Publications by authors named "Susana Frases"

98 Publications

A water-soluble manganese(II) octanediaoate/phenanthroline complex acts as an antioxidant and attenuates alpha-synuclein toxicity.

Biochim Biophys Acta Mol Basis Dis 2022 10 28;1868(10):166475. Epub 2022 Jun 28.

Departamento de Bioquímica, Instituto de Química, Centro de Tecnologia, Cidade Universitária, Universidade Federal do Rio de Janeiro, Brazil; Rede de Micrologia RJ-FAPERJ, Brazil. Electronic address:

The overproduction of reactive oxygen species (ROS) induces oxidative stress, a well-known process associated with aging and several human pathologies, such as cancer and neurodegenerative diseases. A large number of synthetic compounds have been described as antioxidant enzyme mimics, capable of eliminating ROS and/or reducing oxidative damage. In this study, we investigated the antioxidant activity of a water-soluble 1,10-phenantroline-octanediaoate Mn-complex on cells under oxidative stress, and assessed its capacity to attenuate alpha-synuclein (aSyn) toxicity and aggregation, a process associated with increased oxidative stress. This Mn-complex exhibited a significant antioxidant potential, reducing intracelular oxidation and increasing oxidative stress resistance in S. cerevisiae cells and in vivo, in G. mellonella, increasing the activity of the intracellular antioxidant enzymes superoxide dismutase and catalase. Strikingly, the Mn-complex reduced both aSyn oligomerization and aggregation in human cell cultures and, using NMR and DFT/molecular docking we confirmed its interaction with the C-terminal region of aSyn. In conclusion, the Mn-complex appears as an excellent lead for the design of new phenanthroline derivatives as alternative compounds for preventing oxidative damages and oxidative stress - related diseases.
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http://dx.doi.org/10.1016/j.bbadis.2022.166475DOI Listing
October 2022

Comparative Biophysical and Ultrastructural Analysis of Melanins Produced by Clinical Strains of Different Species From the Trichosporonaceae Family.

Front Microbiol 2022 25;13:876611. Epub 2022 Apr 25.

Laboratório de Micologia, Instituto Nacional de Infectologia Evandro Chagas, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil.

Melanin is one of the most studied virulence factors in pathogenic fungi. This pigment protects them from a series of both environmental and host stressors. Among basidiomycetes, and are known to produce melanin in the presence of phenolic precursors. Other species from the Trichosporonaceae family also produce this pigment, but the extent to this production among the clinically relevant species is unknown. For this reason, the aim of this study was to verify the production of melanin by different Trichosporonaceae species of clinical interest and to compare their pigments with the ones from and , which are more prevalent in human infections. Melanin was produced in a minimal medium supplemented with 1 mM L-dihydroxyphenylalanine (L-DOPA). Pigment was evaluated using scanning electron microscopy, Zeta potential measurements, and energy-dispersive X-ray spectroscopy. It was found that, besides and , , , , , , and also produce melanin-like particles in the presence of L-DOPA. Melanin particles have negative charge and are smaller than original cells. Variations in color, fluorescence, and chemical composition was noticed between the studied strains. All melanins presented carbon, oxygen, sodium, and potassium in their composition. Melanins from the most pathogenic species also presented iron, zinc, and copper, which are important during parasitism. Biophysical properties of these melanins can confer to the Trichosporonaceae adaptive advantages to both parasitic and environmental conditions of fungal growth.
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http://dx.doi.org/10.3389/fmicb.2022.876611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9081797PMC
April 2022

Extracellular Vesicles Regulate Biofilm Formation and Yeast-to-Hypha Differentiation in Candida albicans.

mBio 2022 06 14;13(3):e0030122. Epub 2022 Apr 14.

Laboratório de Glicobiologia de Eucariotos, Departamento de Microbiologia Geral, Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.

In this study, we investigated the influence of fungal extracellular vesicles (EVs) during biofilm formation and morphogenesis in Candida albicans. Using crystal violet staining and scanning electron microscopy (SEM), we demonstrated that C. albicans EVs inhibited biofilm formation . By time-lapse microscopy and SEM, we showed that C. albicans EV treatment stopped filamentation and promoted pseudohyphae formation with multiple budding sites. The ability of C. albicans EVs to regulate dimorphism was further compared to EVs isolated from different C. albicans strains, Saccharomyces cerevisiae, and Histoplasma capsulatum. C. albicans EVs from distinct strains inhibited yeast-to-hyphae differentiation with morphological changes occurring in less than 4 h. EVs from S. cerevisiae and H. capsulatum modestly reduced morphogenesis, and the effect was evident after 24 h of incubation. The inhibitory activity of C. albicans EVs on phase transition was promoted by a combination of lipid compounds, which were identified by gas chromatography-tandem mass spectrometry analysis as sesquiterpenes, diterpenes, and fatty acids. Remarkably, C. albicans EVs were also able to reverse filamentation. Finally, C. albicans cells treated with C. albicans EVs for 24 h lost their capacity to penetrate agar and were avirulent when inoculated into Galleria mellonella. Our results indicate that fungal EVs can regulate yeast-to-hypha differentiation, thereby inhibiting biofilm formation and attenuating virulence. The ability to undergo morphological changes during adaptation to distinct environments is exploited by Candida albicans and has a direct impact on biofilm formation and virulence. Morphogenesis is controlled by a diversity of stimuli, including osmotic stress, pH, starvation, presence of serum, and microbial components, among others. Apart from external inducers, C. albicans also produces autoregulatory substances. Farnesol and tyrosol are examples of quorum-sensing molecules (QSM) released by C. albicans to regulate yeast-to-hypha conversion. Here, we demonstrate that fungal EVs are messengers impacting biofilm formation, morphogenesis, and virulence in C. albicans. The major players exported in C. albicans EVs included sesquiterpenes, diterpenes, and fatty acids. The understanding of how C. albicans cells communicate to regulate physiology and pathogenesis can lead to novel therapeutic tools to combat candidiasis.
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http://dx.doi.org/10.1128/mbio.00301-22DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9239257PMC
June 2022

Lessons from protozoans: Phosphate sensing and polyphosphate storage in fungi.

PLoS Pathog 2022 03 3;18(3):e1010298. Epub 2022 Mar 3.

Laboratório de Ultraestrutura Celular Hertha Meyer-Instituto de Biofísica Carlos Chagas Filho-Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.

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http://dx.doi.org/10.1371/journal.ppat.1010298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8893339PMC
March 2022

Harris' hawk () as a source of pathogenic human yeasts: a potential risk to human health.

Future Microbiol 2022 02 19;17:169-175. Epub 2022 Jan 19.

Laboratório de Biofísica de fungos, Instituto de Biofísica Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.

Invasive human fungal infections have been a serious public health problem among immunocompromised patients. Wild bird species are related to the eco-epidemiology of some infectious diseases, mainly Cryptococcosis, Histoplasmosis, Aspergillosis, Chlamydiosis, Salmonellosis and allergic diseases. Falconry is the art of training predators for hunting. Nowadays, birds of prey are used as pets, which brings new sources of infections to humans. We identified fungal pathogenic yeasts, ,  and . Study new environmental niches of human pathogens is vitally important to establish preventive actions with the purpose of minimizing the risks of human contamination. Our work describes yeast microbiota from the excreta of as a potential hazard for human disease.
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http://dx.doi.org/10.2217/fmb-2021-0166DOI Listing
February 2022

Medicines for Malaria Venture COVID Box: a source for repurposing drugs with antifungal activity against human pathogenic fungi.

Mem Inst Oswaldo Cruz 2021 8;116:e210207. Epub 2021 Nov 8.

Universidade Federal do Rio de Janeiro, Instituto de Biofísica Carlos Chagas Filho, Laboratório de Biofísica de Fungos, Rio de Janeiro, RJ, Brasil.

Background: Treatment of mycoses is often ineffective, usually prolonged, and has some side effects. These facts highlight the importance of discovering new molecules to treat fungal infections.

Objectives: To search the Medicines for Malaria Venture COVID Box for drugs with antifungal activity.

Methods: Fourteen human pathogenic fungi were tested against the 160 drugs of this collection at 1.0 µM concentration. We evaluated the ability of the drugs to impair fungal growth, their fungicidal nature, and morphological changes caused to cells.

Findings: Thirty-four molecules (21.25%) presented antifungal activity. Seven are antifungal drugs and one is the agricultural fungicide cycloheximide. The other drugs with antifungal activity included antibiotics (n = 3), antimalarials (n = 4), antivirals (n = 2), antiparasitcs (n = 3), antitumor agents (n = 5), nervous system agents (n = 3), immunosuppressants (n = 3), antivomiting (n = 1), antiasthmatic (n = 1), and a genetic disorder agent (n = 1). Several of these drugs inhibited Histoplasma capsulatum and Paracoccidioides brasiliensis growth (15 and 20, respectively), while Fusarium solani was not affected by the drugs tested. Most drugs were fungistatic, but niclosamide presented fungicidal activity against the three dimorphic fungi tested. Cyclosporine affected morphology of Cryptococcus neoformans.

Main Conclusions: These drugs represent new alternatives to the development of more accessible and effective therapies to treat human fungal infections.
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http://dx.doi.org/10.1590/0074-02760210207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8577065PMC
November 2021

as Cause of Nosocomial Pneumonia in Patient With COVID-19: A Triple Co-infection.

Arch Bronconeumol 2021 Apr 1;57:46-48. Epub 2020 Dec 1.

Laboratório de Biofísica de Fungos, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.

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http://dx.doi.org/10.1016/j.arbres.2020.11.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7705399PMC
April 2021

Candida spp. co-infection in COVID-19 patients with severe pneumonia: Prevalence study and associated risk factors.

Respir Med 2021 11 17;188:106619. Epub 2021 Sep 17.

Laboratório de Biofísica de Fungos, Instituto de Biofísica Carlos Chagas Filho, Universidad Federal Do Rio de Janeiro, Rio de Janeiro, RJ, Brazil. Electronic address:

Background: Invasive fungal infections (IFI) are increasing in prevalence in recent years. In the last few months, the rise of COVID-19 patients has generated a new escalation in patients presenting opportunistic mycoses, mainly by Aspergillus. Candida infections are not being reported yet.

Objectives: We aimed to determine the prevalence of systemic candidiasis in patients admitted to ICUs due to severe pneumonia secondary to SARS-CoV-2 infection and the existence of possible associated risk factors that led these patients to develop candidiasis.

Patients/methods: We designed a study including patients with a confirmed diagnosis of COVID-19.

Results: The prevalence of systemic candidiasis was 14.4%, and the main isolated species were C. albicans and C. parapsilosis. All patients that were tested positive for Candida spp. stayed longer in the ICU in comparison to patients who tested negative. Patients with candidiasis had higher MuLBSTA score and mortality rates and a worse radiological involvement. In our study, Candida spp. isolates were found in patients that were submitted to: tocilizumab, tocilizumab plus systemic steroids, interferon type 1β and Lopinavir-Ritonavir.

Conclusions: Results suggested a high prevalence of systemic candidiasis in severe COVID-19-associated pneumonia patients. Patients with Candidiasis had the worst clinical outcomes. Treatment with tocilizumab could potentialize the risk to develop systemic candidiasis.
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http://dx.doi.org/10.1016/j.rmed.2021.106619DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8445759PMC
November 2021

produces a melanin-like pigment that protects against stress conditions encountered during parasitism.

Future Microbiol 2021 05 7;16:509-520. Epub 2021 May 7.

Departments of Medicine (Division of Infectious Diseases) & Microbiology & Immunology, Albert Einstein College of Medicine, Bronx, NY, USA.

Melanin has been linked to pathogenesis in several fungi. They often produce melanin-like pigments in the presence of L-dihydroxyphenylalanine (L-DOPA), but this is poorly studied in . was grown in minimal medium with or without L-DOPA supplementation and submitted to a chemical treatment with denaturant and hot acid. turned black when grown in the presence of L-DOPA, whereas cells grown without L-DOPA supplementation remained white. Biophysical properties demonstrated that the pigment was melanin. Melanized cells were effectively protected from azoles and amphotericin B, incubation at 42°C and macrophage killing. In the presence of L-DOPA, produces melanin, increases antifungal resistance and enhances host survival.
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http://dx.doi.org/10.2217/fmb-2020-0228DOI Listing
May 2021

Production of Secreted Carbohydrates that Present Immunologic Similarities with the Cryptococcus Glucuronoxylomannan by Members of the Trichosporonaceae Family: A Comparative Study Among Species of Clinical Interest.

Mycopathologia 2021 Jun 6;186(3):377-385. Epub 2021 May 6.

Laboratório de Micologia, Instituto Nacional de Infectologia Evandro Chagas, Fundação Oswaldo Cruz, Av. Brazil 4365-Manguinhos, Fiocruz, Rio de Janeiro, RJ, 21045-900, Brazil.

Glucuronoxylomannan (GXM) participates in several immunoregulatory mechanisms, which makes it an important Cryptococcus virulence factor that is essential for the disease. Trichosporon asahii and Trichosporon mucoides share with Cryptococcus species the ability to produce GXM. To check whether other opportunistic species in the Trichosporonaceae family produce GXM-like polysaccharides, extracts from 28 strains were produced from solid cultures and their carbohydrate content evaluated by the sulfuric acid / phenol method. Moreover, extracts were assessed for cryptococcal GXM cross-reactivity through latex agglutination and lateral flow assay methods. Cryptococcus neoformans and Saccharomyces cerevisiae were used as positive and negative controls, respectively. In addition to T. asahii, the species Trichosporon inkin, Apiotrichum montevideense, Trichosporon japonicum, Trichosporon faecale, Trichosporon ovoides, Cutaneotrichosporon debeurmannianum, and Cutaneotrichosporon arboriformis are also producers of a polysaccharide immunologically similar to the GXM produced by human pathogenic Cryptococcus species. The carbohydrate concentration of the extracts presented a positive correlation with the GXM contents determined by titration of both methodologies. These results add several species to the list of fungal pathogens that produce glycans of the GXM type and bring information about the origin of potential false-positive results on immunological tests for diagnosis of cryptococcosis based on GXM detection.
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http://dx.doi.org/10.1007/s11046-021-00558-wDOI Listing
June 2021

Ultrastructural Study of Surface During Budding Events.

Front Microbiol 2021 1;12:609244. Epub 2021 Mar 1.

Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.

is a fungal pathogen that causes life-threatening infections in immunocompromised individuals. It is surrounded by three concentric structures that separate the cell from the extracellular space: the plasma membrane, the cell wall and the polysaccharide (PS) capsule. Although several studies have revealed the chemical composition of these structures, little is known about their ultrastructural organization and remodeling during budding events. Here, by combining the latest and most accurate light and electron microscopy techniques, we describe the morphological remodeling that occurs among the capsule, cell wall and plasma membrane during budding in . Our results show that the cell wall deforms to generate a specialized region at one of the cell's poles. This region subsequently begins to break into layers that are slightly separated from each other and with thick tips. We also observe a reorganization of the capsular PS around the specialized regions. While daughter cells present their PS fibers aligned in the direction of budding, mother cells show a similar pattern but in the opposite direction. Also, daughter cells form multilamellar membrane structures covering the continuous opening between both cells. Together, our findings provide compelling ultrastructural evidence for surface remodeling during budding, which may have important implications for future studies exploring these remodeled specialized regions as drug-targets against cryptococcosis.
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http://dx.doi.org/10.3389/fmicb.2021.609244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7957021PMC
March 2021

Glycans From Distinct Genotypes Share Structural and Serological Similarities to Glucuronoxylomannan.

Front Cell Infect Microbiol 2020 8;10:565571. Epub 2021 Jan 8.

Laboratório de Bioquímica e Imunologia das Micoses, Departamento de Microbiologia e Parasitologia, Instituto Biomédico, Universidade Federal Fluminense, Niterói, Brazil.

The cell wall is a ubiquitous structure in the fungal kingdom, with some features varying depending on the species. Additional external structures can be present, such as the capsule of (), its major virulence factor, mainly composed of glucuronoxylomannan (GXM), with anti-phagocytic and anti-inflammatory properties. The literature shows that other cryptococcal species and even more evolutionarily distant species, such as the , and can produce GXM-like polysaccharides displaying serological reactivity to GXM-specific monoclonal antibodies (mAbs), and these complex polysaccharides have similar composition and anti-phagocytic properties to cryptococcal GXM. Previously, we demonstrated that the fungus incorporates, surface/secreted GXM of and the surface accumulation of the polysaccharide enhances virulence and . In this work, we characterized the ability of to produce cellular-attached (C-gly-) and secreted (E-gly) glycans with reactivity to GXM mAbs. These C-gly- are readily incorporated on the surface of acapsular cap59; however, in contrast to GXM, C-gly- had no xylose and glucuronic acid in its composition. Mapping of recognized GXM synthesis/export proteins confirmed the presence of orthologs in the database. Evaluation of C-gly and E-gly of from strains of distinct monophyletic clades showed serological reactivity to GXM mAbs, despite slight differences in their molecular dimensions. These C-gly- and E-gly- also reacted with sera of cryptococcosis patients. In turn, sera from histoplasmosis patients recognized glycans, suggesting immunogenicity and the presence of cross-reacting antibodies. Additionally, C-gly- and E-gly- coated cap59 were more resistant to phagocytosis and macrophage killing. C-gly- and E-gly- coated cap59 were also able to kill larvae of . These GXM-like glycans, as well as those produced by other pathogenic fungi, may also be important during host-pathogen interactions, and factors associated with their regulation are potentially important targets for the management of histoplasmosis.
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http://dx.doi.org/10.3389/fcimb.2020.565571DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7874066PMC
June 2021

Prevalence of opportunistic invasive aspergillosis in COVID-19 patients with severe pneumonia.

Mycoses 2021 Feb 3;64(2):144-151. Epub 2020 Dec 3.

Laboratório de Biofísica de Fungos, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil.

Background: As the global coronavirus pandemic (COVID-19) spreads across the world, new clinical challenges emerge in the hospital landscape. Among these challenges, the increased risk of coinfections is a major threat to the patients. Although still in a low number, due to the short time of the pandemic, studies that identified a significant number of hospitalised patients with COVID-19 who developed secondary fungal infections that led to serious complications and even death have been published.

Objectives: In this scenario, we aim to determine the prevalence of invasive fungal infections (IFIs) and describe possible associated risk factors in patients admitted due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.

Patients/methods: We designed an open prospective observational study at the Rey Juan Carlos University Hospital (Mostoles, Spain), during the period from February 1 to April 30, 2020.

Results: In this article, we reported seven patients with COVID-19-associated pulmonary aspergillosis (CAPA) who had a poor prognosis. Severely ill patients represent a high-risk group; therefore, we must actively investigate the possibility of aspergillosis in all of these patients. Larger cohort studies are needed to unravel the role of COVID-19 immunosuppressive therapy as a risk factor for aspergillosis.

Conclusions: As the pandemic continues to spread across the world, further reports are needed to assess the frequency of emergent and highly resistant reemergent fungal infections during severe COVID-19. These coinfections are leading a significant number of patients with COVID-19 to death due to complications following the primary viral disease.
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http://dx.doi.org/10.1111/myc.13219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7753478PMC
February 2021

Systemic mycoses: a potential alert for complications in COVID-19 patients.

Future Microbiol 2020 09;15:1405-1413

Laboratorio de Biofísica de Fungos. Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.

As the global COVID-19 pandemic spreads worldwide, new challenges arise in the clinical landscape. The need for reliable diagnostic methods, treatments and vaccines for COVID-19 is the major worldwide urgency. While these goals are especially important, the growing risk of co-infections is a major threat not only to the health systems but also to patients' lives. Although there is still not enough published statistical data, co-infections in COVID-19 patients found that a significant number of patients hospitalized with COVID-19 developed secondary systemic mycoses that led to serious complications and even death. This review will discuss some of these important findings with the major aim to warn the population about the high risk of concomitant systemic mycoses in individuals weakened by COVID-19.
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http://dx.doi.org/10.2217/fmb-2020-0156DOI Listing
September 2020

Hypervirulence and cross-resistance to a clinical antifungal are induced by an environmental fungicide in Cryptococcus gattii.

Sci Total Environ 2020 Oct 12;740:140135. Epub 2020 Jun 12.

Laboratório de Micologia, Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Minas Gerais, Brazil. Electronic address:

The increasing human population requires ongoing efforts in food production. This is frequently associated with an increased use of agrochemicals, leading to environmental contamination and altering microbial communities, including human fungal pathogens that reside in the environment. Cryptococcus gattii is an environmental yeast and is one of the etiological agents of cryptococcosis. Benomyl (BEN) is a broad-spectrum fungicide used on several crops. To study the effects of agrochemicals on fungal pathogens, we first evaluated the susceptibility of C. gattii to BEN and the interactions with clinical antifungals. Antagonistic interaction between BEN and fluconazole was seen and was strain- and concentration-dependent. We then induced BEN-resistance by culturing strains in increasing drug concentrations. One strain demonstrated to be more resistant and showed increased multidrug efflux pump gene (MDR1) expression and increased rhodamine 6G efflux, leading to cross-resistance between BEN and fluconazole. Morphologically, BEN-adapted cells had a reduced polysaccharide capsule; an increased surface/volume ratio; increased growth rate in vitro and inside macrophages and also higher ability in crossing an in vitro model of blood-brain-barrier. BEN-adapted strain demonstrated to be hypervirulent in mice, leading to severe symptoms of cryptococcosis, early mortality and higher fungal burden in the organs, particularly the brain. The parental strain was avirulent in murine model. In vivo cross-resistance between BEN and fluconazole was observed, with mice infected with the adapted strain unable to present any improvement in survival and behavior when treated with this antifungal. Furthermore, BEN-adapted cells cultured in drug-free media maintained the hypervirulent and cross-resistant phenotype, suggesting a persistent effect of BEN on C. gattii. In conclusion, exposure to BEN induces cross-resistance with fluconazole and increases the virulence of C. gattii. Altogether, our results indicate that agrochemicals may lead to unintended consequences on non-target species and this could result in severe healthy problems worldwide.
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http://dx.doi.org/10.1016/j.scitotenv.2020.140135DOI Listing
October 2020

The mechanical properties of microbial surfaces and biofilms.

Cell Surf 2019 Dec 5;5:100028. Epub 2019 Aug 5.

Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.

Microbes can modify their surface structure as an adaptive mechanism for survival and dissemination in the environment or inside the host. Altering their ability to respond to mechanical stimuli is part of this adaptive process. Since the 1990s, powerful micromanipulation tools have been developed that allow mechanical studies of microbial cell surfaces, exploring little known aspects of their dynamic behavior. This review concentrates on the study of mechanical and rheological properties of bacteria and fungi, focusing on their cell surface dynamics and biofilm formation.
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http://dx.doi.org/10.1016/j.tcsw.2019.100028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7389442PMC
December 2019

Effect of cell geometry in the evaluation of erythrocyte viscoelastic properties.

Phys Rev E 2020 Jun;101(6-1):062403

Instituto de Física, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, 21941-972, Brazil.

The red blood cell membrane-cytoskeleton is a complex structure mainly responsible for giving the cell rigidity and shape. It also provides the erythrocyte with the ability to pass through narrow capillaries of the vertebrate blood circulatory system. Although the red blood cell viscoelastic properties have been extensively studied, reported experimental data differ by up to three orders of magnitude. This could be attributed to the natural cell variability, to the different techniques employed, and also to the models used for the cell response, which are highly dependent on cell geometry. Here, we use two methodologies based on optical tweezers to investigate the viscoelastic behavior of healthy human red blood cells, one applying small cell deformations (microrheology) and another imposing large deformations (tether extraction). We also establish a defocusing microscopy-based method to characterize the cell geometry and thus the erythrocyte form factor, an essential parameter that allows comparisons among the viscoelastic properties at different conditions. Moreover, for small deformations, a soft glassy rheology model is used to discuss the results, while for large deformations two surface shear moduli and one surface viscosity are determined, together with the surface tension and bending modulus of the erythrocyte membrane lipid component. We also show that F-actin is not detected in tethers, although the erythrocyte membrane has physical properties like those of other adherent cells, known to have tethers containing F-actin inside. Altogether, our results show good agreement with the reported literature and we argue that, to properly compare the viscoelastic properties of red blood cells in different situations, the task of cell geometry characterization must be accomplished. This may be especially important when the influence of agents, like the malaria parasite, induces changes in both the geometry and chemical constituents of the erythrocyte membrane. Together, the new methodologies and procedures used in this study would allow the erythrocyte community to better explore the mechanical behavior of red blood cells and may be useful to characterize erythrocyte viscoelasticity changes in several blood diseases.
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http://dx.doi.org/10.1103/PhysRevE.101.062403DOI Listing
June 2020

Membrane Elastic Properties During Neural Precursor Cell Differentiation.

Cells 2020 05 26;9(6). Epub 2020 May 26.

Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-902, RJ, Brazil.

Neural precursor cells differentiate into several cell types that display distinct functions. However, little is known about how cell surface mechanics vary during the differentiation process. Here, by precisely measuring membrane tension and bending modulus, we map their variations and correlate them with changes in neural precursor cell morphology along their distinct differentiation fates. Both cells maintained in culture as neural precursors as well as those plated in neurobasal medium reveal a decrease in membrane tension over the first hours of culture followed by stabilization, with no change in bending modulus. During astrocyte differentiation, membrane tension initially decreases and then increases after 72 h, accompanied by consolidation of glial fibrillary acidic protein expression and striking actin reorganization, while bending modulus increases following observed alterations. For oligodendrocytes, the changes in membrane tension are less abrupt over the first hours, but their values subsequently decrease, correlating with a shift from oligodendrocyte marker O4 to myelin basic protein expressions and a remarkable actin reorganization, while bending modulus remains constant. Oligodendrocytes at later differentiation stages show membrane vesicles with similar membrane tension but higher bending modulus as compared to the cell surface. Altogether, our results display an entire spectrum of how membrane elastic properties are varying, thus contributing to a better understanding of neural differentiation from a mechanobiological perspective.
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http://dx.doi.org/10.3390/cells9061323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7349228PMC
May 2020

Endocytosis and Exocytosis in Are Modulated by Bromoenol Lactone.

Front Cell Infect Microbiol 2020 7;10:39. Epub 2020 Feb 7.

Centro de Ciências da Saúde, Instituto de Microbiologia Paulo de Góes, Universidade Federal Do Rio de Janeiro, Rio de Janeiro, Brazil.

In the protozoan pathogen , endocytosis, and exocytosis occur mainly in the small area of the flagellar pocket membrane, which makes this parasite an interesting model of strikingly polarized internalization and secretion. Moreover, little is known about vesicle recognition and fusion mechanisms, which are essential for both endo/exocytosis in this parasite. In other cell types, vesicle fusion events require the activity of phospholipase A (PLA), including Ca-independent iPLA and soluble, Ca-dependent sPLA. Here, we studied the role of bromoenol lactone (BEL) inhibition of endo/exocytosis in promastigotes of . PLA activities were assayed in intact parasites, in whole conditioned media, and in soluble and extracellular vesicles (EVs) conditioned media fractions. BEL did not affect the viability of promastigotes, but reduced the differentiation into metacyclic forms. Intact parasites and EVs had BEL-sensitive iPLA activity. BEL treatment reduced total EVs secretion, as evidenced by reduced total protein concentration, as well as its size distribution and vesicles in the flagellar pocket of treated parasites as observed by TEM. Membrane proteins, such as acid phosphatases and GP63, became concentrated in the cytoplasm, mainly in multivesicular tubules of the endocytic pathway. BEL also prevented the endocytosis of BSA, transferrin and ConA, with the accumulation of these markers in the flagellar pocket. These results suggested that the activity inhibited by BEL, which is one of the irreversible inhibitors of iPLA2, is required for both endocytosis and exocytosis in promastigotes of .
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http://dx.doi.org/10.3389/fcimb.2020.00039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7020749PMC
June 2021

Surface, adhesiveness and virulence aspects of Candida haemulonii species complex.

Med Mycol 2020 Oct;58(7):973-986

Laboratório de Estudos Avançados de Microrganismos Emergentes e Resistentes, Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.

The emerging opportunistic pathogens comprising the Candida haemulonii complex (C. haemulonii [Ch], C. duobushaemulonii [Cd] and C. haemulonii var. vulnera[Chv]) are notable for their intrinsic antifungal resistance. Different clinical manifestations are associated with these fungal infections; however, little is known about their biology and potential virulence attributes. Herein, we evaluated some surface properties of 12 clinical isolates of Ch (n = 5), Cd (n = 4) and Chv (n = 3) as well as their virulence on murine macrophages and Galleria mellonella larvae. Scanning electron microscopy demonstrated the presence of homogeneous populations among the species of the C. haemulonii complex, represented by oval yeasts with surface irregularities able to form aggregates. Cell surface hydrophobicity was isolate-specific, exhibiting high (16.7%), moderate (25.0%) and low (58.3%) hydrophobicity. The isolates had negative surface charge, except for one. Mannose/glucose- and N-acetylglucosamine-containing glycoconjugates were evidenced in considerable amounts in all isolates; however, the surface expression of sialic acid was poorly detected. Cd isolates presented significantly higher amounts of chitin than Ch and Chv. Membrane sterol and lipid bodies, containing neutral lipids, were quite similar among all fungi studied. All isolates adhered to inert surfaces in the order: polystyrene > poly-L-lysine-coated glass > glass. Likewise, they interacted with murine macrophages in a quite similar way. Regarding in vivo virulence, the C. haemulonii species complex were able to kill at least 80% of the larvae after 120 hours. Our results evidenced the ability of C. haemulonii complex to produce potential surface-related virulence attributes, key components that actively participate in the infection process described in Candida spp.
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http://dx.doi.org/10.1093/mmy/myz139DOI Listing
October 2020

Rheological properties of cryptococcal polysaccharide change with fiber size, antibody binding and temperature.

Future Microbiol 2019 07 25;14:867-884. Epub 2019 Jul 25.

Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.

is the major agent of cryptococcosis. The main virulence factor is the polysaccharide (PS) capsule. Changes in cryptococcal PS properties have been poorly elucidated. We analyzed the mechanical properties of secreted PS and intact capsules, using dynamic light scattering and optical tweezers. Storage and loss moduli showed that secreted PS behaves as a viscoelastic liquid, while capsular PS behaves as a viscoelastic solid. The secreted PS remains as a viscoelastic fluid at different temperatures with thermal hysteresis after 85°C. Antibody binding altered the viscoelastic behavior of both secreted and capsular PS. Deciphering the mechanical aspects of these structures could reveal features that may have consequences in novel therapies against cryptococcosis.
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http://dx.doi.org/10.2217/fmb-2018-0320DOI Listing
July 2019

Interaction with Pantoea agglomerans Modulates Growth and Melanization of Sporothrix brasiliensis and Sporothrix schenckii.

Mycopathologia 2019 Jun 18;184(3):367-381. Epub 2019 Jun 18.

Laboratório de Micologia, Instituto Nacional de Infectologia Evandro Chagas, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil.

Sporothrix brasiliensis and Sporothrix schenckii stand as the most virulent agents of sporotrichosis, a worldwide-distributed subcutaneous mycosis. The origin of Sporothrix virulence seems to be associated with fungal interactions with organisms living in the same environment. To assess this hypothesis, the growth of these two species in association with Pantoea agglomerans, a bacterium with a habitat similar to Sporothrix spp., was evaluated. Growth, melanization, and gene expression of the fungus were compared in the presence or absence of the bacterium in the same culture medium. Both S. brasiliensis and S. schenckii grew in contact with P. agglomerans yielding heavily melanized conidia after 5 days of incubation at 30 °C in Sabouraud agar. This increased melanin production occurred around bacterial colonies, suggesting that fungal melanization is triggered by a diffusible bacterial product, which is also supported by a similar pattern of melanin production during Sporothrix spp. growth in contact with heat-killed P. agglomerans. Growth of P. agglomerans was similar in the presence or absence of the fungus. However, the growth of S. brasiliensis and S. schenckii was initially inhibited, but further enhanced when these species were co-cultured with P. agglomerans. Moreover, fungi were able to use killed bacteria as both carbon and nitrogen sources for growth. Representational difference analysis identified overexpressed genes related to membrane transport when S. brasiliensis was co-cultured with the bacteria. The down-regulation of metabolism-related genes appears to be related to nutrient availability during bacterial exploitation. These findings can lead to a better knowledge on Sporothrix ecology and virulence.
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http://dx.doi.org/10.1007/s11046-019-00350-xDOI Listing
June 2019

Glucuronoxylomannan and Sterylglucoside Are Required for Host Protection in an Animal Vaccination Model.

mBio 2019 04 2;10(2). Epub 2019 Apr 2.

Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, New York, USA

is an encapsulated fungal pathogen that causes meningoencephalitis. There are no prophylactic tools for cryptococcosis. Previously, our group showed that a mutant lacking the gene encoding sterylglucosidase (Δ) induced protection in both immunocompetent and immunocompromised murine models of cryptococcosis. Since sterylglucosidase catalyzes degradation of sterylglucosides (SGs), accumulation of this glycolipid could be responsible for protective immunity. In this study, we analyzed whether the activity of SGs is sufficient for the protective effect induced by the Δ strain. We observed that the accumulation of SGs impacted several properties of the main polysaccharide that composes the fungal capsule, glucuronoxylomannan (GXM). We therefore used genetic manipulation to delete the gene in the acapsular mutant Δ to generate a double mutant (strain Δ/Δ) that was shown to be nonpathogenic and cleared from the lung of mice within 7 days post-intranasal infection. The inflammatory immune response triggered by the Δ/Δ mutant in the lung differed from the response seen with the other strains. The double mutant did not induce protection in a vaccination model, suggesting that SG-related protection requires the main capsular polysaccharide. Finally, GXM-containing extracellular vesicles (EVs) enriched in SGs delayed the acute lethality of against infection. These studies highlighted a key role for GXM and SGs in inducing protection against a secondary cryptococcal infection, and, since EVs notoriously contain GXM, these results suggest the potential use of Δ EVs as a vaccination strategy for cryptococcosis. The number of deaths from cryptococcal meningitis is around 180,000 per year. The disease is the second leading cause of mortality among individuals with AIDS. Antifungal treatment is costly and associated with adverse effects and resistance, evidencing the urgency of development of both therapeutic and prophylactic tools. Here we demonstrate the key roles of polysaccharide- and glycolipid-containing structures in a vaccination model to prevent cryptococcosis.
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http://dx.doi.org/10.1128/mBio.02909-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6445945PMC
April 2019

Surface properties, adhesion and biofilm formation on different surfaces by spp. and .

Biofouling 2018 08 24;34(7):800-814. Epub 2018 Oct 24.

a Laboratório de Estudos Avançados de Microrganismos Emergentes e Resistentes, Departamento de Microbiologia Geral , Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro , Rio de Janeiro , Brazil.

In the present work, some surface properties of the fungi , , , and and their capability to adhere to and form a biofilm on diverse surfaces were evaluated. All four species had high conidial surface hydrophobicity and elevated electronegative zeta potentials. Abundant quantities of melanin were detected at the conidial surface, whereas sialic acid was absent. The numbers of non-germinated and germinated conidia adhered to poly-L-lysine-covered slides was higher than on glass after 4 h of fungi-surface contact. Additionally, after 72 h of interaction a typical biofilm structure had formed. Mature biofilms were also observed after 72 h on a nasogastric catheter (made from polyvinyl chloride), a late bladder catheter (siliconized latex), and a nasoenteric catheter (polyurethane). Interestingly, biofilm biomass increased considerably when the catheters had previously been incubated with serum. These results confirm that spp. are capable of forming biofilms on diverse abiotic surfaces.
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http://dx.doi.org/10.1080/08927014.2018.1503652DOI Listing
August 2018

Trypanosoma cruzi epimastigotes store cholesteryl esters in lipid droplets after cholesterol endocytosis.

Mol Biochem Parasitol 2018 09 19;224:6-16. Epub 2018 Jul 19.

Laboratory of Celullar Ultrastructure Hertha Meyer, Biophysics Institute Carlos Chagas Filho, National Institute of Science and Technology in Structural Biology and Bioimaging, Rio de Janeiro, RJ, Brazil; National Center for Biological Structure and Bioimaging-CENABIO, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil. Electronic address:

The Chagas disease agent Trypanosoma cruzi proliferates in the insect vector as highly endocytic epimastigotes that store nutrients, including lipids in reservosomes (lysosome related compartments). Although nutrient storage is important for epimastigote transformation into infective metacyclics, the epimastigote lipid droplets (LDs) remain uncharacterized. Here, we characterized the epimastigote LDs and examined their relationship with the endocytic pathway. Fluorescence microscopy using BODIPY showed that LDs have high neutral lipid content and harbor Rab18, differently from other lipid-rich organelles (such as reservosomes). Using transmission electron microscopy (TEM), we observed a close relationship between LDs and the endoplasmic reticulum, mitochondria and glycosomes. We developed a reproducible protocol to isolate LDs, and showed (by HTPLC and GC/MS analyses) that they have 89% neutral lipids and 11% phospholipids, which are likely to form the LD monolayer seen by TEM. The LD neutral lipids were mostly sterols, although triacylglycerol, diacylglycerol, monoacylglycerol and free fatty acids (FFA) were also found. Endocytosis of H-labeled cholesterol-BSA showed that internalized cholesterol is stored in LDs mostly in the cholesteryl ester form. Together, these results suggest that exogenous cholesterol internalized by endocytosis reaches the reservosomes and is then stored into LDs after esterification.
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http://dx.doi.org/10.1016/j.molbiopara.2018.07.004DOI Listing
September 2018

A Predicted Mannoprotein Participates in Capsular Structure.

mSphere 2018 04 25;3(2). Epub 2018 Apr 25.

Laboratório de Fungos de Importância Médica e Biotecnológica, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil

The yeast-like pathogen is an etiological agent of cryptococcosis. The major cryptococcal virulence factor is the polysaccharide capsule, which is composed of glucuronoxylomannan (GXM), galactoxylomannan (GalXM), and mannoproteins (MPs). The GXM and GalXM polysaccharides have been extensively characterized; however, there is little information about the role of mannoproteins in capsule assembly and their participation in yeast pathogenicity. The present study characterized the function of a predicted mannoprotein from , designated Krp1. Loss-of-function and gain-of-function mutants were generated, and phenotypes associated with the capsular architecture were evaluated. The null mutant cells were more sensitive to a cell wall stressor that disrupts beta-glucan synthesis. Also, these cells displayed increased GXM release to the culture supernatant than the wild-type strain did. The loss of Krp1 influenced cell-associated cryptococcal polysaccharide thickness and phagocytosis by J774.A1 macrophages in the early hours of interaction, but no difference in virulence in a murine model of cryptococcosis was observed. In addition, recombinant Krp1 was antigenic and differentially recognized by serum from an individual with cryptococcosis, but not with serum from an individual with candidiasis. Taken together, these results indicate that Krp1 is important for the cell wall structure, thereby influencing capsule assembly, but is not essential for virulence has the ability to escape from the host's immune system through poorly understood mechanisms and can lead to the death of healthy individuals. The role of mannoproteins in pathogenicity is not completely understood. The present work characterized a protein, Kpr1, that is essential for the maintenance of main virulence factor, the polysaccharide capsule. Our data contribute to the understanding of the role of Kpr1 in capsule structuring, mainly by modulating the distribution of glucans in cell wall.
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http://dx.doi.org/10.1128/mSphere.00023-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5917426PMC
April 2018

Lack of chitin synthase genes impacts capsular architecture and cellular physiology in .

Cell Surf 2018 Jun 12;2:14-23. Epub 2018 Jun 12.

Instituto de Microbiologia Paulo de Góes, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.

mutants lacking each of the eight putative chitin synthase genes () have been previously generated. However, it is still unclear how deletion of chitin synthase genes affects the cryptococcal capsule. Since the connections between chitin metabolism and capsular polysaccharides in are numerous, we analyzed the effects of deletion of genes on capsular and capsule-related structures of . deletion affected capsular morphology in multiple ways, as determined by scanning electron microscopy and immunofluorescence analysis. Molecular diameter, serological reactivity and export of capsular polysaccharide were also affected in most of the mutants, but the most prominent alterations were observed in the strain. cells lacking genes also had altered formation of extracellular vesicles and variable chitinase activity under stress conditions. These results reveal previously unknown functions of genes that greatly impact the physiology of .
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http://dx.doi.org/10.1016/j.tcsw.2018.05.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7389344PMC
June 2018

Genotypic and Phenotypic Diversity of VGII Clinical Isolates and Its Impact on Virulence.

Front Microbiol 2018 6;9:132. Epub 2018 Feb 6.

Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.

The species complex harbors the main etiological agents of cryptococcosis in immunocompetent patients. molecular type VGII predominates in the north and northeastern regions of Brazil, leading to high morbidity and mortality rates. VGII isolates have a strong clinical relevance and phenotypic variations. These phenotypic variations among species complex isolates suggest that some strains are more virulent than others, but little information is available related to the pathogenic properties of those strains. In this study, we analyzed some virulence determinants of VGII strains (CG01, CG02, and CG03) isolated from patients in the state of Piauí, Brazil. The R265 VGIIa strain, which was isolated from the Vancouver outbreak, differed from CG01, CG02 and CG03 isolates (also classified as VGII) when analyzed the capsular dimensions, melanin production, urease activity, as well as the glucuronoxylomannan (GXM) secretion. Those differences directly reflected in their virulence potential. In addition, CG02 displayed higher virulence compared to R265 (VGIIa) strain in a cryptococcal murine model of infection. Lastly, we examined the genotypic diversity of these strains through Multilocus Sequence Type (MLST) and one new subtype was described for the CG02 isolate. This study confirms the presence and the phenotypic and genotypic diversity of highly virulent strains in the Northeast region of Brazil.
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http://dx.doi.org/10.3389/fmicb.2018.00132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5808156PMC
February 2018

Geometrical Distribution of Mediates Flower-Like Biofilm Development.

Front Microbiol 2017 19;8:2534. Epub 2017 Dec 19.

Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.

Microbial biofilms are highly structured and dynamic communities in which phenotypic diversification allows microorganisms to adapt to different environments under distinct conditions. The environmentally ubiquitous pathogen colonizes many niches of the human body and implanted medical devices in the form of biofilms, an important virulence factor. A new approach was used to characterize the underlying geometrical distribution of cells during the adhesion stage of biofilm formation. Geometrical aspects of adhered cells were calculated from the Delaunay triangulation and Voronoi diagram obtained from scanning electron microscopy images (SEM). A correlation between increased biofilm formation and higher ordering of the underlying cell distribution was found. Mature biofilm aggregates were analyzed by applying an adapted protocol developed for ultrastructure visualization of cryptococcal cells by SEM. Flower-like clusters consisting of cells embedded in a dense layer of extracellular matrix were observed as well as distinct levels of spatial organization: adhered cells, clusters of cells and community of clusters. The results add insights into yeast motility during the dispersion stage of biofilm formation. This study highlights the importance of cellular organization for biofilm growth and presents a novel application of the geometrical method of analysis.
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http://dx.doi.org/10.3389/fmicb.2017.02534DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5742216PMC
December 2017
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