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- ItemAutomated and rapid identification of multidrug resistant Escherichia coli against the lead drugs of acylureidopenicillins, cephalosporins, and fluoroquinolones using specific Raman marker bands(Weinheim : Wiley-VCH-Verl., 2020) Götz, Theresa; Dahms, Marcel; Kirchhoff, Johanna; Beleites, Claudia; Glaser, Uwe; Bohnert, Jürgen A.; Pletz, Mathias W.; Popp, Jürgen; Schlattmann, Peter; Neugebauer, UteA Raman-based, strain-independent, semi-automated method is presented that allows the rapid (<3 hours) determination of antibiotic susceptibility of bacterial pathogens isolated from clinical samples. Applying a priori knowledge about the mode of action of the respective antibiotic, we identified characteristic Raman marker bands in the spectrum and calculated batch-wise weighted sum scores from standardized Raman intensity differences between spectra of antibiotic exposed and nonexposed samples of the same strains. The lead substances for three relevant antibiotic classes (fluoroquinolone ciprofloxacin, third-generation cephalosporin cefotaxime, ureidopenicillin piperacillin) against multidrug-resistant Gram-negative bacteria (MRGN) revealed a high sensitivity and specificity for the susceptibility testing of two Escherichia coli laboratory strains and 12 clinical isolates. The method benefits from the parallel incubation of control and treated samples, which reduces the variance due to alterations in cultivation conditions and the standardization of differences between batches leading to long-term comparability of Raman measurements. © 2020 The Authors. Journal of Biophotonics published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
- ItemAn Innovative Protocol for Metaproteomic Analyses of Microbial Pathogens in Cystic Fibrosis Sputum(Lausanne : Frontiers Media, 2021) Graf, Alexander C.; Striesow, Johanna; Pané-Farré, Jan; Sura, Thomas; Wurster, Martina; Lalk, Michael; Pieper, Dietmar H.; Becher, Dörte; Kahl, Barbara C.; Riedel, KatharinaHallmarks of cystic fibrosis (CF) are increased viscosity of mucus and impaired mucociliary clearance within the airways due to mutations of the cystic fibrosis conductance regulator gene. This facilitates the colonization of the lung by microbial pathogens and the concomitant establishment of chronic infections leading to tissue damage, reduced lung function, and decreased life expectancy. Although the interplay between key CF pathogens plays a major role during disease progression, the pathophysiology of the microbial community in CF lungs remains poorly understood. Particular challenges in the analysis of the microbial population present in CF sputum is (I) the inhomogeneous, viscous, and slimy consistence of CF sputum, and (II) the high number of human proteins masking comparably low abundant microbial proteins. To address these challenges, we used 21 CF sputum samples to develop a reliable, reproducible and widely applicable protocol for sputum processing, microbial enrichment, cell disruption, protein extraction and subsequent metaproteomic analyses. As a proof of concept, we selected three sputum samples for detailed metaproteome analyses and complemented and validated metaproteome data by 16S sequencing, metabolomic as well as microscopic analyses. Applying our protocol, the number of bacterial proteins/protein groups increased from 199-425 to 392-868 in enriched samples compared to nonenriched controls. These early microbial metaproteome data suggest that the arginine deiminase pathway and multiple proteases and peptidases identified from various bacterial genera could so far be underappreciated in their contribution to the CF pathophysiology. By providing a standardized and effective protocol for sputum processing and microbial enrichment, our study represents an important basis for future studies investigating the physiology of microbial pathogens in CF in vivo – an important prerequisite for the development of novel antimicrobial therapies to combat chronic recurrent airway infection in CF.
- ItemSugar-Modified Poly(propylene imine) Dendrimers Stimulate the NF-κB Pathway in a Myeloid Cell Line(Dordrecht [u.a.] : Springer Science + Business Media B.V, 2016) Jatczak-Pawlik, Izabela; Gorzkiewicz, Michal; Studzian, Maciej; Appelhans, Dietmar; Voit, Brigitte; Pulaski, Lukasz; Klajnert-Maculewicz, BarbaraPurpose: Fourth-generation poly(propylene imine) dendrimers fully surface-modified by maltose (dense shell, PPI-m DS) were shown to be biocompatible in cellular models, which is important for their application in drug delivery. We decided to verify also their inherent bioactivity, including immunomodulatory activity, for potential clinical applications. We tested their effects on the THP-1 monocytic cell line model of innate immunity effectors. Methods: To estimate the cytotoxicity of dendrimers the reasazurin assay was performed. The expression level of NF-κB targets: IGFBP3, TNFAIP3 and TNF was determined by quantitative real-time RT-PCR. Measurement of NF-κB p65 translocation from cytoplasm to nucleus was conducted with a high-content screening platform and binding of NF-κB to a consensus DNA probe was determined by electrophoretic mobility shift assay. The cytokine assay was performed to measure protein concentration of TNFalpha and IL-4. Results: We found that PPI-m DS did not impact THP-1 viability and growth even at high concentrations (up to 100 μM). They also did not induce expression of genes for important signaling pathways: Jak/STAT, Keap1/Nrf2 and ER stress. However, high concentrations of 4th generation PPI-m DS (25–100 μM), but not their 3rd generation counterparts, induced nuclear translocation of p65 NF-κB protein and its DNA-binding activity, leading to NF-κB-dependent increased expression of mRNA for NF-κB targets: IGFBP3, TNFAIP3 and TNF. However, no increase in pro-inflammatory cytokine secretion was detected. Conclusion: We conclude that maltose-modified PPI dendrimers of specific size could exert a modest immunomodulatory effect, which may be advantageous in clinical applications (e.g. adjuvant effect in anti-cancer vaccines).
- ItemGas Flow Shaping via Novel Modular Nozzle System (MoNoS) Augments kINPen-Mediated Toxicity and Immunogenicity in Tumor Organoids(Basel : MDPI, 2023) Berner, Julia; Miebach, Lea; Herold, Luise; Höft, Hans; Gerling, Torsten; Mattern, Philipp; Bekeschus, SanderMedical gas plasma is an experimental technology for anticancer therapy. Here, partial gas ionization yielded reactive oxygen and nitrogen species, placing the technique at the heart of applied redox biomedicine. Especially with the gas plasma jet kINPen, anti-tumor efficacy was demonstrated. This study aimed to examine the potential of using passive flow shaping to enhance the medical benefits of atmospheric plasma jets (APPJ). We used an in-house developed, proprietary Modular Nozzle System (MoNoS; patent-pending) to modify the flow properties of a kINPen. MoNoS increased the nominal plasma jet-derived reactive species deposition area and stabilized the air-plasma ratio within the active plasma zone while shielding it from external flow disturbances or gas impurities. At modest flow rates, dynamic pressure reduction (DPR) adapters did not augment reactive species deposition in liquids or tumor cell killing. However, MoNoS operated at kINPen standard argon fluxes significantly improved cancer organoid growth reduction and increased tumor immunogenicity, as seen by elevated calreticulin and heat-shock protein expression, along with a significantly spurred cytokine secretion profile. Moreover, the safe application of MoNoS gas plasma jet adapters was confirmed by their similar-to-superior safety profiles assessed in the hen’s egg chorioallantoic membrane (HET-CAM) coagulation and scar formation irritation assay.
- ItemIn vivo ligamentogenesis in embroidered poly(lactic-co-ε-caprolactone) / polylactic acid scaffolds functionalized by fluorination and hexamethylene diisocyanate cross-linked collagen foams(Berlin ; Heidelberg : Springer, 2022) Kokozidou, Maria; Gögele, Clemens; Pirrung, Felix; Hammer, Niels; Werner, Christian; Kohl, Benjamin; Hahn, Judith; Breier, Annette; Schröpfer, Michaela; Meyer, Michael; Schulze-Tanzil, GundulaAlthough autografts represent the gold standard for anterior cruciate ligament (ACL) reconstruction, tissue-engineered ACLs provide a prospect to minimize donor site morbidity and limited graft availability. This study characterizes the ligamentogenesis in embroidered poly(L-lactide-co-epsilon-caprolactone) (P(LA-CL)) / polylactic acid (PLA) constructs using a dynamic nude mice xenograft model. (P(LA-CL))/PLA scaffolds remained either untreated (co) or were functionalized by gas fluorination (F), collagen foam cross-linked with hexamethylene diisocyanate (HMDI) (coll), or F combined with the foam (F+coll). Cell-free constructs or those seeded for 1 week with lapine ACL ligamentocytes were implanted into nude mice for 12 weeks. Following explantation, cell vitality and content, histo(patho)logy of scaffolds (including organs: liver, kidney, spleen), sulphated glycosaminoglycan (sGAG) contents and biomechanical properties were assessed. Scaffolds did not affect mice weight development and organs, indicating no organ toxicity. Moreover, scaffolds maintained their size and shape and reflected a high cell viability prior to and following implantation. Coll or F+coll scaffolds seeded with cells yielded superior macroscopic properties compared to the controls. Mild signs of inflammation (foreign-body giant cells and hyperemia) were limited to scaffolds without collagen. Microscopical score values and sGAG content did not differ significantly. Although remaining stable after explantation, elastic modulus, maximum force, tensile strength and strain at F-max were significantly lower in explanted scaffolds compared to those before implantation, with no significant differences between scaffold subtypes, except for a higher maximum force in F+coll compared with F samples (in vivo). Scaffold functionalization with fluorinated collagen foam provides a promising approach for ACL tissue engineering.
- ItemROS Pleiotropy in Melanoma and Local Therapy with Physical Modalities(Austin, Tex. : Landes Bioscience, 2021) Sagwal, Sanjeev Kumar; Bekeschus, SanderMetabolic energy production naturally generates unwanted products such as reactive oxygen species (ROS), causing oxidative damage. Oxidative damage has been linked to several pathologies, including diabetes, premature aging, neurodegenerative diseases, and cancer. ROS were therefore originally anticipated as an imperative evil, a product of an imperfect system. More recently, however, the role of ROS in signaling and tumor treatment is increasingly acknowledged. This review addresses the main types, sources, and pathways of ROS in melanoma by linking their pleiotropic roles in antioxidant and oxidant regulation, hypoxia, metabolism, and cell death. In addition, the implications of ROS in various physical therapy modalities targeting melanoma, such as radiotherapy, electrochemotherapy, hyperthermia, photodynamic therapy, and medical gas plasma, are also discussed. By including ROS in the main picture of melanoma skin cancer and as an integral part of cancer therapies, a greater understanding of melanoma cell biology is presented, which ultimately may elucidate additional clues on targeting therapy resistance of this most deadly form of skin cancer.
- ItemThe HIPPO Transducer YAP and Its Targets CTGF and Cyr61 Drive a Paracrine Signalling in Cold Atmospheric Plasma-Mediated Wound Healing(London: Hindawi, 2019) Shome, Debarati; von Woedtke, Thomas; Riedel, Katharina; Masur, KaiReactive species play a pivotal role in orchestrating wound healing responses. They act as secondary messengers and drive redox-signalling pathways that are involved in the homeostatic, inflammatory, proliferative, and remodelling phases of wound healing. The application of Cold Atmospheric Plasma (CAP) to the wound site produces a profusion of short- and long-lived reactive species that have been demonstrated to be effective in promoting wound healing; however, knowledge of the mechanisms underlying CAP-mediated wound healing remains scarce. To address this, an in vitro coculture model was used to study the effects of CAP on wound healing and on paracrine crosstalk between dermal keratinocytes and fibroblasts. Using this coculture model, we observed a stimulatory effect on the migration ability of HaCaT cells that were cocultured with dermal fibroblasts. Additionally, CAP treatment resulted in an upregulation of the HIPPO transcription factor YAP in HaCaTs and fibroblasts. Downstream effectors of the HIPPO signalling pathway (CTGF and Cyr61) were also upregulated in dermal fibroblasts, and the administration of antioxidants could inhibit CAP-mediated wound healing and abrogate the gene expression of the HIPPO downstream effectors. Interestingly, we observed that HaCaT cells exhibited an improved cell migration rate when incubated with CAP-treated fibroblast-conditioned media compared to that observed after incubation with untreated media. An induction of CTGF and Cyr61 secretion was also observed upon CAP treatment in the fibroblast-conditioned media. Finally, exposure to recombinant CTGF and Cyr61 could also significantly improve HaCaT cell migration. In summary, our results validated that CAP activates a regenerative signalling pathway at the onset of wound healing. Additionally, CAP also stimulated a reciprocal communication between dermal fibroblasts and keratinocytes, resulting in improved keratinocyte wound healing in coculture. © 2020 Debarati Shome et al.
- ItemFemtosecond laser induced step-like structures inside transparent hydrogel due to laser induced threshold reduction(San Francisco, California, US : PLOS, 2019) Saerchen, Emanuel; Liedtke-Gruener, Susann; Kopp, Maximilian; Heisterkamp, Alexander; Lubatschowski, Holger; Ripken, TammoIn the area of laser material processing, versatile applications for cutting glasses and transparent polymers exist. However, parasitic effects such as the creation of step-like structures appear when laser cutting inside a transparent material. To date, these structures were only described empirically. This work establishes the physical and chemical mechanisms behind the observed effects and describes the influence of process and material parameters onto the creation of step-like structures in hydrogel, Dihydroxyethylmethacrylat (HEMA). By focusing laser pulses in HEMA, reduced pulse separation distance below 50 nm and rise in pulse energy enhances the creation of unintended step-like structures. Spatial resolved Raman-spectroscopy was used to measure the laser induced chemical modification, which results into a reduced breakdown threshold. The reduction in threshold influences the position of optical breakdown for the succeeding laser pulses and consequently leads to the step-like structures. Additionally, the experimental findings were supplemented with numerical simulations of the influence of reduced damage threshold onto the position of optical breakdown.
- ItemAnalysing Interlinked Frequency Dynamics of the Urban Acoustic Environment(Basel : MDPI AG, 2022) Haselhoff, Timo; Braun, Tobias; Hornberg, Jonas; Lawrence, Bryce T.; Ahmed, Salman; Gruehn, Dietwald; Moebus, SusanneAs sustainable metropolitan regions require more densely built-up areas, a comprehensive understanding of the urban acoustic environment (AE) is needed. However, comprehensive datasets of the urban AE and well-established research methods for the AE are scarce. Datasets of audio recordings tend to be large and require a lot of storage space as well as computationally expensive analyses. Thus, knowledge about the long-term urban AE is limited. In recent years, however, these limitations have been steadily overcome, allowing a more comprehensive analysis of the urban AE. In this respect, the objective of this work is to contribute to a better understanding of the time-frequency domain of the urban AE, analysing automatic audio recordings from nine urban settings over ten months. We compute median power spectra as well as normalised spectrograms for all settings. Additionally, we demonstrate the use of frequency correlation matrices (FCMs) as a novel approach to access large audio datasets. Our results show site-dependent patterns in frequency dynamics. Normalised spectrograms reveal that frequency bins with low power hold relevant information and that the AE changes considerably over a year. We demonstrate that this information can be captured by using FCMs, which also unravel communities of interlinked frequency dynamics for all settings.
- ItemAnalysis of the release characteristics of Cu-treated antimicrobial implant surfaces using atomic absorption spectrometry(New York, NY : Hindawi, 2012) Zietz, C.; Fritsche, A.; Finke, B.; Stranak, V.; Haenle, M.; Hippler, R.; Mittelmeier, W.; Bader, R.New developments of antimicrobial implant surfaces doped with copper (Cu) ions may minimize the risk of implant-associated infections. However, experimental evaluation of the Cu release is influenced by various test parameters. The aim of our study was to evaluate the Cu release characteristics in vitro according to the storage fluid and surface roughness. Plasma immersion ion implantation of Cu (Cu-PIII) and pulsed magnetron sputtering process of a titanium copper film (Ti-Cu) were applied to titanium alloy (Ti6Al4V) samples with different surface finishing of the implant material (polished, hydroxyapatite and corundum blasted). The samples were submersed into either double-distilled water, human serum, or cell culture medium. Subsequently, the Cu concentration in the supernatant was measured using atomic absorption spectrometry. The test fluid as well as the surface roughness can alter the Cu release significantly, whereby the highest Cu release was determined for samples with corundum-blasted surfaces stored in cell medium.