Quality of life and neuropsychiatric disorders in patients with Graves' Orbitopathy: Current concepts

Graves' disease (GD) is an autoimmune chronic thyroiditis frequently associated with development of Graves' orbitopathy (GO) characterized by proptosis, strabismus, impairment of visual function, ocular surface inflammation and dry eye. As consequence, patients with GO experience impairment of quality of life and social function and could develop a neurobehavioral syndrome, ranging from anxious to depressive or psychotic disorders. To date, the pathogenic mechanism underlying neuropsychiatric disorders in patients with GD has not been clearly understood. In fact, the development of neuropsychiatric disorders in patients with GO has been associated with both the detrimental effects of the altered circulating thyroid hormones on the nervous system, and with the psychological discomfort caused by poor quality of life, reduced social interactions and relapsing course of the disease. This paper summarizes current evidence on neuropsychiatric abnormalities in Graves' disease focusing on its impact on QoL and psychosocial function. We remark the importance of a multidisciplinary approach and we emphasize the potential benefit of neuropsychiatric approach on disease perception, patient compliance to medical and/or surgical treatment and clinical outcomes.     

Subclinical cardiovascular disease and Systemic Sclerosis: A comparison between risk charts,quantification of coronary calcium and carotid ultrasonography

Background and objectives

Recently published population-based cohort studies have shown a high prevalence of cardiovascular disease in Systemic Sclerosis (SSc) patients. The aim of this study is to compare three different methods to measure cardiovascular risk in patients with scleroderma.

Dynamic label-free imaging of lipid nanodomains

Lipid rafts are submicron proteolipid domains thought to be responsible for membrane trafficking and signaling. Their small size and transient nature put an understanding of their dynamics beyond the reach of existing techniques, leading to much contention as to their exact role. Here, we exploit the differences in light scattering from lipid bilayer phases to achieve dynamic imaging of nanoscopic lipid domains without any labels. Using phase-separated droplet interface bilayers we resolve the diffusion of domains as small as 50 nm in radius and observe nanodomain formation, destruction, and dynamic coalescence with a domain lifetime of 220 ± 60 ms. Domain dynamics on this timescale suggests an important role in modulating membrane protein function.Cell membranes compartmentalize into lipid domains that enable the selective recruitment of specific proteins (1). These “lipid rafts” have been proposed to control many membrane processes including apical sorting, protein trafficking, and the clustering of proteins during signaling. The dynamic formation and destruction of lipid nanodomains are thought to provide the central mechanism to regulate this wide range of essential processes (2–4). Although many methods now provide strong evidence to support their existence in vivo (5), the combination of nanoscopic size and dynamics on millisecond timescales has placed the direct observation of their behavior beyond the scope of existing techniques. Consequently, although we know they exist, frustratingly little is known regarding their function and dynamics (6).Recent advances in fluorescence nanoscopy provide the only time-dependent information on the behavior of lipid nanodomains (7–9). Stimulated emission depletion–fluorescence correlation spectroscopy has shown cholesterol-mediated hindered nanoscale diffusion of single labeled sphingomyelin lipids that is consistent with the lipid raft hypothesis and transient binding of lipids (9). Superresolution fluorescence microscopy has also revealed protein clusters in cell membranes with 0.5-s temporal resolution (7). All of these experiments, however, are limited in temporal resolution by fluorescence, and must infer lipid nanodomains from the addition of fluorescent labels.Macroscopic phase separation in artificial lipid bilayers has been widely used to help understand the biological implications of domain formation. Different lipid phases can be visualized using fluorescence microscopy with labels that preferentially partition into a specific phase (10–12). This approach is successful for micrometer-sized domains but inevitably fails on the tens to few hundreds of nanometers scale due to limitations in phase specificity, the limited residence time of a label within a specific nanoscopic domain, and the achievable optical resolution (13). The fluorescent probe is itself an additional component that can perturb phase behavior, either directly or through photooxidation (14, 15). As a result, lipid nanodomain dynamics have not been observed directly even in artificial systems, although recent ensemble-based techniques report lipid heterogeneity on the appropriate length scales (13). In addition to fluorescence-based approaches, ellipsometry and reflection interference contrast microscopy have been used to characterize phase separation in lipid bilayers (16, 17), taking advantage of different bilayer thicknesses and refractive indices caused by varying degrees of cholesterol content and lipid packing. Given sufficient sensitivity and resolution, this approach should hold for arbitrarily small domains.We recently developed interferometric scattering microscopy (iSCAT) (18–20) and achieved sensitivity to refractive index perturbations down to the level of a single unlabeled protein molecule in solution with millisecond time resolution (21, 22). Here, we exploit the unique sensitivity of iSCAT to overcome the limitations in temporal resolution and sensitivity to image, track, and characterize lipid nanodomains without requiring any labels. We use droplet interface bilayers (DIBs) as an artificial membrane model (23, 24) with phase-separated lipid mixtures (Fig. 1A). DIBs are formed by the contact of two lipid monolayers; in this case, a monolayer formed at the interface between an aqueous droplet and a solution of phospholipids in oil, and another between a thin hydrogel film and the oil. DIBs are robust, long-lived, and defect-free, show unrestricted diffusion, form gigaohm resistance seals, and are compatible with high-resolution optical microscopy (24).Open in a separate windowFig. 1.Detection of lipid nanodomains using iSCAT. (A) Schematic of a DIB showing ordered (light gray) and disordered (black) phases. The interference between scattered and reflected fields (E_s and E_r) is detected in the far field using a digital camera. (B) The 100-ms TIRF (Top) and iSCAT (Bottom) images of a DIB containing S_o domains within a bulk L_d phase (1:1 DOPC: bSM plus 1 mol% Atto488-DPPE). The static background due to scattering from the agarose substrate can been seen in this raw iSCAT image. This background is subtracted in subsequent images. (C) Time-lapse sequence of iSCAT images of L_o nanodomains appearing from a uniform L_d phase upon cooling of a DIB below the phase transition temperature. The droplet was heated to 45 °C for 10 min. Nanodomains appeared 2–5 min after heating was stopped. Composition, 1:1:1 DPhPC:bSM:Chol. Greyscale values are of the normalized reflected intensity. (D) Trajectories corresponding to average pixel contrast within a 900 × 900-nm window centered on each nanodomain shown in C. Values before the appearance of the domain are representative of the background fluctuations at the position where the domain first becomes visible. (Scale bars: 5 μm.)     

A hypothesis to reconcile the physical and chemical unfolding of proteins

High pressure (HP) or urea is commonly used to disturb folding species. Pressure favors the reversible unfolding of proteins by causing changes in the volumetric properties of the protein–solvent system. However, no mechanistic model has fully elucidated the effects of urea on structure unfolding, even though protein–urea interactions are considered to be crucial. Here, we provide NMR spectroscopy and 3D reconstructions from X-ray scattering to develop the “push-and-pull” hypothesis, which helps to explain the initial mechanism of chemical unfolding in light of the physical events triggered by HP. In studying MpNep2 from Moniliophthora perniciosa, we tracked two cooperative units using HP-NMR as MpNep2 moved uphill in the energy landscape; this process contrasts with the overall structural unfolding that occurs upon reaching a threshold concentration of urea. At subdenaturing concentrations of urea, we were able to trap a state in which urea is preferentially bound to the protein (as determined by NMR intensities and chemical shifts); this state is still folded and not additionally exposed to solvent [fluorescence and small-angle X-ray scattering (SAXS)]. This state has a higher susceptibility to pressure denaturation (lower p_1/2 and larger ΔV_u); thus, urea and HP share concomitant effects of urea binding and pulling and water-inducing pushing, respectively. These observations explain the differences between the molecular mechanisms that control the physical and chemical unfolding of proteins, thus opening up new possibilities for the study of protein folding and providing an interpretation of the nature of cooperativity in the folding and unfolding processes.It is known that proteins are far from equilibrium during folding reactions, and they undergo a wide range of conformational states to reach the global folding minimum. Various physical and chemical strategies, such as the use of high temperature, high pressure, protonation, altered ionic strength, and harsh denaturants, are commonly used to disturb folding species to promote the formation of rarely observed folding intermediates. From the thermodynamic point of view, any perturbing agent affecting protein folding is controlled by Le Chatelier’s principle. For instance, increasing the concentration of urea shifts the folding equilibrium toward the unfolded state because of the increased preferential binding of urea to this state. In the case of pressure, the smaller volume of the unfolded state is favored at high pressure because it only affects the volumetric properties of the molecule.The energy landscape theory of folding assumes that protein folding is the progressive organization of an ensemble of partially folded structures through which the protein passes on its way to a native conformation (1–3). Accordingly, proteins have a rugged funnel-like landscape that is biased toward their native structure due to evolution (3). Obtaining structural and dynamic information on multiple-stage protein intermediates that follow the folding trail may increase our knowledge of protein misfolding, which is associated with amyloidosis, prion formation, and the occurrence of several diseases, including Parkinson’s disease, Huntington’s disease, spongiform encephalopathy, and, more recently, cancer (4–6). In these diseases, proteins tend to traverse the “wrong side of the funnel” (7, 8).The effects of pressure on proteins were discovered in early experiments with egg albumin (9). Currently, pressure is extensively used in various biological and biotechnological applications (10–13) and is one of the most promising variables enabling the structural analysis of these protein substates. The application of pressure to a protein forces the water shell into the protein, thus shifting the equilibrium of the system from the native state to an intermediate or to the unfolded state. Pressure appears to favor water infiltration into the protein and the disassembly of protein cavities (14), leading to increased hydration and decreased partial molar volume. Unlike high temperatures, which cause systematic changes in the total energy and volume of the molecule, high pressure affects only the volumetric properties of the molecule. However, under physiological conditions, water solvation dictates protein folding (15).High pressure can be coupled with various spectroscopic techniques, including methods based on the intrinsic fluorescence of Trp residues (16), Fourier-transform infrared spectroscopy (FTIR) (17), NMR (13, 18, 19), small-angle X-ray scattering (SAXS) (20, 21), microsecond pressure-jump coupled to fluorescence lifetime (22), and, more recently, circular dichroism (CD) (23). To better understand local changes, high-pressure NMR (HP-NMR) is adopted as the most informative approach (18, 19). SAXS is another useful tool for assessing changes in protein folding and the size and shape of macromolecules in solution (24–26).Most denaturants affect protein folding through their binding properties. Urea is one of the most commonly used chemical denaturants for the study of protein folding and thermodynamics. Two main theories exist to explain why urea induces protein denaturation (27, 28). The first theory hypothesizes an indirect mechanism by which urea alters the water structure and leads to hydrophobic group solvation. The second view is based on the direct interaction of urea with the protein. Most studies are based on model compounds and theoretical and modeling approaches, such as molecular dynamics (29–35).The binding of urea to proteins is based on the ability of urea to displace water molecules from the first solvation shell (29), thus increasing the system entropy and weakening the hydrophobic effect (36). Pioneer calorimetric studies on protein–urea interactions were developed by Makhatadze and Privalov (37). Urea is also believed to form hydrogen bonds with the amide unit of peptide bonds, as shown by calorimetric measurements of cyclic dipeptides (36) and H/D exchange by 1D NMR of an alanine-based model compound (38). A two-stage kinetic mechanism for the action of urea has been proposed based on extensive (microsecond) molecular-dynamics simulations (29). However, at equilibrium conditions, experimental insights into the action of urea at the initial stages of denaturation are lacking. A long-standing controversy in the literature concerns the contribution of nonpolar groups, peptide backbone interactions, or both as the driving force behind urea-induced protein denaturation (32, 33, 39, 40). Experimental efforts were mostly based on measurements of transfer free energies (41, 42) of amino acid side chains, peptide backbones, or random-coil polypeptide chains, in which packing defects and the overall 3D architecture of a native polypeptide chain were neglected.Here, we studied Moniliophthora perniciosa necrosis- and ethylene-inducing protein 2 (MpNep2). We systematically studied protein folding in response to chemical (urea), physical (pressure), or both perturbations using HP-NMR spectroscopy and 3D low-resolution shape reconstructions from SAXS data to better understand how each of the two disturbing agents (pressure and urea) affect protein folding and to examine how urea denatures proteins. The use of these synergistic techniques (HP-NMR and SAXS) provides reliable structural information on local/global intermediates within protein energy landscapes. We provide evidence of a conformational state in which urea binds to the protein without promoting full denaturation by populating a dry molten globule (DMG). This state is more sensitive to pressure (lower p_1/2) due to the presence of a pulling force and an increased volume change.