bluuebunny/crossref_metadata_2025 huggingface dataset:
Mapping the interface of alpha globin and eNOS: implications for increasing endogenous NO therapeutically
Authors: Keller TC Stevenson , Swope Nicole K. , Columbus Linda , Isakson Brant E.
Published: June 2021

Nitric oxide signaling is a powerful mechanism for the regulation of blood pressure homeostasis, inducing vasodilation and reducing peripheral resistance. As such, disruption of NO‐inhibitory protein interactions may prove to be a novel therapeutic for the treatment of hypertensive disease states. Focusing on the recent discovery of the alpha globin/eNOS complex in vascular endothelium, we have designed an alpha globin mimetic peptide that binds to eNOS and increases NO signaling and dilation. A key step in the development of therapeutic agents targeting this complex is a high‐resolution structural model of the binding interface of alpha globin and eNOS. Thus, we have used solution NMR to determine the relevant structural details of the alpha globin mimetic peptide known to bind to eNOS. Interestingly, this twenty residue peptide has a stable population of both proline cis and trans isomers, potentially influencing the affinity of the peptide/eNOS interaction. Crosslinking mass spectrometry allows for the mapping of interacting residues between the peptide and eNOS after enzymatic digestion. Using cis and trans proline peptide ensembles, we have generated models of the peptide/eNOS complex through docking simulations which can be verified with full‐length alpha globin. The different conformations adopted by the peptide allows for potential new interaction modes that may not be possible with full alpha globin protein, thereby possibly increasing peptide/protein binding affinity. Determining the binding interface of the alpha globin/eNOS complex allows for targeted searches for molecular inhibitors of this alpha globin/eNOS interaction, in efforts for novel therapeutics to combat vasoconstrictive disease.

Support or Funding Information

American Heart Association Grant 16PRE31180040 (TCSK)

NIH 088554 (BEI)

NIH 087828 (LC)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hepatic Endoplasmic Reticulum Associated Degradation (ERAD) manages FGF21 levels and metabolism via CREBH during fasting‐feeding and growth
Authors: Bhattacharya Asmita , Zhang Kezhong , Qi Ling
Published: June 2021

The liver regulates growth and systemic energy homeostasis through inter‐organ communication via the secretion of growth factors, hormones and peptides. Fibroblast growth factor 21 (FGF21) is a liver‐derived, fasting‐induced hormone with broad effects on growth, nutrient metabolism and insulin sensitivity, and huge potential as an anti‐diabetes/obesity therapeutic. Here, we report the discovery of a novel regulatory mechanism repressing FGF21 expression under growth and fasting‐feeding. The SEL1L‐HRD1 complex is the most conserved branch of mammalian endoplasmic reticulum (ER)‐associated degradation (ERAD) machinery, responsible for the recruitment, ubiquitination and retro‐translocation of ER proteins for cytosolic proteasomal degradation. Mice with liver‐specific deletion of SEL1L exhibit growth retardation and improved metabolism with markedly elevated circulating FGF21, phenocopying FGF21 transgenic mice or pharmacological models. Mechanistically, we show that the SEL1L‐HRD1 ERAD complex controls FGF21 transcription by regulating the turnover (and thus nuclear abundance) of ER‐resident transcription factor CREBH, while having no effect on the other well‐known FGF21 transcription factor PPARα. We further show a physiologically‐regulated, inverse correlation between SEL1L‐HRD1 ERAD and CREBH‐FGF21 levels under fasting‐feeding and growth. This study not only establishes the importance of ERAD in liver in the regulation of systemic energy metabolism, but also reveals a novel hepatic “ERAD‐CREBH‐FGF21” axis directly linking ER protein turnover to gene transcription and systemic metabolic regulation.

Support or Funding Information

AHA predoctoral fellowship

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 Mitophagy by the Parkin Ubiquitin Ligase and PINK1 Ubiquitin Kinase
Authors: Gehring Kalle
Published: June 2021

Familial forms of Parkinson's disease arise from loss of the mitochondrial quality control pathway mediated by PINK1, a protein kinase, and Parkin, an E3 ubiquitin ligase. Together, they regulate the disposal of damaged mitochondria by autophagy (mitophagy). When damaged, mitochondria accumulate PINK1, which phosphorylates ubiquitin to recruit cytosolic Parkin. Normally quiescent due to autoinhibitory interactions, Parkin is activated by both phosphorylation by PINK1 and binding phosphorylated ubiquitin. The molecular mechanisms that activate activate Parkin have been the focus of much speculation. Here, I describe the crystal structure of the active conformation of Parkin that shows phosphorylation of parkin by PINK1 leads to a dramatic conformational change to derepress its E2‐binding and catalytic sites. Using a variety of experimental approaches, the activation pathways mediated by phospho‐ubiquitin binding and Parkin phosphorylation are compared. The study deepens our understanding of the mechanisms of Parkin activation and suggests new approaches for the treatment of Parkinson's disease by activating Parkin with small molecules.

Support or Funding Information

Canadian Institutes of Health Research and the Michael J Fox 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.
Characterization of Bacterial Arginine Kinases in Species from the Order Myxococcales
Authors: Fannin Lindsay , Aryal Manish , Stock Katie , Snider Mark , Fraga Dean
Published: June 2021

Phosphagen kinases are found in many eukaryotes and function in energy buffering within the cell through the exchange of a phosphate group between ATP and one of eight substrates (Ellington, 2001). Arginine kinases (AKs) have been identified in a few bacteria and phylogenetic analysis suggest they arrived through horizontal gene transfer (Andrews et al., 2008; Bragg et al., 2012; Suzuki et al., 2013). Recently, the bacterial AK in the well‐studied soil bacteria, Myxococcus xanthus, was knocked out and the deletion strain characterized. The results were impairment in both recovery from some forms of stress and completing a starvation induced development pathway that leads to spore formation. To update our understanding of the bacterial AKs, we conducted a BLAST search of bacterial genomes and found a total of 37 bacterial species with AKs. The majority of the bacterial AKs were deltaproteobacteria (20) with 5 in the genus Myxococcus. Interestingly, the AKs seen in the Myxococcus species seems to have arrived through two independent HGT events and thus present an opportunity to better understand how two similar evolutionary events (HGT of an AK into a soil bacterial genome) have subsequently evolved. Will the outcomes be the same or different? We will describe the biochemistry and phylogenetics of the AKs found within the deltaproteobacteria order Myxococcales.

Support or Funding Information

Funding provided through 2014 NSF‐PGRP 1444539 and the Whitmore‐Williams Science Scholarship at the College of Wooster.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A New Membrane Potential (ΔΨ)‐Independent Iron Indicator Selectively Detects Mitochondrial Chelatable Iron but Not Calcium in Living Cells
Authors: Hu Jiangting , Nieminen Anna‐Liisa , Kholmukhamedov Andaleb , Lindsey Christopher C. , Beeson Craig C. , Lemasters John J.
Published: June 2021

Mitochondrial chelatable iron contributes to several injury processes, including ischemia/reperfusion injury, oxidative stress and the hepatotoxicity of acetaminophen. To measure mitochondrial chelatable iron in living cells and tissues, we synthesized a new fluorescent Fe2+ indicator, mitoferrofluor (MFeF), which has a reactive group that forms covalent adducts with mitochondrial proteins to allow retention of MFeF after subsequent mitochondrial depolarization (Hepatology 60 [Suppl], 812A). MFeF selectively accumulates in the polarized mitochondria of cultured rat hepatocytes. Here we assessed whether MFeF specifically reports mitochondrial iron independent of calcium. After loading cultured rat hepatocytes with 1 uM MFeF and 500 nM rhodamine 123 (Rh123) or 1 μM calcein‐AM for 30 min, confocal microscopy revealed red MFeF fluorescence that co‐localized with green mitochondrial Rh123. Membrane‐permeant Fe3+/8‐hydroxyquinoline(8‐HQ) (10–50 μM) progressively quenched mitochondrial MFeF fluorescence to ~80% within 15 min, but mitochondrial Rhodamine 123 was unchanged. Fe2+ specific calcein fluorescence in the cytosol was also quenched, confirming that Fe3+/8‐HQ undergoes rapid intracellular reduction to Fe2+. MFeF fluorescence increased when the lipophilic transition metal chelator pyridoxal isonicotinoyl hydrazone (PIH) was added to competitively bind mitochondrial chelatable iron. Importantly, MFeF was retained in mitochondria after mitochondrial de‐energization with CCCP (10 μM), oligomycin (10 μg/ml) and myxothiazol (10 μM), confirming mitochondrial retention of the covalently adducted MFeF. In hepatocytes loaded with MFeF, red fluorescence did not decrease after adding 2 μM of Ca2+‐mobilizing thapsigargin, whereas parallel experiments showed that Ca2+‐indicating mitochondrial Rhod‐2 fluorescence did increase, showing that MFeF is insensitive to physiologically relevant increases of mitochondrial Ca2+. In acetaminophen‐induced hepatotoxicity, progressive quenching of MFeF began at ~4 h after overdose acetaminophen (10 mM) in mouse hepatocytes, signifying increased mitochondrial chelatable Fe2+. Mitochondria then depolarized after ~10 h. Dipyridyl, a membrane‐permeable iron chelator, partially restored MFeF fluorescence. In conclusion, MFeF is a ΔΨ‐independent iron indicator that selectively detects mitochondrial chelatable Fe2+ but not Ca2+.

Support or Funding Information

NIH grants: DK073336, AA021191 and T32 DK083262

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Planar Enteroids Reveal an Autonomous WNT And BMP Circuit Controlling Intestinal Epithelial Growth and Organization
Authors: Thorne Curtis A. , Chen Ina W. , Sanman Laura E. , Cobb Melanie H. , Wu Lani F. , Altschuler Steven J.
Published: June 2021

The epithelial lining of the human intestine is a prime example of tight homeostatic control of cell proliferation, organization and fate determination. As a mechanism for dealing with constant mechanical, chemical and pathogen‐derived insults, the epithelium is in constant turnover, completely renewing every four days. Amazingly, this active process produces multiple cell types at just the right ratios and locations throughout our life span. Failure of this exquisite control is the basis for diseases including Crohn's disease, ulcerative colitis, and cancers of the esophagus, stomach, small and large intestine.

A central challenge is to elucidate mechanisms required for robust control of tissue renewal. Opposing WNT and BMP signaling is essential in establishing epithelial homeostasis. However, it has been difficult to disentangle contributions from multiple sources of morphogenic signals in the tissue. Here, to dissect epithelial‐autonomous morphogenic signaling circuits, we developed a planar enteroid intestinal culture system that recapitulates key properties of the intestinal epithelium, specifically the ability to: maintain proliferative and differentiated zones, self‐renew, polarize and generate major intestinal cell types. We systematically perturb intrinsic and extrinsic sources of WNT and BMP signals to reveal a core morphogenic circuit that controls proliferation, tissue organization and cell fate. Our work demonstrates the ability of intestinal epithelium, even in the absence of 3D tissue architecture, to control its own growth and organization through morphogen‐mediated feedback.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
DETERMINATION OF THE THREE‐DIMENSIONAL STRUCTURE OF FULL‐LENGTH HUMAN EPIDERMAL GROWTH FACTOR RECEPTOR BY CRYO‐ELECTRON TOMOGRAPHY
Authors: Purba Endang R. , Akhouri Reeresh R. , Ofverstedt Lars‐goran , Skoglund Ulf , Maruyama Ichiro
Published: June 2021

Human epidermal growth factor receptor (EGFR) plays vital roles in cellular processes including cell proliferation, survival, motility and differentiation. Mice lacking EGFR die shortly after birth due to multi‐organ failure. Aberrant activation of EGFR is implicated in many human cancers. EGFR is synthesized as a single‐pass transmembrane protein, which consists of an extracellular ligand‐binding domain and an intracellular kinase domain separated by a single transmembrane domain. While crystal structures of EGFR domains are available, three‐dimensional structural determination of the full‐length EGFR has not been successful so far. In the conference, we will present cryo‐electron tomography structures of the intact human EGFR in the presence of bound EGF ligand. We successfully purified the full‐length EGFR with autophosphorylation activity upon EGF stimulation. We then prepared vitrified specimens and collected 30 tilt series projections spanning −60 to +60 degrees, with tilt increments of 0.5 degrees, using a FEI Titan Krios at 39,000× magnification and □1–2 μm underfocus. Tilt series data was analyzed with COMET software. These three‐dimensional structures of the full‐length EGFR are invaluable for understanding of mechanisms underlying activation of EGFR by ligand binding.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Soluble Activin Type IIB Receptor Decoy changes Gene Expression Profiles of Bone Cells in the OIM and not the G610C Mouse Model of Osteogenesis Imperfecta
Authors: OMOSULE CATHERINE LAARIPUOH , Jeong Youngjae , Phillips Charlotte , Pfeiffer Ferris
Published: June 2021

Bone is a highly dynamic tissue which is continually remodeled by osteoblasts and osteoclasts, with regulation by osteocytes. In Osteogenesis Imperfecta (OI), a clinically heterogeneous disorder characterized by bone fragility, the functions of osteoblasts and osteoclasts are thought to be impaired. As a mechanosensitive tissue, bone responds to mechanical loads, some of the strongest of which are from skeletal muscles, by changing its shape.

The inhibition of myostatin, a negative regulator of muscle growth and a member of the TGF‐β superfamily, has been shown to induce muscle growth via hypertrophy. In this study, we investigated the effect of pharmacologically inhibiting myostatin through treatment with the soluble activin receptor type IIB decoy molecule (sActRIIB‐mFc), focusing on the gene expression patterns of bone cells derived from two molecularly distinct mouse models of OI (osteogenesis imperfecta murine [OIM] vs. G610C). Because the molecular defects that give rise to the G610C and OIM models differ and muscle function is differentially impacted, we hypothesize that the two models may respond differently to sActRIIB‐mFc treatment. Homozygous mice (oim/oim) in the OIM model have a single nucleotide deletion in the COL1A2 gene that leads to functional null proα2 (I) collagen chains and the generation of homotrimeric type I collagen, whereas the G610C mouse, heterozygous for a missense mutation in the COL1A2 gene, has a glycine to cysteine substitution at the 610 position of the triple helical domain.

At two months of age, WT, +/G610C, and oim/oim male mice were treated bi‐weekly either with vehicle (Tris‐buffered saline) or sActRIIB‐mFc for 8 weeks. Quantitative RT‐PCR analyses demonstrated that tibias from sActRIIB‐mFc treated oim/oim mice exhibited increased gene expression levels of the markers of osteoblast, osteoclast, and osteocyte function and differentiation analyzed. This contrasts with the significant changes seen in the femoral cortical bone microarchitecture and biomechanical integrity of sActRIIB‐mFc‐treated WT and +/G610C mice, which was not observed in the oim/oim mice.

In conclusion, our findings indicate that sActRIIB‐mFc treatment has differential effects on the G610C and OIM mice, which may in part reflect differences in severity (severe in oim/oim vs. moderately severe in +/G610C), and/or their unique molecular defects.

Understanding the molecular impact of therapeutics is critical in order to develop more effective and targeted treatment for OI.

Support or Funding Information

Leda J. Sears TrustKansas City Area life Sciences Institute Patton Trust Research, Inc. University of Missouri Research Board GrantMarch of Dimes

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Divergent C‐terminal Region of Gα12 and Gα13 Provides Unique Effector Binding and Growth Signaling Mechanisms
Authors: Quick Courtney R. , Stecky Rebecca C. , Mull Makenzy L. , Dover Ellen N. , Meigs Thomas E.
Published: June 2021

Heterotrimeric G proteins of the G12/13 class play key signaling roles in cell growth, oncogenic transformation, migration and invasion, and various other cellular responses. This G protein class is represented by the closely related α subunits Gα12 and Gα13 in mammals, whereas invertebrates encode a single G12/13 homolog. We have sought to identify structural features that evolved in mammalian G12/13 proteins to allow their divergent mechanisms of growth signaling. Previously, we demonstrated that the Drosophila G12/13 homolog Concertina failed to drive growth signaling through serum response factor, and thus we generated a set of evolutionarily guided chimeras in which regions of mammalian Gα12 and Gα13 were replaced by the corresponding invertebrate sequence. These substitutions revealed a non‐conserved, 42‐residue region near the C‐terminus of Gα12 which is necessary for its growth signaling function, whereas the same invertebrate sequence replacement had no effect on Gα13 signaling. In addition, we utilized invertebrate amino acid substitutions to identify specific determinants in this region of Gα12 that are important for its interaction with specific effector proteins. Gα12 mutants uncoupled from signaling through serum response factor are currently being tested for ability to signal through more primitive pathways, including cytoskeletal rearrangements. To further evaluate the functional significance of this 42‐residue region, additional chimeras were constructed to place isolated C‐terminal regions of Gα12 into Gα13. Protein co‐precipitation experiments indicated the divergent 42‐residue region of Gα12 bestowed upon Gα13 the ability to interact with several Gα12‐specific binding partners. These findings indicate that Gα12 and Gα13 evolved to harbor different C‐terminal structural determinants of growth signaling and effector binding that are not present in invertebrate G12/13 homologs.

Support or Funding Information

We acknowledge support from the North Carolina GlaxoSmithKline Foundation, the C.D. Spangler Foundation, and UNC Lineberger Comprehensive Cancer Center.

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 ANKDR49 in evaluating the prognosis of glioma
Authors: Duan Hubin , Hao Chunyan , Wang suping , Wang Huan , Gao Ling , Zheng Xiaojun , Duan Shengqiang , Gao Weiqin , Zhang Ce
Published: June 2021

Objective

To evaluate the role of ANKRD49 in the prognosis of glioma.

Methods

Transcriptome expression data sets and the corresponding clinical information of ANKRD49 gene were downloaded from the website of The Cancer Genome Atlas (TCGA). A total of 558 samples, which contained transcriptional expression data of 548 tumor tissues and 10 normal tissues, were available for this analysis. Human malignant glioma cell lines U251 and U87 were cultured. Total RNA was isolated from indicated cells using TRIzol reagent. Quantitative real‐time PCR was used to analyze ANKRD49 gene expression. Total protein was isolated from indicated cells and West‐blot was used to analyze ANKRD49 protein expression. SPSS 16.0 software was used to analyze experimental data and clinical data.

Results

The expression of ANKRD49 was predominantly higher in glioma samples compared with matched normal samples(fold change =2.11, P=2.99E‐09). The 5‐year overall survival of patients with highly expressed ANKDR49 was distinctively shorter than that of patients with lowly expressed ANKDR49(P<0.01).

Conclusion

ANKRD49 is overexpressed in human glioma tissues, and high expression of ANKRD49 is significantly associated with poor survival. ANKDR49 might be a indicator for evaluating the prognosis of glioma.

Support or Funding Information

This study was supported by the National Natural Science Foundation Youth Fund (Fund No. 30600637), China Postdoctral Foundation(Fund No.2014M561207), the Shanxi Province Basic Research Program Youth Science and Technology Research Fund (Fund No. 2010021034‐4), the Shanxi Scholarship Council of China (Fund No. 2011‐096 and Key Projects No.4), The science and technology research projects of health department of Shanxi Province(Fund No.2016D011119), The Teaching Reform Program of Shanxi Higher Education (No. 2013‐033), The Education Reform Project of Postgraduate Students in Shanxi Province (No. 2017‐027). The Project of “131” Leading Talents of Shanxi Higher Education, The Education Reform Project of Shanxi Medical 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.
Implementing the CURE: Combining Wet‐Lab Protein Biochemistry with Computational Analysis to Provide Gains in Student Learning in the Biochemistry Teaching Lab
Authors: Pikaart Michael
Published: June 2021

Most undergraduates studying biochemistry and molecular biology get their broadest exposure to wet‐lab techniques in protein and nucleic acid chemistry (and, increasingly, computer/visualization) in their upper‐level laboratory courses. These tend to be juniors and seniors with well‐defined career goals. Some of these students will have already have a research background in a traditional one‐to‐one (or one‐to‐few) research mentoring setting, for example a summer research program. This approach has proved effective at increasing student learning and persistence in the sciences. At the same time, extended full‐time PI‐directed research is limited in the number of students served, and can even present a barrier. To broaden the impact of teaching through research, many practitioners have adopted a CURE, or Course‐based Undergraduate Research Experience, approach.

This presentation reports on “BASIL” (Biochemical Authentic Scientific Inquiry Laboratory), a team of faculty who have worked to bring computational and wet‐lab protein science to the biochemistry teaching lab. Together, we have developed a protein biochemistry CURE to determine enzymatic function of proteins of unknown activity. This work leverages the results of the Protein Structure Initiative, a fifteen‐year NIH‐funded effort which concluded in 2015 with the publication and distribution of more than 5000 previously uncharacterized proteins. The great majority of these are “orphans,” with high quality structures and pre‐cloned expression plasmids available, but no research on their enzymatic function or role in native organisms. The BASIL consortium of undergraduate biochemistry faculty and students seeks to identify functional properties of a subset of these uncharacterized proteins, seeking to unify structure and function relationships. Currently, implementable modules are available for faculty who wish to adopt them, and expected student results will be presented.

Support or Funding Information

Supported by NSF IUSE 1709278

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Gene Regulatory Pathways that Modulate Response to Dexamethasone and Daunorubicin in Breast Cancer Cell Lines
Authors: Macias Alejandro Rafael , Medh Rheem
Published: June 2021

Glucocorticoids (GCs) such as Dexamethasone (Dex) are anti‐cancer agents that induce apoptosis (programmed cell death) and counter side effects of chemotherapy through regulation of pro‐ and anti‐apoptotic genes such as E4BP4, FOXO3A, BIM and BIRC3. Anthracyclines, such as Daunorubicin (Dauno), are effective anti‐cancer agents for leukemias, breast cancer and ovarian cancer. Although several regimens combine both classes of chemotherapy agents, Dex has been reported to promote rather than retard breast cancer cell proliferation, and counter the pro‐apoptotic effects of other chemotherapeutic agents. E4BP4, a transcriptional regulator, is upregulated by GCs and is known to mediate Dex‐evoked apoptosis in leukemia cells, but has been reported to promote cell survival in other models of cancer, including breast cancer. The phosphorylation state of E4BP4 and its crosstalk with the FOXO3A and AKT pathways modulate its pro‐survival or pro‐apoptosis functions. We hypothesize that Dex‐evoked upregulation of E4BP4 and its subsequent modulation of proliferative signals may contribute to pro‐survival effects of Dex in breast cancer. In the present study we are investigating the response of breast cancer cell lines MCF‐7 (estrogen receptor positive) and MDA‐MB‐468 (triple negative) to Dex and Dauno (single agent and in combination) with respect to cell viability, apoptosis and regulation of pro‐ and anti‐apoptotic genes including E4BP4, FOXO3A, BIM and BIRC3. MTT cell viability assays determined that MDA‐MB‐468 and MCF‐7 cells are resistant to 1 μM Dex, with viable cells being about 92% and 140% compared to untreated controls, respectively. However, MDA‐MB‐468 cells were sensitive to 1 μM Dauno and in combination with 1 μM Dauno and 10 μM Dex with viability at 54% and 58% compared to control, respectively. To confirm that cell death occurs via apoptosis, cells are stained with Annexin V‐FITC followed by epifluorescence microscopy to detect apoptotic cells. Evaluation of Dex and Dauno‐mediated regulation of gene expression was done via reverse‐transcriptase and end‐point PCR analysis, followed by ImageJ quantitation, or by RT‐qPCR analysis. Our data suggest that Dauno upregulates while Dex downregulates E4BP4 in MDA‐MB‐468 cells. Combined treatment significantly enhances FOXO3 upregulation compared to either single agent, in correlation with increased cell death. Our data suggest that Dex and Dauno may cooperatively promote apoptosis in MDA‐MB‐468 cells.

Support or Funding Information

This project was funded by a NIH SCORE SC3 grant (GM081099) to Dr. Rheem D. Medh and NIGMS BUILD ( 8TL4GM118977) scholarship to Alejandro Macias.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Induction of cancer stem cell sphere explosion by UV irradiation/cold shock or therapeutic chemicals: detection of autofluorescence using visible wavelength
Authors: Chang Nan‐Shan , Chang Katarina T. , Ho Pei‐Chuan
Published: June 2021

We have recently identified a new type of cell death, called “bubbling cell death”. When cells are exposed to UV irradiation followed by incubation at 37°C, they undergo typical apoptosis. However, when the UV‐irradiated cells are incubated at room temperature or down to 4°C, they generate bubbles – one bubble per cell – from the nucleus. The bubbles contain nitric oxide. There is occurrence of no caspase activation, flip‐over of phosphatidylserine, and DNA fragmentation, indicating that bubbling death is a novel type of cell death (Oncotarget 2015;6:8007–18). Here, by time‐lapse microscopy, we demonstrated that when breast cancer 4T1 stem cell spheres were subjected to UV and a brief cold shock (5 min at 4°C), followed by imaging at room temperature. The spheres initially shrank, then slowly expanded and finally exploded, and the cells died. Similar results were observed when the spheres were exposed to an aliquot of cocktail of protease inhibitors and an ERK inhibitor U0126, or an anti‐lung cancer drug ceritinib. Notably, prior to sphere explosion, there was an emission of luminescent light under bright light stimulation. We developed a novel approach to measure visible wavelength‐induced autofluorescence up to energy for DAPI imaging. In addition, a time‐dependent initial increase and then reduction in redox activity and formation of stress fibers occurred. Loss of cellular lipids and nuclear uptake of DAPI first and then propidium iodide prior to death were also observed. The entire course lasts 24–48 hr at room temperature. However, sphere explosion is accelerated dramatically by blebbistatin, an inhibitor of myosin II, suggesting the participation of cytoskeletal proteins in the sphere explosion.

Support or Funding Information

MOST, Taiwan

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 brominated flame retardant tetrabromobisphenol‐A increases DNA methylation at the THY1 (CD90) locus to promote adipogenesis
Authors: Flores E'Lissa
Published: June 2021

Obesity has been classically linked to poor diet/excessive calorie intake and insufficient physical activity. However, an emerging concept is that environmental pollutants may be driving the global obesity epidemic. The rapid increase in the prevalence of obesity in industrialized, developed and even undeveloped countries cannot be strictly accounted for by poor diet and physical inactivity. Environmental chemicals, termed “obesogens” can promote adipogenesis (fat cell formation). Tetrabromobisphenol‐A (TBBPA), a brominated flame retardant found in infant mattresses and electronics such as cell phones, is an obesogens. However, its mechanism of action remains unknown. We hypothesize that TBBPA promotes adipogenesis by decreasing levels of THY1 (CD90), a cell surface protein present on pre‐adipocytes, certain fibroblasts, and stem cells. We previously determined that fibroblasts are heterogeneous for THY1 expression, where only THY1‐/low fibroblasts differentiate into adipocytes, pointing to THY1's crucial role during adipogenesis and cell fate. Using the well‐established mouse pre‐adipocyte cell line (3T3‐L1), we determined that TBBPA reduces THY1 mRNA and protein levels, promoting adipogenesis. Epigenetic changes, such as increases in global DNA methylation are known to occur during adipogenesis and obesity. DNA hypermethylation typically silences gene expression. Therefore, we hypothesized that obesogens, such as TBBPA, alter epigenetic regulation of THY1 to decrease its expression, thereby priming precursor cells to become adipocytes. Using pyrosequencing, we found that CG rich regions on THY1 are hypermethylated during adipogenesis, resulting in decreased THY1 expression and cell differentiation to adipocytes. Using the methylation inhibitor, 5‐aza‐dC, we determined that the adipogenic effects of TBBPA are blunted when methylation is impeded, which results in increased THY1 levels. Our results confirm THY1 plays a critical role in adipogenesis and that its expression is regulated by increases in DNA methylation. Furthermore, TBBPA, a widely‐used flame retardant, increases DNA methylation at the Thy1 locus to promote adipogenesis. Environmental obesogens contribute to the worldwide obesity epidemic and their further study is critical to combating this epidemic.

Support or Funding Information

P30 ES 001247, F31 ES027767‐02

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Resveratrol in Cell Migration and Invasion by Studying the CXCR4‐CXCL12 axis in Breast Cancer Cell Lines
Authors: Arroyo‐Martinez Gerardo Antonio , Figueroa Maria , Muñoz‐Forti Kevin , Trossi Geralin , Robles Jose , Maldonado Alexandra Arroyo , Suarez Edu , Ruiz Abigail
Published: June 2021

Every year, approximately half a million people die due to breast cancer (BC), comprising 16% of the incidence of all cancer occurrences in females. It is the most prevalent invasive cancer among women around the world. In several studies, researchers have suggested the presence of certain chemokines associated with cancer. The chemokine receptor CXCR4, a G‐protein coupled receptor, has a role in trafficking, cell activation, and differentiation in non‐malignant. It has been shown that CXCR4 is overexpressed in cancerous tissues compared to non‐malignant cells. This overexpression has led to an increase in proliferation, migration, and angiogenesis of malignant cells. Tissues with higher expression of the CXCR4 specific ligand CXCL12, are more likely to metastasis to tissue expressing CXCR4. These target tissues include lungs, bone marrow, and lymph nodes. Due to the impact of this disease, there is a need of developing new therapies or adjuvants to target BC. For example, resveratrol is a natural product and a polyphenol derived from grapes, wine, and nuts, among others. Recent studies found that it plays a role in apoptosis, inflammation, and neovascularization. We hypothesized that resveratrol will decrease the expression of CXCR4 in a dose and time‐dependent manner, leading to a decrease in the migration and invasion cellular processes. Using western blot analysis of HS578T (Human Breast Carcinoma) compared with CCD1074Sk (Non‐malignant Human Breast Tissue) cell lines we studied the effects of resveratrol at the CXCR4 protein level and invasion markers involved in metastatic processes. Migration and invasion were assessed using a wound healing assay and matrigel invasion assay, respectively. The preliminary data shows that as the concentration (25, 50, 100, 200μM) increases the cell migration and invasion decreases compared to our control vehicle (EtOH). In addition, we observed a decrease in the expression of pro‐migration and invasion proteins such as N‐cadherin and Cathepsin‐b. This study suggests an effect of resveratrol in cell migration and invasion leading to a decrease of the in‐vitro cell metastatic potential. Currently we are evaluating CXCR4 at the protein expression level to complete our data.

Support or Funding Information

This work was supported by UPR PRISE Program NIH‐NIGMS #2R25GM096955

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
HCT116 Colorectal Cancer Cells Secrete Chemokines Which Induce The Chemotaxis and Intracellular Calcium Mobilization of NK92 Cells. Influence of Dimethyl Fumarate and Monomethyl Fumarate
Authors: Maghazachi Azzam , Elemam Noha , Al‐Jaderi Zaidoon
Published: June 2021

Our group recently reported that pre‐treatment of IL‐2 activated human natural killer (NK) cells with the drugs dimethyl fumarate (DMF) and monomethyl fumarate (MMF) up‐regulated the expression of chemokine receptor CCR10 on the surface of these cells. Ligands for CCR10, namely CCL27 and CCL28 induced the chemotaxis of these cells. Here, we performed bioinformatics analysis to see what chemokines might be secreted from human HCT116 colorectal cancer cells. We observed that in addition to CCL27 and CCL28, HCT116 cells highly express CXCL16 which binds CXCR6. Consequently, we examined whether CXCL16, CCL27 and CCL28 might induce the in vitro chemotaxis of NK92 cells after pre‐treatment with DMF and MMF for 24 h. Incubation of NK92 cells with 100 μM MMF enhanced their chemotaxis towards the 10 ng/ml concentration of CCL27 and towards 10 and 100 ng/ml concentrations of CCL28, whereas pretreatment with 100 μM DMF enhanced their chemotaxis towards the 100 ng/ml concentration of CCL28. Similar to their effects on human IL‐2‐activated NK cells, MMF and DMF up‐regulated the expression of CCR10 on the surface of NK92 cells. Supernatants collected from HCT116 cells after 24 or 48 h incubation also induced the chemotaxis of NK92 cells. CXCL16, CCL27 and CCL28 as well as supernatants collected from HCT116 cells induced the mobilization of intracellular (Ca)+2 in NK92 cells. Cross‐desensitization experiments revealed that supernatants collected after 24 h from HCT116 colorectal cancer cells desensitized the activity of CXCL16 and CCL28 but not CCL27, whereas the 48 h supernatant desensitized the activity of all three chemokines examined. In summary, these results might have important implication in directing the anti‐tumor effectors NK cells towards the sites of tumor growth, particularly colorectal cancers.

Support or Funding Information

This work is supported by grants from the university of Sharjah

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cloning the P53 DNA Binding Domain
Authors: Jones Janae , Faustino Vroniqa , Nogaj Luiza A.
Published: June 2021

P53 is a tumor suppressor protein that binds to the DNA and is responsible for regulating cell growth. Mutations of p53 are associated with numerous types of cancers in patients worldwide. Mutants of p53 affect not only its structure, but its original function, causing p53 to gain oncogenic functions. The focus of this research is to clone the DNA binding domain (DBD) from the wild type p53 as well as its most common mutants and to characterize their p53 DBD. It is proposed that when missense mutations are present within the p53 DBD, p53 will destabilize, leading to its aggregation within the cell. We used molecular cloning techniques such as PCR to isolate the DBD from p53. We also utilized mammalian cell viability assays such as MTT and cytotoxicity to determine the effects of p53 on human cells. PCR results showed that DBD was amplified from full length p53 and cloned into expression vectors pET28 and pcDNA3.1. In our preliminary results both the MTT and cytotoxicity assays showed that WT p53 decreases the viability of mammalian cells. Therefore, we successfully cloned the DBD from p53 and are in the process of characterizing the effects of mutant p53 on cell viability and aggregation propensity.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sirtuin 4 controls leucine metabolism and insulin secretion by reversing effects of reactive metabolites
Authors: Huynh Frank K. , Anderson Kristin A. , Stuart J. Darren , Lin Zhihong , Hirschey Matthew D.
Published: June 2021

Sirtuins are a family of conserved NAD+‐dependent enzymes that remove a variety of post‐translational acyl lysine modifications. By performing this deacylation function, sirtuins can regulate several aspects of metabolism and aging. Recently, we discovered new post‐translational modifications (PTMs) that can be removed by sirtuin 4 (SIRT4). Interestingly, these PTMs can be generated by reactive metabolites produced during leucine oxidation and in the absence of SIRT4, these PTMs accumulate on and inhibit enzymes in the leucine oxidation pathway. These observations suggest that SIRT4 is a key regulator of leucine metabolism through a novel mechanism that responds to levels of reactive metabolites. Accordingly, we found that SIRT4KO mice have dysregulated leucine metabolism. As a consequence of this, SIRT4KO mice have elevated leucine‐stimulated insulin secretion. Long‐term stimulation of insulin secretion by leucine over time in SIRT4KO mice led to elevated plasma insulin levels even in the basal state. This chronic elevation of insulin was associated with an acceleration of aging‐induced insulin resistance in SIRT4KO mice. Interestingly, when leucine was administered as a bolus with glucose, insulin secretion was synergistically increased more than 3‐fold over glucose or leucine alone in aged SIRT4KO mice. Importantly, this elevated insulin secretion resulted in total normalization of glucose tolerance to resemble that of a young mouse and despite extremely high insulin levels, there was no sign of dangerous hypoglycemia. Thus, our data reveal that short‐term inhibition of SIRT4 potentially has therapeutically relevant effects on leucine metabolism and insulin secretion, but a long‐term loss of SIRT4 can drive inappropriate hyperinsulinemia and lead to insulin resistance. Taken together, our data reveal a novel mechanism of metabolic control involving reactive metabolites and position SIRT4 as a key regulator of leucine metabolism and insulin secretion.

Support or Funding Information

We would like to acknowledge funding support from the American Heart Association grants 12SDG8840004 and 12IRG9010008, The Ellison Medical Foundation, the National Institutes of Health and the NIA grant R01AG045351, and the Duke Pepper Older Americans Independence Center (OAIC) Program in Aging Research supported by the National Institute of Aging (P30AG028716‐01). FKH was supported by an American Diabetes Association/Canadian Diabetes Association Post‐doctoral Fellowship (PF‐3‐13‐4342‐FH).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
How Do You Measure Up? BMI versus Waist to Height Ratio
Authors: Smolinski Kasia , Ward Taketa , Hall Brandon , Hurst Sarah Elizabeth
Published: June 2021

For many years, clinical screening rationale for issues such as obesity or type 2 diabetes (T2D) has been the body mass index (BMI) calculation. Our research as well as many others show that a more useful measurement of an individual's health and risk for disease is the waist to height ratio (WtHR). In this study, we measured height, weight, and waist circumference of 65 participants, calculated both BMI and WtHR, and analyzed the data. Using the BMI calculations, our results showed that most males were overweight [F (7, 14) = 3.147, p = 0.03], while most females were classified as normal [F (7, 14) = 3.474, p = 0.02]. However, using WtHR measurements, distributions changed for both men and women. Males were more normal [F (7, 28) = 2.263, p = 0.06], while females were more evenly distributed with some being slender/underweight and others were highly obese [F (7, 28) = 11.77, p < 0.0001]. Evaluating co‐morbidities using both BMI and WtHR, obese males report more medical issues then their normal and overweight peers. For the females, both the obese and highly obese report more co‐morbidities; both sexes had more people who reported no medical issues. Taken together, using more accurate measurements as health indicators will aid physicians and health care professionals in making better, cost‐effective decisions regarding patient treatment. Furthermore, many institutions including NIH have already transitioned into using the WtHR.

Support or Funding Information

Funding provided by donations to The ATP10A Project.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anti‐photoaging activity of sinapic acid in UVB‐irradiated human skin fibroblasts through increasing the expression of PPARδ
Authors: Yeon Dasom , Kim Younghwa , Lee Junsoo
Published: June 2021

Sinapic acid is a phenylpropanoid compound extensively found in spices, citrus and berry fruits, vegetables, cereals, and oilseed crops. Sinapic acid is known to exhibit antioxidant, anti‐inflammatory, anticancer, antimutagenic, antiglycemic, neuroprotective, and antibacterial activities. However, the inhibitory effect of sinapic acid on photoaging has not been fully investigated. The aim of this study was to investigate the protective effect of sinapic acid on UVB‐induced photoaging through PPARδ mediated mechanism in Hs68 cells. Sinapic acid attenuated UVB‐induced expression of MMP‐1 and increased the production of collagen in Hs68 cells. Furthermore, UVB‐induced ROS generation was markedly attenuated by pretreatment with sinapic acid, demonstrating that sinapic acid is to be a potent ROS scavenger. These effects were abolished by treatment with GSK0660, a specific inhibitor of PPARδ. These results suggest that PPARδ is a key regulator of UVB‐induced photoaging and sinapic acid may ameliorate the UVB‐induced photoaging in human skin fibroblasts through modulatiing PPARδ expression.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Betulinic acid alleviates body fat accumulation and dyslipidemia by inhibiting de novo lipogenesis and stimulating lipolysis in vivo
Authors: Kim Hyunkyung , Ryu Yungsun , Go Gwang‐woong
Published: June 2021

Betulinic acid (BA), derived from natural substances especially birch wood bark, is a pentacyclic triterpenic acid. BA has beneficial bioactive effects as an antagonist for tumors, HIV and oxidants. Studies on BA effects against obesity and dyslipidemia have not clearly clarified the lipogenic pathway. This research hypothesized that betulinic acid would improve body fat and dyslipidemia in vivo. Male C57BL/6 mice were randomly assigned to five groups as follows: 1) high cholesterol diet (HCLD, 45% kcal from fat, 1.25% cholesterol) as control, 2) fenofibrate (150 mg/kg b.w + HCLD) as positive control 3) BA5 (BA 5 mg/kg b.w + HCLD), 4) BA15 (BA 15 mg/kg b.w + HCLD) and 5) BA25 (BA 25 mg/kg b.w + HCLD). BA was orally administered for 12 weeks. Body weight of BA25 was decreased compared to the control from 2 weeks to 12 weeks (p < 0.05 and p < 0.01). Fat mass was reduced by approximately 32% in all BA treatment groups (p < 0.05 and p < 0.001). Serum triglyceride was improved by all BA treatments (p < 0.001). Free fatty acid was reduced by all BA treatment groups (p < 0.01 for BA 25). Total cholesterol was diminished by all BA treatments (p < 0.001). LDL‐cholesterol was reduced by BA5 and BA15 (p < 0.05, p < 0.01 respectively). Acetyl‐CoA carboxylase1, a lipogenic enzyme in the liver, was inactivated by BA treatments (p < 0.001 for BA25). Stearoyl CoA desaturase was decreased by BA15 and BA25 (p < 0.01 and p < 0.05 respectively). Fatty acid synthase and sterol regulatory element‐binding protein 1 were reduced by BA although not significantly. Lipase, an enzyme related to lipolysis in white adipose tissue, was increased by BA15 and BA25 (p < 0.01 and p < 0.05 respectively). Phosphorylation hormone sensitive lipase was increased by BA5 (p < 0.01). Inactivating low‐density lipoprotein receptor‐related protein 6 in liver, which plays a major role in lipid homeostasis, was increased by BA25 (p < 0.001). Major factors of dyslipidemia in serum were alleviated by inhibiting lipogenesis in liver and increasing lipolysis in white adipose tissue. It was confirmed that BA is a potential compound for prevention and treatment of metabolic diseases caused by obesity.

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