bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulating Nociceptive Channel Trafficking by Site‐directed mutagenesis of a Calcineurin Interaction Motif
Authors: Chen Annabel
Published: June 2021

Introduction

Transient receptor potential (TRP) channels are a family of receptor proteins regulating pain sensation. TRP receptors are activated by a variety of noxious stimuli, such as capsaicin (chili peppers), heat, and mechanical stress. Chronic TRPV1 stimulation leads to desensitization of the receptor, a process which is mediated by calcium/calmodulin dependent kinases. This attribute, however, has yet to been leveraged for treatment of chronic pain diseases. Here, we identify a C‐terminal phosphorylation site that is involved in regulating TRPV1 trafficking to the cell surface.

Results and Conclusion

In dorsal root ganglion cells, confocal microscopy and western blot show that de‐phosphorylation of threonine 705 is sufficient to induce significant trafficking to the plasma membrane. Mimicking constitutive phosphorylation at that site induces the receptor to be retained on intracellular membranes. TRPV4 ‐ another member of the TRPV family ‐ also has a similar homologous region to TRPV1 that contains two adjacent threonines T739 and T740, both potentially involved in regulating TRPV4 trafficking. Confocal and western blot data show that removing the T740 phosphorylation site is sufficient to significantly increase surface trafficking. However, the phosphomimetic mutation reduced but did not eliminate surface expression. These data lead us to believe that phosphorylation at both threonines is necessary for full internalization of TRPV4 to occur. Together, these data suggest that the highly conserved Calcineurin interaction motif is an important trafficking regulation site for multiple members of the TRP family, and that manipulating key phosphorylation sites is sufficient to significantly change protein trafficking patterns.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Analysis of Spin Probe Viability for Protein Structure Investigation Using Advanced EPR Techniques
Authors: Ebersol Lauren , Bokhari Aneesa , Silakov Alexey
Published: June 2021

An accurate depiction of protein structure and its mobility is necessary to understanding protein function and many methods exist to determine this. Obtaining protein structural characteristic by advanced electron paramagnetic resonance (EPR) spectroscopy techniques like double electron‐electron resonance (DEER) is emerging as a powerful technique that is complementary to other well established structural characterization tools. Such studies most commonly require the ligation of “spin labels” to different sites on the protein, often by a site‐specific reaction between a stable molecule containing a nitroxide radical and an amino acid residue. We seek to develop a robust set of tools for predicting structures of hard‐to‐characterize proteins by combining the distance information obtained from advanced EPR techniques with mobility information obtained from room temperature continuous wave EPR (CW EPR). Many methods of spin‐labeling a protein exist; however, the viability of these specific approaches for performing structural determination techniques like DEER has not yet been explored. The most common spin‐labeling methodology conjugates (1‐oxyl‐2,2,5,5‐tetramethylpyrroline‐3‐methyl) methanethiosulfonate (MTSSL) to a protein via a disulfide bond with a cysteine residue. To achieve this conjugation site‐specifically, all native surface‐located cysteine residues must be removed, which may not be desirable from a structural integrity standpoint. Utilizing unnatural amino acids can alleviate this issue and the spin‐label can be added to the protein via techniques such as click chemistry, a rapid cycloaddition that can occur at physiological conditions. In this work, we compare how different spin labeling techniques perform from an experimental point of view. We seek, overall, to determine a powerful and easy‐to‐use protocol for structural determination. Here, we use T4 Lysozyme as a well‐characterized protein model and compare 4‐alkyl TEMPO installed on the unnatural amino acid p‐‐azidophenylalanine (pAzF) with the aforementioned MTSSL/cysteine label. For this purpose, we use a cysteine‐free version of T4 Lysozyme and genetically encode either cysteine or pAzF to specific positions. The results presented focus on the structural rigidity of the spin labels, a key factor defining the flexibility of certain areas of the protein and the certainty of the distance determination. Moreover, as different spin probes display different spectroscopic characteristics, each spin label is evaluated in terms of DEER sensitivity and efficiency. Finally, we compare different advanced EPR techniques (DEER, DQC, relaxation enhancement) to determine the most robust in distance predictions.

Support or Funding Information

The work is funded by the Pennsylvania State University startup fund of Alexey Silakov.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Optimizing Protein Crystal Formation of a Heterocyclization Domain from Yersiniabactin Synthetase
Authors: Patterson MacKenzie , Dieu Viet , MacRae Mark , Henriquez Bryan , Soule Jessica , Gnann Andrew , Dowling Daniel P.
Published: June 2021

The bacterium Yersinia pestis, known for causing the bubonic plague, is an organism which must scavenge free iron from its host to grow. One method of obtaining iron is by producing a siderophore, yersiniabactin (Ybt), which is a secondary metabolite synthesized by a hybrid polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS). This system utilizes three interesting heterocyclization (Cy) domains to introduce three five‐membered rings that are important for iron affinity. This research project aims to determine the molecular features of the third Cy domain (Cy3) that determine its function using X‐ray crystallography.

Support or Funding Information

Funding for this research was provided in part by the Oracle Education Foundation grant to the College of Science and Mathematics and also by an ASBMB Undergraduate Research Award. Special thanks to the MIT crystallography facility. This research was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number 1R15GM123425‐01 to D.P.D.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evolutionary Conservation of Variant‐dependent Prion‐promoting Hsp40 Functions in Plants
Authors: Brown Rebecca E. , Hines Justin K.
Published: June 2021

The J‐protein (Hsp40) Sis1 is required for the propagation of yeast prions and for the efficient curing of the prion [PSI+] by overexpression of the disaggregase Hsp104. Interestingly, some of the prion‐supporting functions of Sis1 are conserved in homologs from higher organisms: Sis1 homologs from fruit flies and humans have been shown to substitute for Sis1 in yeast in the propagation of some, but not all, yeast prions. The number and diversity of J‐proteins is greatest in the cytosol and the number of J‐protein genes in the eukaryotic genome generally scales with the overall biochemical complexity of the organism and genome size. As such, yeast has 23 J‐proteins, humans have 41, and we recently reported that the model plant Arabidopsis thaliana has 106. Not surprisingly, at least 6 functional homologs of Sis1 have been identified in A. thaliana. Here we present the results of our ongoing investigation, using yeast complementation assays and multiple prions, to examine which prion‐maintaining functions of Sis1 are conserved in various plant Sis1 homologs. Most notably, preliminary results indicate that these homologs give rise to distinct patterns of prion maintenance, indicating that the diversification of Sis1‐homolog structure has created alternative combinations of functions that affect prion‐propagation. In addition to providing information about J‐protein evolution, our investigation is expected to lead to a greater understanding of how the amino acid sequences of various J‐proteins allow for prion‐specific interactions in yeast.

Support or Funding Information

This work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R15GM110606. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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 ER Hsp70 Hspa13 Redirects an Amyloidogenic Protein to Aggregation
Authors: Genereux Joseph
Published: June 2021

The conserved ER Hsp70 chaperone Hspa13/STCH is implicated in cancer and neurodegenerative diseases. While other Hsp70 proteins maintain proteostasis by promoting folding, degradation, or disaggregation of misfolded protein, the biological function of Hspa13 is unknown, in part because it lacks the substrate‐binding domain required for the function of other members of the Hsp70 class. We investigated the intracellular effect of Hspa13 overexpression on the kinetically stable, amyloidogenic protein transthyretin (TTR), finding that Hspa13 promotes the partitioning of TTR into high‐molecular weight, detergent‐resistant aggregates inside living cells. This function is ATP‐independent and opposed by the ER nucleotide exchange factor HYOU1, but unaffected by the primary ER Hsp70 BiP. These results suggest a role for Hspa13 in maintaining ER proteostasis by regulating the aggregation of amyloidogenic protein in the secretory pathway.

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 Parkinson's Disease Protein α‐synuclein Alters the Microenvironment of the Endoplasmic Reticulum in Saccharomyces cerevisiae
Authors: Haak Victoria Melina , McBride Trevor John , Haverly Meredith Julia , Austriaco Nicanor
Published: June 2021

The protein α‐synuclein forms aggregates in human dopaminergic neurons triggering apoptosis and Parkinson's Disease (PD). To better understand the link between protein aggregate formation and cell death, we have over expressed wild type and two mutant forms, A30P and A53T, of human α‐synuclein in the budding yeast, Saccharomyces cerevisiae. Our experiments suggest that these aggregates trigger the unfolded protein response (UPR) in yeast and that different clinically relevant variants of α‐synuclein up‐regulate the UPR to different degrees. Moreover, we show that the overexpression of α‐synuclein alters the redox state and the calcium dynamics of the yeast ER. Finally, our preliminary data suggests that sub‐lethal doses of tunicamycin and β‐mercaptoethanol, drugs that trigger the UPR, may alleviate the aggregation of α‐synuclein. Our results point to possible pharmacological interventions that may lower protein aggregation in PD. [Our laboratory is supported by grant NIGMS R15 GM110578, awarded to N. Austriaco.]

Support or Funding Information

[Our laboratory is supported by grant NIGMS R15 GM110578, awarded to N. Austriaco.]

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PrPc: The Normal Prion
Authors: Wright Collette , Howard Ashley , Lim Shulammite , Lakshman Priya , Loo Cheyenne
Published: June 2021

PrPc is a normal cell‐surface glycoprotein that is conformationally characterized by two alpha helices and two complex‐type N‐linked oligosaccharide chains. This protein is unique in its propensity to misfold into a neurodegenerative disease‐causing proteinaceous infectious particle, known as a prion. PrPc can undergo conversion into PrPSc through spontaneous misfolding, a genetic mutation of the human PRNP gene, or exposure to a prion from an external source. When this happens, its composition shifts from an alpha‐helical, soluble protein low in beta pleated sheets to an insoluble, protease‐resistant particle with a high percentage of beta sheets. This resulting misfolded form, known formally as PrPSc, is pathogenic and aggregates in neural tissue by recruiting and converting more PrPc to PrPSc, ultimately causing neuronal cell dysfunction followed by cell death. The prion's form of “self‐replication” without utilizing genetic material is particularly fascinating, as it appears to defy the central dogma of biology and differs from all other pathogenic particle infection mechanisms. It is notable that some prions have evolved to act as transmissible infectious agents, similar to viruses, despite the absence of genetic material. This distinguishes some prion diseases, such as chronic wasting disease (CWD) and scrapie, from their inherited disease counterparts. The biosynthesis pathway of PrPc is similar to other cellular proteins, with replication occurring on the ribosomes on the endoplasmic reticulum. However, whereas malfunctioning proteins are normally tagged by ubiquitin and then degraded in a proteasome, the ubiquitin‐proteasome complex is inhibited during prion formation, allowing PrPSc to form and survive degradation, leading to its buildup in cytosol and subsequent neurotoxicity. Human and animal prion diseases have been classified into three broad categories‐‐genetic, sporadic, and acquired‐‐based on properties of the PrP proteins and prion infection morphology. Physical symptoms of prion infection include deviant behavior, dementia, ataxia, insomnia, paraplegia, and paresthesias. Commonly discussed prion‐induced diseases (differentiated via host type and slight conformational discrepancies between misfolded proteins) include scrapie, kuru, CJD, bovine spongiform encephalopathy (mad cow disease), and CWD. Here, we present current research on prions, with a focus on CWD, which was first found in Colorado and affects deer and other cervids.

The Olathe North High School Team 1 MSOE Center for BioMolecular Modeling SMART Team used 3‐D modeling and printing technology to examine the structure‐function relationships and histology of PrPc, or the normal cellular prion.

Support or Funding Information

This project is a joint venture between the Milwaukee School of Engineering and the Medical Professions Academy.

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 the Furin‐Cleavable Linker and KDEL Sequence in Cytotoxicity of Recombinant ImmunoDNase
Authors: Baker Jillian
Published: June 2021

The lack of specificity of standard chemotherapies to identify and kill cancer cells has challenged researchers to engineer targeted therapies to replace conventional treatments. One strategy that is currently being optimized to treat cancer is the use of recombinant immunotoxins (RITs). RITs are chimeric proteins that are engineered to contain an antibody conjugated to a toxin. The antibody allows for specific targeting of cancer cells while the toxin domain is internalized by the targeted cell and subsequently induces cell death. These therapies suffer from several problems, including non‐specific toxicities, low anti‐tumor activity, and immunogenicity. A bacterial moiety introduced into a patient with an intact immune system can elicit an immune response that consequently eliminates the possibility of retreatment. Deimmunized, humanized, and fully human immunoconjugates have been engineered, but often lack efficacy. We are using specific sequences known to assist cellular intoxication by Pseudomonas exotoxin A (PE) to enhance the activity of human‐derived immunotoxins. We are developing a recombinant immunotoxin based on human DNase I, using a furin‐cleavable linker and KDEL ER retention sequence adapted from the PE intoxication strategy. We expect this construct to have low immunogenicity, high specificity for dividing cells, and enhanced toxicity relative to the unmodified DNase I construct.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression profile of adiponectin and adiponectin receptors in high fat diet feeding chicken
Authors: Lin YuanYu , Ding ShihTorng
Published: June 2021

Adiponectin is a circulatory cytokine secret from adipose tissue and exerts critical metabolic function in mammals. Avian also expressed adiponectin and adiponectin receptors in peripheral tissues, whereas the expression profiles are very different from mammals and the role of its unique profile is still unknown. The current study explore the role of adiponectin and adiponectin receptors in chicken by high‐fat diet feeding. Twenty Taiwan L2 chickens were challenge with 10% high‐fat diet for 6 weeks. The results are showed that body weight, plasma triglyceride, plasma cholesterol and DPP4 were increased by high‐fat diet treatment. Interestingly, we first demonstrated that chicken macroadiponectin and DsbA‐L (adiponectin multimerization enzyme) were elevated by high‐fat diet feeding. Moreover, the mRNA expression of adiponectin and adiponectin receptors were increased in fat tissues by high‐fat diet treatment. For metabolic‐related genes analysis, FAS, ATGL, LPL and PPAR γ were increased in high‐fat diet group. These results were suggested that high‐fat diet treatment may alter the expression profiles of adiponectin and metabolic genes in chicken fat tissue. In sum, we explore the effect of high‐fat diet in expression profile of adiponectin and its receptors in chicken and provide the new insights for how adipokines modulate physiological function in avian species.

Support or Funding Information

The authors would like to express our gratitude to the laboratory members for their help and input during this study.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Perilipin 5 Protein‐Protein Interactions
Authors: Hughes Erin K. , Tansey John T.
Published: June 2021

Type II diabetes mellitus and non‐alcoholic fatty liver disease are two examples of metabolic diseases that are reaching epidemic proportions in the US. Key to understanding these diseases and their progression is an understanding of neutral lipid metabolism. Perilipins are a class of five conserved proteins that are key regulators of lipid metabolism and are potentially involved in lipid trafficking within cells. The most recently discovered member of the family is perilipin 5, which is expressed most strongly in tissues that are highly oxidative such as heart, oxidative muscle, and fasting liver. Perilipin 5 is predominantly located on the surface of lipid storage droplets, but has also been found in the cytoplasm, nucleus, and the endoplasmic reticulum and mitochondria. We hypothesized that perilipin 5 moves between these cellular pools via interactions with other proteins. Furthermore, we hypothesized that interactions with Rabs were necessary for this trafficking to occur. CHO cells expressing perilipin 5 were assayed using western blotting and were found to express Rab 18. Immunoprecipitations are being used to assay for interactions between perilipin 5, Rab 18, and other proteins. Collectively these data will provide a more detailed picture of perilipin 5 in the cell.

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 novel assay to measure scrambling of natural phospholipids in reconstituted proteoliposomes
Authors: Wang Lei , Pandey Kalpana , Ploier Birgit , Menon Anant K. , Bütikofer Peter
Published: June 2021

Scramblases transport phospholipids between the leaflets of a membrane bilayer in an ATP‐independent and bidirectional way to facilitate membrane growth and maintain lipid homeostasis. Members of two protein families, Class A G protein‐coupled receptors and the TMEM16 family of ion channels, have been identified to act as scramblases. However, the mechanisms by which they translocate phospholipids are still poorly understood. Synthetic glycerophospholipid analogs carrying fluorescent nitrobenzoxadiazole (NBD) groups, such as NBD‐phosphatidylcholine (NBD‐PC), have proven useful to identify and characterize phospholipid scramblases in membrane preparations and reconstituted proteoliposomes.

We now have established a novel biochemical assay to measure the activity of scramblase proteins using radiolabeled natural phosphatidylinositol ([3H]PI) as probe. As sources of scramblase activity we used crude extracts from yeast containing ER membrane proteins, and also purified bovine opsin. We reconstituted symmetric large unilamellar liposomes containing trace amounts of [3H]PI. Addition of PI‐specific phospholipase C (PI‐PLC) resulted in hydrolysis of 46–52% of [3H]PI, i.e. the portion present in the outer leaflet of the bilayer. The time to reach maximal [3H]PI hydrolysis depended on the amount of PI‐PLC used. In contrast, PI‐PLC treatment of [3H]PI‐containing liposomes reconstituted with yeast extract containing scramblase activity or purified opsin resulted in protein‐dependent hydrolysis of 60–80% of [3H]PI, indicating that the [3H]PI located in the inner leaflet of the proteoliposomes became accessible to cleavage by PI‐PLC during the course of the experiment.

To allow direct comparison of opsin's ability to scramble [3H]PI and NBD‐PC, we then prepared opsin proteoliposomes containing both [3H]PI and NBD‐PC and measured scrambling using PI‐PLC and dithionite, respectively, as probes. The results showed that opsin readily scrambled both NBD‐PC and [3H]PI, albeit with different characteristics. The results of these experiments, as well as experiments that directly compare opsin‐mediated scrambling of NBD‐PI and [3H]PI will be presented.

apoprotein

Support or Funding Information

This research is supported by the Velux Stiftung

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Diltiazem Improves Twitch Tension in Dysferlin‐Null BLAJ Mice but Does Not Reduce Contraction‐Induced Muscle Damage In Vivo
Authors: Roche Joseph A. , Begam Morium , Collier Alyssa F.
Published: June 2021

Background

The L‐type Ca2+ channel blocker diltiazem (DTZ) protects dysferlin‐null A/J mice from muscle damage induced by large‐strain eccentric contractions (20 repetitions) (1). One of the disadvantages of studying the A/J mouse strain is that, it does not have a true control mouse strain. Additionally, A/J mice carry several non‐dysferlin mutations that might have an effect on muscle function (2). BLAJ mice, which carry the same dysferlin mutation as A/J mice, albeit in the C57BL/6J background, show lesser muscle damage than A/J mice after large‐strain eccentric contractions (3). We therefore developed a new model of eccentric exercise (40 repetitions of medium‐strain eccentric contractions) that reliably induces sufficient damage in BLAJ mouse muscle, in order to test if DTZ blocks contraction‐induced muscle damage in BLAJ mice.

Methods

We studied the tibialis anterior (TA) muscle in 3–4 month old, male, dysferlin‐null BLAJ mice. We measured contractile torque and exposed the TA muscle to eccentric contractions (one bout; 40 repetitions; 90–160 □ plantarflexion superimposed on maximal tetany of ankle dorsiflexors) with a custom‐built dynamometer. For one group of mice (Vehichle, VEH, N = 6 mice), we gave intraperitoneal injections of distilled water, once daily, for one week prior to eccentric contractions and three days thereafter (10 ul per gram body weight). For another group of mice (DTZ, N = 6 mice), we gave intraperitoneal injections of DTZ (72 mg/kg/day), once daily, following a similar injection schedule as the VEH group (we dissolved 72 mg DTZ in 10 ml distilled water and injected 10 ul per gram body weight). We assessed baseline contractile torque, torque changes after eccentric contractions, and histology of unexercised and exercised TA muscles.

Results

Data are summarized in Table 1. DTZ improved Peak Twitch Torque, but did not provide protection against muscle damage from eccentric contractions.

Conclusion

Diltiazem might not offer protection against all types of contraction‐induced damage to dysferlin‐null muscle.

Translational Relevance

Diltiazem might not be able to protect muscles in patients with dysferlin deficiency if muscle damage exceeds a certain threshold level.

Support or Funding Information

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 Tumor Necrosis Factor‐Alpha in Rheumatoid Arthritis
Authors: Link Tina , Kepner Alisa , Coruso Olivia , Dilip Megha , Jacobson Reagan , Stanovski Lisa
Published: June 2021

Tumor necrosis factor‐alpha (TNF‐α) is a 233 amino acid, 17 kilodalton, homotrimer, bell‐shaped proinflammatory cytokine. TNF‐α is usually secreted from monocytes and macrophages and is also an inducer of many proinflammatory cytokines like IL‐1, IL‐6 and others to help regulate immune cell function. . TNF‐α has two transmembrane cell receptors, TNF‐R1 and TNF‐R2, which have functional binding domains located on the outside of the cell and catalytic signaling domains located inside the cell. TNF‐R1 mediates the activation of a number of transcription factors resulting in an increased inflammatory response, while TNF‐R2 increases the number of T‐cells through a proliferation/survival pathway. A high amount of TNF‐α compared to a low number of its receptors leads to joint inflammation and eventual shock, while a low amount of TNF‐α leads to cachexia. Rheumatoid Arthritis (RA) has many causes including environmental factors, proclivity of smoking, or a genetic history, but in all cases, TNF‐α seems to play a role in disease progression due to its high proteins concentration in advanced disease stages. The source of TNF‐α in joints could be due to the production of the protein by CD4+ T cells in the synovial fluid which has been associated with synoviocyte proliferation, osteoclast activation, and cartilage degradation. Attempts to inhibit TNF‐α are currently centered around general suppression of the protein through TNF‐α inhibitors, but due to the devastating effects these treatments have on the immune system, further studies are considering creating higher numbers of anti‐inflammatory cytokines to create a balance between inflammatory and anti‐inflammatory cytokines, while other studies are attempting to target downstream protein targets of TNF‐α in an attempt to selectively suppress disease symptoms. The Walton High School SMART Team has designed a 3D model of TNF‐α using JMOL to investigate the relationship between structure and function.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Curcumin Suppresses Pro‐Inflammatory Response through Interference of NF‐κB Activation in Human Umbilical Vein Endothelial Cells
Authors: Cheng An‐Chin , Yu Ya‐Mei , Chang Weng‐Cheng
Published: June 2021

Oxidative stress and redox‐sensitive transcription factors are involved in the process of inflammation which plays a central role in the pathophysiology of atherosclerosis. NF‐κB is a redox‐sensitive transcription factor which was activated secondary to excessive mitochondrial ROS production in the endothelium, participating in a various of proinflammatory and prothrombotic reactions in the endothelial cells. In the present study, we found that the production of ROS was induced by IL‐1 β and pretreatment with curcumin could decrease the induction of ROS by IL‐1 β in human umbilical vein endothelial cells (HUVECs). Since atherosclerosis is a chronic inflammatory disease associated with increased oxidative stress in the vascular endothelium, we proposed that the inhibitory effect of curcumin on NF‐κB activation may be due to its antioxidant and anti‐inflammatory properties. We also measured the nuclear translocation of p65 and p50 protein of the NF‐κB family to examined whether the inhibitory effect of curcumin on the cytokine‐induced expression of pro‐inflammatory response is medicated via NF‐κB. Our results indicated that the nuclear translocation of p65 and p50 were induced by IL‐1 β, whereas the pretreatment with curcumin could inhibit the induction of translocation by IL‐1 β in HUVECs. Therefore, we confirmed that curcumin has an anti‐inflammatory effect through the partial interference of NF‐κB activation.

Support or Funding Information

This study was supported by NSC 100‐2313‐B‐309‐003 to A‐C Cheng and NSC 95‐2815‐C‐039‐019‐B to Y‐M Yu.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluating Approaches to Analyze Tissue Score Data
Authors: Meyerholz David , Beck Amanda
Published: June 2021

Histopathology remains an important source of data in the clinical and research settings. However, qualitative evaluation of tissues alone has limitations for translational studies. Recent petitions for enhanced reproducibility in scientific studies have elevated the roles for scoring (semiquantitative and quantitative) of tissues. Effective tissue scoring requires appropriate statistical analysis to validate the group comparisons. Choice of appropriate statistical test is greatly influenced by experimental design (e.g. paired, unpaired, parametric, nonparametric, longitudinal, repeated measures, and multi‐factorial to name a few). We evaluated how the type of statistical analysis could impart highly skewed interpretations of the data. We evaluated mock tissue scores for inflammation representing two groups of tissues. For these ordinal scores, we analyzed the data (n=5/gp) using 4 statistical tests that have been used in the literature for comparing two groups of tissue date: paired T‐test, unpaired T‐test, Mann‐Whitney test and Kolmogorov‐Smirnov test. From these tests, the data produce a wide range of P values of 0.0161, 0.1823, 0.3175, and 0.5238, respectively. The data was further analyzed as to what type of test should have been run to be valid. The data (generated by rolling of dice) failed a normality test (P<0.05, Shapiro‐Wilk test) and given ordinal scoring approach – data analysis should have focused on non‐parametric approaches that showed no significance. Statistical tests that give the best “confidence” in the data interpretation should be preferentially applied to achieve peak validity and reproducibility.

Support or Funding Information

NA

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Predicting Mouse Oocyte Methylome from Polar Body by Single‐cell Whole Genome Bisulfite Sequencing
Authors: Lee Tin‐Lap , Chan Wai‐Yee , Qian Yan , Qin Cao , Yip Kevin Yuk‐Lap , Chow King Lau , Tang Nelson LS. , Liao Jinyue , Luk Alfred Chun Shui , Suen Hoi Ching , Lee Wing Tung , Zhao Ming Peng , Kong Grace Wing Shan , Chung Hoi Sze , Leung David Chan Yiu , Leung Tak Yeung , Li Tin Chiu
Published: June 2021

Evaluating oocyte quality in assisted reproductive technology (ART) is essential to ensure a healthy pregnancy. Currently, morphological assessment on oocyte is applied to predict oocyte and embryo development potential. The method is limited and may not provide an accurate assessment of oocyte quality. Human polar body (PB) is the byproduct of an oocyte maturation. The advent of PB karyotyping has served as an indirect genetic analysis of oocytes and provided metrics of embryonic potential. This prompts the idea of oocyte genome interrogation by studying PB genome, and a recent study has shown transcriptome of PB could accurately reflect the transcriptome landscapes of the sibling oocyte. Yet the epigenetic dynamics of PB and oocyte remains elusive. Here we hypothesized that the methylome profile of PB could be representative of oocyte methylome.

To reveal the epigenome information on PB and oocyte, we performed single‐cell whole genome bisulfite sequencing (scWBS) on mouse metaphase II (MII) oocytes and the associated PBs. Overall, we detected 1,653,985 CpG sites in the oocyte and 55,941 CpG sites in the PB respectively. The genome‐wide methylation level of CpG sites in PB was 73.56%, compared to 54.85% in oocyte. To examine if PB methylome could predict oocyte methylome landscape, all CpG sites covered with more than 3 reads and a window size of 2,000 bp were used to calculate the correlation of methylation level between PB and its sibling oocyte. They showed a moderate Pearson's correlation coefficient (r=0.5295), which was consistent with published data. We also calculated the correlation of methylation level in promoter region, but to our surprise, the correlation was weak (r=0.209). These results suggested that global methylation level in the mouse PB could not be applied to predict it sibling oocyte methylome. In summary, our preliminary data suggests that the correlation of global methylation level between mouse oocyte and its sibling oocyte is not as high as expected, but this conclusion need to be further confirmed by analyzing more samples.

Support or Funding Information

Collaborative Research Fund, Hong Kong Research Grant 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.
Activated mesothelial cells in pleural fluid after lung resection and transplantation
Authors: Kienzle Arne , Servais Andrew B , Alexandra Ysasi B , Gibney Barry C , Cristian Valenzuela D , Wagner Willi L , Ackermann Maximilian , Mentzer Steven J
Published: June 2021

Objectives

The mesothelium, the surface layer of the heart, lung, bowel, liver and tunica vaginalis, is a complex tissue implicated in organ‐specific diseases and regenerative biology. An open question is the mechanism of mesothelial repair after injury. Previous observations indicated that the seeding of denuded mesothelium by free floating mesothelial cells may contribute to mesothelial healing.

Material and Methods

In this study, the pleural fluid of 54 patients after lung resection or transplantation was analysed. Flow cytometry was employed to study the cellular composition of post‐surgical pleural fluid. We used histologically validated mesothelial markers (CD71 and WT1) to estimate the prevalence of mesothelial cells.

Results

The viability of pleural fluid cells approached 100% indicating that the cells were not just dying exfoliated cells. Leukocytes and mesothelial cells were identified in the pleural fluid within the first week after surgery. The leukocyte concentration rapidly declined after postoperative day 1. In contrast, mesothelial cells, identified by CD71 and WT1, peaked on postoperative day 3. Based on optimal sampling and average cell size, we estimate that the post‐operative day 3 pleural fluid contains sufficient numbers of activated mesothelial cells to cover several cm2 of denuded mesothelium. The broad expression of CD71 molecule in postoperative pleural fluid suggests that many of the free‐floating non‐leukocyte cells were activated or proliferative mesothelial cells.

Conclusion

We demonstrated that pleural fluid post lung surgery is a source of mesothelial cells; most of these cells appear to be activated or proliferative mesothelial cells. The observed peak of mesothelial cells on postoperative day 3 is consistent with a potential reparative role of free‐floating mesothelial cells after pulmonary surgery. The high viability of these cells and the convenient drainage chambers used after lung surgery suggests an opportunity for further in vitro studies of these free‐floating mesothelial cells.

Support or Funding Information

Supported in part by NIH Grant HL94567, HL134229, HL007734, CA009535, ES000002, the German Research Foundation (SFB1066), and a Boehringer‐Ingelheim Fonds Travel 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.
Dengue Fever: the Next Global Killer
Authors: Raynes Edilberto A. , Glymph Kelly , Coker Karen , Agazuma Keshia , Michaels Natalie , Menchavez Melinda Roca , Raynes Janika Ziarah Rellama
Published: June 2021

The purpose of the study is to review the epidemiological aspects of the dengue fever infection. The infection is caused by dengue viruses (DENV), which have four different serotypes and antigenically distinct. The DENV is transmitted by a vectorborne day biting mosquito, Aedes agypti. The clinical spectrum ranges from dengue fever to dengue hemorrhagic fever. Based on our review from the World Health Organization, Centers for Diseases Control and Prevention, as well as from other studies, the disease is still more endemic in the Southeast Asia and Western Pacific regions with an estimate of 300 million infections annually, mostly affecting children. Factors that contribute to the emergence of DENV infection worldwide include substandard living conditions, virus evolution, lack of vector control programs, and international travelers. The best measure of DENV control is prevention by increasing awareness of the public through coordinated outreach programs to the community coordinated by various stakeholders. There are still no available vaccines and specific medications to cure. A multidisciplinary collaboration needs to be mobilized to combat the vector.

Support or Funding Information

None

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modulation of striatal dopamine release by nicotinic receptors in adolescent and adult rats
Authors: Pitts Elizabeth , Stowe Taylor , Fennell Ashley , Sexton Lacey , Ferris Mark
Published: June 2021

Adolescence is a developmental period characterized by increased vulnerability to substance use and dependence. However, mechanisms mediating this vulnerability are not well understood. Previous work has shown that locomotor responses in a novel environment predict acquisition of nicotine and cocaine self‐administration in adult rats, with high‐responders (HR) acquiring faster and at lower doses than low‐responders (LR). Additionally, adult rats with higher vulnerability have more sensitive nicotine acetylcholine receptor (nAChR) modulation of striatal dopamine release to phasic firing‐like stimulation, which is known to be important in acquisition of self‐administration. Here we examined how antagonism of nAChRs altered dopamine release in the nucleus accumbens core in adolescent and adult rats using fast scan cyclic voltammetry. We found that non‐selective antagonism of nAChRs, by mecamylamine (2 uM) or a desensitizing dose of nicotine (500 nM), increased dopamine release in HRs to stimulation that models phasic dopamine release, but suppressed dopamine release in LR rats. Further, we report a‐conotoxin PIA (100 nM), an a6‐selective antagonist, produced similar effects as the non‐selective antagonists, indicating that facilitation of dopamine in the HR rats is mediated by a6‐containing nAChR. When dopamine release in adolescent rats was tested under the same parameters, nAChR modulation of dopamine release was similar to HR adult rats, with non‐selective and a6‐selective antagonists facilitating dopamine release following phasic firing‐like stimulation. This suggests a role for a6‐containing nAChR modulation of striatal dopamine release in mediating vulnerability to drug abuse.

Support or Funding Information

This work is supported by NIDA grant R00 DA031791 awarded to MJF

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Synthesis and Evaluation of Symmetrical and Unsymmetrical Diacyl Ureas, Acyl Urea/Carbamate/Thiocarbamate Derivatives and their Evaluation for FAAH inhibition
Authors: El‐Alfy Abir , Johnson Jacklyn , Hernandez Anolan Garcia , Grooms Gregory , Stec Jozef
Published: June 2021

Fatty acid amide hydrolase (FAAH) is a serine hydrolase enzyme that metabolizes various lipophilic transmitters, including oleamide, anandamide, and 2‐arachidonyl glycerol. These neurotransmitters are part of the endocannabinoid system (ECS) that comprises a neuromodulatory network involved in regulation of various physiological functions, including appetite, pain, reward, motor control, memory, and mood. Previous studies have shown that enhancing the ECS offers a potential therapeutic target for various psychiatric disorders. An FAAH inhibitor provides indirect modulation of the system, with the added advantage of avoiding undesirable adverse effects associated with the direct receptor agonists. The objective of this study was to synthesize various oleamide analogs and evaluate them for FAAH inhibition. A total of fifty one compounds (1–51) were synthesized using a one‐pot two‐step synthetic scheme. All compounds were evaluated in the in vitro FAAH inhibition assay using human recombinant FAAH enzyme, at 2–100 uM concentrations. Of these compounds, ten analogs showed >50% FAAH inhibition activity at the 50 uM concentration. These compounds serve as leads for structure activity relationship studies to optimize the inhibitory activity and to help design potent and effective FAAH inhibitors to be evaluated in vivo in various animal models of neuropsychiatric disorders.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Targeted downregulation of MYC through G‐quadruplex stabilization: a novel therapeutic approach for breast cancer and lymphoma
Authors: Chang Katarina T. , Hao Taisen , DeSouza Harriet EJ. , Brooks Tracy Ann
Published: June 2021

MYC, a basic helix‐loop‐helix/leucine zipper transcription factor, is an enigmatic protein with over 30,000 potential binding sites in the human genome, 10–15% of which are generally bound at any one time. It was one of the first proto‐oncogenes to be described and is ultimately disregulated in most tumor types and stages, including estrogen receptor (ER)‐positive breast cancer and non‐Hodgkin's lymphoma. Decreased MYC expression has been shown to significantly impact tumor viability while offering a notable therapeutic window and overall safe profile. As we and other groups have shown, stabilization of the higher order genomic structure – the G‐quadruplex (G4) – in the MYC promoter is an established approach to decreasing transcription and facilitating tumor lysis. We have pioneered a DNA interference (DNAi) approach to take advantage of the “druggability” of the DNA structure, and have previously demonstrated the specificity and efficacy of oligonucleotide (ODN)‐mediated stabilization of the predominant G4 structure within the MYC promoter. In the current report, we further optimize the DNAi, including the evaluation of different ODN lengths, for specifically stabilizing the MYC promoter G4 (as demonstrated by EMSA and chemical footprinting) and modulating promoter activity. The lead DNAi (A‐18) demonstrates anti‐cancer activity in RAJI and CA46 Burkitt's lymphoma cells, and in ER+ MCF‐7 breast cancer cells. Moreover, A‐18 enters the nuclei of cancer cells, where it binds the G4‐forming region of the MYC promoter, in an inducible manner. This clamp has potential as a novel DNAi therapeutic. It is being further explored for its ability to sensitize cancer cells to chemotherapeutic agents, and its formulation for delivery in nanotherapeutic vehicles.

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 the novel anti‐cancer therapeutic ONC201 and related analogs as non‐competitive antagonists of the D2 dopamine receptor
Authors: Cuoco Caroline A. , Free R. Benjamin , Willette Blair K. , Day Marilyn M. , Lane J. Robert , Allen Joshua E. , Sibley David R.
Published: June 2021

ONC201 is the first member of a new class of anti‐cancer small molecules called imipridones to be investigated in clinical trials for select advanced cancers. The anti‐cancer properties of the parent compound were first discovered in a phenotypic screen as a p53‐independent inducer of the immune cytokine TRAIL and tumor cell death. Tumor cell signaling studies revealed that ONC201 causes a dual inactivation of Akt/ERK that drives Foxo3a‐mediated TRAIL gene induction. Recent studies identified that ONC201 also selectively antagonizes D2‐like dopamine receptors (D2R, D3R, D4R) at therapeutically relevant concentrations. Notably, malignant overexpression of the D2R occurs in a variety of cancers and its antagonism imparts pro‐apoptotic and anti‐proliferative effects in tumor cells. In the current study, we sought to characterize the pharmacological properties of ONC201 and several novel analogs (ONC206 and ONC213) on D2‐like receptors. Both functional signaling (β‐arrestin recruitment and cAMP accumulation) and radioligand competition binding assays were employed. Radioligand binding assays with [3H]‐methylspiperone determined that these compounds bind to D2‐like receptors with low μM affinity, consistent with their potencies for anticancer activities. When the compounds were tested in β‐arrestin recruitment assays (D2R, D3R, and D4R) and cAMP accumulation assays (D2R and D4R), all analogs were determined to be antagonists with no measurable agonist efficacy. Importantly, a resolved enantiomer of ONC201 that showed no anti‐cancer activity was found to be inactive in blocking the D2R. To gain further insight into the mode of action of ONC201 and analogs at the D2R, Schild‐like curve shift assays were performed in using the functional assays, where increasing concentrations of compound are used to shift the dopamine concentration‐response curve. Increasing concentrations of all compounds caused both a rightward shift in the dopamine concentration‐response curve and a depression of the maximum response. These data are consistent with a non‐competitive mode of action and could be fitted with an operational model of allostery. These analyses revealed a predominant modulatory effect upon dopamine efficacy and a smaller effect on dopamine potency. Further studies will aim to elucidate the nature in which these analogs interact with D2R binding pockets through site‐directed mutagenesis. Understanding the pharmacological profile of ONC201, including different signaling paradigms, may lead to a better understanding of its mechanism for anticancer targeting.

Support or Funding Information

NINDS IRP

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