bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of ligand‐based inhibitors for protein N‐terminal methyltransferase
Authors: Huang Rong
Published: June 2021

Protein alpha‐N‐terminal methyltransferases (NTMTs) are emerging members of protein methyltransferases and methylate the alpha‐N‐terminal amine of proteins starting with an X‐P‐K/R motif. Since its discovery in 2010, NTMT1 has been implicated in regulating chromatin interactions, mitotic division and DNA damage repair. Therefore, specific NTMT1 inhibitors are important tools to elucidate its biological and pharmacological functions. Our lab has developed the first mechanism‐based inhibitors by covalently connecting both substrates to mimic its ternary complex. However, there is no specific and cell‐potent inhibitor available for NTMT1 until now. Based on our crystal structures of NTMT1 ternary complexes, we identified the unique specificity of NTMT1 substrate binding pocket which is in contract to other protein methyltransferases. Thus, we applied a ligand‐based approach to rationally design peptidomimetic inhibitors and optimized in aid of NTMT1 structural information. Our top synthesized inhibitor has a Ki of 440 nM and its inhibition is through specific targeting the protein substrate binding site instead of the SAM binding site. Moreover, the inhibitor exhibits more than 100‐fold selectivity to NTMTs over nearly 40 other methyltransferases. In summary, we have successfully developed the first potent and specific peptidomimetic inhibitors. Our studies have laid a paradigm for the next optimization towards developing cell‐potent inhibitors to interrogate the biological roles of NTMTs.

Support or Funding Information

NIH R01 GM117275

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Urinary Platelet‐Activating Factor as an Indicator of Interstitial Cystitis/Bladder Pain Syndrome
Authors: McHowat Jane , Isbell T Scott , Campian E Cristian
Published: June 2021

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic inflammatory bladder disease with multiple etiological contributors and risk factors associated with its development, including cigarette smoking. In a recently published manuscript, we have shown that smoking is associated with bladder wall inflammation and accumulation of platelet activating factor (PAF), a potent inflammatory mediator that facilitates transendothelial inflammatory cell migration and reduction in tight junction protein expression, which could contribute to urothelial cell dysfunction. PAF accumulation in response to cigarette smoking is primarily the result of the inhibition of PAF acetylhydrolase (PAF‐AH, the enzyme responsible for PAF hydrolysis and inactivation). The present study was designed to determine whether urine content of PAF or PAF‐AH activity can be used as an indicator of IC/BPS. For stability studies, voided urine specimens were collected from patients with and without a diagnosis of IC/BPS. Samples were stored at room temperature and frozen in liquid nitrogen at intervals up to 60 mins following voiding. PAF content and PAF‐AH activity were measured and normalized to creatinine. Measurement of PAF and PAF‐AH was consistent at all time intervals up to 60 mins, demonstrating stability following voiding. Addition of PAF internal standard to urine samples prior to analysis indicated a recovery of >96% PAF. PAF content in the urine of IC/BPS patients (1.16 ± 0.16 PAF:creatinine ratio, n=6, p<0.05) was significantly greater than that for non‐IC/BPS patients (0.37 ± 0.18 PAF:creatinine ratio, n=5). PAF content was also greater in IC/BPS patients who smoke (1.19 + 0.36 PAF:creatinine ratio, n=4, p<0.05) when compared to non‐IC/BPS patients. PAF‐AH activity was decreased in the urine of IC/BPS patients who were smokers (48.3 ± 2.4 PAF‐AH: creatinine ratio, n=4, p<0.05) or non‐smokers (40.0 ± 7.0 PAF‐AH: creatinine ratio, n=6, p<0.05) when compared to non‐smokers with no IC/BPS (64.6 ± 2.2 PAF‐AH:creatinine ratio, n=5). Our data demonstrate that urine content of PAF and/or PAF‐AH is stable at up to 60 mins storage at room temperature following voiding. Urinary PAF is increased in IC/BPS patients, and is likely the result of PAF‐AH inhibition. Future studies are required to determine the effect of cigarette smoking on the incidence and severity of IC/BPS.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterizing Binding Interactions and Elucidating Structure of Aptamer‐Based Biosensors
Authors: Armstrong Lindsay Therese , Bonham Andrew J.
Published: June 2021

Electrochemical biosensors based on the conformational dynamics of DNA aptamers have found success against a wide variety of proteins, toxins, antibodies, and heavy metals. However, the mechanistic underpinnings of the mechanism by which these surface‐bound DNA molecules change conformation upon target binding, thus changing the dynamics of an appended redox‐active tag and generating a measurable signal, is poorly understood. Our first target for investigation was a previously reported Ricin Chain A binding aptamer biosensor. Here, we have investigated this biosensor using a variety of nucleic acid assays, including PCR‐termination via basepair modification, fluorescence anisotropy, gel mobility shift assays, and FRET tagging to determine 3D orientation. These have allowed us to better characterize the basepair interactions involved in binding targets, as well as offer clues to the changing three dimensional folded structures of these biosensors. These results will help inform the field of biosensors and aptamers in general on strategies for future optimization.

Support or Funding Information

This work is supported by internal grant funding from the Metropolitan State University of Denver.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of Novel Biosensors for Non‐Invasive and Rapid Diagnosis of Celiac Disease
Authors: Nguyen Anna , Bonham Andrew J.
Published: June 2021

There is a pressing need for rapid, non‐invasive, and accurate detection methods to diagnose individuals with the autoimmune disorder celiac disease (CD). It is estimated to affect up to two million Americans, and is caused by an autoimmune response to consuming gluten, resulting in damage to the lining of the gastrointestinal tract that leads to abdominal pain, diarrhea, nausea, and many other complications. Accurate diagnosis of CD remains challenging; the current diagnostic procedure involves multi‐day laboratory protocols, followed by an invasive duodenal biopsy. Here, we demonstrate work towards a blood sample‐based diagnostic tool to allow for early identification of CD in patients. Our method utilizes known autoimmune interactions that occur in the body of an individual with CD; specifically, when they consume gluten, a part of the gluten incorrectly binds to an enzyme in the bloodstream, and the resultant structure that forms between the gluten and the enzyme is targeted by their immune system. Researchers have identified how parts of this novel structure specifically interacts with the autoantibodies involved in CD. These peptide epitopes closely recapitulate the disease state. We have thus created a biosensor that relies on using these peptide epitopes as the capture probe to detect levels of disease‐specific antibodies. This strategy was incorporated into an electrochemical DNA (E‐DNA) biosensor; a common type of biosensor that utilizes small changes in electrical conductivity to measure whether a target molecule is bound or unbound. This sensor design can, in principle, use unprocessed finger‐lancet blood samples to provide a rapid digital readout, allowing for efficient and sensitive detection of CD in patients.

Support or Funding Information

This work is supported by internal grant funding from the Metropolitan State University of Denver.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparative lipidomic profiling of parasite‐infected and non‐infected Northern saw‐whet owls.
Authors: Peros Colin S. , Garrett Teresa A.
Published: June 2021

Haematozoa are present in avian species around the world. In some cases, parasite infection may be fatal. For example, Plasmodium and Leucocytozoon cause avian malaria and anemia, respectively, which can be detrimental to host species. These parasites are responsible for massive reductions in the populations of birds native to Hawaii, the Galapagos, and other archipelagos. However, the same parasites have seemingly benign effects in other avian species. Here, we use liquid chromatography‐mass spectrometry to analyze lipid profiles and study host‐parasite interactions in a population of Northern saw‐whet owls (Aegolius acadicus) known to be infected with Leucocytozoon (49.5%), Plasmodium (10%), Haemoproteus (5%), and Trypanosoma (2.9%). Fragment ions from MS/MS analyses revealed major classes of glycerophospholipids, including phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and phosphatidylcholine (PC). Comparison of the total lipid profiles of infected and non‐infected owls revealed distinct features (p < 0.001) between the two groups in the negative‐ (30) and positive‐ion (376) mode. The lower levels (>1.7‐fold difference) of PC in infected individuals suggests haematozoa infections may influence lipid production, which may impact the health of birds. Further investigations can advance our understanding of bird physiology and the role lipid biomarkers may play in the conservation of parasite‐susceptible avian species.

Support or Funding Information

Supported by National Science Foundation Major Research Instrumentation Grant #1039659.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
P‐cadherin Overexpression Enhances Survival Signaling in Dysplastic Oral Epithelia via the Epidermal Growth Factor Receptor
Authors: Neal Christine , Mody Aaron , Parker Ashley , Cameron Emily , Plager Susan , Lawson Kathryn
Published: June 2021

P‐cadherin is a calcium‐dependent transmembrane protein that mediates cell‐cell adhesion within oral epithelial tissue. Previous histological analyses have suggested that P‐cadherin protein levels increase in oral dysplasia and subsequently decrease in oral squamous cell carcinoma (OSCC). The epidermal growth factor receptor (EGFR) gene has also been shown to undergo amplification in the dysplastic stage of oral tumor development. In comparative studies, we found that levels of P‐cadherin were increased two‐fold in dysplastic oral keratinocytes (DOK) compared to primary human oral keratinocytes (HOK) obtained from normal oral mucosa. We hypothesized that in dysplastic oral epithelia, upregulated P‐cadherin cooperates with amplified EGFR to enhance survival signaling and facilitate tumor progression. To further explore the interaction of P‐cadherin and EGFR, DOK cells were retrovirally infected to generate stably‐selected control and P‐cadherin‐overexpressing cell lines. We have previously demonstrated that the DOK cell line exhibits comparatively high levels of EGFR expression. We found that P‐cadherin overexpression increased basal, as well as EGF‐stimulated EGFR phosphorylation levels. Furthermore, this was accompanied by increased phosphorylation of a downstream effector of EGFR, signal transducer and activator of transcription (STAT3). This increased phosphorylation was not dependent on an increase in total STAT3 protein level. STAT3 phosphorylation suggested activation of a pro‐survival mechanism, a finding that was further supported by P‐cadherin‐dependent increases in the anti‐apoptotic protein Bcl‐xL. When exposed to chemotherapeutic agents such as paclitaxel and carboplatin, P‐cadherin overexpression increased cell survival compared to control cells. P‐cadherin‐overexpressing cells treated with paclitaxel also exhibited decreased apoptosis. Further analysis confirmed that the difference in cell death was not due to altered proliferation of the two cell lines. We also explored the possibility that EGFR was activated by P‐cadherin‐mediated adherens junction formation. In contrast to ligand‐dependent signaling, we observed that calcium‐dependent cadherin engagement suppressed the phosphorylation of both EGFR and STAT3. These studies suggest that the upregulation of P‐cadherin, together with increased EGFR expression, promotes tumor progression in dysplastic oral epithelia through the activation of prosurvival signaling pathways.

Support or Funding Information

Midwestern University Glendale College of Osteopathic Medicine, College of Health Sciences, College of Veterinary Medicine

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
IQGAP1 Binds The Axl Receptor Tyrosine Kinase And Inhibits Its Signaling
Authors: Gorisse Laetitia , Li Zhigang , Hedman Andrew C. , Sacks David B.
Published: June 2021

IQGAP1 is a scaffold protein that integrates cell signaling pathways by binding several growth factor receptors and intracellular signaling molecules (Trends Cell Biol 2015; 25:171–184). The Axl tyrosine kinase receptor is important in hematopoiesis, the innate immune response, platelet aggregation, engulfment of apoptotic cells and cell survival (Cold Spring Harb Perspect Biol 2013; 5:a009076). Binding of growth arrest‐specific protein 6 (Gas6) to immunoglobulin‐like (Ig‐like) domains of Axl activates intracellular signaling. However, the mechanism of inactivation of the Axl receptor is poorly understood. Here, we investigate the interaction between IQGAP1 and Axl. Using GST pulldown assays and immunoprecipitation from mammalian cell lysates, we found that IQGAP1 associates with Axl in cells. Analysis with multiple discrete fragments of each protein revealed that the binding is direct and mediated by the IQ domain of IQGAP1 and the Ig‐like domains of Axl. We observed by proximity ligation assays that Axl:IQGAP1 complexes are located in the cytoplasm, and this interaction is reduced by 64% when cells are incubated with Gas6. Consistent with these findings, reducing IQGAP1 levels in cells by CRISPR/Cas9 enhanced the ability of Gas6 to stimulate both Axl phosphorylation and activation of Akt. Moreover, reduction of IQGAP1 in cells promoted Gas6‐stimulated transcription of matrix metalloproteinase (MMP)‐2, MMP‐3, MMP‐9 and urokinase‐type plasminogen activator genes. Finally, we observed by confocal microscopy that IQGAP1 downregulates the heterodimerization of Axl with the epidermal growth factor receptor. In summary, we identify IQGAP1 as a previously undescribed suppressor of Axl. These findings differ from the previously reported functions for IQGAP1 as a scaffold that promotes signaling by growth factor receptors (J Biol Chem 2007; 292:3273–3289; J Biol Chem 2011; 286:29734–29747; J Biol Chem 2011; 286:15010–15021). Our data expand the repertoire of binding partners for both Axl and IQGAP1, and provide additional insight into the regulation of Axl function.

Support or Funding Information

This work was supported by the National Institutes of Health Intramural Research Program.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Insulin Modulates Hippo Signaling by Impairing YAP Function
Authors: Hedman Andrew , Sayedyahossein Samar , Sacks David
Published: June 2021

Yes‐associated protein (YAP) is the main transcriptional coactivator of the Hippo pathway, which regulates organ size, tissue homeostasis, and regeneration. YAP integrates multiple inputs from different signaling cascades. Recent evidence implicates YAP also in the control of nutrient and energy status of the cell, but the mechanisms underlying this function are unclear. In this study, we show that insulin modulates YAP activity in classic insulin target cells. We found by subcellular fractionation and Western blotting that insulin increases YAP phosphorylation, with a significant decrease in the abundance of YAP in the nuclei of HepG2 liver cells. Further, this regulation is likely mediated through the insulin receptor (IR), as overexpression of IR increased YAP phosphorylation. Proximity ligation assay analysis revealed a marked reduction in the interaction of YAP with TEA domain (TEAD) transcription factors in the nuclei of insulin‐treated cells. Consistent with these findings, insulin significantly impaired YAP/TEAD‐mediated transcription as measured by a dual luciferase assay in both HepG2 and C2C12 muscle cells. Further, quantitative PCR of HepG2 cells treated with insulin revealed reduced transcription of YAP regulated genes. Insulin alters transcription of numerous genes relevant to energy homeostasis, and we observed that knockdown of YAP by siRNA alters expression of genes in gluconeogenesis. Insulin stimulates activation of the phosphoinositide 3‐kinase (PI3K) pathway. The inhibitory effect of insulin on YAP transcription was abrogated by small‐molecule inhibitors of PI3K. Collectively, our results identify insulin as a previously undescribed suppressor of YAP activity and provide insight into an important link between the insulin and Hippo pathways.

Support or Funding Information

This work was supported by the National Institutes of Health Intramural Research Program.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Aqueous Extract of Lavender Demonstrates Immunomuodulatory Effects Through Activation of NFkb.
Authors: Butala Austin Gregory , Byrd Shere , McLain Katherine
Published: June 2021

Lavender flower is commonly used in teas, lotions and other commercial products, and the oil extract and several of its components have been shown to be beneficial in reduction of inflammation. However, the effects of the water soluble components of lavender flowers has not been examined. We hypothesized that aqueous components might also have immunomodulatory effects. In a mouse macrophage cell line (RAW 264.7) we examined TNFa production and the downstream activation of NFkb associated with treatment with aqueous extract of lavender and its primary components, coumarin and herniarin. Unlike oil extract, treatment with aqueous extract for 24 hr resulted in a significant increase in TNFa. Downstream there was also a significant increase in IKK phosphorylation and an increase in NFkb translocation into the nucleus. Individual molecular components of the aqueous extract, herniarin and coumarin did not individually, or in combination, result in similar immunomodulation, and the immune stimulatory effects were not the result of contamination by exogenous endotoxins. We conclude that, similar to other plant derived teas, the aqueous extract of lavender may stimulate immune function, possibly via b‐glucans found in flowering components of the plant.

Support or Funding Information

Supported by the Fort Lewis College Student Competitive 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.
TCL/RhoJ Vesicular Localization is Regulated by GDP‐loading of its Core GTPase Domain
Authors: Tader Brooke R. , Hamann Michael J.
Published: June 2021

TCL (RhoJ) belongs to the Rho family of GTPases, and while it has been shown to contribute to angiogenesis and metastatic melanoma, less is known about its molecular and cellular function. To better understand the inherent cellular biochemistry of TCL, our lab has been performing a detailed structure/function analysis of the GTPase. Previously, the extended N terminus of TCL was found to be important for GTP‐loading and localization to the plasma membrane. The deletion or mutation of this TCL‐specific sequence led to inactivation of TCL and its localizing to intracellular vesicles. To determine if the N‐terminus of TCL contributes to cellular localization by itself, expression plasmids were produced where the first 24 N‐terminal amino acids and last 21 C‐terminal amino acids of TCL were fused to the N‐ and C‐termini (respectively) of the fluorescent protein Venus. Interestingly, expression of the constructs bearing only the C‐terminal tail of TCL or both the N‐ and C‐termini sequestered Venus to the plasma membrane, indicating the C‐terminal tail is potentially sufficient for TCL membrane localization. Additionally, experiments using constitutively active (CA) and dominant negative (DN) mutants showed CA TCL localized to the plasma membrane while DN TCL was vesicular; however, CA TCL where C terminus has been deleted results in diffuse cytoplasmic localization, while the same deletions in the context of the DN mutation lead to a marked vesicular localization. Together, our results suggest TCL is specifically retained to vesicles through an interaction with an as yet unidentified binding partner, and future experiments will use biotinligase tags of CA and DN TCL and BioID procedures to determine what hypothetical partner protein is retaining TCL to vesicular membranes.

Support or Funding Information

Support was provided by the Nielson Foundation, Bemidji, MN and a grant from Regenerative Medicine Minnesota (RMM‐2017‐EP‐04)

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 a Role for TCL/RhoJ in Endocytic Pathways Using Live Cell Imaging
Authors: Simons Beth N. , Baumgartner Claire E. , Hamann Michael J.
Published: June 2021

TCL (or RhoJ) is a plasma membrane localized Rho‐GTPase bearing an additional eighteen N‐terminal amino acids in comparison to its close homologue Cdc42. Previous experiments in the lab have demonstrated that deleting the N‐terminal extension shifts TCL localization to vesicular membranes. To determine how this deletion may influence membrane trafficking of TCL, HeLa cells were transfected with plasmids encoding YFP‐tagged TCL and an N‐terminal deletion mutant (ΔN), and live‐cell imaging was performed using an FV10i fluorescence microscope. The live‐cell imaging demonstrated that wild‐type YFP‐TCL localization was present at the plasma membrane, and it also revealed an organized movement of YFP‐TCL labeled intracellular vesicles. Cells expressing the YFP‐TCL ΔN deletion mutant displayed more of a vesicular localization of the fusion protein, and vesicular movement within the cell was more erratic and random. Previous research has used fluorescent‐labeled transferrin as a marker to investigate a role for TCL in endocytic pathways; therefore, cells were exposed to Alexa 647‐labeled holo‐transferrin to determine if TCL colocalizes with vesicles involved with transferrin receptor uptake and recycling. Colocalization of the two fluorescent labels in time‐lapse images was measured using CellSens software, and initial results show significant colocalization of YFP‐TCL ΔN with transferrin‐labeled vesicles in comparison to wild‐type TCL. However, it is unclear if the alterations in TCL localization are influencing transferrin transport kinetics or if YFP‐TCL ΔN is inappropriately localized to transferrin‐labeled vesicles. Together, these results show TCL has a potential role in vesicle transport, and future experiments will be aimed at determining if it influences vesicle transport kinetics.

Support or Funding Information

Support was provided by the Nielson Foundation, Bemidji, MN and a grant from Regenerative Medicine Minnesota (RMM‐2017‐EP‐04)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Loss of the unique inhibitory G‐protein, Gαz, in the pancreatic β‐cell protects against diet‐induced glucose intolerance by enhancing insulin secretion, but is not β‐cell autonomous
Authors: Reuter Austin , Schaid Michael , Laundre Erin , Harrington Jeff , Wienkes Haley , Mullenberg Cortney , Kimple Michelle
Published: June 2021

The α‐subunit of the heterotrimeric Gz protein, Gαz, is a member of the inhibitory subfamily of heterotrimeric G proteins. As such, it negatively regulates cyclic AMP (cAMP) production. In the insulin‐secreting β‐cell, cAMP potentiates glucose‐stimulated insulin secretion and β‐cell replication and survival. Previous work by our lab has determined that C57BL/6 mice lacking Gαz throughout the body are protected from developing fasting hyperglycemia and glucose intolerance when fed a high‐fat (45 kcal% fat) diet to induce obesity and insulin resistance. Gαz has a limited tissue distribution, but in addition to the pancreatic islets, it is expressed in the brain and platelets. To show the protection from metabolic dysfunction of the Gαz‐null mice is β‐cell autonomous, we created a β‐cell specific‐knockout of Gαz in the C57BL/6 background. Western Blot analysis for Gαz in the hypothalamus, platelets, islets, and kidney confirmed an islet‐specific loss of Gαz in the floxed/Cre‐positive mice as compared to all other genotypes. To characterize any diabetes protection of the β‐cell‐specific Gαz‐null mice, we placed 12‐week‐old mice on either a 45 kcal% high‐fat diet (HFD) or 10 kcal% fat control diet for 28 weeks. Pre‐diet glucose tolerance and insulin tolerance tests were performed, revealing no differences between genotypes. Weekly body weight and fasting blood glucose measurements were performed. Upon study completion, the β‐cell‐specific Gαz‐null mice showed no differences in body weight or insulin sensitivity as compared to their wild‐type counterparts, phenocopying results from the whole‐body Gαz‐null study. However, after glucose challenge, the glucose clearance of Gαz‐null mice fed the HFD was significantly better than wild‐type mice fed the HFD diet. Yet, this protection from glucose intolerance was not as complete as that observed in the whole‐body Gαznull mice, as the β‐cell‐specific Gαz‐null mice had worse glucose tolerance than control‐diet fed mice. Thus, it is possible that loss of Gαz in another cell type is required for full diabetes protection, and experiments are ongoing to confirm the hypothesis it is the α‐cell.

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 Subtle Amino Acid Change Impacts Kinase Function in Dramatically Distinct Ways
Authors: Kunkel Maya T. , Hudson Andrew M. , Brognard John , Newton Alexandra C.
Published: June 2021

Dysregulated protein kinase function is prevalent in cancer. Analyses of disease mutations reveal important insights into structure‐function relationships in kinases. Here we present how mutation of a conserved residue can have vastly different effects on kinase function among distinct kinases. A previous study by Weinreb et al. (Nat Genet. 2014) identified a point mutation within a conserved subdomain of PKD1 (E710D) in polymorphous low‐grade adenocarcinomas of the salivary gland. In their study, in vitro kinase assays revealed increased activity of this mutated PKD1 as well as a change in its subcellular localization. Through use of our D Kinase Activity Reporter (DKAR) to monitor kinase signaling, we confirm that E710D mutation exhibits constitutive activity and we observe that the kinase localization is significantly altered by this subtle amino acid change. Specifically, the mutant PKD1‐E710D protein localizes basally in the nucleus and at Golgi membranes compared to the solely cytoplasmic distribution of wild‐type PKD1. This Glu residue appears as Glu in nearly all of the CAMK family members as well as many members of the AGC family of kinases such as Akt and PKA; interestingly, most of the other members of the AGC family, such as PKC, possess Asp at this site. A search of other kinases mutated in cancer at this conserved residue revealed a Glu to Asp mutation in Akt3 in endometrial cancer. Introduction of this mutation within Akt3, however, does not result in a change in protein localization nor does it result in an active kinase when assessed using our Akt/B Kinase Activity Reporter (BKAR); surprisingly, Akt3‐E275D appears completely inactive. Thus, mutation from Glu to Asp in PKD results in a dramatic conformational alteration of the enzyme leading to constitutive activity, and mutation from Glu to Asp in Akt results in a complete loss of activity. These findings underscore how minute changes (Glu to Asp) can have dramatically different impacts on kinase structure and function, and how alterations of these components can have divergent effects on kinase biology. Further analyses of these mutations in the context of the kinase can yield insight into the role each kinase plays in tumor initiation, progression and metastasis.

Support or Funding Information

This work was supported by NIH P01 DK54441 and NCI R03 CA178524

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deciphering Key Cancer and Inflammation Signaling Pathways with a Novel Homogeneous Bioluminescent Cell Based Kinase Activity Assays
Authors: Hwang Brian (Byounghoon) , Nath Nidhi , Goueli Said , Zegzouti Hicham
Published: June 2021

Signaling pathway activation leads to a multitude of cellular responses including modulation of enzyme activity, altered gene expression, and protein translocation or degradation. Specific phosphorylation events by specific kinases in the cell constitute important nodes in signaling pathways. Altered normal kinase activity leads to several undesirable consequences causing diseases such as cancer and inflammation. Thus, monitoring these signaling events is essential for better understanding of normal cell behavior and disease states. Current assays to analyze cellular kinase activities can be tedious, non‐homogeneous, and not easily adaptable to HTS or suffer from generation of false hits due to fluorescence interference, signal quenching, or require special instruments for detection. To overcome these shortcomings, we developed a simple homogeneous assay platform to detect phosphorylation of specific endogenous proteins in cells. We took advantage of the NanoLuc Binary Technology (NanoBiT), a two‐subunit system based on NanoLuc luciferase that was successfully demonstrated for protein‐protein interaction (PPI) detection. We generated a detection system containing secondary antibody pairs linked to the two Nanoluc components, NanoBiT small and large fragments (11 aa peptide and 18 kDa). Cell lysate containing the phosphorylated protein is incubated with two primary antibodies that recognize separate epitopes on a single protein. These will bring NanoBiT secondary antibodies into proximity to form an active NanoLuc luciferase that makes light in proportion to the amount of target protein. When the primary antibody pair includes a phosphospecific antibody, the luminescence reflects the level of target protein phosphorylation. We tested this system by monitoring the activation of signaling pathways involved in cancer, immune and inflammatory responses (NF‐kB, JAK/STAT, and BTK). With very few steps, we detected the expected biological response of these pathways by monitoring phosphorylation/degradation of IkB, phosphorylation of STAT3, and BTK autophosphorylation. We also tested different small or large molecule inhibitors of these pathways or their corresponding node kinase (IKK, JAKs, and BTK) and obtained the expected pharmacology. The bioluminescent system presented here have many advantages including “Add and Read” format, no cell engineering required as the phosphorylation of endogenous substrate is detected in any cell type including primary cells, and only a simple luminometer is required for detection. Our results demonstrate that this technology can be adapted to any signaling pathway analysis allowing scientists to analyze signaling pathways of interest, study the kinase cellular activity and regulation or identify specific kinase or pathway inhibitors.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of a Computationally‐Based Medicinal Chemistry Course at a Small, Primarily Undergraduate Institution
Authors: Hall Bonnie L.
Published: June 2021

Computational analysis and molecular modeling are essential tools in many disciplines in modern chemistry, and undergraduate students benefit from exposure to these methods. Described here is the development of an active learning Medicinal Chemistry course with an extensive computational component. The course was targeted at upper‐division Chemistry and Biology majors, all of whom had already taken Biochemistry. A Team‐Based Learning (TBL) format was chosen for the course, to leverage both active learning strategies and the power of group learning for the computational work. Overall, the course emphasized the basic chemical and biological principles of Medicinal Chemistry, the process of drug discovery and optimization as well as the power and limitations of computational methods in drug discovery. Software utilized included Marvin Suite, AutoDock, and AutoGrid, as these are all free for academic use. The computers were machines re‐purposed from a computer lab on campus, and had no high‐end processors or other add‐on components. Course details will be discussed, including specific topics and a series of scaffolded molecular modeling exercises utilized in class. Ultimately, students used the content learned in the course to complete an in silico modification of an existing drug, and to predict the effectiveness of the new drug on the original target. Although only limited computational resources were available, student feedback indicates that Medicinal Chemistry was a challenging but rewarding addition to our curriculum.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Using an Innovative Approach to Teach Students How to Communicate About Scientific Topics to Non‐Scientists
Authors: Rowland‐Goldsmith Melissa A. , Bisoffi Marco
Published: June 2021

Regardless what career path biochemistry and molecular biology students take, they will all be required to communicate to non‐scientists. Thus, we are excited to share an innovative approach we used in our upper division cancer biology course to teach this skill. For this key aspect of the course, we used a multi‐faceted approach to teach the students how to communicate with non‐scientists both orally and in written form. To begin, we had three cancer survivors come to the class tell their stories of diagnosis and surviving pancreatic cancer. We then required the students to read three primary journal articles about pancreatic cancer research co‐authored by a pancreatic cancer surgeon/researcher who they would meet after reading these articles. Next, the students volunteered at a pancreatic cancer walk/run event so they could meet additional pancreatic cancer survivors and caregivers. After volunteering at this event on a large university campus, the students met with the author to discuss the details of the three assigned papers. Following this very informative discussion, the students each chose one of the three papers and rewrote it in layman's term meant to inform non‐scientists. Each of the students also gave a short oral presentation about the content of their paper to a panel of five pancreatic cancer survivors. These survivors reviewed each of the presentations using a simple rubric we created. We report that all students learned important skills about how to communicate to non‐scientists. Quantitative assessment of these assignments indicated that the combination of community outreach as well as the involvement of cancer survivors and professional caregivers lead to an elevated sense of importance of ‐ and engagement in ‐ the subject matter. We hypothesize that such inclusive and comprehensive pedagogical approaches are helpful for the students when deciding on and developing their future careers. We also emphasize the merits of implementing our approach in other biochemistry and molecular biology courses in order to deepen the appreciation of the subject matter, foster critical and strategic thinking, and develop enhanced science communication skills. All of these ultimately lead to a better and higher quality learning experience for the students.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Scaffolding soft‐skill development into a two‐semester undergraduate laboratory course
Authors: Grilley Daniel Patrick , Weaver Todd Michael
Published: June 2021

We have designed a two semester sequence of biochemistry labs that provides each biochemistry major a research experience studying the structure‐function relationship of a model enzyme. During this two semester sequence, students master a set of technical skills and develop proficiency in the soft skills that will allow them to thrive in their post‐graduate careers. A firm grasp of the structure‐function paradigm is a central tenet for students within biomolecular science programs. Critically, students need to understand that any molecular model is built on a preponderance of data from overlapping experiments. Allowing students to develop their own data and models requires that a coordinated experiential learning component accompanies lecture content. In addition to studying the structure‐function relationship, the labs also develop many of the top ten soft skills sought after by employers and graduate schools, including data analysis and interpretation, experimental design, teamwork, collaboration, and oral and written communication. This high impact research embedded course progression can implement almost any biological system, provided meaningful data can be generated during a typical lab time. We have found that a two semester approach is critical to adequately prepare students for an independent research project. By using teams of students and building on previous years of data collection, we have been able to reinforce the collaborative nature within science, while generating publication quality results. To force students to consider the big picture of their work we have found it critical that students present formally and informally throughout the course. This heuristic laboratory course has been designed to provide the undergraduate biochemistry students a high impact research experience, while emphasizing soft skills. Our course‐based undergraduate research approach could be adapted to any number of systems.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluation of Real‐time PCR Primer Sets for the Diagnosis of Huanlongbing (HLB) in Citrus Root Tissue
Authors: Avila Courtney J. , Park Jong‐Won , Kunta Madhurababu
Published: June 2021

Through the Step 2 program undergraduate students are placed into internships that will help foster their knowledge and readiness for careers in the field of agriculture and research studies. Collaboration between South Texas College and the diagnostics lab at the Texas A&M Kingsville Citrus Center has allowed student studies within the scope of plant and soil sciences.

Huanglongbing, also known as citrus greening, is one of the most destructive diseases in citrus of which causal agents are unculturable phloem‐limited Gram‐negative Alphaproteobacteria that belongs to the genus ‘Candidatus Liberibacter’. So far, three species of Candidatus Liberibacter, CL asiaticus (CLas), americanus (CLam) and africanus (CLaf), are known to be causing HLB. While CLas is the most widely spread, CLaf and CLam are geographically limited to Africa and South America, respectively. These HLB causing bacteria are transmitted by citrus psyllids, Diaphorina citri in Asia and America and Trioza erytreae in Africa. As there is no known resistant citrus cultivar available for commercial cultivation, the early HLB detection is a critical step to deploy an efficient disease control strategy that is heavily dependent on chemical method to control insect vector population. Currently, symptomatic citrus leaves are the source material for the detection of HLB‐causing bacteria in citrus by real‐time PCR. However, the uneven distribution of HLB‐causing bacteria in a tree canopy can lead to a misdiagnosis. Previous work conducted in our lab indicated that HLB‐positive plant exhibits more uniform distribution of HLB‐causing bacteria in the root system, suggesting that root tissue can be used for HLB diagnosis. However, currently available real‐time PCR primers based on CLas 16s rDNA cannot be used for root HLB test due to non‐target amplification. In this study, we designed new sets of primers and probes based on 16s rDNA of all three HLB‐causing bacteria that can be used for HLB diagnosis with root tissue. BLASTn analysis against prokaryotic ribosomal RNA database after exclusion of 16s rDNAs of HLB causing bacteria revealed that the newly designed forward and reverse primers had ~61% and ~90% sequence identity, respectively, to the prokaryotic 16s rDNA database. The current study evaluated these three new real‐time PCR primer sets, each of which targets CLas, CLam and CLaf, using DNA templates prepared with root DNA extracts.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Are You a Scientist? Exploring Science Identity in a Structural Biology Outreach Program
Authors: Wyatt Brittney N. , Schram Malia , St. Maurice Martin
Published: June 2021

At Marquette University, we developed a research‐intensive outreach lab dedicated to instructing high school students and undergraduate research mentors on techniques used in structural biology. The development of this outreach lab has raised specific questions on the effectiveness of engaging students in science through outreach programs. Specifically, does a semester long science outreach program that mimics a course‐based research experience increase students' perception of themselves as a science person by doing hands on experiments, group discussions and pre/post‐lab assignments? Which students (the high school students or undergraduate research mentors) are more positively influenced, if at all, and have a greater sense of identity in science? We hypothesized that all participants would have a positive gain in science identity but for different reasons. It was thought the high school students would have an increase in science identity due to exposure to an authentic research experience, while the undergraduates' science identity gain would be closely related to the mentoring of the high school students as they perform their experiments. Lab participants of the outreach lab express fluorescent proteins in bacteria and purify these proteins to subsequently determine the protein structure by X‐ray crystallography. These proteins are chosen because of their unique fluorescent properties that allow students to visualize the protein through the various stages of protein purification and protein crystallization. Seven main experiments are performed including: transformation, cell growth and induction, protein purification, screening conditions for crystallization, crystal refinements, and X‐ray diffraction of protein crystals. In order to assess science identity in our students, pre‐ and post‐surveys on how the students view themselves as a science person as well as a pre‐ and post‐exam were given. Due to the small sample size, the data were collected over two years with 22 participants. A focus group was another additional measure that was used to assess the students' perception of the outreach lab and the lab's impact on their science identity. Based on preliminary results, both the high school students and undergraduate research mentors had a positive gain in science identity after completing the outreach lab. Factors such as the type of experiments that were performed and the inclusive nature of the lab provided a space for the students to explore their science identity. This outreach lab can be used to engage both high school students and undergraduates in an authentic research experience that will increase students' engagement in science.

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 a Data Analysis Intensive Course on Student Critical Thinking Skills, Confidence, and Post‐Graduation Success
Authors: Resendes Karen K.
Published: June 2021

Bio 404: Nuclear Structure and Function is an advanced cell and molecular biology course taken by junior and senior undergraduates at Westminster College (New Wilmington, PA). The course averages 10 students, representing majors in Molecular Biology, Biology, Neuroscience and Biochemistry. While the content of the course focuses on the architectural aspects of nuclear cell biology and the molecular understanding of gene expression and genome structure, the major objective of the course is to use primary and secondary literature in order to broaden students' scientific skill set in content comprehension, data analysis, productive scientific discussion, oral presentation, and scientific writing. In order to achieve these objectives the course schedule is divided into four units (Nuclear Periphery, Nuclear Pore, Chromatin and Nuclear Bodies), each of which is comprised of multiple, one‐week modules. Each module is designed to introduce students to a new concept then builds on this knowledge by developing models from data and concludes with presentation and discussion of current research in the field. Specifically, a module consists of three one hour course sessions: first a lecture introducing the week's theme, based on a recent review article on the topic, second a “Work it out Wednesday” session during which students are provided with a packet of data derived from one or more primary research articles accompanied by probing questions about the methodology, results and interpretation, with students working in groups of 3–4 to analyze the data and develop a model that explains the data and it's links back to concepts learned in lecture, and finally the module concludes with a journal club session where one student is responsible for leading a discussion on an assigned primary article that correlates with the information learned in the previous two sessions. Course evaluations from the 2016 offering of the course indicated that students thought the course was well organized, was a valuable learning experience and that the assignments had instructional value; moreover, exam and course grades in 2016 improved over the 2014 offering, which was structured in a more traditional lecture style. As a result of these positive outcomes with the change in course format a more formalized analysis of student gains and perceptions of the course will be performed during the spring 2018 offering. This presentation will consist of an example of a course module as well as preliminary data from the 2018 study. These results will include outcomes from skills tests and perceptions surveys performed at the beginning and middle of the semester, as well as results from alumni surveys from the 2014 and 2016 cohorts.

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