31 research outputs found
Comparison of Metagenomics and Metatranscriptomics Tools: A Guide to Making the Right Choice
The study of microorganisms is a field of great interest due to their environmental (e.g., soil
contamination) and biomedical (e.g., parasitic diseases, autism) importance. The advent of revolutionary
next-generation sequencing techniques, and their application to the hypervariable regions
of the 16S, 18S or 23S ribosomal subunits, have allowed the research of a large variety of organisms
more in-depth, including bacteria, archaea, eukaryotes and fungi. Additionally, together with the
development of analysis software, the creation of specific databases (e.g., SILVA or RDP) has boosted
the enormous growth of these studies. As the cost of sequencing per sample has continuously decreased,
new protocols have also emerged, such as shotgun sequencing, which allows the profiling of
all taxonomic domains in a sample. The sequencing of hypervariable regions and shotgun sequencing
are technologies that enable the taxonomic classification of microorganisms from the DNA present
in microbial communities. However, they are not capable of measuring what is actively expressed.
Conversely, we advocate that metatranscriptomics is a “new” technology that makes the identification
of the mRNAs of a microbial community possible, quantifying gene expression levels and active
biological pathways. Furthermore, it can be also used to characterise symbiotic interactions between
the host and its microbiome. In this manuscript, we examine the three technologies above, and
discuss the implementation of different software and databases, which greatly impact the obtaining of
reliable results. Finally, we have developed two easy-to-use pipelines leveraging Nextflow technology.
These aim to provide everything required for an average user to perform a metagenomic analysis of
marker genes with QIMME2 and a metatranscriptomic study using Kraken2/Bracken.regional Andalusian GovernmentPOSTDOC_21 _0039
Low endogenous NO levels in roots and antioxidant systems are determinants for the resistance of Arabidopsis seedlings grown in Cd
Cadmium (Cd), which is a toxic non-essential heavy metal capable of entering plants and thus the food chain, constitutes a major environmental and health concern worldwide. An understanding of the tools used by plants to overcome Cd stress could lead to the production of food crops with lower Cd uptake capacity and of plants with greater Cd uptake potential for phytoremediation purposes in order to restore soil efficiency in self-sustaining ecosystems. The signalling molecule nitric oxide (NO), whose function remains unclear, has recently been involved in responses to Cd stress. Using different mutants, such as nia1nia2, nox1, argh1-1 and Atnoa1, which were altered in NO metabolism, we analysed various parameters related to reactive oxygen and nitrogen species (ROS/RNS) metabolism and seedling fitness following germination and growth under Cd treatment conditions for seven days. Seedling roots were the most affected, with an increase in ROS and RNS observed in wild type (WT) seedling roots, leading to increased oxidative damage and fitness loss. Mutants that showed lower NO levels in seedling roots under Cd stress were more resistant than WT seedlings due to the maintenance of antioxidant systems which protect against oxidative damage.This study was co-funded by the ERDF and the Science, Innovation and University Ministry (BIO2015-67657-P and PGC2018-098372). L.C. T-C was supported by an FPU fellowship from the Spanish Ministry of Education, Culture and Sports
Development of polymorphic microsatellite markers for the Killarney Fern (Vandenboschia speciosa, Hymenophyllaceae)
Premise of the study:
We characterize 10 microsatellite loci in the endangered fern Vandenboschia speciosa (Hymenophyllaceae), enabling studies on the genetic population structure of this Macaronesian-European species using DNA hypervariable markers.
Methods and Results:
Ten primer sets were developed and tested on 47 individuals in a total of two Iberian populations of V. speciosa. The primers amplified di- and hexanucelotide repeats. The number of alleles ranged from two to eight, and the expected heterozygosity ranged from 0.107 to 0.807 among the populations analyzed.
Conclusions:
The 10 microsatellite markers developed will be useful in characterizing the genetic diversity of V. speciosa and understanding its population structure (including the possible structure between sporophyte and gametophyte phases) and biogeographic history, and will provide important genetic data for the conservation of this species.This study was supported by the Regional Andalusian Government (project P10-RNM-6198). C.G.L. was funded by a postdoctoral grant from the Regional Andalusian Government. S.B.S. and L.T.C. were funded by a Beca-Iniciación a la Investigación grant from the Universidad de Granada during 2012–2013 and 2014–2015, respectively. I.M.A. was funded by a Beca-Colaboración grant (Spanish Government)
Gene network downstream plant stress response modulated by peroxisomal H2O2
Reactive oxygen species (ROS) act as secondary messengers that can be sensed
by specific redox-sensitive proteins responsible for the activation of signal
transduction culminating in altered gene expression. The subcellular site, in
which modifications in the ROS/oxidation state occur, can also act as a specific
cellular redox network signal. The chemical identity of ROS and their subcellular
origin is actually a specific imprint on the transcriptome response. In recent
years, a number of transcriptomic studies related to altered ROS metabolism in
plant peroxisomes have been carried out. In this study, we conducted a metaanalysis
of these transcriptomic findings to identify common transcriptional
footprints for plant peroxisomal-dependent signaling at early and later time
points. These footprints highlight the regulation of various metabolic pathways
and gene families, which are also found in plant responses to several abiotic
stresses. Major peroxisomal-dependent genes are associated with protein
and endoplasmic reticulum (ER) protection at later stages of stress while, at
earlier stages, these genes are related to hormone biosynthesis and signaling
regulation. Furthermore, in silico analyses allowed us to assign human orthologs
to some of the peroxisomal-dependent proteins, which are mainly associated
with different cancer pathologies. Peroxisomal footprints provide a valuable
resource for assessing and supporting key peroxisomal functions in cellular
metabolism under control and stress conditions across species.Spanish Ministry of Science, Innovation and Universities (MCIU)State Research Agency (AEI)FEDER grant PGC2018-098372-B-I00MCIU Research Personnel Training (FPI) grant BES-2016-07651
Mapping the entire functionally active endometrial microbiota
STUDY QUESTION
Does endometrium harbour functionally active microorganisms and whether the microbial composition differs between proliferative and mid-secretory phases?
SUMMARY ANSWER
Endometrium harbours functionally alive microorganisms including bacteria, viruses, archaea and fungi whose composition and metabolic functions change along the menstrual cycle.
WHAT IS KNOWN ALREADY
Resident microbes in the endometrium have been detected, where microbial dysfunction has been associated with reproductive health and disease. Nevertheless, the core microorganismal composition in healthy endometrium is not determined and whether the identified bacterial DNA sequences refer to alive/functionally active microbes is not clear. Furthermore, whether there are cyclical changes in the microbial composition remains an open issue.
STUDY DESIGN, SIZE, DURATION
RNA sequencing (RNAseq) data from 14 endometrial paired samples from healthy women, 7 samples from the mid-secretory phase and 7 samples from the consecutive proliferative phase were analysed for the microbial RNA sequences.
PARTICIPANTS/MATERIALS, SETTING, METHODS
The raw RNAseq data were converted into FASTQ format using SRA Toolkit. The unmapped reads to human sequences were aligned to the reference database Kraken2 and visualised with Krona software. Menstrual phase taxonomic differences were performed by R package metagenomeSeq. The functional analysis of endometrial microbiota was obtained with HUMANn2 and the comparison between menstrual phases was conducted by one-way ANOVA. Human RNAseq analysis was performed using miARma-Seq and the functional enrichment analysis was carried out using gene set enrichment analysis (GSEA; HumanCyc). The integration of metabolic pathways between host and microbes was investigated. The developed method of active microbiota mapping was validated in independent sample set.
MAIN RESULTS AND THE ROLE OF CHANCE
With the novel metatranscriptomic approach, we mapped the entire alive microbiota composing of >5300 microorganisms within the endometrium of healthy women. Microbes such as bacteria, fungi, viruses and archaea were identified. The validation of three independent endometrial samples from different ethnicity confirmed the findings. Significant differences in the microbial abundances in the mid-secretory vs. proliferative phases were detected with possible metabolic activity in the host-microbiota crosstalk in receptive phase endometrium, specifically in the prostanoid biosynthesis pathway and L-tryptophan metabolism.
LARGE SCALE DATA
The raw RNAseq data used in the current study are available at GEO GSE86491 and at BioProject PRJNA379542.
LIMITATIONS, REASONS FOR CAUTION
These pioneering results should be confirmed in a bigger sample size.
WIDER IMPLICATIONS OF THE FINDINGS
Our study confirms the presence of active microbes, bacteria, fungi, viruses and archaea in the healthy human endometrium with implications in receptive phase endometrial functions, meaning that microbial dysfunction could impair the metabolic pathways important for endometrial receptivity. The results of this study contribute to the better understanding of endometrial microbiota composition in healthy women and its possible role in endometrial functions. In addition, our novel methodological pipeline for analysing alive microbes with transcriptional and metabolic activities could serve to inspire new analysis approaches in reproductive medicine.This work is supported by the Spanish Ministry of Economy, Industry and
Competitiveness (MINECO) and European Regional Development Fund (FEDER): grants RYC-2016-21199 and ENDORE SAF2017-87526-
R; FEDER/Junta de Andalucía-Consejería de Economía y Conocimiento: MENDO (B-CTS-500-UGR18) and by the University of Granada
Plan Propio de Investigacio ́n 2016 - Excellence actions: Unit of Excellence on Exercise and Health (UCEES) (SOMM17/6107/UGR).
A.S.-L. and N.M.M. are funded by the Spanish Ministry of Science, Innovation and Universities (PRE2018-0854409 and FPU19/01638). S.A.
has received honoraria for lectures from Merck. The funder had no role in this study
Cd38 deficiency ameliorates chronic graft versus Host disease murine lupus via a b-cell dependent mechanism
Trabajo presentado en el II Congreso investigación PTS, celebrado en Granada (España) del 09 al 11 de febrero de 2022.Absence of mouse cell surface receptor CD38 in Cd38-/- mice suggests that this receptor acts as positive regulator of inflammatory and autoimmune responses. Here we report that in the setting of a chronic graft versus host disease (cGVHD) lupus model induced by the transfer of B6.C-H2bm12/KhEg (bm12) spleen cells into co-isogenic Cd38-/- B6 mice causes milder lupus-like autoimmunity with lower levels of anti-ssDNA autoantibodies than the transfer of bm12 spleen cells into WT B6 mice. I In addition, significantly lower percentages of Tfh cells, as well as GC B cells, plasma cells and T-bet+CD11chi B cells are observed in Cd38-/- mice than in WT mice, while the expansion of Treg cells, and Tfr cells is normal, suggesting that the ability of Cd38-/- B cells to respond to allogeneic help from bm12 CD4+ T cells is greatly diminished. The frequencies of T-bet+CD11chi B cells, which are considered the precursors of the autoantibody secreting cells, correlate with anti-ssDNA autoantibody serum levels, with IL-27, and sCD40L. Proteomics profiling of spleens from WT cGVHD mice reflects a STAT1-driven type I IFN-signature, which is absent in Cd38-/- cGVHD mice. Kidney, spleen and liver inflammation was mild and resolved faster in Cd38-/- cGVHD mice than in WT cGVHD mice. We conclude that in B cells CD38 functions as a modulator receptor that controls autoimmune responses
Methylation and Transcriptome Signatures of Monocytes Reveal Novel Pathways Involved in Giant Cell Arteritis Pathogenesis.
Congresos y Conferencias: Comunicación de Congreso - Oral
CD38 Deficiency Ameliorates Chronic Graft-Versus-Host Disease Murine Lupus via a B-Cell-Dependent Mechanism
© 2021 Martínez-Blanco, Domínguez-Pantoja, Botía-Sánchez, Pérez-Cabrera, Bello-Iglesias, Carrillo-Rodríguez, Martin-Morales, Lario-Simón, Pérez-Sánchez-Cañete, Montosa-Hidalgo, Guerrero-Fernández, Longobardo-Polanco, Redondo-Sánchez, Cornet-Gomez, Torres-Sáez, Fernández-Ibáñez, Terrón-Camero, Andrés-León, O’Valle, Merino, Zubiaur and Sancho.The absence of the mouse cell surface receptor CD38 in Cd38−/− mice suggests that this receptor acts as a positive regulator of inflammatory and autoimmune responses. Here, we report that, in the context of the chronic graft-versus-host disease (cGVHD) lupus inducible model, the transfer of B6.C-H2bm12/KhEg(bm12) spleen cells into co-isogenic Cd38−/− B6 mice causes milder lupus-like autoimmunity with lower levels of anti-ssDNA autoantibodies than the transfer of bm12 spleen cells into WT B6 mice. In addition, significantly lower percentages of Tfh cells, as well as GC B cells, plasma cells, and T-bet+CD11chi B cells, were observed in Cd38−/− mice than in WT mice, while the expansion of Treg cells and Tfr cells was normal, suggesting that the ability of Cd38−/− B cells to respond to allogeneic help from bm12 CD4+ T cells is greatly diminished. The frequencies of T-bet+CD11chi B cells, which are considered the precursors of the autoantibody-secreting cells, correlate with anti-ssDNA autoantibody serum levels, IL-27, and sCD40L. Proteomics profiling of the spleens from WT cGVHD mice reflects a STAT1-driven type I IFN signature, which is absent in Cd38−/− cGVHD mice. Kidney, spleen, and liver inflammation was mild and resolved faster in Cd38−/− cGVHD mice than in WT cGVHD mice. We conclude that CD38 in B cells functions as a modulator receptor that controls autoimmune responses.S and MZ received financial support through “Proyecto del Plan Estatal”: SAF2017–89801-R. The IPBLN-CSIC Proteomics Unit belonged to ProteoRed-ISCIII (PRB2; PRB3) and was supported by grants PT13/0001/0011 (IPBLN-CSIC) and PT17/0019/0010 (CIB-CSIC; IPBLN-CSIC). RM: Project: SAF2017-82905-R. FO'V: Cátedra MIS IMPLANT-UGR. The stay of AC-G in Sancho’s lab was supported by a fellowship-contract JAE-Intro (CSIC). The stay of MD-P in Sancho’s lab was supported by a 1-year post-doctoral fellowship (Reference No. 502492) from the Consejo Nacional de Ciencia y Tecnología (CONACYT) of México. EA-L was recipient of a postdoctoral fellowship from the regional Andalusian Government
Selective inhibition of HDAC6 regulates expression of the oncogenic driver EWSR1-FLI1 through the EWSR1 promoter in Ewing sarcoma
Ewing sarcoma (EWS) is an aggressive bone and soft tissue tumor of children and young adults in which the principal driver is a fusion gene, EWSR1-FLI1. Although the essential role of EWSR1-FLI1 protein in the regulation of oncogenesis, survival, and tumor progression processes has been described in-depth, little is known about the regulation of chimeric fusion-gene expression. Here, we demonstrate that the active nuclear HDAC6 in EWS modulates the acetylation status of specificity protein 1 (SP1), consequently regulating the SP1/P300 activator complex binding to EWSR1 and EWSR1-FLI1 promoters. Selective inhibition of HDAC6 impairs binding of the activator complex SP1/P300, thereby inducing EWSR1-FLI1 downregulation and significantly reducing its oncogenic functions. In addition, sensitivity of EWS cell lines to HDAC6 inhibition is higher than other tumor or non-tumor cell lines. High expression of HDAC6 in primary EWS tumor samples from patients correlates with a poor prognosis in two independent series accounting 279 patients. Notably, a combination treatment of a selective HDAC6 and doxorubicin (a DNA damage agent used as a standard therapy of EWS patients) dramatically inhibits tumor growth in two EWS murine xenograft models. These results could
lead to suitable and promising therapeutic alternatives for patients with EWS.Research in the E.D.A. lab is supported by Asociación Española Contra el Cáncer (AECC), the Ministry of Science of Spain-FEDER (CIBERONC, PI1700464, PI2000003, RD06/0020/0059)S. D.G.D. and L.H.P. are supported by CIBERONC (CB16/12/00361). D.G.D., M.J.R. and L.H.P. are PhD researchers funded by the Consejería de Salud, Junta de Andalucía (PI-0197-2016, ECAI F2-0012-2018 and PI-0013-2018, respectively).Peer reviewe
Role of nitric oxide (NO) in plant response to cadmium and Fusarium oxysporum: Possible crosstalk
In this thesis, we made a bioinformatics analysis of
articles published over the last ten years on the production and/or function of NO in plant responses
to heavy metals, including Cd. This analysis showed that exogenous applications of NO to the
plant protect against heavy metals, particularly Cd, and that, in response to exposure to heavy
metals, particularly Cd, plants initially produce NO, which can act as a signal molecule. At a later
stage, the plant appears to be equipped with mechanisms to control NO levels, thus preventing
further symptoms of toxicity. Using both biochemical techniques, involving modifications in NO
levels through chemical donors and scavengers, and molecular approaches, involving the use of mutants with altered levels of NO, we subsequently analysed the role of NO in Arabidopsis
seedling responses to Cd stress and its relationship to ROS. We also show that prolonged
production of NO in plant responses to Cd can indeed affect antioxidant systems and induce
oxidative stress, suggesting yet again that NO levels need to be strictly regulated in plant responses
to Cd stress in order to prevent further damage to the plant.
Peroxisomes have recently been shown to play a key role in a plant's early response to Cd
stress. While both NO and RNS have been detected in these organelles, little is known about their
effect on peroxisomal metabolism and dynamics. In this thesis, we analysed the role of NO in
peroxisomal metabolism, distribution and dynamics under control conditions and in response to
Cd stress. We showed that NO is involved in changes observed in peroxisomal dynamics which
are necessary for the plant to respond to Cd stress. We also demonstrated the effect of NO on the
oxidative metabolism of peroxisomes and their cellular distribution, as well as on organelledependent
signalling.
In addition, we analysed the little-known role of NO in Arabidopsis-Fusarium oxysporum
interactions. We found that NO metabolism mutants have differential fungal responses with
respect to WT in terms of ROS production, phenols, secondary and iron metabolisms; as well as
defence gene induction Also, nitrate reductase appears to be essential for adequate cell wall
assembly through the regulation of CESA4 and MYB46, given that the cell wall is a key barrier in
the plant’s defence against Fusarium oxysporum.
Finally, pre-treatment with Cd was found to protect plants against Fusarium oxysporum
and to increase their survival. This could be explained by a priming effect, as certain genes are
common to different plant responses to Cd and fungi, particularly Fusarium, suggesting that
crosstalk takes place between both these stress conditions.En esta Tesis, hemos realizado un
análisis mediante bioinformática de los artículos publicados en los últimos diez años donde se
muestra la producción y/o función del NO en la respuesta de la planta a metales pesados, incluido
el Cd; para conocer, en base a los antecedentes disponibles, la posible función del NO en la
respuesta de la planta a los metales pesados. Este análisis mostró la función protectora frente a los metales pesados y en particular frente al Cd, del NO aplicado de manera exógena a la planta.
Además, se encontró que tras la exposición a los metales pesados y en particular al Cd, las plantas
producen NO en la respuesta temprana que podría actuar como molécula señal. Sin embargo, en
etapas posteriores la planta parece que tiene mecanismos para controlar los niveles de NO,
evitando así mayores síntomas de toxicidad. Posteriormente, mediante técnicas tanto bioquímicas
(alterando los niveles de NO mediante donadores y secuestradores químicos) como moleculares
(mediante el uso de los mutantes con niveles alterados de NO disponibles), hemos analizado la
función del NO en la respuesta de plántulas de Arabidopsis al estrés por Cd y su relación con las
ROS. Así, demostramos que efectivamente, una prolongada producción de NO en la respuesta de
la planta al Cd puede afectar a sistemas antioxidantes e inducir un estrés oxidativo, sugiriendo de
nuevo que los niveles de NO deberían ser estrictamente regulados en la respuesta de la planta al
estrés por Cd para evitar mayores daños en la planta.
Recientemente, se ha demostrado que los peroxisomas tienen una función clave en la
respuesta temprana de la planta al estrés por Cd. Si bien, tanto el NO como las RNS se han
detectado dentro de estos orgánulos, su función sobre su metabolismo y dinámica es prácticamente
desconocida. En esta Tesis, hemos analizado la función del NO en el metabolismo, distribución y
dinámica peroxisomal en condiciones control y en respuesta al estrés por Cd. Hemos demostrado
que el NO es necesario para que se produzcan los cambios observados en la dinámica peroxisomal
en la planta en respuesta al Cd; que el NO afecta al metabolismo oxidativo del peroxisoma y a la
distribución de orgánulos dentro de la célula; así como a la señalización dependiente del orgánulo.
Por otro lado, hemos analizado la función del NO en la interacción Arabidopsis-Fusarium
oxysporum, que es prácticamente desconocida. Hemos observado que los mutantes relacionados
con el metabolismo de NO presentan una respuesta al hongo diferencial con respecto al WT, en lo
que se refiere a la producción de ROS, fenoles y metabolismo secundario, metabolismo del hierro
e inducción de genes de defensa. Además, la nitrato reductasa parece ser fundamental para el
ensamblaje adecuado de la pared celular a través de la regulación de CESA4 y MYB46, siendo
esta barrera clave para la defensa de la planta frente a Fusarium oxysporum.
Finalmente, hemos observado que el pretratamiento con Cd protege a las plantas frente
Fusarium oxysporum, incrementando su supervivencia lo que podría ser explicado por un efecto
“priming” ya que como se ha mencionado anteriormente, la respuesta de la planta al Cd y hongos,
en particular a Fusarium, tienen genes en común, sugiriendo una conexión entre ambos tipos de
estrés.Tesis Univ. Granada.Ministerio de Educación, Cultura y Deporte mediante la beca de “Formación de Profesorado Universitario” (FPU 2014)Fondo Europeo de Desarrollo Regional y el Ministerio de Economía, Industria y Competitividad de España (proyectos BIO2015-67657-P y PGC2018-098372-B100)Junta de Andalucía (proyecto EX12-BIO296; grupo no. BIO- 337