Genic SNP markers and legume synteny reveal candidate genes underlying QTL for Macrophomina phaseolina resistance and maturity in cowpea [Vigna unguiculata (L) Walp.]

<p>Abstract</p> <p>Background</p> <p><it>Macrophomina phaseolina </it>is an emerging and devastating fungal pathogen that causes significant losses in crop production under high temperatures and drought stress. An increasing number of disease incidence reports highlight the wide prevalence of the pathogen around the world and its contribution toward crop yield suppression. In cowpea [<it>Vigna unguiculata </it>(L) Walp.], limited sources of low-level host resistance have been identified, the genetic basis of which is unknown. In this study we report on the identification of strong sources of host resistance to <it>M. phaseolina </it>and the genetic mapping of putative resistance loci on a cowpea genetic map comprised of gene-derived single nucleotide polymorphisms (SNPs) and amplified fragment length polymorphisms (AFLPs).</p> <p>Results</p> <p>Nine quantitative trait loci (QTLs), accounting for between 6.1 and 40.0% of the phenotypic variance (R²), were identified using plant mortality data taken over three years in field experiments and disease severity scores taken from two greenhouse experiments. Based on annotated genic SNPs as well as synteny with soybean (<it>Glycine max</it>) and <it>Medicago truncatula</it>, candidate resistance genes were found within mapped QTL intervals. QTL <it>Mac-2 </it>explained the largest percent R²and was identified in three field and one greenhouse experiments where the QTL peak co-located with a SNP marker derived from a pectin esterase inhibitor encoding gene. Maturity effects on the expression of resistance were indicated by the co-location of <it>Mac-6 </it>and <it>Mac-7 </it>QTLs with maturity-related senescence QTLs <it>Mat-2 </it>and <it>Mat-1</it>, respectively. Homologs of the <it>ELF4 </it>and <it>FLK </it>flowering genes were found in corresponding syntenic soybean regions. Only three <it>Macrophomina </it>resistance QTLs co-located with delayed drought-induced premature senescence QTLs previously mapped in the same population, suggesting that largely different genetic mechanisms mediate cowpea response to drought stress and <it>Macrophomina </it>infection.</p> <p>Conclusion</p> <p>Effective sources of host resistance were identified in this study. QTL mapping and synteny analysis identified genomic loci harboring resistance factors and revealed candidate genes with potential for further functional genomics analysis.</p

Fibroblasts Express Immune Relevant Genes and Are Important Sentinel Cells during Tissue Damage in Rainbow Trout (Oncorhynchus mykiss)

Fibroblasts have shown to be an immune competent cell type in mammals. However, little is known about the immunological functions of this cell-type in lower vertebrates. A rainbow trout hypodermal fibroblast cell-line (RTHDF) was shown to be responsive to PAMPs and DAMPs after stimulation with LPS from E. coli, supernatant and debris from sonicated RTHDF cells. LPS was overall the strongest inducer of IL-1β, IL-8, IL-10, TLR-3 and TLR-9. IL-1β and IL-8 were already highly up regulated after 1 hour of LPS stimulation. Supernatant stimuli significantly increased the expression of IL-1β, TLR-3 and TLR-9, whereas the debris stimuli only increased expression of IL-1β. Consequently, an in vivo experiment was further set up. By mechanically damaging the muscle tissue of rainbow trout, it was shown that fibroblasts in the muscle tissue of rainbow trout contribute to electing a highly local inflammatory response following tissue injury. The damaged muscle tissue showed a strong increase in the expression of the immune genes IL-1β, IL-8 and TGF-β already 4 hours post injury at the site of injury while the expression in non-damaged muscle tissue was not influenced. A weaker, but significant response was also seen for TLR-9 and TLR-22. Rainbow trout fibroblasts were found to be highly immune competent with a significant ability to express cytokines and immune receptors. Thus fish fibroblasts are believed to contribute significantly to local inflammatory reactions in concert with the traditional immune cells

Atlantic salmon cardiac primary cultures:An in vitro model to study viral host pathogen interactions and pathogenesis

Development of Salmon Cardiac Primary Cultures (SCPCs) from Atlantic salmon pre-hatch embryos and their application as in vitro model for cardiotropic viral infection research are described. Producing SCPCs requires plating of trypsin dissociated embryos with subsequent targeted harvest from 24h up to 3 weeks, of relevant tissues after visual identification. SCPCs are then transferred individually to chambered wells for culture in isolation, with incubation at 15-22°. SCPCs production efficiency was not influenced by embryo's origin (0.75/ farmed or wild embryo), but mildly influenced by embryonic developmental stage (0.3 decline between 380 and 445 accumulated thermal units), and strongly influenced by time of harvest post-plating (0.6 decline if harvested after 72 hours). Beating rate was not significantly influenced by temperature (15-22°) or age (2-4 weeks), but was significantly lower on SCPCs originated from farmed embryos with a disease resistant genotype (F = 5.3, p<0.05). Two distinct morphologies suggestive of an ex vivo embryonic heart and a de novo formation were observed sub-grossly, histologically, ultra-structurally and with confocal microscopy. Both types contained cells consistent with cardiomyocytes, endothelium, and fibroblasts. Ageing of SCPCs in culture was observed with increased auto fluorescence in live imaging, and as myelin figures and cellular degeneration ultra-structurally. The SCPCs model was challenged with cardiotropic viruses and both the viral load and the mx gene expression were measurable along time by qPCR. In summary, SCPCs represent a step forward in salmon cardiac disease research as an in vitro model that partially incorporates the functional complexity of the fish heart

‘Classical' but not ‘other' mutations of EGFR kinase domain are associated with clinical outcome in gefitinib-treated patients with non-small cell lung cancer

‘Classical' mutations in the EGFR tyrosine kinase domain (exons 18, 19 and 21) have been associated with sensitivity to tyrosine kinase inhibitors (TKIs) in patients with NSCLC. The aim of the current study was to evaluate whether other than the classical G719X, DEL19 and L858R mutations of EGFR confer sensitivity to TKIs. Genomic DNA was extracted from microdissected formalin-fixed paraffin-embedded tumour tissue from 86 patients treated with gefitinib. Exons 18, 19 and 21 were amplified and subjected to direct sequencing. Eleven (13%) patients harboured the classical exon's 18, 19 and 21 mutations, while 14 (16%) had ‘other' variants. There was a significantly higher percentage of ‘never-smoker' patients with ‘classical' EGFR mutations (P=0.002). Among patients with ‘classical' mutations 3 patients achieved PR and 7 SD, while in the ‘other' mutations group 10 patients had SD as best response. In the wild-type group, there were 3 patients with PR and 25 with SD. Median TTP was 16, 64 (P=0.002) and 21 weeks and median survival was 36, 78 and 67 weeks for patients with wild-type, ‘classical' and ‘other' EGFR mutations, respectively. The clinical relevance of ‘other' EGFR mutation variants remains uncertain and requires further assessment in a prospective study

The Rac GTP Exchange Factor TIAM-1 Acts with CDC-42 and the Guidance Receptor UNC-40/DCC in Neuronal Protrusion and Axon Guidance

The mechanisms linking guidance receptors to cytoskeletal dynamics in the growth cone during axon extension remain mysterious. The Rho-family GTPases Rac and CDC-42 are key regulators of growth cone lamellipodia and filopodia formation, yet little is understood about how these molecules interact in growth cone outgrowth or how the activities of these molecules are regulated in distinct contexts. UNC-73/Trio is a well-characterized Rac GTP exchange factor in Caenorhabditis elegans axon pathfinding, yet UNC-73 does not control CED-10/Rac downstream of UNC-6/Netrin in attractive axon guidance. Here we show that C. elegans TIAM-1 is a Rac-specific GEF that links CDC-42 and Rac signaling in lamellipodia and filopodia formation downstream of UNC-40/DCC. We also show that TIAM-1 acts with UNC-40/DCC in axon guidance. Our results indicate that a CDC-42/TIAM-1/Rac GTPase signaling pathway drives lamellipodia and filopodia formation downstream of the UNC-40/DCC guidance receptor, a novel set of interactions between these molecules. Furthermore, we show that TIAM-1 acts with UNC-40/DCC in axon guidance, suggesting that TIAM-1 might regulate growth cone protrusion via Rac GTPases in response to UNC-40/DCC. Our results also suggest that Rac GTPase activity is controlled by different GEFs in distinct axon guidance contexts, explaining how Rac GTPases can specifically control multiple cellular functions

A proteomics approach to decipher the molecular nature of planarian stem cells

Background In recent years, planaria have emerged as an important model system for research into stem cells and regeneration. Attention is focused on their unique stem cells, the neoblasts, which can differentiate into any cell type present in the adult organism. Sequencing of the Schmidtea mediterranea genome and some expressed sequence tag projects have generated extensive data on the genetic profile of these cells. However, little information is available on their protein dynamics. Results We developed a proteomic strategy to identify neoblast-specific proteins. Here we describe the method and discuss the results in comparison to the genomic high-throughput analyses carried out in planaria and to proteomic studies using other stem cell systems. We also show functional data for some of the candidate genes selected in our proteomic approach. Conclusions We have developed an accurate and reliable mass-spectra-based proteomics approach to complement previous genomic studies and to further achieve a more accurate understanding and description of the molecular and cellular processes related to the neoblasts

Time-resolved single-crystal X-ray crystallography

In this chapter the development of time-resolved crystallography is traced from its beginnings more than 30 years ago. The importance of being able to “watch” chemical processes as they occur rather than just being limited to three-dimensional pictures of the reactant and final product is emphasised, and time-resolved crystallography provides the opportunity to bring the dimension of time into the crystallographic experiment. The technique has evolved in time with developments in technology: synchrotron radiation, cryoscopic techniques, tuneable lasers, increased computing power and vastly improved X-ray detectors. The shorter the lifetime of the species being studied, the more complex is the experiment. The chapter focusses on the results of solid-state reactions that are activated by light, since this process does not require the addition of a reagent to the crystalline material and the single-crystalline nature of the solid may be preserved. Because of this photoactivation, time-resolved crystallography is often described as “photocrystallography”. The initial photocrystallographic studies were carried out on molecular complexes that either underwent irreversible photoactivated processes where the conversion took hours or days. Structural snapshots were taken during the process. Materials that achieved a metastable state under photoactivation and the excited (metastable) state had a long enough lifetime for the data from the crystal to be collected and the structure solved. For systems with shorter lifetimes, the first time-resolved results were obtained for macromolecular structures, where pulsed lasers were used to pump up the short lifetime excited state species and their structures were probed by using synchronised X-ray pulses from a high-intensity source. Developments in molecular crystallography soon followed, initially with monochromatic X-ray radiation, and pump-probe techniques were used to establish the structures of photoactivated molecules with lifetimes in the micro- to millisecond range. For molecules with even shorter lifetimes in the sub-microsecond range, Laue diffraction methods (rather than using monochromatic radiation) were employed to speed up the data collections and reduce crystal damage. Future developments in time-resolved crystallography are likely to involve the use of XFELs to complete “single-shot” time-resolved diffraction studies that are already proving successful in the macromolecular crystallographic field.</p

Vivências de familiares de crianças internadas em um Serviço de Pronto-Socorro

A infância apresenta-se como uma fase que exige bastante atenção da família e do serviço de saúde, uma vez que seus integrantes, além de dependerem de familiares, são vulneráveis ao ambiente. Objetivou-se descrever as vivências de familiares de crianças internadas em um serviço de pronto-socorro, discutir como essas vivências influenciam no cotidiano da família e relatar os aspectos que interferem no cuidado de enfermagem. Estudo descritivo, de abordagem qualitativa, desenvolvido em um hospital de urgência da rede privada. Utilizou a técnica de entrevista com dez familiares para produzir os dados. Estes foram submetidos à análise temática, elaborando-se três categorias: vivências do familiar, alterações no cotidiano da família, a fé e a aproximação familiar atuando como agentes facilitadores. Concluiu-se que o ser acompanhante passa por adaptações, ao vivenciar a hospitalização, existindo alterações na rotina familiar. Porém, devido aos conflitos vivenciados pelo familiar, a enfermagem deve compreendê-lo como sujeito do cuidado ampliado

HAD hydrolase function unveiled by substrate screening: enzymatic characterization of Arabidopsis thaliana subclass I phosphosugar phosphatise AtSgpp

[EN] This work presents the isolation and the biochemical characterization of the Arabidopsis thaliana gene AtSgpp. This gene shows homology with the Arabidopsis low molecular weight phosphatases AtGpp1 and AtGpp2 and the yeast counterpart GPP1 and GPP2, which have a high specificity for dl-glycerol-3-phosphate. In addition, it exhibits homology with DOG1 and DOG2 that dephosphorylate 2-deoxy-d-glucose-6-phosphate. Using a comparative genomic approach, we identified the AtSgpp gene as a conceptual translated haloacid dehalogenase-like hydrolase HAD protein. AtSgpp (locus tag At2g38740), encodes a protein with a predicted Mw of 26.7 kDa and a pI of 4.6. Its sequence motifs and expected structure revealed that AtSgpp belongs to the HAD hydrolases subfamily I, with the C1-type cap domain. In the presence of Mg2+ ions, the enzyme has a phosphatase activity over a wide range of phosphosugars substrates (pH optima at 7.0 and K (m) in the range of 3.6-7.7 mM). AtSgpp promiscuity is preferentially detectable on d-ribose-5-phosphate, 2-deoxy-d-ribose-5-phosphate, 2-deoxy-d-glucose-6-phosphate, d-mannose-6-phosphate, d-fructose-1-phosphate, d-glucose-6-phosphate, dl-glycerol-3-phosphate, and d-fructose-6-phosphate, as substrates. AtSgpp is ubiquitously expressed throughout development in most plant organs, mainly in sepal and guard cell. Interestingly, expression is affected by abiotic and biotic stresses, being the greatest under Pi starvation and cyclopentenone oxylipins induction. Based on both, substrate lax specificity and gene expression, the physiological function of AtSgpp in housekeeping detoxification, modulation of sugar-phosphate balance and Pi homeostasis, is provisionally assigned.We acknowledge Professors Montserrat Pages (CSIC Barcelona, Spain), Thomas Kupke (University of Heidelberg, Germany) and Manuel Hernandez (University Polytechnic of Valencia, Spain) for their warm support. 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