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Engagement of sialylated glycans with Siglec receptors on suppressive myeloid cells inhibits anticancer immunity via CCL2

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Cellular & Molecular Immunology 2024年第5期21卷 495-509页

作者： Ronja Wieboldt Michael Sandholzer Emanuele Carlini Chia-wei Lin Anastasiya Börsch Andreas Zingg Didier Lardinois Petra Herzig Leyla Don Alfred Zippelius Heinz Läubli Natalia Rodrigues Mantuano Laboratory for Cancer Immunotherapy Department of BiomedicineUniversity Hospital and University of BaselBaselSwitzerland. Functional Genomics Center Zurich ETH ZurichZurichSwitzerland. Bioinformatics Core Facility Department of BiomedicineUniversity of Basel and Swiss Institute of BioinformaticsBaselSwitzerland. Department of Thoracic Surgery University Hospital BaselBaselSwitzerland. Laboratory of Cancer Immunology Department of BiomedicineUniversity Hospital and University of BaselBaselSwitzerland. Division of Oncology University Hospital BaselBaselSwitzerland.

The overexpression of sialic acids on glycans,called hypersialylation,is a common alteration found in cancer *** glycans can enhance immune evasion by interacting with sialic acid-binding immunoglobulin-like lectin(Siglec)receptors on tumorinfiltrating immune ***,we investigated the effect of sialylated glycans and their interaction with Siglec receptors on myeloid-derived suppressor cells(MDSCs).We found that MDSCs derived from the blood of lung cancer patients and tumor-bearing mice strongly express inhibitory Siglec receptors and are highly *** murine cancer models of emergency myelopoiesis,Siglec-E knockout in myeloid cells resulted in prolonged survival and increased tumor infiltration of activated T *** suppressive myeloid cells by blocking Siglec receptors or desialylation strongly reduced their suppressive *** further identified CCL2 as a mediator involved in T-cell suppression upon interaction between sialoglycans and Siglec receptors on *** results demonstrated that sialylated glycans inhibit anticancer immunity by modulating CCL2 expression.

关键词： sialoglycans Sialic acid-binding immunoglobulin-like lectin tumor microenvironment myeloid derived suppressor cells

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Computational screening of potential drugs against COVID-19 disease: the Neuropilin-1 receptor as molecular target

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VirusDisease 2022年第1期33卷 23-31页

作者： Charoute, Hicham Elkarhat, Zouhair Elkhattabi, Lamiae El Fahime, Elmostafa Oukkache, Naoual Rouba, Hassan Barakat, Abdelhamid Research Unit of Epidemiology Biostatistics and Bioinformatics 1 Place Louis Pasteur Institut Pasteur du Maroc Casablanca 20360 Morocco Laboratory of Genomics and Human Genetics Institut Pasteur du Maroc Casablanca Morocco Molecular Biology and Functional Genomics Platform National Center for Scientific and Technical Research Rabat Morocco Laboratory of Venoms and Toxins Institut Pasteur du Maroc Casablanca Morocco

The transmembrane receptor Neuropilin-1 (NRP-1) was reported to serve as a host cell entry factor for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of COVID-19 disease. Therefore, molecular compounds interfering with SARS-CoV-2 binding to NRP-1 seem to be potential candidates as new antiviral drugs. In this study, NRP-1 receptor was targeted using a library of 1167 compounds previously analyzed in COVID-19 related studies. The results show the effectiveness of Nafamostat, Y96, Selinexor, Ebastine and UGS, in binding to NRP-1 receptor, with docking scores lower than − 8.2 kcal/mol. These molecules interact with NRP-1 receptor key residues, which makes them promising drugs to pursue further biological assays to explore their potential use in the treatment of COVID-19. © 2021, The Author(s), under exclusive licence to Indian Virological Society.

关键词： Antivirals COVID-19 Molecular docking Neuropilin-1 SARS-CoV-2 Virtual screening

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Computational evaluation of natural compounds as potential inhibitors of human PEPCK-M: An alternative for lung cancer therapy

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Advances and Applications in bioinformatics and Chemistry 2019年 12卷 15-32页

作者： Baptista, Luiz Phillippe R. Sinatti, Vanessa V. C. da Silva, Joao H. M. Dardenne, Laurent Emmanuel Guimarães, Ana Carolina Laboratory for Functional Genomics and Bioinformatics Fiocruz Oswaldo Cruz Institute Rio de Janeiro RJ Brazil Group for Computational Modelling Fiocruz Oswaldo Cruz Foundation Eusébio CE Brazil Group for Molecular Modelling of Biologic Systems National Laboratory of Scientific Computing Petrópolis RJ Brazil

Background: Lung cancer is the leading cause of cancer-related death worldwide. Among its subtypes, non-small cell lung cancer (NSCLC) is the most common. Recently, the mitochondrial isoform of the enzyme phosphoenolpyruvate carboxykinase (HsPEPCK-M) was identified as responsible for the metabolic adaptation in the NSCLC allowing tumor growth even under conditions of glucose deficiency. This adaptation is possible due to the role of HsPEPCK-M in gluconeogenesis, converting the oxaloacetate to phosphoenolpyruvate in the presence of GTP, which plays an important role in the energetic support of these tumors. In this context, it was shown that the inhibition or knockdown of this enzyme was able to induce apoptosis in NSCLC under low glucose conditions. Purpose: In this study, novel putative inhibitors were proposed for the human PEPCK-M (HsPEPCK-M) based on a computer-aided approach. Methods: Comparative modeling was used to generate 3D models for HsPEPCK-M. Subsequently, the set of natural compounds of the ZINC database was screened against HsPEPCK-M models using structure-based pharmacophore modeling and molecular docking approaches. The selected compounds were evaluated according to its chemical diversity and clustered based on chemical similarity. Results: The pharmacophore hypotheses, generated based on known PEPCK inhibitors, were able to select 7,124 candidate compounds. These compounds were submitted to molecular docking studies using three conformations of HsPEPCK-M generated by comparative modeling. The aim was to select compounds with high predicted binding affinity for at least one of the conformations of HsPEPCK-M. After molecular docking, 612 molecules were selected as potential inhibitors of HsPEPCK-M. These compounds were clustered according to their structural similarity. Chemical profiling and binding mode analyses of these compounds allowed the proposal of four promising compounds: ZINC01656421, ZINC895296, ZINC00895535 and ZINC02571340. Conclusion:

关键词： Lung cancer Molecular docking Natural products PEPCK-M Pharmacophore modeling Virtual screening

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The enigma of introns: Intronic miRNA-directed mechanisms and alternative splicing diversify alternative oxidase potential in Vitis vinifera

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International journal of biological macromolecules 2025年 316卷 144300页

作者： Shahid Aziz Thais Andrade Germano Antonio Edson Rocha Oliveira José Ednésio da Cruz Freire Matheus Finger Ramos de Oliveira Karine Leitão Lima Thiers Birgit Arnholdt-Schmitt Jose Helio Costa Functional Genomics and Bioinformatics Department of Biochemistry and Molecular Biology Federal University of Ceara Fortaleza Ceara Brazil Non-Institutional Competence Focus (NICFocus) 'Functional Cell Reprogramming and Organism Plasticity' (FunCROP) coordinated from Foros de Vale de Figueira Alentejo Portugal. Electronic address: shahidaziz.cl@***. Non-Institutional Competence Focus (NICFocus) 'Functional Cell Reprogramming and Organism Plasticity' (FunCROP) coordinated from Foros de Vale de Figueira Alentejo Portugal. Nucleus of Experimental Biology University of Fortaleza Fortaleza Ceará Brazil. Biochemistry and Gene Expression Laboratory Superior Institute of Biomedical Sciences State University of Ceará Fortaleza Ceará Brazil. Functional Genomics and Bioinformatics Department of Biochemistry and Molecular Biology Federal University of Ceara Fortaleza Ceara Brazil. Non-Institutional Competence Focus (NICFocus) 'Functional Cell Reprogramming and Organism Plasticity' (FunCROP) coordinated from Foros de Vale de Figueira Alentejo Portugal. Electronic address: biarnaflora@***. Non-Institutional Competence Focus (NICFocus) 'Functional Cell Reprogramming and Organism Plasticity' (FunCROP) coordinated from Foros de Vale de Figueira Alentejo Portugal INCTAgriS - National Institute of Science and Technology in Sustainable Agriculture in the Tropical Semi-Arid Region Brazil. Electronic address: helio.costa@ufc.br.

Alternative oxidase (AOX) transcript levels were associated with efficiently balanced respiration and validated to assist selection on multiple-resilience. Consistently, AOX has also been identified as a target for weakening the survival capabilities of parasites and microorganisms responsible for severe human diseases. Despite the unique features of AOX in Vitis vinifera, particularly intense constitutive expression of AOX2 in the presence of unusually large introns that challenge the dogma of gene expression in eukaryotes, V. vinifera has been overlooked in AOX research. This study uncovered two distinct alternative splicing variants of the AOX: AOX1a-Alternative variant attributed to unusual retention of the intron-4 in the 3´UTR, and AOX2-alternative variant, which is intron-1-dependent, involving the skipping of exon-1. The AOX2-alternative variant differed in that cystine-I changed to serine, which is linked to different metabolite stimulation. However, molecular docking suggested that AOX2 and the variant proteins exhibit the same catalytic activities and binding affinities for ubiquinol. The unique large introns in AOX2 exhibited 16 miRNAs, including the master regulator of development and stress responses, mir-398. Among these, nine were conserved and validated in other plant species, whereas seven were considered potential novel miRNA candidates. Transcriptome analyses revealed down- and up-regulation of AOX1a-Alternative during shrivelling and water deficiency, and up-regulation of AOX2-Alternative with increasing temperatures. Consistent with previous studies, AOX1a and AOX1d were linked to biotic and abiotic stress, whereas AOX2 showed constitutive or developmental regulation. This study encourages hypothesis-driven advanced research on early mechanisms and functionality of newly discovered alternative splicing events and intronic miRNAs. Given functional marker-assisted breeding, it strengthened the requirement to consider overall AOX transcript levels

关键词： Alternative splicing Intron retention Large intron Transcription factors Ubiquinol miRNA

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Single-cell transcriptome analysis dissects lncRNA-associated gene networks in Arabidopsis

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Plant Communications 2024年第2期5卷 94-105页

作者： Zhaohui He Yangming Lan Xinkai Zhou Bianjiong Yu Tao Zhu Fa Yang Liang-Yu Fu Haoyu Chao Jiahao Wang Rong-Xu Feng Shimin Zuo Wenzhi Lan Chunli Chen Ming Chen Xue Zhao Keming Hu Dijun Chen State Key Laboratory of Pharmaceutical Biotechnology School of Life SciencesNanjing UniversityNanjing 210023China Department of Bioinformatics College of Life SciencesZhejiang UniversityHangzhou 310058China Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding/Key Laboratory of Plant Functional Genomics of the Ministry of Education College of AgricultureYangzhou UniversityYangzhou 225009China Co-Innovation Center for Modern Production Technology of Grain Crops of Jiangsu Province/Key Laboratory of Crop Genetics and Physiology of Jiangsu Province Yangzhou UniversityYangzhou 225009China Zhejiang Zhoushan High School Zhoushan 316099China National Key Laboratory for Germplasm Innovation and Utilization for Fruit and Vegetable Horticultural Crops Hubei Hongshan LaboratoryWuhan 430070China

The plant genome produces an extremely large collection of long noncoding RNAs(lncRNAs)that are generally expressed in a context-specific manner and have pivotal roles in regulation of diverse biological ***,we mapped the transcriptional heterogeneity of lncRNAs and their associated gene reg-ulatory networks at single-cell *** generated a comprehensive cell atlas at the whole-organism level by integrative analysis of 28 published single-cell RNA sequencing(scRNA-seq)datasets from juvenile Arabidopsis *** then provided an in-depth analysis of cell-type-related lncRNA signatures that show expression patterns consistent with canonical protein-coding gene *** further demon-strated that the cell-type-specific expression of lncRNAs largely explains their tissue *** addi-tion,we predicted gene regulatory networks on the basis of motif enrichment and co-expression analysis of lncRNAs and mRNAs,and we identified putative transcription factors orchestrating cell-type-specific expression of *** analysis results are available at the single-cell-based plant lncRNA atlas data-base(scPLAD;https://***/scPLAD/).Overall,this work demonstrates the power of inte-grative single-cell data analysis applied to plant lncRNA biology and provides fundamental insights into lncRNA expression specificity and associated gene regulation.

关键词： single-cell transcriptomics long noncoding RNAs lncRNAs gene regulatory networks GRNs plants

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Empowering communities through One Health and ArtScience: An innovative approach to combat Chagas disease in endemic areas of Minas Gerais, Brazil

Public Health Challenges

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Public Health Challenges 2025年第1期4卷

作者： Gonçalves-Oliveira, Jonathan Lopes, Catarina Macedo Gonçalves, Teresa C. M. Sarmento, Suellen Nunes Correia, João Paulo S. O. de Sousa, Rute H. A. Ribeiro, Antônia de Castro Sarquis, Otília Almeida-Silva, Juliana Silva, Sarah C. dos S. Suarez-Fontes, Ana M. Ferreira, Roberto Rodrigues Vieira, Thallyta M. Garzoni, Luciana R. Vannier-Santos, Marcos A. Araujo-Jorge, Tania C. Laboratory of Hantaviruses and Rickettsiosis Oswaldo Cruz Institute (LHR-IOC/Fiocruz) Oswaldo Cruz Foundation Rio de Janeiro Rio de Janeiro Brazil Laboratory Interdisciplinary in Entomological Surveillance in Diptera and Hemiptera Oswaldo Cruz Institute (LIVEDIH-IOC/Fiocruz) Oswaldo Cruz Foundation (Fiocruz) Rio de Janeiro Rio de Janeiro Brazil Laboratory National and International Reference in Triatominae Taxonomy Oswaldo Cruz Institute (LNIRTT-IOC/FIOCRUZ) Oswaldo Cruz Foundation (Fiocruz) Rio de Janeiro Rio de Janeiro Brazil Laboratory of Biology and Parasitology of Reservoir Wild Mammals Oswaldo Cruz Institute (LABPMR-IOC/Fiocruz) Oswaldo Cruz Foundation Rio de Janeiro Rio de Janeiro Brazil Laboratory of Ecoepidemiology of Chagas Disease Oswaldo Cruz Institute (LEDOC-IOC/Fiocruz) Oswaldo Cruz Foundation Rio de Janeiro Rio de Janeiro Brazil Laboratory of Innovations in Therapies Education and Bioproducts Oswaldo Cruz Institute (LITEB-IOC/Fiocruz) Oswaldo Cruz Foundation (Fiocruz) Rio de Janeiro Rio de Janeiro Brazil Laboratory of Functional Genomics and Bioinformatics Oswaldo Cruz Institute (LAGFB-IOC/Fiocruz) Oswaldo Cruz Foundation (Fiocruz) Rio de Janeiro Rio de Janeiro Brazil Center for Biological and Health Science Universidade Estadual de Montes Claros Campus Universitário Prof. Darcy Ribeiro Minas Gerais Montes Claros Brazil

Background: Chagas disease (CD) is a neglected zoonosis that requires study through the One Health approach, as it involves various aspects of animal, environmental, and human health in its transmission cycle. This disease affects 7 million people in 20 countries in Latin America, resulting in approximately seven thousand deaths annually worldwide. Local knowledge is crucial for developing educational strategies to mitigate the risks of disease transmission, particularly in endemic areas. Aims: In this study, we present the experience of integrating five stands with complementary subjects on CD, focusing on environmental, biological, and human life factors, as well as its invertebrate and vertebrate hosts. Methods: This research was conducted as part of the Chagas Express XXI initiative, a social-educational technology conceived as an imaginary train consisting of six thematic “wagons” (education stations) related to CD. The current study took place in the northern region of the state of Minas Gerais, Brazil, focusing on the activities of wagon 4, which involved 649 participants in two municipalities: Espinosa and Montes Claros, both endemic rural and urban areas for CD. Results: Participants’ prior knowledge was assessed through a series of questions, and educational mediators engaged in dialog during voluntary sessions. We found that crucial aspects related to the prevention and control of vector-borne transmissions, such as host and vector diversity, as well as household risks, were neglected in local education activities or unknown to the populations of both municipalities. Conclusions: This study demonstrates that zoonosis, particularly CD, should be included in basic education and health professional training courses, employing strategies that consider the different socio-environmental interfaces and aim to improve human, animal, and environmental living conditions. © 2025 The Authors. Public Health Challenges published by John Wiley & Sons Ltd.

关键词： ArtScience Chagas disease education hosts mammals One Health vectors

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Revelation of potential antiviral activity of Azadirachta indica against Zika virus protease: An integrated molecular docking and dynamic simulation approach

Computational and Structural Biotechnology Reports

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Computational and Structural Biotechnology Reports 2025年 2卷

作者： Md. Habib Ullah Masum Rehana Parvin Syed Mohammad Lokman Zarin Tasnim Md. Mazharul Alam Jannatul Ferdous Department of Genomics and Bioinformatics Faculty of Biotechnology and Genetic Engineering Chattogram Veterinary and Animal Sciences University Khulshi 4225 Bangladesh Department of Pathology and Parasitology Faculty of Veterinary Medicine Chattogram Veterinary and Animal Sciences University Khulshi 4225 Bangladesh Asian University for Women (AUW) Chattogram 4000 Bangladesh Department of Industrial Biotechnology Faculty of Biotechnology and Genetic Engineering Chattogram Veterinary and Animal Sciences University Khulshi 4225 Bangladesh Functional Genomics and Proteomics Laboratory (FGPL) Department of Genetic Engineering and Biotechnology Faculty of Biological Sciences University of Chittagong Chattogram 4331 Bangladesh Department of Obstetrics and Gynecology Chittagong Medical College Hospital Chattogram 4203 Bangladesh

The Zika virus (ZIKV) is an RNA virus that mostly affects humans and primates and is transmitted via Aedes mosquitoes . The emergence of ZIKV has intensified global health concerns, emphasizing the urgent need for effective antiviral therapies. As a vital enzyme in the replication of ZIKV, the NS2B-NS3 protease offers a promising therapeutic target. This study leverages computer-aided drug design to identify bioactive compounds capable of inhibiting the ZIKV NS2B-NS3 protease, thereby disrupting viral replication. Bioactive compounds from Azadirachta indica were screened through virtual approaches, resulting in six with exceptional docking scores (<-5.5 kcal/mol). Analyses of the physicochemical properties validated the drug-like traits of the IMPHY014854 and IMPHY014908, conveying suitable hydrogen bonding profiles and moderate solubility (Log S: −2.22 to −3.60). Evaluations of pharmacokinetics suggested significant gastrointestinal absorption while exposing no permeability across the blood-brain barrier, thereby reducing interactions with the central nervous system. Toxicity evaluations revealed limited toxic effects, while several bioactive compounds exhibited particular toxicological issues. Molecular dynamics simulations further revealed the stability and interaction dynamics of ZIKV protease-ligand complexes, where the IMPHY014854 and IMPHY014908 exhibited significant residue flexibility, compactness, and solvent accessibility profiles over a 100 ns simulation. Principal component analysis revealed conformational distinctions, while Gibbs's free energy analysis demonstrated greater stability for the ZIKV protease-IMPHY014908 complex. This integrative computational strategy highlights the potential of phytochemicals as the ZIKV protease inhibitors and provides a foundation for developing effective antiviral drugs. Further, in vivo studies must be implemented to validate these findings and advance therapeutic prospects against ZIKV.

关键词： Mosquito-borne Phytodynamics Biocomputational Neem plant Protease inhibitor Antiviral drug

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T cell immune responses to SARS-CoV-2 and variants of concern(Alpha and Delta)in infected and vaccinated individuals

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Cellular & Molecular Immunology 2021年第11期18卷 2554-2556页

作者： Stanley C.Jordan Bong-Ha Shin Terry-Ann M.Gadsden Maggie Chu Anna Petrosyan Catherine N.Le Rachel Zabner Jillian Oft Isabel Pedraza Susan Cheng Ashley Vo Noriko Ammerman Jasmine Plummer Shili Ge Max Froch Anders Berg Mieko Toyoda Ruan Zhang Comprehensive Transplant Center Transplant Immunology LaboratoryCedars-Sinai Medical CenterLos AngelesCAUSA Division of Infectious Diseases Cedars-Sinai Medical CenterLos AngelesCAUSA Department of Cardiology Cedars-Sinai Medical CenterLos AngelesCAUSA Department of Bioinformatics&Functional Genomics Cedars-Sinai Medical CenterLos AngelesCAUSA Department of Pathology&Laboratory Medicine Cedars-Sinai Medical CenterLos AngelesCAUSA

Our understanding of immune responses to SARS-CoV-2 and variants of concern(VOCs)has been primarily acquired through analysis of Spike-specific IgG ***,a more comprehensive understanding of the breadth and longevity of immune responses after infection and vaccination requires analysis of cellular ***,we report on T cell immunity in infected and vaccinated individuals,identifying CD4+/CD8+T cell cytokine responses to SARS-CoV-2 and variant peptides(Alpha,B.1.1.7 and Delta,B.1.617.2).

关键词： Alpha infected immunity

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Distinct transcriptional changes in hematopoietic progenitor subsets upon LPS-induced emergency granulopoiesis

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Experimental hematology 2025年 147卷 104792页

作者： Sladjana Kosanovic Karolina Vanickova Mirko Milosevic Irina Ribeiro Bas Mateusz Chilinski Dariusz Plewczynski Petr Danek Jakub Rohlena Katerina Rohlenova Meritxell Alberich-Jorda Department of Hemato-oncology Institute of Molecular Genetics of the Czech Academy of Sciences Videnska 1083 142 20 Prague Czech Republic Faculty of Science Charles University 12800 Prague Czech Republic. Institute of Biotechnology of the Czech Academy of Sciences Prumyslova 595 252 50 Vestec Prague West Czech Republic. Laboratory of Bioinformatics and Computational Genomics Faculty of Mathematics and Information Science Warsaw University of Technology Warsaw Poland Laboratory of Functional and Structural Genomics Centre of New Technologies University of Warsaw Warsaw Poland Section for Computational and RNA Biology Department of Biology University of Copenhagen Copenhagen Denmark. Laboratory of Functional and Structural Genomics Centre of New Technologies University of Warsaw Warsaw Poland. Department of Hemato-oncology Institute of Molecular Genetics of the Czech Academy of Sciences Videnska 1083 142 20 Prague Czech Republic. Childhood Leukaemia Investigation Prague Department of Pediatric Haematology and Oncology 2(nd) Faculty of Medicine Charles University in Prague University Hospital Motol V Uvalu 84 Prague 150 06 Czech Republic. Electronic address: alberich@img.cas.cz.

Emergency granulopoiesis is a critical process by which hematopoietic progenitors and stem cells facilitate enhanced granulocytic production during severe infections. However, the role of distinct multipotent progenitors (MPPs) at early stages of this process remains underexplored. Here, we investigated the contribution of MPPs to granulocytic production following lipopolysaccharide (LPS) administration in wild-type mice, simulating a bacterial infection. Transplantation assays demonstrated that LPS exposure reduces the engraftment capacity of lymphoid-biased MPP4 and enhances lymphoid production, rather than supporting myeloid lineage output. Further, single-cell RNA sequencing (scRNA-seq) of MPPs isolated from control and LPS challenged mice revealed transcriptional reprogramming of non-lineage committed MPPs towards myeloid- and erythroid-biased progenitors. Notably, inflammatory progenitor populations emerged upon activation of LPS-induced emergency granulopoiesis, displaying chromatin accessibility changes that align with a commitment to myeloid and erythroid fates. Pseudotime analysis elucidated cellular trajectories that suggest a developmental pathway where unbiased progenitors, present in non-stress conditions, transition towards myeloid and erythroid lineage outputs upon LPS administration. In line with our functional MPP4 assessment, scRNA-seq suggested that lymphoid-biased progenitors do not transcriptionally rewire during early stages of emergency granulopoiesis. Collectively, our data highlight the critical role of specific MPP subsets in responding to LPS-induced inflammatory signals and underscore the dynamic adaptations that occur during granulocyte production in response to infection.

关键词： ATACseq Emergency Granulopoiesis Hematopoietic stem cell Progenitors lipopolysaccharide scRNAseq

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Identification of Conserved Regulatory Elements in Mammalian Promoter Regions:A Case Study Using the PCK1 Promoter

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genomics, Proteomics & bioinformatics 2008年第3期6卷 129-143页

作者： George E. Liui Matthew T. Weirauch Curtis P. Van Tassell Robert W. Li Tad S. Sonstegard LakshmiK. Matukumal Erin E. Connor Richard W. Hanson Jianqi Yang Bovine Functional Genomics Laboratory Beltsville Agricultural Research Center Beltsville MD 20705 USA Department of Biomolecular Engineering University of California Santa Cruz Santa Cruz CA 95064 USA Department of Bioinformatics and Computational Biology George Mason University Manassas VA 20110 USA Department of Biochemistry Case Western Reserve University School of Medicine Cleveland OH 44106 USA.

A systematic phylogenetic footprinting approach was performed to identify conserved transcription factor binding sites （TFBSs） in mammalian promoter regions using human, mouse and rat sequence alignments. We found that the score distributions of most binding site models did not follow the Gaussian distribution required by many statistical methods. Therefore, we performed an empirical test to establish the optimal threshold for each model. We gauged our computational predictions by comparing with previously known TFBSs in the PCK1 gene promoter of the cytosolic isoform of phosphoenolpyruvate carboxykinase, and achieved a sensitivity of 75% and a specificity of approximately 32% Almost all known sites overlapped with predicted sites, and several new putative TFBSs were also identified. We validated a predicted SP1 binding site in the control of PCK1 transcription using gel shift and reporter assays. Finally, we applied our computational approach to the prediction of putative TFBSs within the promoter regions of all available RefSeq genes. Our full set of TFBS predictions is freely available at http：//***/tfbsConsSites.

关键词： phylogenetic footprinting phosphoenolpyruvate carboxykinase transcription factor binding sites mammalian gene promoters

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