bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 InformationThis 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 InformationSupported 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 InformationSupport 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 InformationSupport 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 InformationThis work was supported by NIH P01 DK54441 and NCI R03 CA178524This 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.