bluuebunny/crossref_metadata_2025_split huggingface dataset:
Optimization of a Bacterial Three‐Hybrid Assay for RNA‐Protein Interactions
Authors: Hegner Courtney L , Wang Clara , Mansky Rachel , Berry Katherine E 
Published: June 2021

Non‐coding RNAs regulate gene expression in every domain of life. In bacteria, non‐coding small RNAs (sRNAs) regulate gene expression in response to stress and are often assisted by protein chaperones. While much is known about the mechanism of a few model RNA chaperone proteins, significant gaps remain in our understanding of how other bacterial RNA‐binding proteins interact with RNA to regulate gene expression. While there are numerous biochemical methods to study bacterial RNA‐protein interactions, new genetic methodologies would be a powerful complement to current tools. We have recently developed a bacterial three‐hybrid (B3H) assay that detects the binding of E. coli sRNAs with multiple RNA chaperone proteins. This assay couples the transcription of a genetic reporter to the interaction of a DNA‐bound “bait” RNA (e.g. an sRNA) and an RNAP‐fused “prey” protein (e.g. Hfq). Successful interaction between RNA bait and protein prey stabilizes RNA polymerase (RNAP) at a test promoter and activates reporter gene expression. This B3H assay offers a straightforward path to identify and interrogate mutations in these RNA‐binding proteins with molecular phenotypes of interest. Currently, only ~40% of known sRNA‐chaperone interactions are detectable in the system. In order to maximize the utility of our bacterial three‐hybrid assay, a larger proportion of known RNA‐protein interactions must be successfully detectable. Current efforts are focused on optimizing the signal of the B3H assay so that weaker RNA‐protein interactions can be reliably detected. Here, we show results from our efforts to improve the assay's signal‐to‐noise ratio by tuning the sequences, spacing, and the concentrations of the B3H assay components. We have observed an improvement in the assay's signal in response to systematic alterations of the assay components. With an optimized B3H assay, we aim to discover new sRNA‐binding proteins and uncover the regulatory roles sRNAs and these proteins play in biology.Support or Funding InformationMount Holyoke College and the Luce FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Binding affinity of TWIST2 wild type and mutant proteins and their potential role in the Barber‐Say Syndrome
Authors: Crespo‐Hernández Noe , Torres‐Bracero Alexandra , Rodriguez‐Pou Yesenia , Cadilla Carmen L 
Published: June 2021

Barber Say Syndrome (BSS) is a rare autosomal dominant disease, classified as an ectodermal dysplasia, affecting the eyelids and skin as well as exhibiting dysmorphic facial features. BSS exhibits variable phenotypes. Molecular analysis of patients with BSS identified mutations in the TWIST2 gene, involving amino acid substitutions of Glutamate 75 to Glutamine (E75Q) or Alanine (E75A).TWIST2 is a transcription factor of the bHLH superfamily, belonging to the class II group. Glutamate 75 is a highly conserved amino acid in the basic region of bHLH proteins, which is responsible for nucleotide binding in both class I and II groups. Both the I and II bHLH classes are known for their specificity towards canonical E‐box (CANNTG) DNA response elements. The E75Q and E75A mutations have been suggested to alter the DNA‐binding activity of TWIST2, leading to both dominant‐negative and gain‐of‐function effects. In this study, we expressed His‐Tagged TWIST2, and the E75Q and E75A mutant proteins, in E. coli plysBL21 cells. Subsequently, proteins were purified by affinity chromatography and yields between 1.2 to 1.7 mg/L of pure recombinant protein were obtained from 2 liter cultures. Protein affinities towards known E‐boxes in the human TNFa, MCK1 and POSTN genes were measured using Biolayer Interferometry technology. TWIST2 wild type equilibrium constants were in the range of 0.003573 to 0.7929 uM. The equilibrium constants of the E75Q and E75A mutants were three to six order of magnitude higher when compared to the wild type protein. Therefore, the TWIST2 wild type protein showed higher binding affinities towards double‐stranded oligonucleotides containing E‐boxes, while the pathological variants found in BSS patients showed lower affinities to the same oligonucleotides. These results suggest that basic region variants of the TWIST2 glutamate 75 may cause gene dysregulation by their reduced capacity to bind their cognate DNA response elements.Support or Funding InformationThese studies were partially supported by grants U54MD007600 (NIMHD RCMI) and R24GM061838 (NIGMS 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.
Hydrogen Sulfide (H2S) Inhibits Insulin Amyloid Fibril Formation
Authors: Torres Alani Aldarondo , Rosario Alomar Manuel F. , Garriga Juan López , Lednev Igor K. 
Published: June 2021

Insulin from bovine pancreas (IBP) is one of about thirty human proteins that form amyloid fibrils. These amyloid fibrils obtain a b‐sheet structure and assemble into insoluble fibrils, causing many amyloidosis diseases. Amyloidosis caused by insulin protein is an acquired systemic disease. Developing procedures to eradicate amyloid fibril precursors is key to detaining amyloid fibril formation. To explore amyloid inhibition, native insulin protein was exposed to different concentrations of H2S, and protein unfolding was induced. Atomic Force Microscopy (AFM) imaging evidenced overlaying spherical structures, as the concentration of H2S increased in each sample as opposed to the fibrillary structures in the control sample. 3D AFM contour plots of the overlaying spheres due to protein‐H2S interaction show smoother peaks of greater height contrary to the sharp smaller peaks of the insulin amyloid fibrils. Results from Far UV‐Circular Dichroism spectra suggest that insulin protein in presence of H2S conserves its native insulin structure. These results are independent of pH value and are observable at physiological pH. Deep Ultraviolet Resonance Raman Spectroscopy further explains protein‐H2S interaction suggesting the formation of trisulfide bridges in insulin‐H2S sphere structures. Results suggest H2S inhibits insulin amyloid fibril formation by detaining the development of insulin fibril precursors.Support or Funding InformationRISE‐E‐BASE Program UPRMNIH Grant Number: 1R25GM127191‐01This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of a Naturally‐occurring Heparin‐binding Peptide Preferentially Targeting the Nucleolus
Authors: Loo Shining , Kam Antony , Tam James P 
Published: June 2021

Organelle‐specific, cell‐penetrating peptides are useful for targeting intracellular proteins. Recent results showed that certain cationic cysteine‐rich peptides (CRPs) can target intracellular proteins, and hold promises for intracellular drug delivery [1]. Previously, we identified a family of CRPs, designated as bleogens, from Pereskia bleo, a medicinal paint found in South‐east Asia [2]. Our results showed that the prototypic bleogen pB1 is a heparin‐binding, anti‐microbial peptide with the potentials of intracellular targeting. Here we report that bleogen pB1 is an organelle‐specific cell‐penetrating peptide that targets the nucleolus. The linear precursor of bleogen pB1 was prepared using a stepwise solid‐phase synthesis and oxidative folded to its native state. We confirmed the purity and integrity of the synthetic bleogen pB1 using reversed‐phase high‐performance liquid chromatography, heparin‐affinity chromatography, and nuclear magnetic resonance spectroscopy. TAMRA‐pB1 was prepared using on‐resin TAMRA labeling at the N‐terminus of bleogen pB1. We demonstrated that TAMRA‐pB1 internalized into living cells via endocytosis using flow cytometry and live‐cell confocal microscopy. The cellular uptake of TAMRA‐pB1 was found to be dependent on glycosaminoglycan expressions. Upon cell entry, TAMRA‐pB1 preferentially targets the nucleus and eventually accumulates in the nucleolus. Taken together, this study highlights the discovery of bleogen pB1 as the first naturally‐occurring cell‐penetrating nucleolus‐targeting CRP. The ability of bleogen pB1 for specific delivery and accumulation of drugs in the nucleolus can be useful to address the problem of efflux‐mediated drug resistance encountered during cancer chemotherapy.Support or Funding InformationThis research was supported in part by Nanyang Technological University Internal Funding ‐ Synzymes and Natural Products (SYNC) and the AcRF Tier 3 funding (MOE2016‐T3‐1‐003).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Antimicrobial VBT:TMQ:DMHDQ (1‐(4‐vinylbenzyl) thymine, vinylbenzyl trimethyl QUAT, and vinylbenzyl dimethylhexadecyl QUAT) terpolymer permanence and performance on hospital scrubs
Authors: Baulsir Elaine , Laemmle Ruby , Jaber Aliya , Yandian Megan , Gurney Richard 
Published: June 2021

Due to the prevalence of nosocomial infections, the need for enhanced precaution between health care provider and patient has increased. In attempt to eliminate scrubs as a transmission vector, this project focused on the methodology behind the integrity of known antibacterial terpolymers on scrub fabric. Terpolymer function of immobilization, water solubility, and antibacterial activity was tested through manipulation of the ratios of 1‐(4‐vinylbenzyl) thymine (VBT), vinylbenzyl trimethyl QUAT (TMQ), and vinylbenzyl dimethylhexadecyl QUAT (DMHDQ), respectively. To begin, solubility testing of varying ratios of VBT: TMQ: DMHDQ was conducted to assess polymeric ability to dissolve in solution for ease in application. This ease in application allows for versatility in use beyond nursing scrubs in less porous surfaces such as light switches and phone screens. In addition to variation in monomer ratios, soaking and pre‐treatment mordants were examined. Electrostatic attraction of an anionic dye, trypan blue, to the polymer on treated scrubs was used for quantification of terpolymer permanence. In a period of time that the toxicology of commercially available unbound quaternary ammonium compounds (QAC) is highly scrutinized, this method addresses the growing concerns associated with excess use of cleaning solutions in attempt to prevent infection. The permanence testing of this terpolymer will determine if this method may be used in application to prevent the spread and increased resistance of bacterium in healthcare settings.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Optimization of Protein Extraction and Identification from the European Green Crab
Authors: Wrobel Neal , Dachel Gabriel , Shipman Richard , Myracle Angela D. , Grant Jennifer E. 
Published: June 2021

Carcinus maenas, or European green crab, is an invasive species off the coast of Maine frequently trapped alongside more desirable crab species, but is too small to provide economic benefits to fishermen. Currently, Marine organisms are widely recognized as intriguing potential sources of peptide antibiotics. Indeed, shells from the green crab may provide an untapped source of protein, potentially providing useful leads for pharmaceutical applications. However, crab shells prose a unique purification problem, in that the hard shells that are rich in hydroxyapatite must first be dissolved. Furthermore, calcium and phosphate must be separated from soluble proteins lest purification strategies result in precipitation of protein‐laden calcium deposits. Here we discuss the optimization of protein extraction protocols, as evidenced by use of the Bradford protein assay and SDS‐PAGE gel analysis. The ultimate goal of this work is to identify novel proteins in these shells by mass spectrometry.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identifying ErbB Proteins as Potential Receptors for Nephronectin during Mouse Corneal Development
Authors: Ponce Aiyana , Ma Justin , Lwigale Peter 
Published: June 2021

Corneal defects are a major cause of blindness, but treatment options remain limited. Advancements require improved understanding of molecular processes involved in corneal development. Corneal development is an intricate process that involves migration, proliferation, and differentiation of embryonic neural stem cells known as neural crest cells. However, very little is known about the molecular mechanisms involved in these processes. Our recent studies identified Nephronectin, an extracellular matrix protein, in the developing cornea. The current study is to determine whether Nephronectin signals through ErbB proteins (ErbB1, ErbB2, ErbB3) during corneal development. To test the hypothesis that neural crest cells co‐localize and interact with nephronectin through ErbB proteins during corneal development, we first isolated cDNA from corneal tissues, then identified the expression of the ErbB genes by reverse transcription polymerase chain reaction (RT‐PCR), and finally determined their localization by in situ hybridization. Our initial analysis by RT‐PCR indicates that ErbB1 is highly expressed during corneal development. Next, we synthesized RNA probes using a cloning vector to analyze their spatial expression on paraffin‐embedded embryonic corneal sections. Our preliminary analyses showed that ErbB1 is expressed in the presumptive corneal stroma and epithelium, suggesting a potential role of Nephronectin/ErbB1 signaling during cell migration, proliferation, or differentiation into corneal cells. Additionally, ErbB2 and ERbB3 are expressed in the epithelium, indicating a role in proliferation. Identifying signaling pathways that govern corneal development can lead to better strategies to repair corneal defects.Support or Funding InformationResearch reported in this abstract was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under linked Award Numbers RL5GM118969, TL4GM118971, and UL1GM118970. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Prediction of Damage‐Associated Non‐Synonymous Single Nucleotide Polymorphisms in the Human LAMB2 Gene: A Computational Study
Authors: Herath Venura , Denipitiyage Shanika 
Published: June 2021

Laminins are a major component of basement membranes, and they consist of a unique combination of three non‐identical sub‐chains, giving rise to at least 15 laminin isoforms. The laminin subunit β2 (LAMB2) gene encodes laminin β2, a sub‐chain of several laminins. LAMB2 has been identified as a risk locus for Pierson syndrome, nephrotic syndrome type 5 as well as chronic kidney disease of unknown etiology (CKDu). We conducted this study to predict damage‐associated non‐synonymous single nucleotide polymorphisms (nsSNPs) in LAMB2. nsSNPs were analyzed using 22 in silico prediction tools based on evolutionary, structural and consensus approaches. All tools showed a significant correlation with each other (p < 0.05) and a significant association between their categorical predictions (p < 0.05). Out of the 853 nsSNPs located in LAMB2, 221 nsSNPs within the functional domains were identified as damaging by ≥10 individual tools and by at least two consensus tools. These nsSNPs were distributed among EGF‐like (136), Laminin IV type B (25) and laminin N‐terminal domains (60). The effects of the damage‐associated nsSNPs located in these domains were found to be more deleterious than those found in other regions (Kruskal‐Wallis test H: 99.570, p = 0.001, df = 3). Overlaps between the identified damage‐associated nsSNPs and post‐translational modification sites were also identified. Future work will include modeling of the predicted damage‐associated nsSNPs, identification of the potential consequences to LAMB2 structure, and its disease association. The predicted damage‐associated nsSNPs will provide insight into the possible causes of LAMB2‐associated diseases.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Plasma Protein Regulation during COPD‐Related Muscle Wasting Using Mass Spectrometry‐Based Proteomics
Authors: Utegg Rachael , Minogue Catherine 
Published: June 2021

Chronic Obstructive Pulmonary Disease (COPD) affects 15.7 million Americans. Skeletal muscle dysfunction is a frequent comorbidity of COPD, which associates with worse prognosis including higher mortality and hospitalization rates, independently of the level of pulmonary disease. Moreover, recovery of skeletal muscle integrity in the context of rehabilitation exercises is associated with improved outcomes. To better understand the mechanisms underlying muscle wasting in COPD patients, a mouse model conditionally over expressing interleukin 13 (IL‐13+) in pulmonary Club cells has been developed. This animal demonstrates a pattern on skeletal muscle wasting that is highly reminiscent of the human counterpart. A comprehensive mass spectrometry‐based proteomics study demonstrated differences in skeletal muscle protein expression in wild type versus IL‐13+ mice. In this study, we used plasma samples to interrogate the proteomic profile of the wild type versus the IL‐13+ mice. Our hypothesis is that the proteomic signature found in skeletal muscle will, to some extent, overlap the pattern present in the plasma from the same animals indicating a systemic readout of skeletal muscle dysfunction. If our hypothesis is confirmed, we plan further validation studies using human plasma samples, which could facilitate early detection and potential interventions to ameliorate this devastating condition and have a significant impact on patients' outcomes.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Stimulation of Gαq Promotes Stress Granule Formation
Authors: Scarlata Suzanne , Qifti Androniqi , Jackson Lela , Singla Ashima , Garwain Osama 
Published: June 2021

During adverse environmental conditions, mammalian cells regulate protein production by sequestering the translation machinery in membraneless organelles (i.e. stress granules) whose formation is carefully regulated. In this study, we show a direct connection between G protein signaling and stress granule formation through phospholipase Cβ (PLCβ). In cells, PLCβ localizes to both the cytoplasm and plasma membrane. We find that a major population of cytosolic PLCβ binds to stress granule proteins; specifically, eIF5A and Ago2, whose RNA‐induced silencing activity is halted under stress. PLCβ1 is activated by Gαq in response to hormones and neurotransmitters and we find that activation of Gαq shifts the cytosolic population of PLCβ1 to the plasma membrane, releasing stress granule proteins. This release is accompanied by the formation of intracellular particles containing stress granule markers, an increase in the size and number of particles, and a shift of cytosolic RNAs to larger sizes consistent with cessation of transcription. Arrest of protein synthesis is seen when the cytosolic level of PLCβ1 is lowered by siRNA or by osmotic stress, but not cold, heat or oxidative stress causes similar behavior. Our results fit a simple thermodynamic model in which eIF5a and its associated proteins partition into particles after release from PLCβ1 due to Gαq stimulation. Taken together, our studies show a link between Gαq‐coupled signals and transcription through stress granule formation.Support or Funding InformationNIH GM116187This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cytokine‐stimulated iNOS Expression Requires Long Chain Fatty Acyl CoA Synthetase (ACSL)
Authors: Ali Yousuf , Weis Margaret Teresa 
Published: June 2021

RationaleACSL catalyzes the ligation of fatty acid to coenzyme A, the first and rate limiting step for fatty acid utilization. Triacsin C is a fungal metabolite which inhibits ACSL1, ACSL3, and ACSL4. Earlier, we showed that Triacsin C reduces the infarct volume and inhibits iNOS expression in the MCAO model of stroke. As iNOS is up‐regulated in stroke, we wished to determine whether the Triacsin C effect was linked to iNOS expression.MethodsbEnd.3 cells were stimulated with cytokine mix, (TNFα, 60 ng/mL; Ilβ,2 ng/mL; IFNγ, 100 units/mL), in the presence or absence of 5μM Triacsin C for 24 hours. RNA was extracted following RNeasy Plus Mini Kit(Qiagaen) instruction. The concentration and purity of RNA was determined by Spectrophometer. 20 μl first strand cDNA was synthesized from 1 μg total RNA for each sample using Superscript III First‐Strand(Invitrogen). One μl cDNA was amplified using SYBR Green PCR Master Mix (applied Biosystems) in CFX96 Touch Real‐Time PCR Detection System (BioRad). β‐actin (cat 330001 PPM02945B) and iNOS (cat 330001PPM02928B) primers were purchased from Qiagen. Expression level in fold change was determined by the comparative threshold cycle method (2−ΔΔCt) with β‐actin as the control gene.ResultsCytomix stimulated iNOS expression in the presence of Triacsin C was 52.8 ± 8.33% of control. (p=0.0005; n=5 independent experiments).ConclusionsOur data confirm that iNOS expression is modulated by ACSL activity, and support our hypothesis that the Triacsin C effect on stroke infarct volume is related to suppression of iNOS expression.Support or Funding InformationDepartment of Pharmaceutical Sciences, Texas Tech University Health Sciences Center, Amarillo, TXThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.