Combined Mutation And Rearrangement Screening by Quantitative PCR High-Resolution Melting: Is It Relevant for Hereditary Recurrent Fever Genes?

The recent identification of genes implicated in hereditary recurrent fevers has allowed their specific diagnosis. So far however, only punctual mutations have been identified and a significant number of patients remain with no genetic confirmation of their disease after routine molecular approaches such as sequencing. The possible involvement of sequence rearrangements in these patients has only been examined in familial Mediterranean fever and was found to be unlikely. To assess the existence of larger genetic alterations in 3 other concerned genes, MVK (Mevalonate kinase), NLRP3 (Nod like receptor family, pyrin domain containing 3) and TNFRSF1A (TNF receptor superfamily 1A), we adapted the qPCR-HRM method to study possible intragenic deletions and duplications. This single-tube approach, combining both qualitative (mutations) and quantitative (rearrangement) screening, has proven effective in Lynch syndrome diagnosis. Using this approach, we studied 113 unselected (prospective group) and 88 selected (retrospective group) patients and identified no intragenic rearrangements in the 3 genes. Only qualitative alterations were found with a sensitivity similar to that obtained using classical molecular techniques for screening punctual mutations. Our results support that deleterious copy number alterations in MVK, NLRP3 and TNFRSF1A are rare or absent from the mutational spectrum of hereditary recurrent fevers, and demonstrate that a routine combined method such as qPCR-HRM provides no further help in genetic diagnosis. However, quantitative approaches such as qPCR or SQF-PCR did prove to be quick and effective and could still be useful after non contributory punctual mutation screening in the presence of clinically evocative signs.     

Trace-Level Determination of Oxolinic Acid and Flumequine in Soil,River Bed Sediment,and River Water Using Microwave-Assisted Extraction and High-Performance Liquid Chromatography with Fluorimetric Detection

This work presents the optimization of analytical procedures for the determination of two antibiotics, oxolinic acid (OA) and flumequine (FL), in bed sediment, river water, and soil samples. Three extraction methods (microwave-assisted extraction (MAE), ultrasonication, and reflux) were tested, and the highest recoveries were obtained with MAE (94 ± 3% and 95 ± 3% for OA and FL, respectively). A solid-phase extraction (SPE) clean-up step was optimized by comparing two polymeric sorbents: Oasis HLB and Oasis MAX. The final extracts were analyzed by liquid chromatography with fluorimetric detection. Limits of detection (LOD) obtained for OA and FL in soil and sediment ranged from 0.3 to 0.5 µg kg^?1. Meanwhile, a novel SPE procedure was also implemented for OA and FL determination in river water samples. It also relied on the use of Oasis MAX, and recovery rates were in the range 90–94%; LODs were 2 ng L^?1 for both OA and FL. These methods were applied for the analysis of samples taken in the Seine River basin (France). The obtained results demonstrated the widespread occurrence of OA and FL, at ng L^?1 and µg kg^?1 levels in water and sediment/soil, respectively, and their persistence in the environment.     

Crown defoliation decreases reproduction and wood growth in a marginal European beech population

Background and AimsAbiotic and biotic stresses related to climate change have been associated with increased crown defoliation, decreased growth and a higher risk of mortality in many forest tree species, but the impact of stresses on tree reproduction and forest regeneration remains understudied. At the dry, warm margin of species distributions, flowering, pollination and seed maturation are expected to be affected by drought, late frost and other stresses, eventually resulting in reproduction failure. Moreover, inter-individual variation in reproductive performance versus other performance traits (growth, survival) could have important consequences for population dynamics. This study investigated the relationships among individual crown defoliation, growth and reproduction in a drought-prone population of European beech, Fagus sylvatica.MethodsWe used a spatially explicit mating model and marker-based parentage analyses to estimate effective female and male fecundities of 432 reproductive trees, which were also monitored for basal area increment and crown defoliation over 9 years.Key ResultsFemale and male fecundities varied markedly between individuals, more than did growth. Both female fecundity and growth decreased with increasing crown defoliation and competition, and increased with size. Moreover, the negative effect of defoliation on female fecundity was size-dependent, with a slower decline in female fecundity with increasing defoliation for the large individuals. Finally, a trade-off between growth and female fecundity was observed in response to defoliation: some large trees maintained significant female fecundity at the expense of reduced growth in response to defoliation, while some other defoliated trees maintained high growth at the expense of reduced female fecundity.ConclusionsOur results suggest that, while decreasing their growth, some large defoliated trees still contribute to reproduction through seed production and pollination. This non-coordinated decline of growth and fecundity at individual level in response to stress may compromise the evolution of stress-resistance traits at population level, and increase forest tree vulnerability.     

A Medicago truncatula NADPH oxidase is involved in symbiotic nodule functioning

The plant plasma membrane-localized NADPH oxidases, known as respiratory burst oxidase homologues (RBOHs), appear to play crucial roles in plant growth and development. They are involved in important processes, such as root hair growth, plant defence reactions and abscisic acid signalling. Using sequence similarity searches, we identified seven putative RBOH-encoding genes in the Medicago truncatula genome. A phylogenetic reconstruction showed that Rboh gene duplications occurred in legume species. We analysed the expression of these MtRboh genes in different M. truncatula tissues: one of them, MtRbohA, was significantly up-regulated in Sinorhizobium meliloti-induced symbiotic nodules. MtRbohA expression appeared to be restricted to the nitrogen-fixing zone of the functional nodule. Moreover, using S. meliloti bacA and nifH mutants unable to form efficient nodules, a strong link between nodule nitrogen fixation and MtRbohA up-regulation was shown. MtRbohA expression was largely enhanced under hypoxic conditions. Specific RNA interference for MtRbohA provoked a decrease in the nodule nitrogen fixation activity and the modulation of genes encoding the microsymbiont nitrogenase. These results suggest that hypoxia, prevailing in the nodule-fixing zone, may drive the stimulation of MtRbohA expression, which would, in turn, lead to the regulation of nodule functioning.     

Peroxynitrite formation and function in plants

Peroxynitrite (ONOO⁻) is a reactive nitrogen species formed when nitric oxide (NO) reacts with the superoxide anion (O₂⁻). It was first identified as a mediator of cell death in animals but was later shown to act as a positive regulator of cell signaling, mainly through the posttranslational modification of proteins by tyrosine nitration. In plants, peroxynitrite is not involved in NO-mediated cell death and its physiological function is poorly understood. However, it is emerging as a potential signaling molecule during the induction of defense responses against pathogens and this could be mediated by the selective nitration of tyrosine residues in a small number of proteins. In this review we discuss the general role of tyrosine nitration in plants and evaluate recent evidence suggesting that peroxynitrite is an effector of NO-mediated signaling following pathogen infection.     

Regulation of epithelial to mesenchymal transition: CK2�� on stage

Protein kinase CK2 participates in the regulation of fundamental cellular processes. Among these processes, cell polarity and cell morphology are controlled by this enzyme probably through the phosphorylation of key proteins. To further study the involvement of CK2 in these processes, we showed that in epithelial cells, the regulatory CK2β subunit was required for LKB1-dependent polarization and cell adhesion. Moreover, CK2β silencing in MCF10A mammary epithelial cells triggered changes in their morphology correlated with the acquisition of mesenchymal phenotype, which were reminiscent to TGFβ-induced epithelial-to-mesenchymal-transition (EMT). TGFβ has emerged as a major inducer of EMT both in vitro and in vivo. We found that among the TGFβ isoforms, TGFβ2 expression was strongly induced in CK2β-knockdown cells. However, the EMT phenotype induced in response to CK2β silencing was not abolished by blocking the TGFβ signaling pathway at TGFβ receptor level, suggesting that alternative pathways might be involved. Given the importance of CK2 in tumorigenesis, a dysregulation of CK2β expression might contribute to EMT induction during cancer progression.     

Phylogeography of the spring and fall waves of the H1N1/09 pandemic influenza virus in the United States

Spatial variation in the epidemiological patterns of successive waves of pandemic influenza virus in humans has been documented throughout the 20th century but never understood at a molecular level. However, the unprecedented intensity of sampling and whole-genome sequencing of the H1N1/09 pandemic virus now makes such an approach possible. To determine whether the spring and fall waves of the H1N1/09 influenza pandemic were associated with different epidemiological patterns, we undertook a large-scale phylogeographic analysis of viruses sampled from three localities in the United States. Analysis of genomic and epidemiological data reveals distinct spatial heterogeneities associated with the first pandemic wave, March to July 2009, in Houston, TX, Milwaukee, WI, and New York State. In Houston, no specific H1N1/09 viral lineage dominated during the spring of 2009, a period when little epidemiological activity was observed in Texas. In contrast, major pandemic outbreaks occurred at this time in Milwaukee and New York State, each dominated by a different viral lineage and resulting from strong founder effects. During the second pandemic wave, beginning in August 2009, all three U.S. localities were dominated by a single viral lineage, that which had been dominant in New York during wave 1. Hence, during this second phase of the pandemic, extensive viral migration and mixing diffused the spatially defined population structure that had characterized wave 1, amplifying the one viral lineage that had dominated early on in one of the world's largest international travel centers.     

Rio1 mediates ATP-dependent final maturation of 40S ribosomal subunits

During the last step in 40S ribosome subunit biogenesis, the PIN-domain endonuclease Nob1 cleaves the 20S pre-rRNA at site D, to form the mature 18S rRNAs. Here we report that cleavage occurs in particles that have largely been stripped of previously characterized pre-40S components, but retain the endonuclease Nob1, its binding partner Pno1 (Dim2) and the atypical ATPase Rio1. Within the Rio1-associated pre-40S particles, in vitro pre-rRNA cleavage was strongly stimulated by ATP and required nucleotide binding by Rio1. In vivo binding sites for Rio1, Pno1 and Nob1 were mapped by UV cross-linking in actively growing cells. Nob1 and Pno1 bind overlapping regions within the internal transcribed spacer 1, and both bind directly over cleavage site D. Binding sites for Rio1 were within the core of the 18S rRNA, overlapping tRNA interaction sites and distinct from the related kinase Rio2. Site D cleavage occurs within pre-40S-60S complexes and Rio1-associated particles efficiently assemble into these complexes, whereas Pno1 appeared to be depleted relative to Nob1. We speculate that Rio1-mediated dissociation of Pno1 from cleavage site D is the trigger for final 18S rRNA maturation.