Publications by authors named "Rafael Prados-Rosales"

52 Publications

The commensal bacterium imprints innate memory-like responses in mononuclear phagocytes.

Gut Microbes 2021 Jan-Dec;13(1):1939598

Inflammation and Macrophage Plasticity Laboratory, CIC bioGUNE-BRTA (Basque Research and Technology Alliance), Derio, Spain.

Gut microbiota is a constant source of antigens and stimuli to which the resident immune system has developed tolerance. However, the mechanisms by which mononuclear phagocytes, specifically monocytes/macrophages, cope with these usually pro-inflammatory signals are poorly understood. Here, we show that innate immune memory promotes anti-inflammatory homeostasis, using as model strains of the commensal bacterium . Priming of monocytes/macrophages with bacteria, especially in its live form, enhances bacterial intracellular survival and decreases the release of pro-inflammatory signals to the environment, with lower production of TNF and higher levels of IL-10. Analysis of the transcriptomic landscape of these cells shows downregulation of pathways associated with the production of reactive oxygen species (ROS) and the release of cytokines, chemokines and antimicrobial peptides. Indeed, the induction of ROS prevents memory-induced bacterial survival. In addition, there is a dysregulation in gene expression of several metabolic pathways leading to decreased glycolytic and respiratory rates in memory cells. These data support commensal microbe-specific metabolic changes in innate immune memory cells that might contribute to homeostasis in the gut.
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http://dx.doi.org/10.1080/19490976.2021.1939598DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8259724PMC
July 2021

Extracellular vesicles in the context of Mycobacterium tuberculosis infection.

Mol Immunol 2021 05 17;133:175-181. Epub 2021 Mar 17.

Department of Preventive Medicine and Public Health and Microbiology, Autonoma University of Madrid, 28029, Madrid, Spain. Electronic address:

The production of extracellular vesicles (EVs) has emerged as an important process in bacterial biology and host-pathogen interactions. Like many other bacteria, mycobacteria, including Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis (TB), produces EVs in vitro and in vivo. These membrane-enclosed nanoparticles enable Mtb to secrete hydrophobic molecules, proteins, lipids and glycolipids in a concentrated and protected manner and engage in remote interactions with the host. The nature of the material secreted in mycobacterial EVs, the functional attributes of these vesicles and their potential as protective antigens have stimulated great interest in the mycobacterial field. Although the field of EVs in mycobacterial infections is developing, it has already uncovered a whole new dimension for Mtb-host interactions potentially relevant to TB pathogenesis. In this mini-review, we discuss the current evidence supporting an important role of mycobacterial EVs in modulating cellular immune response, the challenges and recent advances in understanding the mechanisms of vesicle biogenesis and the implications for development of new preventive and therapeutic tools against TB.
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http://dx.doi.org/10.1016/j.molimm.2021.02.010DOI Listing
May 2021

The Phosphatidyl--Inositol Dimannoside Acyltransferase PatA Is Essential for Mycobacterium tuberculosis Growth and .

J Bacteriol 2021 03 8;203(7). Epub 2021 Mar 8.

Department of Preventive Medicine and Public Health and Microbiology, Autonoma University of Madrid, Madrid, Spain

comprises an unusual cell envelope dominated by unique lipids and glycans that provides a permeability barrier against hydrophilic drugs and is central for its survival and virulence. Phosphatidyl--inositol mannosides (PIMs) are glycolipids considered to be not only key structural components of the cell envelope but also the precursors of lipomannan (LM) and lipoarabinomannan (LAM), important lipoglycans implicated in host-pathogen interactions. Here, we focus on PatA, a membrane-associated acyltransferase that transfers a palmitoyl moiety from palmitoyl coenzyme A (palmitoyl-CoA) to the 6-position of the mannose ring linked to the 2-position of inositol in PIM/PIM We validate that the function of PatA is vital for and We constructed a conditional mutant and showed that silencing is bactericidal in batch cultures. This phenotype was associated with significantly reduced levels of AcPIM, an important structural component of the mycobacterial inner membrane. The requirement of PatA for viability was also demonstrated during macrophage infection and in a mouse model of infection, where a dramatic decrease in viable counts was observed upon silencing of the gene. This is reminiscent of the behavior of PimA, the mannosyltransferase that initiates the PIM pathway, also found to be essential for growth and Altogether, the experimental data highlight the significance of the early steps of the PIM biosynthetic pathway for physiology and reveal that PatA is a novel target for drug discovery programs against this major human pathogen. Tuberculosis (TB) is the leading cause of death from a single infectious agent. The emergence of drug resistance in strains of , the etiologic agent of TB, emphasizes the need to identify new targets and antimicrobial agents. The mycobacterial cell envelope is a major factor in this intrinsic drug resistance. Here, we have focused on the biosynthesis of PIMs, key virulence factors and important components of the cell envelope. Specifically, we have determined that PatA, the acyltransferase responsible for the first acylation step of the PIM synthesis pathway, is essential in These results highlight the importance of early steps of the PIM biosynthetic pathway for mycobacterial physiology and the suitability of PatA as a potential new drug target.
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http://dx.doi.org/10.1128/JB.00439-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8088522PMC
March 2021

Mesoporous Silica Nanoparticles as a Potential Platform for Vaccine Development against Tuberculosis.

Pharmaceutics 2020 Dec 16;12(12). Epub 2020 Dec 16.

Department of Analytical Chemistry, Faculty of Chemistry, Complutense University of Madrid, 28040 Madrid, Spain.

The increasing emergence of new strains of () highly resistant to antibiotics constitute a public health issue, since tuberculosis still constitutes the primary cause of death in the world due to bacterial infection. has been shown to produce membrane-derived extracellular vesicles (EVs) containing proteins responsible for modulating the pathological immune response after infection. These natural vesicles were considered a promising alternative to the development of novel vaccines. However, their use was compromised by the observed lack of reproducibility between preparations. In this work, with the aim of developing nanosystems mimicking the extracellular vesicles produced by mesoporous silica nanoparticles (MSNs) have been used as nanocarriers of immunomodulatory and vesicle-associated proteins (Ag85B, LprG and LprA). These novel nanosystems have been designed and extensively characterized, demonstrating the effectiveness of the covalent anchorage of the immunomodulatory proteins to the surface of the MSNs. The immunostimulatory capacity of the designed nanosystems has been demonstrated by measuring the levels of pro- (TNF) and anti-inflammatory (IL-10) cytokines in exposed macrophages. These results open a new possibility for the development of more complex nanosystems, including additional vesicle components or even antitubercular drugs, thus allowing for the combination of immunomodulatory and bactericidal effects against .
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http://dx.doi.org/10.3390/pharmaceutics12121218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7767215PMC
December 2020

Mesoporous silica nanoparticles containing silver as novel antimycobacterial agents against Mycobacterium tuberculosis.

Colloids Surf B Biointerfaces 2021 Jan 25;197:111405. Epub 2020 Oct 25.

Departament of Analytical Chemistry, Faculty of Chemistry, Complutense University of Madrid, Av. Complutense s/n, 28040, Madrid, Spain. Electronic address:

Tuberculosis remains today a major public health issue with a total of 9 million new cases and 2 million deaths annually. The lack of an effective vaccine and the increasing emergence of new strains of Mycobacterium tuberculosis (Mtb) highly resistant to antibiotics, anticipate a complicated scenario in the near future. The use of nanoparticles features as an alternative to antibiotics in tackling this problem due to their potential effectiveness in resistant bacterial strains. In this context, silver nanoparticles have demonstrated high bactericidal efficacy, although their use is limited by their relatively high toxicity, which calls for the design of nanocarriers that allow silver based nanoparticles to be safely delivered to the target cells or tissues. In this work mesoporous silica nanoparticles are used as carriers of silver based nanoparticles as antimycobacterial agent against Mtb. Two different synthetic approaches have been used to afford, on the one hand, a 2D hexagonal mesoporous silica nanosystem which contains silver bromide nanoparticles distributed all through the silica network and, on the other hand, a [email protected] nanosystem with metallic silver nanoparticles as core and mesoporous silica shell in a radial mesoporous rearrangement. Both materials have demonstrated good antimycobacterial capacity in in vitro test using Mtb, being lower the minimum inhibitory concentration for the nanosystem which contains silver bromide. Therefore, the interaction of this material with the mycobacterial cell has been studied by cryo-electron microscopy, establishing a direct connection between the antimycobactericidal effect observed and the damage induced in the cell envelope.
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http://dx.doi.org/10.1016/j.colsurfb.2020.111405DOI Listing
January 2021

Editorial: Macrophage Metabolism and Immune Responses.

Front Immunol 2020 29;11:1078. Epub 2020 May 29.

Inflammation and Macrophage Plasticity Lab, CIC bioGUNE-BRTA (Basque Research and Technology Alliance), Derio, Spain.

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http://dx.doi.org/10.3389/fimmu.2020.01078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7272707PMC
April 2021

Antimycobacterial Effect of Selenium Nanoparticles on .

Front Microbiol 2020 28;11:800. Epub 2020 Apr 28.

Department of Analytical Chemistry, Faculty of Chemistry, Complutense University of Madrid, Madrid, Spain.

Tuberculosis (TB) remains the leading cause of death from a single infection agent worldwide. In recent years, the occurrence of TB cases caused by drug-resistant strains has spread, and is expected to continue to grow. Therefore, the development of new alternative treatments to the use of antibiotics is highly important. In that sense, nanotechnology can play a very relevant role, due to the unique characteristics of nanoparticles. In fact, different types of nanoparticles have already been evaluated both as potential bactericides and as efficient drug delivery vehicles. In this work, the use of selenium nanoparticles (SeNPs) has been evaluated to inhibit the growth of two types of mycobacteria: () and (). The results showed that SeNPs are able to inhibit the growth of both types of mycobacteria by damaging their cell envelope integrity. These results open a new opportunity for the use of this type of nanoparticles as antimycobacterial agents by themselves, or for the development of novel nanosystems that combine the action of these nanoparticles with other drugs.
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http://dx.doi.org/10.3389/fmicb.2020.00800DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7212347PMC
April 2020

The mitochondrial negative regulator MCJ modulates the interplay between microbiota and the host during ulcerative colitis.

Sci Rep 2020 01 17;10(1):572. Epub 2020 Jan 17.

CIC bioGUNE. Bizkaia Science and Technology Park. bld 801 A, 48160, Derio, Bizkaia, Spain.

Recent evidences indicate that mitochondrial genes and function are decreased in active ulcerative colitis (UC) patients, in particular, the activity of Complex I of the electron transport chain is heavily compromised. MCJ is a mitochondrial inner membrane protein identified as a natural inhibitor of respiratory chain Complex I. The induction of experimental colitis in MCJ-deficient mice leads to the upregulation of Timp3 expression resulting in the inhibition of TACE activity that likely inhibits Tnf and Tnfr1 shedding from the cell membrane in the colon. MCJ-deficient mice also show higher expression of Myd88 and Tlr9, proinflammatory genes and disease severity. Interestingly, the absence of MCJ resulted in distinct microbiota metabolism and composition, including a member of the gut community in UC patients, Ruminococcus gnavus. These changes provoked an effect on IgA levels. Gene expression analyses in UC patients showed decreased levels of MCJ and higher expression of TIMP3, suggesting a relevant role of mitochondrial genes and function among active UC. The MCJ deficiency disturbs the regulatory relationship between the host mitochondria and microbiota affecting disease severity. Our results indicate that mitochondria function may be an important factor in the pathogenesis. All together support the importance of MCJ regulation during UC.
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http://dx.doi.org/10.1038/s41598-019-57348-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6969106PMC
January 2020

Melanin deposition in two species depends on cell-wall composition and flexibility.

J Biol Chem 2020 02 2;295(7):1815-1828. Epub 2020 Jan 2.

Department of Chemistry and Biochemistry, City College of New York and CUNY Institute for Macromolecular Assemblies, New York, New York 10031; Ph.D. Program in Biochemistry, The Graduate Center of the City University of New York, New York, New York 10016; Ph.D. Program in Chemistry, The Graduate Center of the City University of New York, New York, New York 10016. Electronic address:

and are two species complexes in the large fungal genus and are responsible for potentially lethal disseminated infections. These two complexes share several phenotypic traits, such as production of the protective compound melanin. In , the pigment associates with key cellular constituents that are essential for melanin deposition within the cell wall. Consequently, melanization is modulated by changes in cell-wall composition or ultrastructure. However, whether similar factors influence melanization in is unknown. Herein, we used transmission EM, biochemical assays, and solid-state NMR spectroscopy of representative isolates and "leaky melanin" mutant strains from each species complex to examine the compositional and structural factors governing cell-wall pigment deposition in and The principal findings were the following. 1) R265 had an exceptionally high chitosan content compared with H99; a rich chitosan composition promoted homogeneous melanin distribution throughout the cell wall but did not increase the propensity of pigment deposition. 2) Strains from both species manifesting the leaky melanin phenotype had reduced chitosan content, which was compensated for by the production of lipids and other nonpolysaccharide constituents that depended on the species or mutation. 3) Changes in the relative rigidity of cell-wall chitin were associated with aberrant pigment retention, implicating cell-wall flexibility as an independent variable in cryptococcal melanin assembly. Overall, our results indicate that cell-wall composition and molecular architecture are critical factors for the anchoring and arrangement of melanin pigments in both and species complexes.
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http://dx.doi.org/10.1074/jbc.RA119.011949DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7029119PMC
February 2020

Regulation of macrophage activity by surface receptors contained within Borrelia burgdorferi-enriched phagosomal fractions.

PLoS Pathog 2019 11 18;15(11):e1008163. Epub 2019 Nov 18.

Inflammation and Macrophage Plasticity Laboratory, CIC bioGUNE, Derio, Bizkaia, Spain.

Macrophages mediate the elimination of pathogens by phagocytosis resulting in the activation of specific signaling pathways that lead to the production of cytokines, chemokines and other factors. Borrelia burgdorferi, the causative agent of Lyme disease, causes a wide variety of pro-inflammatory symptoms. The proinflammatory capacity of macrophages is intimately related to the internalization of the spirochete. However, most receptors mediating this process are largely unknown. We have applied a multiomic approach, including the proteomic analysis of B. burgdorferi-containing phagosome-enriched fractions, to identify surface receptors that are involved in the phagocytic capacity of macrophages as well as their inflammatory output. Sucrose gradient protein fractions of human monocyte-derived macrophages exposed to B. burgdorferi contained the phagocytic receptor, CR3/CD14 highlighting the major role played by these proteins in spirochetal phagocytosis. Other proteins identified in these fractions include C-type lectins, scavenger receptors or Siglecs, of which some are directly involved in the interaction with the spirochete. We also identified the Fc gamma receptor pathway, including the binding receptor, CD64, as involved both in the phagocytosis of, and TNF induction in response to B. burgdorferi in the absence of antibodies. The common gamma chain, FcγR, mediates the phagocytosis of the spirochete, likely through Fc receptors and C-type lectins, in a process that involves Syk activation. Overall, these findings highlight the complex array of receptors involved in the phagocytic response of macrophages to B. burgdorferi.
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http://dx.doi.org/10.1371/journal.ppat.1008163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6886865PMC
November 2019

Strategies for Membrane Protein Analysis by Mass Spectrometry.

Adv Exp Med Biol 2019 ;1140:289-298

Department of Analytical Chemistry, Faculty of Chemical Sciences, Complutense University of Madrid, Madrid, Spain.

Membrane proteins are of utmost importance in different cellular processes including: cell signaling, substrate transport, homeostasis control, immune surveillance, etc. In addition, they represent between 60% and 70% of the therapeutic targets currently used. Therefore, the identification and characterization of these proteins is crucial in many fields of research. Although proteomics has undergone an extraordinary advance in recent years thanks to the development of mass spectrometry, the methods used for the identification and quantification of soluble proteins generally fail to be used for membrane proteins, mainly due to their hydrophobic character.In this chapter, we revised the different alternatives, modifications and improvements that have been developed over the years with the aim of adapting the methods used in proteomics to the particular study of membrane proteins, thus allowing to increase the number of membrane proteins identified, as well as their coverage.
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http://dx.doi.org/10.1007/978-3-030-15950-4_16DOI Listing
September 2019

The capsule: a cell structure with key implications in pathogenesis.

Biochem J 2019 07 18;476(14):1995-2016. Epub 2019 Jul 18.

CIC bioGUNE, Bizkaia Technology Park, Ed. 801A, Derio 48160, Spain

Bacterial capsules have evolved to be at the forefront of the cell envelope, making them an essential element of bacterial biology. Efforts to understand the (Mtb) capsule began more than 60 years ago, but the relatively recent development of mycobacterial genetics combined with improved chemical and immunological tools have revealed a more refined view of capsule molecular composition. A glycogen-like α-glucan is the major constituent of the capsule, with lower amounts of arabinomannan and mannan, proteins and lipids. The major Mtb capsular components mediate interactions with phagocytes that favor bacterial survival. Vaccination approaches targeting the mycobacterial capsule have proven successful in controlling bacterial replication. Although the Mtb capsule is composed of polysaccharides of relatively low complexity, the concept of antigenic variability associated with this structure has been suggested by some studies. Understanding how Mtb shapes its envelope during its life cycle is key to developing anti-infective strategies targeting this structure at the host-pathogen interface.
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http://dx.doi.org/10.1042/BCJ20190324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6698057PMC
July 2019

Mycobacterium tuberculosis extracellular vesicle-associated lipoprotein LpqH as a potential biomarker to distinguish paratuberculosis infection or vaccination from tuberculosis infection.

BMC Vet Res 2019 Jun 7;15(1):188. Epub 2019 Jun 7.

CIC bioGUNE, Bizkaia Technology Park, Building 801A, 48160, Derio, Bizkaia, Spain.

Background: Both bovine tuberculosis (TB) and paratuberculosis (PTB) are serious and widespread bacterial infections affecting many domestic and wild animal species. However, current vaccines do not confer complete protection and cause interference with other diagnostics tests, including bovine TB. Therefore, the development of "Differentiating Infected from Vaccinated Animals" (DIVA) tests are a pressing need. In this study, we have tested the feasibility of mycobacterial extracellular vesicles (EVs) as potential source of biomarkers to discriminate between Mycobacterium bovis infected, Mycobacterium avium subsp. paratuberculosis (MAP) infected and MAP-vaccinated cows. We have, initially, characterized vesicle production in the two most medically relevant species of mycobacteria for livestock, MAP and M. bovis, for being responsible for tuberculosis (TB) and paratuberculosis (PTB).

Results: Our results indicate that these two species produce EVs with different kinetics, morphology and size distribution. Analysis of the immunogenicity of both type of EVs showed some cross reactivity with sera from PTB+ and TB+ cows, suggesting a limited diagnostic capacity for both EVs. Conversely, we noticed that Mycobacterium tuberculosis (Mtb) EVs showed some differential reactivity between sera from MAP-vaccinated or PTB+ cows from TB+ ones. Mass spectrometry analysis (MS) identified a 19-kDa EV-associated lipoprotein as the main source of the differential reactivity.

Conclusions: LpqH could be a good plasma biomarker with capacity to distinguish PTB+ or MAP-vaccinated cows from cows infected with TB.
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http://dx.doi.org/10.1186/s12917-019-1941-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6555097PMC
June 2019

The structural unit of melanin in the cell wall of the fungal pathogen .

J Biol Chem 2019 07 22;294(27):10471-10489. Epub 2019 May 22.

From the Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, Maryland 21205,

Melanins are synthesized macromolecules that are found in all biological kingdoms. These pigments have a myriad of roles that range from microbial virulence to key components of the innate immune response in invertebrates. Melanins also exhibit unique properties with potential applications in physics and material sciences, ranging from electrical batteries to novel therapeutics. In the fungi, melanins, such as eumelanins, are components of the cell wall that provide protection against biotic and abiotic elements. Elucidation of the smallest fungal cell wall-associated melanin unit that serves as a building block is critical to understand the architecture of these polymers, its interaction with surrounding components, and their functional versatility. In this study, we used isopycnic gradient sedimentation, NMR, EPR, high-resolution microscopy, and proteomics to analyze the melanin in the cell wall of the human pathogenic fungus We observed that melanin is assembled into the cryptococcal cell wall in spherical structures ∼200 nm in diameter, termed melanin granules, which are in turn composed of nanospheres ∼30 nm in diameter, termed fungal melanosomes. We noted that melanin granules are closely associated with proteins that may play critical roles in the fungal melanogenesis and the supramolecular structure of this polymer. Using this structural information, we propose a model for melanization that is similar to the process used in animal melanization and is consistent with the phylogenetic relatedness of the fungal and animal kingdoms.
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http://dx.doi.org/10.1074/jbc.RA119.008684DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6615676PMC
July 2019

Serum IgM Glycosylation Associated with Tuberculosis Infection in Mice.

mSphere 2019 03 27;4(2). Epub 2019 Mar 27.

CIC bioGUNE, Derio, Bizkaia, Spain

Changes in serum glycans discriminate between disease statuses in cancer. A similar connection has not been established in the context of infectious diseases such as tuberculosis (TB). The inflammation arising from infection by may affect host protein glycosylation, thereby providing information about disease status in TB. A mouse model of infection was used to study glycoprotein N-glycosylation in serum. Following digestion of serum glycoproteins with peptide--glycosidase F (PNGase F), released glycans were permethylated and analyzed by multidimensional mass spectrometry (MS). Conditions included naive or BCG-vaccinated animals, which were either uninfected or infected with MS results were validated by lectin blotting. We found that both glycoprotein fucosylation and sialylation were particularly sensitive to infection. We observed that infection elevates serum IgM levels and induces changes in glycosylation that could inform about the disease. We demonstrate that infection influenced host protein glycosylation in a mouse model. The mechanism by which infection modifies glycans in serum proteins is not understood. Investigation of the regulation of such modifications by opens a new field that could lead to the discovery of novel biomarkers. Validation of such findings in human samples will reveal the clinical relevance of these findings.
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http://dx.doi.org/10.1128/mSphere.00684-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6437276PMC
March 2019

Hypoxia Is Not a Main Stress When Is in a Dormancy-Like Long-Chain Fatty Acid Environment.

Front Cell Infect Microbiol 2018 9;8:449. Epub 2019 Jan 9.

Departamento de Medicina Preventiva, Facultad de Medicina, Universidad Autónoma de Madrid, Madrid, Spain.

The capacity of () to sense, respond and adapt to a variable and hostile environment within the host makes it one of the most successful human pathogens. During different stages of infection, is surrounded by a plethora of lipid molecules and current evidence points out the relevance of fatty acids during the infectious process. In this study, we have compared the transcriptional response of to hypoxia in cultures supplemented with a mix of even long-chain fatty acids or dextrose as main carbon sources. Using RNA sequencing, we have identified differential expressed genes in early and late hypoxia, defined according to the Wayne and Hayes model, and compared the results with the exponential phase of growth in both carbon sources. We show that the number of genes over-expressed in the lipid medium was quite low in both, early and late hypoxia, relative to conditions including dextrose, with the exception of transcripts of stable and non-coding RNAs, which were more expressed in the fatty acid medium. We found that and were over-expressed in the early phase of hypoxia, confirming their pivotal role in early adaptation to low oxygen concentration independently of the carbon source. A drastic contrast was found with the transcriptional regulatory factors at early hypoxia. Only 2 transcriptional factors were over-expressed in early hypoxia in the lipid medium compared to 37 that were over-expressed in the dextrose medium. Instead of Rv0081, known to be the central regulator of hypoxia in dextrose, Rv2745c (ClgR), seems to play a main role in hypoxia in the fatty acid medium. The low level of genes associated to the stress-response during their adaptation to hypoxia in fatty acids, suggests that this lipid environment makes hypoxia a less stressful condition for the tubercle bacilli. Taken all together, these results indicate that the presence of lipid molecules shapes the metabolic response of to an adaptive state for different stresses within the host, including hypoxia. This fact could explain the success of to establish long-term survival during latent infection.
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http://dx.doi.org/10.3389/fcimb.2018.00449DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6333855PMC
September 2019

virulence factors, including listeriolysin O, are secreted in biologically active extracellular vesicles.

J Biol Chem 2019 01 30;294(4):1202-1217. Epub 2018 Nov 30.

W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland 21205; Department of Microbiology and Immunology, Bronx, New York 10461; Division of Infectious Diseases of the Department of Medicine, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461. Electronic address:

Outer membrane vesicles produced by Gram-negative bacteria have been studied for half a century but the possibility that Gram-positive bacteria secrete extracellular vesicles (EVs) was not pursued until recently due to the assumption that the thick peptidoglycan cell wall would prevent their release to the environment. However, following their discovery in fungi, which also have cell walls, EVs have now been described for a variety of Gram-positive bacteria. EVs purified from Gram-positive bacteria are implicated in virulence, toxin release, and transference to host cells, eliciting immune responses, and spread of antibiotic resistance. is a Gram-positive bacterium that causes listeriosis. Here we report that produces EVs with diameters ranging from 20 to 200 nm, containing the pore-forming toxin listeriolysin O (LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC). Cell-free EV preparations were toxic to mammalian cells, the murine macrophage cell line J774.16, in a LLO-dependent manner, evidencing EV biological activity. The deletion of increased EV toxicity, suggesting PI-PLC reduced LLO activity. Using simultaneous etabolite, rotein, and ipid traction (MPLEx) multiomics we characterized protein, lipid, and metabolite composition of bacterial cells and secreted EVs and found that EVs carry the majority of listerial virulence proteins. Using immunogold EM we detected LLO at several organelles within infected human epithelial cells and with high-resolution fluorescence imaging we show that dynamic lipid structures are released from during infection. Our findings demonstrate that uses EVs for toxin release and implicate these structures in mammalian cytotoxicity.
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http://dx.doi.org/10.1074/jbc.RA118.006472DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6349127PMC
January 2019

The melanization road more traveled by: Precursor substrate effects on melanin synthesis in cell-free and fungal cell systems.

J Biol Chem 2018 12 1;293(52):20157-20168. Epub 2018 Nov 1.

From the Department of Chemistry and Biochemistry, The City College of New York and CUNY Institute for Macromolecular Assemblies, New York, New York 10031,; the City University of New York, Ph.D. Program in Biochemistry, New York, New York 10036,; the City University of New York, Ph.D. Program in Chemistry, New York, New York 10036, and.

Natural brown-black eumelanin pigments confer structural coloration in animals and potently block ionizing radiation and antifungal drugs. These functions also make them attractive for bioinspired materials design, including coating materials for drug-delivery vehicles, strengthening agents for adhesive hydrogel materials, and free-radical scavengers for soil remediation. Nonetheless, the molecular determinants of the melanin "developmental road traveled" and the resulting architectural features have remained uncertain because of the insoluble, heterogeneous, and amorphous characteristics of these complex polymeric assemblies. Here, we used 2D solid-state NMR, EPR, and dynamic nuclear polarization spectroscopic techniques, assisted in some instances by the use of isotopically enriched precursors, to address several open questions regarding the molecular structures and associated functions of eumelanin. Our findings uncovered: 1) that the identity of the available catecholamine precursor alters the structure of melanin pigments produced either in fungal cells or under cell-free conditions; 2) that the identity of the available precursor alters the scaffold organization and membrane lipid content of melanized fungal cells; 3) that the fungal cells are melanized preferentially by an l-DOPA precursor; and 4) that the macromolecular carbon- and nitrogen-based architecture of cell-free and fungal eumelanins includes indole, pyrrole, indolequinone, and open-chain building blocks that develop depending on reaction time. In conclusion, the availability of catecholamine precursors plays an important role in eumelanin development by affecting the efficacy of pigment formation, the melanin molecular structure, and its underlying scaffold in fungal systems.
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http://dx.doi.org/10.1074/jbc.RA118.005791DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6311522PMC
December 2018

Updates on antibody functions in Mycobacterium tuberculosis infection and their relevance for developing a vaccine against tuberculosis.

Curr Opin Immunol 2018 08 12;53:30-37. Epub 2018 Apr 12.

Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Ave, Bronx, NY 10461, United States; Center for Cooperative Research bioGUNE (CICbioGUNE), Bizkaia Technology Park, 48160 Derio, Bizkaia, Spain.

A more effective vaccine to control tuberculosis (TB), a major global public health problem, is urgently needed. Current vaccine candidates focus predominantly on eliciting cell-mediated immunity but other arms of the immune system also contribute to protection against TB. We review here recent studies that enhance our current knowledge of antibody-mediated functions against Mycobacterium tuberculosis. These findings, which contribute to the increasing evidence that antibodies have a protective role against TB, include demonstrations that firstly distinct human antibody Fc glycosylation patterns, found in latent M. tuberculosis infection but not in active TB, influence the efficacy of the host to control M. tuberculosis infection, secondly antibody isotype influences human antibody functions, and thirdly that antibodies targeting M. tuberculosis surface antigens are protective. We discuss these findings in the context of TB vaccine development and highlight the need for further research on antibody-mediated immunity in M. tuberculosis infection.
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http://dx.doi.org/10.1016/j.coi.2018.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6141321PMC
August 2018

A multi-omic analysis reveals the regulatory role of CD180 during the response of macrophages to Borrelia burgdorferi.

Emerg Microbes Infect 2018 Mar 7;7(1):19. Epub 2018 Mar 7.

Macrophage and Tick Vaccine Laboratory, CIC bioGUNE, 48160, Derio, Bizkaia, Spain.

Macrophages are cells of the innate immune system with the ability to phagocytose and induce a global pattern of responses that depend on several signaling pathways. We have determined the biosignature of murine bone marrow-derived macrophages and human blood monocytes using transcriptomic and proteomic approaches. We identified a common pattern of genes that are transcriptionally regulated and overall indicate that the response to B. burgdorferi involves the interaction of spirochetal antigens with several inflammatory pathways corresponding to primary (triggered by pattern-recognition receptors) and secondary (induced by proinflammatory cytokines) responses. We also show that the Toll-like receptor family member CD180 is downregulated by the stimulation of macrophages, but not monocytes, with the spirochete. Silencing Cd180 results in increased phagocytosis while tempering the production of the proinflammatory cytokine TNF. Cd180-silenced cells produce increased levels of Itgam and surface CD11b, suggesting that the regulation of CD180 by the spirochete initiates a cascade that increases CR3-mediated phagocytosis of the bacterium while repressing the consequent inflammatory response.
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http://dx.doi.org/10.1038/s41426-017-0018-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5841238PMC
March 2018

Innate immune cells for immunotherapy of autoimmune and cancer disorders.

Int Rev Immunol 2017 11 21;36(6):315-337. Epub 2017 Sep 21.

a Millennium Institute on Immunology and Immunotherapy Santiago , Chile.

Modulation of the immune system has been widely targeted for the treatment of several immune-related diseases, such as autoimmune disorders and cancer, due to its crucial role in these pathologies. Current available therapies focus mainly on symptomatic treatment and are often associated with undesirable secondary effects. For several years, remission of disease and subsequently recovery of immune homeostasis has been a major goal for immunotherapy. Most current immunotherapeutic strategies are aimed to inhibit or potentiate directly the adaptive immune response by modulating antibody production and B cell memory, as well as the effector potential and memory of T cells. Although these immunomodulatory approaches have shown some success in the clinic with promising therapeutic potential, they have some limitations related to their effectiveness in disease models and clinical trials, as well as elevated costs. In the recent years, a renewed interest has emerged on targeting innate immune cells for immunotherapy, due to their high plasticity and ability to exert a potent and extremely rapid response, which can influence the outcome of the adaptive immune response. In this review, we discuss the immunomodulatory potential of several innate immune cells, as well as they use for immunotherapy, especially in autoimmunity and cancer.
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http://dx.doi.org/10.1080/08830185.2017.1365145DOI Listing
November 2017

Targeting Innate Immune Cells for Immunotherapy.

J Immunol Res 2017;2017:4271384. Epub 2017 Apr 5.

Millennium Institute on Immunology and Immunotherapy, Santiago, Chile.

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http://dx.doi.org/10.1155/2017/4271384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5396470PMC
January 2019

Enhanced control of Mycobacterium tuberculosis extrapulmonary dissemination in mice by an arabinomannan-protein conjugate vaccine.

PLoS Pathog 2017 03 9;13(3):e1006250. Epub 2017 Mar 9.

Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx NY, United States of America.

Currently there are a dozen or so of new vaccine candidates in clinical trials for prevention of tuberculosis (TB) and each formulation attempts to elicit protection by enhancement of cell-mediated immunity (CMI). In contrast, most approved vaccines against other bacterial pathogens are believed to mediate protection by eliciting antibody responses. However, it has been difficult to apply this formula to TB because of the difficulty in reliably eliciting protective antibodies. Here, we developed capsular polysaccharide conjugates by linking mycobacterial capsular arabinomannan (AM) to either Mtb Ag85b or B. anthracis protective antigen (PA). Further, we studied their immunogenicity by ELISA and AM glycan microarrays and protection efficacy in mice. Immunization with either Abg85b-AM or PA-AM conjugates elicited an AM-specific antibody response in mice. AM binding antibodies stimulated transcriptional changes in Mtb. Sera from AM conjugate immunized mice reacted against a broad spectrum of AM structural variants and specifically recognized arabinan fragments. Conjugate vaccine immunized mice infected with Mtb had lower bacterial numbers in lungs and spleen, and lived longer than control mice. These findings provide additional evidence that humoral immunity can contribute to protection against Mtb.
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http://dx.doi.org/10.1371/journal.ppat.1006250DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5360349PMC
March 2017

The Type of Growth Medium Affects the Presence of a Mycobacterial Capsule and Is Associated With Differences in Protective Efficacy of BCG Vaccination Against Mycobacterium tuberculosis.

J Infect Dis 2016 08 18;214(3):426-37. Epub 2016 Apr 18.

Department of Microbiology and Immunology Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland.

Background: Bacillus Calmette-Guerin (BCG) vaccine is widely used for the prevention of tuberculosis, despite limited efficacy. Most immunological studies of BCG or Mycobacterium tuberculosis strains grow bacteria in the presence of detergent, which also strips the mycobacterial capsule. The impact of the capsule on vaccine efficacy has not been explored.

Methods: We tested the influence of detergent in cultures of BCG and M. tuberculosis strains on the outcome of vaccination experiments on mice and transcriptional responses on M. tuberculosis

Results: Vaccination of mice with encapsulated BCG promoted a more potent immune response relative to vaccination with unencapsulated BCG, including higher polysaccharide-specific capsule antibody titers, higher interferon γ and interleukin 17 splenic responses, and more multifunctional CD4(+) T cells. These differences correlated with variability in the bacterial burden in lung and spleen of mice infected with encapsulated or unencapsulated M. tuberculosis The combination of vaccination and challenge with encapsulated strains resulted in the greatest protection efficacy. The transcriptome of encapsulated M. tuberculosis was similar to that of starvation, hypoxia, stationary phase, or nonreplicating persistence.

Conclusions: The presence of detergent in growth media and a capsule on BCG were associated with differences in the outcome of vaccination, implying that these are important variables in immunological studies.
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http://dx.doi.org/10.1093/infdis/jiw153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4936643PMC
August 2016

Association of Human Antibodies to Arabinomannan With Enhanced Mycobacterial Opsonophagocytosis and Intracellular Growth Reduction.

J Infect Dis 2016 07 7;214(2):300-10. Epub 2016 Apr 7.

Department of Medicine Department of Microbiology and Immunology.

Background: The relevance of antibodies (Abs) in the defense against Mycobacterium tuberculosis infection remains uncertain. We investigated the role of Abs to the mycobacterial capsular polysaccharide arabinomannan (AM) and its oligosaccharide (OS) fragments in humans.

Methods: Sera obtained from 29 healthy adults before and after primary or secondary bacillus Calmette-Guerin (BCG) vaccination were assessed for Ab responses to AM via enzyme-linked immunosorbent assays, and to AM OS epitopes via novel glycan microarrays. Effects of prevaccination and postvaccination sera on BCG phagocytosis and intracellular survival were assessed in human macrophages.

Results: Immunoglobulin G (IgG) responses to AM increased significantly 4-8 weeks after vaccination (P < .01), and sera were able to opsonize BCG and M. tuberculosis grown in both the absence and the presence of detergent. Phagocytosis and intracellular growth inhibition were significantly enhanced when BCG was opsonized with postvaccination sera (P < .01), and these enhancements correlated significantly with IgG titers to AM (P < .05), particularly with reactivity to 3 AM OS epitopes (P < .05). Furthermore, increased phagolysosomal fusion was observed with postvaccination sera.

Conclusions: Our results provide further evidence for a role of Ab-mediated immunity to tuberculosis and suggest that IgG to AM, especially to some of its OS epitopes, could contribute to the defense against mycobacterial infection in humans.
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http://dx.doi.org/10.1093/infdis/jiw141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4918826PMC
July 2016

Functions and importance of mycobacterial extracellular vesicles.

Appl Microbiol Biotechnol 2016 May 29;100(9):3887-92. Epub 2016 Mar 29.

Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Forchheimer Building, Room 411, Bronx, NY, 10461, USA.

The release of cellular factors by means of extracellular vesicles (EVs) is conserved in archaea, bacteria, and eukaryotes. EVs are released by growing bacteria as part of their interaction with their environment and, for pathogenic bacteria, constitute an important component of their interactions with the host. While EVs released by gram-negative bacteria have been extensively studied, the vesicles released by thick cell wall microorganisms like mycobacteria were recognized only recently and are less well understood. Nonetheless, studies of mycobacterial EVs have already suggested roles in pathogenesis, opening exciting new avenues of research aimed at understanding their biogenesis and potential use in antitubercular strategies. In this minireview, we discuss the discovery of mycobacterial vesicles, the current understanding of their nature, content, regulation, and possible functions, as well as their potential therapeutic applications.
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http://dx.doi.org/10.1007/s00253-016-7484-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4879809PMC
May 2016

Activation of Melanin Synthesis in Alternaria infectoria by Antifungal Drugs.

Antimicrob Agents Chemother 2015 Dec 28;60(3):1646-55. Epub 2015 Dec 28.

CNC-Centre for Neurosciences and Cell Biology, University of Coimbra, Coimbra, Portugal FMUC-Faculty of Medicine, University of Coimbra, Coimbra, Portugal

The importance of Alternaria species fungi to human health ranges from their role as etiological agents of serious infections with poor prognoses in immunosuppressed individuals to their association with respiratory allergic diseases. The present work focuses on Alternaria infectoria, which was used as a model organism of the genus, and was designed to unravel melanin production in response to antifungals. After we characterized the pigment produced by A. infectoria, we studied the dynamics of 1,8-dihydroxynaphthalene (DHN)-melanin production during growth, the degree of melanization in response to antifungals, and how melanization affected susceptibility to several classes of therapeutic drugs. We demonstrate that A. infectoria increased melanin deposition in cell walls in response to nikkomycin Z, caspofungin, and itraconazole but not in response to fluconazole or amphotericin B. These results indicate that A. infectoria activates DHN-melanin synthesis in response to certain antifungal drugs, possibly as a protective mechanism against these drugs. Inhibition of DHN-melanin synthesis by pyroquilon resulted in a lower minimum effective concentration (MEC) of caspofungin and enhanced morphological changes (increased hyphal balloon size), characterized by thinner and less organized A. infectoria cell walls. In summary, A. infectoria synthesizes melanin in response to certain antifungal drugs, and its susceptibility is influenced by melanization, suggesting the therapeutic potential of drug combinations that affect melanin synthesis.
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http://dx.doi.org/10.1128/AAC.02190-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4775914PMC
December 2015

Through the wall: extracellular vesicles in Gram-positive bacteria, mycobacteria and fungi.

Nat Rev Microbiol 2015 Oct 1;13(10):620-30. Epub 2015 Sep 1.

Johns Hopkins Bloomberg School of Public Health, 615 North Wolfe Street, Baltimore, Maryland 21205, USA.

Extracellular vesicles (EVs) are produced by all domains of life. In Gram-negative bacteria, EVs are produced by the pinching off of the outer membrane; however, how EVs escape the thick cell walls of Gram-positive bacteria, mycobacteria and fungi is still unknown. Nonetheless, EVs have been described in a variety of cell-walled organisms, including Staphylococcus aureus, Mycobacterium tuberculosis and Cryptococcus neoformans. These EVs contain varied cargo, including nucleic acids, toxins, lipoproteins and enzymes, and have important roles in microbial physiology and pathogenesis. In this Review, we describe the current status of vesiculogenesis research in thick-walled microorganisms and discuss the cargo and functions associated with EVs in these species.
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http://dx.doi.org/10.1038/nrmicro3480DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4860279PMC
October 2015

Structural Characterization of Melanin Pigments from Commercial Preparations of the Edible Mushroom Auricularia auricula.

J Agric Food Chem 2015 Aug 11;63(33):7326-32. Epub 2015 Aug 11.

Goodwin and Wells , New York, New York 10065, United States.

Many of the most widely consumed edible mushrooms are pigmented, and these have been associated with some beneficial health effects. Nevertheless, the majority of the reported compounds associated with these desirable properties are non-pigmented. We have previously reported that melanin pigment from the edible mushroom Auricularia auricula can protect mice against ionizing radiation, although no physicochemical characterization was reported. Consequently, in this study we have characterized commercial A. auricula mushroom preparations for melanin content and carried out structural characterization of isolated insoluble melanin materials using a panel of sophisticated spectroscopic and physical/imaging techniques. Our results show that approximately 10% of the dry mass of A. auricula is melanin and that the pigment has physicochemical properties consistent with those of eumelanins, including hosting a stable free radical population. Electron microscopy studies show that melanin is associated with the mushroom cell wall in a manner similar to that of melanin from the model fungus C. neoformans. Elemental analysis of melanin indicated C, H, and N ratios consistent with 5,6-dihydroxyindole-2-carboxylic acid/5,6-dihydroxyindole and 1,8-dihydroxynaphthalene eumelanin. Validation of the identity of the isolated product as melanin was achieved by EPR analysis. A. auricula melanin manifested structural differences, relative to the C. neoformans melanin, with regard to the variable proportions of alkyl chains or oxygenated carbons. Given the necessity for new oral and inexpensive radioprotective materials coupled with the commercial availability of A. auricula mushrooms, this product may represent an excellent source of edible melanin.
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http://dx.doi.org/10.1021/acs.jafc.5b02713DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4862413PMC
August 2015

Solid-state NMR Reveals the Carbon-based Molecular Architecture of Cryptococcus neoformans Fungal Eumelanins in the Cell Wall.

J Biol Chem 2015 May 30;290(22):13779-90. Epub 2015 Mar 30.

From the Department of Chemistry, City College of New York, Graduate Center and Institute for Macromolecular Assemblies, City University of New York, New York, New York 10031-9101,

Melanin pigments protect against both ionizing radiation and free radicals and have potential soil remediation capabilities. Eumelanins produced by pathogenic Cryptococcus neoformans fungi are virulence factors that render the fungal cells resistant to host defenses and certain antifungal drugs. Because of their insoluble and amorphous characteristics, neither the pigment bonding framework nor the cellular interactions underlying melanization of C. neoformans have yielded to comprehensive molecular-scale investigation. This study used the C. neoformans requirement of exogenous obligatory catecholamine precursors for melanization to produce isotopically enriched pigment "ghosts" and applied 2D (13)C-(13)C correlation solid-state NMR to reveal the carbon-based architecture of intact natural eumelanin assemblies in fungal cells. We demonstrated that the aliphatic moieties of solid C. neoformans melanin ghosts include cell-wall components derived from polysaccharides and/or chitin that are associated proximally with lipid membrane constituents. Prior to development of the mature aromatic fungal pigment, these aliphatic moieties form a chemically resistant framework that could serve as the scaffold for melanin synthesis. The indole-based core aromatic moieties show interconnections that are consistent with proposed melanin structures consisting of stacked planar assemblies, which are associated spatially with the aliphatic scaffold. The pyrrole aromatic carbons of the pigments bind covalently to the aliphatic framework via glycoside or glyceride functional groups. These findings establish that the structure of the pigment assembly changes with time and provide the first biophysical information on the mechanism by which melanin is assembled in the fungal cell wall, offering vital insights that can advance the design of bioinspired conductive nanomaterials and novel therapeutics.
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http://dx.doi.org/10.1074/jbc.M114.618389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4447955PMC
May 2015
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