Multimodal correlative imaging and modelling of phosphorus uptake from soil by hyphae of mycorrhizal fungi.

Phosphorus (P) is essential for plant growth. Arbuscular mycorrhizal fungi (AMF) aid its uptake by acquiring P from sources distant from roots in return for carbon. Little is known about how AMF colonise soil pore-space, and models of AMF-enhanced P-uptake are poorly validated. We used synchrotron X-ray computed tomography to visualize mycorrhizas in soil and synchrotron X-ray fluorescence/X-ray absorption near edge structure (XRF/XANES) elemental mapping for P, sulphur (S) and aluminium (Al) in combination with modelling. We found that AMF inoculation had a suppressive effect on colonisation by other soil fungi and identified differences in structure and growth rate between hyphae of AMF and nonmycorrhizal fungi. Our results showed that AMF co-locate with areas of high P and low Al, and preferentially associate with organic-type P species over Al-rich inorganic P. We discovered that AMF avoid Al-rich areas as a source of P. Sulphur-rich regions were found to be correlated with higher hyphal density and an increased organic-associated P-pool, whilst oxidized S-species were found close to AMF hyphae. Increased S oxidation close to AMF suggested the observed changes were microbiome-related. Our experimentally-validated model led to an estimate of P-uptake by AMF hyphae that is an order of magnitude lower than rates previously estimated - a result with significant implications for the modelling of plant-soil-AMF interactions.

Molecular basis of RNA guanine-7 methyltransferase (RNMT) activation by RAM.

Maturation and translation of mRNA in eukaryotes requires the addition of the 7-methylguanosine cap. In vertebrates, the cap methyltransferase, RNA guanine-7 methyltransferase (RNMT), has an activating subunit, RNMT-Activating Miniprotein (RAM). Here we report the first crystal structure of the human RNMT in complex with the activation domain of RAM. A relatively unstructured and negatively charged RAM binds to a positively charged surface groove on RNMT, distal to the active site. This results in stabilisation of a RNMT lobe structure which co-evolved with RAM and is required for RAM binding. Structure-guided mutagenesis and molecular dynamics simulations reveal that RAM stabilises the structure and positioning of the RNMT lobe and the adjacent α-helix hinge, resulting in optimal positioning of helix A which contacts substrates in the active site. Using biophysical and biochemical approaches, we observe that RAM increases the recruitment of the methyl donor, AdoMet (S-adenosyl methionine), to RNMT. Thus we report the mechanism by which RAM allosterically activates RNMT, allowing it to function as a molecular rheostat for mRNA cap methylation.

Projected Increases in Precipitation Are Expected To Reduce Nitrogen Use Efficiency and Alter Optimal Fertilization Timings in Agriculture in the South East of England.

Nitrogen fertilization is vital for productive agriculture and efficient land use. However, globally, approximately 50% of the nitrogen applied is lost to the environment, causing inefficiencies, pollution, and greenhouse gas emissions. Rainfall and its effect on soil moisture are the major components controlling nitrogen losses in agriculture. Thus, changing rainfall patterns could accelerate nitrogen inefficiencies. We used a mechanistic modeling platform to determine how precipitation-optimal nitrogen fertilization timings and resulting crop nitrogen uptake have changed historically (1950-2020) and how they are predicted to change under the RCP8.5 climate scenario (2021-2069) in the South East of England. We found that historically, neither precipitation-optimal fertilization timings nor resulting plant uptake changed significantly. However, there were large year-to-year variations in both. In the 2030s, where it is projected to get wetter, precipitation-optimal fertilization timings are predicted to be later in the season and the resulting plant uptake noticeably lower. After 2040, the precipitation-optimal uptakes are projected to increase with earlier precipitation-optimal timings closer to historical values, corresponding to the projected mean daily rainfall rates decreasing to the historical values in these growing seasons. It seems that the interannual variation in precipitation-optimal uptake is projected to increase. Ultimately, projected changes in precipitation patterns will affect nitrogen uptake and precipitation-optimal fertilization timings. We argue that the use of bespoke fertilization timings in each year can help recuperate the reduced N uptake due to changing precipitation.

Optimisation of 6-substituted isoquinolin-1-amine based ROCK-I inhibitors.

Rho kinase is an important target implicated in a variety of cardiovascular diseases. Herein, we report the optimisation of the fragment derived ATP-competitive ROCK inhibitors 1 and 2 into lead compound 14A. The initial goal of improving ROCK-I potency relative to 1, whilst maintaining a good PK profile, was achieved through removal of the aminoisoquinoline basic centre. Lead 14A was equipotent against both ROCK-I and ROCK-II, showed good in vivo efficacy in the spontaneous hypertensive rat model, and was further optimised to demonstrate the scope for improving selectivity over PKA versus hydroxy Fasudil 3.

Fragment-based discovery of 6-substituted isoquinolin-1-amine based ROCK-I inhibitors.

Fragment-based NMR screening of a small literature focused library led to identification of a historical thrombin/FactorXa building block, 17A, that was found to be a ROCK-I inhibitor. In the absence of an X-ray structure, fragment growth afforded 6-substituted isoquinolin-1-amine derivatives which were profiled in the primary ROCK-I IMAP assay. Compounds 23A and 23E were selected as fragment optimized hits for further profiling. Compound 23A has similar ROCK-1 affinity, potency and cell based efficacy to the first generation ROCK inhibitors, however, it has a superior PK profile in C57 mouse. Compound 23E demonstrates the feasibility of improving ROCK-1 affinity, potency and cell based efficacy for the series, however, it has a poor PK profile relative to 23A.

Developing a system for in vivo imaging of maize roots containing iodinated contrast media in soil using synchrotron XCT and XRF.

AIMS: We sought to develop a novel experimental system which enabled application of iodinated contrast media to in vivo plant roots intact in soil and was compatible with time-resolved synchrotron X-ray computed tomography imaging. The system was developed to overcome issues of low contrast to noise within X-ray computed tomography images of plant roots and soil environments, the latter of which can complicate image processing and result in the loss of anatomical information. METHODS: To demonstrate the efficacy of the system we employ the novel use of both synchrotron X-ray computed tomography and synchrotron X-ray fluorescence mapping to capture the translocation of the contrast media through root vasculature into the leaves. RESULTS: With the application of contrast media we identify fluid flow in root vasculature and visualise anatomical features, which are otherwise often only observable in ex vivo microscopy, including: the xylem, metaxylem, pith, fibres in aerenchyma and leaf venation. We are also able to observe interactions between aerenchyma cross sectional area and solute transport in the root vasculature with depth. CONCLUSIONS: Our novel system was capable of successfully delivering sufficient contrast media into root and leaf tissues such that anatomical features could be visualised and internal fluid transport observed. We propose that our system could be used in future to study internal plant transport mechanisms and parameterise models for fluid flow in plants. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11104-020-04784-x.

Rapid access towards follow-up NOP receptor agonists using a knowledge based approach.

A knowledge based approach has been adopted to identify novel NOP receptor agonists with simplified hydrophobes. Substitution of the benzimidazol-2-one piperidine motif with a range of hydrophobic groups and pharmacophore guided bio-isosteric replacement of the benzimidazol-2-one moiety was explored. Compound 51 was found to be a high affinity, potent NOP receptor agonist with reduced affinity for the hERG channel.

Utstein-style guidelines on uniform reporting of in-hospital cardiopulmonary resuscitation in dogs and cats. A RECOVER statement.

OBJECTIVE: To provide recommendations for reviewing and reporting clinical in-hospital cardiopulmonary resuscitation (CPR) events in dogs and cats and to establish nonambiguous operational definitions for CPR terminology. DESIGN: Consensus guidelines. SETTING: International, academia, referral practice, general practice, and human medicine. METHODS: An international veterinary Utstein task force was convened in April 2013 in San Francisco to determine the scope of the project, the variables to be reported, their definitions, and a reporting template. Factors that were essential for meaningful data reporting and were amenable to accurate collection (ie, core variables) and additional variables useful for research projects and hypothesis generation (ie, supplemental variables) were defined. Consensus on each item was either achieved during that meeting or during the subsequent online modified Delphi process and dialogue between task force members. RESULTS: Variables were defined and categorized as hospital, animal, event (arrest), and outcome variables. This report recommends a template for standardized reporting of veterinary in-hospital CPR studies involving dogs or cats. Core elements include the suspected cause(s) and location of arrest, first rhythm identified, the occurrence of return of spontaneous circulation (ROSC) of more than 30 seconds (any ROSC) or more than 20 minutes (sustained ROSC), survival to discharge, and functional capacity at discharge. If CPR is discontinued or the patient is euthanized by owner request, a reason is reported. The task force suggests a case report form to be used for individual resuscitation events. CONCLUSIONS: The availability of these veterinary small animal CPR reporting guidelines will encourage and facilitate high-quality veterinary CPR research, improve data comparison between studies and across study sites, and serve as the foundation for veterinary CPR registries.

Determination of protein-ligand binding affinity by NMR: observations from serum albumin model systems.

The usefulness of bovine serum albumin (BSA) as a model protein for testing NMR methods for the study of protein-ligand interactions is discussed. Isothermal titration calorimetry established the binding affinity and stoichiometry of the specific binding site for L-tryptophan, D-tryptophan, naproxen, ibuprofen, salicylic acid and warfarin. The binding affinities of the same ligands determined by NMR methods are universally weaker (larger KD). This is because the NMR methods are susceptible to interference from additional non-specific binding. The L-tryptophan-BSA and naproxen-BSA systems were the best behaved model systems.

Exploring the active site of human factor Xa protein by NMR screening of small molecule probes.

A collection of small molecules (MW < 350 Da) was screened for binding to human factor Xa using saturation transfer difference NMR spectroscopy to detect binding. The NMR screening experiments identified four hits. Binding isotherms constructed from NMR linewidth data showed that the binding affinities of the hits were all in the 30-210 microM range. Competition binding experiments showed that three of the ligands were displaced by a known microM inhibitor of factor Xa. The success of the method for identifying new ligands and the relevance of this information to the design of new factor Xa inhibitors are discussed.