bluuebunny/crossref_metadata_2025_split huggingface dataset:
Chamber specific expression of Myosin heavy chain 7b in the heart of vertebrates
Authors: López‐Unzu Miguel A. , Durán Ana Carmen , Soto‐Navarrete María Teresa , Fernández Borja 
Published: June 2021

In extant vertebrates, myosin heavy chain (MyHC) 6 and 7 are the main isoforms of atrial and ventricular myocardium respectively, whereas MyHC7b has been proposed to be an ancient cardiac isoform only expressed during embryonic development in modern species. The MYHC7b DNA sequence has an intronic miRNA thought to be a regulator of slow‐twitch versus fast‐twitch muscle differentiation.The pan‐MyHC antibodies MF20 and A4.1025 are commonly used to highlight the myocardium in histomorphological studies. In preliminary immunohistochemical studies of the heart of the lesser spotted dogfish (Scyliorhinus canicula; Chondrichthyes), we have observed that while MF20 labels homogeneously all the myocardium, A4.1025 labels the inflow cardiac segments (sinus venosus and atrium) but not the outflow segments (ventricle and conus arteriosus). In order to interpret these results, we have performed western and slot blots from samples of dogfish and hamster (Mesocricetus auratus) hearts, HPLC‐ESI‐MS/MS from dogfish samples, and immunohistochemistry in hearts of representative species of vertebrates, namely elasmobranchs, polypteriforms, acipenseriforms, teleosts and mammals, using MF20 and A4.1025 antibodies.Western and slot blot results confirmed the specificity of MF20 and A4.1025 for MyHC in dogfish, as well as their differential reactivity against different myocardial segments. HPLC‐ESI‐MS/MS using protein databases from Callorhinchus milii (Chondrichthyes) and Chordata revealed the presence of MyHC6 and 7 in all the dogfish myocardial segments, and of MyHC7b only in the outflow segments. Immunohistochemistry showed that while MF20 signals were homogeneous in all the myocardial segments of all the species studied, A4.1025 signals were restricted to the inflow myocardial segments in elasmobranchs, homogeneous in teleosts and acipenseriforms, and low in the ventricle of polypteriforms.From our results it can be inferred that the A4.1025 antibody, as opposed to MF20, has a low affinity for MyHC7b, at least in the dogfish. In addition, we show that MyHC distribution in the cardiac chambers has changed during the evolution of gnathostomes. We raise the hypothesis that in early diverged groups of gnathostomes, MyHC6 and 7 were distributed in all the myocardial segments, whereas MyHC7b was specific for the outflow myocardium. In derived gnathostomes, MyHC6 and 7 became mainly restricted to the atrium and ventricle respectively, whereas MyHC7b disappeared from the heart, acquiring a regulatory role in cardiac and extracardiac myogenesis through its intronic miRNA.Support or Funding InformationThis study was supported by CGL2014‐52356‐P and FPU15/03209This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CRISPR‐Cas9 knock out of gravin variant 1 impairs in vitro angiogenic sprouting
Authors: Ali Ashrifa , Spagnolia Alessandra , Hull Maddysen , Geffre Marcus , Bialk Pawel , Kmiec Eric , Grove Bryon Dennis 
Published: June 2021

Angiogenesis is a highly regulated physiological process that is controlled by several signaling pathways including Src dependent and cAMP dependent pathways. Currently, the mechanisms underlying the coordination and integration of these signaling pathways are unknown, but based on several knockdown studies, we postulate that gravin (AKAP12), which is expressed as two variants in endothelial cells, may serve as a scaffold to coordinate Src and cAMP dependent signaling during angiogenesis. To investigate this hypothesis, we are using gene editing and protein expression approaches to identify the role of each gravin variant and the role of the Src and PKA binding domains in gravin in angiogenesis. Here, we report on the effect of knocking out gravin variant 1 using CRISPR‐Cas9 on angiogenic sprouting in a 3D‐collagen gel model. Immortalized human umbilical vein endothelial cells (HUVEC‐TERT2) were transfected with a CRISPR‐Cas9 construct in which Cas9 was targeted to exon 3. An RFLP assay designed to identify changes in an HpyCH4III site at the targeted double stranded break point identified a positive clone which lacked the restriction site. Subsequent sequencing revealed a single base deletion at the targeted site in exon 3 of the AKAP12 gene. Reverse transcriptase PCR showed a partial loss of variant 1 mRNA and sequencing of the RT‐PCR product confirmed the single base deletion. Western blotting demonstrated loss of variant 1 in the CRISPR‐Cas9 modified cells while immunofluorescence microscopy using an anti‐gravin antibody that recognized the C‐terminal region of both variants revealed approximately a 50% decrease in fluorescence intensity in CRISPR‐Cas9 treated cells. This CRISPR modified cell line, expressing only variant 2 of gravin, showed altered cell morphology and behavior compared to wild type cells. When cultured on collagen gels, variant 1 knockout cultures showed significantly fewer sprouts than wild type cultures. Confocal microscopy revealed that fewer variant 1 knockout cells migrated into collagen gels, resulting in shorter sprouts with fewer branches, and that these cells displayed fewer filopodia than wild type cells. These results provide evidence that variant 1 of gravin plays a role in invasive cell migration and angiogenic sprouting, which are essential cellular events that regulate angiogenesis. Future studies will investigate the mechanism by which variant 1 contributes to angiogenic sprouting and the role that each variant of gravin plays in Src and cAMP dependent signaling in endothelial cell migration, adhesion and angiogenic sprouting.Support or Funding InformationSupported by NIH grants P30GM103329, P20GM103442 and P20GM113123This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of aspirin on melanogenesis in B16F10 cells grown as 3D hanging drop tumor organoids.
Authors: Martin Mike , Lyden Timothy 
Published: June 2021

Aspirin (acetylsalicylic acid)has been shown to significantly reduce the melanin content in murine melanoma cells cultured in 2D. While this phenomenon has been attributed to lower tyrosinase expression and ERK‐induced degradation of MITF, the effect of aspirin treatment on the growth of melanoma cells in 3D, their morphology and melanosome secretion have yet to be characterized. Melanin secretion and gene expression data were generated from three and five, respectively, replicates of the following. Twelve hanging drop (HD) plates were seeded with B16F10 cells (5000 cells/well). Cells in six plates were cultured in DMEM+10% FBS+Pen/Strep (CONTROL; 50 ml/well) at 37C in a 5% CO2 in air environment. Cells in the remaining plates were cultured in the same medium supplemented with aspirin. Medium was changed daily (20 ml/well). Cells and supernatants were harvested (1 plate/treatment) at 24 hour intervals up to and including 144 hrs. of culture. Cells were washed in PBS and stored in either lysis buffer at −20C for Western analysis or RNA‐Later at −80C for qPCR. Supernatants were centrifuged in order to remove cells and 405l absorbance was measured. Aspirin visibly reduced the melanin content in B16F10 cells within 48 hrs. of initial exposure. Supernatants above both ASPIRIN and CONTROL cells contained significantly (P<0.05)more melanin at 120 hrs. of HD culture than any previous time point. The melanin content in ASPIRIN cell supernatants was also significantly (P<0.05) higher than that of CONTROL cell supernatants at 120 hrs. of HD culture. Tyrosinaseexpression and tyrosinaseprotein content were lower (P<0.03) in ASPIRIN as compared to CONTROL cells during the last 72 hrs. of HD culture. MITFexpression was also reduced (P<0.05)in ASPIRIN cells during the last 48 hrs. of HD culture. We have demonstrated that aspirin can reducethe melanin content in B16F10 cells cultured in 3D. This phenomenon may be due to aspirin's downregulation of two genes, tyrosinase and MITF, associated with the melanogenic pathway. Aspirin, however, did not affect melanin secretion by B16F10 cells grown in this HD plate format.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of Genes Involved in Low Temperature Growth of Cronobacter sakazakii ATCC 29544
Authors: LIU JING , Hong Seong Tschool 
Published: June 2021

Cronobacter sakazakii ATCC 29544 is a gram‐negative opportunistic pathogen, which can cause local necrotizing enterocolitis, bacteremia, and meningitis. Powdered infant formula is considered to be the major transmission route for newborn infections. Even though powdered infant formula is stored at a refrigerator temperature (5~10°C), Cronobacter sakazakii can still cause infection. In order to understand its molecular mechanism for low temperature growth, we first constructed random mutant library and screened for no growth at 8 °C. By using the plasmid recovery technique, soluble pyridine nucleotide transhydrogenase (sth), nusB and two ribosomal RNA genes were identified to be responsible for the reduced growth at low temperature. Overall, we report 11 genes that play an important role in the growth of C. sakazakii ATCC 29544 at low temperature.Support or Funding InformationThis research was financially supported by the “Global accompanied growth R&BD program (N042600010)” through the Ministry of Trade, Industry and Energy (MOTIE, Korea).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biochemical Characterization of the Set1 H3K4 Methyltransferase Complexes
Authors: Kim Jaehoon 
Published: June 2021

Methylation on lysine 4 of histone H3 (H3K4) is one of the prominent histone modification marks that correlate strongly with active transcription in eukaryotes. Accumulating studies in metazoans have implicated misregulation of H3K4 methylation in the pathogenesis of cancer and in developmental defects, further emphasizing the importance of understanding the regulation of H3K4 methylation. In budding yeast, a single H3K4 methyltransferase (Set1) complex can methylate all H3K4 in an H2B ubiquitylation‐dependent manner. However, presence of at least six H3K4 methyltransferase (SET1A, SET1B, MLL1, MLL2, MLL3 and MLL4) complexes in mammalian cells has complicated functional characterization of each complex because of their possible redundant and non‐redundant roles. To understand the roles of H3K4 methyltransferases in transcriptional regulation in mammalian cells, we have deployed biochemical analyses with purified human H3K4 methyltransferase complexes. By taking advantage of an in vitro histone methyltransferarse assay employing a reconstituted human H3K4 methyltransferase complexes and a recombinant chromatin template containing fully ubiquitylated H2B, we found that a subset of human H3K4 methyltransferase complexes exhibit H2B ubiquitylation‐dependent H3K4 methylation activity. In addition, we demonstrate different subunit compositions and subunit interaction networks in human H3K4 methyltransferase complexes. Our studies establish minimal components of the H3K4 methyltransferase complexes required for H3K4 methylation and provide a mechanistic basis for H3K4 methylation in mammalian cells.Support or Funding InformationNational Research Foundation (NRF) of Korea (2015R1A1A1A05001593)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A novel approach to identify regulated long non‐coding RNAs in selected pathways
Authors: Okino Steven T. , Kong Michelle , Flynn James M. , Wang Yan 
Published: June 2021

We have developed a highly sensitive RT‐qPCR‐based workflow that quantifies the expression of lncRNAs in a RNA sample. The workflow utilizes a novel reagent that enables streamlined genomic DNA removal, cDNA synthesis and multi‐target pre‐amplification and allows for reliable quantification of as little as 25 pg of RNA (single cell equivalent). This reagent can be used together with pre‐designed qPCR array panels that contain lncRNA targets identified as being involved in specific biological/pathophysiological pathways through perturbation experiments, guilt‐by‐association experiments or literature mining. We demonstrate the utility of this workflow by identifying seven lncRNAs that are regulated in early stem cell differentiation using the NTera2 model system. Two lncRNAs are suppressed and five lncRNAs are induced during the differentiation process and may play a role in the maintenance of the pluripotent state or in stem cell differentiation respectively. Two of the lncRNAs have been previously reported to be involved in stem cell differentiation; the other five lncRNAs appear to be a new finding. We believe that this workflow represents a powerful discovery tool to identify lncRNAs involved in biological/pathophysiological pathways and can promote new insights in lncRNA research.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Micro‐RNA‐205 Regulates Heart Size Through Direct Modulation of the Hippo Pathway
Authors: Weldrick Jonathan James , Kouri Lara , Yi Rui , Burgon Patrick Gary 
Published: June 2021

During fetal development, the mammalian myocardium undergoes a period of hyperplastic growth, which establishes the number of cardiomyocytes in the adult heart. After birth, cardiomyocytes proceed through a final round of cell division in the absence cytokinesis that results in binucleation of >95% of adult cardiomyocytes. Nearly all subsequent increase in myocardial mass is due to myocardial hypertrophy, with extremely low number of new cardiomyocytes being produced throughout post‐natal life. Despite the importance of this phenomenon, little is known about the molecular/genetic basis, especially with regard to the role of micro‐RNAs, for the transition from hyperplastic to hypertrophic‐based myocardial growth.We hypothesize a specific perinatal heart micro‐RNA‐mediated gene program is necessary for the normal transition from a fetal heart to an adult heart gene program.To identify the molecular mechanisms and genetic pathways involved in cardiac myocyte differentiation, RNA was isolated from E19, and 1, 3, 5, 7, 10 and 35‐day old mouse hearts (n=9 hearts/time point pooled). Cardiomyocyte micro‐RNA profiles (n=3 arrays/time point) were measured and bioinformatic analysis was used to identify genes that are transiently and significantly changing (p<0.05, fold change >1.5) during the perinatal period.Our analysis identified microRNA‐205 as a candidate for playing a role in the cardiac transitional program. Previous studies have shown a global knockout of miR‐205 to be neonatally lethal. We observe a transient 20‐fold increase in miR‐205 expression between day 1 and day 5 of post‐natal life, with levels returning to baseline by day 10. In‐situ hybridization revealed miR‐205 expression to be restricted to the epicardium of the heart.Mice harbouring a cardiomyocyte‐specific deletion of miR‐205 using αMHC‐Cre are born healthy with expected Mendelian ratios, and develop through the neonatal period normally. Hearts collected from adult mice show signs of abnormal growth and hypertrophy, up to 50% larger than controls. Previous studies demonstrated that miR‐205 directly targets YAP within the evolutionarily conserved hippo pathway that controls organ size. Hearts lacking miR‐205 exhibit a substantial increase in YAP protein expression. Increased cardiac size has been demonstrated in a constitutively active YAP transgenic mouse model. We conclude that miR‐205 plays a direct role in regulating post‐natal heart size through direct modulation of the Hippo pathway.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effect of 1,4‐dimethylnaphthalene on mir‐166 microRNA
Authors: Alharbi Alhanouf , Campbell Michael 
Published: June 2021

The compound 1,4‐dimethylnaphthalene (DMN) is a natural growth inhibitor produced in dormant potatoes. It prevents potato tubers from sprouting by halting cell division. Recent studies have shown DMN prevents the growth of microbiomes present on the surface of stored potato tubers. However, the effect of DMN on small RNAs has not been yet investigated. The molecular miR166 is a highly conserved class of microRNA in many plants. It is found in meristems, and it modulates transcripts of the III homeodomain‐leucine zipper family genes to control plant growth. High expression of miR166 in Arabidopsis thaliana downregulated transcription factors such as Phavulota (PHV) and Phabulosa (PHB), suppressing growth of various parts of the plant. Therefore, we hypothesized that DMN increases miR166 expression to inhibit sprouting. Potatoes were exposed to DMN or nuclease‐free water (control) for three days. Meristems were isolated, frozen in liquid nitrogen, and stored at −80 C. RNAs were isolated using mirVana kit (Thermofisher.com). Total RNA was measured and converted into cDNAs using stem‐loop primers, specifically made for mir166. TaqMan qtPCR was used to quantify the expression of mir166 in control and treated samples. The results obtained from TaqMan qtPCR showed that the expression of miR166 in the treated sample was equivalent to the control sample, suggesting that DMN did not change miR166 expression in dormant potatoes. Further experiments will determine if DMN alters miR166 expression in potatoes after dormancy terminates.Support or Funding InformationSupported in part by a grant from the 1,4‐Group, Meridian, IDThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Calcineurin Gene Subfunctionalization and Swimming Behavior in Paramecium tetraurelia
Authors: McKenna Strange Madigan , Day Alex , Fraga Dean , Welker Jordan , Gaines Ray , Barrin William 
Published: June 2021

The protein phosphatase calcineurin (PP2B) plays a multifaceted role in the motility of the unicellular Paramecium tetraurelia. The Paramecium tetraurelia genome is the product of at least three whole genome duplications resulting in multiple ohnologs pairs for many genes in the Paramecium genome. The most recent duplication resulted in 7 ohnolog pairs that are virtually identical at the amino acid level. Each pair of ohnologs was tested for their role in the swimming behavior of Paramecium using RNAi to silence each ohnolog pair. The behavioral effects were tested in both wildtype and the mutant strain, Dancer, which displays exaggerated backward swimming due to a reduced ability to close the voltage dependent calcium channel after opening. The results suggest that the calcineurin ohnolog pairs are undergoing subfunctionalization. This conclusion is also supported by a real‐time rtPCR analysis of relative gene expression in log‐phase cells in which the different isoforms display different gene expression levels under these growth conditions. These results will be discussed in the context of gene evolution after duplication.Support or Funding InformationThe College of Wooster Sophomore Research ProgramThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
USP21 and OTUD3 Antagonize Regulatory Ribosomal Ubiquitylation and Ribosome‐associated Quality Control Pathways
Authors: Garshott Danielle M. , Leonard Marilyn , Sundaramoorthy Elayanambi , Bennett Eric J. 
Published: June 2021

Ribosomal translation of messenger RNA into properly decoded polypeptides is a tightly‐controlled process that is regulated by a number of cellular quality control pathways. When ribosomes encounter defective mRNA or otherwise experience a terminal stall in translation, the ribosome‐associated quality control pathway is initiated. Conserved ubiquitylation events on specific 40S ribosomal proteins are required for downstream destruction of the mRNA and nascent peptide as well as recycling of ribosomal subunits. We have previously demonstrated that the E3 ubiquitin ligase, ZNF598, is responsible for regulatory ubiquitylation (RRub) at specific sites on the 40S ribosomal subunit during ribosome‐associated quality control events. Here we demonstrate that the deubiquitylating enzymes USP21 and OTUD3 directly antagonize these regulatory ribosomal ubiquitylation events, and catalyze the deubiquitylation of particular 40S ribosomal proteins. For the majority of cell types, the relative RNA expression and protein levels of ZNF598 as compared to USP21 and OTUD3 is substantially higher for the ligase, suggesting a constrained stoichiometric balance that is required to maintain cellular homeostasis. Overexpression of USP21 or OTUD3 results in enhanced read‐through of polyA sequences that would normally induce ribosomal stalling and abort translation elongation. Our results establish USP21 and OTUD3 catalyzed RRub deubiquitylation as a critical checkpoint on ribosome‐associated quality control.Support or Funding Information

This project described was supported by Grant Number T32 ES007059 from NIH NIEHS and its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIEHS or NIH.
This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Plasma metalloproteinase‐9 (MMP9) changes in acute mild traumatic brain injury (mTBI) and correlates with quantitative EEG
Authors: Hubbard Eric , Dawlaty Jessica , Arakaki Xianghong , Cole Soren , Goldweber Robert , Harrington Michael 
Published: June 2021

Background and ObjectivesWe aim to explore blood plasma levels of matrix metalloproteinase‐9 (MMP9) in acute mild traumatic brain injury (mTBI), and test if MMP9 levels correlate with quantitative electroencephalography (qEEG) during working memory (WM) testing.MethodsStudy participants were recruited from the emergency department of Huntington Memorial Hospital in Pasadena, CA, consisting of thirteen acute mTBI civilian patients and seven controls who were trauma patients without head injury ranging between 18–50 years of age. Blood samples were collected from three time points: within 1 week, 14 days, and 30 days after the injury. To study blood‐brain‐barrier (BBB) integrity, we quantified three MMP9 peptides (SLGPALLLLQK, QLSLPETGELDSATLK, and LGLGADVAQVTGALR) using liquid chromatography and mass spectrometry with stable isotope standards. We also employed qEEG at each visit to investigate alpha frequency power during N‐back WM processing.Results & DiscussionWe detected the presence of all three MMP9 peptides in blood plasma. We observed that MMP9 peptide levels in both mTBI and controls were decreasing in abundance in the 2–4 weeks after injury compared to the first week. Importantly, the MMP9 levels of the LGLGADVAQVTGALR peptide but not the peptides from the pre‐protein, were significantly higher in the mTBI group 2–4 weeks after the injury, consistent with known “secondary injury” phenomena. MMP9 levels correlated with alpha power during WM testing, at the first visit for the controls but not for the mTBI patients, at specific brain regions during different WM load. Elevated MMP9 levels indicate the BBB integrity is compromised acutely after mTBI. The correlations between MMP9 and alpha power during WM that we only found in the trauma controls need further investigation.Support or Funding InformationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.