Mapping surgical coordinates of the sphenopalatine foramen : surgical navigation study

Aims to identify measurements that may help intra operative localisation of the sphenopalatine foramen. The study used three dimensional surgical navigation software to study radiological anatomy, in order to define the distances and angulations between identifiable bony landmarks and the sphenopalatine foramen. The distance from the anterior nasal spine to the sphenopalatine foramen was 59 mm (+4 mm; inter observer variation = 0.866; intra observer variation = 0.822). The distance from the piriform aperture to the sphenopalatine foramen was 48 mm (+4 mm; inter observer variation = 0.828; intra observer variation = 0.779). The angle of elevation from the nasal floor to the sphenopalatine foramen was 22 degrees (+3 degrees; inter observer variation = 0.441; intra observer variation = 0.499). The sphenopalatine foramen is consistently identifiable on three dimensional, reconstructed computed tomography scans. Repeatable measurements were obtained. The centre point of the foramen lies 59 mm from the anterior nasal spine at 22 degrees elevation above the plane of the hard palate and 48 mm from the piriform aperture. We discuss how these data could be used to facilitate intra operative location of the sphenopalatine foramen in difficult cases

Expanding the scope of alkyne-mediated bioconjugations utilizing unnatural amino acids

The importance of bioconjugates within the field of chemistry drives the need for novelmethodologies for their preparation. Well-defined and stable bioconjugates are easily accessible via the utilization of unnatural amino acids (UAAs). As such, we have synthesized and incorporated two new UAAs into green fluorescent protein, and optimized a novel Cadiot-Chodkiewicz bioconjugation, effectively expanding the toolbox of chemical reactions that can be employed in the preparation of bioconjugates

Improvement of Shelf Life for Space Food Through a Hurdle Approach

The processed and prepackaged spaceflight food system is a critical human support system for manned space flights. As missions extend longer and farther from Earth over the next 20 years, strategies to stabilize the nutritional and sensory quality of food must be identified. For a mission to Mars, the space foods themselves must maintain quality for up to 5 years to align with cargo prepositioning scenarios. Optimizing the food system to achieve a 5-year shelf life mitigates the risk of an inadequate food system during extended missions. Because previous attempts to determine a singular pathway to a 5-year shelf life for food were unsuccessful, this investigation combines several approaches, based on science, technological advancement, and past empirical evidence, that will define the prepackaged food system for long duration missions. This study supports the Advanced Food Technology strategic planning process by identifying food processing, packaging, and storage technologies that will be required for exploration missions and the extent that they must be implemented to achieve a 5-year shelf life for the entire food system

Optochemical control of RNA interference in mammalian cells

Short interfering RNAs (siRNAs) and microRNAs (nniRNAs) have been widely used in mammalian tissue culture and model organisms to selectively silence genes of interest. One limitation of this technology is the lack of precise external control over the gene-silencing event. The use of photocleavable protecting groups installed on nucleobases is a promising strategy to circumvent this limitation, providing high spatial and temporal control over siRNA or miRNA activation. Here, we have designed, synthesized and site-specifically incorporated new photocaged guanosine and uridine RNA phosphoramidites into short RNA duplexes. We demonstrated the applicability of these photocaged siRNAs in the light-regulation of the expression of an exogenous green fluorescent protein reporter gene and an endogenous target gene, the mitosis motor protein, Eg5. Two different approaches were investigated with the caged RNA molecules: the light-regulation of catalytic RNA cleavage by RISC and the light-regulation of seed region recognition. The ability to regulate both functions with light enables the application of this optochemical methodology to a wide range of small regulatory RNA molecules

Using Unnatural Amino Acid Mutagenesis To Probe the Regulation of PRMT1

Protein arginine methyltransferase 1 (PRMT1)-dependent methylation contributes to the onset and progression of numerous diseases (e.g., cancer, heart disease, ALS); however, the regulatory mechanisms that control PRMT1 activity are relatively unexplored. We therefore set out to decipher how phosphorylation regulates PRMT1 activity. Curated mass spectrometry data identified Tyr291, a residue adjacent to the conserved THW loop, as being phosphorylated. Natural and unnatural amino acid mutagenesis, including the incorporation of p-carboxymethyl-L-phenylalanine (pCmF) as a phosphotyrosine mimic, were used to show that Tyr291 phosphorylation alters the substrate specificity of PRMT1. Additionally, p-benzoyl-L-phenylalanine (pBpF) was incorporated at the Tyr291 position, and cross-linking experiments with K562 cell extracts identified several proteins (e.g., hnRNPA1 and hnRNP center dot center dot H3) that bind specifically to this site. Moreover, we also demonstrate that Tyr291 phosphorylation impairs PRMT1\u27s ability to bind and methylate both proteins. In total, these studies demonstrate that Tyr291 phosphorylation alters both PRMT1 substrate specificity and protein-protein interacions

Optimization of Solid-Supported Glaser-Hay Reactions in the Microwave

The translation of organometallic reactions into a microwave reactor has numerous advantages. Herein, we describe the application of a previously developed solid-supported Glaser-Hay reaction to microwave conditions. Overall, an array of diynes has been prepared demonstrating the ability to conduct chemoselective reactions in the microwave within 20 min compared to the 16 h thermal conditions. Moreover, non-microwave transparent alkynes have been found to react more quickly, preventing catalyst quenching, and resulting in higher yields

Development of optimized conditions for Glaser-Hay bioconjugations

The efficient preparation of protein bioconjugates represents a route to novel materials, diagnostics, and therapeutics. We previously reported a novel bioorthogonal Glaser-Hay reaction for the preparation of covalent linkages between proteins and a reaction partner; however, deleterious protein degradation was observed under extended reaction conditions. Herein, we describe the systematic optimization of the reaction to increase coupling efficiency and decrease protein degradation. Two optimized conditions were identified varying either the pH of the reaction or the bidentate ligand employed, allowing for more rapid conjugations and/or less protein oxidation. (C) 2017 Elsevier Inc. All rights reserved

Synthesis and Incorporation of Unnatural Amino Acids To Probe and Optimize Protein Bioconjugations

The utilization of unnatural amino acids (UAAs) in bioconjugations is ideal due to their ability to confer a degree of bioorthogonality and specificity. In order to elucidate optimal conditions for the preparation of bioconjugates with UAAs, we synthesized 9 UAAs with variable methylene tethers (2-4) and either an azide, alkyne, or halide functional group. All 9 UAAs were then incorporated into green fluorescent protein (GFP) using a promiscuous aminoacyl-tRNA synthetase. The different bioconjugations were then analyzed for optimal tether length via reaction with either a fluorophore or a derivatized resin. Interestingly, the optimal tether length was found to be dependent on the type of reaction. Overall, these findings provide a better understanding of various parameters that can be optimized for the efficient preparation of bioconjugates

BA11 FKBP5 expression levels correlate with dendritic spine density in postmortem PTSD and controls

AbstractGenetic variants of the immunophilin FKBP5 have been implicated in susceptibility to post-traumatic stress disorder (PTSD) and other stress-related disorders. We examined the relationship between mushroom, stubby, thin and filopodial spine densities measured with Golgi staining and FKBP5 gene expression in the medial orbitofrontal cortex (BA11) in individuals diagnosed with PTSD and normal controls (n = 8/8). ANCOVA revealed PTSD cases had a significantly elevated density of stubby spines (29%, P < 0.037) and a trend for a reduction in mushroom spine density (25%, p < 0.082). Levels of FKBP5 mRNA were marginally elevated in the PTSD cases (z = 1.94, p = 0.053) and levels correlated inversely with mushroom (Spearman's rho = −0.83, p < 0.001) and overall spine density (rho = −0.75, p < 0.002) and directly with stubby spine density (rho = 0.55, p < 0.027). These data suggest that FKBP5 may participate in a cellular pathway modulating neuronal spine density changes in the brain, and that this pathway may be dysregulated in PTSD

Utilization of alkyne bioconjugations to modulate protein function

The ability to introduce or modify protein function has widespread application to multiple scientific disciplines. The introduction of unique unnatural amino acids represents an excellent mechanism to incorporate new functionality; however, this approach is limited by ability of the translational machinery to recognize and incorporate the chemical moiety. To overcome this potential limitation, we aimed to exploit the functionality of existing unnatural amino acids to perform bioorthogonal reactions to introduce the desired protein modification, altering its function. Specifically, via the introduction of a terminal alkyne containing unnatural amino acid, we demonstrated chemically programmable protein modification through the Glaser-Hay coupling to other terminal alkynes, altering the function of a protein. In a proof-of-concept experiment, this approach has been utilized to modify the fluorescence spectrum of green fluorescent protein. (C) 2016 Elsevier Ltd. All rights reserved