Live-cell measurement of protein binding to chromatin allows probing cellular biochemistry in physiological conditions, which are difficult to mimic in vitro. However, different studies have yielded widely discrepant predictions, and so it remains uncertain how to make the measurements accurately. To establish a benchmark we measured binding of the transcription factor p53 to chromatin by three approaches: fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS) and single-molecule tracking (SMT). Using new procedures to analyze the SMT data and to guide the FRAP and FCS analysis, we show how all three approaches yield similar estimates for both the fraction of p53 molecules bound to chromatin (only about 20%) and the residence time of these bound molecules (∼1.8 s). We also apply these procedures to mutants in p53 chromatin binding. Our results support the model that p53 locates specific sites by first binding at sequence-independent sites.

Retinal tissue is damaged during inflammation in Multiple Sclerosis. We assessed molecular changes in inflamed murine retinal cultures by Raman spectroscopy. Partial Least Squares‐Discriminant analysis (PLS‐DA) was able to classify retina cultures as inflamed with high accuracy. Using Multivariate Curve Resolution (MCR) analysis, we deconvolved 6 molecular components suffering dynamic changes along inflammatory process. Those include the increase of immune mediators (Lipoxygenase, iNOS and TNFα), changes in molecules involved in energy production (Cytochrome C, phenylalanine and NADH/NAD+) and decrease of Phosphatidylcholine. Raman spectroscopy combined with multivariate analysis allows monitoring the evolution of retina inflammation.Raman spectroscopy analysis of the Retinal Ganglion Cell layer of the retina. (A) Design of the analysis of the Ganglion cell layer (GCL) and Retinal Nerve Fiber Layer (RNFL) of the retina based in the physical properties of laser light and anatomical structure of retinal layers. (B) Examples of raw Raman spectra from representative retina sample after 10 hours incubation time (black) and LPS treated retina sample after 10 hours incubation time (red) and 12 hours incubation time (blue). (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Background: We aim to identify differentially expressed genes (DEGs) and its pathways associated with clinical activity of relapsing-remitting Multiple Sclerosis (RRMS).