bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ethanol and both Endogenous and Exogenous Reactive Species Accelerate the Mutation Rate of Hepatitis C Virus RNA
Authors: Seronello Scott , Presleigh Kristen , Barlow Miriam , Choi Jinah 
Published: June 2021

Hepatitis C virus (HCV) demonstrates a high level of genetic variability existing as a population of closely related but distinct genetic variants, referred to as quasispecies. Ethanol intake is one of the major cofactors in HCV‐induced pathogenesis and is correlated with increased sequence heterogeneity of HCV RNA in patients, which is likely to promote viral persistence and complicate anti‐HCV therapy. However, the mechanism underlying this interaction between HCV and ethanol is unknown. The purpose of this study was to examine whether ethanol and endogenous and exogenous reactive species can increase the mutation rate of HCV. The NS5A region of Con1 HCV replicon RNA was sequenced after treatment with ethanol, acetaldehyde, exogenous hydrogen peroxide, and L‐buthionine‐S,R‐sulfoximine, which accelerated the mutation rate of HCV RNA over that of the control group within 48 hrs. The dn/ds ratio also increased significantly with the nonsynonymous mutations clustering in the C‐terminal half of NS5A. Half of amino acid substitutions involved serine, threonine, or tyrosine, suggesting possible changes in NS5A phosphorylation. The treatments also induced RNA damage. Therefore, ethanol may synergize with virus‐induced oxidative/nitrosative stress to induce RNA damage that amplifies the error rate of the viral replicase increasing the heterogeneity of HCV RNA.This work was supported by funds from UC Merced.
Dose dependent effects of dominant‐negative K6W‐ubiquitin: Construction of mini‐genes that encode multiple copies to K6W‐ubiquitin
Authors: Shang Fu , Guo Weimin , Taylor Allen , Bian Qingning 
Published: June 2021

PurposeWe previously demonstrated that K6W‐ubiquitin behaves as a dominant negative inhibitor of the ubiquitin proteasome pathway (UPP). The objective of this work is to construct mini‐genes that encode multiple copies K6W‐ubiquitin for effectively inhibiting the function of the UPP for investigating the roles of the UPP in vivo and in vitro.MethodsMouse cDNA for ubiquitin was used as a template to generate mini‐genes that encode 1, 2, 4 and 8 copies of K6W‐ubiquitin, respectively. To test the effects of K6W‐ubiquitin on UPP‐mediated degradation, HeLa cells were co‐transfected with plasmids that encode K6W‐ubiquitin and a plasmid that encodes UbG76V GFP. Levels of GFP were monitored under fluorescent microscope and by Western blotting.ResultsSimilar to endogenous poly‐ubiquitin, the tandem poly‐K6W‐ubiquitin was processed into mono ubiquitin in the cells. As compared on the basis of same amount DNA, levels of mono‐K6W‐ubiquitin and high mass ubiquitin conjugates were proportional to copies of K6W‐ubiquitin in the mini‐genes. Expression of K6W‐ubiquitin in HeLa cells resulted in a dose‐dependent inhibition of UPP‐mediated degradation of UbG76V GFP and TNFα‐induced degradation of I‐κB.ConclusionsK6W‐ubiquitin inhibits UPP‐mediated protein degradation in a dose dependent manner. Mini‐genes that encode multiple copies of K6W‐ubiquitin are unique tools for investigating the functions of the UPP.
SBP1 Genetically Interacts With WHI3, a Cell Cycle Control Gene in Saccharyomyces cerevisiae
Authors: Segal Scott , Theede Kelly 
Published: June 2021

Proper control of cell cycle is crucial allows cells to grow to the appropriate size in the budding yeast, Saccharyomyces cerevisiae, and to ensure cancer formation does not occur in humans. Although there are a number of cell cycle checkpoints which control passage through the cell cycle, the most critical of these is the G1/S checkpoint, also known as START. Upon passage through the G1/S checkpoint, the cell is committed to replicate its DNA and undergo a round of cell division. In yeast, expression of the cyclin Cln3p, a mammalian cyclin homolog, is necessary to progress through the G1/S checkpoint. Cln3p accumulates to high levels during late G1phase and forms a complex with Cdc28p. The Cln3p/CDC28 complex then enters the nucleus and drives the cell through the G1/S checkpoint.In order to control Cln3p levels through early and middle G1 phase, such that the cell proceeds through the G1/S checkpoint at the appropriate time, it is thought that Whi3p, an RNA recognition motif (RRM) containing protein, translationally represses the CLN3 mRNA. Recently, a genetic interaction was identified between WHI3 and SBP1. SBP1 encodes a single stranded RNA binding protein Sbp1p which has been shown to work in conjunction with Dhh1p repress global mRNA translation under physiological stress. Therefore, it is possible Whi3p may work in conjunction with Sbp1p to translationally repress the CLN3 mRNA during early G1 phase. In this work, we show that overexpression of Sbp1p has a significant effect in whi3Δ cells. First, overexpression of Sbp1p in whi3Δ cells results in a significant growth defect at 37° C. Second, overexpression of Sbp1p in whi3Δ cells results in increased cell size as compared to whi3Δ cells. Next, we will investigate whether Sbp1p is necessary for CLN3 mRNA translational repression.
Cholesterol‐depletion in human blood‐derived neutrophils by methyl‐β‐cyclodextrin leads to the formation of neutrophil extracellular traps (1001.5)
Authors: Neumann Ariane , Brogden Graham , Brodesser Susanne , Naim Hassan Y. , von Köckritz‐Blickwede Maren 
Published: June 2021

The formation of neutrophil extracellular traps (NETs) as an innate immune defence mechanism has been shown to be the result of a novel cell death process called NETosis. Mechanisms leading to the release of NETs are still not completely understood. The objective of the study was to investigate the role of cholesterol in the formation of NETs.To this end, primary human neutrophils were treated with different concentrations of methy‐β‐cyclodetxrin (MβCD) to deplete cholesterol from the cell. The effect of MβCD on the lipid composition of the cells was determined using TLC. The identities of lipids separated by TLC were confirmed by MS/MS. Furthermore, the formation of NETs was studied using immunofluorescence microscopy.Treatment of neutrophils with MβCD revealed distinct changes in the lipid composition. These changes were statistically significant for cholesterol; other lipid classes like phosphatidylethanolamine and sphingomyelin were only slightly affected. Interestingly, neutrophils treated with MβCD showed a dose‐ and time‐dependent formation of NETs. In contrast, the treatment of neutrophils with N‐butyl‐deoxynojirimycin to reduce the levels of glycosphingolipids had no effect on NET formation, thus confirming a cholesterol‐specific effect.In conclusion, this study shows that MβCD treatment of neutrophils leads to the depletion of cholesterol and thereby facilitates formation of NETs.
Role of ICAM‐1 hypoglycosylation in atherosclerotic plaque formation (1003.1)
Authors: Vallejo Matthew , Scott David , Patel Rakesh 
Published: June 2021

N‐glycosylation is an ubiquitous post‐translational modification of proteins secreted from cells or expressed on the cell surface. N‐glycan maturation occurs sequentially moving from high mannose to hybrid to complex glycoforms. We recently showed that that during chronic vascular inflammation that underlies atherosclerosis, endothelial cell surface proteins are hypoglycosylated, characterized by increases in high mannose / hybrid glycoforms. Importantly, these epitopes provide ligands for monocyte rolling and adhesion. The protein(s) or adhesion molecule(s) that harbor high mannose epitopes to mediate monocyte rolling and adhesion was determined in this study. Evidence is provided that that a high mannose glycoform of intercellular adhesion molecule‐1 (ICAM‐1), referred to as HM‐ICAM‐1, is produced in activated endothelial cells. HM‐ICAM‐1 formation was also indicated on human atherosclerotic lesions by proximity ligation assay analysis. Moreover, a role for HM‐ICAM‐1 in mediating monocyte rolling and adhesion was demonstrating in cell culture by expressing ICAM‐1 in Cos‐1 cells in the presence of absence of selective inhibitors of N‐glycan processing (kifunensine or swainsonine) which result in high mannose N‐glycan accumulation. Interestingly, at equal surface expression levels, HM‐ICAM‐1 was more effective at binding monocytes than WT (complex glycosylated ICAM‐1). Finally, we present data showing the effects of mutation of all eight individual N‐glycan binding sites on ICAM‐1 and effects on monocyte rolling and adhesion. The assessment of the specific role of each N‐glycan site in ICAM‐1 may lead to future N‐glycan targeted therapies to modulate atherogenic inflammation.Grant Funding Source: Supported in part by by NIH T32 HL007457, Mechanisms of Hypertension and Cardiovascular Disease
N‐glycan analysis of IgG by enzymatic digestion with Remove‐iT® Endo S and PNGase F (1004.1)
Authors: McLeod Elizabeth , McClung Colleen , Bielik Alicia , Magnelli Paula , Guthrie Ellen 
Published: June 2021

Objective: The objective of this study was to compare the release of N‐glycans from different sources of IgG using a novel endoglycosidase with a chitin‐binding domain tag (Remove‐iT Endo S) and PNGase F. Biotechnology companies are currently developing a growing number of monoclonal IgG antibodies as therapeutic agents. Critical for the structure and biological activity of the molecule is the N‐glycan moiety attached to the asparagine 297 residue in the constant domain of the antibody. There are many variables in cell culture systems that can greatly influence the heterogeneity of the glycans on IgG. For this reason it has become increasingly important to monitor the glycosylation profiles of these biotherapeutics in the production process.Methods: Remove‐iT Endo S (also known as Endoglycosidase S) was cloned from Streptococcus pyogenes and overexpressed in E. coli as a fusion to the chitin‐binding domain. IgG samples were enzymatically deglycosylated under native conditions using Remove‐iT Endo S and PNGase F Glycerol Free. The deglycosylated IgG samples were then analyzed by SDS‐PAGE gel shift analysis and ESI‐TOF MS to estimate the degree of deglycosylation. In addition, the glycans released from IgG following deglycosylation by both enzymes were analyzed using nano LC‐TOF MS.Results: Remove‐iT Endo S has a high specificity for removing the N‐glycan moiety of IgG after the first N‐acetylglucosamine (GlcNAc) residue, leaving only the GlcNAc with or without a core fucose residue. However, PNGase F cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid and complex oligosaccharides from N‐linked glycoproteins.Conclusion: Remove‐iT Endo S is a more robust enzyme for the method described herein as it completely removes the sugar residues from IgG. Conversely, the PNGase F glycerol free digest does not result in a complete digestion under native conditions.
ArtinM activates CD4+ T cells through recognition of N‐glycans of CD3γ chain (1004.3)
Authors: da Silva Thiago , Souza Maria , Roque Barreira Maria Cristina 
Published: June 2021

ArtinM, from Artocarpus heterophyllus, a lectin that binds to the manotriose core of N‐glycans, modulates immunity toward Th1 axis and confers resistance to intracellular pathogens. These properties were credited to the ArtinM binding to TLR2 N‐glycans on innate immune cells, with the consequent production of IL‐12. In this work we investigated the ArtinM effects on murine CD4+ T cells. ArtinM stimulated CD4+ T cells released high levels of IL‐2, IFN‐γ, IL‐6 and IL‐17A and increased the expression of CD25 and CTLA‐4. These effects were inhibited by D‐mannose. To determine the ArtinM glycotarget(s) on CD4+ T cells, specific antibodies for CD3εγ, CD3ε, CD28, CD45 and CD4 were assayed for the inhibition of the responses to ArtinM. Only anti‐CD3εγ antibody prevented the cytokines secretion and the ArtinM binding to CD4+ T cells. Because anti‐CD3ε had no effect on the responses to ArtinM, we inferred that CD3γ glycan(s) is(are) targeted by ArtinM and accounts for its ability to activate CD4+ T cells. By using pharmacological inhibitors of signaling molecules, we verified that PI3K, PTK and p42/44MAPK participate in the cytokine production induced by ArtinM. We conclude that ArtinM (1) activates CD4+ T cells through binding to glycans of CD3γ chain and (2) promotes naive CD4+ T cells differentiation into Th1 and Th17. We postulate that the effects exerted on CD4+ T cells contribute for the immunomodulatory activity of ArtinM.Grant Funding Source: Supported by FAPESP, CAPES, CNPq, FAEPA, FINEP
Specificity of glycosaminoglycan‐protein interactions: the role of desolvation (1007.5)
Authors: Sarkar Aurijit , Desai Umesh 
Published: June 2021

Why do most glycosaminoglycan (GAG)‐protein interactions lack specificity? We hypothesized that desolvation of highly charged groups, which is highly unfavorable, plays an as‐yet‐unrecognized dominant role. We performed molecular dynamics (MD) simulations of water binding to antithrombin (AT) and thrombin (T), two prototypic ‘specific’ and ‘non‐specific’ GAG‐binding proteins respectively, to parse the role of desolvation energy (DE). The DE for Arg/Lys sidechains was a substantial ~4.5 kcal/mol, which counteracts enthalpic gains during hydrogen‐bond formation with GAG sulfates. This DE was irrespective of the location ‐ within the heparin‐binding site (HBS) or elsewhere ‐ on either protein, suggesting that hydrogen‐bonding by Arg/Lys residues with GAG sulfates does not explain the interaction specificity. However, DE for selected uncharged sidechain‐ and/or backbone‐ donors may play a dominant role. Specifically, Asn45 of AT contributes singularly to GAG recognition with a much‐reduced DE, while no such equivalent uncharged residue exists for T. These results find support in mutagenesis and crystallography studies reported in literature. The key finding that GAG specificity for proteins arises from hydrogen‐bonding with a select group of uncharged donors due to lower DE penalty is being extended to the serpin family of proteins.Grant Funding Source: Supported by NIH grants R01 HL090586 and P01 HL107152 to URD, and NCCR grant S10 RR027411 to VCU.
Endo F3 glycosynthase mutant enables chemoenzymatic synthesis of core fucosylated triantennary complex type glycopeptides and glycoproteins (1007.7)
Authors: Giddens John , Lomino Joseph , Wang Lai‐Xi 
Published: June 2021

Chemoenzymatic synthesis of glycoproteins, which enables the production of glycoproteins with homogenous glycans, is an essential tool for structural and functional studies of glycoproteins. The endo‐β‐N‐acetylglucosaminidase from Elizabethkingia meningosepticum, Endo F3, is an endoglycosidase that can hydrolyze complex type N‐glycans and also possess transglycosylation activity. We have generated glycosynthase mutants by site‐directed mutagenesis and have found that the glycosynthases were capable of efficiently transferring complex type N‐glycans to GlcNAc‐peptide/protein without product hydrolysis. Our experimental data have further shown that the Endo‐F3 glycosynthases are able to use tri‐antennary glycan oxazolines as substrates for transglycosylation, which were unable to achieve by previously reported endoglycosidase mutants derived from Endo‐A, Endo‐M, Endo‐D and Endo‐S. The usefulness of the Endo‐F3 mutants was demonstrated by the synthesis of CD52 antigen and by the efficient Fc glycosylation remodeling of monoclonal antibody rituximab. The binding of these novel glycoforms of rituximab to FcγRIIIa and galectin‐3 is being analyzed. This chemoenzymatic approach permits an efficient synthesis of core fucosylated bi‐ and tri‐antennary glycopeptides and the glycosylation remodeling of intact monoclonal antibodies, making it possible to probe the structural and functional roles of the attached glycans in more details.Grant Funding Source: Supported by NIH R01 GM080374
Endoplasmic reticulum stress induces alveolar epithelial cell apoptosis through the activation of the unfolded protein response (1010.10)
Authors: Nguyen Hang , Dang MyTrang , Ono Shinji , Morimoto Konosuke , Uhal Bruce 
Published: June 2021

STUDY OBJECTIVE: Our prior work showed that ER stress‐induced apoptosis of AECs is regulated by the autocrine angiotensin (ANG)II/ANG1‐7 system. MG132 or surfactant protein C (SP‐C) BRICHOS domain mutation G100S induced apoptosis in human AECs by activating cathepsin D or decreasing ACE‐2. This study tested the hypothesis that ER stress‐induced apoptosis of human AECs might be mediated by the activation of the UPR thus regulates the ANGII/ANG1‐7 system.METHODS: A549 cells were challenged with proteasome inhibitor MG132 or SP‐C BRICHOS domain mutant G100S. The level of UPR markers and angiotensin components were measured by Western blotting. AEC apoptosis was measured by caspase activation and nuclear fragmentation.RESULTS: MG132‐ or G100S mutation‐induced ER stress activated the UPR pathways (eIF2α, ATF4, IRE1, XBP1), which led to an increase of cathepsin D or a decrease of ACE‐2. These changes were inhibited by the presence of UPR inhibitors, thus blocking the AEC apoptosis caused by the ER stress.CONCLUSIONS: These data show that ER stress induces apoptosis in human AECs through UPR‐mediated pathways that in turn regulate the autocrine ANGII/ANG1‐7 system. It is also demonstrated that ER stress‐induced AEC apoptosis can be blocked by inhibition of the UPR pathways.Grant Funding Source: PHS HL‐45136
Effects of exposure to bisphenol A on hepatocytes and pre‐adipocytes in vitro (1010.7)
Authors: Macis Elias , Kostrominova Tatiana 
Published: June 2021

In recent years, environmental pollutants/endocrine disrupting chemicals (EDCs) have received special attention as metabolic disruptors that produce adverse developmental, reproductive, neurological, cardiovascular, metabolic and immune effects. Bisphenol A (BPA) is one of the most abundant EDCs present in the environment. We focused our study on the effects produced by the exposure to BPA on cell viability, apoptosis and DNA fragmentation in FaO hepatocytes and 3T3 pre‐adipocytes in vitro. In particular, we were interested whether there are considerable differences in the sensitivity to cytotoxic effects of BPA in hepatocytes and pre‐adipocytes when evaluated under the same experimental conditions. Exposure to 10 and 100 μM of BPA showed that although morphology of both cell lines was affected after 24 and 48 hours of exposure, the effect on 3T3 pre‐adipocytes was more pronounced. Most probably exposure to BPA attenuated cell growth or caused cell detachment since the number of viable cells attached to the substrate did not show statistically significant differences when evaluated by trypan blue exclusion method. Several methods for apoptotic DNA fragmentation evaluation were compared. Qiagen DNeasy kit for DNA isolation showed the best detection sensitivity. Exposure to 100 μM of BPA for 48 hours showed presence of apoptotic DNA ladder when evaluated by gel electrophoresis.Support: IUN and IUSM‐NW.Grant Funding Source: Undergraduate Research Fund IUN
Magnolol affects the expression of polyamine biosynthesis and catabolism‐linked proteins in PC3 human prostate cancer cells in vitro (1011.1)
Authors: McKeown Brendan , Hurta Robert 
Published: June 2021

This study examined the effect of magnolol on the expression of proteins involved in polyamine biosynthesis/catabolism in PC3 prostate cancer cells in vitro. Magnolol, 80 μM for 6 hours, decreased cellular viability by ~50%. Magnolol exposure decreased expression of ornithine decarboxylase (ODC), a key protein in polyamine biosynthesis, and antizyme‐inhibitor (AZI) with no apparent change to expression of antizyme (AZ), an ODC inhibitor. Expression of spermidine/spermine N1‐acetyltransferase, a key protein in polyamine catabolism, was also decreased. These magnolol‐mediated changes to ODC & AZI expression suggest that changes in polyamine biosynthesis would occur. An AZI/AZ ratio decrease in response to magnolol favors growth repression of PC3 cells. Cellular signaling proteins were also evaluated, with no apparent change occurring in ERK1, ERK2, p‐ERK1, p‐ERK2, p38, p‐JNK2, PI3Kp110 & AKT expression. Decreased expression of p‐p38, JNK1, JNK2, PI3Kp85, p‐PI3Kp85 & p‐AKT and increased expression of p‐JNK1 were observed. This study demonstrates that magnolol can alter the behavior of human prostate cancer cells in vitro by affecting activities associated with growth regulation. (Supported by the Jeanne and J.‐Louis Levesque Foundation.)Grant Funding Source: Supported by the Jeanne and J.‐Louis Levesque Foundation
Phosphatidic acid activates mTORC1 by displacing the inhibitor DEPTOR (1013.19)
Authors: Plaisier Christina , Yoon Mee Sup , Wu Cong , Truong Nga , Chen Jie 
Published: June 2021

The lipid second messenger, phosphatidic acid (PA), is a critical mediator of mitogenic activation of the mammalian target of rapamycin complex 1 (mTORC1). Previously we showed that PA binds to mTOR and activates mTORC1, but the mechanism of this activation remains incompletely understood. Here we propose a novel mechanism of mTORC1 activation by PA. Using mass spectrometry analysis to identify mTOR interactions regulated by PA, followed by confirmation by co‐immunoprecipitation, we discovered that the interaction between mTOR and its inhibitor DEPTOR was disrupted by PA. Dissociation of DEPTOR by PA coincided with activation of mTORC1. Phospholipase D1 (PLD1) catalyzes the production of PA and is also an established mediator of mitogenic activation of mTORC1. Treatment of cells with phorbol myristate acetate and lysophosphatidic acid, known to activate PLD1, recapitulated the DEPTOR displacement and mTORC1 activation achieved with exogenous PA, and this effect was decreased by PLD1 inhibition. Furthermore, by examining a panel of PA molecules with various fatty acid chains, we found that only the PA species known to be produced by PLD displaced DEPTOR and activated mTORC1 both in vitro and in cells. Taken together, our results strongly suggest that PA produced by PLD1 regulates mTORC1 activity by displacing the inhibitor, DEPTOR.Grant Funding Source: Supported by NIH GM089771 and AR048914 grants to J. Chen.
Plasma membrane recovery kinetics of a microfluidic intracellular delivery platform (1013.2)
Authors: Poceviciute Roberta , Sharei Armon , Jensen Klavs , Langer Robert 
Published: June 2021

Intracellular delivery of macromolecules continues to be a challenge in both research and therapeutic applications. Recently, physical delivery methods, such as electroporation and sonoporation, have emerged that provide direct access to the cytoplasm by disrupting the plasma membrane. For a rapid development of such methods the mechanisms behind their action need to be understood. We use the microfluidic shear‐based CellSqueeze delivery platform to examine the kinetics of plasma membrane recovery after disruption and its dependence on the calcium content in the surrounding buffer. Delivery experiments with HeLa cells show that the pores close ten times faster when calcium is present in the surrounding buffer. If we eliminate calcium from the buffer, we are able to improve delivery efficiency five‐fold without sacrificing cellular viability. Membrane repair characteristics initially observed in HeLa cells are shown to translate to primary naïve murine T cells, suggesting that calcium may have a similar role across many cell types. Subsequent manipulation of other delivery parameters enables the delivery of larger macromolecules, such as antibodies, into naïve t cells. The work provides insight into the membrane repair process in response to mechanical disruption and could potentially enable the development of improved delivery methods.Grant Funding Source: Supported by National Institutes of Health Grants RC1 EB011187‐02 and R01GM101420‐01A1