De Novo Assembly and Functional Annotation of the Olive (Olea europaea) Transcriptome

Olive breeding programmes are focused on selecting for traits as short juvenile period, plant architecture suited for mechanical harvest, or oil characteristics, including fatty acid composition, phenolic, and volatile compounds to suit new markets. Understanding the molecular basis of these characteristics and improving the efficiency of such breeding programmes require the development of genomic information and tools. However, despite its economic relevance, genomic information on olive or closely related species is still scarce. We have applied Sanger and 454 pyrosequencing technologies to generate close to 2 million reads from 12 cDNA libraries obtained from the Picual, Arbequina, and Lechin de Sevilla cultivars and seedlings from a segregating progeny of a Picual × Arbequina cross. The libraries include fruit mesocarp and seeds at three relevant developmental stages, young stems and leaves, active juvenile and adult buds as well as dormant buds, and juvenile and adult roots. The reads were assembled by library or tissue and then assembled together into 81 020 unigenes with an average size of 496 bases. Here, we report their assembly and their functional annotation.     

Candida albicans stimulates in vivo differentiation of haematopoietic stem and progenitor cells towards macrophages by a TLR2‐dependent signalling

Toll‐like receptors (TLRs) are expressed by haematopoietic stem and progenitor cells (HSPCs), and may play a role in haematopoiesis in response to pathogens during infection. We have previously demonstrated that (i) inactivated yeasts of Candida albicans induce in vitro differentiation of HSPCs towards the myeloid lineage, and (ii) soluble TLR agonists induce in vivo their differentiation towards macrophages. In this work, using an in vivo model of HSPCs transplantation, we report for the first time that HSPCs sense C. albicans in vivo and subsequently are directed to produce macrophages by a TLR2‐dependent signalling. Purified lineage‐negative cells (Lin^?) from bone marrow of C57BL/6 mice (CD45.2 alloantigen) were transplanted into B6Ly5.1 mice (CD45.1 alloantigen), which were then injected with viable or inactivated C. albicans yeasts. Transplanted cells were detected in the spleen and in the bone marrow of recipient mice, and they differentiate preferentially to macrophages, both in response to infection or in response to inactivated yeasts. The generation of macrophages was dependent on TLR2 but independent of TLR4, as transplanted Lin^? cells from TLR2^?/? mice did not give rise to macrophages, whereas Lin^? cells from TLR4^?/? mice generated macrophages similarly to control cells. Interestingly, the absence of TLR2, or in a minor extent TLR4, gives Lin^? cells an advantage in transplantation assays, as increases the percentage of transplanted recovered cells. Our results indicatethat TLR‐mediated recognition of C. albicans by HSPCs may help replace and/or increase cells that constitute the first line of defence against the fungus, and suggest that TLR‐mediated signalling may lead to reprogramming early progenitors to rapidly replenishing the innate immune system and generate the most necessary mature cells to deal with the pathogen.     

Avoiding pitfalls in bone marrow engraftment analysis: a case study highlighting the weakness of using buccal cells for determining a patient's constitutional genotype after hematopoietic stem cell transplantation

Background aimsAn accurate and reliable assessment of bone marrow engraftment (BME) after hematopoietic stem cell transplantation (HSCT) is based on the ability to distinguish between recipient and donor cells at selected polymorphic short tandem repeat (STR) DNA loci. Buccal cells are an important source of DNA for determining the recipient's constitutional genotype, particularly in patients transplanted before the STR evaluation.MethodsGenomic DNA was extracted from the recipient buccal cells and from isolated CD3⁺ (T-cell lymphocyte) and CD33⁺ (myelocyte) cells after HSCT. BME analysis was performed using a STR-based polymerase chain reaction amplification method followed by fragment-size analysis for assessing the recipient-derived or donor-derived composition of cell lineage-specific peripheral blood DNA.ResultsWe identified three cases of complete buccal epithelial cell engraftment after HSCT detected by BME analysis, potentially leading to misinterpretation of testing results if these cells were used as the sole source for determining the recipient's genotype.ConclusionsThese cases suggest that complete engraftment of buccal epithelial cells may be a common finding in patients receiving HSCT, drawing attention to important issues such as the type of samples used for determining a patient's constitutional genotype that may confound testing results. This study also highlights the need for careful interpretation of the BME testing results in the context of the clinical findings.     

Co-regulation of senescence-associated genes by oncogenic homeobox proteins and polycomb repressive complexes

Cellular senescence is a stable cell cycle arrest that can be induced by stresses such as telomere shortening, oncogene activation or DNA damage. Senescence is a potent anticancer barrier that needs to be circumvented during tumorigenesis. The cell cycle regulator p16^INK4a is a key effector upregulated during senescence. Polycomb repressive complexes (PRCs) play a crucial role in silencing the INK4/ARF locus, which encodes for p16^INK4a, but the mechanisms by which PRCs are recruited to this locus as well as to other targets remain poorly understood. Recently we discovered the ability of the homeobox proteins HLX1 (H2.0-like homeobox 1) and HOXA9 (Homeobox A9) to bypass senescence. We showed that HLX1 and HOXA9 recruit PRCs to repress INK4a, which constitutes a key mechanism explaining their effects on senescence. Here we provide evidence for the regulation of additional senescence-associated PRC target genes by HLX1 and HOXA9. As both HLX1 and HOXA9 are oncogenes implicated in leukemogenesis, we discuss the implications that the collaboration between Homeobox proteins and PRCs has for senescence and cancer.     

An Abscisic Acid-Independent Oxylipin Pathway Controls Stomatal Closure and Immune Defense in Arabidopsis

Plant stomata function in innate immunity against bacterial invasion and abscisic acid (ABA) has been suggested to regulate this process. Using genetic, biochemical, and pharmacological approaches, we demonstrate that (i) the Arabidopsis thaliana nine-specific-lipoxygenase encoding gene, LOX1, which is expressed in guard cells, is required to trigger stomatal closure in response to both bacteria and the pathogen-associated molecular pattern flagellin peptide flg22; (ii) LOX1 participates in stomatal defense; (iii) polyunsaturated fatty acids, the LOX substrates, trigger stomatal closure; (iv) the LOX products, fatty acid hydroperoxides, or reactive electrophile oxylipins induce stomatal closure; and (v) the flg22-mediated stomatal closure is conveyed by both LOX1 and the mitogen-activated protein kinases MPK3 and MPK6 and involves salicylic acid whereas the ABA-induced process depends on the protein kinases OST1, MPK9, or MPK12. Finally, we show that the oxylipin and the ABA pathways converge at the level of the anion channel SLAC1 to regulate stomatal closure. Collectively, our results demonstrate that early biotic signaling in guard cells is an ABA-independent process revealing a novel function of LOX1-dependent stomatal pathway in plant immunity.     

The Fusarium oxysporum gnt2, Encoding a Putative N-Acetylglucosamine Transferase,Is Involved in Cell Wall Architecture and Virulence

With the aim to decipher the molecular dialogue and cross talk between Fusarium oxysporum f.sp. lycopersci and its host during infection and to understand the molecular bases that govern fungal pathogenicity, we analysed genes presumably encoding N-acetylglucosaminyl transferases, involved in glycosylation of glycoproteins, glycolipids, proteoglycans or small molecule acceptors in other microorganisms. In silico analysis revealed the existence of seven putative N-glycosyl transferase encoding genes (named gnt) in F. oxysporum f.sp. lycopersici genome. gnt2 deletion mutants showed a dramatic reduction in virulence on both plant and animal hosts. Δgnt2 mutants had αalterations in cell wall properties related to terminal αor β-linked N-acetyl glucosamine. Mutant conidia and germlings also showed differences in structure and physicochemical surface properties. Conidial and hyphal aggregation differed between the mutant and wild type strains, in a pH independent manner. Transmission electron micrographs of germlings showed strong cell-to-cell adherence and the presence of an extracellular chemical matrix. Δgnt2 cell walls presented a significant reduction in N-linked oligosaccharides, suggesting the involvement of Gnt2 in N-glycosylation of cell wall proteins. Gnt2 was localized in Golgi-like sub-cellular compartments as determined by fluorescence microscopy of GFP::Gnt2 fusion protein after treatment with the antibiotic brefeldin A or by staining with fluorescent sphingolipid BODIPY-TR ceramide. Furthermore, density gradient ultracentrifugation allowed co-localization of GFP::Gnt2 fusion protein and Vps10p in subcellular fractions enriched in Golgi specific enzymatic activities. Our results suggest that N-acetylglucosaminyl transferases are key components for cell wall structure and influence interactions of F. oxysporum with both plant and animal hosts during pathogenicity.     

Associations between Level and Change in Physical Function and Brain Volumes

Background

Higher levels of fitness or physical function are positively associated with cognitive outcomes but the potential underlying mechanisms via brain structure are still to be elucidated in detail. We examined associations between brain structure and physical function (contemporaneous and change over the previous three years) in community-dwelling older adults.

Methodology/Principal Findings

Participants from the Lothian Birth Cohort 1936 (N=694) underwent brain MRI at age 73 years to assess intracranial volume, and the volumes of total brain tissue, ventricles, grey matter, normal-appearing white matter, and white matter lesions. At ages 70 and 73, physical function was assessed by 6-meter walk, grip strength, and forced expiratory volume. A summary ‘physical function factor’ was derived from the individual measures using principal components analysis. Performance on each individual physical function measure declined across the three year interval (p<0.001). Higher level of physical function at ages 70 and 73 was associated with larger total brain tissue and white matter volumes, and smaller ventricular and white matter lesion volumes (standardized β ranged in magnitude from 0.07 to 0.17, p<0.001 to 0.034). Decline in physical function from age 70 to 73 was associated with smaller white matter volume (0.08, p<0.01, though not after correction for multiple testing), but not with any other brain volumetric measurements.

Conclusions/Significance

Physical function was related to brain volumes in community-dwelling older adults: declining physical function was associated with less white matter tissue. Further study is required to explore the detailed mechanisms through which physical function might influence brain structure, and vice versa.