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- ItemDifferent storage conditions influence biocompatibility and physicochemical properties of iron oxide nanoparticles(Basel : Molecular Diversity Preservation International (MDPI), 2015) Zaloga, Jan; Janko, Christina; Agarwal, Rohit; Nowak, Johannes; Müller, Robert; Boccaccini, Aldo R.; Lee, Geoffrey; Odenbach, Stefan; Lyer, Stefan; Alexiou, ChristophSuperparamagnetic iron oxide nanoparticles (SPIONs) have attracted increasing attention in many biomedical fields. In magnetic drug targeting SPIONs are injected into a tumour supplying artery and accumulated inside the tumour with a magnet. The effectiveness of this therapy is thus dependent on magnetic properties, stability and biocompatibility of the particles. A good knowledge of the effect of storage conditions on those parameters is of utmost importance for the translation of the therapy concept into the clinic and for reproducibility in preclinical studies. Here, core shell SPIONs with a hybrid coating consisting of lauric acid and albumin were stored at different temperatures from 4 to 45 °C over twelve weeks and periodically tested for their physicochemical properties over time. Surprisingly, even at the highest storage temperature we did not observe denaturation of the protein or colloidal instability. However, the saturation magnetisation decreased by maximally 28.8% with clear correlation to time and storage temperature. Furthermore, the biocompatibility was clearly affected, as cellular uptake of the SPIONs into human T-lymphoma cells was crucially dependent on the storage conditions. Taken together, the results show that the particle properties undergo significant changes over time depending on the way they are stored.
- ItemRevealing the Chemical Composition of Birch Pollen Grains by Raman Spectroscopic Imaging(Basel : Molecular Diversity Preservation International (MDPI), 2022) Stiebing, Clara; Post, Nele; Schindler, Claudia; Göhrig, Bianca; Lux, Harald; Popp, Jürgen; Heutelbeck, Astrid; Schie, Iwan W.The investigation of the biochemical composition of pollen grains is of the utmost interest for several environmental aspects, such as their allergenic potential and their changes in growth conditions due to climatic factors. In order to fully understand the composition of pollen grains, not only is an in-depth analysis of their molecular components necessary but also spatial information of, e.g., the thickness of the outer shell, should be recorded. However, there is a lack of studies using molecular imaging methods for a spatially resolved biochemical composition on a single-grain level. In this study, Raman spectroscopy was implemented as an analytical tool to investigate birch pollen by imaging single pollen grains and analyzing their spectral profiles. The imaging modality allowed us to reveal the layered structure of pollen grains based on the biochemical information of the recorded Raman spectra. Seven different birch pollen species collected at two different locations in Germany were investigated and compared. Using chemometric algorithms such as hierarchical cluster analysis and multiple-curve resolution, several components of the grain wall, such as sporopollenin, as well as the inner core presenting high starch concentrations, were identified and quantified. Differences in the concentrations of, e.g., sporopollenin, lipids and proteins in the pollen species at the two different collection sites were found, and are discussed in connection with germination and other growth processes.
- ItemConsensusPrime—A Bioinformatic Pipeline for Ideal Consensus Primer Design(Basel : MDPI, 2022) Collatz, Maximilian; Braun, Sascha D.; Monecke, Stefan; Ehricht, RalfBackground: High-quality oligonucleotides for molecular amplification and detection procedures of diverse target sequences depend on sequence homology. Processing input sequences and identifying homogeneous regions in alignments can be carried out by hand only if they are small and contain sequences of high similarity. Finding the best regions for large and inhomogeneous alignments needs to be automated. Results: The ConsensusPrime pipeline was developed to sort out redundant and technical interfering data in multiple sequence alignments and detect the most homologous regions from multiple sequences. It automates the prediction of optimal consensus primers for molecular analytical and sequence-based procedures/assays. Conclusion: ConsensusPrime is a fast and easy-to-use pipeline for predicting optimal consensus primers that is executable on local systems without depending on external resources and web services. An implementation in a Docker image ensures platform-independent executability and installability despite the combination of multiple programs. The source code and installation instructions are publicly available on GitHub.
- ItemLinear and non-linear optical imaging of cancer cells with silicon nanoparticles(Basel : Molecular Diversity Preservation International (MDPI), 2016) Tolstik, Elen; Osminkina, Liubov A.; Akimov, Denis; Gongalsky, Maksim B.; Kudryavtsev, Andrew A.; Timoshenko, Victor Yu.; Heintzmann, Rainer; Sivakov, Vladimir; Popp, JürgenNew approaches for visualisation of silicon nanoparticles (SiNPs) in cancer cells are realised by means of the linear and nonlinear optics in vitro. Aqueous colloidal solutions of SiNPs with sizes of about 10–40 nm obtained by ultrasound grinding of silicon nanowires were introduced into breast cancer cells (MCF-7 cell line). Further, the time-varying nanoparticles enclosed in cell structures were visualised by high-resolution structured illumination microscopy (HR-SIM) and micro-Raman spectroscopy. Additionally, the nonlinear optical methods of two-photon excited fluorescence (TPEF) and coherent anti-Stokes Raman scattering (CARS) with infrared laser excitation were applied to study the localisation of SiNPs in cells. Advantages of the nonlinear methods, such as rapid imaging, which prevents cells from overheating and larger penetration depth compared to the single-photon excited HR-SIM, are discussed. The obtained results reveal new perspectives of the multimodal visualisation and precise detection of the uptake of biodegradable non-toxic SiNPs by cancer cells and they are discussed in view of future applications for the optical diagnostics of cancer tumours.
- ItemFunctional Delineation of a Protein–Membrane Interaction Hotspot Site on the HIV-1 Neutralizing Antibody 10E8(Basel : Molecular Diversity Preservation International (MDPI), 2022) Insausti, Sara; Garcia-Porras, Miguel; Torralba, Johana; Morillo, Izaskun; Ramos-Caballero, Ander; de la Arada, Igor; Apellaniz, Beatriz; Caaveiro, Jose M. M.; Carravilla, Pablo; Eggeling, Christian; Rujas, Edurne; Nieva, Jose L.Antibody engagement with the membrane-proximal external region (MPER) of the envelope glycoprotein (Env) of HIV-1 constitutes a distinctive molecular recognition phenomenon, the full appreciation of which is crucial for understanding the mechanisms that underlie the broad neutralization of the virus. Recognition of the HIV-1 Env antigen seems to depend on two specific features developed by antibodies with MPER specificity: (i) a large cavity at the antigen-binding site that holds the epitope amphipathic helix; and (ii) a membrane-accommodating Fab surface that engages with viral phospholipids. Thus, besides the main Fab–peptide interaction, molecular recognition of MPER depends on semi-specific (electrostatic and hydrophobic) interactions with membranes and, reportedly, on specific binding to the phospholipid head groups. Here, based on available cryo-EM structures of Fab–Env complexes of the anti-MPER antibody 10E8, we sought to delineate the functional antibody–membrane interface using as the defining criterion the neutralization potency and binding affinity improvements induced by Arg substitutions. This rational, Arg-based mutagenesis strategy revealed the position-dependent contribution of electrostatic interactions upon inclusion of Arg-s at the CDR1, CDR2 or FR3 of the Fab light chain. Moreover, the contribution of the most effective Arg-s increased the potency enhancement induced by inclusion of a hydrophobic-at-interface Phe at position 100c of the heavy chain CDR3. In combination, the potency and affinity improvements by Arg residues delineated a protein–membrane interaction site, whose surface and position support a possible mechanism of action for 10E8-induced neutralization. Functional delineation of membrane-interacting patches could open new lines of research to optimize antibodies of therapeutic interest that target integral membrane epitopes.
- ItemDNA-Biofunctionalization of CTAC-Capped Gold Nanocubes(Basel : MDPI, 2020) Slesiona, Nicole; Thamm, Sophie; Stolle, H. Lisa K.S.; Weißenborn, Viktor; Müller, Philipp; Csáki, Andrea; Fritzsche, WolfgangClinical diagnostics and disease control are fields that strongly depend on technologies for rapid, sensitive, and selective detection of biological or chemical analytes. Nanoparticles have become an integral part in various biomedical detection devices and nanotherapeutics. An increasing focus is laid on gold nanoparticles as they express less cytotoxicity, high stability, and hold unique optical properties with the ability of signal transduction of biological recognition events with enhanced analytical performance. Strong electromagnetic field enhancements can be found in close proximity to the nanoparticle that can be exploited to enhance signals for e.g., metal-enhanced fluorescence or Raman spectroscopy. Even stronger field enhancements can be achieved with sharp-edged nanoparticles, which are synthesized with the help of facet blocking agents, such as cetyltrimethylammonium bromide/chloride (CTAB/CTAC). However, chemical modification of the nanoparticle surface is necessary to reduce the particle’s cytotoxicity, stabilize it against aggregation, and to bioconjugate it with biomolecules to increase its biocompatibility and/or specificity for analytical applications. Here, a reliable two-step protocol following a ligand exchange with bis (p-sulfonatophenyl) phenyl phosphine (BSPP) as the intermediate capping-agent is demonstrated, which results in the reliable biofunctionalization of CTAC-capped gold nanocubes with thiol-modified DNA. The functionalized nanocubes have been characterized regarding their electric potential, plasmonic properties, and stability against high concentrations of NaCl and MgCl2.
- ItemVibrational Spectroscopic Investigation of Blood Plasma and Serum by Drop Coating Deposition for Clinical Application(Basel : Molecular Diversity Preservation International (MDPI), 2021) Huang, Jing; Ali, Nairveen; Quansah, Elsie; Guo, Shuxia; Noutsias, Michel; Meyer-Zedler, Tobias; Bocklitz, Thomas; Popp, Jürgen; Neugebauer, Ute; Ramoji, AnuradhaIn recent decades, vibrational spectroscopic methods such as Raman and FT-IR spectroscopy are widely applied to investigate plasma and serum samples. These methods are combined with drop coating deposition techniques to pre-concentrate the biomolecules in the dried droplet to improve the detected vibrational signal. However, most often encountered challenge is the inhomogeneous redistribution of biomolecules due to the coffee-ring effect. In this study, the variation in biomolecule distribution within the dried-sample droplet has been investigated using Raman and FT-IR spectroscopy and fluorescence lifetime imaging method. The plasma-sample from healthy donors were investigated to show the spectral differences between the inner and outer-ring region of the dried-sample droplet. Further, the preferred location of deposition of the most abundant protein albumin in the blood during the drying process of the plasma has been illustrated by using deuterated albumin. Subsequently, two patients with different cardiac-related diseases were investigated exemplarily to illustrate the variation in the pattern of plasma and serum biomolecule distribution during the drying process and its impact on patient-stratification. The study shows that a uniform sampling position of the droplet, both at the inner and the outer ring, is necessary for thorough clinical characterization of the patient’s plasma and serum sample using vibrational spectroscopy.
- ItemTime Optimization of Seed-Mediated Gold Nanotriangle Synthesis Based on Kinetic Studies(Basel : MDPI, 2021) Podlesnaia, Ekaterina; Csáki, Andrea; Fritzsche, WolfgangThe synthesis of shape-anisotropic plasmonic nanoparticles such as gold nanotriangles is of increasing interest. These particles have a high potential for applications due to their notable optical properties. A key challenge of the synthesis is usually the low reproducibility. Even the optimized seed-based methods often lack in the synthesis yield or are labor- and time-consuming. In this work, a seed-mediated synthesis with high reproducibility is replicated in order to determine the necessary reaction time for each step. Online monitoring of the reaction mixtures by UV–VIS spectroscopy is used as a powerful tool to track the evolution of the synthesis. The kinetics of the individual stages is elucidated by real-time investigations. As a consequence, the complete synthesis could be optimized and can now be realized in a single day instead of three without any loss in the resulting sample quality.
- ItemPresence of β-Lactamase-producing Enterobacterales and Salmonella Isolates in Marine Mammals(Basel : Molecular Diversity Preservation International (MDPI), 2021) Grünzweil, Olivia M.; Palmer, Lauren; Cabal, Adriana; Szostak, Michael P.; Ruppitsch, Werner; Kornschober, Christian; Korus, Maciej; Misic, Dusan; Bernreiter-Hofer, Tanja; Korath, Anna D. J.; Feßler, Andrea T.; Allerberger, Franz; Schwarz, Stefan; Spergser, Joachim; Müller, Elke; Braun, Sascha D.; Monecke, Stefan; Ehricht, Ralf; Walzer, Chris; Smodlaka, Hrvoje; Loncaric, IgorMarine mammals have been described as sentinels of the health of marine ecosystems. Therefore, the aim of this study was to investigate (i) the presence of extended-spectrum β-lactamase (ESBL)- and AmpC-producing Enterobacterales, which comprise several bacterial families important to the healthcare sector, as well as (ii) the presence of Salmonella in these coastal animals. The antimicrobial resistance pheno- and genotypes, as well as biocide susceptibility of Enterobacterales isolated from stranded marine mammals, were determined prior to their rehabilitation. All E. coli isolates (n = 27) were screened for virulence genes via DNA-based microarray, and twelve selected E. coli isolates were analyzed by whole-genome sequencing. Seventy-one percent of the Enterobacterales isolates exhibited a multidrug-resistant (MDR) pheno- and genotype. The gene blaCMY (n = 51) was the predominant β-lactamase gene. In addition, blaTEM-1 (n = 38), blaSHV-33 (n = 8), blaCTX-M-15 (n = 7), blaOXA-1 (n = 7), blaSHV-11 (n = 3), and blaDHA-1 (n = 2) were detected. The most prevalent non-β-lactamase genes were sul2 (n = 38), strA (n = 34), strB (n = 34), and tet(A) (n = 34). Escherichia coli isolates belonging to the pandemic sequence types (STs) ST38, ST167, and ST648 were identified. Among Salmonella isolates (n = 18), S. Havana was the most prevalent serotype. The present study revealed a high prevalence of MDR bacteria and the presence of pandemic high-risk clones, both of which are indicators of anthropogenic antimicrobial pollution, in marine mammals.
- ItemLink to glow - iEDDA conjugation of a Ruthenium(II) tetrazine complex leading to dihydropyrazine and pyrazine complexes with improved 1O2 formation ability(Amsterdam : Elsevier, 2022) Müller, Carolin; Wintergerst, Pascal; Nair, Shruthi Santhosh; Meitinger, Nicolas; Rau, Sven; Dietzek-Ivanšić, BenjaminThe synthesis and photophysical properties of the Ru-polypyridyl type complex [(tbbpy)2Ru(bptz)]2+ (Ru-bptz, tbbpy: 4,4’-di-tert-butyl-2,2’-bipyridine, bptz: 2,6-dipyrido-1,2,4,5-tetrazine), and the complexes [(tbbpy)2Ru(L)]2+ formed by inverse electron demand Diels Alder reaction (iEDDA) of Ru-bptz with with alkenes and alkynes, where L is 3,6-dipyrido-2,5-dihydropyridazine (bpdhpn) or 3,6-dipyrido-pyridazine (bppn) are described. A combination of steady-state and time-resolved spectroscopy complemented by the computation of state-specific absorption properties by means of time-dependent density functional theory reveals that the intense visible absorption band stems from Ru → tbbpy and Ru → L metal-to-ligand charge-transfer (MLCT) excitations. The studies show that lowest-lying L-centered MLCT states (3MLCTL) show comparably low emission quantum yields (3–9%) and lifetimes (90–150 ns). This correlates with the singlet oxygen generation ability, following the trend: Ru-bppn > Ru-bpdhpn > Ru-bptz.