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- ItemSurface-Enhanced Raman Spectroscopy to Characterize Different Fractions of Extracellular Vesicles from Control and Prostate Cancer Patients(Basel : MDPI, 2021) Osei, Eric Boateng; Paniushkina, Liliia; Wilhelm, Konrad; Popp, Jürgen; Nazarenko, Irina; Krafft, ChristophExtracellular vesicles (EVs) are membrane-enclosed structures ranging in size from about 60 to 800 nm that are released by the cells into the extracellular space; they have attracted interest as easily available biomarkers for cancer diagnostics. In this study, EVs from plasma of control and prostate cancer patients were fractionated by differential centrifugation at 5000× g, 12,000× g and 120,000× g. The remaining supernatants were purified by ultrafiltration to produce EV-depleted free-circulating (fc) fractions. Spontaneous Raman and surface-enhanced Raman spectroscopy (SERS) at 785 nm excitation using silver nanoparticles (AgNPs) were employed as label-free techniques to collect fingerprint spectra and identify the fractions that best discriminate between control and cancer patients. SERS spectra from 10 µL droplets showed an enhanced Raman signature of EV-enriched fractions that were much more intense for cancer patients than controls. The Raman spectra of dehydrated pellets of EV-enriched fractions without AgNPs were dominated by spectral contributions of proteins and showed variations in S-S stretch, tryptophan and protein secondary structure bands between control and cancer fractions. We conclude that the AgNPs-mediated SERS effect strongly enhances Raman bands in EV-enriched fractions, and the fractions, EV12 and EV120 provide the best separation of cancer and control patients by Raman and SERS spectra.
- ItemPEGylation of Guanidinium and Indole Bearing Poly(methacrylamide)s - Biocompatible Terpolymers for pDNA Delivery(Weinheim : Wiley-VCH, 2021) Cokca, Ceren; Hack, Franz J.; Costabel, Daniel; Herwig, Kira; Hülsmann, Juliana; Then, Patrick; Heintzmann, Rainer; Fischer, Dagmar; Peneva, KalinaThis study describes the first example for shielding of a high performing terpolymer that consists of N-(2-hydroxypropyl)methacrylamide (HPMA), N-(3-guanidinopropyl)methacrylamide (GPMA), and N-(2-indolethyl)methacrylamide monomers (IEMA) by block copolymerization of a polyethylene glycol derivative – poly(nona(ethylene glycol)methyl ether methacrylate) (P(MEO9MA)) via reversible addition–fragmentation chain transfer (RAFT) polymerization. The molecular weight of P(MEO9MA) is varied from 3 to 40 kg mol–1 while the comonomer content of HPMA, GPMA, and IEMA is kept comparable. The influence of P(MEO9MA) block with various molecular weights is investigated over cytotoxicity, plasmid DNA (pDNA) binding, and transfection efficiency of the resulting polyplexes. Overall, the increase in molecular weight of P(MEO9MA) block demonstrates excellent biocompatibility with higher cell viability in L-929 cells and an efficient binding to pDNA at N/P ratio of 2. The significant transfection efficiency in CHO-K1 cells at N/P ratio 20 is obtained for block copolymers with molecular weight of P(MEO9MA) up to 10 kg mol–1. Moreover, a fluorescently labeled analogue of P(MEO9MA), bearing perylene monoimide methacrylamide (PMIM), is introduced as a comonomer in RAFT polymerization. Polyplexes consisting of labeled block copolymer with 20 kg mol–1 of P(MEO9MA) and pDNA are incubated in Hela cells and investigated through structured illumination microscopy (SIM).
- ItemLong-Term Sinonasal Carriage of Staphylococcus aureus and Anti-Staphylococcal Humoral Immune Response in Patients with Chronic Rhinosinusitis(Basel : MDPI, 2021) Thunberg, Ulrica; Hugosson, Svante; Ehricht, Ralf; Monecke, Stefan; Müller, Elke; Cao, Yang; Stegger, Marc; Söderquist, BoWe investigated Staphylococcus aureus diversity, genetic factors, and humoral immune responses against antigens via genome analysis of S. aureus isolates from chronic rhinosinusitis (CRS) patients in a long-term follow-up. Of the 42 patients who provided S. aureus isolates and serum for a previous study, 34 could be included for follow-up after a decade. Clinical examinations were performed and bacterial samples were collected from the maxillary sinus and nares. S. aureus isolates were characterized by whole-genome sequencing, and specific anti-staphylococcal IgG in serum was determined using protein arrays. S. aureus was detected in the nares and/or maxillary sinus at both initial inclusion and follow-up in 15 of the 34 respondents (44%). Three of these (20%) had S. aureus isolates from the same genetic lineage as at inclusion. A low number of single-nucleotide polymorphisms (SNPs) were identified when comparing isolates from nares and maxillary sinus collected at the same time point. The overall change of antibody responses to staphylococcal antigens over time showed great variability, and no correlation was found between the presence of genes encoding antigens and the corresponding anti-staphylococcal IgG in serum; thus our findings did not support a role, in CRS, of the specific S. aureus antigens investigated.
- ItemThe Pheno- and Genotypic Characterization of Porcine Escherichia coli Isolates(Basel : MDPI, 2021) Bernreiter-Hofer, Tanja; Schwarz, Lukas; Müller, Elke; Cabal-Rosel, Adriana; Korus, Maciej; Misic, Dusan; Frankenfeld, Katrin; Abraham, Kerstin; Grünzweil, Olivia; Weiss, Astrid; Feßler, Andrea T.; Allerberger, Franz; Schwarz, Stefan; Szostak, Michael P.; Ruppitsch, Werner; Ladinig, Andrea; Spergser, Joachim; Braun, Sascha D.; Monecke, Stefan; Ehricht, Ralf; Loncaric, IgorEscherichia (E.) coli is the main causative pathogen of neonatal and post-weaning diarrhea and edema disease in swine production. There is a significant health concern due to an increasing number of human infections associated with food and/or environmental-borne pathogenic and multidrug-resistant E. coli worldwide. Monitoring the presence of pathogenic and antimicrobial-resistant E. coli isolates is essential for sustainable disease management in livestock and human medicine. A total of 102 E. coli isolates of diseased pigs were characterized by antimicrobial and biocide susceptibility testing. Antimicrobial resistance genes, including mobile colistin resistance genes, were analyzed by PCR and DNA sequencing. The quinolone resistance-determining regions of gyrA and parC in ciprofloxacin-resistant isolates were analyzed. Clonal relatedness was investigated by two-locus sequence typing (CH clonotyping). Phylotyping was performed by the Clermont multiplex PCR method. Virulence determinants were analyzed by customized DNA-based microarray technology developed in this study for fast and economic molecular multiplex typing. Thirty-five isolates were selected for whole-genome sequence-based analysis. Most isolates were resistant to ampicillin and tetracycline. Twenty-one isolates displayed an ESBL phenotype and one isolate an AmpC β-lactamase-producing phenotype. Three isolates had elevated colistin minimal inhibitory concentrations and carried the mcr-1 gene. Thirty-seven isolates displayed a multi-drug resistance phenotype. The most predominant β-lactamase gene classes were blaTEM-1 (56%) and blaCTX-M-1 (13.71%). Mutations in QRDR were observed in 14 ciprofloxacin-resistant isolates. CH clonotyping divided all isolates into 51 CH clonotypes. The majority of isolates belonged to phylogroup A. Sixty-four isolates could be assigned to defined pathotypes wherefrom UPEC was predominant. WGS revealed that the most predominant sequence type was ST100, followed by ST10. ST131 was detected twice in our analysis. This study highlights the importance of monitoring antimicrobial resistance and virulence properties of porcine E. coli isolates. This can be achieved by applying reliable, fast, economic and easy to perform technologies such as DNA-based microarray typing. The presence of high-risk pathogenic multi-drug resistant zoonotic clones, as well as those that are resistant to critically important antibiotics for humans, can pose a risk to public health. Improved protocols may be developed in swine farms for preventing infections, as well as the maintenance and distribution of the causative isolates.
- ItemRaman Stable Isotope Probing of Bacteria in Visible and Deep UV-Ranges(Basel : MDPI, 2021) Azemtsop Matanfack, Georgette; Pistiki, Aikaterini; Rösch, Petra; Popp, JürgenRaman stable isotope probing (Raman-SIP) is an excellent technique that can be used to access the overall metabolism of microorganisms. Recent studies have mainly used an excitation wavelength in the visible range to characterize isotopically labeled bacteria. In this work, we used UV resonance Raman spectroscopy (UVRR) to evaluate the spectral red-shifts caused by the uptake of isotopes (13C, 15N, 2H(D) and 18O) in E. coli cells. Moreover, we present a new approach based on the extraction of labeled DNA in combination with UVRR to identify metabolically active cells. The proof-of-principle study on E. coli revealed heterogeneities in the Raman features of both the bacterial cells and the extracted DNA after labeling with 13C, 15N, and D. The wavelength of choice for studying 18O- and deuterium-labeled cells is 532 nm is, while 13C-labeled cells can be investigated with visible and deep UV wavelengths. However, 15N-labeled cells are best studied at the excitation wavelength of 244 nm since nucleic acids are in resonance at this wavelength. These results highlight the potential of the presented approach to identify active bacterial cells. This work can serve as a basis for the development of new techniques for the rapid and efficient detection of active bacteria cells without the need for a cultivation step.
- ItemGrasshopper herbivory immediately affects element cycling but not export rates in an N‐limited grassland system(Ithaca, NY : ESA, 2021) Potthast, Karin; Meyer, Stefanie; Tischer, Alexander; Gleixner, Gerd; Sieburg, Anne; Frosch, Torsten; Michalzik, BeateAs a cause of ecosystem disturbances, phytophagous insects are known to directly influence the element and organic matter (OM) cycling in ecosystems by their defoliation and excretion activity. This study focuses on the interplay between short-term, insect herbivory, plant responses to feeding activity, rhizosphere processes, and belowground nutrient availability under nutrient-poor soil conditions. To test the effects of insect herbivory on OM and nutrient cycling in an N-limited pasture system, mesocosm laboratory experiments were conducted using Dactylis glomerata as common grass species and Chorthippus dorsatus, a widespread grasshopper species, to induce strong defoliating herbivory. 13CO2 pulse labeling was used together with labeled 15N feces to trace the fate of C in soil respiration at the beginning of herbivory, and of C and N in above- and belowground plant biomass, grasshopper, feces, bulk soil, soil microbial biomass, throughfall solutions, and soil solutions. Within five days, herbivory caused a reduction in aboveground grass biomass by about 34%. A linear mixed-effects model revealed that herbivory significantly increased total dissolved C and N amounts in throughfall solutions by a factor of 4–10 (P < 0.05) compared with the control. In total, 27.6% of the initially applied feces 15N were translocated from the aboveground to the belowground system. A significant enrichment of 15N in roots led to the assumption that feces-derived 15N was rapidly taken up to compensate for the frass-related foliar N losses in light of N shortage. Soil microorganisms incorporated newly available 13C; however, the total amount of soil microbial biomass remained unaffected, while the exploitative grass species rapidly sequestered resources to facilitate its regrowth after herbivory attack. Heavy herbivory by insects infesting D. glomerata-dominated, N-deficient grasslands remarkably impacted belowground nutrient cycling by an instant amplification of available nutrients, which led to an intensified nutrient competition between plants and soil microorganisms. Consequently, these competitive plant–soil microbe interactions accelerated N cycling and effectively retained herbivory-mediated C and N surplus release resulting in diminished N losses from the system. The study highlighted the overarching role of plant adaptations to in situ soil fertility in short-term ecosystem disturbances.
- ItemBiochemical Analysis of Leukocytes after In Vitro and In Vivo Activation with Bacterial and Fungal Pathogens Using Raman Spectroscopy(Basel : MDPI, 2021) Pistiki, Aikaterini; Ramoji, Anuradha; Ryabchykov, Oleg; Thomas-Rueddel, Daniel; Press, Adrian T.; Makarewicz, Oliwia; Giamarellos-Bourboulis, Evangelos J.; Bauer, Michael; Bocklitz, Thomas; Popp, Juergen; Neugebauer, UteBiochemical information from activated leukocytes provide valuable diagnostic information. In this study, Raman spectroscopy was applied as a label-free analytical technique to characterize the activation pattern of leukocyte subpopulations in an in vitro infection model. Neutrophils, monocytes, and lymphocytes were isolated from healthy volunteers and stimulated with heat-inactivated clinical isolates of Candida albicans, Staphylococcus aureus, and Klebsiella pneumoniae. Binary classification models could identify the presence of infection for monocytes and lymphocytes, classify the type of infection as bacterial or fungal for neutrophils, monocytes, and lymphocytes and distinguish the cause of infection as Gram-negative or Gram-positive bacteria in the monocyte subpopulation. Changes in single-cell Raman spectra, upon leukocyte stimulation, can be explained with biochemical changes due to the leukocyte’s specific reaction to each type of pathogen. Raman spectra of leukocytes from the in vitro infection model were compared with spectra from leukocytes of patients with infection (DRKS-ID: DRKS00006265) with the same pathogen groups, and a good agreement was revealed. Our study elucidates the potential of Raman spectroscopy-based single-cell analysis for the differentiation of circulating leukocyte subtypes and identification of the infection by probing the molecular phenotype of those cells.
- 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.
- 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.
- 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.