Publications by authors named "Rodrigo Pulgar"

21 Publications

  • Page 1 of 1

Tomato Cultivars With Variable Tolerances to Water Deficit Differentially Modulate the Composition and Interaction Patterns of Their Rhizosphere Microbial Communities.

Front Plant Sci 2021 13;12:688533. Epub 2021 Jul 13.

Laboratorio de Genómica y Genética de Interacciones Biológicas (LG2IB), Instituto de Nutrición y Tecnología de los Alimentos, Universidad de Chile, Santiago, Chile.

Since drought is the leading environmental factor limiting crop productivity, and plants have a significant impact in defining the assembly of plant-specific microbial communities associated with roots, we aimed to determine the effect of thoroughly selected water deficit tolerant and susceptible cultivars on their rhizosphere microbiome and compared their response with plant-free soil microbial communities. We identified a total of 4,248 bacterial and 276 fungal different operational taxonomic units (OTUs) in soils by massive sequencing. We observed that tomato cultivars significantly affected the alpha and beta diversity of their bacterial rhizosphere communities but not their fungal communities compared with bulk soils (BSs), showing a plant effect exclusively on the bacterial soil community. Also, an increase in alpha diversity in response to water deficit of both bacteria and fungi was observed in the susceptible rhizosphere (SRz) but not in the tolerant rhizosphere (TRz) cultivar, implying a buffering effect of the tolerant cultivar on its rhizosphere microbial communities. Even though water deficit did not affect the microbial diversity of the tolerant cultivar, the interaction network analysis revealed that the TRz microbiota displayed the smallest and least complex soil network in response to water deficit with the least number of connected components, nodes, and edges. This reduction of the TRz network also correlated with a more efficient community, reflected in increased cooperation within kingdoms. Furthermore, we identified some specific bacteria and fungi in the TRz in response to water deficit, which, given that they belong to taxa with known beneficial characteristics for plants, could be contributing to the tolerant phenotype, highlighting the metabolic bidirectionality of the holobiont system. Future assays involving characterization of root exudates and exchange of rhizospheres between drought-tolerant and susceptible cultivars could determine the effect of specific metabolites on the microbiome community and may elucidate their functional contribution to the tolerance of plants to water deficit.
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http://dx.doi.org/10.3389/fpls.2021.688533DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8313812PMC
July 2021

Increased dietary availability of selenium in rainbow trout (Oncorhynchus mykiss) improves its plasma antioxidant capacity and resistance to infection with Piscirickettsia salmonis.

Vet Res 2021 May 1;52(1):64. Epub 2021 May 1.

Laboratorio de Genómica y Genética de Interacciones Biológicas (LG2IB), Instituto de Nutrición y Tecnología de los Alimento, Universidad de Chile, El Líbano, Macul, 5524, Santiago, Chile.

Salmonid Rickettsial Septicaemia (SRS), caused by Piscirickettsia salmonis, is the most important infectious disease in the Chilean salmon farming industry. An opportunity to control this disease is to use functional micronutrients to modulate host mechanisms of response to the infection. Since P. salmonis may affect the host antioxidant system in salmonids, particularly that dependent on selenium (Se), we hypothesized that fish's dietary selenium supplementation could improve the response to the bacterial infection. To address this, we defined a non-antibiotic, non-cytotoxic concentration of selenium to evaluate its effect on the response to in vitro infections of SHK-1 cells with P. salmonis. The results indicated that selenium supplementation reduced the cytopathic effect, intracellular bacterial load, and cellular mortality of SHK-1 by increasing the abundance and activity of host glutathione peroxidase. We then prepared diets supplemented with selenium up to 1, 5, and 10 mg/kg to feed juvenile trout for 8 weeks. At the end of this feeding period, we obtained their blood plasma and evaluated its ability to protect SHK-1 cells from infection with P. salmonis in ex vivo assays. These results recapitulated the observed ability of selenium to protect against infection with P. salmonis by increasing the concentration of selenium and the antioxidant capacity in fish's plasma. To the best of our knowledge, this is the first report of the protective capacity of selenium against P. salmonis infection in salmonids, becoming a potential effective host-directed dietary therapy for SRS and other infectious diseases in animals at a non-antibiotic concentration.
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http://dx.doi.org/10.1186/s13567-021-00930-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8088010PMC
May 2021

Genistein Activates Transcription Factor EB and Corrects Niemann-Pick C Phenotype.

Int J Mol Sci 2021 Apr 19;22(8). Epub 2021 Apr 19.

Departament of Gastroenterology, School of Medicine, Pontificia Universidad Católica de Chile, Santiago 3580000, Chile.

Niemann-Pick type C disease (NPCD) is a lysosomal storage disease (LSD) characterized by abnormal cholesterol accumulation in lysosomes, impaired autophagy flux, and lysosomal dysfunction. The activation of transcription factor EB (TFEB), a master lysosomal function regulator, reduces the accumulation of lysosomal substrates in LSDs where the degradative capacity of the cells is compromised. Genistein can pass the blood-brain barrier and activate TFEB. Hence, we investigated the effect of TFEB activation by genistein toward correcting the NPC phenotype. We show that genistein promotes TFEB translocation to the nucleus in HeLa TFEB-GFP, Huh7, and SHSY-5Y cells treated with U18666A and NPC1 patient fibroblasts. Genistein treatment improved lysosomal protein expression and autophagic flux, decreasing p62 levels and increasing those of the LC3-II in NPC1 patient fibroblasts. Genistein induced an increase in β-hexosaminidase activity in the culture media of NPC1 patient fibroblasts, suggesting an increase in lysosomal exocytosis, which correlated with a decrease in cholesterol accumulation after filipin staining, including cells treated with U18666A and NPC1 patient fibroblasts. These results support that genistein-mediated TFEB activation corrects pathological phenotypes in NPC models and substantiates the need for further studies on this isoflavonoid as a potential therapeutic agent to treat NPCD and other LSDs with neurological compromise.
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http://dx.doi.org/10.3390/ijms22084220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8073251PMC
April 2021

Pharmacological iron-chelation as an assisted nutritional immunity strategy against Piscirickettsia salmonis infection.

Vet Res 2020 Oct 28;51(1):134. Epub 2020 Oct 28.

Laboratorio de Genómica y Genética de Interacciones Biológicas (LG2IB), Instituto de Nutrición y Tecnología de los Alimento, Universidad de Chile, Av. El Líbano 5524, Macul, 7830490, Santiago, Chile.

Salmonid Rickettsial Septicaemia (SRS), caused by Piscirickettsia salmonis, is a severe bacterial disease in the Chilean salmon farming industry. Vaccines and antibiotics are the current strategies to fight SRS; however, the high frequency of new epizootic events confirms the need to develop new strategies to combat this disease. An innovative opportunity is perturbing the host pathways used by the microorganisms to replicate inside host cells through host-directed antimicrobial drugs (HDAD). Iron is a critical nutrient for P. salmonis infection; hence, the use of iron-chelators becomes an excellent alternative to be used as HDAD. The aim of this work was to use the iron chelator Deferiprone (DFP) as HDAD to treat SRS. Here, we describe the protective effect of the iron chelator DFP over P. salmonis infections at non-antibiotic concentrations, in bacterial challenges both in vitro and in vivo. At the cellular level, our results indicate that DFP reduced the intracellular iron content by 33.1% and P. salmonis relative load during bacterial infections by 78%. These findings were recapitulated in fish, where DFP reduced the mortality of rainbow trout challenged with P. salmonis in 34.9% compared to the non-treated group. This is the first report of the protective capacity of an iron chelator against infection in fish, becoming a potential effective host-directed therapy for SRS and other animals against ferrophilic pathogens.
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http://dx.doi.org/10.1186/s13567-020-00845-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7592559PMC
October 2020

Microbial communities from arid environments on a global scale. A systematic review.

Biol Res 2020 Jul 6;53(1):29. Epub 2020 Jul 6.

Laboratorio de Bioinformática y Expresión Génica, Instituto de Nutrición y Tecnología de los Alimentos, Universidad de Chile, Santiago, Chile.

Arid environments are defined by the lack of water availability, which is directly related to the mean annual precipitation (MAP), and high values of solar irradiation, which impacts the community composition of animals, plants, and the microbial structure of the soil. Recent advances in NGS technologies have expanded our ability to characterize microbiomes, allowing environmental microbiologists to explore the complete microbial structure. Intending to identify and describe the state-of-the-art of bacterial communities in arid soils at a global scale, and to address the effect that some environmental features may have on them, we performed a systematic review based on the PRISMA guideline. Using a combination of keywords, we identified a collection of 66 studies, including 327 sampled sites, reporting the arid soil bacterial community composition by 16S rDNA gene high-throughput sequencing. To identify factors that can modulate bacterial communities, we extracted the geographical, environmental, and physicochemical data. The results indicate that even though each sampled site was catalogued as arid, they show wide variability in altitude, mean annual temperature (MAT), soil pH and electric conductivity, within and between arid environments. We show that arid soils display a higher abundance of Actinobacteria and lower abundance of Proteobacteria, Cyanobacteria, and Planctomycetes, compared with non-arid soil microbiomes, revealing that microbial structure seems to be strongly modulated by MAP and MAT and not by pH in arid soils. We observed that environmental and physicochemical features were scarcely described among studies, hence, we propose a reporting guideline for further analysis, which will allow deepening the knowledge of the relationship between the microbiome and abiotic factors in arid soil. Finally, to understand the academic collaborations landscape, we developed an analysis of the author's network, corroborating a low degree of connectivity and collaborations in this research topic. Considering that it is crucial to understand how microbial processes develop and change in arid soils, our analysis emphasizes the need to increase collaborations between research groups worldwide.
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http://dx.doi.org/10.1186/s40659-020-00296-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7336661PMC
July 2020

Fungal Diversity Analysis of Grape Musts from Central Valley-Chile and Characterization of Potential New Starter Cultures.

Microorganisms 2020 Jun 24;8(6). Epub 2020 Jun 24.

Laboratorio de Bioinformática y Expresión Génica, Instituto de Nutrición y Tecnología de los Alimentos (INTA), Universidad de Chile, Santiago 7810000, Chile.

Autochthonous microorganisms are an important source of the distinctive metabolites that influence the chemical profile of wine. However, little is known about the diversity of fungal communities associated with grape musts, even though they are the source of local yeast strains with potential capacities to become starters during fermentation. By using internal transcribed spacer (ITS) amplicon sequencing, we identified the taxonomic structure of the yeast community in unfermented and fermented musts of a typical L. var. Sauvignon blanc from the Central Valley of Chile throughout two consecutive seasons of production. Unsurprisingly, represented the most abundant fungal genus in unfermented and fermented musts, mainly due to the contribution of (42.7%) and (80%). Unfermented musts were highly variable between seasons and showed higher values of fungal diversity than fermented musts. Since microbial physiological characterization is primarily achieved in culture, we isolated nine species belonging to six genera of fungi from the unfermented must samples. All isolates were characterized for their potential capacities to be used as new starters in wine. Remarkably, only could co-exist with a commercial strain under fermentative conditions, representing a feasible candidate strain for wine production.
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http://dx.doi.org/10.3390/microorganisms8060956DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7356840PMC
June 2020

An acylhydroquinone derivative produces OXPHOS uncoupling and sensitization to BH3 mimetic ABT-199 (Venetoclax) in human promyelocytic leukemia cells.

Bioorg Chem 2020 07 18;100:103935. Epub 2020 May 18.

Instituto de Química de Recursos Naturales, Universidad de Talca, Chile; Network for Snake Venom Research and Drug Discovery, Santiago, Chile. Electronic address:

Since cancer cells have different mitochondrial bioenergetic requirements than non-cancerous cells, therapeutic inhibition of its mitochondrial functionality continues to be an important target for anticancer drug discovery. In this study, a series of acylhydroquinones with different acyl-chain length, and their chlorinated derivatives, in the aromatic ring, synthesized by Fries rearrangement under microwave irradiation, were evaluated for their anticancer activity in two leukemia cell lines. Findings from the primary and secondary screening of the 18 acylhydroquinones, tested at 5 µM on acute promyelocytic leukemia HL-60 and acute lymphoblastic leukemia CEM cells lines, identified an acylchlorohydroquinone (12) with a highly selective anti-proliferative effect toward HL-60 cells. This compound induced S-phase arrest in the cell cycle progression of HL-60 cells with insignificant toxicity on leukemic CEM cells and non-cancerous Hs27 cells. In HL-60 leukemic cells, 12 triggered increased mitochondrial NADH oxidation, increased respiration in presence of oligomycin (state 4o), mitochondrial depolarization, and ROS production, suggesting an uncoupling of OXPHOS. This provoked a metabolic adaptation dependent on AMPK/ACC/autophagy axis, having the mitochondrial β-oxidation a pro-survival role since the combination of 12 and etomoxir, a carnitine palmitoyl-transferase (CPT) inhibitor promoted extensive HL-60 cell death. Finally, 12-induced metabolic stress sensitized to HL-60 cells to cell death by the FDA-approved anti-leukemic drug ABT-199, a BH3 mimetic. Therefore, our results suggest that acylchlorohydroquinone is a promising scaffold in anti-promyelocytic leukemia drug research.
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http://dx.doi.org/10.1016/j.bioorg.2020.103935DOI Listing
July 2020

PCR-RFLP Detection and Genogroup Identification of in Field Samples.

Pathogens 2020 May 8;9(5). Epub 2020 May 8.

Laboratorio de Bioinformática y Expresión Génica, Instituto de Nutrición y Tecnología de los Alimentos (INTA), Universidad de Chile, Santiago 7830490, Chile.

, the causative agent of piscirickettsiosis, is genetically divided into two genomic groups, named after the reference strains as LF-89-like or EM-90-like. Phenotypic differences have been detected between the genogroups, including antibiotic susceptibilities, host specificities and pathogenicity. In this study, we aimed to develop a rapid, sensitive and cost-effective assay for the differentiation of the genogroups. Using an in silico analysis of the 16S rDNA digestion patterns, we have designed a genogroup-specific assay based on PCR-restriction fragment length polymorphism (RFLP). An experimental validation was carried out by comparing the restriction patterns of 13 strains and 57 field samples obtained from the tissues of dead or moribund fish. When the bacterial composition of a set of field samples, for which we detected mixtures of bacterial DNA, was analyzed by a high-throughput sequencing of the 16S rRNA gene amplicons, a diversity of taxa could be identified, including pathogenic and commensal bacteria. Despite the presence of mixtures of bacterial DNA, the characteristic digestion pattern of the genogroups could be detected in the field samples without the need of a microbiological culture and bacterial isolation.
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http://dx.doi.org/10.3390/pathogens9050358DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7281544PMC
May 2020

Transcriptomic Changes of During Intracellular Growth in a Salmon Macrophage-Like Cell Line.

Front Cell Infect Microbiol 2019 9;9:426. Epub 2020 Jan 9.

Laboratorio de Bioinformática y Expresión Génica, Instituto de Nutrición y Tecnología de los Alimentos (INTA), Universidad de Chile, Santiago, Chile.

is the causative agent of Piscirickettsiosis, a systemic infection of salmonid fish species. infects and survives in its host cell, a process that correlates with the expression of virulence factors including components of the type IVB secretion system. To gain further insights into the cellular and molecular mechanism behind the adaptive response of during host infection, we established an model of infection using the SHK-1 cell line from Atlantic salmon head kidney. The results indicated that in comparison to uninfected SHK-1 cells, infection significantly decreased cell viability after 10 days along with a significant increment of genome equivalents. At that time, the intracellular bacteria were localized within a spacious cytoplasmic vacuole. By using a whole-genome microarray of LF-89, the transcriptome of this bacterium was examined during intracellular growth in the SHK-1 cell line and exponential growth in broth. Transcriptome analysis revealed a global shutdown of translation during intracellular growth and suggested an induction of the stringent response. Accordingly, key genes of the stringent response pathway were up-regulated during intracellular growth as well as at stationary phase bacteria, suggesting a role of the stringent response on bacterial virulence. Our results also reinforce the participation of the Dot/Icm type IVB secretion system during infection and reveals many unexplored genes with potential roles in the adaptation to intracellular growth. Finally, we proposed that intracellular alternates between a replicative phase and a stationary phase in which the stringent response is activated.
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http://dx.doi.org/10.3389/fcimb.2019.00426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6964531PMC
September 2020

Comparative Genomic Analysis of Three Salmonid Species Identifies Functional Candidate Genes Involved in Resistance to the Intracellular Bacterium .

Front Genet 2019 5;10:665. Epub 2019 Aug 5.

Instituto de Nutrición y Tecnología de los Alimentos, Universidad de Chile, Santiago, Chile.

is the etiologic agent of salmon rickettsial syndrome (SRS) and is responsible for considerable economic losses in salmon aquaculture. The bacterium affects coho salmon (CS; ), Atlantic salmon (AS; ), and rainbow trout (RT; ) in several countries, including Norway, Canada, Scotland, Ireland, and Chile. We used Bayesian genome-wide association study analyses to investigate the genetic architecture of resistance to in farmed populations of these species. Resistance to SRS was defined as the number of days to death and as binary survival (BS). A total of 828 CS, 2130 RT, and 2601 AS individuals were phenotyped and then genotyped using double-digest restriction site-associated DNA sequencing and 57K and 50K Affymetrix® Axiom® single nucleotide polymorphism (SNP) panels, respectively. Both traits of SRS resistance in CS and RT appeared to be under oligogenic control. In AS, there was evidence of polygenic control of SRS resistance. To identify candidate genes associated with resistance, we applied a comparative genomics approach in which we systematically explored the complete set of genes adjacent to SNPs, which explained more than 1% of the genetic variance of resistance in each salmonid species (533 genes in total). Thus, genes were classified based on the following criteria: i) shared function of their protein domains among species, ii) shared orthology among species, iii) proximity to the SNP explaining the highest proportion of the genetic variance, and iv) presence in more than one genomic region explaining more than 1% of the genetic variance within species. Our results allowed us to identify 120 candidate genes belonging to at least one of the four criteria described above. Of these, 21 of them were part of at least two of the criteria defined above and are suggested to be strong functional candidates influencing resistance. These genes are related to diverse biological processes, such as kinase activity, GTP hydrolysis, helicase activity, lipid metabolism, cytoskeletal dynamics, inflammation, and innate immune response, which seem essential in the host response against infection. These results provide fundamental knowledge on the potential functional genes underpinning resistance against in three salmonid species.
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http://dx.doi.org/10.3389/fgene.2019.00665DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6690157PMC
August 2019

FR58P1a; a new uncoupler of OXPHOS that inhibits migration in triple-negative breast cancer cells via Sirt1/AMPK/β1-integrin pathway.

Sci Rep 2018 09 4;8(1):13190. Epub 2018 Sep 4.

Instituto de Química de Recursos Naturales and Programa de Investigación Asociativa en Cáncer Gástrico, Universidad de Talca, casilla 747, Talca, Chile.

Highly malignant triple-negative breast cancer (TNBC) cells rely mostly on glycolysis to maintain cellular homeostasis; however, mitochondria are still required for migration and metastasis. Taking advantage of the metabolic flexibility of TNBC MDA-MB-231 cells to generate subpopulations with glycolytic or oxidative phenotypes, we screened phenolic compounds containing an ortho-carbonyl group with mitochondrial activity and identified a bromoalkyl-ester of hydroquinone named FR58P1a, as a mitochondrial metabolism-affecting compound that uncouples OXPHOS through a protonophoric mechanism. In contrast to well-known protonophore uncoupler FCCP, FR58P1a does not depolarize the plasma membrane and its effect on the mitochondrial membrane potential and bioenergetics is moderate suggesting a mild uncoupling of OXPHOS. FR58P1a activates AMPK in a Sirt1-dependent fashion. Although the activation of Sirt1/AMPK axis by FR58P1a has a cyto-protective role, selectively inhibits fibronectin-dependent adhesion and migration in TNBC cells but not in non-tumoral MCF10A cells by decreasing β1-integrin at the cell surface. Prolonged exposure to FR58P1a triggers a metabolic reprograming in TNBC cells characterized by down-regulation of OXPHOS-related genes that promote cell survival but comprise their ability to migrate. Taken together, our results show that TNBC cell migration is susceptible to mitochondrial alterations induced by small molecules as FR58P1a, which may have therapeutic implications.
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http://dx.doi.org/10.1038/s41598-018-31367-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6123471PMC
September 2018

Microbiome analysis and bacterial isolation from Lejía Lake soil in Atacama Desert.

Extremophiles 2018 Jul 23;22(4):665-673. Epub 2018 Apr 23.

Laboratorio de Bioinformática y Expresión Génica, INTA-Universidad de Chile, El Líbano, 5524, Santiago, Chile.

As a consequence of the severe climatic change affecting our entire world, many lakes in the Andes Cordillera are likely to disappear within a few decades. One of these lakes is Lejía Lake, located in the central Atacama Desert. The objectives of this study were: (1) to characterize the bacterial community from Lejía Lake shore soil (LLS) using 16S rRNA sequencing and (2) to test a culture-based approach using a soil extract medium (SEM) to recover soil bacteria. This extreme ecosystem was dominated by three phyla: Bacteroidetes, Proteobacteria, and Firmicutes with 29.2, 28.2 and 28.1% of the relative abundance, respectively. Using SEM, we recovered 7.4% of the operational taxonomic units from LLS, all of which belonged to the same three dominant phyla from LLS (6.9% of Bacteroidetes, 77.6% of Proteobacteria, and 15.3% of Firmicutes). In addition, we used SEM to recover isolates from LLS and supplemented the culture medium with increasing salt concentrations to isolate microbial representatives of salt tolerance (Halomonas spp.). The results of this study complement the list of microbial taxa diversity from the Atacama Desert and assess a pipeline to isolate selective bacteria that could represent useful elements for biotechnological approaches.
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http://dx.doi.org/10.1007/s00792-018-1027-6DOI Listing
July 2018

Genomic-Based Restriction Enzyme Selection for Specific Detection of Piscirickettsia salmonis by 16S rDNA PCR-RFLP.

Front Microbiol 2016 9;7:643. Epub 2016 May 9.

Laboratorio de Bioinformática y Expresión Génica, Instituto de Nutrición y Tecnología de los Alimentos, Universidad de ChileSantiago, Chile; Fondap Center for Genoma RegulationSantiago, Chile; Laboratorio de Genómica Aplicada, Instituto de Nutrición y Tecnología de los Alimentos, Universidad de ChileSantiago, Chile.

The gram negative facultative bacterium P. salmonis is the etiological agent of Salmonid Rickettsial Septicaemia (SRS), a severe disease that causes important economic losses in the global salmon farmer industry. Despite efforts to control this disease, the high frequency of new epizootic events indicate that the vaccine and antibiotics treatments have limited effectiveness, therefore the preventive and diagnostic approaches must be improved. A comparison of several methodologies for SRS diagnostic indicate differences in their specificity and its capacity to detect other bacteria coexisting with P. salmonis in culture media (contamination) and fish samples (coinfection), aspects relevant for research, vaccine development and clinical diagnostic. By computer-simulation analyses, we identified a group of restriction enzymes that generate unique P. salmonis 16S rDNA band patterns, distinguishable from all other bacteria. From this information, we designed and developed a PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) assay, which was validated using 16S rDNA universal primers and restriction enzyme PmaCI for the amplification and digestion, respectively. Experimental validation was performed by comparing the restriction pattern of P. salmonis with the restriction patterns generated by bacteria that cohabit with P. salmonis (fish bacterial isolates and culture media contaminants). Our results indicate that the restriction enzyme selection pipeline was suitable to design a more specific, sensible, faster and cheaper assay than the currently used P. salmonis detection methodologies.
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http://dx.doi.org/10.3389/fmicb.2016.00643DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4860512PMC
May 2016

Complete genome sequence of Microbacterium sp. CGR1, bacterium tolerant to wide abiotic conditions isolated from the Atacama Desert.

J Biotechnol 2015 Dec 29;216:149-50. Epub 2015 Oct 29.

Laboratorio de Bioinformática y Expresión Génica, INTA-Universidad de Chile, El Líbano 5524 Santiago, Chile; Fondap Center for Genome Regulation (CGR), Avenida Blanco Encalada 2085 Santiago, Chile. Electronic address:

Microbacterium sp. CGR1 (RGM2230) is an isolate from the Atacama Desert that displays a wide pH, salinity and temperature tolerance. This strain exhibits riboflavin overproducer features and traits for developing an environmental arsenic biosensor. Here, we report the complete genome sequence of this strain, which represents the first genome of the genus Microbacterium sequenced and assembled in a single contig. The genome contains 3,634,864bp, 3299 protein-coding genes, 45 tRNAs, six copies of 5S-16S-23S rRNA and a high genome average GC-content of 68.04%.
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http://dx.doi.org/10.1016/j.jbiotec.2015.10.020DOI Listing
December 2015

Identification and molecular characterization of five putative toxins from the venom gland of the snake Philodryas chamissonis (Serpentes: Dipsadidae).

Toxicon 2015 Dec 26;108:19-31. Epub 2015 Sep 26.

Laboratorio de Neuroetología, Programa de Fisiología y Biofísica, Facultad de Medicina, Universidad de Chile, Casilla 70005, Correo 7, Santiago, Chile; Department of Biosciences, Centre for Ecological and Evolutionary Synthesis, University of Oslo, PB1066 Blinder, 0316 Oslo, Norway. Electronic address:

Philodryas chamissonis is a rear-fanged snake endemic to Chile. Its bite produces mild to moderate symptoms with proteolytic and anti-coagulant effects. Presently, the composition of the venom, as well as, the biochemical and structural characteristics of its toxins, remains unknown. In this study, we cloned and reported the first full-length sequences of five toxin-encoding genes from the venom gland of this species: Type III snake venom metalloprotease (SVMP), snake venom serine protease (SVSP), Cysteine-rich secretory protein (CRISP), α and β subunits of C-type lectin-like protein (CLP) and C-type natriuretic peptide (NP). These genes are highly expressed in the venom gland and their sequences exhibited a putative signal peptide, suggesting that these are components of the venom. These putative toxins had different evolutionary relationships with those reported for some front-fanged snakes, being SVMP, SVSP and CRISP of P. chamissonis closely related to the toxins present in Elapidae species, while NP was more related to those of Viperidae species. In addition, analyses suggest that the α and β subunits of CLP of P. chamissonis might have a α-subunit scaffold in common with Viperidae species, whose highly variable C-terminal region might have allowed the diversification in α and β subunits. Our results provide the first molecular description of the toxins possibly implicated in the envenomation of prey and humans by the bite of P. chamissonis.
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http://dx.doi.org/10.1016/j.toxicon.2015.09.032DOI Listing
December 2015

Complete genome sequence of Piscirickettsia salmonis LF-89 (ATCC VR-1361) a major pathogen of farmed salmonid fish.

J Biotechnol 2015 Oct 26;212:30-1. Epub 2015 Jul 26.

Laboratorio de Bioinformática y Expresión Génica, INTA-Universidad de Chile, El Líbano 5524, Santiago, Chile; Fondap Center for Genome Regulation (CGR), Avenida Blanco Encalada 2085, Santiago, Chile. Electronic address:

Piscirickettsia salmonis, the causative agent of salmonid rickettsial septicemia (SRS), is a significant threat to the healthy and sustainable production of salmonid farming industry. This Gram-negative bacterium, originally isolated from a coho salmon in Southern Chile, produces a systemic infection characterized by colonization of several fish organs. P. salmonis is able to infect, survive, and replicate inside salmonid macrophages however little is known about its mechanisms of pathogenesis. Here, we present the whole genome sequence and annotation of the P. salmonis reference strain LF-89 (ATCC VR-1361). The genome contains one circular chromosome of 3,184,851 bp and three plasmids, pPSLF89-1 (180,124 bp), pPSLF89-2 (33,516 bp) and pPSLF89-3 (51,573 bp). A total of 2850 protein-coding genes, 56 tRNAs and six copies of 5S-16S-23S rRNA.
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http://dx.doi.org/10.1016/j.jbiotec.2015.07.017DOI Listing
October 2015

Transcriptional response of Atlantic salmon families to Piscirickettsia salmonis infection highlights the relevance of the iron-deprivation defence system.

BMC Genomics 2015 Jul 4;16:495. Epub 2015 Jul 4.

Laboratorio de Bioinformática y Expresión Génica, Instituto de Nutrición y Tecnología de los Alimentos (INTA), Universidad de Chile, El Líbano 5524, Santiago, Chile.

Background: Piscirickettsiosis or Salmonid Rickettsial Septicaemia (SRS) is a bacterial disease that has a major economic impact on the Chilean salmon farming industry. Despite the fact that Piscirickettsia salmonis has been recognized as a major fish pathogen for over 20 years, the molecular strategies underlying the fish response to infection and the bacterial mechanisms of pathogenesis are poorly understood. We analysed and compared the head kidney transcriptional response of Atlantic salmon (Salmo salar) families with different levels of susceptibility to P. salmonis infection in order to reveal mechanisms that might confer infection resistance.

Results: We ranked forty full-sibling Atlantic salmon families according to accumulated mortality after a challenge with P. salmonis and selected the families with the lowest and highest cumulative mortalities for microarray gene expression analysis. A comparison of the response to P. salmonis infection between low and high susceptibility groups identified biological processes presumably involved in natural resistance to the pathogen. In particular, expression changes of genes linked to cellular iron depletion, as well as low iron content and bacterial load in the head kidney of fish from low susceptibility families, suggest that iron-deprivation is an innate immunity defence mechanism against P. salmonis. To complement these results, we predicted a set of iron acquisition genes from the P. salmonis genome. Identification of putative Fur boxes and expression of the genes under iron-depleted conditions revealed that most of these genes form part of the Fur regulon of P. salmonis.

Conclusions: This study revealed, for the first time, differences in the transcriptional response to P. salmonis infection among Atlantic salmon families with varied levels of susceptibility to the infection. These differences correlated with changes in the abundance of transcripts encoding proteins directly and indirectly involved in the immune response; changes that highlighted the role of nutritional immunity through iron deprivation in host defence mechanisms against P. salmonis. Additionally, we found that P. salmonis has several mechanisms for iron acquisition, suggesting that this bacterium can obtain iron from different sources, including ferric iron through capturing endogenous and exogenous siderophores and ferrous iron. Our results contribute to determining the underlying resistance mechanisms of Atlantic salmon to P. salmonis infection and to identifying future treatment strategies.
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http://dx.doi.org/10.1186/s12864-015-1716-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4490697PMC
July 2015

Comparative gene expression analysis of Dtg, a novel target gene of Dpp signaling pathway in the early Drosophila melanogaster embryo.

Gene 2014 Feb 7;535(2):210-7. Epub 2013 Dec 7.

Laboratorio de Bioinformática y Expresión Génica, INTA-Universidad de Chile, El Líbano 5524, Santiago, Chile; Fondap Center for Genome Regulation (CGR), Universidad de Chile, Santiago, Chile. Electronic address:

In the early Drosophila melanogaster embryo, Dpp, a secreted molecule that belongs to the TGF-β superfamily of growth factors, activates a set of downstream genes to subdivide the dorsal region into amnioserosa and dorsal epidermis. Here, we examined the expression pattern and transcriptional regulation of Dtg, a new target gene of Dpp signaling pathway that is required for proper amnioserosa differentiation. We showed that the expression of Dtg was controlled by Dpp and characterized a 524-bp enhancer that mediated expression in the dorsal midline, as well as, in the differentiated amnioserosa in transgenic reporter embryos. This enhancer contained a highly conserved region of 48-bp in which bioinformatic predictions and in vitro assays identified three Mad binding motifs. Mutational analysis revealed that these three motifs were necessary for proper expression of a reporter gene in transgenic embryos, suggesting that short and highly conserved genomic sequences may be indicative of functional regulatory regions in D. melanogaster genes. Dtg orthologs were not detected in basal lineages of Dipterans, which unlike D. melanogaster develop two extra-embryonic membranes, amnion and serosa, nevertheless Dtg orthologs were identified in the transcriptome of Musca domestica, in which dorsal ectoderm patterning leads to the formation of a single extra-embryonic membrane. These results suggest that Dtg was recruited as a new component of the network that controls dorsal ectoderm patterning in the lineage leading to higher Cyclorrhaphan flies, such as D. melanogaster and M. domestica.
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http://dx.doi.org/10.1016/j.gene.2013.11.032DOI Listing
February 2014

Genes encoding novel secreted and transmembrane proteins are temporally and spatially regulated during Drosophila melanogaster embryogenesis.

BMC Biol 2009 Sep 22;7:61. Epub 2009 Sep 22.

Laboratorio de Bioinformática y Expresión Génica, INTA-Universidad de Chile, Millennium Nucleus Center for Genomics of the Cell (CGC), Santiago, Chile.

Background: Morphogenetic events that shape the Drosophila melanogaster embryo are tightly controlled by a genetic program in which specific sets of genes are up-regulated. We used a suppressive subtractive hybridization procedure to identify a group of developmentally regulated genes during early stages of D. melanogaster embryogenesis. We studied the spatiotemporal activity of these genes in five different intervals covering 12 stages of embryogenesis.

Results: Microarrays were constructed to confirm induction of expression and to determine the temporal profile of isolated subtracted cDNAs during embryo development. We identified a set of 118 genes whose expression levels increased significantly in at least one developmental interval compared with a reference interval. Of these genes, 53% had a phenotype and/or molecular function reported in the literature, whereas 47% were essentially uncharacterized. Clustering analysis revealed demarcated transcript groups with maximum gene activity at distinct developmental intervals. In situ hybridization assays were carried out on 23 uncharacterized genes, 15 of which proved to have spatiotemporally restricted expression patterns. Among these 15 uncharacterized genes, 13 were found to encode putative secreted and transmembrane proteins. For three of them we validated our protein sequence predictions by expressing their cDNAs in Drosophila S2R+ cells and analyzed the subcellular distribution of recombinant proteins. We then focused on the functional characterization of the gene CG6234. Inhibition of CG6234 by RNA interference resulted in morphological defects in embryos, suggesting the involvement of this gene in germ band retraction.

Conclusion: Our data have yielded a list of developmentally regulated D. melanogaster genes and their expression profiles during embryogenesis and provide new information on the spatiotemporal expression patterns of several uncharacterized genes. In particular, we recovered a substantial number of unknown genes encoding putative secreted and transmembrane proteins, suggesting new components of signaling pathways that might be incorporated within the existing regulatory networks controlling D. melanogaster embryogenesis. These genes are also good candidates for additional targeted functional analyses similar to those we conducted for CG6234.See related minireview by Vichas and Zallen: http://www.jbiol.com/content/8/8/76.
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http://dx.doi.org/10.1186/1741-7007-7-61DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2761875PMC
September 2009

CCS and SOD1 mRNA are reduced after copper supplementation in peripheral mononuclear cells of individuals with high serum ceruloplasmin concentration.

J Nutr Biochem 2008 Apr 1;19(4):269-74. Epub 2007 Aug 1.

Laboratory of Micronutrients, University of Chile (INTA), Santiago, Chile.

The limits of copper homeostatic regulation in humans are not known, making it difficult to define the milder effects of early copper excess. Furthermore, a robust assay to facilitate the detection of early stages of copper excess is needed. To address these issues, we assessed changes in relative mRNA abundance of methallothionein 2A (MT2A), prion (PrP), amyloid precursor-like protein 2 (APLP2), Cu/Zn superoxide dismutase (SOD1) and its copper chaperone (CCS) in peripheral mononuclear cells (PMNCs) from healthy adults representing the 5% highest and lowest extremes in the distribution curve of serum ceruloplasmin (Cp) concentrations of 800 individuals. The intracellular Cu content was also determined. PMNCs were isolated from individuals before and after exposure to a single daily dose of 10 mg Cu (as CuSO(4)) for 2 months. Results showed that although there were fluctuations in serum Cp values of the samples assessed before copper exposure, no significant differences were observed in cell copper content or in the relative abundance of MT2A, PrP and APLP2 transcripts in PMNCs. Also, these values were not modified after copper supplementation. However, CCS and SOD1 mRNA levels were reduced in PMNCs after copper supplementation in the individuals with the high Cp values, suggesting that they should be further explored as biomarkers of moderate copper overload in humans.
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http://dx.doi.org/10.1016/j.jnutbio.2007.04.003DOI Listing
April 2008

Gene expression profiling in wild-type and metallothionein mutant fibroblast cell lines.

Biol Res 2006 ;39(1):125-42

Department of Nutritional Science and Toxicology, University of California, Berkeley, Berkeley, CA 94720, USA.

The role of metallothioneins (MT) in copper homeostasis is of great interest, as it appears to be partially responsible for the regulation of intracellular copper levels during adaptation to extracellular excess of the metal. To further investigate a possible role of MTs in copper metabolism, a genomics approach was utilized to evaluate the role of MT on gene expression. Microarray analysis was used to examine the effects of copper overload in fibroblast cells from normal and MT I and II double knock-out mice (MT-/-). As a first step, we compared genes that were significantly upregulated in wild-type and MT-/- cells exposed to copper. Even though wild-type and mutant cells are undistinguishable in terms of their morphological features and rates of growth, our results show that MT-/- cells do not respond with induction of typical markers of cellular stress under copper excess conditions, as observed in the wild-type cell line, suggesting that the transcription initiation rate or the mRNA stability of stress genes is affected when there is an alteration in the copper store capacity. The functional classification of other up-regulated genes in both cell lines indicates that a large proportion (>80%) belong to two major categories: 1) metabolism; and 2) cellular physiological processes, suggesting that at the transcriptional level copper overload induces the expression of genes associated with diverse molecular functions. These results open the possibility to understand how copper homeostasis is being coordinated with other metabolic pathways.
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http://dx.doi.org/10.4067/s0716-97602006000100015DOI Listing
February 2007
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