bluuebunny/crossref_metadata_2025 huggingface dataset:
Scientific Community Outreach: Promoting Science to Students at the K‐12 Level and STEM Students in ASBMB UPR‐RP
Authors: Navarro Edwin D. , Perez‐Oquendo Mabel G. , Ramirez‐Lugo Juan
Published: June 2021

Objectives

The American Society for Biochemistry and Molecular Biology (ASBMB) Student Chapter at the University of Puerto Rico‐Rio Piedras Campus (UPR‐RP) is devoted on promoting science in communities inside and outside of our university. The primary goals of the chapter are: 1) to increase the interest in science of students in the K‐12 level and 2) to provide STEM students in the University with the tools and guidance to improve their academic and professional development.

Methods

To do this we offer outreach activities to students at the K‐ 12 level. These activities are comprised of experiments related to different fields of science such as biology, chemistry, physics, biotechnology and environmental science. The idea is to illustrate scientific concepts in an easy, and hands‐on way. Therefore, students can better understand these concepts, making biochemistry and molecular biology accessible to all. Aside of the outreach activities offered in schools, we also collaborate with other associations, such as the American Chemistry Society (ACS) and the American Association for the Advancement of Science (AAAS), in local activities at the ACS Chemistry Festival and AAAS Annual Meeting, respectively. Members of the ASBMB student chapter are also encouraged to participate in all outreach events so they can develop their oral presentation skills. For students in STEM fields, we offer a variety of workshops and seminars including: how to make personal statements and curriculum vitae, how to seek out research opportunities and internship opportunities, graduate and professional school panels, as well as journal clubs.

Conclusion/Future Direction

The collection of the activities and experiences that we offer to students in K‐12 level, STEM students and ASBMB members result in a successful promotion of science community at a variety of levels. Our hope is to develop an interest in science in students at the K‐12 level who are involved in our outreach activities and to facilitate the professional development of college students (both STEM students and ASBMB members).

Support or Funding Information

ASBMB Regional Meeting 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.
Patterns of Conduit Artery Shear Stress Across the Menopause Transition
Authors: Somani Yasina B , Moore David J , Kim Jin‐Kwang , Gonzales Joaquin U , Elavsky Steriani , Proctor David N
Published: June 2021

Introduction

Disturbed patterns of conduit artery shear, characterized by greater oscillatory (bi‐directional) and retrograde (backward) shear are associated with endothelial dysfunction. Moreover, declines in endothelial function can take place rapidly across the menopause transition, placing women at heightened risk for development of cardiovascular disease.

Methods

Conduit artery shear patterns were measured in women at varying stages of the menopause transition. Resting brachial artery and common femoral artery mean blood velocities and diameters were measured via Doppler ultrasound in early perimenopausal, late perimenopausal, and early postmenopausal women.

Results

Brachial artery oscillatory shear was significantly higher in early postmenopausal women (n=15, 0.17 ± 0.08 a.u.) than both early (n=12, 0.08 ± 0.05 a.u., p=0.002) and late (n=8, 0.08 ± 0.04 a.u., p= 0.007) perimenopausal women, and retrograde shear was significantly higher in early postmenopausal vs. early perimenopausal women (−9.74 ± 6.48 s−1 vs. −4.81 ± 3.05 s−1, p= 0.03). Femoral artery retrograde and oscillatory shear were greater, respectively, in early postmenopausal women (n=15, −6.78 ± 2.91 s−1; 0.19 ± 0.08 a.u.) than early perimenopausal women (n=14, −4.07 ± 2.21 s−1, p=0.03; 0.11 ± 0.08 a.u., p=0.01). Further, Pearson correlation analyses revealed significant associations between follicle stimulating hormone (FSH) and both retrograde and oscillatory shear, respectively, in the brachial (R=−0.37, p=0.04; R=0.5, p=0.005) and common femoral artery (R=−0.4, p=0.03; R=0.45, p=0.01).

Discussion

Our findings suggest that increases to conduit artery retrograde and oscillatory shear take place during the menopause transition and these increases are associated with FSH, a marker of reproductive age.

Support or Funding Information

Social Sciences Research Institute and Center on Population Health and Aging at Penn State, M01 RR‐10732 (General Clinical Research Center), K12 HD055882

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Spearmint Extract Containing Rosmarinic Acid on Physical and Executive Functioning Following a Tactical Operation
Authors: Hoffman Jay R. , Ben‐Moshe Yuval , Hoffman Mattan W. , Ostfeld Ishay
Published: June 2021

This investigation examined the effect of a proprietary spearmint extract containing rosmarinic acid (PSE) on physical, cognitive and executive functioning following a high‐risk tactical operation while sleep deprived for 24‐hours. Ten operators (35.1 ± 5.2 y; 177.6 ± 5.3 cm; 81.3 ± 9.3 kg) from an elite counter‐terrorism unit volunteered to participate in this randomized, double‐blind, parallel design study. Participants were randomized into either PSE or placebo (PL) group and ingested 900 mg·day−1 of PSE or an equivalent amount of PL for 17‐days. PRE testing occurred prior to supplementation, while POST testing occurred within 1‐hour of the operation's conclusion. Magnitude based inferences indicated that differences between PSE and PL in jump power, reactive agility, eye‐hand coordination, and cognition were unclear. However, subjective feelings of energy, alertness and focus were very likely, likely and possibly better for PSE than PL, respectively. No difference (p = 0.64) in identifying the correct target was noted between groups, however all participants in PSE correctly identified the target, while only 60% of participants in PL correctly identified the target at POST. Although the results of this study do not provide conclusive evidence in regards to the efficacy of PSE, it does suggest that further research is warranted using a larger sample of participants.

Support or Funding Information

This study was supported by Kemin Foods, L.C. (Des Moines, IA).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Crystal structure of an assembly intermediate of respiratory Complex II
Authors: Sharma Pankaj , Maklashina Elena , Cecchini Gary , Iverson Tina
Published: June 2021

In approximately 10% of flavoenzymes, the flavin is covalently attached. Despite the importance of this covalent cofactor for a number of biological functions, the mechanism of covalent flavinylation is currently poorly understood. In this work, we determined the crystal structure of a captured assembly intermediate of covalent flavinylation using the Escherichia coli Complex II FrdA subunit crosslinked with its assembly factor, SdhE. Two global conformational changes in FrdA subunit were observed as compared to prior structures of the assembled Complex II protein: rotation of the capping domain, and the destabilization of two large loops proximal to the active site of the FrdA subunit. Together, these changes create a tunnel to the active site. This FrdA‐SdhE structure was used as a basis to develop a mechanism of covalent flavinylation that is supported by spectroscopic and kinetic analyses.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
L‐lactate Increases Apoptosis in Dopaminergic‐Like PC12 Cells
Authors: LaLonde Zoë M , Behnke Jessica E , Chapp Andrew D , Shan Zhiying Jenny , Chen Qing Hui
Published: June 2021

Lactate has been shown to be excitatory through activation of ligand gated calcium channels. Studies have demonstrated that extracellular lactate is capable of activating N‐methyl‐D‐aspartate receptors (NMDAR), acidifying neurons and glial cells, which may contribute to excitotoxicity. Since cerebrospinal fluid (CSF) lactate concentrations are elevated in several neurodegenerative disorders, including Parkinson's disease, we tested the hypothesis that increased L‐lactate may be a contributing factor to excitotoxicity of dopaminergic like PC12 cells. Nerve growth factor derived PC12 cells were exposed to increasing concentrations of L‐lactate (0mM, 1.5mM, 2mM, 3mM, 4mM and 8mM) for 4 hrs which increased cell death in a dose dependent manner, analyzed via flow cytometry using propidium iodide (PI) exclusion. The (EC) 50 value was found to be 3.30 mM. The percent of cells staining positive for PI were significantly increased (P < 0.05): control, 0 mM L‐lactate, 15.0 +/− 0.5 % (n=4) and 4 mM L–lactate, 31.6 +/− 4.6% (n=4). This data suggests that elevated CSF lactate may be a contributing factor to dopaminergic cell death in Parkinson's disease.

Support or Funding Information

Funding support: AHA 16PRE27780121, Andrew Chapp, R15‐HL129213, Zhiying Shan; R15‐HL122952, Qing‐Hui Chen. Michigan Technological University Undergraduate Research Internship Program.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Increased Levels of Interleukin‐6 (IL‐6) in Andean Males with Chronic Mountain Sickness and Sea‐Level Participants After One Day at High Altitude May Reflect Differences in IL‐6 Regulation
Authors: Heinrich Erica C , Anza‐Ramirez Cecilia , Macarlupu Jose‐Luis , Corante Noemi , Vizcardo‐Galindo Gustavo , Villafuerte Francisco C , Powell Frank L , Simonson Tatum S
Published: June 2021

The gene encoding interleukin‐6 (IL‐6) shows signals of evolutionary selection in Tibetans and Andeans and could be a target of convergent adaptation to high altitude. Furthermore, IL‐6 is elevated in sea‐level residents upon acute high‐altitude exposure, is involved in both the inflammatory and hypoxia‐response pathways via HIF‐1a and NFκB regulation, and plays a role in regulating hematopoiesis. We hypothesized that high‐altitude adapted populations have variants associated with reduced IL6 expression that prevent chronic systemic inflammation in response to chronic hypoxaemia. We examined the sequence of the IL6 gene region in both Tibetans (n = 27) and and Andeans (n = 32) and measured IL‐6 protein levels in male Andean highlanders with and without Excessive Erythrocytosis (EE) (Hb ≥ 21 g/dL) in Cerro de Pasco, Peru (CDP, 4,350 m) as well as healthy male sojourners before and after travel to Barcroft Station, White Mountain Research Center (3,800 m). Plasma IL‐6 was significantly higher in Andean men with EE (healthy: 1.0 ± 0.4 pg/mL, EE: 2.5 ± 2.0 pg/mL; p = 0.02). IL‐6 levels also increased in sea‐level participants from 1.2 ± 0.6 to 2.3 ± 0.7 pg/mL (p = 0.01) after one day at altitude. Elevated IL‐6 protein in Andeans with EE could result from increased IL‐6 release from monocytes and vascular cells in response to more severe hypoxemia, the degree of inflammatory responsiveness, and/or increased blood vessel sheer stress imposed by increased blood viscosity. Elevated IL‐6 could also exacerbate CMS by further stimulating hematopoiesis. Sequence data revealed an uncommon promoter gain of function (rs1800795, c.‐237C>G, c.‐274C>G) and promoter loss of function (rs1800797, c.‐661A>G, c.‐698A>G) variant present in all 27 Tibetans as well as a shared missense variant (rs13306435, c.258T>A, c.486T>A) in 11/37 Andeans and 3/27 Tibetans; analysis of sequence data from subjects at White Mountain are underway. Since EE is uncommon in Tibetan highlanders, it is possible that these unique variants may afford protection against inflammatory responses and/or polycythemia at high altitude.

Support or Funding Information

This study was supported by an NIH NRSA Individual Postdoctoral Fellowship (Parent F32) to ECH (F32HL131218), NIH R00HL118215 and American Physiological Society Giles F. Filley Memorial Award to TSS. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. The UPCH team was supported by a Wellcome Trust PHTM grant (107544/Z/15/Z) to FCV.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ganoderma lucidum extract decreases inflammatory breast cancer progression via translational control
Authors: Martinez‐Montemayor Michelle M. , Schneider Robert J. , Zhang Chunling , Andrade Jorge , Suarez‐Arroyo Ivette J.
Published: June 2021

Inflammatory Breast Cancer is a highly aggressive and chemo/radiation resistant cancer in which cells invade the vascular and lymphatic systems via generation of tumor emboli; a process regulated by selective mRNA translation. Accordingly, our objective is to characterize the role of Ganoderma lucidum extract (GLE) in translational regulation. In IBC, the mammalian target of rapamycin (mTOR) pathway is hyperactivated, while the eukaryotic initiation factor 4G (eIF4GI) is overexpressed. mTOR regulates translation initiation, and together with eIF4G, both regulate resistance to therapy of IBC cells, cancer cell survival and invasion. We previously showed that GLE reduces cap‐dependent mRNA translation in part through the inactivation (dephosphorylation) of mTOR, thereby activating eIF4E sequestration in IBC cells and tumors. GLE also induces cancer cell death, inhibits cancer cell invasion and synergizes with conventional chemotherapy to reduce IBC progression, while not affecting non‐cancerous cells. However the role of GLE on translational control in advanced BC has not been studied. We hypothesize that GLE regulates translation via translation initiation inhibition. Genome‐wide translation and transcription profiling was conducted in SUM‐149 IBC cells treated with vehicle or GLE for 24h. Analysis of actively translated mRNAs revealed that 689 genes showed differential expression by GLE treatment. Ingenuity pathway analysis revealed that the majority of these mRNAs are involved in the eIF2 and mTOR pathways, which control translation. Future directions include performing quantitative PCR arrays to validate the microarray results. This study is relevant to our long‐term goal of contributing to the discovery of therapeutics to reduce mortality of IBC.

Support or Funding Information

This project was sponsored by Title V PPOHA and Title‐V‐Cooperative #P031S130068 US Department of Education #P031M105050, NIH/RCMI #8G12MD007583, NIH/NIGMS #1SC3GM111171 to MMM, NCI #R01CA178509 to RJS, and the Breast Cancer Research Foundation (BCRF‐16–143) to RJS. The content is solely the responsibility of the authors and does not necessarily represent the official views of the supporting agencies.

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 Pulmonary Valve Anatomy and Morphometry
Authors: Holda Jakub , Holda Mateusz Krystian , Tyrak Kamil , Klimek‐Piotrowska Wieslawa
Published: June 2021

Background

There is a strong clinical need of accurate pulmonary valve anatomy knowledge. Thus, the aim of this study is to present detailed morphometric description of the human pulmonary valve.

Methods

A total of 200 autopsied human hearts from healthy donors (26.0% females) were investigated. We have measured several pulmonary valve parameters and noted the presence of calcifications, semilunar valve nodules and leaflets' fenestrations. This study has been conducted in conformance with the Basic Principles of the Declaration of Helsinki and their expansion.

Results

Among study material 199 pulmonary valves were tricuspid and only one was bicuspid. The mean diameter of the pulmonary valve was 20.0±4.1 mm in the frontal plane and 18.0±4.0 mm in the sagittal plane. The mean distance from the free margin to the attachment line of the leaflet (leaflet height) in its central part was 15.6±3.4 mm for left, 15.1±3.1 mm for anterior and 15.4±2.8 mm for right leaflet. The mean distance from the bottom of the pulmonary sinus to the sino‐tubular junction for the left, anterior and right sinuses were: 19.0±3.8 mm, 19.1±3.7 mm and 18.4±4.1 mm, respectively. The distance from the free margin of the leaflet to the pulmonary sinus wall (in closed valve) was 15.9±2.8 mm for left, 15.5±2.9 mm for anterior and 16.1±2.8 mm for right leaflet. No statistically significant differences in size of leaflets and sinuses were found (p>0.05). Nodule of semilunar valve in the left, anterior and right leaflet was observed in 71.0%, 85.5% and 85.5% of hearts respectively. In 52.5% of cases nodules were presented in all three leaflets. In 22.0% of cases fenestrations of at least one leaflet were seen. The myocardial sleeve from the right ventricle tissue was situated higher than the bottom of the right pulmonary sinus in 87.5% of cases. The mean height of the myocardial sleeve extension onto right pulmonary sinus was 3.4±1.5 mm. Calcifications of pulmonary valve were observed only in 2.0% of cases.

Conclusion

The pulmonary valve has almost always three leaflets, which are of comparable size (p>0.05). The myocardial sleeve of right ventricle tissue entering the pulmonary valve sinus is present in 87.5% of cases and should be taken into consideration during pulmonary valve surgical procedures. The presence of leaflets' fenestrations may be responsible for valvular leak.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
mTORC1 controls GSK3β nuclear localization
Authors: Antonescu Costin N. , Bautista Stephen , Boras Ivan , Vissa Adriao , Mecica Noa , Yip Christopher M. , Kim Peter
Published: June 2021

Glycogen synthase kinase 3β (GSK3β) is a serine/threonine kinase that regulates numerous cellular processes such as apoptosis, self‐renewal, growth, and metabolism. GSK3β phosphorylates over 100 substrates ranging from cytosolic to nuclear substrate targets, including c‐myc, a key ongogenic transcription factor, leading to control of c‐myc stability. GSK3β is itself regulated by phosphorylation on S9 by signals such as Akt (activated within the phosphatidylinositol‐3kinase, PI3K, signaling pathway), resulting in reduced GSK3β kinase activity. Given the wide range of substrates and the incomplete regulation of GSK3β by S9 phosphorylation, it is likely that other mechanisms gate GSK3β function.

GSK3β may be localized within different cellular compartments such early endosomes, the nucleus, and in multivesicular bodies. However, how GSK3β localization is regulated, and how this may control GSK3β function is poorly understood, which we have examined here. Specifically, we examined how mTORC1, a key cellular sensor of mitogenic and metabolic signals, controls GSK3β localization and function. Using pharmacological inhibitors of mTORC1 and GSK3β, as well as siRNA gene silencing approaches, we uncovered that GSK3β‐mediated c‐myc degradation is negatively regulated by mTORC1. Using fluorescence microscopy approaches, we find that inhibition of PI3K‐Akt‐mTOR signaling axis, and metabolic insufficiency (e.g. amino acid deficiency), elicit GSK3β nuclear translocation from the cytosol, suggesting that mTORC1 mediates GSK3β nucleocytoplasmic shuttling. Consistent with regulation of GSK3β by mTORC1, we also uncovered that GSK3β localizes to LAMP1 positive late endosome/lysosomes, and perturbations of late endosome/lysosome membrane traffic impacted nuclear localization of GSK3β. This suggests that late endosomes/lysosomal compartments serve as organizing platforms, integrating mitogenic and metabolic signals via mTORC1 to elicit GSK3β cytosolic retention, thus impacting access of GSK3β to nuclear targets and controlling c‐myc degradation. Understanding how mTORC1 dependent signals regulate GSK3b subcellular localization to control GSK3β function may give useful insight to the development of drugs and therapies targeting GSK3β for the treatment of cancer cell growth and survival.

Support or Funding Information

This work is supported by a Discovery Grant from the Natural Science and Engineering Research Council (of Canada), an Early Researcher Award from the Ontario Ministry of Research, Innovation and Science, and a New Investigator Award from the Canadian Institutes of Health Research to C.N.A.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Localized immune transcriptional response to intratumorally administrated TLR7 agonist conjugates
Authors: Echeagaray‐Bredesen Oscar E , Vallez Nick , Mendez Natalie , Huang Ching‐Hsin , Gude Natalie , Kummel Andrew
Published: June 2021

Induced or circumstantial immunosuppression present opportunities for cancer development, and progression. Recently, multiple therapies have been developed to harness the immune system's ability to selectively eradicate cancer cells. These approaches have primarily targeted features of the adaptive immune response such as checkpoint blockade and vaccination of tumorigenic peptides. Toll‐Like receptors (TLRs) are innate immune receptors that recognize molecules broadly shared by various pathogens. TLR pathways are increasingly targeted in cancer immunotherapy approaches seeking to overcome immune‐evasion by cancer cells. Here, we hypothesize that activation of TLR7, an endosomal receptor which recognizes pathogen associated nucleic acids, can mitigate tumor immunosuppression. TLR7 agonist (TLR7‐A) conjugated to silica nanoparticles will be used as an adjuvant in a mouse colon adenocarcinoma (CT26 cell line derived) in vivo tumor model. Transcriptional targets of adaptive immune cells (CD3, CD4, CD8, Granzyme B and Perforin), antigen presenting cells (MHCII, CD80, CD86 and CD40) and inflammatory cytokine response (TNFα, IL‐6, IP‐10, MCP1, MIP1α and IFNγ) were measured by qPCR in tumor tissue following 21 days of treatment. We found that treatment with TLR7‐A unconjugated and conjugated increased localized transcript levels of inflammatory cytokines and chemokines: IL‐6, IP‐10, MCP1, MIP1α and IFNγ. This project will provide valuable insights on the distinct transcriptional immune interplay in response to TLR7‐A treatment (localized vs systemic), support the decision‐making process of subsequent experimental approaches and evaluate TLR7‐A conjugate's potential for treatment.

Support or Funding Information

This research was supported by the National Cancer Institute of the National Institutes of Health under award numbers:

U54CA132384 & U54CA132379

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mast Cell Numbers In Rat Livers In An Acute Model Of Fat Embolism Are Reduced By Losartan But Not By Captopril
Authors: Siddiqi Ahsan , Hamdan Hana , Siddiqi Saba , Pour Mohammad , Randall Zachary , Leupold Brad , Arif Dauod , Poisner Alan , Hamidpour Soheila , Molteni Agostino
Published: June 2021

Introduction

Fat embolism (FE) induced by IV injection of triolein (T) causes vasculitis, septal inflammation and fibrosis in rat lungs within 48 hrs (1). The damage is mitigated by two drugs acting on the renin‐angiotensin system (RAS): captopril and losartan (2). In the same organ, mast cell numbers are reduced in the lungs by losartan but not by captopril (3). This study examines whether captopril and losartan also modify the mast cell response in the liver.

Methods

Sprague‐Dawley rats (280–300g) were treated with 0.2ml iv (T) n=18 or saline n=7. One hour later 0.2ml of saline (n=6) ip, captopril (150 mg/kg n=6) or losartan (10mg/kg n=6) were given. Saline treated rats received ip, one hour later, the same does of saline. 48 hours later all the rats were necropsied after isoflurane anesthesia, lungs fixed in 10% formalin and H&E stained for morphology and CD117 for mast cells. Two pathologists unaware of the slides identity took at random 10 photographs of all the slides at 400× and separately counted the mast cells.

Results

As previously reported, 48 hours after triolein the lungs showed severe histopathological damage that was prevented by both RAS drugs and was associated with a high number of mast cells after captopril treatment but not by losartan (3). Similar results were observed in the liver where the mast cells after 48 hours of triolein treatment were statistically significantly increased (p=0.019) and even more when triolein treatment was associated with captopril (p=0.009). Mast cells numbers in the liver of losartan treated rats was however similar to that of controls and no statistically significant difference was observed. The overwhelming majority of mast cells were observed within the portal tract approximating 78%. The remaining were spread out within the hepatic parenchyma.

Conclusion

Mast cells may be involved in the pathological damage found after FE as suggested by their increase in number in the lungs and the liver after T and their reduction during protective treatment with losartan. However, the lack of effect of captopril on mast cell number at this acute period both in lungs and the liver may suggest that some other pathway may be involved in mast cell recruitment such as angiotensin 1–7.

Support or Funding Information

Supported by the Catherine T. Geldmacher Foundation, St. Louis MO.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cultivation establishment and antiproliferative activity from Asclepias subulata, for the development of a phytopharmaceutical
Authors: Armenta Hector Enrique Bustamante , Elías Jesús López , Rodríguez Julio César , Valenzuela Luisa Alondra Rascón , Zepeda Ramón Enrique Robles
Published: June 2021

Cancer is one of the leading causes of death in the world, this disease is commonly treated with non‐specific therapies, an alternative is the use of phytopharmaceuticals. Asclepias subulata is a medicinal plant, whose extracts report a high antiproliferative activity and selectivity on human carcinogenic cell lines. For the eventual development of a phytopharmaceutical from A. subulata, it is necessary to establish the conditions for the domesticated cultivation of this wild species. The aim of this work was the cultivation establishment of A. subulata and determine the antiproliferative activity of the plant under water stress. The seeds were collected and germinated in a greenhouse. Once germinated, were transplanted to the field, under four different conditions: Drought stress (DS), Drought stress with gel (DG), Irrigation (I) and Irrigation with gel (IG). Monthly for a year, aerial biomass and water stress were evaluated for each treatment. In addition, ethanolic extracts were obtained to determine the profile of cardenolides (HPLC) and antiproliferative activity on the cell lines A549 and ARPE‐19. The results obtained from aerial biomass in April‐December were, for DH, DG, I and IG were 75.9 ± 27.1, 83.1 ± 31.1, 323.8 ± 139.5 and 335.4 ± 93.0 g / plant respectively. The IG treatment was the one with more production of aerial biomass. The ethanolic extracts showed strong antiproliferative activity with values of IC50 <3.5 μg / mL, on the A549 cell line. On the other hand the ARPE‐19 cell line show IC50 >200 μg / mL, all these data can indicate the selectivity of the ethanolic extracts towards human cancer cell lines. We did not significate differences between treatments or season in the antiproliferative activity. Additional studies are required to determine other factors could be influencing the production of cardenolides.

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 Novel Vitamin D Receptor Antagonists that Mediate Suppression of Vitamin D Signaling
Authors: Staniszewski Lech J.P. , Shahani Pritika , Heck Michael , Hasan Dania S. , Wagner Carl , Jurutka Peter W.
Published: June 2021

Antagonists are chemical compounds that bind to receptors without eliciting activation of signaling, therefore the biochemical response to the receptor ligand can be blocked or significantly decreased. Receptor antagonists display varying levels of binding affinity but do not produce the agonist‐mediated response upon binding. The vitamin D receptor (VDR) binds with high affinity to its endocrine agonist, 1,25‐dihydroxyvitamin D3 (1,25D) to regulate the expression of a suite of genes in target tissues including kidney, intestine, and bone to control biological processes such as calcium and phosphate bone mineral homeostasis. In conditions of VDR hyperactivity, excess vitamin D production, or significantly increased intake via diet or supplementation, there are risks associated with potential resultant hypercalcemia including kidney stones, GI dysfunction, behavioral changes such as depression, and cardiac problems. In the present study, we synthesized novel VDR antagonists to create synthetic ligands that have high affinity for VDR, and consequently inhibit the activation of 1,25D‐VDR regulated genes that lead to hypercalcemia. It is well established that agonist‐bound VDR forms a functional DNA‐binding heterodimer with the retinoid X receptor (RXR) that associates with vitamin D responsive elements (VDREs) to either induce or repress transcription. Therefore, we exploited this agonist‐dependent VDR‐RXR heterodimerization pathway as an initial screening assay to evaluate the efficacy of our putative VDR antagonists. The initial test employed the mammalian two‐hybrid assay (M2H), which can assess if the novel antagonists compete with 1,25D and bind to VDR to prevent the formation of a heterodimer with RXR, a prerequisite step in VDR signaling. A second assay utilized VDRE‐linked luciferase reporter plasmids to measure antagonist activity in the more natural environment of the VDRE DNA platform. This assay was followed by a third screen to determine the specificity of antagonist binding to VDR versus other closely related receptors in the nuclear receptor superfamily. Results from these assays revealed that two novel analogs bind as bona fide antagonists to the VDR, albeit with different affinity. Our findings also suggest that additional chemical modification of antagonists may result in analogs with enhanced ability to suppress 1,25D‐VDR activation. Thus, this study further broadens our understanding of VDR‐ligand binding interactions, and may facilitate the development of novel therapeutic compounds with the potential for clinical applications in treating VDR‐directed hypercalcemia.

Support or Funding Information

School of Mathematical and Natural Sciences at Arizona State 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.
Discovery of a Plant‐derived Cell‐penetrating Proteasome Inhibitor
Authors: Kam Antony , Loo Shining , Tam James P
Published: June 2021

Plant‐derived cysteine‐rich peptides (CRPs) are known to play a role in plant defence against fungi and insects [1]. An increasing body of evidence, however, showed that certain CRPs display cell‐penetrating functions, and could be exploited for therapeutics targeting intracellular proteins [2]. Hibiscus sabdariffa, commonly known as roselle, is a shrub cultivated in South‐east Asia. We previously reported the discovery of CRPs from roselle, which we designated as roseltides (rT1‐rT7) [3]. Roseltides possess an evolutionary conserved six‐cysteine motif typical of hevein‐like peptides, but without a chitin‐binding domain. Roseltide rT1 is a human neutrophil elastase inhibitor, which in part, correlates to the ethnomedicinal use of roselle. Here we report that roseltide rT7, the second most abundant CRP in roselle, is a cell‐penetrating peptide and a proteasome inhibitor. To prepare the synthetic roseltide rT7, we used a stepwise solid‐phase peptide synthesis, followed by oxidative folding, to afford a native‐folded rT7. We showed that the synthetic and native roseltide rT7 are indistinguishable using reversed‐phase high‐performance liquid chromatography and nuclear magnetic resonance spectroscopy. To gain insight into its cell‐penetrating properties, we selectively labeled roseltide rT7 at its N‐terminus with a fluorescent probe (TAMRA‐rT7). Using flow cytometry and live‐cell confocal microscopy, we showed that TAMRA‐rT7 is cell membrane permeable via the endocytotic pathway. Roseltide rT7 was found to inhibit the chymotrypsin‐like activities of human 20S‐proteasome. Taken together, this study highlights the discovery of roseltide rT7 as the first plant‐derived cell‐penetrating proteasome inhibitor that can be potentially useful in the development of therapeutic biologics for cancer, inflammatory, and heart diseases.

Support or Funding Information

This research was supported in part by Nanyang Technological University Internal Funding ‐ Synzymes and Natural Products (SYNC) and the AcRF Tier 3 funding (MOE2016‐T3‐1‐003).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differentiation of Sickle Cell Zygosity Utilizing a Sodium Metabisulfite Method
Authors: Le Austin , Uppugunduri Manoja , Korducki John , Randolph Tim
Published: June 2021

Hemoglobinopathies are the most prevalent monogenetic disorder worldwide, and sickle cell disease is the most prevalent and severe structural hemoglobinopathy. Globally, over 85% of the sickle cell gene penetrance occurs in sub‐Saharan Africa (64.4%) and Arab‐India (22.7%) which contains the poorest countries on earth. Diagnosing sickle cell disease remains problematic in these populations where poverty severely limits clinic budgets, country infrastructure, and healthcare education making the use of modern diagnostic methods impractical. By modifying an established sodium metabisulfite method to detect sickle cells, we developed a simple, rapid, cost‐effective, microscopic method to distinguish the homozygous genotype (SS) seen in sickle cell disease (SCD) from the heterozygous genotype (AS) seen in sickle cell trait (SCT). We hypothesized that the two zygosities can be differentiated by observing the number, intensity, and rate of sickle cell formation over time. A solution of 2% sodium metabisulfite was prepared and 15 μL was mixed with 15 uL of patient blood and 5uL of the mixture was transferred to a microscope slide for evaluation using 100x oil immersion brightfield microscopy. Sickle cells were counted in one hour intervals for three hours and judged for sickling intensity on a 0–4+ Likert Scale. It was determined that 4+ sickle cells at 3 hours incubation optimally differentiated SS from AS genotypes. SS samples had an average of 135, 4+ sickle cells (N=10) compared to 12, 4+ sickle cells in the sickle cell trait samples (N=5) at three hours. Although more data are being collected, this inexpensive method can potentially be used to diagnose SCD and SCT, benefiting countries where there are limited resources.

Support or Funding Information

AL is supported by DeNardo Education and Research Foundation Fellowship.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Role of Reactive Oxygen Species in Crotalus atrox Venom‐induced Cell Death
Authors: Brown Lindsay
Published: June 2021

Venomous snake bites impact humans all around the world. Anti‐venom treatments mitigate systemic effects such as vascular hemorrhage, platelet aggregation inhibition, and the activation of inflammatory mediators. However, hemorrhagic snake venom also causes a loss of cellular adhesion to extracellular matrix components resulting in massive local tissue damage. To better understand the mechanism in which venom induces local tissue damage human embryonic kidney cells (HEKS) were grown on PEI then stimulated with 500μg/ml Crotalus atrox venom for 4 and 10 hours. Alamar Blue assays were used to measure cell viability and results suggest a 14.5±8.6% (p<0.05) and 58.95±10.7% (p<0.05) reduction in cell viability at 4 and 10 hours, respectively. Cells stimulated with 500μg/ml venom for 10 hours stained 97.6±2.2% (p<0.05) positive for Trypan blue, suggesting the venom reduces membrane integrity. HEK cells stimulated with 500μg/ml venom for 10 hours in the presence of 200 units PEG‐catalase increased viability by 44.1±5.1% (p>0.001) compared to cells stimulated with venom alone. 2′,7′‐Dichlorofluorescin‐diacetate (DCF‐DA) was used to quantify reactive oxygen species during venom stimulation. HEK cells stimulated with 50μg/ml Crotalus atrox venom for 4 hours returned 1.3×106±6.5×104 relative fluorescent units (RFU) compared to the 3.3×103±1.9×102 for non‐stimulated controls (p<0.05), a 433‐fold increase suggesting reactive oxygen species such as hydrogen peroxide play a critical role in Crotalus atrox venom induced cell death.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Serum Lipoproteins Regulate Hypoxia‐Inducible Factors Under Normoxia
Authors: Espenshade Peter , Shao Wei , Hwang Jiwon
Published: June 2021

Hypoxia‐inducible factor (HIF) transcription factors are central regulators of oxygen homeostasis, controlling energy utilization, erythrogenesis, angiogenesis and cancer metabolism. Oxygen negatively regulates HIF activity by promoting HIFα prolyl hydroxylation and proteasome‐dependent degradation. Using human pancreatic adenocarcinoma cells, we demonstrate that serum lipoproteins are an independent regulator of HIF under normoxia. Decreasing extracellular lipid supply inhibited HIF prolyl hydroxylation, leading to accumulation of HIFα comparable to hypoxia with activation of downstream target genes. Addition of unsaturated fatty acids suppressed this signal, which required an intact mitochondrial respiratory chain. A survey of different cell lines showed that this signaling pathway functions in a wide range of tissues. These studies show that lipoprotein and oxygen supply are distinct signals integrated to control HIF activity and identify serum lipids as a potential modulator of HIF pathway activity.

Support or Funding Information

National Institutes of Health, HL077588 and GM126088

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protective Effect of Oxytocin Against Apoptosis and Oxidative Stress: Role of Extracellular Signal Regulating Kinases
Authors: Havranek Tomas , Alanazi Mohammed M. , Bakos Jan , Bacova Zuzana , Cubeddu Luigi X. , Castejon Ana Maria
Published: June 2021

Neuropeptide oxytocin plays a role in the neuronal proliferation, differentiation and affects related morphological changes of neuronal cells. It has been suggested that oxytocin has protective effect against inflammation and consequences of oxidative stress. However, mechanisms of its action at the molecular level are not fully understood. The aim of the present study was to determine oxytocin anti‐apoptotic effects and potential inhibition of oxidative stress in glioblastoma U87‐MG cell line. We have clearly shown that activation of oxytocin receptors dose‐dependently stimulated proliferation of U‐87MG cells. Incubation of cells in the presence of oxytocin increased ERK1/2 phosphorylation, which was reduced by oxytocin receptor antagonist. Our results also revealed that in the U87‐MG cell‐line, hydrogen peroxide (H2O2) increased the intracellular reactive oxygen species (ROS) production in a dose‐dependent manner. Interestingly, cells treated with H2O2 in the presence of oxytocin showed a significant decline in the ROS production. We have also confirmed that oxytocin mediates its protective effect in U87‐MG cells through activation of the MAPK/ERK ½ signaling pathway. The protective effect of oxytocin has been observed against consequences of oxidative stress in peroxide model and apoptosis induced by camptothecin, as well. To our best knowledge this is the first study demonstrating that there is interference in production of ROS and activation of oxytocin receptors in neuronal cells. Overall, the present data indicate that oxytocin stimulates proliferation and protects glial cells against apoptosis and oxidative stress.

Support or Funding Information

1‐ President's Faculty Research & Development (PFRDG) Grant ‐ 2015–2016

2‐ Saudi Arabian Cultural Mission (SACM) Graduate Students Research Grant

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mathematical model of megalin trafficking in differentiated proximal tubule cells and its application in Dent disease
Authors: Shipman Katherine , Long Kimberly R , Rbaibi Youssef , Baty Catherine J , Weisz Ora A
Published: June 2021

The polarized epithelial cells that comprise the proximal tubule (PT) have a specialized apical endocytic pathway that allows for high‐capacity endocytosis which is necessary to recover essential nutrients and to maintain a protein‐free urine. Megalin, a multi‐ligand receptor at the apical surface of the epithelial cells, binds proteins in the ultrafiltrate and internalizes them via receptor‐mediated endocytosis. Ligands are sorted from receptors in endocytic compartments, and the receptors are recycled back to the surface. The molecular identities of the compartments involved in sorting and recycling in PT cells and the kinetics of megalin trafficking through them are unknown. When endocytosis in the proximal tubule is dysfunctional, tubular proteinuria results. Dent disease, an X‐linked disorder characterized by tubular proteinuria that progresses to renal failure, is caused by mutations in the Cl‐/H+ exchanger CLC‐5. Loss of CLC‐5 has been shown to reduce endocytic uptake and to decrease megalin protein expression without altering mRNA levels. However, the mechanism by which this occurs is unclear. We previously discovered that OK cells cultured under continuous fluid shear stress develop morphological and functional features similar to that of the PT in vivo, including high apical endocytic capacity and increased megalin expression. We used data obtained through biochemical approaches to estimate endocytic and recycling rates and the half‐life of surface megalin in untreated and CLC‐5 knockdown OK cells. These rates were used to construct a simplified model of megalin trafficking in differentiated PT cells. We present an ordinary differential equation (ODE) model of megalin trafficking describing surface and internalized pools of megalin with estimated kinetic parameters. We observed a greater fraction of internalized megalin compared to the surface pool due to an endocytic rate that is much faster than the recycling rate. We have quantified decreases in megalin expression and surface half‐life with loss of CLC‐5 expression. Current and future work includes quantifying alterations in megalin trafficking in CRISPR/Cas9 mediated CLC‐5 KO models and defining the structure and markers of the apical endocytic pathway by quantitative imaging to expand our model to include additional trafficking steps. This model will then be used to predict which steps in megalin trafficking are altered in Dent disease.

Support or Funding Information

ASN Pre‐Doctoral Fellowship, NIH R01‐DK101484, NIH P30‐DK079307

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Role of Beta‐2 Adrenergic Receptors in Cardiac Bioenergetics Following Severe Burns
Authors: El Ayadi Amina , Prasai Anesh , Jay Jayson , Bhattari Nisha , Guilory Ashley , Herndon David , Porter Craig , Finnerty Celeste
Published: June 2021

Severe burn injury is characterized by increased energy expenditure, and altered mitochondrial function is thought to be central to the hypermetabolic response to burns. As catecholamines are an important driver of the hypermetabolic stress response to burn, we set out to determine the role of adrenergic signaling through Beta‐2 adrenergic receptors in cardiac mitochondrial function following burn. We hypothesized that a loss of adrenergic signaling would attenuate cardiac mitochondrial dysfunction in response to burns.

We used beta ‐2 adrenergic receptor knockout mice (ADRB2‐KO) and wild type controls (WT). Mice received a 30% of total body surface area scald burn or a sham burn and were sacrificed one week following injury. Hearts were excised and mitochondrial function was measured in saponin‐permeabilized myofiber bundles by high‐resolution respirometry. Data were analyzed using two‐way ANOVA and Bonferroni post‐test to compare all groups.

Mitochondrial respiratory capacity was significantly reduced in both WT (P<0.01) and KO (P<0.01) mice following burn, although there was no difference between WT and KO mice. In WT animals, respiration coupled to ATP production was significantly lower in the burn animals compared to shams (p< 0.05). In contrast, respiration coupled to ATP production was not different in sham and burn treated ADRB2‐KO mice. Mitochondrial flux control ratios (leak and couple respiration normalized to maximal uncoupled respiration) were significantly higher in burned WT mice compared to sham (P<0.05). However, mitochondrial flux control was not significantly altered by burn in KO mice.

Collectively, these data underscore the role of catecholamines and adrenergic stress in the metabolic response to burns. Specifically, ablation of beta 2 adrenergic signaling in the heart prevents cardiac mitochondrial dysfunction in response to severe burn trauma.

Support or Funding Information

This study was supported by grants from the National Institute of Health (P50‐GM60338, R01‐GM56687, and GM 112936‐01), Shriners Hospitals for Children (80100, 87300, 71008, 70900, 85116, 71000), and SSF grant for Craig Porter. This study was also conducted with the support of the Institute for Translational Sciences at the University of Texas Medical Branch, supported in part by a Clinical and Translational Science Award (UL1TR000071) from the National Center for Advanc‐ing Translational Sciences, National Institutes of Health.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Partial purification of a Leishmania tarentolae protein that binds a putative 5S rRNA promoter element
Authors: Okezie Oneybuchi , Jagus Rosemary , Pitula Joseph
Published: June 2021

Kinetoplastids are a group of protozoan parasites that have been intensively studied due to their adverse impact on agriculture and human health. As in higher eukaryotes, 5S rRNAs in kinetoplastids are derived from RNA polymerase III (pol III) transcription. Despite the presence of pol III activity for 5S rRNA transcription characterization of the RNA pol III promoters of kinetoplastids and the transcription factors (TF) binding them remains meager. Thus, we have initiated a biochemical approach towards the identification of TFIII proteins from the model kinetoplastid Leishmania tarentolae. Specifically, we have employed a putative 5S rDNA pol III promoter sequence as the target for TFIII‐specific interaction by gel shift analysis. Our experiments show that this promoter sequence is specifically recognized by at least one protein factor from a L. tarentolae nuclear extract, as demonstrated through competition assays with various 5S rDNA‐specific constructs. In addition, we have employed double stranded DNA affinity chromatography followed by gel shift analysis as an initial step in purification of this putative 5S rDNA gene‐specific binding protein factor.