Normally, repeated voluntary movements to the same target are nearly identical to one another. If, however, the cerebellum is damaged, then repeated movements to the same targets have variable trajectories and end in different places. To investigate why cerebellar damage makes movements variable, we record in a rhesus monkey from the type of neurons in the cerebellum that we know most about, those that influence rapid eye movements (saccades). These neurons are in the medial nucleus of the cerebellum and send their axons to the saccade control center in the brainstem. The burst of action potentials fired by a single saccade-related cerebellar neuron can be long, short, fast, or slow. How do these variable signals from the cerebellum help create identical saccades? One commonly accepted idea is that the variability for each cerebellar neuron is not correlated with the variability of other cerebellar neurons. The uncorrelated variability of many cerebellar neurons produces a consistent summed signal that produces identical saccades. If this is true, then why are saccades variable when we damage the cerebellum? We propose an alternative hypothesis: the variability of saccade-related neurons is correlated. This correlation produces a variable summed output from the cerebellum for identical saccades. We think that the saccade-to-saccade variability of cerebellar output compensates for variability in the motor commands from the cerebral cortex. The signals from both the cerebellum and the cortex vary but their sum is consistent because they vary in opposite directions. Without cerebellar output, only the variable motor command from the cortex drives saccades. Variable commands make variable saccades. To test our proposal, we record from several cerebellar neurons simultaneously to measure how correlated their variability is. If our proposal is correct, then the variability of cerebellar neurons is not random. It serves a previously unsuspected function to create consistent saccades. 

Targeted gene modification and correction is a transformative technology that is currently progressing from basic research and development to applications in medicine, industry and agriculture. Gene targeting systems include four types of targeted nucleases: LAGLIDADG homing endonucleases (LHEs), zinc-finger nucleases, TAL effector nucleases, and clustered regularly interspaced short palindromic repeats/CRISPR-associated endonucleases (CRISPR/Cas9). Each of these systems has the ability to make double strand breaks in DNA. LHEs are promising tools for gene targeting due to their small size (~35 kDa) and high specificity (14-20 bp). One specific LAGLIDADG homing endonuclease is I-LtrWI. To fully understand the LHE family, wildtype, and six variants of I-LtrW1 were overexpressed and purified by nickel affinity chromatography. Using site-directed mutagenesis, variants of I-LtrW1 were generated with an amino acid residue change in the catalytic sites. The purified enzymes were assayed for activity using supercoiled DNA containing a target DNA sequence specific to I-LtrW1. The activity was monitored by agarose gel electrophoresis in an effort to understand catalytically important residues. The results will be used to develop a general method for converting LHEs into LHE nickases, enzymes that make single-strand breaks in target DNA sequences.

Thioester reactions are essential for performing all types of biochemical and organic processes. The reactive C-S bond serves as a gateway to other acyl groups in a multitude of organic chemistry reactions, and thioesters are necessary for processes such as thioester exchange, Claisen condensations, and hydrolysis. These reactions are typically enzyme-catalyzed and are vital for controlling levels of acyl CoAs, S-palmitoylation, and for fatty acid biosynthesis. Due to the strong reactive center of a thioester, the mechanism for thioester hydrolysis is not as extensively studied or completely known as the mechanism for the oxoester. There are two types of mechanisms that are focused on when it comes to thioester hydrolysis; a step-wise mechanism with formation of a tetrahedral intermediate or a concerted mechanism where the nucleophile and leaving group leave simultaneously. This research concentrates on comparing the hydrolysis mechanism of the thioester formylthiocholine with the oxoester analog, formyl choline, which is synthesized in the lab. In addition to synthesizing formyl choline, the isotopic derivated deutero-formyl choline was made. To study the transition-state structure, deutero-formyl choline (d-Fch) and hydrogen-formyl choline (h-Fch) are mixed together and reacted in acidic, basic, and neutral conditions. Kinetic isotope effect values are obtained by measuring the rates of hydrolysis by NMR spectroscopy. To date, the KIEs for the formyl-H have been determined in basic conditions and yield an inverse KIE. This suggests that the rate determining step is during the formation of the tetrahedral intermediate, where the carbonyl-C undergoes an sp2-sp3 conversion. This knowledge provides initial data for the comparison with the thioester to understand the mechanism of hydrolysis.