Role of clathrin in dense core vesicle biogenesis

The dense-core vesicles (DCVs) of neuroendocrine cells are a rich source of bioactive molecules such as peptides, hormones, and neurotransmitters, but relatively little is known about how they are formed. Using fractionation profiling, a method that combines subcellular fractionation with mass spectrometry, we identified ∼1200 proteins in PC12 cell vesicle-enriched fractions, with DCV-associated proteins showing distinct profiles from proteins associated with other types of vesicles. To investigate the role of clathrin in DCV biogenesis, we stably transduced PC12 cells with an inducible shRNA targeting clathrin heavy chain, resulting in ∼85% protein loss. DCVs could still be observed in the cells by electron microscopy, but mature profiles were ∼4-fold less abundant than in mock-treated cells. By quantitative mass spectrometry, DCV-associated proteins were found to be reduced ∼2-fold in clathrin-depleted cells as a whole and ∼5-fold in vesicle-enriched fractions. Our combined datasets enabled us to identify new candidate DCV components. Secretion assays revealed that clathrin depletion causes a near-complete block in secretagogue-induced exocytosis. Taken together, our data indicate that clathrin has a function in DCV biogenesis beyond its established role in removing unwanted proteins from the immature vesicle.This work was funded by grants from the Wellcome Trust: 086598 (to M.S.R.), 100140 (Wellcome Trust Strategic Award), and 093026 (for the FEI Tecnai G2 Spirit BioTWIN transmission EM); and by a National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases grant (R01DK102496) to A.B

Requirements modelling and formal analysis using graph operations

The increasing complexity of enterprise systems requires a more advanced analysis of the representation of services expected than is currently possible. Consequently, the specification stage, which could be facilitated by formal verification, becomes very important to the system life-cycle. This paper presents a formal modelling approach, which may be used in order to better represent the reality of the system and to verify the awaited or existing system’s properties, taking into account the environmental characteristics. For that, we firstly propose a formalization process based upon properties specification, and secondly we use Conceptual Graphs operations to develop reasoning mechanisms of verifying requirements statements. The graphic visualization of these reasoning enables us to correctly capture the system specifications by making it easier to determine if desired properties hold. It is applied to the field of Enterprise modelling

Effects of dietary phytoestrogens on plasma testosterone and triiodothyronine (T3) levels in male goat kids

<p>Abstract</p> <p>Background</p> <p>Exposure to xenoestrogens in humans and animals has gained increasing attention due to the effects of these compounds on reproduction. The present study was undertaken to investigate the influence of low-dose dietary phytoestrogen exposure, i.e. a mixture of genistein, daidzein, biochanin A and formononetin, on the establishment of testosterone production during puberty in male goat kids.</p> <p>Methods</p> <p>Goat kids at the age of 3 months received either a standard diet or a diet supplemented with phytoestrogens (3 - 4 mg/kg/day) for ~3 months. Plasma testosterone and total and free triiodothyronine (T₃) concentrations were determined weekly. Testicular levels of testosterone and cAMP were measured at the end of the experiment. Repeated measurement analysis of variance using the MIXED procedure on the generated averages, according to the Statistical Analysis System program package (Release 6.12, 1996, SAS Institute Inc., Cary, NC, USA) was carried out.</p> <p>Results</p> <p>No significant difference in plasma testosterone concentration between the groups was detected during the first 7 weeks. However, at the age of 5 months (i.e. October 1, week 8) phytoestrogen-treated animals showed significantly higher testosterone concentrations than control animals (37.5 nmol/l vs 19.1 nmol/l). This elevation was preceded by a rise in plasma total T₃that occurred on September 17 (week 6). A slightly higher concentration of free T₃was detected in the phytoestrogen group at the same time point, but it was not until October 8 and 15 (week 9 and 10) that a significant difference was found between the groups. At the termination of the experiment, testicular cAMP levels were significantly lower in goats fed a phytoestrogen-supplemented diet. Phytoestrogen-fed animals also had lower plasma and testicular testosterone concentrations, but these differences were not statistically significant.</p> <p>Conclusion</p> <p>Our findings suggest that phytoestrogens can stimulate testosterone synthesis during puberty in male goats by increasing the secretion of T₃; a hormone known to stimulate Leydig cell steroidogenesis. It is possible that feedback signalling underlies the tendency towards decreased steroid production at the end of the experiment.</p

Conservation and divergence within the clathrin interactome of <i>Trypanosoma cruzi</i>

Trypanosomatids are parasitic protozoa with a significant burden on human health. African and American trypanosomes are causative agents of Nagana and Chagas disease respectively, and speciated about 300 million years ago. These parasites have highly distinct life cycles, pathologies, transmission strategies and surface proteomes, being dominated by the variant surface glycoprotein (African) or mucins (American) respectively. In African trypanosomes clathrin-mediated trafficking is responsible for endocytosis and post-Golgi transport, with several mechanistic aspects distinct from higher organisms. Using clathrin light chain (TcCLC) and EpsinR (TcEpsinR) as affinity handles, we identified candidate clathrin-associated proteins (CAPs) in Trypanosoma cruzi; the cohort includes orthologs of many proteins known to mediate vesicle trafficking, but significantly not the AP-2 adaptor complex. Several trypanosome-specific proteins common with African trypanosomes, were also identified. Fluorescence microscopy revealed localisations for TcEpsinR, TcCLC and TcCHC at the posterior region of trypomastigote cells, coincident with the flagellar pocket and Golgi apparatus. These data provide the first systematic analysis of clathrin-mediated trafficking in T. cruzi, allowing comparison between protein cohorts and other trypanosomes and also suggest that clathrin trafficking in at least some life stages of T. cruzi may be AP-2-independent

Properties of Graphene: A Theoretical Perspective

In this review, we provide an in-depth description of the physics of monolayer and bilayer graphene from a theorist's perspective. We discuss the physical properties of graphene in an external magnetic field, reflecting the chiral nature of the quasiparticles near the Dirac point with a Landau level at zero energy. We address the unique integer quantum Hall effects, the role of electron correlations, and the recent observation of the fractional quantum Hall effect in the monolayer graphene. The quantum Hall effect in bilayer graphene is fundamentally different from that of a monolayer, reflecting the unique band structure of this system. The theory of transport in the absence of an external magnetic field is discussed in detail, along with the role of disorder studied in various theoretical models. We highlight the differences and similarities between monolayer and bilayer graphene, and focus on thermodynamic properties such as the compressibility, the plasmon spectra, the weak localization correction, quantum Hall effect, and optical properties. Confinement of electrons in graphene is nontrivial due to Klein tunneling. We review various theoretical and experimental studies of quantum confined structures made from graphene. The band structure of graphene nanoribbons and the role of the sublattice symmetry, edge geometry and the size of the nanoribbon on the electronic and magnetic properties are very active areas of research, and a detailed review of these topics is presented. Also, the effects of substrate interactions, adsorbed atoms, lattice defects and doping on the band structure of finite-sized graphene systems are discussed. We also include a brief description of graphane -- gapped material obtained from graphene by attaching hydrogen atoms to each carbon atom in the lattice.Comment: 189 pages. submitted in Advances in Physic

Anti-diabetic effect of a preparation of vitamins, minerals and trace elements in diabetic rats: a gender difference

BACKGROUND: Although multivitamin products are widely used as dietary supplements to maintain health or as special medical food in certain diseases, the effects of these products were not investigated in diabetes mellitus, a major cardiovascular risk factor. Therefore, here we investigated if a preparation of different minerals, vitamins, and trace elements (MVT) for human use affects the severity of experimental diabetes. METHODS: Two days old neonatal Wistar rats from both genders were injected with 100 mg/kg of streptozotocin or its vehicle to induce diabetes. At week 4, rats were fed with an MVT preparation or vehicle for 8 weeks. Well established diagnostic parameters of diabetes, i.e. fasting blood glucose and oral glucose tolerance test were performed at week 4, 8 and 12. Moreover, serum insulin and blood HbA1c were measured at week 12. RESULTS: An impaired glucose tolerance has been found in streptozotocin-treated rats in both genders at week 4. In males, fasting blood glucose and HbA1c were significantly increased and glucose tolerance and serum insulin was decreased at week 12 in the vehicle-treated diabetic group as compared to the vehicle-treated non-diabetic group. All of the diagnostic parameters of diabetes were significantly improved by MVT treatment in male rats. In females, streptozotocin treatment resulted in a less severe prediabetic-like phenotype as only glucose tolerance and HbA1c were altered by the end of the study in the vehicle-treated diabetic group as compared to the vehicle-treated non-diabetic group. MVT treatment failed to improve the diagnostic parameters of diabetes in female streptozotocin-treated rats. CONCLUSION: This is the first demonstration that MVT significantly attenuates the progression of diabetes in male rats with chronic experimental diabetes. Moreover, we have confirmed that females are less sensitive to STZ-induced diabetes and MVT preparation did not show protection against prediabetic state. This may suggest a gender difference in the pathogenesis of diabetes

Performance of CMS muon reconstruction in pp collision events at sqrt(s) = 7 TeV

The performance of muon reconstruction, identification, and triggering in CMS has been studied using 40 inverse picobarns of data collected in pp collisions at sqrt(s) = 7 TeV at the LHC in 2010. A few benchmark sets of selection criteria covering a wide range of physics analysis needs have been examined. For all considered selections, the efficiency to reconstruct and identify a muon with a transverse momentum pT larger than a few GeV is above 95% over the whole region of pseudorapidity covered by the CMS muon system, abs(eta) < 2.4, while the probability to misidentify a hadron as a muon is well below 1%. The efficiency to trigger on single muons with pT above a few GeV is higher than 90% over the full eta range, and typically substantially better. The overall momentum scale is measured to a precision of 0.2% with muons from Z decays. The transverse momentum resolution varies from 1% to 6% depending on pseudorapidity for muons with pT below 100 GeV and, using cosmic rays, it is shown to be better than 10% in the central region up to pT = 1 TeV. Observed distributions of all quantities are well reproduced by the Monte Carlo simulation.Comment: Replaced with published version. Added journal reference and DO

Performance of CMS muon reconstruction in pp collision events at sqrt(s) = 7 TeV

The performance of muon reconstruction, identification, and triggering in CMS has been studied using 40 inverse picobarns of data collected in pp collisions at sqrt(s) = 7 TeV at the LHC in 2010. A few benchmark sets of selection criteria covering a wide range of physics analysis needs have been examined. For all considered selections, the efficiency to reconstruct and identify a muon with a transverse momentum pT larger than a few GeV is above 95% over the whole region of pseudorapidity covered by the CMS muon system, abs(eta) < 2.4, while the probability to misidentify a hadron as a muon is well below 1%. The efficiency to trigger on single muons with pT above a few GeV is higher than 90% over the full eta range, and typically substantially better. The overall momentum scale is measured to a precision of 0.2% with muons from Z decays. The transverse momentum resolution varies from 1% to 6% depending on pseudorapidity for muons with pT below 100 GeV and, using cosmic rays, it is shown to be better than 10% in the central region up to pT = 1 TeV. Observed distributions of all quantities are well reproduced by the Monte Carlo simulation.Comment: Replaced with published version. Added journal reference and DO

Performance of the CMS Cathode Strip Chambers with Cosmic Rays

The Cathode Strip Chambers (CSCs) constitute the primary muon tracking device in the CMS endcaps. Their performance has been evaluated using data taken during a cosmic ray run in fall 2008. Measured noise levels are low, with the number of noisy channels well below 1%. Coordinate resolution was measured for all types of chambers, and fall in the range 47 microns to 243 microns. The efficiencies for local charged track triggers, for hit and for segments reconstruction were measured, and are above 99%. The timing resolution per layer is approximately 5 ns