Modulating the G-Quadruplex and Duplex DNA Binding by Controlling the Charge of Fluorescent Molecules.

Two fluorescent and non-toxic spirobifluorene molecules bearing either positive (Spiro-NMe3) or negative (Spiro-SO3) charged moieties attached to the same aromatic structure have been investigated as binders for DNA. The novel Spiro-NMe3 containing four alkylammonium substituents interacts with G-quadruplex (G4) DNA structures and shows preference for G4s over duplex by means of FRET melting and fluorescence experiments. The interaction is governed by the charged substituents of the ligands as deduced from the lower binding of the sulfonate analogue (Spiro-SO3). On the contrary, Spiro-SO3 exhibits higher binding affinity to duplex DNA structure than to G4. Both molecules show a moderate quenching of the fluorescence upon DNA binding. The confocal microscopy evaluation shows the internalization of both molecules in HeLa cells and their lysosomal accumulation.

Self-Assembled Monolayers of Arylazopyrazoles on Glass and Silicon Oxide: Photoisomerization and Photoresponsive Wettability.

Surface coatings that respond to external influences and change their physical properties upon application of external stimuli are of great interest, with light being a particularly desirable choice. Photoswitches such as azobenzenes have been employed in a range of photoresponsive coatings. One striking change in physical property of many photoresponsive coatings is their responsive wettability upon illumination. In this work, we present photoswitchable self-assembled monolayers based on arylazopyrazoles (AAPs). In solution, AAPs offer significant improvements in terms of the photostationary state, thermal stability, and fatigue resistance. The AAP photoswitch is coupled to triethoxysilanes for an easy, one-step functionalization of glass and silicon oxide surfaces. We show the synthesis of AAP-based silanes and the successful surface functionalization, and we confirm the excellent photoswitchability of the AAPs in a self-assembled monolayer upon alternating irradiation with UV (365 nm) and green (520 nm) light. The self-assembled monolayers are investigated by UV/vis spectroscopy, X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS), and contact angle goniometry. We furthermore investigate the effect of substitution of the AAPs on the photoresponsive wetting behavior and compare this with density functional theory (DFT) calculations of the dipole moments of the AAPs.

An Arylazopyrazole-Based N-Heterocyclic Carbene as a Photoswitch on Gold Surfaces: Light-Switchable Wettability, Work Function, and Conductance.

A novel photoresponsive and fully conjugated N-heterocyclic carbene (NHC) has been synthesized that combines the excellent photophysical properties of arylazopyrazoles (AAPs) with an NHC that acts as a robust surface anchor (AAP-BIMe). The formation of self-assembled monolayers (SAMs) on gold was proven by ToF-SIMS and XPS, and the organic film displayed a very high stability at elevated temperatures. This stability was also reflected in a high desorption energy, which was determined by temperature-programmed SIMS measurements. E-/Z-AAP-BIMe@Au photoisomerization resulted in reversible alterations of the surface energy (i.e. wettability), the surface potential (i.e. work function), and the conductance (i.e. resistance). The effects could be explained by the difference in the dipole moment of the isomers. Furthermore, sequential application of a dummy ligand by microcontact printing and subsequent backfilling with AAP-BIMe allowed its patterning on gold. To the best of our knowledge, this is the first example of a photoswitchable NHC on a gold surface. These properties of AAP-BIMe@Au illustrate its suitability as a molecular switch for electronic devices.

Integrin α2ß1 Represents a Prognostic and Predictive Biomarker in Primary Ovarian Cancer.

Currently, the same first-line chemotherapy is administered to almost all patients suffering from primary ovarian cancer. The high recurrence rate emphasizes the need for precise drug treatment in primary ovarian cancer. Being crucial in ovarian cancer progression and chemotherapeutic resistance, integrins became promising therapeutic targets. To evaluate its prognostic and predictive value, in the present study, the expression of integrin α2ß1 was analyzed immunohistochemically and correlated with the survival data and other therapy-relevant biomarkers. The significant correlation of a high α2ß1-expression with the estrogen receptor alpha (ERα; p = 0.035) and epithelial growth factor receptor (EGFR; p = 0.027) was observed. In addition, high α2ß1-expression was significantly associated with a low number of tumor-infiltrating immune cells (CD3 intratumoral, p = 0.017; CD3 stromal, p = 0.035; PD-1 intratumoral, p = 0.002; PD-1 stromal, p = 0.049) and the lack of PD-L1 expression (p = 0.005). In Kaplan-Meier survival analysis, patients with a high expression of integrin α2ß1 revealed a significant shorter progression-free survival (PFS, p = 0.035) and platinum-free interval (PFI, p = 0.034). In the multivariate Cox regression analysis, integrin α2ß1 was confirmed as an independent prognostic factor for both PFS (p = 0.021) and PFI (p = 0.020). Dual expression of integrin α2ß1 and the hepatocyte growth factor receptor (HGFR; PFS/PFI, p = 0.004) and CD44v6 (PFS, p = 0.000; PFI, p = 0.001; overall survival [OS], p = 0.025) impaired survival. Integrin α2ß1 was established as a prognostic and predictive marker in primary ovarian cancer with the potential to stratify patients for chemotherapy and immunotherapy, and to design new targeted treatment strategies.

A cyclodextrin surfactant for stable emulsions with an accessible cavity for host-guest complexation.

Although cyclodextrins (CDs) have been used as additives to small molecule surfactants, there are only a few examples of this cyclic oligosaccharide being used as a surfactant template. Herein, we present a novel ionic ß-CD derivative that shows significant surface activity and stabilizes oil-in-water emulsions prepared with hexadecane as a model oil. The macrocyclic surfactant strongly reduces the oil-water surface tension with increasing concentration and shows a critical micelle concentration (CMC) around 5 mM. We demonstrate the stability of the oil-in-water emulsions and verify the possibility to address the emulsion droplet surface using molecular recognition of the ß-CD cavity at the oil-water interface.

Functionalized Polymers Enhance Permeability of Antibiotics in Gram-negative MDR Bacteria and Biofilms for Synergistic Antimicrobial Therapy.

The emergence of multi-drug resistant pathogenic bacteria constitutes a key threat to global health. Infections caused by multi-drug resistant Gram-negative bacteria are particularly challenging to treat due to the ability of pathogens to prevent antibiotic penetration inside the bacterial membrane. Antibiotic therapy is further rendered ineffective due to biofilm formation where the protective Extracellular Polymeric Substance (EPS) matrix limits the diffusion of antibiotics inside the biofilm. We hypothesized that careful engineering of chemical groups on polymer scaffolds could enable polymers to penetrate the barriers of Gram-negative bacterial membrane and biofilm matrix. Here, we present the use of engineered polymeric nanoparticles in combination with antibiotics for synergistic antimicrobial therapy. These polymeric nanoparticles enhance the accumulation of antibiotics inside Gram-negative bacteria and biofilm matrix, resulting in increased potency of antibiotics in combination therapy. Sub-lethal concentrations of engineered polymeric nanoparticles reduce the antibiotic dosage by 32-fold to treat MDR bacteria and biofilms. Tailoring of chemical groups on polymers demonstrate a strong-structure activity relationship in generating additive and synergistic combinations with antibiotics. This study demonstrates the ability of polymeric nanoparticles to 'rejuvenate' antibiotics rendered ineffective by resistant bacteria and provides a rationale to design novel compounds to achieve effective antimicrobial combination therapies.

Self-assembled multilayer surfaces of highly fluorescent spirobifluorene-based dye for label-free protein recognition.

The preparation of smart surfaces for protein detection is a challenging field of research. With the aim to achieve label-free detection in the solid state, we report on the organic surface functionalization for protein recognition without the need of previous chemical modification of the fluorophore. Layer-by-layer deposition of polyelectrolyte poly(vinyl benzyl tetramethylammonium) chloride (p(VBTMA)Cl) and a tetrasulfonate water-soluble low molecular weight fluorophore (1) based on spirobifluorene leads to modified glass and quartz substrates with outstanding photophysical properties in response to bovine serum albumin (BSA). The absorbance, photoluminescence as well as the fluorescence lifetimes were recorded for all surfaces. The surface structure and height of the different number of bilayers polymer/fluorophore were characterized by atomic force microscopy and ellipsometry. The results show linear trends in the absorption, fluorescence and height of the multilayer with increasing number of functionalization steps. Upon incubation with BSA the multilayer shows an increase in fluorescence up to 3-fold, which is also detectable with the naked eye. In conclusion, we report an easy, fast and biocompatible approach for the construction of protein sensors by self-assembly.

Immune Heterogeneity Between Primary Tumors and Corresponding Metastatic Lesions and Response to Platinum Therapy in Primary Ovarian Cancer.

CD3+ and CD8+ lymphocytes are well known prognostic markers in primary ovarian cancer. In contrast, the predictive value of the immune infiltrate concerning treatment response and the involvement of immune heterogeneity between primary and metastatic lesions are poorly understood. In this study, the immune infiltrate of 49 primary tumors and 38 corresponding lesions in the omentum (n = 23) and the peritoneum (n = 15) was immunohistochemically analyzed and correlated with clinicopathological factors and platinum-sensitivity. Immune heterogeneity was observed between paired primary and metastatic lesions for all immune cell phenotypes. The stromal immune infiltrate was higher in the omental lesions than in the primary tumors, which was reflected by CD45 (p=0.007), CD3 (p=0.005), CD8 (p=0.012), and PD-1 (programmed cell-death protein 1) (p=0.013). A higher stromal infiltrate of both CD45+ and CD3+ cells in the omental lesions was associated with the detection of lymph node metastasis (CD45, p=0.018; CD3, p=0.037). Platinum-sensitive ovarian cancers revealed a higher intratumoral CD8+ infiltrate in the peritoneal lesions compared to the primary tumors (p=0.045). In contrast, higher counts of stromal PD-1+ cells in the peritoneal lesions have been associated with reduced platinum-sensitivity (p=0.045). Immune heterogeneity was associated with platinum response and might represent a selection marker for personalized therapy.

A highly fluorescent water soluble spirobifluorene dye with a large Stokes shift: synthesis, characterization and bio-applications.

The first water-soluble spirobifluorene derivative has been synthesized, which exhibits high fluorescence quantum yield and a large Stokes shift (>100 nm). Proteins induce changes in the emission color, allowing to reach the nanomolar detection limit. Cellular uptake and cytotoxicity studies in living cells revealed its biocompatibility, indicating potential application for live cell imaging.

Development of a reliable and accurate algorithm to quantify the tumor immune stroma (QTiS) across tumor types.

The tumor microenvironment plays an important role in the tumor biology. Overall survival of tumor patients after resection is influenced by tumor-infiltrating lymphocytes (TILs) as a component of the tumor stroma. However, it is not clear how to assess TILs in the tumor stroma due to heterogeneous methods in different cancer types. Therefore, we present a novel Quantification of the Tumor immune Stroma (QTiS) Algorithm to reliably and accurately quantify cells in the tumor stroma. Immunohistochemical staining of CD3 and CD8 cells in sections of metastatic colorectal cancer (mCRC), ovarian cancer (OvCa), hepatocellular carcinoma (HCC), and pancreatic ductal adenocarcinoma (PDAC), alltogether N = 80, was performed. Hot spots of infiltrating immune cells are reported in the literature. Reliability of the hot spot identification of TILs was examined by two blinded observers. Accuracy was tested in 1 and 3 hot spots using computed counting methods (ZEN 2 software counting (ZC), ImageJ software with subjective threshold (ISC) and ImageJ with color deconvolution (IAC)) and compared to manual counting. All tumor types investigated showed an accumulation of TILs in the tumor stroma (peri- and intratumoral). Reliability between observers indicated a high level consistency. Accuracy for CD8+/CD3+ ratio and absolute cell count required 1 and 3 hot spots, respectively. ISC was found to be the best for paraffin sections, whereas IAC was ideal for frozen sections. ImageJ software is cost-effective and yielded the best results. In conclusion, an algorithm for quantification of tumoral stroma could be established. With this QTiS Algorithm counting of tumor stromal cells is reliable, accurate, and cost-effective.

Oxidation differentially modulates the recombinant voltage-gated Na(+) channel α-subunits Nav1.7 and Nav1.8.

Voltage-gated Na(+) channels regulate neuronal excitability by generating the upstroke of action potentials. The α-subunits Nav1.7 and Nav1.8 are required for normal function of sensory neurons and thus for peripheral pain processing, but also for an increased excitability leading to an increased pain sensitivity under several conditions associated with oxidative stress. While little is known about the direct effects of oxidants on Nav1.7 and Nav1.8, a recent study on mouse dorsal root ganglion neurons suggested that oxidant-induced alterations of nociceptor excitability are primarily driven by Nav1.8. Here we performed whole-cell patch clamp recordings to explore how oxidation modulates functional properties of recombinant Nav1.7 and Nav1.8 channels. The strong oxidant chloramine-T (ChT) at 100 and 500µM induced a shift of the voltage-dependency of activation towards more hyperpolarized potentials. While fast inactivation was stabilized by 100µM ChT, it was partially removed by 500µM ChT on both α-subunits (Nav1.7