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Highlights: [•] The principles underlying most commonly used microRNA target prediction algorithms. [•] An evaluation of the experimental data that supports each computational approach. [•] An evaluation of microRNA target databases, their strengths and weaknesses. [Copyright &y& Elsevier]

CTCF is a highly conserved, ubiquitously expressed DNA-binding protein that has widespread capabilities in gene regulation. CTCF plays important roles in cell growth regulatory processes and epigenetic functions. Ectopic expression of CTCF results in severe cell growth inhibition at multiple points within the cell cycle, indicating that CTCF levels must be stringently monitored. We have investigated the subcellular localization of CTCF in detail. Interestingly, we observe that CTCF shows a dynamic cell cycle-dependent distribution. Immunofluorescent staining reveals that in interphase CTCF is a nuclear protein, which is mainly excluded from the nucleolus. Strikingly, CTCF is associated with the centrosome during mitosis, especially from metaphase to anaphase. At telophase, CTCF dissociates from the centrosome and localizes to the midbody and the reformed nuclei. The association of CTCF with centrosomes and the midbody is further confirmed by biochemical fractionation. Moreover, subcellular fractions of CTCF show cell cycle and organelle-specific posttranslational modifications, suggesting different roles for CTCF at different stages of the cell cycle. [Copyright &y& Elsevier]

Abstract Spred, like Sprouty (Spry) and also Sef proteins, have been identified as important regulators of receptor tyrosine kinase (RTK)-mediated MAPK/ERK-signaling in various developmental systems, controlling cellular processes such as proliferation, migration and differentiation. Spreds are widely expressed during early embryogenesis, and in the eye lens, become more localised in the lens epithelium with later development, overlapping with other antagonists including Spry. Given the synexpression of Spreds and Spry in lens, in order to gain a better understanding of their specific roles in regulating growth factor mediated-signaling and cell behavior, we established and characterised lines of transgenic mice overexpressing Spred1 or Spred2 , specifically in the lens. This overexpression of Spreds resulted in a small lens phenotype during ocular morphogenesis, retarding its growth by compromising epithelial cell proliferation and fiber differentiation. These in situ findings were shown to be dependent on the ability of Spreds to suppress MAPK-signaling, in particular FGF-induced ERK1/2-signaling in lens cells. This was validated in vitro using lens epithelial explants, that highlighted the overlapping role of Spreds with Spry2, but not Spry1. This study provides insights into the putative function of Spreds and Spry in situ , some overlapping and some distinct, and their importance in regulating lens cell proliferation and fiber differentiation contributing to lens and eye growth. Highlights • Spred1 and Spred2 have the same impact on lens cellular processes. • Spreds, like Spry2, but not Spry1 can block FGF-induced ERK1/2-signaling in lens cells. • Spreds, like Spry2, but not Spry1 can block FGF-induced lens epithelial cell proliferation. • Spreds can negatively modulate the rate of FGF-induced lens fiber differentiation. • Spred overexpression in lens retards lens growth. [ABSTRACT FROM AUTHOR]

Despite the major success obtained by the use of tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML), resistances to therapies occur due to mutations in the ABL-kinase domain of the BCR-ABL oncogene. Amongst these mutations, the “gatekeeper” T315I is a major concern as it renders leukemic cells resistant to all licenced TKI except Ponatinib. We report here that Fourier transform infrared (FTIR) microspectroscopy is a powerful methodology allowing rapid and direct identification of a spectral signature in single cells expressing T315I-mutated BCR-ABL. The specificity of this spectral signature is confirmed using a Dox-inducible T315I-mutated BCR-ABL-expressing human UT-7 cells as well as in murine embryonic stem cells. Transcriptome analysis of UT-7 cells expressing BCR-ABL as compared to BCR-ABL T315I clearly identified a molecular signature which could be at the origin of the generation of metabolic changes giving rise to the spectral signature. Thus, these results suggest that this new methodology can be applied to the identification of leukemic cells harbouring the T315I mutation at the single cell level and could represent a novel early detection tool of mutant clones. It could also be applied to drug screening strategies to target T315I-mutated leukemic cells. [ABSTRACT FROM AUTHOR]

RNA sequencing has revealed a striking diversity in transcriptomic complexity, to which alternative splicing is a major contributor. Intron retention (IR) is a conserved form of alternative splicing that was originally overlooked in normal mammalian physiology and development, due mostly to difficulties in its detection. IR has recently been revealed as an independent mechanism of controlling and enhancing the complexity of gene expression. IR facilitates rapid responses to biological stimuli, is involved in disease pathogenesis, and can generate novel protein isoforms. Many challenges, however, remain in detecting and quantifying retained introns and in determining their effects on cellular phenotype. In this review, we provide an overview of these challenges, and highlight approaches that can be used to address them. [ABSTRACT FROM AUTHOR]

Cellular therapies utilizing T cells expressing chimeric antigen receptors (CARs) have garnered significant interest due to their clinical success in hematological malignancies. Unfortunately, this success has not been replicated in solid tumors, with only a small fraction of patients achieving complete responses. A number of obstacles to effective CAR-T cell therapy in solid tumors have been identified including tumor antigen heterogeneity, poor T cell fitness and persistence, inefficient trafficking and inability to penetrate into the tumor, immune-related adverse events due to on-target/off-tumor toxicity, and the immunosuppressive tumor microenvironment. Many preclinical studies have focused on improvements to CAR design to try to overcome some of these hurdles. However, a growing body of work has also focused on the use of local and/or regional delivery of CAR-T cells as a means to overcome poor T cell trafficking and inefficient T cell penetration into tumors. Most trials that incorporate locoregional delivery of CAR-T cells have targeted tumors of the central nervous system - repurposing an Ommaya/Rickham reservoir for repeated delivery of cells directly to the tumor cavity or ventricles. Hepatic artery infusion is another technique used for locoregional delivery to hepatic tumors. Locoregional delivery theoretically permits increased numbers of CAR-T cells within the tumor while reducing the risk of immune-related systemic toxicity. Studies to date have been almost exclusively phase I. The growing body of evidence indicates that locoregional delivery of CAR-T cells is both safe and feasible. This review focuses specifically on the use of locoregional delivery of CAR-T cells in clinical trials. [Display omitted] [ABSTRACT FROM AUTHOR]

Epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) principally contributes to the pathogenesis of fibrotic cataract. Sprouty (Spry) and Spred proteins are receptor tyrosine kinase (RTK) antagonists that can regulate RTK-mediated signaling pathways, such as the MAPK/ERK1/2-signaling pathway. The present study examines the ability of Spry and Spred to inhibit TGFβ-induced EMT in LECs. LECs explanted from postnatal-day-21 Wistar rats were transduced with adenoviral vectors coding for Spry1, Spry2 or Spred2, and subsequently treated with or without TGFβ2. Immunofluorescent labeling of explants for the epithelial membrane marker β-catenin, and the mesenchymal marker alpha-smooth muscle actin (α-sma), were used to characterize the progression of EMT. Western blotting was used to quantify levels of α-sma and ERK1/2-signaling. Overexpression of Spry or Spred in LECs was sufficient to suppress EMT in response to TGFβ, including a block to cell elongation, β-catenin delocalization and α-sma accumulation. Spry and Spred were also shown to significantly block ERK1/2 phosphorylation for up to 18 h of TGFβ treatment but did not impair the earlier activation of ERK1/2 at 20 min. These findings suggest that Spry and Spred may not directly impact ERK1/2-signaling activated by the serine/threonine kinase TGFβ receptor, but may selectively target later ERK1/2-signaling driven by downstream RTK-mediated signaling. Taken together, our data establish Spry and Spred antagonists as potent negative regulators of TGFβ-induced EMT that can regulate ERK1/2-signaling in a temporal manner. A greater understanding of how Spry and Spred regulate the complex signaling interactions that underlie TGFβ-induced EMT will be essential to facilitate the development of novel therapeutics for different pathologies driven by EMT, including fibrotic forms of cataract. • Overexpression of Spry or Spred in lens epithelia can suppress EMT. • Spry or Spred block ERK1/2 phosphorylation in lens EMT. • ERK1/2 activity in lens EMT is temporally regulated by Spry and Spred. • Spry is a more potent suppressors of ERK1/2-signaling than Spred2 in lens EMT. • ERK2 is preferentially suppressed over ERK1 by Spry in lens EMT. [ABSTRACT FROM AUTHOR]

Abstract: Gene mutations in cardiac troponin I (cTnI) account for up to 5% of genotyped families with familial hypertrophic cardiomyopathy (FHC). Little is known about how cTnI mutations cause disease. Five lines of transgenic mice were generated which overexpress the human disease-causing cTnI gene mutation, Gly203Ser (designated cTnI-G203S), in a cardiac-specific manner. Mice were compared to transgenic mice that overexpress normal cTnI (cTnI-wt) and non-transgenic littermates (NTG). cTnI-G203S mice developed all the characteristic features of FHC by age 21 weeks. Left ventricular hypertrophy was observed on echocardiography (1.25±0.05 mm vs. 0.86±0.02 mm in cTnI-wt, P <0.01), associated with a significant 4-fold increase in RNA markers of hypertrophy, ANF and BNP. Myocyte hypertrophy, myofiber disarray and interstitial fibrosis were observed in cTnI-G203S mice. Expression of the cTnI-G203S mutation in neonatal cardiomyocytes resulted in a significant increase in myocyte volume, and reduced interactions with both troponins T and C. Ca2+ cycling was abnormal in adult cardiomyocytes extracted from cTnI-G203S mice, with a prolonged decay constant in Ca2+ transients and a reduced decay constant in response to caffeine treatment. Mice with the cTnI-G203S gene mutation develop all the phenotypic features of human FHC. The cTnI-G203S mutation disrupts interactions with partner proteins, and results in intracellular Ca2+ dysregulation early in life, suggesting a pathogenic role in development of FHC. [Copyright &y& Elsevier]

Abstract: The aqueous humour of the eye is a rich source of growth factors, many of which have been shown to be lens cell mitogens; however, the identity of the endogenous mitogen(s) for lens cells is still unknown. As a first approach to identify the mechanisms by which these aqueous humour-derived growth factors induce lens cell proliferation, the present study set out to examine MAPK/ERK1/2 and PI3-K/Akt signalling associated with lens cell proliferation. Using a lens explant system, we examined the effects of different lens mitogens (aqueous humour, FGF, PDGF, IGF and EGF) using 5′–2′-bromo-deoxyuridine incorporation. In addition, we adopted immunolabelling techniques to compare the roles that the ERK1/2 and PI3-K signalling pathways play in regulating lens cell proliferation. We showed that the aqueous humour, and all the other growth factors examined, could activate ERK1/2 and PI3-K/Akt signalling. By targeting these pathways using specific pharmacological inhibitors, we were able to show that both ERK1/2 and PI3-K signalling are required for growth factor-induced lens cell proliferation, and that there was a strong correlation between the spatial distribution of proliferating cells in lens explants with ERK1/2 labelling. Furthermore, our blocking studies confirmed that PI3-K/Akt signalling can act upstream of ERK1/2, potentiating ERK1/2 phosphorylation in growth factor-induced lens cell proliferation. A better understanding of the signalling pathways required for aqueous humour-induced lens cell proliferation may ultimately allow us to identify the mitogen(s) that are important for regulating lens cell proliferation in situ. [Copyright &y& Elsevier]