The colorectal adenoma-carcinoma sequence has provided a paradigmatic framework for understanding the successive somatic genetic changes and consequent clonal expansions leading to cancer. As for most cancer types, however, understanding of the earliest phases of colorectal neoplastic change, which may occur in morphologically normal tissue, is comparatively limited. Here, we whole genome sequenced hundreds of normal crypts from 42 individuals. Signatures of multiple mutational processes were revealed, some ubiquitous and continuous, others only found in some individuals, in some crypts or during certain periods of life. Likely driver mutations were present in ~1% of normal colorectal crypts in middle-aged individuals, indicating that adenomas and carcinomas are rare outcomes of a pervasive process of neoplastic change across morphologically normal colorectal epithelium. Colorectal cancers exhibit substantially elevated mutation burdens relative to normal cells. Sequencing normal colorectal cells provides quantitative insights into the genomic and clonal evolution of cancerdriver mutations, which conceivably are morphologically indistinguishable from normal cells, are similarly unclear. In large part, these deficiencies are due to the technical challenge of identifying somatic mutations in normal tissues, which are composed of myriad microscopic cell clones. Several different approaches have been adopted to address this 4-14 , revealing signatures of common somatic mutational processes in normal cells of the small and large intestine, liver, blood, skin, and nervous system. Thus far, however, studies have not been of sufficient scale to characterise variation in signature activity or detect less frequent processes 4-14. Remarkably high proportions of normal skin, oesophageal, and endometrial epithelial cells have been shown to be members of clones already carrying driver mutations 10,11,15,16 , and large mutant clones have been detected in blood 17-20. The extent of this phenomenon in the colon, an organ with a high cancer incidence, has not been investigated. Colonic epithelium is a contiguous cell sheet organised into ~15,000,000 crypts each composed of ~2,000 cells 21. Towards the base of each crypt resides a small number of stem cells ancestral to the maturing and differentiated cells in the crypt 22. These stem cells stochastically replace one another through a process of neutral drift 23,24 such that all stem cells, and thus all cells, in a crypt derive from a single ancestor stem cell that existed in recent years 25-27. The somatic mutations that were present in this ancestor are thus found in all ~2,000 descendant cells and can be revealed by DNA sequencing of an individual crypt. These stem cells are thought to be the cells of origin of colorectal cancers 28. To characterise the earliest stages of colorectal carcinogenesis, somatic mutation burdens, mutational signatures, clonal dynamics, and the frequency of driver mutations in normal colorectal epithelium were explored by sequencing individual colorect...
26The colorectal adenoma-carcinoma sequence has provided a paradigmatic framework for 27 understanding the successive somatic genetic changes and consequent clonal expansions 28 leading to cancer. As for most cancer types, however
The peripheral nervous system has a limited innate capacity for self-repair following injury, and surgical intervention is often required. For injuries greater than a few millimeters autografting is standard practice although it is associated with donor site morbidity and is limited in its availability. Because of this, nerve guidance conduits (NGCs) can be viewed as an advantageous alternative, but currently have limited efficacy for short and large injury gaps in comparison to autograft. Current commercially available NGC designs rely on existing regulatory approved materials and traditional production methods, limiting improvement of their design. The aim of this study was to establish a novel method for NGC manufacture using a custom built laser-based microstereolithography (μSL) setup that incorporated a 405 nm laser source to produce 3D constructs with ∼ 50 μm resolution from a photocurable poly(ethylene glycol) resin. These were evaluated by SEM, in vitro neuronal, Schwann and dorsal root ganglion culture and in vivo using a thy-1-YFP-H mouse common fibular nerve injury model. NGCs with dimensions of 1 mm internal diameter × 5 mm length with a wall thickness of 250 μm were fabricated and capable of supporting re-innervation across a 3 mm injury gap after 21 days, with results close to that of an autograft control. The study provides a technology platform for the rapid microfabrication of biocompatible materials, a novel method for in vivo evaluation, and a benchmark for future development in more advanced NGC designs, biodegradable and larger device sizes, and longer-term implantation studies.
Summary Inflammatory bowel disease (IBD) is a chronic inflammatory disease associated with increased risk of gastrointestinal cancers. We whole-genome sequenced 446 colonic crypts from 46 IBD patients and compared these to 412 crypts from 41 non-IBD controls from our previous publication on the mutation landscape of the normal colon. The average mutation rate of affected colonic epithelial cells is 2.4-fold that of healthy colon, and this increase is mostly driven by acceleration of mutational processes ubiquitously observed in normal colon. In contrast to the normal colon, where clonal expansions outside the confines of the crypt are rare, we observed widespread millimeter-scale clonal expansions. We discovered non-synonymous mutations in ARID1A , FBXW7 , PIGR , ZC3H12A , and genes in the interleukin 17 and Toll-like receptor pathways, under positive selection in IBD. These results suggest distinct selection mechanisms in the colitis-affected colon and that somatic mutations potentially play a causal role in IBD pathogenesis.
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