bluuebunny/crossref_metadata_2025 huggingface dataset:
Setting Up an In Vitro System for Echinoderm Radial Nerve Cord Explants
Authors: Díaz Eduardo Andres Quesada , Figueroa‐Delgado Paola , Castro‐Ruiz Christian , García‐Arrarás José E.
Published: June 2021

Among animal phyla, some of the least studied nervous systems are those of the phylum Echinodermata. Part of the problem lies in that most of their nervous components are embedded in the body wall that has calcareous skeletal components. We have now developed a dissection technique for successful isolation of the radial nerve cords (RNCs) and an in vitro system where the isolated RNCs can be cultured. This in vitro system is thus amenable to experimental manipulations aimed at characterizing the nervous system of the sea cucumber Holothuria glaberrima and to extend our studies on its regeneration capabilities. In this procedure, the body wall containing the RNCs is surgically excised and digested with collagenase producing RNCs explants that can be kept in culture for up to 2 weeks. We have performed histological studies to determine changes in the RNC anatomy during culture. We have also performed immunohistochemistry using neuronal and collagen markers to identify specific cellular populations. Labeling with a nuclear dye (DAPI) shows the presence of cells throughout the cultured period with a slight increase in nuclear counts in the medial and lateral regions of the RNC neuropile through days 3 to 7 when compared to in vivo RNCs (control). Neuronal markers, RN1 monoclonal antibody and anti‐GFSKLYFamide polyclonal antibody, identified nerve fibers and, neuron‐like cell bodies along the RNC periphery, as has been documented previously in in vivo studies. In addition, anti‐collagen marker IBA2 highlighted the demarcation between the ectoneural and hyponeural subdivisions of RNC explants kept for at least 3 days in culture. Our results showed that the central nervous system components of an echinoderm can be kept alive, maintaining its structure and morphology for various length of time. These RNC explant cultures provide a unique tool to study cellular and molecular basis of processes occurring in nervous systems such as neuronal regeneration and degeneration.

Support or Funding Information

RISE Program: 5R25GM061151‐17

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 Androgen Availability on the Activation of Neuroprotective Pathways Following Traumatic Brain Injury
Authors: De La Cruz Lianne , Duncan Kelli
Published: June 2021

Following traumatic brain injury (TBI), steroid hormones are neuroprotective. Specifically, estrogens decrease apoptotic signaling, neuroinflammation, oxidative stress, and have been shown to normalize glutamate concentrations. Furthermore, androgens also regulate neuroinflammation and mediate gliosis. However, the mechanisms underlying these influences are poorly understood. Cyclooxygenase (COX‐2) and prostaglandin E2 (PGE2) activation appear to be necessary for the injury induced upregulation of estrogen synthesis. This sex specific increase in estrogens promotes neuroprotection by increasing estrogen receptor‐α dependent decreases in neuroinflammation presumably by altering the activity of protein kinase A (PKA) or phosphoinositide‐3‐kinase (PI3K). While there is a considerable amount known about the role of estrogens in this pathway, very little is known about how androgens interact with estrogens to promote neuroprotection. The goal of this study was to further investigate the effect of androgens specifically, but also to identify if there are any interactions between androgens and estrogens on the activation of the COX‐2/PGE2 pathway. Prior to the concussive injury protocol, adult male and female zebra finches (Taeniopygia guttata) were implanted with androgens or an androgen antagonist (flutamide) and collected 1 week later. Activation of the COX‐2/PGE2 pathway was measured using analysis of quantitative polymerase chain reaction and immunohistochemistry. Preliminary data suggest that androgens are involved in estrogen mediated neuroprotection following injury and suggest a novel role for androgens following TBI.

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 DNA sequence, endosomal pH, and lipid rafts/caveolae on uptake of human cathelicidin LL‐37/dsDNA complexes by monocytic cells
Authors: Rhodenizer Emma , Cho Katriel , Li Kelly , Craig Maria
Published: June 2021

LL‐37, the only human homolog of the class of cathelicidin cationic peptides, has many beneficial roles in the immune system as an antimicrobial and pathogen‐sensing molecule. However, altered levels of LL‐37 have been linked to a growing number of diseases, including autoimmune diseases, cancer, cystic fibrosis, and asthma. LL‐37 forms complexes with bacterial and self‐DNA that are internalized by many immune cell types, and formation of these complexes both protects DNA from degradation by nucleases and increases internalization of LL‐37. LL‐37 and self‐DNA uptake appears be related to autoimmune disease because it is a pathway by which the immune system may recognize and respond to self‐DNA. LL‐37 can bind to DNA and RNA and bring them into endosomes, activating innate immune receptors known as toll‐like receptors 3 and 9 (TLR3 and TLR9). In order to examine this mechanism, we have used fluorescence microscopy to observe the uptake of 5‐FAM‐LC‐LL‐37 (10 μM) into THP‐1 monocytic cells in the absence and presence of 24‐mer dsDNA oligonucleotides (0.6 μM). We investigate sequence effects and observe that complexes formed with CpG and poly A enhance LL‐37 uptake, but that GpC and poly G are less effective. We have also examined the effect of endosome acidification on uptake and intracellular association of LL‐37 and dsDNA by performing uptake in the presence or absence of inhibitors of endosome acidification. To investigate the possible role of cholesterol‐rich lipid rafts and caveolae in uptake, we pre‐treated the cells with the cholesterol‐depleting agents methyl‐β‐cyclodextrin (MCD) and nystatin known to disrupt raft/caveolar structures. The mechanism for LL‐37/DNA uptake is an interesting area of study that may reveal ways to facilitate LL‐37‐mediated immune responses and help to create more specialized medicines.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
LPS decreases the expression of Monocarboxylate transporter‐1 (MCT1) in mouse monocytes
Authors: Ochrietor Judith D , Al‐Khatab Ryan
Published: June 2021

The molecule Toll‐like Receptor‐4 (TLR4) recognizes Gram negative bacteria, specifically the lipopolysaccharide component (LPS) and initiates a signal transduction cascade leading to an inflammatory response. Recent studies by this laboratory suggest the cell adhesion glycoprotein Basigin (CD147) interacts with TLR4 via their transmembrane domains. The role of Basigin in mouse retina has been well‐studied and includes an association with Monocarboxylate Transporter 1 (MCT1) for metabolic support of photoreceptor neurons. The role of Basigin in innate immune responses is less‐well studied. Therefore, the response of Basigin to LPS was assessed and compared to that of TLR4. The response of MCT1 to LPS was also assessed. It was hypothesized that Basigin and MCT1 expression would increase in response to LPS. To test this, RAW 264.7 mouse monocytes were incubated in the presence of 1 mg/mL LPS in serum‐free medium for various times ranging from 2–24 hours. Control cells received PBS. The cells were collected, and RNA was isolated for gene expression analyses via quantitative‐reverse transcription PCR (q‐RT‐PCR). The expression of TLR4 decreased at 2 hours and increased at 24 hours of LPS induction, when compared to control samples, although no changes were determined to be statistically significant. The expression of Basigin was similar between control and LPS‐treated cells at all time points, except for 24 hours of induction, when Basigin expression was significantly lower in LPS‐treated cells. MCT1 expression was also similar between control and LPS‐treated cells at all time points, except for 24 hours of induction, when MCT1 expression was significantly lower in LPS‐treated cells. These findings do not support the hypothesis. The data for TLR4 expression in response to LPS in mouse monocytes are consistent with published reports. Although it was hypothesized that metabolic responses would increase in monocytes to support immune mechanisms, the data for MCT1 suggests that metabolism is reduced in activated monocytes, perhaps as a shift of resources from general cellular maintenance to induction of an innate immune response. The observed decrease in Basigin expression also did not support the hypothesis and suggests that the molecule is either regulated directly by the innate immune response or indirectly via its role in metabolism through MCT1. Future studies will assess the expression of Basigin and MCT1 at the protein level to better analyze the role of metabolism in the innate immune response.

Support or Funding Information

UNF Office of Academic Affairs

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Homogenized mouse neural retina extracts stimulate an immune response in the mouse monocyte RAW 264.7 cell line
Authors: Ray Ashton Nicole , Ochrietor Judith D
Published: June 2021

There are two main protein products of the Basigin gene. One protein, known as Basiginvariant‐1 is expressed in the neural retina, specifically by the photoreceptor neurons. The other protein, known as Basigin‐variant‐2 is expressed throughout the body, including monocytes, as well as Müller glial cells and the retinal pigmented epithelium of the eye. Studies by this laboratory using recombinant proteins indicate that the two Basigin gene products interact via their extracellular domains and that the region of the Ig0 domain of Basigin‐variant‐1 used to interact with Basigin‐variant‐2 stimulates a proinflammatory response in monocytes. The purpose of the present study was to determine the biological relevance of this proinflammatory response using mouse neural retina samples rather than recombinantly‐generated proteins. It was hypothesized that mouse neural retina extracts would induce an immune response in mouse monocytes. Mouse neural retinas were obtained using an approved protocol and homogenized in phosphate buffered saline (PBS). The mouse neural retina extracts were then incubated with mouse monocytic RAW 264.7 cells. After 24 hours, the cell culture medium was collected and assayed for the expression of the pro‐inflammatory cytokine interleukin‐6 (IL‐6) via an ELISA and the cells were collected for RNA isolation and subsequent quantitative reverse transcription PCR (q‐RT‐PCR). Cells treated with a similar volume of PBS served as the controls. It was determined that incubation of the neural retina extracts with the RAW 264.7 cells elicits a proinflammatory response in the monocytes and that the expression of the innate immune molecule TLR4 significantly increases in expression in those cells, when compared to the response observed with PBS treatment. The data suggest a biological relevance for the observation that recombinant Basigin‐variant‐1 interacts with Basigin‐variant‐2 to stimulate a proinflammatory response in monocytes. This study is important for a better understanding of immune response aspects of diabetic retinopathy and other diseases in which the retinal pigmented epithelium is compromised and can allow the infiltration of monocytes into the neural retina.

Support or Funding Information

UNF Office of Academic Affairs

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Synthesis and Evaluation of a Photoswitchable COX‐2 Inhibitor
Authors: Denison Madeline , Streu Craig Nolan
Published: June 2021

The research being presented herein is focused on making a drug that competitively and selectively inhibits cyclooxygenase‐2 (COX‐2) in response to a given wavelength of light, thereby reducing side effects. Cyclooxygenase‐2 is an enzyme primarily responsible for synthesizing prostaglandin, which generates inflammation in response to an infection or injury. Overproduction of prostaglandin can lead to painful inflammation like rheumatoid arthritis and osteoarthritis. Therefore, a COX‐2 inhibitor can solve this issue of inflammation by inhibiting the synthesis of prostaglandin.

Before the advent of selective COX‐2 inhibitors, drugs targeting prostaglandin production were not as selective and also inhibited Cyclooxygenase‐1 (COX‐1), which could lead to unwanted side effects, like ulcers and gastrointestinal toxicity. To address this problem, “coxib” drugs were created to selectively inhibit COX‐2. While these drugs worked well in reducing inflammation, long‐term use was found to increase the risk of adverse vascular side effects. These side effects are thought to arise, in part, as a result of the role of COX‐2 in the maintenance of the vascular system. The research being presented aims to eliminate this side effect by making a drug that can target COX‐2 at the site of inflammation, without affecting the vascular system.

The drug being synthesized is an azologue of a validated COX‐2 selective inhibitor. Azologues are modified to contain a nitrogen‐nitrogen double bond between two aromatic rings. Molecules containing this moiety can change conformation trans to cis or cis to trans in response to near UV or visible light, respectively. It is this change in conformation that creates a difference in target binding in the presence or absence of the appropriate wavelength of light. The target compound was designed to exist in the trans conformation in the absence of light. In silico binding analysis suggests that this is the active conformation of the inhibitor. The synthesis of the target molecule will be disclosed and its photophysical characterization by UV/Vis spectrophotometry will be outlined.

Support or Funding Information

Albion College Foundation for Undergraduate Research, Scholarship and Creativity Activity

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Quantitative Survey of NAD+ Flux in Aged Mice
Authors: McReynolds Melanie R. , Chellappa Karthikeyani , Chu Qingwei , Baur Joseph A. , Rabinowitz Joshua
Published: June 2021

Decline of NAD+ is associated with aging and disease; hence this critical metabolite is proposed as a master regulator of age‐dependent pathology. NAD+ is found in all living cells and is an essential coenzyme that broadly impacts metabolism and protein covalent modification. NAD+ depletion contributes to mitochondrial dysfunction, which is a classic hallmark of aging. Redox state and the rates of synthesis and degradation of the NAD+ chemical backbone govern the cellular concentration of NAD+. NAD+ decline during aging reflects depletion of the total NAD(H) pool, rather a shift in the NADH/NAD+ ratio, suggesting defective NAD+ synthesis and over‐consumption of NAD+ by cleaving enzymes. Concentration measurements are ill‐suited to distinguish these possibilities. Thus, there is a critical need to examine NAD+ metabolic flux, i.e. the rates of production and consumption of the NAD+ chemical backbone. To this end, we used isotope‐labeled NAD+ precursors, mass spectrometry, and quantitative modeling to determine NAD+ fluxes in young and aged mice. Tissue‐specific measurements of NAD+ labeling enabled organ‐specific assessment of NAD+ fluxes. These experiments identified accelerated NAD+ production and consumption in aging, and thus rational targets for intervention. Understanding of NAD+ fluxes will lay the foundation for understanding how NAD+ homeostasis is achieved: What are the primary control mechanisms that determine proper NAD+ concentration and fluxes? How do these go wrong in aging and disease?

Support or Funding Information

HHMI Hanna H. Gray Fellows Program Burroughs Wellcome Fund Postdoctoral Enrichment Program

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Novel CBWD Gene Products Augment Mammalian Zinc Homeostasis
Authors: Ogo Ogo Agbor , Obochi Godwin Oche , Valentine Ruth , Ford Dianne
Published: June 2021

The CBWD genes (coding for COBW domain‐containing proteins) was previously identified through an in silico search of the human genome for the presence of a zinc‐responsive element known as Zinc Transcriptional Regulatory Element (ZTRE)1. The human CBWD gene family consist of six isoforms with different chromosomal and cytogenetic locations and exhibit a remarkable nucleotide and amino acid sequence conservation. The prokaryotic homologues of CBWD proteins belonging to the GOG0523 family of proteins have been reported to have roles in zinc homeostasis2, however, no known role has yet been documented for the mammalian types. The discovery that the zinc‐sensitive element (ZTRE) occurred in multiple copies of the CBWD genes was intriguing and triggered further investigations into a likely role for this gene family in mammalian cellular zinc handling and/or trafficking. Moreover, we reported a protein that acts at the ZTRE site to mediate regulation of zinc homeostatic genes and found CBWD genes to be a prominent feature3. Using Caco‐2 cells as in vitro model of human enterocytes, we demonstrate the ability of CBWD genes to respond to changes in zinc content at both transcriptional and post‐transcriptional levels evidenced by decreased transcript and overexpressed protein abundance in response to increased extracellular zinc concentration. We also show that increased expression of CBWD protein through a transgene extended cellular tolerance to extreme windows of zinc availability, demonstrating the potential of the human CBWD gene products to augment cellular zinc buffering. This potential buffering ability of CBWD gene products may be effected through cellular zinc redistribution evidenced by a less punctate zinc distribution in cells made to overexpress CBWD protein compared to control cells. Furthermore, overexpression of COBW protein attenuated the responsiveness of a zinc‐sensitive metallothionein promoter, indicating an action consistent with a role in zinc trafficking or buffering. These findings give plausible evidence in support of the involvement of the mammalian CBWD gene products in cellular zinc homeostasis.

Support or Funding Information

This work was supported by the Nigerian Tertiary Education Trust Fund through Benue State University, Makurdi, Benue State Nigeria

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anti‐apoptotic effect of Korean ginseng extract on aging‐induced ovary via inhibition of p63 signaling pathway in mouse
Authors: Song Ji‐Hyeon , Chei Sungwoo , Seo Young‐Jin , Lee Kippeum , Jin Heegu , Oh Hyun‐Ji , Oh Jae‐Hoon , Jang Hoon , Choi Youngsok , Lee Boo‐Yong
Published: June 2021

Background

The problem of women's subfertility has been steadily increasing worldwide, and many studies are under way to treat it. The ovary is a key element in the problem of subfertility, and it is very important to maintain integrity. Recently, the pregnancy age of women is increasing, and so the function of the ovary is deteriorated with aging. Korean ginseng has been widely used as a herb medicine and it is known to contain many beneficial biological properties. We investigated the effect of Korean ginseng extract (KGE) on ovary function of female.

Methods

The ovaries were harvested from 6‐month‐old female mice and cultured in the KGE‐treated media for 24 hours. After that, RNA was extracted from the control and KGE‐treated groups and microarray was performed. In addition, based on the microarray data, the gene expression was verified by qPCR.

Results

Aging dramatically increased expression of apoptosis regulatory factors in ovary, but expression of these factors was significantly reduced in the KGE‐treated groups. Expression levels of apoptosis regulatory factors such as p63, NOXA, BCL2 and BAX were increased in the ovary with aging. KGEs reduced the expression level of these factors. Consequently, KGEs helps to maintain ovary function by suppressing apoptotic damage caused by aging.

Conclusions

We suggest that KGEs protect against aging‐induced ovary damage by inhibiting apoptosis signaling pathway.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cardiometabolic Profile after Pediatric Cancer Treatment: Insight into HDL Composition and Nutritional Intake
Authors: Belanger Veronique , Diallo‐Blais Simon Saliou , Robert Carol‐Ann , St‐Martin Juliette Sauve , Beaulieu‐Gagnon Sabrina , Drouin Simon , Bertout Laurence , Bouchard Isabelle , Laverdiere Caroline , Sinnett Daniel , Marcil Valérie
Published: June 2021

Few studies have investigated the impact of pediatric cancer treatment on cardiometabolic health, although chemotherapy in children can cause rapid weight gain, transient hypertension, elevated blood glucose and hyperlipidemia. Besides, we have previously shown that young survivors of childhood leukemia are at increased risk of having the metabolic syndrome, dyslipidemia, hypertension and altered high‐density lipoprotein (HDL) composition. This study aims to examine the cardiometabolic profile of children after cancer treatment in relation to their diet, as well as HDL2 and HDL3 composition.

Methods

Patients were recruited at Sainte‐Justine University Health Center in Montreal as part of the VIE Program (Valorization, Implication, Education). Fasting blood samples were collected and we assessed participants' anthropometric, metabolic, and biochemical profiles. Three day food records were used to evaluate dietary intake. HDL2 and HDL3 fractions were isolated from plasma by ultracentrifugation, and total protein and lipids were measured by colorimetric reactions.

Results

To date, 63 patients (44.4% boys) were recruited. Mean age was 11.7 ± 5.6 years and 46.0% were treated for acute lymphoblastic leukemia. The time elapsed since the last treatment was 17.0 ± 9.7 months. We found that 25.4% of participants (n=16/63) had hypertension, 22.2% (n=14/63) were obese and 8.9% (n=5/56) were insulin resistant. One third of patients had dyslipidemia (n=21/63), a risk that was increased by age (OR: 1.31, 95%CI: 1.16–1.50, P<0.01). Patients over 10 years old had insufficient intakes of several nutrients (vitamins A and E, folate, magnesium, zinc). Compared to healthy children, the proportion of esterified cholesterol in HDL2 was lower (18.6 ± 1.2% vs. 11.9 ± 1.4%, P<0.01), but protein content was higher (41.1 ± 1.6% vs. 48.8 ± 1.0, P<0.01). The proportion of esterified cholesterol was also lower in HDL3 fractions of patients treated for cancer (12.6 ± 1.3% vs. 10.1 ± 0.7%, P<0.01).

Conclusion

These results indicate cardiometabolic disturbances and anomalies in HDL composition in children shortly after cancer treatment. They emphasize the need for nutritional interventions in this high‐risk population.

Support or Funding Information

This work is funded by The Foundation Centre de cancérologie Charles‐Bruneau and The Joy of Eating Better Foundation and the CHU Sainte‐Justine Foundation.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lipid rafts transduce force to TREK‐1 channels via phospholipase D
Authors: Petersen E. Nicholas , Gudheti Manasa , Pavel Mahmud Arif , Murphy Keith R , Ja William , Jorgensen Erik , Hansen Scott
Published: June 2021

The transduction of force into a biological signal is critical to all living organisms. Recently, disruption of ordered lipids has emerged as an ‘atypical’ force sensor in biological membranes; however, disruption has yet to link with canonical channel mechanosensation. We show here that force‐induced disruption and lipid mixing activates TREK‐1. This activation is dependent on PLD2 which transduces the force into the chemical signal phosphatidic acid that is then sensed by TREK‐1 with a latency of <3 ms. TREK‐1 then produces a mechanically induced change in membrane potential. These experiments establish the ordered lipid as the force sensor, PLD2 as the primary chemical transducer, and the ‘mechanosensitive’ ion channel TREK‐1 as a downstream effector of mechanical transduction. Confirming a central role for PA singling in force transduction, genetic deletion of PLD2 decreases mechanosensitivity and pain thresholds in D. melanogaster.

Support or Funding Information

NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE

NATIONAL INSTITUTE OF AGEING

HOWARD HUGHES MEDICAL INSTITUTE

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
2‐Amino Muramyl Dipeptide Derivatives: Chemical probes to assay the stability and activation of NOD2
Authors: Brown Ashley R , Ukaegbu Ophelia , Grimes Catherine Leimkuhler
Published: June 2021

Mutations of the cytosolic protein nucleotide‐binding oligomerization domain‐containing protein 2 (NOD2), which detects the synthetic bacterial cell wall fragment muramyl dipeptide (MDP), are implicated in making individuals more susceptible to Crohn's disease (CD). However, the mode by which these mutations affect the pathogenesis of this disease is not fully understood. Interestingly, studies suggest NOD2 positively and negatively regulates the expression of toll‐like receptors (TLRs) and the consequent production of inflammatory molecules upon activation. Like NOD2, TLRs can detect bacterial fragments and produce an inflammatory response via the NF‐kB pathway, though through distinct mechanisms. Stabilizing the NOD2 Crohn's mutants through protein‐ligand binding can be a potential therapeutic target for CD. A library of MDP derivatives with altering functionality at the 2‐amino position were synthesized in order to investigate the stabilization imparted to NOD2 upon binding. A simplified 7‐step synthetic route to prepare 2‐amino MDP and subsequent functionalization of the molecule with mild NHS coupling or acetylating conditions is utilized. Compounds are then tested for stabilization with the cellular thermal shift assay (CESTA). In this cell‐based assay, binding of a ligand to a protein stabilizes a protein and protects it against thermal degradation at increased temperatures, resulting in a thermal shift (ΔTm) when compared to the unbound protein. The activation of the NF‐kB pathway by these molecules is tested concurrently, using a luciferase reporter assay to determine if changes in functionalization also yield a modulated immune response.

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 Mammalian UDP‐Galactose 4′‐Epimerase (GalE) Is Required for Cell Surface Glycome Structure and Function
Authors: Florwick Alyssa T , Broussard Alex , Desbiens Chelsea , Nischan Nicole , Robertson Corrina , Guan Ziqiang , Kohler Jennifer , Wells Lance , Boyce Michael
Published: June 2021

Nucleotide‐sugars are required for glycosylation reactions in mammals, but it is largely unknown how their levels are dynamically regulated in response to cellular signals. The UDP‐galactose 4’‐epimerase (GalE) has recently emerged as a potential regulator of nucleotide‐sugar flux in response to stimuli such as feeding, ischemia and endoplasmic reticulum (ER) stress. The importance of this enzyme is underscored by human patients with hypomorphic GalE alleles, constituting a disease termed type III galactosemia. Critically, the molecular underpinnings of galactosemic pathophysiology are unclear.

Because it controls the levels of multiple substrates in several signaling contexts, GalE is an ideal candidate for studying the importance of nucleotide‐sugar regulation in both normal and disease physiology. In this work, we generated GalE−/− human cell lines using CRISPR‐Cas9 mutagenesis. We characterized the effects of GalE loss on nucleotide‐sugar metabolism, protein glycosylation, and death receptor function. Our results demonstrate that loss of the GalE enzyme effects dramatic dysfunction in nucleotide‐sugar metabolism along with protein and lipid glycosylation. We have observed mis‐glycosylation of various cell‐surface proteins. GalE−/− human cells exhibit dysregulated cellular signaling, which we hypothesize is due to mis‐glycosylation of cell‐surface receptors.

Support or Funding Information

Research in the Boyce Lab has been supported by NIH grants R01GM118847 and R01GM117473 and the Rita Allen Foundation Scholars program

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Targeting the O‐GlcNAc Transferase to Specific Proteins Using RNA Aptamers
Authors: Zhu Yi , Hart Gerald
Published: June 2021

O‐linked β‐D‐N‐acetylglucosamine (O‐GlcNAc) is an abundant post‐translational modification (PTM) of thousands of proteins that regulates signaling, transcription and cytoskeletal functions. A major roadblock toward elucidating O‐GlcNAc's functions is our inability to alter the levels of O‐GlcNAcylation on a single protein at a time. Herein, we describe a novel method to alter the O‐GlcNAcylation of a specific protein. We designed series of modulated RNA aptamers that are able to induce the proximity between β‐catenin and O‐GlcNAc transferase (OGT). These aptamers are able to increase O‐GlcNAcylation of β‐catenin by ~6‐fold in HEK293T cells without affecting the O‐GlcNAcylation of other proteins. Increased O‐GlcNAcylation of β‐catenin concomitantly altered the phosphorylation state at the degradation‐related sites (Ser33, Ser37, Thr41 and Ser45), indicating a cross‐talk between O‐GlcNAcylation and phosphorylation in regulating the stability and transcriptional activity of β‐catenin. Detailed analyses of a series of similar modulated aptamers indicate that the distance and spatial alignment between OGT and β‐catenin in the induced proximity are critical considerations when designing the modulated aptamers. This modulated aptamer design is also able to increase O‐GlcNAcylation on some other cellular proteins. Taken together, our research provides a novel tool to study how O‐GlcNAcylation specifically regulates individual proteins, establishes a novel method to induce proximity between cellular components, as well as providing insights on how O‐GlcNAcylation regulates the function of β‐catenin.

Support or Funding Information

Supported by NIH P01HL107153, & R01GM116891, the Georgia Research Alliance and the Univ. of Georgia. Dr. Hart receives a share of royalty received by the university on sales of the CTD 110.6 antibody, which are managed by JHU.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MicroRNA Sequencing of Discoid Lupus Erythematosus Skin
Authors: Calderon Irene , Mina Rina
Published: June 2021

Background

Discoid lupus erythematous (DLE) is an autoimmune skin condition that causes profound disfigurement. Environmental effects are linked to increased risk and worsening disease off activity in DLE. MicroRNA (miRNA) are increasingly found to have important roles in the environmental modulation of inflammation and cellular differentiation in autoimmune diseases.

Objective

The goal of the pilot study was to comprehensively identify the miRNA transcriptome in DLE.

Methods

The specimens were from African American women (DLE=4, normal=4) and were age‐matched [median age=55 years (range=50–61), median DLE duration=18 years]. Subjects with DLE did not have concomitant systemic lupus and were not on systemic immunosuppression during skin biopsy. Skin specimens were composed of keratinocytes, epidermal appendages, vasculature, dermal fibroblasts and skin homing immune cell populations. We extracted total RNA using a method that preserved small RNAs (Ambion). RNA was prepared for sequencing with the Small RNA Sample Prep Kit,□□ □□ adapters were ligated to approximately 1 microgram of total RNA. Each library was loaded on a single Illumina lane at 20 picogram and subjected to 86 cycles of sequencing on HiSeq. We generated 52.4 million raw and 15.3 million qualified reads. We mapped qualified reads to miRNA precursors, functional non‐coding RNAs and build 37 (hg19) of the human genome. The adapter‐trimmed, qualified reads were distributed around an average length of 21 nucleotides. The number of reads and mapping proportions in the DLE group approximate that in the normal skin groups. We detected mature miRNAs derived from 1240 known miRNA precursor families. These were represented by at least one read in the cumulative data set. We analyzed the data from our next generation sequencing based on relative comparisons of normalized read counts between DLE and normal skin to detect relative changes. Standardized fold changes of miRNAs were calculated as mean differences in read counts divided by pooled standard deviations in the log scale.

Results

We identified 18 known miRNAs that were significantly over or under expressed in active DLE lesions versus normal skin (p‐values < 0.05). Among these are mir‐146a, mir‐223, mir‐21, mir‐23b. These miRNAs are linked to IFN‐regulation, hypomethylation, ultraviolet exposure, differentiation of the keratinocytes, epidermal dendritic cells, and T cells, and estrogen response.

Conclusion

From this discovery‐based study, we have identified several miRNAs that may play an important role in epigenetic regulation in DLE that ultimately affects disease severity.

Support or Funding Information

CTSA of University of Cincinnati

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Src and cortactin are involved in pemphigus skin blistering
Authors: Kugelmann Daniela , Rötzer Vera , Walter Elias , Egu Desalegn Tadesse , Vielmuth Franziska , Vargas‐Robles Hilda , Schnoor Michael , Hertl Michael , Rottner Klemens , Spindler Volker , Waschke Jens
Published: June 2021

Autoantibodies against desmoglein (Dsg) 1 and Dsg3 primarily cause blister formation in the autoimmune disease pemphigus vulgaris (PV). Src was proposed to contribute to loss of keratinocyte cohesion. However, the role and underlying mechanisms are unclear. In keratinocytes, cell cohesion in response to autoantibodies was reduced in a Src‐dependent manner by two patient‐derived PV‐IgG fractions as well as by AK23, but not by a third PV‐IgG fraction, although Src was activated by all autoantibodies. Loss of cell cohesion was progredient and AK23 similar to PV‐IgG interfered with reconstitution of cell cohesion after Ca2+‐switch, indicating that the autoantibodies also interfered with desmosome assembly. Dsg3 co‐localized along cell contacts and interacted with the Src substrate cortactin. Concomitantly, cell adhesion was impaired in keratinocytes isolated from cortactin‐deficient mice in comparison to keratinocytes isolated from wildtype (wt). AK23‐induced loss of cell cohesion was Src‐dependent only when cortactin was expressed. Similarly, AK23 impaired reconstitution of desmosomal adhesion in Src‐dependent fashion only in the presence of cortactin and AK23‐induced skin blistering was abolished by Src inhibition in wt but not cortactin‐deficient mice. However, in human epidermis, PV‐IgG‐induced skin blistering and ultrastructural alterations of desmosomes were not affected by Src inhibition. Our data suggest that Src and cortactin are involved in pemphigus skin blistering but the contribution of Src is variable.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Novel Junctional and Nuclear Roles of the Mitochondrial Protein Mitofusin 2 During Inflammation
Authors: Kim Young‐Mee , Krantz Sarah , Toth Peter T. , Rehman Jalees
Published: June 2021

Background

Severe inflammation can cause widespread damage to the endothelium and result in fatal illnesses such as acute lung injury (ALI) or acute respiratory distress syndrome (ARDS). Vascular endothelial barrier dysfunction is the major vascular complication in ALI. One of the key mechanisms of barrier disruption is the disassembly of adherens junction (AJ) complexes consisting of VE‐cadherin as well as accessory proteins such as β‐catenin and p120‐catenin. We observed that Mfn2, which has previously been primarily studied as a mitochondrial protein, is also located at the endothelial plasma membrane and co‐localizes with VE‐cadherin as well as translocates into nucleus under inflammatory condition, suggesting novel roles in AJs.

Methods and Results

By using three‐dimensional structured illumination microscopy (3D‐SIM) which provides high spatial resolution (□130 nm), we studied Mfn2 localization in human lung microvascular ECs (HLMVECs). Mfn2 predominantly co‐localizes with the mitochondrial membrane protein Tom20 but it also co‐localizes at the plasma membrane with VE‐cadherin in resting ECs. However, upon pro‐inflammatory stimulation with TNF‐α, Mfn2 is dissociated from AJs and no longer co‐localizes with VE‐cadherin. Mechanistically, Mfn2 directly interacts with VE‐cadherin and β‐catenin in resting ECs as assessed by immunoprecipitation assay and a proximity ligation assay (PLA). To determine whether Mfn2 affects endothelial barrier integrity and function, Mfn2 was depleted in HLMVECs with a doxycycline inducible lentiviral shMfn2 RNA. Mfn2 silencing disrupted AJ junctions as visualized by VE‐cadherin and β‐catenin immunostaining. Mfn2 depletion significantly increased transendothelial permeability of ECs as assessed by a FITC‐conjugated albumin tracer assay and transendothelial resistance (TER). We also identified novel binding partners of Mfn2 using the high affinity tandem mass spectrometry (LC‐MS/MS). The analysis of gene ontology indicates that Mfn2 interacts with cell‐cell interaction proteins including cadherin binding and nuclear factor binding proteins with >1.5‐fold increase. Finally, the complex of Mfn2 and β‐catenin translocates into the nucleus upon TNF‐α stimulation.

Conclusion

These data suggest that Mfn2 may directly regulate AJ stability in ECs by interacting with the AJ complex proteins VE‐cadherin and β‐catenin. Mfn2 may act as a co‐regulator with β‐catenin, a key transcription factor in inflammatory condition. These studies enhance our understanding of how the “mitochondrial” protein Mfn2 regulates inflammatory activation via novel “non‐mitochondrial” functions.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Aerobic glycolysis is essential to remodel the epigenetic landscape and initiate transcription during T cell activation
Authors: Coffer Paul , Russo Laura , Vastert Bas , Mokry Michal , Burgering Boudewijn , Mocholi Enric
Published: June 2021

Recent work has started to provide evidence that cellular metabolism is critical in the regulation of immune cell differentiation and function, thereby influencing the final outcome of both the adaptive and innate immune response. Upon engagement of the T Cell Receptor (TCR) after antigen recognition, T cells rapidly upregulate aerobic glycolysis, a process often termed the Warburg effect. It has become clear that this switch to aerobic glycolysis is essential for T cell proliferation, differentiation, and function. However, glycolysis is extremely inefficient for ATP production, suggesting that likely has alternative roles during T cells activation.

Using a combination of RNAseq, ChIP‐seq, and metabolomics, our results show for the first time that after TCR engagement, pyruvate produced by induction of glycolysis is essential to remodel the human CD4+ T cell epigenome. Pyruvate is translocated to the nucleus where it is converted to acetyl‐CoA and can act as a co‐factor for histone acetyltransferases (HATs) resulting in chromatin remodeling and subsequent expression of T cell activation markers. Preventing TCR‐induced pyruvate generation severely impacts T cell activation and transcriptional reprogramming. We further show that inflammatory microenvironment can reprogram CD4+ T cell metabolism and through epigenetic reprogramming can thereby trigger aberrant, pathogenic gene expression.

Taken together our findings demonstrate a direct link between cellular metabolism, chromatin remodeling, and T cell function/fate. Understanding the influence of metabolic alterations in inflammatory environments and there impact on chromatin remodeling may result in the design of novel interventions to specifically target these pathways and alter T cell function.

Support or Funding Information

This work was supported by a grant from the Dutch Arthritis Society (ReumaNederland)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Can enhancing the expression and activity of the Cystic Fibrosis Transmembrane Regulator improve efficacy of gene therapy for Cystic Fibrosis?
Authors: Woodall Maximillian , Tarran Robert , Baines Deborah
Published: June 2021

Cystic Fibrosis, an autosomal recessive genetic disease, is the most common cause of respiratory disease in children of the Caucasian population. Due to the monogenetic nature of the disease and the accessibility of the airways to nebulizers, it is a desirable candidate for gene therapy/editing. It has been suggested that ~10% of endogenous cystic fibrosis transmembrane regulator (CFTR) expression within target cells in vivo is sufficient for prevention of lung disease. With gene editing technology gaining traction it is important for CF researchers to understand how much functional CFTR is required to prevent the development of lung disease and how to make this more achievable.

To answer this question we used two models: cultures of mixed CF:non‐CF primary epithelial cells (a model for gene correction) and cultures of CF cells transduced with CFTR under expression of a high activity promoter (a model for gene therapy). Cultures were grown on semipermeable membranes and differentiated at airway liquid interface. Ussing chamber, Airway surface liquid height and immunohistochemistry were performed as functional readouts. It was observed that essential CFTR functions increased in a linear fashion in CF:WT primary epithelial mixes (CFTRinhIsc μA/cm2: R2=0.91, n=28) whilst only ~16% transduction of a CF culture with CFTR fully restored ion channel activity and airway surface liquid height (n=6).

Our data shows that overexpression of CFTR is a more effective method for restoring function in a CF culture. Therefore, we have since developed an enhanced CFTR gene with ~7 fold greater processing in to protein and dramatically increased anion transport activity as shown by a YFP quenching assay (n=3). It is our hope that this enhanced gene could be used as a tool for a more effective gene therapy.

Support or Funding Information

Cystic Fibrosis Trust UK

St. George's University London

University of North Carolina, Chapel Hill

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Rb S249 as an Emerging Biomarker in the Prognosis of Prostate Cancer
Authors: Camacho‐Hernández Elaine , Feliciano‐Rivera Maylee , Rodriguez‐Lopez Xavier , Perez‐Morales Jaileene , Chardon‐Colon Camille , Caban‐Rivera Jennifer , Santiago‐Cardona Pedro
Published: June 2021

Biomarkers have become a potential tool in the diagnosis of cancer patients, allowing a more accurate diagnosis and treatment. Prostate cancer is the second most common type of cancer in men worldwide. In prostate cancer, it is important to identify which patients would benefit from early treatment. Here we report the combination of phosphorylation of the retinoblastoma protein (Rb) on serine 249 (Rb S249) wit with p39 expression as biomarkers for predicting prostate cancer. We performed an immunohistochemical analysis of Rb S249 and p39 expression. Our results indicate that there is an inverse relationship between Rb S249 and prostate adenocarcinoma. Hence, Rb S249 could help in determining a more accurate prognosis for prostate cancer patients.

Support or Funding Information

Funded by NIH‐NIGMS #2R25GM096955

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the Expression of Wnt5a Isoforms in Urothelial Carcinoma
Authors: Mehlman Amy N , Malgor Ramiro , Coschigano Karen T
Published: June 2021

It is projected that there will be over 81,000 new bladder cancer diagnoses in 2018 in the United States alone. In fact, 1 in 27 men will develop bladder cancer in their lifetime. Despite its prevalence, little is known about this cancer's molecular mechanism. Research performed both at Ohio University and by other scientists has linked a protein, Wnt5a, to urothelial carcinoma (UC), which is the most common form of bladder cancer. This protein has been shown to be involved in an array of diseases, including several types of cancer. Recent research has suggested that two Wnt5a isoforms, Wnt5a‐Short (S) and Wnt5a‐Long (L), may play differing roles in a variety of cancers, with Wnt5a‐S promoting cellular proliferation and Wnt5a‐L suppressing cellular proliferation. Based on this and the research linking Wnt5a to UC, we want to investigate the roles of the Wnt5a isoforms in UC cell lines and human UC biopsies. We extracted RNA from human UC cell lines derived from low and high grade tumors to quantify the amount of each Wnt5a isoform and total Wnt5a expression using real‐time reverse transcription / polymerase chain reaction (RT‐RT/PCR). Trends in expression for Wnt5a‐L and Wnt5a‐Total were extremely similar, suggesting that Wnt5a‐L is the predominantly expressed isoform in urothelial cells. Wnt5a‐L RNA expression was highest in a cell line derived from a high grade tumor but also lowest in a cell line derived from a second high grade tumor. In contrast, expression of Wnt5a‐S RNA was highest in cell lines derived from high grade tumors in comparison to low grade tumors. Linking this to previous research published by our lab, the pattern of Wnt5a‐S expression was similar to that of vimentin, a marker of epithelial mesenchymal transition (EMT) linked to metastasis. We are currently performing immunohistochemistry (IHC) and laser microdissection (LMD) to visualize the location of Wnt5a isoform expression in human UC biopsies from both low grade and high grade tumors. Our hypothesis is that high grade tumors will express higher levels of the Wnt5a‐S isoform than low grade tumors; in contrast, high grade tumors will express lower levels of Wnt5a‐L than low grade tumors. An affirmative outcome of our hypothesis would suggest that the Wnt5a‐S isoform promotes oncogenic properties in UC, while the Wnt5a‐L isoform promotes tumor suppressive properties. Future experiments will be aimed at determining the molecular pathway(s) through which each isoform acts in order to advance diagnostic, prognostic, and therapeutic approaches to UC.

Support or Funding Information

Funding and support were provided in part by Ohio University Heritage College of Osteopathic Medicine and its Research and Scholarly Advancement Fellowship (RSAF), the Diabetes Institute at Ohio University, as well as by an Ohio University Student Enhancement Award (SEA).

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