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  1. Article ; Online: Development and assessment of a multiepitope synthetic antigen for the diagnosis of Dengue virus infection.

    da Silva, Isis Botelho Nunes / de Moraes Rodrigues, Juliano / Batista, Ramon Cid Gismonti / Gomes, Vivian Dos Santos / Chacon, Clarissa de Souza / Almeida, Marcius da Silva / de Araujo, Talita Stelling / Ortiz da Silva, Bianca / Castiñeiras, Terezinha Marta Pereira Pinto / Ferreira Junior, Orlando da Costa / Carneiro, Fabiana Avila / Montero-Lomeli, Monica

    The Brazilian journal of infectious diseases : an official publication of the Brazilian Society of Infectious Diseases

    2024  Volume 28, Issue 3, Page(s) 103746

    Abstract: Immunodiagnostic tests for detecting dengue virus infections encounter challenges related to cross-reactivity with other related flaviviruses. Our research focuses on the development of a synthetic multiepitope antigen tailored for dengue ... ...

    Abstract Immunodiagnostic tests for detecting dengue virus infections encounter challenges related to cross-reactivity with other related flaviviruses. Our research focuses on the development of a synthetic multiepitope antigen tailored for dengue immunodiagnostics. Selected dengue epitopes involved structural linearity and dissimilarity from the proteomes of Zika and Yellow fever viruses which served for computationally modeling the three-dimensional protein structure, resulting in the design of two proteins: rDME-C and rDME-BR. Both proteins consist of seven epitopes, separated by the GPGPG linker, and a carboxy-terminal 6 × -histidine tag. The molecular weights of the final proteins rDME-C and rDME-BR are 16.83 kDa and 16.80 kDa, respectively, both with an isoelectric point of 6.35. The distinguishing factor between the two proteins lies in the origin of their epitope sequences, where rDME-C is based on the reference dengue proteome, while rDME-BR utilizes sequences from prevalent Dengue genotypes in Brazil from 2008 to 2019. PyMol analysis revealed exposure of epitopes in the secondary structure. Successful expression of the antigens was achieved in soluble form and fluorescence experiments indicated a disordered structure. In subsequent testing, rDME-BR and rDME-C antigens were assessed using an indirect Elisa protocol against Dengue infected serum, previously examined with a commercial diagnostic test. Optimal concentrations for antigens were determined at 10 µg/mL for rDME-BR and 30 µg/mL for rDME-C, with serum dilutions ranging from 1:50 to 1:100. Both antigens effectively detected IgM and IgG antibodies in Dengue fever patients, with rDME-BR exhibiting higher sensitivity. Our in-house test showed a sensitivity of 77.3 % and 82.6 % and a specificity of 89.4 % and 71.4 % for rDME-C and rDEM-BR antigens. No cross-reactivity was observed with serum from Zika-infected mice but with COVID-19 serum samples. Our findings underscore the utility of synthetic biology in crafting Dengue-specific multiepitope proteins and hold promise for precise clinical diagnosis and monitoring responses to emerging Dengue vaccines.
    Language English
    Publishing date 2024-05-01
    Publishing country Brazil
    Document type Journal Article
    ZDB-ID 2041400-6
    ISSN 1678-4391 ; 1413-8670
    ISSN (online) 1678-4391
    ISSN 1413-8670
    DOI 10.1016/j.bjid.2024.103746
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    Zs.A 5633: Show issues Location:
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  2. Article ; Online: Mutations in asparaginase II from E. coli and implications for inactivation and PEGylation.

    de Magalhães, Mariana T Q / de Araújo, Talita Stelling / Silva, Bruno Marques / Icart, Luis Peña / Scapin, Sandra M N / da Silva Almeida, Marcius / Lima, Luís Maurício T R

    Biophysical chemistry

    2023  Volume 299, Page(s) 107041

    Abstract: All clinically-used asparaginases convert L-asparagine (L-Asn) to l-aspartate (L-Asp) and l-glutamine (L-Gln) to L-glutamate (L-Glu), which has been useful in reducing bioavailable asparagine and glutamine in patients under treatment for acute ... ...

    Abstract All clinically-used asparaginases convert L-asparagine (L-Asn) to l-aspartate (L-Asp) and l-glutamine (L-Gln) to L-glutamate (L-Glu), which has been useful in reducing bioavailable asparagine and glutamine in patients under treatment for acute lymphoblastic leukemia. The E. coli type 2 L-asparaginase (EcA2) can present different sequences among varying bacterial strains, which we hypothesized that might affect their biological function, stability and interchangeability. Here we report the analysis of two EcA2 provided by the public health system of a middle-income country. These enzymes were reported to have similar specific activity in vitro, whereas they differ in vivo. Protein sequencing by LC-MS-MS and peptide mapping by MALDI-ToF-MS of their tryptic digests revealed that Aginasa™ share similar sequence to EcA2 from E. coli strain BL21(DE3), while Leuginase™ has sequence equivalent to EcA2 from E. coli strain AS1.357. The two amino acid differences between Aginasa™ (64D and 252 T) and Leuginase™ (64 N and 252S) resulted in structural divergences in solution as accessed by small-angle X-ray scattering and molecular dynamics simulation trajectories. The conformational variability further results in dissimilar surface accessibility with major consequences for PEGylation, as well as different susceptibility to degradation by limited proteolysis. The present results reveal that the sequence variations between these two EcA2 variants results in conformational changes associated with differential conformational plasticity, potentially affecting physico-chemical and biological properties, including proteolytic and immunogenic silent inactivation.
    MeSH term(s) Humans ; Amino Acid Sequence ; Asparaginase/chemistry ; Escherichia coli/genetics ; Mutation ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; Polyethylene Glycols/chemistry ; Polyethylene Glycols/metabolism
    Chemical Substances Asparaginase (EC 3.5.1.1) ; Polyethylene Glycols (3WJQ0SDW1A)
    Language English
    Publishing date 2023-05-13
    Publishing country Netherlands
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 185052-0
    ISSN 1873-4200 ; 0301-4622
    ISSN (online) 1873-4200
    ISSN 0301-4622
    DOI 10.1016/j.bpc.2023.107041
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    Zs.A 1147: Show issues Location:
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  3. Article ; Online: The

    de Araujo, Talita Stelling / Barbosa, Glauce Moreno / Sanches, Karoline / Azevedo, Jéssica M / Dos Santos Cabral, Katia Maria / Almeida, Marcius S / Almeida, Fabio C L

    Biomolecular NMR assignments

    2021  Volume 15, Issue 2, Page(s) 341–345

    Abstract: During the past 17 years, the coronaviruses have become a global public emergency, with the first appearance in 2012 in Saudi Arabia of the Middle East respiratory syndrome. Among the structural proteins encoded in the viral genome, the nucleocapsid ... ...

    Abstract During the past 17 years, the coronaviruses have become a global public emergency, with the first appearance in 2012 in Saudi Arabia of the Middle East respiratory syndrome. Among the structural proteins encoded in the viral genome, the nucleocapsid protein is the most abundant in infected cells. It is a multifunctional phosphoprotein involved in the capsid formation, in the modulation and regulation of the viral life cycle. The N-terminal domain of N protein specifically interacts with transcriptional regulatory sequence (TRS) and is involved in the discontinuous transcription through the melting activity of double-stranded TRS (dsTRS).
    MeSH term(s) Middle East Respiratory Syndrome Coronavirus ; Models, Molecular ; Nuclear Magnetic Resonance, Biomolecular ; Nucleocapsid Proteins/chemistry ; Protein Domains
    Chemical Substances Nucleocapsid Proteins
    Language English
    Publishing date 2021-04-29
    Publishing country Netherlands
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2388861-1
    ISSN 1874-270X ; 1874-2718
    ISSN (online) 1874-270X
    ISSN 1874-2718
    DOI 10.1007/s12104-021-10027-6
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article ; Online: Biophysical characterization of two commercially available preparations of the drug containing Escherichia coli L-Asparaginase 2.

    de Araújo, Talita Stelling / Scapin, Sandra M N / de Andrade, William / Fasciotti, Maíra / de Magalhães, Mariana T Q / Almeida, Marcius S / Lima, Luís Maurício T R

    Biophysical chemistry

    2021  Volume 271, Page(s) 106554

    Abstract: The hydrolysis of asparagine and glutamine by L-asparaginase has been used to treat acute lymphoblastic leukemia for over four decades. Each L-asparaginase monomer has a long loop that closes over the active site upon substrate binding, acting as a lid. ... ...

    Abstract The hydrolysis of asparagine and glutamine by L-asparaginase has been used to treat acute lymphoblastic leukemia for over four decades. Each L-asparaginase monomer has a long loop that closes over the active site upon substrate binding, acting as a lid. Here we present a comparative study of two commercially available preparations of the drug containing Escherichia coli L-Asparaginase 2 (EcA2), performed by a comprehensive array of biophysical and biochemical approaches. We report the oligomeric landscape and conformational and dynamic plasticity of E. coli type 2 L-asparaginase present in two different formulations, and its relationship with L-aspartic acid, which is present in Aginasa, but not in Leuginase. The L-Asp present in Aginasa formulation was found to provide to EcA2 a resistance to in vitro proteolysis. EcA2 shows a composition of monomers and oligomers up to tetramers, which is mostly not altered in the presence of L-Asp. Ion-mobility spectrometry-mass spectrometry reveals two conformers for the monomeric EcA2, and that monomeric species has sufficient capacity for selective binding to L-Asp and L-Glu. The N-terminal loop of the EcA2 present in Leuginase, which is part of the active site is disordered, but it gets ordered in the presence of L-Asp, while L-Glu only does so to a limited extent. These data provide new insights on the mechanistic of ligand recognition by EcA2, and the impact of formulation in its conformational diversity landscape.
    MeSH term(s) Asparaginase/chemistry ; Asparaginase/metabolism ; Escherichia coli/enzymology ; Protein Conformation
    Chemical Substances Asparaginase (EC 3.5.1.1)
    Language English
    Publishing date 2021-02-05
    Publishing country Netherlands
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 185052-0
    ISSN 1873-4200 ; 0301-4622
    ISSN (online) 1873-4200
    ISSN 0301-4622
    DOI 10.1016/j.bpc.2021.106554
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

    Zs.A 1147: Show issues Location:
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  5. Article: Insights into the specificity for the interaction of the promiscuous SARS-CoV-2 nucleocapsid protein N-terminal domain with deoxyribonucleic acids

    Caruso, Icaro Putinhon / dos Santos Almeida, Vitor / do Amaral, Mariana Juliani / de Andrade, Guilherme Caldas / de Araújo, Gabriela Rocha / de Araújo, Talita Stelling / de Azevedo, Jéssica Moreira / Barbosa, Glauce Moreno / Bartkevihi, Leonardo / Bezerra, Peter Reis / dos Santos Cabral, Katia Maria / de Lourenço, Isabella Otênio / Malizia-Motta, Clara L.F. / de Luna Marques, Aline / Mebus-Antunes, Nathane Cunha / Neves-Martins, Thais Cristtina / de Sá, Jéssica Maróstica / Sanches, Karoline / Santana-Silva, Marcos Caique /
    Vasconcelos, Ariana Azevedo / da Silva Almeida, Marcius / de Amorim, Gisele Cardoso / Anobom, Cristiane Dinis / Da Poian, Andrea T. / Gomes-Neto, Francisco / Pinheiro, Anderson S. / Almeida, Fabio C.L.

    International journal of biological macromolecules. 2022 Apr. 01, v. 203

    2022  

    Abstract: The SARS-CoV-2 nucleocapsid protein (N) is a multifunctional promiscuous nucleic acid-binding protein, which plays a major role in nucleocapsid assembly and discontinuous RNA transcription, facilitating the template switch of transcriptional regulatory ... ...

    Abstract The SARS-CoV-2 nucleocapsid protein (N) is a multifunctional promiscuous nucleic acid-binding protein, which plays a major role in nucleocapsid assembly and discontinuous RNA transcription, facilitating the template switch of transcriptional regulatory sequences (TRS). Here, we dissect the structural features of the N protein N-terminal domain (N-NTD) and N-NTD plus the SR-rich motif (N-NTD-SR) upon binding to single and double-stranded TRS DNA, as well as their activities for dsTRS melting and TRS-induced liquid-liquid phase separation (LLPS). Our study gives insights on the specificity for N-NTD(-SR) interaction with TRS. We observed an approximation of the triple-thymidine (TTT) motif of the TRS to β-sheet II, giving rise to an orientation difference of ~25° between dsTRS and non-specific sequence (dsNS). It led to a local unfavorable energetic contribution that might trigger the melting activity. The thermodynamic parameters of binding of ssTRSs and dsTRS suggested that the duplex dissociation of the dsTRS in the binding cleft is entropically favorable. We showed a preference for TRS in the formation of liquid condensates when compared to NS. Moreover, our results on DNA binding may serve as a starting point for the design of inhibitors, including aptamers, against N, a possible therapeutic target essential for the virus infectivity.
    Keywords DNA ; RNA ; Severe acute respiratory syndrome coronavirus 2 ; dissociation ; liquids ; nucleocapsid ; nucleocapsid proteins ; oligonucleotides ; pathogenicity ; separation ; therapeutics ; thermodynamics ; transcription (genetics) ; viruses
    Language English
    Dates of publication 2022-0401
    Size p. 466-480.
    Publishing place Elsevier B.V.
    Document type Article
    ZDB-ID 282732-3
    ISSN 1879-0003 ; 0141-8130
    ISSN (online) 1879-0003
    ISSN 0141-8130
    DOI 10.1016/j.ijbiomac.2022.01.121
    Database NAL-Catalogue (AGRICOLA)

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  6. Article ; Online: Insights into the specificity for the interaction of the promiscuous SARS-CoV-2 nucleocapsid protein N-terminal domain with deoxyribonucleic acids.

    Caruso, Icaro Putinhon / Dos Santos Almeida, Vitor / do Amaral, Mariana Juliani / de Andrade, Guilherme Caldas / de Araújo, Gabriela Rocha / de Araújo, Talita Stelling / de Azevedo, Jéssica Moreira / Barbosa, Glauce Moreno / Bartkevihi, Leonardo / Bezerra, Peter Reis / Dos Santos Cabral, Katia Maria / de Lourenço, Isabella Otênio / Malizia-Motta, Clara L F / de Luna Marques, Aline / Mebus-Antunes, Nathane Cunha / Neves-Martins, Thais Cristtina / de Sá, Jéssica Maróstica / Sanches, Karoline / Santana-Silva, Marcos Caique /
    Vasconcelos, Ariana Azevedo / da Silva Almeida, Marcius / de Amorim, Gisele Cardoso / Anobom, Cristiane Dinis / Da Poian, Andrea T / Gomes-Neto, Francisco / Pinheiro, Anderson S / Almeida, Fabio C L

    International journal of biological macromolecules

    2022  Volume 203, Page(s) 466–480

    Abstract: The SARS-CoV-2 nucleocapsid protein (N) is a multifunctional promiscuous nucleic acid-binding protein, which plays a major role in nucleocapsid assembly and discontinuous RNA transcription, facilitating the template switch of transcriptional regulatory ... ...

    Abstract The SARS-CoV-2 nucleocapsid protein (N) is a multifunctional promiscuous nucleic acid-binding protein, which plays a major role in nucleocapsid assembly and discontinuous RNA transcription, facilitating the template switch of transcriptional regulatory sequences (TRS). Here, we dissect the structural features of the N protein N-terminal domain (N-NTD) and N-NTD plus the SR-rich motif (N-NTD-SR) upon binding to single and double-stranded TRS DNA, as well as their activities for dsTRS melting and TRS-induced liquid-liquid phase separation (LLPS). Our study gives insights on the specificity for N-NTD(-SR) interaction with TRS. We observed an approximation of the triple-thymidine (TTT) motif of the TRS to β-sheet II, giving rise to an orientation difference of ~25° between dsTRS and non-specific sequence (dsNS). It led to a local unfavorable energetic contribution that might trigger the melting activity. The thermodynamic parameters of binding of ssTRSs and dsTRS suggested that the duplex dissociation of the dsTRS in the binding cleft is entropically favorable. We showed a preference for TRS in the formation of liquid condensates when compared to NS. Moreover, our results on DNA binding may serve as a starting point for the design of inhibitors, including aptamers, against N, a possible therapeutic target essential for the virus infectivity.
    MeSH term(s) Binding Sites ; COVID-19/virology ; DNA/chemistry ; DNA/metabolism ; Gene Expression Regulation, Viral ; Host-Pathogen Interactions ; Humans ; Hydrogen Bonding ; Models, Molecular ; Nucleic Acids/chemistry ; Nucleic Acids/metabolism ; Nucleocapsid Proteins/chemistry ; Nucleocapsid Proteins/metabolism ; Protein Binding ; Protein Interaction Domains and Motifs ; RNA/chemistry ; RNA/metabolism ; SARS-CoV-2/physiology ; Spectrum Analysis ; Structure-Activity Relationship
    Chemical Substances Nucleic Acids ; Nucleocapsid Proteins ; RNA (63231-63-0) ; DNA (9007-49-2)
    Language English
    Publishing date 2022-01-22
    Publishing country Netherlands
    Document type Journal Article
    ZDB-ID 282732-3
    ISSN 1879-0003 ; 0141-8130
    ISSN (online) 1879-0003
    ISSN 0141-8130
    DOI 10.1016/j.ijbiomac.2022.01.121
    Database MEDical Literature Analysis and Retrieval System OnLINE

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