The VEGF-A exon 8 splicing-sensitive fluorescent reporter mouse is a novel tool to assess the effects of splicing regulatory compounds in vivo.

Vascular endothelial growth factor (VEGF)-A is differentially spliced to give two functionally different isoform families; pro-angiogenic, pro-permeability VEGF-Axxx and anti-angiogenic, anti-permeability VEGF-Axxxb. VEGF-A splicing is dysregulated in several pathologies, including cancer, diabetes, and peripheral arterial disease. The bichromatic VEGF-A splicing-sensitive fluorescent reporter harboured in a transgenic mouse is a novel approach to investigate the splicing patterns of VEGF-A in vivo. We generated a transgenic mouse harbouring a splicing-sensitive fluorescent reporter designed to mimic VEGF-A terminal exon splicing (VEGF8ab) by insertion into the ROSA26 genomic locus. dsRED expression denotes proximal splice site selection (VEGF-Axxx) and eGFP expression denotes distal splice site selection (VEGF-Axxxb). We investigated the tissue-specific expression patterns in the eye, skeletal muscle, cardiac muscle, kidney, and pancreas, and determined whether the splicing pattern could be manipulated in the same manner as endogenous VEGF-A by treatment with the SRPK1 inhibitor SPHINX 31. We confirmed expression of both dsRED and eGFP in the eye, skeletal muscle, cardiac muscle, kidney, and pancreas, with the highest expression of both fluorescent proteins observed in the exocrine pancreas. The ratio of dsRED and eGFP matched that of endogenous VEGF-Axxx and VEGF-Axxxb. Treatment of the VEGF8ab mice with SPHINX 31 increased the mRNA and protein eGFP/dsRED ratio in the exocrine pancreas, mimicking endogenous VEGF-A splicing. The VEGF-A exon 8 splicing-sensitive fluorescent reporter mouse is a novel tool to assess splicing regulation in the individual cell-types and tissues, which provides a useful screening process for potentially therapeutic splicing regulatory compounds in vivo.

ELF3 is an antagonist of oncogenic-signalling-induced expression of EMT-TF ZEB1.

Background: Epithelial-to-mesenchymal transition (EMT) is a key step in the transformation of epithelial cells into migratory and invasive tumour cells. Intricate positive and negative regulatory processes regulate EMT. Many oncogenic signalling pathways can induce EMT, but the specific mechanisms of how this occurs, and how this process is controlled are not fully understood. Methods: RNA-Seq analysis, computational analysis of protein networks and large-scale cancer genomics datasets were used to identify ELF3 as a negative regulator of the expression of EMT markers. Western blotting coupled to siRNA as well as analysis of tumour/normal colorectal cancer panels was used to investigate the expression and function of ELF3. Results: RNA-Seq analysis of colorectal cancer cells expressing mutant and wild-type ß-catenin and analysis of colorectal cancer cells expressing inducible mutant RAS showed that ELF3 expression is reduced in response to oncogenic signalling and antagonizes Wnt and RAS oncogenic signalling pathways. Analysis of gene-expression patterns across The Cancer Genome Atlas (TCGA) and protein localization in colorectal cancer tumour panels showed that ELF3 expression is anti-correlated with ß-catenin and markers of EMT and correlates with better clinical prognosis. Conclusions: ELF3 is a negative regulator of the EMT transcription factor (EMT-TF) ZEB1 through its function as an antagonist of oncogenic signalling.

Protein metabolism in burned rats.

Incorporation of [2-14C]glycine was used to estimate serum protein synthesis in four groups of rats. These were the control (group C); 20% body surface burn (group B); 20% burn, seeded with Pseudomonas aeruginosa (group BI); and burned-infected treated topically with mafenide (alpha-amino-p-toluenesulfonamide) acetate (group BIS), a treatment which controls P, aeruginosa burn-wound infection in humans. On the 6th day postburn the relative specific activities of all fractions were increased in the order BI greater than BIS greater than B greater than C, as were the concentrations of the globulins; Serum albumin concentration fell, being lowest in BI. Tissue albumin contents, measured by radioimmunoassay, of eviscerated blood-free bodies of rats were (mg/100 g rat wt): C, 207; B, 294; BI, 256. Analyses of individual tissues showed that the difference was due to increased albumin content in the burn-wound area. The tissue albumin was of normal molecular size and was immunologically reactive. We conclude that the prolonged hypoalbuminemia following burn injury is not a consequence of impaired albumin synthesis, but a result of altered compartmentation.