bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanistic and Transcriptome‐wide interrogation of eukaryotic translation initiation factor 3 (eIF3)
Authors: Aitken Colin Echeverría , DiNardo Acadia , Yoo Yoon , Stanciu Andrei
Published: June 2021

Translation initiation is the rate‐limiting and most‐regulated phase of translation, and its misregulation has been implicated in myriad maladies, including cancer and neurodegenerative disease. In eukaryotes, initiation is a complex pathway requiring the participation of at least twelve initiation factors (eIFs). Eukaryotic translation initiation factor 3 (eIF3) is the largest and most complex of these factors and appears to participate in events throughout the pathway.

In particular, eIF3 is a pivotal player in the recruitment of mRNA to the translation pre‐initiation complex (PIC). Biochemical and genetic studies have identified interactions of eIF3 with the PIC near the mRNA entry and exit channels of the ribosome; structural studies have revealed that eIF3 binds the solvent face of the small (40S) ribosomal subunit, projecting arms into both mRNA channels and ultimately encircling the PIC.

We recently probed the effects of a library of S. cerevisiae eIF3 functional variants using an in‐vitro reconstituted system. These experiments demonstrated that mutations throughout eIF3 disrupt its interaction with the PIC and diminish its ability to accelerate recruitment of a native yeast mRNA. Using a series of model mRNAs to control the amount of nucleotide sequence in either the mRNA entry or exit channel, we further revealed a critical role for eIF3 in stabilizing mRNA at the exit channel. Together, these experiments are consistent with the entry‐ and exit‐channel arms of eIF3 playing distinct roles during PIC formation, mRNA recruitment, and scanning.

We have also investigated the collaboration of eIF3 with Rps3/uS3 in stabilizing mRNA within the mRNA entry channel. We found that Rps3/uS3 residues previously implicated in the helicase activity of the elongating ribosome stabilize mRNA binding within the ribosomal entry channel and enhance the effects of eIF3 on mRNA binding in both channels. More recently, we have observed that eIF3i and eIF3g contribute to the PIC‐dependent enhancement of eIF4A ATPase activity, which recent work suggests may function to remodel the mRNA entry channel.

Our current work is focused on dissecting the interactions between eIF3, the PIC, and other initiation factors in both the mRNA entry and exit channels, as well as on the inter‐subunit face of the PIC, using both in‐vitro and next‐generation sequencing approaches. In particular, we are exploring the genome‐wide translational effects of depleting or disrupting the eIF3 complex, using ribosome profiling. In parallel, we are investigating the effects of mutations to the 40S protein Rps2 on the PIC:mRNA interaction, as well as how mutations to Rps2 and Rps3 affect the sensitivity of this interaction to mutations within the eIF3 complex.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular Dissection of the Mechanism of Eukaryotic Initiation Factor 3 (eIF3)
Authors: Yoo Kyungyoon , Aitken Colin Echeverría
Published: June 2021

Eukaryotic translation initiation involves a complex series of events culminating in the assembly of a ribosome containing the methionyl initiator tRNA (Met‐tRNA) positioned over the AUG start codon of an mRNA. This process is the rate‐limiting and most regulated step of translation. It begins with the formation of a pre‐initiation complex (PIC) on the small (40S) ribosomal subunit. This PIC is then recruited to the 5′ end of an mRNA, from which it scans in the 5′ to 3′ direction to identify the start (AUG) codon. This convoluted pathway is mediated by multiple protein factors called eukaryotic initiation factors (eIFs). Previous genetic and biochemical studies have shown that eIF3, the largest of these protein factors, participates in events throughout the pathway. However, characterization of eIF3's role has been challenged by this pervasive participation and its complicated architecture: eIF3 is a multisubunit complex containing at least five essential subunits, complicating mutational analysis. To circumvent this problem, we are reconstituting yeast eIF3 by recombinant expression from bacterial strains. Previous structural and biochemical studies have demonstrated the feasibility of this approach. We aim to couple it with an easily manipulatable in vitro reconstituted system to enable systematic dissection of the entire complex. Our initial studies will focus on a set of mutations targeted at understanding the role of eIF3 in mRNA recruitment: truncation of the eIF3c Nterminal domain, which has been seen to promote accurate start‐codon selection through interaction with eIF1; isolation of the eIF3a/c heterodimer that stabilizes mRNA at the ribosomal exit channel; truncation of the eIF3b C‐terminal domain, which promotes the formation of the eIF3b‐g‐i module found near the mRNA entry channel; and truncation of the eIF3b RRM domain. The effects of these mutations on component events during translation initiation will shine new light on the role of eIF3 and its subunits.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cloning of the Escherichia coli methyl‐accepting chemotaxis protein Tar and characterization of nickel repellent interactions
Authors: Coleman Aja Kiana , Healy Frank
Published: June 2021

Sensory systems are vital to organismal survival and are used to interpret and detect the composition of the environment. Microorganisms such as bacteria have evolved sensory systems to detect and respond to their chemical surroundings. Escherichia coli possess transmembrane chemoreceptors known as methyl‐accepting chemotaxis proteins that bind attractant and repellent chemicals, directing swimming behavior. The E. coli chemoreceptor Tar binds the attractant aspartic acid and nickel ion repellent molecules. An important aspect of Tar is that within the periplasmic space, a region between the inner cytoplasmic membrane and the bacterial outer membrane, is a binding domain for aspartate. The binding of aspartate can initiate a stimulus‐response that ultimately affects the cell motility and migration and allows the cell to detect further changes in the concentration of aspartate and respond. Transmembrane biochemical studies have elucidated Tar‐aspartate interactions, however little is known regarding Tar‐nickel interactions. To investigate this, PCR was used to amplify the tar gene from wild‐type E. coli genomic DNA. Amplification products were ligated into the low copy number plasmid vector pACYC184 and the desired E. coli transformants were selected. Defects in nickel repellent sensing of an E. coli Δtar strain were complemented with pACYC184::tar and Tar mutants were screened utilizing a soft agar motility assay. This experimental platform will be used to identify Tar residues required for nickel binding by random mutagenesis of the periplasmic loop region of plasmid‐encoded tar. Nucleotide sequencing and biochemical studies will allow us to identify and characterize the nickel ion repellent binding interaction within the Tar periplasmic region. These results may provide insights into mechanistic differences between attractant and repellent chemosensing and intracellular signaling processes. Additionally, this work may broaden our fundamental understanding of receptor‐ligand interactions.

Support or Funding Information

Ronald E. McNair Scholars 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.
Turnip mosaic virus genome‐linked protein inhibits in vitro activity of ricin A‐chain
Authors: Domashevskiy Artem V , Singh Shaneen
Published: June 2021

Ricin is a plant derived protein toxin produced by the castor bean plant (Ricinus communis). The Centers for Disease Control (CDC) classifies ricin as a Category B biological agent. Currently, there is neither an effective vaccine that can be used to protect against ricin exposure nor a therapeutic to reverse the effects once exposed. Here we quantitatively characterize interactions between catalytic ricin A‐chain (RTA) and a viral genome‐linked protein (VPg) from turnip mosaic virus (TuMV). VPg and its N‐terminal truncated variant, VPg1‐110, bind to RTA and abolish ricin's catalytic depurination of 28S rRNA in vitro and in a cell‐free rabbit reticulocyte translational system. RTA and VPg bind in a 1 to 1 stoichiometric ratio, and their binding affinity increases ten‐fold as temperature elevates (5 °C to 37 °C). RTA‐VPg binary complex formation is enthalpically driven and favored by entropy, resulting in an overall favorable energy, ΔG = −39.8 kJ/mol. Molecular modeling supports our experimental observations and predicts a major contribution of electrostatic interactions, suggesting an allosteric mechanism of downregulation of RTA activity through conformational changes in RTA structure, and/or disruption of binding with the ribosomal stalk. Fluorescence anisotropy studies show that heat affects the rate constant and the activation energy for the RTA‐VPg complex, Ea = −62.1 kJ/mol. The thermodynamic and kinetic findings presented here are an initial lead study with promising results and provides a rational approach for synthesis of therapeutic peptides that successfully eliminate toxicity of ricin, and other cytotoxic RIPs.

Support or Funding Information

National Institutes of Health (NIH) and National Institute of Allergy and Infectious Diseases (NIAID): 1R15AI124044

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression, Purification and Analysis of Recombinant Proteins Responsible for the Functional Properties of a Tough Biological Glue
Authors: Falconer Rebecca , Smith Andrew
Published: June 2021

The slug Arion subfuscus secretes a defensive glue that is very strong despite being composed of 97% water. The overarching goal for analyzing this glue is to guide the development of a biomimetic hydrogel in hopes of replacing staples and stitches in surgical practice. Previous research has identified a group of 11 proteins (all around size 15kDa) that are unique to the glue and are integral for adhesion. The goal of this research was to express and purify recombinant versions of all of these proteins. We then tested whether the proteins could stiffen gels, and whether they formed oligomers. Ligation independent cloning was used to insert cDNA coding for each protein into a plasmid vector. The plasmid contained a 6His‐tag to facilitate purification, and the coding sequence for Protein G to decrease toxicity of the protein to the bacteria. After amplification and purification of the recombinant plasmids, the plasmids were used to transform an expression vector designed to facilitate disulfide bond formation (SHuffleT cells, NEB). After triggering overexpression, the proteins were isolated by binding to nickel resins. All eleven recombinant proteins were successfully purified in this way. Five of the proteins were analyzed. In preliminary results, they had the expected gel‐stiffening activity. Additionally, the recombinant proteins were analyzed by size‐exclusion chromatography to determine the proteins' tendency to oligomerize. The proteins primarily formed dimers at the pH of slug glue, with some trimer formation. Future work will test whether oligomerization is necessary for gel‐stiffening.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating Pal's Interaction with Peptidoglycan
Authors: Lewis Sean D , Liu Xinbei , Ward Zack , LaClair Ciara , Michel Lea Vacca
Published: June 2021

Peptidoglycan associated lipoprotein (Pal) has been shown to exhibit a unique dual orientation in Escherichia coli (E. coli). That is, Pal has two subpopulations, one surface exposed subpopulation and one periplasmic subpopulation. The periplasmic Pal subpopulation binds tightly (but noncovalently) to peptidoglycan. Both subpopulations of Pal are thought to be released from E. coli under certain conditions, and released Pal has been shown to be implicated in the clinical condition of sepsis. This study uses site‐directed mutagenesis and ultracentrifugation to elucidate the detailed structural interaction between Pal and peptidoglycan, with the long‐term goal of understanding the role of that interaction in Pal's release from E. coli. Preliminary data suggest several key residues in Pal dictate the Pal‐peptidoglycan interaction.

Support or Funding Information

Rochester Institute of Technology, College of Science

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Probing the Effect of the Pal‐Peptidoglycan Interaction on Pal Release from Escherichia coli
Authors: Liu Xinbei , Lewis Sean D , Ward Zachary , LaClair Ciara , Michel Lea Vacca
Published: June 2021

Peptidoglycan‐associated lipoprotein, Pal, is inserted into the outer membrane of Escherichia coli (E. coli) and other Gram‐negative bacteria. Under certain conditions, including several animal models of sepsis, Pal is released from E. coli and is thought to be involved in the clinical condition of sepsis. As its name infers, Pal is tethered to the peptidoglycan layer. We propose that the strong noncovalent Pal‐peptidoglycan interaction may have an effect on Pal's ability to be released from E. coli. We tested several site‐directed mutants of Pal with altered affinities to peptidoglycan to determine the effect of those mutations on Pal's release from E. coli. Preliminary results suggest the Pal‐peptidoglycan interaction, alone, does not dictate its release from E. coli.

Support or Funding Information

Rochester Institute of Technology; The Rochester Institute of Technology Research Initiative for Scientific Enhancement (RIT‐RISE)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Elucidation of the Substrate Binding Site of the Yeast Zinc Metalloprotease, Ste24
Authors: Theisen Chelsea , Hsu Erh‐Ting , Wiley Patty , Vervacke Jeffrey , Distefano Mark , Hrycyna Christine
Published: June 2021

The Ste24 zinc metalloprotease from S. cerevisiae is an intriguing integral membrane protease whose precise mechanism of action has yet to be explored fully at a molecular level. This protease is unique in that it recognizes only isoprenylated substrates and performs two distinct cleavage reactions sequentially at structurally unrelated sites in the same substrate molecule, notably the mating pheromone a‐factor in yeast. The recent X‐ray structure of yeast Ste24 revealed unique features that define a novel class of membrane protease that does not resemble any other class of intramembrane proteases described to date. The 7 transmembrane helices of Ste24 form a voluminous water‐filled intramembrane barrel‐shaped chamber that is capped at both ends. Notably, the metalloprotease active site faces the chamber interior so that substrate entry and exit is restricted and must occur through one of four side portals in the protein. Since the structure provided only minimal mechanistic information, we use it to guide our studies to define how the farnesylated yeast mating pheromone a‐factor enters and exits the chamber, how it is recognized, and how Ste24 mediates catalysis. Herein, we aim to identify the residues that comprise the substrate binding site for isoprenylated a‐factor in Ste24. Mutant forms of Ste24 generated by site‐directed mutagenesis were characterized for protease activity by a radioactive coupled methyltransferase‐protease assay and assayed for substrate binding using a photoactivatable analog a‐factor containing a biotin moiety. Substrate binding was assessed by immunoblot analysis using Neutravidin‐HRP. Tandem MS/MS methods are being developed to accurately identify the labeled amino acids in or near the binding site of the labeled proteins. Preliminary results suggest that a region surrounding the active site, lying between two portal sites within the chamber, appear to be important for a‐factor binding for the first C‐terminal cleavage reaction. Further studies will aim to determine residues important for binding for the second proteolytic cleavage reaction.

Support or Funding Information

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.
Probing the Pal‐peptidoglycan interaction
Authors: Ward Zachary , Liu Xinbei , Lewis Sean D , LaClair Ciara , Michel Lea Vacca
Published: June 2021

Sepsis can be caused by a bacterial infection in the human bloodstream, upon which the immune system is over‐activated causing hyper‐inflammation throughout the body. One of the molecules in Escherichia coli (E. coli) that is thought to contribute to the over‐exuberant immune response is peptidoglycan‐associated lipoprotein (PAL). PAL binds tightly to peptidoglycan in the E. coli cell, but has been shown to be released from E. coli under certain conditions. We propose that modulating the PAL‐peptidoglycan interaction would have an effect on E. coli's ability to release PAL. Here, we describe an enzyme linked immunosorbent assay in which we measure and quantify changes to the PAL‐peptidoglycan interaction. Preliminary data suggest we were able to dramatically decrease binding of PAL to peptidoglycan with a single amino acid change in PAL.

Support or Funding Information

Rochester Institute of Technology

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exploring the functional role of Nlrp12's interaction with Hck
Authors: Zhang Yue , Okamoto Curtis
Published: June 2021

The members of the family of Nucleotide‐binding oligomerization domain‐Like Receptor proteins (NLR) are composed of a N‐terminal domain, a central nucleotide‐binding domain, and a C‐terminal ligand‐binding domain. They are generally known to be involved in regulating NF‐kB pathway. A yeast two‐hybrid screen was performed with the pyrin plus nucleotide binding domain of NLR family pyrin domain‐containing12 protein (Nlrp12) as a bait and human leucocyte proteins a prey. It revealed that Hck, a src non‐receptor tyrosine kinase family member was the top hit. Further experiments confirmed that: 1) the C‐terminal 40 amino acids of Hck specifically binds to Nlrp12's pyrin plus nucleotide binding domains; 2) the last 30 amino acids of Hck is sufficient to bind to Nlrp12's pyrin plus nucleotide binding domains, but neither to Nlrp12's pyrin domain alone nor Nlrp12's nucleotide‐binding domain alone; and, 3) the last 40 amino acids of dephosphorylated, but not phosphorylated, Hck preferentially binds to full‐length Nlrp12. Nlrp12 co‐immunoprecipitated with Hck when both are heterologously overexpressed in 293T cells. Using the same system, Nlrp12 and Hck are co‐localized by immunofluorescence. In addition, in Raw 264.7, U937, and THP‐1 cells, endogenous Hck co‐immunoprecipitated with transfected Nlrp12. Since Csk is a tyrosine kinase of members of the src family that specifically binds to the tyrosine negative regulatory residue at the C‐terminal of src family kinases and phosphorylates Hck to inactivate it in mammalian cells, it is hypothesized that Nlrp12 competes with Csk binding to Hck, thereby activating Hck. Since Nlrp12 also inhibits the NF‐kB pathway, an alternative hypothesis is that Hck rescues cells from Nlrp12's inhibition of NF‐kB activity. An NF‐kB luciferase assay confirmed that overexpression of Hck alone does not inhibit NF‐kB, but overexpression of Hck plus Nlrp12 rescued Nlrp12's inhibition of the NF‐kB pathway.

Support or Funding Information

USC School of Pharmacy Mutual Funds 2017–2019

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Coenzyme A Persulfide Prioritizes Sulfide over Butyrate Oxidation
Authors: Landry Aaron P. , Ballou David P. , Banerjee Ruma
Published: June 2021

The human gut microbiota metabolizes fiber remnants from the diet to produce large quantities of butyrate, the primary energy source for colonic epithelial cells. In the mitochondria of these cells, short‐chain acyl‐CoA dehydrogenase (ACADS) is a flavoenzyme that catalyzes the initial step of butyrate oxidation and relays the resulting electrons to the coenzyme Q10 pool. When ACADS is isolated from native or recombinant sources, a vibrant green color is observed in the protein that corresponds to a charge transfer complex between the FAD cofactor and coenzyme A persulfide (CoASSH). The CoASSH‐bound form of ACADS is enzymatically inactive, and previous studies demonstrated that butyrate oxidation is impaired in colonocytes exposed to hydrogen sulfide (H2S), suggesting that CoASSH may be generated under these conditions. H2S is produced at high concentrations by sulfate‐reducing bacteria in the colon, and its toxic accumulation is prevented by sulfide quinone oxidoreductase (SQR), a mitochondrial membrane‐bound flavoenzyme that oxidizes H2S to a persulfide with concomitant reduction of coenzyme Q10. SQR is remarkably promiscuous, and is capable of transferring the sulfane sulfur from H2S oxidation to a number of low molecular weight thiol acceptors. We thus hypothesized that the source of CoASSH, and therefore ACADS inhibition, is H2S oxidation via SQR activity utilizing CoA as an acceptor. We demonstrated that CoA was a competent small molecule sulfur acceptor using the steady‐state SQR assay, while the kinetics of sulfur transfer from H2S to CoA were characterized using stopped flow spectroscopy. CoASSH generated in situ by SQR led to a charge transfer complex in ACADS, providing further evidence that CoASSH is the persulfide product of the SQR reaction. As both ACADS and SQR relay electrons to coenzyme Q10, competition over the coenzyme Q10 substrate would inhibit the effective clearance of the respiratory toxin, H2S. These studies implicate the inhibition of ACADS by CoASSH as a potential modulatory mechanism to prioritize sulfide clearance by reserving the coenzyme Q10 pool for SQR.

Support or Funding Information

This work was supported in part by the National Institutes of Health (GM112455 to Ruma Banerjee and F32GM122357 to Aaron P. Landry).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Probing the SpNOX substrate binding site for NADPH vs. NADH affinity
Authors: Notice Brittany D , Scott James , Gonzalez Victoria , Thomas Sarah , Vermot Annelise , Petit‐Hartlein Isabelle , Fieschi Franck , Smith Susan M.E.
Published: June 2021

NADPH oxidases (NOXes) are a family of eukaryotic enzymes that produce reactive oxygen species (ROS). These transmembrane enzymes move electrons from the substrate NADPH across the membrane to the final electron acceptor O2, forming the superoxide anion O2 −. SpNOX is a recently discovered prokaryotic NOX homolog that, unlike eukaryotic versions, is robust to bacterial expression and is active in detergent solution, making it a good model system for study of the NOX family. Eukaryotic NOXes use NADPH exclusively as a substrate, while SpNOX has similar affinity for either NADH and NADPH. Tyrosine 353 in SpNOX occupies the same position in space as a well‐conserved arginine residue in eukaryotic NOX; by homology to other NADPH binding sites this R should interact with both the adenine ring and the extra phosphate group of NADPH, explaining the eukaryotic NOX preference for NADPH. We have created Y353R and Y353A mutations in SpNOX to probe the effects of this position on the affinity of SpNOX for NADPH vs. NADH. These results will further define the NOX substrate binding site, crucial for drug discovery.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of a Rapid, DNA‐Based Field Test for Detection of Ranavirus, an Emerging Amphibian Disease
Authors: Angel Allyssa , Reyes Patricia , Gubler Ueli , Monsen Kirsten , Hazard Lisa , Goodey Nina
Published: June 2021

A key problem when developing molecular diagnostic techniques is procuring target detection probes. The need of a stable and selective probe is necessary for accurate target identification in disease detection. Antibodies are widely used but they are unstable at ambient temperatures and are expensive and time consuming to develop. The use of aptamers (short ssDNA) as molecular probes provides several advantages over protein‐based methods because they are more stable, easily selected and modifiable. The need of a reliable and stable probe is necessary for accurate detection tests in the field for disease monitoring. Currently, field methods are limited in the time and expertise required to obtain results. Our objective is to develop an aptamer probe with high affinity and specificity for Ranavirus virion particles and recombinant Ranavirus major capsid protein (MCP) through an iterative process known as SELEX. We developed two SELEX methods utilizing either spin concentrators or protein affinity chromatography to successfully enrich a starting library of 1014 non‐specific aptamers to those specific to Ranavirus virion particles and MCP, respectively. After 6 rounds of selection, we have isolated 24 potential Ranavirus probes and subsequently cloned into a TOPO_BluntII vector for sequencing. Aptamer sequences will be analyzed for common structural motifs between virion specific aptamers and MCP specific aptamers, to identify possible common binding sites for these two respective targets. The binding affinities of selected aptamer probes will be measured by Surface Plasma Resonance (SPR) using a streptavidin modified biosensor chip and biotinylated aptamers. We expect binding affinities of aptamers to Ranavirus targets to be within the nanomolar range, demonstrating the aptamers high affinity and specificity. Ultimately, the selected aptamers will be incorporated as molecular probes for Ranavirus into a field usable rapid pathogen test that will surpass the limitations of current field tests.

Support or Funding Information

The New Jersey Water Resources Research Institute (NJWRRI) The PSEG Institute for Sustainability Studies

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Electrochemical DNA Biosensors for Detection of Mannose‐capped Lipoarabinomannan
Authors: Sodia Tyler , Bonham Andrew J
Published: June 2021

Mycobacterium Tuberculosis (TB) is one of the world's most prevalent bacterial pathogens. It is estimated that almost 10 million cases of TB emerge every year, and roughly one‐fifth of these cases are fatal. The current detection and diagnosis of TB is done primarily via two methods; the TB skin test and the TB blood tests. Neither of these tests can differentiate between latent TB infection and TB disease. In order to differentiate these states, time‐consuming sputum tests are required, which rely on culturing the mycobacterium. Designing a sensitive serologic biosensor would dramatically decrease the time line of diagnosis and therefore improve patient outcomes. One possible avenue for improved detection lies in the cell wall of TB, which includes many complex glycolipids—many of which are believed to have immunopathogenic mechanisms in physiologic pathways. Mannose‐capped lipoarabinomannan (ManLAM) is one of the most prevalent of these glycolipids, and presents a novel target as a bio‐marker for the sensitive detection of TB and related Mycobacterium strains. Here, we have utilized an existing aptamer sequence that binds to ManLAM to generate a sensitive electrochemical, DNA‐based biosensor for the detection of TB. This biosensor is able to adopt multiple different folded conformations, only one of which presents the core aptamer sequence in a state capable of binding ManLAM. An appended redox‐active tag (methylene blue) generates a measurable difference in electrochemical current upon this conformational change, providing a sensitive and quantitative measurement of ManLAM concentration. Such biosensors may ultimately allow rapid, on site, diagnosis of TB infection within the time constraints of patient‐doctor interaction.

Support or Funding Information

This work is supported by internal grant funding from the Metropolitan State University of Denver.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phage Display Selection and Identification of Novel Pancreatic Cancer Targeting Peptides
Authors: Asar Mallika , Soendergaard Mette
Published: June 2021

Pancreatic ductal adenocarcinoma (PDA) is currently the fourth leading cause of cancer deaths in the U.S. It has been recently estimated that the disease causes approximately 30,000 deaths per year in the U.S., and that pancreatic cancer patients exhibit a 5‐year survival rate of only 3%. The high lethality of pancreatic cancer is due to the fact that most cases are diagnosed at advanced metastatic stages. While there are diagnostic methods in use clinically, most are inadequate, potentially leading to delayed diagnosis and further spreading of the cancer prior to treatment. Due to insufficient diagnostic techniques, it is imperative to develop new methods, which may facilitate early diagnosis. One such modality employs the use of peptides with high binding affinity and specificity to a pancreatic cancer biomarker.

Bacteriophage (phage) display technology is a high throughput method for discovering peptides that confer specific binding properties to a single molecular target. The peptides are typically displayed on the distal end of coat protein III of the Fd filamentous phage. Here we report using phage display technology to select peptides with specific binding to human pancreatic cancer cells. To do so, a naïve fUSE5 15‐mer library (kind gift from Dr. George Smith) was negatively selected against normal pancreatic ductal cells (hTERT‐hPNE). Unbound cells were collected, amplified by infection of E. coli K91BK overnight, and isolated using polyethylene glycol (PEG), NaCl precipitation and centrifugation. Amplified phage were then subjected to four rounds of positive selection against pancreatic cancer cells (Mia‐Paca‐2) grown to 80–90% confluency. Between each round of selection, phage were collected, amplified in E. coli K91BK and isolated as described above. Stringency was increased in the fourth round of selection by performing the selection against cells grown to 60% confluency. After the final round of selection, phage DNA was extracted and polymerase chain reaction (PCR) amplicons were identified by paired‐end (2×150bp) next generation sequencing (MiSeq) followed by translation and sorting of the sequences (Genewiz, NJ). The “top” 10 peptides identified by this method will next be screened in cell binding assays to determine individual binding constants. Peptides that exhibit high binding affinity and specificity for Mia‐Paca‐2 cells may subsequently be employed as probes in the detection of pancreatic cancer.

Support or Funding Information

Western Illinois University Research Council Grant

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Metabolomic Responses to Burn Injury in a Rodent Model
Authors: Alkhalil Abdulnaser , Ball Robert , Day Anna , Kumar Raina , Hammamieh Rasha , Garg Guarav , Moffatt Lauren T , Shupp Jeffrey W
Published: June 2021

Management of total body surface area (TBSA) burn injuries remains clinically challenging and is an understudied area. In order to explore the role of supplementary nutrient interventions and supportive tissue building component administration in the clinic to improve wound healing and patient survivability, we assessed metabolomic of a rodent model for burn injury of 30% TBSA. Nine mice were subjected to a 30% TBSA by submersion in 100 °C water. Mice sera or skin biopsies (4 mm) were taken on euthanasia at hour 2, 6, and 24 after injury (n=3 at each time point). An equal number of mice underwent sham procedures and biopsy collection for each time point with similar administration of anesthetic and fluid resuscitation. Samples were analyzed by ultra‐performance liquid chromatography‐quadrupole time‐of‐flight mass spectrometry (UPLCQ‐TOF‐MS). Multivariate analysis, the principal components analysis (PCA), was calculated using stats and pathway analysis using metaboanalyst R packages to analyze metabolic changes. Large changes in metabolite presence were detected in sham treated animals in both specimen types relative to baseline. These changes are caused by animal preparation steps and anesthesia were characterized by comparative analysis of results of specimens from thermally challenged animals relative to either baseline or to sham of the same time point across the study to identify burn associated changes. Metabolite responses in serum were more rapid than changes in skin, and while burn caused larger changes during early time points (1 and 2 hours post injury) in positively charged metabolites relative to negatively charged ones in serum samples, and to a lesser degree in sham treated skin samples, no difference was seen in burned skin samples. Pathway enrichment analysis of metabolites common to all time points from skin identified glycerophospholipid, ascorbate and aldarate, and phenylalanine metabolism pathways as the top three affected pathways. In serum, arachidoinc acid, starch and sucrose, and galactose metabolism were the top three pathways. These results underscore the differences in the kinetics and specimen‐dependent nature of metabolome responses.

Support or Funding Information

None

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 Fucosyltransferase Activity via MSn
Authors: Ashline David , Reinhold Vernon
Published: June 2021

Fucosyltransferases add fucose residues to oligosaccharides. Resulting fucosylated epitopes include the common core fucosylation of N‐glycans, as well as important ligands such as Lewis structures and H antigens. Many of these structural motifs can be found on N‐linked glycans, O‐linked glycans, and glycosphingolipids. In complex biological samples, multiple fucosyltransferases, as well as multiple substrate specificities of individual fucosyltransferases, can result in multiple isomeric forms within individual glycan compositions. Intact mass analysis alone will not distinguish these isomers. Chromatographic separation may or may not resolve these structures, and, will not provide direct structure identification.

Typically, MS/MS analysis of fucosylated N‐linked glycans can distinguish core fucosylation from antennal fucosylation. Finer details such as distinguishing Lewis structures from H antigens is a more difficult problem, as fucosylated lactosamine antennae fragments are produced from both structures. Disassembling these structural fragments to a further stage, MS3, in an ion trap, can easily distinguish Lewis from H antigen structures, providing direct empirical evidence of structure.

We demonstrate MSn data from a variety of samples. Fucosyltransferase activity was assessed through transfection of fucosyltransferase (3,4,5,6,7, and 9) genes into mesenchymal stem cells, with comparison to non‐transfected cells as a control. MCF‐7 and MDA‐MB‐231 cultured cells were also interrogated for fucosylation isomers. Glycophorin C was also interrogated, with fucosylation on polylactosamine structures.

For cultured cells, glycolipids were extracted via solvent extraction. Proteins were enzymatically digested, followed by enzymatic release of N‐linked glycans. Released N‐glycans were separated from O‐glycopeptides; O‐glycans were released by reductive b‐elimination. Purified glycan and glycosphingolipid fractions were permethylated. Glycan samples were directly infused into the mass spectrometer via nano‐ESI. Precursor ion selection was made manually.

Fucosyltransferase‐transfected mesenchymal stem cells were interrogated for synthesis of Lewis X structures, with data shown to indicate that different fucosyltransferases show differing preferences for synthesizing Lewis X vs. sialylated Lewis X.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Examining the Accuracy of Monosaccharide Structures Calculated with Density Functional Theory by Experimental Comparisons
Authors: Ziperman Emily D. , Calixte Emvia I. , McCutcheon Meg , Pulipaka Srinivas , Gallagher Elyssia S.
Published: June 2021

Glycans are prominent in biological processes. Glycan structural characterizations can have significant implications for disease progression, e.g. cancer metastasis.1 Glycans are challenging to analyze with traditional chemical techniques due to a lack in diversity of functional groups; thus, computational modeling of glycans can help bridge this gap in knowledge regarding glycan structures. However, it is necessary that the computational models adequately represent carbohydrate structures and align with experimental results. Experimentally, mass spectrometry (MS) is one technique that is used for glycan characterization. In MS analyses, glycans are often analyzed as ion adducts. A group of computational methods and basis sets have been used to compare the energies of a subset of monosaccharides2, but this work has not been expanded to the ion adducts detected with MS, nor has it included experimental comparison. Herein, we optimized the geometry of both anomers of fucose and glucose in neutral, protonated, ammoniated, and sodiated forms using Density Functional Theory with the B3LYP or B3PW91 methods and 15 basis sets. Three basis sets (cc‐pVQZ, GEN, and GENECP) were unsuccessful in running calculations to completion. For the remaining computations, we examined values from the output files for variations between methods and basis sets. If computational methods yield accurate structures, the calculated values for molecular parameters, e.g. dipole moment, should be similar. Yet, if there are incorrect approximations in the basis set, the calculated electron density around the molecule will be incorrect and alter the values of the parameters using that specific functional. Greater variation occurred in the average dipole moments calculated for all methods and basis sets of fucose (standard deviation (SD) = 1.5) compared to glucose (SD = 0.68). The addition of ion adducts increased error in dipole moment calculations compared to the neutral molecules. The variation in these results suggests that some methods and basis sets are not reliable when modelling carbohydrates. Currently, molecular dynamics runs using GROMACS are being done to obtain over 50 different coordinate sets of each system for input into MOBCAL.3 MOBCAL will be used to obtain theoretical collisional cross sections (CCS) which will be compared with experimentally measured CCS by ion mobility MS. These results will illustrate which computational methods yield accurate carbohydrate structures, allowing future application of this work to address the biological utility of glycans.

Support or Funding Information

Baylor University Office of the Vice Provost for Research and the Undergraduate Research and Scholarly Achievement Small 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.
Unveiling the Mechanotransduction Mechanism of Substrate Stiffness‐modulated Cancer Cell Motility via ROCK1 and ROCK2 Differentially Regulated Manner
Authors: Peng Yueting , Chen Yu , Qin Xiang , Li Shun , Liu Yiyao
Published: June 2021

In addition to soluble factors and stromal cells, the mechanical properties of extracellular matrix (ECM) are considered to be an important component of tumor microenvironment. In particular, substrate stiffness is a mechanical characteristic of the ECM by which certain anchorage cells sense and respond to a variety of cellular behaviors. At the same time, clinical studies have observed that substrate stiffness was increased during tumorigenesis, which could promote tumor malignant transformation and metastasis. However, the biomechanical behavior of tumor cells and the underlying mechanotransduction pathways remain unclear. Here, using stiffness‐regulable polyacrylamide (PAA) substrates to simulate the tissue stiffness at different progress stages of breast cancer in vitro, we found that moderate substrate stiffness induced the emergence of malignant phenotypes and further unregulated breast cancer cell motility. Subcellular structure observation showed that moderate substrate stiffness promote the formation of leading‐edge protrusion by accelerating focal adhesion maturation and polarizing intracellular traction force distribution. Moreover, the substrate stiffness directly activated integrin β1 and focal adhesion kinase (FAK), which accelerate focal adhesion (FA) maturation and induce the downstream cascades of intracellular signals of the RhoA/ROCK pathway. Interestingly, the differential regulatory mechanism between two ROCK isoforms (ROCK1 and ROCK2) in cell motility and mechanotransduction was clearly identified. ROCK1 phosphorylated the myosin‐regulatory light chain (MRLC) and facilitated the generation of traction force, while ROCK2 phosphorylated cofilin and regulated the cytoskeletal remodeling by suppressing F‐actin depolymerization. The ROCK isoforms differentially regulated the pathways of RhoA/ROCK1/p‐MLC and RhoA/ROCK2/p‐cofilin in a coordinate fashion to modulate breast cancer cell motility in a substrate stiffness‐dependent manner via integrin β1‐activated FAK signaling. Our findings provide new insights into the mechanisms of matrix mechanical property‐induced cancer cell migration and malignant behaviors. Taming these physical forces by implanting scaffolds with specific stiffness or by inhibiting specific ROCK isoforms could have potential implications for improving therapeutic outcomes in cancer.

Support or Funding Information

This work was supported, in part or in whole, by the National Natural Science Foundation of China (11772088, 31470906, 31700811, 11802056, and 31800780).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Function of Yeast Bax Inhibitor BXI1 in Redox and Calcium Homeostasis of the Endoplasmic Reticulum and Programmed Cell Death in Saccharomyces cerevisiae
Authors: Jacob Walter , Alisch Joseph , McDonough Liam , Abaherah Ibrahim , Andrews Nick , Bittner Michael , Raffa Amanda , Kalhorn Jack , Austriaco Nicanor
Published: June 2021

Yeast Bax Inhibitor‐1 (BXI1/YBH3) encodes a protein (Bxi1p) that belongs to the Bax Inhibitor family of proteins. In mammals, this family of proteins has cytoprotective properties that are most evident in paradigms of endoplasmic reticulum (ER) stress and have been linked to several human cancers. Both the crystal structure of a prokaryotic member of the family (BsYetJ) and our own published studies suggest that yeast Bxi1p is a pH‐sensitive calcium leak channel localized to the ER membrane. It is involved in the unfolded protein response (UPR). Bxi1p is thought to act via a mechanism involving altered calcium dynamics. We now show that cells lacking BXI1 have an altered redox microenvironment in the ER. We also report that cells lacking BXI1 accumulate more calcium in the ER than wild type cells. Together, our data suggest that Bxi1p is involved in regulating the ER microenvironment and, consequently, programmed cell death.

Support or Funding Information

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

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interplay of reactive oxygen species, apoptosis and necroptosis in atypical cell death of Tak1‐deficient macrophages and mouse mortality
Authors: Lopez‐Perez Wilfred , Sakamachi Yosuke , Sai Kazuhito , Mihaly September , Takaesu Giichi , Ninomiya‐Tsuji Jun
Published: June 2021

TAK1 is an intracellular signaling intermediate of TNF signaling pathway, in which it mediates inflammatory responses and plays a critical role in blocking TNF‐induced cell death. Tak1‐deficiency makes most cell types hyper‐sensitive to TNF‐induced apoptosis, which is mediated by caspase 8 with typical apoptotic features, e.g. caspase 3 activation and membrane blebbing. On the contrary, Tak1‐deficienct macrophages die with features closer to necrosis accompanied with marginal activation of caspases. TNF‐induced necrosis, known as necroptosis, is mediated by a protein kinase complex within which receptor interacted protein kinase 3 (RIPK3) plays a critical role. However, Tak1‐deficient macrophages only marginally activates RIPK3 pathway, and their cell death was not rescued by deletion of Ripk3. In the current study, we attempted to define the mechanism of this atypical Tak1‐deficient macrophage death. We found that mitochondria‐derived reactive oxygen species (ROS) were highly upregulated by ablation of TAK1 in macrophages. Unexpectedly, even though they lack typical apoptosis and necroptosis features, we found that double but not any single deletion of caspase 8 and RIPK3 largely abolished elevation of ROS and cell death in Tak1‐deficient macrophages. Interestingly, blockade of ROS diminished Tak1 deficiency‐induced marginal activation of the caspase and RIPK3 pathways. These demonstrate that ablation of TAK1 elicits a feedforward loop involving caspase 8, RIPK3 and mitochondrial ROS leading to atypical macrophage death. We also characterized mice having Casp8, Ripk3 and Tak1 gene deletion in the in vivo settings. Tak1 deletion caused animal mortality within several days with tissue injuries in the liver and the intestine. Compound double deletion of Casp8 and Ripk3 prolonged Tak1‐deficient animal life for an additional several days, but was insufficient for complete rescue. Thus, caspase 8 and RIPK3‐independent pathway(s) are also involved in Tak1‐deficient animal mortality.

Support or Funding Information

This work was supported by National Institutes of Health Grant GM068812 and GM112986 (J.N‐T). Y.S. and W.L‐P were partly supported by National Institutes of Health Training Grant T32 ES007046.

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