bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of O‐GlcNAc Hydrolase with Phosphomimetic Mutations
Authors: Hinkle Megan , Crawford Garland
Published: June 2021

O‐linked beta N‐acetylglucosamine (O‐GlcNAc) hydrolase, or OGA, is an enzyme involved in post‐translational modification. This enzyme hydrolyzes a single O‐GlcNAc from serine or threonine residues on target proteins located in the cytosol of metazoan organisms. OGA is the only protein known to carry out this function in the context of cytosolic proteins. This exclusivity of function makes OGA a likely target of post‐translational modifications in order to regulate its activity. Our aim is to generate a phosphomimetic OGA with altered enzymatic activity by using site‐directed mutagenesis to convert a single serine or threonine to an aspartic acid. The end goal of this research being to introduce the mutated OGA gene into a mammalian cell system to study the relationship between OGA phosphorylation and its activity. Analysis by Western blot will reveal the extent to which the mutants alter OGA expression levels and O‐GlcNAc modification levels in the cell. Additionally, interactions of OGA with known binding partners, such as OGT, the enzyme which adds the O‐GlcNAc sugar to target proteins, can be studied with co‐immunoprecipitation. This work lays the foundation for identifying potential phosphorylation states of OGA and would aid in elucidating the regulatory pathways of this enzyme.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Screening Point Mutations of the O‐GlcNAc Hydrolase Enzyme, OGA, to Investigate Potential Regulation
Authors: Crawford Garland
Published: June 2021

The O‐GlcNAcase enzyme (OGA) hydrolyzes the glycosidic linkage between a GlcNAc and the hydroxyl group on serine and threonine residues on target proteins. OGA is the sole enzyme responsible for removing this cytosolic, post‐translational modification which makes the enzyme itself a likely target for post‐translation modification. Several groups using proteomic analysis have identified phosphorylation sites on OGA, but the functional consequences of these changes have not been fully investigated. In an effort to characterize potential sites of regulation, a plasmid containing 6xHis‐OGA was altered by site‐directed mutagenesis to produce phosphomimetics by substituting an aspartic acid or glutamic acid for a serine or threonine. These mutant OGA enzymes were expressed in E.coli and purified using a two‐step purification of Ni2+‐metal affinity followed by size exclusion. Enzyme activities were investigated using a paranitrophenol‐GlcNAc substrate and were compared to the wild‐type enzyme. The goal of this research is to better understand the function and regulation of OGA, and the role that this important enzyme plays in signaling cascades.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
High Throughput Pancreatic Islet Microtissue 3D Characterization for Compound Screening
Authors: Villani Thomas , Johnson Michael , Gardner Graeme
Published: June 2021

In the last few years several companies have developed 3D in vitro cell culture models and have demonstrated that these 3D models more accurately depict in vivo characteristics compared to traditional 2D monolayer cell culture. Because of this improved in vivo relevancy, these models are being rapidly adopted in the drug discovery space as they can more accurately predict in vivo efficacy and toxicological characteristics. Of the different types of 3D cell culture models being used today, pancreatic islet microtissues have been shown to be a powerful tool for assessing the effectiveness of compounds on diabetes. However, current imaging approaches (e.g. confocal microscopy, wide‐field microscopy) are limited in their ability to completely characterize these 3D models as light attenuation limits imaging depth to approx. 1–3 cell layers. Therefore, current imaging techniques are not able to completely characterize the effect of compounds on beta cell proliferation. Through this work, pancreatic islet microtissues of approx. 250 μm diameter were exposed to several compounds that induce beta cell proliferation and the microtissues were then stained for EdU or Ki67, glucagon, insulin and DAPI. The labeled microtissues were then rendered transparent with a rapid and plate‐compatible tissue clearing technique that allows for the microtissues to be imaged in their entirety using high content confocal imaging. It was shown through this work that the addition of a tissue clearing technique resulted in a 3‐fold increase in the number of cells characterized in the microtissues compared to traditional wide‐field or confocal microscopy.

Support or Funding Information

This work was support by Visikol Inc.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Highly efficient long‐acting cocaine hydrolases as a treatment for cocaine addiction
Authors: Zhan Chang‐Guo , Zheng Fang
Published: June 2021

Aims

Enzyme therapy using a highly efficient exogenous cocaine‐metabolizing enzyme is recognized as a promising new therapeutic strategy for cocaine abuse. Our recently designed highly efficient, long‐acting cocaine hydrolases (LA‐CocHs), engineered from human butyrylcholinesterase, have a considerably improved catalytic efficiency against cocaine and a significantly improved biological half‐life. Further, we want to know whether LA‐CocHs are effective in blocking the physiological and psychological effects of cocaine. Based on the animal data, we may select the LA‐CocH with the best overall in vivo profile for further development.

Methods

Structure and mechanism‐based computational design was followed by laboratory experimental studies. LA‐CocHs were produced in a bioreactor using stable cell lines that we developed previously, and were purified. The purified LA‐CocHs were used to test their actual in vivo activities in animals (rats and monkeys), including pharmacokinetics (PK), pharmacodynamics (PD), hyperactivity, drug discrimination, and drug self‐administration studies.

Results

We have successfully designed, prepared, and characterized a set of LA‐CocHs, including CocH5‐Fc(6M). According to the animal data (including those from extensive PK/PD, locomotor activity, drug discrimination, and drug self‐administration tests), a single dose of LA‐CocH was able to effectively accelerate cocaine metabolism in animals even after 30 days and, thus, block cocaine‐induced physiological effects and toxicity for a long period of time. In consideration of the general observation that the biological half‐life of a protein drug in humans is generally longer than that in animals, CocH5‐Fc(6M) examined in this study could allow dosing once every month for cocaine addiction treatment in humans.

Conclusion

The animal data suggest that CocH5‐Fc(6M) has the desirable potency of a therapeutic enzyme. Hence, CocH5‐Fc(6M) could be developed further to become the desirable enzyme therapy for cocaine addiction treatment.

Support or Funding Information

This work was supported by an NIDA Translational Avant‐Garde Award (grant UH2/UH3 DA041115) and NIDA R01 grants (DA035552, DA032910, DA013930, and DA025100).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protein kinase C ζ‐dependent upregulation of GDNF by metformin
Authors: Katila Nikita , Choi Dong‐Young , Bhurtel Sunil
Published: June 2021

Parkinson's disease is the second most common neurodegenerative disorder characterized by prominent loss of the dopaminergic neurons in the substantia nigra (SN). The substantial demise of the dopaminergic neurons in the SN is followed by depletion of dopamine in the striatum manifesting typical motor symptoms including resting tremor, bradykinesia, and rigidity. In spite of effort to develop therapies for the disease, there is still no way to cure or halt the neurodegeneration. Recently, we have demonstrated that metformin rescued the nigrostriatal dopaminergic pathway from MPTP neurotoxicity by inducing neurotrophic factors. In this study, we attempted to address how metformin upregulates neurotrophic factors in the brain which might contribute to the neuroprotection. We observed the increase in the levels of brain derived neurotrophic factor (BDNF) and glial cell derived neurotrophic factor (GDNF) in the SN and striatum of C57BL6 mice via immunohistochemistry and western blotting. These results were recapitulated in vitro study using SH‐SY5Y cells. Upregulation of the neurotrophic factors was accompanied by the activation of AKT/ERK/CREB pathway. Furthermore, we found that inhibition of protein kinase (PK) C ζ attenuated metformin‐induced GDNF elevation, but not BDNF. Tyrosine hydroxylase phosphorylation by metformin was also blocked by PKC ζ inhibition. These results suggest that metformin increase GDNF through induction of PKC ζ activity, which might exert neuroprotection. Although further research is required to identify the metformin's action mechanism, metformin might be a potential neuroprotective therapy for Parkinson's disease treatment.

Support or Funding Information

This work was supported by the Korea Foundation for the Advancement of Science & Creativity (KOFAC) grant funded by the Korean Government.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating Phosphorylation Differences in FFA4 Short and Long Isoforms
Authors: Senatorov Ilya S. , Cheshmehkani Ameneh , Moniri Nader H.
Published: June 2021

The G protein‐coupled receptor Free Fatty Acid Receptor 4 (FFA4) is an attractive drug target due to its profound influence on a variety of endocrine, inflammatory, and metabolic processes. Humans contain a unique long splice variant of FFA4, which contains an additional 16 amino acid insert within the 3rd intracellular loop, and has not been found to be expressed in any other mammals. Although the shorter FFA4 isoform (FFA4‐S) is ubiquitously expressed, human FFA4‐L has only been found in colorectal tissue. More interestingly, although both isoforms are agonized by the same ligands, upon agonism, FFA4‐L has been demonstrated to possess an intrinsic bias towards β‐arrestin signaling rather than coupling to G proteins. Since it is known that receptor phosphorylation is required for interaction with β‐arrestin, the purpose of the current study was to determine whether any differences exist between the long and short isoform of FFA4 in terms of basal, homologous, or heterologous phosphorylation.

Methods

Whole cell phosphorylation assays using 32P‐labeled orthophosphate were performed in HEK293 cells transiently expressing FFA4 isoforms or mutants. Inquiries into G‐protein receptor kinase (GRK) activity were performed using small interfering RNA (siRNA) followed by whole cell phosphorylation assay. Inquiries into protein kinase A (PKA) or protein kinase C (PKC) activity utilized the adenylyl cyclase activator forskolin to induce PKA activity, PKA inhibitor H‐89, PKC activator phorbol‐myristol acetate (PMA), and PKC inhibitor BIMII. Arrestin recruitment assays were conducted using yellow fluorescence protein (YFP) tagged arrestin‐3 and visualized via confocal microscopy. Truncated mutants were generated by sequential deletions via overlap primers and PCR mutagenesis.

Results

Similar to our previously reported results with FFA4‐S, homologous phosphorylation of FFA4‐L is mediated by GRK6, whereas heterologous phosphorylation is mediated by PKC. FFA4‐L has lower basal phosphorylation compared to FFA4‐S. Truncation of FFA4‐L at S356 fully inhibits receptor phosphorylation and abrogates arrestin recruitment, matching reported observations for FFA4‐S at corresponding S340.

Conclusion

The same kinases are responsible for homologous and heterologous phosphorylation of FFA4‐S and FFA4‐L, while basal phosphorylation of FFA4‐L remains lower compared to FFA4‐S. The arrestin sensor for both isoforms is located on the C‐terminal tail of the receptor following S340 (FFA4‐S) or S356 (FFA4‐L).

Support or Funding Information

NIH/NIDDK DK098730 to NHM and Diabetes Action Research and Education Foundation grants to NHM

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Signaling Bias and Antagonism of Pilocarpine for M3 Muscarinic Acetylcholine Receptor
Authors: Pronin Alexey N. , Wang Kelly , Slepak Vladlen Z.
Published: June 2021

Pilocarpine is a prototypical drug used to treat glaucoma and dry mouth since the 19th century and classified as either a full or partial muscarinic agonist. Here, we report several unexpected results pertaining to its interaction with its main therapeutic target ‐ muscarinic M3 receptor (M3R). We found that pilocarpine was 1,000 times less potent in stimulating mouse eye pupil constriction than muscarinic agonists oxotremorin‐M (Oxo‐M) or carbachol (CCh), even though all three ligands have similar Kd values for M3R. In contrast to CCh or Oxo‐M, pilocarpine does not induce Ca2+ mobilization via endogenous M3R in HEK293T or mouse insulinoma MIN6 cells. Pilocarpine also fails to stimulate insulin secretion, and instead, antagonizes insulinotropic effect of Oxo‐M and CCh‐induced Ca2+ upregulation. However, in HEK293T or CHO‐K1 cells overexpressing M3R, pilocarpine induces Ca2+ transients like those recorded with another Gq‐coupled muscarinic receptor, M1R. Stimulation of cells overexpressing M1R or M3R with CCh resulted in a similar reduction in PIP2. In contrast to CCh, pilocarpine stimulated PIP2 hydrolysis only in cells overexpressing M1R, but not M3R. Moreover, pilocarpine blocked CCh‐stimulated PIP2 hydrolysis in M3R‐overexpressing cells, thus, it acted as an antagonist. Pilocarpine activates ERK1/2 in MIN6 cells. The stimulatory effect on ERK1/2 was blocked by the Src family kinase inhibitor PP2, indicating that the action of pilocarpine on endogenous M3R is biased toward b‐arrestin. Taken together, our findings show that pilocarpine can act as either an agonist or antagonist of M3R, depending on the cell type, expression level and signaling pathway downstream of this receptor.

Support or Funding Information

This work was supported by the National Institutes of Health Grants RO1DK105427 and RO1DK111538

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 soluble epoxide hydrolase attenuates pathological vascularization by preventing astrocyte loss in a retinopathy of prematurity model in mice
Authors: Hu Jiong , Bibli Sofia Iris , Wittig Janina , Lin Jihong , Popp Rüdiger , Hammes Hans‐Peter , Fleming Ingrid
Published: June 2021

Retinopathy of prematurity (ROP) is a leading cause of childhood blindness. Several signalling pathways have been implicated in the process of ROP but a novel drugable target is the cytochrome P450 (CYP)/soluble epoxide hydrolase (sEH) pathway. The latter enzymes play a crucial role in biological processes by generating bioactive epoxides and diols from endogenous polyunsaturated fatty acids (PUFAs). This study aimed to characterize the effects of the newly identified sEH‐derived w‐3 PUFA diol; 19,20‐dihydroxydocosapentaenoic acid (DHDP) in a mouse ROP model.

ROP in mice was achieved by placing pups in 75% oxygen for 5 days from postnatal day (P) 7 until P12, before moving animals back to room air for an additional 5 days. This procedure resulted in the development of avascular areas that were significantly larger in retinas from sEH−/− mice than in their wild‐type (WT) littermates at P17. Avascularization was accompanied by angiogenesis in the form of characteristic vascular “tufts” which were more evident in retinas from sEH−/− mice. Although there was no difference in vascular obliteration, a marked decrease in the astrocyte network was observed in sEH−/− mice after exposure to high oxygen. Mechanistically, astrocyte loss in hyperoxic conditions was a consequence of elevated mitochondrial (mt) DNA damage mediated by increased interaction of the PARP‐1 DNA binding domain with mtDNA, as revealed by chromatin immunoprecipitation studies. Exposure of astrocytes to 75% O2 in vitro resulted in elevated PARP‐1 cleavage/activation; a phenomenon reversed in the presence of 19,20‐DHDP. Administration of 19,20‐DHDP during hyperoxia preserved the complex of presenilin 1 with mitochondrial carrier homolog 1 (MTCH‐1) in the outer mitochondria membrane, inhibiting the translocation of the MTCH‐1 to the inner mitochondria membrane and subsequently inhibiting PARP‐1 cleavage. The intravitreal injection of 19,20‐DHDP at day 7 significantly prevented astrocyte loss caused by high O2 levels at day 8, and reduced both the avascular area as well as vascular tuft formation at P17 in the sEH−/− mice.

Our date indicate that the sEH dependent generation of 19,20‐DHDP from w‐3PUFA is required for retinal astrocyte survival and preservation of DNA integrity under hyperoxic conditions.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lodoxamide protects against LXR‐mediated steatosis through G protein‐coupled receptor 35 in Hep3B cells and primary mouse hepatocytes
Authors: Nam So‐Yeon , Im Dong‐soon
Published: June 2021

Background

G protein‐coupled receptor 35 has been an orphan receptor, although synthetic agonists and antagonists have been developed. Cromolyn and lodoxamide, mast cell stabilizers, were reported as agonists. Recently, cromolyn exhibited antifibrotic effects through actions on hepatocytes and stellate cells. However, the function of GPR35 in hepatocytes has not been investigated.

METHODS

Using an in vitro model of liver X receptor (LXR)‐mediated hepatocellular steatosis, the roles of lodoxamide and GPR35 were investigated in human and mouse hepatocytes.

RESULTS

T0901317, a specific LXR activator, induced lipid accumulation in Hep3B human hepatoma cells. This lipid accumulation was inhibited by lodoxamide, the most potent agonist of GPR35. The protective effects of lodoxamide were inhibited by CID2745687, a specific GPR35 antagonist or by siRNA trnasfection. T0901317 treatment induced expression of SREBP‐1c, a key transcription factor for lipid synthesis. Lodoxamide inhibited the induction of SREBP‐1c proteins in a GPR35‐dependent manner. Using specific inhibitors of cellular signaling components, lodoxamide‐induced inhibition of lipid accumulation was found to be mediated through p38 MAPKs and JNK, but not through Gi/o proteins, PI3K, and ERKs. Furthermore, the protective effect of lodoxamide was confirmed in mouse primary hepatocytes.

CONCLUSION

Therefore, the present data suggest that GPR35 may function to protect against lipid accumulation in the liver.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Genetic Polymorphisms on the Sulfation of 17α‐Ethinylestradiol (EE2) by Human Cytosolic Sulfotransferase SULT1E1
Authors: El Daibani Amal A. , Alherz Fatemah A. , Abunnaja Maryam S. , Liu Ming
Published: June 2021

In human, previous pharmacokinetic studies have shown the metabolism of 17α‐ethinylestradiol (EE2) through sulfation, with estrogen sulfotransferase (SULT1E1). Since SULT1E1 is a major responsible enzyme that mediates the biotransformation of endogenous estrogens and estrogen‐like drugs, genetic polymorphisms of SULT1E1 may significantly impact the 17α‐ethinylestradiol‐sulfating activity by SULT1E1. Subsequently, this study was performed to investigate the implications of SULT1E1 genetic polymorphisms in the sulfation of 17α‐ethinylestradiol. By using the site directed mutagenesis technique, five selected recombinant SULT1E1 allozymes were generated, expressed and purified. Compared with the wild‐type SULT1E1, purified allozymes of SULT1E1 were shown to exhibit significantly distinct sulfating activity with 17α‐Ethinylestradiol as a substrate. In summary, the obtained results provide evidence related to differential metabolism of 17α‐ethinylestradiol in individuals with different SULT1E1 genotypes, which may be useful for further in‐vivo study contributed to the therapeutic‐ as well as adverse effects of 17α‐ethinylestradiol through sulfation.

Support or Funding Information

The College of Pharmacy, University of Toledo

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Synergistic neuroprotective effect by combination treatment with DAPK1 and Pin1 inhibitor on ischemic stroke
Authors: Won Jinyoung , Kang Hyunbon , Hong Yonggeun
Published: June 2021

DAPK1 (death‐associated protein kinase 1) and Pin1 (peptidyl‐prolyl isomerase 1) are positive mediators of the neuronal death signaling pathway in cerebral ischemia. Pin1 induces the cis‐trans conversion on a phosphorylated serine/threonine (pS/T) amino acid residue of target protein that is regulated by the calcium/calmodulim (CaM)‐dependent kinase termed DAPK1. Whereas DAPK1 and Pin1 activation is required for neuronal death induced by ischemia/reperfusion (I/R) injury, this mechanism has not been elucidated. Thus, the purpose of this study was to determine the effects of DAPK1/Pin1 on pathogenesis of cerebral ischemia. Ischemic stroke model was induced by middle cerebral artery occlusion (MCAo). Experimental animals were randomly divided into three groups; vehicle (control), single inhibitor (DAPK1 inhibitor or Pin1 inhibitor), inhibitor combination (DAPK1 inhibitor and Pin1 inhibitor). Inhibitor groups including single injection and combined injection groups showed the decrease of neurological deficit and the altered body weight compared to control. Also, the low mortality rates were shown in inhibitor groups. In addition, the expansion of ischemic core from the striatum to the cerebral cortex was inhibited in inhibitor groups. Particularly, combination group showed significantly decreased ischemic lesions compared to control group. These results indicated that suppression of DAPK1 and Pin1 catalytic activity could enhance the neuroprotective effect against ischemic insult.

Support or Funding Information

Funding: NRF‐2013R1A2A2A01067169, NRF‐2017R1A2A2A01067169, KRIBB‐KGM4611714

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Microarray Analysis of Long Non‐coding RNA and mRNA Expression Profiles in Diabetic Cardiomyopathy Using Human iPSCs‐Derived Cardiomyocytes.
Authors: Pant Tarun , Mishra Manoj , Bai Xiaowen , Bosnjak Zeljko J. , Ge Zhi‐Dong , Dhanasekaran Anuradha
Published: June 2021

Long non‐coding RNAs (lncRNAs) are 200 nucleotides long and represent the non–protein‐coding transcripts of our genome capable of regulating vital cell processes like transcription, translation and chromosome remodeling. Studies have reported the association of these deregulated non‐Coding RNAs (ncRNAs) with the development of diabetes. However, the potential role of these lncRNAs and their changing expression profiles in Diabetic Cardiomyopathy (DCM) has not been explored. Advancing field of stem cell technology offers us the opportunity to use human induced pluripotent stem cell‐derived cardiomyocytes ( hiPSC‐CMs) for disease modeling. We hypothesized that disease phenotype can alter the expression profile of these lncRNAs. In the present study utilizing the previously established in vitro DCM model, we investigated the deregulated genome profile. We performed (lncRNA and mRNA) microarray analysis on the disease modeled hiPSC‐CMs.

Results

The array data revealed around 2046 lncRNAs to be differentially regulated between diseased and control group. Genome profiling of mRNA found 1582 probes with significant differential expression. In addition, Pathway and Gene ontology (G.O) analysis for differentially expressed mRNAs indicated that both Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) to be upregulated in the entire DCM group, distinct pathways which are associated with DCM were also investigated.

Conclusion

These results provide a better understanding about deregulation of lncRNAs that are associated with disease pathogenesis.In the future, these lncRNAs can not only serve as early diagnostic biomarkers but also as potential therapeutic targets.

Support or Funding Information

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 Pathway for Fatty Acid: Palmitate Transport from Plasma into Fat Vacuoles and Mitochondria in the Heart
Authors: Bassingthwaighte James B. , Barr Nathan
Published: June 2021

As a part of a multi‐university, multi‐investigator program on integrated function of the heart, we are targeting the metabolism and regulation of the endothelial cell (EC) and the cardiomyocyte (CM) and the interactions between these two cell types. Models for blood flow, glucose, fatty acid and for oxygen are essential steps. Initial models are “eternal” in that there is no protein anabolism or catabolism, thus all enzymes remain at fixed concentrations. The emphasis is on the physico‐chemical balances (charge, ionic regulation, mass, volume, energy, reducing equivalents, carbon, oxygen, phosphorus and so on), on the limitations to the kinetics imposed by the energetics, and the responses to transients rather than steady state analysis. Fatty acid, the prime substrate for cardiac metabolism, is tightly bound to as many as 12 sites on plasma albumin, yet is about 1/3 extracted during a single capillary passage. The modeling of palmitate (a 16:0 fatty acid) flux starts with the release from these sites, the rebinding at multiple sites, diffusion in the plasma, and uptake across the capillary endothelial plasmalemma by a fatty acid transporter, e.g. CD36, facilitated diffusion by fatty acid binding protein (FABP) across the 1 micron thick EC, then facilitated or diffusive transport into the interstitial fluid space to the CM surface and transmembrane passage. The next stage, acylCoA formation, leads 2 ways. One is to di‐ and tri‐acyl glycerol (TAG) and lipid vacuole formation and the reverse, a storage and buffering system. The other is for acyl‐CoA to cross the mitochondrial membrane, undergo beta‐oxidation and insert the two‐carbon products into the tricarboxylic acid (TCA) cycle to provide energy for ATP generation through oxidative phosphorylation, thereby setting the balance between oxygen delivery by flow and ATP generation. The models serve for data analysis and hypothesis testing, account for the kinetics of enzyme binding and the resultant concentration‐dependent substrate capacitance (cellular volume of distribution), and provide mass balance. They serve as working hypotheses until disproven, and are “open source” on our website (www.physiome.org), so that they can be tested and improved by the scientific community. Such models will serve as tools to link overall cellular function to the underlying physiology and its regulation at the proteomic and genetic levels.

Support or Funding Information

These efforts are a part of the Physiome Project sponsored by NIH/NHLBI U01‐HL122199 Cardiac Energy Grid and our Simulation Resource (http://www.physiome.org).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biomarkers and components of the interactive Physical and Cognitive Exercise System (iPACES™ v2.0) for Mild Cognitive Impairment (MCI): Cortisol, Dehydroepiandrosterone (DHEA‐S), and Insulin‐like Growth Factor (IGF1)
Authors: Wall Kathryn Mary , Stark Jessica , Karla‐Lall Anvit , Schillaci Alexa , Christian Hannah , Doty Carolyn , McLaren Elizabeth , Saulnier Tobi , Cohen Brian D. , Anderson‐Hanley Cay
Published: June 2021

Dementia cases are on the rise among our aging global population, and thus there is increasing urgency to identify efficacious interventions for preserving or ameliorating cognitive decline. Physical exercise, cognitive training and combined physical and cognitive interventions have been found to slow the decline of cognitive abilities in those with mild cognitive impairment (MCI) but the biological mechanisms underlying these changes need further clarification. This quasi‐experimental within‐subjects design compared changes in biomarkers during two‐week exposures to components of the interactive physical and cognitive exercise system (iPACES™). Participants were evaluated over eight weeks at two‐week intervals. Biomarker levels of Cortisol, IGF1 and DHEAS were assessed at each evaluation through saliva (passive drool collection). Each saliva sample was analyzed using enzyme‐linked immunosorbent assays (ELISAs). Additionally, each sample was analyzed using bicinchoninic acid (BCA) protein assay to normalize protein concentration. Cognitive outcomes were also assessed, focusing on executive function via Stroop, Trails, and Flanker tests. This pilot study enrolled 14 older adults, 13 of which met criteria for MCI (MoCA<26), with ten study completers. Paired t‐tests revealed significant (ps<.05) cognitive improvements in Stroop and Flanker from baseline during all active component conditions (e.g., pedaler, game, and iPACES™), but not during placebo. Biomarkers did not change significantly during placebo or pedaler conditions, but during the game intervention, DHEAS declined significantly (p=.04), and during iPACES™ intervention, DHEAS declined further and IGF1 declined significantly (p=.05), consistent with prior research (McTiernan, et al., 2005; Yuichiro, et al., 2010). Furthermore, the changes (from baseline to the end of the 8‐week trial) for cognition were moderately correlated with biomarker changes (r's ranging from −.22 to −.48), suggesting a link between neurobiological mechanisms and cognitive outcomes. Further research is needed to replicate and extend this pilot research; in particular, it would be useful to compare such interventions in a randomized controlled trial.

Support or Funding Information

National Institute on Aging STTR Grant #R41AG053120 and Union College Undergraduate Research 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.
Effect of chronic lactate administration prior to endurance training on mitochondrial adaptations in mouse skeletal muscle
Authors: Takahashi Kenya , Kitaoka Yu , Matsunaga Yutaka , Hatta Hideo
Published: June 2021

Lactate is considered as not only an oxidizable fuel, but also a signal molecule inducing mitochondrial biogenesis. In this study, we hypothesized that lactate administration prior to training augments mitochondrial adaptations. ICR male mice were randomly assigned to Con (n=9), Tr (n=7) or La+Tr (n=8) group. Mice in Tr and La+Tr were performed treadmill running (20 m/min, 60 min) immediately after administration of PBS (same volume as lactate) or sodium lactate (1 mg/g BW) via i.p. injection for 21 consecutive days. Mice in Con were administrated same volume of PBS on the same days. All the mice were performed lactate tolerance test (LTT: i.p. injection of 1 mg/g BW of sodium lactate) 24h after final training. Blood samples were collected pre, 5, 15, 30, 60 and 180 minutes after the lactate administration. Soleus and plantaris muscle were taken for the biochemical analysis 24h after LTT. As a result, some mitochondrial enzyme activities in soleus and plantaris muscle were significantly higher in La+Tr than Tr (CS: P<0.05, β‐HAD: P=0.05, COX: P<0.01 in soleus, CS: n.s., β‐HAD: n.s., COX: P<0.05 in plantaris). However, mitochondrial OXPHOS protein content was not different between Tr and La+Tr. MCT1 protein content was significantly higher in La+Tr than Con (+18%; P<0.01) in plantaris muscle, but not MCT4. As a result of LTT, blood lactate concentrations after lactate administration were significantly lower in Tr (60 min: P<0.05 vs. Con) and La+Tr (30 min: p<0.01, 60 min: P<0.01 vs. Con) than Con. These data indicate that lactate upregulates mitochondria functionally, and that lactate administration increased MCT1 protein content leading to lower blood lactate concentration after lactate administration.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ER stress dictates inflammatory, but not hormonal, lipolytic triggers in adipocytes
Authors: Schertzer Jonathan D. , Foley Kevin , Duggan Brittany , Heal Mark , Chi Wendy , Barra Nicole
Published: June 2021

Objective

Obesity is characterized by inflammation that can impair endocrine control of cell metabolism. The triggers and stress responses of these immune‐mediated defects are ill‐defined. There is a reciprocal relationship between inflammation and lipolysis in adipocytes, but hormones and adrenergic signals can also stimulate lipolytic programs in adipocytes. This study aimed to identify the cell stress responses that differentiate inflammatory and hormonal triggers of lipolysis in adipocytes.

Methods

Lipolysis was measured by glycerol release from 3T3‐L1 adipocytes treated with inhibitors of ER stress, inositol‐requiring protein 1α (IRE1α) or tyrosine kinases before stimulation with inflammatory lypolytic stimuli. The inflammatory stimuli included bacterial peptidoglycan, lipopolysaccharide, and tumor necrosis factor. The Hormonal or adrenergic stimuli tested included isoproterenol and forskolin. Inflammation was characterized by Il6 secretion and NF‐κβ activity.

Results

Inhibition of the kinase activity of IRE1α was sufficient to block lipolysis and Il6 secretion caused by thapsigargin‐induced ER‐stress in adipocytes. Inhibition of IRE1α kinase activity blocked augmented lipolysis from inflammatory, but not hormonal stimuli. Inhibition of IRE1α also blocked augmented Il6 secretion from all inflammatory stimuli. Inhibition of IRE1α blocked the increased NF‐κB activity from all inflammatory stimuli except for lipopolysaccharide. Inhibition of ABL kinases with imatinib did not alter lipolysis. Inhibition of IRE1α RNase activity did not alter lipolysis. The potent RIPK2 inhibitor ponatinib blocked lipolysis, Il6 secretion, and NF‐κβ activation stimulated by peptidoglycan, but did not alter lipolysis from other inflammatory stimuli, despite attenuated Il6 secretion.

Conclusions

The ER stress sensor IRE1α is essential for inflammation‐induced lipolysis and Il6 secretion in adipocytes. Neither RIPK2 nor NF‐κβ can fully capture the IRE1α lipolyitic pathway from inflammatory stimuli. These findings show that IRE1α discriminates inflammation‐induced lipolysis linked to ER stress from hormonal or adrenergic triggers of adipocyte lipolysis.

Support or Funding Information

This research is supported by operating grants to JDS from the Natural Sciences and Engineering Research Council (NSERC). JDS held CDA Scholar (SC‐5‐12‐3891‐JS) and CIHR New Investigator awards (MSH‐136665) and holds a Canada Research Chair in Metabolic Inflammation. BD was supported by an Ontario Graduate Scholarship (OGS). KF is supported by an NSERC 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.
Targeting the Prolactin Receptor Signaling Using an Antipsychotic Drug to Suppress Pancreatic Cancer
Authors: Dandawate Prasad , Kaushik Gaurav , Subramaniam Dharmalingam , Ramamoorthy Prabhu , Ghosh Chandrayee , Choudhury Sonali , Standing David , Dhar Animesh , Thomas Sufi M. , Santimukul Santra , Padhye Subhash , Tawfik Ossama , Weir Scott , Jensen Roy A. , Anant Shrikant
Published: June 2021

Pancreatic cancer (PCa) is deadly cancer with an abysmal five‐year survival rate of < 6%. PCa has a bad prognosis and is associated with resistance to standard of care chemotherapeutic gemcitabine and high recurrence rate after surgery. Unfortunately, gemcitabine remains the drug of choice for metastatic pancreatic adenocarcinoma, with minor improvements in survival rate. Hence, there is a need for novel therapeutic interventions for the treatment of PCa. Here we report that prolactin receptor (PRLR) is overexpressed in both PCa tissues and in cell lines. When it is bound by the ligand prolactin (PRL), various downstream events such as JAK‐STAT and ERK‐MAPK signaling pathways are activated. In PCa cell lines, PRL treatment induced dose‐ and time‐dependent STAT3, and ERK1/2 phosphorylation. Moreover, while PRL did not affect PCa cell proliferation, it significantly enhanced pancosphere formation suggesting that PRL affects stem cells. We manipulated PRLR levels using the CRISPR/Cas9 system. This resulted in the lower colony and spheroid formation capacity, decreased migration in vitro as well as reduced tumor‐forming capacity in an orthotopic model of PCa in C57BL/6 mice. Based on these results, we propose that PRL signaling is a novel target for therapeutic intervention. Currently, there are no known small molecules targeting PRL‐PRLR signaling, and X‐ray crystal structure of the intracellular domain of PRLR is not available. Hence, we obtained a homology model for the C‐terminal intracellular region of the receptor and performed a virtual screening for small molecules in silico. In this, we demonstrated that penfluridol (a first‐generation antipsychotic drug) interacts with the JAK2 binding site of the receptor. Penfluridol is known for its long half‐life and is used in the treatment of chronic schizophrenia. We experimentally confirmed binding of penfluridol to PRLR in the cells by using the cellular thermal shift assay (CETSA). Penfluridol treatment decreased PRL‐induced STAT3 and ERK phosphorylation, spheroid formation as well as the migration of PCa cells. We observed a significant increase in autophagy based on increased levels of LC3B, p62 and enhanced expression of autophagy‐related genes ATG‐5, −7 and −12 after penfluridol treatment. Penfluridol treatment also significantly decreased tumor burden in orthotopic PCa model and induced the expression of autophagic related proteins. Taken together, these results suggest that PRL‐PRLR signaling is important for PCa growth and repurposing penfluridol to target PRL‐signaling an effective therapeutic targeting strategy for treating the disease.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparison of Isolation, Characterization and Differentiation of Human Umbilical Cord Mesenchymal Stromal Cells in a traditional 2D culture versus a 3D culture.
Authors: Jirakittisonthon Thitikan , Murnane Joan , Snyder Larry , Cromer Adrienne , Weiss Mark
Published: June 2021

Mesenchymal stromal cells (MSCs) are attractive candidates for regenerative medicine since they appear to be safe and effective for modulating diseases with an inflammatory component such as osteoarthritis and Crohn's disease. The immune modulating and paracrine effects noted clinically cannot be explained solely by cellular engraftment since a fraction of the injected MSCs can be recovered one month after injection. Traditional two‐dimensional (2‐D) MSC culture evaluates cells expanding along a flat surface. In contrast, their natural environment, e.g., in vivo, is complex and three‐dimensional (3‐D). We speculate that 3‐D cell culture may more accurately reflect in vivo cellular behavior. The objective of this study was to characterize human umbilical cord‐MSCs cultured in a traditional 2‐D on tissue culture plastic versus a 3‐D hydrogel model. We compared expression of cell surface receptors (Flow Cytometry), metabolic activity (MTT Assay), and histological cellular differentiation of HUC‐MSCs isolated from 2‐D culture conditions to 3‐D hydrogels. We found trilineage differentiation potential and mesenchymal surface markers similar to that observed in 2D culture. This pilot work, together with published spheroid MSC culture work, suggests that surface interactions and cell‐cell interactions brought about by 3D culture influences cell physiology but doesn't interfere with the cells meeting the ISCT minimal MSC definition.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Probiotic Metabolites Promote Stem Cell Proliferation and Differentiation
Authors: Wu Danielle , Fu Richard , Scoffield Jessica , Kannappan Ramaswamy
Published: June 2021

Probiotic metabolites (PM) impart numerous health benefits in human and may have a potential to induce cell proliferation and differentiation. We tested the effects of PM released by probiotics found in yogurts on stem cell proliferation and differentiation. Five different brands of yogurts (Oikos, Stonyfield, Yoplait, Chobani and Activia) often sold in the supermarkets were tested in this study. Yogurt cultures analyzed by Gram‐staining and DNA sequencing of 16s rRNA gene identified that these yogurts are not equally supplemented and include different types and various amounts of probiotics. The probiotics were cultured and the condition media containing PMs were collected and used in our study. To evaluate the effect of PM on stem cells, human induced pluripotent stem cells (iPSC) were treated with probiotic conditioned media. We found that probiotic Lactobacillus reuteri conditioned media (LRCM) induced iPSC proliferation at lower doses but inhibited cell proliferation at higher doses. Additionally, expression of the proliferating cell nuclear antigen (PCNA) and pluripotent stem cell factors, OCT4, SOX2 and NANOG transcripts was upregulated in iPSCs treated with LRCM. iPS cells exposed to LRCM during in vitro differentiation led to enhanced expression of myocyte markers, Nkx2.5 and cTnT. Consistently, sodium butyrate, a defined bacterial metabolite (short chain fatty acid), had similar effect on iPSC proliferation and expression of pluripotent markers and myocyte markers. In summary, we have demonstrated that probiotics in different brands of yogurts are diverse in their numbers, viability and composition. Further, our results show that PMs can potentiate iPS proliferation and induce cardiomyocyte differentiation. Our findings have laid a foundation for further investigation of how probiotics and its metabolites from yogurt affect human health and disease.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Doxorubicin‐induced fetal glomerular mesangial cell apoptosis involves NADPH oxidase‐dependent reactive oxygen species generation
Authors: Matthews Anberitha T. , Soni Hitesh , Buddington Randal K. , Adebiyi Adebowale
Published: June 2021

Animal studies have shown that chemotherapeutic agent doxorubicin (DOX) causes fetal toxicity, including renal abnormalities. Although DOX‐associated nephropathy includes oxyradical stress and glomerular injury, its effect on fetal glomerular cells remains unclear. This study aimed to examine the effect of DOX on fetal glomerular mesangial cell (FE‐GMC) survival. Furthermore, we tested the hypothesis that NADPH oxidase (NOX)‐dependent reactive oxygen species generation contributes to DOX‐induced alterations in FE‐GMCs. Confocal microscopy and high‐performance liquid chromatography revealed that the level of 2‐hydroxyethidium was increased in DOX‐treated cells when compared to DMSO controls, which was reversed by selective NOX inhibitor, apocynin. Apocynin also attenuated DOX‐induced caspase 3/7 activation in FE‐GMCs. These data suggest that DOX induces apoptosis in porcine FE‐GMCs by promoting NOX‐dependent oxidative stress. Caspase‐mediated cell death may contribute to the toxic effects of DOX on the developing kidney.

Support or Funding Information

R01DK101668; R01DK101668‐04S1

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Nicotine Attenuates Acetate‐Induced Increase of Cytosolic Reactive Oxygen Species in Dopaminergic‐Like PC12 Cells
Authors: Behnke Jessica E. , Chapp Andrew D. , Shan Zhiying , Chen Qing‐Hui
Published: June 2021

We have previously reported that the major ethanol metabolite, acetate (6 mM), significantly (p<0.05) increases levels of cytosolic calcium in neural growth factor (NGF) derived PC12 cells measured in real time with Fluo‐4AM. Literature suggests that increased intracellular calcium through glutamatergic channels binds with calmodulin to trigger a cascade leading to the formation of nitric oxide (NO), which can contribute to cell death. We therefore hypothesized that treatment of PC12 cells with acetate (6 mM) would elicit an increase in the levels of cytosolic reactive oxygen species (ROS). Furthermore, it has been shown that nicotine induces protection against glutamate cytotoxicity, although the mechanism is still not fully understood. NGF derived PC12 cells exposed to acetate (6 mM) for 4 hrs had a significant (p<0.05) increase in ROS positive staining cells relative to control (44.3 ± 1.8 % (N=5) vs 33.3 ± 1.5 % (N=3)), measured using flow cytometry with an oxidative stress detection probe (CellROX Orange, Thermofisher). Co‐application of acetate (6 mM) and nicotine (10 μM) was able to significantly (p<0.05) attenuate the acetate‐induced increase in cytosolic ROS back to baseline levels. Percent cells with positive staining are: control (28.6 ± 2.1 %, N=3), acetate (6 mM) (47.5 ± 5.4 %, N=3) and acetate (6 mM) and nicotine (10 μM) (24.04 ± 3.8 %, N=5). This data suggests that nicotine's neuroprotective effect on glutamatergic cytotoxicity may be due, at least partially, to nicotine's ability to reduce the amount of NO formed intracellularly.

Support or Funding Information

Funding support: AHA 16PRE27780121, Andrew Chapp, R15‐HL129213, Zhiying Shan; R15‐HL122952, Qinghui Chen

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