bluuebunny/crossref_metadata_2025 huggingface dataset:
Proteomic and Phosphoproteomic Signatures of Severe Alcoholic Hepatitis
Authors: Hardesty Josiah , Warner Jeffrey C. , Warner Dennis C. , Jacobs Jon , McClain Craig C. , Kirpich Irina A
Published: June 2021

Background

The increasing prevalence of alcoholic liver disease (ALD) presents a large global healthcare burden. Currently, no therapeutics are available and the mechanisms underlying ALD development and progression are not well understood. Alcoholic hepatitis (AH) is a severe stage of ALD with a high mortality. Thus, identifying new signaling pathways involved in AH development, as well as novel biomarkers of AH progression are of a great clinical importance. The current study was aimed to determine hepatic proteomic and phosphoproteomic signatures and impaired metabolic pathways in severe AH.

Methods

Liquid chromatography and tandem mass spectrometry analysis were performed to examine proteomic and phosphoproteomic changes in explanted liver tissues obtained from AH patients (n=6) and patients without ALD (controls, n=12). A Student's two‐tailed unpaired t‐test was used to determine statistical significance between the two groups. MetaCore ontology analyses (false‐discovery rate < 0.05) were performed to identify proteomic changes, transcription factors and metabolic/signaling pathways altered in AH patients.

Results

3,288 peptides and 5,923 phospho‐peptides were significantly changed in AH liver samples compared to controls. The most diminished transcription factors in AH patients were hepatocyte nuclear factor 4‐alpha (HNF4a), hepatocyte nuclear factor 1‐alpha (HNF1a), and thyroid hormone receptor‐beta (THRb) as well as their target genes (45, 26, and 17 genes decreased in the networks, respectively). In contrast, the expression of transcription factors related to inflammation such as nuclear factor kappa B subunit 1 and 2 (NFkB1, NFkB2), as well as specificity protein 3 (SP3), were increased as were their target genes (9, 16, and 32 genes increased in each network, respectively). Further, the pathway involved in phagocytic bacterial clearance was significantly diminished in AH patients compared to control (p=4.2e‐8), while the neutrophil extracellular trap pathway was elevated (p=1.6e‐8). In addition, proteins involved in hepatocyte maturation (p=1.4e‐6) and glutathione metabolism (p=5.1e‐5) pathways were downregulated in AH patients.

Conclusions

Liver proteomic and phosphoproteomic analysis identified multiple alterations in transcriptional networks in AH patients characterized by diminished expression of transcription factors such as HNF4a, HNF1a, and THRb and elevation of NFkB1/2, and SP3. Further investigations are required to uncover new mechanisms in AH development and progression, as well as to identify novel biomarkers and therapeutic targets.

Support or Funding Information

NIH NIAAA RO1 AA024102NIH NIGMS P20 GM113226

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 Association of Fibroblast Growth Factor 5 (FGF5), Fibroblast Growth Factor Receptor 1 (FGFR1), and Prolactin (PRL) with Hair Length in Scottish Highland Cattle
Authors: Li Haoju , Lipsett Koren A
Published: June 2021

Investigating genetic disorders in large mammals is important because large mammals mimic humans due to their size, lifespan and physiology. Therefore, genetic mutations attributed to mammal disease can be correlated to similar human genetic diseases. Fibroblast growth factor family (FGF) and its receptor (FGFR1) are responsible for cell growth and wound healing. Prolactin (PRL) and its receptor (PRLR) are also in charge of the growth hormone and responsible for milk production in mammals. Previous research has shown that mutations in the FGF5 and FGFR1 genes result in abnormal hair growth in mice, dogs, sheep and donkeys. In addition, deficiency in dairy production and abnormally long hair are the result of mutations in PRL and PRLR. By defining a mutation associated with abnormal hair growth, and by extension, unregulated cell growth, abnormal growth of cancer cells can be further understood. However, to date no studies have found mutations in these genes in cattle. The Scottish Highland cattle is the only cattle breed with long hair (year‐round). In this study, FGF5, FGFR1, PRL were sequenced using our experimental cattle and control cattle. Our results showed that single nucleotide polymorphisms (SNPs) exist between the long‐hair cattle and control cattle, however, the mutations are silent mutations.

To further investigate, we expanded our research to the intronic regions and other genes through whole genome (WGS) sequencing by Illumina. We processed the raw mega‐data utilizing Amazon Web Service (AWS) including EC2, S3 coupled with Dragen by Edico Genome to map, align, and complete variant calling of the WGS from Scottish Highland cattle. This work generated BAM and VCF files that are being used for further variant analysis. Here, we report our progress leading to the identification of variants that may be responsible for long hair growth in Scottish Highland cattle.

Support or Funding Information

Provost's Senior Research Fund, X‐SIG Fund, & Albaugh Fund, Gettysburg College, Gettysburg, PA 17325

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Transforming Growth Factor Beta Induces Fibroblasts to Express and Release the Immunomodulatory Protein PD‐L1 into Extracellular Vesicles
Authors: Kang Jeong‐han , Jung Mi‐Yeon , Leof Edward B
Published: June 2021

Transforming growth factor beta (TGF‐β) is an enigmatic protein with various roles in normal tissue homeostasis/development as well as the development and/or progression of cancer, wound healing, fibrotic disorders, and immune modulation, to name a few. As TGF‐β is causal to various fibroproliferative disorders featuring localized or systemic tissue/organ fibrosis as well as the activated stroma observed in numerous malignancies, characterizing the pathways and players mediating its action is fundamental. To that end, in the current study we found that (i) TGF‐β induces the expression of the immunoinhibitory molecule Programmed Death‐Ligand 1 (PD‐L1) in human and murine fibroblasts in a Smad2/3‐dependent manner; (ii) profibrotic TGF‐β signaling is dependent upon PD‐L1 expression; and (iii) following treatment with TGF‐β, human lung fibroblasts secrete PD‐L1 into extracellular vesicles (EVs) capable of inhibiting T‐cell proliferation in response to T cell receptor stimulation. These findings provide new insights and potential targets for a variety of fibrotic and/or malignant diseases.

Support or Funding Information

This work was supported by Public Health Service grants GM‐55816 and GM‐54200 from the National Institute of General Medical Sciences.

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 Impact of Cancer‐Associated Mutations in Thyroid Hormone Receptor (TR) on TR Localization
Authors: Malapati Sri Harshini , Punzalan Paige , Allison Lizabeth
Published: June 2021

The thyroid hormone receptor (TR)—a nuclear receptor that is essential for the proper regulation of metabolism and development, as it regulates gene expression in response to thyroid hormone—contains nuclear localization signals (NLSs) and nuclear export signals (NESs) that allow for TR transport into and out of the nucleus, respectively. Previous research has shown that nuclear import, nuclear retention, and nuclear export of TR are associated with modulation of gene expression, the alteration of which can contribute to various diseases, including Resistance to Thyroid Hormone (RTH) Syndrome, type II diabetes mellitus, and several types of cancer. There are three main subtypes of TR: TRα1, TRβ1, and TRβ2. Here, through mammalian cell transfection of expression plasmids for green fluorescent protein (GFP)‐tagged TR and fluorescence microscopy, we examined particular groups of TRα1 mutations that were observed in patients with hepatocellular carcinoma and renal carcinoma and are associated with NLSs and NESs of TRα1. More specifically, we calculated the nuclear‐to‐cytoplasmic ratios of TRα1 variants to determine the impact of TRα1 mutations on receptor localization within HeLa cells. We found that, while wild‐type TR has a primarily nuclear localization with a smooth distribution in human cells grown in culture, the TRα1 mutations S40T, K136R, L251P, V390A and A225G, D246N, G350K lead to significantly decreased nuclear‐to‐cytoplasmic TRα1 ratios (greater cytoplasmic distribution) and formation of TRα1 aggregates. We observed that the distribution of a TRα1 mutant with just one of the four mutations (L251P) is very similar to the distribution of the TRα1 mutant with all four mutations (S40T, K136R, L251P, V390A), suggesting that L251P plays a major role in the mislocalization of TRα1. In addition, we found that TRα1 with a G24E mutation had a significantly greater cytoplasmic distribution, compared to wild‐type TRα1. In conclusion, our findings suggest that certain TRα1 mutations that are associated with carcinoma lead to TRα1 mislocalization, which may alter the expression of thyroid hormone‐dependent genes. These results contribute to an improved understanding of TR structure and function, which could lead to novel strategies for discovering therapies for TR‐related illnesses.

Support or Funding Information

NIH #2R15DK058028‐04

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ouabain Regulated Phosphoproteome Reveals Molecular Mechanisms Behind Na, K‐ATPase Control of Cell Adhesion, Proliferation and Survival
Authors: Panizza Elena , Svensson Daniel , Zhang Liang , Fontana Jacopo Maria , Akkuratov Evgeny , Scott Lena , Brismar Hjalmar , Lehtiö Janne , Aperia Anita
Published: June 2021

Ouabain is a cardiotonic steroid with a history of medical uses. It acts as a ligand to the ubiquitous ion pump Na+,K+‐ATPase which it inhibits at high concentrations. At lower concentrations it triggers calcium oscillations and activation of MAP kinases, stimulates cell proliferation as well as adhesion and acts anti‐apoptotic. In light of this, it has been proposed that the pump may have a dual function as a signaling transducer. To investigate this, we have analyzed the phosphoproteome of COS‐7 kidney cells after treatment with sub‐saturating levels (100 nM) of ouabain, which were high enough to induce Ca2+ oscillations but below those needed to modulate the ion pumping function of Na+,K+‐ATPase. Gene ontology analysis was performed to find distinct cellular processes regulated by ouabain after 10 and 20 min of treatment in the phopshoproteomic analysis. The analysis revealed 2580 ouabain‐regulated phosphorylation sites, many of which were associated with cell adhesion and proliferation. Two of the regulated phospho‐proteins were the inositol triphosphoate receptor (InsP3) and stromal interaction molecule (STIM), both of which are essential for ouabain‐induced calcium oscillations. We used siRNA and Western blotting for target confirmation. One confirmed target, calcium/calmodulin‐dependent protein kinase II gamma (CAMK2γ), were shown to be involved in the anti‐apoptotic effect of ouabain; siRNA silencing of CaMK2γ in primary renal cells eliminates the protective effect of ouabain against apoptosis induced by either serum starvation or glucose. In conclusion, our findings support a role for Na+,K+‐ATPase as a signal transducer that serves to protect cell and tissue integrity.

Support or Funding Information

This study was supported by the Swedish Research Council and Erling‐Persson Family Foundation. D.S. is supported by a Novo Nordisk postdoctoral fellowship run in partnership with Karolinska Institutet.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Nuclear Receptor Corepressor 1 Regulates Thyroid Hormone Receptor Nuclear Retention
Authors: Sun Xiaopeng , Allison Lizabeth
Published: June 2021

The thyroid hormone receptor (TR) mediates gene transcription in response to thyroid hormone (T3) and significantly affects cell growth, development and metabolism. Although TR shuttles rapidly between the nucleus and cytoplasm, it is primarily nuclear localized at steady state. A shift in the balance between export and import leading to TR mislocalization may correlate with certain cancers or endocrine disorders. Previously, we have identified multiple nuclear localization signals and nuclear export signal within TR that interact with importins and exportins, respectively, to mediate shuttling. Given that TR contains multiple, competing import and export signals, the question arises: what factors are involved in nuclear retention of TR? In addition to DNA‐binding sites, the lab's prior research indicated that nuclear coactivator MED1 facilitates TR nuclear localization. Thus, we hypothesize that certain transcriptional coregulators, including Nuclear Receptor Corepressor 1 (NCoR1), contribute to TR nuclear retention. Using transient transfection assays and fluorescence microscopy, we measured the fluorescent intensity of GFP or mCherry‐tagged TR in the nucleus (N) and cytoplasm (C) of HeLa cells, and then calculated the N/C ratio as an indicator of intracellular localization patterns. We discovered that when NCoR1 is knocked down by shRNA, the amount of TR in the nucleus decreased significantly (lower N/C ratio). Inversely, when NCoR1 is over‐expressed, the amount of TR in the nucleus significantly increased (higher N/C ratio). An even more dramatic cytosolic shift was observed for a TR mutant that cannot bind to NCoR1 (AHT‐TR). Taken together, our results suggest that NCoR1 is a key regulator of TR nuclear retention. This finding will contribute to the development of a dynamic mathematical model that aims to predict TR's N/C ratio during the interaction with different complexes and TR localization's effect on transcriptional output of T3‐dependent genes.

Support or Funding Information

NIH 2R15DK058028

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Assembly and Annotation of the Starfish Egg Transcriptome: The Search for the Elusive Molecular Controllers of Fertilization
Authors: Bates Lauren , Wiseman Emily , Kitson Jamie , Dunn Leah , Baghban Mehrasa , Carroll David
Published: June 2021

Fertilization is the process of sperm‐egg interaction leading to a permanent block to polyspermy and re‐initiation of the cell cycle. Release of Ca2+ from the endoplasmic reticulum is the integral molecular mechanism responsible for activation of fertilization; however, the molecular players involved in the initiation and regulation of the Ca2+ release are not well‐established in any organism. Research in the fertilization field has been hampered due to difficulty in studying the sperm‐egg cell system. Eggs and sperm of the starfish Patiria miniata present an attractive model to study the molecular aspects of fertilization due to the ease of obtaining millions of viable sperm and eggs, as well as, their amenability to in vitro fertilization and biochemical assays. However, the lack of specific antibodies for the identification of proteins in the starfish model system has delayed discovery. To address this deficiency, we produced the first P. miniata egg transcriptome by de novo RNA sequencing, which will allow us to predict which proteins could be present and function in controlling the fertilization signaling pathway. The goal of this project is to annotate the starfish egg transcriptome and categorize the transcripts into known protein families. We present evidence for the presence of all major signaling pathways and confirm biologically‐relevant RNAs via RT‐PCR against freshly isolated starfish egg RNA. This work will be used to establish a potential proteome for the identification of proteins that may function in the fertilization signaling pathway.

Support or Funding Information

National Institutes of Health R15

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of GDP‐dependent Interactions Between TCL and Potential Effector Proteins
Authors: Tader Brooke R , Hamann Michael J
Published: June 2021

TCL/RhoJ belongs to the Rho family of GTPases and has been shown to contribute to tumor‐associated angiogenesis as well as metastatic melanoma. While the role of TCL in these pathological events is becoming clearer, less is known about its biochemistry and molecular/cellular function. To better understand the cellular events controlled by TCL, experiments in our lab have focused on where TCL localizes to in cells based upon its nucleotide loading status and prenyl modifications. Using constitutively active (CA) and dominant negative (DN) mutants of TCL, we have shown that GTP‐loaded TCL localizes to the plasma membrane while GDP‐loaded TCL is more vesicular in appearance. Clearly, localization of CA TCL to the plasma membrane is also dependent on prenylation of the TCL CAAX motif as mutation of this signal sequence results in diffuse cytoplasmic localization. However, the same CAAX mutation in the context of the DN mutation lead to a marked vesicular localization. Together, our results have led us to hypothesize that TCL is specifically retained to vesicular membranes through an interaction with an unidentified binding partner, and current experiments using biotin‐ligase tags of CA and DN TCL and BioID procedures are aimed at identifying a vesicle‐bound partner protein. We anticipate that a more complete understanding of how TCL functions at vesicular and plasma membranes will help identify its potential role in vesicular trafficking in pathology‐related processes.

Support or Funding Information

This work was supported by Bemidji State University Biology Department and the College of Business, Mathematics, and Science. Support was also provided through the Neilson Foundation, Bemidji, MN and Regenerative Medicine Minnesota (RMM‐2017‐EP‐04).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interplay of Soluble, Mechanical and Redox Signaling in Airway Remodeling During Asthma
Authors: Al‐Azzam Nosayba Zakariya , Gombedza Farai , Snyderman Matthew , Ghebreigziabher Samarawit , Covington Kayla , Thodeti Charles , Paruchuri Sailaja
Published: June 2021

NADPH oxidase 4 (NOX4) is a member of the NADPH oxidase family, encoded by the NOX4 gene, and has been implicated in TGF‐β1‐induced human cardiac fibroblast to myofibroblast differentiation1. NOX4 has been shown to potentiate lung fibroblast differentiation in vitro and its transcript level is enhanced in fibroblasts isolated from patients with idiopathic pulmonary fibrosis (IPF) compared to normal fibroblasts2. Our lab has previously identified that Transient Receptor Potential Vanilloid 4 (TRPV4), a mechano‐sensitive ion channel, is involved in D. farinae‐induced airway remodeling during asthma in vivo and myofibroblast differentiation in vitro. However, the role of NOX4 in asthma, its interaction with TRPV4, and the mechanism of action still remains elusive. In the present study, we show that NOX4 is up‐regulated during lung remodeling in asthma and fibroblast differentiation in a TRPV4‐dependent pathway. TGF‐β1 treatment up‐regulated NOX4 gene expression and function. Further, employing antioxidants and NOX4 siRNA, our results reveal that NOX4 is involved in TGF‐β1‐induced α smooth muscle actin (αSMA), SM22, fibronectin (FN), myocardin‐related transcription factor‐A (MRTF‐A) and plasminogen activator inhibitor ‐1 (PAI‐1) expression in human lung fibroblasts (hLF). Interestingly, TRPV4 inhibition led to attenuation in TGF‐β1‐mediated NOX4 expression and fibroblast differentiation. Furthermore, NOX4 transcripts were up‐regulated in lung tissues in response to D. farinae in an asthma model supporting our in vitro findings. More importantly, TRPV4−/− mice were protected from D. farinae‐induced asthma and exhibited lower levels of NOX4 gene expression in response to D. farinae, further suggesting that TRPV4 promotes airway remodeling through NOX4.

Support or Funding Information

James Foght research support

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 NDM‐X, a mononuclear zinc metallo‐β‐lactamase derived from a di‐zinc enzyme
Authors: Montgomery Jonathan , Shurina Ben , VanPelt Jamie , Page Richard
Published: June 2021

Over the years, the continued use of antibiotics has caused an evolutionary defense mechanism in bacteria to emerge, and the resulting antibiotic resistance has become a serious issue. These antibiotic resistant bacteria employ enzymes known as β‐lactamases. These β‐lactamases are able to hydrolyze the β‐lactam ring of many antibiotics such as penicillin and carbapenems, rendering the antibiotic ineffective at killing bacteria. New Delhi metallo‐β‐lactamases (NDM), which utilize a di‐zinc catalytic center, have been observed to undergo several mutations which have led to increased thermal stability and increased Zn2+ affinity when there are low concentrations of Zn2+ present. These mutations have been suggested to enable NDM to exhibit activity in both di‐zinc and mono‐zinc states. Additionally, NDM poses a particular threat as it is able to act on a wide range of current antibiotics. One strategy for combating these β‐lactamases is the use of small molecule inhibitors that will prevent the enzyme from hydrolyzing β‐lactam containing antibiotics, allowing for their continued use. A major challenge is presented in solving the structure of NDM with an inhibitor bound prior to hydrolysis. In order to solve this problem, our lab has engineered a new version of NDM through site directed mutagenesis. We engineered a mono‐zinc version of NDM that has an approximate 10,000 fold reduction in activity while maintaining the global architecture of NDM. The structure of this new NDM variant, NDM‐X, has been solved to 1.6 Å resolution and, due to the reduced catalytic activity, could be used in crystal soaking experiments to obtain the structural information of these protein‐substrate interactions in a pre‐hydrolyzed state. These pre‐hydrolysis enzyme‐substrate complexes may allow for further development and modification of proposed inhibitors to better combat antibiotic resistance.

Support or Funding Information

NIH R35 GM128595

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Physiological cardiac hypertrophy activates PPARγ/PGC‐1α axis and up‐regulates perilipin family
Authors: Soñanez‐Organis José Guadalupe , Verduzco‐Avila Denisse , Padilla‐Bórquez Diana Lourdes , Del Carmen Castro‐Meza Susana , Rosas‐Rodríguez Jesús Alfredo , Godoy‐Lugo José Arquímides , Ortiz Rudy M
Published: June 2021

Peroxisome proliferator‐activated receptor γ (PPARγ) and peroxisome proliferator‐activated receptor gamma coactivator 1‐α (PGC‐1α) are coordinately up‐regulated in hypertrophic cardiomyopathy and cooperate to mediate changes in lipid metabolism and storage that are characteristic of and contribute to common forms of heart disease. Previous results in our laboratory suggest that PPARγ regulates the shift in cardiac metabolism during physiological cardiac hypertrophy. On the other hand, the perilipin family (Plin: 1–5) is involved in the formation of lipid droplets in various tissues, and it has been demonstrated that Plin5 participates protecting the heart during myocardial ischemia and diabetic cardiomyopathy. However, the participation of PPARγ/PGC‐1α and the Plin family in physiologic hypertrophy induced by pregnancy and its reversible process (postpartum) are not well defined. We evaluated PPAR (alpha, delta, gamma) transcriptional activity, and PGC‐1α and Plin family gene expression along with triglyceride content before, during, and after pregnancy in the left ventricle of rat. The activity of PPARγ increased 1.6‐fold during pregnancy, and decreased to basal levels postpartum. Expression of mRNA for PGC‐1α increased 18‐ and 11‐fold during pregnancy and postpartum, respectively. Plin1, Plin2 y Plin5 transcript increased 9‐ to 16‐fold and 2‐ to 11‐fold during pregnancy and postpartum, respectively, while expression of Plin3 did not change and Plin4 was not detected. The results demonstrate that in, pregnancy‐induced, physiological cardiac hypertrophy activates the expression of genes involved in lipid storage suggesting that the shift in cardiac metabolism is mediated by the activation of PPARγ/PGC‐1α.

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 a moderate physical training protocol on the metabolism hepatic of malnourished juvenile rats
Authors: Lima Talitta Ricarlly , Freitas Cristiane Moura , Silva‐Filho Reginaldo Correia , Braz Glauber Rudá Feitoza , Lagranha Claudia Jacques , Fernandes Mariana Pinheiro
Published: June 2021

The purpose of this study was evaluate the effects of the moderate physical training on the hepatic metabolism of juvenile rats submitted to protein restriction during critical periods of development. Wistar rats received Normoprotein (protein17%) and Low protein (protein8%) diets throughout gestation and lactation. After weaning (21 days), pups received Labina®. The offspring was submitted to the incremental aerobic run test from the 26th, 27th and 28th days and subdivided into two additional groups according to the practice or not the physical training (Normoprotein Sedentary‐NS; Normoprotein Trained‐NT; Low protein Sedentary‐LS; Low protein Trained‐LT). The animals of NT and LT groups underwent a moderate physical training (60min day/5days/ 4 weeks). At 60 days, the male were euthanized and the liver removed to biochemistry analyzes. Our results showed a decrease on mitochondrial oxygen consumption after ADP addition (State 3, 53%, p<0.01) and respiratory control ratio (RCR, 52%, p<0.0001), additionally we observe increase on mitochondrial swelling (40%, p<0.001) and ROS production (36%, p<0.01) in LS group when compared to the NS group. The levels of lipid peroxidation (42%, p<0.0001) and protein oxidation (81%, p<0.0001) were increased and SH‐content (41%, p<0.0001) was decreased in the LS group when compared to the NS group. The antioxidant enzymes activities as SOD (33%, p<0.0001), CAT (48%, p<0.0001), GPx (49%, p<0.001) and GST (64%, p<0.01) were decreased in the LS group without altering the GSH levels. The metabolic enzymes activities showed a decrease on the CS (28%, p<0.05), no change on PFK1, decrease on G6PDH (54%, p<0.001) and increase on β‐HAD (2.1 fold, p<0.0001) in the LS group when compared to the NS group. In the LT group we observe that moderate physical training induced a significant effect on the all‐hepatic metabolism pathways. Taken together, our results suggest that the physical exercise acted as a non‐pharmacological therapeutic agent capable to minimizing the effects of a protein restriction in the early life of these animals.

Support or Funding Information

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior ‐ Brasil (CAPES) ‐ Finance Code 001 and Fundação de Amparo a Ciência e Tecnologia do Estado de Pernambuco (FACEPE)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Thyroid hormone regulation of retinoic acid synthesis in brown adipose tissue
Authors: Olson Charles Johan Henrik , Krois Charles Robert
Published: June 2021

In mice, alterations to vitamin A status through gene knockout or treatment with pharmacological doses of retinoic acid (RA), the active form of vitamin A, affect overall energy metabolism and body weight. Generally, increases in RA lead to increased metabolism and weight loss, and reductions in RA lead to weight gain. One specific effect is that within brown adipose tissue impaired RA synthesis impairs the tissue's ability to generate heat and thus an organism's ability to maintain normal body weight and temperature. We therefore hypothesize that in a cell model of brown adipose tissue, thyroid hormone (T3), a potent activator of brown adipose function, will also increase RA synthesis through induction of retinol and retinal dehydrogenases. Together, these enzymes synthesize RA from the precursor retinol. Overall, our work contributes to a better understanding of factors that impact both obesity and metabolic disease.

Support or Funding Information

Minnesota State University, MankatoUndergraduate Research Grant

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dietary iron restriction improvement mitochondrial dysfunction and oxidative stress, and iron supplementation increases oxidative damages in kidneys mitochondria of streptozotocin‐diabetic rats
Authors: Peña‐Montes Donovan , Huerta‐Cervantes Maribel , Trujillo Xóchitl , Huerta Miguel , Montoya‐Pérez Rocío , Cortés‐Rojo Christian , Salgado‐Garciglia Rafael , Saavedra‐Molina Alfredo
Published: June 2021

Diabetes mellitus (DM) is a multifactorial disease, characterized by a chronic increment in blood glucose (hyperglycemia). In parallel with the growing DM pandemic, there is an increasing burden of DM complications occurring in the majority that suffering, such as diabetic kidney disease (DKD). Furthermore, the chronic hyperglycemia of diabetes results in mitochondrial dysfunctionality followed by an increase in mitochondrial reactive oxygen species (ROS) production and mitochondrial oxidative stress (OE). On the other hand, iron plays an important role in all organism for the maintaining of the physiological homeostasis. However, in diabetes has been reported a disruption of iron metabolism leading to an iron accumulation in kidney. The aim of this study was to evaluate the effect of chronic iron restriction in the diet, and iron supplementation in mitochondrial function and oxidative stress in kidneys mitochondria employing male Wistar rat with experimental diabetes. 28 rats of two‐old months were divided randomly into four groups diabetic rats were administered with a single dose of STZ, (45 mg/Kg of BW in citrate buffer, pH 4.5). Experimental diabetes mellitus with STZ, increase non‐heme iron content in whole‐kidney mitochondria and chronic iron restriction restores oxygen rate consumption (OCR) and GSH:GSSG ratio, and prevents the increment in lipid‐peroxidation and decrease mitochondrial ROS production compared with the diabetic group with a standard normal diet. Meanwhile chronic iron supplementation increases the oxidative damages. Taken together, these results suggest that experimental DM increase mitochondrial iron, and iron restriction in the diet restores mitochondrial functionality and prevents mitochondrial oxidative damage in rat kidneys.

Support or Funding Information

The authors acknowledge to CONACYT (DPM) and Coordinación de Investigación Científica. UMSNH grants (ASM‐2.16).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression of Iron Regulatory Proteins in Healthy and Osteoarthritic Chondrocytes
Authors: Rose Brandon J , Behrens Nathan F , Harris Shelby T , Maloy Sarah A , Nelson Tanner K , Smith Jacob F , Starr Evan G , Woolstenhulme Jonathan B , Watt Richard K , Kooyman David L
Published: June 2021

Iron dysregulation is a known risk factor in the progression of osteoarthritis and is a feature of comorbid syndromes Alzheimer's disease, insulin resistance, and age‐related macular degeneration. Little, however, is known about iron homeostasis in healthy chondrocytes or whether iron dysregulation occurs in osteoarthritis. To gain information on this matter, we performed surgical destabilization of the medial meniscus on the right knee of 8‐week‐old mice. An untreated sham group served as the control. We found and document for the first time that chondrocytes produce hepcidin, and that both hepcidin and ferroportin expression is downregulated in osteoarthritic cartilage to a statistically significant extent. We found, however, that there was no statistically significant change in ferritin expression in osteoarthritic vs. healthy chondrocytes. We submit that this data, in conjunction with observed iron metabolism trends in other tissues, suggests a role for iron dysregulation in the pathogenesis of osteoarthritis.

Support or Funding Information

Office of Research and Creative Activities, Brigham Young University

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Antioxidant Potential of Artocarpusaltilis Leaf Powder in Alloxan Induced Diabetic Rat
Authors: Ajayi Olubunmi Bolanle , Arowolo Godwin Oluwaseun , Akomolafe Seun Funmilola
Published: June 2021

The possible protective effect of Artocarpus altilis leaf powder on diabetes‐induced oxidative stress was evaluated in alloxan‐induced diabetic male rat. Albino rats each weighing about 100 ± 25g were divided into five groups (A–E) (where n=8). Group A is the control fed with normal diet, Group D was pre‐treated with supplemented 5% A. altilis leaf powder for four weeks before induction with alloxan. Thereafter, groups B, C, D, and E were induced with 75mg/kg body weight of alloxan. However, group C was treated after induction with 5% A. altilis leaf powder supplemented diet and E with metformin. Significant increase (P<0.05) in lipid peroxidation and decrease in Superoxide dismutase (SOD), Catalase (CAT) and reduced glutathione (GSH), were observed in the diabetic control compared with the control rats. However, pretreatment with A. altilis leaf powder supplemented diet (Grp. D) and treatment of diabetic rats with same diet (Grp. C) resulted in a reduced lipid peroxidation and significant increase (P<0.05) in the activity of SOD, CAT, and GSH concentration in the serum, liver, and kidney of these group of rats compared with the diabetic control. These findings indicate that A. altilis leaf powder supplemented diet has a potential to reduce free radical mediated oxidative stress to cells in experimental diabetic rats. The pretreated A. altilis leaf powder supplemented diet was found to be more effective in restoring the values of these parameters to normal values, thus lending credence to its folkloric usage in the management and/or control of diabetes mellitus and possible use as a nutraceutical

Support or Funding Information

nil

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Physico‐chemical characteristics and antioxidant capacity of chickpea and wheat flour blends
Authors: Sidhu Jiwan S. , Zafar Tasleem A. , Almusallam Abdulwahab S. , Ali Muslim , Al‐Othman Amani
Published: June 2021

Type‐2 diabetes is becoming a serious metabolic disease in the State of Kuwait. We wanted to make use of chickpea flour which is known to offer many benefits to the diabetic person especially in maintaining their blood sugar levels in acceptable range. The chemical composition and antioxidant capacity of white flour (WF), whole wheat flour (WWF) and chickpea flour (BF), in addition to the effect of replacement of WF and WWF with BF on the Texture Profile Analysis, and objective color of these blends, in terms of L* a* b* values were also investigated. Trolox Equivalent Antioxidant Capacity (TEAC), Malondialdehyde (MDA) and Total Phenolic Content (TPC) values varied significantly among all the three flour samples. WWF had the highest TEAC (117.42/100g) value, followed by WF (73.98/100g) and BF (60.67/100g). The TPC content also followed the similar trend, as the Gallic Acid Equivalent (GAE) values were the highest (8.67 GAE/g) for WWF, followed by WF (5.65GAE/g) and BF (3.45 GAE/g). Compared with WF and WWF, the b* values for BF were the highest (16.89) indicating higher yellowness in this sample. The TPA carried on the WF, WWF and their blends with BF showed significant variations in hardness and compressibility values, dough becoming less hard and less compressible when the replacement of WF and WWF with BF was increased from 0 to 40%. To overcome the deleterious effects of adding BF to WF and WWF on the rheological properties will be an interesting challenge, when a good quality pan bread or Arabic flat bread have to be produced during the follow up study.

Support or Funding Information

(Study funded by Research Administration, Kuwait University, Project No. WF01/13)(No human subjects used in this study)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interactions of Perilipin 5 with Cytoskeletal Elements
Authors: Austin Rachel L , Tansey John T
Published: June 2021

Cardiovascular diseases, obesity, and diabetes are disease that are affecting the American population at alarming rates. Central to these diseases is lipid metabolism and lipid storage. Perilipin 5 is a protein found on the surface of lipid droplets within cells, but has also been found within the cytoplasm, mitochondria, and nucleus, and is implicated in the mobilization of lipid droplets. This mobilization is required to associate triglycerides with mitochondria, where beta oxidation occurs. Cellular trafficking of lipids is also aided by cytoskeletal elements – like actin filaments. These filaments provide a mode for transportation for molecules within the cell. We hypothesize perilipin 5 will interact with cytoskeletal elements. To test this, we are conducting immmunofluorescence on cultured cells expressing perilipin 5 and stained with antibodies against perilipin 5 and fluorescent actin filaments. Evidence of this interactions will add more information about the role of Perilipin 5 in fatty acid oxidation, the dysfunction of lipolysis, and its associated diseases.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Crystal Structure of Poly(Aspartic Acid) Hydrolase‐1
Authors: Bolay Austin L , Salvo Henry F , Wallen Jamie R , Weiland Mitch H
Published: June 2021

Poly(aspartic acid) (PAA) is a biodegradable synthetic polymer that is easily produced through heating aspartic acid followed by the subsequent addition of sodium hydroxide. Polymer applications range from drug delivery and biomimetics to hygiene products. Partial biodegradation of PAA is accomplished using poly(aspartic acid) hydrolase‐1 (PAAH‐1) which selectively cleaves a specific cross‐link. E. coli expressions of recombinant PAAH‐1 showed the production of significant amounts of protein which could be purified using a single Ni‐NTA column. Analysis of the PAAH‐1 sequence using the Phyre2 Protein Fold Recognition Server resulted in a 33% similarity to a “putative” poly(3‐hydroxybutyrate) depolymerase belonging to a family of enzymes that hydrolyze carboxylic ester bonds; however, no known structure of PAAH‐1 has been reported. Protein crystallization screening at the Hauptman‐Woodward Research Institute resulted in numerous protein crystallization hits being identified. Crystallization experiments were optimized in‐house and the structure was determined at 2.45Å using molecular replacement. The progress of PAAH‐1 characterization and its structure will be discussed.

Support or Funding Information

NSF‐IUSE Grant: DUE 1611988

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dystrophin Promotes Colorectal Carcinoma Metastasis and Exosome Secretion via Protein‐Protein Interaction with Rab GTPase 3C
Authors: Chen Chi‐Long , Chang Yu‐Chan , Hsiao Michael
Published: June 2021

RAB GTPases is involved in membrane trafficking (vesicle formation) and cell motility. Recently, exocytotic RABs has been noted that the exocytosis by RABs is correlated with drug resistance and immune response in tumorigenesis. However, the molecular mechanisms of exocytotic RABs in colorectal tumorigenicity remain unknown. RAB GTPase 3C (RAB3C) has been identified as the key component for exosome formation and exocytosis. In clinical colorectal carcinoma patient cohort, high RAB3C expression maintained its independent prognostic significance for both overall survival and disease‐free survival. We further established RAB3C‐based proteomics datasets and mass spectrometry analyses and found dystrophin (DMD) is upregulated in RAB3C ectopically overexpressed colon cancer cell lines. High DMD expression levels were found correlated with poor survival in colorectal cancer patient cohorts. Moreover, Dystrophin‐complex components including dystrobrevin (DTNA) and syntrophin (SNTA1/SNTB1) also showed the same prognosis trends in colorectal cancer patients. We further found that DMD is a rare mutant event in colorectal clinical cohort by next generation sequencing analysis. Our results showed that DMD could be stabilized by RAB3C through protein‐protein interaction. Besides, our results revealed RAB3C co‐localized with dystrophin complex by immunohistochemistry staining analysis. Merged DMD with RAB3C serves as a significant independent prognostic factor in colorectal cancer and is associated with several clinical parameters. In addition, dystrophin complex decreased cell membrane integrity and altered calcium channel activity for exocytosis. In summary, our results identified DMD is a new functional binding protein for RAB3C that affected colorectal carcinogenesis. These findings suggest a new therapeutic strategy for targeting cancer‐specific protein‐protein interactions in the treatment patients with colorectal cancer.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of omega‐3 and omega‐6 fatty acids on nitric oxide and IL‐6 production by RAW 264.7 macrophages
Authors: Danchine Veronika Denise , Caldari Cristina
Published: June 2021

Chronic low‐grade inflammation, or meta‐inflammation, which may occur in obese subjects, is related to the infiltration of macrophages into adipose tissue (AT) as adipocyte number and size increase. The increased count in fat cells is correlated with an increase in adipose tissue macrophages (ATMs). These ATMs can secrete pro‐inflammatory cytokines which can have effects on the surrounding adipocytes. Omega‐3 fatty acids (FAs) reduce inflammation through direct competition with omega‐6 FAs, the precursors of the inflammatory eicosanoids, as well as through changes in gene expression of inflammatory molecules. Less is known about the effects of omega FAs on pro‐inflammatory cytokine production by immune cells, such as macrophages. The objective of this research was to determine the effects of omega‐3 and omega‐6 FAs on the production of nitric oxide (NO), a signaling molecule secreted by activated macrophages which is involved in mediating inflammation and production of the pro‐inflammatory cytokine interleukin‐6 (IL‐6), in lipopolysaccharide (LPS)‐activated macrophages. RAW 264.7 (murine macrophages) were incubated with 100 μM of sodium salts of linoleic acid (LA; C18:2, omega‐6), arachidonic acid (ARA; C20:4, omega‐6), or eicosapentaenoic acid (EPA; C20:5, omega‐3) for 24 h. After pre‐incubation with FAs macrophages were stimulated with 0.01 μg/ml of LPS for 6 h. Conditioned media was collected for NO and IL‐6 quantification via Griess assay and ELISA, respectively. FA treatments were cytoprotective, resulting in higher percentages of live cells than both control and LPS‐treated cells (Trypan blue exclusion method; p<0.0001, two‐way ANOVA). LPS‐stimulated macrophages pre‐incubated with LA, ARA, or EPA produced less NO than LPS‐stimulated macrophages without FA pre‐incubation (p=0.0001, two‐way ANOVA). Pre‐incubation of macrophages with LA, ARA, or EPA decreased IL‐6 production in comparison to LPS‐stimulated macrophages, with omega‐6 FAs having a more pronounced effect than omega‐3 FAs (p<0.0001, two‐way ANOVA). In conclusion, both omega‐6 and ‐3 FAs decreased activation of murine macrophages and decreased production of the inflammatory molecules NO and IL‐6, with omega‐6 FAs having a more marked effect than omega‐3 FAs. The stronger anti‐inflammatory effect of omega‐6 FAs on murine macrophages, compared to omega‐3 FAs, warrants further investigation.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Endothelial Extracellular Vesicle Production and Cargo Property are Altered after Inflammatory Injury
Authors: Chatterjee Victor , Yang Xiaoyuan , Yuan Sarah Y.
Published: June 2021

Extracellular vesicles (EVs) play an important role in cell‐cell communication and signal transduction by transferring bioactive cargo contents between cells or tissues. During inflammation, the vascular endothelium is activated and releases vasoactive EVs that alter vascular homeostasis via paracrine or autocrine pathways. In this study, we examined the molecular property and mechanisms of production of EVs derived from endothelial cells (EC) during septic or sterile (thermal injury) inflammation, and during stimulation by inflammatory agonists, including tumor necrosis alpha (TNFa), interleukins (ILs), lipopolysaccharide (LPS) and thrombin. EVs isolated from plasma or cell culture conditioned media were analyzed by flow cytometry using cell specific markers. Size‐based gating of microvesicles (MV) was performed using a mixture of standardized beads of different sizes and absolute MV numbers calculated based on reference beads of known concentration. Immuno‐capture of smaller exosomes was performed with anti‐CD9 coated beads, probed for proteins of interest and visualized by flow cytometry. The results revealed that mice subjected to cecal ligation and puncture‐induced septic injury or thermal injury had increased CD144+ EVs but decreased endothelial nitric oxide synthase (eNOS)+ EVs in plasma as compared to sham‐treated mice. Likewise, endothelial cells stimulated by TNFa, IL‐1b or thrombin release EVs containing a higher level of caveolin‐1 (Cav‐1) and lower level of eNOS compared to non‐stimulated cells. Pre‐treatment with the ROCK inhibitor Y27632 prevented this response. Furthermore, EV‐EC interactions were observed under confocal microscopy. In endothelial cells, internalization of inflammatory EVs induced eNOS translocation from the cell membrane to cytosol. These findings suggest that inflammation alters endothelial production of EVs and their cargo property and function.

Support or Funding Information

This work was supported by NIH grants HL070752, HL126646 and GM097270

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