@article{scheidemantle_duan_lodge_cummings_hilovsky_pham_wang_kennedy_liu_2024, title={Data-dependent and -independent acquisition lipidomics analysis reveals the tissue-dependent effect of metformin on lipid metabolism}, volume={20}, ISSN={["1573-3890"]}, DOI={10.1007/s11306-024-02113-2}, abstractNote={Despite the well-recognized health benefits, the mechanisms and site of action of metformin remains elusive. Metformin-induced global lipidomic changes in plasma of animal models and human subjects have been reported. However, there is a lack of systemic evaluation of metformin-induced lipidomic changes in different tissues. Metformin uptake requires active transporters such as organic cation transporters (OCTs), and hence, it is anticipated that metformin actions are tissue-dependent. In this study, we aim to characterize metformin effects in non-diabetic male mice with a special focus on lipidomics analysis. The findings from this study will help us to better understand the cell-autonomous (direct actions in target cells) or non-cell-autonomous (indirect actions in target cells) mechanisms of metformin and provide insights into the development of more potent yet safe drugs targeting a particular organ instead of systemic metabolism for metabolic regulations without major side effects. To characterize metformin-induced lipidomic alterations in different tissues of non-diabetic male mice and further identify lipids affected by metformin through cell-autonomous or systemic mechanisms based on the correlation between lipid alterations in tissues and the corresponding in-tissue metformin concentrations. A dual extraction method involving 80% methanol followed by MTBE (methyl tert-butyl ether) extraction enables the analysis of free fatty acids, polar metabolites, and lipids. Extracts from tissues and plasma of male mice treated with or without metformin in drinking water for 12 days were analyzed using HILIC chromatography coupled to Q Exactive Plus mass spectrometer or reversed-phase liquid chromatography coupled to MS/MS scan workflow (hybrid mode) on LC-Orbitrap Exploris 480 mass spectrometer using biologically relevant lipids-containing inclusion list for data-independent acquisition (DIA), named as BRI-DIA workflow followed by data-dependent acquisition (DDA), to maximum the coverage of lipids and minimize the negative effect of stochasticity of precursor selection on experimental consistency and reproducibility. Lipidomics analysis of 6 mouse tissues and plasma allowed a systemic evaluation of lipidomic changes induced by metformin in different tissues. We observed that (1) the degrees of lipidomic changes induced by metformin treatment overly correlated with tissue concentrations of metformin; (2) the impact on lysophosphatidylcholine (lysoPC) and cardiolipins was positively correlated with tissue concentrations of metformin, while neutral lipids such as triglycerides did not correlate with the corresponding tissue metformin concentrations; (3) increase of intestinal tricarboxylic acid (TCA) cycle intermediates after metformin treatment. The data collected in this study from non-diabetic mice with 12-day metformin treatment suggest that the overall metabolic effect of metformin is positively correlated with tissue concentrations and the effect on individual lipid subclass is via both cell-autonomous mechanisms (cardiolipins and lysoPC) and non-cell-autonomous mechanisms (triglycerides).}, number={3}, journal={METABOLOMICS}, author={Scheidemantle, Grace and Duan, Likun and Lodge, Mareca and Cummings, Magdalina J. and Hilovsky, Dalton and Pham, Eva and Wang, Xiaoqiu and Kennedy, Arion and Liu, Xiaojing}, year={2024}, month={May} }
 @misc{hilovsky_hartsell_young_liu_2024, title={Stable Isotope Tracing Analysis in Cancer Research: Advancements and Challenges in Identifying Dysregulated Cancer Metabolism and Treatment Strategies}, volume={14}, ISSN={["2218-1989"]}, DOI={10.3390/metabo14060318}, abstractNote={Metabolic reprogramming is a hallmark of cancer, driving the development of therapies targeting cancer metabolism. Stable isotope tracing has emerged as a widely adopted tool for monitoring cancer metabolism both in vitro and in vivo. Advances in instrumentation and the development of new tracers, metabolite databases, and data analysis tools have expanded the scope of cancer metabolism studies across these scales. In this review, we explore the latest advancements in metabolic analysis, spanning from experimental design in stable isotope-labeling metabolomics to sophisticated data analysis techniques. We highlight successful applications in cancer research, particularly focusing on ongoing clinical trials utilizing stable isotope tracing to characterize disease progression, treatment responses, and potential mechanisms of resistance to anticancer therapies. Furthermore, we outline key challenges and discuss potential strategies to address them, aiming to enhance our understanding of the biochemical basis of cancer metabolism.}, number={6}, journal={METABOLITES}, author={Hilovsky, Dalton and Hartsell, Joshua and Young, Jamey D. and Liu, Xiaojing}, year={2024}, month={Jun} }
 @article{mcpherson_van gorder_hilovsky_jamali_keliinui_suzawa_bland_2024, title={Synchronizing Drosophila larvae with the salivary gland reporter Sgs3-GFP for discovery of phenotypes in the late third instar stage}, volume={512}, ISSN={["1095-564X"]}, DOI={10.1016/j.ydbio.2024.05.002}, abstractNote={The larval stage of the Drosophila melanogaster life cycle is characterized by rapid growth and nutrient storage that occur over three instar stages separated by molts. In the third instar, the steroid hormone ecdysone drives key developmental processes and behaviors that occur in a temporally-controlled sequence and prepare the animal to undergo metamorphosis. Accurately staging Drosophila larvae within the final third instar is critical due to the rapid developmental progress at this stage, but it is challenging because the rate of development varies widely across a population of animals even if eggs are laid within a short period of time. Moreover, many methods to stage third instar larvae are cumbersome, and inherent variability in the rate of development confounds some of these approaches. Here we demonstrate the usefulness of the Sgs3-GFP transgene, a fusion of the Salivary gland secretion 3 (Sgs3) and GFP proteins, for staging third instar larvae. Sgs3-GFP is expressed in the salivary glands in an ecdysone-dependent manner from the midpoint of the third instar, and its expression pattern changes reproducibly as larvae progress through the third instar. We show that Sgs3-GFP can easily be incorporated into experiments, that it allows collection of developmentally-equivalent individuals from a mixed population of larvae, and that its use enables precise assessment of changing levels of hormones, metabolites, and gene expression during the second half of the third instar.}, journal={DEVELOPMENTAL BIOLOGY}, author={McPherson, W. Kyle and Van Gorder, Elizabeth E. and Hilovsky, Dalton L. and Jamali, Leila A. and Keliinui, Cami N. and Suzawa, Miyuki and Bland, Michelle L.}, year={2024}, month={Aug}, pages={35–43} }
 @article{chen_chen_ruszczycky_hilovsky_hostetler_liu_zhou_chang_2024, title={Variation in Biosynthesis and Metal-Binding Properties of Isonitrile-Containing Peptides Produced by Mycobacteria versus Streptomyces}, volume={3}, ISSN={["2155-5435"]}, DOI={10.1021/acscatal.4c00645}, abstractNote={A number of bacteria are known to produce isonitrile-containing peptides (INPs) that facilitate metal transport and are important for cell survival; however, considerable structural variation is observed among INPs depending on the producing organism. While nonheme iron 2-oxoglutarate-dependent isonitrilases catalyze isonitrile formation, how the natural variation in INP structure is controlled and its implications for INP bioactivity remain open questions. Herein, total chemical synthesis is utilized with X-ray crystallographic analysis of mycobacterial isonitrilases to provide a structural model of substrate specificity that explains the longer alkyl chains observed in mycobacterial versus Streptomyces INPs. Moreover, proton NMR titration experiments demonstrate that INPs regardless of the alkyl chain length are specific for binding copper instead of zinc. These results suggest that isonitrilases may act as gatekeepers in modulating the observed biological distribution of INP structures, and this distribution may be primarily related to differing metal transport requirements among the producing strains.}, journal={ACS CATALYSIS}, author={Chen, Tzu-Yu and Chen, Jinfeng and Ruszczycky, Mark W. and Hilovsky, Dalton and Hostetler, Tyler and Liu, Xiaojing and Zhou, Jiahai and Chang, Wei-chen}, year={2024}, month={Mar} }
 @article{phan_manley_skirboll_cha_hilovsky_chang_thompson_liu_makris_2023, title={Excision of a Protein-Derived Amine for <i>p</i>-Aminobenzoate Assembly by the Self-Sacrificial Heterobimetallic Protein CADD}, volume={62}, ISSN={["1520-4995"]}, url={https://doi.org/10.1021/acs.biochem.3c00406}, DOI={10.1021/acs.biochem.3c00406}, abstractNote={Chlamydia protein associating with death domains (CADD), the founding member of a recently discovered class of nonheme dimetal enzymes termed hemeoxygenase-like dimetaloxidases (HDOs), plays an indispensable role in pathogen survival. CADD orchestrates the biosynthesis of p-aminobenzoic acid (pABA) for integration into folate via the self-sacrificial excision of a protein-derived tyrosine (Tyr27) and several additional processing steps, the nature and timing of which have yet to be fully clarified. Nuclear magnetic resonance (NMR) and proteomics approaches reveal the source and probable timing of amine installation by a neighboring lysine (Lys152). Turnover studies using limiting O2 have identified a para-aminobenzaldehyde (pABCHO) metabolic intermediate that is formed on the path to pABA formation. The use of pABCHO and other probe substrates shows that the heterobimetallic Fe/Mn form of the enzyme is capable of oxygen insertion to generate the pABA-carboxylate.}, number={22}, journal={BIOCHEMISTRY}, author={Phan, Han N. and Manley, Olivia M. and Skirboll, Sydney S. and Cha, Lide and Hilovsky, Dalton and Chang, Wei-chen and Thompson, Peter M. and Liu, Xiaojing and Makris, Thomas M.}, year={2023}, month={Nov}, pages={3276–3282} }