Functional divergence in the role of N-linked glycosylation in smoothened signaling

The G protein-coupled receptor (GPCR) Smoothened (Smo) is the requisite signal transducer of the evolutionarily conserved Hedgehog (Hh) pathway. Although aspects of Smo signaling are conserved from Drosophila to vertebrates, significant differences have evolved. These include changes in its active sub-cellular localization, and the ability of vertebrate Smo to induce distinct G protein-dependent and independent signals in response to ligand. Whereas the canonical Smo signal to Gli transcriptional effectors occurs in a G protein-independent manner, its non-canonical signal employs Gαi. Whether vertebrate Smo can selectively bias its signal between these routes is not yet known. N-linked glycosylation is a post-translational modification that can influence GPCR trafficking, ligand responsiveness and signal output. Smo proteins in Drosophila and vertebrate systems harbor N-linked glycans, but their role in Smo signaling has not been established. Herein, we present a comprehensive analysis of Drosophila and murine Smo glycosylation that supports a functional divergence in the contribution of N-linked glycans to signaling. Of the seven predicted glycan acceptor sites in Drosophila Smo, one is essential. Loss of N-glycosylation at this site disrupted Smo trafficking and attenuated its signaling capability. In stark contrast, we found that all four predicted N-glycosylation sites on murine Smo were dispensable for proper trafficking, agonist binding and canonical signal induction. However, the under-glycosylated protein was compromised in its ability to induce a non-canonical signal through Gαi, providing for the first time evidence that Smo can bias its signal and that a post-translational modification can impact this process. As such, we postulate a profound shift in N-glycan function from affecting Smo ER exit in flies to influencing its signal output in mice

Expression and Characterization of Drosophila Signal Peptide Peptidase-Like (sppL), a Gene That Encodes an Intramembrane Protease

Intramembrane proteases of the Signal Peptide Peptidase (SPP) family play important roles in developmental, metabolic and signaling pathways. Although vertebrates have one SPP and four SPP-like (SPPL) genes, we found that insect genomes encode one Spp and one SppL. Characterization of the Drosophila sppL gene revealed that the predicted SppL protein is a highly conserved structural homolog of the vertebrate SPPL3 proteases, with a predicted nine-transmembrane topology, an active site containing aspartyl residues within a transmembrane region, and a carboxy-terminal PAL domain. SppL protein localized to both the Golgi and ER. Whereas spp is an essential gene that is required during early larval stages and whereas spp loss-of-function reduced the unfolded protein response (UPR), sppL loss of function had no apparent phenotype. This was unexpected given that genetic knockdown phenotypes in other organisms suggested significant roles for Spp-related proteases

Quality of Life After Sentinel Lymph Node Biopsy or Axillary Lymph Node Dissection in Stage I/II Breast Cancer Patients: A Prospective Longitudinal Study

Background:\ud Breast cancer patients’ quality of life (QoL) after surgery has been reported to improve significantly over time. Little is known about QoL recovery after sentinel lymph node biopsy (SLNB) in comparison to axillary lymph node dissection (ALND).\ud \ud Methods:\ud 175 of 195 stage I/II breast cancer patients completed the EORTC QLQ-C30: one day before surgery (T0) and after 6 (T1), 26 (T2), 52 (T3) and 104 (T4) weeks. Of these, 54 patients underwent SLNB, 56 SLNB+ALND and 65 ALND. General linear models and paired T-tests between T0–T4 and T1–T4 were computed. Complications, radiotherapy and systemic therapy were added to the model.\ud \ud Results:\ud Significant time effects were found on physical, role and emotional functioning. Physical and role functioning decreased between T0 and T1. At T4, SLNB patients’ functioning had increased to their T0 level; ALND (+/– SLNB) patients’ functioning had increased, but had not improved to T0 level. Emotional functioning increased linearly between T0 and T4. At T4, emotional functioning was significantly higher in all groups as compared with T0. No significant group or interaction (time × group) effects were found. Complications and chemotherapy had a significant negative effect on role, emotional and cognitive functioning. Complications had a significant effect on social functioning also. Effect sizes varied between 0.00 and 0.06.\ud \ud Conclusion:\ud Two years post surgery, breast cancer patients’ QoL is comparable to that shortly before surgery. Women rated their emotional functioning as even better. SLNB is not associated with a better QoL than ALND. However, undergoing systemic therapy and/or experiencing complications affects QoL negatively

pp→pK+Λpp \to pK^{+}\Lambda reaction in an effective Lagrangian model

We investigate the $pp \to pK^{+}\Lambda$ reaction within an effective Lagrangian model where the contributions to the amplitudes are taken into account within the tree level. The initial interaction between the two nucleons is modeled by the exchange of $\pi$, $\rho$, $\omega$ and $\sigma$ mesons and the $\Lambda K^{+}$ production proceeds via the excitation of the $N^*$(1650), $N^*$(1710), $N^*$(1720) baryonic resonances. The parameters of the model at the nucleon-nucleon-meson vertices are determined by fitting the elastic nucleon-nucleon scattering with an effective interaction based on the exchange of these four mesons, while those at the resonance vertices are calculated from the known decay widths of the resonances as well as the vector meson dominance model. Available experimental data is described well by this approach. The one-pion-exchange diagram dominates the production process at both higher and lower beam energies. The $\rho$ and $\omega$ meson exchanges make negligible contributions. However, the $\sigma$-exchange processes contribute substantially to the total cross sections at lower beam energies. The excitation of the $N^*$(1710) and $N^*$(1650) resonances dominate this reaction at beam momenta above and below 3 GeV/c respectively. The interference among the amplitudes of various resonance excitation processes is significant. For beam energies very close to the $K^{+}$ production threshold the hyperon-proton final state interaction effects are quite important. The data is selective about the model used to describe the low energy scattering of the two final state baryons.Comment: Revised version, to appear in Phys. Rev.

Midline Signalling Systems Direct the Formation of a Neural Map by Dendritic Targeting in the Drosophila Motor System

During embryonic development of the motor system of Drosophila, motorneurons target their dendrites to different regions along the body axis in response to midline guidance cues

A Small Molecule Inhibitor of Signal Peptide Peptidase Inhibits Plasmodium Development in the Liver and Decreases Malaria Severity

The liver stage of Plasmodium's life cycle is the first, obligatory step in malaria infection. Decreasing the hepatic burden of Plasmodium infection decreases the severity of disease and constitutes a promising strategy for malaria prophylaxis. The efficacy of the gamma-secretase and signal peptide peptidase inhibitor LY411,575 in targeting Plasmodium liver stages was evaluated both in human hepatoma cell lines and in mouse primary hepatocytes. LY411,575 was found to prevent Plasmodium's normal development in the liver, with an IC50 of approximately 80 nM, without affecting hepatocyte invasion by the parasite. In vivo results with a rodent model of malaria showed that LY411,575 decreases the parasite load in the liver and increases by 55% the resistance of mice to cerebral malaria, one of the most severe malaria-associated syndromes. Our data show that LY411,575 does not exert its effect via the Notch signaling pathway suggesting that it may interfere with Plasmodium development through an inhibition of the parasite's signal peptide peptidase. We therefore propose that selective signal peptide peptidase inhibitors could be potentially used for preventive treatment of malaria in humans

Periodic actin structures in neuronal axons are required to maintain microtubules

Axons are the cable-like neuronal processes wiring the nervous system. They contain parallel bundles of microtubules as structural backbones, surrounded by regularly-spaced actin rings termed the periodic membrane skeleton (PMS). Despite being an evolutionarily-conserved, ubiquitous, highly-ordered feature of axons, the function of PMS is unknown. Here we studied PMS abundance, organisation and function, combining versatile Drosophila genetics with super-resolution microscopy and various functional readouts. Analyses with 11 different actin regulators and 3 actin-targeting drugs suggest PMS to contain short actin filaments which are depolymerisation resistant and sensitive to spectrin, adducin and nucleator deficiency - consistent with microscopy-derived models proposing PMS as specialised cortical actin. Upon actin removal we observed gaps in microtubule bundles, reduced microtubule polymerisation and reduced axon numbers suggesting a role of PMS in microtubule organisation. These effects become strongly enhanced when carried out in neurons lacking the microtubule-stabilising protein Short stop (Shot). Combining the aforementioned actin manipulations with Shot deficiency revealed a close correlation between PMS abundance and microtubule regulation, consistent with a model in which PMS-dependent microtubule polymerisation contributes to their maintenance in axons. We discuss potential implications of this novel PMS function along axon shafts for axon maintenance and regeneration

Seasonal pattern of regional carbon balance in the central Rocky Mountains from surface and airborne measurements

[1] High-elevation forests represent a large fraction of potential carbon uptake in North America, but this uptake is not well constrained by observations. Additionally, forests in the Rocky Mountains have recently been severely damaged by drought, fire, and insect outbreaks, which have been quantified at local scales but not assessed in terms of carbon uptake at regional scales. The Airborne Carbon in the Mountains Experiment was carried out in 2007 partly to assess carbon uptake in western U.S. mountain ecosystems. The magnitude and seasonal change of carbon uptake were quantified by (1) paired upwind-downwind airborne CO2 observations applied in a boundary layer budget, (2) a spatially explicit ecosystem model constrained using remote sensing and flux tower observations, and (3) a downscaled global tracer transport inversion. Top-down approaches had mean carbon uptake equivalent to flux tower observations at a subalpine forest, while the ecosystem model showed less. The techniques disagreed on temporal evolution. Regional carbon uptake was greatest in the early summer immediately following snowmelt and tended to lessen as the region experienced dry summer conditions. This reduction was more pronounced in the airborne budget and inversion than in flux tower or upscaling, possibly related to lower snow water availability in forests sampled by the aircraft, which were lower in elevation than the tower site. Changes in vegetative greenness associated with insect outbreaks were detected using satellite reflectance observations, but impacts on regional carbon cycling were unclear, highlighting the need to better quantify this emerging disturbance effect on montane forest carbon cycling

A Concerted Action of Engrailed and Gooseberry-Neuro in Neuroblast 6-4 Is Triggering the Formation of Embryonic Posterior Commissure Bundles

One challenging question in neurogenesis concerns the identification of cues that trigger axonal growth and pathfinding to form stereotypic neuronal networks during the construction of a nervous system. Here, we show that in Drosophila, Engrailed (EN) and Gooseberry-Neuro (GsbN) act together as cofactors to build the posterior commissures (PCs), which shapes the ventral nerve cord. Indeed, we show that these two proteins are acting together in axon growth and midline crossing, and that this concerted action occurs at early development, in neuroblasts. More precisely, we identified that their expressions in NB 6-4 are necessary and sufficient to trigger the formation of the PCs, demonstrating that segmentation genes such as EN and GsbN play a crucial role in the determination of NB 6-4 in a way that will later influence growth and guidance of all the axons that form the PCs. We also demonstrate a more specific function of GsbN in differentiated neurons, leading to fasciculations between axons, which might be required to obtain PC mature axon bundles