Deoxygenation of Unhindered Alcohols via Reductive Dealkylation of Derived Phosphate Esters

Primary alcohols can be deoxygenated cleanly and in yields of 60–95% by reduction of derived diphenyl phosphate esters with lithium triethylborohydride in tetrahydrofuran at room temperature. Selective deoxygenation of a primary alcohol in the presence of a secondary alcohol was demonstrated. The two-step process can be performed in one pot, making it simple and convenient

Chemical Factors that Control Lignin Polymerization

Lignin is a complex, branched polymer that reinforces plant tissue. Understanding the factors that govern lignin structure is of central importance to the development of technologies for converting lignocellulosic biomass into fuels because lignin imparts resistance to chemical, enzymatic, and mechanical deconstruction. Lignin is formed by enzymatic oxidation of phenolic monomers (monolignols) of three main types, guaiacyl (G), syringyl (S), and <i>p</i>-hydroxyphenyl (H) subunits. It is known that increasing the relative abundance of H subunits results in lower molecular weight lignin polymers and hence more easily deconstructed biomass, but it is not known why. Here, we report an analysis of frontier molecular orbitals in mono-, di-, and trilignols, calculated using density functional theory, which points to a requirement of strong p-electron density on the reacting phenolic oxygen atom of the neutral precursor for enzymatic oxidation to occur. This model is consistent with a proton-coupled electron transfer (PCET) mechanism and for the first time explains why H subunits in certain linkages (β–β or β–5) react poorly and tend to “cap” the polymer. In general, β–5 linkages with either a G or H terminus are predicted to inhibit elongation. More broadly, the model correctly accounts for the reactivity of the phenolic groups in a diverse set of dilignols comprising H and G subunits. Thus, we provide a coherent framework for understanding the propensity toward growth or termination of different terminal subunits in lignin

Bilayer Thickness Mismatch Controls Domain Size in Model Membranes

The observation of lateral phase separation in lipid bilayers has received considerable attention, especially in connection to lipid raft phenomena in cells. It is widely accepted that rafts play a central role in cellular processes, notably signal transduction. While micrometer-sized domains are observed with some model membrane mixtures, rafts much smaller than 100 nmbeyond the reach of optical microscopyare now thought to exist, both in vitro and in vivo. We have used small-angle neutron scattering, a probe free technique, to measure the size of nanoscopic membrane domains in unilamellar vesicles with unprecedented accuracy. These experiments were performed using a four-component model system containing fixed proportions of cholesterol and the saturated phospholipid 1,2-distearoyl-<i>sn</i>-glycero-3-phosphocholine (DSPC), mixed with varying amounts of the unsaturated phospholipids 1-palmitoyl-2-oleoyl-<i>sn</i>-glycero-3-phosphocholine (POPC) and 1,2-dioleoyl-<i>sn</i>-glycero-3-phosphocholine (DOPC). We find that liquid domain size increases with the extent of acyl chain unsaturation (DOPC:POPC ratio). Furthermore, we find a direct correlation between domain size and the mismatch in bilayer thickness of the coexisting liquid-ordered and liquid-disordered phases, suggesting a dominant role for line tension in controlling domain size. While this result is expected from line tension theories, we provide the first experimental verification in free-floating bilayers. Importantly, we also find that changes in bilayer thickness, which accompany changes in the degree of lipid chain unsaturation, are entirely confined to the disordered phase. Together, these results suggest how the size of functional domains in homeothermic cells may be regulated through changes in lipid composition

Characterization of Indole-3-acetic Acid Biosynthesis and the Effects of This Phytohormone on the Proteome of the Plant-Associated Microbe <i>Pantoea</i> sp. YR343

Indole-3-acetic acid (IAA) plays a central role in plant growth and development, and many plant-associated microbes produce IAA using tryptophan as the precursor. Using genomic analyses, we predicted that <i>Pantoea</i> sp. YR343, a microbe isolated from <i>Populus deltoides</i>, synthesizes IAA using the indole-3-pyruvate (IPA) pathway. To better understand IAA biosynthesis and the effects of IAA exposure on cell physiology, we characterized proteomes of <i>Pantoea</i> sp. YR343 grown in the presence of tryptophan or IAA. Exposure to IAA resulted in upregulation of proteins predicted to function in carbohydrate and amino acid transport and exopolysaccharide (EPS) biosynthesis. Metabolite profiles of wild-type cells showed the production of IPA, IAA, and tryptophol, consistent with an active IPA pathway. Finally, we constructed an Δ<i>ipdC</i> mutant that showed the elimination of tryptophol, consistent with a loss of IpdC activity, but was still able to produce IAA (20% of wild-type levels). Although we failed to detect intermediates from other known IAA biosynthetic pathways, this result suggests the possibility of an alternate pathway or the production of IAA by a nonenzymatic route in <i>Pantoea</i> sp. YR343. The Δ<i>ipdC</i> mutant was able to efficiently colonize poplar, suggesting that an active IPA pathway is not required for plant association

Characterization of Indole-3-acetic Acid Biosynthesis and the Effects of This Phytohormone on the Proteome of the Plant-Associated Microbe <i>Pantoea</i> sp. YR343

Indole-3-acetic acid (IAA) plays a central role in plant growth and development, and many plant-associated microbes produce IAA using tryptophan as the precursor. Using genomic analyses, we predicted that <i>Pantoea</i> sp. YR343, a microbe isolated from <i>Populus deltoides</i>, synthesizes IAA using the indole-3-pyruvate (IPA) pathway. To better understand IAA biosynthesis and the effects of IAA exposure on cell physiology, we characterized proteomes of <i>Pantoea</i> sp. YR343 grown in the presence of tryptophan or IAA. Exposure to IAA resulted in upregulation of proteins predicted to function in carbohydrate and amino acid transport and exopolysaccharide (EPS) biosynthesis. Metabolite profiles of wild-type cells showed the production of IPA, IAA, and tryptophol, consistent with an active IPA pathway. Finally, we constructed an Δ<i>ipdC</i> mutant that showed the elimination of tryptophol, consistent with a loss of IpdC activity, but was still able to produce IAA (20% of wild-type levels). Although we failed to detect intermediates from other known IAA biosynthetic pathways, this result suggests the possibility of an alternate pathway or the production of IAA by a nonenzymatic route in <i>Pantoea</i> sp. YR343. The Δ<i>ipdC</i> mutant was able to efficiently colonize poplar, suggesting that an active IPA pathway is not required for plant association

The in vivo structure of biological membranes and evidence for lipid domains

<div><p>Examining the fundamental structure and processes of living cells at the nanoscale poses a unique analytical challenge, as cells are dynamic, chemically diverse, and fragile. A case in point is the cell membrane, which is too small to be seen directly with optical microscopy and provides little observational contrast for other methods. As a consequence, nanoscale characterization of the membrane has been performed ex vivo or in the presence of exogenous labels used to enhance contrast and impart specificity. Here, we introduce an isotopic labeling strategy in the gram-positive bacterium <i>Bacillus subtilis</i> to investigate the nanoscale structure and organization of its plasma membrane in vivo. Through genetic and chemical manipulation of the organism, we labeled the cell and its membrane independently with specific amounts of hydrogen (H) and deuterium (D). These isotopes have different neutron scattering properties without altering the chemical composition of the cells. From neutron scattering spectra, we confirmed that the <i>B</i>. <i>subtilis</i> cell membrane is lamellar and determined that its average hydrophobic thickness is 24.3 ± 0.9 Ångstroms (Å). Furthermore, by creating neutron contrast within the plane of the membrane using a mixture of H- and D-fatty acids, we detected lateral features smaller than 40 nm that are consistent with the notion of lipid rafts. These experiments—performed under biologically relevant conditions—answer long-standing questions in membrane biology and illustrate a fundamentally new approach for systematic in vivo investigations of cell membrane structure.</p></div

Characterization of Indole-3-acetic Acid Biosynthesis and the Effects of This Phytohormone on the Proteome of the Plant-Associated Microbe <i>Pantoea</i> sp. YR343

Indole-3-acetic acid (IAA) plays a central role in plant growth and development, and many plant-associated microbes produce IAA using tryptophan as the precursor. Using genomic analyses, we predicted that <i>Pantoea</i> sp. YR343, a microbe isolated from <i>Populus deltoides</i>, synthesizes IAA using the indole-3-pyruvate (IPA) pathway. To better understand IAA biosynthesis and the effects of IAA exposure on cell physiology, we characterized proteomes of <i>Pantoea</i> sp. YR343 grown in the presence of tryptophan or IAA. Exposure to IAA resulted in upregulation of proteins predicted to function in carbohydrate and amino acid transport and exopolysaccharide (EPS) biosynthesis. Metabolite profiles of wild-type cells showed the production of IPA, IAA, and tryptophol, consistent with an active IPA pathway. Finally, we constructed an Δ<i>ipdC</i> mutant that showed the elimination of tryptophol, consistent with a loss of IpdC activity, but was still able to produce IAA (20% of wild-type levels). Although we failed to detect intermediates from other known IAA biosynthetic pathways, this result suggests the possibility of an alternate pathway or the production of IAA by a nonenzymatic route in <i>Pantoea</i> sp. YR343. The Δ<i>ipdC</i> mutant was able to efficiently colonize poplar, suggesting that an active IPA pathway is not required for plant association

Detecting lateral lipid organization in <i>B</i>. <i>subtilis</i>.

<p>(a) Schematic of the experiment. Neutron contrast in the membrane was controlled using different blends of hydrogen (H)- and deuterium (D)-fatty acids (FAs). The control mixture consisted of anteiso-pentadecanoic acid (a15:0) and normal-hexadecanoic acid (n16:0), each 30% H and 70% D (inset to <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002214#pbio.2002214.g003" target="_blank">Fig 3b</a>, red curve). This mixture is contrast-matched to 85% D₂O and produces only background scattering whether or not the lipids are uniformly distributed (lower left and lower center panels). The experimental mixture contained the same FAs, but with a different isotopic distribution, i.e., n16:0 being 100% D and a15:0 being 40/60 H/D. In <i>B</i>. <i>subtilis</i>, this mixture produces an overall ratio of 30/70 H/D because a15:0 is more abundant (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002214#pbio.2002214.g002" target="_blank">Fig 2c</a>, bottom). If the lipids are uniformly distributed the membrane will be contrast-matched and produces no net scattering above background (indistinguishable from the control, lower center panel). However, if the lipids are organized in a manner that the high-melting n16:0 and low-melting a15:0 in membrane lipids are nonuniformly distributed, neutron contrast and a scattering signal will arise—illustrated in the lower right panel of (a) and in (c), which have patches enriched in n16:0. Just as these patches create contrast visible to the eye in the illustration, they create contrast visible to neutrons in the experiment in the form of excess scattering. (b) Small-angle neutron scattering (SANS) spectra of cerulenin-treated <i>B</i>. <i>subtilis</i> Δ<i>yusL</i>, incorporating experimental and control mixtures. The experimental mixture displays clear excess scattering (inset) that can result only from the nonuniform distribution of lipids with different contrast. From the relation ℓ = 2π/<i>q</i>, it can be inferred that the excess scattering in the range <i>q</i> = 0.01–0.15 Å⁻¹ corresponds to structures with length scales of 3–40 nm. Error bars correspond to ± <i>σ</i>. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002214#pbio.2002214.s016" target="_blank">S4 Data</a>. Gas chromatography/mass spectrometry (GC/MS) analyses of membrane FAs are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002214#pbio.2002214.s010" target="_blank">S10 Fig</a>. (c) Representation of the membrane having visible lipid patches (of arbitrary structure), with the buffer, cell wall, and cytoplasmic contents all being contrast-matched.</p

Contrast variation small-angle neutron scattering (SANS) reveals the structure of the <i>B</i>. <i>subtilis</i> membrane.

<p>(a) Schematic of the hydrogen (H)-labeled membrane as seen by neutrons. The cell wall and cytoplasmic contents are contrast-matched at 85% D₂O, hence invisible to neutrons, while the membrane with incorporated H-fatty acids (FAs) stands out in strong contrast. (b) SANS data plotted as scattered intensity, <i>I(q)</i>, as a function of scattering wavevector, <i>q</i> (Å⁻¹), from the <i>B</i>. <i>subtilis</i> membrane. Error bars correspond to ± <i>σ</i>. The experimental data are shown superimposed with the best-fit (red solid line) using a lamellar form factor,[<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002214#pbio.2002214.ref045" target="_blank">45</a>] revealing an average hydrophobic thickness of 24.3 ± 0.9 Å, consistent with a fluid-phase lipid bilayer (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002214#pbio.2002214.s015" target="_blank">S3 Data</a>). Gas chromatography/mass spectrometry (GC/MS) analysis of the membrane FAs is shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002214#pbio.2002214.g002" target="_blank">Fig 2c</a> with peak integrals in Table E in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002214#pbio.2002214.s012" target="_blank">S1 Text</a>.</p

VACNFs penetrate <i>Populus</i> epidermis without damaging cells.

<p>(a) and (b) Micrographs of transverse sections through a <i>Populus</i> leaf showing individual nanofibers penetrating the cuticle and epidermis. The arrow in (a) shows a nanofiber reaching the base of an epidermal cell without penetrating the underlying palisade cell. Arrows in (b) indicate a nanofiber traversing the cytoplasm of the cell. (c) DIC micrograph of a leaf epidermis showing that carbon nanofiber impalement occurs in a grid pattern, similar to the original chip. (d) DIC micrograph of the same leaf depicted in (c) with the focal plane moved into the underlying palisade layer showing the absence of over-penetrant fibers. Results are representative of leaves from 2 separate plants. (e) and (f) Bright-field micrographs of <i>Populus</i> leaves after staining with DAB to detect H₂O₂ production. Leaves penetrated by carbon nanofibers (e) show no DAB staining and are similar to untreated areas of the leaf (f). The boxed inset in (e) shows a magnified image of the nanofiber-treated area, with black arrows indicating the location of carbon nanofibers in this image. (g) and (h) Leaves wounded with a cork borer (g) or abraded with carborundum (h) show areas stained dark brown by DAB deposition in reaction to H₂O₂ produced in the wound response. Black arrows in (h) indicate carborundum powder remaining on the leaf. Dashed arrows in (g) point to the cut edge of the leaf and solid arrows indicate staining with DAB. Similar results were obtained with leaves from 3 separate plants. (i) and (j) Transverse sections of a <i>Populus</i> leaf after carborundum abrasion, showing areas of (i) severe and (j) mild epidermal damage. The black arrow in (j) points to grit particles within the palisade layer. (k) DIC micrograph of a leaf epidermis after carborundum treatment showing abraded epidermal cells (denoted by white arrows). (l) DIC micrograph of the same leaf depicted in (k) with the focal plane moved into the underlying palisade layer, showing the presence of embedded grit particles (denoted by white arrows). Images (a), (b), (i) and (j) were obtained from thin sections of fixed (formalin), embedded (paraffin) and stained (toluidine blue) tissue; images (c), (d), (k) and (l) were obtained from fixed (ethanol-acetic acid) and cleared (chloral hydrate) tissue; images in (e)–(h) were obtained from stained (DAB) and decolorized (boiling ethanol) tissue. Details are provided in <i>Materials and Methods</i>.</p