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: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: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: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:To further differentiate the gene expression of HPL-cultured or FBS-cultured ASC sheets, we have employed microarray analysis. ASCs were isolated from the subcutaneous fat tissue of four non-diabetic, non-smoking female donors with an average age of 45 y (32–57 y) and an average body mass index of 24.6 (21.0–26.6). Significantly up-regulated or down-regulated genes are highlighted in the HPL-cultured ASC sheets. Pathway enrichment analysis showcasing the top 10 hallmark gene-sets from MSigDB significantly enriched in up-regulated (top) or down-regulated (bottom) genes. The heatmap represented the expression patterns of angiogenesis-related genes between the HPL-cultured and FBS-cultured ASC sheets.
Project description:Top-down and bottom-up protein analysis of venom and saliva of solenodon. Venom and saliva was separated by HPLC and either directly analysis by HR FT MS/MS (Top-down) or further decomplexed by SDS-PAGE followed by in-gel trypsine digestion and HR LC-MS/MS analysis (bottom-up, Venom only). For shotgun bottom-up comparison of venom and saliva proteins, samples were directly reduced, alkylated, digested with trypsin and measured by HPLC-MS/MS. Additional bottom-up analysis was performed from bioactivity guided fractionation experiments.
