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1. Don’t Drink and Fly: Alcohol Resistance Behavior in Drosophila Informs us about Genes that Contribute to Alcoholism

   Echeverria, Jennifer S.; Key, S. Catherine (April 2022, Advances in biology laboratory education)

   Thueck, Suzanne; Ettinger, Audrey J. (Ed.)

   Many undergraduate students understand that model organisms are important for understanding how biology works, but may not make the connection that animal models such as Drosophila melanogaster can be used to understand such human conditions as Alcohol Use Disorder (AUD) and Fetal Alcohol Syndrome (FAS). To address this knowledge gap, we introduced an inquiry-based laboratory module in which students perform hands-on Ethanol Behavior Mobility Assays (EMBAs) using flies with either different Alcohol Dehydrogenase (ADH) alleles or different developmental exposure to ethanol. The lab module contains a bioinformatic component for students to explore the evolutionary conservation of the ADH gene between flies and humans. The implementation of this exercise in a sophomore/junior-level Genetics course led to a high level of student satisfaction and a more integrated view of the role of model organisms in studying AUD and FAS. Funding acknowledgements: ABLE Roberta Williams Laboratory Teaching Initiative Grant and NSF HBCU-UP TIP Grant # 1912188. 

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2. Don’t Drink and Fly: Alcohol Resistance Behavior in Drosophila Informs us about Genes that Contribute to Alcoholism

   Echeverria, Jennifer S.; Key, S. Catherine (April 2022, Advances in biology laboratory education)

   Thuecks, Suzanne; Ettinger, Audrey J. (Ed.)

   Many undergraduate students understand that model organisms are important for understanding how biology works, but may not make the connection that animal models such as Drosophila melanogaster can be used to understand such human conditions as Alcohol Use Disorder (AUD) and Fetal Alcohol Syndrome (FAS). To address this knowledge gap, we introduced an inquiry-based laboratory module in which students perform hands-on Ethanol Behavior Mobility Assays (EMBAs) using flies with either different Alcohol Dehydrogenase (ADH) alleles or different developmental exposure to ethanol. The lab module contains a bioinformatic component for students to explore the evolutionary conservation of the ADH gene between flies and humans. The implementation of this exercise in a sophomore/junior-level Genetics course led to a high level of student satisfaction and a more integrated view of the role of model organisms in studying AUD and FAS. Funding acknowledgements: ABLE Roberta Williams Laboratory Teaching Initiative Grant and NSF HBCU-UP TIP Grant # 1912188. 

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3. Knockout of a secondary alcohol dehydrogenase gene in Nocardia cholesterolicum NRRL 5767 by CRISPR-Cas9 technology

   Huang, J.K.; Samassekou, K.; Seiver, J.; McClenahan, S.; Holt, S.; Wen, L. (October 2019, 2019 ACS Midwest Regional Meeting)

   In literature, Nocardia cholesterolicum NRRL 5767 (NC NRRL5767) is well-known for its ability to transform ~95% of added oleic acid, an abundant agricultural commodity, to value-added product of 10-hydroxystearic acid (10-HSA). A small amount of unwanted 10-ketostearic acid (10-KSA) was also produced. This microbe also transforms ~80% of added linoleic acid to 10-hydroxy-12(Z)-octadecenoic acid (10-OH-12-OD) (an isomer of ricinoleic acid) with minor 10-oxo-12(Z)-octadecenoic acid (10-oxo-12-OD). The conversion of oleic acid to 10-HSA and then to 10-KSA (or linoleic acid to 10-OH-12-OD and then to 10-oxo-12-OD) is catalyzed by oleate hydratase and secondary alcohol dehydrogenase (2o-ADH), respectively. The objective of this project was to knockout the 2o-ADH gene in NC NRRL5767 so that the sole biotransformation product from oleic acid would be 10-HSA. Here, we report construction of CRISPR/Cas9/sgRNA chimeric plasmid that specifically target 5’ coding region of the 2o-ADH gene by Golden Gate Assembly. The construct was confirmed by DNA sequencing and transformed into NC NRRL 5767 via electroporation. The transformants were selected by apramycin resistance and screened for the presence of the target insert (crRNA) by PCR. The ability of the selected transformants to transform oleic acid to 10-HSA was screened by TLC and further confirmed by GC-MS. Our results showed that two of the transformants produced only 10-HSA with no detectable 10-KSA from oleic acid suggesting successful knockout of the 2o-ADH gene. Final confirmation came from the isolation of genomic DNA from these two transformants and the wild type NC NRRL5767 (used as DNA template) and using 17 primers (locate at different positions along the 2o-ADH gene and the 5’ upstream of this gene) for PCR. To our best knowledge, this is the first report to knockout the target gene in Nocardia species by CRISPR-Cas9 technology. 

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4. Supercharging Prions via Amyloid‐Selective Lysine Acetylation

   https://doi.org/10.1002/anie.202103548  

   Baumer, Katelyn M.; Cook, Christopher D.; Zahler, Collin T.; Beard, Alexandra A.; Chen, Zhijuan; Koone, Jordan C.; Dashnaw, Chad M.; Villacob, Raul A.; Solouki, Touradj; Wood, John L.; et al (May 2021, Angewandte Chemie International Edition)

   Abstract Repulsive electrostatic forces between prion‐like proteins are a barrier against aggregation. In neuropharmacology, however, a prion's net charge (Z) is not a targeted parameter. Compounds that selectively boost prionZremain unreported. Here, we synthesized compounds that amplified the negative charge of misfolded superoxide dismutase‐1 (SOD1) by acetylating lysine‐NH₃⁺in amyloid‐SOD1, without acetylating native‐SOD1. Compounds resembled a “ball and chain” mace: a rigid amyloid‐binding “handle” (benzothiazole, stilbene, or styrylpyridine); an aryl ester “ball”; and a triethylene glycol chain connecting ball to handle. At stoichiometric excess, compounds acetylated up to 9 of 11 lysine per misfolded subunit (ΔZ_fibril=−8100 per 10³subunits). Acetylated amyloid‐SOD1 seeded aggregation more slowly than unacetylated amyloid‐SOD1 in vitro and organotypic spinal cord (these effects were partially due to compound binding). Compounds exhibited reactivity with other amyloid and non‐amyloid proteins (e.g., fibrillar α‐synuclein was peracetylated; serum albumin was partially acetylated; carbonic anhydrase was largely unacetylated). 

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5. Real-time monitoring of dynamic isomer populations with CI-SLIM IMS-MS

   https://doi.org/10.5194/egusphere-egu24-6074  

   Riva, M; Gerber, S; Claflin, M; Mettke, P; Frauenheim, M; Rice, R; Gold, A; Surratt, J; Yatsyna, V; Graf, S; et al (April 2024, EGU24)

   Ion mobility spectrometry (IMS) using Structures for Lossless Ion Manipulations (SLIM) is an emerging powerful tool for rapid isomer separations. This technology offers high mobility resolution due to prolonged ion mobility path lengths that are achieved on a small form factor separation device. In this work, we interface SLIM IMS separation with a chemical ionization source, which allows one to sample from the gas- and particle-phases directly. As such, one can monitor dynamic isomer populations in ambient air in real-time without prior sample preparation. This technology opens the door to new possibilities in atmospheric chemistry where isomer distribution is expected to play a key role in gas phase processes and in the formation of organic aerosols. The CI-IMS-TOF instrument built by TOFWERK produces ions via a two-step chemical ionization process, which involves 1) producing reagent ions and 2) ionizing neutral analyte molecules via the reagent ions through either charge transfer or adduct formation. Once generated, these secondary ions travel into the SLIM IMS region, where a series of DC- and AC-electrodes on printed circuit boards create a traveling wave driving force. As the ions travel through the helium buffer gas, they separate based on their rotationally averaged collision cross-sections. In the present work, various experiments were performed using an aerosol flow tube reactor and an atmospheric simulation chamber to recreate atmospheric conditions. Gas-phase oxidation of isoprene was used to explore the capabilities of the CI-SLIM IMS-MS under atmospheric relevant conditions. Firstly, the most important oxidation products produced from the OH-oxidation of isoprene were used including methacrolein, methyl vinyl ketone, isoprene epoxy diols (IEPOX), isoprene hydroxy hydroperoxide (1,2 and 4,3-ISOPOOH), and other C5H10O3 reactive uptake products as a single component or as a mixture to first evaluate the capabilities of the CI-SLIM IMS-MS at resolving the different isomers. Secondly, the reactive uptake of IEPOX onto acidic particles and OH-initiated oxidation (low and high NO regimes) of isoprene were studied to characterize the dynamic of the isomers generated within the simulation chamber under various environmental conditions. Results will be presented to demonstrate the capabilities of the newly developed CI-SLIM IMS-MS at resolving isomers in real time. How to cite: Riva, M., Gerber, S., Claflin, M., Mettke, P., Frauenheim, M., Rice, R., Gold, A., Surratt, J., Yatsyna, V., Graf, S., Canagaratna, M., Herrmann, H., Rohner, U., Kamrath, M., and Lopez-Hilfiker, F.: Real-time monitoring of dynamic isomer populations with CI-SLIM IMS-MS, EGU General Assembly 2024, Vienna, Austria, 14–19 Apr 2024, EGU24-6074, https://doi.org/10.5194/egusphere-egu24-6074, 2024. 

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