bluuebunny/crossref_metadata_2025 huggingface dataset:
Metformin inhibits autophagy and mitophagy in cardiomyocytes
Authors: Chang Michael , Pinkhasova Polina , Kobayashi Tamayo , Patel Rahul , Cohen Max , Mehta Puja , Zhang Yawen , Kobayashi Satoru , Liang Qiangrong
Published: June 2021

Metformin, a first‐line medication for the treatment of type 2 diabetes mellitus, has been suggested by several clinical studies to be cardioprotective. However, the mechanisms underlying metformin's cardioprotection remain debated. One current hypothesis proposes that metformin protects the heart by increasing autophagy in cardiomyocytes, leading to increased removal of protein aggregates and damaged organelles including mitochondria. In this study, we tested this hypothesis using both in vitro and in vivo models. Rat H9C2 cardiac myoblasts were treated with 3 mM metformin for 24 hours and autophagy/mitophagy were determined by Western blot analysis of LC3‐II, a well‐established marker of autophagic vesicles. Surprisingly, metformin significantly reduced LC3‐II levels in either total cell lysates or mitochondrial fractions; and lysosomal inhibitors increased LC3‐II in metformin‐treated cells to a less degree as compared with vehicle‐treated cells. These results suggest that metformin inhibited rather than stimulated autophagy and mitophagy, contrary to the current belief. This was confirmed by a novel dual fluorescent mitophagy reporter showing reduced mitochondrial fragments being degraded in the lysosomes in the presence of metformin. The reduced mitophagy was also found in the heart from the mitophagy reporter mice that received 200 mg/kg of metformin through gavage. Collectively, these results indicate that the cardioprotective benefit of metformin observed in many different models may be through mechanisms other than increased autophagy and mitophagy.

Support or Funding Information

This work was supported by the National Institutes of Health National Center for Research Resources.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Assembly of Fragment Ends After PCR: An Efficient and Accurate Multi‐part DNA Assembly Method for Large DNA Sequence
Authors: Lin Yibin
Published: June 2021

Recent development of DNA assembly technologies has spurred myriad advances in synthetic biology. But new tools are always required for complicated scenarios. Here, we have developed an alternative DNA assembly method named AFEAP cloning (Assembly of Fragment Ends After PCR), which allows scarless, modular, and reliable construction of biological pathways and circuits from basic genetic parts. The method requires two‐round of PCRs followed by ligation of the sticky ends of DNA fragments. The first PCR yields linear DNA fragments and is followed by a second asymmetric (one primer) PCR that inserts overlapping overhangs at both sides of each DNA fragment. The resulting products of the second PCR are then annealed and ligated with T4 DNA ligase, followed by bacterial transformation to yield the desired plasmids. We characterized its capability and limitations and demonstrated its application to assemble DNA with varying scenarios. Under the optimized conditions, AFEAP cloning allows assembly of an 8 kb plasmid from 1–12 fragments with high accuracy (between 80 and 100%), and 8.0, 11.6, 19.6, 28, and 35.6 kb plasmids from five fragments at 91.67, 91.67, 88.33, 86.33, and 81.67% fidelity, respectively. AFEAP cloning also is capable to construct bacterial artificial chromosome (BAC, 200 kb) with a fidelity of 46.7%. AFEAP cloning provides a powerful, efficient, seamless, and sequence‐independent DNA assembly tool for multiple fragments up to 12 and large DNA up to 200 kb that expands synthetic biologist's toolbox.

Support or Funding Information

This work was supported by National Modern Agriculture Industry Technique Systems (CARS‐02) to Jingao Dong and Starting Grant from Hebei Agricultural University to Fanli Zeng (grant number ZD201622).

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 Role of Epigenetic Factors in Age‐Dependent Changes in Drosophila Melanogaster Visual Behavior and Photoreceptor Neuron Gene Expression.
Authors: Escobedo Spencer
Published: June 2021

Age is a major risk factor for ocular disease. The goal of our lab is to understand the gene expression changes that occur with age. Our previous work shows that there is a visual senescence, the decrease in visual function with age, in Drosophila melanogaster. We have also shown that this age‐dependent decrease in visual function correlates with changes in gene expression. Epigenetic factors have been proposed to play a key role in maintaining ocular function with age. However, the epigenetic mechanisms that are involved in age‐dependent gene expression changes have yet to be identified. To address these questions we use Drosophila photoreceptor neurons as a model of the aging eye. We have conducted an RNAi screen of photoreceptor‐specific knockdown of these epigenetic factors which enables us to identify if they are necessary for visual senescence. Factors that are necessary for visual senescence can be further assessed for their role in gene regulation through photoreceptor‐specific RNA sequencing. This will identify the target genes of these factors, revealing potential mechanisms of age‐dependent regulation of gene expression. One specific factor is Brahma, the catalytic subunit of BAF, an ATP‐dependent chromatin remodeling complex. Preliminary data in the lab shows an enhanced visual senescence in Drosophila expressing a dominant negative Brahma in photoreceptors. Our current work is identifying the gene expression changes in these flies by RNA sequencing.

Support or Funding Information

Research reported in this poster was supported by the National Eye Institute of the National Institutes of Health under award number R01EY024905

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
JARID2 Regulates Skeletal Muscle Differentiation through Regulation of Canonical Wnt Signaling Pathway
Authors: Adhikari Abhinav , Davie Judy
Published: June 2021

Myogenesis is a tightly regulated process that is dependent on the precise and dynamic integration of signals from various signaling pathways including the Notch, Sonic hedgehog and Wnt signaling pathways. The Wnt signaling pathway is an essential signaling pathway that has been shown to have diverse roles during both embryonic muscle development and adult muscle regeneration. In this study, we sought to understand the role of JARID2 ‐ an evolutionarily conserved, inactive histone demethylase that often has been shown to function in association with a transcription repressive complex, polycomb repressive complex‐2 (PRC2), during muscle differentiation. Utilizing the widely used C2C12 cell model system, we report that JARID2 is required for muscle differentiation and it regulates muscle differentiation through modulation of Wnt signaling. We found that JARID2 regulates the myogenic regulatory factors (MRFs), MyoD and myogenin. We discovered that JARID2/PRC2 not only regulates expression of the MRFs directly through tri‐methylation of lysine 27 of histone 3 (H3K27me3) but also indirectly, through regulation of canonical Wnt signaling. JARID2 controls MRF expression indirectly through direct repression of the Wnt antagonist gene, secreted frizzled‐related protein‐1 (Sfrp1). We found that activation of canonical Wnt signaling through inhibition of GSK3b rescues the differentiation block of JARID2 depleted cells through the restoration of MyoD and myogenin expression. JARID2 depleted cells showed a reduction in the nuclear levels of the canonical‐Wnt signaling pathway effector, b‐catenin, and we found that inhibition of GSK3b induced translocation of b‐catenin into the nucleus of these cells. Using chromatin immunoprecipitation (ChIP) assays, we also show that b‐catenin positively regulates the transcription of MyoD through direct binding in the distal regulatory region (DRR) of the MyoD promoter. Thus, we establish here that JARID2 and the PRC2 complex are essential regulators of the initiation of skeletal muscle differentiation through modulation of the Wnt signaling pathway. In myoblasts, PRC2 represses the MRFs but also represses Wnt antagonists, which allows the Wnt pathway to activate MyoD and initiate differentiation.

Support or Funding Information

NIH/NIAMS AR068622

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Preventing Autoimmunity in Transcription‐Dependent CRISPR‐Cas Systems
Authors: Johnson Kaitlin , Learn Brian , Bailey Scott
Published: June 2021

The CRISPR‐Cas systems are a collection of RNA‐based adaptive immune systems that protect many prokaryotes from invasion by viruses and plasmids. CRISPR‐Cas systems are very diverse and can be organized into six types and more than 15 subtypes. Type III systems are unique as their targeting mechanism requires transcription of the target DNA. Type III effector complexes use small CRISPR RNAs (crRNAs) to identify complementary transcripts. Transcript binding simultaneously activates two nuclease domains, leading to the degradation of both the bound transcript and adjacent DNA. Like all other immune systems, CRISPR‐Cas systems must differentiate between self and non‐self in order to prevent autoimmunity, which could arise from targeting of the host CRISPR‐array. Type III systems use two mechanisms to identify self‐transcripts. Studies of the Type III‐A system from Staphylococcus epidermidis revealed that base pairing between the crRNA and the sequence flanking the 3′ end of RNA target site prevents autoimmunity. However, a study of the Type III‐B system from Pyrococcus furiosus revealed that a short sequence motif flanking the RNA target sequence is required for immunity and the absence of this motif in CRISPR arrays prevents autoimmunity. Neither of these mechanisms is well understood biochemically and it is not clear if these mechanisms are conserved within the subtypes. To address these points, we monitored RNA‐activated DNA cleavage by the Type III‐B effector complex from Thermotoga maritima with purified components. A series of RNA transcripts containing various target 3′‐flanking sequences were tested for their ability to activate DNA cleavage. We find that two factors are important in preventing autoimmunity in the T. maritima Type III‐B system: (1) base pairing between the crRNA and the target 3′‐flanking sequence of the transcript and (2) the presence of a G, which does not base pair with the crRNA, immediately adjacent to the 3′‐end of the RNA target sequence. These data suggest that the mechanisms used to avoid autoimmunity by Type III systems are not subtype‐specific and that identification of self transcripts is complex, involving base pairing and specific sequence recognition.

Support or Funding Information

This work was supported by National Institutes of Health grant GM097330 to S.B.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
NAD+ Bound N‐terminal Domain of CARDS Toxin Increases IL‐1β Secretion
Authors: Woods Allison Taylor , Segovia Jesus , Galaleldeen Ahmad
Published: June 2021

Mycoplasma pneumoniae (Mp) is an atypical bacterium that is linked to various respiratory diseases such as walking pneumonia and asthma. Upon infection, Mp produces a 591‐aa virulence factor known as Community Acquired Respiratory Distress Syndrome Toxin (CARDS TX). The crystal structure of this 591‐aa cytotoxin reveals a triangular molecule comprised of an N‐terminal ADP‐ribosylating domain and a C‐terminal tandem β‐trefoil domain that is responsible for vacuolation of the host's cells. Based on structural and sequence homology to other ADP ribosylating toxins, the NAD+ binding site has been predicted, yet none of the published structures of the CARDS toxin contain NAD+. The loop connecting the N− and the C‐termini is anchored using a disulfide bond that surrounds the NAD+ binding site which might sterically hinder NAD+ binding. An N‐terminal truncation mutant (NTTM) lacking the disulfide bond has been cloned and the protein was expressed, purified and crystallization trials are underway. The NTTM was also used to treat U937 cells in presence and absence of NAD+ to test the effect of the NTTM on cytokine secretion. The data show that NTTM causes an increase in the secretion of IL‐1β and TNF‐α in presence of NAD+ suggesting an increase in the inflammatory response to the NTTM in presence of NAD+.

Support or Funding Information

St. Mary's University Biaggini Funds and The San Antonio Livestock Expedition Funds

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 Effect of Organophosphate (OP)‐Induced Structural Changes in Acetylcholinesterase on Kinetics of OP Inhibition and Oxime Reactivation
Authors: Yu Wanlu , Kovalevsky Andrey , Blumenthal Donald K. , Cheng Xiaolin , Gerlits Oksana , Fajer Mikolai , Ho Kwok‐Yiu , Taylor Palmer , Radic Zoran
Published: June 2021

Organophosphate (OP) pesticides and nerve warfare agents covalently inhibit human acetylcholinesterase (hAChE) compromising cholinergic neurotransmission that can lead to fatal outcome. Effective therapy of OP intoxication requires that both, excess OP is removed from circulation and that activity of OP inhibited tissue hAChE is recovered. For both reactions nucleophilic oxime reactivation can play an essential role. For oxime assisted OP bioscavenging it is also important that the rate of inhibition of bioscavenger AChE is not compromised. Recently determined structures of diethylphosphorylated hAChE (POX‐hAChE) formed by paraoxon (POX) inhibition, both our unpublished and PDB released (5HF5 and 5HF8) show significant structural changes of the acyl pocket, immediately close to the active Ser203 when compared to the non‐inhibited enzyme. Those are likely to compromise nucleophilic oxime attack and rate of reactivation, but could also influence rate of hAChE inhibition by POX. Inhibition of AChE by dimethylphosphate analogue of POX (dmPOX) does not lead to conformational changes in the conjugated AChE (dmPOX‐AChE). In order to investigate effect of conformational changes in POX‐hAChE on rates of POX inhibition and oxime reactivation we studied dependence of rates of those reactions on reaction temperatures compared to dmPOX reactions. No difference was observed in temperature dependences of inhibition by diethyl‐ and dimethylphosphates. However, we observed strong temperature dependence of the oxime 2PAM reactivation, where reactivation of POX‐hAChE at 37 °C appeared an order of magnitude faster than the reaction at 22 °C. Reactivation of dimethylphosphorylated hAChE (dmPOX‐hAChE), as well as both dimetyhyl‐ and diethyl‐ phosphorylated Y337A/F338A hAChE mutant by 2PAM was significantly less temperature dependent, consistent with the absence of compromising conformational changes in dmPOX‐hAChE and in the mutant. This result indicates that formation of distorted acyl pocket loop in POX‐hAChE is not limiting for inhibition reaction but affects rates of oxime reactivation. Ability of POX‐hAChE to change acyl pocket loop conformation at the higher temperature allows for faster oxime reactivation and emphasizes the importance of conformational flexibility of hAChE for the efficient oxime reactivation.

Support or Funding Information

This work was supported by the CounterACT Program, National Institutes of Health Office of the Director, Grant 1U01NS083451 from NINDS.

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 JAVA Based Computational Tool for Pairwise Comparison of Protein Backbone Folds in Liganded and Apo 3D Structures of the alpha/beta Hydrolase Fold Proteins
Authors: Zheng Zihan , Rohrer Jacqueline , RADIC ZORAN
Published: June 2021

Cholinesterases, carboxylesterases and lipases are some of prominent catalytically active proteins with specifically similar, globular tertiary structure fold termed “alpha/beta hydrolase fold”. Ligand binding, both reversible and covalent, frequently results in small, yet systematic and potentially functionally important changes in the conformation of their alpha carbon backbones that can be easily overseen by commonly used overlays based on overall minimization of backbone atom RMSD analysis. In particular, backbone conformations of cholinesterases ‐ acetylcholinesterase and butyrylcholinesterase ‐ show only small conformational changes. This is revealed in more than 200 PDB deposited cholinesterase X‐ray structures in complexes with structurally diverse ligands that affect their function.

We developed a novel, reference point based principle for overlay‐independent pairwise comparison of liganded and non‐liganded alpha carbon backbone conformations from respective PDB (Protein Data Bank) deposited 3D structure entries and encoded it in JAVA based computer algorithm for quick analysis. Comparisons are based on differences in distances between each alpha carbon and chosen reference point in each pair of compared structures, as well as on differences in the angle between center of mass, reference point and each of alpha carbons in the comparison. Combination of the two comparison criteria reveals subset of backbone alpha carbons in two structures that maintains best their relative positions in the 3D space and that can be used as tethering points for overlay of compared structures. Created overlay has capacity to reveal small but systematic local changes in the backbone conformation frequently masked by the overall RMSD minimization approach.

Our systematic analysis of a number of PDB deposited alpha/beta hydrolase fold structures revealed small, yet functionally important conformational changes in their backbone consistent with experimental observations and instrumental in revealing molecular mechanisms in respective catalytic reactions. These findings are important for complete molecular target template characterization in the structure based development of novel antidotes of exposure to organophosphates which covalently inhibit this family of enzymes.

Support or Funding Information

This work was supported by the CounterACT Program, National Institutes of Health Office of the Director, Grant R21 NS098998 from NINDS.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dissecting the molecular basis of a phenylketonuria‐causing mutation in phenylalanine hydroxylase
Authors: Khan Crystal A. , Meisburger Steve P. , Ando Nozomi , Fitzpatrick Paul F.
Published: June 2021

Phenylketonuria (PKU) is a metabolic disorder in which the conversion of excess dietary phenylalanine to tyrosine is disrupted. Phenylalanine hydroxylase (PheH) is responsible for this hydroxylation reaction and uses tetrahydrobiopterin (BH4) and molecular oxygen as cosubstrates. The prevalence of PKU in the U.S. is 1 in 10,000 newborns and was the leading cause of mental retardation prior to the discovery of treatment by a protein‐restricted diet. R68S PheH has been shown to cause a mild form of PKU but the mechanism of action has been controversial. Studies have reported that mutations to Arg68 cause the enzyme to become fully active and lose sensitivity to phenylalanine as an allosteric activator. Arg68 is located in the regulatory domain of PheH, which is responsible for the activation of the enzyme. The current model for activation states that the regulatory domains of two subunits on the tetrameric PheH exist in a monomer‐dimer equilibrium and the dimer is stabilized by binding of phenylalanine. We characterized R68S PheH and found that the R68S PheH is fully active compared to the wild‐type enzyme. Sedimentation velocity experiments showed the isolated regulatory domain of R68S PheH does experience a concentration‐dependent shift from a monomer to a dimer; however, phenylalanine had no effect on this shift. The mutant protein displayed a fluorescence emission spectrum different from that of the wild‐type protein that is unchanged in the presence of phenylalanine. SAXS experiments showed no transitions when the protein was treated with 1 mM phenylalanine. Limited proteolysis experiments showed R68S PheH was more proteolytically sensitive than wild‐type PheH. The data suggest that R68S PheH exhibits constitutively active behavior due to the destabilization of the regulatory domain, which causes the protein to be more proteolytically sensitive.

Support or Funding Information

NIH F31GM116452

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Introducing allosteric regulation into homing endonucleases via tryptophan modification
Authors: Danon Sonja , Kaiser Brett K.
Published: June 2021

Homing endonucleases (HEGs) are DNA cleaving enzymes present in microbial genomes that recognize unusually long DNA target sites (20–22 bp). Given their high specificity for DNA targets, these enzymes may be extremely useful genome editing reagents. Introducing allosteric control into these enzymes would enable them to have a broader applicability in genome editing approaches. Previous studies have shown that tryptophan residues near the active site of enzymes can play an important role in enzyme activity. Mutating these residues to glycine results in loss of enzymatic activity and can be rescued by the addition of indole in solution. However, this approach hasn't been demonstrated with DNA cleaving enzymes. The goal of this study is to test whether this approach can be used to introduce allosteric regulation into HEGs. We generated tryptophan to glycine single point mutations in I‐SmaMI, a homing endonuclease of the LAGLIDADG family, based on the structure of I‐SmaMI bound to DNA. We are expressing these proteins in bacteria and utilizing an in vitro cleavage assay to assess the level of activity of the mutants in the presence and absence of indole. These studies may have important genome editing applications and are likely to provide insight into the mechanism by which HEGs bind and cleave DNA.

Support or Funding Information

Murdock Charitable Trust

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 High‐Throughput Assay to Measure Phosphoenolpyruvate Carboxykinase
Authors: Li Shunan , Cheung Keith , Tchaga Grigoriy , Yuan Gordon , Xu Jianping
Published: June 2021

Phosphoenolpyruvate Carboxykinase (PEPCK EC 4.1.1.32) catalyzes the conversion of oxaloacetate (OAA) into phosphoenolpyruvate (PEP) in the presence of GTP. In humans, two isoforms of PEPCK are found: cytosolic form (PEPCK‐C, also called PCK1) and mitochondrial form (PEPCK‐M, also called PCK2). PEPCK‐C (PCK1) is a rate‐controlling enzyme in gluconeogenesis. Recent studies found abnormal concentrations of PEPCK in diabetic mice. Overexpression of PEPCK attenuates insulin signaling and decreases hepatic insulin sensitivity in transgenic mice. Our high throughput assay for Phosphoenolpyruvate Carboxykinase activity provides a valuable tool for both mechanistic and therapeutic studies of diabetes as well as avenue for targeting PEPCK activity.

The Phosphoenolpyruvate Carboxykinase activity assay is developed by using a set of specific enzymes that convert Phosphoenolpyruvate (PEP) and carbonate into a series of intermediates and hydrogen peroxide, which in turn, reacts with a probe generating a colorimetric signal (OD570 nm). The color intensity is directly proportional to the amount of active Phosphoenolpyruvate Carboxykinase. This assay is simple, sensitive, high‐throughput adaptable and can detect less than 10 μU of Phosphoenolpyruvate Carboxykinase activity per sample.

In conclusion, a Phosphoenolpyruvate Carboxykinase (PEPCK) Activity Assay kit is developed. The kit provides easy and robust tool for measuring PEPCK activity in biological and purified samples (BioVision, Inc. Cat. # K359‐100).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Validation of Cdc42 Inhibition as the Mechanism of Action of the Anti‐metastatic Drug MBQ‐167, Using Budding Yeast as a Model System
Authors: Rivera‐Robles Michael John , Medina‐Velázquez Julia , Asencio‐Torres Gabriela , González‐Crespo Sahily , Rymond Brian C. , Rodríguez‐Medina José R. , Dharmawardhane Suranganie
Published: June 2021

Cdc42 (Cell division control protein 42 homolog), a Rho GTPase, is highly conserved among eukaryotes in its structure and function. Mechanical or chemical cues in the microenvironment stimulate the localized activation of Cdc42 to rearrange the actin cytoskeleton and regulate cell polarity and motility. Its role in cell polarization was first discovered in the budding yeast Saccharomyces cerevisiae, but was subsequently shown to also establish directional motility in animal cells. Accordingly, in cancer, Cdc42 promotes migration, invasion, and spread of tumor cells. Thus, we targeted Cdc42 as a therapeutic strategy to treat metastatic breast cancer, and designed and characterized the small molecule MBQ‐167 as a potent inhibitor against Cdc42 and the homolog Rac. We recently reported that MBQ‐167 inhibits cancer cell and tumor growth and spread in‐vitro and in‐vivo using a mouse xenograft model of metastatic breast cancer. Since budding yeast express a single Cdc42 gene and do not express Rac, we used this well characterized model of polarization to validate the MBQ‐167 specificity for Cdc42 inhibition. Using a tetracycline regulated transcriptional inactivation (Tet‐Off) system in the promoter region of the yeast CDC42 gene, we knocked‐down Cdc42 supplementing the media with doxycycline (DOX; IC=10μg/μL). Next, we analyzed the growth, budding pattern, and Cdc42 activity of yeast exposed to DOX, MBQ‐167, or in combination. As expected, maximum growth, doubling time and budding polarity of yeast with the Tet‐Off system were reduced when treated with DOX (EC50=10μg/mL). The same effect was seen with MBQ‐167 (ED50=100μM) alone and in combination with DOX. Ultimately, pull‐downs of the active Cdc42‐GTP complex confirmed our hypothesis that MBQ‐167 disrupts cell polarity through impeding Cdc42 activation. Hence, we have validated MBQ‐167 as a Cdc42 inhibitor in another biological context and present a method to screen Cdc42 inhibitors as potential therapeutics against cancer metastasis.

Support or Funding Information

This work is partially supported by grants from the Susan G Komen for the Cure and the Puerto Rico Science and Technology Research Trust (S.D.), NIH/NIGMS P20GM103475 (JRRM), and NIH/NIGMS‐RISE R25 GM061838 (MJR)

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 kinetic assay for non‐automated drug screening
Authors: Zem Gregory C. , Chimayan A , Aleksanyan V , Gordon J , Gomez F , Seyedroudbari A , Chang J , Botello T , Tan N , Arefin D , Tobar D , Khachekian A , Gama L , Durodola E , Batty J , Plascencia C , Barillas L , Roverud A , Kreuz S , Sarkisyan L , Lee F , Munoz J , Reque L , Abed V , Kinog L , Oppenheimer SB
Published: June 2021

A non‐automated kinetic assay has been used in this laboratory to screen reagents that unclump fixed yeast, a model for unclumping human cancer cells, human blood cells and pathogens (FASEB J, 26:62ab; 28:234ab; 29:65ab; 31:539ab). Fixed yeast is the material of choice because its surface properties are similar to live cells without unknowns associated with live cell metabolism (Acta Histochem 104: 99–106). Here the assay is tested to determine if it also can be used to assess cell clumping instead of unclumping, of importance in many drug screening venues, using a calcium induced flocculation model. In addition, the precision of the assay is tested using reagents that do not clump or unclump cells. O.05 ml of Prefer (Anatech Ltd, Battle Creek, MI) fixed yeast (Saccharomyces cerevisae) were added to 1 ml droplets of deionized water on glass microscope slides. The percentages of single yeast and clumps (2 or more cells) were assessed with light microscopes (100X, 200X) before and after addition of 1, 2 or 3 mg of calcium sulfate dihydrate (droplets with no additions served as controls) and at 20 min, 40 min and 60 min with agitation. The results showed that calcium sulfate flocculated the yeast in a concentration and time dependent manner. For example for calcium sulfate, 1, 2 and 3 mg per ml, in 30 trials, with p values generally below 0.05 and tight standard error bars, the percentages of single yeast at 60 min were: 1 mg/ml 40 percent vs 67 percent in controls, 2 mg/ml 27% vs 67 percent in controls, 3 mg/ml 14 percent vs 72 percent in controls. The assay, therefore, assesses the kinetics of cell clumping (flocculation). When 3 reagents, D‐melizitose, D‐melibiose, L‐ascorbic acid were tested at 1, 2 and 3 mg per ml at 20, 40 and 60 min, the assay picked up little or no effect on the yeast. For example in 90 trials with tight error bars each time point showed little to no significant difference between experimentals and controls. The results indicate that the assay kinetically measures cell clumping and is reproducible and precise in generating data. The non‐automated assay is ideal for drug screening by hundreds of investigators at a time especially when expense is a consideration.

Support or Funding Information

CSUN Foundation and Corporation Accounts, U.S. Presidential Award

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanistic Studies of Drug‐Like Inhibitors of P‐glycoprotein using ATPase Assays, Electron Spin Resonance Spectroscopy and Cancer Cell Models
Authors: Chen Gang , Ballou‐Crawford Jett , Aljowni Maha , Lippert Alexander , Wise John G. , Vogel Pia D.
Published: June 2021

Multidrug resistance (MDR) in cancer and other diseases is frequently associated with transmembrane efflux proteins, one of which is P‐glycoprotein (P‐gp). Over‐expression of P‐gp in cancer cells increases the efflux of therapeutic drugs rendering them ineffective. In order to re‐sensitize multidrug resistant cancers to chemotherapy, we have found inhibitors of P‐gp by in silico screening methods (Brewer et al., Molecular pharmacology 86, 716–726, 2014), and several of these inhibitors were shown to successfully overcome MDR in the drug resistant DU145TXR prostate cancer cell line (Follit et al., Pharmacol. Res. Perspect. 3, e00170, 2015). In this study, we expanded our cell culture screening to evaluate 25 new compounds identified by our in silico screening method and report that over 30% of these compounds re‐sensitized DU145TXR cancer cells to chemotherapy, while not exhibiting significant toxicity to the non‐cancerous human lung fibroblast (HFL‐1) cell line. The best of the identified hit compounds were further investigated with regard to the mechanism of inhibition using ATPase activity assays and electron spin resonance spectroscopy (ESR). Several of the best compounds were found to inhibit ATPase activity. Using a spin‐labeled ATP analog and ESR spectroscopy, we assessed the effects of the identified inhibitors on ATP binding to P‐gp. Variants of one of the original hit compounds, SMU 29, that were computationally predicted to interact more strongly with P‐gp have been synthesized and were analyzed here for their effects on P‐gp ATPase activity and ATP binding characteristics.

Support or Funding Information

This work is supported by NIH NIGMS [R15GM094771‐02] to John G. Wise, SMU University Research Council, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas, as well as the Hamilton Undergraduate Research Scholars Program and the SMU Summer Research Assistantship and Undergraduate Research Assistantship Programs.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Monitoring immune cells‐mediated cytolysis through impedance technology allows kinetic analysis of reagent efficacy
Authors: Cerignoli Fabio , Xi Biao , Guenther Garret , Muir Lincoln , Lamarche Brandon , Abassi Yama
Published: June 2021

In vitro characterization of immunotherapy reagents efficacy and combination synergies is necessary before moving to expensive animal models and clinical studies. Current standard assays like Chromium‐51 release, ATP‐based luminescence cell monitoring or flow cytometry are difficult to implement in a high throughput environment and are based on end point methodologies that are unable to capture the full dynamic of the immune response. Here we validate an impedance‐based platform for monitoring cytotoxic activity of immune cells in the context of cancer immunotherapy assays. The technology detects cell death and proliferation of adherent cells by measuring changes in conductance of microelectrodes embedded in 96 and 384‐wells cell culture plates, without the use of labeling or cell modification. We present a large set of validation including CD19 Car T models, EpCAM/CD3 BiTE antibodies and checkpoint combination therapies with anti PD‐1/PD‐L1 and CTLA‐4 antibodies. Data comparison with Annexin V staining/Flow Cytometry shows perfect correlation between the drop in impedance signal and % of apoptotic cells. Long time monitoring (> 7 days) in combination with low effector:target ratios reveal the occurrence of resistance mechanisms in target cells and exhaustion in effector cells. Such dynamic response has been observed in animal models and patient clinical trials, but it is difficult to monitor in short time assays. Overall, our results demonstrate the value of such label‐free approach in measuring the cytotoxic response across the temporal scale, an aspect that is otherwise very difficult to assess with more canonical end point assays but that is highly relevant when testing combinations. Thanks to the availability of 384‐wells format and minimal sample handling, the technology is ideally suited for applications in large reagent validation screening or therapeutic protocol validation directly on patient samples.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Discovery of novel glucose‐6‐phosphate dehydrogenase activators to correct G6PD deficiency
Authors: HWANG SUNHEE
Published: June 2021

Glucose‐6‐phosphate dehydrogenase (G6PD) deficiency, one of the most common human genetic disorders, is caused by over 160 different point mutations and results in a variety of health problems, including hemolytic anemia and neurological damage. As G6PD is a major source of protective anti‐oxidant through NADPH production, G6PD deficiency likely contributes to the severity of many other acute and chronic diseases associated with increased oxidative stress. As no medications are available to treat G6PD deficiency directly, we sought to identify a small molecule that increases catalysis of G6PD mutant enzymes. Crystal structure and mutagenesis analyses of one common mutant (Canton, R459L) provided insight into the functional defect of the enzyme. Using high‐throughput screening, we identified AG1, a small molecule that increases the activity of the wild‐type, the Canton mutant and several other common human G6PD mutants as well. AG1 reduced oxidative stress and increased NADPH levels in vivo, in zebrafish, and prevented chloroquine‐induced hemolysis of human erythrocytes. Our study suggests that a pharmacological agent, of which AG1 may be a lead, will likely alleviate the clinical problems associated with multiple variants of G6PD deficiency.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cell Cycle Regulation and Hypoxic Adaptation in the Pathogenic Yeast Cryptococcus neoformans
Authors: Kawamoto Susumu , Moranova Zuzana , Virtudazo Eric , Ohusu Misako , Raclavsky Vladislav
Published: June 2021

We have reported that the cell cycle behavior of the pathogenic yeast Cryptococcus neoformans (C. neoformans) is different from the cell cycle control exhibited by the model yeast Saccharomyces cerevisiae, and also have reported the molecular characterization and physiological roles of the two main eukaryotic cell cycle genes, C. neoformans cyclin dependent kinase 1 (CnCdk1) and cyclin homologues. We also identified components of the G2‐M control machinery, Wee1 kinase and Cdc25 phosphatase homologues, in C. neoformans. Wee1 and Cdc25 are involved in the inhibition and activation, respectively, of the Cdk1‐cyclin complex at the mitotic entry checkpoint in eukaryotes.

On the other hand, in the obligate aerobic pathogenic yeast C. neoformans, limited aeration has been demonstrated to cause slowdown in proliferation and delayed budding, resulting eventually in a unique unbudded G2‐arrest. The ability to adapt to decreased oxygen levels during pathogenesis has been identified as a virulence factor in C. neoformans. We have identified and characterized the gene that is necessary for the proliferation slowdown and G2‐arrest caused by limited aeration. Random mutants were prepared and screened for lack of typical slowdown of proliferation under limited aeration. The gene coding for a zinc‐finger transcription factor was identified in mutants showing most distinctive phenotype. Targeted deletion strain and reconstituted strain were prepared to characterize and confirm the gene functions. This gene was also identified in a parallel studies as homologous both to calcineurin responsive (Crz1) and PKC1‐dependent (SP1‐like) transcription factors. We have confirmed the role of the cryptococcal homologue of CRZ1/SP1‐like transcription factor in cell integrity, and newly demonstrated its role in slowdown of proliferation and survival under reduced aeration, in biofilm formation and in susceptibility to fluconazole. Our data demonstrate a tight molecular link between slowdown of proliferation during hypoxic adaptation and maintenance of cell integrity in C. neoformans and present a new role for the CRZ1 family of transcription factors in fungi. The exact positioning of this protein in cryptococcal signalling cascades remains to be clarified.

Support or Funding Information

This work was partially supported by Grants‐in‐Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan and Cooperative Research Program of Medical Mycology Research Center, Chiba 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.
Inhibition of Autophagy by Mevalonate Pathway Inhibitors, a New Therapeutic Approach to sensitize Glioblastoma Cells to Temozolomide Induced Apoptosis
Authors: Shojaei Shahla , Alizadeh Javad , Thliveris James , Koleini Navid , Kardami Elissavet , Hatch Grant , Xu FY , Hombach‐Klonisch Sabine , Klonisch Thomas , Ghavami Saeid
Published: June 2021

Glioblastoma multiforme (GBM) is the deadliest brain tumor with an approximate 20 month survival rate after diagnosis and treatment. Temozolomide (TMZ), the chemotherapeutic drug of choice for GBM, is an alkylating agent that causes DNA damage. TMZ treatment results in the induction of apoptosis in GBM cells, however, it induces autophagy and consequently chemoresistance. Statins are mevalonate (MEV) cascade inhibitors with beneficial effects on the enhancement of the survival rate of patients with different types of cancer, particulary if consumed long term before diagnosis of cancer. Here, we determined the effect of simvastatin (Simva), a blood brain barrier permeable statin, on the sensitization of GBM cells to TMZ induced apoptosis through inhibition of autophagy flux. We pretreated two GMB cell lines, U251 and U87 cells, with low doses of Simva (1 and 2.5 μM, respectively) with or without different intermediates of the mevalonate cascade: MEV (2.5 mM), Farnesyl pyrophosphate (FPP) (15 μM), Geranylgeranyl pyrophosphate (GGPP) (15 μM) and cholesterol (50 μM) for 4 h and then treated cells with TMZ (100 μM) for 48–96 h. A signficiantly reduced viability and increase in the population of apoptotic dead cells were observed in GBM cells treated with the combination of Simva‐TMZ compared to cells treated with TMZ alone. Addition of MEV, FPP, GGPP and cholesterol did not attenuate these effects significantly. Sima‐TMZ treatment did not alter the total cholesterol pool in U87 and U251 cells compared to controls. Western blot analysis, immunocytochemistry and transmission electron microscopy revealed that the combination of Simva with TMZ inhibited autophagic flux. Overall, the results suggest that sensitization of GBM cells to TMZ‐induced apoptosis by Simva is independent on the cholesterol biosynthetic pathway but may involve inhibition of autophagy flux.

Support or Funding Information

Supported by funding from a Mitacs Accelerate PDF Award & a Health Science Center Foundation Operating 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.
Muscarinic Antagonist Mediated Activation of ERK Signaling Depends on β‐arrestin Recruitment to Augment Axonal Outgrowth in Neurons
Authors: Sabbir Mohammad Golam , Fernyhough Paul
Published: June 2021

Objective

One of the major cellular effectors activated by G protein coupled receptors is extracellular signal‐regulated kinase (ERK). The ERK signaling cascade regulates a variety of cellular processes including growth and proliferation. Both G protein and β‐arrestin mediated signaling pathways lead to ERK activation by phosphorylation through different kinases. Recently, we have shown muscarinic acetylcholine type 1 receptor (M1R) antagonists, muscarinic toxin 7 (MT7) and pirenzepine, induced elevated neurite outgrowth and protected from small and large fiber neuropathy in adult sensory neurons in various animal models. Thus, we hypothesized that the neuritogenic effect of muscarinic antagonists was mediated by M1R‐ERK signaling.

Methodology

We have used extensive two dimensional isoelectric focusing/SDS‐PAGE combined with analysis using multiple phospho‐epitope specific antibodies to study ERK1/2 phosphorylation and activation of its downstream nuclear effector ‐ cyclic response element binding protein (CREB) in cultured adult rat primary dorsal root ganglion (DRG) neurons. Activation of CREB is known for neuroprotective and growth promoting effects. In addition, we have used CRISPR/Cas9 based β‐arrestin1/2 and GαS/L/Q/11/12/13 null HEK293 cell lines to dissect the downstream signaling events following antagonist binding to M1R.

Results

MT7 and pirenzepine‐mediated M1R‐ERK signaling induced a significant increase in activation‐loop specific phosphorylation (T202/Y204) of ERK1/2 and significantly elevated pCREB(S133) levels in cultured sensory neurons. Further, using M1R expressing β‐arrestin1/2 and GαS/L/Q/11/12/13 null HEK293 cell lines, we have shown that MT7 or pirenzepine mediated ERK1/2 activation is positively dependent on β‐arrestin recruitment to M1R but negatively regulated by G proteins. We have identified multiple charged fractions of ERK1/2 modulated by muscarinic antagonist treatment that is indicative of complex dynamics related to allosteric drug binding.

Conclusions

Our study provides evidence that the neuritogenic effect of MT7 and pirenzepine may be mediated by selective blockade of M1R and downstream activation of ERK‐CREB signaling. We also give insights into allosteric drug binding and complex dynamics of ERK activation. In addition, our study also indicates that removal of negative regulation by G proteins on antagonist‐M1R‐ERK signaling could be exploited for improved therapy in neuropathic diseases.

Support or Funding Information

Supported by CIHR grant # MOP‐130282 (PF)

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 the putative GTPase activity of the Ellis van Creveld protein 1
Authors: Nguyen Giang T. , Odunuga Odutayo O.
Published: June 2021

Ellis van Creveld (EvC) syndrome is a genetic disorder characterized by mild short stature, postaxial polydactyly, dysplastic nails and teeth. Loss‐of‐function EVC1 and EVC2 proteins resulting from mutations in EVC1 and EVC2 genes, respectively, have been identified as the primary causes of EvC syndrome. Essential to their function is the physical interaction of EVC1 and EVC2 proteins at the base of the primary cilium, where this complex regulates the Sonic and Indian hedgehog (Hh) signaling systems. Recent studies have revealed that this EVC1‐EVC2 protein complex is tethered to the base of the primary cilium by another complex comprising of two newly identified proteins, EFCAB7 and IQCE. Together, this multiprotein complex positively regulates downstream processes after signaling activation by hedgehog proteins. The presence of a putative P‐loop sequence in EVC1 strongly suggests that its interaction with EVC2 is dependent on nucleotide hydrolysis. Using protein fragments, we have experimental evidence that suggest that EVC1 possesses an intrinsic GTPase activity conferred by the presence of the P‐loop. This observation may provide insight into the function and regulation of the EVC1‐EVC2 complex in hedgehog signaling pathways.

Support or Funding Information

Welch Foundation Grant # AN‐0008

FRG‐RCA Grant, SFASU

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