bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects of psychological stress in histone acetylation (537.2)
Authors: Gómez Padilla Paola , Colón Caraballo Mariano , Rivera Ivelisse , Appleyard Caroline , Flores Idhaliz 
Published: June 2021

Recently, epigenetic factors, including histone modifications, and environmental factors have shown to play an active role in endometriosis. We have recently shown that the levels of histone de‐acetylates (HDACs) are higher and that histone acetylation marks are decreased in endometriosis lesions. Also, stress contribute to the development of higher number of lesions and inflammatory parameters are exacerbated. Psychological stress has shown to cause changes in the epigenome. We sought to test the hypothesis that in endometriotic tissue from rats exposed to stress, histone acetylation is modulated, which would impact gene expression and lead to higher severity of symptoms. This randomized controlled study consists of two groups of rats, both with surgically induced endometriosis: the experimental group, which was exposed to a 10‐day swim stress protocol and a control group, which was not exposed to stress. On day 60, all rats were sacrificed and examined for the presence of endometriotic vesicles, which were processed for protein extraction and western blot analysis for HDACs, and for acetylated H3K9 and H4K8, as well as the housekeeping gene GAPDH. Densitometry and statistical analyses were performed to the bands. We have standardized the conditions for Western blot analysis of HDAC2 (57kD), H3K9ac and H3K8ac (~20Kd). Our preliminary results identified a specific HDAC2 band, which is expressed at the highest level by a rat exposed to stress, characterized by higher number of lesions and worse inflammatory parameters. Next, we will conduct Western Blot for HDACs and histone marks in both groups and will evaluate possible correlations between protein levels and inflammatory parameters in this rat model. These studies may produce evidence in support of a possible role of stress in modulation of epigenetic markers in endometriosis. Due to the fact that HDACs can be targeted using inhibitors, which can become potential new therapies, these results have a high translational potential.Grant Funding Source: Supported by: NIH Grant #R25GM096955 and (NCCAM) #3 NC1R15AT006373.
Hypoxia during embryonic development of myocardium (542.1)
Authors: Nanka Ondrej , Krejci Eliska , Barth Jeremy , Sedmera David 
Published: June 2021

Our previous results demonstrate that normal embryonic development involves physiological tissue hypoxia. Through analysis of avian embryos we found that hypoxic conditions caused alteration in myocardial architecture. We also found that the extent and distribution of hypoxic regions differed in the embryos in relation to HIF1α levels. Now we have used microarray analysis to examine how gene expression levels are affected under hypoxic conditions at embryonic day 8 in comparison to embryos at normoxic conditions.Chicken eggs were incubated at 38□C under normoxic (21% O2) conditions for the first 48 hrs and subsequently under hypoxic (16% O2) conditions. Controls were incubated in normoxia only. RNA was extracted from isolated normal and hypoxic chick embryonic ventricles. cRNA was hybridized to Affymetrix Chicken Genome array according to manufacturer protocols. We identified 164 transcripts that were differentially expressed between the normoxic and hypoxic group (fc>1.5 and p<0.05). Analysis of gene ontology information for the subset of transcripts up‐regulated by hypoxia (89) detected functional relationships to cell growth, cell differentiation, muscle contraction and signal transduction. Similar ontology analyses of transcripts down‐regulated by hypoxia (75) detected functional associations with cell communication, ion transport, and signal transduction.Grant Funding Source: Supported by Charles University PRVOUK 1105‐280002 and GACR P302/11/1308; NIH grants GM103342,103499
The type I TGFß receptor ALK‐1 and it’s ligands promote angiogenesis (544.2)
Authors: Duffy Iain , Zundell Joseph , Robinson Jesse , Hawker James 
Published: June 2021

Control of angiogenesis is critical in diseases such as cancer, heart disease, or wound repair. Growth factors such as transforming growth factor beta (TGFß) and fibroblast growth factor 2 (FGF‐2) contribute to angiogenesis. The TGFß family signaling receptor Activin‐Like Kinase 1 Receptor (ALK‐1) is expressed in coronary venular endothelial cells (CVEC) at near confluent cell densities on gelatin, but not collagen but it is induced by TGFß + FGF‐2 during tube formation in collagen. Other genes induced during tube formation were Smad 6 and Fibroblast Growth Factor Receptor 1 (FGFR1). By contrast, ALK‐5, a related TGFß type 1 receptor, and TGFßRII were expressed in these cells in both gelatin and collagen. ALK‐1 is also expressed in other EC types, smooth muscle cells, cardiac fibroblasts, embryonic fibroblasts (3T3) but not epithelial cells. Three ligands: TGFß, activin, and bone morphogenetic protein 9 (BMP‐9), have been shown to bind ALK‐1. We show that endogenous activin binds ALK 1 and can be displaced by exogenous TGFß. It may function to regulate control of ALK 1 activation. The response of endothelial cells to various ALK‐1 ligands may depend on the cell matrix of endothelial cells and which other co‐receptors (type II receptors and endoglin) are expressed. We are: 1) testing expression of various ALK‐1 ligands and co‐receptors in endothelial cells under various conditions; and 2) we are generating mutants of ALK‐1, ALK‐5, and endoglin to further test their roles in angiogenesis.Grant Funding Source: Research support was from AHA‐TX and Dept Veteran’s Affairs grants (JH)
Metaanalysis of post‐translational modifications on proteasomal proteins (555.17)
Authors: Hornbeck Peter 
Published: June 2021

The 26S proteasome plays a critical in regulating protein degradation in multiple cellular compartments. The activity of the proteasome must respond to short term and long‐term cellular and environmental inputs efficiently. The control of phosphorylation and other post‐translational modifications (PTMs) is a central component of cellular signaling, yet little is known about the location, extent and possible roles of proteasomal PTMs. We analyzed data in PhosphoSitePlus that has been curated from >15,000 peer‐reviewed low‐throughput papers, over 100 publications of high‐throughput PTM analyses, and data from over 7,000 mass‐spec analyses performed at Cell Signaling Technology. Only experimentally verified PTMs are included in the study. They include > 150,000 phosphorylation, 50,000 ubiquitinylation, 24,000 acetylation, and ~ 8,000 methylation sites. We have analyzed the location and distribution of multiple types of PTMs on proteasome subunits and associated proteins, and have identified specific residues that are heavily modified. For example, PSMA3 is heavily phosphorylated on Ser250, but not on Tyr residues, while PSMA2 is heavily phosphorylated on four tyrosine residues including Tyr683 but not on serine residues. In contrast phosphorylation occurs infrequently on PSMA1 and PSMA4. Analysis of the topology of PTMs on available molecular structures reveal that some of the observed tyrosine and serine phosphorylation sites occur on interface residues that could potentially regulate processes such as opening the catalytic chamber and interacting with the regulatory subunits. As a contrast to the PTM landscape of the proteasome, and to place it in perspective with another cellular structure, a similar analysis of the PTM profile of nuclear pore complex will be presented.Grant Funding Source: NCI, NIGMS, NIAAA
Gene loops enhance mRNA export in yeast (560.8)
Authors: Singh Badri , Hampsey Michael 
Published: June 2021

Extraordinary progress has been made in recent years toward understanding the structure of the eukaryotic genome and the mechanisms that underlie gene expression. Missing from the picture, though, is a fundamental understanding of the three‐dimensional organization of the genome and how these structures affect gene expression. We are investigating the higher order structure of genes transcribed by RNA polymerase II (RNAP II) in the yeast Saccharomyces cerevisiae. Using a powerful technique, called “chromosome conformation capture”, designed to probe the three‐dimensional architecture of the genome, we have detected gene loops that specifically juxtapose promoter and terminator regions. Gene loops are formed on actively transcribed genes and are dependent upon components of both the initiation and pre‐mRNA 3’‐end processing machineries. However, neither the structural requirements for formation and maintenance of gene loops, nor the functions of gene loops are well understood. Here, we report that gene loops are RNA‐dependent and facilitate export of mRNA from the nucleus to the cytoplasm. Further, our systematic analysis of genetic and physical requirements for looping (1) Define the transcription and mRNA export factors TREX‐1 and TREX‐2 as required for formation and/or maintenance of gene loops; (2) Determine the function of gene loops by defining how gene loops, the nuclear pore and mRNA export are interconnected. Our working hypothesis is that all protein‐encoding genes form loops. Looping is not essential, but rather facilitates mRNA export by interaction with nuclear pore complex. This is consistent with (i) coupling of export to transcription of nascent mRNA and (ii) interaction of actively transcribed genes to the nuclear pore. Our results define a fundamental process involved in gene expression and illuminate the pathway by which transcription is coupled to mRNA export.Grant Funding Source: Supported by NIH
Understanding the functional interplay between DNA demethylation and transcription (561.1)
Authors: Wang Dong 
Published: June 2021

Dynamic regulation and faithful maintenance of proper DNA methylation patterns are essential for many cellular functions. 5‐Hydroxylmethylcytosine (5hmC), 5‐formylcytosine (5fC) and 5‐carboxylcytosine, newly discovered oxidized forms of 5‐methylcytosine (5mC) are involved in active DNA demethylation process. The roles of these oxidized species of 5mC in epigenetic and transcription regulation have been an area of intensive study recently. Here we report a systematic study of the effects of five different forms of cytosine in DNA on mammalian and yeast RNA polymerase II transcription elongation, providing new insights into potential functional interplay between cytosine methylation status and transcription. In addition, we will discuss our recent effort in developing new chemical tools that target the formylcytosine sites and modulate 5fC‐related biological processes.Grant Funding Source: NIH GM085136, NIH GM102362
Understanding the role of pea enation mosaic virus mRNA 3’ untranslated region in translation initiation (566.5)
Authors: Liu Qiao , Goss Dixie 
Published: June 2021

Most eukaryotic mRNAs use two key elements at opposite ends of the mRNA, the 5’ m7GpppN cap and the 3’ poly adenosine (A) tail, to recruit translation initiation machinery and circularize mRNA. Pea Enation Mosaic Virus (PEMV) mRNA lacks both a 5’ m7GpppN cap and a 3’ poly (A) tail. A novel type of cap independent translation elements (PTE) is found in the 3’UTR and is essential for translation initiation, the detailed mechanisms of initiation are not well known. Direct protein fluorescence quenching and labeled RNA fluorescence anisotropy were used to investigate the binding affinity of PTE fragment with two important eukaryotic initiation factors, eIF4E (Kd=~7.2nM) and eIF4F (Kd=~8.4nM). These binding affinities are as high as those for eIF4E and eIF4F binding with the cap analogue m7GTP and much higher than those for m7GpppG. With the fluorescence anisotropy assays, we also demonstrated that eIF4A, eIF4B and Poly A tail binding protein (PABP), which can form a stable complex with eIF4F in vivo, only slightly increased the binding affinity of eIF4F with the PTE RNA (Kd<5nM). These binding affinities suggest the PTE element can sequester eIFs from the host cell. The role of eIF/PTE binding in ribosome assembly is being investigated.Grant Funding Source: NSF MCB 1157632
A computational evaluation of the steric and electronic contributions to the stability of the structures of α− and β−D‐glucopyanose Part 2: natural bond orbital analysis of monosubstituted cyclohexanes and pyrans (593.3)
Authors: Baello Rofe , Abdulsalam Maryam , Balilli Rahih , Brutofsky Christian , Kasner Marc 
Published: June 2021

The anomeric effect refers to the tendency of an electronegative substituent at C2 in a pyran to be lower in energy when in the axial orientation than in the equatorial orientation. By examining the geometric and electronic parameters of a sequence of monosubstituted tetrahydro‐2H‐pyrans (‐CH3 ‐OH and ‐CH2OH) and comparing them to a set of similarly substituted cyclohexanes the steric factors can be evaluated and the magnitude of the electronic interaction can be evaluated. The changes in the bond lengths and bond angles that occur when an oxygen is substituted for a CH2 in the pairs of molecules (cyclohexanes versus the pyrans) show that steric interactions cannot explain all the structural stabilities and energies. Natural Bond Orbital analysis shows that donation of electron density from the ring oxygen occurs in all three pyrans) but, as expected, is most significant in 2‐hydroxyl tetrahydro‐2H‐pyran. There appears to be little or no donation of electron density is the substituted cyclohexanes .Recognizing that the cyclic configuration of glucopyranose is a substituted tetrahydro‐2H‐pyran and that the ‐CH2OH substituent plays a major role in determining the energetic stability of the compound, analysis of this series of compounds can determine the significance of many of the electronic interaction in glucopyranose . The NBO analysis helps to quantify the details of the electronic interactions that contribute to the geometrical structure of α− and β−D‐glucopyranose.
The nuclear accumulation of alpha‐synuclein is mediated by importin alpha and promotes neurotoxicity in nucleus (596.6)
Authors: Ma Kai Li , Chen Nai Hong 
Published: June 2021

α‐synuclein (α‐syn), a 14 kDa presynaptic protein, is widely involved in the Parkinson’s disease (PD) pathogenesis. It has been noted that the nuclear import of α‐syn may be mediated by importin α and that both the amino acid residues 1‐60 and 103‐140 of α‐syn were indispensable for its nuclear import. After imported into the nucleus, the accumulated α‐syn played a toxic role in the PC12 cells. Thus, it can be concluded that α‐syn can accumulate in nucleus, which is mediated by importin α, and promote neurotoxicity in nucleus.7
Determining the role of melanopsin C‐tail in deactivation and trafficking using chimeric constructs (596.7)
Authors: Ortiz Elelbin , Cameron Evan , Robinson Phyllis 
Published: June 2021

Melanopsin is a non‐image forming G protein‐coupled receptor expressed in intrisically photosensitive retinal ganglion cells in the vertebrate retina. These cells are involved in functions such as the photoentrainment of circadian rhythm and the pupillary light reflex. The deactivation of melanopsin occurs through the phosphorylation of the C‐tail followed by the binding of a β‐arrestin molecule. The binding of β‐arrestin allows for endocytosis of GPCRs after inactivation. However, preliminary data suggests that melanopsin is not internalized. We hypothesize that the C‐tail of melanopsin plays a key role in the prevention of internalization.Angiotensin II type 1A receptor (ATII1AR) and β‐2‐andrenergic receptor (B2AR) are GPCRs that bind β‐arrestin but differ in their trafficking via endocytic vesicles. In this study, the melanopsin C‐tail was replaced with either ATII1AR and B2AR C‐tail through cloning. The construction of the chimeras has been confirmed by DNA sequencing.Our next step is to introduce our plasmids into Human Embryonic Kidney (HEK) cells. These constructs will then be assayed for trafficking using immuno flow cytometry and immunohistochemistry. We hypothesize that the chimeric constructs will undergo endocytotic pathways similar to ATII1AR and B2AR. This would allow us to conclude that the melanopsin c‐tail is essential in maintaining melanopsin in the cell membrane.Grant Funding Source: Supported by HHMI, MARC U*STAR Program at UMBC, and a National Eye Institute grant to P.R.R.
Clathrin and its adaptor AP1 are required for apical sorting of GPI‐anchored proteins in biosynthetic and recycling routes in MDCK cells (598.4)
Authors: Watanabe Reika , Castillon Guillaume 
Published: June 2021

To fulfill their functions, epithelial cells establish asymmetric protein distributions in apical and basolateral plasma membranes. To date, the role of clathrin and its adaptor, the AP1 complex, has been documented exclusively for basolateral protein trafficking in mammalian epithelial cells. We found that the apical localization of endogenous glycosylphosphatidylinositol (GPI)‐anchored proteins is severely affected by gene silencing of the clathrin heavy chain and components of the AP1 adaptor complex in MDCK cells. In these cells, apical targeting is affected not only for GPI‐ anchored proteins but also for endogenous secretory proteins and the apical‐destined t‐SNARE, syntaxin 3. Analysis of apical secretion of lysoGPI‐APs reveals that the biosynthetic delivery of GPI‐APs toward apical plasma membrane is impaired in these cells. Furthermore, apical recycling and transcytosis from basolateral to apical membranes of GPI‐APs are also impaired in these cells. Our results formally demonstrate the importance of biosynthetic and endocytic routes for establishment and maintenance of apical localization of GPI‐APs in polarized MDCK cells.Grant Funding Source: Swiss National Sciences Foundation Assistant Professorship
Stearoyl‐CoA desaturase‐3 mediates the regulation of adipose and hepatic murine lipid metabolism (605.1)
Authors: Bond Laura , Burhans Maggie , Ntambi James 
Published: June 2021

Stearoyl‐CoA desaturase (SCD) is a critical enzyme involved in de novo lipogenesis and catalyzes the Δ9 desaturation of saturated fatty acids. Four SCD isoforms have been identified in mouse (SCD1‐4); SCD isoform 3 exhibits unique substrate specificity and tissue expression. While SCD1, 2, and 4 convert palmitate and stearate to palmitoleate and oleate, respectively, SCD3 preferentially desaturates palmitate. SCD3 expression is restricted to skin, harderian glands, preputial glands and differentiating adipocytes. To determine the role of SCD3 in murine lipid metabolism, we created a mouse model in which SCD3 is globally knocked out. Despite the limited tissue expression of this isoform, SCD3‐/‐ mice exhibit a global phenotype when fed a lipogenic low‐fat high‐carbohydrate diet. In contrast to SCD1 deletion, which protects mice from diet‐induced obesity and hepatic steatosis, deletion of SCD3 deletion predisposes mice to increased hepatic triglyceride accumulation under high‐carbohydrate feeding compared to SCD3flox mice. Gene expression studies in white adipose tissue reveal that SCD3 deletion and high‐carbohydrate feeding cause a transcriptional remodeling of subcutaneous white adipose tissue. Expression of genes involved in de novo lipogenesis, fatty acid oxidation, and uncoupling are simultaneously increased in the subcutaneous depot of SCD3‐/‐ mice.Grant Funding Source: Supported by NIH National Research Service Award T32 GM07215.
Catalytically important residues in the S. cerevisiae lysophospholipid acyltransferase, Lpt1p (605.15)
Authors: Oelkers Peter , Renauer Paul 
Published: June 2021

Members of the membrane bound o‐acyltransferase family mediate reactions in phospholipid and triglyceride synthesis, cholesterol esterification, and protein modification. Aside from a conserved histidine, the amino acids critical for catalysis in this family remain to be conclusively determined. Thus, the reaction mechanism remains undetermined. We used bioinformatics to identify residues highly conserved among homologs spanning three kingdoms and to perform site directed mutagenesis of the respective residues in the S. cerevisiae homolog, LPT1. Eight residues were identified which have R‐groups containing hydroxyl, carboxyl, and primary amine groups. Initial assays suggest that an acid / base mechanism is utilized by Lpt1p to esterify lysophosphatidylcholine with oleoyl‐CoA. To further examine Lpt1’s mechanism, utilization of three 18‐carbon monounsaturated acyl‐CoA species with differing double bond locations was assayed in vitro. Interestingly, location of the double bond at position Δ11 lowered the apparent Vmax by six fold and at position Δ6 lowered it two fold compared to the Δ9 species. No activity was detected with a 24‐carbon, Δ9 acyl‐CoA species. Together, these data suggest in Lpt1p, the acyl‐CoA substrate lies in a protein furrow, uniquely suited to accommodate acyl‐CoA species with less than 24 carbons and a double bond nine carbons from the thioester.Grant Funding Source: Supported by the University of Michigan Office of Research and Sponsored Programs
Discovery of novel monosaccharides in animal glycans: natural occurrence of N‐glycolylhexosamines (607.6)
Authors: Bergfeld Anne , Lawrence Roger , Diaz Sandra , Pearce Oliver , van Vleet Jeremy , Esko Jeffrey , Choudhury Biswa , Varki Ajit 
Published: June 2021

Metabolism of the major mammalian sialic acid N‐glycolylneuraminic acid (Neu5Gc) generates N‐glycolylglucosamine (GlcNGc) as a byproduct of its degradative pathway (1). Mammalian cells cultured with exogenous chemically synthesized GlcNGc were found to synthesize UDP‐GlcNGc (2). Chemically synthesized N‐glycolylgalactosamine (GalNGc) was also utilized by mammalian cells to synthesize UDP‐GalNGc and UDP‐GlcNGc, and these served as precursors for assembly of most major glycan classes (3). We asked if these novel reactions could also happen naturally. Comparing wild‐type with Neu5Gc‐deficient mice, we demonstrate that Neu5Gc metabolism is indeed a source of naturally‐occurring UDP‐GalNGc and UDP‐GlcNGc. These endogenously derived N‐glycolylated aminosugars are also naturally incorporated into animal glycans such as chondroitin sulfates (CS), O‐glycans, and N‐glycans. In contrast, humans lack Neu5Gc biosynthesis and should therefore be devoid of GalNGc and GlcNGc in glycans, and also be deficient in any as yet unknown functions these glycans may mediate. Comparisons of human and chimpanzee samples indeed show the predicted difference. However, there is detectable trace amounts of GalNGc in human CS, likely derived from Neu5Gc‐containing animal‐derived foods, particularly of mammalian origin.(1) Bergfeld et al., J Biol Chem. 2012 Aug 17;287(34):28865‐81 (2) Macauley et al., J Biol Chem. 2012 Aug 17;287(34):28882‐97 (3) Bergfeld et al., J Biol Chem. 2012 Aug 17;287(34):28898‐916Grant Funding Source: This work was supported by NIH grant R01 GM32373.