Neurofibromatosis Type 1 Diagnosed in a Child Based on Multiple Juvenile Xanthogranulomas and Juvenile Myelomonocytic Leukemia

An association between juvenile xanthogranuloma (JXG), neurofibromatosis type 1 (NF1), and juvenile myelomonocytic leukemia (JMML) has been described in the literature but has only been documented in approximately 20 cases. We diagnosed a patient with NF1 at 25 months of age, before any cutaneous stigmata of this disease had appeared, because we decided to screen for the NF1 gene mutation because of his presentation with multiple JXGs and moderate macrocephaly (2.5 standard deviations) at 9 months of age and JMML diagnosed at 20 months of age. The child is well today after treatment with chemotherapy and allogenic bone marrow transplantation. With increased awareness, patients with JXG and NF1 who develop symptoms possibly related to JMML, such as paleness, skin bleeding, cough, unexplained fever, and hepatosplenomegaly, should be further evaluated. We also emphasize that multiple JXG lesions can be an early marker of NF1.     

Identification of Cellular Voids in the Human Otic Capsule

Journal of the Association for Research in Otolaryngology - The otic capsule consists of dense highly mineralized compact bone. Inner ear osteoprotegerin (OPG) effectively inhibits perilabyrinthine...     

H-Ras forms dimers on membrane surfaces via a protein–protein interface

The lipid-anchored small GTPase Ras is an important signaling node in mammalian cells. A number of observations suggest that Ras is laterally organized within the cell membrane, and this may play a regulatory role in its activation. Lipid anchors composed of palmitoyl and farnesyl moieties in H-, N-, and K-Ras are widely suspected to be responsible for guiding protein organization in membranes. Here, we report that H-Ras forms a dimer on membrane surfaces through a protein–protein binding interface. A Y64A point mutation in the switch II region, known to prevent Son of sevenless and PI3K effector interactions, abolishes dimer formation. This suggests that the switch II region, near the nucleotide binding cleft, is either part of, or allosterically coupled to, the dimer interface. By tethering H-Ras to bilayers via a membrane-miscible lipid tail, we show that dimer formation is mediated by protein interactions and does not require lipid anchor clustering. We quantitatively characterize H-Ras dimerization in supported membranes using a combination of fluorescence correlation spectroscopy, photon counting histogram analysis, time-resolved fluorescence anisotropy, single-molecule tracking, and step photobleaching analysis. The 2D dimerization K_d is measured to be ∼1 × 10³ molecules/µm², and no higher-order oligomers were observed. Dimerization only occurs on the membrane surface; H-Ras is strictly monomeric at comparable densities in solution. Analysis of a number of H-Ras constructs, including key changes to the lipidation pattern of the hypervariable region, suggest that dimerization is a general property of native H-Ras on membrane surfaces.In mammalian signal transduction, Ras functions as a binary switch in fundamental processes including proliferation, differentiation, and survival (1). Ras is a network hub; various upstream signaling pathways can activate Ras-GDP to Ras-GTP, which subsequently selects between multiple downstream effectors to elicit a varied but specific biochemical response (2, 3). Signaling specificity is achieved by a combination of conformational plasticity in Ras itself (4, 5) and dynamic control of Ras spatial organization (6, 7). Isoform-specific posttranslational lipidation targets the main H-, N-, and K-Ras isoforms to different subdomains of the plasma membrane (8–10). For example, H-Ras localizes to cholesterol-sensitive membrane domains, whereas K-Ras does not (11). A common C-terminal S-farnesyl moiety operates in concert with one (N-Ras) or two (H-Ras) palmitoyl groups, or with a basic sequence of six lysines in K-Ras4B (12), to provide the primary membrane anchorage. Importantly, the G-domain (residues 1–166) and the hypervariable region (HVR) (residues 167–189) dynamically modulate the lipid anchor localization preference to switch between distinct membrane populations (13). For example, repartitioning of H-Ras away from cholesterol-sensitive membrane domains is necessary for efficient activation of the effector Raf and GTP loading of the G-domain promotes this redistribution by a mechanism that requires the HVR (14). However, the molecular details of the coupling between lipid anchor partitioning and nucleotide-dependent protein–membrane interactions remain unclear.In addition to biochemical evidence for communication between the C-terminal membrane binding region and the nucleotide binding pocket, NMR and IR spectroscopic observations suggest that the HVR and lipid anchor membrane insertion affects Ras structure and orientation (15–17). Molecular dynamics (MD) modeling of bilayer-induced H-Ras conformations has identified two nucleotide-dependent states, which differ in HVR conformation, membrane contacts, and G-domain orientation (18). In vivo FRET measurements are consistent with a reorientation of Ras with respect to the membrane upon GTP binding (19, 20). Further modeling showed that the membrane binding region and the canonical switch I and II regions communicate across the protein via long-range side-chain interactions (21) in a conformational selection mechanism (22). Whereas these allosteric modes likely contribute to Ras partitioning and reorientation in vivo, direct functional consequences on Ras protein–protein interactions are poorly understood.Members of the Ras superfamily of small GTPases are widely considered to be monomeric (23). However, several members across the Ras GTPase subfamilies are now known to dimerize (24–28), and a class of small GTPases that use dimerization instead of GTPase activating proteins (GAPs) for GTPase activity has been identified (29). Recently, semisynthetic natively lipidated N-Ras was shown to cluster on supported membranes in vitro, in a manner broadly consistent with molecular mechanics (MM) modeling of dimers (30). For Ras, dimerization could be important because Raf, which is recruited to the membrane by binding to Ras, requires dimerization for activation. Soluble Ras does not activate Raf in vitro (31), but because artificial dimerization of GST-fused H-Ras leads to Raf activation in solution, it has been hypothesized that Ras dimers exist on membranes (32). However, presumed dimers were only detected after chemical cross-linking (32), and the intrinsic oligomeric properties of Ras remain unknown.Here, we use a combination of time-resolved fluorescence spectroscopy and microscopy to characterize H-Ras(C118S, 1–181) and H-Ras(C118S, 1–184) [referred to as Ras(C181) and Ras(C181,C184) from here on] anchored to supported lipid bilayers. By tethering H-Ras to membranes at cys181 (or both at cys181 and cys184) via a membrane-miscible lipid tail, we eliminate effects of lipid anchor clustering while preserving the HVR region between the G-domain and the N-terminal palmitoylation site at cys181 (or cys184), which is predicted to undergo large conformational changes upon membrane binding and nucleotide exchange (18). Labeling is achieved through a fluorescent Atto488-linked nucleotide. Fluorescence correlation spectroscopy (FCS) and time-resolved fluorescence anisotropy (TRFA) show that H-Ras forms surface density-dependent clusters. Photon counting histogram (PCH) analysis and single-molecule tracking (SMT) reveal that H-Ras clusters are dimers and that no higher-order oligomers are formed. A Y64A point mutation in the loop between beta strand 3 (β3) and alpha helix 2 (α2) abolishes dimer formation, suggesting that the corresponding switch II (SII) region is either part of, or allosterically coupled to, the dimer interface. The 2D dimerization K_d is measured to be on the order of 1 × 10³ molecules/µm², within the broad range of Ras surface densities measured in vivo (10, 33–35). Dimerization only occurs on the membrane surface; H-Ras is strictly monomeric at comparable densities in solution, suggesting that a membrane-induced structural change in H-Ras leads to dimerization. Comparing singly lipidated Ras(C181) and doubly lipidated Ras(C181,C184) reveals that dimer formation is insensitive to the details of HVR lipidation, suggesting that dimerization is a general property of H-Ras on membrane surfaces.     

Capillary transit time heterogeneity and flow-metabolism coupling after traumatic brain injury

Most patients who die after traumatic brain injury (TBI) show evidence of ischemic brain damage. Nevertheless, it has proven difficult to demonstrate cerebral ischemia in TBI patients. After TBI, both global and localized changes in cerebral blood flow (CBF) are observed, depending on the extent of diffuse brain swelling and the size and location of contusions and hematoma. These changes vary considerably over time, with most TBI patients showing reduced CBF during the first 12 hours after injury, then hyperperfusion, and in some patients vasospasms before CBF eventually normalizes. This apparent neurovascular uncoupling has been ascribed to mitochondrial dysfunction, hindered oxygen diffusion into tissue, or microthrombosis. Capillary compression by astrocytic endfeet swelling is observed in biopsies acquired from TBI patients. In animal models, elevated intracranial pressure compresses capillaries, causing redistribution of capillary flows into patterns argued to cause functional shunting of oxygenated blood through the capillary bed. We used a biophysical model of oxygen transport in tissue to examine how capillary flow disturbances may contribute to the profound changes in CBF after TBI. The analysis suggests that elevated capillary transit time heterogeneity can cause critical reductions in oxygen availability in the absence of ‘classic'' ischemia. We discuss diagnostic and therapeutic consequences of these predictions.     

Social and emotional factors as predictors of poor outcomes following cardiac surgery

 Open in a separate windowOBJECTIVESExisting risk prediction models in cardiac surgery stratify individuals based on their predicted risk, including only medical and physiological factors. However, the complex nature of risk assessment and the lack of parameters representing non-medical aspects of patients’ lives point towards the need for a broader paradigm in cardiac surgery. Objectives were to evaluate the predictive value of emotional and social factors on 4 outcomes; death within 90 days, prolonged stay in intensive care (≥72 h), prolonged hospital admission (≥10 days) and readmission within 90 days following cardiac surgery, as a supplement to traditional risk assessment by European System for Cardiac Operative Risk Evaluation (EuroSCORE).METHODSThe study included adults undergoing cardiac surgery in Denmark 2014–2017 including information on register-based socio-economic factors, and, in a nested subsample, self-reported symptoms of anxiety and depression. Logistic regression analyses were conducted, adjusted for EuroSCORE, of variables reflecting social and emotional factors.RESULTSAmongst 7874 included patients, lower educational level (odds ratio 1.33; 95% confidence interval 1.17–1.51) and living alone (1.25; 1.14–1.38) were associated with prolonged hospital admission after adjustment for EuroSCORE. Lower educational level was also associated with prolonged intensive care unit stay (1.27; 1.00–1.63). Having a high income was associated with decreased odds of prolonged hospital admission (0.78; 0.70–0.87). No associations or predictive value for symptoms of anxiety or depression were found on any outcomes.CONCLUSIONSSocial disparity is predictive of poor outcomes following cardiac surgery. Symptoms of anxiety and depression are frequent especially amongst patients with a high-risk profile according to EuroSCORE.Subj collection105, 123