bluuebunny/crossref_metadata_2025_split huggingface dataset:
Multiplex Imaging of Activity‐Dependent Changes in Neuronal Redox Dynamics Using Compartment‐Specific Redox Probes
Authors: Radhakrishnan Saranya , Norley Jake , Leroy Nathan , Tantama Mathew 
Published: June 2021

Reactive oxygen species (ROS) are integral components of redox signaling in cells and mediate physiologically essential signaling across compartments. Under pathological conditions, excessive presence of ROS can lead to oxidative stress that damages cellular components and leads to cellular dysfunction and death. To understand dynamic redox changes and identify the mechanisms that disrupt redox homeostasis, we need high‐precision tools that can be used to define redox processes in live cells under physiological and pathophysiological conditions.Genetically encoded fluorescent sensors that measure cellular redox status can be targeted to specific subcellular compartments to study the redox changes in intracellular compartments during live‐cell imaging. Reduction‐oxidation‐sensitive green fluorescent proteins (roGFPs) are redox sensors designed by modifying a green fluorescent protein to encode two cysteines engineered proximal to the chromophore, and a change in green fluorescence is observed upon reduction of the disulfide bond formed by the cysteines. However, it has remained technically challenging to quantitatively correlate spatially distinct redox dynamics with subcellular resolution.To address this challenge, we developed genetically‐encoded sensors that can be used along with roGFP to visualize redox changes in multiple compartments within the same cell. Our approach utilized Förster‐type resonance energy transfer (FRET) in a spectral relay strategy that extends the fluorescence emission of the redox‐sensitive green fluorescent protein (roGFP) into red emission wavelengths. We targeted the sensors to mitochondria and cytosol in primary hippocampal neurons and monitored sensor response to oxidizing and reducing agents. Upon neuronal stimulation of the neurons with glutamate, we observed an increase in mitochondrial oxidation accompanied by a cytosolic reduction. Pre‐treatment with antioxidants like MitoTEMPO and N‐Acetyl Cysteine attenuated both the neuron‐activity dependent cytosolic reduction and mitochondrial oxidation implying that these redox changes were coupled. In a mixed culture system, astrocytes are known to have a neuroprotective effect from oxidative insults and glutamate excitotoxicity. We targeted the redox sensors in a neuron‐astrocyte co‐culture system to observe the neuroprotective effect of astrocytes on neuronal redox dynamics. Using this sensor in combination with the roGFP, we can conduct live‐cell imaging with spatio‐temporal accuracy to delineate sub‐cellular changes in redox dynamics that result in pathological oxidative stress.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Increasing Efficiency of Inducing Pluripotency with a Novel Cell Penetrating Peptide‐Adaptor System
Authors: Oja Laura‐Maria , Clearman Kelsey R. , Hudson Martin L. , McMurry Jonathan L. 
Published: June 2021

Somatic stem cells have been used to treat a number of disorders for decades but are limited in their ability to differentiate and self‐renew. Induced pluripotent stem cells (iPSCs) not only have the ability to generate cells of all three germ layers, but bypass the ethical concerns related to embryonic stem cells. They also hold the prospect of utilizing a patient's own cells, thus avoiding host‐vs‐graft issues. In 2006, Takahashi and Yamanaka discovered that expression of only four genes were required to induce pluripotency: Oct4, Sox2, Klf4 and c‐Myc (OSKM). Since then, pluripotency has been induced most effectively by transfection or transduction of these genes. However, efficiencies are low, expression is difficult to control, and off‐target effects are a concern. Inducing pluripotency by exogenous delivery of OSKM proteins using cell‐penetrating peptides (CPPs), while promising, is also inefficient due to of poor penetration and entrapment in endosomes. Our novel CPP‐adaptor system overcomes entrapment by relying on the calcium flux during endosomal trafficking. The adaptor, TAT‐CaM, is a recombinant protein consisting of commonly used CPP, TAT, fused to calmodulin. The protein of interest (the ‘cargo’) has an engineered calmodulin binding site (CBS). TAT‐CaM binds purified CBS‐cargo outside the cell where the Ca2+ concentration is relatively high, but after CPP‐mediated endocytosis, cargo dissociates from TAT‐CaM inside endosomes as calcium concentrations drop, leaving the CPP‐adaptor trapped but cargo free to escape to the cytoplasm and beyond. We have used our adaptor system to deliver submicromolar doses of CBS‐Oct4 protein, inducing pluripotency as measured by expression of stem state markers and alkaline phosphatase staining. CBS‐Sox2 and CBS‐Klf4 have also been delivered and while insufficient to induce pluripotency, play an efficiency enhancing role. While not directly comparable to published results, CPP‐mediated efficiency as measured expression of stem‐state markers is higher than current transfection‐ and transduction‐mediated methods. Parameters such as concentration and timing of doses and molar ratios of OSK to each other and TAT‐CaM affect efficiency. iPSCs created via our CPP‐adaptor system can be cultured, expanded over multiple passages and differentiated into other cell types such as cardiomyocytes. Success in this work will lead to a virus‐ and nucleic acid‐free, nontoxic, tunable method for inducing pluripotency.Support or Funding InformationThis work was supported by NIH grant R15GM120691 and R15NS100632This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Size of protein is a major factor that affects retention on preparative IMAC columns
Authors: ODUNUGA ODUTAYO O , TOVAR CARMEL N 
Published: June 2021

Immobilized metal affinity chromatography (IMAC) is a specific high‐capacity technique used in large‐scale purification of proteins. IMAC exploits the ability of immobilized metal ions to form coordination bonds with atoms in the side chains of certain amino acids. The technique is generally robust. However, several factors still affect column binding capacity, retention, yield and purity of proteins during IMAC. It was observed that the recovery of 6 × histidine, (His)6‐tagged proteins from metal affinity columns differ significantly depending on the size of the protein. To test this observation, we determined the effect of protein size, flow‐rate, number and position of (His)6 tag on the retention of highly expressing proteins on commercial Ni2+ and Co2+ IMAC columns. All experiments were performed in phosphate buffer to eliminate interference of amine‐containing buffers with the binding of the (His)6 tag to the columns. Column retention was determined as the ratio of protein of interest in the supernatant (input) to flow‐through (output). Data obtained suggest that regardless of the flow‐rate, (His)6 tag position and number, the size of protein is a major factor affecting column retention and therefore recovery during column IMAC purification. Small and medium‐sized proteins (~50 kDa) have higher column retention than bigger proteins, resulting in higher recovery. These outcomes provide important information to consider when performing IMAC.Support or Funding InformationWelch Foundation Grant # AN‐0008 SFASU CoSM SURE 2018This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Single Neuron and Microglia Gene Expression Networks Demonstrate Cellular Subphenotype Shifts and Altered Glial‐Neuronal Signaling in Solitary Nucleus during Alcohol Withdrawal: A Time Series
Authors: O'Sullivan Sean J , Park James , Mcintosh‐Clark Damani , Schwaber James S 
Published: June 2021

Alcohol use disorder remains a major societal and healthcare challenge motivating investigation into novel mechanisms of alcohol addiction. Negative‐reinforcement models of addiction postulate that avoidance of negative physical and emotional symptoms during withdrawal drive drug‐seeking. Alcohol withdrawal syndrome is characterized by autonomic dysregulation and negative emotion implicating the strongly connected amygdala and solitary nucleus (NTS) involved in autonomic inputs, outputs, and emotional states in the pathophysiology of alcohol withdrawal. Our previous work demonstrates a neuroinflammatory state in the amygdala during alcohol withdrawal, which we speculate contributes to the negative physical and emotional withdrawal symptoms and consequently drug‐seeking. Here, we investigated the effect of alcohol withdrawal on gene expression in single glucagon‐like peptide 1 (GLP‐1) neurons, adrenergic neurons, and microglia in the NTS. GLP‐1 neurons in this region receive autonomic inputs via vagal afferents and signal to amygdala neurons co‐expressing GLP‐1 receptors and corticotropin‐releasing factor. NTS adrenergic neurons respond to higher‐order inputs to maintain cardiovascular homeostasis. Control, ethanol‐dependent, and rats undergoing ethanol withdrawal at 8, 32, and 176 hour time points were sampled. Single cells were harvested using using laser‐capture microdissection, and microfluidic RT‐qPCR was employed to generate a high‐dimensional dataset. Cellular subphenotypes were characterized and local glial‐neuronal signaling networks were described based on gene networks that defined these subphenotypes. We found an increase in inflammatory subphentoypes in the 8 and 32 hour withdrawal condition with the 176 hour withdrawal condition demonstrating decreased GABA receptor expression. Preproglucagon (Gcg), the GLP‐1 precursor transcript, was found to be upregulated only in alcohol withdrawal conditions. The levels of this transcript opposed levels of the norepinephrine precursor tyrosine hydroxylase transcript suggesting these two signaling ligands have oppositional regulation in alcohol withdrawal.Support or Funding InformationU01HL133360‐01This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Simulating protein and nucleic acid sequence co‐evolution
Authors: Camenares Devin 
Published: June 2021

Protein structures and interactions can be predicted by identifying co‐evolution of residues, both within the same protein or across different proteins and nucleic acids. While conserved residues might represent invariant features of a protein's structure, co‐evolving residue pairs can suggest a sequence‐specific and consequential interaction. Different computational approaches have been used to detect co‐evolution in sequence alignments, but biological data is often used as a benchmark. Such evaluation is important due in part to the confounding presence of indirect co‐evolution. However, using biological data as a benchmark is problematic, because not all the interactions and evolutionary constraints can be known a priori. Instead, the ideal benchmark would be a set of sequences generated with constraints such that they simulate co‐evolution. This was achieved using new cross‐platform, user friendly software tools, which were then used to benchmark and evaluate existing detection methods. This new software can generate sequences in which intramolecular, intermolecular, and indirect co‐evolution can be simulated. Systematic tuning of these evolutionary constraints shed new light on how they drive co‐evolution between residues. Better understanding how to detect co‐evolution and the residue interactions they predict can lead to a wide range of insights important for synthetic biologists interested in engineering new, orthogonal interactions between two proteins or nucleic acids.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Automating the design of structure‐switching aptamer biosensors
Authors: Haider Austin , Bulow Aviva , Bonham Andrew J 
Published: June 2021

For both personalized medicine and academic research, there is pressing need for tools to enable the rapid interrogation of an array of biological targets associated with diseased states. Electrochemical, DNA‐based (E‐DNA) biosensors utilize the dynamic nature of nucleotide polymers to quantify the concentration of a specific peptide, substrate, or heavy‐metal species contained in a biological sample. A critical limitation of E‐DNA biosensors is the complexity associated with designing a nucleotide sequence that maximizes sensitivity while maintaining specificity. This dilemma is further complicated by the vast number of conformations that can exist for a given sequence. While great strides have been made in computationally predicting the folded structures that DNA and RNA‐based biosensors may assume, interpreting whether such predicted structures would be effective biosensors and using that knowledge to design functional E‐DNA biosensors ab initio remains challenging, suggesting the utility of an in silico approach to E‐DNA sensor design. We have created a program called Fealden, which automates the process of aptamer‐based biosensor generation. Fealden utilizes several programmatic paradigms to build an ideal E‐DNA biosensor sequence; the validity of this sequence is then assessed by assigning thermodynamic values and conformational distance metrics to each possible fold. Using these metrics, Fealden can provide the user with several candidates for an E‐DNA aptamer biosensor that are sensitive to the target of interest. Fealden has already been applied to optimize the detection of the antibiotic tobramycin with minimal human interaction. This work provides insights into applying an algorithmic approach to utilize the wide pool of existing aptamers as E‐DNA biosensors in practical applications spanning such diverse fields as food safety, environmental monitoring, and clinical diagnostics.Support or Funding InformationThis 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.
Curcumin Induces Apoptosis by regulating the cell cycle proteins in MDA‐MB‐231 Breast CancerCells
Authors: Ali Mohammed Showkat , Smiley Rebecca 
Published: June 2021

BackgroundCurcumin, a natural polyphenolic compound found in turmeric, has potential cancertreatment efficacy, including apoptosis‐induction, in many cancer cell types. Cancer cells arecharacteristically anti‐apoptotic, highly proliferative andcontinue to live indefinitely. Controlof cell cycle progression and the initiation of apoptosis in breast cancercells is considered to be a potentially effective strategy for the prevention/treatment of the disease. The triple‐negative breast cancer cell line MDA‐MB‐231 does not express progesterone, estrogen, or human epidermal growth factor receptors, is highly aggressive and is difficult to treat. This study aims to elucidate the mechanism of curcumin‐induced apoptosis and its effects on cell cycle proteins in the MDA‐MB‐213 breast cancer cell line.MethodsApoptosis was measured by flow cytometric analysis of AnnexinV/Propidium Iodide expression. Cell cycle arrest was measured by Western Blot analyses of p21Waf‐1, p53 and retinoblastoma protein pRb, as well as the cyclin dependent kinases Cyclin B1, and Cyclin D1.ResultsFlow cytometry data suggest that curcumin induces both early and late stage apoptosis in MDA‐MB‐231 cells. Cell cycle inhibitory protein p21Waf‐1 was significantly increased in curcumin‐treated cells compared to untreated cells. Tumor suppressor protein p53 and Cyclin D1 were significantly down‐regulated (p=.0.0001 and p=0.014, respectively), pRb, another tumors uppressor protein, was unchanged, and Cyclin B1 was upregulated (p=0.0006).ConclusionGiven that upregulation of p21Waf‐1 can lead to apoptosis and can disrupt CDKs/cyclin complexes necessary for cell cycle progression, and that down‐regulation of Cyclin D1, which is required for progression through the G1/S phase and whose over expression is associated with most breast cancers, can lead to cell‐cycle arrest at G1, curcumin appears to be effective in both apoptosis induction, demonstrated also in the flow cytometric results, and in cell‐cycle arrest at G1. The upregulation of cyclinB1, which is involved in the G2/M phase transition, and the down‐regulation ofp53 by curcumin, indicate that additional mechanisms are involved with curcumin exposure. Our findings will aid in the development of future therapeutic strategies, including curcumin as a novel treatment supplement, to effectively induce both cell‐cycle arrest and apoptosis in breast tumors.Disclaimer: The views expressed in this manuscript are those of the author and do not reflect the official policy of the Department of Army, Department of Defense, or U.S. Government.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cell Growth Inhibition and Transglutaminase 2 Expression in Human Erythroleukemia Cells (K562) Following Exposure to Retinoic Acid and Sodium Butyrate
Authors: Spencer Gabi Rose , Birckbichler Paul J 
Published: June 2021

Transglutaminase (TG‐2) is a calcium‐dependent enzyme that has been linked to numerous biological functions including cell adhesion, extracellular matrix, stabilization, wound healing, receptor signaling, cellular proliferation, and cellular motility. This enzyme is also thought to play a role in cancer, as TG‐2 expression is elevated in several types of cancer cells. Some studies have also shown that patients with neurodegenerative diseases like Parkinson's, and Alzheimer's have exhibited elevated levels of TG‐2 which may be linked to the formation of protein aggregates that cause these diseases. The goal of this study was to explore the biological responses of human erythroleukemia cells (K562) to retinoic acid and sodium butyrate. The cells were treated with DMSO (20 uL), sodium butyrate (100 uM), retinoic acid (20 uM), or a combination of sodium butyrate (100 uM) and retinoic acid (20 uM). Preliminary data has shown that TG‐2 is expressed more in cells treated with sodium butyrate, while retinoic acid does not have an effect on TG‐2 expression. The control cells grew continually over the 72‐hour observation period, while the growth of cells treated with sodium butyrate and the mix of both chemicals were slowed to approximately 50% of the growth of control cells. Immunofluorescent photo‐microscopy was used to measure the expression of TG‐2. The cells were also tested for apoptosis using fluorescent microscopy to differentiate between viable, necrotic, and apoptotic cells. Future research will attempt to localize the actual location of the TG‐2 when treated with both chemicals, and if it promotes or suppresses the viability of the cancer cells. Studies are also underway to determine the amount of TG‐2 that is present in each of the samples.Support or Funding InformationFunding provided by Department of Chemistry, Slippery Rock UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Benzimidazole scaffolds as potential anticancer agents: Synthesis and Biological evaluation
Authors: VEMANA HARI PRIYA , Barasa Leonard , Surubhotla Nirupama , Kong Jing , Ha Sin Sil , Palaguachi Christian , Croft John L. , Yoganathan Sabesan , Dukhande Vikas V. 
Published: June 2021

Cancer continues to be one of the leading causes of mortality worldwide. Current anti‐cancer agents have a major limitation of drug resistance. Continued search for newer and safer anticancer drugs is critical and this led us to synthesize a small library of structurally unique benzimidazoles with anticancer potential. Various analogs of these compounds were derived from indole‐2‐carboxylic acid, fatty acids and alpha amino acids, providing a panel of diverse structures. Synthesized compounds were tested in three different cancer cell lines‐HepG2 (human hepatocellular carcinoma), HeLa (human cervical cancer), A549 (human lung carcinoma) and one transformed cell line HEK293 (human embryonic kidney). Many of the compounds tested induced cytotoxicity in a concentration‐dependent manner. Cell viability studies using MTT assay showed that indole and fatty acid based benzimidazoles were most potent followed by amino acid derivatives. Presence of protective functional groups decreased activity of the compounds. In order to investigate the mechanism of the cytotoxicity of the compounds, cleaved caspase 3 expression was measured and a fluorescence‐based apoptosis‐necrosis assay was performed. These results confirmed the presence of apoptosis process in cell death induced by the benzimidazole compounds. Furthermore, cell cycle alterations accompanied the cell death. In addition to anticancer activity, several of these compounds also showed promising antibacterial activity against S. aureus and S. epidermidis. Taken together, our findings demonstrate that the novel benzimidazole compounds could serve as potential leads for the further development of new anticancer agents.Support or Funding InformationFunding for this research was provided by College of Pharmacy and Health Sciences, St John's University, Queens, NYThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identifying novel molecular vulnerabilities to PTK2/FAK inhibition in G α q‐driven uveal melanoma using a kinome‐wide CRISPR/Cas9 screen
Authors: Kishore Ayush , Feng Xiaodong , Arang Nadia , Wu Xingyu , Pachter Jonathan , Schlaepfer David D. , Tamayo Pablo , Cheng Qianming , Gutkind J. Silvio 
Published: June 2021

Uveal melanoma (UM) is the most common primary cancer of the eye in adults. It is diagnosed in about 2,500 adults in the United States every year. Approximately 50% of UM patients develop liver metastasis within 5–10 years after diagnosis, independently of successful treatment of the primary lesions. Activating mutations in GNAQ and GNA11 (encoding GTPase deficient/constitutively active Gαq proteins) were identified in ~90% of UM; where they act as driver oncogenes. MEK inhibition with selumetinib or trametinib has nearly no impact on the overall survival of UM patients. Therefore, there is an urgent need to understand mechanistically how Gαq promotes UM progression in order to develop new therapeutics. In a recent study, we have performed an integrated bioinformatics analysis of gene interaction networks and identified PTK2, encoding the tyrosine kinase: Focal Adhesion Kinase (FAK) as a candidate gene highly predicted to contribute to Gαq‐driven UM progression. Using a variety of biochemical and cell‐based assays, we established that FAK is indeed a pivotal signaling node downstream of oncogenic Gαq that mediates UM proliferation through the activity of the growth‐promoting transcription factor Yap. Notably, our studies with UM xenograft mouse models demonstrated that FAK blockade by VS‐4718, a new generation orally‐bioavailable FAK inhibitor, dramatically reduced tumor volume. The major objective of the current study is to further delineate the Gαq‐FAK signaling axis with interest in identifying novel synthetic lethal interactions to FAK inhibition in UM. To this end, we conducted a kinome‐wide CRISPR/Cas9 screen using VS‐4718 in UM cell line OMM1.5 to pinpoint molecular vulnerabilities in the Gαq‐FAK onco‐circuitry. Positive hits from our gene‐drug screen will be presented, which shed light on the Gαq‐FAK network and may serve as potential co‐targets to FAK inhibition in UM clinical therapy.Support or Funding InformationCancer Biology, Informatics & Omics Training ProgramThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.