bluuebunny/crossref_metadata_2025 huggingface dataset:
TNFα Impairs Cerebral Blood Flow Autoregulation in Pregnant Rats
Authors: Duncan Jeremy W. , Warrington J. Paula , Drummond Heather A. , Granger Joey P. , Ryan Michael J.
Published: June 2021

Neurological symptoms are often reported among preeclamptic patients and cerebrovascular events account for ~40% of all preeclamptic/eclamptic‐related deaths. While cerebral blood flow (CBF) autoregulation is impaired in preeclamptic (PE) women as well as in placental ischemic rats which develop a PE‐like syndrome, the underlying mechanisms responsible for abnormal CBF autoregulation are unclear. Placental ischemic rats have higher levels of circulating cytokines, and we previously reported that tumor necrosis factor – alpha (TNFα), at levels that mimic TNFα in human preeclampsia, increases blood brain barrier (BBB) permeability and cerebral edema in pregnant rats. Therefore, we tested the hypothesis that TNFα alters CBF autoregulation in pregnant rats. To test this hypothesis, normal pregnant Sprague Dawley rats were infused with TNFα (200ng/day) and cerebral blood flow was measured by laser Doppler flowmetry under anesthesia. Rats were intubated and ventilated in order to maintain constant blood gasses and mean arterial pressure was elevated step‐wise from 100–190 mmHg by infusion of phenylephrine to determine changes in CBF. TNFα was found to impair CBF autoregulation in pregnant rats where CBF was significantly increased in TNFα‐infused rats at 180 mmHg (225±13 vs 173±8) and 190 mmHg (253±17 vs 188±9) compared to normal pregnant rats with no significant difference in peak CO2 between the two groups at 180 mmHg (44.9±0.8 vs 45.4±0.9 mmHg) or 190 mmHg (43±0.8 vs 45.3±0.9). Autoregulatory index (percent change in blood flow divided by percent change in perfusion pressure) was also significantly increased in TNFα‐infused rats at 180 mmHg (1.56±0.17 vs 0.91±0.11) and 190 mmHg (1.7±0.07 vs 0.99±0.11) compared to normal pregnant rats. These data suggest that circulating TNFα from the ischemic placenta could contribute to cerebrovascular complications during pregnancy and postpartum. TNFα could serve as a novel pharmacological target to ameliorate CBF autoregulation in preeclampsia.

Support or Funding Information

This study was supported by National Institutes of Health Grants, HLHL51971, HL136684‐A0, and an NRSA Institutional Training Grant, T32HL105324.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of the OVLT‐gut microbiome axis on the development of DOCA‐salt hypertension
Authors: Collister John , Nahey David , Hartson Rochelle , Youmans Bonnie , Johnson Tim
Published: June 2021

Limited studies suggest an association of gut microbiome changes with certain forms of hypertension, but a cause/effect relationship has yet to be fully defined. The deoxycorticosterone acetate (DOCA)‐salt model of hypertension in rats is known to have a neurogenic component linked to increased sympathetic nervous system activity. Indeed, we have recently shown the hypertensive response in DOCA‐treated rats requires the organum vasculosum of the lamina terminalis (OVLT), a circumventricular organ that has direct connections to the paraventricular nucleus (PVN) to increase sympathetic activity. Furthermore, celiac ganglionectomy also supports a role of splanchnic sympathetic innervation to the gut in the hypertensive response to DOCA. Taken together, we hypothesize an OVLT‐sympathetic‐gut (OSG) microbiome axis is involved in the development of hypertension. Having previously reported that the OVLT plays a significant role in the development of DOCA‐salt hypertension, the present study was designed to test the hypothesis that the OVLT mediates changes in the gut microbiome associated with concomitant hypertension. In uninephrectomized OVLT lesioned (OVLTx; n = 5) and SHAM (n = 2) Sprague‐Dawley rats consuming a 2% NaCl diet and 0.9% NaCl drinking solution, 24‐hour mean arterial pressure (MAP) was recorded telemetrically 5 days before and 21 days after DOCA implantation (100 mg/rat; SQ). Microbiome analyses were performed on regional gut samples at the end of the study and compared to normal rats (n=2). The chronic pressor response to DOCA was attenuated in OVLTx rats such that MAP increased to 153±4 mmHg in SHAM rats by Day 21 of DOCA compared to 127±5 mmHg in OVLTx rats. Moreover, DOCA hypertension in SHAM rats was associated with decreased levels of Lactobacillus species in the jejunum and ileum, and increased Escherichia levels in the cecum compared with normal rats. However, these changes in bacterial genera were mitigated and offset by lesion of the OVLT and these bacterial populations in OVLT lesioned rats with attenuated hypertension more closely resembled those in normal control rats. We conclude that DOCA‐salt hypertension is associated with specific microbiome changes in the gut, and the attenuated hypertensive effects of DOCA‐salt in OVLT lesioned rats is mediated in part through counteracting changes in these bacterial populations.

Support or Funding Information

University of Minnesota Grant‐In‐Aid # 107426

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Gut Microbiota Effects On Bone Density Are Dependent On T‐ and B‐ lymphocytes
Authors: Rios‐Arce Naiomy Deliz , Schepper Jonathan , McCabe Laura R. , Parameswaran Narayanan
Published: June 2021

Recent studies have shown that gut microbiota is an important regulator of bone density. In addition, it is well known that the gut microbiota can modulate the immune system. However, whether the effects of the gut microbiota on bone health involve the lymphocytes is still unknown. We hypothesized that modulation of gut microbiota will alter bone density, and this effect will be prevented in the absence of lymphocytes. To test our hypothesis, we treated Rag knockout (T and B lymphocyte deficient) mice and the corresponding C57BL/6J wild‐type mice (males, 12 weeks of age) with antibiotics (ABX) ampicillin (1g/L) and neomycin (0.5g/L) in the water for two weeks. After two weeks, mice received water without antibiotics for another 4 weeks to allow the repopulation of gut microbiota. Bone volume fractions (BVF) of the femur were analyzed using microcomputed tomography system. Fecal samples were used to confirm microbiota depletion after 2 weeks and repopulation after 6 weeks. Repopulation of the gut microbiota in the WT‐ABX group significantly decreased femoral trabecular BVF by 24% compared to the non‐ABX treated WT group (BVF/BW WT:.98, WT‐ABX:.74, n= 14–17). In the Rag KO mice, however, repopulation of the gut microbiota following ABX treatment did not affect femoral BVF compared to the respective control group (BVF/BW KO: 1.59, KO‐ABX: 1.49, n=7–11). Analysis of serum bone remodeling parameters indicated significantly lower serum osteocalcin levels (~ 37% of control levels) in WT‐ABX treated mice, while no changes were seen in the RAG‐KO group. Our study demonstrates that repopulation of the gut microbiota can be detrimental to the bone and that this effect is dependent on the T and B lymphocytes.

Support or Funding Information

Porter Fellowship awarded to Naiomy‐Deliz Rios Arce. National Institutes of Health Grants RO1 DK101050 and RO1 AT007695‐01

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Alcohol‐Induced Gut Lymphatic Leak Results in Adipocentric Immunometabolic Dysregulation in Female Rats
Authors: Souza‐Smith Flavia M. , Simon Liz , Siggins Robert , Molina Patricia E.
Published: June 2021

Previously we demonstrated that alcohol induces mesenteric lymphatic vessel (MLV)hyperpermeability, perilymphatic adipose tissue (PLAT) inflammation, and impaired insulin signaling in male rats. MLV play an important role in the trafficking of gut mucosal derived dendritic cells (DC) to mesenteric lymph nodes (MLN). Leakage of DC from MLV has been reported in infectious states. DC promote regulatory T cell (Treg) expansion in adipose tissue and this mechanism has been proposed to be a driver of age‐associated insulin resistance. We hypothesize that alcohol‐induced MLV hyperperemeability results in DC leakage to PLAT and this contributes to immunometabolicdys function in chronic alcohol fed animals. To test this hypothesis, female Fisher 344 rats were randomized to Lieber‐DeCarli liquid diet (36% of calories from alcohol) for 10 weeks or pair‐fed control groups. PLAT, MLN, and peripheral blood lymphocytes (PBL) were isolated for phenotyping by flow cytometry. Alcohol‐fedanimals had increased PLAT Tregs and CD4 T cells and decreased % Tregs in PBLs. These findings suggest that alcohol‐induced lymphatic leakage is associated with DC deviation into PLAT that we speculate mediates Treg expansion and initiatesmetabolic dysregulation. In conjunction with our previous findings, these results support an alcohol‐induced immune cell deviation from the gutlymphnode pathway that promotes PLAT immunometabolic dysregulation and couldpotentially have additional implications on induction of mucosal immunity.

Support or Funding Information

Supported by LSUHSC Department of Physiology.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of germinated brown rice on the LPS‐induced inflammation in adipocytes
Authors: Park Mi‐Young , Lee Young Min
Published: June 2021

Germinated brown rice (GBR, Orysa sartiva L.) has been reported to have anti‐obesity and anti‐inflammatory effects. However, the mechanisms underlying the anti‐inflammatory effect of GBR on adipocytes are not fully understood. The aim of this study was to explore the anti‐inflammatory mechanisms of GBR on lipopolysaccharide (LPS)‐stimulated 3T3‐L1 adipocytes. 3T3‐L1 adipocytes were pretreated with GBR extracts (0–20 mg/ml) 1h before LPS stimulation. The mRNA expressions of adipokines and Toll‐like receptor 4 (TLR4) were measured by RT‐PCR. The protein expressions of TLR4‐related molecules were detected by western blotting and nuclear factor‐κB (NF‐κB) activation was measured. Our results showed that GBR extract dose‐dependently inhibited mRNA expressions of LPS‐induced tumor necrosis factor‐α (TNF‐α), interleukin‐6 (IL‐6), and monocyte chemoattractant protein‐1 (MCP‐1). GBR extract was found to inhibit LPS‐induced mRNA expression of TLR4 and protein expressions of both myeloid differentiation factor 88 (MyD88) and TNF receptor‐associated factor 6 (TRAF6). Furthermore, GBR extract significantly inhibited extracellular receptor‐activated kinase (ERK) phosphorylation and NF‐κB activation. These results suggest that GBR extract has the anti‐inflammatory property on LPS‐induced inflammation via inhibiting TLR4 signaling including ERK and NF‐κB signaling pathways in adipocytes. (This research was supported by the Soonchunhyang University Research Fund.)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Peripheral blood mitochondrial DNA copy number, a potential marker of non‐alcoholic fatty liver disease?
Authors: Tapia Maria Azucena Pescador , Fragoso‐Bargas Nicolas , Rodríguez‐Ríos Dalia , Lazo‐de‐la‐Vega‐Monroy María Luisa , del Rocío Ibarra‐Reynoso Lorena , Ruíz‐Noa Yeniley , del Carmen Preciado‐Puga Mónica , Velazquez‐Villafaña Marion , Jordan‐Pérez Benjamín , Garnelo‐Cabañes Serafin , García‐Ramírez Juana Rosalba , Lund Gertrud
Published: June 2021

Introduction

Nonalcoholic fatty liver disease (NAFLD) includes a spectrum of histological findings ranging from simple steatosis (SS) to non‐alcoholic steatohepatitis (NASH). Although often considered the hepatic manifestation of metabolic syndrome (MS), longitudinal studies suggest that NAFLD precedes MS. To date, liver biopsy is the most accurate method to differentiate between SS and NASH. However, recent studies indicate an association between mitochondrial DNA (mtDNA) copy number and NAFLD severity in liver biopsies. If a similar association exists in peripheral blood, mtDNA copy number could represent a non‐invasive alternative for differentiating NAFLD disease stages.

Given the above, we analyzed whether mtDNA copy number differs in individuals discordant for NAFLD and MS. We also probed for correlations between liver and peripheral blood mtDNA copy number, in addition to associations between NAFLD and mtDNA copy number.

Objective

To analyze whether mtDNA copy number is a useful blood‐based marker of NAFLD severity.

Materials and methods

Cohort = 73 peripheral blood samples and liver biopsies obtained from patients that were operated for gallstones; individuals were separated into 4 groups: controls (n = 27), NAFLD (n = 14), MS (n = 9) and MS + NAFLD (n = 23). NAFLD severity was determined according to the parameters described by Kleiner; mtDNA copy number was determined by semiquantitative PCR.

Results

We found no significant differences in mtDNA copy number across the four groups analyzed (i.e. control, NAFLD, MS, MS+NAFLD). However, we did find an association between mtDNA copy number and NAFLD severity. In particular, we found that a significant increase in mtDNA copy number in SS relative to control samples, while mtDNA copy number was significantly reduced in NASH relative to SS.

Conclusion

Akin to the previously published data of mtDNA copy number in liver biopsies of individuals with NAFLD, we uncovered a dynamic relationship between mtDNA copy number and NAFLD severity in peripheral blood samples. This suggests that mtDNA copy number may be a promising blood‐based biomarker for differentiating between SS and NASH. To address this issue, we are currently probing for correlations between liver and peripheral blood mtDNA copy number.

Support or Funding Information

Mexican National Council for Science and Technology (CONACyT).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Proteomic Analyses of Plant Exosomes
Authors: Özdemir Bihter , Çimen Hüseyin
Published: June 2021

Extracellular vesicles (EVs) have been identified as high potential vesicles for intercellular communication. Although their physiological roles have not been identified clearly, they are spherical, non‐organelle lipid membrane vesicles. Recent studies show that mammalian EVs contain RNA, proteins, and lipids implicated to be essential for their transfer [1]. EVs have three subgroups, which are apoptotic bodies, ectosomes, and exosomes. Exosome is the smallest EV with 50–100 nm diameter and their content is specific to the originated cell. Exosomes play the critical role in cell‐to‐cell communication and promote cellular activities such as stimulation of immune response, lysosome‐like activities or transfer of the antigens to other cells [2]. They have also a potential for biomarker design and cellular targeting based on their RNA and protein specification [3]. Plant cells are also demonstrated to produce exosomes with limited knowledge [4]. Given the available databases for animal exosomes, there is no database for plant exosomes up to date. We present our initial results for protein contents of plant exosomes to be used for the construction of such a database. In this study, Apium graveolens (Celery) exosomes were characterized by mass spectrometry‐based proteomic analysis. Following exosome isolation by using in‐house generated procedure from celery extract, proteins were precipitated with cold acetone. The peptide fractions obtained with filter‐aided sample preparation (FASP) analyzed with nanoLC‐captivespray‐qTOF‐MS/MS (Thermo Dionex RSLC3000 coupled with Bruker Compact). Data were submitted to MASCOT database against green plants to identify proteins.

Support or Funding Information

We would like to thank Prof. Dr. Fikrettin Şahin, Dr. P. Neslihan Taşlı, Kaan Kırbaş and members of YediPROT laboratory for their contribution to this project. This project was supported by Yeditepe University.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hyperinsulinemia‐Induced Changes In Chromatin Acetylation In Triple Negative Breast Cancer
Authors: Senapati Parijat , Cordova Jaime , Ann David K. , Seewaldt Victoria , Schones Dustin E.
Published: June 2021

Obesity‐associated hyperinsulinemia or insulin‐resistance is considered as a poor prognostic factor for triple negative breast cancer (TNBC), a particularly aggressive breast cancer subtype, constituting about 15–20% of all breast cancers. As a growth factor, insulin can activate the PI3K/AKT/mTOR pathway that is associated with the increased proliferation rate of TNBC cells. While this association has been recognized for some time, the mechanistic role of insulin‐resistance in promoting TNBC remains unclear. mTOR signaling enhances mitochondrial biogenesis and activity thereby potentially increasing acetyl‐CoA precursors; pyruvate and citrate. Acetyl‐CoA, in turn, can be utilized by nuclear acetyltransferases for histone acetylation. We hypothesize that hyperinsulinemia‐induced metabolic changes lead to altered chromatin structure in TNBC through genome‐wide changes in histone acetylation. To test this hypothesis, we used quantitative ChIP‐seq (ChIP‐Rx), RNA‐seq, western blot and immunofluorescence analyses on MDA‐MB‐231 (TNBC) cells treated with insulin. Insulin treatment leads to global increase in chromatin‐associated histone acetylation levels, particularly at H3K9. Using inhibitors targeting kinases in the PI3K/AKT/mTOR signaling pathway, we demonstrate its role in insulin‐induced histone hyperacetylation. ChIP‐Rx confirmed the global increase in histone acetylation at gene promoters that could potentially alter chromatin accessibility and/or gene expression. Comparison of ChIP‐seq and RNA‐seq data revealed that insulin upregulated genes accumulate higher levels of H3K9ac as compared to downregulated genes after insulin treatment. In addition, insulin induces higher levels of reactive oxygen species and DNA damage foci in cells. Moreover, peripheral blood mononuclear cells from insulin‐resistant women show higher levels of H3K9ac and DNA damage marker gamma‐H2AX relative to those from insulin‐sensitive women. These results demonstrate the significance of targeting hyperinsulinemia‐induced processes that lead to chromatin dysfunction through mitochondria in TNBC.

Support or Funding Information

This work was supported by the NCI (R01 CA220693) and a City of Hope CCSG pilot award.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Antibiotic Suceptibility Studies and Protein Degradation by Fungi Growing in Aerobic and Anaerobic Conditions
Authors: Mercado Benjamin , Bayman Paul
Published: June 2021

Fusarium species cause a broad spectrum of infections including superficial infections in healthy people or disseminated infections in immunocompromised patients. Many of these infections have been associated with proteases secreted by fungi to degrade keratin, an important component of the skin and other tissues. Ischemia and abscess are complications associated to the lack of oxygen in severe mycosis caused by Fusarium. However studies of protein degradation by fungi have been done only in aerobic condition. In this study we hypothesized that protease production would be greater in anaerobic than in aerobic condition. 14 Fusarium isolated from environmental samples were grown in Gelatin Media enriched with sodium nitrate to compare their ability to degrade proteins in anaerobic vs. aerobic conditions. Also antibiotic susceptibility was tested using Fluconazole, Itraconazole and Myconazole in aerobic and anaerobic conditions. All fungi tested showed higher Hydrolysis Capacity Index under anaerobic conditions. In aerobic conditions almost all the fungi tested showed similar colony sizes and halo of protein degradation. On other hand, under anaerobic conditions smallest colony sizes exhibited greater halos of protein degradation. Similarly, all fungi tested were more susceptible to antibiotics in anaerobic conditions too. This study showed high protein degradation in anaerobic conditions. Usually fungal contribution to degradation is ignored due to their low presence in environmental or clinical samples under anaerobic conditions. Our results suggest that Fusarium population could have a role or an impact in anaerobic environments such as ischemia or abscess where protein degradation takes place. Future works includes identification of proteins secreted under anaerobic conditions.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Enhanced Chemotactic Response of MDA‐MB‐231 Breast Cancer Cells Towards Stable Gradients of EGF
Authors: Schwarz Jan , Kuttenberger Verena , Baumann Nina , Horn Elias , Zantl Roman
Published: June 2021

Chemotactic cell migration is a basic mechanism underlying cancer cell invasion and hence metastasis. In this regard, growth factors such as epidermal growth factor (EGF) have been identified as guidance cues. Using ibidi's μ‐Slide Chemotaxis we confirm the growth factor EGF as potent guidance cue for directional MDA‐MB‐231 migration in a physiological 3D environment. Moreover, we find enhanced sensitivity towards gradients of EGF in serum free media supplemented with defined protein composition (UltraCULTURETM). Combining defined, physiological migration conditions and enhanced sensitivity allowed us to quantify EGF dependent MDA‐MB‐231 chemotaxis in a detailed manor. MDA‐MB‐231 cells showed increased directional and kinetic response towards temporal and stable spatial gradients of EGF. While EGF guided directional migration was highly concentration dependent with a concentration optimum of 10 ng/mL, we found the chemokinetic effect induced by the presence of EGF being concentration independent. Those results suggest differential response of the migration and signal interpretation machinery of MDA‐MB‐231 cells. Both, blocking the ligand binding domain of the EGF‐Receptor (EGFR) by a blocking antibody and inhibition of the kinase domain of EGFR by a small molecule inhibitor (AG1478) led to a reduction in EGF induced directional sensing and migration velocity. Additionally, low, non‐effective single doses of antibody and small molecule inhibitor showed synergistically effective inhibition if combined. Those results support EGF being a potent guidance cue for MDA‐MB‐231 cell migration and help to understand the mechanisms behind chemotactic migration driven cancer metastasis

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
SOCS3 down‐regulates ROS signaling and inhibits M1 and M2 human macrophage polarization
Authors: Lee Choong‐Eun , Yoon Hye‐young , Jeong Hana
Published: June 2021

Initially found as feed‐back regulators of Jak/STAT pathways during cytokine signal transduction, the suppressors of cytokine signaling (SOCS) proteins have been recognized as modulators of inflammation through the regulation of macrophage activity. Macrophages are critical role players in immune‐inflammatory reactions to combat pathogen and to restore tissue homeostasis through the balanced action of distinct phenotypes: pro‐inflammatory M1 vs antiinflammatory M2. While anti‐inflammatory effect of SOCS1 through the inhibition TLR and NF‐kB signaling is well established, the role of SOCS3 in macrophage function has been controversial, being pro‐inflammatory or anti‐inflammatory. In the present work, we have studied regulatory functions of SOCS3 in inflammation employing M1 vs M2 differentiation systems and investigated the regulatory mechanisms involved. Human promonocytic THP1 cells were differentiated by PMA treatment and subject to polarization to M1 vs M2 phenotypes with LPS vs Dex stimulation, respectively for 24 to 48 h. Under such M1 or M2 differentiation condition, intracellular ROS generation and MAPK activation, albeit with different kinetics, were induced which preceded the pro‐inflammatory or anti‐inflammatory markers, respectively. Over‐expression of SOCS3 attenuated the LPS‐ or Dex‐induced ROS generation and suppressed both M1 and M2 differentiation as measured by the expression of proinflammatory (IL‐1beta, TNF‐alpha, and IL‐6) and anti‐inflammatory (TGF‐beta and IL‐10) cytokines. On the contrary, shRNA‐mediated SOCS3 knock‐down has shown the opposite effects. As a mechanism of ROS down‐regulation by SOCS3, we have observed the upregulation of anti‐oxidant factors such as Nrf‐2, Trx, SOD, and Prx in SOCS3 over‐expressing cells. Among MAPK members regulated by ROS signal, SOCS3 inhibited p38 activity critical for IL‐10 production during M2 differentiation with a prominent upregulation of MKP1. As SOCS3 suppressed STAT activation and GILZ expression important for M1 and M2 differentiation, the role of ROS signal in the regulation of these downstream transcription factors is also under investigation.

Support or Funding Information

< Supported by NRF grants 2015‐003291 and 2016‐911262 >

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The role of yeast RNA polymerase I initiation factor Core Factor subunit Rrn7 in promoter open complex formation
Authors: Jackobel Ashleigh J. , Han Yan , He Yuan , Knutson Bruce A.
Published: June 2021

A central component in S. cerevisiae RNA polymerase I (Pol I) pre‐initiation complex (PIC) formation and transcriptional regulation is the evolutionarily conserved initiation factor termed Core Factor (CF). CF is a heterotrimer composed of Rrn6, Rrn11, and the TFIIB‐related factor Rrn7. Recent Cryo‐EM structures of the Pol I initiation complex reveal that CF resembles a right hand grasping promoter DNA in which key contacts are mediated by Rrn11 and Rrn7 (Han et al., eLife, 2017). Similar to its Pol II counterpart TFIIB, CF subunit Rrn7 consists of an N‐terminal zinc ribbon (ZR) domain followed by a linker region and two cyclin fold repeats. We systematically mutagenized the Rrn7 ZR and linker domains to further understand their role in Pol I transcription initiation. We found that ZR and linker mutants are still able to form the CF complex and assemble a PIC, yet are defective in promoter opening. These findings, paired with the Cryo‐EM structure, suggest that CF acts as a ratchet to drive promoter DNA into the active site of Pol I and that the Rrn7 ZR and linker domains play a dynamic role in ATP‐independent promoter opening.

Support or Funding Information

This work was supported by grants awarded to BAK: National Cancer Institute (5K22CA184235), Alexandrine and Alexander L. Sinsheimer Scholar award, and SUNY Research Foundation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The novel cardio‐oncology drug, NP‐6A4, regulates the growth of multiple breast cancer cell lines through distinct cell‐specific mechanisms
Authors: Pulakat Lakshmi , Belenchia Anthony M , Toedebusch Ryan G
Published: June 2021

While chemotherapeutic agents (anthracyclins, kinase‐inhibitors) have improved 5‐year breast cancer survival, their cardiovascular toxicity cause alarming increase in life‐threatening heart disease for millions of cancer survivors. Therefore, developing new cardio‐oncology drugs to protect heart during and after chemotherapy is an immediate critical need. Although literature indicate that Angiotensin II type 2 receptor AT2R attenuates cardiac damage, to date there are no AT2R agonists that are used in clinic to treat heart disease. NP‐6A4 is an AT2R peptide agonist that has recently received orphan drug designation for pediatric cardiomyopathy from the FDA. Our previous studies indicated that NP‐6A4 treatment (1.8mg/kg/day) could improve cardiac function and attenuate cardiomyopathy in diabetic rats and protect human cardiovascular cells from acute nutrient stress. We hypothesized that since AT2R is a regulator of cell growth, NP‐ 6A4‐AT2R signaling will attenuate breast cancer cell growth. Here we report NP‐6A4 treatment of triple negative breast cancer cells (MDA‐MB‐231) and estrogen positive breast cancer cells (MCF‐7) resulted in significant (p<0.05) suppression of cell index (as determined by real time cell analyzer), and survival (as determined by MTS assay), albeit the underlying mechanisms were different. NP‐6A4 treatment of MCF‐7 cells augmented Tamoxifen‐induced inhibition of cell growth (p<0.05), and suppressed proliferation marker Ki67 (20%) and phosphokinases CREB (32%), Jun N‐terminal kinase (JNK) (51%) and Erk1/2 (41%). In MDA‐MB‐231 cells, NP‐6A4 treatment caused increase in apoptotic marker Caspase 3 (2.3‐fold increase), but suppressed its inhibitor phosphokinase p38 (37%) (p<0.05), and also suppressed phosphokinases CREB (30%), JNK (51%) and Erk1/2 (33%). However, doxorubicin (anthracycline)‐induced cell death was not reversed by NP‐6A4. NP‐6A4 treatment increased different tumor suppressors: ATIP 1 mRNA in MCF‐7 and ATIP‐3 mRNA in MDA‐MB‐231 (p<0.05). These data suggest that NP‐6A4 differentially regulates human breast cancer cell growth and has high potential as a synergistic cardio‐oncology drug for breast cancer treatment.

Support or Funding Information

This work was supported by NIH grants: 1R01HL118376‐01 and 1R01HL138988‐01A1

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cooperative Inhibition of Wnt/β‐Catenin Signaling by Klotho and Vitamin D: Implications for Chemoprevention
Authors: Khan Sameera , Khan Zainab , Whitfield Graham , Haussler Mark , Jurutka Peter
Published: June 2021

The Klotho gene has been closely associated with delayed onset of aging. Inhibition of the klotho protein promotes aging‐like phenotypes, while its overexpression has been found to extend lifespan in mice. The single‐pass transmembrane isoform of klotho (m‐klotho) forms a receptor complex with the fibroblast growth factor (FGF) receptor to create a high‐affinity binding site for FGF23, a hormone involved in phosphate homeostasis and suppressing vitamin D activation. In contrast, secreted klotho (s‐klotho) forms via ectodomain shedding of the membrane‐bound isoform or through alternative splicing of the Klotho gene. It exhibits pleiotropic functions presumably via its weak glycosidase activity, including the inhibition of the insulin/insulin‐like growth factor 1. The proposed anti‐aging properties of s‐klotho have already been demonstrated by its endocrine role in reducing oxidative stress and promoting cardiovascular protection. Due to its potential role as a tumor‐suppressor, we have focused on how s‐klotho interacts with a known chemopreventative agent and steroid hormone, 1,25‐dihydroxyvitamin D3 (1,25 D), and its nuclear receptor (VDR) to inhibit the activity of target proto‐oncogenes. One such target for 1,25D/klotho interaction is β‐catenin. Mutations in the Wnt/β‐catenin signaling pathway have been implicated in numerous cancers, particularly colorectal neoplasia. In this study, the activity of β‐catenin was analyzed in vitro using transcriptional luciferase assays and western blotting as a response to modulated levels of exogenous klotho, 1,25D, and VDR. In addition, quantitative polymerase chain reaction (qPCR) was utilized to evaluate the ability of s‐klotho and 1,25D to inhibit the expression of known β‐catenin target genes including cell cycle regulators such as cyclin D1 and c‐myc, as well as the apoptosis inhibitor survivin. Our results revealed that s‐klotho stimulated VDR transactivation while inhibiting β‐catenin activity independently as well as synergistically with VDR and 1,25D. These findings were further supported by the 1,25D/klotho‐mediated down‐regulation of cyclin D1, c‐myc, and survivin expression. The dual isoforms of klotho exhibited functional differences, as lower concentrations of m‐klotho were found to boost β‐catenin activity, implicating the secreted isoform of klotho in directing its hypothesized anti‐aging properties. Our findings indicate that the crosstalk between vitamin D and klotho may promote healthful aging in part due to their cooperative tumor‐suppressive role and inhibition of factors implicated in oncogenic signaling.

Support or Funding Information

This project was funded by the New College of Interdisciplinary Arts and Sciences at Arizona State University and the National Institute of Health.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sestrin2 Supports Lung Tumor Growth in Early Stages of Cancer Development in Mice but Might Play a Tumor Suppressive Role in the Late Stages of Carcinogenesis
Authors: Budanov Andrei Vladimirovich , Ding Boxiao , Parmigiani Anita , Chumakov Peter Mikhailovich , Lee Jun Hee , Chawla Ayesha , Grossman Steven
Published: June 2021

The goal of this study was to understand the role of the p53‐inducible SESN1 and SESN2 genes in the development of lung cancer. Our studies are based on the development of mouse models employing inducible activation of the Kras oncogene in mice deficient for Sesn1 or Sesn2 genes. These mice develop lung cancers in response to injection with Adeno‐Cre virus.

The members of the mammalian Sestrin gene family (SESN1, SESN2 and SESN3) encode evolutionary conserved proteins called sestrins that play an important role in regulating cell viability and metabolism in response to stress. The major activity of Sestrins is modulation of mammalian target of rapamycin complexes 1 and 2 (mTORC1/2), which are responsible for regulation of cell growth, metabolism, autophagy and viability. Strikingly, two of three members of the mammalian sestrin family, i.e. SESN1 and SESN2, are targets of transcription factor p53 which plays a major role in tumor suppression. We found that both SESN1 and SESN2 genes are downregulated in the majority of human lung cancers. Therefore, we proposed that SESN1 and SESN2 might be responsible for tumor suppressive activities of p53. However, inactivation of a single Sesn2 gene or both Sesn1 and Sesn2 genes did not affect the number of tumors in mice. Moreover, we observed that Sestrin2‐positive tumors were larger, indicating that Sestrin2 supports tumor growth during early stages of lung carcinogenesis. These effects correlated with higher activity of AKT kinase in the Sestrin2‐proficient mice as compared to Sestrin2‐deficient counterparts. Being a major regulator of cell growth and proliferation, AKT can be a critical target of Sestrin2 in regulating of tumor growth. Nevertheless, we have also demonstrated that silencing of Sestrin2 in human lung adenocarcinoma cells accelerates growth of tumor xenografts in nude mice and provides resistance to anticancer genotoxic drugs.

Therefore, we conclude that Sestrin2 support tumor growth at early stages of cancer development, but might suppress carcinogenesis in the late stages. Moreover, tumors with low expression of Sestrin2 may be selected during chemo‐ and radiotherapy.

Support or Funding Information

The studies were supported by the Grant 17‐14‐01420 from the Russian Science Foundation to AB and NCI CA172660 to SG.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modulatory effect of Cashew (Anacardium occidentale L.) nut supplemented diet on fertility activity of clomiphene citrate in male rats
Authors: Akomolafe Seun
Published: June 2021

Context

Increasing concern has been expressed about the declining sperm count of humans and the potential fertility effects of clomiphene citrate, a synthetic oestrogen‐antagonist on human reproductive health in the last few decades.

Aim

This study aims to investigate the influence of cashew nut supplemented diet on fertility activity of clomiphene citrate in male rats.

Methods

The rats were divided into six groups (n=6): rats fed basal diet; rats fed basal diet and then given clomiphene citrate (cc) orally; rats fed diet supplemented with 10% processed cashew nut and given cc orally; and rat fed diet supplemented with 20% processed cashew nut and given cc orally; rats fed diet supplemented with 10% processed cashew nut and rat fed diet supplemented with 20% processed cashew nut.

Results

The results revealed that there was a significant (p<0.05) improvement of total testosterone level and epididymal sperm count, viability and progressive motility in all the groups, in comparison to control, with more significance in combination therapy group. Similarly, increased testicular and epididymal total antioxidant capacity (TAC), nitric oxide (NO), total thiol (T‐SH), nonprotein thiol (NPSH) levels, Glutathione‐S‐transferase (GST) and steroidogenic enzymes activities with concomitant decrease in reactive oxygen species (ROS) level, thiobarbituric acid reactive substances (TBARS) production and arginase activity were observed in all the groups when compared with control, with more significance in combination therapy group.

Conclusion

Therefore, the combination therapy with cashew nut boosts the fertility potentials of clomiphene citrate. Thus, this combination therapy can be used to treat male partners suffering from infertility.

Support or Funding Information

nil

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CRY1 dysregulation leads to hyperglycemia in diabetic mice
Authors: KIM YEYOUNG , LEE GUNG , KIM JAEBUM
Published: June 2021

Hepatic glucose production (HGP) is essential for maintenance of normal glucose homeostasis. Glucagon promotes hepatic gluconeogenesis to increase the level of blood glucose upon fasting. However, during the postprandial state, insulin suppresses HGP via FOXO1 regulation. In diabetes, HGP dysregulation and insulin resistance contributes to hyperglycemia. Recently, it has been reported that CRY1 could act as a potential suppressor of hepatic gluconeogenesis by FOXO1 protein degradation. We previously reported that the level of CRY1 protein was decreased in the liver of obese and diabetic animals. However, the underlying mechanism for hepatic CRY1 dysregulation has not been thoroughly elucidated. In order to find out which factors are involved in CRY1 degradation, we screened CRY1 binding proteins in the liver of diabetic mice. We discovered that several candidate proteins appear to physically associate with CRY1 to mediate its stability. Our data support that CRY1 binding proteins could regulate CRY1 protein stability, providing a clue to understand the mechanism for hyperglycemia through CRY1 dysregulation in the liver of diabetic mice.

Support or Funding Information

This work was supported by NRF of Korea Grant MSIP 2011‐0018312 and BK21.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biomedical Status of Women Attending In‐Vitro Fertilization Clinics in Nigeria
Authors: Gbenle Oluwasegun Adekunle , Igwo‐Ezikpe Miriam N. , Taiwo Idowu A. , Anyashina Olumuyiwa A. , Ofoma Osita N. , Ikwuazom Okwunna O.
Published: June 2021

Infertility constitutes a major problem in many developing countries like Nigeria where itsprevalence is significant. The relationship between the endocrinological, physiological and environmental determinants of conception in IVF among Nigerian infertile women have not been well elucidated; hence, the need for the biomedical status of women attending IVF Clinics in Nigeria.

Blood samples of infertile women (n=200) undergoing in‐vitro fertilization were obtained from different fertility clinics in Nigeria. Questionnaires were distributed to participants to obtain physiological characteristics such as age, blood group, genotype and Rhesus factor. The levels of reproductive hormones (Luteinizing Hormone, Follicle Stimulating Hormone and Prolactin) were analysed using a micro‐plate reader while the levels of heavy metals (Mercury, Lead, Cadmium and Chromium) in the blood samples was determined using atomic absorption spectroscopy.

There was significant (p< 0.05) difference in the levels of Luteinizing Hormone, Follicle Stimulating Hormone and Prolactin between the age groups of the infertile women. There was higher non significant (p>0.05) levels of the three reproductive hormones in the negative Rhesus factor subjects compared to positive Rhesus factor. However, there was no significant (p<0.5) difference in the three reproductive hormones in relation to the blood grouped subjects. There was significant (p<0.5) difference between the levels of the reproductive hormones and age. There were more negative correlations between the levels of reproductive hormones and levels of heavy metals whereas there was no significantdifference between the levels of heavy metals and age of infertile women.

The study suggests that IVF patients with a specific blood group do not have a higher incidence of reduced fertility in response to ovarian stimulation. It also suggests no relationship between Rhesus factor and infertility but age and levels of heavy metals may affect female infertility.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Analysis of Alaskan Wild Bog Blueberries Anthocyanins in the Mitigation of Type II Diabetes.
Authors: Callahan Brandaise , Collin Aline , Kuhn Thomas
Published: June 2021

Alaska natives were recently found to have the fastest increase in diagnosed type II diabetes (T2D) cases in the U.S. population due to the influence of western diets. A common manifestation of T2D is insulin resistance which alters the body's regulation of insulin and glucose metabolism. Alaskan blueberries have been shown to have a higher content of antioxidants in the form of anthocyanins compared to other varieties. Dietary anthocyanins have have shown to act on the insulin transduction pathway and reduce insulin resistance. Our research focuses on the anthocyanins present in Alaska wild bog blueberries and their potency to prevent and reduce insulin resistance and therefore mitigate T2D development.

The experimental design encompassed differentiated 3T3‐L1 adipocytes exposed to palmitate for 16 hr, which reduces insulin‐dependent glucose uptake, in the presence or absence of anthocyanins. Our study focused on the phosphorylation of AS160, a pivotal substrate in the insulin transduction pathway, as a response of the cells to insulin. Anthocyanin fractions (ANC) (10 ug/mL) were applied for 1 hr prior to palmitate (0.75 mM, 16 h) and were present throughout the stress period. Total cell lysates were analyzed by western blotting to determine the presence of phosphorylated AS160.

Each condition was run in duplicate and two western blots were run. The software ImageJ was used to obtain relative levels of p‐AS160. Values were obtained from the ratio of insulin stimulated over basal levels of AS160.

The control, (no palmitate or ANCs), had the highest amount of phosphorylated AS160 (1.925, p <0.05). Cells stressed with palmitate had the least AS160 phosphorylation levels (0.791 p= 0.05). Importantly, cells treated with ANCs prior to stress exhibited 1.047 (p=0.11) of phosphorylated AS160.

The data supports the hypothesis that the ANCs used prior to stressing the cells mitigates insulin resistance. Future work will look at an intervention paradigm and focus on a comparative analysis of prevention and intervention.

Support or Funding Information

Work reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under three linked awards number RL5GM118990, TL4GM118992 and 1UL1GM118991. The work is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Punica Granatum Rind Extract Promotes Mouse Liver Mitochondrial Function and Attenuates Paraquat Toxicity in a Caenorhabditis Elegans Model
Authors: Mowery Colin , Ramanathan Ranjith , Johny Anup
Published: June 2021

Punica granatum (pomegranate) has drawn the interest of the scientific community due to its rich array of phenolic compounds and antioxidant capacity. Evidence supports the fruit's role in preventing free radical damage in humans. Although various studies have concluded the beneficial effects of pomegranate juice, limited studies have characterized the antioxidant effects of its rind (outer covering/peel; PRE). The goal of our study was to characterize the effects of PRE on oxidative stress. We utilized mouse liver mitochondria and paraquat (PQ) toxicity in a Caenorhabditis elegans (C. elegans) model to determine the beneficial effects of PRE. Mitochondria were isolated from C57 male mice liver tissue (6 weeks of age). Phosphatidylcholine/cholesterol liposomes (Lp) were used to induce oxidative stress. Samples were then incubated at 37□ for 2 hours with or without the addition of PRE at two levels (PRE1 and PRE2 represent 60 and 120 μM Trolox equivalent/mL, respectively). Oxygen consumption rate (OCR; nmol O2 consumed/min per mg mitochondria) was used to express the activity of NADH‐CoQ oxidoreductase, succinate dehydrogenase, and cytochrome c oxidase (complex I, II, and IV of electron transport chain, respectively). Antioxidant level in PRE was determined by DPPH‐, ABTS‐ and Folin‐Ciocalteu assays. The experiment was repeated four times. Pre‐incubation of mitochondria with Lp decreased (p < 0.05) OCR by 50% compared with control mitochondria without Lp. However, incubation of Lp with PRE2 increased (p < 0.05) OCR for complex II and IV. No significant changes (p > 0.05) in OCR were observed for complex I with incubation of Lp and PRE 1 or PRE2. Using C. elegans, an oxidative stress resistance assay was performed to explore PREs effects on paraquat toxicity. Wild‐type adult/L4 hermaphrodites were placed in 96‐well plates. PQ was dissolved to a final concentration of 50 μM and half concentration of PRE1 was added for experimental groups. Resistance to oxidative stress was measured through percentage survival at hour time intervals. After 5 hours, mean survival for PQ alone was 61.91±6.56%; PQ groups supplemented with PRE had a mean survival of 88.29±6.18%. The current study suggests that PRE may play a role in preserving mitochondrial function during oxidative stress and attenuates PQ toxicity.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.