The formation of acetylcholine receptor clusters visualized with quantum dots

Background: Motor innervation of skeletal muscle leads to the assembly of acetylcholine receptor (AChR) clusters in the postsynaptic membrane at the vertebrate neuromuscular junction (NMJ). Synaptic AChR aggregation, according to the diffusion-mediated trapping hypothesis, involves the establishment of a postsynaptic scaffold that "traps" freely diffusing receptors into forming high-density clusters. Although this hypothesis is widely cited to explain the formation of postsynaptic AChR clusters, direct evidence at molecular level is lacking. Results: Using quantum dots (QDs) and live cell imaging, we provide new measurements supporting the diffusion-trap hypothesis as applied to AChR cluster formation. Consistent with published works, experiments on cultured Xenopus myotomal muscle cells revealed that AChRs at clusters that formed spontaneously (pre-patterned clusters, also called hot spots) and at those induced by nerve-innervation or by growth factor-coated latex beads were very stable whereas diffuse receptors outside these regions were mobile. Moreover, despite the restriction of AChR movement at sites of synaptogenic stimulation, individual receptors away from these domains continued to exhibit free diffusion, indicating that AChR clustering at NMJ does not involve an active attraction of receptors but is passive and diffusion-driven. Conclusion: Single-molecular tracking using QDs has provided direct evidence that the clustering of AChRs in muscle cells in response to synaptogenic stimuli is achieved by two distinct cellular processes: the Brownian motion of receptors in the membrane and their trapping and immobilization at the synaptic specialization. This study also provides a clearer picture of the "trap" that it is not a uniformly sticky area but consists of discrete foci at which AChRs are immobilized

Z-Selectivity in Olefin Metathesis with Chelated Ru Catalysts: Computational Studies of Mechanism and Selectivity

The mechanism and origins of Z-selectivity in olefin metathesis with chelated Ru catalysts were explored using density functional theory. The olefin approaches from the “side” position of the chelated Ru catalysts, in contrast to reactions with previous unchelated Ru catalysts that favor the bottom-bound pathway. Steric repulsions between the substituents on the olefin and the N-substituent on the N-heterocyclic carbene ligand lead to highly selective formation of the Z product

Crosslinking-induced endocytosis of acetylcholine receptors by quantum dots

In a majority of patients with myasthenia gravis (MG), anti-acetylcholine receptor (AChR) antibodies target postsynaptic AChR clusters and thus compromise the membrane integrity of neuromuscular junctions (NMJs) and lead to muscle weakness. Antibody-induced endocytosis of AChRs in the postsynaptic membrane represents the initial step in the pathogenesis of MG; however, the molecular mechanisms underlying AChR endocytosis remain largely unknown. Here, we developed an approach to mimic the pathogenic antibodies for inducing the crosslinking and internalization of AChRs from the postsynaptic membrane. Using biotin-α-bungarotoxin and quantum dot (QD)-streptavidin, cell-surface and internalized AChRs could be readily distinguished by comparing the size, fluorescence intensity, trajectory, and subcellular localization of the QD signals. QD-induced AChR endocytosis was mediated by clathrin-dependent and caveolin-independent mechanisms, and the trafficking of internalized AChRs in the early endosomes required the integrity of microtubule structures. Furthermore, activation of the agrin/MuSK (muscle-specific kinase) signaling pathway strongly suppressed QD-induced internalization of AChRs. Lastly, QD-induced AChR crosslinking potentiated the dispersal of aneural AChR clusters upon synaptic induction. Taken together, our results identify a novel approach to study the mechanisms of AChR trafficking upon receptor crosslinking and endocytosis, and demonstrate that agrin-MuSK signaling pathways protect against crosslinking-induced endocytosis of AChRs. © 2014 Lee et al.published_or_final_versio

The function of Shp2 tyrosine phosphatase in the dispersal of acetylcholine receptor clusters

<p>Abstract</p> <p>Background</p> <p>A crucial event in the development of the vertebrate neuromuscular junction (NMJ) is the postsynaptic enrichment of muscle acetylcholine (ACh) receptors (AChRs). This process involves two distinct steps: the local clustering of AChRs at synapses, which depends on the activation of the muscle-specific receptor tyrosine kinase MuSK by neural agrin, and the global dispersal of aneural or "pre-patterned" AChR aggregates, which is triggered by ACh or by synaptogenic stimuli. We and others have previously shown that tyrosine phosphatases, such as the SH2 domain-containing phosphatase Shp2, regulate AChR cluster formation in muscle cells, and that tyrosine phosphatases also mediate the dispersal of pre-patterned AChR clusters by synaptogenic stimuli, although the specific phosphatases involved in this latter step remain unknown.</p> <p>Results</p> <p>Using an assay system that allows AChR cluster assembly and disassembly to be studied separately and quantitatively, we describe a previously unrecognized role of the tyrosine phosphatase Shp2 in AChR cluster disassembly. Shp2 was robustly expressed in embryonic Xenopus muscle in vivo and in cultured myotomal muscle cells, and treatment of the muscle cultures with an inhibitor of Shp2 (NSC-87877) blocked the dispersal of pre-patterned AChR clusters by synaptogenic stimuli. In contrast, over-expression in muscle cells of either wild-type or constitutively active Shp2 accelerated cluster dispersal. Significantly, forced expression in muscle of the Shp2-activator SIRPα1 (signal regulatory protein α1) also enhanced the disassembly of AChR clusters, whereas the expression of a truncated SIRPα1 mutant that suppresses Shp2 signaling inhibited cluster disassembly.</p> <p>Conclusion</p> <p>Our results suggest that Shp2 activation by synaptogenic stimuli, through signaling intermediates such as SIRPα1, promotes the dispersal of pre-patterned AChR clusters to facilitate the selective accumulation of AChRs at developing NMJs.</p

The Transmission Dynamics of Tuberculosis in a Recently Developed Chinese City

BACKGROUND: Hong Kong is an affluent subtropical city with a well-developed healthcare infrastructure but an intermediate TB burden. Declines in notification rates through the 1960s and 1970s have slowed since the 1980s to the current level of around 82 cases per 100 000 population. We studied the transmission dynamics of TB in Hong Kong to explore the factors underlying recent trends in incidence. METHODOLOGY/PRINCIPAL FINDINGS: We fitted an age-structured compartmental model to TB notifications in Hong Kong between 1968 and 2008. We used the model to quantify the proportion of annual cases due to recent transmission versus endogenous reactivation of latent infection, and to project trends in incidence rates to 2018. The proportion of annual TB notifications attributed to endogenous reactivation increased from 46% to 70% between 1968 and 2008. Age-standardized notification rates were projected to decline to approximately 56 per 100 000 in 2018. CONCLUSIONS/SIGNIFICANCE: Continued intermediate incidence of TB in Hong Kong is driven primarily by endogenous reactivation of latent infections. Public health interventions which focus on reducing transmission may not lead to substantial reductions in disease burden associated with endogenous reactivation of latent infections in the short- to medium-term. While reductions in transmission with socio-economic development and public health interventions will lead to declines in TB incidence in these regions, a high prevalence of latent infections may hinder substantial declines in burden in the longer term. These findings may therefore have important implications for the burden of TB in developing regions with higher levels of transmission currently.published_or_final_versio

New Insights into Traffic Dynamics: A Weighted Probabilistic Cellular Automaton Model

From the macroscopic viewpoint for describing the acceleration behavior of drivers, this letter presents a weighted probabilistic cellular automaton model (the WP model, for short) by introducing a kind of random acceleration probabilistic distribution function. The fundamental diagrams, the spatio-temporal pattern are analyzed in detail. It is shown that the presented model leads to the results consistent with the empirical data rather well, nonlinear velocity-density relationship exists in lower density region, and a new kind of traffic phenomenon called neo-synchronized flow is resulted. Furthermore, we give the criterion for distinguishing the high-speed and low-speed neo-synchronized flows and clarify the mechanism of this kind of traffic phenomena. In addition, the result that the time evolution of distribution of headways is displayed as a normal distribution further validates the reasonability of the neo-synchronized flow. These findings suggest that the diversity and randomicity of drivers and vehicles has indeed remarkable effect on traffic dynamics.Comment: 12 pages, 5 figures, submitted to Europhysics Letter

Cutaneous Conditions Leading to Dermatology Consultations in the Emergency Department

<p>Introduction: We established the most common cutaneous diseases that received dermatology consultation in the adult emergency department (ED) and identified differentiating clinical characteristics of dermatoses that required hospital admission.</p> <p>Methods: A retrospective chart review of 204 patients presenting to the ED who received dermatology consultations at Los Angeles County/University of Southern California Medical Center, an urban tertiary care teaching hospital.</p> <p>Results: Of all patients, 18% were admitted to an inpatient unit primarily for their cutaneous disease, whereas 82% were not. Of nonadmitted patients, the most commonly diagnosed conditions were eczematous dermatitis not otherwise specified (8.9%), scabies (7.2%), contact dermatitis (6.6%), cutaneous drug eruption (6.0%), psoriasis vulgaris (4.2%), and basal cell carcinoma (3.6%). Of patients admitted for their dermatoses, the most highly prevalent conditions were erythema multiforme major/Stevens-Johnson syndrome (22%), pemphigus vulgaris (14%), and severe cutaneous drug eruption (11%). When compared with those of nonadmitted patients, admitted skin conditions were more likely to be generalized (92% vs 72%; P = 0.0104), acute in onset (< 1 month duration) (81% vs 51%; P = 0.0005), painful (41% vs 15%; P = 0.0009), blistering (41% vs 7.8%; P < 0.0001), and ulcerated or eroded (46% vs 7.8%; P < 0.0001). They were more likely to involve the mucosa (54% vs 7.2%; P < 0.0001) and less likely to be pruritic (35% vs 58%; P = 0.0169).</p> <p>Conclusion: We have described a cohort of patients receiving dermatologic consultation in the ED of a large urban teaching hospital. These data identify high-risk features of more severe skin disease and may be used to refine curricula in both emergency and nonemergency cutaneous disorders for emergency physicians. [West J Emerg Med. 2011;12(4):551–555.]</p

Differential effects of neurotrophins and schwann cell-derived signals on neuronal survival/growth and synaptogenesis

Recent studies have shown that the survival of mammalian motoneurons in vitro is promoted by neurotrophins (NTs) and cAMP. There is also evidence that neurotrophins enhance transmitter release.Wethus investigated whether these agents also promote synaptogenesis. Cultured Xenopus spinal cord neurons were treated with a mixture of BDNF, glia-derived neurotrophic factor, NT-3, and NT-4, in addition to forskolin and IBMX or the cell-permeant form of cAMP, to elevate the cAMP level. The outgrowth and survival of neurons were dramatically increased by this trophic stimulation. However, when these neurons were cocultured with muscle cells, the trophic agents resulted in a failure of synaptogenesis. Specifically, the induction ofAChreceptor (AChR) clustering in cultured muscle cells was inhibited at nerve–muscle contacts, in sharp contrast to control, untreated cocultures. Because AChR clustering induced by agrin or growth factor-coated beads in muscle cells was unaffected by trophic stimulation, its effect on synaptogenesis is presynaptic in origin. In the control, agrin was deposited along the neurite and at nerve–muscle contacts. This was significantly downregulated in cultures treated with trophic stimuli. Reverse transcriptase-PCR analyses showed that this decrease in agrin deposition was caused by an inhibition of agrin synthesis by trophic stimuli. Both agrin synthesis and induction of AChR clustering were restored under trophic stimulation when Schwann cell-conditioned medium was introduced. These results suggest that trophic stimulation maintains spinal neurons in the growth state, and Schwann cell-derived factors allow them to switch to the synaptogenic state

Z-Selective Ethenolysis with a Ruthenium Metathesis Catalyst: Experiment and Theory

The Z-selective ethenolysis activity of chelated ruthenium metathesis catalysts was investigated with experiment and theory. A five-membered chelated catalyst that was successfully employed in Z-selective cross metathesis reactions has now been found to be highly active for Z-selective ethenolysis at low ethylene pressures, while tolerating a wide variety of functional groups. This phenomenon also affects its activity in cross metathesis reactions and prohibits crossover reactions of internal olefins via trisubstituted ruthenacyclobutane intermediates. In contrast, a related catalyst containing a six-membered chelated architecture is not active for ethenolysis and seems to react through different pathways more reminiscent of previous generations of ruthenium catalysts. Computational investigations of the effects of substitution on relevant transition states and ruthenacyclobutane intermediates revealed that the differences of activities are attributed to the steric repulsions of the anionic ligand with the chelating groups