Amina S. Woods, PhD - US grants

As the brain is over 50% lipids, and traumatic brain injury (TBI) is the main cause of death among young adults and TBI victims often become drug addicts; We studied qualitative and quantitative lipids changes in brain tissue of a rat model of controlled cortical impact (CCI), over time (1, 3 and 7days post trauma), to understand progression of the pathology. The animals were divided into two groups control and CCI brains. We found lipid biomarkers that are reflected in CSF and plasma, and thus could be used for an early and rapid non invasive diagnosis and monitoring. Lipids distribution was mapped by mass spectrometric lipid imaging, to trace and follow lipids changes location in various areas of the brain. We invented a peptide drug, that when administered intravenously 30 minutes post trauma, stopped or significantly diminished progression of the damage. Intranasal administration within the same time frame gave an improved outcome, but was less effective. As our data turned out to be very promising, we believe that further work on the peptide drug would be beneficial. As the project is work intensive and requires expensive supplies, we are seeking funding for further testing of various dosages and regimen.

Our preliminary data shows a statistically significant decrease in several sphingomyelin(SM) species, accompanied by an increase in several ceramides species in the adult mice group chronically exposed to alcohol as compared to the adult animal group exposed to water. While in the adolescent mice, there was a non statiscally significant trend, towards an increase in one sphingomyelin specie as compared to water and a slight decrease in ceramides. So we see far less differences between the alcohol and water groups. Our data suggest that alcohol consumption leads to SM hydrolysis, which generates Ceramides. Our imaging suggests that the bregma containing the striatum is most affected.

Previously, we used gold nanoparticles suspended in a buffer. However, metallic particles are not stable in suspension making homogeneous deposition challenging Jackson SN, Ugarov M, Post J, Egan T, Langlais D, Schultz JA and Woods AS. A study of Phospholipids by Ion Mobility TOFMS. JASMS 19, 16551662 (2008). An alternative deposition technique is the implantation of nanoparticles. Implantation is a dry and uniform deposition technique, which initially employed a massive cluster ion source. The gold cluster ion currents were insufficient for implanting large areas, and therefore not suited for full organ imaging Fernandez-Lima FA, Post J, DeBord JD, Eller MJ, Verkhoturov SV, Della-Negra S, Woods AS, and Schweikert EA. Analysis of native biological surfaces using a 100kV Massive Gold Cluster Source. Anal Chem. 83, 8448-8453 (2011). Tissue was implanted with a NPlanter (Ionwerks, Houston, TX). A silver target is inserted into a particle source NanoGen50. These nanoparticles (NP) are generated by expulsion from the metals surface using magnetron sputtering, and then allowed to grow by condensation in a refinement zone (particles are 0.5-15 nm in diameter). The size is selected with a quadrupole mass filter coupled to the magnetron, so that we can insure that the NP size is reproducible. Presently we are using AgNP 6-7 nm in diameter, so we always have the same size nanoparticles implanted in different tissue sections, making implantation reproducible. Finally, the particle beam can be deviated with electro-optics over a minimum of 400 square millimeters (20 x 20), allowing implantation of a whole tissue section at once. We use the equivalent of 2-4 monolayers. It takes 18 minutes to implant each tissue section. By using the same size NP particles and the same number of monolayers. We have standardized the implantation procedure and made it reproducible. Thus making comparison of tissue implanted at various time possible, as they are more likely to give reproducible data. In addition a tissue section can be reimplanted for in-depth profiling. So far we have been able to reimplant and reimage a tissue section nine times in a row. Each image is of an area 10-15 nanometer deeper. However we still are in the process of confirming the depth. We also were able to image the same tissue section five times in a row after a single implantation. The implanter reduces the inherent variability between operators and reduces the influence of humidity and temperature fluctuations, which makes it consistent across time, samples, and operators. Silver implantation is a dry method, and therefore does not cause any blurring or analyte migration which may occur with solvent dissolved matrix. We found that hand spraying with an artistic airbrush is a fairly reproducible method for depositing organic matrices with minimal lipid disruption. However it can be a work intensive and time consuming process that requires an experienced hand. While in implantation, the rapid automated rastering step achieves excellent uniformity in matrix implantation by precisely covering the whole implanted areas. Total control of the amount of particles implanted at each point also contributes to the improved uniformity and reproducibility of the implantation. The quantity of silver and quality of coverage can be validated through measurement.

Our work targets the receptors involved in addiction, to explain how their structure dictates how they interact with other receptors and what makes these interactions specific. Our data suggest that heteromer formation, mainly involves linear motifs found in disordered regions of proteins. Local disorder imparts plasticity to linear motifs. Many molecular recognition of proteins occur between short linear segments, known as LMs. Interaction of short continuous epitopes are not constrained by sequence and have the advantage of resulting in interactions with micromolar affinities which suites transient, reversible complexes such as receptor heteromers. Electrostatic Interactions between epitopes of the GPCR involved, is the Key step in driving heteromer formation forward. The first step in heteromerization, involves phosphorylating the Ser/Thr, in an epitope containing a casein kinase 1/2 (CK1/2)-consensus site. Our data suggests that dopaminergic neurotransmission, through cAMP dependent Protein kinase A (PKA) slows down heteromerization. The negative charge, acquired by the phosphorylation of a Ser/Thr in a PKA consensus site in the Arg rich epitope affects the activity of the receptors involved in heteromerization by causing allosteric conformational changes, due to the repulsive effect generated by the negatively charged phosphate. In addition to modulating heteromerization, it affects the stability of the heteromers interactions and heir binding affinity. So here we have an instance where phosphorylation is not just an on/off switch, instead by weakening the noncovalent bond, heteromerization acts like a rheostat that controls the stability of the heteromer through activation or inhibition of adenylate cyclase by the neurotransmitter Dopamine depending on which Dopamine receptor it docks at. Complex molecular and cellular mechanisms regulate G protein-coupled receptors (GPCRs). It is suggested that proteins intrinsically disordered regions (IDRs) are to play a role in GPCRsintra and extracellular regions plasticity, due to their potential for post-translational modification and interaction with other proteins. These regions are defined as lacking a stable three-dimensional (3D) structure. They are rich in hydrophilic cand charged, amino acids and are capable to assume different conformations which allow them to interact with multiple partners. In this study we analyzed 75 GPCR involved in synaptic transmission using computational tools for sequence-based prediction of IDRs within a protein. We also evaluated putative ligand-binding motifs using receptor sequences. The disorder analysis indicated that neurotransmitter GPCRs have a significant amount of disorder in their N-terminus, third intracellular loop (3IL) and C-terminus. About 31%, 39% and 53% of human GPCR involved in synaptic transmission are disordered in these regions. Thirty-three percent of receptors show at least one predicted PEST motif, this being statistically greater than the estimate for the rest of human GPCRs. About 90% of the receptors had at least one putative site for dimerization in their 3IL or C-terminus. ELM instances sampled in these domains were 14-3-3, SH3, SH2 and PDZ motifs. In conclusion, the increased flexibility observed in GPCRs, added to the enrichment of linear motifs, PEST and heteromerization sites, maybe critical for the nervous systems functional plasticity. Certain amino acid residues and posttranslational modifications play an important role in the formation of noncovalent complexes (NCXs) by electrostatic interactions. Electrospray ionization mass spectrometry(ESI-MS) is the most widely used MS technique for the study of NCXs, due to its softer ionization process and compatibility with the solution phase of NCX mixtures. In order to locate the site where interactions are forming in the NCXs involving phosphopeptides and adjacent arginines, tandem mass spectrometry studies using collision-induced dissociation (CID) and electron transfer dissociation (ETD) were performed on NCXs at different charge states. CID fragmentation revealed two dissociation pathways:one in which the electrostatic interaction is disrupted and another in which the covalent bond attaching the phosphate group to the amino acid residue is cleaved, while the electrostatic interaction is maintained. ETD and sequential ETD/ETD, and CID/ETD allow the determination of the NCX interaction site. These results confirmed the involvement of the phosphorylated amino acid and at least two adjacent arginines as the binding site.