Heterologous Expression and Engineering Studies of Labyrinthopeptins, Class III Lantibiotics from Actinomadura namibiensis

SummaryLabyrinthopeptins are class III lantibiotics produced by the actinomycete Actinomadura namibiensis. The most characteristic structural feature is the posttranslationally installed triamino triacid labionin with a quaternary α-carbon. In addition to the unique structure, labyrinthopeptin A2 possess remarkable antiviral and antiallodynic biological activities. To harness the substrate tolerance of the biosynthetic machinery, we developed an efficient system for the generation of labyrinthopeptin analogs. Streptomyces lividans was used as a heterologous host since the natural producer Actinomadura namibiensis remained genetically intractable. Generation of a library of 39 mutants allowed identification of variable and invariable regions in the labyrinthopeptin structures. Additional data on the flexibility of the biosynthetic machinery were provided by in vitro experiments. This study is detailed investigation on the potential to generate analogs of class III lantibiotics by genetic engineering

Activation-induced chromatin reorganization in neurons depends on HDAC1 activity

Spatial chromatin organization is crucial for transcriptional regulation and might be particularly important in neurons since they dramatically change their transcriptome in response to external stimuli. We show that stimulation of neurons causes condensation of large chromatin domains. This phenomenon can be observed in vitro in cultured rat hippocampal neurons as well as in vivo in the amygdala and hippocampal neurons. Activity-induced chromatin condensation is an active, rapid, energy-dependent, and reversible process. It involves calcium-dependent pathways but is independent of active transcription. It is accompanied by the redistribution of posttranslational histone modifications and rearrangements in the spatial organization of chromosome territories. Moreover, it leads to the reorganization of nuclear speckles and active domains located in their proximity. Finally, we find that the histone deacetylase HDAC1 is the key regulator of this process. Our results suggest that HDAC1-dependent chromatin reorganization constitutes an important level of transcriptional regulation in neurons.publishedVersio

Activation-induced chromatin reorganization in neurons depends on HDAC1 activity

Spatial chromatin organization is crucial for transcriptional regulation and might be particularly important in neurons since they dramatically change their transcriptome in response to external stimuli. We show that stimulation of neurons causes condensation of large chromatin domains. This phenomenon can be observed in vitro in cultured rat hippocampal neurons as well as in vivo in the amygdala and hippocampal neurons. Activity-induced chromatin condensation is an active, rapid, energy-dependent, and reversible process. It involves calcium-dependent pathways but is independent of active transcription. It is accompanied by the redistribution of posttranslational histone modifications and rearrangements in the spatial organization of chromosome territories. Moreover, it leads to the reorganization of nuclear speckles and active domains located in their proximity. Finally, we find that the histone deacetylase HDAC1 is the key regulator of this process. Our results suggest that HDAC1-dependent chromatin reorganization constitutes an important level of transcriptional regulation in neurons.This work was supported by the National Science Centre grant nos. UMO-2015/18/E/NZ3/00730 (A.M., A.G., H.S.N., E.J. and Y.Y.), 2014/15/N/NZ2/00379 and 2017/24/T/NZ2/00307 (P.T.), 2019/35/O/ST6/02484 (D.P. and G.B.), and 2014/14/M/NZ4/00561 (K.H.O. and R.K.F.). B.W. and B.G. were supported by the Foundation for Polish Science TEAM-TECH Core Facility project “NGS platform for comprehensive diagnostics and personalized therapy in neuro-oncology,” Foundation for Polish Science co-financed by the European Union under the European Regional Development Fund (TEAM to D.P.). A.M.G. was supported by the H2020-MSCA-COFUND-2014 grant Bio4Med (GA no. 665735).Peer reviewe

Multidifferential study of identified charged hadron distributions in ZZ-tagged jets in proton-proton collisions at s=\sqrt{s}=13 TeV

Jet fragmentation functions are measured for the first time in proton-proton collisions for charged pions, kaons, and protons within jets recoiling against a $Z$ boson. The charged-hadron distributions are studied longitudinally and transversely to the jet direction for jets with transverse momentum 20 $< p_{\textrm{T}} < 100$ GeV and in the pseudorapidity range $2.5 < \eta < 4$. The data sample was collected with the LHCb experiment at a center-of-mass energy of 13 TeV, corresponding to an integrated luminosity of 1.64 fb$^{-1}$. Triple differential distributions as a function of the hadron longitudinal momentum fraction, hadron transverse momentum, and jet transverse momentum are also measured for the first time. This helps constrain transverse-momentum-dependent fragmentation functions. Differences in the shapes and magnitudes of the measured distributions for the different hadron species provide insights into the hadronization process for jets predominantly initiated by light quarks.Comment: All figures and tables, along with machine-readable versions and any supplementary material and additional information, are available at https://cern.ch/lhcbproject/Publications/p/LHCb-PAPER-2022-013.html (LHCb public pages

Study of the B−→Λc+Λˉc−K−B^{-} \to \Lambda_{c}^{+} \bar{\Lambda}_{c}^{-} K^{-} decay

The decay $B^{-} \to \Lambda_{c}^{+} \bar{\Lambda}_{c}^{-} K^{-}$ is studied in proton-proton collisions at a center-of-mass energy of $\sqrt{s}=13$ TeV using data corresponding to an integrated luminosity of 5 $\mathrm{fb}^{-1}$ collected by the LHCb experiment. In the $\Lambda_{c}^+ K^{-}$ system, the $\Xi_{c}(2930)^{0}$ state observed at the BaBar and Belle experiments is resolved into two narrower states, $\Xi_{c}(2923)^{0}$ and $\Xi_{c}(2939)^{0}$, whose masses and widths are measured to be $$m(\Xi_{c}(2923)^{0}) = 2924.5 \pm 0.4 \pm 1.1 \,\mathrm{MeV}, \\ m(\Xi_{c}(2939)^{0}) = 2938.5 \pm 0.9 \pm 2.3 \,\mathrm{MeV}, \\ \Gamma(\Xi_{c}(2923)^{0}) = \phantom{000}4.8 \pm 0.9 \pm 1.5 \,\mathrm{MeV},\\ \Gamma(\Xi_{c}(2939)^{0}) = \phantom{00}11.0 \pm 1.9 \pm 7.5 \,\mathrm{MeV},$$ where the first uncertainties are statistical and the second systematic. The results are consistent with a previous LHCb measurement using a prompt $\Lambda_{c}^{+} K^{-}$ sample. Evidence of a new $\Xi_{c}(2880)^{0}$ state is found with a local significance of $3.8\,\sigma$, whose mass and width are measured to be $2881.8 \pm 3.1 \pm 8.5\,\mathrm{MeV}$ and $12.4 \pm 5.3 \pm 5.8 \,\mathrm{MeV}$, respectively. In addition, evidence of a new decay mode $\Xi_{c}(2790)^{0} \to \Lambda_{c}^{+} K^{-}$ is found with a significance of $3.7\,\sigma$. The relative branching fraction of $B^{-} \to \Lambda_{c}^{+} \bar{\Lambda}_{c}^{-} K^{-}$ with respect to the $B^{-} \to D^{+} D^{-} K^{-}$ decay is measured to be $2.36 \pm 0.11 \pm 0.22 \pm 0.25$, where the first uncertainty is statistical, the second systematic and the third originates from the branching fractions of charm hadron decays.Comment: All figures and tables, along with any supplementary material and additional information, are available at https://cern.ch/lhcbproject/Publications/p/LHCb-PAPER-2022-028.html (LHCb public pages

Measurement of the ratios of branching fractions R(D∗)\mathcal{R}(D^{*}) and R(D0)\mathcal{R}(D^{0})

The ratios of branching fractions $\mathcal{R}(D^{*})\equiv\mathcal{B}(\bar{B}\to D^{*}\tau^{-}\bar{\nu}_{\tau})/\mathcal{B}(\bar{B}\to D^{*}\mu^{-}\bar{\nu}_{\mu})$ and $\mathcal{R}(D^{0})\equiv\mathcal{B}(B^{-}\to D^{0}\tau^{-}\bar{\nu}_{\tau})/\mathcal{B}(B^{-}\to D^{0}\mu^{-}\bar{\nu}_{\mu})$ are measured, assuming isospin symmetry, using a sample of proton-proton collision data corresponding to 3.0 fb${ }^{-1}$ of integrated luminosity recorded by the LHCb experiment during 2011 and 2012. The tau lepton is identified in the decay mode $\tau^{-}\to\mu^{-}\nu_{\tau}\bar{\nu}_{\mu}$. The measured values are $\mathcal{R}(D^{*})=0.281\pm0.018\pm0.024$ and $\mathcal{R}(D^{0})=0.441\pm0.060\pm0.066$, where the first uncertainty is statistical and the second is systematic. The correlation between these measurements is $\rho=-0.43$. Results are consistent with the current average of these quantities and are at a combined 1.9 standard deviations from the predictions based on lepton flavor universality in the Standard Model.Comment: All figures and tables, along with any supplementary material and additional information, are available at https://cern.ch/lhcbproject/Publications/p/LHCb-PAPER-2022-039.html (LHCb public pages

Effect of manufacturing temperature and storage duration on stability of chemically defined media measured with LC-MS/MS

Krattenmacher F, Heermann T, Calvet A, Krawczyk B, Noll T. Effect of manufacturing temperature and storage duration on stability of chemically defined media measured with LC-MS/MS. JOURNAL OF CHEMICAL TECHNOLOGY AND BIOTECHNOLOGY. 2019;94(4):1144-1155.BACKGROUND Chemically defined media (CDM) are used routinely in industrial settings for the production of biopharmaceuticals from mammalian cells, so attention has shifted to understanding their chemical behavior and impact on process robustness. In this context, one particular problem is the risk of cross reactivity and instability in complex mixtures of chemicals at high concentrations. RESULTS In order to characterize potentially unstable medium compounds in feed media, we developed and validated a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) method to simultaneously quantify the majority of medium compounds. We used this application advantage to provide the following insights into molecular processes occurring in CDM prepared at different temperatures and during storage: Concentration profiles of critical compounds in CDM are independent of preparation temperature and storage material but show significant alterations over time. In particular l-cysteine, l-proline, vitamin B-6, thiamine and cyanocobalamin were found to be rather unstable. Using mixed mode chromatography allows expansion of the method to cover additional analytes of interest, as demonstrated by inclusion of seven additional CDM reaction products. CONCLUSION The successful development of a new LC-MS/MS method allowed us to show the impact of storage duration on the stability of CDM compounds. Furthermore, the results suggest that essential parameters for medium preparation (e.g. temperature) and storage upscaling (e.g. vessel material) are not impacting upon the chemical composition of CDM. (c) 2018 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry

Involvement and Unusual Substrate Specificity of a Prolyl Oligopeptidase in Class III Lanthipeptide Maturation

Lanthipeptides represent an important group of ribosomally synthesized and post-translationally modified peptides (RiPPs). Commonly, in the last steps of their maturation, a part of the peptide, termed the leader, is removed, providing the active compound. This contribution describes for the first time the identification of a protease involved in the removal of the leader peptide of a class III lanthipeptide. Four putative class III biosynthetic gene clusters were identified in bacterial genomes, each containing a gene encoding a prolyl oligopeptidase (POP). Further in vitro investigations of the gene cluster from Kribbella flavida, involving reconstitution of the biosynthesis of the new lanthipeptide flavipeptin, proved that a POP-type FlaP protease is responsible for leader removal. Interestingly, detailed in vitro studies of the substrate specificity revealed that FlaP is specific to the post-translationally modified peptide and can discriminate between N- and C-terminal rings. Therefore, it has been shown for the first time that factors other than size and amino acid sequence might be involved in substrate recognition by POPs

A Wind Tunnel Experimental Study of Icing on NACA0012 Aircraft Airfoil with Silicon Compounds Modified Polyurethane Coatings

Ice formation on the aerodynamic surfaces of an aircraft is regarded as a major problem in the aerospace industry. Ice accumulation may damage parts, sensors and controllers and alter the aerodynamics of the airplane, leading to a range of undesired consequences, including flight delays, emergency landings, damaged parts and increased energy consumption. There are various approaches to reducing ice accretion, one of them being the application of icephobic coatings. In this work, commercially available polyurethane-based coatings were modified and deposited on NACA 0012 aircraft airfoils. A hybrid modification of polyurethane (PUR) topcoats was adopted by the addition of nanosilica and three-functional spherosilicates (a variety of silsesqioxane compound), which owe their unique properties to the presence of three different groups. The ice accretion on the manufactured nanocomposites was determined in an icing wind tunnel. The tests were performed under three different icing conditions: glaze ice, rime ice and mixed ice. Furthermore, the surface topography and wetting behavior (static contact angle and contact angle hysteresis) were investigated. It was found that the anti-icing properties of polyurethane nanocomposite coatings strongly depend on the icing conditions under which they are tested. Moreover, the addition of nanosilica and spherosilicates enabled the reduction of accreted ice by 65% in comparison to the neat topcoat