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The use of fluorescent probes in a multitude of applications is still an expanding field. This review covers the recent progress made in small molecular, spirocyclic xanthene-based probes containing different heteroatoms (e.g., oxygen, silicon, carbon) in position 10′. After a short introduction, we will focus on applications like the interaction of probes with enzymes and targeted labeling of organelles and proteins, detection of small molecules, as well as their use in therapeutics or diagnostics and super-resolution microscopy. Furthermore, the last part will summarize recent advances in the synthesis and understanding of their structure–behavior relationship including novel computational approaches.

Background Exosomes consist of membrane vesicles that are secreted by several cell types, including tumors and have been found in biological fluids. Exosomes interact with other cells and may serve as vehicles for the transfer of protein and RNA among cells. Methods SKOV3 exosomes were labelled with carboxyfluoresceine diacetate succinimidyl-ester and collected by ultracentrifugation. Uptake of these vesicles, under different conditions, by the same cells from where they originated was monitored by immunofluorescence microscopy and flow cytometry analysis. Lectin analysis was performed to investigate the glycosylation properties of proteins from exosomes and cellular extracts. Results In this work, the ovarian carcinoma SKOV3 cell line has been shown to internalize exosomes from the same cells via several endocytic pathways that were strongly inhibited at 4°C, indicating their energy dependence. Partial colocalization with the endosome marker EEA1 and inhibition by chlorpromazine suggested the involvement of clathrin-dependent endocytosis. Furthermore, uptake inhibition in the presence of 5-ethyl-N-isopropyl amiloride, cytochalasin D and methyl-beta-cyclodextrin suggested the involvement of additional endocytic pathways. The uptake required proteins from the exosomes and from the cells since it was inhibited after proteinase K treatments. The exosomes were found to be enriched in specific mannose- and sialic acid-containing glycoproteins. Sialic acid removal caused a small but non-significant increase in uptake. Furthermore, the monosaccharides D-galactose, α-L-fucose, α-D-mannose, D-N-acetylglucosamine and the disaccharide β-lactose reduced exosomes uptake to a comparable extent as the control D-glucose. Conclusions In conclusion, exosomes are internalized by ovarian tumor cells via various endocytic pathways and proteins from exosomes and cells are required for uptake. On the other hand, exosomes are enriched in specific glycoproteins that may constitute exosome markers. This work contributes to the knowledge about the properties and dynamics of exosomes in cancer.

We report on generation of pulsed broadband terahertz radiation utilizing the inverse spin hall effect in Fe/Pt bilayers on MgO and sapphire substrates. The emitter was optimized with respect to layer thickness, growth parameters, substrates and geometrical arrangement. The experimentally determined optimum layer thicknesses were in qualitative agreement with simulations of the spin current induced in the ferromagnetic layer. Our model takes into account generation of spin polarization, spin diffusion and accumulation in Fe and Pt and electrical as well as optical properties of the bilayer samples. Using the device in a counterintuitive orientation a Si lens was attached to increase the collection efficiency of the emitter. The optimized emitter provided a bandwidth of up to 8 THz which was mainly limited by the low-temperature-grown GaAs (LT-GaAS) photoconductive antenna used as detector and the pulse length of the pump laser. The THz pulse length was as short as 220 fs for a sub 100 fs pulse length of the 800 nm pump laser. Average pump powers as low as 25 mW (at a repetition rate of 75 MHz) have been used for terahertz generation. This and the general performance make the spintronic terahertz emitter compatible with established emitters based on optical rectification in nonlinear crystals.

Background Exosomes are small membrane vesicles with a size of 40-100 nm that are released by different cell types from a late endosomal cellular compartment. They can be found in various body fluids including plasma, malignant ascites, urine, amniotic fluid and saliva. Exosomes contain proteins, miRNAs and mRNAs (exosome shuttle RNA, esRNA) that could serve as novel platform for diagnosis. Method We isolated exosomes from amniotic fluid, saliva and urine by differential centrifugation on sucrose gradients. Marker proteins were identified by Western blot and FACS analysis after adsorption of exosomes to latex beads. We extracted esRNA from exosomes, carried out RT-PCR, and analyzed amplified products by restriction length polymorphism. Results Exosomes were positive for the marker proteins CD24, CD9, Annexin-1 and Hsp70 and displayed the correct buoyant density and orientation of antigens. In sucrose gradients the exosomal fractions contained esRNA that could be isolated with sufficient quantity for further analysis. EsRNAs were protected in exosomes from enzymatic degradation. Amniotic fluid esRNA served as template for the typing of the CD24 single nucleotide polymorphism (rs52812045). It also allowed sex determination of the fetus based on the detection of the male specific ZFY gene product. Conclusions Our data demonstrate that exosomes from body fluids carry esRNAs which can be analyzed and offers access to the transcriptome of the host organism. The exosomal lipid bilayer protects the genetic information from degradation. As the isolation of exosomes is a minimally invasive procedure, this technique opens new possibilities for diagnostics.

Mechanisms behind how the immune system signals to the brain in response to systemic inflammation are not fully understood. Transgenic mice expressing Cre recombinase specifically in the hematopoietic lineage in a Cre reporter background display recombination and marker gene expression in Purkinje neurons. Here we show that reportergene expression in neurons is caused by intercellular transfer of functional Cre recombinase messenger RNA from immune cells into neurons in the absence of cell fusion. In vitro purified secreted extracellular vesicles (EVs) from blood cells contain Cre mRNA, which induces recombination in neurons when injected into the brain. Although Cre-mediated recombination events in the brain occur very rarely in healthy animals, their number increases considerably in different injury models, particularly under inflammatory conditions, and extend beyond Purkinje neurons to other neuronal populations in cortex, hippocampus, and substantia nigra. Recombined Purkinje neurons differ in their miRNA profile from their nonrecombined counterparts, indicating physiological significance. These observations reveal the existence of a previously unrecognized mechanism to communicate RNA-based signals between the hematopoietic system and various organs, including the brain, in response to inflammation.

Spintronic ferromagnetic/non-magnetic heterostructures are novel sources for the generation of THz radiation based on spin-to-charge conversion in the layers. The key technological and scientific challenge of THz spintronic emitters is to increase their intensity and frequency bandwidth. Our work reveals the factors to engineer spintronic Terahertz generation by introducing the scattering lifetime and the interface transmission for spin polarized, non-equilibrium electrons. We clarify the influence of the electron-defect scattering lifetime on the spectral shape and the interface transmission on the THz amplitude, and how this is linked to structural defects of bilayer emitters. The results of our study define a roadmap of the properties of emitted as well as detected THz-pulse shapes and spectra that is essential for future applications of metallic spintronic THz emitters.

Background The circulating metabolome, reflecting underlying cellular processes and disease biology, has not been fully characterized in patients with idiopathic pulmonary fibrosis (IPF). We evaluated whether circulating levels of metabolites correlate with the presence of IPF, with the severity of IPF, or with the risk of clinically relevant outcomes among patients with IPF. Methods We analyzed enrollment plasma samples from 300 patients with IPF in the IPF-PRO Registry and 100 individuals without known lung disease using a set of targeted metabolomics and clinical analyte modules. Linear regression was used to compare metabolite and clinical analyte levels between patients with IPF and controls and to determine associations between metabolite levels and measures of disease severity in patients with IPF. Unadjusted and adjusted univariable Cox regression models were used to evaluate associations between circulating metabolites and the risk of mortality or disease progression among patients with IPF. Results Levels of 64 metabolites and 5 clinical analytes were significantly different between patients with IPF and controls. Among analytes with greatest differences were non-esterified fatty acids, multiple long-chain acylcarnitines, and select ceramides, levels of which were higher among patients with IPF versus controls. Levels of the branched-chain amino acids valine and leucine/isoleucine were inversely correlated with measures of disease severity. After adjusting for clinical factors known to influence outcomes, higher levels of the acylcarnitine C:16-OH/C:14-DC were associated with all-cause mortality, lower levels of the acylcarnitine C16:1-OH/C14:1DC were associated with all-cause mortality, respiratory death, and respiratory death or lung transplant, and higher levels of the sphingomyelin d43:2 were associated with the risk of respiratory death or lung transplantation. Conclusions IPF has a distinct circulating metabolic profile characterized by increased levels of non-esterified fatty acids, long-chain acylcarnitines, and ceramides, which may suggest a more catabolic environment that enhances lipid mobilization and metabolism. We identified select metabolites that were highly correlated with measures of disease severity or the risk of disease progression and that may be developed further as biomarkers. Trial registration ClinicalTrials.gov; No: NCT01915511; URL: www.clinicaltrials.gov .

We report on the determination of the spin Hall angle in ultra-clean, defect-reduced epitaxial Pt films. By applying vector network analyzer ferromagnetic resonance spectroscopy to a series of single crystalline Fe (12 nm) /Pt (tPt) bilayers we determine the real part of the spin mixing conductance (4.4 0.2) × 1019 m−2 and reveal a very small spin diffusion length in the epitaxial Pt (1.1 0.1) nm film. We investigate the spin pumping and ISHE in a stripe microstucture excited by a microwave coplanar waveguide antenna. By using their different angular dependencies, we distinguish between spin rectification effects and the inverse spin Hall effect. The relatively large value of the spin Hall angle (5.7 1.4)% shows that ultra-clean e-beam evaporated non-magnetic materials can also have a comparable spin-to-charge current conversion efficiency as sputtered high resistivity layers.

Background BI 836880 is a humanized bispecific nanobody® that binds to and blocks vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2). A comprehensive biomarker and modeling approach is presented here that supported dose finding for BI 836880. Methods Two Phase I dose-escalation studies (1336.1 [NCT02674152], 1336.6 [NCT02689505]) assessed BI 836880 in adults with confirmed locally advanced or metastatic solid tumors, refractory to standard therapy or for which standard therapy was not reliably effective. Two dosing schedules were investigated, 3 weeks (q3w) or once weekly (qw), starting at a dose of 40 mg. In a comprehensive biomarker approach, soluble pharmacodynamic markers (free and total plasma VEGF-A and Ang-2), as well as circulating angiogenic factors (soluble VEGF3, soluble Tie2 and placenta growth factor, amongst others) were analyzed to assess target engagement in peripheral blood for q3w doses. A Population based pharmacokinetics/pharmacodynamics (PopPK/PD) model was built using the limited Phase I dataset to support dose finding by simulations. In order to demonstrate drug activity in the tumor, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was applied. Results DCE-MRI scans supported target engagement in the tumor. Free VEGF-A was depleted at all doses, whereas free Ang-2 decreased dose-dependently, reaching depletion in most patients from 360 mg q3w onwards. While total VEGF-A levels increased in a dose-dependent manner, reaching saturation at 360 mg q3w, total Ang-2 levels increased, but did not plateau. Angiogenic biomarkers showed changes from doses ≥ 360 mg q3w. PopPK/PD modeling showed that doses ≥ 360 mg q3w led to > 90% inhibition of free Ang-2 at steady-state in most patients. By increasing the dose to ≥ 500 mg q3w, > 90% of patients are expected to achieve this level. Conclusions The comprehensive analyses of multiple target engagement markers support BI 836880 720 mg q3w as a biologically relevant monotherapy dose schedule. Trial registration : NCT02674152 and NCT02689505.

BI 1595043 is an oral vanin‐1 and vanin‐2 inhibitor, which demonstrated promising effects on epithelial cell protection and reduction of inflammatory mediators in preclinical studies, as well as an acceptable safety profile in a previous single‐rising‐dose trial. Here, we report the results of a double‐blind, randomized, placebo‐controlled, multiple rising dose study, which investigated the safety, tolerability, pharmacokinetics, and pharmacodynamics of BI 1595043 in healthy male volunteers following oral administration of single and multiple rising doses over 18 days in total. Thirty subjects were treated (18–50 years of age; body mass index: 18.5–29.9 kg/m2); each dose group included 10 subjects, of which eight were administered BI 1595043 (15, 30, or 60 mg) and two were administered placebo after an overnight fast of ≥ 10 h. With multiple rising doses, BI 1595043 appeared to effectively inhibit the conversion of pantetheine to pantothenic acid. BI 1595043 achieved rapid absorption after administration (median time from dosing to maximum measured concentration of the analyte in plasma: ~1 h) and plasma concentrations generally increased in a dose‐proportional manner, with the majority excreted in urine within the first 24 h after dosing. Most adverse events reported were of mild or moderate severity, e.g., headache, dizziness, and abdominal discomfort. However, this study was temporarily halted after ophthalmologic adverse events judged to be drug‐related by the investigator were reported in six subjects treated with BI 1595043. The study was discontinued prematurely due to the sponsor's decision to terminate the development of BI 1595043 in all indications.