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- ItemQuantification of Dolichyl Phosphates Using Phosphate Methylation and Reverse-Phase Liquid Chromatography-High Resolution Mass Spectrometry(Columbus, Ohio : American Chemical Society, 2023) Kale, Dipali; Kikul, Frauke; Phapale, Prasad; Beedgen, Lars; Thiel, Christian; Brügger, BrittaDolichyl monophosphates (DolPs) are essential lipids in glycosylation pathways that are highly conserved across almost all domains of life. The availability of DolP is critical for all glycosylation processes, as these lipids serve as membrane-anchored building blocks used by various types of glycosyltransferases to generate complex post-translational modifications of proteins and lipids. The analysis of DolP species by reverse-phase liquid chromatography-mass spectrometry (RPLC-MS) remains a challenge due to their very low abundance and wide range of lipophilicities. Until now, a method for the simultaneous qualitative and quantitative assessment of DolP species from biological membranes has been lacking. Here, we describe a novel approach based on simple sample preparation, rapid and efficient trimethylsilyl diazomethane-dependent phosphate methylation, and RPLC-MS analysis for quantification of DolP species with different isoprene chain lengths. We used this workflow to selectively quantify DolP species from lipid extracts derived of Saccharomyces cerevisiae, HeLa, and human skin fibroblasts from steroid 5-α-reductase 3- congenital disorders of glycosylation (SRD5A3-CDG) patients and healthy controls. Integration of this workflow with global lipidomics analyses will be a powerful tool to expand our understanding of the role of DolPs in pathophysiological alterations of metabolic pathways downstream of HMG-CoA reductase, associated with CDGs, hypercholesterolemia, neurodegeneration, and cancer.
- ItemGlycerylphytate as an ionic crosslinker for 3D printing of multi-layered scaffolds with improved shape fidelity and biological features(London : Royal Society of Chemistry, 2020) Mora-Boza, A.; Włodarczyk-Biegun, M.K.; Del Campo, A.; Vázquez-Lasa, B.; Román, J.S.The fabrication of intricate and long-term stable 3D polymeric scaffolds by a 3D printing technique is still a challenge. In the biomedical field, hydrogel materials are very frequently used because of their excellent biocompatibility and biodegradability, however the improvement of their processability and mechanical properties is still required. This paper reports the fabrication of dual crosslinked 3D scaffolds using a low concentrated (<10 wt%) ink of gelatin methacryloyl (GelMA)/chitosan and a novel crosslinking agent, glycerylphytate (G1Phy) to overcome the current limitations in the 3D printing field using hydrogels. The applied methodology consisted of a first ultraviolet light (UV) photopolymerization followed by a post-printing ionic crosslinking treatment with G1Phy. This crosslinker provides a robust framework and avoids the necessity of neutralization with strong bases. The blend ink showed shear-thinning behavior and excellent printability in the form of a straight and homogeneous filament. UV curing was undertaken simultaneously to 3D deposition, which enhanced precision and shape fidelity (resolution ≈150 μm), and prevented the collapse of the subsequent printed layers (up to 28 layers). In the second step, the novel G1Phy ionic crosslinker agent provided swelling and long term stability properties to the 3D scaffolds. The multi-layered printed scaffolds were mechanically stable under physiological conditions for at least one month. Preliminary in vitro assays using L929 fibroblasts showed very promising results in terms of adhesion, spreading, and proliferation in comparison to other phosphate-based traditional crosslinkers (i.e. TPP). We envision that the proposed combination of the blend ink and 3D printing approach can have widespread applications in the regeneration of soft tissues.
- ItemTime-resolved luminescence detection of peroxynitrite using a reactivity-based lanthanide probe(Cambridge : RSC, 2020) Breen, Colum; Pal, Robert; Elsegood, Mark R.J.; Teat, Simon J.; Iza, Felipe; Wende, Kristian; Buckley, Benjamin R.; Butler, StephenPeroxynitrite (ONOO-) is a powerful and short-lived oxidant formed in vivo, which can react with most biomolecules directly. To fully understand the roles of ONOO- in cell biology, improved methods for the selective detection and real-time analysis of ONOO- are needed. We present a water-soluble, luminescent europium(iii) probe for the rapid and sensitive detection of peroxynitrite in human serum, living cells and biological matrices. We have utilised the long luminescence lifetime of the probe to measure ONOO- in a time-resolved manner, effectively avoiding the influence of autofluorescence in biological samples. To demonstrate the utility of the Eu(iii) probe, we monitored the production of ONOO- in different cell lines, following treatment with a cold atmospheric plasma device commonly used in the clinic for skin wound treatment. This journal is © The Royal Society of Chemistry.
- ItemA non-cytotoxic resin for micro-stereolithography for cell cultures of HUVECs(Basel : MDPI, 2020) Männel, Max J.; Fischer, Carolin; Thiele, JulianThree-dimensional (3D) printing of microfluidic devices continuously replaces conventional fabrication methods. A versatile tool for achieving microscopic feature sizes and short process times is micro-stereolithography (µSL). However, common resins for µSL lack biocompatibility and are cytotoxic. This work focuses on developing new photo-curable resins as a basis for µSL fabrication of polymer materials and surfaces for cell culture. Different acrylate-and methacrylate-based compositions are screened for material characteristics including wettability, surface roughness, and swelling behavior. For further understanding, the impact of photo-absorber and photo-initiator on the cytotoxicity of 3D-printed substrates is studied. Cell culture experiments with human umbilical vein endothelial cells (HUVECs) in standard polystyrene vessels are compared to 3D-printed parts made from our library of homemade resins. Among these, after optimizing material composition and post-processing, we identify selected mixtures of poly(ethylene glycol) diacrylate (PEGDA) and poly(ethylene glycol) methyl ethyl methacrylate (PEGMEMA) as most suitable to allow for fabricating cell culture platforms that retain both the viability and proliferation of HUVECs. Next, our PEGDA/PEGMEMA resins will be further optimized regarding minimal feature size and cell adhesion to fabricate microscopic (microfluidic) cell culture platforms, e.g., for studying vascularization of HUVECs in vitro. © 2020 by the authors.
- ItemStability of hepatitis E virus at high hydrostatic pressure processing(Amsterdam : Elsevier, 2021) Johne, R.; Wolff, A.; Gadicherla, A.K.; Filter, M.; Schlüter, O.Hepatitis E virus (HEV) is the causative agent of acute and chronic hepatitis in humans. The zoonotic HEV genotype 3 is the main genotype in Europe. The foodborne transmission via consumption of meat and meat products prepared from infected pigs or wild boars is considered the major transmission route of this genotype. High hydrostatic pressure processing (HPP) is a technique, which can be used for inactivation of pathogens in food. Here, preparations of a cell culture-adapted HEV genotype 3 strain in phosphate-buffered saline (PBS) were subjected to HPP and the remaining infectivity was titrated in cell culture by counting fluorescent foci of replicating virus. A gradual decrease in infectivity was found by application of 100 to 600 MPa for 2 min. At 20 °C, infectivity reduction of 0.5 log10 at 200 MPa and 1 log10 at 400 MPa were observed. Slightly higher infectivity reduction of 1 log10 at 200 MPa and 2 log10 at 400 MPa were found by application of the pressure at 4 °C. At both temperatures, the virus was nearly completely inactivated (>3.5 log10 infectivity decrease) at 600 MPa; however, low amounts of remaining infectious virus were observed in one of three replicates in both cases. Transmission electron microscopy showed disassembled and distorted particles in the preparations treated with 600 MPa. Time-course experiments at 400 MPa showed a continuous decline of infectivity from 30 s to 10 min, leading to a 2 log10 infectivity decrease at 20 °C and to a 2.5 log10 infectivity decrease at 4 °C for a 10 min pressure application each. Predictive models for inactivation of HEV by HPP were generated on the basis of the generated data. The results show that HPP treatment can reduce HEV infectivity, which is mainly dependent on pressure height and duration of the HPP treatment. Compared to other viruses, HEV appears to be relatively stable against HPP and high pressure/long time combinations have to be applied for significant reduction of infectivity.
- ItemRedox-triggerable firefly luciferin-bioinspired hydrogels as injectable and cell-encapsulating matrices(Cambridge : RSC Publ., 2022) Jin, Minye; Gläser, Alisa; Paez, Julieta I.Stimuli-responsive hydrogels are smart materials that respond to variations caused by external stimuli and that are currently exploited for biomedical applications such as biosensing, drug delivery and tissue engineering. The development of stimuli-responsive hydrogels with defined user control is relevant to realize materials with advanced properties. Recently, our group reported firefly luciferin-inspired hydrogel matrices for 3D cell culture. This platform exhibited advantages like rapid gelation rate and tunability of mechanical and biological properties. However, this first molecular design did not allow fine control of the gelation onset, which restricts application as a cell-encapsulating matrice with injectable and processable properties. In this article, we endow the firefly luciferin-inspired hydrogels with redox-triggering capability, to overcome the limitations of the previous system and to widen its application range. We achieve this goal by introducing protected macromers as hydrogel polymeric precursors that can be activated in the presence of a mild reductant, to trigger gel formation in situ with a high degree of control. We demonstrate that the regulation of molecular parameters (e.g., structure of the protecting group, reductant type) and environmental parameters (e.g., pH, temperature) of the deprotection reaction can be exploited to modulate materials properties. This redox-triggerable system enables precise control over gelation onset and kinetics, thus facilitating its utilization as an injectable hydrogel without negatively impacting its cytocompatibility. Our findings expand the current toolkit of chemically-based stimuli-responsive hydrogels.
- ItemNanoparticles for Directed Immunomodulation: Mannose-Functionalized Glycodendrimers Induce Interleukin-8 in Myeloid Cell Lines(Columbus, Ohio : American Chemical Society, 2021) Jatczak-Pawlik, Izabela; Gorzkiewicz, Michał; Studzian, Maciej; Zinke, Robin; Appelhans, Dietmar; Klajnert-Maculewicz, Barbara; Pułaski, ŁukaszNew therapeutic strategies for personalized medicine need to involve innovative pharmaceutical tools, for example, modular nanoparticles designed for direct immunomodulatory properties. We synthesized mannose-functionalized poly(propyleneimine) glycodendrimers with a novel architecture, where freely accessible mannose moieties are presented on poly(ethylene glycol)-based linkers embedded within an open-shell maltose coating. This design enhanced glycodendrimer bioactivity and led to complex functional effects in myeloid cells, with specific induction of interleukin-8 expression by mannose glycodendrimers detected in HL-60 and THP-1 cells. We concentrated on explaining the molecular mechanism of this phenomenon, which turned out to be different in both investigated cell lines: in HL-60 cells, transcriptional activation via AP-1 binding to the promoter predominated, while in THP-1 cells (which initially expressed less IL-8), induction was mediated mainly by mRNA stabilization. The success of directed immunomodulation, with synthetic design guided by assumptions about mannose-modified dendrimers as exogenous regulators of pro-inflammatory chemokine levels, opens new possibilities for designing bioactive nanoparticles. © 2021 The Authors. Published by American Chemical Society.
- Item3D Quantification of Vascular-Like Structures in z Stack Confocal Images(Amsterdam : Elsevier, 2020) Bonda, Ulrich; Jaeschke, Anna; Lighterness, Anthony; Baldwin, Jeremy; Werner, Carsten; De-Juan-Pardo, Elena M.; Bray, Laura J.Optical slice microscopy is commonly used to characterize the morphometric features of 3D cellular cultures, such as in vitro vascularization. However, the quantitative analysis of those structures is often performed on a single 2D maximum intensity projection image, limiting the accuracy of data obtained from 3D cultures. Here, we present a protocol for the quantitative analysis of z stack images, utilizing Fiji, Amira, and WinFiber3D. This protocol facilitates the in-depth examination of vascular-like structures within 3D cell culture models.