Modeling canopy-induced turbulence in the Earth system: a unified parameterization of turbulent exchange within plant canopies and the roughness sublayer (CLM-ml v0)

Land surface models used in climate models neglect the roughness sublayer and parameterize within-canopy turbulence in an ad hoc manner. We implemented a roughness sublayer turbulence parameterization in a multilayer canopy model (CLM-ml v0) to test if this theory provides a tractable parameterization extending from the ground through the canopy and the roughness sublayer. We compared the canopy model with the Community Land Model (CLM4.5) at seven forest, two grassland, and three cropland AmeriFlux sites over a range of canopy heights, leaf area indexes, and climates. CLM4.5 has pronounced biases during summer months at forest sites in midday latent heat flux, sensible heat flux, gross primary production, nighttime friction velocity, and the radiative temperature diurnal range. The new canopy model reduces these biases by introducing new physics. Advances in modeling stomatal conductance and canopy physiology beyond what is in CLM4.5 substantially improve model performance at the forest sites. The signature of the roughness sublayer is most evident in nighttime friction velocity and the diurnal cycle of radiative temperature, but is also seen in sensible heat flux. Within-canopy temperature profiles are markedly different compared with profiles obtained using Monin–Obukhov similarity theory, and the roughness sublayer produces cooler daytime and warmer nighttime temperatures. The herbaceous sites also show model improvements, but the improvements are related less systematically to the roughness sublayer parameterization in these canopies. The multilayer canopy with the roughness sublayer turbulence improves simulations compared with CLM4.5 while also advancing the theoretical basis for surface flux parameterizations

Optimizing RNA Isolation and Histology Protocols for Characterization of Preclinical Models of Tendon Disease

Tendons join muscle to bone and are essential for posture and movement. Tenocytes are the resident tendon cells. Isolating RNA from small animal models such as mice can be difficult, especially from tendon, as much of the time, they are extremely small samples. Downstream analysis such as qPCR and especially RNA sequencing require greater quantity and quality of RNA than is often achievable. RNA degradation is extremely rapid following tendon isolation, with RNA quality decreasing as time increases between the collection of tendons and RNA isolation. Even tendons whose RNA was isolated immediately following tendon isolation had imperfect quality. The goals for this project were to first, find and optimize a reproducible method for isolating RNA from mouse tendons for downstream qPCR and next generation sequencing that is replicable for people with varying degrees of experience, and second, optimize a method of sectioning mouse joints for the examination of tendon tissue structure

Bed-load effects on hydrodynamics of rough-bed open-channel flows

Peer reviewedPublisher PD

Engineering a Seven Enzyme Biotransformation using Mathematical Modelling and Characterized Enzyme Parts (article)

This is the final version. Available on open access from Wiley via the DOI in this recordThe dataset associated with this article is located in ORE at: https://doi.org/10.24378/exe.1623Multi‐step enzyme reactions offer considerable cost and productivity benefits. Process models offer a route to understanding the complexity of these reactions, and allow for their optimization. Despite the increasing prevalence of multi‐step biotransformations, there are few examples of process models for enzyme reactions. From a toolbox of characterized enzyme parts, we demonstrate the construction of a process model for a seven enzyme, three step biotransformation using isolated enzymes. Enzymes for cofactor regeneration were employed to make this in vitro reaction economical. Good modelling practice was critical in evaluating the impact of approximations and experimental error. We show that the use and validation of process models was instrumental in realizing and removing process bottlenecks, identifying divergent behavior, and for the optimization of the entire reaction using a genetic algorithm. We validated the optimized reaction to demonstrate that complex multi‐step reactions with cofactor recycling involving at least seven enzymes can be reliably modelled and optimized.Biotechnology & Biological Sciences Research Council (BBSRC)GlaxoSmithKlin

Highly thermostable carboxylic acid reductases generated by ancestral sequence reconstruction (article)

This is the final version. Available on open access from Nature Research via the DOI in this recordThe research data supporting this publication are openly available in ORE at https://doi.org/10.24378/exe.2003Carboxylic acid reductases (CARs) are biocatalysts of industrial importance. Their properties, especially their poor stability, render them sub-optimal for use in a bioindustrial pipeline. Here, we employed ancestral sequence reconstruction (ASR) – a burgeoning engineering tool that can identify stabilizing but enzymatically neutral mutations throughout a protein. We used a three-algorithm approach to reconstruct functional ancestors of the Mycobacterial and Nocardial CAR1 orthologues. Ancestral CARs (AncCARs) were confirmed to be CAR enzymes with a preference for aromatic carboxylic acids. Ancestors also showed varied tolerances to solvents, pH and in vivo-like salt concentrations. Compared to well-studied extant CARs, AncCARs had a Tm up to 35 °C higher, with half-lives up to nine times longer than the greatest previously observed. Using ancestral reconstruction we have expanded the existing CAR toolbox with three new thermostable CAR enzymes, providing access to the high temperature biosynthesis of aldehydes to drive new applications in biocatalysis.Glaxosmithkline Research & Development Lt

Structural and biochemical characterisation of Archaeoglobus fulgidus esterase reveals a bound CoA molecule in the vicinity of the active site

A new carboxyl esterase, AF-Est2, from the hyperthermophilic archaeon Archaeoglobus fulgidus has been cloned, over-expressed in Escherichia coli and biochemically and structurally characterized. The enzyme has high activity towards short- to medium-chain pnitrophenyl carboxylic esters with optimal activity towards the valerate ester. The AF-Est2 has good solvent and pH stability and is very thermostable, showing no loss of activity after incubation for 30 min at 80 °C. The 1.4 Å resolution crystal structure of AF-Est2 reveals Coenzyme A (CoA) bound in the vicinity of the active site. Despite the presence of CoA bound to the AF-Est2 this enzyme has no CoA thioesterase activity. The pantetheine group of CoA partially obstructs the active site alcohol pocket suggesting that this ligand has a role in regulation of the enzyme activity. A comparison with closely related α/β hydrolase fold 2 enzyme structures shows that the AF-Est2 has unique structural features that allow CoA binding. A comparison of the structure of AF-Est2 with the human carboxyl esterase 1, which has CoA thioesterase activity, reveals that CoA is bound to different parts of the core domain in these two enzymes and approaches the active site from opposite directions.This work was supported by the Hotzyme project (grant agreement no. 265933) financed by the European Union 7th Framework Programme FP7/2007-2013. WF is funded by a BBSRC PhD studentship. MI would like to thank the BBSRC funded ERA-IB grant BB/L002035/1 and the University of Exeter for support. The authors would like to thank the Diamond Synchrotron Light Source for access to beamline I03 (proposals No. MX8889 and No. MX11945) and the beamline scientists for assistance. The work of ML was funded by the Graduate School VLAG Wageningen, the Netherlan

Production of the Extremolyte Cyclic 2,3-Diphosphoglycerate Using Thermus thermophilus as a Whole-Cell Factory (article)

This is the final version. Available on open access from Frontiers Media via the DOI in this recordData Availability Statement: The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author. Data has been deposited with the University of Exeter ORE https://doi.org/10.24378/exe.3683Osmolytes protect microbial cells against temperature, osmolarity and other stresses. The osmolyte cyclic 2,3-diphosphoglycerate, originally isolated from the thermophilic archaeon Methanothermus fervidus, naturally protects cellular proteins under extreme conditions. The biosynthetic pathway for cyclic 2,3-diphosphoglycerate has been introduced into the thermophilic bacterium Thermus thermophilus. The two enzymes in this synthetic pathway, 2-phosphoglycerate kinase and cyclic diphosphoglycerate synthetase, were incorporated into a newly designed modular BioBricks vector. The expression of this two-enzyme cascade resulted in the whole cell production of cyclic 2,3 diphosphoglycerate. In vivo production of cyclic 2,3-diphosphoglycerate was confirmed by mass spectrometry to a concentration up to 650 µM. This study demonstrates the feasibility of using this well studied thermophilic bacterium as a host in a whole-cell factory approach to produce cyclic 2,3 diphosphoglycerate. This raises the potential for commercialisation of cDPG for cosmetic and healthcare applications. Our work demonstrates the application of Thermus thermophilus as an alternative host for other high value small organic molecules of industrial interest.Biotechnology and Biological Sciences Research Council (BBSRC)University of Exete

Polycyclic aromatic hydrocarbons (PAHs) and estrogenic compounds in experimental flue gas streams

The importance of combustion processes as a source of substances with estrogenic activity in the environment was investigated. Wood (nontreated and treated with wood preservatives), barbecue charcoal, meat, and kitchen waste were combusted in a laboratory-scale incinerator. Flue gas emissions (particulates and gaseous pollutants) were trapped in polyurethane foam cartridges. The cartridges were subjected to Soxhlet extraction and part of the extracts redissolved in dimethylsulfoxide (DMSO) for analyses of estrogenic activity by means of the yeast-based human estrogen receptor (hER) bioassay. A synthetic estrogen, 17-alpha-ethinylestradiol (EE2), was used as the reference estrogenic compound. Part of the extracts was analyzed for the 16 USEPA priority polycyclic aromatic hydrocarbons (PAHs). Estrogenic compounds in the flue gas (wood) were as high as 234 +/- 25 ng m(-3) EE2 equivalent compared with 27 to 81 ng m(-3) EE2 equivalent in flue gas from combustion of barbecue charcoal. Concentrations of polycyclic aromatic hydrocarbons in both flue gas streams were in the range of 21000 +/- 2000 and 240 +/- 110 ng m(-3), respectively. In general, the concentrations of EE2 equivalent in the flue gas samples were at least a factor of 1000 lower than total PAH concentration. The EE2 levels were not related to the concentration of PAHs in any flue gas sample