bluuebunny/crossref_metadata_2025 huggingface dataset:
Allosteric Regulation of Protein Kinases Using Optogenetics
Authors: Shaaya Mark , Huyot Vincent , Zhurikhina Anastasya , Tsygankov Denis , Natarajan Viswanathan , Karginov Andrei
Published: June 2021

Activation of single protein kinase can result in a very distinct signaling output depending on the duration, location, and frequency of its activation in the cell. However, interrogation of kinase‐mediated processes remains challenging due to the paucity of tools that precisely regulate kinases in space and time. To overcome existing limitations, we developed a novel approach for light‐mediated regulation of kinases. We engineered a light‐regulated allosteric switch (LightR), a small domain based on Vivid (VVD) photoreceptor, that undergoes conformational changes upon illumination with blue light. Insertion of LightR at a specific location in the catalytic domain of Src kinase enables light‐mediated control of its activity. In the dark, engineered LightR‐Src is inactive. Illumination of cells with blue light leads to robust activation of the kinase. Activation of LightR‐Src is specific, fast, and reversible. This allows us to control duration of kinase activation as well as induce repeated cycles of kinase activation/inactivation in living cells. Moreover, the level of activation can be controlled by changing light intensity. Specific modifications of the LightR domain allow us to modulate the activation/inactivation kinetics and the level of activity of LightR‐Src, enabling additional “tuning” of the engineered kinase and its optimization for different applications. Activation of LightR‐Src in living cells induces robust cell spreading and protrusive activity, which are known Src‐induced phenotypes described in previous studies. Importantly, the morphological changes stop as soon as the kinase is inactivated, demonstrating reversibility of the manipulated signaling. Repeated activation/inactivation of LightR‐Src induces cycles of morphodynamic activity of the cells, showing the capability of this tool to mimic oscillation of kinase signaling in living cells. Localized illumination of cells induces local formation of protrusions and cell polarization towards the light, demonstrating spatial regulation of LightR‐Src. Our study also reveals that continuous local activation of LightR‐Src at the cell periphery induced local oscillations of protrusive activity with defined periodicity. Insertion of LightR domain at a homologous position in Abl kinase also enabled light‐mediated regulation of its activity. These results, and the high structural similarity among catalytic domains of kinases, suggest broad applicability of this tool for regulation of different kinases. Furthermore, this approach could be potentially employed to control the activity of other enzymes in time and space.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Utilizing an Optogenetic System for Manipulating Protein Localization in Fission Yeast
Authors: Mehdi Maahum , Goss John Woodward
Published: June 2021

The cellular function of a protein is largely dependent upon proper localization, particularly in complex protein networks that regulate stages of cell division. Many of these regulatory proteins are highly conserved and essential for cell viability. As a result, manipulating protein function in vivo often involves controlling expression levels using inducible promoters or generating point mutations to disrupt function, both of which have limited temporal resolution for observing phenotypes. An emerging technology to manipulate the function of essential proteins involves the field of optogenetics. This approach utilizes light‐inducible dimerization modules to enable rapid, reversible spatial and temporal manipulation of in vivo protein localization. Transiently altering protein localization to temporarily disrupt protein function can provide insight into how that protein contributes to a cellular pathway.

In this study, we utilize molecular cloning to adapt a CRY2‐CIBN optogenetic light‐inducible dimerization system into common fission yeast expression vectors. These vectors enable us to incorporate genetically encoded cryptochrome fusion tags onto genes of interest in their native locus of the fission yeast genome using homologous recombination. We can then induce dimerization of the CRY2‐CIBN domains in the resulting tagged‐proteins in a rapid and reversible manner using 488nm light, which alters their spatio‐temporal localization in vivo allowing for direct observation of the downstream effects on cellular processes in real time. We utilize this system to manipulate localization of an essential regulatory kinase involved in cytokinesis as a method to further characterize its contribution to successful cell division. This optogenetic system is a valuable tool for studying the fission yeast cell cycle that enables researchers to better understand the role proteins play in regulation of this essential, highly conserved process.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Convergence of Wnt, Growth Factor and Trimeric G‐protein Signals on the Signaling Scaffold Daple
Authors: Ear Jason , Aznar Nicolas , Dunkel Ying , Sun Nina , Satterfield Kendall , He Fang , Lopez‐Sanchez Inmaculada , Ghassemian Majid , Sahoo Debashis , Kufareva Irina , Ghosh Pradipta
Published: June 2021

Aberrant signal transduction is a hallmark of cancer biology. Pathways commonly deregulated include G‐protein signaling, Wnt signaling, and Receptor Tyrosine kinase (RTK) signaling. While typically studied individually, a growing amount of evidence indicate that these pathways are intertwined. Recent studies have demonstrated that Daple (CCDC88A) is multimodular domain containing protein that can tether various pathways together, and plays a major role in colorectal cancer (CRCs) progression. Daple is a Dishevelled (Dvl)‐ and Frizzled (FZD) ‐binding protein and a guanine‐nucleotide exchange factor (GEF) for the trimeric G protein, Gαi. Daple enhances β‐catenin‐independent (non‐canonical) Wnt signaling through its ability to activate the pathway downstream of FZD7 upon Wnt5a stimulation. Here we identified that Daple is a substrate of multiple RTKs and non‐RTKs, and hence, can serve as a point of convergence for all three signaling cascades. We show that phosphorylation by both RTKs and non‐RTKs near the atypical PDZ‐binding motif (PBM) on Daple dissociated the Daple:Dvl complexes and augmented the ability of Daple to bind and activate Gαi; which potentiated β‐catenin‐independent Wnt signals and triggered epithelial‐mesenchymal transition (EMT) in a manner similar to that triggered by Wnt5a/FZD7. Although Daple acts as a tumor suppressor in the healthy colon, concurrent upregulation of Daple and the prototype RTK, epidermal growth factor receptor (EGFR), in colorectal tumors from patients was associated with poor prognosis. We conclude that Daple‐dependent activation of Gαi and enhancement of β‐catenin‐independent Wnt signals is not just triggered by Wnt5a/FZD7 to suppress tumorigenesis, but also is hijacked by growth factor‐RTKs to stoke tumor progression. Thus, this work defines a previously unknown crosstalk paradigm amongst growth factor RTKs, trimeric G‐proteins, and Wnt/FZD in cancer biology.

Support or Funding Information

This work was supported by NIH grants CA100768, CA160911 and DK099226 (to P.G). P.G. was also supported by the American Cancer Society (ACS‐IRG 70‐002) and by the UC San Diego Moores Cancer Center. J.E is supported by an NIH‐institutional training grant (2T32CA067754‐21A1). I.K. was supported by NIH grants GM071872, AI118985, and GM117424. I.L‐S was supported by a fellowship from the American Heart Association (AHA #14POST20050025). F.H was supported by the State Scholarship Fund of China Scholarship Council (No.201208510048) and fund from West China Hospital, Sichuan University, PR China, during her tenure as a visiting professor to UCSD.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Chronic Beta2‐Adrenergic Receptor Stimulation Improves Whole‐Body Glucose Homeostasis through Skeletal Muscle Metabolic Reprogramming
Authors: Meister Jaroslawna , Bone Derek , Knudsen Jonas Roland , Lee Regina , Cohen Amanda , Kleinert Maximilian , Berdeaux Rebecca , Richter Erik , Wess Jürgen
Published: June 2021

Type 2 diabetes (T2D) has emerged as a major health problem worldwide. Since skeletal muscle (SKM) is the major site of insulin‐stimulated glucose disposal in the body, SKM insulin resistance plays a crucial role in the pathogenesis of this disorder. Drugs that could enhance or mimic insulin action in SKM hold great therapeutic potential for the treatment of obesity and T2D.

Previous work carried out with cultured SKM cells indicates that activation of SKM β2‐adrenergic receptors (β2‐ARs) can promote glucose uptake. In rodent models of T2D, chronic administration of clenbuterol, a selective β2‐AR agonist, greatly improves glucose tolerance. However, the mechanism and in vivo tissue(s) responsible for the actions of clenbuterol are still unclear. This study was designed to test the hypothesis that the beneficial metabolic effects of clenbuterol observed in vivo are mediated by SKM β2‐ARs. For this purpose, we generated mice that lack β2‐ARs or the Gαs subunit selectively in SKM (SKM‐β2‐AR‐KO and SKM‐Gs‐KO mice, respectively). We also generated transgenic mice expressing a Gs‐DREADD selectively in SKM (SKM‐Gs‐DREADD mice).

As expected, chronic administration of clenbuterol via drinking water improved glucose tolerance in wild‐type (WT) mice without affecting glucose‐stimulated insulin secretion or insulin tolerance. The SKM selectivity of this chronic clenbuterol effect was confirmed using SKM‐β2‐KO, SKM‐Gs‐KO, and SKM‐Gs‐DREADD mice. RNA‐seq and functional data indicated increases in SKM glucose accumulation, glycolysis, and glycogen synthesis following chronic treatment, suggesting a switch of SKM metabolism towards enhanced glucose utilization.

Multiple components of known, but also less studied metabolic pathways seem to be simultaneously involved in mediating the beneficial metabolic effects of chronic β2‐AR stimulation.

Our study represents a striking example of the complexity of β2‐AR signaling in vivo. Further analysis of β2‐AR signaling pathways in SKM could help identify potential drug targets for new T2D treatments.

Support or Funding Information

This research was funded by the Intramural Research Program of the NIDDK.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of a phospholipase C‐like protein (TbPI‐PLC2) from Trypanosoma brucei
Authors: Negrao Nuria Waddington , King‐Keller Sharon , Huang Guozhong , Docampo Roberto
Published: June 2021

Trypanosoma brucei is the causative agent of African Trypanosomiasis, a deadly disease affecting humans and cattle. There are very few drugs to treat the disease and evidence of mounting resistance raises the need for new drug development. The inositol 1,4,5 triphosphate/diacylglycerol (IP3/DAG) pathway regulates important processes in many organisms. T. brucei has an active IP3 receptor, localized to the acidocalcisome, that is essential for infection in mice. In previous work (King‐Keller et al., Eukaryot. Cell 14:486–494, 2015) we characterized a phosphoinositide phospholipase C (TbPI‐PLC1, Tb927.11.5970) from T. brucei that contains a domain organization characteristic of PI‐PLCs, such as X and Y catalytic domains, an EF‐hand calcium‐binding motif, and a C2 domain, but lacks a pleckstrin homology (PH) domain. In addition, TbPI‐PLC1 contains an N‐terminal myristoylation consensus sequence only found in trypanosomatid PI‐PLCs. Here we report the presence of a second PI‐PLC‐like protein (TbPI‐PLC2, Tb927.6.2090) that is very similar to TbPI‐PLC1 but lacks the Y catalytic domain and the C2 domain and possesses instead a PDZ domain. Recombinant TbPI‐PLC2 hydrolyzes neither phosphatidylinositol (PI) nor phosphatidylinositol 4,5‐bisphosphate (PIP2), and does not modulate TbPI‐PLC1 activity. However, knockdown of TbPI‐PLC2 expression alone or together with downregulation of TbPI‐PLC1 expression by RNAi resulted in growth inhibition. This is in contrast with the lack of effect of downregulation of expression of TbPI‐PLC1 alone. TbPI‐PLC2 has a plasma membrane and intracellular localization and it might be involved in IP3 binding as has been reported for the phospholipase C‐related catalytically inactive protein 1 (PRIP‐1) of mammalian cells (Uji et al., Life Sci 72:443–453, 2002). The PDZ domain could be involved in this binding and this is being investigated.

Support or Funding Information

NIH grant # AI077538

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mitochondrial adenylate kinase 4 regulates dual AMPK and mTORC1 activation
Authors: Gonzales Carlos , Nguyen Anh Phuong , Abdullah Amelia , Meraz Ryan , Castellanos Ernesto , Nittayo Benjamin , Lanning Nathan
Published: June 2021

In order to maintain cellular homeostasis, mitochondria integrate bioenergetic, metabolic, and redox cues. Signals both converge on and emanate from mitochondria to respond to varied cellular stressors. Therefore, mitochondrial proteins must play critical roles in maintaining cellular homeostasis. In a search for mitochondrial proteins that impact cellular bioenergetics, we identified the adenylate kinase, AK4, as the most potent regulator of ATP levels out of >1,000 mitochondrial proteins. We also found AK4 expression to significantly impact AMKP and mTORC1 activation status as well as cells' ability to respond to energetic stressors. Suppressing AK4 expression increases steady‐state cellular ATP levels by up to 30%, alters cellular respiration metabolite pools, and results in concomitant activation of both AMPK and mTORC1 signaling. Together, these effects of suppressed AK4 expression allow cells to thrive in the context of nutrient deprivation‐induced stress. The profound effects of AK4 expression on bioenergetics, survival under stress, and dual activation of the AMPK and mTORC1 energy sensing pathways position AK4 as a potential master regulator of mitochondria‐dependent cellular homeostasis.

Support or Funding Information

NIH 1R15GM123382 CSUPERB New Investigator Grant

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Using embryonic zebrafish to evaluate the effects of exposure to novel bisphenol analogues
Authors: Bruton Laura N. , Zhou Xinrui , Timmons Shannon C. , Morrissette Jeffrey M.
Published: June 2021

Bisphenol A (BPA) is a well‐established endocrine disruptor that has been shown to induce developmental and behavioral changes in both human and animal models. Gestational exposure of embryonic zebrafish to low nanomolar doses of bisphenol A (or S) altered hypothalamic neurogenesis during development and induced hyperactive swimming behaviors. There are many potential bisphenol analogues, but little is known about their toxicity or ability to act as endocrine disruptors. We have synthesized a small library of novel bisphenol analogues and are testing their toxicity in a zebrafish larval toxicity assay. We are also using an ELISA‐based estrogen binding assay to assess the analogues' affinity for estrogen receptors. We will be exposing zebrafish embryos to ranging concentrations of bisphenol analogues during time periods of 10–16 hpf (hours post‐fertilization), 16–24 hpf, and 24–36 hpf. At 5 dpf (days post‐fertilization) zebrafish locomotor activity in control and analogue exposed fish will be assessed using video‐tracking software quantifying bursts of hyperactivity. Results from these experiments will be discussed.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization and expression of Rhizophora mangle RmMT2, a type 2 metallothionein gene, in response to heavy metal exposure
Authors: Snyder Ian D. , Acacio Samantha , Trabing Rebecca M. , Shor Audrey C.
Published: June 2021

Mangrove forests are important to the coastal ecosystems for their preservation of coastline landmass and offering habitats for marine organisms. Mangrove ecosystems also play a significant role in phytoremediation, or the uptake of toxic metals from the surrounding environment. This process is likely due to the expression of proteins known as metallothionines, a class of cysteine rich proteins whose sulfhydryl groups chelate and deactivate toxic metal ions. This study sought to better understand the expression and regulation of RmMT2A as a function of exposure to heavy metals. It was hypothesized that Rhizophora mangle, the red mangrove, possess the MT2A protein to combat abiotic stress, and its regulation is directly correlated with increasing concentrations of toxic metals in the surrounding environment. To investigate these hypotheses, R. mangle seedlings were exposed to increasing concentrations of toxic metals (CdCl2 and Pb(C2H3O2)2) for 3, 7, and 12 days, at concentrations 0 μM, 2 μM, 10 μM, 20 μM and 40 μM. Leaf samples were harvested, and total mRNA was extracted. Qualitative PCR was performed confirming the presence and expression of the RmMT2A gene and real‐time quantitative PCR assessed expression as a function of increased heavy metal exposure. Subsequent analysis will determine the metal‐binding properties of the GST‐RmMT2A fusion protein as well as the stress resistance properties of the gene. Metal tolerance was measured in Escherichia coli BL21 (DE3) transformed with RmMT2A. These data may suggest that the RmMT2A gene plays an important role in the efficiency by which R. mangle play as biogeochemical barriers to heavy metals.

Support or Funding Information

This research was funded by the Department of Mathematics and Science, Saint Leo University.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Relaxation of bladder smooth muscle by flavonoids from Sophora flavescens via direct activation of the large‐conductance calcium‐activated potassium channel.
Authors: Han Jisun , Lee Sojung , Lee Narasaem , Jo Hee‐Ji , Park Chul‐seung
Published: June 2021

The large‐conductance calcium‐activated potassium channel (BKCa channel) plays critical roles in smooth muscle relaxation. In urinary bladder, BKCa channel activity underlies the maintenance of the resting membrane potential and the phasic relaxation. We recently reported kurarinone, a flavonoid found in S. flavescens, as a potent activator of the BKCa channel and thus a strong relaxant of bladder smooth muscle (Lee et al., Mol. Pharmacol., 2016). Since the potentiating effects of flavonoids with similar chemical structures vary significantly, we further screen the flavonoids of S. flavescens. We newly identified three more flavonoids from S. flavescens as potent activator of BKCa channel. In particular, kuraridinol, a prenylated chalcone, strongly activates BKCa from the extracellular side by stabilizing its open conformation. This compound markedly affects the gating kinetics of the channel by stimulating faster opening and slower closing. In order to identify new potent activators of BKCa channel from other natural sources, we are currently screening a flavonoid library using a cell‐based fluorescence assay. Thus, the flavonoids can be an excellent natural source of BKCa channel activators that may be of therapeutic benefit for targeting channel activity.

Support or Funding Information

Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries(IPET) through Agri‐Bio industry Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs(MAFRA)(2017‐352)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An Astrocyte Model Looking Toward Understanding Neurodeneration
Authors: Corcoran Ellen , Hemkin Sheryl
Published: June 2021

Astrocytes, the most numerous subtype of glial cell in the central nervous system, have been previously identified as key regulators of neural systems due to their ability to maintain neurochemical homeostasis in the central nervous system. There is growing evidence that astrocytes not only have a supportive role in regulation, but are active partners in the information transfer processes within the brain. A healthy astrocyte can, under unknown circumstances, be transformed to a pathogenic or “reactive” astrocyte. This astrocyte form has been linked to neurodegeneration and enigmatic diseases such as Alzheimer's Disease, Parkinson's Disease, and Amyotrophic Lateral Sclerosis. It is postulated that intercellular signals shuttled through the astrocyte may be at least partially responsible for this neural degeneration, and extracellular stimuli to the astrocyte, such as by glutamate, ATP or TNFα, may cause changes in the oscillatory behavior of the cytosolic calcium and thus cause the downstream signaling. This work proposes a new model to help gain insight as to neuroinflammatory signals alter intracellular calcium dynamics, disrupt astrocyte metabolism, and ultimately facilitate neurodegeneration.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biochemical Characterization of the Drosophila Methionine Sulfoxide Reductase A
Authors: Tarafdar Sreya , Kim Geumsoo , Rusan Nasser , Levine Rodney
Published: June 2021

Stereospecific reduction of S‐methionine sulfoxide to methionine is enzymatically catalyzed by methionine sulfoxide reductase A (MsrA) which plays an important role in defense against oxidative stress. Recent work from our laboratory has shown that mammalian MsrA is not only a reductase, but also a methionine oxidase, capable of stereospecifically catalyzing both the oxidation of Met in proteins to S‐methionine sulfoxide (S‐MetO) as well as reduction of S‐MetO back to Met. We reported that mammalian MsrA stereospecifically and selectively oxidizes Met77 in calcium‐bound calmodulin and can fully reduce it as well. The control mechanism that prevents futile cycling is hypothesized to be through interaction with a postulated regulatory protein. Thus, cyclic oxidation and reduction of methionines in proteins by MsrA could function as a redox‐based mechanism of cellular regulation. Our aim in this study was to elucidate the physiological significance of methionine sulfoxide reductase A mediated reversible oxidation of calmodulin Met77 in Drosophila. However, we found that Drosophila MsrA, unlike its mammalian counterpart, is not a methionine oxidase. This led us to explore the mechanistic details of the enzyme. Using a double alkylation approach with HPLC‐mass spectrometric sequencing, we found that the active site cysteine residue in Drosophila MsrA becomes locked in a disulfide bond with the terminal cysteine residue of the protein and thus cannot mediate oxidation. We also created a mutant Drosophila MsrA lacking the two C‐terminal cysteine residues, reasoning that the lack of the resolving cysteines may allow the active site cysteine to mediate both oxidation and reduction of its substrates. However, this mutant Drosophila MsrA also lacked oxidase activity, despite not being able to form a disulfide bond with the active site cysteine. We are currently determining the solution structure of the Drosophila MsrA to understand the structural basis for its difference in catalytic capability from the mammalian enzyme.

Support or Funding Information

This research was supported by the Intramural Research Program of the NIH, NHLBI

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of Batrachochytrium dendrobatidis inhibiting isolates via challenge against salamander skin microbiota.
Authors: Erdman Fantasia Marie , Hill Aubree , Russell Gabrielle , Griffith John , Wharton Megan , Walker Donald
Published: June 2021

Rapid declines in global amphibian populations can be partially attributed to chytridiomycosis caused by the fungus Batrachochytrium dendrobatidis (Bd). Due to how quickly it has spread worldwide, and the number of amphibians affected, this fungus is of increasing concern in both the herpetological and mycological communities. We characterized the cutaneous microbiome of Appalachian salamanders, which persist in the presence of the fungus despite high rates of infection in co‐occurring frog populations. The cutaneous microbiome of vertebrates is known as the first line of defense against invading pathogens. We hypothesized that these salamanders might have resident bacteria on their skin with the ability to inhibit Bd growth. Our objectives were to 1) isolate resident bacteria from salamander skin swabs, 2) challenge these isolates against Bd, and 3) genotype all inhibitory isolates. During the summer of 2016, 82 swabs were collected from three salamander genera including Desmognathus (n=36), Eurycea (n=20), and Plethodon (n=26). To isolate cutaneous bacteria, swabs were streaked on Reasoner's 2A agar plates that we checked daily for two weeks. Colonies with unique morphology were isolated onto secondary plates until pure cultures were obtained. To challenge the isolates against Bd, zoospores were harvested from cultures that had been aged 5–7 days, counted with a hemocytometer, and diluted to approximately 1,000,000 cells/milliliter (mL). Tryptone agar plates were then flooded with 1.0 mL of standardized zoospore solution and dried for 45 minutes in a laminar flow hood. Bacterial isolates were streaked onto each plate in co‐culture with Bd. Plates were incubated at room temperature for four days, examined for noticeable zones of inhibition (ZOI), measured in millimeters. Isolates that produced a measurable ZOI were photographed and will be screened in further testing against Bd. We used direct colony PCR and Sanger sequencing to identify all inhibitory isolates. Presently, 63 of the 472 acquired isolates have been challenged, 16 of which produced a ZOI. The challenge phase is ongoing and we anticipate discovery of additional candidate probiotic species. In addition, we plan to test for possible synergistic inhibition by challenging candidates in polyculture.

Support or Funding Information

Great Smokey Mountains Conservation Association and Tennessee Herpetological Society

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Metabolism of Fluoropyrimidine Anticancer Drugs by the Human Gut Microbiome
Authors: Spanogiannopoulos Peter , Patteron Andrew D. , Turnbaugh Peter J.
Published: June 2021

The treatment of cancer is notoriously difficult due to the high toxicity of the current medications and unpredictable variations between patients. Genetic polymorphisms within the human genome are certainly important, but they often fail to explain most of the observed variation in treatment outcomes in patients. We propose that the human gut microbiome is a major contributor to the inter‐individual variation in anticancer drug response and hypothesize that this microbial reservoir harbors determinants of resistance towards anticancer drugs, including genes responsible for drug metabolism. We are focusing on the oral cancer drug capecitabine (CAP), a prodrug of 5‐fluorouracil (5FU), due to its widespread administration for colorectal and other cancers, the unexplained differences in drug efficacy and toxicity, and studies suggesting that the mechanism of action and metabolism of this class of compounds (fluoropyrimidines) is conserved in bacteria. Here, we assessed the reciprocal interactions between CAP and 5FU and the human gut microbiome. Ex vivo incubations of human gut microbial communities demonstrated that CAP and 5FU impacts the cellular integrity and growth of gut bacteria. We determined the minimum inhibitory concentration of 5FU versus a diverse collection of 50 individual human gut bacterial type strains. Interestingly, gut bacteria showed a remarkable high variation of sensitivity towards 5FU, ranging over four orders of magnitude. Using a bioassay, we screened 5FU‐resistant gut bacteria for their ability to inactivate 5FU. Several gut bacteria, including Escherichia coli, were capable of 5FU inactivation. Mass spectrometry analysis showed that E. coli converts 5FU to the less active metabolite dihydrofluorouracil (DHFU). This activity is dependent on the preTA operon, which encodes the enzyme dihydropyrimidine dehydrogenase (DPD), as a preTA‐knockout strain of E. coli fails to metabolize 5FU. Orthologs of DPD are found in other gut bacterial species and their ability to metabolize 5FU is being investigated. Such microbial enzymes from the gut microbiome may influence the pharmacokinetics and toxicity of anticancer drugs. Additional studies in animal models and human cohorts are needed to evaluate the clinical relevance of these findings and their utility in improving clinical medicine.

Support or Funding Information

Peter Spanogiannopoulos is supported by a Canadian Institutes of Health Research Postdoctoral Fellowship.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Twin Talk Series: A novel method to foster peer‐to‐peer pedagogical training
Authors: Brown Ashley K. , Martínez‐Gálvez Gabriel , Coffman Kirsten E. , Matchett William E. , Horazdovsky Bruce F.
Published: June 2021

Mayo Clinic annually trains over 1000 predoctoral students and postdoctoral research fellows in medicine and biomedical sciences. However, training opportunities are limited for those interested in teaching and mentoring methodologies. Therefore, Mayo Clinic predoctoral students sought to provide trainee‐led learning opportunities for fellow trainees. A survey was sent to all Mayo Clinic predoctoral and postdoctoral trainees to determine preferences for pedagogy education formats. In response to survey results, the Twin Talk Series was developed. A Twin Talk is a set of one‐hour talks comprised of a Take‐in session and one or more Try‐out session(s). At the Take‐in session, a content expert discusses a pedagogical technique, and at the following Try‐out session(s), trainee volunteers practice the technique among themselves. Eight Twin Talks have been organized to date on several teaching techniques and pedagogical development topics. The impact of each Twin Talk, including understanding, format, and interest level, was evaluated through survey tools. Survey results suggested trainees favored in‐person pedagogy expert presenters during the Take‐in session over video conferences. Furthermore, modeling of the teaching technique by the expert presenter during the Take‐in session was well‐received. However, participation in the follow‐up Try‐out practice session was limited. Overall, the Twin Talk Series is a promising method for trainee‐led, peer‐to‐peer pedagogy education, particularly at institutions with limited access to pedagogical resources. The Twin Talk Series will continue to be refined to increase trainee participation, particularly in the follow‐up Try‐out sessions, and to provide relevant opportunities for trainees to practice teaching.

Support or Funding Information

Mayo Foundation of Medical Education and Research

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Elucidating the Role of a Putative Monooxygenase in the Catabolism of Nicotinic Acid and Nicotine by Bacillus niacini
Authors: Fine Sydney , Snider Mark J.
Published: June 2021

N‐Heteroaromatic compounds (NHACs) comprise a class of molecules that are commonly found in pharmaceuticals, personal care products, and pesticides and are known to have toxic effects on the environment. Intriguingly, certain species of soil‐dwelling bacteria have the innate ability to degrade particular NHACs. A better understanding of how these soil bacteria degrade NHACs has important implications in the development of bioremediation techniques to remove toxic compounds from the environment. This investigation focuses on developing preexisting hypotheses in regards to how Bacillus niacini, a ubiquitous species of soil bacteria, degrades nicotinic acid (NA) and nicotine, both of which are NHACs. It has been hypothesized that the non‐canonical NA degradation pathway in B. niacini involves a reaction in which 2,6‐dihydroxynicotinic acid undergoes oxidative decarboxylation to form 2,3,6‐trihydroxypyridine by a enzyme termed NicC‐like. The identity of the nicC‐like gene has yet to be established. A novel transformation technique was developed to generate a B. niacini knock out variant lacking the putative nicC gene. Though there are hypotheses regarding the identity of the nicC‐like gene and its involvement in NA catabolism, sequence analysis indicates that the nicC‐like gene is only 21% similar to the nicC gene involved in NA degradation in Pseudomonas putida. In contrast, the nicC‐like gene is 24% similar to HspB, which encodes an enzyme involved in nicotine degradation. Additionally, the structure and the associated catabolic processes of nicotine and NA are similar, suggesting that the novel nicC‐like gene in B. niacini might play a role in either nicotine or NA degradation. Preliminary HPLC evidence indicates that B. niacini does not degrade nicotine. Thus, the genes currently hypothesized to encode the genes necessary for NA catabolism do not code for nicotine degradation enzymes. Characterizing the reaction catalyzed by the putative NicC‐like enzyme and determining whether or not B. niacini catabolizes nicotine has led to an improved understanding about the genes necessary for NA degradation by this particular species of pervasive soil bacteria. Continued contributions to this field may eventually lead to the development of techniques to remove NHACs from the environment.

Support or Funding Information

Henry J. Copeland Fund for Independent Study at the College of Wooster

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Chebulic Acid against t‐BHP‐induced effect via Modulations of Nrf2 and Its Related Enzymes in HepG2 cells
Authors: Lee Kwangwon , Jung Hye‐Lim , Yang Sung‐Yong
Published: June 2021

Chebulic acid, phenolic compound isolated from the ripe fruits of Terminalia chebular Retz. shows diverse biological effects. The purpose of this study is to investigate protective effects of chebulic acid against tert‐butyl hydroperoxide (t‐BHP) induced oxidative stress and its mechanism related to Nrf2 signal pathway in HepG2 cells. Chebulic acid attenuated cell death and production of reactive oxygen species in t‐BHP‐induced HepG2 cells and increased intracellular glutathione (GSH) content and the ratio of GSH to glutathione disulfide (GSSG). Chebulic acid also upregulated the activity of heme oxygenase‐1 (HO‐1). The treatment with chebulic acid increased the translocation of Nrf2 into the nucleus and the expression of Nrf2 target genes such as γ‐glutamate cysteine ligase and HO‐1 in a dose‐dependent manner. In addition, the exposure of chebulic acid enhanced the phosphorylation of mitogen‐activated protein kinases (MAPK). The overall result is that chebulic acid has cytoprotective effect on t‐BHP‐induced hepatotoxicity in HepG2 cells through Nrf2‐mediated antioxidant enzymes and MAPK.

Support or Funding Information

This work was supported by a Grant from the Institute of Life Science and Natural Resources, Korea University.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ubiquinone is a Key Antioxidant during Long Chain Fatty Acid Metabolism in Escherichia coli
Authors: Agrawal Shashank , Jaswal Kanchan , Shiver Anthony , Balecha Himanshi , Patra Tapas , Chaba Rachna
Published: June 2021

Long chain fatty acids (LCFAs) are a rich source of energy for several bacteria including many important pathogens. However, LCFAs also induce oxidative stress. It is thus important to understand the reason for LCFA mediated oxidative stress and the strategies employed by bacteria to counteract this stress. In this study, we establish that fatty acid uptake and degradation is the reason for LCFA mediated oxidative stress in Escherichia coli. Our genetic screen in E. coli on the LCFA, oleate, and its comparison with published genome‐wide screens on multiple carbon sources reveals that among various electron transport chain (ETC) components, genes involved in the biosynthesis of ubiquinone, an electron carrier in ETC are highly required for growth in LCFAs compared to other carbon sources. Detail genetic and biochemical experiments suggest that the increased requirement of ubiquinone on oleate is to counter elevated levels of reactive oxygen species (ROS) generated by LCFA degradation. Additionally, we find that among various oxidative stress combat players in E. coli, ubiquinone is the major antioxidant and acts as the cell's first line of defense against LCFA‐induced oxidative stress. Intriguingly, we find that whereas the requirement of other ETC components is inversely correlated with the energy yield of non‐fermentable carbon sources, the requirement of ubiquinone correlates with oxidative stress. Our results thus suggest that the known electron carrier function of ubiquinone cannot solely explain the antioxidant role of ubiquinone. The mechanism by which ubiquinone combats ROS would depend on the major site of ROS formation during LCFA degradation. We suggest that FadE, a flavoenzyme involved in β‐oxidation that reduces FAD to FADH2 could be a predominant site of ROS formation during LCFA metabolism. It is likely that ubiquinone enables the rapid transfer of electrons from FadE to ETC thereby limiting ROS formation. In this direction, we are currently trying to establish the biochemical activity of FadE and its role in re‐oxidizing FADH2. Taken together, our studies on the role of ubiquinone during LCFA metabolism would provide a rationale to investigate the contribution of this key ETC component in managing oxidative stress in LCFA‐utilizing pathogenic bacteria.

Support or Funding Information

This work was supported by start‐up funds from IISER‐Mohali to Rachna Chaba and was partly funded by CSIR Govt. of India to Rachna Chaba.

Shashank Agrawal : Supported by a fellowship from IISER‐Mohali for doctoral work.

Kanchan Jaswal : Recipient of a DST‐Inspire fellowship for doctoral work.

Himanshi Balecha : Supported by a fellowship from DST‐Inspire for undergraduate studies.

Tapas Patra : Supported by a fellowship from IISER‐Mohali for postdoctoral work

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Long‐Chain Acyl‐CoA synthetase 6 deficiency reduces the omega‐3 fatty acid DHA in the brain and disrupts motor control
Authors: Ellis Jessica M. , Fernandez Regina F. , Zhao Yingwei , Counihan Jessica L. , Nomura Daniel K. , Chester Julia A.
Published: June 2021

Docosahexaenoic acid (DHA) is an omega‐3 fatty acid that is highly abundant in the brain and thought to confer neuroprotection against many diseases and disorders through its anti‐inflammatory and antioxidant actions, yet the fundamental mechanisms regulating the enrichment of DHA in brain remain enigmatic. The metabolism of fatty acids at the cellular level is controlled, in part, by a family of enzymes called Acyl‐CoA Synthetases (ACSs) which perform an energy‐requiring reaction that constitutes the first and required step for nearly every cellular lipid metabolic pathway. Of the identified ACSs, one in particular, Acsl6, appears to be brain specific and its manipulation has been shown to regulate DHA metabolism in vitro. To determine the role of Acsl6 in vivo, we generated a conditional Acsl6‐deficient mouse model (Acsl6−/−). The loss of mouse Acsl6 decreases cerebellar DHA‐containing phospholipid species by 35–72%, and increases arachidonic acid‐, an omega‐6 fatty acid, containing phospholipids 120–200%. Despite the loss of DHA in brain phospholipids, the Acsl6−/− and control inflammatory response to an acute pro‐inflammatory lipopolysaccharide intraperitoneal injection was similar. However, Acsl6−/− mice present with motor dysfunction as evidenced by impaired performance in a wire hang test and reduce startle response to acoustic stimuli. Acsl6−/− brains have reduced glutathione, increased mRNA expression of the oxidative stress‐related transcription factor Nrf2, and increased GFAP protein abundance. Together these data suggest that ACSL6 is required for enriching the brain with the neuroprotective omega‐3 fatty acid, DHA, and that the loss of Acsl6 impairs motor function, increases oxidative stress and promotes astrocyte reactivity in the brain.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MED17 is Phosphorylated at S53 by CK2 for Transcriptional Activation of Lipogenic Genes in Response to Insulin
Authors: Viscarra Jose A. , Wang Yuhui , Sul Hei Sook
Published: June 2021

De novo lipogenesis is under tight regulation by nutritional status and hormonal conditions. Many of the enzymes involved in this process, such as fatty acid synthase (FASN), are regulated at the transcriptional level by insulin signaling cascades. We previously showed that USF1 is a key transcription factor for FASN activation that, by binding to the −65 E‐box, leads to the recruitment of various other lipogenic transcription factors for insulin mediated transcriptional activation. We show here that USF1 directly interacts with the Mediator subunit MED17 at the FASN promoter for recruitment of the Mediator complex, as well as POL II and other general transcription machinery. Additionally, we show that MED17 is phosphorylated at S53 by casein kinase 2 (CK2) in the livers of fed mice or insulin‐stimulated hepatocytes, but not in the livers of fasted mice or untreated hepatocytes, and that this phosphorylation event is required for the FASN promoter activation in response to insulin. To assess whether this newly identified pathway becomes dysregulated in obesity, we next examined phosphorylation of MED17 in the liver of leptin‐deficient and insulin resistant ob/ob mice and, indeed, we detected chronic phosphorylation of S53 even in livers of fasted mice. These results demonstrate that phosphorylation of MED17 at S53 by CK2 is required for the transcriptional activation of lipogenic genes in response to insulin, and furthermore, that dysregulation of this pathway may contribute to the chronic activation of lipogenesis for insulin resistance.

Support or Funding Information

RO1DK081098 (NIH)

F32DK105671 (NIH)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Novel Multi‐domain Phosphatidylinositol Transfer Protein/Oxysterol Binding Protein Senses Specific Phosphoinositide Pools On Toxoplasma Dense Granules
Authors: Grabon Aby , Bankaitis Vytas A.
Published: June 2021

The Apicomplexan Toxoplasma gondii is a common parasite of humans and other mammals and causes severe disease in immunocompromised individuals. Apicomplexa are obligate intracellular pathogens, and their infection of host cells requires the orchestrated secretion of proteins from several highly specialized secretory organelles, the regulation of which is incompletely understood. Studies in other eukaryotic systems have established that membrane trafficking through distal stages of the secretory pathway is regulated by phosphoinositide (PIP) signaling networks and that phosphatidylinositol (PtdIns) transfer proteins (PITPs) are important components of these signaling circuits. We used PIP‐specific biosensors to map phosphoinositide pools on Toxoplasma membranes, and identified at least two distinct pools of PtdIns(4)P: one on the Golgi/early secretory membranes, and one associated with Apicomplexan‐specific secretory organelles called dense granules (GRA3(+)). Studies in yeast demonstrate that PITPs potentiate the activities of PtdIns kinases (PIKs) to produce privileged PIP pools on secretory membranes, and PtdIns(4)P‐mediated pro‐secretory activities are countered by the antagonistic activities of specific members of the oxysterol binding protein (OSBP) superfamily. We have identified a Toxoplasma multi‐domain protein, the PITP membrane protein (PIMP), that couples a START‐type PITP domain, a pleckstrin homology (PH) domain, and a C‐terminal OSBP domain. PIMP is a bona fide PITP that transfers PtdIns and phosphatidylcholine (PtdCho) between membranes in vitro, and potentiates PIK activity in vivo when expressed in yeast. Of the four known Toxoplasma PITP genes, it is the only one capable of doing so. The PIMP PH domain specifically binds PtdIns(4)P and PtdIns(4,5)P2 in vivo, and recognizes these pools on dense granules in intracellular parasites. Full length PIMP localizes to (GRA3+) dense granules, and this association is mediated by PH domain PIP binding. PIMP is a physical platform for integration of multiple lipid signals on specialized secretory membranes in Toxoplasma and is the first known example of a PITP coupled to an OSBP domain. As dense granules are unique to Apicomplexa and required to support intracellular proliferation of the parasites, dense granule signaling networks would make good therapeutic targets for Apicomplexan infections.

Support or Funding Information

This research was funded by NIH grant R01‐GM112591, RO1‐GM44530 and grant BE‐0017 from the Robert A. Welch Foundation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.