Project description:The role of stem cells in solid tumors remains controversial. In colorectal cancers (CRC), this is complicated by the conflicting ‘top-down’ or ‘bottom-up’ hypothesis of cancer initiation. We profiled the expressions of genes from the top (T) and bottom (B) fractions of the crypt in morphologically normal-appearing colonic mucosa (M) and contrasted this to that of matched mucosa adjacent to tumors (MT) in twenty three sporadic CRC patients. In thirteen patients, the genetic distance (M-MT) between the B fractions is smaller than the distance between the T fractions indicating that the expressions of significant genes diverge further in the top fractions (B<T). In the remaining ten patients, the reverse is observed (B>T). Taking genetic divergence as an intermediate endpoint, the data indicates that it is equally likely that CRC initiates from ‘top-down’ via dedifferentiated colonocytes or ‘bottom-up’ via dysregulated intestinal stem cells. This has important ramification for subsequent therapeutic considerations.
Project description:Eutrophication can lead to an uncontrollable increase in algal biomass, which has repercussions for the entire microbial and pelagic community. Studies have shown how nutrient enrichment affects microbial species succession, however details regarding the impact on community functionality are rare. Here, we applied a metaproteomic approach to investigate the functional changes to algal and bacterial communities, over time, in oligotrophic and eutrophic conditions, in freshwater microcosms. Samples were taken early during algal and cyanobacterial dominance and later under bacterial dominance. 1048 proteins, from the two treatments and two timepoints, were identified and quantified by their exponentially modified protein abundance index. In oligotrophic conditions, Bacteroidetes express extracellular hydrolases and Ton-B dependent receptors to degrade and transport high molecular weight compounds captured while attached to the phycosphere. Alpha- and Beta-proteobacteria were found to capture different substrates from algal exudate (carbohydrates and amino acids, respectively) suggesting resource partitioning to avoid direct competition. In eutrophic conditions, environmental adaptation proteins from cyanobacteria suggested better resilience compared to algae in a low carbon nutrient enriched environment. This study provides insight into differences in functional microbial processes between oligo- and eutrophic conditions at different timepoints and highlights how primary producers control bacterial resources in freshwater environments.
Project description:The neocortex is functionally organized into layers. Layer four receives the densest bottom up sensory inputs, while layers 2/3 and 5 receive top down inputs that may convey predictive information. A subset of cortical somatostatin (SST) neurons, the Martinotti cells, gate top down input by inhibiting the apical dendrites of pyramidal cells in layers 2/3 and 5, but it is unknown whether an analogous inhibitory mechanism controls activity in layer 4. Using high precision circuit mapping, in vivo optogenetic perturbations, and single cell transcriptional profiling, we reveal complementary circuits in the mouse barrel cortex involving genetically distinct SST subtypes that specifically and reciprocally interconnect with excitatory cells in different layers: Martinotti cells connect with layers 2/3 and 5, whereas non-Martinotti cells connect with layer 4. By enforcing layer-specific inhibition, these parallel SST subnetworks could independently regulate the balance between bottom up and top down input.
Project description:MALDI imaging mass spectrometry (MALDI IMS) is a powerful tool for the visualization of proteins in tissues and has demonstrated considerable diagnostic and prognostic value. One main challenge is that the molecular identity of such potential biomarkers mostly remains unknown. We introduce a method that removes this issue by systematically identifying the proteins embedded in the MALDI matrix using a combination of bottom-up and top-down proteomics. The analyses of ten human tissues lead to the identification of 1,400 abundant and soluble proteins constituting the set of proteins detectable by MALDI IMS including >90% of all IMS biomarkers reported in the literature. Top-down analysis of the matrix proteome identified 124 mostly N- and C-terminally fragmented proteins indicating considerable protein processing activity in tissues. This work presents a generic method and near complete list of MALDI IMS biomarkers that will become a valuable resource for the IMS community. Detailed description of the bioinformatics pipeline can be found in pipeline.txt. Briefly, different Mascot Distiller, Mascot and Scaffold versions have been used for the bottom-up and top-down data.
Project description:In the present work we developed a sample preparation approach for the combined bottom-up and top-down proteomics analysis of small open reading frame encoded proteins (SEP). Key improvements were made by the application of solid phase extraction (SPE) supported enrichment of LMW proteins, followed by two-dimensional LC-MS top-down analysis encompassing both HCD and EThcD ion activation. Bottom-up experiments were used to support and confirm top-down data interpretation. This strategy allowed for the top-down characterisation 36 proteoforms mapping to 12 SEP of the archaea Methanosarcina mazei, and for the first time the identification of posttranslational modifications in these microproteins.
Project description:Fast photochemical oxidation of proteins (FPOP) footprinting is a structural mass spectrometry method that maps proteins by fast and irreversible chemical reactions. The position of oxidative modification reflects solvent accessibility and site reactivity and thus provides information about protein conformation, structural dynamics, and interactions. Bottom-up mass spectrometry is an established standard method to analyze FPOP samples. In the bottom-up approach, all forms of the protein are digested together by a protease of choice, which results in mixture of peptides from various subpopulations of proteins with varying degrees of photochemical oxidation. But, once the protein is already oxidized at least once, the spatial distribution of any consecutive oxidation no longer truly reflects initial protein solvent accessibility and residue reactivity, because the site preference of further oxidation may also be influenced by the changes caused by the prior oxidation. Thus, it would be interesting to obtain an assessment of the protein structure based on the FPOP data, by analyzing only singly oxidized protein populations. This requires utilization of more specific top-down mass spectrometry approaches. The key element of any top-down experiment is selection of a suitable method of ion isolation, excitation and fragmentation. Here we employ and compare collision-induced dissociation (CID), electron-transfer dissociation (ETD), and electron-capture dissociation (ECD) combined with multi continuous accumulation of selected ions (CASI). Singly oxidized subpopulation of FPOP labeled ubiquitin was used to optimize the method. The usefulness was then demonstrated further by using it to visualize structural changes induced by cofactor removal from holo/apo myoglobin system. The top-down data were compared with the literature and with the bottom-up data set obtained on the same samples. The top-down results were found to be in a good agreement, which indicates that monitoring singly FPOP oxidized ion population by top-down is a functional workflow for oxidative protein footprinting.
Project description:This bottom-up analysis is the supplementary data file to the top-down analysis deposited with identifier PXD014660. The peptide data are used to support assignments of modifications detected in top-down. Details are described in the associated manuscript.
Project description:We investigated a contaminant-degrading microbial community by sequencing total RNA (without rRNA depletion) from microcosms containing sediment from a hypoxic contaminated aquifer fed with isotopically labeled toluene.