Interplay of Protein and DNA Structure Revealed in Simulations of the lac Operon

The E. coli Lac repressor is the classic textbook example of a protein that attaches to widely spaced sites along a genome and forces the intervening DNA into a loop. The short loops implicated in the regulation of the lac operon suggest the involvement of factors other than DNA and repressor in gene control. The molecular simulations presented here examine two likely structural contributions to the in-vivo looping of bacterial DNA: the distortions of the double helix introduced upon association of the highly abundant, nonspecific nucleoid protein HU and the large-scale deformations of the repressor detected in low-resolution experiments. The computations take account of the three-dimensional arrangements of nucleotides and amino acids found in crystal structures of DNA with the two proteins, the natural rest state and deformational properties of protein-free DNA, and the constraints on looping imposed by the conformation of the repressor and the orientation of bound DNA. The predicted looping propensities capture the complex, chain-length-dependent variation in repression efficacy extracted from gene expression studies and in vitro experiments and reveal unexpected chain-length-dependent variations in the uptake of HU, the deformation of repressor, and the folding of DNA. Both the opening of repressor and the presence of HU, at levels approximating those found in vivo, enhance the probability of loop formation. HU affects the global organization of the repressor and the opening of repressor influences the levels of HU binding to DNA. The length of the loop determines whether the DNA adopts antiparallel or parallel orientations on the repressor, whether the repressor is opened or closed, and how many HU molecules bind to the loop. The collective behavior of proteins and DNA is greater than the sum of the parts and hints of ways in which multiple proteins may coordinate the packaging and processing of genetic information. © 2013 Czapla et al

Lac repressor mediated DNA looping: Monte Carlo simulation of constrained DNA molecules complemented with current experimental results

Tethered particle motion (TPM) experiments can be used to detect time-resolved loop formation in a single DNA molecule by measuring changes in the length of a DNA tether. Interpretation of such experiments is greatly aided by computer simulations of DNA looping which allow one to analyze the structure of the looped DNA and estimate DNA-protein binding constants specific for the loop formation process. We here present a new Monte Carlo scheme for accurate simulation of DNA configurations subject to geometric constraints and apply this method to Lac repressor mediated DNA looping, comparing the simulation results with new experimental data obtained by the TPM technique. Our simulations, taking into account the details of attachment of DNA ends and fluctuations of the looped subsegment of the DNA, reveal the origin of the double-peaked distribution of RMS values observed by TPM experiments by showing that the average RMS value for anti-parallel loop types is smaller than that of parallel loop types. The simulations also reveal that the looping probabilities for the anti-parallel loop types are significantly higher than those of the parallel loop types, even for loops of length 600 and 900 base pairs, and that the correct proportion between the heights of the peaks in the distribution can only be attained when loops with flexible Lac repressor conformation are taken into account. Comparison of the in silico and in vitro results yields estimates for the dissociation constants characterizing the binding affinity between O1 and Oid DNA operators and the dimeric arms of the Lac repressor. © 2014 Biton et al

Combined Tumor Cell-Based Vaccination and Interleukin-12 Gene Therapy Polarizes the Tumor Microenvironment in Mice

Tumor progression depends on tumor milieu, which influences neovasculature formation and immunosuppression. Combining immunotherapy with antiangiogenic/antivascular therapy might be an effective therapeutic approach. The aim of our study was to elaborate an anticancer therapeutic strategy based on the induction of immune response which leads to polarization of tumor milieu. To achieve this, we developed a tumor cell-based vaccine. CAMEL peptide was used as a B16-F10 cell death-inducing agent. The lysates were used as a vaccine to immunize mice bearing B16-F10 melanoma tumors. To further improve the therapeutic effect of the vaccine, we combined it with interleukin (IL)-12 gene therapy. IL-12, a cytokine with antiangiogenic properties, activates nonspecific and specific immune responses. We observed that combined therapy is significantly more effective (as compared with monotherapies) in inhibiting tumor growth. Furthermore, the tested combination polarizes the tumor microenvironment, which results in a switch from a proangiogenic/immunosuppressive to an antiangiogenic/immunostimulatory one. The switch manifests itself as a decreased number of tumor blood vessels, increased levels of tumor-infiltrating CD4+, CD8+ and NK cells, as well as lower level of suppressor lymphocytes (Treg). Our results suggest that polarizing tumor milieu by such combined therapy does inhibit tumor growth and seems to be a promising therapeutic strategy

Radiometric Calibration Network for Vicarious Calibration of Earth Observing Imagers in the Reflected Solar

A challenge for the scientific community in respect to the ever-increasing number of Earth observing satellite sensors is to ensure that the absolute radiometric calibration of the sensors is harmonized to the same SI-traceable scale. Assessing the post-launch radiometric calibration is the responsibility of each sensor team and typically involves simulating the top-of-atmosphere signal from in-situ and atmospheric measurements. As this is done on an individual sensor-by-sensor basis often using a single ground site, radiometric biases can exist between sensors. In an effort to minimize these biases, the Committee on Earth Observation Satellites (CEOS) Working Group on Calibration and Validation (WGCV) Infrared Visible Optical Sensors (IVOS) is currently working to develop the prototype radiometric calibration network, RadCalNet. This network will standardize methodology and processing streams for participating ground sites. The current RadCalNet working group consists of members from the National Aeronautics and Space Administration (NASA, USA), the Centre National d’Etudes Spatiales (CNES, France), the European Space Agency (ESA), the National Physical Laboratory (NPL, UK), the University of Arizona (USA), and the Academy of Opto Electronics (AOE, China). Four radiometric calibration test sites in the USA, France, China, and Namibia are being used as test cases for the collection of surface reflectance and atmospheric data, which are then converted to top-of-atmosphere (TOA) reflectance for comparison with a limited number of satellite sensors. The goal of the RadCalNet working group is to provide via a public website site the hyper-spectral (in the range 400 nm to 1000 nm and at specific sites up to 2500 nm) top-of-atmosphere reflectance at 30-minute intervals for a nadir viewing sensor for the 4 sites. RadCalNet recently completed its Beta Testing Phase and examples from that testing phase as well as a description of the method for providing uncertainties and ensuring SI-traceability are presented