Mining of unexplored habitats for novel chitinases—chiA as a helper gene proxy in metagenomics

The main objective of this study was to assess the abundance and diversity of chitin-degrading microbial communities in ten terrestrial and aquatic habitats in order to provide guidance to the subsequent exploration of such environments for novel chitinolytic enzymes. A combined protocol which encompassed (1) classical overall enzymatic assays, (2) chiA gene abundance measurement by qPCR, (3) chiA gene pyrosequencing, and (4) chiA gene-based PCR-DGGE was used. The chiA gene pyrosequencing is unprecedented, as it is the first massive parallel sequencing of this gene. The data obtained showed the existence across habitats of core bacterial communities responsible for chitin assimilation irrespective of ecosystem origin. Conversely, there were habitat-specific differences. In addition, a suite of sequences were obtained that are as yet unregistered in the chitinase database. In terms of chiA gene abundance and diversity, typical low-abundance/diversity versus high-abundance/diversity habitats was distinguished. From the combined data, we selected chitin-amended agricultural soil, the rhizosphere of the Arctic plant Oxyria digyna and the freshwater sponge Ephydatia fluviatilis as the most promising habitats for subsequent bioexploration. Thus, the screening strategy used is proposed as a guide for further metagenomics-based exploration of the selected habitats

The Pseudomonas aeruginosa Transcriptome in Planktonic Cultures and Static Biofilms Using RNA Sequencing

In this study, we evaluated how gene expression differs in mature Pseudomonas aeruginosa biofilms as opposed to planktonic cells by the use of RNA sequencing technology that gives rise to both quantitative and qualitative information on the transcriptome. Although a large proportion of genes were consistently regulated in both the stationary phase and biofilm cultures as opposed to the late exponential growth phase cultures, the global biofilm gene expression pattern was clearly distinct indicating that biofilms are not just surface attached cells in stationary phase. A large amount of the genes found to be biofilm specific were involved in adaptation to microaerophilic growth conditions, repression of type three secretion and production of extracellular matrix components. Additionally, we found many small RNAs to be differentially regulated most of them similarly in stationary phase cultures and biofilms. A qualitative analysis of the RNA-seq data revealed more than 3000 putative transcriptional start sites (TSS). By the use of rapid amplification of cDNA ends (5′-RACE) we confirmed the presence of three different TSS associated with the pqsABCDE operon, two in the promoter of pqsA and one upstream of the second gene, pqsB. Taken together, this study reports the first transcriptome study on P. aeruginosa that employs RNA sequencing technology and provides insights into the quantitative and qualitative transcriptome including the expression of small RNAs in P. aeruginosa biofilms

The management of acute venous thromboembolism in clinical practice. Results from the European PREFER in VTE Registry

Venous thromboembolism (VTE) is a significant cause of morbidity and mortality in Europe. Data from real-world registries are necessary, as clinical trials do not represent the full spectrum of VTE patients seen in clinical practice. We aimed to document the epidemiology, management and outcomes of VTE using data from a large, observational database. PREFER in VTE was an international, non-interventional disease registry conducted between January 2013 and July 2015 in primary and secondary care across seven European countries. Consecutive patients with acute VTE were documented and followed up over 12 months. PREFER in VTE included 3,455 patients with a mean age of 60.8 ± 17.0 years. Overall, 53.0 % were male. The majority of patients were assessed in the hospital setting as inpatients or outpatients (78.5 %). The diagnosis was deep-vein thrombosis (DVT) in 59.5 % and pulmonary embolism (PE) in 40.5 %. The most common comorbidities were the various types of cardiovascular disease (excluding hypertension; 45.5 %), hypertension (42.3 %) and dyslipidaemia (21.1 %). Following the index VTE, a large proportion of patients received initial therapy with heparin (73.2 %), almost half received a vitamin K antagonist (48.7 %) and nearly a quarter received a DOAC (24.5 %). Almost a quarter of all presentations were for recurrent VTE, with >80 % of previous episodes having occurred more than 12 months prior to baseline. In conclusion, PREFER in VTE has provided contemporary insights into VTE patients and their real-world management, including their baseline characteristics, risk factors, disease history, symptoms and signs, initial therapy and outcomes

Genetic differentiation among brazilian populations of Euschistus heros (Fabricius) Heteroptera: Pentatomidae) based on RAPD analysis.

O percevejo marrom da soja, Euschistus heros (Fabricius), possui ampla distribuição na região sojicola do Brasil, sendo mais importante na região central do País. Tem sido o alvo principal das aplicações de inseticidas dirigidas contra o complexo de pentatomídeos praga. A variabilidade nos RAPD entre e dentro das populações do percevejo marrom foi determinada. Amostras da espécie foram coletadas nos campos de soja de Ubiratã (PR), Londrina (PR), Centenário do Sul (PR), Cândido Mota (SP), Ponta Porã (MS) e Sapezal (MT). O DNA genômico foi extraído da cabeça para minimizar a contaminação do DNA proveniente dos endoparasitóides e parasitas que ocorrem na hemocele. O DNA foi amplificado com iniciadores de 10 nucleotídeos. Quinze iniciadores produziram 246 bandas. A similaridade genética foi obtida com base na freqüência alélica dos RAPD utilizando-se a distância de Nei 1972. Os percevejos coletados da mesma região geográfica apresentaram a maior similaridade genética. As populações de Londrina e Centenário do Sul foram geneticamente mais próximas que as restantes. Também, percevejos coletados em Cândido Mota foram próximos das populações de Ponta Porã. A população geográfica de Sapezal foi a mais divergente das outras. As fêmeas e os machos agruparam-se em grupos diferentes dentro de cada população geográfica, o que significa que os RAPD possibilitam a diferenciação dos sexos. Não foram observados indivíduos de uma população agrupando-se com indivíduos de outra região. O número de loci polimórficos das diferentes populações variou entre 40,6% e 52,1%. Os índices de fluxo gênico de E. heros (Nm = 0,8307) foram menores que os observados anteriormente para Anticarsia gemmatalis Hübner e Helicoverpa armigera (Hübner) sugerindo que as populações do pentatomídeo apresentam maior isolamento geográfico que nos referidos noctuídeos, por exemplo

The impact of fungicides on Nomuraea rileyi(farlow) Samson epizootics and on populations of Anticarsia gemmatalis Hübner (Lepidoptera: noctuidae), on soybean.

The fungus Nomuraea rileyi (Farlow) Samson is one of the most important natural enemies of soybean caterpillars, mainly under humid weather conditions. Outbreaks of the fungus Microsphaera diffusa Cooke & Peck have demanded fungicide applications on soybeans, which could result in outbreaks of noctuid populations by reduction of the natural inocula of N. rileyi. The recommended fungicides have shown to be detrimental to beneficial fungi, reducing infection, delaying epizootics, and resulting in increased host population densities. In laboratory assays, benomyl, difenoconazole, sulphur and carbendazim affected conidial germination of N. rileyi, being the latter less deleterious. To assess the impact of fungicides used to control M. diffusa, on N. rileyi, two tests were carried on, spraying difenoconazole (75 g a.i./ha) and benomyl (262.5 g a.i./ha) on soybean plots. In the 1997/98 trial, fungicide was sprayed once on soybean plants at R1-R2 developmental stages. In the 1998/99 test, two applications were made, when plants were at V5 and V7 developmental stages, respectively. The number of VBC larvae was significantly higher in the fungicide treated plots than in the control plots. In the 1997/98 test, benomyl treated plots resulted in higher populations of VBC than in the control or in the difenoconazole plots. In the 1998/99 test, VBC population was higher from 7 to 12 days after the first application, and remained high until 19 days after the second application. In general, fungicide treatments delayed the begining of N. rileyi epizootics from 2 to 14 days.200

Metagenomic comparison of microbial communities inhabiting confined and unconfined aquifer ecosystems

A metagenomic analysis of two aquifer systems located under a dairy farming region was performed to examine to what extent the composition and function of microbial communities varies between confined and surface-influenced unconfined groundwater ecosystems. A fundamental shift in taxa was seen with an overrepresentation of Rhodospirillales, Rhodocyclales, Chlorobia and Circovirus in the unconfined aquifer, while Deltaproteobacteria and Clostridiales were overrepresented in the confined aquifer. A relative overrepresentation of metabolic processes including antibiotic resistance (β-lactamase genes), lactose and glucose utilization and DNA replication were observed in the unconfined aquifer, while flagella production, phosphate metabolism and starch uptake pathways were all overrepresented in the confined aquifer. These differences were likely driven by differences in the nutrient status and extent of exposure to contaminants of the two groundwater systems. However, when compared with freshwater, ocean, sediment and animal gut metagenomes, the unconfined and confined aquifers were taxonomically and metabolically more similar to each other than to any other environment. This suggests that intrinsic features of groundwater ecosystems, including low oxygen levels and a lack of sunlight, have provided specific niches for evolution to create unique microbial communities. Obtaining a broader understanding of the structure and function of microbial communities inhabiting different groundwater systems is particularly important given the increased need for managing groundwater reserves of potable water.Renee J. Smith, Thomas C. Jeffries, Ben Roudnew, Alison J. Fitch, Justin R. Seymour, Marina W. Delpin, Kelly Newton, Melissa H. Brown, and James G. Mitchel

Nutrient regime regulates complex transcriptional start site usage within a Pseudoalteromonas chitinase gene cluster

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