bluuebunny/crossref_metadata_2025_split huggingface dataset:
Interplay between the Thioredoxin System and the ASK‐1 Signaling Pathway in Testicular Ischemia Reperfusion Injury
Authors: Al‐Kandari Nora Yaqoub , Al‐Maghrebi May 
Published: June 2021

Testicular ischemia reperfusion injury (tIRI) has been suggested through several studies as the underlying mechanism for testicular torsion and detorsion (TTD) due to the excessive generation of reactive oxygen species (ROS) leading to germ cell apoptosis (GCA). The aim of the study is to explore the mechanism of the apoptosis signal‐regulating kinase 1 (ASK1) signaling pathway during tIRI by using its specific inhibitor, NQDI‐1. We also investigated the effect of NQDI‐1 on the expression of the thioredoxin (Trx) system, a major source of ROS. Male Sprague‐Dawley rats (n=36, 250–300 g) were divided equally into three experimental groups: sham, tIRI and tIRI + NQDI‐1 (10 mg/kg). During tIRI, the testicular artery was occluded for 1 hour followed by 4 hours reperfusion. The NQDI1 was injected intraperitoneally 30 minutes post ischemia. Histological analyses were used to evaluate testicular morphological changes as well as spermatogenesis. Gene expression, protein immunoexpression and enzyme activities were assessed by real‐time PCR, ELISA, IHC and colorimetric assays. Following tIRI, histological examination revealed impairment in spermatogenesis represented by a significant decrease in the Johnsen score. Real‐time PCR confirmed the upregulation of the pro‐apoptosis genes BAD and BAX and the downregulation of anti‐apoptosis gene Bcl2, which were accompanied by activation of the executioner caspase‐3 in the tIRI group. The levels of the antioxidants GSH, SOD, as well as thioredoxin reductase (TrxR) enzyme were also reduced upon tIRI induction. Immunoexpression of ph‐p38 and ph‐ASK1 were increased significantly after tIRI indicating the activation of ASK1 signalling pathway, while a remarkable decrease in Trx was simultaneously observed. The above damaging consequences of tIRI were attenuated after NQDI1 treatment obtaining normalized values similar to sham. Our findings suggest that the redox status of the cell is cooperatively regulated with ASK1 signaling to maintain cellular homeostasis during testicular oxidative stress. Additionally, the interplay between the Trx system and ASK1 suggests their potential role in the pathogenesis of tIRI.Support or Funding InformationThe project was supported by the College of Graduate studies and Research Administration, Kuwait University through grant # YM 09/17 and OMICSRU/RCF project grant # SRUL02/13.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Reprogramming of Colonic Cell Metabolism by H2S
Authors: Vitvitsky Victor , Libiad Marouane , Bostelaar Trever , Maebius Allison , Lee Ho‐Joon , Lyssiotis Costas A. , Banerjee Ruma 
Published: June 2021

Synthesis of H2S by gut microbiota leads to routine colonic cells exposure to this respiratory toxin. Herein we studied the effect of H2S on metabolism and proliferation in human colonic cells in culture at sulfide concentrations up to 300 μM, which represents a physiologically relevant exposure for these cells. H2S caused a marked activation of glycolysis and inhibition of proliferation. Between 5–20 μM H2S the oxygen consumption rate (OCR) was activated, while OCR was inhibited at higher concentrations. A high‐capacity mitochondrial sulfide oxidation pathway housed in colonocytes supports O2‐dependent H2S clearance and the absence of the committing enzyme (sulfide quinone reductase) in the pathway leads to inhibition of OCR even at the lowest H2S concentration that was tested. H2S triggers persulfidation of numerous protein targets that are enriched in energy metabolism pathways. The antiproliferative effect of H2S exposure results from electron acceptor insufficiency that ensues from H2S oxidation activity and inhibition of respiration. Under these conditions, reductive carboxylation of a‐ketoglutarate is enhanced and exogenous uridine and aspartate alleviated H2S‐imposed growth restriction. Overexpression of the sulfide oxidation enzymes in colon cancer tissue and cells confers a growth advantage during H2S exposure compared to non‐malignant cells. These results predict that the stress response in colonocytes triggered by microbial H2S exposure involves metabolic reprogramming to recycle electron acceptors.Support or Funding InformationNIH (GM112455 to R.B.), AACR NextGen Grant for Transformative Cancer Research (17‐20‐01‐LYSS) to C.A.L., UMCCC Core Grant (P30 CA046592) to R.B. and C.A.L.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Metabolic Tracing and Quantification of β‐Alanine in Wheat
Authors: Reinhart Peter E , Rouhier Kerry A 
Published: June 2021

There are few plants that come close to having the same global importance as wheat. While mainly grown in the middle latitudes and northern hemisphere, the growth range of wheat nearly encompasses the entire globe. As a grain, wheat provides essential phytochemicals and dietary fiber for the human diet in addition to vitamins, minerals, and nutrients. Due to its widespread relevance, wheat was chosen as a system to study the synthesis of β‐alanine, a key precursor for the synthesis of vitamin B5 (pantothenate) and subsequently coenzyme A, which is an essential coenzyme in lipid and carbohydrate metabolism. While the synthesis of β‐alanine is well characterized in bacterials systems, it is less so in plants. Currently the proposed precursors include polyamines, uracil, and propionate and appear to be plant‐specific. Recent data from our lab suggests that β‐alanine can be synthesized from isoleucine via the propionyl‐CoA pathway in the model plant Arabidopsis thaliana. To determine whether this proposed pathway is plant‐specific, we investigated the metabolic profiles of wheat seedlings as a proxy for monocotyledon plants using gas chromatography‐mass spectrometry (GC‐MS). β‐Alanine was found in lower amounts than in Arabidopsis, however, subsequent metabolic tracing studies using 13C‐labeled precursors showed that wheat synthesized β‐alanine from both isoleucine and propionate as seen in Arabidopsis. While other proposed synthetic routes seem to be plant specific, these findings not only support the hypothesis that isoleucine functions as a precursor in the synthesis of β‐alanine but also that it also occurs in both monocotyledon and dicotyledon plants.Support or Funding InformationKenyon Summer Science Scholars ProgramThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The conserved hydrophobic sequence LLI of yeast Spo7 is required for its regulatory role in Nem1‐Spo7 phosphatase function
Authors: Mirheydari Mona , Han Gil‐Soo , Carman George M 
Published: June 2021

Nem1 (catalytic subunit) and Spo7 (regulatory subunit) form a nuclear/endoplasmic reticulum‐associated protein phosphatase complex that regulates the phosphorylation state and function of phosphatidate phosphatase Pah1 in yeast. By dephosphorylating Pah1, the Nem1‐Spo7 phosphatase facilitates the association of Pah1 with the membrane for its function to convert phosphatidate to diacylglycerol for triacylglycerol synthesis. In this work, a mutagenesis analysis of Spo7 was conducted to gain information on the structural requirements for the protein's role in regulating Nem1‐Spo7 phosphatase function. A nested deletion analysis of the N‐terminal region of Spo7 indicated that amino acid residues 46–56 were required for Nem1‐Spo7 function as scored by a temperature sensitivity assay of yeast cells common to that exhibited by cells lacking Pah1. A conserved hydrophobic sequence LLI is contained within this region, and the importance of this sequence to Spo7 function was examined by site‐directed mutagenesis. Whereas all three hydrophobic amino acids affected Nem1‐Spo7 function, Leu‐54 had the greatest effects on lipid metabolism. Cells carrying the L54R mutation had much reduced levels of triacylglycerol and elevated levels of phospholipids and fatty acids, indicating a defect in the dephosphorylation and function of Pah1 phosphatidate phosphatase activity. The importance of Leu‐54 to the interactions of Spo7 with Nem1 and/or Pah1 are currently under investigation.Support or Funding InformationSupported by NIH grant GM050679This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Leukotriene D4 and prostaglandin E2 synergism in inflammation and asthma
Authors: Chachkovskyy Tatiana , Kondeti Vinay , Thodeti Charles K. , Paruchuri Sailaja 
Published: June 2021

Mast cells (MCs) are effector cells in asthma and their activation causes secretion of cysteinyl leukotrienes (cys‐LTs) and prostaglandins (PGs). MCs not only secrete these mediators, but they also possess receptors for them, to perceive their signals. Cys‐LTs are potent bronchoconstrictors, powerful inducers of vascular leakage, potentiators of airway hyper‐responsiveness and play an important role in asthma and other inflammatory disorders. On the other hand, Prostaglandin E2 (PGE2) function in the field of asthma is controversial acting both as pro and anti‐inflammatory, depending mainly on the receptor/s through which PGE2 exerts its effect. Our results indicate that cys‐LTs together with PGE2 synergistically potentiated vascular inflammation through their action on MCs (enhanced calcium flux, c‐Fos, COX‐2, PGD2 and Macrophage Inflammatory Protein‐1b (MIP‐1b; CCL4) generation). Interestingly, LTD4‐PGE2 synergism is blocked only by combined treatment of CysLT1R antagonist (MK571/singulair) and EP3 antagonist (L‐798), suggesting the need for a combination of CysLT1R antagonists and EP3 blockers to treat inflammation in asthma. Furthermore, LTD4+PGE2 synergism also potentiates pulmonary inflammation in D. farinae‐induced airway remodeling during asthma in vivo (recruitment of immune cells, goblet cell metaplasia, up‐regulation of inflammatory transcripts). Our data suggests that LTD4 has a potential to switch PGE2 signals from EP2/Gs/cAMP/PKA pathway to EP3/Gi/cGMP/PKG axis, generating pro‐inflammatory signals and MC activation via PPARƔ, COX‐2 and PGD2. Our studies carry substantial pathogenic and therapeutic implications for asthma and allergic diseases, and provide the basis for development and translation of future therapeutic molecules to target inflammation.Support or Funding InformationThis work is supported by NIH R15 grant (1R15HL133918), and James Foght Assistant Professor 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.
Mapping Perilipin 5 interactions via Protein Crosslinking Studies
Authors: Forney Nathan B , Tansey John T 
Published: June 2021

Many human health issues in the United States are related to lipid metabolism, heart disease, obesity, type II diabetes, and non‐alcoholic fatty liver disease among them. Nearly all cells store neutral lipids in lipid storage droplets. Perilipins are a family of proteins known to associate with these lipid droplets. In particular, perilipin 5 is more strongly expressed in highly oxidative tissues such as cardiac tissues and fasting liver. Previously, we have identified a cytosolic pool of perilipin 5 which exists on small, lipoprotein‐like lipid droplets. Based on that work we hypothesized that quantification of perilipin 5 on these lipid droplets could identify interaction partners and help provide clues to perilipin function. To test this, cells expressing perilipin 5 are disrupted and lipid droplets are isolated. Bis[sulfosuccinimidyl] suberate (BS3) is used to crosslink proteins on these microdroplets. These cross‐linked proteins can then be separated by SDS‐PAGE and proteins crosslinked to perilipin 5 identified by western blotting. Overall, this data will allow for identification of perilipin 5 interaction partners on these structures and help identify if perilipin 5 acts as a monomer or multimer on lipid droplets.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
High‐Resolution Chronology of Murine Biological Responses to 70% Partial Hepatectomy
Authors: Barnhart Benjamin Karl , Hengstler Jan G , Hoek Jan B , Vadigepalli Rajanikanth 
Published: June 2021

The regenerating liver orchestrates an organ‐wide reprogramming of transcription. This results in a precise recovery of liver mass by regulating not only cell cycle progression, but a diverse set of cell functions, metabolic, catabolic, immune, and lysosomal. Here, we attempt to create an organ‐scale, high‐resolution chronology of major biological processes affected by liver regeneration after 70% partial hepatectomy (PHx), with attention given to cell‐type population dynamics. A 70% PHx was performed on C57BL/6 mice and total RNA was isolated from liver tissue at 8 time points spanning 1 hour to 1 week post‐operation. A microarray (GeneChip™ Mouse Genome 430 2.0, Applied Biosystems) was used to quantify transcripts from the whole genome. Differential expression was calculated at each time point, and visualized using a novel pattern‐based approach developed by our lab. Sub‐clusters determined by patterns of expression were evaluated using the Database for Annotation, Visualization and Integrated Discovery (DAVID) bioinformatics resource (6.8), with specific focus on the biological process information from the Gene Ontology Consortium. Highly enriched annotations for specific processes were used as the basis for process entries on the biological timeline. Cell‐specific markers were identified among the differentially expressed genes to show the dynamics of macrophage and hepatic stellate cell activation. While pro‐inflammatory macrophage activity coincides with HSC activation, pro‐restorative macrophages activity grows slowly during recovery. Taken together, this detailed chronology of biological processes reveals the coordination of responses after partial hepatectomy, specifically the timing and coordination of non‐parenchymal cell activity. This information is critical for understanding how global transcriptional changes can regenerate and regulate liver mass, as well as for designing computational simulations at the molecular, cellular and organ‐level scales.Support or Funding InformationResearch was supported by NIH grant: 5U01EB023224‐02This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Human brain microvascular endothelial cells produce CXCL9 after IFN gamma stimulation through JAK2/STAT1 Activation
Authors: Winford Edric , Davis Stephanie , Pennypacker Keith 
Published: June 2021

BackgroundEmergent large vessel occlusion (ELVO) is the deadliest form of stroke and is caused by a blockage within a major a cerebral artery, usually the middle cerebral artery. This condition triggers edema, water movement into the brain, thus pressuring the brain producing massive damage resulting in disability and death. Leukemia inhibitory factor (LIF), a neuroprotective and anti‐inflammatory cytokine, decreases neurodegeneration and increases survival in a rat model of ELVO. Previously we have reported IFNγ is the primary inflammatory mediator in this stroke model and treatment with LIF decreases its expression. This study examined the presence of IFNγ on human endothelial cells by measuring the expression of the IFNγ‐induced CXCL9 and determine LIF's ability to block IFNγ signaling.MethodsFor cells under normoxia conditions, cells were plated and allowed to mature for 7 days, then stimulated with 1 ng/ml IFNγ. At time points 6, 24, 48 and 72 hours after stimulation the supernatant was taken and used for ELISA to determine the amount of CXCL9 released. For OGD conditions, cells were allowed to grow for a week and given glucose free media and placed in oxygen free chamber for 6 hours. The cells were then perfused with media with glucose and 1 ng/ml IFNγ. The supernatant was collected the same time points previously mentioned and CXCL9 was determined as stated previously. To determine the effects inhibitors had on CXCL9 release after being treated with inhibitors to JAK2 and STAT1 under normoxic conditions, cells were treated with IFNy the same as above along coupled with 50 μM and 100 M of fludarabine and AG490.ResultsExposing cerebral microvascular endothelial cells to 1 ng/ml IFNγ significantly induced expression of CXCL9 under both normoxia and oxygen‐glucose deprivation (OGD). Inhibitors to JAK2 and STAT1 significantly blocked the induction of CXCL9. Under normoxic conditions, the addition of LIF (200 ng/ml) with IFNγ significantly augmented the increase in CXCL9 in endothelial cells exposed to OGD.ConclusionCerebral microvascular endothelial cells are a major source of CXCL9, which is upregulated by IFNγ and LIF in a JAK2/STAT1‐dependent manner. The LIF signal transduction pathway in these cells has previously been shown to be dependent upon kinases such as ERK and Akt. CXCL9 is known to break down the blood‐brain barrier, which leads to edema. One potential explanation for this induction of CXCL9 is to open the blood‐brain barrier and allow for anti‐inflammatory leukocytes to enter the ischemic brain and promote repair after stroke.Support or Funding InformationThis project was funded by NINDS Award # 5R0INS091146‐04This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Menthol Inhibits UGT2B10‐catalyzed Detoxification of the Potent Tobacco‐carcinogen, NNAL
Authors: Kozlovich Shannon , Blot William J , Chen Gang , Lazarus Philip 
Published: June 2021

Menthol may be added to tobacco products as either naturally occurring L‐menthol or synthesized racemic DL‐menthol and is excreted as a menthol glucuronide (MG). The most potent tobacco carcinogen is 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK). NNK is extensively metabolized to enantiomers [(R) or (S)] of the equally carcinogenic chiral 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanol (NNAL). Both (R)‐ and (S)‐NNAL have been shown to be carcinogenic. Detoxification of (R)‐ and (S)‐NNAL through glucuronidation on NNAL's pyridine ring nitrogen (NNAL‐N‐gluc) is primarily mediated by UGT2B10. Menthol has been shown to inhibit NNAL‐N‐gluc formation in human liver microsomes (HLM). The goal of the present study was to elucidate the impact of menthol on NNAL‐N‐gluc formation in vivo. In vitro inhibition assays using HLM or microsomes from UGT2B10 over‐expressing HEK293 cells were performed with racemic (R/S)‐NNAL as substrate with each menthol enantiomer added separately as inhibitor. In vivo data were gathered from analysis of MG, NNAL, and NNAL metabolites from 50 menthol smokers and 50 non‐menthol smokers. Samples were obtained from the Southern Community Cohort Study. Both L‐ and D‐menthol enantiomers were inhibitors of UGT2B10 in vitro with IC50 values of 236 and 202 μM, respectively. In vivo D‐MG levels, when present, were far below the levels of L‐MG observed in smokers of both menthol or non‐menthol cigarettes, with D‐MG comprising an average of 1.3% of total MG. Urinary L‐MG was detectable in 98 subjects and levels were not significantly different between menthol and non‐menthol smokers (p=0.50). When stratified by tertiles of urinary MG levels, NNAL‐N‐gluc levels were highest in smokers with the lowest MG levels, with NNAL‐N‐gluc levels inversely associated with MG levels in smokers (p<0.05). These data suggest that menthol inhibits a major detoxification pathway of the tobacco carcinogen, NNK. The relatively high levels of urinary menthol in smokers of both menthol and non‐menthol cigarettes raise the possibility that menthol branding may not be the best approach to studying the impact of menthol as a tobacco additive.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 and Characterization of Clinically Relevant Non‐Small Cell Lung Cancer Mouse Model with Near‐Infrared Fluorescent 3D Spheroids
Authors: Huang Yingbo , Lu Yifan , Vadlamudi Mallika , Guo Xin 
Published: June 2021

BackgroundLung cancer claims the highest mortality among all cancers in the US and leads the development of new cancer cases in developing countries. Pre‐clinical mouse models hold an essential role in guiding the development of new therapeutics for lung cancer [1]. Injection of suspended cancer cells into the lung is one of the most common approaches to build an orthotopic lung cancer model, but carries several major disadvantages, including the omission of a primary solid tumor and the artificial leakage of the injected cancer cells [2][3]. These drawbacks limit the application of such model in evaluating potential therapeutics for metastasized lung cancer. Herein, tumor spheroids were injected transpleurally to develop an orthotopic lung cancer model in mice, which then progressed through all the four clinical stages of non‐small cell lung cancer. Moreover, the near‐infrared fluorescent signal of the tumor cells allows efficient and convenient tracking of the cancer progression compared to traditional imaging methods, such as CT/PET and bioluminescence.PurposeThe purpose of this study is to build a more clinically relevant, orthotopic lung cancer model by inoculating 3D spheroids of lung cancer cells into the mouse lung.MethodThe 3D spheroids were cultured by seeding human lung cancer cell line A549‐iRFP in Corning 96 multicellular microplate. The cells stably expressed infrared fluorescent protein, whose signal was detected on an Odyssey CLx imaging machine (lex = 690 nm, lem = 700 nm) to image the tumor growth in vivo. Tumor inoculation was performed by transpleural surgery on the left side of the athymic nude mice.ResultsThe fluorescence of the spheroids was proportional to their volume and viability (R2=0.9805 and 0.9191). The mice implanted with tumor spheroid showed tumor growth confined to the inoculation side of the lung. Furthermore, the in vivo imaging showed cancer development from a primary tumor to metastasis within one month. The anatomical observations and ex vivo imaging confirmed the tumor location. In comparison, mice that were injected with suspended cancer cells displayed perfused the fluorescent signal on both sides of the lung indicating premature leakage of the inoculated cancer cells.ConclusionThe mice inoculated with suspended cancer cells displayed artificial leakage of the injected cells inside the lung, which decreased its clinical relevance of mimicking lung cancer progression. In contrast, mice inoculated with the 3D cancer cell spheroids better mimic the progression of non‐small cell lung cancer in the clinic.
imageThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.