Factors Determining the Superior Performance of Lipid/DNA/Protammine Nanoparticles over Lipoplexes.

The utility of using a protammine/DNA complex coated with a lipid envelope made of cationic 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) for transfecting CHO (Chinese hamster ovary cells), HEK293 (human embryonic kidney cells), NIH 3T3 (mouse embryonal cells), and A17 (murine cancer cells) cells was examined. The widely used DOTAP/DNA lipoplex was employed as a reference. In all the tested cell lines lipid/protamine/DNA (LPD) nanoparticles were more e!cient in transfecting cells than lipoplexes even though the lipid composition of the lipid envelope was the same in both devices. Physical!chemical properties were found to control the ability of nanocarriers to release DNA upon interaction with cellular membranes. LPD complexes easily release their DNA payload, while lipoplexes remain largely intact and accumulate at the cell nucleus. Collectively, these data explain why LPD nanoparticles often exhibit superior performances compared to lipoplexes in trasfecting cells and represent a promising class of nanocarriers for gene delivery

Computational saturation mutagenesis to predict structural consequences of systematic mutations in the beta subunit of RNA polymerase in Mycobacterium leprae

ABSTRACT In contrast to the situation with tuberculosis, rifampin resistance in leprosy may remain undetected due to the lack of rapid and effective diagnostic methods. A quick and reliable method is essential to determine the impacts of emerging detrimental mutations. The functional consequences of missense mutations within the β-subunit of RNA polymerase in Mycobacterium leprae ( M. leprae ) contribute to phenotypic rifampin resistance outcomes in leprosy. Here we report in-silico saturation mutagenesis of all residues in the β-subunit of RNA polymerase to all other 19 amino acid types and predict their impacts on overall thermodynamic stability, on interactions at subunit interfaces, and on β-subunit-RNA and rifampin affinities using state-of-the-art structure, sequence and normal mode analysis-based methods. A total of 21,394 mutations were analysed, and it was noted that mutations in the conserved residues that line the active-site cleft show largely destabilizing effects, resulting in increased relative solvent accessibility and concomitant decrease in depth of the mutant residues. The mutations at residues S437, G459, H451, P489, K884 and H1035 are identified as extremely detrimental as they induce highly destabilizing effects on the overall stability, nucleic acid and rifampin affinities. Destabilizing effects were predicted for all the experimentally identified rifampin-resistant mutations in M. leprae indicating that this model can be used as a surveillance tool to monitor emerging detrimental mutations conferring rifampin resistance in leprosy. AUTHOR SUMMARY Emergence of primary and secondary drug resistance to rifampin in leprosy is a growing concern and poses threat to the leprosy control and elimination measures globally. In the absence of an effective in-vitro system to detect and monitor phenotypic rifampin resistance in leprosy, most of the diagnosis relies on detecting mutations in the drug resistance determining regions of the rpoB gene that encodes the β subunit of RNA polymerase in M. leprae . Few labs in the world perform mouse food pad propagation of M. leprae in the presence of drugs (rifampin) to determine growth patterns and confirm resistance, however the duration of these methods lasts from 8 to 12 months making them impractical for diagnosis. Understanding molecular mechanisms of drug resistance is vital to associating mutations to clinical resistance outcomes in leprosy. Here we propose an in-silico saturation mutagenesis approach to comprehensively elucidate the structural implications of any mutations that exist or can arise in the β subunit of RNA polymerase in M. leprae . Most of the predicted mutations may not occur in M. leprae due to fitness costs but the information thus generated by this approach help decipher the impacts of mutations across the structure and conversely enable identification of stable regions in the protein that are least impacted by mutations (mutation coolspots) which can be a choice for small molecule binding and structure guided drug discovery

Anatomy of protein disorder, flexibility and disease-related mutations.

Integration of protein structural information with human genetic variation and pathogenic mutations is essential to understand molecular mechanisms associated with the effects of polymorphisms on protein interactions and cellular processes. We investigate occurrences of non-synonymous SNPs in ordered and disordered protein regions by systematic mapping of common variants and disease-related SNPs onto these regions. We show that common variants accumulate in disordered regions; conversely pathogenic variants are significantly depleted in disordered regions. These different occurrences of pathogenic and common SNPs can be attributed to a negative selection on random mutations in structurally highly constrained regions. New approaches in the study of quantitative effects of pathogenic-related mutations should effectively account for all the possible contexts and relative functional constraints in which the sequence variation occurs.This research was supported by the Biotechnology and Biological Sciences Research Council (BB/H018409/1 to FF), the British Heart Foundation (FS/12/41/29724 to AF and FF) and the Leukaemia & Lymphoma Research (to FF). SSC is funded by a Leukaemia & Lymphoma Research Gordon Piller PhD Studentship

Observation of associated near-side and away-side long-range correlations in √sNN=5.02 TeV proton-lead collisions with the ATLAS detector

Two-particle correlations in relative azimuthal angle (Δϕ) and pseudorapidity (Δη) are measured in √sNN=5.02  TeV p+Pb collisions using the ATLAS detector at the LHC. The measurements are performed using approximately 1  μb-1 of data as a function of transverse momentum (pT) and the transverse energy (ΣETPb) summed over 3.1<η<4.9 in the direction of the Pb beam. The correlation function, constructed from charged particles, exhibits a long-range (2<|Δη|<5) “near-side” (Δϕ∼0) correlation that grows rapidly with increasing ΣETPb. A long-range “away-side” (Δϕ∼π) correlation, obtained by subtracting the expected contributions from recoiling dijets and other sources estimated using events with small ΣETPb, is found to match the near-side correlation in magnitude, shape (in Δη and Δϕ) and ΣETPb dependence. The resultant Δϕ correlation is approximately symmetric about π/2, and is consistent with a dominant cos⁡2Δϕ modulation for all ΣETPb ranges and particle pT

Jet energy measurement with the ATLAS detector in proton-proton collisions at root s=7 TeV

The jet energy scale and its systematic uncertainty are determined for jets measured with the ATLAS detector at the LHC in proton-proton collision data at a centre-of-mass energy of √s = 7TeV corresponding to an integrated luminosity of 38 pb-1. Jets are reconstructed with the anti-kt algorithm with distance parameters R=0. 4 or R=0. 6. Jet energy and angle corrections are determined from Monte Carlo simulations to calibrate jets with transverse momenta pT≥20 GeV and pseudorapidities {pipe}η{pipe}<4. 5. The jet energy systematic uncertainty is estimated using the single isolated hadron response measured in situ and in test-beams, exploiting the transverse momentum balance between central and forward jets in events with dijet topologies and studying systematic variations in Monte Carlo simulations. The jet energy uncertainty is less than 2. 5 % in the central calorimeter region ({pipe}η{pipe}<0. 8) for jets with 60≤pT<800 GeV, and is maximally 14 % for pT<30 GeV in the most forward region 3. 2≤{pipe}η{pipe}<4. 5. The jet energy is validated for jet transverse momenta up to 1 TeV to the level of a few percent using several in situ techniques by comparing a well-known reference such as the recoiling photon pT, the sum of the transverse momenta of tracks associated to the jet, or a system of low-pT jets recoiling against a high-pT jet. More sophisticated jet calibration schemes are presented based on calorimeter cell energy density weighting or hadronic properties of jets, aiming for an improved jet energy resolution and a reduced flavour dependence of the jet response. The systematic uncertainty of the jet energy determined from a combination of in situ techniques is consistent with the one derived from single hadron response measurements over a wide kinematic range. The nominal corrections and uncertainties are derived for isolated jets in an inclusive sample of high-pT jets. Special cases such as event topologies with close-by jets, or selections of samples with an enhanced content of jets originating from light quarks, heavy quarks or gluons are also discussed and the corresponding uncertainties are determined. © 2013 CERN for the benefit of the ATLAS collaboration

Measurement of the inclusive and dijet cross-sections of b-jets in pp collisions at sqrt(s) = 7 TeV with the ATLAS detector

The inclusive and dijet production cross-sections have been measured for jets containing b-hadrons (b-jets) in proton-proton collisions at a centre-of-mass energy of sqrt(s) = 7 TeV, using the ATLAS detector at the LHC. The measurements use data corresponding to an integrated luminosity of 34 pb^-1. The b-jets are identified using either a lifetime-based method, where secondary decay vertices of b-hadrons in jets are reconstructed using information from the tracking detectors, or a muon-based method where the presence of a muon is used to identify semileptonic decays of b-hadrons inside jets. The inclusive b-jet cross-section is measured as a function of transverse momentum in the range 20 < pT < 400 GeV and rapidity in the range |y| < 2.1. The bbbar-dijet cross-section is measured as a function of the dijet invariant mass in the range 110 < m_jj < 760 GeV, the azimuthal angle difference between the two jets and the angular variable chi in two dijet mass regions. The results are compared with next-to-leading-order QCD predictions. Good agreement is observed between the measured cross-sections and the predictions obtained using POWHEG + Pythia. MC@NLO + Herwig shows good agreement with the measured bbbar-dijet cross-section. However, it does not reproduce the measured inclusive cross-section well, particularly for central b-jets with large transverse momenta.Comment: 10 pages plus author list (21 pages total), 8 figures, 1 table, final version published in European Physical Journal

Search for the neutral Higgs bosons of the minimal supersymmetric standard model in pp collisions at root s=7 TeV with the ATLAS detector

A search for neutral Higgs bosons of the Minimal Supersymmetric Standard Model (MSSM) is reported. The analysis is based on a sample of proton-proton collisions at a centre-of-mass energy of 7TeV recorded with the ATLAS detector at the Large Hadron Collider. The data were recorded in 2011 and correspond to an integrated luminosity of 4.7 fb-1 to 4.8 fb-1. Higgs boson decays into oppositely-charged muon or τ lepton pairs are considered for final states requiring either the presence or absence of b-jets. No statistically significant excess over the expected background is observed and exclusion limits at the 95% confidence level are derived. The exclusion limits are for the production cross-section of a generic neutral Higgs boson, φ, as a function of the Higgs boson mass and for h/A/H production in the MSSM as a function of the parameters mA and tan β in the mhmax scenario for mA in the range of 90GeV to 500 GeV. Copyright CERN

In Silico and In Vitro Investigations of the Mutability of Disease-Causing Missense Mutation Sites in Spermine Synthase

Spermine synthase (SMS) is a key enzyme controlling the concentration of spermidine and spermine in the cell. The importance of SMS is manifested by the fact that single missense mutations were found to cause Snyder-Robinson Syndrome (SRS). At the same time, currently there are no non-synonymous single nucleoside polymorphisms, nsSNPs (harmless mutations), found in SMS, which may imply that the SMS does not tolerate amino acid substitutions, i.e. is not mutable.To investigate the mutability of the SMS, we carried out in silico analysis and in vitro experiments of the effects of amino acid substitutions at the missense mutation sites (G56, V132 and I150) that have been shown to cause SRS. Our investigation showed that the mutation sites have different degree of mutability depending on their structural micro-environment and involvement in the function and structural integrity of the SMS. It was found that the I150 site does not tolerate any mutation, while V132, despite its key position at the interface of SMS dimer, is quite mutable. The G56 site is in the middle of the spectra, but still quite sensitive to charge residue replacement.The performed analysis showed that mutability depends on the detail of the structural and functional factors and cannot be predicted based on conservation of wild type properties alone. Also, harmless nsSNPs can be expected to occur even at sites at which missense mutations were found to cause diseases