Synthesis and Characterization of Uniform Macromolecules and Data Storage in Small Compounds

Inspiriert durch die hoch komplexe und sequenzdefinierte Struktur von Biomakromolekülen, wie zum Beispiel Desoxynukleinsäure (DNS), hat sich mit der Synthese und Untersuchung uniformer, sequenzdefinierter Makromoleküle nicht-natürlichen Ursprungs ein neues Forschungsgebiet entwickelt. Da klassische Polymerchemie durch Dispersität und Strukturverteilung geprägt ist, beschränkte sich die Synthese von uniformen, sequenzdefinierten Strukturen lange Zeit auf Biopolymere. In dieser Arbeit wird die Synthese von uniformen poly(ethylenglycol)-block-poly(ε caprolacton) (PEG-b-PCL) Blockcopolymeren (BCP) beschrieben, die zur Untersuchung der dispersitätsabhängigen Struktur Eigenschafts-Beziehungen verwendet werden. Des Weiteren wurde die Anwendung von komplexen kleinen organischen Molekülen als potenzielle Medien für Datenspeicherung auf Oberflächen untersucht. Drei uniforme BCP, die in der Länge des hydrophoben PCL-Blocks variieren, wurden über die Kopplung von uniformen PEG- und PCL-Homopolymeren gebildet, welche über ein iteratives exponentielles Wachstum hergestellt wurden. Ringöffnende Polymerisation von ε-Caprolacton mit Methoxy-OEG als Makroinitiator lieferte Vergleichsmoleküle mit identischem Mn und einer engen Molmassenverteilung (Đ = 1.06). Die thermischen Eigenschaften wurde mittels dynamischer Differenzkalorimetrie (DSC) und das Phasenseparationsverhalten der BCP über Kleinwinkel-Röntgenstreuung (engl. small-angle X-ray scattering, SAXS) untersucht. Die Ergebnisse zeigen eine eindeutige konstitutions- und dispersitätsabhängige Struktur-Eigenschaftsbeziehung anhand der Kristallisationstemperatur Tc sowie ein unterschiedliches Phasenseparationsverhalten des kleinsten BCP in Abhängigkeit von der Dispersität. In dem zweiten Teil der Arbeit wurden kleine komplexe organische Moleküle für die Anwendung als potenzielle Datenspeichermedien auf einer Oberfläche untersucht. Die jeweiligen Verbindungen wurden in einer einstufigen Synthese unter Verwendung der Passerini-Dreikomponenten-Reaktion (P-3KR) in Kombination mit einer Hetero-Michael-Addition synthetisiert. Unter Verwendung einer Datenbank aus 708 kommerziell erhältlichen Komponenten, sind mit dem beschriebenen Ansatz 2.63 Mio einzigartige Strukturen potenziell zugänglich. Diese Anzahl an Permutationen entspricht einer Datenspeicherkapazität von 21 Bit pro Molekül. Die Moleküle wurden mit einem Pipettierroboter auf eine Glasoberfläche gedruckt und mittels hochauflösender Matrix-assistierter Laser-Desorption-Ionisation (MALDI) Tandem Massenspektrometie (MS/MS) anhand des resultierenden Fragmentmusters eindeutig identifiziert. In einem weiteren Ansatz wurde eine schnelle und effiziente Methode zur Speicherung von Informationen in kommerziell erhältlichen Verbindungen demonstriert. In mehreren Molekülmischungen wurde ein 625 bit QR-code gespeichert und mittels Gaschromatographie (GC) ausgelesen und, unterstützt durch ein eigens programmiertes Computerskript, mit 100%iger Genauigkeit entschlüsselt

Molecular data storage with zero synthetic effort and simple read-out

Compound mixtures represent an alternative, additional approach to DNA and synthetic sequence-defined macromolecules in the field of non-conventional molecular data storage, which may be useful depending on the target application. Here, we report a fast and efficient method for information storage in molecular mixtures by the direct use of commercially available chemicals and thus, zero synthetic steps need to be performed. As a proof of principle, a binary coding language is used for encoding words in ASCII or black and white pixels of a bitmap. This way, we stored a 25 × 25-pixel QR code (625 bits) and a picture of the same size. Decoding of the written information is achieved via spectroscopic (1H NMR) or chromatographic (gas chromatography) analysis. In addition, for a faster and automated read-out of the data, we developed a decoding software, which also orders the data sets according to an internal “ordering” standard. Molecular keys or anticounterfeiting are possible areas of application for information-containing compound mixtures

Synthesis and characterization of uniform OCL-OEG block cooligomers

Block copolymers are an interesting class of materials, offering the opportunity to form nanostructured morphologies, making them suitable for a broad range of applications in nanotechnology, medicine, or biotechnology. Especially for the pharmaceutical sector, a uniform structure and a distinct structure–property relationship is desirable to manufacture highly reproducible and tailor-made materials. Herein, we report the synthesis and characterization of uniform (Đ = 1.01) oligo(ε-caprolactone)-oligo(ethylene glycol) (OCL-OEG) block co-oligomers (BCOs). Three different BCOs, varying in the length of the hydrophobic caprolactone segment, were obtained via Steglich esterification of the corresponding homo oligomers. A clear dispersity and composition dependent structure–property relationship based on the thermal properties is observed, compared to identical structures similar in M$_n$ and dispersities of Đ = 1.06, obtained via ring-opening polymerization (ROP) of ε-caprolactone. In addition, increased long-range-order distances L$_0$ with increasing dispersity of the BCOs are found for the formed morphologies after solvent vapor annealing (SVA). These results highlight the importance of uniform structures for a better understanding of the structure–property relationship of block copolymers

Role of IFN-gamma and IL-6 in a protective immune response to Yersinia enterocolitica in mice

<p>Abstract</p> <p>Background</p> <p><it>Yersinia </it>outer protein (Yop) H is a secreted virulence factor of <it>Yersinia enterocolitica </it>(Ye), which inhibits phagocytosis of Ye and contributes to the virulence of Ye in mice. The aim of this study was to address whether and how YopH affects the innate immune response to Ye in mice.</p> <p>Results</p> <p>For this purpose, mice were infected with wild type Ye (pYV⁺) or a YopH-deficient Ye mutant strain (Δ<it>yopH</it>). CD11b⁺cells were isolated from the infected spleen and subjected to gene expression analysis using microarrays. Despite the attenuation of Δ<it>yopH in vivo</it>, by variation of infection doses we were able to achieve conditions that allow comparison of gene expression in pYV⁺and Δ<it>yopH </it>infection, using either comparable infection courses or splenic bacterial burden. Gene expression analysis provided evidence that expression levels of several immune response genes, including IFN-γ and IL-6, are high after pYV⁺infection but low after sublethal Δ<it>yopH </it>infection. In line with these findings, infection of IFN-γR^-/-and IL-6^-/-mice with pYV⁺or Δ<it>yopH </it>revealed that these cytokines are not necessarily required for control of Δ<it>yopH</it>, but are essential for defense against infection with the more virulent pYV⁺. Consistently, IFN-γ pretreatment of bone marrow derived macrophages (BMDM) strongly enhanced their ability in killing intracellular Ye bacteria.</p> <p>Conclusion</p> <p>In conclusion, this data suggests that IFN-γ-mediated effector mechanisms can partially compensate virulence exerted by YopH. These results shed new light on the protective role of IFN-γ in Ye wild type infections.</p

Immune Evasion by Yersinia enterocolitica: Differential Targeting of Dendritic Cell Subpopulations In Vivo

CD4+ T cells are essential for the control of Yersinia enterocolitica (Ye) infection in mice. Ye can inhibit dendritic cell (DC) antigen uptake and degradation, maturation and subsequently T-cell activation in vitro. Here we investigated the effects of Ye infection on splenic DCs and T-cell proliferation in an experimental mouse infection model. We found that OVA-specific CD4+ T cells had a reduced potential to proliferate when stimulated with OVA after infection with Ye compared to control mice. Additionally, proliferation of OVA-specific CD4+ T cells was markedly reduced when cultured with splenic CD8α+ DCs from Ye infected mice in the presence of OVA. In contrast, T-cell proliferation was not impaired in cultures with CD4+ or CD4−CD8α− DCs isolated from Ye infected mice. However, OVA uptake and degradation as well as cytokine production were impaired in CD8α+ DCs, but not in CD4+ and CD4−CD8α− DCs after Ye infection. Pathogenicity factors (Yops) from Ye were most frequently injected into CD8α+ DCs, resulting in less MHC class II and CD86 expression than on non-injected CD8α+ DCs. Three days post infection with Ye the number of splenic CD8α+ and CD4+ DCs was reduced by 50% and 90%, respectively. The decreased number of DC subsets, which was dependent on TLR4 and TRIF signaling, was the result of a faster proliferation and suppressed de novo DC generation. Together, we show that Ye infection negatively regulates the stimulatory capacity of some but not all splenic DC subpopulations in vivo. This leads to differential antigen uptake and degradation, cytokine production, cell loss, and cell death rates in various DC subpopulations. The data suggest that these effects might be caused directly by injection of Yops into DCs and indirectly by affecting the homeostasis of CD4+ and CD8α+ DCs. These events may contribute to reduced T-cell proliferation and immune evasion of Ye

Expression of Transketolase like gene 1 (TKTL1) predicts disease-free survival in patients with locally advanced rectal cancer receiving neoadjuvant chemoradiotherapy

<p>Abstract</p> <p>Background</p> <p>For patients with locally advanced rectal cancer (LARC) neoadjuvant chemoradiotherapy is recommended as standard therapy. So far, no predictive or prognostic molecular factors for patients undergoing multimodal treatment are established. Increased angiogenesis and altered tumour metabolism as adaption to hypoxic conditions in cancers play an important role in tumour progression and metastasis. Enhanced expression of Vascular-endothelial-growth-factor-receptor <it>(VEGF-R</it>) and Transketolase-like-1 (<it>TKTL1</it>) are related to hypoxic conditions in tumours. In search for potential prognostic molecular markers we investigated the expression of <it>VEGFR-1</it>, <it>VEGFR-2 </it>and <it>TKTL1 </it>in patients with LARC treated with neoadjuvant chemoradiotherapy and cetuximab.</p> <p>Methods</p> <p>Tumour and corresponding normal tissue from pre-therapeutic biopsies of 33 patients (m: 23, f: 10; median age: 61 years) with LARC treated in phase-I and II trials with neoadjuvant chemoradiotherapy (cetuximab, irinotecan, capecitabine in combination with radiotherapy) were analysed by quantitative PCR.</p> <p>Results</p> <p>Significantly higher expression of <it>VEGFR-1/2 </it>was found in tumour tissue in pre-treatment biopsies as well as in resected specimen after neoadjuvant chemoradiotherapy compared to corresponding normal tissue. High <it>TKTL1 </it>expression significantly correlated with disease free survival. None of the markers had influence on early response parameters such as tumour regression grading. There was no correlation of gene expression between the investigated markers.</p> <p>Conclusion</p> <p>High <it>TKTL-1 </it>expression correlates with poor prognosis in terms of 3 year disease-free survival in patients with LARC treated with intensified neoadjuvant chemoradiotherapy and may therefore serve as a molecular prognostic marker which should be further evaluated in randomised clinical trials.</p

Restricting Glycolysis Preserves T Cell Effector Functions and Augments Checkpoint Therapy

Tumor-derived lactic acid inhibits T and natural killer (NK) cell function and, thereby, tumor immunosurveillance. Here, we report that melanoma patients with high expression of glycolysis-related genes show a worse progression free survival upon anti-PD1 treatment. The non-steroidal anti-inflammatory drug (NSAID) diclofenac lowers lactate secretion of tumor cells and improves anti-PD1-induced T cell killing in vitro. Surprisingly, diclofenac, but not other NSAIDs, turns out to be a potent inhibitor of the lactate transporters monocarboxylate transporter 1 and 4 and diminishes lactate efflux. Notably, T cell activation, viability, and effector functions are preserved under diclofenac treatment and in a low glucose environment in vitro. Diclofenac, but not aspirin, delays tumor growth and improves the efficacy of checkpoint therapy in vivo. Moreover, genetic suppression of glycolysis in tumor cells strongly improves checkpoint therapy. These findings support the rationale for targeting glycolysis in patients with high glycolytic tumors together with checkpoint inhibitors in clinical trials