TY - JOUR A1 - Bröker, Katharine A1 - Sinelnikov, Evgeny A1 - Gustavus, Dirk A1 - Schumacher, Udo A1 - Pörtner, Ralf A1 - Hoffmeister, Hans A1 - Lüth, Stefan A1 - Dammermann, Werner T1 - Mass Production of Highly Active NK Cells for Cancer Immunotherapy in a GMP Conform Perfusion Bioreactor JF - Frontiers in Bioengineering and Biotechnology N2 - NK cells have emerged as promising candidates for cancer immunotherapy, especially due to their ability to fight circulating tumor cells thereby preventing metastases formation. Hence several studies have been performed to generate and expand highly cytotoxic NK cells ex vivo, e.g., by using specific cytokines to upregulate both their proliferation and surface expression of distinct activating receptors. Apart from an enhanced activity, application of NK cells as immunotherapeutic agent further requires sufficient cell numbers and a high purity. All these parameters depend on a variety of different factors including the starting material, additives like cytokines as well as the culture system. Here we analyzed PBMC-derived NK cells of five anonymized healthy donors expanded under specific conditions in an innovative perfusion bioreactor system with respect to their phenotype, IFN gamma production, and cytotoxicity in vitro. Important features of the meander type bioreactors used here are a directed laminar flow of medium and control of relevant process parameters. Cells are cultivated under "steady state" conditions in perfusion mode. Our data demonstrate that expansion of CD3(+) T cell depleted PBMCs in our standardized system generates massive amounts of highly pure (>85%) and potent anticancer active NK cells. These cells express a variety of important receptors driving NK cell recruitment, adhesion as well as activation. More specifically, they express the chemokine receptors CXCR3, CXCR4, and CCR7, the adhesion molecules L-selectin, LFA-1, and VLA-4, the activating receptors NKp30, NKp44, NKp46, NKG2D, DNAM1, and CD16 as well as the death ligands TRAIL and Fas-L. Moreover, the generated NK cells show a strong IFN gamma expression upon cultivation with K562 tumor cells and demonstrate a high cytotoxicity toward leukemic as well as solid tumor cell lines in vitro. Altogether, these characteristics promise a high clinical potency of thus produced NK cells awaiting further evaluation. KW - natural killer cells (NK cells) KW - cytotoxicity KW - tumor immunity KW - immunotherapy KW - perfusion bioreactor KW - GMP KW - mass production process Y1 - 2019 U6 - https://doi.org/10.3389/fbioe.2019.00194 SN - 2296-4185 VL - 7 PB - Frontiers Research Foundation CY - Lausanne ER - TY - JOUR A1 - Löpfe, Moira A1 - Duss, Anja A1 - Zafeiropoulou, Katerina-Alexandra A1 - Bjoergvinsdottir, Oddny A1 - Eglin, David A1 - Fortunato, Giuseppino A1 - Klasen, Jürgen A1 - Ferguson, Stephen J. A1 - Würtz-Kozak, Karin A1 - Krupkova, Olga T1 - Electrospray-Based Microencapsulation of Epigallocatechin 3-Gallate for Local Delivery into the Intervertebral Disc JF - Pharmaceutics N2 - Locally delivered anti-inflammatory compounds can restore the homeostasis of the degenerated intervertebral disc (IVD). With beneficial effects on IVD cells, epigallocatechin 3-gallate (EGCG) is a promising therapeutic candidate. However, EGCG is prone to rapid degradation and/or depletion. Therefore, the purpose of this study was to develop a method for controlled EGCG delivery in the degenerated IVD. Primary IVD cells were isolated from human donors undergoing IVD surgeries. EGCG was encapsulated into microparticles by electrospraying of glutaraldehyde-crosslinked gelatin. The resulting particles were characterized in terms of cytocompatibility and anti-inflammatory activity, and combined with a thermoresponsive carrier to produce an injectable EGCG delivery system. Subsequently, electrospraying was scaled up using the industrial NANOSPIDER (TM) technology. The produced EGCG microparticles reduced the expression of inflammatory (IL-6, IL-8, COX-2) and catabolic (MMP1, MMP3, MMP13) mediators in pro-inflammatory 3D cell cultures. Combining the EGCG microparticles with the carrier showed a trend towards modulating EGCG activity/release. Electrospray upscaling was achieved, leading to particles with homogenous spherical morphologies. In conclusion, electrospray-based encapsulation of EGCG resulted in cytocompatible microparticles that preserved the activity of EGCG and showed the potential to control EGCG release, thus favoring IVD health by downregulating local inflammation. Future studies will focus on further exploring the biological activity of the developed delivery system for potential clinical use. KW - degenerative disc disease KW - inflammation KW - drug delivery KW - EGCG KW - microparticles KW - injectable biomaterial KW - electrospraying Y1 - 2019 U6 - https://doi.org/10.3390/pharmaceutics11090435 SN - 1999-4923 VL - 11 IS - 9 PB - MDPI CY - Basel ER - TY - JOUR A1 - Miele, Vincent A1 - Guill, Christian A1 - Ramos-Jiliberto, Rodrigo A1 - Kéfi, Sonia T1 - Non-trophic interactions strengthen the diversity-functioning relationship in an ecological bioenergetic network model JF - PLoS Computational Biology : a new community journal N2 - Ecological communities are undeniably diverse, both in terms of the species that compose them as well as the type of interactions that link species to each other. Despite this long recognition of the coexistence of multiple interaction types in nature, little is known about the consequences of this diversity for community functioning. In the ongoing context of global change and increasing species extinction rates, it seems crucial to improve our understanding of the drivers of the relationship between species diversity and ecosystem functioning. Here, using a multispecies dynamical model of ecological communities including various interaction types (e.g. competition for space, predator interference, recruitment facilitation in addition to feeding), we studied the role of the presence and the intensity of these interactions for species diversity, community functioning (biomass and production) and the relationship between diversity and functioning. Taken jointly, the diverse interactions have significant effects on species diversity, whose amplitude and sign depend on the type of interactions involved and their relative abundance. They however consistently increase the slope of the relationship between diversity and functioning, suggesting that species losses might have stronger effects on community functioning than expected when ignoring the diversity of interaction types and focusing on feeding interactions only. Y1 - 2020 U6 - https://doi.org/10.1371/journal.pcbi.1007269 SN - 1553-7358 VL - 15 IS - 8 PB - PLoS CY - San Fransisco ER - TY - JOUR A1 - Koetz, Joachim T1 - The Effect of Surface Modification of Gold Nanotriangles for Surface-Enhanced Raman Scattering Performance JF - Nanomaterials N2 - A surface modification of ultraflat gold nanotriangles (AuNTs) with different shaped nanoparticles is of special relevance for surface-enhanced Raman scattering (SERS) and the photo-catalytic activity of plasmonic substrates. Therefore, different approaches are used to verify the flat platelet morphology of the AuNTs by oriented overgrowth with metal nanoparticles. The most important part for the morphological transformation of the AuNTs is the coating layer, containing surfactants or polymers. By using well established AuNTs stabilized by a dioctyl sodium sulfosuccinate (AOT) bilayer, different strategies of surface modification with noble metal nanoparticles are possible. On the one hand undulated superstructures were synthesized by in situ growth of hemispherical gold nanoparticles in the polyethyleneimine (PEI)-coated AOT bilayer of the AuNTs. On the other hand spiked AuNTs were obtained by a direct reduction of Au³⁺ ions in the AOT double layer in presence of silver ions and ascorbic acid as reducing agent. Additionally, crumble topping of the smooth AuNTs can be realized after an exchange of the AOT bilayer by hyaluronic acid, followed by a silver-ion mediated reduction with ascorbic acid. Furthermore, a decoration with silver nanoparticles after coating the AOT bilayer with the cationic surfactant benzylhexadecyldimethylammonium chloride (BDAC) can be realized. In that case the ultraviolet (UV)-absorption of the undulated Au@Ag nanoplatelets can be tuned depending on the degree of decoration with silver nanoparticles. Comparing the Raman scattering data for the plasmon driven dimerization of 4-nitrothiophenol (4-NTP) to 4,4′-dimercaptoazobenzene (DMAB) one can conclude that the most important effect of surface modification with a 75 times higher enhancement factor in SERS experiments becomes available by decoration with gold spikes. KW - undulated KW - spiked and crumble gold nanotriangles KW - SERS enhancement factor KW - dimerization of 4-nitrothiophenol KW - AOT bilayer KW - PEI coating Y1 - 2020 U6 - https://doi.org/10.3390/nano10112187 SN - 2079-4991 VL - 10 IS - 11 PB - MDPI CY - Basel ER - TY - JOUR A1 - Rieck, Christoph Paul Kurt A1 - Geiger, Daniel A1 - Munkert, Jennifer A1 - Messerschmidt, Katrin A1 - Petersen, Jan A1 - Strasser, Juliane A1 - Meitinger, Nadine A1 - Kreis, Wolfgang T1 - Biosynthetic approach to combine the first steps of cardenolide formation in Saccharomyces cerevisiae JF - Microbiologyopen N2 - A yeast expression plasmid was constructed containing a cardenolide biosynthetic module, referred to as CARD II, using the AssemblX toolkit, which enables the assembly of large DNA constructs. The genes cloned into the vector were (a) a Δ5‐3β‐hydroxysteroid dehydrogenase gene from Digitalis lanata, (b) a steroid Δ5‐isomerase gene from Comamonas testosteronii, (c) a mutated steroid‐5β‐reductase gene from Arabidopsis thaliana, and (d) a steroid 21‐hydroxylase gene from Mus musculus. A second plasmid bearing an ADR/ADX fusion gene from Bos taurus was also constructed. A Saccharomyces cerevisiae strain bearing these two plasmids was generated. This strain, termed “CARD II yeast”, was capable of producing 5β‐pregnane‐3β,21‐diol‐20‐one, a central intermediate in 5β‐cardenolide biosynthesis, starting from pregnenolone which was added to the culture medium. Using this approach, five consecutive steps in cardenolide biosynthesis were realized in baker's yeast. Y1 - 2019 U6 - https://doi.org/10.1002/mbo3.925 SN - 2045-8827 VL - 8 IS - 12 PB - Wiley CY - Hoboken ER - TY - JOUR A1 - Werger, Luise A1 - Bergmann, Joana A1 - Weber, Ewald A1 - Heinze, Johannes T1 - Wind intensity affects fine root morphological traits with consequences for plant-soil feedback effects JF - Annals of Botany Plants N2 - Wind influences the development, architecture and morphology of plant roots and may modify subsequent interactions between plants and soil (plant–soil feedbacks—PSFs). However, information on wind effects on fine root morphology is scarce and the extent to which wind changes plant–soil interactions remains unclear. Therefore, we investigated the effects of two wind intensity levels by manipulating surrounding vegetation height in a grassland PSF field experiment. We grew four common plant species (two grasses and two non-leguminous forbs) with soil biota either previously conditioned by these or other species and tested the effect of wind on root:shoot ratio, fine root morphological traits as well as the outcome for PSFs. Wind intensity did not affect biomass allocation (i.e. root:shoot ratio) in any species. However, fine-root morphology of all species changed under high wind intensity. High wind intensity increased specific root length and surface area and decreased root tissue density, especially in the two grasses. Similarly, the direction of PSFs changed under high wind intensity in all four species, but differences in biomass production on the different soils between high and low wind intensity were marginal and most pronounced when comparing grasses with forbs. Because soils did not differ in plant-available nor total nutrient content, the results suggest that wind-induced changes in root morphology have the potential to influence plant–soil interactions. Linking wind-induced changes in fine-root morphology to effects on PSF improves our understanding of plant–soil interactions under changing environmental conditions. KW - Wind KW - root traits KW - root morphology KW - specific root length KW - plant–soil feedback Y1 - 2020 U6 - https://doi.org/10.1093/aobpla/plaa050 SN - 2041-2851 VL - 12 IS - 5 PB - Oxford University Press CY - Oxford ER - TY - JOUR A1 - Knudsen, Erlend Moster A1 - Heinold, Bernd A1 - Dahlke, Sandro A1 - Bozem, Heiko A1 - Crewell, Susanne A1 - Gorodetskaya, Irina V. A1 - Heygster, Georg A1 - Kunkel, Daniel A1 - Maturilli, Marion A1 - Mech, Mario A1 - Viceto, Carolina A1 - Rinke, Annette A1 - Schmithusen, Holger A1 - Ehrlich, Andre A1 - Macke, Andreas A1 - Lüpkes, Christof A1 - Wendisch, Manfred T1 - Meteorological conditions during the ACLOUD/PASCAL field campaign near Svalbard in early summer 2017 JF - Atmospheric chemistry and physics N2 - The two concerted field campaigns, Arctic CLoud Observations Using airborne measurements during polar Day (ACLOUD) and the Physical feedbacks of Arctic planetary boundary level Sea ice, Cloud and AerosoL (PASCAL), took place near Svalbard from 23 May to 26 June 2017. They were focused on studying Arctic mixed-phase clouds and involved observations from two airplanes (ACLOUD), an icebreaker (PASCAL) and a tethered balloon, as well as ground-based stations. Here, we present the synoptic development during the 35-day period of the campaigns, using near-surface and upper-air meteorological observations, as well as operational satellite, analysis, and reanalysis data. Over the campaign period, short-term synoptic variability was substantial, dominating over the seasonal cycle. During the first campaign week, cold and dry Arctic air from the north persisted, with a distinct but seasonally unusual cold air outbreak. Cloudy conditions with mostly low-level clouds prevailed. The subsequent 2 weeks were characterized by warm and moist maritime air from the south and east, which included two events of warm air advection. These synoptical disturbances caused lower cloud cover fractions and higher-reaching cloud systems. In the final 2 weeks, adiabatically warmed air from the west dominated, with cloud properties strongly varying within the range of the two other periods. Results presented here provide synoptic information needed to analyze and interpret data of upcoming studies from ACLOUD/PASCAL, while also offering unprecedented measurements in a sparsely observed region. Y1 - 2018 U6 - https://doi.org/10.5194/acp-18-17995-2018 SN - 1680-7316 SN - 1680-7324 VL - 18 IS - 24 SP - 17995 EP - 18022 PB - Copernicus CY - Göttingen ER - TY - JOUR A1 - Runge, Alexandra A1 - Grosse, Guido T1 - Mosaicking Landsat and Sentinel-2 Data to Enhance LandTrendr Time Series Analysis in Northern High Latitude Permafrost Regions JF - Remote Sensing N2 - Permafrost is warming in the northern high latitudes, inducing highly dynamic thaw-related permafrost disturbances across the terrestrial Arctic. Monitoring and tracking of permafrost disturbances is important as they impact surrounding landscapes, ecosystems and infrastructure. Remote sensing provides the means to detect, map, and quantify these changes homogeneously across large regions and time scales. Existing Landsat-based algorithms assess different types of disturbances with similar spatiotemporal requirements. However, Landsat-based analyses are restricted in northern high latitudes due to the long repeat interval and frequent clouds, in particular at Arctic coastal sites. We therefore propose to combine Landsat and Sentinel-2 data for enhanced data coverage and present a combined annual mosaic workflow, expanding currently available algorithms, such as LandTrendr, to achieve more reliable time series analysis. We exemplary test the workflow for twelve sites across the northern high latitudes in Siberia. We assessed the number of images and cloud-free pixels, the spatial mosaic coverage and the mosaic quality with spectral comparisons. The number of available images increased steadily from 1999 to 2019 but especially from 2016 onward with the addition of Sentinel-2 images. Consequently, we have an increased number of cloud-free pixels even under challenging environmental conditions, which then serve as the input to the mosaicking process. In a comparison of annual mosaics, the Landsat+Sentinel-2 mosaics always fully covered the study areas (99.9–100 %), while Landsat-only mosaics contained data-gaps in the same years, only reaching coverage percentages of 27.2 %, 58.1 %, and 69.7 % for Sobo Sise, East Taymyr, and Kurungnakh in 2017, respectively. The spectral comparison of Landsat image, Sentinel-2 image, and Landsat+Sentinel-2 mosaic showed high correlation between the input images and mosaic bands (e.g., for Kurungnakh 0.91–0.97 between Landsat and Landsat+Sentinel-2 mosaic and 0.92–0.98 between Sentinel-2 and Landsat+Sentinel-2 mosaic) across all twelve study sites, testifying good quality mosaic results. Our results show that especially the results for northern, coastal areas was substantially improved with the Landsat+Sentinel-2 mosaics. By combining Landsat and Sentinel-2 data we accomplished to create reliably high spatial resolution input mosaics for time series analyses. Our approach allows to apply a high temporal continuous time series analysis to northern high latitude permafrost regions for the first time, overcoming substantial data gaps, and assess permafrost disturbance dynamics on an annual scale across large regions with algorithms such as LandTrendr by deriving the location, timing and progression of permafrost thaw disturbances KW - time series analysis KW - data fusion KW - disturbance tracking KW - permafrost KW - permafrost thaw Y1 - 2020 U6 - https://doi.org/10.3390/rs12152471 SN - 2072-4292 VL - 12 IS - 15 PB - MDPI CY - Basel ER - TY - JOUR A1 - He, Hai A1 - Höper, Rune A1 - Dodenhöft, Moritz A1 - Marlière, Philippe A1 - Bar-Even, Arren T1 - An optimized methanol assimilation pathway relying on promiscuous formaldehyde-condensing aldolases in E. coli JF - Metabolic Engineering N2 - Engineering biotechnological microorganisms to use methanol as a feedstock for bioproduction is a major goal for the synthetic metabolism community. Here, we aim to redesign the natural serine cycle for implementation in E. coli. We propose the homoserine cycle, relying on two promiscuous formaldehyde aldolase reactions, as a superior pathway design. The homoserine cycle is expected to outperform the serine cycle and its variants with respect to biomass yield, thermodynamic favorability, and integration with host endogenous metabolism. Even as compared to the RuMP cycle, the most efficient naturally occurring methanol assimilation route, the homoserine cycle is expected to support higher yields of a wide array of products. We test the in vivo feasibility of the homoserine cycle by constructing several E. coli gene deletion strains whose growth is coupled to the activity of different pathway segments. Using this approach, we demonstrate that all required promiscuous enzymes are active enough to enable growth of the auxotrophic strains. Our findings thus identify a novel metabolic solution that opens the way to an optimized methylotrophic platform. KW - Pathway design KW - Promiscuous enzymes KW - Formaldehyde assimilation KW - Serine cycle KW - Growth selection Y1 - 2020 U6 - https://doi.org/10.1016/j.ymben.2020.03.002 SN - 1096-7176 SN - 1096-7184 VL - 60 SP - 1 EP - 13 PB - Elsevier CY - Amsterdam [u.a.] ER - TY - JOUR A1 - He, Hai A1 - Noor, Elad A1 - Ramos-Parra, Perla A. A1 - García-Valencia, Liliana E. A1 - Patterson, Jenelle A. A1 - Díaz de la Garza, Rocío I. A1 - Hanson, Andrew D. A1 - Bar-Even, Arren T1 - In Vivo Rate of Formaldehyde Condensation with Tetrahydrofolate JF - Metabolites N2 - Formaldehyde is a highly reactive compound that participates in multiple spontaneous reactions, but these are mostly deleterious and damage cellular components. In contrast, the spontaneous condensation of formaldehyde with tetrahydrofolate (THF) has been proposed to contribute to the assimilation of this intermediate during growth on C1 carbon sources such as methanol. However, the in vivo rate of this condensation reaction is unknown and its possible contribution to growth remains elusive. Here, we used microbial platforms to assess the rate of this condensation in the cellular environment. We constructed Escherichia coli strains lacking the enzymes that naturally produce 5,10-methylene-THF. These strains were able to grow on minimal medium only when equipped with a sarcosine (N-methyl-glycine) oxidation pathway that sustained a high cellular concentration of formaldehyde, which spontaneously reacts with THF to produce 5,10-methylene-THF. We used flux balance analysis to derive the rate of the spontaneous condensation from the observed growth rate. According to this, we calculated that a microorganism obtaining its entire biomass via the spontaneous condensation of formaldehyde with THF would have a doubling time of more than three weeks. Hence, this spontaneous reaction is unlikely to serve as an effective route for formaldehyde assimilation. KW - one-carbon metabolism KW - spontaneous reaction KW - auxotrophy KW - serine cycle KW - phenotypic phase plane Y1 - 2019 U6 - https://doi.org/10.3390/metabo10020065 SN - 2218-1989 VL - 10 IS - 65 PB - MDPI CY - Basel ER -