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In this work, we established an efficient process for the production of itaconate from the regionally sourced industrial side-stream molasses using Ustilago cynodontis and Ustilago maydis. While being relatively cheap and more environmentally friendly than refined sugars, there are some major challenges to overcome when working with molasses. Some of those challenges are a high nitrogen load, unknown impurities in the feedstock, and high amounts of ill-favoured carbon sources, such as sucrose or lactate. We could show that the activity of the sucrose-hydrolysing enzyme invertase plays a crucial role in the efficiency of the process and that the fructose utilisation differs between the two strains used in this work. Thus, with a higher invertase activity, the ability to convert fructose into the desired product itaconate, and an overall higher tolerance towards undesired substances in molasses, U. maydis is better equipped for the process on the alternative feedstock molasses than U. cynodontis. The established process with U. maydis reached competitive yields of up to 0.38 g g -1 and a titre of more than 37 g L -1 . This shows that an efficient and cost-effective itaconate production process is generally feasible using U. maydis, which has the potential to greatly increase the sustainability of industrial itaconate production., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Next-generation bioprocesses of a future bio-based economy will rely on a flexible mix of readily available feedstocks. Renewable energy can be used to generate sustainable CO 2 -derived substrates. Metabolic engineering already enables the functional implementation of different pathways for the assimilation of C1 substrates in various microorganisms. In addition to feedstocks, the benchmark for all future bioprocesses will be sustainability, including the avoidance of CO 2 emissions. Here we review recent advances in the utilization of C1-compounds from different perspectives, considering both strain and bioprocess engineering technologies. In particular, we evaluate methanol as a co-feed for enabling the CO 2 emission-free production of acetyl-CoA-derived compounds. The possible metabolic strategies are analyzed using stoichiometric modeling combined with thermodynamic analysis and prospects for industrial-scale implementation are discussed., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Dirofilaria immitis, also known as heartworm, is a major parasitic threat for dogs and cats around the world. Because of its impact on the health and welfare of companion animals, heartworm disease is of huge veterinary and economic importance especially in North America, Europe, Asia and Australia. Within the animal health market many different heartworm preventive products are available, all of which contain active components of the same drug class, the macrocyclic lactones. In addition to compliance issues, such as under-dosing or irregular treatment intervals, the occurrence of drug-resistant heartworms within the populations in the Mississippi River areas adds to the failure of preventive treatments. The objective of this review is to provide an overview of the disease, summarize the current disease control measures and highlight potential new avenues and best practices for treatment and prevention., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Single-cell analysis in microfluidic cultivation devices bears a great potential for the development and optimization of industrial bioprocesses. High parallelization allows running a large number of cultivation experiments simultaneously even under quick alteration of environmental conditions. For example, the impact of changes in media composition on cell growth during classical batch cultivation can be easily resolved. A missing link for the scalability of microfluidic experiments is, however, their complete characterization via conventional performance indicators such as product titer and productivity. While existing mass spectrometry technology is not yet sufficiently coupled with microfluidics, optical methods like enzymatic assays or fluorescence sensors are promising alternatives but require further improvement to generate quantitative measurements of extracellular metabolites., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

This is the description of a 58-year-old female patient presenting 8 months after a horse riding accident with significant pain and inability to walk independently. Imaging revealed a large osseous defect of the lateral tibia plateau which was not united posteriorly. The patient refused knee replacement and we developed a patient specific two-step procedure for her. Step 1: Filling of the defect with a large cortico-cancellous autograft from the posterior iliac crest; step 2: Transplantation of a fresh large osteochondral shell allograft (FLOCSAT). The postoperative protocol included continuous passive motion (CPM), partial weight bearing for three months, and physiotherapy. Based on the concept of immuno-privileged cartilage tissue, the patient did not get any immuno-suppressive therapy. Pain-, activity of daily living, Lysholm and Tegner scores were evaluated before defect filling surgery with autograft, before allograft transplantation, and at 12 and 24 months after allograft transplantation. There were no complications. Radiographic analyses with plain films and CT scans revealed solid osseous integration within 3 month. The patient regained excellent functionality in both, activities of daily living and sports (back to horse riding, trampolin jumping). Knee arthroscopy after 1year showed excellent condition of the lateral meniscus and the cartilage of the lateral tibia plateau. Chimerism/DNA analysis of a cartilage biopsy showed, that at 1year 32% of the donor cells have been already replaced by the patient's own cells. To our knowledge, this is the first case of a patient who sustained such a large defect during a tibia plateau fracture, and got successfully treated with a fresh large osteochondral shell allograft transplantation in a two-step procedure., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Due to the rising interest in Europe to treat large cartilage defects with osteochondrale allografts, research aims to find a suitable solution for long-term storage of osteochondral allografts. This is further encouraged by the fact that legal restrictions currently limit the use of the ingredients from animal or human sources that are being used in other regions of the world (e.g. in the USA). Therefore, the aim of this study was A) to analyze if a Lactated Ringer (LR) based solution is as efficient as a Dulbecco modified Eagle's minimal essential medium (DMEM) in maintaining chondrocyte viability and B) at which storage temperature (4°C vs. 37°C) chondrocyte survival of the osteochondral allograft is optimally sustained., Methods: 300 cartilage grafts were collected from knees of ten one year-old Black Head German Sheep. The grafts were stored in four different storage solutions (one of them DMEM-based, the other three based on Lactated Ringer Solution), at two different temperatures (4 and 37°C) for 14 and 56days. At both points in time, chondrocyte survival as well as death rate, Glycosaminoglycan (GAG) content, and Hydroxyproline (HP) concentration were measured and compared between the grafts stored in the different solutions and at the different temperatures., Results: Independent of the storage solutions tested, chondrocyte survival rates were higher when stored at 4°C compared to storage at 37°C both after short-term (14days) and long-term storage (56days). At no point in time did the DMEM-based solution show a superior chondrocyte survival compared to lactated Ringer based solution. GAG and HP content were comparable across all time points, temperatures and solutions., Conclusion: LR based solutions that contain only substances that are approved in Germany may be just as efficient for storing grafts as the USA DMEM-based solution gold standard. Moreover, in the present experiment storage of osteochondral allografts at 4°C was superior to storage at 37°C., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Vector-borne diseases are responsible for significant health problems in humans, as well as in companion and farm animals. Killing the vectors with ectoparasitic drugs before they have the opportunity to pass on their pathogens could be the ideal way to prevent vector borne diseases. Blocking of transmission might work when transmission is delayed during blood meal, as often happens in ticks. The recently described systemic isoxazolines have been shown to successfully prevent disease transmission under conditions of delayed pathogen transfer. However, if the pathogen is transmitted immediately at bite as it is the case with most insects, blocking transmission becomes only possible if ectoparasiticides prevent the vector from landing on or, at least, from biting the host. Chemical entities exhibiting repellent activity in addition to fast killing, like pyrethroids, could prevent pathogen transmission even in cases of immediate transfer. Successful blocking depends on effective action in the context of the extremely diverse life-cycles of vectors and vector-borne pathogens of medical and veterinary importance which are summarized in this review. This complexity leads to important parameters to consider for ectoparasiticide research and when considering the ideal drug profile for preventing disease transmission., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The use of autologous cells for the coating of implant surfaces presents a promising tool to attenuate foreign body reaction and inflammation. However, insertion forces that occur especially during implantation of electrodes into the narrow cochlea may strip off cells from the surface. Thus, implant surfaces should be ideally structured in a way that protects the cell coating from mechanical removal during implantation. The structuring of implant surfaces may also direct cells towards desired functions to further enhance their performance and clinical suitability. In this study, grid-like square cavities were generated on thermoplastic polyurethane (TPU) surfaces using a combination of femtosecond laser ablation and replication methods. Afterwards, they were tested as potential scaffolds for human bone marrow-derived mesenchymal stem cells (MSCs) in order to use it on neural prostheses. Structured and non-structured TPU allowed proper adhesion and survival of MSCs. Surface structuring resulted in regulation of over 500 genes. Many of the upregulated genes are known to be involved in anti-inflammatory, anti-fibrotic and wound healing processes whereas genes relevant for mesenchymal differentiation programs were downregulated. The enhanced secretion of two representative factors (prostaglandin E2 and interleukin-1 receptor antagonist, respectively) was confirmed by ELISA and the downregulation of other genes involved in adipogenic and osteogenic differentiation were confirmed by gene expression analysis for a cultivation period of up to 21 days. In addition, mRNA of the surface antigens CD24 and ENDOGLIN (CD105) as representative factors for stemness did not show notable variation between cultivation on structured versus non-structured TPU or between 7 versus 21days of cultivation. Thus, surface topography of TPU seems to be a powerful tool to protect cells from mechanical forces during insertion and to influence cell behaviour., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The soil bacterium Corynebacterium glutamicum is one of the best-studied production hosts for industrial biotechnology, and it is primarily used for the large-scale production of essential amino acids, such as l-lysine. For rational strain development, detailed knowledge of intracellular protein concentration is crucial to determine metabolic capacities and limitations. We developed a QconCAT approach for the accurate absolute quantification of key enzymes of C. glutamicum glycolysis and anaplerosis. Following well-defined batch cultivations, 10 metabolic enzymes were quantified, accounting for approximately 6% of the total cell dry weight. Copy numbers per cell ranged from 36,700±3500 for phosphofructokinase (PFK) to 507,700±40,300 for enolase (ENO), which is considerably lower than the corresponding data obtained from Saccharomyces cerevisiae. Moreover, accurate measurement of the biovolume permitted an estimation of molar concentrations of intracellular enzyme catalysts ranging from 7.6±1.9μM (PFK) to 105.2±28.6μM (ENO). Finally, model-assisted data evaluation demonstrates that our method provides an important cornerstone toward a more detailed mechanistic understanding of C. glutamicum metabolism., Biological Significance: Determination of absolute protein amounts using quantitative concatemers (QconCAT's) has already been successfully demonstrated for various species including human, animal and yeast. Interestingly, application of the QconCAT methodology for the determination of cytoplasmic enzyme concentrations in a prokaryote has not been described so far. This study is concerned with a novel targeted approach for the absolute quantification of 10 key enzymes from the central carbon metabolism of the industrially important organism Corynebacterium glutamicum. We demonstrate a method that enables complete cell lysis of this robust soil bacterium, thus allowing for accurate quantification of cytoplasmic enzymes. By linking measured enzyme amounts with respective biovolume data, intracellular enzyme concentrations were estimated, which are of special importance for any systems biology approach studying C. glutamicum's metabolism at the mechanistic level. To our knowledge this is the first report of applying the QconCAT methodology for determining intracellular enzyme concentrations in a prokaryote., (Copyright © 2014. Published by Elsevier B.V.)

Sustained progress in metabolic engineering methodologies has stimulated new efforts toward optimizing fungal production strains such as through metabolite analysis of Penicillium chrysogenum industrial-scale processes. Accurate intracellular metabolite quantification requires sampling procedures that rapidly stop metabolism (quenching) and avoid metabolite loss via the cell membrane (leakage). When sampling protocols are validated, the quenching efficiency is generally not quantitatively assessed. For fungal metabolomics, quantitative biomass separation using centrifugation is a further challenge. In this study, P. chrysogenum intracellular metabolites were quantified directly from biomass extracts using automated sampling and fast filtration. A master/slave bioreactor concept was applied to provide industrial production conditions. Metabolic activity during sampling was monitored by 13C tracing. Enzyme activities were efficiently stopped and metabolite leakage was absent. This work provides a reliable method for P. chrysogenum metabolomics and will be an essential base for metabolic engineering of industrial processes., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Selected reaction monitoring allows quantitative measurements of proteins over several orders of magnitude in complex biological samples. Here we present a targeted approach for quantification of 19 enzymes from Corynebacterium glutamicum applying isotope dilution mass spectrometry coupled to high performance liquid chromatography (IDMS-LC-MS/MS). Investigations of protein dynamics upon growth on acetate and glucose as sole carbon source shows highly stable peptide amounts for enzymes of the central carbon metabolism during the transition phase and after substrate depletion. However significant adaptations of protein amounts are observed between both growth conditions well agreeing with known changes in metabolic fluxes. Time-resolved measurements of protein expression after metabolic switch from glycolytic to gluconeogenetic conditions reveal fast responses in protein synthesis rates for glyoxylate shunt enzymes., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Mechanistic (also called kinetic) models quantitatively describe dynamic and steady states of biochemical pathways. They are based on network structure (stoichiometry), regulatory information (enzyme inhibitors and activators) and the corresponding reaction kinetics. Although this approach to understand and predict the behavior of biochemical networks has now been in use for almost half a century, its experimental foundation has dramatically changed in the data-rich age of systems biology. Large mechanistic models, ranging up to the genome scale, are now being built and lots of data are available to validate and test them. From the broad scope of possible modeling applications, this survey focuses on the recent developments and central problems of metabolic network modeling in the field of bioprocess development for industrial biotechnology., (Copyright © 2011 Elsevier Ltd. All rights reserved.)