CARPET: a web-based package for the analysis of ChIP-chip and expression tiling data

Summary: CARPET (Collection of Automated Routine Programs for Easy Tiling) is a set of Perl, Python and R scripts, integrated on the Galaxy2 web-based platform, for the analysis of ChIP-chip and expression tiling data, both for standard and custom chip designs. CARPET allows rapid experimental data entry, simple quality control, normalization, easy identification and annotation of enriched ChIP-chip regions, detection of the absolute or relative transcriptional status of genes assessed by expression tiling experiments and, more importantly, it allows the integration of ChIP-chip and expression data. Results can be visualized instantly in a genomic context within the UCSC genome browser as graph-based custom tracks through Galaxy2. All generated and uploaded data can be stored within sessions and are easily shared with other users. Availability: http://bio.ifom-ieo-campus.it/galaxy Contacts: [email protected] lucilla.luzi@if om-ieo-campus.i

The endless frontier? The recent increase of R&D productivity in pharmaceuticals

Background: Studies on the early 2000s documented increasing attrition rates and duration of clinical trials, leading to a representation of a "productivity crisis" in pharmaceutical research and development (R&D). In this paper, we produce a new set of analyses for the last decade and report a recent increase of R&D productivity within the industry. Methods: We use an extensive data set on the development history of more than 50,000 projects between 1990 and 2017, which we integrate with data on sales, patents, and anagraphical information on each institution involved. We devise an indicator to quantify the novelty of each project, based on its set of mechanisms of action. Results: First, we investigate how R&D projects are allocated across therapeutic areas and find a polarization towards high uncertainty/high potential reward indications, with a strong focus on oncology. Second, we find that attrition rates have been decreasing at all stages of clinical research in recent years. In parallel, for each phase, we observe a significant reduction of time required to identify projects to be discontinued. Moreover, our analysis shows that more recent successful R&D projects are increasingly based on novel mechanisms of action and target novel indications, which are characterized by relatively small patient populations. Third, we find that the number of R&D projects on advanced therapies is also growing. Finally, we investigate the relative contribution to productivity variations of different types of institutions along the drug development process, with a specific focus on the distinction between the roles of Originators and Developers of R&D projects. We document that in the last decade Originator-Developer collaborations in which biotech companies act as Developers have been growing in importance. Moreover, we show that biotechnology companies have reached levels of productivity in project development that are equivalent to those of large pharmaceutical companies. Conclusions: Our study reports on the state of R&D productivity in the bio-pharmaceutical industry, finding several signals of an improving performance, with R&D projects becoming more targeted and novel in terms of indications and mechanisms of action

The genomic landscape of 8-oxodG reveals enrichment at specific inherently fragile promoters

8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) is the most common marker of oxidative stress and its accumulation within the genome has been associated with major human health issues such as cancer, aging, cardiovascular and neurodegenerative diseases. The characterization of the different genomic sites where 8-oxodG accumulates and the mechanisms underlying its formation are still poorly understood. Using OxiDIP-seq, we recently derived the genome-wide distribution of 8-oxodG in human non-tumorigenic epithelial breast cells (MCF10A). Here, we identify a subset of human promoters that accumulate 8-oxodG under steady-state condition. 8-oxodG nucleotides co-localize with double strand breaks (DSBs) at bidirectional and CG skewed promoters and their density correlate with RNA Polymerase II co-occupancy and transcription. Furthermore, by performing OxiDIP-seq in quiescent (G0) cells, we found a strong reduction of oxidatively-generated damage in the majority of 8-oxodG-positive promoters in the absence of DNA replication. Overall, our results suggest that the accumulation of 8-oxodG at gene promoters occurs through DNA replication-dependent or -independent mechanisms, with a possible contribution to the formation of cancer-associated translocation events

The emerging role of p53 in stem cells

Among the hundreds of oncogenes and tumor suppressors that have been identified in the past 50 years, p53 is probably the best characterized; nevertheless, new functions are constantly being discovered. As a tumor suppressor, p53 regulates cellular responses to different stress stimuli by inducing reversible cell cycle arrest and DNA repair, or triggering senescence or apoptosis. Recent findings on the regulation of stem cell (SC) division and reprogramming suggest the intriguing possibility that p53 also carries out its tumor suppression function by regulating SC homeostasis. Specifically, p53 activation may counteract SC expansion by several emerging mechanisms including restriction of self-renewing divisions, inhibition of symmetric division and block of reprogramming of somatic/progenitor cells into SCs

The Eps15 Homology (Eh) Domain-Based Interaction between Eps15 and Hrb Connects the Molecular Machinery of Endocytosis to That of Nucleocytosolic Transport

The Eps15 homology (EH) module is a protein–protein interaction domain that establishes a network of connections involved in various aspects of endocytosis and sorting. The finding that EH-containing proteins bind to Hrb (a cellular cofactor of the Rev protein) and to the related protein Hrbl raised the possibility that the EH network might also influence the so-called Rev export pathway, which mediates nucleocytoplasmic transfer of proteins and RNAs. In this study, we demonstrate that Eps15 and Eps15R, two EH-containing proteins, synergize with Hrb and Hrbl to enhance the function of Rev in the export pathway. In addition, the EH-mediated association between Eps15 and Hrb is required for the synergistic effect. The interaction between Eps15 and Hrb occurs in the cytoplasm, thus pointing to an unexpected site of action of Hrb, and to a possible role of the Eps15–Hrb complex in regulating the stability of Rev

Modeling cell proliferation in human acute myeloid leukemia xenografts

Motivation: Acute myeloid leukemia (AML) is one of the most common hematological malignancies, characterized by high relapse and mortality rates. The inherent intra-tumor heterogeneity in AML is thought to play an important role in disease recurrence and resistance to chemotherapy. Although experimental protocols for cell proliferation studies are well established and widespread, they are not easily applicable to in vivo contexts, and the analysis of related time-series data is often complex to achieve. To overcome these limitations, model-driven approaches can be exploited to investigate different aspects of cell population dynamics. Results: In this work, we present ProCell, a novel modeling and simulation framework to investigate cell proliferation dynamics that, differently from other approaches, takes into account the inherent stochasticity of cell division events. We apply ProCell to compare different models of cell proliferation in AML, notably leveraging experimental data derived from human xenografts in mice. ProCell is coupled with Fuzzy Self-Tuning Particle Swarm Optimization, a swarm-intelligence settings-free algorithm used to automatically infer the models parameterizations. Our results provide new insights on the intricate organization of AML cells with highly heterogeneous proliferative potential, highlighting the important role played by quiescent cells and proliferating cells characterized by different rates of division in the progression and evolution of the disease, thus hinting at the necessity to further characterize tumor cell subpopulations. Availability and implementation: The source code of ProCell and the experimental data used in this work are available under the GPL 2.0 license on GITHUB at the following URL: https://github.com/aresio/ProCell

WDR5 inhibition halts metastasis dissemination by repressing the mesenchymal phenotype of breast cancer cells

Background: Development of metastases and drug resistance are still a challenge for a successful systemic treatment in breast cancer (BC) patients. One of the mechanisms that confer metastatic properties to the cell relies in the epithelial-to-mesenchymal transition (EMT). Moreover, both EMT and metastasis are partly modulated through epigenetic mechanisms, by repression or induction of specific related genes. Methods: We applied shRNAs and drug targeting approaches in BC cell lines and metastatic patient-derived xenograft (PDX) models to inhibit WDR5, the core subunit of histone H3 K4 methyltransferase complexes, and evaluate its role in metastasis regulation. Result: We report that WDR5 is crucial in regulating tumorigenesis and metastasis spreading during BC progression. In particular, WDR5 loss reduces the metastatic properties of the cells by reverting the mesenchymal phenotype of triple negative- and luminal B-derived cells, thus inducing an epithelial trait. We also suggest that this regulation is mediated by TGF\u3b21, implying a prominent role of WDR5 in driving EMT through TGF\u3b21 activation. Moreover, such EMT reversion can be induced by drug targeting of WDR5 as well, leading to BC cell sensitization to chemotherapy and enhancement of paclitaxel-dependent effects. Conclusions: We suggest that WDR5 inhibition could be a promising pharmacologic approach to reduce cell migration, revert EMT, and block metastasis formation in BC, thus overcoming resistance to standard treatments

Diabetes Causes Bone Marrow Autonomic Neuropathy and Impairs Stem Cell Mobilization via Dysregulated p66Shc and Sirt1

Diabetes compromises the bone marrow (BM) microenvironment and reduces circulating CD34 + cells. Diabetic autonomic neuropathy (DAN) may impact the BM, because the sympathetic nervous system (SNS) is prominently involved in BM stem cell trafficking. We hypothesize that neuropathy of the BM affects stem cell mobilization and vascular recovery after ischemia in diabetes. We report that, in patients, cardiovascular DAN was associated with fewer circulating CD34 + cells. Experimental diabetes (STZ and Ob/Ob ) or chemical sympathectomy in mice resulted in BM autonomic neuropathy, impaired Lin - cKit + Sca1 + (LKS) cell and endothelial progenitor cells (EPC, CD34 + Flk1 + ) mobilization and vascular recovery after ischemia. DAN increased expression of p66Shc and reduced expression of Sirt1 in mice and humans. p66Shc KO in diabetic mice prevented DAN in the BM, and rescued defective LKS cell and EPC mobilization. Hematopoietic Sirt1 KO mimicked the diabetic mobilization defect, while hematopoietic Sirt1 overexpression in diabetes rescued defective mobilization and vascular repair. Through p66Shc and Sirt1 , diabetes and sympathectomy elevated the expression of various adhesion molecules, including CD62L . CD62L KO partially rescued the defective stem/progenitor cell mobilization. In conclusion, autonomic neuropathy in the BM impairs stem cell mobilization in diabetes with dysregulation of the lifespan regulators p66Shc and Sirt1

Exploiting the tunability of stimulated emission depletion microscopy for super-resolution imaging of nuclear structures

Imaging of nuclear structures within intact eukaryotic nuclei is imperative to understand the effect of chromatin folding on genome function. Recent developments of super-resolution fluorescence microscopy techniques combine high specificity, sensitivity, and less-invasive sample preparation procedures with the sub-diffraction spatial resolution required to image chromatin at the nanoscale. Here, we present a method to enhance the spatial resolution of a stimulated-emission depletion (STED) microscope based only on the modulation of the STED intensity during the acquisition of a STED image. This modulation induces spatially encoded variations of the fluorescence emission that can be visualized in the phasor plot and used to improve and quantify the effective spatial resolution of the STED image. We show that the method can be used to remove direct excitation by the STED beam and perform dual color imaging. We apply this method to the visualization of transcription and replication foci within intact nuclei of eukaryotic cells