Plant-Expressed Cocaine Hydrolase Variants of Butyrylcholinesterase Exhibit Altered Allosteric Effects of Cholinesterase Activity and Increased Inhibitor Sensitivity

Butyrylcholinesterase (BChE) is an enzyme with broad substrate and ligand specificities and may function as a generalized bioscavenger by binding and/or hydrolyzing various xenobiotic agents and toxicants, many of which target the central and peripheral nervous systems. Variants of BChE were rationally designed to increase the enzyme’s ability to hydrolyze the psychoactive enantiomer of cocaine. These variants were cloned, and then expressed using the magnICON transient expression system in plants and their enzymatic properties were investigated. In particular, we explored the effects that these site-directed mutations have over the enzyme kinetics with various substrates of BChE. We further compared the affinity of various anticholinesterases including organophosphorous nerve agents and pesticides toward these BChE variants relative to the wild type enzyme. In addition to serving as a therapy for cocaine addiction-related diseases, enhanced bioscavenging against other harmful agents could add to the practicality and versatility of the plant-derived recombinant enzyme as a multivalent therapeutic

Near-unity nuclear polarization with an open-source 129Xe hyperpolarizer for NMR and MRI

The exquisite NMR spectral sensitivity and negligible reactivity of hyperpolarized xenon-129 (HP129Xe) make it attractive for a number of magnetic resonance applications; moreover, HP129Xe embodies an alternative to rare and nonrenewable 3He. However, the ability to reliably and inexpensively produce large quantities of HP129Xe with sufficiently high 129Xe nuclear spin polarization (PXe) remains a significant challenge—particularly at high Xe densities. We present results from our “open-source” large-scale (∼1 L/h) 129Xe polarizer for clinical, preclinical, and materials NMR and MRI research. Automated and composed mostly of off-the-shelf components, this “hyperpolarizer” is designed to be readily implementable in other laboratories. The device runs with high resonant photon flux (up to 200 W at the Rb D1 line) in the xenon-rich regime (up to 1,800 torr Xe in 500 cc) in either single-batch or stopped-flow mode, negating in part the usual requirement of Xe cryocollection. Excellent agreement is observed among four independent methods used to measure spin polarization. In-cell PXe values of ∼90%, ∼57%, ∼50%, and ∼30% have been measured for Xe loadings of ∼300, ∼500, ∼760, and ∼1,570 torr, respectively. PXe values of ∼41% and ∼28% (with ∼760 and ∼1,545 torr Xe loadings) have been measured after transfer to Tedlar bags and transport to a clinical 3 T scanner for MR imaging, including demonstration of lung MRI with a healthy human subject. Long “in-bag” 129Xe polarization decay times have been measured (T1 ∼38 min and ∼5.9 h at ∼1.5 mT and 3 T, respectively)—more than sufficient for a variety of applications

Genetic effects on gene expression across human tissues

Characterization of the molecular function of the human genome and its variation across individuals is essential for identifying the cellular mechanisms that underlie human genetic traits and diseases. The Genotype-Tissue Expression (GTEx) project aims to characterize variation in gene expression levels across individuals and diverse tissues of the human body, many of which are not easily accessible. Here we describe genetic effects on gene expression levels across 44 human tissues. We find that local genetic variation affects gene expression levels for the majority of genes, and we further identify inter-chromosomal genetic effects for 93 genes and 112 loci. On the basis of the identified genetic effects, we characterize patterns of tissue specificity, compare local and distal effects, and evaluate the functional properties of the genetic effects. We also demonstrate that multi-tissue, multi-individual data can be used to identify genes and pathways affected by human disease-associated variation, enabling a mechanistic interpretation of gene regulation and the genetic basis of diseas

Genetic effects on gene expression across human tissues

Characterization of the molecular function of the human genome and its variation across individuals is essential for identifying the cellular mechanisms that underlie human genetic traits and diseases. The Genotype-Tissue Expression (GTEx) project aims to characterize variation in gene expression levels across individuals and diverse tissues of the human body, many of which are not easily accessible. Here we describe genetic effects on gene expression levels across 44 human tissues. We find that local genetic variation affects gene expression levels for the majority of genes, and we further identify inter-chromosomal genetic effects for 93 genes and 112 loci. On the basis of the identified genetic effects, we characterize patterns of tissue specificity, compare local and distal effects, and evaluate the functional properties of the genetic effects. We also demonstrate that multi-tissue, multi-individual data can be used to identify genes and pathways affected by human disease-associated variation, enabling a mechanistic interpretation of gene regulation and the genetic basis of disease

KINETICS AND STRUCTURAL CHARACTERIZATION OF LIPOLYTIC ENZYMES FOR HYDROLYZING POULTRY FEATHERS

The processing of chicken feathers poses a challenge for the poultry industry. In the USA, near 9 billion chickens are slaughtered annually, which generates 5 million tons of recalcitrant feathers to manage. Enzymatic processing of feathers has promise to increase economic potential of the feathers, however, the abundant presence of lipids on feathers serve as a likely barrier. The genomes of a feather- degrading actinomycete and related microorganisms were mined for lipolytic genes candidates, which resulted in the identification of 16 active enzymes. SFK3309 from Streptomyces fradiae var. k11 was the top performing enzyme when tested against the synthetic lipase substrate p-nitrophenyl palmitate (pNPP). SFK3309 also displayed activity against feather lipids. Wax esters are the most abundant lipid class in feather lipids and a colorimetric assay was modified to perform kinetics experiments of SFK3309 against cetyl-palmitate. The Km and Kcat were calculated to be 850 μM and 11.63 s-1, respectively. Structural studies were undertaken for SFK3309, though diffracting protein crystals were unable to be produced. Thermal-shift assays suggest the presence of a hydrophobic patch on the protein surface that may interfere with protein being monodispersed in solution. Yet, some additives hold promise at stabilizing the enzyme in solution. A different lipase from S. fradiae var. k11, SFK3087, which lacked wax ester hydrolase activity, was explored using protein engineering to improve industrial characteristics of the enzyme and initiate work in expanding the substrate specificity and performance towards wax esters. The mutation Y83A increased the thermostability of SFK3087 by 3.76°C, but enzyme activity against pNPP dropped sharply. Saturation mutagenesis was performed on the serine at residue 119 and only large, aromatic residues maintained the same stability seen in wild type. The S119F mutation had a slight increase of thermostability (0.31°C), and an overall improvement in enzyme performance against pNPP, as represented by a Kcat/Km increase of 47%. A more drastic protein engineering scheme may be necessary to incorporate wax ester specificity in an enzyme. However, our work establishes feather-degrading microorganisms produce lipolytic enzymes capable of hydrolyzing wax esters and lays the foundation for further investigation on economic impact lipids have in feather processing