bluuebunny/crossref_metadata_2025 huggingface dataset:
Authentic research in the teaching laboratory at a large university: comparative studies of malate dehydrogenase isoforms in trypanosomes
Authors: Springer Amy
Published: June 2021

Offering authentic research experiences to all Biochemistry and Molecular Biology (BMB) majors at a large university can be challenging. BMB at UMass Amherst has developed a series of Course‐Based Undergraduate Research experiences (CUREs), required for all majors. One of these focuses on studying Malate dehydrogenase (MDH) isoforms in Trypanosoma brucei, a protozoan parasite that causes African sleeping sickness. This organism utilizes oxidative phosphorylation of amino acids during growth in its Tsetse fly host, but is strictly glycolytic during growth in the mammalian bloodstream, thus understanding metabolic regulation in T. brucei is important to elucidating how the parasite proliferates in the host. T. brucei has three functional MDH isoforms localized to mitochondria, cytoplasm or glycosomes, respectively. MDH is an important enzyme that catalyzes the oxidation of oxaloacetate to malate, concomitant with the reduction of NAD+ to NADH. It highly conserved and important not only as part of the citric acid cycle reactions but also as part of gluconeogenesis and of the malate aspartate shuttle used to maintain redox balance. The wealth of structural and functional information about MDH from model organisms is used to inform studies of the orthologs from a range of evolutionarily distinct organisms, currently being conducted by a consortium of collaborating faculty from various colleges and universities out as part of the protein‐centric MDH CURE community (MCC). This collaboration gives faculty and students access to community beyond the students and instructors in the course. We have generated clones for recombinant expression of all three trypanosome MDHs and have begun kinetic analyses on the cytoplasmic and glycosomal isoforms, and are optimizing expression levels for the mitochondrial form. Students can design a projects de novo or choose to continue a project from students in a previous semester. During the term students give a presentation and have many writing assignments, with opportunities for peer review and revision, all teams present their results in a department‐wide poster session at the end of the semester. Projects a have included comparisons between recombinant forms with C‐ or N‐terminal tags and between different isoforms, or orthologs of different organisms, function at different temperature, pH or ionic strength. These studies provide tools for further analysis of biochemical and kinetic properties of the T. brucei enzymes and to perform comparative analyses with well characterized mammalian and plant MDH enzymes.

Support or Funding Information

NSF IUSE “An Interdisciplinary Faculty Community Using a Protein‐focused Course Based Undergraduate Research Experience (CURE) to Improve Student Learning,”

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
3D Model of Human O‐N‐Acetylglucosamine Hydrolase
Authors: Hawkins Darrius C. , Tasie Ihuoma , Gayle Ashley , Billings Ryan , Harris Aliah , Jones Candace , Van Stry Melanie
Published: June 2021

The post‐translational modification (PTM) of proteins enable cells to react promptly to internal and external signals through direct and progressive control of protein function. Addition of O‐linked‐n‐acetylglucosamine (O‐GlcNAc) to serine and threonine residues of cytoplasmic, nuclear and mitochondrial proteins is a nourishment and stress response PTM. O‐GlcNAcylation has been linked to several diseases such as diabetes and cancer. For example, increased O‐GlcNAcylation is directly linked to insulin resistance and to hyperglycemia‐induced glucose toxicity, two characteristics of diabetes and diabetic complications. O‐GlcNAc transferase (OGT) and O‐GlcNAcase (OGA) are enzymes that control the dynamic cycling of this PTM. Here, we have constructed a 3D model of human OGA bound to the transition state analog Thiamet‐G using Jmol and the PDB file 5M7S, a truncated version of the enzyme called Split1 (Roth et al. Nat. Chem. Biol. 2017). This protein is a functional homodimer that is stabilized by a helical bundle formed by helices from each subunit. Thiamet‐G fits in the substrate binding pocket formed by Cys215, Tyr298, and Trp278 and interacts with Gly67, Lys98, Asn280, Glu295, and Asn313 to form hydrogen bonds. Thiamet‐G also interacts with the catalytic residues Glu174 and Glu175. The focus of this project was to explore the protein structure of OGA and design and build a physical model that illustrated key functional features of the protein.

Support or Funding Information

Funded in part by NSF‐DUE 1725940 for the CREST Project.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Epigallocatechin‐3‐gallate Suppressed Methylglyoxal‐Induced Apoptosis in SH‐SY5Y Cells
Authors: Cheng An‐Chin , Lee Ming‐Fen , Huang Tzu‐Li , Liou Yi‐Siou , Pan Min‐Hsiung
Published: June 2021

Glycation is an endogenous process that generates a class of compounds known as advanced glycation end products (AGEs). AGEs are found in age‐related intracellular protein deposits of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Green tea polyphenols have a role in the reversal of age‐related loss of neuronal plasticity and recovery after neuronal lesions. There are several polyphenolic catechins in green tea, where epigallocatechin‐3‐gallate (EGCG) is both the most abundant and most biologically active form. EGCG could exhibit protective effects against AGEs‐induced injury in neuronal cells through its antioxidative properties, suggesting a beneficial role for this tea catechin against neurodegenerative diseases. In the current study, efforts were made to investigate the effects of EGCG on MG‐induced apoptosis. SH‐SY5Y cell were treated with different concentrations of EGCG followed by treatment with MG. Our data exhibited that both the cleaved PARP and cleaved‐caspase‐3 were downregulated by EGCG, and the MG‐induced reduction of Bcl‐2 expression were reversed by EGCG treatment. Moreover, EGCG could protect cells from death via reverse MG‐induced promoting of ERK/JNK pathway, and associated with reduced p53 accumulation. Our results implicate that EGCG could suppress MG‐induced apoptosis.

Support or Funding Information

This study was supported by NSC 100‐2313‐B‐309‐003 to A‐C Cheng

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Induction of immunogenic cell death of cancer cell by using chemical detergent treatment and its activation of immune system
Authors: Kimura Tsuyoshi , Nakamra Naoko , Hashimoto Yoshihide , Kishida Akio
Published: June 2021

Allogenic whole cancer cell vaccines have been proposed for cancer immunotherapy. Immunogenic cell death (ICD) is one of programed cell death and the ICD induced cells show high immunogenicity and can activate immune system effectively. So, ICD induced cancer cell is expected as an autologous cell vaccine. As ICD induction method, chemical and physical methods have been proposed. Among them, high hydrostatic pressure (HHP) treatment can induce ICD of cancer cell. The HHP‐ICD induced cancer cells show higher immune activation than other methods. However, the ICD induction efficiency is still low. Therefore, in order to develop simple and effective ICD induction method, we investigated ICD induction method by using various detergents, such as SDS, Triton X‐100 and Tween 20. HeLa cells were treated with detergents at various concentrations. For the Tween 20 treated HeLa cell, the calreticulin and HMGB1 translocations were confirmed by confocal laser microscopic observation (Fig. 1). This result indicates that immunogenic cell death of HeLa cells was induced by the detergent treatment. For SDS and Triton X‐100, although the cell death was induced, the calreticulin and HMGB1 translocations were not confirmed. The ICD induced HeLa cells were effectively phagocytosed by macrophage‐like cells and the effective secretion of TNF‐alpha from the macrophage‐like cells also confirmed by ELIZA assay (Fig. 2). From these results, it was suggested that the ICD inducted cancer cells by using Tween 20 could activate immune system.

Support or Funding Information

JSPS Grant‐in‐Aid for Scientific Research (B)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modulation of Phospho Tensin Homolog (PTEN) in Cancer Cells and Lipid Peroxides in Peripheral Blood Mononuclear Cells (PBMCs) Following Exposure to Flavonoids
Authors: Garcia Alekzander Sky , Myles Elbert , Boadi William Y.
Published: June 2021

Plant flavonoids have been shown to offer more protective health benefits against oxidative deoxyribonucleic acid (DNA) damages caused by γ‐ray radiation, UV irradiation, chemicals, and endogenous oxidative stress. The above compounds have been shown to be capable of modulating the activity of enzymes, and affect the behavior of many cell systems. While these may account for the anti‐mutagenic activities of flavonoids in experimental systems, relatively little is known about the mechanisms of the modulation of PTEN activity in cancer cells and the levels of lipid peroxides in peripheral blood mononuclear cells (PMBCs). The objectives of this study was to investigate the effects of three flavonoids: quercetin, kaempferol and genistein on the expression levels of both phospho‐ and total‐PTEN in breast cancer (BT‐549), lung cancer (A‐549) and human preadipocytes (3T3‐L1 which was used as controls). Furthermore, the effects of the flavonoids on lipid peroxides in PMBCs were investigated. Cells were seeded and were exposed to each of the flavonoid at concentrations of 0, 5, 10, 15, 20 and 25 μM and the phosphorylation of PTEN at Ser380, total PTEN levels, and lipid peroxides (i.e., malonaldehyde (MDA before and after the oxidative stress)) levels were measured. The results indicate the flavonoids increased both phospho‐PTEN‐ and total‐PTEN levels increased in a dose‐dependent manner. The effect of quercetin was more pronounced followed by genistein and kaempferol. Exposure of PMBCs cells to the various flavonoids at the different concentrations lowered the MDA levels compared to their respective controls. Levels of MDA in quercetin treated samples were lower compared to kaempferol and genistein. The findings suggest that the flavonoids play an important role in controlling oxidative stress in cells.

Support or Funding Information

National Institutes of Health: 2T34GM007663

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inductively Coupled Plasma Optical Emission Spectrometry (ICP‐OES) Analysis of Lead Bioaccumulation in Chlamydomonas reinhardtii and the Role of Cia7 Gene
Authors: Vazquez Cano Erick U. , Reyes Victoria , Ynalvez Ruby A.
Published: June 2021

The indiscriminate use of heavy metals in human activities has become a threat for the biochemical equilibrium and geochemical cycles of nature. To survive in heavy‐metal polluted environments, some microorganisms have developed a variety of detoxifying mechanisms. An excellent model organism for the study of heavy metal tolerance and homeostasis is Chlamydomonas reinhardtii, a unicellular green haploid microalga. The purpose of this research project was to identify the function of the novel gene Cia7 in C. reinhardtii, and specifically, to determine if it has a role in heavy metal tolerance. It was hypothesized that wild type C. reinhardtii (CC4425) would exhibit less bioaccumulation and higher resistance heavy metal stress; while a mutant strain in which Cia7 has been suppressed (CC5013), would exhibit inhibited growth and higher metal bioaccumulation compared to the wild type. In this study, both strains of C. reinhardtii were treated with either 0 μM or 150 μM of Pb(NO3)2 solution. Lead bioaccumulation after three days of treatment was determined by inductively coupled plasma optical emission spectrometry (ICP‐OES). Chlorophyll concentration and cell growth analysis were also performed. Both strains of C. reinhardtii were treated with increasing concentrations of lead, and absorbance readings at 663, 645, 730 nm were taken on 3‐day intervals over a 15‐day treatment period. Results from ICP‐OES analysis showed no difference in lead bioaccumulation between both strains. The wild type strain (CC4425) demonstrated a relatively higher growth rate than the mutant (CC5013). Chlorophyll concentration and growth in the wild type strain decrease as the concentration of lead in solution increases. On the other hand, chlorophyll concentration and growth in the mutant strain was observed to increase with a higher treatment of lead.

Support or Funding Information

ACT on Ideas ‐ Texas A&M International University/College of Arts and Sciences ‐ Texas A&M International 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.
Determining the Role, Expression and Interactions of FAP‐1 in S. cerevisiae, Cultivated in a Nitrogen‐limited Media
Authors: Velez Astrid Carolina Rodriguez , Matos Elsie Pares
Published: June 2021

The protein FAP‐1 was found in the cytoplasm of Saccharomyces cerevisiae, and it confers resistance to the immunosuppressive drug and antifungal Rapamycin, by competing with the drug for the binding site, which is mutually exclusive for both, thus preventing the formation of the Fpr1p‐Rapamycin complex. This complex inhibits the products of the TORC1 which regulates essential pathways that control some cytoplasmic and nuclear events, and some nutrient regulated genes. In preliminary studies, it has been observed that FAP1 needs to be overexpressed in other to confer this resistance to the yeast. The complex FAP1:Fpr1p has been also observed in the absent of Rapamycin, especially when some nutrient levels are low. By using these observations, we proposed that the concentration of FAP1 might be inversely proportional to the availability of nutrients in culturing media. To prove this hypothesis, the strain CFY7‐PJ69‐6A of Saccharomyces cerevisiae will be cultured in Nitrogen‐limited media and at different time periods. The technique qPCR was used to quantify the amount of fap1 RNA available at different concentrations of ammonium sulfate (the nitrogen source), and at incubation points of 3 hrs, 6 hrs, 9 hrs and 12 hrs. We found that the expression of fap1 is inversely proportional to the availability of nitrogen in culturing media, whereas the growth and division was directly proportional to the nitrogen availability. The expression levels observed with the qPCR could be useful to optimize bioprocesses at industry by monitoring FAP1 in order to determine the availability of target nutrients in media in yeast cultures. Currently, we are interested in the identification of protein‐DNA interactions via Chromatin Immunoprecipitation, to determine which genes this transcription factor might regulate.

Support or Funding Information

This work is supported by the NIH‐MARC‐U‐STAR Grants: 5T34GM008419‐25 and 2T34GM008419‐26, and Puerto Rico's Louis Stoke Alliance for Minority Participation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Distinct Roles of Dietary Fat and Sugar in the Development of Obesity, Insulin Resistance, Atherosclerosis and Cardiac Dysfunction in LDL Receptor Knockout Mice
Authors: Perazza Laís Rossi , Daniel Noemie , Dubois Marie Julie , Pilon Geneviève , Mitchelle Patricia , Le Quang Khai , Lachance Dominic , Plante Eric , Varin Thibaut , Bouchareb Rihab , Mathieu Patrick , Pouliot Yves , Gauthier Sylvie , Roy Denis , Asselin Claude , Blais Mylène , Lessard Martin , Marette André
Published: June 2021

The increased consumption of high caloric foods has been associated with the obesity epidemic and a higher incidence of cardiometabolic disease, but the specific role of dietary fat and sugar remains unclear. There is growing evidence that high intake of added sugar contributes to cardiovascular disease (CVD) but whether this is independent from high fat content in the diet remains to be established. To this end, our objective was to determine the impact of high sucrose intake on the features of metabolic syndrome and CVD in a mouse model of atherosclerosis.

We used atherosclerosis prone LDLr−/− ApoB100/100 (LRKOB100) mouse fed either a low‐fat/high‐sucrose (LFHS) or a high‐fat/low‐sucrose (HFLS) diet supplemented with 0.2% cholesterol for 24 weeks.

Body weight gain and whole‐body fat mass were greater in HFLS‐fed vs LFHS‐fed mice. HFLS feeding promoted higher liver triglyceride (TG) deposition and plasma hypercholesterolemia when compared to LFHS feeding. HFLS‐fed animals were more insulin resistant than LFHS as revealed by a higher HOMA‐IR index. Unexpectedly, while LFHS‐fed mice were less prone to metabolic impairments, they had significant atherosclerosis development as revealed by extensive aortic plaque formation in comparison to HFLS‐fed mice. Consistent with atherosclerotic plaque formation, echocardiography further suggested that LFHS‐fed LRKOB100 mice developed left ventricle eccentric hypertrophy (LVEH) after only 12 weeks of feeding as compared to HFLS‐fed mice. Determination of the inflammatory status by multiplex analysis revealed that HFLS‐fed mice had elevated hepatic IFN‐γ, circulating MCP‐1 and PAI‐1. Conversely, LFHS‐related atherosclerosis was linked with increased hepatic levels of IL‐1β, IL‐6, RANTES and TNF‐α. In addition to hepatic inflammation, LFHS fed animals showed higher amount of TNF‐α and IFN‐γ in visceral adipose tissue, consistent with activation of metabolic inflammation.

Our results indicate that dietary sugar and fat have distinct roles in the development of cardiometabolic diseases in LRKOB100 mice. While high fat intake promotes obesity and metabolic alterations, high sugar intake promotes atherosclerosis and cardiac dysfunction, with a major effect on the hepatic inflammatory profile. Further investigations are needed to unravel the underlying mechanisms by which high sugar intake promotes the atherosclerotic process.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Antioxidant effect of Justicia spicigera in liver from diabetic rats
Authors: Murillo‐Villicaña Marina , Martínez‐Mora José A. , Noriega‐Cisneros Ruth , Manzo‐Avalos Salvador , Salgado‐Garciglia Rafael , Cortés‐Rojo Christian , Montoya‐Pérez Rocío , Saavedra‐Molina Alfredo
Published: June 2021

The oxidative stress is a factor that contributes to the development of complications in the diabetes disease; however, its effects can be counteracted by the use of exogenous antioxidants, which are found in some plants. That is why people look to the traditional medicine in search of the therapeutic treatment. Justicia spicigera has been demonstrated to have the capacity of reduce glycemia levels; nevertheless the effects in non‐insulin dependent‐organs like liver, has not been reported. During administration of the ethanol extract for 30 days, the blood glucose and weight data were taken every 5 days. Once the treatment concludes, the rats were sacrificed. The corporal weight, blood glucose, cholesterol‐Very Low Density Lipoprotein (VLDL), triglycerides, total lipids and liver profile; were diminished in the diabetic condition and normalized with the application of ethanol extract of J. Spicigera (EJS). Meanwhile catalase activity shown a significant increase in control diabetic rats and a decrease in their activity with the application of the extract, without any effect in normoglucemic rats. There is also a tendency to increase the GSH/GSSG ratio and a decrease in total glutathione levels in diabetic rats. In conclusion, the ethanol extract of Justicia spicigera is considered to reduce oxidative stress in liver in STZ‐induced diabetes rats.

Support or Funding Information

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the elongated cell phenotype of Escherichia coli overexpressing the lysophospholipase PldB
Authors: Georgiou George S. , Garrett Teresa A.
Published: June 2021

The overexpression of the enzyme PldB leads to increased levels of the headgroup‐acylated glycerophospholipid, acyl phosphatidylglycerol (acyl PG), in the membranes of Escherichia coli. The role of acyl PG in E. coli has yet to be fully determined. Interestingly, PldB overexpression leads to an elongated phenotype, suggesting a role for acyl PG in cell length and division. To gain a better understanding of this phenotype, wildtype MG1655 and cells lacking the chromosomal pldB (ΔpldB) were transformed with an empty arabinose‐inducible plasmid (pBAD), and a plasmid containing pldB (pPldB). These four strains (MG1655/pBAD, MG1655/pPldB, ΔpldB/pBAD, and ΔpldB/pPldB) were grown to mid‐log phase and imaged via scanning electron microscopy. Quantification of cell lengths from SEM images showed a significant difference in the average length of the wild‐type strain, MG1655/pBAD (1.85 μm ± 0.46 μm), versus MG1655/pPldB (2.69 μm ± 1.12 μm) and ΔpldB/pPldB (3.02 μm ± 2.42 μm). Furthermore, many of the elongated cells appeared to contain no cleavage furrows suggesting that they failed to complete cellular division. Visualization of the chromosomal DNA by staining with DAPI revealed that PldB overexpressing strains contained multiple unsegregated nucleoids within individual cells, providing more evidence that PldB overexpression leads to irregularities in cellular division. Future research will focus on separation of the inner and outer membranes of E. coli overexpressing acyl‐PG in order to assess the lipid compositions of each respective membrane and elucidate where acyl‐PG migrates and integrates once formed. Additionally, the contractile Z‐ring of E. coli overexpressing acyl‐PG will be visualized via fluorescence microscopy to further investigate the relationship between acyl‐PG and cellular division.

Support or Funding Information

Supported by National Science Foundation Research at Undergraduate Institutions grant #1516805.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulating the activity of Akt by inhibiting the pleckstrin homology domain‐PtdIns(3,4,5)P3 interaction using flavonoids
Authors: Kang Yerin , Lee Woonwoo , Yoon Youngdae
Published: June 2021

Since over‐activation of cellular signalling was frequently observed from a wide range of cancer types, the down‐regulation of signalling is regarded as a goal for drug development. A serine‐threonine kinase, AKT, playing a pivotal role in tumour progression was also over‐activated in cancer cells; thereby AKT was believed as a critical therapeutic target for cancer intervention. In this study, we targeted the pleckstrin homology (PH) domain to down‐regulate AKT activity via inhibiting phosphatidylinositol‐3,4,5‐triphosphate (PIP3) binding using by small natural molecules, flavonoids. The inhibition rates of flavonoids were evaluated by liposome pull‐down assay coupled with fluorescence spectrophotometry, and several flavonoids including 3,6‐dihydroxyflavone and 6,2′‐dihydroxyflavone showed about 40 and 50 % of inhibition at 10μM. Moreover, it was confirmed that flavonoids targeting PH domain‐PIP3 interaction decreased the activity of AKT in cellular levels. And then, we further analyzed the relationship between structure and activity using QSAR to find optimum structural moiety because of the structural similarity of flavonoids. Although it needed more study to derive optimum moiety of flavonoids as anticancer drugs for targeting AKT, we believed that this finding would provide a strong evidence to regulate AKT activity by targeting the regulatory domain instead of catalytic kinase domain.

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 Effects of the Senescent Preadipocyte Secretome on Skeletal Muscle Cells
Authors: White Thomas A. , Mazula Daniel , Schafer Marissa , Giorgadze Nino , Aversa Zaira , Kirkland James L. , LeBrasseur Nathan K.
Published: June 2021

Adipocytes accumulate within and around skeletal muscle with advancing age. Adipose progenitor cells, preadipocytes, are prone to cellular senescence, a state of stable growth arrest associated with the secretion of cytokines, chemokines and matrix remodeling proteins, collectively referred to as the senescence‐associated secretory phenotype (SASP). Considerable evidence implicates the SASP in age‐related tissue inflammation and degeneration; however, its impact on skeletal muscle is poorly understood. Herein, we examined the effects of conditioned media (CM) collected from healthy human (control CM) and senescent by irradiation (senescent CM) preadipocytes on human skeletal muscle cells (SkMCs). Compared to control CM, senescent CM had more than 10‐fold higher concentrations of IL‐6, IL‐1β, IL‐8, PAI‐1, MCP‐1 and TNFα as quantified by a multiplexed antibody‐based assay. Senescent CM increased expression of IL‐6 (5‐fold), IL‐1α (1.6‐fold), IL‐1β (3.5‐fold), MCP‐1 (2.5‐fold), PAI‐1 (1.7‐fold) and MMP‐3 (3‐fold) in differentiated SkMCs relative to control CM as measured by qPCR (all p < 0.05). Senescent CM increased proliferation of undifferentiated SKMCs by 20%; however, inhibited differentiation relative to control CM per the fusion index. Preliminary data further suggest senescent CM compromises insulin‐ and IGF‐1‐mediated activation of Akt. Collectively, our data suggest that the SASP of senescent preadipocytes may drive inflammation in SkMCs and compromise their differentiation and, potentially, response to metabolic an anabolic stimuli. These effects may contribute to age‐related changes in skeletal muscle health and regeneration.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Engineering riboswitch‐based whole cell biosensors in Escherichia coli
Authors: Zhao Maryann , Page Katharine , Liu Jane
Published: June 2021

In vitro diagnostics (IVD) have become increasingly popular tool for tackling global health concerns, but conventional IVD tests, such as microscopy and nucleic‐acid amplification methods, are often expensive and complex to use, limiting their use in low‐resource settings. Whole‐cell biosensors in bacterial systems offer an attractive solution as an easy‐to‐use, portable device that non‐specialists could use for clinical purposes outside clinical locations. However, a strategy is needed to engineer ligand specific sensors for new targets. An appealing candidate for a sensing module is the riboswitch. Genetic RNA switches, termed riboswitches, are genetic regulatory elements found in the 5′‐UTR (untranslated region) of some prokaryotic mRNA and contain two functional components: an aptamer binding domain and an expression platform that modulates downstream gene expression. We developed an engineering platform coupling genetic selections and Fluorescence‐Activated Cell Sorting (FACS) screens to identify novel riboswitches from a 108 random‐sequence library in which we replaced the aptamer sequence of the ThiM#2 riboswitch with a degenerate 40‐nucleotide sequence. In a proof‐of‐principle validation of the platform, we identified novel riboswitches for the small molecule theophylline, which are distinct from the existing synthetic theophylline switches. Our best riboswitch hit displays a 2.3‐fold activation in response to theophylline and does not respond to the structurally unrelated molecule thiamine. We performed directed evolution on this riboswitch hit by mutagenizing its sequence and performing additional rounds of selections and screens, resulting in a variant with a 3.6‐fold activation. Current efforts are focused on assessing the efficacy of the selection platform when isolating riboswitches for dopamine. Furthermore, a variant of the engineering platform is being used side‐by‐side to compare if a dopamine riboswitch can be selected from a library composed of an in vitro selected aptamer coupled with a 15‐nucleotide degenerate sequence. We anticipate that these efforts will provide general guidelines toward the development of novel riboswitches for a variety of natural and non‐natural ligands.

Support or Funding Information

NSF CBET 1258307

Arnold and Mabel Beckman Foundation

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Vasopressin infusion throughout pregnancy causes placental pathology in mice consistent with preeclampsia
Authors: Gibson‐Corley Katherine N. , Sandgren Jeremy A. , Perschbacher Katherine J. , Zhang Shao Y. , Deng Guorui , Santillan Donna A. , Santillan Mark K. , Grobe Justin L.
Published: June 2021

Human preeclampsia (PE) is associated with elevated secretion of arginine vasopressin (AVP), and chronic infusion of AVP into pregnant mice is sufficient to model PE by causing hypertension, renal glomerular endotheliosis, proteinuria, fetal placental hypoxia, and growth restriction. Early stages of PE are associated with defective trophoblast invasion of maternal spiral arteries, leading to decreased artery diameter and placental oxygenation. AVP infusion (24 ng/hr, sc) reduced placental growth factor mRNA and protein at gestational day (GD) 17.5. Therefore, we performed histopathologic analyses of placentas collected from mice at GD12.5 infused with saline or AVP, with the hypothesis that AVP leads to early placental PE phenotypes. This preliminary cohort demonstrated an increased urine protein content (saline n=7 27.1±2.7, AVP n=11 44.1±2.5 mg/mL; p<0.01), trends toward elevated mid‐gestational systolic blood pressure (104±2.1, 110±2.2 mmHg; p=0.09), and similar changes in heart rate (Δ from baseline: 37.1±15.5, 38.8±11.8 bpm; p=0.93). GD12.5 placentas were then routinely processed and stained with HE or cytokeratin‐8 to examine morphological changes induced by AVP. AVP infusion had no significant effects junctional zone (saline n=11 383±18, AVP n=13 376±15 μm; p=0.76) or decidua layer (652±21, 585±33 μm; p=0.12) thicknesses but significantly reduced labyrinth layer thickness (727±26, 640±13 μm; p<0.05). AVP caused a reduction in average maximum spiral artery diameter within the decidua (161±12, 125±7 μm; p<0.05) and reduced total spiral artery number (8.1±0.8, 4.8±0.4, p<0.01), but no difference in the maximum invasion depth of CK8‐positive trophoblasts (348±22, 302±23 μm; p=0.16). We conclude that AVP infusion is sufficient to induce key mid‐gestational features of PE in pregnant mice, including reduced spiral artery diameter. Such morphological changes may be associated with the reduced placental growth factor expression in this model.

Support or Funding Information

National Institutes of Health (GM067795, HL134850, HL084207), the American Heart Association (13SDG143400012, 15SFRN23480000, 15SFRN23730000, 15SFRN23760002, 15SFRN23860007, 16PRE30980043, 15UFEL25850040, 14PRE20380401), the American Physiological Society (UGREF & UGSRF programs), University of Iowa Center for Hypertension Research, and the Roy J. Carver Trust.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Chemorepulsion as a novel therapeutic concept to inhibit pancreatic cancer metastasis
Authors: Niclou Benoit , Li Xiaoran , Zessler Astrid , Adam Rosalyn , Briscoe David , Bielenberg Diane
Published: June 2021

Pancreatic ductal adenocarcinoma (PDAC) has a high mortality rate primarily due to the absence of effective pharmacologic treatments and the limited effects of surgical resection in the early stages of the disease. The most frequent cause of death from the disease is due to its tendency to metastasize to the liver, at which point the chances of long‐term survival drop significantly. Therefore, there is an urgent need to find a drug therapy that not only affects the primary tumor, but perhaps more importantly, prevents the formation of metastases in the liver. Neuropilin 2 (Nrp2) is a transmembrane co‐receptor protein that binds a secreted protein called semaphorin 3F (S3F). Nrp2 is not expressed in the liver but is highly overexpressed in PDAC cells and PDAC‐associated endothelial cells. Based on these observations, we hypothesized that S3F expression in the liver could repel metastasizing PDAC cells. As part of a series of prevention trials, we performed an IV injection of S3F expressing adenovirus in mice and thereby induced the expression of the protein in the liver. Three days later we performed an orthotopic injection of syngeneic PDAC cells. For our intervention trials, we inverted the sequence of tumor and virus injections. Our results showed a significant reduction in tumor metastases in the experimental SEMA3F‐treated group compared to control virus treated mice. But the actions of Nrp2 are not limited to its ability to mediate repulsion. The tumor microvessel density and hence the overall PDAC tumor size was also reduced, thereby illustrating the systemic anti‐angiogenic effects of S3F‐mediated signaling via Nrp2. The action of Nrp2 on tumor growth was further elucidated by repeating the same experimental procedure in Nrp2‐deficient mice. Again, we saw a reduction in size and microvessel density within the primary tumor, which validates our initial hypothesis that Nrp2 is necessary for tumor growth and angiogenesis in PDAC. In summary the therapeutic effects of inhibiting Nrp2 are twofold: First and foremost, we have shown that S3F expression in the liver will repel Nrp2‐positive PDAC cells, as well as reduce tumor growth in the pancreas compared to the control. Finally, we have also demonstrated the anti‐angiogenic effects of S3F.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of inflammation and genes associations with Prostate cancer risk.
Authors: ANDAVOLU RADHIKA GADE , Rodriguez Olivia , Shaaf Emon GADE
Published: June 2021

The pathogenesis of cancer involves environmental factors in addition to hereditary factors. Recent studies have pointed to a potential role for inflammation in prostatic carcinogenesis and tumor progression. An important factor is the development of chronic inflammation in the prostate due to a number of potential causes including infections, dietary factors, hormonal changes and/or other unknown environmental exposures. In this study we investigated the possible association between single nucleotide polymorphisms in the interleukin IL‐1B (C>T), tumor necrosis factor (308G>A), RANTES‐403, GSTP1 exon 5 polymorphic site (Ile105àVal), PPARG (Pro12Ala) genes also two STR polymorphic marker GSTP1 (penta‐nucleotide repeat at promoter regions) and a pyrimidine‐rich penta‐nucleotide repeat length polymorphism, CD4‐1188 (TTTTC) and the risk for developing prostate cancer. Our study is comprised of 134 prostate cancer cases and age, gender and race matched 178 normal healthy controls. These genes are known to be important for inflammation of prostate and common allelic variants have been shown to have a biological effect. Hence, we searched for a possible association between SNP polymorphisms and STR polymorphic allele gene frequencies obtained in prostate cancer patients and compared with those of a normal control population, looking for allelic associations between any of these polymorphic loci and prostate cancer risk. Strong association between GSTP1, CD4, RANTES (p<0.001), a high variation in allelic frequency of mutant IL1B, TNF and PPARG (p=NS) and prostate cancer risk are observed. Genetic associations of disease and genetic variations in inflammatory genes are often relatively modest, it is likely that polymorphisms in multiple inflammatory genes cooperate in an additive or synergistic manner to impact disease risk Because genetic associations of disease and genetic variations in inflammatory genes are often relatively modest, it is likely that polymorphisms in multiple inflammatory genes cooperate in an additive or synergistic manner to impact disease risk. The possible role of inflammation and gene associations with prostate cancer risk will be discussed.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Toll‐like Receptor‐4 Activation Improves the Immunosuppressive Properties of Cancer Cells‐Derived Exosomes
Authors: Domenis Rossana , Marinò Daniele , Cifù Adriana , Fabris Martina , Niazi Kayvan R. , Soon‐Shiong Patrick , Curcio Francesco
Published: June 2021

Tumor cells actively produce and release exosomes to promote tumor growth and progression. Upon the contact with target cells, exosomes alter phenotypic and functional features of recipients, reprogramming them into active contributors to metastasis and immunosuppression. Overexpression of Toll‐like receptor‐4 has been implicated in the development of several types of cancer, since its activation increase their tumorigenic potential of cancer cells.

The purpose of this study is to investigate the immunosuppressive properties of cancer cell‐derived exosomes released upon TLR4 activation by LPS.

Cancer cell lines (primary colon SW480, metastatic colon SW620, metastatic breast MDA‐MG‐231 and glioma U87) were treated with LPS (1μg/ml) for 24h and then cultured in exosome‐free medium for 48h. Exosomes were isolated from cells supernatants by polymer precipitation method (Exoquick), quantified by determining the activity of Acetyl‐CoA Esterase (Exocet test) and validated by exosome‐specific tetraspanins expression by western blot and flow cytometry analysis. The expression of immunosuppressive factors on exosomes surface was evaluated by flow cytometry and Multiplex ELISA. Exosomes immuneregulatory properties were assessed by in vitro functional assays on immune cell subpopulations isolated from healthy donor's blood.

We demonstrated that the treatment with LPS enhances the immunosuppressive potential of cancer cells‐derived exosomes, although differences were observed depending on the origin of the tumor. The expression of TGFβ and MICA/B was increased on surface of exosomes released from LPS‐stimulated cells. Moreover, exosomes became able to inhibit the proliferation of activated CD3+ cells, increase T cells apoptosis and promote the conversion of CD4+ in regulatory T cells.

Our data suggest that the activation of TLR4 contributes to tumor progression by promoting the release of more effective immunosuppressive exosomes, which allow tumor cells to escape immune surveillance.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Chemotherapeutic tolerability and Estrogen dose response in the B6;129‐Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) mouse model
Authors: Naden Jamie , Melton Michele , Bast Athena , Gardner Jermaine
Published: June 2021

The B6;129‐Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) knockout mouse was developed by backcrossing an IL2rg (common gamma) knockout model to a RAG2 (recombinase activating gene) knockout model. The resulting mouse lacks various cytokines including IL‐2, IL‐4, IL‐7, IL‐9 and IL‐15. In addition, this model lacks B cells, T cells, NK cells and has a deficit in lymphocyte development. This model was developed to provide another immunodeficient option for the oncology and immunology fields. The literature supports better tolerability of DNA damaging oncology treatments for models that do not carry the SCID mutation. We have already reported in a white paper that the R2G2 mouse model is more tolerant of whole body radiation than a model with the SCID mutation. Herein we describe a study examining chemotherapeutic tolerability of common DNA damaging oncology drugs including 5‐fluorouracil (5‐FU), doxorubicin (Doxo), and cyclophosphamide (CTX) (n = 10 per group). 5‐FU was given at 30, 60 or 100 mg/kg, intraperitoneally, twice weekly for five weeks. Doxo was given at 2 or 5 mg/kg, intraperitoneally, once weekly for three weeks. CTX was given at 100 or 140 mg/kg intraperitoneally, once weekly for three weeks. The low dose was chosen based on the average dose used in immunodeficient mouse models and the high dose was chosen based on the average dose used in immunocompetent mouse models. This was done to determine if the R2G2 mouse model can tolerate higher doses of these chemotherapeutic agents compared to other immunodeficient mouse models. Results show that the R2G2 mouse model tolerates higher doses of these chemotherapeutic drugs than doses found in the literature for SCID models. Exogenous estrogen tolerance is another common concern in oncology research as some immunodeficient mouse models cannot tolerate the subcutaneous estrogen pellets, developing negative secondary effects resulting in removal from study. We performed an estrogen pellet dose response study using four doses of 60‐day release 17‐β estradiol pellets at 0.18, 0.36, 0.72, and 1.7 mg/pellet (n = 10 per group). R2G2 mice show dose dependent effects of estrogen on morbidity. These data will allow researchers to determine the optimal dose for use in the R2G2 model. In conclusion, these data support that the R2G2 mouse model may be a good alternative to SCID models when administering DNA damaging chemotherapies or when estrogen supplementation is required for xenograft growth.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Elucidation of self‐mediated enhancement of dopamine transport by the dopamine transporter which can be modulated by extracellular gate and N‐terminal residues
Authors: Henry L. Keith , Allen Michael Dean , Shetty Madhur , Cwikla Allison , Nies Isaac
Published: June 2021

The dopamine (DA) and serotonin (5‐HT) transporters (DAT and SERT respectively) are members of the SLC6 family of neurotransmitter:sodium symporters (NSSs). DAT and SERT clear substrate from perisynaptic spaces by the generally accepted alternating access mechanism in which the transporters transition between outward‐facing and inward‐facing conformational states. This mechanism involves a sequential closing and opening of extracellular (EC) and intracellular (IC) gates along the permeation pathway. The EC and IC gates are almost exclusively composed of acidic and basic residues capable of interacting through salt bridge formation, closing the primary substrate binding site to the intracellular and extracellular milieus. Previously, we identified that the human SERT acidic/basic residue pair is critical for the recognition of 3,4‐methylenedioxymethamphetamine (MDMA) as a substrate. In contrast, mutations in the EC gate had no effect on 5‐HT recognition. Our current efforts focus on determining if the EC gate of the human DAT has similar mechanistic characteristics for substrate selectivity. While investigating that possibility, we identified a previously unreported phenomenon of self‐mediated enhancement of DA transport in hDAT expressing cells. In homologous competitive uptake experiments utilizing [3H]DA, a 200% to 300% increase in [3H]DA uptake is observed upon addition of micromolar concentrations of unlabeled DA. This enhancement is further modulated by the identity of the acidic EC gate residue. Substituting the Asp with the polar amino acid Asn (D476N) produces a 600% increase in [3H]DA uptake. Experiments utilizing heterologous competition with [3H]DA and the known hDAT substrates 1‐methyl‐4‐phenylpyridinium (MPP+), 4‐(4‐dimethylamino)phenyl‐1‐methylpyridinium

(APP+) and tyramine indicate that the enhancement effect is not exclusive to DA nor is it observed with all DAT substrates. Notably, elimination of phosphorylation at N‐terminal residue Thr53 virtually eliminates the DA self‐enhancement phenotype. The apparent ability of DA to enhance its own uptake is consistent with reports proposing a second substrate binding site (S2) that can act as a trigger for release of substrate from the primary DA binding site (S1). However, the presence of this S2 site remains controversial. Continued investigation of this phenomenon may provide insight into molecular determinants on both the substrate and transporter that are important for substrate identification and translocation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
D2 Autoreceptor Signaling is Depressed by Neurotensin Released from Discrete Inputs to Midbrain Dopamine Neurons.
Authors: Tschumi Christopher W. , Beckstead Michael J.
Published: June 2021

Midbrain dopamine neurons play physiological roles in many processes including reward learning and motivated behavior. Neurotensin (NT) is a neuropeptide which acutely modulates midbrain dopamine neuron excitability through multiple mechanisms, including a decrease in dopamine D2 autoreceptor‐mediated inhibition. While there are dense neurotensinergic inputs to the midbrain that arise from many different brain regions, most studies to date have relied on exogenous application of NT and NT ligands to determine physiological consequences of NT signaling. As such, the mechanisms involved in release and signaling of endogenous NT from different sources are poorly understood. Here we used transgenic mice that express channelrhodopsin in neurons that also either express NT or the dopamine transporter. We then combined patch clamp electrophysiology of dopamine neurons in midbrain slices with blue light stimulation to specifically activate either neurotensinergic or dopamine neurons (respectively). We found that low frequency stimulation of dopamine neurons alone was sufficient to induce long‐term depression (LTD) of D2 autoreceptor inhibitory postsynaptic currents (IPSCs) and that this LTD could be blocked by either chelation of calcium in the patched cell or by preincubating the slice with a non‐selective NT type 1 and 2 receptor antagonist. It is believed that dopamine neurons do not synthesize NT, but rather take up NT via internalization of NT receptors. Consistent with this, in mice that expressed channelrhodopsin in neurotensinergic neurons, blue light did not cause a channelrhodopsin‐mediated excitation of dopamine neurons. However, low frequency stimulation of neurotensinergic neurons did result in a depression of D2‐IPSCs (though to a lesser extent than stimulation of dopamine neurons) and this depression was blocked by preincubating the slice with a non‐selective NT type 1 and 2 receptor antagonist. Together, these results suggest that neurotensin could be released either by dopamine neurons themselves to act as a retrograde messenger or released by non‐dopaminergic neurons to act as an anterograde messenger, and in both cases can cause LTD of D2 autoreceptor signaling. These findings are the first steps in defining the circuit‐specific NT‐induced plasticity which regulates dopamine neuron activity.

Support or Funding Information

This work was supported by National Institute on Drug Abuse Grant R01 DA32701 (M.J.B.), as well as funds from the Presbyterian Health Foundation and the Oklahoma Center for Adult Stem Cell Research.

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