bluuebunny/crossref_metadata_2025 huggingface dataset:
Unstructured Proteins in Biological Structures: The Case of Human Teeth from a Protein Chemist's Perspective
Authors: Sharma Vaibhav , Srinivasan Alagiri , Roychoudhury Ajoy , Nikolajeff Fredrik , Kumar Saroj
Published: June 2021

Proteins are the biomacromolecules that work downstream to genes in every living system. It is these protein molecules that are responsible for a plethora of functions in our body, ranging from building skeleton tissues to transmitting signal from one place to others. There was a period when biologists have the view that for a protein to function, the structure is of supreme importance. Nevertheless, this structure‐function paradigm is fairly obeyed by most of the proteins, if not all of them. Last two decades have witnessed the increased reports of proteins that do not have a stable three‐dimensional structure but are very much indispensable for the cellular and biological activities. These unstructured proteins are now known as “Intrinsically disordered proteins” (IDPs). Interestingly, these IDPs function not only in isolation (e.g. transcription factors) but helps in the formation of hard tissues like teeth, bone, and mollusks shell, by governing a process called biomineralization. Biomineralization is the process by which living organisms produce minerals, often to harden or stiffen existing tissues. Such tissues are called mineralized tissues.

We have isolated total protein extracts from a healthy molar, premolar, canine and incisor teeth. We studied their protein secondary structural characteristics using various biophysical techniques. We have also investigated the effects of these protein extracts on mineralization of calcium phosphate through in vitro biomineralization assay followed by size‐based measurement analysis via Nanoparticle tracking analysis (NTA). The biophysical studies confirmed the presence of IDPs in the majority within these protein extracts. Moreover, these extracts were shown to elevate calcium phosphate biomineralization in a time‐dependent manner. This is the first time the effect of the whole protein extract was taken into account which in our view more closely imitates the in vivo process of tooth biomineralization. Furthermore, we also evaluated the differential proteome profile of the individual type of human teeth through Mass spectroscopy‐based method. The comparative qualitative and quantitative data of proteins from these different human teeth were obtained. The implication of these results will be discussed.

Support or Funding Information

There was no support or funding from any organization

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ordered and Disordered Segments of Amyloid‐β Drive Sequential Steps of the Toxic Pathway
Authors: Maiti Sudipta , Maity Barun Kumar , Das Anand Kant , Dey Simli , Moorthi Ullhas Kaarthi , Kaur Amandeep , Dey Arpan , Surendran Dayana , Pandit Rucha , Kallianpur Mamata , Chandra Bappaditya , Chandrakesan Murlidharan , Arumugam Senthil
Published: June 2021

While the roles of intrinsically disordered protein domains in driving many interactions are increasingly well‐appreciated, the mechanism of toxicity of disease‐causing disordered proteins remains poorly understood. A prime example is Alzheimer's disease (AD) associated amyloid beta (Aβ). Aβ oligomers are highly toxic partially structured peptide assemblies with a distinct ordered region (residues ~10–40) and a shorter disordered region (residues ~1–9). Here, we investigate the role of this disordered domain and its relation to the ordered domain in the manifestation of toxicity through a set of Aβ fragments and stereo‐isomers designed for this purpose. We have measured their effects on lipid membranes and cultured neurons, probing their toxicity, intracellular distributions, and specific molecular interactions using the techniques of confocal imaging, lattice light sheet imaging, fluorescence lifetime imaging, and fluorescence correlation spectroscopy (FCS). Remarkably, we find that neither part ‐ Aβ10–40 or Aβ1–9, is toxic by itself. The ordered part (Aβ10–40) is the major determinant of how Aβ attaches to lipid bilayers, enters neuronal cells, and localizes primarily in the late endosomal compartments. However, once Aβ enters the cell, it is the disordered part (only when it is connected to the rest of the peptide) which has a strong and stereospecific interaction with an unknown cellular component, as demonstrated by distinct changes in the fluorescence lifetime of a fluorophore attached to the N‐terminal. This interaction appears to commit Aβ to the toxic pathway. Our findings correlate well with Aβ sites of familial AD mutations, a significant fraction of which cluster in the disordered region. We conclude that while the ordered region dictates attachment and cellular entry, the key to toxicity lies in the ordered part presenting the disordered part for a specific cellular interaction.

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 Protective Effect of DAS and DADS Against Doxorubicin Induced Toxicity
Authors: Taylor LaShaundra , Miles Jana , Webster Camille , Hudson Alicia , Soliman Karam , Flores‐Rozas Hernan , Darling‐Reed Selina
Published: June 2021

Purpose

Cancer survival rates are increasing but the mechanisms involved in the adverse effects of cancer therapy such as doxorubicin are still being investigated along with treatment for these effects. Doxorubicin, an established chemotherapeutic agent used for various cancers, has limited use due to cardiotoxicity. The objective of this study, is to determine how garlic constituents, Diallyl sulfide (DAS) and Diallyl disulfide (DADS) may decrease the adverse effects of doxorubicin exposure.

With a S. cerevisiae deletion library, a genome wide screen was conducted to detect mutants sensitive to doxorubicin exposure. A mutant deleted for the gene enconding for Mac1, a copper sensing transcription factor, was found to be highly sensitive. In S. cerevisiae the MAC1 gene is activated upon copper deficiency, resulting in the induction of copper transporters. Other doxorubicin sensitive strains included the sod1 mutant and its copper chaperone ccs1 mutant, both of which are associated with MAC1. The sensitivity of the mutant strains to doxorubicin, was in part due to the drug's capacity to generate reactive oxygen species (ROS). To test if garlic constituents, DAS and DADS could reduce the sensitivity of the mutants to ROS, the viability of the sensitive yeast strains were determined by exposing them to doxorubicin in the presence or absence of DAS and DADS.

Methods

Yeast survival assays were completed to determine the sensitivity of wt, mac1, ccs1, and sod1 strains to doxorubicin, menadione and etoposide, co‐treated with DAS and DADS. Statistical significance was set at p<0.05.

Results

mac1 shows sensitivity to doxorubicin and etoposide but not menadione. Menadione contains a quinone ring like doxorubicin, and acts through the induction of reactive oxygen species. Etoposide, a chemotherapeutic agent acts only through inhibition of DNA topoisomerase II. The sensitivity of mac1 to both etoposide and doxorubicin, shows that doxorubicin's sensitivity to mac1 is through its action on topoisomerase II. Unlike DAS, DADS was able to decrease the sensitivity of mac1 to doxorubicin and etoposide. Menadione has greater toxicity to ccs1 and sod1 than doxorubicin and etoposide, with DAS and DADS both increasing the survival of all chemicals to ccs1 and sod1.

Conclusion

Garlic constituents, das and dads may decrease the adverse effects of doxorubicin exposure, improving the quality of life of patients expose to doxorubicin.

Support or Funding Information

Research supported by National Institutes of Health (NIH) under Award Number G12MD007582

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Macromolecular Crowding on Alcohol Dehydrogenase and Citrate Synthase Kinetics
Authors: Slade Kristin , Chung Charmaine , Jackson Jasmine
Published: June 2021

Most of our understanding of enzyme kinetics is based on experiments conducted in dilute solution. However, these conditions do not accurately represent the crowded cellular environment, which contains high concentrations (300–400g/L) of various macromolecules such as proteins, carbohydrates and ribosomes. This crowding reduces the volume of solvent available for other molecules in the solution and has previously been shown to impact the behavior of enzymes. To study these potential consequences, the Michaelis‐Menten kinetics of yeast alcohol dehydrogenase (YADH) and citrate synthase (CS) were monitored under crowded conditions. Spectroscopic assays were performed in the presence and absence of high concentrations of dextran or glucose, the small‐molecule counterpart. This study also explored the effects of mixed crowding by combining equal ratios of different sizes of dextran. Overall, the effects of crowding depend on the size and concentration of the crowding agent. Furthermore, the results demonstrated that, in addition to merely excluded volume effects, chemical interactions from crowders must also be considered.

Support or Funding Information

Camille and Henry Drefyus Foundation.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Examination of specific binding activity of packaging sequence RNAs to the SARS‐CoV nucleocapsid by using cell‐based in vivo assay for RNA‐Protein interaction
Authors: Woo Jiwon , Cho Yong‐Eun
Published: June 2021

Severe acute respiratory syndrome coronavirus (SARS‐CoV) is the etiological agent of SARS, an emerging disease characterized by atypical pneumonia. The SARS‐CoV nucleocapsid protein serves multiple functions in viral replication, transcription and the formation of viral genome complex. Coronaviruses specifically package genomic RNA into assembled virions, little is known about the specific interaction between Nucleocapsid protein and packaging RNA sequence in previously. Herein, we exploited a cell‐based translation inhibition assay to elucidate the protein‐RNA interaction in living cells. Our results demonstrated that the packaging signal is pivotal in the specific interaction of the nucleocapsid and viral genomic RNA for incorporation into virions. We identified that the specific conserved RNA sequence of the nsp12 encoding vRNA of SARS‐CoV, and conserved RNAs of other corona viruses are major determinant sequences for the N‐protein interaction, respectively. In addition, using deletion mapping of the N‐protein, we also found that the specific in vivo RNA binding activity of the Nucleocapsid protein of the SARS‐CoV requires both the N‐terminal and C‐terminal domain. These results provide the first evidence for a specific interaction of the SARS‐CoV Nucleocapsid protein with the its viral packaging RNA sequence in vivo that will be useful as a valuable research tool for further detailed studies and providing new targets for antiviral therapies.

Support or Funding Information

This work was supported by the Brain Korea 21 PLUS Project for Medical Science, Yonsei University. In addition, this work was supported by a grant from the National Research Foundation of Korea (NRF‐2017R1D1A1B03030315).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Assessing anti‐cancer potential of newly synthesized staurosporine analogs
Authors: Hurysz Brianna , McFadden Marissa , Pelkey Erin , Mowery Patricia
Published: June 2021

The discovery of staurosporine in 1977 and subsequent finding that it was a potent inhibitor of protein kinase C suggested that it might have anti‐cancer potential. Subsequently staurosporine was found to be a pan‐kinase inhibitor, making it to toxic for drug development, however, derivatives have been found with increased specificity and have been tested in clinical trials. The structure of staurosporine can be divided into three parts: a pyran ring that bridges the indole nitrogen atoms, an indolocarbazole central core, and a fused pyrrol‐2‐one. We focus on the synthesis and biological activity of the pyrrolinone group and bis‐indole motifs. Using a cytotoxicity assays we have analyzed structure‐function activity of our derivatives and endeavor to synthetic new targets with increased potency. We recently reported on the synthesis and biological activity of a 1st generation of analogs with low micromolar potency. Our findings suggested that biologically effective compounds require: a) an indole cis to the carbonyl oxygen atom on the 3‐pyrrolin‐2‐one central core ring, b) a flexible, rather than a planar, fused analog, c) aryl/heteroaryl groups at both the C3 and C4 position of the 3‐pyrrolin‐2‐one core, and d) multiple methoxy groups on the C4 ring. Building upon this work, we now report on a 2nd generation of compounds with nanomolar cytotoxic activity.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluation of Ant‐Lion Larvae Extract As Alpha‐Glucosidase and Alpha‐Amylase Inhibitor
Authors: Adeyanju Muinat Moronke , OLANIYI Omolayo Omobolawa , RASAK Olashile Fatimat , ADEOSUN Oyewale Oluwapelumi , ASHIDI Joseph Senu , OBUOTOR Efere Martins
Published: June 2021

Diabetes mellitus is a serious condition with potentially devastating complications that affects all age groups worldwide with the percentage of affected people increasing at an alarming rate. Inhibition of alpha‐amylase and alpha‐glucosidase are the most important strategies in controlling the postprandial blood glucose and invariably for the treatment of disorders in carbohydrate uptake, such as diabetes and obesity. There had been several efforts geared towards sourcing anti‐diabetic therapeutics, the most studied being plants and have been reported to effectively do this but quite a number of them are toxic to humans. Thus we investigated alternative means of managing the ailment using ant lion larvae extract. In this study, alpha‐amylase and alpha‐glucosidase inhibitory potentials of the ant‐lion larvae extract were investigated. About 0.50 g of live ant‐lion larvae samples were hand‐picked from the soil within the school premises and homogenized in 25 ml of 100 mM phosphate buffer at pH 6.0. The protein concentration of the extract was determined while alpha‐glucosidase and alpha‐amylase inhibitory activities were determined by calculating the percentage inhibition and the inhibitory concentrations at 50 percent (IC50) of the crude extract. The values were compared to that of acarbose, a standard inhibitor and the mode of inhibition determined. The protein concentration of the extract was 5.256 ± 0.062 mg/ml with an IC50 value of 13.329 ±1.739 mg/ml for alpha‐glucosidase and 0.710±0.040 mg/ml for alpha‐amylase inhibitory activities while acarbose gave lower IC50 values of 0.620±0.090 mg/ml and 0.399±0.004 mg/ml for alpha‐glucosidase and alpha‐amylase respectively. The extract was noted to exhibit uncompetitive inhibition for both enzymes, mode of action attractive for drug design. It can therefore be concluded that the crude ant‐lion larvae extract could be more potent if purified and therapeutically exploited in the management of Diabetes mellitus or other related diseases.

Support or Funding Information

Self Supported

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
SP101, a novel synthetic compound displays survivin suppression, apoptosis and tumor inhibition in both the EGFR‐wild type and ‐T790M of non‐small cell lung cancer
Authors: Chao Jui‐I
Published: June 2021

Gefitinib is a tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR‐TKI) which is currently used therapy in non‐small cell lung cancer (NSCLC) patients; however, drug resistance of cancer patients treatment is a critical problem. Here, we show a novel synthetic compound, dodecyl‐4‐(4‐(3‐(4‐(3‐chloro‐4‐fluorophenylamino)‐7‐methoxyquinazolin‐6‐yloxy)propyl)piperazin‐1‐yl)‐4‐oxobutanoate derivative from gefitinib, which is called SP101 that attenuated drug resistance in both the EGFR‐wild type and ‐T790M of non‐small cell lung cancer. SP101 inhibited EGFR kinase activity in the gefitinib‐resistant lung cancer cells. SP101 induced cancer cell death and apoptosis in both the A549 (EGFR‐wild type) and H1975 (EGFR‐T790M) lung cancer cells. SP101 inhibited anti‐apoptotic Survivin protein levels but conversely induced active caspase 3 proteins for apoptosis induction. The ectopic expression of survivin by a survivin‐expressed vector decreased the SP101‐induced cell death. The cell death was higher after treatment with SP101 than that gefitinib in the drug‐resistant H1975 cells. SP101 inhibited tumor growth and substantially reduced survivin protein but conversely elicited active caspase 3 in the xenograft human H1975 lung tumors of nude mice. Moreover, SP101 markedly inhibited the patient‐derived xenografted patient's lung tumors in nude mice. We demonstrate a novel compound SP101 that can reduce the drug resistance of the EGFR mutation at T790M as well as the EGFR wild type in lung cancer cells.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Nicotinic acetylcholine receptor silent agonists modulate inflammation
Authors: Simard Alain , Richter Katrin , Roy Danika , Espinosa Eduardo Soto , Roy Patrick , Godin Jean‐Rémi , Grau Veronika , Quadri Marta , Horenstein Nicole , Papke Roger
Published: June 2021

Nicotinic acetylcholine receptors (nAChRs) have been increasingly studied for their immune regulatory properties. nAChRs are best known to function as ligand‐gated ion channels in neurons, however, recent evidence suggests that nicotine modulates inflammation by desensitizing non‐neuronal nAChRs, rather than by inducing channel opening. The objective of this study was to determine whether nAChR‐dependent anti‐inflammatory properties are due to channel desensitization. To this end, we made use of various nAChR ligands, including: 1) nicotine, a nAChR non‐selective agonist that also desensitizes the receptors; 2) AR‐R17779, an α7 nAChR subtype‐selective agonist; 3) α‐bungarotoxin, an α7 and α9 nAChR‐selective competitive antagonist, 4) mecamylamine, a non‐selective and non‐competitive antagonist; 5) N,N‐diethyl‐N’‐phenylpiperazine (DEPP)‐based silent agonists that selectively induce the desensitized state of α7 nAChR while producing little or no channel opening. We first evaluated the effects of these various nAChR ligands on the modulation of mouse and human monocyte/macrophage numbers, phenotype and cytokine production. Monocyte phenotype, cellular viability, proliferation and cytokine production were assessed by flow cytometry. In bone marrow‐derived monocyte/macrophage (BMDM) cell cultures, nicotine, mecamylamine and silent agonists reduced pro‐inflammatory monocyte numbers by affecting their viability and proliferation. The silent agonists also significantly reduced both LPS‐ and ATP‐induced cytokine production by mouse BMDMs. In human peripheral blood mononuclear cells (PBMCs), the silent agonists tested had modest effects on monocyte numbers and phenotype. However, LPS‐ and ATP‐induced cytokine production by human blood‐derived immune cells (PBMCs and whole blood) was inhibited by silent agonists. Taken together, our results suggest that nAChRs modulate pro‐inflammatory monocyte numbers and function via receptor desensitization rather than channel opening. nAChR silent agonists may thus be a novel source of anti‐inflammatory compounds that could be used for the treatment of inflammatory disorders.

Support or Funding Information

This work was supported by the MS Society of Canada, the New Brunswick Health Research Foundation and NIH.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biosynthesis of IAA and its role as signal molecule in the phytopathogenic bacterium Pseudomonas savastanoi
Authors: Calvillo Adrian Pintado , Pastor Victoria , Cerezo Miguel , Flors Victor , Ramos Cayo
Published: June 2021

The strains of Pseudomonas savastanoi are responsible for the formation of tumors or excrescences in the aerial parts of woody plants, belong to the Pseudomonas syringaecomplex and are classified in the pathovars savastanoi (Psv, olive), nerii (Psn, oleander), retacarpa (Psr, Spanish broom) and fraxini (Psf, ash). The production of indole‐3‐acetic acid (IAA) in Psv and Psn is a factor of pathogenicity essential for the correct development of tumors in olive and oleander. In addition, IAA has been described as a signaling molecule capable of modifying gene expression of other virulence factors, such as the type III and type VI secretion systems. Most strains of P. savastanoi produce IAA from tryptophan through the indole‐3‐acetamide pathway. However, the iaaMHoperon is not present in Psf and it is not frequent in other pathovars of the P. syringae complex. Previously, our research group showed that a Psv ΔiaaMH mutant drastically reduces IAA levels and does not induce olive tumors. This mutant produces an IAA concentration in culture supernatant similar to produce by strains of Psf and other strains of P. syringae lacking these genes. These results suggest that the existence of an alternative route of IAA biosynthesis in Pseudomonas. In order to identify an alternative route, a comparative genomic analysis of P. savastanoi and other strains in the P. syringae complex was carried out. Currently, Psv mutants have been constructed in genes potentially involved in the biosynthesis of IAA. Additionally, a metabolic analysis of indolic compounds has been conducted to determine possible intermediates of this route. Finally, we carried out a comparative transcriptomic analysis (RNAseq) of the wild type Psv and its mutant Psv ΔiaaMHand was determined the impact of the addition of exogenous IAA to cultures of P. savastanoi. In summary, this work aims to identify new routes of IAA biosynthesis in P. savastanoi and to study regulatory roles of the phytohormone, IAA, on the expression of other virulence factors in Pseudomonas.

Support or Funding Information

AGL2017‐82492‐C2‐1‐R (MINECO‐FEDER)

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 Study of the Impact of Genetic Polymorphism of Leptin Gene G2548A on Obesity and Its Related Traits
Authors: Kamalakannan Theekshitha , Robert Roshan Sam , Haqh Mohammed Shadabul
Published: June 2021

Background

Leptin is a neuroendocrine protein hormone with 167 amino acids mainly produced by adipose tissue that regulates food intake and energy expenditure to maintain relative stability of body weight and energy storage over long period of time. A single nucleotide polymorphism in leptin, consisting of G to A substitution at nucleotide −2548 upstream from ATG start site (LEP G‐2548A, rs7799039) has been found to influence leptin expression, possibly at the transcriptional level and was associated with increased leptin secretion in adipocytes. However, these high levels of leptin do not serve their function in controlling the obesity of these individuals possibly due to a condition known as ‘leptin resistance’.

Aim

Assess the risk of the genetic polymorphism of leptin gene G‐2548A on obesity.

Material and Method

A case‐control study which consisted of 66 individuals of different BMI (46 normal, 16 overweight and 4 obese). The different genotypes of leptin G‐2548A were determined by restriction fragment length polymorphism polymerase reaction after DNA extraction from saliva samples.

Results

A high risk of obesity was seen among carrier of AA genotype compared to other genotypes (GG&GA) (OR=2.6, 95% CI= 0.9–7.5). Furthermore AA genotype was found to be a risk factor for abdominal obesity (OR=1.3, 95%CI= 0.5–3.7).

Conclusion

Carrier of AA genotype of leptin G‐2548A polymorphism are at risk of obesity.

Support or Funding Information

Rak Medical and Health Sciences 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.
Adenomatous Polyposis Coli‐like protein (APCLP) Functions as a Novel Negative Regulator of NF‐kB Signaling in Colon Cancer Cells
Authors: Martin Matthew , Mundade Rasika , Lu Tao
Published: June 2021

Colon cancer (CRC) is the second leading cause of cancer related deaths in the United States. CRC is marked by aberrantly activated signaling of the nuclear factor of kappaB (NF‐kB), a family of transcription factors which regulate wide varieties of cellular processes. Despite recent advances in comprehension of players of NF‐kB signaling, deeper understanding of its regulation is imperative for the development of novel cancer therapeutics. Using the powerful validation‐based insertional mutagenesis (VBIM) technique, we recently discovered APCLP as a novel negative regulator of NF‐kB. The objective of this study is to elucidate the role APCLP in regulating NF‐kB signaling in CRC at the molecular and biological levels and to understand the mechanism by which this regulation occurs. To determine the biological effect of APCLP on NF‐kB signaling, we used lentiviral vectors to either overexpress or knockdown (shRNA) APCLP in human CRC cell lines (HT‐29, HCT116, DLD‐1). We show that overexpression of APCLP decreased the NF‐kB activity, reduced cellular proliferation, migratory ability, as well as anchorage‐independent growth of cells while knockdown of APCLP had an inverse effect. Furthermore, in vivo experiments in a xenograft mouse model confirmed that APCLP overexpression impeded whereas shRNA knockdown promoted tumor growth. To study the mechanism by which APCLP regulated NF‐kB signaling, we conducted co‐Immunoprecipitation experiments and confirmed that APCLP and the major subunit of NF‐kB, p65, may complex or bind directly to each other. Studies are ongoing regarding the mechanism of interaction between APCLP and p65. In summary, discovery of APCLP and understanding of its molecular mechanism and biological function are significant because the knowledge acquired from this study could lead to utilization of APCLP as a potential biomarker and therapeutic target in CRC as well as other cancers that are driven by hyperactivated NF‐kB.

Support or Funding Information

This work is supported by NIH‐NIGMS Grant (# 1R01GM120156‐01A1 to TL), NIH‐NCI Grant (# 1R03 CA223906‐01 to TL), V foundation Kay Yow Cancer Fund (Grant 4486242 to TL), and 100 VOH Grant (# 2987613 to TL).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Celecoxib Impairs Differentiation of Primary Human Skeletal Myoblasts
Authors: Geddis Alyssa , Kolb Alexander , Matheny Ronald
Published: June 2021

Non‐steroidal anti‐inflammatory drugs (NSAIDs) are medications commonly consumed for their anti‐inflammatory, analgesic, and antipyretic effects. NSAIDs act by inhibiting cyclooxygenase (COX) enzymes, which mediate the synthesis of pro‐inflammatory prostaglandins from substrates such as arachidonic acid. Despite the widespread use of NSAIDs, the risks of taking NSAIDs are only beginning to be defined; in particular, their effects on highly metabolic tissues such as skeletal muscle. We therefore sought to determine whether celecoxib, an NSAID selective for COX2, impaired the differentiation and hypertrophy of cultured primary human skeletal myoblasts. Myoblasts were exposed to celecoxib at concentrations from 50 μM – 0.08 μM and allowed to differentiate for 72 hours. Vehicle‐treated myotubes showed a robust fusion index and myotube area as determined by immunocytochemistry and confocal microscopy; however, the fusion indices and areas of myotubes exposed to 50 μM and 25 μM celecoxib were significantly reduced (90% reduced fusion index and 89% reduced myotube area in cells exposed to 50 μM celecoxib; P<0.001, and 33% reduced fusion index and 25% reduced myotube area in cells exposed to 25 μM celecoxib; P<0.001). The total nuclei present across all treatments did not differ. Myoblasts treated with NS‐398 (a COX2‐selective pharmacological inhibitor) showed no impaired differentiation at any concentration. These observations were associated with dose‐dependent decreases in expression of myosin heavy chain mRNA transcripts of slow (Myh7) and developmental (Myh3 and Myh8) myosins, and exposure to 50 μM celecoxib virtually eliminated expression of all myosin heavy chain transcripts (P<0.001 for all). Phosphorylation of AKT (S473), p70S6K (T389), and S6 (S235/236) were dose‐dependently reduced in celecoxib‐treated myotubes (P<0.05), whereas administration of NS‐398 had no effect on phosphorylation of any of these molecules at any concentration. These data reveal that celecoxib inhibits differentiation of primary human skeletal myoblasts, possibly through a mechanism that regulates the activation of molecules involved in protein synthesis. Celecoxib‐mediated impairment of human myoblast differentiation may be independent of its actions on COX2, because treatment with NS‐398 did not alter differentiation. The exact mechanism through which celecoxib acts to alter muscle cell differentiation and physiology remains to be established.

The views expressed in this abstract are those of the authors and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government. This abstract has been approved for public release with unlimited distribution.

Support or Funding Information

US Army Military operational Medicine Research 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.
The Large Tumor Suppressor (LATS) and 14‐3‐3 Regulate Mixed Lineage Kinase 3 (MLK3) Subcellular Localization
Authors: Kasturirangan Srimathi , Chadee Deborah N
Published: June 2021

Mixed Lineage Kinase 3 (MLK3) is a mitogen‐activated protein (MAP) kinase kinase kinase (MAP3K) that activates multiple MAPK signaling pathways in response to cytokines and stress stimuli. MLK3 is important for ovarian cancer cell proliferation and invasion. However, few upstream regulators of MLK3 are known, and MLK3 subcellular localization in ovarian cells is not well understood. LATS (Large Tumor Suppressor), a Ser/Thr kinase, is activated by cell‐cell contact to inhibit proliferation, and also regulates apoptosis and mitotic exit. We observed that confluent ovarian epithelial cells had high levels of phosphorylated, activated LATS (p‐LATS). Furthermore, ovarian cancer cells had substantially lower amounts of p‐LATS in comparison to ovarian epithelial cells. Using confocal microscopy and cell fractionation, we found that MLK3 is both cytoplasmic and nuclear in ovarian cells. Co‐immunoprecipitation assays indicated that LATS interacts with MLK3 and promotes MLK3/14‐3‐3 association and MLK3 cytoplasmic localization; thereby linking the MAPK and Hippo signaling pathways. In conclusion, our results identify LATS as a novel regulator of MLK3 that affects MLK3 subcellular localization in ovarian epithelial cells.

Support or Funding Information

R15‐CA199164

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigation of Differential Post Transcriptional Regulation of 4E‐BP1 and 4E‐BP2 in Eukaryotic Cells Under Stress
Authors: Gobbooru Shruthi , Low Woon‐Kai , Kommaraju Sai Shilpa
Published: June 2021

eIF4E‐binding proteins (eIF4E‐BPs or 4E‐BPs) are a family of proteins that inhibit the initiation of eukaryotic translation. They bind to eIF4E and inhibit the formation of eIF4F complex which is crucial for cap‐dependent translation initiation. There are 3 isotypes of 4E‐BPs, 4E‐BP1, 4E‐BP2 and 4E‐BP3. They are nearly 60% identical at the amino acid level. According to the NCBI reference sequences, 4E‐BP2 has a long 3′ UTR (6.8kb) whereas 4E‐BP1 (428 bp) and 4E‐BP3 (315 bp) do not. The presence of this relatively longer 3′ UTR could indicate the presence of cis‐acting regulatory elements (CREs) that are specific to 4E‐BP2. Under various cellular stress conditions, global translation is shut down to conserve energy. To examine the role of 3′ UTRs in promoting the availability of these 4E‐BPs, during stress, to decrease the cap‐dependent translation, 4E‐BP1 and 4E‐BP2 were both studied in relation to their gene expression (transcription and translation) under various stress conditions (ER stress, oxidative stress), cell cycle arrest and mTOR inhibition (conditions of growth arrest and inhibition, respectively). Under conditions of acute ER stress, 4E‐BP1 showed nearly 3‐fold increased expression than in control treatment (DMSO) whereas 4E‐BP2c and 4E‐BP3 did not show significant increase using qPCR. No changes in 4E‐BP1 protein levels (controls vs various conditions of stress) were detected whereas different 4E‐BP2 protein levels were seen under various conditions of cellular stress. Future studies will include investigation of promoter activity of these three gene products. The data suggests possible differential regulation of 4E‐BPs under cell stress condition.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
FGFR2 and TRPA1 Interaction in Lung Cancer
Authors: Kyriakopoulou Eleni , Gamper Nikita
Published: June 2021

Lung cancer is the leading cause of cancer‐related mortality worldwide. The most common type of lung cancer is lung adenocarcinoma (LUAD) accounting for approximately 40% of all cases. Recent studies unravelled the oncogenic role of the Fibroblast Growth Factor Receptor‐2 (FGFR2) in LUAD. Moreover, it has been shown that the Transient Receptor Potential Ankyrin‐1 (TRPA1), a non‐selective cation channel commonly recognised for its role in somatosensation, is involved in lung malignancies, yet the exact mechanism of this involvement remains unknown. Thus, the aim of this study was to test for potential interaction between FGFR2 and TRPA1 in transfected HEK‐293T cells and LUAD cells as well as to map the binding sites on both proteins. Protein interaction techniques, including immunoprecipitation and Proximity Ligation Assay (PLA) in HEK‐293T cells overexpressing FGFR2 and TRPA1, revealed complex formation between the two proteins. Microscale Thermophoresis (MST) performed on purified proteins indicated direct interaction (Kd = 122.8 ± 23.4 nM) and involvement of the N‐terminal Ankyrin repeat (AR) domain of TPRA1 in the interaction with FGFR2. This result was verified by immunoprecipitation experiments performed on HEK‐293T cells overexpressing FGFR2 and truncated versions of TRPA1. On the other hand, mutations on the C‐terminal proline‐rich motif of FGFR2 pulled down TRPA1 less effectively compared to the wild type C‐terminal domain of FGFR2. These results suggest the direct binding through the AR domain of TRPA1 and the Proline‐rich motif of FGFR2. Complex formation was also shown in a LUAD cell line (HCC‐515) that endogenously expresses both proteins. In conclusion, these results describe for the first time the novel interaction between a tyrosine kinase receptor and an ion channel that could have an effect in signal transduction and subsequently in cancer progression.

Support or Funding Information

University of Leeds, BBSRC

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Does DMD‐10 independently affect levels of GLR‐1?
Authors: Nammour Josette , McGehee Annette
Published: June 2021

We aim to study the mechanisms that affect the Caenorhabditis elegans glutamate receptor GLR‐1. The roles of genes that may be involved in the regulation of GLR‐1 levels are assessed using GLR‐1‐dependent behavioral tests such as spontaneous reversals assays and nose touch assays. Results from our laboratory have shown that dmd‐10 mutants exhibit a low rate of spontaneous reversals when compared to the wildtype strain. We generated rescue strains by cloning DMD‐10 and microinjecting it into dmd‐10 mutant worms to assess the effect of wildtype DMD‐10. The constructs developed for microinjection contained the dmd‐10 gene being driven by either the glr‐1 promoter (Pglr‐1::dmd‐10) or its own promoter (Pdmd‐10::dmd‐10). Pglr‐1 rescue strains, which consist of the dmd‐10 mutant and wildtype DMD‐10 expressed in cells that express GLR‐1, were compared to the dmd‐10 mutant strain and wildtype using behavioral assays. Pglr‐1 rescue strains did not exhibit behavioral differences from dmd‐10 mutant worms. Pdmd‐10 rescue strains have been generated and are currently being tested for behavioral rescue. Additionally, we are developing constructs that will allow us to visualize the location of dmd‐10 neurons in the wildtype worms using the red fluorescent protein, mCherry.

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 potential role of IDA‐1 and neuromodulatory signaling in GLR‐1 regulation
Authors: McLaughlin Shannon
Published: June 2021

In organisms, behavior can be a reflection of environment and nervous system activity. Previous studies have shown that the DAF‐7 signaling pathway in C. elegans is affected by the environment, such as inadequate food supply or high temperature. The DAF‐7 signaling pathway also regulates the glutamate receptor GLR‐1, and increased amounts of GLR‐1 occur in C. elegans strains with mutations in the genes of the DAF‐7 signaling pathway. Additionally, spontaneous reversal behavior depends on GLR‐1 and increased reversals are evident in the daf‐7 mutants. The mechanism by which the DAF‐7 pathway affects GLR‐1 levels is currently unknown. Our research focuses on understanding this mechanism by investigating the role of the candidate gene ida‐1. IDA‐1 was identified because of its involvement in regulating presynaptic neurotransmission and its presence in neurons along the ventral nerve cord (VNC) that express GLR‐1. To test if IDA‐1 was involved in the DAF‐7 pathway regulation of GLR‐1 an assessment of spontaneous reversals in single and double mutants was performed along with fluorescence imaging. Results supported the idea that IDA‐1 plays a role in the DAF‐7 pathway regulation of GLR‐1. To follow up on IDA‐1's potential regulation of GLR‐1 levels, the involvement of IDA‐1 in neuromodulatory signaling was examined. IDA‐1 is normally found on the surface of dense core vesicles (DCV's) and is involved in normal neuropeptide and DCV signaling. To investigate if IDA‐1 is working through the DCV pathway to regulate GLR‐1 levels several strains will be obtained with mutations in various places in the DCV pathway. Future directions will include analysis of these mutants using fluorescence imaging as well as direct protein level analysis through immunoblotting.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Arl4A‐PAK1 complex establishes a positive‐feedback loop contributing to PAK1 activation for cell migration
Authors: Lee Fang‐Jen Scott , Chen Kuan‐Jung , Chiang Tsai‐Chen , Yu Chia‐Jung
Published: June 2021

Cell migration requires coordination of multiple signaling pathways involving in membrane dynamic and cytoskeleton rearrangement. Arf‐like small GTPase Arl4A has been shown to modulate the Golgi organization and actin cytoskeleton remodeling. However, the evidences for Arl4A function in cell migration are insufficient. Here, we report that Arl4A acts with a serine/threonine protein kinase PAK1 to modulate cell migration. We first observe that Arl4A directly interacts with PAK1, and recruits PAK1 to the plasma membrane in a GTP‐dependent manner. Unexpectedly, we found that PAK1 can recruit a cytoplasmic myristoylation‐deficient Arl4A‐G2A mutant, but not GTP‐binding defective Arl4A‐T34N mutant, to the plasma membrane. Thus, these results suggest that there is a positive feedback loop between Arl4A and PAK1, which mutually recruit each other to the plasma membrane. Furthermore, we found that Arl4A promotes cell migration in a GTP‐dependent manner and Arl4A‐PAK1 interaction is critical for Arl4A‐induced cell migration, which is independent of Rac‐PAK1 interaction. Overall, this study shows that Arl4A plays important role for cell migration through direct interaction of PAK1 without effecting Rac1 activation. Our data presented here provide evidence for a novel mechanism for PAK1 regulation and activation.

Support or Funding Information

This work was supported by grants from the NHRI in Taiwan (NHRI‐EX106‐10601B1) to F.‐J. S. Lee

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Immune response mediated by NRLP3 expression in children infected with Respiratory Syncytial Virus
Authors: NETO ERASMO ASSUMPCAO , DE ANGELIS KATIA , DE PAULA VIEIRA RODOLFO , CONSOLIM‐COLOMBO FERNANDA MARCIANO
Published: June 2021

OBJECTIVE

To evaluate the expression of the NRLP3 gene present in the inflammatory pathway and its regulation against pediatric infection with Respiratory Syncytial Virus (RSV)

METHODS

: 31 patients younger than 2 years of age were divided into RSV (n = 11) and control (n = 20) groups. Five cc of venous blood was collected in EDTA tube. The tubes were centrifuged and, after separation of the blood constituents, the buff coat was added and RNA latter Total RNA was isolated. The amount and quality (280/260) of RNA were assessed by reading spectrophotometry at Nanodrop Total sample RNA was retranscribed and the cDNA/reaction was used for relative expression analysis of the NLRP3 inflammation gene by means of gene‐specific type‐specific fluorogenic probes and qPCR. The ExpressionSuite software in the Cloud Platform was used to obtain the raw data (Ct). The GAPDH gene was used as endogenous control for raw data normalization (Ct). The gene expression data were analyzed using the comparative method of Ct

RESULTS

The analysis of the data obtained in the qPCR technique for the expression of the NRLP3 gene was that in the patients infected with Respiratory Syncytial Virus it has a lower expression of it in relation to the control patients. Statistical analysis was performed using GraphPad Prism® software, using t test. presenting (P <0.05) between the groups compared.

CONCLUSION

With the present analysis of the studied study we can observe a lower expression of NRLP3 in pediatric patients infected with RSV in relation to the control patients without infection, within this result we can collaborate with the literature in the description of the behavior of the NRLP3 gene that is not yet well defined as in some articles demonstrate in the bronchial epithelial cells, that when the infection is expected an increase in the expression of the gene and consequently of its products the pro‐inflammatory cytokines and the caspases for induction to cell death. However in our study we have seen that the decrease in NRLP3 expression in bloodstream cells may demonstrate a mechanism of escape of the virus so that the cell does not produce pro‐inflammatory cytokines and caspases for induction of cell death so that it can maintain alive and replicating for longer.

Support or Funding Information

FAPESP

Fold change of expression mRNA

image

Fold change of expression mRNA

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Temporal inhibition of ERK Activity by Optogenetic Control of MAPK Phosphatase 3
Authors: Sharum Savanna R. , Mondal Payel , Cho Kelly , Zhang Kai
Published: June 2021

The functional outcome of the Ras/ERK pathway is encoded by both signal amplitude and temporal kinetics. While a suite of optogenetic strategies allow for precise activation of the Ras/ERK pathway, inhibition of this pathway remains challenging because most negative regulators do not function through subcellular protein translocation or dimerization, features often utilized in common optogenetic strategies. Here, we developed an optogenetic split protein system to accomplish reversible inhibition of ERK activity using the MAPK phosphatase 3 (opto‐sMKP3). Opto‐sMKP3 reversibly inhibits ERK activity under patterned blue light stimulation. Scanning of a 6‐hour pERK inhibition window revealed that equal reduction of PC12 cell differentiation occurs regardless of the placement of the window, indicating that persistent pERK activity is necessary for maximum PC12 cell differentiation. In combination with current optogenetic strategies, opto‐sMKP3 provides an emerging modality to achieve bi‐directional control of the Ras/ERK pathway and helps identify how signaling dynamics encodes functional outcome.

Support or Funding Information

SRS thanks the support of the Westcott Bioscience Fellowship from the Department of Biochemistry at UIUC. KZ thanks the support from the School of Molecular and Cellular Biology at UIUC.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bio‐risk assessment research on genetically engineered cyanobacteria for sustainable biofuels
Authors: Nguyen Thu , Barnes Cherrelle , Sherazi Sana , Agola Jason , Greene Lesley , Lee James W
Published: June 2021

Genetically engineered (GE) cyanobacteria are an attractive photosynthetic platform for sustainable biofuels and bioproducts. A number of biofuels such as ethanol, isobutanol, and 1‐butanol have been experimentally produced using GE cyanobacteria. Even though this approach is promising, there are still questions regarding the biosafety of these GE organisms. What happens to the plasmids that are used for introducing transgenes into the cyanobacterial cells? Studies have shown that E. coli can transfer plasmids into cyanobacteria. We are interested in knowing if the reverse direction is possible? Furthermore, can the transgenes be horizontally transferred into other organisms after they are already integrated into the genomic DNA of the cyanobacteria? These questions need to be addressed before this approach can be commercialized. In order to answer these questions, we conducted an experiment by co‐incubating wild‐type E. coli DH5α with different sets of GE Thermosynechococcus elongatus BP1 carrying either plasmid transgenes or solely integrated transgenes. The transfer event was monitored using selection markers. Our results showed that the transgenes in the form of plasmids were transferred from these GE cyanobacteria into wild‐type E. coli DH5α even without the help of a nitrocellulose membrane while there was no detectable transfer event for the integrated transgenes observed so far. In addition, the frequency of the plasmid transfer event appeared to decrease gradually over time. These results suggested that even though GE cyanobacteria carrying integrated transgenes possess a more permanent change in the genomic DNA, the bio‐risk of these GE organisms in the context of gene sharing is less severe than plasmids‐carrying GE cyanobacteria.

Support or Funding Information

This work is supported by Biotechnology Risk Assessment Grant Program competitive grant award no. 2016‐33522‐25624 from the U.S. Department of Agriculture to JWL and LHG.

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