Roots-eye view: using microdialysis and microCT to non-destructively map root nutrient depletion and accumulation zones

Improvement in fertiliser use efficiency is a key aspect for achieving sustainable agriculture in order to minimise costs, greenhouse gas emissions and pollution from nutrient runoff. To optimise root architecture for nutrient uptake and efficiency we need to understand what the roots encounter in their environment. Traditional methods of nutrient sampling such as salt extractions can only be done at the end of an experiment, are impractical for sampling locations precisely and give total nutrient values which can overestimate the nutrients available to the roots. In contrast, microdialysis provides a non-invasive, continuous method for sampling available nutrients in the soil. Here for the first time we have used microCT imaging to position microdialysis probes at known distances from the roots and then measured the available nitrate and ammonium. We found that nitrate accumulated close to roots while ammonium was depleted demonstrating that this combination of complementary techniques provides a unique ability to measure root-available nutrients non-destructively and in almost real-time

Toward a Molecular Understanding of the Mechanism of Cryopreservation by Polyampholytes: Cell Membrane Interactions and Hydrophobicity

Cryopreservation enables long-term preservation of cells at ultralow temperatures. Current cryoprotective agents (CPAs) have several limitations, making it imperative to develop CPAs with advanced properties. Previously, we developed a novel synthetic polyampholyte-based CPA, copolymer of 2-(dimethylamino)­ethyl methacrylate (DMAEMA) and methacrylic acid­(MAA) (poly­(MAA-DMAEMA)), which showed excellent efficiency and biocompatibility. Introduction of hydrophobicity increased its efficiency significantly. Herein, we investigated the activity of other polyampholytes. We prepared two zwitterionic polymers, poly­(sulfobetaine) (SPB) and poly­(carboxymethyl betaine) (CMB), and compared their efficiency with poly­(MAA-DMAEMA). Poly-SPB showed only intermediate property and poly-CMB showed no cryoprotective property. These data suggested that the polymer structure strongly influences cryoprotection, providing an impetus to elucidate the molecular mechanism of cryopreservation. We investigated the mechanism by studying the interaction of polymers with cell membrane, which allowed us to identify the interactions responsible for imparting different properties. Results unambiguously demonstrated that polyampholytes cryopreserve cells by strongly interacting with cell membrane, with hydrophobicity increasing the affinity for membrane interaction, which enables it to protect the membrane from various freezing-induced damages. Additionally, cryoprotective polymers, especially their hydrophobic derivatives, inhibit the recrystallization of ice, thus averting cell death. Hence, our results provide an important insight into the complex mechanism of cryopreservation, which might facilitate the rational design of polymeric CPAs with improved efficiency

Self-Assembled Peptide-Based System for Mitochondrial-Targeted Gene Delivery: Functional and Structural Insights

Human mitochondrial dysfunction can lead to severe and often deadly diseases, for which there are no known cures. Although the targeted delivery of therapeutic gene to mitochondria is a promising approach to alleviate these disorders, gene carrier systems for the selective delivery of functional DNA into the mitochondria of living mammalian cells are currently unavailable. Here we rationally developed dual-domain peptides containing DNA-condensing/cell-penetrating/endosome-disruptive and mitochondria-targeting sequences. Secondary structures of the dual-domain peptides were analyzed, and variations in the physicochemical properties (stability, size, and ζ potential) of peptide/DNA complexes were studied as a function of peptide-to-DNA ratio and serum addition. An optimized formulation, identified through qualitative and quantitative studies, fulfills the fundamental prerequisites for mitochondria-specific DNA delivery, successfully transfecting a high proportion (82 ± 2%) of mitochondria in a human cell line with concomitant biocompatibility. Nuclear magnetic resonance studies confirmed the effectiveness of our bipartite peptide design with segregated functions: a helical domain necessary for mitochondrial import and an unstructured region for interaction with DNA involving lysine residues. Further analyses revealed that the lysine-specific interaction assisted the self-organization of the peptide and the DNA cargo, leading to a structural arrangement within the formed complex that is crucial for its biological efficiency. Thus the reported gene vector represents a new and reliable tool to uncover the complexity of mitochondrial transfection

Structural Analysis of the End Groups and Substructures of Commercial Poly(ethylene terephthalate) by Multiple-WET ¹H/¹³C NMR

Structural Analysis of the End Groups and Substructures of Commercial Poly(ethylene terephthalate) by Multiple-WET ¹H/¹³C NM

Niizalactams A–C, Multicyclic Macrolactams Isolated from Combined Culture of <i>Streptomyces</i> with Mycolic Acid-Containing Bacterium

A terrestrial bacterium, <i>Streptomyces</i> sp. NZ-6, produced niizalactams A–C (<b>1</b>–<b>3</b>), unprecedented di- and tricyclic macrolactams, by coculturing with the mycolic acid-containing bacterium <i>Tsukamurella pulmonis</i> TP-B0596. Their complete structures, including absolute configurations, were elucidated on the basis of spectroscopic data and chemical derivatization. Their unique skeletons are proposed to be biosynthesized from a common 26-membered macrolactam intermediate by S_N2 cyclization or an intramolecular Diels–Alder reaction

The differences in the chemical shifts between E12A and WT at pH 7.

<p>The differences in the chemical shifts of (A) CA, (B) CB, (C) CO, (D) H, and (E) N are indicated. Some residue numbers are shown. The values for residue 12 were out of the shown range and were -3.2 (A) and -9.9 (B), respectively. The value for residue 9 was -0.8 (D). The locations of the helices are shown at the top of each figure with black bars for helices 2, 3, and 5, and red bars for helices 1 and 4, based on the structure of p17 (PDB: 2H3F). The values of chemical shifts are shown for E12A (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167176#pone.0167176.s005" target="_blank">S1 Table</a>).</p

Thermodynamic parameters for the urea denaturation of p17.

<p>Thermodynamic parameters for the urea denaturation of p17.</p

NMR structure of p17 (PDB: 2H3F).

<p>Red color: α1–2 loop, helices 3 and 5. The side chains for E12 and H89 are shown in blue. This figure was prepared using MOLMOL[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167176#pone.0167176.ref062" target="_blank">62</a>].</p

Discovery of Key Dioxygenases that Diverged the Paraherquonin and Acetoxydehydroaustin Pathways in <i>Penicillium brasilianum</i>

Paraherquonin (<b>1</b>), a fungal meroterpenoid produced by <i>Penicillium brasilianum</i> NBRC 6234, possesses a unique, highly congested hexacyclic molecular architecture. Here we identified the biosynthetic gene cluster of <b>1</b> (the <i>prh</i> cluster) and elucidated the pathway up to berkeleydione (<b>2</b>), which serves as the key intermediate for the biosynthesis of <b>1</b> as well as many other meroterpenoids. Interestingly, the nonheme iron and α-ketoglutarate-dependent dioxygenase PrhA constructs the cycloheptadiene moiety to afford <b>2</b> from preaustinoid A1 (<b>6</b>), probably via the homoallyl-homoallyl radical rearrangement. Additionally, another fungal strain, <i>P. brasilianum</i> MG11, which produces acetoxydehydroaustin instead of <b>1</b>, was found to have a gene cluster nearly identical to the <i>prh</i> cluster. The dioxygenase encoded by the cluster shares 92% sequence identity with PrhA, and also accepts <b>6</b> but produces preaustinoid A3 (<b>17</b>) with a spiro-lactone system, generating a diverging point for the two different meroterpenoid pathways in the same species

Hinduchelins A–D, Noncytotoxic Catechol Derivatives from <i>Streptoalloteichus hindustanus</i>

Four new catechol derivatives, hinduchelins A–D (<b>1</b>–<b>4</b>), composed of 2,3- dihydroxybenzoic acid, threonine, and decarboxylated phenylalanine, were isolated from <i>Streptoalloteichus hindustanus</i>. Their structures and absolute configurations were elucidated by interpretation of NMR and HRMS data and quantum chemical ECD calculations. The iron-binding properties of the compounds were evaluated by a pyoverdine production assay in <i>Pseudomonas aeruginosa</i>, and compound <b>4</b> showed moderate ability to induce pyoverdine production at 50 μM. None of the compounds were cytotoxic toward HL-20, A549, SMMC-7721, MCF-7, and SW-480 tumor cell lines