Reactive species such as free radicals are constantly generated in vivo and DNA is the most important target of oxidative stress. Oxidative DNA damage is used as a predictive biomarker to monitor the risk of development of many diseases. The comet assay is widely used for measuring oxidative DNA damage at a single cell level. The analysis of comet assay output images, however, poses considerable challenges. Commercial software is costly and restrictive, while free software generally requires laborious manual tagging of cells. This paper presents OpenComet, an open-source software tool providing automated analysis of comet assay images. It uses a novel and robust method for finding comets based on geometric shape attributes and segmenting the comet heads through image intensity profile analysis. Due to automation, OpenComet is more accurate, less prone to human bias, and faster than manual analysis. A live analysis functionality also allows users to analyze images captured directly from a microscope. We have validated OpenComet on both alkaline and neutral comet assay images as well as sample images from existing software packages. Our results show that OpenComet achieves high accuracy with significantly reduced analysis time.

Although oxidative stress has been shown to induce senescence and replication stress independently, no study has implicated unresolved replication stress as the driver for cellular senescence in response to oxidative stress. Using cells exposed to increasing concentrations of hydrogen peroxide, we show that sub-lethal amount of exogenous hydrogen peroxide induces two waves of DNA damage. The first wave is rapid and transient while the second wave coincides with the cells transition from the S to the G2/M phases of cell cycle. Subsequently, cells enter growth arrest accompanied by the acquisition of senescence-associated characteristics. Furthermore, a p53-dependent decrease in Rad51, which is associated with the formation of DNA segments with chromatin alterations reinforcing senescence, and Lamin B1 that is involved in chromatin remodeling, is observed during the establishment of the senescent phenotype. On the other hand, increase in senescence associated-β-Gal activity, a classical marker of senescence and HMGA2, a marker of the senescence-associated heterochromatin foci, is shown to be independent of p53. Together, our findings implicate replication stress-induced endogenous DNA damage as the driver for the establishment of cellular senescence upon sub-lethal oxidative stress, and implicate the role of p53 in some but not all hallmarks of the senescent phenotype.

Targeting undruggable intracellular proteins with peptides: novel on-target macrocyclic peptide inhibitors of KRAS with broad inhibition of proliferation of multiple KRAS-dependent cancer cell lines.

BackgroundActivation of PPARs has been reported to inhibit the proliferation of malignant cells from different lineages. They are involved in transcription regulation of genes upon activation by a ligand. The binding of PPARs to the promoter sequence either represses or activates the gene. Hence, PPARs represent promising targets for cancer treatment because of their anti-proliferative and pro-apoptotic activities. Here we computationally identified PPAR binding regions in NHE1 and MnSOD. We further validated the predictions in vitro.ResultsOur results computationally predicted the presence of 2 PPRE motifs in NHE1 and 3 PPRE motifs in MnSOD. We experimentally confirmed the true motifs and their regulation by PPAR.ConclusionOur results suggest that both NHE1 and MnSOD have PPRE binding motif in their upstream/promoter region and hence are regulated by PPAR upon ligand binding.

An oncogenic mutant-specific druggable allosteric pocket captured by MD simulations.

Here we provide evidence to link sub-lethal oxidative stress to lysosome biogenesis. Exposure of cells to sub-lethal concentrations of exogenously added hydrogen peroxide resulted in cytosol to nuclear translocation of the Transcription Factor EB (TFEB), the master controller of lysosome biogenesis and function. Nuclear translocation of TFEB was dependent upon the activation of a cathepsin-caspase 3 signaling pathway, downstream of lysosomal membrane permeabilization and accompanied by a significant increase in lysosome numbers as well as induction of TFEB-dependent lysosome-associated genes expression such as Ctsl, Lamp2 and its spliced variant Lamp2a, Neu1, Ctsb, Sqstm1, and Atg9b. The effects of sub-lethal oxidative stress on lysosomal gene expression and biogenesis were rescued upon gene silencing of caspase 3 and TFEB. Notably, caspase 3 activation was not associated with phenotypic hallmarks of apoptosis, evidenced by the absence of caspase 3 substrate cleavage, such as PARP, Lamin A/C or gelsolin. Taken together, these data demonstrate for the first time an unexpected and non-canonical role of a cathepsin-caspase 3 axis in the nuclear translocation of TFEB leading to lysosome biogenesis under conditions of sub-lethal oxidative stress.

Peroxisome proliferator-activated receptor (PPAR) belongs to the nuclear hormone receptor superfamily. Recently published reports demonstrate the importance of a direct repeat 2 (DR2) as a PPARγ-responsive element in addition to the canonical direct repeat 1 (DR1) Peroxisome proliferator response elements (PPREs). However, a comprehensive and systematic approach to constructing de novo disease-specific gene networks for PPARγ is lacking, especially one that includes PPARγ target genes containing either DR1 or DR2 site within their promoter region. Here, we computationally identified 1154 PPARγ direct target genes and constructed the PPARγ disease gene network, which revealed 138 PPARγ target genes that are associated with 65 unique diseases. The network shows that PPARγ target genes are highly associated with cancer and neurological diseases. Thirty-eight PPARγ direct target genes were found to be involved in prostate cancer and two key (hub) PPARγ direct target genes, PRKCZ and PGK1, were experimentally validated to be repressed upon PPARγ activation by its natural ligand, 15d-PGJ(2) in three prostrate cancer cell lines. We proposed that PRKCZ and PGK1 could be novel therapeutic targets for prostate cancer. These investigations would not only aid in understanding the molecular mechanisms by which PPARγ regulates disease targets but would also lead to the identification of novel PPARγ gene targets.

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