Biological Replicas Research Articles

Polyglutamine Atrophin provokes neurodegeneration inDrosophilaby repressingfat

Large alterations in transcription accompany neurodegeneration in polyglutamine (polyQ) diseases. These pathologies manifest both general polyQ toxicity and mutant protein-specific effects. In this study, we report that the fat tumour suppressor gene mediates neurodegeneration induced by the polyQ protein Atrophin. We have monitored early transcriptional alterations in a Drosophila model of Dentatorubral-pallidoluysian Atrophy and found that polyQ Atrophins downregulate fat. Fat protects from neurodegeneration and Atrophin toxicity through the Hippo kinase cascade. Fat/Hippo signalling does not provoke neurodegeneration by stimulating overgrowth; rather, it alters the autophagic flux in photoreceptor neurons, thereby affecting cell homeostasis. Our data thus provide a crucial insight into the specific mechanism of a polyQ disease and reveal an unexpected neuroprotective role of the Fat/Hippo pathway.

Axonal Transport Proteomics Reveals Mobilization of Translation Machinery to the Lesion Site in Injured Sciatic Nerve

Investigations of the molecular mechanisms underlying responses to nerve injury have highlighted the importance of axonal transport systems. To obtain a comprehensive view of the protein ensembles associated with axonal transport in injured axons, we analyzed the protein compositions of axoplasm concentrated at ligatures following crush injury of rat sciatic nerve. LC-MS/MS analyses of iTRAQ-labeled peptides from axoplasm distal and proximal to the ligation sites revealed protein ensembles transported in both anterograde and retrograde directions. Variability of replicates did not allow straightforward assignment of proteins to functional transport categories; hence, we performed principal component analysis and factor analysis with subsequent clustering to determine the most prominent injury-related transported proteins. This strategy circumvented experimental variability and allowed the extraction of biologically meaningful information from the quantitative neuroproteomics experiments. 299 proteins were highlighted by principal component analysis and factor analysis, 145 of which correlate with retrograde and 154 of which correlate with anterograde transport after injury. The analyses reveal extensive changes in both anterograde and retrograde transport proteomes in injured peripheral axons and emphasize the importance of RNA binding and translational machineries in the axonal response to injury.

Influence of cell geometry and number of replicas in the reproducibility of whole cell FTIR analysis

Fourier Transform InfraRed (FTIR) spectroscopy is an increasingly used technique in biology, especially for whole cell metabolomic fingerprint. The reproducibility of this technique is influenced by a large number of factors such as the physiological state of cells, sample manipulation and growth conditions. Evidence exists suggesting that the cell shape and dimension can be further elements to consider in whole cell FTIR analysis. In this study we aimed to address the effect of cell geometry on the FTIR spectra and to define the extent of variability occurring between machine and biological replicas with a standardized protocol. The yeast species Saccharomyces cerevisiae (large oval-shaped cells) and Debaryomyces hansenii (small round shaped cells) were employed for their different morphology. Thirty machine replicas of each were analyzed separately and after averaging in groups of three, showing a three to four-fold reduction of the variability. Similarly, a two-fold reduction of variability was observed when thirty biological replicas of the two yeast species were analyzed. The optimal number of replicas to average was then estimated with a bootstrap-like procedure in which biological and machine replicas were randomly resampled 2000 times and averaged in groups spanning from 2 to 12 replicas. This simulation has shown that little if any advantage can be obtained by increasing the number of replicas over five and that the variability exhibited by the small regular cells of D. hansenii was always roughly half of that displayed by the large S. cerevisiae cells, confirming the results obtained with standard non-bootstrapped averages.

Optimising oligonucleotide array design for ChIP-on-chip

The sequencing of whole genomes has allowed for custom-made genome-wide microarray assays such as the ChIP-on-chip. With this technology we can detect e.g. transcription factor binding sites over an entire genome. In principle, an accurate detection is only limited by the resolution of the chipdesign, i.e. the tiling density of the oligonucleotides. However, the inherent noise of the DNA hybridisation severely hampers the interpretation of the results. We mined existing ChIP-on-chip datasets to identify the main sources of noise arising from the sequence selection. We found that limiting intervals must be imposed on 1)the melting temperature, 2)the lengths of the probes, 3)palindromic sequences and 4)the sequence uniqueness relative to the rest of the genome. Based on this knowledge we developed an oligonucleotide array design algorithm [1] to generate a genome-wide array design for any given genome at a given tiling density. To obtain unique sequences we invented a novel approach for selecting the sequences. Using an augmented suffix-array implementation we score sequences by their content of sequence-unique subsequences and select preferentially the sequences with the highest content of unique subsequences. We have tested our design algorithm using different parameter settings in a fractional factorial test setup, in effect testing eight different parameter combinations. The tests were designed for the mouse genome on the 2.18 M feature array from Nimblegen and performed under true ChIP-on-chip experimental conditions using mouse TBP ChIP samples for the hybridisations. Test hybridisations were performed for three biological replicas, each hybridised three times, to estimate the variance across both biological and technical replicas. From the tested designs we deduce the effect of each parameter on the resulting signal and coverage of the design. We correlate the effects and interactions of the probe properties on the probe level (signal intensities) as well as on the design level (quality measures for the whole data set). From this analysis we quantify the effect of each parameter, thus allowing us to choose the design parameter settings that optimise the signal-to-noise ratio, while maintaining a high coverage of the genome. Using our design algorithm and the optimised parameter settings we can produce a genome-wide microarray design with low noise and high coverage for any sequenced genome.

A quality-controlled microarray method for gene expression profiling

Gene expression profiling on microarrays is widely used to measure the expression of large numbers of genes in a single experiment. Because of the high cost of this method, feasible numbers of replicates are limited, thus impairing the power of statistical analysis. As a step toward reducing technically induced variation, we developed a procedure of sample preparation and analysis that minimizes the number of sample manipulation steps, introduces quality control before array hybridization, and allows recovery of the prepared mRNA for independent validation of results. Sample preparation is based on mRNA separation using oligo(dT) magnetic beads, which are subsequently used for first-strand cDNA synthesis on the beads. cDNA covalently bound to the magnetic beads is used as template for second-strand cDNA synthesis, leaving the intact mRNA in solution for further analysis. The quality of the synthesized cDNA can be assessed by quantitative polymerase chain reaction using 3′- and 5′-specific primer pairs for housekeeping genes such as glyceraldehyde-3-phosphate dehydrogenase. Second-strand cDNA is chemically labeled with fluorescent dyes to avoid dye bias in enzymatic labeling reactions. After hybridization of two differently labeled samples to microarray slides, arrays are scanned and images analyzed automatically with high reproducibility. Quantile-normalized data from five biological replica display a coefficient of variation ⩽45% for 90% of profiled genes, allowing detection of twofold changes with false positive and false negative rates of 10% each. We demonstrate successful application of the procedure for expression profiling in plant leaf tissue. However, the method could be easily adapted for samples from animal including human or from microbial origin.