bluuebunny/crossref_metadata_2025_split huggingface dataset:
Changes in the Circular RNA Transcriptome During Inorganic Arsenic‐Induced Epithelial‐to‐Mesenchymal Transition
Authors: Eleazer Rebekah , Andreeva Kalina , Fondufe‐Mittendorf Yvonne 
Published: June 2021

Inorganic arsenic (iAs) found in the environment is associated with diseases such as cancer. Chronic, low dose exposure to iAs induces cellular transformation and epithelial‐to‐mesenchymal transition (EMT), a hallmark of metastasis. Since iAs is not genotoxic, we hypothesize that iAs drives carcinogenesis by altering gene expression though epigenetic mechanisms. One class of epigenetic gene regulators recently discovered are circular RNAs (circRNAs), which are covalently closed and highly stable isoforms derived from precursor messenger RNAs (mRNAs). They are generated from actively transcribed genes and their biogenesis competes with that of mRNA. circRNAs are highly conserved across eukaryotes however the function of this new class of regulators is still unclear. Our lab and others have shown alterations to the epigenome as driving iAs‐mediated carcinogenesis. However the relevance of the expression of circRNAs in this process is not understood. Interestingly, our preliminary data suggest that circRNAs are differentially expressed during this process. We therefore aim to identify and profile the changes in circRNA expression that occur during iAs‐induced EMT. We have treated BEAS‐2B cells with iAs over a time‐course, measuring changes in EMT‐markers over time. We already have shown that cells move from pre‐EMT to mid EMT to late‐EMT and finally back through MET during iAs‐mediated carcinogenesis. This model was used to profile differential expression of circRNAs. To accomplish this, total RNA was isolated and enriched for circRNAs with an RNase R digest. For gene‐specific analyses, divergent primers designed to amplify across the circRNA backsplice junctions were used in reverse transcription quantitative polymerase chain reaction to identify differentially expressed circRNA transcripts. In our preliminary gene‐specific approach, we have identified a circRNA containing the 3′ untranslated region of neural cell adhesion molecule 2 (NCAM2) in BEAS‐2B cells. NCAM2 is a cell‐to‐cell adhesion molecule involved in neuronal growth and migration, and is known to be upregulated during EMT. We observed that during iAs‐mediated EMT, while the expression of the linear NCAM2 transcript increased, there was a concurrent decrease in the expression of the 3′ untranslated region. These results indicate that circRNA expression is altered during iAs‐induced EMT and suggests that iAs promotes carcinogenesis by altering steady state competition between linear and circular RNA production. We are currently using next generation sequencing to fully profile the circRNA transcriptome as cells undergo iAs‐induced EMT to gain a broader understanding of the functional significance of circRNA in iAs‐induced EMT.Support or Funding InformationThis work was supported by NIEHS grant (R01‐ES024478).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Analysis of cell‐type restricted transcriptional networks in human photoreceptor neurons
Authors: Schumacker Scott , Enke Ray A 
Published: June 2021

The retina is a layered neuronal tissue lining the posterior portion of the eye that converts photons of light into visual images in the brain. Rod and cone photoreceptors are highly specialized light sensitive neurons that initiate this process of phototransduction. Though they are similar cell types, rods and cones have distinct functionalities, synaptic connections, and are affected by different blinding disease alleles. In order to characterize cell‐type specific transcriptional networks in human photoreceptors, rod and cone‐enriched samples were biopsied from the eyes of 7 post‐mortem human donors. Total RNAs were extracted from retinal samples using a Qiagen AllPrep Mini Kit. High quality RNAs were prepared into cDNA libraries representing cellular mRNAs and sequenced using an Illumina NextSeq 500 sequencer. Sequencing reads were assessed, trimmed and visualized using FastQC, Trimmomatic, and MultiQC softwares respectively. High quality reads were then aligned to the human hg38 reference transcriptome using the high speed pseudoaligner Kallisto. The sleuth statistical model and RStudio package was used to quantify read alignments and calculate differentially expressed transcripts (DETs) between rod and cone‐enriched sample groups. These analyses identified 10,875 DETs between sample groups with a FDR adjusted p‐value ≤0.05. Of these DETs, 4,365 are transcribed at higher levels in rod‐enriched retinal tissue while 6,519 demonstrate increased transcription in the cone‐enriched retina. We are currently testing the hypothesis that differential patterns of DNA methylation contribute to cell‐type restricted transcription in photoreceptor neurons. These data will contribute to a better understanding of epigenetic regulation of gene expression networks in the human retina.Support or Funding InformationThis research is supported by a JMU 4‐VA Collaborative Research Grant, Burroughs Wellcome Fund Grant #1017506, and NIH grant #1 R15 EY028725‐01A1 awarded to RAE.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
High‐throughput analysis of CRISPR RNA seed sequence identity
Authors: Phan Phong T , Sashital Dipali 
Published: June 2021

The type I‐E CRISPR–Cas (clustered regularly interspaced short palindromic repeat–CRISPR associated) system allows bacteria to fight off bacteriophage and other invader threats. During CRISPR interference, CRISPR‐derived RNAs (crRNAs) guide Cas effector proteins to base pair with complementary sequences in the foreign dsDNA, which leads to the cleavage of the DNA target. Target binding occurs directionally from the 5′‐end of the crRNA guide, and the first several base pairs formed between the crRNA and DNA are important to ensure binding. Mutation within this “seed” sequence often reduces the level of CRISPR interference and promotes an alternative “priming” mechanism. During priming, segments of the foreign DNA are inserted into the CRISPR array to create templates for new crRNAs. Our previous results have shown that for some crRNA sequences, mutations within the seed region can be tolerated. Here, we systematically tested the effects of the crRNA seed sequences on CRISPR interference and priming activities. Using partially randomized seed libraries, we identified the position and nucleotide‐specific effects on the mismatch tolerance for four Escherichia coli strains expressing crRNAs with different seed sequences. We show that G/C‐rich seed sequences are more tolerant of mutations than A/U‐rich seed sequences. We observe that guanine mismatches cause the strongest CRISPR immunity defects for all seed sequences tested. G/C‐rich crRNAs may promote CRISPR immunity against mutated sequences at higher rates due to the lower probability of encountering guanine mismatches. Our results indicate that crRNA sequences with higher G/C content may provide stronger immunity than A/U‐rich crRNAs.Support or Funding InformationR01 GM115874This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Genetic Delivery of RNA Therapeutics to Alter the Expression of Oncogenic Transcripts in Glioblastoma
Authors: Hicks Martin J 
Published: June 2021

Glioblastoma multiforme (GBM) is an incurable and aggressive type of brain tumor. It is the most common central nervous system malignancy with a median survival of only 14 months. It is characterized by increased activation of one or more tyrosine kinase receptors, particularly epidermal growth factor receptor (EGFR). This receptor is dysregulated in about 60% of GBM tumors. EGFR amplification, over‐expression and constitutive activation leads to uncontrolled growth and proliferation of GBM. Although a great deal is known about the aberrant biology exhibited by EGFR‐activated GBM, the application of therapies against the biologic processes is limited by the blood‐brain barrier, which restricts systemically administered therapies from reaching the brain. Although anti‐sense RNAs and small interfering RNAs can be used to target and silence gene expression, exogenously expressed RNAs are susceptible to extracellular nucleases as well as activation of cellular immunity against foreign nucleic acids. To bypass these degradatory mechanisms, we take advantage of a natural noncoding RNA gene architecture and the miRNA expression pathway along with an anti‐sense targeted approach to design and deliver DNA that encodes RNA therapeutics that alter EGFR expression. In addition, we make use of a polycistronic delivery system to express RNAs targeting splicing and alternative poly‐A signal/G‐rich elements of the EGFR transcript. DNA delivery vectors encoding RNA therapeutics were transfected into human GBM cell lines. Analysis of RNA transcripts demonstrates changes in expression of the EGFR target. Results show that our vectors were expressed at high levels with subsequent reduction in full‐length EGFR mRNA expression and concomitant activation of alternative therapeutic isoforms. Current strategies include using the polycistronic delivery mechanism to target additional oncogenic transcripts and adapting to a mouse model of GBM.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Saccharomyces cerevisiae as a viable model for the overexpression and purification of proteins involved in iron homeostasis
Authors: Hati Debolina 
Published: June 2021

Iron is indispensable for the eukaryotic cell but excess of iron is toxic. Disruptions in iron metabolism leads to numerous human diseases, hence the study of iron regulation and metabolism is of high importance. Saccharomyces cerevisiae is used as a model system in our lab to study iron trafficking and homeostasis pathways to better understand iron regulation at the cellular and molecular level. In yeast, two paralogous transcriptional activators, Aft1 and Aft2, play a central role in iron regulation by activating the transcription of target genes in response to iron deprivation. Monothiol glutaredoxins Grx3 and Grx4 that utilize glutathione to bind [2Fe‐2S] clusters interact specifically with Aft1 and Aft2, transferring an Fe‐S cluster to Aft1/2 that inhibits its DNA binding activity. Aft1 is considered the primary regulator of iron homeostasis because aft1Δ mutants exhibit a stronger iron deficiency phenotype than aft2Δ mutants. Aft1 and Aft2 share 39% sequence homology in their N‐terminal DNA binding domains. The only available structure is of a truncated version of Aft2 that contains this homologous portion and includes the domains responsible for DNA binding and iron sensing. Aft1/2 have been shown to be very unstable and difficult to overexpress and purify in Escherichia coli. Hence we optimized the growth conditions to overexpress Aft1 in its native organism, Saccharomyces cerevisiae, and further try to purify Aft1 from the same. Further studies will be carried out in order to investigate the in‐vitro interaction between full length Aft1/2 and Grx3/4‐Fra2 to better understand the mechanism of Aft1/2 inhibition at the molecular level.Support or Funding InformationSupported by Dr. Caryn E. Outten's Research FundingThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Closing the GAP Between IQGAP1 and its Yeast Homologue
Authors: Levitus Ricki Pearl , Hoeprich Gregory , Goode Bruce 
Published: June 2021

IQGAP1 is actin binding protein localized to the leading edge of mammalian cells and plays an important role in cell migration. This process is common to many healthy cells including epithelial cells and neurons, but abnormal cell migration is a key contributor to metastasis of cancerous cells. Understanding the molecular basis of actin regulation during cell migration has the potential to shed light on how these cancerous abnormalities arise. Previous studies have shown increasing actin density at the leading edge when IQGAP1 is knocked down. This phenotype is consistent with knocking down Capping Protein, which blocks actin assembly. We hypothesize IQGAP1 similarly blocks actin assembly. To test this, we investigated the functions of mammalian IQGAP1 on actin assembly in vitro. Using total internal reflection fluorescence (TIRF) microscopy and bulk fluorescence assays we measured actin assembly in the presence of IQGAP1 to assess its effects. We show for the first time IQGAP1 attenuating actin elongation on single actin filaments. Our results are also consistent with the current working hypothesis that mammalian IQGAP1 attenuates actin elongation rates through a capping mechanism. Further, we provide evidence that mammalian IQGAP1 bundles actin filaments in vitro, validating previous results. Lastly, Iqg1 was identified in the yeast model organismS. Cerevisiae with sequence homology to mammalian IQGAP1. Yeast are an ideal model organism for answering fundamental questions about the function of IQGAP1 in vivo as this project expands, but in vitro functional studies are required first to show similarity between yeast Iqg1 and mammalian IQGAP1. Moving forward, we plan to repeat the in vitro experiments above with the yeast homologue to confirm that the functions of mammalian IQGAP1 are evolutionarily conserved across species.Support or Funding InformationThis research was conducted as a part of the Brandeis MRSEC SMURF Fellowship (Brandeis NSF MRSEC, DMR‐1420382).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Using Site‐directed Mutagenesis alongside a Collagen binding assay to reveal the role of Polycystic Kidney Disease domain in ColH Collagenase
Authors: Caviness Perry , Mima Takehiko , Matsushita Osamu , Sakon Joshua 
Published: June 2021

Hathewaya histolyticum, one of the causative agents of Gas Gangrene and previously known as Clostridium histolyticum, secretes two collagen dismantling enzymes ColG and ColH as part of its pathology. Both ColG and ColH have segmental structure consisting of a catalytic domain, polycystic kidney disease domains (single in ColG; double in ColH) and collagen binding domains (double in ColG; single in ColH). Their ability to seek out and bind to loosely wound regions of collagen make the non‐catalytic portions of ColG and ColH promising drug delivery tools. Crystal structure of dual CBDs found in ColG suggest that ColG may wedge between two collagen molecules to aid in fibril rearrangement and collagen hydrolysis while SAXS derived envelope for ColH PKD‐CBD:[GPRG(POG)12]3 suggest that ColH interacts with only collagen molecule. This may suggest that the two work together to synergistically degrade collagen. SAXS envelopes of ColH PKD‐CBD:[GPRG(POG)12]3 show that the PKD domain makes contact with the collagenous peptide. ColH PKD domains 1 and 2 contain regions that are thought to aid in collagen binding for ColH. Collagen binding assay for ColH PKD‐CBD segment bound to type 1 collagen fibrils in a biphasic manner, with one having a higher affinity (KD = 3.39 × 10−7 M) and the other having a low affinity (KD = 2.11 × 10−6 M). The PKD‐CBD segment of ColH binds to collagen much tighter than just the CBD (KD = 1.59 × 10−5). Aromatic residues (Tyr780, His782, Tyr796 and Tyr801) located on the potential collagen binding cleft of the PKD domain in Col H PKD‐CBD were mutated to Ser using site directed mutagenesis. In addition an expected tighter binding ColH PKD‐CBD mutant was developed by mutating two non‐aromatic residues (Val778 and Lys785) on the potential collagen binding cleft to Tyr. Val778 and Lys785 were chosen based on a sequence alignment study. Collagen binding assay of the ColH PKD‐CBD four Ser mutant shows a loss of one of the phases of binding and has a KD consistent with the lower affinity phase of the wild type ColH PKD‐CBD (KD = 1.87 × 10−6). Collagen binding assay of the tighter binding ColH PKD‐CBD shows a biphasic plot with the second phase binding much tighter than the wild type (KD = 5 × 10−8). Fusion proteins containing binding segments from ColG and ColH have been used to anchor bFGF and PTH antagonist at their receptors to stimulate healing of bone fractures, enhance bone allografts for spinal fusion and reverse alopecia. Our study would reveal knowledge on how ColH binds to collagen which would help the development of fusion proteins containing ColH binding segments with a higher efficacy.Support or Funding InformationNational Institutes of Health. Grant Number: 8P30GM103450Arkansas Biosciences InstituteThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Presence of UCP2 and Amylin in human Alzheimer's disease brains
Authors: Saunders Dakarai , Clemons Tameka 
Published: June 2021

Although the role of amyloid beta in Alzheimer's disease has been studied for decades, the link between amyloid beta and amylin is fairly new. Amylin is a protein that is co‐secreted from pancreatic beta cells alongside insulin and has been shown to cross the blood‐brain barrier and impact the formation of amyloid plaques. The normal function of amylin in the brain is directly correlated to metabolism because amylin is considered a satiety protein. The output involved in the metabolic process involves oxidative phosphorylation and includes a set of proteins known as uncoupling proteins (UCPs), which are proteins that directly impact oxidative phosphorylation. Traditionally, UCPs are known to impact the ATP levels that occur in oxidative phosphorylation. However, recently it has been postulated that UCPs may have an additional role that involves cell signaling. In fact, UCP2 has been shown to be present in pancreatic beta cells and to have diminished protein levels in Alzheimer's disease brains. The experiments conducted in this study analyzed the relationship between UCP2 and amylin. Specifically, immunohistochemistry experiments that examine both the presence of amylin and UCP2 in Alzheimer's disease brains were conducted. Western Blot analysis experiments were also conducted to compare the findings from the immunohistochemistry experiments. The initial studies suggest that the role of UCP2 may influence the role of amylin or the role of amylin may influence the role of UCP2.Support or Funding InformationNational Science Foundation Research Initiation Award #24430This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Predominant Beta Subunit Isoform of the Na+, K+‐ATPase in Photoreceptor Neurons Undergoes S‐Palmitoylation
Authors: Sechrest Emily , Sokolov David , Murphy Joseph , Kolandaivelu Saravanan 
Published: June 2021

Many proteins undergo post‐translational lipid modifications, which are often essential to aid in subcellular trafficking, protein‐protein interactions, and membrane association. S‐palmitoylation is a unique post‐translational lipid modification which involves reversible attachment of the 16‐ carbon fatty acid palmitate to cysteine residues on proteins. In the retina, photoreceptor neurons are responsible for capturing photons of light and converting them into electrical signals, which are subsequently sent to the brain for visual processing. Many crucial phototransduction proteins in photoreceptors, including rhodopsin, phosphodiesterase‐6, and the transducin complex, have been shown to require post‐translational lipidation for proper function. To better understand the importance of “palmitoyl” lipid modifications in retinal photoreceptor cells, our lab employed acyl‐resin assisted capture (acyl‐RAC) followed by mass‐spectrometry analysis to isolate and identify proteins in retinal tissue which undergo S‐palmitoylation. From the mass‐spectrometry data pool, our lab chose several candidate proteins to study in more detail, including the β2‐ subunit of the Na+, K+‐ATPase (ATP1B2). ATP1B2 has been shown to be the predominant β‐subunit isoform of the Na+, K+‐ATPase in photoreceptor cells and is thought to be involved in maturation and trafficking of its catalytic α‐subunit counterpart. Additionally, ATP1B2 has been shown to be required by the retinal‐specific protein retinoschisin‐1 (RS1) for its localization and association with photoreceptor and bipolar cell membranes. Although it is known that the Na+, K+‐ATPase regulates the photocurrent in photoreceptors needed to send visual information to downstream neurons, the distinct role of ATP1B2 remains unknown. Acyl‐RAC followed by immunoblot served as a complementary approach to confirm that ATP1B2 undergoes palmitoylation, whereas its homolog, the β1 isoform, does not. After palmitoylation prediction software analysis, we utilized mammalian cell culture and in vivo subretinal injection/electroporation to demonstrate that ATP1B2 is palmitoylated on the 10th amino acid. Additionally, click chemistry with the palmitate analog 17‐Octadecynoic acid, was used as an independent approach to validate ATP1B2 palmitoylation. Altogether, these data confirm that ATP1B2 is post‐translationally lipid modified by palmitoylation on the 10th amino acid. However, the functional role of palmitoylation of ATP1B2 remains unknown and warrants further investigation.Support or Funding InformationNIH 1RO1EY028959‐01, WVU bridge funding, and intramural RFDG funding award (SK)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.