A general framework for optimization of probes for gene expression microarray and its application to the fungus Podospora anserina

<p>Abstract</p> <p>Background</p> <p>The development of new microarray technologies makes custom long oligonucleotide arrays affordable for many experimental applications, notably gene expression analyses. Reliable results depend on probe design quality and selection. Probe design strategy should cope with the limited accuracy of <it>de novo </it>gene prediction programs, and annotation up-dating. We present a novel <it>in silico </it>procedure which addresses these issues and includes experimental screening, as an empirical approach is the best strategy to identify optimal probes in the <it>in silico </it>outcome.</p> <p>Findings</p> <p>We used four criteria for <it>in silico </it>probe selection: cross-hybridization, hairpin stability, probe location relative to coding sequence end and intron position. This latter criterion is critical when exon-intron gene structure predictions for intron-rich genes are inaccurate. For each coding sequence (CDS), we selected a sub-set of four probes. These probes were included in a test microarray, which was used to evaluate the hybridization behavior of each probe. The best probe for each CDS was selected according to three experimental criteria: signal-to-noise ratio, signal reproducibility, and representative signal intensities. This procedure was applied for the development of a gene expression Agilent platform for the filamentous fungus <it>Podospora anserina </it>and the selection of a single 60-mer probe for each of the 10,556 <it>P. anserina </it>CDS.</p> <p>Conclusions</p> <p>A reliable gene expression microarray version based on the Agilent 44K platform was developed with four spot replicates of each probe to increase statistical significance of analysis.</p

Comparative expression of cell wall related genes in four maize RILs and one parental line of variable lignin content and cell wall degradability

A comparison of gene expression in maize between the parental line F271 and four RILs derived from the cross F288 x F271 was investigated based on hybridization on the 17,555 probes Affymetrix micro-array, targeting nearly one third of the genes present in maize genomes. The parental line had unfavorable alleles for cell wall degradability traits at the major QTL position in bin 6.06, while the set of RILs had both the favorable allele and high cell wall degradability. 360 genes were differentially expressed in the four RIL in comparison to F271, including nine genes underlying the major QTL position and 36 underlying two other QTL positions. However, their proposed function (whenever is described) do not allow us to firmly consider their involvement in the observed variation of cell wall related traits. Only a few genes involved in monolignol biosynthesis or polymerization located elsewhere in the genome were differentially expressed between the four RILs and F271, corroborating with the fact that these genes are probably not involved in major determinants of cell wall degradability in the studied set of lines. Among the investigated regulation factors, three ZmMYB, one NAC and one C3HC4 zinc finger were differentially expressed between the four RILs and F271, but they were not located in bin 6.06. Notwithstanding, the obtained results especially strengthened the probable involvement of these genes in maize secondary wall assembly and/ or lignification

Comparative expression of cell wall related genes in four maize RILs and one parental line of variable lignin content and cell wall degradability

A comparison of gene expression in maize between the parental line F271 and four RILs derived from the cross F288 x F271 was investigated based on hybridization on the 17,555 probes Affymetrix micro-array, targeting nearly one third of the genes present in maize genomes. The parental line had unfavorable alleles for cell wall degradability traits at the major QTL position in bin 6.06, while the set of RILs had both the favorable allele and high cell wall degradability. 360 genes were differentially expressed in the four RIL in comparison to F271, including nine genes underlying the major QTL position and 36 underlying two other QTL positions. However, their proposed function (whenever is described) do not allow us to firmly consider their involvement in the observed variation of cell wall related traits. Only a few genes involved in monolignol biosynthesis or polymerization located elsewhere in the genome were differentially expressed between the four RILs and F271, corroborating with the fact that these genes are probably not involved in major determinants of cell wall degradability in the studied set of lines. Among the investigated regulation factors, three ZmMYB, one NAC and one C3HC4 zinc finger were differentially expressed between the four RILs and F271, but they were not located in bin 6.06. Notwithstanding, the obtained results especially strengthened the probable involvement of these genes in maize secondary wall assembly and/ or lignification

Global Analysis of Extracytoplasmic Stress Signaling in Escherichia coli

The Bae, Cpx, Psp, Rcs, and σE pathways constitute the Escherichia coli signaling systems that detect and respond to alterations of the bacterial envelope. Contributions of these systems to stress response have previously been examined individually; however, the possible interconnections between these pathways are unknown. Here we investigate the dynamics between the five stress response pathways by determining the specificities of each system with respect to signal-inducing conditions, and monitoring global transcriptional changes in response to transient overexpression of each of the effectors. Our studies show that different extracytoplasmic stress conditions elicit a combined response of these pathways. Involvement of the five pathways in the various tested stress conditions is explained by our unexpected finding that transcriptional responses induced by the individual systems show little overlap. The extracytoplasmic stress signaling pathways in E. coli thus regulate mainly complementary functions whose discrete contributions are integrated to mount the full adaptive response

Comparative transcriptomics of drought responses in Populus: a meta-analysis of genome-wide expression profiling in mature leaves and root apices across two genotypes

<p>Abstract</p> <p>Background</p> <p>Comparative genomics has emerged as a promising means of unravelling the molecular networks underlying complex traits such as drought tolerance. Here we assess the genotype-dependent component of the drought-induced transcriptome response in two poplar genotypes differing in drought tolerance. Drought-induced responses were analysed in leaves and root apices and were compared with available transcriptome data from other <it>Populus </it>species.</p> <p>Results</p> <p>Using a multi-species designed microarray, a genomic DNA-based selection of probesets provided an unambiguous between-genotype comparison. Analyses of functional group enrichment enabled the extraction of processes physiologically relevant to drought response. The drought-driven changes in gene expression occurring in root apices were consistent across treatments and genotypes. For mature leaves, the transcriptome response varied weakly but in accordance with the duration of water deficit. A differential clustering algorithm revealed similar and divergent gene co-expression patterns among the two genotypes. Since moderate stress levels induced similar physiological responses in both genotypes, the genotype-dependent transcriptional responses could be considered as intrinsic divergences in genome functioning. Our meta-analysis detected several candidate genes and processes that are differentially regulated in root and leaf, potentially under developmental control, and preferentially involved in early and long-term responses to drought.</p> <p>Conclusions</p> <p>In poplar, the well-known drought-induced activation of sensing and signalling cascades was specific to the early response in leaves but was found to be general in root apices. Comparing our results to what is known in arabidopsis, we found that transcriptional remodelling included signalling and a response to energy deficit in roots in parallel with transcriptional indices of hampered assimilation in leaves, particularly in the drought-sensitive poplar genotype.</p

Proteomics-based study of morphine effects on primary human brain microvascular endothelial cells

Morphine is a common antinociceptive drug, whose transport to the central nervous system (CNS) requires crossing the blood-brain barrier (BBB). However, its administration is associated with side effects reducing its efficiency, but the molecular processes are still not well understood. The present study evaluated the molecular mechanisms associated to morphine exposure on primary human brain microvascular endothelial cells (HBMECs) with a combination of mass spectrometry-based proteomics and pathway enrichment strategies. Pathway enrichment unveiled several affected pathways, including the NRF-2 pathway involved in oxidative stress response. We also underlined mitochondria dysfunctions, which could be related to oxidative stress. In addition, morphine ability to modulate HBMECs' secretion of TNF-α and IL-6 was observed, as well as an alteration of proteins expression involved in junctional complexes and cell adhesion. In conclusion, our study indicated morphine-induced modulations in redox homeostasis, immune response and barrier integrity in HBMECs, encouraging further investigation

Depletion of abundant plasma proteins for extracellular vesicle proteome characterization: benefits and pitfalls

Blood extracellular vesicles (EVs) play essential roles in cell–cell communication and their molecular cargo is a promising source of disease biomarkers. However, proteomic characterization of plasma-derived EVs is challenged by the presence of highly abundant plasma proteins, which limits the detection of less abundant proteins, and by the low number of EVs in biological fluids. The aim of this study was to investigate if the removal of abundant plasma proteins prior to EV isolation could improve plasma-derived EV characterization by LC–MS/MS and expand the proteome coverage. Plasma depletion was performed using a single-use spin column and EVs were isolated from only 100 µL of non-depleted and depleted plasma by size exclusion chromatography. Afterwards, EVs were characterized by nanoparticle tracking analysis and mass spectrometry–based proteomics using a data-independent acquisition approach. Depleted plasma-derived EVs had higher particle concentrations and particle-to-protein ratios. Depletion did increase the protein coverage with a higher number of identifications in EVs from depleted plasma (474 proteins) than from non-depleted (386 proteins). However, EVs derived from non-depleted plasma carried a slightly higher number of common EV markers. Overall, our findings suggest that plasma depletion prior to EV isolation by size exclusion chromatography provides higher yield and protein coverage, but slightly lower identification of EV markers. This study also showed the possibility to characterize the proteome of EVs derived from small plasma volumes, encouraging the clinical feasibility of the discovery of EV biomarkers

Neurovascular Unit-Derived Extracellular Vesicles: From Their Physiopathological Roles to Their Clinical Applications in Acute Brain Injuries

Extracellular vesicles (EVs) form a heterogeneous group of membrane-enclosed structures secreted by all cell types. EVs export encapsulated materials composed of proteins, lipids, and nucleic acids, making them a key mediator in cell&ndash;cell communication. In the context of the neurovascular unit (NVU), a tightly interacting multicellular brain complex, EVs play a role in intercellular communication and in maintaining NVU functionality. In addition, NVU-derived EVs can also impact peripheral tissues by crossing the blood&ndash;brain barrier (BBB) to reach the blood stream. As such, EVs have been shown to be involved in the physiopathology of numerous neurological diseases. The presence of NVU-released EVs in the systemic circulation offers an opportunity to discover new diagnostic and prognostic markers for those diseases. This review outlines the most recent studies reporting the role of NVU-derived EVs in physiological and pathological mechanisms of the NVU, focusing on neuroinflammation and neurodegenerative diseases. Then, the clinical application of EVs-containing molecules as biomarkers in acute brain injuries, such as stroke and traumatic brain injuries (TBI), is discussed

Improving the dielectric losses of (Ba,Sr)TiO3 thin films using a SiO2 buffer layer

The efficient use of ferroelectric thin films in rF agile devices faces several limits. One of them is the dielectric losses which are usually above 1%, i.e. above the threshold as set by the electronic industry..

Substantial reduction of the dielectric losses of Ba0.6Sr0.4TiO3 thin films using a SiO2 barrier layer

The efficient use of ferroelectric thin films in radio-frequency agile devices faces several limitations. One of them is imposed by the dielectric losses which are usually above 1%, i.e. above the threshold as set by the electronic industry. Following the same route as for bulk ceramics, we have processed composite stacks made of BST/SiO2 multilayers using radio-frequency magnetron sputtering..