bluuebunny/crossref_metadata_2025 huggingface dataset:
Presence of UCP2 and Amylin in human Alzheimer's disease brains
Authors: Saunders Dakarai , Clemons Tameka
Published: June 2021

Although the role of amyloid beta in Alzheimer's disease has been studied for decades, the link between amyloid beta and amylin is fairly new. Amylin is a protein that is co‐secreted from pancreatic beta cells alongside insulin and has been shown to cross the blood‐brain barrier and impact the formation of amyloid plaques. The normal function of amylin in the brain is directly correlated to metabolism because amylin is considered a satiety protein. The output involved in the metabolic process involves oxidative phosphorylation and includes a set of proteins known as uncoupling proteins (UCPs), which are proteins that directly impact oxidative phosphorylation. Traditionally, UCPs are known to impact the ATP levels that occur in oxidative phosphorylation. However, recently it has been postulated that UCPs may have an additional role that involves cell signaling. In fact, UCP2 has been shown to be present in pancreatic beta cells and to have diminished protein levels in Alzheimer's disease brains. The experiments conducted in this study analyzed the relationship between UCP2 and amylin. Specifically, immunohistochemistry experiments that examine both the presence of amylin and UCP2 in Alzheimer's disease brains were conducted. Western Blot analysis experiments were also conducted to compare the findings from the immunohistochemistry experiments. The initial studies suggest that the role of UCP2 may influence the role of amylin or the role of amylin may influence the role of UCP2.

Support or Funding Information

National Science Foundation Research Initiation Award #24430

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Predominant Beta Subunit Isoform of the Na+, K+‐ATPase in Photoreceptor Neurons Undergoes S‐Palmitoylation
Authors: Sechrest Emily , Sokolov David , Murphy Joseph , Kolandaivelu Saravanan
Published: June 2021

Many proteins undergo post‐translational lipid modifications, which are often essential to aid in subcellular trafficking, protein‐protein interactions, and membrane association. S‐palmitoylation is a unique post‐translational lipid modification which involves reversible attachment of the 16‐ carbon fatty acid palmitate to cysteine residues on proteins. In the retina, photoreceptor neurons are responsible for capturing photons of light and converting them into electrical signals, which are subsequently sent to the brain for visual processing. Many crucial phototransduction proteins in photoreceptors, including rhodopsin, phosphodiesterase‐6, and the transducin complex, have been shown to require post‐translational lipidation for proper function. To better understand the importance of “palmitoyl” lipid modifications in retinal photoreceptor cells, our lab employed acyl‐resin assisted capture (acyl‐RAC) followed by mass‐spectrometry analysis to isolate and identify proteins in retinal tissue which undergo S‐palmitoylation. From the mass‐spectrometry data pool, our lab chose several candidate proteins to study in more detail, including the β2‐ subunit of the Na+, K+‐ATPase (ATP1B2). ATP1B2 has been shown to be the predominant β‐subunit isoform of the Na+, K+‐ATPase in photoreceptor cells and is thought to be involved in maturation and trafficking of its catalytic α‐subunit counterpart. Additionally, ATP1B2 has been shown to be required by the retinal‐specific protein retinoschisin‐1 (RS1) for its localization and association with photoreceptor and bipolar cell membranes. Although it is known that the Na+, K+‐ATPase regulates the photocurrent in photoreceptors needed to send visual information to downstream neurons, the distinct role of ATP1B2 remains unknown. Acyl‐RAC followed by immunoblot served as a complementary approach to confirm that ATP1B2 undergoes palmitoylation, whereas its homolog, the β1 isoform, does not. After palmitoylation prediction software analysis, we utilized mammalian cell culture and in vivo subretinal injection/electroporation to demonstrate that ATP1B2 is palmitoylated on the 10th amino acid. Additionally, click chemistry with the palmitate analog 17‐Octadecynoic acid, was used as an independent approach to validate ATP1B2 palmitoylation. Altogether, these data confirm that ATP1B2 is post‐translationally lipid modified by palmitoylation on the 10th amino acid. However, the functional role of palmitoylation of ATP1B2 remains unknown and warrants further investigation.

Support or Funding Information

NIH 1RO1EY028959‐01, WVU bridge funding, and intramural RFDG funding award (SK)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Optimization of Cell Free Protein Synthesis: Development of a Fusion Protein Fitness Strategy
Authors: Travieso Tatianna , Willis Caroline , Rojas Maria , Eckdahl Anthony , Roland Kyla , Edman Elise , Tovar Sofia Aguilera , Hanlan Nicklaus , Ong Shi Qing , Wagner Jacob , Poet Jeffrey L. , Eckdahl Todd T. , Heyer Laurie J. , Campbell A. Malcolm
Published: June 2021

Our undergraduate researchers work in the field of metabolic engineering to harness metabolic pathways for the production of useful metabolites with applications in the biofuel, pharmaceutical, or industrial chemical industries. We published a metabolic engineering approach called programmed evolution that introduces variation in regulatory elements for the expression of enzymes that control orthogonal metabolic pathways. Programmed evolution employs a riboswitch‐based fitness module to transduce the production of a desired metabolite into antibiotic resistance. We validated programmed evolution by optimizing the conversion of caffeine to theophylline by caffeine demethylase, but we recognized several limitations to the widespread use of our approach, including crosstalk between orthogonal and native bacterial metabolism, potential toxicity of metabolites, and poor representation of large variation spaces. To avoid these limitations, we are developing an in vitro approach to programmed evolution. For cell‐free protein synthesis (CFPS), we found supercoiled plasmid was a much better template than linear PCR amplicon. We designed, constructed, and have begun testing a novel fitness module that responds to the production of theophylline produced by the action of caffeine demethylase produced by CFPS. The fitness module encodes a fusion protein composed of three functional units. The amino portion contains a Gal4 DNA binding domain that will target binding sites on a library of DNA regulatory elements for the expression of caffeine demethylase. The carboxyl terminus includes streptavidin which will bind to avidin beads for physical separation of selected DNA regulatory elements. Sandwiched in between these two domains is GFP, which serves as a biosensor. Using this in vitro selection strategy, we plan to pursue the optimization of CFPS for the discovery of efficient metabolic pathways of interest.

Support or Funding Information

NSF RUI grant MCB‐1613203 to Davidson College and MCB‐1613281 to Missouri Western State University, Davidson College Martin Genomics Program

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Antimicrobial Activity of 3, 4‐Annelated Coumarin‐Benzodiazepine‐Systems
Authors: Govori Sevdije , Ibrahimi Hamide , Daci Majlinda , Haziri Arben
Published: June 2021

This study presents the synthesis of 7‐iminobenzo[b]chromeno[4,3‐e][1,4]diazepin‐6(7H)‐ones (3–4) by simple reaction of 4‐chloro‐2‐oxo‐2H‐chromene‐3‐carbonitrile with o‐phenylidenediamine. The compounds (3–4) were characterized by elemental analysis data, IR, 1H NMR and mass spectral data. The novel heterocyclic compounds were evaluated for antimicrobial activity against Escherichia coli and Staphylococus aureus using the Kirby‐Bayer method. The novel compounds demonstrated moderate antimicrobial activity.

Support or Funding Information

Funding: Support for this study was provided by the Ministry of Education, Science and Technology of the Republic of Kosovo. Grant number:2‐5521‐2

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Alcohol + PLD = Phosphatidylethanol, a Long‐Term Alcohol Biomarker
Authors: Neilsen Paul O. , Johnson L. Jeff , Rzepecki Piotr , Davies Sean
Published: June 2021

Heavy alcohol consumption—both chronic and binge, increases the risk of digestive tract tumors, coronary artery disease, and other health problems. Better biomarkers for consumption and ethanol‐induced organ damage are needed. Direct biomarkers including phosphatidylethanol (PEth), ethyl glucuronide (EtG) and ethylsulfate (EtS) are gaining increased use and acceptance. We first prepared bioanalytical tools and assays then recruited 9 alcohol‐consuming volunteers (2 Heavy, 4 Moderate, 3 Light) in a pilot study. We measured four abundant PEth species by mass spectrometry and anti‐PEth IgG antibodies by ELISA. We found a high correlation between total PEth measured and expected PEth levels depending on amount and timing of ethanol consumption. Interestingly, we observed differences in PEth molecular species based on the amount of alcohol consumed, and even differences between the two heaviest drinkers—one of which drank in a more regular pattern while the other had a number of binge episodes. These observations led to a clinical study where alcohol was administered in both binge and consistent patterns with abstinence periods monitored by continuous alcohol monitoring (CAM) ankle bracelets. We sampled urine for EtG/EtS; serum/plasma for both carbohydrate deficient transferrin (%CDT) and anti‐PEth IgG; and whole blood for PEth. We found the direct biomarkers (PEth, EtG/EtS) were superior to indirect biomarkers (%CDT and anti‐PEth IgG) for detecting alcohol consumption for both drinking patterns at 1, 7 and 21 days post‐consumption. Interestingly, we found that plasma anti‐PEth IgG levels followed blood alcohol concentration in binge‐drinking volunteers indicating preformed, bound IgG is displaced by high blood alcohol levels. Our results indicate that PEth is a valuable biomarker for detecting both acute and consistent alcohol consumption; and that this unique lipid merits further study to better understand its formation, elimination, and impact on the human immune system and alcohol‐related disorders.

Support or Funding Information

Supported by National Institutes of Health, NIAAA, Contract, HHSN267200800020C to PON.

The content of this communication is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute on Alcohol Abuse and Alcoholism nor the National Institutes of Health.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Fluorogenic Atom Transfer Radical Polymerization as a Strategy for Biomolecular Detection
Authors: Tahseen Danyal , Cooley Christina B
Published: June 2021

The sensitive detection of analytes is valuable in various chemical and biomedical contexts. This sensitivity can be conferred by signal amplification, where the readout magnifies the signal of an otherwise low level of analyte. The Cooley Lab's approach to signal amplification involves using fluorogenic radical polymerizations, where the polymers' fluorescence is a quantifiable proxy for presence of an analyte. This approach also enables the qualitative appreciation of analyte presence since the fluorescence is visible to the naked eye.

In order to use this technology to detect water‐soluble analytes such as biomolecules, we are conjugating the polymerization initiator to a model protein. This involves developing the fluorogenic polymerization in biologically relevant conditions such as an aqueous medium, so that polymers can be grafted directly from the biomolecule. The choice of an atom transfer radical polymerization (ATRP) facilitates the controllable formation of fluorogenic polymers in aqueous media as a function of both analyte concentration and reaction time. The development of the polymerization towards a biomolecular detection assay will be discussed, with emphasis on optimization of the reaction conditions, synthesis and characterization of biomolecule‐initiator conjugates, and fluorescence detection.

Support or Funding Information

San Antonio Area Foundation, American Chemical Society Petroleum Research Fund, Trinity University

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A New Synthetic Route to Cycloheptatrienylidene Fluorophores
Authors: David Navindra , Hyatt Ivan Fabe Dempsey
Published: June 2021

Luminescent molecules can lose their fluorescent properties through aggregation caused quenching (ACQ). ACQ is caused when these molecules, in high concentration, undergo π‐π stacking. Previous methods have been developed that utilize Aggregate Induced Emission (AIE) mechanisms to avoid the π‐π stacking. Analyzing how to turn on this AIE mechanism through aggregation with a metal can stop this stacking phenomena and allow the molecule to exhibit fluorescence in a concentrated solution. One known fluorophore that exhibits AIE properties are cycloheptatrienylidenes (CHT). The project described herein uses hypervalent iodine in a new methodology to synthesize CHTs. A faster and cheaper way to synthesize asymmetric CHTs is being investigated for the generation of AIE materials. Currently, methodologies of producing symmetrical cycloheptatrienylidene fluorophores are expensive, time consuming, and involve multiple steps. Ongoing reactions involve synthesizing the CHT using a modified Catellani reaction. The Catellani reaction has eliminated a number of steps in creating the precursor to the CHT. Production of AIE fluorophores that will “turn‐on” fluorescence after coming into contact with metals could be used to activate endogenous metals in the body which could help to target malignancies such as cancer.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Experimental Computational Evaluation of Biological Immunofluorescence Data
Authors: Hietpas Taylor
Published: June 2021

Optical spectroscopy and fluorescence have become important techniques for elucidating molecular biological function. Two‐photon and confocal microscopy have particularly enabled increased potential for data acquisition and cellular resolution. With this increased potential has developed a need for efficient and reliable methods for evaluating these large data sets. Historically, researchers have relied upon fundamental image processing techniques and manually implemented image modification to obtain interpretations of their data. While these methods have proven successful, increased computation power and well‐established statistical segmentation protocols have enabled a renaissance of sorts in biological image analysis. In our lab, lymphatic and macrophage immunofluorescence data or endogenous fluorescence from transgenic reporters is most commonly acquired via either confocal or two‐photon microscopy of whole mount corneal and skin tissues. Manual analysis of these large and complex data sets is time consuming and technically challenging. This project has allowed us to explore more sophisticated methods of image processing and analysis. First, K‐means clustering algorithms were implemented to evaluate efficacy of unsupervised partitioning and segmentation of these image data sets. While K‐Means has proven effective at segmentation in many disciplines, the task of segmenting noise and autofluorescence has in these types of data has proven subtler than expected. As such, further methodologies are being investigated including Nonnegative Matrix Factorization, Spectral Phasor Analysis.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Activating transcription factor 4‐dependent B cell translocation gene‐1 induction promotes ferroptosis
Authors: Cho Il Je , Kim Jae Kwang , Kim Eun Ok , Park Sang Mi , Ki Sung Hwan , Kim Sang Chan
Published: June 2021

Ferroptosis is a special type of programmed cell death that is characterized by iron‐dependent lipid peroxidation. The present study investigated the role of B cell translocation gene‐1 (BTG‐1) on sulfur amino acid deficiency (SAAD)‐mediated cell death. SAAD in HepG2 cells depleted the reduced glutathione, accelerated lipid peroxidation, increased cyclooxygenase 2 expression, and promoted cell death. SAAD‐mediated cell death was specifically inhibited by pretreatment with ferrostain or defroxamine, chemical inhibitors of ferroptosis. In parallel with cell death, SAAD increased expression of microtubule‐associated protein light chain 3‐II and facilitated acridine orange positive autophagic flux. Inhibition of autophagy accelerated the SAAD‐mediated cell death. Moreover, SAAD induced BTG‐1 expression in HepG2 cells. By using BTG‐1 promoter haboring reporter gene and siRNA, activating transcription factor 4 (ATF4) was identified as an essential transcription factor for SAAD‐mediated BTG‐1 induction. Knock out of BTG‐1 gene by CRISPR/Cas9 system decreased the cytotoxicity by SAAD. Furthermore, BTG‐1 deficiency increased microtubule‐associated protein light chain 3‐II and autophagic flux. Thus, present study demonstrates that SAAD induces ferroptosis, and the ATF4‐dependent BTG‐1 induction contributes to accelerate ferroptosis by inhibiting autophagy.

Support or Funding Information

This study was supported by the National Research Foundation of Korea (NRF) funded by Korea government (MSIP)(Grant No. 2018R1A5A2025272 and 2018R1A2B6007514).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Chloroform Fraction of Adenopus Breviflorus Benth Fruit on Opening of Rat Liver Mitochondrial Permeability Transition Pore, Mitochondrial Atpase and Cytochrome C Release
Authors: Oyedeji Tolulope A , Akobi Chibuzor I , Onireti Daniel O , Olorunsogo Olufunso O
Published: June 2021

Bioactive agents from medicinal plants that can induce the intrinsic pathway of apoptosis in tumours by opening of the mitochondrial permeability transition (MPT) pore maybe developed into drugs useful in chemotherapy. The fruit of Adenopus breviflorus (A. breviflorus), a tropical medicinal plant found in the Western part of Nigeria, West Africa, is widely used in folklore medicine against the outbreak of measles, and in the treatment of gonorrhoea in men. However, its use in the treatment of tumours has not been well elucidated. Therefore, this study aim at assessing the in vitro effect of varying concentration of the chloroform fraction of A. breviflorus (CFAB) on the opening of rat liver MPT pore, mitochondrial ATPase (mATPase) and cytochrome C (Cyt C) release. Rat liver mitochondria were isolated by differential centrifugation. Mitochondrial Permeability Transition, mATPase activity and Cyt C release were determined spectrophotometrically. The results obtained from this study show that the varying concentration of CFAB, significantly induced opening of MPT pore in rat liver mitochondria in a concentration‐dependent manner. Specifically, the pore was opened by 15.0, 18.8. 18.9 and 19.2 folds at 50, 150, 250 and 350 μg/mL, respectively. Also, the CFAB also stimulated mATPase activity by 4.4, 2.5, 3.3 and 22.4% at 50, 150, 250 and 350 μg/mL, respectively. Furthermore, the varying concentration of CFAB significantly (p < 0.05) stimulated Cyt C release in concentration‐dependent manner. This study therefore suggests that the chloroform fraction of A. breviflorus may possess potent bioactive agents that could induce the intrinsic pathway of apoptosis through the induction of the mitochondrial permeability transition pore, stimulation of mitochondrial ATPase and the release of cytochrome c.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effects of Epigenetic Modifiers on PD‐L1 and HLA Class I Expression on Tumor Cells
Authors: Maini Trisha , Sternberg Lauren , O'Donnell Robert W.
Published: June 2021

Expression of PD‐L1 on various tumor cell lines leads to the inhibition of T cell cytotoxicity. When this takes place, the T cells are unable to combat the tumor cells and cancer persists. In the same regard, increased expression of human leukocyte antigen (HLA) allows tumor cells to be more easily detected by these T cells. In previous research, epigenetic modifiers including the DNA methylation inhibitor 5‐Azacytidine (5‐AzaC) and the histone deacetylase inhibitor vorinostat, were shown to increase HLA expression. However, epigenetic modifiers have also been shown by others to upregulate PD‐L1 expression. We hope to elucidate whether or not PD‐L1 expression is upregulated along with HLA expression upon exposure to these epigenetic modifiers. If this were to occur, then the effects of both PD‐L1 and HLA would essentially negate each other. Two cell lines that are known to express PD‐L1 are MCF‐7 and MDA‐MB‐231. Our initial results confirm that PD‐L1 is expressed on the MDA‐MB‐231 cell lines, as demonstrated by flow cytometry. Our results have also shown that two other cell lines, MDA‐MB‐435 and HL‐60, do not express PD‐L1 and that exposure with the epigenetic modifier 5‐AzaC did not cause either cell line to express PD‐L1. Nonetheless, there was still an up regulation of MHC Class I antigens following a 48 hour exposure to a non‐cytotoxic dose of 5‐AzaC. Future experiments are planned to optimize the doses of 5‐AzaC and vorinostat to maximize HLA expression while not increasing or inducing the expression of PD‐L1.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effect of Sodium Glucose Transporter 2 Inhibitors on Proliferation and Growth Factor Signaling Pathways in Triple Negative Breast Cancer
Authors: Ware Kierra , Smith Tunde , Brown Del‐Vecie , Hill Dorian , Stewart LaMonica
Published: June 2021

Triple negative breast cancer (TNBC) is an aggressive form of breast cancer that does not express the estrogen receptor, progesterone receptor, and the HER2 receptor. Since it does not express these receptors, TNBC does not respond to many of the standard therapies for breast cancer. To decrease the number of deaths associated with breast cancer, we must identify therapeutic strategies that effectively prevent the growth and progression of TNBC. Recent studies from our laboratory and others show that the sodium glucose transporter 2 (SGLT2) inhibitor canagliflozin reduces proliferation of human prostate cancer cell lines. To determine whether SGLT2 inhibitors can also suppress TNBC growth, we tested the effect of canagliflozin and other SGLT2 inhibitors on proliferation and protein expression within the BT‐549 human breast cancer cells. Presto Blue assays revealed that two SGLT2 inhibitors, canagliflozin and ipragliflozin, significantly inhibited BT‐549 cell proliferation. However, the SGLT2 inhibitor empagliflozin produced little to no change in cell proliferation. We next examined the effect of SGLT2 inhibitors on Akt and Erk ½ MAP kinase, two proteins that promote TNBC growth. Western blot analysis revealed that canagliflozin reduced phosphorylation of Akt and Erk ½ within BT‐549 cells. Therefore, the ability of canagliflozin to block BT‐549 proliferation may be due in part to canagliflozin‐mediated decreases in Akt and Erk activity. The epidermal growth factor receptor (EGFR) activates both Akt and Erk ½ signaling in BT‐549 cells. To determine whether inhibition of EGFR signaling would alter the response to canagliflozin, we tested the combined effect of canagliflozin and EGFR inhibitors on BT‐549 cell proliferation. Canagliflozin alone was more effective at reducing BT‐549 cell proliferation than the EGFR inhibitors gefitinib and lapatinib. However, the combined effect of canagliflozin and gefitinib as well as the combined effect of canagliflozin and lapatinib was greater than that produced by either compound alone. These data suggest that combination treatments involving canagliflozin and EGFR inhibitors may serve as an effective therapy for patients with TNBC.

Support or Funding Information

NIH grant 5T34GM007663

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Gemcitabine Sensitivity is Improved in Pancreatic Cancer by CYR61/CCN1‐Depletion‐Mediated Upregulation of dCK and Suppression of CTGF
Authors: Ghosh Arnab , Maity Gargi , Gupta Vijaylaxmi , Haque Inamul , Sarkar Sandipto , Das Amlan , Dhar Kakali , Bhavanasi Sneha , Gunewardena Sumedha S , Von Hoff Daniel D , Mallik Sanku , Kambhampati Suman , Banerjee Sushanta , Banerjee Snigdha
Published: June 2021

Introduction

Pancreatic ductal adenocarcinoma (PDAC) develops resistance (both extrinsic and intrinsic) by preventing chemotherapeutic agents from entering inside the cells by promoting desmoplastic reactions (DR) and metabolic malfunctions of the drugs. Resistance to gemcitabine (GEM) is associated with these responses. However, the mechanism by which these resistant pathways function in the pancreatic cancer cells remains poorly understood.

Objective

In this study, we have shown that CYR61/CCN1‐signaling plays a vital role in making pancreatic cancer cells resistant to GEM in vitro and in‐vivo (tumor xenograft model).

Result

We have shown that the catastrophic effect of GEM could significantly be increased in GEM‐resistant PDAC cells when CYR61/CCN1 is depleted, while this effect can be suppressed in GEM‐sensitive neoplastic cells by treating them with CYR61/CCN1 recombinant protein. On the other hand, non‐transformed pancreatic cells, which are sensitive to GEM, cannot be resistant to GEM by CYR61/CCN1 protein treatment, showing a unique feature of CYR61/CCN‐signaling that only influences PDAC cells to become resistant. Additionally, we demonstrated that CYR61/CCN1 suppresses the expression of the GEM‐activating enzyme dCK while it induces the expression of a DR‐promoting factor CTGF (connective tissue growth factor) in pancreatic cancer cells in vitro and in vivo.

Conclusion

dCK and CTGF pathway mediated mechanisms for GEM resistance may act as a novel target for CYR61/CCN1, that protect pancreatic cancer cells from GEM. Collectively, these studies reveal a novel paradigm in which CYR61/CCN1 regulates both extrinsic and intrinsic GEM‐resistance in PDAC cells by employing unique signaling pathways.

Support or Funding Information

The work is supported by Merit review grant from Department of Veterans Affairs (Sushanta K. Banerjee, 5I01BX001989‐04 and Snigdha Banerjee, I01BX001002‐05).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Enteropathogenic E. coli Hijacks Programmed Host‐Cell Death Pathways by Interfering with the Higher Order Oligomerization of Immune System Proteins
Authors: Monserrat‐Martinez Ana , Redpath Gregory , Govendir Matt Alfred , O'Carroll Ailis , de Andrés Murillo Andrea , Sierecki Emma , Gambin Yann
Published: June 2021

During an infection, pathogens operate by hijacking or promoting crucial intracellular pathways within their hosts. In the case of Enteropathogenic Escherichia coli (EPEC) infection, bacterial proteins interact with host proteins to alter programmed cell death pathways. Recently, it has been found that many proteins from the immune system polymerise to transduce these death signals, forming higher order assembly structures.

The aim of this project is to study how EPEC effector proteins interfere with the higher order assembly of immune proteins involved in signal transduction in inflammation, apoptosis and cell survival pathways.

A selection of immune system proteins involved in signal transduction in different cell pathways was chosen to perform an in vitro screen against EPEC effector proteins by confocal spectroscopy. It was observed that espF (a bacterial effector protein) was able to change TRAF2 oligomerization/aggregation propensity. TRAF2 is a cytosolic protein involved in signal transduction in the cell death pathway. In cases of infection or stress, the protein forms higher order assembly structures to transduce the signal downstream. Further experiments performed in HeLa cells transfected with TRAF2 showed an increase in these structures in stressed cells when compared with the control group. When espF and TRAF2 were co‐transfected, co‐localization of both proteins was observed in distinct punctate intracellular structures reminiscent of higher order assemblies, together with other changes that are still under investigation.

The bacterial protein espF appears to be hijacking the cell death pathway when there is an EPEC infection by avoiding TRAF2 self‐aggregation. Understanding precisely this host‐pathogen interaction could give us information useful for future drug development against EPEC infection.

Support or Funding Information

EMBL Australia, University International Postgraduate Award (UIPA), University of New South Wales

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dissecting the mechanism of host shutoff by SARS coronavirus
Authors: Nag Anita , Gomez Garret G.
Published: June 2021

Down regulation of host gene expression during viral infection, known as host shutoff, involves destabilization of host mRNA. Several viruses, including Kaposi's sarcoma virus, Epstein B virus, severe acute respiratory syndrome coronavirus (SARS CoV) and Middle Eastern respiratory syndrome coronavirus inhibit host gene expression by stalling the ribosome on mRNA and subsequently cleaving and degrading host mRNA. In SARS‐CoV, nsp1 is the key factor in virus‐induced down‐regulation of host gene expression. In infected cells, nsp1 engages in a multi‐pronged mechanism to inhibit host gene expression by binding to the 40S ribosomal subunit to stall further ribosome assembly and inducing endonucleolytic cleavage and degradation of host mRNAs. We identified a previously undetected mechanism by which nsp1 exploits the nuclear pore complex and disrupts nuclear‐cytoplasmic transport of biomolecules. Using quantitative proteomics we identified members of the nuclear pore complex from nsp1‐associated protein assembly. Expression of nsp1 in HEK cells disrupts Nup93 localization around the nuclear envelope without triggering proteolytic degradation of the protein while other nucleoporins and the nuclear lamina remain unperturbed, suggesting significant alteration of the nuclear pore complex in the presence of nsp1. Furthermore, Nup93 reconvenes to the nuclear pore when nsp1 is removed. Consistent with its role in host shutoff, nsp1 alters the nuclear‐cytoplasmic distribution of a RNA binding protein, nucleolin. Our results suggest that nsp1 alone can regulate multiple steps of gene expression including nuclear‐cytoplasmic transport.

Support or Funding Information

SC INBRE Bioinformatics Pilot Project AwardSCICU

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Translational Fusion of a G‐protein Coupled‐Receptor from the Hookworm Ancylostoma ceylanicum Expressed in Caenorhabditis elegans
Authors: Norman Brandon , DeLong Lauren , Dickey Myra , Jewell Mollie , Erickson Patti T. , Ratnappan Ramesh , O'Halloran Damien , Hawdon John M.
Published: June 2021

Hookworms are infectious parasites that affect more than 500 million people worldwide, causing extensive morbidity and economic burden in developing countries. Mass drug administration is a current control method, but resistance is of concern. Many hookworm species, including those in the genus Ancylostoma, are specialists and require specific host signals to continue development upon infection. Little is known about the signaling mechanisms, although evolutionary conservation between parasitic nematodes and the model organism C. elegans suggests that G‐protein coupled receptors (GPCRs) expressed in amphid neurons are chemosensory detection candidates. The amphid neurons of C. elegans are exposed to the environment, even during the dauer stage, an alternate phase of larval development that is similar to the parasitic infective juvenile stage. Previous experiments involving A. ceylanicum transcriptional GFP fusions in C. elegans identified putative GPCR genes expressed in neurons during dauer stage. To confirm that hookworm proteins are neuronally localized under dauer conditions, a translational GFP fusion of a putative hookworm GPCR was generated. The entire coding sequence, plus introns, was cloned using homologous recombination‐based, yeast assembly methods to create a C‐terminal GFP fusion. The putative A. ceylanicum promoter consisted of the upstream intergenic region, which is approximately 5000 base pairs, and the transcriptional terminator from C. elegans unc‐54 was used. To generate the expression plasmid, amplicons containing homology to the adjacent segment in the assembly were amplified using polymerase chain reaction (PCR) and transformed into yeast. The plasmid was verified by junction‐confirming PCR analysis, restriction enzyme mapping, and sequencing. After microinjection, GFP expression in amphid neurons of stably transformed C. elegans during the dauer stage would support the hypothesis that this putative GPCR plays a role in reception of host signaling molecules and may provide a possible therapeutic target.

Support or Funding Information

This work was supported by the Salisbury University Faculty Mini‐Grant Program.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Determination of Antimicrobial Activity and Secondary Metabolites in Acacia rigidula and Acacia berlandieri
Authors: Gonzalez David Israel
Published: June 2021

There is continuous interest in the antimicrobial activities of different extracts obtained from plants especially traditional medicinal plants. There are more than 20,000 species of plants used in traditional medicines. Therapeutic drugs can be derived from natural products, which are usually secondary metabolites and their derivatives. Drugs derived from these secondary metabolites can be used to fight against the increased presence of antibiotic‐resistant bacteria. Therefore, finding new antimicrobial agents is important for the continuing development of new therapeutic drugs. In this study, plants found in South Texas have been a focus of interest in terms of antimicrobial activity present in their secondary metabolites. Studies have indicated that Acacia species, such as Acacia nilotica, have displayed antimicrobial activity. The objectives of this study were to determine the presence of antimicrobial activities and qualitatively determine the secondary metabolites of plant extracts from Acacia rigidula (Blackbrush) and Acacia berlandieri (Guajillo). The extracts were subjected to acetone, methanol, acetic acid to extract secondary metabolites. To test the antimicrobial activities of the species, the disk diffusion method was employed against gram‐positive and gram‐negative bacteria such as: Escherichia coli, Staphylococcus aureus, Serratia marcescens, Enterococcus faecalis, and Enterobacter aerogenes. Qualitative determinations of the presence of phenols, tannins, terpenoids, alkaloids, and flavonoids were done. Thus, this study will report the antimicrobial activities of A. rigidula and A. berlandieri extracts. Preliminary results of the disk diffusion method indicate the presence of antimicrobial activity against E. coli found in the bark of A. rigidula extracted with acetic acid and the leaves of A. berlandieri extracted with acetone. The zone of inhibition measured was 14.9 mm and 15.1 mm respectively. To compare with a positive control, streptomycin displayed a zone of inhibition of 16.5 mm against the E. Coli confirming the antimicrobial activity from the two species. Future results will establish the antimicrobial activity further once tested against different bacteria. This study will also report the secondary metabolites present in the extracts understudy. The findings of this study will provide researchers with the basis to derive potential medications against antibiotic‐resistant bacteria.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Generation of a Difluoromethylornithine (DFMO) Resistant Cell Line in a Polyamine Depletion Strategy
Authors: Noureddine Sarah , Dobrovolskaite Aiste , Phanstiel Otto , von Kalm Laurence
Published: June 2021

Polyamines are essential growth factors for normal and malignant cells. Cells obtain polyamines via biosynthesis or transport from the extracellular environment and polyamine depletion therapy is an attractive option to inhibit the growth of cancer cells. Previous reports have indicated that depletion therapy (e.g. androgen depletion in prostate cancer) can lead to epigenetic changes resulting in resistance to the therapy and aggressive metastatic cancer. We were interested to know if polyamine depletion driven by treatment with the polyamine biosynthesis inhibitor difluoromethylornithine (DFMO) could lead to similar outcomes. The pancreatic cancer cell line Panc‐1 was passaged for 10 generations on IC50 and IC75 concentrations of DFMO, after which a decreased sensitivity to DFMO was observed. Our results indicate the need for further investigation into the mechanism of resistance to DFMO, and it's potential consequences for clinical outcomes.

Support or Funding Information

University of Central Florida, College of Sciences Seed Award

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Docosahexaenoic acid (DHA) as an adjunctive therapeutic agent for the treatment of cancer
Authors: Rahman Mizanur , Ouhtit Allal , Saleh Haissam Abou
Published: June 2021

Breast and prostate cancers are the leading causes of cancer‐related death among women and men respectively. A significant challenge in treating these cancers is the limited array of therapeutic options and the rapid development of resistance to existing agents as well as severe side effects associated with these agents. Thus, finding new natural compounds would of great interest to improve the effectiveness of current agents, decrease the emergence of resistance, and increase side effects‐free survival. To this end, docosahexaenoic acid (DHA), a marine derived non toxic natural lipid (approved by FDA as generally recognized as safe (GRAS)), has been shown to enhance the cytotoxicity of various anticancer drugs to a variety of cancer cell lines or tumors in animal models. We first demonstrated if DHA can enhance the breast cancer cell killing by doxorubicin (Dox) using human breast adenocarcinoma cell line. Interestingly, DHA augmented the Dox induced inhibition of breast cancer cell proliferation, and invasion and Dox induced stimulation of cell cycle arrest at G2/M phase and apoptosis. Moreover, DHA boosted the chemosensitization of doxorubicin‐resistant human breast adenocarcinoma to doxorubicin. This improved chemosensitization was accompanied with enhanced accumulation of doxorubicin, reduction of P‐glycoprotein (P‐gp)‐ a classical multi drug resistance (MDR) transporter, and TG2‐ a tumor survival factor under stress condition, and induction of lipid peroxidation, and pro‐apoptotic markers Bak1 and caspase 3 in drug resistant breast cancer cell. In a separate study, we also determined if DHA can augment the anticancer effect of an FDA approved histone deacetylase inhibitor, valproic acid (VPA). Several phase 1 and 2 clinical trials demonstrated that VPA delayed the prostate cancer recurrence but exhibiting severe side effects at its effective dose. Using in vitro cell culture model, we demonstrated that VPA dose could be reduced down to half when combined with DHA to achieve same level of prostate cancer cell killing as seen with VPA alone at high dose. Our findings demonstrated that DHA might be a promising natural adjunctive therapeutic natural compound to improve the efficacy of existing cancer drugs and to reduce the toxic side effects associated with these drugs. Further preclinical, clinical, and mechanistic studies are required to establish the DHA as an adjunctive natural therapy to augment the efficacy of existing cancer drugs.

Support or Funding Information

Qatar University grant QUUG‐CAS‐DBES‐15/16‐23

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Fatty Acid‐Induced Hepatocellular Carcinoma Growth is Mediated by Decreasing Mitochondrial H2O2 Emission Coupled to Increased Glutathione Levels
Authors: Turnbull Patrick C. , Dehghani Ali C. , Perry Christopher G. R.
Published: June 2021

Rationale and Hypothesis

High fat diets are associated with increased hepatocellular carcinoma (HCC) risk, as well as increased HCC growth rates. Accelerated HCC growth in response to fatty acid challenges has been attributed to a variety of signalling pathways, but the role of metabolic and redox flexibility in mediating a pro‐growth environment in this context has not been examined. Here we examined the direct effect of fatty acid challenges on HCC growth in relation to altered metabolic and redox homeostasis.

Experimental approach

The HCC cell line HepG2 was incubated with 0μM, 50μM and 100μM palmitoylcarnitine (PCarn) for up to 48 hrs. We measured clonogenic survival, mitochondrial H2O2 emission and glutathione following PCarn incubations with and without the glutathione depleting agent buthionine sulfoximine (BSO) and inhibition of uncoupling protein‐2 (UCP2) activity by genipin.

Results

100μM PCarn increased clonogenic survival in HepG2 by 8% more than control (p<0.05) at 48 hrs which represents a marked early effect considering the population doubling time of HepG2 cells is ~48 hrs. This was associated with an increase in both reduced and oxidized glutathione at both 24 and 48 hrs (p<0.05). Depleting glutathione with BSO prevented PCarn‐stimulated growth (p<0.05). In a separate experiment, acute incubations of 100μM PCarn increased H2O2 emission within the first 10 minutes (p<0.05) followed by a decrease in H2O2 at 1 hr that remained lower at 24 hrs (p<0.05). The acute increase in H2O2 followed by a more chronic depression in H2O2 suggested PCarn might have triggered a compensatory mechanism. In support of this notion, inhibition of UCP2 with genipin sensitized HepG2 cells to PCarn‐induced decreases in clonogenic survival (p<0.05) without a change in UCP2 protein content.

Conclusion

Collectively, this data suggests that PCarn‐induced HCC growth is in part attributed to elevated glutathione. Increases in glutathione may be a result of UCP2 rapidly attenuating PCarn‐induced H2O2 emission which may permit greater glutathione synthetic rates and creation of a pro‐growth redox environment.

Support or Funding Information

Funding was provided to C.G.R.P. by National Science and Engineering Research Council (#436138‐2013) with infrastructure supported by Canada Foundation for Innovation, Ontario Research Fund and the James H. Cummings Foundation. P. C. T. was supported by an NSERC CGS‐D scholarship.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inflammatory and Nutritional Biomarkers in the Plasma of Women in Zambia Reflect Low DHA and HIV Status
Authors: Kisling Sophia , Majid Sonoor , Wood Charles , Chunda Catherine , DiRusso Concetta
Published: June 2021

Docosahexanenoic Acid (DHA) is a fatty acid that is abundant in the brain, retina, and cerebral cortex. It is critical in fetal brain development and is implicated in resolution of inflammation. In recent years, there has been a growing concern that low dietary levels of DHA in pregnancy may lead to poor birth outcomes and delayed or reduced cognitive function in children after birth. Uncontrolled HIV infection may lead to cognitive impairments as well as reduced immune function. Therefore, for pregnant women living with food insecurity and HIV infection, these problems may be compounded and health risks to their infants high. To evaluate these concerns, a pre‐clinical trial was conducted in pregnant women in Zambia, a country where malnutrition and HIV are endemic. The study participants included 91 women in the second trimester of pregnancy, 46 of which were HIV seropositive and 45 HIV seronegative. Among the HIV positive participants 11 had high viral load. The overarching study goal was to determine if plasma DHA levels were low relative to global averages and if they correlated with inflammation and HIV status. Hematological assessment included measurement of biomarkers for nutritional status, HIV status and viral load, as well as inflammation. Plasma lipids were assessed using the Folch extraction method, fatty acid methyl esters were prepared and analyzed by gas chromatography/mass spectrometry. Protein biomarkers data were measured using commercial ELISA kits. Overall, DHA levels were low at 2.16±0.75 mass percent of total fatty acids but did not vary significantly with HIV status. Averages of the nutritional biomarkers insulin and leptin were low and high, respectively, compared to global averages when comparing all participants. Insulin was not significantly different based on HIV status. Leptin was reduced in HIV seropositive participants compared to those who were seronegative and this correlated, as expected, with pre‐pregnancy BMI. For the inflammatory markers CRP and LCN2, only LCN2 was significantly different dependent on HIV status, and levels were on average lower in seropositive individuals. Within the HIV positive participants, the 11 with high vial load did not have levels of insulin, leptin, CRP or LCN2 that were significantly different from those with low viral load. These data demonstrate that select protein biomarkers for inflammation and nutrition are variably affected by HIV and nutritional status. Currently, these studies are being expanded to understand the role of dietary fatty acids in contributing to health outcomes in pregnancy and dependent upon HIV status.

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