Molecular phylogeny and divergence times of drosophilid species

The phylogenetic relationships and divergence times of 39 drosophilid species were studied by using the coding region of the Adh gene. Four genera--Scaptodrosophila, Zaprionus, Drosophila, and Scaptomyza (from Hawaii)--and three Drosophila subgenera--Drosophila, Engiscaptomyza, and Sophophora--were included. After conducting statistical analyses of the nucleotide sequences of the Adh, Adhr (Adh-related gene), and nuclear rRNA genes and a 905-bp segment of mitochondrial DNA, we used Scaptodrosophila as the outgroup. The phylogenetic tree obtained showed that the first major division of drosophilid species occurs between subgenus Sophophora (genus Drosophila) and the group including subgenera Drosophila and Engiscaptomyza plus the genera Zaprionus and Scaptomyza. Subgenus Sophophora is then divided into D. willistoni and the clade of D. obscura and D. melanogaster species groups. In the other major drosophilid group, Zaprionus first separates from the other species, and then D. immigrans leaves the remaining group of species. This remaining group then splits into the D. repleta group and the Hawaiian drosophilid cluster (Hawaiian Drosophila, Engiscaptomyza, and Scaptomyza). Engiscaptomyza and Scaptomyza are tightly clustered. Each of the D. repleta, D. obscura, and D. melanogaster groups is monophyletic. The splitting of subgenera Drosophila and Sophophora apparently occurred about 40 Mya, whereas the D. repleta group and the Hawaiian drosophilid cluster separated about 32 Mya. By contrast, the splitting of Engiscaptomyza and Scaptomyza occurred only about 11 Mya, suggesting that Scaptomyza experienced a rapid morphological evolution. The D. obscura and D. melanogaster groups apparently diverged about 25 Mya. Many of the D. repleta group species studied here have two functional Adh genes (Adh-1 and Adh-2), and these duplicated genes can be explained by two duplication events.      

Aromatic trap analysis of free radicals production in experimental collagen-induced arthritis in the rat: protective effect of glycosaminoglycans treatment

Many findings demonstrated that Glycosaminoglycans (GAGs) and Proteoglycans (PGs) possess antioxidant activity. Collagen-induced arthritis (CIA) is an experimental animal model similar to human rheumatoid arthritis (RA) in which free radicals are involved. Sodium salicylate can be used as a chemical trap for hydroxyl radicals (OH •), the most damaging reactive oxygen species (ROS), yielding 2,5-dihydroxybenzoic acid), (2,5-DHBA) and 2,3-dihydroxybenzoic acid (2,3-DHBA). The measurement of these two acids in the plasma allows to indirectly assess the production of OH • radicals. The aim of the study was to investigate the effect of hyaluronic acid (HYA) (30 mg/kg i.p.) or chondroitin-4-sulphate (C4S) (30 mg/kg i.p.), on free radical production in Lewis rats subjected to CIA. After the immunization with bovine collagen type II in complete Freund's adjuvant, rats developed an erosive hind paw arthritis, that produced high plasma OH • levels assayed as 2,3-DHBA and 2,5-DHBA, primed lipid peroxidation, evaluated by analyzing conjugated dienes (CD) in the articular cartilage; decreased the concentration of endogenous vitamin E (VE) and catalase (CA) in the joint cartilage; enhanced macrophage inflammatory protein-2 (MIP-2) serum levels and increased elastase (ELA) evaluated as an index of activated leukocyte polymophonuclear (PMNs) accumulation in the articular joints. The administration of HYA and C4S starting at the onset of arthritis (day 11) for 20 days, limited inflammation and the clinical signs in the knee and paw, reduced OH • production, decreased CD levels, partially restored the endogenous antioxidants VE and CA, reduced MIP-2 serum levels and limited PMNs infiltration. The results indicate that the GAGs HYA and C4S significantly reduce free radical production in CIA and could be used as a tool to investigate the role of antioxidants in RA.     

Evolutionary implications of intron–exon distribution and the properties and sequences of the RPL10A gene in eukaryotes

The RPL10A gene encodes the RPL10 protein, required for joining 40S and 60S subunits into a functional 80S ribosome. This highly conserved gene, ubiquitous across all eukaryotic super-groups, is characterized by a variable number of spliceosomal introns, present in most organisms. These properties facilitate the recognition of orthologs among distant taxa and thus comparative studies of sequences as well as the distribution and properties of introns in taxonomically distant groups of eukaryotes. The present study examined the multiple ways in which RPL10A conservation vs. sequence changes in the gene over the course of evolution, including in exons, introns, and the encoded proteins, can be exploited for evolutionary analysis at different taxonomic levels. At least 25 different positions harboring introns within the RPL10A gene were determined in different taxa, including animals, plants, fungi, and alveolates. Generally, intron positions were found to be well conserved even across different kingdoms. However, certain introns seemed to be restricted to specific groups of organisms. Analyses of several properties of introns, including insertion site, phase, and length, along with exon and intron GC content and exon–intron boundaries, suggested biases within different groups of organisms. The use of a standard primer pair to analyze a portion of the intron-containing RPL10A gene in 12 genera of green algae within Chlorophyta is presented as a case study for evolutionary analyses of introns at intermediate and low taxonomic levels. Our study shows that phylogenetic reconstructions at different depths can be achieved using RPL10A nucleotide sequences from both exons and introns as well as the amino acid sequences of the encoded protein.     

A Novel Immunomodulatory Hemocyanin from the Limpet Fissurella latimarginata Promotes Potent Anti-Tumor Activity in Melanoma

Hemocyanins, the huge oxygen-transporting glycoproteins of some mollusks, are used as immunomodulatory proteins with proven anti-cancer properties. The biodiversity of hemocyanins has promoted interest in identifying new anti-cancer candidates with improved immunological properties. Hemocyanins promote Th1 responses without known side effects, which make them ideal for long-term sustained treatment of cancer. In this study, we evaluated a novel hemocyanin from the limpet/gastropod Fissurella latimarginata (FLH). This protein has the typical hollow, cylindrical structure of other known hemocyanins, such as the keyhole limpet hemocyanin (KLH) and the Concholepas hemocyanin (CCH). FLH, like the KLH isoforms, is composed of a single type of polypeptide with exposed N- and O-linked oligosaccharides. However, its immunogenicity was significantly greater than that of KLH and CCH, as FLH induced a stronger humoral immune response and had more potent anti-tumor activity, delaying tumor growth and increasing the survival of mice challenged with B16F10 melanoma cells, in prophylactic and therapeutic settings. Additionally, FLH-treated mice demonstrated increased IFN-γ production and higher numbers of tumor-infiltrating CD4⁺ lymphocytes. Furthermore, in vitro assays demonstrated that FLH, but not CCH or KLH, stimulated the rapid production of pro-inflammatory cytokines (IL-6, IL-12, IL-23 and TNF-α) by dendritic cells, triggering a pro-inflammatory milieu that may explain its enhanced immunological activity. Moreover, this effect was abolished when deglycosylated FLH was used, suggesting that carbohydrates play a crucial role in the innate immune recognition of this protein. Altogether, our data demonstrate that FLH possesses increased anti-tumor activity in part because it activates a more potent innate immune response in comparison to other known hemocyanins. In conclusion, FLH is a potential new marine adjuvant for immunization and possible cancer immunotherapy.     

Development of alcoholic and malolactic fermentations in highly acidic and phenolic apple musts

This work reports the influence of the high acidity and high phenolic content in apple musts on the development of alcoholic and malolactic fermentations and on the final chemical and microbiological composition of the ciders. Four different musts were obtained by pressing several varieties and proportions of cider apples from the Basque Country (Northern Spain). Specially acidic and phenolic varieties were selected. Three musts were obtained in experimental stations and the fourth one, in a cider factory following usual procedures. The evolution of these musts was monitored during five months by measuring 18 parameters throughout eight samplings. In the most acidic of the three experimental musts, yeasts were added to complete the alcoholic fermentation. In the rest of the musts, alcoholic and malolactic fermentations took place spontaneously due to natural microflora and no chemical was added to control these processes. Malolactic fermentation (MLF) finished before alcoholic fermentation in the three tanks obtained in experimental stations, even in the most acidic and phenolic one (pH 3.18, 1.78 g tannic acid/l). After four months, these ciders maintained low levels of lactic acid bacteria (10(4)CFU/ml) and low content of acetic acid (<0.60 g/l). Both fermentations began simultaneously in the must obtained in the cider factory, but MLF finished 10 days after alcoholic fermentation. Subsequently, this must maintained a high population of lactic acid bacteria (>10(6)CFU/ml), causing a higher production of acetic acid (>1.00 g/l) than in the other ciders. These results show the possible advantages of MLF finishing before alcoholic fermentation.     

Recombination Blurs Phylogenetic Groups Routine Assignment in Escherichia coli: Setting the Record Straight

The characterization of population structures plays a main role for understanding outbreaks and the dynamics of bacterial spreading. In Escherichia coli, the widely used combination of multiplex-PCR scheme together with goeBURST has some limitations. The purpose of this study is to show that the combination of different phylogenetic approaches based on concatenated sequences of MLST genes results in a more precise assignment of E. coli phylogenetic groups, complete understanding of population structure and reconstruction of ancestral clones. A collection of 80 Escherichia coli strains of different origins was analyzed following the Clermont and Doumith''s multiplex-PCR schemes. Doumith''s multiplex-PCR showed only 1.7% of misassignment, whereas Clermont''s-2000 protocol reached 14.0%, although the discrepancies reached 30% and 38.7% respectively when recombinant C, F and E phylogroups were considered. Therefore, correct phylogroup attribution is highly variable and depends on the clonal composition of the sample. As far as population structure of these E. coli strains, including 48 E. coli genomes from GenBank, goeBURST provides a quite dispersed population structure; whereas NeighborNet approach reveals a complex population structure. MLST-based eBURST can infer different founder genotypes, for instance ST23/ST88 could be detected as the founder genotypes for STC23; however, phylogenetic reconstructions might suggest ST410 as the ancestor clone and several evolutionary trajectories with different founders. To improve our routine understanding of E. coli molecular epidemiology, we propose a strategy based on three successive steps; first, to discriminate three main groups A/B1/C, D/F/E and B2 following Doumith''s protocol; second, visualization of population structure based on MLST genes according to goeBURST, using NeighborNet to establish more complex relationships among STs; and third, to perform, a cost-free characterization of evolutionary trajectories in variants emerging along the clonal expansion using parsimony methods of phylogenetic analysis.     

Validated predictive modelling of the environmental resistome

Multi-drug-resistant bacteria pose a significant threat to public health. The role of the environment in the overall rise in antibiotic-resistant infections and risk to humans is largely unknown. This study aimed to evaluate drivers of antibiotic-resistance levels across the River Thames catchment, model key biotic, spatial and chemical variables and produce predictive models for future risk assessment. Sediment samples from 13 sites across the River Thames basin were taken at four time points across 2011 and 2012. Samples were analysed for class 1 integron prevalence and enumeration of third-generation cephalosporin-resistant bacteria. Class 1 integron prevalence was validated as a molecular marker of antibiotic resistance; levels of resistance showed significant geospatial and temporal variation. The main explanatory variables of resistance levels at each sample site were the number, proximity, size and type of surrounding wastewater-treatment plants. Model 1 revealed treatment plants accounted for 49.5% of the variance in resistance levels. Other contributing factors were extent of different surrounding land cover types (for example, Neutral Grassland), temporal patterns and prior rainfall; when modelling all variables the resulting model (Model 2) could explain 82.9% of variations in resistance levels in the whole catchment. Chemical analyses correlated with key indicators of treatment plant effluent and a model (Model 3) was generated based on water quality parameters (contaminant and macro- and micro-nutrient levels). Model 2 was beta tested on independent sites and explained over 78% of the variation in integron prevalence showing a significant predictive ability. We believe all models in this study are highly useful tools for informing and prioritising mitigation strategies to reduce the environmental resistome.     

人肺腺癌细胞分化相关基因cDNAs的克隆

在用10^-5 mol/L全反式维甲酸(RA)诱导人肺腺癌细胞系GLC-82分化的基础上,以M13噬菌粒pSPORT1为载体,应用定向克隆技术,分别构建了未经RA诱导和RA诱导1d及4d细胞的3个cDNA文库.以含重组子的诱导文库单链DNA为靶标(Target)同未诱导文库的cDNA驱除子(Driver)进行消减杂交,富集RA特异性单链DNA,将富集的单链DNA回复为双链后转化感受态菌,建立细胞诱导分化过程中活化表达基因的cDNA消减文库,得到124个cDNA消减克隆.经同源性分析和与文库总cDNA作Southern印迹杂交,进而与RA诱导前后细胞的RNA作Northern印迹杂交,筛选出2个(RA5,RA28)诱导后呈早期瞬时表达和1个(RA42)呈早期并持续表达的cDNA克隆,cDNA全长分别为1.8,1.5和0.7kb.序列测定及初步功能分析结果表明,RA5,RA28和RA42这3个首次报道的序列,可能是人肺腺癌细胞分化相关基因的cDNA克隆.