Biology subjects

Bruno, V.

Publications and source records attributed to Bruno, V..

3 recordsLinked to original sources

bioRxiv2018

How fluorescent tags modify oligomer size distributions of the Alzheimer-peptide Aβ(1-40)

Within the complex aggregation process of A{beta}-peptides into fibrils, oligomeric species, play a central role and reveal fundamental properties of the underlying mechanism of aggregation. In particular, low molecular weight aggregates have attracted increasing interest because of their role in cytotoxicity and neuronal apoptosis, typical of aggregation related diseases. One of the main techniques used to characterize such early stages of aggregation is fluorescence spectroscopy. To this end, A{beta}-peptide chains are functionalized with fluorescent tags, often covalently bound to the disordered N-terminus region of the peptide, with the assumption that functionalization and presence of the fluorophore will not modify the process of self-assembly nor the final fibrillar structure. Up to date, experimental findings reveal size distributions of thermodynamically stable oligomers ranging from very narrow distributions of dimers to octamers, to very broad distributions up to 50-mers. In the present investigation we systematically study the effects of five of the most commonly used fluorophores on the aggregation of A{beta}(1-40)-peptides. Time-resolved and single-molecule fluorescence spectroscopy have been chosen to monitor the oligomer populations at different fibrillation times, TEM, AFM and X-ray diffraction to investigate the structure of mature fibrils. While the structures of the mature fibrils were only slightly affected by the fluorescent tags, the sizes of the detected oligomeric species varied significantly depending on the chosen fluorophore. In particular, we relate the presence of high molecular weight oligomers (as found for the fluorophores HiLyte 647, Atto 647N and Atto 655) to net-attractive, hydrophobic fluorophore-peptide interactions, which are weak in the case of HiLyte 488, and Atto 488. The latter form low molecular weight oligomers only. Our findings reveal the potentially high impact of the properties of fluorophores on transient aggregates which needs to be included in the interpretation of experimental data of oligomers of fluorescently labeled peptides.

biophysics↗

bioRxiv2018

Targeted enrichment outperforms other enrichment techniques and enables more multi-species RNA-Seq analyses

Enrichment methodologies enable analysis of minor members in multi-species transcriptomic analyses. We compared standard enrichment of bacterial and eukaryotic mRNA to targeted enrichment with Agilent SureSelect (AgSS) capture for Brugia malayi, Aspergillus fumigatus, and the Wolbachia endosymbiont of B. malayi (wBm). Without introducing significant systematic bias, the AgSS quantitatively enriched samples, resulting in more reads mapping to the target organism. The AgSS-enriched libraries consistently had a positive linear correlation with its unenriched counterpart (r2=0.559-0.867). Up to a 2,242-fold enrichment of RNA from the target organism was obtained following a power law (r2=0.90), with the greatest fold enrichment achieved in samples with the largest ratio difference between the major and minor members. While using a single total library for prokaryote and eukaryote in a single sample could be beneficial for samples where RNA is limiting, we observed a decrease in reads mapping to protein coding genes and an increase of multi-mapping reads to rRNAs in AgSS enrichments from eukaryotic total RNA libraries as opposed to eukaryotic poly(A)-enriched libraries. Our results support a recommendation of using Agilent SureSelect targeted enrichment on poly(A)-enriched libraries for eukaryotic captures and total RNA libraries for prokaryotic captures to increase the robustness of multi-species transcriptomic studies.

genomics↗

bioRxiv2017

Transcriptome analysis reveals the differences between cellular response to ribosomal stress and translational stress

Ribosome biogenesis is an essential metabolic process of a growing cell. Cells need to continuously synthesize new ribosomes in order to make new proteins than can support building biomass and cell division. It is obvious that in the absence of ribosome biogenesis, cell growth will stop and cell division will stall. However, it is not clear whether cell growth stops due to reduced protein synthesis capacity (translational stress) or due to activation of signaling specific to ribosome biogenesis abnormalities (ribosomal stress). To understand the signaling pathways leading to cell cycle arrest under ribosomal and translational stress conditions, we performed time series RNA-seq experiments of cells at different time of ribosomal and translational stress. We found that expression of ribosomal protein genes follow different course over the time of these two stress types. In addition, ribosomal stress is sensed early in the cell, as early as 2hr. Up-regulation of genes responsive to oxidative stress and over representation of mRNAs for transcription factors responsive to stress was detected in cell at 2hr of ribosomal protein depletion. Even though, we detected phenotypic similarities in terms of cell separation and accumulation in G1 phase cells during inhibition of ribosome formation and ribosome function, different gene expression patterns underlie these phenotypes, indicating a difference in causalities of these phenotypes. Both ribosomal and translational stress show common increased expression of stress responsive gene expression, like Crz1 target gene expression, signature of oxidative stress response and finally membrane or cell wall instability. We speculate that cell membrane and cell wall acts as major stress sensor in the cell and adjust cellular metabolism accordingly. Any change in membrane lipid composition, or membrane protein oxidation, or decrease or increase in intracellular turgor pressure causes stress in cell membrane. Cell membrane or cell wall stress activates and/or inactivates specific signaling pathway which triggers stress responsive gene expression and adaptation of cellular behavior accordingly.

genomics↗