bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation and Functional Studies of Tribbles homolog 2 (TRIB2) in Ovarian Granulosa Cells
Authors: Warma Aly , Price Christopher A. , Silversides David W. , Lussier Jacques G. , Ndiaye Kalidou
Published: June 2021

Tribbles homolog (TRIB) 1, 2 and 3 represent atypical members of the serine/threonine kinase superfamily and are homologs of Drosophila tribbles. TRIB2 mRNA is rapidly induced by mitogens, has a short half‐life, and is expressed in a cell‐specific manner. We previously identified TRIB2 as a differentially expressed gene in granulosa cells (GC) of bovine preovulatory follicles. This study aimed to further investigate TRIB2 mRNA and protein regulation, to identify its binding partners, and study its function in GC of bovine dominant follicles. Granulosa cells were obtained from follicles at different developmental stages: small follicles (SF: 2–4 mm), dominant follicles (DF) at day 5 of the estrous cycle (day 0 = day of the estrous), ovulatory follicles (OF) 24 h following injection of an ovulatory dose of hCG, and corpus luteum (CL) at day 5 of the estrous cycle. RT‐qPCR analyses showed greatest expression of TRIB2 in GC of DF, while the weakest expression was in OF (P < 0.0001). Temporal expression of TRIB2 mRNA was further studied in follicular walls (granulosa and theca cells) obtained from ovulatory follicles recovered at 0, 6, 12, 18 and 24 h after hCG injection. There was a 20‐ and 166‐fold reduction of TRIB2 steady‐state mRNA levels in follicular walls, respectively at 6 and 24 hours post‐hCG as compared to 0 hour (P < 0.001). Additionally, we have generated specific anti‐TRIB2 polyclonal antibodies that confirmed, in western blot analyses, TRIB2 downregulation by LH/hCG at the protein level. Yeast two‐hybrid screening of a DF‐cDNA library as well as functional studies using the CRISPR‐Cas9 approach are currently being used to identify TRIB2 binding partners and its function in GC. These results support a physiologically relevant role of TRIB2 in follicular development and GC function.

Support or Funding Information

Research supported by internal funds from Université de Montréal to KN and NSERC of Canada grant # 104199 to JGL.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Optimization and Initiation of a Genome‐Wide Forward Genetic Death Screen for the Lacritin Receptor Complex using the Brunello CRISPR/Cas 9 sgRNA Library
Authors: Teixeira Karina Luiza Dias , Mao Leran , Doench John , Laurie Gordon W.
Published: June 2021

Introduction

Ocular tears refract 80% of light and are essential for epithelial homeostasis. 7% of the world's population suffer from tear insufficiency associated with compromised visual acuity and chronic ocular surface inflammation. Lacritin, a glycoprotein constituent of tears, restores homeostasis in a mouse dry eye model, and in human corneal epithelial cells (HCET) subjected to dry eye‐like IFN‐g and TNF‐a stress. It does so by transiently stimulating autophagy and restoring oxidative phosphorylation. Lacritin also promotes basal tearing by increasing the sensitivity of ion channel TRPM8 on corneal sensory nerves to tear drying. Less well understood is the receptor complex, likely inclusive of a GPCR ‐ based on pertussis toxin and MK912 sensitivity, active N‐24, but not inactive C‐25, lacritin truncation mutant inhibition (64%) of 125 I‐iodoclonidine binding to purified alpha‐2C adrenergic receptor (ADRA2C), and function loss after pooled siRNA knockdown of ADRA2C. ADRA2C is known to complex with ADRA2A, which in turn can modulate TRPM8. Yet, lacritin is unlike any other ADRA2C agonist, requires prior binding to heparanase modified syndecan‐1 (SDC1), failed to inhibit 125 Iiodoclonidine binding to ADRA2C and did not activate a beta‐annexin based ADRA2C Tango assay.

Goal

Our goal is to identify the lacritin GPCR(s) using a forward genome‐wide CRISPR/Cas9 screen in HCET cells stressed with interferon‐gamma (IFNG) and tumor necrosis factor (TNF).

Methods

Initial experiments took advantage of GFP expressing pRosetta to establish optimal HCET viral infection and polybrene concentration. Next, we optimized HCET infection with the Brunello human CRISPR/Cas9 lentiviral library (Addgene #73179) and selection with puromycin, followed by INFG/TNF. For the latter, Alamar Blue and Colony Assays such to identify INFG/TNF levels that killed all cells yet made possible recovery by lacritin using the N‐94 synthetic peptide representing lacritin's C‐terminal active domain. Deep sgRNA sequencing will be performed on genomic DNA from cells stressed with INFG/TNF without or with N‐94.

Results

For HCET cells, the optimal polybrene concentration was 4 mg/ml that yielded an infection efficiency of ~97% as determined by FACS. The optimal volume of Brunello lentiviral library to obtain 30–50% of infection efficiency was 200 μl. 2 μg/ml was best for puromycin, 1000 U/ml for INFG, 100 ng/ml for TNF and 1000 nM for N‐94 recovery of ~50% of cells. The library consists of 76,441 sgRNAs targeting 19,114 human genes (including ADRA2C, ADRA2A, TRPM8 and SDC1) plus 1000 non‐targeting sgRNA controls. The forward screen is now underway.

Support or Funding Information

NIH R01 EY024327

NIH R01 EY026171

Uva Pinn Scholar 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.
Modeling SLC26A4 Associated Hearing Loss Using Zebrafish as a Model System
Authors: Scott Ashley Jennie , Koleilat Alaa , Schimmenti Lisa
Published: June 2021

Two out of every thousand newborns are born deaf or hard of hearing. Sixty percent of hearing loss has an identifiable genetic cause. One of the most common forms of genetic hearing loss is Pendred syndrome. Disease causing genetic alterations or pathogenic variants in SLC26A4 are associated with Pendred Syndrome, a disorder characterized by bilateral sensorineural hearing loss and occasionally thyroid goiter. Patients with SLC26A4 pathogenic variants have an enlarged vestibular aqueduct, a malformation of the temporal bone. Pendrin, the protein product of SLC26A4, is a solute carrier. Pendrin is expressed in the inner ear, thyroid, and kidney. In the inner ear, pendrin modulates the transport of bicarbonate and chloride, allowing bicarbonate to leave the supporting cells of the inner ear and chloride to enter. This helps maintain a relatively neutral pH environment in the inner ear. It is not known how the abnormal SLC26A4 protein products modulate hearing loss and contribute to temporal bone abnormalities. The goal of this study was to model SLC26A4 associated genetic hearing loss in zebrafish and elucidate a mechanism responsible for hearing loss in patients with pathogenic variants in SLC26A4. To model the human condition, slc26a4 mutations in zebrafish were generated via transcription activator like effector nucleases (TALENs) at the site of the the most common SLC26A4 pathogenic variants. We obtained a 23bp deletion mutation in exon 10 of slc26a4. We hypothesized that fish with biallelic slc26a4 mutations would closely recapitulate the human phenotype and have significant hearing loss and structural ear malformations. To test for deafness in zebrafish, we developed a hearing assessment which exposes larval or adult fish to specific sound stimuli while recording startle responses. To quantitatively study the slc26a4 mutant ear phenotype, we took measurements of the posterior and anterior otolith area and the ear area using bright field microscopy. Results indicate that homozygous embryos tested at 5 days post fertilization, the day of hearing onset in zebrafish, have hearing that was not different that wildype, however adult fish with two copies of mutated slc26a4 have frequency specific reduced responses at 400 Hz, 80db. These results indicate that slc26a4 mutants have progressive frequency specific hearing loss. Bright field imaging and measurement analysis showed that slc26a4 mutant larvae have significantly smaller standardized posterior otolith areas, anterior otolith areas, and ear areas on average compared to wildtype at 5dpf. This closely matches the human phenotype, as humans with SLC26A4 pathogenic variants may have progressive hearing loss and temporal bone abnormalities.

Support or Funding Information

Mayo Center for Individualized Medicine

Mayo Clinic Department of Otorhinolaryngology

NIH 5R25GM075148‐11

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Measuring the cAMP/PKA signaling pathway in stress‐induced sleep using C. elegans as a model organism
Authors: Schuck Ryan , Cianciulli Alana , Buerkert Thomas , Li Edwin , Nelson Matthew
Published: June 2021

The cyclic adenosine monophosphate (cAMP)/Protein Kinase A (PKA) pathway promotes arousal in nematodes, fruit flies and mammals. Caenorhabditis elegans displays unique forms of sleep: developmentally timed sleep (DTS) and stress‐induced sleep (SIS). The cAMP/PKA pathway is downregulated during both DTS and SIS; however, where in the nervous system this is occurring is unknown. C. elegans provides a unique avenue for studying the ubiquitous cAMP/PKA pathway with single cell resolution in a living animal. To do this, we have introduced the red light‐activated adenylate cyclase, ilac22, and the Förster resonance energy transfer (FRET)‐based cAMP biosensor, epac1‐camps, into the C. elegans system. We have found that activation of IlaC in the DVA interneuron is sufficient to disrupt DTS, SIS and non‐physiological quiescence induced by over‐expression of neuropeptides. Next, we have expressed epac1‐camps in the DVA and ASI neurons and are currently quantifying cAMP during SIS. Our preliminary results indicate that cAMP is increased in the DVA during sleep relative to awake individuals, and we are beginning to quantify cAMP in the ASI neurons to test if this pattern is conserved. PKA‐mediated phosphorylation of the metabolic regulator adenosine monophosphate‐activated protein kinase (AMPK) inhibits its activity. To determine if AMPK is functioning downstream of cAMP/PKA, we are quantifying AMPK‐phosphorylation during SIS using temporal western blot analysis.

Support or Funding Information

NIH ‐ National Institute of General Medical Sciences – 1R15GM122058‐01 (PI: M. Nelson)

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 5‐IP7 in the Regulation of Gene Expression
Authors: Sahu Soumyadip , Gu Chunfang , Shears Stephen B.
Published: June 2021

Inositol pyrophosphates (PP‐IPs) are unique, evolutionarily conserved signaling molecules, which act at the interface between signal transduction and cellular metabolism. 5‐IP7 (5‐diphosphoinositol 1,2,3,4,6‐pentakisphosphate) is the most physiologically abundant PP‐IP (cytoplasmic concentration: 1–2 μM). 5‐IP7 is a highly electronegative molecule with five monophosphate groups and one bisphosphate group, all packed around a six‐carbon inositol ring. Reported functions of 5‐IP7 include non‐enzymatic pyrophosphorylation of proteins and non‐covalent binding to the highly electropositive domains in certain proteins (e.g., pleckstrin homology domains). Here, we provide evidence of a new mechanism of action for a PP‐IP, namely, as an enzyme inhibitor. The enzyme in question, encoded by NUDT3, catalyzes mRNA 5′‐decapping activity. NUDT3 knockdown in MCF7 breast cancer cells cause an increase in the mRNA stability of several ‘migration marker proteins’, namely, β6‐integrin, fibronectin, lipocalin and S100A8, which promote an elevated migratory phenotype compared to the wild type (WT) MCF7 cells. The NUDT3 gene product also hydrolyzes 5‐IP7, so we hypothesized that a persistent elevation of cellular levels of that PP‐IP would competitively inhibit mRNA decapping, and hence promote the expression of migration marker proteins. To pursue this idea, we used HCT116 colon cancer cells and HEK293 kidney epithelial cells in which we knocked out the IP7Ks that metabolize 5‐IP7, thereby increasing levels of this PP‐IP by 2 to 3‐fold. In the current study, qRT‐PCR analysis of IP7K−/− HCT116 cells showed an increase in the mRNA levels of β6‐integrin by 2 ± 0.2‐fold, fibronectin by 2.5 ± 0.4‐fold, lipocalin by 5 ± 0.4‐fold, and S100A8 by 3 ± 0.4‐fold compared to WT cells (data are expressed as mean ± SE, n=5, p ≤ 0.01). Knockout of IP7K in HEK293 cells resulted in elevation of mRNA levels of β6‐integrin by 6 ± 0.5‐fold, fibronectin by 3 ± 0.6‐fold, lipocalin by 7.5 ± 0.4‐fold, and S100A8 by 3.7 ± 0.35‐fold vs. WT HEK293 cells (data are expressed as mean ± SE, n=5, p ≤ 0.01). We are now developing techniques to load WT cells with 5‐IP7 to more directly test our idea that this PP‐IP has a novel, enzymatic function as a regulator of expression of pro‐migratory genes that may have relevance to tumor metastasis.

Support or Funding Information

Intramural Research Program Funding for NIEHS/NIH.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of Probiotic Bacteria from the Cutaneous Microbiome of Endangered Tennessee Bats
Authors: Wharton Megan , Bowers Olivia , Jones Ben , Moore Andrew , Grisnik Matthew , Munafo John , Walker Donald
Published: June 2021

Since its arrival in 2006, the fungal agent of White‐Nose Syndrome (WNS), Psuedogymnascus destructans (Pd), has rapidly spread across the eastern United States. As a result, millions of native bats have been killed. This fungal pathogen infects bats in cave systems during their hibernation, causing disruption of natural physiological processes. Notably, the affected species experience an increased frequency of arousal from hibernation, which causes them to consume more of their reserved energy, eventually causing the bat to die of starvation. WNS, which is capable of producing a near 100% mortality rate within the bat's roost during a given hibernation cycle, has been confirmed to infect nine bat species present in the Eastern United States, three of which are federally listed as endangered. The presence of Pd has also been observed on six additional bat species without causing diagnostic symptoms. To provide treatment for Pd, current options include the use of anti‐fungal and volatile compounds as well as naturally occurring probiotic bacteria. The primary objectives of this research are 1) to evaluate and determine if the resident cutaneous microbiome of bats displays anti‐fungal activity against this fungal pathogen, and 2) to identify key probiotic bacterial strains for future biocontrol assessment. Currently, 355 bacterial isolates have been isolated into pure culture from 78 bat skin swab samples. It has been determined that through the use of microbiological challenge assays of the bacterial isolates against Pd, that 111 cultures from bat skin exhibit production of diffusible organic compounds and metabolites that display anti‐fungal activity against Pd. An isolate from the Actinobacteria (genus Streptomyces) has been identified to produce volatile organic compounds that completely inhibit fungal growth. Identification of the bioactive compound(s) is under investigation.

Support or Funding Information

Support and funding were provided by the Tennessee Wildlife Resources Agency.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Diet and fitness genetic screening an approach to understand genetic diversity and optimize wellness in Saudi Arabia
Authors: Dhawi Faten
Published: June 2021

The revolution of genetic mapping era and the whole genome project made it possible to enhance the quality of life and maintain health and well being. Our aim was to a review the knowledge and acceptance of genetic test to enhance the quality of life among the population of Saudi Arabia. The descriptive analysis for questionnaire consisted of 27 questions scored for 302 respondents gathered electronically. The respondents' demographics showed 50% in the age of 18–25 years and 50% of respondents from 18–60 years old know about genetic test concept. Over 50% of respondents were interested in performing genetic test to enhance health while 40% were interested in performing genetic test to enhance fitness. Specific questions related to response to coffee, macro and micro nutrition elements and enzyme activity scored less than 50% of concept understanding among respondents. These results represent a contribution to the genetic test discussion on the relevance of genetic testing validity and acceptance among Saudi Arabia population. These results might help in producing specific guidelines on genetic testing and genomic analysis and help of implementation of fitness and future health plan in cooperation of Saudi genome projects. The future study will focus on the consanguinity on population structure and genetic frequency related to specific diet or fitness.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Integrating Undergraduate Molecular Biology and Biochemistry: the Development of a Program Capstone Laboratory Based on Authentic Research
Authors: Crawford Garland
Published: June 2021

The Departments of Chemistry and Biology at Mercer University jointly oversee the Biochemistry and Molecular Biology degree. Recently, the program underwent a review and subsequent reorganization. As a part of these changes, a new capstone laboratory was developed to integrate techniques from traditional molecular biology and biochemistry labs into a single, semester‐long project that provides an authentic research experience for all majors. Students design primers for site‐directed mutatgenesis to introduce a single amino acid change for an enzyme of interest. Students gain experience with PCR, cloning, DNA purification, and sequence analysis. Following confirmation of their mutation, students express the wild‐type and mutated enzymes as a 6xHis‐tagged fusion proteins which they purify and characterize using a paranitrophenol‐linked substrate. Enzyme activity of the mutated protein is compared to that of the wild‐type enzyme. In addition to meeting the traditional objectives for a biochemistry laboratory, students receive the added benefit of exposure to a semester‐long research project with clear experimental connections between techniques.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
How to make red yeast: An undergraduate laboratory using CRISPR/Cas9 technology to target ADE2 in budding yeast
Authors: Callahan Kevin P.
Published: June 2021

The ability of CRISPR/Cas9 to edit genomic sequence in a multitude of organisms has revolutionized modern science and is an ideal topic to engage undergraduate students. The ease and speed of this technology combined with the short generation time, well‐characterized genome, established protocols, and relatively limited financial demands of Saccharomyces cerevisiae (bakers yeast) make for a practical undergraduate laboratory. Students, working in pairs were assigned the task of replacing the chromosomal copy of ADE2 with HIS3. Over the course of several weeks students cloned the ADE2 gRNA into an expression vector, PCR amplified a HIS3 construct for homologous recombination, and transformed these components into yeast. ADE2 encodes for Phosphoribosylaminoimidazole carboxylase, an enzyme that catalyzes a reaction in the de novo synthesis of purine nucleotides. Disruption of this gene results in the accumulation of red pigment in the yeast cells. Students were thus able to screen for disruption of the ADE2 gene and subsequent replacement with HIS3 with a specific growth phenotype: red yeast capable of growing on media lacking histidine. The methods used in this lab included: basic cell culture, primer design, PCR, plasmid isolation, transformation, restriction digestion, ligation and DNA sequencing. To reinforce scientific writing and analysis skills the culmination of this project was a manuscript describing their results and a final class lab meeting where each group presented their data and conclusions. Informal assessments indicated that participating in this lab increased student's interest in attending graduate schools and pursing careers in biomedical 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.
Gamification of learning in an introductory cell biology class
Authors: Beaulieu Elaine , Petit‐Turcotte Caroline
Published: June 2021

Students taking introductory cell biology need to master a large amount of new vocabulary and course content before they can achieve a deeper understanding of fundamental concepts. Games are notoriously good at engaging their target audience and keeping players on task, and if carefully designed, games have the potential to be tremendous pedagogical tools. In order to facilitate learning for our students, we are developing an online game based on cognitive science, which is designed to encourage students to engage with the class material.

A pilot trivia game (https://www.biolingo.ca/) designed to test interest in such a game in our students was released in March 2017, with a bank of over 250 questions covering the class content (Bloom levels 1 and 2), which students were at liberty to use as a study tool. Students were able to choose between subject categories and a 15 questions quiz would be randomly generated from the question bank. Students were given access to the game 3 weeks before the final exam, playing time was not limited and quizzes were completed on a voluntary basis. On the final exam, students were asked to self‐report how much time they spent on the game and all 491 students answered. Students were grouped according to two categories: their use of the game (>3h were considered high users, 1–3h mid‐users, <1h low users and 0h are non users) and their exam result (A>80%, B = 70–79%, C= 60–69%, D= 50–59%, E= 40–49%, F<40%). The majority of students (67.3% of all A students, 54.2% of all B students, 54% of all C students, 58.2% of all D students, 56,8% of all E students and 33.3% of F students) accessed the game at least once. Interestingly, the proportion of mid‐users and high‐users was the greatest in F students (23.3% and 13.3% of all F students respectively) and comparatively low in A students (10% and 0.3% respectively).

Clearly, students were curious about the online game and were eager to try it. It is also interesting to see that struggling students seemed to be eager to use a tools that could help them practice and improve their learning. Further studies using a modified version of the pilot game, designed to stimulate distributed learning and repeat testing throughout the term, as well as tying the students' game performance to their course mark, will help us research the potential for increased learning through the carefully designed gamification of content.

Support or Funding Information

Teaching and learning support services (TLSS) University of Ottawa and eCampusOntario

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Johns Hopkins Science Policy Group: Training scientists to be effective advocates and communicators
Authors: Matney Chanel , Sima Richard , Carlson Jenny , Cairns Leah , Wood Kaitlin , Pham Daniel
Published: June 2021

Legislative and regulatory affairs in science policy play a central role in shaping the practice of biomedical research. However, there are limited opportunities allowing scientists to practice techniques in effective policy advocacy. Since its inception in 2016, the Johns Hopkins Science Policy Group has provided trainees in the biological and health sciences with immersive training and professional development opportunities in advocacy, policy analysis, and strategic communications. Our student‐led initiatives, such as our flagship “Eat and Advocate” program, trains researchers to engage their Congressional representatives with targeted advocacy messages promoting pro‐science legislation. Trainees attending our advocacy training seminars learn how to communicate the value of federally‐funded research to policy‐makers, honing their science outreach and communication skills. Further, our members develop educational content, including fact sheets, infographics, message templates, and call‐to‐action memos, to incentive fellow researchers to engage in civic action supporting pro‐research legislative agendas. Past student‐led projects have provided training and professional development opportunities through advocacy initiatives on NIH appropriations, the Affordable Care Act, 21st Century Cures, the Paris Climate Accord, prescription drug pricing regulation, and the Clean Water Act of 2015. The Johns Hopkins Science Policy Group is a model of a highly effective trainee‐led program that is capable of training researchers in 1) organizational leadership by leading grassroots advocacy initiatives 2) strategic communications in advocacy, policy analysis, and science communication 3) civic engagement and public outreach.

Support or Funding Information

Johns Hopkins Graduate Student Association, Johns Hopkins School of Public Health Student Assembly, and the Johns Hopkins University Alumni Association

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exploring the Role of Trehalose‐6‐phosphate Synthase in Oxidative Stress Tolerance of Fusarium verticillioides
Authors: McMillan Sarah D. , Oberlie Nicole R. , Brown Daren W. , McQuade Kristi L.
Published: June 2021

Fusarium verticillioides is a filamentous fungus that infects maize and synthesizes fumonisins, a family of toxic and carcinogenic secondary metabolites. We are studying the role of trehalose in stress response in F. verticillioides. Trehalose‐6‐phosphate (T6P) synthase catalyzes the first step in trehalose synthesis, and a mutant lacking the gene that codes for T6P synthase does not produce trehalose or fumonisins. We report here that treatment with menadione or hydrogen peroxide inhibits germination of the mutant strain (Δtps1) more than the wild‐type strain. Whereas hydrogen peroxide delays germination, menadione reduces both the rate and percent germination, and significantly inhibits hyphal branching of both strains. To explore whether the sensitivity of Δtps1 to oxidative stress is caused by the lack of trehalose or the absence of a secondary function of the T6P synthase protein, we are studying mutants that express a catalytically inactive T6P synthase.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dynamic Actin Reorganization and Vav/Cdc42‐dependent Actin Polymerization Promote Macrophage Aggregated LDL Uptake and Catabolism
Authors: Singh Rajesh K. , Haka Abigail S. , Bhardwaj Priya , Maxfield Frederick R.
Published: June 2021

During atherosclerosis, low density lipoproteins (LDL) accumulate in the arteries, where they become modified, aggregated and retained. Such deposits of aggregated LDL (agLDL) can be recognized by macrophages, which attempt to digest and clear them. AgLDL catabolism promotes internalization of cholesterol and foam cell formation, which is an important component of atherosclerosis. Characterization and therapeutic blockade of this process will likely delay disease progression. When macrophages interact with agLDL in vitro, they form a novel extracellular, hydrolytic compartment, the lysosomal synapse, aided by local actin polymerization to digest agLDL. Here, we demonstrate in vivo that atherosclerotic plaque macrophages contacting cholesterol rich LDL deposits polymerize actin and form a strikingly similar compartment. Live cell imaging revealed macrophage cortical F‐actin depolymerization driven by PIP2 hydrolysis occurs in parallel to actin polymerization at the lysosomal synapse and is required for its formation. Using siRNA‐mediated silencing, pharmacological inhibition, genetic knockout and stable overexpression, we elucidate key roles for Cdc42 Rho GTPase and guanine nucleotide exchange factor Vav in promoting actin polymerization at the lysosomal synapse, and we rule out roles for Rac1 and Rac2. These data indicate that dynamic actin rearrangement and polymerization via Vav and Cdc42 aids compartment formation, catabolism of agLDL and promotes foam cell formation.

Support or Funding Information

The project described was supported by National Institutes of Health grants R37‐DK27083 and R01‐HL093324. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. R.K.S. is an American Heart Association Stanley Stahl Postdoctoral Fellow (ID: 15POST22990022).

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 PhoP/PhoQ two‐component regulatory system influences on Escherichia coli headgroup‐acylated glycerophospholipids
Authors: DiNardo Acadia S. , Garrett Teresa A.
Published: June 2021

Headgroup‐acylated glycerophospholipids (GPLs) are a class of low abundance membrane lipids that are thought to play a roll in cell division and membrane integrity in Gram‐negative bacteria, such as Escherichia coli. The PhoP/PhoQ two‐component regulatory system is known to aid in virulence of pathogenic E. coli and responds to low Mg2+ concentrations. Previous research has suggested a protein activated by the PhoP/PhoQ system increases synthesis of acyl phosphatidylglycerol (acyl PG), a type of headgroup‐acylated GPL. Currently, though, the synthase of another headgroup‐acylated GPL, N‐acyl phosphatidylethanolamines (N‐acyl PE), is unknown. In order to determine if the PhoP/PhoQ system affects levels of acyl PGs, as well as N‐acyl PEs, wild‐type MG1655 E. coli with an empty pBAD 33.1 plasmid were grown in N‐minimal media with Mg2+ concentrations of either 8μM or 10mM. To confirm the activation of the PhoP/PhoQ system when cells are grown in 8μM Mg2+ media a reporter strain of E. coli, pNL3, was used. A β‐galactosidase assay of the pNL3 stain confirmed the activation of the PhoP/PhoQ system in low Mg2+ conditions through a 3.8 fold increase of activity in 8μM Mg2+ conditions compared to growths in 10mM Mg2+. In the MG1655 wild‐type strain, three normalized N‐acyl PE and four normalized acyl PG species were measured, utilizing reverse phase column mass spectrometry, and summed in each Mg2+ condition. N‐acyl PE and acyl PG levels in the 8μM Mg2+ growth was double that of the MG1655 grown in 10mM Mg2+. These data suggest that the PhoP/PhoQ system may be implicated in the synthesis of N‐acyl PEs and acyl PGs. Further research is required to verify the correlation between the activated PhoP/PhoQ system in N‐acyl PE and acyl PG synthase, as well as identify the protein specifically responsible for the synthesis of N‐acyl PEs.

Support or Funding Information

Supported by National Science Foundation Major Research Instrumentation grant #1039659 and Research at Undergraduate Institution 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.
Native Gel Electrophoresis Reveals Partial Physical Association of band 3 and aquaporins on the Erythrocyte Membrane
Authors: Hsu Kate , Li Ying‐Syuan , Lin Hui‐Ju
Published: June 2021

Band 3 is the most abundant membrane protein in human red cells. Band 3 primarily functions as a bi‐directional anion transporter for Cl− and HCO3− to support blood CO2 exchange. Because CO2 mainly exists in the form of HCO3− (aq) in the human body, the rate of CO2 blood respiration is dependent on the intraerythrocytic reaction CO2(g) + H2O □ HCO3− (aq) + H+ (aq). By FLIM‐FRET, we recently uncovered the existence of mobile interaction between band 3 and water channel AQP1. Their interaction could be disrupted when red cell vesicles were put in a hypotonic milieu, suggesting AQP1 bearing a functional role in the band 3‐central CO2 transport metabolon.

Objective

As there are at least two types of band 3 protein complexes on the erythrocyte membrane, the goal of this study was to probe the physical associations between aquaporins and different band 3 complexes.

Method

We utilized semi‐native and native gel electrophoresis to identify aquaporin complexes and to differentiate their associations with the different band 3 complexes.

Results

Water channel AQP1 and aquaglyceroporin AQP3 complexes were both identified in red cell ghosts by semi‐native gel electrophoresis. Some of the aquaporin complexes were found to be structurally linked to band 3 protein complexes.

Conclusion

The physical association between AQP1 and band 3 and that between AQP3 and band 3 can be differentiated by semi‐native gel electrophoresis. Erythrocyte aquaporins also appear physically linked to other membrane‐associated proteins in the human red cells.

Support or Funding Information

MOST 106‐2320‐B‐195‐002 from Taiwan Ministry of Science & Technology

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 Effect of Spices on Cancer Incidence in Peru
Authors: Perez Patricia , Solomon Rahia
Published: June 2021

Breast and cervical cancer are a major concern for women all throughout the globe. Statistically over the last 30 years, there have been an increased incidence of breast (3.1%) and cervical cancer (0.6%) worldwide (WHO). Nutrition, natural remedies, and lifestyles of the Peruvian women were explored in an attempt to understand the effect nutrition and natural remedies have on women's health. It can be argued that nutrition plays a large role in the overall health of a person, therefore supplements, food, and spices may affect the frequency of cancer amongst Peruvian women. Patients and community members around Cusco, Peru were interviewed about their health, diets, and cooking habits. Using survey methods, we focused mainly on spices since they have been known to contain medicinal compounds. In analyzing the data from the responses about spice usage, it is clear that most spices used in the tested population are derived from meat (protein), which means that the protein was processed to be made into a broth or a dried additive. Seventeen people mentioned vegetables such as garlic, onions, parsley, tomatoes, olives, and peppers. Eight people discussed oregano as one of their most useful spice. Based on the collected survey results, oregano and other commonly used spices will undergo extraction and be tested for their effect on mammalian cells. Following, MTT and cytotoxicity assays will be conducted to help us understand the role these compounds play on mammalian cell viability.

Support or Funding Information

Mount Saint Mary's 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.
Impaired Insulin‐like Growth Factor‐1 function in X‐linked Adrenoleukodystrophy patients
Authors: DHAUNSI GURSEV S.
Published: June 2021

Introduction

X‐linked Adrenoleukodystrophy (X‐ALD), a common peroxisomal disorder is characterized by elevated very long chain fatty acids (VLCFA) levels in tissues and body fluids of patients. X‐ALD patients present with adrenal insufficiency, demyelination and progressive mental retardation and IGF‐1 has been reported to alleviate demyelination and neurodegeneration in an animal model of X‐ALD. We examined the effect of VLCFA on IGF‐1 function in human skin fibroblasts.

Methods

Human skin fibroblasts grown from skin explants were cultured in growth medium. Cell cultures were treated with varying concentrations of IGF‐1 and/or VLCFA (Lignoceric acid; C24:0, hexadecanoic acid; C26:0 ) for 24–72 hrs. DNA synthesis into cultured fibroblasts was measured and total RNA was extracted for RT‐PCR analysis of IGF‐1 receptor and GAPDH. Cell homogenates were prepared for western blotting and protein measurements. Levels of IGF‐1 and VLCFA were measured in blood samples of 10 patients with confirmed diagnosis of X‐ALD.

Results

Both VLCFAs used in this study significantly (p <0.01) reduced the IGF‐1–induced DNA synthesis in cultured skin fibroblasts. Lignoceric acid as well as hexadecanoic acid markedly reduced the gene expression of IGF‐1 receptor. IGF‐1 receptor protein was also significantly reduced following 48 hrs of treatment with VLCFAs. X‐ALD patients were observed to have significantly lower levels of plasma IGF‐1 as compared to healthy controls.

Conclusions

This study suggests that abnormally elevated VLCFA in X‐ALD patients might impair IGF‐1 function through downregulation of IGF‐1 receptor thereby providing a new insight into molecular mechanisms of X‐ALD pathogenesis.

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 Zinc on Mitochondrial Morphology
Authors: Knies Katherine , Lu Qiping , Li Yang
Published: June 2021

Zinc (Zn2+) is involved in normal and abnormal cellular physiology. Cellular insults may cause zinc overload, which may affect mitochondrial functioning. Mitochondrial functioning influences mitochondrial morphology. Normal mitochondrial morphology has a tubular appearance, while abnormal mitochondrial morphology has a blob or donut appearance. In this study, it was addressed whether mitochondrial stress and insults caused by zinc resulted in abnormal mitochondrial morphology. To indicate mitochondrial morphology, live HeLa cells were stained with either MitoFluor Red 589 or MitoTracker Red. The two different treatments applied to the cells were zinc chloride pyrithione or pyrithione. Zinc chloride pyrithione was the experimental treatment and pyrithione was the vehicle in this study. These treatments were applied for 30 minutes. Images were taken before and after treatment to assess different parameters of morphology. The parameters examined were the length, diameter, surface area, mitochondrial morphology, and the number of mitochondria per area. Pyrithione did not have a significant effect on the measured parameters. In the zinc chloride pyrithione treated cells, the morphology changed from the tubular shape to donut and blob shape. This indicated that there was a rise in mitochondrial stress. There was also a reduction in mitochondrial number within zinc chloride pyrithione treated cells. This finding indicated that there was a higher amount of mitophagy, which is a form of autophagy that removes defective mitochondria, in these cells.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Theasinensins A Against Methylglyoxal‐Induced Neurotoxicity via Autophagy in SH‐SY5Y Cells
Authors: Cheng An‐Chin , Lee Ming‐Fen , Huang Tzu‐Li , Cai Kuan‐Ci , Pan Min‐Hsiung
Published: June 2021

Methylglyoxal (MG) is a highly reactive dicarbonyl aldehyde. MG has been proved to be toxic to neuron and may be the reason of many neurodegenerative diseases. Tea polyphenols have notable effects against various types of cancers, blood pressure, cardiovascular problems, arthritis, and atherosclerosis without inducing major toxicity consequences. Tea polyphenols have a role in the reversal of age‐related loss of neuronal plasticity and recovery after neuronal lesions. Theasinensins are a group of tea polyphenols different from green tea catechins, and they are considered as bioactive compounds in Oolong tea. During enzymatic oxidation, EGCG is fermentated and several B‐ring homodimers: theasinensins A (TSA) intermediates generated. TSA had been found to have many bioactivities, such as anti‐oxidation. However, the effect of the neuroprotective effect of this tea polyphenol remains unclear. The aim of this study is to investigate the neuroprotective effects and the molecular mechanism of TSA against MG‐induced toxicity in SH‐SY5Y cell model. The cell viability assay demonstrated that TSA treatment protected cells from MG‐induced neurotoxicity. The result of acridine orange (AO) stain, detecting AO fluoresce intensity and the level of LC3, showed that TSA could increase intracellular AVO value and induce autophagy in MG‐treated neuron cell. We expect that TSA could be reagents to protect neurodegenerative diseases.

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.
β‐Catenin Deficiency in Hepatocytes Aggravates Hepatocarcinogenesis Driven by Oncogenic β‐Catenin and MET
Authors: Liang Yan , Feng Yun , Zong Min , Wei Xu‐Fu , Lee Jin , Feng Yukuan , Li Hairi , Yang Guang‐Shun , Wu Zhong‐Jun , Fu Xiang‐Dong , Feng Gen‐Sheng
Published: June 2021

Both activating and inactivating mutations in ctnnb1, encoding β‐catenin, have been implicated in liver tumorigenesis in humans and mice, although the underlying mechanisms are not fully understood. Herein we show that deletion of endogenous β‐catenin in hepatocytes aggravated hepatocellular carcinoma (HCC) development driven by an oncogenic version of β‐catenin (CAT) in combination with HGF receptor MET. Although the mitogenic signaling and cell cycle progression was modestly impaired after CAT/MET transfection, the β‐catenin‐deficient livers displayed changes in transcriptomes, increased DNA damage response, expanded Sox9+ cells, and upregulation of pro‐tumorigenic cytokines including IL‐6 and TGF‐β1. These events eventually exacerbated CAT/MET‐driven hepatocarcinogenesis in β‐catenin‐deficient livers, featured by upregulation of Erk, Akt and Wnt/β‐catenin signaling and cyclin D1 expression. The resultant mouse tumors showed similar transcriptomes to human HCC samples with concomitant CTNNB1 mutations and MET overexpression. These data argue that while dominantly activating mutants of β‐catenin are oncogenic, inhibiting the oncogenic signaling pathway generates a prooncogenic microenvironment, which may facilitate HCC recurrence following a targeted therapy of the primary tumor. Therefore, an effective therapeutic strategy must require disruption of the oncogenic signaling in tumor cells and also suppression of the secondary tumor‐promoting stromal effects in the liver microenvironment.

Support or Funding Information

This project was supported by NIHR01CA176012 and R01CA188506 (to G.S.F.). Yan Liang was supported by a pre‐doctoral fellowship of FISP.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Putative MicroRNA Regulatory Networks in Hepatic Stellate Cells Underlying Chronic Ethanol‐Mediated Impairment of Liver Regeneration after Partial Hepatectomy
Authors: Parrish Austin , Juskeviciute Egle , Hoek Jan , Vadigepalli Rajanikanth
Published: June 2021

microRNAs (miRNAs) are a class of small, non‐coding RNAs ~22 nucleotides in length that are involved in post‐transcriptional regulation of a wide variety of processes critical for normal cell function, such as development, cell differentiation, cell proliferation, and cell death. There remains, however, a comparative lack of knowledge on the precise role and function of miRNAs in the context of alcoholic liver disease. In this study, we sought to characterize global miRNA expression patterns in the rat liver for both control and ethanol‐fed animals before and after 70% partial hepatectomy (PHx). Chronic ethanol consumption using the Lieber‐DeCarli diet leads to a maladaptive response across multiple organ systems, and is sufficient by itself to alter normal miRNA expression dynamics in the liver. This adaptation to ethanol consumption has also been shown to alter miRNA expression in response to PHx when compared with control animals. Additionally, the regenerative capacity of the liver in response to injury is significantly decreased by chronic ethanol consumption. Previous research has identified several miRNAs that may be key regulators of liver regeneration, such as miR‐21, whose role in liver regeneration and liver disease has been well‐studied by our lab and others. By using the NanoString nCounter expression system, we obtained an un‐biased view of changes in miRNA expression. Our results show that the differentially expressed miRNAs exhibit complex expression dynamics during liver regeneration, and that many of these miRNAs appear to be strongly correlated with each other, suggesting the possibility of co‐regulatory networks. This is further supported by our results showing that alteration of the expression of a single miRNA, in this case inhibition of miR‐21, is capable of affecting the expression of multiple other miRNAs in both correlated and anti‐correlated fashions. Functional analysis of these dysregulated miRNAs suggests potential mechanisms involved in hepatic stellate cell activity, which was further supported by gene set enrichment analysis using previously published data involving stellate cell‐specific miRNAs. In conclusion, we have identified a network of miRNAs whose differential expression may regulate liver regeneration via alteration of hepatic stellate cell function.

Support or Funding Information

Research was supported by the following NIH grants from NIAAA: R01 AA018873, U01EB023224, F31 AA024969‐01, and T32 AA007463.

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