Publications by authors named "Ana Marcu"

23 Publications

  • Page 1 of 1

HLA Ligand Atlas: a benign reference of HLA-presented peptides to improve T-cell-based cancer immunotherapy.

J Immunother Cancer 2021 Apr;9(4)

Clinical Neuroscience Center and Department of Neurosurgery, University Hospital and University of Zurich, Zurich, Switzerland.

Background: The human leucocyte antigen (HLA) complex controls adaptive immunity by presenting defined fractions of the intracellular and extracellular protein content to immune cells. Understanding the benign HLA ligand repertoire is a prerequisite to define safe T-cell-based immunotherapies against cancer. Due to the poor availability of benign tissues, if available, normal tissue adjacent to the tumor has been used as a benign surrogate when defining tumor-associated antigens. However, this comparison has proven to be insufficient and even resulted in lethal outcomes. In order to match the tumor immunopeptidome with an equivalent counterpart, we created the HLA Ligand Atlas, the first extensive collection of paired HLA-I and HLA-II immunopeptidomes from 227 benign human tissue samples. This dataset facilitates a balanced comparison between tumor and benign tissues on HLA ligand level.

Methods: Human tissue samples were obtained from 16 subjects at autopsy, five thymus samples and two ovary samples originating from living donors. HLA ligands were isolated via immunoaffinity purification and analyzed in over 1200 liquid chromatography mass spectrometry runs. Experimentally and computationally reproducible protocols were employed for data acquisition and processing.

Results: The initial release covers 51 HLA-I and 86 HLA-II allotypes presenting 90,428 HLA-I- and 142,625 HLA-II ligands. The HLA allotypes are representative for the world population. We observe that immunopeptidomes differ considerably between tissues and individuals on source protein and HLA-ligand level. Moreover, we discover 1407 HLA-I ligands from non-canonical genomic regions. Such peptides were previously described in tumors, peripheral blood mononuclear cells (PBMCs), healthy lung tissues and cell lines. In a case study in glioblastoma, we show that potential on-target off-tumor adverse events in immunotherapy can be avoided by comparing tumor immunopeptidomes to the provided multi-tissue reference.

Conclusion: Given that T-cell-based immunotherapies, such as CAR-T cells, affinity-enhanced T cell transfer, cancer vaccines and immune checkpoint inhibition, have significant side effects, the HLA Ligand Atlas is the first step toward defining tumor-associated targets with an improved safety profile. The resource provides insights into basic and applied immune-associated questions in the context of cancer immunotherapy, infection, transplantation, allergy and autoimmunity. It is publicly available and can be browsed in an easy-to-use web interface at .
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http://dx.doi.org/10.1136/jitc-2020-002071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8054196PMC
April 2021

Mass spectrometry-based identification of a B-cell maturation antigen-derived T-cell epitope for antigen-specific immunotherapy of multiple myeloma.

Blood Cancer J 2020 02 28;10(2):24. Epub 2020 Feb 28.

University Hospital Tübingen, Clinical Collaboration Unit Translational Immunology, German Cancer Consortium (DKTK), Tübingen, Germany.

The B-cell maturation antigen (BCMA) is currently being evaluated as promising tumor-associated surface antigen for T-cell-based immunotherapy approaches, such as CAR T cells and bispecific antibodies, in multiple myeloma (MM). Cytotoxic T cells bearing BCMA-specific T-cell receptors might further allow targeting HLA-presented antigens derived from the intracellular domain of BCMA. By analyzing a mass spectrometry-acquired immunopeptidome dataset of primary MM samples and MM cell lines for BCMA-derived HLA ligands, we identified the naturally presented HLA-B*18-restricted ligand P(BCMA). Additionally, P(BCMA) was identified on primary CLL samples, thereby expanding the range for possible applications. P(BCMA) induced multifunctional BCMA-specific cells de novo from naïve CD8 T cells of healthy volunteers. These T cells exhibited antigen-specific lysis of autologous peptide-loaded cells. Even in the immunosuppressive context of MM, we detected spontaneous memory T-cell responses against P(BCMA) in patients. By applying CTLA-4 and PD-1 inhibition in vitro we induced multifunctional P(BCMA)-specific CD8 T cells in MM patients lacking preexisting BCMA-directed immune responses. Finally, we could show antigen-specific lysis of autologous peptide-loaded target cells and even MM.1S cells naturally presenting P(BCMA) using patient-derived P(BCMA)-specific T cells. Hence, this BCMA-derived T-cell epitope represents a promising target for T-cell-based immunotherapy and monitoring following immunotherapy in B-cell malignancy patients.
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http://dx.doi.org/10.1038/s41408-020-0288-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7048774PMC
February 2020

Prostaglandin E in a TLR3- and 7/8-agonist-based DC maturation cocktail generates mature, cytokine-producing, migratory DCs but impairs antigen cross-presentation to CD8 T cells.

Cancer Immunol Immunother 2020 Jun 25;69(6):1029-1042. Epub 2020 Feb 25.

Laboratory for Stem Cell Processing and Cellular TherapyUniversity Medical Center, Children's Hospital, Würzburg, Germany.

Mature dendritic cells (DCs) represent cellular adjuvants for optimal antigen presentation in cancer vaccines. Recently, a combination of prostaglandin E (PGE) with Toll-like receptor agonists (TLR-P) was proposed as a new standard to generate superior cytokine-producing DCs with high migratory capacity. Here, we compare TLR-P DCs with conventional DCs matured only with the proinflammatory cytokines TNFα and IL-1ß (CDCs), focussing on the interaction of resulting DCs with CD8 T-cells. TLR-P matured DCs showed elevated expression of activation markers such as CD80 and CD83 compared to CDCs, together with a significantly higher migration capacity. Secretion of IL-6, IL-8, IL-10, and IL-12 was highest after 16 h in TLR-P DCs, and only TLR-P DCs secreted active IL-12p70. TLR-P DCs as well as CDCs successfully primed multifunctional CD8 T-cells from naïve precursors specific for the peptide antigens Melan-A, NLGN4X, and PTP with comparable priming efficacy and T-cell receptor avidity. CD8 T-cells primed by TLR-P DCs showed significantly elevated expression of the integrin VLA-4 and a trend for higher T-cell numbers after expansion. In contrast, TLR-P DCs displayed a substantially reduced capability to cross-present CMVpp65 protein antigen to pp65-specific T cells, an effect that was dose-dependent on PGE during DC maturation and reproducible with several responder T-cell lines. In conclusion, TLR-P matured DCs might be optimal presenters of antigens not requiring processing such as short peptides. However, PGE seems less favorable for maturation of DCs intended to process and cross-present more complex vaccine antigens such as lysates, proteins or long peptides.
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http://dx.doi.org/10.1007/s00262-019-02470-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7223547PMC
June 2020

Guidance Document: Validation of a High-Performance Liquid Chromatography-Tandem Mass Spectrometry Immunopeptidomics Assay for the Identification of HLA Class I Ligands Suitable for Pharmaceutical Therapies.

Mol Cell Proteomics 2020 03 14;19(3):432-443. Epub 2020 Jan 14.

Department of Immunology, Institute for Cell Biology, University of Tübingen, Tübingen, Germany; German Cancer Research Center (DKFZ) partner site and German Cancer Consortium (DKTK) Tübingen, Tübingen, Germany. Electronic address:

For more than two decades naturally presented, human leukocyte antigen (HLA)-restricted peptides (immunopeptidome) have been eluted and sequenced using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Since, identified disease-associated HLA ligands have been characterized and evaluated as potential active substances. Treatments based on HLA-presented peptides have shown promising results in clinical application as personalized T cell-based immunotherapy. Peptide vaccination cocktails are produced as investigational medicinal products under GMP conditions. To support clinical trials based on HLA-presented tumor-associated antigens, in this study the sensitive LC-MS/MS HLA class I antigen identification pipeline was fully validated for our technical equipment according to the current US Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines.The immunopeptidomes of JY cells with or without spiked-in, isotope labeled peptides, of peripheral blood mononuclear cells of healthy volunteers as well as a chronic lymphocytic leukemia and a bladder cancer sample were reliably identified using a data-dependent acquisition method. As the LC-MS/MS pipeline is used for identification purposes, the validation parameters include accuracy, precision, specificity, limit of detection and robustness.
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http://dx.doi.org/10.1074/mcp.C119.001652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7050110PMC
March 2020

Identification of HCMV-derived T cell epitopes in seropositive individuals through viral deletion models.

J Exp Med 2020 03;217(3)

Institute of Virology, Medical Center University of Freiburg, Freiburg, Germany.

In healthy individuals, immune control of persistent human cytomegalovirus (HCMV) infection is effectively mediated by virus-specific CD4+ and CD8+ T cells. However, identifying the repertoire of T cell specificities for HCMV is hampered by the immense protein coding capacity of this betaherpesvirus. Here, we present a novel approach that employs HCMV deletion mutant viruses lacking HLA class I immunoevasins and allows direct identification of naturally presented HCMV-derived HLA ligands by mass spectrometry. We identified 368 unique HCMV-derived HLA class I ligands representing an unexpectedly broad panel of 123 HCMV antigens. Functional characterization revealed memory T cell responses in seropositive individuals for a substantial proportion (28%) of these novel peptides. Multiple HCMV-directed specificities in the memory T cell pool of single individuals indicate that physiologic anti-HCMV T cell responses are directed against a broad range of antigens. Thus, the unbiased identification of naturally presented viral epitopes enabled a comprehensive and systematic assessment of the physiological repertoire of anti-HCMV T cell specificities in seropositive individuals.
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http://dx.doi.org/10.1084/jem.20191164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7062530PMC
March 2020

PathBank: a comprehensive pathway database for model organisms.

Nucleic Acids Res 2020 01;48(D1):D470-D478

Department of Plant Breeding, Wageningen University and Research, 6708 PB Wageningen, Gelderland, Netherlands.

PathBank (www.pathbank.org) is a new, comprehensive, visually rich pathway database containing more than 110 000 machine-readable pathways found in 10 model organisms (Homo sapiens, Bos taurus, Rattus norvegicus, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, Saccharomyces cerevisiae, Escherichia coli and Pseudomonas aeruginosa). PathBank aims to provide a pathway for every protein and a map for every metabolite. This resource is designed specifically to support pathway elucidation and pathway discovery in transcriptomics, proteomics, metabolomics and systems biology. It provides detailed, fully searchable, hyperlinked diagrams of metabolic, metabolite signaling, protein signaling, disease, drug and physiological pathways. All PathBank pathways include information on the relevant organs, organelles, subcellular compartments, cofactors, molecular locations, chemical structures and protein quaternary structures. Each small molecule is hyperlinked to the rich data contained in public chemical databases such as HMDB or DrugBank and each protein or enzyme complex is hyperlinked to UniProt. All PathBank pathways are accompanied with references and detailed descriptions which provide an overview of the pathway, condition or processes depicted in each diagram. Every PathBank pathway is downloadable in several machine-readable and image formats including BioPAX, SBML, PWML, SBGN, RXN, PNG and SVG. PathBank also supports community annotations and submissions through the web-based PathWhiz pathway illustrator. The vast majority of PathBank's pathways (>95%) are not found in any other public pathway database.
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http://dx.doi.org/10.1093/nar/gkz861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6943071PMC
January 2020

Therapeutic vaccine strategies to induce tumor-specific T-cell responses.

Bone Marrow Transplant 2019 08;54(Suppl 2):806-809

University Children's Hospital Würzburg, Würzburg, Germany.

Haploidentical stem cell transplantation is increasingly used worldwide as an alternative donor transplantation method. Although novel preparative regimens and T-cell deletion techniques have improved engraftment rates and viral safety, relapses of the underlying leukemia/lymphoma are still frequent, thus representing a significant and unsolved problem. Recent technological advances now enable us to individually decipher the MHC-associated immunopeptidome of cancer cells in reasonable time. These tumor-specific peptides can then be used to skew the early immune reconstitution toward anti-leukemia T-cell responses. In this meeting contribution, we summarize recent innovations in the field and present preliminary data on using this technique for cancer epitope discovery in a paradigmatic pediatric brain tumor with very low mutational burden.
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http://dx.doi.org/10.1038/s41409-019-0619-9DOI Listing
August 2019

The HLA ligandome landscape of chronic myeloid leukemia delineates novel T-cell epitopes for immunotherapy.

Blood 2019 02 10;133(6):550-565. Epub 2018 Dec 10.

Department of Hematology and Oncology, University Hospital Tübingen, Tübingen, Germany.

Antileukemia immunity plays an important role in disease control and maintenance of tyrosine kinase inhibitor (TKI)-free remission in chronic myeloid leukemia (CML). Thus, antigen-specific immunotherapy holds promise for strengthening immune control in CML but requires the identification of CML-associated targets. In this study, we used a mass spectrometry-based approach to identify naturally presented HLA class I- and class II-restricted peptides in primary CML samples. Comparative HLA ligandome profiling using a comprehensive dataset of different hematological benign specimens and samples from CML patients in deep molecular remission delineated a panel of novel frequently presented CML-exclusive peptides. These nonmutated target antigens are of particular relevance because our extensive data-mining approach suggests the absence of naturally presented BCR-ABL- and ABL-BCR-derived HLA-restricted peptides and the lack of frequent tumor-exclusive presentation of known cancer/testis and leukemia-associated antigens. Functional characterization revealed spontaneous T-cell responses against the newly identified CML-associated peptides in CML patient samples and their ability to induce multifunctional and cytotoxic antigen-specific T cells de novo in samples from healthy volunteers and CML patients. Thus, these antigens are prime candidates for T-cell-based immunotherapeutic approaches that may prolong TKI-free survival and even mediate cure of CML patients.
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http://dx.doi.org/10.1182/blood-2018-07-866830DOI Listing
February 2019

TAPBPR mediates peptide dissociation from MHC class I using a leucine lever.

Elife 2018 11 28;7. Epub 2018 Nov 28.

Department of Pathology, University of Cambridge, Cambridge, United Kingdom.

Tapasin and TAPBPR are known to perform peptide editing on major histocompatibility complex class I (MHC I) molecules; however, the precise molecular mechanism(s) involved in this process remain largely enigmatic. Here, using immunopeptidomics in combination with novel cell-based assays that assess TAPBPR-mediated peptide exchange, we reveal a critical role for the K22-D35 loop of TAPBPR in mediating peptide exchange on MHC I. We identify a specific leucine within this loop that enables TAPBPR to facilitate peptide dissociation from MHC I. Moreover, we delineate the molecular features of the MHC I F pocket required for TAPBPR to promote peptide dissociation in a loop-dependent manner. These data reveal that chaperone-mediated peptide editing on MHC I can occur by different mechanisms dependent on the C-terminal residue that the MHC I accommodates in its F pocket and provide novel insights that may inform the therapeutic potential of TAPBPR manipulation to increase tumour immunogenicity.
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http://dx.doi.org/10.7554/eLife.40126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6307860PMC
November 2018

Mapping the tumour human leukocyte antigen (HLA) ligandome by mass spectrometry.

Immunology 2018 07 8;154(3):331-345. Epub 2018 May 8.

Interfaculty Institute for Cell Biology, Department of Immunology, University of Tübingen, Tübingen, Germany.

The entirety of human leukocyte antigen (HLA)-presented peptides is referred to as the HLA ligandome of a cell or tissue, in tumours often termed immunopeptidome. Mapping the tumour immunopeptidome by mass spectrometry (MS) comprehensively views the pathophysiologically relevant antigenic signature of human malignancies. MS is an unbiased approach stringently filtering the candidates to be tested as opposed to epitope prediction algorithms. In the setting of peptide-specific immunotherapies, MS-based strategies significantly diminish the risk of lacking clinical benefit, as they yield highly enriched amounts of truly presented peptides. Early immunopeptidomic efforts were severely limited by technical sensitivity and manual spectra interpretation. The technological progress with development of orbitrap mass analysers and enhanced chromatographic performance led to vast improvements in mass accuracy, sensitivity, resolution, and speed. Concomitantly, bioinformatic tools were developed to process MS data, integrate sequencing results, and deconvolute multi-allelic datasets. This enabled the immense advancement of tumour immunopeptidomics. Studying the HLA-presented peptide repertoire bears high potential for both answering basic scientific questions and translational application. Mapping the tumour HLA ligandome has started to significantly contribute to target identification for the design of peptide-specific cancer immunotherapies in clinical trials and compassionate need treatments. In contrast to prediction algorithms, rare HLA allotypes and HLA class II can be adequately addressed when choosing MS-guided target identification platforms. Herein, we review the identification of tumour HLA ligands focusing on sources, methods, bioinformatic data analysis, translational application, and provide an outlook on future developments.
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http://dx.doi.org/10.1111/imm.12936DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6002237PMC
July 2018

The natural HLA ligandome of glioblastoma stem-like cells: antigen discovery for T cell-based immunotherapy.

Acta Neuropathol 2018 06 20;135(6):923-938. Epub 2018 Mar 20.

Laboratory of Molecular Neuro-Oncology, Department of Neurology, Clinical Neuroscience Center, University Hospital Zurich and University of Zurich, Frauenklinikstrasse 26, 8091, Zurich, Switzerland.

Glioblastoma is the most frequent malignant primary brain tumor. In a hierarchical tumor model, glioblastoma stem-like cells (GSC) play a major role in tumor initiation and maintenance as well as in therapy resistance and recurrence. Thus, targeting this cellular subset may be key to effective immunotherapy. Here, we present a mass spectrometry-based analysis of HLA-presented peptidomes of GSC and glioblastoma patient specimens. Based on the analysis of patient samples (n = 9) and GSC (n = 3), we performed comparative HLA peptidome profiling against a dataset of normal human tissues. Using this immunopeptidome-centric approach we could clearly delineate a subset of naturally presented, GSC-associated HLA ligands, which might serve as highly specific targets for T cell-based immunotherapy. In total, we identified 17 antigens represented by 41 different HLA ligands showing natural and exclusive presentation both on GSC and patient samples. Importantly, in vitro immunogenicity and antigen-specific target cell killing assays suggest these peptides to be epitopes of functional CD8+ T cell responses, thus rendering them prime candidates for antigen-specific immunotherapy of glioblastoma.
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http://dx.doi.org/10.1007/s00401-018-1836-9DOI Listing
June 2018

HMDB 4.0: the human metabolome database for 2018.

Nucleic Acids Res 2018 01;46(D1):D608-D617

International Agency for Research on Cancer (IARC), 150 Cours Albert Thomas, 69372 Lyon CEDEX 08, France.

The Human Metabolome Database or HMDB (www.hmdb.ca) is a web-enabled metabolomic database containing comprehensive information about human metabolites along with their biological roles, physiological concentrations, disease associations, chemical reactions, metabolic pathways, and reference spectra. First described in 2007, the HMDB is now considered the standard metabolomic resource for human metabolic studies. Over the past decade the HMDB has continued to grow and evolve in response to emerging needs for metabolomics researchers and continuing changes in web standards. This year's update, HMDB 4.0, represents the most significant upgrade to the database in its history. For instance, the number of fully annotated metabolites has increased by nearly threefold, the number of experimental spectra has grown by almost fourfold and the number of illustrated metabolic pathways has grown by a factor of almost 60. Significant improvements have also been made to the HMDB's chemical taxonomy, chemical ontology, spectral viewing, and spectral/text searching tools. A great deal of brand new data has also been added to HMDB 4.0. This includes large quantities of predicted MS/MS and GC-MS reference spectral data as well as predicted (physiologically feasible) metabolite structures to facilitate novel metabolite identification. Additional information on metabolite-SNP interactions and the influence of drugs on metabolite levels (pharmacometabolomics) has also been added. Many other important improvements in the content, the interface, and the performance of the HMDB website have been made and these should greatly enhance its ease of use and its potential applications in nutrition, biochemistry, clinical chemistry, clinical genetics, medicine, and metabolomics science.
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http://dx.doi.org/10.1093/nar/gkx1089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5753273PMC
January 2018

DrugBank 5.0: a major update to the DrugBank database for 2018.

Nucleic Acids Res 2018 01;46(D1):D1074-D1082

Department of Biological Sciences, University of Alberta, Edmonton, AB T6G 2E9, Canada.

DrugBank (www.drugbank.ca) is a web-enabled database containing comprehensive molecular information about drugs, their mechanisms, their interactions and their targets. First described in 2006, DrugBank has continued to evolve over the past 12 years in response to marked improvements to web standards and changing needs for drug research and development. This year's update, DrugBank 5.0, represents the most significant upgrade to the database in more than 10 years. In many cases, existing data content has grown by 100% or more over the last update. For instance, the total number of investigational drugs in the database has grown by almost 300%, the number of drug-drug interactions has grown by nearly 600% and the number of SNP-associated drug effects has grown more than 3000%. Significant improvements have been made to the quantity, quality and consistency of drug indications, drug binding data as well as drug-drug and drug-food interactions. A great deal of brand new data have also been added to DrugBank 5.0. This includes information on the influence of hundreds of drugs on metabolite levels (pharmacometabolomics), gene expression levels (pharmacotranscriptomics) and protein expression levels (pharmacoprotoemics). New data have also been added on the status of hundreds of new drug clinical trials and existing drug repurposing trials. Many other important improvements in the content, interface and performance of the DrugBank website have been made and these should greatly enhance its ease of use, utility and potential applications in many areas of pharmacological research, pharmaceutical science and drug education.
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http://dx.doi.org/10.1093/nar/gkx1037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5753335PMC
January 2018

PAMDB: a comprehensive Pseudomonas aeruginosa metabolome database.

Nucleic Acids Res 2018 01;46(D1):D575-D580

Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD 21209, USA.

The Pseudomonas aeruginosaMetabolome Database (PAMDB, http://pseudomonas.umaryland.edu) is a searchable, richly annotated metabolite database specific to P. aeruginosa. P. aeruginosa is a soil organism and significant opportunistic pathogen that adapts to its environment through a versatile energy metabolism network. Furthermore, P. aeruginosa is a model organism for the study of biofilm formation, quorum sensing, and bioremediation processes, each of which are dependent on unique pathways and metabolites. The PAMDB is modelled on the Escherichia coli (ECMDB), yeast (YMDB) and human (HMDB) metabolome databases and contains >4370 metabolites and 938 pathways with links to over 1260 genes and proteins. The database information was compiled from electronic databases, journal articles and mass spectrometry (MS) metabolomic data obtained in our laboratories. For each metabolite entered, we provide detailed compound descriptions, names and synonyms, structural and physiochemical information, nuclear magnetic resonance (NMR) and MS spectra, enzymes and pathway information, as well as gene and protein sequences. The database allows extensive searching via chemical names, structure and molecular weight, together with gene, protein and pathway relationships. The PAMBD and its future iterations will provide a valuable resource to biologists, natural product chemists and clinicians in identifying active compounds, potential biomarkers and clinical diagnostics.
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http://dx.doi.org/10.1093/nar/gkx1061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5753269PMC
January 2018

nmrML: A Community Supported Open Data Standard for the Description, Storage, and Exchange of NMR Data.

Anal Chem 2018 01 14;90(1):649-656. Epub 2017 Dec 14.

European Bioinformatics Institute (EMBL-EBI), European Molecular Biology Laboratory , Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, U.K.

NMR is a widely used analytical technique with a growing number of repositories available. As a result, demands for a vendor-agnostic, open data format for long-term archiving of NMR data have emerged with the aim to ease and encourage sharing, comparison, and reuse of NMR data. Here we present nmrML, an open XML-based exchange and storage format for NMR spectral data. The nmrML format is intended to be fully compatible with existing NMR data for chemical, biochemical, and metabolomics experiments. nmrML can capture raw NMR data, spectral data acquisition parameters, and where available spectral metadata, such as chemical structures associated with spectral assignments. The nmrML format is compatible with pure-compound NMR data for reference spectral libraries as well as NMR data from complex biomixtures, i.e., metabolomics experiments. To facilitate format conversions, we provide nmrML converters for Bruker, JEOL and Agilent/Varian vendor formats. In addition, easy-to-use Web-based spectral viewing, processing, and spectral assignment tools that read and write nmrML have been developed. Software libraries and Web services for data validation are available for tool developers and end-users. The nmrML format has already been adopted for capturing and disseminating NMR data for small molecules by several open source data processing tools and metabolomics reference spectral libraries, e.g., serving as storage format for the MetaboLights data repository. The nmrML open access data standard has been endorsed by the Metabolomics Standards Initiative (MSI), and we here encourage user participation and feedback to increase usability and make it a successful standard.
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http://dx.doi.org/10.1021/acs.analchem.7b02795DOI Listing
January 2018

PHAST, PHASTER and PHASTEST: Tools for finding prophage in bacterial genomes.

Brief Bioinform 2019 07;20(4):1560-1567

PHAST (PHAge Search Tool) and its successor PHASTER (PHAge Search Tool - Enhanced Release) have become two of the most widely used web servers for identifying putative prophages in bacterial genomes. Here we review the main capabilities of these web resources, provide some practical guidance regarding their use and discuss possible future improvements. PHAST, which was first described in 2011, made its debut just as whole bacterial genome sequencing and was becoming inexpensive and relatively routine. PHAST quickly gained popularity among bacterial genome researchers because of its web accessibility, its ease of use along with its enhanced accuracy and rapid processing times. PHASTER, which appeared in 2016, provided a number of much-needed enhancements to the PHAST server, including greater processing speed (to cope with very large submission volumes), increased database sizes, a more modern user interface, improved graphical displays and support for metagenomic submissions. Continuing developments in the field, along with increased interest in automated phage and prophage finding, have already led to several improvements to the PHASTER server and will soon lead to the development of a successor to PHASTER (to be called PHASTEST).
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http://dx.doi.org/10.1093/bib/bbx121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6781593PMC
July 2019

A Web Tool for Generating High Quality Machine-readable Biological Pathways.

J Vis Exp 2017 02 8(120). Epub 2017 Feb 8.

Department of Computer Science, University of Alberta; Department of Biological Sciences, University of Alberta;

PathWhiz is a web server built to facilitate the creation of colorful, interactive, visually pleasing pathway diagrams that are rich in biological information. The pathways generated by this online application are machine-readable and fully compatible with essentially all web-browsers and computer operating systems. It uses a specially developed, web-enabled pathway drawing interface that permits the selection and placement of different combinations of pre-drawn biological or biochemical entities to depict reactions, interactions, transport processes and binding events. This palette of entities consists of chemical compounds, proteins, nucleic acids, cellular membranes, subcellular structures, tissues, and organs. All of the visual elements in it can be interactively adjusted and customized. Furthermore, because this tool is a web server, all pathways and pathway elements are publicly accessible. This kind of pathway "crowd sourcing" means that PathWhiz already contains a large and rapidly growing collection of previously drawn pathways and pathway elements. Here we describe a protocol for the quick and easy creation of new pathways and the alteration of existing pathways. To further facilitate pathway editing and creation, the tool contains replication and propagation functions. The replication function allows existing pathways to be used as templates to create or edit new pathways. The propagation function allows one to take an existing pathway and automatically propagate it across different species. Pathways created with this tool can be "re-styled" into different formats (KEGG-like or text-book like), colored with different backgrounds, exported to BioPAX, SBGN-ML, SBML, or PWML data exchange formats, and downloaded as PNG or SVG images. The pathways can easily be incorporated into online databases, integrated into presentations, posters or publications, or used exclusively for online visualization and exploration. This protocol has been successfully applied to generate over 2,000 pathway diagrams, which are now found in many online databases including HMDB, DrugBank, SMPDB, and ECMDB.
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http://dx.doi.org/10.3791/54869DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5409186PMC
February 2017

YMDB 2.0: a significantly expanded version of the yeast metabolome database.

Nucleic Acids Res 2017 01 28;45(D1):D440-D445. Epub 2016 Nov 28.

Departments of Computing Science, University of Alberta, Edmonton, AB T6G 2E8, Canada

YMDB or the Yeast Metabolome Database (http://www.ymdb.ca/) is a comprehensive database containing extensive information on the genome and metabolome of Saccharomyces cerevisiae Initially released in 2012, the YMDB has gone through a significant expansion and a number of improvements over the past 4 years. This manuscript describes the most recent version of YMDB (YMDB 2.0). More specifically, it provides an updated description of the database that was previously described in the 2012 NAR Database Issue and it details many of the additions and improvements made to the YMDB over that time. Some of the most important changes include a 7-fold increase in the number of compounds in the database (from 2007 to 16 042), a 430-fold increase in the number of metabolic and signaling pathway diagrams (from 66 to 28 734), a 16-fold increase in the number of compounds linked to pathways (from 742 to 12 733), a 17-fold increase in the numbers of compounds with nuclear magnetic resonance or MS spectra (from 783 to 13 173) and an increase in both the number of data fields and the number of links to external databases. In addition to these database expansions, a number of improvements to YMDB's web interface and its data visualization tools have been made. These additions and improvements should greatly improve the ease, the speed and the quantity of data that can be extracted, searched or viewed within YMDB. Overall, we believe these improvements should not only improve the understanding of the metabolism of S. cerevisiae, but also allow more in-depth exploration of its extensive metabolic networks, signaling pathways and biochemistry.
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http://dx.doi.org/10.1093/nar/gkw1058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5210545PMC
January 2017

Heatmapper: web-enabled heat mapping for all.

Nucleic Acids Res 2016 07 17;44(W1):W147-53. Epub 2016 May 17.

Department of Computing Science, University of Alberta, Edmonton, AB T6G 2E8, Canada Department of Biological Sciences, University of Alberta, Edmonton, AB T6G 2E9, Canada National Institute for Nanotechnology, 11421 Saskatchewan Drive, Edmonton, AB T6G 2M9, Canada

Heatmapper is a freely available web server that allows users to interactively visualize their data in the form of heat maps through an easy-to-use graphical interface. Unlike existing non-commercial heat map packages, which either lack graphical interfaces or are specialized for only one or two kinds of heat maps, Heatmapper is a versatile tool that allows users to easily create a wide variety of heat maps for many different data types and applications. More specifically, Heatmapper allows users to generate, cluster and visualize: (i) expression-based heat maps from transcriptomic, proteomic and metabolomic experiments; (ii) pairwise distance maps; (iii) correlation maps; (iv) image overlay heat maps; (v) latitude and longitude heat maps and (vi) geopolitical (choropleth) heat maps. Heatmapper offers a number of simple and intuitive customization options for facile adjustments to each heat map's appearance and plotting parameters. Heatmapper also allows users to interactively explore their numeric data values by hovering their cursor over each heat map cell, or by using a searchable/sortable data table view. Heat map data can be easily uploaded to Heatmapper in text, Excel or tab delimited formatted tables and the resulting heat map images can be easily downloaded in common formats including PNG, JPG and PDF. Heatmapper is designed to appeal to a wide range of users, including molecular biologists, structural biologists, microbiologists, epidemiologists, environmental scientists, agriculture/forestry scientists, fish and wildlife biologists, climatologists, geologists, educators and students. Heatmapper is available at http://www.heatmapper.ca.
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http://dx.doi.org/10.1093/nar/gkw419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4987948PMC
July 2016

PHASTER: a better, faster version of the PHAST phage search tool.

Nucleic Acids Res 2016 07 3;44(W1):W16-21. Epub 2016 May 3.

Department of Computing Science, Edmonton, AB T6G 2E8, Canada Department of Biological Sciences, University of Alberta, Edmonton, AB T6G 2E9, Canada National Institute for Nanotechnology, 11421 Saskatchewan Drive, Edmonton, AB T6G 2M9, Canada

PHASTER (PHAge Search Tool - Enhanced Release) is a significant upgrade to the popular PHAST web server for the rapid identification and annotation of prophage sequences within bacterial genomes and plasmids. Although the steps in the phage identification pipeline in PHASTER remain largely the same as in the original PHAST, numerous software improvements and significant hardware enhancements have now made PHASTER faster, more efficient, more visually appealing and much more user friendly. In particular, PHASTER is now 4.3× faster than PHAST when analyzing a typical bacterial genome. More specifically, software optimizations have made the backend of PHASTER 2.7X faster than PHAST, while the addition of 80 CPUs to the PHASTER compute cluster are responsible for the remaining speed-up. PHASTER can now process a typical bacterial genome in 3 min from the raw sequence alone, or in 1.5 min when given a pre-annotated GenBank file. A number of other optimizations have also been implemented, including automated algorithms to reduce the size and redundancy of PHASTER's databases, improvements in handling multiple (metagenomic) queries and higher user traffic, along with the ability to perform automated look-ups against 14 000 previously PHAST/PHASTER annotated bacterial genomes (which can lead to complete phage annotations in seconds as opposed to minutes). PHASTER's web interface has also been entirely rewritten. A new graphical genome browser has been added, gene/genome visualization tools have been improved, and the graphical interface is now more modern, robust and user-friendly. PHASTER is available online at www.phaster.ca.
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http://dx.doi.org/10.1093/nar/gkw387DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4987931PMC
July 2016

Inhibitory effect of phenothiazine- and phenoxazine-derived chloroacetamides on Leishmania major growth and Trypanosoma brucei trypanothione reductase.

Eur J Med Chem 2016 Jan 23;108:436-443. Epub 2015 Nov 23.

Institute of Pharmaceutical and Medicinal Chemistry, University of Muenster, Corrensstraße 48, D-48149 Muenster, Germany. Electronic address:

A number of phenothiazine-, phenoxazine- and related tricyclics-derived chloroacetamides were synthesized and evaluated in vitro for antiprotozoal activities against Leishmania major (L. major) promastigotes. Several analogs were remarkably potent inhibitors, with antileishmanial activities being comparable or superior to those of the reference antiprotozoal drugs. Furthermore, we explored the structure-activity relationships of N-10 haloacetamides that influence the potency of such analogs toward inhibition of L. major promastigote growth in vitro. With respect to the mechanism of action, selected compounds were evaluated for time-dependent inactivation of Trypanosoma brucei trypanothione reductase. Our results are indicative of a covalent interaction which could account for potent antiprotozoal activities.
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http://dx.doi.org/10.1016/j.ejmech.2015.11.023DOI Listing
January 2016

ECMDB 2.0: A richer resource for understanding the biochemistry of E. coli.

Nucleic Acids Res 2016 Jan 19;44(D1):D495-501. Epub 2015 Oct 19.

Department of Computing Science, University of Alberta, Edmonton, AB, T6G 2E9, Canada Department of Biological Sciences, University of Alberta, Edmonton, AB, T6G 2E8, Canada National Institute for Nanotechnology, 11421 Saskatchewan Drive, Edmonton, AB, T6G 2M9, Canada

ECMDB or the Escherichia coli Metabolome Database (http://www.ecmdb.ca) is a comprehensive database containing detailed information about the genome and metabolome of E. coli (K-12). First released in 2012, the ECMDB has undergone substantial expansion and many modifications over the past 4 years. This manuscript describes the most recent version of ECMDB (ECMDB 2.0). In particular, it provides a comprehensive update of the database that was previously described in the 2013 NAR Database Issue and details many of the additions and improvements made to the ECMDB over that time. Some of the most important or significant enhancements include a 13-fold increase in the number of metabolic pathway diagrams (from 125 to 1650), a 3-fold increase in the number of compounds linked to pathways (from 1058 to 3280), the addition of dozens of operon/metabolite signalling pathways, a 44% increase in the number of compounds in the database (from 2610 to 3760), a 7-fold increase in the number of compounds with NMR or MS spectra (from 412 to 3261) and a massive increase in the number of external links to other E. coli or chemical resources. These additions, along with many other enhancements aimed at improving the ease or speed of querying, searching and viewing the data within ECMDB should greatly facilitate the understanding of not only the metabolism of E. coli, but also allow the in-depth exploration of its extensive metabolic networks, its many signalling pathways and its essential biochemistry.
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http://dx.doi.org/10.1093/nar/gkv1060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4702790PMC
January 2016

Autophagic digestion of Leishmania major by host macrophages is associated with differential expression of BNIP3, CTSE, and the miRNAs miR-101c, miR-129, and miR-210.

Parasit Vectors 2015 Jul 31;8:404. Epub 2015 Jul 31.

Institute for Molecular Infection Biology, University of Wuerzburg, Josef-Schneider-Str. 2/D15, 97080, Wuerzburg, Germany.

Background: Autophagy participates in innate immunity by eliminating intracellular pathogens. Consequently, numerous microorganisms have developed strategies to impair the autophagic machinery in phagocytes. In the current study, interactions between Leishmania major (L. m.) and the autophagic machinery of bone marrow-derived macrophages (BMDM) were analyzed.

Methods: BMDM were generated from BALB/c mice, and the cells were infected with L. m. promastigotes. Transmission electron microscopy (TEM) and electron tomography were used to investigate the ultrastructure of BMDM and the intracellular parasites. Affymetrix chip analyses were conducted to identify autophagy-related messenger RNAs (mRNAs) and microRNAs (miRNAs). The protein expression levels of autophagy related 5 (ATG5), BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), cathepsin E (CTSE), mechanistic target of rapamycin (MTOR), microtubule-associated proteins 1A/1B light chain 3B (LC3B), and ubiquitin (UB) were investigated through western blot analyses. BMDM were transfected with specific small interfering RNAs (siRNAs) against autophagy-related genes and with mimics or inhibitors of autophagy-associated miRNAs. The infection rates of BMDM were determined by light microscopy after a parasite-specific staining.

Results: The experiments demonstrated autophagy induction in BMDM after in vitro infection with L. m.. The results suggested a putative MTOR phosphorylation-dependent counteracting mechanism in the early infection phase and indicated that intracellular amastigotes were cleared by autophagy in BMDM in the late infection phase. Transcriptomic analyses and specific downregulation of protein expression with siRNAs suggested there is an association between the infection-specific over expression of BNIP3, as well as CTSE, and the autophagic activity of BMDM. Transfection with mimics of mmu-miR-101c and mmu-miR-129-5p, as well as with an inhibitor of mmu-miR-210-5p, demonstrated direct effects of the respective miRNAs on parasite clearance in L. m.-infected BMDM. Furthermore, Affymetrix chip analyses revealed a complex autophagy-related RNA network consisting of differentially expressed mRNAs and miRNAs in BMDM, which indicates high glycolytic and inflammatory activity in the host macrophages.

Conclusions: Autophagy in L. m.-infected host macrophages is a highly regulated cellular process at both the RNA level and the protein level. Autophagy has the potential to clear parasites from the host. The results obtained from experiments with murine host macrophages could be translated in the future to develop innovative and therapeutic antileishmanial strategies for human patients.
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http://dx.doi.org/10.1186/s13071-015-0974-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4521392PMC
July 2015