Developmental dosing with a MEK inhibitor (PD0325901) rescues myopathic features of the muscle-specific but not limb-specific Nf1 knockout mouse

Neurofibromatosis Type 1 (NF1) is a common autosomal dominant genetic disorder While NF1 is primarily associated with predisposition for tumor formation, muscle weakness has emerged as having a significant impact on quality of life. NF1 inactivation is linked with a canonical upregulation Ras-MEK-ERK signaling. This in this study we tested the capacity of the small molecule MEK inhibitor PD0325901 to influence the intramyocellular lipid accumulation associated with NF1 deficiency. Established murine models of tissue specific Nf1 deletion in skeletal muscle (Nf1_MyoD^?/?) and limb mesenchyme (Nf1_Prx1^?/?) were tested.Developmental PD0325901 dosing of dams pregnant with Nf1_MyoD^?/? progeny rescued the phenotype of day 3 pups including body weight and lipid accumulation by Oil Red O staining. In contrast, PD0325901 treatment of 4?week old Nf1_Prx1^?/? mice for 8?weeks had no impact on body weight, muscle wet weight, activity, or intramyocellular lipid. Examination of day 3 Nf1_Prx1^?/? pups showed differences between the two tissue-specific knockout strains, with lipid staining greatest in Nf1_MyoD^?/? mice, and fibrosis higher in Nf1_Prx1^?/? mice.These data show that a MEK/ERK dependent mechanism underlies NF1 muscle metabolism during development. However, crosstalk from Nf1-deficient non-muscle mesenchymal cells may impact upon muscle metabolism and fibrosis in neonatal and mature myofibers.     

Arterial stenosis in antiphospholipid syndrome: Update on the unrevealed mechanisms of an endothelial disease

First described in 1983, antiphospholipid syndrome (APS) is an autoimmune condition characterized by the occurrence of recurrent arterial and/or venous thrombosis, and/or pregnancy morbidity, in the setting of persistent presence of antiphospholipid antibodies (aPL). While thrombosis is the most well-known pathogenic mechanism in this disorder, the relevance of some other mechanisms such as arterial stenosis is being increasingly recognized. Arterial stenosis has been first described in the renal arteries in patients with APS, however intracranial and coeliac arteries can also be involved with various and treatable clinical manifestations. The underlying pathophysiology of this stenotic arterial vasculopathy is not fully understood but some recent studies revealed new insights into the molecular mechanism behind this endothelial cell activation in APS. In this review, we discuss these newly discovered mechanisms and highlight the diagnostic and therapeutic modalities of the APS related arterial stenosis.     

Neural coding underlying the cue preference for celestial orientation

Diurnal and nocturnal African dung beetles use celestial cues, such as the sun, the moon, and the polarization pattern, to roll dung balls along straight paths across the savanna. Although nocturnal beetles move in the same manner through the same environment as their diurnal relatives, they do so when light conditions are at least 1 million-fold dimmer. Here, we show, for the first time to our knowledge, that the celestial cue preference differs between nocturnal and diurnal beetles in a manner that reflects their contrasting visual ecologies. We also demonstrate how these cue preferences are reflected in the activity of compass neurons in the brain. At night, polarized skylight is the dominant orientation cue for nocturnal beetles. However, if we coerce them to roll during the day, they instead use a celestial body (the sun) as their primary orientation cue. Diurnal beetles, however, persist in using a celestial body for their compass, day or night. Compass neurons in the central complex of diurnal beetles are tuned only to the sun, whereas the same neurons in the nocturnal species switch exclusively to polarized light at lunar light intensities. Thus, these neurons encode the preferences for particular celestial cues and alter their weighting according to ambient light conditions. This flexible encoding of celestial cue preferences relative to the prevailing visual scenery provides a simple, yet effective, mechanism for enabling visual orientation at any light intensity.The blue sky is a rich source of visual cues that are used by many animals during orientation or navigation (1, 2). Besides the sun, celestial phenomena, such as the skylight intensity gradient or the more complex polarization pattern, can serve as references for spatial orientation (3–5). Polarized skylight is generated by scattered sunlight in the atmosphere, and to a terrestrial observer, the resulting alignment of the electric field vectors extends across the entire sky, forming concentric circles around the position of the sun (Fig. 1A). A similar distribution of brightness and polarization pattern is also created around the moon (6). Although this nocturnal pattern is 1 million-fold dimmer than the daylight pattern (6), some animals, such as South African ball-rolling dung beetles, can use this lunar polarization pattern for orientation (7). To avoid competition for food at the dung pile, these beetles detach a piece of dung, shape it into a ball, and roll it away along a straight-line path. For this type of straight-line orientation, nocturnal beetles seem to rely exclusively on celestial cues (8), such as the moon or polarized light.Open in a separate windowFig. 1.Celestial cue preference in dung beetles under a natural sky. (A) Schematic illustration of the polarization pattern around a celestial body (sun or moon). Change of direction in diurnal (D, Left) and nocturnal (N, Right) beetles rolling under a sun-lit (B) or moon-lit (C) sky. The change of direction was calculated as the angular difference between two consecutive rolls, either without manipulation (control, ●) or when the sun or moon was reflected to the opposite sky hemisphere between the two rolls (test, ○). The mean directions (μ) are indicated by black (control) or red (test) lines, and error bars indicate circular SDs. (B) Without manipulation, both species kept the direction [P < 0.001 by V test; μ_diurnal (±SD) = 2.6° ± 17.98°, n = 20; μ_nocturnal = −8.7° ± 38.34°, n = 20). When the sun was reflected to the opposite sky hemisphere (and the real sun was shaded), both species responded to this change (P < 0.001 by V-test; μ_diurnal = 178.9° ± 54.6°, n = 20; μ_nocturnal = 163.8° ± 46.58°, n = 20). (C) Under the moon in the control experiments, both species showed a constant rolling direction (P < 0.001 by V test; μ_diurnal = 3.1° ± 35.39°, n = 20; μ_nocturnal = −3.3° ± 37.87°, n = 20). When the moon was reflected to the opposite sky hemisphere, the diurnal species followed the position change of the moon (P = 0.002 by V test; μ = 179.5° ± 72.37°, n = 20), whereas the nocturnal species continued rolling in the original rolling direction (P < 0.001 by V test; μ = −13.4° ± 74.27°, n = 20).As with all nocturnal animals, night-active beetles have to overcome a major challenge: They need to maintain high orientation precision even under extremely dim light conditions. Indeed, recent experiments have shown that nocturnal dung beetles orient at night with the same precision as their diurnal relatives during the day (9), an ability partly due to the fact that their eyes are considerably more sensitive than the eyes of species that are active at brighter light levels (10–12). Nonetheless, for each species, orientation precision relies on being tuned to the most reliable celestial compass cue that is available during the animal’s normal activity window. How salient are these cues for nocturnal and diurnal species? Do diurnal species have a different celestial cue preference than nocturnal species? If so, how are these preferences represented neurally in the brain?In this study, we present a detailed picture of how the orientation systems of two closely related nocturnal and diurnal animals have been adapted to the ambient light conditions, combining behavioral experiments from the field with electrophysiological investigations of the underlying neural networks. Using behavioral experiments, we show that nocturnal dung beetles switch from a compass that uses a discrete celestial body (the sun) during the day to a celestial polarization compass for dim light orientation at night, whereas diurnal beetles use a celestial body (the sun or moon) for orientation at all light levels. In a second step, we simulated these skylight cues (the sun or moon and the polarization pattern) while electrophysiologically recording responses from neurons in the dung beetle’s central complex, a brain area that has been suggested to house the internal compass for celestial orientation (13, 14). These neural data precisely matched the cue preferences observed in behavioral field trials and show how an animal’s visual ecology influences the neural activity of its sky compass neurons. Our results also reveal, for the first time to our knowledge, how a weighting of celestial orientation cues could be neurally encoded in an animal brain.     

Seasonal fluxes of carbonyl sulfide in a midlatitude forest

Carbonyl sulfide (OCS), the most abundant sulfur gas in the atmosphere, has a summer minimum associated with uptake by vegetation and soils, closely correlated with CO₂. We report the first direct measurements to our knowledge of the ecosystem flux of OCS throughout an annual cycle, at a mixed temperate forest. The forest took up OCS during most of the growing season with an overall uptake of 1.36 ± 0.01 mol OCS per ha (43.5 ± 0.5 g S per ha, 95% confidence intervals) for the year. Daytime fluxes accounted for 72% of total uptake. Both soils and incompletely closed stomata in the canopy contributed to nighttime fluxes. Unexpected net OCS emission occurred during the warmest weeks in summer. Many requirements necessary to use fluxes of OCS as a simple estimate of photosynthesis were not met because OCS fluxes did not have a constant relationship with photosynthesis throughout an entire day or over the entire year. However, OCS fluxes provide a direct measure of ecosystem-scale stomatal conductance and mesophyll function, without relying on measures of soil evaporation or leaf temperature, and reveal previously unseen heterogeneity of forest canopy processes. Observations of OCS flux provide powerful, independent means to test and refine land surface and carbon cycle models at the ecosystem scale.Carbonyl sulfide (OCS) is the most abundant sulfur gas in the atmosphere (1), and biogeochemical cycling of OCS affects both the stratosphere and the troposphere. The tropospheric OCS mixing ratio is between 300 and 550 parts per trillion (ppt) (1) (10⁻¹² mol OCS per mol dry air), decreasing sharply with altitude in the stratosphere (2). In times of low volcanic activity, the sulfur budget and aerosol loading of the stratosphere are largely controlled by transport and photooxidation of OCS from the troposphere (3). The processes regulating emission and uptake of OCS are thus important factors in determining how changes in climate and land cover may affect the stratospheric sulfate layer.Oceans are the dominant source of atmospheric OCS (4), with smaller emissions from anthropogenic and terrestrial sources, such as wetlands and anoxic soils (e.g., refs. 5 and 6) and oxic soils during times of heat or drought stress (e.g., refs. 7 and 8). The terrestrial biosphere is the largest sink for OCS (1, 4, 9, 10) with uptake by both oxic soils (e.g., ref. 11) and vegetation (e.g., ref. 9). Once OCS molecules pass through the stomata of leaves, the uptake rate of OCS is controlled by reaction with carbonic anhydrase (CA) within the mesophyll, to produce H₂S and CO₂. CA is the same enzyme that hydrolyzes carbon dioxide (CO₂) in the first chemical step of photosynthesis (12).Studies considering the large-scale atmospheric variability of OCS have linked OCS fluxes and the photosynthetic uptake of CO₂ for regional and global scales (1, 4, 13). Leaf-scale studies have confirmed the OCS link to photosynthesis (14, 15). Initial OCS ecosystem flux estimations were made using flask sampling followed by analysis via gas chromatography–mass spectrometry (GC-MS) (13, 16), but these studies did not have sufficient resolution to examine daily or hourly controls on the OCS flux. Laser spectrometers have been developed (17, 18) to enable direct, in situ measurement of OCS fluxes by eddy covariance, and measurements of OCS ecosystem fluxes have been reported, for periods of up to a few weeks, above arid forests (19) and an agricultural field (8, 20).Net carbon exchange in terrestrial ecosystems [net ecosystem exchange (NEE)] can be measured by eddy flux methods. NEE may be regarded as the sum of two gross fluxes: gross ecosystem productivity (GEP) and ecosystem respiration (R_eco). GEP is the light-dependent part of NEE, estimated by subtracting daytime ecosystem respiration (R_eco), computed by extrapolation of the temperature dependence of nighttime NEE (NEE – R_eco = GEP) (e.g., refs. 21–24). At night, NEE includes all autotrophic and heterotrophic respiration processes. During the day, GEP approximates the carboxylation rate minus photorespiration at the ecosystem scale (25). Extrapolation of nighttime R_eco introduces major uncertainty in the interpretation of GEP, which could be reduced, and the ecological significance of GEP increased, by developing independent methods of measuring rates of photosynthetic processes. As shown below, fluxes of OCS give more direct information on one of the major controls on GEP, stomatal conductance, rather than GEP itself, providing a powerful means for testing and improving ecosystem models and for scaling up leaf-level processes to the whole ecosystem.Here we describe the factors controlling the hourly, daily, seasonal, and total fluxes of OCS in a forest ecosystem, using a year (2011) of high-frequency, direct measurements at Harvard Forest, MA. We report the seasonal cycle, the response to environmental conditions, and the total deposition flux of OCS throughout the year 2011. We compare these fluxes to corresponding measurements of CO₂ flux and to simulations using the Simple Biosphere model (SiB3).     

Local and global structural drivers for the photoactivation of the orange carotenoid protein

Photoprotective mechanisms are of fundamental importance for the survival of photosynthetic organisms. In cyanobacteria, the orange carotenoid protein (OCP), when activated by intense blue light, binds to the light-harvesting antenna and triggers the dissipation of excess captured light energy. Using a combination of small angle X-ray scattering (SAXS), X-ray hydroxyl radical footprinting, circular dichroism, and H/D exchange mass spectrometry, we identified both the local and global structural changes in the OCP upon photoactivation. SAXS and H/D exchange data showed that global tertiary structural changes, including complete domain dissociation, occur upon photoactivation, but with alteration of secondary structure confined to only the N terminus of the OCP. Microsecond radiolytic labeling identified rearrangement of the H-bonding network associated with conserved residues and structural water molecules. Collectively, these data provide experimental evidence for an ensemble of local and global structural changes, upon activation of the OCP, that are essential for photoprotection.Photosynthetic organisms have evolved a protective mechanism known as nonphotochemical quenching (NPQ) to dissipate excess energy, thereby preventing oxidative damage under high light conditions (1). In plants and algae, NPQ involves pH-induced conformation changes in membrane-embedded protein complexes and enzymatic interconversion of carotenoids (2, 3). Cyanobacteria, in contrast, use a relatively simple NPQ mechanism governed by the water soluble orange carotenoid protein (OCP). The OCP is composed of an all α-helical N-terminal domain (NTD) consisting of two discontinuous four-helix bundles and a mixed α/β C-terminal domain (CTD), which is a member of the widely distributed nuclear transport factor 2-like superfamily (Fig. S1A) (4, 5). There are two regions of interaction between the NTD and CTD (4, 5): the major interface, which buries 1,722 Å of surface area, and the interaction between the N-terminal alpha-helix (αA) and the CTD (minor interface) (Fig. S1A). A single noncovalently bound keto-carotenoid [e.g., echinenone (ECN)] spans both domains in the structure of the resting (inactive) form of the protein (OCP^O).Open in a separate windowFig. S1.Structure of the OCP. (A) Crystal structure of Synechocystis OCP (PDB ID code 3MG1) consisting of two domains, NTD and CTD as described in the main text introduction, which form major and minor interfaces. (B) Amino acid residues within 3.9 Å of the carotenoid are shown by sticks. (C) Surface-bound water molecules at the major interface are shown in slate-colored spheres in Synechocystis OCP (PDB ID code 3MG1). This layer of water molecules fully or partially eclipses other water molecules, which are either conserved or found to be at the same location (within 0.5 Å) in the crystal structures of A. maxima and Synechocystis OCP (Fig. 4 A and B. Removal of the slate-colored spheres, exposing partially buried water, is shown in orange in D. The fully buried waters (red spheres) are invisible in the surface diagram of OCP. (E) Cross-sectional view to show the position of fully and partially buried structural waters in OCP^O. (F) Details of water–protein H-bonding network in water cluster 1 at the major interface. The absolutely conserved R155 is closely surrounded (<3.2 Å, capable of forming H-bond) by a number of buried (HOH1151,1200, and 1671) water molecules, which are involved in dense residue-water interactions as discussed in the main text. Similar H-bonding networks are also observed in the water clusters 2 and 3 (6). Exposure to blue light converts OCP^O to the active (red) form, OCP^R (7). OCP^R is involved in protein–protein interactions with the phycobilisome (PB) (5) and the fluorescence recovery protein (FRP), which converts OCP^R back to OCP^O (8). The OCP^R form is therefore central to the photoprotective mechanism, and determining the exact structural changes that accompany its formation are critical for a complete mechanistic understanding of the reversible quenching process in cyanobacteria. Although crystal structures exist of both the (inactive) OCP^O (4, 5) and the active NTD (effector domain) form of the protein (9), crystallization of the activated, full-length OCP^R has not been achieved. To identify the protein structural changes that occur after absorption of light by the OCP’s ECN chromophore, we undertook a hybrid approach to structurally characterize OCP^R in solution.In Synechocystis OCP^O, the 4-keto group on the “β1” ring of ECN is H-bonded to two conserved residues, Y201 and W288, in a hydrophobic pocket in the core of the CTD (Fig. S1B) (5). The other end of the carotenoid is positioned between the two four-helix bundles of the NTD. Several conserved residues within 3.9 Å of the carotenoid are known to interact with its extensive conjugation and result in fine tuning of the spectral characteristics of the OCP (Fig. S1B) (4, 5); these residues have been implicated in photochemical function via mutagenesis studies (5). A recent study of the OCP bound to the carotenoid canthaxanthin (OCP-CAN) showed that photoactivation of the OCP results in a substantial translocation (12 Å) of the carotenoid deeper into the NTD (9). Mutational analyses of the full-length OCP and biochemical studies on the constitutively active NTD [commonly known as the red carotenoid protein (RCP)] suggested that the NTD and CTD at least partially separate, resulting in the breakage of an interdomain salt-bridge (R155–E244) upon photoactivation (9–12). Together, the previous studies suggest that large-scale protein structural changes in the OCP accompany carotenoid translocation upon light activation; however, such changes in the context of the full-length protein have yet to be experimentally demonstrated. Here, we report use of X-ray radiolytic labeling with mass spectrometry (XF-MS) and hydrogen/deuterium exchange with mass spectrometry (HDX-MS), which detect residue-specific changes (13–15), to investigate the structural changes that occur during OCP photoactivation. In conjunction with small angle X-ray scattering (SAXS), which enables characterization of global conformational changes in the solution state (16), we show that dissociation of the NTD and CTD is complete in photoactivated OCP. This separation is accompanied by an unfolding of the N-terminal α-helix that is associated with the CTD in the resting state. We also pinpoint changes in specific amino acids and structurally conserved water molecules, providing insight into the signal propagation pathway from carotenoid to protein surface upon photoactivation. Collectively, these data provide a comprehensive view of both global and local intraprotein structural changes in the OCP upon photoactivation that are essential to a mechanistic understanding of cyanobacterial NPQ.     

A new finite element approach for near real‐time simulation of light propagation in locally advanced head and neck tumors

Background and Objectives

Several clinical studies suggest that interstitial photodynamic therapy (I‐PDT) may benefit patients with locally advanced head and neck cancer (LAHNC). For I‐PDT, the therapeutic light is delivered through optical fibers inserted into the target tumor. The complex anatomy of the head and neck requires careful planning of fiber insertions. Often the fibers' location and tumor optical properties may vary from the original plan therefore pretreatment planning needs near real‐time updating to account for any changes. The purpose of this work was to develop a finite element analysis (FEA) approach for near real‐time simulation of light propagation in LAHNC.

Methods

Our previously developed FEA for modeling light propagation in skin tissue was modified to simulate light propagation from interstitial optical fibers. The modified model was validated by comparing the calculations with measurements in a phantom mimicking tumor optical properties. We investigated the impact of mesh element size and growth rate on the computation time, and defined optimal settings for the FEA. We demonstrated how the optimized FEA can be used for simulating light propagation in two cases of LAHNC amenable to I‐PDT, as proof‐of‐concept.

Results

The modified FEA was in agreement with the measurements (P = 0.0271). The optimal maximum mesh size and growth rate were 0.005–0.02 m and 2–2.5 m/m, respectively. Using these settings the computation time for simulating light propagation in LAHNC was reduced from 25.9 to 3.7 minutes in one case, and 10.1 to 4 minutes in another case. There were minor differences (1.62%, 1.13%) between the radiant exposures calculated with either mesh in both cases.

Conclusions

Our FEA approach can be used to model light propagation from diffused optical fibers in complex heterogeneous geometries representing LAHNC. There is a range of maximum element size (MES) and maximum element growth rate (MEGR) that can be used to minimize the computation time of the FEA to 4 minutes. Lasers Surg. Med. 47:60–67, 2015. © 2015 The Authors. Lasers in Surgery and Medicine Published by Wiley Periodicals, Inc.