Understanding oligonucleotide synthesis

Oligonucleotides are synthesised almost exclusively via the solid-supported phosphoramidite method. However popular this method may be, the expensive reagents used in large excess during the synthesis as well as the large amounts of organic and aqueous solvents and purification steps makes the scale-up of oligonucleotide synthesis costly and possibly harmful to the environment. The therapeutic use of anti-sense oligonucleotides (ASOs) is hindered by their susceptibility to nuclease catalysed hydrolysis and to overcome this problem ASOs have been modified commonly by the introduction of a phosphorothioate backbone. This research aims to provide a better understanding of some of the more problematic stages of the synthesis cycle, the formation of the sulfurizing agent and sulfurisation of inter-nucleotide phosphite linkages, in order to make this method more sustainable and efficient. The investigation of the activation, alcoholysis and hydrolysis of the phosphoramidites 2´-methoxy-5´-O-DMT-uridine 3´-CE phosphoramidite (UAm) and di-tert-butyl N,N-di-isopropyl phosphoramidite (DBAm) using several tetrazole activators found that complete conversion of the phosphoramidite UAm to products required an excess of activator and that this was due to the generation of di-isopropyl amine during coupling. Conductivity measurements show that the amine deprotonates the acidic activator and that the ammonium and tetrazolide ions that are subsequently formed strongly ion pair (Kip = 6540 M-1) removing free activator from solution. The tetrazole-catalysed reaction of phosphoramidites with oxygen nucleophiles was found to be first order with respect to phosphoramidite and activator and the nucleophilic displacement of the di-isopropyl amine group by the tetrazoyl group at phosphorus is rate-limiting. Investigation into the 3-picoline-catalysed ageing of the sulfur transfer reagent phenylacetyl disulfide (PADS) has shown that the process is overall second order and is proportional to the concentration of PADS and 3-picoline. Deuterium exchange experiments show that ageing proceeds via abstraction of the methylene CH2 protons of PADS via an E1cB-type decomposition of the PADS molecule generating a disulfide anion and a ketene by-product which was trapped using an intra-molecular [2+2]-cycloaddition reaction. Mass spectrometry data shows that disulfide anions act as nucleophiles with PADS molecules to generate polysulfides which are the active sulfur transfer reagents in aged PADS solutions. Using pyridines that are less basic than 3-picoline causes the rate of degradation of PADS to become slower, indicating the possibility that the rate-limiting step of this process is the generation of the disulfide anion. The rate of sulfurisation of phosphites by both ‘fresh’ and ‘aged’ PADS in the presence of 3-picoline was found to be first order with respect to phosphite, PADS and 3-picoline at low concentrations of each. However, the rate of the reaction becomes independent of base when using aged PADS in the presence of high 3-picoline concentration. Brönsted correlations for the sulfurisation of alkyl phosphites using fresh PADS give a βnuc value of 0.51, consistent with a mechanism involving nucleophilic attack by the phosphite on the disulfide bond of PADS to generate a phosphonium ion intermediate. This degrades to the phosphorothioate product via a base-catalysed mechanism which has been confirmed by removal of the methylene protons from the PADS molecule. Comparison of the βnuc values seen when altering the pKa of the pyridine catalyst used shows that the rate of the reaction of fresh PADS is much more sensitive to the pKa of the pyridine than is aged PADS (βnuc = 0.43 and 0.26 for fresh and aged PADS respectively). This suggests that in the case of aged PADS, the phosphite attacks the sulfur atom adjacent to the carboxylate group in the polysulfide chain. This generates a phosphonium intermediate which can be broken down via a much more facile S-S bond fission, as opposed to the C-S bond fission as seen in when using fresh PADS

Algunos aspectos de las relaciones entre establecidos y marginados: el modelo Maycomb

Este ensayo de sociología de los procesos sociales contemporáneos fue publicado originalmente en 1990 y recogido por Cas Wouters en el cuarto volumen de la compilación de trabajos de Norbert Elias que editó en la imprenta de University College Dublin. Se trata de un corolario de la “investigación sociológica sobre los problemas de la vida en comunidad” que, bajo el título de Establecidos y marginados, fue publicada originalmente en 1965 por la editorial londinense de Frank Cass & Co. Una traducción española de este libro apareció durante el año 2016 en el Fondo de Cultura Económica. Por su valor metodológico para la comprensión de una de las características repetitivas de las sociedades humanas se ofrece este ensayo a los lectores de la Revista de Santander en su primera traducción castellana, debida a Marina González de Cala y Amelia Acebedo Silva.&nbsp

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

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

Funder: U.S. Department of Energy through the LANL/LDRD ProgramFunder: G. T. Seaborg InstitutePhosphorus (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

Measurement of micro-scale soil deformation around roots using four-dimensional synchrotron tomography and image correlation

This study applied time lapse (four-dimensional) synchrotron X-ray computed tomography to observe micro-scale interactions between plant roots and soil. Functionally contrasting maize root tips were repeatedly imaged during ingress into soil columns of varying water content and compaction. This yielded sequences of three-dimensional densiometric data, representing time-resolved geometric soil and root configurations at the micronmetre scale. These data were used as inputs for two full-field kinematic quantification methods, which enabled the analysis of three-dimensional soil deformation around elongating roots. Discrete object tracking was used to track rigid mineral grains, while continuum digital volume correlation was used to track grey-level patterns within local sub-volumes. These techniques both allowed full-field soil displacements to be quantified at an intra-rhizosphere spatial sampling scale of less than 300 µm. Significant differences in deformation mechanisms were identified around different phenotypes under different soil conditions. A uniquely strong contrast was observed between intact and de-capped roots grown in dry, compacted soil. This provides evidence that functional traits of the root cap significantly reduce the amount of soil disturbance per unit of root elongation, with this effect being particularly significant in drier soil.</jats:p

Significance of root hairs at the field scale - modelling root water and phosphorus uptake under different field conditions

Background and aims: root hairs play a significant role in phosphorus (P) extraction at the pore scale. However, their importance at the field scale remains poorly understood.Methods: this study uses a continuum model to explore the impact of root hairs on the large scale uptake of P, comparing root hair influence under different agricultural scenarios. High vs low and constant vs decaying P concentrations down the soil profile are considered, along with early vs late precipitation scenarios.Results: simulation results suggest root hairs accounted for 50% of total P uptake by plants. Furthermore, a delayed initiation time of precipitation potentially limits the P uptake rate by over 50% depending on the growth period. Despite the large differences in the uptake rate, changes in the soil P concentration in the domain due to root solute uptake remains marginal when considering a single growth season. However, over the duration of six years, simulation results showed that noticeable differences arise over time.Conclusion: root hairs are critical to P capture, with uptake efficiency potentially enhanced by coordinating irrigation with P application during earlier growth stages of crops

Significance of root hairs at the field scale – modelling root water and phosphorus uptake under different field conditions

Abstract Background and aims Root hairs play a significant role in phosphorus (P) extraction at the pore scale. However, their importance at the field scale remains poorly understood. Methods This study uses a continuum model to explore the impact of root hairs on the large-scale uptake of P, comparing root hair influence under different agricultural scenarios. High vs low and constant vs decaying P concentrations down the soil profile are considered, along with early vs late precipitation scenarios. Results Simulation results suggest root hairs accounted for 50% of total P uptake by plants. Furthermore, a delayed initiation time of precipitation potentially limits the P uptake rate by over 50% depending on the growth period. Despite the large differences in the uptake rate, changes in the soil P concentration in the domain due to root solute uptake remains marginal when considering a single growth season. However, over the duration of 6 years, simulation results showed that noticeable differences arise over time. Conclusion Root hairs are critical to P capture, with uptake efficiency potentially enhanced by coordinating irrigation with P application during earlier growth stages of crops. </jats:sec

Multimodal correlative imaging 1 and modelling of phosphorus 2 uptake from soil by hyphae of mycorrhizal fungi

• Phosphorus (P) is essential for plant growth. Arbuscular mycorrhizal fungi (AMF) aid its uptake by acquiring 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 (SXRCT) to visualize mycorrhizae in soil, and synchrotron X-ray fluorescence (XRF/XANES) elemental mapping for phosphorus (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. Results showed that AMF co-locate with areasof high P and low Al, andpreferentially associate with organic-type P species in preference to Al-rich inorganic P.• We discovered that AMF avoid Al-rich areas as a source of P. S-rich regions 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 modelling of plant-soil-AMF interactions.<br/

Dataset for: Multimodal correlative imaging and modelling of phosphorus uptake from soil by hyphae of mycorrhizal fungi

Dataset for the paper titled Multimodal correlative imaging and modelling of phosphorus uptake from soil by hyphae of mycorrhizal fungi. Accepted and in press at New Phytologist. This Dataset contains XCT imaging, XRF, XANES and modelling data to recreate the figures in the publication. Since the XCT imaging data is large, only csvs and single images as a result of image processing are included. If you would like the raw XCT data, please contact Tiina Roose ([email protected]). Links to the sequencing data depository in NCBI Sequence Read Archive (SRA) accession number PRJNA498673are also included . </span