Targeting CDC25B-CDK2/Cyclin A Activity Using Chemical Biology Approaches

The cell cycle is a fundamental process of cell biology, and its progression is highly regulated. A critical mode of regulation for proper advancement of the cell cycle is the activation of the CDKs by the CDC25 family of dual specificity phosphatases. The CDC25 proteins are often overexpressed or misregulated in cancer, resulting in dysregulated cell growth, genomic instability and evasion of apoptosis. The oncogenic role of the CDC25 proteins has inspired over two decades of drug discovery efforts to inhibit their enzymatic activity. Despite these efforts, no therapeutic agents targeting family of CDC25 phosphatases emerged. In order to identify new classes of CDC25B inhibitors, new approaches to target CDC25 are needed. We have employed a novel approach to inhibit the CDC25 family member CDC25B by targeting its interaction with its native substrate, the CDK2/Cyclin A complex. We used two different methods, fragment-based drug discovery and “gray-box” high-throughput screening, to identify inhibitors of the CDC25B-CDK2/Cyclin A protein-protein interaction. Using NMR- based fragment based screening, we identified a small molecule ligand of the CDC25B catalytic domain. We solved the co-crystal structure with this ligand bound to CDC25B, and used this structure to develop more potent analogs. We have shown that fragment-derived compounds can disrupt the CDC25B-CDK2/Cyclin A interaction and inhibit CDC25B catalytic activity. To our knowledge, our inhibitor-bound crystal structure of CDC25B is the first crystal structure with CDC25B bound to a small molecule ligand. We have also developed several protein-protein interaction assays to quantify the interaction between CDC25B and CDK2/Cyclin A. We employed these assays in three high- throughput screens to identify several classes of CDC25B-CDK2/Cyclin A protein-protein interaction inhibitors. The inhibitors we identified do not target CDC25B, but disrupt the protein- protein interaction by targeting CDK2/Cyclin A. Importantly, we have developed a high quality screening assay for the identification of CDC25B-CDK2/Cyclin A interaction inhibitors. This assay will be useful for future drug discovery efforts targeting the CDC25B-CDK2/Cyclin A interaction. In summary, we have developed two new approaches to inhibit CDC25B. These results pave the way towards the development of new chemical probes and potential therapeutic agents targeting CDC25B.PhDMolecular and Cellular PathologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/113311/1/lundgeo_1.pd

Solution NMR studies reveal no global flexibility in the catalytic domain of CDC25B

The CDC25B phosphatase is a critical regulator of the cell cycle and has been validated as an important therapeutic target in cancer. Previous studies using molecular dynamics simulations have concluded that the catalytic domain of CDC25B may experience a significant degree of dynamics or be partially disordered in solution, a finding that has a pronounced impact on the structure‐based development of CDC25B inhibitors. We have probed the backbone dynamics of the CDC25B catalytic domain in solution using NMR relaxation experiments and found that the core of the protein is relatively rigid and does not experience any large‐scale dynamics over a broad range of time scales. Furthermore, based on residual dipolar coupling measurements we have concluded that the conformation in solution is very similar to that observed in the crystal form. Importantly, these findings rationalize the application of the CDC25B crystal structure in structure‐based drug development. Proteins 2014; 82:2889–2895. © 2014 Wiley Periodicals, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/109315/1/prot24581-sup-0001-suppinfo01.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/109315/2/prot24581.pd

System and market integration of wind power in Denmark

Denmark has more than 10 years’ of experience with a wind share of approximately 20 per cent. During these 10 years, electricity markets have been subject to developments with a key focus on integrating wind power as well as trading electricity with neighbouring countries. This article introduces a methodology to analyse and understand the current market integration of wind power and concludes that the majority of Danish wind power in the period 2004–2008 was used to meet the domestic demand. Based on a physical analysis, at least 63 per cent of Danish wind power was used domestically in 2008. To analyse the remaining 37 per cent, we must apply a market model to identify cause–effect relationships. The Danish case does not illustrate any upper limit for wind power integration, as also illustrated by Danish political targets to integrate 50 per cent by 2020. In recent years, Danish wind power has been financed solely by the electricity consumers, while maintaining production prices below the EU average. The net influence from wind power has been as low as 1–3 per cent of the consumer price. Keywords: Wind power, Wind power integration, Wind power cost, Energy system analysis, Electricity market

Quantification of Three-Dimensional Cell-Mediated Collagen Remodeling Using Graph Theory

Background: Cell cooperation is a critical event during tissue development. We present the first precise metrics to quantify the interaction between mesenchymal stem cells (MSCs) and extra cellular matrix (ECM). In particular, we describe cooperative collagen alignment process with respect to the spatio-temporal organization and function of mesenchymal stem cells in three dimensions. Methodology/Principal Findings: We defined two precise metrics: Collagen Alignment Index and Cell Dissatisfaction Level, for quantitatively tracking type I collagen and fibrillogenesis remodeling by mesenchymal stem cells over time. Computation of these metrics was based on graph theory and vector calculus. The cells and their three dimensional type I collagen microenvironment were modeled by three dimensional cell-graphs and collagen fiber organization was calculated from gradient vectors. With the enhancement of mesenchymal stem cell differentiation, acceleration through different phases was quantitatively demonstrated. The phases were clustered in a statistically significant manner based on collagen organization, with late phases of remodeling by untreated cells clustering strongly with early phases of remodeling by differentiating cells. The experiments were repeated three times to conclude that the metrics could successfully identify critical phases of collagen remodeling that were dependent upon cooperativity within the cell population. Conclusions/Significance: Definition of early metrics that are able to predict long-term functionality by linking engineere

The Role of Diabetes Co-Morbidity for Tuberculosis Treatment Outcomes: A Prospective Cohort Study from Mwanza, Tanzania.

Due to the association between diabetes and pulmonary tuberculosis (TB), diabetes may threaten the control of TB. In a prospective cohort study nested in a nutrition trial, we investigated the role of diabetes on changes in anthropometry, grip strength, and clinical parameters over a five months follow-up period. Among pulmonary TB patients with known diabetes status, we assessed anthropometry and clinical parameters (e.g. haemoglobin) at baseline and after two and five months of TB treatment. A linear mixed-effects model (repeated measurements) was used to investigate the role of diabetes during recovery. Of 1205 TB patients, the mean (standard deviation) age was 36.6 (13.0) years, 40.9% were females, 48.9% were HIV co-infected, and 16.3% had diabetes. TB patients with diabetes co-morbidity experienced a lower weight gain at two (1.3 kg, CI95% 0.5; 2.0, p = 0.001) and five months (1.0 kg, CI95% 0.3; 1.7, p = 0.007). Similarly, the increase in the level of haemoglobin was lower among TB patients with diabetes co-morbidity after two (Δ 0.6 g/dL, CI95% 0.3; 0.9 p < 0.001) and five months (Δ 0.5 g/dL, CI95% 0.2; 0.9 p = 0.004) of TB treatment, respectively. TB patients initiating TB treatment with diabetes co-morbidity experience delayed recovery of body mass and haemoglobin, which are important for the functional recovery from disease

CLIVAR Mode Water Dynamics Experiment (CLIMODE) fall 2006 R/V Oceanus voyage 434 November 16, 2006–December 3, 2006

CLIMODE (CLIVAR Mode Water Dynamic Experiment) is a research program designed to understand and quantify the processes responsible for the formation and dissipation of North Atlantic subtropical mode water, also called Eighteen Degree Water (EDW). Among these processes, the amount of buoyancy loss at the ocean-atmosphere interface is still uncertain and needs to be accurately quantified. In November 2006, cruise 434 onboard R/V Oceanus traveled in the region of the separated Gulf Stream and its recirculation, where intense oceanic heat loss to the atmosphere in the winter is believed to trigger the formation of EDW. During this cruise, the surface mooring F that was anchored in the core of the Gulf Stream was replaced by a new one, as well as two subsurface moorings C and D located on the southeastern edge of the stream. Surface drifters, ARGO and bobbers RAFOS floats were deployed, CTD profiles and water samples were also carried out. This array of instruments will permit a characterization of EDW with high spatial and temporal resolutions and accurate in-situ measurements of air-sea fluxes in the EDW formation region. The present report documents this cruise, the methods and locations for the deployments of instruments and some evaluation of the measurements from these instruments.Funding was provided by the National Science Foundation under contract No. OCE04-2453

A Bichromatic Incidence Bound and an Application

We prove a new, tight upper bound on the number of incidences between points and hyperplanes in Euclidean d-space. Given n points, of which k are colored red, there are O_d(m^{2/3}k^{2/3}n^{(d-2)/3} + kn^{d-2} + m) incidences between the k red points and m hyperplanes spanned by all n points provided that m = \Omega(n^{d-2}). For the monochromatic case k = n, this was proved by Agarwal and Aronov. We use this incidence bound to prove that a set of n points, no more than n-k of which lie on any plane or two lines, spans \Omega(nk^2) planes. We also provide an infinite family of counterexamples to a conjecture of Purdy's on the number of hyperplanes spanned by a set of points in dimensions higher than 3, and present new conjectures not subject to the counterexample.Comment: 12 page