Targeting the Interaction between the SH3 Domain of Grb2 and Gab2

Gab2 is a scaffolding protein, overexpressed in many types of cancers, that plays a key role in the formation of signaling complexes involved in cellular proliferation, migration, and differentiation. The interaction between Gab2 and the C-terminal SH3 domain of the protein Grb2 is crucial for the activation of the proliferation-signaling pathway Ras/Erk, thus representing a potential pharmacological target. In this study, we identified, by virtual screening, seven potential inhibitor molecules that were experimentally tested through kinetic and equilibrium binding experiments. One compound showed a remarkable effect in lowering the affinity of the C-SH3 domain for Gab2. This inhibitory effect was subsequently validated in cellula by using lung cancer cell lines A549 and H1299. Our results are discussed under the light of previous works on the C-SH3:Gab2 interaction

Histone Acetylation Defects in Brain Precursor Cells: A Potential Pathogenic Mechanism Causing Proliferation and Differentiation Dysfunctions in Mitochondrial Aspartate-Glutamate Carrier Isoform 1 Deficiency

Mitochondrial aspartate-glutamate carrier isoform 1 (AGC1) deficiency is an ultra-rare genetic disease characterized by global hypomyelination and brain atrophy, caused by mutations in the SLC25A12 gene leading to a reduction in AGC1 activity. In both neuronal precursor cells and oligodendrocytes precursor cells (NPCs and OPCs), the AGC1 determines reduced proliferation with an accelerated differentiation of OPCs, both associated with gene expression dysregulation. Epigenetic regulation of gene expression through histone acetylation plays a crucial role in the proliferation/differentiation of both NPCs and OPCs and is modulated by mitochondrial metabolism. In AGC1 deficiency models, both OPCs and NPCs show an altered expression of transcription factors involved in the proliferation/differentiation of brain precursor cells (BPCs) as well as a reduction in histone acetylation with a parallel alteration in the expression and activity of histone acetyltransferases (HATs) and histone deacetylases (HDACs). In this study, histone acetylation dysfunctions have been dissected in in vitro models of AGC1 deficiency OPCs (Oli-Neu cells) and NPCs (neurospheres), in physiological conditions and following pharmacological treatments. The inhibition of HATs by curcumin arrests the proliferation of OPCs leading to their differentiation, while the inhibition of HDACs by suberanilohydroxamic acid (SAHA) has only a limited effect on proliferation, but it significantly stimulates the differentiation of OPCs. In NPCs, both treatments determine an alteration in the commitment toward glial cells. These data contribute to clarifying the molecular and epigenetic mechanisms regulating the proliferation/differentiation of OPCs and NPCs. This will help to identify potential targets for new therapeutic approaches that are able to increase the OPCs pool and to sustain their differentiation toward oligodendrocytes and to myelination/remyelination processes in AGC1 deficiency, as well as in other white matter neuropathologies

Notch versus the proteasome: what is the target of γ-secretase inhibitor-I?

γ-Secretase inhibitors are new anti-cancer agents targeting Notch signaling. Their specificity for Notch is as yet unclear. Han and colleagues investigated the effects of Z-LeuLeuNleu-CHO on growth of breast cancer cells. The results demonstrated a reduction in cell viability primarily via proteasome inhibition independent of Notch activity. Currently, γ-secretase inhibitors in clinical trials are structurally distinct from Z-LeuLeuNleu-CHO. Their effects on the proteasome are yet to be determined. However, findings from Han and colleagues pose two critical questions: Is the level of proteasomal activity in breast tumors the driving force for growth? What does the Notch pathway contribute to this growth

Retinoic acid-induced 1 gene haploinsufficiency alters lipid metabolism and causes autophagy defects in Smith-Magenis syndrome

Smith-Magenis syndrome (SMS) is a neurodevelopmental disorder characterized by cognitive and behavioral symptoms, obesity, and sleep disturbance, and no therapy has been developed to alleviate its symptoms or delay disease onset. SMS occurs due to haploinsufficiency of the retinoic acid-induced-1 (RAI1) gene caused by either chromosomal deletion (SMS-del) or RAI1 missense/nonsense mutation. The molecular mechanisms underlying SMS are unknown. Here, we generated and characterized primary cells derived from four SMS patients (two with SMS-del and two carrying RAI1 point mutations) and four control subjects to investigate the pathogenetic processes underlying SMS. By combining transcriptomic and lipidomic analyses, we found altered expression of lipid and lysosomal genes, deregulation of lipid metabolism, accumulation of lipid droplets, and blocked autophagic flux. We also found that SMS cells exhibited increased cell death associated with the mitochondrial pathology and the production of reactive oxygen species. Treatment with N-acetylcysteine reduced cell death and lipid accumulation, which suggests a causative link between metabolic dyshomeostasis and cell viability. Our results highlight the pathological processes in human SMS cells involving lipid metabolism, autophagy defects and mitochondrial dysfunction and suggest new potential therapeutic targets for patient treatment

Next Generation European Research Vessels: Current Status and Foreseeable Evolution

The European research vessel fleet plays a vital role in supporting scientific research and development not just in Europe but also across the globe. This document explores how the fleet has developed since the publication of the European Marine Board Position Paper 10 (EMB PP 10) "European Ocean Research Fleets – Towards a Common Strategy and Enhanced Use" (Binot et al., 2007). It looks at the current fleet and its equipment and capabilities (Chapter 2), the deep sea (Chapter 3) and Polar regions (Chapter 4) as study areas of ever- increasing importance for science and for the vessels that explore them, the role that research vessels play in the wider ocean observing landscape (Chapter 5), the importance of training personnel for research vessels (Chapter 6), and considers management of the European research vessel fleet (Chapter 7). This Position Paper considers what has changed since 2007, what the status is in 2019, and future directions for the European fleet, with a 10-year horizon to 2030. This Position Paper finds that the current European research vessel fleet is highly capable, and is able to provide excellent support to European marine science and wider scientific research and can lead on the world stage. However, with a typical life expectancy of a research vessel of 30 years, the fleet is ageing and urgently requires further investment and reinvestment to continue to be as efficient and capable as the scientific community expects and requires. The capabilities of the fleet have increased considerably since 2007, and vessels have kept up with fast-paced technological developments. The demand for complex and highly capable vessels will continue, and research vessel designs and the fleet as a whole will need to keep pace in order to remain fit-for-purpose and continue to be a key player globally. There is huge diversity in vessel types and designs in terms of capabilities and equipment, management structures and processes, and training possibilities. While it would not be possible or appropriate to highlight any one approach as the only one to use, a growing trend in collaboration through community groups, agreements, legal entities and funded projects now enables more strategic thinking in the development of these vital infrastructures. However, some issues remain in enabling equal access to research vessel time for all researchers across Europe regardless of country, and regardless of whether or not that country owns a suitable research vessel for their scientific needs

HER2 Phosphorylation Is Maintained by a PKB Negative Feedback Loop in Response to Anti-HER2 Herceptin in Breast Cancer

A feedback loop maintains HER2 receptor signalling and cell survival in response to Herceptin treatment in HER2-positive breast cancers, but this Herceptin resistance may be bypassed by pan-HER inhibitors

Coding by Choice: A Transitional Analysis of Social Participation Patterns and Programming Contributions in the Online Scratch Community

While massive online communities have drawn the attention of researchers and educators on their potential to support active collaborative work, knowledge sharing, and user-generated content, few studies examine participation in these communities at scale. The little research that does exist attends almost solely to adults rather than communities to support youths’ learning and identity development. In this chapter, we tackle two challenges related to understanding social practices that support learning in massive social networking forums where users engage in design. We examined a youth programmer community, called Scratch.mit.edu, that garners the voluntary participation of millions of young people worldwide. We report on site-wide distributions and patterns of participation that illuminate the relevance of different online social practices to ongoing involvement in the online community. Drawing on a random sample of more than 5000 active users of Scratch.mit.edu over a 3-month time period in early 2012, we examine log files that captured the frequency of three types of social practices that contribute to enduring participation: DIY participatory activities, socially supportive actions, and socially engaging interactions. Using latent transition analysis, we found (1) distinct patterns of participation (classes) across three time points (e.g., high networkers who are generally active, commenters who focus mainly on social participation, downloaders engaging in DIY participatory activities), (2) unique migration changes in class membership across time, (3) relatively equal gender representation across these classes, and (4) importance of membership length (or age) in terms of class memberships. In the discussion, we review our approach to analysis and outline implications for the design and study of online communities and tools for youth

Suppression of apoptosis inhibitor c-FLIP selectively eliminates breast cancer stem cell activity in response to the anti-cancer agent, TRAIL

Introduction It is postulated that breast cancer stem cells (bCSCs) mediate disease recurrence and drive formation of distant metastases - the principal cause of mortality in breast cancer patients. Therapeutic targeting of bCSCs however, is hampered by their heterogeneity and resistance to existing therapeutics. In order to identify strategies to selectively remove bCSCs from breast cancers, irrespective of their clinical subtype, we sought an apoptosis mechanism that would target bCSCs yet would not kill normal cells. Suppression of the apoptosis inhibitor cellular FLICE-Like Inhibitory Protein (c-FLIP) partially sensitizes breast cancer cells to the anti-cancer agent Tumour Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL). Here we demonstrate in breast cancer cell lines that bCSCs are exquisitely sensitive to the de-repression of this pro-apoptotic pathway, resulting in a dramatic reduction in experimental metastases and the loss of bCSC self-renewal. Methods Suppression c-FLIP was performed by siRNA (FLIPi) in four breast cancer cell lines and by conditional gene-knockout in murine mammary glands. Sensitivity of these cells to TRAIL was determined by complementary cell apoptosis assays, including a novel heterotypic cell assay, while tumour-initiating potential of cancer stem cell subpopulations was determined by mammosphere cultures, aldefluor assay and in vivo transplantation. Results Genetic suppression of c-FLIP resulted in the partial sensitization of TRAIL-resistant cancer lines to the pro-apoptotic effects of TRAIL, irrespective of their cellular phenotype, yet normal mammary epithelial cells remained refractory to killing. While 10%-30% of the cancer cell populations remained viable after TRAIL/FLIPi treatment, subsequent mammosphere and aldefluor assays demonstrated that this pro-apoptotic stimulus selectively targeted the functional bCSC pool, eliminating stem cell renewal. This culminated in an 80% reduction in primary tumours and a 98% reduction in metastases following transplantation. The recurrence of residual tumour initiating capacity was consistent with the observation that post-treated adherent cultures re-acquired bCSC-like properties in vitro. Importantly however this recurrent bCSC activity was attenuated following repeated TRAIL/FLIPi treatment. Conclusions We describe an apoptotic mechanism that selectively and repeatedly removes bCSC activity from breast cancer cell lines and suggest that a combined TRAIL/FLIPi therapy could prevent metastatic disease progression in a broad range of breast cancer subtypes. [PROVISIONAL