44 research outputs found
Regulation of cadherin adhesion at intercellular junctions
No full textAdhesion proteins maintain cell-cell interactions, which are critical for tissue formation and the hierarchical organization of all multicellular organisms, and among them, cadherins are the major transmembrane cell-cell adhesion proteins in all vertebrate tissues. Regulation of cadherin mediated adhesion at cell-cell junctions is crucial to our understanding of development and disease. This thesis focuses on the regulation of cadherin adhesion, which can be influenced by its extracellular domain interactions, ligand or antibody binding, post translational modifications, or inside out signaling from cytoplasmic binding proteins.
In this thesis, micropipette-based adhesion frequency measurements of cadherin-mediated, cell-cell binding kinetics identified a unique kinetic signature that appears to reflect both adhesive (trans) bonds between cadherins on opposing cells and lateral (cis) interactions between cadherins on the same cell. These kinetic measurements were used to assess the impact of confinement within narrow adhesion zones on the assembly of intercellular adhesions. Specifically, a unique kinetic signature suggested the formation of lateral interactions that were not detected in solution binding assays. Mutations postulated to disrupt lateral cadherin association altered the kinetic signature, but did not affect cadherin binding affinity. Perturbed kinetics further correlated with altered cadherin clustering at cell-cell junctions, wound healing dynamics, and paracellular permeability.
Adhesion frequency measurements were used to demonstrate the allosteric regulation of cadherin adhesive function. In this thesis, measured kinetics of cadherin-mediated intercellular adhesion demonstrated quantitatively that activating anti-E-cadherin monoclonal antibodies or the dephosphorylation of a cytoplasmic binding partner, p120 catenin, increased the homophilic binding affinity of E-cadherin on Colo 205 cells. Further studies of Colo 205 cells demonstrated that four treatments, which similarly altered p120 catenin phosphorylation resulted in quantitatively similar enhancement in E-cadherin affinity.
Using this approach, I further investigated the effect of N-linked and O-linked glycosylation on E-cadherin activity and function. Results revealed that, contrary to the influence of glycosylation on N-cadherin function, N-glycosylation of E-cadherin in the EC4 and EC5 domains negatively regulated cadherin adhesion, by altering binding kinetics and clustering at cell-cell junctions. This suggests the influence of N-glycosylation depends on its position in the cadherin ectodomain.
In conclusion, this dissertation describes studies which elucidated different mechanisms regulating cadherin adhesive function. Results showed that cadherin binding is regulated by its ectodomain interactions at cell-cell junctions, by glycosylation, and by allosteric inside-out signaling. These findings were enabled by the adhesion frequency measurements, which enabled quantitative assessment of cadherin binding function, in the native context of the cell membrane and cytosolic binding partners.Submission published under a 24 month embargo labeled 'U of I Access', the embargo will last until 2018-08-01The student, Nitesh Shashikanth, accepted the attached license on 2016-06-15 at 11:56.The student, Nitesh Shashikanth, submitted this Dissertation for approval on 2016-06-15 at 12:01.This Dissertation was approved for publication on 2016-06-16 at 14:36.DSpace SAF Submission Ingestion Package generated from Vireo submission #9668 on 2016-11-10 at 12:24:30Made available in DSpace on 2016-11-10T18:39:07Z (GMT). No. of bitstreams: 2
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Previous issue date: 2016-06-16Embargo set by: Seth Robbins for item 95428
Lift date: 2018-11-10T18:39:22Z
Reason: Author requested U of Illinois access only (OA after 2yrs) in Vireo ETD systemEmbargo set by: Seth Robbins for item 95428
Lift date: 2018-11-10T18:43:22Z
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Differentiation of the 50B11 dorsal root ganglion cells into NGF and GDNF responsive nociceptor subtypes
No full textThe embryonic rat dorsal root ganglion (DRG) neuron-derived 50B11 cell line is a promising sensory neuron model expressing markers characteristic of NGF and GDNF-dependent C-fibre nociceptors. Whether these cells have the capacity to develop into distinct nociceptive subtypes based on NGF- or GDNF-dependence has not been investigated. Here we show that by augmenting forskolin (FSK) and growth factor supplementation with NGF or GDNF, 50B11 cultures can be driven to acquire differential functional responses to common nociceptive agonists capsaicin and ATP respectively. In addition, to previous studies, we also demonstrate that a differentiated neuronal phenotype can be maintained for up to 7 days. Western blot analysis of nociceptive marker proteins further demonstrates that the 50B11 cells partially recapitulate the functional phenotypes of classical NGF-dependent (peptidergic) and GDNF-dependent (non-peptidergic) neuronal subtypes described in DRGs. Further, 50B11 cells differentiated with NGF/FSK, but not GDNF/FSK, show sensitization to acute prostaglandin E2 treatment. Finally, RNA-Seq analysis confirms that differentiation with NGF/FSK or GDNF/FSK produces two 50B11 cell subtypes with distinct transcriptome expression profiles. Gene ontology comparison of the two subtypes of differentiated 50B11 cells to rodent DRG neurons studies shows significant overlap in matching or partially matching categories. This transcriptomic analysis will aid future suitability assessment of the 50B11 cells as a high-throughput nociceptor model for a broad range of experimental applications. In conclusion, this study shows that the 50B11 cell line is capable of partially recapitulating features of two distinct types of embryonic NGF and GDNF-dependent nociceptor-like cells. Matusica Dusan, Canlas Jastrow, Martin M Alyce, Wei Yingkai, Marri Shashikanth, Erickson Andelain, Barry M Christine, Brierley M Stuart, Best G Oliver, Michael Z Michael, Voelcker H Nicolas, Keating J Damien, and Haberberger V Raine
Satellite-assisted content delivery network using MobilityFirst future internet architecture
No full textThis thesis presents the design of a satellite-assisted CDN that utilizes the MobilityFirst Future Internet Architecture (MF-FIA) for content delivery. Current CDN solutions require an overlay control framework on top of IP to deal with content caching and delivery. However these features can be integrated into the network layer in an ICN based design. The proposed CDN solution uses MobilityFirst, a representative example of ICN to show the benefit of an integrated network layer solution. The framework uses in-network caches to reduce the latency of content delivery relative to overlay CDN designs. Content requests are efficiently routed to the nearest content source using the MobilityFirst routing protocol. As content providers are aware of content popularity and its distribution, the framework supports a pushing mechanism where content providers can pro-actively push popular contents to designated cache locations. This can be efficiently done using services such as multicast or broadcast which are natively supported in MobilityFirst. The routing mechanism can choose to deliver contents either through a terrestrial network or through a broadcast medium such as a satellite. Finally the proposed architecture uses self-certifying content names that enable efficient content validation. A proof of concept prototype was created to demonstrate the feasibility of this solution and to conduct performance studies on the effectiveness of content caching in the network. The prototype includes Click based MobilityFirst routers, a GNRS, a content provider and a Click based satellite emulation. The benefit of in-network caches was studied by requesting contents with popularity drawn from a Zipf distribution. Results obtained with the prototype system demonstrated that clients experienced reduced delay due to in-network caches and that a fairly high cache hit ratio was achieved in some content distributions even when the cache only stored a small portion of the total contents. The evaluation was also used to study the impact of key parameters such as the Zipf distribution parameter and cache size on metrics such as delay and cache hit ratio.M.S.Includes bibliographical referencesby Shashikanth Rangan Penugond
Circular RNAs drive oncogenic chromosomal translocations within the MLL recombinome in leukemia
No full textPublished: June 8, 2023The first step of oncogenesis is the acquisition of a repertoire of genetic mutations to initiate and sustain the malignancy. An important example of this initiation phase in acute leukemias is the formation of a potent oncogene by chromosomal translocations between the mixed lineage leukemia (MLL) gene and one of 100 translocation partners, known as the MLL recombinome. Here, we show that circular RNAs (circRNAs)-a family of covalently closed, alternatively spliced RNA molecules-are enriched within the MLL recombinome and can bind DNA, forming circRNA:DNA hybrids (circR loops) at their cognate loci. These circR loops promote transcriptional pausing, proteasome inhibition, chromatin re-organization, and DNA breakage. Importantly, overexpressing circRNAs in mouse leukemia xenograft models results in co-localization of genomic loci, de novo generation of clinically relevant chromosomal translocations mimicking the MLL recombinome, and hastening of disease onset. Our findings provide fundamental insight into the acquisition of chromosomal translocations by endogenous RNA carcinogens in leukemia.Vanessa M. Conn, Marta Gabryelska, John Toubia, Kirsty Kirk, Laura Gantley, Jason A. Powell, Go, khan Cildir, Shashikanth Marri, Ryan Liu, Brett W. Stringer, Scott Townley, Stuart T. Webb, He Lin, Saumya E. Samaraweera, Sheree Bailey, Andrew S. Moore, Mellissa Maybury, Dawei Liu, Alex D. Colella, Timothy Chataway, Craig T. Wallington-Gates, Lucie Walters, Jane Sibbons, Luke A. Selth, Vinay Tergaonkar, Richard J. D, Andrea, Stuart M. Pitson, Gregory J. Goodall, and Simon J. Con
Semi-automated genomic newborn screening highlights complexities in reporting
No full textOnlinrPublNewborn screening programs are instrumental in the early detection of treatable conditions in the first days of life. By integrating genomic approaches, there is potential to expand the range of conditions included in these programs. As part of a research study, NewbornsInSA, we validated a genomic newborn screening workflow. Analysis of whole-genome sequencing data was restricted to a virtual panel of 613 genes, selected through engagement with local clinical teams. We assessed the workflow’s performance using retrospective samples with known variant status. To reduce manual curation time, bioinformatics scripts were developed to auto-classify cases into those with no findings and those requiring manual review. We report on early findings from applying this workflow to a prospectively recruited cohort in which five reportable findings have been made to date. We discuss reporting challenges encountered in genes associated with multiple conditions, with incomplete penetrance, or variants associated with only mild phenotypes.Ayesha Chowdhury, Shashikanth Marri, Lucy Anastasi, Alex Ashenden, Tomas Rozek, Jinghua Feng, Lucas DeJong, Rosalie Kenyon, Dominik Kaczorowski, Hung Nguyen, Khoa Lam, Kirsty Stallard, Tracy Merlin, Enzo Ranieri, Sunita De Sousa, Nicholas Smith, Abhi Kulkarni, Benjamin Saxon, Drago Bratkovic, Christopher Barnett, Carol Wai-Kwan Siu, Hamish S. Scott, Jovanka King and Karin S. Kassah
Deriving accurate microbiota profiles from human samples with low bacterial content through post-sequencing processing of Illumina MiSeq data
No full textPublished: 5 May 2015Background: The rapid expansion of 16S rRNA gene sequencing in challenging clinical contexts has resulted in a growing body of literature of variable quality. To a large extent, this is due to a failure to address spurious signal that is characteristic of samples with low levels of bacteria and high levels of non-bacterial DNA. We have developed a workflow based on the paired-end read Illumina MiSeq-based approach, which enables significant improvement in data quality, post-sequencing. We demonstrate the efficacy of this methodology through its application to paediatric upper-respiratory samples from several anatomical sites. Results: A workflow for processing sequence data was developed based on commonly available tools. Data generated from different sample types showed a marked variation in levels of non-bacterial signal and ‘contaminant’ bacterial reads. Significant differences in the ability of reference databases to accurately assign identity to operational taxonomic units (OTU) were observed. Three OTU-picking strategies were trialled as follows: de novo, open-reference and closed-reference, with open-reference performing substantially better. Relative abundance of OTUs identified as potential reagent contamination showed a strong inverse correlation with amplicon concentration allowing their objective removal. The removal of the spurious signal showed the greatest improvement in sample types typically containing low levels of bacteria and high levels of human DNA. A substantial impact of pre-filtering data and spurious signal removal was demonstrated by principal coordinate and co-occurrence analysis. For example, analysis of taxon co-occurrence in adenoid swab and middle ear fluid samples indicated that failure to remove the spurious signal resulted in the inclusion of six out of eleven bacterial genera that accounted for 80% of similarity between the sample types. Conclusions: The application of the presented workflow to a set of challenging clinical samples demonstrates its utility in removing the spurious signal from the dataset, allowing clinical insight to be derived from what would otherwise be highly misleading output. While other approaches could potentially achieve similar improvements, the methodology employed here represents an accessible means to exclude the signal from contamination and other artefacts.Jake Jervis-Bardy, Lex E X Leong, Shashikanth Marri, Renee J Smith, Jocelyn M Choo, Heidi C Smith-Vaughan, Elizabeth Nosworthy, Peter S Morris, Stephen O'Leary, Geraint B Rogers and Robyn L Mars
Identification of microRNA biomarkers of response to neoadjuvant chemoradiotherapy in esophageal adenocarcinoma using next generation sequencing
No full textPublished Online: 9 July 2018Background. Clinical trials report improved overall survival following neoadjuvant chemoradiotherapy in patients undergoing surgery for esophageal adenocarcinoma, with a 10–15% survival improvement. MicroRNAs (miRNAs) are small noncoding RNAs that are known to direct the behavior of cancers, including response to treatment. We investigated the ability of miRNAs to predict outcomes after neoadjuvant chemoradiotherapy. Methods. Endoscopic biopsies from esophageal adenocarcinomas were obtained before neoadjuvant chemoradiotherapy and esophagectomy. miRNA levels were measured in the biopsies using next generation sequencing and compared with pathological response in the surgical resection, and subsequent survival. miRNA ratios that predicted pathological response were identified by Lasso regression and leave-one-out crossvalidation. Association between miRNA ratio candidates and relapse-free survival was assessed using Kaplan–Meier analysis. Cox regression and Harrell’s C analyses were performed to assess the predictive performance of the miRNAs. Results. Two miRNA ratios (miR-4521/miR-340-5p and miR-101-3p/miR-451a) that predicted the pathological response to neoadjuvant chemoradiotherapy were found to be associated with relapse-free survival. Pretreatment expression of these two miRNA ratios, pretreatment tumor differentiation, posttreatment AJCC histopathological tumor regression grading, and posttreatment tumor clearance/ margins were significant factors associated with survival in Cox regression analysis. Multivariate analysis of the two ratios together with pretherapy factors resulted in a risk prediction accuracy of 85% (Harrell’s C), which was comparable with the prediction accuracy of the AJCC treatment response grading (77%). Conclusions. miRNA-ratio biomarkers identified using next generation sequencing can be used to predict disease free survival following neoadjuvant chemoradiotherapy and esophagectomy in patients with esophageal adenocarcinoma.Karen Chiam, George C. Mayne, David I. Watson, Richard J. Woodman, Tim F. Bright, Michael Z. Michael, Christos S. Karapetis, Tanya Irvine, Wayne A. Phillips, Richard Hummel, Tingting Wang, Letitia K. Pimlott, Shashikanth Marri, David StJ. Astill, Andrew R. Ruszkiewicz, Sarah K. Thompson, and Damian J. Husse
Expression of microRNA in human retinal pigment epithelial cells following infection with Zaire ebolavirus
No full text© The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Objective: Survivors of Ebola virus disease (EVD) are at risk of developing blinding intraocular inflammation—or uveitis—which is associated with retinal pigment epithelial (RPE) scarring and persistence of live Zaire ebolavirus (EBOV) within the eye. As part of a large research project aimed at defining the human RPE cell response to being infected with EBOV, this work focused on the microRNAs (miRNAs) associated with the infection.
Results: Using RNA-sequencing, we detected 13 highly induced and 2 highly repressed human miRNAs in human ARPE-19 RPE cells infected with EBOV, including hsa-miR-1307-5p, hsa-miR-29b-3p and hsa-miR-33a-5p (up-regulated), and hsa-miR-3074-3p and hsa-miR-27b-5p (down-regulated). EBOV-miR-1-5p was also found in infected RPE cells. Through computational identification of putative miRNA targets, we predicted a broad range of regulatory activities, including effects on innate and adaptive immune responses, cellular metabolism, cell cycle progression, apoptosis and autophagy. The most highly-connected molecule in the miR-target network was leucine-rich repeat kinase 2, which is involved in neuroinflammation and lysosomal processing. Our findings should stimulate new studies on the impact of miRNA changes in EBOV-infected RPE cells to further understanding of intraocular viral persistence and the pathogenesis of uveitis in EVD survivors
The Expression Profiles and Deregulation of UDP-Glycosyltransferase (UGT) Genes in Human Cancers and Their Association with Clinical Outcomes
No full textThe human UDP-glycosyltransferase (UGTs) superfamily has 22 functional enzymes that play a critical role in the metabolism of small lipophilic compounds, including carcinogens, drugs, steroids, lipids, fatty acids, and bile acids. The expression profiles of UGT genes in human cancers and their impact on cancer patient survival remains to be systematically investigated. In the present study, a comprehensive analysis of the RNAseq and clinical datasets of 9514 patients from 33 different TCGA (the Genome Cancer Atlas) cancers demonstrated cancer-specific UGT expression profiles with high interindividual variability among and within individual cancers. Notably, cancers derived from drug metabolizing tissues (liver, kidney, gut, pancreas) expressed the largest number of UGT genes (COAD, KIRC, KIRP, LIHC, PAAD); six UGT genes (1A6, 1A9, 1A10, 2A3, 2B7, UGT8) showed high expression in five or more different cancers. Kaplan–Meier plots and logrank tests revealed that six UGT genes were significantly associated with increased overall survival (OS) rates [UGT1A1 (LUSC), UGT1A6 (ACC), UGT1A7 (ACC), UGT2A3 (KIRC), UGT2B15 (BLCA, SKCM)] or decreased OS rates [UGT2B15 (LGG), UGT8 (UVM)] in specific cancers. Finally, differential expression analysis of 611 patients from 12 TCGA cancers identified 16 UGT genes (1A1, 1A3, 1A6, 1A7, 1A8, 1A9, 1A10, 2A1, 2A3, 2B4, 2B7, 2B11, 2B15, 3A1, 3A2, UGT8) that were up/downregulated in at least one cancer relative to normal tissues. In conclusion, our data show widespread expression of UGT genes in cancers, highlighting the capacity for intratumoural drug metabolism through the UGT conjugation pathway. The data also suggests the potentials for specific UGT genes to serve as prognostic biomarkers or therapeutic targets in cancers
Resistance to pentamidine is mediated by AdeAB, regulated by AdeRS, and influenced by growth conditions in<i>Acinetobacter baumannii</i>ATCC 17978
No full textAbstractIn recent years, effective treatment of infections caused byAcinetobacter baumanniihas become challenging due to the ability of the bacterium to acquire or up-regulate antimicrobial resistance determinants. Two component signal transduction systems are known to regulate expression of virulence factors including multidrug efflux pumps. Here, we investigated the role of the AdeRS two component signal transduction system in regulating the AdeAB efflux system, determined whether AdeA and/or AdeB can individually confer antimicrobial resistance, and explored the interplay between pentamidine resistance and growth conditions inA. baumanniiATCC 17978. Results identified that deletion ofadeRSaffected resistance towards chlorhexidine and 4’,6-diamidino-2-phenylindole dihydrochloride, two previously defined AdeABC substrates, and also identified an 8-fold decrease in resistance to pentamidine. Examination of ΔadeA, ΔadeBand ΔadeABcells augmented results seen for ΔadeRSand identified a set of dicationic AdeAB substrates. RNA-sequencing of ΔadeRSrevealed transcription of 290 genes were ≥2-fold altered compared to the wildtype. Pentamidine shock significantly increasedadeAexpression in the wildtype, but decreased it in ΔadeRS, implying that AdeRS activatesadeABtranscription in ATCC 17978. Investigation under multiple growth conditions, including the use of Biolog phenotypic microarrays, revealed resistance to pentamidine in ATCC 17978 and mutants could be altered by bioavailability of iron or utilization of different carbon sources. In conclusion, the results of this study provide evidence that AdeAB in ATCC 17978 can confer intrinsic resistance to a subset of dicationic compounds and in particular, resistance to pentamidine can be significantly altered depending on the growth conditions.</jats:p
