ABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
The media formulations necessary for deriving and sustaining organoids from epithelial tissues such as prostate, colon, gastric, liver, pancreas, and others have been established. Critical components of organoid media are a set of growth factors that include R-spondins and BMP signalling antagonists such as Noggin or Gremlin 1. Currently, the practical limitations for formulating organoid media of reproducible potency and larger-scale media production that have hampered further technological applications of organoid technology include: the cost of growth factors such as R-spondins and Gremlin 1/Noggin and their production as defined specific activities free of contaminants that may affect organoid growth. Here we report the production of highly pure recombinant Gremlin 1 and R-spondin 1 from bacterial expression for use in organoid media. We detail the workflow for Gremlin 1 and R-spondin 1 expression, purification, quantification of cellular activity, quality control and use in media formulated for culturing organoids derived from a number of tissues. The development of precisely formulated, cost-effective media of defined specific activity will engender the development of novel applications for organoid technology.
Subject(s)
Cell Culture Techniques/economics , Culture Media/chemistry , Intercellular Signaling Peptides and Proteins/chemistry , Organoids/growth & development , Animals , Bacteria/genetics , Bacteria/metabolism , Bone Morphogenetic Proteins/antagonists & inhibitors , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Culture Techniques/methods , Culture Media/economics , Gene Transfer Techniques , Humans , Intercellular Signaling Peptides and Proteins/economics , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Mice , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Thrombospondins/genetics , Thrombospondins/metabolismABSTRACT
An 84-year-old man was admitted with 4 days of postprandial emesis. Gastroscopy revealed the presence of a large gallstone in the duodenal bulb causing gastric outlet obstruction. The patient was diagnosed with Bouveret's syndrome. Treatment consisted of gastrotomy with removal of the gallstone.
Subject(s)
Gallstones/diagnosis , Gallstones/surgery , Gastric Outlet Obstruction/diagnosis , Gastric Outlet Obstruction/surgery , Aged, 80 and over , Gallstones/complications , Gastric Outlet Obstruction/etiology , Gastroscopy , Humans , Male , Treatment Outcome , Vomiting/diagnosis , Vomiting/etiology , Vomiting/surgeryABSTRACT
The GTPase Ras is a molecular switch engaged downstream of G-protein-coupled receptors and receptor tyrosine kinases that controls multiple cell-fate-determining signalling pathways. Ras signalling is frequently deregulated in cancer, underlying associated changes in cell phenotype. Although Ca(2+) signalling pathways control some overlapping functions with Ras, and altered Ca(2+) signalling pathways are emerging as important players in oncogenic transformation, how Ca(2+) signalling is remodelled during transformation and whether it has a causal role remains unclear. We have investigated Ca(2+) signalling in two human colorectal cancer cell lines and their isogenic derivatives in which the allele encoding oncogenic K-Ras (G13D) was deleted by homologous recombination. We show that agonist-induced Ca(2+) release from the endoplasmic reticulum (ER) intracellular Ca(2+) stores is enhanced by loss of K-Ras(G13D) through an increase in the Ca(2+) content of the ER store and a modification of the abundance of inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) subtypes. Consistently, uptake of Ca(2+) into mitochondria and sensitivity to apoptosis was enhanced as a result of K-Ras(G13D) loss. These results suggest that suppression of Ca(2+) signalling is a common response to naturally occurring levels of K-Ras(G13D), and that this contributes to a survival advantage during oncogenic transformation.
