Search Results
Surface-Enhanced Raman Spectroscopy to Characterize Different Fractions of Extracellular Vesicles from Control and Prostate Cancer Patients
Extracellular 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.
Influence of redox stress on crosstalk between fibroblasts and keratinocytes
Although the skin is constantly subjected to endogenous and exogenous stress, it maintains a homeostatic state through wound repair and regeneration pathways. Treatment for skin diseases and injury requires a significant understanding of the various mechanisms and interactions that occur within skin cells. Keratinocytes and fibroblasts interact with each other and act as key players in the repair process. Although fibroblasts and keratinocytes are widely studied in wound healing and skin remodeling under different conditions, the influence of redox stress on keratinocyte-fibroblast crosstalk has not been thoroughly investigated. In this study, we used cold atmospheric plasma (CAP) to generate and deliver oxidative stress to keratinocytes and fibroblasts and to assess its impact on their interactions. To this end, we used a well-established in vitro 3D co-culture model imitating a realistic scenario. Our study shows that low CAP exposure is biocompatible and does not affect the viability or energetics of fibroblasts and keratinocytes. Exposure to low doses of CAP enhanced the proliferation rate of cells and stimulated the expression of key genes (KGF, MMP2, GMCSF, IL-6, and IL-8) in fibroblasts, indicating the activation and initiation of the skin repair process. Additionally, enhanced migration was observed under co-culture conditions under the given redox stress conditions, and expression of the upstream regulator and the effectors of the Hippo pathway (YAP and CYR61, respectively), which are associated with enhanced migration, were elevated. Overall, this study reinforces the application of CAP and redox stress in skin repair physiology.
Particulate Matter Dispersion Modeling in Agricultural Applications: Investigation of a Transient Open Source Solver
Agriculture is a major emitter of particulate matter (PM), which causes health problems and can act as a carrier of the pathogen material that spreads diseases. The aim of this study was to investigate an open-source solver that simulates the transport and dispersion of PM for typical agricultural applications. We investigated a coupled Eulerian–Lagrangian solver within the open source software package OpenFOAM. The continuous phase was solved using transient large eddy simulations, where four different subgrid-scale turbulence models and an inflow turbulence generator were tested. The discrete phase was simulated using two different Lagrangian solvers. For the validation case of a turbulent flow of a street canyon, the flowfield could be recaptured very well, with errors of around 5% for the non-equilibrium turbulence models (WALE and dynamicKeq) in the main regions. The inflow turbulence generator could create a stable and accurate boundary layer for the mean vertical velocity and vertical profile of the turbulent Reynolds stresses R11. The validation of the Lagrangian solver showed mixed results, with partly good agreements (simulation results within the measurement uncertainty), and partly high deviations of up to 80% for the concentration of particles. The higher deviations were attributed to an insufficient turbulence regime of the used validation case, which was an experimental chamber. For the simulation case of PM dispersion from manure application on a field, the solver could capture the influence of features such as size and density on the dispersion. The investigated solver is especially useful for further investigations into time-dependent processes in the near-source area of PM sources.
Eosinophils and Neutrophils-Molecular Differences Revealed by Spontaneous Raman, CARS and Fluorescence Microscopy
Leukocytes are a part of the immune system that plays an important role in the host's defense against viral, bacterial, and fungal infections. Among the human leukocytes, two granulocytes, neutrophils (Ne) and eosinophils (EOS) play an important role in the innate immune system. For that purpose, eosinophils and neutrophils contain specific granules containing protoporphyrin-type proteins such as eosinophil peroxidase (EPO) and myeloperoxidase (MPO), respectively, which contribute directly to their anti-infection activity. Since both proteins are structurally and functionally different, they could potentially be a marker of both cells' types. To prove this hypothesis, UV-Vis absorption spectroscopy and Raman imaging were applied to analyze EPO and MPO and their content in leukocytes isolated from the whole blood. Moreover, leukocytes can contain lipidic structures, called lipid bodies (LBs), which are linked to the regulation of immune responses and are considered to be a marker of cell inflammation. In this work, we showed how to determine the number of LBs in two types of granulocytes, EOS and Ne, using fluorescence and coherent anti-Stokes Raman scattering (CARS) microscopy. Spectroscopic differences of EPO and MPO can be used to identify these cells in blood samples, while the detection of LBs can indicate the cell inflammation process.
Cold atmospheric plasma is a potent tool to improve chemotherapy in melanoma in vitro and in vivo
Malignant melanoma is a devastating disease. Because of its aggressiveness, it also serves as a model tumor for investigating novel therapeutic avenues. In recent years, scientific evidence has shown that cold atmospheric plasma (CAP) might be a promising modality in cancer therapy. In this study, we aimed to evaluate the effect of CAP generated by an argon plasma jet alone or in combination with dacarbazine (DAC) on melanoma cells in vitro and in vivo. The effects of the CAP on inducing lipid peroxidation and nitric oxide production were higher in B16 melanoma cells in comparison to non-malignant L929 cells. Assays on cell growth, apoptosis, and expression of genes related to, e.g., autophagic processes, showed CAP to have a substantial impact in melanoma cells while there were only minoreffects in L929 cells. In vivo, both CAP monotherapy and combination with DAC significantly decreased tumor growth. These results suggest that CAP not only selectively induces cell death in melanoma but also holds promises in combination with chemotherapy that might lead to improved tumor control. © 2020 by the authors.
Starch and Glycogen Analyses : Methods and Techniques
For complex carbohydrates, such as glycogen and starch, various analytical methods and techniques exist allowing the detailed characterization of these storage carbohydrates. In this article, we give a brief overview of the most frequently used methods, techniques, and results. Furthermore, we give insights in the isolation, purification, and fragmentation of both starch and glycogen. An overview of the different structural levels of the glucans is given and the corresponding analytical techniques are discussed. Moreover, future perspectives of the analytical needs and the challenges of the currently developing scientific questions are included.
The Effect of Diet and Farm Management on N2O Emissions from Dairy Farms Estimated from Farm Data
The 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.
On the liquid chemistry of the reactive nitrogen species peroxynitrite and nitrogen dioxide generated by physical plasmas
Cold physical plasmas modulate cellular redox signaling processes, leading to the evolution of a number of clinical applications in recent years. They are a source of small reactive species, including reactive nitrogen species (RNS). Wound healing is a major application and, as its physiology involves RNS signaling, a correlation between clinical effectiveness and the activity of plasma‐derived RNS seems evident. To investigate the type and reactivity of plasma‐derived RNS in aqueous systems, a model with tyrosine as a tracer was utilized. By high‐resolution mass spectrometry, 26 different tyrosine derivatives including the physiologic nitrotyrosine were identified. The product pattern was distinctive in terms of plasma parameters, especially gas phase composition. By scavenger experiments and isotopic labelling, gaseous nitric dioxide radicals and liquid phase peroxynitrite ions were determined as dominant RNS. The presence of water molecules in the active plasma favored the generation of peroxynitrite. A pilot study, identifying RNS driven post‐translational modifications of proteins in healing human wounds after the treatment with cold plasma (kINPen), demonstrated the presence of in vitro determined chemical pathways. The plasma‐driven nitration and nitrosylation of tyrosine allows the conclusion that covalent modification of biomolecules by RNS contributes to the clinically observed impact of cold plasmas. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.
Impact of Camera Viewing Angle for Estimating Leaf Parameters of Wheat Plants from 3D Point Clouds
Estimation 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.
Comparison of Multiscale Imaging Methods for Brain Research
A major challenge in neuroscience is how to study structural alterations in the brain. Even small changes in synaptic composition could have severe outcomes for body functions. Many neuropathological diseases are attributable to disorganization of particular synaptic proteins. Yet, to detect and comprehensively describe and evaluate such often rather subtle deviations from the normal physiological status in a detailed and quantitative manner is very challenging. Here, we have compared side-by-side several commercially available light microscopes for their suitability in visualizing synaptic components in larger parts of the brain at low resolution, at extended resolution as well as at super-resolution. Microscopic technologies included stereo, widefield, deconvolution, confocal, and super-resolution set-ups. We also analyzed the impact of adaptive optics, a motorized objective correction collar and CUDA graphics card technology on imaging quality and acquisition speed. Our observations evaluate a basic set of techniques, which allow for multi-color brain imaging from centimeter to nanometer scales. The comparative multi-modal strategy we established can be used as a guide for researchers to select the most appropriate light microscopy method in addressing specific questions in brain research, and we also give insights into recent developments such as optical aberration corrections.