bluuebunny/crossref_metadata_2025 huggingface dataset:
OspC's Pivotal Role in Lyme Disease Infection and Transmission
Authors: Shabbir Sanah , Spanel Jack , Wagoner Natalie
Published: June 2021

The Olathe North MSOE Center for BioMolecular Modeling SMART Team used 3‐D modeling and printing technology to examine structure‐function of OspC. Lyme disease is a tick‐borne illness caused by the spirochetal bacterium Borrelia burgdorferi. It is the most commonly occurring vector‐borne disease in the United States and if not treated, Lyme disease can cause carditis, arthritis and neurological symptoms. The surface of B. burgdorferi is covered by surface lipoproteins. One of them is the outer surface lipoprotein C (OspC). The ospC gene is located on a plasmid and expressed by the bacteria during their transmission from ticks to warm‐blooded hosts. OspC is critical for colonization of the host and begins to be synthesized by the bacteria when the blood and its nutrients enters the tick's midgut upon a tick bite. The presence of OspC promotes the movement of B. burgdorferi from the midgut to the salivary glands, allowing the bacteria to be injected with the tick saliva into the host. The tick salivary protein Salp15 binds to portions of OspC and interferes with the initial host immune response, which allows the bacteria to spread to different tissues. OspC also binds plasminogen which is activated by host to the protease plasmin. The resulting coat of plasmin breaks down host tissues and offers the bacteria a pathway to disseminate further. The OspC protein is a homodimer consisting of a bundle of multiple alpha helices as well as a single beta‐pleated sheet. A region facing away from the bacterial membrane is negatively charged and may bind positively charged host ligands. This feature is unique to OspC variants that are known to cause invasive human disease. The precise biological function of OspC is unclear and its relationship between OspC variant and pathogenicity is not well understood. To further study this, mutations within, e.g,, the above mentioned negatively charged region may affect the bacteria's ability bind Salp15 or plasminogen and thus disrupt the overall effectiveness of Borrelia burgdorferi to be transmitted and cause disease.

Support or Funding Information

Olathe North High School SMART Team

Sponsor: Chris Elniff

Mentor: Dr. Wolfram Zückert, PhD, Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural And Functional Versatility of Interferon‐Inducible GTPases
Authors: Yin Qian , Roy Sayantan , Wang Bing , Tian Yuan , Yang Wei
Published: June 2021

GTP triphosphatases (GTPases) have been meticulously documented for their roles to regulate multiple cellular processes spanning from cell mobility, membrane fusion and fission, to cytokinesis and vesicle transport. Interferon (IFN)‐inducible GTPases, consisting of more than forty members in human and mice, are among the most highly expressed interferon stimulated genes (ISGs), sometimes accounting for twenty percent of all proteins induced by IFN‐γ. Recent genetic and cell studies start to reveal the important roles of IFN‐inducible GTPases in restriction and elimination of pathogens, yet the molecular mechanisms are largely unclear. Using a combination of biochemical, structural, biophysical, and computational tools, we are investigating the functional forms of two IFN‐inducible GTPases, GBP2 and IRGM, and the mechanisms governing their activation. We established the relationship between nucleotide binding and the oligomeric status of GBP2. A combination of X‐ray crystallography, electron microscopy, and molecular dynamics captured GBP2 conformational changes along the full trajectory of GTP binding and hydrolysis. Structure‐guided mutagenesis validated the roles of key residues in nucleotide binding pocket. In contrast to the dynamic nature of GBP2, IRGM assumed a highly stable oligomeric state that possessed intrinsic affinity towards lipids. In conclusion, GTP binding and hydrolysis regulate the oligomeric status of interferon‐inducible GTPases and subsequential effector molecule binding.

Support or Funding Information

NIAID R00AI108793 “Dissecting inflammasome anatomy: mechanistic studies and potential intervention”; Florida State University startup funds

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Elementary Steps in Plant‐Pathogen Recognition Revealed by Enhancing the Stability and Structure of Domains of Plant Innate Immune Receptors
Authors: Eisenstein Edward , Li Jia , Walsh Scott T.R. , Bera Asim , Bakalarz Janet , Weatherwax Colleen , Pierce Brian , Parsons James
Published: June 2021

Pathogens elicit an immune response in plants through the recognition of the avirulent effectors by multi‐domain intracellular disease resistance receptors. The nature and specificity of plant‐pathogen interactions and the molecular mechanism of eliciting an immune response is being explored using the N protein from Nicotianaglutinosa. The N protein is an archetype of the nucleotide binding (NB), leucine rich repeat (LRR) family of innate immune receptors (NLRs), and the interaction with its avirulent effector, the helicase domain (HEL) of the replicase protein of tobacco mosaic virus (TMV) triggers a classic hypersensitive cell death response in plants. An initial step in activating cell death pathways upon pathogen recognition is a structure change that enables NLR assembly through their N‐terminal Toll‐Interlukin1 Receptor‐like (TIR) domains. Stabilized domains with increased tendency to self‐associate have been constructed by focused mutagenesis guided by computational modeling of a 3.0 Å crystal structure of the N‐TIR domain. The energetics of TIR dimerization is being correlated with the severity of the hypersensitive response by measuring salicylic acid using LC‐MS to examine the hypothesis that receptor assembly is correlated with NLR signaling. Yeast surface display is being used to probe the manner whereby host NLRs recognize pathogenic determinants, and reveals that the interaction of N with its pathogen effector, the HEL domain of TMV, is mediated via its LRR domain. Binding analyses show that a stabilized LRR domain binds HEL about 250‐fold more strongly than a non‐virulent homologue that does not display a hypersensitive response. Variants of the full‐length N protein are being rebuilt from the individually stabilized TIR and LRR domains optimized for functional analyses to enable biochemical and structural investigations of plant NLR receptors.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of a Novel In Situ Activity Assay for Lysyl Oxidase Like‐2 (LOXL2)
Authors: Wang Huilei , Abouelkheir Mahmoud , Poe Alan , Hong Yurie , Jandu Sandeep , Nandakumar Kavitha , Santhanam Lakshmi
Published: June 2021

Lysyl oxidase like‐2 (LOXL2) is a member of the lysyl oxidase (LOX) super family, which are copper‐dependent amine oxidases that initialize collagen and elastin cross‐linking to form a stable and organized matrix. Augmented LOXL2 expression is highly associated with diverse disease processes with a known element of matrix accumulation including fibrosis, pulmonary hypertension, and cancer metastasis. To date, lack of sensitive and specific assays that can detect LOXL2 catalytic function in situ in the extracellular matrix (ECM) of cells and tissue has limited the ability to fully elucidate the role of LOXL2 in pathobiological mechanisms.

Current methods to detect LOXL2 enzymatic activity are inefficient and limited in scope. The most widely used assay is hydrogen peroxide based: oxidation of the LOX active site occurs when substrate is added to samples, and releases hydrogen peroxide. The level of hydrogen peroxide is detected using an HRP‐coupled system, that utilizes the conversion of Amplex Red to the fluorescent end product, resorufin. While the results are semi‐quantitative, this assay has limited dynamic range and cannot distinguish hydrogen peroxide arising from other pathways. Moreover, the assay requires LOXL2 in solution. The harsh conditions required to obtain a solubilized ECM sample, where the pertinent LOXL2 activity occurs, cause LOXL2 unfolding and loss of activity, rendering this assay unfeasible. Thus, there is a need to develop a sensitive, specific, and practicable in situ activity assay that overcomes these limitations.

This study provides a proof of concept for a novel in situ activity assay for LOXL2. Here, we developed an assay to fluorescently label the products of LOXL2 catalysis. Specifically, LOXL2 catalyzes an oxidative de‐amination of lysine and hydroxylysine residues (found on collagen and elastin) to highly reactive semi‐aldehydes called allysines. In this assay, we labeled the newly generated allysine residues with biotin hydrazide using 1‐ethyl‐3‐(3‐dimethyl‐aminopropyl) carbodiimide hydrochloride (EDC) crosslinker. This biotinylation allows for a robust method of measuring LOXL2 activity through western blotting, immunoprecipitation and immunofluorescence imaging using biotin‐streptavidin interactions.

Support or Funding Information

Funded by MedImmune research funds.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lipophilic Modification of an Anti‐Cancer Stem Cell Agent Improves Pharmacokinetic and Anti‐Cancer Properties
Authors: Morla Shravan , Afosah Daniel K. , O'Hara Connor , Boothello Rio , Patel Bhaumik B. , Sakagami Masahiro , Desai Umesh R.
Published: June 2021

Current anti‐cancer agents target bulk tumor cells but do not eradicate the rare, quiescent, cancer stem cells (CSCs). CSCs are being increasingly recognized as the reason for cancer relapse. Targeting CSCs has become a major area of interest in the field of anti‐cancer drug discovery and several clinical trials focused on CSC targeting are in progress. Previously, we identified a CSC selective agent G2.2 (Fig. 1), which inhibited CSC spheroidal growth with a median IC50 (±1SEM) s of 193 μM±58 μM. G2.2 was found to exhibit robust anti‐CSC properties in colorectal cancer xenograft models in vivo. Mechanistically, G2.2 is a mimetic of heparan sulfate hexasaccharide (HS06) and is perhaps the only non‐saccharide heparan sulfate mimetic identified to date to exhibit considerable anti‐cancer promise. Preliminary pharmacokinetic (PK) studies showed that G2.2 exhibited a half‐life of nearly 4 hours following IV administration and 6% bioavailability following oral administration in rats.

To improve upon G2.2's PK properties, we hypothesized lipid‐modification of G2.2 would enhance penetration across GI barrier while also simultaneously enhancing its potency through better targeting of plasma‐membrane growth factor receptors – the presumed targets of G2.2 and HS06. Several lipid‐modified agents were synthesized. These modifications resulted in improvement of PK properties (bioavailability by greater than 4‐fold). Further, the analogs also showed up to ~ 53‐fold (NCI‐H508 spheroids) enhanced inhibition potency against CSCs in in‐vitro spheroid cell‐based assays. In fact, these lipid‐modified G2.2 analogs inhibited several (>10) colorectal cancer spheroids with median IC50 values (±1SEM) ranging from 8 μM±3μM to 68 μM±14μM. Considering that G2.2 is a mimetic of heparan sulfate, these results present a simple avenue to enhance drug‐like properties of such highly charged agents. The results also suggest that the lipid‐modified analogs may also serve as probes of molecular processes governing the CSC phenotype.

Support or Funding Information

This work was funded by the NHLBI grant 1P01HL107152 given to Umesh R. Desai.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Methamphetamine alters the expression of Toll‐like receptor 4 on microglia‐like cells
Authors: Rivera Dormarie E. , Martinez Luis R.
Published: June 2021

Methamphetamine (METH) is a major public health and safety problem in the United States. METH is a strong central nervous system (CNS) stimulant that mimics the pharmacological effects of cocaine. METH abuse can also alter biological processes and immune functions necessary for host defense. Microglia are key cellular mediators of the innate immune system in the CNS and can be activated by METH in a dose‐dependent manner through the Toll‐like receptor (TLR) 4/MD2 complex. This complex could also be stimulated in microglia by interactions with pathogen‐associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS) resulting in neuroinflammation and neuronal damage. Moreover, minocycline, a semi‐synthetic tetracycline derivative antimicrobial drug, inhibits microglial activation. Therefore, we hypothesized that METH impairs the microglial expression of TLR4. NR‐9460 cells, a murine microglial line derived from wild‐type mouse brain tissue, were treated with METH or minocycline for 2 h followed by incubation with LPS for 24 h. Then, flow cytometry, confocal microscopy, and western blot analyses were performed to determine the effect of METH on the expression of TLR4 receptors on microglia‐like cells. We found that METH alone increases the expression and distribution of TLR4 receptors on the surface of microglia‐like cells and changes the cellular morphology. Interestingly, after stimulation of METH–treated microglia with LPS, the TLR4 expression and distribution on the surface of these cells increases, however, the protein expression of the receptor showed no difference in total protein lysates. Our findings suggest that METH use may impair the cellular production of TLR4 in microglia. In fact, it is conceivable that METH interferes with the signaling pathways and inflammatory responses important to prevent and eradicate CNS infection. We believe this interdisciplinary project is of considerable significance in the fields of neurosciences, immunology, and drug abuse. We anticipate that our findings will result in a deeper understanding of neuroinflammation and CNS microbial infection control in METH users.

Support or Funding Information

This work was supported by NIGMS‐NIH grants: 1R15GM117501‐01A1 (LRM), and UPR‐Ponce Research Initiative for Scientific Enhancement (R25GM096955). The authors also acknowledge the support from the Border Biomedical Research Center (BBRC) at the University of Texas at El Paso (NIMHD Grant 2G12MD007592).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparing ROS Production in Murine Microglial Cells in Response to Two Different Types of Particulate Matter
Authors: Merrill Luke Lee , Buchmann Kelsey , Street Mikayla , Stadem Nathan , Fortuna Tyler , Mazzer Paula
Published: June 2021

Alzheimer's and other related neurodegenerative diseases affect nearly 24.3 million individuals globally‐a number that will likely increase with the rising world population [1]. In a recent survey, a panel of experts discussed existing data and concluded that there are higher rates of dementia in developed regions than developing regions. A link between air pollution and neurodegeneration has been established [2–4]. We are looking at the ability of two different types of particulate matter, diesel exhaust and urban dust, to activate microglial cells and determine if there is an increase in oxidative stress when the microglial cells are exposed to these types of particulate matter in vitro. We utilize fluorescence and ELIZA kits to measure production of TNF‐alpha and hydrogen peroxide.

Support or Funding Information

Research reported in this publication was supported by an institutional development award from the National Institute of general medical sciences of the National Institutes of Health under grant number P20GM103443.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cell Death Induced by Airborne Particulate Matter in Murine Microglial Cells
Authors: Street Mikayla Elizabeth , Merrill Luke , Mazzer Paula
Published: June 2021

Airborne particulate matter has been linked to neurodegeneration in recent epidemiological studies. Previous work in our lab has shown that different types of particulate matter elicit different reactions. We set out to investigate two different particulate matter types in microglial cells to see whether they would have the same effect in different types of neurological cells. We combined caspase‐3 assay with lipid profiling to determine the mode of cell death induced by several different types of airborne particulate matter. Some types of particulate matter induced cell death via apoptosis and others induced cell death by neuroinflammatory methods.

Support or Funding Information

Research reported in this publication was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103443. The content is solely the responsibility of the authors and does not necessarily represent the official views of the 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.
Time‐Resolved Shotgun Lipidomic Analysis of Murine Astrocyte Cells Exposed to Airborne Particulate Matter
Authors: Travis Meghan , Vettrus Noah , Johansen‐Sallee Anthony , Mazzer Paula
Published: June 2021

Millions of people around the world that are affected by neurodegenerative disease. A potential correlation has been found between neurodegenerative disease and particulate pollution. Several studies have shown oxidative stress in the brain and induction of neuroinflammation due to airborne particulate matter. Our lab has shown that urban dust induces necrosis, and diesel exhaust particulate matter induces apoptosis. I studied the the lipid profile of murine astrocyte cells exposed to urban dust and diesel exhaust at several different time points. I used shotgun lipidomics to identify all the lipid chemical species present in the cell samples. The samples were run through the 12T Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (COSMIC). A key difference between the treated and untreated samples was the presence of certain ceramides. We believe these ceramides might be associated with ceramide signaling pathways in cell death.

Support or Funding Information

Research reported in this publication was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103443. The content is solely the responsibility of the authors and does not necessarily represent the official views of the 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.
The Toxicity of Electronic Cigarette Vapor on Human Oral Cells
Authors: Urena Jose Francisco , Ebersol Lauren , Silakov Alexey , Lambert Joshua
Published: June 2021

Electronic cigarettes (ECs) have emerged as a popular alternative to conventional cigarettes. These devices operate by using resistive heating to vaporize a fluid consisting of a propylene glycol‐glycerol base, flavorings, and nicotine. Although considered safer than conventional cigarettes, recent studies have shown that EC vapor contains harmful compounds such as free radicals and carbonyls. A limited number of studies have shown that EC liquid and vapor can cause oxidative stress and cell death in vitro. While the majority of in vitro studies have focused on the toxicity in respiratory tract cell lines, the tissues in the oral cavity, which are exposed to high local concentrations of EC vapor, have been under‐studied. Our objective was to investigate the ability of EC vapor to induce cytotoxicity and oxidative stress in human oral cell lines. HGF‐1 human gingival fibroblasts and SCC‐25 human oral squamous cell carcinoma cells were exposed to a panel of commercially‐available fruit and tobacco‐flavored EC vapors using an air‐liquid interface exposure model, which mimics physiological exposures in vitro. Cytotoxicity and markers of oxidative stress were assessed after ecologically‐relevant exposure protocols. Cytotoxic EC vapors caused a greater than 30% reduction in cell viability. Nicotine was shown to ablate the effects of the cytotoxic EC vapors. There was no difference in the cytotoxic response to EC vapor between the cell lines tested. Treatment of SCC‐25 cells with a cytotoxic EC vapor caused a significant increase in intracellular ROS after a single 15 puff exposure. This increase occurred for both nicotine‐containing and nicotine‐free vapors. The differential effects of nicotine on cytotoxicity and oxidative stress suggest that either (1) cytotoxicity is not driven by ROS or (2) that nicotine protects cells by a mechanism that does not impact intracellular ROS levels. Ongoing studies are investigating the role of nicotine in this aerosolized system and the relationship between EC vapor constituents and cytotoxicity in human oral cells.

Support or Funding Information

Penn State Tobacco Center of Regulatory Science; Penn State Department of Food Science

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modulating Human Follicle Stimulating Hormone Signaling Activity Using Negative Allosteric Modulators
Authors: Love Jillian R , Cohen Brian D
Published: June 2021

Human follicle stimulating hormone (hFSH) is a gonadotropin hormone involved in maturation of ovarian follicles and estrogen production in females and spermatogenesis in males. Its release is regulated via the hypothalamic‐pituitary‐gonadal axis. Proper regulation and function of hFSH in females is necessary for fertility, so its manipulation has been a focus in female fertility treatments and contraception. The hFSH receptor (hFSHR) is a GPCR found in granulosa cells in females and Sertoli cells in males and is responsible for initiating a complex downstream signaling cascade in both cell types. It has recently been discovered that hFSHR is capable of biased signaling, meaning it may be possible to selectively activate some downstream signaling pathways without affecting others. This biased signaling may be achieved through the addition of negative allosteric modulators (NAM's), notably the small molecules ADX68692 and ADX68693. Both NAM's have been shown to decrease FSH‐dependent ovulation in vivo although with differing effects on steroid hormone production. The specific molecular mechanisms by which this occurs is unclear. Utilizing the human granulosa cell line hGrC1 we have observed differences in p44/42 MAPK signaling after treatment with NAM's. In control conditions, activation of p42/44 MAP kinase peaked five minutes following FSH stimulation, but in the presence of NAM's there was a continuous increase in signaling over thirty minutes. Additionally, signaling through protein kinase A (PKA) in the presence of ADX68692 showed increased activation of some substrates but in the presence of ADX68693 basal levels of the same substrates were initially higher and showed a decrease over time. Interestingly, some PKA substrates showed no difference in hFSH activation between untreated and NAM treated samples. The effects of treatment with NAM's show their applicability in the development of novel non‐steroidal contraceptive methods that could be associated with fewer side effects. Studying the complex biased signaling mechanisms of hFSHR could identify ways to manipulate hFSHR for not only improved methods of contraception but also fertility treatments.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Fyn is recruited to specialized clathrin coated pits and regulates EGF receptor signaling
Authors: Antonescu Costin N , Lucarelli Stefanie , Judge Gurjeet , Abousawan John , DiGiovanni Laura , Vural Dafne , Hong Wanjin , Kim Peter K
Published: June 2021

The Epidermal Growth Factor (EGF) Receptor (EGFR) is a key receptor tyrosine kinase that controls many facets of cell physiology, including proliferation, survival and migration. EGF stimulation elicits EGFR phosphorylation and/or membrane recruitment of Grb2, Gab1 and phosphatidylinositol‐3‐kinase (PI3K), resulting in production of phosphatidylinositol‐3,4,5‐trisphosphate (PIP3), leading to Akt activation. Concomitantly upon ligand binding, EGFR is simultaneously recruited to clathrin‐coated pits (CCPs), eventually leading to receptor endocytosis. We previously uncovered that perturbation of clathrin, but not of receptor endocytosis, impairs EGF‐stimulated activation of Akt signaling (Garay et al. 2015. Mol Biol Cell.26:3504–19). Moreover, we found that some key EGFR signaling intermediates such as phosphorylated Gab1 are enriched with some CCPs. We thus proposed that in addition to their function as endocytic portals, some CCPs have a direct role in controlling EGFR signaling. How CCPs and clathrin directly control the spatiotemporal properties of EGFR signaling at the plasma membrane prior to receptor endocytosis had remained poorly understood. We uncovered that perturbations of TOM1L1 (a clathrin‐binding regulator of Src‐family kinases) and the TOM1L1‐binding Src‐family kinase Fyn impaired EGF‐stimulated Akt phosphorylation, similarly to the effects of clathrin perturbations. We found that TOM1L1 and Fyn are enriched within a subset of CCPs that also contain EGFR, and that perturbation of TOM1L1 prevented Fyn recruitment to CCPs. Importantly, TOM1L1‐ or Fyn‐positive CCPs exhibit unique properties, such as distinct lifetimes, compared to other CCPs. Thus, our results indicate that a subset of CCPs is specialized by enrichment with key signaling regulators to control EGFR signaling, a phenomenon required for subsequent Akt activation. Given the importance of EGFR in driving growth and survival of certain types of cancer, obtaining a better understanding of how CCPs organize key EGFR signals in space and time within the PM may lead to the development of novel anti‐cancer treatments.

Support or Funding Information

Funding for this research was provided by an Operating Grant (no. 125854), a Project Grant and a New Investigator Salary Award from the Canadian Institutes of Health Research, as well as an Early Researcher Award (Ontario Ministry of Research, Innovation and Science) to C.N.A., and an Ontario Graduate Scholarship to S.L.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cardiac Fibroblast Regulation of Matrix Assembly and Inflammation
Authors: Tallquist Michelle D.
Published: June 2021

Heart failure is a progressive disease that is often exacerbated by inappropriate cardiac remodeling in response to stress. The excess deposition of extracellular matrix (ECM) by fibroblasts, known as fibrosis, is often coincident with reduced cardiac output . While the detrimental effects of overactive ECM secretion are appreciated, our current understanding of the function of the cardiac fibroblast in the absence of stress is limited. Using a genetic approach in mice to deplete hearts of cardiac fibroblasts, we have investigated how loss of fibroblasts affects heart morphogenesis in the neonate and the adult. In the absence of fibroblasts at postnatal day 5, we observe limited production of ECM especially type I collagen. In the absence of ECM, cardiomyocytes bypass the growth arrest that usually occurs in the first week of postnatal growth and continue to undergo cytokinesis. These data suggest that ECM content may restrict cardiomyocyte growth and loss of ECM may be permissive for continued cardiomyocyte proliferation. To investigate the role of fibroblasts in heart homeostasis and inflammation, we also depleted cardiac fibroblasts from adult hearts. Although subtle alterations in cardiac structure were observed, hearts with 70–80% reduction in fibroblasts were functionally indistinguishable from controls suggesting that type I collagen is more stable than previously thought. Finally, we will describe how the loss of fibroblasts impacts inflammatory responses after injury. While an inflammatory response after acute injury is essential for clearing dead tissue, and some fibrosis may be necessary to maintain the tensile strength of injured myocardium, a sustained inflammatory response can lead to inappropriate fibroblast activation. After injury we observed increased cardiomyocyte hypertrophy and reduced infiltration of neutrophils, monocytes, and T and B cells. Cardiac fibroblast specific translatome analysis revealed that chemokine expression was dynamic after myocardial infarction. In the first four days after injury, Cxcl4, Cxcl10, and Ccl5 were increased, while Ccl17 expression increased and Ccl11 decreased after 8 days of injury. Our results suggest that fibroblasts are essential for ECM production and fibroblast depletion leads to changes in cardiomyocyte biology and inflammation after injury.

Support or Funding Information

NHLBI Grant HL144067

AHA 17GRNT33660474

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 effects of Se‐allylselenocysteine (ASC), an analogue of garlic compound, on DMBA/TPA‐induced skin tumorigenesis
Authors: Cheng An‐Chin , Lee Ming‐Fen , Pan Min‐Hsiung
Published: June 2021

Se‐allylselenocysteine (ASC), an analogue of garlic compound, has been shown to inhibit mammary carcinogenesis in vivo and cell growth in vitro. The two‐stage skin carcinogenesis model is an excellent in vivo model which is induced by 7, 12‐dimethylbenz[a]anthracene (DMBA) /12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) in ICR mice, and can well annotate multistep process of carcinogenesis including initiation, promotion and progression. Mice at 6 weeks old were started on 200 nmol DMBA in 200 μL acetone; control mice received 200 μL acetone alone. One week after initiation, the mice were treated topically with 200 μL acetone or promoted with TPA (5 nmol in 200 μL acetone) twice a week for 20 weeks. In the other two groups, the mice were treated with ASC (1 and 5 μmol in 200 μL acetone) 30 min before each TPA treatment. Tumors of at least 1 mm in diameter were counted and recorded every week. The results were expressed as the average number of tumors per mouse, percentage of tumor‐bearing mice, and average tumor weight per mouse. In our finding, topical application of ASC on the dorsal skin of DMBA‐initiated and TPA‐promoted mice could significantly accelerate skin tumorigenesis and raise tumor multiplicity than positive control group (DMBA+TPA). The number of tumors that were 1–3 mm, 3–5 mm and >5 mm in size per mouse was increased in a dose‐dependent manner in the ASC‐pretreated groups. It's probably because ASC needs metabolizing in vivo to have the inhibitory effect of cancer. However, the study is based on the model of applying drug on mouse skin. The pro‐inflammatory activity of ASC can be demonstrated by its effect on COX‐2 expression in TPA‐induced mouse skin.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Garlic, Ginger and Tumeric Extracts Alter Cell Cycle Arrest through Estrogen Pathway
Authors: Ebuehi Osaretin Albert Taiwo
Published: June 2021

Garlic (Allium sativum), ginger (Zingiber officinale)) and turmeric (Curcuma longa) possess diverse nutritional, gastronomic and therapeutic functions. The effects of administration of aqueous garlic and ginger extracts, and acetone extract of tumeric at different concentrations from 4.88–1,250μg/ml were tested against the human (MCF‐7) adenocarcinoma breast cancer cell line. The phytochemical examination of these plants showed the presence of alkaloids, flavonoids, and phenol in the plant extracts, while tannins were present in garlic and ginger extracts. Terpenoids was present in garlic and turmeric extracts, cardiac glycoside was present in ginger and turmeric, steroid and saponin were exclusively present in turmeric, while anthroquinone was not detected. The radical scavenging properties of extracts were examined against 2,2‐Diphenyl‐1‐Picrylhydrazyl, Nitric Oxide (NO) and reducing power assay show significant antioxidant activity in all plant extracts along the concentration gradient. Gas chromatography‐mass spectrometry screening of the extracts identifies various biomolecules that might be responsible for anti‐carcinogenicity. The percentage viability of human adenocarcinoma breast cancer cell line (MCF‐7) was determined using MTT test, the percentage viability of cell increase across all concentrations (4.88–1250μg/ml) above 100% original viability. Cisplatin (standard drug) used as a control increase in the same pattern parallel to the garlic extract increase from 0.98 to 250 μg/ml. This result suggests that the effects of garlic, ginger and turmeric extracts do not significantly inhibit carcinogenesis / pathogenesis of human breast cancer rather promote proliferation of breast cancer cell probably due to it lower concentration of the extract that cannot effectively act on the oncogene/tumour suppressor, cyclins E and D1, and p27 of the cell. Data of study indicate that garlic, gingrer and turmeric extracts allow free promotion of the estrogen stimulation that leads to increase cellular proliferation via upregulation of cyclin D1 levels and CDK4/6 activity, as well as c‐myc, and cyclin E/CDK2 levels.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Glia Maturation Factor‐Antibody Injection Reduces Behavioral Impairment, Neuro Inflammation and Amyloid Pathology in 5XFAD Mice Brains
Authors: AHMED MOHAMMAD EJAZ , Thangavel Ramasamy , Selvakumar Govindhasamy Pushpavathi , Kempuraj Duraisamy , Dubova Iuliia , Zahoor Haris , Raikwar Sudhanshu P , Mentor Shireen , Zaheer Smita , Iyer Shankar S , Zaheer Asgar
Published: June 2021

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the accumulation of beta‐amyloid (Aβ) plaques and neurofibrillary tangles (NFTs) in the brain. Emerging evidence suggested that in neurodegenerative disease, aggregation of these proteins can promotes neuroinflammation and neurodegeneration. Glia maturation factor (GMF), a neuroinflammatory protein isolated and cloned in our laboratory play an important role in the pathogenesis of AD. To study the relationship between overexpression of GMF, glial activation and neuroinflammation, we used 9‐month‐old 5XFAD mice, a transgenic model of AD. We intravenously (iv) injected single dose of GMF antibody and found that antibody injection leads to: improvements in behavioral deficits, decreased activation of glial cells and reduced expression of pro‐inflammatory cytokines and amyloid pathology. We report that aberrant expression of GMF, GFAP, IBA1, and co‐localization of GMF with GFAP, IBA1 and Aβ significantly more in 5XFAD mice as compared with wild type mice. However, following GMF antibody injection, we found significant reduced expression of GMF, GFAP, IBA1 and Aβ in the cortex region of 5XFAD mice. We conclude that GMF‐antibody may be used to reduce neuroinflammation in neurodegenerative diseases including Alzheimer's disease.

Support or Funding Information

National Institutes of Health Grants NS073670, AG048205 and Veterans Affairs Merit Award I01BX002477 to AZ.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Na,K‐ATPase β2‐subunit/AMOG as a regulator of Merlin/NF2 signaling in the cerebellum
Authors: Langhans Sigrid A , Litan Alisa , Li Zhiqin , Tokhtaeva Elmira , Kelly Patience , Vagin Olga
Published: June 2021

The Na,K‐ATPase, consisting of a catalytic α‐subunit and a regulatory β‐subunit, is a ubiquitously expressed ion pump that carries out the transport of Na+ and K+ across the plasma membranes of most animal cells. In addition to its pump function, Na,K‐ATPase serves as a signaling scaffold and a cell adhesion molecule. Of three b‐subunit isoforms, β1 is found almost in all tissues, while β2 expression is mostly restricted to brain and muscle. In cerebellar granule cells, the b2‐subunit, also known as Adhesion Molecule on Glia (AMOG), has been linked to neuron‐astrocyte adhesion and granule cell migration, suggesting its role in cerebellar development. Nevertheless, little is known about molecular pathways that link the b2‐subunit to its cellular functions. Using cerebellar granule precursor cells, we found that the β2‐subunit, but not the b1‐subunit, negatively regulates expression of a key activator of the Hippo/YAP signaling pathway, Merlin/neurofibromin‐2 (NF2). The knockout of the β2‐subunit in mice or its knockdown in cerebellar granule cell precursors resulted in increased Merlin/NF2 expression and affected down‐stream targets of Hippo signaling, i.e. increased YAP phosphorylation and decreased expression of N‐Ras. Further, the β2‐subunit knockdown altered the kinetics of Epidermal Growth Factor Receptor (EGFR) signaling in a Merlin‐dependent mode and impaired EGF‐induced reorganization of the actin cytoskeleton. Therefore, our studies for the first time provide a functional link between the Na,K‐ATPase β2‐subunit and Merlin/NF2 and suggest a role for the β2‐subunit in regulating cytoskeletal dynamics and Hippo/YAP signaling during neuronal differentiation.

Support or Funding Information

This work was supported by funds from NIGMS‐8P20GM103464, the Nemours Foundation and the DO Believe Foundation. The Biomolecular Core Lab is supported by grants NIGMS P30GM114736 (COBRE) and NIGMS‐P20GM103446 (INBRE).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of flavor chemicals commonly found in E‐vaping liquids on IL‐8 increase and barrier function in human epithelial cells
Authors: Ware Blue , Waters Alexander , Roecklein‐Canfield Jennifer
Published: June 2021

The number of individuals using e‐cigarettes or e‐vaporizers has significantly increased, within the last decade. Many use these products as a way stop smoking cigarettes, as they are advertised as a safe alternative. Recent studies, however, have shown that e‐liquids can contain numerous flavor chemicals at various quantities, since there is currently no government regulation determining how much of these chemicals can be added to e‐liquids. While recent studies have analyzed the contents of e‐liquids, almost nothing is known about the harmful effects of these flavor chemicals on lung cell health. The goal of this study was to evaluate barrier function and the release of pro‐inflammatory cytokines, specifically interleukin‐8 (IL‐8), in human epithelial cells in response to three different e‐vaping flavoring chemicals. The barrier function is highly regulated and acts as an obstacle against harmful gases and vapors. In response to cellular stresses, the function of the barrier is negatively impacted, which increases pollutants access to the submucosal membrane, resulting in an inflammatory response. Inflammation can often lead to irreversible scarring that can damage lung epithelial cells.. This research analyzes how various concentrations of cinnamaldehyde, benzaldehyde and vanillan, which have been found in e‐liquids at high concentrations, affect epithelial cell health. After exposure the relative expression of IL‐8 were measured using qPCR and ELISA. The barrier function was analyzed using Multiplex in order to look at tight junctions (TJs) in relation to epithelial barrier function. These findings will be used to elucidate the harmful effects flavor chemicals, in e‐liquids, have on lung cell 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.
Attenuated Innate Immunity in Mouse Embryonic Stem Cells as a Self‐Protective Mechanism for Early Embryogenesis
Authors: Chen Bohan , Guo Yan‐Lin
Published: June 2021

Embryonic stem cells (ESCs) have been intensively studied as a promising cell source for regenerative medicine. However, their immunological property is an important yet poorly understood issue in the therapeutic application of ESCs and in developmental biology. Innate immune responses are critical defense mechanisms that are well‐developed in somatic cells, however, a series of our recent studies demonstrated that ESCs are deficient in innate immune responses to viral/bacterial infections and inflammatory cytokines. These findings raised an intriguing question about the rationale for ESCs, the progenitors of all tissue cells, to not have an innate immunity that has adapted so well in somatic cells. Immune and inflammatory responses have been viewed as a double‐edged sword. On the one hand, they defend the organism against pathogens; on the other hand, they can cause collateral damage to the tissue cells, such as cell cycle inhibition and even cell death. From this perspective, we hypothesize that the attenuated innate immunity could be a self‐protective mechanism for ESCs to avoid the cytotoxic effect of immune/inflammatory responses. To test this hypothesis, we differentiated ESCs to fibroblast‐like cells (ESC‐FBs), which have gained partial innate immunity. In comparative analyses, we exposed ESCs and ESC‐FBs to several inflammatory conditions, including inflammatory cytokines (TNFα and IFNγ), lipopolysaccharide (LPS), the conditioned medium collected from LPS‐stimulated macrophages, and heat killed bacteria. The effects were assessed by cell viability analysis and the induction of inflammatory genes. In all conditions tested, ESCs were less susceptible to the cytotoxic effects of the inflammatory insults and showed diminished induction of inflammatory genes than ESC‐FBs. At the molecular level, the signaling pathways that mediate the effects of LPS, TNFα and IFNγ are mostly defective in ESCs. We conclude that the attenuated innate immune responses could be an adaptive mechanism for ESCs to avoid the cytotoxicity caused by immune reactions resulting from embryo implantation or pathogen infection at the early stage of embryogenesis.

Support or Funding Information

This work was supported by National Institutes of Health Grants R15GM109299 and R15GM128196 (to Y‐L. G.). We thank Mississippi‐INBRE for the use of the imaging facility, funded by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103476

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dissecting the Role of ArhGAP12 in Natural Killer Cell‐Mediated Cytotoxicity
Authors: Heyblom Raini A. , Overlee Brittany L. , Billadeau Daniel B.
Published: June 2021

The immune system consists of two broad branches: innate and adaptive. In the innate branch of the immune system, one will find natural killer (NK) cells, which are responsible for recognizing and killing virally infected or cancerous cells within the human body. Numerous immunodeficiency syndromes arise from mutations in genes that encode proteins essential to NK cells including those involved in NK cell development and effector functions. Among the most important NK cell effector functions is cell‐mediated cytotoxicity. Our lab has been interested in understanding the molecular machinery that regulates this process, in particular those regulated by the small GTP‐binding proteins of the Rho family. With this in mind, the ArhGAP12 protein, which functions as a GTPase activating protein for members of the Rho family, was tested in NK cells to see if it had an effect on cytotoxicity. Significantly, knockdown of ArhGAP12 in NK cells resulted in diminished NK cell‐mediated cytotoxicity. In addition, we found that ArhGAP12 is tyrosine phosphorylated following stimulation through the NKG2D+2B4 activating receptors, indicating that it is linked to this important signaling pathway. As ArhGAP12 is a multi‐modular signaling protein containing a src‐homology 3 (SH3), WW, pleckstrin homology (PH), and GAP domains, we performed proteomics to identify potential interacting partners using the SH3 domain as bait. We found that ArhGAP12 interacts strongly with multiple regulators of F‐actin dynamics including WAVE2, WASP, Evl and VASP. Taken together, these results suggest that ArhGAP12 is linked to activating receptors on NK cells, associates with F‐actin regulators, and is involved in NK cell‐mediated cytotoxicity.

Support or Funding Information

Mayo Clinic Graduate School of Biomedical Sciences SURF Program and the R01 grant AI120949 to D.D.B.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of resistance‐induced heat on mice skin
Authors: Monte‐Alto‐Costa Andrea , dos Anjos Raquel Anacleto
Published: June 2021

The continuous exposure to solar radiation happens to most living beings, their interaction with humans can be both positive or negative. Much of the research into its effects refers to ultraviolet (UV) and its consequences, such as skin damage and aging, with little emphasis on the effects of heat alone. The aim of this work was to investigate macroscopic, microscopic and biochemical effects of heat‐induced thermal exposure on mouse skin. Mice were divided into control (23° C – room temperature) and exposed (43° C) groups, with heat exposure induced by resistance plate in a thermal chamber for 30 minutes for 14 days on alternate days and at the end skin samples were collected for analysis. A thickness analysis of the skin layers was performed, where we observed an increase in the thickness of the dermis as well as the epidermis in the groups exposed to heat. In addition, there was an increase in the elastic and collagen fibers deposition in the 43° C group when compared to the control group. We performed a protein quantification for type I collagen, and observed a decrease in pre collagen in the 43° C group, while the mature collagen showed a slight increase in the same group, suggesting a possible renewal of this collagen, stimulated by heat. We also investigated the levels of oxidative stress in these conditions, and observed that reduction in lipid peroxidation, nitrite levels and free radicals in groups exposed to high temperature. With all these findings our results suggest that heat, without radiation, can behave as an important factor of skin anti‐aging.

Support or Funding Information

Supported by: FAPERJ and CNPq

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.