Al-Nahrain University / College of Medicine
MBChB, MSc Histopathology, PhD Molecular Pathology
Ahmed K. Mahdi, Juan F. Medrano, and Pablo J. Ross MDPI AG
We investigated the possibility of single-step genome editing in small ruminants by CRISPR-Cas9 zygote electroporation. We targeted SOCS2 and PDX1 in sheep embryos and OTX2 in goat embryos, utilizing a dual sgRNA approach. Gene editing efficiency was compared between microinjection and three different electroporation settings performed at four different times of embryo development. Electroporation of sheep zygotes 6 h after fertilization with settings that included short high-voltage (poring) and long low-voltage (transfer) pulses was efficient at producing SOCS2 knock-out blastocysts. The mutation rate after CRISPR/Cas9 electroporation was 95.6% ± 8%, including 95.4% ± 9% biallelic mutations; which compared favorably to 82.3% ± 8% and 25% ± 10%, respectively, when using microinjection. We also successfully disrupted the PDX1 gene in sheep and the OTX2 gene in goat embryos. The biallelic mutation rate was 81 ± 5% for PDX1 and 85% ± 6% for OTX2. In conclusion, using single-step CRISPR-Cas9 zygote electroporation, we successfully introduced biallelic deletions in the genome of small ruminant embryos.
Marco Youssef William Zaki, Ahmed Khairallah Mahdi, Gillian Lucinda Patman, Anna Whitehead, João Pais Maurício, Misti Vanette McCain, Despina Televantou, Sameh Abou-Beih, Erik Ramon-Gil, Robyn Watson,et al. Springer Science and Business Media LLC
AbstractThe prevalence of obesity and non-alcoholic fatty liver disease (NAFLD) associated hepatocellular carcinoma (HCC) is rising, even in the absence of cirrhosis. We aimed to develop a murine model that would facilitate further understanding of NAFLD-HCC pathogenesis. A total of 144 C3H/He mice were fed either control or American lifestyle (ALIOS) diet, with or without interventions, for up to 48 weeks of age. Gross, liver histology, immunohistochemistry (IHC) and RNA-sequencing data were interpreted alongside human datasets. The ALIOS diet promoted obesity, elevated liver weight, impaired glucose tolerance, non-alcoholic fatty liver disease (NAFLD) and spontaneous HCC. Liver weight, fasting blood glucose, steatosis, lobular inflammation and lipogranulomas were associated with development of HCC, as were markers of hepatocyte proliferation and DNA damage. An antioxidant diminished cellular injury, fibrosis and DNA damage, but not lobular inflammation, lipogranulomas, proliferation and HCC development. An acquired CD44 phenotype in macrophages was associated with type 2 diabetes and NAFLD-HCC. In this diet induced NASH and HCC (DINAH) model, key features of obesity associated NAFLD-HCC have been reproduced, highlighting roles for hepatic steatosis and proliferation, with the acquisition of lobular inflammation and CD44 positive macrophages in the development of HCC—even in the absence of progressive injury and fibrosis.
Leqian Yu, Yulei Wei, Hai-Xi Sun, Ahmed K. Mahdi, Carlos A. Pinzon Arteaga, Masahiro Sakurai, Daniel A. Schmitz, Canbin Zheng, Emily D. Ballard, Jie Li,et al. Elsevier BV
Chiao-En Wu, Arman Esfandiari, Yi-Hsuan Ho, Nan Wang, Ahmed Khairallah Mahdi, Erhan Aptullahoglu, Penny Lovat, and John Lunec Springer Science and Business Media LLC
Background:Cutaneous melanoma is the most serious skin malignancy and new therapeutic strategies are needed for advanced melanoma. TP53 mutations are rare in cutaneous melanoma and hence activation of wild-type p53 is a potential therapeutic strategy in cutaneous melanoma. Here, we investigated the WIP1 inhibitor, GSK2830371, and MDM2–p53 binding antagonists (nutlin-3, RG7388 and HDM201) alone and in combination treatment in cutaneous melanoma cell lines and explored the mechanistic basis of these responses in relation to the genotype and induced gene expression profile of the cells.Methods:A panel of three p53WT (A375, WM35 and C8161) and three p53MUT (WM164, WM35-R and CHL-1) melanoma cell lines were used. The effects of MDM2 and WIP1 inhibition were evaluated by growth inhibition and clonogenic assays, immunoblotting, qRT–PCR gene expression profiling and flow cytometry.Results:GSK2830371, at doses (⩽10 μM) that alone had no growth-inhibitory or cytotoxic effects on the cells, nevertheless significantly potentiated the growth-inhibitory and clonogenic cell killing effects of MDM2 inhibitors in p53WT but not p53MUT melanoma cells, indicating the potentiation worked in a p53-dependent manner. The siRNA-mediated knockdown of p53 provided further evidence to support the p53 dependence. GSK2830371 increased p53 stabilisation through Ser15 phosphorylation and consequent Lys382 acetylation, and decreased ubiquitination and proteasome-dependent degradation when it was combined with MDM2 inhibitors. These changes were at least partly ATM mediated, shown by reversal with the ATM inhibitor (KU55933). GSK2830371 enhanced the induction of p53 transcriptional target genes, cell cycle arrest and apoptosis.Conclusions:GSK2830371, a WIP1 inhibitor, at doses with no growth-inhibitory activity alone, potentiated the growth-inhibitory and cytotoxic activity of MDM2 inhibitors by increasing phosphorylation, acetylation and stabilisation of p53 in cutaneous melanoma cells in a functional p53-dependent manner.