Avoiding cytotoxicity of transposases by dose-controlled mRNA delivery

The Sleeping Beauty (SB) transposase and its newly developed hyperactive variant, SB100X, are of increasing interest for genome modification in experimental models and gene therapy. The potential cytotoxicity of transposases requires careful assessment, considering that residual integration events of transposase expression vectors delivered by physicochemical transfection or episomal retroviral vectors may lead to permanent transposase expression and resulting uncontrollable transposition. Comparing retrovirus-based approaches for delivery of mRNA, episomal DNA or integrating DNA, we found that conventional SB transposase, SB100X and a newly developed codon-optimized SB100Xo may trigger premitotic arrest and apoptosis. Cell stress induced by continued SB overexpression was self-limiting due to the induction of cell death, which occurred even in the absence of a co-transfected transposable element. The cytotoxic effects of SB transposase were strictly dose dependent and heralded by induction of p53 and c-Jun. Inactivating mutations in SB’s catalytic domain could not abrogate cytotoxicity, suggesting a mechanism independent of DNA cleavage activity. An improved approach of retrovirus particle-mediated mRNA transfer allowed transient and dose-controlled expression of SB100X, supported efficient transposition and prevented cytotoxicity. Transposase-mediated gene transfer can thus be tuned to maintain high efficiency in the absence of overt cell damage

High-molecular-weight, water-soluble polyammonium compounds

High-mol. wt., water-sol. compds., useful for nonsticky coatings on elec. conductive paper are manufd. by polymn. of 0.01-5 mol.% Z(CO2CH2CR:CR'R2)2 (Z = (CH2)n, (CH:CH)m, O(CH2CH2)p O, arylene, cycloalkylene, n = 0-20; m = 1-4, p = 0-5, R, R1, R2 = H, C1-6 alkyl, C5-6 cycloalkyl), e.g. diallyl maleinate, and a dialkenyldialkylammonium compd., e.g. diallyldimethylammonium chloride, in H2O in an inert atm. at 50-100 Deg. [on SciFinder (R)

Protein transduction from retroviral Gag precursors

Retroviral particles assemble a few thousand units of the Gag polyproteins. Proteolytic cleavage mediated by the retroviral protease forms the bioactive retroviral protein subunits before cell entry. We hypothesized that this process could be exploited for targeted, transient, and dose-controlled transduction of nonretroviral proteins into cultured cells. We demonstrate that gammaretroviral particles tolerate the incorporation of foreign protein at several positions of their Gag or Gag-Pol precursors. Receptor-mediated and thus potentially cell-specific uptake of engineered particles occurred within minutes after cell contact. Dose and kinetics of nonretroviral protein delivery were dependent upon the location within the polyprotein precursor. Proteins containing nuclear localization signals were incorporated into retroviral particles, and the proteins of interest were released from the precursor by the retroviral protease, recognizing engineered target sites. In contrast to integration-defective lentiviral vectors, protein transduction by retroviral polyprotein precursors was completely transient, as protein transducing retrovirus-like particles could be produced that did not transduce genes into target cells. Alternatively, bifunctional protein-delivering particle preparations were generated that maintained their ability to serve as vectors for retroviral transgenes. We show the potential of this approach for targeted genome engineering of induced pluripotent stem cells by delivering the site-specific DNA recombinase, Flp. Protein transduction of Flp after proteolytic release from the matrix position of Gag allowed excision of a lentivirally transduced cassette that concomitantly expresses the canonical reprogramming transcription factors (Oct4, Klf4, Sox2, c-Myc) and a fluorescent marker gene, thus generating induced pluripotent stem cells that are free of lentivirally transduced reprogramming genes

Higher-order correlations between different moments of two flow amplitudes in Pb−-Pb collisions at sNN=5.02\sqrt{s_{\rm NN}}=5.02 TeV

International audienceThe correlations between different moments of two flow amplitudes, extracted with the recently developed asymmetric cumulants, are measured in Pb$-$Pb collisions at $\sqrt{s_{\rm NN}} = 5.02$ TeV recorded by the ALICE detector at the LHC. The magnitudes of the measured observables show a dependence on the different moments as well as on the collision centrality, indicating the presence of non-linear response in all even moments up to the eighth. Furthermore, the higher-order asymmetric cumulants show different signatures than the symmetric and lower-order asymmetric cumulants. Comparisons with state-of-the-art event generators using two different parameterizations obtained from Bayesian optimization show differences between data and simulations in many of the studied observables, indicating a need for further tuning of the models behind those event generators. These results provide new and independent constraints on the initial conditions and transport properties of the system created in heavy-ion collisions

First measurement of the ∣t∣|t|-dependence of incoherent J/ψ\psi photonuclear production

International audienceThe first measurement of the cross section for incoherent photonuclear production of J/$\psi$ vector meson as a function of the Mandelstam $|t|$ variable is presented. The measurement was carried out with the ALICE detector at midrapidity, $|y|<0.8$, using ultra-peripheral collisions of Pb nuclei at a centre-of-mass energy per nucleon pair $\sqrt{s_{\mathrm{NN}}} = 5.02$ TeV. This rapidity interval corresponds to a Bjorken-$x$ range $(0.3$$-$$1.4)\times 10^{-3}$. Cross sections are reported in five $|t|$ intervals in the range $0.04<|t|<1$~GeV$^2$ and compared to the predictions of different models. Models that ignore quantum fluctuations of the gluon density in the colliding hadron predict a $|t|$-dependence of the cross section much steeper than in data. The inclusion of such fluctuations in the same models provides a better description of the data

Data-driven precision determination of the material budget in ALICE

The knowledge of the material budget with a high precision is fundamental for measurements of direct photon production using the photon conversion method due to its direct impact on the total systematic uncertainty. Moreover, it influences many aspects of the charged-particle reconstruction performance. In this article, two procedures to determine data-driven corrections to the material-budget description in ALICE simulation software are developed. One is based on the precise knowledge of the gas composition in the Time Projection Chamber. The other is based on the robustness of the ratio between the produced number of photons and charged particles, to a large extent due to the approximate isospin symmetry in the number of produced neutral and charged pions. Both methods are applied to ALICE data allowing for a reduction of the overall material budget systematic uncertainty from 4.5% down to 2.5%. Using these methods, a locally correct material budget is also achieved. The two proposed methods are generic and can be applied to any experiment in a similar fashion.The knowledge of the material budget with a high precision is fundamental for measurements of direct photon production using the photon conversion method due to its direct impact on the total systematic uncertainty. Moreover, it influences many aspects of the charged-particle reconstruction performance. In this article, two procedures to determine data-driven corrections to the material-budget description in ALICE simulation software are developed. One is based on the precise knowledge of the gas composition in the Time Projection Chamber. The other is based on the robustness of the ratio between the produced number of photons and charged particles, to a large extent due to the approximate isospin symmetry in the number of produced neutral and charged pions. Both methods are applied to ALICE data allowing for a reduction of the overall material budget systematic uncertainty from 4.5% down to 2.5%. Using these methods, a locally correct material budget is also achieved. The two proposed methods are generic and can be applied to any experiment in a similar fashion

Measurement of Non-prompt D0\rm D^0-meson Elliptic Flow in Pb-Pb Collisions at sNN\sqrt{s_{\rm NN}} = 5.02 TeV

International audienceThe elliptic flow ($v_2$) of $\rm D^0$ mesons from beauty-hadron decays (non-prompt $\rm D^0$) was measured in midcentral (30-50%) Pb-Pb collisions at a centre-of-mass energy per nucleon pair $\sqrt{s_{\rm NN}}$ = 5.02 TeV with the ALICE detector at the LHC. The $\rm D^0$ mesons were reconstructed at midrapidity ($|y|<0.8$) from their hadronic decay $\mathrm{D^0 \to K^-\pi^+}$, in the transverse momentum interval $2 < p_{\rm T} < 12$ GeV/$c$. The result indicates a positive $v_2$ for non-prompt $\rm D^0$ mesons with a significance of 2.7$\sigma$. The non-prompt $\rm D^0$-meson $v_2$ is lower than that of prompt non-strange D mesons with 3.2$\sigma$ significance in $2 < p_{\rm T} < 8$ GeV/$c$, and compatible with the $v_2$ of beauty-decay electrons. Theoretical calculations of beauty-quark transport in a hydrodynamically expanding medium describe the measurement within uncertainties

Skewness and kurtosis of mean transverse momentum fluctuations at the LHC energies

International audienceThe first measurements of skewness and kurtosis of mean transverse momentum ($\langle p_\mathrm{T}\rangle$) fluctuations are reported in Pb$-$Pb collisions at $\sqrt{s_\mathrm{NN}}$ = 5.02 TeV, Xe$-$Xe collisions at $\sqrt{s_\mathrm{NN}}$$=$ 5.44 TeV and pp collisions at $\sqrt{s} = 5.02$ TeV using the ALICE detector. The measurements are carried out as a function of system size $\langle \mathrm{d}N_\mathrm{ch}/\mathrm{d}\eta\rangle_{|\eta|<0.5}^{1/3}$, using charged particles with transverse momentum ($p_\mathrm{T}$) and pseudorapidity ($\eta$), in the range $0.2 < p_\mathrm{T} < 3.0$ GeV/$c$ and $|\eta| < 0.8$, respectively. In Pb$-$Pb and Xe$-$Xe collisions, positive skewness is observed in the fluctuations of $\langle p_\mathrm{T}\rangle$ for all centralities, which is significantly larger than what would be expected in the scenario of independent particle emission. This positive skewness is considered a crucial consequence of the hydrodynamic evolution of the hot and dense nuclear matter created in heavy-ion collisions. Furthermore, similar observations of positive skewness for minimum bias pp collisions are also reported here. Kurtosis of $\langle p_\mathrm{T}\rangle$ fluctuations is found to be in good agreement with the kurtosis of Gaussian distribution, for most central Pb$-$Pb collisions. Hydrodynamic model calculations with MUSIC using Monte Carlo Glauber initial conditions are able to explain the measurements of both skewness and kurtosis qualitatively from semicentral to central collisions in Pb--Pb system. Color reconnection mechanism in PYTHIA8 model seems to play a pivotal role in capturing the qualitative behavior of the same measurements in pp collisions