Undergraduate Research Experience Publications

The treatment of orofacial pain disorders is poor. Both opioids and serotonin agonists are commonly used; however, they produce dangerous and unpleasant side effects.

Therefore, there is an urgent need to identify new pharmacological treatments that can resolve orofacial pain. Moreover, a treatment that engages multiple mechanisms using one compound may be advantageous. Fatty acid amide hydrolase (FAAH) and soluble epoxide hydrolase (sEH) are two enzymes that can regulate both pain and inflammation via independent pathways. Small molecules that inhibit both enzymes simultaneously were previously synthesized and produced antinociception in vivo . Quinolinyl-based dual inhibitors of FAAH and sEH can inhibit acute inflammatory pain in rats. Here, following on these findings, we generated 7 new isoquinolinyl- and 7 pyridinyl-based analogs and tested their inhibition at both enzymes. Structure-activity relationship study coupled with docking experiments, revealed that the isoquinoline moiety is well-tolerated in the binding pockets of both enzymes, yielding several analogs with nanomolar activity in enzymatic assays. All newly synthesized analogs were assessed in the solubility assay at pH 7.4, and we determined that isoquinolinyl- and substituted pyridinyl-analogs exhibit limited solubility under the experimental conditions. The most potent inhibitor, 4f , with IC ₅₀ values in the low nanomolar range for both enzymes, was evaluated in a plasma stability assay in human and rat plasma where it showed a moderate stability. Primary binding assays revealed that 4f does not engage any opioid or serotonin receptors. A high dose (3 mg/kg) of 4f reversed orofacial hyperalgesia following pretreatment with nitroglycerin and orofacial injection of formalin; however, this same dose did not inhibit acute orofacial inflammatory pain or restore pain-depressed wheel running. These findings indicate that simultaneous inhibition of FAAH and sEH using isoquinolinyl-based dual inhibitors may only reverse certain components of orofacial hyperalgesia.

Oxygen transport membranes find broad usage in many technologies, but due to the harsh conditions under which
such technologies operate, detailed structural analysis under operational conditions has been limited. This work details the in situ neutron and synchrotron diffraction of the industrially relevant family of compounds, La_0.9Sr_0.1Co_1−yFe_yO_3−δ   ( y = 0, 0.25, 0.75, 1), under reductive and oxidative conditions at elevated temperatures ranging from 723 to 1123 K. Quantitative Rietveld refinements determine the molar fraction of all crystalline intermediates and products that form during the reactions of La _0.9 Sr _0.1 Co _{1 − y} Fe _y O _{3 − δ} ( y = 0, 0.25, 0.75, 1) with methane and then air. Coupling in situ diffraction analysis with catalytic product analysis of the partial oxidation of methane allows for the catalytically active phases to be determined. This work shows the strength of in situ diffraction under extreme conditions and the insights it can give about reactions in the solid state that have previously been elusive.

Recently, considerable uncertainty has arisen concerning the appropriate susceptibility testing for cefiderocol in gram-negative bacilli, particularly in the context of its application to Acinetobacter spp. The optimal method for assessing the susceptibility levels of Acinetobacter spp. to cefiderocol remains a subject of debate due to substantial disparities observed in the values obtained through various testing procedures. This study employed four minimum inhibitory concentration (MIC) methodologies and the disk diffusion to assess the susceptibility of twenty-seven carbapenem resistant (CR)- Acinetobacter strains to cefiderocol. The results from our study reveal significant variations in the minimum inhibitory concentration (MIC) values obtained with the different methods and in the level of agreement in interpretation categories between the different MIC methods and the disk diffusion test. Among the MIC methods, there was relatively more consistency in reporting the interpretation categories. For European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints, the categorical agreement (CA) for MIC methods ranged between 66.7 and 81.5%. On the other hand, the essential agreement (EA) values were as low as 18.5–29.6%. The CA between MIC methods and disk diffusion was 81.5%. These results emphasize the need for a reliable, accurate, and clinically validated methodology to effectively assess the susceptibility of Acinetobacter spp. to cefiderocol. The wide variability observed in our study highlights the importance of standardizing the susceptibility testing process for cefiderocol to ensure consistent and reliable results for clinical decision-making.

RNA Binding Proteins regulate, in part, alternative pre-mRNA splicing and, in turn, gene expression patterns. Polypyrimidine tract binding proteins PTBP1 and PTBP2 are paralogous RNA binding proteins sharing 74% amino acid sequence identity. Both proteins contain four structured RNA-recognition motifs (RRMs) connected by linker regions and an N-terminal region. Despite their similarities, the paralogs have distinct tissue-specific expression patterns and can regulate discrete sets of target exons. How two highly structurally similar proteins can exert different splicing outcomes is not well understood. Previous studies revealed that PTBP2 is post-translationally phosphorylated in the unstructured N-terminal, Linker 1, and Linker 2 regions that share less sequence identity with PTBP1 signifying a role for these regions in dictating the paralogs distinct splicing activities. To this end, we conducted bioinformatics analysis to determine evolutionary conservation of RRMs vs linker regions in PTBP1 and PTBP2 across species. To determine the role of PTBP2 unstructured regions in splicing activity, we created hybrid PTBP1-PTBP2 constructs that had counterpart PTBP1 regions swapped to an otherwise PTBP2 protein and assayed on differentially regulated exons. We also conducted molecular dynamics studies to investigate how negative charges introduced by phosphorylation in PTBP2 unstructured regions can alter their physical properties. Collectively, results from our studies reveal an important role for PTBP2 unstructured regions and suggest a role for phosphorylation in the differential splicing activities of the paralogs on certain regulated exons.