific linkage analysis using mAbs in a high-throughput system. This new approach will help develop more advanced versions of future high throughput glycome profiling methods that can be used to separate and characterize oligosaccharides present in biomarkers for diagnostic applications.

59 BASIC BIOLOGICAL SCIENCES↗

Convergent behavior of extended stalk regions from staphylococcal surface proteins with widely divergent sequence patterns

Staphylococcus epidermidis and Staphylococcus aureus are highly problematic bacteria in hospital settings. A major challenge is their ability to form biofilms on abiotic or biotic surfaces. Biofilms are well-organized, multicellular bacterial aggregates that resist antibiotic treatment and often lead to recurrent infections. Bacterial cell wall-anchored (CWA) proteins are important players in biofilm formation and infection. Many have putative stalk-like regions or regions of low complexity near the cell wall-anchoring motif. Recent work demonstrated the strong propensity of the stalk region of S. epidermidis accumulation-associated protein (Aap) to remain highly extended under solution conditions that typically induce compaction. This behavior is consistent with the expected function of a stalk-like region that is covalently attached to the cell wall peptidoglycan and projects the adhesive domains of Aap away from the cell surface. In this study, we evaluate whether the ability to resist compaction is a common theme among stalk regions from various staphylococcal CWA proteins. Circular dichroism spectroscopy was used to examine secondary structure changes as a function of temperature and cosolvents along with sedimentation velocity analytical ultracentrifugation, size-exclusion chromatography, and SAXS to characterize structural characteristics in solution. All stalk regions tested are intrinsically disordered, lacking secondary structure beyond random coil and polyproline type II helix, and they all sample highly extended conformations. Remarkably, the Ser-Asp dipeptide repeat region of SdrC exhibited nearly identical behavior in solution when compared to the Aap Pro/Gly-rich region, despite highly divergent sequence patterns, indicating conservation of function by various distinct staphylococcal CWA protein stalk regions.

59 BASIC BIOLOGICAL SCIENCES↗

Application of Rotating Wall Vessel (RWV) Cell Culture for Pancreas Islet Cell Transplantation

Type I insulin-dependent diabetes mellitus (IDDM) remains a major cause of morbidity and mortality in both pediatric and adult populations, despite significant advances in medical management. While insulin therapy treats symptoms of acute diabetes, it fails to prevent chronic complications such as microvascular disease, blindness, neuropathy, and chronic renal failure. Strict control of blood glucose concentrations delays but does not prevent the onset and progression of secondary complications. Although, whole pancreas transplantation restores physiological blood glucose levels, a continuous process of allograft rejection causes vascular and exocrine-related complications. Recent advances in methods for isolation and purification of pancreatic islets make transplantation of islet allografts an attractive alternative to whole pancreas transplantation. However, immunosuppressive drugs are necessary to prevent rejection of islet allografts and many of these drugs are known to be toxic to the islets. Since auto-transplants of isolated islets following total pancreatectomy survive and function in vivo, it is apparent that a major obstacle to successful clinical islet transplantation is the immunogenicity of the islet allografts.

Rutzky, Lynne P.↗

Surface Generation and Cartesian Mesh Support

This document serves as the final report for the grant titled Surface Generation and Cartesian Mesh Support . This completed work was in algorithmic research into automatically generating surface triangulations from CAD geometries. NASA's OVERFLOW and Cart3D simulation packages use surface triangulations as an underlying geometry description and the ability to automatically generate these from CAD files (without translation) substantially reduces both the wall-clock time and expertise required to get geometry out of CAD and into mesh generation. This surface meshing was exercised greatly during the Shuttle investigation during the last year with success. The secondary efforts performed in this grant involve work on a visualization system cut-cell handling for Cartesian Meshes with embedded boundaries.

Haimes, Robert↗

Suppression of antigen-specific lymphocyte activation in modeled microgravity

Various parameters of immune suppression are observed in lymphocytes from astronauts during and after a space flight. It is difficult to ascribe this suppression to microgravity effects on immune cells in crew specimens, due to the complex physiological response to space flight and the resultant effect on in vitro immune performance. Use of isolated immune cells in true and modeled microgravity in immune performance tests, suggests a direct effect of microgravity on in vitro cellular function. Specifically, polyclonal activation of T-cells is severely suppressed in true and modeled microgravity. These recent findings suggest a potential suppression of oligoclonal antigen-specific lymphocyte activation in microgravity. We utilized rotating wall vessel (RWV) bioreactors as an analog of microgravity for cell cultures to analyze three models of antigen-specific activation. A mixed-lymphocyte reaction, as a model for a primary immune response, a tetanus toxoid response and a Borrelia burgdorferi response, as models of a secondary immune response, were all suppressed in the RWV bioreactor. Our findings confirm that the suppression of activation observed with polyclonal models also encompasses oligoclonal antigen-specific activation.

NASA Discipline Cell Biology↗

Outstanding questions on xylan biosynthesis

Xylan is the second most abundant polysaccharide in plant biomass. It is a crucial component of cell wall structure as well as a significant factor contributing to biomass recalcitrance. Xylan consists of a linear chain of β-1,4-linked xylosyl residues that are often substituted with glycosyl side chains, such as glucuronosyl/methylglucuronosyl and arabinofuranosyl residues, and acetylated at O-2 and/or O-3. Xylan from gymnosperms and dicots contains a unique reducing end tetrasaccharide sequence that is not detected in xylan from grasses, bryophytes and seedless vascular plants. Grass xylan is heavily decorated at O-3 with arabinofuranosyl residues that are frequently esterified with hydroxycinnamates. Further, genetic and biochemical studies have uncovered a number of genes involved in xylan backbone elongation and acetylation, xylan glycosyl substitutions and their modifications, and the synthesis of the unique xylan reducing end tetrasaccharide sequence, but some outstanding issues on the biosynthesis of xylan still remain unanswered. Here, we provide a brief overview of xylan structure and focus on discussion of the current understanding and open questions on xylan biosynthesis. Further elucidation of the biochemical mechanisms underlying xylan biosynthesis will not only shed new insights into cell wall biology but also provide molecular tools for genetic modification of biomass composition tailored for diverse end uses.

59 BASIC BIOLOGICAL SCIENCES↗

Formation and differentiation of three-dimensional rat marrow stromal cell culture on microcarriers in a rotating-wall vessel

Using a high aspect ratio vessel (HARV), this study investigated the formation of 3-D rat marrow stromal cell culture on microcarriers and the expression of bone-related biochemical markers under conditions of simulated microgravity. In addition, it calculated the shear stresses imparted on the surface of microcarriers of different densities by the medium fluid in an HARV. Secondary rat marrow stromal cells were cultured on two types of microcarriers, Cytodex-3 beads and modified bioactive glass particles. Examination of cellular morphology by scanning electron microscopy revealed the presence of three-dimensional multicellular aggregates consisting of multiple cell-covered Cytodex-3 microcarriers bridged together. Mineralization was observed in the aggregates. Spherical cell-bead aggregates were observed in an HARV, while cell-bead assemblies were mostly loosely packed in a chain-like or branched structure in a cell bag. The expressions of alkaline phosphatase activity, collagen type I, and osteopontin were shown via the use of histochemical staining, immunolabeling, and confocal scanning electron microscopy. Using a numerical approach, it was found that at a given rotational speed and for a given culture medium, a larger density difference between the microcarrier and the culture medium (e.g., a modified bioactive glass particle) imparted a higher maximum shear stress on the microcarrier.

Dextrans↗