bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of RNA Polymerase III Transcription Factors BRF1 and BRF2 by Cancer Stem Cell Signaling Pathways
Authors: Cabarcas‐Petroski Stephanie M. , Schramm Laura
Published: June 2021

The direct link between deregulation of RNA polymerase III (pol III) transcription and the progression and maintenance of cancer cells is well‐established. The small untranslated RNAs which are produced as a result of RNA pol III transcription, including U6 snRNA and tRNAs, play a role in the proliferative capacity of a cancer cell. The requirement of TFIIIB for proper RNA pol III transcription has led researchers to investigate the role of TFIIIB subunits in cancer development. To date, there is evidence demonstrating that the TFIIIB factors, BRF1 and BRF2, play a significant role in cancer development including the classification of BRF2 as an oncogene. Using genomic analysis, we investigated potential factors contributing to deregulation of BRF1 and BRF2. We performed a search for putative transcription factor binding sites within the BRF1 and BRF2 promoters. Specifically, we sought to determine if there were binding sites for transcription factors that have been shown to play a role in the maintenance of cancer stem cells (CSCs). CSCs represent a subpopulation of cells that are responsible for tumor initiation, formation, maintenance and metastasis. CSC specific signaling pathways identified to date include: JAK/STAT, Hedgehog, Wnt, Notch, PI3K/PTEN and NF‐kB. Our search identified CSC related binding sites within the BRF1 and BRF2 promoters that could establish a link between CSC signaling pathways and RNA pol III deregulation. Additionally, utilizing additional genomic analysis from previously published datasets, we correlated BRF1 and BRF2 expression in isolated CSC's. Taken together, these data demonstrate a potential link for BRF1 and BRF2 regulation by CSC associated signaling molecules which could contribute to deregulation of RNA pol III transcription.

Support or Funding Information

Sandra E. Gleason Faculty Enhancement 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.
Isoforms of human CD46 Produced by Alternative Splicing Adopt Different Quaternary Structures
Authors: Wu Eugene Y. , Romanoff Emily L. , Woon Jesse W.
Published: June 2021

Alternative splicing can produce multiple mRNA transcripts from a single gene sequence, thereby expanding the number of protein sequences encoded by the genome of a cell. Protein isoforms from alternative splicing may have different functions owing to their different sequences, but examples of alternative splicing producing isoforms with different structures are lacking. CD46 (membrane cofactor protein) is a cell surface glycoprotein that helps protect human cells from destruction by the complement system and a receptor for several human pathogens. It is alternatively spliced into two main isoforms in the extracellular region (BC and C) that differ by the presence or absence of 15 amino acids. Based on existing structural data, we hypothesized that the C isoforms of CD46 form oligomers while the BC isoforms exist as monomers on the cell surface. Proteins on the HeLa cervical cell surface were treated using a membrane‐impermeable crosslinker, then subjected to denaturing gel electrophoresis and western blotting. HeLa cells express both BC and C isoforms of CD46, but only the C isoform changed its electrophoretic mobility to a higher molecular weight after crosslinking. A549 lung epithelial cells express only the BC isoforms, and the molecular weights of their CD46 proteins were unaffected by the crosslinker. These results indicate that CD46 can exist as monomeric and oligomeric spliceoforms. We show that alternative splicing can produce proteins with different quaternary structures from the same gene.

Support or Funding Information

Funded by University of Richmond School of Arts & Sciences

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 pro‐pol Frameshifting Efficiencies for Naturally Occurring Variants of HTLV‐1
Authors: White Emily A. , Mouzakis Kathryn , Williams Marcus , Nash Hannah , Hamilton Adam , Harrison Jacob , Eades Austin , Daily Leandrew , Contreras Uriah , Cooper‐Sansone Andrew , Chadeayne Devon , Banks Terrance , Abrams Tara , Joe Natalie , Mylroie Elena
Published: June 2021

Human T‐cell leukemia Virus Type 1 (HTLV‐1) utilizes two ‐1 programmed ribosomal frameshifts (PRFs) to translate its enzymatic proteins. This translational mechanism induces a change in the ribosomal reading frame during elongation, which allows for the production of proteins from alternate reading frames within a single transcript. Frameshifting is utilized by many retroviruses and is often required for successful replication. Each frameshift site includes three components: a heptad “slippery sequence”, a spacer, and a downstream structure. Preliminary data from our group suggests that the HTLV‐1 pro‐pol frameshift site structure is a pseudoknot. Interestingly, multiple HTLV‐1 isolates include point‐mutations within this pseudoknot structure. How these mutations affect the ‐1 PRF efficiency is uncertain. Here, we investigate the impact of several pseudoknot point‐mutations on the HTLV‐1 pro‐pol frameshift efficiency. Site‐directed mutagenesis was used to insert these mutations into a dual‐luciferase vector that includes the HTLV‐1 pro‐pol frameshift site. The mutated DNA for each variant was transcribed, translated, and used in a dual‐luciferase assay, allowing calculation of the in‐vitro ‐1 PRF efficiencies for each mutant. Our preliminary data suggests that these point‐mutations create significant differences in the ‐1 PRF efficiency.

Support or Funding Information

NIH NIGMS SCORE SC2 Award (Award# 1SC2GM121197‐01) and Research Corporation for Science Advancement Cottrell Scholar Award (Award #23983)

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 Time Course Study Examining the Effects of Tributyltin Exposures on eIF4E
Authors: Brown Shyretha , Whalen Margaret
Published: June 2021

Tributyltin (TBT) is an environmental contaminant which is found in human blood (ranging as high as 261 nM) and other tissues due to its lipophilic nature, and it has been shown to increase incidences of tumors in mammals. TBT alters secretion of pro‐inflammatory cytokines such as interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL‐1β), and interleukin 6 (IL‐6) from human lymphocytes. Previous ex vivo studies have shown that when TBT is introduced as a stress inducer to the cellular environment, the production (secretion and/or intracellular levels) of IL‐1β and IL‐6 was increased in human lymphocytes, and ERK1/2 and p38 MAPK pathways were shown to be necessary for this TBT effect. An additional study indicated that TBT‐induced increases in IL‐1β and IL‐6 production cannot be explained by increases in their respective mRNA at concentrations below 50 nM. Thus, further studies were carried out to address whether the changes in IL‐1β and IL‐6 production induced by TBT might be due to alterations of mRNA translation. The ERK1/2 pathway has been noted to catalyze the phosphorylation (activation) of eukaryotic initiation factor 4E (eIF4E), which is a key player of binding the 5′ cap of mRNAs. As previous studies have indicated a role for the ERK1/2 pathway in the TBT‐induced increases in IL‐1β and IL‐6 production, we carried out initial studies to examine the levels and phosphorylation state of eIF4E after 10 min, 30 min, 6 h, and 24 h exposures to TBT in monocyte‐depleted peripheral blood mononuclear cells (MD‐PBMCs) using western blot analysis. Results suggested that increased phosphorylation (activation) of eIF4E was seen after 10 min exposure to TBT and maintained after 24 h exposure. These results may indicate that eIF4E is a key factor for regulating translation when MD‐PBMCs are exposed to TBT.

Support or Funding Information

Supported by NIH grant U54CA163066.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of Notch binding protiens and localization of Notch to focal adhesions
Authors: Havel Shelby Lee , Gazdik Tana Ranae , Wood Travis Lee , LaFoya Bryce , Albig Allan
Published: June 2021

The Notch signaling pathway begins at the cell surface when Notch receptors interact with Notch ligands on adjacent cells triggering an intramolecular cleavage that releases the NICD (or Notch Intracellular Domain fragment) that then enters the nucleus to drive transcription. In addition to this cannonical Notch mechanism, our lab previously demonstrated that Notch signaling is modified by integrin ligation, via a previously unknown non‐cannonical mechanism. In unpublished research performed by LaFoya et al, SRC kinase has now been determined to bind to, and phosphorylate a small peptide near the ankyrin domain of Notch 1, resulting in modification of Notch signaling. The goal of our project is to expand upon these results by discovering other proteins that may interact with this region of Notch. To accomplish this goal, we constructed a fusion protein of the Notch peptide and the BirA protein, and performed a proximity biotin ligation screen in 293T cells for interacting proteins. Biotinylated proteins were isolated by streptavidin magnetic beads, and identified by MS/MS. Amongst the many hits, we identified members of the SRC family of kinases and several proteins commonly associated with focal adhesions and/or tight junctions. Western blotting confirmed the presence of focal adhesion proteins α‐actinin 1 and α‐actinin 4, and tight junction protiens cingulin, and angiomotin, in the biotin precipitated fractions. Subsequent co‐immunoprecipitation experiments in HMEC endothelial cells positively determined an interaction between the NICD domain and α‐actinin1, but failed to confirm interactions with the other proteins. The functional significance of this Notch‐actinin interaction is currently unknown, although given that SRC and actinin both localize to focal adhesions, we speculate that Notch may localize to focal adhesions where it is phosphorylated by SRC kinases.

Support or Funding Information

NIH Grant 2R15GM102852‐02

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
DNA binding properties of the transcription factor optix
Authors: Rodriguez Jessica Marie
Published: June 2021

DNA binding proteins such as transcription factors are key determinants of cellular state and have been shown to control cell differentiation. Evaluating the specificity profile of DNA binding proteins is a nontrivial challenge that hinders the ability to decipher gene regulatory networks or engineer molecules that act on genomes. optix is a transcription factors that has been previously identified as a master regulator of red color patterns in the butterfly (Heliconius) developing wing. However the direct gene targets of optix and their effects in final wing coloration remain to be determined. Based on this, we want to determine the intrinsic DNA‐binding preferences of optix. We are currently working on cloning, over‐expressing and purifying full‐length optix and optix's DNA‐binding domain, the homeodomain (HD). We will determine the in vitro DNA‐binding specificity of optix protein of Heliconius erato using Cognate Site Identification by high‐throughput SELEX. Our results will enable us to bioinformatically predict the genomic targets of optix and to better understand its role in wing color patterning.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression and Characterization of the Receptor Tyrosine Kinase Tie2
Authors: Kennedy Madison Arza , Wu Yunjin , Grunseth Adam , Sohl Christal D.
Published: June 2021

Nearly one‐third of human kinases are implicated in disease, and this enzyme class has proved to be an enormously successful drug target for combating many diseases. Kinases are key regulators in signaling pathways responsible for such processes as growth, proliferation, and angiogenesis. Tie2 is an endothelium‐specific receptor tyrosine kinase (RTK) responsible for angiogenesis and vasculature maintenance. Mutations in this enzyme can cause venous malformations for which no current therapies are available, and Tie2 has been proposed to be a target for anticancer therapies. However, the molecular mechanisms of Tie2 activity are not well understood. Here, we developed a robust purification strategy using heterologous expression in insect cells, optimizing expression and purification with a post‐infection incubation time of 72 hours and utilization of affinity chromatography. We have created autophosphorylation assays to establish catalytic rates of wild‐type Tie2 and mutants implicated in disease. We hypothesize that mutant Tie2 will have faster rates of autophosphorylation, likely through stabilization of the active conformation of the protein. These studies in kinase activity can improve our understanding of the catalytic features of wild‐type and mutant Tie2, and can inform the development of therapeutic targets for vascular disease.

Support or Funding Information

NIH

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A synergistic combination of Huperzine A, Convolvulus pluricaulis and Celastrus paniculatus promote cognitive function and health
Authors: Ahmad Imtiaz , Swaroop Anand , Bagchi Debasis
Published: June 2021

Approximately 56 billion dollars are spent as a result of traumatic brain injury and about 90,000 Americans suffer from long‐term disability. Uprising stress, busy lifestyle and unhealthy food habits are the major causes of traumatic brain injury. The majority of the treatment strategies are based on the improvement of cholinergic function in the brain and one of the emerging therapeutic target is to enhance the acetylcholine level in the brain. Standardized herbal extracts including Huperzia serrata, Convolvulus pluricaulis (Shankhapushpi; SP) and Celastrus paniculatus (Jyotismati; JY) are long been known to attenuate brain function and well‐being. Research studies have demonstrated the versatile roles of these novel phytochemicals as natural acetylcholinesterase inhibitors and neuroprotectants, and exhibited their efficacy in the management of neurological or memory impairment, dementia, Parkinson's and Alzheimer's disease. In the recent past, we developed a novel, standardized Huperzia serrata extract, CogniUp™ enriched with 1% Huperzine A, which demonstrated broad spectrum safety and neuroprotective abilities. In this study, we developed a unique combination of CogniUp, SP and JY (MZ001), which can effectively and synergistically inhibit acetylcholinesterase (AChE) inhibition. Based on our initial assessment, concentration‐dependent AChE inhibition kinetics was assessed individually using 0, 3.0, 6.0, 9.0, 12.0 and 24.0 mg SP/ml, while AChE inhibition kinetics was assessed individually using 0, 0.5, 1.0, 2.0, 4.0 and 8.0 mg JY/ml. Simultaneously, concentration‐dependent AChE inhibition kinetics was assessed individually using 0, 0.0625, 0.125, 0.25 and 0.5 μg CogniUp/ml. Several combinations were assessed and ultimately, we found that a combination of SP, JY and CogniUp (12 mg/ml, 4 mg/ml and 0.125 μg/ml) provided the most efficacious and synergistic AChE inhibition. No significant adverse events were observed. Further studies are in progress to establish the therapeutic efficacy of our unique proprietary combination‐MZ001 containing SP, JY and CogniUp.

Support or Funding Information

This project was funded by Cepham Inc., Piscataway, NJ

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 luminal kinase regulates sarcoplasmic reticulum calcium cycling and heart disease
Authors: Pollak Adam , Wiley Sandra , Dixon Jack
Published: June 2021

The mechanisms and machinery regulating calcium cycling through the sarcoplasmic reticulum (SR) remain incompletely understood, and further insight is critical for discerning normal and diseased heart function. An overlooked component is luminal SR protein phosphorylation. Our lab recently identified a protein kinase in the secretory pathway lumen, Fam20C, which phosphorylates Ser within Ser‐x‐Glu/pSer motifs. Fam20C is responsible for generating the majority of the secretory phosphoproteome. However, Fam20C's role in cardiac function is currently undefined.

Here we show that cardiac histidine‐rich calcium binding‐protein (HRC) is a Fam20C substrate. Importantly, a common HRC Ser96Ala genetic mutation causes deadly arrhythmias in patients. The functional significance of this mutation is to block Fam20C phosphorylation at Ser96. Furthermore, several other calcium regulating luminal SR proteins are Fam20C substrates, suggesting that Fam20C plays a broad and impactful role in SR function.

We developed a cardiac‐specific Fam20C mouse knockout (KO) model. Using transverse aortic constriction (TAC), we show that Fam20C protects against heart failure. Fam20C KO mice show altered levels of SR calcium handling proteins during TAC, suggesting that calcium cycling through the SR is perturbed. Furthermore, we demonstrate that contractility is altered in Fam20C KO mice.

In summary, we introduce Fam20C as a novel yet critical regulator of SR calcium cycling and cardiac biology. The phosphorylation of SR luminal cardiac proteins by Fam20C plays an important role in SR calcium dynamics and heart disease.

Support or Funding Information

NHLBI RUTH L. KIRSCHSTEIN NRSA F32 HL136122‐01

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Assessment of Visual Literacy Skill Gains in Graduate Biochemistry
Authors: Procko Kristen , Dean Diane M.
Published: June 2021

Biochemistry II, a culminating course of the biochemistry master's degree program at the University of Saint Joseph, emphasizes protein structure, folding, catalysis, and mechanism. Accordingly, the course has a significant molecular modeling component. Students are taught to render and manipulate proteins in PyMOL through tutorials that span the basics of the program, measurement and evaluation of potential molecular interactions, assessment of mutations, and prediction of protein function based on structural features. To evaluate students' progress in their ability to interpret visual representations of macromolecules, we designed a study using several of the overarching visual literacy themes outlined by the BioMolViz group (BioMolViz.org). At the beginning of the course, students complete a pretest in which they view macromolecule structures and answer questions that encompass several of the overarching visual literacy themes to evaluate their baseline biomolecular visualization skills. At the end of the semester, students complete an exit evaluation, which contains unique questions that mirror the first assignment. Students demonstrated improvement in each of the visual literacy themes evaluated, with the most significant gains in the structure‐function relationship and structural model skepticism areas. Here, we present the results of the assessment from three unique sections of the course, along with an overview of the tutorials and assignments being used to build visual literacy skills. Likewise, we will discuss revised course materials addressing areas in which students did not initially achieve the expected gains.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Kaposi's Sarcoma‐Associated Herpesvirus (KSHV) role in preventing apoptosis: The vBCL‐2 and human BIK association
Authors: Yeager Chyenne , Canfield Jennifer
Published: June 2021

Kaposi's Sarcoma‐Associated Herpesvirus (KSHV) is a harmful oncogenic virus that leads to skin cancer commonly found in immunocompromised individuals. KSHV evades host defense systems by targeting signals transduction pathways to suppress autophagy and apoptosis. The BCL‐2 family of proteins controls commitment to apoptosis by regulating mitochondrial outer membrane permeabilization (MOMP). Research has been conducted examining the specific mechanisms and localization of different proteins in the BCL‐2 family such as BAK and BAX, but other proteins like BIK ( BCL‐2 interacting killer) have been far less studied. BIK is a pro‐apoptotic tumor suppressor that is critical n apoptosis induced by cytokines, toxins and viral invasion. Anti‐apoptotic Bcl‐2 family members competitively bind pro‐apoptotic proteins, like BIK, and suppress apoptosis. Although BIK is a known therapeutic target, little is known about the association of BIK with the KSHV BCL‐2 homology, vBCL‐2. In order to learn more about this association, we overexpressed vBCL‐2 in HEK293 cells and carried out subcellular fractionation to localize the protein. We analyzed the physical interaction human BIK and vBCL‐2 using immunoprecipitation under different cellular conditions. We examined the apoptosis mechanism using isolated mitochondria to measure the induction of cytochrome c release in the presence another BCL‐2 family member, BIM, from cells expressing MCL‐1 and MCL‐1 andvBCL‐2. By titrating in BIM and BIK, we can analyze the BIK/vBCL‐2 interactions. Results of these experiments will help elucidate the unique relationship between vBCL‐2 and BIK in the context of mitochondria mediated apoptosis. Definitively elucidating the mechanism, localization, and protein interactions in apoptotic and non‐apoptotic cell death in the presence and absence of vBCL‐2 will guide future therapeutic approaches against KSHV and possibly elucidate other non‐resistant mechanisms to kill cancer cells.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Apoptotic Effect of Methylglyoxal in Pancreatic RINm5F Cells; Reversal by INGAP‐P
Authors: Gonzales Kerald , Su Jing , Mungre Sue
Published: June 2021

Diabetes mellitus is a metabolic disease characterized by high level of blood sugar. In a healthy person, the hormone insulin from β‐cells of pancreatic islets regulates blood sugar level. In diabetic patients, there is a continuous loss of pancreatic β‐cells mass, dysfunction of β‐cells that result in deficiency of insulin. Methylglyoxal (MG), a normal by product of glycolysis, is in high in a diabetic person. MG causes oxidative stress to β‐cells leading to cell death. A linear pentadecapeptide segment of Islet Neogenesis Associated Protein ‐ Peptide (INGAP‐P), Ac‐IGLHDPSHGTLPNGS‐NH2, has been shown to induce proliferation of β‐cells through transdifferentiation of matured ductal cells. The goal of this research was to determine whether INGAP‐P protects RINm5F cells, an islet β‐cell line, from the detrimental effects of MG. Cells were treated with different concentrations of MG in the absence or presence of INGAP‐P. Cell viability of RINm5F cells after treatment was measured by MTT, [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide], assay at 570 nm. MG at 40μM caused 53% cell death in 24 hours. When treated with INGAP‐P at 1μM for 24 hours cell viability increased by 20%. When cells were treated with MG and INGAP‐P together, RINm5F cells showed no cell death suggesting that INGAP‐P protects the cells from MG. We are currently determining whether the cell death is due to apoptosis and how INGAP‐P protects RINm5F cells from the effect of MG. We will study the expression of Bax and Bcl2 in RINm5F cells treated with MG and INGAP‐P. The findings from this research will provide insights into the mode of action of INGAP‐P and guide the design of new synthetic drug molecules for diabetes that act in a similar mechanism.

Support or Funding Information

US Department of Education Title III 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.
Exploration of Novel Markers of Posterior Capsular Opacification
Authors: Jackson Erin , Shihan Mahbubul , Wang Yan , Duncan Melinda
Published: June 2021

Cataract, the leading cause of blindness worldwide, is defined as the clouding of the ocular lens. While cataract surgery is commonly performed by removing the lens and replacing it with an intraocular lens (IOL), it only temporarily cures cataract and actually causes a fibrotic condition called posterior capsular opacification (PCO). PCO, or secondary cataract, results from the migration and proliferation of lens capsule cells remaining post cataract surgery (PCS). While laser treatments can cure PCO, this incurs risks to vision. Thus, a comprehensive understanding of the signaling pathways leading to lens epithelial cell (LEC) fibrosis must be reached in order to prevent PCO. Previous research discovered that over‐activation of transforming growth factor beta (TGF‐β) signaling leads to increased fibrosis. The bone morphogenetic protein (BMP) signaling pathway is important for normal cell behavior and, in other cell types, controls fibrosis by inhibiting TGF‐β signaling. This work explores possible relationships between BMP and TGF‐β signaling during PCO pathogenesis. RNA sequencing and immunostaining revealed that many fibrotic markers are upregulated by 48 hours PCS including alpha smooth muscle actin (αSMA), collagen I, and lysyl oxidase, an extracellular matrix (ECM) protein that plays a role in the cross‐linking of collagen‐1. These upregulations occur concurrently with increases in phosphorylated SMAD3 (pSMAD3), a marker of the TGF‐β signaling pathway which is known to mediate LEC fibrosis. Additionally, a decrease in pSMAD1/5/8, major effectors of BMP signaling, was observed at the same time point consistent with the known requirement for BMP signaling in normal lens epithelial cell biology. Notably, the mRNA for Gremlin, a BMP antagonist, upregulates several hundred‐fold in LECs by 48 hours PCS, while Gremlin protein levels upregulate in lens cells sharply within six hours PCS and reach high levels at 48 hours PCS, providing a possible explanation for the attenuation of pSMAD1/5/8 observed. These data suggest that the upregulation of Gremlin PCS may be a critical factor in the downregulation of BMP signaling and the upregulation of TGF‐β signaling necessary for the fibrotic response leading to PCO.

Support or Funding Information

The National Eye Institute grant EY015279 as well as the University of Delaware Summer Scholars program funded this work.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification and characterization of protein changes in the Drosophila brain upon Wolbachia infection
Authors: Lucas Amber , Adams Haili , Winegardner Nicole , Sullivan William , Minden Jonathan S.
Published: June 2021

Many bacterial hosts in recent years have been shown to have a profound effect on host behavior, and the endosymbiotic alphaproteobacteria Wolbachia is no exception. Present in at least 40% of the insect population, the maternally transmitted Wolbachia modulates behaviors such as male aggression, mate selection, and migration. Wolbachia have also been shown to change host response to olfactory cues, influence life span, and prevent infection by certain pathogens. This last feature of Wolbachia, specifically the prevention of infection by viral pathogens such as Dengue, Zika, and Chikungunya in mosquitos, has become of recent interest as a potential mechanism to suppress transmission of these disease‐causing viruses to humans.

To tease apart the mechanisms by which Wolbachia influences host behavior and viral suppression, Drosophila, a genetically tractable model organism that is commonly used to delve into the molecular and cell biology of the symbiont behavioral, was used. A proteomic screen of changes in the Drosophila brain upon Wolbachia infection was performed using 2D Differential Gel Electrophoresis and LC‐MS. Protein abundance changes and post translational modification changes were observed and compared in both Drosophila simulans and Drosophila melanogaster as well as between male and female populations. Post translational modification changes were observed in multiple metabolic proteins and proteins responsible for neurotransmitter synthesis. This study describes the protein and post translational changes identified after Wolbachia infection and how they might contribute to behavioral changes in the host.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modeling Phage Survival in Limiting Bacterial Growth
Authors: Tomassi Nicole
Published: June 2021

Phages are viruses that infect bacteria and play a key role in ecosystems, including the human body. The resources available in the environment affect bacterial growth, which controls the production of phage particles. The impact of this pipeline in the ecological coexistence of phage and bacteria, however, remains unclear. Here we address this problem introducing a new mathematical model for E. coli bacteria and T4 phage—two reference model systems found in humans. First, experimental data is analyzed using linear regressions to determine the dependency of the phage production parameters as a function of the bacterial growth rate, which led to high R‐squared values and statistically significant p‐values. Then, a Lotka‐Volterra predator‐prey model was extended to include the bacterial dependency on resources as well as the phage dependency on bacterial growth. Standard tools in dynamical systems were applied to study the equilibrium and stability of the system analytically and numerically. Our preliminary analysis suggests that the coexistence of phage and bacteria depends on the initial conditions of the system, contrary to the classical Lotka‐Volterra model. Additional analysis will be necessary to determine the biological implications of this result and its potential biomedical application, for example, in phage therapy.

Support or Funding Information

Funded by a grant from the National Institute of General Medical Sciences of the National Institutes of Health: SDSU MARC U*STAR 5T34GM008303‐28

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
HIV‐1 Infected NSG‐BLT Humanized Mice as a Model for HIV‐Associated Lung Complications
Authors: Geohring Jason L. , Cota‐Gomez Adela
Published: June 2021

Human immunodeficiency virus (HIV) is an incurable and life‐threatening disease impacting over 36 million people world‐wide. Antiretroviral treatment (ART) suppresses the virus and allows many patients to live longer lives. However, even patients undergoing ART are susceptible to complications in many organ systems including the lung. HIV has been successfully linked to pulmonary complications including: tuberculosis, cytomegalovirus, Hodgkin's lymphoma, chronic obstructive pulmonary disease (COPD), and pulmonary arterial hypertension (PAH). Some of these diseases have a higher prevalence amongst patients infected with HIV. For example, in HIV patients, the incidence of PAH is 1:200, which is approximately 300‐fold higher than idiopathic PAH in the general population. Due to the limited host specificity of HIV, there are not many well‐developed animal models to study these complications. However, previous research in NOD‐Scid IL‐2 receptor γ chain knockout mice engrafted with human hematopoietic stem cells (hCD34+) and surgically implanted with human fetal thymic and liver tissue (NSG‐BLT mice) has shown disseminated HIV infection, making NSG‐BLT mice a promising in vivo model. In this study, we aimed to confirm HIV dissemination to the lungs in these mice and to examine pulmonary pathologies after an acute infection period. We infected NSG‐BLT mice with HIV for 5, 10, or 15 weeks and looked for viral RNA and pro‐viral DNA in the lungs using qPCR and nested PCR, respectively. To begin investigating possible lung pathology, we measured cytokines from lung tissue and cell‐free bronchoalveolar (BAL) fluid using an ELISA‐based protein array. Of the many proteins that were dysregulated in HIV infected mice, RAGE and IL‐25 (also known as IL‐17) are of particular interest because they have been implicated in endothelial dysfunction or vascular remodeling. This suggests a role of these proteins in HIV‐directed lung pathology in these mice. We are currently confirming the dysregulation of these genes by qPCR. In conclusion, NSG‐BLT mice constitute a useful model for studying pulmonary dissemination and pathologies during HIV infection.

Support or Funding Information

National Institutes of Health (NIH) 1R01HL125050‐01

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Selenium and Exendin‐4 Combination is a Promising Therapeutic Approach for Diabetes Mellitus
Authors: Moustafa Mohamed E. , Assi Layal Abou , Najem Sonia Abou , Merii Mohamed , Hasan Randa , Barakat Ghinwa , Dhaini Hassan R. , El‐Sabban Marwan
Published: June 2021

Previous studies suggested that selenium (Se) or exendin‐4 (Ex4) exerts antidiabetic effects. The mechanisms of the effects of Se, Ex4 and their combination on diabetes require further investigation. This study examined the effects of Se, Ex4, insulin or their combinations on hyperglycemia and on the expression of insulin‐signaling transducers: insulin receptor substrate 1 (IRS‐1), phosphoinositide‐3‐kinase (PI3K), and serine/threonine‐specific protein kinase (AKT) and glucose transporter‐2 (GLUT2) in diabetic rats. Diabetic rats were injected intraperitoneally twice daily with 4 U insulin/Kg body weight or 0.03 μg exendin‐4/Kg body weight, and/or treated with 5 ppm Se as sodium selenite in drinking water. All treatment modalities were continued for 6 days. Se/Ex4 combination lowered blood glucose level to 142 ± 7.0 mg/dL, increased mRNA levels of IRS‐1, PI3K, AKT and GLUT2 in the liver, and GLUT2 mRNA level in the kidneys. Se treatment of diabetic rats alone resulted in a significant increase in IRS‐1, AKT and GLUT2 mRNA expression levels in the kidneys. The hepatic protein levels of AKT were increased by all treatment modalities, whereas a significant increase in GLUT2 was found in rats treated with Se alone and Se/Ex4 combination as compared to untreated diabetic rats. Se/Ex4 combination was effective in controlling hyperglycemia and increased the levels of AKT and GLUT2 in the liver, and thus is a promising therapeutic approach for diabetes.

Support or Funding Information

National Council for Scientific Research (CNRS), Lebanon, research fund to M.E.M. (Grant No. 01‐11‐08)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Crystallographic and enzyme kinetic analyses of the human Inositol Polyphosphate Multikinase (IPMK)
Authors: Blind Ray
Published: June 2021

Inositol polyphosphate multikinase (IPMK, IPK2) is a nuclear, non‐canonical PI3‐kinase and a member of the IPK‐superfamily of inositol kinases. IPMK also has important non‐catalytic roles in p53, mTOR, AMPK and TRAF6 signaling, mediated largely by two disordered domains that decorate the well‐ordered ATP‐grasp kinase domain of IPMK. Ablation of IPMK disordered domains alters non‐catalytic IPMK functions, however it is unknown if these domains intrinsically alter the PI3‐kinase activity of IPMK. We present enzyme kinetic analyses of a highly engineered human IPMK devoid of all disordered domains (ΔIPMK) that has a 10‐fold increase in the apparent KM/kcat for PI(4,5)P2 (PIP2) compared to wild‐type, suggesting that the disordered domains inhibit IPMK activity in vitro. Further, the apparent KM for ATP is 17‐fold worse for ΔIPMK compared to wild type, indicating that sequences in the disordered domains affect ATP binding. We present the 2.9Å crystal structure of this human IPMK construct, showing that the conserved ATP‐grasp fold is maintained in ΔIPMK and providing structural rationales for the observed kinetic behavior. Taken together, these studies suggest that in addition to mediating protein‐protein interactions, the disordered domains of human IPMK impart intrinsic regulatory capacity to the PI3‐kinase activity of human IPMK.

Support or Funding Information

Journal of Biological Chemistry Herb Tabor Award, American Cancer Society Research Scholar Award, Vanderbilt Ingram Cancer Center Ambassador Discovery Award, Jimmy V Foundation for Cancer Research V‐Scholar Award, Vanderbilt Diabetes Center Discovery Award, Vanderbilt Diabetes Research and Training Center Pilot and Feasibility Grant, and an American Cancer Society Institutional 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.
The Glycerophosphocholine Acyltransferase, Gpc1, Impacts PC Remodeling and Stationary Phase Cell Viability in Saccharomyces cerevisiae
Authors: Anaokar Sanket Pramod , Kodali Ravindra , Jonik Benjamin , Nikiforov Alexiy , Lager Ida , Stymne Sten , de Kroon A.I.P.M. (toon) , Patton‐Vogt Jana
Published: June 2021

Phosphatidylcholine (PC) is the most abundant glycerophospholipid found in most eukaryotic membranes. Changes in its synthesis, turnover and remodeling play a crucial role in maintaining membrane homeostasis. PC species vary depending upon the acyl chains attached at the sn‐1 and sn‐2 position of the glycerol backbone. The fatty acid components of yeast PC are relatively simple, consisting of mostly C16:0, C16:1, C18:0 and C18:1 combined to produce PC species in which either one or both acyl chains is monounsaturated. Thus, the four major PC species found in yeast consist predominantly of the following chain combinations: two C16:1 to produce 32:2 PC, C16:0 and C16:1 to produce 32:1PC, C16:1 and C18:1 to produce 34:2PC, C16:0 and C18:1 or 18:0 and C16;1 to produce 34:1PC. Acyl chain composition is important as it affects the physical properties of membranes such as thickness, curvature and fluidity. We have recently reported on the characterization of glycerophosphocholine acyltransferase, Gpc1. Gpc1 acylates glycerophosphocholine (GPC), the product of the complete deacylation of PC, to produce lysophosphatidylcholine (LysoPC). LysoPC can then be converted to PC by the lysophospholipd acyltransferase, Ale1. In order to investigate the role of Gpc1 in PC remodeling, we performed PC species analyses on strains lacking or overexpressing GPC1. Our data indicates that the loss of GPC1 results in a decrease in PC species with a single unsaturation (32:1 and 34:1) and an increase in PC species with two unsaturations (32:2 and 34:2). Conversely, the overexpression of GPC1 results in an increase in singly unsaturated PC species (32:1 and 34:1). Expression analyses indicate that the GPC1 is upregulated upon attenuation of PC and sphingolipid biosynthetic pathways. Finally, preliminary phenotypic studies indicate that while loss of GPC1 does not affect log phase doubling time, it does result in a decrease in long‐term cell survival. Overall, we provide evidence that the remodeling of PC to more saturated species by Gpc1 is regulated in coordination with other lipid biosynthetic pathways and impacts stationary phase cell viability.

Support or Funding Information

We acknowledge support from National Institute of Health Grant NIH R15 GM104876 and Duquesne 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.
Structure and Function of Lipins: Key Enzymes in Triglyceride Metabolism
Authors: Airola Michael
Published: June 2021

Triglycerides serve as a major reservoir for energy and elevated levels of triglycerides strongly correlate with increased risk for cardiovascular disease and diabetes. Triglyceride biosynthesis is carried out by a series of enzymes, with lipins catalyzing the penultimate step. Lipins are a family of evolutionarily conserved lipid phosphatases that convert phosphatidic acid to diacylglycerol. In mammals, lipins also function in the nucleus as transcriptional co‐activators to upregulate fatty acid oxidation genes. Accordingly, expression levels and mutations in lipins can affect whole‐body energy metabolism, obesity, adipocyte differentiation, and myopathy.

Lipins are structurally uncharacterized and contain several unique but highly conserved protein domains. The lack of homology and structural information has hampered our understanding of how lipins can participate as both enzymes in lipid metabolism and in the nucleus to regulate gene expression.

We will present new structural information that identifies a novel protein fold/domain in mammalian lipins whose molecular function was not previously determined. This domain forms a tightly associated dimer. The dimer interface is conserved among lipin paralogs that suggests a role in homo and hetero‐dimerization. A large surface patch of this domain is also highly conserved that suggests a potential binding interface for DNA, membranes, or proteins. Functional assays testing these hypotheses will be discussed.

Support or Funding Information

American Heart Association Scientist Development Award #17SDG33410860

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Polymorphic Pseudogenization of SIGLEC12 in Humans: Relationship to Late Stage Cancer Progression
Authors: Siddiqui Shoib Sarwar , Do Raymond , Zhang Wu , Lenz Heinz‐Josef , Johnson‐Pais Teresa , Leach Robin , Fraser Gary , Wang Charles , Varki Nissi , Varki Ajit
Published: June 2021

Human Siglec‐XII (a receptor encoded by the SIGLEC12 gene) is expressed in tissue macrophages and some epithelial cell surfaces. Humans (but not the closely related “great apes”) harbor a universally fixed missense mutation that eliminated the sialic acid binding property of this protein, a canonical functional feature of all other human Siglecs. Population analysis of the SIGLEC12 locus identified a polymorphic frameshift mutation, which leads to truncation of the Siglec‐XII polypeptide, and loss of expression. The homozygous null state of this mutation is present in all human populations with an average frequency of ~40%, and the locus was independently identified as undergoing an unexplained “negative selective sweep”, apparently favoring the null state. In the current study, immunohistochemistry using a monoclonal antibody to Siglec‐XII showed that while some epithelia in ~35% of normal human tissue samples from human autopsies express Siglec‐XII on epithelial cells (approximately the expected rate based on the frequency of the null state), high expression was observed in about 80% of samples from advanced carcinomas. Genomic studies on population cohorts with a low frequency of carcinomas (Seventh‐day Adventists) and in early stage prostate cancers diagnosed clinically by PSA expression did not show any correlation of disease risk or early‐stage progression with the SIGLEC12 frameshift mutation. In contrast, analysis of two advanced colorectal cancer cohorts (TRIBE and FIRE3) demonstrated that more than 80% of the patients had wild‐type SIGLEC12. Moreover, in one of the colorectal cancer cohorts (FIRE3), the presence of wild‐type SIGLEC12 was correlated with significantly decreased survival. Meanwhile, gene expression data comparing a tumor cell line transfected with a human SIGLEC12 cDNA with sham‐transfected cells shows upregulation of pathways associated with cancer progression. Taken together, all of these evolutionary clues and experimental findings suggest that while SIGLEC12 likely lost its function in the immune system of humans prior to the origin of our species, its persistence in some humans could be associated with the pathobiology of late stage carcinomas. In this regard, we have taken advantage of the expression of wild type Siglec‐XII on urinary epithelial cells, to develop a simple urine test to check for the expression status of SIGLEC12. This approach can be combined with future genomic analyses and signaling studies, to explore this unusual human specific evolution of an apparent evolutionary liability.

Support or Funding Information

The G. Harold & Leila Y. Mathers Charitable Foundation 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.
Rapid Evolution of Bacterial Exotoxin B Subunits Independent of A subunits: Sialic Acid Binding Preferences Correlate with Host Range and Intrinsic Toxicity
Authors: KHAN NAAZNEEN , Verhagen Andrea , Khedri Zahra , Diaz Sandra , Varki Nissi , Paton Adrienne , Beddoe Travis , Paton James , Varki Ajit
Published: June 2021

Several well‐known AB bacterial toxins have cytotoxic A subunits that are delivered to the cytosol following B subunit binding to terminal sialic acid‐presenting glycans on host cell surfaces. Phylogenetic analysis of known and predicted B subunits in available genomes showed a poor correlation with that of associated A subunits, or even with that of the bacterial species phylogeny. In some cases, such as an exported protein of Yersinia pestis (YpB) there also appears to be no associated A subunit encoded in the genome. Y. pestis is the etiologic agent of Plague, responsible for major devastating epidemics in human history and is of global importance to public health and biodefense. Plague can never be eradicated because of the adaptability of the organism to infect more than 200 known host species. We noted that YpB shares 58% identity/79% similarity with the homopentameric B subunit of Subtilase cytotoxin, an AB5 toxin of E. coli, which also has 48% identity/68% similarity with that of the A2B5 S. Typhi typhoid toxin. We previously studied binding of such toxin B5 pentamers to a sialoglycan microarray and shown selective binding to different kinds of sialic acids that happen to be enriched in corresponding host cells, such as N‐acetylneuraminic acid (Neu5Ac; prominent in humans) or N‐glycolylneuraminic acid (Neu5Gc; prominent in ruminant mammals and rodents). Consistent with the much broader host range of Y. pestis, we have now found that YpB binds to essentially all types of sialic acids studied including Neu5Ac, Neu5Gc and their modified versions, and even to Kdn (2‐keto‐3‐deoxy‐D‐glycero‐D‐galacto‐nononic acid) a sialic acid prominent in reptiles and amphibians. Notably, YpB toxin alone can cause cytotoxicity that is abolished by mutations eliminating sialic acid recognition, suggesting that both uncontrolled cell proliferation and cell death might be mediated via lectin‐like cross‐linking of cell surface glycoproteins. These findings may help explain the wide host range of Y. pestis and could be important for understanding pathogenic mechanisms. Taken together, the data suggest Red Queen effects that are driving ongoing rapid evolution of both host sialic acids and pathogen toxin binding properties. Further studies of this clade of B5 toxins are revealing additional correlations of sialic acid binding preferences with host range and intrinsic toxicity, and molecular modeling studies are underway to define amino acid residues that determine binding differences.

Support or Funding Information

NIH U01 CA199792

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