bluuebunny/crossref_metadata_2025_split huggingface dataset:
Crosstalk Between Tetraspanin‐Interacting Protein Igsf3 and Sphingolipid Metabolism
Authors: Schweitzer Kelly , Ni Kevin , Petrache Irina 
Published: June 2021

RationaleBuilding on our finding that the tetraspanin‐interacting protein Igsf3 controls glucosylceramide levels in lung endothelial and epithelial cells, we investigated the mechanism and functional impact of Igsf3 regulation of sphingolipid metabolism. We focused on tetraspanin‐controlled cell functions, such as cell migration and adhesion and effectors such as the tight junction proteins, including claudins.MethodsPrimary rat lung endothelial cells and human bronchial epithelial cells (Beas2b) were stably transfected with lentiviral IGSF3 shRNA (R‐shIGSF3 and B2B‐shIGSF3, respectively) or control shRNA or with pIGSF3‐V5His overexpression plasmid. Primary human lung microvascular cells (HMVECL) were treated with IGSF3 siRNA or non‐targeting siRNA. Gene expression profiles were investigated using TaqMan Low Density Arrays (TLDA) for genes involved in the sphingolipid metabolism in epithelial cells with IGSF3 knockdown and in lymphocytes lacking IGSF3. Cell migration and cell adhesion assays were performed using standard methods.ResultsLoss of IGSF3 in B2B‐shIGSF3 and in lymphocytes, in parallel with a dysregulation of glucosylceramide levels, was associated with significant increases in Dihydroceramide Desaturase 1 (DEGS1, p<0.05, p<0.0001), Neutral Sphingomyelinase (SMPD2, p=0.07, p<0.05), Acid Ceramidase (ASAH1, p<0.0001, p<0.01), and Glucosylceramide Synthase (UGCG, p<0.01; ns) mRNA. Functionally, all approaches to decrease IGSF3 caused a significant delay in cell migration, whereas cells overexpressing IGSF3 had increased migration compared to controls (p<0.001). The decrease in migration was attenuated with pharmacological inhibition of Ugcg (p<0.001). In addition, adherent or non‐adherent cells with decreased IGSF3 expression exhibited enhanced cell adhesion to fibronectin (p<0.0001) or to other cells (p<0.01), respectively, which was attenuated with pharmacological inhibition of Asah1 (p<0.05) and was associated with increased expression of claudin 6 (CLDN6, p<0.05, p<0.05, respectively), and decreased expression of claudin 7 (CLDN7, p<0.001, p<0.0001, respectively) mRNA.ConclusionsThe tetraspanin‐interacting protein IGSF3 promotes cell migration and inhibits cell adhesion through inhibition of glucosylceramide synthase and acid ceramidase, respectively. Conditions characterized by loss of IGSF3, through an upregulation of sphingolipid production towards glycosphingolipids and decreased cell migration, may impair functions that require proper cell migration, such as lung tissue repair.Support or Funding InformationFunding: Wollowick Chair of Pulmonary Research, Department of Medicine Research Microgrant, National Jewish HealthThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating Impact of Mycobacterial Physiology on Mycobacteriophage Life Cycles by Mass Spectrometry
Authors: Li Yi , Clase Kari L. 
Published: June 2021

Phage‐bacteria interaction plays an important role in both ecology and evolution. Phages lyse bacterial cells to keep nutrient and energy flows of the environment. Arm‐race between phages and bacteria drives evolution of both phages and bacteria. Current phage infection model demonstrates two typical types of phage‐bacteria interaction: lytic and lysogenic cycles. However, the model emphasizes the actions of individual phage particle and bacterial cell. Several researches have unveiled that phage‐bacteria interaction is behavior of both phage and bacterial communities, and a portion of bacterial cells can turn unsusceptible for phage infection. Since phage infection is initiated after the attachment of phage particles to bacterial cells, bacterial surface properties may impact phage‐bacteria interaction. Mycobacterium smegmatis (M. smegmatis) has a thick lipid‐rich cell envelop. Previous researches elucidated that glycopeptidolipids (GPLs) in cell envelope are involved in cell surface properties, receptors of phages and colony morphology of M. smegmatis. Therefore, GPLs may impact phage‐bacteria interaction. Besides GPLs, unique lipids including monomycoloyl trehaloses (MMTs) and triacyl glycerols (TAGs) of M. smegmatis may also impact phage‐bacteria interaction. As morphologies of both M. smegmatis cell and colony change with cell growth phase, composition of envelop lipids (GPLs, MMTs, TAGs etc.) and proteins may change with growth phage, which ultimately lead to alteration of phage‐bacteria interaction. To explore how the mycobacterial physiology affects mycobacteriophage life cycles, the impact of bacterial lipids and proteins composition change on phage and bacterial populations need to be investigated.Five mycobacteriophages from diverse clusters were selected to infect M. smegmatis cell cultures at early exponential and stationary phases for ten hours. Populations of both phages and bacterial cells were determined over time. After four and ten hours of infection, total proteins and lipids were extracted from subsamples of phage‐bacteria mixture, and further analyzed by mass spectrometry. The overall objective of this project is to figure out how phage life cycle alters when bacterial growth phase changes, and which proteins and lipids may be involved. The findings will provide a better understanding of phage‐host interactions over time and contribute towards novel applications in biotechnology.Support or Funding InformationHoward Hughes Medical Institute, Science Education Alliance; Polytechnic Institute, Purdue University; Department of Agricultural and Biological Engineering, Purdue University; Biotechnology Innovation and Regulatory Science Center, Purdue UniversityThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Examination of Piccolo Splice Variants in Regulated Insulin Secretion from Pancreatic β‐Cells
Authors: Quintana Victoria G. , Salamon Rebecca , Fenster Steven D. 
Published: June 2021

Insights regarding the regulated secretion of insulin from pancreatic β‐cells has implications for understanding diabetes and regulation of glucose metabolism. Piccolo is a high‐molecular weight protein and part of a multi‐molecular complex involved in insulin secretion. Interestingly, genetic variants of the Piccolo gene (PCLO) have been modestly associated with increased risk of early‐onset type II diabetes among Pima Indians. Piccolo may regulate insulin secretion through a novel mechanism by serving as a Ca2+ sensor that interacts with L‐type voltage‐dependent Ca2+ channels and other molecules associated with insulin secretory vesicles. The purpose of this study is to examine the role of Piccolo and its splice variants in insulin secretion. Piccolo was originally identified as a protein involved in regulation of neurotransmitter secretion at presynaptic active zones. In neurons, PCLO undergoes extensive alternative splicing. Two major splice variants have been found in both neurons and pancreatic cells: isoform‐1 (I‐1) and isoform‐2 (I‐2). I‐1 has two Ca2+‐binding C2 domains, C2A and C2B, while I‐2 includes only C2A. Alternative splicing also leads to additional minor splice variants with altered Ca2+‐binding properties. Specifically, an exon‐skipping event results in a splice variant that lacks exon 16, a 27‐nucleotide (nt) exon within the C2A domain. Exclusion of this exon (i.e. short variant) enhances the Ca2+‐ and phospholipid binding affinity of C2A motif of rat Piccolo. In this study, we sought to examine the role of Piccolo and its splice variants in Ca2+‐mediated insulin secretion in rat INS‐832/13 pancreatic β‐cells. We demonstrate that INS‐832/13 cells express both I‐1 and I‐2 and also the long and short variant of the C2A domain. In addition, we used CRISPR‐CAS gene editing to selectively target exon 1 of PCLO to examine how loss of function of Piccolo may affect glucose‐stimulated insulin secretion. Finally, we examined the effect of overexpression of the short or long C2A‐domain on insulin secretion.Support or Funding InformationResearch in this study was funded by NIH‐Maximizing Access to Research Careers Undergraduate Student Training in Academic Research (MARC U*STAR) award number T34GM092711, start‐up funds provided to Dr. Steven Fenster (Fort Lewis College), and funds received from SSNAP (Science Scholars the Native American Path) by SACNAS (Dr. Les Sommerville, Fort Lewis College). We would also like to thank Dr. Christopher Newgaard at Duke University for supplying the pancreatic cells and culturing procedure.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Great, Green, Glowing Worms: The Insulin/IGF‐1 Signaling Pathway Regulates C. Elegan Feeding Behavior
Authors: Gousy Nicole , Quadros‐Mennella Princy 
Published: June 2021

Type II diabetes mellitus is caused by cellular insulin resistance resulting in increased blood glucose levels. Insulin exerts its actions through a signaling cascade that can be modeled in the Caenorhabditis elegan. The Insulin/IGF‐1 signaling (IIS) pathway found in C. elegans contains the same key genes that are involved in human insulin signaling. These genes include the Daf‐2 gene, encoding for an insulin‐like receptor, the Daf‐16 gene, encoding for transcription factors, and Age‐1 gene, encoding for the phosphatidylinositol 3‐kinase (PI(3)K). When C. elegans sense food in their environment, insulin is released, initiating the IIS pathway, resulting in food uptake via pharyngeal pumping. This study will explore the effects that these specific genes have on feeding behaviors based on how knockout mutations of these genes affect the insulin signaling pathway. To study the pumping rate as in indicator of insulin signaling, we will measure the amount of E. coli in their intestines. The greater the rate of pumping, the higher the concentration of E. coli within the intestines. The worms will be fed GFP‐expressing E. coli so that when placed under a confocal microscope, the E. coli within the intestines will fluoresce. From there the intensity of fluorescence can be analyzed for fluorescence intensity using image analysis software and will be compared between the different mutants. We hypothesize that the Daf‐2 and Age‐1 mutants will show fluorescence indicative of little feeding activity since both of these genes further activate components of the pathway to instigate feeding. However, we hypothesize that the Daf‐16 mutants will show fluorescence similar to that of the wildtype nematodes. This is because Daf‐16 works as a negative inhibitor towards the signaling pathway. This research is significant because it can provide how mutations in homologous genes can contribute to type II diabetes.Support or Funding InformationWe would like to acknowledge the help of Dr. Yadilette Rivera Colon, for her expertise, as well as Emily Burns for her lab assistance. In addition, we would like to thank Eileen Stern for her financial contribution under the Stern Scholarship for Undergraduate Researchers.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Perturbation of FoxO1 expression levels in 3T3‐L1 pre‐adipocytes using cinnamon extract
Authors: Dingess Alexandria , Bova Kaitlin , Aulthouse Amy , Stockert Amy 
Published: June 2021

Type 2 diabetes and obesity are among the most prevalent chronic diseases in the United States. Recent research has suggested that these disease states lead to numerous changes in the transcriptome that can be both causal or progressive in nature. Cinnamon has been explored as a potential therapy for type 2 diabetes based on observed decreases in blood glucose in some clinical studies, although its therapeutic benefits are still challenged and its mechanisms of action are still unknown. Aqueous cinnamon extract contains polyphenols, which have been shown to elicit epigenetic changes in several studies. Several key metabolic regulators, thought to be sensitive to epigenetic changes have been identified as players in these disease states. Metabolic regulator FoxO1 is one potential protein sensitive to cinnamon treatment. In addition to metabolic control, FoxO1 has been implemented in numerous other processes including aging, cell death, and lipid localization. Here we present the changes in FoxO1 expression in 3T3‐L1 pre‐adipocytes during early and mid‐stage differentiation both in the presence and absence of cinnamon extract. We have identified increases in FoxO1 expression during mid‐stage differentiation when treated with cinnamon extract. Together with clinical data that demonstrates a phenotype of decreased blood glucose levels in some type 2 diabetic patients when treated with cinnamon, these data suggest the involvement of FoxO1 in the anti‐diabetic mechanisms of cinnamon.Support or Funding InformationOhio Northern University Raabe College of Pharmacy Bower, Bennett and Bennett Research AwardThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sustained O‐GlcNAcylation Amplifies Erk Signaling
Authors: Cork Gentry K. , Tan Ee Phie , Thompson Jeffrey , McGreal Steven , Machecek Miranda , Kelsh John , Peterson Kenneth , Apte Udayan , Slawson Chad 
Published: June 2021

O‐linked N‐Acetylglucosamine, more commonly referred to as O‐GlcNAc, is a single glycosidic post‐translational modification. OGT (O‐GlcNAc Transferase) and OGA (O‐GlcNAcase) are the sole enzymes responsible for the addition (O‐GlcNAcylation) and removal of O‐GlcNAc, respectively. O‐GlcNAc, OGA, and OGT are present in all multicellular organisms and organ tissues. Importantly, OGT knockouts are embryonically lethal, demonstrating the requirement for O‐GlcNAcylation in early organismal development. Like most intracellular post‐translational modifications, O‐GlcNAc regulates a number of cellular pathways including mitochondrial respiration, cell cycle progression, and transcription. Recently, we demonstrated that prolonged treatment of SY5Y neuroblastoma cells with Thiamet‐G (TMG), an OGA inhibitor, caused a dramatic reprogramming of the transcriptome. Therefore, we performed next generation RNA sequencing to investigate the transcriptome changes from isolated liver tissues from mice treated with TMG for two weeks. We also performed the same analysis for liver tissue containing an OGT knockout (OGT‐KO) to compare the effects of sustained O‐GlcNAcylation to the loss of O‐GlcNAcylation. Gene Set Enrichment Analysis (GSEA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed differences in expression for gene sets between the two groups. Further analysis of the data demonstrated an inverse correlation of transcriptional changes for several gene families between TMG‐treated and OGT‐KO. MAPK/ERK signaling genes were among these families, which we found to be of interest. ERK (extracellular regulated protein kinase) phosphorylates a wide variety of protein substrates, regulating processes such as apoptosis, transcription, and cell cycle progression. OGT‐KO was predicted to down‐regulate this pathway while our previous data predicted TMG to up‐regulate the pathway. To test this observation, we grew HeLa cells in TMG for three weeks and serum starved them overnight. The following day, we returned the serum to the media and harvested the lysate at 0, 5,10, 20, 60, and 120 minute time points. When we performed a western blot analysis of the results, the TMG‐treated group demonstrated dramatic amplification of pERK. This observation demonstrates a novel role for O‐GlcNAc as a regulator of the ERK signaling pathway.Support or Funding InformationNIH NIDDK R01 DK100595This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Combining mass spectrometry and glycan array data to explore invertebrate glycomes
Authors: Wilson Iain , Eckmair Barbara , Hykollari Alba , Yan Shi , Vanbeselaere Jorick , Paschinger Katharina 
Published: June 2021

As our glycoanalytical possibilities increase and the amounts of glycomic data on non‐mammalian species accumulate, we can start to consider whether the structures of glycoconjugates from invertebrates can be fitted into a sort of glycophylogeny and which protein receptors recognise them. Our recent data show that N‐glycans from a variety of nematode, mollusc and insect species contain a range of core and antennal modifications with fucose, galactose, glucuronic acid, sulphate, phosphoethanolamine, aminoethylphosphonate and phosphorylcholine. On one hand, we employ an off‐line HPLC/MALDI‐TOF‐MS workflow to assess the actual glycan structures; on the other, beyond previous Western blotting data, we are now printing invertebrate N‐glycomes in a microarray format. Thereby, we are uncovering interactions with different lectins, pentraxins and antisera. This approach should allow us to explore whether and how invertebrate glycomes, especially from medically or biotechnologically relevant species, interact with mammalian immune systems.Support or Funding InformationThis work is funded by the Austrian Science Fund (FWF)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Understanding Heparin/Hparan Sulfate Biosynthetic Pathway in the Generation of Antithrombin Binding Motif using Combinatorial Virtual Library Screening (CVLS)
Authors: Bi Yiling , Sankaranarayanan Nehru Viji , Kuberan Balagurunathan , Desai Umesh R. 
Published: June 2021

Heparin/Heparan sulfate glycosaminoglycans (HS‐GAG), is an essential sulfated polysaccharide that regulates multiple biological and pathological processes. Previous biochemical studies have narrowed down antithrombin III binding heparin/heparan sulfate pentasaccharide binding motif responsible for its anticoagulant activity. However, the synthetic sequence did not elucidate how the binding motif is generated in the HS‐GAG biosynthetic pathway. Therefore, novel approaches are required to understand the generation of antithrombin binding domain in the biological system. Here, we utilized computational modeling to understand how different HS biosynthetic enzymes evolve and coordinate together to generate antithrombin binding domain. We constructed a virtual library of tetrasaccharide and hexasaccharide HS sequences with different disaccharide combinations and modifications. Based on 3‐O‐sulfotransferase isoform 1 (3OST‐1) crystal structure and the Combinatorial Virtual Library Screening (CVLS), we found that pentasaccharide could be the minimum recognition motif for 3OST‐1 enzyme. Also, through enzyme‐substrate specificity analysis, we found that 3OST‐1 preferred 6‐O‐sulfo, N‐sulfoglucosamine (GlcNS6S) to N‐sulfoglucosamine (GlcNS), while 2‐O‐sulfation was dispensable, suggesting that 6‐O‐sulfation occur before 3‐O‐sulfation and facilitate 3OST‐1 recognition. In comparison study with 3OST‐3a, which only generates glycoprotein D binding site, we also rationalized the positioning of glucuronic acid (GlcA) instead of the iduronic acid (IdoA) at the non‐reducing end of the modification site. A chemoenzymatic approach was exploited to further validate our findings. Our study used antithrombin domain generation as a test case, and could pave the way to study other GAG biosynthetic enzymes to advance our knowledge of the HS‐GAG biosynthetic pathway.Support or Funding InformationNIH P01 (HL107152) Programs of Excellence in Glycosciences (PEG)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Beyond ERAD: N‐glycanase will bring you to tears
Authors: Tambe Mitali , Ng Bobby , Freeze Hudson 
Published: June 2021

Patients with mutations in NGLY1 cannot make tears (alacrima), have global developmental delay, movement disorder and liver dysfunction. N‐glycanase 1 (NGLY1) de‐glycosylates misfolded N‐glycosylated proteins in the cytoplasm as part of the ERAD pathway prior to their proteasomal degradation. Surprisingly, NGLY1‐deficient patient cells do not accumulate cytoplasmic misfolded N‐glycoproteins, suggesting a more complex function. Interestingly, new studies show NGLY1 is needed for transcription factor NFE2L1/NRF1 activation and normal mitochondrial function.Using WT and NGLY1‐deficient mouse embryonic fibroblasts (MEFs) we found that NGLY1‐deficient cells were resistant to hypotonic lysis compared to WT. The same was seen in NGLY1‐patient fibroblasts. Additionally, MEFs deficient in both NGLY1 and ENGase (another deglycosylation enzyme) were even more resistant to hypotonic lysis compared to NGLY1‐deficient cells. Water influx and cell swelling precedes cell lysis. We found that NGLY1‐deficient MEFs swell slower than WT MEFs. Since aquaporins (AQP) transport water, we hypothesized that AQP levels might be disrupted in NGLY1‐deficient cells. We found both AQP1 mRNA and protein were reduced in NGLY1‐deficient MEFs. shRNA knockdown of AQP1 in WT MEFs decreased hypotonic lysis, suggesting AQP1 is associated with hypotonic lysis. NGLY1 shRNA and CRISPR studies confirmed that NGLY1 regulates AQP1 levels and hypotonic cell lysis. Preliminary studies show that complementing NGLY1‐deficient MEFs with NGLY1 increases AQP1 and restores hypotonic lysis. Current efforts are directed towards understanding whether NGLY1 enzyme activity is necessary for AQP regulation. 13 types of AQP are expressed in different cell types. Our preliminary data suggest that AQP11 levels are disrupted in NGLY1‐deficient patient cells and CRISPR cells, suggesting that NGLY1 might regulate different types of AQP.We have identified a novel function of NGLY1, i.e. to directly/indirectly regulate AQPs. This finding may relate to NGLY1‐deficient patients' inability to make tears. Future efforts will try to identify NGLY1‐dependent transcription factors responsible for regulating AQPs. This work is supported by the Bertrand Might Research Fund and NGLY1.org.Support or Funding InformationThis work is supported by the Bertrand Might Research Fund.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.