RGS1 Modulates Autophagic and Metabolic Programs and Is a Critical Mediator of Human Regulatory T Cell Function.

Regulatory T cells (Tregs) are critical mediators of immune tolerance and play a diametric role in cancer and autoimmunity. Tumor-infiltrating Tregs are often associated with poor prognosis in solid tumors because their enrichment in the tumor microenvironment contributes to immunosuppression. Conversely, dysregulation in the Treg compartment can disrupt self-tolerance, leading to autoimmunity. In the present study, we describe what is, to our knowledge, a novel regulator of Tregs, the GTPase activator regulator of G protein 1 (RGS1), demonstrating that RGS1-deficient human Tregs show downregulation of Treg-associated genes and are less immunosuppressive. These RGS1-deficient Tregs exhibit perturbations to the FOXP3-c-MYC transcriptional axis and downstream metabolic and autophagy programs by shifting their energy demands toward glycolysis and rendering them less autophagic. Taken together, RGS1 may serve as an apical node of Treg function by regulating the FOXP3-c-MYC transcriptional axis, thereby providing a therapeutic rationale for targeting RGS1 for treatment of cancer and autoimmune diseases.

Comparative Study on Interface Fracture of 4th Generation 3-Steps Adhesive and 7th Generation Universal Adhesive.

The purpose of this paper is to compare the fracture behavior of interfaces obtained using fourth-generation and universal dental adhesives. The study relies on optic and SEM to evaluate the dentin-adhesive-restoration material interface of the samples and also on FEA simulation of fracture behavior. Specimen fabrication relied on 20 extracted teeth, in which class I cavities were created according to a protocol established based on the rules of minimally invasive therapy. For the direct adhesive technique, the adhesives used were: three-step All Bond, three-batch A and one-step Clearfil Universal Bond Quick-batch B. The restoration was performed with the same composite for both adhesives: Gradia direct posterior. The simulation used a 3D reconstructed molar on which geometric operations were performed to obtain an assembly that replicated a physical specimen. Material properties were applied to each component based on the information found in the literature. A simplified model for crack propagation was constructed, and using the fracture mechanics tool in Ansys 2019, the stress intensity factors that act at the crack tip of the adhesive interface were obtained. Mechanical simulation and microscopic investigation showed us how the interface of the dentine-adhesive-filling material performed in cases of both dental adhesives and for a certain loading condition. Important differences were identified among the adhesives, the fourth generation being superior to the fourth generation especially due to the separate steps in which the tooth surface was prepared for adhesion.

Adhesive-Ceramic Interface Behavior in Dental Restorations. FEM Study and SEM Investigation.

The purpose of this study is to identify the stress levels that act in inlay and onlay restorations, according to the direction and value of the external force applied. The study was conducted using the Finite Element Method (FEM) of three types of ceramics: pressed lithium disilicate and monolith, zirconia, and three different adhesive systems: self-adhesive, universal, and dual-cure cements. In addition to FEM, the inlay/onlay-dental structure interface analysis was performed by means of Scanning Electron Microscopy (SEM). The geometric models were reconstructed based on computer tomography images of an undamaged molar followed by geometrical procedures of inducing the inlay and onlay reconstructions. The two functional models were then simulated for different orientations of external force and different material properties, according to the considered adhesives and ceramics. The Scanning Electron Microscopy (SEM) was conducted on 30 extracted teeth, divided into three groups according to the adhesive cement type. Both FEM simulation and SEM investigations reveal very good mechanical behavior of the adhesive-dental structure and adhesive-ceramic interfaces for inlay and onlay reconstructions. All results lead to the conclusion that a physiological mastication force applied, regardless of direction, cannot produce a mechanical failure of either inlay or onlay reconstructions. The adhesive bond between the restorations and the dental structure can stabilize the ceramic restorations, resulting in a higher strength to the action of external forces.

Large Scale Chromosome Folding Is Stable against Local Changes in Chromatin Structure.

Characterizing the link between small-scale chromatin structure and large-scale chromosome folding during interphase is a prerequisite for understanding transcription. Yet, this link remains poorly investigated. Here, we introduce a simple biophysical model where interphase chromosomes are described in terms of the folding of chromatin sequences composed of alternating blocks of fibers with different thicknesses and flexibilities, and we use it to study the influence of sequence disorder on chromosome behaviors in space and time. By employing extensive computer simulations, we thus demonstrate that chromosomes undergo noticeable conformational changes only on length-scales smaller than 105 basepairs and time-scales shorter than a few seconds, and we suggest there might exist effective upper bounds to the detection of chromosome reorganization in eukaryotes. We prove the relevance of our framework by modeling recent experimental FISH data on murine chromosomes.

Paired arrangement of kinetochores together with microtubule pivoting and dynamics drive kinetochore capture in meiosis I.

Kinetochores are protein complexes on the chromosomes, whose function as linkers between spindle microtubules and chromosomes is crucial for proper cell division. The mechanisms that facilitate kinetochore capture by microtubules are still unclear. In the present study, we combine experiments and theory to explore the mechanisms of kinetochore capture at the onset of meiosis I in fission yeast. We show that kinetochores on homologous chromosomes move together, microtubules are dynamic and pivot around the spindle pole, and the average capture time is 3-4 minutes. Our theory describes paired kinetochores on homologous chromosomes as a single object, as well as angular movement of microtubules and their dynamics. For the experimentally measured parameters, the model reproduces the measured capture kinetics and shows that the paired configuration of kinetochores accelerates capture, whereas microtubule pivoting and dynamics have a smaller contribution. Kinetochore pairing may be a general feature that increases capture efficiency in meiotic cells.

Microscopy comparative evaluation of the SE systems adhesion to normal and sclerotic dentin.

The purpose of this study was in vitro evaluation and comparison of the adhesion of self-etch (SE) adhesive systems applied on normal and sclerotic dentin. For this study, Class 5 cavities were prepared on sound teeth as well as on teeth with sclerotic dentin. They were then restored by means of the SE 2-step OptiBond XTR (Kerr) and SE 1-step Bond Force (Tokuyama Dental) adhesive systems, as well as the Estelite Sigma Quick (Tokuyama Dental) composite resin. For teeth with sclerotic dentin, the hypermineralized superficial layer was removed by means of round bur on low speed, than the adhesive systems and composite resin were applied. These teeth were prepared for microscopic study according to the protocol specific to each microscope. SEM (scanning electron microscopy) examination reveals that on normal and sclerotic dentin, OptiBond XTR and Bond Force form hybrid layers with about the same thickness, greater in normal dentin, but only OptiBond XTR pervades into the dentinal tubules, both in normal and sclerotic dentin. However, TEM (transmission electron microscopy) examination of Bond Force reveals that it penetrates into the dentinal tubules as well, but only in the case of normal dentin. The thickness of the hybrid layers resulting from the application of the SE adhesive systems to sound dentin is different from the thickness of the hybrid layers obtained when the same adhesive systems have been applied to sclerotic dentin.

Microscopic aspects of the hybrid layer formed by the SE 1-step Futurabond M (Voco) adhesive system applied to normal and sclerotic dentin.

STUDY OBJECTIVES: In vitro evaluation and comparison of the adhesion of a generation-7 adhesive system to normal and sclerotic dentin. MATERIALS AND METHODS: For this study, sound teeth as well as teeth with sclerotic dentin, which had been extracted for periodontal reasons, were prepared. Class 5 cavities were prepared, then restored by means of the SE 1-step Futurabond M (Voco) adhesive system, as well as the Estelite Sigma Quick (Tokuyama Dental) composite resin. For teeth with sclerotic dentin, the hypermineralized superficial layer was removed by means of round bur on low speed, then the adhesive system and composite resin were applied. These teeth were prepared for microscopic study according to the protocol specific to each microscope. For the study involving the confocal microscope, the adhesive was mixed with the Evans Blue dye before being applied to the tooth, then the same protocol was followed. RESULTS: When applied to normal dentin, Futurabond M (Voco), the generation-7 adhesive system, forms a hybrid layer with a depth of 20-25 µm, while it can be noted that it pervades 6-8 µm into the dentinal tubules. When applied to sclerotic dentin, it was noted that the adhesive system does not pervade into the tubules, with an approximately 10-15 µm depth of the hybrid layer. CONCLUSIONS: The adhesion to sclerotic dentin shows particular aspects. When it is desired to employ generation-7 adhesive systems (SE 1-step) on sclerotic dentin, the therapeutic approach needs to include the following supplementary stages: removal of the superficial hypermineralized layer, as well as predemineralization with 37% phosphoric acid; they are the only stages that might improve the adhesion to this substrate.

Kinetic control of nucleosome displacement by ISWI/ACF chromatin remodelers.

Chromatin structure is dynamically organized by chromatin remodelers, motor protein complexes which move and remove nucleosomes. The regulation of remodeler action has recently been proposed to underlie a kinetic proofreading scheme which combines the recognition of histone-tail states and the ATP-dependent loosening of DNA around nucleosomes. Members of the ISWI-family of remodelers additionally recognize linker length between nucleosomes. Here, we show that the additional proofreading step involving linker length alone is sufficient to promote the formation of regular arrays of nucleosomes. ATP-dependent remodeling by bidirectional motors is shown to reinforce positioning as compared to statistical positioning.

Thermal and mechanical denaturation properties of a DNA model with three sites per nucleotide.

In this paper, we show that the coarse grain model for DNA, which has been proposed recently by Knotts et al. [J. Chem. Phys. 126, 084901 (2007)], can be adapted to describe the thermal and mechanical denaturation of long DNA sequences by adjusting slightly the base pairing contribution. The adjusted model leads to (i) critical temperatures for long homogeneous sequences that are in good agreement with both experimental ones and those obtained from statistical models, (ii) a realistic step-like denaturation behaviour for long inhomogeneous sequences, and (iii) critical forces at ambient temperature of the order of 10 pN, close to measured values. The adjusted model furthermore supports the conclusion that the thermal denaturation of long homogeneous sequences corresponds to a first-order phase transition and yields a critical exponent for the critical force equal to σ = 0.70. This model is both geometrically and energetically realistic, in the sense that the helical structure and the grooves, where most proteins bind, are satisfactorily reproduced, while the energy and the force required to break a base pair lie in the expected range. It therefore represents a promising tool for studying the dynamics of DNA-protein specific interactions at an unprecedented detail level.

Comparison of kinetic and dynamical models of DNA-protein interaction and facilitated diffusion.

It has long been asserted that proteins such as transcription factors may locate their target in DNA sequences at rates that surpass by several orders of magnitude the three-dimensional diffusion limit thanks to facilitated diffusion, that is, the combination of one-dimensional (sliding along the DNA) and three-dimensional diffusion. This claim has been supported throughout the years by several mass action kinetic models, while the dynamical model we proposed recently (J. Chem. Phys. 2009, 130, 015103) suggests that acceleration of targeting due to facilitated diffusion cannot be large. In order to solve this apparent contradiction, we performed additional simulations to compare the results obtained with our model to those obtained with the kinetic model of Klenin et al. (Phys. Rev. Lett. 2006, 96, 018104). We show in this paper that the two models actually support each other and agree in predicting a low efficiency for facilitated diffusion. Extrapolation of these results to real systems even indicates that facilitated diffusion necessarily slows down the targeting process compared to three-dimensional diffusion.

Modeling of 2-D DNA display.

2-D display is a fast and economical way of visualizing polymorphism and comparing genomes, which is based on the separation of DNA fragments in two steps, first according to their size and then to their sequence composition. In this article, we present an exhaustive study of the numerical issues associated with a model aimed at predicting the final absolute locations of DNA fragments in 2-D display experiments. We show that simple expressions for the mobility of DNA fragments in both dimensions allow one to reproduce experimental final absolute locations better than experimental uncertainties. However, our simulations also point out that the results of 2-D display experiments are not sufficient to determine the best set of parameters for the modeling of fragments separation in the second dimension and that additional detailed measurements of the mobility of a few sequences are necessary to achieve this goal. We hope that this work will help in establishing simulations as a powerful tool to optimize experimental conditions without having to perform a large number of preliminary experiments and to estimate whether 2-D DNA display is suited to identify a mutation or a genetic difference that is expected to exist between the genomes of closely related organisms.

Description of nonspecific DNA-protein interaction and facilitated diffusion with a dynamical model.

We propose a dynamical model for nonspecific DNA-protein interaction, which is based on the "bead-spring" model previously developed by other groups, and investigate its properties using Brownian dynamics simulations. We show that the model successfully reproduces some of the observed properties of real systems and predictions of kinetic models. For example, sampling of the DNA sequence by the protein proceeds via a succession of three-dimensional motion in the solvent, one-dimensional sliding along the sequence, short hops between neighboring sites, and intersegmental transfers. Moreover, facilitated diffusion takes place in a certain range of values of the protein effective charge, that is, the combination of one-dimensional sliding and three-dimensional motion leads to faster DNA sampling than pure three-dimensional motion. At last, the number of base pairs visited during a sliding event is comparable to the values deduced from single-molecule experiments. We also point out and discuss some discrepancies between the predictions of this model and some recent experimental results as well as some hypotheses and predictions of kinetic models.

Dynamical versus statistical mesoscopic models for DNA denaturation.

We recently proposed a dynamical mesoscopic model for DNA, which is based, like the statistical ones, on site-dependent finite stacking and pairing enthalpies. In the present paper, we first describe how the parameters of this model are varied to get predictions in better agreement with experimental results that were not addressed up to now, like mechanical unzipping, the evolution of the critical temperature with sequence length and temperature resolution. We show that the model with the new parameters provides results that are in quantitative agreement with those obtained from statistical models. Investigation of the critical properties of the dynamical model suggests that DNA denaturation looks like a first-order phase transition in a broad temperature interval, but that there necessarily exists, very close to the critical temperature, a crossover to another regime. The exact nature of the melting dynamics in this second regime still has to be elucidated. We finally point out that the descriptions of the physics of the melting transition inferred from statistical and dynamical models are not completely identical and discuss the relevance of our model from the biological point of view.