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Thioredoxin (Trx) overexpression is known to be a cause of chemotherapy resistance in various tumor entities. However, Trx effects on resistance are complex and depend strictly on tissue type. In the present study, we analyzed the impact of the Trx system on intrinsic chemoresistance of human glioblastoma multiforme (GBM) cells to cytostatic drugs. Resistance of GBM cell lines and primary cells to drugs and signaling inhibitors was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Impact of Trx inhibition on apoptosis was investigated by proteome profiling of a subset of proteins and annexin V apoptosis assays. Trx-interacting protein (TXNIP) was overexpressed by transfection and protein expression was determined by immunoblotting. Pharmacological inhibition of Trx by 1-methyl-2-imidazolyl-disulfide (PX-12) reduced viability of three GBM cell lines, induced expression of active caspase-3, and reduced phosphorylation of AKT-kinase and expression of β-catenin. Sensitivity to cisplatin could be restored by both PX-12 and recombinant expression of the upstream Trx inhibitor TXNIP, respectively.
In addition, PX-12 also sensitized primary human GBM cells to temozolomide. Combined inhibition of Trx and the phosphatidylinositide 3-kinase (PI3K) pathway resulted in massive cell death. We conclude that the Trx system and the PI3K pathway act as a sequential cascade and could potentially present a new drug target.
Multidrug resistance (MDR) in tumors and pathogens remains a major problem in the efficacious treatment of patients by reduction of therapy options and subsequent treatment failure. Various mechanisms are described to be involved in the development of MDR with overexpression of ATP-binding cassette (ABC) transporters reflecting the most extensively studied. These membrane transporters translocate a wide variety of substrates utilizing energy from ATP hydrolysis leading to decreased intracellular drug accumulation and impaired drug efficacy. One treatment strategy might be inhibition of transporter-mediated efflux by small molecules. Isocoumarins and 3,4-dihydroisocoumarins are a large group of natural products derived from various sources with great structural and functional variety, but have so far not been in the focus as potential MDR reversing agents. Thus, three natural products and nine novel 3,4-dihydroisocoumarins were designed and analyzed regarding cytotoxicity induction and inhibition of human ABC transporters P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1) and breast cancer resistance protein (BCRP) in a variety of human cancer cell lines as well as the yeast ABC transporter Pdr5 in Saccharomyces cerevisiae. Dual inhibitors of P-gp and BCRP and inhibitors of Pdr5 were identified, and distinct structure-activity relationships for transporter inhibition were revealed. The strongest inhibitor of P-gp and BCRP, which inhibited the transporters up to 80 to 90% compared to the respective positive controls, demonstrated the ability to reverse chemotherapy resistance in resistant cancer cell lines up to 5.6-fold. In the case of Pdr5, inhibitors were identified that prevented substrate transport and/or ATPase activity with IC50 values in the low micromolar range. However, cell toxicity was not observed. Molecular docking of the test compounds to P-gp revealed that differences in inhibition capacity were based on different binding affinities to the transporter. Thus, these small molecules provide novel lead structures for further optimization.
One-step preparation of bilayered films from kraft lignin and cellulose acetate to mimic tree bark
(2020)
This contribution presents the development of a dry-cast method for the one-step preparation of bio-based films from wood polymers that mimic the bilayered structure of tree bark, the natural protective layer of the tree. In a simplified view, natural bark can be considered as the superposition of an external homogeneous and non-porous layer (outer bark) and a porous substructure layer (inner bark). This work is a first step for the future development of bio-based biomimetic wood coatings. The film had a bark-like appearance and its total density, bulk density and porosity were similar to values measured in natural bark. Furthermore, the structural characteristics of the studied film, namely specific surface area (BET) and pore size distribution, as well as the performance of the water adsorption ability were investigated and discussed.
Stable recombinant mammalian cells are of growing importance in pharmaceutical biotechnology production scenarios for biologics such as monoclonal antibodies, growth and blood factors, cytokines and subunit vaccines. However, the establishment of recombinant producer cells using classical stable transfection of plasmid DNA is hampered by low stable gene transfer efficiencies. Consequently, subsequent selection of transgenic cells and the screening of clonal cell populations are time- and thus cost-intensive. To overcome these limitations, expression cassettes were embedded into transposon-derived donor vectors. Upon the co-transfection with transposase-encoding constructs, elevated vector copy numbers stably integrated into the genomes of the host cells are readily achieved facilitating under stringent selection pressure the establishment of cell pools characterized by sustained and high-yield recombinant protein production. Here, we discuss some aspects of transposon vector technologies, which render these vectors promising candidates for their further utilization in the production of biologics.
Enhancing DPCD in Liquid Products by Mechanical Inactivation Effects: Assessment of Feasibility
(2020)
The enhancement of standard dense phase carbon dioxide (DPCD) pasteurization by additional mechanical effects was assessed in this work. These effects were induced during pasteurization by the sudden depressurization in a narrow minitube. The high flow velocities, moderate pressures (40–80 bar) and low temperatures (25–45 °C) lead to intense degasification and shear stress. The inactivation of the test microorganism Escherichia coli DH5α (E. coli DH5α) was determined before and after depressurization in the minitube, representing entirely chemical DPCD via dissolved CO2 and total inactivation comprising the effects of dissolved CO2 and mechanical effects, respectively. Compared to conventional DPCD pasteurization, which is mostly attributed to chemical effects, the additional mechanical effects increased the inactivation efficiency considerably.
Starmerella bombicola is known to produce sub‐terminally hydroxylated lactonic sophorolipids (SLs), while Candida kuoi synthesizes acidic open chain SLs with terminally hydroxylated fatty acids. Upon feeding glucose and fatty alcohols both strains form long‐chain nonionic SLs. According to structure elucidation the SLs consist of a hydroxylated fatty acid esterified with fatty alcohol and linked via a glycoside bond to the diacetylated sophorose unit. Palmityl, stearyl, and oleyl alcohols lead to products with lipid chain lengths of C32 or C36. Oleyl alcohol is the preferred substrate leading to 45 g L−1 of the double unsaturated C36 SL with S. bombicola and 20 g L−1 with C. kuoi. Scale up from shake flask to 1.5 L fermentations is possible and 65 g L−1 long‐chain SLs are obtained with S. bombicola within 7 days. Mixed feeding of oleic acid and a variety of fatty alcohols leads to new long‐chain SLs. In the presence of oleic acid the yeasts do not oxidize the fatty alcohol and thus the production of biosurfactants with tailored chain length is possible. The long‐chain SLs show good emulsification ability of water/paraffin oil mixtures at low energy input and reduced interfacial tension significantly.
Practical Applications: Sophorolipids are produced by fermentation on industrial scale focusing on cleaning and detergent applications. Mainly lactonic or anionic open‐chain forms are used today. The new long‐chain SLs presented in this manuscript are accessible with existing production technology and can be produced with high titers from cost‐efficient renewable raw materials. In contrast to the commercial products the long‐chain SLs are more hydrophobic and exhibit a strong emulsification behavior. Therefore they have the potential to broaden the application range of SLs in future. They may be useful as novel emulsifiers for cosmetic creams and lotions, pharmaceutical ointments and food products or may find application in oil spill remediation.
Polyimides rank among the most heat-resistant polymers and find application in a variety of fields, including transportation, electronics, and membrane technology. The aim of this work is to study the structural, thermal, mechanical, and gas permeation properties of polyimide based nanocomposite membranes in flat sheet configuration. For this purpose, numerous advanced techniques such as atomic force microscopy (AFM), SEM, TEM, TGA, FT-IR, tensile strength, elongation test, and gas permeability measurements were carried out. In particular, BTDA–TDI/MDI (P84) co-polyimide was used as the matrix of the studied membranes, whereas multi-wall carbon nanotubes were employed as filler material at concentrations of up to 5 wt.% All studied films were prepared by the dry-cast process resulting in non-porous films of about 30–50 μm of thickness. An optimum filler concentration of 2 wt.% was estimated. At this concentration, both thermal and mechanical properties of the prepared membranes were improved, and the highest gas permeability values were also obtained. Finally, gas permeability experiments were carried out at 25, 50, and 100 ◦C with seven different pure gases. The results revealed that the uniform carbon nanotubes dispersion lead to enhanced gas permeation properties.
AbstractThis paper discusses the comparison of two methods to achieve thermal comfort utilising air conditioning (AC) system in a small indoor space – adaptive control and fuzzy control. Thermal comfort indoors is performed to provide comfortability individually or for a group of people. Due to the small indoor space which usually a bit cramped, crowded and less airy, the ambience can be very uncomfortable either for doing sedentary or active work, thus the AC system can be very useful to provide thermal comfort. Both methods can be utilised depending on how thermal comfort is viewed and how the level of thermal comfort is decided. Every method has its own advantage and limitations, and will be covered in this paper as well.
Linoleic acid hydroperoxides are versatile intermediates for the production of green note aroma compounds and bifunctional ω-oxo-acids. An enzyme cascade consisting of lipoxygenase, lipase and catalase was developed for one-pot synthesis of 13-hydroperoxyoctadecadienoic acid starting from safflower oil. Reaction conditions were optimized for hydroperoxidation using lipoxygenase 1 from Glycine max (LOX-1) in a solvent-free system. The addition of green surfactant Triton CG-110 improved the reaction more than two-fold and yields of >50% were obtained at linoleic acid concentrations up to 100 mM. To combine hydroperoxidation and oil hydrolysis, 12 lipases were screened for safflower oil hydrolysis under the reaction conditions optimized for LOX-1. Lipases from Candida rugosa and Pseudomonas fluorescens were able to hydrolyze safflower oil to >75% within 5 h at a pH of 8.0. In contrast to C. rugosa lipase, the enzyme from P. fluorescens did not exhibit a lag phase. Combination of P. fluorescens lipase and LOX-1 worked well upon LOX-1 dosage and a synergistic effect was observed leading to >80% of hydroperoxides. Catalase from Micrococcus lysodeikticus was used for in-situ oxygen production with continuous H2O2 dosage in the LOX-1/lipase reaction system. Foam generation was significantly reduced in the 3-enzyme cascade in comparison to the aerated reaction system. Safflower oil concentration was increased up to 300 mM linoleic acid equivalent and 13-hydroperoxides could be produced in a yield of 70 g/L and a regioselectivity of 90% within 7 h.
Abstract
Due to their pronounced bioactivity and limited availability from natural resources, metabolites of the soft coral Pseudopterogorgia elisabethae, such as erogorgiaene and the pseudopterosines, represent important target molecules for chemical synthesis. We have now developed a particularly short and efficient route towards these marine diterpenes exploiting an operationally convenient enantioselective cobalt‐catalyzed hydrovinylation as the chirogenic step. Other noteworthy C−C bond forming transformations include diastereoselective Lewis acid‐mediated cyclizations, a Suzuki coupling and a carbonyl ene reaction. Starting from 4‐methyl‐styrene the anti‐tubercular agent (+)‐erogorgiaene (>98 % ee) was prepared in only 7 steps with 46 % overall yield. In addition, the synthesis of the pseudopterosin A aglycone was achieved in 12 steps with 30 % overall yield and, surprisingly, was found to exhibit a similar anti‐inflammatory activity (inhibition of LPS‐induced NF‐κB activation) as a natural mixture of pseudopterosins A−D or iso‐pseudopterosin A, prepared by β‐D‐xylosylation of the synthetic aglycone.
In the last decade, the utilization of waste by-product apple pomace has been extensively researched (due to its difficult disposal) and currently finds beneficial usage in various industries; as substrate for microbial growth or recovery of pectin, xyloglucan and polyphenols. In this research apple juice was produced at pilot scale. Furthermore, apple pomace was employed as substrate for the production of pectin, biofuel (pellets) and concentrated apple pomace extract. Extensive mass and heat balances were conducted to evaluate the feasibility of this approach on industrial scale. The produced pellets had very similar characteristics to wood pellets (net calorific value of 20.3 MJ/kg). Dried apple pomace contained 11.9% of pectin. Fed-batch cultivation of baker´s yeast with apple pomace extract demonstrated a potential for partial substitution of molasses in industrial bioprocesses. This concept shows how a zero discharge biorefinery process converts waste from apple juice production into three valuable products enabling connections between different industries.
Due to reasons of sustainability and conservation of resources, polyurethane (PU)-based systems with preferably neutral carbon footprints are in increased focus of research and development. The proper design and development of bio-based polyols are of particular interest since such polyols may have special property profiles that allow the novel products to enter new applications. Sophorolipids (SL) represent a bio-based toolbox for polyol building blocks to yield diverse chemical products. For a reasonable evaluation of the potential for PU chemistry, however, further investigations in terms of synthesis, derivatization, reproducibility, and reactivity towards isocyanates are required. It was demonstrated that SL can act as crosslinker or as plasticizer in PU systems depending on employed stoichiometry. (ω-1)-hydroxyl fatty acids can be derived from SL and converted successively to polyester polyols and PU. Additionally, (ω-1)-hydroxyl fatty acid azides can be prepared indirectly from SL and converted to A/B type PU by Curtius rearrangement.
Electroplating generates high volumes of rinse water that is contaminated with heavy metals. This study presents an approach for direct metal recovery and recycling from simulated rinse water, made up of an electroplating electrolyte used in industry, using reverse osmosis (RO). To simulate the real industrial application, the process was examined at various permeate fluxes, ranging from 3.75 to 30 L·m−2·h−1 and hydraulic pressures up to 80 bar. Although permeance decreased significantly with increasing water recovery, rejections of up to 93.8% for boric acid, >99.9% for chromium and 99.6% for sulfate were observed. The final RO retentate contained 8.40 g/L chromium and was directly used in Hull cell electroplating tests. It was possible to deposit cold-hued chromium layers under a wide range of relevant current densities, demonstrating the reusability of the concentrate of the rinsing water obtained by RO.
Despite intensive research over the last three decades, it has not yet been possible to bring an effective vaccine against human immunodeficiency virus (HIV) and the resulting acquired immunodeficiency syndrome (AIDS) to market. Virus-like particles (VLP) are a promising approach for efficient and effective vaccination and could play an important role in the fight against HIV. For example, HEK293 (human embryo kidney) cells can be used to produce virus-like particles. In this context, given the quality-by-design (QbD) concept for manufacturing, a digital twin is of great importance for the production of HIV-Gag-formed VLPs. In this work, a dynamic metabolic model for the production of HIV-Gag VLPs was developed and validated. The model can represent the VLP production as well as the consumption or formation of all important substrates and metabolites. Thus, in combination with already described process analytical technology (PAT) methods, the final step towards the implementation of a digital twin for process development and design, as well as process automation, was completed.
The development and adoption of digital twins (DT) for Quality-by-Design (QbD)-based processes with flexible operating points within a proven acceptable range (PAR) and automation through Advanced Process Control (APC) with Process Analytical Technology (PAT) instead of conventional process execution based on offline analytics and inflexible process set points is one of the great challenges in modern biotechnology. Virus-like particles (VLPs) are part of a line of innovative drug substances (DS). VLPs, especially those based on human immunodeficiency virus (HIV), HIV-1 Gag VLPs, have very high potential as a versatile vaccination platform, allowing for pseudotyping with heterologous envelope proteins, e.g., the S protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As enveloped VLPs, optimal process control with minimal hold times is essential. This study demonstrates, for the first time, the use of a digital twin for the overall production process of HIV-1 Gag VLPs from cultivation, clarification, and purification to lyophilization. The accuracy of the digital twins is in the range of 0.8 to 1.4% in depth filtration (DF) and 4.6 to 5.2% in ultrafiltration/diafiltration (UFDF). The uncertainty due to variability in the model parameter determination is less than 4.5% (DF) and less than 3.8% (UFDF). In the DF, a prediction of the final filter capacity was demonstrated from as low as 5.8% (9mbar) of the final transmembrane pressure (TMP). The scale-up based on DT in chromatography shows optimization potential in productivity up to a factor of 2. The schedule based on DT and PAT for APC has been compared to conventional process control, and hold-time and process duration reductions by a factor of 2 have been achieved. This work lays the foundation for the short-term validation of the DT and PAT for APC in an automated S7 process environment and the conversion from batch to continuous production.
Different mechanisms mediate the toxicity of RNA. Genomic retroviral mRNA hijacks infected host cell factors to enable virus replication. The viral genomic RNA of the human immunodeficiency virus (HIV) encompasses nine genes encoding in less than 10 kb all proteins needed for replication in susceptible host cells. To do so, the genomic RNA undergoes complex alternative splicing to facilitate the synthesis of the structural, accessory, and regulatory proteins. However, HIV strongly relies on the host cell machinery recruiting cellular factors to complete its replication cycle. Antiretroviral therapy (ART) targets different steps in the cycle, preventing disease progression to the acquired immunodeficiency syndrome (AIDS). The comprehension of the host immune system interaction with the virus has fostered the development of a variety of vaccine platforms. Despite encouraging provisional results in vaccine trials, no effective vaccine has been developed, yet. However, novel promising vaccine platforms are currently under investigation.
In this work, supported cellulose acetate (CA) mixed matrix membranes (MMMs) were prepared and studied concerning their gas separation behaviors. The dispersion of carbon nanotube fillers were studied as a factor of polymer and filler concentrations using the mixing methods of the rotor–stator system (RS) and the three-roll-mill system (TRM). Compared to the dispersion quality achieved by RS, samples prepared using the TRM seem to have slightly bigger, but fewer and more homogenously distributed, agglomerates. The green γ-butyrolactone (GBL) was chosen as a polyimide (PI) polymer-solvent, whereas diacetone alcohol (DAA) was used for preparing the CA solutions. The coating of the thin CA separation layer was applied using a spin coater. For coating on the PP carriers, a short parameter study was conducted regarding the plasma treatment to affect the wettability, the coating speed, and the volume of dispersion that was applied to the carrier. As predicted by the parameter study, the amount of dispersion that remained on the carriers decreased with an increasing rotational speed during the spin coating process. The dry separation layer thickness was varied between about 1.4 and 4.7 μm. Electrically conductive additives in a non-conductive matrix showed a steeply increasing electrical conductivity after passing the so-called percolation threshold. This was used to evaluate the agglomeration behavior in suspension and in the applied layer. Gas permeation tests were performed using a constant volume apparatus at feed pressures of 5, 10, and 15 bar. The highest calculated CO2/N2 selectivity (ideal), 21, was achieved for the CA membrane and corresponded to a CO2 permeability of 49.6 Barrer.
Despite great efforts to develop a vaccine against human immunodeficiency virus (HIV), which causes AIDS if untreated, no approved HIV vaccine is available to date. A promising class of vaccines are virus-like particles (VLPs), which were shown to be very effective for the prevention of other diseases. In this study, production of HI-VLPs using different 293F cell lines, followed by a three-step purification of HI-VLPs, was conducted. The quality-by-design-based process development was supported by process analytical technology (PAT). The HI-VLP concentration increased 12.5-fold while >80% purity was achieved. This article reports on the first general process development and optimization up to purification. Further research will focus on process development for polishing and formulation up to lyophilization. In addition, process analytical technology and process modeling for process automation and optimization by digital twins in the context of quality-by-design framework will be developed.
The oxidation of cumene and following cleavage of cumene hydroperoxide (CHP) with sulfuric acid (Hock rearrangement) is still, by far, the dominant synthetic route to produce phenol. In 2020, the global phenol market reached a value of 23.3 billion US$ with a projected compound annual growth rate of 3.4% for 2020–2025. From ecological and economical viewpoints, the key step of this process is the cleavage of CHP. One sought-after way to likewise reduce energy consumption and waste production of the process is to substitute sulfuric acid with heterogeneous catalysts. Different types of zeolites, silicon-based clays, heteropoly acids, and ion exchange resins have been investigated and tested in various studies. For every type of these solid acid catalysts, several materials were found that show high yield and selectivity to phenol. In this mini-review, first a brief introduction and overview on the Hock process is given. Next, the mechanism, kinetics, and safety aspects are summarized and discussed. Following, the different types of heterogeneous catalysts and their performance as catalyst in the Hock process are illustrated. Finally, the different approaches to substitute sulfuric acid in the synthetic route to produce phenol are briefly concluded and a short outlook is given.
Abstract
(−)‐Menthol is one of the most popular aroma compounds worldwide. While in the past mostly extracted from mint plants, today (−)‐menthol synthesis from other raw materials is becoming more relevant. Common starting materials for menthol synthesis are m‐cresol, citral and myrcene, but also substrates like menthone, mono‐ and bicyclic terpenes and terpenoids have been used for this purpose in the past. As for many applications (−)‐menthol of high purity is required, asymmetric syntheses and enantiomeric resolution of obtained raw products are applied for menthol production. This review gives an overview on the most important synthetic menthol production processes of the companies Symrise, Takasago and BASF and relevant literature in the field of menthol synthesis with a focus on the last 20 years.