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- ItemMonoclonal Antibodies 13A4 and AC133 Do Not Recognize the Canine Ortholog of Mouse and Human Stem Cell Antigen Prominin-1 (CD133)(San Francisco, California, US : PLOS, 2016) Thamm, Kristina; Graupner, Sylvi; Werner, Carsten; Huttner, Wieland B.; Corbeil, Denis; Nabi, Ivan RThe pentaspan membrane glycoprotein prominin-1 (CD133) is widely used in medicine as a cell surface marker of stem and cancer stem cells. It has opened new avenues in stem cell-based regenerative therapy and oncology. This molecule is largely used with human samples or the mouse model, and consequently most biological tools including antibodies are directed against human and murine prominin-1. Although the general structure of prominin-1 including its membrane topology is conserved throughout the animal kingdom, its primary sequence is poorly conserved. Thus, it is unclear if anti-human and -mouse prominin-1 antibodies cross-react with their orthologs in other species, especially dog. Answering this issue is imperative in light of the growing number of studies using canine prominin-1 as an antigenic marker. Here, we address this issue by cloning the canine prominin-1 and use its overexpression as a green fluorescent protein fusion protein in Madin-Darby canine kidney cells to determine its immunoreactivity with antibodies against human or mouse prominin-1. We used immunocytochemistry, flow cytometry and immunoblotting techniques and surprisingly found no cross-species immunoreactivity. These results raise some caution in data interpretation when anti-prominin-1 antibodies are used in interspecies studies.
- ItemGraphene oxide functional nanohybrids with magnetic nanoparticles for improved vectorization of doxorubicin to neuroblastoma cells(Basel : MDPI AG, 2019) Lerra, L.; Farfalla, A.; Sanz, B.; Cirillo, G.; Vittorio, O.; Voli, F.; Grand, M.L.; Curcio, M.; Nicoletta, F.P.; Dubrovska, A.; Hampel, S.; Iemma, F.; Goya, G.F.With the aim to obtain a site-specific doxorubicin (DOX) delivery in neuroblastoma SH-SY5Y cells, we designed an hybrid nanocarrier combining graphene oxide (GO) and magnetic iron oxide nanoparticles (MNPs), acting as core elements, and a curcumin–human serum albumin conjugate as functional coating. The nanohybrid, synthesized by redox reaction between the MNPs@GO system and albumin bioconjugate, consisted of MNPs@GO nanosheets homogeneously coated by the bioconjugate as verified by SEM investigations. Drug release experiments showed a pH-responsive behavior with higher release amounts in acidic (45% at pH 5.0) vs. neutral (28% at pH 7.4) environments. Cell internalization studies proved the presence of nanohybrid inside SH-SY5Y cytoplasm. The improved efficacy obtained in viability assays is given by the synergy of functional coating and MNPs constituting the nanohybrids: while curcumin moieties were able to keep low DOX cytotoxicity levels (at concentrations of 0.44–0.88 µM), the presence of MNPs allowed remote actuation on the nanohybrid by a magnetic field, increasing the dose delivered at the target site.
- ItemOut of the lab and into the bathroom: Evening short-term exposure to conventional light suppresses melatonin and increases alertness perception(Basel : MDPI AG, 2013) Wahnschaffe, A.; Haedel, S.; Rodenbeck, A.; Stoll, C.; Rudolph, H.; Kozakov, R.; Schoepp, H.; Kunz, D.Life in 24-h society relies on the use of artificial light at night that might disrupt synchronization of the endogenous circadian timing system to the solar day. This could have a negative impact on sleep-wake patterns and psychiatric symptoms. The aim of the study was to investigate the influence of evening light emitted by domestic and work place lamps in a naturalistic setting on melatonin levels and alertness in humans. Healthy subjects (6 male, 3 female, 22-33 years) were exposed to constant dim light (<10 lx) for six evenings from 7:00 p.m. to midnight. On evenings 2 through 6, 1 h before habitual bedtime, they were also exposed to light emitted by 5 different conventional lamps for 30 min. Exposure to yellow light did not alter the increase of melatonin in saliva compared to dim light baseline during (38 ± 27 pg/mL vs. 39 ± 23 pg/mL) and after light exposure (39 ± 22 pg/mL vs. 44 ± 26 pg/mL). In contrast, lighting conditions including blue components reduced melatonin increase significantly both during (office daylight white: 25 ± 16 pg/mL, bathroom daylight white: 24 ± 10 pg/mL, Planon warm white: 26 ± 14 pg/mL, hall daylight white: 22 ± 14 pg/mL) and after light exposure (office daylight white: 25 ± 15 pg/mL, bathroom daylight white: 23 ± 9 pg/mL, Planon warm white: 24 ± 13 pg/mL, hall daylight white: 22 ± 26 pg/mL). Subjective alertness was significantly increased after exposure to three of the lighting conditions which included blue spectral components in their spectra. Evening exposure to conventional lamps in an everyday setting influences melatonin excretion and alertness perception within 30 min.
- ItemInteraction between immobilized polyelectrolyte complex nanoparticles and human mesenchymal stromal cells(Auckland : DOVE Medical Press, 2014) Woltmann, B.; Torger, B.; Müller, M.; Hempel, U.Background: Implant loosening or deficient osseointegration is a major problem in patients with systemic bone diseases (eg, osteoporosis). For this reason, the stimulation of the regional cell population by local and sustained drug delivery at the bone/implant interface to induce the formation of a mechanical stable bone is promising. The purpose of this study was to investigate the interaction of polymer-based nanoparticles with human bone marrow-derived cells, considering nanoparticles' composition and surface net charge. Materials and methods: Polyelectrolyte complex nanoparticles (PECNPs) composed of the polycations poly(ethyleneimine) (PEI), poly(L-lysine) (PLL), or (N,N-diethylamino)ethyldextran (DEAE) in combination with the polyanions dextran sulfate (DS) or cellulose sulfate (CS) were prepared. PECNPs' physicochemical properties (size, net charge) were characterized by dynamic light scattering and particle charge detector measurements. Biocompatibility was investigated using human mesenchymal stromal cells (hMSCs) cultured on immobilized PECNP films (5-50 nmol·cm-2) by analysis for metabolic activity of hMSCs in dependence of PECNP surface concentration by MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt) assay, as well as cell morphology (phase contrast microscopy). Results: PECNPs ranging between ~50 nm and 150 nm were prepared. By varying the ratio of polycations and polyanions, PECNPs with a slightly positive (PEC+NP) or negative (PEC-NP) net charge were obtained. The PECNP composition significantly affected cell morphology and metabolic activity, whereas the net charge had a negligible influence. Therefore, we classified PECNPs into "variant systems" featuring a significant dose dependency of metabolic activity (DEAE/CS, PEI/DS) and "invariant systems" lacking such a dependency (DEAE/DS, PEI/CS). Immunofluorescence imaging of fluorescein isothiocyanate isomer I (FITC)-labeled PECNPs suggested internalization into hMSCs remaining stable for 8 days. Conclusion: Our study demonstrated that PECNP composition affects hMSC behavior. In particular, the PEI/CS system showed biocompatibility in a wide concentration range, representing a suitable system for local drug delivery from PECNP-functionalized bone substitute materials.
- ItemIn vitro model of metastasis to bone marrow mediates prostate cancer castration resistant growth through paracrine and extracellular matrix factors(San Francisco, CA : Public Library of Science, 2012) Lescarbeau, R.M.; Seib, F.P.; Prewitz, M.; Werner, C.; Kaplan, D.L.The spread of prostate cancer cells to the bone marrow microenvironment and castration resistant growth are key steps in disease progression and significant sources of morbidity. However, the biological significance of mesenchymal stem cells (MSCs) and bone marrow derived extracellular matrix (BM-ECM) in this process is not fully understood. We therefore established an in vitro engineered bone marrow tissue model that incorporates hMSCs and BM-ECM to facilitate mechanistic studies of prostate cancer cell survival in androgen-depleted media in response to paracrine factors and BM-ECM. hMSC-derived paracrine factors increased LNCaP cell survival, which was in part attributed to IGFR and IL6 signaling. In addition, BM-ECM increased LNCaP and MDA-PCa-2b cell survival in androgen-depleted conditions, and induced chemoresistance and morphological changes in LNCaPs. To determine the effect of BM-ECM on cell signaling, the phosphorylation status of 46 kinases was examined. Increases in the phosphorylation of MAPK pathway-related proteins as well as sustained Akt phosphorylation were observed in BM-ECM cultures when compared to cultures grown on plasma-treated polystyrene. Blocking MEK1/2 or the PI3K pathway led to a significant reduction in LNCaP survival when cultured on BM-ECM in androgen-depleted conditions. The clinical relevance of these observations was determined by analyzing Erk phosphorylation in human bone metastatic prostate cancer versus non-metastatic prostate cancer, and increased phosphorylation was seen in the metastatic samples. Here we describe an engineered bone marrow model that mimics many features observed in patients and provides a platform for mechanistic in vitro studies.
- ItemAntimicrobial Efficacy of Two Surface Barrier Discharges with Air Plasma against In Vitro Biofilms(San Francisco, CA : Public Library of Science, 2013) Matthes, R.; Bender, C.; Schlüter, R.; Koban, I.; Bussiahn, R.; Reuter, S.; Lademann, J.; Weltmann, K.-D.; Kramer, A.The treatment of infected wounds is one possible therapeutic aspect of plasma medicine. Chronic wounds are often associated with microbial biofilms which limit the efficacy of antiseptics. The present study investigates two different surface barrier discharges with air plasma to compare their efficacy against microbial biofilms with chlorhexidine digluconate solution (CHX) as representative of an important antibiofilm antiseptic. Pseudomonas aeruginosa SG81 and Staphylococcus epidermidis RP62A were cultivated on polycarbonate discs. The biofilms were treated for 30, 60, 150, 300 or 600 s with plasma or for 600 s with 0.1% CHX, respectively. After treatment, biofilms were dispensed by ultrasound and the antimicrobial effects were determined as difference in the number of the colony forming units by microbial culture. A high antimicrobial efficacy on biofilms of both plasma sources in comparison to CHX treatment was shown. The efficacy differs between the used strains and plasma sources. For illustration, the biofilms were examined under a scanning electron microscope before and after treatment. Additionally, cytotoxicity was determined by the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay with L929 mouse fibroblast cell line. The cell toxicity of the used plasma limits its applicability on human tissue to maximally 150 s. The emitted UV irradiance was measured to estimate whether UV could limit the application on human tissue at the given parameters. It was found that the UV emission is negligibly low. In conclusion, the results support the assumption that air plasma could be an option for therapy of chronic wounds.
- ItemVinculin binding angle in podosomes revealed by high resolution microscopy(San Francisco, CA : Public Library of Science, 2014) Walde, M.; Monypenny, J.; Heintzmann, R.; Jones, G.E.; Cox, S.Podosomes are highly dynamic actin-rich adhesive structures formed predominantly by cells of the monocytic lineage, which degrade the extracellular matrix. They consist of a core of F-actin and actin-regulating proteins, surrounded by a ring of adhesion-associated proteins such as vinculin. We have characterised the structure of podosomes in macrophages, particularly the structure of the ring, using three super-resolution fluorescence microscopy techniques: stimulated emission depletion microscopy, structured illumination microscopy and localisation microscopy. Rather than being round, as previously assumed, we found the vinculin ring to be created from relatively straight strands of vinculin, resulting in a distinctly polygonal shape. The strands bind preferentially at angles between 116° and 135°. Furthermore, adjacent vinculin strands are observed nucleating at the corners of the podosomes, suggesting a mechanism for podosome growth.
- ItemGeometry-Driven Cell Organization Determines Tissue Growths in Scaffold Pores: Consequences for Fibronectin Organization(San Francisco, CA : Public Library of Science, 2013) Joly, P.; Duda, G.N.; Schöne, M.; Welzel, P.B.; Freudenberg, U.; Werner, C.; Petersen, A.To heal tissue defects, cells have to bridge gaps and generate new extracellular matrix (ECM). Macroporous scaffolds are frequently used to support the process of defect filling and thus foster tissue regeneration. Such biomaterials contain micro-voids (pores) that the cells fill with their own ECM over time. There is only limited knowledge on how pore geometry influences cell organization and matrix production, even though it is highly relevant for scaffold design. This study hypothesized that 1) a simple geometric description predicts cellular organization during pore filling at the cell level and that 2) pore closure results in a reorganization of ECM. Scaffolds with a broad distribution of pore sizes (macroporous starPEG-heparin cryogel) were used as a model system and seeded with primary fibroblasts. The strategies of cells to fill pores could be explained by a simple geometrical model considering cells as tensioned chords. The model matched qualitatively as well as quantitatively by means of cell number vs. open cross-sectional area for all pore sizes. The correlation between ECM location and cell position was higher when the pores were not filled with tissue (Pearson's coefficient ρ = 0.45±0.01) and reduced once the pores were closed (ρ = 0.26±0.04) indicating a reorganization of the cell/ECM network. Scaffold pore size directed the time required for pore closure and furthermore impacted the organization of the fibronectin matrix. Understanding how cells fill micro-voids will help to design biomaterial scaffolds that support the endogenous healing process and thus allow a fast filling of tissue defects.
- ItemSleep apnea-hypopnea quantification by cardiovascular data analysis(San Francisco, CA : Public Library of Science (PLoS), 2014) Camargo, S.; Riedl, M.; Anteneodo, C.; Kurths, J.; Penzel, T.; Wessel, N.Sleep disorders are a major risk factor for cardiovascular diseases. Sleep apnea is the most common sleep disturbance and its detection relies on a polysomnography, i.e., a combination of several medical examinations performed during a monitored sleep night. In order to detect occurrences of sleep apnea without the need of combined recordings, we focus our efforts on extracting a quantifier related to the events of sleep apnea from a cardiovascular time series, namely systolic blood pressure (SBP). Physiologic time series are generally highly nonstationary and entrap the application of conventional tools that require a stationary condition. In our study, data nonstationarities are uncovered by a segmentation procedure which splits the signal into stationary patches, providing local quantities such as mean and variance of the SBP signal in each stationary patch, as well as its duration L. We analysed the data of 26 apneic diagnosed individuals, divided into hypertensive and normotensive groups, and compared the results with those of a control group. From the segmentation procedure, we identified that the average duration 〈L〉, as well as the average variance 〈σ2〉, are correlated to the apnea-hypoapnea index (AHI), previously obtained by polysomnographic exams. Moreover, our results unveil an oscillatory pattern in apneic subjects, whose amplitude S∗ is also correlated with AHI. All these quantities allow to separate apneic individuals, with an accuracy of at least 79%. Therefore, they provide alternative criteria to detect sleep apnea based on a single time series, the systolic blood pressure.
- ItemGuidance of mesenchymal stem cells on fibronectin structured hydrogel films(San Francisco, California, US : PLOS, 2014) Kasten, Annika; Naser, Tamara; Brüllhoff, Kristina; Fiedler, Jörg; Müller, Petra; Möller, Martin; Rychly, Joachim; Groll, Jürgen; Brenner, Rolf E.; Engler, Adam J.Designing of implant surfaces using a suitable ligand for cell adhesion to stimulate specific biological responses of stem cells will boost the application of regenerative implants. For example, materials that facilitate rapid and guided migration of stem cells would promote tissue regeneration. When seeded on fibronectin (FN) that was homogeneously immmobilized to NCO-sP(EO-stat-PO), which otherwise prevents protein binding and cell adhesion, human mesenchymal stem cells (MSC) revealed a faster migration, increased spreading and a more rapid organization of different cellular components for cell adhesion on fibronectin than on a glass surface. To further explore, how a structural organization of FN controls the behavior of MSC, adhesive lines of FN with varying width between 10 µm and 80 µm and spacings between 5 µm and 20 µm that did not allow cell adhesion were generated. In dependance on both line width and gaps, cells formed adjacent cell contacts, were individually organized in lines, or bridged the lines. With decreasing sizes of FN lines, speed and directionality of cell migration increased, which correlated with organization of the actin cytoskeleton, size and shape of the nuclei as well as of focal adhesions. Together, defined FN lines and gaps enabled a fine tuning of the structural organization of cellular components and migration. Microstructured adhesive substrates can mimic the extracellular matrix in vivo and stimulate cellular mechanisms which play a role in tissue regeneration.