Immune Cells Invade the Collateral Circulation during Human Stroke: Prospective Replication and Extension

It remains unclear if principal components of the local cerebral stroke immune response can be reliably and reproducibly observed in patients with acute large-vessel-occlusion (LVO) stroke. We prospectively studied a large independent cohort of n = 318 consecutive LVO stroke patients undergoing mechanical thrombectomy during which cerebral blood samples from within the occluded anterior circulation and systemic control samples from the ipsilateral cervical internal carotid artery were obtained. An extensive protocol was applied to homogenize the patient cohort and to standardize the procedural steps of endovascular sample collection, sample processing, and laboratory analyses. N = 58 patients met all inclusion criteria. (1) Mean total leukocyte counts were significantly higher within the occluded ischemic cerebral vasculature (I) vs. intraindividual systemic controls (S): +9.6%, I: 8114/µL ± 529 vs. S: 7406/µL ± 468, p = 0.0125. (2) This increase was driven by neutrophils: +12.1%, I: 7197/µL ± 510 vs. S: 6420/µL ± 438, p = 0.0022. Leukocyte influx was associated with (3) reduced retrograde collateral flow (R² = 0.09696, p = 0.0373) and (4) greater infarct extent (R² = 0.08382, p = 0.032). Despite LVO, leukocytes invade the occluded territory via retrograde collateral pathways early during ischemia, likely compromising cerebral hemodynamics and tissue integrity. This inflammatory response can be reliably observed in human stroke by harvesting immune cells from the occluded cerebral vascular compartment.     

Fluorescence Lifetime Imaging Microscopy (FLIM) of Intracellular Transport by Means of Doubly Labelled siRNA Architectures

For monitoring the intracellular pathway of small interfering RNA (siRNA), both strands were labelled at internal positions by two ATTO dyes as an interstrand Förster resonance energy transfer pair. siRNA double strands show red emission and a short donor lifetime as readout, whereas siRNA antisense single strands show green emission and a long donor lifetime. This readout signals if GFP silencing can be expected (green) or not (red). We attached both dyes to three structurally different alkyne anchors by postsynthetic modifications. There is only a slight preference for the ribofuranoside anchors with the dyes at their 2’-positions. For the first time, the delivery and fate of siRNA in live HeLa cells was tracked by fluorescence lifetime imaging microscopy (FLIM), which revealed a clear relationship between intracellular transport using different transfection methods and knockdown of GFP expression, which demonstrates the potential of our siRNA architectures as a tool for future development of effective siRNA.     

Getting a Grip on the Undrugged: Targeting β-Catenin with Fragment-Based Methods

Aberrant WNT pathway activation, leading to nuclear accumulation of β-catenin, is a key oncogenic driver event. Mutations in the tumor suppressor gene APC lead to impaired proteasomal degradation of β-catenin and subsequent nuclear translocation. Restoring cellular degradation of β-catenin represents a potential therapeutic strategy. Here, we report the fragment-based discovery of a small molecule binder to β-catenin, including the structural elucidation of the binding mode by X-ray crystallography. The difficulty in drugging β-catenin was confirmed as the primary screening campaigns identified only few and very weak hits. Iterative virtual and NMR screening techniques were required to discover a compound with sufficient potency to be able to obtain an X-ray co-crystal structure. The binding site is located between armadillo repeats two and three, adjacent to the BCL9 and TCF4 binding sites. Genetic studies show that it is unlikely to be useful for the development of protein–protein interaction inhibitors but structural information and established assays provide a solid basis for a prospective optimization towards β-catenin proteolysis targeting chimeras (PROTACs) as alternative modality.     

Synthesis of carbon nanotubes with and without catalyst particles

The initial development of carbon nanotube synthesis revolved heavily around the use of 3d valence transition metals such as Fe, Ni, and Co. More recently, noble metals (e.g. Au) and poor metals (e.g. In, Pb) have been shown to also yield carbon nanotubes. In addition, various ceramics and semiconductors can serve as catalytic particles suitable for tube formation and in some cases hybrid metal/metal oxide systems are possible. All-carbon systems for carbon nanotube growth without any catalytic particles have also been demonstrated. These different growth systems are briefly examined in this article and serve to highlight the breadth of avenues available for carbon nanotube synthesis.     

Moving Fourier Analysis for Locally Stationary Processes with the Bootstrap in View

We introduce a moving Fourier transformation for locally stationary time series, which captures the time‐varying spectral density in a similar manner as the classical Fourier transform does for stationary time series. In particular, the resulting Fourier coefficients as well as moving local periodograms are shown to be (almost all) asymptotically uncorrelated. The moving local periodogram is obtained by thinning the local periodogram to avoid multiple information present at different but close points in time. We obtain consistent estimators for the local spectral density at each point in time by smoothing the moving local periodogram. Furthermore, the moving Fourier coefficients, respectively periodograms, are well suited to adapt stationary frequency domain bootstrap methods to the locally stationary case. For the wild time frequency toggle bootstrap, it is shown that the corresponding bootstrap covariance of a global locally stationary bootstrap samples captures the time‐varying covariance structure of the underlying locally stationary time series correctly. Furthermore, this bootstrap in addition to adaptations of other frequency domain bootstrap methods is used in a simulation study to obtain uniform confidence bands for the time‐varying autocorrelation at lag 1. Finally, this methodology is applied to a wind data set.     

Spherical polymer brushes with vinylimidazolium-type poly(ionic liquid) chains as support for metallic nanoparticles

We present here the synthesis of spherical polyelectrolyte brushes by photo-emulsion polymerization with a solid polystyrene core (diameter ～ 100 nm) onto which chains of vinylimidazolium-type poly(ionic liquid)s have been densely grafted. The as-synthesized brush particles were employed as nanoreactors for the generation and immobilization of metal nanoparticles. Transmission electron microscopy characterization shows that Au and Pd nanoparticles with diameter of 2.1 ± 0.2 nm and 2.5 ± 0.3 nm are homogeneously embedded inside the PIL brushes, respectively. The study of swelling behavior of the brush particles before and after metal deposition indicates obviously ion-specific effect. The composite particles exhibit a long-term colloidal stability in aqueous solutions as well as in ionic liquids. Catalytic activity of the as-synthesized metal nanocomposite particles is investigated by using the reduction of 4-nitrophenol with NaBH₄ as a model reaction, which can be compared directly with reported systems. In addition, it is found that the rate constant k_app of PIL-metal nanocatalyst could be modulated by salt concentration.     

Bestimmung von reduziertem und oxydiertem Glutathion in Weizenmehlen und -teigen

Zusammenfassung Für die quantitative Analyse von reduziertem (GSH) und oxydiertem (GSSG) Glutathion in Cerealien wurde das folgende Verfahren entwickelt: Extraktion eines Mehles oder Teigs und Carboxymethylierung des GSH, Gelfiltration an Sephadex G-50, Chromatographic an Dowex 50 WX8 und nach alkalischer Spaltung Bestimmung des abgetrennten GSSG mit 5,5-Dithio-bis(2-nitrobenzoesaure), Abtrennung des carboxymethylierten GSH an Dowex 1X8 und Bestimmung mit 2,4,6-Trinitrobenzoesulfonsäure. In drei Sorten von Weizenmehlen wurden 0,27–0,46 mol/g GSH and 0,26–0,38 mol/g GSSG gefunden. Beim Kneten (5 min) verschwand das GSH und das GSSG nahm entsprechend zu.

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Determination of reduced and oxidised glutathione in wheat flours and doughs

Summary A quantitative method for the analysis of reduced (GSH) and oxidised (GSSG) glutathione in cereals has been developed. The steps are: Extraction of the flour or dough and carboxymethylation of GSH, gel filtration on Sephadex G-50, chromatography on Dowex 50 WX8 and after basic cleavage determination of the separated GSSG with 5,5-dithio-bis(2-nitrobenzoic acid), separation of carboxymethylated GSH on Dowex 1X8 and determination with 2,4,6-trinitrobenzenesulfonic acid. When analysing for the amount of GSH and GSSG in flours of three varieties of wheat the former ranged from 0.27 to 0.46 mol/g and the latter from 0.26 to 0.38 mol/g. After kneading for 5 min to a dough the GSH disappeared with a corresponding increase in GSSG.

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Herrn Prof. Dr. L. Acker zu seinem 65. Geburtstag gewidmet

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Wir danken der AIF und dem Forschungskreis der Ernährungsindustrie für die finanzielle Unterstützung derArbeit. Frl. Ch. Hopfer danken wir für die geschickte experimentelle Mitarbeit     

Establishment of new genetic traits in a microbial biofilm community

Conjugational transfer of the TOL plasmid (pWWO) was analyzed in a flow chamber biofilm community engaged in benzyl alcohol degradation. The community consisted of three species, Pseudomonas putida RI, Acinetobacter sp. strain C6, and an unidentified isolate, D8. Only P. putida RI could act as a recipient for the TOL plasmid. Cells carrying a chromosomally integrated lacIq gene and a lacp-gfp-tagged version of the TOL plasmid were introduced as donor strains in the biofilm community after its formation. The occurrence of plasmid-carrying cells was analyzed by viable-count-based enumeration of donors and transconjugants. Upon transfer of the plasmids to the recipient cells, expression of green fluorescence was activated as a result of zygotic induction of the gfp gene. This allowed a direct in situ identification of cells receiving the gfp-tagged version of the TOL plasmid. Our data suggest that the frequency of horizontal plasmid transfer was low, and growth (vertical transfer) of the recipient strain was the major cause of plasmid establishment in the biofilm community. Employment of scanning confocal laser microscopy on fixed biofilms, combined with simultaneous identification of P. putida cells and transconjugants by 16S rRNA hybridization and expression of green fluorescence, showed that transconjugants were always associated with noninfected P. putida RI recipient microcolonies. Pure colonies of transconjugants were never observed, indicating that proliferation of transconjugant cells preferentially took place on preexisting P. putida RI microcolonies in the biofilm.