TP53 harbors somatic mutations in more than half of human tumors with some showing characteristic mutation spectra that have been linked to environmental exposures. In bladder cancer, a unique distribution of mutations amongst several codons of TP53 has been hypothesized to be caused by environmental carcinogens including 4-aminobiphenyl (4-ABP). 4-ABP undergoes metabolic activation to N-hydroxy-4-aminobiphenyl (N-OH-4-ABP) and forms pre-mutagenic adducts in DNA, of which N-(deoxyguanosin-8-yl)-4-ABP (dG-C8-4-ABP) is the major one. Human TP53 knock-in mouse embryo fibroblasts (HUFs) are a useful model to study the influence of environmental carcinogens on TP53-mutagenesis. By performing the HUF immortalization assay (HIMA) TP53-mutant HUFs are generated and mutations can be identified by sequencing. Here we studied the induction of mutations in human TP53 after treatment of primary HUFs with N-OH-4-ABP. In addition, mutagenicity in the bacterial lacZ reporter gene and the formation of dG-C8-4-ABP, measured by 32 P-postlabelling analysis, were determined in N-OH-4-ABP-treated primary HUFs. A total of 6% TP53-mutants were identified after treatment with 40 μM N-OH-4-ABP for 24 hr (n = 150) with G>C/C>G transversion being the main mutation type. The mutation spectrum found in the TP53 gene of immortalized N-OH-4-ABP-treated HUFs was unlike the one found in human bladder cancer. DNA adduct formation (~40 adducts/108 nucleotides) was detected after 24 hr treatment with 40 μM N-OH-4-ABP, but lacZ mutagenicity was not observed. Adduct levels decreased substantially (sixfold) after a 24 hr recovery period indicating that primary HUFs can efficiently repair the dG-C8-4-ABP adduct possibly before mutations are fixed. In conclusion, the observed difference in the N-OH-4-ABP-induced TP53 mutation spectrum to that observed in human bladder tumors do not support a role of 4-ABP in human bladder cancer development.
Background: The incidence of colorectal cancer (CRC) is increasing in developing countries, yet limited research on the CRC- associated microbiota has been conducted in these areas, in part due to scarce resources, facilities, and the difficulty of fresh or frozen stool storage/transport. Here, we aimed (1) to establish a broad representation of diverse developing countries (Argentina, Chile, India, and Vietnam); (2) to validate a ‘resource-light’ sample-collection protocol translatable in these settings using guaiac faecal occult blood test (gFOBT) cards stored and, importantly, shipped internationally at room temperature; (3) to perform initial profiling of the collective CRC-associated microbiome of these developing countries; and (4) to compare this quantitatively with established CRC biomarkers from developed countries.
Methods: We assessed the effect of international storage and transport at room temperature by replicating gFOBT from five UK volunteers, storing two in the UK, and sending replicates to institutes in the four countries. Next, to determine the effect of prolonged UK storage, DNA extraction replicates for a subset of samples were performed up to 252 days apart. To profile the CRC-associated microbiome of developing countries, gFOBT were collected from 41 treatment-naïve CRC patients and 40 non-CRC controls from across the four institutes, and V4 16S rRNA gene sequencing was performed. Finally, we constructed a random forest (RF) model that was trained and tested against existing datasets from developed countries.
Results: The microbiome was stably assayed when samples were stored/transported at room temperature and after prolonged UK storage. Large-scale microbiome structure was separated by country and continent, with a smaller effect from CRC. Importantly, the RF model performed similarly to models trained using external datasets and identified similar taxa of importance (Parvimonas, Peptostreptococcus, Fusobacterium, Alistipes, and Escherichia).
Conclusions: This study demonstrates that gFOBT, stored and transported at room temperature, represents a suitable method of faecal sample collection for amplicon-based microbiome biomarkers in developing countries and suggests a CRC-faecal microbiome association that is consistent between developed and developing countries.
Methods for highly multiplexed RNA imaging are limited in spatial resolution, and thus in their ability to localize transcripts to nanoscale and subcellular compartments. We adapt expansion microscopy, which physically expands biological specimens, for long-read untargeted and targeted in situ RNA sequencing. We applied untargeted expansion sequencing (ExSeq) to mouse brain, yielding readout of thousands of genes, including splice variants and novel transcripts. Targeted ExSeq yielded nanoscale-resolution maps of RNAs throughout dendrites and spines in neurons of the mouse hippocampus, revealing patterns across multiple cell types; layer-specific cell types across mouse visual cortex; and the organization and position-dependent states of tumor and immune cells in a human metastatic breast cancer biopsy. Thus ExSeq enables highly multiplexed mapping of RNAs, from nanoscale to system scale.
Summary: In situ sequencing of physically expanded specimens enables multiplexed mapping of RNAs at nanoscale, subcellular resolution.
Discovery: The Rosetta team in Nature Genetics have demonstrated that the amino acid transporter SLC7A5 is required for efficient growth of KRAS-mutant colorectal cancer (CRC). SLC7A5 mediates the transmembrane trafficking of glutamine in exchange for essential amino acids that sustain cell growth, also referred to as an antiporter* in the paper, and has been shown to be critical for tumour development in early and late-stage mouse models of CRC.
Methods: This was achieved using a combination of approaches, including 3D cell culture (organoids), the generation of genetically engineered mouse models (GEMMs) [early and late stage carcinoma] and the analysis of human CRC samples.
*An antiporter (also called exchanger or counter-transporter) is a cotransporter and integral membrane protein involved in secondary active transport of two or more different molecules or ions across a phospholipid membrane such as the plasma membrane in opposite directions, one into the cell and one out of the cell.
Identification of early processes leading to complex tissue pathologies, such as inflammatory bowel diseases, poses a major scientific and clinical challenge that is imperative for improved diagnosis and treatment. Most studies of inflammation onset focus on cellular processes and signaling molecules, while overlooking the environment in which they take place, the continuously remodeled extracellular matrix. In this study, we used colitis models for investigating extracellular–matrix dynamics during disease onset, while treating the matrix as a complete and defined entity. Through the analysis of matrix structure, stiffness and composition, we unexpectedly revealed that even prior to the first clinical symptoms, the colon displays its own unique extracellular–matrix signature and found specific markers of clinical potential, which were also validated in human subjects. We also show that the emergence of this pre-symptomatic matrix is mediated by subclinical infiltration of immune cells bearing remodeling enzymes. Remarkably, whether the inflammation is chronic or acute, its matrix signature converges at pre-symptomatic states. We suggest that the existence of a pre-symptomatic extracellular–matrix is general and relevant to a wide range of diseases.