bluuebunny/crossref_metadata_2025 huggingface dataset:
Targeting Aspartate Transcarbamoylase in Staphylococcus aureus: A Novel Therapeutic Approach for Sepsis
Authors: Patel Chandni , Vaishnav Asmita , Brunzelle Joseph S. , Guy‐Evans Hedeel I. , Edwards Brian FP. , Evans David R.
Published: June 2021

Sepsis is one of the leading causes of death in the United States. More than 1.5 million people get sepsis and 250,000 people die from sepsis each year. Staphylococcus aureus happens to be most common cause of healthcare‐associated infections. Research and development of new antibiotics against S. aureus is a high priority due to its multi‐drug resistance. The levels of pyrimidines in blood are too low to sustain the growth of bacteria, so they must rely on pyrimidine biosynthesis. Previous studies have shown that a defect in several pyrimidine biosynthetic enzymes resulted in 1000‐fold decrease in the titer of bacteria growing in blood. Aspartate transcarbamoylase (ATC) catalyzes the first committed step of the de novo pyrimidine pathway, making it a good drug target. S. aureus ATC containing his‐tag was overexpressed in Escherichia coli and purified using nickel affinity column chromatography. S. aureus ATC has a Km of 2.4 mM for carbamoyl phosphate and 4.5 mM for aspartate, with a maximal velocity of 296 μmol/min/mg. Cross‐linking data suggests that S. aureus ATC is a trimer of 33 kDa and it does not associate with DHO, the subsequent enzyme in the pathway, as seen in Pseudomonas aeruginosa, Aquifex aeolicus, and several other ATC. We have successfully solved the preliminary structure of S. aureus ATC at 2.27Å resolution. Once our preliminary structure has been further refined, it will serve as the basis for the design of novel non‐competitive inhibitors that block domain closure of ATC, an essential step in catalysis.

Support or Funding Information

American Heart Association Predoctoral Fellowship, Interdisciplinary Biomedical Sciences Fellowship, Wayne State University School of Medicine, Wayne State University Graduate School & Office of VP for Research

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Overexpression of Insulin‐like Growth Factor Binding Protein 1 (IGFBP1) Generates Tamoxifen Resistance in Breast Cancer Cells
Authors: Kieltyka Jacqueline , Zheng Yan , Hobbs Alvaro , Houston Kevin D.
Published: June 2021

Tamoxifen, a selective estrogen receptor modulator and antagonist for estrogen receptor alpha (ERα) in breast tissue, is a commonly prescribed adjuvant treatment for patients presenting with ERα‐positive breast cancer. Acquired resistance to tamoxifen remains a clinically relevant challenge for many patients receiving treatment and some patients with ERα‐positive breast cancer are innately resistant to tamoxifen treatment. The molecular mechanisms underlying both acquired and innate tamoxifen resistance are not well understood. Previous studies have shown that, in addition to ERα inhibition, tamoxifen activates the G‐protein‐coupled estrogen receptor 1 (GPER1) and activation of this receptor is associated with the development of tamoxifen resistance. GPER1 mediates the extracellular accumulation of the insulin‐like growth factor binding protein 1 (IGFBP‐1) after tamoxifen treatment resulting in the inhibition of the IGF‐1R signaling in breast cancer cells. We hypothesize sustained exposure of breast cancer cells to IGFBP‐1 will result in the development of tamoxifen resistance. Stable transfection of an IGFBP‐1 expression vector was used for sustained exposure of MCF‐7 breast cancer cells. IGF‐1R expression was decreased in MCF‐7 cells stably transfected with the IGFBP‐1 expression vector similar to IGF‐1R expression in tamoxifen resistant breast cancer cells. Similar to tamoxifen‐resistant MCF‐7 cells, the IGFBP‐1 overexpressing MCF‐7 cells also have elevated levels of activated ERK1/2. Finally, cell viability assays revealed that sustained IGFBP‐1 overexpression decreased breast cancer sensitivity to tamoxifen treatment. Taken together, these results suggest that elevated levels of IGFBP‐1 contribute to the development of tamoxifen resistance in breast cancer cells.

Support or Funding Information

Research reported in this publication was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103451.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of Hydrogen Sulfide Levels by Cannabinoids in the Bovine Isolated Retina
Authors: Bush Leah , Robinson Jenaye , Kutse Seun , Ebuzoeme Christabel , Onyenaka Collins , Akpan Idongesit , Opere Catherine , Ohia Sunny , Njie‐Mbye Ya Fatou
Published: June 2021

Both endocannabinoids and hydrogen sulfide (H2S) have been reported to exert neuroprotective actions in neural tissues (Biermann et al., Mol Vis. 2011;17:1275–86; Nucci et al., Invest Ophthalmol Vis Sci. 2007;48(7):2997–3004). It remains to be determined if these modulators can interact with each other and/or share a common pathway in their neuroprotective actions.

Purpose

The aim of the present study was, therefore, to investigate the pharmacological actions of endocannabinoids on the biosynthesis of H2S in isolated bovine neural retina.

Methods

Isolated bovine retinae were homogenized and exposed to different concentrations of methanandamide (1 nM – 10 uM) and 2‐arachidonyl glycerol (2‐AG) (1 – 3 uM) for 90 minutes at 37ºC. Retinal homogenates were then incubated with ethylenediaminetetraacetic acid (EDTA) (1%), Zinc acetate (1%), and Borate buffer (pH 11) for 30 mins followed by 10‐minute incubation with N, N dimethyl p‐phenylenediamine (20 mM) and FeCl3 (300 mM). Samples were then centrifuged at 5000 g for three minutes and supernatant was assessed for H2S content using the well‐established Methylene Blue method.

Results

Both methanandamide and 2‐AG elicited a concentration‐dependent decrease in basal H2S production in the isolated retina. For instance, methanandamide (10 μM) significantly (p <0.05) reduced basal production of H2S by 30% when compared to controls. Likewise, 2‐AG (10 μM) also elicited a 30% decrease in basal retinal H2S concentration when compared to controls.

Conclusions

We conclude that exogenously applied endocannabinoids can alter the basal production of H2S in the isolated neural retina. The observed effect of endocannabinoids on the pathway leading to H2S biosynthesis suggests that a cross‐talk exists between both neuromodulators in the retina.

Support or Funding Information

Supported by NIH/NEI Research Grant 1R15EY022215‐01

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The 4 Position of 5‐HT2A Structural Agonists Imparts the Anti‐Inflammatory Propensity Responsible for Reducing Airway Hyperrresponsiveness in Allergic Asthma
Authors: Flanagan Thomas Wayne , Landry Alexus Nichol , Hibicke Meghan , Nichols Charles David
Published: June 2021

The 5‐HT2A receptor is the most widely expressed serotonin receptor in mammals. While distribution occurs largely in the central nervous system, the 5‐HT2A receptor has been detected in platelets, endothelial and epithelial cells, and lymphatic tissue. Most commonly viewed as the classical target of psychedelic compounds, our lab has made significant progress in understanding how activity at the 5‐HT2A receptor profoundly activates anti‐inflammatory pathways. Here we have generated a chronic asthma model to examine the therapeutic potential of 5‐HT2A receptor activation in persistent allergic airways disease. Our studies show that nasal administration of the 5‐HT2A agonist (R)‐DOI attenuates the elevated airway responsiveness, goblet cell hyperplasia, collagen deposition, and airway structural remodeling that accompanies persistent allergen challenge. Additionally, we have used an acute model in rodents to interrogate structural components of known psychedelics most critical for their anti‐inflammatory effects. Our studies indicate that the 4 position of 5‐HT2A structural agonists contains the chief mediator for reducing bronchial hyperresponsiveness in asthma. This ultimately suggests that a blood‐brain inpenetrant molecule can be developed that lacks the psychoactivity of classical psychedelics but maintains therapeutic efficacy.

Support or Funding Information

This work was supported, in part, by research funds from the American Asthma Foundation Scholar Grant Award to C.N., The Betsey Gordon Foundation, and the Eleusis Benefit Corporation, PBC.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Activity of Single and Double‐modified Salinomycin Analogs against Primary Acute Lymphoblastic Cells In Vitro
Authors: Urbaniak Alicja Joanna , Delgado Magdalena Rose , Antoszczak Michał , Borgström Björn , Strand Daniel , Huczyñski Adam , Chambers Timothy C.
Published: June 2021

Salinomycin (SAL) and monensin (MON) are polyether ionophore antibiotics known from their anti‐cancer activity against various types of cancer cells, including those that display multi‐drug resistance as well as cancer stem cells. In order to increase the biological activity profile and reduce toxicity against normal cells, while retaining the activities in the micromolar range, a library of novel SAL analogs has been synthesized. This included esters and amides single‐modified in C1 position as well as double‐modified derivatives additionally acylated in the C20 position (Fig. 1). The aim of the study was to evaluate the activity of newly synthesized SAL derivatives (12 single‐modified and 12 double‐modified) towards primary acute lymphoblastic leukemia cells. MON (IC50 = 1.2 ± 0.8 μM) and nine SAL derivatives (IC50 in the range of 1.2 ± 0.02 to 1.9 ± 0.7 μM) were more potent than SAL (IC50 = 2.4 ± 0.7 μM) in cell viability assays. Moreover, the most potent SAL analogs induced characteristics of apoptotic cell death and increased expression of p53. In further studies, SAL acted synergistically with the Bcl‐2 inhibitor ABT‐263, whereas 2,2,2‐trifluoroethyl ester, the most active analog of SAL, antagonized ABT‐263. This is intriguing and suggests that the parent compound may differ mechanistically from its derivatives. The present findings indicate that SAL derivatives constitute promising lead compounds for targeting acute lymphoblastic leukemia. Further study and development of these novel cancer therapeutics with potential clinical application is warranted.

Support or Funding Information

TCC acknowledges support from Arkansas Breast Cancer Research Program and Chancellors Circle Award from the UAMS College of Medicine. MA thanks the Polish Science Centre (NCN) for financial support by grant SONATA (2016/23/D/ST5/00242) and fellowship ETIUDA (2014/12/T/ST5/00710). We thank the Swedish Research Council, and the Cancer Foundation for funding.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of Amino Acids in Muscle and Blood from Patients with Pancreatic Cancer‐ Induced Cachexia
Authors: Talbert Erin E. , Chakedis Jeffery , Guttridge Denis C.
Published: June 2021

Cachexia, or weight loss, is a profound side effect of cancer. Patients that suffer from cachexia have poor treatment tolerance, poor post‐surgical outcomes, poor survival, and poor quality of life. Despite decades of research, we lack a pharmacological intervention to treat cachexia, as mechanisms driving cachexia remain incompletely understood. Further, while the significance of cancer cachexia is clear, this syndrome remains poorly recognized, and a biomarker of cancer cachexia would be a valuable tool to improve the recognition of this syndrome. Because cachexia is ultimately a metabolic syndrome, we hypothesized that a metabolomics approach could identify new potential mechanisms of cachexia while also identifying potential biomarkers. Using a targeted mass spectrometry approach, we sought to distinguish cachectic pancreatic cancer patients from those without cancer or weight stable pancreatic cancer patients. Surprisingly, amino acid content of muscle from both cachectic and weight stable cancer patients was pronouncedly decreased compared to control patients. Similarly, plasma amino acid content was also generally decreased in both weight stable and cachectic cancer patients. Only differential levels of gamma‐amino‐N‐butryic acid (GABA) distinguished cachectic cancer patients from weight stable cancer patients and controls. In summary, the majority of changes we detected were specific to pancreatic cancer rather than cachexia, emphasizing the need to include cancer patients with and without cachexia in the study design.

Support or Funding Information

Support was provided through a Pilot and Feasibility Grant from the Mayo Clinic Metabolomics Resource Core through grant number U24DK100469 from the National Institute of Diabetes and Digestive and Kidney Diseases. Additional funding was provided by NIH R01CA180057 (DCG) and American Cancer Society Postdoctoral Fellowship PF‐15‐156‐01‐CSM (EET).

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 mechanosensitive ion channel Piezo1 in Autosomal Dominant Polycystic Kidney Disease (ADPKD)
Authors: Chebib Fouad , Beyder Arthur , Wang Xiaofang , Alcaino Constanza , Ehrlich Barbara , Torres Vicente
Published: June 2021

Disruption of intracellular calcium likely underlies the upregulation of cyclic AMP (cAMP) signaling and the proliferative and secretory phenotype of the cystic epithelium in ADPKD. However, the mechanisms by which polycystins reduce cytoplasmic calcium remain elusive. Impaired flow‐induced calcium entry through ciliary polycystin complex has been previously proposed as a pathogenic mechanism, but newer evidence suggests the involvement of polycystins in modulation of other mechanosensitive ion channels such as Piezo1, the newly‐identified stretch‐activated non‐selective cation channel. We hypothesized that polycystins, through their physical and functional interaction with Piezo1, modulate various cellular responses to mechanical stimulation.

Piezo1 was expressed in both wild type mouse inner medullary collecting duct cells (mIMCD3) cells and PKD2 null cells (~3 fold higher than wild type). Wild type mIMCD3 cells were stably transfected with genetically encoded biosensors, jRGeco1a and Flamindo2, to study spatiotemporal dynamics of calcium and cAMP during live cell imaging, respectively. Piezo1 activation by Yoda1 increased cytoplasmic calcium and simultaneously lowered forskolin‐induced cAMP (Figure 1A). Additionally, exerting physiological fluid shear stress (FSS) (ibidi pump system) led to reduction in cAMP (Figure 1B). Both Yoda1 and FSS lead to an increase in ATP release in mIMCD3 cells (Figure 1C). Furthermore, Yoda1 significantly reduced forskolin‐induced in vitro cystogenesis by 68% in WT and by 48% in PKD2 null mIMCD3 cells (Figure 1D).

These results suggest that Piezo1 is involved in renal cystogenesis by controlling FSS‐induced calcium entry. Further studies are required to determine in vivo effect of Piezo1 on renal cystogenesis.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Targeting Iron Metabolism for Colorectal Cancer Treatment
Authors: Xue Xiang , Shah Yatrik
Published: June 2021

Hypoxia is a key feature of most solid tumors including colorectal cancer (CRC). Hypoxia‐inducible factors (HIFs) are the major transcription factors that respond to hypoxia and are essential for tumor survival and growth. However, the main downstream pathways of HIF‐mediated tumor growth are not clear. We show that HIF‐mediated dysregulation of intratumoral intestinal iron homeostasis through induction of the iron uptake transporter divalent metal transporter 1 (DMT1) and a ferrireductase six‐transmembrane epithelial antigen of prostate 4 (STEAP4) is a major mechanism leading to CRC. Decreasing intratumoral iron by low iron diet or germ‐line Dmt1 knockout inhibits, whereas genetic overexpression of STEAP4 promotes CRC progression. If this is a druggable pathway and could serve as a therapeutic method for CRC is not known. We generated a mouse model with a temporal and conditional disruption of Dmt1 gene in the intestine (VilERT2 Dmt1F/F) by crossing Dmt1F/F mice with Villin‐CreERT2 transgenic mice, carrying a tamoxifen‐inducible Cre. Colorectal tumors were first established in this mouse model by one dose of azoxymethane (AOM) and three cycles of 1.5% dextran sulfate sodium (DSS), and then mice were treated with three daily doses of 100 mg/kg tamoxifen to disrupt Dmt1 in established colorectal tumor epithelium. One month later tumor number, tumor size at 2–3 mm and 3–4 mm, and tumor burden were significantly reduced in VilERT2 Dmt1F/F mice compared to their littermate control (Dmt1F/F). Consistent with the findings in human CRC, the gene expression of Dmt1 was increased in the tumor tissues compared to normal adjacent tissues in littermate control mice, and significantly decreased in both normal and tumor tissues in VilERT2 Dmt1F/F mice. Immunofluorescence staining revealed that cell proliferation, as determined by bromodeoxyuridine (BrdU) and Ki67 staining, was not changed, but cell apoptosis assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and cleaved caspase 3 (cCasp3) staining was increased in colorectal tumor tissues from VilERT2 Dmt1F/F mice compared to control mice. In addition, tumor vascularization and macrophage infiltration were not altered. These results demonstrate that Dmt1 is critical for tumor growth in the AOM‐DSS‐induced mouse colitis‐tumor model, and targeting iron metabolism is a feasible strategy for CRC treatment.

Support or Funding Information

X.X. was supported by NIH Grant 1K01DK114390‐01 and a Research Scholar Award from the American Gastroenterological Association. Y.M.S. was supported by NIH Grants CA148828 and DK095201.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Quantification of Striatal Dopamine Receptors in a Monogamous Bird
Authors: Eisenman Lauren Elizabeth , Coleman Melissa
Published: June 2021

Little is known about the neural mechanisms that underlie monogamous behavior. In this study, dopamine 1 receptor (D1R) and dopamine 2 receptor (D2R) reorganization in the nucleus accumbens and other striatal tissue was evaluated as a possible physiological basis of monogamous behaviors. Previous research in a monogamous mammal, the prairie vole, has shown that striatal D1R activation is implicated in maintaining pair‐bonds, and is upregulated in pair‐bonded voles versus un‐pair bonded voles. The same research team showed that D2R activation plays an important role in forming the pair‐bond, however there was no significant increase in D2R in pair‐bonded voles versus monogamously naïve voles (Aragona et al., 2006). Dopamine is also implicated in a monogamous bird, the zebra finch (Taeniopygia guttata); after pair‐bonding, dopamine levels rise in the nucleus accumbens (Banerjee et al., 2013). Furthermore, previous research in the Coleman lab has shown that D2R activation by a systemic, subcutaneous D2R agonist can induce a partner preference in finches, like in prairie voles. These similar underpinnings with the D2R indicate that D1R upregulation may also occur in zebra finches. To determine if there is D1R upregulation in zebra finches, we used quantitative real‐time PCR to quantify striatal D1R and D2R expression in pair‐bonded and un‐pair bonded birds, as well as in naïve birds that were directly infused with a D2R agonist in the ventral medial striatum, the location of the nucleus accumbens. We found no statistically significant upregulation of either D1R nor D2R between pair‐bonded and naïve birds. However, there was an upregulation of D2R in the upper striatum of birds that were directly infused with D2R agonist. Furthermore, we found an upregulation of tyrosine hydroxylase (TH), a precursor enzyme in the production of dopamine, in striatal tissue of pair‐bonded birds. These findings suggest that zebra finches may not rely on physiological reorganization of dopamine receptors, but instead on increased dopamine levels in the ventral medial striatum created by an upregulation of TH.

Support or Funding Information

APS IOSP Undergraduate Summer Research Fellowship IOS‐1238831

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cortactin's Influence on Mitochondrial Bioenergetics in the Diabetic Heart: A Novel Viewpoint
Authors: Durr Andrya Jean , Shepherd Danielle Lee , Hathaway Quincy Alexander , Hollander John Michael
Published: June 2021

Cortactin, a protein which controls the cytoskeleton, has been suggested to impact mitochondrial function by influencing the outer mitochondrial membrane during fusion and fission. Cortactin has been shown to interact directly with Coronin 1b by affecting Arp 2/3 during actin formation and branching, but it is currently unknown how cortactin dynamics affect the structure and function of mitochondria in the heart during type 2 diabetes mellitus. The goal of the study was to visualize changes in cytoskeletal proteins in the type 2 diabetic heart, and determine whether genetic alteration of cortactin levels impacts the mitochondrion on a molecular level. We hypothesized that cytoskeletal proteins associated with mitochondrial dynamics would be significantly altered in the type 2 diabetic state, and that specific changes in cortactin would have a direct effect on mitochondrial bioenergetics and function. Western blots were utilized to visualize cortactin, coronin1b and phosphor‐coronin1b levels in type 2 diabetic human patient heart and db/db mouse cardiac tissue. Results indicated that in both type 2 diabetic human patient and db/db cardiac tissue, cortactin and coronin1b levels are dysregulated. Cortactin levels were significantly increased (P<0.05) as were coronin1b levels in db/db mouse cardiac tissue (P<0.05). Phosphorylated coronin1b was significantly decreased in human type 2 diabetic cardiac tissue (P<0.05). To determine whether increased cortactin levels observed in the type 2 diabetic state can influence the mitochondrion, we utilized an HL‐1 cardiomyocyte model in which cortactin was either knocked down or overexpressed. Mitochondria were isolated post transfection with an expression vector for cortactin and analyzed via flow cytometry for size and internal complexity, JC‐1 staining for membrane potential, electron transport chain complex activities, and bioenergetics using the Seahorse. Cortactin overexpression and knockdown significantly altered the membrane potential of the mitochondria (P<0.05). Further, in cells overexpressing cortactin, exogenous fatty acid utilization was decreased (P<0.05), and cells appeared to be completely dependent of endogenous free fatty acids (FFA) for respiration (P<0.05). Taken together, our data indicate that cortactin is significantly increased in the type 2 diabetic heart of both human patient and mouse cardiac tissue. Further, these data indicate that increased cortactin levels associated with the type 2 diabetic heart may contribute to changes in mitochondrial function by influencing membrane potential and their capacity to utilize FFA during respiration.

Support or Funding Information

Support: NIH R01 HL128485; AHA 17PRE33660333

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Daily and seasonal rhythms of interleukin 6 and cortisol levels in saliva and some lifestyle habits of medical students in Poland
Authors: Kanikowska Dominka , Rutkowski Rafał , Sikorska Dorota , Orzechowska Zofia , Sato Maki , Brêborowicz Andrzej , Witowski Janusz
Published: June 2021

Objective

A primary aim of this study was to establish possible daily and seasonal rhythms of interleukin 6 and cortisol in the saliva using 15 healthy female volunteers.

Methods

All of the females were at the same stage of their menstrual cycle (follicular or luteal) during the two seasons tested. Twelve saliva samples were taken at 20 h, 22 h, 24 h, 02 h, 04 h, 06 h, 08 h, 10 h, 12 h, 14 h, 18 h and 20 h the next day, during one of the days of the two seasons tested (in January/February and May/June). Additionally, each subject filled out a questionnaire regarding her lifestyle habits. The volunteers answered questions regarding their sleep time, food and fluid intake, and the amount of physical activity undertaken on a weekday and weekend of each season tested. The study adhered to the Principles of the Declaration of Helsinki.

Results

There is a statistical difference between cortisol concentrations during the seasons, with a higher level of cortisol during the summer. However, there was no statistical significance regarding the concentration of interleukin 6. A significant positive correlation between the increased physical activity and the knowledge regarding a healthy lifestyle was found (P = 0.005).

Conclusions

This study indicates that cortisol concentration in the saliva shows statistically significant differences between the seasons. Some aspects of lifestyle, especially related to sleep and eating patterns, differ significantly between weekdays and weekends.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
UCHL1 regulates Interleukin‐6 expression in skeletal muscles
Authors: Li Yifan , Gao Hongbo , Wu Penglong , Wang Xuejun
Published: June 2021

Chronic low grade inflammation is a hallmark of aging and has been linked to aging related dysfunction and disease. As the largest tissue mass, skeletal muscles express and release several inflammatory cytokines including interleukin‐6 (IL‐6). With altered function and metabolism, aged muscles can be a major source of inflammatory cytokine in aging. Ubiquitin C‐terminal Hydrolase L1 (UCHL1) was originally found in the brain. Our recent study revealed that UCHL1 is inducible and upregulated in skeletal muscles in mice by denervation and lipopolysaccharide (LPS). In differentiated C2C12 cells. UCHL1 gene knockdown using siRNA significantly increased Interleukin 6 (IL‐6) level in the media. Consistent with this in vitro data, the plasma level of IL‐6 was also higher in mice with global or muscle specific UCHL1 gene knockout. Moreover, UCHL1 gene knockdown in C2C12 significantly reduced the protein level of IκB, a key inhibitory molecule for the activation of NF‐kB signaling pathway. Reduced protein level of IκB was also observed in muscles from UCHL1 knockout mice. These data suggest a potential novel mechanism by which UCHL1 controls IL‐6 expression in skeletal muscle by stabilizing IκB and thus inhibiting activation of NF‐kB pathway. Given the impaired UCHL1 function in the brain with aging, it is plausible to hypothesize that aging may also reduce the UCHL1 control of NF‐kB activation in skeletal muscle, leading to muscle‐derived chronic inflammation in aging.

Support or Funding Information

This study is supported by NIH grant 1R15HL118696 and 1R03AG051926, the USD BBS graduate program, USD CBBRe pilot grant, and USD PQCD pilot grant.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Liposomal Delivery of miR‐29b Restored PRMT ‐1 and PRMT‐5 Expression and Histone Methylation in Mice Exposed to Perinatal Inflammation
Authors: Sugar Sophia S. , Heyob Kathryn M. , Lee Robert M. , Rogers Lynette K.
Published: June 2021

Background

Bronchopulmonary dysplasia (BPD) continues to be a significant pulmonary complication for preterm infants, but the mechanisms associated with altered lung development remain poorly understood. Our mouse model of perinatal inflammation mimics the pathophysiology observed in infants with severe BPD. We have previously reported decreased pulmonary miR‐29b expression, and that restoration of miR‐29b expression improves lung development. The current studies tested the hypothesis that decreased expression of miR‐29b was associated with decreases in expression of key protein methyltransferases (PRMTs), and that restoration of miR‐29b would restore expression.

Methods

Pregnant C3H/HeN mice received an intraperitoneal LPS injection on E16 and newborn pups were exposed in 85% oxygen from birth to 14 days of life. On postnatal day 3, PBS containing a liposomal preparation of miR‐29b or an empty liposome was administered intranasally. On postnatal 28, mouse lung tissues were analyzed for changes in PRMT‐1, PRMT‐5, and symmetrical and asymmetrical dimethylation of histone 4, arginine 3 by western blot.

Results

Perinatal inflammation resulted in decreased expression of PRMT‐1 and PRMT‐5 which led to a decrease in histone 4, arginine 3 symmetrical dimethylation. Liposomal delivery of miR‐29b restored PRMT‐1 and PRMT‐5 expression and partially attenuated the decrease in histone 4 symmetrical methylation.

Conclusion

Our data suggest that restoration of miR‐29b may prevent aberrant expression of matrix proteins by increasing histone 4, arginine 3 dimethylation and promoting gene silencing.

Support or Funding Information

The authors gratefully acknowledge funding from the NIH/NICHD R01HD0880833

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Novel role of CREB in regulating maturation of alveolar macrophages and thereby in maintaining lung‐fluid balance
Authors: Rochford Ian P. , Joshi Jagdish P. , Mehta Dolly P.
Published: June 2021

Alveolar macrophages (AM) represent a specialized subpopulation of lung resident macrophages responsible for coordinating both the initiation and resolution of inflammatory lung injury. Analysis of the dynamics of lung resident macrophage populations using LyzM‐GFP mice (green myeloid cells) over the course of five days post‐injury by nebulized LPS showed a gradual decline in AM during injury phase but AM expanded and returned to baseline levels by 5th day post injury. While evidence suggests that recruited bone marrow monocytes can adopt AM phenotype following their differentiation into interstitial macrophage mechanisms reconstituting AM remains elusive. CREB, a cAMP‐Response Element Binding protein, is known to transcribe genes which regulate several cellular processes including cell differentiation, angiogenesis and anti‐inflammatory signaling. Mice lacking CREB rapidly die after birth primarily due to impairment of lung function. Thus, we generated mice lacking CREB in myeloid cells (CREBΔMye) by crossing CREBfl/fl mice with LyzM‐Cre mice to test the hypothesis that CREB regulates AM generation and thereby maintains lung‐fluid homeostasis. We show that in contrast to CREBfl/fl mice, CREBΔMye lungs developed lung edema basally. LPS induced significantly more edema formation at 4h in CREBΔMye than control mice. Whereas the edema resolved fully in control mice at 24h CREBΔMye lungs remained edematous. FACS analysis showed ~50% reduction in AM in CREBΔMye lungs (SiglecF+/CD11b‐/CD11c+ cells) basally and at 24 h following LPS‐induced lung injury. Intriguingly, we found increase in MHCII+/Siglec+/CD11clo lung cell population in CREBΔMye lungs suggesting loss of CREB in myeloid cells led to generation of incompetent pre‐AM/interstitial phenotype. Bone marrow derived macrophages from floxed and CREB lacking mice indicated dysregulation of several glycolytic enzymes, including hexokinase and pyruvate dehydrogenase. Further studies are being carried out to fully characterize the transcriptome of CREB lacking lung macrophages and whether CREB regulation of metabolic reprogramming is required for full differentiation of Pre‐AM/interstitial macrophages into AMs.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dishevelled has YAP nuclear export function in a tumour suppressor context dependent manner
Authors: Kang Hee Eun , Lee Yoonmi
Published: June 2021

The Hippo signaling is an evolutionary conserved pathway that inhibits cell proliferation by contact inhibition, and whose loss leads to organ growth and cancer development. The Yes‐associated protein (YAP) transcription co‐activator is a key regulator of the Hippo pathway. Phosphorylation‐dependent YAP translocation is a well‐known intracellular mechanism of the Hippo pathway, however the responsible molecular effectors by which the YAP leads cytosolic translocation remain undefined. Recent findings indicate that oncogenic YAP paradoxically suppresses canonical Wnt activity.

We found that DVL, a scaffolding protein of the Wnt pathway as well as a key regulator of Wnt‐independent epithelial polarity, is a molecular effector for nuclear‐cytoplasmic shuttling of YAP in a YAP‐phosphorylation dependent manner. Mutational inactivation of the nuclear export signal in DVL leads to nuclear YAP retention, with increases TEAD transcriptional activity. DVL is also required for YAP intracellular dynamics induced by E‐cadherin, α‐catenin, or AMPK activation. Importantly, the nuclear‐cytoplasmic trafficking is largely dependent on the p53‐Lats2 or LKB1‐AMPK tumor suppressor axes, which determine YAP phosphorylation status. Conversely, loss of p53 or LKB1 relieves DVL‐linked reciprocal inhibition between the canonical Wnt pathway and nuclear YAP activity. Our observations provide novel mechanistic insights into controlled proliferation coupled with epithelial polarity during development and human cancer.

Support or Funding Information

NFR‐2017R1A2B3002241 funded by the Korean government (MSIP) andBK 21 PLUS Project, Yonsei University College of Dentistry, Seoul, Korea.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibitory action of cTPx II on respiratory growth of yeast as a possible physiological function
Authors: Cha Mee‐Kyung , Kim Kyu‐Jung , Hong Seung‐Keun , Oh Young‐Mee , Kim Il‐Han
Published: June 2021

A new type of peroxidase (“thiol peroxidase”; TPx) having cysteine as the primary site of catalysis has been discovered from prokaryotes to eukaryotes. Five TPx homologues were identified in Saccharomyces cerevisiae. Previously, we found that cTPx II, one of cytoplasmic form of yeast TPx isoenzymes, was highly expressed in the ΔRas2 mutant cells compared to their wild‐typed cells (YPK9) and that its expression is under negative control of Ras2 signaling. In this communication, we reported that Ras2‐delected yeast (ΔRas2 mutant) shows a growth deficiency in a non‐fermentable YPGE media as previously reported. The double mutant cell lacking both Ras2 and cTPx II (ΔRas2/ΔcTPx II) was restored to its respiratory growth. The cTPx II gene was able significantly to complement the double mutant phenotype when cTPx II was introduced into the mutant by transformation. The complementation of the ΔRas2/ΔcTPx II mutant phenotype by cTPx II gene provides the genetic evidence that cTPx II is related to the inability of respiratory growth of ΔRas2 mutant. Taken together, we, for the first time, suggest an inhibitory action of cTPx II on respiratory growth of yeast under negative control of Ras2 signaling pathway.

Support or Funding Information

This work was financially supported by NRF (2015R1D1A3A01018288).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ABCD: The Language of Replication Protein A (RPA)
Authors: Schuld Molly , Perez Jose , Anderson C. , Bhatia R. , Ehlers M. , Leger K. , Lentz H. , Marsh S. , Haralson B. , Puls A. , Sheikh S. , Smith A. , Spellecy M. , Sweeney M. , Ya A. , Antony Edwin
Published: June 2021

RPA is a ssDNA binding protein whose function is essential to DNA replication, recombination, and repair. RPA coordinates DNA repair processes responsible for maintaining genomic integrity, and hence is an attractive target for oncology. RPA interacts with ssDNA, and recruits other proteins onto ssDNA. RPA binds to ssDNA very tightly. Current research focuses on determining how an ssDNA‐RPA complex is removed from the DNA by weaker DNA binding enzymes. Structurally, RPA is a heterotrimer made up of three subunits (RPA70, RPA32 and RPA14). The DNA binding function of RPA is carried out by four distinct DNA binding domains (DBDs), A (residues 182 – 305), B (residues 306 – 424), C (residues 425 – 623) and D (residues 46 – 175), that directly attach to the single stranded DNA (ssDNA). DBDs A, B and C reside in the RPA70 subunit and are connected by flexible linkers. DBD‐D resides in the RPA32 subunit. The heterotrimer is held together as a complex through interactions between DBD‐C (RPA70), DBD‐D (RPA32) and the RPA14 subunit. Scientists use unnatural amino acids and chemical fluorophores to capture how each DBD binds and disconnects from ssDNA. This approach shows that DBD‐A binds rapidly to ssDNA, but detaches quickly, while DBD‐D binds more slowly to ssDNA, but is stable. Ronald Reagan High School's SMART (Students Modeling A Research Topic) Team has designed a model of RPA using 3D printing technology to investigate its structure and function. Additional research on RPA structure and mutations may prove helpful in determining cause and risk for cancer and developing potential treatments.

Support or Funding Information

The MSOE Center for BioMolecular Modeling would like to acknowledge and thank the National Institutes of Health Science Education Partnership Award (NIH‐SEPA 1R25OD010505‐01) and the National Institutes of Health Clinical and Translational Science Award (NIH‐CTSA UL1RR031973) for their support in funding the 2017–2018 SMART Team program.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Atypical use of audience response system provides opportunity to formatively assess teaching and improve learning outcomes
Authors: Hopper Mari K , Wright Serena M , Carroll Megan , Bauer Erich , Carlos W. Graham
Published: June 2021

PURPOSE

Although audience response systems (ARS) are frequently used to formatively assess student learning, this technology is rarely employed to formatively assess teaching. Aim of study was implementation of novel approach using ARS to assess teaching methods in order to enact rapid improvement.

METHODS

Second‐year students of large multi‐center medical school participated anonymously. Study involved nine sequential, three‐hour pulmonary class sessions delivered live on one campus and livestreamed to others. Following each session, ARS delivered three Likert scale and one open‐ended question: 1)What degree have you utilized higher order skills; 2)Rate your level of engagement; 3)Estimate percentage of time focused; and 4)What went well/suggestions for improvement. Following each session, course leaders considered student input and adjustments made before delivery of next session. Faculty also quantified parameters including number of cases, key concepts, videos, and slides per session and “Bloomed” embedded questions.

RESULTS

Approximately 60% of students responded to ARS questions. Results indicated consistent improvement from session one to nine in all parameters: number of students perceiving very high use of higher order skills increased from 10% to 30%, students rating the session as highly engaging rose from 55% initially to 80% for final session, and ability to remain focused for more than 70% of the class time rose from 55% of students to over 75%. Comparison of student perceptions to objective measures is underway.

CONCLUSION

Innovative ARS use allowed faculty to immediately gather feedback from diverse students at distributed locations. Feedback guided change leading to improved student engagement, focus, and utilization of higher order skills. Students were also highly appreciative of opportunity to impact change in the class while participating.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Improving Cardiac Function and Body Composition through Incentive‐Based Smartphone Application in Sedentary Overweight Adults
Authors: Choi Myung Dong , Standridge Austin , Landis‐Piwowar Kristin
Published: June 2021

Smartphones evolved from simple calling, texting to streaming video and tracking your daily physical activity. Previous studies support the use of financial incentives in increasing daily physical activity, leading to an increase in human health and lifestyle behavior modification can reduce the risk of cardiovascular disease. However, there is a lack of evidence that smartphone apps which provide incentives, are associated with health promotion.

Purpose

The aim of this study is to determine the effects of an incentive based smartphone app to promote physical activity and improve cardiopulmonary health and body composition in sedentary overweight individuals.

Methods

Twenty sedentary overweight adults (body mass index≥25) were randomly assigned to a control group (20.4±1.3 yrs, n=10) or an intervention group (27.5±12.7 yrs, n=10). The intervention group downloaded an incentive‐based step counter app (Carrot wellness app) on their smartphone. If the daily step goal was met in the intervention group, they were rewarded with a coin that could potentially be traded in for gift cards. The control group downloaded a step counter app (Google Fit app), which does not provide any incentives. Data was collected twice: pre‐ and post‐ten weeks Carrot wellness app usage. Pre and post‐testing evaluations included blood lipid profile, body mass index (BMI), body fat percentage (% body fat), maximal oxygen consumption (VO2max) and cardiac output at 50% of a subject's VO2max. A one‐way ANCOVA was used to determine differences in variables between the groups.

Results

There were significant differences in BMI (P=0.017), body weight (P=0.045), cardiac output (P=0.002) and stroke volume (p=0.002) between the control and the intervention group. However, there were no significant differences in blood lipid profile (e.g. high density lipoprotein, low density lipoprotein, and total cholesterol) or VO2max.

Conclusions

Ten weeks of incentive‐based step counter app usage may be beneficial to improve body composition and submaximal cardiac function in sedentary overweight adults. These findings may support that providing incentives to sedentary adults for exercising may contribute to further health improvements. However, there is no significant improvement on blood lipids and maximal cardiovascular fitness. Overall, our study may indicate that incentive‐based health promoting smartphone apps can be especially helpful for those sedentary adults having difficulty self‐motivating themselves to exercise.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
No changes observed in Circulating Tumor Necrosis Factor‐alpha or receptors following Prolonged Exercise in a Cold Environment
Authors: Jajtner Adam R , Arroyo Eliott , Followay Brittany N , Vaughan Jeremiah A , Laudato Joseph A , Gibson Brandon M , Glickman Ellen L
Published: June 2021

Tumor Necrosis Factor‐alpha (TNF‐α) is a pro‐inflammatory cytokine that is known to increase during exercise in the heat, however, has not been examined in a cold environment.

OBJECTIVE

The purpose of this study was to examine the circulating concentration of TNF‐α and the corresponding expression of TNF‐receptor 1 and 2 (TNFr1 and TNFr2) on classical monocytes following aerobic exercise in a cold environment.

METHODS

Seven recreationally active men (25.0 ± 3.2 yrs; 1.82 ± 0.06 m; 85.3 ± 7.6 kg; 4.04 ± 0.40 L·min−1) completed three experimental visits: a VO2max test, and cycling in 5°C (LT) and 23°C (MT) in a counterbalanced fashion. Cycling consisted of 60 minutes at 60% of their previously determined VO2max (TC60), and a time to exhaustion trial at 90% of their VO2max (TTE). Rectal core temperature was monitored and recorded continuously, while blood samples were taken prior to exercise (PRE), at TC60, TTE and after one hour of recovery (REC). Samples were analyzed for circulating TNF‐α via ELISA, while a subset of participants (n=4) were assessed for TNFr1 and TNFr2 expression on classical monocytes (CD14++/CD16−) via flow cytometry. Non‐normally distributed data were LN transformed, and area under the curve (AUC) was calculated via the trapezoidal method. Data were analyzed using a within‐subjects repeated measures ANOVA, and a paired samples t‐test. Data are presented as mean ± SD.

RESULTS

No significant interaction (F = 0.811, p = 0.504, η2p = 0.119) nor main effects of time (F = 0.670, p = 0.581, η2p = 0.100) or trial (F = 4.093, p = 0.090, η2p = 0.406) were observed for circulating TNF‐α concentration. Additionally, no significant difference was observed between trials for TNF‐α AUC (p = 0.083). No significant interaction (F = 0.811, p = 0.519, η2p = 0.213), nor main effects of time (F = 1.144, p = 0.383, η2p = 0.276) or trial (F = 0.303, p = 0.621, η2p = 0.092) were observed for TNFr1. No significant difference was observed between trials for TNFr1 AUC (p = 0.117). No significant interaction (F = 0.196, p = 0.896, η2p = 0.061), nor main effects of time (F = 0.974, p = 0.447, η2p = 0.245) or trial (F = 0.320, p = 0.611, η2p = 0.096) were observed for TNFr2. Additionally, no differences were observed between trials for TNFr2 AUC (p= 0.236).

CONCLUSIONS

Neither 60 minutes of moderate intensity exercise, nor exercise to exhaustion at a high intensity were sufficient to induce a response in circulating TNF‐α, nor classical monocyte expression of TNFr1 or TNFr2. Furthermore, exercise in the cold does not appear to modify this response.

Support or Funding Information

This study was partially funded by the Kent State University Research Council.

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