33 research outputs found
GoArrays: highly dynamic and efficient microarray probe design
MOTIVATION: The use of oligonucleotide microarray technology requires a very
detailed attention to the design of specific probes spotted on the solid phase.
These problems are far from being commonplace since they refer to complex
physicochemical constraints. Whereas there are more and more publicly available
programs for microarray oligonucleotide design, most of them use the same
algorithm or criteria to design oligos, with only little variation. RESULTS: We
show that classical approaches used in oligo design software may be inefficient
under certain experimental conditions, especially when dealing with complex
target mixtures. Indeed, our biological model is a human obligate parasite, the
microsporidia Encephalitozoon cuniculi. Targets that are extracted from
biological samples are composed of a mixture of pathogen transcripts and host
cell transcripts. We propose a new approach to design oligonucleotides which
combines good specificity with a potentially high sensitivity. This approach is
original in the biological point of view as well as in the algorithmic point of
view. We also present an experimental validation of this new strategy by
comparing results obtained with standard oligos and with our composite oligos.
A specific E.cuniculi microarray will overcome the difficulty to discriminate
the parasite mRNAs from the host cell mRNAs demonstrating the power of the
microarray approach to elucidate the lifestyle of an intracellular pathogen
using mix mRNAs
Impact of oil on bacterial community structure in bioturbated sediments
Oil spills threaten coastlines where biological processes supply essential ecosystem services. Therefore, it is crucial to understand how oil influences the microbial communities in sediments that play key roles in ecosystem functioning. Ecosystems such as sediments are characterized by intensive bioturbation due to burrowing macrofauna that may modify the microbial metabolisms. It is thus essential to consider the bioturbation when determining the impact of oil on microbial communities. In this study, an experimental laboratory device maintaining pristine collected mudflat sediments in microcosms closer to true environmental conditions - with tidal cycles and natural seawater - was used to simulate an oil spill under bioturbation conditions. Different conditions were applied to the microcosms including an addition of: standardized oil (Blend Arabian Light crude oil, 25.6 mg.g21 wet sediment), the common burrowing organism Hediste (Nereis) diversicolor and both the oil and H. diversicolor. The addition of H. diversicolor and its associated bioturbation did not affect the removal of petroleum hydrocarbons. After 270 days, 60% of hydrocarbons had been removed in all microcosms irrespective of the H. diversicolor addition. However, 16S-rRNA gene and 16S-cDNA T-RFLP and RT-PCR-amplicon libraries analysis showed an effect of the condition on the bacterial community structure, composition, and dynamics, supported by PerMANOVA analysis. The 16S-cDNA libraries from microcosms where H. diversicolor was added (oiled and un-oiled) showed a marked dominance of sequences related to Gammaproteobacteria. However, in the oiled-library sequences associated to Deltaproteobacteria and Bacteroidetes were also highly represented. The 16S-cDNA libraries from oiled-microcosms (with and without H. diversicolor addition) revealed two distinct microbial communities characterized by different phylotypes associated to known hydrocarbonoclastic bacteria and dominated by Gammaproteobacteria and Deltaproteobacteria. In the oiled-microcosms, the addition of H. diversicolor reduced the phylotype-richness, sequences associated to Actinobacteria, Firmicutes and Plantomycetes were not detected. These observations highlight the influence of the bioturbation on the bacterial community structure without affecting the biodegradation capacities
Effective degradation of organophosphate ester flame retardants and plasticizers in coastal sediments under high urban pressure
Empirical evidence of the effective degradation at environmentally relevant conditions of organophosphate esters (OPEs) flame retardants and plasticizers in coastal sediments from an impacted area in the NW Mediterranean Sea is provided. Half-lives varied from 23.3 to 77.0 (abiotic conditions) and from 16.8 to 46.8 days (biotic conditions), depending on the compound, highlighting the relevant role of microbial assemblages enhancing OPE degradation. After an immediate significant reduction of the bacterial abundance due to OPE addition to the sediment at the very beginning of the experiment, the observed biodegradation was associated to a general stimulation of the growth of the bacterial community during a first period, but without a marked change of the structure of the community. However, OPE contamination induced a decrease on the diversity of the bacterial community in the coastal sediment, noticeable after 14 days of incubation. It is likely that on one side the contamination had favoured the growth of some bacterial groups maybe involved in the biodegradation of these compounds but, on the other side, had also impacted some sensitive bacteria. The estimated half-lives fill a data gap concerning OPE degradation rates in marine sediments and will be valuable data for the refinement of OPE chemical risk assessment in marine environments, particularly on impacted sites
Ecological significance of Synergistetes in the biological treatment of tuna cooking wastewater by an anaerobic sequencing batch reactor
Lab-scale 2L-anaerobic sequencing batch reactor was operated under mesothermic conditions. The degradation of protein-rich organic matter was determined by chemical oxygen demand, biogas production, and protein-removal activity over the operation. The structure of the microbial community was determined by qPCR and next-generation sequencing on 16S rRNA genes. At the steady state, a very efficient removal of protein (92 %) was observed. Our results demonstrate a decrease of archaeal and bacterial abundance over time. Members of the phylum Synergistetes, with a peculiar emphasis for those pertaining to families Dethiosulfovibrionaceae and Aminiphilaceae, are of major ecological significance regarding the treatment of this industrial wastewater. The prominent role to be played by members of the phylum Synergistetes regarding protein and/or amino acid degradation is discussed
Probe design strategies for oligonucleotide microarrays
Chapter 6Oligonucleotide microarrays have been widely used for gene detection and/or quantification of gene expression in various samples ranging from a single organism to a complex microbial assemblage. The success of a microarray experiment, however, strongly relies on the quality of designed probes. Consequently, probe design is of critical importance and therefore multiple parameters should be considered for each probe in order to ensure high specificity, sensitivity, and uniformity as well as potentially quantitative power. Moreover, to assess the complete gene repertoire of complex biological samples such as those studied in the field of microbial ecology, exploratory probe design strategies must be also implemented to target not-yet-described sequences. To design such probes, two algorithms, KASpOD and HiSpOD, have been developed and they are available via two user-friendly web services. Here, we describe the use of this software necessary for the design of highly effective probes especially in the context of microbial oligonucleotide microarrays by taking into account all the crucial parameters