bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cloning and Characterization of Putative Histidinol‐Phosphatase not called by Machine Annotation
Authors: Ellis Samuel 
Published: November 2023

The generation of genomic information has increased exponentially in the past decade, and while this has aided biological research immensely, it has also been shown to be problematic. The information being collected is compiled at a rate much faster than it can be interpreted, and this has led to potential annotation errors in genomic databases. The Kyoto Encyclopedia of Genes and Genomes (KEGG) is a widely used genomic database that records compilations of biochemical pathways. KEGG uses the genome of the bacterium Escherichia coli as a reference genome for some of these pathways. The biosynthesis of the amino acid histidine is a complex, multistep process that requires the use of a multitude of enzymes to catalyze a sequence of biochemical reactions. E. coli utilize a fused, bifunctional imidazoleglycerol‐phosphate dehydratase/histidinol‐phosphatase encoded by the HisB gene to catalyze the sixth and eighth steps in the pathway. However, other bacteria may be able to catalyze these steps using two separate proteins instead of a single fused protein similar to the one utilized by E. coli. The deep‐sea autotrophic bacterium Thiomicrospira crunogena has been proposed to have evolved to catalyze these two steps in this manner by using a separated pair of proteins, encoded by the Tcr_0356 and Tcr_1966 genes, instead of a fused protein. As a consequence, when the genome of T. crunogena was compared against the reference genome, KEGG determined that this organism cannot perform the phosphatase activity in the eighth step and therefore machine calling a histidine auxotroph. By expressing and isolating the proteins and performing biochemical assays, evidence has been collected revealing a protein hypothesized to be a histidinol‐phosphatase. A malachite green phosphatase assay was used to assess the phosphatase activity of the protein encoded by Tcr_0356, while an IGP‐Dehydratase assay was used to assess activity of the protein encoded by the Tcr_1966 gene. The implications of this study further solidify the need for accurate annotations in databases in order to maintain the integrity of genomic data.Support or Funding InformationThis work is based upon ideas generated and supported by the National Science Foundation under Grant Number 0954829, funding the Microbial Genome Annotation network.
Identifying the Metal That Activates the Prenyltransferase That Catalyzes Formation of Geranyl Diphosphate in the Diatom Pseudo‐nitzschia multiseries
Authors: Tran Hai , Savage Thomas 
Published: November 2023

Domoic acid (DA) is a neurotoxin synthesized by marine diatoms of the genus Pseudo‐nitzschia which causes amnesiac shellfish poisoning in humans and death of marine wildlife. Geranyl diphosphate (GPP), a key intermediate in domoic acid biosynthesis, is formed from the “head‐to‐tail” condensation of isopentenyl diphosphate and dimethylallyl diphosphate in a reaction catalyzed by a member of the prenyltransferase family of enzymes. These prenyltransferases can generate different chain‐length products, including geranyl diphosphate (C10), farnesyl diphosphate (FPP, C15), and geranylgeranyl diphosphate (GGPP, C20), all of which are precursors to terpenoid natural products. Prenyltransferases require a divalent metal cation for activity. Previous work has shown that a single prenyltransferase can generate multiple products (GPP, FPP, and GGPP), and that the product distribution will vary depending on the species of divalent cation used to support the reaction (Mg2+, Mn2+, or Co2+). Here, we measured the levels of Mg2+, Mn2+, and Co2+ in Pseudo‐nitzschia cells to determine which ion is most likely to support GPP formation. Three samples of Pseudo‐nitzschia multiseries cultured in sea water were harvested by centrifugation. Samples were digested with HNO3 before analysis using atomic absorption spectroscopy. The average concentration of Mg2+ present, per cell and per μm3, is over 100‐fold higher than the concentrations of Mn2+ and Co2+. This is consistent with results from studies in other systems, suggesting that Mg2+ is the main cofactor that activates the prenyltransferase that generates GPP for domoic acid biosynthesis in Pseudo‐nitzschia.Support or Funding InformationThis project is supported by the SURE (Summer Undergraduate Research Experience) Award Program from the College of Natural Science and Mathematics, California State University Sacramento.
Lipoprotein Lipase regulates the expression of genes responsible for cellular cholesterol uptake and efflux in human and mouse macrophages.
Authors: Moctar Khadija , Madhwani Kimberly Rose R. , Kim Aidan J.M , Medh Jheem D. 
Published: November 2023

BackgroundCellular cholesterol accumulation is the net result of receptor‐mediated cellular uptake and transporter‐mediated efflux. Scavenger receptors (CD36, SR‐AI, SR‐BI) mediate the cellular uptake of cholesterol whereas the ATP‐binding cassette transporter A1 (ABCA1) mediates the unidirectional efflux of excess cholesterol. SR‐BI also mediates bidirectional cholesterol transport. Lipoprotein lipase (LPL) is secreted by macrophages and catalyzes the hydrolysis of circulating triglycerides. Macrophage LPL expression was shown to be inversely correlated with ABCA1 levels. We hypothesized that LPL may regulate the expression of macrophage scavenger receptors as well.ObjectiveTo examine if the expression of macrophage scavenger receptors and cholesterol transporter are regulated by LPL. Regulation of gene expression was studied in LPL‐knock‐down THP‐1 macrophages and LPL‐over‐expressing mouse macrophages. Additionally we are preparing recombinant wild type and mutant LPL to determine which structural components of LPL modulate cholesterol efflux.MethodsTHP‐1 monocytes were cultured and were induced to differentiate into macrophages with phorbol 12‐myristate 13‐acetate. Thioglycolate‐stimulated macrophages were isolated from the peritoneal cavity of transgenic mice with macrophage‐specific LPL overexpression. The expression of CD36, SR‐AI, SR‐BI and ABCA1 was compared in normal and LPL‐(THP‐1) or LPL+ (mice) macrophages by RT‐PCR analysis. b‐actin served as a control for RNA mass. Gel images were quantified by ImageJ analysis.ResultsSilencing of the LPL gene in THP‐1 macrophages correlated with a significant increase in ABCA1 expression (161% of WT). This was corroborated in mouse macrophages since overexpression of LPL resulted in a 50% decrease in ABCA1 expression. The expression of scavenger receptors SR‐BI and CD36 was significantly lower in LPL‐THP‐1 cells compared to WT macrophages (55% and 60%, respectively, compared to WT). Contrary to results in THP‐1 cells, LPL‐overexpressing mouse macrophages showed a decrease in SR‐BI levels (20% of WT), but no change in CD36 expression. The expression of SR‐AI was not influenced by LPL silencing or overexpression.ConclusionManipulation of LPL expression modulates the transcription of genes relevant to cholesterol accumulation in isolated THP‐1 cells. In LPL+ transgenic mice, a decrease in SR‐BI may be due to the influence of other organs in cholesterol homeostasis and SR‐BI's role in cholesterol efflux.Support or Funding InformationAcknowledgements: This work was supported by National Institutes of Health Awards R15HL083946, SC3GM095413, GM008395 and GM063787.
Cardiolipin Content has a Stronger Influence than Acyl Chain Composition on Select Membrane Properties of Biomimetic Mitochondrial Membranes
Authors: Pennington Edward R 
Published: November 2023

Cardiolipin (CL), the hallmark phospholipid of the mitochondria, plays a fundamental role in maintaining mitochondrial inner membrane structure and function. In several ailments including cardiovascular diseases and Barth Syndrome, in which mitochondrial dysfunction contributes towards disease pathology, there is loss of CL content and remodeling of CL's acyl chains. However, it remains unclear how CL content and acyl chain composition impact the biophysical lipid organization of the inner mitochondrial membrane and thereby protein activity. Therefore, we discriminated how CL content and acyl chain composition influence distinct membrane properties of biomimetic mitochondrial membranes. In monolayer studies, the loss of tetralinoleoyl [(18:2)4] CL promoted lipid‐lipid miscibility, while replacement of (18:2)4CL acyl chains with tetraoleoyl [(18:1)4] CL or tetradocosahexaenoyl [(22:6)4] CL had no influence on lipid‐lipid miscibility. Replacement of (18:2)4CL acyl chains with tetramyristoyl [(14:0)4] CL, which has limited biological relevance for humans, promoted lipid‐lipid immiscibility. Supporting studies in bilayers revealed that substitution of (18:2)4CL with (18:1)4CL or (22:6)4CL lowered the phase transition temperature of phosphatidylcholine vesicles, whereas (14:0)4CL had no effect. Finally, quantitative confocal imaging of giant unilamellar vesicles (GUVs) showed that loss of CL content had a stronger influence than remodeling of CL acyl chains. Taken together, we propose that loss of (18:2)4CL content may have a stronger influence on select biophysical properties of membranes than CL acyl chain composition, which has implications for mitochondrial inner membrane structure/function and the design of CL‐specific therapeutics.Support or Funding InformationResearch was supported by NIH R01HL123647 (D.B. and S.R.S), an East‐West Collaboration Award (S.R.S. and A.K.), NIH R15HL122922 (D.B. and S.R.S.), Brody Brothers Foundation Award 213147 (S.R.S.), NIH K25GM100480 (S.R.S. and P. Wang), and NIH R01AT008375 (S.R.S.).
Impact of altering the headgroup‐acylated glycerophospholipid levels on Escherichia coli cells deficient in phosphatidylserine and phosphatidylethanolamine
Authors: Oke Oluwadamilola , Garrett Teresa A 
Published: November 2023

Escherichia coli contains a variety of glycerophospholipids (GPLs). Phosphatidylethanolamine (PE) is particularly important as it is the only zwitterionic phospholipid and makes up 70–80% of total GPLs. It is formed from CDP‐diacylglycerol by the action of phosphatidylserine synthase (Pss) and phosphatidylserine decarboxylase (Psd). Pss condenses CDP‐diacylglycerol and serine to form phosphatidylserine (PS) and releases cytidine monophosphate. PS is a short‐lived intermediate that is rapidly decarboxylated to PE by the Psd enzyme. This study investigates the impact of altering headgroup‐acylated GPLs (HAGPLs) has on E. coli that are deficient in Pss and Psd. Overexpression of the E. coli genes pldB and pagP or the Arabidopsis thaliana gene At1g78690, elevates the levels of the HAGPL acyl phosphatidylglycerol (acyl PG). The overexpression of the human calcium independent N‐acyl transferase (iNAT) increases the levels of the HAGPL N‐acyl phosphatidylethanolamine (N‐acyl PE) in the cells. We transformed E. coli with temperature sensitive mutations in pss and psd with plasmids that contain pldB, pagP, At1g78690 or iNAT behind an arabinose inducible promotor. For each strain, a variety of cellular phenotypes, such as temperature sensitivity, cell morphology, antibiotic resistance, membrane permeability, membrane fluidity and lipid composition, were determined. Expression of At1g78690 promotes reversion of the temperature sensitive phenotype of the pss mutant by, we hypothesize, creating conditions under which compensatory mutations are more likely to support high temperature growth. Expression of pagP results in a longer cell phenotype that will be further investigated using scanning electron microscopy. Additional experiments will use fluorescently‐tagged proteins involved in cell division to determine if the increased cell length is the result of cell division defects promoted by accumulation of HAGPLs.Support or Funding InformationNational Science Foundation Research at Undergraduate Institutions Grant #1516805 and #1152463, and National Science Foundation Major Instrumentations Grant #1039659
Modulation of Endothelial Membrane Biomechanics by Oxidized Phospholipids: A Combined Experimental and Computational Approach
Authors: Ayee Manuela A.A. , LeMaster Elizabeth , Shentu Tzu Pin , Barbera Nicolas , Akpa Belinda S , Levitan Irena 
Published: November 2023

Elevated concentrations of biologically active oxidized phospholipids (oxPCs) are known to exert various pathophysiological effects, including disruption of the endothelial barrier and induction of monocyte adhesion to endothelial cells. These effects are further implicated in a range of disorders, including certain cardiovascular, metabolic, and immunological diseases. The specific mechanisms by which oxPCs modulate membrane barrier function are poorly understood.We investigated the differential influence of two bioactive oxidized phospholipids on model bilayer properties, membrane packing, fluidity, elastic properties, and endothelial cell biomechanics both experimentally, using Laurdan multi‐photon imaging and atomic force microscopy (AFM) microindentation experiments, and computationally, using coarse‐grained molecular dynamics simulations. Specifically, we focused on two major oxidation products of 1‐palmitoyl‐2‐arachidonoyl‐sn‐glycero‐3‐phosphocholine (PAPC), which are known to exert multiple pathophysiological effects. The oxidized phospholipids 1‐palmitoyl‐2‐(5‐oxovaleroyl)‐sn‐glycero‐3‐phosphocholine (POVPC ‐ aldehyde‐terminated) and 1‐palmitoyl‐2‐glutaroyl‐sn‐glycero‐3‐phosphocholine (PGPC ‐ carboxyl‐terminated) have been found to induce inflammation, cell proliferation, and cell death.Our molecular simulations of multicomponent phospholipid bilayers predicted differential bilayer perturbation effects of the two oxidized phospholipids based on the chemical identities of their truncated tails. We showed that for the analogous oxPCs incorporated in a phosphatidylcholine bilayer containing sphingomyelin, the carboxylic analog, PGPC, experienced more significant reorientation of the truncated tail and displacement of the oxidized lipid from the plane of the bilayer. These phenomena induced greater perturbation of the structure of the lipid bilayer than the aldehyde analog, POVPC. Decreased bilayer packing and increased water permeation was observed, which was consistent with Laurdan imaging results. Spectral analysis of bilayer undulations further indicated increased elasticity of bilayer systems containing both oxPCs. Computational predictions of larger membrane perturbation by PGPC corresponded with greater stiffness observed for PGPC‐treated endothelial cells measured by cellular elastic moduli using AFM. These analyses confirmed the paradoxical relationship observed between membrane softening by oxPCs and increased overall cell stiffness in the presence of POVPC and PGPC.Support or Funding InformationNIH grants: T32 HL‐82547, HL‐073965, HL‐083298; AHA MWA Pre‐doctoral Fellowship: 14PRE20490156
Role of Na‐H Exchanger‐2 in Experimental Colitis: Mechanism of Regulation of Expression
Authors: Khan Islam , Soleiman Amal Ali , Thameem Farook 
Published: November 2023

BackgroundThe Na‐H exchanger (NHE) perform an electroneutral uptake of NaCl and water from the lumen of the gastrointestinal tract. There are multiple distinct NHE isoforms, some of which show an altered expression in the inflammatory bowel diseases (IBD). In this study, we examined a role of NHE‐2 in experimental colitis.MethodsColitis was induced in male Sprague‐Dawley rats by intra‐rectal administration of trinitrobenezenesulfonic acid (TNBS). On day 6 post‐TNBS, the animals were sacrificed, colon and ileal segments were taken out, cleaned with phosphate buffered saline and used in this study.ResultsThere was a significant decrease in the level of NHE‐2 protein as measured by ECL western blot and confocal immunofluorescence micrometry. The levels of NHE‐2 mRNA and heteronuclear RNA measured by an end‐point RT‐PCR and the real time PCR were also decreased significantly in the inflamed colon. However there was no change in the level of NHE‐2 protein in response to in‐vitro TNF‐α treatment of an uninflamed rat colon. These changes were selective and localized to colon as actin, an internal control remained unchanged. The confocal fluorescence microscopy revealed co‐localization of NHE‐2 and NHE‐3 in the brush borders of the colonic epithelial cells. Inflamed colon showed a significant increase in the myeloperoxidase activity and colon hypertrophy, and a significant decrease in the body weight and goblet cells' mucin staining. These changes were not conspicuous in the non‐inflamed ileum.ConclusionsThese findings demonstrate suppression of NHE‐2 expression on the brush borders in the colonic epithelial cells which is regulated transcriptionally. However a role of TNF‐α in the regulation of NHE‐2 is discounted in the present model of colitis. This decrease in the NHE‐2 expression will lead to a loss of electrolyte and water uptake thus contributing to the symptoms associated with IBD.Support or Funding InformationThe Kuwait University, Research Grant No. [YM 12/15]. Prof Khan, Anatomy for his help in tissue block preparation for histochemistry and confocal microscopy. Research Core Facility, Health Sciences, the Kuwait University for using their facility to conduct confocal immunofluorescence experiments.
Control of PI4P turnover by endogenous OSBP for fast cholesterol transport at membrane contact sites
Authors: Mesmin Bruno , Bigay Joëlle , Polidori Joël , Lacas‐Gervais Sandra , Antonny Bruno 
Published: November 2023

We have demonstrated previously that Oxysterol‐Binding Protein (OSBP) counterexchanges phosphatidylinositol 4‐phosphate (PI4P) for cholesterol at membrane contact sites between the endoplasmic reticulum (ER) and the Golgi. Because PI4P is produced in the Golgi but consumed by a specific phosphatase when it reaches the ER, we have proposed that OSBP uses PI4P as a fuel to direct cholesterol export from the ER, against its cellular concentration gradient. By using a potent OSBP inhibitor, we now provide evidence that the sole activity of endogenous OSBP strongly influences PI4P turnover and intracellular distribution of cholesterol. We show that OSBP constantly consumes more than half of the total Golgi PI4P. We also found that blocking the PI4‐kinase PI4KIIIβ, which typically operates in the vicinity of OSBP, triggers PI4P oscillations across the Golgi. These oscillations result from the production of PI4P by the distant PI4‐kinase PI4KIIα, and PI4P consumption by OSBP. Collectively, these data illustrate the spatiotemporal coupling between PI4P turnover and OSBP‐mediated cholesterol transport in a cellular context.Support or Funding InformationThis work is supported by the Agence nationale de la recherche (ANR‐15‐CE11‐0027‐02).
Interaction between a 70‐kDA heat shock protein and phosphatidylserine in stressed human cells
Authors: Bilog Andrei D , Labanieh Cedra , Nikolaidis Nikolas 
Published: November 2023

Seventy‐kilodalton heat shock proteins (Hsp70s) are molecular chaperones playing essential roles in protein homeostasis and cellular survival. Apart from their indispensable roles in cellular homeostasis specific Hsp70s also localize at the plasma membrane and bind to several lipids, including phosphatidylserine (PS). The interaction of Hsp70s with PS regulates important processes, like microautophagy and cell death. However, the conditions that trigger and promote the interaction between Hsp70s and intracellularly localized PS in human cells remain unknown. To shed light to this newly described property of Hsp70s, we tested whether heat‐shock, a stress known to affect both protein expression and membrane lipid composition, triggers the binding of HspA1A, a stress inducible Hsp70, to PS. To this end, we first determined whether HspA1A and the C2 domain of lactadherin (Lact‐C2), a known PS‐biosensor, compete for binding to intracellular PS. Specifically, HeLa cells were subjected to mild heat‐shock and the competition was assessed by quantifying the amount of membrane localized HspA1A in the presence or absence of the Lact‐C2 protein. These imaging experiments revealed that in the absence of Lact‐C2 HspA1A's membrane localization increases continuously after heat‐shock and reaches a maximal value at 8 hours during recovery. In the presence of Lact‐C2, however, HspA1A's membrane localization was minimal and did not show an increase during recovery after heat‐shock. These experiments were verified using cell surface biotinylation. In these experiments, Hek293 cells, after heat‐shock were incubated with membrane impermeable biotin, lysed, and incubated with streptavidin agarose beads, and the biotinylated proteins were analyzed by western blots and quantified using densitometry. Collectively, these results strongly suggest that HspA1A binds to intracellular PS, and that HspA1A's membrane localization and anchorage depends on its interaction with PS. These observations institute PS as a new and dynamic partner in the cellular stress response and establish the required foundation to directly assess the biological implications of this newly described and largely uncharacterized function of Hsp70s.Support or Funding InformationThis project was supported by funds from NIH and CSUF to NN
Understanding Ethanol Hexanoyl Transferase (Eht1) Localization to Lipid Droplets and the Endoplasmic Reticulum in Saccharomyces cerevisiae
Authors: Zhu Jie , Wheeldon Ian 
Published: November 2023

Lipid droplets (LDs) are ubiquitous organelles that play central roles in energy and lipid metabolism. They have a unique structure: a core of neutral lipids, composed of tryacylglyceride (TAG) and sterol esters (SEs), surrounded by a phospholipid monolayer. LDs are also decorated with proteins that mediate important metabolic functions and inter‐organelle communications. However, the mechanisms protein targeting to LDs are not well understood and still under investigation. It is understood that proteins with a membrane‐embedded, hydrophobic hairpin motif can access LDs from the endoplasmic reticulum (ER) either during LD formation or through ER‐LD membrane bridges. Another proposed mechanism is that proteins are able to access and bind to LD surface from cytosol through amphipathic helices, hydrophobic domains, lipid modifications, and protein‐protein interactions. Our protein of interest is Eht1 an acyl‐coenzyme A: ethanol O‐acyltransferase from Saccharomyces cerevisiae. Our data shows that Eht1 localizes to the ER and subsequently to LDs; however, it lacks hydrophobic hairpin motifs, has no clear transmembrane domains, and there is no evidence of amphipathic terminal helices or other known LD targeting domains. The aim of this work is to understand the trafficking mechanisms of Eht1 from ER to LDs in S. cerevisiae. Structure and function analysis suggests that both N‐ and C‐termini are crucial for its membrane association. In addition, fluorescence microscopy analysis suggests that the ER translocon may be related to the translocation process of Eht1. Furthermore, to understand how Eht1 interactions with other proteins contribute to the trafficking, we conducted a series of immunoprecipitation experiments and identified associated proteins by mass spectrometry (MS). We are currently exploring the genetic interactions between the MS‐identified proteins and Eht1 through the generation of knockout library using CRISPR/Cas9 genome editing.Support or Funding InformationThis work was supported the National Science Foundation and the University of California, Riverside.