Publications by authors named "Mônica Vieira"

83 Publications

Changes in blood donation and utilization secondary to Covid-19 outbreak.

Transfus Apher Sci 2021 Feb 17:103102. Epub 2021 Feb 17.

Fundação Pró-Sangue São Paulo Hemocenter, São Paulo, Brazil; Laboratory of Medical Investigation in Pathogenesis and Targeted Therapy in Onco-Immuno-Hematology (LIM-31), Department of Hematology, Hospital das Clínicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, Brazil.

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http://dx.doi.org/10.1016/j.transci.2021.103102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7888989PMC
February 2021

The Impact of [C16Pyr][Amp] on the Aggressiveness in Breast and Prostate Cancer Cell Lines.

Int J Mol Sci 2020 Dec 16;21(24). Epub 2020 Dec 16.

Research Centre in Health and Environment (CISA), School of Health (ESS), Polytechnic Institute of Porto (P.PORTO), 4200-072 Porto, Portugal.

Breast (BrCa) and prostate (PCa) cancers are the most common malignancies in women and men, respectively. The available therapeutic options for these tumors are still not curative and have severe side effects. Therefore, there is an urgent need for more effective antineoplastic agents. Herein, BrCa, PCa, and benign cell lines were treated with two ionic liquids and two quinoxalines and functional experiments were performed-namely cell viability, apoptosis, cytotoxicity, and colony formation assays. At the molecular level, an array of gene expressions encompassing several molecular pathways were used to explore the impact of treatment on gene expression. Although both quinoxalines and the ionic liquid [C2OHMIM][Amp] did not show any effect on the BrCa and PCa cell lines, [C16Pyr][Amp] significantly decreased cell viability and colony formation ability, while it increased the apoptosis levels of all cell lines. Importantly, [C16Pyr][Amp] was found to be more selective for cancer cells and less toxic than cisplatin. At the molecular level, this ionic liquid was also associated with reduced expression levels of , , and , in both BrCa and PCa cell lines. Hence, [C16Pyr][Amp] was shown to be a promising anticancer therapeutic agent for BrCa and PCa cell lines.
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http://dx.doi.org/10.3390/ijms21249584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7765672PMC
December 2020

Phagocyte Escape of : The Role of TLRs and NLRs.

Front Immunol 2020 6;11:571816. Epub 2020 Oct 6.

Institut Pasteur, Microbiology Department, Unité Biologie et Génétique de la Paroi Bactérienne, Paris, France.

The spirochetal bacteria spp. are causative agents of leptospirosis, a globally neglected and reemerging zoonotic disease. Infection with these pathogens may lead to an acute and potentially fatal disease but also to chronic asymptomatic renal colonization. Both forms of disease demonstrate the ability of leptospires to evade the immune response of their hosts. In this review, we aim first to recapitulate the knowledge and explore the controversial data about the opsonization, recognition, intracellular survival, and killing of leptospires by scavenger cells, including platelets, neutrophils, macrophages, and dendritic cells. Second, we will summarize the known specificities of the recognition or escape of leptospire components (the so-called microbial-associated molecular patterns; MAMPs) by the pattern recognition receptors (PRRs) of the Toll-like and NOD-like families. These PRRs are expressed by phagocytes, and their stimulation by MAMPs triggers pro-inflammatory cytokine and chemokine production and bactericidal responses, such as antimicrobial peptide secretion and reactive oxygen species production. Finally, we will highlight recent studies suggesting that boosting or restoring phagocytic functions by treatments using agonists of the Toll-like or NOD receptors represents a novel prophylactic strategy and describe other potential therapeutic or vaccine strategies to combat leptospirosis.
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http://dx.doi.org/10.3389/fimmu.2020.571816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7573490PMC
May 2021

Virulent Induce Cytotoxic Effects in Human Platelets Through Direct Interactions.

Front Microbiol 2020 24;11:572972. Epub 2020 Sep 24.

Laboratório de Desenvolvimento de Vacinas, Butantan Institute, São Paulo, Brazil.

Leptospirosis is a prevalent zoonotic disease, caused by bacteria of the genus . Leptospirosis frequently leads to hemostatic disturbances, and the severe cases are marked by hemorrhages and low platelet number in circulation, which is associated with the patients' poor outcomes. Nevertheless, -platelet interactions remain poorly explored. In this study, we performed a series of experiments evaluating whether leptospires induce human platelet aggregation, activation, and morphological changes. Platelets were incubated with virulent and the platelet outcomes were assessed by aggregometry, flow cytometry, and scanning and transmission electron microscopy. Our results show that leptospires alone do not induce platelet aggregation and activation, and induce platelet cytotoxic effects instead, by clearly inducing platelet disruption and detachment. We show for the first time that virulent leptospires do interact directly with platelets, an event that could trigger pathophysiological effects during the infection. This study might serve as a basis for the development of novel treatments for the disease.
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http://dx.doi.org/10.3389/fmicb.2020.572972DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7552899PMC
September 2020

Structural analysis of CACHE domain of the McpA chemoreceptor from Leptospira interrogans.

Biochem Biophys Res Commun 2020 12 21;533(4):1323-1329. Epub 2020 Oct 21.

Laboratório de Desenvolvimento de Vacinas, Instituto Butantan, Avenida Vital Brasil, 1500, 05503-900, São Paulo, SP, Brazil.

Leptospira is a genus of spirochete bacteria highly motile that includes pathogenic species responsible to cause leptospirosis disease. Chemotaxis and motility are required for Leptospira infectivity, pathogenesis, and invasion of bacteria into the host. In prokaryotes, the most common chemoreceptors are methyl-accepting chemotaxis proteins that have a role play to detect the chemical signals and move to a favorable environment for its survival. Here, we report the first crystal structure of CACHE domain of the methyl-accepting chemotaxis protein (McpA) of L. interrogans. The structural analysis showed that McpA adopts similar α/β architecture of several other bacteria chemoreceptors. We also found a typical dimerization interface that appears to be functionally crucial for signal transmission and chemotaxis. In addition to McpA structural analyses, we have identified homologous proteins and conservative functional regions using bioinformatics techniques. These results improve our understanding the relationship between chemoreceptor structures and functions of Leptospira species.
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http://dx.doi.org/10.1016/j.bbrc.2020.10.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7744396PMC
December 2020

Oxidative DNA damage is increased in older adults with a major depressive episode: A preliminary study.

J Affect Disord 2021 01 24;279:106-110. Epub 2020 Sep 24.

Campbell Family Mental Health Research Institute, Centre for Addiction and Mental Health, Toronto, ON, Canada; Adult Neurodevelopment and Geriatric Psychiatry Division, Centre for Addiction and Mental Health, Toronto, ON, Canada; Department of Psychiatry, Faculty of Medicine, University of Toronto, Toronto, ON, Canada. Electronic address:

Background: DNA oxidative damage is a marker of increased oxidative stress activity. Elevated DNA oxidative damage has been associated with major depressive disorder in young adults, but there is no information about DNA oxidative damage in late-life depression. This study aims to evaluate whether older adults with late-life depression (LLD) has increased DNA oxidative damage compared to healthy older adults.

Methods: We included 92 participants (57 with LLD [73.2 ± 7.7 years-old] and 35 non-depressed subjects (Controls) [70.5 ± 7.4 years-old]). We analyzed the plasma 8‑hydroxy-2'-deoxyguanosine (8-oxo-dG), a marker of DNA oxidation, using a commercially-available ELISA assay.

Results: LLD participants had significantly higher 8-oxo-DG levels compared to controls (P<0.001). 8-oxo-dG levels were significantly correlated with depressive symptoms as assessed by the Hamilton Depression Rating Scale (rho=0.34, p<0.001). The plasma levels of 8-OHdG were not significantly correlated with other clinical, neurocognitive, and demographic variables.

Limitations: Our current results are limited by the relatively small sample size, cross-sectional design, and the recruitment of participants in tertiary center for assessment and treatment of LLD.

Conclusions: Older adults with LLD have increased DNA oxidative damage. Our findings provide additional evidence for elevated oxidative stress activity in LLD and the possible activation of age-related biological pathways and enhanced biological aging changes in LLD.
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http://dx.doi.org/10.1016/j.jad.2020.09.084DOI Listing
January 2021

Recycling Old Antibiotics with Ionic Liquids.

Antibiotics (Basel) 2020 Sep 4;9(9). Epub 2020 Sep 4.

Ciências Químicas e das Biomoléculas/CISA, Escola Superior de Saúde-Instituto Politécnico do Porto, Rua Dr. António Bernardino de Almeida 400, P-4200-072 Porto, Portugal.

Antibiotics are considered one of the great "miracles" of the 20th century. Now in the 21st century in the post-antibiotic era, the miracle is turning into a nightmare, due to the growing problem of the resistance of microorganisms to classic antimicrobials and the non-investment by the pharmaceutical industry in new antimicrobial agents. Unfortunately, the current COVID-19 pandemic has demonstrated the global risks associated with uncontrolled infections and the various forms of impact that such a pandemic may have on the economy and on social habits besides the associated morbidity and mortality. Therefore, there is an urgent need to recycle classic antibiotics, as is the case in the use of ionic liquids (ILs) based on antibiotics. Thus, the aim of the present review is to summarize the data on ILs, mainly those with antimicrobial action and especially against resistant strains. The main conclusions of this article are that ILs are flexible due to their ability to modulate cations and anions as a salt, making it possible to combine the properties of both and multiplying the activity of separate cations and anions. Also, these compounds have low cost methods of production, which makes it highly attractive to explore them, especially as antimicrobial agents and against resistant strains. ILs may further be combined with other therapeutic strategies, such as phage or lysine therapy, enhancing the therapeutic arsenal needed to fight this worldwide problem of antibacterial resistance. Thus, the use of ILs as antibiotics by themselves or together with phage therapy and lysine therapy are promising alternatives against pathogenic microorganisms, and may have the possibility to be used in new ways in order to restrain uncontrolled infections.
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http://dx.doi.org/10.3390/antibiotics9090578DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7558273PMC
September 2020

The Leptospira interrogans LIC10774 is a multifunctional surface protein that binds calcium and interacts with host components.

Microbiol Res 2020 May 19;235:126470. Epub 2020 Mar 19.

Laboratório de Desenvolvimento de Vacinas, Instituto Butantan, 05503-900, São Paulo, Brazil; Programa de Pós-Graduação Interunidades em Biotecnologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, 05508-900, São Paulo, Brazil. Electronic address:

Leptospirosis is a global re-emerging zoonosis, caused by pathogenic bacteria of the genus Leptospira. Humans are infected mainly through contact with contaminated water or soil. The understanding of the molecular mechanisms of leptospirosis through the characterization of unknown outer membrane proteins may contribute to the development of new treatments, diagnostic methods and vaccines. We have identified using bioinformatics analysis a protein that is encoded by the gene LIC10774, predicted to be localized at the leptospiral outer membrane and exhibit beta-roll folding. Surface exposure was confirmed by flow cytometry, ELISA and immunofluorescence-based confocal microscopy. Through circular dichroism spectroscopy and hydrophobic dye binding we have shown that rLIC10774 binds calcium ions, which imposes changes to secondary and tertiary structures. The recombinant protein was capable of binding to several host extracellular matrix and serum components. Therefore, we describe LIC10774 as a calcium-binding protein exposed in the outer surface of pathogenic leptospires with possible multifunctional roles in adhesion to host tissues, evasion of the immune system and participation in dissemination processes during leptospirosis. In addition, we hypothesize that the calcium binding is important for temperature-dependent functional roles during leptospirosis.
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http://dx.doi.org/10.1016/j.micres.2020.126470DOI Listing
May 2020

The interplay between host haemostatic systems and spp. infections.

Crit Rev Microbiol 2020 Mar 6;46(2):121-135. Epub 2020 Mar 6.

Lab. Desenvolvimento de Vacinas, Butantan Institute, Sao Paulo, Brazil.

Hemostasis is a defence mechanism that protects the integrity of the vascular system and is comprised of the coagulation cascade, fibrinolysis, platelet aggregation, and vascular endothelium. Besides the primary function in preserving the vascular integrity, the haemostatic system cooperates with immune and inflammatory processes to eliminate invading pathogens during microbial infections. Under pathological manifestations, hemostasis must therefore interact in a coordinated manner with inflammatory responses and immune reactions. Several pathogens can modulate these host-derived countermeasures by specifically targeting certain haemostatic components for their own benefit. Thus, the ability to modulate host defence systems has to be considered as an essential bacterial virulence mechanism. Complications that bacterial pathogens can induce are therefore often the consequence of evoked host responses. A comprehensive understanding of the molecular mechanisms triggered in infectious processes may help to develop prophylactic methods and novel therapies for the patients suffering from a particular infectious disease. This review aims to provide a critical updated compiling of recent studies on how the pathogenic can interact with and manipulate the host haemostatic systems and the consequences for leptospirosis pathogenesis.
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http://dx.doi.org/10.1080/1040841X.2020.1735299DOI Listing
March 2020

Leptospira interrogans Bat proteins impair host hemostasis by fibrinogen cleavage and platelet aggregation inhibition.

Med Microbiol Immunol 2020 Apr 20;209(2):201-213. Epub 2020 Feb 20.

Lab. Desenvolvimento de Vacinas, Instituto Butantan, São Paulo, Brazil.

Leptospirosis is a worldwide spread zoonosis, caused by pathogenic Leptospira. Evidences suggest that compromised hemostasis might be involved in the leptospirosis pathophysiology. In the genome of L. interrogans serovar Copenhageni, we found two genes coding for proteins which comprise von Willebrand factor (VWF) A domains (BatA and BatB). As VWF A domains exhibit multiple binding sites which contributes to human VWF hemostatic functions, we hypothesized that the L. interrogans BatA and BatB proteins could be involved in the hemostatic impairment during leptospirosis. We have cloned, expressed in Escherichia coli, and purified recombinant BatA and BatB. The influence of recombinant BatA and BatB on different in vitro hemostatic assays evaluating the enzymatic activity, platelet aggregation and fibrinogen integrity was investigated. We describe BatB as a new serine protease which is able to cleave thrombin chromogenic substrate, fibrin, fibrinogen, gelatin and casein; while BatA is active only towards fibrinogen. BatA and BatB interfere with the platelet aggregation induced by VWF/ristocetin and thrombin. Our results suggest an important role of the L. interrogans serovar Copenhageni Bat proteins in the hemostasis dysfunction observed during leptospirosis and contribute to the understanding of the leptospirosis pathophysiological mechanisms.
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http://dx.doi.org/10.1007/s00430-020-00664-4DOI Listing
April 2020

Nursing technician training: qualification profile.

Cien Saude Colet 2020 Jan 20;25(1):67-78. Epub 2019 Sep 20.

Escola Nacional de Saúde Pública Sergio Arouca, Fundação Oswaldo Cruz. R. Leopoldo Bulhões 1480, Manguinhos. 21041-210  Rio de Janeiro  RJ  Brasil.

The identification of the current vocational training profile of nursing aides and technicians becomes a central element in understanding the dynamics of their qualification in several states, aiming to expose both the existence of trends for under- and overqualification and the participation of the public sector in the offer and expansion of nursing courses in the country. The article explores three relevant aspects of vocational training based on the results found in the research "Nursing Profile in Brazil (FIOCRUZ/COFEN)": the level of schooling/qualification; the geographical distribution and the governmental participation in the consolidation of the current situation. This is an analytical study based on the interpretation of indicators identified by Pearson's Asymmetry Coefficient. The study uses the database generated by the research, as well as data from MEC/Inep and IBGE. The achieved results establish relations between the characteristics of training, distribution of NA&T in all Brazilian states with the phenomenon of overqualification, besides revealing an apparent separation of the Federal Education Network from the actual demand for nursing technicians in the country.
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http://dx.doi.org/10.1590/1413-81232020251.27652019DOI Listing
January 2020

Oxidative Stress Modulation and Radiosensitizing Effect of Quinoxaline-1,4-Dioxides Derivatives.

Anticancer Agents Med Chem 2020 ;20(1):111-120

Centro de Investigacao em Saude Ambiental (CISA), Escola Superior de Saude do Porto, Politecnico do Porto, Porto, Portugal.

Background: Quinoxaline-1,4-dioxide (QNX) derivatives are synthetic heterocyclic compounds with multiple biological and pharmacological effects.

Objective: In this study, we investigated the oxidative status of quinoxaline-1,4-dioxides derivatives in modulating melanoma and glioma cell lines, based on previous results from the research group and their capability to promote cell damage by the production of Reactive Oxygen Species (ROS).

Methods: Using in vitro cell cultures, the influence of 2-amino-3-cyanoquinoxaline-1,4-dioxide (2A3CQNX), 3- methyl-2-quinoxalinecarboxamide-1,4-dioxide (3M2QNXC) and 2-hydroxyphenazine-1,4-dioxide (2HF) was evaluated in metabolic activity, catalase activity, glutathione and 3-nitrotyrosine (3-NT) quantitation by HPLC in malignant melanocytes (B16-F10, MeWo) and brain tumor cells (GL-261 and BC3H1) submitted to radiotherapy treatments (total dose of 6 Gy).

Results: 2HF increased the levels of 3-NT in non-irradiated MeWo and glioma cell lines and decreased cell viability in these cell lines with and without irradiation.

Conclusion: Quinoxaline-1,4-dioxides derivatives modulate the oxidative status in malignant melanocytes and brain tumor cell lines and exhibited a potential radiosensitizer in vitro action on the tested radioresistant cell lines.
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http://dx.doi.org/10.2174/1871520619666191028091547DOI Listing
January 2021

Adjuvanted leptospiral vaccines: Challenges and future development of new leptospirosis vaccines.

Vaccine 2019 07 8;37(30):3961-3973. Epub 2019 Jun 8.

Laboratório Especial de Desenvolvimento de Vacinas - Centro de Biotecnologia - Instituto Butantan, Avenida Vital Brazil, 1500, 05503-900 São Paulo, SP, Brazil. Electronic address:

Leptospirosis is a neglected infectious disease of global importance. Vaccination is the most viable strategy for the control of leptospirosis, but in spite of efforts for the development of an effective vaccine against the disease, few advances have been made, and to date, bacterin is the only option for prevention of leptospirosis. Bacterins are formulations based on inactivated leptospires that present a series of drawbacks, such as serovar-dependence and short-term immunity. Therefore, bacterins are not widely used in humans, and only Cuba, France and China have these vaccines licensed for at-risk populations. The development of recombinant DNA technology emerges as an alternative to solve the problem. Recombinant protein-based vaccines or DNA vaccines seem to be an attractive strategy, but the use of adjuvants is critical for achievement of a protective immune response. Adjuvants are capable of enhancing and/or modulating immune responses by exposing antigens to antigen-presenting cells. In the last years, several components have been tested as adjuvants, such as aluminum salts, oil based-emulsion adjuvants, bacteria-derived components and liposomes. This review highlights the use of adjuvants in the multiple vaccine approaches that have been used for leptospirosis and their most important immunological aspects. Immune response data generated by these strategies can contribute to the understanding of the immune mechanisms involved in protection against leptospirosis, and consequently, the development of effective vaccines against this disease. This is the first review on leptospiral vaccines focusing on adjuvant aspects.
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http://dx.doi.org/10.1016/j.vaccine.2019.05.087DOI Listing
July 2019

Quinoxaline-1,4-dioxide derivatives inhibitory action in melanoma and brain tumor cells.

Future Med Chem 2019 04 9;11(7):645-657. Epub 2019 Apr 9.

Centro de Investigação em Saúde Ambiental (CISA), Escola Superior de Saúde do Porto, Politécnico do Porto, Porto, Portugal.

Quinoxaline-1,4-dioxide derivatives are synthetic heterocyclic compounds with multiple biological and pharmacological effects. In this study, we investigated the bioactivity of five quinoxaline-1,4-di--oxides derivatives in different animal cell lines. Using cell cultures, we evaluated the influence of quinoxaline-1,4-dioxide, 2-methylquinoxaline-1,4-dioxide, 2-amino-3-cyanoquinoxaline-1,4-dioxide, 3-methyl-2-quinoxalinecarboxamide-1,4-dioxide and 2-hydroxyphenazine--dioxide (2HF) in the viability, migration and proliferation of nonmalignant (3T3-L1 and human dermal microvascular endothelial cell) and malignant (B16-F10, MeWo, GL-261 and BC3H1) cell lines. The viability IC concentrations for each quinoxaline-1,4-di--oxide derivative were calculated, and a concomitant reduction of migration and proliferation was observed mainly in malignant cell lines. 2HF exhibited potent anti-viability, anti-migration and anti-proliferative actions selectively in tumor cells, nevertheless more studies are required to further investigate 2HF promising biologic effects.
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http://dx.doi.org/10.4155/fmc-2018-0251DOI Listing
April 2019

Phenotypic characterization and virulence-related properties of Escherichia albertii strains isolated from children with diarrhea in Brazil.

Pathog Dis 2019 03;77(2)

Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina (UNIFESP-EPM), Universidade Federal de São Paulo, Rua Botucatu, 862, 3° andar, CEP 04023-062, São Paulo, SP, Brazil.

Escherichia albertii are emerging enteropathogens, whose identification is difficult, as they share biochemical characteristics and some virulence-related genes with diarrheagenic Escherichia coli (DEC). Studies on phylogeny, phenotypic characteristics and potential virulence factors of human E. albertii strains are scarce. In this study, we identified by multiplex PCR five E. albertii among 106 strains isolated from diarrheic children in São Paulo, Brazil, which were previously classified as atypical enteropathogenic E. coli. All strains were investigated regarding their phylogeny, biochemical properties, virulence-related properties, antimicrobial resistance and presence of putative virulence-related genes. All strains belonged to different E. albertii lineages and adhered to and produced attaching and effacing lesions on HeLa cells. Three strains invaded Caco-2 cells, but did not persist intracellularly, and three formed biofilms on polystyrene surfaces. All strains were resistant to few antibiotics and only one carried a self-transmissible resistance plasmid. Finally, among 38 DEC and 18 extraintestinal pathogenic E. coli (ExPEC) virulence-related genes searched, six and three were detected, respectively, with paa and cdtB being found in all strains. Despite the limited number of strains, this study provided additional knowledge on human E. albertii virulence potential, showing that they share important virulence factors with DEC and ExPEC.
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http://dx.doi.org/10.1093/femspd/ftz014DOI Listing
March 2019

Distribution of the pilS gene in Escherichia coli pathovars, its transfer ability and influence in the typical enteropathogenic E. coli adherence phenotype.

Int J Med Microbiol 2019 Jan 11;309(1):66-72. Epub 2018 Dec 11.

Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil. Electronic address:

Typical enteropathogenic Escherichia coli strains (tEPEC) cause attaching/effacing lesions in eukaryotic cells and produce the bundle-forming pilus (BFP), which interweaves and aggregates bacteria, resulting in the localized adherence (LA) pattern on eukaryotic cells. Previously, we identified tEPEC strains (serotype O119:H6) that exhibited LA simultaneously with an aggregative adherence (AA)-like pattern (LA/AA-like+). Remarkably, AA is characteristically produced by strains of enteroaggregative E. coli (EAEC), another diarrheagenic E. coli pathovar. In one LA/AA-like + strain (Ec404/03), we identified a conjugative plasmid containing the pil operon, which encodes the Pil fimbriae. Moreover, a pil operon associated with an AA pattern and plasmid transfer had been previously described in the EAEC C1096 strain. In this study, we investigated the occurrence of the two pilS alleles (pilS and pilS) in tEPEC strains of different serotypes, origins and years of isolation. We also examined the potential relationship of pilS with the AA-like phenotype, its ability to be transferred by conjugation, and occurrence among strains of the other E. coli pathovars. The pilS alleles were found in 90 (55.2%) of 163 tEPEC strains, with pilS occurring more often (30.7%) than pilS (25.1%). About 21 tEPEC serotypes carried pilS. The pilS alleles were found in tEPEC strains from Chile, Peru and different Brazilian cities, with the oldest strain being isolated in 1966. No absolute correlation was found between the presence of pilS and the AA-like pattern. Conjugative pilS transfer was detected in 26.2% of pilS+ strains and in 65.1% of pilS+ strains, but only pilS+ transconjugants were AA-like+, thus suggesting that the latter allele might need a different genetic background to express this phenotype. pilS was found in all other E. coli pathovars, where it was most prevalent in enterotoxigenic E. coli. More studies are needed to understand the mechanisms involved in the regulation of Pil expression and production.
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http://dx.doi.org/10.1016/j.ijmm.2018.12.001DOI Listing
January 2019

Heparin-Binding Protein Release Is Strongly Induced by Leptospira Species and Is a Candidate for an Early Diagnostic Marker of Human Leptospirosis.

J Infect Dis 2019 02;219(6):996-1006

Division of Infection Medicine, Department of Clinical Sciences, Lund University, Lund, Sweden.

Background: Leptospirosis, caused by spirochetes of the genus Leptospira, is one of the most widespread zoonoses worldwide. Efficient diagnostic methods for early diagnosis of leptospirosis are still lacking, and acute disease presents with nonspecific symptomatology and is often misdiagnosed. The leptospires pathogenic processes and virulence mechanisms remain virtually unknown. In severe infections, hemostatic impairment is frequently observed, and pathophysiological complications often develop when the host response is modulated by the pathogen. The neutrophil heparin-binding protein (HBP) is an inflammatory mediator and potent inducer of vascular leakage.

Results: In this study, we found that leptospires and their secreted products induce the release of HBP from stimulated neutrophils through a controlled degranulation mechanism. We acknowledged 2 leptospiral proteins as able to induce HBP degranulation. These findings have clinical implications, as high levels of HBP were detected in serum from patients with leptospirosis, especially at the early phase of the disease.

Conclusion: In conclusion, we describe a new mechanism by which the leptospirosis pathophysiological complications may arise, such as vascular leakage and edema formation. We also propose HBP as a new early screening biomarker for human leptospirosis.
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http://dx.doi.org/10.1093/infdis/jiy589DOI Listing
February 2019

Neutrophils engage the kallikrein-kinin system to open up the endothelial barrier in acute inflammation.

FASEB J 2019 02 3;33(2):2599-2609. Epub 2018 Oct 3.

Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden.

Neutrophil recruitment and plasma exudation are key elements in the immune response to injury or infection. Activated neutrophils stimulate opening of the endothelial barrier; however, the underlying mechanisms have remained largely unknown. In this study, we identified a pivotal role of the proinflammatory kallikrein-kinin system and consequent formation of bradykinin in neutrophil-evoked vascular leak. In mouse and hamster models of acute inflammation, inhibitors of bradykinin generation, and signaling markedly reduced plasma exudation in response to chemoattractant activation of neutrophils. The neutrophil-driven leak was likewise suppressed in mice deficient in either the bradykinin B receptor or factor XII (initiator of the kallikrein-kinin system). In human endothelial cell monolayers, material secreted from activated neutrophils induced cytoskeletal rearrangement, leading to paracellular gap formation in a bradykinin-dependent manner. As a mechanistic basis, we found that a neutrophil-derived heparin-binding protein (HBP/azurocidin) displaced the bradykinin precursor high-molecular-weight kininogen from endothelial cells, thereby enabling proteolytic processing of kininogen into bradykinin by neutrophil and plasma proteases. These data provide novel insight into the signaling pathway by which neutrophils open up the endothelial barrier and identify the kallikrein-kinin system as a target for therapeutic interventions in acute inflammatory reactions.-Kenne, E., Rasmuson, J., Renné, T., Vieira, M. L., Müller-Esterl, W., Herwald, H., Lindbom, L. Neutrophils engage the kallikrein-kinin system to open up the endothelial barrier in acute inflammation.
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http://dx.doi.org/10.1096/fj.201801329RDOI Listing
February 2019

Oxidative stress markers imbalance in late-life depression.

J Psychiatr Res 2018 07 20;102:29-33. Epub 2018 Mar 20.

Department of Psychiatry and Behavioral Sciences, McMaster University, Hamilton, Canada.

Background: Oxidative stress has been implicated in the pathophysiology of mood disorders in young adults. However, there is few data to support its role in the elderly. The primary aim of this study was to evaluate whether subjects with late-life depression (LLD) presented with changes in oxidative stress response in comparison with the non-depressed control group. We then explored how oxidative stress markers associated with specific features of LLD, in particular cognitive performance and age of onset of major depressive disorder in these individuals.

Methods: We included a convenience sample of 124 individuals, 77 with LLD and 47 non-depressed subjects (Controls). We measure the plasma levels of 6 oxidative stress markers: thiobarbituric acid reactive substances (TBARS), protein carbonil content (PCC), free 8-isoprostane, glutathione peroxidase (GPx) activity, glutathione reductase (GR) activity, and glutathione S-transferase (GST) activity.

Results: We found that participants with LLD had significantly higher free 8-isoprostane levels (p = 0.003) and lower glutathione peroxidase activity (p = 0.006) compared to controls. Free 8-isoprostane levels were also significantly correlated with worse scores in the initiation/perseverance (r = -0.24, p = 0.01), conceptualization (r = -0.22, p = 0.02) sub-scores, and the total scores (r = -0.21, p = 0.04) on the DRS.

Conclusions: Our study provides robust evidence of the imbalance between oxidative stress damage, in particular lipid peroxidation, and anti-oxidative defenses as a mechanism related to LLD, and cognitive impairment in this population. Interventions aiming to reduce oxidative stress damage can have a potential neuroprotective effect for LLD subjects.
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http://dx.doi.org/10.1016/j.jpsychires.2018.02.023DOI Listing
July 2018

Proteomics as a tool to understand Leptospira physiology and virulence: Recent advances, challenges and clinical implications.

J Proteomics 2018 05 1;180:80-87. Epub 2018 Mar 1.

Laboratório Especial de Desenvolvimento de Vacinas, Instituto Butantan, Avenida Vital Brazil, 1500, 05503-900 Sao Paulo, SP, Brazil. Electronic address:

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http://dx.doi.org/10.1016/j.jprot.2018.02.025DOI Listing
May 2018

Leptospira interrogans outer membrane protein LipL21 is a potent inhibitor of neutrophil myeloperoxidase.

Virulence 2018 01;9(1):414-425

a Department of Clinical Sciences, Lund, Division of Infection Medicine , Lund University , Lund , Sweden.

Leptospirosis is a widespread zoonotic and neglected infectious disease of human and veterinary concern that is caused by pathogenic Leptospira species. After entrance in the host, pathogenic leptospires evade the host natural defense mechanisms in order to propagate and disseminate to multiple organs. Myeloperoxidase is an enzyme stored in neutrophils azurophilic granules, and is released upon neutrophil activation to produce mainly hypochlorous acid, a strong oxidant and potent antimicrobial agent. In the present investigation, we studied the modulation of myeloperoxidase activity by L. interrogans serovar Copenhageni. We show that leptospires and their culture supernatants are able to inhibit both peroxidase and chlorination activities of myeloperoxidase, without interfering with neutrophil degranulation. By leptospiral outer membrane protein extraction and fractionation, we identified the proteins LipL21 and LipL45 as myeloperoxidase inhibitors, constituting new Leptospira virulence factors. Accordingly, we propose a function for the protein LipL21, one of the most expressed leptospiral outer membrane proteins. Our results show a novel innate immune evasion mechanism by which leptospires interfere with the host response in order to cope with the host oxidative stress and efficiently achieve dissemination and colonization.
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http://dx.doi.org/10.1080/21505594.2017.1407484DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5955187PMC
January 2018

Captive wild birds as reservoirs of enteropathogenic E. coli (EPEC) and Shiga-toxin producing E. coli (STEC).

Braz J Microbiol 2017 Oct - Dec;48(4):760-763. Epub 2017 Jun 2.

Departamento de Patologia. Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo (USP), São Paulo, Brazil. Electronic address:

Psittacine birds have been identified as reservoirs of diarrheagenic Escherichia coli, a subset of pathogens associated with mortality of children in tropical countries. The role of other orders of birds as source of infection is unclear. The aim of this study was to perform the molecular diagnosis of infection with diarrheagenic E. coli in 10 different orders of captive wild birds in the state of São Paulo, Brazil. Fecal samples were analyzed from 516 birds belonging to 10 orders: Accipitriformes, Anseriformes, Columbiformes, Falconiformes, Galliformes, Passeriformes, Pelecaniformes, Piciformes, Psittaciformes and Strigiformes. After isolation, 401 E. coli strains were subjected to multiplex PCR system with amplification of genes eae and bfp (EPEC), stx1 and stx2 for STEC. The results of these tests revealed 23/401 (5.74%) positive strains for eae gene, 16/401 positive strains for the bfp gene (3.99%) and 3/401 positive for stx2 gene (0.75%) distributed among the orders of Psittaciformes, Strigiformes and Columbiformes. None of strains were positive for stx1 gene. These data reveal the infection by STEC, typical and atypical EPEC in captive birds. The frequency of these pathotypes is low and restricted to few orders, but the data suggest the potential public health risk that these birds represent as reservoirs of diarrheagenic E. coli.
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http://dx.doi.org/10.1016/j.bjm.2017.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5628295PMC
May 2018

Pandemic extra-intestinal pathogenic Escherichia coli (ExPEC) clonal group O6-B2-ST73 as a cause of avian colibacillosis in Brazil.

PLoS One 2017 8;12(6):e0178970. Epub 2017 Jun 8.

Department of Pathology, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, Brazil.

Extra-intestinal pathogenic Escherichia coli (ExPEC) represent an emerging pathogen, with pandemic strains increasingly involved in cases of urinary tract infections (UTIs), bacteremia, and meningitis. In addition to affecting humans, the avian pathotype of ExPEC, avian pathogenic E. coli (APEC), causes severe economic losses to the poultry industry. Several studies have revealed overlapping characteristics between APEC and human ExPEC, leading to the hypothesis of a zoonotic potential of poultry strains. However, the description of certain important pandemic clones, such as Sequence Type 73 (ST73), has not been reported in food sources. We characterized 27 temporally matched APEC strains from diverse poultry farms in Brazil belonging to the O6 serogroup because this serogroup is frequently described as a causal factor in UTI and septicemia in humans in Brazil and worldwide. The isolates were genotypically characterized by identifying ExPEC virulence factors, phylogenetically tested by phylogrouping and multilocus sequence type (MLST) analysis, and compared to determine their similarity employing the pulsed field gel electrophoresis (PFGE) technique. The strains harbored a large number of virulence determinants that are commonly described in uropathogenic E. coli (UPEC) and sepsis associated E. coli (SEPEC) strains and, to a lesser extent in neonatal meningitis associated E. coli (NMEC), such as pap (85%), sfa (100%), usp (100%), cnf1 (22%), kpsMTII (66%), hlyA (52%), and ibeA (4%). These isolates also yielded a low prevalence of some genes that are frequently described in APEC, such as iss (37%), tsh, ompT, and hlyF (8% each), and cvi/cva (0%). All strains were classified as part of the B2 phylogroup and sequence type 73 (ST73), with a cluster of 25 strains showing a clonal profile by PFGE. These results further suggest the zoonotic potential of some APEC clonal lineages and their possible role in the epidemiology of human ExPEC, in addition to providing the first description of the O6-B2-ST73 clonal group in poultry.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0178970PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5464619PMC
September 2017

Infection Interferes with the Prothrombinase Complex Assembly during Experimental Leptospirosis.

Front Microbiol 2017 28;8:500. Epub 2017 Mar 28.

Laboratorio Especial de Desenvolvimento de Vacinas, Instituto ButantanSão Paulo, Brazil; Programa de Pós-Graduação Interunidades em Biotecnologia, Instituto de Ciências Biomédicas, Universidade de São PauloSão Paulo, Brazil.

Leptospirosis is a worldwide zoonotic and neglected infectious disease of human and veterinary concern, caused by pathogenic species. Although bleeding is a common symptom of severe leptospirosis, the cause of hemorrhage is not completely understood. In severe infections, modulation of hemostasis by pathogens is an important virulence mechanism, and hemostatic impairments such as coagulation/fibrinolysis dysfunction are frequently observed. Here, we analyze the coagulation status of experimentally infected hamsters in an attempt to determine coagulation interferences and the origin of leptospirosis hemorrhagic symptomatology. Hamsters were experimentally infected with . The lungs, kidneys, and livers were collected for culture, histopathology, and coagulation assays. infection disturbs normal coagulation in the organs of animals. Our results suggest the presence of a thrombin-like factor or FX activator, which is able to activate FII in the leptospirosis organ extracts. The activity of those factors is accelerated in the prothrombinase complex. Additionally, we show for the first time that live leptospires act as a surface for the prothrombinase complex assembly. Our results contribute to the understanding of leptospirosis pathophysiological mechanisms and may open new routes for the discovery of novel treatments in the severe manifestations of the disease.
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http://dx.doi.org/10.3389/fmicb.2017.00500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5368274PMC
March 2017

Development of a new HPLC-based method for 3-nitrotyrosine quantification in different biological matrices.

J Chromatogr B Analyt Technol Biomed Life Sci 2017 Mar 24;1046:48-57. Epub 2017 Jan 24.

Ciências Químicas e das Biomoléculas, Centro de Investigação em Saúde e Ambiente, Escola Superior de Saúde do Porto, Instituto Politécnico do Porto, Portugal; I3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal. Electronic address:

Background: The nitration of tyrosine residues in proteins is associated with nitrosative stress, resulting in the formation of 3-nitrotyrosine (3-NT). 3-NT levels in biological samples have been associated with numerous physiological and pathological conditions. Hence several attempts have been made in order to develop methods that accurately quantify 3-NT in these matrices. The aim of this study was to develop a simple, rapid, low-cost and sensitive high-performance liquid chromatography (HPLC)-based 3-NT quantification method.

Methods: All experiments were performed on an Hitachi LaChrom Elite HPLC system. The method was validated according to International Conference on Harmonisation (ICH) guidelines for serum samples. Additionally, other biological matrices were tested, namely whole blood, urine, B16 F-10 melanoma cell line, growth medium conditioned with the same cell line, bacterial and yeast suspensions.

Results: From all the protocols tested, the best results were obtained using 0.5% CHCOOH:MeOH:HO (15:15:70) as mobile phase, with detection at wavelengths 215, 276 and 356nm, at 25°C, and using a flow rate of 1mLmin. By using this protocol, it was possible to obtain a linear calibration curve, limits of detection and quantification in the order of μgL, and a short analysis time (<15min per sample). The developed protocol allowed the successful detection and quantification of 3-NT in all biological matrices tested, with detection at 356nm.

Conclusion: This method, successfully developed and validated for 3-NT quantification, is simple, cheap and fast. These features render this method a suitable option for analysis of a wide range of biological matrices, being a promising useful tool for both research and diagnosis activities.
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http://dx.doi.org/10.1016/j.jchromb.2017.01.035DOI Listing
March 2017

Melanoma and obesity: Should antioxidant vitamins be addressed?

Life Sci 2016 Nov 23;165:83-90. Epub 2016 Sep 23.

Ciências Químicas e das Biomoléculas, Centro de Investigação em Saúde e Ambiente, Escola Superior de Saúde, Instituto Politécnico do Porto, Portugal; I3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal. Electronic address:

Melanoma is an aggressive form of skin cancer refractory to conventional therapies. Obesity has reached epidemic dimensions acting as a risk factor for several cancer types, such as melanoma. Several reactive species of oxygen are also involved in melanoma initiation and progression. Low levels of antioxidant content and/or activity in lightly pigmented cells could expose them to an extremely oxidative environment and rise the susceptibility to oxidative damage and consequently loss of cell homeostasis. Despite the knowledge about melanoma biology, pathogenesis and developed therapies, is extremely important to understand the antioxidant modulation of melanoma under an environment of obesity, especially the effect of some natural compounds of the diet, such as antioxidant vitamins A, C and E and selenium in order to establish alternatives to conventional therapies, which are known to be ineffective against melanoma.
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http://dx.doi.org/10.1016/j.lfs.2016.09.015DOI Listing
November 2016

Modulation of Hemostatic and Inflammatory Responses by Leptospira Spp.

PLoS Negl Trop Dis 2016 05 11;10(5):e0004713. Epub 2016 May 11.

Department of Clinical Sciences, Lund, Division of Infection Medicine, Lund University, Lund, Sweden.

Leptospirosis is a worldwide spread zoonotic and neglected infectious disease of human and veterinary concern that is caused by pathogenic Leptospira species. In severe infections, hemostatic impairments such as coagulation/fibrinolysis dysfunction are frequently observed. These complications often occur when the host response is controlled and/or modulated by the bacterial pathogen. In the present investigation, we aimed to analyze the modulation of the hemostatic and inflammatory host responses by the bacterial pathogen Leptospira. The effects of leptospires and their secreted products on stimulation of human intrinsic and extrinsic pathways of coagulation were investigated by means of altered clotting times, assembly and activation of contact system and induction of tissue factor. We show that both extrinsic and intrinsic coagulation cascades are modulated in response to Leptospira or leptospiral secreted proteins. We further find that the pro-inflammatory mediator bradykinin is released following contact activation at the bacterial surface and that pro-coagulant microvesicles are shed from monocytes in response to infection. Also, we show that human leptospirosis patients present higher levels of circulating pro-coagulant microvesicles than healthy individuals. Here we show that both pathways of the coagulation system are modulated by leptospires, possibly leading to altered hemostatic and inflammatory responses during the disease. Our results contribute to the understanding of the leptospirosis pathophysiological mechanisms and may open new routes for the discovery of novel treatments for the severe manifestations of the disease.
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http://dx.doi.org/10.1371/journal.pntd.0004713DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4864083PMC
May 2016

Evaluation of two novel leptospiral proteins for their interaction with human host components.

Pathog Dis 2016 07 28;74(5). Epub 2016 Apr 28.

Centro de Biotecnologia, Instituto Butantan, Avenida Vital Brazil, 1500, 05503-900, São Paulo, SP, Brazil Programa de Pós-Graduação Interunidades em Biotecnologia, Instituto de Ciências Biomédicas, USP, Avenida Prof. Lineu Prestes, 1730, 05508-900, São Paulo, SP, Brazil

Pathogenic species of the genus Leptospira are the etiological agents of leptospirosis, the most widespread zoonosis. Mechanisms involved in leptospiral pathogenesis are not well understood. By data mining the genome sequences of Leptospira interrogans we have identified two proteins predicted to be surface exposed, LIC10821 and LIC10064. Immunofluorescence and proteinase K assays confirmed that the proteins are exposed. Reactivity of the recombinant proteins with human sera has shown that rLIC10821, but not rLIC10064, is recognized by antibodies in confirmed leptospirosis serum samples, suggesting its expression during infection. The rLIC10821 was able to bind laminin, in a dose-dependent fashion, and was called Lsa37 (leptospiral surface adhesin of 37 kDa). Studies with human plasma components demonstrated that rLIC10821 interacts with plasminogen (PLG) and fibrinogen (Fg). The binding of Lsa37 with PLG generates plasmin when PLG activator was added. Fibrin clotting reduction was observed in a thrombin-catalyzed reaction, when Fg was incubated with Lsa37, suggesting that this protein may interfere in the coagulation cascade during the disease. Although LIC10064 protein is more abundant than the corresponding Lsa37, binding activity with all the components tested was not detected. Thus, Lsa37 is a novel versatile adhesin that may mediate Leptospira-host interactions.
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http://dx.doi.org/10.1093/femspd/ftw040DOI Listing
July 2016

Genetic relatedness and virulence properties of enteropathogenic Escherichia coli strains of serotype O119:H6 expressing localized adherence or localized and aggregative adherence-like patterns on HeLa cells.

Int J Med Microbiol 2016 May 27;306(3):152-64. Epub 2016 Feb 27.

Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo-Escola Paulista de Medicina, São Paulo, Brazil. Electronic address:

Enteropathogenic Escherichia coli (EPEC) induce attaching and effacing (A/E) lesions in enterocytes and produce the bundle-forming pilus (BFP) contributing to the localized adherence (LA) pattern formation on HeLa cells. Enteroaggregative E. coli (EAEC) produce aggregative adherence (AA) on HeLa cells and form prominent biofilms. The ability to produce LA or AA is an important hallmark to classify fecal E. coli isolates as EPEC or EAEC, respectively. E. coli strains of serotype O119:H6 exhibit an LA+ phenotype and have been considered as comprising a clonal group of EPEC strains. However, we have recently identified O119:H6 EPEC strains that produce LA and an AA-like pattern concurrently (LA/AA-like+). In this study, we evaluated the relatedness of three LA/AA-like+ and three LA+ O119:H6 strains by comparing their virulence and genotypic properties. We first found that the LA/AA-like+ strains induced actin accumulation in HeLa cells (indicative of A/E lesions formation) and formed biofilms on abiotic surfaces more efficiently than the LA+ strains. MLST analysis showed that the six strains all belong to the ST28 complex. All strains carried multiple plasmids, but as plasmid profiles were highly variable, this cannot be used to differentiate LA/AA-like+ and LA+ strains. We further obtained their draft genome sequences and the complete sequences of four plasmids harbored by one LA/AA-like+ strain. Analysis of these sequences and comparison with 37 fully sequenced E. coli genomes revealed that both O119:H6 groups belong to the E. coli phylogroup B2 and are very closely related with only 58-67 SNPs found between LA/AA-like+ and LA+ strains. Search of the draft sequences of the six strains for adhesion-related genes known in EAEC and other E. coli pathotypes detected no genes specifically present in LA/AA-like+ strains. Unexpectedly however, we found that a large plasmid distinct from pEAF is responsible for the AA-like phenotype of the LA/AA-like+ strains. Although we have not identified any plasmid genes specifically present in all LA/AA-like+ strains and absent in the LA+ strains, these results suggest the presence of an unknown mechanism to promote the AA-like pattern production and biofilm formation by the LA/AA-like+ strains. Because their ability to produce A/E lesions and biofilm concomitantly could exacerbate the clinical condition of the patient and lead to persistent diarrhea, the mechanism underlying the enhanced biofilm formation by the LA/AA-like+ O119:H6 strains and their spread and involvement in severe diarrheal diseases should be more intensively investigated.
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http://dx.doi.org/10.1016/j.ijmm.2016.02.008DOI Listing
May 2016

3-Nitrotyrosine quantification methods: Current concepts and future challenges.

Biochimie 2016 Jun 26;125:1-11. Epub 2016 Feb 26.

Ciências Químicas e das Biomoléculas, Centro de Investigação em Saúde e Ambiente, Escola Superior de Tecnologia da Saúde do Porto, Instituto Politécnico do Porto, Portugal; I3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal. Electronic address:

Background: Measurement of 3-nitrotyrosine (3-NT) in biological samples can be used as a biomarker of nitrosative stress, since it is very stable and suitable for analysis. Increased 3-NT levels in biological samples have been associated with several physiological and pathological conditions. Different methods have been described for the detection and quantification of this molecule, such as (i) immunological methods; (ii) liquid chromatography, namely high-pressure liquid chromatography (HPLC)-based methods that use ultraviolet-visible (UV/VIS) absorption, electrochemical (ECD) and diode array (DAD) detection, liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS); (iii) gas chromatography, such as gas chromatography-mass spectrometry (GC-MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS).

Methods: A literature review on nitrosative stress, protein nitration, as well as 3-NT quantification methods was carried out.

Results: This review covers the different methods for analysis of 3-NT that have been developed during the last years as well as the latest advances in this field. Overall, all methods present positive and negative aspects, although it is clear that chromatography-based methods present good sensitivity and specificity. Regarding this, GC-based methods exhibit the highest sensibility in the quantification of 3-NT, although it requires a prior time consuming derivatization step. Conversely, HPLC does not require such derivatization step, despite being not as accurate as GC.

Conclusion: It becomes clear that all the methods described during this literature review, although accurate for 3-NT quantification, need to be improved regarding both sensitivity and specificity. Moreover, optimization of the protocols that have been described is clearly needed.
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http://dx.doi.org/10.1016/j.biochi.2016.02.011DOI Listing
June 2016