Evaluation of Established Methods for DNA Extraction and Primer Pairs Targeting 16S rRNA Gene for Bacterial Microbiota Profiling of Olive Xylem Sap.

Next-generation sequencing has revolutionized our ability to investigate the microbiota composition of diverse and complex environments. However, a number of factors can affect the accuracy of microbial community assessment, such as the DNA extraction method, the hypervariable region of 16S rRNA gene targeted, or the PCR primers used for amplification. The aim of this study was to assess the influence of commercially available DNA extraction kits and different primer pairs to provide a non-biased vision of the composition of bacterial communities present in olive xylem sap.

Complete Circularized Genome Data of Two Spanish strains of (IVIA5235 and IVIA5901) Using Hybrid Assembly Approaches.

is an economically important plant pathogenic bacterium of global importance associated, since 2013, with a devastating epidemic in olive trees in Italy. Since then, several outbreaks of this pathogen have been reported in other European member countries including Spain, France, and Portugal. In Spain, the three major subspecies (subsp.

The Influence of Different Maternal Microbial Communities on the Development of Infant Gut and Oral Microbiota.

Very few studies have analyzed how the composition of mother's microbiota affects the development of infant's gut and oral microbiota during the first months of life. Here, microbiota present in the mothers' gut, vagina, breast milk, oral cavity, and mammary areola were compared with the gut and oral microbiota of their infants over the first six months following birth. Samples were collected from the aforementioned body sites from seven mothers and nine infants at three different time points over a 6-month period.

Near-Bottom Hypoxia Impacts Dynamics of Bacterioplankton Assemblage throughout Water Column of the Gulf of Finland (Baltic Sea).

Over the past century the spread of hypoxia in the Baltic Sea has been drastic, reaching its 'arm' into the easternmost sub-basin, the Gulf of Finland. The hydrographic and climatological properties of the gulf offer a broad suite of discrete niches for microbial communities. The current study explores spatiotemporal dynamics of bacterioplankton community in the Gulf of Finland using massively parallel sequencing of 16S rRNA fragments obtained by amplifying community DNA from spring to autumn period.

Influence of edaphic, climatic, and agronomic factors on the composition and abundance of nitrifying microorganisms in the rhizosphere of commercial olive crops.

The microbial ecology of the nitrogen cycle in agricultural soils is an issue of major interest. We hypothesized a major effect by farm management systems (mineral versus organic fertilizers) and a minor influence of soil texture and plant variety on the composition and abundance of microbial nitrifiers. We explored changes in composition (16S rRNA gene) of ammonia-oxidizing archaea (AOA), bacteria (AOB), and nitrite-oxidizing bacteria (NOB), and in abundance of AOA and AOB (qPCR of amoA genes) in the rhizosphere of 96 olive orchards differing in climatic conditions, agricultural practices, soil properties, and olive variety.

Redox-specialized bacterioplankton metacommunity in a temperate estuary.

This study explored the spatiotemporal dynamics of the bacterioplankton community composition in the Gulf of Finland (easternmost sub-basin of the Baltic Sea) based on phylogenetic analysis of 16S rDNA sequences acquired from community samples via pyrosequencing. Investigations of bacterioplankton in hydrographically complex systems provide good insight into the strategies by which microbes deal with spatiotemporal hydrographic gradients, as demonstrated by our research. Many ribotypes were closely affiliated with sequences isolated from environments with similar steep physiochemical gradients and/or seasonal changes, including seasonally anoxic estuaries.

Anthropogenic land use shapes the composition and phylogenetic structure of soil arbuscular mycorrhizal fungal communities.

Arbuscular mycorrhizal (AM) fungi play an important role in ecosystems, but little is known about how soil AM fungal community composition varies in relation to habitat type and land-use intensity. We molecularly characterized AM fungal communities in soil samples (n = 88) from structurally open (permanent grassland, intensive and sustainable agriculture) and forested habitats (primeval forest and spruce plantation). The habitats harboured significantly different AM fungal communities, and there was a broad difference in fungal community composition between forested and open habitats, the latter being characterized by higher average AM fungal richness.

Arbuscular mycorhizal fungi associated with the olive crop across the Andalusian landscape: factors driving community differentiation.

Background: In the last years, many olive plantations in southern Spain have been mediated by the use of self-rooted planting stocks, which have incorporated commercial AMF during the nursery period to facilitate their establishment. However, this was practised without enough knowledge on the effect of cropping practices and environment on the biodiversity of AMF in olive orchards in Spain.

Methodology/principal Findings: Two culture-independent molecular methods were used to study the AMF communities associated with olive in a wide-region analysis in southern Spain including 96 olive locations.

Species richness of arbuscular mycorrhizal fungi: associations with grassland plant richness and biomass.

Although experiments show a positive association between vascular plant and arbuscular mycorrhizal fungal (AMF) species richness, evidence from natural ecosystems is scarce. Furthermore, there is little knowledge about how AMF richness varies with belowground plant richness and biomass. We examined relationships among AMF richness, above- and belowground plant richness, and plant root and shoot biomass in a native North American grassland.

Role of mitochondria-cytoskeleton interactions in respiration regulation and mitochondrial organization in striated muscles.

The aim of this work was to study the regulation of respiration and energy fluxes in permeabilized oxidative and glycolytic skeletal muscle fibers, focusing also on the role of cytoskeletal protein tubulin βII isotype in mitochondrial metabolism and organization. By analyzing accessibility of mitochondrial ADP, using respirometry and pyruvate kinase-phosphoenolpyruvate trapping system for ADP, we show that the apparent affinity of respiration for ADP can be directly linked to the permeability of the mitochondrial outer membrane (MOM). Previous studies have shown that MOM permeability in cardiomyocytes can be regulated by VDAC interaction with cytoskeletal protein, βII tubulin.

Changes in the transcriptome of the human endometrial Ishikawa cancer cell line induced by estrogen, progesterone, tamoxifen, and mifepristone (RU486) as detected by RNA-sequencing.

Background: Estrogen (E2) and progesterone (P4) are key players in the maturation of the human endometrium. The corresponding steroid hormone modulators, tamoxifen (TAM) and mifepristone (RU486) are widely used in breast cancer therapy and for contraception purposes, respectively.

Methodology/principal Findings: Gene expression profiling of the human endometrial Ishikawa cancer cell line treated with E2 and P4 for 3 h and 12 h, and TAM and RU486 for 12 h, was performed using RNA-sequencing.

Research resource: small RNA-seq of human granulosa cells reveals miRNAs in FSHR and aromatase genes.

The granulosa cells in the mammalian ovarian follicle respond to gonadotropin signaling and are involved in the processes of folliculogenesis and oocyte maturation. Studies on gene expression and regulation in human granulosa cells are of interest due to their potential for estimating the oocyte viability and in vitro fertilization success. However, the posttranscriptional gene expression studies on micro-RNA (miRNA) level in the human ovary have been scarce.

Global sampling of plant roots expands the described molecular diversity of arbuscular mycorrhizal fungi.

We aimed to enhance understanding of the molecular diversity of arbuscular mycorrhizal fungi (AMF) by building a new global dataset targeting previously unstudied geographical areas. In total, we sampled 96 plant species from 25 sites that encompassed all continents except Antarctica. AMF in plant roots were detected by sequencing the nuclear SSU rRNA gene fragment using either cloning followed by Sanger sequencing or 454-sequencing.

Characterization of the vaginal micro- and mycobiome in asymptomatic reproductive-age Estonian women.

The application of high-throughput sequencing methods has raised doubt in the concept of the uniform healthy vaginal microbiota consisting predominantly of lactobacilli by revealing the existence of more variable bacterial community composition. As this needs to be analyzed more extensively and there is little straightforward data regarding the vaginal mycobiome of asymptomatic women we aimed to define bacterial and fungal communities in vaginal samples from 494 asymptomatic, reproductive-age Estonian women. The composition of the vaginal microbiota was determined by amplifying bacterial 16S rRNA and fungal internal transcribed spacer-1 (ITS-1) regions and subsequently sequencing them using 454 Life Sciences pyrosequencing.

Communities of arbuscular mycorrhizal fungi detected in forest soil are spatially heterogeneous but do not vary throughout the growing season.

Despite the important ecosystem role played by arbuscular mycorrhizal fungi (AMF), little is known about spatial and temporal variation in soil AMF communities. We used pyrosequencing to characterise AMF communities in soil samples (n = 44) from a natural forest ecosystem. Fungal taxa were identified by BLAST matching of reads against the MaarjAM database of AMF SSU rRNA gene diversity.

Mucin5B expression by lung alveolar macrophages is increased in long-term smokers.

This study investigated the expression of MUC5B by AMs in the lungs of cigarette smokers and nonsmokers. We analyzed MUC5B expression by measuring the levels of apomucin and mRNA in human BALF cells from 50 subjects (20 nonsmokers, 17 patients with CB, and 13 patients with COPD). apoMUC5B was observed in BALF mononuclear cells in 60% of all subjects, but a significantly higher frequency of apoMUC5B(+) cells was found in subjects with CB (95% CI, 4.

Plant species richness belowground: higher richness and new patterns revealed by next-generation sequencing.

Variation in plant species richness has been described using only aboveground vegetation. The species richness of roots and rhizomes has never been compared with aboveground richness in natural plant communities. We made direct comparisons of grassland plant richness in identical volumes (0.

Mitochondria-cytoskeleton interaction: distribution of β-tubulins in cardiomyocytes and HL-1 cells.

Mitochondria-cytoskeleton interactions were analyzed in adult rat cardiomyocytes and in cancerous non-beating HL-1 cells of cardiac phenotype. We show that in adult cardiomyocytes βII-tubulin is associated with mitochondrial outer membrane (MOM). βI-tubulin demonstrates diffused intracellular distribution, βIII-tubulin is colocalized with Z-lines and βIV-tubulin forms microtubular network.

Genes targeted by the estrogen and progesterone receptors in the human endometrial cell lines HEC1A and RL95-2.

Background: When the steroid hormones estrogen and progesterone bind to nuclear receptors, they have transcriptional impact on target genes in the human endometrium. These transcriptional changes have a critical function in preparing the endometrium for embryo implantation.

Methods: 382 genes were selected, differentially expressed in the receptive endometrium, to study their responsiveness of estrogen and progesterone.

Large-scale parallel 454 sequencing reveals host ecological group specificity of arbuscular mycorrhizal fungi in a boreonemoral forest.

* Knowledge of the diversity of arbuscular mycorrhizal fungi (AMF) in natural ecosystems is a major bottleneck in mycorrhizal ecology. Here, we aimed to apply 454 sequencing--providing a new level of descriptive power--to assess the AMF diversity in a boreonemoral forest. * 454 sequencing reads of the small subunit ribosomal RNA (SSU rRNA) gene of Glomeromycota were assigned to sequence groups by blast searches against a custom-made annotated sequence database.

Identification of the gene transcription repressor domain of Gli3.

Gli transcription factors are downstream targets of the Hedgehog signaling pathway. Two of the three Gli proteins harbor gene transcription repressor function in the N-terminal half. We have analyzed the sequences and identified a potential repressor domain in Gli2 and Gli3 and have tested this experimentally.

Sponge OAS has a distinct genomic structure within the 2-5A synthetase family.

2',5'-Oligoadenylate synthetases (2-5A synthetases, OAS) are enzymes that play an important role in the interferon-induced antiviral defense mechanisms in mammals. Sponges, the evolutionarily lowest multicellular animals, also possess OAS; however, their function is presently unclear. Low homology between primary structures of 2-5A synthetases from vertebrates and sponges renders their evolutionary relationship obscure.

Combination of native and denaturing PAGE for the detection of protein binding regions in long fragments of genomic DNA.

Background: In a traditional electrophoresis mobility shift assay (EMSA) a 32P-labeled double-stranded DNA oligonucleotide or a restriction fragment bound to a protein is separated from the unbound DNA by polyacrylamide gel electrophoresis (PAGE) in nondenaturing conditions. An extension of this method uses the large population of fragments derived from long genomic regions (approximately 600 kb) for the identification of fragments containing protein binding regions. With this method, genomic DNA is fragmented by restriction enzymes, fragments are amplified by PCR, radiolabeled, incubated with nuclear proteins and the resulting DNA-protein complexes are separated by two-dimensional PAGE.

Corticosterone actions on the hippocampal brain-derived neurotrophic factor expression are mediated by exon IV promoter.

Brain-derived neurotrophic factor (BDNF) expression is strongly regulated by adrenocorticosteroids via activated gluco- and mineralocorticoid receptors. Four separate promoters are located upstream of the BDNF noncoding exons I to IV and may thus be involved in adrenocorticosteroid-mediated gene regulation. In adrenalectomised rats, corticosterone (10 mg/kg s.