The Transcriptional Complex Sp1/KMT2A by Up-Regulating Restrictive Element 1 Silencing Transcription Factor Accelerates Methylmercury-Induced Cell Death in Motor Neuron-Like NSC34 Cells Overexpressing SOD1-G93A

Methylmercury (MeHg) exposure has been related to amyotrophic lateral sclerosis (ALS) pathogenesis and molecular mechanisms of its neurotoxicity has been associated to an overexpression of the Restrictive Element 1 Silencing Transcription factor (REST). Herein, we evaluated the possibility that MeHg could accelerate neuronal death of the motor neuron-like NSC34 cells transiently overexpressing the human Cu2+/Zn2+superoxide dismutase 1 (SOD1) gene mutated at glycine 93 (SOD1-G93A). Indeed, SOD1-G93A cells exposed to 100 nM MeHg for 24 h showed a reduction in cell viability, as compared to cells transfected with empty vector or with unmutated SOD1 construct. Interestingly, cell survival reduction in SOD1-G93A cells was associated with an increase of REST mRNA and protein levels. Furthermore, MeHg increased the expression of the transcriptional factor Sp1 and promoted its binding to REST gene promoter sequence. Notably, Sp1 knockdown reverted MeHg-induced REST increase. Co-immunoprecipitation experiments demonstrated that Sp1 physically interacted with the epigenetic writer Lysine-Methyltransferase-2A (KMT2A). Moreover, knocking-down of KMT2A reduced MeHg-induced REST mRNA and protein increase in SOD1-G93A cells. Finally, we found that MeHg-induced REST up-regulation triggered necropoptotic cell death, monitored by RIPK1 increased protein expression. Interestingly, REST knockdown or treatment with the necroptosis inhibitor Necrostatin-1 (Nec) decelerated MeH-induced cell death in SOD1-G93A cells. Collectively, this study demonstrated that MeHg hastens necroptotic cell death in SOD1-G93A cells via Sp1/KMT2A complex, that by epigenetic mechanisms increases REST gene expression

Molecular Interactions in and Transport Properties of Densely Cross-linked Networks: A Time-resolved FTIR Spectroscopy Investigation

Transport of water in an epoxy network with high cross-link d. was investigated at several water vapor activities by time-resolved FT-IR spectroscopy and gravimetric measurements. The anal. of the IR spectra provided information about the mol. interactions occurring in the system. In particular, several interaction complexes were identified and their structures were proposed. An est. was made of the concn. of the various water species present in the system, based on the knowledge of the resp. molar absorptivities. An excellent agreement between the spectroscopic and gravimetric detns. of sorbed water was found throughout. The evolution of the different water species was monitored by resolving the complex profile of the water spectrum in the nOH frequency range. This information, coupled with the results of the gravimetric anal., was used to evaluate the effect of polymer/penetrant H-bonding interactions on the diffusion process of water mols. Transport of the different water species was found to follow a Fickian behavior characterized by an effective diffusion coeff. which increases with total water concn

Exploring doxorubicin localization in eluting TiO2 nanotube arrays through fluorescence correlation spectroscopy analysis

Drug elution properties of TiO2 nanotube arrays have been largely investigated by means of solely macroscopic observations. Controversial elution performances have been reported so far and a clear comprehension of these phenomena is still missing as a consequence of a lack of molecular investigation methods. Here we propose a way to discern drug elution properties of nanotubes through the evaluation of drug localization by Fluorescence Correlation Spectroscopy (FCS) analysis. We verified this method upon doxorubicin elution from differently loaded TiO2 nanotubes. Diverse elution profiles were obtained from nanotubes filled by soaking and wet vacuum impregnation methods. Impregnated nanotubes controlled drug diffusion up to thirty days, while soaked samples completed elution in seven days. FCS analysis of doxorubicin motion in loaded nanotubes clarified that more than 90% of drugs dwell preferentially in inter-nanotube spaces in soaked samples due to decorrelation in a 2D fashion, while a 97% fraction of molecules showed 1D mobility ascribable to displacements along the nanotube vertical axis of wet vacuum impregnated nanotubes. The diverse drug localizations inferred from FCS measurements, together with distinct drug-surface interaction strengths resulting from diverse drug filling techniques, could explain the variability in elution kinetics

Fluorescence correlation spectroscopy in semiadhesive wall proximity

With examination of diffusion in heterogeneous media through fluorescence correlation spectroscopy, the temporal correlation of the intensity signal shows a long correlation tail and the characteristic diffusion time results are no longer easy to determine. Excluded volume and sticking effects have been proposed to justify such deviations from the standard behavior since all contribute and lead to anomalous diffusion mechanisms. Usually, the anomalous coefficient embodies all the effects of environmental heterogeneity providing too general explanations for the exotic diffusion recorded. Here, we investigated whether the reason of anomalies could be related to a lack of an adequate interpretative model for heterogeneous systems and how the presence of obstacles on the detection volume length scale could affect fluorescence correlation spectroscopy experiments. We report an original modeling of the autocorrelation function where fluorophores experience reflection or adsorption at a wall placed at distances comparable with the detection volume size. We successfully discriminate between steric and adhesion effects through the analysis of long time correlations and evaluate the adhesion strength through the evaluation of probability of being adsorbed and persistence time at the wall on reference data. The proposed model can be readily adopted to gain a better understanding of intracellular and nanoconfined diffusion opening the way for a more rational analysis of the diffusion mechanism in heterogeneous systems and further developing biological and biomedical application

A closed form for fluorescence correlation spectroscopy experiments in submicrometer structures

Fluorescence correlation spectroscopy (FCS) is a powerful technique for measuring low concentrations of fluorescent molecules and their diffusion coefficients in an open detection volume. However, in several practical cases, when FCS measurements are carried out in small compartments like microchannels, neglecting boundary effects could lead to erroneous results. Here, a close form solution is proposed to explicitly account for the presence of walls located at a distance comparable with the characteristic detection volume lengths. We derive a one-dimensional diffusion constrained model and then generalize the solution to the two- and the three-dimensional constrained cases. We further indicate within which limits the standard autocorrelation function (ACF) model gives reliable results in microconfinement. Our model relies just on the assumption of elastic hits at the system walls and succeeds in describing the ACF of fluorescent probes confined along one direction. Through the analysis of FCS experimental data, we are able to predict the correct shape of the ACF in channels of micrometric and submicrometric width and measure the extent of lateral confinement. In addition, it permits the investigation of microstructured material features such as cages and cavities having dimensions on the micrometric range. On the basis of the proposed model, we also show in which conditions confinement could generate an apparent time dependent probe mobility, thus allowing a proper interpretation of the transport process taking place in submicrometric compartments

Fluorescence Correlation Spectroscopy of Nanomaterials

The first in-depth treatment of the synthesis, processing, and characterization of nanomaterials using lasers, ranging from fundamentals to the latest research results, this handy reference is divided into two main sections. After introducing the concepts of lasers, nanomaterials, nanoarchitectures and laser-material interactions in the first three chapters, the book goes on to discuss the synthesis of various nanomaterials in vacuum, gas and liquids. The second half discusses various nanomaterial characterization techniques involving lasers, from Raman and photoluminescence spectroscopies to light dynamic scattering, laser spectroscopy and such unusual techniques as laser photo acoustic, fluorescence correlation spectroscopy, ultrafast dynamics and laser-induced thermal pulses. The specialist authors adopt a practical approach throughout, with an emphasis on experiments, set-up, and results. Each chapter begins with an introduction and is uniform in covering the basic approaches, experimental setups, and dependencies of the particular method on different parameters, providing sufficient theory and modeling to understand the principles behind the technique

Gas sorption and transport in syndiotactic polystyrene with nanoporous crystalline phase.

In this contribution we analyze sorption and transport of several gases in semicryst. syndiotactic polystyrene with nanoporous cryst. d form. Investigation was performed on amorphous samples and on samples characterized by different degrees of crystallinity. Sorption isotherms of carbon dioxide, nitrogen and oxygen in the cryst. phase have been detd. starting from exptl. results obtained for semicryst. and amorphous samples. Corresponding isosteric heats of sorption were evaluated for the cryst. and amorphous phase. Permeation tests were also performed to gather information on mass transport properties of semicryst. samples, evaluating av. diffusivities of carbon dioxide and oxygen, in the limit of small concns. as function of degree of crystallinity