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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.
- ItemCombined In Vitro Toxicity and Immunogenicity of Cold Plasma and Pulsed Electric Fields(Basel : MDPI, 2022) Wolff, Christina M.; Kolb, Juergen F.; Bekeschus, SanderIn modern oncology, therapies are based on combining monotherapies to overcome treatment resistance and increase therapy precision. The application of microsecond-pulsed electric fields (PEF) is approved to enhance local chemotherapeutic drug uptake within combination electrochemotherapy regimens. Reactive oxygen species (ROS) have been implicated in anticancer effects, and cold physical plasma produces vast amounts of ROS, which have recently been shown to benefit head and neck cancer patients. PEF and cold plasma technology have been linked to immunogenic cell death (ICD) induction, a regulated cell death accompanied by sterile inflammation that promotes antitumor immunity. To this end, we investigated the combined effect of both treatments regarding their intracellular ROS accumulation, toxicity, ICD-related marker expression, and optimal exposure sequence in a leukemia model cell line. The combination treatment substantially increased ROS and intracellular glutathione levels, leading to additive cytotoxic effects accompanied by a significantly increased expression of ICD markers, such as the eat-me signal calreticulin (CRT). Preconditioned treatment with cold plasma followed by PEF exposure was the most potent treatment sequence. The results indicate additive effects of cold plasma and PEF, motivating further studies in skin and breast tumor models for the future improvement of ECT in such patients.
- ItemCharacterization of Staphylococci and Streptococci Isolated from Milk of Bovides with Mastitis in Egypt(Basel : MDPI, 2020) Ahmed, Wedad; Neubauer, Heinrich; Tomaso, Herbert; El Hofy, Fatma Ibrahim; Monecke, Stefan; Abdeltawab, Ashraf Awad; Hotzel, HelmutThe aim of this study was to characterize staphylococci and streptococci in milk from Egyptian bovides. In total, 50 milk samples were collected from localities in the Nile Delta region of Egypt. Isolates were cultivated, identified using matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF MS), and antibiotic susceptibility testing was performed by the broth microdilution method. PCR amplifications were carried out, targeting resistanceassociated genes. Thirty-eight Staphylococcus isolates and six Streptococcus isolates could be cultivated. Staphylococcus aureus isolates revealed a high resistance rate to penicillin, ampicillin, clindamycin, and erythromycin. The mecA gene defining methicillin-resistant Staphylococcus aureus, erm(C) and aac-aphD genes was found in 87.5% of each. Coagulase-negative staphylococci showed a high prevalence of mecA, blaZ and tetK genes. Other resistance-associated genes were found. All Streptococcus dysgalactiae isolates carried blaZ, erm(A), erm(B), erm(C) and lnuA genes, while Streptococcus suis harbored erm(C), aphA-3, tetL and tetM genes, additionally. In Streptococcus gallolyticus, most of these genes were found. The Streptococcus agalactiae isolate harbored blaZ, erm(B), erm(C), lnuA, tetK, tetL and tetM genes. Streptococcus agalactiae isolate was analyzed by DNA microarray analysis. It was determined as sequence type 14, belonging to clonal complex 19 and represented capsule type VI. Pilus and cell wall protein genes, pavA, cadD and emrB/qacA genes were identified by microarray analysis. © 2020 by the authors.
- ItemPhenotypic and Molecular Detection of Biofilm Formation in Staphylococcus aureus Isolated from Different Sources in Algeria(Basel : MDPI, 2020) Achek, Rachid; Hotzel, Helmut; Nabi, Ibrahim; Kechida, Souad; Mami, Djamila; Didouh, Nassima; Tomaso, Herbert; Neubauer, Heinrich; Ehricht, Ralf; Monecke, Stefan; El-Adawy, HosnyStaphylococcus aureus is an opportunistic bacterium causing a wide variety of diseases. Biofilm formation of Staphylococcus aureus is of primary public and animal health concern. The purposes of the present study were to investigate the ability of Staphylococcus aureus isolated from animals, humans, and food samples to form biofilms and to screen for the presence of biofilmassociated and regulatory genes. In total, 55 Staphylococcus aureus isolated from sheep mastitis cases (n = 28), humans (n = 19), and from food matrices (n = 8) were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The ability of Staphylococcus aureus for slime production and biofilm formation was determined quantitatively. A DNA microarray examination was performed to detect adhesion genes (icaACD and biofilmassociated protein gene (bap)), genes encoding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs), regulatory genes (accessory gene regulator (agr) and staphylococcal accessory regulator (sarA)), and the staphylococcal cassette chromosome mec elements (SCCmec). Out of 55 Staphylococcus aureus isolates, 39 (71.0%) and 23 (41.8%) were producing slime and biofilm, respectively. All Staphylococcus aureus strains isolated from food showed biofilm formation ability. 52.6% of the Staphylococcus aureus strains isolated from sheep with mastitis, and 17.9% of isolates from humans, were able to form a biofilm. Microarray analysis typed the Staphylococcus aureus into 15 clonal complexes. Among all Staphylococcus aureus isolates, four of the human isolates (21.1%) harbored the mecA gene (SCCmec type IV) typed into 2 clonal complexes (CC22-MRSA-IV and CC80-MRSA-IV) and were considered as methicillin-resistant, while two of them were slime-producing. None of the isolates from sheep with mastitis harbored the cna gene which is associated with biofilm production. The fnbB gene was found in 100%, 60% and 40% of biofilm-producing Staphylococcus aureus isolated from food, humans, and sheep with mastitis, respectively. Three agr groups were present and agr group III was predominant with 43.6%, followed by agr group I (38.2%), and agr group II (18.2%). This study revealed the capacity of Staphylococcus aureus isolates to form biofilms and highlighted the genetic background displayed by Staphylococcus aureus isolates from different sources in Algeria. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.
- ItemRelease of Bioactive Molecules from Graphene Oxide-Alginate Hybrid Hydrogels: Effect of Crosslinking Method(Basel : MDPI, 2023) Madeo, Lorenzo Francesco; Curcio, Manuela; Iemma, Francesca; Nicoletta, Fiore Pasquale; Hampel, Silke; Cirillo, GiuseppeTo investigate the influence of crosslinking methods on the releasing performance of hybrid hydrogels, we synthesized two systems consisting of Graphene oxide (GO) as a functional element and alginate as polymer counterpart by means of ionic gelation (physical method, 𝐻𝑃𝐴−𝐺𝑂) and radical polymerization (chemical method, 𝐻𝐶𝐴−𝐺𝑂). Formulations were optimized to maximize the GO content (2.0 and 1.15% for 𝐻𝑃𝐴−𝐺𝑂 and 𝐻𝐶𝐴−𝐺𝑂, respectively) and Curcumin (CUR) was loaded as a model drug at 2.5, 5.0, and 7.5% (by weight). The physico-chemical characterization confirmed the homogeneous incorporation of GO within the polymer network and the enhanced thermal stability of hybrid vs. blank hydrogels. The determination of swelling profiles showed a higher swelling degree for 𝐻𝐶𝐴−𝐺𝑂 and a marked pH responsivity due to the COOH functionalities. Moreover, the application of external voltages modified the water affinity of 𝐻𝐶𝐴−𝐺𝑂, while they accelerated the degradation of 𝐻𝑃𝐴−𝐺𝑂 due to the disruption of the crosslinking points and the partial dissolution of alginate. The evaluation of release profiles, extensively analysed by the application of semi-empirical mathematical models, showed a sustained release from hybrid hydrogels, and the possibility to modulate the releasing amount and rate by electro-stimulation of 𝐻𝐶𝐴−𝐺𝑂.
- ItemNew Approach against Chondrosoma Cells—Cold Plasma Treatment Inhibits Cell Motility and Metabolism, and Leads to Apoptosis(Basel : MDPI, 2022) Nitsch, Andreas; Strakeljahn, Silas; Jacoby, Josephine M.; Sieb, Konrad F.; Mustea, Alexander; Bekeschus, Sander; Ekkernkamp, Axel; Stope, Matthias B.; Haralambiev, Lyubomir(1) Background: Chondrosarcoma (CS) is a malignant primary bone tumor with a carti-laginous origin. Its slow cell division and severely restricted vascularization are responsible for its poor responsiveness to chemotherapy and radiotherapy. The decisive factor for the prognosis of CS patients is the only adequate therapy—surgical resection. Cold atmospheric pressure plasma (CAP) is emerging as a new option in anti-cancer therapy. Its effect on chondrosarcomas has been poorly investigated. (2) Methods: Two CS cell lines—SW 1353 and CAL 78—were used. Various assays, such as cell growth kinetics, glucose uptake, and metabolic activity assay, along with two different apoptosis assays were performed after CAP treatment. A radius cell migration assay was used to examine cell motility. (3) Results: Both cell lines showed different growth behavior, which was taken into account when using the assays. After CAP treatment, a reduction in metabolic activity was observed in both cell lines. The immediate effect of CAP showed a reduction in cell numbers and in influence on this cell line’s growth rate. The measurement of the glucose concentration in the cell culture medium showed an increase after CAP treatment. Live-dead cell imaging shows an increase in the proportion of dead cells over the incubation time for both cell lines. There was a significant increase in apoptotic signals after 48 h and 72 h for both cell lines in both assays. The migration assay showed that CAP treatment inhibited the motility of chondrosarcoma cells. The effects in all experiments were related to the duration of CAP exposure. (4) Conclusions: The CAP treatment of CS cells inhibits their growth, motility, and metabolism by initiating apoptotic 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.
- ItemFYCO1 Increase and Effect of Arimoclomol–Treatment in Human VCP–Pathology(Basel : MDPI, 2022) Guettsches, Anne-Katrin; Meyer, Nancy; Zahedi, René P.; Evangelista, Teresinha; Muentefering, Thomas; Ruck, Tobias; Lacene, Emmanuelle; Heute, Christoph; Gonczarowska-Jorge, Humberto; Schoser, Benedikt; Krause, Sabine; Hentschel, Andreas; Vorgerd, Matthias; Roos, AndreasDominant VCP–mutations cause a variety of neurological manifestations including inclusion body myopathy with early–onset Paget disease and frontotemporal dementia 1 (IBMPFD). VCP encodes a ubiquitously expressed multifunctional protein that is a member of the AAA+ protein family, implicated in multiple cellular functions ranging from organelle biogenesis to ubiquitin–dependent protein degradation. The latter function accords with the presence of protein aggregates in muscle biopsy specimens derived from VCP–patients. Studying the proteomic signature of VCP–mutant fibroblasts, we identified a (pathophysiological) increase of FYCO1, a protein involved in autophagosome transport. We confirmed this finding applying immunostaining also in muscle biopsies derived from VCP–patients. Treatment of fibroblasts with arimoclomol, an orphan drug thought to restore physiologic cellular protein repair pathways, ameliorated cellular cytotoxicity in VCP–patient derived cells. This finding was accompanied by increased abundance of proteins involved in immune response with a direct impact on protein clearaqnce as well as by elevation of pro–survival proteins as unravelled by untargeted proteomic profiling. Hence, the combined results of our study reveal a dysregulation of FYCO1 in the context of VCP–etiopathology, highlight arimoclomol as a potential drug and introduce proteins targeted by the pre–clinical testing of this drug in fibroblasts.
- ItemHeat Shock Protein 27 Affects Myeloid Cell Activation and Interaction with Prostate Cancer Cells(Basel : MDPI, 2022) Singer, Debora; Ressel, Verena; Stope, Matthias B.; Bekeschus, SanderHeat shock proteins are cytoprotective molecules induced by environmental stresses. The small heat shock protein 27 (Hsp27) is highly expressed under oxidative stress conditions, mediating anti-oxidative effects and blocking apoptosis. Since medical gas plasma treatment subjects cancer cells to a multitude of reactive oxygen species (ROS), inducing apoptosis and immunomodulation, probable effects of Hsp27 should be investigated. To this end, we quantified the extracellular Hsp27 in two prostate cancer cell lines (LNCaP, PC-3) after gas plasma-induced oxidative stress, showing a significantly enhanced release. To investigate immunomodulatory effects, two myeloid cell lines (THP-1 and HL-60) were also exposed to Hsp27. Only negligible effects on viability, intracellular oxidative milieu, and secretion profiles of the myeloid cells were found when cultured alone. Interestingly, prostate cancer-myeloid cell co-cultures showed altered secretion profiles with a significant decrease in vascular endothelial growth factor (VEGF) release. Furthermore, the myeloid surface marker profiles were changed, indicating an enhanced differentiation in co-culture upon Hsp27 treatment. Finally, we investigated morphological changes, proliferation, and interaction with prostate cancer cells, and found significant alterations in the myeloid cells, supporting the tendency to differentiate. Collectively, our results suggest an ambiguous effect of Hsp27 on myeloid cells in the presence of prostate cancer cells which needs to be further investigated.
- ItemThe Anticancer Efficacy of Plasma-Oxidized Saline (POS) in the Ehrlich Ascites Carcinoma Model In Vitro and In Vivo(Basel : MDPI, 2021) Brito, Walison Augusto Silva; Freund, Eric; Nascimento, Thiago Daniel Henrique do; Pasqual-Melo, Gabriella; Sanches, Larissa Juliani; Dionísio, Joyce Hellen Ribeiro; Fumegali, William Capellari; Miebach, Lea; Cecchini, Alessandra Lourenço; Bekeschus, SanderCold physical plasma, a partially ionized gas rich in reactive oxygen species (ROS), is receiving increasing interest as a novel anticancer agent via two modes. The first involves its application to cells and tissues directly, while the second uses physical plasma-derived ROS to oxidize liquids. Saline is a clinically accepted liquid, and here we explored the suitability of plasma-oxidized saline (POS) as anticancer agent technology in vitro and in vivo using the Ehrlich Ascites Carcinoma (EAC) model. EAC mainly grows as a suspension in the peritoneal cavity of mice, making this model ideally suited to test POS as a putative agent against peritoneal carcinomatosis frequently observed with colon, pancreas, and ovarium metastasis. Five POS injections led to a reduction of the tumor burden in vivo as well as in a decline of EAC cell growth and an arrest in metabolic activity ex vivo. The treatment was accompanied by a decreased antioxidant capacity of Ehrlich tumor cells and increased lipid oxidation in the ascites supernatants, while no other side effects were observed. Oxaliplatin and hydrogen peroxide were used as controls and mediated better and worse outcomes, respectively, with the former but not the latter inducing profound changes in the inflammatory milieu among 13 different cytokines investigated in ascites fluid. Modulation of inflammation in the POS group was modest but significant. These results promote POS as a promising candidate for targeting peritoneal carcinomatosis and malignant ascites and suggest EAC to be a suitable and convenient model for analyzing innovative POS approaches and combination therapies.