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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.
- ItemImpact of Camera Viewing Angle for Estimating Leaf Parameters of Wheat Plants from 3D Point Clouds(Basel : MDPI, 2021) Li, Minhui; Shamshiri, Redmond R.; Schirrmann, Michael; Weltzien, CorneliaEstimation of plant canopy using low-altitude imagery can help monitor the normal growth status of crops and is highly beneficial for various digital farming applications such as precision crop protection. However, extracting 3D canopy information from raw images requires studying the effect of sensor viewing angle by taking into accounts the limitations of the mobile platform routes inside the field. The main objective of this research was to estimate wheat (Triticum aestivum L.) leaf parameters, including leaf length and width, from the 3D model representation of the plants. For this purpose, experiments with different camera viewing angles were conducted to find the optimum setup of a mono-camera system that would result in the best 3D point clouds. The angle-control analytical study was conducted on a four-row wheat plot with a row spacing of 0.17 m and with two seeding densities and growth stages as factors. Nadir and six oblique view image datasets were acquired from the plot with 88% overlapping and were then reconstructed to point clouds using Structure from Motion (SfM) and Multi-View Stereo (MVS) methods. Point clouds were first categorized into three classes as wheat canopy, soil background, and experimental plot. The wheat canopy class was then used to extract leaf parameters, which were then compared with those values from manual measurements. The comparison between results showed that (i) multiple-view dataset provided the best estimation for leaf length and leaf width, (ii) among the single-view dataset, canopy, and leaf parameters were best modeled with angles vertically at -45⸰_ and horizontally at 0⸰_ (VA -45, HA 0), while (iii) in nadir view, fewer underlying 3D points were obtained with a missing leaf rate of 70%. It was concluded that oblique imagery is a promising approach to effectively estimate wheat canopy 3D representation with SfM-MVS using a single camera platform for crop monitoring. This study contributes to the improvement of the proximal sensing platform for crop health assessment. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.
- ItemThe Effect of Diet and Farm Management on N2O Emissions from Dairy Farms Estimated from Farm Data(Basel : MDPI, 2021) Menardo, Simona; Lanza, Giacomo; Berg, WernerThe N2O emissions of 21 dairy farms in Germany were evaluated to determine the feasi-bility of an estimation of emissions from farm data and the effects of the farm management, along with possible mitigation strategies. Emissions due to the application of different fertilisers, manure storage and grazing were calculated based on equations from the IPCC (Intergovernmental Panel of Climate Change) and German emission inventory. The dependence of the N2O emissions on fertiliser type and quantity, cultivated crops and diet composition was assessed via correlation analysis and linear regression. The N2O emissions ranged between 0.11 and 0.29 kg CO2eq per kilogram energy-corrected milk, with on average 60% resulting from fertilisation and less than 30% from fertiliser storage and field applications. The total emissions had a high dependence on the diet composition; in particular, on the grass/maize ratio and the protein content of the animal diet, as well as from the manure management. A linear model for the prediction of the N2O emissions based on the diet composition and the fertilisation reached a predictive power of R2 = 0.89. As a possible mitigation strategy, the substitution of slurry for solid manure would reduce N2O emissions by 40%. Feeding cows maize-based diets instead of grass-based diets could reduce them by 14%. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.
- ItemQuantification of EGFR-HER2 Heterodimers in HER2-Overexpressing Breast Cancer Cells Using Liquid-Phase Electron Microscopy(Basel : MDPI, 2021) Peckys, Diana B.; Gaa, Daniel; de Jonge, NielsCurrently, breast cancer patients are classified uniquely according to the expression level of hormone receptors, and human epidermal growth factor receptor 2 (HER2). This coarse classification is insufficient to capture the phenotypic complexity and heterogeneity of the disease. A methodology was developed for absolute quantification of receptor surface density ρR, and molecular interaction (dimerization), as well as the associated heterogeneities, of HER2 and its family member, the epidermal growth factor receptor (EGFR) in the plasma membrane of HER2 overexpressing breast cancer cells. Quantitative, correlative light microscopy (LM) and liquid-phase electron microscopy (LPEM) were combined with quantum dot (QD) labeling. Single-molecule position data of receptors were obtained from scanning transmission electron microscopy (STEM) images of intact cancer cells. Over 280,000 receptor positions were detected and statistically analyzed. An important finding was the subcellular heterogeneity in heterodimer shares with respect to plasma membrane regions with different dynamic properties. Deriving quantitative information about EGFR and HER2 ρR, as well as their dimer percentages, and the heterogeneities thereof, in single cancer cells, is potentially relevant for early identification of patients with HER2 overexpressing tumors comprising an enhanced share of EGFR dimers, likely increasing the risk for drug resistance, and thus requiring additional targeted therapeutic strategies.
- ItemIn Vitro Examinations of Cell Death Induction and the Immune Phenotype of Cancer Cells Following Radiative-Based Hyperthermia with 915 MHz in Combination with Radiotherapy(Basel : MDPI, 2021) Hader, Michael; Streit, Simon; Rosin, Andreas; Gerdes, Thorsten; Wadepohl, Martin; Bekeschus, Sander; Fietkau, Rainer; Frey, Benjamin; Schlücker, Eberhard; Gekle, Stephan; Gaipl, Udo S.Multimodal tumor treatment settings consisting of radiotherapy and immunomodulating agents such as immune checkpoint inhibitors are more and more commonly applied in clinics. In this context, the immune phenotype of tumor cells has a major influence on the anti-tumor immune response as well as the composition of the tumor microenvironment. A promising approach to further boost anti-tumor immune responses is to add hyperthermia (HT), i.e., heating the tumor tissue between 39 °C to 45 °C for 60 min. One key technique is the use of radiative hyperthermia systems. However, knowledge is limited as to how the frequency of the used radiative systems affects the immune phenotype of the treated tumor cells. By using our self-designed in vitro hyperthermia system, we compared cell death induction and expression of immune checkpoint molecules (ICM) on the tumor cell surface of murine B16 melanoma and human MDA-MB-231 and MCF-7 breast cancer cells following HT treatment with clinically relevant microwaves at 915 MHz or 2.45 GHz alone, radiotherapy (RT; 2 × 5 Gy or 5 × 2 Gy) alone or in combination (RHT). At 44 °C, HT alone was the dominant cell death inductor with inactivation rates of around 70% for B16, 45% for MDA-MB-231 and 35% for MCF-7 at 915 MHz and 80%, 60% and 50% at 2.45 GHz, respectively. Additional RT resulted in 5-15% higher levels of dead cells. The expression of ICM on tumor cells showed time-, treatment-, cell line- and frequency-dependent effects and was highest for RHT. Computer simulations of an exemplary spherical cell revealed frequency-dependent local energy absorption. The frequency of hyperthermia systems is a newly identified parameter that could also affect the immune phenotype of tumor cells and consequently the immunogenicity of tumors.
- ItemCruciate Ligament Cell Sheets Can Be Rapidly Produced on Thermoresponsive poly(glycidyl ether) Coating and Successfully Used for Colonization of Embroidered Scaffolds(Basel : MDPI, 2021) Zahn, Ingrid; Stöbener, Daniel David; Weinhart, Marie; Gögele, Clemens; Breier, Annette; Hahn, Judith; Schröpfer, Michaela; Meyer, Michael; Schulze-Tanzil, GundulaAnterior cruciate ligament (ACL) cell sheets combined with biomechanically competent scaffolds might facilitate ACL tissue engineering. Since thermoresponsive polymers allow a rapid enzyme-free detachment of cell sheets, we evaluated the applicability of a thermoresponsive poly(glycidyl ether) (PGE) coating for cruciate ligamentocyte sheet formation and its influence on ligamentocyte phenotype during sheet-mediated colonization of embroidered scaffolds. Ligamentocytes were seeded on surfaces either coated with PGE or without coating. Detached ligamentocyte sheets were cultured separately or wrapped around an embroidered scaffold made of polylactide acid (PLA) and poly(lactic-co-ε-caprolactone) (P(LA-CL)) threads functionalized by gas-phase fluorination and with collagen foam. Ligamentocyte viability, protein and gene expression were determined in sheets detached from surfaces with or without PGE coating, scaffolds seeded with sheets from PGE-coated plates and the respective monolayers. Stable and vital ligamentocyte sheets could be produced within 24 h with both surfaces, but more rapidly with PGE coating. PGE did not affect ligamentocyte phenotype. Scaffolds could be colonized with sheets associated with high cell survival, stable gene expression of ligament-related type I collagen, decorin, tenascin C and Mohawk after 14 d and extracellular matrix (ECM) deposition. PGE coating facilitates ligamentocyte sheet formation, and sheets colonizing the scaffolds displayed a ligament-related phenotype.
- ItemIndicative Marker Microbiome Structures Deduced from the Taxonomic Inventory of 67 Full-Scale Anaerobic Digesters of 49 Agricultural Biogas Plants(Basel : MDPI, 2021) Hassa, Julia; Klang, Johanna; Benndorf, Dirk; Pohl, Marcel; Hülsemann, Benedikt; Mächtig, Torsten; Effenberger, Mathias; Pühler, Alfred; Schlüter, Andreas; Theuerl, SusanneThere are almost 9500 biogas plants in Germany, which are predominantly operated with energy crops and residues from livestock husbandry over the last two decades. In the future, biogas plants must be enabled to use a much broader range of input materials in a flexible and demand-oriented manner. Hence, the microbial communities will be exposed to frequently varying process conditions, while an overall stable process must be ensured. To accompany this transition, there is the need to better understand how biogas microbiomes respond to management measures and how these responses affect the process efficiency. Therefore, 67 microbiomes originating from 49 agricultural, full-scale biogas plants were taxonomically investigated by 16S rRNA gene amplicon sequencing. These microbiomes were separated into three distinct clusters and one group of outliers, which are characterized by a specific distribution of 253 indicative taxa and their relative abundances. These indicative taxa seem to be adapted to specific process conditions which result from a different biogas plant operation. Based on these results, it seems to be possible to deduce/assess the general process condition of a biogas digester based solely on the microbiome structure, in particular on the distribution of specific indicative taxa, and without knowing the corresponding operational and chemical process parameters. Perspectively, this could allow the development of detection systems and advanced process models considering the microbial diversity.
- ItemThe Role of Streptococcus spp. in Bovine Mastitis(Basel : MDPI, 2021) Kabelitz, Tina; Aubry, Etienne; van Vorst, Kira; Amon, Thomas; Fulde, MarcusThe Streptococcus genus belongs to one of the major pathogen groups inducing bovine mastitis. In the dairy industry, mastitis is the most common and costly disease. It not only negatively impacts economic profit due to milk losses and therapy costs, but it is an important animal health and welfare issue as well. This review describes a classification, reservoirs, and frequencies of the most relevant Streptococcus species inducing bovine mastitis (S. agalactiae, S. dysgalactiae and S. uberis). Host and environmental factors influencing mastitis susceptibility and infection rates will be discussed, because it has been indicated that Streptococcus herd prevalence is much higher than mastitis rates. After infection, we report the sequence of cow immune reactions and differences in virulence factors of the main Streptococcus species. Different mastitis detection techniques together with possible conventional and alternative therapies are described. The standard approach treating streptococcal mastitis is the application of ß-lactam antibiotics. In streptococci, increased antimicrobial resistance rates were identified against enrofloxacin, tetracycline, and erythromycin. At the end, control and prevention measures will be considered, including vaccination, hygiene plan, and further interventions. It is the aim of this review to estimate the contribution and to provide detailed knowledge about the role of the Streptococcus genus in bovine mastitis.
- 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.