bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Mechanism of Protein Kinase A in Fibrolamellar Hepatocellular Carcinoma
Authors: faradonbeh mahsa shirani
Published: June 2021

Fibrolamellar Hepatocellular Carcinoma (FL‐HCC) is a rare form of liver cancer that affects adolescents and young adults without a prior history of liver disease. Chemotherapy is ineffective in treating FL‐HCC, and surgical resection of the primary tumor and metastases is currently the only semi‐effective treatment. Reoccurrence rates in FL‐HCC are extremely high.

A 400kB deletion in chromosome 19 results in the fusion of the first exon of a heat shock protein, DNAJB1, with the catalytic subunit of protein kinase A, PRKACA (1). The new chimeric protein, DNAJB1‐PRKACA, retains the full catalytic activity of protein kinase A. We have performed partial biophysical characterization of DNAJB1‐PRKACA in comparison to PRKACA. Our preliminary results indicate the structural stability of DNAJB1‐PRKACA may differ from that of PRKACA. We also report the DNAJB1‐PRKACA chimera forms a stable complex with its regulatory domains, and differences in structural stability may extend to the chimera/regulatory domain complex. Surface plasmon resonance (SPR) does not reveal any difference in the binding affinity of DNAJB1‐PRKACA and wild type PRKACA to their Regulatory subunit type Iβ.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Oxidative Metabolism and Comparative Analysis of Synthetic Cannabinoid N‐(1‐adamantyl)‐1‐(5‐fluoropentyl)indazole‐3‐carboxamide (5F‐AKB‐48) and the Unfluorinated Analog AKB‐48
Authors: Yarbrough Azure Leigh , Pinson Anna , Prather Paul L. , Fantegrossi William E. , Bush John M. , Fujiwara Ryoichi , Radominska‐Pandya Anna
Published: June 2021

Recently, the rising frequency in the abuse of synthetic cannabinoids (SCBs) has resulted in the occurrence of numerous severe adverse effects. We have previously shown that several early generations of SCBs undergo extensive metabolism by cytochrome P450s (P450s) and that their metabolites retain biological activity at CB1 and CB2 receptors. In this study, we examined the oxidative metabolism of 5F‐AKB‐48, one of a new structural generation of SCBs. Activity towards 5F‐AKB‐48 was investigated using various human microsomes followed by studies utilizing multiple recombinant human P450s. Screening with these enzymes showed that only 4, CYP2D6, −2J2, −3A4, and −3A5, biosynthesized the 2 major products, mono‐ and dihydroxylated 5F‐AKB‐48, with activities in the range of 2–5 pmol/min/pmol CYP. CYP3A5 formed the most dihydroxylated metabolite, whereas CYP2J2, highly expressed in cardiovascular tissues, showed the biosynthesized the most monohydroxylated derivative. CYP2D6 produced only monohydroxylated derivatives whereas CYP3A4 produced mostly dihydroxylated ones. To elucidate the roles of CYP3A4 and CYP3A5, additional enzymatic reactions utilized genotyped HLMs containing normal activity for CYP3A4 but low or high activities of CYP3A5. Those experiments resulted in significantly different patterns of oxidized metabolites. An UPLC equipped with UV/Vis detector and/or MS/MS were used to analyze the reactions. The profiles of the products biosynthesized were significantly different between the fluorinated and unfluorinated analogs. As this structural difference is known to increase potency for CB1 receptors, understanding the involved mechanism is essential. In conclusion, extensive metabolic studies of SCBs, including investigation of the mechanism(s) of action leading to toxicity, could result in the development of life saving, efficacious treatments for SCB abuse.

Support or Funding Information

(INBRE 117‐1006096 2018 AP and NIH/NIDA DA039143 to ARP and PLP)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Do Lactic Acid Bacteria produce antifungal bacteriocins?
Authors: Turcotte Madison , Allen Mary E , Piefer Andrew J
Published: June 2021

Bacteriocins are small antimicrobial peptides that are produced by bacteria and are known to inhibit the growth of closely related or non‐related bacterial strains without causing harm to the producer strain. Bacteriocins are categorized into several classes and lactic acid bacteria (LAB) are known to produce class IIa bacteriocins which are described as small, heat‐stable peptides. LAB produced bacteriocins could create a wide range of applications in food preservation, which may present a safer alternative than currently used chemical preservatives. The mold Fusarium graminearum frequently contaminates barley malt, which along with water, yeast, and hops form the ingredients of beer. Hydrophobic compounds produced by F. graminearum causes beer to retain high levels of carbon dioxide, which results in gushing, or excessive foaming when bottled. Gushing can damage the reputation of a brewery. Thus, malt producers are searching for a way to control this grain contaminant. Recently, several studies demonstrating potential antifungal properties of bacteriocins were reported. Using a sourdough starter, we were able to isolate environmental strains of LAB by growing them anaerobically on MRS agar medium. We will then test their potential antifungal properties against F. graminearum using traditional microbiological methods. If we can determine that bacteriocins produced by LAB are in fact antifungal, we will then use PCR to rapidly detect bacteriocin genes that might be present in these strains. Potential bacteriocins will be exposed to a variety of experimental conditions such as protease treatment, pH, and detergents for further classification and characterization.

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‐Fibrotic Effects of HDAC8 Inhibitor in Bleomycin‐Induced Pulmonary Fibrosis Mouse Model–Whole Transcriptome Analysis Using RNA‐seq
Authors: Saito Shigeki , Zhuang Yan , Lasky Joseph , Liu Yaozhong
Published: June 2021

Background

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive lung disease with high mortality. Current therapies for IPF have only limited efficacy, thus there is a critical need for more effective medications. We have previously reported that HDAC8 inhibition represses TGF‐β1‐induced fibroblast‐myofibroblast differentiation in lung fibroblasts and that an HDAC8‐selective inhibitor NCC170 ameliorates bleomycin‐induced pulmonary fibrosis in mice. Here we report our recent analyses of RNA‐seq data derived from the NCC170‐treated mice.

Methods

On Day 0, mice were given Bleomycin or PBS (=vehicle) via oropharyngeal aspiration. On Day 7–13, mice were given NCC170 or DMSO (=vehicle) daily via intraperitoneal injection. On Day 14, lungs were harvested and total RNA was isolated for RNA‐seq. Differentially expressed genes between the PBS+DMSO group (n=4) and the Bleomycin+DMSO group (n=4) as well as between the Bleomycin+DMSO group and the Bleomycin+NCC170 group (n=4) were identified using DESeq2. Differentially regulated pathways between these groups were identified using Generally Applicable Gene‐set Enrichment (GAGE) analysis. To confirm the findings from RNA‐seq and GAGE analyses, THP‐1 cells (human monocytic cells) were differentiated into macrophages with PMA, and then treated with HDAC8 siRNA or scramble siRNA. The effects of HDAC8 knockdown on expressions of pro‐fibrotic cytokines were assessed using immunoblots.

Results

The comparison between the PBS+DMSO group and the Bleomycin+DMSO group revealed that the Bleomycin treatment upregulated 3330 genes (>1.5 fold change) and downregulated 2598 genes (<0.66 fold change) with FDR<0.05. The comparison between the Bleomycin+DMSO group and the Bleomycin+NCC170 group revealed that the NCC170 treatment upregulated 182 genes and downregulated 454 genes. GAGE analysis suggested that NCC170 repressed bleomycin‐induced activation of several pathways, including well expected pathways (e.g., extracellular matrix ‐ receptor interaction) as well as novel pathways (e.g., innate immunity). Our in vitro study confirmed that HDAC8 knockdown repressed pro‐fibrotic cytokines (e.g., IL‐6) in PMA‐treated THP‐1 cells.

Conclusion

Our RNA‐seq analysis provided novel insights into the mechanism through which HDAC8 inhibition exerts anti‐fibrotic effects in a bleomycin‐induced pulmonary fibrosis mouse model, a widely‐used animal model relevant to IPF. The role of HDAC8 in innate immunity during pulmonary fibrosis development should be investigated further.

Support or Funding Information

Wetmore 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.
ERK Controls Myosin Phosphatase and Cell Migration through RSK Phosphorylation of MYPT1
Authors: Mendoza Michelle C , Samson Shiela , Elliott Andrew , Carney Keith
Published: June 2021

Cell migration is essential to embryonic development, wound healing, and cancer cell dissemination. Cells move via leading edge protrusion, substrate adhesion, and retraction of the cell's rear. However, the signaling mechanisms by which extracellular cues control the actomyosin cytoskeleton to induce motility are poorly understood. The growth factor‐responsive and oncogenically activated ERK kinase promotes motility by signaling for actin polymerization‐mediated edge protrusion. ERK has previously been proposed to also control the contractile machinery through myosin light chain kinase (MLCK). Here, we show that ERK indeed signals to myosin, but rather through its substrate, p90 ribosomal S6 kinase (RSK). By probing the signaling and migration dynamics of a cell line panel, we found that RSK phosphorylates the myosin phosphatase targeting subunit (MYPT1) at Ser507, which promotes Rho kinase (ROCK) interaction with and inhibition of MYPT1. By inhibiting the myosin phosphatase, ERK and RSK promote the myosin II–mediated tension necessary for lamella expansion and protrusion and retraction persistence. This level of myosin activation promotes optimal edge dynamics for cell migration, rather than inducing a strong contractile response. In conclusion, our findings suggest that ERK activity can coordinately amplify both protrusive and contractile forces for optimal cell motility.

Support or Funding Information

K01CA168850, ALA Research Grant, and American Cancer Society RSG CSM130435 to MCM

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 Role of eIF2α in the Collagen Production in Hepatic Stellate Cell is Associated with Autophagic and Apoptotic Signaling
Authors: Qi‐Lin Liu , Teng Lin Hua , Liu Ze‐Feng , Li Fu‐Jian , Chen Li , Zhang Guo
Published: June 2021

Background & Aims

Studies have documented cellular apoptosis, autophagy and endoplasmic reticulum (ER) stress participate in the liver fibrogenesis. However, the specific mechanism remains unclear. This study was aimed to determine whether and how eIF2α protein play a role in the collagen generation in hepatic stellate cell (HSC) via autophagic signaling events.

Methods

HSC‐LX2 cell line was activated by transforming growth factor beta1 (TGF‐β1) in the presence of eIF2α plasmid or vehicle control. Real‐time fluorescence quantitative PCR was then performed to compare the differential mRNA level of COL1, eIF2α, CHOP, Bcl‐2 and Bax. Western Blot was performed to analyze the protein expression of α‐SMA, COL1, LC3, P62, eIF2α, P‐eIF2α and Bcl‐2 in LX2 cells. Meanwhile, the numbers of apoptotic cells were measured by flow cytometry.

Results

Compare to vehicle control, the ER stress hallmarks of eIF2α, CHOP were at lower level in HSC‐LX2 cells in TGF‐β1 treated condition. While transfected eIF2α plasmid in HSC‐LX2 cells, it was found that CHOP and BAX significantly increased in these cells but substantial reduction in LC3, P62, Bcl‐2, α‐SMA and COL1 v.s. control(<0.05). Moreover, thus exogenously enhancing of eIF2α gene was found to induce apoptosis of HSC‐LX2 cells with a 50% increment (<0.05).

Conclusions

Overexpression of eIF2α in HSCs may trigger autophagy inhibition and apoptosis promotion, thereby reduce collagen production in these cells, which provide a potential therapeutic strategy for hepatic fibrosis.

Support or Funding Information

Supported by NSFC Grants 81360077, 81860654, 30630145, 81172260 and GXNSFC Grants 2015GXNSFDA139022 to Guo Zhang

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 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 Information

The 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 Information

NIH (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 Information

Kenyon Summer Science 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.
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 Information

Supported by NIH grant GM050679

This 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 Information

This 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 Information

Research was supported by NIH grant: 5U01EB023224‐02

This 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

Background

Emergent 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.

Methods

For 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.

Results

Exposing 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.

Conclusion

Cerebral 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 Information

This project was funded by NINDS Award # 5R0INS091146‐04

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

Background

Lung 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.

Purpose

The 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.

Method

The 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.

Results

The 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.

Conclusion

The 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.

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This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of Lymphangiogenesis in Adipose Tissue Upon Local, Induced VEGF‐D Overexpression
Authors: Chakraborty AA , Rizwan K , Khetan JN , Scogin CK , Rutkowski JM
Published: June 2021

Inflammatory adipose tissue expansion is the root of the metabolic syndrome in obesity. Insulin‐resistant adipocytes and dysregulated lipolysis results in increased circulating lipids, ectopic lipid deposition, and systemic insulin resistance. Lymphangiogenesis ‐ expansion of the lymphatic vessel network ‐ is a necessary process in clearing fluid and immune cells during inflammation. Lymphatic capillaries are, however, largely absent from murine adipose tissues making induced lymphangiogenesis a potential target in adipose inflammation. We have previously described a mouse model of inducible, adipose tissue‐specific expression of murine VEGF‐D that exhibits de novo lymphangiogenesis in white and brown adipose tissue. Our previous studies suggest that enhanced adipose lymphangiogenesis reduces obesity‐associated inflammation and improves adipose metabolism. We have observed that induced adipose lymphangiogenesis is only robust after nearly 4 months of elevated VEGF‐D levels and the extent varies across adipose depots in Adipo‐VD mice. The mechanism of VEGF‐D initiated lymphatic formation in each adipose depot is therefore potentially different, with processes involving either sprouting or lymphvasculogenesis. Our current work quantifies the lymphangiogenic time‐course, studying adipose tissues at 1, 2, 3, and 4, months following VEGF‐D induction. Comparison across adipose depots, sex, and diet (chow versus 60% kcal from fat diet) are made using qPCR for lymphatic markers as well as immunofluorescence of lymphatic structures on fixed and whole mount tissues. Correlation of adipose tissue macrophage infiltration/accumulation with lymphatic density are also performed. In addition to a quantified time course, our whole mount imaging and lineage tracing suggest that lymphangiogenesis in subcutaneous adipose depots – the white inguinal and brown interscapular – may arise from dermal lymphatic endothelium. Understanding how lymphatics grow and impact adipose tissue health thus provides a novel target in obesity's metabolic syndrome.

Support or Funding Information

LIPEDEMA 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.
Hyperhomocysteinemia and the effects of Lactobacillus rhamnosus GG on cardiac functions in CBS+/− mice
Authors: George Akash K , Singh Mahavir , Majumder Avisek , Homme Rubens P , Metreveli Naira , Pushpakumar Sathnur B , Tyagi Suresh C
Published: June 2021

Hyperhomocysteinemia (HHcy) is one of the prominent risk factors for precipitating adverse cardiac events. Among the known causes, dysregulated homocysteine (Hcy) metabolism especially during dietary deficiency of the vitamins ‘B’ complex is a key cause of HHcy in susceptible population. Lactobacillus rhamnosus GG is a widely used probiotic, and its salutary effects have been extensively documented on host metabolism. The main objective of this study was to investigate the role of HHcy on cardiovascular dysfunction and the effect of Lactobacillus rhamnosus GG on Hcy metabolism. In this study, CBS+/− mice representing HHcy and C57BL/6J (wild type; WT) were administered orally Lactobacillus rhamnosus GG @ 2.5×105 CFU once daily for 12 weeks. The animal groups were: Group‐I: WT + regular chow diet, Group‐II: WT + regular chow diet + Lactobacillus rhamnosus GG, Group‐III: CBS+/− + High Methionine Diet (HMD), and Group‐IV: CBS+/− + HMD + Lactobacillus rhamnosus GG. In CBS+/− + HMD mice, angiography revealed reduced coronary vascularity and impaired cardiac functions which were improved with Lactobacillus rhamnosus GG treatment. Further experimentations on isolated adult cardiomyocytes from the CBS+/− + HMD + Lactobacillus rhamnosus GG lent credible support to the observed beneficial effects of the probiotic on cardiac functions. In addition, Lactobacillus rhamnosus GG administration reduced HHcy‐induced oxidative and ER stress levels in the CBS+/− + HMD mice that were revealed via expression analyses of the key marker proteins in the cardiac tissues of probiotic treated mice groups. Our findings indicated that Lactobacillus rhamnosus GG administration helped mitigate the adverse cardiac effects as induced by chronic HHcy environment.

Support or Funding Information

The work was supported by grants from the National Institute of Health (HL‐74815, HL‐139047, and AR071789).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Influence of resistance exercise training in diabetic hypertrophy renal: The role of mTOR and Acetyl CoA Carboxylase
Authors: Jorge Luciana , Silva Kleiton A. S. , Reinecke Natalia , Da Silva Germano Jorge Maria Lucia , Schor Nestor
Published: June 2021

Diabetic kidney disease (DKD) is a progressive complication that develops from diabetes. An important change observed is renal hypertrophy associated with the mTOR pathway. On the other hand, increasing evidence indicates that the reduction in the phosphorylation of Acetyl‐CoA Carboxylase (ACC) may be important in the development of Renal Fibrosis. Therefore, it is fundamental to understand non‐pharmacological intervention as the effect of resistance training (RET) to prevent these complications.

In this study, our aim is evaluating the RET on the renal hypertrophy.

Male Wistar rats were divided into 4 groups: control (C=8), control trained (CT=8), diabetic (D=8) and diabetic trained (DT=8). DM (glycaemia>250mg/dL) was induced by Streptozotocin (STZ) (50mg/kg, i.v.). Trained groups were submitted to a resistance exercise training protocol on Ladder climb (8wk). The expression of Akt/mTOR pathway was evaluated by two technical, western blotting and multiplex. Immunohistochemical was used to analyze the expression by ACC. A hypertrophy renal index was calculated by the reason the kidney weight/body weight. The histomorphology was analyzed by hematoxylin and eosin stain.

RET improved renal parameters in the DT group showing a decreased proteinuria (DS:15±1.5; DT:16±2.0; CS:38±3; CT:16±2mg/24h). The renal expression of mTOR and Akt, was increased in the DS animals, and the early RET was able to prevent, the same results were observed in the hypertrophy renal index (p<0,05). The ACC was decreased in DS group and RET normalized this expression (p<0,05). Surprisingly, when analyzing the morphology was not find any difference between the groups.

Our findings showed that RET was able to prevent renal hypertrophy, molecularly, and improvement of the renal parameters induced by experimental diabetes. These results demonstrate that the RET the clinical application in early diabetic patients may help prevent and/or treat the renal hypertrophy.

Support or Funding Information

Sources of Funding This study was supported by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico (CNPq).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Muscle Metaboreflex Induced Increases in Effective Arterial Elastance
Authors: Mannozzi Joseph T , Al‐Hassan Mohamed , Lessanework Beruk B , Alvarez Alberto , O'Leary Donal S
Published: June 2021

Dynamic exercise elicits robust changes in sympathetic activity in part due to accumulation of metabolites within the active skeletal muscle which activates group III and IV afferents triggering the muscle metaboreflex. This reflex raises cardiac output via tachycardia, increased ventricular contractility, and central blood volume mobilization which improves perfusion to the active skeletal muscle. An important component of maintaining exercise performance is optimal ventricular ‐ vascular coupling which can change with exercise, aging, as well as pathophysiological conditions. The effect of muscle metaboreflex activation (MMA) on ventricular ‐ vascular interactions is unknown. We hypothesized that MMA increases Effective Arterial Elastance (Ea). We utilized two previously published methods of evaluating Ea (End Systolic Pressure / Stroke Volume (M1) and Heart Rate x Vascular Resistance (M2)) at rest, during mild treadmill exercise, and during MMA induced via partial reductions in hindlimb blood flow imposed during exercise. At rest Ea averaged 3.0 ± 0.1 and 3.0 ± 0.2 mmHg/mL for M1 and M2, respectively. With the transition from rest to mild exercise little change in Ea occurred, whereas with subsequent MMA, Ea increased significantly by 33.6% ± 2.7 and 20.5% ± 2.3 for M1 and M2. We conclude that MMA elicits robust increases in Ea which parallel the substantial increases in ventricular performance thereby optimizing ventricular ‐ vascular coupling.

Support or Funding Information

Supported by HL‐55473, HL‐126706, HL120822 and R25 GM058905

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Skin Erythema and Blood Flow Responses to Acute Ultraviolet Radiation Exposure
Authors: Berry Craig W. , Hill Imani N. , Wolf S. Tony , Stanhewicz Anna E. , Kenney W. Larry
Published: June 2021

Acute exposure to ultraviolet (UV)‐B light elicits an inflammatory response in the skin, resulting in erythema (reddening) and increased skin blood flow over the following 24 hours. However, the time course of, and relation between, these responses is unclear. Likewise, it is unknown whether these responses differ following exposure to broad spectrum (UV‐AB) vs. UV‐B light, as well as how application of sunscreen on the skin impacts these two responses.

Purpose

To examine (1) the time course of erythema and skin blood flow responses over 8 h following acute UV‐B and UV‐AB exposure, and (2) the impact of sunscreen application on those responses. We hypothesized that (1) the erythema and blood flow responses would track each other, (2) both responses would be higher following UV‐AB compared to UV‐B exposure, and (3) sunscreen would attenuate responses to both UV‐AB and UV‐B.

Methods

The ventral aspect of both forearms of 10 healthy young adults (25±4 yrs; 6M/4F) were exposed to either UV‐AB (10 mJ/cm2; 6 mJ/cm2 UVA + 4 mJ/cm2 UV‐B) or UV‐B (4 mJ/cm2). Two 2‐cm2 sites on each arm were randomly chosen for acute UV exposure with or without pre‐treatment with topical SPF‐50 sunscreen. Erythema index (EI; reflectance spectrometry) and red cell flux (laser‐Doppler flowmetry) were measured at each site before, immediately after, and 2, 4, 6, and 8 h post‐exposure. Cutaneous vascular conductance was calculated (CVC=flux/MAP) at each time point and both EI and CVC were expressed as change from baseline.

Results

EI increased linearly after exposure to UV‐B (p<0.01 at 4, 6, and 8 h post‐exposure) and UV‐AB (p=0.02 at 6 h, p<0.01 at 8 h). Unlike the immediate increase in EI, no increase was seen for CVC until 4 h post‐UV‐B (p<0.01) and UV‐AB (p=0.06). Sunscreen effectively blocked the EI response to both UV‐B and UV‐AB exposures (p>0.05) and prevented, the CVC responses to UV‐B (p<0.01 at 6 and 8 hours) and UV‐AB (p=0.03 at 8 h).

Conclusion

Exposure to UV‐B or UV‐AB induced an immediate and linear increase in EI, but a delayed (by 4 h) increase in CVC. Sunscreen prevented the EI response and blunted the rise in CVC. These data suggest that the inflammatory response to acute UV exposure is characterized by an immediate erythema response and a delayed blood flow response in the cutaneous microvasculature and that sunscreen may protect against both responses.

Support or Funding Information

ACSM Foundation Doctoral Student 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.