The Sinorhizobium meliloti RNA chaperone Hfq influences central carbon metabolism and the symbiotic interaction with alfalfa

<p>Abstract</p> <p>Background</p> <p>The bacterial Hfq protein is able to interact with diverse RNA molecules, including regulatory small non-coding RNAs (sRNAs), and thus it is recognized as a global post-transcriptional regulator of gene expression. Loss of Hfq has an extensive impact in bacterial physiology which in several animal pathogens influences virulence. <it>Sinorhizobium meliloti </it>is a model soil bacterium known for its ability to establish a beneficial nitrogen-fixing intracellular symbiosis with alfalfa. Despite the predicted general involvement of Hfq in the establishment of successful bacteria-eukaryote interactions, its function in <it>S. meliloti </it>has remained unexplored.</p> <p>Results</p> <p>Two independent <it>S. meliloti </it>mutants, 2011-3.4 and 1021Δ<it>hfq</it>, were obtained by disruption and deletion of the <it>hfq </it>gene in the wild-type strains 2011 and 1021, respectively, both exhibiting similar growth defects as free-living bacteria. Transcriptomic profiling of 1021Δ<it>hfq </it>revealed a general down-regulation of genes of sugar transporters and some enzymes of the central carbon metabolism, whereas transcripts specifying the uptake and metabolism of nitrogen sources (mainly amino acids) were more abundant than in the wild-type strain. Proteomic analysis of the 2011-3.4 mutant independently confirmed these observations. Symbiotic tests showed that lack of Hfq led to a delayed nodulation, severely compromised bacterial competitiveness on alfalfa roots and impaired normal plant growth. Furthermore, a large proportion of nodules (55%-64%) elicited by the 1021Δ<it>hfq </it>mutant were non-fixing, with scarce content in bacteroids and signs of premature senescence of endosymbiotic bacteria. RT-PCR experiments on RNA from bacteria grown under aerobic and microoxic conditions revealed that Hfq contributes to regulation of <it>nifA </it>and <it>fixK1/K2</it>, the genes controlling nitrogen fixation, although the Hfq-mediated regulation of <it>fixK </it>is only aerobiosis dependent. Finally, we found that some of the recently identified <it>S. meliloti </it>sRNAs co-inmunoprecipitate with a FLAG-epitope tagged Hfq protein.</p> <p>Conclusions</p> <p>Our results support that the <it>S. meliloti </it>RNA chaperone Hfq contributes to the control of central metabolic pathways in free-living bacteria and influences rhizospheric competence, survival of the microsymbiont within the nodule cells and nitrogen fixation during the symbiotic interaction with its legume host alfalfa. The identified <it>S. meliloti </it>Hfq-binding sRNAs are predicted to participate in the Hfq regulatory network.</p

Applied methodology based on HEC-HMS for reservoir filling estimation due to soil erosion

Authors propose a beneficial methodology for hydrological planning in their study. Prospective evaluations of the basins' net capacity can be done using the technique presented. The HEC-HMS (Hydrologic Modelling System) software can be used to estimate in a basin, the sediment emitted. For a certain precipitation, this methodology allows estimating, within a certain range, the gradual blockage of a reservoir, and even a projected date for total blockage. This has some applications to adopt corrective measures that prevent or delay the planned blockage deadlines. The model is of the semi-distributed type, estimating the generation and emission of sediments by sub-basins. The integration of different return periods in HEC-HMS with a semi-distributed model by sub-basins and the application of a mathematical model are the differentiating element of this research. The novelty of this work is to allow prognosing the reservoir sedimentation rate of basins in a local and regional scale with a medium and large temporary framework. The developed methodology allows public institutions to take decisions concerning hydrological planning. It has been applied to the case of "Charco Redondo"reservoir, in Cádiz, Andalusia, in southern Spain. Applying the methodology to this case, an average soil degradation of the reservoir basin has been estimated. Therefore, it is verified that in 50 years the reservoir is expected to lose 8.4% of its capacity

CFTR/ENaC-dependent regulation of membrane potential during human sperm capacitation is initiated by bicarbonate uptake through NBC

To fertilize an egg, sperm must reside in the female reproductive tract to undergo several maturational changes that are collectively referred to as capacitation. From a molecular point of view, the HCO3-dependent activation of the atypical soluble adenylyl cyclase (ADCY10) is one of the first events that occurs during capacitation and leads to the subsequent cAMP-dependent activation of protein kinase A (PKA). Capacitation is also accompanied by hyperpolarization of the sperm plasma membrane. We previously reported that PKA activation is necessary for CFTR (cystic fibrosis transmembrane conductance regulator channel) activity and for the modulation of membrane potential (Em). However, the main HCO3 transporters involved in the initial transport and the PKA-dependent Em changes are not well known nor characterized. Here, we analyzed how the activity of CFTR regulates Em during capacitation and examined its relationship with an electrogenic Na/HCO3 cotransporter (NBC) and epithelial Na channels (ENaCs). We observed that inhibition of both CFTR and NBC decreased HCO3 influx, resulting in lower PKA activity, and that events downstream of the cAMP activation of PKA are essential for the regulation of Em. Addition of a permeable cAMP analog partially rescued the inhibitory effects caused by these inhibitors. HCO3 also produced a rapid membrane hyperpolarization mediated by ENaC channels, which contribute to the regulation of Em during capacitation. Altogether, we demonstrate for the first time, that NBC cotransporters and ENaC channels are essential in the CFTR-dependent activation of the cAMP/PKA signaling pathway and Em regulation during human sperm capacitation.Fil: Puga Molina, Lis del Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Pinto, Nicolás Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Torres, Nicolás I.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: González Cota, Ana, L.. University of Washington; Estados UnidosFil: Luque, Guillermina Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Balestrini, Paula Ania. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Romarowski, Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Krapf, Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Santi, Celia M.. University of Washington; Estados UnidosFil: Treviño, Claudia L.. Universidad Nacional Autónoma de México; MéxicoFil: Darszon, Alberto. Universidad Nacional Autónoma de México; MéxicoFil: Buffone, Mariano Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

Tumor-Experienced Human NK Cells Express High Levels of PD-L1 and Inhibit CD8+ T Cell Proliferation

Natural Killer (NK) cells play a key role in cancer immunosurveillance. However, NK cells from cancer patients display an altered phenotype and impaired effector functions. In addition, evidence of a regulatory role for NK cells is emerging in diverse models of viral infection, transplantation, and autoimmunity. Here, we analyzed clear cell renal cell carcinoma (ccRCC) datasets from The Cancer Genome Atlas (TCGA) and observed that a higher expression of NK cell signature genes is associated with reduced survival. Analysis of fresh tumor samples from ccRCC patients unraveled the presence of a high frequency of tumor-infiltrating PD-L1+ NK cells, suggesting that these NK cells might exhibit immunoregulatory functions. In vitro, PD-L1 expression was induced on NK cells from healthy donors (HD) upon direct tumor cell recognition through NKG2D and was further up-regulated by monocyte-derived IL-18. Moreover, in vitro generated PD-L1hi NK cells displayed an activated phenotype and enhanced effector functions compared to PD-L1- NK cells, but simultaneously, they directly inhibited CD8+ T cell proliferation in a PD-L1-dependent manner. Our results suggest that tumors might drive the development of PD-L1-expressing NK cells that acquire immunoregulatory functions in humans. Hence, rational manipulation of these regulatory cells emerges as a possibility that may lead to improved anti-tumor immunity in cancer patients.Fil: Sierra, Jessica Mariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Secchiari, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Nuñez, Sol Yanel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Raffo Iraolagoitia, Ximena Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Ziblat, Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Friedrich, Adrián David. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Cátedra de Inmunología; ArgentinaFil: Regge, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Santilli, Maria Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Torres, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Gantov, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Trotta, Aldana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Ameri, Carlos Enrique. Hospital Alemán; ArgentinaFil: Vitagliano, Gonzalo. Hospital Alemán; ArgentinaFil: Ríos Pita, Hernando. Hospital Alemán; ArgentinaFil: Rico, Luis. Hospital Alemán; ArgentinaFil: Rovegno, Agustín. Centro de Educaciones Médicas e Investigación Clínica "Norberto Quirno"; ArgentinaFil: Richards, Nicolás. Centro de Educaciones Médicas e Investigación Clínica "Norberto Quirno"; ArgentinaFil: Domaica, Carolina Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Zwirner, Norberto Walter. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Fuertes, Mercedes Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

A survey of sRNA families in α-proteobacteria

We have performed a computational comparative analysis of six small non-coding RNA (sRNA) families in α-proteobacteria. Members of these families were first identified in the intergenic regions of the nitrogen-fixing endosymbiont S. meliloti by a combined bioinformatics screen followed by experimental verification. Consensus secondary structures inferred from covariance models for each sRNA family evidenced in some cases conserved motifs putatively relevant to the function of trans-encoded base-pairing sRNAs i.e., Hfq-binding signatures and exposed anti Shine-Dalgarno sequences. Two particular family models, namely αr15 and αr35, shared own sub-structural modules with the Rfam model suhB (RF00519) and the uncharacterized sRNA family αr35b, respectively. A third sRNA family, termed αr45, has homology to the cis-acting regulatory element speF (RF00518). However, new experimental data further confirmed that the S. meliloti αr45 representative is an Hfq-binding sRNA processed from or expressed independently of speF, thus refining the Rfam speF model annotation. All the six families have members in phylogenetically related plant-interacting bacteria and animal pathogens of the order of the Rhizobiales, some occurring with high levels of paralogy in individual genomes. In silico and experimental evidences predict differential regulation of paralogous sRNAs in S. meliloti 1021. The distribution patterns of these sRNA families suggest major contributions of vertical inheritance and extensive ancestral duplication events to the evolution of sRNAs in plant-interacting bacteria

A non-canonical RNA silencing pathway promotes mRNA degradation in basal fungi

The increasing knowledge on the functional relevance of endogenous small RNAs (esRNAs) as riboregulators has stimulated the identification and characterization of these molecules in numerous eukaryotes. In the basal fungus Mucor circinelloides, an emerging opportunistic human pathogen, esRNAs that regulate the expression of many protein coding genes have been described. These esRNAs share common machinery for their biogenesis consisting of an RNase III endonuclease Dicer, a single Argonaute protein and two RNA-dependent RNA polymerases. We show in this study that, besides participating in this canonical dicer-dependent RNA interference (RNAi) pathway, the rdrp genes are involved in a novel dicer-independent degradation process of endogenous mRNAs. The analysis of esRNAs accumulated in wild type and silencing mutants demonstrates that this new rdrp-dependent dicer-independent regulatory pathway, which does not produce sRNA molecules of discrete sizes, controls the expression of target genes promoting the specific degradation of mRNAs by a previously unknown RNase. This pathway mainly regulates conserved genes involved in metabolism and cellular processes and signaling, such as those required for heme biosynthesis, and controls responses to specific environmental signals. Searching the Mucor genome for candidate RNases to participate in this pathway, and functional analysis of the corresponding knockout mutants, identified a new protein, R3B2. This RNase III-like protein presents unique domain architecture, it is specifically found in basal fungi and, besides its relevant role in the rdrp-dependent dicer-independent pathway, it is also involved in the canonical dicer-dependent RNAi pathway, highlighting its crucial role in the biogenesis and function of regulatory esRNAs. The involvement of RdRPs in RNA degradation could represent the first evolutionary step towards the development of an RNAi mechanism and constitutes a genetic link between mRNA degradation and post-transcriptional gene silencing

The Fourteenth Data Release of the Sloan Digital Sky Survey: First Spectroscopic Data from the extended Baryon Oscillation Spectroscopic Survey and from the second phase of the Apache Point Observatory Galactic Evolution Experiment

The fourth generation of the Sloan Digital Sky Survey (SDSS-IV) has been in operation since July 2014. This paper describes the second data release from this phase, and the fourteenth from SDSS overall (making this, Data Release Fourteen or DR14). This release makes public data taken by SDSS-IV in its first two years of operation (July 2014-2016). Like all previous SDSS releases, DR14 is cumulative, including the most recent reductions and calibrations of all data taken by SDSS since the first phase began operations in 2000. New in DR14 is the first public release of data from the extended Baryon Oscillation Spectroscopic Survey (eBOSS); the first data from the second phase of the Apache Point Observatory (APO) Galactic Evolution Experiment (APOGEE-2), including stellar parameter estimates from an innovative data driven machine learning algorithm known as "The Cannon"; and almost twice as many data cubes from the Mapping Nearby Galaxies at APO (MaNGA) survey as were in the previous release (N = 2812 in total). This paper describes the location and format of the publicly available data from SDSS-IV surveys. We provide references to the important technical papers describing how these data have been taken (both targeting and observation details) and processed for scientific use. The SDSS website (www.sdss.org) has been updated for this release, and provides links to data downloads, as well as tutorials and examples of data use. SDSS-IV is planning to continue to collect astronomical data until 2020, and will be followed by SDSS-V.Comment: SDSS-IV collaboration alphabetical author data release paper. DR14 happened on 31st July 2017. 19 pages, 5 figures. Accepted by ApJS on 28th Nov 2017 (this is the "post-print" and "post-proofs" version; minor corrections only from v1, and most of errors found in proofs corrected

Influence of the length of hospitalisation in post-discharge outcomes in patients with acute heart failure: Results of the LOHRCA study

Objective: To investigate the relationship between length of hospitalisation (LOH) and post-discharge outcomes in acute heart failure (AHF) patients and to ascertain whether there are different patterns according to department of initial hospitalisation. Methods: Consecutive AHF patients hospitalised in 41 Spanish centres were grouped based on the LOH (15 days). Outcomes were defined as 90-day post-discharge all-cause mortality, AHF readmissions, and the combination of both. Hazard ratios (HRs), adjusted by chronic conditions and severity of decompensation, were calculated for groups with LOH >6 days vs. LOH <6 days (reference), and stratified by hospitalisation in cardiology, internal medicine, geriatrics, or short-stay units. Results: We included 8563 patients (mean age: 80 (SD = 10) years, 55.5% women), with a median LOH of 7 days (IQR 4–11): 2934 (34.3%) had a LOH 15 days. The 90-day post-discharge mortality was 11.4%, readmission 32.2%, and combined endpoint 37.4%. Mortality was increased by 36.5% (95%CI = 13.0–64.9) when LOH was 11–15 days, and by 72.0% (95%CI = 42.6–107.5) when >15 days. Conversely, no differences were found in readmission risk, and the combined endpoint only increased 21.6% (95%CI = 8.4–36.4) for LOH >15 days. Stratified analysis by hospitalisation departments rendered similar post-discharge outcomes, with all exhibiting increased mortality for LOH >15 days and no significant increments in readmission risk. Conclusions: Short hospitalisations are not associated with worse outcomes. While post-discharge readmissions are not affected by LOH, mortality risk increases as the LOH lengthens. These findings were similar across hospitalisation departments

Natural resource exploitation in Western Sahara: new research directions

The authors wish to thank the Leverhulme Trust (through their Early Career scheme) and the Spanish Ministry of Economy, Industry and Competitivity, (CSO2017-86986-P, AEI/FEDER, UE) for funding this research. We would also like to thank Patricia Lalonde for her translations and editorial work, as well as all participants of the 'Analysis of the Management and Exploitation of Natural Resources in Situations of Conflict: The Case of Western Sahara' project (funded by the aforementioned ministry), for their constructive comments on earlier iterations of this paper.This review article provides an overview of research to date with an explicit focus on natural resource exploitation in Western Sahara. It integrates findings from various perspectives and disciplines, and synthesises the research done with a view to revealing gaps and, therefore, potential new research directions. As the issue of natural resource exploitation in Western Sahara has been conceptualised in very different ways and from the perspectives of a variety of disciplines, the authors have opted for a semi-systematic review of the work done encompassing academic, non-academic, and activist backgrounds.Leverhulme TrustSpanish Ministry of Economy, Industry and Competitivity (AEI/FEDER, UE) CSO2017-86986-

RNAi-Based Functional Genomics Identifies New Virulence Determinants in Mucormycosis

Mucorales are an emerging group of human pathogens that are responsible for the lethal disease mucormycosis. Unfortunately, functional studies on the genetic factors behind the virulence of these organisms are hampered by their limited genetic tractability, since they are reluctant to classical genetic tools like transposable elements or gene mapping. Here, we describe an RNAi-based functional genomic platform that allows the identification of new virulence factors through a forward genetic approach firstly described in Mucorales. This platform contains a whole-genome collection of Mucor circinelloides silenced transformants that presented a broad assortment of phenotypes related to the main physiological processes in fungi, including virulence, hyphae morphology, mycelial and yeast growth, carotenogenesis and asexual sporulation. Selection of transformants with reduced virulence allowed the identification of mcplD, which encodes a Phospholipase D, and mcmyo5, encoding a probably essential cargo transporter of the Myosin V family, as required for a fully virulent phenotype of M. circinelloides. Knock-out mutants for those genes showed reduced virulence in both Galleria mellonella and Mus musculus models, probably due to a delayed germination and polarized growth within macrophages. This study provides a robust approach to study virulence in Mucorales and as a proof of concept identified new virulence determinants in M. circinelloides that could represent promising targets for future antifungal therapies