Publications by authors named "Juan Anguita"

93 Publications

Peripheral blood mononuclear cells (PBMC) microbiome is not affected by colon microbiota in healthy goats.

Anim Microbiome 2021 Apr 14;3(1):28. Epub 2021 Apr 14.

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

Background: The knowledge about blood circulating microbiome and its functional relevance in healthy individuals remains limited. An assessment of changes in the circulating microbiome was performed by sequencing peripheral blood mononuclear cells (PBMC) bacterial DNA from goats supplemented or not in early life with rumen liquid transplantation.

Results: Most of the bacterial DNA associated to PBMC was identified predominantly as Proteobacteria (55%) followed by Firmicutes (24%), Bacteroidetes (11%) and Actinobacteria (8%). The predominant genera found in PBMC samples were Pseudomonas, Prevotella, Sphingomonas, Acinetobacter, Corynebacterium and Ruminococcus. Other genera such as Butyrivibrivio, Bifidobacterium, Dorea and Coprococcus were also present in lower proportions. Several species known as blood pathogens or others involved in gut homeostasis such as Faecalibacterium prausnitzii were also identified. However, the PBMC microbiome phylum composition differed from that in the colon of goats (P ≤ 0.001), where Firmicutes was the predominant phylum (83%). Although, rumen liquid administration in early-life altered bacterial community structure and increased Tlr5 expression (P = 0.020) in colon pointing to higher bacterial translocation, less than 8% of OTUs in colon were also observed in PBMCs.

Conclusions: Data suggest that in physiological conditions, PBMC microbiome differs from and is not affected by colon gut microbiota in small ruminants. Although, further studies with larger number of animals and covering other animal tissues are required, results point to a common circulating bacterial profile on mammals being phylum Proteobacteria, and genera Pseudomonas and Prevotella the most abundants. All suggest that PBMC microbiome in healthy ruminants could be implicated in homeostatic condition. This study expands our knowledge about PBMC microbiome contribution to health in farm animals.
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http://dx.doi.org/10.1186/s42523-021-00091-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8048065PMC
April 2021

Bovine Neutrophils Release Extracellular Traps and Cooperate With Macrophages in subsp. clearance .

Front Immunol 2021 17;12:645304. Epub 2021 Mar 17.

Animal Health Department, NEIKER-Basque Institute for Agricultural Research and Development-Basque Research and Technology Allianca (BRTA), Derio, Spain.

subsp. (Map) is the underlying pathogen causing bovine paratuberculosis (PTB), an enteric granulomatous disease that mainly affects ruminants and for which an effective treatment is needed. Macrophages are the primary target cells for Map, which survives and replicates intracellularly by inhibiting phagosome maturation. Neutrophils are present at disease sites during the early stages of the infection, but seem to be absent in the late stage, in contrast to healthy tissue. Although neutrophil activity has been reported to be impaired following Map infection, their role in PTB pathogenesis has not been fully defined. Neutrophils are capable of releasing extracellular traps consisting of extruded DNA and proteins that immobilize and kill microorganisms, but this mechanism has not been evaluated against Map. Our main objective was to study the interaction of neutrophils with macrophages during an mycobacterial infection. For this purpose, neutrophils and macrophages from the same animal were cultured alone or together in the presence of Map or Bacillus-Calmette-Guérin (BCG). Extracellular trap release, mycobacteria killing as well as IL-1β and IL-8 release were assessed. Neutrophils released extracellular traps against mycobacteria when cultured alone and in the presence of macrophages without direct cell contact, but resulted inhibited in direct contact. Macrophages were extremely efficient at killing BCG, but ineffective at killing Map. In contrast, neutrophils showed similar killing rates for both mycobacteria. Co-cultures infected with Map showed the expected killing effect of combining both cell types, whereas co-cultures infected with BCG showed a potentiated killing effect beyond the expected one, indicating a potential synergistic cooperation. In both cases, IL-1β and IL-8 levels were lower in co-cultures, suggestive of a reduced inflammatory reaction. These data indicate that cooperation of both cell types can be beneficial in terms of decreasing the inflammatory reaction while the effective elimination of Map can be compromised. These results suggest that neutrophils are effective at Map killing and can exert protective mechanisms against Map that seem to fail during PTB disease after the arrival of macrophages at the infection site.
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http://dx.doi.org/10.3389/fimmu.2021.645304DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010319PMC
March 2021

Borrelia burgdorferi infection induces long-term memory-like responses in macrophages with tissue-wide consequences in the heart.

PLoS Biol 2021 01 4;19(1):e3001062. Epub 2021 Jan 4.

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

Lyme carditis is an extracutaneous manifestation of Lyme disease characterized by episodes of atrioventricular block of varying degrees and additional, less reported cardiomyopathies. The molecular changes associated with the response to Borrelia burgdorferi over the course of infection are poorly understood. Here, we identify broad transcriptomic and proteomic changes in the heart during infection that reveal a profound down-regulation of mitochondrial components. We also describe the long-term functional modulation of macrophages exposed to live bacteria, characterized by an augmented glycolytic output, increased spirochetal binding and internalization, and reduced inflammatory responses. In vitro, glycolysis inhibition reduces the production of tumor necrosis factor (TNF) by memory macrophages, whereas in vivo, it produces the reversion of the memory phenotype, the recovery of tissue mitochondrial components, and decreased inflammation and spirochetal burdens. These results show that B. burgdorferi induces long-term, memory-like responses in macrophages with tissue-wide consequences that are amenable to be manipulated in vivo.
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http://dx.doi.org/10.1371/journal.pbio.3001062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7808612PMC
January 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

A structurally unique Fusobacterium nucleatum tannase provides detoxicant activity against gallotannins and pathogen resistance.

Microb Biotechnol 2020 Dec 18. Epub 2020 Dec 18.

Inflammation and Macrophage Plasticity lab, CIC bioGUNE-BRTA (Basque Research and Technology Alliance), Derio, 48160, Spain.

Colorectal cancer pathogenesis and progression is associated with the presence of Fusobacterium nucleatum and the reduction of acetylated derivatives of spermidine, as well as dietary components such as tannin-rich foods. We show that a new tannase orthologue of F. nucleatum (TanBF ) has significant structural differences with its Lactobacillus plantarum counterpart affecting the flap covering the active site and the accessibility of substrates. Crystallographic and molecular dynamics analysis revealed binding of polyamines to a small cavity that connects the active site with the bulk solvent which interact with catalytically indispensable residues. As a result, spermidine and its derivatives, particularly N -acetylated spermidine, inhibit the hydrolytic activity of TanBF and increase the toxicity of gallotannins to F. nucleatum. Our results support a model in which the balance between the detoxicant activity of TanBF and the presence of metabolic inhibitors can dictate either conducive or unfavourable conditions for the survival of F. nucleatum.
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http://dx.doi.org/10.1111/1751-7915.13732DOI Listing
December 2020

Replacing the Rhamnose-Xylose Moiety of QS-21 with Simpler Terminal Disaccharide Units Attenuates Adjuvant Activity in Truncated Saponin Variants.

Chemistry 2021 Mar 4;27(14):4731-4737. Epub 2021 Feb 4.

Chemical Immunology Laboratory, Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Biscay Science and Technology Park, Building 801A, 48160, Derio, Spain.

Adjuvants are key immunostimulatory components in vaccine formulations, which improve the immune response to the co-administered antigen. The saponin natural product QS-21 is one of the most promising immunoadjuvants in the development of vaccines against cancer and infectious diseases but suffers from limitations that have hampered its widespread human use. Previous structure-activity relationship studies have identified simplified saponin variants with truncated carbohydrate chains, but have not focused on the influence of the linear oligosaccharide domain of QS-21 in adjuvant activity. Herein, an expeditious 15-step synthesis of new linear trisaccharide variants of simplified QS-21-derived adjuvants is reported, in which the complex terminal xylose-rhamnose moiety has been replaced with commercially available, simpler lactose and cellobiose disaccharides in a β-anomeric configuration. In vivo immunological evaluation of the synthetic saponins showed attenuated antibody responses, highlighting the negative impact of such carbohydrate modifications on adjuvant activity, which could be associated with higher saponin conformational flexibility.
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http://dx.doi.org/10.1002/chem.202004705DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7986102PMC
March 2021

A combined transcriptomic approach to identify candidates for an anti-tick vaccine blocking B. afzelii transmission.

Sci Rep 2020 11 18;10(1):20061. Epub 2020 Nov 18.

Center for Experimental and Molecular Medicine, Amsterdam Infection and Immunity, Amsterdam UMC, Location Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

Ixodes ricinus is the vector for Borrelia afzelii, the predominant cause of Lyme borreliosis in Europe, whereas Ixodes scapularis is the vector for Borrelia burgdorferi in the USA. Transcription of several I. scapularis genes changes in the presence of B. burgdorferi and contributes to successful infection. To what extend B. afzelii influences gene expression in I. ricinus salivary glands is largely unknown. Therefore, we measured expression of uninfected vs. infected tick salivary gland genes during tick feeding using Massive Analysis of cDNA Ends (MACE) and RNAseq, quantifying 26.179 unique transcripts. While tick feeding was the main differentiator, B. afzelii infection significantly affected expression of hundreds of transcripts, including 465 transcripts after 24 h of tick feeding. Validation of the top-20 B. afzelii-upregulated transcripts at 24 h of tick feeding in ten biological genetic distinct replicates showed that expression varied extensively. Three transcripts could be validated, a basic tail protein, a lipocalin and an ixodegrin, and might be involved in B. afzelii transmission. However, vaccination with recombinant forms of these proteins only marginally altered B. afzelii infection in I. ricinus-challenged mice for one of the proteins. Collectively, our data show that identification of tick salivary genes upregulated in the presence of pathogens could serve to identify potential pathogen-blocking vaccine candidates.
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http://dx.doi.org/10.1038/s41598-020-76268-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7674437PMC
November 2020

Lessons from Bacillus Calmette-Guérin: Harnessing Trained Immunity for Vaccine Development.

Cells 2020 09 16;9(9). Epub 2020 Sep 16.

Inflammation and Macrophage Plasticity Laboratory, CIC bioGUNE-Basque Research and Technology Alliance (BRTA), Parque Tecnológico de Bizkaia, 48160 Derio, Spain.

Vaccine design traditionally focuses on inducing adaptive immune responses against a sole target pathogen. Considering that many microbes evade innate immune mechanisms to initiate infection, and in light of the discovery of epigenetically mediated innate immune training, the paradigm of vaccine design has the potential to change. The Bacillus Calmette-Guérin (BCG) vaccine induces some level of protection against Mycobacterium tuberculosis (Mtb) while stimulating trained immunity that correlates with lower mortality and increased protection against unrelated pathogens. This review will explore BCG-induced trained immunity, including the required pathways to establish this phenotype. Additionally, potential methods to improve or expand BCG trained immunity effects through alternative vaccine delivery and formulation methods will be discussed. Finally, advances in new anti-Mtb vaccines, other antimicrobial uses for BCG, and "innate memory-based vaccines" will be examined.
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http://dx.doi.org/10.3390/cells9092109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564904PMC
September 2020

Silencing hepatic MCJ attenuates non-alcoholic fatty liver disease (NAFLD) by increasing mitochondrial fatty acid oxidation.

Nat Commun 2020 07 3;11(1):3360. Epub 2020 Jul 3.

Department of Medicine, Immunobiology Division, University of Vermont, Burlington, VT, 05405, USA.

Nonalcoholic fatty liver disease (NAFLD) is considered the next major health epidemic with an estimated 25% worldwide prevalence. No drugs have yet been approved and NAFLD remains a major unmet need. Here, we identify MCJ (Methylation-Controlled J protein) as a target for non-alcoholic steatohepatitis (NASH), an advanced phase of NAFLD. MCJ is an endogenous negative regulator of the respiratory chain Complex I that acts to restrain mitochondrial respiration. We show that therapeutic targeting of MCJ in the liver with nanoparticle- and GalNAc-formulated siRNA efficiently reduces liver lipid accumulation and fibrosis in multiple NASH mouse models. Decreasing MCJ expression enhances the capacity of hepatocytes to mediate β-oxidation of fatty acids and minimizes lipid accumulation, which results in reduced hepatocyte damage and fibrosis. Moreover, MCJ levels in the liver of NAFLD patients are elevated relative to healthy subjects. Thus, inhibition of MCJ emerges as an alternative approach to treat NAFLD.
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http://dx.doi.org/10.1038/s41467-020-16991-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7334216PMC
July 2020

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

Ixodes scapularis saliva components that elicit responses associated with acquired tick-resistance.

Ticks Tick Borne Dis 2020 05 3;11(3):101369. Epub 2020 Jan 3.

Section of Infectious Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06420, United States; Howard Hughes Medical Institute. Chevy Chase, MD, 20815, United States. Electronic address:

Ticks and tick-borne diseases are on the rise world-wide and vaccines to prevent transmission of tick-borne diseases is an urgent public health need. Tick transmission of pathogens to the mammalian host occurs during tick feeding. Therefore, it is reasoned that vaccine targeting of tick proteins essential for feeding would thwart tick feeding and consequently prevent pathogen transmission. The phenomenon of acquired tick-immunity, wherein, repeated tick infestations of non-natural hosts results in the development of host immune responses detrimental to tick feeding has served as a robust paradigm in the pursuit of tick salivary antigens that may be vaccine targeted. While several salivary antigens have been identified, immunity elicited against these antigens have only provided modest tick rejection. This has raised the possibility that acquired tick-immunity is directed against tick components other than tick salivary antigens. Using Ixodes scapularis, the blacklegged tick, that vectors several human pathogens, we demonstrate that immunity directed against tick salivary glycoproteins is indeed sufficient to recapitulate the phenomenon of tick-resistance. These observations emphasize the utility of tick salivary glycoproteins as viable vaccine targets to thwart tick feeding and direct our search for anti-tick vaccine candidates.
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http://dx.doi.org/10.1016/j.ttbdis.2019.101369DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7382422PMC
May 2020

Exploiting structure-activity relationships of QS-21 in the design and synthesis of streamlined saponin vaccine adjuvants.

Chem Commun (Camb) 2020 Jan;56(5):719-722

Chemical Immunology Lab, Centre for Cooperative Research in Biosciences, CIC-bioGUNE, 48160 Derio, Biscay, Spain.

We report the design, synthesis, immunological evaluation, and conformational analysis of new saponin variants as promising vaccine adjuvants. These studies have provided expedient synthetic access to streamlined adjuvant-active saponins and yielded molecular-level insights into saponin conformation that correlated with their in vivo adjuvant activities.
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http://dx.doi.org/10.1039/c9cc07781bDOI Listing
January 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

miR-873-5p targets mitochondrial GNMT-Complex II interface contributing to non-alcoholic fatty liver disease.

Mol Metab 2019 11 16;29:40-54. Epub 2019 Aug 16.

Liver disease Laboratory, Liver metabolism Laboratory, CIC bioGUNE, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), 48160, Derio, Bizkaia, Spain. Electronic address:

Objective: Non-alcoholic fatty liver disease (NAFLD) is a complex pathology in which several dysfunctions, including alterations in metabolic pathways, mitochondrial functionality and unbalanced lipid import/export, lead to lipid accumulation and progression to inflammation and fibrosis. The enzyme glycine N-methyltransferase (GNMT), the most important enzyme implicated in S-adenosylmethionine catabolism in the liver, is downregulated during NAFLD progression. We have studied the mechanism involved in GNMT downregulation by its repressor microRNA miR-873-5p and the metabolic pathways affected in NAFLD as well as the benefit of recovery GNMT expression.

Methods: miR-873-5p and GNMT expression were evaluated in liver biopsies of NAFLD/NASH patients. Different in vitro and in vivo NAFLD murine models were used to assess miR-873-5p/GNMT involvement in fatty liver progression through targeting of the miR-873-5p as NAFLD therapy.

Results: We describe a new function of GNMT as an essential regulator of Complex II activity in the electron transport chain in the mitochondria. In NAFLD, GNMT expression is controlled by miR-873-5p in the hepatocytes, leading to disruptions in mitochondrial functionality in a preclinical murine non-alcoholic steatohepatitis (NASH) model. Upregulation of miR-873-5p is shown in the liver of NAFLD/NASH patients, correlating with hepatic GNMT depletion. Importantly, NASH therapies based on anti-miR-873-5p resolve lipid accumulation, inflammation and fibrosis by enhancing fatty acid β-oxidation in the mitochondria. Therefore, miR-873-5p inhibitor emerges as a potential tool for NASH treatment.

Conclusion: GNMT participates in the regulation of metabolic pathways and mitochondrial functionality through the regulation of Complex II activity in the electron transport chain. In NAFLD, GNMT is repressed by miR-873-5p and its targeting arises as a valuable therapeutic option for treatment.
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http://dx.doi.org/10.1016/j.molmet.2019.08.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6728756PMC
November 2019

Gut microbiome and serum metabolome analyses identify molecular biomarkers and altered glutamate metabolism in fibromyalgia.

EBioMedicine 2019 Aug 18;46:499-511. Epub 2019 Jul 18.

Exosomes Laboratory, CIC bioGUNE, CIBERehd, Bizkaia Technology Park, Derio, Spain; Metabolomics Platform, CIC bioGUNE, CIBERehd, Bizkaia Technology Park, Derio, Spain; IKERBASQUE, Basque Foundation for Science, Bilbao, Spain. Electronic address:

Background: Fibromyalgia is a complex, relatively unknown disease characterised by chronic, widespread musculoskeletal pain. The gut-brain axis connects the gut microbiome with the brain through the enteric nervous system (ENS); its disruption has been associated with psychiatric and gastrointestinal disorders. To gain an insight into the pathogenesis of fibromyalgia and identify diagnostic biomarkers, we combined different omics techniques to analyse microbiome and serum composition.

Methods: We collected faeces and blood samples to study the microbiome, the serum metabolome and circulating cytokines and miRNAs from a cohort of 105 fibromyalgia patients and 54 age- and environment-matched healthy individuals. We sequenced the V3 and V4 regions of the 16S rDNA gene from faeces samples. UPLC-MS metabolomics and custom multiplex cytokine and miRNA analysis (FirePlex™ technology) were used to examine sera samples. Finally, we combined the different data types to search for potential biomarkers.

Results: We found that the diversity of bacteria is reduced in fibromyalgia patients. The abundance of the Bifidobacterium and Eubacterium genera (bacteria participating in the metabolism of neurotransmitters in the host) in these patients was significantly reduced. The serum metabolome analysis revealed altered levels of glutamate and serine, suggesting changes in neurotransmitter metabolism. The combined serum metabolomics and gut microbiome datasets showed a certain degree of correlation, reflecting the effect of the microbiome on metabolic activity. We also examined the microbiome and serum metabolites, cytokines and miRNAs as potential sources of molecular biomarkers of fibromyalgia.

Conclusions: Our results show that the microbiome analysis provides more significant biomarkers than the other techniques employed in the work. Gut microbiome analysis combined with serum metabolomics can shed new light onto the pathogenesis of fibromyalgia. We provide a list of bacteria whose abundance changes in this disease and propose several molecules as potential biomarkers that can be used to evaluate the current diagnostic criteria.
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http://dx.doi.org/10.1016/j.ebiom.2019.07.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6710987PMC
August 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

Counterattacking the tick bite: towards a rational design of anti-tick vaccines targeting pathogen transmission.

Parasit Vectors 2019 May 14;12(1):229. Epub 2019 May 14.

Amsterdam UMC, Location AMC, Center for Experimental and Molecular Medicine, Amsterdam, The Netherlands.

Hematophagous arthropods are responsible for the transmission of a variety of pathogens that cause disease in humans and animals. Ticks of the Ixodes ricinus complex are vectors for some of the most frequently occurring human tick-borne diseases, particularly Lyme borreliosis and tick-borne encephalitis virus (TBEV). The search for vaccines against these diseases is ongoing. Efforts during the last few decades have primarily focused on understanding the biology of the transmitted viruses, bacteria and protozoans, with the goal of identifying targets for intervention. Successful vaccines have been developed against TBEV and Lyme borreliosis, although the latter is no longer available for humans. More recently, the focus of intervention has shifted back to where it was initially being studied which is the vector. State of the art technologies are being used for the identification of potential vaccine candidates for anti-tick vaccines that could be used either in humans or animals. The study of the interrelationship between ticks and the pathogens they transmit, including mechanisms of acquisition, persistence and transmission have come to the fore, as this knowledge may lead to the identification of critical elements of the pathogens' life-cycle that could be targeted by vaccines. Here, we review the status of our current knowledge on the triangular relationships between ticks, the pathogens they carry and the mammalian hosts, as well as methods that are being used to identify anti-tick vaccine candidates that can prevent the transmission of tick-borne pathogens.
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http://dx.doi.org/10.1186/s13071-019-3468-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6518728PMC
May 2019

Preliminary Evaluation of Tick Protein Extracts and Recombinant Ferritin 2 as Anti-tick Vaccines Targeting in Cattle.

Front Physiol 2018 5;9:1696. Epub 2018 Dec 5.

Institute for Parasitology and Tropical Veterinary Medicine, Freie Universität Berlin, Berlin, Germany.

Anti-tick vaccines have the potential to be an environmentally friendly and cost-effective option for tick control. In vaccine development, the identification of efficacious antigens forms the major bottleneck. In this study, the efficacy of immunization with recombinant ferritin 2 and native tick protein extracts (TPEs) against infestations in calves was assessed in two immunization experiments. In the first experiment, each calf ( = 3) was immunized twice with recombinant ferritin 2 from (IrFER2), TPE consisting of soluble proteins from the internal organs of partially fed females, or adjuvant, respectively. In the second experiment, each calf ( = 4) was immunized with protein extracts from the midgut (ME) of partially fed females, the salivary glands (SGE) of partially fed females, a combination of ME and SGE, or adjuvant, respectively. Two weeks after the booster immunization, calves were challenged with 100 females and 200 nymphs. Blood was collected from the calves before the first and after the second immunization and fed to females and nymphs using an artificial tick feeding system. The two calves vaccinated with whole TPE and midgut extract (ME) showed hyperemia on tick bite sites 2 days post tick infestation and exudative blisters were observed in the ME-vaccinated animal, signs that were suggestive of a delayed type hypersensitivity (DTH) reaction. Significantly fewer ticks successfully fed on the three animals vaccinated with TPE, SGE, or ME. Adults fed on the TPE and ME vaccinated animals weighed significantly less. Tick feeding on the IrFER2 vaccinated calf was not impaired. The feeding of serum or fresh whole blood collected from the vaccinated animals did not significantly affect tick feeding success. Immunization with native TPEs thus conferred a strong immune response in calves and significantly reduced the feeding success of both nymphs and adults. feeding of serum or blood collected from vaccinated animals to ticks did not affect tick feeding, indicating that antibodies alone were not responsible for the observed vaccine immunity.
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http://dx.doi.org/10.3389/fphys.2018.01696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6290058PMC
December 2018

Bacterial tannases: classification and biochemical properties.

Appl Microbiol Biotechnol 2019 Jan 20;103(2):603-623. Epub 2018 Nov 20.

Laboratorio de Biotecnología Bacteriana, Instituto de Ciencia y Tecnología de Alimentos y Nutrición, ICTAN-CSIC, Juan de la Cierva 3, 28006, Madrid, Spain.

Tannin acyl hydrolases, also known as tannases, are a group of enzymes critical for the transformation of tannins. The study of these enzymes, which initially evolved in different organisms to detoxify and/or use these plant metabolites, has nowadays become relevant in microbial enzymology research due to their relevant role in food tannin transformation. Microorganisms, particularly bacteria, are major sources of tannase. Cloning and heterologous expression of bacterial tannase genes and structural studies have been performed in the last few years. However, a systematic compilation of the information related to all recombinant tannases, their classification, and characteristics is missing. In this review, we explore the diversity of heterologously produced bacterial tannases, describing their substrate specificity and biochemical characterization. Moreover, a new classification based on sequence similarity analysis is proposed. Finally, putative tannases have been identified in silico for each group of tannases taking advantage of the use of the "tannase" distinctive features previously proposed.
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http://dx.doi.org/10.1007/s00253-018-9519-yDOI Listing
January 2019

MiR-873-5p acts as an epigenetic regulator in early stages of liver fibrosis and cirrhosis.

Cell Death Dis 2018 09 20;9(10):958. Epub 2018 Sep 20.

CIC bioGUNE, Centro de Investigación Cooperativa en Biociencias, Derio, Bizkaia, Spain.

Glycine N-methyltransferase (GNMT) is the most abundant methyltransferase in the liver and a master regulator of the transmethylation flux. GNMT downregulation leads to loss of liver function progressing to fibrosis, cirrhosis, and hepatocellular carcinoma. Moreover, GNMT deficiency aggravates cholestasis-induced fibrogenesis. To date, little is known about the mechanisms underlying downregulation of GNMT levels in hepatic fibrosis and cirrhosis. On this basis, microRNAs are epigenetic regulatory elements that play important roles in liver pathology. In this work, we aim to study the regulation of GNMT by microRNAs during liver fibrosis and cirrhosis. Luciferase assay on the 3'UTR-Gnmt was used to confirm in silico analysis showing that GNMT is potentially targeted by the microRNA miR-873-5p. Correlation between GNMT and miR-873-5p in human cholestasis and cirrhosis together with miR-873-5p inhibition in vivo in different mouse models of liver cholestasis and fibrosis [bile duct ligation and Mdr2 (Abcb4) mouse] were then assessed. The analysis of liver tissue from cirrhotic and cholestatic patients, as well as from the animal models, showed that miR-873-5p inversely correlated with the expression of GNMT. Importantly, high circulating miR-873-5p was also detected in cholestastic and cirrhotic patients. Preclinical studies with anti-miR-873-5p treatment in bile duct ligation and Mdr2 mice recovered GNMT levels in association with ameliorated inflammation and fibrosis mainly by counteracting hepatocyte apoptosis and cholangiocyte proliferation. In conclusion, miR-873-5p emerges as a novel marker for liver fibrosis, cholestasis, and cirrhosis and therapeutic approaches based on anti-miR-873-5p may be effective treatments for liver fibrosis and cholestatic liver disease.
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http://dx.doi.org/10.1038/s41419-018-1014-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6148053PMC
September 2018

Repurposing ciclopirox as a pharmacological chaperone in a model of congenital erythropoietic porphyria.

Sci Transl Med 2018 09;10(459)

Protein Stability and Inherited Disease Laboratory, CIC bioGUNE, 48160 Derio, Spain.

Congenital erythropoietic porphyria is a rare autosomal recessive disease produced by deficient activity of uroporphyrinogen III synthase, the fourth enzyme in the heme biosynthetic pathway. The disease affects many organs, can be life-threatening, and currently lacks curative treatments. Inherited mutations most commonly reduce the enzyme's stability, altering its homeostasis and ultimately blunting intracellular heme production. This results in uroporphyrin by-product accumulation in the body, aggravating associated pathological symptoms such as skin photosensitivity and disfiguring phototoxic cutaneous lesions. We demonstrated that the synthetic marketed antifungal ciclopirox binds to the enzyme, stabilizing it. Ciclopirox targeted the enzyme at an allosteric site distant from the active center and did not affect the enzyme's catalytic role. The drug restored enzymatic activity in vitro and ex vivo and was able to alleviate most clinical symptoms of congenital erythropoietic porphyria in a genetic mouse model of the disease at subtoxic concentrations. Our findings establish a possible line of therapeutic intervention against congenital erythropoietic porphyria, which is potentially applicable to most of deleterious missense mutations causing this devastating disease.
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http://dx.doi.org/10.1126/scitranslmed.aat7467DOI Listing
September 2018

CD8 T Cell Responses to an Immunodominant Epitope within the Nonstructural Protein NS1 Provide Wide Immunoprotection against Bluetongue Virus in IFNAR Mice.

J Virol 2018 08 31;92(16). Epub 2018 Jul 31.

Center for Animal Health Research, INIA-CISA, Valdeolmos, Madrid, Spain

The development of vaccines against bluetongue, a prevalent livestock disease, has been focused on surface antigens that induce strong neutralizing antibody responses. Because of their antigenic variability, these vaccines are usually serotype restricted. We now show that a single highly conserved nonstructural protein, NS1, expressed in a modified vaccinia Ankara virus (MVA) vector can provide multiserotype protection in IFNAR 129 mice against bluetongue virus (BTV) that is largely dependent on CD8 T cell responses. We found that the protective antigenic capacity of NS1 resides within the N terminus of the protein and is provided in the absence of neutralizing antibodies. The protective CD8 T cell response requires the presence of a specific peptide within the N terminus of NS1, since its deletion ablates the efficacy of the vaccine formulation. These data reveal the importance of the nonstructural protein NS1 in CD8 T cell-mediated protection against multiple BTV serotypes when vectorized as a recombinant MVA vaccine. Conventional vaccines have controlled or limited BTV expansion in the past, but they cannot address the need for cross-protection among serotypes and do not allow distinguishing between infected and vaccinated animals (DIVA strategy). There is a need to develop universal vaccines that induce effective protection against multiple BTV serotypes. In this work we have shown the importance of the nonstructural protein NS1, conserved among all the BTV serotypes, in CD8 T cell-mediated protection against multiple BTV serotypes when vectorized as a recombinant MVA vaccine.
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http://dx.doi.org/10.1128/JVI.00938-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6069212PMC
August 2018

Plasticity in early immune evasion strategies of a bacterial pathogen.

Proc Natl Acad Sci U S A 2018 04 2;115(16):E3788-E3797. Epub 2018 Apr 2.

Department of Veterinary Medicine, University of Maryland, College Park, MD 20742;

is one of the few extracellular pathogens capable of establishing persistent infection in mammals. The mechanisms that sustain long-term survival of this bacterium are largely unknown. Here we report a unique innate immune evasion strategy of , orchestrated by a surface protein annotated as BBA57, through its modulation of multiple spirochete virulent determinants. BBA57 function is critical for early infection but largely redundant for later stages of spirochetal persistence, either in mammals or in ticks. The protein influences host IFN responses as well as suppresses multiple host microbicidal activities involving serum complement, neutrophils, and antimicrobial peptides. We also discovered a remarkable plasticity in BBA57-mediated spirochete immune evasion strategy because its loss, although resulting in near clearance of pathogens at the inoculum site, triggers nonheritable adaptive changes that exclude detectable nucleotide alterations in the genome but incorporate transcriptional reprograming events. Understanding the malleability in spirochetal immune evasion mechanisms that ensures their host persistence is critical for the development of novel therapeutic and preventive approaches to combat long-term infections like Lyme borreliosis.
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http://dx.doi.org/10.1073/pnas.1718595115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5910839PMC
April 2018

Microglial immune response is impaired against the neurotropic fungus Lomentospora prolificans.

Cell Microbiol 2018 08 22;20(8):e12847. Epub 2018 Apr 22.

Fungal and Bacterial Biomics Research Group, Department of Immunology, Microbiology and Parasitology, Faculty of Science and Technology, University of the Basque Country (UPV/EHU), Leioa, Spain.

Lomentospora (Scedosporium) prolificans is an opportunistic pathogen capable of causing invasive infections in immunocompromised patients. The fungus is able to disseminate via the bloodstream finally arriving at the central nervous system producing neurological symptoms and, in many cases, patient death. In this context, microglial cells, which are the resident immune cells in the central nervous system, may play an important role in these infections. However, this aspect of anti-L. prolificans immunity has been poorly researched to date. Thus, the interactions and activity of microglial cells against L. prolificans were analysed, and the results show that there was a remarkable impairment in their performance regarding phagocytosis, the development of oxidative burst, and in the production of pro-inflammatory cytokines, compared with macrophages. Interestingly, L. prolificans displays great growth also when challenged with immune cells, even when inside them. We also proved that microglial phagocytosis of the fungus is highly dependent on mannose receptor and especially on dectin-1. Taken together, these data provide evidence for an impaired microglial response against L. prolificans and contribute to understanding the pathobiology of its neurotropism.
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http://dx.doi.org/10.1111/cmi.12847DOI Listing
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

Identification of a highly active tannase enzyme from the oral pathogen Fusobacterium nucleatum subsp. polymorphum.

Microb Cell Fact 2018 Feb 26;17(1):33. Epub 2018 Feb 26.

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

Background: Tannases are tannin-degrading enzymes that have been described in fungi and bacteria as an adaptative mechanism to overcome the stress conditions associated with the presence of these phenolic compounds.

Results: We have identified and expressed in E. coli a tannase from the oral microbiota member Fusobacterium nucleatum subs. polymorphum (TanB). TanB is the first tannase identified in an oral pathogen. Sequence analyses revealed that it is closely related to other bacterial tannases. The enzyme exhibits biochemical properties that make it an interesting target for industrial use. TanB has one of the highest specific activities of all bacterial tannases described to date and shows optimal biochemical properties such as a high thermal stability: the enzyme keeps 100% of its activity after prolonged incubations at different temperatures up to 45 °C. TanB also shows a wide temperature range of activity, maintaining above 80% of its maximum activity between 22 and 55 °C. The use of a panel of 27 esters of phenolic acids demonstrated activity of TanB only against esters of gallic and protocatechuic acid, including tannic acid, gallocatechin gallate and epigallocatechin gallate. Overall, TanB possesses biochemical properties that make the enzyme potentially useful in biotechnological applications.

Conclusions: We have identified and characterized a metabolic enzyme from the oral pathogen Fusobacterium nucleatum subsp. polymorphum. The biochemical properties of TanB suggest that it has a major role in the breakdown of complex food tannins during oral processing. Our results also provide some clues regarding its possible participation on bacterial survival in the oral cavity. Furthermore, the characteristics of this enzyme make it of potential interest for industrial use.
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http://dx.doi.org/10.1186/s12934-018-0880-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5828091PMC
February 2018

Quantum DNA Sequencing: A Peek Into a Dystopic Future?

Bioessays 2018 03 29;40(3). Epub 2018 Jan 29.

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

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http://dx.doi.org/10.1002/bies.201700248DOI Listing
March 2018